Summary Repeated and prolonged use of fluconazole in treating candidosis leads to drug resistance. The aim of this study was to confirm the relationship between T916C (Y257H) mutation in Candida albicans ERG11 and fluconazole resistance. We replaced one copy of ERG11 with ERG11 containing the T916C mutation in C. albicans CAI4 and expressed ERG11 with the T916C mutation in Saccharomyces cerevisiae INVSc1. The MIC values were two‐ to four‐fold greater in CAI4 transformants with than without the T916C mutation and 128 and 32 μg ml −1 for S. cerevisiae INVSc1‐containing ERG11 with and without the T916C mutation. T916C mutation may be associated with fluconazole resistance in C. albicans .
Objective To investigate the performance of DNA microarray in identifying 6 common Candida spp. and validating ERG11 mutations resulting in fluconazolc-resistance in Candida albicans. Methods Oligonucleotide probes were designed and synthesized targeting the species-specific sequence in the internal transcribed spacer 2 (ITS2) region of rDNA of Candida albicans, Candida tropicalis, Candida glabrata, Candida dubliniensis, Candida parapsilosis and Candida krusei, as well as 6 sequences embracing the following mutations respectively in ERG11 gene leading to fluconazole-resistance, i.c., T541C, A 1090G, C1361T, G1537A, G1547A, and T1559C, then arranged onto a chip. Twelve 50-base-pair oligonucleotides were artificially synthesized based on the above specific sequences, and utilized to hybridize with the DNA microarray. Thirty-lbur Candida strains, including 29 C. albicans, 1 Candida tropicalis, 1 Candida glabrata,1 Candida dubliniensis, 1 Candida parapsilosis and 1 Candida krusei, were detected with microarray. Genomic DNA was extracted from these tested strains and underwent multiple PCR for the amplification of ITS2 region and ERGI 1 gene. Sequencing was performed to analyze the sequence of ERG11 in 29 strains of C. albicans and the results were compared with those of DNA microarray hybridization. Results Multiple PCR successfully produced ITS2 fragment of 307-415 bp from all the 34 strains, as well as ERG11 fragment of 1712 bp from 29 C. albicans strains. DNA microarray hybridization offered the same results in species identification of the 34 strains with their given information, as well as in mutation detection of the 29 strains of C. albicans with ERG11 sequencing results. Also, the 6 synthesized oligonucleotides containing the muta- tions were identified precisely as T541C, A1090G, C1361T, G1537A, G1547A, and T1559C, and the 6 species specific oligonucleotieds were identified correctly as C. albicans, C. tropicalis, C. glabrata, C. dubliniensis, C. parapsilosis and C. krusei Both the sensitivity and the specificity of the microarray were 100%. Conclu- sion DNA microarray is a quite reliable method to identify Candida spp. and fluconazole resistance-associ- ated mutations in the ERG11 gene of C. albicans.
Key words:
Oligonucleotide array sequence analysis; Candida; Mycological typing techniques; DNA mutation analysis; ERG11 genes
Probing relevant proteomic biomarkers may facilitate effective pancreatic adenocarcinoma (PDAC) diagnosis, treatment and prevention. Here, we developed a protein-based prognostic model for PDAC by using relevant proteomic biomarkers data from The Cancer Genome Atlas (TCGA).We obtained PDAC's proteomic and clinical data from TCGA and used various analytical tools to identify differentially expressed proteins between normal and cancer tissues. We constructed our protein-based prognostic model and confirmed its accuracy using receiver operating characteristic curve and Kaplan-Meier survival analyses. We elucidated clinical factor-signature protein correlations by clinical correlation assessments and protein coexpression networks. We also used immunohistochemistry (protein expression assessment), Gene Set Enrichment Analysis (protein role identification) and CIBERSORT (infiltrating immune cell distribution assessment).CIITA, BRAF_pS445, AR, YTHDF2, IGFBP2 and CDK1_pT14 were identified as PDAC-associated prognostic proteins. All risk scores calculated using our model provided 1-, 3-, 5-year survival probability at 70 % accuracy. The reliability of our model was validated by the GEO as well. In high- and low-risk groups, age, sex, T- and N- stage disparities were significant, and prognostic and coexpressed proteins correlated. PDAC tissues demonstrated significant CDK1_pT14 overexpression but significant BRAF_pS445, YTHDF2, and IGFBP2 underexpression. Downstream proteins of BRAF were validated by IHC. Low-risk tissues demonstrated more naïve B cells, eosinophils, activated NK cells and regulatory T cells, whereas high-risk tissues demonstrated more activated memory T cells, monocytes, neutrophils, dendritic cells and resting NK cells.Our protein-based prognostic model for PDAC, along with six signature proteins, might aid in predicting PDAC prognosis and therapeutic targets.
Sir, A 56-year-old Chinese Han woman presented to us with a febrile illness and a rapid progressing of erythematous, maculopapular rash involving her entire body 14 days after taking methazolamide with topical timolol maleate for her secondary glaucoma. Clinical examination revealed swollen eyelids, moderate hyperemia, and purulent discharge in her eyes, hemorrhagic crusts over lips, various sizes of erythematous eruptions and vesicles around her face, trunk and extremities, and a temperature of 103.82°F. Nikolsky sign was positive. She disclosed allergies to sulfanilamide antibiotics. The patient was diagnosed with toxic epidermal necrolysis (TEN) associated with methazolamide treatment. Intravenous methylprednisolone was administered, combining with fresh frozen plasma and immunoglobulin intravenously. During the therapy, her condition continued to progress [Fig. 1]. HLA-B5901 was detected positive in this patient. After about 2 weeks of hospitalization, skin eruptions of her upper trunk dried, and crust obviously while lesions of lower limbs still remained in serious condition [Fig. 2]. The dosage of intravenous methylprednisolone was then gradually tapered off. On the 25th day of hospitalization, the patient was discharged with erosions healed and epithelialized.Figure 1: On the 8th day of hospitalization, the skin, and mucosa lesions involved almost her entire body, accompanied with extensive denudation, erosion, and obvious exudationFigure 2: On the 16th day of hospitalization, skin eruptions of upper trunk dried and crust obviously while lesions of her lower limbs still remained in serious conditionTEN is a rare but severe mucocutaneous reaction, primarily due to drug intake. The similar, but the mild condition is Stevens–Johnson syndrome (SJS). The most common drugs associated with SJS/TEN are antibiotics such as sulfonamides, nonsteroidal anti-inflammatory drugs, and anticonvulsants. Methazolamide is an inhibitor of carbonic anhydrase commonly used to treat glaucoma. Severe cutaneous reactions to methazolamide were rarely reported. The first report was published in Japanese literature in 1989[1] and since then 32 cases of SJS/TEN associated with methazolamide treatment have been reported.[234] A correlation between HLA-B59 and methazolamide-induced SJS/TEN was first suggested by Shirato in 1997.[3] Later in 2010, Kim et al. further noted that HLA-B5901, which is specific to the Japanese or Korean population is correlated strongly with methazolamide-induced SJS/TEN. B59 is an HLA-B serotype and has been observed mainly in Asians. Its frequency was estimated to be only 1.8% in Japanese and 2.1% in Koreans.[4] Immediate discontinuation of the causative agents and full dosage of corticosteroids at an early stage is the key principle in the management of SJS/TEN. Intravenous immunoglobulin has also been recommended in recent years. Recent researches stressed the need of intensive supportive care, aiming at satisfying the nutritional requirements, maintaining electrolyte balance, and preventing severe secondary infection. Plasmapheresis is considered to be a safe intervention to treat extremely ill TEN patients. Through plasmapheresis, circulating antigens, autoantibodies, immune complexes, and other toxic substances can be removed, leading to shorter periods and less severity of the disease. This is the first report of methazolamide-induced TEN of a Chinese Han female with positive HLA-B5901 typing, which strongly suggests a possible relationship between HLA-B5901 and methazolamide-induced SJS/TEN. HLA-B5901 could be a useful marker to predict methazolamide-induced SJS/TEN in Chinese or Han people. Financial support and sponsorship Nil. Conflicts of interest The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Monitoring and controlling the freezing process and thermal properties of foods is an important means to understand and maintain product quality. Saccharides were used in this study to regulate the gelation of liquid egg yolks induced by freeze‒thawing; the selected saccharides included sucrose, L-arabinose, xylitol, trehalose, D-cellobiose, and xylooligosaccharides. The regulatory effects of saccharides on frozen egg yolks were investigated by characterizing their thermal and rheological properties and structural changes. The results showed that L-arabinose and xylitol were effective gelation regulators. After freeze‒thawing, the sugared egg yolks exhibited a lower consistency index and fewer rheological units than those without saccharides, indicating controlled gelation. Weaker aggregation of egg yolk proteins was confirmed by smaller aggregates observed by confocal laser scanning microscopy and smaller particle sizes. Saccharides alleviated the freeze-induced conversion of α-helices to β-sheets in egg yolk proteins, exposing fewer Trp residues. Overall, L-arabinose showed the greatest improvement in regulating the gelation of egg yolks, followed by xylitol, which is correlated with its low molecular weight.
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