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Jiang, L.

Publications and source records attributed to Jiang, L..

At least 73 records · Page 4Linked to original sources
bioRxiv2025

FAM19A4 Enhances Neutrophil Respiratory Burst via p38 MAPK in Lethal Sepsis

Sepsis causes high mortality and resource strain, with neutrophil-derived reactive oxygen species (ROS) contributing to excessive inflammation. The secretory protein FAM19A4 modulates ROS release, but its role in sepsis was unclear. We measured elevated FAM19A4 levels in septic patients and cecal ligation and puncture (CLP) mice, correlating with increased mortality. Fam19a4-/- mice subjected to CLP showed significantly improved survival and attenuated multiorgan injury without impaired peritoneal bacterial clearance or altered circulating neutrophil counts. Deficiency reduced the cell counts of neutrophil (Ly6G+) and macrophage (F4/80+) in lungs and liver, diminished systemic ROS production tracked by bioluminescence, and decreased neutrophil extracellular trap (NET) formation in serum and lung tissue. In vitro, FAM19A4 enhanced neutrophil phagocytosis and ROS generation but did not affect lipopolysaccharide-induced chemotaxis. Mechanistically, bulk RNA sequencing, western blotting, and the p38 inhibitor SB203580 demonstrated that FAM19A4 drives neutrophil ROS release specifically through p38 MAPK signaling activation. These results indicate that FAM19A4 is upregulated during sepsis and exacerbates outcomes by enhancing neutrophil ROS production via p38 MAPK, representing a promising therapeutic target for this condition.

molecular biology↗
bioRxiv2025

The transcriptional regulatory circuit as a driver and therapeutic target in CML blast crisis

Patients with blast crisis in chronic myeloid leukemia (CML) exhibit an extremely poor prognosis, with median survival of only a few months and limited therapeutic options. Dysregulated transcriptional regulation plays a pivotal role in driving blast crisis progression. In this study, we performed histone modification and transcriptomic high-throughput sequencing on bone marrow cells derived from chronic and blast-phase CML patients, identifying a CML blast-specific transcriptional regulatory circuit comprising MEF2C, MYB, MEIS1, and ZEB2. These transcription factors (TFs) form protein complexes that co-bind to shared enhancers or promoters, reciprocally enhancing each others transcriptional activity and that of their downstream targets. Mechanistically, this circuit synergistically reprograms chromatin accessibility and transcriptional landscapes in blast cells, parallel activation of purine metabolism and Rho GTPase signaling pathways which exhibit functional crosstalk. Functional validation demonstrated that silencing components of this circuit or its downstream core targets significantly suppressed CML blast progression in vitro and in vivo. Notably, mebendazole (MBZ), a clinically approved inhibitor targeting MYB protein stability, recapitulated these inhibitory effects. Our findings establish the MEF2C-MYB-MEIS1-ZEB2 transcriptional circuit as a central driver of CML blast crisis and position MBZ as a high-potential translational strategy for targeting this lethal phase of the disease. Key PointsO_LIMEF2C, MYB, MEIS1, and ZEB2 form the CML blast-crisis transcriptional circuit, synergistically reprogramming chromatin and transcription. C_LIO_LIMebendazole inhibits CML blast progression by impairing the circuit, offering a rapid translational strategy for this lethal phase. C_LI

cancer biology↗
bioRxiv2025

Symmetric brain-liver circuits mediate lateralized regulation of hepatic glucose output

Neural lateralization is well recognized in the control of contralateral somatic movement, yet its relevance to visceral organ regulation remains poorly understood. This study aimed to determine whether the central nervous system exerts lateralized control over hepatic glucose metabolism and to localize the site of peripheral sympathetic crossover to the liver. Pseudorabies virus (PRV) tracing revealed bilateral projections from the lateral paragigantocellular nucleus (LPGi) with preferential innervation of the contralateral hepatic lobes. Unilateral LPGi activation elevated systemic glucose by enhancing glycogenolysis and gluconeogenesis specifically in contralateral lobes, whereas bilateral activation produced additive effects. Following unilateral hepatic denervation, contralateral LPGi activation induced metabolic compensation in the remaining innervated lobes, characterized by increased norepinephrine release, glucose production, and glycogen depletion. Whole-mount tissue clearing and dual viral tracing localized the sympathetic crossover to the porta hepatis. Developmental analysis showed that lobar-specific innervation along the vasculature emerges by postnatal week 2. These findings demonstrate that the brainstem can exert lobe-specific, lateralized control of hepatic glucose metabolism via bilaterally projecting brain - liver sympathetic pathways. This contralateral regulation arises from a peripheral decussation at the porta hepatis, and the compensatory activation observed after denervation reveals an intrinsic neuroadaptive mechanism that helps safeguard systemic glucose homeostasis. HighlightsO_LIBrain-liver sympathetic projections exhibit predominant contralateral innervation C_LIO_LIUnilateral LPGi activation drives glucose production in contralateral hepatic lobes C_LIO_LIUnilateral denervation augments contralateral LPGi-mediated metabolic compensation C_LIO_LISympathetic crossover to the liver localizes at the porta hepatis C_LI

neuroscience↗
bioRxiv2025

PIWIL3-piRNA Pathway Is Essential for Rabbit Oogenesis and Embryogenesis via Broad Regulation of the Transcriptome and Proteome

Female infertility represents a significant challenge in reproductive medicine. Although it is known to be primarily due to oogenic and early embryonic failures, the molecular mechanisms underlying these failures remain elusive. The Piwi-piRNA pathway is crucial for gametogenesis in diverse organisms. Yet, its role in mammalian female fertility is unclear. This is partly because most studies are done in mice, which contain only PIWIL1, PIWIL2, and PIWIL4 that are essential for spermatogenesis but not for oogenesis. PIWIL3 emerges in higher mammals and is highly expressed in human oocytes. Interestingly, female PIWIL3-knockout golden hamsters exhibit only reduced fertility without detectable defects in oogenesis. This has left the function of PIWIL3 in higher mammals, including humans, unexplored. Here, we discovered that PIWIL3 in the rabbit (Oryctolagus cuniculus) shares high homology with human PIWIL3. It is the predominant PIWI protein in oocytes in both species. Using CRISPR-cas9-mediated knockout, we demonstrated that rabbit PIWIL3 is essential for female fertility. Its loss leads to severe defects in oogenesis. Moreover, embryos lacking maternal PIWIL3 arrest developmentally at the 8-cell stage. Mechanistically, rabbit PIWIL3 binds [~]18-nucleotide piRNAs, mirroring the behavior of human PIWIL3, and is critical for piRNA biogenesis. Moreover, it regulates transcriptomic and proteomic landscapes and silences a broad array of transposons during late oogenesis yet activates another set of transposons during early embryogenesis. These findings establish PIWIL3 as a pivotal dual regulator of gene expression and transposon activity, which is essential for oogenesis and early embryogenesis in non-rodent mammals, potentially including humans.

developmental biology↗
bioRxiv2025

Pan-cancer analysis reveals that tumor microbiomes generate specific epitopes through transcriptional reprogramming

The therapeutic promise of tumor microbial peptides faces a critical safety paradox: tumors and peritumor tissues harbor compositionally similar microbiomes, yet safe immunotherapy demands tumor-specific targeting. We performed comprehensive transcriptomic analysis of microbial communities from 1,868 tumor and peritumor samples across 11 cancer types. Although 88.5% of microbial species are present in both tissue types, tumor and peritumor tissues exhibit profound transcriptional divergence: only 34.1% of microbial genes are expressed in both tissue types, and merely 18.2% of predicted HLA-binding peptides overlap. This transcriptional divergence represents microbial transcriptional reprogramming (MTR) in tumor tissues, through which microbes activate distinct genomic regions while adapting to tumor microenvironmental pressures, thereby generating tumor-specific peptide repertoires. Using Microbial Transcriptional Reprogramming Index (MiTRI) to quantify MTR, we found that higher MiTRI values correlate with expanded immunogenic peptide repertoires. Mass spectrometry-based immunopeptidomics confirmed that 25% of naturally presented microbial peptides originate from MTR regions. Crucially, in microsatellite-stable colorectal cancer, neo-adjuvant therapy responders exhibited significantly higher MiTRI values. T cell receptors (TCRs) recognized MTR-derived epitopes at 2.10-fold higher rates than genome-wide epitopes, accounting for 79.1% of clonally expanded TCRs. These findings establish MTR as the principle that ensures the tumor-specificity and safety of micorbiome-directed immunotherapy.

cancer biology↗
bioRxiv2025

Modeling Alzheimer's Disease with APOE4 Neuron-Glial Brain Assembloids Reveals IGFBPs as Therapeutic Targets

Alzheimers disease (AD) research has been hindered by the lack of models that faithfully recapitulate the full profile of disease progression in a human genetic background. We developed a 3D assembloid model ("Masteroid") using iPSC-derived neurons, astrocytes, and microglia from APOE4/4 and isogenic control lines. Neurons were seeded with tau oligomers, then combined with astrocytes and microglia to form mature 3D Masteroids, followed by amyloid-{beta} oligomer exposure. After four weeks, AD-Masteroids exhibited hallmark pathologies, including extracellular amyloid-{beta} deposits, intracellular tau aggregation, neurodegeneration, astrogliosis, and microglial activation, with APOE4 exacerbating all phenotypes. Single-cell RNA sequencing further identified novel roles of IGFBP pathways in amyloid-{beta} and tau-mediated pathology. This innovative platform provides a robust system to dissect cellular and molecular mechanisms of AD progression and offers a powerful tool for therapeutic discovery. HighlightsO_LIThe 3D human neuron-glia assembloid ("Masteroid"), composed of neurons, astrocytes, microglia, and oligodendrocytes, faithfully recapitulates human brain ultrastructure and intercellular interactions. C_LIO_LIExposure to oligomeric tau and A{beta} induced hallmark Alzheimers pathologies, including amyloid deposition, tau aggregation, neurodegeneration, and gliosis. C_LIO_LIThe APOE4 genotype exacerbated all pathological features, highlighting its role in driving multicellular interactions that accelerate disease progression. C_LIO_LIThe IGF signaling axis was identified as a key mediator of A{beta}- and tau-induced pathology and a potential therapeutic target. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=183 SRC="FIGDIR/small/683162v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@f91740org.highwire.dtl.DTLVardef@165726eorg.highwire.dtl.DTLVardef@13b957forg.highwire.dtl.DTLVardef@d40307_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗
bioRxiv2025

A tailored variant filtering procedure for multi-breed and multi-species unbalanced animal SNP collections

Technological advancements and decreasing costs of whole-genome sequencing have generated a huge amount of resequencing data. Large-sized datasets, spanning several species and/or populations can now be assembled easily. However, they vary widely in geographical provenance, and sample sizes, with taxonomic groups varying from single to hundreds of entries. Consequently, standard filtering approaches may bias the representation of groups or gene pools. Commonly adopted variant filters relying on minor allele frequency (MAF) and linkage disequilibrium (LD) are not adequate because LD patterns and allele frequencies differ substantially within datasets representing both local and global diversity of multiple populations and species. Thus, by using the VarGoats 1000 goat genome project, we devised a novel approach which avoids the biases of the standard filtering procedures by adopting within-population subsampling, minor allele count (MAC) and marker spacing (bp-space) as filters. Starting from a quality-filtered dataset of >28M SNPs from 1372 animals, we generated a dataset of <14M markers and 750 individuals, complying with the initial requirements and facilitating further computational steps.

bioinformatics↗
bioRxiv2025

Sphingosine kinase 2 suppresses neutrophil responses to promote viral persistence while attenuating immune pathology

Chronic virus infections often suppress immune cell functions which helps in restricting immune pathology but leads to viral persistence. However, the underlying mechanisms are incompletely understood. We recently found that sphingosine kinase 2 (SphK2)-deficient (Sphk2-/-) mice succumbed to lymphocytic choriomeningitis virus (LCMV) infection due to immune pathology. In addition to heightened T cell immunity, a notable increase of neutrophils was observed in LCMV-infected Sphk2-/- mice. Depletion of neutrophils increased the viability of virus-infected Sphk2-/- mice, supporting a role of SphK2-deficient neutrophils in viral immune pathogenesis. Further, SphK2-deficient neutrophils expressed lower levels of the immune suppressive marker CD244 during infection. Importantly, adoptively transferred SphK2-deficient neutrophils demonstrated intrinsic regulation of CD244 and improved virus-specific T cell responses, resulting in diminished viral burden. Transcriptomic analysis revealed increased expression of pro-inflammatory and antiviral genes in SphK2-deficient neutrophils. These results indicate that SphK2 promotes suppressive neutrophil responses and regulates neutrophil-associated immune pathology during a persistent infection. Our findings may help design new immune therapeutics to control chronic viral diseases. SignificanceNeutrophils are the sentinels of the innate immune system; they can reshape innate and adaptive immune responses. During chronic illnesses, such as persistent viral infections, neutrophils can suppress the host immune response and help in disease progression. Here, we demonstrate regulation of neutrophil expansion and functions by sphingosine kinase 2 (SphK2) during LCMV infection. SphK2-deficient neutrophils express a reduced level of inhibitory receptor CD244, exert immune stimulatory effects on T cells, and promote virus clearance. Further, transcriptomic analysis reveals that SphK2 deficiency leads to the development of proinflammatory neutrophils. Our study identifies SphK2, a host factor, as being critical for neutrophil suppression that regulates dysfunctional T cell response and virus persistence.

immunology↗
bioRxiv2025

Polyclonal origins of human premalignant colorectal lesions

Cancer is generally thought to be caused by expansion of a single mutant cell1. However, analyses of early colorectal cancer lesions suggest that tumors may instead originate from multiple, genetically distinct cell populations2,3. Detecting polyclonal tumor initiation is challenging in patients, as it requires profiling early-stage lesions before clonal sweeps obscure diversity. To investigate this, we analyzed normal colorectal mucosa, benign and dysplastic premalignant polyps, and malignant adenocarcinomas (123 samples) from six individuals with familial adenomatous polyposis (FAP). Individuals with FAP have a germline heterozygous APC mutation, predisposing them to colorectal cancer and numerous premalignant polyps by early adulthood4. Whole-genome and/or whole-exome sequencing revealed that many premalignant polyps--40% with benign histology and 28% with dysplasia--were composed of multiple genetic lineages that diverged early, consistent with polyclonal origins. This conclusion was reinforced by whole-genome sequencing of single crypts from multiple polyps in additional patients which showed limited sharing of mutations among crypts within the same lesion. In some cases, multiple distinct APC mutations co-existed in different lineages of a single polyp, consistent with polyclonality. These findings reshape our understanding of early neoplastic events, demonstrating that tumor initiation can arise from the convergence of diverse mutant clones. They also suggest that cell-intrinsic growth advantages alone may not fully explain tumor initiation, highlighting the importance of microenvironmental and tissue-level factors in early cancer evolution.

cancer biology↗
bioRxiv2025

STX4 is indispensable for mitochondrial homeostasis in skeletal muscle

BackgroundMitochondrial homeostasis is vital for optimal skeletal muscle integrity. Mitochondrial quality control (MQC) mechanisms that are essential for maintaining proper functions of mitochondria include mitochondrial biogenesis, dynamics and mitophagy. Previously, Syntaxin 4 (STX4) traditionally considered a cell surface protein known for glucose uptake in skeletal muscle, was also identified at the outer mitochondrial membrane. STX4 enrichment was sufficient to reverse Type 2 diabetes-associated mitochondrial damage in skeletal muscle by inactivation of mitochondrial fission. However, whether STX4 could modulate skeletal muscle mitochondrial homeostasis through MQC mechanisms involving mitochondrial biogenesis or mitophagy remains to be determined. MethodsTo determine the requirements of STX4 in mitochondrial structure, function and MQC processes of biogenesis and mitophagy, we implemented our in-house generated inducible skeletal muscle-specific STX4-knockout (skmSTX4-iKO) mice (Stx4fl/fl; Tg(HSA-rtTA/TRE-Cre)/B6) and STX4-depleted immortalized L6.GLUT4myc myotubes via siRNA knockdown (siSTX4). ResultsWe found that non-obese skmSTX4-iKO male mice (>50% reduced STX4 abundance, Soleus and Gastrocnemius ***p<0.001, Tibialis anterior (TA) ****p<0.0001) developed insulin resistance (**p<0.01), together with reduced energy expenditure (AUC *p<0.05), respiratory exchange ratio (AUC **p<0.01), and grip strength (*p<0.05). STX4 ablation in muscle also impaired mitochondrial oxygen consumption rate (****p<0.0001). Mitochondrial morphological damage was heterogenous in STX4 depleted muscle, presenting with small fragmented mitochondria (****p<0.0001) and deceased electron transport chain (ETC) abundance (CI ***p<0.001, CII *p<0.05, CIV **p<0.01) in oxidative soleus muscle, while glycolytic TA fibers display enlarged swollen mitochondria (****p<0.0001) with no change in ETC abundance. Notably, >60% reduction of STX4 in siSTX4 L6.GLUT4myc myotubes (****p<0.0001) also decreased ETC abundance (CI ****p<0.0001, CII ****p<0.0001, CIV *p<0.05) without changes in mitochondrial glucose metabolism, as shown by [U-13C] glucose isotope tracing. For MQC, both skmSTX4-iKO male mice (*p<0.05) and siSTX4 L6.GLUT4myc myotubes (*p<0.05) showed decreased mitochondrial DNA levels alongside reduced mRNA expression of mitochondrial biogenesis genes Ppargc1a (PGC1-, *p<0.05) and Tfam (*p<0.05) in skmSTX4-iKO soleus muscle and PGC1- (mRNA *p<0.05, protein ***p<0.001), NRF1 (mRNA and protein *p<0.05) and Tfam (mRNA *p<0.05) in siSTX4 L6.GLUT4myc myotubes. Furthermore, live cell imaging using mt-Keima mitophagy biosensor in siSTX4 L6.GLUT4myc cells revealed significantly impaired mitochondrial turnover by mitophagy (*p<0.05) and mitochondria-lysosome colocalization (*p<0.05). STX4 depletion also reduced canonical mitophagy markers, PINK1 and PARKIN in both skmSTX4-iKO muscle (PARKIN *p<0.05, PINK1 **p<0.01) and siSTX4 L6.GLUT4myc myotubes (PARKIN ****p<0.0001, PINK1 *p<0.05). ConclusionsOur study demonstrated STX4 as a key mitochondrial regulator required for mitochondrial homeostasis in skeletal muscle.

cell biology↗
bioRxiv2025

Novel Function of Arylalkalamine N-acetyltransfe- rase-1 in Modulating Host Coagulation and Blood Feeding in Aedes aegypti

During blood feeding, mosquitoes introduce substantial quantities of salivary proteins into their hosts to facilitate feeding. Arylalkylamine N-acetyltransferase-1 (aaNAT1) is a salivary protein prominently produced in the salivary glands of Aedes aegypti. Previous research has demonstrated that mosquitoes with AaaaNAT1 mutations fail to efficiently inhibit host blood coagulation; however, the precise regulatory mechanisms remain unknown. This study identified the primary metabolites and proteins interacting with AaaaNAT1 through integrated transcriptomic, proteomic, and metabolomic analyses. Combined with isothermal titration calorimetry (ITC), immunohistochemistry, ELISA, platelet function assays, and coagulation tests, we further established that AaaaNAT1 binds norepinephrine, reducing host platelet aggregation and thereby limiting activation of the host coagulation cascade and fibrinolytic system. Additionally, behavioral investigations showed that AaaaNAT1 knockdown in Aedes aegypti significantly prolonged the time required for female mosquitoes to locate a host and successfully obtain blood, while also markedly reducing blood consumption. This effect may stem from the inability of aaNAT1-depleted mosquitoes to effectively suppress host blood aggregation during feeding, as well as the downregulation of octopamine following AaaaNAT1 knockdown. Therefore, this study reveals a novel role for aaNAT1 distinct from its known functions in pigmentation and immunity, advancing our understanding of aaNAT1s role in insects.

molecular biology↗
bioRxiv2025

Developmental expression of risk genes implicates the age of onset for neuropsychiatric disorders

The functional effects of genetic variants associated with complex diseases exhibit pronounced spatiotemporal specificity. Although spatially resolved studies have advanced, their temporal dynamics remain poorly characterized. Here, we present an analytical framework integrating developmental gene expression with genome-wide association studies to decipher age-specific windows during which genetic variants exert their effects and to elucidate underlying mechanisms. Applying this framework to five major neuropsychiatric disorders, we uncover a fundamental principle: the peak incidence of a disease precisely coincides with the developmental window of peak expression of its associated risk genes in the prefrontal cortex. These risk windows are characterized by distinct biological processes; for instance, childhood risk for attention-deficit/hyperactivity disorder aligns with a peak in presynaptic machinery gene expression, whereas late-life risk for Alzheimers disease corresponds to heightened immune-related gene activity. Leveraging this principle of temporal convergence significantly improves the prioritization of disease genes. Our work establishes the developmental basis for the age of onset of complex diseases, providing a temporal roadmap for understanding disease mechanisms and developing age-appropriate therapeutic strategies.

genetics↗
bioRxiv2025

Seq2Karyotype (S2K): A Method for in-silico Karyotyping Using Single-Sample Whole-Genome Sequencing Data

DNA abnormalities characterized by cytogenetic imaging at the single cell resolution, i.e. karyotyping, have long served as cancer diagnostic and prognostic biomarkers. To enable in-silico karyotyping using unpaired whole-genome sequencing data, we developed Seq2Karyotype (S2K), a tool that fits karyotype models with clonality estimation based on read-depth and allelic imbalance in a bulk sample and supports visualization-guided refinement. Analysis on 19 adult and pediatric cancer cell lines revealed unexpected intratumoral heterogeneity involving multiple copy number variation (CNV) states including whole-genome duplication, which were validated by imaging and single-cell omics profiling. Analyses on patient samples showed high concordance with clinical cytogenetic reports for acute myeloid leukemia, and revealed evolutionary trajectories from multi-region metastatic neuroblastomas implicating reversion. These findings highlight extensive and dynamic intratumoral heterogeneity contributed by CNV in both cell line models and patient samples, which may inform future research on tumor evolution under selective pressure such as drug exposure.

bioinformatics↗
bioRxiv2025

Deep Learning of Functional Perturbations from Condensate Morphology

Biomolecular condensates compartmentalize the interior of living cells to spatiotemporally organize complex functions, yet linking molecular interactions within condensates to their mesoscale organization remains a major challenge. To bridge this gap, we developed a neural network-based framework - Deep-Phase - that uses microscopy images to quantitatively classify condensate morphology changes resulting from pharmacological alterations in associated biochemical processes. We use Deep-Phase to precisely quantify time- and concentration-dependent structural perturbations to the multiphase nucleolus and show that they are tightly coupled to potencies of drugs inhibiting rRNA transcription and processing. Applying Deep-Phase in a chemical screen, we identify a unique nucleolar morphology and discover a role for a DNA topoisomerase in rRNA processing. Mechanistic studies of this morphology provide insights into how interfaces between nucleolar subcompartments are maintained. We demonstrate Deep-Phases adaptability to diverse cell lines, labels, and condensates, offering a powerful platform for uncovering cellular organizing principles and therapeutic targets. HighlightsO_LIA deep learning framework, Deep-Phase, classifies and quantifies drug-induced changes in morphologies of nucleoli, nucleolar speckles, and viral cytoplasmic condensates, directly from images. C_LIO_LITime- and concentration-dependent morphological responses to perturbation predict associated disruptions in RNA transcription and processing. C_LIO_LIUsing Deep-Phase in a high-content small molecule screen reveals a unique nucleolar morphology induced by TOP1 inhibition. C_LIO_LITOP1 inhibition leads to reduced levels and processing of large ribosomal subunit precursors and provides a mechanism for maintenance of nucleolar phase boundaries. C_LI

molecular biology↗
bioRxiv2025

Extreme Small-World, Modular, and Rich-Club Topology of Single-Neuron Networks in Mouse Primary Visual Cortex

Understanding whether the canonical topologies of macroscale connectomes, such as small-world architecture, hub dominance, rich-club cores, and modularity, extend to local cortical microcircuitry has remained challenging due to limitations in simultaneously recording large neuronal populations in vivo. Here, using ultra-large-scale, high-resolution calcium imaging, we tracked spontaneous activity from approximately 2,000 neurons across the mouse primary visual cortex (V1). Across multiple mice and correlation thresholds, V1 neuronal networks exhibited hallmark characteristics of efficient brain organization, but in a markedly intensified form compared to macroscopic brain networks. Local clustering coefficients remained an order of magnitude above random levels, while characteristic path lengths approached those observed in random networks, yielding an exceptionally high small-world index that substantially exceeded typical values previously reported at macroscopic scales. Degree distributions followed a power-law, identifying highly connected hub neurons whose interconnections formed a robust rich-club integrative core. Community detection analyses showed robust modularity upon pruning weak connections, indicating functionally specialized neuronal clusters interconnected predominantly through hubs. These findings provide one of the first direct in vivo evidence that single cortical microcircuits not only recapitulate but intensify network topologies observed at macroscopic scale, implying evolutionarily conserved design principles underlying brain organization from neurons to systems.

neuroscience↗
bioRxiv2025

Determine Interaction Affinity Changes of the SUMO E1 Activating Enzymes During SUMO Activation using Quantitative FRET technology

The heterodimeric E1 complex, Aos1-Uba2, catalyzes the first adenylation activation of the SUMO1 peptide in the SUMOylation cascade. The reaction affinity and dynamics of the Aos1-Uba2 heterodimer during the first step activation have yet to be determined. The Kd for the Aos1-Uba2 interaction provides a unique perspective for the activation step of the ubiquitin-like protein conjugation cascade. Here, we report for the first time the determination of the Aos1 and Uba2 interaction dissociation constant (Kd) and kinetics using the qFRET assay. We also used the SPR method to verify the interaction Kd between Aos1 and Uba2. We also determined the kinetics changes of Aos1-Uba2 when SUMOs and ATP were added to the reaction in real time. The results showed that forming a thioester bond between SUMO1 and Uba2 increases the FRET signal, indicating that the E1 heterodimer is more stable and bound to each other in SUMO and ATP. These results suggest that the qFRET method can be used to determine protein interaction affinity changes and track real-time changes in protein conformation and dynamics changes during biochemical reactions.

bioengineering↗
bioRxiv2025

Determine Protein Interaction Affinity without Purification Reveals a Two-enzyme Co-recognition of SUMOylation Substrate using Quantitative FRET (qFRET) Technology

Protein-protein interaction is the most fundamental process in life, and the quantification and manipulation of protein interaction activation or inhibition are critical for a comprehensive understanding of physiological processes and the development of new medicines that modulate protein-protein interactions. Due to its essential roles in physiology and pathology, many methodologies and technologies have been developed to determine the interaction events and parameters of interaction affinity. However, due to the nature of these methodologies, interactive partner needs to be purified, and large quantities of proteins are needed. This significantly limits quantitatively understanding of protein interaction and its application in therapeutics development. Recently, a quantitative FRET(qFRET) method was developed to determine purified protein-protein interaction affinity for purified proteins in vitro. Here, for the first time, we report the development of a high-throughput approach to determining the protein interaction affinity between two un-purified proteins using a quantitative FRET assay(qFRET). Two proteins, SUMO E2 conjugating enzyme, Ubc9, and RanGap1c, were first employed for the methodology development. We then determined the interaction affinities of Ubc9 and PIAS1 with influenza A virus NS1 protein without purifying two interactive partners. We found that both enzymes have high affinities to interaction affinity to the NS1, and this provides a kinetics explanation for the SUMOylation two-enzyme co-recognition mechanism to achieve high substrate specificity in vivo. The protein interaction affinity determination without purification provides several advantages over the current methods, including high accuracy, no tedious protein purification process, and high-throughput assay. The novel approach, so-called High-throughput determination of protein interaction affinity, KD, without purification using quantitative FRET assay, and scientific discoveries, representing a significant advancement in technology and scientific discovery, holds great potential for large-scale protein-protein interaction affinity determinations in vitro and in vivo. TeaserA novel qFRET technology for protein interaction affinity determination without protein purification for two-enzyme recognition.

bioengineering↗