PK-15细胞的ISG15基因敲除促进PRV的复制

本试验旨在研究干扰素刺激基因15（interferon-stimulated gene 15,ISG15）敲除对猪伪狂犬病病毒（PRV）复制的影响。通过CRISPR/Cas9技术构建猪ISG15基因敲除猪肾上皮（PK-15）细胞系,利用CCK-8试剂盒检测PK-15敲除ISG15基因对细胞活力的影响,采用间接免疫荧光技术检测PK-15以及PK15-ISG15^-/-细胞感染PRV的增殖差异,通过RT-qPCR检测PRV-EP0、PRV-gE、PRV-VP16和IFN-β的转录水平,Western blot检测PRV-gE和ISG15的蛋白表达水平,以及通过病毒噬斑检测对子代病毒感染力的影响。结果表明,sgRNA1和sgRNA2均成功敲除ISG15基因;CCK-8试剂盒检测细胞活力结果表明,敲除ISG15基因对PK-15细胞活力无影响;间接免疫荧光检测结果表明,PRV感染后,PK15-ISG15^-/-细胞中的荧光强度明显高于PK-15细胞;RT-qPCR和Western blot结果表明,敲除ISG15可以促进PRV的转录和蛋白表达;病毒噬斑试验进一步显示,敲除ISG15可以促进PRV的复制。另外,RT-qPCR结果显示,敲除ISG15可以抑制PRV感染引起的IFN-β转录上调。本研究成功构建了PK15-ISG15^-/-细胞系,并通过PRV感染试验证实ISG15基因可以抑制PRV在PK-15细胞中的增殖,并推测这种抑制作用可能与IFN通路有关。     

Nitrogen-fixation dynamics in a cut-and-carry silvopastoral system in the subhumid conditions of Guadeloupe, French Antilles

This paper summarizes several studies on N recycling in a tropical silvopastoral system for assessing the ability of the system to increase soil fertility and insure sustainability. We analyzed the N₂ fixation pattern of the woody legume component (Gliricidia sepium), estimated the recycling rate of the fixed N in the soil, and measured N outputs in tree pruning and cut grass (Dichanthium aristatum). With this information, we estimated the N balance of the silvopastoral system at the plot scale. The studies were conducted in an 11-year-old silvopastoral plot established by planting G. sepium cuttings at 0.3 m × 2 m spacing in natural grassland. The plot was managed as a cut-and-carry system where all the tree pruning residues (every 2-4 months) and cut grass (every 40-50 days) were removed and animals were excluded. No N fertilizer was applied. Dinitrogen fixation, as estimated by the ¹⁵N natural abundance method, ranged from 60-90% of the total N in aboveground tree biomass depending on season. On average, 76% of the N exports from the plot in tree pruning (194 kg [N] ha^–1 yr^–1) originated from N₂ fixation. Grass production averaged 13 Mg ha^–1 yr^–1 and N export in cut grass was 195 kg [N] ha^–1 yr^–1. The total N fixed by G. sepium, as estimated from the tree and grass N exports and the increase in soil N content, was about 555 kg [N] ha^–1 yr^–1. Carbon sequestration averaged 1.9 Mg [C] ha^–1 yr^–1 and soil organic N in the 0-0.2 m layer increased at a rate of 166 kg [N] ha^–1 yr^–1, corresponding to 30% of N₂ fixation by the tree. Nitrogen released in nodule turnover (10 kg [N] ha^–1 yr^–1) and litter decomposition (40 kg [N] ha^–1 yr^–1) contributed slightly to this increase, and most of the recycled N came from the turnover or the activity of other below-ground tree biomass than nodules. This revised version was published online in June 2006 with corrections to the Cover Date.     

Nitrogen contribution by multipurpose trees to rice and cowpea in an alley cropping system in Sierra Leone

In an alley cropping experiment, a study was carried out on N₂ fixation by Gliricidia sepium, nitrogen (N) accumulation by prunings of Gliricidia, Senna siamea (formerly Cassia siamea) and Gmelina arborea, and the N contribution to associated crops of rice and cowpea.Total N accumulated by the hedgerow trees ranged from 297–524 kg N ha^–1 on average but varied between tree species and depended on the growing season. Gliricidia sepium accumulated 370 kg N ha^–1 on average and more than half of this came from fixation. Senna siamea and Gmelina arborea served as reference trees for estimating N₂ fixation. The estimates of N₂ fixation using Gmelina as a reference gave higher estimates than those using Senna.Although the dry matter and nitrogen yields of prunings from the hedgerow trees were high, their relative nitrogen contribution to the associated crops was generally low ranging from 5 to 29%. Higher crop yields and nitrogen contribution were observed with Gliricidia sepium prunings. The low N contribution from prunings was attributed to the lack of synchronization between the N released from the prunings and the crop's demand for N.     

Biological nitrogen fixing capacity and biomass production of different understorey pastures in a Pinus radiata-pasture agroforestry system in New Zealand

Quantitative field measurements of biological nitrogen fixation (BNF) and biomass production by four different understorey pastures in a Pinus radiata-pasture agroforestry system were determined over a period of one year. The trees were two years old at the beginning of this study and the understorey pastures were being cut and removed for silage. The BNF was determined using the ¹⁵N dilution technique. Pastures of ryegrass+clover, cocksfoot+clover, phalaris+clover and lucerne were used. Substantial amounts of BNF were found (71 to 230 kg N ha^–1 year^–1) with lucerne showing the highest N fixation. However, lucerne derived only 71 to 72% of its N from the atmosphere (%Ndfa) during the spring/summer period compared to 83–97% with clovers, thus the net N demand from the soil was substantially higher with lucerne. This caused increased N stress to the trees. Clover in ryegrass+clover pasture fixed more N than the other grass+clover pastures. Although pasture position in relation to trees did not affect annual pasture total DMY and %Ndfa, pastures north of tree row grew better than those in other positions. Trees significantly affected the BNF of legumes and the botanical composition of pastures with highest BNF and legume production occurring in pastures midway between two rows of trees. These results suggest that it would be advantageous to evaluate different legumes and grasses for tolerance of shade and moisture stress in future studies. As the trees studied were only 1.5 to 3 m in height, their effects on BNF, seasonal pasture biomass production and botanical composition are expected to increase with tree dominance in the ecosystem with time. Amounts of N fixed were related to the productivity (i.e. dry matter and N yield) and seasonal persistence of the legumes. The productivity was high in spring and summer and low in autumn and winter.     

Carbon and nitrogen dynamics under windrowed residues during the establishment phase of a second-rotation hoop pine plantation in subtropical Australia

The dynamics of carbon (C) and nitrogen (N), derived from the decomposition of windrowed harvest residues, was examined in the establishment phase of a second rotation (2R) hoop pine (Araucaria cunninghamii Aiton ex A. Cunn) plantation in subtropical Queensland, Australia. Following harvesting and site preparation, when residues were formed into windrows, in situ N mineralisation was measured in positions along the three tree-planting rows formed between the windrows. The position above the windrow had a higher nitrification rate than the other positions, averaging about 18 kg N ha⁻¹/month compared with 12 and 9 Kg N ha⁻¹ for the positions between and below the windrow positions, respectively. This position also had consistently greater soil moisture.

Macroplots were formed extending 5 m above and 10 m below a windrow. Windrowed residues within the macroplots were replaced by ¹⁵N-labelled material comprising hoop pine foliage, branch and stem. Hoop pine trees were planted within each macroplot with foliar samples taken at 12 and 24 months. Differences in foliar ¹⁵N enrichment between positions within macroplots were <1‰. Soil samples were taken from positions along the macroplots at 6-monthly intervals. Samples revealed an initial release of labile C and N but soil δ¹⁵N showed that residue-derived N was largely immobilised within the windrows for the 30-month sampling period. Whilst the use of windrows may act as a barrier to the down-slope movement of water, the residue N within the windrows may not be available to the trees of the following rotation for a considerable period following planting. Trees closest to the windrows may be able to introduce roots under the windrows thereby gaining access to the available N, but trees in the central tree planting row are unlikely to derive any significant benefit from the decomposition of windrowed residues.     

Heterotrophic and autotrophic nitrification in two acid pasture soils

Laboratory incubation experiments, using ¹⁵N-labeling techniques and simple analytical models, were conducted to measure heterotrophic and autotrophic nitrification rates in two acid soils (pH 4.8-5.3; 1/5 in H₂O) with high organic carbon contents (6.2-6.8% in top 5 cm soil). The soils were from pastures located near Maindample and Ruffy in the Northeast Victoria, Australia. Gross rates of N mineralization, nitrification and immobilization were measured. The gross rates of autotrophic nitrification were 0.157 and 0.119 μg N g⁻¹ h⁻¹ and heterotrophic nitrification rates were 0.036 and 0.009 μg N g⁻¹ h⁻¹ for the Maindample and Ruffy soils, respectively. Heterotrophic nitrification accounted for 19% and 7% of the total nitrification in the Maindample and Ruffy soils, respectively. The heterotrophic nitrifiers used organic N compounds and no as the substrate for nitrification.     

N tracing models with a Monte Carlo optimization procedure provide new insights on gross N transformations in soils

¹⁵N tracing studies in combination with analyses via process-based models are the current “state-of-the-art” technique to quantify gross nitrogen (N) transformation rates in soils. A crucial component of this technique is the optimization algorithm which primarily decides how many model parameters can simultaneously be estimated. Recently, we published a Markov chain Monte Carlo (MCMC) method which has the potential to simultaneously estimate large number of parameters in ¹⁵N tracing models [Müller et al., 2007. Estimation of parameters in complex ¹⁵N tracing models by Monte Carlo sampling. Soil Biology & Biochemistry 39, 715-726].Here, we present the results of a reanalysis of datasets by Kirkham and Bartholomew [1954. Equations for following nutrient transformations in soil, utilizing tracer data. Soil Science Society of America Proceedings 18, 33-34], Myrold and Tiedje [1986. Simultaneous estimation of several nitrogen cycle rates using ¹⁵N: theory and application. Soil Biology & Biochemistry 18, 559-568] and Watson et al. [2000. Overestimation of gross N transformation rates in grassland soils due to non-uniform exploitation of applied and native pools. Soil Biology & Biochemistry 32, 2019-2030] using the MCMC technique. Analytical solutions such as the ones derived by Kirkham and Bartholomew [1954. Equations for following nutrient transformations in soil, utilizing tracer data. Soil Science Society of America Proceedings 18, 33-34] result in gross rates without uncertainties. We show that the analysis of the same data sets with the MCMC method provides standard deviations for gross N transformations. The standard deviations are further reduced if realistic data uncertainties are considered. Reanalyzing data by Myrold and Tiedje [1986. Simultaneous estimation of several nitrogen cycle rates using ¹⁵N: theory and application. Soil Biology & Biochemistry 18, 559-568] (Capac soil) resulted in a model fit similar to the one of the original analysis but with more precise estimates of gross N transformations. In addition, our analysis showed that small N transformations such as heterotrophic nitrification, which was neglected in the original analysis, could be quantified for this soil. Watson et al. [2000. Overestimation of gross N transformation rates in grassland soils due to non-uniform exploitation of applied and native pools. Soil Biology & Biochemistry 32, 2019-2030] provided evidence of a non-uniform exploitation of applied and native N that led to an overestimation of gross N transformations. Reanalyzing the data (CENIT soil, low N application) with the Müller et al. [2007. Estimation of parameters in complex ¹⁵N tracing models by Monte Carlo sampling. Soil Biology & Biochemistry 39, 715-726] model where oxidation was set to Michaelis-Menten kinetics resulted in a satisfactory fit between modeled and observed data, indicating that the observed artifact by Watson et al. [2000. Overestimation of gross N transformation rates in grassland soils due to non-uniform exploitation of applied and native pools. Soil Biology & Biochemistry 32, 2019-2030] was mainly due to inappropriate kinetic settings. Our study shows that the combination of a MCMC method with ¹⁵N tracing models is able to consider more complex and possibly more realistic models and kinetic settings to estimate gross N transformation rates and thus overcomes restriction of previous ¹⁵N tracing techniques.     

IL-15过表达对猪骨骼肌细胞成肌分化的影响

【目的】研究肌肉因子IL-15（interleukin 15）对猪骨骼肌成肌细胞增殖与凋亡的影响,为进一步研究IL-15在动物肌肉品质调控和骨骼肌疾病治疗提供依据。【方法】构建IL-15过表达慢病毒载体GV-492-IL-15,体外无菌分离培养猪骨骼肌卫星细胞,诱导分化,并通过免疫荧光染色进行成肌细胞验证。将成肌细胞转染IL-15过表达重组慢病毒载体,试验分别设置空白对照组（Control）、转染阴性对照病毒组（IL-15-）和转染GV-492-IL-15（IL-15+）慢病毒试验组（n=3）。培养72 h后,收集细胞和培养上清液。分别采用实时定量PCR（qRT-PCR）和Western Blot技术分析目的基因和蛋白的表达情况,采用ELISA试剂盒分析培养液中IL-15含量,采用CCK-8试剂盒分析成肌细胞活力,采用流式细胞术分析细胞周期和细胞凋亡,采用Western Blot技术检测与细胞凋亡密切相关的caspase-3蛋白表达水平的变化。【结果】（1）经鉴定后的质粒转染293T细胞,细胞内可观察到明显的绿色荧光,经Western Blot检测,可以观察到20 kD附近处有特征条带;（2）分离培养的猪骨骼肌卫星细胞呈梭形或纺锤形,诱导后可分化为呈管状的成肌细胞。将分化后的成肌细胞,进行α-SMA单克隆抗体免疫荧光染色,视野中90%的细胞呈阳性反应,胞浆染成红色,表明细胞为骨骼肌成肌细胞。（3）转染GV-492-IL-15慢病毒后,与对照细胞组相比,成肌细胞内IL-15 mRNA和蛋白相对表达量均极显著升高（P<0.001）,但培养液中IL-15蛋白水平变化不大（P>0.05）。CCK-8结果显示,过表达IL-15可增强细胞的增殖能力（P<0.05）。与对照组相比,转染GV-492-IL-15慢病毒的细胞早期凋亡率差异不显著（P>0.05）,但细胞晚期凋亡率显著下降（P<0.05）。与对照组相比,转染慢病毒组细胞中caspase-3蛋白有下降的趋势,但差异不显著（P>0.05）。此外,转染IL-15过表达慢病毒可使G1期细胞比例显著下降,S期和G2/M期细胞比例显著升高（P<0.05）。【结论】在正常生理条件下,IL-15是定位在细胞内并发挥作用的,IL-15过表达对猪骨骼肌成肌细胞早期凋亡没有显著影响,但可以抑制其晚期凋亡,并促进细胞增殖。这一研究将为IL-15正向调控猪骨骼肌肌肉品质和治疗相关肌肉疾病提供技术和理论依据。     

Fecal carriage of multi-drug resistant and extended spectrum β-lactamases producing E. coli in household pigeons,Bangladesh

Antibiotic resistance and ESBL constitute a risk to human and animal health. Birds residing close to humans could mirror the spectrum of human associated antibiotic resistance. Household pigeons were screened in Bangladesh to shed light on human associated, as well as, environmental antibiotic resistance. Escherichia coli from pigeons (n = 150) were tested against 11 antibiotics. 89% E. coli isolates were resistant to one or more critically important human antibiotics like ampicillin, cefadroxil, mecillinam, ciprofloxacin, gentamicin and tigecycline. No carbapenamase-producers were detected and the lower ESBL prevalence (5%) in pigeons. ESBL-producing E. coli isolates had bla_CTX-M-15 genes. Pigeons shared some bacterial clones and had bird associated sequence types like E. coli ST1408. Fecal carriage of bacteria resistance of critically important human antibiotics, together with examples of shared genotypes among pigeons, indicate the human-birds and bird to bird transmissions are important in the epidemiology of antibiotic resistance.     

4个山羊品种GDF9、BMP15、FSHR基因的多态性分析

采用RFLP和SSCP 2种方法研究了生长分化因子9(GDF9)基因、骨形态发生蛋白15(BMP15)基因和促卵泡素受体(FSHR)基因在河北绒山羊、美姑黑山羊、承德黑山羊、河南槐山羊4个山羊品种中的多态性.结果显示,在4个山羊品种中未检测到GDF9的突变位点;BMP15基因出现1处突变位点(890 bp处,G→T),但未引起基因型改变;FSHR基因出现3种基因型HH、HK、KK,存在1处突变位点(1 568 bp处,C→G).河南槐山羊Hardy-Weinberg不平衡,且为中度多态.表明检测的FSHR基因在4个山羊品种中存在多态性.