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1.
Cell ; 182(1): 73-84.e16, 2020 07 09.
Article in English | MEDLINE | ID: mdl-32425270

ABSTRACT

The COVID-19 pandemic urgently needs therapeutic and prophylactic interventions. Here, we report the rapid identification of SARS-CoV-2-neutralizing antibodies by high-throughput single-cell RNA and VDJ sequencing of antigen-enriched B cells from 60 convalescent patients. From 8,558 antigen-binding IgG1+ clonotypes, 14 potent neutralizing antibodies were identified, with the most potent one, BD-368-2, exhibiting an IC50 of 1.2 and 15 ng/mL against pseudotyped and authentic SARS-CoV-2, respectively. BD-368-2 also displayed strong therapeutic and prophylactic efficacy in SARS-CoV-2-infected hACE2-transgenic mice. Additionally, the 3.8 Å cryo-EM structure of a neutralizing antibody in complex with the spike-ectodomain trimer revealed the antibody's epitope overlaps with the ACE2 binding site. Moreover, we demonstrated that SARS-CoV-2-neutralizing antibodies could be directly selected based on similarities of their predicted CDR3H structures to those of SARS-CoV-neutralizing antibodies. Altogether, we showed that human neutralizing antibodies could be efficiently discovered by high-throughput single B cell sequencing in response to pandemic infectious diseases.


Subject(s)
Antibodies, Monoclonal/isolation & purification , Antibodies, Neutralizing/isolation & purification , B-Lymphocytes/immunology , Coronavirus Infections/immunology , Pneumonia, Viral/immunology , Single-Cell Analysis , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , Antibodies, Neutralizing/administration & dosage , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/metabolism , COVID-19 , Convalescence , High-Throughput Nucleotide Sequencing , Humans , Mice , Pandemics , Sequence Analysis, RNA , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , VDJ Exons
2.
Appl Microbiol Biotechnol ; 108(1): 396, 2024 Jun 26.
Article in English | MEDLINE | ID: mdl-38922447

ABSTRACT

The human gut microbiota refers to a diverse community of microorganisms that symbiotically exist in the human intestinal system. Altered microbial communities have been linked to many human pathologies. However, there is a lack of rapid and efficient methods to assess gut microbiota signatures in practice. To address this, we established an appraisal system containing 45 quantitative real-time polymerase chain reaction (qPCR) assays targeting gut core microbes with high prevalence and/or abundance in the population. Through comparative genomic analysis, we selected novel species-specific genetic markers and primers for 31 of the 45 core microbes with no previously reported specific primers or whose primers needed improvement in specificity. We comprehensively evaluated the performance of the qPCR assays and demonstrated that they showed good sensitivity, selectivity, and quantitative linearity for each target. The limit of detection ranged from 0.1 to 1.0 pg/µL for the genomic DNA of these targets. We also demonstrated the high consistency (Pearson's r = 0.8688, P < 0.0001) between the qPCR method and metagenomics next-generation sequencing (mNGS) method in analyzing the abundance of selected bacteria in 22 human fecal samples. Moreover, we quantified the dynamic changes (over 8 weeks) of these core microbes in 14 individuals using qPCR, and considerable stability was demonstrated in most participants, albeit with significant individual differences. Overall, this study enables the simple and rapid quantification of 45 core microbes in the human gut, providing a promising tool to understand the role of gut core microbiota in human health and disease. KEY POINTS: • A panel of original qPCR assays was developed to quantify human gut core microbes. • The qPCR assays were evaluated and compared with mNGS using real fecal samples. • This method was used to dynamically profile the gut core microbiota in individuals.


Subject(s)
Bacteria , Feces , Gastrointestinal Microbiome , Real-Time Polymerase Chain Reaction , Humans , Real-Time Polymerase Chain Reaction/methods , Gastrointestinal Microbiome/genetics , Feces/microbiology , Bacteria/genetics , Bacteria/classification , Bacteria/isolation & purification , Metagenomics/methods , High-Throughput Nucleotide Sequencing/methods , Sensitivity and Specificity , DNA Primers/genetics , DNA, Bacterial/genetics
3.
Int J Mol Sci ; 25(14)2024 Jul 09.
Article in English | MEDLINE | ID: mdl-39062755

ABSTRACT

Opsins are a class of transmembrane proteins encoded by opsin genes, and they play a variety of functional roles. Short wavelength-sensitive opsin 2 (sws2), one of the five classes of visual opsin genes, mainly senses blue light. Previous research has indicated that sws2 is essential for melanocyte formation in fish; however, its specific role in skin color differentiation remains to be elucidated. Here, we identified the sws2 gene in a prized reef-dwelling fish, Plectropomus leopardus. The full-length P. leopardus sws2 gene encodes a protein consisting of 351 amino acids, and exhibits substantial homology with other fish species. The expression of the sws2 gene was widespread across P. leopardus tissues, with high expression in eye and skin tissues. Through immunohistochemistry and in situ hybridization analyses, we discovered that the sws2 gene was primarily localized in the rod and cone cells of the retina, and epidermal cells of the skin. Furthermore, dsRNA interference was used for sws2 gene knockdown in living P. leopardus to elucidate its function in skin color differentiation. Black-color-related genes, melanin contents, and tyrosinase activity in the skin significantly decreased after sws2 knockdown (p < 0.05), but red-color-related genes and carotenoid and lutein contents significantly increased (p < 0.05). Retinoic acid injection produced the opposite results. Our results suggested that the sws2 gene influences P. leopardus skin color regulation by affecting vitamin synthesis and melanin-related gene expression levels. This study establishes a foundation for elucidating the molecular mechanisms by which sws2 regulates melanocyte formation in fish skin.


Subject(s)
Melanins , Skin , Tretinoin , Animals , Melanins/biosynthesis , Melanins/metabolism , Tretinoin/metabolism , Skin/metabolism , Fish Proteins/genetics , Fish Proteins/metabolism , Skin Pigmentation/genetics , Opsins/metabolism , Opsins/genetics , Gene Expression Regulation
4.
Article in English | MEDLINE | ID: mdl-37184922

ABSTRACT

Two related anaerobic strains, designated as SWB101512T and SWB19611, were isolated from the bronchoalveolar lavage fluid of two lung cancer patients. Cells were Gram-stain-positive, non-motile and non-spore-forming. Growth could be observed at 26-45 °C (optimum, 37 °C), pH 5.0-8.5 (optimum, pH 7.0) and with 0.5-2.0 % (v/w) NaCl (optimum, 1.0%). The 16S rRNA gene sequences of SWB101512T and SWB19611 showed the highest similarities to Denitrobacterium detoxificans DSM 21843T (91.1 and 91.3 %, respectively). The phylogenetic tree based on the 16S rRNA gene sequences and the core genome sequences demonstrated that the two strains clustered together and formed a distinct lineage within the family Eggerthellaceae. The DNA G+C contents of strains SWB101512T and SWB19611 were 62.0 and 61.9 mol%, respectively. The predominant cellular fatty acids of strains SWB101512T and SWB19611 were C16 : 0 DMA (27.8 and 28.8 %, respectively). The respiratory menaquinone in both strains was menaquinone 6 and the polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, two phospholipids, three glycolipids and three unidentified lipids. Based on evidence from phenotypic, chemotaxonomic and genomic analyses, a new genus and species belonging to the family Eggerthellaceae, named Curtanaerobium respiraculi gen. nov., sp. nov. is proposed. The type strain is SWB101512T (=GDMCC 1.2991T=JCM 35330T).


Subject(s)
Actinobacteria , Fatty Acids , Humans , Fatty Acids/chemistry , Phylogeny , Base Composition , RNA, Ribosomal, 16S/genetics , Anaerobiosis , Bronchoalveolar Lavage Fluid , DNA, Bacterial/genetics , Sequence Analysis, DNA , Bacterial Typing Techniques , Phospholipids/chemistry , Bacteria, Anaerobic/genetics , Actinobacteria/genetics , China
5.
Mol Cell Proteomics ; 20: 100066, 2021.
Article in English | MEDLINE | ID: mdl-33631294

ABSTRACT

Plague is a zoonotic disease that primarily infects rodents via fleabite. Transmission from flea to host niches requires rapid adaption of Yersinia pestis to the outer environments to establish infection. Here, quantitative proteome and secretome analyses of Y. pestis grown under conditions mimicking the two typical niches, i.e., the mammalian host (Mh) and the flea vector (Fv), were performed to understand the adaption strategies of this deadly pathogen. A secretome of Y. pestis containing 308 proteins has been identified using TMT-labeling mass spectrometry analysis. Although some proteins are known to be secreted, such as the type III secretion substrates, PsaA and F1 antigen, most of them were found to be secretory proteins for the first time. Comparative proteomic analysis showed that membrane proteins, chaperonins and stress response proteins are significantly upregulated under the Mh condition, among which the previously uncharacterized proteins YP_3416∼YP_3418 are remarkable because they cannot only be secreted but also translocated into HeLa cells by Y. pestis. We further demonstrated that the purified YP_3416 and YP_3418 exhibited E3 ubiquitin ligase activity in in vitro ubiquitination assay and yp_3416∼3418 deletion mutant of Y. pestis showed significant virulence attenuation in mice. Taken together, our results represent the first Y. pestis secretome, which will promote the better understanding of Y. pestis pathogenesis, as well as the development of new strategies for treatment and prevention of plague.


Subject(s)
Bacterial Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Yersinia pestis/metabolism , Yersinia pestis/pathogenicity , Animals , Bacterial Proteins/genetics , Female , HeLa Cells , Humans , Mice, Inbred BALB C , Mutation , Plague , Proteomics , Secretome , Ubiquitin-Protein Ligases/genetics , Virulence , Yersinia pestis/genetics
6.
Appl Environ Microbiol ; 85(12)2019 06 15.
Article in English | MEDLINE | ID: mdl-30979834

ABSTRACT

Many genes in the bacterial pathogen Yersinia pestis, the causative agent of three plague pandemics, remain uncharacterized, greatly hampering the development of measures for plague prevention and control. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been shown to be an effective tool for gene knockdown in model bacteria. In this system, a catalytically dead Cas9 (dCas9) and a small guide RNA (sgRNA) form a complex, binding to the specific DNA target through base pairing, thereby impeding RNA polymerase binding and causing target gene repression. Here, we introduce an optimized CRISPRi system using Streptococcus pyogenes Cas9-derived dCas9 for gene knockdown in Y. pestis Multiple genes harbored on either the chromosome or plasmids of Y. pestis were efficiently knocked down (up to 380-fold) in a strictly anhydrotetracycline-inducible manner using this CRISPRi approach. Knockdown of hmsH (responsible for biofilm formation) or cspB (encoding a cold shock protein) resulted in greatly decreased biofilm formation or impaired cold tolerance in in vitro phenotypic assays. Furthermore, silencing of the virulence-associated genes yscB or ail using this CRISPRi system resulted in attenuation of virulence in HeLa cells and mice similar to that previously reported for yscB and ail null mutants. Taken together, our results confirm that this optimized CRISPRi system can reversibly and efficiently repress the expression of target genes in Y. pestis, providing an alternative to conventional gene knockdown techniques, as well as a strategy for high-throughput phenotypic screening of Y. pestis genes with unknown functions.IMPORTANCEYersiniapestis is a lethal pathogen responsible for millions of human deaths in history. It has also attracted much attention for potential uses as a bioweapon or bioterrorism agent, against which new vaccines are desperately needed. However, many Y. pestis genes remain uncharacterized, greatly hampering the development of measures for plague prevention and control. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been successfully used in a variety of bacteria in functional genomic studies, but no such genetic tool has been reported in Y. pestis Here, we systematically optimized the CRISPRi approach for use in Y. pestis, which ultimately repressed target gene expression with high efficiency in a reversible manner. Knockdown of functional genes using this method produced phenotypes that were readily detected by in vitro assays, cell infection assays, and mouse infection experiments. This is a report of a CRISPRi approach in Y. pestis and highlights the potential use of this approach in high-throughput functional genomics studies of this pathogen.


Subject(s)
CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , Gene Expression , Gene Knockdown Techniques/methods , Yersinia pestis/genetics , Streptococcus pyogenes
7.
J Biol Chem ; 292(13): 5488-5498, 2017 Mar 31.
Article in English | MEDLINE | ID: mdl-28196868

ABSTRACT

The type III secretion system is a highly conserved virulence mechanism that is widely distributed in Gram-negative bacteria. It has a syringe-like structure composed of a multi-ring basal body that spans the bacterial envelope and a projecting needle that delivers virulence effectors into host cells. Here, we showed that the Yersinia inner rod protein YscI directly interacts with the needle protein YscF inside the bacterial cells and that this interaction depends on amino acid residues 83-102 in the carboxyl terminus of YscI. Alanine substitution of Trp-85 or Ser-86 abrogated the binding of YscI to YscF as well as needle assembly and the secretion of effectors (Yops) and the needle tip protein LcrV. However, yscI null mutants that were trans-complemented with YscI mutants that bind YscF still assembled the needle and secreted Yops, demonstrating that a direct interaction between YscF and YscI is critical for these processes. Consistently, YscI mutants that did not bind YscF resulted in greatly decreased HeLa cell cytotoxicity. Together, these results show that YscI participates in needle assembly by directly interacting with YscF.


Subject(s)
Bacterial Proteins/metabolism , Type III Secretion Systems/biosynthesis , Yersinia pestis/chemistry , Binding Sites/genetics , Cell Death , HeLa Cells , Humans , Mutagenesis, Site-Directed , Protein Binding , Type III Secretion Systems/chemistry , Type III Secretion Systems/toxicity , Yersinia pestis/pathogenicity
8.
Infect Immun ; 86(6)2018 06.
Article in English | MEDLINE | ID: mdl-29610260

ABSTRACT

Recent studies revealed that acetylation is a widely used protein modification in prokaryotic organisms. The major protein acetylation acetyltransferase YfiQ and the sirtuin-like deacetylase CobB have been found to be involved in basic physiological processes, such as primary metabolism, chemotaxis, and stress responses, in Escherichia coli and Salmonella However, little is known about protein acetylation modifications in Yersinia pestis, a lethal pathogen responsible for millions of human deaths in three worldwide pandemics. Here we found that Yp_0659 and Yp_1760 of Y. pestis encode the major protein acetylation acetyltransferase YfiQ and the sirtuin-like deacetylase CobB, respectively, which can acetylate and deacetylate PhoP enzymatically in vitro Protein acetylation impairment in cobB and yfiQ mutants greatly decreased bacterial tolerance to cold, hot, high-salt, and acidic environments. Our comparative transcriptomic data revealed that the strongly decreased tolerance to stress stimuli was probably related to downregulation of the genes encoding the heat shock proteins (HtpG, HslV, HslR, and IbpA), cold shock proteins (CspC and CspA1), and acid resistance proteins (HdeB and AdiA). We found that the reversible acetylation mediated by CobB and YfiQ conferred attenuation of virulence, probably partially due to the decreased expression of the psaABCDEF operon, which encodes Psa fimbriae that play a key role in virulence of Y. pestis This is the first report, to our knowledge, on the roles of protein acetylation modification in stress responses, biofilm formation, and virulence of Y. pestis.


Subject(s)
Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial/physiology , Sirtuins/metabolism , Yersinia pestis/metabolism , Acetyltransferases , Bacterial Proteins/genetics , Biofilms/growth & development , Gene Deletion , HeLa Cells , Humans , Hydrogen-Ion Concentration , Sirtuins/genetics , Sodium Chloride , Stress, Physiological , Temperature , Virulence , Yersinia pestis/genetics , Yersinia pestis/physiology
9.
J Infect Dis ; 215(7): 1107-1110, 2017 Apr 01.
Article in English | MEDLINE | ID: mdl-28498995

ABSTRACT

We performed Ebola virus disease diagnosis and viral load estimation for Ebola cases in Sierra Leone during the late stage of the 2014-2015 outbreak (January-March 2015) and analyzed antibody and cytokine levels and the viral genome sequences. Ebola virus disease was confirmed in 86 of 1001 (9.7%) patients, with an overall case fatality rate of 46.8%. Fatal cases exhibited significantly higher levels of viral loads, cytokines, and chemokines at late stages of infection versus early stage compared with survivors. The viruses converged in a new clade within sublineage 3.2.4, which had a significantly lower case fatality rate.


Subject(s)
Ebolavirus/genetics , Hemorrhagic Fever, Ebola/epidemiology , Hemorrhagic Fever, Ebola/immunology , Viral Load , Antibodies, Viral/blood , Cytokines/blood , Disease Outbreaks , Genome, Viral , Humans , Sierra Leone/epidemiology , Survivors
10.
Infect Immun ; 85(8)2017 08.
Article in English | MEDLINE | ID: mdl-28533472

ABSTRACT

Pathogenic yersiniae harbor a type III secretion system (T3SS) that injects Yersinia outer protein (Yop) into host cells. YopK has been shown to control Yop translocation and prevent inflammasome recognition of the T3SS by the innate immune system. Here, we demonstrate that YopK inhibits bacterial adherence to host cells by binding to the extracellular matrix adaptor protein matrilin-2 (MATN2). YopK binds to MATN2, and deleting amino acids 91 to 124 disrupts binding of YopK to MATN2. A yopK null mutant exhibits a hyperadhesive phenotype, which could be responsible for the established Yop hypertranslocation phenotype of yopK mutants. Expression of YopK, but not YopKΔ91-124, in a yopK mutant restored the wild-type phenotypes of adhesion and Yop translocation, suggesting that binding to MATN2 might be essential for YopK to inhibit bacterial adhesion and negatively regulate Yop translocation. A green fluorescent protein (GFP)-YopK fusion specifically binds to the endogenous MATN2 on the surface of HeLa cells, whereas GFP-YopKΔ91-124 cannot. Addition of purified YopK protein during infection decreased adhesion of Y. pestis to HeLa cells, while YopKΔ91-124 protein showed no effect. Taking these results together, we propose a model that the T3SS-secreted YopK hinders bacterial adhesion to HeLa cells by binding to MATN2, which is ubiquitously exposed on eukaryotic cells.


Subject(s)
Bacterial Adhesion , Bacterial Outer Membrane Proteins/metabolism , Yersinia pestis/metabolism , Animals , Bacterial Outer Membrane Proteins/genetics , Bacterial Translocation , HeLa Cells , Humans , Matrilin Proteins/metabolism , Mice , Mutation , Phagocytosis , Phenotype , Type III Secretion Systems/metabolism , Yersinia pestis/chemistry , Yersinia pestis/genetics , Yersinia pestis/pathogenicity
11.
Int J Med Microbiol ; 307(1): 64-74, 2017 Jan.
Article in English | MEDLINE | ID: mdl-27876297

ABSTRACT

Pneumonic plague is the most deadly form of infection caused by Yersinia pestis and can progress extremely fast. However, our understanding on the host transcriptomic response to pneumonic plague is insufficient. Here, we used RNA-sequencing technology to analyze transcriptomic responses in mice infected with fully virulent strain 201 or EV76, a live attenuated vaccine strain lacking the pigmentation locus. Approximately 600 differentially expressed genes (DEGs) were detected in lungs from both 201- and EV76-infected mice at 12h post-infection (hpi). DEGs in lungs of 201-infected mice exceeded 2000 at 48hpi, accompanied by sustained large numbers of DEGs in the liver and spleen; however, limited numbers of DEGs were detected in those organs of EV-infected mice. Remarkably, DEGs in lungs were significantly enriched in critical immune responses pathways in EV76-infected but not 201-infected mice, including antigen processing and presentation, T cell receptor signaling among others. Pathological and bacterial load analyses confirmed the rapid systemic dissemination of 201-infection and the confined EV76-infection in lungs. Our results suggest that fully virulent Y. pestis inhibits both the innate and adaptive immune responses that are substantially stimulated in a self-limited infection, which update our holistic views on the transcriptomic response to pneumonic plague.


Subject(s)
Adaptive Immunity , Gene Expression Profiling , Host-Pathogen Interactions , Immune Evasion , Immunity, Innate , Plague/pathology , Yersinia pestis/pathogenicity , Animals , Bacterial Load , Disease Models, Animal , Female , Liver/microbiology , Liver/pathology , Lung/microbiology , Lung/pathology , Mice , Spleen/microbiology , Spleen/pathology , Time Factors , Yersinia pestis/immunology
12.
Plant Physiol ; 167(3): 639-49, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25635113

ABSTRACT

Transcription factors control important gene networks, altering the expression of a wide variety of genes, including those of agronomic importance, despite often being expressed at low levels. Detecting transcription factor proteins is difficult, because current high-throughput methods may not be sensitive enough. One-dimensional, silicon-substrate photonic crystal (PC) arrays provide an alternative substrate for printing multiplexed protein microarrays that have greater sensitivity through an increased signal-to-noise ratio of the fluorescent signal compared with performing the same assay upon a traditional aminosilanized glass surface. As a model system to test proof of concept of the silicon-substrate PC arrays to directly detect rare proteins in crude plant extracts, we selected representatives of four different transcription factor families (zinc finger GATA, basic helix-loop-helix, BTF3/NAC [for basic transcription factor of the NAC family], and YABBY) that have increasing transcript levels during the stages of seedling cotyledon development. Antibodies to synthetic peptides representing the transcription factors were printed on both glass slides and silicon-substrate PC slides along with antibodies to abundant cotyledon proteins, seed lectin, and Kunitz trypsin inhibitor. The silicon-substrate PC arrays proved more sensitive than those performed on glass slides, detecting rare proteins that were below background on the glass slides. The zinc finger transcription factor was detected on the PC arrays in crude extracts of all stages of the seedling cotyledons, whereas YABBY seemed to be at the lower limit of their sensitivity. Interestingly, the basic helix-loop-helix and NAC proteins showed developmental profiles consistent with their transcript patterns, indicating proof of concept for detecting these low-abundance proteins in crude extracts.


Subject(s)
Cotyledon/metabolism , Glycine max/growth & development , Photons , Protein Array Analysis , Seedlings/growth & development , Silicon/pharmacology , Transcription Factors/metabolism , Antibodies/pharmacology , Cotyledon/drug effects , Cotyledon/genetics , Cross Reactions/immunology , Crystallization , Epitopes/metabolism , Fluorescence , Gene Expression Profiling , Gene Expression Regulation, Plant/drug effects , Genes, Developmental , Genes, Plant , Peptides/immunology , Plant Extracts/metabolism , Plant Proteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Seedlings/drug effects , Seedlings/metabolism , Glycine max/drug effects , Glycine max/metabolism , Transcription Factors/genetics
13.
Cell Microbiol ; 17(4): 473-85, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25298072

ABSTRACT

Pathogenic Yersinia species evolved a type III secretion system that injects a set of effectors into the host cell cytosol to promote infection. One of these effectors, Yersinia protein kinase A (YpkA), is a multidomain effector that harbours a Ser/Thr kinase domain and a guanine dissociation inhibitor (GDI) domain. The intercellular targets of the kinase and GDI domains of YpkA were identified to be Gαq and the small GTPases RhoA and Rac1, respectively, which synergistically induce cytotoxic effects on infected cells. In this study, we demonstrate that vasodilator-stimulated phosphoprotein (VASP), which is critical for regulation of actin assembly, cell adhesion and motility, is a direct substrate of YpkA kinase activity. Ectopic co-expression of YpkA and VASP in HEK293T cells leads to the phosphorylation of VASP at S157, and YpkA kinase activity is essential for VASP phosphorylation at this site. Moreover, YpkA directly phosphorylates VASP in in vitro kinase assay. YpkA-mediated VASP phosphorylation significantly inhibits actin polymerization and promotes the disruption of actin cytoskeleton, which inhibits the phagocytosis. Taken together, our study found a novel molecular mechanism used by YpkA to disrupt cytoskeleton dynamics, thereby promoting the anti-phagocytosis ability of pathogenic Yersiniae.


Subject(s)
Bacterial Proteins/metabolism , Cell Adhesion Molecules/metabolism , Cytoskeleton/metabolism , Host-Pathogen Interactions , Microfilament Proteins/metabolism , Phosphoproteins/metabolism , Protein Processing, Post-Translational , Protein Serine-Threonine Kinases/metabolism , Yersinia/enzymology , Animals , Cell Line , Humans , Mice , Phosphorylation
14.
J Immunol ; 192(2): 704-13, 2014 Jan 15.
Article in English | MEDLINE | ID: mdl-24337746

ABSTRACT

Innate immune cells, including neutrophils and macrophages, are critically involved in host antimicrobial defense responses. Intrinsic regulatory mechanisms controlling neutrophil and macrophage activities are poorly defined. In this study, we found that IL-17A, a natural signal factor, could provide protection against early pneumonic plague inflammation by coordinating the functions of neutrophils and programming of macrophages. The IL-17A level is promptly increased during the initial infection. Importantly, abrogation of IL-17A or IL-17AR significantly aggravated the infection, but mIL-17A treatment could significantly alleviate inflammatory injury, revealing that IL-17A is a critical requirement for early protection of infection. We also demonstrated that IL-17A was predominantly produced by CD11b(+)Ly6G(+) neutrophils. Although IL-17A could not significantly affect the antimicrobial responses of neutrophils, it could target the proinflammatory macrophage (M1) programming and potentiate the M1's defense against pneumonic plague. Mechanistically, IFN-γ treatment or IFN-γ-activated M1 macrophage transfer could significantly mitigate the aggravated infection of IL-17A(-/-) mice. Finally, we showed that IL-17A and IFN-γ could synergistically promote macrophage anti-infection immunity. Thus, our findings identify a previously unrecognized function of IL-17A as an intrinsic regulator in coordinating neutrophil and macrophage antimicrobial activity to provide protection against acute pneumonic plague.


Subject(s)
Interferon-gamma/immunology , Interferon-gamma/metabolism , Interleukin-17/immunology , Interleukin-17/metabolism , Macrophages/immunology , Neutrophils/immunology , Plague/immunology , Animals , Antigens, Ly/immunology , Antigens, Ly/metabolism , CD11b Antigen/immunology , CD11b Antigen/metabolism , Inflammation/immunology , Inflammation/metabolism , Macrophages/metabolism , Mice , Neutrophils/metabolism , Plague/metabolism , Receptors, Interleukin-17/immunology , Receptors, Interleukin-17/metabolism
15.
Anal Chem ; 86(17): 8805-13, 2014 Sep 02.
Article in English | MEDLINE | ID: mdl-25098859

ABSTRACT

We demonstrate the first use of smartphone spectrophotometry for readout of fluorescence-based biological assays. We evaluated the smartphone fluorimeter in the context of a fluorescent molecular beacon (MB) assay for detection of specific nucleic acid sequences in a liquid test sample and compared performance against a conventional laboratory fluorimeter. The capability of distinguishing a one-point mismatch is also demonstrated by detecting single-base mutation in target nucleic acids. Our approach offers a route toward portable biomolecular assays for viral/bacterial pathogens, disease biomarkers, and toxins.


Subject(s)
Cell Phone , Chemistry Techniques, Analytical/instrumentation , Chemistry Techniques, Analytical/methods , Nucleic Acids/analysis , Spectrometry, Fluorescence/instrumentation , Base Pair Mismatch , Base Sequence , Fluorescent Dyes/chemistry , MicroRNAs/analysis , MicroRNAs/genetics , Molecular Probes/chemistry , Nucleic Acid Hybridization , Nucleic Acids/chemistry
17.
J Virol ; 87(22): 12260-9, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24006436

ABSTRACT

Yep-phi is a T7-related bacteriophage specific to Yersinia pestis, and it is routinely used in the identification of Y. pestis in China. Yep-phi infects Y. pestis grown at both 20°C and 37°C. It is inactive in other Yersinia species irrespective of the growth temperature. Based on phage adsorption, phage plaque formation, affinity chromatography, and Western blot assays, the outer membrane proteins of Y. pestis Ail and OmpF were identified to be involved, in addition to the rough lipopolysaccharide, in the adsorption of Yep-phi. The phage tail fiber protein specifically interacts with Ail and OmpF proteins, and residues 518N, 519N, and 523S of the phage tail fiber protein are essential for the interaction with OmpF, whereas residues 518N, 519N, 522C, and 523S are essential for the interaction with Ail. This is the first report to demonstrate that membrane-bound proteins are involved in the adsorption of a T7-related bacteriophage. The observations highlight the importance of the tail fiber protein in the evolution and function of various complex phage systems and provide insights into phage-bacterium interactions.


Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Bacteriophage T7/metabolism , Host-Pathogen Interactions , Plague/microbiology , Porins/metabolism , Virulence Factors/metabolism , Yersinia pestis/metabolism , Adsorption , Amino Acid Sequence , Blotting, Western , China , Chromatography, Affinity , Lipopolysaccharides/metabolism , Molecular Sequence Data , Plague/metabolism , Sequence Homology, Amino Acid , Yersinia pestis/pathogenicity
18.
Analyst ; 139(22): 5954-63, 2014 Nov 21.
Article in English | MEDLINE | ID: mdl-25265458

ABSTRACT

We demonstrate photonic crystal enhanced fluorescence (PCEF) microscopy as a surface-specific fluorescence imaging technique to study the adhesion of live cells by visualizing variations in cell-substrate gap distance. This approach utilizes a photonic crystal surface incorporated into a standard microscope slide as the substrate for cell adhesion, and a microscope integrated with a custom illumination source as the detection instrument. When illuminated with a monochromatic light source, angle-specific optical resonances supported by the photonic crystal enable efficient excitation of surface-confined and amplified electromagnetic fields when excited at an on-resonance condition, while no field enhancement occurs when the same photonic crystal is illuminated in an off-resonance state. By mapping the fluorescence enhancement factor for fluorophore-tagged cellular components between on- and off-resonance states and comparing the results to numerical calculations, the vertical distance of labelled cellular components from the photonic crystal substrate can be estimated, providing critical and quantitative information regarding the spatial distribution of the specific components of cells attaching to a surface. As an initial demonstration of the concept, 3T3 fibroblast cells were grown on fibronectin-coated photonic crystals with fluorophore-labelled plasma membrane or nucleus. We demonstrate that PCEF microscopy is capable of providing information about the spatial distribution of cell-surface interactions at the single-cell level that is not available from other existing forms of microscopy, and that the approach is amenable to large fields of view, without the need for coupling prisms, coupling fluids, or special microscope objectives.


Subject(s)
Crystallization , Microscopy, Fluorescence/methods , Photons , 3T3 Cells , Animals , Biosensing Techniques , Culture Media , Mice
19.
Org Biomol Chem ; 12(44): 8919-29, 2014 Nov 28.
Article in English | MEDLINE | ID: mdl-25270760

ABSTRACT

Five new cardenolide lactates (1­5) and one new dioxane double linked cardenolide glycoside (17) along with 15 known compounds (6­16 and 18­21) were isolated from the ornamental milkweed Asclepias curassavica. Their structures were elucidated by extensive spectroscopic methods (IR, UV, MS, 1D- and 2D-NMR). The molecular structures and absolute configurations of 1­3 and 17 were further confirmed by single-crystal X-ray diffraction analysis. Simultaneous isolation of dioxane double linked cardenolide glycosides (17­21) and cardenolide lactates (1­5) provided unique chemotaxonomic markers for this genus. Compounds 1­21 were evaluated for the inhibitory activities against DU145 prostate cancer cells. The dioxane double linked cardenolide glycosides showed the most potent cytotoxic effect followed by normal cardenolides and cardenolide lactates, while the C21 steroids were non-cytotoxic. Enzymatic assay established a correlation between the cytotoxic effects in DU145 cancer cells and the Ki for the inhibition of Na(+),K(+)-ATPase. Molecular docking analysis revealed relatively strong H-bond interactions between the bottom of the binding cavity and compounds 18 or 20, and explained why the dioxane double linked cardenolide glycosides possessed higher inhibitory potency on Na(+),K(+)-ATPase than the cardenolide lactate.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Asclepias/chemistry , Cardenolides/pharmacology , Enzyme Inhibitors/pharmacology , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/isolation & purification , Cardenolides/chemistry , Cardenolides/isolation & purification , Cell Line, Tumor , Cell Proliferation/drug effects , Crystallography, X-Ray , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/isolation & purification , Humans , Models, Molecular , Molecular Conformation , Molecular Structure , Sodium-Potassium-Exchanging ATPase/metabolism , Structure-Activity Relationship
20.
BMC Complement Med Ther ; 24(1): 243, 2024 Jun 22.
Article in English | MEDLINE | ID: mdl-38909225

ABSTRACT

BACKGROUND: Cucurbita pepo cv Dayangua (CPD) is an edible plant with diverse pharmacological properties. The current research on CPD has primarily focused on initial investigations of its chemical composition and pharmacological effects, and no comprehensive toxicity assessment has been conducted to date. METHODS: In the present study, the toxicity of CPD was evaluated through both acute and sub-chronic oral toxicity tests in mice. 16S rDNA sequencing was used to analyze the composition of the gut microbiota of mice at different time points to observe the effect of CPD on these microbial communities. RESULTS: In the acute toxicity test, CPD exhibited low toxicity, with a median lethal dose (LD50) > 2000 mg/kg. The sub-chronic toxicity test indicated that CPD administration at doses of 200, 400, and 600 mg/kg did not cause mortality or significant organ damage in mice. Furthermore, analysis of the gut microbiota after gavage administration of CPD at 400 and 600 mg/kg revealed an improved abundance of some beneficial gut bacteria. CONCLUSIONS: In summary, no acute or sub-chronic toxic effects were observed in mice following the oral administration of CPD. CPD did not affect the structure and diversity of the gut microbiota and may contribute to an increase in the number of beneficial gut bacteria.


Subject(s)
Cucurbita , Gastrointestinal Microbiome , Animals , Gastrointestinal Microbiome/drug effects , Mice , Male , Plant Extracts/pharmacology , Plant Extracts/toxicity , Female , Toxicity Tests, Acute
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