Biology subjects

Qiu, H.

Publications and source records attributed to Qiu, H..

At least 19 recordsLinked to original sources

bioRxiv2026

Intrinsic Ionic Mechanisms Underlying Burst Firing in Spontaneously Active Dorsal Horn Parvalbumin Interneurons

Parvalbumin-expressing interneurons (PVINs) in the spinal dorsal horn play a key role in preventing touch inputs from engaging nociceptive pathways through fast inhibitory control. Although most PVINs are typically quiescent and require external input to fire, a subset of these neurons exhibits spontaneous activity, including isolated spikes and bursts. The mechanisms underlying this behavior in spontaneously active PVINs (spPVINs) remain unclear. To address this, we developed a stochastic two-compartment Hodgkin-Huxley (HH) type model, consisting of a soma and an axon initial segment (AIS), to investigate the effects of synaptic noise and intrinsic electrical properties of spPVINs in driving their spontaneous firing. The model incorporates two subthreshold currents: the M-type K+ current (Im) and the hyperpolarization-activated current (Ih). The model revealed that, in the presence of Ornstein-Uhlenbeck noise, Ih promotes spontaneous firing by enhancing noise-driven depolarizations. Model simulations closely reproduced the firing patterns of spPVINs observed experimentally, including irregular spiking and bursting. Bifurcation analysis showed that varying the applied current can lead to transitions between quiescent, tonic, and elliptic bursting regimes, allowing stochastic fluctuations to switch between these states, which gives rise to irregular spontaneous activity. Reducing the conductance of Im and increasing the conductance of the fast Na+ current (INa) both enlarge the elliptic bursting regime, thereby enabling noise to promote bursting. Interestingly, when the model was incorporated into a circuit, the downstream inhibitory effects of spPVINs were enhanced by the expression of Ih. Taken together, these results demonstrate how intrinsic electrical properties of spPVINs shape their firing dynamics and circuit activity.

neuroscience↗

bioRxiv2026

Mistrafficking of KCC2 promotes hyperexcitability in hippocampal circuitry in a murine model of Christianson Syndrome

How endosomal trafficking shapes inhibitory neurotransmission remains poorly understood, despite both processes being linked independently to epilepsy and neurodevelopmental disease. Christianson syndrome (CS), an X-linked neurodevelopmental disorder, represents a potential tractable condition to investigate such coupled processes. CS is caused by loss-of-function mutations in the SLC9A6 gene which encodes the organellar (Na+, K+)/H+ exchanger NHE6 isoform whose loss overacidifies recycling endosomes and disrupts cargo delivery. Prior work has emphasized NHE6's role in excitatory neurons and glia; whether endosomal dysfunction reshapes inhibitory circuits and thereby drives the early treatment-resistant epilepsy that defines CS has not been addressed. Using Nhe6-/Y mice, we identify a previously unrecognized endosomal-to-inhibitory axis: NHE6 is required for the surface delivery and stability of the neuron-specific potassium-chloride co-transporter KCC2 (encoded by SLC12A5) that sets the driving force for GABAergic inhibition. Loss of NHE6 produces developmental downregulation and mistargeting of KCC2 in hippocampal neurons, impaired chloride homeostasis, and circuit-level hyperexcitability in response to a subthreshold convulsant challenge. These results recast CS epilepsy not as a downstream consequence of excitatory dysfunction but as a primary failure of inhibitory development driven by endosomal mistrafficking. More broadly, they establish endosomal pH regulation as a determinant of KCC2 biology; a node implicated across genetic and acquired epilepsies. Pharmacologically restoring KCC2 function therefore offers a mechanism-based therapeutic strategy for CS, and likely for the broader class of disorders in which endosomal trafficking and inhibitory imbalance converge.

neuroscience↗

bioRxiv2026

Peripheral nerve injury reallocates primary afferent input through spinal parvalbumin microcircuits

Complex neural functions rely on finely tuned circuits in which the recruitment of local interneurons gates the flow of information, determining whether an input is relayed, amplified, or suppressed. Somatosensory information, such as touch or pain, is processed through such complex circuits in the dorsal horn of the spinal cord. There, local inhibitory interneurons play a key role in the proper segregation of touch and pain inputs. After nerve injury-evoked neuropathic pain, loss of inhibition impairs the function of these circuits, resulting in mechanical allodynia, where innocuous touch is perceived as painful. Disinhibition can be attributed to pruning of inhibitory synapses, reduced intrinsic excitability of inhibitory neurons, or weakened excitatory drive from primary afferents. Yet the complexity of the excitatory drive onto inhibitory neurons, and its potential modification after nerve injury, remains largely unexplored. Here we examined the nature of the synaptic drive from low-threshold A{beta} mechanoreceptors (A{beta}-LTMRs) onto parvalbumin-expressing interneurons (PVNs), and how this recruitment is affected by nerve injury. A{beta}-LTMR stimulation evoked excitatory responses in a subset of PVNs, which is expected given the exclusively excitatory nature of primary afferent inputs. However, an unexpected subset of PVNs displayed inhibitory responses, suggesting the recruitment of a feedforward inhibitory circuit. We reconciled these observations by showing that A{beta}-LTMRs engaged PVNs through both direct excitation and feedforward inhibition, which are differentially distributed between the inhibitory (iPVN) and excitatory (ePVN) subpopulation. Indeed, our results show that under naive conditions, A{beta} input preferentially recruited iPVNs, while ePVNs were predominantly suppressed by A{beta}-driven feedforward inhibition mediated by a previously unrecognized Complexin-1 (Cplx1)-expressing inhibitory interneuron. After peripheral nerve injury, this balance becomes functionally redistributed. A{beta}-to-iPVN transmission showed increased failure rates and impaired temporal precision, whereas A{beta} drive onto ePVNs shifted from inhibition toward excitation. Notably, these functional changes occurred despite preserved afferent connectivity, synapse density, and spontaneous synaptic events, indicating that the dynamic reallocation of circuit recruitment occurs in the absence of structural changes. To test the behavioral consequences of these two populations, we used chemogenetic approaches and found that iPVNs suppress, whereas ePVNs promote, mechanical hypersensitivity. Together, these findings show that nerve injury functionally reallocates primary afferent drive away from inhibitory and toward excitatory spinal PVNs, establishing functional reallocation of afferent input as a mechanism of spinal disinhibition and a key determinant of mechanical allodynia.

neuroscience↗

bioRxiv2026

Astrocytic FMRP regulates the function of spinal parvalbumin-expressing neurons in Fragile X Syndrome

Tactile hypersensitivity is a common symptom of Fragile X Syndrome (FXS) characterized by over-responsiveness to innocuous touch or textures. Yet, the neural circuitry underlying this altered sensory processing remains incompletely understood. Previous studies have focused on cortical and peripheral neuron dysfunction. However, the spinal dorsal horn circuits, which make up the first central site of somatosensory integration, also comprises elements that could contribute to the hypersensitivity to innocuous stimuli, yet its involvement in this pathology has remained incompletely understood. Here, we show that spinal parvalbumin (PV)-expressing interneurons (PVNs), an inhibitory population that gates touch sensory input from activating nociceptive pathways, are dysfunctional in FXS. Using a mouse model of FXS (global Fmr1 knockout; gKO), we showed that dorsal horn PVNs exhibit impaired function characterized by reduced PV expression and inability to sustain tonic firing. To determine whether these deficits arise through cell-intrinsic mechanisms, we selectively deleted Fmr1 in PVNs. This deletion failed to reproduce the molecular and electrophysiological changes observed in the gKO mice. In contrast, astrocyte-specific deletion of Fmr1 recapitulated key features of the gKO mice, including decreased PV expression and firing. To understand the biophysical basis of this firing deficit, we simulated spinal PVNs using conductance-based Hodgkin-Huxley type modelling. Surprisingly, modifying intrinsic membrane conductances alone was insufficient to account for the experimental data. In fact, PVN firing required incorporation of an additional calcium-dependent extrinsic synaptic component consistent with the experimental finding that astrocytic, but not PVN-specific, loss of FMRP reproduced the PVN phenotype in gKO mice. Together, these findings show that these deficits in spinal PVNs arise primarily from loss of FMRP in astrocytes rather than in PVNs themselves. It reveals astrocyte-dependent dysfunction of dorsal horn inhibitory circuits as a previously uncharacterized consequence of FXS.

neuroscience↗

bioRxiv2026

Lineage Memory and Convergent Adaptation Dictate the Single-Cell and Spatial Architecture of Brain Malignancies

How diverse cancer lineages navigate the draconian central nervous system environment--whether constrained by ancestral ontogeny or driven by convergent adaptation--remains a fundamental biological paradox. To decode this, we integrated extensive in-house and public resources to construct the largest single-cell atlas of primary and secondary brain malignancies (>550,000 cells). We demonstrate that malignant cells balance strict lineage imprinting with shared brain-adaptive programs, powerfully converging upon a dominant pan-cancer mesenchymal (MES)-like state alongside a SYT1+ pioneer subpopulation exploiting neuronal mimicry. Concurrently, the microenvironment undergoes lineage-constrained divergence. The myeloid landscape shifts from a resident-dominated architecture in gliomas to extensive blood-borne infiltration in metastases. Lymphoid responses parallel this, crowning laryngocarcinoma as an ultra-hot subtype and revealing that massive T cell influx in highly infiltrated niches is paradoxically driven into terminal exhaustion, contrasting with severe immune exclusion in cold tumors. Furthermore, stromal-vascular adaptation couples pro-angiogenic tip-endothelial activation with a dynamic opposition between structural extracellular matrix (ECM) deposition and focal proteolytic degradation. Anchored by the pan-cancer MES adaptation, we leveraged subcellular spatial transcriptomics to redefine the microenvironment around four MES-like architectural niches, mapping an evolutionary trajectory from perivascular entry to immunosuppressive stromal remodeling. Finally, projecting these architectures onto independent clinical cohort establishes the MES-S2 proliferative niche as the primary driver of severe clinical deterioration across both transcriptomic and proteomic dimensions.

Cancer Biology↗

bioRxiv2026

Coordinated immune-epithelial dynamics in the nasal epithelium protect against respiratory virus infection

Epithelial remodeling is a hallmark of host antiviral responses that strengthen barrier defenses against infection. Our current understanding of viral pathophysiology within the nasal epithelium, the initial site of most respiratory viral infections, remains incomplete due to limited understanding of the native tissue architecture and protective epithelial remodeling during immune events. Leveraging coronavirus disease 2019 (COVID-19) as a model, we applied spatial multi-omics on nasal cross-sectional tissues to characterize the immune-epithelial landscape during infection, identifying a coordinated increase in goblet cells and suppressive macrophages associated with elevated IL13 in the immune compartment. Our results further reveal that goblet cell and suppressive macrophage enrichment are spatially linked in proximity to IL13-expressing CD4 T cells, consistent with IL13-driven remodeling in situ. Using a primary human nasal air-liquid interface model, we demonstrate that IL13 alone is sufficient to remodel epithelial composition and morphology, subsequently restricting viral infection by reshaping the apical mucus barrier of the nasal epithelium. Our findings uncover a spatially organized, IL13-driven circuit for immune-epithelial remodeling as a protective barrier against respiratory viral infections.

immunology↗

bioRxiv2026

Genome-scale functional mapping of the mammalian whole brain with in vivo Perturb-seq

Functional genomics studies have provided critical insights into cell type-specific gene regulatory programs, but to date most have been conducted in wild-type tissues or cell cultures. Here, we present a gene expression functional atlas across the mouse brain. We use an enhanced in vivo Perturb-seq platform to analyze transcriptome-wide responses to loss of 1,947 disease-associated genes, profiling over 7.7 million cells spanning major brain regions and neuronal populations. We find striking cell-type-specific essentiality and transcriptional programs and show that closely related disease genes such as two NMDA receptor subunits can drive opposing transcriptional programs. Together, this work reveals insights into the genetics and mechanisms of neurodevelopmental, psychiatric, and neurodegenerative diseases in vivo, paving the way for the design of future genetic medicine.

genomics↗

bioRxiv2026

In vivo multiomic Perturb-seq with enhanced nuclear gRNA capture

In vivo CRISPR screening with joint transcriptomic and chromatin readouts has been limited by inefficient recovery of gRNAs from nuclei. Here, we developed in vivo multiomic Perturb-seq, an effective platform combining nuclear transcript anchoring with gRNA-specific capture and amplification to enable high-fidelity, high-recovery gRNA assignment and scalable perturbation-resolved single-nucleus multiomics. Applying this platform to interrogate neurodevelopmental disorder risk genes in the developing cortex reveals cell-type-specific transcriptomic and epigenomic perturbation phenotypes.

genomics↗

bioRxiv2026

Single-Cell and Spatial Methods for Multimodal Functional Glycan Profiling in Tissues

Glycans regulate multiple physiological processes, including immune recognition and cancer progression. In disease, altered glycan landscapes are interpreted by human lectins. Functional glycan-lectin interactions are difficult to profile because glycans are not genome-encoded and their changes are poorly captured by existing multimodal methods. We present two platforms, single-cell outlining and transcriptome sequencing (scGOAT-seq) and GlycoScope, which use human lectins to enable functional glycan accessibility into single-cell and spatial multiomic measurements. ScGOAT-seq quantifies lectin-accessible glycan states with gene expression, while GlycoScope enables multiplexed in situ co-detection of glycans and proteins in tissues. Applying these approaches to immune cells, we identify stimulus-specific glycan remodeling and show that distinct Siglec-ligand-defined programs stratify immune activation states not captured by traditional methods; in follicular lymphoma, GlycoScope, resolves spatial glycan programs associated with malignant B cells and localized immune microenvironments. The presented methods provide a general framework for integrating functional glycan accessibility into single-cell and spatial multiomics.

molecular biology↗

bioRxiv2026

Pharmacological Inhibition of SLC33A1 Promotes Endoplasmic Reticulum Hyperoxidation and Induces Adaptive IRE1/XBP1s Signaling

The endoplasmic reticulum (ER) transporter solute carrier family 33 member 1 (SLC33A1) has emerged as an attractive therapeutic target in etiologically diverse diseases, ranging from lung cancer to neurodegenerative disorders. Yet, no pharmacologic SLC33A1 modulators have been described. Here, we show that the small molecule IXA4, a highly selective activator of the adaptive IRE1/XBP1s signaling arm of the unfolded protein response (UPR), binds to SLC33A1 and inhibits its activity. Genetic depletion of SLC33A1 phenocopies the selective induction of IRE1/XBP1s signaling brought about by IXA4 treatment. Chemoproteomic analyses and cryo-electron microscopy show that IXA4 binds SLC33A1 within the central channel to inhibit transport of its substrate metabolite(s). Binding of IXA4 to SLC33A1 leads to the accumulation of oxidized glutathione within the ER, hyperoxidizing the ER lumen and inducing activation of adaptive IRE1/XBP1s signaling. Consistent with this function, we find that pharmacologic inhibition of SLC33A1 with IXA4 selectively reduces viability of KEAP1-deficient lung adenocarcinoma cells that have elevated levels of glutathione, mimicking the sensitivity of these cells to genetic deletion of SLC33A1. Our work demonstrates a new physiologic role of SLC33A1 in regulation of ER redox homeostasis and designates IXA4 as a pharmacologic inhibitor of SLC33A1 that can be used to evaluate the biological impact and therapeutic utility of SLC33A1 inhibition in homeostasis and in disease.

cell biology↗

bioRxiv2026

Development of KASP Molecular Markers and Construction of Fingerprinting for Cowpea (Vigna unguiculata (L.) Walp) unguiculata Based on ddRAD-Seq

Cowpea (Vigna unguiculata (L.) Walp.) is a globally important legume crop. However, the scarcity of efficient molecular markers has hindered molecular breeding efforts and the protection of plant breeders rights. In this study, we employed double-digest restriction-site associated DNA sequencing (ddRAD-seq) to characterize the genetic diversity of 19 core cowpea germplasm accessions. We generated 791,621 SNPs, from which 13,469 high-quality SNPs were filtered. Population structure and phylogenetic analyses revealed that these accessions cluster into three distinct groups. To facilitate cost-effective and rapid genotyping, we developed a panel of KASP (Kompetitive Allele Specific PCR) markers. Through rigorous screening for polymorphism and stability, we identified six core KASP markers located in exonic regions. These six markers alone were sufficient to discriminate all 19 accessions. Based on these core markers, we constructed a unique DNA fingerprinting profile and assigned specific QR codes for each accession. This study demonstrates that extracting core KASP markers from ddRAD-seq data is a powerful strategy for germplasm identification. The developed fingerprinting system provides a robust, low-cost tool for seed purity testing, variety authentication, and marker-assisted selection in cowpea breeding programs.

plant biology↗

bioRxiv2026

Elimination of intramuscular immunoglobulin accumulation alleviates Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disorder due to loss of dystrophin, a cytoskeletal protein critical for muscle integrity and functionality. Despite recent therapeutic advances, there remains a significant unmet need for more effective and accessible therapeutics. Here, we discovered an early accumulation of immunoglobulin G (IgG) in the sarcolemma, which exacerbates tissue inflammation and disease progression. The IgG accumulation primarily resulted from ectopic localization of Fc{gamma}R1, a high-affinity IgG Fc receptor, on dystrophin-deficient myofibers. In two independent murine models, eliminating IgG accumulation via B cell depletion provided sustained benefit to alleviate DMD progression. Our findings uncover a novel disease accelerator in DMD and demonstrate the potential to target this mechanism as therapeutics for broader population of patients with DMD.

immunology↗

bioRxiv2026

BiOmics: A Foundational Agent for Grounded and Autonomous Multi-omics Interpretation

While AI has automated bioinformatic workflows, biological interpretation remains fragmented and often disconnected from mechanistic insights. Existing AI is bifurcated between statistical "black-box" models that lack logical grounding and simple agents restricted to shallow knowledge retrieval. To bridge this divide, we introduce BiOmics, a foundational agent that synthesizes multi-omics data with adaptive knowledge for biological interpretation. BiOmics introduces a novel dual-track architecture comprising a harmonized explicit reasoning space for grounded logic and a unified latent embedding space for high-dimensional association mapping. This architecture enables a transformative "Retrieving-Reasoning-Predicting" paradigm for purposeful, cross-scale inference traversing the biological hierarchy, from molecular variants to disease phenotypes. Empirical evaluations demonstrate that BiOmics surpasses state-of-the-art AI agents and specialized algorithms, markedly augmenting the granularity and depth of biological insights. Specifically, BiOmics exhibits unique superiority in uncovering indirect pathogenic variants, achieving reference-free cell annotation, and prioritizing drug repurposing candidates tailored to specific datasets. BiOmics further enriches the interpretive landscape of biological entities, leveraging its reasoning-grounded knowledge graph to uncover deep functional contexts. Ultimately, BiOmics provides a versatile engineering foundation to transition AI for Science from descriptive "data fitting" to autonomous, knowledge-driven interpretation.

bioinformatics↗

bioRxiv2025

A changing hierarchy of environmental time cues guide development of the migratory pest moth Loxostege sticticalis

Insects use environmental cycling cues to align developmental progression. In holometabolous insects, the progression from larva to adulthood entails decision points, i.e., larval diapause and adult eclosion, that engage circadian timing mechanisms. What environmental time cues dynamically interact to regulate these developmental checkpoints lying at the intersection of the orthogonal timing systems, remains elusive. We used the seasonally migratory insect Loxostege sticticalis to disentangle the influence of photoperiod regimes and temperature conditions at distinct checkpoints of larval growth and metamorphosis. We here employed sensory conflict paradigms to reveal that the relative dominance of these cues changes over ontogeny. Our results confirm that diapause is induced primarily by the photoperiod during early larval development, rather than by the diel thermoperiod. In contrast, the circadian clock-controlled adult eclosion, normally locked to the first hour of the daybreak, is jointly controlled by light and temperature cycles. An increase in the constant temperature advanced the peak timing of daily eclosion. On the other hand, the interval timing mechanism regulating the duration of the eclosion event is tuned specifically by the night temperatures. Together, photoperiod dominates early developmental decisions, but thermoperiod gains weight at later stages. This shifting hierarchy provides a mechanistic framework for understanding phenological plasticity. Since the timing of eclosion presents a window of increased vulnerability for this pest insect, our results could help to optimize the daily timing of pest management intervention under variable thermal and light conditions.

ecology↗

bioRxiv2025

Cutting Through the Artifacts: Dissecting gRNA Impurities with FUSS-seq

CRISPR-based therapeutics rely on guide RNAs (gRNAs) and the Cas9 endonuclease for precise gene editing. Ensuring gRNA purity and base-level sequence integrity is essential for clinical translation. While industry-standard practice relies on liquid chromatography-high-resolution mass spectrometry to assess oligonucleotide identity and purity, more recent FDA guidance recommends complementary base-by-base sequence analysis (FDA CBER Webinar, 2024). In this study, we evaluated next-generation sequencing (NGS) strategies for characterizing chemically synthesized gRNAs. We found that the widely used SMARTer assay, while capable of producing sequenceable libraries, introduced substantial artifacts during library preparation. These included truncated scaffold species at oligo(A) stretches in the scaffold region and 5'(n-1) deletions within the spacer sequence. Although absent in the original gRNA, these artifacts accounted for over 10% of the sequencing reads, creating the false appearance of impurities. Through experimental and computational approaches, we traced these artifacts to mispriming by template-switching oligonucleotides (TSOs). Importantly, these artifacts occur during sequencing, and although they do not reflect real gRNA impurities, they compromise assay accuracy and can obscure true sequence impurities. To overcome these limitations, we developed FUSS-seq (Full-length Uncoupled Second-strand Synthesis followed by sequencing), a novel assay that integrates principles from 5' RACE with a modified TSO bearing a 3' polymerase-blocking moiety. FUSS-seq markedly reduced artifacts and increased full-length gRNA recovery, providing a more accurate and lower-bias method for gRNA purity assessment. This approach supports improved Chemistry Manufacturing and Controls (CMC) characterization of gRNAs and strengthens the analytical toolkit needed for reliable CRISPR-based therapeutic development.

molecular biology↗

bioRxiv2025

Integrating Human Genetics and Protective Genome Editing to Enable ADGRE2-Directed AML Therapy

Acute myeloid leukemia (AML) remains a major therapeutic challenge due to extensive disease heterogeneity and lack of cancer-specific antigens. ADGRE2 has emerged as a promising AML target with broad expression in AML patient blast and leukemic stem cell-enriched populations. However, comparable expression in healthy hematopoietic stem and progenitor cells (HSPCs) and myeloid lineages suggests a high susceptibility to on-target, off-tumor myelotoxicity with ADGRE2-targeted therapies. Guided by human genetics data identifying loss-of-function variants, we evaluated whether ADGRE2 is dispensable in hematopoietic stem cells as a protective approach for transplant-based shielding from ADGRE2-directed therapies. Using CRISPR-Cas9 and adenine base editors, we achieved high-efficiency ADGRE2 knockout (>94%) in HSPCs with corresponding protein loss without impairing cell viability, differentiation, and cytokine release in vitro, or long-term engraftment, multilineage differentiation, and persistence of gene editing in mouse xenografts. We also developed novel ADGRE2-specific chimeric antigen receptor (CAR) T cells that demonstrated potent cytotoxicity against AML cells, even at low antigen levels. Together, these findings establish ADGRE2 as a compelling AML target and provide a framework for hematopoietic stem cell transplant with protective gene editing to enable ADGRE2-directed immunotherapies while minimizing myelotoxicity.

cancer biology↗

bioRxiv2025

SANTON: Sequencing Analysis Toolkits for Off-target Nomination

BackgroundGenome-wide off-target nomination and screening sequencing methods, such as synthetic oligonucleotide-based sequencing and whole genome-based sequencing, evaluate the precision and safety of gene-editing technologies. However, there remains a lack of comprehensive bioinformatics tools for analyzing the sequencing data generated by off-target nomination assays for various gene editors, including Cas9, Cas12a, and base editors. ResultsWe introduce a Sequencing Analysis Toolkits for Off-target Nomination (SANTON) for the identification and quantification of potential off-target sites using datasets generated from synthetic oligonucleotide-based sequencing and whole genome-based sequencing (e.g., Digenome-seq) methods. By applying SANTON to a Cas9 treated oligo synthesization-based sequencing data, a comprehensive set of potential off-target sites are evaluated in which the top off-target sites displayed highly constituency with a published study using in vivo method (e.g, GUIDE-seq). Utilizing SANTON to previously published Digenome-seq datasets, we identified more potential off-target regions than previous studies, including ones showing significantly higher cleavage level than the on-target site. To the best of our knowledge, SANTON is the first public package capable of analyzing synthetic oligonucleotide-based sequencing data and the first to specific optimized for the analysis of whole genome-based sequencing analysis for Cas12a and base editors. We demonstrated the capability of SANTON to effectively analyze complex cleavage patterns by a diverse editing system. ConclusionsSANTON provides a powerful computational solution to support genome editing research, facilitating more reliable and comprehensive off-target profiling.

bioinformatics↗

bioRxiv2025

A Mechanism Based Pharmacokinetic/Pharmacodynamic Analysis of Polymyxin B-Based Combination Therapy Against Carbapenem-Resistant Klebsiella pneumoniae Isolates with Diverse Phenotypic and Genotypic Resistance Mechanisms

Increased resistance to {beta}-lactams/{beta}-lactamase inhibitor by mutations in {beta}-lactamases gene, porin mutations and efflux pumps complicates the management of carbapenem-resistant Klebsiella pneumoniae (CRKP). Polymyxin B (PMB) based combination therapy is considered as a best alternative treatment for those middle and low-income countries that cannot access to the latest medicines. Its crucial to know both phenotypic and genotypic characteristics of a pathogen to understand the killing effect of each drug and its combinations. Hence, our objective of this study was to incorporate mechanistic insights gained from resistance mechanisms to develop a mechanism based pharmacokinetic/pharmacodynamic model (MBM). Six clinical CRKP isolates were used for static concentration time kill (SCTK) assays to evaluate the rate and extent of killing by monotherapy, double and triple combinations using PMB, meropenem and fosfomycin. A MBM was developed using the SCTK data in S-ADAPT. The MBM estimated lower maximum killing rate constant of PMB (3.61 h-{superscript 1}) in an isolate with non-functional MgrB and high-level phenotypic resistance. Based upon model discrimination and PMBs outer membrane disruption, mechanistic synergy was included in 3 isolates which has porin mutations. Mechanistic synergy of PMB was 83-88% with meropenem and 81-98% with fosfomycin. The PMB concentration required to achieve 50% of synergy was 0.48-0.64 mg/L. Simulations with a lower PMB regimen (1mg/kg q12h) and fosfomycin (8g q8h) showed >73% reduction in area under the bacterial load-versus-time curve for four isolates. The triple combination showed 67.7% reduction in non-carbapenamase producing isolate. This study demonstrates that a low dosing regimen of PMB can produce synergistic effects in combination therapy and might be effective in managing infections caused by CRKP, including PMB resistant isolates. Author summaryAntimicrobial resistance is a major concern in treating infectious diseases. CRKP bacterial isolates are resistant to most of the novel antimicrobial agents. One of the primary resistant mechanisms is restricting the permeability of drugs to the site of action. Polymyxin B, an antimicrobial agent, disrupts the bacterial outer membrane, enhancing the permeability of other drugs. When using combination therapy with polymyxin B, selecting drugs with different mechanisms of action is crucial to enhance synergistic effects and improve treatment efficacy. In our study we chose to evaluate the efficacy of meropenem and fosfomycin in double and triple combination therapies. Mechanism based models (MBMs) are the strongest tool to analyse the time course bacterial load data. Our study provides insights into applying available phenotypic and genotypic information to refine and enhance the accuracy of MBMs. Final model simulations revealed that low exposure of polymyxin B below the nephrotoxic threshold was sufficient to produce synergistic effects when combined with fosfomycin and meropenem. Additionally, we found that polymyxin B combination with fosfomycin was more effective compared to meropenem in treating CRKP.

pharmacology and toxicology↗