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1.
Blood ; 136(7): 898-908, 2020 08 13.
Article in English | MEDLINE | ID: mdl-32374827

ABSTRACT

Drug-induced bleeding disorders contribute to substantial morbidity and mortality. Antithrombotic agents that cause unintended bleeding of obvious cause are relatively easy to control. However, the mechanisms of most drug-induced bleeding disorders are poorly understood, which makes intervention more difficult. As most bleeding disorders are associated with the dysfunction of coagulation factors, we adapted our recently established cell-based assay to identify drugs that affect the biosynthesis of active vitamin K-dependent (VKD) coagulation factors with possible adverse off-target results. The National Institutes of Health (NIH) Clinical Collection (NCC) library containing 727 drugs was screened, and 9 drugs were identified, including the most commonly prescribed anticoagulant warfarin. Bleeding complications associated with most of these drugs have been clinically reported, but the pathogenic mechanisms remain unclear. Further characterization of the 9 top-hit drugs on the inhibition of VKD carboxylation suggests that warfarin, lansoprazole, and nitazoxanide mainly target vitamin K epoxide reductase (VKOR), whereas idebenone, clofazimine, and AM404 mainly target vitamin K reductase (VKR) in vitamin K redox cycling. The other 3 drugs mainly affect vitamin K availability within the cells. The molecular mechanisms underlying the inactivation of VKOR and VKR by these drugs are clarified. Results from both cell-based and animal model studies suggest that the anticoagulation effect of drugs that target VKOR, but not VKR, can be rescued by the administration of vitamin K. These findings provide insights into the prevention and management of drug-induced bleeding disorders. The established cell-based, high-throughput screening approach provides a powerful tool for identifying new vitamin K antagonists that function as anticoagulants.


Subject(s)
Anticoagulants/isolation & purification , Anticoagulants/pharmacology , Hemorrhagic Disorders/chemically induced , High-Throughput Screening Assays/methods , Vitamin K/metabolism , 4-Hydroxycoumarins/adverse effects , 4-Hydroxycoumarins/isolation & purification , 4-Hydroxycoumarins/pharmacology , Animals , Anticoagulants/adverse effects , Blood Coagulation/drug effects , Cell Culture Techniques/methods , Drug Evaluation, Preclinical/methods , HEK293 Cells , Hep G2 Cells , Humans , Indenes/adverse effects , Indenes/isolation & purification , Indenes/pharmacology , Male , Metabolic Networks and Pathways/drug effects , Mice , Mice, Inbred BALB C , Off-Label Use , Vitamin K/adverse effects , Vitamin K/antagonists & inhibitors , Vitamin K/isolation & purification , Vitamin K/pharmacology , Vitamin K Epoxide Reductases/antagonists & inhibitors , Vitamin K Epoxide Reductases/metabolism
2.
FASEB J ; 33(5): 5876-5886, 2019 05.
Article in English | MEDLINE | ID: mdl-30721625

ABSTRACT

Fibronectin type III domain containing 5 (Fndc5) is a transmembrane protein highly expressed in the skeletal muscle. It was reported that exercise promotes the shedding of the extracellular domain of Fndc5, generating a circulating peptide (irisin) that cross-talks to adipose tissues to convert lipid-storing white adipocytes to energy-catabolizing beige adipocytes. However, the requirement of Fndc5 in mediating the beneficial effect of exercise remains to be determined. Here, we created a mouse model of Fndc5 mutation through transcription activator-like effector nuclease-mediated DNA targeting. The Fndc5 mutant mice have normal skeletal muscle development, growth, regeneration, as well as glucose and lipid metabolism at resting state, even when fed a high-fat diet. In response to running exercise, however, the Fndc5 mutant mice exhibit reduced glucose tolerance and insulin sensitivity and have lower maximal oxygen consumption compared with the exercised wild-type mice. Mechanistically, Fndc5 mutation attenuates exercise-induced browning of white adipose tissue that is crucial for the metabolic benefits of physical activities. These data provide genetic evidence that Fndc5 is dispensable for muscle development and basal metabolism but essential for exercise-induced browning of white adipose tissues in mice.-Xiong, Y., Wu, Z., Zhang, B., Wang, C., Mao, F., Liu, X., Hu, K., Sun, X., Jin, W., Kuang, S. Fndc5 loss-of-function attenuates exercise-induced browning of white adipose tissue in mice.


Subject(s)
Adipose Tissue, Brown/physiology , Adipose Tissue, White/physiology , Fibronectins/genetics , Physical Conditioning, Animal , Animals , Blood Glucose/analysis , Female , Fibronectins/physiology , Insulin Resistance , Lipid Metabolism , Male , Mice , Muscle, Skeletal/metabolism , Mutation , Obesity/metabolism , Oxygen Consumption , Regeneration , Transcription Factors/metabolism , Uncoupling Protein 1/metabolism
3.
FASEB J ; 33(8): 9672-9684, 2019 08.
Article in English | MEDLINE | ID: mdl-31162944

ABSTRACT

Skeletal muscles contain heterogeneous myofibers that are different in size and contractile speed, with type IIb myofiber being the largest and fastest. Here, we identify methyltransferase-like 21e (Mettl21e), a member of newly classified nonhistone methyltransferases, as a gene enriched in type IIb myofibers. The expression of Mettl21e was strikingly up-regulated in hypertrophic muscles and during myogenic differentiation in vitro and in vivo. Knockdown (KD) of Mettl21e led to atrophy of cultured myotubes, and targeted mutation of Mettl21e in mice reduced the size of IIb myofibers without affecting the composition of myofiber types. Mass spectrometry and methyltransferase assay revealed that Mettl21e methylated valosin-containing protein (Vcp/p97), a key component of the ubiquitin-proteasome system. KD or knockout of Mettl21e resulted in elevated 26S proteasome activity, and inhibition of proteasome activity prevented atrophy of Mettl21e KD myotubes. These results demonstrate that Mettl21e functions to maintain myofiber size through inhibiting proteasome-mediated protein degradation.-Wang, C., Zhang, B., Ratliff, A. C., Arrington, J., Chen, J., Xiong, Y., Yue, F., Nie, Y., Hu, K., Jin, W., Tao, W. A., Hrycyna, C. A., Sun, X., Kuang, S. Methyltransferase-like 21e inhibits 26S proteasome activity to facilitate hypertrophy of type IIb myofibers.


Subject(s)
Cell Differentiation/drug effects , Methyltransferases/metabolism , Muscular Atrophy/metabolism , Myofibrils/metabolism , Animals , Blotting, Western , Bortezomib/therapeutic use , Cell Differentiation/genetics , Cells, Cultured , Female , Immunoprecipitation , Methyltransferases/genetics , Mice , Mice, Knockout , Muscle Fibers, Skeletal/drug effects , Muscle Fibers, Skeletal/metabolism , Muscle, Skeletal/cytology , Muscle, Skeletal/metabolism , Muscular Atrophy/pathology , Mutation/genetics , Myoblasts/drug effects , Myoblasts/metabolism , Myofibrils/drug effects , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/metabolism , Real-Time Polymerase Chain Reaction , Sequence Analysis, RNA
4.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1863(4): 409-419, 2018 Apr.
Article in English | MEDLINE | ID: mdl-29341928

ABSTRACT

The thermogenic activities of brown and beige adipocytes can be exploited to reduce energy surplus and counteract obesity. Recent RNA sequencing studies have uncovered a number of long noncoding RNAs (lncRNAs) uniquely expressed in white and brown adipose tissues (WAT and BAT), but whether and how these lncRNAs function in adipogenesis remain largely unknown. Here, we report the identification of a novel brown adipocyte-enriched LncRNA (AK079912), and its nuclear localization, function and regulation. The expression of AK079912 increases during brown preadipocyte differentiation and in response to cold-stimulated browning of white adipocytes. Knockdown of AK079912 inhibits brown preadipocyte differentiation, manifested by reductions in lipid accumulation and down-regulation of adipogenic and BAT-specific genes. Conversely, ectopic expression of AK079912 in white preadipocytes up-regulates the expression of genes involved in thermogenesis. Mechanistically, inhibition of AK079912 reduces mitochondrial copy number and protein levels of mitochondria electron transport chain (ETC) complexes, whereas AK079912 overexpression increases the levels of ETC proteins. Lastly, reporter and pharmacological assays identify Pparγ as an upstream regulator of AK079912. These results provide new insights into the function of non-coding RNAs in brown adipogenesis and regulating browning of white adipocytes.


Subject(s)
Adipocytes, Brown/cytology , Adipocytes, Brown/metabolism , Adipocytes, White/metabolism , Cell Differentiation/genetics , RNA, Long Noncoding/metabolism , Thermogenesis/genetics , Animals , Cold Temperature , Gene Knockdown Techniques , Mice, Inbred C57BL , Organelle Biogenesis , PPAR gamma/metabolism , RNA, Long Noncoding/genetics , Up-Regulation/genetics
5.
Plant Cell Physiol ; 58(11): 1924-1933, 2017 Nov 01.
Article in English | MEDLINE | ID: mdl-29016977

ABSTRACT

Sesquiterpenes are one of the most important defensive secondary metabolite components of agarwood. Agarwood, which is a product of the Aquilaria sinensis response to external damage, is a fragrant and resinous wood that is widely used in traditional medicines, incense and perfume. We previously reported that jasmonic acid (JA) plays an important role in promoting agarwood sesquiterpene biosynthesis and induces expression of the sesquiterpene synthase ASS1, which is a key enzyme that is responsible for the biosynthesis of agarwood sesquiterpenes in A. sinensis. However, little is known about this molecular regulation mechanism. Here, we characterized a basic helix-loop-helix transcription factor, AsMYC2, from A. sinensis as an activator of ASS1 expression. AsMYC2 is an immediate-early jasmonate-responsive gene and is co-induced with ASS1. Using a combination of yeast one-hybrid assays and chromatin immunoprecipitation analyses, we showed that AsMYC2 bound the promoter of ASS1 containing a G-box motif. AsMYC2 activated expression of ASS1 in tobacco epidermis cells and up-regulated expression of sesquiterpene synthase genes (TPS21 and TPS11) in Arabidopsis, which was also promoted by methyl jasmonate. Our results suggest that AsMYC2 participates in the regulation of agarwood sesquiterpene biosynthesis in A. sinensis by controlling the expression of ASS1 through the JA signaling pathway.


Subject(s)
Plant Proteins/metabolism , Sesquiterpenes/metabolism , Thymelaeaceae/metabolism , Transcription Factors/metabolism , Acetates/metabolism , Acetates/pharmacology , Alkyl and Aryl Transferases/genetics , Alkyl and Aryl Transferases/metabolism , Arabidopsis/genetics , Cyclopentanes/metabolism , Cyclopentanes/pharmacology , Gene Expression Regulation, Plant , Helix-Loop-Helix Motifs , Oxylipins/metabolism , Oxylipins/pharmacology , Plant Proteins/genetics , Plants, Genetically Modified , Promoter Regions, Genetic , Thymelaeaceae/drug effects , Thymelaeaceae/genetics , Transcription Factors/genetics
6.
J Cell Sci ; 128(12): 2340-50, 2015 Jun 15.
Article in English | MEDLINE | ID: mdl-25948585

ABSTRACT

Rett syndrome (RTT) is a progressive neurological disorder caused by mutations in the X-linked protein methyl-CpG-binding protein 2 (MeCP2). The endogenous function of MeCP2 during neural differentiation is still unclear. Here, we report that mecp2 is required for brain development in zebrafish. Mecp2 was broadly expressed initially in embryos and enriched later in the brain. Either morpholino knockdown or genetic depletion of mecp2 inhibited neuronal differentiation, whereas its overexpression promoted neuronal differentiation, suggesting an essential role of mecp2 in directing neural precursors into differentiated neurons. Mechanistically, her2 (the zebrafish ortholog of mammalian Hes5) was upregulated in mecp2 morphants in an Id1-dependent manner. Moreover, knockdown of either her2 or id1 fully rescued neuronal differentiation in mecp2 morphants. These results suggest that Mecp2 plays an important role in neural cell development by suppressing the Id1-Her2 axis, and provide new evidence that embryonic neural defects contribute to the later motor and cognitive dysfunctions in RTT.


Subject(s)
Cell Differentiation , Embryo, Nonmammalian/cytology , Genes, erbB-2 , Inhibitor of Differentiation Protein 1/antagonists & inhibitors , Methyl-CpG-Binding Protein 2/metabolism , Neurons/cytology , Zebrafish/metabolism , Animals , Animals, Genetically Modified/embryology , Animals, Genetically Modified/genetics , Animals, Genetically Modified/metabolism , Base Sequence , Blotting, Western , Brain/cytology , Brain/metabolism , Cells, Cultured , Chromatin Immunoprecipitation , Embryo, Nonmammalian/metabolism , Immunoenzyme Techniques , Immunoprecipitation , Inhibitor of Differentiation Protein 1/genetics , Inhibitor of Differentiation Protein 1/metabolism , Methyl-CpG-Binding Protein 2/genetics , Mice , Molecular Sequence Data , Neurogenesis/physiology , Neurons/metabolism , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Nucleic Acid , Zebrafish/embryology , Zebrafish/genetics , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism
7.
J Cell Sci ; 127(Pt 12): 2761-70, 2014 Jun 15.
Article in English | MEDLINE | ID: mdl-24727614

ABSTRACT

Protein tyrosine phosphatases (PTPs) are involved in hematopoiesis, but the function of many PTPs is not well characterized in vivo. Here, we have identified Ptpn9a, an ortholog of human PTPN9, as a crucial regulator of erythroid cell development in zebrafish embryos. ptpn9a, but not ptpn9b, was expressed in the posterior lateral plate mesoderm and intermediate cell mass - two primitive hematopoietic sites during zebrafish embryogenesis. Morpholino-mediated knockdown of ptpn9a caused erythrocytes to be depleted by inhibiting erythroid cell maturation without affecting erythroid proliferation and apoptosis. Consistently, both dominant-negative PTPN9 (with mutation C515S) and siRNA against PTPN9 inhibited erythroid differentiation in human K562 cells. Mechanistically, depletion of ptpn9 in zebrafish embryos in vivo or in K562 cells in vitro increased phosphorylated STAT3, and the hyper-phosphorylated STAT3 entrapped and prevented the transcription factors GATA1 and ZBP-89 (also known as ZNF148) from regulating erythroid gene expression. These findings imply that PTPN9 plays an important role in erythropoiesis by disrupting an inhibitory complex of phosphorylated STAT3, GATA1 and ZBP-89, providing new cellular and molecular insights into the role of ptpn9a in developmental hematopoiesis.


Subject(s)
Erythroid Cells/enzymology , Protein Processing, Post-Translational , Protein Tyrosine Phosphatases, Non-Receptor/physiology , STAT3 Transcription Factor/metabolism , Zebrafish Proteins/metabolism , Zebrafish Proteins/physiology , Zebrafish/physiology , Animals , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/enzymology , Erythropoiesis , GATA1 Transcription Factor/metabolism , Gastrulation , Gene Expression , Gene Expression Regulation, Developmental , Humans , K562 Cells , Phosphorylation , Transcription Factors/metabolism , Zebrafish/embryology
8.
Yi Chuan ; 38(11): 1004-1011, 2016 11 20.
Article in English | MEDLINE | ID: mdl-27867150

ABSTRACT

MeCP2 mutations are associated with the Rett syndrome (RTT). Currently, there is an urgent need for new animal models for RTT as the existing MeCP2 knockout mouse models fail to fully mimic the pathogenesis and symptoms of RTT patients. In order to investigate the role of MeCP2 in brain development and RTT pathogenesis, we aimed to set up the MeCP2-null rat model using the CRISPR/Cas9 technology. Firstly we constructed the MeCP2 targeting vector and then microinjected Cas9 mRNA and sgRNA mixtures into fertilized ova of SD rats. The sgRNA was designed to target the exon 2 of MeCP2. Next, knockout rats were confirmed using DNA sequencing and Western blotting. Lastly, phenotypes including growth and behaviors of MeCP2 knockout rats were analyzed. The results indicated that the MeCP2 knockout rats showed body weight loss, anxiety tendency and cognitive deficits. The MeCP2-null rat model established in this study recapitulates the major symptoms of RTT patients and provides an alternative tool for future studies of MeCP2 functions.


Subject(s)
Disease Models, Animal , Methyl-CpG-Binding Protein 2/genetics , Rett Syndrome/genetics , Animals , Base Sequence , Gene Knockout Techniques , Humans , Male , Methyl-CpG-Binding Protein 2/metabolism , Molecular Sequence Data , Rats , Rats, Sprague-Dawley , Rett Syndrome/metabolism
9.
Zhongguo Zhong Yao Za Zhi ; 41(22): 4096-4102, 2016 Nov.
Article in Zh | MEDLINE | ID: mdl-28933073

ABSTRACT

The application of proteomics in the research of traditional Chinese medicine (TCM) is very extensive, and there have been many successful cases. In this paper, the previous studies on the complex system of TCM by using proteomics technology were reviewed, and the authors proposed to set up a special subject on proteomics in TCM, which is called TCM proteomics. In this paper, the research strategies and the future research directions of TCM proteomics were reviewed and discussed, which may provide some ideas for the researchers of TCM proteomics.


Subject(s)
Medicine, Chinese Traditional/trends , Proteomics/trends , Drugs, Chinese Herbal , Humans , Research Design
10.
J Biol Chem ; 288(7): 4602-12, 2013 Feb 15.
Article in English | MEDLINE | ID: mdl-23283965

ABSTRACT

Mitochondrial superoxide flashes reflect a quantal, bursting mode of reactive oxygen species (ROS) production that arises from stochastic, transient opening of the mitochondrial permeability transition pore (mPTP) in many types of cells and in living animals. However, the regulatory mechanisms and the exact nature of the flash-coupled mPTP remain poorly understood. Here we demonstrate a profound synergistic effect between mitochondrial Ca(2+) uniport and elevated basal ROS production in triggering superoxide flashes in intact cells. Hyperosmotic stress potently augmented the flash activity while simultaneously elevating mitochondrial Ca(2+) and ROS. Blocking mitochondrial Ca(2+) transport by knockdown of MICU1 or MCU, newly identified components of the mitochondrial Ca(2+) uniporter, or scavenging mitochondrial basal ROS markedly diminished the flash response. More importantly, whereas elevating Ca(2+) or ROS production alone was inefficacious in triggering the flashes, concurrent physiological Ca(2+) and ROS elevation served as the most powerful flash activator, increasing the flash incidence by an order of magnitude. Functionally, superoxide flashes in response to hyperosmotic stress participated in the activation of JNK and p38. Thus, physiological levels of mitochondrial Ca(2+) and ROS synergistically regulate stochastic mPTP opening and quantal ROS production in intact cells, marking the flash as a coincidence detector of mitochondrial Ca(2+) and ROS signals.


Subject(s)
Calcium/metabolism , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Reactive Oxygen Species/metabolism , Superoxides/metabolism , Biological Transport , Cells, Cultured/cytology , HeLa Cells , Humans , Kinetics , Membrane Potentials , Microscopy, Confocal/methods , Mitochondrial Permeability Transition Pore , Osmosis , Oxidative Stress , RNA Interference , Signal Transduction
11.
Vaccines (Basel) ; 12(4)2024 Apr 15.
Article in English | MEDLINE | ID: mdl-38675799

ABSTRACT

Most available neutralizing antibodies are ineffective against highly mutated SARS-CoV-2 Omicron subvariants. Therefore, it is crucial to develop potent and broad-spectrum alternatives to effectively manage Omicron subvariants. Here, we constructed a high-diversity nanobody phage display library and identified nine nanobodies specific to the SARS-CoV-2 receptor-binding domain (RBD). Five of them exhibited cross-neutralization activity against the SARS-CoV-2 wild-type (WT) strain and the Omicron subvariants BA.1 and BA.4/5, and one nanobody demonstrated marked efficacy even against the Omicron subvariants BQ.1.1 and XBB.1. To enhance the therapeutic potential, we engineered a panel of multivalent nanobodies with increased neutralizing potency and breadth. The most potent multivalent nanobody, B13-B13-B13, cross-neutralized all tested pseudoviruses, with a geometric mean of the 50% inhibitory concentration (GM IC50) value of 20.83 ng/mL. An analysis of the mechanism underlying the enhancement of neutralization breadth by representative multivalent nanobodies demonstrated that the strategic engineering approach of combining two or three nanobodies into a multivalent molecule could improve the affinity between a single nanobody and spike, and could enhance tolerance toward escape mutations such as R346T and N460K. Our engineered multivalent nanobodies may be promising drug candidates for treating and preventing infection with Omicron subvariants and even future variants.

12.
Vaccines (Basel) ; 11(2)2023 Feb 06.
Article in English | MEDLINE | ID: mdl-36851249

ABSTRACT

Most neutralizing antibodies neutralize the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by directly blocking the interactions between the spike glycoprotein receptor-binding domain (RBD) and its receptor, human angiotensin-converting enzyme 2 (ACE2). Here, we report a novel nanobody (Nb) identified by an RBD-ACE2 competitive panning method that could specifically bind to the RBD of SARS-CoV-2 with a high affinity (EC50 = 0.03 nM) and successfully block the binding between the RBD and ACE2 recombinant protein. A structural simulation of the RBD-VHH complex also supports a mechanism of the Nb to block the interaction between the RBD and ACE2. A pseudovirus assay of the Nb showed it could neutralize the WT pseudovirus with high potency (IC50 = 0.026 µg/mL). Furthermore, we measured its binding to phages displaying RBDs of different SARS-CoV-2 variants and found that it could bind to recombinant phages displaying the RBD of beta and delta variants. This study also provides a method of phage library competitive panning, which could be useful for directly screening high-affinity antibodies targeting important functional regions.

14.
Proc Natl Acad Sci U S A ; 106(12): 4882-7, 2009 Mar 24.
Article in English | MEDLINE | ID: mdl-19225110

ABSTRACT

Mutations of MECP2 (Methyl-CpG Binding Protein 2) cause Rett syndrome. As a chromatin-associated multifunctional protein, how MeCP2 integrates external signals and regulates neuronal function remain unclear. Although neuronal activity-induced phosphorylation of MeCP2 at serine 421 (S421) has been reported, the full spectrum of MeCP2 phosphorylation together with the in vivo function of such modifications are yet to be revealed. Here, we report the identification of several MeCP2 phosphorylation sites in normal and epileptic brains from multiple species. We demonstrate that serine 80 (S80) phosphorylation of MeCP2 is critical as its mutation into alanine (S80A) in transgenic knock-in mice leads to locomotor deficits. S80A mutation attenuates MeCP2 chromatin association at several gene promoters in resting neurons and leads to transcription changes of a small number of genes. Calcium influx in neurons causes dephosphorylation at S80, potentially contributing to its dissociation from the chromatin. We postulate that phosphorylation of MeCP2 modulates its dynamic function in neurons transiting between resting and active states within neural circuits that underlie behaviors.


Subject(s)
Chromatin/metabolism , Methyl-CpG-Binding Protein 2/metabolism , Neurons/metabolism , Phosphoserine/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Antibodies, Phospho-Specific/metabolism , Brain/metabolism , Gene Expression Regulation , Gene Knock-In Techniques , Methyl-CpG-Binding Protein 2/chemistry , Mice , Molecular Sequence Data , Motor Activity , Mutation/genetics , Phosphorylation , Promoter Regions, Genetic/genetics , Protein Binding , Rats
15.
Front Microbiol ; 13: 968036, 2022.
Article in English | MEDLINE | ID: mdl-36071962

ABSTRACT

To combat the continued pandemic of COVID-19, multiplex serological assays have been developed to comprehensively monitor the humoral immune response and help to design new vaccination protocols to different SARS-CoV-2 variants. However, multiplex beads and stably transfected cell lines require stringent production and storage conditions, and assays based on flow cytometry is time-consuming and its application is therefore restricted. Here, we describe a phage display system to distinguish the differences of immune response to antigenic domains of multiple SARS-CoV-2 variants simultaneously. Compared with linear peptides, the recombinant antigens displayed on the phage surface have shown some function that requires the correct folding to form a stable structure, and the binding efficiency between the recombinant phage and existing antibodies is reduced by mutations on antigens known to be important for antigen-antibody interaction. By using Phage display mediated immuno-multiplex quantitative PCR (Pi-mqPCR), the binding efficiency between the antibody and antigens of different SARS-CoV-2 variants can be measured in one amplification reaction. Overall, these data show that this assay is a valuable tool to evaluate the humoral response to the same antigen of different SARS-CoV-2 variants or antigens of different pathogens. Combined with high-throughput DNA sequencing technology, this phage display system can be further applied in monitoring humoral immune response in a large population before and after vaccination.

16.
Nat Commun ; 13(1): 6672, 2022 11 05.
Article in English | MEDLINE | ID: mdl-36335128

ABSTRACT

Dual-specificity phosphatase 6 (DUSP6) serves a specific and conserved function on the dephosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). We previously identified Dusp6 as a regenerative repressor during zebrafish heart regeneration, therefore we propose to investigate the role of this repressor in mammalian cardiac repair. Utilizing a rat strain harboring Dusp6 nonsense mutation, rat neutrophil-cardiomyocyte co-culture, bone marrow transplanted rats and neutrophil-specific Dusp6 knockout mice, we find that Dusp6 deficiency improves cardiac outcomes by predominantly attenuating neutrophil-mediated myocardial damage in acute inflammatory phase after myocardial infarction. Mechanistically, Dusp6 is transcriptionally activated by p38-C/EBPß signaling and acts as an effector for maintaining p-p38 activity by down-regulating pERK and p38-targeting phosphatases DUSP1/DUSP16. Our findings provide robust animal models and novel insights for neutrophil-mediated cardiac damage and demonstrate the potential of DUSP6 as a therapeutic target for post-MI cardiac remodeling and other relevant inflammatory diseases.


Subject(s)
Myocardial Infarction , Animals , Mice , Rats , Dual Specificity Phosphatase 6 , Mice, Knockout , Myocardial Infarction/genetics , Myocardium , Myocytes, Cardiac , Neutrophils
17.
Vaccine ; 39(8): 1241-1247, 2021 02 22.
Article in English | MEDLINE | ID: mdl-33516600

ABSTRACT

Without approved vaccines and specific treatments, COVID-19 is spreading around the world with above 26 million cases and approximately 864 thousand deaths until now. An efficacious and affordable vaccine is urgently needed. The Val308 - Gly548 of spike protein of SARS-CoV-2 linked with Gln830 - Glu843 of Tetanus toxoid (TT peptide) (designated as S1-4) and without TT peptide (designated as S1-5) were expressed and renatured. The antigenicity and immunogenicity of S1-4 were evaluated by Western Blotting (WB) in vitro and immune responses in mice, respectively. The protective efficiency was measured preliminarily by microneutralization assay (MN50). The soluble S1-4 and S1-5 protein was prepared to high homogeneity and purity. Adjuvanted with Alum, S1-4 protein stimulated a strong antibody response in immunized mice and caused a major Th2-type cellular immunity supplemented with Th1-type immunity. Furthermore, the immunized sera could protect the Vero E6 cells from SARS-CoV-2 infection with neutralizing antibody titer 256. Recombinant SARS-CoV-2 RBD with a built in T helper epitope could stimulate both strong humoral immunity supplemented with cellular immunity in mice, demonstrating that it could be a promising subunit vaccine candidate.


Subject(s)
Antibodies, Viral/immunology , COVID-19 Vaccines/immunology , Epitopes, T-Lymphocyte/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Neutralizing/immunology , Antibody Formation , COVID-19 , Female , Humans , Mice , Mice, Inbred BALB C , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics
19.
Nat Commun ; 11(1): 1620, 2020 03 27.
Article in English | MEDLINE | ID: mdl-32221306

ABSTRACT

Since 2002, beta coronaviruses (CoV) have caused three zoonotic outbreaks, SARS-CoV in 2002-2003, MERS-CoV in 2012, and the newly emerged SARS-CoV-2 in late 2019. However, little is currently known about the biology of SARS-CoV-2. Here, using SARS-CoV-2 S protein pseudovirus system, we confirm that human angiotensin converting enzyme 2 (hACE2) is the receptor for SARS-CoV-2, find that SARS-CoV-2 enters 293/hACE2 cells mainly through endocytosis, that PIKfyve, TPC2, and cathepsin L are critical for entry, and that SARS-CoV-2 S protein is less stable than SARS-CoV S. Polyclonal anti-SARS S1 antibodies T62 inhibit entry of SARS-CoV S but not SARS-CoV-2 S pseudovirions. Further studies using recovered SARS and COVID-19 patients' sera show limited cross-neutralization, suggesting that recovery from one infection might not protect against the other. Our results present potential targets for development of drugs and vaccines for SARS-CoV-2.


Subject(s)
Antibodies, Viral/immunology , Betacoronavirus/physiology , Broadly Neutralizing Antibodies/immunology , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization , Angiotensin-Converting Enzyme 2 , Betacoronavirus/chemistry , Betacoronavirus/immunology , COVID-19 , Calcium Channels/metabolism , Cathepsin L/metabolism , Cathepsins/antagonists & inhibitors , Cathepsins/metabolism , Cell Fusion , Coronavirus Infections/immunology , Cross Reactions , Endocytosis , Giant Cells/physiology , HEK293 Cells , Humans , Neutralization Tests , Pandemics , Peptidyl-Dipeptidase A/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Pneumonia, Viral/immunology , Protein Domains , Protein Multimerization , Receptors, Virus/metabolism , Severe acute respiratory syndrome-related coronavirus/immunology , SARS-CoV-2 , Severe Acute Respiratory Syndrome/immunology , Spike Glycoprotein, Coronavirus/chemistry , Trypsin/metabolism
20.
Phytomedicine ; 53: 193-204, 2019 Feb.
Article in English | MEDLINE | ID: mdl-30668399

ABSTRACT

BACKGROUND: Flavanomarein is the main component of Coreopsis tinctoria Nutt. (C. tinctoria), which is a globally well-known flower tea that has a distinct flavor and many beneficial health effects, such as antioxidant activities. We aimed to explore the effect of flavanomarein on a 6-hydroxydopamine (6-OHDA)-lesioned cell model of oxidative stress. METHODS: In this study, we used 6-OHDA-lesioned PC12 cells and primary cortical neurons to investigate the protective effects of flavanomarein and its potential mechanism. RESULTS: The results indicated that pretreatment with flavanomarein (25, 50, or 100 µM for 24 h) significantly increased the cell viability, reduced the lactate dehydrogenase (LDH) release and improved the mitochondrial membrane potential (∆Ψm) and mitochondrial impairment. Additionally, flavanomarein markedly reduced the gene expression of tumor necrosis factor (TNF)-α and protein kinase C ζ (PKC-ζ), the nuclear translocation of p65, and the levels of p-AMPK-α and acetyl-p53. Flavanomarein also elevated the gene expression of P85α, PKC-ß1, and Bcl-2, the protein expression of Sirt1 and ICAD, and the phosphorylation level of AKT. CONCLUSIONS: Together, these results suggest that flavanomarein protects PC12 cells and primary cortical neurons from 6-OHDA-induced neurotoxicity by upregulating the PI3K/AKT signaling pathway and attenuating the nuclear factor kappa B (NF-κB) signaling pathway. Therefore, our study provides evidence that may aid in the development of a potential compound against 6-OHDA toxicity.


Subject(s)
Flavanones/pharmacology , Neurons/drug effects , Neuroprotective Agents/pharmacology , Oxidative Stress/drug effects , Oxidopamine/toxicity , Animals , Cell Survival/drug effects , Gene Expression Regulation/drug effects , Membrane Potential, Mitochondrial/drug effects , Mitochondria/drug effects , Mitochondria/metabolism , Mitochondria/pathology , NF-kappa B/metabolism , Neurons/metabolism , Neurons/pathology , Oxidative Stress/genetics , PC12 Cells , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Rats , Rats, Sprague-Dawley , Sirtuin 1/genetics , Sirtuin 1/metabolism
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