[Mechanism of reduced insecticidal protein expression in Bt cotton under high temperature and drought based on proteomics.] / 基于蛋白质组学的高温干旱下Bt棉杀虫蛋白表达下降机制.

To provide theoretical basis for the safety of insecticidal efficiency in Bt cotton, the effects of high temperature and drought stress on insecticidal protein expression and protein diffe-rently expression profile was studied. In this study, the Bt cotton cultivar Sikang 3 was used as expe-rimental material, with two treatments (40% field capacity and 38 ℃, HD, and 60% field capacity and 32 ℃, CK). Differences in proteomics of Bt cotton between HD and CK were compared using label-free quantitative proteomics technology. The results showed that high temperature and drought caused a significant reduction of insecticidal protein content in bolls, with a decrease of 38.2 ng·g-1 FM. The analysis of differential protein expression by label-free quantitative proteomic approach showed that 83 proteins were significantly up-regulated, but 104 proteins were significantly down-regulated in HD stressed cotton plants compared with CK. 122 new proteins were detected and 167 proteins expression was not observed under stressed conditions. Results from the enrichment analysis of differently expressed protein between two treatments showed that 14 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways were affected under stress. Three KEGG pathways were related to the Bt protein synthesis and degradation: carbohydrate digestion and absorption pathway, protein export pathway, and protein processing in endoplasmic reticulum pathway. In the carbohydrate digestion and absorption pathway, the starch hydrolysis ability of HD treated cotton plants increased, while the ability to phosphorylate the hexoses, fructose and glucose decreased. In protein export pathway, the peptide synthesis in HD treatment was not significantly affected, while the process of transferring peptides into the endoplasmic reticulum was prohibited. In the protein processing in endoplasmic reticulum pathway, the ability of ubiquitin mediated proteolysis was increased in HD treatment.

A Trichophyton Rubrum Infection Model Based on the Reconstructed Human Epidermis - Episkin®.

BACKGROUND: Trichophyton rubrum represents the most common infectious fungus responsible for dermatophytosis in human, but the mechanism involved is still not completely understood. An appropriate model constructed to simulate host infection is the prerequisite to study the pathogenesis of dermatophytosis caused by T. rubrum. In this study, we intended to develop a new T. rubrum infection model in vitro, using the three-dimensional reconstructed epidermis - EpiSkin ®, and to pave the way for further investigation of the mechanisms involved in T. rubrum infection. METHODS: The reconstructed human epidermis (RHE) was infected by inoculating low-dose (400 conidia) and high-dose (4000 conidia) T. rubrum conidia to optimize the infection dose. During the various periods after infection, the samples were processed for pathological examination and scanning electron microscopy (SEM) observation. RESULTS: The histological analysis of RHE revealed a fully differentiated epidermis with a functional stratum corneum, which was analogous to the normal human epidermis. The results of hematoxylin and eosin staining and the periodic acid-Schiff staining showed that the infection dose of 400 conidia was in accord with the pathological characteristics of host dermatophytosis caused by T. rubrum. SEM observations further exhibited the process of T. rubrum infection in an intuitionistic way. CONCLUSIONS: We established the T. rubrum infection model on RHE in vitro successfully. It is a promising model for further investigation of the mechanisms involved in T. rubrum infection.

Effect of Culture Supernatant Derived from Trichophyton Rubrum Grown in the Nail Medium on the Innate Immunity-related Molecules of HaCaT.

BACKGROUND: Trichophyton rubrum is superficial fungi characteristically confined to dead keratinized tissues. These observations suggest that the soluble components released by the fungus could influence the host immune response in a cell in contact-free manner. Therefore, this research aimed to analyze whether the culture supernatant derived from T. rubrum grown in the nail medium could elicit the immune response of keratinocyte effectively. METHODS: The culture supernatants of two strains (T1a, T XHB ) were compared for the ß-glucan concentrations and their capacity to impact the innate immunity of keratinocytes. The ß-glucan concentrations in the supernatants were determined with the fungal G-test kit and protein concentrations with bicinchoninic acid protein quantitative method, then HaCaT was stimulated with different concentrations of culture supernatants by adopting morphological method to select a suitable dosage. Expressions of host defense genes were assessed by quantitative polymerase chain reaction after the HaCaT was stimulated with the culture supernatants. Data were analyzed with one-way analysis of variance, followed by the least significant difference test. RESULTS: The T. rubrum strains (T1a and T XHB ) released ß-glucan of 87.530 ± 37.581 pg/ml and 15.747 ± 6.453 pg/ml, respectively into the media. The messenger RNA (mRNA) expressions of toll-like receptor-2 (TLR2), TLR4, and CARD9 were moderately up-regulated in HaCaT within 6-h applications of both supernatants. HaCaT cells were more responsive to T1a than T XHB . The slight increase of dendritic cells-specific intercellular adhesion molecule 3-grabbing nonintegrin expression was faster and stronger, induced by T1a supernatant than T XHB . The moderate decreases of RNase 7, the slight up-regulations of Dectin-1 and interleukin-8 at the mRNA level were detected only in response to T1a rather than T XHB . After a long-time contact, all the elevated defense genes decreased after 24 h. CONCLUSION: The culture supernatant of T. rubrum could directly and transiently activate the innate immune response of keratinocytes.

[Evolution of hepatitis B virus quasispecies during antiviral therapy in one chronic hepatitis B patient].

OBJECTIVE: To investigate the evolution of hepatitis B virus (HBV) quasispecies in one patient during lamivudine (LAM) monotherapy and switching to entecavir (ETV) rescue treatment. METHODS: Serum samples were taken at seven different time points during antiviral therapy (0, 24, 48, 60, 72, 96, 152 weeks, respectively), the HBV DNA polymerase gene was amplified, cloned and sequenced to analyze the amino acid substitutions within HBV DNA polymerase gene and distribution of virus quasispecies. Quantitative detection of the HBV wild strains and total virus was performed by amplification refractory mutation system real-time PCR (ARMS-PCR). RESULTS: Three mutation patterns detected during antiviral therapy in the patient: rtM204V, rtM204V+rtL180M and rtM204I. The HBV quasispecies were found always in dynamic variation. The HBV populations were completely replaced with the LAM-resistant variants when the viral breakthrough was encountered during LAM monotherapy. Interestingly, the wild-type variants presented gradually dominant (79.3%) with the decline of HBV DNA load after switching to ETV rescue administration. ARMS-PCR results showed that the wild-type variants account ed for 68.55% of the HBV populations at baseline and this proportion declined to 0.21% when the viral breakthrough emerged under LAM therapy. The wild-type variants gradually increased from week 24 after switching to ETV rescue therapy and the proportion of HBV wild-type variants in the population fluctuated between 16.01% to 26.93%. CONCLUSIONS: The distribution of virus quasispecies were always in dynamic variation during sequential therapy with nucleotide analogs in chronic hepatitis B patients. Different patterns of dynamic HBV quasispecies may have different contribution in ETV resistance in LMV refractory patients with ETV administration.