Biology subjects

Ye, L.

Publications and source records attributed to Ye, L..

At least 91 records · Page 5Linked to original sources

bioRxiv2022

A Bispecific Antibody Targeting RBD and S2 Potently Neutralizes SARS-CoV-2 Omicron and Other Variants of Concern

Emerging severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) variants, especially the Omicron variant, have impaired the efficacy of existing vaccines and most therapeutic antibodies, highlighting the need for additional antibody-based tools that can efficiently neutralize emerging SARS-CoV-2 variants. The use of a "single" agent to simultaneously target multiple distinct epitopes on the spike is desirable to overcome the neutralizing escape of SARS-CoV-2 variants. Herein, we generated a human-derived IgG-like bispecific antibody (bsAb), Bi-Nab35B5-47D10, which successfully retained the specificity and simultaneously bound to the two distinct epitopes on RBD and S2. Bi-Nab35B5-47D10 showed improved spike binding breadth among wild-type (WT) SARS-CoV-2, variants of concern (VOCs) and variants being monitored (VBMs) compared with its parental mAbs. Furthermore, pseudotyped virus neutralization demonstrated that Bi-Nab35B5-47D10 can efficiently neutralize VBMs including Alpha (B.1.1.7), Beta (B.1.351) and Kappa (B.1.617.1) and VOCs including Delta (B.1.617.2), Omicron BA.1 and Omicron BA.2. Crucially, Bi-Nab35B5-47D10 substantially improved neutralizing activity against Omicron BA.1 (IC50= 27.3 ng/mL) and Omicron BA.2 (IC50= 121.1 ng/mL) compared with their parental mAbs. Therefore, Bi-Nab35B5-47D10 represents a potential effective countermeasure against SARS-CoV-2 Omicron and other variants of concern. ImportanceThe new highly contagious SARS-CoV-2 Omicron variant caused substantial breakthrough infections and has become the dominant strain in countries across the world. Omicron variants usually bear high mutations in the spike protein and exhibit considerable escape of most potent neutralization monoclonal antibodies and reduced efficacy of current COVID-19 vaccines. The development of neutralizing antibodies with potent efficacy against the Omicron variant is still an urgent priority. Here, we generated a bsAb, Bi-Nab35B5-47D10, that simultaneously targets SARS-CoV-2 RBD and S2 and improved neutralizing potency and breadth against SARS-CoV-2 WT and the tested variants compared with their parental antibodies. Notably, Bi-Nab35B5-47D10 has more potent neutralizing activity against the VOC Omicron pseudotyped virus. Therefore, Bi-Nab35B5-47D10 is a feasible and potentially effective strategy to treat and prevent COVID-19.

immunology↗

bioRxiv2022

Double knockout CRISPR screen in cancer resistance to T cell cytotoxicity

Immunotherapy has transformed cancer treatments; however, a large fraction of patients encounter resistance. Such resistance is mediated by complex factors, often involving interactions between multiple genes. Thus, it is crucially important to identify genetic interactions between genes that are significantly mutated in cancer patients and those involved in immune responses, ideally the ones that currently have chemical compounds for direct targeting. To systematically interrogate such genetic interactions that mediate cancer cells response to T cell killing, we designed an asymmetric CRISPR/Cas9 dual perturbation library targeting the matched combinations between significantly mutated tumor suppressors and immune resistance genes. We performed a combinatorial double knockout screen on 1,159 gene pairs and identified those where joint loss-of-function renders altered cellular response to T cell cytotoxicity. With individual double knockout constructs we validated these genetic interactions including Jak1-Trp53, Jak1-Kmt2d, and Ifngr1-Kmt2d. Interactions between significantly mutated tumor suppressors and potentially druggable immune resistance genes may offer insights on potential new concepts of how to target clinically relevant cancer mutations with currently available agents. This study also provides a technology platform and an asymmetric CRISPR double knockout library for interrogating genetic interactions between cancer mutations and immune resistance pathways under various settings.

genomics↗

bioRxiv2022

Targeting fatty acid beta-oxidation impairs monocyte differentiation and prolongs heart allograft survival

AO_SCPLOWBSTRACTC_SCPLOWMonocytes play an important role in the regulation of alloimmune responses after heart transplantation (HTx). Recent studies have highlighted the importance of immunometabolism in the differentiation and function of myeloid cells. While the importance of glucose metabolism in monocyte differentiation and function has been reported, a role for fatty acid {beta}-oxidation (FAO) has not been explored. Heterotopic HTx was performed using hearts from Balb/c donor mice implanted into C57Bl/6 recipient mice and treated with etomoxir (eto), an irreversible inhibitor of carnitine palmitoyltransferase 1 (Cpt1), a rate-limiting step of FAO, or vehicle control. FAO inhibition prolonged HTx survival, reduced early T cell infiltration/ activation and reduced dendritic cell (DC) and macrophage infiltration to heart allografts of eto-treated HTx recipients. ELISPOT demonstrated eto-treated HTx were less reactive to activated donor antigen presenting cells. FAO inhibition reduced monocyte-to-DC and monocyte-to-macrophage differentiation in vitro and in vivo. Further, FAO inhibition did not alter the survival of heart allografts when transplanted into Ccr2-deficient recipients, suggesting the effects of FAO inhibition on reduced immune cell infiltration and increased heart allograft survival were dependent on monocyte mobilization. Finally, we confirmed the importance of FAO on monocyte differentiation in vivo using conditional deletion of Cpt1a. Our findings demonstrate that targeting FAO attenuates alloimmunity after HTx, in part through impairing monocyte differentiation.

immunology↗

bioRxiv2022

Hedgehogs are the major amplifying hosts of severe fever with thrombocytopenia syndrome virus

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne bandavirus mainly transmitted by Haemaphysalis longicornis in East Asia, mostly in rural areas. To date, the amplifying host involved in the natural transmission of SFTSV remains unidentified. Our epidemiological field survey conducted in endemic areas in China showed that hedgehogs were widely distributed, had heavy tick infestations, and had high SFTSV seroprevalence and RNA prevalence. After experimental infection of Erinaceus amurensis and Atelerix albiventris hedgehogs with SFTSV, robust but transitory viremias were detected, which lasted for around nine to eleven days. The infected hedgehogs experienced light weight loss and histopathology of the spleen showed hemorrhagic necrosis and lymphopenia, with infected hedgehogs recovering after viral clearance. Remarkably, SFTSV transmission cycle between hedgehogs and nymph/adult H. longicornis was easily accomplished under laboratory condition with 100% efficiency. Furthermore, naive H. longicornis ticks could be infected by SFTSV-positive ticks co-feeding on naive hedgehogs, with transstadial transmission of SFTSV also confirmed. We also found that SFTSV viremia remained high in hedgehogs during hibernation, suggesting that this mechanism might contribute to the persistence of SFTSV from one year to the next. Of concern, we recently found evidence of the natural circulation of SFTSV in the urban area of Beijing City in China involving H. longicornis ticks and E. amurensis hedgehogs. Our study suggests that the hedgehogs are the major wildlife amplifying hosts of SFTSV and that urban outbreaks of SFTSV might occur in the future.

microbiology↗

bioRxiv2022

Control of Leaf Width by the APC/CTAD1-WL1-NAL1 Pathway in Rice

Leaf morphology is one of the most important features of the ideal plant architecture. However, the genetic and molecular mechanisms controlling leaf morphology in crops remain largely unknown, despite their central importance. Here we demonstrate that the APC/CTAD1-WL1-NAL1 pathway regulates leaf width in rice, and mutation of WL1 leads to width leaf variation. WL1 interacts with TAD1 and is degraded by APC/CTAD1, with the loss of TAD1 function resulting in narrow leaves. The WL1 protein directly binds to the regulatory region of NAL1 and recruits the corepressor TOPLESS-RELATED PROTEIN to inhibit NAL1 expression by down-regulating the level of histone acetylation of chromatin. Furthermore, biochemical and genetic analyses revealed that TAD1, WL1, and NAL1 function in a common pathway to control leaf width. Our study establishes an important framework for the APC/CTAD1-WL1-NAL1 pathway-mediated control of leaf width in rice and introduces novel perspectives for using this regulatory pathway for improving crop plant architecture.

plant biology↗

bioRxiv2022

Hbxip (Lamtor5) is essential for embryogenesis and regulates embryonic stem cell differentiation through activating mTORC1

Hbxip, also named Lamtor5, has been well characterized as a transcriptional coactivator in various cancers. However, the role of Hbxip in normal development remains unexplored. Here, we demonstrated that homozygous knockout of Hbxip leads to embryonic lethality, with retarded growth around E7.5. Using Hbxip knockout embryonic stem cells (ESCs), we showed that depletion of Hbxip compromises the self-renewal of ESCs, with reduced expression of pluripotency genes, reduced cell proliferation, and decreased colony forming capacity. In addition, Hbxip-/- ESCs are defective in differentiation, particularly ectodermal and mesodermal differentiation. Consistently, Hbxip-/- epiblast fails to differentiate properly, indicated by sustained expression of Oct4 in E8.5 Hbxip-/- epiblast. Mechanistically, in ESCs, Hbxip interacts with other components of the Ragulator complex, which is required for mTORC1 activation by amino acids. Importantly, ESCs depleted of Ragulator subunits, Lamtor3 or Lamtor4, display differentiation defects similar to those of Hbxip-/- ESCs. Moreover, Hbxip-/-, p14-/-, and p18-/- mice, lacking subunits of the Ragulator complex, also share similar phenotypes, embryonic lethality and retarded growth around E7-8. Thus, we conclude that Hbxip plays a pivotal role in the development and differentiation of the epiblast, as well as the self-renewal and differentiation of ESCs, through activating mTORC1 signaling.

developmental biology↗

bioRxiv2021

Monospecific and bispecific monoclonal SARS-CoV-2 neutralizing antibodies that maintain potency against B.1.617

COVID-19 pathogen SARS-CoV-2 has infected hundreds of millions and caused over 5 million deaths to date. Although multiple vaccines are available, breakthrough infections occur especially by emerging variants. Effective therapeutic options such as monoclonal antibodies (mAbs) are still critical. Here, we report the development, cryo-EM structures, and functional analyses of mAbs that potently neutralize SARS-CoV-2 variants of concern. By high-throughput single cell sequencing of B cells from spike receptor binding domain (RBD) immunized animals, we identified two highly potent SARS-CoV-2 neutralizing mAb clones that have single-digit nanomolar affinity and low-picomolar avidity, and generated a bispecific antibody. Lead antibodies showed strong inhibitory activity against historical SARS-CoV-2 and several emerging variants of concern. We solved several cryo-EM structures at [~]3 [A] resolution of these neutralizing antibodies in complex with prefusion spike trimer ectodomain, and revealed distinct epitopes, binding patterns, and conformations. The lead clones also showed potent efficacy in vivo against authentic SARS-CoV-2 in both prophylactic and therapeutic settings. We also generated and characterized a humanized antibody to facilitate translation and drug development. The humanized clone also has strong potency against both the original virus and the B.1.617.2 Delta variant. These mAbs expand the repertoire of therapeutics against SARS-CoV-2 and emerging variants.

immunology↗

bioRxiv2021

A human monoclonal antibody potently pan-neutralizes SARS-CoV-2 VOCs by targeting RBD invariant sites

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of novel corona virus disease (COVID-19). The neutralizing monoclonal antibodies (mAbs) targeting the receptor binding domain (RBD) of SARS-CoV-2 are among the most promising strategies to prevent and treat COVID-19. However, SARS-CoV-2 variants of concern (VOCs) profoundly reduced the efficacies of most of mAbs and vaccines approved for clinical use. Herein, we demonstrated mAb 35B5 efficiently neutralizes both wild-type (WT) SARS-CoV-2 and VOCs, including B.1.617.2 (delta) variant, in vitro and in vivo. Cryo-electron microscopy (cryo-EM) revealed that 35B5 neutralizes SARS-CoV-2 by targeting a unique epitope that avoids the prevailing mutation sites on RBD identified in circulating VOCs, providing the molecular basis for its pan-neutralizing efficacy. The 35B5-binding epitope could also be exploited for the rational design of a universal SARS-CoV-2 vaccine.

immunology↗

bioRxiv2021

Severe fever with thrombocytopenia syndrome virus and parthenogenetic Asian longhorned tick Haemaphysalis longicornis (Acari: Ixodidae)

Severe fever with thrombocytopenia syndrome virus (SFTSV) is spreading rapidly in Asia. It is transmitted by Haemaphysalis longicornis (Asian longhorned tick, ALT), which has both parthenogenetically and sexually reproducing populations. Parthenogenetic populations were found in at least 15 provinces in China and strongly correlated with the distribution of SFTS. The distribution of SFTS cases was however poorly correlated with the distribution of populations of bisexual ALTs. Phylogeographic analysis suggested that the parthenogenetic population spread much faster than bisexual population because colonization is independent of sexual reproduction. A higher proportion of parthenogenetic ALTs were collected from migratory birds captured at an SFTS endemic area, implicating the contribution to the long-range movement of ALTs in China. The SFTSV susceptibility of parthenogenetic females was like that of bisexual females under laboratory conditions. These results suggest that parthenogenetic ALT, probably transported by migratory birds, play a major role in the rapid spread of SFTSV. Article Summary LineThe parthenogenetic population of Asian longhorned tick is broadly distributed in China and plays a major role in the long-distance spread of SFTSV and perhaps future invasion of countries outside of Asia.

evolutionary biology↗

bioRxiv2021

Hybrid ssDNA repair templates enable high yield genome engineering in primary cells for disease modeling and cell therapy manufacturing

CRISPR-Cas9 offers unprecedented opportunities to modify genome sequences in primary human cells to study disease variants and reprogram cell functions for next-generation cellular therapies. CRISPR has several potential advantages over widely used retroviral vectors including: 1) site-specific transgene insertion via homology directed repair (HDR), and 2) reductions in the cost and complexity of genome modification. Despite rapid progress with ex vivo CRISPR genome engineering, many novel research and clinical applications would be enabled by methods to further improve knock-in efficiency and the absolute yield of live knock-in cells, especially with large HDR templates (HDRT). We recently reported that Cas9 target sequences (CTS) could be introduced into double-stranded DNA (dsDNA) HDRTs to improve knock-in, but yields and efficiencies were limited by toxicity at high HDRT concentrations. Here we developed a novel system that takes advantage of lower toxicity with single-stranded DNA (ssDNA). We designed hybrid ssDNA HDRTs that incorporate CTS sites and were able to boost knock-in percentages by >5-fold and live cell yields by >7-fold relative to dsDNA HDRTs with CTS. Knock-in efficiency and yield with ssCTS HDRTs were increased further with small molecule inhibitor combinations to improve HDR. We demonstrate application of these methods across a variety of target loci, knock-in constructs, and primary human cell types to reach ultra-high HDR efficiencies (>80-90%) which we use for pathogenic gene variant modeling and universal gene replacement strategies for IL2RA and CTLA4 mutations associated with mendelian immune disorders. Finally, we develop a GMP-compatible method for fully non-viral CAR-T cell manufacturing, demonstrating knock-in efficiencies of 46-62% and generating yields of >1.5 x 109 CAR+ T cells, well above current doses for adoptive cellular therapies. Taken together, we present a comprehensive non-viral approach to model disease associated mutations and re-write targeted genome sequences to program immune cell therapies at a scale compatible with future clinical application.

synthetic biology↗

bioRxiv2021

DENIES: A deep learning based two-layer predictor for enhancing the identification of enhancers and their strength with DNA shape information

The identification of enhancers has always been an important task in bioinformatics owing to their major role in regulating gene expression. For this reason, many computational algorithms devoted to enhancer identification have been put forward over the years. To boost the performance of their methods, more features are extracted from the single DNA sequences and integrated to develop an ensemble classifier. Nevertheless, the sequence-derived features used in previous studies can hardly provide the 3D structure information of DNA sequences, which is regarded as an important factor affecting the binding preferences of transcription factors to regulatory elements like enhancers. Given that, we here propose SENIES, a DNA shape enhanced deep learning predictor, for the identification of enhancers and their strength. The predictor consists of two layers where the first layer is for enhancer and non-enhancer identification, and the second layer is for predicting the strength of enhancers. Besides utilizing two common sequence-derived features (i.e. one-hot and k-mer) as input, it introduces DNA shape for describing the 3D structures of DNA sequences. Performance comparison with state-of-the-art methods conducted on the same datasets demonstrates the effectiveness and robustness of our method. The code implementation of our predictor is publicly available at https://github.com/hlju-liye/SENIES.

bioinformatics↗

bioRxiv2021

PLURIPOTENT STEM CELL-DERIVED CARDIOVASCULAR PROGENITORS DIFFFERENTIATED ON LAMININ 221 REGENERATE AND IMPROVE FUNCTION OF INFARCTED SWINE HEARTS

BackgroundIschemic heart disease is a huge global burden where patients often have irreversibly damaged heart muscle. State-of-the-art technology using stem cell-derived products for cellular therapy could potentially replace damaged heart muscle for regenerative cardiology. Methods and ResultsPluripotent human embryonic stem cells (hESCs) were differentiated on a laminin LN521+221 matrix to cardiovascular progenitors (CVPs). Global transcriptome analyses at multiple time points by single-cell RNA-sequencing demonstrated high reproducibility (R2 > 0.95) between two hESCs lines. We identified several CVP signature genes as quality batch control parameters which are highly specific to our CVPs as compared to canonical cardiac progenitor genes. A total of 200 million CVPs were injected into the infarcted region caused by permanent ligation of the coronary arteries of 10 immunosuppressed pigs and maintained for 4- and 12-weeks post transplantation. The transplanted cells engrafted and proliferated in the infarcted area as indicated by IVIS imaging, histology staining and spatial transcriptomic analysis. Spatial transcriptomic analysis at 1 week following transplantation showed that the infarcted region expressed human genes in the same area as immunohistology sections. Heart function was analyzed by magnetic resonance imaging (MRI) and computerized tomography (CT). Functional studies revealed overall improvement in left ventricular ejection fraction by 21.35 {+/-} 3.3 %, which was accompanied by significant improvements in ventricular wall thickness and wall motion, as well as a reduction in infarction size after CVP transplantation as compared to medium control pigs (P < 0.05). Immunohistology analysis revealed maturation of the CVPs to cardiomyocytes (CMs) where the human grafts aligned with host tissue forming end-to-end connections typical for heart muscle. Electrophysiology analyses revealed electric continuity between injected and host tissue CMs. Episodes of ventricular tachyarrhythmia (VT) over a period of 25 days developed in four pigs, one pig had persistent VT, while the rest remained in normal sinus rhythm. All ten pigs survived the experiment without any VT-related death. ConclusionsWe report a highly reproducible, chemically defined and fully humanized differentiation method of hESCs for the generation of potent CVPs. This method may pave the way for lasting stem cell therapy of myocardial infarction (MI) in humans. Clinical PerspectiveO_ST_ABSWhat is New?C_ST_ABSO_LIWe present a highly reproducible, chemically defined and fully humanized laminin-based differentiation method for generation of large amounts of cardiovascular progenitors (CVP); 20 million cells in a 10 cm2 culture dish which were used for a preclinical study in pigs. C_LIO_LITransplantation of the CVPs into the myocardial infarcted pig hearts yields maturation of the progenitor cells to cardiomyocytes (CMs) and improved cardiac function (21.35 {+/-} 3.3 % LVEF improvement) using only 200 million CVPs. C_LIO_LITemporary episodes of ventricular arrhythmia (50%) were observed after CVP transplantation. No fatal ventricular arrhythmia occurred. C_LI What are the clinical implications?O_LIOur laminin-based approach generated potent CVPs in vivo and largely restored function of the damaged heart. C_LIO_LICardiovascular progenitors may provide a new and safe therapeutic strategy for myocardial infarction. C_LIO_LIThe results may have a significant impact on regenerative cardiology. C_LI

cell biology↗

bioRxiv2021

Maxent modeling for predicting the potential distribution of global talaromycosis

Talaromycosis, an invasive mycosis caused by Talaromyces marneffei (Tm), has rapidly increased in recent years, becoming an emerging pathogenic fungal disease. However, The driving factors and potential distribution of global talaromycosis is still unclear. Here, we developed maxent ecology model using environmental variables, Rhizomys distribution and HIV/AIDS epidemic to forecast ecological niche of talaromycosis worldwhile, as well as Identify the drivering factors. The constructed model had excellent performance with the area under the curve (AUC) of the receiver operating curve (ROC) of 0.997 in training data and 0.991 in testing data. Our model revealed that Rhizomys distribution, mean temperature of warmest quarter, precipitation of wettest month, HIV/AIDS epidemic and mean temperature of driest quarter were the top 5 important variables affecting talaromycosis distribution. In addition to traditional talaromycosis epidemic areas (South of the Yangtze River in China, Southeast Asian and North and Northeast India), our model also identified other potential epidemic regions, inculding parts of the North of the Yangtze River, Central America, West Coast of Africa, East Coast of South America, the Korean Peninsula and Japan. Our findings has redefined global talaromycosis, discovered hidden high-risk areas and prorvided insights about driving factors of talaromycosis distribution, which will help inform surveillance strategies and improve the effectiveness of public health interventions against Tm infections. Author SummaryOur study aims to explore the spatial ecology of talaromycosis worldwhile. The diseases burden of Talaromycosis, a neglected zoonotic disease, is continuously rising in recent years because of the sheer size of susceptible population in the setting of increased globalization, rising HIV prevalence, and emerging iatrogenic immunodeficiency conditions. Here, we used historic reported talaromycosis cases from 1964 to 2017, combined with environmental factors, Rhizomys distribution and HIV/AIDS epidemic to build an maxent ecology model to define the ecological niche of talaromycosis, then predicting the potential distribution of the disease. The ecological niche of talaromycosis is characterized by a concentrated distribution, which can be cognitively divided into two regions: traditional talaromycosis epidemic areas (South of the Yangtze River in China, Southeast Asian and North and Northeast India), while other potential epidemic regions were predicted in parts of the North of the Yangtze River, Central America, West Coast of Africa, East Coast of South America, the Korean Peninsula and Japan. Our model also identified 5 driving factors affecting talaromycosis distribution. These findings will help demonstrate the global distribution of talaromycosis, discover hidden high-risk areas, and improve the effectiveness of public health interventions against Tm infections.

ecology↗

bioRxiv2021

Talaromyces marneffei promotes M2 polarization of human macrophages by downregulating SOCS3 expression and activating TLR9 pathway

Little is known about how Talaromyces marneffei, a thermally dimorphic fungus that causes substantial morbidity and mortality in Southeast Asia, evades the human immune system. Polarization of macrophages into fungal-inhibiting M1 and fungal-promoting M2 types has been shown to play an important role in the innate immune response against fungal pathogens. This mechanism has not been defined for T. marneffei. Here, we demonstrated that T. marneffei promotes its survival in human macrophages by inducing them towards M2 polarization. Our investigations of the mechanism revealed that T. marneffei infection led to SOCS3 protein degradation by inducing tyrosine phosphorylation, thereby relieving the inhibitory effect of SOCS3 on p-STAT6, a key factor for M2 polarization. Our SOCS3-overexpression experiments showed that SOCS3 is a positive regulator of M1 polarization and plays an important role in limiting M2 polarization. Furthermore, we found that inhibition of TLR9 pathway partially blocked T. marneffei-induced M2 polarization and significantly enhanced the killing activity of macrophages against T. marneffei. Collectively, these results reveal a novel mechanism by which T. marneffei evades the immune response of human macrophages.

microbiology↗

bioRxiv2021

Learning association for single-cell transcriptomics by integrating profiling of gene expression and alternative polyadenylation

Single-cell RNA-sequencing (scRNA-seq) has enabled transcriptome-wide profiling of gene expressions in individual cells. A myriad of computational methods have been proposed to learn cell-cell similarities and/or cluster cells, however, high variability and dropout rate inherent in scRNA-seq confounds reliable quantification of cell-cell associations based on the gene expression profile alone. Lately bioinformatics studies have emerged to capture key transcriptome information on alternative polyadenylation (APA) from standard scRNA-seq and revealed APA dynamics among cell types, suggesting the possibility of discerning cell identities with the APA profile. Complementary information at both layers of APA isoforms and genes creates great potential to develop cost-efficient approaches to dissect cell types based on multiple modalities derived from existing scRNA-seq data without changing experimental technologies. We proposed a toolkit called scLAPA for learning association for single-cell transcriptomics by combing single-cell profiling of gene expression and alternative polyadenylation derived from the same scRNA-seq data. We compared scLAPA with seven similarity metrics and five clustering methods using diverse scRNA-seq datasets. Comparative results showed that scLAPA is more effective and robust for learning cell-cell similarities and clustering cell types than competing methods. Moreover, with scLAPA we found two hidden subpopulations of peripheral blood mononuclear cells that were undetectable using the gene expression data alone. As a comprehensive toolkit, scLAPA provides a unique strategy to learn cell-cell associations, improve cell type clustering and discover novel cell types by augmentation of gene expression profiles with polyadenylation information, which can be incorporated in most existing scRNA-seq pipelines. scLAPA is available at https://github.com/BMILAB/scLAPA.

bioinformatics↗

bioRxiv2020

Overexpression of EiKCS confers paraquat-tolerance in rice (Oryza sativa L.) by promoting polyamine pathway

Paraquat is an important bipyridine herbicide by acting on the photosynthetic system of the plants and generating reactive oxygen species leading to cell death, whereas the mechanism of the paraquat resistance remains to be explored. In this study, a putative paraquat-resistant gene EiKCS from goosegrass (Eleusine indica L.) was isolated and overexpressed in a transgenic rice (Oryza sativa L.). This transgenic rice (KCSox) was treated by exogenous spermidine and paraquat and then was analyzed by qualitative and quantitative proteomics. Overexpressing of EiKCS enhanced paraquat tolerance in KCSox by the accumulation of endogenous polyamines whose dominant presences of polyamines benzoylation derivatizations in rice were C18H20N2O2, C28H31N3O3, and C38H42N4O4. The mechanism underlying the improving tolerance enhanced antioxidant capacity of ROS systems and light-harvesting in photosynthesis in KCSox rice leaves to reducing paraquat toxicity. The protein {beta}-Ketoacyl-CoA Synthase (EiKCS) encoded by the EiKCS gene promoted the synthesis and metabolism of proteins of the polyamine pathway. Three cofactors CERs were identified and positively correlated with the function of EiKCS on very-long-chain fatty acids (VLCFAs) biosynthesis via promoting the polyamine pathway and inhibiting the links with the TCA pathway and fatty acid pathway to responding to the paraquat tolerance in the KCSox rice, which also caused the prolongation of the overproduction of spermine and a transient increase of intracellular malondialdehyde (MDA). These results expanded the polyamines pathway manipulated in cereals using genetic engineering to clarify the mechanism of paraquat-tolerance. One Sentence SummaryA putative paraquat-resistant EiKCS gene from the goosegrass overexpressing in the rice resulted in the accumulation of polyamines, especially the spermine, and promoted the proteins in polyamine pathways by its EiKCS protein under paraquat stress.

physiology↗

bioRxiv2020

Ligand Modulation of the Conformational Dynamics of the A2A Adenosine Receptor Revealed by Single-Molecule Fluorescence

G protein-coupled receptors (GPCRs) are the largest class of transmembrane proteins, making them an important target for therapeutics. Activation of these receptors is modulated by orthosteric ligands, which stabilize one or several states within a complex conformational ensemble. The intra-and inter-state dynamics, however, is not well documented. Here, we used single-molecule fluorescence to measure ligand-modulated conformational dynamics of the adenosine A2A Receptor (A2AR) on nanosecond to millisecond timescales. Experiments were performed on detergent-purified A2R in either the ligand-free (apo) state, or when bound to an inverse, partial or full agonist ligand. Single-molecule Forster resonance energy transfer (smFRET) was performed on detergent-solubilized A2AR to resolve active and inactive states via the separation between transmembrane (TM) helices 4 and 6. The ligand-dependent changes of the smFRET distributions are consistent with conformational selection and with inter-state exchange lifetimes [≥] 3 ms. Local conformational dynamics around residue 229 on TM6 was measured using Fluorescence Correlation Spectroscopy (FCS), which captures dynamic quenching due to photoinduced electron transfer (PET) between a covalently-attached dye and proximal aromatic residues. Global analysis of PET-FCS data revealed fast (150-350 ns), intermediate (50-60 s) and slow (200-300 s) conformational dynamics in A2AR, with lifetimes and amplitudes modulated by ligands and a G-protein mimetic (mini-Gs). Most notably, the agonist binding and the coupling to mini-Gs accelerates and increases the relative contribution of the sub-microsecond phase. Molecular dynamics simulations identified three tyrosine residues (Y112, Y288, and Y290) as being responsible for the dynamic quenching observed by PET-FCS and revealed associated helical motions around residue 229 on TM6. This study provides a quantitative description of conformational dynamics in A2AR and supports the idea that ligands bias not only GPCR conformations but also the dynamics within and between distinct conformational states of the receptor.

biophysics↗

bioRxiv2020

A Transcriptional Switch Governing Fibroblast Plasticity Underlies Reversibility of Chronic Heart Disease

In diseased organs, stress-activated signaling cascades alter chromatin, triggering broad shifts in transcription and cell state that exacerbate pathology. Fibroblast activation is a common stress response that worsens lung, liver, kidney and heart disease, yet its mechanistic basis remains poorly understood1,2. Pharmacologic inhibition of the BET family of transcriptional coactivators alleviates cardiac dysfunction and associated fibrosis, providing a tool to mechanistically interrogate maladaptive fibroblast states and modulate their plasticity as a potential therapeutic approach3-8. Here, we leverage dynamic single cell transcriptomic and epigenomic interrogation of heart tissue with and without BET inhibition to reveal a reversible transcriptional switch underlying stress-induced fibroblast activation. Transcriptomes of resident cardiac fibroblasts demonstrated robust and rapid toggling between the quiescent fibroblast and activated myofibroblast state in a manner that directly correlated with BET inhibitor exposure and cardiac function. Correlation of single cell chromatin accessibility with cardiac function revealed a novel set of reversibly accessible DNA elements that correlated with disease severity. Among the most dynamic elements was an enhancer regulating the transcription factor MEOX1, which was specifically expressed in activated myofibroblasts, occupied putative regulatory elements of a broad fibrotic gene program, and was required for TGF{beta}-induced myofibroblast activation. CRISPR interference of the most dynamic cis-element within the enhancer, marked by nascent transcription, prevented TGF{beta}-induced activation of Meox1. These findings identify MEOX1 as a central regulator of stress-induced myofibroblast activation associated with cardiac dysfunction. The plasticity and specificity of the BET-dependent regulation of MEOX1 in endogenous tissue fibroblasts provides new trans- and cis- targets for treating fibrotic disease.

molecular biology↗