[Research progress on the mechanisms of delayed encephalopathy in acute carbon monoxide poisoning].

In our country, there are a large number of carbon monoxide poisoning patients every winter. 10% to 30% of patients with acute carbon monoxide poisoning develop acute carbon monoxide poisoning delayed encephalopathy after a "false recovery period" of about 2 to 60 days. The morbidity and mortality rates of the disease are extremely high, but there is still no effective treatment for this condition. The pathogenesis of the disease is complex, and there is no clear conclusion yet. After consulting a large number of recent relevant literatures, this article reviews the main research results of the pathogenesis of the disease so far, with an aim to facilitate its early clinical diagnosis and correct treatment.

Construction and expression of prokaryotic expression vectors fused with genes of Magnaporthe oryzae effector proteins and mCherry.

The aim of the current study was to investigate the prokaryotic expression of the Magnaporthe oryzae effector genes BAS1 and BAS4 fused to the fluorescent protein mCherry. Based on previous polymorphic analysis of BAS1 and BAS4 in rice blast strains using PCR, blast strains containing the PCR products of BAS1 and BAS4 were selected for liquid culture for total RNA extraction. For PCR analysis, cDNA was selected as a template to amplify the coding region of BAS1 and BAS4, the plasmid pXY201 was selected as template to amplify the mCherry sequence, and the three sequences were cloned into pMD®19-T vectors. Positive recombinant plasmids were digested using two restriction enzymes and the cleaved fragments of BAS1 and mCherry and BAS4 and mCherry were ligated to pGEX-4T-1 vectors and expression was induced using IPTG. The PCR results showed that the sequence sizes of BAS1, BAS4, and mCherry were 348, 309, and 711 bp, respectively, and these were cloned into pMD®19-T vectors. After digestion and gel purification, the fragments of BAS1 and mCherry, BAS4 and mCherry were ligated into pGEX-4T-1 vectors and expressed in Escherichia coli BL21 competent cells. The expressed proteins were approximately 60 kDa, corresponding to their theoretical size. Prokaryotic expression products of BAS1 and BAS4 fused to mCherry were presented in this study, providing a base for constructing prokaryotic expression vectors of pathogen effector genes fused to mCherry, which will contribute to further study of the subcellular localization, function, and protein interactions of these effectors.

Construction of overexpression vectors of Magnaporthe oryzae genes BAS1 and BAS4 fusion to mCherry and screening of overexpression strains.

The aim of this study was to construct overexpression vectors and selecting strains of the Magnaporthe oryzae effectors BAS1 and BAS4. Primer pairs of BAS1, BAS4, and mCherry were designed based on their known nucleotide sequences. The coding sequences of BAS1 and BAS4 were amplified, and the pXY201 plasmid was selected as a template to amplify the mCherry sequence. Fragments of BAS1 and mCherry, and BAS4 and mCherry were ligated into the pCAMBIA1302 vector. The recombinant pCAMBIA-BAS1-mCherry and pCAMBIA-BAS4-mCherry plasmids were transformed into E. coli DH5α competent cells. Transformants were screened by PCR, and plasmids from the positive transformants were extracted by enzymatic digestion to obtain pCAMBIA-BAS1-mCherry and pCAMBIA-BAS4-mCherry. The pCAMBIA-BAS1-mCherry and pCAMBIA-BAS4-mCherry plasmids were transformed into protoplasts of rice blast strains and the transformed strains were screened by PCR using primer pairs against the hygromycin gene. The result showed that the PCR products corresponded with the theoretical sizes. RT-PCR was used to analyze the expression of BAS1 and BAS4 in five transformed strains of BAS1 and BAS4, and the result showed that the higher expression level of the two genes was occurred in five transformant strains comparing to wild-type strain A3467-40 (the strain containing BAS1 and BAS4), but there was no difference among the five overexpression strains. The sporulation and spore germination of transformed strains was higher than in wild type strain, and there was no difference in the germination time. Construction of overexpression vectors and strains of M. oryzae effectors BAS1 and BAS4 provide reference material for other new effectors.

Expression of Magnaporthe oryzae genes encoding cysteine-rich proteins secreted during nitrogen starvation and interaction with its host, Oryza sativa.

Previous studies have shown that the blast fungus, Magnaporthe oryzae, may experience nitrogen starvation during infection of its plant host (rice,Oryza sativa). Here, we studied the expression of seven genes encoding cysteine-rich proteins with N-terminal signal peptides during nitrogen limitation and throughout the infection process. Some genes were upregulated to a greater extent in weak pathogenic strains than in strong pathogenic strains when they were cultured in complete media, and the expression of some genes was higher in both weak and strong pathogenic strains cultured in 1/10-N and nitrogen starvation media. Furthermore, the expression of these genes was upregulated to different extents in the early stages of M. oryzae infection. These data demonstrate that the genes of interest are highly expressed in weak and strong pathogenic strains cultured under nitrogen limitation and at the early stage of the infection process. This indicates that cysteine-rich secreted proteins in the blast fungus might be involved in establishing disease in the host and that they are sensitive to nitrogen levels. Thus, their role in sensing nitrogen availability within the host is implied, which provides a basis for further functional identification of these genes and their products during plant infection.

Comparison of cytological adequacy in ultrasound-guided fine-needle aspiration of thyroid nodules with different numbers of needle passes.

OBJECTIVE: The aim of this study was to compare the cytological adequacy rates of different needle passes in ultrasound-guided fine-needle aspiration biopsy of thyroid nodules and, thus, to help establish the criterion for selecting the number of needle passes according to the characteristics of thyroid nodules. PATIENTS AND METHODS: This single-center and randomized prospective study involved 207 consecutive patients with 240 solid or predominantly solid thyroid nodules. These nodules were randomly divided into a 1-pass group, a 2-pass group, and a 3-pass group. Then the nodules were sent for cytopathological diagnosis, and cytological results were classified according to the Bethesda classification. Bethesda I was defined as inadequate, and Bethesda Ⅱ-Ⅵ were defined as adequate. Then the cytological adequacy rates of different groups were compared. RESULTS: In total, 221 nodule specimens were considered as adequate and 19 nodule specimens inadequate. The overall adequacy rate was 92.1%. However, there were no significant differences among the 1, 2, and 3-pass groups in terms of adequacy rates (91.3%, 92.5%, and 92.5%, respectively). CONCLUSIONS: The number of needle passes does not significantly affect the cytological adequacy in ultrasound-guided fine-needle aspiration of solid or predominantly solid thyroid nodules. The cytological adequacy of one-needle pass is comparable to those of two and three-needle passes.