Biology subjects

Fan, H.

Publications and source records attributed to Fan, H..

At least 19 recordsLinked to original sources

bioRxiv2026

A Refined Developmental Transcriptomic Landscape Reveals Four Coordinated Programs in Chlamydia trachomatis

The developmental cycle of Chlamydia trachomatis requires coordinated transitions between infectious elementary bodies (EBs) and replicative reticulate bodies (RBs), yet the temporal organization of the underlying transcriptional programs remains incompletely resolved. We performed high-coverage RNA sequencing from 0 to 32 h post-infection (hpi), with closely spaced early sampling, and used unsupervised clustering of temporal expression profiles to identify four coordinated programs: immediate-early host adaptation and developmental program, RB formation and maintenance, RB proliferation and secondary differentiation, and EB formation and invasion. The immediate-early program peaked at 1 hpi and contained a strong representation of inclusion-membrane and intracellular-trafficking genes, together with the developmental regulator gene euo. The RB formation and maintenance program peaked at approximately 3 hpi and sustained expression throughout much of the cycle, encompassing genes that support transcription, translation, and core metabolism. The RB proliferation and secondary differentiation program combined DNA replication and cell-division genes with alternative sigma factors, partner-switching components, and type III secretion apparatus genes, consistent with developmental preparation during proliferation. The EB formation and invasion program contained genes associated with structural maturation, host-cell interaction, and infectious competence. These findings distinguish transient host-adaptation expression from sustained early biosynthetic expression, separate RB establishment from population expansion, and differentiate regulatory preparation for the RB-to-EB transition from later EB maturation. Together, the four programs define partially overlapping transcriptional priorities that provide a framework for investigating how gene expression coordinates chlamydial growth, differentiation, and preparation for the next infection cycle.

microbiology↗

bioRxiv2026

Inhibitory Evoked Potentials as a Spatially Dependent Intraoperative Marker of Clinical Tremor Reduction

Deep brain stimulation (DBS) of the ventral intermediate nucleus (Vim) of the thalamus may be used to treat medication refractory essential tremor. Using recordings from in vivo human Vim neurons, our previous work has suggested that evoked potentials (that we termed quasi-evoked inhibition) ~2 ms following high frequency microstimulation pulses may be related to inhibitory synapses onto the Vim. Here, we investigate whether (i) quasi-evoked inhibition is related to clinical tremor reduction, and (ii) if quasi-evoked inhibition is dependent on the stimulation location within the Vim. By developing an objective determination of the presence or absence of quasi-evoked inhibition and utilizing accelerometer recordings, we showed that recordings with quasi-evoked inhibition at 100 Hz microstimulation exhibit greater tremor reduction than those without (P < 0.05, BF > 30). The number of stimulation pulses with quasi-evoked inhibition is also correlated with tremor reduction (rho = 0.18, P < 0.05) at all stimulation frequencies >=100 Hz. Furthermore, by analyzing microelectrode trajectories reconstructed from structural MRIs, we found that proximity to the ventral caudal border (P < 0.005) and to a previously established sweet spot (P < 0.05) are anti-correlated with the number of stimulation pulses with quasi-evoked inhibition. Our findings suggest that quasi-evoked inhibition is a potential biomarker of tremor reduction by means of network inhibition, and the more posterior regions of the Vim may allow for better recruitment of inhibition. This may be useful for closed-loop stimulation design.

neuroscience↗

bioRxiv2026

A curated human lactylome and protein language model framework enable accurate prediction and reveal local determinants of lysine lactylation

Lysine lactylation is a dynamic post-translational modification that can alter protein function and has been implicated in diverse physiological and pathological processes. Accurate identification of lactylation sites is therefore important for defining its regulatory landscape and for generating testable hypotheses about lactylation-associated mechanisms. Here, we introduce CLEAR-Lactyl and AttentionKla. CLEAR-Lactyl is a curated benchmark dataset of human lysine lactylation comprising 16,604 positive sites. AttentionKla is a deep learning framework trained on CLEAR-Lactyl that employs a pre-trained protein language model fine-tuned with LoRA; it significantly outperforms existing tools, and the factors contributing to its performance gain have been dissected through comprehensive ablation studies. Its utility in predicting novel lactylation sites and in sequence-directed modulation of lactylation levels has been experimentally validated in cellular assays. Together, CLEAR-Lactyl and AttentionKla provide a powerful platform for lysine lactylation research and offer an extensible framework for the precise modulation of other post-translational modifications.

bioinformatics↗

bioRxiv2026

Regulation of Chlamydia trachomatis infection in the female genital tract by type I and type II interferons

Following an intravaginal inoculation with Chlamydia trachomatis, mice deficient in type I interferon receptor IFNR1 (IFNR1-/-) significantly increased the yield of live chlamydiae on days 3 & 5 but reduced it to the level of wild-type mice by day 7, while mice deficient in type II interferon receptor IFN{gamma}R1 (IFN{gamma}R1-/-) significantly increased the chlamydial yield by day 5 and the increase persisted throughout the remainder of the infection course. These observations reveal a temporal division of labor between type I & II interferons in regulating C. trachomatis infection in the female genital tract. Interestingly, mice deficient in both IFNR1 & IFN{gamma}R1 exhibited higher mortality and shed more chlamydial organisms than IFN{gamma}R1-/- mice by week 6, suggesting that IFNR1 remains critical for inhibiting C. trachomatis at late stages. An anti-IFNR1 antibody blockade significantly increased chlamydial yields in IFN{gamma}R1-/- mice, suggesting that the anti-chlamydial activities of type I & II interferon systems are both distinct and overlapping throughout the infection course. Furthermore, the anti-chlamydial activity of type I interferon signaling is localized to the lower vagina, while that of type II interferon signaling is localized to the upper vagina. Thus, we have demonstrated that type I & II interferons function complementarily and synergistically in time and space to control C. trachomatis infection, laying the foundation for further elucidating the mechanisms of IFN regulation of chlamydial infection and for developing interventional and preventive strategies against C. trachomatis in the female genital tract. ImportanceLack of information on the precise roles of type I and type II interferons during chlamydial infection has hindered the development of interferon-based strategies to prevent chlamydial infection and pathogenicity. The current study has revealed a temporal division of labor between type I & type II interferons in regulating chlamydial infection in the female genital tract, with type I acting earlier than type II during the innate phase. Nevertheless, type I remains critical for cooperating with type II to suppress chlamydia 6 weeks after infection. Finally, type I interferons seem to mainly target chlamydial infection in the lower vagina, while type II interferons target the infection in the upper vagina. These new findings on the distinct and overlapping roles of type I and type II interferons in regulating chlamydial infection may guide the development of interferon-based precision strategies to reduce chlamydial infection and pathogenicity in the female genital tract.

immunology↗

bioRxiv2026

Using spIsoNet to address the preferred-orientation problem in cryoEM reconstructions

Cryogenic electron microscopy (cryoEM) is now routinely used for high-resolution structure determination of biological macromolecules. However, many biological specimens exhibit varying degrees of preferred orientation on cryoEM grids, resulting in uneven sampling of three-dimensional Fourier space. This orientation bias produces anisotropic reconstruction artifacts and, in severe cases, can exacerbate particle misalignment during iterative refinement, thereby limiting the success rate of near-atomic resolution cryoEM structure determination. This protocol provides a practical guide for applying spIsoNet, a self-supervised deep-learning method, to mitigate preferred-orientation issues in cryoEM reconstructions. We describe two complementary workflows: (1) map Anisotropy Correction to correct anisotropic artifacts of cryoEM maps and (2) particle Misalignment Correction, which integrates spIsoNet with RELION external reconstruction to improve particle-pose estimation. We demonstrate these workflows using two influenza hemagglutinin (HA) trimer datasets representing moderate and severe degrees of preferred-orientation bias. The protocol includes installation instructions, parameter-selection guidance, quality-control checkpoints and troubleshooting advice, and can typically be completed in [~]7 hours on a workstation equipped with four NVIDIA A100 GPUs. Together, these workflows provide step-by-step guidance for using the open-source spIsoNet software to mitigate the preferred-orientation problem directly from experimental data.

biophysics↗

bioRxiv2026

Confidence-supported label-free metabolic imaging with FPhaS phase autofluorescence microscopy

Label-free optical redox imaging utilizes endogenous NAD(P)H and FAD autofluorescence to evaluate metabolism in living specimens. The conventional optical redox ratio collapses these two channels into a single value; however, it does not indicate whether a pixel has sufficient photon support or the cellular context necessary for quantitative aggregation. To address this limitation, we introduce FPhaS, a fixed-calibration phase- autofluorescence framework that integrates quantitative phase imaging (QPI) with simultaneous label-free autofluorescence multi-harmonic microscopy (SLAM), using fluorescence lifetime imaging (FLIM) solely for validation. Because QPI and SLAM are acquired with the same objective, a unified non-biological calibration aligns phase-derived structural data with the autofluorescence frame, yielding a residual error of 0.39 pixels. This calibration is maintained across all biological specimens. This shared geometric reference enables local evaluation of structural and metabolic information, rather than comparing approximately aligned images. FPhaS decomposes the data into cell presence, ratio credibility, and confidence-supported pooling. We validated FPhaS on A549 cells under high and low-photon conditions; the framework is designed to generalize to other cell and tissue types. Confidence-weighted intensity redox estimates were compared with lifetime-derived measurements within mask-locked cellular regions. Concordance improved exclusively when both the denominator photon support and an independent structural criterion were satisfied. The same reference layer generated cell-level descriptors of metabolic content, metabolic-structural organization, and measurement reliability, while also constraining the CombinedWLS reconstruction under diminished fluorescence acquisition. FPhaS redefines label-free metabolic imaging from producing comprehensive ratio maps to identifying regions where optical evidence substantiates quantitative inference.

bioengineering↗

bioRxiv2026

Protocol for scalable generation of uniform 3D porcine oviduct epithelial cell spheroids

While previous in vitro porcine (Sus scrofa) oviduct spheroid models often rely on manual selection of the formed aggregates, here, we establish a scalable and reproducible system for generating oviduct spheroids. We describe steps for isolating primary secretory and ciliated oviduct epithelial cells and their assembling them into spheroids by forced aggregation using the AggreWell platform. This enables the mass production of uniformly sized spheroids ready for use in a range of downstream applications, such as sperm-oviduct co-incubation experiments. For complete details on the use and execution of this protocol, please refer to Molina et al.1 Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/731731v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@20f94aorg.highwire.dtl.DTLVardef@ce80eeorg.highwire.dtl.DTLVardef@172c631org.highwire.dtl.DTLVardef@31f420_HPS_FORMAT_FIGEXP M_FIG C_FIG Before you beginThis protocol describes the isolation of primary porcine oviduct epithelial cells and their use in generating a three-dimensional (3D) spheroid model in vitro. The protocol is specifically designed to enrich and preserve ciliated oviduct epithelial cells, which are critical for physiologically relevant sperm-oviduct interaction studies yet are frequently underrepresented in conventional in vitro oviduct models. To minimize the de-differentiation of ciliated cells during culture, a specialized Incubation Medium is employed to maintain epithelial cell viability and ciliary function. The relatively brief spheroid formation period (48 h) further supports preservation of ciliary structure and activity prior to downstream functional applications. This protocol consists of four major steps. Steps 1 and 2 cover oviductal epithelial cell isolation through mechanical and enzymatic digestion, step 3 describes the spheroid formation and culture conditions, and step 4 applies the spheroid model to sperm co-incubations or immunofluorescent staining. Although optimized for the porcine isthmus, this protocol may also be suitable for the cells from the ampulla and adaptable to other large animal species.

Cell Biology↗

bioRxiv2026

Protein Phosphatase 2A Orchestrates Mitochondrial Dynamics via MAPK Signaling in heart

Heart as one high ATP consuming organ accounts for 5% of the total oxygen demands. The central question of heart health is how mitochondria fit its needs. Impaired mitochondrial dynamics (fission and fusion) have been observed in failing heart, but whether and how phosphorylation events involved in mitochondrial quality control are still imperceptive. The phosphatase 2A catalytic subunit (PP2A c) cardiac-specific knockout mouse (KO), which exhibited a hypertrophic cardiomyopathy phenotype, was studied. We profiled the pattern of morphological and functional alteration of cardiac mitochondria that appeared during postnatal development. Increased heterogeneity of mitochondria and a decreased ATP yield was displayed. Notably, a fission procedure escalated. To illustrate the protagonist of the mitochondrial dynamics, we applied a high-throughput spectrometry-based phosphoproteomic screening following by GO and KEGG pathway annotations for 788 phosphosites, accounting for 90 proteins. Results suggested that the MAPK signaling may be a predominant factor associated with those mitochondrial alternations in KO hearts. Furthermore, we identified hyperphosphorylated ERK2 accumulated into the nucleus regarding PP2Ac depletion. Consequently, Fis1 expression was accelerated at the transcriptional level which facilitated recruitment of Drp1 onto the outer mitochondrial membrane. The mitochondrial fission towards shifting led to excessed mitophagy and is considered the culprit in early mortality. These findings are indicative of the fundamental role of PP2A in mitochondrial dynamics regulation and cardiomyopathy progression. During the progression of heart failure, the phospho-regulation of ERK2 could be a novel therapeutic approach to prevent or attenuate adverse hypertrophic cardiomyopathy.

cell biology↗

bioRxiv2026

3D, multi-omic imaging reveals molecular biomarkers of the pre-metastatic niche in lung cancer

The recurrence rate following complete surgical resection of primary non-small cell lung cancer is as high as 55%, yet no approach currently exists to evaluate the risk of local recurrence. The premetastatic paradigm is the recognition that metastasis is preceded by reprogramming naive tissues to prime a microenvironment for tumor cell survival and subsequent reactivation. Identification of biomarkers of the pre-metastatic niche would allow us to evaluate a patients risk of local relapse in the normal lung parenchyma surrounding the resected tumor. We designed a workflow incorporating in vivo modelling, radiology, and deep learning-guided three-dimensional (3D) imaging, spatial proteomics, and transcriptomics to identify previously unreported signals associated with the early transformation of the lung parenchyma announcing regional metastasis. We curated biorepository spanning timepoints before and after resection of primary Lewis Lung Carcinoma (LLC) tumors. Using radiology and cellular resolution 3D histology, we calculated the number and distribution of metastases in mouse lungs and developed an algorithm to guide placement of spatial proteomics and transcriptomics to regions containing early micro-metastases and the pre-metastatic microenvironment. Molecular and tissue features associated with presence, size, and location of metastases guided the identification of both myeloid (F4/80) and senescent (p16/p21) cell signatures in the premetastatic and metastatic environments. Finally, multiparametric flow cytometry of metastatic lungs in a senescence reporter GEMM (tdTomato-p16 INKA mice) resolved senescent cells including alveolar macrophages as the cellular phenotypes associated with these early premetastatic signatures. Altogether, this work highlights a novel AI-assisted approach for detection of biomarkers of tissue remodeling during lung cancer invasion.

bioengineering↗

bioRxiv2026

Reproducible Tools and Enhanced Computational Workflows for Batch Effect Evaluation of High-Throughput Data Using BatchQC

Batch effect correction is a common and often necessary step in data analysis to reduce bias due to technical and experimental factors when combining multiple batches of data. The severity of the batch effects dictates the correction strategy; therefore, a careful assessment of each datasets batch effects is necessary. BatchQC is an R package that provides reproducible tools and visualizations for quantitatively and qualitatively addressing batch effects across a broad range of data types. BatchQC integrates with standardized Bioconductor data structures and features an object-oriented design, enabling the application of workflows that can freely evaluate and process data within and outside the package tools. Common batch evaluation methods, along with novel quantitative metrics, help determine the benefits of batch correction for each dataset and enable direct comparisons between methods. Here, we present BatchQC as the first comprehensive batch-correction R package, with independent tools, reproducible workflows, visualization, and novel statistics.

bioinformatics↗

bioRxiv2026

Chlamydial Histones Control Developmental Fitness in the Next Infection Cycle

The unique chlamydial developmental cycle comprises three stages: primary differentiation of infectious elementary bodies (EBs) into reticulate bodies (RBs), RB replication, and secondary differentiation into progeny EBs. Extensive chromosome remodeling during RB-to-EB differentiation is thought to be mediated by the histones HctA and HctB. Here, we used an inducible CRISPR interference system to repress hctA, hctB, or both genes during development in Chlamydia trachomatis. Surprisingly, repression of either histone gene alone or in combination caused only modest reductions in EB yield and did not prevent nucleoid condensation during the parental developmental cycle. In contrast, when progeny EBs generated under histone-repressing conditions were used to initiate secondary infections in the absence of inducer, histone deficiency during EB maturation profoundly impaired fitness in the next infection cycle. Secondary cultures initiated with HctA-deficient EBs exhibited a delayed onset of genome replication, consistent with inefficient primary EB-to-RB differentiation, whereas combined repression of hctA and hctB caused both delayed genome replication and persistently reduced genome accumulation, indicative of defects in RB formation and subsequent growth. Repression of hctB alone did not measurably affect genome replication in secondary cultures. Together, these findings reveal a transgenerational role for chlamydial histones and establish chromosome organization during EB maturation as a key determinant of developmental fitness across infection cycles. IMPORTANCEChlamydial histones HctA and HctB are unusual among bacterial chromatin-binding proteins in that they share sequence homology with mammalian histones and are developmentally regulated during the formation of infectious particles. Here, we show that reduced expression of HctA and HctB has only modest effects on genome condensation and EB production, consistent with partial functional redundancy between the two histones and suggesting that additional chromatin factors contribute to EB chromosome compaction. In contrast, deficiency of HctA and HctB during EB maturation has profound consequences in the next infection cycle, impairing primary EB-to-RB differentiation and subsequent RB growth. These findings reveal a previously unrecognized transgenerational role for chlamydial histones and establish chromosome organization during EB maturation as a key determinant of developmental fitness across infection cycles.

microbiology↗

bioRxiv2026

Identification of collaborative cross mouse strains susceptible to chlamydial induction of hydrosalpinx

Sexually transmitted infection with Chlamydia trachomatis can cause pathology, such as hydrosalpinx, in the upper genital tract, leading to infertility. To investigate how genetic variation affects chlamydial pathogenicity, we screened five strains of Collaborative Cross (CC) mice for susceptibility to intravaginal infection and hydrosalpinx induction by C. muridarum, a mouse-adapted chlamydial species used extensively to reveal chlamydial pathogenic mechanisms. In terms of susceptibility to genital chlamydial infection, the five CC strains fell into two categories: CC011 and CC012 were resistant, while CC037, CC042 & CC080 were susceptible. The resistant strains shed significant levels of live organisms from the genital tract for only [~]2 weeks, whereas the susceptible strains shed for [~]4 weeks. However, the resistant CC012 mice developed the highest level of hydrosalpinx, while the susceptible CC042 mice were fully resistant to hydrosalpinx induction. None of the CC mice developed spontaneous hydrosalpinx in the absence of chlamydial infection. The above results, validated macroscopically and microscopically, indicate no correlation between pathology and genital infection, as observed in other inbred mice. Nevertheless, among the two infection-resistant strains, CC012 developed more severe hydrosalpinx than CC011, and hydrosalpinx was positively correlated with live organism shedding from rectal swabs but not from vaginal swabs, supporting a potential role of gastrointestinal chlamydia in chlamydial genital pathogenicity. The above results lay a foundation for using CC mice to further map the genetic determinants that regulate host susceptibility to chlamydial infection and pathogenicity in the female genital tract.

microbiology↗

bioRxiv2026

Dynamic control of Raf-ERK signaling modulates neuronal activity across biological scales

Neuronal activity robustly engages the extracellular signal-regulated kinase (ERK) signaling pathway through Ca2+-dependent mechanisms; however, whether ERK can acutely and causally modulates ongoing neuronal activity remains unsolved due to complex upstream regulation and diverse subcellular functions. Here, we directly address this question using an optogenetic ERK activator, opto-miniRaf, that enables selective, rapid, graded, and reversible control of ERK signaling. Combining this AAV-compatible system with calcium imaging and electrophysiology, we interrogate ERK functions across biological scales, from cultured neurons, acute brain slices, and the intact brain. Acute optogenetic activation of ERK enhances synchronized network burst activity in cultured rat cortical neurons and increases calcium activity of cortical pyramidal neurons in awake and moving mice following non-invasive light stimulation. Together, these results establish ERK signaling as an acute modulator of neuronal and network activity, positioning opto-miniRaf as a generalizable platform for precise spatiotemporal control of intracellular kinase signaling in complex biological systems.

neuroscience↗

bioRxiv2025

Comparative Transcriptomics Reveals Genes Commonly Induced by Distinct Stressors in Chlamydia

Chlamydia trachomatis is a leading cause of urogenital infections that can result in serious long-term complications. This obligate intracellular bacterium undergoes a biphasic developmental cycle alternating between the infectious elementary body and the replicative reticulate body and can enter a persistent state in response to adverse environmental conditions. Although transcriptomic reprogramming is central to chlamydial stress adaptation and persistence, how responses differ across biologically distinct stressors remains incompletely defined. Here, we performed a comparative reanalysis of five published, high-quality C. trachomatis RNA-Seq datasets generated under prolonged interferon-{gamma} treatment, tryptophan starvation, iron starvation, penicillin exposure, or acute heat shock. Global transcriptomic analyses reveal extensive stress-specific reprogramming and a clear separation between the transcriptome induced by heat shock and those induced by chronic stresses. Transcriptomic overlap observed among chronic stress conditions is substantially reduced when the heat shock transcriptome is included, indicating that shared transcriptional features are stressor-dependent. Unexpectedly, tryptophan starvation and iron starvation exhibit particularly close transcriptomic similarity, consistent with regulatory cross-talk mediated by the iron-dependent transcriptional repressor YtgR. In contrast, interferon-{gamma} induces a distinct but partially overlapping transcriptome, likely reflecting activation of additional host-mediated antimicrobial mechanisms beyond tryptophan deprivation. Together, these findings demonstrate that adaptation to different biological stressors in C. trachomatis is driven by distinct transcriptomic reprogramming, while consistently involving a subset of functions that may represent points of vulnerability for disrupting chlamydial persistence.

microbiology↗

bioRxiv2025

IsoNet2 determines cellular structures at submolecular resolution without averaging

AbstractWe introduce IsoNet2, an end-to-end self-supervised deep-learning method that directly reconstructs high-quality 3D densities from cryogenic electron tomography. A unified network simultaneously performs denoising, contrast transfer function correction, and missing-wedge restoration, achieving [~]20 [A] resolution without averaging. A feature-rich GUI enables rapid, dataset-specific fine-tuning for end-users. IsoNet2 resolves domain organization in HIV capsid proteins, tRNA occupancy in individual ribosomes, and in situ architectures of mitochondrial respiration-related complexes, enabling atomic-level interpretation of cellular environments.

cell biology↗

bioRxiv2025

Outward-facing P-glycoprotein bound to drug displays nucleotide-dependent drug egress mechanism

ABCB1/P-glycoprotein requires coordination between ligand movement in the trans-membrane cavity and ATP binding and hydrolysis at the nucleotide binding domains, yet the sequence of these events remains incompletely resolved. Here, atomistic simulations with paclitaxel and verapamil across outward facing P-glycoprotein ensembles in various nucleotide states were integrated with analysis of transmembrane domain contacts, geometric solvent/ligand pathways, extracellular gating distances, and nucleotide coordi-nation characterization. Collectively, our simulations delineate a mechanistic framework for P-glycoprotein in which ATP binding organizes an outward-facing ensemble and sequential hydrolysis at the two nucleotide-binding sites drives substrate progression toward egress. Asymmetry in the nucleotide state is correlated to distinct motions within the transmembrane domains, which suggests a possible transient extracellular gating mechanism that enables staged release and limits re-entry of the ligand. The resulting model connects nucleotide chemistry to transmembrane domain motions that regulate gate dynamics and may guide experiments and development of modulators for ABC exporter activity in physiological and pharmacological contexts.

molecular biology↗

bioRxiv2025

Drug-bound P-glycoprotein reveals nucleotide-dependent ligand pockets and ingress channels

P-glycoprotein, an ATP-binding cassette exporter, coordinates drug recognition in a central cavity with nucleotide handling at nucleotide binding domains, yet the sequence and coupling of these events remain incompletely defined. Here, we report an integrated computational analysis of inward facing P-glycoprotein ensembles across varying drug locations, nucleotide states, and starting conformations. The sampled inward-facing ensembles of P-glycoprotein display continuous ligand cavity, reshaped by nucleotide occupancy, which enables polyspecific ligand recognition while transmitting allosteric signals to the nucleotide binding domains through intracellular coupling loops. Asymmetry between the two nucleotide binding sites follows from this communication, which alters the nucleotide coordination to potentially affect the catalytic progression. Our simulations reveal dynamic cavity access routes from cytosol and inner membrane, together with transient secondary structure within the interdomain flexible linker. Overall, our study gives insights into how ligand redistribution, asymmetric nucleotide coordination, and linker flexibility may correlate to form a unified view of early transport steps, yielding hypotheses for mutational and kinetic evaluation and informing strategies for small-molecule modulation of transporter function in cellular contexts where multidrug transport affects therapeutic response.

molecular biology↗

bioRxiv2025

Synergistic role of riboflavin-auxotrophic Enterococcus for MR1 expression and intra-tumoral mucosal associated invariant T (MAIT) cell activation

The role of mucosal invariant T cells (MAITs), in the lung tumor microenvironment re-mains poorly understood, especially in the setting of immune checkpoint inhibitors. We identified intratumoral MAIT cells from paired single cell RNA and TCR sequencing datasets of tumor infil-trating CD3 T cells isolated from non-small cell lung cancer tumors in patients receiving neoadju-vant PD-1 blockade therapy. MAIT cells were subclustered to identify conventional MAIT-associ-ated TCR clonotypes predicted to recognize intratumoral bacteria, which we then tested for func-tional recognition using a MAIT TCR capture functional assay. Strikingly, although not directly recognized by MAIT cells, previously identified probiotic Enterococcus spp and detected in the intratumoral microbiome of lung cancer patients, selectively synergized with exogenous riboflavin biosynthesis-derived metabolites to induce expression of MR1 by antigen presenting cells, includ-ing dendritic cells, B cells and mononuclear phagocytes. Boosting of MR1 cell surface expression resulted from perturbation of endo-lysosomal vacuolar pathway by Enterococcus and recycling of early endosomal MR1 to the cytoplasmic membrane. Riboflavin auxotrophic Enterococcus spp may therefore exercise their beneficial immunomodulatory functions upon immune checkpoint blockade treatment, at least in part, by promoting intratumoral MR1 expression and innate like T cell activation. Our results indicate that composition of the intratumoral microbiome during im-mune checkpoint inhibitor treatment has the potential to impact the function of human intratumoral MAIT cells.

immunology↗