Biology subjects

Kulkarni, S.

Publications and source records attributed to Kulkarni, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Anvaya: phylogenomic analysis of chromosome fusion-and-mixing characters and detection of shared whole-genome duplication

Chromosome-scale conservation of gene linkage (macrosynteny) provides phylogenetic characters largely independent of sequence data. Fusion of ancestral linkage groups, and especially the subsequent interspersion (mixing) of their genes, is argued to be effectively irreversible, and such synapomorphic events have been used to resolve deep, contentious nodes. I present Anvaya, a dependency-light, open-source toolkit of two Python programs with companion threshold-calibration tools which takes reciprocal-best-hit (RBH) synteny data directly to a phylogenetic analysis that codes fusion and mixing as discrete characters under explicit optimality criteria. The syntenybuilder module builds RBH orthology tables from genome assemblies, using either a designated reference genome or a multi-genome consensus procedure that reconstructs linkage groups de novo, requiring no curated reference set. The character-maps module codes every pairwise linkage-group co-occurrence as unfused, fused-clean or fused-and-mixed from gene order and a Fisher's exact test, then searches for the most parsimonious trees under an unordered (Fitch/Sankoff) and an ordered, irreversible (Camin-Sokal) cost model, optionally under a backbone constraint. Every tree is accompanied by ancestral states, synapomorphies mapped onto clades, consistency and retention indices, bootstrap support, and a permutation test of phylogenetic signal. Anvaya also detects whole-genome and segmental duplication from an intragenomic comparison and recovers the ohnologs that reciprocal best hit structurally drops, which can otherwise render a retained linkage group invisible. Outputs include vector figures and matrices in NEXUS and TNT/Hennig86 formats for verification in PAUP* and TNT. On synthetic data, consensus reconstruction resists a conflicting genome, rescue reverses a total false negative, and the diagnostics flag a deliberately homoplastic character. Across thirteen acarine and outgroup genomes, weakly supported associations concentrate in the most distant genome and in poorly assembled regions. In two duplicated chelicerates, ohnolog rescue recovered 12.5% (Limulus polyphemus) and 2.8% (Mastigoproctus giganteus) and restored three Limulus chromosomes that otherwise carried none.

evolutionary biology↗

bioRxiv2026

Thermoregulatory roles of preoptic area histamine H1 receptor-expressing neurons

The preoptic area (POA) is a key regulator of body temperature, orchestrating behavioral and physiological thermoregulatory functions, including fever, torpor, and homeostatic responses to ambient temperature. Histamine regulates body temperature across animal taxa, including mammals, lower vertebrates, and invertebrates. Hrh1, which encodes the histamine H1 receptor, is expressed in several neuronal populations in the POA with limited overlap with other POA thermoregulatory neurons. Acute activation of POAHrh1 neurons increased body temperature and physical activity. Acute inhibition attenuated the dark phase increase in body temperature without affecting physical activity. There was no effect of inhibition on body temperature during the light phase, fever, cold exposure, or warm exposure. Assessing behavioral thermoregulation, we found that POAHrh1 neurons stimulated nest building in both sexes, possibly contributing to cold-induced nesting behavior. Through projections to the arcuate, dorsomedial hypothalamic and tuberomammillary nuclei, POAHrh1 neurons can increase body temperature and physical activity. Overall, POAHrh1 neurons participate in several thermoregulatory functions, including daily body temperature rhythm regulation, physical activity, and nest building behavior.

neuroscience↗

bioRxiv2026

Biosynthetic Olfactory System from Multiplexed Engineered Microbes (BOSMEM)

Whole-cell biosensors can detect many types of chemicals in complex environments, but existing designs force a tradeoff in multiplexed sensing: separating sensor strains into individual reservoirs limits how many analytes can be monitored at once, while pooling strains in open co-culture lets faster-growing strains take over within days, corrupting the signal. The microfluidic "mother machine" has been used for over a decade, mostly to study individual bacterial strains with single-cell resolution, but never to house multiple engineered strains simultaneously for sensing applications. We repurpose this device to hold a library of genetically distinct biosensor strains, physically isolating each strain in its own growth channel while continuous flow keeps every strain fed. Each strain reports on a target chemical through a unique combination of three fluorescent proteins, so seven distinguishable signals emerge from only three optical channels. We characterized turn-on and turn-off kinetics of several biosensors in the mother machine. Over a multi-day experiment with repeated chemical exposures, biosensors housed this way matched the expected on/off state and held stable population ratios, while biosensors in an open batch co-culture drifted and lost reliable signal. This establishes mother-machine-based multiplexing as a scalable route to long-term chemical monitoring with living sensors.

bioengineering↗

bioRxiv2026

Ancestral genomic stasis and coordinated genome erosion underlies asymmetric acarine diversification

Mites and ticks (Acari) constitute one of the most species-rich and ecologically diverse arachnid radiations, yet their interrelationships and genomic mechanisms underlying their diversification remain poorly resolved. Here, we reconstructed the evolutionary relationships and trajectory of Parasitiformes using phylogenetics and comparative genomics to test the phylogenetic signal in rare genomic changes. Maximum likelihood and macrosynteny analysis revealed multiple shared chromosomal fusion-with-mixing events that unite Opilioacaridae and Ixodida as sister taxa, with Mesostigmata as a derived sister clade, overturning the prior basal placement of Opilioacaridae as a slowly evolving "living fossil" lineage. We show that major parasitiform lineages diversified through asymmetric, large-scale genome and regulatory rewiring. Opilioacaridae genome retains ancestral chelicerate genomic features, including an intact, contiguous Hox gene cluster, high repetitive-element content, and a conserved microRNA repertoire, all shared with Ixodida. By contrast, the hyperdiverse Mesostigmata shows marked genome compaction, with shortened introns, disrupted and fragmented Hox clusters, and widespread loss of microRNA families, despite retention of developmentally essential miRNAs. Comparative analysis of gene-family evolution further identifies lineage-specific expansions in Ixodida associated with blood feeding and immune evasion, alongside independent, convergent expansion of cuticle-protein gene families in ectoparasitic Mesostigmata. These findings demonstrate that contrasting modes of genome evolution, long-term structural stasis versus rapid compaction, together with convergent gene-family expansion linked to independent origins of parasitism, can coexist within a single arachnid radiation, offering new insight into the molecular basis of developmental regulatory evolution and adaptive diversification in Acari.

evolutionary biology↗

bioRxiv2026

Efficient exploration of sequence space enables rapid generation of functional genome editors

The problem of how protein sequences translate into defined functions remains largely unsolved despite decades of progress. New methods to efficiently explore protein sequence space will help to shed light on these sequence-function relationships, particularly for complex protein function. Here, we describe an approach to create novel, functional proteins through the integration of deep mutational scanning, structural analysis, and evolutionary mining within prompts for a generative protein language model (PLM). We demonstrate the utility of this approach with the generation of novel compact RNA-guided nucleases. This approach is highly efficient, resulting in active nucleases with [~]40% sequence divergence relative to natural proteins and activity equivalent to or exceeding by up to [~]3X that of other compact nucleases at multiple endogenous loci in human cells. The approach described here is rapidly deployable and produces new sequences that will serve as scaffolds for further exploration of complex protein functionality, as well as substrates for novel genome engineering applications.

synthetic biology↗

bioRxiv2026

Noise-induced temporary threshold shift in macaques disrupts electrophysiological temporal processing despite recovery of cochlear sensitivity and preserved ribbon synapse counts

Noise exposure can produce lasting auditory dysfunction in the absence of permanent threshold shifts or hair cell loss, yet the functional consequences of temporary threshold shift (TTS) remain poorly defined in translational models. We assessed auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs) in rhesus macaques (n = 13) at 2 and 9-10 months following a single moderate noise exposure that induced TTS. Previous histological analyses of these macaques showed no significant loss of hair cells or ribbon synapses but revealed persistent broadening of inner and outer hair cell ribbon-volume distributions. After exposure, DPOAE amplitudes and thresholds and ABR thresholds returned to pre-exposure values and showed low-frequency enhancement at later time points. Suprathreshold click- and tone-evoked ABR amplitudes were largely preserved or enhanced after exposure, consistent with compensatory gain. In contrast, macaque-specific chirp-evoked ABRs showed modest amplitude reductions and latency prolongation across waves, indicating altered neural synchrony at standard stimulus presentation rates, but with variable time courses. More temporally demanding paradigms revealed persistent impairments. ABRs to faster click rates and shorter paired-click intervals showed reduced adaptability in response amplitude and timing after normalization, with deficits persisting through 9-10 months. Increased inner hair cell ribbon-volume variability was more consistently associated with temporal response measures, including latency, paired-click recovery, and rate adaptation, than with amplitude-based ABR measures. Together, these findings reveal a lasting dissociation between response magnitude and fidelity after TTS: suprathreshold responses may be preserved or enhanced, while neural synchrony and temporal adaptability remain impaired. Increased presynaptic ribbon volume variability may serve as a structural marker of synaptic remodeling accompanying hidden auditory dysfunction, rather than as a direct determinant of suprathreshold response magnitude. Temporally demanding ABR paradigms may supplement threshold-based diagnostics for detecting persistent noise-induced auditory dysfunction.

neuroscience↗

bioRxiv2026

Percolation-inspired criticality in complement activation: universal scaling and transport-limited complement surface amplification

The collective spatial phenomenon of complement protein opsonization on nanoparticle surfaces is a key component of the immune response to viruses, engineered nanoparticles, and diseased cells. Recent work showed this opsonization follows a sharp, percolation-like transition versus the spacing d between surface-bound attachment sites, leaving two open questions: 1) whether the transition exhibits hallmarks of true criticality, such as diverging susceptibility, and 2) whether it can be distinguished from an alternative first-order cooperative (Hill-type) process producing an equally sharp threshold without true criticality. Here, we resolve both questions using a hierarchical statistical-mechanics treatment spanning stochastic, mean-field, and spatial reaction-diffusion models. The variance of two order parameters, peak complement activity and activation lifetime, diverges near threshold and sharpens systematically with system size, the defining signature of a critical point rather than a smooth cooperative response. Extending the analysis across site spacing and intrinsic kinetic rate constants traces a two-dimensional locus of critical points with consistent critical exponents throughout, establishing a single, robust universality class. The mean-field dynamic exponent for activation lifetime agrees quantitatively with the exact value predicted for the general epidemic process. Finally, a reaction-diffusion model of the nanoparticle surface shows the critical locus is set by a diffusion-limited length scale, establishing complement percolation as a fundamentally transport-limited surface reaction. These results place complement activation within the percolation universality class and identify the physical parameters, diffusion, catalysis, and decay, that govern its critical threshold, with direct implications for rational design of complement-evading nanomaterials, immunology, and evolutionary biology.

biophysics↗

bioRxiv2026

Ebola virus exploits host lncRNA LINC01740 to enhance ATF3 and suppress antiviral immune responses

Ebola virus (EBOV) infection causes severe hemorrhagic fever marked by dysregulated cytokine production, impaired antiviral defenses, and multi-organ failure. Macrophages are primary targets of EBOV, and viral replication profoundly alters macrophage transcriptional programs, driving hyperinflammation. Although long non-coding RNAs (lncRNAs) are increasingly recognized as regulators of immunity and viral pathogenesis, their roles in EBOV infection remain poorly understood. We performed comprehensive transcriptomic profiling of primary human monocyte-derived macrophages infected with the highly pathogenic EBOV Mayinga variant. Infection triggered extensive remodeling of both coding and non-coding transcriptomes, including hundreds of differentially expressed lncRNAs. Functional analysis of neighboring protein-coding genes of EBOV-induced lncRNAs (EVILs) revealed enrichment of pathways linked to cytokine signaling, transcriptional regulation, and cell signaling, all of which are central to Ebola virus disease (EVD) pathogenesis. Among the most strongly induced EVILs, LINC01740 and its neighboring protein-coding gene, Activating Transcription Factor 3 (ATF3), were significantly upregulated. Antisense oligonucleotide-mediated inhibition of LINC01740 reduced ATF3 mRNA and protein levels. CRISPR/Cas13d-mediated knockdown of ATF3 restored type I interferon (IFN-I) signaling and antiviral gene expression in EBOV-infected macrophages. Mechanistically, ATF3 functions as a negative regulator of IFN-I and type I interferon-stimulated gene expression, thereby suppressing antiviral immune responses in EBOV-infected macrophages. Together, these findings identify a previously unrecognized LINC01740-ATF3-IFN-I regulatory axis that EBOV exploits to promote immune suppression and viral replication.

immunology↗

bioRxiv2026

The Aorta-Gonad-Mesonephros niche shapes the functions of yolk sac-derived macrophages involved in hematopoietic stem and progenitor cell generation ex vivo

Hematopoietic stem cells (HSCs) generated from induced pluripotent stem cells (iPSCs) offer a promising patient-specific alternative to allogeneic transplantation, yet current differentiation protocols fail to fully recapitulate in vivo HSC maturation. During mouse development, yolk sac (YS)-derived macrophages populate the aorta-gonad-mesonephros (AGM) region at the time of HSC emergence, but the mechanisms by which they support ex vivo hematopoietic stem and progenitor cell (HSPC) generation remain poorly defined. Bulk RNA sequencing revealed that mature AGM CD206 macrophages upregulate pro-inflammatory cytokines and the adhesion molecule F4/80. Using F4/80 knockout embryos, we identify a previously unreported, niche-specific role for F4/80 in restraining the frequency and colony-forming activity of HSPC subsets in the AGM, while supporting endothelial cell maintenance; this effect was absent in the YS. Lineage-tracing with a Cdh5-CreERT2;Rosa26LSL-tdTomato pulse-chase system confirmed that both CD206 and CD206- AGM cells originate from early YS-derived endothelial precursors, with no evidence of local macrophage generation within the AGM. Functional co-culture assays further demonstrated that the ability of CD206 macrophages to enhance the progenitor potential of hemogenic endothelium is AGM-specific and not an intrinsic, ontogeny-determined property, as YS macrophages failed to confer the same benefit even when paired with AGM endothelial cells, and AGM macrophages were ineffective with YS endothelium. Differential expression and NicheNet ligand-receptor interaction analyses identified a small set of AGM-restricted macrophage genes - including Mmp2, Nrep, Ccl2, and Cxcl16 - which are predicted to interact with both endothelial and cluster cells during endothelial-to-hematopoietic transition. Together, these findings establish that AGM macrophages acquire niche-specific transcriptional and functional properties upon entry into the aortic microenvironment, independent of their YS origin, and identify candidate macrophage-derived factors and a novel regulatory role for F4/80 in shaping HSPC output. These insights may guide the refinement of iPSC-based HSC differentiation protocols through the targeted, temporally controlled addition of macrophage-associated signals.

developmental biology↗

bioRxiv2026

Expression patterns and interaction profiles of heterotrimeric transducin subunits in the retina of the European robin ( Erithacus rubecula )

The heterotrimeric G-protein transducin (Gt) is mediating phototransduction in rod and cone cells of the vertebrate retina, but its expression patterns in migratory songbirds is unknown. We characterised Gt expression in the European robin, a night-migratory songbird known for its light-dependent magnetoreception. One well-supported hypothesis of magnetoreception involves radical-pair formation in the blue light receptor cryptochrome type 4a. The - and {gamma}-subunits of cone specific transducin have been identified as possible interaction partners of cryptochrome 4a. Therefore, we analysed the expression patterns of various G-protein subunits in bird photoreceptors by combining single cell RNA sequencing and immunohistochemistry. Protein-protein interaction was tested by pulldown, co-immunoprecipitation, and NanoBiT luminescence assays. G-protein subunits Gt{beta}1 and Gt{beta}3 are predominantly expressed in rods and cones, and Gt{gamma}T2 was the principal isoform in cones, whereas Gt{gamma}11 was associated with rods. In contrast, we did not detect Gt{gamma}10 expression in either photoreceptor type. Interaction assays demonstrated that all three {beta}{gamma} combinations; {beta}{gamma}T2, {beta}{gamma}10, and {beta}{gamma}11, can associate in vitro. These findings indicate that {beta}{gamma} dimer formation in vivo is likely constrained by the photoreceptor-specific expression of the respective subunits. The absence of Gt{gamma}10 expression in rods and cones does not support a role in photoreceptor-based magnetoreception.

Molecular Biology↗

bioRxiv2026

Shape, Shrink, Spheroid: A DIY High-throughput Spheroid Generation Device

3D spheroids, which closely replicate three-dimensional cell-cell and cell-extracellular matrix interactions, offer superior predictive capabilities compared to conventional 2D monolayer cultures, positioning them as forward-looking platforms in drug testing, cancer biology, and regenerative medicine. However, high-throughput generation of uniform sized spheroids is still a technological challenge. In one hand, the use of conventional ultra-low attachment (ULA) multiwell plates for this purpose is labour intensive and complex. On the other hand, the use of microfabricated facilities demands cutting-edge infrastructure such as clean room, photolithography, and microfluidic setup which are often unavailable for the resource constrained laboratories. In this study, we addressed these problems by developing a low-cost Do-It-Yourself (DIY), polydimethylsiloxane (PDMS) and agarose-based spheroid generation device, capable of producing and maintaining hundreds of spheroids with minimal user intervention. We have demonstrated two variants based on their size, termed here as S1 and S2 devices which fit into 6-well and 12-well plates, and can generate 600 and 1200 uniform-sized spheroids respectively. We validated our device with various cell lines including primary and cancerous cell lines. We further demonstrated the drug testing capabilities of the device by estimating the IC50 value of the anticancer drug Temozolomide on U87-MG. The value was comparable with the same obtained from the spheroids generated using conventional ULA plates. Additional attachment of a perfusion system made the device suitable for long-term spheroid culture without much user intervention. Furthermore, the devices can also be used for the production of spheroids with gradually changing diameters in a controlled manner, resembling a size gradient. This feature is useful for checking the effect of drugs on different-sized spheroids and for co-culturing spheroids with varying cell densities, mimicking the disease architecture. We have co-cultured two types of the placental trophoblast cells, i.e., extravillous trophoblast (HTR-8) and syncytiotrophoblast (BeWo) with varying densities. In summary, this paper demonstrates a unique DIY method for a high-throughput uniform-sized spheroid generation at a fraction of cost which can be deployed to resource-constrained labs.

bioengineering↗

bioRxiv2026

Proteome wide serology reveals immune defined subtypes of gastrointestinal disease in systemic sclerosis

BackgroundGastrointestinal (GI) involvement in systemic sclerosis (SSc) affects up to 90% of patients and is a major driver of morbidity and mortality. Despite its clinical importance, GI disease in SSc is highly heterogeneous, with upper and lower GI manifestations representing distinct phenotypic extremes whose underlying immunologic basis remains poorly defined. MethodsWe performed unbiased, proteome-wide autoantibody profiling using a human protein microarray comprising >21,000 full-length proteins (>80% of the human proteome). Sera from patients with SSc and isolated upper GI dysmotility (n=23), isolated lower GI dysmotility (n=17), and non-SSc controls (n=20) were analyzed. Enriched autoantibodies were identified using Fishers exact test, and unsupervised clustering was applied to define serology-based patient subsets and relate immune signatures to clinical phenotypes. ResultsDistinct autoantibody profiles differentiated patients with upper versus lower GI disease. Upper GI-predominant SSc was characterized by enrichment of previously unreported autoantibodies, including those targeting TiSSc1/2 (newly identified proteins encoded within the MIRLET7BHG locus), FAM9C, SPATA20, FAM110D, EMILIN1, CARD14, SMN1, KCTD7, and PHYHD1, whereas lower GI disease was associated with antibodies against HAO2, KLHL7, SUFU, APPL1, BNIP2, UCHL3, ZNF385A, LIMD1, MAGEA9, and PPP2R3C. Serology-driven clustering identified four reproducible subgroups with distinct patterns of GI, pulmonary, vascular, and autonomic involvement, defining clinically meaningful disease phenotypes that extend beyond traditional anatomic classification. ConclusionsProteome-scale serological profiling reveals previously unrecognized autoimmune signatures underlying GI heterogeneity in SSc. These findings support a shift from anatomy-based to serology-defined classification of SSc GI disease and provide a foundation for biomarker development, patient stratification, and precision medicine approaches in this population.

immunology↗

bioRxiv2026

Enteric neurons modulate colorectal cancer cell cycle through a PCSK1 - Methionine-Enkephalin Axis

Background and AimsThe tumor microenvironment in colorectal cancer (CRC) is richly innervated, yet the contribution of the enteric nervous system (ENS) to CRC biology remains poorly defined. ENS neurons express proenkephalin (PENK), which can be processed by proprotein convertase 1/3 (PCSK1) to generate Methionine-enkephalin (M-ENK), a bioactive peptide with growth-regulatory potential. We hypothesized that an ENS-derived PCSK1-M-ENK axis restrains CRC proliferation through opioid growth factor receptor (OGFr) signaling and is modulated by stress-associated glucocorticoid receptor (GR) signaling and GLP1 receptor (GLP1R) activity. MethodsPublicly available human CRC single-cell RNA-sequencing datasets were analyzed for OGFr expression. PCSK1 and M-ENK expression in murine ENS and tumor-associated tissue was assessed by immunofluorescence. Functional studies were performed using murine CRC organoids, and primary murine ENS neurons in mono- and co-culture. CRC proliferation was quantified by EdU incorporation following treatment with recombinant M-ENK, recombinant PCSK1, OGFr synthetic ligand naloxone, or PCSK1 inhibitors. Effects of dexamethasone and liraglutide on PCSK1 expression in ENS-containing murine tissue were evaluated. ResultsOGFr was enriched in CRC cells and positively associated with KRAS gene expression. A subset of adult murine colonic myenteric neurons expressed PCSK1 and M-ENK. M-ENK dose-dependently suppressed proliferation of CRC organoid cells. ENS neurons also suppressed CRC proliferation in a PCSK1-dependent manner. Dexamethasone reduced, whereas liraglutide increased, PCSK1 expression. ConclusionsThese findings define a previously unrecognized ENS-derived neuro-oncologic pathway that is associated with reduced CRC cell proliferation and identify the GR/GLP1R-PCSK1-M-ENK axis as a potentially actionable therapeutic node. SummaryThis study identifies a neuronal PCSK1 - M-ENK pathway in the ENS that directly suppresses colorectal cancer growth through local OGFr activation, revealing a previously unrecognized neuropeptidergic mechanism of tumor control within the intestinal microenvironment.

cancer biology↗

bioRxiv2026

Loss of enteric BDNF TrkB signaling and VIPergic dysfunction underlie gastrointestinal dysmotility in a Mecp2-null mouse model of Rett syndrome

Gastrointestinal (GI) dysmotility is a highly prevalent and clinically significant feature of Rett syndrome (RTT), yet its underlying mechanisms remain poorly defined. Here, we investigated these mechanisms of GI dysmotility in a Mecp2-null mouse model of RTT. First, we observed that MeCP2 was expressed in murine myenteric ganglia, including in enteric neurons and that Mecp2-null males developed maturation-associated functional regression in their GI motility. In dysmotile mice, longitudinal muscle-myenteric plexus tissue showed marked reductions in enteric Bdnf isoforms IV, VI, and II, whereas expression of the BDNF receptor isoforms TrkB.FL and TrkB.T1 was not significantly altered, consistent with reduced enteric BDNF-TrkB signaling. Despite impaired GI motility, Mecp2-null mice showed no significant changes in total enteric neuronal density, nitrergic neuronal abundance, or expression of Nos1, Chat, and Uchl1. In contrast, Vip expression was significantly reduced, while expression of VIP receptor genes: Vipr1 and Vipr2 was increased, indicating disrupted VIPergic signaling. Integration with publicly available enteric single-cell/nucleus datasets and targeted qRT-PCR further suggested altered inhibitory neuronal subtype composition, with reduced Vip+ Cartpt+ signatures and increased Nfia expression, suggesting that MeCP2 loss differentially affects distinct inhibitory neuronal subpopulations. Finally, conditional loss of TrkB.FL in neural crest-derived cells reduced Vip expression without recapitulating the full Mecp2-null VIPergic phenotype, indicating that impaired BDNF-TrkB signaling contributes to, but does not completely explain, the GI dysmotility in this model of RTT. Together, these findings identify enteric BDNF-TrkB and VIPergic dysfunction as key mechanisms underlying GI dysmotility in RTT.

neuroscience↗

bioRxiv2026

Molecular basis of promiscuous chemokine-engagement by the Duffy antigen receptor

The Duffy blood group antigen, encoded by a seven transmembrane protein known as Duffy Antigen Receptor for Chemokines (DARC), or Atypical Chemokine Receptor 1 (ACKR1), serves as a key receptor for the malarial parasite, Plasmodium vivax, on erythrocytes. DARC exhibits remarkable functional divergence compared to prototypical chemokine receptors and other G protein-coupled receptors (GPCRs) as it does not engage canonical signal-transducers such as G-proteins, GPCR regulatory kinases (GRKs), and {beta}-arrestins. DARC is a highly promiscuous receptor interacting with several homeostatic and inflammatory C-C and C-X-C subtype chemokines, and similar to other chemokine receptors, these interactions are modulated by post-translational tyrosine sulfation in the N-terminus. A single nucleotide polymorphism in DARC leading to Gly42Asp substitution forms the basis for Fya vs. Fyb Duffy blood group antigen classification with differential impact on Plasmodium vivax infection and cancer progression. However, the molecular basis of promiscuous chemokine-binding by DARC and the modulation by receptor sulfation and Fya/Fyb allelic variation remains unclear. Here, we design a sortase-mediated chemical-ligation strategy to generate purified DARC with naturally-occurring tyrosine sulfation at the N-terminus, and determine high-resolution cryo-EM structures in complex with a C-C type chemokine, CCL7, and a C-X-C type chemokine, CXCL8. We observe that similar to CCL7, CXCL8 engages with DARC primarily through the N-terminus of the receptor, which is in stark contrast with the two-site binding mechanism displayed by other chemokine receptors. Interestingly, the E-L-R motif in CXCL8 engages a pseudo-R-D motif in DARC leading to superficial engagement unlike CXCR2, a prototypical chemokine receptor sharing the same agonist. Moreover, tyrosine sulfation and Fyb allelic variation leads to a repositioning of the receptor N-terminus on the core domain of CXCL8, resulting in distinct interaction network imparting greater binding affinity. Taken together, our study presents novel insights into promiscuous chemokine-binding to DARC, tyrosine sulfation-mediated fine-tuning of chemokine engagement, and a generalizable sortase-mediated chemical-ligation platform applicable to other chemokine receptors.

biochemistry↗

bioRxiv2026

Benchmarking within-sample minority variant detection with short-read sequencing in M. tuberculosis

MotivationLow-frequency (minority) variants--variants detectable within-sample at low allele frequencies--are relevant in several areas of research and health, from cancer to pathogen heteroresistance. There is uncertainty around the optimal bioinformatic approach to accurately and reproducibly distinguish low-frequency variants from sequencing or mapping errors. To address this, we benchmarked seven variant callers on precision, recall, and false positive characteristics for detecting low-frequency variants using simulated short-read whole-genome sequencing data for 700 Mycobacterium tuberculosis strains. We developed a new low-frequency error model for filtering the output of the best-performing tool using read mapping and quality metrics. ResultsWe simulated 378 unique variants across five genomic backgrounds spanning four lineages. Variants were simulated to represent three genomic region categories, 10 allele frequencies and five sequencing depths. FreeBayes, a haplotype-based variant caller, achieved the highest pooled F1 score of the seven tools in drug resistance regions (average F1=0.86) and its higher performance held across genomic context and background. Across tools, we identified lower performance in repetitive (low mappability) regions, and strong reference bias in low-frequency variant calling. We validated variant caller performance on in-vitro strain mixtures substantiating our ranking. The error model excludes <1% of true variants identified by FreeBayes on average per strain, and excludes 97% of FPs on average per strain when combined with low mappability region and rRNA gene masking. Our analysis informs best practices for low-frequency variant calling, including tool choice, masking, and filtering. We also provide a new error model that excludes false low-frequency variant calls from FreeBayes output. Availability and implementationAll relevant code is available at https://github.com/shandu-m/benchmark-minority-variants-Mtb. Supplementary InformationSupplementary Files 1-3 respectively are attached with this submission.

bioinformatics↗

bioRxiv2026

Eye-Tracking-BIDS: the Brain Imaging Data Structure extended to gaze position and pupil data

The Brain Imaging Data Structure (BIDS) is a widely adopted, community-driven standard to organize neuroimaging data and metadata. Although numerous extensions have been developed to incrementally extend coverage to new modalities and data types, an unambiguous, granular specification for eye-tracking recordings is lacking. Here, we present how BIDS will structure data and metadata produced by eye-tracking devices, including gaze position and pupil data. In addition to prescribing the organization of the unprocessed (raw) recordings and associated metadata as produced by the device, BEP20 also resolves gaps in current BIDS specifications beyond the scope of eye tracking. In particular, it adds a mechanism for including asynchronous model parameters and messages, such as contextual information, statuses, and events, such as triggers, generated by the device. BEP20 includes examples that illustrate its applicability in various experimental settings. This BIDS extension provides a robust standard that supports the development of self-adaptive, open, and automated eye-tracking data structures, thereby bolstering transparency and reliability of results in this field.

neuroscience↗

bioRxiv2026

Computational insights on the competition between electrotaxis and durotaxis

Understanding how cells migrate in response to external cues has important implications for biology, medicine, and bioengineering. Chemical, mechanical, and electrical signals are the primary drivers of directed cell migration, and each has been extensively studied over the past decades. Among them, chemical cues were the first to be investigated and remain the most widely studied due to their undeniable role in in vivo guidance. Mechanical signals--particularly substrate stiffness gradients--have gained prominence for their ubiquity across cell types and their potential to direct migration. More recently, growing evidence suggests that electrotaxis offers a highly precise and programmable means to guide cell movement. Despite this, these cues are often studied in isolation, whereas in vivo they typically coexist and interact. Using wellestablished biophysical models, we investigate how mechanical and electrical signals cooperate and how they can be engineered to compete for control over cell migration. We demonstrate that an electric field can override and even reverse durotaxis, with outcomes that depend strongly on the specific cell type. To address this large variability in controlling cell migration, we propose particular steps toward further exploration. To support such future research, we provide a freely available platform for predicting electro-mechanical interactions in cell migration, based on a given cells sensing and signaling characteristics, which could tailor the mechanical and electrical signals that arise naturally during organ development, cancer invasion, or tissue regeneration.

biophysics↗