Biology subjects

Li, S.

Publications and source records attributed to Li, S..

At least 775 records · Page 43Linked to original sources

bioRxiv2023

TRPV1 Opening is Stabilized Equally by Its Four Subunits

Capsaicin receptor TRPV1 is a nociceptor for vanilloid molecules such as capsaicin and resiniferatoxin (RTX). Even though cryo-EM structures of TRPV1 in complex with these molecules are available, how their binding energetically favors the open conformation is not known. Here we report an approach to control the number of bound RTX molecules (0-to-4) in functional mouse TRPV1. The approach allowed direct measurements of each of the intermediate open states under equilibrium conditions at both macroscopic and single-molecule levels. We found that RTX binding to each of the four subunits contributes virtually the same activation energy, which we estimated to be 1.86 kcal/mol and found to arise predominately from destabilizing the closed conformation. We further showed that sequential bindings of RTX increase open probability without altering single-channel conductance, confirming that there is likely a single open-pore conformation for TRPV1 activated by RTX.

biophysics↗

bioRxiv2023

Preclinical Basis of the Efficacy and Pharmacodynamics of Finotonlimab, a Humanized Anti-PD-1 Monoclonal Antibody with Potent Implications for Clinical Benefit

BackgroundThe antibodies of programmed cell death protein 1 (PD-1) and its ligand (PD-L1) have dramatically changed the treatment landscapes for patients with cancer. Clinical uses of PD-1 antibodies have greatly improved the overall survival and durable responses in patients across selected tumor types. MethodsWe describe the preclinical characterization of Finotonlimab, a humanized anti-PD-1 antibody, by head to head comparison with Nivolumab or Pembrolizumab. Herein, we characterized the in vitro and in vivo efficacy, PK, PD and Fc mediated effector function of Finotonlimab. The single-agent anti-tumor activity of Finotonlimab was evaluated using humanized mouse models and a human PBMC reconstituted mouse model. Furthermore, in cynomolgus monkeys, comparative PK measurements confirmed better PK profiles of Finotonlimab than that of Pembrolizumab and Nivolumab. ResultsOur data showed Finotonlimab bind to human PD-1 with significantly high affinity and effectively inhibited its interaction with its ligands, PD-L1 and PD-L2, and thus could effectively stimulate the human T cell functions in vitro and exhibited significant antitumor efficacy in vivo. In addition, Finotonlimab showed minimal impact on Fc receptor dependent effector cell activation, which may contribute to the killing of PD-1+ T cells. In cynomolgus monkeys, Finotonlimab exhibited a non-linear pharmacokinetics (PK) profile in a dose-dependent manner, and approximately 90% of consistent receptor occupancy period was observed at 168 h after a single administration of 1 mg/kg. Following a 13-week successive administration of Finotonlimab, a pharmacodynamics study indicated a sustained mean receptor occupancy of [≥] 93% of PD-1 molecules on circulating T cells in cynomolgus monkeys up to 8 weeks even at 3 mg/kg. ConclusionsTaken together, these preclinical data are encouraging and provide a basis for the efficacy and pharmacodynamics of Finotonlimab in clinical trials.

immunology↗

bioRxiv2023

Effect of oncolytic virotherapy on immune microenvironment in immune subtypes identified in gastric cancer and hepatocellular carcinoma

Tumor occurrence and progression are significantly influenced by immunity, and the immune infiltration and immune-related gene expression in solid tumors are closely correlated to the response of patients to immunotherapy. In this study, the level of tumor infiltrating immune cells in gastric cancer and hepatocellular carcinoma samples from the TCGA database were assessed using ssGSEA, and the tumor samples were divided into two subtypes (Imm_H and Imm_L) with different immune cell infiltration level. The differences in immune cell percentage and immune checkpoint gene expression between the two subtypes indicated that the Imm_H group had higher levels of immune infiltration, but also more infiltrated immunosuppressive cells and higher mRNA levels of immune checkpoint genes. Then the immune subtype-specific gene network was built and the main modules representing the genes and functions that differ between the two immune subtypes were identified. To explore the effect of oncolytic virus on tumor immune microenvironment, we constructed the previously developed synthetic adenovirus containing the synthetic sensory switch gene circuit, assessed the antitumor effect in mouse models, and measured the proportion of different cell types by single-cell RNA sequencing. The results showed that synthetic oncolytic virus inhibited tumor development and altered the proportion of infiltrating immune cells, suggesting that synthetic oncolytic virus may have different mechanism on the two immune subtypes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=168 HEIGHT=200 SRC="FIGDIR/small/524374v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@dffa9dorg.highwire.dtl.DTLVardef@be8e2eorg.highwire.dtl.DTLVardef@68acf6org.highwire.dtl.DTLVardef@33862f_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2023

Primate superior colliculus is engaged in abstract higher-order cognition

Categorization is a fundamental cognitive process by which the brain assigns stimuli to behaviorally meaningful groups. Investigations of visual categorization in primates have identified a hierarchy of cortical areas that are involved in the transformation of sensory information into abstract category representations. However, categorization behaviors are ubiquitous across diverse animal species, even those without a neocortex, motivating the possibility that subcortical regions may contribute to abstract cognition in primates. One candidate structure is the superior colliculus (SC), an evolutionarily conserved midbrain region that, although traditionally thought to mediate only reflexive spatial orienting, is involved in cognitive tasks that require spatial orienting. Here, we reveal a novel role of the primate SC in abstract, higher-order visual cognition. We compared neural activity in the SC and the posterior parietal cortex (PPC), a region previously shown to causally contribute to category decisions, while monkeys performed a visual categorization task in which they report their decisions with a hand movement. The SC exhibits stronger and shorter-latency category encoding than the PPC, and inactivation of the SC markedly impairs monkeys category decisions. These results extend SCs established role in spatial orienting to abstract, non-spatial cognition.

neuroscience↗

bioRxiv2023

Discovering dynamic plant enzyme complexes in yeast for novel alkaloid pathway identification from a medicinal plant kratom

Discovering natural product biosynthetic pathways from medicinal plants is challenging and laborious, largely due to the complexity of the transcriptomics-driven pathway prediction process. Here we developed a novel approach that captures the protein-level connections between enzymes for pathway discovery with improved accuracy. We proved that heterologous protein-protein interaction screening in yeast enabled the efficient discovery of both dynamic plant enzyme complexes and the pathways they organize. This approach discovered complexes and pathways in the monoterpene indole alkaloid metabolism of a medicinal plant, kratom with high success rate. Screening using a strictosidine {beta}-D-glucosidase (MsSGD1) against 19 medium-chain dehydrogenase/reductases (MsMDRs) identified five MsSGD1-MsMDR complexes. Three out of the five interacting MsMDRs were then proven functional, while the remaining 14 non-interacting candidates did not show obvious activities. The work discovered three branched pathways by combining transcriptomics, metabolomics, and heterologous PPI screening and demonstrated a new plant pathway discovery strategy.

synthetic biology↗

bioRxiv2023

Phosphorylation and Lamin B receptor modulate the accumulation of PERIOD protein foci

Circadian clock drives the 24h rhythm in our behavior and physiology. Previously, we have shown that inner nuclear membrane protein Lamin B receptor (LBR) regulates circadian rhythm in human cells and Drosophila, while the underlying mechanism is unclear (Lin et al., 2014). A very recent study reported that the circadian clock protein PERIOD is organized into discrete foci at the nuclear envelope in fly circadian neurons, which is believed to be important for controlling the subcellular localization of clock genes. Loss of LBR leads to disruption of these foci, but how they are regulated is yet unknown. Here we found that LBR likely facilitates PER foci accumulation by destabilizing the catalytic subunit of protein phosphatase 2A, MICROTUBULE STAR (MTS). MTS is known to dephosphorylate PER and hampers the accumulation of PER foci. On the other hand, the circadian kinase DOUBLETIME (DBT) which phosphorylates PER enhances the accumulation of the foci. These foci are likely phase-separated condensates, the formation of which mediated by intrinsically disordered region in PER. Taken together, here we demonstrate a key role for phosphorylation in promoting the accumulation of PER foci, while LBR modulates this process by impinging on the circadian phosphatase MTS.

cell biology↗

bioRxiv2023

Loss of PTPMT1 limits mitochondrial utilization of carbohydrates and leads to muscle atrophy and heart failure

While mitochondria in different tissues have distinct preferences for energy sources, they are flexible in utilizing competing substrates for metabolism according to physiological and nutritional circumstances. However, the regulatory mechanisms and significance of metabolic flexibility are not completely understood. Here we report that the deletion of PTPMT1, a mitochondria-based phosphatase, critically alters mitochondrial fuel selection - the utilization of pyruvate, a key mitochondrial substrate derived from glucose (the major simple carbohydrate), is inhibited, whereas the fatty acid utilization is enhanced. PTPMT1 knockout does not impact the development of the skeletal muscle or heart. However, the metabolic inflexibility ultimately leads to muscular atrophy, heart failure, and sudden death. Mechanistic analyses reveal that the prolonged substrate shift from carbohydrates to lipids causes oxidative stress and mitochondrial destruction, which in turn results in marked accumulation of lipids and profound damage in the knockout muscle cells and cardiomyocytes. Interestingly, PTPMT1 deletion from the liver or adipose tissue does not generate any local or systemic defects. These findings suggest that PTPMT1 plays an important role in maintaining mitochondrial flexibility and that their balanced utilization of carbohydrates and lipids is essential for both the skeletal muscle and the heart despite the two tissues having different preferred energy sources.

cell biology↗

bioRxiv2023

Subclonal somatic copy number alterations emerge and dominate in recurrent osteosarcoma

Multiple large-scale tumor genomic profiling efforts have been undertaken in osteosarcoma, however, little is known about the spatial and temporal intratumor heterogeneity and how it may drive treatment resistance. We performed whole-genome sequencing of 37 tumor samples from eight patients with relapsed or refractory osteosarcoma. Each patient had at least one sample from a primary site and a metastatic or relapse site. We identified subclonal copy number alterations in all but one patient. We observed that in five patients, a subclonal copy number clone from the primary tumor emerged and dominated at subsequent relapses. MYC gain/amplification was enriched in the treatment-resistant clone in 6 out of 7 patients with more than one clone. Amplifications in other potential driver genes, such as CCNE1, RAD21, VEGFA, and IGF1R, were also observed in the resistant copy number clones. Our study sheds light on intratumor heterogeneity and the potential drivers of treatment resistance in osteosarcoma. SignificanceSubclonal copy number clones emerged and dominated in relapsed osteosarcoma, with MYC gain/amplification being the defining characteristic in our cohort. Selective pressure from neoadjuvant chemotherapy revealed this clone at the time of primary resection, highlighting that genomic profiling at this time may identify clones that are selected for, or determine innate resistance to primary chemotherapy.

cancer biology↗

bioRxiv2023

Generalized tree structure to annotate untargeted metabolomics and stable isotope tracing data

In untargeted metabolomics, multiple ions are often measured for each original metabolite, including isotopic forms and in-source modifications, such as adducts and fragments. Without prior knowledge of the chemical identity or formula, computational organization and interpretation of these ions is challenging, which is the deficit of previous software tools that perform the task using network algorithms. We propose here a generalized tree structure to annotate ions to relationships to the original compound and infer neutral mass. An algorithm is presented to convert mass distance networks to this tree structure with high fidelity. This method is useful for both regular untargeted metabolomics and stable isotope tracing experiments. It is implemented as a Python package (khipu), and provides a JSON format for easy data exchange and software interoperability. By generalized pre-annotation, khipu makes it feasible to connect metabolomics data with common data science tools, and supports flexible experimental designs.

bioinformatics↗

bioRxiv2023

A simple and efficient in planta transformation method based on the active regeneration capacity of plants

Plant genetic transformation strategies serve as essential tools for the genetic engineering and advanced molecular breeding of plants. However, the complicated operational protocol and low efficiency of the current transformation strategies restrict the genetic modification of most plant species. This paper describes the development of a Regenerative Activity-dependent in Planta Injection Delivery (RAPID) method based on the active regeneration capacity of plants. In this method, Agrobacterium tumefaciens was delivered to plant meristems via injection for inducing transfected renascent tissues. Stable transgenic plants were obtained by subsequent vegetative propagation of the positive renascent tissues. The method was successfully applied for the transformation of plants with strong regeneration capacity, including different genotypes of sweet potato (Ipomoea batatas), potato (Solanum tuberosum), and bayhops (I. pes-caprae). Compared to the traditional transformation methods, RAPID has a markedly high transformation efficiency (up to ~ 40%), shorter duration (less than 4 weeks), and does not require tissue culture procedures. The RAPID method therefore overcomes the limitations of traditional methods for achieving rapid in planta transformation, and can be potentially applied to a wide range of plant species that are capable of active regeneration.

plant biology↗

bioRxiv2022

Cataloguing and profiling of the gut virome in Chinese populations uncover extensive viral signatures across common diseases

The gut viral community has been linked to human physiology and health, but our knowledge of its genetic and functional contents and disease dependence is far from complete. Here, we collected 11,327 bulk or viral metagenomes from fecal samples from large-scale Chinese populations to establish a Chinese gut virus catalogue (cnGVC) comprising 67,096 nonredundant viral genomes. This catalogue included [~]70% of novel viruses that are not represented in existing gut viral databases, and allowed us to characterize the functional diversity and specificity of the gut virome. Using cnGVC, we 1) profiled the gut virome in large-scale populations and evaluated their sex- and age-related variations, 2) investigated the diversity and compositional patterns of the gut virome across common diseases by analyzing 6,314 bulk metagenomes spanning 28 disease or unhealthy statuses, and 3) identified a large number of universal viral signatures of diseases and validated their predictive ability for health status. Overall, our resources and results would contribute to the grand effort of expanding the knowledge of the human gut virome and addressing a full picture of the associations between viruses and common diseases.

microbiology↗

bioRxiv2022

Visualization of mitochondrial cristae and mtDNA evolvement and interactions with super-resolution microscopy

Mitochondrial cristae host the respiratory chain complexes composed of mitochondrial DNA (mtDNA)-encoded and nuclear-encoded proteins and are responsible for ATP production. Movement of mtDNA located in the matrix is limited due to blockade by the cristae; yet, the dynamic interaction between the inner membrane and mtDNA remains unknown due to the insufficient spatiotemporal resolution of conventional microscopy and the lack of appropriate in vivo probes targeted to the mitochondrial inner membrane. Here, we developed a novel fluorescence probe to visualize the inner membrane using low-power stimulated emission depletion (STED) microscopy. Dual-color imaging of the inner membrane and mtDNA demonstrated that mtDNA is more likely to spread at mitochondrial tips or branch points under an overall even distribution. Interestingly, exploration of forming this distribution propensity uncovered that the mitochondrial dynamics are closely related to the location of mtDNA, and further insight found that fusion always occurs near mtDNA in order to minimize the pressure for cristae remodeling. In healthy cells, mitochondrial dynamics based on cristae remodeling promotes the even distribution of mtDNA, on the contrary, when cristae structure fails in apoptosis and ferroptosis, leading to mtDNA distribution disorder. Observation of active changes during apoptosis further captured the dynamic process of inner membrane herniation and mtDNA leakage along with cristae remodeling. Under ferroptosis, the mitochondria shrank into ellipsoids and mtDNA converged at the center of mitochondria. The rich dynamics between the cristae and mtDNA, revealed at unprecedented spatiotemporal resolution, show the motive and outgrowth of mtDNA distribution.

biophysics↗

bioRxiv2022

Autophagy inspects Rim4-mRNA interaction to safeguard programmed meiotic translation

Autophagy can degrade RNA, with an intricate preference for specific RNAs. However, the mechanism remains unclear. During yeast meiosis, autophagy is active, while a subset of transcripts needs to survive until their programmed translation during and at the end of meiotic divisions. Thus, the challenge here is how meiotic autophagy temporally spares specific mRNAs. Rim4, a meiosis-specific RNA binding protein (RBP), sequesters a specific set of mid-late meiotic transcripts during early meiosis to suppress premature translation. Recently, we reported that autophagy degrades Rim4, while the underlying mechanism and the fate of transcripts in complex with Rim4 remain uncharacterized. Here, we show that Rim4 utilizes a nuclear localization signal (NLS) to enter the nucleus to load its mRNA substrates before the nuclear export. Once in the cytoplasm, the active autophagy spares Rim4-mRNA. Using combined genetic, biochemical, and cell imaging approaches, we show that autophagy selectively degrades Rim4 during meiotic divisions in an Atg11-dependent manner upon Rim4-bound mRNAs released for translation; meanwhile, released mRNAs also become sensitive to autophagy. In vitro, purified Rim4 and its RRM-motif-containing variants, but not Rim4-mRNA complex, activate Atg1 kinase activity in meiotic cell lysates and in the immunoprecipitated Atg1 complex, suggesting that the conserved RRMs are involved in stimulating Atg1 and hence selective autophagy. These findings indicate that autophagy inspects Rim4-mRNA interaction to ensure meiotic stage-specific translation.

cell biology↗

bioRxiv2022

Cdc14 spatiotemporally regulates Rim4-mRNA complex assembly and stability during meiosis

In budding yeast, Rim4 sequesters a subset of meiotic transcripts and essentially suppresses their translation until being degraded at the end of meiosis I. We found that Rim4 loads mRNAs in the nucleus as a prerequisite for relocating Rim4 into the cytoplasm, where mRNAs protect Rim4 from autophagy. Nonetheless, the underlying mechanism remains unknown. Using genetic, biochemical, and cell imaging approaches, here, we revealed that phosphorylation states regulate Rim4s intracellular interactions with mRNAs and the yeast 14-3-3 proteins Bmh1 and Bmh2. Our data showed that Rim4 forms a heterotrimeric complex with Bmh1 and Bmh2 via multiple phosphorylated sites with the consensus of a PKA kinase target. Remarkably, the Bmh1/2-Rim4 complex excludes mRNAs and is resistant to autophagy. We further found that Cdc14, a conserved cell cycle phosphatase, binds to a canonical Cdc14 docking site (PxL) in Rim4s C-terminal low complexity domain (LCD) to de-phosphorylate Rim4 at multiple sites, resulting in Bmh1/2-Rim4 disassembly. Notably, before meiotic cell divisions, Cdc14 primarily resides in the nucleus, where mRNAs are transcribed. Therefore, Cdc14-triggered Bmh1/2 dissociation facilitates the nuclear Rim4 to target and sequester the nascent mRNAs. In contrast, during the meiotic divisions, Rim4-sequestered mRNAs are released for translation, while Cdc14 mediates Rim4-Bmh1/2 disassembly in the cytoplasm due to its temporary cytoplasmic relocation at the anaphases; subsequently, loss of protection from mRNAs and Bmh1/2 leads to autophagy-mediated Rim4 degradation at this stage. We conclude that phosphorylation states spatiotemporally regulate Rim4s meiotic interactions, subcellular localization, and stability, regulated by Cdc14.

cell biology↗

bioRxiv2022

Restoring glucose homeostasis with stomach-derived human insulin-secreting organoids

Gut stem cells are accessible by biopsy and propagate robustly in culture, offering an invaluable resource for autologous cell therapies. Insulin-producing cells can be induced in mouse gut, but it has not been possible to generate abundant and durable insulin-secreting cells from human gut tissues to evaluate their potential as a cell therapy for diabetes. Here we describe a protocol to differentiate cultured human gastric stem cells (hGSCs) into pancreatic islet-like organoids containing gastric insulin-secreting (GINS) cells that resemble {beta}-cells in molecular hallmarks and function. Sequential activation of the inducing factors NGN3 and PDX1-MAFA led hGSCs onto a novel differentiation path, including a SOX4High endocrine and GalaninHigh GINS precursor, before adopting {beta}-cell identity, at efficiencies close to 70%. GINS organoids acquired glucose-stimulated insulin secretion in 10 days and restored glucose homeostasis for over 100 days in diabetic mice after transplantation, providing proof of concept for a new approach to treat diabetes.

cell biology↗

bioRxiv2022

Striated muscle-specific base editing enables correction of mutations causing dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is the second most common cause for heart failure with no cure except a high-risk heart transplantation. Approximately 30% of DCM patients harbor heritable mutations which are amenable to CRISPR-based gene therapy1. However, challenges related to delivery of the editing complex and off-target concerns hamper the broad applicability of CRISPR agents in the heart2. We employed a combination of the viral gene transfer vector AAVMYO with superior targeting specificity of heart muscle tissue3 and CRISPR base editors to repair patient mutations in the cardiac splice factor Rbm20, which cause aggressive and arrhythmogenic DCM4. Using optimized conditions, we could improve splice defects in human iPSC-derived cardiomyocytes (iPSC-CMs) and repair >70% of cardiomyocytes in two Rbm20 knock-in mouse models that we generated to serve as an in vivo platform of our editing strategy. Treatment of juvenile mice restored the localization defect of RBM20 in 75% of cells and splicing of RBM20 targets including TTN. Three months after injection, cardiac dilation and ejection fraction reached wildtype levels. Single-nuclei RNA sequencing (snRNA-seq) uncovered restoration of the transcriptional profile across all major cardiac cell types and whole-genome sequencing (WGS) revealed no evidence for aberrant off-target editing. Our study highlights the potential of base editors combined with AAVMYO to achieve gene repair for treatment of hereditary cardiac diseases.

genetics↗

bioRxiv2022

Simultaneous inhibition of DNA-PK and Pol{Theta} improves integration efficiency and precision of genome editing

Genome editing tools, especially CRISPR/Cas9-based strategies, have transformed biomedical research and opened opportunities for developing curative treatments for genetic diseases. Despite rapid progress, low efficiency of targeted DNA integration and generation of undesired mutations represent major limitations for genome editing applications. Both issues arise from the interplay between the main DNA Double-Strand Break (DSB) repair pathways, Homology-Directed Repair (HDR), Non-Homologous End Joining (NHEJ), and Microhomology-Mediated End Joining (MMEJ). To improve efficiencies of targeted CRISPR-Cas9 genome editing, we screened a large compound library. This led to the discovery of AZD7648, a DNA-dependent protein kinase (DNA-PK) inhibitor and potent enhancer of CRISPR-Cas9-mediated integration. We demonstrated that AZD7648 increased HDR and decreased mutagenic NHEJ repair, thus resulting in improved performance of precise gene editing. Furthermore, we observed additional improvement of integration efficiency by impairing MMEJ repair through DNA polymerase {ominus} (Pol{ominus}) inhibition. Combined treatment with AZD7648 and Pol{ominus} inhibitors (which we named 2iHDR) substantially increased precision of templated insertions, with efficiencies of up to 80%, and nearly no formation of undesired Insertion-Deletions (InDels). Importantly, 2iHDR also decreased Cas9-associated off-target activity, dramatically improving the performance and fidelity of CRISPR-Cas9 gene editing.

molecular biology↗

bioRxiv2022

Sfrp2 is a multifunctional regulator of rodent color patterns

Animal pigment patterns are excellent models to elucidate mechanisms of biological organization. Although theoretical simulations, such as Turing reaction-diffusion systems, recapitulate many animal patterns, they are insufficient to account for those showing a high degree of spatial organization and reproducibility. Here, we compare the coats of the African striped mouse (Rhabdomys pumilio) and the laboratory mouse (Mus musculus) to study the molecular mechanisms controlling stripe pattern formation. By combining transcriptomics, mathematical modeling, and mouse transgenics, we show that Sfrp2 regulates the distribution of hair follicles and establishes an embryonic prepattern that foreshadows pigment stripes. Moreover, by developing and employing in vivo gene editing experiments in striped mice, we find that Sfrp2 knockout is sufficient to alter the stripe pattern. Strikingly, mutants also exhibit changes in coat color, revealing an additional function of Sfrp2 in regulating hair color. Thus, a single factor controls coat pattern formation by acting both as an orienting signaling mechanism and a modulator of pigmentation. By uncovering a multifunctional regulator of stripe formation, our work provides insights into the mechanisms by which spatial patterns are established in developing embryos and the molecular basis of phenotypic novelty.

developmental biology↗