Biology subjects

Lee, H.

Publications and source records attributed to Lee, H..

At least 253 records · Page 14Linked to original sources

bioRxiv2023

Enhancing CRISPR prime editing by reducing misfolded pegRNA interactions

CRISPR prime editing (PE) requires a Cas9 nickase-reverse transcriptase fusion protein (known as PE2) and a prime editing guide RNA (pegRNA), an extended version of a standard guide RNA (gRNA) that both specifies the intended target genomic sequence and encodes the desired genetic edit. Here we show that sequence complementarity between the 5 and the 3 regions of a pegRNA can negatively impact its ability to complex with Cas9, thereby potentially reducing PE efficiency. We demonstrate this limitation can be overcome by a simple pegRNA refolding procedure, which improved ribonucleoprotein-mediated PE efficiencies in zebrafish embryos by up to nearly 25-fold. Further gains in PE efficiencies of as much as 6-fold could also be achieved by introducing point mutations designed to disrupt internal interactions within the pegRNA. Our work defines simple strategies that can be implemented to improve the efficiency of PE.

genomics↗

bioRxiv2023

Plug-and-play protein biosensors using aptamer-regulated in vitro transcription

Molecular biosensors that accurately measure protein concentrations without external equipment are critical for solving numerous problems in diagnostics and therapeutics. Modularly transducing the binding of protein antibodies, protein switches or aptamers into a useful output remains challenging. Here, we develop a biosensing platform based on aptamer-regulated transcription in which aptamers integrated into transcription templates serve as inputs to molecular circuits that can be programmed to a produce a variety of responses. We modularly design molecular biosensors using this platform by swapping aptamer domains for specific proteins and downstream domains that encode different RNA transcripts. By coupling aptamer-regulated transcription with diverse transduction circuits, we rapidly construct analog protein biosensors or digital protein biosensors with detection ranges that can be tuned over two orders of magnitude. Aptamer-regulated transcription is a straightforward and inexpensive approach for constructing programmable protein biosensors suitable for diverse research and diagnostic applications. One sentence summaryWe develop a modular platform for biosensing across a wide dynamic range using aptamer-regulated transcription to detect different proteins and molecular circuits to process the RNA transcript outputs.

bioengineering↗

bioRxiv2023

Evolving Membrane-Associated Accessory Protein Variants for Improved Adeno-Associated Virus Production

Manufacturing sufficient Adeno-Associated Virus (AAV) to meet current and projected clinical needs is a significant hurdle to the growing gene therapy industry. The recently discovered membrane-associated accessory protein (MAAP) is encoded by an alternative open reading frame in the AAV cap gene that is found in all presently reported natural serotypes. Recent evidence has emerged supporting a functional role of MAAP in AAV egress, though the underlying mechanisms of MAAP function remain unknown. Here, we show that inactivation of MAAP from AAV2 by a single point mutation that is silent in the VP1 ORF (AAV2-{Delta}MAAP) decreased exosome-associated and secreted vector genome production. We hypothesized that novel MAAP variants could be evolved to increase AAV production and thus subjected a library encoding over 1E6 MAAP protein variants to five rounds of packaging selection into the AAV2-{Delta}MAAP capsid. Between each successive packaging round, we observed a progressive increase in both overall titer and ratio of secreted vector genomes conferred by the bulk selected MAAP library population. Next-generation sequencing uncovered enriched mutational features, and a resulting selected MAAP variant containing missense mutations and a novel C-terminal domain increased overall GFP transgene packaging in AAV2, AAV6, and AAV9 capsids.

bioengineering↗

bioRxiv2023

Single-cell analysis of the nervous system at small and large scales with instant partitions

Single-cell RNA sequencing is a new frontier across all biology, particularly in neuroscience. While powerful for answering numerous neuroscience questions, limitations in sample input size, and initial capital outlay can exclude some researchers from its application. Here, we tested a recently introduced method for scRNAseq across diverse scales and neuroscience experiments. We benchmarked against a major current scRNAseq technology and found that PIPseq performed similarly, in line with earlier benchmarking data. Across dozens of samples, PIPseq recovered many brain cell types at small and large scales (1,000-100,000 cells/sample) and was able to detect differentially expressed genes in an inflammation paradigm. Similarly, PIPseq could detect expected and new differentially expressed genes in a brain single cell suspension from a knockout mouse model; it could also detect rare, virally-la-belled cells following lentiviral targeting and gene knockdown. Finally, we used PIPseq to investigate gene expression in a nontraditional model species, the little skate (Leucoraja erinacea). In total, PIPSeq was able to detect single-cell gene expression changes across models and species, with an added benefit of large scale capture and sequencing of each sample.

neuroscience↗

bioRxiv2023

Radial glia control microglial differentiation via integrin avb8-dependent trans-activation of TGFB1

Microglia diversity emerges from interactions between intrinsic genetic programs and environment-derived signals, but how these processes unfold and interact in the developing brain remains unclear. Here, we show that radial glia-expressed integrin beta 8 (ITGB8) expressed in radial glia progenitors activates microglia-expressed TGF{beta}1, permitting microglial development. Domain-restricted deletion of Itgb8 in these progenitors establishes complementary regions with developmentally arrested "dysmature" microglia that persist into adulthood. In the absence of autocrine TGF{beta}1 signaling, we find that microglia adopt a similar dysmature phenotype, leading to neuromotor symptoms almost identical to Itgb8 mutant mice. In contrast, microglia lacking the TGF{beta} signal transducers Smad2 and Smad3 have a less polarized dysmature phenotype and correspondingly less severe neuromotor dysfunction. Finally, we show that non-canonical (Smad-independent) signaling partially suppresses disease and development associated gene expression, providing compelling evidence for the adoption of microglial developmental signaling pathways in the context of injury or disease.

immunology↗

bioRxiv2023

Plant-to-plant reactive oxygen signal transmission via a Cuscuta bridge

Cuscuta, commonly known as dodder, is a parasitic plant that thrives by attaching itself to the stems of other plants. It is found across the globe and is notorious for obstructing crop growth as a weed. Over the past decade, Cuscuta has been used to gain insights into plant-plant interactions and molecular trafficking. Here, we report that two plants connected via a Cuscuta bridge can exchange rapid systemic calcium, electric, and reactive oxygen species signals. These findings suggest that plant interactions with Cuscuta may have beneficial effects to plants, enabling them to rapidly communicate with each other.

plant biology↗

bioRxiv2023

PAK3 downregulation induces cognitive impairment following cranial irradiation

Cranial irradiation is used for prophylactic brain radiotherapy as well as treatment of primary brain tumors. Despite its high efficiency, it often induces unexpected side effects, including cognitive dysfunction. Herein, we observed that mice exposed to cranial irradiation exhibited cognitive dysfunction, including altered spontaneous behavior, decreased spatial memory, and reduced novel object recognition. Analysis of actin cytoskeleton revealed that ionizing radiation (IR) disrupted the filamentous/globular actin (F/G-actin) ratio and downregulated the actin turnover signaling pathway p21-activated kinase 3 (PAK3)-LIM kinase 1 (LIMK1)-cofilin. Furthermore, we found that IR could upregulate microRNA-206-3p (miR-206-3p) targeting PAK3. As the inhibition of miR-206-3p through antagonist (antagomiR), IR-induced disruption of PAK3 signaling is restored. In addition, intranasal administration of antagomiR-206-3p recovered IR-induced cognitive impairment in mice. Our results suggest that cranial irradiation-induced cognitive impairment could be ameliorated by regulating PAK3 through antagomiR-206-3p, thereby affording a promising strategy for protecting cognitive function during cranial irradiation, and promoting quality of life in patients with radiation therapy.

neuroscience↗

bioRxiv2023

Decision-consistent bias mediated by drift dynamics of human visual working memory

Sensory evidence tends to be fleeting, often unavailable when we categorize or estimate world features. To overcome this, our brains sustain sensory information in working memory. Although keeping that information accurate while acting on it is vital, humans display two canonical biases: estimates are biased toward a few stimuli ("stimulus-specific bias") and prior decisions ("decision-consistent bias"). Integrative--especially neural mechanistic--accounts of these biases remain scarce. Here, we identify drift dynamics toward discrete attractors as a common source of both biases in orientation estimation, with decisions further steering memory states. Behavior and neuroimaging data reveal how these biases co-evolve through the decision-steered attractor dynamics. Task-optimized recurrent neural networks suggest neural mechanisms that enable categorical decisions to emerge from working memory for continuous stimuli while updating its trajectory, warping decision-consistent biases under stimulus-specific drift.

neuroscience↗

bioRxiv2023

A Platform for Controlled Template-Independent Enzymatic Synthesis of RNA Oligonucleotides and Therapeutics

Therapeutic RNA oligonucleotides have shown tremendous potential to manage and treat disease, yet current manufacturing methods cannot deliver on this promise. Here, we report the development and optimization of a novel, aqueous-based, template-independent enzymatic RNA oligonucleotide synthesis platform as an alternative to traditional chemical methodologies. Our platform is made possible by reversible terminator nucleoside triphosphates and an enzyme capable of their incorporation. We show that many common therapeutic RNA modifications are compatible with our process and demonstrate the enzymatic synthesis of natural and modified oligonucleotides in both liquid and solid phases. Our platform offers many unique advantages over chemical synthesis, including the realization of a more sustainable process to produce therapeutic RNA oligonucleotides. One-Sentence SummaryAn enzyme and novel nucleotide building blocks are used to synthesize RNA oligonucleotides template independently under aqueous conditions.

synthetic biology↗

bioRxiv2023

Tiny Earth CURE improves student persistence in science

Course-based undergraduate research experiences (CUREs) enhance student retention in STEM, particularly among students who belong to historically excluded communities. Yet the mechanisms by which CUREs contribute to student integration and persistence are poorly understood. Utilizing the Tripartite Integration Model of Social Influence (TIMSI), this longitudinal study examines how Tiny Earth, an antibiotic-discovery CURE, impacts students scientific self-efficacy, scientific identity, endorsement of scientific community values, and intentions to persist in science. The study also explores how gains in TIMSI factors vary as a function of student demographics and course characteristics. Results of pre-and post-course measurements show that scientific self-efficacy and identity increased among students in Tiny Earth, and some student demographics and course characteristics moderated these gains. Gains in scientific self-efficacy, identity, and values correlated with gains in persistence intentions, whereas student demographics and course characteristics did not. Results of this study show that the Tiny Earth CURE enhanced students integration into the scientific community, which was linked to intentions of students of both historically underrepresented and majority groups to persist in STEM. We discuss how courses that provide opportunity to learn science skills in the context of a CURE can contribute toward enlarging and diversifying the STEM workforce.

scientific communication and education↗

bioRxiv2023

Discrete projections from MCH neurons mediate anti-parkinsonian effects of acupuncture

Parkinsons disease (PD) presents with typical motor dysfunction and non-motor symptoms, including memory loss. Although acupuncture is suggested as an alternative therapy for PD, its neuroanatomical mechanisms remain unclear. We demonstrate that acupuncture ameliorates both motor and memory deficits in PD mice through activation of melanin-concentrating hormone (MCH) neurons in the lateral hypothalamus and zona incerta (LH/ZI)--MCHLH/ZI-- via nerve conduction. We identify two distinct subpopulations of MCHLH/ZI projecting to the substantia nigra and hippocampus, each of which is responsible for controlling motor and memory function. This effect can be attributed to MCH-mediated recovery from dopaminergic neurodegeneration, reactive gliosis, and impaired hippocampal synaptic plasticity. Collectively, MCHLH/ZI constitutes not only the neuroanatomical basis of acupuncture but also a potential cellular target for treating both motor and non-motor PD symptoms. One-Sentence SummaryAcupuncture alleviates both motor and non-motor symptoms in Parkinsons disease by activating two distinct MCH projections.

neuroscience↗

bioRxiv2023

Resource competition can explain simplicity in microbial community assembly

Predicting the composition and diversity of communities is a central goal in ecology. While community assembly is considered hard to predict, laboratory microcosms often follow a simple assembly rule based on the outcome of pairwise competitions. This assembly rule predicts that a species that is excluded by another species in pairwise competition cannot survive in a multispecies community with that species. Despite the empirical success of this bottom-up prediction, its mechanistic origin has remained elusive. In this study, we elucidate how this simple pattern in community assembly can emerge from resource competition. Our geometric analysis of a consumer-resource model shows that trio community assembly is always predictable from pairwise outcomes when one species grows faster than another species on every resource. We also identify all possible trio assembly outcomes under three resources and find that only two outcomes violate the assembly rule. Simulations demonstrate that pairwise competitions accurately predict trio assembly with up to 100 resources and the assembly of larger communities containing up to twelve species. We then further demonstrate accurate quantitative prediction of community composition using harmonic mean of pairwise fractions. Finally, we show that cross-feeding between species does not decrease assembly rule prediction accuracy. Our findings highlight that simple community assembly can emerge even in ecosystems with complex underlying dynamics. SignificanceMultispecies microbial communities play an essential role in the health of ecosystems ranging from the ocean to the human gut. A major challenge in microbial ecology is to understand and predict which species can coexist within a community. While a simple empirical rule utilizing only pairwise outcomes successfully predicts multispecies laboratory communities, its mechanistic origin has remained unexplained. Here, we find that the observed simplicity can emerge from competition for resources. Using a generic consumer-resource model, we demonstrate that community assembly of highly complex ecosystems is nevertheless well predicted by pairwise competitions. Our results argue that community assembly can be surprisingly simple despite the potential complexity associated with competition and crossfeeding of many different resources by many different species.

ecology↗

bioRxiv2023

Effect of Terpenes from Poria Cocos: Verifying Modes of Action against Alzheimer's disease Using Molecular Docking, Drug-induced Transcriptomes and Diffusion Network

Poria cocos is used to treat various diseases because of its unique terpenes. However, no study verifying its modes of action using only its compounds based on in silico experiments has been conducted. Here, molecular docking and drug-induced transcriptome analyses were performed to confirm the modes of action of Poria cocos. Additionally, a diffusion network analysis method was proposed to confirm its modes of action against Alzheimers. First, Poria cocos terpenes were collected from standard databases for molecular docking. They were then used for large-scale molecular docking using druggable proteomes, and the modes of action of lanostane and seco-lanostane, unique to Poria cocos, were confirmed. Additionally, the brain cell line was treated with Poria cocos to produce Poria cocos-induced transcriptome data, and the transcriptome-based modes of action of Poria cocos were confirmed. Finally, a diffusion network was constructed using Alzheimers, which acts on both modes of action, and submodules of Alzheimers with which terpenes interact were identified. It was confirmed that Poria cocos acts on the Wnt signaling pathway, Zn-to-anterograde axonal transport, autophagy impairment, insulin and AGE-RAGE signaling pathways, and apoptosis mechanisms. This study identified the modes of action of Poria cocos using biological data and in silico methods.

bioinformatics↗

bioRxiv2023

A Unifying Principle for the Functional Organization of Visual Cortex

A key feature of many cortical systems is functional organization: the arrangement of neurons with specific functional properties in characteristic spatial patterns across the cortical surface. However, the principles underlying the emergence and utility of functional organization are poorly understood. Here we develop the Topographic Deep Artificial Neural Network (TDANN), the first unified model to accurately predict the functional organization of multiple cortical areas in the primate visual system. We analyze the key factors responsible for the TDANNs success and find that it strikes a balance between two specific objectives: achieving a task-general sensory representation that is self-supervised, and maximizing the smoothness of responses across the cortical sheet according to a metric that scales relative to cortical surface area. In turn, the representations learned by the TDANN are lower dimensional and more brain-like than those in models that lack a spatial smoothness constraint. Finally, we provide evidence that the TDANNs functional organization balances performance with inter-area connection length, and use the resulting models for a proof-of-principle optimization of cortical prosthetic design. Our results thus offer a unified principle for understanding functional organization and a novel view of the functional role of the visual system in particular.

neuroscience↗

bioRxiv2023

Evolutionary spread of protein L-(iso)aspartyl O-methyltransferases guides the discovery of distinct isoaspartate-containing peptides, pimtides

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a structurally diverse class of natural products with a distinct biosynthetic logic, the enzymatic modification of genetically encoded precursor peptides. Although their structural and biosynthetic diversity remains largely underexplored, the identification of novel subclasses with unique structural motifs and biosynthetic pathways has been challenging. Here, we report that protein L-(iso)aspartyl O-methyltransferases (PIMTs) present in several RiPP subclasses are highly homologous. Importantly, we discovered that the apparent evolutionary transmission of the PIMT gene could serve as a basis to identify a novel RiPP subclass. Biochemical and structural analyses suggest that these homologous PIMTs commonly convert aspartate to isoaspartate via aspartyl-O-methyl ester and aspartimide intermediates, and often require cyclic or hairpin-like structures for modification. By conducting homology-based bioinformatic analysis of PIMTs, we identified over 2,800 biosynthetic gene clusters (BGCs) for known RiPP subclasses in which PIMTs install a secondary modification, and over 1,500 BGCs in which PIMTs function as a primary modification enzyme, thereby defining a new RiPP subclass, named pimtides. Our results suggest that the genome mining of proteins with secondary biosynthetic roles could be an effective strategy for discovering novel biosynthetic pathways of RiPPs. Insert Table of Contents artwork here O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/540355v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1f8afadorg.highwire.dtl.DTLVardef@1d5b2f3org.highwire.dtl.DTLVardef@d78d2eorg.highwire.dtl.DTLVardef@176070a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Delayed blood pressure recovery after exercise stress test is associated with autonomic dysfunction and pulse pressure in a middle-aged healthy group

BackgroundDelayed heart rate (HR) and blood pressure recovery after exercise test is known as the reliable indexes of autonomic dysfunction. Here we tried to evaluate the serial changes in various indicators during exercise test and correlations among them in a normotensive healthy middle-aged group. MethodsA total of 122 patients without hypertension or diabetes was enrolled (mean age, 55.6 {+/-} 11.0; male, 56.6 %; mean blood pressure, 124.8 {+/-} 16.6 / 81.5 {+/-} 9.6 mmHg). Treadmill test was performed for evaluation of chest pain. Patients with coronary artery disease, positive treadmill test result, left ventricular dysfunction or renal failure were excluded. Heart rate recovery was calculated by subtracting the HR in the first or second minute of recovery period from the HR of peak exercise (HRR1 or HRR2). Systolic blood pressure in the 4th minute of recovery stage (SBPR4) was used to show delayed blood pressure recovery. ResultsMetabolic equivalents (METs) and HR in stage 2 to 4 were significantly correlated with both HRR1 and HRR2. Multiple regression analysis of HRR revealed significant correlation of METs and SBPR4. SBPR4 was significantly correlated with both HRR1 and HRR2 (HRR1, r=-0.376, p<0.001; HRR2, r=-0.244, p=0.008). SBPR4 revealed significant correlation with SBP in the baseline to stage 3 and pulse pressure (r=0.406, p<0.001). ConclusionsDelayed BP recovery after peak exercise test revealed significant association with autonomic dysfunction and pulse pressure in normotensive middle-aged healthy group. It can be a simple and useful marker of autonomic dysfunction and arterial stiffness.

neuroscience↗

bioRxiv2023

Discovery and characterization of highly potent and selective covalent inhibitors of SARS-CoV-2 PLpro

Coronavirus infections, such as the global COVID-19 pandemic, have had a profound impact on many aspects of our daily life including working style, economy, and the healthcare system. To prevent the rapid viral transmission and speed up recovery from the infection, many academic organizations and industry research labs have conducted extensive research on discovering new therapeutic options for SARS-CoV-2. Among those efforts, RNA-dependent RNA polymerase (RdRp) inhibitors such as Remdesivir, Molnupiravir and 3CLpro inhibitor such as Nirmatrelvir (Paxlovid) have been widely used as the therapeutic options. Given the recent emergence of several new variants that caused a resurgence of the virus, it would be beneficial to discover more diverse therapeutic options with novel anti-viral mechanisms. In this regard, PLpro has been highlighted since it, along with 3CLpro, is one of the two most important proteases that are required for SARS-CoV-2 viral processing. While 3CLpro inhibitors were extensively investigated in the light of Emergency Use Authorizations of Nirmatrelvir, PLpro inhibitors have not been thoroughly investigated even preclinically. Thus, discovery efforts on antivirals acting against PLpro will be valuable. PLpro inhibitors may exert their activity by inhibiting viral replication and enhancing the host defense system through blocking virus-induced cell signaling events for evading host immune response. In this study, we report the discovery and development of two covalent irreversible PLpro inhibitors, HUP0109 and its deuterated analog DX-027, out of our quest for novel anti-COVID 19 therapeutic agents for the past two and half years. HUP0109 selectively targets the viral catalytic cleft of PLpro and covalently modifies its active site cysteine residue (C111). Promising results from preclinical evaluation suggest that DX-027 can be developed as a potential COVID-19 treatment.

pharmacology and toxicology↗

bioRxiv2023

Changes to Astrocyte-associated Protein Expression at Different Timepoints of Cuprizone Treatment

Glial cells, including astrocytes, microglia, and oligodendrocytes, are brain cells that support and dynamically interact with neurons and each other. These intercellular dynamics undergo changes during stress and disease states. In response to most forms of stress, astrocytes will undergo some variation of activation, meaning upregulation in certain proteins expressed and secreted and either upregulations or downregulations to various constitutive and normal functions. While types of activation are many and contingent on the particular disturbance that triggers these changes, there are two main overarching categories that have been delineated thus far: A1 and A2. Named in the convention of microglial activation subtypes, and with the acknowledgement that the types are not completely distinct or completely comprehensive, the A1 subtype is generically associated with toxic and pro-inflammatory factors, and the A2 phenotype is broadly associated with anti-inflammatory and neurogenic factors. The present study served to measure and document dynamic changes in these subtypes at multiple timepoints using an established experimental model of cuprizone toxic demyelination. The authors found increases in proteins associated with both cell types at different timepoints, with protein increases in the A1 marker C3d and the A2 marker Emp1 in the cortex at one week and protein increases in Emp1 in the corpus callosum at three days and four weeks. There were also increases in Emp1 staining specifically colocalized with astrocyte staining in the corpus callosum at the same timepoints as the protein increases, and in the cortex weeks later at four weeks. C3d colocalization with astrocytes also increased most at four weeks. This indicates simultaneous increases of both types of activation as well as the likely existence of astrocytes expressing both markers. The authors also found the increase in two A1 associated proteins (TNF alpha and C3d) did not show a linear relationship in line with findings from other research and indicating a more complex relationship between cuprizone toxicity and astrocyte activation. The increases in TNF alpha and IFN gamma did not occur at timepoints preceding increases in C3d and Emp1, showing that other factors also precipitate the subtypes associated (A1 for C3d and A2 for Emp1). These findings add to the body of research showing the specific early timepoints at which A1 and A2 markers are most increased during the course of cuprizone treatment, including the fact that these increases can be non-linear in the case of Emp1. This provides additional information on optimal times for targeted interventions during the cuprizone model.

neuroscience↗