Biology subjects

Ji, K.

Publications and source records attributed to Ji, K..

15 recordsLinked to original sources

bioRxiv2026

Thermodynamic Diversity in Staphylococcal Nitrate Sensors with a Conserved Binding Pocket

Nitrate is an important oxyanion in bacterial physiology, with major roles as a nitrogen source for assimilation and as an alternative electron acceptor in anaerobic respiration. Accordingly, diverse nitrate-responsive regulatory systems sense nitrate availability and control the expression of genes involved in nitrate transport and metabolism. In the NreABC system, the soluble GAF-like domain protein NreA functions as a cellular nitrate sensor. To date, our molecular understanding of nitrate recognition by NreA has been derived primarily from studies of the Staphylococcus carnosus protein, with less known about homologues from other species. Here, we identified 736 NreA homologues through bioinformatic analysis of the GAF-like domain superfamily and selected five staphylococcal representatives for thermodynamic characterization by isothermal titration calorimetry. Despite conservation of the nitrate-binding pocket, these homologues resolved into high- and low-affinity groups with distinct thermodynamic profiles. Comparative structural modeling and chimeragenesis of a low-affinity homologue revealed that the C-terminal region is a non-coordinating determinant of nitrate-binding affinity. Together, these findings show that a conserved nitrate-binding pocket preserves recognition, while distal sequence variation shapes thermodynamics and tunes the nitrate concentration range over which NreA sensors can function.

biochemistry↗

bioRxiv2026

The Staphylococcus aureus carotenoid staphyloxanthin modifies the structure of phosphoglycerol lipid bilayers

Staphyloxanthin (STX) is a carotenoid synthesized by the human pathogen Staphylococcus aureus. The golden color of this bacterium is due to this carotenoid. STX protects Staphylococcus aureus from oxidative stress by scavenging free radical species. Furthermore, STX has been shown to mechanically strengthen the Staphylococcus aureus membrane and to form microdomains that recruit antibiotic-resistance factors. Thus, inhibition of STX is a promising strategy for intervening against multidrug-resistant strains of this pathogen. However, the molecular mechanisms by which STX regulates the membrane structure and function of Staphylococcus aureus remain unclear. More specifically, the localization of STX within phosphatidylglycerol (PG) bilayers, the primary phospholipid of this bacteriums membrane, and how this localization drives macroscopic biophysical changes remain unresolved questions. Here, we addressed this issue by integrating molecular dynamics (MD) simulations, X-ray scattering experiments, and fluorescence spectroscopy. We developed an atomistic model of STX, which was validated against X-ray scattering data and which is suitable for all-atom MD simulations. We demonstrate that STX significantly increases lipid packing and acyl chain order of STX-PG bilayer mixtures. In addition, STX self-assembles into clusters, where the long and rigid conjugated triterpenoid chain interdigitates across both leaflets, modifying locally the density of the surrounding PG molecules. These findings provide a molecular explanation to the reduced headgroup spacing and core dynamics observed in fluorescence experiments and are consistent with the formation of structurally-distinct STX-enriched microdomains. Notably, STX reduces the gel-to-liquid crystalline phase transition temperature, indicating a general stabilizing effect for the fluid phase of PG lipids of varying length. Overall, our findings provide molecular insights into how STX enhances membrane mechanical integrity. It will be highly interesting to establish how the membrane remodeling effects observed here connect with STXs dual roles, acting as an antioxidant and preventing pore formation and other mechanical perturbations induced by antimicrobial molecules.

biophysics↗

bioRxiv2026

Comprehensive transcriptomic and proteomic profiling of mTOR-related epilepsy

Malformations of cortical development (MCDs) are a major cause of drug-resistant epilepsy (DRE) in children. A subset of MCDs, including focal cortical dysplasia type II, tuberous sclerosis complex, and hemimegalencephaly, shares characteristic histopathological features and somatic mutations in genes within the PI3K-AKT-mTOR signaling cascade, and is termed mTORopathies. However, the molecular mechanisms that drive epileptogenesis and consequent uncontrollable seizures in these disorders remain poorly understood. Here, we performed integrated genomic, transcriptomic, and proteomic analyses of 60 surgical brain specimens from 42 patients with MCDs and 18 with mesial temporal lobe epilepsy. Targeted genomic sequencing identified 19 somatic mutations in PI3K-AKT-mTOR pathway genes, including novel variants in AKT3 (L51H) and MTOR (A512T). RNA sequencing comparing mTORopathy brains with controls, along with cross-validation using an independent public dataset, identified 287 consistently differentially expressed genes that were consistently altered. Downregulated genes were enriched in oxidative phosphorylation (OXPHOS) and mitochondrial metabolic pathways, whereas upregulated genes were associated with gliogenesis and cellular senescence. Proteomic profiling identified 44 differentially expressed proteins, including significant reductions in OXPHOS-related proteins (COX5B, NDUFS4, GOT2, RAB27B, and DKK3). Our study provides a patient-matched, multi-omics dataset of human mTOR-related epilepsies from a single-institution cohort. Integrated analyses across transcriptomic and proteomic layers highlight impairment of OXPHOS as a molecular hallmark of mTORopathies, Integrated transcriptomic and proteomic analyses identified impaired OXPHOS as a molecular hallmark, potentially contributing to focal hypometabolism in mTORopathy. These findings offer mechanistic insights into epileptogenesis and provide a valuable resource for understanding molecular pathologies and developing mechanism-based therapies for mTOR-related epilepsy.

neuroscience↗

bioRxiv2026

Mechanical Stiffening Promotes Growth, Invasion, and Mitogen-activated protein kinase kinase (MEK) Inhibitor Resistance in 3D Plexiform Neurofibroma Cultures

Plexiform neurofibromas with neurofibromatosis type I (pNF1s) are benign peripheral nerve sheath tumors caused by NF1 loss, leading to dysregulated RAS/mitogen-activated protein kinase (MAPK) signaling. While mitogen-activated protein kinase kinase (MEK) inhibitors, selumetinib and mirdametinib, can reduce tumor volume, surgical resection remains the primary treatment for immediate debulking and symptom relief. Complete removal is often limited by tumor infiltration along nerve plexuses, and residual tumors may undergo postsurgical tissue remodeling, producing localized regions of stiffened extracellular matrix (ECM). The impact of ECM stiffness on pNF1 progression and drug response is unknown. Using patient-derived pNF1 tumor cells cultured in 3D hydrogels with defined stiffness (1.5 kPa soft, 7 kPa stiff), we found that stiff ECM promoted spread morphology, increased growth, and progressive intracellular softening. Stiff ECM reduced lysyl oxidase (LOX) expression, reflecting mechanoadaptive ECM remodeling, and upregulated P-glycoprotein, leading to decreased sensitivity to selumetinib. These results provide the first evidence that ECM stiffening, such as that arising from postsurgical remodeling, directly drives pNF1 progression and therapeutic resistance. Our findings highlight mechanobiology as a key regulator of tumor behavior and support targeting ECM mechanics to improve clinical outcomes in NF1 patients.

cancer biology↗

bioRxiv2026

MEIS1 is Required for Establishing Bergmann Glia-Specific Properties in the Developing Cerebellum

Compared to normal multipolar astrocytes, Bergmann glial cells (BGs), specifically differentiated astrocytes in the cerebellum, possess unique unipolar morphology and additional cellular functions. However, the molecular mechanisms that confer BG-specific properties onto normal multipolar astrocytes remain unknown. Here, we show that the transcription factor, MEIS1, is involved in BGs acquiring their unique characteristics. Targeted disruption of Meis1 in the whole cerebellum or astroglial lineage cells resulted in a marked reduction of BGs accompanied by an increase in multipolar astrocytes in mice. Postnatal deletion of Meis1 in Bergmann glia-like progenitors (BGLPs), which produce both BGs and multipolar astrocytes, suppressed their differentiation into BGs while promoting into multipolar astrocytes. Single-cell RNA sequencing, immunohistochemistry, and ChIP-Atlas analyses indicated that MEIS1 directly upregulates expression of BG-specific genes, including Vimentin and Zeb2, which are known to contribute to the correct localization and the unipolar process formation of BGs. These findings suggest that MEIS1 promotes the endowment of BG-specific properties to astrocytes by controlling the expression of BG-specific genes, thereby ensuring proper differentiation of BGLPs into BGs.

developmental biology↗

bioRxiv2026

An ancestral haplotype of P2RX5 yields a B-cell surface marker and a promising multi-lineage immunotherapy target

While CD19- and BCMA-directed immunotherapies have improved outcomes for B-lymphoid and plasma cell malignancies, frequent relapses with antigen loss/downregulation highlight the need for new targets. Here, using transcriptomic datasets and newly-developed monoclonal antibodies, we show that P2RX5, long considered a pseudogene in humans, encodes a stable protein in 80% of individuals of African descent carrying the ancestral haplotype. Like CD19, P2RX5 displays B-cell lineage-restricted expression in normal tissues. Unlike CD19, P2RX5 is expressed not only in B-cell neoplasms, but also in T-cell leukemia (T-ALL) and multiple myeloma (MM). We developed P2RX5-directed bispecific T-cell engagers and CAR T cells, which killed T-ALL cells with no evidence of T-cell fratricide. These agents were non-inferior to FDA-approved CD19- and BCMA-directed immunotherapeutics in cell culture and xenograft models of Burkitt lymphoma and MM, while maintaining potency against CD19- and BCMA-negative variants. Hence, P2RX5 is a unique multi-lineage target for frontline or salvage immunotherapy.

cancer biology↗

bioRxiv2025

Context-Dependent Effects of Midkine on Plexiform Neurofibroma Growth and Drug Response in 3D Coculture Models

Midkine (MDK) is a heparin-binding growth factor that promotes tumor growth in many cancers and may contribute to type I neurofibromatosis (NF1) tumor by stimulating Schwann cell proliferation and supporting neurofibroma growth. Here, we investigated the role of midkine in plexiform neurofibroma with NF1 (pNF1) using both monoculture and neuron-tumor coculture system with biomimetic neuronal axons engineered to release midkine in 3D. In monoculture, midkine enhanced the growth of pNF1 tumor cells, consistent with its tumor-promoting role in several cancers. In contrast, in 3D coculture, midkine did not increase tumor growth and failed to alter response to selumetinib, the FDA-approved treatment for pNF1. These findings suggest that while midkine promotes tumor growth, its effect may be buffered or neutralized in a neuron-associated microenvironment, underscoring the importance of physiologically relevant models for evaluating therapeutic targets in NF1.

cancer biology↗

bioRxiv2025

An ultra-long acting insulin enables glucose-synchronised release

Delayed and weak glucose-responsive kinetics, coupled with short plasma exposure and complicated formulation, remain major obstacles to the clinical translation of glucose-responsive insulin formulations. Here we report a rapidly absorbable, ultra-long acting, and glucose-responsive insulin analogue via engineering recombinant human insulin with two phenylboronic acids. The insulin analogue has a long circulating half-life (t1/2{beta} [~]150 h) due to its unique, unprecedented, glucose-responsive binding to blood circulation-associated proteins. This interaction drives the formation of a unique systemic reservoir that allows synchronised dynamic response in insulin levels to glucose fluctuations. In type 1 diabetic mice and minipigs, a single subcutaneous dose can maintain normoglycaemia for over one week (47.5-fold compared to insulin glargine). After the last injection of four consecutive weekly administrations in mice, the glucose-lowering effect can last 800 hours. Administering this glucose-responsive insulin analogue with a fully automated insulin delivery system can leverage dual closed loops and achieve a glucose time in range of over 85% and a coefficient of variation of approximately 30%, better than the recommended targets for human therapy. Importantly, no toxicity was identified in the study.

bioengineering↗

bioRxiv2025

NitrOFF: An engineered fluorescent biosensor to illuminate nitrate transport in living cells

The duality of nitrate is nowhere best exemplified than in human physiology - a detrimental pollutant but also a protective nutrient and signaling ion - particularly as connected to reactive nitrogen oxides. Aside from limited insights into nitrate uptake and storage, foundational nitrate biology has lagged. Genetically encoded fluorescent biosensors can address this gap with real-time imaging. However, imaging technologies for mammalian cell applications remain rare. Here, we set out to design and engineer a two-domain chimera fusing the split green fluorescent protein EGFP and the nitrate recognition domain NreA from Staphylococcus carnosus. Over 7 rounds of directed evolution, 15 mutations were accumulated resulting in the functional biosensor NitrOFF. NitrOFF has a high degree of allosteric communication between the domains reflected in a turn-off intensiometric response (Kd {approx} 9 {micro}M). This was further reinforced by X-ray crystal structures of apo and nitrate bound NitrOFF, which revealed that the two domains undergo a large-scale conformational rearrangement that changes the relative positioning of the EGFP and NreA domains by 68.4{degrees}. Such a dramatic difference was triggered by the formation of a long helix at the engineered linker connecting the two domains, peeling the {beta}7 strand off the EGFP and thus extinguishing the fluorescence upon nitrate binding. Finally, as a proof-of-concept, we highlighted the utility of this first-generation biosensor to monitor exogenous nitrate uptake and modulation in a human embryonic kidney (HEK) 293 cell line. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/644677v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1ba853eorg.highwire.dtl.DTLVardef@81bed9org.highwire.dtl.DTLVardef@18a313org.highwire.dtl.DTLVardef@17a5e80_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Phase-field Approach to Cellular Blebbing

Bulges in the plasma membrane of cells known as blebs can form spontaneously in a wide range of biological processes but what controls their shape and stability remains incompletely understood. To address this, we introduce a dual phase-field model with coupled order parameters representing the cell cortex and plasma membrane that can quantitatively model blebbing in three dimensions. Simulations and analysis of the model reveal that, depending on whether blebbing occurs by detachment of the plasma membrane or rupture of the actin cortex, blebs can form discontinuously through a saddle node bifurcation or continuously with increasing cortical tension. The model predictions are in good quantitative agreement with existing experimental data for laser-induced cortex rupture.

biophysics↗

bioRxiv2024

A Ketogenic Diet Sensitizes Pancreatic Cancer to Inhibition of Glutamine Metabolism

Pancreatic cancer is the third leading cause of cancer death in the United States, and while conventional chemotherapy remains the standard treatment, responses are poor. Safe and alternative therapeutic strategies are urgently needed1. A ketogenic diet has been shown to have anti-tumor effects across diverse cancer types but will unlikely have a significant effect alone. However, the diet shifts metabolism in tumors to create new vulnerabilities that can be targeted (1). Modulators of glutamine metabolism have shown promise in pre-clinical models but have failed to have a marked impact against cancer in the clinic. We show that a ketogenic diet increases TCA and glutamine-associated metabolites in murine pancreatic cancer models and under metabolic conditions that simulate a ketogenic diet in vitro. The metabolic shift leads to increased reliance on glutamine-mediated anaplerosis to compensate for low glucose abundance associated with a ketogenic diet. As a result, glutamine metabolism inhibitors, such as DON and CB839 in combination with a ketogenic diet had robust anti-cancer effects. These findings provide rationale to study the use of a ketogenic diet with glutamine targeted therapies in a clinical context. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=186 SRC="FIGDIR/small/604377v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1ebeefforg.highwire.dtl.DTLVardef@97e2ceorg.highwire.dtl.DTLVardef@1ab45c3org.highwire.dtl.DTLVardef@1713540_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical Abstract Description: Mechanistic rationale for combining a ketogenic diet and glutamine metabolism inhibitors. The combination of low glucose from a ketogenic diet and pharmacologic glutamine inhibition impairs nutrient input to mitochondria, reducing cancer growth.

cancer biology↗

bioRxiv2023

A new vulnerability to BET inhibition due to enhanced autophagy in BRCA2 deficient pancreatic cancer

Pancreatic cancer is one of the deadliest diseases in human malignancies. Among total pancreatic cancer patients, [~]10% of patients are categorized as familial pancreatic cancer (FPC) patients, carrying germline mutations of the genes involved in DNA repair pathways (e.g., BRCA2). Personalized medicine approaches tailored toward patients mutations would improve patients outcome. To identify novel vulnerabilities of BRCA2-deficient pancreatic cancer, we generated isogenic Brca2-deficient murine pancreatic cancer cell lines and performed high-throughput drug screens. High-throughput drug screening revealed that Brca2-deficient cells are sensitive to Bromodomain and Extraterminal Motif (BET) inhibitors, suggesting that BET inhibition might be a potential therapeutic approach. We found that BRCA2 deficiency increased autophagic flux, which was further enhanced by BET inhibition in Brca2-deficient pancreatic cancer cells, resulting in autophagy-dependent cell death. Our data suggests that BET inhibition can be a novel therapeutic strategy for BRCA2-deficient pancreatic cancer.

cancer biology↗

bioRxiv2022

Chaperones Hsc70 and Hsp70 Play Distinct Roles in Bocaparvovirus Minute Virus of Canines Infection

Minute virus of canines (MVC) belongs to the genus Bocaparvovirus and reproduces rapidly in its permissive cells Walter Reed/3873D (WRD). The crosstalk between MVC and WRD is poorly characterized in terms of molecular requirements and mechanisms. Here, we identified two novel cellular proteins Hsc70 and Hsp70 that interact with both NS1 and VP2 via mass spectrometry (MS), co-immunoprecipitation (Co-IP) and confocal immunofluorescence assays (IFA). Hsp70 was upregulated upon MVC infection. Respective silencing of Hsc70 and Hsp70 led to contrasting results at nearly every stage of MVC life cycle, including virus entry, transcription, translation, replication and production. Strikingly, transfection with low and high dose of pFlag-Hsp70 contributed to opposing impacts on viral protein levels and virus production possibly through a ubiquitin-dependent manner, indicating that MVC is quite sensitive to the levels of Hsp70. Treatment with quercetin and VER155008, two Hsp70 family inhibitors, both significantly decreased viral replication and particle levels. Together, these results illustrated that both Hsc70 and Hsp70 are involved in MVC life cycle, and targeting to Hsp70 family may represent a novel anti-MVC mechanism.

molecular biology↗

bioRxiv2019

Circular RNA profiling in the oocyte and cumulus cells reveals that circARMC4 is essential for porcine oocyte maturation

Thousands of circular RNAs (circRNAs) have been recently discovered in cumulus cells and oocytes from several species. However, the expression and function of circRNA during porcine oocyte meiotic maturation have been never examined. Here, we separately identified 7,067 and 637 circRNAs in both the cumulus cell and the oocyte via deep sequencing and bioinformatic analysis. Further analysis revealed that a faction of circRNAs is differentially expressed (DE) in a developmental stage-specific manner. The host genes of DE circRNAs are markedly enriched to multiple signaling pathways associated with cumulus cell function and oocyte maturation. Additionally, most DE circRNAs harbor several miRNA targets, suggesting that these DE circRNAs potentially act as miRNA sponge. Importantly, we found that maternal circARMC4 knockdown by siRNA microinjection caused a severely impaired chromosome alignment, and significantly inhibited first polar body extrusion and early embryo development. Taken together, these results demonstrate for the first time that circRNAs are abundantly and dynamically expressed in a developmental stage-specific manner in cumulus cells and oocytes, and maternally expressed circARMC4 is essential for porcine oocyte meiotic maturation and early embryo development.

developmental biology↗

bioRxiv2017

FGF21 regulates melanogenesis in alpaca melanocytes via ERK1/2-Mediated MITF downregulation

Fibroblast growth factor 21 (FGF21) is known as a metabolic regulator to regulate the metabolism of glucose and lipids. However, the underlying mechanism of FGF21 on melanin synthesis remains unknown. Therefore, the current study investigates the effect of FGF21 on melanogenesis in alpaca melanocytes. We transfected the FGF21 into alpaca melanocytes, then detected the melanin contents, protein and mRNA levels of pigmentation-related genes in order to determine the melanogenesis-regulating pathway of FGF21. The results showed that FGF21 overexpression suppressed melanogenesis and decreased the expression of the major target genes termed microphthalmia-associated transcription factor (MITF) and its downstream genes, including tyrosinase (TYR) and tyrosinase-related protein 2 (TRP2). However FGF21 increased the expression of phospho-extracellular signal-regulated kinase (p-Erk1/2). In contrast, FGF21-siRNA, a small interference RNA mediating FGF21 silencing, abolished the inhibition of melanogenesis. Altogether, FGF21 may decrease melanogenesis in alpaca melanocytes via ERK activation and subsequent MITF downregulation, which is then followed by the suppression of melanogenic enzymes and melanin production.

cell biology↗