Biology subjects

Zeng, S.

Publications and source records attributed to Zeng, S..

At least 55 records · Page 3Linked to original sources

bioRxiv2023

Phosphorylation and O-GlcNAcylation at the same alpha-synuclein site generate distinct fibril structures

-Synuclein (-syn) forms amyloid fibrils that are critical in the progression of Parkinsons disease (PD) and serves as the pathological hallmark of PD. Different posttranslational modifications (PTMs) have been identified at multiple sites of -syn, influencing its conformation, aggregation and function. Here, we investigate how disease-related phosphorylation and O-GlcNAcylation at the same -syn site (S87) affect fibril structure and neuropathology. Using semi-synthesis, we obtained homogenous -syn monomer with site-specific phosphorylation (pS87) and O-GlcNAcylation (gS87) at S87, respectively. Cryo-EM analysis revealed that pS87 and gS87 -syn form two novel but distinct fibril structures. The GlcNAc situated at S87 establishes interactions with K80 and E61, inducing a unique iron-like fold with the GlcNAc molecule on the iron handle. While, phosphorylation at the same site prevents a lengthy C-terminal region including residues 73-140 from incorporating into the fibril core due to electrostatic repulsion. Instead, the N-terminal half (1-72) shapes a novel arch-like fibril structure. We further show that both pS87 and gS87 -syn fibrils display reduced neurotoxicity and propagation activity compared with unmodified -syn fibril. Our findings demonstrate that different PTMs at the same site can produce distinct fibril structures, which emphasizes the precise regulation of PTMs to amyloid fibril formation and pathology.

biochemistry↗

bioRxiv2023

Base editing strategies to convert CAG to CAA diminish the disease-causing mutation in Huntington's disease

An expanded CAG repeat in the huntingtin gene (HTT) causes Huntingtons disease (HD). Since the length of uninterrupted CAG repeat, not polyglutamine, determines the age-at-onset in HD, base editing strategies to convert CAG to CAA are anticipated to delay onset by shortening the uninterrupted CAG repeat. Here, we developed base editing strategies to convert CAG in the repeat to CAA and determined their molecular outcomes and effects on relevant disease phenotypes. Base editing strategies employing combinations of cytosine base editors and gRNAs efficiently converted CAG to CAA at various sites in the CAG repeat without generating significant indels, off-target edits, or transcriptome alterations, demonstrating their feasibility and specificity. Candidate BE strategies converted CAG to CAA on both expanded and non-expanded CAG repeats without altering HTT mRNA and protein levels. In addition, somatic CAG repeat expansion, which is the major disease driver in HD, was significantly decreased by a candidate BE strategy treatment in HD knock-in mice carrying canonical CAG repeats. Notably, CAG repeat expansion was abolished entirely in HD knock-in mice carrying CAA-interrupted repeats, supporting the therapeutic potential of CAG-to-CAA conversion base editing strategies in HD and potentially other repeat expansion disorders.

genetics↗

bioRxiv2023

ABI5-FLZ13 Module Transcriptionally Represses Growth-related Genes to Delay Seed Germination in Response to ABA

The bZIP transcription factor ABSCISIC ACID INSENSITIVE5 (ABI5) is a master regulator determining seed germination and postgerminative growth in response to abscisic acid (ABA), but the detailed molecular mechanism underlying the repression function of ABI5 in plant growth remains to be characterized. In this study, we used proximity labeling (PL) to map the neighboring proteome of ABI5 and identified FCS-LIKE ZINC FINGER PROTEIN 13 (FLZ13) as an up-to-now unknown ABI5-interacting partner. Phenotypic analysis of flz13 mutants and FLZ13-overexpressing lines demonstrated that FLZ13 acts as a positive regulator of ABA signaling. Interestingly, transcriptomic analysis showed that ABI5 and FLZ13 co-downregulate the expression of ABA-repressed and growth-related genes involved in chlorophyll biosynthesis, photosynthesis and cell wall organization, thereby repressing seed germination and seedling establishment in response to ABA. Further genetic analysis proved that FLZ13 works synergistically with ABI5 to regulate seed germination. Collectively, our study reveals a previously uncharacterized transcriptional regulatory mechanism regarding ABA-inhibited seed germination and seedling establishment.

plant biology↗

bioRxiv2022

GLKs directly regulate carotenoid biosynthesis via interacting with GBFs in nuclear condensates in plants

Carotenoids are vital photosynthetic pigments for plants and provide essential nutrients for humans. However, our knowledge of the regulatory control of carotenoid biosynthesis remains limited. Golden2-like transcription factors (GLKs) are widely recognized as essential and conserved factors for chloroplast development and the major regulators of chlorophyll biosynthesis. Yet the molecular mechanisms by which GLKs transcriptionally activate their target genes are unclear. Here, we report that GLKs directly regulate carotenoid biosynthesis in a G-box Binding Factor (GBF)-dependent manner. Both in vitro and in vivo studies reveal that GLKs physically interact with GBFs. Through the direct binding of GBFs to the G-box motif, the GLK-GBF regulatory module transcriptionally activates phytoene synthase (PSY), the gene encoding the rate-limiting enzyme for carotenoid biosynthesis. The ability of GLKs to promote carotenoid and chlorophyll biosynthesis is greatly diminished in the Arabidopsis gbf1/2/3 triple knockout mutants, showing the requirement of GBFs for GLK function. GLKs and GBFs form liquid-liquid phase separation-mediated nuclear condensates as the compartmented and concentrated transcriptional complexes. Our findings uncover a novel and conserved regulatory module for photosynthetic pigment biosynthesis through formation of GLK-GBF transcriptional complexes and nuclear biomolecular condensates in plants. One-sentence summaryGLKs transcriptionally regulate photosynthetic pigment synthesis in a GBF-dependent manner and are associated with the formation of phase separation-mediated nuclear condensates.

plant biology↗

bioRxiv2022

Early life adversity and adult social relationships have independent effects on survival in a wild animal model of aging

Does social isolation in adulthood predict survival because socially isolated individuals are already unhealthy due to adversity earlier in life (health selection)? Or do adult social environments directly cause poor health and increased mortality risk ("social causation")? These alternative hypotheses are difficult to disentangle in humans because prospective data on survival and the environment for both early life and adulthood are rarely available. Using data from the baboon population of Amboseli, Kenya, a model for human behavior and aging, we show that early adversity and adult social isolation contribute independently to reduced adult survival, in support of both health selection and social causation. Further, strong social bonds and high social status can buffer some negative effects of early adversity on survival. These results support a growing change in perspective, away from "either-or" hypotheses and towards a multi-causal perspective that points to multiple opportunities to mitigate the effects of social adversity. TeaserEarly life environments and adult social bonds have strong, but largely independent effects on survival in wild baboons.

animal behavior and cognition↗

bioRxiv2022

The thermodynamic state of aerobic glycolytic flux plays a critical role in stabilizing aerobic glycolytic flux of cancer cells

Inhibition of aerobic glycolysis (AG) is a strategy for cancer treatment, but its clinical translation has not been successful, suggesting that the mechanism underlying the inhibition of AG is not completely understood. We introduce thermodynamic state of AG (TSAG) into AG flux control, which refers the actual change of Gibbs free energy ({Delta}G) of reactions along the pathway. By using PKM2 knockdown and knockout models, we found that TSAG is resistant to PKM2 perturbation and it can effectively counteract the inhibitory effect of PKM2 perturbation on AG flux. Moreover, we deciphered the interrelationship between TSAG, AG rate, intermediate concentrations, and PK activity, revealed that TSAG is a system property of AG independent of cancer cells pathophysiological features and it plays important roles in stabilizing AG flux responding to perturbation. We propose that both inhibition of glycolytic enzyme and disruption of TSAG are required for effective AG inhibition.

cancer biology↗

bioRxiv2022

A Novel Prognosis Model based on Comprehensive Analysis of Pyroptosis-Related Genes in Breast Cancer

BackgroundPyroptosis plays a dual role in cancer. It can not only induce chronic tumor necrosis, but also stimulate acute inflammation to enhance immune response. However, the study of cell pyroptosis in Breast Cancer (BC) is still limited. MethodsA total of 26 pyroptosis-related differential genes were obtained, and PPI network and correlation analysis were demonstrated as well. Besides, 3 pyroptosis-related prognostic genes were collected by the univariate COX analysis. Through these 3 genes, patients were clustered into 2 sub-types by the Consensus clustering, then the EM and XMeans were also carried out to recheck the result. Furthermore, the significant prognosis-related genes were obtained by the Random Forest and LASSO analysis. Based on the above, the corresponding weights were calculated through the univariate and multivariate Cox analysis, and the prognostic model was constructed. In addition, the ROC curve, risk curve, PCA, t-SNE, COX, GO, KEGG and ssGSEA were analyzed, respectively. ResultsThrough the verification of 3 different clustering algorithms, 2 sub-types of BC were obtained. Furthermore, ELOVL2, IGLV6-57, FGBP1, HLA-DPB2 with weights of -0.183, -0.101, -0.227 and -0.254 were employed to establish the prognostic model. Validation shows that our model has good effect and the Go KEGG and Immune microenvironment analysis showed the enrichment of the differential genes. ConclusionOur study identified 4 genes that are closely correlated with the overall survival of BC patients, and the prognostic model can effectively divide patients into high- and low-risk groups, which may have certain guiding significance for prognosis.

bioinformatics↗

bioRxiv2022

Myeloid-biased HSC require Semaphorin 4A from the bone marrow niche for self-renewal under stress and life-long persistence

Tissue stem cells are hierarchically organized. Those that are most primitive serve as key drivers of regenerative response but the signals that selectively preserve their functional integrity are largely unknown. Here, we identify a secreted factor, Semaphorin 4A (Sema4A), as a specific regulator of myeloid-biased hematopoietic stem cells (myHSC), which are positioned at the top of the HSC hierarchy. Lack of Sema4A leads to exaggerated myHSC (but not downstream "balanced" HSC) proliferation after acute inflammatory stress, indicating that Sema4A enforces myHSC quiescence. Strikingly, aged Sema4A knock-out myHSC expand but almost completely lose reconstitution capacity. The effect of Sema4A is non cell-autonomous, since upon transplantation into Sema4A-deficient environment, wild-type myHSC excessively proliferate but fail to engraft long-term. Sema4A constrains inflammatory signaling in myHSC and acts via a surface receptor Plexin-D1. Our data support a model whereby the most primitive tissue stem cells critically rely on a dedicated signal from the niche for self-renewal and life-long persistence.

cell biology↗

bioRxiv2021

Direct regulation of cell cycle regulatory gene expression by NtrX to promote Sinorhizobium meliloti cell division

Cell division of the alfalfa symbiont, Sinorhizobium meliloti, is regulated by the CtrA signaling network. The gene expression of regulatory proteins in the network is affected by nutrient signaling. In this study, we found that NtrX, one of the regulators of nitrogen metabolic response, can directly regulate the expression of several regulatory genes from the CtrA signaling network. Three sets of S. meliloti ntrX mutants, including the plasmid insertion strain, the depletion strain and the substitution of the 53rd aspartate (ntrXD53E) from a plasmid in the wild-type strain (Sm1021), showed similar cell division defects, such as slow growth, abnormal morphology of partial cells and delayed DNA synthesis. Transcript quantitative evaluation indicated that the transcription of genes such as ctrA and gcrA was up-regulated, while the transcription of genes such as dnaA and ftsZl was down-regulated in the insertion mutant and the strain of Sm1021 expressing ntrXD53E. Correspondingly, inducible transcription of ntrX activates the expression of dnaA and ftsZ1, but represses ctrA and gcrA in the depletion strain. The expression levels of CtrA and GcrA were confirmed by western blotting, which were consistent with the transcription data. The transcriptional regulation of these genes requires phosphorylation of the conserved 53rd aspartate in the NtrX protein. The NtrX protein binds directly to the promoter regions of ctrA, gcrA, dnaA and ftsZ1 by recognizing the characteristic sequence CAAN2-5TTG Our findings reveal that NtrX is a novel transcriptional regulator of the CtrA signaling pathway genes, and positively affects bacterial cell division, associated with nitrogen metabolism. IMPORTANCESinorhizobium meliloti infects the host alfalfa and induces formation of nitrogen-fixing nodules. Proliferation of rhizobia in plant tissues and cells is strictly controlled in the early stage of symbiotic interactions. However, the control mechanism is not very clear. Cell division of S. meliloti in the free-living state is regulated by the CtrA signaling network, but molecular mechanisms by which the CtrA system is associated with environmental nutrient signals (e.g., ammonia nitrogen) need to be further explored. This study demonstrates that NtrX, a regulator of nitrogen metabolism, required for symbiotic nodulation and nitrogen fixation by S. meliloti 1021, can act as a transcriptional regulator of the CtrA signaling system. It may link nitrogen signaling to cell cycle regulation in Rhizobium species.

microbiology↗

bioRxiv2021

Polypeptides IGF-1C and P24 synergistically promote osteogenic differentiation of bone marrow mesenchymal stem cells in vitro through the p38 and JNK signaling pathways

ObjectivesInsulin-like growth factor-1 (IGF-1) and bone morphogenetic protein 2 (BMP-2) both promote osteogenesis of bone marrow mesenchymal stem cells (BMSCs). IGF-1C, the C domain peptide of IGF-1, and P24, a BMP-2-derived peptide, both have similar biological activities as their parent growth factors. This study aimed to investigate the effects and their mechanisms of polypeptides IGF-1C and P24 on the osteogenic differentiation of BMSCs. MethodsThe optimum concentrations of IGF-IC and P24 were explored. The effects of the two polypeptides on the proliferation and osteogenic differentiation of BMSCs were examined using the Cell Counting Kit-8(CCK-8), Alkaline phosphatase (ALP) staining, ALP activity assay, alizarin red S staining, qPCR, and western blotting. In addition, specific pathway inhibitors were utilized to explore whether p38 and JNK pathways were involved in this process. ResultsThe optimal concentrations of action were both 50 g/ml. IGF-1C and P24 synergistically promoted the proliferation of BMSCs, increased ALP activity and the formation of calcified nodules and upregulated the mRNA and protein levels of osterix (Osx), runt-related transcription factor 2 (Runx2), and osteocalcin (Ocn), phosphorylation level of p38 and JNK proteins also improved. Inhibition of the pathways significantly reduced the activation of p38 and JNK, blocked the expression of Runx2 while inhibiting ALP activity and the formation of calcified nodules. ConclusionsThese findings suggest IGF-1C and P24 synergistically promote the osteogenesis of BMSCs through activation of p38 and JNK signal pathways.

cell biology↗

bioRxiv2021

MAPK Pathway Inhibition as A Rational Therapeutic Strategy for MiR-138-5p/PAQR3 Dysregulation-mediated Epirubicin Resistance in Triple-negative Breast Cancer

Anthracyclines, such as epirubicin, activate the mitogen-activated protein kinase (MAPK) pathway in breast cancer (BC). Better predictors of tumor response are needed to guide de-intensification of anthracyclines when used in BC treatment. Here, we aimed to see if MAPK activation was responsible and targetable for epirubicin resistance, and to explore mechanisms and predictive markers for resistance. MAPK pathway inhibitors were used to ameliorate epirubicin resistance. Negative regulators of MAPK were screened to identify the essential gene cascades required for activation of the pathway. In vitro and in vivo approaches were applied to investigate epirubicin resistance. The regulatory miRNA was identified through bioinformatics-based screening and luciferase reporter assay. 114 estrogen receptor negative patients who received epirubicin monotherapy were included to evaluate predictive markers for tumor response. The MAPK pathway was activated in cells resistant to epirubicin; MAPK inhibition ameliorated epirubicin resistance. In triple-negative breast cancer (TNBC), progestin and adipoQ receptor 3 (PAQR3) were the most significantly decreased negative MAPK regulators. PAQR3 increased epirubicin sensitivity and suppressed MAPK activation in resistant cells. MiR-138-5p was increased in epirubicin resistant cells, and was shown to downregulate PAQR3, causing resistance in epirubicin resistant cells. PAQR3 was an independent factor in predicting response to epirubicin (OR=4.86, 95%CI=1.13-20.87, P=0.034), and possessed a high negative predictive value (NPV) (0.93; 95%CI=0.83-0.97). In addition, PAQR3 exhibited a better predictive value in patients older than 50 years (12.92 (95% CI, 1.43-116.78, P = 0.023). The combinative use of PAQR3 and topoisomerase II- (TOP2A) led to an increased specificity (0.70; 95%CI=0.61-0.79), when compared with either PAQR3 or TOP2A alone. MAPK inhibition ameliorated epirubicin resistance. MiR-138-5p-induced PAQR3 reduction causes epirubicin resistance in TNBC via activation of MAPK cascades. PAQR3 is an independent and favorable predictor response to epirubicin in BC.

cancer biology↗

bioRxiv2021

Significant East Asian affinity of Chinese Hui genomic structure suggesting their predominant cultural diffusion model in the genetic formation process

Ancestral origin and genomic history of Chinese Hui people remain to be explored due to the paucity of genome-wide data. Some evidence argued that an eastward migration of Central Asian given rise to modern Hui people, which was inferred as the demic diffusion hypothesis, and others favored the cultural diffusion hypothesis that posited indigenous East Asian adopted Muslim-like culture and formed the modern culturally different populations. However, the extent to which the observed Huis genetic structure was mediated by the movement of people or the assimilation of Muslim culture also remains one of the most contentious puzzles. Analyses of over 700K SNPs in 109 western Chinese individuals (49 Sichuan Hui and 60 geographically close Nanchong Han) together with the available ancient and modern Eurasians allowed us to fully explore the genomic makeup and origin of Huis and neighboring Hans. The results of the traditional and formal admixture-statistics (PCA, ADMIXTURE, and allele-sharing-based f-statistics) illuminated a strong genomic affinity between Sichuan Hui and Neolithic-to-modern Northern East Asians, which suggested massive gene influx from East Asian into Sichuan Hui people. Three-way admixture models in the qpWave/qpAdm analyses further revealed a small stream of gene influx from western Eurasian related to French or Andronovo into these Hui people, which was further directly confirmed via the admixture event from the temporally different western sources to Hui people in the qpGraph-based phylogenetic model, suggesting the key role of cultural diffusion model in the genetic formation of the modern East Asian Hui. ALDER-based admixture date estimation showed that this observed western Eurasian admixture signal was introduced into East Asian Hui during the historic periods, concordant with the extensive western-eastern communication in the Silk Road and historically documented Huis migration history. Summarily, although significant cultural differentiation among Hui and their neighbors existed, our genomic analysis showed their strong affinity with modern and ancient Northern East Asians. Our results supported that modern Chinese Hui arose from the mixture of minor western Eurasian ancestry and predominantly East Asian ancestry.

genetics↗

bioRxiv2020

NtrX systemically controls transcription of the CtrA system genes to regulate Rhizobium cell division

Cell division of the alfalfa symbiont, Sinorhizobium meliloti, is regulated by a signaling network centered on CtrA. The gene expression of regulatory proteins in the network can be regulated by nutrient signaling systems. In this study, we found that NtrX, one of the regulators of nitrogen metabolic response, can directly regulate the expression of several regulatory genes in the CtrA signaling network. Three groups of S. meliloti ntrX mutants showed similar cell division defects, such as slow growth, abnormal morphology of some cells and delayed DNA synthesis. Quantitative RT-PCR assays indicated that in these mutants, the transcription of genes such as ctrA and gcrA was up-regulated, while the transcription of genes such as dnaA and ftsZ1 was down-regulated. Western blotting showed that the CtrA and GcrA proteins were apparently increased in the mutants. The 53rd aspartate conserved in NtrX homologs can be phosphorylated in vitro and in vivo. The phosphorylated NtrX protein can bind directly to the promoter regions of ctrA, gcrA, dnaA and ftsZ1 by recognizing the characteristic sequence CAAN1-5TTG. Therefore, phosphorylation of NtrX is essential for cell cycle regulation of S. meliloti. We expressed the NtrX protein carrying a phosphorylation site substitution in Agrobacterium tumefaciens and found that the expressed strains had different growth phenotypes, suggesting that NtrX also regulates cell division in this bacterial species. Our findings reveal that NtrX acts as a transcriptional regulator that positively affects bacterial cell division, associated with nitrogen metabolism. IMPORTANCESinorhizobium meliloti infects the host legume alfalfa and induces the formation of nitrogen-fixing nodules. The proliferation of rhizobia in plant tissues and cells is strictly controlled in the early stage of the interaction between symbiotic partners. However, the control mechanism is not very clear. Cell division of S. meliloti in the free-living state is regulated by the CtrA signaling network, but the molecular mechanisms by which the CtrA system is associated with environmental nutrient signals (e.g., ammonia nitrogen) need to be further explored. This study demonstrates that NtrX, a regulator of nitrogen metabolism, required for symbiotic nodulation and nitrogen fixation by S. meliloti 1021, can act as a transcriptional regulator of the CtrA signaling system. It may link nitrogen signaling to cell cycle regulation in Rhizobium species.

molecular biology↗

bioRxiv2020

Integrative proteomic and phosphoproteomic analysis of granulosa cells during follicular atresia in porcine

Ovarian follicular atresia is a natural physiological process; however, the mechanism is not fully understood. In this study, quantitative proteomic and phosphoproteomic analyses of granulosa cells (GC) in healthy (H), slightly atretic (SA), and atretic follicles (A) of porcine were performed by TMT labeling, enrichment of phosphopeptides and LC-MS/MS analysis. In total, 6,201 proteins were quantified and 4,723 phosphorylation sites of 1,760 proteins were quantified. In total, 24 (11 up, 13 down) and 50 (29 up, 21 down) proteins with a fold change (FC) > 5 were identified in H/SA and H/A, respectively. In addition, there were 20 (H/SA, up) and 39 (H/A, up) phosphosites with an FC > 7, that could serve as potential biomarkers for distinguishing different quality categories of follicles. Western blotting and immunofluorescence confirmed the reliability of the proteomic analysis. Some key proteins (e.g., MIF, beta catenin, integrin {beta}2), phosphosites (e.g., S76 of caspase6, S22 and S636 of lamin A/C), pathways (e.g., apoptosis, regulation of actin cytoskeleton pathway), transcription factors (e.g., STAT5A, FOXO1, and BCLAF1), and kinases (e.g., PBK, CDK5, CDK12, AKT3) involved in atresia process were revealed via further analysis of the differentially expressed proteins (DEPs) and phosphorylated proteins (DEPPs). Collectively, the proteomic and phosphoproteomic profiling and functional research in the current study comprehensively analyzed the dynamic changes in protein expression and phosphorylation during follicular atresia and provided some new explanations regarding the regulation of this process.

cell biology↗

bioRxiv2020

Prolonged inhibitory effect of repeated period tibial nerve stimulation on the micturition reflex in the rat

BackgroundThe aims of this study was determine if stimulation of tibial nerve afferents could induce a prolonged modulation of the micturition reflex in the rats. MethodsFifteen female Sprague Dawley rats (250-350 g) were fully decorticated and paralysed for the study. Tibial nerve stimulation (TNS) was delivered by inserting two pairs of needle electrodes close to the nerves at the level of the medial malleolus. Constant flow cystometries (0.07 ml/min) at about 10 min interval were performed and the micturition threshold volume (MTV) was recorded and used as the dependent variable. After 4 - 5 stable control recordings, the tibial nerves of both sides were stimulated continuously for 5 min at 10 Hz, 3 times threshold for -motor axons. Six times of same stimulation were applied repeatedly with an interval of 5 min between the stimulations. The mean MTV was compiled from several cystometries in each half hour before the TNS and during, after 6 periods TNS. ResultsDuring the experiment, all the animals survived in a good condition with reasonably stable micturition reflexes, a significant increase in MTV was revealed after TNS. The best effect (mean 178%) occurred during the first 30 min after 6 periods of stimulation. This clear threshold increase remained for at least 5 h. ConclusionsA prolonged increase in MTV was demonstrated by a short periods of TNS repeatedly. This post stimulation modulatory effects of micturition reflex would provide a theoretical explanation for the clinical beneficial effect of TNS in patients with overactive bladder (OAB).

neuroscience↗

bioRxiv2020

Super-resolution segmentation network for reconstruction of packed neurites

Neuron reconstruction can provide the quantitative data required for measuring the neuronal morphology and is crucial in the field of brain research. However, the difficulty in reconstructing packed neuritis, wherein massive labor is required for accurate reconstruction in most cases, has not been resolved. In this work, we provide a fundamental pathway for solving this challenge by proposing the use of the super-resolution segmentation network (SRSNet) that builds the mapping of the neurites in the original neuronal images and their segmentation in a higher-resolution space. SRSNet focuses on enlarging the distances between the boundaries of the packed neurites producing the high-resolution segmentation images. Thus, in the construction of the training datasets, only the traced skeletons of neurites are required, which vastly increase the usability of SRSNet. From the results of the experiments conducted in this work, it has been observed that SRSNet achieves accurate reconstruction of packed neurites where the other state-of-the-art methods fail.

neuroscience↗

bioRxiv2020

Nature and effective range of non-cell autonomous activator and inhibitor peptides specifying plant stomatal patterning

Stomata are epidermal valves that facilitate gas exchange between plants and their environment. Stomatal patterning is regulated by EPIDERMAL PATTERING FACTOR (EPF)-family of secreted peptides: EPF1 enforcing stomatal spacing, whereas EPF-LIKE9, also known as Stomagen, promoting stomatal development. It remains unknown, however, how far these signaling peptides act. Utilizing Cre-Lox recombination-based mosaic sectors that overexpress either EPF1 or Stomagen in Arabidopsis cotyledons, we reveal a range within the epidermis and across the cell layers in which these peptides influence patterns. To quantitatively determine their effective ranges, we developed a computational pipeline, SPACE (Stomata Patterning AutoCorrelation on Epidermis), that describes probabilistic two-dimensional stomatal distributions based upon spatial autocorrelation statistics used in Astrophysics. The SPACE analysis shows that, whereas both peptides act locally, the inhibitor, EPF1, exerts longer-range effects than the activator, Stomagen. Furthermore, local perturbation of stomatal development has little influence on global two-dimensional stomatal patterning. Our findings conclusively demonstrate the nature and extent of EPF peptides as non-cell autonomous local signals and provides a means to quantitatively characterize complex spatial patterns in development. Summary StatementNon-cell autonomous effects of activator and inhibitor peptides on 2-D spatial patterning of stomata were quantitatively characterized using chimeric sectors and a SPACE computational pipeline.

plant biology↗

bioRxiv2020

Blocking antibodies against SARS-CoV-2 RBD isolated from a phage display antibody library using a competitive biopanning strategy

The infection of the novel coronavirus SARS-CoV-2 have caused more than 150,000 deaths, but no vaccine or specific therapeutic antibody is currently available. SARS-CoV-2 relies on its spike protein, in particular the receptor binding domain (RBD), to bind human cell receptor angiotensin-converting enzyme 2 (ACE2) for viral entry, and thus targeting RBD holds the promise for preventing SARS-CoV-2 infection. In this work, a competitive biopanning strategy of a phage display antibody library was applied to screen blocking antibodies against RBD. High-affinity antibodies were enriched after the first round using a standard panning process in which RBD-His recombinant protein was immobilized as a bait. At the next two rounds, immobilized ACE2-Fc and free RBD-His proteins were mixed with the enriched phage antibodies. Antibodies binding to RBD at epitopes different from ACE2-binding site were captured by the immobilized ACE2-Fc, forming a "sandwich" complex. Only antibodies competed with ACE2 for recognizing RBD at the same or similar epitopes can bind to the free RBD-His in the supernatant and be subsequently separated by the Ni-NTA magnetic beads. Top 1 lead from the competitive biopanning of a synthetic antibody library, Lib AB1, was produced as the full-length IgG1 format. It was proved to competitively block the binding of RBD to ACE2 protein, and potently inhibit SARS-CoV-2 pseudovirus infection of ACE2-overexpressing Hela cells with IC50 values of 12nM. Nevertheless, top 1 lead from the standard biopanning of Lib AB1, can only bind to RBD in vitro but not have the blocking or neutralization activity. Our strategy can efficiently isolate the blocking antibodies of RBD, and it would speed up the discovery of neutralizing antibodies against SARS-CoV-2.

microbiology↗