Biology subjects

Shi, L.

Publications and source records attributed to Shi, L..

At least 127 records · Page 7Linked to original sources

bioRxiv2024

A subtle modification of modafinil-based DAT inhibitors changes conformational preference

Modafinil analogs with either a sulfoxide or sulfide moiety have improved binding affinities at the human dopamine transporter (hDAT) compared to modafinil, with lead sulfoxide-substituted analogs showing characteristics of atypical inhibition (e.g., JJC8-091). Interestingly, the only distinction between sulfoxide and sulfide substitution is the presence of one additional oxygen atom. To elucidate why such a subtle difference in ligand structure can result in different typical or atypical profiles, we investigated two pairs of analogs. Our quantum mechanical calculations revealed a more negatively charged distribution of electrostatic potential surface of the sulfoxide substitution. Using molecular dynamics simulations, we demonstrated that sulfoxide-substituted modafinil analogs have a propensity to attract more water into the binding pocket. They also exhibited a tendency to dissociate from Asp79 and form a new interaction with Asp421, consequently promoting an inward-facing conformation of DAT. In contrast, sulfide-substituted analogs did not display these effects. These findings deepen our understanding of the functionally relevant conformational spectrum of DAT.

biophysics↗

bioRxiv2024

Unlocking the Potential of High-Quality Dopamine Transporter Pharmacological Data: Advancing Robust Machine Learning-Based QSAR Modeling

The dopamine transporter (DAT) plays a critical role in the central nervous system and has been implicated in numerous psychiatric disorders. The ligand-based approaches are instrumental to decipher the structure-activity relationship (SAR) of DAT ligands, especially the quantitative SAR (QSAR) modeling. By gathering and analyzing data from literature and databases, we systematically assemble a diverse range of ligands binding to DAT, aiming to discern the general features of DAT ligands and uncover the chemical space for potential novel DAT ligand scaffolds. The aggregation of DAT pharmacological activity data, particularly from databases like ChEMBL, provides a foundation for constructing robust QSAR models. The compilation and meticulous filtering of these data, establishing high-quality training datasets with specific divisions of pharmacological assays and data types, along with the application of QSAR modeling, prove to be a promising strategy for navigating the pertinent chemical space. Through a systematic comparison of DAT QSAR models using training datasets from various ChEMBL releases, we underscore the positive impact of enhanced data set quality and increased data set size on the predictive power of DAT QSAR models.

bioinformatics↗

bioRxiv2024

Engineering customized viral receptors for various coronaviruses

Coronaviruses display versatile receptor usage, yet in-depth characterization of coronaviruses lacking known receptor identities has been impeded by the absence of feasible infection models1,2. Here, we developed an innovative strategy to engineer functional customized viral receptors (CVRs). The modular design relies on building receptor frameworks comprising various function modules and generating specific epitope-targeting viral binding domains. We showed the key factors for CVRs to efficiently facilitate spike cleavage, membrane fusion, pseudovirus entry, and authentic virus propagation for various coronaviruses, resembling their native receptors. Applying this strategy, we delineated the accessible receptor binding epitopes for functional SARS-CoV-2 CVR design and elucidated the mechanism of entry supported by an amino-terminus domain (NTD) targeting S2L20-CVR. Furthermore, we created CVR-expressing cells for assessing antibodies and inhibitors against 12 representative coronaviruses from six subgenera, most of which lacking known receptors. Notably, a pan-sarbecovirus CVR supported entry of various sarbecoviruses, as well as propagation of a replicable HKU3 pseudovirus and the authentic strain RsHuB2019A3. Through combining an HKU5-specific CVR with reverse genetics, we successfully rescued and cultured wild-type and fluorescence protein-incorporated HKU5, a receptor-unidentified merbecovirus. Our study demonstrated the great potential of CVR strategy in establishing native receptor-independent infection models, paving the way for studying various viruses that are challenging to culture due to the lack of susceptible cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/583237v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@7cb9ccorg.highwire.dtl.DTLVardef@8a2855org.highwire.dtl.DTLVardef@5e14f0org.highwire.dtl.DTLVardef@b0b993_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphic abstractC_FLOATNO C_FIG

microbiology↗

bioRxiv2024

A comprehensive personal omics clinical interpreter based on genomic and transcriptomic profiles

Advances in precision medicine rely on the accurate identification and analysis of molecular alterations for personalized diagnostic, prognostic, and therapeutic decision-making. A critical obstacle is the integration of heterogeneous interpretations of clinically actionable alterations from various knowledgebases. Here, we present the Personal Omics Interpreter (POI), a web-based application engineered to aggregate and interpret therapeutic options, including targeted, immunological, and chemotherapeutic agents, by leveraging personal genomic and transcriptomic profiles. POI employs the Precision Medicine Knowledgebase (PreMedKB), an updated harmonized resource we previously reported, to annotate the clinically actionable somatic variants. It further incorporates a predictive algorithm to broaden therapeutic options according to established gene-gene interactions and offers insights into phenotypic responses of chemotherapeutic agents through phasing germline diplotypes. Validated against three cohort datasets encompassing over 22,000 cancer patients, POI demonstrates consistently high matching rates (94.7 [~] 95.6%) between patients and suggested therapies, highlighting its potential in supporting precision-driven informed treatment strategies.

genomics↗

bioRxiv2024

High-precision photoacoustic neural modulation uses a non-thermal mechanism

Neuromodulation is a powerful tool for fundamental studies in neuroscience and potential treatments of neurological disorders. Both photoacoustic (PA) and photothermal (PT) effects have been harnessed for non-genetic high-precision neural stimulation. Using a fiber-based device excitable by a nanosecond pulsed laser and a continuous wave laser for PA and PT stimulation, respectively, we systematically investigated PA and PT neuromodulation at single neuron level. Our results show that the laser energy needed for PA neurostimulaion is 1/40 of that needed for PT stimulation to achieve the same level of cell response recorded by Ca2+ imaging. The threshold energy for PA stimulation is found to be further reduced in neurons overexpressing mechano-sensitive channels, indicating the direct involvement of mechano-sensitive channels in PA stimulation. Electrophysiology study of single neurons upon PA and PT stimulation was performed by patch clamp recordings. Electrophysiological features stimulated by PA are distinct from those induced by PT, confirming that PA and PT stimulations operate through distinct mechanisms. These insights offer a foundation for rational design of more efficient and safer non-genetic neural modulation approaches.

neuroscience↗

bioRxiv2024

Virus-free CRISPR knock-in of a chimeric antigen receptor into KLRC1 generates potent GD2-specific natural killer cells

Natural killer (NK) cells are an appealing off-the-shelf, allogeneic cellular therapy due to their cytotoxic profile. However, their activity against solid tumors remains suboptimal in part due to the upregulation of NK-inhibitory ligands, such as HLA-E, within the tumor microenvironment. Here, we utilize CRISPR-Cas9 to disrupt the KLRC1 gene (encoding the HLA-E-binding NKG2A receptor) and perform non-viral insertion of a GD2-targeting chimeric antigen receptor (CAR) within NK cells isolated from human peripheral blood. Genome editing with CRISPR/Cas9 ribonucleoprotein complexes yields efficient genomic disruption of the KLRC1 gene with 98% knockout efficiency and specific knock-in of the GD2 CAR transgene as high as 23%, with minimal off-target activity as shown by CHANGE-Seq, in-out PCR, and next generation sequencing. KLRC1-GD2 CAR NK cells display high viability and proliferation, as well as precise cellular targeting and potency against GD2+ human melanoma cells. Notably, KLRC1-GD2 CAR NK cells overcome HLA-E-based inhibition by HLA-E-expressing, GD2+ melanoma cells. Using a single-step, virus-free genome editing workflow, this study demonstrates the feasibility of precisely disrupting inhibitory signaling within NK cells via CRISPR/Cas9 while expressing a CAR to generate potent allogeneic cell therapies against HLA-E+ solid tumors.

bioengineering↗

bioRxiv2024

Far-red chemigenetic biosensors for multi-dimensional and super-resolved kinase activity imaging

Fluorescent biosensors revolutionized biomedical science by enabling the direct measurement of signaling activities in living cells, yet the current technology is limited in resolution and dimensionality. Here, we introduce highly sensitive chemigenetic kinase activity biosensors that combine the genetically encodable self-labeling protein tag HaloTag7 with bright far-red-emitting synthetic fluorophores. This technology enables five-color biosensor multiplexing, 4D activity imaging, and functional super-resolution imaging via stimulated emission depletion (STED) microscopy.

bioengineering↗

bioRxiv2024

Sarbecovirus RBD indels and specific residues dictating ACE2 multi-species adaptiveness

Sarbecoviruses exhibit varying abilities in using angiotensin-converting enzyme 2 (ACE2) receptor1-3. However, a comprehensive understanding of their multi-species ACE2 adaptiveness and the underlying mechanism remains elusive, particularly for many sarbecoviruses with various receptor binding motif (RBM) insertions/deletions (indels)4-11. Here, we analyzed RBM sequences from 268 sarbecoviruses categorized into four RBM indel types. We extensively examined the capability of 14 representative sarbecoviruses and their derivatives in using ACE2 orthologues from 51 bats and five non-bat mammals. We revealed that most sarbecoviruses with longer RBMs (type-I), present broad ACE2 tropism, whereas viruses with single deletions in Region 1 (type-II) or Region 2 (type-III) generally exhibit narrow ACE2 tropism, typically favoring their hosts ACE2. Sarbecoviruses with double region deletions (type-IV) exhibit a complete loss of ACE2 usage. Subsequent investigations unveiled that both loop deletions and critical RBM residues significantly impact multi-species ACE2 tropism in different ways. Additionally, fine mapping based on type-IV sarbecoviruses elucidated the role of several clade-specific residues, both within and outside the RBM, in restricting ACE2 usage. Lastly, we hypothesized the evolution of sarbecovirus RBM indels and illustrated how loop length, disulfide, and adaptive mutations shape their multi-species ACE2 adaptiveness. This study provides profound insights into the mechanisms governing ACE2 usage and spillover risks of sarbecoviruses.

microbiology↗

bioRxiv2024

Linkers in bitopic agonists shape bias profile among transducers for the dopamine D2 and D3 receptors

Bitopic ligands bind both orthosteric and allosteric or secondary binding sites within the same receptor, often resulting in improvement of receptor selectivity, potency, and efficacy. In particular, for both agonists and antagonists of the dopamine D2 and D3 receptors (D2R and D3R), the primary therapeutic targets for several neurological and neuropsychiatric disorders, bitopic ligand design has proved advantageous in achieving better pharmacological profiles in vitro. Although the two pharmacophores within a bitopic ligand are typically considered the main drivers of conformational change for a receptor, the role of the linker that connects the two has not yet been systematically studied for its relevance in receptor activity profiles. Here, we present a comprehensive analysis of sumanirole and PF592,379-based indole-containing bitopic compounds in agonist activity at D2R and D3R, with a focus on linker chemical space and stereochemistry achieved through testing seven distinct chirally resolved linkers. The current study examines the structure activity relationships (SAR) of these linkers extensively, beyond the conventional level, by characterizing activation of all putative transducers over a 44 min time course. Our multiparametric analysis provides previously unappreciated clarity of linker-dependent effects, highlighting the utility of this applied comprehensive approach and the significance of linker type in the shaping of transducer bias profiles.

pharmacology and toxicology↗

bioRxiv2024

Development of deaminase-free T-to-S base editor and C-to-G base editor by engineered human uracil DNA glycosylase

DNA base editors could enable direct editing of adenine (A), cytosine (C), or guanine (G), but there is no base editor for direct thymine (T) editing currently. Here, by fusing Cas9 nickase (nCas9) with engineered human uracil DNA glycosylase (UNG) variants, we developed a deaminase-free glycosylase-based thymine base editor (gTBE) with the ability of direct T editing. By several rounds of UNG mutagenesis via rational screening, we demonstrated that gTBE with engineered UNG variants could achieve T editing efficiency by up to 81.5%. Furthermore, the gTBE exhibited high T-to-S (i.e., T-to-C or T-to-G) conversion ratio with up to 0.97 in cultured human cells. Using similar strategy, we developed a deaminase-free cytosine base editor (gCBE) facilitating specifically direct C editing by engineered UNG with mutations different from gTBE. Thus, we provide two novel base editors, gTBE and gCBE, with corresponding engineered UNG variants, broadening the targeting scope of base editors.

molecular biology↗

bioRxiv2023

Toward Best Practice in Identifying Subtle Differential Expression with RNA-seq: A Real-World Multi-Center Benchmarking Study Using Quartet and MAQC Reference Materials

Translating RNA-seq into clinical diagnostics requires ensuring the reliability of detecting clinically relevant subtle differential expressions, such as those between different disease subtypes or stages. Moreover, cross-laboratory reproducibility and consistency under diverse experimental and bioinformatics workflows urgently need to be addressed. As part of the Quartet project, we presented a comprehensive RNA-seq benchmarking study utilizing Quartet and MAQC RNA reference samples spiked with ERCC controls in 45 independent laboratories, each employing their in-house RNA-seq workflows. We assessed the data quality, accuracy and reproducibility of gene expression and differential gene expression and compared over 40 experimental processes and 140 combined differential analysis pipelines based on multiple ground truths. Here we show that real-world RNA-seq exhibited greater inter-laboratory variations when detecting subtle differential expressions between Quartet samples. Experimental factors including mRNA enrichment methods and strandedness, and each bioinformatics step, particularly normalization, emerged as primary sources of variations in gene expression and have a more pronounced impact on the subtle differential expression measurement. We underscored the pivotal role of experimental execution over the choice of experimental protocols, the importance of strategies for filtering low-expression genes, and optimal gene annotation and analysis tools. In summary, this study provided best practice recommendations for the development, optimization, and quality control of RNA-seq for clinical diagnostic purposes.

genomics↗

bioRxiv2023

The in vitro functional profiles of fentanyl and nitazene analogs at the μ-opioid receptor - high efficacy is dangerous regardless of signaling bias

Novel synthetic opioids (NSOs), including both fentanyl and non-fentanyl analogs that act as the -opioid receptor (MOR) agonists, are associated with serious intoxication and fatal overdose. Previous studies proposed that G protein biased MOR agonists are safer pain medications, while other evidence indicates that low intrinsic efficacy at MOR better explains reduced opioid side effects. Here, we characterized the in vitro functional profiles of various NSOs at MOR using adenylate cyclase inhibition and {beta}-arrestin2 recruitment assays, in conjunction with the application of the receptor depletion approach. By fitting the concentration-response data to the operational model of agonism, we deduced the intrinsic efficacy and affinity for each opioid in the Gi protein signaling and {beta}-arrestin2 recruitment pathways. Compared to the reference agonist DAMGO, we found that several fentanyl analogs were more efficacious at inhibiting cAMP production, whereas all fentanyl analogs were less efficacious at recruiting {beta}-arrestin2. In contrast, the non-fentanyl 2-benzylbenzimidazole (i.e., nitazene) analogs were highly efficacious and potent in both the cAMP and {beta}-arrestin2 assays. Our findings suggest that the high intrinsic efficacy of the NSOs in Gi protein signaling is a common property that may underlie their high risk of intoxication and overdose, highlighting the limitation of using in vitro functional bias to predict the adverse effects of opioids. Instead, our results show that, regardless of bias, opioids with sufficiently high intrinsic efficacy can be lethal, especially given the extremely high potency of many of these compounds that are now pervading the illicit drug market.

pharmacology and toxicology↗

bioRxiv2023

Deep generative model deciphers derailed trajectories in acute myeloid leukemia

Biological insights often depend on comparing conditions such as disease and health, yet we lack effective computational tools for integrating single-cell genomics data across conditions or characterizing transitions from normal to deviant cell states. Here, we present Decipher, a deep generative model that characterizes derailed cell-state trajectories. Decipher jointly models and visualizes gene expression and cell state from normal and perturbed single-cell RNA-seq data, revealing shared and disrupted dynamics. We demonstrate its superior performance across diverse contexts, including in pancreatitis with oncogene mutation, acute myeloid leukemia, and gastric cancer.

bioinformatics↗

bioRxiv2023

Unraveling activation-related rearrangements and intrinsic divergence from ligand-induced conformational changes of the dopamine D3 and D2 receptors

Effective rational drug discovery targeting a specific protein hinges on understanding their functional states and distinguishing it from homologs. However, for the G protein coupled receptors, both the activation-related conformational changes (ACCs) and the intrinsic divergence among receptors can be misled or obscured by ligand-induced conformational changes (LCCs). Here, we unraveled ACCs and intrinsic divergence from LCCs of the dopamine D3 and D2 receptors (D3R and D2R), by analyzing their experimentally determined structures and the molecular dynamics simulation results of the receptors bound with different ligands. In addition to the ACCs common to other aminergic receptors, we revealed unique ACCs for these two receptors including TM5e and TM6e shifting away from TM2e and TM3e, with a subtle rotation of TM5e. In identifying intrinsic divergence, we found pronounced outward tilting of TM6e in the D2R compared to the D3R in both experimental structures and simulations with ligands in different scaffolds. This tilting was drastically reduced in the simulations of the receptors bound with nonselective full agonist quinpirole, suggesting a misleading impact of LCCs. Further, in the quinpirole-bound simulations, TM1 showed a greater disparity between these receptors, indicating that LCCs may obscure intrinsic divergence. In addition, our analysis showed that the impact of the nonconserved TM1 propagated to conserved Trp7.40 and Glu2.65, both are ligand binding residues. We also found that the D2R exhibited heightened flexibility compared to the D3R in the extracellular portions of TMs 5, 6, and 7, potentially associated with its greater ligand binding site plasticity. Our results lay the groundwork for crafting ligands specifically targeting D2R or D3R with more precise pharmacological profiles.

biophysics↗

bioRxiv2023

A Fuzzy sequencer for rapid DNA fragment counting and genotyping

High-throughput sequencing technologies generate a vast number of DNA sequence reads simultaneously, which are subsequently analyzed using the information contained within these fragmented reads. The assessment of sequencing technology relies on information efficiency, which measures the amount of information entropy produced per sequencing reaction cycle. In this study, we propose a fuzzy sequencing strategy that exhibits information efficiency more than twice of currently prevailing cyclic reversible terminator sequencing methods. To validate our approach, we developed a fully functional and high-throughput fuzzy sequencer. This sequencer implements a highly efficient fluorogenic sequencing-by-synthesis chemistry and underwent testing across various application scenarios, including copy-number variation detection, noninvasive prenatal testing, transcriptome profiling, mutation genotyping, and metagenomic profling. Our findings unequivocally demonstrate that the fuzzy sequencing strategy outperforms existing methods in terms of information efficiency and delivers accurate resequencing results with faster turnaround times. One Sentence SummaryA fuzzy sequencer can exceed current limit of information efficiency of DNA sequencers for resequencing applications.

genomics↗

bioRxiv2023

DrugMap: A quantitative pan-cancer analysis of cysteine ligandability

Cysteine-focused chemical proteomic platforms have accelerated the clinical development of covalent inhibitors of a wide-range of targets in cancer. However, how different oncogenic contexts influence cysteine targeting remains unknown. To address this question, we have developed DrugMap, an atlas of cysteine ligandability compiled across 416 cancer cell lines. We unexpectedly find that cysteine ligandability varies across cancer cell lines, and we attribute this to differences in cellular redox states, protein conformational changes, and genetic mutations. Leveraging these findings, we identify actionable cysteines in NF{kappa}B1 and SOX10 and develop corresponding covalent ligands that block the activity of these transcription factors. We demonstrate that the NF{kappa}B1 probe blocks DNA binding, whereas the SOX10 ligand increases SOX10-SOX10 interactions and disrupts melanoma transcriptional signaling. Our findings reveal heterogeneity in cysteine ligandability across cancers, pinpoint cell-intrinsic features driving cysteine targeting, and illustrate the use of covalent probes to disrupt oncogenic transcription factor activity.

systems biology↗

bioRxiv2023

Integrating large-scale meta-GWAS and PigGTEx resources to decipher the genetic basis of complex traits in pig.

Understanding the molecular and cellular mechanisms that underlie complex traits in pigs is crucial for enhancing their genetic improvement program and unleashing their substantial potentials in human biomedicine research. Here, we conducted a meta-GWAS analysis for 232 complex traits with 28.3 million imputed whole-genome sequence variants in 70,328 individuals from 14 pig breeds. We identified a total of 6,878 genomic regions associated with 139 complex traits. By integrating with the Pig Genotype-Tissue Expression (PigGTEx) resource, we systemically explored the biological context and regulatory circuits through which these trait-associated variants act and finally prioritized 16,664 variant-gene-tissue-trait circuits. For instance, rs344053754 regulates the expression of UGT2B31 in the liver by affecting the activity of regulatory elements and ultimately influences litter weight at weaning. Furthermore, we investigated the conservation of genetic and regulatory mechanisms underlying 136 human traits and 232 pig traits. Overall, our multi-breed meta-GWAS in pigs provides invaluable resources and novel insights for understanding the regulatory and evolutionary mechanisms of complex traits in both pigs and humans.

genetics↗

bioRxiv2023

Herpes zoster mRNA vaccine induces superior vaccine immunity over licensed vaccine in mice and rhesus macaques

Herpes zoster remains an important global health issue and mainly occurs in aged and immunocompromised individuals with an early exposure history to Varicella Zoster Virus (VZV). Although the licensed vaccine Shingrix has a remarkably high efficacy, undesired reactogenicity and increasing global demand causing vaccine shortage urged the development of improved or novel VZV vaccines. In this study, we developed a novel VZV mRNA vaccine candidate (named as ZOSAL) containing sequence-optimized mRNAs encoding full-length glycoprotein E encapsulated in an ionizable lipid nanoparticle. In mice and rhesus macaques, ZOSAL demonstrated superior immunogenicity and safety in multiple aspects over Shingrix, especially in the induction of strong T cell immunity. Transcriptomic analysis revealed that both ZOSAL and Shingrix could robustly activate innate immune compartments, especially Type-I IFN signaling and antigen processing/presentation. Multivariate correlation analysis further identified several early factors of innate compartments that can predict the magnitude of T cell responses, which further increased our understanding of the mode of action of two different VZV vaccine modalities. Collectively, our data demonstrated the superiority of VZV mRNA vaccine over licensed subunit vaccine. The mRNA platform therefore holds prospects for further investigations in next-generation VZV vaccine development.

immunology↗