Detection of genes involved in biodegradation and biotransformation in microbial communities by using 50-mer oligonucleotide microarrays

To effectively monitor biodegrading populations, a comprehensive 50-mer-based oligonucleotide microarray was developed based on most of the 2,402 known genes and pathways involved in biodegradation and metal resistance. This array contained 1,662 unique and group-specific probes with <85% similarity to their nontarget sequences. Based on artificial probes, our results showed that under hybridization conditions of 50 degrees C and 50% formamide, the 50-mer microarray hybridization can differentiate sequences having <88% similarity. Specificity tests with representative pure cultures indicated that the designed probes on the arrays appeared to be specific to their corresponding target genes. The detection limit was approximately 5 to 10 ng of genomic DNA in the absence of background DNA and 50 to 100 ng of pure-culture genomic DNA in the presence of background DNA or 1.3 x 10(7) cells in the presence of background RNA. Strong linear relationships between the signal intensity and the target DNA and RNA were observed (r(2) = 0.95 to 0.99). Application of this type of microarray to analyze naphthalene-amended enrichment and soil microcosms demonstrated that microflora changed differently depending on the incubation conditions. While the naphthalene-degrading genes from Rhodococcus-type microorganisms were dominant in naphthalene-degrading enrichments, the genes involved in naphthalene (and polyaromatic hydrocarbon and nitrotoluene) degradation from gram-negative microorganisms, such as Ralstonia, Comamonas, and Burkholderia, were most abundant in the soil microcosms. In contrast to general conceptions, naphthalene-degrading genes from Pseudomonas were not detected, although Pseudomonas is widely known as a model microorganism for studying naphthalene degradation. The real-time PCR analysis with four representative genes showed that the microarray-based quantification was very consistent with real-time PCR (r(2) = 0.74). In addition, application of the arrays to both polyaromatic-hydrocarbon- and benzene-toluene-ethylbenzene-xylene-contaminated and uncontaminated soils indicated that the developed microarrays appeared to be useful for profiling differences in microbial community structures. Our results indicate that this technology has potential as a specific, sensitive, and quantitative tool in revealing a comprehensive picture of the compositions of biodegradation genes and the microbial community in contaminated environments, although more work is needed to improve detection sensitivity.     

Salinity Regulation of the Interaction of Halovirus SNJ1 with Its Host and Alteration of the Halovirus Replication Strategy to Adapt to the Variable Ecosystem

Halovirus is a major force that affects the evolution of extreme halophiles and the biogeochemistry of hypersaline environments. However, until now, the systematic studies on the halovirus ecology and the effects of salt concentration on virus-host systems are lacking. To provide more valuable information for understanding ecological strategies of a virus-host system in the hypersaline ecosystem, we studied the interaction between halovirus SNJ1 and its host Natrinema sp.J7-2 under various NaCl concentrations. We found that the adsorption rate and lytic rate increased with salt concentration, demonstrating that a higher salt concentration promoted viral adsorption and proliferation. Contrary to the lytic rate, the lysogenic rate decreased as the salt concentration increased. Our results also demonstrated that cells incubated at a high salt concentration prior to infection increased the ability of the virus to adsorb and lyse its host cells; therefore, the physiological status of host cells also affected the virus-host interaction. In conclusion, SNJ1 acted as a predator, lysing host cells and releasing progeny viruses in hypersaline environments; in low salt environments, viruses lysogenized host cells to escape the damage from low salinity.     

OsGL1-3 is Involved in Cuticular Wax Biosynthesis and Tolerance to Water Deficit in Rice

Cuticular wax covers aerial organs of plants and functions as the outermost barrier against non-stomatal water loss. We reported here the functional characterization of the Glossy1(GL1)-homologous gene OsGL1-3 in rice using overexpression and RNAi transgenic rice plants. OsGL1-3 gene was ubiquitously expressed at different level in rice plants except root and its expression was up-regulated under ABA and PEG treatments. The transient expression of OsGL1-3–GFP fusion protein indicated that OsGL1-3 is mainly localized in the plasma membrane. Compared to the wild type, overexpression rice plants exhibited stunted growth, more wax crystallization on leaf surface, and significantly increased total cuticular wax load due to the prominent changes of C₃₀–C₃₂ aldehydes and C₃₀ primary alcohols. While the RNAi knockdown mutant of OsGL1-3 exhibited no significant difference in plant height, but less wax crystallization and decreased total cuticular wax accumulation on leaf surface. All these evidences, together with the effects of OsGL1-3 on the expression of some wax synthesis related genes, suggest that OsGL1-3 is involved in cuticular wax biosynthesis. Overexpression of OsGL1-3 decreased chlorophyll leaching and water loss rate whereas increased tolerance to water deficit at both seedling and late-tillering stages, suggesting an important role of OsGL1-3 in drought tolerance.     

Specific inhibition effects of N-pentafluorobenzyl-1-deoxynojirimycin on human CD4+ T cells

We first synthesized N-pentafluorobenzyl-1-deoxynojirimycin (5F-DNM), one new derivative of 1-deoxynojirimycin (DNM). Effects on human peripheral blood mononuclear cells (PMBC) and secretion of cytokines from human PBMC by 5F-DNM were investigated. It was first found that 5F-DNM remarkably inhibited the secretion of interleukin-4 (IL-4) and had a specific inhibition on the expression of CD4 molecules. 5F-DNM, much less toxic than cyclosporin A, might be used as a new candidate of immunosuppressant for specifically treating Th2-mediated immune diseases.     

Validation Study to Determine the Accuracy of Widespread Promoterless EGFP Reporter at Assessing CRISPR/Cas9-Mediated Homology Directed Repair

An accurate visual reporter system to assess homology-directed repair (HDR) is a key prerequisite for evaluating the efficiency of Cas9-mediated precise gene editing. Herein, we tested the utility of the widespread promoterless EGFP reporter to assess the efficiency of CRISPR/Cas9-mediated homologous recombination by fluorescence expression. We firstly established a promoterless EGFP reporter donor targeting the porcine GAPDH locus to study CRISPR/Cas9-mediated homologous recombination in porcine cells. Curiously, EGFP was expressed at unexpectedly high levels from the promoterless donor in porcine cells, with or without Cas9/sgRNA. Even higher EGFP expression was detected in human cells and those of other species when the porcine donor was transfected alone. Therefore, EGFP could be expressed at certain level in various cells transfected with the promoterless EGFP reporter alone, making it a low-resolution reporter for measuring Cas9-mediated HDR events. In summary, the widespread promoterless EGFP reporter could not be an ideal measurement for HDR screening and there is an urgent need to develop a more reliable, high-resolution HDR screening system to better explore strategies of increasing the efficiency of Cas9-mediated HDR in mammalian cells.     

布氏田鼠摄食量、累积摄食量与日龄的关系

布氏田鼠 (Ｍｉｃｒｏｔｕｓｂｒａｎｄｔｉ)是内蒙古典型草原区的主要害鼠 ,其危害方式主要表现之一为与牲畜争夺牧草资源[1,4 ,11,12 ] 。准确地测定其日食量(ＤＦＣ)与累积摄食量 (ＣＦＣ) ,对于定量地衡量该鼠的危害程度 ,进一步推算鼠害防治的经济阈值有着重要的意义。一些学者着手有关该鼠食性与食量 ,以及非取食性牧草消耗量的研究工作[3 ,7～ 9] 。但由于受实验手段的限制 ,一直未能解决布氏田鼠日食量、累积摄食量与田鼠年龄的关系等难题。本文以测定典型草原区布氏田鼠日食量与体重的关系 ,结合布氏田鼠体重与年龄的数学关系方程 …     

蛋白质错误折叠循环扩增及其在朊病毒蛋白检测中的应用研究进展

朊病毒蛋白（prion protein,PrP）是传染性海绵状脑病的病原体,其检测是该病诊断的重要依据。该文从原理、方法、影响因素和检测应用方面对蛋白质错误折叠循环扩增（protein mis-folding cyclic amplification,PMCA）这种朊病毒蛋白新型检测技术做了介绍,旨在为朊病毒蛋白的检测和发病机制研究提供理论参考。     

Using cassava distiller’s dried grains as carbon and microbe sources to enhance denitrification of nitrate-contaminated groundwater

Nitrate removal from synthetic and real groundwater was investigated by using cassava distiller’s dried grains (CDDG), which served as sole carbon source as well as the only microbe seed. It was found that remarkably higher total nitrogen removal efficiency (96.8±0.6 %) could be reached; the accumulation of nitrite and the releases of organic compounds, meanwhile, were insignificant in the denitrification process. Scanning electron microscope (SEM) analysis showed that CDDG were degraded during the denitrification process. Further investigation showed that CDDG were anaerobically hydrolyzed and acidified to butyric acid, acetic acid, and carbohydrate, which could be utilized directly as the reducing equivalent providers for denitrification by the microorganisms separated from CDDG. Microbial community analysis revealed that the fungi and bacteria present in the original CDDG functioned as the denitrifiers, which mainly consisted of Aspergillus (69.8 %) and Rhizomucor (15.9 %) in the fungi community and Burkholderia (20.6 %) and Rhizobium (15.9 %) in the bacteria community, respectively. Finally, the use of CDDG as both carbon and microbial sources for real groundwater denitrification was testified to be feasible and safe with a total nitrogen removal efficiency of around 100 %.     

Interpretation of gas residence time distributions in large airlift tower loop reactors

Gas-residence time distribution (RTD) response curves measured in a 23 m high pilot plant airlift tower loop reactor, which consisted of a riser, a special downcomer construction and at the top of the riser a large head. The measurements were evaluated by means of a deterministic dispersion model, which yielded the following particular parameters for the riser, downcomer and the head: Gas-Bo numbers, gas-mean residence times, gas holdups, liquid velocities, gas and liquid circulation times as well as a fraction of the large and small bubbles in a model medium (water) and during cultivation of baker's yeast.List of Symbols A cross section - Bo Bodenstein number - Bo _d (= l _d w _G,d /D _d ) - Bo _h (= l _h w _G,h /D _h ) - Bo _r (= l _r w _G,r /D _r ) - D longitudinal dispersion coefficient - E gas holdup - E(t) RTD-density function - L, l length parameter - q fraction of the gas throughput which is not recirculated (approximately equal to fraction of the large bubbles) - r fraction of the throughput which is recirculated (approximately equal to the fraction of the small bubbles) - t _c circulation time - t _cL liquid circulation time - t _c,L ^* liquid circulation time calculated from the measured w _Ld in the downcomer - V ^h hydrodynamical calculated gas-liquid volume - V _d ^h (=V _d+0.75/2 V _k ) - V _k ^h =(0.25V _k ) - V _r ^h = (V _r+0.75/2 V _k ) - V _L liquid volume - V _G dispersed gas volume - V _G ^* gas throughput at the gas distributor (given in m³/h) under standard conditions, 1 bar and 25°C) - V _G,d ^* gas throughput in downcomer (=V _G ^* ) - V _G,h ^* gas throughput in head (=V _G ^* ) - V _G,r ^* gas throughput in riser (V _G ^* (1+) - w _g gas velocity - w _G,rel relative gas velocity with respect to the liquid velocity w _L - w _G,d gas velocity in the downcomer (=w _G,rel –w _Ld ) - w _G,h gas velocity in the head (=w _G,rel ) (since wLh = o) - w _G,r gas velocity in the riser (=w _G,rel +w _Lr ) - w _L liquid velocity - w _L,d liquid velocity in the downcomer measured with mass flow meter - w _sg ·w _SL superficial gas and liquid velocities - first moment of the response curve - mean residence time Indices d downcomer - G gas phase - h head - L liquid phase - r riser - h hydrodynamic (upper position) Dedicated to the 65th birthday of Proffessor Fritz Wagner.The authors gratefully acknowledge the financial support by the Krupp Industrietechnik, Grevenbroich and the support of Pleser Co, Darmstadt. H. M. Rüffer thanks the Verband der Chemischen Industrie for a Fond der Chemie scholarship, and W. Liwei thanks the government of Lower Saxony for a graduate scholarship.     

Structural and functional insights into the modulation of the activity of a flax cytokinin oxidase by flax rust effector AvrL567-A

During infection, plant pathogens secrete effector proteins to facilitate colonization. In comparison with our knowledge of bacterial effectors, the current understanding of how fungal effectors function is limited. In this study, we show that the effector AvrL567-A from the flax rust fungus Melampsora lini interacts with a flax cytosolic cytokinin oxidase, LuCKX1.1, using both yeast two-hybrid and in planta bimolecular fluorescence assays. Purified LuCKX1.1 protein shows catalytic activity against both N6-(Δ2-isopentenyl)-adenine (2iP) and trans-zeatin (tZ) substrates. Incubation of LuCKX1.1 with AvrL567-A results in increased catalytic activity against both substrates. The crystal structure of LuCKX1.1 and docking studies with AvrL567-A indicate that the AvrL567 binding site involves a flexible surface-exposed region that surrounds the cytokinin substrate access site, which may explain its effect in modulating LuCKX1.1 activity. Expression of AvrL567-A in transgenic flax plants gave rise to an epinastic leaf phenotype consistent with hormonal effects, although no difference in overall cytokinin levels was observed. We propose that, during infection, plant pathogens may differentially modify the levels of extracellular and intracellular cytokinins.