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PURPOSE: The aim of this study was to develop and validate a method to generate phenol red thread tests (PRTT) due to the lack of availability of commercial PRTT. METHODS: White cotton thread was dyed with phenol red (pH indicator) for 48 h, dried, cut, and sterilized. To validate its wicking ability, the thread was inserted into solutions of varying pH, flanking the pH of healthy tears, for different time intervals. To assess its diagnostic utility, PRTTs were performed in vivo on wildtype and a murine model of evaporative dry eye, acyl-coA: wax alcohol acyltransferase 2 knockout (Awat2 KO) mice. RESULTS: Two batches of PRTT were produced that had a similar appearance and function to the commercial product. In vitro testing revealed no significant differences in the wicking kinetics at any time point across the pH solutions for batch 1 and only one difference for batch 2 (pH 7.4 vs 7.8 at 5 s, P = 0.029). When comparing both batches, similar wicking kinetics were found with only two significant differences identified (pH 7.6 at 40 s and pH 7.8 at 35 s, P < 0.01). In vivo, our PRTT yielded similar measurements to the commercial PRTT in wildtype and Awat2 KO mice and detected a significant increase in aqueous tear volume in the Awat2 KO mice (commercial: P = 0.015, our PRTT: P = 0.002). CONCLUSION: Our method provides a reproducible diagnostic test that performs similarly to its commercial counterpart in a relevant dry eye model indicating that it can serve as a valid and reliable replacement.
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Aspergillus vadensis CBS 113365, a close relative of A. niger, has been suggested as a more favourable alternative for recombinant protein production as it does not acidify the culture medium and produces very low levels of extracellular proteases. The aim of this study was to investigate the underlying cause of the non-amylolytic and non-proteolytic phenotype of A. vadensis CBS 113365. Our results demonstrate that the non-functionality of the amylolytic transcription factor AmyR in A. vadensis CBS 113365 is primarily attributed to the lack of functionality of its gene's promoter sequence. In contrast, a different mechanism is likely causing the lack of PrtT activity, which is the main transcriptional regulator of protease production. The findings presented here not only expand our understanding of the genetic basis behind the distinct characteristics of A. vadensis CBS 113365, but also underscore its potential as a favourable alternative for recombinant protein production.
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Aspergillus , Proteínas Fúngicas , Aspergillus/genética , Aspergillus/metabolismo , Aspergillus/enzimología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regiones Promotoras Genéticas , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteolisis , Péptido Hidrolasas/metabolismo , Péptido Hidrolasas/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , TransactivadoresRESUMEN
OBJECTIVE: To survey genome-scale protease profiles regulated by the Aspergillus niger transcription factor PrtT and further controlled by carbon sources. RESULTS: The PrtT disruption mutant (delprtT) and overexpression (OEprtT) strains were successfully generated and further confirmed by phenotypic and protease activity analysis. RNA-seq analysis of WT and mutants identified 32 differentially expressed protease genes, which mostly belonged to serine-type peptidases, aspartic-type endopeptidases, aminopeptidases and carboxypeptidases. Furthermore, based on the MEME predicted motif analysis of the PrtT promoter, EMSA and phenotypic and qRT-PCR analyses confirmed that the carbon metabolism regulator AmyR directly regulated the protease genes and their regulatory factor PrtT. CONCLUSION: Thirty-two PrtT-regulated protease genes were identified by RNA-seq, and the secondary carbon source regulator AmyR was found to have a negative regulatory effect on the expression of PrtT and its target protease genes.
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Aspergillus niger/crecimiento & desarrollo , Proteínas Fúngicas/genética , Perfilación de la Expresión Génica/métodos , Péptido Hidrolasas/genética , Transactivadores/genética , Factores de Transcripción/genética , Aspergillus niger/genética , Aspergillus niger/metabolismo , Carbono/metabolismo , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Mutación , Péptido Hidrolasas/metabolismo , Fenotipo , Regiones Promotoras Genéticas , Análisis de Secuencia de ARNRESUMEN
BACKGROUND: Translocated chromosomal duplications occur spontaneously in many organisms; segmental duplications of large chromosomal regions are expected to result in phenotypic changes because of gene dosage effects. Therefore, experimentally generated segmental duplications in targeted chromosomal regions can be used to study phenotypic changes and determine the functions of unknown genes in these regions. Previously, we performed tandem duplication of a targeted chromosomal segment in Aspergillus oryzae. However, in tandem chromosomal duplication, duplication of chromosomal ends and multiple chromosomal duplication are difficult. In this study, we aimed to generate fungal strains with a translocated duplication or triplication of a targeted chromosomal region via break-induced replication. RESULTS: Double-strand breaks were introduced into chromosomes of parental strains by treating protoplast cells with I-SceI meganuclease. Subsequently, strains were generated by nonreciprocal translocation of a 1.4-Mb duplicated region of chromosome 2 to the end of chromosome 4. Another strain, containing a triplicated region of chromosome 2, was generated by translocating a 1.4-Mb region of chromosome 2 onto the ends of chromosomes 4 and 7. Phenotypic analyses of the strains containing segmental duplication or triplication of chromosome 2 showed remarkable increases in protease and amylase activities in solid-state cultures. Protease activity was further increased in strains containing the duplication and triplication after overexpression of the transcriptional activator of proteases prtT. This indicates that the gene-dosage effect and resulting phenotypes of the duplicated chromosomal region were enhanced by multiple duplications, and by the combination of the structural gene and its regulatory genes. Gene expression analysis, conducted using oligonucleotide microarrays, showed increased transcription of a large population of genes located in duplicated or triplicated chromosomal regions. CONCLUSION: In this study, we performed translocated chromosomal duplications and triplications of a 1.4-Mb targeted region of chromosome 2. Strains containing a duplication of chromosome 2 showed significant increases in protease and amylase activities; these enzymatic activities were further increased in the strain containing a triplication of chromosome 2. This indicates that segmental duplications of chromosomes enhance gene-dosage effects, and that the resulting phenotypes play important phenotypic roles in A. oryzae.
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To evaluate tear production in the common mynah ( Acridotheres tristis ) using the phenol red thread test (PRTT) and to make a comparison of measurements with the PRTT placed in the fornices of lower and upper eyelids, tear production of both eyes in 22 healthy adult captive mynah birds was evaluated. After positioning of threads in the fornices of upper and lower eyelids, the PRTT values of the birds were 17.5 ± 3.1 mm/15 s and 19.2 ± 2.5 mm/15 s, respectively. A significant difference was found between PRTT values for upper eyelids and lower eyelids (P = 0.01). This study provides novel data for normal reference ranges of PRTT values in healthy common mynah birds and shows that a difference is found depending on where the PRTT thread is placed.
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Técnicas de Diagnóstico Oftalmológico/veterinaria , Fenolsulfonftaleína , Estorninos/fisiología , Lágrimas/fisiología , AnimalesRESUMEN
PrtT is a fungal-specific transcription activator of extracellular proteases in Aspergilli. In this study, the roles of the PrtT homolog from Penicillum oxalicum was investigated by transcription profiling in combination with electrophoretic mobility shift assay (EMSA). The prtT deletion dramatically reduced extracellular protease activities and caused intracellular nutrient limitation when cultured on casein as the sole carbon source. PrtT was found to directly regulate the expression of an intracellular peptidase encoding gene (tripeptidyl-peptidase) and the gene encoding the extracellular dipeptidyl-aminopeptidase V, in addition to the expected extracellular peptidase genes (carboxypeptidase and aspergillopepsin). Five amylase genes (α-amylase, glucoamylase, α-glucosidase) and three major facilitator superfamily transporter genes related to maltose, monosaccharide and peptide transporting were also confirmed as putative targets of PrtT by EMSA. In contrast, the transcription levels of other genes encoding polysaccharide degrading enzymes (e.g. cellulases) and most iron or multidrug transporter encoding genes were up- or down-regulated in the ΔprtT mutant due to nutrient limitation resulting from the reduced usage of the sole carbon source, casein. These results deepen the understanding of the interaction of regulation systems for nitrogen and carbon catabolism, which benefit strain improvement of P. oxalicum for industrial enzyme production.