Biology subjects

Dong, Y.

Publications and source records attributed to Dong, Y..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

Ultrahigh-Throughput Forster Resonance Energy Transfer-Activated Droplet Sorting for Terminator Polymerase Engineering

Droplet microfluidic screening systems enable high-throughput, labor-saving enzyme directed evolution by employing fluorescence, absorbance, and Raman-activated sorting strategies for library screening. Forster resonance energy transfer (FRET) - a nanoscale technique for monitoring intramolecular/intermolecular conformational changes - is yet to be integrated into this process. We upgraded a single-channel sorter to a dual-channel one without redesigning the microscopy setup, which can monitor FRET signals during enzyme reactions in droplets at kilohertz rates. We applied this upgraded sorter to improve the incorporation efficiency of KOD DNA polymerase towards reversible terminator deoxyribonucleotides, a property crucial for its application in next-generation sequencing (NGS). Our data show that a single-round sorting can achieve 30-fold enrichment of active variants. Five KOD variants enabling 100-cycle single-end runs of DNA sequencing were identified using a novel cyclic reversible termination (CRT) substrate featuring a terminator group {approx} 5-fold bulkier than the classical azidomethyl moiety. We also engineered a metagenome-derived novel polymerase, and a variant achieving 90% terminator incorporation efficiency within 2 minutes was identified after two rounds of enrichment. In sum, we provide a practical setup for dual-channel FRET-based droplet sorting, and demonstrate its ability in terminator polymerases engineering, thereby broaden the scope of microfluidic applications.

biochemistry↗

bioRxiv2025

Windows to the goal: Pupillary working memory signatures prospectively adapt to task demands

The pupillary light response was once considered a brainstem reflex, but newer findings indicate that pupil dilation can also reflect content held in mind with working memory (WM). This suggests that WM may recruit even the earliest sensorimotor apparatus for maintenance. Here, we tested two boundaries of this pupillary WM response: whether it generalizes beyond low-level stimuli and whether it adapts to changing behavioral goals. Namely, we tested whether the pupils reflect remembered brightness for real-world scene images, and whether the effect varies when different features dimensions are emphasized for the memory test (i.e., visual detail vs. semantic category). We found a feature-specific pupillary WM effect for remembering natural scenes, but only when the task encouraged a visual maintenance strategy. Rather than a retrospective echo of sensory-evoked stimulus features, the pupillary WM response prospectively adapts to how the memory content will be used.

neuroscience↗

bioRxiv2025

Ecology of hydrocarbon degradation in widespread viruses

Hydrocarbons are vital for energy production and lead to serious environmental issues due to pollution. Microorganisms largely drive the production and degradation of hydrocarbons, yet little is known about viral contributions to hydrocarbon degradation. Here we identified 786 viral contigs from IMG/VR(v4), encoding five aerobic (alkB, ladA, almA, ndoB and dszC) and three anaerobic (bssA, ebdA and abcA) hydrocarbon-degrading genes (HDGs). vOTUs encoding HDGs span 26 distinct viral families, including 249 are mainly associated with host-associated environment and 463 are derived from aquatic habitat, respectively, implying that these viruses are broadly engaged in hydrocarbon-degradation processes across the entire biosphere. alkB (35%) and almA (28%) was tended to be encoded by Schizomimiviridae, bssA was tended to be encoded by T5-like bacteriophages (29%) and SPO1-like bacteriophages (22%), suggesting that the carriage of HDGs by viruses exhibits taxonomic specificity. With respect to host associations, Pseudomonadota and Bacillota constitute the two main potential host lineages, associated with 268 and 71 vOTUs, respectively. The profiling of ecological footprint reveals that viruses contribute a total of up to 30% gene abundance and 32% transcribing activity to hydrocarbon degradation in the global ocean. This study systematically revealed the distribution, diversity, virus-host correlation and activity of virus-encoded HDGs, underscoring the significant role of viruses in hydrocarbon metabolism on a global scale.

ecology↗

bioRxiv2025

Development of Long-Term Human Adipocyte Organoids Manifesting Aging in Response to Intermittent Hypoxia

Obstructive sleep apnea (OSA) (and consequent intermittent hypoxia (IH)) is increasingly recognized as a driver of adipose tissue dysfunction, insulin resistance, and aging. However, current in vitro experimental models inadequately capture the long-term effects of IH on human adipocytes. Here, we report the development and optimization of a robust long-term human adipocyte organoid culture system that faithfully recapitulates IH-induced adipocyte aging in vitro. Human stromal vascular fraction (SVF) cells, isolated from subcutaneous abdominal fat biopsies, were embedded in Matrigel and seeded into Biofloat U-bottom 96-well plates. Using a 1:1 Matrigel-cell mixture and optimized seeding volumes (5-20 {micro}L), we established adipocyte organoids that formed within 10-12 days and remained viable with stable morphology for up to 90 days or more. Matrigel was essential for organoid integrity, while alternative matrices such as gelatin and low-melting agarose failed to support proper organoid formation. Subcutaneous preadipocyte medium with 10% FBS from ZenBio was superior to "Advanced/F12K" medium for adipogenic differentiation and long-term maintenance. To model OSA-related hypoxic stress, we exposed organoids to intermittent hypoxia using a programmable hypoxia chamber. IH treatment suppressed adipogenesis, as shown by reduced lipid accumulation, downregulation of adipogenic markers (e.g., PPAR{gamma}, adiponectin, FABP4), and smaller intracellular lipid droplets. Transmission electron microscopy (TEM) revealed IH-induced structural abnormalities, including ER fragmentation, mitochondrial disruption, nuclear enlargement, and heterochromatin formation, all of which are hallmarks of cellular aging. Furthermore, IH upregulated HIF1, H2AX, and aging-associated histone methylation markers (H3K9me3, H3K79me3, H4K20me3), as well as extracellular matrix remodeling proteins such as fibronectin and LOX. Insulin signaling was also impaired, evidenced by decreased phosphorylation of PI3K and AKT. Collectively, these results establish a reliable platform for long-term human adipocyte organoid culture and demonstrate its utility in modeling IH-induced adipocyte dysfunction and aging. This system offers a physiologically relevant tool for mechanistic studies and preclinical therapeutic screening targeting hypoxia-related metabolic disorders.

cell biology↗

bioRxiv2025

IgG-Bridging-Seeded Synergistic Aggregation of SARS-CoV-2 Spikes Underlies Potent Neutralization by A Low-Affinity Antibody

Mechanistic studies of viral neutralization typically prioritize high-affinity antibodies, relegating low-affinity binders to the sidelines. We report P5-1C8, a Class 1 SARS-CoV-2 antibody that exemplifies this underexplored "low-affinity yet high-potency" phenotype, retaining strong neutralization of Omicron JN.1 despite markedly weakened trimer binding (KD = 225 nM; IC50 = 0.06 nM). Structural and biophysical analyses reveal that P5-1C8 engages WT and BA.1 spikes through canonical intra-spike bivalency, but with JN.1 it induces aggregation. Using virion-like nanoparticles displaying multiple spikes, we show that IgG remains bound with no detectable dissociation and triggers pronounced aggregation. Coarse-grained molecular dynamics delineate the stepwise pathway in which weak IgG-spike contacts seed aggregation via transient inter-spike bridging. Together, these findings establish the first mechanistic framework demonstrating how weak-binding antibodies can nonetheless achieve potent neutralization through higher-order aggregation, thereby expanding the conceptual landscape of antibody function and opening new directions for antibody evaluation and design. Graphical AbstractLow-affinity antibodies are frequently disregarded in discovery pipelines. This work reports P5-1C8, a Class 1 SARS-CoV-2 antibody with weak trimer binding (KD-to-IC50 > 3,700-fold) yet potent neutralization of Omicron JN.1. Structural, biophysical, functional and coarse-grained simulations collectively demonstrate that transient inter-spike IgG bridging seeds higher-order aggregation, which in turn drives neutralization and provides a mechanistic framework. O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/691454v1_ufig1.gif" ALT="Figure 1000"> View larger version (79K): org.highwire.dtl.DTLVardef@1d4f132org.highwire.dtl.DTLVardef@1274400org.highwire.dtl.DTLVardef@e186a6org.highwire.dtl.DTLVardef@4f1870_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

NanoporeDB: A Structural Resource Of Multimeric Protein Nanopores In Single-Molecule Sensing

Protein nanopores are essential molecular gateways in biology and have inspired transformative technologies in biosensing and single-molecule sequencing. While this technology has transformed genomics and biosensing, the discovery of novel nanopore scaffolds remains limited due to the scarcity of experimentally resolved pore structures. Here, we present NanoporeDB, an open-access structural resource comprising over 6,600 high-confidence multimeric models across four representative pore types. Candidate proteins were systematically mined from large-scale datasets, including the AlphaFold Protein Structure Database, UniRef90, and MGnify90, and assembled using AlphaFold-Multimer and AlphaFold3. We performed membrane embedding, pore axis annotation, and constriction profiling to enable functional interpretation. NanoporeDB features an interactive web interface with 3D visualization and quantitative metrics such as insertion depth, tilt angle, and pore geometry. This resource provides a structural foundation for advancing nanopore-based molecular sensing, precision diagnostics, and synthetic biology.

bioinformatics↗

bioRxiv2025

Orchestrating Spatial Transcriptomics Analysis with Bioconductor

Spatial transcriptomics technologies provide spatially-resolved measurements of gene expression through assays that can either target selected genes or capture transcriptome-wide expression profiles. The complexity and variability of these technologies and their associated data necessitate multi-step workflows integrating diverse computational methods and software packages. We provide a freely accessible, open-source, continuously updated and tested online book containing reproducible code examples, datasets, and discussion about data analysis workflows for spatial omics data using Bioconductor in R, including interoperability with Python.

bioinformatics↗

bioRxiv2025

Single-Cell Multi-Omics Dissects Transcript Isoform and Immune Repertoire Dynamics in Human Immunosenescence

Immunosenescence, a major hallmark of systemic aging, refers to the progressive functional decline of the immune system. This decline not only compromises host defense and immunological memory but also fuels chronic inflammation and tissue degeneration (collectively known as inflammaging). While single-cell RNA sequencing (scRNA-seq) has revealed transcriptomic alterations in immune aging, analyses restricted to transcript abundance fail to capture deeper regulatory layers, such as transcript isoform diversity and the remodeling of immune receptor repertoires. To address this, we present the human peripheral immune single-cell multi-omics atlas that integrates gene expression, transcript isoforms diversity, and immune receptor repertoires. By combining single-cell full-length transcriptome sequencing (scCycloneSEQ), short-read scRNA-seq, and single-cell immune receptor sequencing (scTCR/BCR-seq), we systematically profiled peripheral blood mononuclear cells (PBMCs) from healthy young and elderly donors. Our analyses uncovered extensive age-related remodeling of immune cell composition, functional states, and TCR/BCR diversity. Notably, we identified in CD4 effector memory T cells exhibited widespread differential isoform usage (DIU), 3'UTR length variation, and a marked reshaping of cytotoxic T lymphocyte (CTL) clonotypes--all closely associated with aging-related inflammation and cellular senescence. This multi-omics atlas delineates key molecular features of immunosenescence and provides a high-resolution resource for deciphering the regulatory architecture underlying immune aging.

immunology↗

bioRxiv2025

Reprogrammed SimCells for Antimicrobial Therapy

Antimicrobial resistance (AMR) is a critical global health challenge. In this study, we developed a novel platform based on chromosome-free and non-replicating simple cells (SimCells, size 1-2 {micro}m) and mini-SimCells (size 100-400 nm) for targeted pathogen elimination. Engineered with surface-displayed nanobodies, SimCells and mini-SimCells selectively bind bacteria expressing specific antigens (e.g., OmpA in E. coli). The selective interactions facilitate close SimCell-pathogen proximity, enabling two antimicrobial mechanisms: direct injection of toxic effectors into bacterial cytoplasm via a heterologous expression of type VI secretion system (T6SS), and enzymatic conversion of aspirin into catechol by engineered salicylate hydroxylase, leading to sustained local production of hydrogen peroxide (H2O2). Our results demonstrate that both reprogrammed SimCells and mini-SimCells can eliminate target E. coli with high specificity and efficiency. Multi-dose reprogrammed mini-SimCell treatment led to a 103-fold selective reduction of targeted bacteria in mixed microbial communities, with minimal disruption to non-target bacteria. We demonstrate that reprogrammed mini-SimCells, engineered with nanobody targeting outer membrane protein OmpA of the clinically relevant multidrug resistant pathogen E. coli ST131, achieved elimination efficiencies over 97% at 24 and 48 hrs. This modularised plug-and-play antimicrobial platform provides a highly specific, efficient and adaptable solution for combating diverse AMR pathogens. Significance StatementAntimicrobial-resistant (AMR) bacteria cause millions of deaths worldwide annually, representing a critical global health challenge. In this study, we developed a novel therapeutic platform using two types of chromosome-free, non-replicating engineered bacterial cells: SimCells (1-2 {micro}m) and mini-SimCells (100-400 nm). These SimCells were engineered to selectively bind to targeted E. coli, facilitating precise delivery of toxic proteins via a Type VI secretion system (T6SS) and localised generation of hydrogen peroxide from aspirin. We demonstrate that mini-SimCells eliminated over 97% of a targeted AMR strain within 48 hours. Moreover, multiple-dose administration achieved a selective 103-fold reduction of targeted E. coli in mixed microbial communities. This modular plug-and-play platform offers an adaptable solution against diverse multi-drug-resistant pathogens.

synthetic biology↗

bioRxiv2025

Shape, Strain, and Stability: Epithelia Under High Strain

Mechanical forces play crucial roles in epithelia tissue morphogenesis and function, yet individual cells often respond differently to the same external force. Strain heterogeneity, the non-uniform deformation of cells under uniform external mechanical stimuli, may underlie tissue-level robustness and guide morphogenetic outcomes. In this study, we applied a defined uniaxial strain to Xenopus laevis epithelial explants and investigated how strain was distributed at the cellular level. Live imaging and quantitative analysis revealed that despite a uniform tissue-level strain, cellular strain was heterogeneous, suggesting variable responses to a uniform mechanical stimulus. To understand the source of this variability, we used an image analysis pipeline to explore multiple aspects of cell morphology. We found that cell intrinsic material properties, represented by Poisson index, had the strongest correlation with strain heterogeneity, suggesting a dominant role in variable mechanical response. We further analyzed how force was distributed at the cellular level using a vinculin force sensor and laser ablation. These experiments demonstrated that forces are primarily transmitted through the medio-apical actomyosin cortex whereas junctional actomyosin facilitates dissipation and remodeling. These findings provide new insights into the physical principles that underlie epithelial resilience and adaptive remodeling, highlighting the importance of distinct functions of junctional and medio-apical actomyosin networks in mechanical adaptation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/687918v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1d27474org.highwire.dtl.DTLVardef@1db811forg.highwire.dtl.DTLVardef@1fa36dforg.highwire.dtl.DTLVardef@288107_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

ISKNV-pORF71 hijacks outer mitochondrial membrane-localized VDAC2 to the nucleus, enhancing apoptosis by disrupting mitochondrial membrane permeability

Infectious spleen and kidney necrosis virus (ISKNV), the type species of the genus Megalocytivirus within the family Iridoviridae, is one of the most devastating pathogens affecting global teleost populations. Our previous study confirmed that ISKNV-pORF71 (p71/VP71) is a viral virulence factor by constructing a recombinant virus with ISKNV orf071 deletion (ISKNV-{Delta}71). In the present study, we further found that compared with ISKNV-WT, the ISKNV-{Delta}71 mutant is more capable of maintaining mitochondrial membrane potential, alleviating mitochondrial membrane permeabilization, preserving mitochondrial membrane integrity, sustaining lower cytochrome c release, and thereby reducing the apoptosis rate of infected cells. Further investigations demonstrated a strong interaction between p71 and the outer mitochondrial membrane (OMM)-localized voltage-dependent anion channel 2 (VDAC2). At the subcellular level, p71 and mandarin fish VDAC2 (mfVDAC2) are originally localized to the nucleus and mitochondria, respectively. However, in cells co-transfected with p71 and mfVDAC2, robust nuclear translocation of mfVDAC2 was verified. Endogenous mfVDAC2 was also confirmed to undergo nuclear translocation upon ISKNV-WT infection. In the zebrafish infection model, zebrafish VDAC2 (zfVDAC2)--but not zfVDAC-1 or zfVDAC-3--was observed to translocate into the nucleus through interaction with p71. Using CRISPR/Cas9 technology, VDAC2-knockout zebrafish were successfully constructed. Infection experiments showed that ISKNV-{Delta}71 exhibits significantly reduced lethality to both wild-type zebrafish and zfVDAC2-knockout zebrafish. In conclusion, our findings reveal that p71 exerts its function during ISKNV infection by hijacking VDAC2 into the nucleus, which alters mitochondrial membrane permeability and enhances ISKNV-induced apoptosis. The phenotypic characteristics of VDAC2 knockout (vdac-/-) zebrafish were also thoroughly analyzed and discussed. Author summaryVDAC2, a well-characterized cellular protein localized to the outer mitochondrial membrane (OMM), plays critical roles in viral pathogenesis and antiviral immune escape. Previous studies have shown that VDAC2 interacts directly or indirectly with viral proteins or functions as a functional receptor to modulate viral pathogenesis; however, few studies have demonstrated that VDAC2 is hijacked from its native OMM to other organelles. Our study here provides robust evidence that cellular VDAC2 is rerouted from its original OMM to the nucleus through interaction with ISKNV p71, thereby altering viral pathogenesis. This work offers novel insights into the role of VDAC2 in pathogen virulence. As far as we know, this is the first report demonstrating that host VDAC2 is hijacked into the nucleus by a viral protein to subsequently modulate the viral life cycle. Collectively, these findings advance our understanding of the active regulation of host functional proteins by iridoviruses.

microbiology↗

bioRxiv2025

Thyrotropin-releasing hormone protects hippocampal neurons against glutamate toxicity via phosphatidylinositol 3-kinase/AKT pathway and new protein synthesis

Thyrotropin-releasing hormone is best known as a neuropeptide that stimulates the release of thyroid-stimulating hormone and prolactin in hypothalamic-pituitary-thyroid (HPT) axis. Independent from its activity in the HPT axis, TRH also exerts strong neuroprotective activity against neurodegenerative diseases such as Alzheimers disease, epilepsy and traumatic brain injury. Although multiple factors have been linked to its neuroprotective action, the cellular mechanism of TRH neuroprotection is still not clear. Here we show that TRH protects hippocampal neurons against glutamate toxicity via phosphatidylinositol 3-kinase (PI3K)/AKT pathway and new protein synthesis. Both adeno-associated virus (AAV) mediated TRH transduction and TRH peptide given exogenously over 24 hours period of time inhibit glutamate-induced lactate dehydrogenase (LDH) release. This effect is not mediated by the decreased intracellular calcium response as TRH treatment (24 hours) has no effect on glutamate-induced increase in intracellular calcium nor the calpain activity. While TRH treatment (10 minutes) significantly inhibits glutamate-induced increase in intracellular calcium, no protective effect is observed when TRH is applied 30 minutes before or after glutamate stimulation. Instead, PI3K inhibitor LY294002 but not mitogen-activated protein kinase (MAPK)/Extracellular signal-regulated kinase (ERK)1/2 inhibitor U0126 completely inhibits the protective effect of TRH. LY294002 also blocks TRH induced AKT activation. In addition, protein synthesis inhibitor cycloheximide inhibits the protective effect of TRH. Taken together, these results suggest PI3K/AKT signaling pathway and new protein synthesis are involved in the protective effect of TRH against glutamate toxicity, thereby providing mechanistic support for its action in neurodegenerative diseases. HighlightsTRH has strong neuroprotective activity against neurodegenerative diseases such as traumatic brain injury and Alzheimers disease. Understanding the cellular mechanism for TRH neuroprotection might aid developing novel treatment strategy. In the present study we demonstrate that TRH neuroprotection is mediated via PI3K/AKT signaling pathway and new protein synthesis. This finding provides mechanistic support for the action of TRH in traumatic brain injury and other neurodegenerative diseases.

neuroscience↗

bioRxiv2025

The scaffold protein PRR14L links the PP2A-TACC3 axis to mitotic fidelity and sensitivity to MPS1 inhibition

Aneuploidy is a hallmark of cancer and is a potential vulnerability that can be selectively targeted. To systematically identify genes that affect the incidence and fitness of aneuploid cells, we conducted a genome-wide CRISPR/Cas9 screen using NMS-P715, an inhibitor of the spindle assembly checkpoint (SAC) kinase MPS1/TTK. In this study, we identified a number of genes known to regulate aneuploidy and mitosis, and subsequently focused on PRR14L, a ubiquitously expressed gene previously implicated in chronic myelomonocytic leukemia (CMML). Proximity labeling of PRR14L using TurboID revealed several cell division proteins, including the PP2A-B56 phosphatase complex and the spindle assembly factor TACC3, as PRR14L-interacting proteins. Loss of PRR14L prolongs SAC-dependent mitotic arrest in response to microtubule depolymerization but, paradoxically, leads to catastrophic mitotic errors upon SAC abrogation by MPS1 inhibitors. A model derived from our findings provides a rationale for exploiting MPS1 inhibition as a potential vulnerability in cancers containing either PRR14L loss of function mutations or FGFR-TACC3 fusions. Significance StatementO_LIAneuploidy is a hallmark of cancer. Whether aneuploidy can be selectively targeted is not known. C_LIO_LIUtilizing a CRISPR/Cas9 screen, the authors found that loss of the gene PRR14L sensitizes cells to aneuploidy induction. Taking advantage of live cell imaging and proximity labeling, they linked PRR14L to TACC3 and mitosis. C_LIO_LIThese findings suggest that spindle checkpoint inhibitors may have therapeutic potential in cancers with either loss-of-function PRR14L and/or gain of function TACC3 mutations. C_LI

cell biology↗

bioRxiv2025

Genetic and developmental constraints drive parallelism in flower evolution

Evolution repeatedly gives rise to similar phenotypes, suggesting that constraints and biases make some evolutionary trajectories more likely than others, yet the mechanisms underlying this predictability remain poorly understood. Using repeated transitions to self-fertilization in a model Brassicaceae genus, we show that independent reductions in petal size arise from dosage changes at the same pleiotropic growth regulator, JAGGED (JAG). Petal development exhibits particularly high sensitivity to JAG dosage, resulting in organ-specific morphological shifts with minimal effects on other organs and limited pleiotropy. In both selfing species, these changes were drawn from functionally divergent variants whose polymorphism patterns are consistent with long-term maintenance under weak selection. Our results demonstrate how developmental network architecture and the maintenance of functional variation at pleiotropic loci combine to channel evolution toward predictable outcomes.

evolutionary biology↗

bioRxiv2025

From Signal to Symphony: Exploring 2D Sequence Representations for Protein Function Prediction

Predicting protein function from its primary sequence is a fundamental challenge in computational biology. While deep learning has excelled, the optimal representation of sequence data remains an open question. This study explores protein sonification--the conversion of amino acid sequences into 2D spectrograms--as a representation for this task. To facilitate this investigation, we developed a benchmark dataset of 18,000 sequences spanning 12 functionally diverse protein classes. Our systematic evaluation suggests that the structural transformation from a 1D sequence to a 2D spectrogram may be a key contributor to the models predictive performance. This observation is supported by ablation studies where models using either purely visual or acoustic features from the spectrogram demonstrated effective standalone performance, suggesting that the representation itself is a key source of this capability. For instance, a model using a sonification map without explicit biophysical meaning achieved 81.08% accuracy, while our biophysically-informed model reached 84.00%, indicating that such domain knowledge may offer a modest performance benefit. When trained from scratch on our dataset, our fusion model achieved performance comparable to or slightly exceeding that of standard transformer architectures like ESM-2 and ProtBERT, suggesting its potential for data efficiency in this specific context. The models potential for generalizability was further supported by its performance on the external CARE enzyme classification benchmark, where it achieved 90.44% accuracy. Finally, as a proof-of-concept, we explore the utility of our encoding to guide a diffusion model in generating novel GFP variants, which were assessed for structural viability using computational methods. Our work provides evidence suggesting that the utility of sonification in this context may stem largely from its representational structure, offering a perspective on feature engineering for biological sequences.

bioinformatics↗

bioRxiv2025

Hydration Network Drives Activation and G Protein Selectivity in GPR174

G protein-coupled receptor 174 (GPR174), a key modulator of autoimmune responses, maintains immune homeostasis through distinct G protein signaling pathways, particularly Gs and Gi. Although the structural mechanism of lysophosphatidylserine (LysoPS)-activated GPR174 in the Gs pathway has been characterized, how hydration-mediated interactions influence GPR174 activation and signaling selectivity remains unclear. Here, we determined high-resolution cryo-electron microscopy (cryo-EM) structures of LysoPS-activated GPR174 bound to Gs (2.0 [A]) and Gi (3.4 [A]), revealing a continuous hydration-mediated signaling transduction network that bridges the sodium-binding pocket, NPxxY and DRY motifs, and the G protein-binding interface. This network stabilizes the active-state conformation of GPR174 and dynamically reshapes the intracellular cavity, thereby enabling differential engagement of Gs and Gi. Molecular dynamics simulations and functional assays demonstrated that the hydration network is essential for receptor activation and selectively modulates G protein coupling. To evaluate its conservation, we performed sequence alignment and structural analysis across class A GPCRs, defining three hydration cavities: the conserved water cavity (CWC), the junction water cavity (JWC), and the extended water cavity (EWC), whose hydration is determined by residue properties at position 5.58. Together, our study reveals a hydration-driven molecular mechanism that underlies the activation of GPR174 and its dual G protein selectivity. These findings advance the understanding of hydration-mediated signaling in GPR174 and provide a framework for investigating water-mediated regulation across class A GPCRs.

biophysics↗

bioRxiv2025

Flexible gaze reinstatement during working memory for natural scenes

The information we hold in mind with working memory (WM) may propagate beyond the cortex and out to the peripheral motor system. For instance, directionally biased hand and eye movements can express simple visuo-spatial features during WM maintenance. If such sensorimotor inflections are a form of WM representation, they may also express more multifaceted and task-adaptive information than just spatial location. Here, we ask whether gaze patterns carry item-specific detail about complex stimuli held in mind, and whether gaze prospectively adapts to the behavioral task demands. To do this, we tracked human eye movements (male and female) during WM for naturalistic images, and we manipulated which image features were most task-relevant (visual or semantic). In two experiments, we found that the eyes moved in complex, item-specific spatiotemporal sequences during WM maintenance. Delay period gaze patterns retraced the scanpaths from stimulus encoding, echoing the gaze reinstatement that aligns with neural pattern reactivation during long-term memory retrieval. Therefore, gaze patterns during WM may reinstate visual encoding processes for maintenance. We also used time-resolved representational similarity analyses, guided by convolutional neural network modeling, to test the specificity of WM gaze patterns across a trial. We found that gaze representations were more distinct when the task prioritized visual detail, and also ramped up in anticipation of the WM probe. Therefore, oculomotor WM signals are malleable to when and how the content will be used. These results suggest that the earliest levels of visual processing reflect prospectively-oriented and functionally flexible WM content representations. SignificanceThe eyes are an outwardly accessible extension of the brain. They are the first stop for visual processing, but may also offer a rich window into underlying cognition. For instance, pupil dilation can reflect changing cognitive load, and eye movement biases can reflect basic spatial information about content held in mind with working memory. Working memory is a critical neuro-cognitive function, but its physiological bases remain under debate. Here, we show that eye movements code for more than simple spatial WM, and instead express intricate patterns that read out the complex, real-world stimuli held in mind. This work shows how cognitive states influence oculomotor functions, and also advances theories of WM representation to include the earliest stages of sensory processing.

neuroscience↗

bioRxiv2025

Flexible Working Memory in the Peripheral Nervous System

Working memory (WM) representations that are distributed across the brain can be flexibly recruited to best guide behavior1-4. For instance, information may be represented relatively more strongly in visual cortex when a WM task requires fine visual detail, or more strongly in motor cortex when a specific response can be prepared5-10. If WM drives goal-oriented actions, we might also expect such task-dependent signals to propagate to the peripheral effectors that realize WM commands. Accordingly, oculomotor signatures like gaze biases can track simple visuo-spatial WM features11,12, but their functional flexibility is unclear. Here, we test the idea that WM content is adaptively distributed across the nervous system according to behavioral demands. We ask whether patterns in both eye and hand movements can express visual WM stimulus features, and whether the distribution of such activity shifts with the task context. In a delayed recall task, we manipulated how human participants reported their memory: they would either draw a line or adjust a visible response wheel to match a remembered orientation. Via continuous eye- and stylus-tracking, we found that remembered orientations were decodable from small inflections in both gaze and hand movements during the WM maintenance period. Moreover, this decoding strength varied by response format. Gaze patterns tracked memorized features relatively better in the wheel condition (vs. draw), while hand movements were better in the draw condition (vs. wheel). Therefore, visually encoded WM contents may be adaptively allocated to task-relevant motor effectors, balancing WM representations across peripheral activity according to behavioral needs.

neuroscience↗