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3-methyl-4-nitrophenol (PNMC), a degradation product of organophosphorus insecticides and a byproduct of fuel combustion, exerting endocrine-disrupting effects. However, its impact on the meiotic process of oocytes remains unclear. In the present study, we investigated the effects of PNMC on meiotic maturation of mouse oocytes in vitro and related mechanisms. Morphologically, PNMC-exposure affected germinal vesicle breakdown (GVBD) and polar body extrusion (PBE) in mouse oocytes. Proteomic analysis suggested that PNMC-exposure altered oocyte protein expression that are associated with cytoskeleton, mitochondrial function and oxidative stress. Further studies demonstrated that PNMC-exposure disrupted spindle assembly and chromosome alignment, caused sustained activation of spindle assembly checkpoint (SAC), and arrested meiosis in oocytes. Specifically, PNMC-exposure interfered with the function of microtubule organizing centers (MTOCs) by significantly reducing phosphorylated mitogen activated protein kinase (p-MAPK) expression and disrupting the localization of Pericentrin and p-Aurora A, leading to spindle assembly failure. Besides, PNMC-exposure also increased α-tubulin acetylation, decreased microtubule stability. Moreover, PNMC-exposure impaired mitochondrial function, evidenced by abnormal mitochondrial distribution, decreased mitochondrial membrane potential and ATP levels, release of Cytochrome C into the cytoplasm, and elevated ROS levels. As a result, exposure to PNMC caused DNA damage and early apoptosis in oocytes. Fortunately, melatonin was able to promote oocyte maturation by removing the excessive ROS and enhancing mitochondrial function. These results highlight the adverse effects of PNMC on meiotic maturation, and underscore the protective role of melatonin against PNMC-induced damage.
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Meiosis , Melatonina , Mitocondrias , Oocitos , Huso Acromático , Animales , Oocitos/efectos de los fármacos , Melatonina/farmacología , Ratones , Mitocondrias/efectos de los fármacos , Femenino , Huso Acromático/efectos de los fármacos , Meiosis/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Disruptores Endocrinos/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Antioxidantes , Insecticidas/toxicidadRESUMEN
OBJECTIVES: Sarcopenia is associated with functional disability in older adults. However, no consistent conclusions have been reached considering the differences in the measurement and criteria of sarcopenia. We aimed to examine the association between sarcopenia status and functional disability based on China Health and Retirement Longitudinal Study (CHARLS). DESIGN: A nationally representative longitudinal study. SETTING AND PARTICIPANTS: Participants aged at least 60 years old from the CHARLS 2015y were included. MEASUREMENTS: Sarcopenia was assessed according to the Asian Working Group for Sarcopenia 2019 algorithm. The outcomes of this study were basic activities of daily living (ADLs) and instrumental activities of daily living (IADLs). The logistic regression model was conducted to analyze the cross-sectional and longitudinal associations between sarcopenia status and ADLs and IADLs disability. RESULTS: In the cross-sectional study, 37.2% of the 6893 participants were defined as having sarcopenia. Any form of sarcopenia was associated with ADLs and IADLs disability. During three years of follow-up, 786 (16.5%) participants developed new-onset ADLs disability, and 980 (22.5%) participants developed new-onset IADLs disability. Compared with the no-sarcopenia, participants with possible sarcopenia (OR: 1.62, 95%CI: 1.34-1.95), sarcopenia (OR: 1.58, 95%CI: 1.18-2.11), or total sarcopenia (OR: 1.58, 95%CI: 1.34-1.88) had a higher risk of ADLs disability. While, the risk of IADLs disability for participants with possible sarcopenia (OR: 1.68, 95%CI: 1.41-2.00), sarcopenia (OR: 1.87, 95%CI: 1.40-2.51), or total sarcopenia (OR: 1.71, 95%CI: 1.45-2.00) was still significantly increased. With statistical interaction between sarcopenia status and residence or sex in ADLs and IADLs disability, older adults in urban, with ORs ranging from 2.14 to 2.44, were at a higher risk of functional disability than those in rural. Possible sarcopenia was associated with a much higher risk of ADLs disability (OR: 1.68, 95%CI: 1.26-2.25) in males and a higher risk of IADLs disability (OR: 1.98, 95%CI: 1.56-2.52) in females. CONCLUSIONS: Sarcopenia was associated with an increased risk of ADLs and IADLs disability among older Chinese adults. Even possible sarcopenia still significantly impacted ADLs and IADLs disability, and this association varied by sex and residence.
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Actividades Cotidianas , Sarcopenia , Femenino , Masculino , Humanos , Persona de Mediana Edad , Anciano , Estudios Transversales , Estudios Longitudinales , Sarcopenia/complicaciones , Sarcopenia/epidemiología , AlgoritmosRESUMEN
Vegetable oil is an important part of the human diet and has multiple industrial uses. The rapid increase in vegetable oil consumption has necessitated the development of viable methods for optimizing the oil content of plants. The key genes regulating the biosynthesis of maize grain oil remain mostly uncharacterized. In this study, by analyzing oil contents and performing bulked segregant RNA sequencing and mapping analyses, we determined that su1 and sh2-R mediate the shrinkage of ultra-high-oil maize grains and contribute to the increase in the grain oil content. Functional kompetitive allele-specific PCR (KASP) markers developed for su1 and sh2-R detected su1su1Sh2Sh2, Su1Su1sh2sh2, and su1su1sh2sh2 mutants among 183 sweet maize inbred lines. An RNA sequencing (RNA-seq) analysis indicated that genes differentially expressed between two conventional sweet maize lines and two ultra-high-oil maize lines were significantly associated with linoleic acid metabolism, cyanoamino acid metabolism, glutathione metabolism, alanine, aspartate, and glutamate metabolism, and nitrogen metabolism. A bulk segregant analysis and sequencing (BSA-seq) analysis identified another 88 genomic intervals related to grain oil content, 16 of which overlapped previously reported maize grain oil-related QTLs. The combined analysis of BSA-seq and RNA-seq data enabled the identification of candidate genes. The KASP markers for GRMZM2G176998 (putative WD40-like beta propeller repeat family protein), GRMZM2G021339 (homeobox-transcription factor 115), and GRMZM2G167438 (3-ketoacyl-CoA synthase) were significantly related to maize grain oil content. Another candidate gene, GRMZM2G099802 (GDSL-like lipase/acylhydrolase), catalyzes the final step of the triacylglycerol synthesis pathway and was expressed at significantly higher levels in the two ultra-high-oil maize lines than in the two conventional sweet maize lines. These novel findings will help clarify the genetic basis of the increased oil production in ultra-high-oil maize lines with grain oil contents exceeding 20%. The KASP markers developed in this study may be useful for breeding new high-oil sweet maize varieties.
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KEY MESSAGE: The novel ZmR1CQ01 allele for maize anthocyanin synthesis was identified, and the biological function and regulatory molecular mechanisms of three ZmR1 alleles were unveiled. Anthocyanins in maize are valuable to human health. The R1 gene family is one of the important regulatory genes for the tissue-specific distribution of anthocyanins. R1 gene allelic variations are abundant and its biological function and regulatory molecular mechanisms are not fully understood. By exploiting genetic mapping and transgenic verification, we found that anthocyanin pigmentation in maize leaf midrib was controlled by ZmR1 on chromosome 10. Allelism test of maize zmr1 EMS mutants confirmed that anthocyanin pigmentation in leaf sheath was also controlled by ZmR1. ZmR1CQ01 was a novel ZmR1 allelic variation obtained from purple maize. Its overexpression caused the whole maize plant to turn purple. ZmR1B73 allele confers anthocyanin accumulation in near ground leaf sheath rather than in leaf midribs. The mRNA expression level of ZmR1B73 was low in leaf midribs, resulting in no anthocyanin accumulation. ZmR1B73 overexpression promoted anthocyanin accumulation in leaf midribs. Loss of exon 5 resulted in ZmR1ZN3 allele function destruction and no anthocyanin accumulation in leaf midrib and leaf sheath. DNA affinity purification sequencing revealed 1010 genes targeted by ZmR1CQ01, including the bz2 in anthocyanin synthesis pathway. RNA-seq analysis showed 55 genes targeted by ZmR1CQ01 changed the expression level significantly, and the expression of genes encoding key enzymes in flavonoid and phenylpropanoid biosynthesis pathways were significantly up-regulated. ZmR1 functional molecular marker was developed. These results revealed the effects of transcriptional regulation and sequence variation on ZmR1 function and identified the genes targeted by ZmR1CQ01 at the genome-wide level.
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Antocianinas , Zea mays , Alelos , ADN , Regulación de la Expresión Génica de las Plantas , Pigmentación/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , ARN Mensajero , Zea mays/genética , Zea mays/metabolismoRESUMEN
Cadmium (Cd) accumulation in maize grains is detrimental to human health. Developing maize varieties with low Cd content is important for safe consumption of maize grains. However, the key genes controlling maize grain Cd accumulation have not been cloned. Here, we identified one major locus for maize grain Cd accumulation (qCd1) using a genome-wide association study (GWAS) and bulked segregant RNA-seq analysis with a biparental segregating population of Jing724 (low-Cd line) and Mo17 (high-Cd line). The candidate gene ZmHMA3 was identified by fine mapping and encodes a tonoplast-localized heavy metal P-type ATPase transporter. An ethyl methane sulfonate mutant analysis and an allelism test confirmed that ZmHMA3 influences maize grain Cd accumulation. A transposon in intron 1 of ZmHMA3 is responsible for the abnormal amino acid sequence in Mo17. Based on the natural sequence variations in the ZmHMA3 gene of diverse maize lines, four PCR-based molecular markers were developed, and these were successfully used to distinguish five haplotypes with different grain Cd contents in the GWAS panel and to predict grain Cd contents of widely used maize inbred lines and hybrids. These molecular markers can be used to breed elite maize varieties with low grain Cd contents.
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ATPasas Tipo P , Contaminantes del Suelo , Cadmio/metabolismo , Grano Comestible/metabolismo , Estudio de Asociación del Genoma Completo , Fitomejoramiento , Zea mays/genética , Zea mays/metabolismoRESUMEN
Salt stress is a major devastating abiotic factor that affects the yield and quality of maize. However, knowledge of the molecular mechanisms of the responses to salt stress in maize is limited. To elucidate the genetic basis of salt tolerance traits, a genome-wide association study was performed on 348 maize inbred lines under normal and salt stress conditions using 557 894 single nucleotide polymorphisms (SNPs). The phenotypic data for 27 traits revealed coefficients of variation of >25%. In total, 149 significant SNPs explaining 6.6%-11.2% of the phenotypic variation for each SNP were identified. Of the 104 identified quantitative trait loci (QTLs), 83 were related to salt tolerance and 21 to normal traits. Additionally, 13 QTLs were associated with two to five traits. Eleven and six QTLs controlling salt tolerance traits and normal root growth, respectively, co-localized with QTL intervals reported previously. Based on functional annotations, 13 candidate genes were predicted. Expression levels analysis of 12 candidate genes revealed that they were all responsive to salt stress. The CRISPR/Cas9 technology targeting three sites was applied in maize, and its editing efficiency reached 70%. By comparing the biomass of three CRISPR/Cas9 mutants of ZmCLCg and one zmpmp3 EMS mutant with their wild-type plants under salt stress, the salt tolerance function of candidate genes ZmCLCg and ZmPMP3 were confirmed. Chloride content analysis revealed that ZmCLCg regulated chloride transport under sodium chloride stress. These results help to explain genetic variations in salt tolerance and provide novel loci for generating salt-tolerant maize lines.
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Estudio de Asociación del Genoma Completo , Zea mays , Disección , Fenotipo , Polimorfismo de Nucleótido Simple/genética , Tolerancia a la Sal/genética , Plantones/genética , Zea mays/genéticaRESUMEN
Na+, K+ and pH homeostasis are important for plant life and they are controlled by the monovalent cation proton antiporter (CPA) superfamily. The roles of ZmCPAs in salt tolerance are not fully elucidated. In this study, we identified 35 ZmCPAs comprising 13 Na+/H+ exchangers (ZmNHXs), 16 cation/H+ exchanger (ZmCHXs), and 6 K+ efflux antiporters (ZmKEAs). All ZmCPAs have transmembrane domains and most of them were localized to plasma membrane or tonoplast. ZmCHXs were specifically highly expressed in anthers, while ZmNHXs and ZmKEAs showed high expression in various tissues. ZmNHX5 and ZmKEA2 were up-regulated in maize seedlings under both NaCl and KCl stresses. Yeast complementation experiments revealed the roles of ZmNHX5, ZmKEA2 in NaCl tolerance. Analysis of the maize mutants further validated the salt tolerance functions of ZmNHX5 and ZmKEA2. Our study highlights comprehensive information of ZmCPAs and provides new gene targets for salt tolerance maize breeding.
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Antiportadores , Tolerancia a la Sal , Antiportadores/genética , Antiportadores/metabolismo , Cationes Monovalentes/metabolismo , Fitomejoramiento , Protones , Tolerancia a la Sal/genética , Zea mays/genética , Zea mays/metabolismoRESUMEN
Waxy maize has many excellent characteristics in terms of its nutritional and economic value. In recent decades, the waxy maize germplasm has increased dramatically as a result of different selection methods. We collected 200 waxy maize inbred accessions from different origins to study their genetic diversity and phylogenetic relationships, and to identify new waxy mutations. A simple sequence repeat (SSR) analysis revealed wide genetic diversity among the 200 waxy maize accessions. The maize accessions were clustered into three groups. We sequenced the waxy gene from the first to the 14th exon. Nucleotide variation analysis of 167 waxy maize and 14 flint maize lines revealed some nucleotide differences in the waxy gene among different waxy maize groups, and much narrower nucleotide diversity in waxy maize than in flint maize. In a phylogenetic analysis, waxy maize carrying the same mutation allele clustered together, and waxy maize carrying different mutation alleles distributed in different groups; waxy maize was intermixed with flint maize in each branch, and wx-D7 waxy maize separated significantly from waxy maize lines carrying wx-D10, wx-124 and wx-hAT mutant alleles. The wx-hAT was a new waxy mutation identified in this study. It consisted of a 2286-bp transposon inserted into the middle of exon three of the waxy gene. A PCR marker specific for the wx-hAT allele was developed. These results will be useful for the utilization and preservation of the waxy maize germplasm, and the PCR marker has potential uses in waxy maize breeding programs.
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Genes de Plantas/genética , Zea mays/genética , Alelos , Sitios Genéticos/genética , Variación Genética , Filogenia , Proteínas de Plantas/genética , Reacción en Cadena de la Polimerasa , Polimorfismo Genético/genética , Alineación de Secuencia , Almidón Sintasa/genéticaRESUMEN
Mammalian target of rapamycin (mTOR), a serine/threonine kinase orchestrating cellular metabolism, is a crucial immune system regulator. However, it remains unclear how mTOR regulates dendritic cell (DC) function in vivo, especially DC-T cell encounters, a critical step for initiating adaptive immune responses. We dynamically visualized DC-T contacts in mouse lymph node using confocal microscopy and established an encounter model to characterize the effect of mTOR inhibition on DC-T cell encounters using DC morphology. Quantitative data showed mTOR inhibition via rapamycin altered DC shape, with an increased form factor (30.17%) and decreased cellular surface area (20.36%) and perimeter (22.43%), which were associated with Cdc42 protein downregulation (52.71%). Additionally, DCs adopted a similar morphological change with Cdc42 inhibition via ZCL278 as that observed with mTOR inhibition. These morphologically altered DCs displayed low encounter rates with T cells. Time-lapse imaging data of T cell motility supported the simulated result of the encounter model, where antigen-specific T cells appeared to reduce arrest in the lymph nodes of rapamycin-pretreated mice relative to the untreated group. Therefore, mTOR inhibition altered DC morphology in vivo and decreased the DC-T cell encounter rate, as well as Cdc42 inhibition. By establishing an encounter model, our study provides an intuitive approach for the early prediction of DC function through morphological quantification of form factor and area.
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Ganglios Linfáticos/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Benzamidas/metabolismo , Comunicación Celular , Diferenciación Celular , Movimiento Celular , Células Dendríticas/metabolismo , Regulación hacia Abajo , Femenino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Sirolimus/metabolismo , Linfocitos T/metabolismo , Tiourea/análogos & derivados , Tiourea/metabolismo , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Plant height (PH) is a key factor in maize (Zea mays L.) yield, biomass, and plant architecture. We investigated the PH of diverse maize inbred lines (117 temperate lines, 135 tropical lines) at four growth stages using unmanned aerial vehicle high-throughput phenotypic platforms (UAV-HTPPs). We extracted PH data using an automated pipeline based on crop surface models and orthomosaic model. The correlation between UAV and manually measured PH data reached 0.95. Under temperate field conditions, temperate maize lines grew faster than tropical maize lines at early growth stages, but tropical lines grew faster at later growth stages and ultimately became taller than temperate lines. A genome-wide association study identified 68 unique quantitative trait loci (QTLs) for seven PH-related traits, and 35% of the QTLs coincided with those previously reported to control PH. Generally, different QTLs controlled PH at different growth stages, but eight QTLs simultaneously controlled PH and growth rate at multiple growth stages. Based on gene annotations and expression profiles, we identified candidate genes controlling PH. The PH data collected by the UAV-HTPPs were credible and the genetic mapping power was high. Therefore, UAV-HTPPs have great potential for use in studies on PH.
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Estudios de Asociación Genética , Sitios de Carácter Cuantitativo , Carácter Cuantitativo Heredable , Tecnología de Sensores Remotos/instrumentación , Tecnología de Sensores Remotos/métodos , Zea mays/genética , Mapeo Cromosómico , Ambiente , Heterogeneidad Genética , FenotipoRESUMEN
Maize is a globally important crop that was a classic model plant for genetic studies. Here, we report a 2.2 Gb draft genome sequence of an elite maize line, HuangZaoSi (HZS). Hybrids bred from HZS-improved lines (HILs) are planted in more than 60% of maize fields in China. Proteome clustering of six completed sequenced maize genomes show that 638 proteins fall into 264 HZS-specific gene families with the majority of contributions from tandem duplication events. Resequencing and comparative analysis of 40 HZS-related lines reveals the breeding history of HILs. More than 60% of identified selective sweeps were clustered in identity-by-descent conserved regions, and yield-related genes/QTLs were enriched in HZS characteristic selected regions. Furthermore, we demonstrated that HZS-specific family genes were not uniformly distributed in the genome but enriched in improvement/function-related genomic regions. This study provides an important and novel resource for maize genome research and expands our knowledge on the breadth of genomic variation and improvement history of maize.
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Genoma de Planta , Zea mays/genética , China , Mapeo Cromosómico , Análisis por Conglomerados , Variación Genética , Genómica , Filogenia , Fitomejoramiento , Proteínas de Plantas/genética , Sitios de Carácter Cuantitativo , Zea mays/clasificaciónRESUMEN
Salt stress is a major abiotic factor limiting maize yield. To characterize the mechanism underlying maize salt tolerance, we compared the seedling root proteomes of salt-tolerant Jing724 and salt-sensitive D9H. The germination rate and growth parameter values (weight and length) were higher for Jing724 than for D9H under saline conditions. Using an iTRAQ-based method, we identified 513 differentially regulated proteins (DRPs), with 83 and 386 DRPs specific to Jing724 and D9H, respectively. In salt-stressed Jing724, the DRPs were primarily associated with the pentose phosphate pathway, glutathione metabolism, and nitrogen metabolism. Key DRPs, such as glucose-6-phosphate 1-dehydrogenase, NADPH-producing dehydrogenase, glutamate synthase, and glutamine synthetase, were identified based on pathway enrichment and protein-protein interaction analyses. Moreover, salt-responsive proteins in Jing724 seedlings were implicated in energy management, maintenance of redox homeostasis, detoxification of ammonia, regulation of osmotic homeostasis, stress defense and adaptation, biotic cross-tolerance, and regulation of gene expression. Quantitative analyses of superoxide dismutase activity, malondialdehyde content, relative electrolyte leakage, and proline content were consistent with the predicted changes based on DRP functions. Furthermore, changes in the abundance of eight representative DRPs were correlated with the corresponding mRNA levels. Our results may be useful for elucidating the molecular networks mediating salt tolerance.
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Genotipo , Proteómica/métodos , Tolerancia a la Sal/genética , Zea mays/genética , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/metabolismo , Proteoma/análisis , ARN Mensajero/análisis , Plantones/químicaRESUMEN
BACKGROUND: Salt stress significantly restricts plant growth and production. Maize is an important food and economic crop but is also a salt sensitive crop. Identification of the genetic architecture controlling salt tolerance facilitates breeders to select salt tolerant lines. However, the critical quantitative trait loci (QTLs) responsible for the salt tolerance of field-grown maize plants are still unknown. RESULTS: To map the main genetic factors contributing to salt tolerance in mature maize, a double haploid population (240 individuals) and 1317 single nucleotide polymorphism (SNP) markers were employed to produce a genetic linkage map covering 1462.05 cM. Plant height of mature maize cultivated in the saline field (SPH) and plant height-based salt tolerance index (ratio of plant height between saline and control fields, PHI) were used to evaluate salt tolerance of mature maize plants. A major QTL for SPH was detected on Chromosome 1 with the LOD score of 22.4, which explained 31.2% of the phenotypic variation. In addition, the major QTL conditioning PHI was also mapped at the same position on Chromosome 1, and two candidate genes involving in ion homeostasis were identified within the confidence interval of this QTL. CONCLUSIONS: The detection of the major QTL in adult maize plant establishes the basis for the map-based cloning of genes associated with salt tolerance and provides a potential target for marker assisted selection in developing maize varieties with salt tolerance.
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Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo , Tolerancia a la Sal/genética , Zea mays/fisiología , Marcadores Genéticos , Zea mays/genéticaRESUMEN
The nuclear-encoded DAG-like (DAL) gene family plays critical roles in organelle C-to-U RNA editing in Arabidopsis thaliana. However, the origin, diversification and functional divergence of DAL genes remain unclear. Here, we analyzed the genomes of diverse plant species and found that: DAL genes are specific to spermatophytes, all DAL genes share a conserved gene structure and protein similarity with the inhibitor I9 domain of subtilisin genes found in ferns and mosses, suggesting that DAL genes likely arose from I9-containing proproteases via exon shuffling. Based on phylogenetic inference, DAL genes can be divided into five subfamilies, each composed of putatively orthologous and paralogous genes from different species, suggesting that all DAL genes originated from a common ancestor in early seed plants. Significant type I functional divergence was observed in 6 of 10 pairwise comparisons, indicating that shifting functional constraints have contributed to the evolution of DAL genes. This inference is supported by the finding that functionally divergent amino acids between subfamilies are predominantly located in the DAL domain, a critical part of the RNA editosome. Overall, these findings shed light on the origin of DAL genes in spermatophytes and outline functionally important residues involved in the complexity of the RNA editosome.
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Genes de Plantas , Familia de Multigenes/genética , Filogenia , Proteínas de Plantas/genética , Tracheophyta/genética , Metilación de ADN , Evolución Molecular , Edición de ARN , Zea mays/genéticaRESUMEN
S-type cytoplasmic male sterility (CMS-S) is the largest group among the three major types of CMS in maize. CMS-S exhibits fertility instability as a partial fertility restoration in a specific nuclear genetic background, which impedes its commercial application in hybrid breeding programs. The fertility instability phenomenon of CMS-S is controlled by several minor quantitative trait locus (QTLs), but not the major nuclear fertility restorer (Rf3). However, the gene mapping of these minor QTLs and the molecular mechanism of the genetic modifications are still unclear. Using completely sterile and partially rescued plants of fertility instable line (FIL)-B, we performed bulk segregant RNA-Seq and identified six potential associated genes in minor effect QTLs contributing to fertility instability. Analyses demonstrate that these potential associated genes may be involved in biological processes, such as floral organ differentiation and development regulation, energy metabolism and carbohydrates biosynthesis, which results in a partial anther exsertion and pollen fertility restoration in the partially rescued plants. The single nucleotide polymorphisms (SNPs) identified in two potential associated genes were validated to be related to the fertility restoration phenotype by KASP marker assays. This novel knowledge contributes to the understanding of the molecular mechanism of the partial fertility restoration of CMS-S in maize and thus helps to guide the breeding programs.
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In vivo optical spatio-temporal imaging of the tumor microenvironment is useful to explain how tumor immunotherapies work. However, the lack of fluorescent antigens with strong immunogenicity makes it difficult to study the dynamics of how tumors are eliminated by any given immune response. Here, we develop an effective fluorescent model antigen based on the tetrameric far-red fluorescent protein KatushkaS158A (tfRFP), which elicits both humoral and cellular immunity. We use this fluorescent antigen to visualize the dynamic behavior of immunocytes as they attack and selectively eliminate tfRFP-expressing tumors in vivo; swarms of immunocytes rush toward tumors with high motility, clusters of immunocytes form quickly, and numerous antigen-antibody complexes in the form of tfRFP(+) microparticles are generated in the tumor areas and ingested by macrophages in the tumor microenvironment. Therefore, tfRFP, as both a model antigen and fluorescent reporter, is a useful tool to visualize specific immune responses in vivo.
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Antígenos de Neoplasias/análisis , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/inmunología , Neoplasias/química , Neoplasias/inmunología , Animales , Modelos Animales de Enfermedad , Microscopía Intravital , Ratones Endogámicos C57BL , Proteína Fluorescente RojaRESUMEN
Neutrophils and monocytes/macrophages (MMs) play important roles in the development of cell-mediated delayed type hypersensitivity (DTH). However, the dynamics of neutrophils and MMs during the DTH reaction and how the immunosuppressant rapamycin modulates their behavior in vivo are rarely reported. Here, we take advantage of multi-scale optical imaging techniques and a footpad DTH reaction model to non-invasively investigate the dynamic behavior and properties of immune cells from the whole field of the footpad to the cellular level. During the classic elicitation phase of the DTH reaction, both neutrophils and MMs obviously accumulated at inflammatory foci at 24 h post-challenge. Rapamycin treatment resulted in advanced neutrophil recruitment and vascular hyperpermeability at an early stage (4 h), the reduced accumulation of neutrophils (> 50% inhibition ratio) at 48 h, and the delayed involvement of MMs in inflammatory foci. The motility parameters of immune cells in the rapamycin-treated reaction at 4 h post-challenge displayed similar mean velocities, arrest durations, mean displacements, and confinements as the classic DTH reaction at 24 h. These results indicate that rapamycin treatment shortened the initial preparation stage of the DTH reaction and attenuated its intensity, which may be due to the involvement of T helper type 2 cells or regulatory T cells.
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Hipersensibilidad Tardía/patología , Inmunosupresores/farmacología , Sirolimus/farmacología , Animales , Humanos , Macrófagos/inmunología , Ratones Endogámicos C57BL , Microscopía Confocal , Neutrófilos/inmunología , Imagen Óptica/métodos , Factores de TiempoRESUMEN
The ability of cell-penetrating peptides (CPPs) to deliver a range of membrane-impermeable molecules into living cells makes them attractive potential vehicles for therapeutics. However, in vivo, the efficiency of CPP delivery to the cytosol remains unsatisfactory owing to endosomal entrapment and/or systemic toxicity, which severely restrict their bioavailability and efficacy in in vivo applications. In this study, we developed a series of novel chimeras consisting of various numbers of d- and l-arginine residues and investigated their cellular uptake behaviors and systemic toxicities. We demonstrated that the intracellular distribution, uptake efficiency, and systemic toxicity of these oligoarginines were all significantly affected by the number of d-arginine residues in the peptide sequence. We also found that a hybrid peptide, (rR)(3)R(2), possessed low systemic toxicity, high uptake efficiency, and, remarkably, achieved efficient cytosolic delivery not only in cultured cells but also in living tissue cells in mice after intravenous injection, implying that this heterogeneous motif might have promising applications in the delivery of cargoes of small sizes directed to cytosolic targets in vivo. Our studies into the uptake mechanism of (rR)(3)R(2) indicate that its cellular uptake was not affected by pharmacological or physical inhibitors of endocytosis but by the elimination of the membrane potential, suggesting that (rR)(3)R(2) does not enter the cells via endocytosis but rather through direct membrane translocation driven by the membrane potential. The results here might provide useful guidelines for the design and application of CPPs in drug delivery.
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Péptidos de Penetración Celular/administración & dosificación , Portadores de Fármacos/administración & dosificación , Oligopéptidos/administración & dosificación , Animales , Arginina/química , Transporte Biológico , Membrana Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/farmacocinética , Citosol/metabolismo , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Colorantes Fluorescentes/administración & dosificación , Colorantes Fluorescentes/farmacocinética , Células HEK293 , Células HeLa , Humanos , Ratones , Oligopéptidos/química , Oligopéptidos/farmacocinética , Rodaminas/administración & dosificación , Rodaminas/farmacocinéticaRESUMEN
In this study, we describe a new strategy for labeling and tracking lysosomes with a cell-permeable fluorescent activity-based probe (CpFABP) that is covalently bound to select lysosomal proteins. Colocalization studies that utilized LysoTracker probes as standard lysosomal markers demonstrated that our novel probe is effective in specifically labeling lysosomes in various kinds of live cells. Furthermore, our studies revealed that this probe has the ability to label fixed cells, permeabilized cells, and NH4Cl-treated cells, unlike LysoTracker probes, which show ineffective labeling under the same conditions. Remarkably, when applied to monitor the process of lysosome-dependent apoptosis, our probe not only displayed the expected release of lysosomal cathepsins from lysosomes into the cytosol but also revealed additional information about the location of the cathepsins during apoptosis, which is undetectable by other chemical lysosome markers. These results suggest a wide array of promising applications for our probe and provide useful guidelines for its use as a lysosome marker in lysosome-related studies.