Combining affinity enrichment,cross‐linking with photo amino acids,and mass spectrometry for probing protein kinase D2 interactions

We present a novel approach that relies on the affinity capture of protein interaction partners from a complex mixture, followed by their covalent fixation via UV‐induced activation of incorporated diazirine photoreactive amino acids (photo‐methionine and photo‐leucine). The captured protein complexes are enzymatically digested and interacting proteins are identified and quantified by label‐free LC/MS analysis. Using HeLa cell lysates with photo‐methionine and photo‐leucine‐labeled proteins, we were able to capture and preserve protein interactions that are otherwise elusive in conventional pull‐down experiments. Our approach is exemplified for mapping the protein interaction network of protein kinase D2, but has the potential to be applied to any protein system. Data are available via ProteomeXchange with identifiers PXD005346 (photo amino acid incorporation) and PXD005349 (enrichment experiments).     

Life cycle assessment of Australian automotive door skins

Background, aim, and scope  Policy initiatives, such as the EU End of Life Vehicle (ELV) Directive for only 5% landfilling by 2015, are increasing the pressure for higher material recyclability rates. This is stimulating research into material alternatives and end-of-life strategies for automotive components. This study presents a Life Cycle Assessment (LCA) on an Australian automotive component, namely an exterior door skin. The functional unit for this study is one door skin set (4 exterior skins). The material alternatives are steel, which is currently used by Australian manufacturers, aluminium and glass-fiber reinforced polypropylene composite. Only the inputs and outputs relative to the door skin production, use and end-of-life phases were considered within the system boundary. Landfill, energy recovery and mechanical recycling were the end-of-life phases considered. The aim of the study is to highlight the most environmentally attractive material and end-of-life option. Methods  The LCA was performed according to the ISO 14040 standard series. All information considered in this study (use of fossil and non fossil based energy resources, water, chemicals etc.) were taken up in in-depth data. The data for the production, use and end-of-life phases of the door skin set was based upon softwares such as SimaPro and GEMIS which helped in the development of the inventory for the different end-of-life scenarios. In other cases, the inventory was developed using derivations obtained from published journals. Some data was obtained from GM-Holden and the Co-operative research Centre for Advanced Automotive Technology (AutoCRC), in Australia. In cases where data from the Australian economy was unavailable, such as the data relating to energy recovery methods, a generic data set based on European recycling companies was employed. The characterization factors used for normalization of data were taken from (Saling et. al. Int J Life Cycle Assess 7(4):203–218 2002) which detailed the method of carrying out an LCA. Results  The production phase results in maximum raw material consumption for all materials, and it is higher for metals than for the composite. Energy consumption is greatest in the use phase, with maximum consumption for steel. Aluminium consumes most energy in the production phase. Global Warming Potential (GWP) also follows a trend similar to that of energy consumption. Photo Oxidants Creation Potential (POCP) is the highest for the landfill scenario for the composite, followed by steel and aluminium. Acidification Potential (AP) is the highest for all the end-of-life scenarios of the composite. Ozone Depletion Potential (ODP) is the highest for the metals. The net water emissions are also higher for composite in comparison to metals despite high pollution in the production phases of metallic door skins. Solid wastes are higher for the metallic door skins. Discussion  The composite door skin has the lowest energy consumption in the production phase, due to the low energy requirements during the manufacturing of E-glass and its fusion with polypropylene to form sheet molding compounds. In general, the air emissions during the use phase are strongly dependent on the mass of the skins, with higher emissions for the metals than for the composite. Material recovery through recycling is the highest in metals due to efficient separation techniques, while mechanical recycling is the most efficient for the composite. The heavy steel skins produce the maximum solid wastes primarily due to higher fuel consumption. Water pollution reduction benefit is highest in case of metals, again due to the high efficiency of magnetic separation technique in the case of steel and eddy current separation technique in the case of aluminium. Material recovery in these metals reduces the amount of water needed to produce a new door skin set (water employed mainly in the ingot casting stage). Moreover, the use of heavy metals, inorganic salts and other chemicals is minimized by efficient material recovery. Conclusions  The use of the studied type of steel for the door skins is a poor environmental option in every impact category. Aluminium and composite materials should be considered to develop a more sustainable and energy efficient automobile. In particular, this LCA study shows that glass-fiber composite skins with mechanical recycling or energy recovery method could be environmentally desirable, compared to aluminium and steel skins. However, the current limit on the efficiency of recycling is the prime barrier to increasing the sustainability of composite skins. Recommendations and perspectives  The study is successful in developing a detailed LCA for the three different types of door skin materials and their respective recycling or end-of-life scenarios. The results obtained could be used for future work on an eco-efficiency portfolio for the entire car. However, there is a need for a detailed assessment of toxicity and risk potentials arising from each of the four different types of door skin sets. This will require greater communication between academia and the automotive industry to improve the quality of the LCA data. Sensitivity analysis needs to be performed such as the assessment of the impact of varying substitution factors on the life cycle of a door skin. Incorporation of door skin sets made of new biomaterials need to be accounted for as another functional unit in future LCA studies. Discussion contributions to this article from the readership would the highly welcome. The authors     

Phototransformation of the Red Light Sensor Cyanobacterial Phytochrome 2 from Synechocystis Species Depends on Its Tongue Motifs

Phytochromes are photoreceptors using a bilin tetrapyrrole as chromophore, which switch in canonical phytochromes between red (P_r) and far red (P_fr) light-absorbing states. Cph2 from Synechocystis sp., a noncanonical phytochrome, harbors besides a cyanobacteriochrome domain a second photosensory module, a P_r/P_fr-interconverting GAF-GAF bidomain (SynCph2(1-2)). As in the canonical phytochromes, a unique motif of the second GAF domain, the tongue region, seals the bilin-binding site in the GAF1 domain from solvent access. Time-resolved spectroscopy of the SynCph2(1-2) module shows four intermediates during P_r → P_fr phototransformation and three intermediates during P_fr → P_r back-conversion. A mutation in the tongue''s conserved PRXSF motif, S385A, affects the formation of late intermediate R3 and of a P_fr-like state but not the back-conversion to P_r via a lumi-F-like state. In contrast, a mutation in the likewise conserved WXE motif, W389A, changes the photocycle at intermediate R2 and causes an alternative red light-adapted state. Here, back-conversion to P_r proceeds via intermediates differing from SynCph2(1-2). Replacement of this tryptophan that is ∼15 Å distant from the chromophore by another aromatic amino acid, W389F, restores native P_r → P_fr phototransformation. These results indicate large scale conformational changes within the tongue region of GAF2 during the final processes of phototransformation. We propose that in early intermediates only the chromophore and its nearest surroundings are altered, whereas late changes during R2 formation depend on the distant WXE motifs of the tongue region. Ser-385 within the PRXSF motif affects only late intermediate R3, when refolding of the tongue and docking to the GAF1 domain are almost completed.     

聚球藻(Synechococcus leopoliensis625)藻胆体-类囊体膜光化学活性和亚显微结构的研究

聚球藻藻胆体-类囊体膜吸收光谱中有五个吸收峰,它们位于420nm,438nm,490nm,624nm 和678nm。放氧速率为161—179mol O2/mg Chl.hr.电镜照片中显示在类囊体膜上有大量藻胆体。     

Colorimetric assay for sensitive poly(styrene sulfonate) quantification in a template directed polyelectrolyte-assembling process

Template directed Layer-by-layer (LbL) technology recently moved into the center of scientific attention, particularly as a versatile tool for bioencapsulation purposes. Its major advantages can be found in the striking simplicity of tuning wall properties and the complete control over layer thickness and permeability. Yet, for the most commonly applied pair of polyelectrolytes, poly(allylamine) hydrochloride (PAH) and poly(styrene sulfonate) sodium salt (PSS), the mandatory control of the successful deposition on plane and colloidal surfaces is currently only attainable by means of sophisticated and expensive equipment. Here we describe an alternative quantification method based on a simple colorimetric assay using the Bradford reagent, a cost-effective commercially available dye, and standard laboratory technical devices. The binding of the dye to PSS causes a distinct shift of the absorption maximum from 465 to 680 nm, providing a method for spectral quantification of submicrogram amounts of dissolved PSS during LbL coating with significant accuracy and excellent reproducibility. The method was successfully employed to quantify accurate polyelectrolyte loadings on several particles that have a general importance as LbL templates. Thus, this method can be recommended as standard laboratory technique for control of LbL encapsulation and will considerably broaden the applicability of this promising technology in biotechnology.     

Chromatic photoacclimation, photosynthetic electron transport and oxygen evolution in the Chlorophyll d-containing oxyphotobacterium Acaryochloris marina

Changes in photosynthetic pigment ratios showed that the Chlorophyll d-dominated oxyphotobacterium Acaryochloris marina was able to photoacclimate to different light regimes. Chl d per cell were higher in cultures grown under low irradiance and red or green light compared to those found when grown under high white light, but phycocyanin/Chl d and carotenoid/Chl d indices under the corresponding conditions were lower. Chl a, considered an accessory pigment in this organism, decreased respective to Chl d in low irradiance and low intensity non-white light sources. Blue diode PAM (Pulse Amplitude Modulation) fluorometry was able to be used to measure photosynthesis in Acaryochloris. Light response curves for Acaryochloris were created using both PAM and O₂ electrode. A linear relationship was found between electron transport rate (ETR), measured using a PAM fluorometer, and oxygen evolution (net and gross photosynthesis). Gross photosynthesis and ETR were directly proportional to one another. The optimum light for white light (quartz halogen) was about 206 ± 51 μmol m^− 2 s^− 1 (PAR) (Photosynthetically Active Radiation), whereas for red light (red diodes) the optimum light was lower (109 ± 27 μmol m^− 2 s^− 1 (PAR)). The maximum mean gross photosynthetic rate of Acaryochloris was 73 ± 7 μmol mg Chl d^− 1 h^− 1. The gross photosynthesis/respiration ratio (P_g/R) of Acaryochloris under optimum conditions was about 4.02 ± 1.69. The implications of our findings will be discussed in relation to how photosynthesis is regulated in Acaryochloris.     

沿海防护林四个树种根系分布对盐胁迫的响应

研究盐分胁迫下植物根系的分布及生长特性对沿海防护林树种的筛选具有重要意义。以8年生女贞(Ligustrum lucidum)、洋白蜡(Fraxinus pennsylvanica)、木麻黄(Casuarina equiestifolia)和墨西哥落羽杉(Taxodium mucronatum)等4个亚热带沿海防护林树种为对象,对其根系生长特性与土壤盐度关系、根系生物量分配格局、功能根的分布特征以及根系的比根长和根长密度进行了比较。结果表明:1)土壤盐分对植物根系垂直生长与分布具有强烈的制约作用,根系的水平伸展能力与树种根系垂直生长呈负相关;2)不同树种适应盐分胁迫能力不同,导致各自功能根的生物量分配格局差异;3)根径级越小,其吸收水分和营养物质的能力越强;根径级越大,固定和支持植株的能力越强;4)墨西哥落羽杉盐分阈值0.45%,根系垂直分布在0～95cm的土层中,具有良好的抗台风和耐盐土能力;木麻黄和洋白蜡2个树种的主根系长度分别为25cm和29cm,支撑根的比例分别为87.2%和56.3%,因此也具有一定的抗台风能力,可以栽植在盐度0.36%的沿海滩涂上;女贞盐分阈值0.21%,垂直根系为20cm,支撑根比例51.0%,不适合栽植于高盐分的台风分布地理区域。     

Enhancing the growth of Physcomitrella patens by combination of monochromatic red and blue light - a kinetic study

In the current work we demonstrate the relevance of monochromatic light conditions in moss plant cell culture. Light intensity and illumination wavelength are important cultivation parameters due to their impact on growth and chlorophyll formation kinetics of the moss Physcomitrella patens. This moss was chosen as a model organism due to its capability to produce complex recombinant pharmaceutical proteins. Filamentous moss cells were cultivated in mineral medium in shaking flasks. The flasks were illuminated by light emitting diodes (LED) providing nearly monochromatic red and blue light as well as white light as a reference. A maximum growth rate of 0.78 day((1) was achieved under additional CO(2) aeration and no growth inhibition was observed under high light illumination. The application of dual red and blue light is the most effective way to reach high growth and chlorophyll formation rates while minimizing energy consumption of the LEDs. These observations are discussed as effects of photo sensory pigments in the moss. The combination of monochromatic red and blue light should be considered when a large scale process is set up.