Biology subjects

Ouyang, H.

Publications and source records attributed to Ouyang, H..

30 records · Page 2Linked to original sources

bioRxiv2020

Obstructed tear duct causes epiphora and precocious eyelid opening due to disruption of Prickle 1-mediated Wnt/PCP signaling

The tear drainage apparatus evolved in terrestrial animals serving as conduits for tear flow. Obstruction of tear drainage causes a range of ocular surface disorders. Hitherto, genetics of tear duct development and obstruction has been scarcely explored. Here we report that a severe Prickle 1 hypomorph mouse line exhibited epiphora. This phenotype was due to blockage of the tear drainage by the incompletely formed nasolacrimal duct (NLD) and lacrimal canaliculi (CL). Further analysis revealed that the precocious eyelid opening, previously observed in the same type of Prickle 1 mutants, is also caused by tear duct dysplasia. A comparison of wild type, the Prickle 1 hypomorph and null mutants revealed a dose-dependent requirement of Prickle 1 for tear duct outgrowth. As a key component of a set of six Wnt/PCP core proteins, Prickle 1 usually works together with other PCP components. An investigation of expression of Wnt/PCP core genes demonstrated three of the six PCP components in tear duct, supporting the notion of context-dependent organization of PCP protein complexes. Furthermore, expression of Fgfr2/Fgf10 and p63 genes, mutations of which are associated with NLD and CL hypoplasia in human, were not altered in Prickle 1 mutant mice. Lastly, we showed that Prickle 1 expression in developing tear drainage system is conserved between mouse and human despite anatomical differences. Altogether, the study uncovered how obstruction of the tear drainage could lead to a complex ocular surface disorder, which may have genetic implications in human ocular health.

genetics↗

bioRxiv2020

Introduction of the Aspergillus fumigatus α-1,2-mannosidase MsdS into Trichoderma reesei leads to abnormal polarity and improves the ligno-cellulose degradation

-1,2-Mannosidase is an important enzyme essential for N-glycan processing and plays a significant role in the biosynthesis and organization of fungal cell wall. Lacking of -1,2-mannosidase leads to cell wall defect in yeast and filamentous fungi. Trichoderma reesei is known to be non-toxic to human, and its N-glycan on secreted glycoprotein is Man8GlcNAc2. To evaluate the significance of the N-glycan processing in T. reesei, in this study Aspergillus fumigatus -1, 2-mannosidase MsdS, an enzyme that cleaves N-linked Man8GlcNAc2 in Golgi to produce Man6GlcNAc2 on secreted glycoprotein, was introduced into T. reesei. The msdS-expressing strain Tr-MsdS produced a major glycoform of Man6GlcNAc2 on its secreted glycoproteins, instead of Man8GlcNAc2 in the parent strain. Although the cell wall content of msdS-expressing strain Tr-MsdS was changed, it appeared that the cell wall integrity was not affected. However, phenotypes such as increased conidiation, multiple budding and random branching were observed in strain Tr-MsdS. In addition, expression of MsdS into T. ressei also affected protein secretion and improved the ligno-cellulose degradation of T. reesei. Our results indicate that processing of the N-glycan is species-specific and plays an important role in protein secretion in T. reesei, specially cellulases. Also, our results provide a new strategy to improve cellulases production by interfering the N-glycan processing in T. reesei. ImportanceFor the first time, the N-glycan processing is shown to play an important role in polarized growth and protein secretion in T. reesei. In addition, our results show that alterated N-glycan processing enhances cellulose degradation, which provides a strategy to improve cellulases production in T. reesei.

bioengineering↗

bioRxiv2020

Ontogenesis of the tear drainage system requires Prickle 1-controlled polarized basement membrane (BM) deposition

In terrestrial animals, lacrimal drainage apparatus evolved to serve as conduits for tear flow. Little is known about the ontogenesis of this system. Here, we investigated tear duct origin, developmental course, genetic and cellular determinants in mouse. We report that primordial tear duct (PTD) originates from junction epithelium of the joining maxillary and lateral nasal processes, which reshapes into future tear duct branches. We identified Prickle 1 as a hallmark for tear duct outgrowth, ablation of which stalled duct elongation. In particular, the disruption of basement membrane (BM) with cytoplasmic accumulation of laminin suggests aberrant protein trafficking. Mutant embryoid bodies (EBs) derived from iPSCs recapitulate BM phenotype of the PTD exhibiting defective visceral endoderm (VE), which normally expresses high level of Prickle 1. Furthermore, replenishing mutant VE with Prickle 1 completely rescued BM but not cell polarity. Taken together, our results reveal a distinct role of Prickle 1 in regulating polarized BM secretion and deposition in precedently uncharacterized tear drainage system and VE, which is independent of apicobasal polarity establishment.

developmental biology↗

bioRxiv2020

AfRip3, a RIP3-like kinase, is identified as a key modulator of necroptotic death in Aspergillus fumigatus

Aspergillus fumigatus exhibits autophagic and necroptotic process when its GPI anchor synthesis is suppressed. A putative kinase (AFUA_6G02590) is found to be overexpressed in response to GPI anchor suppression and identified as a RIP3-like protein, namely AfRip3. To elucidate its function, in this study a Afrip3-overexpressing strain OE-Afrip3 was constructed. Although OE-Afrip3 strain exhibited an increased cell death, neither apoptotic nor autophagic process was activated. Our evidences demonstrated that overexpression of Afrip3 gene in A. fumigatus only led to necroptosis, while the Afrip3-knockout mutant was unable to activate necroptotic process. Further analysis revealed that both JNK and SMase pathways were activated in OE-Afrip3 strain, by which an increase of reactive oxygen species (ROS) was induced. We also showed that expression of Afrip3 gene was induced by Ca2+. In addition, eEF1B{gamma} and adenylylsulfate kinase (ASK) were identified as potential candidates to interact with AfRip3. These results indicate that AfRip3 is a key modulator that activates necroptotic process in A. fumigatus, which can be induced by Ca2+ and in turn activate JNK (c-Jun NH2-terminal kinase) and SMase (sphingomyelinase) pathway. Our findings suggest that necroptotic pathway in A. fumigatus is distinct from that in mammalian cell and may provide a new strategy for development of anti-fungal drug. Author summaryAspergillus fumigatus is a human fungal pathogen and causes invasive aspergillosis (IA) in immunocompromised patients with high mortality (30-95%). Development of novel therapies is urgently needed. In this study, we confirm AfRip3 (AFUA_6G02590), a RIP3-like protein, is a key modulator that activates necroptotic process in A. fumigatus. We also find that cytosolic Ca2+ can induce the expression of Afrip3 and activated AfRip3 in turn activate JNK (c-Jun NH2-terminal kinase) and SMase (sphingomyelinase) pathway. Our findings suggest that necroptotic pathway in A. fumigatus is distinct from that in mammalian cell and may provide a new strategy for development of anti-fungal drug.

microbiology↗

bioRxiv2019

Widespread arginine phosphorylation in human cells - a novel protein PTM revealed by mass spectrometry

Arginine phosphorylation (pArg) is recently discovered as a ubiquitous protein N- phosphorylation in bacteria. However, its prevalence and roles in mammalian cells remain largely unknown due to the lack of established workflow and the inherent lability of the phosphoramidate (P-N) bond. Emerging evidence suggests that N-phosphorylation may extensively exist in eukaryotes and play crucial roles. We report an experimental phosphoproteomic workflow, which for the first time allowed to reveal the widespread occurrence of pArg in human cells by mass spectrometry. By virtue of this approach, we identified 152 high-confidence pArg sit]es derived from 118 proteins. Remarkably, the discovered phosphorylation motif and gene ontology of pArg hint a possible cellular function of arginine phosphorylation by regulating the favorability of propeptide convertase substrate. The generated extensive data set should enable a better understanding of the biological functions of eukaryotic pArg in the future.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=37 SRC=\"FIGDIR/small/725291v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (13K):\norg.highwire.dtl.DTLVardef@77b2d0org.highwire.dtl.DTLVardef@7c76f4org.highwire.dtl.DTLVardef@1658d6borg.highwire.dtl.DTLVardef@27a2e2_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2018

Super-resolution Molecular Map of Basal Foot Reveals Novel Cilium in Airway Multiciliated Cells

Motile cilia are beating machines that play a critical role in airway defense. During airway cell differentiation, hundreds of motile cilia are templated from basal bodies that extend a basal foot, an appendage that links motile cilia together to ensure beating coordination. This assembly has thus far escaped structural analysis because its size is below the resolution limit. Here, we determine the molecular architecture and identify basal foot proteins using a super-resolution-driven approach. Quantitative super-resolution image analysis shows that the basal foot is organized in three main regions linked by elongated coiled-coil proteins. FIB-SEM tomography and comparative super-resolution mapping of basal feet reveal that, among hundreds of motile cilia of an airway cell, a hybrid cilium with features of primary and motile cilia is harbored. The hybrid cilium is conserved in mammalian multiciliated cells and originates from parental centrioles. We further demonstrate that this novel cilium is a signalling centre whose cellular position is dependent on flow.

cell biology↗

bioRxiv2018

Development of whole-porcine monoclonal antibodies with potent neutralization activity against classical swine fever virus (CSFV) from single B cells

Classical swine fever (CSF) is a highly contagious swine disease found worldwide that has caused devastating economic losses. However, there are few efficacious mAbs against the CSF virus (CSFV) that can be used for treatment because most mAbs against CSFV are derived from mouse hybridoma cells and these murine mAbs have disadvantages of inefficient effector functions elicitations and high immunogenicity in vivo. Accordingly, we characterized whole-porcine anti-CSFV neutralizing mAbs (NAbs) isolated directly from single B cells sorted from a CSFV-vaccinated pig using the fluoresceinated conserved linear neutralizing epitope of the CSFV E2 protein and fluorophore conjugated goat anti-pig IgG. Immunoglobulin (Ig) genes were isolated via nested PCR, and two porcine mAbs termed HK24 and HK44 were produced. We determined that these mAbs can bind to E2 protein and recognize sites within this major antigenic epitope. In addition, we found that mAbs HK24 and HK44 exhibit potent neutralizing activity against CSFV, and they can protect PK-15 cells from infections in vitro with potent IC50 values of 9.3 g/ml and 0.62 g/ml, respectively. Notably, we demonstrated that these two mAbs can be used as novel reagents for detecting virus infection. These data suggest that our results not only provide a method for efficiently obtaining mAbs against CSFV but also offer promising mAb candidates for development of antibody-based diagnostic and antiviral agents.\n\nImportanceNeutralizing monoclonal antibodies (NAbs) can prevent and may slow the spread of virus infection. The discovery of NAbs that recognize classical swine fever virus (CSFV) necessitates new technologies because the NAbs produced by immunization and hybridoma technology could not be transferred to in vivo research. Multiple full-length human therapeutic antibodies have been produced via single-cell polymerase chain reactions but whole-porcine NAbs for CSFV have not been generated. In this study, two whole-porcine mAbs, named HK24 and HK44, were isolated from epitope-specific single B cells. We demonstrate that these two mAbs have potent neutralizing activity against CSFV and can protect cells against viral infection. Therefore, they may facilitate the development of vaccines or antiviral drugs that offer the advantages of stability and low immunogenicity.

molecular biology↗

bioRxiv2018

Genetically modified pigs are protected from classical swine fever virus

Classical swine fever (CSF) caused by classical swine fever virus (CSFV) is among the most detrimental diseases, and leads to significant economic losses in the swine industry. Despite efforts by many government authorities try to stamp out the disease from national pig populations, the disease remains widespread. Here, antiviral small hairpin RNAs (shRNAs) were selected and then inserted at the porcine ROSA26 (pROSA26) locus via a CRISPR/Cas9-mediated knock-in strategy. Finally, anti-CSFV transgenic (TG) pigs were produced by somatic nuclear transfer (SCNT). Importantly, in vitro and in vivo viral challenge assays demonstrated that these TG pigs could effectively limit the growth of CSFV and reduced CSFV-associated clinical signs and mortality, and the disease resistance was stably transmitted to F1-generation. The use of these TG pigs can improve the well-being of livestock and substantially reduce virus-related economic losses. Additionally, this antiviral approach may provide a reference for future antiviral research.\n\nAuthor summaryClassical swine fever (CSF), caused by classical swine fever virus (CSFV), and is a highly contagious, often fatal porcine disease with significant economic losses. Due to its economic importance to the pig industry, the biology and pathogenesis of CSFV have been investigated extensively. Despite efforts by many government authorities to stamp out the disease from national pig populations, the disease remains widespread in some regions and seems to be waiting for the reintroduction and the next round of disease outbreaks. These highlight the necessity and urgency of developing more effective approaches to eradicate the challenging CSFV. In this study, we successfully produced anti-CSFV transgenic pigs and confirmed that these transgenic pigs could effectively limit the growth of CSFV in vivo and in vitro and that the disease resistance traits in the TG founders can be stably transmitted to their F1-generation offspring. This study suggests that these TG pigs can improve the well-being of livestock and contribute to offer potential benefits over commercial vaccination. The use of these TG pigs can improve the well-being of livestock and substantially reduce CSFV-related economic losses.

genomics↗

bioRxiv2018

High resolution profile of body wide pathological changes induced by abnormal elastin metabolism in Loxl1 knockout mice

Abnormal ECM caused serious body wide diseases and elastin is one of the important ECM components. But its systemic function still has not yet been thoroughly illustrated due to limitations related to novel research technologies. To uncover the functions of elastin, a new method for body-wide organ transcriptome profiling, combined with single-cell mass cytometry of the blood, was developed. A body-wide organ transcriptomic (BOT) map was created by performing RNA-seq of 17 organs from both Loxl1 knockout (KO) and wide type (WT) mice. The BOT results showed a systematic up-regulation of genes related to immune response and proliferation process in multiple tissues of the KO mice; histological and immune staining also confirmed the hyperplasia and infiltration of local immune cells in the vagina, small intestine, and liver tissues of KO mice. Furthermore, using 32 markers, CYTOF mass cytometry analysis of the immune cell subpopulations from the peripheral blood revealed apparent systemic immune changes in the KO mice; data showed an activated NK cells and T cells with a higher expression of CD44 and CD38, and a suppressed B cells, macrophages and neutrophils with lower expressions of CD62L, CD44 and IL6. More interestingly, these findings also correlated well with the data obtained from cancer patient databases; tumor patients had higher mutation frequency of Loxl1, and the Loxl1-mutant tumor patients had up-regulated immune process, cell proliferation and decreased survival rate. Thus, this research provided a powerful strategy to screen body-wide organ functions of a particular gene; the findings also illustrated the important biological roles of elastin on multiple organ cells and systemic immunity. These strategy and discoveries are both of important value for the understanding of ECM biology and multi-organ cancer pathology.

cancer biology↗

bioRxiv2018

Identification and Structure-Activity Relationship of HDAC6 Zinc-finger Ubiquitin Binding Domain Inhibitors

HDAC6 plays a central role in the recruitment of protein aggregates for lysosomal degradation, and is a promising target for combination therapy with proteasome inhibitors in multiple myeloma. Pharmacologically displacing ubiquitin from the zinc-finger ubiquitin-binding domain (ZnF-UBD) of HDAC6 is an underexplored alternative to catalytic inhibition. Here, we present the discovery of a HDAC6 ZnF-UBD-focused chemical series and its progression from virtual screening hits to low micromolar inhibitors. A carboxylate mimicking the C-terminal extremity of ubiquitin, and an extended aromatic system stacking with W1182 and R1155 are necessary for activity. One of the compounds induced a conformational remodeling of the binding site where the primary binding pocket opens-up onto a ligand-able secondary pocket that may be exploited to increase potency. The preliminary structure-activity relationship accompanied by nine crystal structures should enable further optimization into a chemical probe to investigate the merit of targeting the ZnF-UBD of HDAC6 in multiple myeloma and other diseases.

biophysics↗

bioRxiv2018

Cell atlas of human uterus

The human uterus is a highly dynamic tissue that undergoes repeated damage repair and regeneration during the menstrual cycle, which make it ideal model to study tissue regeneration and pathological process. Stem/progenitors were speculated to be involved in the regeneration of endometrial epithelial and pathogenesis of endometriosis. But the identity, microenvironment and regulatory mechanisms of the uterus epithelial stem/progenitors in vivo remain unclear. Here, we dissected the cell heterogeneities of the full-thickness human uterus epithelial cells (11 clusters), stroma cells (6 clusters), endothelial cells (5 clusters), smooth muscle cells (2 clusters), myofibroblasts (2 clusters) and immune cells (6 clusters) from 2735 single cell by single cell RNA-seq. Further analysis identified a unique ciliated epithelial cell cluster showing characteristics of stem/progenitors with properties of epithelial-mesenchymal transition (EMT) that mainly localized in the upper functionalis of the endometrium. Ordering the cell subpopulations along the pseudo-space revealed cell clusters possess cellular states of stress, inflammation and apoptosis in the upper functionalis cellular ecosystem of the endometrium. Connectivity map between the human uterus subpopulations revealed potential inflammatory (cytokines and chemokines) and developmental (WNT, FGF, VEGF) signals within the upper functionalis cellular ecosystem of the endometrium, especially from other epithelial clusters, regulating cell plasticity of the EMT-epithelial clusters. This study reconstructed the heterogeneities, space-specific distribution and connectivity map of human uterus atlas, which would provide insight in the regeneration of uterus endometria and reference for the pathogenesis of uterus.

cell biology↗

bioRxiv2018

Local Delivery of Stromal Cell-Derived Factor-1α Improves the Pregnancy Rate of Injured Uterus through the Promotion of Endometrial and Vascular Regeneration

Severe infection and mechanical injury of the uterus may lead to infertility and miscarriage. Currently, there is a lack of effective treatment modality for functional repair of uterine injury. To address this clinical challenge, this study aimed to develop a chemotactic composite scaffold by incorporating recombinant human stromal cell-derived factor-1 (rhSDF-1) into a silk fibroin-bacterial cellulose (SF-BC) membrane carrier. A rat model of uterine injury was utilized for this study, which was composed of three groups: blank control, implantation with SF-BC only or SF-BC loaded with rhSDF-1. The tissue regeneration efficacy of the three groups were analyzed and compared. The results showed that SF-BC loaded with rhSDF-1 significantly enhanced endometrial regeneration and arteriogenesis of the injured rat uterus, which led to improved pregnancy outcomes, thus indicating much promise for functional uterine repair and regeneration.

bioengineering↗