Biology subjects

Chen, P.

Publications and source records attributed to Chen, P..

At least 199 records · Page 11Linked to original sources

bioRxiv2020

lncRNA-RMST functioned as a SOX2 transcription co-regulator to regulate miR-1251 in the progression of Hirschsprung's disease

Hirschsprungs disease (HSCR) is a congenital disorder characterized by the absence of enteric neural crest cells (ENCCs). Non-coding RNAs including long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) have been authenticated as important regulators of biological functions. We conducted a microarray analysis and found lncRNA Rhabdomyosarcoma 2-associated transcript (RMST) was down-regulated in the stenotic segment of HSCR patients. MiR-1251 is transcribed from the intron region of RMST and was also low-expressed. When the expression of RMST or miR-1251 was reduced, the cell proliferation and migration were attenuated. However, RMST didnt affect the expression of miR-1251 directly found in this study. Through bioinformatic analysis, transcription factor SOX2 was predicted to bind to the promoter region of miR-1251 which was confirmed by CHIP assay. Herein, we demonstrated that RMST exerted as a co-regulator of SOX2 to regulate the expression of miR-1251. Furtherly, AHNAK was proved to be the target gene of miR-1251 in this study. Taken together, we revealed the role of RMST/SOX2/miR-1251/AHNAK pathway in the occurrence of Hirschsprungs disease and provided a potential therapeutic target for this disease. SUMMARY STATEMENTHirschsprung disease (HSCR) is characterized by a deficit in enteric neurons, however, the underlying mechanism remains unclear. This study revealed the role of lnc-RMST during the occurrence of HSCR.

cell biology↗

bioRxiv2020

Scribble mutation disrupts convergent extension and apical constriction during mammalian neural tube closure

Morphogenesis of the vertebrate neural tube occurs by elongation and bending of the neural plate, tissue shape changes that are driven at the cellular level by polarized cell intercalation and cell shape changes, notably apical constriction and cell wedging. Coordinated cell intercalation, apical constriction, and wedging undoubtedly require complex underlying cytoskeletal dynamics and remodeling of adhesions. Mutations of the gene encoding Scribble result in neural tube defects in mice, however the cellular and molecular mechanisms by which Scrib regulates neural cell behavior remain unknown. Analysis of Scribble mutants revealed defects in neural tissue shape changes, and live cell imaging of mouse embryos showed that the Scrib mutation results in defects in polarized cell intercalation, particularly in rosette resolution, and failure of both cell apical constriction and cell wedging. Scrib mutant embryos displayed aberrant expression of the junctional proteins ZO-1, Par3, Par6, E- and N-cadherins, and the cytoskeletal proteins actin and myosin. These findings show that Scribble has a central role in organizing the molecular complexes regulating the morphomechanical neural cell behaviors underlying vertebrate neurulation, and they advance our understanding of the molecular mechanisms involved in mammalian neural tube closure. HighlightsO_LIPolarized cell intercalation is lost in Scrib mutant embryos C_LIO_LIScrib mutation has specific effects on rosette formation and resolution C_LIO_LIScrib mutation disrupts apical constriction and cell shape changes necessary for neural tube closure C_LIO_LIAdherens and tight junction composition is altered in the neural epithelial cells of Scrib mutants C_LI

developmental biology↗

bioRxiv2020

Extracellular histones stimulate collagen expression and potentially promote liver fibrogenesis via TLR4-MyD88 signalling pathway

BACKGROUNDLiver fibrosis progressing to liver cirrhosis and hepatic carcinoma is very common and causes more than one million deaths annually. Fibrosis develops from recurrent liver injury but the molecular mechanisms are not fully understood. Recently, the Toll-like receptor (TLR) 4-MyD88 signalling pathway has been reported to contribute to fibrosis. Extracellular histones are the ligands of TLR4 but their roles in liver fibrosis have not been investigated. AIMThis study aims to investigate the roles and potential mechanisms of extracellular histones in liver fibrosis. METHODSIn vitro, the LX2 cells, a human hepatic stellate cell (HSC) line, were treated with histones in the presence or absence of non-anticoagulant heparin (NAHP) for neutralising histones or TLR4-blocking antibody. The cells resultant expression of collagen I was detected using Western blotting and immunofluorescent staining. In vivo, the CCl4-induced liver fibrosis model was generated in male 6 week old ICR mice and in TLR4 or MyD88 knockout and parental mice. Circulating histones were detected and the effect of NAHP was evaluated. RESULTSExtracellular histones strongly stimulated LX2 cells to produce collagen I. The histone-enhanced collagen expression was significantly reduced by NAHP and TLR4 blocking antibody. In CCl4-treated wild type mice, circulating histones were dramatically increased and maintained high levels during the whole course of fibrosis-induction. Injection of NAHP not only reduced alanine aminotransferase (ALT) and liver injury scores, but also significantly reduced fibrogenesis. Since the TLR4-blocking antibody reduced histone-enhanced collagen I production in HSC, the CCl4 model with TLR-4 and MyD88 knockout mice was used to demonstrate the roles of the TLR4-MyD88 signalling pathway in CCl4-induced liver fibrosis. The levels of liver fibrosis were indeed significantly lower than in these knockout mice than the wild type parental mice. CONCLUSIONThis study demonstrated that extracellular histones are able to stimulate HSC to produce collagen I and TLR4 is involved in this process. The in vivo findings support the novel concept that high levels of circulating histones potentially stimulate TLR4 receptor to enhance fibrogenesis via the TLR4-MyD88 signalling pathway. NAHP detoxify extracellular histones and thus has a potential therapeutic role by reducing liver injury and fibrogenesis. Core tipThis work fills the gap between recurrent liver injury and liver fibrosis. When liver cells die, histones will be released. High levels of extracellular histones not only cause a secondary liver injury, but also activate the TLR4-MyD88 signalling pathway to enhance collagen I production and liver fibrosis. Binding of non-anticoagulant heparin (NAHP) to extracellular histones reduces histone toxicity, alleviates liver injury and prevent histones from activating the TLR4-MyD88 signalling pathway. These results may explain why NAHP reduces liver fibrosis in this animal model although further investigations are required. ARTICLE HIGHLIGHTSO_ST_ABSResearch backgroundC_ST_ABSCurrently, the molecular mechanisms of liver fibrosis are not fully understood. Recurrent liver injury or inflammation initiate wound healing process along with fibrogenesis. However, what initiates this process is not clear. When cells die, damage-associated molecular patterns (DAMPs) will be released. Histones are the most abundant DAMP and are also the ligands for TLR4, which in turn has been demonstrated to be involved in bile duct ligation-induced liver fibrosis. Lipopolysaccharides (LPS) has been proposed to be a ligand for TLR4. Since recurrent liver injury does not naturally produce LPS but abundant extracellular histones, this study sought to investigate the potential roles of extracellular histones as TLR4 ligands in liver fibrosis. Research motivationSince our laboratory has been involved in studying the roles of DAMPs in critical illnesses, extracellular histones in liver fibrosis is of great interest in terms of biological and clinical significance. Research objectivesOur study aimed to clarify the roles of extracellular histones in fibrogenesis in vitro and in vivo. Research methodsIn our study, a HSC cell line and animal models of liver fibrosis were employed. Intervention studies with NAHP to detoxify histones and TLR4 blocking antibodies to inhibit TLR4 was performed. In addition, TLR4 and MyD88 knockout mice were used to support that the theory that the TLR4-MyD88 signalling pathway is involved in liver fibrosis in the CCl4 mouse model. Research resultsO_LIDemonstrated that high levels of circulating histones exist when fibrosis is induced by CCl4 in the mouse model. C_LIO_LIDemonstrated in vitro that extracellular histones are able to stimulate HSC cells to produce collagen I. C_LIO_LIDemonstrated that NAHP is able to inhibit histone-enhanced collagen production in the HSC cell line, and reduce liver injury and fibrosis in the animal model. C_LIO_LIDemonstrated that TLR4 is involved in histone-enhanced collagen I production in HSC cells. In vivo, the TLR4-MyD88 signalling pathway was demonstrated to be involved in liver fibrosis, but whether circulating histones are the major activators of the pathway is not fully clear. C_LI Research conclusionRecurrent liver injury releases extracellular histones which potentially activate TLR4-MyD88 signalling pathway to promote liver fibrosis. The ability of NAHP to detoxify circulating histones holds the potential for the treatment of liver injury and prevention of liver fibrosis. Research perspectivesFuture studies demonstrating the contributions of circulating histones in activating the TLR4-MyD88 signalling pathway in vivo will validate their role in liver fibrosis. Development of better anti-histone reagents to reduce liver injury and prevent liver fibrosis will hold great potential in the management of diseases with recurrent liver injury.

pathology↗

bioRxiv2020

SARS-CoV-2 spike D614G variant exhibits highly efficient replication and transmission in hamsters

SARS-CoV-2 causes disease varying in severity from asymptomatic infections to severe respiratory distress and death in humans. The viral factors which determine transmissibility and pathogenicity are not yet clearly characterized. We used the hamster infection model to compare the replication ability and pathogenicity of five SARS-CoV-2 strains isolated from early cases originating in Wuhan, China, in February, and infected individuals returning from Europe and elsewhere in March 2020. The HK-13 and HK-95 isolates showed distinct pathogenicity in hamsters, with higher virus titers and more severe pathological changes in the lungs observed compared to other isolates. HK-95 contains a D614G substitution in the spike protein and demonstrated higher viral gene expression and transmission efficiency in hamsters. Intra-host diversity analysis revealed that further quasi species were generated during hamster infections, indicating that strain-specific adaptive mutants with advantages in replication and transmission will continue to arise and dominate subsequent waves of SARS-CoV-2 dissemination.

microbiology↗

bioRxiv2020

piRNA-mediated gene regulation and adaptation to sex-specific transposon expression in D. melanogaster male germline

Small non-coding piRNAs act as sequence-specific guides to repress complementary targets in Metazoa. Prior studies in Drosophila ovaries have demonstrated the function of piRNA pathway in transposon silencing and therefore genome defense. However, the ability of piRNA program to respond to different transposon landscape and the role of piRNAs in regulating host gene expression remain poorly understood. Here, we comprehensively analyzed piRNA expression and defined the repertoire of their targets in Drosophila melanogaster testes. Comparison of piRNA programs between sexes revealed sexual dimorphism in piRNA programs that parallel sex-specific transposon expression. Using a novel bioinformatic pipeline, we identified new piRNA clusters and established complex satellites as dual-strand piRNA clusters. While sharing most piRNA clusters, two sexes employ them differentially to combat sex-specific transposon landscape. We found several host genes targeted by piRNAs in testis, including CG12717/pita, a SUMO protease gene. piRNAs encoded on Y chromosome silence pita, but not its paralog, to exert sex- and paralog-specific gene regulation. Interestingly, pita is targeted by endogenous siRNAs in a sibling species, Drosophila mauritiana, suggesting distinct but related silencing strategies invented in recent evolution to regulate a conserved protein-encoding gene.

molecular biology↗

bioRxiv2020

RDC complex executes a dynamic piRNA program during Drosophila spermatogenesis to safeguard male fertility

piRNAs are small non-coding RNAs that guide the silencing of transposons and other targets in animal gonads. In Drosophila female germline, many piRNA source loci dubbed piRNA clusters lack hallmarks of active genes and exploit an alternative path for transcription, which relies on the Rhino-Deadlock-Cutoff (RDC) complex. It remains to date unknown how piRNA cluster transcription is regulated in the male germline. We found that components of RDC complex are expressed in male germ cells during early spermatogenesis, from germline stem cells (GSCs) to early spermatocytes. RDC is essential for expression of dual-strand piRNA clusters and transposon silencing in testis; however, it is dispensable for expression of Y-linked Suppressor of Stellate piRNAs and therefore Stellate silencing. Despite intact Stellate repression, rhi mutant males exhibited compromised fertility accompanied by germline DNA damage and GSC loss. Thus, piRNA-guided repression is essential for normal spermatogenesis beyond Stellate silencing. While RDC associates with multiple piRNA clusters in GSCs and early spermatogonia, its localization changes in later stages as RDC concentrates on a single X-linked locus, AT-chX. Dynamic RDC localization is paralleled by changes in piRNA cluster expression, indicating that RDC executes a fluid piRNA program during different stages of spermatogenesis.

molecular biology↗

bioRxiv2020

Pathogenicity, immunogenicity, and protective ability of an attenuated SARS-CoV-2 variant with a deletion at the S1/S2 junction of the spike protein

SARS-CoV-2 contains a PRRA polybasic cleavage motif considered critical for efficient infection and transmission in humans. We previously reported that virus variants with spike protein S1/S2 junction deletions spanning this motif are attenuated. Here we characterize a further cell-adapted SARS-CoV-2 variant, Ca-DelMut. Ca-DelMut replicates more efficiently than wild type or parental virus in cells, but causes no apparent disease in hamsters, despite replicating in respiratory tissues. Unlike wild type virus, Ca-DelMut does not induce proinflammatory cytokines in hamster infections, but still triggers a strong neutralizing antibody response. Ca-DelMut-immunized hamsters challenged with wild type SARS-CoV-2 are fully protected, demonstrating sterilizing immunity.

microbiology↗

bioRxiv2020

Single-Cell Transcriptomics Identifies Dysregulated Metabolic Programs of Aging Alveolar Progenitor Cells in Lung Fibrosis

Aging is a critical risk factor in progressive lung fibrotic diseases such as idiopathic pulmonary fibrosis (IPF). Loss of integrity of type 2 alveolar epithelial cells (AEC2s) is the main causal event in the pathogenesis of IPF. To systematically examine the genomic program changes of AEC2s with aging and lung injury, we performed unbiased single cell RNA-seq analyses of lung epithelial cells from either uninjured or bleomycin-injured young and old mice. Major lung epithelial cell types were readily identified with canonical cell markers in our dataset. Heterogenecity of AEC2s was apparent, and AEC2s were then classified into three subsets according to their gene signatures. Genes related to lipid metabolism and glycolysis were significantly altered within these three clusters of AEC2s, and also affected by aging and lung injury. Importantly, IPF AEC2s showed similar genomic programming and metabolic changes as that of AEC2s from bleomycin injured old mouse lungs relative to controls. Furthermore, perturbation of both lipid metabolism and glycolysis significantly changed progenitor renewal capacity in 3-Demensional organoid culture of AEC2s. Taken togather, this work identified metabolic defects of AEC2s in aging and during lung injury. Strategies to rectify these altered programs would promote AEC2 renewal which in turn improves lung repair. One sentence summaryMetabolic defects of alveolar progenitors in aging and during lung injury impair their renewal.

cell biology↗

bioRxiv2020

TLR7 and RIG-I dual-adjuvant loaded nanoparticles drive broadened and synergistic responses in dendritic cells and boost cellular immunity in influenza vaccination

Although the existing flu vaccines elicit strong antigen-specific antibody responses, they fail to provide effective, long term protection - partly due to the absence of robust cellular memory immunity. We hypothesized that co-administration of combination adjuvants, mirroring the flu-virus related innate signaling pathways, could elicit strong cellular immunity. Here, we show that the small molecule adjuvant R848 and the RNA adjuvant PUUC, targeting endosomal TLR7s and cytoplasmic RLRs respectively, when delivered together in polymer nanoparticles (NP), elicits a broadened immune responses in mouse bone marrow-derived dendritic cells (mBMDCs) and a synergistic response in both mouse and human plasmacytoid dendritic cells (pDCs). In mBMDCs, NP-R848-PUUC induced both NF-{kappa}B and interferon signaling. Interferon responses to co-delivered R848 and PUUC were additive in human peripheral blood mononuclear cells (PBMCs) and synergistic in human FLT3-differentiated mBMDCs and CAL-1 pDCs. Vaccination with NPs loaded with H1N1 Flu antigen, R848, and PUUC increased percentage of CD8+ T-cells in the lungs, percentage of antigen-specific CD4+T-cells in the spleen, and enhanced overall cytokine-secreting T cell percentages upon antigen restimulation. Also in the spleen, T lymphopenia, especially after in vitro restimulation, was observed. Our results demonstrate that simultaneous engagement of TLR7 and RIG-I pathways using particulate carriers is a potential approach to improve cellular immunity in flu vaccination. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/207423v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1599111org.highwire.dtl.DTLVardef@1431395org.highwire.dtl.DTLVardef@baa40dorg.highwire.dtl.DTLVardef@16877cf_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2020

Definition and Signatures of Lung Fibroblast Populations in Development and Fibrosis in Mice and Men

The heterogeneity of fibroblasts in the murine and human lung during homeostasis and disease is increasingly recognized. It remains unclear if the different phenotypes identified to date are characteristic of unique subpopulations with unique progenitors or whether they arise by a process of differentiation from common precursors. Our understanding of this ubiquitous cell type is limited by an absence of well validated, specific, markers with which to identify each cell type and a clear consensus on the distinct populations present in the lung. Here we describe single cell RNA sequencing (scRNA-seq) analysis on mesenchymal cells from the murine lung throughout embryonic (E) development (E9.5 - 17.5), at post-natal day (P1 - 15), as well as in the adult and the aged murine lungs before and after bleomycin-induced fibrosis. We carried out complementary scRNA-seq on human lung tissue from a P1 lung, a month 21 lung and lung tissue from healthy donors and patients with idiopathic pulmonary fibrosis (IPF). The murine and human data were supplemented with publicly available scRNA-seq datasets. We consistently identified lipofibroblasts, myofibroblasts, pericytes, mesothelial cells and smooth muscle cells. In addition, we identified a novel population delineated by Ebf1 (early B-cell factor 1) expression and an intermediate subtype. Comparative analysis with human mesenchymal cells revealed homologous mesenchymal subpopulations with remarkably conserved transcriptomic signatures. Comparative analysis of changes in gene expression in the fibroblast subpopulations from age matched non-fibrotic and fibrotic lungs in the mouse and human demonstrates that many of these subsets contribute to matrix gene expression in fibrotic conditions. Subtype selective transcription factors were identified and putative divergence of the clusters during development were delineated. Prospective isolation of these fibroblast subpopulations, localization of signature gene markers, and lineage-tracing each cluster are under way in the laboratory. This analysis will enhance our understanding of fibroblast heterogeneity in homeostasis and fibrotic disease conditions.

cell biology↗

bioRxiv2020

Peopling of Tibet Plateau and multiple waves of admixture of Tibetans inferred from both modern and ancient genome-wide data

Archeologically attested human occupation on the Tibet Plateau (TP) can be traced back to 160 thousand years ago (kya, Xiahe) via archaic people and 30~40 kya via anatomically modern human in Nwya Devu. However, the past human movements and peopling of the TP keep in its infancy in the modern/ancient DNA studies. Here, we performed the first modern/ancient genomic meta-analysis among 3,017 Paleolithic to present-day eastern Eurasian genomes (2,444 modern individuals from 183 populations (including 98 Ü-Tsang/Ando/Kham Tibetans) and 573 ancients (including 161 Chinese ancients first meta-analyzed here)). Closer genetic connection between ancient-modern highland Tibetans and lowland island/coastal Neolithic northern East Asians was identified, reflecting the main ancestry of high-altitude Tibeto-Burman speakers originated from the ancestors of Houli/Yangshao/Longshan ancients in the middle and lower Yellow River basin, consistent with the common North-China origin of Sino-Tibetan language and dispersal pattern of millet farmers. Although the shared common northern East Asian lineage between Tibetans and lowland East Asians, we still identified genetic differentiation between Highlanders and lowland northern East Asians, the former harboring more deeply diverged Hoabinhian/Onge ancestry and the latter possessing more modern Neolithic southern East Asian and Siberian ancestry, which suggested the co-existence of Paleolithic and Neolithic ancestries in modern and Neolithic East Asian Highlanders. Tibetans from Ü-Tsang/Ando/Kham Tibetan regions showed strong population stratifications consistent with their cultural backgrounds and geographic terrains (showed as barriers for human movements): stronger Chokhopani affinity in Ü-Tsang Tibetans, more western Eurasian ancestry in Ando and greater Neolithic southern East Asian ancestry in Kham Tibetan. Modern combined ancient genomes documented multiple waves of human migrations in TP past: the first layer of local Hunter-Gatherer mixed with Qijia Farmer arose the Chokhopani-associated Proto-Tibetan-Burman, admixture with the additional genetic materials from the western Eurasian steppe, Yellow River and Yangtze River respectively gave rise to modern Ando, Ü-Tsang and Kham Tibetans.Competing Interest StatementThe authors have declared no competing interest.View Full Text

genetics↗

bioRxiv2020

Trantrace: a reliable, traceable, and collaborative solution to streamline the translation of emerging biomedical resources

BackgroundPrecision medicine is gaining popularity in routine health care, which heavily relies on the interpretation of emerging biomedical databases and professional guidelines. Translation of those biomedical materials, that is crucial to deliver research discovery to health interventions in non-native English-speaking countries, remains a large amount of work for biomedical practitioners. ResultsWe presented an application called Trantrace to facilitate the routine of large-scale long-running translation. Especially for the case that many people joined multiple translation projects, it has a rigorous task division and cooperation process: assignment, translation, review, release, and iteration. Trantrace empowers users to build own projects with different permissions, version control all the translation operation without specific skills, and further improve the translation efficiency through real-time task management tools. ConclusionsBy providing a working platform for collaborative translation and enabling long-time version correction, the Trantrace contributes towards the creation of a multilingual biomedical knowledge commons, which would be a valuable aid for personalized therapies. The source code is freely accessible at https://github.com/sgi-drylab/trantrace.

bioinformatics↗

bioRxiv2020

Mass-spectrometry-based near complete draft of the Saccharomyces cerevisiae proteome

Proteomics approaches designed to catalogue all open reading frames (ORFs) under a defined set of growth conditions of an organism have flourished in recent years. However, no proteome has been sequenced completely so far. Here we generate the largest yeast proteome dataset, including 5610 identified proteins using a strategy based on optimized sample preparation and high-resolution mass spectrometry. Among the 5610 identified proteins, 94.1% are core proteins, which achieves near complete coverage of the yeast ORFs. Comprehensive analysis of missing proteins in our dataset indicate that the MS-based proteome coverage has reached the ceiling. A review of protein abundance shows that our proteome encompasses a uniquely broad dynamic range. Additionally, these values highly correlate with mRNA abundance, implying a high level of accuracy, sensitivity and precision. We present examples of how the data could be used, including re-annotating gene localization, providing expression evidence of pseudogenes. Our near complete yeast proteome dataset will be a useful and important resource for further systematic studies.

biochemistry↗

bioRxiv2020

Visualizing dendritic characteristics of corticotropin-releasing hormone neurons at single-cell resolution in the whole mouse brain

Corticotropin-releasing hormone (CRH) is an important neuromodulator with wide distribution in the brain. Here, we screened the CRH-IRES-Cre;Ai32 mouse line to reveal the morphologies of individual CRH neurons throughout the mouse brain by using fluorescence micro-optical sectioning tomography (fMOST) system. Diverse dendritic morphologies and projection fibers were found in various brain regions. Reconstructions showed hypothalamic CRH neurons had the smallest somatic volumes and simplest dendritic branches, and CRH neurons in several regions shared a bipolar morphology. Further investigations in the medial prefrontal cortex unveiled somatic depth-dependent morphologies that exhibited three types of connections and CRH neurons in the anterior parvicellular area of hypothalamus had fewer and smaller Herring bodies whereas in the periventricular area had more and larger Herring bodies that were present within fibers projecting to the third ventricle. Our findings provide the most comprehensive intact morphologies of CRH neurons throughout the mouse brain that is currently available.

neuroscience↗

bioRxiv2020

Single cell reconstruction of human basal cell diversity in normal and IPF lung.

RationaleDeclining lung function in patients with interstitial lung disease is accompanied by epithelial remodeling and progressive scarring of the gas-exchange region. There is a need to better understand the contribution of basal cell hyperplasia and associated mucosecretory dysfunction to the development of idiopathic pulmonary fibrosis (IPF). ObjectivesWe sought to decipher the transcriptome of freshly isolated epithelial cells from normal and IPF lung to discern disease-dependent changes within basal stem cells. MethodsSingle cell RNA sequencing was used to map epithelial cell types of the normal and IPF human airway. Organoid and ALI cultures were used to investigate functional properties of basal cell subtypes. Measurements and Main ResultsWe found that basal cells included multipotent and secretory primed subsets in control adult lung tissue. Secretory primed basal cells include an overlapping molecular signature with basal cells obtained from distal lung tissue of IPF lungs. We confirmed that NOTCH2 maintains undifferentiated basal cells and restrict basal-to-ciliated differentiation, and present evidence that NOTCH3 functions to restrain secretory differentiation. ConclusionsBasal cells are dynamically regulated in disease and are specifically biased towards expansion of the secretory primed basal cell subset in idiopathic pulmonary fibrosis. Modulation of basal cell plasticity may represent a relevant target for therapeutic intervention in IPF.

cell biology↗

bioRxiv2020

Immobilization, Direct Electrochemistry and Electrocatalysis of Hemoglobin on Peptide-Carbon Nanotube Modified Electrode

The direct electrochemistry of hemoglobin (Hb) immobilized on a glassy carbon electrode (GCE) by mixing with a hybrid dispersion of self-assembling peptide EFK8 and single-walled carbon nanotubes (SWNT) was investigated and shown to have effective properties as a biosensor for hydrogen peroxide (H2O2). Preliminary experiments involving cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) showed that the presence of SWNTs in the modifying peptide layer on GCE significantly enhanced the electrochemical response of the electrode for the ferricyanide/ferrocyanide redox couple. Also, when more layers of the peptide-SWNT dispersion were applied to the surface, the electrochemical response of the GCE was further increased. This behavior was then exploited to fabricate a biosensor for H2O2 by mixing hemoglobin with the hybrid peptide-SWNT dispersion and casting this mixture on a glassy carbon electrode. AFM imaging, CV and EIS analysis revealed successful immobilization of Hb on the electrode and the ability of the electrode to enable direct electron transfer from Hb to the electrode surface. In particular, CVs obtained in 0.1M PBS (pH 7.0) showed that the immobilized Hb retained its bio-catalytic activity for Fe ions, indicating that it had not denatured and the hybrid layer remained biocompatible with Hb. Furthermore, this electrode was found to accurately measure the H2O2 concentration over the range from 20 to 9.6x102 M in 0.1M PBS (pH 7.0) under mediatorless conditions using both CV and amperometry techniques. In this way, the EFK8-SWNT hybrid layer shows promise as a biocompatible layer for simple non-covalent enzyme immobilization and the basis of a mediatorless biosensor.

bioengineering↗

bioRxiv2020

Annexina2 as atarget protein for chlorogenic acidin human lung cancerA549cells

Chlorogenic acid, an important active component of coffee with anti-tumor activities, has been found for many years. However, the lack of understanding about its target proteins greatly limits the exploration of its anti-tumor molecular mechanism and clinical application. Here, in vitro and animal experiments showed that chlorogenic acid had a significant inhibitory effect on the proliferation of A549 cells. Using the spontaneous fluorescence characteristic of chlorogenic acid to screen the target proteins cleverly to avoid the problem of chemical modification increasing false positive, we identify and verify annexin A2 (ANXA2) as a covalent binding target of chlorogenic acid in A549 cells. Then, we discover that chlorogenic acid as an inhibitor of the binding of ANXA2 to p50 subunit inhibited the expression of downstream anti-apoptotic genes cIAP1 and cIAP2 of NF-κB signaling pathway in A549 cells in vitro and vivo. Moreover, we find chlorogenic acid hindered the binding of ANXA2 and actin maybe involved in the impediment of tumor cell cycle and migration. Thus, this work demonstrates that chlorogenic acid, as a binding ligand of ANXA2, decrease the expression of NF-κB downstream anti-apoptotic genes, inhibiting the proliferation of A549 cells in vivo and vitro.View Full Text

pharmacology and toxicology↗

bioRxiv2020

Discovery of a molecular glue promoting CDK12-DDB1 interaction to trigger Cyclin K degradation

Molecular glues are small molecules that exert their biologic or therapeutic activities by inducing gain-of-function interactions between pairs of proteins. In particular, molecular-glue degraders, which mediate interactions between target proteins and components of the ubiquitin proteasome system to cause targeted protein degradation, hold great promise as a unique modality for therapeutic targeting of proteins that are currently intractable. Here, we report a new molecular glue HQ461 discovered by high-throughput screening of small molecules that inhibited NRF2 activity. Using unbiased loss-of-function and gain-of-function genetic screening followed by biochemical reconstitution, we show that HQ461 acts by promoting interaction between CDK12 and DDB1-CUL4-RBX1 E3 ubiquitin ligase, leading to polyubiquitination and proteasomal degradation of CDK12s interacting protein Cyclin K (CCNK). Degradation of CCNK mediated by HQ461 compromised CDK12 function, leading to reduced phosphorylation of CDK12 substrate, downregulation of DNA damage response genes, and cell death. Structure-activity relationship analysis of HQ461 revealed the importance of a 5-methylthiazol-2-amine pharmacophore and resulted in an HQ461 derivate with improved potency. Our studies reveal a new molecular glue that engages its target protein directly with DDB1 to bypass the requirement of a substrate-specific receptor, presenting a new strategy for targeted protein degradation.

biochemistry↗