Biology subjects

Williams, B.

Publications and source records attributed to Williams, B..

At least 19 recordsLinked to original sources

bioRxiv2026

A conformational switch governs the dual role of liquid-liquid phase separation in amyloid fibrillization

Biomolecular condensates provide dynamic environments that store and organize proteins, yet the fundamental principles determining whether condensation promotes or suppresses irreversible protein aggregation remain unclear. Here, we show that the receptor-interacting protein kinase 3 (RIPK3) RHIM domain follows distinct self-assembly pathways into condensates or amyloid fibrils depending on its conformational state. We find that predominantly folded proteins undergo liquid-liquid phase separation (LLPS), forming reversible condensates that kinetically suppress fibril formation. Partial unfolding instead promotes direct fibrillization that bypasses LLPS, whereas induced condensate formation via increasing ionic strength or addition of molecular crowder delays amyloid formation. In contrast, when the protein is predominantly unfolded, LLPS accelerates fibrillization through condensate-interface-mediated nucleation. These findings establish protein conformation as a determinant of whether condensates suppress or promote amyloid assembly, revealing a dual role for phase separation in regulating functional amyloid formation and providing a framework that connects condensate dynamics with cellular functions.

biophysics↗

bioRxiv2026

Xylazine Reshapes the Sleep and Respiratory Consequences of Chronic Fentanyl Exposure in a Sex-Dependent Manner

Recent shifts in the illicit fentanyl supply have led to widespread exposure to xylazine, yet the biological consequences of chronic fentanyl-xylazine co-exposure remain poorly understood. Here, we compared the behavioral and molecular effects of chronic exposures to fentanyl and xylazine both alone and in combination in male and female mice. Combined fentanyl-xylazine produced distinct, sex-dependent alterations in sleep architecture, including changes in NREMS recovery, REMS dynamics, and reduced sleep bout duration compared with fentanyl alone. The 2-adrenergic receptor antagonist yohimbine partially reversed these sleep alterations in a sex-dependent manner. Fentanyl-xylazine also exacerbated respiratory depression relative to fentanyl alone, particularly in males. In contrast, despite its prevalence in humans no skin lesions were observed. Molecular analyses revealed that fentanyl-xylazine co-administration leads to increased peripheral Fibroblast growth factor 21, a hepatokine linked to reduced opioid preference and alcohol consumption. Together, these findings demonstrate that xylazine substantially alters the physiological and neurobiological consequences of chronic fentanyl exposure in a sex-dependent manner, providing a preclinical framework for understanding the growing public health impact of fentanyl-xylazine co-use.

neuroscience↗

bioRxiv2026

Caffeinated Coli: Inspiring the Next Generation of Scientists through Synthetic Biology

The Caffeinated Coli Educational Module brings inquiry-driven learning to high schools, introducing students to scientific research, synthetic biology, and genetic engineering. The module focuses on the exploration of a genetically engineered strain of E. coli modified to grow exclusively on caffeine and, as such, can be used as a measurement device to determine the amount of caffeine in a liquid or beverage. Students conduct two bioassay experiments using these bacteria. During this process, they learn to create cultures and then measure bacterial growth followed by calculating the caffeine concentrations of unknown samples using their own data. This flexible module contains five weeks of original lectures and student learning materials that follow Next Generation Science Standards (NGSS), allowing the content to be adapted for basic, intermediate, or advanced biology courses. Upon completion of the module, students and teachers expressed that the most memorable aspects of the module include collaboration with peers, hands-on learning of content, and the opportunity to interact with the professor/mentors through office hours. Already implemented in 8 Texas high schools over the past two academic years, our module inspires STEM learning while bringing 21st century biology research to new audiences.

scientific communication and education↗

bioRxiv2026

An in vitro regeneration system with efficient rooting in sweet orange (Citrus sinensis) supports recovery of transgenic plants

In vitro regeneration of Citrus plants is a widely used method, however, induction of adventitious roots from regenerated shoots remains a major bottleneck, limiting the recovery of healthy plants for commercial production and genomic research for crop improvement. We established an in vitro regeneration system producing profuse, healthy roots for sweet orange (Citrus sinensis cv. Benyenda) by optimising combinations and concentrations of auxins. Prior to optimising the rooting media (RTMs), we obtained a shoot regeneration rate of 90.6% from sweet orange epicotyl explants using a cytokinin, 6-benzylaminopurine (BAP). Across twelve auxin-supplemented RTMs containing different concentrations of indole-3-butyric acid (IBA) and/or 1-naphthaleneacetic acid (NAA), rooting percentages ranged from 8 - 87.5%. The combination of IBA 1.0 mg L-1 and NAA 0.1 mg L-1 promoted the best overall performance, 75 {+/-} 7.2% rooting percentage with healthy, callus-free roots ([≥]5 cm in length), whereas other RTMs with other auxin combinations induced callus and limited root elongation. The best-performing SRM and RTM were subsequently used for selection and recovery of transgenic sweet orange lines carrying an empty CRISPR/Cas9 construct, resulting in an 4.8% transformation efficiency. Both transgenic and non-transgenic rooted plantlets were successfully acclimatised under glasshouse conditions with a survival rate of 90%. This enhanced regeneration system overcomes rooting bottleneck and improves plant survival,enabling faster recovery of transgenic citrus lines within four months. It supports accelerated development for commercial applications and advances in citrus genetic improvement.

plant biology↗

bioRxiv2026

A dish-to-biobank framework links β-cell nutrient-stress programs to genetic and dietary risk for Type 2 Diabetes

Type 2 diabetes (T2D) arises from genetic susceptibility and chronic metabolic stress, but whether these converge on shared molecular programs in human populations remains unclear. Here, we develop a dish-to-biobank framework linking controlled {beta}-cell perturbation to population-scale disease genetics through the circulating plasma proteome, and apply it to T2D. scRNA-seq of human stem cell-derived islets under factorial glucose and palmitate exposure identifies their combination (glucolipotoxicity) as the condition eliciting the strongest SC-{beta} cell transcriptional response, with glucolipotoxicity-upregulated genes uniquely enriched for T2D heritability, monogenic diabetes genes, and rare-variant burden signals. CRISPR knockout of {beta}-cell identity regulators PAX6 and PDX1 aligns with this program, establishing convergence of environmental and genetic perturbations on a shared disease-relevant state. We then used the plasma proteome as an accessible population-scale readout of these experimentally defined {beta}-cell stress programs, scoring 45,956 UK Biobank White British participants. We define heritable stress signatures that associate with refined carbohydrate and saturated fat intake, and undergo trans-tissue genetic regulation, with a subset of variants showing diet-dependent effects. Together, these findings establish glucolipotoxicity as a genetically anchored model of {beta}-cell dysfunction and provide a generalizable framework for linking controlled cellular perturbations to human disease genetics at population scale.

genetics↗

bioRxiv2026

Distinct cell-type contributions and network topography of theta-nested gamma oscillations in the medial entorhinal cortex

Theta-nested gamma oscillations in the medial entorhinal cortex (mEC) are essential for spatial coding and memory, but the underlying cellular mechanisms remain unclear. We combined optogenetics, whole-cell electrophysiology, intracellular voltage imaging, and local field potential (LFP) recordings in acute slices from CaMKII-ChR2 mice to investigate how excitation and inhibition shape theta-gamma coupling in layer II/III mEC. During theta-frequency stimulation, fast-spiking interneurons received strong gamma-frequency excitation and fired rhythmic bursts, whereas stellate and pyramidal neurons fired more sparsely and were dominated by gamma-frequency inhibition. This sparse firing could support the selective firing of grid cells. Excitatory post-synaptic currents in interneurons preceded inhibitory currents and LFP gamma by [~]3 ms, supporting a pyramidal-interneuron network gamma (PING) mechanism. Pyramidal neurons fired on the descending phase of the gamma cycle, whereas stellate cells and fast-spiking interneurons fired before and after the trough, respectively. Intracellular voltage imaging revealed network gamma synchronization among excitatory neurons at a population level, with topographic clustering of subthreshold membrane potentials, but not spike timing, while individual neurons often skipped gamma cycles. These findings identify the dominant role of reciprocal E-I interactions in generating theta-nested gamma oscillations and highlight distinct cell-type contributions to the temporal dynamics of the mEC. Further, a biophysically realistic computational model predicted gamma cycle skipping in stellate cells and burst firing in fast-spiking interneurons during PING. Our experimental and computational results provide mechanistic insight into how the intrinsic properties of mEC cell types generate oscillatory activity in a manner that could support grid cell function and spatial computation. Significance StatementTheta-nested gamma oscillations in the medial entorhinal cortex (mEC) are essential for spatial navigation and memory, yet the underlying circuit mechanisms remain incompletely understood. Using optogenetics, voltage imaging, electrophysiology, and biophysically realistic computational modeling, we show that reciprocal interactions between excitatory neurons and fast-spiking interneurons generate robust gamma oscillations through a pyramidal-interneuron network gamma (PING) mechanism. We reveal cell-type specific differences in gamma phase-locking and demonstrate that principal neurons synchronize at the population-level across large laminar distances (up to 800 {micro}m). Further, the voltage activity, but not spike timing, of principal neurons exhibited topographic clustering. These findings clarify how local circuits in mEC generate temporal dynamics critical for grid cell coding and further constrain computational models of spatial representation.

neuroscience↗

bioRxiv2026

Coral restoration alters reef soundscapes but machine learning and manual analyses suggest different recovery rates

Coral restoration is recognised as a critical tool to mitigate pantropical degradation of reef ecosystems. Robust monitoring of restoration progress is crucial for projects to evaluate their success, improve practice, and share knowledge. However, traditional visual surveys often fail to capture the full impact of coral restoration on reef function. Therefore, we employed Passive Acoustic Monitoring (PAM) to assess whether the soundscape of a coral restoration site in the Seychelles differs from adjacent healthy and degraded reference reefs. We applied two methods of soundscape analysis: manual detection of unidentified fish sounds; and machine learning-based Uniform Manifold Approximation and Projection analysis. Results were approach-specific: the manual approach highlighted similarities in fish calls between the restoration site and the healthy reference reef, while the machine learning approach extracted broader soundscape patterns, clustering the restoration site alongside the degraded reference reef. Although this is a single-site study, these findings suggest that a) coral restoration alters reef soundscapes, though recovery time may be taxon-specific, and b) multiple metrics are needed to bridge single-taxon and broad soundscape scales. This study contributes to the evolving field of soundscape ecology in coral reef ecosystems, highlighting the utility of PAM in monitoring changes to reef function through coral restoration.

ecology↗

bioRxiv2026

Mechanism of Gating and Isoform-Specific Inhibition in Renal CLC Chloride Channels

Hyponatremia is a prevalent disorder marked by excess water retention and substantial morbidity, motivating interest in the CLC-Ka chloride channel as a therapeutic target. Selectively inhibiting CLC-Ka without affecting the closely related CLC-Kb is essential for preventing serious side effects. However, developing isoform-selective inhibitors has been challenging because most small molecules do not distinguish between CLC-Ka and CLC-Kb, and the basis for selectivity in the few known exceptions remains unclear. The small molecule BIM1 preferentially inhibits CLC-Ka over CLC-Kb, providing an opportunity to dissect isoform-specific pharmacology. To investigate this mechanism, we determined cryo-EM structures of BIM1 and BIM15, a related nonselective analog, bound to a CLC-K variant engineered to match the human CLC-Ka binding pocket. Structural and computational analyses reveal that inhibition and isoform selectivity are anchored by interactions with a conserved lysine, with surrounding binding-site residues subtly tuning the local electrostatic environment to promote or disfavor these contacts. These analyses further identify a dynamic extracellular loop that intermittently occludes the shared pathway accessing the inhibitor-binding site and pore. Bound BIM15 engages this gating loop more extensively than BIM1, suggesting that differential loop engagement contributes to inhibitor selectivity, a prediction validated by mutagenesis. Because loop dynamics block the pore, we examined the structural impact of Ca{superscript 2}, which favors opening, and found the gating loop ordered and withdrawn from the pathway. Together, these findings define how binding-site microenvironments and gating-loop dynamics shape isoform-specific inhibition and pore access in CLC-K channels. Significance StatementHyponatremia is a major clinical problem with limited therapeutic options. The kidney chloride channel CLC-Ka is an attractive drug target, but its high sequence identity to CLC-Kb has hindered the development of isoform-selective inhibitors needed for safe therapy. A low-micromolar CLC-Ka-selective inhibitor had been identified, providing a foothold for drug development, but the structural basis of its selectivity was unknown. Here, by integrating cryo-EM structures with molecular dynamics simulations, we define the inhibitor-binding site and reveal the mechanism that enables preferential CLC-Ka inhibition. We further show that a dynamic extracellular loop functions as a gating element shaping inhibitor access and engagement. These findings establish a mechanistic foundation for developing improved treatments for hyponatremia.

biophysics↗

bioRxiv2026

TLR7 inhibition limits cardiac ischemic injury by disrupting ITGAM-dependent immune-endothelial interaction

Percutaneous coronary intervention (PCI) limits ischemic myocardial infarction but also triggers ischemia-reperfusion (I/R) injury in part driven by innate immune activation. Here, we identify Toll-like receptor 7 (TLR7), an endosomal sensor of single-stranded RNA, as a mediator of post-ischemic inflammation and myocardial damage. Pharmacological inhibition of TLR7 with enpatoran reduced myocardial inflammation and infarct size and improved cardiac function in a mouse model of I/R injury when administered before, during, or shortly after ischemia. Single-nucleus RNA sequencing revealed coordinated post-I/R expansion of myeloid cells and distinct inflammatory endothelial subsets enriched for leukocyte-interaction programs, with marked upregulation of Itgam in cardiac leukocytes and endothelial cells and in circulating monocytes. Circulating ITGAM+ monocytes were similarly increased in patients with ST-segment elevation myocardial infarction 24 hours after coronary stenting. Mechanistically, TLR7 activation induced Itgam expression in endothelial cells and leukocytes and promoted their adhesion via ITGAM-ICAM1 interaction under physiological shear stress, whereas ITGAM neutralization disrupted this interaction, reduced immune cell infiltration, and limited ischemic injury. These findings define a TLR7-ITGAM signaling axis as a key driver of endothelial-leukocyte crosstalk in myocardial I/R injury and support TLR7 inhibition as a promising therapeutic strategy to mitigate acute myocardial infarction.

immunology↗

bioRxiv2026

Circulating miR-4532 is associated with loss of ambulation in dysferlinopathy

BackgroundLimb-girdle muscular dystrophies (LGMDs) are inherited myopathies characterized mainly by progressive weakness of the proximal muscles of the shoulder and pelvic girdle areas, leading to functional decline and eventual loss of independent ambulation. Dysferlinopathy (LGMD2B) is an autosomal recessive LGMD subtype, is caused by mutations in the DYSF gene that lead to lack of dysferlin which results in muscle death and chronic muscle fiber degeneration. Preservation of ambulation is a key clinical milestone, as loss of independent gait markedly reduces quality of life and complicates care management. Although patients often perceive functional decline before their initial clinical presentation, current clinical assessments typically detect disease progression after substantial muscle damage has occurred. MethodsIn this multigroup case-control study, we profiled plasma miRNAs from 49 genetically confirmed dysferlinopathy patients (24 ambulatory, 25 non-ambulatory) and 25 age- and sex-matched healthy controls. Total RNA was extracted from blood samples and hybridized to Affymetrix GeneChip miRNA 3.1 arrays. After quality control and filtering, differential expression analysis was performed using linear models for microarrays, adjusting for age and sex, with a false discovery rate cutoff of 10%. Results14 miRNAs were significantly altered between dysferlinopathy patients and controls. Notably, miR-4532 was upregulated in ambulatory patients relative to controls, whereas it was downregulated in non-ambulatory patients compared with ambulatory patients, although expression levels remained higher than in controls. Levels of miR-4532 were positively associated with circulating monocyte levels in ambulatory patients only. ConclusionThese results suggest that miR-4532 may be a circulating marker associated with ambulatory status in dysferlinopathy. Its known involvement in inflammatory signaling and muscle regeneration pathways underscores its potential as an early indicator for disease activity.

bioinformatics↗

bioRxiv2025

Accessible AI Enhances Monitoring of Coral Seeding Devices in Reef Restoration

1. Coral seeding devices (CSDs) - tools designed to deliver sexually propagated corals to target locations - offer a promising means to increase coral abundance on degraded reefs. However, evaluating CSD effectiveness for coral reef restoration across wide areas and over many years is limited by the lack of robust and efficient monitoring approaches. Large-area reef imagery offers an attractive potential solution, but manual detection of CSDs within imagery is slow and limits the scalability of CSD monitoring. 2. We investigated whether machine learning classifiers could accurately detect CSDs in reef orthoimages and tested the performance of classifiers created following minimal manual annotation effort. Using freely available software, we first evaluated classifier performance in a single-site experiment using an orthoimage containing 989 CSDs deployed in Palau. We then also evaluated performance in a multi-site experiment using orthoimages containing a different CSD design deployed across seven sites in the central Great Barrier Reef, Australia. 3. In Experiment 1, classifiers trained on just 30 CSDs annotated within 10 minutes achieved mean recall and precision of 96.7% and 97.1% respectively, reducing manual annotation time by 95.6% whilst still detecting 98.8% of the number of devices found manually. Larger training sets yielded less reliable classifiers and required more manual effort. In Experiment 2, classifiers trained on 30 CSD annotations from one site performed excellently across seven orthoimages from multiple reefs, achieving mean recall and precision of 99.2% and 93.3%. 4. We present evidence that CSD classifiers can be highly effective across both single- and multi-site CSD deployments. In using a free and user-friendly software, we also demonstrate their accessibility to reef restoration practitioners. To facilitate wider uptake of CSD monitoring, we provide a step-by-step protocol for implementing CSD classifiers. By improving access to efficient, direct assessment of intervention outcomes, this method can play a vital role in guiding the enhancement of approaches aiming to restore coral reefs.

ecology↗

bioRxiv2025

Hundred-Picolitre Droplet on Demand Tape Drive and XES Prototypes for Time-Resolved Serial Crystallography at VMXi, Diamond Light Source

Time resolved X-ray crystallography is experiencing a resurgence, in part, because of serial methods that readily allow scientists to create stop-motion movies of macromolecular function of photoactivation, enzyme catalysed reactions, and ligand-induced conformational changes triggering further downstream signalling events. While some reactions can be initiated with light, either naturally or using photocaged compounds, a more generally applicable approach is to mix microcrystals with reagents at varying time points prior to exposure to the X-ray beam. A powerful approach has been to combine droplet on demand tape drive sample delivery with X-ray emission spectroscopy (XES) that correlates atomic structure with electronic states of metal ions within the sample. To our knowledge, such a combined methodology has not been deployed previously at a synchrotron beamline and has been restricted to XFELs. Here we describe prototype experiments along the development pathway to a combined droplet on demand diffraction and XES system at the microfocus beamline VMXi at Diamond Light Source. We demonstrate the collection of a high-quality serial diffraction data set from microcrystals within hundreds of picolitre-volume droplets deposited on a moving tape. In separate experiments at VMXi, we collected XES data from microcrystals of a copper enzyme delivered using a high viscosity extruder. Together, these results demonstrate the feasibility of combined droplet on demand serial crystallography and XES experiments using a third-generation synchrotron beamline; project work currently underway at Diamond Light Source. SynopsisWe describe proof of concept experiments towards correlated serial crystallography (SSX) and X-ray emission spectroscopy (XES) from microcrystals at a microfocus synchrotron beamline. A droplet on demand tape drive system delivers microcrystals to the beam within well-separated, hundreds of picolitre-volume droplets while XES allows validation of redox states of metals within protein crystals.

biophysics↗

bioRxiv2025

Design and development of a SARS and MERS Combination Vaccine

This work was conducted during the COVID-19 pandemic prior to the licensing of any vaccines against COVID-19. Although several COVID-19 vaccines are now commercially available, this research on the development of a combination Severe Acute Respiratory Syndrome-associated Coronavirus (SARS-CoV), SARS-coronavirus 2 (SARS-CoV-2), and Middle Eastern Respiratory Syndrome (MERS) is still relevant and shows how a combination vaccine can be designed, produced and rapidly tested in the laboratory. We present the development of a combination vaccine designed to provide immunity against Severe Acute Respiratory Syndrome-associated Coronavirus (SARS-CoV), SARS-coronavirus 2 (SARS-CoV-2), and Middle Eastern Respiratory Syndrome (MERS). The primary objective of this vaccine design is twofold: to mitigate the burden of coronavirus and to address the specific vulnerability of regions prone to recurrent MERS outbreaks. Our combination vaccine incorporates antigenic components from zoonotic sources, specifically SARS-CoV, SARS-CoV-2, and MERS. We assess the impact of combining different Spike proteins S1 subunit antigens, due to its recognised immunogenic potential, and adjuvants on serum antibody titres, virus neutralizing capabilities, and inter-antigen immune responses. We report a robust and broad antibody response against SARS-CoV-2 and related coronaviruses, which was amplified by different adjuvant formulations, including alum, MPLA, CpG, and Squalene-in-Water Emulsion.

immunology↗

bioRxiv2025

Evidence of ecosystem process recovery across a large-scale coral reef restoration programme using AI accelerated soundscape analysis

1.Coral reef restoration efforts are on the increase globally. However, reporting on the ecological outcomes of these efforts is rare and typically focuses on coral related metrics. As a result, understanding of whether restoration can recover broader aspects of reef functioning remains limited. In this study we use passive acoustic monitoring coupled with human-in-the-loop artificial intelligence to analyse >12 months of soundscape recordings from 45 sites across five biogeographically independent regions to investigate the impact of active restoration on reef functioning. We trained and rigorously evaluated machine learning models to identify 34 biological sound types within this data, generating >912,000 high-confidence detections. These detections were used to infer four key functions across healthy, degraded, early-stage (<3 months) and mid-stage (32-53 months) restored reefs. Restoration significantly enhanced: (i) biological sounds at night, key to recruiting juvenile fish; (ii) diversity of biological sounds, an indicator of fish community diversity; and (iii) snapping shrimp activity, an indicator of bioturbation. However, effects varied by region, and audible parrotfish grazing, key to algal control and bioerosion, did not differ among habitat types in four of the five regions. Our findings provide evidence that restoration can support recovery of broader ecosystem functioning when carefully implemented in the right contexts. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=174 SRC="FIGDIR/small/678197v4_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@7d5462org.highwire.dtl.DTLVardef@2efa7dorg.highwire.dtl.DTLVardef@3f4b9eorg.highwire.dtl.DTLVardef@17d829c_HPS_FORMAT_FIGEXP M_FIG C_FIG

ecology↗

bioRxiv2025

Fast spiking interneurons autonomously generate fast gamma oscillations in the medial entorhinal cortex with excitation strength tuning ING-PING transitions

Gamma oscillations (40-140 Hz) play a fundamental role in neural coordination, facilitating communication and cognitive functions in the medial entorhinal cortex (mEC). While previous studies suggest that pyramidal-interneuron network gamma (PING) and interneuron network gamma (ING) mechanisms contribute to these oscillations, the precise role of inhibitory circuits remains unclear. Using optogenetic stimulation and whole-cell electrophysiology in acute mouse brain slices, we examined synaptic input and spike timing in neurons across layer II/III mEC. We found that fast-spiking interneurons exhibited robust gamma-frequency firing, while excitatory neurons engaged in gamma cycle skipping. Stellate and pyramidal cells received minimal recurrent excitation, whereas fast-spiking interneurons received strong excitatory input. Both excitatory neurons and fast-spiking interneurons received gamma frequency inhibition, emphasizing the role of recurrent inhibition in gamma rhythm generation. Notably, gamma activity was reduced, but persisted after AMPA/kainate receptor blockade, indicating that interneurons can sustain gamma oscillations independently through an ING mechanism. Selective activation of PV+ interneurons confirmed their ability to sustain fast gamma inhibition autonomously. To further assess the interplay of excitation and inhibition, we developed computational network models constrained by our experimental data. Simulations revealed that weak excitatory input to interneurons supports fast ING-dominated rhythms ([~]100-140 Hz), while strengthening excitatory drive induces a transition to slower PING-dominated oscillations (60-100 Hz). These findings highlight the dominant role of inhibitory circuits in sustaining gamma rhythms, demonstrate how excitation strength tunes the oscillatory regime, and refine models of entorhinal gamma oscillations critical for spatial memory processing. Significance StatementGamma oscillations in the medial entorhinal cortex (mEC) are essential for spatial navigation and memory, yet the mechanisms underlying their generation remain unresolved. Combining optogenetics, whole-cell electrophysiology, and computational modeling, we show that fast-spiking interneurons can autonomously sustain gamma rhythms via interneuron network gamma (ING). Blocking excitatory input reduced, but did not abolish gamma-frequency inhibition, and selective activation of PV+ interneurons confirmed their capacity to generate fast gamma independently. Modeling revealed that excitatory strength tunes the oscillatory regime, with weak excitation favoring fast ING and stronger excitation inducing slower pyramidal-interneuron network gamma (PING). These findings refine entorhinal gamma models and suggest a hybrid mechanism for switching between faster and slower gamma critical for spatial computation.

neuroscience↗

bioRxiv2025

ARAP2 regulates responses to interferon-gamma by restricting SOCS1

Interferon-gamma (IFN{gamma}) is critical for immunity against intra-macrophagic pathogens, signaling through the JAK-STAT pathway to induce a tyrosine-phosphorylation cascade that ensures a potent immune response. Excessive JAK-STAT signaling can drive hyperinflammation and autoimmunity, and thus signaling is tightly and selectively regulated by the IFN{gamma}-inducible protein, Suppressor of Cytokine Signaling 1 (SOCS1). SOCS1 inhibits signaling by directly blocking JAK kinase activity. Here we identified a SOCS1-interacting partner, ARAP2 that fine-tunes SOCS1 function. We report that tyrosine 415 in ARAP2 binds the SOCS1-Src Homology 2 (SH2) domain and limits the ability of SOCS1 to inhibit IFN{gamma} signaling. Our findings show that ARAP2 promotes the IFN{gamma} response through a phosphorylation dependent interaction with the negative regulator SOCS1.

cell biology↗

bioRxiv2025

Structural Studies of an Anti-necroptosis Viral:Human Functional Hetero-amyloid M45:RIPK3 using SSNMR

The formation of RIP-homotypic interaction motif (RHIM)-based heteromeric amyloid assemblies between effector proteins such as RIPK1, ZBP1, or TRIF and the kinase RIPK3 serve as regulating signals for the necroptosis process, a key element of innate immune defense. Murine cytomegalovirus (MCMV) expresses the M45-encoded viral inhibitor of RIP activation (vIRA) which inhibits necroptosis in a RHIM-dependent manner. A pivotal question is how viral M45 forms hetero-amyloids with RIPK3 to effectively create an inhibitory assembly. We report a novel high-resolution structure of the M45:RIPK3 complex where M45 and RIPK3 alternately stack in an amyloid-state structure. Mutagenesis of the residues flanking the IQIG tetrad in M45 results in specific impacts on co-assembly with RIPK3, indicating an extended interface in the heteromeric fibrils. Other key interactions support the formation of stable viral:host fibrils. The M45: RIPK3 hetero-amyloid is likely to act as an anti-necroptotic signal by competing with formation of other pro-necroptotic species and introducing a barrier to RIPK3 autophosphorylation. Significance StatementThis study investigates the structural biology of the necroptotic pathway, an understudied programmed cell death mechanism that plays a crucial role in innate immunity and has implications for infectious diseases, cell cycle regulation, and cancer. We present the high-resolution structure of a cross-species hetero-amyloid in which M45, a murine cytomegalovirus (MCMV) protein, co-assembles with human RIPK3 to inhibit necroptosis by competing with pro-necroptotic amyloids. Using solid-state NMR, cryo-EM, mutagenesis, and biophysical analyses, we uncover a novel structural paradigm for cross-species hetero-amyloids, shedding light on viral strategies to manipulate host immunity and protein interactions.

biophysics↗

bioRxiv2025

CTCF directly binds G-quadruplex structures to regulate genome topology and gene expression

DNA G-quadruplexes (G4s) are non-B form secondary DNA structures that are highly conserved across evolution. G4 structures occupy key regulatory sites in the mammalian genomes and are implicated in several cellular processes. However, the mechanisms by which G4s contribute to distinct facets of genome function are not well understood. Here, we conduct a proteomics screen with G4s of diverse topologies to uncover novel G4 binding activities in genomic regulators of nucleosome remodeling, paraspeckle assembly, RNA splicing, and 3D genome organization. Among the most prominent hits, we identify the genomic architectural protein, CTCF, as one of the strongest G4 binders. Building on this discovery, we perform extensive biochemical validation of CTCF-G4 interaction and identify CTCF mutants, with pronounced affinity for G4s over its consensus DNA motif. By implementing well-established approaches and developing new G4 mapping tools, we define a comprehensive catalog of genomic G4s and investigate their association with CTCF binding. Using genetic reconstitution of mouse embryonic stem cells with a G4-specific CTCF mutant, we ascribe genomic functions to CTCF-G4 interactions in regulation of CTCF occupancy, chromatin looping and gene expression. Interestingly, our studies reveal a subset of G4-linked chromatin loop anchors that form "persistent loops" which are retained even upon CTCF depletion. Collectively, our work establishes the G4 binding activity of CTCF and provides new insights into the functional significance of G4 structures.

molecular biology↗