Biology subjects

Mi, L.

Publications and source records attributed to Mi, L..

11 recordsLinked to original sources

bioRxiv2026

Programmable and Switchable RNA Scaffolds for Synthetic Condensate Engineering in Mammalian Cells

The ability to engineer synthetic biomolecular condensates in living cells offers new opportunities to control intracellular organization, yet robust and programmable RNA-based systems have remained limited. Here, we introduce genetically encoded, modular platforms that generate RNA-driven condensates using nanostar-derived scaffolds. Systematic comparison of repeat-based and de novo designs identified nanostar variants that reliably assemble nuclear condensates in mammalian cells. Unexpectedly, condensate formation in cells is governed primarily by double-stranded RNA stems that recruit endogenous RNA-binding proteins, rather than by the kissing-loop interactions that drive assembly in vitro. This mechanistic shift highlights the divergence between cellular and in vitro environments and accounts for the limited orthogonality among scaffolds. Sequence refinement to reduce nonspecific pairing improved homotypic assembly and enhanced orthogonality. We further demonstrated functional compartmentalization by recruiting protein and RNA clients to modulate their stability and activity, and we incorporated an acyclovir-responsive allosteric switch to achieve reversible, small-molecule control of condensation. Together, this work establishes a versatile RNA-based toolkit for constructing programmable cellular compartments, advancing strategies for controlling RNA-protein organization and enabling new biosensing and therapeutic applications.

bioengineering↗

bioRxiv2025

DPCMHC: efficient prediction of MHC-peptide binding affinity by deep learning based on dual-padding convolution

Predicting peptide binding affinities to major histocompatibility complex class I (MHCI) is a crucial challenge in immunological bioinformatics and is essential for identifying neoantigens in personalized cancer vaccines. Current deep learning methods often struggle, especially with 10-mer and 11-mer peptides. To address this, we developed DPCMHC, an advanced deep learning model featuring an embedding module, a dual-padding convolutional module, a BiLSTM module, and an output module. This design enhances the models understanding of amino acid sequences and their lengths. DPCMHC effectively captures the information on the beginning and end of amino acids, as well as the diverse sizes of adjacent amino acids. Using concatenation, the model extracts continuous sequence information. We rigorously evaluated DPCMHC on three benchmark datasets, demonstrating its superior or comparable performance to existing state-of-the-art methods. Our validation results confirm that DPCMHC is a robust and efficient tool for predicting MHC-peptide binding affinities.

bioinformatics↗

bioRxiv2025

A Population-scale Single-cell Spatial Transcriptomic Atlas of the Human Cortex

The genetic and spatial determinants of cell type diversity in human cerebral cortices remain poorly defined. Here, we present a population-level single-cell spatial transcriptomic atlas of human cortices from 71 donors across the lifespan. We identified 906 layer-specific genes showing conserved and divergent laminar expression patterns between humans and other species. Spatial analysis revealed neuronal vulnerability and glial activation during aging, together with a decline in the proportion of superficial SST neurons and their interactions with other cells. Disease-associated genes exhibited high cell-type and layer-specific expression, implicating the pathogenic role of spatially specific gene expression. Spatial cis-eQTL analysis identified regulatory variants linked to genes related to diseases like Tourette syndrome. Cross-species comparison demonstrated glial expansion in the human cortex, accompanied by enhanced neuron-glia communication via the neuregulin signaling. Together, we provide a comprehensive single-cell atlas of the human cortex that is essential for understanding aging, evolution, and disease pathogenesis.

neuroscience↗

bioRxiv2025

Longitudinal and large-scale monitoring of transcriptome and RBP-RNA interactome in living cells by engineered protein nanocages

Nondestructive sequencing of RNA from live cells is essential for monitoring and understanding dynamic biological processes. However, most existing RNA sequencing methods rely on cell lysis or fixation, limiting their applicability for longitudinal studies. Here, we introduce POND-seq (Protein nanocage-empOwered Non-Destructive sequencing), a novel approach that employs secretory protein nanocages fused with RNA-binding proteins (RBPs) to capture the RBP-RNA interactome and transcriptome in live cells. POND-seq reliably identifies RNA targets of canonical RBPs across multiple cell types. By fusing poly(A)-binding protein (PABPC1) to the nanocage, we demonstrate that POND-seq can monitor transcriptomic changes in response to signaling stimuli and selectively capture cell-type-specific transcriptomes from mixed populations. Additionally, POND-seq facilitates the dissection of RNA-binding domains and key amino acid residues critical for RBP-RNA interactions. We further highlight its utility in large-scale screening, offering compelling evidence for the pathogenicity of FMR1 variants. POND-seq represents a transformative advancement in RNA biology, cell biology and precision medicine, enabling unprecedented insights into cellular dynamics and disease mechanisms.

molecular biology↗

bioRxiv2024

A portable electrochemical DNA sensor for sensitive and tunable detection of piconewton-scale cellular forces

Cell-generated forces are a key player in cell biology, especially during cellular shape formation, migration, cancer development, and immune response. A new type of label-free smartphone-based electrochemical DNA sensor is developed here for cellular force measurement. When cells apply tension forces to the DNA sensors, the rapid rupture of DNA duplexes allows multiple redox reporters to reach the electrode and generate highly sensitive electrochemical signals. The sensitivity of these portable sensors can be further enhanced by incorporating a CRISPR-Cas12a system. Meanwhile, the threshold force values of these DNA-based sensors can be rationally tuned based on the force application geometries and also DNA intercalating agents. Overall, these highly sensitive, portable, cost-efficient, and easy-to-use electrochemical sensors can be powerful tools for detecting different cell-generated molecular forces. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/586508v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@e9bb33org.highwire.dtl.DTLVardef@10114d0org.highwire.dtl.DTLVardef@1fd925dorg.highwire.dtl.DTLVardef@942a20_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

SmartEM: machine-learning guided electron microscopy

Connectomics provides nanometer-resolution, synapse-level maps of neural circuits to understand brain activity and behavior. However, few researchers have access to the high-throughput electron microscopes necessary to generate enough data for whole brain or even whole circuit reconstruction. To date, machine-learning methods have been used after the collection of images by electron microscopy (EM) to accelerate and improve neuronal segmentation, synapse reconstruction and other data analysis. With the computational improvements in processing EM images, acquiring EM images has now become the rate-limiting step in automated connectomics. Here, in order to speed up EM imaging, we integrate machine-learning into real-time image acquisition in a single-beam scanning electron microscope. This SmartEM approach allows an electron microscope to perform intelligent, data-aware imaging of specimens. SmartEM saves time by allocating the proper imaging time for each region of interest - first scanning all pixels rapidly, then rescanning more slowly only the small subareas where a higher quality signal is required. We demonstrate that SmartEM achieves up to a ~7-fold acceleration of image acquisition time for connectomic samples using a commercial single-beam SEM in samples from nematodes, mice and human brain. We apply this fast imaging method to reconstruct a portion of mouse cerebral cortex with an accuracy comparable to traditional electron microscopy.

neuroscience↗

bioRxiv2023

Multiplexed Sequential Imaging in Living Cells with Orthogonal Fluorogenic RNA Aptamer/Dye Pairs

Single-cell detection of multiple target analytes is an important goal in cell biology. However, due to the spectral overlap of common fluorophores, multiplexed fluorescence imaging beyond two-to-three targets inside living cells remains a technical challenge. Herein, we introduce a multiplexed imaging strategy that enables live-cell target detection via sequential rounds of imaging-and-stripping process, which is named as "sequential Fluorogenic RNA Imaging-Enabled Sensor" (seqFRIES). In seqFRIES, multiple orthogonal fluorogenic RNA aptamers are genetically encoded inside cells, and then the corresponding cell membrane permeable dye molecules are added, imaged, and rapidly removed in consecutive detection cycles. As a proof-of-concept, we have identified in this study five in vitro orthogonal fluorogenic RNA aptamer/dye pairs (>10-fold higher fluorescence signals), four of which can be used for highly orthogonal and multiplexed imaging in living bacterial and mammalian cells. After further optimizing the cellular fluorescence activation and deactivation kinetics of these RNA/dye pairs, the whole four-color semi-quantitative seqFRIES process can now be completed in [~]20 min. Meanwhile, seqFRIES-mediated simultaneous detection of two critical signaling molecules, guanosine tetraphosphate and cyclic diguanylate, was also achieved within individual living cells. We expect our validation of this new seqFRIES concept here will facilitate the further development and potential broad usage of these orthogonal fluorogenic RNA/dye pairs for highly multiplexed and dynamic cellular imaging and cell biology studies.

bioengineering↗

bioRxiv2023

Targeted RNA Condensation in Living Cells via Genetically Encodable Triplet Repeat Tags

Living systems contain various functional membraneless organelles that can segregate selective proteins and RNAs via liquid-liquid phase separation. Inspired by nature, many synthetic compartments have been engineered in vitro and in living cells, mostly focused on protein-scaffolded systems. Herein, we introduce a nature-inspired genetically encoded RNA tag to program cellular condensate formations and recruit non-phase-transition target RNAs to achieve functional modulation. In our system, different lengths of CAG-repeat tags were tested as the self-assembled scaffold to drive multivalent condensate formation. Various selective target messenger RNAs and noncoding RNAs can be compartmentalized into these condensates. With the help of fluorogenic RNA aptamers, we have systematically studied the formation dynamics, spatial distributions, sizes, and densities of these cellular RNA condensates. The regulation functions of these CAG-repeat tags on the cellular RNA localization, lifetime, RNA-protein interactions, and gene expression have also been investigated. Considering the importance of RNA condensation in both health and disease conditions, these genetically encodable modular and self-assembled tags can be potentially widely used for chemical biology and synthetic biology studies.

bioengineering↗

bioRxiv2022

Genetically Encoded RNA-based Bioluminescence Resonance Energy Transfer (BRET) Sensors

RNA-based nanostructures and molecular devices have become popular for developing biosensors and genetic regulators. These programmable RNA nanodevices can be genetically encoded and modularly engineered to detect various cellular targets and then induce output signals, most often a fluorescence readout. Although powerful, the high reliance of fluorescence on the external excitation light raises concerns on its high background, photobleaching, and phototoxicity. Bioluminescence signals can be an ideal complementary readout for these genetically encoded RNA nanodevices. However, RNA-based real-time bioluminescent reporters have been rarely developed. In this study, we reported the first type of genetically encoded RNA-based bioluminescence resonance energy transfer (BRET) sensors that can be used for real-time target detection in living cells. By coupling a luciferase bioluminescence donor with a fluorogenic RNA-based acceptor, our BRET system can be modularly designed to image and detect various cellular analytes. We expect this novel RNA-based bioluminescent system can be potentially used broadly in bioanalysis and nanomedicine for engineering biosensors, characterizing cellular RNA-protein interactions, as well as high-throughput screening or in vivo imaging.

bioengineering↗

bioRxiv2021

Genetic diversity, evolutionary dynamics and pathogenicity of ferret badger rabies virus variants in mainland China, 2008-2018

In contrast to dog associated human rabies cases decline year by year due to the rabies vaccination coverage rates increase in China, ferret badger (FB, Melogale moschata)-associated human rabies cases emerged in the 1990s, and are now an increasingly recognized problem in southeast China. To investigate epidemiology, temporal evolution dynamics, transmission characterization and pathogenicity of FB-associated rabies viruses (RABVs), from 2008 to 2018, we collected 3,622 FB brain samples in Jiangxi and Zhejiang Province, and detected 112 RABV isolates. Four FB-related lineages were identified by phylogenetic analysis (lineages A-D), the estimated Times to Most Recent Common Ancestor were 1941, 1990, 1937 and 1997 for lineages A-D respectively. Furthermore, although no FB-associated human rabies case has been reported there apart from Wuyuan area, FB-RABV isolates are mainly distributed in Jiangxi Province. Pathogenicity of FB-RABVs was assessed using peripheral inoculation in mice and in beagles with masseter muscles, mortality-rates ranging from 20% to 100% in mice and 0 to 20% in beagles in the groups infected with the various isolates. Screening of sera from humans with FB bites and no postexposure prophylaxis to rabies, revealed that 5 of 9 were positive for neutralizing antibodies of RABV. All the results above indicated that FB-RABV variants caused a lesser pathogenicity in mice, beagles and even humans. Vaccination in mice suggests that inactivated vaccine or recombinant subunit vaccine products can be used to control FB- associated rabies, however, oral vaccines for stray dogs and wildlife need to be developed and licensed in China urgently. IMPORTANCEIn recent years FB-associated rabies virus has been identified as a major life-threatening pathogen in some districts in China. To understand the risk to public health and the contemporary dynamics, the present study conducted extensive investigations on FB rabies, distribution, virus isolation, phylogeny analysis, pathogenicity determination of various FB rabies virus strains, besides, serologic epidemiology survey to those whom bit by FB was also collected. The results show that the majority of FBs dwell in southeast China, like Jiangxi and Zhejiang Province, Phylogenetic analysis indicates that all isolated FB RABVs evolved from dogs, and the FB RABV can separate into 4 distinct lineages distributed relatively independent in different areas. The isolate strains differ in pathogenicity, although they have relatively lower pathogenicity compared to dog rabies virus according to our study, the need for further study to licensed oral vaccines and FB RABV pathogenesis is emphasized in order to control rabies.

pathology↗

bioRxiv2018

Is it actionable? An Evaluation of the Rapid PCR-Based Blood Culture Identification Panel on the Management of Gram-Positive and Gram-Negative Blood Stream Infection

BackgroundThere is growing interest in the use of rapid blood culture identification (BCID) panels in antimicrobial stewardship programs (ASP). While many studies have looked at its clinical and economic utility, its comparative utility in gram-positive and gram-negative blood stream infections (BSI) have not been as well characterized.\n\nMethodsThe study was a quasi-experimental retrospective study at the Mayo Clinic in Phoenix, Arizona. All adult patients with positive blood cultures before BCID implementation (June 2015 to December 2015) and after BCID implementation (June 2016 to December 2016) were included. The outcomes of interest included: time to first appropriate antibiotic escalation, time to first appropriate antibiotic de-escalation, time to organism identification, LOS, infectious disease consultation, discharge disposition, and in-hospital mortality.\n\nResultsIn total, 203 patients were included in this study. There was a significant difference in the time to organism identification between pre- and post-BCID cohorts (27.1h vs. 3.3h, p<0.0001). BCID did not significantly reduce the time to first appropriate antimicrobial escalation or de-escalation for either GP-BSIs or GN-BSIs. Providers were more likely to escalate antimicrobial therapy in GP-BSIs after gram stain and more likely to de-escalate therapy in GN-BSIs after susceptibilities. While there were no significant differences in changes in antimicrobial therapy after organism identification by BCID, over a quarter of providers (28.1%) made changes after organism identification.\n\nConclusionsWhile BCID significantly reduced the time to identification for both GP-BSIs and GN-BSIs, BCID did not reduce the time to first appropriate antimicrobial escalation and de-escalation.

microbiology↗