Microbial Communities and Functional Genes Associated with Soil Arsenic Contamination and the Rhizosphere of the Arsenic-Hyperaccumulating Plant Pteris vittata L.

To understand how microbial communities and functional genes respond to arsenic contamination in the rhizosphere of Pteris vittata, five soil samples with different arsenic contamination levels were collected from the rhizosphere of P. vittata and nonrhizosphere areas and investigated by Biolog, geochemical, and functional gene microarray (GeoChip 3.0) analyses. Biolog analysis revealed that the uncontaminated soil harbored the greatest diversity of sole-carbon utilization abilities and that arsenic contamination decreased the metabolic diversity, while rhizosphere soils had higher metabolic diversities than did the nonrhizosphere soils. GeoChip 3.0 analysis showed low proportions of overlapping genes across the five soil samples (16.52% to 45.75%). The uncontaminated soil had a higher heterogeneity and more unique genes (48.09%) than did the arsenic-contaminated soils. Arsenic resistance, sulfur reduction, phosphorus utilization, and denitrification genes were remarkably distinct between P. vittata rhizosphere and nonrhizosphere soils, which provides evidence for a strong linkage among the level of arsenic contamination, the rhizosphere, and the functional gene distribution. Canonical correspondence analysis (CCA) revealed that arsenic is the main driver in reducing the soil functional gene diversity; however, organic matter and phosphorus also have significant effects on the soil microbial community structure. The results implied that rhizobacteria play an important role during soil arsenic uptake and hyperaccumulation processes of P. vittata.Arsenic (As) is an abundant and widespread trace metalloid element present in virtually all environmental media and is well known to be carcinogenic even at low levels (24). Arsenic contaminations in soil and groundwater have been reported in many parts of the world (2, 29, 34). Recently, in parts of Asia, including China, chronic drinking of arsenic-contaminated groundwater has caused endemic arsenicosis, which has become a major threat to public health (36). Soil arsenic contamination also affects the physiology, growth, and grain quality of crops. For example, high arsenic concentrations were found in rice seeds from Chenzhou, Hunan province, which exceeded the maximal permissible limit of 0.5 mg/kg (dry weight) (21). Hence, remediation of arsenic-contaminated soil and water is one of the major challenges in environmental science and public health. Low-cost, efficient, and environmentally friendly remediation technologies to remove arsenic from contaminated soil and water are urgently needed.Phytoremediation, the use of plants to restore contaminated soil, has attracted great attention recently. A pivotal step toward the phytoremediation of arsenic-contaminated soils is the discovery of the arsenic hyperaccumulator Pteris vittata L. (Chinese brake fern), which possesses high arsenic tolerance and produces a large biomass. This plant species holds great promise for the phytoremediation of arsenic-contaminated soils. It was shown previously that the leaflets of P. vittata were able to accumulate about 100-fold of arsenic from soils (22). Plant arsenic uptake depends mainly on the arsenic source and bioavailability (25). P. vittata remediates arsenic contamination mainly by taking up arsenate [As(V)] via phosphate transport systems, whereas arsenite [As(III)] is very slowly taken up by P. vittata, at 1/10 of the rate of that for arsenate in the absence of phosphate (41). However, the uptake mechanisms still remain largely unknown.Microorganisms play a crucial role in arsenic geochemical cycling through microbial transformation processes, including reduction, oxidation, and methylation (2, 11, 31, 33, 40). Although the impacts of microbial metabolisms were previously reported to be associated with arsenic cycling of soil and water (7, 29), little is known about how rhizobacterial communities of P. vittata respond to arsenic. Recently, we found that inoculating arsenic resistance bacteria increased the arsenic accumulation efficiency of P. vittata by 13 to 110% (46). Therefore, rhizobacteria may play an important role during arsenic uptake and accumulation processes by P. vittata. Thus, it is important to elucidate the microbially diverse populations and functional genes associated with arsenic mobility and transport in the P. vittata rhizosphere. However, to fully understand the ecology of such complex rhizosphere-contaminated soils, it is necessary to analyze different microbial populations simultaneously.Our hypothesis is that the arsenic-hyperaccumulating ability of P. vittata is due to the interactions among plants, rhizobacteria, and arsenic. A study of microbial communities present in the plant rhizosphere is important to illustrate the mechanisms of arsenic hyperaccumulation in P. vittata. Thus, the objectives of this research were to understand how microbial metabolic diversities, communities, and functional genes/relative abundances were affected by soil arsenic contamination and the P. vittata rhizosphere environment. To determine the soil microbial metabolic diversity, the Biolog system (Biolog, Carlsbad, CA) was used to analyze the sole-carbon-source-utilizing capabilities of the soil microbial communities. For functional gene analysis, a high-density, sensitive, oligonucleotide-based microarray (GeoChip 3.0) was used. GeoChip-based technologies have revealed the structure, metabolic activity, and dynamics of microbial communities from complex environments, such as soil, sediments, and groundwater (10, 38, 39, 45, 48). Our results provide evidence that changes of microbial community structure, functional gene distribution, and microbial metabolic diversity are associated with the soil arsenic level and the rhizosphere effect of P. vittata and suggest that plant phytoremediation is an interactive process among plants, microorganisms, and soil contaminants.     

PMS‐1077, a PAF antagonist,induced differentiation of HL‐60 cells with its novel activity

The cell differentiation‐inducing effect of 2‐N,N‐diethylaminocarbonyloxymethyl‐1 ‐diphenylmethyl‐4‐(3,4,5‐trimethoxybenzoyl) piperazine, hydrochloride (PMS‐1077) was determined in human leukaemic HL‐60 cells with profiling of cell proliferation, analysis of cell cycling, characterization of expression of various CD molecules and determination of phagocytotic activity of differentiated HL‐60 cells. After treatment with PMS‐1077, HL‐60 cells exhibited a decreased cell viability during which cell cycle was arrested in G₀‐/G₁‐phase. Flow cytometric analysis showed CD11b and CD14 were up‐regulated, whereas CD15 was unaffected. Together with the finding that PMS‐1077‐treated HL‐60 cells exhibited activities of differentiation by examining their ability of phagocytosing latex beads, an antiproliferative effect and a differentiation‐inducing role were determined for PMS‐1077 in HL‐60 cells.     

Protein structure prediction enhanced with evolutionary diversity: SPEED

For naturally occurring proteins, similar sequence implies similar structure. Consequently, multiple sequence alignments (MSAs) often are used in template‐based modeling of protein structure and have been incorporated into fragment‐based assembly methods. Our previous homology‐free structure prediction study introduced an algorithm that mimics the folding pathway by coupling the formation of secondary and tertiary structure. Moves in the Monte Carlo procedure involve only a change in a single pair of ?,ψ backbone dihedral angles that are obtained from a Protein Data Bank‐based distribution appropriate for each amino acid, conditional on the type and conformation of the flanking residues. We improve this method by using MSAs to enrich the sampling distribution, but in a manner that does not require structural knowledge of any protein sequence (i.e., not homologous fragment insertion). In combination with other tools, including clustering and refinement, the accuracies of the predicted secondary and tertiary structures are substantially improved and a global and position‐resolved measure of confidence is introduced for the accuracy of the predictions. Performance of the method in the Critical Assessment of Structure Prediction (CASP8) is discussed.     

Discriminative learning for protein conformation sampling

Protein structure prediction without using templates (i.e., ab initio folding) is one of the most challenging problems in structural biology. In particular, conformation sampling poses as a major bottleneck of ab initio folding. This article presents CRFSampler, an extensible protein conformation sampler, built on a probabilistic graphical model Conditional Random Fields (CRFs). Using a discriminative learning method, CRFSampler can automatically learn more than ten thousand parameters quantifying the relationship among primary sequence, secondary structure, and (pseudo) backbone angles. Using only compactness and self-avoiding constraints, CRFSampler can efficiently generate protein-like conformations from primary sequence and predicted secondary structure. CRFSampler is also very flexible in that a variety of model topologies and feature sets can be defined to model the sequence-structure relationship without worrying about parameter estimation. Our experimental results demonstrate that using a simple set of features, CRFSampler can generate decoys with much higher quality than the most recent HMM model.     

Redifferentiation of human hepatoma cells (SMMC-7721) induced by two new highly oxygenated bisabolane-type sesquiterpenes

Bisabolane-type sesquiterpenes are a class of biologically active compounds that has antitumour, antifungal, antibacterial, antioxidant and antivenom properties. We investigated the effect of two new highly oxygenated bisabolane-type sesquiterpenes (HOBS) isolated from Cremanthodium discoideum (C. discoideum) on tumour cells. Our results showed that HOBS induced morphological differentiation and reduced microvilli formation on the cell surface in SMMC-7721 cells. Flow cytometry analysis demonstrated that HOBS could induce cell-cycle arrest in the G1 phase. Moreover, HOBS was able to increase tyrosine-α-ketoglutarate transaminase activity, decrease α foetoprotein level and γ-glutamyl transferase activity. In addition, we found that HOBS inhibited the anchorageindependent growth of SMMC-7721 cells in a dose-dependent manner. Taken together, all the above observations indicate that HOBS might be able to normalize malignant SMMC-7721 cells by inhibiting cell proliferation and inducing redifferentiation.     

Myristoylation-regulated direct interaction between calcium-bound calmodulin and N-terminal region of pp60v-src

pp60v-src tyrosine protein kinase was suggested to interact with Ca2+-bound calmodulin (Ca2+/CaM) through the N-terminal region based on its structural similarities to CAP-23/NAP-22, a myristoylated neuron-specific protein, whose myristoyl group is essential for interaction with Ca2+/CaM; (1) the N terminus of pp60v-src is myristoylated like CAP-23/NAP-22; (2) both lysine residues are required for the myristoylation-dependent interaction and serine residues that are thought to regulate the interaction through the phosphorylations located in the N-terminal region of pp60v-src. To verify this possibility, we investigated the direct interaction between pp60v-src and Ca2+/CaM using a myristoylated peptide corresponding to the N-terminal region of pp60v-src. The binding assay indicated that only the myristoylated peptide binds to Ca2+/CaM, and the non-myristoylated peptide is not able to bind to Ca2+/CaM. Analyses of the binding kinetics revealed two independent reactions with the dissociation constants (KD) of 2.07 x 10(-9)M (KD1) and 3.93 x 10(-6)M (KD2), respectively. Two serine residues near the myristoyl moiety of the peptide (Ser2, Ser11) were phosphorylated by protein kinase C in vitro, and the phosphorylation drastically reduced the interaction. NMR experiments indicated that two molecules of the myristoylated peptide were bound around the hydrophobic clefts of a Ca2+/CaM molecule. The small-angle X-ray scattering analyses showed that the size of the peptide-Ca2+/CaM complex is 2-3A smaller than that of the known Ca2+/CaM-target molecule complexes. These results demonstrate clearly the direct interaction between pp60v-src and Ca2+/CaM in a novel manner different from that of known Ca2+/CaM, the target molecules, interactions.     

RAPTOR: optimal protein threading by linear programming

This paper presents a novel linear programming approach to do protein 3-dimensional (3D) structure prediction via threading. Based on the contact map graph of the protein 3D structure template, the protein threading problem is formulated as a large scale integer programming (IP) problem. The IP formulation is then relaxed to a linear programming (LP) problem, and then solved by the canonical branch-and-bound method. The final solution is globally optimal with respect to energy functions. In particular, our energy function includes pairwise interaction preferences and allowing variable gaps which are two key factors in making the protein threading problem NP-hard. A surprising result is that, most of the time, the relaxed linear programs generate integral solutions directly. Our algorithm has been implemented as a software package RAPTOR-RApid Protein Threading by Operation Research technique. Large scale benchmark test for fold recognition shows that RAPTOR significantly outperforms other programs at the fold similarity level. The CAFASP3 evaluation, a blind and public test by the protein structure prediction community, ranks RAPTOR as top 1, among individual prediction servers, in terms of the recognition capability and alignment accuracy for Fold Recognition (FR) family targets. RAPTOR also performs very well in recognizing the hard Homology Modeling (HM) targets. RAPTOR was implemented at the University of Waterloo and it can be accessed at http://www.cs.uwaterloo.ca/~j3xu/RAPTOR_form.htm.     

Fold recognition by predicted alignment accuracy

One of the key components in protein structure prediction by protein threading technique is to choose the best overall template for a given target sequence after all the optimal sequence-template alignments are generated. The chosen template should have the best alignment with the target sequence since the three-dimensional structure of the target sequence is built on the sequence-template alignment. The traditional method for template selection is called Z-score, which uses a statistical test to rank all the sequence-template alignments and then chooses the first-ranked template for the sequence. However, the calculation of Z-score is time-consuming and not suitable for genome-scale structure prediction. Z-scores are also hard to interpret when the threading scoring function is the weighted sum of several energy items of different physical meanings. This paper presents a support vector machine (SVM) regression approach to directly predict the alignment accuracy of a sequence-template alignment, which is used to rank all the templates for a specific target sequence. Experimental results on a large-scale benchmark demonstrate that SVM regression performs much better than the composition-corrected Z-score method. SVM regression also runs much faster than the Z-score method.     

Dynamics of dendritic spines and their afferent terminals: spines are more motile than presynaptic boutons

Previous work has established that dendritic spines, sites of excitatory input in CNS neurons, can be highly dynamic, in later development as well as in mature brain. Although spine motility has been proposed to facilitate the formation of new synaptic contacts, we have reported that spines continue to be dynamic even if they bear synaptic contacts. An outstanding question related to this finding is whether the presynaptic terminals that contact dendritic spines are as dynamic as their postsynaptic targets. Using multiphoton time-lapse microscopy of GFP-labeled Purkinje cells and DiI-labeled granule cell parallel fiber afferents in cerebellar slices, we monitored the dynamic behavior of both presynaptic terminals and postsynaptic dendritic spines in the same preparation. We report that while spines are dynamic, the presynaptic terminals they contact are quite stable. We confirmed the relatively low levels of presynaptic terminal motility by imaging parallel fibers in vivo. Finally, spine motility can occur when a functional presynaptic terminal is apposed to it. These analyses further call into question the function of spine motility, and to what extent the synapse breaks or maintains its contact during the movement of the spine.