The anatomy of organogenesis: novel solutions to old problems

Understanding anatomical aspects of mammalian organ development, in both normal and mutant animals, is important for basic biology and also for regenerative medicine and tissue engineering. The size and complexity of developing organs, together with variations in their detailed anatomy, has made the obtaining of high-resolution time-courses of anatomical change difficult to obtain. The fact that organ development tends to use the same genes as early embryogenesis also makes genetic manipulation difficult, as so many mutant embryos die before organogenesis begins. These problems have seriously hampered the study of organogenesis. Here, we describe three significant advances that promise solutions: (1) the production of GFP-reporter mice that can be used for high-resolution live-imaging of small tissues as they grow, (2) RNA interference, which allows the manipulation of specific genes at any stage of organ development, and (3) optical projection tomography, which allows medium-resolution three-dimensional images of complete embryos to be obtained easily. We finish by looking ahead to the prospects of uniting these three technologies to allow accurate, high-throughput screening of mutants and automated comparison of biological data with computer predictions.     

Quantitative measurement of damage caused by 1064-nm wavelength optical trapping of Escherichia coli cells using on-chip single cell cultivation system

We quantitatively examined the possible damage to the growth and cell division ability of Escherichia coli caused by 1064-nm optical trapping. Using the synchronous behavior of two sister E. coli cells, the growth and interdivision times between those two cells, one of which was trapped by optical tweezers, the other was not irradiated, were compared using an on-chip single cell cultivation system. Cell growth stopped during the optical trapping period, even with the smallest irradiated power on the trapped cells. Moreover, the damage to the cell's growth and interdivision period was proportional to the total irradiated energy (work) on the cell, i.e., irradiation time multiplied by irradiation power. The division ability was more easily affected by a smaller energy, 0.36 J, which was 30% smaller than the energy that adversely affected growth, 0.54 J. The results indicate that the damage caused by optical trapping can be estimated from the total energy applied to cells, and furthermore, that the use of optical trapping for manipulating cells might cause damage to cell division and growth mechanisms, even at wavelengths under 1064 nm, if the total irradiation energy is excessive.     

Allene oxide synthase from Arabidopsis thaliana (CYP74A1) exhibits dual specificity that is regulated by monomer-micelle association

We investigate the effects of detergent on the kinetics and oligomeric state of allene oxide synthase (AOS) from Arabidopsis thaliana (CYP74A1). We show that detergent-free CYP74A1 is monomeric and highly water soluble with dual specificity, but has relatively low activity. Detergent micelles promote a 48-fold increase in k(cat)/K(m) (to 5.9 x 10(7)M(-1)s(-1)) with concomitant changes in the spin state equilibrium of the haem-iron due to the binding of a single detergent micelle to the protein monomer, which is atypical of P450 enzymes. This mechanism is shown to be an important determinant of the substrate specificity of CYP74A1. CYP74A1 may be suited for structural resolution of the first plant cytochrome P450 and its 9-AOS activity and behaviour in vitro has implications for its role in planta.     

A label-free continuous total-internal-reflection-fluorescence-based immunosensor

In this study, we continuously monitored, second-by-second, concentration changes of two different carbohydrates (maltose and panose) by using monoclonal antibodies in an optical immunosensor based on total internal reflection fluorescence. Earlier studies have demonstrated that these antibodies increase their intrinsic tryptophan fluorescence upon binding of carbohydrate antigens. Using the four immobilized monoclonal antibodies with low affinities (K(d)>10(-6)M), fast kinetics (k(off)>1s(-1)), and high reversibility gave opportunities for developing a continuous immunosensor without any need for regeneration. Since intrinsic fluorescence was used, no extrinsic labeling was necessary. Sensitivity was in the range of 1-5 microM for panose, and 10-15 microM for maltose and the loss of intensity was as low as 3.5% per hour during measurements. Calculations of DeltaH degrees and DeltaS degrees from the temperature dependence of K(d) indicated an enthalpic driven antigen-antibody binding event that is diminished upon antibody immobilization. We feel certain that weakly interacting antibodies can be used in future applications for continuous monitoring where there is a need to achieve instantaneous information on the concentration of an analyte.     

Impact of moisture and UV degradation on Beauveria bassiana (Balsamo) Vuillemin conidial viability in turfgrass

Beauveria bassiana conidial viability in turfgrass was evaluated using a two-component nucleic acid stain and fluorescence microscopy. Turfgrass samples along with the top 5 cm of soil were used for conidial extraction and viability evaluation on 1, 2, 3, 7, 14 and 21 days after treatment. There were no differences in conidial viability between two Orthoptera strains, 3622 and 5977, and both strains were able to persist in a sandy loam soil for up to three weeks after application. High and low irrigation levels were applied to each of the two strains and results show that higher irrigation (5.1 cm/week) maintains conidial viability better than a low irrigation level (2.5 cm/week). Mean conidial viability was approximately 8–12% greater in plots with the high irrigation regime. Rather than significantly increase soil moisture levels, it is hypothesized that the greater amount of irrigation helps to move the conidia deeper into the thatch layer and soil profile, an area that provides protection from damaging surface temperatures and UV exposure. Rainfall that occurred during the beginning of the 2005 test minimized the irrigation effect, and the irrigation treatment differences were more pronounced after 48 h. Four different UV protectants were evaluated for an impact on conidial viability of strain 3622. Two protectants, an optical brightener and magnesium silicate clay, when added to an emulsifiable oil formulation, significantly increased conidial viability on all evaluation dates. The clay particles act as a sunlight blocker while the optical brightener absorbs UV light. The combined protection from the oil and the additive increased conidial viability by approximately 10% on all evaluation dates. Results from this study provide insight into ways to increase entomopathogenic fungal viability under field conditions.     

Cooperative responses of multiple kinesins to variable and constant loads

Microtubule-dependent transport is most often driven by collections of kinesins and dyneins that function in either a concerted fashion or antagonistically. Several lines of evidence suggest that cargo transport may not be influenced appreciably by the combined action of multiple kinesins. Yet, as in previous optical trapping experiments, the forces imposed on cargos will vary spatially and temporally in cells depending on a number of local environmental factors, and the influence of these conditions has been largely overlooked. Here, we characterize the dynamics of structurally defined complexes containing multiple kinesins under the controlled loads of an optical force clamp. While demonstrating that there are generic kinetic barriers that restrict the ability of multiple kinesins to cooperate productively, the spatial and temporal properties of applied loads is found to play an important role in the collective dynamics of multiple motor systems. We propose this dependence has implications for intracellular transport processes, especially for bidirectional transport.     

小型猪冠脉支架模型中即刻OCT检查评价支架贴壁不良的安全性和有效性

目的评价中华小型猪冠状动脉支架植入术后即刻行光学相干断层成像扫描（OCT）的安全性和有效性。方法健康中华小型猪22只,经股动脉途径行右冠状动脉西罗莫司药物洗脱支架植入术,术后即刻行OCT检查评价支架贴壁不良情况,同时记录OCT检查时间和相关并发症。结果 OCT共检查25段支架,失败3例（1例指引导丝断裂于支架内,2例发生冠脉痉挛导致阻断球囊送入困难未能成像）,OCT检查并发症包括一过性ST段抬高（5例）和室颤（3例）,死亡1例,平均手术时间49.7±12.9 min,其中OCT检查耗时17.9±4.9 min,OCT共检测522 mm支架,定量测量4514个支架丝,其中66个存在贴壁不良,即刻支架贴壁不良率1.46%。结论利用冠脉内OCT技术可以有效评价支架术后即刻贴壁不良情况,安全性良好。     

光电阅读机在大人群现场调查中数据处理及质量控制 的应用

目的:探讨光电阅读机在大样本现场调查项目质量控制和数据录入中的作用.方法:使用光电阅读机读入《子宫颈癌危险因素调查表》信息并进行表格质量评估,核查调查表中调查对象编码缺失、重复及进行读入信息一致性比对.结果:在录入的70160份数据中,调查对象编码缺失率为0.87％,调查对象编码重复率为0.79％,信息一致率为99.47％.3个项目点连续三个月质量控制,表格不合格率略有下降.结论:在大人群现场调查项目中通过光电阅读机进行质量控制及数据录入,能快速准确的将纸质信息转化为电子信息,并对电子信息进行核查,能迅速将调查表评估结果和建议反馈给调查表填写人员,及时指导调查员改进调查项目的编写方法和调查表内容的填写方法,从而提高了调查表信息采集质量,促进项目科研数据真实可信.     

Experimental study on the influence of bioturbation performed by Nephtys caeca (Fabricius) and Nereis virens (Sars) annelidae on the distribution of dinoflagellate cysts in the sediment

Dinoflagellates include noxious microalgae responsible for the formation of toxic red tides and the poisoning of molluscs and crustaceans, resulting in important economic losses. As a consequence, the life cycle of these algae has been extensively studied, but the dormancy phase (cyst) in the sediment record is little known. In the intertidal zone, bioturbation, an important biological process resulting from the activities of benthic fauna, significantly influences the movement of particles in the sediments. Laboratory experiments have allowed comparing and quantifying the movements of fluorescent microspheres resulting from the activity of two polychaetes annelidae, Nereis virens and Nephtys caeca. The particles, which simulate 45 µm diameter dinoflagellate cysts, are deposited in flat aquaria at the surface or deep in the sediment. Photographs of the aquaria were taken at regular intervals, to observe, in a non-destructive manner, the movement of the particles and to calculate, using adapted software, the optical reworking coefficient (ORC) over time. A difference appears between the movements of the particles generated by both species of polychaetes. Nereis virens create “permanent” galleries that carry the microspheres deeply in the sediment during the digging, bioirrigation and feeding, and Nephtys caeca homogenize the particles in the first centimetres of sediment during its erratic movements. The study shows that the bioturbation generated by these organisms can modify the distribution of the 45 µm diameter dinoflagellate cysts in the sedimentary column, burying them or raising them back to the water-sediment interface.     

Structure and elastic properties of tunneling nanotubes

We investigate properties of a reported new mechanism for cell–cell interactions, tunneling nanotubes (TNT’s). TNT’s mediate actin-based transfer of vesicles and organelles and they allow signal transmission between cells. The effects of lateral pulling with polystyrene beads trapped by optical tweezers on TNT’s linking separate U-87 MG human glioblastoma cells in culture are described. This cell line was chosen for handling ease and possible pathology implications of TNT persistence in communication between cancerous cells. Observed nanotubes are shown to have the characteristic features of TNT’s. We find that pulling induces two different types of TNT bifurcations. In one of them, termed V-Y bifurcation, the TNT is first distorted into a V-shaped form, following which a new branch emerges from the apex. In the other one, termed I-D bifurcation, the pulled TNT is bent into a curved arc of increasingly broader span. Curves showing the variation of pulling force with displacement are obtained. Results yield information on TNT structure and elastic properties. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.