Biology subjects

Kim, K.

Publications and source records attributed to Kim, K..

At least 73 records · Page 4Linked to original sources

bioRxiv2026

Collagen type I promotes pancreatic tumor growth and limits immune cell infiltration

Solid tumors are often characterized by a dense extracellular matrix (ECM) that contributes to increased tissue stiffness. Collagen type I is the main component of the ECM and its abundance in tumors is frequently associated with poor prognosis. In vitro studies suggest that a high collagen density promotes tumor invasion and modulates immune responses. However, recent in vivo findings have questioned the pro-tumorigenic role of collagen type I. In this study, we investigate the role of collagen for pancreatic tumor growth and immune cell infiltration using conditional collagen type I knockout mice and transgenic collagenase-resistant mice. Preventing collagen type I significantly reduces intratumoral collagen content and tumor growth. This reduction is accompanied increased infiltration of natural killer (NK) cells, a higher CD8/CD4 T cell ratio, and decreased numbers of monocytic myeloid-derived suppressor cells (MDSCs). Conversely, collagenase-resistant mice develop collagen-dense tumors and display enhanced tumor growth. These mice also generally exhibit opposing effects on cell infiltration, including a lower CD8/CD4 ratio and increased MDSC abundance. These findings are further supported by analyses of publicly available human cancer datasets, which confirm an association between collagen type I levels and immune cell infiltration. Overall, our results demonstrate a pronounced pro-tumorigenic role of collagen type I in pancreatic cancer, which is associated with modulation of the tumor immune microenvironment. This study highlights the importance of extracellular matrix components as key regulators of tumor progression and anti-tumor immunity.

cancer biology↗

bioRxiv2026

Lrat-Cre Exhibits Widespread Expression Beyond Hepatic Stellate Cells Across Multiple Tissues

Hepatic stellate cells (HSCs) play a central role in liver fibrosis, shifting from quiescent vitamin A-storing cells to activated, myofibroblast-like cells that secrete collagen and other profibrotic factors1. HSCs have thus become a major focus in liver fibrosis research, and several Cre driver lines have been created to target HSCs in mice. However, early Cre lines had significant limitations. Glial fibrillary acidic protein (Gfap)-Cre labels only a subset of HSCs and also induces recombination in cholangiocytes2. Collagen type I alpha 1 (Col1a1)-Cre and alpha-smooth muscle actin (SMA)-Cre/CreERT2 primarily label activated myofibroblasts and broadly mark portal fibroblasts and vascular smooth muscle cells3,4. Platelet-derived growth factor receptor beta (Pdgfr{beta})-Cre reliably labels HSCs but also recombines pericytes and smooth muscle cells, limiting its specificity5. The introduction of lecithin-retinol acyltransferase (Lrat)-Cre marked a major advance, offering highly specific labeling of quiescent and activated HSCs and rapidly becoming the most widely used driver for HSC tracing and genetic perturbation2. However, the extrahepatic expression of Lrat-Cre remains incompletely understood. This is a critical limitation, given that liver biology is closely coordinated with other organs to maintain systemic metabolism. Addressing these gaps is essential for the accurate interpretation of HSC-specific genetic models in liver biology.

pathology↗

bioRxiv2026

DelPi Learns Generalizable Peptide-Signal Correspondence for Mass Spectrometry-Based Proteomics

Peptide identification in mass spectrometry-based proteomics has traditionally relied on handcrafted features or simplified probabilistic approaches that limit the interpretation of structured peptide evidence. We present DelPi, an open-source peptide identification framework that learns generalizable peptide-signal correspondence from raw spectra through self-supervised pre-training followed by task-specific fine-tuning. With model distillation enabling practical deployment, DelPi expands the interpretation of peptide evidence across data-independent and data-dependent acquisition while maintaining robust false discovery control.

bioinformatics↗

bioRxiv2026

Graft-Induced Reprogramming Under Arterial Hemodynamic Stress is Associated with a Shared Proteomic Phenotype in Arterial and Venous Conduits Following Coronary Artery Bypass Grafting

BackgroundAutologous venous conduits remain the mainstay of coronary artery bypass grafting (CABG); however, their long-term patency is limited by adverse vascular remodeling. While arterial and venous grafts undergo structural and molecular adaptation to arterial hemodynamics, the mechanisms underlying successful adaptation remain poorly characterized owing to the scarcity of functioning human graft tissues. MethodsWe analyzed bypass grafts explanted en bloc during orthotopic heart transplantation, comprising patent vein grafts (VGP, n=19), occluded vein grafts (VGO, n=9), and patent arterial grafts (AGP, n=13), alongside freshly harvested controls (vein, n=23; artery, n=11). Quantitative histomorphometry, label-free LC-MS/MS proteomics, pathway analysis, and immunohistochemistry were performed. ResultsWhile patent arterial and venous grafts exhibited medial elastin reinforcement, adventitial neovascularization, and organized intimal remodeling, occluded vein grafts demonstrated elastic layer disruption, medial degeneration, and collagen-dense fibrosis. Proteomic analysis revealed a convergent adaptive signature in patent grafts enriched for RNA metabolism, protein synthesis, cytoskeletal dynamics, and extracellular matrix organization, with shared activation of NR4A3, STAT1, and EGFR signaling. Vein-specific adaptation involved SRC-PTGES activation with concomitant STAT3 suppression, whereas arterial grafts engaged IGF1-RUNX2-associated proliferative programs. Failed venous grafts exhibited angiotensin-driven fibroinflammatory remodeling. Endothelial nitric oxide synthase expression was markedly upregulated in the luminal endothelium and adventitial neovessels of patent grafts. ConclusionsDespite distinct vascular origins, arterial and venous grafts converged toward a shared adaptive phenotype characterized by structural maturation and vasoprotective signaling. Targeting common adaptive pathways--including NR4A3, STAT1, EGFR, and adventitial eNOS--may offer therapeutic strategies to enhance long-term graft durability. WHAT ARE THE CLINICAL IMPLICATIONS?This study provides human tissue-based evidence that patent coronary bypass grafts share a common adaptive remodeling program, irrespective of whether the conduit is arterial or venous. Rather than simple venous "arterialization," successful grafts converged on a phenotype characterized by coordinated biosynthetic activity, structural maturation, and vasoprotective signaling, whereas occluded vein grafts exhibited a distinct angiotensin-driven fibroinflammatory remodeling program. Importantly, patent grafts demonstrated conserved upregulation of NR4A3, STAT1, EGFR, and endothelial nitric oxide synthase (eNOS) in both luminal and adventitial endothelial compartments, identifying molecular features that distinguish adaptive from maladaptive remodeling in humans. These findings are clinically relevant because they highlight shared, potentially targetable pathways that may support graft durability across conduit types and provide mechanistic support for surgical strategies that preserve perivascular adipose tissue during vein harvesting. Moreover, the association of graft failure with angiotensin-centered extracellular matrix remodeling suggests a modifiable biological axis that warrants further investigation. Together, this work establishes a human molecular framework for graft adaptation that may inform future biomarker development, therapeutic targeting, and precision strategies aimed at improving outcomes after coronary artery bypass grafting.

pathology↗

bioRxiv2026

Spatiotemporally distinctive astrocytic and neuronal responses to repetitive intracortical microstimulation

Astrocytes are increasingly recognized as active modulators of neuronal synaptic transmission. Intracortical microstimulation (ICMS) is widely used to manipulate neuronal activity, yet the accompanying astrocytic responses remain poorly characterized. Using dual-color in vivo two-photon calcium imaging to simultaneously monitor neurons and astrocytes, we show that ICMS elicits astrocytic activation with spatiotemporal features that diverge from those of neurons. Astrocytes were recruited at stimulation intensities as low as 10A, thresholds sufficient to activate neurons, indicating that astrocytes robustly sense electrical perturbation. Unlike neurons, however, astrocytic responses were spatially heterogeneous and temporally variable across trials. At higher stimulation intensities (>=50A), astrocytic responsiveness, i.e., response peak amplitude, and number of responsive trials, progressively attenuated across repeated trials, in contrast to the stable and consistent neuronal responses. Although neuronally driven, astrocytes exhibited a distinct response profile under the same stimulation parameter, revealing a unique component of electrically evoked cortical activity that underscores the importance of incorporating glial physiology into future neuroprosthetic strategies.

neuroscience↗

bioRxiv2025

Master clock-thalamic-prefrontal circuit controls circadian social priority

Social behaviors arise from complex brain operations that adapt to changing social contexts and internal states. However, how internal states guide the resolution of competing social goals remains unclear. Here, we show that prioritization between competing instincts follows a circadian pattern via a circuit linking the suprachiasmatic nucleus (SCN) to the medial prefrontal cortex (mPFC). Specifically, SCNVIP neurons project to Vipr2-expressing neurons in the nucleus reuniens (RE), which innervate the mPFC to regulate social prioritization. Vipr2 signaling in the RE is essential for circadian modulation of appetitive and consummatory social behaviors, aligning social behaviors with clock-regulated reproductive system. Overall, this SCN-RE-mPFC circuit provides a neural principle through which the master clock coordinates the prefrontal cortex to resolve a conflict of motivated behaviors by referencing an internal state.

neuroscience↗

bioRxiv2025

Anatomical and molecular organization of the nociceptive medial thalamus

The medial thalamus (MTh) is a critical hub for integrating the affective and motivational dimensions of pain, yet its cellular and circuit organization remains poorly defined. Here, we combine activity-dependent genetic tagging, whole-brain clearing and light-sheet imaging, single-cell transcriptomics, and viral circuit tracing to build a comprehensive anatomical and molecular atlas of nociceptive and opioidergic neurons within the MTh. We show that acute and chronic pain activate distinct, spatially localized microdomains across multiple MTh subnuclei, including the central medial, interanteromedial, mediodorsal, and xiphoid nuclei. Activity-dependent tagging reveals a stable ensemble of nociceptive neurons that remains engaged across acute and chronic pain states, suggesting persistent thalamic encoding of affective nociception. Using fluorescent in situ hybridization, we find that most nociceptive-activated neurons express Oprm1, and that nearly all Oprm1+ neurons are activated by nociceptive stimuli, identifying a pain-active, {micro}-opioid receptor-expressing population in MTh (MThMOR). Rabies-based tracing demonstrates that MThMOR neurons receive convergent inputs from nociceptive and inhibitory regions including the anterior cingulate cortex, periaqueductal gray, parabrachial nucleus, and habenula, while projecting broadly to cortical, subcortical, and brainstem structures involved in pain, arousal, and motivation. Enkephalinergic inputs to MTh arise from distinct, non-nociceptive populations, suggesting parallel pathways for nociceptive transmission and endogenous opioid modulation. Together, these findings define the anatomical and molecular organization of the nociceptive medial thalamus and highlight MThMOR neurons as a cell-type-specific substrate for the development of targeted opioid-based pain therapies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=57 SRC="FIGDIR/small/692629v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@a1e6b3org.highwire.dtl.DTLVardef@13112eborg.highwire.dtl.DTLVardef@d125caorg.highwire.dtl.DTLVardef@107fd80_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

Differential targeting of human pyroptotic Caspase-5 and Caspase-4 by Shigella OspC2 and OspC3

Pyroptosis is an inflammatory cell death pathway that is a key defense mechanism of intestinal epithelial cells. To successfully establish an infection, intracytosolic Gram-negative pathogens must block this host response. Indeed, Shigella OspC3 suppresses epithelial pyroptosis by targeting and inactivating Caspase-4 (CASP4). Here, we demonstrate that OspC2, which shares 96% identity with OspC3, targets Caspase-5 (CASP5), a close paralog of CASP4. Through a combination of yeast two-hybrid, transfection, and bacterial infection assays, we show that the distinct pyroptotic caspase specificities of OspC2 and OspC3 are determined by a short -helical region, designated the Pyroptotic Caspase Specificity (PCS) domain. This domain is located upstream from the Ankyrin Rich Repeat (ARR) region previously established to promote OspC3 binding to CASP4. Swapping PCS domains between OspC2 and OspC3 is sufficient to redirect their caspase targeting. Evidence for CASP5-driven pyroptosis in response to infection has not yet been established. However, CASP5 displays signatures of positive selection at residues predicted to interact with the PCS domain of OspC2. Notably, the introduction of orangutan-specific residues into human CASP5 disrupt its interaction with and modification by OspC2, demonstrating that CASP5 natural variation can cause key functional differences in this host-microbe molecular interaction. These findings highlight the evolutionary interplay between bacterial effectors and host proteins, support a likely role for CASP5 in responding to Gram-negative bacteria, and identify promising therapeutic targets for enhancing epithelial defense against bacterial pathogens. ImportanceShigella species are human-specific Gram-negative pathogens that establish a replicative niche in intestinal epithelial cells by blocking pyroptosis, a key inflammatory cell death pathway. We reveal that two closely related Shigella type III secreted effectors, OspC2 and OspC3, specifically inactivate the human caspase paralogs CASP5 and CASP4, respectively. This specificity is determined by their newly identified Pyroptotic Caspase Specificity (PCS) domains. In addition, we find that positively selected residues in CASP5 alter the OspC2/CASP5 interaction, underscoring the impacts of ongoing evolutionary arms races between bacterial effectors and host immune proteins. By elucidating the molecular basis of caspase targeting and adaptation, this work provides new insight into the diversification of host defense mechanisms and identifies potential therapeutic targets for enhancing epithelial resistance to bacterial infection.

microbiology↗

bioRxiv2025

A foundation model for microbial growth dynamics

Microbial growth dynamics contain rich information about microbial populations, which support applications from antibiotic testing to microbiome engineering. However, the high dimensionality of growth data and the scarcity of large, task-specific datasets have limited generalizable modeling analysis across systems. Here, we develop a foundation model for microbial growth dynamics. It is a large-scale, self-supervised representation model trained on [~]370,000 experimental and simulated growth curves spanning diverse microbial species, environmental conditions, and community contexts. The model learns lower-dimensional latent embeddings that capture essential dynamical features of raw growth data and enable accurate reconstruction of these data. The concise representations enhance predictive performance in diverse downstream applications. Using these embedding, we achieve few-shot learning for antibiotic classification and concentration prediction, accurate forecasting of simulated and experimental communities, and inference of total abundance from relative-abundance data. By extracting transferable representations from heterogeneous datasets, our model provides a general framework for analyzing and predicting microbial community dynamics from limited measurements.

synthetic biology↗

bioRxiv2025

Aggressive Neuroblastomas Start Growing after Infancy

In neuroblastoma, population screening during infancy failed to lower mortality because it primarily detected biologically indolent tumors rather than aggressive, life-threatening disease. The failure may reflect limited screening sensitivity, or disease onset after infancy among aggressive tumors. Here, we used an epigenetic mitotic clock based on fluctuating CpG DNA methylation to estimate patient-specific tumor mitotic ages and calendar ages in a cohort of unscreened children diagnosed with neuroblastoma. Aggressive cancers (stage 4) primarily started growing after the first year of life, making them undetectable by screening during infancy. In contrast, biologically more indolent tumors (stages 1, 2, 3 and 4S) often started growing in utero or during the first year of life, and affected children had better survival outcomes. Due to a short preclinical detectable phase of aggressive neuroblastomas, reducing mortality through screening is impractical as it would require frequent screening among older children. Patient-specific tumor-age estimation may help refine screening windows and improve early-detection strategies in other cancers where screening has so far failed to yield substantial mortality reductions.

cancer biology↗

bioRxiv2025

Structure of bacteriophage P1 head provides insight into capsid polymorphism

The highly regulated assembly of different proteins forming mature P1 myophage capsid show a unique polymorphism where the virion population is primarily divided in brackets of two different capsid sizes. The contrasting capsids, sized 95nm (capsid_L), triangulation number (T)=13 and 68nm (capsid_S) with T=7, both in Dextro format and have the same protein forming the phage head with similar intra and inter capsomeric interactions. Comparative study of the electron density maps of the capsids reveals the presence of protein appendages DarA and Hdf below the 5-fold symmetric region inside capsid_L that anchor the phage dsDNA to the capsid-shell. Such densities are also noticed in large capsids depleted off their dsDNA but not observed in capsid_S. Deficiency of these appendages in capsid_S results in a smaller sized dsDNA packed inside capsid_S. Despite missing out on nearly 60% of the phage dsDNA virions with capsid_S has all the essential genes responsible for the formation of a fully mature thermodynamically stable virion particle with structurally identical tail and baseplate as of capsid_L. Co-existence of both stable conformation in a single lysate is a unique phenomenon in the phage community and suggest that DarA and Hdf play an important role in phage capsid morphogenesis.

biophysics↗

bioRxiv2025

Effects of a novel proteasome inhibitor, UR238 on the tumor immune microenvironment and growth in epithelial ovarian cancer

High-grade serous ovarian cancer (HGSOC) is a deadly gynecologic malignancy, often diagnosed at an advanced stage and most patients will experience recurrence and resistance to platinum-based chemotherapy. While there are few targeted therapies available for HGSOC, there are no effective immunotherapies available to treat this disease. The ubiquitin-proteasome system (UPS) maintains cellular protein homeostasis by degrading misfolded or damaged proteins. In epithelial ovarian cancer (EOC) elevated expression of proteasome subunit PSMB4 correlates with epithelial ovarian cancer growth and poor prognosis. A proteasome inhibitor has yet to be approved for EOC treatment despite evidence of activity in phase-1/2 clinical trials. Limitations of past generation proteosome inhibitors include sub-optimal solid tumor/tissue penetration due to boronic acid functionality, and poor solubility. Here, we show that a novel proteasome inhibitor, UR238, suppresses viability of several EOC cell lines and reduces tumor burden in both murine xenograft and rat syngeneic models. In a rat EOC model, UR238 treatment reduced tumor burden, shifted the predominantly suppressive immune microenvironment to an inflammatory phenotype with reduced suppressive macrophages, and decreased PD-L1 expression on myeloid cells. Additionally, in a mouse model, UR238 also induced robust immune changes in HGSOC bearing mice without a corresponding reduction in tumor burden. Our data highlights the promise of UR238 for the treatment of ovarian cancers as a single-agent or in combination with immunotherapies, where modulating the immune composition of these tumors will be important in improving outcomes for ovarian cancer patients.

immunology↗

bioRxiv2025

Multimodal Approach for Identification and Validation of Hepatocellular Carcinoma Targets for Radiotheranostics

Identifying tumor selective targets is critical for the development of precision diagnostic and therapeutic agents in oncology. Despite advances in precision oncology elsewhere, there are no FDA-approved hepatocellular carcinoma (HCC)-selective treatments. HCC is the most common type of liver cancer and accounts for significant morbidity and mortality worldwide. Here, we sought to integrate bulk (371 cases) and single cell RNA sequencing (scRNAseq, n=2 datasets, 34 cases, 102,956 cells) of patient samples to enrich for molecules that are overexpressed in HCC, which could serve as HCC-selective targets. To guide definitions of tumor and normal cell clusters with higher fidelity, we also imported a normal liver scRNAseq dataset. Using this integrated approach, we identified several HCC-selective plasma membrane molecules. To validate these targets, we performed immunohistochemical staining of HCC and normal tissue microarrays and confirmed HCC-selective staining of identified targets. Next, we verified the presence of these targets in several commercially available HCC cell lines by flow cytometry and western blot. Finally, we designed, engineered, and tested novel antibody-based positron emission tomography (immunoPET) agents to these targets in various murine models of liver cancer. Our findings confirm that we can leverage this multimodal approach to identify and validate of HCC-selective targets, which can be used to develop tumor-selective diagnostic and therapeutic radiopharmaceuticals, or radiotheranostics, and other precision oncology agents. One Sentence SummaryA multimodal pipeline defines and validates tumor-selective surface targets for radiotheranostic use in hepatocellular carcinoma.

cancer biology↗

bioRxiv2025

Neat1 alleviates ischemic injury in skeletal muscles

Peripheral artery disease (PAD) has historically been regarded as an age-related vascular disorder; however, recent attention has shifted toward the myopathic components of the disease. Conventional interventions, such as revascularization, have had limited success in reversing muscle pathology or preventing adverse outcomes like amputation. Hypoxia-inducible factors (HIFs) are key regulators of cellular responses to ischemia, including the promotion of angiogenesis and glycolysis. While pharmacological stabilization of HIF proteins represents a promising therapeutic strategy, its efficacy is diminished in aged muscles due to their intrinsically low basal HIF expression. We hypothesized that long noncoding RNAs (lncRNAs) might enhance the hypoxic response in aged muscle through post-transcriptional regulation of HIF expression. Our study identified a long noncoding RNA Neat1, as a critical mediator of the hypoxia-induced stress response, including upregulation of Hif1. In a murine hindlimb ischemia model, Neat1 knockout mice exhibited extensive necrosis following femoral artery ligation, whereas Neat1 overexpression conferred protection against ischemic injury. Mechanistically, we found that Neat1 regulates the stability of Hif1a mRNA, as Hif1a transcript levels were significantly reduced in Neat1-deficient muscle cells. Importantly, aged muscles displayed a blunted hypoxic response due to diminished Hif1a expression--an effect that was reversed through Neat1 overexpression, resulting in improved resistance to ischemic damage. In summary, our findings highlight Neat1 as a novel regulator of muscle adaptation to hypoxia in aging. Enhancing Neat1 expression may represent a promising therapeutic strategy for improving ischemic outcomes in patients with peripheral artery disease.

cell biology↗

bioRxiv2025

Modular evolution and strain-specific partnerships: how Hamiltonella defensa shapes defense and symbiont communities in aphids

Insects frequently carry maternally-transmitted endosymbionts that mediate ecological interactions, including resource acquisition and host defense. All pea aphids (Acyrthosiphon pisum), for example, carry the obligate nutritional symbiont Buchnera, while most have one or more of the seven heritable facultative symbionts, which play diverse roles. A common symbiont, Hamiltonella defensa, confers protection against the parasitic wasp Aphidius ervi via a toxin-bearing bacteriophage called APSE, with strain-level variation in protection best predicted by APSE variant. Yet, little is known about Hamiltonella/APSE strain variation in the field, resulting in an incomplete understanding of the full arsenal of symbiont defenses and how these change over space and time. Here, we characterized Hamiltonella/APSE diversity in over 3,000 field-collected aphids from two North American populations across multiple years. We identified bacterial strains representing five Hamiltonella clades, seven APSE variants, and numerous toxin alleles, resulting in at least 38 distinct combinations. We found that APSEs moved laterally among Hamiltonella strains more readily than toxins among phage backbones, together generating modular defensive diversity. Hamiltonella strains exhibited strain-specific coinfection preferences with other symbionts, particularly Fukatsuia, Rickettsia, and Rickettsiella, indicating strain-level structuring of heritable bacterial communities. Geographic and temporal analyses revealed dramatic regional differences and rapid population turnover, with combinations conferring intermediate laboratory protection dominating despite the decline of highly protective strains, suggesting ecological success goes beyond parasitoid resistance alone. This extensive cryptic diversity provides standing genetic variation enabling rapid evolutionary responses to biological control efforts and anthropogenic stressors, including climate change, with implications for pest management and host adaptation.

evolutionary biology↗

bioRxiv2025

Molecular mechanism of naturally-encoded signaling-bias at the complement anaphylatoxin receptors

The conceptual framework of biased signaling has revolutionized our understanding of GPCR signaling and regulatory paradigms, and greatly impacted the efforts focused on the discovery of GPCR-targeted therapeutics. However, the mechanistic basis of biased signaling remains primarily defined based on synthetic ligands and receptor mutants with relatively limited progress in understanding naturally-encoded signaling-bias. Here, we present fundamental molecular and structural insights into naturally-encoded signaling-bias at the complement anaphylatoxin C5a receptors namely, C5aR1 and C5aR2. We first discover that C5a-d-Arg, the naturally-occurring version of C5a lacking the terminal arginine, exhibits robust G-protein signaling-bias at C5aR1, characterised by attenuated {beta}arr recruitment. This signaling-bias manifests in both cytokine release from primary human immune cells, and in vivo, during neutrophil mobilization. We combine the cryo-EM structures of C5a/C5a-d-Arg-C5aR1 complexes with MD simulation, site-directed mutagenesis, and cellular experiments to elucidate that the G-protein-bias exhibited by C5a-d-Arg results from a distinct orientation of TM7 and helix 8 in C5aR1 leading to inefficient GRK recruitment and receptor phosphorylation. Next, we determine the first cryo-EM structures of C5aR2, a naturally-encoded {beta}-arrestin-biased receptor, in an apo state, complexed with the natural agonists C5a and C5a-d-Arg, and three peptide agonists including a first-in-class, newly discovered C5aR2-selective agonist, R8Y. These structural snapshots reveal key differences between the binding of C5a and C5a- d-Arg to C5aR1 and C5aR2, and provide a molecular basis of functional specialization at these two receptors. Moreover, the structural insights also allow us to decipher the molecular basis of naturally-encoded signaling-bias at C5aR2 originating from a shallower cytoplasmic interface with hydrophobic interior pocket that is not permissive to efficient G-protein-coupling and activation. Finally, we also engineer and characterize loss-of-function and gain-of-function variants of C5aR1 and C5aR2, which in turn corroborate and validate the structural observations presented here. Collectively, our findings offer crucial insights into previously lacking molecular mechanisms of the naturally-encoded signaling-bias at GPCRs, which have broad implications not only for the general framework of biased-signaling, but also for novel therapeutic design.

biochemistry↗

bioRxiv2025

Quantitative phase deformability cytometry (QP-DC) for precise and clinically relevant multiparametric immune cell profiling

Imaging flow cytometry enables the detailed analysis of cell morphology and internal structures through high-throughput cell imaging, and quantitative phase imaging (QPI)-based microfluidic approaches have extended this by providing label-free measures such as dry mass and refractive index (RI). Building on these developments, we present quantitative phase deformability cytometry (QP-DC), which integrates QPI with deformability cytometry to simultaneously measure morphology, mechanics, and intrinsic biophysical parameters such as mass density and dry mass. Numerical refocusing ensures in-focus images independent of axial position, improving precision in contour detection and feature extraction. Using microspheres and whole blood, we validated QP-DC and then applied it to neutrophils under lipopolysaccharide (LPS) stimulation and from patients with systemic lupus erythematosus (SLE). QP-DC revealed LPS-induced reductions in neutrophil mass density and identified heterogeneous subpopulations in SLE. These results demonstrate the capability of QP-DC for precise biophysical and mechanical characterization, offering significant potential for research and clinical diagnostics.

biophysics↗

bioRxiv2025

Lysosomal permeabilization by Group A Streptococcus releases proteins into the macrophage cytosol

The human-specific bacterial pathogen Group A Streptococcus (GAS) is a significant cause of morbidity and mortality due to its ability to cause severe invasive infection. Although macrophages are important for controlling GAS infection, we and others have demonstrated that GAS can persist in macrophages by perforating the phagolysosome using the pore-forming toxin streptolysin O (SLO). In this study, we examined how phagosomal perforation releases lysosomal and bacterial proteins into the cytosol and alters cytosolic protein content in the macrophage. Using IL-1{beta} as a measure of intracellular pathogen detection, we confirmed that cytosolic preparations from macrophages infected with either wild-type (WT) or SLO-deficient ({Delta}SLO) bacteria contained new proteins that are absent in uninfected cytosol controls. Proteomic analysis revealed distinct cytosolic protein profiles in both WT- and {Delta}SLO-infected macrophages. M1 protein was detected only in the cytosol of WT-infected macrophages and corresponded with the IL-1{beta} response, indicating SLO-mediated release of M1 protein from the phagosome, and providing a mechanism for cytosolic recognition of this virulence factor. Unexpectedly, cytosolic extracts of both WT- and {Delta}SLO-infected macrophages contained all histone proteins, suggesting that nucleosomal complexes are released into the cytosol during GAS infection. Our work reveals both a mechanism for the activation of the inflammatory response on a cellular level, and the surprising consequences of phagosomal perforation in GAS infections. These responses may collectively contribute to the pathologies observed during severe invasive GAS infection, and can help inform therapies aimed at improving macrophage function and patient outcomes. ImportanceThe post-pandemic increase in cases of invasive Group A Streptococcus (GAS) infection, along with its sudden onset and severity of symptoms, underscores the need to understand GAS survival in host cells. Macrophages are key first responder immune cells essential for limiting the spread of GAS infection. As professional phagocytes, macrophages typically engulf bacteria in phagosomes and digest them using proteolytic enzymes. However, the human-specific pathogen GAS has acquired several evasion tactics through co-evolution with our immune system. In previous work, we have shown that GAS survives digestion by perforating the phagosome. In this study, we determined that this perforation allows both host and bacterial proteins to leak into the cytosol. These leaked proteins can dictate the subsequent response of the macrophage and may contribute to the pathology observed in invasive infection. Understanding this response can lead to improved therapies for GAS infections.

microbiology↗