昆虫病原菌(Isaria fumosorosea)查尔酮异构酶基因CHI克隆及生物信息学分析

本研究旨在对昆虫病原菌玫烟色棒束孢(Isaria fumosorosea)查尔酮异构酶基因CHI进行克隆、测序及相关生物信息学分析.序列分析结果显示:IfCHI基因编码区全长为1152bp,编码383个氨基酸,理论等电点(pI)D为9.72,属不稳定水溶性蛋白,其二级结构为混合型,蛋白质溶剂可及性主要分为三类,三级结构由α-螺旋、β-折叠、β-转角和无规则卷曲组成,同源序列分析及多重序列比对分析存在明显的差异.该昆虫病原菌玫烟色棒束孢IfCHI编码基因的成功克隆及生物信息学分析,为进一步研究病原菌CHI基因的遗传特性和表达机制以及为寻找更多的查尔酮类化合物提供了理论基础.     

Human recombinant IL-10 reduces xenogenic cytotoxicity via macrophage M2 polarization

Xenotransplantation has been considered an alternative to the moderate shortage of donor organs for transplantation. To achieve successful xenotransplatation, there is the need to overcome immune rejection. Although, hyperacute rejection has been overcome by α1,3-galactosyltransferase knockout pig, cellular immune rejection remains as a subsequent barrier. Interleukin-10 (IL-10) is known as an anti-inflammatory and immunomodulatory cytokine which has been shown to limit inflammatory responses by inhibiting macrophage activation in several animal experiments. To study the effect of human IL-10 (hIL-10) on pig-to-human xenotransplantation, porcine kidney epithelial cell line (PK(15)) expressing hIL-10 was established. The cytotoxicity of macrophages decreased by hIL-10 from transgenic cells. Furthermore, there is a decreased production of pro-inflammatory cytokines, tumor necrosis factor-α and interleukin-23, and increased anti-inflammatory cytokines like IL-10, but not transforming growth factor beta, in the presence of hIL-10. Also, macrophage polarization toward M2-like phenotype were induced by hIL-10 from transgenic PK(15) cells. Finally, we suggest that the cytotoxicity of human macrophages was reduced by hIL-10 from transgenic cells, inducing M2-like macrophage polarization. Therefore, these results show that hIL-10 transgenic pig can be used as a model to overcome acute immune rejection in pig-to-human xenotransplantation.     

Genomic Consequences of Long-Term Population Decline in Brown Eared Pheasant

Population genetic theory and empirical evidence indicate that deleterious alleles can be purged in small populations. However, this viewpoint remains controversial. It is unclear whether natural selection is powerful enough to purge deleterious mutations when wild populations continue to decline. Pheasants are terrestrial birds facing a long-term risk of extinction as a result of anthropogenic perturbations and exploitation. Nevertheless, there are scant genomics resources available for conservation management and planning. Here, we analyzed comparative population genomic data for the three extant isolated populations of Brown eared pheasant (Crossoptilon mantchuricum) in China. We showed that C. mantchuricum has low genome-wide diversity and a contracting effective population size because of persistent declines over the past 100,000 years. We compared genome-wide variation in C. mantchuricum with that of its closely related sister species, the Blue eared pheasant (C. auritum) for which the conservation concern is low. There were detrimental genetic consequences across all C. mantchuricum genomes including extended runs of homozygous sequences, slow rates of linkage disequilibrium decay, excessive loss-of-function mutations, and loss of adaptive genetic diversity at the major histocompatibility complex region. To the best of our knowledge, this study is the first to perform a comprehensive conservation genomic analysis on this threatened pheasant species. Moreover, we demonstrated that natural selection may not suffice to purge deleterious mutations in wild populations undergoing long-term decline. The findings of this study could facilitate conservation planning for threatened species and help recover their population size.     

Distinct Modes of Transmission of Tuberculosis in Aboriginal and Non-Aboriginal Populations in Taiwan

Tuberculosis incidence among aborigines is significantly higher than for Han Chinese in Taiwan, but the extent to which Mycobacterium tuberculosis (MTB) strain characteristics contribute to this difference is not well understood. MTB isolates from aborigines and Han Chinese living in eastern and southern Taiwan, the major regions of aborigines, were analyzed by spoligotyping and 24-loci MIRU-VNTR. In eastern Taiwan, 60% of aboriginal patients were ≤20 years old, significantly younger than the non-aboriginal patients there; aborigines were more likely to have clustered MTB isolates than Han Chinese (odds ratio (OR) = 5.98, p<0.0001). MTB lineages with high clustering were EAI (54.9%) among southern people, and Beijing (62.5%) and Haarlem (52.9%) among eastern aborigines. Resistance to first-line drugs and multidrug resistance (MDR) were significantly higher among eastern aborigines (≥15%) than in any other geographic and ethnic group (p<0.05); MDR was detected in 5 of 28 eastern aboriginal patients ≤20 years old. Among patients from the eastern region, clustered strains (p = 0.01) and aboriginal ethnicity (p = 0.04) were independent risk factors for MDR. The lifestyles of aborigines in eastern Taiwan may explain why the percentage of infected aborigines is much higher than for their Han Chinese counterparts. The significantly higher percentage of the MDR-MTB strains in the aboriginal population warrants close attention to control policy and vaccination strategy.     

CFS-1686 Causes Cell Cycle Arrest at Intra-S Phase by Interference of Interaction of Topoisomerase 1 with DNA

CFS-1686 (chemical name (E)-N-(2-(diethylamino)ethyl)-4-(2-(2-(5-nitrofuran-2-yl)vinyl)quinolin-4-ylamino)benzamide) inhibits cell proliferation and triggers late apoptosis in prostate cancer cell lines. Comparing the effect of CFS-1686 on cell cycle progression with the topoisomerase 1 inhibitor camptothecin revealed that CFS-1686 and camptothecin reduced DNA synthesis in S-phase, resulting in cell cycle arrest at the intra-S phase and G1-S boundary, respectively. The DNA damage in CFS-1686 and camptothecin treated cells was evaluated by the level of ATM phosphorylation, γH2AX, and γH2AX foci, showing that camptothecin was more effective than CFS-1686. However, despite its lower DNA damage capacity, CFS-1686 demonstrated 4-fold higher inhibition of topoisomerase 1 than camptothecin in a DNA relaxation assay. Unlike camptothecin, CFS-1686 demonstrated no activity on topoisomerase 1 in a DNA cleavage assay, but nevertheless it reduced the camptothecin-induced DNA cleavage of topoisomerase 1 in a dose-dependent manner. Our results indicate that CFS-1686 might bind to topoisomerase 1 to inhibit this enzyme from interacting with DNA relaxation activity, unlike campothecin''s induction of a topoisomerase 1-DNA cleavage complex. Finally, we used a computer docking strategy to localize the potential binding site of CFS-1686 to topoisomerase 1, further indicating that CFS-1686 might inhibit the binding of Top1 to DNA.     

Validation of a chloroquine-induced cell death mechanism for clinical use against malaria

An alternative antimalarial pathway of an ‘outdated'' drug, chloroquine (CQ), may facilitate its return to the shrinking list of effective antimalarials. Conventionally, CQ is believed to interfere with hemozoin formation at nanomolar concentrations, but resistant parasites are able to efflux this drug from the digestive vacuole (DV). However, we show that the DV membrane of both resistant and sensitive laboratory and field parasites is compromised after exposure to micromolar concentrations of CQ, leading to an extrusion of DV proteases. Furthermore, only a short period of exposure is required to compromise the viability of late-stage parasites. To study the feasibility of this strategy, mice malaria models were used to demonstrate that high doses of CQ also triggered DV permeabilization in vivo and reduced reinvasion efficiency. We suggest that a time-release oral formulation of CQ may sustain elevated blood CQ levels sufficiently to clear even CQ-resistant parasites.Along with improvements in vector control, surveillance/diagnosis and treatment accessibility, the development of new drugs to counteract the problem of drug resistance remains integral to the eradication agenda.¹ Efforts to develop novel antimalarials have been promising,^{2, 3} and drugs designed specifically to reverse drug resistance are also being uncovered.⁴ However, novel chemical entities are expensive to test and take considerable time before they can be deployed. In comparison, alternative strategies to fully exploit the existing arsenal of antimalarials (largely already affordable and accessible) are likely to be relatively expedient and cost-effective.We had previously demonstrated the existence of a novel parasite programmed cell death (PCD) mechanism that was induced by high concentrations of chloroquine (CQ) and shown that clan CA cysteine proteases were key mediators of the pathway.⁵ We had also observed that the permeabilization of the parasite digestive vacuole (DV) was an important upstream trigger of this pathway and that other lysosomotropic compounds that are not parasite-specific could similarly destabilize the DV to initiate parasite PCD.⁶ We hypothesize that by altering the dosing regimen or formulation of CQ, it might be possible to reinstate CQ into antimalarial chemotherapy by making use of this novel mechanism.⁷In this present study, we begin by showing evidence that CQ treatment is able to result in the extrusion of DV proteases into the parasite cytoplasm. Second, we validate the existence of this PCD pathway in multiple laboratory strains and field isolates to suggest its clinical relevance and universality. Third, we investigate the minimum concentration and duration required for CQ to trigger PCD to determine if the pharmacokinetics of the current CQ regimen might be suitable for initiating PCD. Finally, we make use of two murine malaria models to demonstrate that a short exposure to high levels of CQ is able to induce parasite DV permeabilization in vivo and that this procedure reduces parasite viability.     

Chlorination of cellulose with N-chlorosuccinimide-triphenylphosphine under homogeneous conditions in lithium chloride-N,N-dimethylacetamide.

Microcrystalline cellulose was chlorinated with N-chlorosuccinimide-triphenylphosphine under homogeneous conditions in LiCl-N,N-dimethylacetamide. At the early stage of the reaction only replacement of the 6-hydroxyl groups with chlorine was observed, and 3-hydroxyl groups were replaced at a lower rate with Walden inversion. The effects of reaction conditions on the extent of chlorination were studied in detail. More than two equivalents of chlorination reagents per glucose residue were necessary to attain a high degree of substitution (ds) by chlorine, and the maximum ds attained was 1.86. Chlorinated disaccharides were found in the hydrolyzates of chlorodeoxycelluloses hydrolyzed under mild conditions, and their structures were studied by mass spectrometry.     

Intracellular cholesterol mobilization involved in the ABCA1/apolipoprotein-mediated assembly of high density lipoprotein in fibroblasts

Differential regulation has been suggested for cellular cholesterol and phospholipid release mediated by apolipoprotein A-I (apoA-I)/ABCA1. We investigated various factors involved in cholesterol mobilization related to this pathway. ApoA-I induced a rapid decrease of the cellular cholesterol compartment that is in equilibrium with the ACAT-accessible pool in cells that generate cholesterol-rich HDL. Pharmacological and genetic inactivation of ACAT enhanced the apoA-I-mediated cholesterol release through upregulation of ABCA1 and through cholesterol enrichment in the HDL generated. Pharmacological activation of protein kinase C (PKC) also decreased the ACAT-accessible cholesterol pool, not only in the cells that produce cholesterol-rich HDL by apoA-I (i.e., human fibroblast WI-38 cells) but also in the cells that generate cholesterol-poor HDL (mouse fibroblast L929 cells). In L929 cells, the PKC activation caused an increase in apoA-I-mediated cholesterol release without detectable change in phospholipid release and in ABCA1 expression. These results indicate that apoA-I mobilizes intracellular cholesterol for the ABCA1-mediated release from the compartment that is under the control of ACAT. The cholesterol mobilization process is presumably related to PKC activation by apoA-I.     

Calicivirus 3C-like proteinase inhibits cellular translation by cleavage of poly(A)-binding protein

Caliciviruses are single-stranded RNA viruses that cause a wide range of diseases in both humans and animals, but little is known about the regulation of cellular translation during infection. We used two distinct calicivirus strains, MD145-12 (genus Norovirus) and feline calicivirus (FCV) (genus Vesivirus), to investigate potential strategies used by the caliciviruses to inhibit cellular translation. Recombinant 3C-like proteinases (r3CL(pro)) from norovirus and FCV were found to cleave poly(A)-binding protein (PABP) in the absence of other viral proteins. The norovirus r3CL(pro) PABP cleavage products were indistinguishable from those generated by poliovirus (PV) 3C(pro) cleavage, while the FCV r3CL(pro) products differed due to cleavage at an alternate cleavage site 24 amino acids downstream of one of the PV 3C(pro) cleavage sites. All cleavages by calicivirus or PV proteases separated the C-terminal domain of PABP that binds translation factors eIF4B and eRF3 from the N-terminal RNA-binding domain of PABP. The effect of PABP cleavage by the norovirus r3CL(pro) was analyzed in HeLa cell translation extracts, and the presence of r3CL(pro) inhibited translation of both endogenous and exogenous mRNAs. Translation inhibition was poly(A) dependent, and replenishment of the extracts with PABP restored translation. Analysis of FCV-infected feline kidney cells showed that the levels of de novo cellular protein synthesis decreased over time as virus-specific proteins accumulated, and cleavage of PABP occurred in virus-infected cells. Our data indicate that the calicivirus 3CL(pro), like PV 3C(pro), mediates the cleavage of PABP as part of its strategy to inhibit cellular translation. PABP cleavage may be a common mechanism among certain virus families to manipulate cellular translation.     

Role of kinetic intermediates in the folding of leech carboxypeptidase inhibitor

The oxidative folding and reductive unfolding pathways of leech carboxypeptidase inhibitor (LCI; four disulfides) have been characterized in this work by structural and kinetic analysis of the acid-trapped folding intermediates. The oxidative folding of reduced and denatured LCI proceeds rapidly through a sequential flow of 1-, 2-, 3-, and 4-disulfide (scrambled) species to reach the native form. Folding intermediates of LCI comprise two predominant 3-disulfide species (designated as III-A and III-B) and a heterogeneous population of scrambled isomers that consecutively accumulate along the folding reaction. Our study reveals that forms III-A and III-B exclusively contain native disulfide bonds and correspond to stable and partially structured species that interconvert, reaching an equilibrium prior to the formation of the scrambled isomers. Given that these intermediates act as kinetic traps during the oxidative folding, their accumulation is prevented when they are destabilized, thus leading to a significant acceleration of the folding kinetics. III-A and III-B forms appear to have both native disulfides bonds and free thiols similarly protected from the solvent; major structural rearrangements through the formation of scrambled isomers are required to render native LCI. The reductive unfolding pathway of LCI undergoes an apparent all-or-none mechanism, although low amounts of intermediates III-A and III-B can be detected, suggesting differences in protection against reduction among the disulfide bonds. The characterization of III-A and III-B forms shows that the former intermediate structurally and functionally resembles native LCI, whereas the III-B form bears more resemblance to scrambled isomers.