Biology subjects

Peng, R.

Publications and source records attributed to Peng, R..

At least 19 recordsLinked to original sources

bioRxiv2026

Combining an autoactivated cGAS mutant with membrane-tethered IL-12 via mRNA lipid nanoparticles promotes systemic tumor control

Local tumor therapies rarely induce systemic immunity capable of controlling metastatic disease. Here, we engineer an mRNA lipid nanoparticle (LNP) platform to coordinate innate immune sensing and T cell activation in situ through co-delivery of an autoactivated cyclic GMP-AMP synthase mutant (cGASR241E) and membrane-tethered IL-12 (IL-12TM). cGASR241E mRNA LNPs autonomously generated cyclic GMP-AMP in vivo, activating host STING signaling and initiating antitumor responses. Co-administration with IL-12TM mRNA LNPs promoted localized cytokine production, reduced regulatory T cells, and restored effector function in tumor-infiltrating T cells. This combination elicited systemic antitumor immunity and suppressed distal tumors across multiple mouse models. The therapeutic efficacy required CD8+ T cells and IFNg; signaling in host, supporting a model in which innate DNA sensing is coupled to IL-12-mediated T cell functional reinforcement. In addition, the combination enhanced responsiveness to PD-L1 blockade. Together, these findings establish a clinically translatable strategy to integrate cGAS-STING activation with cytokine-driven T cell programming to drive systemic tumor control.

cancer biology↗

bioRxiv2026

Biologically grounded cell profiling across microscopy modalities

Microscopy-based cell profiling has broad applications in biological discovery, disease characterization, and phenotypic drug screening. Modern microscopy continues to push the limits of resolution, speed, depth and throughput, but better imaging does not automatically lead to better biomedical discovery and translation. A key bottleneck is feature representation: existing features are either handcrafted or learned as black-box embeddings and often lack explicit biological meaning. Here we propose biological grounding as a first principle for cell profiling and implement it through MorphAgent, an AI agent framework in which each quantitative feature originates from a biological hypothesis and is anchored to a cellular structure or process. By integrating biological knowledge, multimodal reasoning and automated validation, MorphAgent makes biologically grounded feature design systematic and scalable. We show biological grounding fundamentally changes the properties of cell profiling features. Across three microscopy modalities, MorphAgent produces compact, expressive and transferable features for drug screening, cell state characterization and disease morphology analysis. In wide-field Cell Painting, a common assay for phenotypic drug screening, it improves perturbation-retrieval mean average precision by 48% over CellProfiler and 20% over DeepProfiler while using substantially fewer dimensions. In confocal mitochondrial imaging, biologically grounded features transfer across independently acquired datasets and imaging resolutions, supporting accurate aging-state classification. In structured illumination microscopy (SIM) imaging of Tau-labeled samples, biologically grounded super resolution features reveal nanoscale morphologies inaccessible at conventional resolution, enhancing the discrimination of disease-associated mutations. Moreover, biologically grounded features provide a hierarchical organization of cellular morphology, enabling multilevel alignment with transcriptomics data and facilitating mechanistic understanding. Biologically grounded cell profiling thus brings AI reasoning closer to biological mechanisms, advancing biomedical discovery and translation.

bioinformatics↗

bioRxiv2026

miRstring: An RNA language model enables mature miRNA decoding and artificial small RNA design across species

MicroRNAs (miRNAs) are processed from structured precursors and subsequently loaded into Argonaute proteins to repress target mRNAs. Yet generalized computational frameworks capable of decoding mature miRNAs from precursor context remain limited, constraining both cross-species annotation and rational artificial miRNA design. Here, we present miRstring, a biogenesis-aware RNA language framework that decodes the four boundaries defining the miRNA/miRNA duplex. Trained on 77,708 miRNA precursors spanning 414 species, miRstring outperforms existing methods under family- and species-held-out evaluations and accurately identifies the first nucleotide of mature miRNAs. Importantly, its attention mechanism highlights the miRNA/ miRNA* boundary sites cleaved by endonucleases, indicating that the model captures biologically meaningful features. Furthermore, we employed miRstring to design optimal pre-miRNA scaffolds for artificial miRNAs and validated its efficacy in repressing target mRNAs. Taken together, miRstring establishes a scalable route from cross-species mature-miRNA annotation to predictive design, enabling artificial intelligence-driven miRNA engineering and extending computational miRNA analysis toward broadly applicable small-RNA biotechnology.

bioinformatics↗

bioRxiv2026

Ultrasensitive, Low-Input Detection of Avocado Sunblotch Viroid via RPA-CRISPR and Nanopore-Array Single-Bead Fluorescence Readout

Rapid and sensitive detection of plant pathogens, such as the Avocado Sunblotch Viroid (ASBVd), is essential for early disease management and agricultural biosecurity. Yet, most current diagnostic methods not only require relatively large sample inputs but also often lack the ultrasensitivity required for reliable detection with scarce or minimally collected plant material. Here, we report a novel low-input but ultrasensitive diagnostic platform that integrates isothermal recombinase polymerase amplification (RPA), CRISPR-Cas12a detection, and a solid-state nanopore array for the detection of ASBVd. The system leverages CRISPR-Cas12a collateral cleavage activity to generate single-bead fluorescent signals, which are captured by a nanopore array through pressure-driven blockage. Our platform achieves a detection limit down to 1.68 copies/L while using only 40 nL of bead-fluorophore mixture per readout, which is over 100-fold less than conventional assays based on fluorescent readout using an imaging reader, enabling detection from minimal avocado sample collection. We demonstrate robust binary classification of ASBVd-positive and -negative samples from multiple avocado tissue types and orchards in California. The assay requires just 60 minutes and operates entirely under isothermal conditions, avoiding the need for bulky PCR instruments and supporting on-site deployment with minimal equipment. This method provides a promising platform for field-deployable, ultrasensitive, and low-input diagnostics of viroids and other low-titer pathogens in plant or clinical settings.

bioengineering↗

bioRxiv2025

Shape-Dependent Toxicity of Gold Nanoparticles in Microalgae: Distinct Cellular and Molecular Responses

The ecotoxicological effects of engineered nanoparticles in aquatic environments are influenced not only by composition and size but also by particle morphology, yet shape dependent interactions with primary producers, remain poorly understood. In this study, we evaluated the cellular and molecular responses of freshwater algae (Chlamydomonas reinhardtii) following exposure to 100 nm silica-coated gold nanospheres (AuNP) and nanostars (AuNS) across multiple concentrations. Exposure to 10 mg/L AuNS for 72 h results in significantly stronger inhibition of algal growth and photosynthetic activity compared to the same concentration of AuNP. Morphometric profiling reveals that AuNS induced pronounced structural injury, including cell enlargement, debris production, and disruption of subcellular organization than AuNP. Confocal imaging suggested this heightened toxicity may stem from distinct internalization patterns, with AuNP primarily adhering to chloroplast surfaces, whereas AuNS penetrated more deeply into intracellular compartments. RNA sequencing identified 9 upregulated and 38 downregulated differentially expressed genes (DEGs) in the 10 mg/L AuNP treated cells, impairing photosynthesis and energy storage via the photosystem II subunit S1 (PSBS1)/ early light-inducible protein (ELI3) pathway. In contrast, the AuNS group exhibits 246 upregulated and 145 downregulated DEGs, affecting membrane integrity and nitrogen metabolism through the nitrate reductase (NIT1)/ aminomethyl transferase (AMT1)/ protein kinase domain-containing protein (A0A2K3CRU5) pathway. These results demonstrate that nanoparticle morphology can drive divergent toxicity mechanisms in algal cells. Our findings highlight the necessity of incorporating NPs morphology into environmental risk assessments and suggest that safer nanomaterial design should consider shape-dependent interactions with aquatic microorganisms. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/694526v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@29fbccorg.highwire.dtl.DTLVardef@5221a7org.highwire.dtl.DTLVardef@1f6249org.highwire.dtl.DTLVardef@6dc054_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

Silicon Micropillar-Enhanced CRISPR Biosensor for Rapid and Sensitive Detection of Drug-Resistant Bacteria

1The growing threat of antibiotic-resistant pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA), underscores the urgent need for rapid, sensitive, and field-deployable diagnostic technologies. Here, we present a silicon micropillar-enhanced CRISPR biosensor that integrates high-aspect-ratio microstructures with a one-pot RPA/CRISPR-Cas12a assay for ultrasensitive and specific detection of MRSA. Micropillar arrays with fixed diameters and varying heights (100 {micro}m, 300 {micro}m, and 500 {micro}m) were fabricated via deep reactive ion etching and functionalized for surface probe immobilization. Suboptimal crRNA design was employed to modify Cas12a activation kinetics, enabling declined trans-cleavage and enhanced end-point signal accumulation. The 500 {micro}m micropillar configuration demonstrated a tenfold improvement in sensitivity compared to the 100 {micro}m array, with a limit of detection reaching 103 CFU mL-1. The platform also showed high specificity against non-target bacterial strains. These findings highlight the potential of combining microstructured chips with one-pot CRISPR diagnostics to advance next-generation point-of-care tools for infectious disease monitoring. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=184 SRC="FIGDIR/small/694528v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@5f5f7corg.highwire.dtl.DTLVardef@175cb35org.highwire.dtl.DTLVardef@7043e5org.highwire.dtl.DTLVardef@79aa15_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

CryoSift - An accessible and automated CNN-driven tool for cryo-EM 2D class selection

Single-particle cryo-electron microscopy (cryo-EM) has become an essential tool in structural biology. However, automating repetitive tasks remains an ongoing challenge in cryo-EM dataset processing. Here, we present a platform-independent convolutional neural network (CNN) tool for assessing the quality of 2D averages to enable automatic selection of suitable particles for high-resolution reconstructions, termed CryoSift. We integrate CryoSift into a fully automated processing pipeline using the existing cryosparc-tools library. Our integrated and customizable 2D assessment workflow enables high-throughput processing that accommodates experienced to novice cryo-EM users. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=183 SRC="FIGDIR/small/667259v2_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@1fdac98org.highwire.dtl.DTLVardef@14e3c7corg.highwire.dtl.DTLVardef@16520bforg.highwire.dtl.DTLVardef@48fa0f_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2025

Rational design of respiratory syncytial virus dimeric F-subunit vaccines in protein and mRNA forms

BackgroundRespiratory syncytial virus (RSV) poses a significant public health threat, particularly to children and the elderly. Two protein-based vaccines and one mRNA vaccine have been approved, all targeting the prefusion conformation of the fusion (F) glycoprotein. However, it has been reported that the F protein transitions to the post-fusion state during storage, resulting in a reduction of the vaccines immunogenicity. MethodsIn this study, we engineered novel pre-F-based antigens to preserve pre-F-specific immunodominant epitopes while eliminating sub-potent ones. Based on this, we constructed a series of single-chain dimers and selected the one with the highest expression yield and melting temperature (Tm). Next, we created a heterodimer, scDimer AB. Structural and protein characterization analyses were conducted to verify our design. All monomeric and scDimer antigens were used to immunize rodent models. Additionally, we prepared the antigens in mRNA form and immunized BALB/c mice. Finally, we combined both antigen forms, administering intramuscular mRNA priming followed by intranasal protein delivery in mice. In all immunization strategies, viral challenges were performed in animals to evaluate the immunologic protective effects. FindingsThrough rational design, we developed a monomeric and two single-chain dimeric (scDimer) proteins with the expected characteristics, including complete II, V, and O epitopes and a partial III epitope. The scDimers elicited stronger binding and neutralizing antibody responses in rodent models compared to the monomer, and they also boosted T cell responses when combined with appropriate adjuvants. After three doses of scDimer immunization, live RSV was barely detectable in the tissues of infected animals. The copies of RNA encoding N-gene were significantly reduced in the immunized groups compared to the PBS-injected control groups. We also engineered mRNA versions of the antigens and verified their protective efficacy in mice. Notably, there were no significant differences between intranasal boost and intramuscular two doses after RSV challenged, suggesting that intranasal boost provided equivalent protection to intramuscular vaccination and could reduce the risk of vaccine-enhanced disease (VED) potentially. InterpretationThe scDimer-based RSV vaccines effectively protected rodents from RSV infections, highlighting their clinical potential. Our antigen design removed certain suboptimal epitope regions, enhancing the efficiency of antigen presentation and increasing the proportion of the most potent pre-F-specific neutralizing antibodies. This approach provides a novel perspective for future vaccine design. FundingNational Key R&D Program of China, National Science Foundation of China, Young Scientists in Basic Research, Chinese Academy of Sciences, and Special Program of China National Tobacco Corporation. RESEARCH IN CONTEXTO_ST_ABSEvidence before this studyC_ST_ABSThe licensed respiratory syncytial virus (RSV) vaccines were based on RSV pre-F structure. We conducted a PubMed search for articles published in English from databases inception to March 31, 2025, using the search terms "RSV" (interchanged with "respiratory syncytial virus"), "pre-F" (interchanged with "prefusion F") and "vaccine". Our search revealed that most of the articles focus on combining pre-F with RSV G protein or other proteins to form viral-like particles (VLPs) or nanoparticles. The second-largest category of articles examined the antibodies level elicited by the pre-F antigens in animal model or some special cohorts. Other studies addressed topics such as optimization of injection dosage and immunization strategies, methodologies for detecting pre-F-specific antibodies, and the development of mRNA-LNP vaccines. Notably, only 4 articles described novel designs of RSV pre-F antigens, all of which were trimeric and based exclusively on the RSV A subtype F protein sequence. Added value of this studyTo our knowledge, our design is the first single-chain dimer version of RSV pre-F antigen. The engineered antigens demonstrated high yield, thermostability, and immunogenicity comparable to that of DS-Cav1. Additionally, we developed the first chimeric RSV-antigen covering both subtype A and B, thereby broadening the potential protective spectrum. We further validated the immunogenicity of our designs in mRNA form and as a protein-mRNA combination. This research emphasizes the potential of constructing and combining minimized, stable antigen modules, offering a novel approach for future vaccine development. Implications of all the available evidenceIn this study, we present a novel approach for constructing and combining minimized, stable antigen modules for future vaccine design. The strong immunogenicity of our antigens underscores their potential for clinical application. Furthermore, given the structural similarities between the RSV antigen and those of other Pneumoviruses, such as human metapneumovirus (hMPV), our design strategy could be extended to the development of multivalent vaccines.

immunology↗

bioRxiv2025

CRISPR-On-Beads: A Simple and Sensitive Approach for Bacterial DNA Detection

CRISPR/Cas12a, recognized for its high efficiency and specificity in nucleic acid recognition, has found extensive applications in infectious disease sensing. Nonetheless, a significant challenge persists in devising a simple method for the rapid, convenient, and accurate visualization of detection results. In this study, we demonstrate an innovative and simple CRISPR-on-beads assay for visual detection of methicillin-resistant Staphylococcus aureus (MRSA). This approach combines isothermal target amplification, CRISPR/LbCas12a-mediated cleavage, and magnetic bead-based probe binding for signal amplification, enabling rapid and convenient identification of bacterial DNA. Our results show that this detection method exhibits both high sensitivity and specificity, with a detection limit as low as 30 CFU/L. This newly developed biosensing approach can easily be integrated with a portable fluorescence microscope for medical diagnostics in resource-limited settings.

bioengineering↗

bioRxiv2025

Dissecting dynamic gene regulatory network using transformer-based temporal causality analysis

The AI Virtual Cell (AIVC) framework promises to revolutionize biological research through high-fidelity simulations of cellular behaviors and responses to perturbations. Central to realizing this vision is the ability to model cell differentiation dynamics, which requires accurate inference of gene regulatory network (GRN) that govern cell fate decisions. However, existing computational approaches rely on static GRN models that fail to capture the dynamic changes of regulatory relationships during differentiation, limiting their utility for simulating developmental processes and predicting perturbation outcomes. Here, we present CellProphet, an interpretable AI model that infers dynamic GRN by integrating temporal causality with transformer self-attention mechanism. CellProphet captures time-lagged dependencies between transcription factor (TF) expression and target gene activation while providing interpretable regulatory weights, enabling both accurate prediction and mechanistic insight. When benchmarked against nine state-of-the-art methods across seven differentiation datasets, CellProphet achieves superior performance in all evaluation metrics. Applied to mouse embryonic stem cell differentiation, CellProphet identifies both well-known and potentially novel TFs with substantially high sensitivity and successfully reconstructs dynamic regulatory relations validated through multi-modal epigenomic data. In mouse hematopoietic differentiation, CellProphet accurately predicts cell fate transitions and gene expression changes following in silico perturbation of key TFs Gata1 and Spi1, demonstrating its capability for virtual experimentation. These results establish CellProphet as a foundational tool for the AIVC framework, enabling researchers to decode the dynamic regulatory logic of differentiation, accelerate discovery of key regulatory factors, and design targeted cellular interventions for widespread applications.

bioinformatics↗

bioRxiv2024

Cardiolipin-mimic lipid nanoparticles without antibody modification delivered senolytic in-vivo CAR-T therapy for inflamm-aging

mRNA-based in vivo CAR T cell engineering offers advantages over ex vivo therapies, including streamlined manufacturing and transient expression. However, current delivery requires antibody-modified vehicles with manufacturing challenges. In this study, inspired by cardiolipin, we identified a cardiolipin-like di-phosphoramide lipid that improved T cell transfection without targeting ligands, both in vivo and in vitro. The T cell-favored tropism is likely due to the lipids packing, shape, and rigidity. Encapsulating circular RNA further prolonged mRNA expression in the spleen and T cells. Using PL40 lipid nanoparticles, we delivered mRNA encoding a CAR targeting the senolytic and inflammatory antigen urokinase-type plasminogen activator receptor (uPAR), alleviating uPAR-related liver fibrosis and rheumatoid arthritis (RA). Single cell sequencing in humans confirmed uPARs relevance to senescence and inflammation in RA. To further enhance clinical translation, we screened and humanized scFvs against uPAR, establishing PL40 mRNA encoding a circular human uPAR CAR, with potential for treating aging-inflamed disorders. One Sentence SummaryWeve developed a unique class of Cardiolipin-mimic lipids that facilitate mRNA delivery to T cells in vivo without the need for antibody modification, enhancing the treatment of liver fibrosis and rheumatoid arthritis through circular CAR uPAR RNA and propelling the clinical application of humanized CAR against human uPAR.

bioengineering↗

bioRxiv2024

Multi-Channel Funnel Adapted Sensing Tube (MFAST) for the Simple and Duplex Detection of Parasites

Leishmaniasis poses a significant global health threat, infecting millions of people annually, particularly in tropical and subtropical regions. Timely and accurate detection of Leishmania species is crucial for effective treatment and control of this debilitating disease. This study introduces the Multi-channel Funnel Adapted Sensing Tube (MFAST) chip, an innovative diagnostic tool designed for the rapid detection of Leishmania panamensis. MFAST is fabricated through 3D printing and sacrificial molding of acrylonitrile butadiene styrene (ABS) and the reagents are transported between the reservoirs by gravity. We combine experiments and finite element analysis to reduce the leaking issues and facilitate smoother fluid flow, improving the overall performance of the device. Highly sensitive and specific RPA-CRISPR/Cas12a assay is utilized in the chip, achieving a detection limit as low as 1,000 parasites/mL (detecting as few as 5 parasites per reaction). The multi-channel design enables duplex detection, allowing for simultaneous identification of both L. braziliensis and L. panamensis through distinct channels. Furthermore, stability tests indicate that lyophilized reagents retain functionality for up to 15 days when stored at 4 {degrees}C, underscoring the potential of this chip for practical diagnostic applications in low-resource settings.

bioengineering↗

bioRxiv2024

Durable Antimicrobial Microstructure Surface (DAMS) Enabled by 3D-Printing and ZnO Nanoflowers

A.Numerous studies have been trying to create nanomaterials based antimicrobial surfaces to combat the growing bacterial infection problems. Mechanical durability has become one of the major challenges to applying those surfaces in real life. In this study, we demonstrate the Durable Antimicrobial Microstructures Surface (DAMS) consisting of DLP 3D printed microstructures and zinc oxide (ZnO) nanoflowers. The microstructures serve as a protection armor for the nanoflowers during abrasion. The antimicrobial ability was tested by immersing in 2E8 CFU/mL Escherichia coli (E. coli) suspension and then evaluated using electron microscopy. Compared to the bare control, our results show that the DAMS reduces bacterial coverage by more than 90% after 12 hrs of incubation and approximately 50% after 48 hrs of incubation before abrasion. Importantly, bacterial coverage is reduced by approximately 50% after 2 min of abrasion with a tribometer, and DAMS remains effective even after 6 min of abrasion. These findings highlight the potential of DAMS as an affordable, scalable, and durable antimicrobial surface for various biomedical applications.

bioengineering↗

bioRxiv2024

Asymmetric Allostery in Estrogen Receptor-α Homodimers Drives Responses to the Ensemble of Estrogens in the Hormonal Milieu

The estrogen receptor- (ER) is thought to function only as a homodimer, but responds to a variety of environmental, metazoan, and therapeutic estrogens at sub-saturating doses, supporting binding mixtures of ligands as well as dimers that are only partially occupied. Here, we present a series of flexible ER ligands that bind to receptor dimers with individual ligand poses favoring distinct receptor conformations --receptor conformational heterodimers--mimicking the binding of two different ligands. Molecular dynamics simulations showed that the pairs of different ligand poses changed the correlated motion across the dimer interface to generate asymmetric communication between the dimer interface, the ligands, and the surface binding sites for epigenetic regulatory proteins. By examining binding of the same ligand in crystal structures of ER in the agonist versus antagonist conformers, we also showed that these allosteric signals are bidirectional. The receptor conformer can drive different ligand binding modes to support agonist versus antagonist activity profiles, a revision of ligand binding theory that has focused on unidirectional signaling from ligand to the coregulator binding site. We also observed differences in the allosteric signals between ligand and coregulator binding sites in the monomeric versus dimeric receptor, and when bound by two different ligands, states that are physiologically relevant. Thus, ER conformational heterodimers integrate two different ligand-regulated activity profiles, representing new modes for ligand-dependent regulation of ER activity. SignificanceThe estrogen receptor- (ER) regulates transcription in response to a hormonal milieu that includes low levels of estradiol, a variety of environmental estrogens, as well as ER antagonists such as breast cancer anti-hormonal therapies. While ER has been studied as a homodimer, the variety of ligand and receptor concentrations in different tissues means that the receptor can be occupied with two different ligands, with only one ligand in the dimer, or as a monomer. Here, we use X-ray crystallography and molecular dynamics simulations to reveal a new mode for ligand regulation of ER activity whereby sequence-identical homodimers can act as functional or conformational heterodimers having unique signaling characteristics, with ligand-selective allostery operating across the dimer interface integrating two different signaling outcomes.

biophysics↗

bioRxiv2024

The Structure of the Drp1 Lattice on Membrane

Mitochondrial health relies on the membrane fission mediated by dynamin-related protein 1 (Drp1). Previous structural studies of Drp1 on remodeled membranes were hampered by heterogeneity, leaving a critical gap in the understanding of the mitochondrial fission mechanisms. Here we present a cryo-electron microscopy structure of full-length human Drp1 decorated on membrane tubules. Using the reconstruction of average subtracted tubular regions (RASTR) technique, we report that Drp1 forms a locally ordered lattice along the tubule without global helical symmetry. The filaments in the lattice are similar to dynamin rungs with conserved stalk interactions. Adjacent filaments are connected by GTPase domain interactions in a novel stacked conformation. We identified two states of the Drp1 lattice among the heterogenous dataset representing conformational changes around hinge 1. Additionally, we observed contact between Drp1 and membrane that can be assigned to the variable domain sequence. Together these structures revealed a putative mechanism by which Drp1 constricts mitochondria membranes in a stepwise, "ratchet" manner. SUMMARYThis study provides new insights into the structure of Drp1 on lipid membranes. A locally ordered Drp1 lattice structure is solved and reveals intermolecular contacts and conformational rearrangements that suggest a mechanism for constriction of mitochondrial membranes.

biochemistry↗

bioRxiv2023

Pneumatic Nano-Sieve for CRISPR-based Detection of Drug-resistant Bacteria

The increasing prevalence of antibiotic-resistant bacterial infections, particularly methicillin-resistant Staphylococcus aureus (MRSA), presents a significant public health concern. Timely detection of MRSA is crucial to enable prompt medical intervention, limit its spread, and reduce antimicrobial resistance. Here, we introduce a miniaturized nano-sieve device featuring a pneumatically-regulated chamber for highly efficient MRSA purification from human plasma samples. By using packed magnetic beads as a filter and leveraging the deformability of the nano-sieve channel, we achieve an on-chip concentration factor of 15 for MRSA. We integrated this device with recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas detection system, resulting in an on-chip limit of detection (LOD) of approximately 100 CFU/mL. This developed approach provides a rapid, precise, and centrifuge-free solution suitable for point-of-care diagnostics, with the potential to significantly improve patient outcomes in resource-limited medical conditions.

bioengineering↗

bioRxiv2023

Superhydrophobic Rotation-Chip for Computer-Vision Identification of Drug-Resistant Bacteria

The transport, distribution, and mixing of microfluidics often require additional instruments, such as pumps and valves, which are not feasible when operated in point-of-care (POC) settings. Here, we present a simple microfluidic pathogen detection system known as Rotation-Chip that transfers the reagents between wells by manually rotating two concentric layers without using external instruments. The Rotation-Chip is fabricated by a simple computer numerical control (CNC) machining process and is capable of carrying out 60 multiplexed reactions with a simple 30-degree or 60-degree rotation. Leveraging superhydrophobic coating, a high fluid transport efficiency of 92.78% is achieved without observable leaking. Integrated with an intracellular fluorescent assay, an on-chip detection limit of 1.8x106 CFU/mL is achieved for ampicillin-resistant Escherichia coli (E. coli), which is similar to our off-chip results. We also develop a computer vision method to automatically distinguish positive and negative samples on the chip, showing 100% accuracy. Our Rotation-Chip is simple, low-cost, high-throughput, and can display test results with a single chip image, ideal for various multiplexing POC applications in resource-limited settings.

bioengineering↗

bioRxiv2022

Quartet DNA reference materials and datasets for comprehensively evaluating germline variants calling performance

Current methods for evaluating the accuracy of germline variant calls are restricted to easy-to-detect high-confidence regions, thus ignoring a substantial portion of difficult variants beyond the benchmark regions. We established four DNA reference materials from immortalized cell lines derived from a Chinese Quartet including parents and monozygotic twins. We integrated benchmark calls of 4.2 million small variants and 15,000 structural variants from multiple platforms and bioinformatic pipelines for evaluating the reliability of germline variant calls inside the benchmark regions. The genetic built-in-truth of the Quartet family design not only improved sensitivity of benchmark calls by removing additional false positive variants with apparently high quality, but also enabled estimation of the precision of variants calls outside the benchmark regions. Batch effects of variant calling in large-scale DNA sequencing efforts can be effectively identified with the concurrent use of the Quartet DNA reference materials along with study samples, and can be alleviated by training a machine learning model with the Quartet reference datasets to remove potential artifact calls. Matched RNA and protein reference materials were also established in the Quartet project, thereby enabling benchmark calls constructed from DNA reference materials for evaluation of variants calling performance on RNA and protein data. The Quartet DNA reference materials from this study are a resource for objective and comprehensive assessment of the accuracy of germline variant calls throughout the whole-genome regions.

bioinformatics↗