The Functional Role of a Conserved Loop in EAL Domain-Based Cyclic di-GMP-Specific Phosphodiesterase

EAL domain-based cyclic di-GMP (c-di-GMP)-specific phosphodiesterases play important roles in bacteria by regulating the cellular concentration of the dinucleotide messenger c-di-GMP. EAL domains belong to a family of (β/α)₈ barrel fold enzymes that contain a functional active site loop (loop 6) for substrate binding and catalysis. By examining the two EAL domain-containing proteins RocR and PA2567 from Pseudomonas aeruginosa, we found that the catalytic activity of the EAL domains was significantly altered by mutations in the loop 6 region. The impact of the mutations ranges from apparent substrate inhibition to alteration of oligomeric structure. Moreover, we found that the catalytic activity of RocR was affected by mutating the putative phosphorylation site (D56N) in the phosphoreceiver domain, with the mutant exhibiting a significantly smaller Michealis constant (K_m) than that of the wild-type RocR. Hydrogen-deuterium exchange by mass spectrometry revealed that the decrease in K_m correlates with a change of solvent accessibility in the loop 6 region. We further examined Acetobacter xylinus diguanylate cyclase 2, which is one of the proteins that contains a catalytically incompetent EAL domain with a highly degenerate loop 6. We demonstrated that the catalytic activity of the stand-alone EAL domain toward c-di-GMP could be recovered by restoring loop 6. On the basis of these observations and in conjunction with the structural data of two EAL domains, we proposed that loop 6 not only mediates the dimerization of EAL domain but also controls c-di-GMP and Mg²⁺ ion binding. Importantly, sequence analysis of the 5,862 EAL domains in the bacterial genomes revealed that about half of the EAL domains harbor a degenerate loop 6, indicating that the mutations in loop 6 may represent a divergence of function for EAL domains during evolution.The cyclic dinucleotide cyclic di-GMP (c-di-GMP) has emerged as a major bacterial messenger for mediating a variety of cellular functions that range from virulence expression and biofilm formation (5, 14, 30). The cellular concentration of c-di-GMP is controlled by the GGDEF domain proteins with diguanylate cyclase (DGC) activity and the EAL domain proteins with c-di-GMP-specific phosphodiesterase (PDE) activity. GGDEF domains catalyze the synthesis of c-di-GMP from GTP, whereas EAL domains catalyze the hydrolysis of c-di-GMP to generate the linear 5′-pGpG. Although a family of HD-GYP domain proteins has also been found as c-di-GMP-specific PDEs, the overwhelmingly large number of genes encoding the EAL domains in bacterial genomes suggests that the EAL domains are the major PDEs for maintaining the cellular c-di-GMP concentration. Remarkably, multiple copies of EAL domain-encoding genes are usually found in bacterial cells, with as many as 21 in Pseudomonas aeruginosa and 32 in Vibrio cholerae. Although many of the EAL domains were found to function as PDE domains for c-di-GMP degradation, emerging evidence suggests that some EAL domains function as ligand- or protein-binding domains without catalytic activity (24, 28, 40).The detailed structure and catalytic mechanism of the EAL domains have started to be elucidated recently. The crystal structures of two proteins with EAL domains, TdEAL and YkuI, have been determined (Protein Data Bank accession nos. 2BAS, 2R6O, and 2w27) (23). EAL domains adopt a (β/α)₈ barrel fold that contains two extended strands, including an antiparallel strand. The (β/α)₈ barrel fold, first found in triosephosphate isomerase, has been observed in a diversity of enzymes that include many hydrolyases and isomerases (34). Similar to other (β/α)₈ barrel fold enzymes, the catalytic residues of the EAL domain are located at the C-terminal ends of the β-strands and the beginning of the β→α loops connecting the β-strands and α-helices. In the proposed mechanism, EAL domains catalyze the hydrolysis of c-di-GMP by using a Mg²⁺ ion and a general base catalyst (Glu) for generating the nucleophilic H₂O (28). The catalytic mechanism is supported by the crystal structure of the YkuI-substrate binary complex (Protein Data Bank accession no. 2w27) and the model of the TdEAL-substrate complex (23, 28). Both structures showed that the EAL domains bind c-di-GMP in such a configuration that the scissile phosphorus-oxygen bond aligns linearly with the attaching water and the general base catalyst. The catalytic mechanism can account for the lack of catalytic activity for most known inactive EAL domains, with the loss of enzymatic activity arising from the absence of the general base catalyst and/or the residues that coordinate the Mg²⁺ ion (28).It is well-known that many (β/α)₈ barrel fold enzymes contain a flexible active site loop between the β6 strand and α6 helix (34). Despite the diverse reactions catalyzed by (β/α)₈ barrel fold enzymes, this extended loop, often referred to as loop 6, plays an important role as a functional lid for substrate sequestering, solvent exclusion, and product release (15). The loop was found to facilitate substrate binding and conformational transition in tryptophan synthase (3, 4) and functions as a lid for substrate sequestering during catalysis in inosine 5′-monophosphate dehydrogenase (22). Notably, it was shown that the loop sways from the active site in the nonactive structure of ribulose-1,5-bisphosphate carboxylase but folds over to shield the active site from the solvent in the activated structure (21). Similar functions have also been proposed for loop 6 in other (β/α)₈ barrel fold enzymes, such as triosephosphate isomerase and phosphoriboxyl anthranilate isomerase (15, 25, 26). Hence, it seems that the functional role of loop 6 has been well preserved in (β/α)₈ barrel fold enzymes during evolution. The (β/α)₈ barrel folded EAL domains also contain an eight-residue loop between the β6 strand and α6 helix that seems to be critical for catalysis. Schmidt and coworkers (32) first noticed that the catalytically active EAL domains seem to contain a conserved motif that was later confirmed to contain loop 6 [DFG(T/A)GYSS] and one of the residues (Asp) for Mg²⁺ binding (28, 32). We previously noticed that mutation of the essential catalytic residues is usually accompanied by the degeneration of loop 6 in catalytically inactive EAL domains (28). Moreover, we observed that the mutation of a residue interacting with loop 6 in the EAL domain-containing RocR abolished enzymatic activity, which led us to postulate a critical role for loop 6 in catalysis (28).To elucidate the precise role played by loop 6 in c-di-GMP hydrolysis, we examined three EAL domain-containing proteins that include RocR, PA2567, and A. xylinus DGC2. The residues of loop 6 [DFG(A/T)SYSS] in RocR and PA2567 are well conserved, as observed in other catalytically active EAL domains. We show that mutations in the loop 6 region in RocR and PA2567 had significant effect on the structure and catalysis of the EAL domain. By using the method of hydrogen-deuterium (H/D) exchange-coupled mass spectrometry, we demonstrated that a single remote mutation in the phosphoreceiver domain of RocR caused correlated changes in loop 6 conformation and catalytic properties. We further show that the catalytic activity of the inactive EAL domain of A. xylinus DGC2 can be recovered by restoring loop 6. The functional roles of loop 6 in EAL domains in substrate binding and catalysis were discussed in conjunction with the structural data for two EAL domains.     

Soil drenching of paclobutrazol: An efficient way to improve quinoa performance under salinity

Salinity extent and severity is rising because of poor management practices on agricultural lands, possibility lies to grow salt‐tolerant crops with better management techniques. Therefore, a highly nutritive salt‐tolerant crop quinoa with immense potential to contribute for future food security was selected for this investigation. Soil drenching of paclobutrazol (PBZ; 20 mg l^?1) was used to understand the ionic relations, gaseous exchange characteristics, oxidative defense system and yield under saline conditions (400 mM NaCl) including normal (0 mM NaCl) and no PBZ (0 mg l^?1) as controls. The results revealed that salinity stress reduced the growth and yield of quinoa through perturbing ionic homeostasis with the consequences of overproduction of reactive oxygen species (ROS), oxidative damages and reduced photosynthesis. PBZ improved the quinoa performance through regulation of ionic homeostasis by decreasing Na⁺, Cl^?, while improving K⁺, Mg²⁺ and Ca²⁺ concentration. It also enhanced the antioxidative system including ascorbic acid, phenylalanine ammonia‐lyase, polyphenol oxidase and glutathione peroxidase, which scavenged the ROS (H₂O₂ and O₂^?‐) and lowered the oxidative damages (malondialdehyde level) under salinity in roots and more specifically in leaf tissues. The photosynthetic rate and stomatal conductance consequently improved (16 and 21%, respectively) in salt‐stressed quinoa PBZ‐treated compared to the non‐treated ones and contributed to the improvement of panicle length (33%), 100‐grain weight (8%) and grain yield (38%). Therefore, PBZ can be opted as a shotgun approach to improve quinoa performance and other crops under high saline conditions.     

Effect of herbivory on the growth and photosynthesis of replanted Calligonum caput-medusae saplings in an infertile arid desert

Replanted Calligonum caput-medusae saplings in the Tarim River watershed face short-term and frequent herbivory by goats, which can result in either growth inhibition or stimulation. The effects of herbivory on shrub saplings are unclear. We simulated herbivory with clipping to test two hypotheses. We hypothesized that (1) moderate herbivory may positively affect replanted shrub saplings due to overcompensatory growth and compensatory photosynthesis and that (2) high amounts of defoliation may change water availability and impair photosynthesis and growth of saplings. We applied four defoliation treatments (0, 30, 50, and 70 %) to 2-year-old C. caput-medusae saplings to test the effects of herbivory. Moderately defoliated (~30 %) saplings grew faster and had higher photosynthetic performance than controls; however, defoliation of 50 % or more reduced growth due to undercompensatory photosynthesis and reduced water availability. Non-photochemical quenching by thermal dissipation provided photoprotection when absorbed light energy used in PSII photochemistry was inhibited, reducing excess excitation energy and allowing saplings with high amounts of defoliation to maintain adequate photosystem functioning. This suggests that moderate herbivory of replanted shrubs used as forage in arid ecological restoration projects is feasible, but that uncontrolled grazing should be forbidden.     

Construction of BIBAC and BAC libraries from a variety of organisms for advanced genomics research

Large-insert BAC (bacterial artificial chromosome) and BIBAC (binary BAC) libraries are essential for modern genomics research for all organisms. We helped pioneer the BAC and BIBAC technologies, and by using them we have constructed hundreds of BAC and BIBAC libraries for different species of plants, animals, marine animals, insects, algae and microbes. These libraries have been used globally for different aspects of genomics research. Here we describe the procedure with the latest improvements that we have made and used for construction of BIBAC libraries. The procedure includes the preparation of BIBAC vectors, the preparation of clonable fragments of the desired size from the source DNA, the construction and transformation of BIBACs and, finally, the characterization and assembly of BIBAC libraries. We also specify the modifications necessary for construction of BAC libraries using the protocol. The entire protocol takes ～7 d.     

塔里木河下游植物群落的物种数量变化与生态系统动态研究

物种多样性是指物种及其集合体的生物学多样性。物种多样性研究的核心是物种的数量变化和物种的生物学多样性程度。本文根据野外采集的数据 ,运用Simpson指数、McIn tosh指数以及Margalef指数 ,对塔里木河下游英苏、阿布达勒、喀尔达依、阿拉干、依干不及麻等地区的物种多样性指数分别进行了计算 ,并根据计算得到的生物多样性指数探讨了干旱区退化生态系统的物种多样性以及在干旱区物种多样性与生态系统稳定性之间的关系。结果表明 ,塔里木河下游从英苏至依干不及麻 ,Simpson多样性指数的变化范围为 0 82～ 0 2 6 ,McIn tosh均匀度指数的变化范围为 0 6 0～ 0 1 8,Margalef丰富度指数的变化范围为 1 4 7～ 0 38,物种数的变化范围是 9～ 2。分析表明塔里木河下游生态系统退化十分严重 ,并据此讨论了塔里木河下游生态系统退化的特征、演替动态及稳定性     

A low‐cost,automated parasite diagnostic system via a portable,robotic microscope and deep learning

Manual hand counting of parasites in fecal samples requires costly components and substantial expertise, limiting its use in resource‐constrained settings and encouraging overuse of prophylactic medication. To address this issue, a cost‐effective, automated parasite diagnostic system that does not require special sample preparation or a trained user was developed. It is composed of an inexpensive (~US$350), portable, robotic microscope that can scan over the size of an entire McMaster chamber (100 mm²) and capture high‐resolution (~1 μm lateral resolution) bright field images without need for user intervention. Fecal samples prepared using the McMaster flotation method were imaged, with the imaging region comprising the entire McMaster chamber. These images are then automatically segmented and analyzed using a trained convolution neural network (CNN) to robustly separate eggs from background debris. Simple postprocessing of the CNN output yields both egg species and egg counts. The system was validated by comparing accuracy with hand‐counts by a trained operator, with excellent performance. As a further demonstration of utility, the system was used to conveniently quantify drug response over time in a single animal, showing residual disease due to Anthelmintic resistance after 2 weeks.     

荒漠防护林典型树种液流特征及其对环境因子的响应

利用基于热补偿理论的SF300分体液流仪对干旱荒漠区人工防护林典型树种(俄罗斯杨、胡杨、榆树、沙枣)树干液流全天候监测,自动气象站同步记录相关环境因子变化。研究表明:①4种防护林树种茎干液流日变化除沙枣树外均存在明显昼夜节律,液流速度在同属种间差异较小,在不同属种间差异显著,俄罗斯杨的日平均液流速度可以达到沙枣的13.8倍,耗水量排序为俄罗斯杨胡杨榆树沙枣树。②水分充足条件下,增加实验地灌溉量使4种树木蒸腾受到抑制,液流流速降低,水分利用效率降低。③液流流速因所处树干径向位点不同而存在差异,俄罗斯杨、榆树、沙枣液流速度表现出由形成层到髓心的递减趋势,胡杨树干径向位点液流没有表现一定规律。④树干液流流速与环境因子进行相关分析,通过逐步回归分析建立了4个典型树种茎干液流速度与环境因子关系估算模型,分析认为4种树木的环境敏感性排序为俄罗斯杨榆树胡杨沙枣。     

大鼠回肠肌间神经丛一氧化氮合酶和乙酰胆碱酯酶双重显示法研究

本文用一氧化氮合酶和乙酰胆碱酯酶双重显示法,对大鼠回肠肌间神经丛进行了组织化学观察,结果发现三种不同染色的神经元：（１）乙酰胆碱酯酶阳性神经元（占８２％）;（２）一氧化氮合酶阳性神经元（占１６％）;（３）一氧化氮合酶和乙酰胆碱酯酶阳性神经元（占２％）。以上结果提示,一氧化氮可以与乙酰胆碱共存于大鼠回肠肌间神经丛的少数神经元内。本文还对肠肌间神经丛内神经元的类型和一氧化氮的作用进行了讨论。     

基于WRF模式评估土地利用/覆被变化的气候和水文效应

目前针对土地利用/覆被变化(LUCC)导致产汇流等水文过程(直接水文效应)变化的研究较多,而对于LUCC导致区域气候变化引起的径流变化(间接水文效应)研究却鲜有报道。采用天气预报模式(Weather Research and Forecasting Model,WRF模式)和弹性系数等方法研究了沂河流域1990—2010年LUCC产生的间接水文效应。结果表明:WRF模式对研究区气温具有较好的模拟能力,模拟值与实测值的相关系数较高(0.86—0.97,P0.001);虽然模式对降水的模拟精度低于气温,但模拟值与实测值之间的相关系数(0.41—0.91)均达到了P0.05显著性水平。近20年来,研究区LUCC主要是从旱地向建设用地(747.3km~2)和裸地(132.4km~2)转化的过程。LUCC引起2013年1月和10月气温增加了0.2℃,导致7月气温减小了0.2℃,而4月气温基本稳定。LUCC对1月、4月和10月降水变化的影响很弱,而对7月降水变化影响较大,表现在使其减少了23.7mm。弹性分析表明,1960—2013年,流域年均降水和气温变化1%,将引起年径流量分别变化2.4%和1.8%。1990—2010年,LUCC引起2013年沂河流域降水和气温变化使得径流量分别改变了18.4%和1.7%。     

Interference of the Histone Deacetylase Inhibits Pollen Germination and Pollen Tube Growth in Picea wilsonii Mast

Histone deacetylase (HDAC) is a crucial component in the regulation of gene expression in various cellular processes in animal and plant cells. HDAC has been reported to play a role in embryogenesis. However, the effect of HDAC on androgamete development remains unclear, especially in gymnosperms. In this study, we used the HDAC inhibitors trichostatin A (TSA) and sodium butyrate (NaB) to examine the role of HDAC in Picea wilsonii pollen germination and pollen tube elongation. Measurements of the tip-focused Ca²⁺ gradient revealed that TSA and NaB influenced this gradient. Immunofluorescence showed that actin filaments were disrupted into disorganized fragments. As a result, the vesicle trafficking was disturbed, as determined by FM4-64 labeling. Moreover, the distribution of pectins and callose in cell walls was significantly altered in response to TSA and NaB. Our results suggest that HDAC affects pollen germination and polarized pollen tube growth in Picea wilsonii by affecting the intracellular Ca²⁺ concentration gradient, actin organization patterns, vesicle trafficking, as well as the deposition and configuration of cell wall components.