Biology subjects

Tan, S.

Publications and source records attributed to Tan, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Aging-induced hepatocyte CD44 drives IL6/STAT3 signaling and associates with impaired neighboring T cell function.

Liver cancer incidences increase dramatically beyond 55 years of age, suggesting that age-associated changes contribute critically to tumor initiation. However, the mechanisms linking liver aging and cancer initiation are not well defined. This study investigates the role of CD44, a marker of liver tumor-initiating cells (TIC), in age-associated liver pathophysiology. Aged livers showed accumulation of CD44-expressing hepatocytes exhibiting enrichment of immune modulatory genes and activation of the immunosuppressive IL6/JAK/STAT3 pathway. Indeed, in adoptive transfer assays, antigen-exposed CD8+ T cells mounted a lower IFN-{gamma} response in aged livers than in young livers, indicating an immunosuppressive aged milieu. Concordantly, spatial analyses showed that the proximal neighbourhoods of Cd44-expressing hepatocytes are enriched in T cells exhibiting reduced cytokine and chemokine gene expression. Finally, hepatocyte-specific knock out of Cd44 mitigated the IL6/JAK/STAT3 gene signature in aged livers. Overall, these findings suggest that CD44 expression in aged hepatocytes promotes activation of the immunosuppressive IL6/JAK/STAT3 pathway and this is associated with impaired T cell effector function.

cell biology↗

bioRxiv2025

Multi-Timescale Neural Adaptation Failure as a Mechanistic Signature of Major Depression

Major depression (MD) is usually characterized in terms of momentary abnormalities, such as negative bias or emotional blunting, derived from paradigms that measure only the first response to a stimulus. Yet depressive experience in daily life is repetitive and cumulative. Here we test the hypothesis that a failure of neural adaptation across repetitions is a core feature of MD. Using a repeated negative-stimulus paradigm, we quantified adaptation at micro (within-trial) and macro (across-session) scales via electroencephalography in 28 MD patients and 34 healthy controls, measuring both conventional event-related potentials and large-scale traveling-wave propagation. MD patients exhibited pervasive maladaptation: neural rigidity at the micro scale (absent repetition suppression) and progressive regulatory depletion at the macro scale (escalating emotional reactivity with declining cognitive control). Across analyses, adaptation-based metrics revealed larger group differences than single-shot indices, and classifiers built on adaptation features--especially traveling-wave dynamics--outperformed single-shot models (task AUC = 0.83 vs 0.78) and generalized to two independent datasets (up to AUC = 0.91). These findings challenge conventional symptom-based characterizations of MD, establishing adaptation failure--not initial reactivity--as the mechanistically primary pathological signature. This reframing provides a unified mechanistic explanation for rumination and cognitive inflexibility while offering a standardized, low-cost biomarker requiring minimal electrodes ([≤]8 channels), enabling large-scale screening in resource-limited settings.

neuroscience↗

bioRxiv2025

The CCC complex directs phagosomal maturation and bactericidal activity in macrophages through PI(3)P regulation

The evolutionarily conserved COMMD/CCDC22/CCDC93 (CCC) complex regulates endolysosomal function and is required for effective antibacterial immunity. However, the mechanisms linking the CCC complex to macrophage function remain poorly understood. Using bone marrow-derived macrophages, we found that CCC deficiency impaired bacterial clearance and disrupted phagosome maturation, as evidenced by defective phagosomal membrane remodeling, reduced phagosome-endoplasmic reticulum contacts, and impaired phagosome-lysosome fusion. These defects were independent of the CCC complex interaction partner, Retriever. CCC-deficient macrophages exhibited excessive phosphatidylinositol 3-phosphate (PI(3)P) accumulation on phagosomal membranes due to markedly reduced recruitment of the PI(3)P phosphatase MTMR2. Restoration of normal PI(3)P levels rescued bactericidal activity, establishing aberrant PI(3)P regulation as the principal defect affecting CCC-deficient macrophages. These findings identify a previously unrecognized Retriever-independent function of the CCC complex in regulating PI(3)P dynamics during phagosome maturation, a process essential for efficient bacterial clearance and innate immune defense.

cell biology↗

bioRxiv2025

Serine/threonine protein kinase phosphorylation of DosR alters target gene transcription mechanics and regulates Mycobacterium tuberculosis sensitivity to nitric oxide stress

Successful host colonization by bacterial pathogens requires appropriate response and adaptation to environmental signals encountered during infection, with two-component systems (TCSs) and serine/threonine protein kinases (STPKs) being two important signal transduction mechanisms. Mycobacterium tuberculosis (Mtb) possesses similar numbers of STPKs (11) and TCSs (12), but if and how these two regulatory systems coordinate to enable Mtb adaptation in response to key environmental cues remains poorly understood. Here, we identify extensive interactions between STPKs and TCSs, with a subset of STPKs demonstrating interactions with multiple TCS response regulators. STPK phosphorylation of DosR, the response regulator of the key nitric oxide (NO)/hypoxia-responsive TCS DosRS(T), decreased its binding to target promoter DNA and its ability to activate steady-state gene transcription, in marked contrast with the opposite phenotypes observed with the activated, phospho-aspartic acid form of DosR. Strikingly, a {Delta}STPK Mtb mutant exhibited increased DosR regulon transcription at lower NO levels than wild type Mtb, illustrating how STPK phosphorylation of a TCS RR may act to restrict and fine-tune conditions in which activation occurs. Together, our results support a functional relationship between STPKs and TCSs, and shed light on the mechanisms underpinning STPK-TCS interplay. AUTHOR SUMMARYMycobacterium tuberculosis (Mtb) is the bacterium that causes tuberculosis, which remains the largest cause of death from an infectious disease globally. Successful host colonization by Mtb requires that the bacteria appropriately sense and respond to changes encountered in its local microenvironment throughout the course of infection. Here, we provide evidence for the interplay between two key signal transduction regulatory mechanisms - two-component systems (TCSs) and serine/threonine protein kinases (STPKs). Focusing on the DosRS(T) TCS that is crucial in the response of Mtb to the critical environmental signals of nitric oxide (NO) and hypoxia, we reveal that STPK phosphorylation of the DosR regulator decreases target gene promoter binding and the activation of steady-state transcription. Further, an Mtb mutant that was disrupted in an STPK that phosphorylates DosR exhibited increased DosR target gene expression at lower NO concentrations than wild type Mtb. These results indicate that STPK phosphorylation serves as an additional regulatory layer for TCSs, adjusting the DosR concentration range under which full activation of the TCS occurs.

microbiology↗

bioRxiv2025

Direct visualization of bacterial transcripts in the infected lung illuminates spatiotemporal environmental adaptation of Mycobacterium tuberculosis

Spatiotemporal environmental variation results in marked heterogeneity in bacterial infection progression and disease outcome, with vital consequences for treatment success. For the globally important pathogen Mycobacterium tuberculosis (Mtb), while the pronounced intra-host spatial heterogeneity in lesion immune cell composition and phenotype has been well-described, the highly complex Mtb cell envelope has presented a particular challenge for the required equivalent insight into bacterial heterogeneity. Here, we develop hybridization chain reaction- fluorescence in situ hybridization (HCR-FISH)-based methodology for Mtb mRNA visualization in the context of intact lung and lesion architecture. In combination with a Mtb transcriptional/translational activity reporter, we reveal spatiotemporal differences in gene expression relating to Mtb lipid metabolism, response to key environmental signals, and the ESX-1 type VII secretion system. Our results establish a framework for in situ analysis of Mtb mRNA, opening the path to elucidating critical bacterial drivers that underlie the marked heterogeneity in Mtb-host interactions.

microbiology↗

bioRxiv2025

Comparative transcriptomics of lateral hypothalamic cell types reveals conserved growth hormone-tachykinin dynamics in feeding

The lateral hypothalamus (LH) is a highly heterogeneous brain region regulating hunger and motivated behaviors. In zebrafish, the LH shows distinct neural activity across hunger, feeding, and satiety states. However, the functional and evolutionary conservation of relevant neural circuits remain unclear. Using integrative transcriptomics of zebrafish and mouse LH, we identify conserved cellular clusters with shared molecular markers, particularly within GABAergic neurons. We highlight a conserved GABAergic population expressing tachykinin and growth hormone receptors, which is responsive to food cues and modulated by hunger and feeding states. This cluster may mediate acute appetite-enhancing effects of growth hormone. In both species, feeding elevates growth hormone receptor and tachykinin expression and activates these neurons, while human growth hormone increases their activity and food intake in zebrafish. These findings suggest a conserved neural mechanism by which metabolic hormones influence feeding behavior. Our comparative LH atlas highlights the evolutionary biology of appetite regulation and the integration of hormonal and neural signals driving energy homeostasis.

neuroscience↗

bioRxiv2025

Increased proportion of growth-arrested bacilli in acidic pH adaptation promotes Mycobacterium tuberculosis treatment survival

The ability of Mycobacterium tuberculosis (Mtb) to adapt its growth behavior in response to host environments promotes survival against immune and drug stressors. However, how these behaviors shift at the single-cell level remains poorly understood. Here, we show that Mtb adapts to acidic conditions by increasing the proportion of bacteria in a growth-arrested state, rather than uniformly slowing the growth rate of the entire population. This non-growing subpopulation exhibits greater tolerance to ethambutol. Clinical strains displayed higher proportions of growth-arrested cells under both neutral and acidic conditions, suggesting that growth arrest serves as a bet-hedging strategy during infection. Though the PhoPR two-component system contributes to regulating this non-growing state, we show that it is a partial regulator of the non-growing bacterial subpopulation and that additional transcriptional regulators are involved. Our study demonstrates that non-growing subpopulations of Mtb provide fitness benefits and are an active adaptation to environmental cues and not a passive consequence of stressors.

microbiology↗

bioRxiv2025

Phosphorylation by the PknB serine/threonine kinase stimulates dimer formation by WalR

WalKR is a two-component system that regulates cell wall homeostasis and other processes in low-GC Gram-positive bacteria. It is essential for viability in Staphylococcus aureus and regulates genes that encode the autolysins and other critical proteins. The sensor kinase WalK phosphorylates WalR at aspartic acid residue 53 (D53) in the receiver domain, stimulating promoter DNA binding. Unlike other response regulators, WalR is thought to have a second kinase, the serine/threonine kinase PknB, which phosphorylates the receiver domain at threonine residue 101. We previously reported that a walR mutation that changed T101 to a methionine conferred low level resistance to vancomycin and greatly increased susceptibility to tunicamycin along with several other phenotypic traits. In this work we demonstrate similarities between pknB null and the WalRT101M mutants that support a regulatory role for PknB-mediated phosphorylation of WalR. Using a combination of in vitro and in vivo approaches, we confirm the specificity of this posttranslational modification and confirm that phosphorylation at both D53 and T101 is important for dimer formation. Importantly, using an in silico approach, we have discovered that phosphorylation at T101 creates an intermolecular hydrogen bond between the phosphate group and E108, that strengthens contacts along the WalR dimerization interface. This is the first time that a clear molecular role has been assigned to this posttranslational modification and it is the strongest molecular evidence to date for a direct functional relationship between PknB and WalR. We propose that, together with primary activation by WalK, PknB serves as a second potentiating input into the WalKR system, stimulating WalR dimer formation, and target DNA binding and tethering this transcriptional event to important extracellular stimuli.

microbiology↗

bioRxiv2025

BRD4 binds the nucleosome via both histone and DNA interactions

BRD4, a bromodomain and extraterminal (BET) family transcriptional regulator of cell cycle progression, cell differentiation and cancer development, is believed to be recruited to chromatin via interactions between its tandem bromodomains (BD1 and BD2) and acetylated histone tails. Although extensive studies have explained how individual BRD4 bromodomains bind to acetylated peptides and how BET inhibitors interfere with such interactions, equivalent studies of full-length BRD4 protein with the nucleosome have been lacking. Our cryo-EM structure of the BRD4 short (BRD4-S) isoform bound to a nucleosome diacetylated on histone H4 shows how BRD4 BD1 engages both the H4 tail and nucleosomal DNA. Unexpectedly, our biochemical studies indicate that BRD4 uses basic regions outside of the bromodomains to bind nucleosomes tightly even in the absence of histone acetylation. Our results further show that histone H4 acetylation influences the conformation of the BRD4/nucleosome complex.

molecular biology↗

bioRxiv2025

Movi Color: fast and accurate long-read classification with the move structure

The number of reference genomes is rapidly increasing, thanks to advances in long-read sequencing and assembly. While these collections can improve the sensitivity and specificity of classification methods, this requires highly efficient compressed indexes. K-mer-based approaches like Kraken 2 are efficient but limit the analysis to a fixed k-mer length. This is hard for the user to set ahead of time, and suboptimal settings can harm sensitivity and specificity. Methods that use compressed full-text indexes like SPUMONI2 and Cliffy lift this constraint, but are less efficient than k-mer-based tools. Further, these methods either cannot report a full listing of genomes where a match occurs, or cannot scale to large reference databases. We propose new methods and algorithms that use compressed full-text indexes to enable multi-class and taxonomic classification. Unlike past compressed-indexing methods for classification, ours uses the move structure, which is extremely fast thanks to its locality of reference. Our method, called Movi Color, augments the main table of the Movi index. Specifically, Movi Color assigns a "color" to each run of the Burrows-Wheeler Transform according to the subset of genomes from which the run suffixes originated. When the reference is highly repetitive - as is typical when indexing pangenomes or reference databases - only certain colors occur, creating opportunities to compress the index. For species-level classification, Movi Color achieves over 1.6x higher precision and 2x higher recall than Kraken 2 and Metabuli. At the genus level, it achieves 70% higher precision and 80% higher recall. Movi Colors read processing time is 7-20x faster than Metabuli and is a comparable to Kraken 2. Although Movi Color uses more memory than both Kraken 2 and Metabuli, its speed-accuracy trade-off makes it well-suited for real-time or high-throughput scenarios.

bioinformatics↗

bioRxiv2025

Roles of histone chaperone Nap1 and histone acetylation in regulating phase-separation of chromatin arrays

Chromatin condensation is dynamically regulated throughout the cell cycle and plays key roles in modulating gene accessibility at the highest physical level in a cell. The DNA-histone dynamics in short- and long-range inter-nucleosomal interactions are central to the regulation mechanisms of chromatin condensation which remain under active investigation. We employed 12-mer nucleosome arrays to investigate the roles of histone chaperone Nap1, and histone H3 and H4 N-terminal tails and their acetylation in regulating the formation and the properties of phase-separated chromatin condensates. These arrays form liquid-like condensate droplets under a physiological salt condition of 150 mM NaCl. According to our results from bright-field microscopy, fluorescence recovery after photobleaching, optical super-resolution imaging, and microrheology with optical tweezers, histone H4 tail lysine residues are the main drivers of liquid-liquid phase separation of chromatin arrays. We also found that the condensed liquid-like droplets contain both a mobile fraction and a relatively immobile structural scaffold and that histone chaperone Nap1 and histone H3 tail acetylation facilitate DNA-histone dynamics within the structural scaffold to lower the overall viscosity of the droplets. These results suggest that histone chaperone and histone H3/H4 tails play critical roles in regulating chromatin condensation and gene accessibility in condensed chromatin.

biophysics↗

bioRxiv2025

Deep three-photon functional imaging in the mouse cortex with adaptive optics

Three-photon (3P) microscopy enables functional non-invasive single-cell resolution imaging at greater depths than any other technique. A key challenge of deep imaging is tissue-induced optical aberration, which reduces the excitation confinement. Adaptive optics use deformable mirrors to compensate for optical distortions, hence correcting these aberrations. Here, we present a practical adaptive optics-assisted 3P imaging system for functional imaging in the mouse brain during behavior. We introduce hierarchical corrections that sequentially target aberrations caused by the microscope system, the cranial window, and tissue depth. We demonstrate the utility of this strategy in the prelimbic cortex, where large vasculature near the midline causes aberrations, and in the lateral somatosensory cortex, where side access leads to distinct wavefront distortions. In both regions, adaptive optics significantly improved imaging performance, restoring cellular visibility near vasculature and enhancing signal-to-noise ratio. Our work provides a practical framework for utilizing adaptive optics to improve 3P imaging during behavior.

neuroscience↗

bioRxiv2025

The ribosomal protein RPS6A modulates auxin signalling and root development in Arabidopsis

Protein biosynthesis by the ribosome is a fundamental biological process in living systems. Recent studies suggest that ribosomal subunits also play essential roles in cell growth and differentiation beyond their roles in protein translation. The ribosomal subunit RPS6 has been studied for more than 50 years in various organisms, but little is known about its specific roles in a certain signalling pathway. In this study, we focused on the functions of Arabidopsis RPS6A in auxin-related root growth and development. The rps6a mutant exhibited a series of auxin-deficient phenotypes, such as shortened primary root length, reduced lateral root number, and defective vasculatures. Treatment of the rps6a mutant with various concentrations of auxin and its analogues did not restore the root defect phenotype, suggesting a defect in the auxin signalling pathway. Further cell biological and global transcriptome analyses revealed that auxin signalling was weakened in the rps6a mutant and that there was a reduced abundance of PIN-FORMED (PIN) auxin transporters. Our work provides insights into the role of the protein biosynthesis pathway involved in auxin signalling.

plant biology↗

bioRxiv2025

Emergence of binding and catalysis from a designed generalist binding protein

The evolution of binding and catalysis played a central role in the emergence of life. While natural proteins have finely tuned affinities for their primary ligands, they also bind weakly and promiscuously to other molecules, which serve as starting points for stepwise, incremental evolution of entirely new specificities. Thus, modern proteins emerged from the joint exploration of sequence and structural space. The ability of natural proteins to bind small molecule fragments in well-defined geometries has been widely evaluated using methods including crystallographic fragment screening. However, this approach had not been applied to de novo proteins. Here, we apply this method to explore the binding specificity of a de novo small molecule-binding protein ABLE. As in Nature, we found ABLE was capable of forming weak complexes, which were excellent starting points for designing entirely new functions, including a binder of a turn-on fluorophore and a highly efficient Kemp eliminase enzyme (kcat/KM = 2,200,000 M-1s-1) approaching the diffusion limit. This work illustrates how simultaneous consideration of both sequence and chemical structure diversity can guide the emergence of new function in designed proteins.

biophysics↗

bioRxiv2025

Single-AAV CRISPR editing of skeletal muscle in non-human primates with NanoCas, an ultracompact nuclease

CRISPR gene editing is a transformative technology for addressing genetic diseases, but delivery constraints have largely limited its therapeutic applications to liver-targeted and ex vivo therapies. Here, we present the discovery and engineering of NanoCas, an ultracompact CRISPR nuclease capable of extending CRISPRs reach in vivo beyond liver targets. We experimentally screened 176 ultracompact CRISPR systems found in metagenomic data and applied protein engineering approaches to enhance the editing efficiency of NanoCas. The optimized NanoCas exhibits potent editing capabilities across various cell systems and tissues in vivo when administered via adeno-associated viral (AAV) vectors. This is accomplished despite NanoCas being approximately one-third the size of conventional CRISPR nucleases. In proof-of-concept experiments, we observed robust editing with our optimized NanoCas in mouse models targeting Pcsk9, a gene involved in cholesterol regulation, and targeting exon splice sites in dystrophin to address Duchenne muscular dystrophy (DMD) mutations. We further tested the efficacy of our NanoCas system in vivo in non-human primates (NHPs) resulting in editing levels above 30% in muscle tissues. The compact size of NanoCas, in combination with robust nuclease editing, opens the door for single-AAV editing of non-liver tissues in vivo, including the use of newer editing modalities such as reverse transcriptase (RT) editing, base editing, and epigenetic editing.

bioengineering↗

bioRxiv2025

Cancer cells transfer invasive properties through microRNAs contained in collagen-tracks

Invasion is a prerequisite for metastasis formation. During tumor development, the extracellular matrix (ECM) is remodeled in part through overexpression of type I collagen, increasing tumor microenvironment stiffness, and facilitating cancer dissemination. During breast cancer cell migration, we observed membrane debris left behind, attached to the collagen fibrils, along the migration path. We named these structures collagen-tracks. These collagen-tracks can be deposited in 3D matrices in vitro and in vivo and their formation is stimulated by the interaction between the ECM and matrix receptors, such as the discoidin-domain receptor (DDR1). However, they are different from structures already known to be involved in cell-cell communication such as exosomes and migrasomes, due to their specific nucleic acid and protein contents. When deposited by highly invasive breast cancer cells, internalized collagen-tracks reprogram non-invasive cells into highly pro-metastatic ones by inducing a partial epithelial-mesenchymal transition (EMT). This cell reprogramming is dependent on specific miRNAs present in the collagen-tracks, that are necessary to promote ECM degradation, increase cell motility and invasiveness. Collagen-tracks thus represent a new form of cell-cell communication important for driving tumor invasion that could be targeted to prevent metastasis.

cell biology↗

bioRxiv2024

mRNA-1273 vaccines adapted to JN.1 or KP.2 elicit cross-neutralizing responses against the JN.1 sublineages of SARS-CoV-2 in mice

The continued diversification of SARS-CoV-2 omicron lineage has given rise to the JN.1 variant and descendant strains (KP.2, KP.3, and XEC) that have prolonged the JN.1 infection wave. JN.1 and KP.2 show decreased susceptibility to neutralization sera in recipients of XBB.1.5 vaccine boosters, supporting the recent authorization of JN.1- and KP.2-matched mRNA vaccines in the United States, Europe, and other regions. We evaluated the immunogenicity of two updated monovalent variant-containing formulations of mRNA-1273 vaccines encoding the spike protein of the omicron subvariants JN.1 (mRNA-1273.167) and KP.2 (mRNA-1273.712) as compared with the monovalent XBB.1.5 vaccine (mRNA-1273.815). The vaccines were administered either as a two-dose primary series in naive mice or as a booster (third) dose in mice previously immunized with two-dose primary series of mRNA-1273 (ancestral strain). The neutralizing antibody response elicited by these vaccines against JN.1 subvariants (KP.3 and LA.2) and the recombinant strain (XEC), which achieved dominance in the United States during late 2024, was evaluated. Primary series immunization with either JN.1- or KP.2-matched vaccine elicited robust neutralizing antibody titers against the matched strains and effectively cross-neutralized KP.3, LA.2, and XEC, but not the antigenically distant XBB.1.5. Similarly, JN.1- and KP.2-matched vaccines administered as a booster (third) dose increased titers against the corresponding strains and JN.1-related subvariants, but not against XBB.1.5. These data suggest these strains are antigenically similar with relatively few spike differences between JN.1 and KP.2/JN.1-related subvariants. Our results demonstrate the potency of JN.1- and KP.2-containing mRNA-1273 vaccines in neutralizing the matched variants and their utility in cross-neutralizing JN.1-related subvariants KP.3, LA.2, and XEC. Taken together, these data suggest that the licensed JN.1 and KP.2 mRNA vaccines are likely to continue to protect against the emerging strains as the JN.1 lineage further evolves.

immunology↗

bioRxiv2024

Differentiating Peromyscus leucopus bone marrow-derived macrophages for characterization of responses to Borrelia burgdorferi and lipopolysaccharide

Currently, most tools utilized in host-pathogen interaction studies depend on the use of human or mouse (Mus musculus) cells and tissues. While these species have led to countless breakthroughs in our understanding of infectious disease, there are undoubtably important biological processes that are missed by limiting studies to these two vertebrate species. For instance, it is well-established that a common North American deermouse, Peromyscus leucopus, has unique interactions with microbes, which likely shape its ability to serve as a critical reservoir for numerous zoonotic pathogens, including a Lyme disease spirochete, Borrelia burgdorferi. In this work, we expand the immunological toolkit to study P. leucopus biology by performing the first differentiation of deermouse bone marrow to macrophages using P. leucopus M-CSF producing HEK293T cells. We find that P. leucopus BMDMs generated through this method behave broadly very similarly to C57BL/6J macrophages generated with L-929 supernatant, though RNA sequencing revealed modest differences in transcriptomic responses to B. burgdorferi and lipopolysaccharide. In particular, differences in Il10 induction and caspase expression were observed between the species.

immunology↗