Biology subjects

Qiu, R.

Publications and source records attributed to Qiu, R..

At least 19 recordsLinked to original sources

bioRxiv2026

One placement, many fields: robust voxel-wise mapping with multichannel TMS

Understanding the human brain at the individual level is central to understanding its function and a prerequisite for personalized therapy. Transcranial magnetic stimulation (TMS) can map structure-function relationships at high resolution by regressing trial-wise variation in the TMS-induced electric field (E-field) against a response such as motor evoked potentials (MEPs). This requires distinct E-fields across pulses at different cortical locations, which single-coil TMS (sTMS) achieves by physically repositioning the coil (typically supported by neuronavigation software such as InVesalius Navigator; Souza et al., 2018)--increasingly reliant on robotic guidance and capped by the coil geometry. Multichannel TMS (mTMS) instead steers the E-field electronically via independent currents in several coils, opening a design space of high variability without physical repositioning. We adapt determinant-based sequence optimization from discrete sTMS coil-placement sets to the continuous mTMS current space (D-optimal design), identifying pulse sequences whose induced cortical E-fields are as mutually dissimilar as biophysical constraints allow. We benchmarked optimized mTMS sequences against random baselines and optimized sTMS placements in virtual motor mapping across multiple head models, assessing three mTMS array geometries (5-channel planar, 6- and 12-channel spherical), two figure-of-eight sTMS coils, and varying pulse counts, response noise, and cortical depth. D-optimal mTMS currents substantially outperformed random currents and optimized sTMS at matched pulse counts. The gain was modest without noise but increased significantly with it: at the highest noise level, optimized sTMS failed to converge after 100 pulses (~20-22 mm error), whereas all mTMS arrays reached median errors at or below 6 mm. mTMS also recovered deep targets better than sTMS (up to 35 mm skin-cortex distance, versus 25 mm) and performed uniformly across the region of interest. A coil-space scheme based only on the magnetic vector potential, precomputable once per array without per-subject MRI, captured most of the benefit, matching subject-specific optimization at 100 pulses. mTMS thus makes voxel-wise mapping robust throughout a region of interest without coil repositioning.

neuroscience↗

bioRxiv2026

Real-time posterior probabilistic TMS motor mapping with robust individualized stopping criteria

Background. Electric-field-informed regression provides high-resolution transcranial magnetic stimulation (TMS) motor maps, but conventional nonlinear fitting is too slow to update the map during acquisition, so sessions rely on predefined stimulation budgets, despite substantial inter-individual variability in the data required for stable localization. Objective. To develop a real-time analysis updating a posterior probability map between pulses and compare its hotspots with finger-tapping functional MRI (fMRI). Methods. We analyzed two independent cohorts (12 and 13 participants, one newly collected), analyzing each hand muscle independently. We replaced the conventional per-element sigmoid fit with Spearman's rank correlation coefficient and transformed the resulting score map into a posterior probability map using marginal-likelihood evidence. Acquisition stopped once posterior mass within 5 mm of the hotspot passed a preset confidence level. Results. The Spearman and linear scores updated the region of interest within milliseconds, versus tens of seconds for sigmoid fitting. Spearman more closely matched the full-data sigmoid hotspot in recorded sessions and converged faster and more precisely to the true source in virtual subjects. Across both fast scores, at a confidence level of 0.85, the stopping criterion was met after a median of 74-159 pulses, with 26-454 across individual problems. FDI hotspots lay 8.3 {+/-} 4.3 mm from the fMRI center of gravity, in the precentral hand region. Conclusions. Rank correlation reduces computation time without loss of accuracy relative to sigmoid fitting. The resulting posterior probability map provides a session-specific stopping criterion in place of a fixed pulse budget.

neuroscience↗

bioRxiv2026

Ayahuasca plant genomes reveal retrotransposon-derived centromeres and contrasting routes to DMT and β-carbolines

Ayahuasca, used religiously for centuries by Amazonian Indigenous peoples and increasingly studied for its therapeutic potential, owes its psychoactivity to two plants: Psychotria viridis supplies N,N-dimethyltryptamine (DMT), whereas Banisteriopsis caapi supplies beta-carbolines that render DMT orally active. Here we show that the two plants have distinct genome histories: P. viridis is hexaploid (2n = 6x = 66), consistent with autopolyploidy, and B. caapi is diploid (2n = 2x = 20). CENH3-bound regions of both species are nonetheless enriched in CRM-type Ty3/Gypsy retrotransposons, with additional LINE elements and CRM-derived satellites in B. caapi. In P. viridis, a single multifunctional methyltransferase catalyzes both N-methylations of tryptamine to DMT. In B. caapi, beta-carboline biosynthesis combines dedicated enzymes with an acetaldehyde-dependent Pictet-Spengler cyclization that can proceed non-enzymatically, followed by enantioselective oxidation to harmaline. These resources link the ethnobotanical history of ayahuasca to its molecular basis, providing genomes of both source plants, the genetic structure of traditionally recognized cultivated varieties, and the enzymes behind the alkaloids long combined in this decoction.

genomics↗

bioRxiv2026

Autophagosome-tethering compounds selectively lower mutant ataxin-3 in preclinical models of spinocerebellar ataxia type 3

Spinocerebellar ataxia type 3 (SCA3) is most common subtype of autosomal dominant spinocerebellar ataxia worldwide and the most prevalent CAG expansion disorder in the east Asian population, but disease-modifying therapies remain unavailable. SCA3 is caused by the accumulation of polyglutamine- (polyQ-) expanded mutant ataxin-3 (mAtxn3), while the wild-type ataxin-3 (wtAtxn3) plays an important cellular role. Thus, allele-selective clearance of mAtxn3 without affecting wtAtxn3 is highly desired for SCA3 treatment. To achieve this goal, we tested previously reported compounds that have been shown to selectively degrade the Huntingtons disease causing mutant HTT protein by tethering the expanded polyQ to the autophagosome protein LC3. Here, we demonstrated that two of these compounds, AN2 and GW5074, selectively reduced both soluble and aggregated mAtxn3 with expanded polyQ (84Q), but not wtAtxn3, in both cellular and mouse models of SCA3. Mechanistically, both compounds lowered mAtxn3 by enhancing its autophagic degradation without obvious activation of global autophagy. At the molecular level, both compounds increased mAtxn3-LC3 interaction and colocalization to enhance the allele-selectively autophagic degradation of mAtxn3. Phenotypically, both compounds rescued cell viability and ATP content of SCA3 cellular models. In SCA3 patient pluripotent stem cells (iPSCs) derived cortical neurons, both compounds allele-selectively reduced mAtxn3 and partially rescued neurite shortening. Importantly, in a well-established SCA3 transgenic mouse model (MJD84.2), intracerebroventricular, intraperitoneal, and oral administration allele-selectively reduced mAtxn3 in multiple brain regions and rescued motor function deficits. Oral dosing achieved nanomolar cerebellar exposure and showed no overt liver, kidney, or glial toxicity over 28 days. Analyses of 1303 Chinese patients with SCA3 and ATXN3 constructs of different polyglutamine lengths further supported the compounds polyQ length-dependent activity with relative sparing of normal-length ataxin-3. Together, these findings provide preclinical proof of concept that utilizing orally available small molecule compounds to allele-selectively enhance mAtxn3 autophagic degradation for SCA3 treatment. One-sentence summarySmall-molecule autophagosome-tethering compounds selectively lower mutant ataxin-3 through an LC3- and autophagy-dependent mechanism and improve disease-related phenotypes in preclinical models of SCA3.

neuroscience↗

bioRxiv2026

A direct, MFRN-independent Fe(II) transfer pathway at mitochondria-lysosome contacts

Mitochondrial iron homeostasis is fundamental to respiration and redox balance, and its dysregulation is implicated in neurodegeneration, cardiomyopathy, and metabolic diseases. Although lysosomes harbor the major cellular iron reservoir, the prevailing model holds that mitochondria acquire Fe(II) directly from the cytosolic labile iron pool (LIP) via MFRN transporters. Here, we challenge the canonical view by identifying a direct, MFRN-independent Fe(II) transfer pathway at mitochondria-lysosome contacts (MLCs). This VPS39/TOMM22/SFXN1-coordinated pathway enables lysosome-to-mitochondria Fe(II) flux bypassing the cytosolic LIP. Using live-cell structured illumination microscopy (SIM), we visualize direct Fe(II) transfer specifically occurring at MLCs. Multiple lines of evidence confirm that VPS39 and TOMM22 stabilize MLCs, while SFXN1 serves as the core effector protein for this MLC-dependent Fe(II) transport. Notably, SFXN1 knockdown markedly reduces mitochondrial Fe(II) levels independent of its established serine transport function. This pathway reveals a major route for mitochondrial Fe(II) acquisition to support redox homeostasis.

cell biology↗

bioRxiv2026

GESTURE: unsupervised genotype-specific behavioral phenotyping in rodents via graph-based representation learning

Objective quantification of complex animal behavior is fundamental to neuroscience and pharmacology, yet extracting biologically meaningful insights from high-dimensional pose data remains challenging. This is especially true for subtle phenotypic differences caused by genetic mutations, which frequently evade conventional evaluation metrics. Here, we introduce GESTURE, an unsupervised, graph-based deep generative framework that autonomously discovers and quantifies behavioral structure from raw pose dynamics. Analyzing mice with motor dysfunction alongside wild-type controls, GESTURE identifies a shared vocabulary of behavioral motifs. We show that genotype-specific differences arise from the differential usage of these motifs, yielding distinct "behavioral fingerprints" that reliably separate genotypes without supervision. By modeling behavior as a sequence rather than a static partition, GESTURE measures temporal organization directly: affected animals held motifs longer and transitioned more predictably, revealing a slowing and stereotyping of behavioral sequences rather than a simple reduction in activity. Importantly, GESTUREs graph-based representation enables training across multiple recordings and embedding behaviors into a shared latent space, supporting robust cross-animal comparisons and future cross-experiment alignment. Furthermore, automatically derived metrics of behavioral divergence track the temporal dynamics of expert-annotated disability scores, reaching agreement comparable to independent human raters. Finally, node- and edge-level explainability analyses indicate that the models latent representations are shaped by a biologically plausible focus on the animals core motor scaffold. Together, these results position GESTURE as an interpretable and scalable framework for automated behavioral phenotyping, linking genetic perturbation to quantitative behavioral phenotypes. Author summaryHow can we measure, objectively, the subtle changes in behavior caused by a disease-causing mutation? Such changes are hard for human observers to judge consistently, and scoring them by hand is slow and does not scale. We studied mice carrying a mutation in a calcium-channel gene. In people, mutations in the same gene cause episodic ataxia and related movement disorders. We developed GESTURE, which learns recurring patterns of movement directly from video of a freely moving mouse, without being told what to look for. It treats the body as a set of connected landmarks and follows how their arrangement changes over time, discovering a shared vocabulary of movements. Affected and healthy mice drew on the same vocabulary but used it differently. Each animals pattern of use formed a distinctive "behavioral fingerprint", and these fingerprints alone identified which animals carried the mutation --in every case. They also revealed something a simple activity measure would miss: affected mice hold each movement longer, and string movements together more predictably. A severity score read automatically from them matched expert judgement as closely as two trained raters matched each other, and returned the same answer every time.

neuroscience↗

bioRxiv2026

A confound-diagnostic toolkit for in silico perturbation with single-cell foundation models

Deleting a gene token from a cells input sequence offers a convenient native strategy for in silico perturbation, but the resulting embedding delta may not represent a biological knockout response. Apparent effects can instead reflect gene identity, universal responsiveness, limited tokenization coverage, library-size contamination, or circular state scoring. Here, we present a confound-diagnostic framework combining held-out increment testing, responsiveness adjustment, coverage gating, library-size diagnostics, and de-circularized state-shift analysis, together with a numerically matched reimplementation of frozen Geneformers perturbation engine. Across Frangieh and Replogle datasets and linear and nonlinear readouts, the native embedding delta provided no reproducible held-out improvement beyond gene identity. Signal-injection calibration showed that the test detected injected residual signal, whereas native increments remained below its detection floor. Matched controls traced apparent positives to raw-count library-size structure, broad responsiveness, and self-referential scoring, while coverage constrained perturbation applicability and estimate stability without establishing biological specificity. This model-adaptable framework helps determine when foundation-model perturbation readouts warrant biological interpretation. MotivationFoundation-model in silico perturbation could predict perturbation effects when matched experimental data are unavailable. However, in zero-shot settings, embedding-derived responses may reflect gene identity, universal responsiveness, tokenization limits, library-size artifacts, or circular state scoring rather than biological knockout effects. We therefore developed a reusable confound-diagnostic framework that applies matched controls to test whether native perturbation readouts contain information beyond these confounds and warrant biological interpretation.

bioinformatics↗

bioRxiv2026

Excess p50 induces Arp1-dependent dynactin clusters containing the assembly factor VezA

Dynactin, a complex essential for cytoplasmic dynein function, contains an Arp1 mini-filament with a pointed-end sub-complex including Arp11 and a shoulder sub-complex including p50 and p150. We recently identified in Aspergillus nidulans a vezatin homolog VezA that enhances dynactin assembly, but VezA does not co-localize with dynactin, suggesting a transient interaction. It was found that overexpression of p50 separates the shoulder from the mini-filament and causes late-onset neurodegeneration in mice, but its cellular effects in the context of VezA need to be studied. Here we found astonishingly that p50 overexpression in A. nidulans causes Arp11, p50 (but not p150), and VezA to form co-localized clusters whose presence depends on Arp1. Formation of the VezA cluster also depends on Arp11, and VezA significantly enhances the intensity of Arp11-GFP clusters but not that of p50-GFP clusters. Moreover, an evolutionarily conserved region of p50 (aa17-25) forming a beta-sheet structure with Arp1 as revealed in previous cryo-EM studies is critical for the formation of Arp11, p50 and VezA clusters. These results suggest that excess p50 induces cluster formation driven by p50-Arp1 interactions, and that this assembly intermediate without the full shoulder traps VezA, which is involved in the Arp11-Arp1-p50 interactions.

cell biology↗

bioRxiv2026

Optimal Pre-Experimental Coil Sequence Selection for TMS Motor Mapping

Transcranial magnetic stimulation (TMS) motor mapping increasingly relies on electric-field (E-field) modeling to localize cortical targets, but many candidate coil placements induce highly redundant cortical patterns. We frame prospective coil-sequence design as a subset-selection problem and compare farthest-point sampling, determinant-based objectives, and related controls in virtual mapping experiments across 12 realistic head models. Across convergence, matrix-diagnostic, and manifold-coverage analyses, the best-performing objectives combined low between-stimulation redundancy with preserved inter-element separability on the cortex; objectives that maximized one of these at the expense of the other fell below random sampling. An RBF-kernelized D-optimal objective matched FPS in mapping accuracy while using [~]15 fewer unique scalp positions per 100 pulses, suggesting reduced arm-reconfiguration cost on robotic TMS platforms. A singular-spectrum analysis of the candidate library provides a low-cost a-priori indicator that predicts when discretization choices fall below the diversity floor required for stable mapping. These results recast prospective TMS mapping as a limited manifold-coverage problem and offer concrete design rules for sample-efficient, robotically efficient sequence planning. Algorithm implementations are available as part of the open-source pynibs tool-box at https://gitlab.gwdg.de/tms-localization/pynibs/-/tree/dev/pynibs/optimization.

neuroscience↗

bioRxiv2026

Liver sinusoidal endothelial cells integrate metabolic and immune signals for MAPK-dependent BMP6 regulation and hepcidin induction

Liver sinusoidal endothelial cells (LSECs) separate the blood from the hepatic parenchyma and thus are at the frontline as scavengers of blood-borne waste, pathogens and metabolic stimuli. LSECs are also critical for sensing systemic iron availability by controlling the synthesis of bone morphogenetic protein (BMP) 6, which is essential for hepcidin expression in hepatocytes. Hepcidin maintains systemic iron homeostasis by inhibiting dietary iron uptake and iron release from iron recycling macrophages. Hepcidin is also an acute-phase protein and its activation by inflammation requires active BMP signaling. It is incompletely understood how signals derived from inflammation, cellular damage and iron are integrated by the liver to assure adequate hepcidin expression. Here, we show that Bmp6 expression is activated in primary LSEC cultures upon their exposure to damage-associated molecular patterns (DAMPs), such as heme and myoglobin, pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS) and Fibroblast-Stimulating Lipopeptide-1 (FSL1), or oxidative stress inducers (H2O2). All regulatory cues converge at the MAPK signaling pathway, although the specific signaling branches involved are stimulus-specific. Notably, Bmp6 upregulation in LSECs in response to all signals tested is strongly enhanced by the hepatocyte secretome. As hepatocytes critically depend on active BMP/SMAD signaling to control hepcidin activation, our results reveal that multiple sources of signaling input activating Bmp6 in LSECs and hepcidin in hepatocytes serve to determine BMP/SMAD signaling strength. Furthermore, our findings identify hypoferremia, the result of high hepcidin levels due to elevated Bmp6, as a convergent response in conditions of inflammation, oxidative stress and cellular damage. HighlightsO_LIDAMPs (heme and myoglobin), PAMPs (LPS) and oxidative stress activate Bmp6 mRNA expression via the MAPK signaling pathway C_LIO_LIThe TLR/MAPK/BMP6 regulatory axis integrates inflammatory and iron signals C_LIO_LIOur work uncovers a novel connection between innate immune sensing, oxidative stress and hepatic iron homeostasis C_LI

cell biology↗

bioRxiv2026

Metabolic dehalogenation of halomethanes by mercury methylators

Mercury (Hg) methylators play key roles in the global Hg cycle. Nonetheless, the Hg methylators are widely present in Hg-absent environments and the Hg methylation is a cometabolic process without cell growth, which leave the origin and evolution of the Hg methylation as a mystery. Here, we reported the CCl4/trihalomethane-to-dihalomethane dehalogenation by model Hg methylators in a metabolic way. Heterologously-expressed HgcAB catalyzed both the CCl4 dechlorination and Hg methylation. Halomethanes were shown to sustain the Hg-methylation community without adding external carbon source, electron donor and acceptor. Metadata analyses suggested the halomethane dehalogenation potential of Hg methylators at the global scale. These results, together with much higher global flux and kinetic Vmax of the metabolic halomethanes dehalogenation relative to the co-metabolic Hg methylation, suggested halomethanes as the potential HgcAB substrates for the evolution of Hg methylation.

microbiology↗

bioRxiv2026

Population Structure and Antimicrobial Resistance Gene Transfer of Respiratory Escherichia coli Isolated from Swine in China

ABSTRACPorcine respiratory diseases caused by extraintestinal pathogenic Escherichia coli (ExPEC) pose a severe threat to swine production and public health; however, research on respiratory tract-isolated ExPEC remains limited. This study comprehensively analyzed the genomic characteristics and antibiotic resistance gene (ARG) transfer potential of 441 ExPEC strains isolated from porcine lungs across 21 Chinese provinces (including 53 newly isolated strains from 2022-2024 and 388 NCBI-deposited strains). Phylogenetic analysis revealed that 84% of the isolates belonged to phylogroups A, B1, and C, with ST410, ST101, and ST88 as the predominant STs. The strains exhibited extensive ARG diversity, harboring 111 distinct ARG subtypes, with sul2 (81.4%), floR (73.5%), and tet (A) (68.0%) being the most prevalent. Importantly, critical "last-resort" antibiotic resistance genes (e.g., blaNDM-1/5, the mcr family, and tet (X4)) were also detected. Notably, 77.2% of the ARGs presented horizontal transfer potential, with plasmids (especially IncF family replicons) serving as core vectors, followed by integrons and transposons. Cooccurrence network analysis identified aph (3)-Ib, aph (6)-Id, sul2, and floR as core subnetworks driving multidrug resistance dissemination. Pangenomic analysis confirmed an open genome architecture, with core genes accounting for only 6%, reflecting the strains capacity to acquire exogenous genetic material via horizontal transfer. From the One Health perspective, these transferable ARGs can spread to the environment and humans through fecal discharge and the food chain. These findings underscore the importance of monitoring and controlling ExPEC infections in swine, as such strains can as reservoirs of ARGs, pose potential risks to human health, and may even act as sources of pathogenic agents responsible for human infections. IMPORTANCEPorcine respiratory ExPEC-induced diseases threaten swine production and public health, yet respiratory tract-isolated ExPEC research remains scarce. This study comprehensively analyzed 441 porcine lung ExPEC strains across 21 Chinese provinces, uncovering their dominant phylogroups, high ARG diversity (111 subtypes) and the presence of "last-resort" antibiotic resistance genes. We identified 77.2% of ARGs with horizontal transfer potential, plasmids (especially IncF family) as core vectors, and a core ARG subnetwork driving multidrug resistance. The open pangenome (6% core genes) highlights ExPECs strong capacity to acquire exogenous genes. These findings fill the research gap of respiratory ExPEC, clarify ARG transmission mechanisms in swine ExPEC, and provide critical genomic data for One Health-based AMR surveillance and control, guiding targeted strategies to mitigate ARG spread from swine to humans and the environment.

microbiology↗

bioRxiv2026

Spatial multi-omics unveils the monoclonal origin, neuroendocrine plasticity, and microenvironment niches in combined small cell lung cancer

Combined small-cell lung cancer (cSCLC) is a rare and aggressive subtype of small-cell lung cancer (SCLC) characterized by mixed histology comprising SCLC and non-small cell lung cancer (NSCLC) or large cell neuroendocrine carcinoma (LCNEC) components. Despite its histological heterogeneity and even poorer prognosis than de novo SCLC, cSCLC is clinically managed as pure SCLC, largely due to the lack of molecular insights into its biology, lineage plasticity, and tumor microenvironment (TME). Here, we perform multi-omics profiling, including spatially-resolved whole-exome sequencing (WES), spatial transcriptomics (ST) and single-nucleus RNA sequencing (snRNA-seq), across 19 treatment-naive cSCLC tumors spanning all major histological subtypes. Our analysis reveals that SCLC and NSCLC/LCNEC components share a monoclonal origin, with histological divergence characterized by distinct mutation and copy number alteration patterns. ST and snRNA-seq uncover spatially exclusive or interspersed tumor domains, with distinct TME compositions and immune landscapes. Notably, fibroblast-rich regions enriched for an aggressive fibroblast subtype form boundaries between tumor domains, potentially influencing immune TME and treatment responses. We identify extensive lineage plasticity within cSCLC, including active LUAD-to-SCLC transdifferentiation and SCLC subtype coexistence, suggesting transitional cellular states not captured by traditional diagnostics. Leveraging these insights, we developed the cSCLC Detector, a sensitive mutation-based diagnostic assay that improves the detection of cSCLC in tissue and liquid biopsy samples. Our findings offer critical insights into cSCLC lineage plasticity, cellular evolution, and microenvironmental interactions, underscoring the need for tailored treatment strategies and diagnostic frameworks for this aggressive cancer subtype.

cancer biology↗

bioRxiv2026

Structural rearrangements underlying the activation of STIM1 by ER calcium depletion

In resting cells, STIM1, the dimeric ER Ca2+ sensor that controls store-operated Ca2+ entry (SOCE), is held in a Ca2+-bound inactive state by multiple intramolecular restraints, or brakes. Receptor-evoked release of Ca2+ from the ER causes a large conformational change in STIM1 that releases the brakes and exposes the CRAC activation domain (CAD), enabling it to bind and open store-operated Orai1 channels in the plasma membrane. We performed single-molecule FRET measurements with purified STIM1 to better understand how Ca2+ release from the luminal domain of STIM1 drives the conformational changes in the cytosolic domain that underlie CAD release. We find that Ca2+ removal releases the CAD from CC11 (the CC1 clamp) without obligatory formation of the CC1 coiled-coil that has been associated with CAD release in cells. Surprisingly, the CAD rearranges dramatically during release, as the two hairpin protomers that create its characteristic V-shaped structure are spread apart. Locking the two protomers together by cysteine crosslinking prevents CAD release, suggesting that the CAD must rearrange to escape the CC1 clamp. Our data support a model in which ER depletion-induced dimerization of the luminal SAM domains stabilizes an intermediate 3-helix bundle structure arising from helical interactions of CC12 and CC13 with CC11, thereby releasing the CC1 clamp and allowing the CAD to escape through a fold-out mechanism, with subsequent formation of the CC1 coiled-coil enabling the CAD to revert to its original shape to activate Orai1 in vivo.

physiology↗

bioRxiv2025

Non-Carbohydrate Inhibitors of Sialic Acid-binding Immunomodulatory-type Lectin-7 (Siglec-7) Discovered from Genetically Encoded Bicyclic Peptide Libraries

Glycan-binding proteins (GBP) are among the most difficult to drug targets. This deficiency delays clinical progress for therapeutically important GBPs. We employed bicyclic genetically encoded libraries (BiGELs), produced by chemical modification of phage-displayed libraries of peptides with two-fold symmetric linchpins, to discover inhibitors of therapeutically relevant Siglec-7:GD3 interactions. Next-generation sequencing (NGS) analysis of panning of BiGEL against Siglec-7 yielded 815 candidates from which 23 hits yielded KD = 1-100 {micro}M as determined by surface plasmon resonance (SPR). Competitive enzyme-linked immunosorbent assays (ELISA) identified a subset of leads that disrupted the Siglec-7:GD3 interaction with IC50= 3-300 {micro}M. Machine learning models trained on NGS datasets identified additional inhibitors with equivalent potency. Alanine scans of 8c (SWCRPATVNC, IC50 = 3.8 {micro}M) and 12c (SFCHYPTHVC, IC50= 11 {micro}M), identified key residues as crucial for activity. Ring reshaping studies of compound 8c highlighted the critical role of bicyclic topology produced by analogue 46e (SAAAAAWCRPATVNC, IC50= 9.5 {micro}M). Multivalent display of the lead bicycles alongside [~]100 glycans in Liquid glycan Array (LiGA), made it possible to compare the binding of bicycles and glycans to Siglec-7 expressed on CHO, Jurkat, and Raji cells. LiGA assays confirmed binding of the bicycles to Siglec-7 but revealed considerable non-specific interactions with receptor-negative cells. Saturation transfer difference nuclear magnetic resonance (STD-NMR) revealed 46e binds to Siglec-7 at a site distinct from the V-Ig domain, suggesting it might inhibit binding of glycans to the glycan-binding site of Siglec-7 via an allosteric site. Together these results demonstrate that BiGEL enables the discovery of bicyclic peptides for undruggable Siglec targets but highlights future challenges in molecular discoveries that aim to identify small, non-carbohydrate inhibitors of GBPs.

biochemistry↗

bioRxiv2025

BRD2 bridges TFIID and histone acetylation to promote transcriptional initiation

Members of the bromodomain and extraterminal domain (BET) protein family play a central role in transcription by RNA Polymerase II (Pol II). Small-molecule inhibitors that block interaction between BET bromodomains and acetylated histones have been developed to achieve therapeutic benefit. However, the BET protein BRD4 does not require bromodomains to perform its major transcriptional elongation function, and the mechanisms by which other BET proteins regulate transcription remain incompletely understood. Addressing the disparity between pan-BET degraders and BRD4-specific depletion, we report that the BET protein BRD2 generally functions to promote transcriptional initiation in a bromodomain-dependent manner at both promoters and enhancers in human cells. We demonstrate that BRD2 bromodomains preferentially bind to tetra-acetylated histones harboring MOF-mediated H4K16ac, while the BRD2 C terminal domain facilitates recruitment of TFIID. Our studies provide mechanistic insight into the distinct roles of BRD2 in transcriptional initiation through the recruitment of TFIID and BRD4 in transcriptional elongation through the recruitment of CDK9 and controlling proper regulation of gene expression.

biochemistry↗

bioRxiv2025

The role of intrinsically disordered domains in regulating G protein coupled receptor signaling

The 2A adrenergic receptor (2AAR) is a clinically important target for various diseases including hypertension, diabetes and chronic pain. Here, using single-molecule fluorescence resonance energy transfer imaging, we show how agonist-specific activation dynamics in both structured transmembrane domain (TMD) and intrinsically disorders regions (IDRs) of 2AAR lead to diverse signaling profiles. Through seven pairs of strategically designed fluorophore labels, we systematically investigate the real-time conformational changes of 2AAR. Our study reveals unique TM6 dynamics in 2AAR, featured by a high energy barrier for agonist-induced outward movements essential for activation. In contrast, we identify agonist-specific conformational dynamics of a partially disordered extracellular loop (ECL2), highlighting its role as a dynamic regulatory module that controls receptor function. Moreover, we characterize the conformational landscapes of the long third intracellular loop (ICL3), revealing its compact structural features and membrane-proximal localization in the basal state, where it acts as a negative allosteric regulator in transducer coupling. Furthermore, we identify multiful functional sub-states of ICL3 that are dynamically modulated by both kinase phosphorylation and drug efficacy. These findings offer previously underappreciated structural and dynamic insights into 2AAR function governed by both TMD and IDRs, and may open up new avenues for the development of better therapeutics.

biophysics↗

bioRxiv2025

A Two-Step Synthesis of Covalent Genetically-Encoded Libraries of Peptide-Derived Macrocycles (cGELs) enables use of electrophiles with diverse reactivity

Genetically-encoded libraries of peptide-derived macrocycles containing electrophile warheads (cGELs) can be used to identify potent and selective covalent ligands for protein targets. Such cGELs are synthesized either by incorporation of unnatural amino acids that display mild electrophiles on their side chains or by chemical post-translational modification (cPTM) of mRNA or phage-displayed peptide libraries. Here we investigate fundamental barriers to the synthesis of cGELs. We observe that a previously reported cPTM that proceeds in neutral-to-basic conditions creates mixtures of regioisomers. The complexity of the resulting mixture scales with the electrophilicity of the warhead used in the linker, with some electrophiles being not suitable for use under basic conditions. In contrast, use of a Knorr-pyrazole cPTM enables attachment of electrophiles in acidic pH, thus preventing unwanted reactions with nucleophilic sidechains. The Electrophile is activated only upon mixture with the desired protein target in neutral pH. We use this approach to generate a cGEL with alkyne-bearing macrocycles and use it to identify covalent macrocyclic ligands for pyruvate kinase 2 (PKM2). Our results suggest that construction of cGELs should be performed in conditions that silence the electrophiles (e.g., acidic environment) to prevent unwanted side reactions. In addition to the Knorr-pyrazole method, many other biocompatible bond-forming processes that proceed in mildly acidic pH are likely to be equally effective in constructing cGELs.

biochemistry↗