Biocontrol: Endophytic bacteria could be crucial to fight soft rot disease in the rare medicinal herb,Anoectochilus roxburghii

Microbial destabilization induced by pathogen infection has severely affected plant quality and output, such as Anoectochilus roxburghii, an economically important herb. Soft rot is the main disease that occurs during A. roxburghii culturing. However, the key members of pathogens and their interplay with non-detrimental microorganisms in diseased plants remain largely unsolved. Here, by utilizing a molecular ecological network approach, the interactions within bacterial communities in endophytic compartments and the surrounding soils during soft rot infection were investigated. Significant differences in bacterial diversity and community composition between healthy and diseased plants were observed, indicating that the endophytic communities were strongly influenced by pathogen invasion. Endophytic stem communities of the diseased plants were primarily derived from roots and the root endophytes were largely derived from rhizosphere soils, which depicts a possible pathogen migration image from soils to roots and finally the stems. Furthermore, interactions among microbial members indicated that pathogen invasion might be aided by positively correlated native microbial members, such as Enterobacter and Microbacterium, who may assist in colonization and multiplication through a mutualistic relationship in roots during the pathogen infection process. Our findings will help open new avenues for developing more accurate strategies for biological control of A. roxburghii bacterial soft rot disease.     

Arbuscular mycorrhizal fungi protect a subtropical tree species exposed to simulated acid rain by accelerating photosynthetic ability,antioxidant enzymes and osmolyte accumulation

丛枝菌根真菌对其宿主光合能力、抗氧化酶和渗透物质积累的促进作用 及其抗酸雨机制的探讨 酸雨在中国南方发生频繁,对亚热带树种生长具有明显抑制作用。以往研究表明,丛枝菌根真菌(AM真菌)可以缓解酸雨对宿主植物的胁迫效应。榉树(Zelkova serrata)为中国南方主要经济树种之一,其如何与共生AM真菌协同、增强其抗酸雨胁迫的能力是本项研究所要探讨的关键科学问题。通过温室控制实验,将榉树幼苗随机接受4个水平的AM真菌接种处理(接种灭菌菌种;单独接种Rhizophagus intraradices;单独接种Diversispora versiformis;接种这两种菌种的混合菌种)和3个pH水平(pH2.5、pH4.0和pH5.6)的硫酸型酸雨和硝酸型酸雨处理组成的12个处理组合,同时测定其生长、光合性能、抗氧化酶、渗透调节和土壤酶的响应格局。研究发现酸雨处理显著降低了非菌根榉树幼苗的总干重、总叶绿素含量、叶片净光合速率和可溶性蛋白的含量;接种AM真菌,特别是接种混合菌种,显著提高了强酸胁迫下榉树幼苗的总干重、光合性能、丙二醛、过氧化物酶、超氧化物歧化酶、可溶性蛋白和根系酸性磷酸酶活性。此外,菌根效应依赖于AM真菌的种类和酸胁迫的梯度。本研究 结果表明,AM真菌对榉树幼苗抗酸胁迫的调控作用主要源于调节宿主植株光合能力、抗氧化酶和渗透物质的积累。榉树与其共生AM真菌在应对酸胁迫上协同机制的解析为该树种在中国南方酸雨区的栽培提供理论基础、具有重要的实践指导意义。     

Effect of temperature on tether extraction, surface protrusion, and cortical tension of human neutrophils

Neutrophil rolling on endothelial cells, the initial stage of its migrational journey to a site of inflammation, is facilitated by tether extraction and surface protrusion. Both phenomena have been studied extensively at room temperature, which is considerably lower than human body temperature. It is known that temperature greatly affects cellular mechanical properties such as viscosity. Therefore, we carried out tether extraction, surface protrusion, and cortical tension experiments at 37 degrees C with the micropipette aspiration technique. The experimental temperature was elevated using a custom-designed microscope chamber for the micropipette aspiration technique. To evaluate the constant temperature assumption in our experiments, the temperature distribution in the whole chamber was computed with finite element simulation. Our simulation results showed that temperature variation around the location where our experiments were performed was less than 0.2 degrees C. For tether extraction at 37 degrees C, the threshold force required to pull a tether (40 pN) was not statistically different from the value at room temperature (51 pN), whereas the effective viscosity (0.75 pN.s/microm) decreased significantly from the value at room temperature (1.5 pN.s/microm). Surface protrusion, which was modeled as a linear deformation, had a slightly smaller spring constant at 37 degrees C (40 pN/microm) than it did at room temperature (56 pN/microm). However, the cortical tension at 37 degrees C (5.7+/-2.2 pN/microm) was substantially smaller than that at room temperature (23+/-8 pN/microm). These data clearly suggest that neutrophils roll differently at body temperature than they do at room temperature by having distinct mechanical responses to shear stress of blood flow.     

Activity based fingerprinting of proteases using FRET peptides

We have successfully developed a protease assay using fluorescence resonance energy transfer based peptide libraries, which allows not only general detection of enzymatic activities, but more importantly substrate fingerprinting of proteases from different classes. The method allows the generation of substrate fingerprints of a protease from both the nonprime and prime sites. Therefore, it is well suited for profiling of major metalloproteases such as thermolysin and MMPs. We envisage that this method will provide a useful tool in the emerging field of Catalomics for high-throughput studies of proteases.     

Encapsulation of proteins in biodegradable polymeric microparticles using electrospray in the Taylor cone-jet mode

Solvent extraction (or evaporation from a W(1)/O/W(2)-dispersion), coacervation, and spray drying methods are commonly employed to encapsulate protein drugs in polymeric microparticles for sustained delivery applications. To overcome the limitations of these methods, a novel electrospray method was developed to encapsulate a model protein drug-bovine serum albumin (BSA) in biodegradable polymeric microparticles and examine the feasibility of the process in not denaturing the protein. Microparticles of approximately 20 microm diameter with corrugated surfaces and smooth surfaces were observed by scanning electron microscope. Confocal laser scanning microscope images showed that BSA was distributed evenly in microparticles. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was employed to investigate the protein integrity of BSA released from the polymer matrix after 38 days. No protein degradation was observed during the 38 days release. The secondary structure of released BSA was characterized by Fourier transform infrared (FTIR) and circular dichroism (CD), which suggested that the released BSA was almost identical to native BSA. The encapsulation efficiency could reach 76% by adjusting the amount of the additive Pluronic F127 and processing parameters. The release profile could be tailored by the fabrication process and the sustained release of BSA could endure for more than 1 month. More than 80% of the bioactivity of BSA (evaluated by BSA ELISA kit) could be maintained after releasing from polymer matrix. Findings of the present study demonstrate that this novel electrospray method is a promising approach to encapsulate bioactive materials such as proteins, enzymes, antibiotics, and DNA fragments in biodegradable polymeric particles.     

Differential effects of endoplasmic reticulum stress-induced autophagy on cell survival

Autophagy is a cellular response to adverse environment and stress, but its significance in cell survival is not always clear. Here we show that autophagy could be induced in the mammalian cells by chemicals, such as A23187, tunicamycin, thapsigargin, and brefeldin A, that cause endoplasmic reticulum stress. Endoplasmic reticulum stress-induced autophagy is important for clearing polyubiquitinated protein aggregates and for reducing cellular vacuolization in HCT116 colon cancer cells and DU145 prostate cancer cells, thus mitigating endoplasmic reticulum stress and protecting against cell death. In contrast, autophagy induced by the same chemicals does not confer protection in a normal human colon cell line and in the non-transformed murine embryonic fibroblasts but rather contributes to cell death. Thus the impact of autophagy on cell survival during endoplasmic reticulum stress is likely contingent on the status of cells, which could be explored for tumor-specific therapy.     

Methods of using click chemistry in the discovery of enzyme inhibitors

This protocol describes the step-by-step procedures for the efficient assembly of bidentate inhibitor libraries of a target enzyme, using the so-called 'click chemistry' between an alkyne-bearing core group and an azide-modified peripheral group, followed by direct biological screening for the identification of potential 'hits'. The reaction is highlighted by its modularity, high efficiency (approximately 100% yield in most cases) and tolerance toward many functional groups present in the fragments, as well as biocompatibility (typically carried out in aqueous conditions with small amounts of biocompatible catalysts). The approach consists of three steps: (i) chemical synthesis of alkyne-bearing protein tyrosine phosphatase or matrix metalloprotease core groups and diverse azide-modified peripheral groups; (ii) click chemistry to assemble the bidentate inhibitor libraries; and (iii) direct screening of the libraries with target enzymes using 384-well microplate assays. Following the chemical synthesis of the core and peripheral groups and optimization of the click chemistry conditions (approximately 1 week), steps (ii) and (iii) take 3 d to complete (approximately 1-2 d for library assembly and 1 d for inhibitor screening).     

Genetic differentiation of Arthrobacter population from heavy metal-contaminated environment

Six samples containing extremely high concentration of Pb, Zn, and Cd were obtained from the layers of 5–10 cm and 25–30 cm three tailing piles, with ages of about 10, 20 and more than 80 years, respectively. Then, 48 bacterial strains were obtained from these samples, and subsequently their phylogenetic positions were determined by analysis on the partial sequence of 16S rRNA gene (fragment length ranging from 474 to 708 bp). These isolates were members of the Arthrobacter genus, phylogenetically close to A. keyseri and A. ureafaciens, with sequence ranging from 99.1% to 100%. Furthermore, genetic variation between subpopulations from different samples was revealed by analysis on their randomly amplified polymorphic DNA profile. Nei genetic distance showed that the greatest differentiation occurred between subpopulation A and C. Notably, either genetic distance between subpopulations from the layers of 5–10 cm and 25–30 cm of each tailing pile or between same layers of different tailing pile increased with the history of tailings. Moreover, correlation analysis showed that soluble Pb has a significantly negative relationship with Nei’ gene diversity of subpopulation. It was assumed that soluble Pb may be responsible for the reduced genetic diversity of the Arthrobacter population. Our data provided evidence that genetic differentiation of microbial populations was consistent with the changes of environmental factors, particularly heavy metals. Translated from Acta Ecologica Sinica, 2005, 25(10): 2569–2573 [译自: 生态学报]     

Monthly change of community structure of zoobenthos in Xiangxi Bay after impoundment of the Three Gorges Teservoir, China

Xiangxi Bay, a typical bay of the Three Gorges Reservoir (TGR) in China, was formed in June 2003 after the impoundment of the TGR. We investigated the community structure of zoobenthos in Xiangxi Bay from August 2003 to August 2004. Benthic abundance and biomass were calculated per unit area of sediment. A total of 26 taxa were identified. They included two families of Lamellibranchia (7.7% of the total taxa number), two families of Oligochaeta (50%), Chironomidae (38.5%), and one other animal (3.8%). The average density and biomass were 276 ind./m² and 0.301 g/m², respectively. Oligochaetes and chironomids constituted the major groups, and Limnodrilus hoffmeisteri, Procladius sp., Branchiura sowerbyi, Tubificidae sp1. and Polypedilum scalaenum group sp. were the common taxa in this reservoir system. Among them, L. hoffmeisteri and Procladius sp. were the dominant species, and contributed 37.0% and 28.3% of the total density, respectively. The standing crop of zoobenthos in Xiangxi Bay was relative low, compared with other similar reservoirs. Our studies demonstrate that the standing crop increased gradually with time. The total density and biomass in August 2004 were 8.8 and 14.3 times those of the initial impoundment, separately. Oligochaetes did not become the sole dominant group, indicating that the sediment loading in Xiangxi Bay may be not serious. __________ Translated from Acta Hydrobiologica Sinica, 2006, 30(1): 64–69 [译自: 水生生物学报]     

Stimulation of ATG12-ATG5 conjugation by ribonucleic acid

The ubiquitin-like conjugation reactions, ATG8/microtubule-associated protein 1 light chain 3/MAP1LC3 (LC3) to phosphatidylethanolamine (PE) and ATG12 to ATG5, are biochemical hallmarks for autophagy, a cellular process that degrades bulk cellular proteins and organelles. The two conjugation reactions share the same E1-like enzyme ATG7 but have different E2-like enzymes, ATG3 for LC3-PE and ATG10 for ATG12-ATG5. In cells, ATG12-ATG5 conjugation appears to be required for LC3-PE conjugation. Previously, in vitro reconstitution of LC3-PE conjugation, but not the upstream ATG12-ATG5 conjugation, was reported. In this study, we describe for the first time the de novo reconstitution of mammalian ATG12-ATG5 conjugation by using purified recombinant proteins. We show that ATG7, ATG10 and ATP as an energy source are all essential for ATG12-ATG5 conjugation, and mutation of the specific lysine residue of ATG5 for ATG12 conjugation abrogates the reaction. Furthermore, a potent stimulating activity for ATG12-ATG5 conjugation was detected in mammalian cell extracts, and was surprisingly identified as ribosomes. Our detail biochemical analyses indicate that the ribonucleic acid (RNA) component of ribosomes is both necessary and sufficient for this stimulation.