Biology subjects

Tang, W.

Publications and source records attributed to Tang, W..

At least 109 records · Page 6Linked to original sources

bioRxiv2022

Additive and Dominant Loci Jointly Pyramiding the Grain Quality of Hybrid Rice

Hybrid rice has an advantage in its heterosis, resulting in high yield and resistance to biotic and abiotic stress. However, genetic improvement of grain quality is more challenging in hybrid rice than in inbred rice due to additional complexity, such as the dominant effect. It is critical to identify a path to efficiently develop inbreds and identify their superior crosses for hybrid production. Here, we developed a pipeline for joint analysis of phenotypes, effects, and generations (JPEG) and analysed 113 inbred varieties as male parents, five tester varieties as female parents, and their 565 (113x5) hybrid testcrosses for 12 grain quality traits, including grain length and width, chalkiness, and amylose content. A total of 1,619,588 single nucleotide polymorphisms were obtained for the parent varieties and inferred for the hybrids using whole-genome sequencing with an average sequencing depth of 11x. Genome-wide association studies with JPEG identified 128 loci associated with at least one of the 12 traits. There were 44 and 97 loci with additive and dominant effects, respectively, including 13 overlaps. Among the 128 associated loci, 42 loci are located in or near (<200 kb) 17 known genes. Pyramiding genetic loci with additive and/or dominant effects in genomic selection dramatically improves the accuracy of predicting hybrid performances. More than 45% of genetic variation was explained by the identified markers for all traits except one (30%). These results demonstrate that pyramiding genetically identified additive and dominant loci can afford substantial power for predicting hybrid performances, presenting opportunities to identify superior crosses from existing phenotypes and genotypes of inbreds and hybrids.

plant biology↗

bioRxiv2022

Human statistical learning dynamically shapes the hippocampal processing of temporal associations

We investigated the neural basis of chunking during statistical learning (SL). Behavioral evidence suggests that a common mechanism in learning and memory can serve to combine smaller units into larger ones to facilitate sensory and higher-level processing. And yet, the neural underpinnings of this mechanism remain unclear. Drawing insights from previous findings of neural codes in the hippocampus, we propose a computational model to account for the temporal chunking process in SL for sequential inputs. We operationalize chunking into a hidden Markov model (HMM) that incorporates two core principles: (1) the hidden states represent serial order rather than specific visual features, and (2) the formation of temporal chunks leads to autocorrelated brain activity. We show with numeric simulations that the HMM can decode embedded triplet representations when both assumptions hold. Applying the HMM to functional neuroimaging data from subjects performing a visual SL task, we show that decoding was successful (1) for triplet sequences but not random sequences, (2) at the later stage but not earlier stage of learning, and (3) in the hippocampus but not in the early visual cortex. These results provide evidence for a hippocampal representation of generalized temporal structure emerged from sequential visual input, shedding light on the chunking mechanism for SL. SignificanceIn statistical learning (SL), individuals develop internal representations of patterns after brief exposure to structured stimuli. People tend to recognize frequently co-occurring items as a single unit. This process, known as "chunking", is understood to play an important role in facilitating sensory processing for learning. However, its neural underpinnings remain unclear. In this study we draw insights from hippocampal coding theories and introduce a chunking model focusing on generalized presentations for SL. With functional neuroimaging data from human subjects performing a visual learning task, the chunking model successfully decoded the temporal regularities embedded in the sequential inputs. This model and related findings provide critical evidence for a chunking process underlying SL as well as its representation in the human hippocampus.

neuroscience↗

bioRxiv2022

Dissecting β-Cardiac Myosin and Cardiac Myosin-Binding Protein C Interactions using a Nanosurf Assay

Cardiac myosin-binding protein C (cMyBP-C) regulates cardiac contractility by slowing shortening velocity and sensitizing the thin filament to calcium. cMyBP-C has been shown to interact with the proximal myosin S2 tail and the thin filament. However, the relative contribution of these interactions to the collective modulation of actomyosin ensemble function remains unclear. Hence, we developed a "nanosurf" assay as a model system to interrogate cMyBP-C interactions with actin and/or myosin. Synthetic thick filaments were generated using recombinant human {beta}-cardiac myosin subfragments (HMM or S1) attached to DNA nanotubes, with 14 or 28 nm spacing, corresponding to the 14.3 nm myosin spacing found in native thick filaments. In vitro motility assays with myosin bound to the surrounding surface, exhibit enhanced thin filament interactions with synthetic thick filaments. No significant differences were observed in mean thin filament velocities between 14 and 28 nm spacing, consistent with our previous results for myosin V, VI, and {beta}-cardiac myosin S1. Our nanosurf assay demonstrates the slowing of actomyosin motility by cMyBP-C. Alternating {beta}-cardiac myosin HMM and cMyBP-C N-terminal fragments, C0-C2 or C1-C2, every 14 nm on the nanotube, reduced the mean thin filament velocity 4-6 fold relative to myosin alone. Interestingly, similar inhibition was observed using a {beta}-cardiac myosin S1 construct, which lacks the S2 region proposed to interact with cMyBP-C, suggesting the actin-cMyBP-C interactions may dominate the inhibitory mechanism. No significant inhibition of thin filament velocity was observed with a C0-C1f fragment, lacking the majority of the M-domain, supporting the importance of this domain for inhibitory interaction(s). A phosphomimetic C0-C2 fragment showed a 3-fold higher velocity compared to its phosphonull counterpart, further highlighting phosphorylation-dependent regulation via the M-domain. Together, we have established the nanosurf assay as a tool to precisely manipulate spatially dependent cMyBP-C binding partner interactions, shedding light on the molecular regulation of {beta}-cardiac myosin contractility. STATEMENT OF SIGNIFICANCECardiac myosin-binding protein C (cMyBP-C) is the most frequently mutated protein associated with hypertrophic cardiomyopathy (HCM), a common cause of sudden cardiac death. Despite the importance of cMyBP-C in cardiac contractility, the mechanisms underlying this regulation are unclear due to experimental challenges in studying the complex, transient, weak interactions of cMyBP-C with the contractile proteins of the sarcomere. In this study, we created a nanosurf synthetic DNA thick filament assay to dissect the cMyBP-C interactions with actin and human {beta}-cardiac myosin. We demonstrate actomyosin inhibition by cMyBP-C fragments regardless of recombinant human {beta}-cardiac myosin subfragment (HMM or S1) and highlight the importance of the cMyBP-C M-domain using cMyBP-C fragments and phosphomimetics.

biophysics↗

bioRxiv2022

The infiltration pattern of microenvironmental cells and different immune escape mechanisms in colorectal cancer

BackgroundThe tumour microenvironment (TME) plays a crucial role in tumour progression and metastasis. However, the infiltration pattern of TME cells in CRC samples and the immune escape mechanism underneath have not been studied sufficiently. MethodsTranscriptomic data from public datasets were retrieved online. In total, 1802 samples from the microarray dataset and 619 samples from the TCGA dataset were enrolled. The ssGSEA algorithm and unsupervised clustering were used for TME cells infiltration speculation and infiltration pattern recognition. ResultsCRC samples can be classified into three distinct TME cell subtypes. Subtype 1, the immune-active subtype, was characterised by high infiltration of activated adaptive immune cells. Subtype 2, the immune-desert subtype, featured high tumour purity and low infiltration of immune and stromal cells. And subtype 3, stroma-rich subtype, had high infiltration of stromal cells. The stroma-rich subtype conferred a significantly worse prognosis. Three subtypes had different immune escape mechanisms. The immune-active subtype has the highest immune checkpoint expression level. In comparison, the immune-desert subtype had the lowest immunogenicity and the defective antigen presentation. And the stroma-rich subtype lacked activated immune cells. ConclusionsDistinct TME cell subtypes and immune escape mechanisms may provide inspiration and direction for further researches on CRC immunotherapy.

cancer biology↗

bioRxiv2022

Heterotrimeric G protein α subunit (GPA1) regulates the response to low-nitrogen stress in Arabidopsis by interacting with AtNRT1.4 and AtATG8a

Efficient nitrogen absorption and utilization are important factors for higher plants to increase yield and reduce eutrophication (caused by excessive use of nitrogen fertilizers). Heterotrimeric G proteins, including three subunits of , {beta}, and {gamma}, participate in the pathway regulating nitrogen absorption and utilization in plants. However, the regulatory mechanism remains largely obscured. In this study, our results revealed that the G protein subunit (AtGPA1) mutant gpa1-4 was tolerant to low-nitrogen stress in Arabidopsis. AtGPA1 was shown to directly interact with a nitrate transporter (AtNRT1.4) and a key autophagy-related protein (AtATG8a) on the plasma membrane using the yeast hybrid system and pull-down assay (in vitro) and BiFC assay (in vivo). GUS staining and subcellular localization showed that AtGPA1 and AtNRT1.4 were co-expressed in roots and leaf veins and on the plasma membrane. Under low-nitrate conditions, the single mutant gpa1-4 and NRT1.4RNAi plants (AtNRT1.4RNA interference plants) and the double mutant NRT1.4RNAi/gpa1-4 plants (AtNRT1.4RNA interference plants on a gpa1-4 background) were healthier than the wild type plants. Moreover, the phenotype of the double mutant NRT1.4RNAi/gpa1-4 plants was closer to that of the NRT1.4RNAi plants compared to that of the gpa1-4 mutants. The results of the nitrate efflux rate assay in roots were consistent with the phenotypic changes under low-nitrogen conditions. These results indicated that AtGPA1 is an upstream factor that regulated the response to low-nitrogen stress through interaction with AtNRT1.4. In addition, we found that transgenic plants overexpressing AtATG8a were more tolerant to low-nitrogen stress, and their phenotype was similar to that of gpa1-4 mutants and double mutant ATG8aOX/gpa1-4 plants (AtATG8a overexpressing plants on a gpa1-4 mutant background). Further, autophagosome observations were consistent with the phenotypes in mutant plants, indicating that AtGPA1 regulated the response to low-nitrogen stress in Arabidopsis plants by affecting the autophagosome assembly. Our findings may provide a new model for improving nitrogen-use efficiency through genetical modification to boost crop yields. One sentence summaryAtGPA1 negative regulates low nitrogen stress response by interaction with a nitrate transporter, AtNRT1.4 and an autophagy-related protein, AtATG8a in Arabidopsis.

physiology↗

bioRxiv2022

An organoid CRISPRi screen revealed that SOX9 primes human fetal lung tip progenitors to receive WNT and RTK signals

The balance between self-renewal and differentiation in human fetal lung epithelial progenitors controls the size and function of the adult organ. Moreover, progenitor cell gene regulation networks are employed by both regenerating and malignant lung cells, where modulators of their effects could potentially be of therapeutic value. Details of the molecular networks controlling human lung progenitor self-renewal remain unknown. We performed the first CRISPRi screen in primary human lung organoids to identify transcription factors controlling progenitor self-renewal. We show that SOX9 promotes proliferation of lung progenitors and inhibits precocious airway differentiation. Moreover, by identifying direct transcriptional targets using Targeted DamID we place SOX9 at the centre of a transcriptional network which amplifies WNT and RTK signalling to stabilise the progenitor cell state. In addition, the proof-of-principle CRISPRi screen and Targeted DamID tools establish a new approach for using primary human organoids to elucidate detailed functional mechanisms underlying normal development and disease. HighlightsO_LIA pooled CRISPRi screen in human fetal lung organoids identified transcription factors controlling progenitor cell self-renewal. C_LIO_LISOX9 promotes tip progenitor cell proliferation and supresses precocious airway differentiation. C_LIO_LITargeted DamID (TaDa) identified SOX9 direct binding targets, revealing that SOX9 lies at the intersection of WNT and RTK signalling. C_LIO_LISOX9 and ETVs co-regulate the human fetal lung progenitor self-renewal programme. C_LI

developmental biology↗

bioRxiv2022

Stabilization of E-cadherin adhesions by COX-2/GSK3β signaling is a targetable pathway in metastatic breast cancer

Metastatic progression and treatment-resistance of breast cancer has been associated with epithelial-mesenchymal-transition including downregulation of E-cadherin (CDH1) expression, which can be initiated by inflammatory mediators such as COX-2. Recently, E-cadherin-mediated, cluster-based metastasis and treatment resistance has become more appreciated, though the mechanisms that maintain E-cadherin expression in this context are unknown. Through studies of inflammatory breast cancer and an in vitro tumor cell emboli culture paradigm, we identified a role for COX-2, a target gene of C/EBP{delta}, or its metabolite PGE2 in promoting protein stability of E-cadherin, {beta}-catenin and p120 catenin through inhibition of GSK3{beta}, without affecting CDH1 mRNA. The COX-2 inhibitor celecoxib downregulated E-cadherin complex proteins and caused cell death. Co-expression of E-cadherin and COX-2 was seen in breast cancer patients with poor outcome and, along with inhibitory GSK3{beta} phosphorylation, in patient-derived xenografts of triple negative breast cancer. Celecoxib alone decreased E-cadherin protein expression within xenograft tumors, reduced circulating tumor cells and clusters, and in combination with paclitaxel attenuated or regressed lung metastases. This study uncovered a mechanism by which metastatic breast cancer cells can maintain E-cadherin-mediated cell-cell adhesions and cell survival, suggesting that patients with COX-2+/E-cadherin+ breast cancer may benefit from targeting of the PGE2 signaling pathway.

cancer biology↗

bioRxiv2022

High seroprevalence of SARS-CoV-2 in white-tailed deer (Odocoileus virginianus) at one of three captive cervid facilities in Texas

Free-ranging white-tailed deer (Odocoileus virginanus) across the United States are increasingly recognized as involved in SARS-CoV-2 transmission cycles. Through a cross-sectional study of 80 deer at three captive cervid facilities in central and southern Texas, we provide evidence of 34 of 36 (94.4%) white-tailed deer at a single captive cervid facility seropositive for SARS-CoV-2 by neutralization assay (PRNT90), with endpoint titers as high as 1280. In contrast, all tested white-tailed deer and axis deer (Axis axis) at two other captive cervid facilities were seronegative, and SARS-CoV-2 RNA was not detected in respiratory swabs from deer at any of the three facilities. These data support transmission among captive deer that cannot be explained by human contact for each infected animal, as only a subset of the seropositive does had direct human contact. The facility seroprevalence was more than double of that reported from wild deer, suggesting that the confined environment may facilitate transmission. Further exploration of captive cervids and other managed animals for their role in the epizootiology of SARS-CoV-2 is critical for understanding impacts on animal health and the potential for spillback transmission to humans or other animal taxa. ImportanceAs SARS-CoV-2 vaccine coverge of the human population increases and variants of concern continue to emerge, identification of the epidemiologic importance of animal virus reservoirs is critical. We found that nearly all (94.4%) of the captive white-tailed deer at a cervid facility in central Texas had neutralizing antibodies for SARS-CoV-2. This seroprevalence is over double than that which has been reported from free-ranging deer from other regions of the US. Horizontal transmission among deer may be facilitated in confinement. Tracking new infections among wild and confined deer is critical for understanding the importance of animal reservoirs for both veterinary and human health.

zoology↗

bioRxiv2022

Transcription shapes 3D chromatin organization by interacting with loop-extruding cohesin complexes

Cohesin folds mammalian interphase chromosomes by extruding the chromatin fiber into numerous loops. "Loop extrusion" can be impeded by chromatin-bound factors, such as CTCF, which generates characteristic and functional chromatin organization patterns. It has been proposed that transcription relocalizes or interferes with cohesin, and that active promoters are cohesin loading sites. However, the effects of transcription on cohesin have not been reconciled with observations of active extrusion by cohesin. To determine how transcription modulates extrusion, we studied mouse cells in which we could alter cohesin abundance, dynamics, and localization by genetic knockouts of the cohesin regulators CTCF and Wapl. Through Hi-C experiments, we discovered intricate, cohesin-dependent contact patterns near active genes. Chromatin organization around active genes exhibited hallmarks of interactions between transcribing RNA polymerases (RNAPs) and extruding cohesins. These observations could be reproduced by polymer simulations in which RNAPs were "moving barriers" to extrusion that obstructed, slowed, and pushed cohesins. The simulations predicted that preferential loading of cohesin at promoters is inconsistent with our experimental data. Additional ChIP-seq experiments showed that the putative cohesin loader Nipbl is not predominantly enriched at promoters. Therefore, we propose that cohesin is not preferentially loaded at promoters and that the barrier function of RNAP accounts for cohesin accumulation at active promoters. Altogether, we find that RNAP is a new type of extrusion barrier that is not stationary, but rather, translocates and relocalizes cohesin. Loop extrusion and transcription might interact to dynamically generate and maintain gene interactions with regulatory elements and shape functional genomic organization. Significance StatementLoop extrusion by cohesin is critical to folding the mammalian genome into loops. Extrusion can be halted by CTCF proteins bound at specific genomic loci, which generates chromosomal domains and can regulate gene expression. However, the process of transcription itself can modulate cohesin, thus refolding chromosomes near active genes. Through experiments and simulations, we show that transcribing RNA polymerases (RNAPs) act as "moving barriers" to loop-extruding cohesins. Unlike stationary CTCF barriers, RNAPs actively relocalize cohesins, which generates characteristic patterns of spatial organization around active genes. Our model predicts that the barrier function of RNAP can explain why cohesin accumulates at active promoters and provides a mechanism for clustering active promoters. Through transcription-extrusion interactions, cells might dynamically regulate functional genomic contacts.

genomics↗

bioRxiv2021

Nucleocapsid 203 mutations enhance SARS-CoV-2 immune evasion

Previous work indicated that the nucleocapsid 203 mutation increase the virulence and transmission of the SARS-CoV-2 Alpha variant. However, Delta later outcompeted Alpha and other lineages, promoting a new wave of infections. Delta also possesses a nucleocapsid 203 mutation, R203M. Large-scale epidemiological analyses suggest a synergistic effect of the 203 mutation and the spike L452R mutation, associated with Delta expansion. Viral competition experiments demonstrate the synergistic effect in fitness and infectivity. More importantly, we found that the combination of R203M and L452R brings in a 3.2-fold decrease in neutralizing titers to the neutralizing serum relative to L452R-only virus. R203M/L452R show an increased fitness after the initiation of global vaccination programmes, possibly associated with the enhanced immune evasion. Another rapidly emerging variant Omicron also bears the 203 mutation. Thus, we proposed that nucleocapsid mutations play an essential role for the rise and predominance of variants in concern.

microbiology↗

bioRxiv2021

CV-containing vesicle budding from chloroplast inner envelope membrane is mediated by clathrin but inhibited by GAPC

Clathrin-mediated vesicular formation and trafficking are highly conserved in eukaryotic cells and are responsible for molecular cargo transport and signal transduction among organelles. It remains largely unknown whether clathrin-coated vesicles can be generated from chloroplasts. CHLOROPLAST VESICULATION (CV)-containing vesicles (CVVs) generate from chloroplasts and mediate chloroplast degradation under abiotic stress. In this study, we showed that CV interacted with the clathrin heavy chain (CHC) and induced vesicle budding from the chloroplast inner envelope membrane. Defects on CHC2 and the dynamin-encoding DRP1A gene affected CVV budding and releasing from chloroplast. CHC2 is also required for CV-induced chloroplast degradation and hypersensitivity to water stress. Moreover, GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPC) interacts with CV and impairs the CV-CHC2 interaction. GAPC1 overexpression inhibited CV-mediated chloroplast degradation and hypersensitivity to water stress. CV silencing alleviated the hypersensitivity of gapc1gapc2 plant to water stress. Together, our work revealed a pathway of clathrin-assisted CVV budding from the chloroplast inner envelope membrane, which mediated the stress-induced chloroplast degradation and stress response.

plant biology↗

bioRxiv2021

Impaired astrocytic Ca2+ signalling in awake Alzheimer's disease transgenic mice

Increased astrocytic Ca2+ signaling related to amyloid plaques has been shown in Alzheimers disease mouse models, but to date no reports have characterized behaviorally induced astrocytic Ca2+ signalling in such mice without the confounding effects of anesthesia. Here, we employ an event-based algorithm to assess astrocytic Ca2+ signals in the neocortex of awake-behaving tg-ArcSwe mice and non-transgenic wildtype littermates while monitoring pupil responses and behavior. We demonstrate an attenuated astrocytic Ca2+ response to locomotion and an uncoupling of pupil responses and astrocytic Ca2+ signalling in 15-months old plaque-bearing mice. This points to a potential decoupling of neuromodulatory activation and astrocytic Ca2+ activity, which may account for some of the cognitive dysfunctions observed in Alzheimers disease.

neuroscience↗

bioRxiv2021

Proximity labeling identifies LOTUS domain proteins that promote the formation of perinuclear germ granules in C. elegans

The germline produces gametes that transmit genetic and epigenetic information to the next generation. Maintenance of germ cells and development of gametes require germ granules--well-conserved membraneless and RNA-rich organelles. The composition of germ granules is elusive owing to their dynamic nature and their exclusive expression in the germline. Using C. elegans germ granule, called P granule, as a model system, we employed a proximity-based labeling method in combination with mass spectrometry to comprehensively define its protein components. This set of experiments identified over 200 proteins, many of which contain intrinsically disordered regions. An RNAi-based screen identified factors that are essential for P granule assembly, notably EGGD-1 and EGGD-2, two previously uncharacterized LOTUS-domain proteins. Loss of eggd-1 and eggd-2 results in separation of P granules from nuclear envelope, germline atrophy and reduced fertility. We show that intrinsically disordered regions of EGGD-1 are required to anchor EGGD-1 to the nuclear periphery while its LOTUS domains are required to promote perinuclear localization of P granules. Together, our work expands the repertoire of P granule constituents and provides new insights into the role of LOTUS-domain proteins in germ granule organization.

cell biology↗

bioRxiv2021

Defining the Sensitivity Landscape of 74,389 EGFR Variants to Tyrosine Kinase Inhibitors

BackgroundTyrosine kinase inhibitors (TKIs) therapy is a standard treatment for patients with advanced non-small-cell lung carcinoma (NSCLC) when activating epidermal growth factor receptor (EGFR) mutations are detected. However, except for the well-studied EGFR mutations, most EGFR mutations lack treatment regimens. MethodsWe constructed two EGFR variant libraries containing substitutions, deletions, or insertions using the saturation mutagenesis method. All the variants were located in the EGFR mutation hotspot (exons 18-21). The sensitivity of these variants to afatinib, erlotinib, gefitinib, icotinib, and osimertinib was systematically studied by determining their enrichment in massively parallel cytotoxicity assays using an endogenous EGFR-depleted cell line, PC9. ResultsA total of 3,914 and 70,475 variants were detected in the constructed EGFR Substitution-Deletion (Sub-Del) and exon 20 Insertion (Ins) libraries, accounting for 99.3% and 55.8% of the designed variants, respectively. Of the 3,914 Sub-Del variants, 813 were highly enriched in the reversible TKI (erlotinib, gefitinib, icotinib) cytotoxicity assays and 51 were enriched in the irreversible TKI (afatinib, osimertinib) cytotoxicity assays. For the 70,475 Ins variants, insertions at amino acid positions 770-774 were highly enriched in all the five TKI cytotoxicity assays. Moreover, the top 5% of the enriched insertion variants included a glycine or serine insertion at high frequency. ConclusionsWe present a comprehensive reference for the sensitivity of EGFR variants to five commonly used TKIs. The approach used here should be applicable to other genes and targeted drugs.

genetics↗

bioRxiv2021

Bcl-3 suppresses Th9 differentiation by regulating glutamine utilization

Bcl-3 is an atypical member of the I{kappa}B protein family that plays important and diverse roles in both innate and adaptive immunity, including Th17-dependent autoimmunity models in mice. When naive mouse splenic CD4+ T cells were cultured under Th17 conditions in vitro, we unexpectedly found that the most highly differentially expressed gene between wild type and Bcl-3-deficient (KO) Th17 cells encoded the cytokine IL-9. We therefore investigated the role of Bcl-3 in Th9 cell differentiation. When naive CD4+ T cells were cultured under Th9-polarizing conditions in vitro, the extent of Th9 differentiation observed in wild type cells was increased in Bcl-3 KO cells and conversely was decreased in cells overexpressing Bcl-3. The suppressive effect of Bcl-3 on Th9 differentiation was cell-autonomous, and NF-{kappa}B inhibitors abolished increased Th9 differentiation in Bcl-3 KO cells. Consistent with this, in the Th9 transfer model of OVA-induced allergic airway inflammation, mice receiving Bcl-3 KO cells had greater immune cell infiltration in the lung than mice receiving wild type cells. Mechanistically, unsupervised transcriptomic analysis revealed differentially expressed genes in KO cells, including the glutamine transporter Slc1a5, which was downregulated. The functional significance of this was suggested by the ability of increasing concentrations of glutamine in the media to reduce the difference in Th9 differentiation between WT and KO cells. Our results suggest a novel role for Bcl-3 as a negative regulator of Th9 differentiation, in part by limiting glutamine accessibility through downregulation of Slc1a5.

immunology↗

bioRxiv2021

Acquisition of alveolar fate and differentiation competence by human fetal lung epithelial progenitor cells

Variation in lung alveolar development is strongly linked to disease susceptibility. However, the cellular and molecular mechanisms underlying alveolar development are difficult to study in humans. Using primary human fetal lungs we have characterized a tip progenitor cell population with alveolar fate potential. These data allowed us to benchmark a self-organising organoid system which captures key aspects of lung lineage commitment and can be efficiently differentiated to alveolar type 2 cell fate. Our data show that Wnt and FGF signalling, and the downstream transcription factors NKX2.1 and TFAP2C, promote human alveolar or airway fate respectively. Moreover, we have functionally validated cell-cell interactions in human lung alveolar patterning. We show that Wnt signalling from differentiating fibroblasts promotes alveolar type 2 cell identity, whereas myofibroblasts secrete the Wnt inhibitor, NOTUM, providing spatial patterning. Our organoid system recapitulates key aspects of human lung development allowing mechanistic experiments to determine the underpinning molecular regulation.

developmental biology↗

bioRxiv2021

Microfluidic Streptomyces Cultivation for Whole Lifecycle Characterization and Phenotypic Assays Enabled by Nanogap-stabilized Air-Water Interface

Streptomyces is a model filamentous prokaryote to study multicellular differentiation and a rich reservoir for antibiotics discovery. In their natural conditions, Streptomyces grows at the interface of porous soil, air, and water. The morphological development of Streptomyces is traditionally performed on agar plates and mostly studied at the population levels. However, the detailed lifecycle of Streptomyces has not been well studied due to its complexity and lack of research tools which can mimic their natural conditions in the soil. Here, we developed a simple assembled microfluidic device for cultivation and the entire lifecycle observation of Streptomyces development from single-cell level. The microfluidic device composed of a microchannel for loading samples and supplying nutrients, microwell arrays for seeding and growth of single spores, and air-filled chambers aside of the microwells that facilitate growth of aerial hyphae and spores. A unique feature of this device is that each microwell is surrounded by a 1.5 {micro}m gap connected to an air-filled chamber which provide stabilized water-air interface. We used this device to observe the development of single Streptomyces spores and found that unlike those in bulk liquid culture, Streptomyces can differentiate at water-air interfaces in microscale liquid culture. Finally, we demonstrated that phenotypic A-Factor assay can be performed at defined time point of its lifecycle. This microfluidic device could become a robust tool for studying Streptomyces multi-cellular differentiation and interaction at single cell level. IMPORTANCEWe describe a microfluidic device that mimics the natural porous environment for the growth and development of Streptomyces, the model system for bacterial multicellularity. The microfluidic device is used for cultivation and the entire lifecycle observation of Streptomyces development from single-cell level, including growth of aerial filaments. The aerial hyphae development of Streptomyces at the water-air interface was observed at real time in the microfluidic device. The early growth, opportunistic transformation (in the gap), and merging of aerial hyphae of Streptomyces in the microfluidic device were observed for the first time. It will play an important role in finding single-cell heterogeneity to study secondary metabolites related to the complex lifecycle of Streptomyces.

developmental biology↗

bioRxiv2021

CoVac501, a self-adjuvanting peptide vaccine conjugated with TLR7 agonists, against SARS-CoV-2 induces protective immunity

Safe, economical and effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are needed to achieve adequate herd immunity and halt the pandemic. We have constructed a novel SARS-CoV-2 vaccine, CoVac501, which is a self-adjuvanting peptide vaccine conjugated with Toll-like receptor 7 (TLR7) agonists. The vaccine contains two immunodominant peptides screened from receptor-binding domain (RBD) and is fully chemically synthesized. And the vaccine has optimized nanoemulsion formulation, outstanding stability and safety. In non-human primates (NHPs), CoVac501 elicited high and persistent titers of RBD-specific and protective neutralizing antibodies (NAbs), which were also effective to RBD mutations. CoVac501 was found to elicit the increase of memory T cells, antigen-specific CD8+ T cell responses and Th1-biased CD4+ T cell immune responses in NHPs. More importantly, the sera from the immunized NHPs can prevent infection of live SARS-CoV-2 in vitro. One-Sentence SummaryA novel SARS-CoV-2 vaccine we developed, CoVac501, which is a fully chemically synthesized and self-adjuvanting peptides conjugated with TLR7 agonists, can induce high-efficient humoral and cellular immune responses against SARS-CoV-2.

immunology↗