RESUMEN
Fungal spores are common airborne allergens, and fungal richness has been implicated in allergic disease. Amplicon sequencing of environmental DNA from air samples is a promising method to estimate fungal spore richness with semi-quantification of hundreds of taxa and can be combined with quantitative PCR to derive abundance estimates. However, it remains unclear how the choice of air sampling method influences these estimates. This study compared active sampling with a portable impactor and passive sampling with a passive trap over different durations to estimate fungal spore richness and the abundance of allergenic taxa. Air sampling was conducted indoors and outdoors at 12 residences, including repeated measurements with a portable impactor and passive traps with 1-day and 7-day durations. ITS2 amplicon sequence data were transformed to spore equivalents estimated by quantitative PCR, repeated active samples were combined, and abundance-based rarefaction was performed to standardize sample coverage for estimation of genus-level richness and spore abundance. Rarefied fungal richness was similar between methods indoors but higher for passive traps with a 7-day duration outdoors. Rarefied abundance of allergenic genera was similar between methods but some genera had lower abundance for passive traps with a 1-day duration, which differed indoors and outdoors indicating stochasticity in the collection of spores on collocated samplers. This study found that similar estimates of fungal spore richness and abundance of allergenic taxa can be obtained using a portable impactor or a passive trap within one day and that increased passive sample duration provides limited additional information.
Asunto(s)
Alérgenos , Hongos , Esporas Fúngicas/genética , Hongos/genética , Microbiología del Aire , Monitoreo del AmbienteRESUMEN
Rice blast caused by Magnaporthe oryzae is a dangerous threat to rice production and food security worldwide. Breeding and proper deployment of resistant varieties are effective and environmentally friendly strategies to manage this notorious disease. However, a highly dynamic and quickly evolved rice blast pathogen population in the field has made disease control with resistance germplasms more challenging. Therefore, continued monitoring of pathogen dynamics and application of effective resistance varieties are critical tasks to prolong or sustain field resistance. Here, we report a team project that involved evaluation of rice blast resistance genes and surveillance of M. oryzae field populations in Taiwan. A set of International Rice Research Institute-bred blast-resistant lines (IRBLs) carrying single blast resistance genes was utilized to monitor the field effectiveness of rice blast resistance. Resistance genes such as Ptr (formerly Pita2) and Pi9 exhibited the best and most durable resistance against the rice blast fungus population in Taiwan. Interestingly, line IRBLb-B harboring the Pib gene with good field protection has recently shown susceptible lesions in some locations. To dissect the genotypic features of virulent isolates against the Pib resistance gene, M. oryzae isolates were collected and analyzed. Screening of the AvrPib locus revealed that the majority of field isolates still maintained the wild-type AvrPib status but eight virulent genotypes were found. Pot3 insertion appeared to be a major way to disrupt the AvrPib avirulence function. Interestingly, a novel AvrPib double-allele genotype among virulent isolates was first identified. Pot2 repetitive element-based polymerase chain reaction (rep-PCR) fingerprinting analysis indicated that mutation events may occur independently among different lineages in different geographic locations of Taiwan. This study provides our surveillance experience of rice blast disease and serves as the foundation to sustain rice production.
Asunto(s)
Magnaporthe , Oryza , Magnaporthe/genética , Enfermedades de las Plantas/microbiología , Oryza/genética , Oryza/microbiología , Taiwán , FitomejoramientoRESUMEN
Airborne fungal spores are important aeroallergens that are remarkably diverse in terms of taxonomic richness. Indoor fungal richness is dominated by outdoor fungi and is geographically patterned, but the influence of natural landscape is unclear. We aimed to elucidate the relationship between indoor fungal spore richness and natural landscape by examining the amount of surrounding forest cover. Passive sampling of airborne fungal spores was conducted in 24 schools in Taiwan during hot and cool seasons, and amplicon sequencing was used to study fungal spore (genus) richness targeting the internal transcribed spacer 2 (ITS2) region. In total, 693 fungal genera were identified, 12 of which were ubiquitous. Despite overall similarity of fungal spore richness between seasons, Basidiomycota and Ascomycota richness increased during the hot and cool seasons, respectively. Fungal spore richness in schools had a strong positive correlation with the amount of surrounding forest cover during the cool season, but not during the hot season. Fungal assemblages in schools were more similar during the hot season due to the increased ubiquity of Agaricomycetes genera. These observations indicate dispersal limitation at the kilometer scale during the cool season and increased long-distance dispersal during the hot season. Several allergenic fungi were commonly identified in schools, including some previously overlooked by conventional methods, which may be targeted as sensitizing agents in future investigations into atopic conditions. More generally, the relative importance of fungal spore richness in the development, chronicity, and severity of atopic conditions in children requires investigation.
Asunto(s)
Alérgenos , Bosques , Microbiología del Aire , Alérgenos/genética , Niño , Hongos/genética , Humanos , Instituciones Académicas , Estaciones del Año , Esporas FúngicasRESUMEN
Rice blast is a serious threat to global rice production. Large-scale and long-term cultivation of rice varieties with a single blast resistance gene usually leads to breakdown of resistance. To effectively control rice blast in Taiwan, marker-assisted backcrossing was conducted to develop monogenic lines carrying different blast resistance genes in the genetic background of an elite japonica rice cultivar, Kaohsiung 145 (KH145). Eleven International Rice Research Institute (IRRI)-bred blast-resistant lines (IRBLs) showing broad-spectrum resistance to local Pyricularia oryzae isolates were used as resistance donors. Sequencing analysis revealed that the recurrent parent, KH145, does not carry known resistance alleles at the target Pi2/9, Pik, Pita, and Ptr loci. For each IRBL × KH145 cross, we screened 21 to 370 (average of 108) plants per generation from the BC1F1 to BC3F1/BC4F1 generation. A total of 1,499 BC3F2/BC4F2 lines carrying homozygous resistance alleles were selected and self-crossed for four to six successive generations. The derived lines were also evaluated for background genotype using genotyping by sequencing, for blast resistance under artificial inoculation and natural infection conditions, and for agronomic performance in multiple field trials. In Chiayi and Taitung blast nurseries in 2018 to 2020, Pi2, Pi9, and Ptr conferred high resistance, Pi20 and Pik-h moderate resistance, and Pi1, Pi7, Pik-p, and Pik susceptibility to leaf blast; only Pi2, Pi9, and Ptr conferred effective resistance against panicle blast. The monogenic lines showed agronomic traits, yield, and grain quality similar to those of KH145, suggesting the potential of growing a mixture of lines to achieve durable resistance in the field.
Asunto(s)
Resistencia a la Enfermedad/genética , Magnaporthe , Oryza , Enfermedades de las Plantas , Genotipo , Oryza/genética , Oryza/microbiología , Fitomejoramiento , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiologíaRESUMEN
BACKGROUND: Phalaenopsis is one of the important ornamental plants worldwide. It plays the most significant role in flower exportation in Taiwan. However, the yellow leaf disease caused by Fusarium spp. has reduced the orchid flower yield 10-50 % yearly. Varieties resistant to yellow leaf disease associated with Fusarium is urgently needed for orchid growers and breeders, and is the ultimate solution for the long-term goal. To achieve this, phenotyping is the first step and the most necessary information for further studies, such as resistance gene identification, quantitative trait loci identification, and genome-wide association study. RESULTS: The inoculation of Fusarium was performed in either abbreviated stem or detached leaf, and the pros and cons were compared. The former is the general method of phenotyping for estimating the tolerance to yellow leaf disease of Phalaenopsis, but it is time-consuming and spacy, and thus not suitable for the assessment of large numbers of samples. In contrast, the latter not only showed a similar trend of disease severity with time reduced to only one fourth of the former one but also less space needed. CONCLUSIONS: This solution allows a better phenotyping approach for the fast detection of yellow leaf disease associated with Fusarium in a large number of Phalaenopsis samples.
RESUMEN
Bananas are among the world's most important cash and staple crops but are threatened by various devastating pathogens. The phytohormone salicylic acid (SA) plays a key role in the regulation of plant immune response. Tracking the expression of SA-responsive marker genes under pathogen infection is important in pathogenesis elucidation. However, the common SA-responsive marker genes are not consistently induced in different banana cultivars or different organs. Here, we conducted transcriptome analysis for SA response of a banana cultivar, 'Pei-Chiao' (Cavendish, AAA genome), and identified three genes, MaWRKY40, MaWRKY70, and Downy Mildew Resistant 6 (DMR6)-Like Oxygenase 1 (MaDLO1) that are robustly induced upon SA treatment in both the leaves and roots. Consistent induction of these three genes by SA treatment was also detected in both the leaves and roots of bananas belonging to different genome types such as 'Tai-Chiao No. 7' (Cavendish, AAA genome), 'Pisang Awak' (ABB genome), and 'Lady Finger' (AA genome). Furthermore, the biotrophic pathogen cucumber mosaic virus elicited the expression of MaWRKY40 and MaDLO1 in infected leaves of susceptible cultivars. The hemibiotrophic fungal pathogen Fusarium oxysporum f. sp. cubense tropical race 4 (TR4) also consistently induced the expression of MaWRKY40 and MaDLO1 in the infected roots of the F. oxysporum f. sp. cubense TR4-resistant cultivar. These results indicate that MaWRKY40 and MaDLO1 can be used as reliable SA-responsive marker genes for the study of plant immunity in banana. Revealing SA-responsive marker genes provides a stepping stone for further studies in banana resistance to pathogens.
Asunto(s)
Musa , Productos Agrícolas , Inmunidad , Musa/genética , Enfermedades de las Plantas , Ácido SalicílicoRESUMEN
The heterothallic basidiomycetous fungus Cryptococcus neoformans has two mating types, MATa and MATα. Morphological progression of bisexual reproduction in C. neoformans is as follows: yeast to hyphal transition, filament extension, basidium formation, meiosis, and sporulation. C. neoformans Cdk-related kinase 1 (CRK1) is a negative regulator of bisexual mating. In this study, we characterized the morphological features of mating structures in the crk1 mutant and determined the genetic interaction of CRK1 in the regulatory networks of sexual differentiation. In the bilateral crk1 mutant cross, despite shorter length of filaments than in the wild-type cross, dikaryotic filaments and other structures still remained intact during bisexual mating, but the timing of basidium formation was approximately 18 h earlier than in the cross between wild type strains. Furthermore, gene expression analyses revealed that CRK1 modulated the expression of genes involved in the progression of hyphal elongation, basidium formation, karyogamy and meiosis. Phenotypic results showed that, although deletion of C. neoformans CRK1 gene increased the efficiency of bisexual mating, filamentation in the crk1 mutant was blocked by MAT2 or ZNF2 mutation. A bioinformatics survey predicted the C. neoformans GATA transcriptional factor Gat1 as a potential substrate of Crk1 kinase. Our genetic and phenotypic findings revealed that C. neoformansGAT1 and CRK1 formed a regulatory circuit to negatively regulate MAT2 to control filamentation progression and transition during bisexual mating.
Asunto(s)
Cryptococcus neoformans/genética , Quinasas Ciclina-Dependientes/genética , Genes del Tipo Sexual de los Hongos/genética , Diferenciación Sexual/genética , Cryptococcus neoformans/crecimiento & desarrollo , Cryptococcus neoformans/patogenicidad , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica/genética , Hifa/genética , Hifa/crecimiento & desarrollo , Meiosis/genética , Mutación/genética , Fosforilación/genética , Reproducción/genéticaRESUMEN
Bifunctional fusion protein design has been widely utilized as a strategy to increase the efficacy of protein therapeutics. Previously, we proposed a novel application of the bifunctional fusion protein design through the introduction of proinsulin-transferrin (ProINS-Tf) fusion protein as a liver-specific protein prodrug to achieve a glucose-lowering effect in type 1 diabetic mice. In this report, we studied the binding characteristics of this activated fusion protein to the insulin receptor to elucidate its mechanism in eliciting insulin receptor-mediated signaling. We found that, with the assistance of the transferrin moiety binding to the transferrin receptor, the activated ProINS-Tf exhibited significantly higher binding affinity to the insulin receptor compared with the native insulin, resulting in a prolonged and stronger Akt phosphorylation. This enhanced induction by activated ProINS-Tf overcame insulin resistance in palmitate-treated HepG2 cells. ProINS-Tf also demonstrated a better glucose-lowering effect than native insulin, even with a much lower dose and less frequent injections, in non-obese diabetic mice with insulin resistance symptoms. The activated ProINS-Tf, serving as a bivalent protein molecule, could be a new insulin analog to overcome insulin resistance, which is associated with several diseases, including type 2 diabetes and non-alcoholic fatty liver disease.
Asunto(s)
Antígenos CD/genética , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Resistencia a la Insulina/genética , Insulina/farmacología , Receptor de Insulina/genética , Transferrina/genética , Animales , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Modelos Animales de Enfermedad , Glucosa/metabolismo , Células Hep G2 , Humanos , Hipoglucemiantes/farmacología , Insulina/genética , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos NOD , Proinsulina/genética , Proinsulina/farmacología , Unión Proteica/efectos de los fármacos , Receptores de Transferrina/genética , Transferrina/farmacologíaRESUMEN
Hemophilia B is a severe blood clotting disorder caused by the deficiency of factor IX (FIX). FIX is not bioavailable when given orally due to poor stability and permeability in the gastrointestinal tract. The feasibility of fusing FIX with transferrin (Tf) to enhance the oral bioavailability of FIX is explored. Seven recombinant fusion proteins (rFIX-Tf) with different linkers were constructed and expressed in HEK293 cells and characterized by in vitro transcytosis and transferrin receptor (TfR) binding assay in Caco-2 cells and a one-stage clotting assay. The in vivo efficacy study was performed using a tail-bleeding model in hemophilia B mice. Fusion proteins rFIX-Tf/G2 and rFIX-Tf/SVSQ were most permeable and showed a specific binding ability to TfR in Caco-2 cells. Both proteins retained FIX activity in clotting generation. The in vivo efficacy study showed that both proteins by intravenous injection significantly reduced blood loss. Most significantly, rFIX-Tf/G2 demonstrated anti-bleeding activity when administered orally. Our results showed that the fusion protein technique with Tf could be potentially used for oral delivery of FIX and the linker between FIX and Tf in the fusion protein is crucial. rFIX-Tf/G2 appears to be the most promising fusion protein as potential oral therapeutics for hemophilia B.
Asunto(s)
Factor IX/genética , Hemofilia B/tratamiento farmacológico , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Transferrina/genética , Administración Oral , Animales , Coagulación Sanguínea/efectos de los fármacos , Pruebas de Coagulación Sanguínea , Células CACO-2 , Cromatografía en Gel , Modelos Animales de Enfermedad , Expresión Génica , Ingeniería Genética , Hemofilia B/sangre , Hemofilia B/genética , Humanos , Ratones , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
Sordaria fimicola, a coprophilous ascomycete, is a homothallic fungus that can undergo sexual differentiation with cellular and morphological changes followed by multicellular tissue development to complete its sexual cycle. In this study, we identified and characterized the blue-light photoreceptor gene in S. fimicola The S. fimicola white collar-1 photoreceptor (SfWC-1) contains light-oxygen-voltage-sensing (LOV), Per-Arnt-Sim (PAS), and other conserved domains and is homologous to the WC-1 blue-light photoreceptor of Neurospora crassa The LOV domain of Sfwc-1 was deleted by homologous recombination using Agrobacterium-mediated protoplast transformation. The Sfwc-1(Δlov) mutant showed normal vegetative growth but produced less carotenoid pigment under illumination. The mutant showed delayed and less-pronounced fruiting-body formation, was defective in phototropism of the perithecial beaks, and lacked the fruiting-body zonation pattern compared with the wild type under the illumination condition. Gene expression analyses supported the light-induced functions of the Sfwc-1 gene in the physiology and developmental process of perithecial formation in S. fimicola Moreover, green fluorescent protein (GFP)-tagged SfWC-1 fluorescence signals were transiently strong upon light induction and prominently located inside the nuclei of living hyphae. Our studies focused on the putative blue-light photoreceptor in a model ascomycete and contribute to a better understanding of the photoregulatory functions and networks mediated by the evolutionarily conserved blue-light photoreceptors across diverse fungal phyla.IMPORTANCESordaria sp. has been a model for study of fruiting-body differentiation in fungi. Several environmental factors, including light, affect cellular and morphological changes during multicellular tissue development. Here, we created a light-oxygen-voltage-sensing (LOV) domain-deleted Sfwc-1 mutant to study blue-light photoresponses in Sordaria fimicola Phototropism and rhythmic zonation of perithecia were defective in the Sfwc-1(Δlov) mutant. Moreover, fruiting-body development in the mutant was reduced and also significantly delayed. Gene expression analysis and subcellular localization study further revealed the light-induced differential gene expression and cellular responses upon light stimulation in S. fimicola.
Asunto(s)
Cuerpos Fructíferos de los Hongos/crecimiento & desarrollo , Proteínas Fúngicas/genética , Fotorreceptores Microbianos/genética , Procesos Fototróficos/genética , Sordariales/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Cuerpos Fructíferos de los Hongos/genética , Proteínas Fúngicas/metabolismo , Fotorreceptores Microbianos/metabolismo , Sordariales/crecimiento & desarrollo , Sordariales/efectos de la radiación , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Zizania latifolia Turcz., which is mainly distributed in Asia, has had a long cultivation history as a cereal and vegetable crop. On infection with the smut fungus Ustilago esculenta, Z. latifolia becomes an edible vegetable, water bamboo. Two main cultivars, with a green shell and red shell, are cultivated for commercial production in Taiwan. Previous studies indicated that cultivars of Z. latifolia may be related to the infected U. esculenta isolates. However, related research is limited. The infection process of the corn smut fungus Ustilago maydis is coupled with sexual development and under control of the mating type locus. Thus, we aimed to use the knowledge of U. maydis to reveal the mating system of U. esculenta. We collected water bamboo samples and isolated 145 U. esculenta strains from Taiwan's major production areas. By using PCR and idiomorph screening among meiotic offspring and field isolates, we identified three idiomorphs of the mating type locus and found no sequence recombination between them. Whole-genome sequencing (Illumina and PacBio) suggested that the mating system of U. esculenta was bipolar. Mating type locus 1 (MAT-1) was 552,895â¯bp and contained 44% repeated sequences. Sequence comparison revealed that U. esculenta MAT-1 shared high gene synteny with Sporisorium reilianum and many repeats with Ustilago hordei MAT-1. These results can be utilized to further explore the genomic diversity of U. esculenta isolates and their application for water bamboo breeding.
Asunto(s)
Genes Fúngicos , Genes del Tipo Sexual de los Hongos , Poaceae/microbiología , Ustilago/genética , Asia , Interacciones Huésped-Patógeno , Enfermedades de las Plantas/microbiología , Secuenciación Completa del GenomaRESUMEN
The Magnaporthe oryzae avirulence gene AvrPib is required for the resistance mediated by its cognate resistance gene Pib, which has been intensively used in indica rice breeding programs in many Asian countries. However, the sequence diversity of AvrPib among geographically distinct M. oryzae populations was recently shown to be increasing. Here, we selected a field population consisting of 248 rice blast isolates collected from a disease hotspot in Philippine for the analysis of AvrPib haplotypes and their pathogenicity against Pib. We found that all of the isolates were virulent to Pib and each of them contained an insertion of Pot3 transposon in AvrPib. Moreover, Pot3 insertion was detected in different genomic positions, resulting in three different AvrPib haplotypes, designated avrPib-H1 to H3. We further conducted a genome-wide Pot2 fingerprinting analysis by repetitive element palindromic polymerase chain reaction (PCR) and identified seven different lineages out of 47 representative isolates. The isolates belonging to the same lineage often had the same AvrPib haplotype. In contrast, the isolates having the same AvrPib haplotypes did not always belong to the same lineages. Both mating types MAT1-1 and MAT1-2 were identified in the population in Bohol and the latter appeared dominant. On the host side, we found that 32 of 52 released rice varieties in the Philippines contained Pib diagnosed by PCR gene-specific primers and DNA sequencing of gene amplicons, suggesting that it was widely incorporated in different rice varieties. Our study highlights the genetic dynamics of rice blast population at both the AvrPib locus and the genome-wide levels, providing insight into the mechanisms of the mutations in AvrPib leading to the breakdown of Pib-mediated resistance in rice.
Asunto(s)
Magnaporthe/genética , Oryza/microbiología , Enfermedades de las Plantas/microbiología , Elementos Transponibles de ADN , Resistencia a la Enfermedad/genética , Variación Genética , Magnaporthe/patogenicidad , Mutagénesis Insercional , Oryza/genética , Filipinas , Enfermedades de las Plantas/genética , VirulenciaRESUMEN
BACKGROUND: Rice blast, caused by Magnaporthe oryzae, is an important rice disease occurring in all rice-growing areas. To manage blast disease effectively and in an environmentally friendly way, it is important to continually discover diverse resistant resources for breeding. In this study, genome-wide association study (GWAS) was used to map genes/loci resistant to rice blast in the open-access rice diversity panel 1 (RDP1), previously genotyped with a 44K single-nucleotide polymorphism array. Two geographically and genetically different M. oryzae isolates from Taiwan, D41-2 and 12YL-DL3-2, were used to challenge RDP1. Infected leaves were visually rated for lesion type (LT) and evaluated for proportion of diseased leaf area (%DLA) by image analysis software. RESULTS: A total of 32 quantitative trait loci (QTLs) were identified, including 6 from LT, 30 from DLA, and 4 from both LT and DLA. In all, 22 regions co-localized with previously reported resistance (R) genes and/or QTLs, including two cloned R genes, Pita and Ptr; 19 mapped R loci, and 20 QTLs. We identified 100 candidate genes encoding leucine-rich repeat-containing proteins, transcription factors, ubiquitination-related proteins, and peroxidases, among others, in the QTL intervals. Putative resistance and susceptibility haplotypes of the 32 QTL regions for each tested rice accessions were also determined. CONCLUSIONS: By using Taiwanese M. oryzae isolates and image-based phenotyping for detailed GWAS, this study offers insights into the genetics underlying the natural variation of blast resistance in RDP1. The results can help facilitate the selection of desirable donors for gene/QTL validation and blast resistance breeding.
RESUMEN
BACKGROUND: The coprophilous ascomycete Sordaria fimicola usually reproduces sexually. Sexual differentiation in S. fimicola is accompanied by cellular and morphological changes, followed by multicellular tissue development to complete the sexual cycle. Although the morphological features of the sexual reproductive structure in S. fimicola have been well characterized, little is known about the nuclear dynamics and organization during these processes. Therefore, in this study, we successfully developed an Agrobacterium-mediated protoplast transformation protocol and generated histone H2B-mCherry-labeled S. fimicola strains. The life cycle of S. fimicola begins with germination of the ascospore and ends with the formation and discharge of new ascospores from the mature black sexual fruiting bodies, the so-called perithecia. The nuclear dynamics of the fluorescently labeled strains were examined during ascospore germination, hyphal elongation, and hyphal fusion using fluorescent microscopy. RESULTS: Live imaging revealed that the nuclei in the germlings and fusion hyphae during the pre-contact interaction are located adjacent to the tip. CONCLUSIONS: This is the first report of the application of a fluorescence labeling technique in S. fimicola. This application will help researchers gain a better understanding of nuclear distribution and investigate the protein-protein interaction networks during fruiting body formation for advanced molecular genetic studies in S. fimicola.
RESUMEN
Cell-penetrating peptides (CPPs) have become a novel drug delivery system due to their distinct advantages, including high cell transmembrane potency and ability to carry cargo molecules inside cells. However, owing to their cationic charge and non-specificity characteristics, the clinical application of CPPs is limited. In the current study, we engineered a reversibly activatable cell-penetrating peptide (RACPP), containing oligoarginine fused to a pH-sensitive masking sequence via a polyglycine linker ((HE)10G5R6 or HE-CPP) with ultra-pH-sensitivity. The HE-CPP sequence was coupled to the surface of polyethyleneglycol-polylactic acid (PEG-PLA) polymer micelles (PMs-HE-CPP) to realize improve specificity and targeted delivery of encapsulated paclitaxel (PTX). PTX/PMs-HE-CPP showed the satisfactory encapsulated efficiency, loading capacity, size distribution as well as reversible charge-conversion in response to the surrounding pH. The zeta potential of PMs-HE-CPP was negative at pHâ¯7.5, moderately positive at pHâ¯6.5, and even more positive at a lower pH. Coumarin 6-loaded PMs-HE-CPP (C6/PMs-HE-CPP) showed enhanced tumor cellular uptake at a mildly acidic tumor microenvironment (pHâ¯6.5) via energy-dependent and clathrin-mediated endocytosis. Furthermore, PTX/PMs-HE-CPP had significantly higher cytotoxicity toward mice breast cancer (4T1) cells at pHâ¯6.5 versus at pHâ¯7.4. In vivo imaging studies in 4T1-BALB/c tumor xenograft models confirmed the tumor-targeting characteristic of PMs-HE-CPP. PTX/PMs-HE-CPP also exhibited improved anti-tumor efficacy against unmodified polymer micelles and Taxol® in this tumor model. Accordingly, not only do RACPPs show the great potential to endow CPPs with specificity and reversible net-charge converting characteristic, they are also able to improve the targeting effect of nanoparticles.
Asunto(s)
Sistemas de Liberación de Medicamentos , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Paclitaxel/administración & dosificación , Polímeros/química , Animales , Antineoplásicos Fitogénicos/administración & dosificación , Antineoplásicos Fitogénicos/farmacología , Péptidos de Penetración Celular/química , Endocitosis/efectos de los fármacos , Femenino , Concentración de Iones de Hidrógeno , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Micelas , Nanopartículas , Paclitaxel/farmacología , Poliésteres/química , Polietilenglicoles/química , Microambiente Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We examined the genomes of 100 isolates of Magnaporthe oryzae (Pyricularia oryzae), the causal agent of rice blast disease. We grouped current field populations of M. oryzae into three major globally distributed groups. A genetically diverse group, clade 1, which may represent a group of closely related lineages, contains isolates of both mating types. Two well-separated clades, clades 2 and 3, appear to have arisen as clonal lineages distinct from the genetically diverse clade. Examination of genes involved in mating pathways identified clade-specific diversification of several genes with orthologs involved in mating behavior in other fungi. All isolates within each clonal lineage are of the same mating type. Clade 2 is distinguished by a unique deletion allele of a gene encoding a small cysteine-rich protein that we determined to be a virulence factor. Clade 3 isolates have a small deletion within the MFA2 pheromone precursor gene, and this allele is shared with an unusual group of isolates we placed within clade 1 that contain AVR1-CO39 alleles. These markers could be used for rapid screening of isolates and suggest specific events in evolution that shaped these populations. Our findings are consistent with the view that M. oryzae populations in Asia generate diversity through recombination and may have served as the source of the clades 2 and 3 isolates that comprise a large fraction of the global population.
Asunto(s)
Magnaporthe/genética , Genes Fúngicos , Variación Genética , Genoma Fúngico , Genómica , Magnaporthe/clasificación , Oryza/microbiología , Enfermedades de las Plantas/microbiologíaRESUMEN
A recombinant proinsulin-transferrin fusion protein (ProINS-Tf) has been previously reported to be a novel long-lasting INS analog, acting specifically on the inhibition of hepatic glucose output. In this study, we investigated the biodistribution, activation and tissue retention of ProINS-Tf to elucidate its liver targeted anti-diabetic mechanism. The biodistribution study revealed that ProINS-Tf exhibited liver specific accumulation after a single intravenous injection, whereas transferrin (Tf) or insulin (INS) showed relatively even distribution among different organs. The conversion of inactive ProINS-Tf into an active immune-reactive INS-Tf form (irINS-Tf) via a Tf receptor (TfR) mediated process only occurred in the liver, but not in other organs. In addition, ProINS-Tf demonstrated a prolonged retention in the liver after an intravenous injection, suggesting the enhanced association of the bifunctional active form, irINS-Tf, within liver cells. Taken together, our results indicate that ProINS-Tf is a highly liver-targeted INS prodrug with a combination of 3 specific actions in liver cells: (1) TfR-mediated binding and uptake of the prodrug on the cell surface, (2) liver-specific, TfR-mediated conversion of the prodrug into its active form, and (3) the bifunctional binding of the active fusion protein to both Tf and INS receptors in the liver to achieve prolonged retention and thus enhanced anti-diabetic activities.
Asunto(s)
Hipoglucemiantes/farmacocinética , Insulina/farmacocinética , Hígado/metabolismo , Profármacos/farmacocinética , Proinsulina/farmacocinética , Proteínas Recombinantes de Fusión/farmacocinética , Transferrina/farmacocinética , Animales , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Femenino , Masculino , Ratones , Receptor de Insulina/metabolismo , Receptores de Transferrina/metabolismo , Distribución TisularRESUMEN
Proinsulin-transferrin fusion protein (ProINS-Tf) has been designed and successfully expressed from the mammalian HEK293 cells (HEK-ProINS-Tf). It was found that HEK-ProINS-Tf could be converted into an activated form in the liver. Furthermore, HEK-ProINS-Tf was demonstrated as an extra-long acting insulin analogue with liver-specific insulin action in streptozotocin (STZ)-induced type 1 diabetic mice. However, due to the low production yield from transfected HEK293 cells, there are other interesting features, including the oral bioavailability, which have not been fully explored and characterized. To improve the protein production yield, an alternative protein expression system, ExpressTec using transgenic rice (Oryza sativa L.), was used. The intact and active rice-derived ProINS-Tf (ExpressTec-ProINS-Tf) was successfully expressed from the transgenic rice expression system. Our results suggested that, although the insulin-like bioactivity of ExpressTec-ProINS-Tf was slightly lower in vitro, its potency of in vivo blood glucose control was considerably stronger than that of HEK-ProINS-Tf. The oral delivery studies in type 1 diabetic mice demonstrated a prolonged control of blood glucose to near-normal levels after oral administration of ExpressTec-ProINS-Tf. Results in this report suggest that ExpressTec-ProINS-Tf is a promising insulin analog with advantages including low cost, prolonged and liver targeting effects, and most importantly, oral bioactivity.
Asunto(s)
Diabetes Mellitus Experimental/tratamiento farmacológico , Hipoglucemiantes/administración & dosificación , Proinsulina/administración & dosificación , Transferrina/administración & dosificación , Administración Oral , Animales , Glucemia/metabolismo , Células HEK293 , Humanos , Hipoglucemiantes/uso terapéutico , Masculino , Ratones , Ratones Endogámicos C57BL , Oryza/genética , Proinsulina/genética , Proinsulina/uso terapéutico , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapéutico , Transferrina/genética , Transferrina/uso terapéuticoRESUMEN
Polyelectrolyte complexes (PECs) are self-assembling nano-sized constructs that offer several advantages over traditional nanoparticle carriers including controllable size, biodegradability, biocompatibility, and lack of toxicity, making them particularly appealing as tools for drug delivery. Here, we discuss potential application of PECs for drug delivery to the slightly acidic tumor microenvironment, a pH in the range of 6.5-7.0. Poly(l-glutamic acid) (En), poly(l-lysine) (Kn), and a copolymer composed of histidine-glutamic acid repeats ((HE)n) were studied for their ability to form PECs, which were analyzed for size, polydispersity, and pH sensitivity. PECs showed concentration dependent size variation at residue lengths of E51/K55 and E135/K127, however, no complexes were observed when E22 or K21 were used, even in combination with the longer chains. (HE)20/K55 PECs could encapsulate daunomycin, were stable from pH 7.4-6.5, and dissociated completely between pH 6.5-6.0. Conversely, the E51-dauno/K55 PEC dissociated between pH 4.0 and 3.0. These values for pH-dependent particle dissociation are consistent with the pKa's of the ionizable groups in each formulation and indicate that the specific pH-sensitivity of (HE)20-dauno/K55 PECs is mediated by incorporation of histidine. This response within a pH range that is physiologically relevant to the acidic tumors suggests a potential application of these PECs in pH-dependent drug delivery.
Asunto(s)
Aminoácidos , Aniones , Cationes , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos , Concentración de Iones de Hidrógeno , Polielectrolitos , Aminoácidos/química , Aniones/química , Cationes/química , Péptidos/química , Polielectrolitos/químicaRESUMEN
Fc fusion protein technology has been successfully used to generate long-acting forms of several protein therapeutics. In this study, a novel Fc-based drug carrier, single chain Fc-dimer (sc(Fc)2), was designed to contain two Fc domains recombinantly linked via a flexible linker. Since the Fc dimeric structure is maintained through the flexible linker, the hinge region was omitted to further stabilize it against proteolysis and reduce FcγR-related effector functions. The resultant sc(Fc)2 candidate preserved the neonatal Fc receptor (FcRn) binding. sc(Fc)2-mediated delivery was then evaluated using a therapeutic protein with a short plasma half-life, human growth hormone (hGH), as the protein drug cargo. This novel carrier protein showed a prolonged in vivo half-life and increased hGH-mediated bioactivity compared to the traditional Fc-based drug carrier. sc(Fc)2 technology has the potential to greatly advance and expand the use of Fc-technology for improving the pharmacokinetics and bioactivity of protein therapeutics.