Quantitative Deep Sequencing Reveals Dynamic HIV-1 Escape and Large Population Shifts during CCR5 Antagonist Therapy In Vivo

High-throughput sequencing platforms provide an approach for detecting rare HIV-1 variants and documenting more fully quasispecies diversity. We applied this technology to the V3 loop-coding region of env in samples collected from 4 chronically HIV-infected subjects in whom CCR5 antagonist (vicriviroc [VVC]) therapy failed. Between 25,000–140,000 amplified sequences were obtained per sample. Profound baseline V3 loop sequence heterogeneity existed; predicted CXCR4-using populations were identified in a largely CCR5-using population. The V3 loop forms associated with subsequent virologic failure, either through CXCR4 use or the emergence of high-level VVC resistance, were present as minor variants at 0.8–2.8% of baseline samples. Extreme, rapid shifts in population frequencies toward these forms occurred, and deep sequencing provided a detailed view of the rapid evolutionary impact of VVC selection. Greater V3 diversity was observed post-selection. This previously unreported degree of V3 loop sequence diversity has implications for viral pathogenesis, vaccine design, and the optimal use of HIV-1 CCR5 antagonists.     

Remote sensing of larch phenological cycle and analysis of relationships with climate in the Alpine region

This research aims at developing a remote sensing technique for monitoring the interannual variability of the European larch phenological cycle in the Alpine region of Aosta Valley (Northern Italy) and to evaluate its relationships with climatic factors. Phenological field observations were conducted in eight test sites from 2005 to 2007 to determine the dates of completion of different phenological phases. MODerate Resolution Imaging Spectrometer (MODIS) 250 m 16‐days normalized difference vegetation index (NDVI) time series were fitted with double logistic curves and the dates corresponding to different features of the curves were determined. Comparison with field data showed that the features of the fitted NDVI curve that allowed the best estimate of the start and end of the growing season were the zeroes of its third derivative (MAE of 6 and 4 days, respectively). The start and end of season were also estimated with the spring warming (SW) and growing season index (GSI) phenological models. MODIS start and end of season dates generally agreed with those obtained by the SW and GSI climate‐driven phenological models. However, phenological models provided erroneous results when applied in years with anomalous meteorological conditions. The relationships between interannual variability of the larch phenological cycle and climate were investigated by comparing the mean start and end of season yearly anomalies with air temperature anomalies. A strong linear relationship (R²=0.91) was found between mean spring temperatures and mean start of season dates, with an increase of 1 °C in mean spring temperature leading to a 7‐day anticipation of mean larch bud‐burst date. Leaf coloring dates were found to be best related with mean September temperature (R²=0.77), but with higher spring temperatures appearing to lead to earlier leaf coloring.     

百合组织培养和遗传转化的研究进展

百合是单子叶球茎类观赏花卉,具有食用和药用的作用。本文综述了国内外百合组织培养和遗传转化研究进展。包括营养器官、生殖器官和原生质体再生系统的建立。详细介绍了基因转化的方法,例如农杆菌、基因枪、电激穿孔等。浅析百合外源基因的表达,例如GUS、半夏凝集素基因、几丁质酶基因等。同时,讨论了百合遗传转化中存在的问题,以期为百合基因工程方面的研究提供依据。     

锁阳多糖提取工艺的研究

以浸膏得率和浸膏中多糖质量分数综合评价,利用L9(34)正交实验从水提和醇沉两方面优选锁阳中水溶性多糖最佳提取工艺条件,为锁阳活性多糖的开发提供科学依据和实验基础。最佳水提工艺条件为:m(锁阳):v(水)=1:12,回流提取0.5 h,提取2次;最佳醇沉工艺条件为:在m(生药):v(水提液)=1 g:2 mL溶液中加入乙醇,使φ(乙醇)=70%,静置6 h。     

中国木层孔菌属三个新记录种

报道了木层孔菌属Phellinus 3个中国新记录种。赤杨木层孔菌Phellinus alni典型特征为菌盖具有较宽的同心环带和清晰的密集环纹菌核;黑木层孔菌P. nigricans具有较大的担孢子;东方木层孔菌P. orienticus具有平伏的子实体,担孢子相对较小。对这3个种进行了详细的描述和显微结构绘图。     

平地无风条件下蒙古栎阔叶床层的火行为Ⅱ.火焰长度和驻留时间影响因子分析与预测模型

以帽儿山地区蒙古栎凋落叶的含水率、载量和床层高度为控制变量,模拟野外凋落叶床层状态,进行了100次平地无风条件下的室内点烧试验,分析含水率、载量和床层高度对火焰长度和驻留时间的影响,并建立了多元线性预测模型.结果表明： 含水率与火焰长度呈极显著线性负相关(P<0.01),与驻留时间的线性关系并不显著(P>0.05);载量、床层高度与火焰长度和驻留时间均呈极显著线性正相关(P<0.01).床层高度与含水率、载量的交互作用对火焰长度有显著影响;含水率与载量、床层高度的交互作用对驻留时间有显著影响.火焰长度预测模型的预测效果较好,能解释火焰长度83.3%的变异,平均绝对误差为7.8 cm,平均相对误差为16.2%;驻留时间预测模型的效果略差,仅能解释驻留时间54%的变异,平均绝对误差为9.2 s,平均相对误差为18.6%.     

Assessing vesicoureteral reflux in live inbred mice via ultrasound with a microbubble contrast agent

Vesicoureteral reflux (VUR) is a common pediatric anomaly linked to renal scarring and hypertension. Although there are many mouse VUR models, cystograms have previously only been performed in euthanized animals, thus preventing serial assessments for VUR in the same animal and not delineating "live" physiology. Our purpose was to develop a live murine cystogram assay that could be used serially to track reflux. We injected microbubbles via transurethral catheters into bladders of C57BL6/J and C3H/HeJ inbred mouse strains that are known to have low and high VUR rates, respectively. We performed ultrasound to visualize microbubbles in the renal pelvis to determine feasibility of the procedure. We then repeated the microbubble ultrasound using a transducer allowing for visualization of both kidneys and ureters simultaneously and for 3 dimensional (3D) reconstruction. We then performed "euthanized" cystograms on all mice for comparison. C3H/HeJ mice had a strong and persistent microbubble signal in the renal pelvis and ureters bilaterally with low-contrast infusion volumes (<100 μl) and similarly showed bilateral reflux by euthanized cystograms. With larger infused volumes (≥150 μl), C57BL6/J mice had small volumes of microbubbles in the renal pelvis that cleared quickly and did not show reflux on euthanized cystograms. Thus, using animal models of known VUR, we demonstrate the utility of contrast-enhanced ultrasound to visualize reflux in live mice.     

Melatonin enhances hydrogen peroxide-induced apoptosis in human promyelocytic leukaemia HL-60 cells

Melatonin is an indoleamine secreted by the pineal gland that shows multiple tasks. This ubiquitously acting free radical scavenger has recently been shown to stimulate the production of reactive oxygen species (ROS) in tumour cells, making them undergo apoptosis, whilst it prevents apoptosis in healthy cells. The mechanisms by which melatonin exerts these dual actions are, however, not yet clearly understood. Thus, the aim of this study was to further investigate how melatonin can enhance oxidative stress-induced apoptosis in a leukaemia cell line. The results show that melatonin increased the apoptotic effects of H(2)O(2) in human myeloid HL-60 cells as assessed by cellular viability, mitochondrial permeability transition induction, mitochondrial membrane depolarization, ROS generation, caspases 3, 8 and 9 activity, phosphatidylserine externalization, and DNA fragmentation techniques. When healthy leucocytes were exposed to H(2)O(2), melatonin increased the viability of the cells. Taken together, the findings indicate that melatonin is a potential physiological tool capable of protecting healthy cells from chemotherapy-induced ROS production as well as inducing tumour cell death. Because cancer cells manifest increased oxidative stress as a result of their elevated metabolism, the use of melatonin may be useful in impairing their ROS buffering capacity.     

Long Term Expression of Drosophila melanogaster Nucleoside Kinase in Thymidine Kinase 2-deficient Mice with No Lethal Effects Caused by Nucleotide Pool Imbalances

Mitochondrial DNA depletion caused by thymidine kinase 2 (TK2) deficiency can be compensated by a nucleoside kinase from Drosophila melanogaster (Dm-dNK) in mice. We show that transgene expression of Dm-dNK in Tk2 knock-out (Tk2^−/−) mice extended the life span of Tk2^−/− mice from 3 weeks to at least 20 months. The Dm-dNK^+/−Tk2^−/− mice maintained normal mitochondrial DNA levels throughout the observation time. A significant difference in total body weight due to the reduction of subcutaneous and visceral fat in the Dm-dNK^+/−Tk2^−/− mice was the only visible difference compared with control mice. This indicates an effect on fat metabolism mediated through residual Tk2 deficiency because Dm-dNK expression was low in both liver and fat tissues. Dm-dNK expression led to increased dNTP pools and an increase in the catabolism of purine and pyrimidine nucleotides but these alterations did not apparently affect the mice during the 20 months of observation. In conclusion, Dm-dNK expression in the cell nucleus expanded the total dNTP pools to levels required for efficient mitochondrial DNA synthesis, thereby compensated the Tk2 deficiency, during a normal life span of the mice. The Dm-dNK^+/− mouse serves as a model for nucleoside gene or enzyme substitutions, nucleotide imbalances, and dNTP alterations in different tissues.