Highly efficient rescue of dengue virus using a co-culture system with mosquito/mammalian cells

The production rate of dengue viruses (DENVs), especially low-passage virus isolates, is low, and, therefore, the isolates are generally used only after several passages. However, in vitro passages could induce mutation(s). In this study, we established a system for the characterization of low-passage viral isolates using an infectious cDNA clone. We used R05-624, a plaque derived from type 2 (DENV-2) Thai strain, for the construction of the cDNA clone, named pmMW/R05-624. We found that transfection of both of mammalian Vero cells and mosquito C6/36 cells with viral RNA derived from the cDNA clone produced a significant amount of progeny virus: 3.2 × 10⁶ focus-forming units (FFU) production per ml of cultured fluid only 3 days after transfection with 2 μg RNA. Conversely, no detectable level of viruses was produced by conventional methods using a single cell line, Vero or C6/36. When this system was applied for the characterization of eight low-passage clinical viral isolates by placing their 5′-half or 3′-half in the above cDNA clone, we found that all the isolates, except for L04-225, produced similar levels of progeny virus. Among a total of eight cDNA clones reconstructed with the NS4A-3′NCR region derived from L04-225, one clone carried an insertion and produced a low level of progeny virus. Thus, our system to efficiently rescue clinical samples or low-passage viral isolates could be useful for assessing the virological and molecular characteristics of DENV that could be related to disease pathogenesis.     

公立综合性医院在突发灾难事件医疗救援中的伦理实践

突发灾难中的医疗救援不同于常规的医疗救治,其具有显著的特殊性。而这种特殊性不仅体现在救援组织、医疗技术上,还体现在公立医院医疗救援应遵守的伦理原则上。突发灾难事件下公立综合性医院应突破因情况特殊性而带来的医疗救援伦理局限性,使其得到充分合理转化和运用。     

Interspecific hybridization of <Emphasis Type="Italic">Prunus persica</Emphasis> with <Emphasis Type="Italic">P. armeniaca</Emphasis> and <Emphasis Type="Italic">P. salicina</Emphasis> using embryo rescue

Embryo rescue technique was used successfully to produce interspecific hybrids by crossing peach (P. persica) as a female parent with apricot (P. armeniaca) and plum (P. salicica). In those crosses that had ‘Yuhualu’ or ‘Zhonghuashoutao’ as female parents, hybrid embryos aborted from the 7th or 8th week after pollination mainly due to post-pollination incompatibility. An embryo rescue protocol was established to rescue such embryos and recover hybrid plants. Modified half-strength MS medium containing 4 mg l⁻¹ 6-BA and 0.5 mg l⁻¹ IBA produced up to 90% germination in the embryos. Modified MS medium with 1.0 mg l⁻¹ 6-BA and 1.0 mg l⁻¹ IBA gave the highest bud induction and multiplication whereas modified MS medium containing 0.5 mg l⁻¹ IAA and 0.2 mg l⁻¹ NAA gave the best rooting percentage. All the hybrids obtained using this embryo rescue technique were verified using simple sequence repeat (SSR) markers. A series of pollen treatments were carried out to partially overcome pre-pollination incompatibility, and it was found accidentally that pollen treatment with electrostatic field not only improved pollen germination but also increased the multiplication coefficient of embryo-induced shoots.     

无核葡萄胚挽救影响因素的研究

分别以‘底莱特’和‘大粒红无核’自交、‘红宝石无核’ב新郁’和‘爱莫无核’ב黑奥林’胚珠为材料进行无核葡萄胚挽救影响因素的研究。结果显示:(1)‘底莱特’自交和‘红宝石无核’ב新郁’胚珠剥胚后萌发率分别为11.4%和10.0%,高于横切胚珠情况下的萌发率(5.5%、0.6%);(2)用Nitsch培养基培养的‘大粒红无核’自交和‘爱莫无核’ב黑奥林’胚珠的胚萌发率分别为9.5%和5.6%,胚状体诱导率分别为6.3%和3.9%,明显高于ER培养基的萌发率。结果表明:剥胚有利于无核葡萄胚的萌发;Nitsch比ER培养基更有利于胚萌发和胚状体诱导;同时,由于杂交亲本染色体倍性的差异导致胚萌发率不同,表现为:二倍体无核自交>二倍体无核×二倍体有核>二倍体无核×四倍体有核。     

Gene flow, effective population size and selection at major histocompatibility complex genes: brown trout in the Hardanger Fjord, Norway

Brown trout populations in the Hardanger Fjord, Norway, have declined drastically due to increased exposure to salmon lice from salmonid aquaculture. We studied contemporary samples from seven populations and historical samples (1972 and 1983) from the two largest populations, one of which has declined drastically whereas the other remains stable. We analysed 11 microsatellite loci, including one tightly linked to the UBA gene of the major histocompatibility class I complex (MHC) and another locus linked to the TAP2A gene, also associated with MHC. The results revealed asymmetric gene flow from the two largest populations to the other, smaller populations. This has important conservation implications, and we predict that possible future population recoveries will be mediated primarily by the remaining large population. Tests for selection suggested diversifying selection at UBA, whereas evidence was inconclusive for TAP2A. There was no evidence for temporally fluctuating selection. We assessed the distribution of adaptive divergence among populations. The results showed the most pronounced footprints of selection between the two largest populations subject to the least immigration. We suggest that asymmetric gene flow has an important influence on adaptive divergence and constrains local adaptive responses in the smaller populations. Even though UBA alleles may not affect salmon louse resistance, the results bear evidence of adaptive divergence among populations at immune system genes. This suggests that similar genetic differences could exist at salmon louse resistance loci, thus rendering it a realistic scenario that differential population declines could reflect differences in adaptive variation.     

梅与山桃远缘杂交亲和性初步研究

为培育早花抗寒梅花新品种,以梅(Prunus mume)品种‘江梅’(P.mume‘Jiangmei’)、‘淡丰后’(P.mume‘Dan Fenghou’)与山桃(P.davidiana)、‘白花’山桃(P.davidiana‘Alba’)为亲本进行杂交试验,记录种间杂交结实率,观察花粉管生长,对未成熟胚进行培养。结果表明:(1)梅与山桃、‘白花’山桃杂交结实率很低,‘江梅’ב白花’山桃未结果,结实率最高的组合为‘淡丰后’×山桃,也仅有7.4%,且杂交果实的果核内部分胚干瘪、败育。(2)山桃和‘白花’山桃的花粉在梅柱头上都能正常萌发,但花粉管生长受抑制,多数花粉管到达花柱中部即弯曲、缠绕、断裂,花粉管生长过程中有大量的胼胝质产生,表现较低的杂交亲和性,但不同种间杂交亲和程度又有所不同。(3)通过未成熟胚培养获得了杂种苗。研究表明,梅与同属种杂交存在不亲和性,幼胚拯救是获得梅与李属其他种远缘杂交杂种苗的有效途径。     

Consequences of interspecific hybridization and virus infection on the growth and fecundity of three threatened coastal Lepidium (Brassicaceae) species from New Zealand

Lepidium castellanum, L. juvencum and L. oleraceum are threatened coastal cresses endemic to New Zealand. These three species were selfed and interspecific hybrids generated for examination of hybrid fitness and inbreeding depression. In controlled glasshouse experiments, the interspecific hybrids and selfed progeny were inoculated with a strain of the introduced Turnip mosaic virus (TuMV) previously isolated from wild populations of L. aegrum. Experiments tested the hypothesis that heterosis in the interspecific hybrids provides a gain in TuMV resistance in comparison to selfed plants. We show that interspecific hybrids of three genetically distinct species of Lepidium increased plant performance and reduced susceptibility to the effects of the TuMV. We suggest that interspecific hybridization could be implemented as a conservation management strategy and that a broader outlook may be required to mitigate the negative impacts of introduced pathogens on threatened species.     

医学干预对武警某部军事训练伤的影响

目的：探讨医学干预对南方某武警部队战士训练伤发生情况的影响。方法：随机将南方某武警部队的一大队,按照干预措施施训,作为干预组;另一大队按照以往常规方法进行训练,不加任何干预措施,作为对照组。观察6个月内的两大队（干预组和对照组）的训练伤发生情况。结果：骨关节和软组织损伤为常见。实施医疗干预可有效降低军事训练伤的发生率（17．95％〈29．18％）,其主要效果在于大幅度降低了骨关节损伤的发生率（6．53％〈10.31％）和软组织损伤的发生率（5．59％〈9．92％）。结论：实施医学干预可有效降低我武警部队军事训练伤的发生率。     

Studies on the specificity of unprocessed and mature forms of phytanoyl-CoA 2-hydroxylase and mutation of the iron binding ligands

The mature form of phytanoyl-coenzyme A 2-hydroxylase (PAHX), a nonheme Fe(II)- and 2-oxoglutarate-dependent oxygenase, catalyzes the alpha-hydroxylation of phytanoyl-CoA within peroxisomes. Mutations in PAHX result in some forms of adult Refsum's disease. Unprocessed PAHX (pro-PAHX) contains an N-terminal peroxisomal targeting sequence that is cleaved to give mature PAHX (mat-PAHX). Previous studies have implied a difference in the substrate specificity of the unprocessed and mature forms of PAHX. We demonstrate that both forms are able to hydroxylate a range of CoA derivatives, but under the same assay conditions, the N-terminal hexa-His-tagged unprocessed form is less active than the nontagged mature form. Analyses of the assay conditions suggest a rationale for the lack of activity previously reported for some substrates (e.g. isovaleryl-CoA) for the (His)6pro-PAHX. Site-directed mutagenesis was used to support proposals for the identity of the iron binding ligands (His-175, Asp-177, His-264) of the 2-His-1-carboxylate motif of PAHX. Mutation of other histidine residues (His-213, His-220, His-259) suggested that these residues were not involved in Fe(II) binding.     

The particle inflow gun can be used to co-transform Paramecium using Tungsten particles

A particle inflow gun (PIG) was constructed and tested for its utility to transform Paramecium using tungsten or gold as the DNA carrier particle. In the first set of experiments we transformed Paramecium with a plasmid containing the neomycin-resistance gene, obtaining a transformation efficiency of 0.31+/-0.14% (mean+/-SD) for tungsten particles and 1.30+/-0.29% for gold particles. Plasmid DNA precipitated upon tungsten was shown to be stable for transformation purposes for up to 1 h prior to use and had no detectable effects on transformation efficiency. In addition, we demonstrated that at high frequency (71+/-20%) a Paramecium mutant strain could be phenotypically rescued by co-transformation with a second plasmid containing the selectable neomycin-resistance gene. The PIG coupled with tungsten particles as the carrier offers a low-cost alternative for biolistic transformation of Paramecium.