Biology subjects

Zhang, S.

Publications and source records attributed to Zhang, S..

At least 325 records · Page 18Linked to original sources

bioRxiv2025

Mitochondrial Genome Variants and Nuclear Mitochondrial DNA Segments in 7331 Individuals from NyuWa and 1KGP

Dysfunctional mitochondria are implicated in various diseases, while little is known about comprehensive characterization of mitochondrial DNA (mtDNA) in Chinese population. Here, we conducted a systematic analysis of mtDNA from 7331 samples, comprising 4129 Chinese samples (NyuWa) and 3202 samples from the 1000 Genomes Project (1KGP). We identified 7216 distinct high-quality mtDNA variants and classified them into 22 macrohaplogroups, and detected 1466 distinct nuclear mitochondrial DNA segments (NUMTs), with 88 mtDNA variants and 642 NUMTs being specific to NyuWa. The genome-wide association analyses revealed that 12 mtDNA variants were significantly correlated with 199 nuclear DNA variants. Our findings revealed that all individuals in both NyuWa and 1KGP harbored common NUMTs, and one-fifth possessed ultra-rare NUMTs, which tended to insert into nuclear gene regions. Compared to 1KGP, significant enrichment of nuclear breakpoints in long interspersed nuclear elements (LINEs) was observed for rare NUMTs in NyuWa. Overall, this study represents the first comprehensive profiling the landscape of Chinese NUMTs and offers the most extensive resource of Chinese mtDNA variants and NUMTs based on high-depth WGS to date, providing valuable reference resources for genetic research on mtDNA-related diseases.

genomics↗

bioRxiv2025

Cell layer-specific modification of cell wall is associated with exo-mesocarp split in pistachio (Pistacia vera L.)

Pistachio (Pistacia vera) is a drought and salinity-tolerant perennial whose fruit features a fleshy exo-mesocarp, or "hull," that protects the kernel. Hull development and degradation are key to kernel quality, yet the anatomy and mechanisms driving hull breakdown during late-stage development remain largely unknown. Here, we show that the hull contains anatomically distinct layers of hypodermal parenchyma and filler parenchyma. Using a combination of transcriptome analyses and immunohistochemistry, we show that changes in pectin associated gene expression and modification of this polysaccharide are involved in hull cell size increase, loss of cell-cell adhesion, and softening. Anatomical analysis shows that filler parenchyma expands during late-stage hull development while hypodermal parenchyma remains constant in size. Field data suggest that irrigation and humidity affect pistachio hull split, implicating a role for water status in cell expansion. In summary, the complex interplay between molecular, cellular, and environmental changes suggests that cell layer-specific modifications of the cell wall are linked to exo-mesocarp splitting, forming a model for understanding the mechanism of fruit split during ripening in non-berry fruit crops. HighlightCell-layer specific modifications of the cell wall are associated with cell expansion and loss of cell-cell adhesion, leading to hull split during late-stage pistachio fruit development.

plant biology↗

bioRxiv2025

Comparative Analysis of Neonatal Hypomyelination Models Using Spatial Transcriptomics

White matter injury (WMI) is a major cause of morbidity in premature infants, contributing to 5%-10% of cerebral palsy cases and up to 50% of cognitive and behavioral deficits in the United States. Two commonly used preclinical models, intermittent hypoxia (IH) and hypoxia-ischemia (HI) are widely employed to investigate the effects of WMI. The internal capsule (IC) and corpus callosum (CC) are major white matter tracts undergoing active myelination during the neonatal period, making them particularly vulnerable to hypoxic insults. This study aims to compare the effects of IH and HI models on myelination as well as the involvement of inflammatory cells in the IC and CC. We evaluated five oligodendrocyte (OL) subtypes, along with astrocytes, microglia, and activated microglia in IC and CC at postnatal day 12 (P12) and day 20 (P20) using spatial transcriptomics (CosMx, Novogene). For the HI model, C57BL/6 mice at P10 underwent permanent ligation of the left carotid artery followed by 45 minutes of hypoxia (8% O2 / 92% N2). For the IH model, P3 mice were exposed to 5% O2 / 95% N2, twice daily for five consecutive days. Animals were euthanized at P12 and P20, perfused transcardially, and brains were post-fixed in 4% paraformaldehyde, dehydrated in an ethanol series, embedded in paraffin, and coronally sectioned at 7 m. Slides were submitted for CosMx spatial transcriptomic analysis (NanoString Technologies), and data analysis was performed using the Seurat package in RStudio. Our results demonstrate that IH and HI models affect OL populations differently, and these effects vary by brain region. In the IC, the IH model caused earlier and more pronounced changes in OL differentiation-related gene expression compared to HI. In contrast, the CC was more affected by HI. Moreover, in the HI group, mature OL s in both regions showed reduced expression of myelination-associated genes. This was accompanied by greater activation of inflammatory cells and increased intercellular communication between these cells and mature OLs, potentially contributing to the observed hypomyelination. Overall, our study provides critical insights into how each model of neonatal hypoxia differentially impacts white matter development. This knowledge can help refine preclinical strategies and guide therapeutic research tailored to the underlying pathology of each model.

neuroscience↗

bioRxiv2025

Fibulin-4 is highly expressed in metastatic breast cancer and can serve as a target of peptide-based imaging probes and experimental therapeutics

We have previously reported a cyclic peptide CRAGVGRGC (termed BLMP6) that homes to disseminating tumor cells in mouse cancer models and could be used for metastasis detection and intervention. Here, based on BLMP6 similarity to latent transforming growth factor beta binding protein 4 (LTBP4), we discovered fibulin-4 as a BLMP6 target. We show that BLMP6 mimics the LTBP4 domain binding to fibulin-4 and selectively binds to fibulin-4 in vitro. Fibulin-4 knockout in mouse 4T1 cancer cells abrogated BLMP6 homing to lung metastases. Fibulin-4 expression was found to be increased in invasive and metastatic human breast cancer. AZDye555 fluorophore-labeled BLMP6 was developed as a reagent selectively binding to invasive and metastatic human breast cancer cells in tissue sections and homing to MDA-MB-231 metastases in mice. We show that radiolabeling BLMP6 with 68Ga can be used for the detection of MDA-MB-231 metastases. We designed a peptide-drug conjugate consisting of monomethyl auristatin E (MMAE) and BLMP6 that preferentially kills aggressive cancer cells. Cytotoxicity of MMAE-BLMP6 against MDA-MB-231 tumors was confirmed in vivo. In an immunocompetent mouse model of B16F10 experimental lung metastases, treatment with MMAE-BLMP6 suppressed metastasis growth and improved survival. There was also a trend for metastasis suppression and survival improvement in the MDA-MB-231 experimental metastasis model. Our results suggest that fibulin-4 and BLMP6 may be further developed for the detection and targeting of metastatic human cancers. Statement of significanceThis study identifies fibulin-4 as a protein highly expressed in breast cancer metastasis. It evaluates the application of peptide conjugates targeting fibulin-4 in mouse models as non-invasive probes for metastasis detection and cytotoxic drug delivery.

cancer biology↗

bioRxiv2025

Attomolar Detection of HIV-1 with Label-Free RCA-rCRISPR on Smartphone

Human Immunodeficiency Virus-1 (HIV) remains a major global public health challenge, having led to over 42.3 million deaths since its discovery in the early 1980s. Despite progress in prevention and treatment, around 60% of people with HIV (PWH) remain undiagnosed in resource-limited regions, disproportionately affecting vulnerable populations and underserved communities across the world. This illustrates the critical need for accessible, accurate, and equipment-free diagnostic tools to enhance detection and thus provide opportunities to curb its spread. Here, we developed a low-cost, robust, and label-free rolling circle amplification (RCA)-rCRISPR diagnostic platform for detecting HIV viral load with minimal instrumentation. Our strategy, combining the integration of RNA-detecting RCA reaction with plasmid reporter-based ratiometric CRISPR (rCRISPR), enables sensitive detection of unprocessed RNA targets without the need for intensive sample pre-treatment. This label-free RCA-rCRISPR diagnostic platform detected HIV RNA down to single-digit aM sensitivity (~3000 copies/mL) from PWH-derived HIV samples ex vivo. Unlike typical RCA, which requires sample fragmentations to break long RNA target sequences, our design harnesses the triple functions of the phi29 DNA polymerase (namely exonuclease activity, polymerization, and strand displacement), enabling the detection of the entire HIV genome without pre-fragmentation. For point-of-care (POC) applications, we constructed an all-in-one smartphone-based minigel electrophoresis device to facilitate equipment-free HIV viral load testing, making it accessible to resource-limited communities. Additionally, the assay has demonstrated the ability for point mutation detection (BRAF mutation in canine urothelial carcinoma), showcasing the robustness of our strategy for broad disease diagnostic applications.

bioengineering↗

bioRxiv2025

Proof-of-Concept in a Murine Model of Treatment of Thrombotic Thrombocytopenic Purpura Using Engineered Red Blood Cells

Thrombotic Thrombocytopenic Purpura (TTP) is caused by congenital or acquired deficiency of ADAMTS13, a metalloproteinase that cleaves von Willebrand Factor (vWF) multimers. Current treatments--plasma exchange and immunosuppression--are costly and associated with significant morbidity therefore, alternative strategies are needed. We developed the kitJak2 platform for producing genetically engineered lab-grown red blood cells (lgRBCs) as drug delivery vectors. We hypothesized that membrane-bound ADAMTS13 displayed on lgRBCs could provide a durable treatment for TTP. To test this, we engineered erythroid cells expressing both wild-type and mutant variants MDTCS fragments of ADAMTS13, conferring resistance to autoantibodies. Flow cytometry and FRET-based assays confirmed robust membrane expression and enzymatic activity. Importantly, mutant MDTCS variants retained catalytic activity in the presence of plasma from TTP patients, whereas wild-type variants were inhibited. For in vivo evaluation, we generated transgenic mice expressing MDTCS ADAMTS13 on their RBC membranes. These mice exhibited normal RBC half-lives and stable, catalytically active ADAMTS13 expression. Using a murine model of TTP--where ADAMTS13 knockout mice challenged with recombinant human vWF (rhvWF) develop thrombocytopenia and schistocytes--we demonstrated that transfusion of ADAMTS13-expressing RBCs significantly mitigated disease, preventing platelet loss and schistocyte formation. This confirms that membrane-bound MDTCS ADAMTS13 cleaves circulating rhvWF under physiological flow conditions in vivo. Finally, employing our KitJak2 platform, we generated human enucleated lgRBCs expressing high levels of catalytically active ADAMTS13. This novel work establishes proof-of-concept that membrane-anchored ADAMTS13-expressing lab- grown RBCs may offer a feasible and effective therapeutic approach for both congenital and acquired TTP.

cell biology↗

bioRxiv2025

VAMPIRE: Analyzing variation and motif pattern in tandem repeats

Tandem repeats (TRs) are pervasive in eukaryotic genomes and play key roles in genome organization, evolution, and function, particularly in complex regions such as centromeres and subtelomeres. Although long-read sequencing technologies have improved the resolution of these regions, existing methods remain limited in their ability to systematically and accurately characterize large-scale TRs. Here, we introduce VAMPIRE, a k-mer-based computational tool for comprehensive TR discovery, annotation, and quantification. Unlike previous methods, VAMPIRE enables reference-free, fine-grained decomposition of both simple and complex TRs, capturing motif variation in sequence, length, and structure with high sensitivity and scalability. Applied to complete telomere-to-telomere (T2T) human and nonhuman primate (NHP) genome assemblies, VAMPIRE reveals previously unrecognized high-order repeat inversions within human centromeres--an underappreciated evolutionary mechanism contributing to centromere diversity. Additionally, the tool identifies lineage-specific and expanded TRs, including human-specific STR/VNTR expansions and NHP-specific subtelomeric heterochromatin (e.g., pCht/StSat), underscoring their dynamic turnover and structural complexity. VAMPIRE provides a robust and scalable framework for TR analysis in the era of long-read sequencing, with broad utility across human genetics, evolutionary biology, and the study of complex TRs in non-model organisms.

genomics↗

bioRxiv2025

Purification of post-transcriptionally modified tRNAs for enhanced cell-free translation systems

Transfer RNAs (tRNAs) are utilized by the ribosome to decode the nucleic acid alphabet. tRNA structure, stability, aminoacylation efficiency, and decoding efficacy are governed by their extensive post-transcriptional modifications. In most studies, individual tRNAs are generated using in vitro transcription, which produces tRNAs devoid of these critical site-specific modifications, negatively affecting translation yields and fidelity. To address this, we have developed a purification method which couples tRNA overexpression to DNA hybridization-based purification. Using this approach, we produced native tRNAs from E. coli in high yield and purity while retaining their complement of native post-transcriptional modifications and translational activity. We extend this technique to the purification of [Formula] and [Formula], tRNAs of critical importance for genetic code expansion. We confirmed that both [Formula] and [Formula] contain native E. coli post-transcriptional modifications and provide the first complete modification profiles of each. Moreover, we found that in vivo-generated [Formula] significantly outperforms its in vitro-generated counterpart in amber codon suppression in cell-free translation reactions. Finally, we purified an engineered variant of E. coli [Formula], extending our studies to synthetic tRNAs. We present a flexible method which generates modified tRNAs in high yield and purity, addressing a critical and persistent challenge in RNA biochemistry. This toolkit enables future structural and cell-free studies through scalable access to native and engineered tRNAs, advancing the broader field of translation and synthetic biology.

biochemistry↗

bioRxiv2025

Single-cell imaging reveals spontaneous phenotypic sorting and bet-hedging in developing biofilms

How phenotypic heterogeneity shapes biofilm architecture and development remains poorly understood. Motivated by this, we developed imaging tools to track intracellular levels of c-di-GMP, a key second messenger that controls the motile-to-sessile transition, in developing Vibrio cholerae biofilms at single-cell resolution. We show that c-di-GMP levels spontaneously bifurcate into a bimodal distribution that forms a spatially sorted pattern: High-c-di-GMP cells dominate the biofilm core while low-c-di-GMP cells localize to the periphery. Combining single-lineage tracing, mutant analysis, and agent-based modeling, we reveal that this pattern arises from differential viscosity and surface friction mediated by matrix-dependent interactions between cells and their microenvironments. We demonstrate that this heterogeneity and phenotypic sorting enable continuous emergence and shedding of planktonic cells, enhancing fitness in fluctuating environments. Our findings uncover a differential drag mechanism for pattern formation in multicellular systems, and expand the classical picture of biofilm lifecycle by highlighting the functional significance of phenotypic heterogeneity.

biophysics↗

bioRxiv2025

Advancing Ligand Binding Affinity Prediction with Cartesian Tensor-Based Deep Learning

0.We present PBCNet2.0, a cartesian tensor-based Siamese Neural Network for protein-ligand relative binding affinity prediction. Trained on 8.6 million protein-ligand complex structure pairs, PBCNet2.0 achieves zero-shot performance comparable to computationally intensive physics-based simulations. Our prioritization experiments show that PBCNet2.0 speeds up binding affinity optimization by 718% while reducing resource use by 41%. Through extensive retrospective experiments, we demonstrate that PBCNet2.0 intrinsically comprehends protein-ligand interactions, showing high sensitivity to intermolecular interactions and exceptional perception of spatial geometric information. Strikingly, PBCNet2.0 exhibits an emergent capability to predict affinity changes induced by binding residue variations, highlighting its potential for identifying resistance mutation. We prospectively validated these capabilities on two targets ENPP1 and ALDH1B1, where PBCNet2.0 successfully identified affinity shifts arising from subtle molecular interactions and conformational differences, and pinpointed critical binding residues with an 83% hit rate. This combination of computational efficiency, spatial geometric perception of binding site, and generalizable affinity prediction establishes PBCNet2.0 as a transformative tool for developing pharmacological probes for all human proteins.

molecular biology↗

bioRxiv2025

Convergent and divergent brain-cognition relationships during development revealed by cross-sectional and longitudinal analyses in the ABCD Study

How brain networks and cognition co-evolve during development remains poorly understood. Here, we use resting-state functional magnetic resonance imaging (rs-fMRI) and cognitive data at baseline and Year 2 of 2,949 individuals in the Adolescent Brain Cognitive Development (ABCD) Study to examine how stable and changing features of brain network organization predict cognitive development during early adolescence. We find that baseline resting-state functional connectivity (FC) more strongly predicts future cognitive ability than baseline cognitive ability. Models trained on baseline FC to predict baseline cognition generalize better to Year 2 FC and cognition, suggesting that brain-cognition relationships strengthen over time. Intriguingly, baseline FC outperforms longitudinal FC change in predicting future cognitive ability. One potential reason is the lower reliability of FC change compared to baseline FC: ICC = 0.24 vs. 0.56. However, reducing baseline FCs reliability by shortening scan duration only partially narrows the predictive gap, suggesting reliability alone cannot be the full explanation. Furthermore, neither baseline FC nor FC change meaningfully predicts longitudinal change in cognitive ability. We also identify converging and diverging predictive network features across cross-sectional and longitudinal models of brain-cognition relationships, revealing a multivariate twist on Simpsons paradox. Together, these findings suggest that during early adolescence, stable individual differences in brain functional network organization play a more critical role than dynamic changes in shaping future cognitive outcomes.

neuroscience↗

bioRxiv2025

Developmental dynamic transcriptomics reveals multiple effectors and transcription factors critical for Ditylenchus destructor parasitism

Plant-parasitic nematodes (PPNs) pose a major threat to global agricultural production, yet fundamental research on their biology remains limited. The origin and evolutionary trajectory of PPNs remain elusive, largely due to the scarcity of chromosome-level genomic data. Among them, migratory PPNs are considered a key transitional form between free-living and obligate parasitic lifestyles, as they exhibit both plant parasitism and fungal feeding behaviors. In this study, we assembled a chromosome-level genome of the sweet potato rot nematode Ditylenchus destructor and confirmed the presence of four chromosomes through Hi-C scaffolding and karyotype analysis. Comparative genomic analysis with two other migratory PPNs, Bursaphelenchus xylophilus and Aphelenchoides besseyi, revealed that the Nigon elements in B. xylophilus are largely conserved with those of the model organism Caenorhabditis elegans, while D. destructor and A. besseyi exhibit extensive Nigon element rearrangements. These rearrangements were strongly correlated with patterns of protein sequence collinearity. Moreover, transcriptomic profiling across five developmental stages of D. destructor identified numerous stage-specific effectors and transcription factors. Functional analysis via RNA interference demonstrated that many of these genes play essential roles in embryogenesis and parasitic activity. Together, our results provide valuable genomic and transcriptomic resources for studying PPNs, uncovering critical insights into their genome evolution and parasitism-related gene functions, and laying a crucial foundation for advancing the understanding of PPN biology and their impact on agricultural systems.

genomics↗

bioRxiv2025

Cell-surface proteomic profiling identifies CD72 as a regulator of microglial tiling

Microglial tiling--the phenomenon of consistent cell-to-cell distances and non-overlapping processes--is regarded as a qualitative indicator of homeostasis, but mechanisms of microglial tiling are unknown. We used cell-surface proximity labeling and mass spectrometry to profile the microglial cell-surface proteome in an in vitro model of homeostatic glial physiology and used single-cell RNA sequencing and public databases to identify candidate cell-surface proteins that might modulate tiling. We designed an image-based functional assay which measures six morphological/spatial readouts to screen these proteins for modulation of tiling. CD72, a coreceptor to the B cell receptor that is expressed by microglia, disrupted tiling; we validated its effects in vitro and in situ in organotypic hippocampal brain slices. Phosphoproteomic studies revealed that CD72 modulates pathways associated with cell adhesion, repulsive receptors, microglial activation, and cytoskeletal organization. These results lay the groundwork for further investigation of the functional roles of tiling in homeostasis and disease.

neuroscience↗

bioRxiv2025

Biomni: A General-Purpose Biomedical AI Agent

Biomedical research underpins progress in our understanding of human health and disease, drug discovery, and clinical care. However, with the growth of complex lab experiments, large datasets, many analytical tools, and expansive literature, biomedical research is increasingly constrained by repetitive and fragmented workflows that slow discovery and limit innovation, underscoring the need for a fundamentally new way to scale scientific expertise. Here, we introduce Biomni, a general-purpose biomedical AI agent designed to autonomously execute a wide spectrum of research tasks across diverse biomedical subfields. To systematically map the biomedical action space, Biomni first employs an action discovery agent to create the first unified agentic environment - mining essential tools, databases, and protocols from tens of thousands of publications across 25 biomedical domains. Built on this foundation, Biomni features a generalist agentic architecture that integrates large language model (LLM) reasoning with retrieval-augmented planning and code-based execution, enabling it to dynamically compose and carry out complex biomedical workflows - entirely without relying on predefined templates or rigid task flows. Systematic benchmarking demonstrates that Biomni achieves strong generalization across heterogeneous biomedical tasks - including causal gene prioritization, drug repurposing, rare disease diagnosis, micro-biome analysis, and molecular cloning - without any task-specific prompt tuning. Real-world case studies further showcase Biomnis ability to interpret complex, multi-modal biomedical datasets and autonomously generate experimentally testable protocols. Biomni envisions a future where virtual AI biologists operate alongside and augment human scientists to dramatically enhance research productivity, clinical insight, and healthcare. Biomni is ready to use at https://biomni.stanford.edu, and we invite scientists to explore its capabilities, stress-test its limits, and co-create the next era of biomedical discoveries.

bioinformatics↗

bioRxiv2025

Germline-targeting HIV Envelope SOSIP immunization more frequently elicits broadly-neutralizing antibody precursor responses in infant compared to juvenile rhesus macaques

A vaccine capable of inducing broadly neutralizing antibodies (bnAbs) is essential for effective prevention against HIV in children and adolescents. Germline-targeting vaccine strategies aim to stimulate bnAb precursor B cells through carefully designed immunogens, such as the stabilized SOSIP trimers, which mimic native HIV envelope (Env) proteins while presenting key neutralizing epitopes to germline B cell receptors. Given the ability of children living with HIV to develop bnAbs earlier and at a higher frequency than adults, we compared the immunogenicity of a CD4 binding site (CD4bs) bnAb germline-targeting SOSIP trimer immunization strategy in infant (n = 5) and juvenile (n = 4) rhesus macaques (RMs). Animals received 3 doses of the germline-targeting BG505 GT1.1 immunogen, followed by 3 boosts of wild-type BG505 SOSIP, each adjuvanted with the TLR7/8 agonist, 3M-052-SE. After 1.5 years, the RMs were further boosted with a mixed clade B Env trimer nanoparticle to enhance heterologous virus neutralization responses. This germline-targeting strategy induced equivalent titers of neutralizing antibodies in both groups of RMs, yet the infants exhibited a higher magnitude of vaccine-specific IgG binding. Notably, after 3 doses of BG505 GT1.1 SOSIP, infants had higher BG505 GT1.1-specific IgD- B cells. Upon completion of the vaccine regimen, 4 of 5 infants developed a CD4bs bnAb precursor response detectable in serum compared to only 1 of 4 juveniles. Finally, administration of the mixed clade B nanoparticle was able to increase the breadth of antibody responses in 3 of 5 infants and 2 of 4 juveniles. These results suggest that immunization in early-life may enhance bnAb induction and highlight the potential for future pediatric HIV-1 vaccine strategies. Author SummaryVaccines that can generate antibody responses that have breadth and potency in neutralization can protect children and adolescents from acquiring multi-strain viruses like HIV. Increasingly, vaccine strategies have become better tailored to elicit these broadly neutralizing antibodies (bnAbs), such as the germline-targeting HIV envelope antigens, which have shown great promise. To determine if we could elicit protective antibody responses against HIV in early life, we immunized infant and juvenile monkeys using the same vaccine strategy. In this study, we found that while both infants and juveniles developed similar levels of antibodies that could neutralize HIV, the infants showed stronger antibody responses in several key areas. Moreso, infants developed the type of early antibody response that researchers believe is needed to eventually evolve into broadly neutralizing antibodies. These findings suggest that initiating HIV vaccination earlier in life may offer a better chance at generating the bnAbs needed to protect against various HIV strains. Our work highlights the potential benefits of initiating an HIV vaccine regimen in childhood.

immunology↗

bioRxiv2025

Co-transcriptional folding orchestrates sequential multi-effector sensing by a glycine tandem riboswitch

Riboswitches are non-coding RNA motifs that regulate gene expression in response to ligand binding. The glycine tandem riboswitch (GTR) is notable because it comprises two distinct glycine aptamers that interact extensively. These inter-aptamer contacts drive conformational changes in the downstream expression platform to control gene expression. Despite extensive studies, the role of glycine and RNA folding pathways in co-transcriptional regulation remains unclear. Here, we integrate single-molecule kinetic analysis, co-transcriptional RNA structure probing, and computational modeling to reveal that the GTR processes multiple molecular inputs sequentially, guided by polymerase pausing. Our findings elucidate its stepwise 5-to-3 folding pathway and demonstrate how sequential glycine binding to each aptamer, K+ binding to a kink-turn, non-native RNA folding intermediates, inter-aptamer docking that drives binding site pre-organization, and modulation by the transcription factor NusA collectively orchestrate co-transcriptional gene regulation. These results support a model in which glycine binding cooperativity arises through non-equilibrium mechanisms rather than a classical concerted model.

molecular biology↗

bioRxiv2025

Automated and parallelized microfluidic generation of large and precisely-defined lipid nanoparticle libraries

Building on the success of lipid nanoparticles (LNPs) in vaccines, LNPs are being developed for a broad set of therapeutic applications by changing both the structures of the lipids used to formulate each LNP and their relative proportions. Because lipid synthesis and in vivo screening have been parallelized using combinatorial chemistry and LNP barcoding respectively, the manual and sequential microfluidic formulation of LNPs has become the rate-limiting step in the discovery process. In this work, we present a high-throughput, automated microfluidic platform capable of generating large, precisely-defined LNP libraries in parallel at a rate of one distinct formulation every three seconds. Each formulation is defined by varying the reagent flow ratios into one of eight microscale mixers using litho-graphically encoded fluidic resistors and dynamically controlled external pressure supplies. The microfluidic chip is integrated with custom frobotic plate handling for the rapid collection of each distinct formulation. Using this platform, we produce a library of 96 formulations, which we profile physicochemically and evaluate in terms of both in vitro and in vivo transfection.

bioengineering↗

bioRxiv2025

Map functional insulators with cross-platform Hi-C meta-analysis

CTCF forms the boundaries of topologically associating domains (TADs) but their insulating function in transcription is controversial. Here we report that functional insulators (FINs) shall be determined not only by their ability to form static loops or boundaries, but also by the dynamic context if their flanking sequences form new loops upon boundary loss. We performed a meta-analysis of nine independent Hi-C and micro-C datasets upon acute CTCF- or cohesin protein-depletion and found that only at several hundred loci, newly gained enhancers-promoters (E-P) loops can be reproducibly observed upon CTCF depletion. The new E-P loops are cohesin-dependent and associated with recurrent gene activation. These findings allow us to map FINs and define their direct target genes genome-wide. FINs are mostly present in euchromatin, do not overlap with TAD boundaries, and can be perturbed by Wapl-depletion or CTCF-displacement. We therefore conclude that FINs, but not TAD boundaries, are bona fide insulators.

genomics↗