Genetic mutation analysis at early stages of cell line development using next generation sequencing

A central goal for most biopharmaceutical companies is to reduce the development timeline to reach clinical proof of concept. This objective requires the development of tools that ensure the quality of biotherapeutic material destined for the clinic. Recent advances in high throughput protein analytics provide confidence in our ability to assess productivity and product quality attributes at early stages of cell line development. However, one quality attribute has, until recently, been absent from the standard battery of analytical tests facilitating informed choices early in cell line selection: genetic sequence confirmation. Techniques historically used for mutation analysis, such as detailed mass spectrometry, have limitations on the sample number and turnaround times making it less attractive at early stages. Thus, we explored the utility of Next‐Generation Sequencing (NGS) as a solution to address these limitations. Amplicon sequencing is one such NGS technique that is robust, rapid, sensitive, and amenable to multiplexing, all of which are essential attributes for our purposes. Here we report a NGS method based upon amplicon sequencing that has been successfully incorporated into our cell line development workflow alongside other high‐throughput protein analytical assays. The NGS method has demonstrated its value by identifying at least one Chinese hamster ovary (CHO) clone expressing a variant form of the biotherapeutic in each of the four clinical programs in which it has been utilized. We believe this sequence confirmation method is essential to safely accelerating the time to clinical proof of concept of biotherapeutics, and guard against delays related to sequence mutations. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:813–817, 2016     

Embryonic development of adriatic sturgeon,Acipenser naccarii (Bonaparte 1836), in farming conditions: a guide

The sequence and timing of the embryonic development of Adriatic sturgeon (Acipenser naccarii) are described at a constant temperature (17 ± 1 °C), from fertilisation to hatch in a hatchery. Fertilised eggs, obtained from one female and two males, were held in a flow‐through system. On the first day, embryos were sampled every 20 min during the first 12 h, and every 40 min in the following 12 h. Embryos were sampled hourly on the second day. On the third and fourth days, embryos were sampled every 2 h, and every 3 h on the fifth and sixth days. The first cleavage furrow appeared at 1 h 40 min postfertilisation. Blastulation concluded at 12 HPF (hours postfertilisation), when the dorsal blastopore lip formed. The completion of gastrulation was at 18 HPF, closing of the neural tube at 23 HPF and formation of the s‐shape heart at 31 HPF. The mass hatch occurred at 120 HPF. A. naccarii is a relatively common sturgeon species in aquaculture and possesses similar developmental patterns to those of other sturgeons; hence, we can use this species as a model for further studies about sturgeon species which are more difficult to breed artificially. Information about the embryonic development of the Adriatic sturgeon could assist restocking programmes.     

Phenological growth stages of nashi tree (Pyrus pyrifolia): codification and description according to the BBCH scale

The phenological growth stages of nashi tree were firstly described here using the BBCH scale. Based on this general scale, nashi phenology showed 8 of the 10 principal stages (0–9): bud, leaf and shoot development, inflorescence emergence, flowering, fruit development, fruit maturity and senescence. A schematic representation of the chronological progression of principal growth stages of nashi is also shown. The codification of the different growth stages is important for correct timing of general orchard management, particularly for disease and pest management. Besides, it will help farmers to efficiently schedule and manage nashi cultivation, as well as to improve knowledge dissemination among scientists around the World.     

How predation by Podisus nigrispinus is influenced by developmental stage and density of its prey Alabama argillacea

The functional response of a predator to the density of its prey is affected by several factors, including the prey's developmental stage. This study evaluated the functional response of Podisus nigrispinus (Dallas) (Hemiptera: Heteroptera: Pentatomidae) females to fourth instars and pupae of Alabama argillacea (Hübner) (Lepidoptera: Noctuidae), an important pest of cotton (Gossypium hirsutum L., Malvaceae) in Brazil. The prey were exposed to the predator for 12 and 24 h, and in densities of 1, 6, 12, 18, 24, and 30 items per predator female. The predation data were subjected to polynomial regression logistic analysis to determine the type of functional response. Holling and Rogers' equations were used to estimate parameters such as attack rate and handling time. Podisus nigrispinus females showed functional response types II and III by preying on larvae and pupae, respectively. The attack rate and handling time did not differ between the 12 and 24 h exposure times. Predation rate was higher at higher larval and pupal densities; predation was highest at a density of 30 prey items per female, and it was similar at 18 and 24 prey per predator. Understanding the interaction of predators and their food resources helps to optimize biological control strategies. It also helps the decision‐making and the improvement of release techniques of P. nigrispinus in the field.     

A life‐history perspective on the demographic drivers of structured population dynamics in changing environments

Current understanding of life‐history evolution and how demographic parameters contribute to population dynamics across species is largely based on assumptions of either constant environments or stationary environmental variation. Meanwhile, species are faced with non‐stationary environmental conditions (changing mean, variance, or both) created by climate and landscape change. To close the gap between contemporary reality and demographic theory, we develop a set of transient life table response experiments (LTREs) for decomposing realised population growth rates into contributions from specific vital rates and components of population structure. Using transient LTREs in a theoretical framework, we reveal that established concepts in population biology will require revision because of reliance on approaches that do not address the influence of unstable population structure on population growth and mean fitness. Going forward, transient LTREs will enhance understanding of demography and improve the explanatory power of models used to understand ecological and evolutionary dynamics.     

内镜黏膜下剥离术治疗消化系统早癌及癌前病变的临床疗效

目的:探讨内镜黏膜下剥离术(ESD)对消化道早癌及癌前病变的治疗效果。方法:选择2013年8月至2014年8月在我院接受治疗的消化道肿瘤患者79例作为研究对象,根据手术方法不同将所选患者分为ESD组(49例)和对照组(30例)。ESD组患者采用内镜黏膜下剥离术治疗,对照组采用传统手术治疗。观察并比较两组患者的手术时间、治愈性切除率、整块完整切除率、术后并发症的发生率及复发、转移情况。结果:ESD组患者的手术时间少于对照组,差异具有统计学意义(P0.05);两组患者手术治愈性切除率均为100%,差异无统计学意义(P0.05);ESD组手术整块完整切除率(63.27%)低于对照组(86.67%),差异具有统计学意义(P0.05)。ESD组患者术后并发症的发生率(4.08%)显著低于对照组(13.3%),差异具有统计学意义(P0.05)。两组患者术后一年内均未出现原发病灶转移及复发。结论:ESD治疗消化道早癌及癌前病变的临床疗效较好,与传统手术相比,ESD手术并发症较少、且安全性较高,更加适宜临床推广及应用。     

Long-term survival of haptophyte and prasinophyte resting stages in marine sediment

Resting stages of marine phytoplankton have been shown to have potential for long-term survival and to remain viable in marine sediments for up to about a century. This study documents for the first time long-term survival in haptophytes and prasinophytes, by germination of resting stages of Isochrysis galbana and Mantoniella squamata from up to 40-year-old sediment layers. Germination was induced by setting up sediment slurries in L1 medium at 15°C. Cyst formation was induced in culture strains acquired from the germinations by keeping mixtures of strains in the light or dark at salinities of 20 or 30. The identity of the two species was confirmed by light and electron microscopy as well as LSU and SSU rDNA-based phylogenetic analyses.     

籼粳稻杂交衍生RIL系的苗期抗旱性评价

为鉴定籼粳稻杂交衍生系的苗期抗旱性,以课题组自育的高代抗逆品系ZD15为母本、籼稻品种IR29为父本,以及杂交衍生的重组自交系群体120份为试验材料,利用PEG-6000对各材料苗期进行干旱胁迫处理,测定根长、根冠比、地上部鲜重、地下部鲜重、地上部干重和地下部干重;利用PEG-6000对各材料芽期进行干旱胁迫处理,测定芽鞘长和芽长。采用主成分分析和隶属函数法对各材料的抗旱性进行综合评价,根据综合抗旱D值可将122份材料分成3类,D值在0.201～0.400之间的有33份,属于不抗旱材料;D值在0.401～0.600之间的有79份,属于中等抗旱材料;D值在0.601～0.800之间的有10份,属于抗旱材料。利用D值进行逐步回归分析,结果表明根长、根冠比、地上部鲜重、地下部鲜重、地上部干重、地下部干重、芽鞘长和芽长8个性状均可作为水稻苗期抗旱性的评价指标。本研究筛选出的抗旱材料,可作为育种中间材料进一步培育,或作为育种资源加以利用,以丰富本区水稻育种的资源库。     

Evaluation of Phytochemical Compositions and Biological Properties of Achillea gypsicola at Different Phenological Stages

Phytochemicals, which are commonly found at different levels in many medicinal plants, are natural strong antioxidants used in traditional medicine. In this research, determination of differences of phytochemical compositions and biological properties were aimed as periodically (pre‐, full and post flowering) and daily (6 am, 1 pm and 8 pm) in Achillea gypsicola Hub.‐Mor . The volatile oils belonging to A. gypsicola were obtained by hydrodistillation and analyzed by gas chromatography‐flame ionization detection (GC‐FID) and gas chromatography‐mass spectrometry (GC/MS). The antimicrobial activities of the volatile oils were determined with disc diffusion method. The microdilution method was used to determine minimum inhibitory concentration (MIC). Total phenolic and flavonoid contents were determined by spectrophotometric methods and antioxidant capacities were evaluated by 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) free radical, reducing power (RP) and metal chelating activity (MCA) assay. In addition, the phenolic acid and flavonoid compositions were evaluated by reversed phase‐high‐performance liquid chromatography (RP‐HPLC). This study presented a comprehensive report for the first time on evaluation of the phytochemical composition and the biological properties of A. gypsicola at different phenological stages. Thirty‐two compounds, containing the major component as camphor, 1,8‐cineole and borneol, were detected. Designated harvest time for the highest yield of volatile oils was found to be at full flowering stage‐1 pm. It has been observed that the volatile oil composition changes periodically and even daily. Also, in this research, menthol and menthone were found as the composition of volatile oil in Achillea species for the first time. Full flowering stage was found as the richest period in terms of phenolic acid and flavonoid compositions of A. gypsicola for the first time. The species examined in this research showed a high antioxidant and antimicrobial activity in comparison to other studies with Achillea species. The volatile oils exhibited high performances with range of inhibition zones (8.3–42.3 mm) and minimum inhibitory concentration values (2.25—144 μg/ml). Besides, a high correlation between antioxidant activity and phenolic content of A. gypsicola was found. These results suggest that A. gypsicola can be used as a safe source in the cosmetic, food and pharmaceutical industries.     

红颈常室茧蜂蛹发育的形态特征

红颈常室茧蜂Peristenus spretus Chen et van Achterberg是一种绿盲蝽若虫的优势内寄生蜂,本研究利用超景深三维显微系统、显微镜观察了红颈常室茧蜂老熟幼虫发育特征、蛹形态特征及蛹发育后期卵巢的超微结构。结果表明:红颈常室茧蜂蛹期为12～15 d,其中雌蛹期比雄蛹期长2～3 d。根据其形态特征,可将蛹划分为预蛹期、第一蛹阶段、第二蛹阶段、第三蛹阶段。蛹期发育前9 d,蛹期组织结构开始分化形成,成蜂的外部形态基本显现,但性别难以区分;蛹期发育至10～14 d,可通过幼蜂体表颜色的变化或腹部末端的形状区分性别,在相同的适宜温度条件下,雌蜂比雄蜂颜色深,雌蜂腹部末端比较尖,有突出的产卵瓣,雄峰腹部末端钝圆,无凸起;雌蜂卵巢在化蛹后的第11天开始形成,卵巢初期的形状如细丝,此时卵巢管已开始初步分化,但卵室尚未分化;蛹发育至第12天时,卵巢管逐渐加粗,卵室开始分化;第13天时,卵巢管、卵室数量逐渐增多;第14天卵巢基部开始有少量成熟卵子产生,呈乳白色。10～12 d,雄蜂腹部末端绒毛逐渐增多,体壁变硬,逐渐角质化,虫体可活动,雄蜂胸部略带黑色,翅逐渐伸展开,整个虫体呈浅红棕色。本研究结果明确了红颈常室茧蜂蛹发育的形态变化过程,提出了蛹期雌雄区分的方法,叙述了蛹后期卵巢发育特征,为研究该蜂繁殖机理奠定了形态学基础。