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Shen, J.

Publications and source records attributed to Shen, J..

At least 217 records · Page 12Linked to original sources
bioRxiv2020

Inhibitory and Transport Mechanisms of the Human Cation-Chloride Cotransport KCC1

Secondary active cation-chloride cotransporters (CCCs) catalyze electroneutral symport of Cl- with Na+ and/or K+ across membranes1,2. CCCs are fundamental in cell volume homeostasis, transepithelia ion movement, maintenance of intracellular Cl- concentration, and inhibitory synaptic transmission3-6. K+-Cl- cotransport 1 (KCC1) was first characterized in red blood cells and later in many other cell types as a crucial player in regulatory volume decrease in defense against cell swelling upon hypotonic challenges7,8. Here we present two cryo-EM structures of human KCC1: one captured in an inward-open state and another arrested in an outward-open state by a small molecule inhibitor. KCC1 can surprisingly adopt two distinct dimeric architectures via homotypic association of different protein domains and conversion between these two forms of dimers may entail dynamic formation and rupture of two interdigitating regulatory cytoplasmic domains. The inhibitor wedges into and forces open an extracellular ion permeation path and arrests KCC1 in an outward-open conformation. Concomitantly, the outward-open conformation involves inward movement of the transmembrane helix 8 and occlusion of the intracellular exit by a conserved short helix within the intracellular loop 1. Our structures provide a blueprint for understanding the mechanisms of CCC transporters and their inhibition by small molecule compounds.

biophysics↗
bioRxiv2020

Assessment of Proton-Coupled Conformational Dynamics of SARS and MERS Coronavirus Papain-like Proteases: Implication for Designing Broad-Spectrum Antiviral Inhibitors

Broad-spectrum antiviral drugs are urgently needed to stop the COVID-19 pandemic and prevent future ones. The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is related to SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), which have caused the previous outbreaks. The papain-like protease (PLpro) is an attractive drug target due to its essential roles in the viral life cycle. As a cysteine protease, PLpro is rich in cysteines and histidines and their protonation/deprotonation modulates catalysis and conformational plasticity. Here we report the pKa calculations and assessment of the proton-coupled conformational dynamics of SARS-CoV-2 in comparison to SARS-CoV and MERS-CoV PLpros using a newly developed GPU-accelerated implicit-solvent continuous constant pH molecular dynamics method with an asynchronous replica-exchange scheme. The calculated pKa’s support the catalytic roles of the Cys-His-Asp triad. We also found that several residues can switch protonation states at physiological pH, among which is C270/271 located on the flexible blocking loop 2 (BL2) of SARS-CoV-2/CoV PLpro. Simulations revealed that the BL2 conformational dynamics is coupled to the titration of C271/270, in agreement with the crystal structures of SARS-CoV-2 PLpro. Simulations also revealed that BL2 in MERS-CoV PLpro is very flexible, sampling both open and closed states despite the lack of an analogous cysteine. Our work provides a starting point for more detailed mechanistic studies to assist structure-based design of broad-spectrum inhibitors against CoV PLpros.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗
bioRxiv2020

SARS-CoV-2 envelope protein causes acute respiratory distress syndrome (ARDS)-like pathological damage and constitutes an antiviral target

Cytokine storm and multi-organ failure are the main causes of SARS-CoV-2-related death. However, the origin of the virus excessively damaging abilities remains unknown. Here we show that the SARS-CoV-2 envelope (2-E) protein alone is sufficient to cause acute respiratory distress syndrome (ARDS)-like damage in vitro and in vivo. Overexpression of 2-E protein induced rapid pyroptosis-like cell death in various susceptible cells and robust secretion of cytokines and chemokines in macrophages. Intravenous administration of purified 2-E protein into mice caused ARDS-like pathological damage in lung and spleen. Overexpressed 2-E protein formed cation channels in host cell membranes, eventually leading to membrane rupture. Newly identified channel inhibitors exhibited potent anti-SARS-CoV-2 activity and excellent protective effects against the 2-E-induced damage both in vitro and in vivo. Importantly, their channel inhibition, cell protection and antiviral activities were positively correlated with each other, supporting 2-E is a promising drug target against SARS-CoV-2.

microbiology↗
bioRxiv2020

TRPM7 kinase-mediated immunomodulation in macrophage plays a central role in magnesium ion-induced bone regeneration

The use of magnesium ion (Mg2+)-modified biomaterials in bone regeneration is a promising and cost-effective therapeutic. Despite the widespread observation on the osteogenic effects of Mg2+, the diverse roles played by Mg2+ in the complex biological process of bone healing have not been systematically dissected. Here, we reveal a previously unknown biphasic mode of action of Mg2+ in bone repair. In the early inflammation phase, Mg2+ primarily targets the monocyte-macrophage lineage to promote their recruitment, activation, and polarization. We showed that an increase in extracellular Mg2+ contributes to an upregulated expression of transient receptor potential cation channel member 7 (TRPM7) and a TRPM7-dependent influx of Mg2+ in the monocyte-macrophage lineage, resulting in the cleavage and nuclear accumulation of TRPM7-cleaved kinase fragments (M7CKs). This then triggers the phosphorylation of Histone H3 at serine 10, in a TRPM7-dependent manner at the promoters of inflammatory cytokines like IL-8, leading to the formation of a pro-osteogenic immune microenvironment. In the later active repair/remodeling phase of bone healing, however, continued exposure of Mg2+ and IL-8 leads to over activation of NF-{kappa}B signaling in macrophages, turning the immune microenvironment into pro-osteoclastogenesis. Moreover, the presence of Mg2+ at this stage also decelerates bone maturation through the suppression of hydroxyapatite precipitation. The negative effects of Mg2+ on osteogenesis can override the initial pro-osteogenic benefits of Mg2+, as we found prolonged delivery of Mg2+ compromises overall bone formation. Taken together, this study establishes a paradigm shift in understanding the diverse and multifaceted roles of Mg2+ in bone healing.

cell biology↗
bioRxiv2020

Efficacy of transoral laser surgery versus linear accelerator radiotherapy for the treatment of T1 glottic carcinoma: a meta-analysis of oncologic outcomes

ObjectiveA meta-analysis was conducted to compare oncologic outcomes for patients of T1 glottic carcinoma who were treated with transoral laser surgery (TLS) or linear accelerator radiotherapy (linac RT). MethodsAll related studies published up to September 2019 were acquired by searching Pubmed, EMBASE, and Cochrane, with the index words: glottic, vocal, laryngeal, radiation, radiotherapy, irradiation, laser, surgery, cordectomy, carcinoma and cancer. Relative studies which compared oncologic outcomes between linac RT and TLS were included. Sensitivity analysis were performed to evaluate heterogeneity. ResultsA total of twelve eligible studies were included for the analysis, which contained three prospective studies and nine retrospective studies. Patients who underwent TLS had increased overall survival (OR = 1.40, 95% CI=1.02-1.94, P=0.04) and laryngeal preservation (OR = 5.37, 95% CI = 3.05-9.44, P < 0.00001) versus who underwent linac RT. No statistical difference was observed between TLS group and linac RT group in terms of local control (OR=0.88, 95% CI = 0.62-1.24, P = 0.47), disease-specific survival (OR = 0.61, 95% CI = 0.26-1.43, P = 0.26), and disease-free survival (OR = 1.63, 95% CI = 0.70-3.81, P = 0.14). ConclusionsThe results of this meta-analysis indicate that there were clinical benefits for patients with glottic carcinoma after TLS compared with linac RT with respect to overall survival and laryngeal preservation. However, more multi-center randomized controlled trials would be urgently needed to prove these differences.

cancer biology↗
bioRxiv2020

Cryo-EM structures reveal transcription initiation steps by yeast mitochondrial RNA polymerase

Cryo-EM structures of transcription pre-initiation complex (PIC) and initiation complex (IC) of yeast mitochondrial RNA polymerase show fully resolved transcription bubbles and explain promoter melting, template alignment, DNA scrunching, transition into elongation, and abortive synthesis. Promoter melting initiates in PIC with MTF1 trapping the -4 to -2 non-template (NT) bases in its NT-groove. Transition to IC is marked by a large-scale movement that aligns the template with RNA at the active site. RNA synthesis scrunches the NT strand into an NT-loop, which interacts with centrally positioned MTF1 C-tail. Steric clashes of the C-tail with RNA:DNA and NT-loop, and dynamic scrunching-unscrunching of DNA explain abortive synthesis and transition into elongation. Capturing the catalytically active IC-state with UTPS poised for incorporation enables modeling toxicity of antiviral nucleosides/nucleotides.

biophysics↗
bioRxiv2020

Discovery of baicalin and baicalein as novel, natural product inhibitors of SARS-CoV-2 3CL protease in vitro

Human infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cause coronavirus disease 19 (COVID-19) and there is currently no cure. The 3C-like protease (3CLpro), a highly conserved protease indispensable for replication of coronaviruses, is a promising target for development of broad-spectrum antiviral drugs. To advance the speed of drug discovery and development, we investigated the inhibition of SARS-CoV-2 3CLpro by natural products derived from Chinese traditional medicines. Baicalin and baicalein were identified as the first non-covalent, non-peptidomimetic inhibitors of SARS-CoV-2 3CLpro and exhibited potent antiviral activities in a cell-based system. Remarkably, the binding mode of baicalein with SARS-CoV-2 3CLpro determined by X-ray protein crystallography is distinctly different from those of known inhibitors. Baicalein is perfectly ensconced in the core of the substrate-binding pocket by interacting with two catalytic residues, the crucial S1/S2 subsites and the oxyanion loop, acting as a "shield" in front of the catalytic dyad to prevent the peptide substrate approaching the active site. The simple chemical structure, unique mode of action, and potent antiviral activities in vitro, coupled with the favorable safety data from clinical trials, emphasize that baicalein provides a great opportunity for the development of critically needed anti-coronaviral drugs.

molecular biology↗
bioRxiv2020

Plastome phylogenomic study of Gentianeae (Gentianaceae): widespread gene tree discordance and its association with evolutionary rate heterogeneity of plastid genes

BackgroundPlastome-scale data have been prevalent in reconstructing the plant Tree of Life. However, phylogenomic studies currently based on plastomes rely primarily on maximum likelihood (ML) inference of concatenated alignments of plastid genes, and thus phylogenetic discordance produced by individual plastid genes has generally been ignored. Moreover, structural and functional characteristics of plastomes indicate that plastid genes may not evolve as a single locus and are experiencing different evolutionary forces, yet the genetic characteristics of plastid genes within a lineage remain poorly studied. ResultsWe sequenced and annotated ten plastome sequences of Gentianeae. Phylogenomic analyses yielded robust relationships among genera within Gentianeae. We detected great variation of gene tree topologies and revealed more than half of the genes, including one (atpB) of the three widely used plastid markers (rbcL, atpB and matK) in phylogenetic inference of Gentianeae, are likely contributing to phylogenetic ambiguity of Gentianeae. Estimation of nucleotide substitution rates showed extensive rate heterogeneity among different plastid genes and among different functional groups of genes. Comparative analysis suggested that the ribosomal protein (RPL and RPS) genes and the RNA polymerase (RPO) genes have higher substitution rates and genetic variations in Gentianeae. Our study revealed that just one (matK) of the three (matK, ndhB and rbcL) widely used markers show high phylogenetic informativeness (PI) value. Due to the high PI and lowest gene-tree discordance, rpoC2 is advocated as a promising plastid DNA barcode for taxonomic studies of Gentianeae. Furthermore, our analyses revealed a positive correlation of evolutionary rates with genetic variation of plastid genes, but a negative correlation with gene-tree discordance under purifying selection. ConclusionsOverall, our results demonstrate the heterogeneity of nucleotide substitution rates and genetic characteristics among plastid genes providing new insights into plastome evolution, while highlighting the necessity of considering gene-tree discordance into phylogenomic studies based on plastome-scale data.

evolutionary biology↗
bioRxiv2020

Glycyrrhizic acid improves cognitive levels of aging mice by regulating T/B cell proliferation

Dear Editor,Licorice is the root of Glycyrrhiza glabra L. (Leguminosae), which grows in various warm climates such as the Middle East, Asia, and Southern Europe. It is one of the oldest known medicinal herbs and is referred to as "the father of herbal medicine." Glycyrrhizic acid (GA) (Fig. 1A), a triterpenoid saponin, is a major component of licorice. It has a variety of pharmacological activities such as anti-inflammatory, antioxidant, anticancer, neuroprotective, and immune effects, among others.[1]. Previous studies indicated that GA produces robust neuroprotection via the modulation of anti-apoptotic and pro-apoptotic factors, primarily through the ERK signaling pathway and its anti-inflammatory properties against high-mobility group box 1 phosphorylation and the suppression of inflammatory cytokine induction [2-4]. These results were based on pathological models. Although numerous pathways have been implicated in the neuroprotective effects of GA, the molecular mechanisms are not yet completely understood. In this study, we aimed to investigate the effects of GA in preventing age-related immune involution and cognitive disorders, the relationship between immune involution and cognition, and the underlying molecular mechanisms. O_FIG O_LINKSMALLFIG WIDTH=141 HEIGHT=200 SRC="FIGDIR/small/008821v1_fig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@5a123dorg.highwire.dtl.DTLVardef@163a085org.highwire.dtl.DTLVardef@1bfadb6org.highwire.dtl.DTLVardef@125b2ae_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Glycyrrhizic acid ameliorated cognitive declines in aged mice, improved CBF and increased T and B cell numbers. A. The chemical structure of glycyrrhizic acid (GA). B. Escape latencies from six consecutive daily tests. C. The first platform-finding latencies in the probe trial. D. The time spent in the target quadrant in the probe trial. E. The target platform crossing times in the probe trial. F. Images of CBF in C57BL/6 mice. G. Quantification of blood perfusion in the ipsilateral hemisphere. H. Bubble diagram showing GA treatment influence on hematopoietic cell lineage. I. Heatmap showing actively expressed genes related to T cells in the hematopoietic cell lineage of control and GA-treated C57BL/6 mice. J. Representative FACS plots showing CD3e+ T and CD45R/B220+ B cells in the spleens of C57BL mice; bar graphs showing the statistical analysis of T and B cells in the spleens of C57BL mice. K. Representative FACS plots showing CD3e+ T and CD45R/B220+ B cells in the blood of C57BL mice; Bar graphs showing the statistical results of T and B cell analyses in the blood of C57BL mice. GA (5mg/kg, once every other day); Data represent mean {+/-} SEM (n = 8 per group). *P < 0.05, **P < 0.01, ***P < 0.001 vs. Ctrl. C_FIG

animal behavior and cognition↗
bioRxiv2020

Metformin improves cognition of aged mice by promoting cerebral angiogenesis and neurogenesis

The cerebral microvasculature is essential for preservation of normal cerebral function. Age-related decreases of neurogenesis and cognitive function are accompanied by reduced blood flow and a decline in neural stem cell (NSC) number. Here, we report that metformin administered by tail vein injection enhanced cognition in aged but not young mice in a dose-dependent manner. Further, metformin restored cerebral blood flow and brain vascular density and promoted neurogenic potential of the subependymal zone/subventricular zone both in vivo and in vitro. RNA-Seq result indicated that metformin could enhance glycolysis in blood, with an increase in relative mRNA expression of the enzyme in the glycolysis pathway from hippocampal tissue of metformin-treated mice. Mechanistically, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in the glycolysis pathway, may contribute to angiogenic and neurogenic potentials of NSCs. Interestingly, examination of peripheral blood mononuclear cells from people of various ages showed that mRNA expression of GAPDH gradually decreased with age, while its expression level positively correlated with cognitive levels. Our results indicate that metformin represents a candidate pharmacological approach for recruitment of NSCs in aged mouse brain by enhancing glycolysis and promoting neurovascular generation, a strategy that might be of therapeutic value for anti-aging in humans. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/006767v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1f8fc8aorg.highwire.dtl.DTLVardef@18be6c8org.highwire.dtl.DTLVardef@1dc815forg.highwire.dtl.DTLVardef@1b19557_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗
bioRxiv2020

Automated design and optimization of multitarget schizophrenia drug candidates by deep learning

Complex neuropsychiatric diseases such as schizophrenia require drugs that can target multiple G protein-coupled receptors (GPCRs) to modulate complex neuropsychiatric functions. Here, we report an automated system comprising a deep recurrent neural network (RNN) and a multitask deep neural network (MTDNN) to design and optimize multitargeted antipsychotic drugs. The system successfully generates novel molecule structures with desired multiple target activities, among which high-ranking compound 3 was synthesized, and demonstrated potent activities against dopamine D2, serotonin 5-HT1A and 5-HT2A receptors. Hit expansion based on the MTDNN was performed, 6 analogs of compound 3 were evaluated experimentally, among which compound 8 not only exhibited specific polypharmacology profiles but also showed antipsychotic effect in animal models with low potential for sedation and catalepsy, highlighting their suitability for further preclinical studies. The approach can be an efficient tool for designing lead compounds with multitarget profiles to achieve the desired efficacy in the treatment of complex neuropsychiatric diseases. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/999615v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@17046d8org.highwire.dtl.DTLVardef@3b4c2aorg.highwire.dtl.DTLVardef@10590e9org.highwire.dtl.DTLVardef@6cc609_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗
bioRxiv2020

Auto-inhibitory regulation of DNA binding by the C-terminal tails of the mitochondrial transcription factors Mtf1 and TFB2M

The structurally homologous Mtf1 and TFB2M proteins serve as transcription initiation factors of the Saccharomyces cerevisiae and human mitochondrial RNA polymerases, respectively. These transcription factors directly interact with the non-template strand of the transcription bubble to drive promoter melting. Given the key roles of Mtf1 and TFB2M in promoter-specific transcription initiation, it is expected that the DNA binding activity of the mitochondrial transcription factors would be regulated to prevent DNA binding at inappropriate times. However, there is little information on how mitochondrial DNA transcription is regulated. While studying the C-tail deletion mutants of Mtf1 and TFB2M, we stumbled upon a new finding that suggested that the flexible C-tail region of these factors autoregulates their DNA binding activity. Quantitative DNA binding studies with fluorescence anisotropy-based titrations show that Mtf1 with an intact C-tail has no affinity for the DNA but the deletion of C-tail greatly increases the DNA binding affinity. Similar observations were made with TFB2M, although autoinhibition by the C-tail of TFB2M was not as absolute as in Mtf1. Analysis of available TFB2M structures show that the C-tail makes intramolecular interactions with the DNA binding groove in the free factor, which we propose masks the DNA binding activity. Further studies show that the RNA polymerase relieves autoinhibition by interacting with the C-tail and engaging it in complex formation. Thus, our biochemical and structural analysis identify previously unknown autoinhibitory and activation mechanisms of mitochondrial transcription factors that regulate the DNA binding activity and aid in specific assembly of the initiation complexes.

biochemistry↗
bioRxiv2020

Structure and mechanism of a primate ferroportin

Ferroportin is the only cellular iron exporter in human and essential for iron homoeostasis. Mutations in ferroportin are associated with hemochromatosis or ferroportin diseases characterized by a paradoxical combination of anemia and abnormal accumulation of iron in cells. Ferroportin is also the target of hepcidin, which is a hormone that downregulates ferroportin activity. However, due to a lack of three-dimensional structures, the mechanism of iron transport by ferroportin and its regulation by hepcidin remains unclear. Here we present the structure of a ferroportin from the primate Philippine tarsier (TsFpn) at 3.0 [A] resolution determined by cryo-electron microscopy. TsFpn has a structural fold common to major facilitator superfamily of transporters and the current structure is in an outward-open conformation. The structure identifies two potential ion binding sites with each site coordinated by two residues. Functional studies demonstrate that TsFpn is a H+/Fe2+ antiporter and that transport of one Fe2+ is coupled to the transport of two H+ in the opposite direction such that the transport cycle is electroneutral. Further studies show that the two ion binding sites affect transport of H+ and Fe2+ differently. The structure also provides mechanistic interpretation for mutations that cause ferroportin diseases.

biochemistry↗
bioRxiv2020

Hippo signaling promotes Ets21c-dependent apical cell extrusion in the Drosophila wing disc

Cell extrusion is a crucial regulator of epithelial tissue development and homeostasis. Epithelial cells undergoing apoptosis, bearing pathological mutations, and possessing developmental defects are actively extruded toward elimination. However, the molecular mechanisms of Drosophila epithelial cell extrusion are not fully understood. Here, we report that activation of the conserved Hippo (Hpo) signaling pathway induces both apical and basal cell extrusion in the Drosophila wing disc epithelia. We show that canonical Yorki targets Diap1, and that dMyc and Cyclin E are not required for either apical or basal cell extrusion induced by activation of this pathway. Another target gene, bantam, is only involved in basal cell extrusion, suggesting novel Hpo-regulated apical cell extrusion mechanisms. Using RNA-Seq analysis, we found that JNK signaling is activated in the extruding cells. We provide genetic evidence that JNK signaling activation is both sufficient and necessary for Hpo-regulated cell extrusion. Furthermore, we demonstrate that the ETS-domain transcription factor Ets21c, an ortholog of proto-oncogenes FLI1 and ERG, acts downstream of JNK signaling to mediate apical cell extrusion. Our findings reveal a novel molecular link between Hpo signaling and cell extrusion. SUMMARY STATEMENTActivation of Hippo signaling induces cell extrusion in the Drosophila wing epithelia, in which bantam mediates basal cell extrusion and Ets21c mediates apical cell extrusion.

developmental biology↗
bioRxiv2020

A Myosin-7B dependent endocytosis pathway mediates cellular entry of α-Synuclein fibrils and polycation-bearing cargos

Cell-to-cell transmission of misfolding-prone -Synuclein (-Syn) has emerged as a key pathological event in Parkinsons disease. This process is initiated when -Syn-bearing fibrils enter cells via clathrin-mediated endocytosis, but the underlying mechanisms are unclear. Using a CRISPR-mediated knockout screen, we identify SLC35B2 and Myosin-7B (MYO7B) as critical endocytosis regulators for -Syn preformed fibrils (PFF). We show that SLC35B2, as a key regulator of heparan sulfate proteoglycan (HSPG) biosynthesis, is essential for recruiting -Syn PFF to the cell surface because this process is mediated by interactions between negatively charged sugar moieties of HSPGs and clustered K-T-K motifs in -Syn PFF. By contrast, MYO7B regulates -Syn PFF entry by maintaining a plasma-membrane-associated actin network that controls membrane dynamics. Without MYO7B or actin filaments, many clathrin-coated pits fail to be severed from the membrane, causing accumulation of large clathrin-containing scars on the cell surface. Intriguingly, the requirement for MYO7B in endocytosis is only restricted to -Syn PFF and other cargos that enter cells via HSPGs. Thus, by identifying new regulatory factors for -Syn PFF endocytosis, our study defines a mechanistically distinct clathrin-mediated endocytosis pathway that requires additional force generated by MYO7B and actin filaments. SignificanceThe spreading of misfolded protein aggregates such as -Synuclein preformed fibrils (-Syn PFF) from cell to cell is a pathologic hallmark associated with the progression of many neurodegenerative diseases, but it is unclear how mammalian cells take up these large protein aggregates to initiate this prion-like protein transmission process. Here we define the mechanism of -Syn PFF endocytosis using a combination of genetic, biochemical, and live-cell imaging techniques. Our study reveals how -Syn PFF binds to the cell surface heparan sulfate proteoglycans using two lysine-bearing motifs and then enters cells following a Myosin-7B- and actin-dependent endocytosis mechanism that is specifically tailored for polycation-bearing cargos.

cell biology↗
bioRxiv2020

Prediction of meiosis-essential genes based upon the dynamic proteomes responsive to spermatogenesis

Mammalian meiosis is a specific cell division process during sexual reproduction, whereas a comprehensive proteome of the different meiotic stages has not been systematically investigated. Here, we isolated different types of spermatocytes from the testes of spermatogenesis-synchronized mice and quantified the corresponding proteomes with high-resolution mass spectrometry. A total of 8,002 proteins were identified in nine types of germ cells, and the protein signatures of spermatogenesis were characterized using the dynamic proteomes. A supervised machine learning package, FuncProFinder, was developed to predict meiosis-essential candidate genes based on changes in their protein abundance. Of the candidates without functional annotation, four of the ten genes with the highest prediction scores, Zcwpw1, Tesmin, 1700102P08Rik, and Kctd19, were validated as meiosis-essential genes using knockout mouse models. The proteomic analysis of spermatogenic cells provides a solid foundation for studying the mechanism of mammalian meiosis.

developmental biology↗
bioRxiv2020

Discovery of an evolutionarily conserved smooth muscle cell-specific lncRNA CARMN

Differentiated vascular smooth muscle cells (VSMCs) are critical in maintaining vascular homeostasis by expressing a unique repertoire of contractile genes. Despite the well-defined coding transcriptome in differentiated VSMCs, little is known about the non-coding gene expression signature. Herein, by de novo analyzing publicly available RNA-seq and single cell RNA-seq datasets generated from different tissues and cell types, we unambiguously identified CARMN (CARdiac Mesoderm Enhancer-associated Non-coding RNA) as an evolutionarily conserved, SMC-specific lncRNA. CARMN was initially annotated as the host gene of MIR143/145 cluster and recently reported to play roles in cardiac differentiation. Here, we generated a Carmn GFP knock-in reporter mouse model and confirmed its specific expression in SMCs in vivo. In addition, we found Carmn is transcribed independently from Mir143/145 and only expressed transiently in embryonic cardiomyocytes and thereafter becomes restricted to adult SMCs in both human and mouse. Furthermore, we demonstrated that CARMN expression is not only dramatically decreased in human vascular diseases but functionally critical in maintaining VSMC contractile phenotype in vitro. In conclusion, we provided the first evidence showing that CARMN is an evolutionarily conserved SMC-specific lncRNA, down-regulated in different human vascular diseases, and a key lncRNA for maintaining SMC contractile phenotype.

developmental biology↗