Evaluation of the environmental specificity of Fluorescence In Situ Hybridization (FISH) using Fluorescence-Activated Cell Sorting (FACS) of probe (PSE1284)-positive cells extracted from rhizosphere soil

We explicitly tested for the first time the ‘environmental specificity’ of traditional 16S rRNA-targeted Fluorescence In Situ Hybridization (FISH) through comparison of the bacterial diversity actually targeted in the environment with the diversity that should be exactly targeted (i.e. without mismatches) according to in silico analysis. To do this, we exploited advances in modern Flow Cytometry that enabled improved detection and therefore sorting of sub-micron-sized particles and used probe PSE1284 (designed to target Pseudomonads) applied to Lolium perenne rhizosphere soil as our test system. The 6-carboxyfluorescein (6-FAM)-PSE1284-hybridized population, defined as displaying enhanced green fluorescence in Flow Cytometry, represented 3.51 ± 1.28% of the total detected population when corrected using a nonsense (NON-EUB338) probe control. Analysis of 16S rRNA gene libraries constructed from Fluorescence Activated Cell Sorted-recovered fluorescent populations (n = 3), revealed that 98.5% (Pseudomonas spp. comprised 68.7% and Burkholderia spp. 29.8%) of the total sorted population was specifically targeted as evidenced by the homology of the 16S rRNA sequences to the probe sequence. In silico evaluation of probe PSE1284 with the use of RDP-10 probeMatch justified the existence of Burkholderia spp. among the sorted cells. The lack of novelty in Pseudomonas spp. sequences uncovered was notable, probably reflecting the well-studied nature of this functionally important genus. To judge the diversity recorded within the FACS-sorted population, rarefaction and DGGE analysis were used to evaluate, respectively, the proportion of Pseudomonas diversity uncovered by the sequencing effort and the representativeness of the Nycodenz^® method for the extraction of bacterial cells from soil.     

Use of the β‐function to estimate the skewness of species responses

Abstract. Response of a species to an environmental variable may be modeled and predicted using a wide spectrum of different functions. Contrary to other functions (Gaussian, polynomial etc), all parameters of the β‐function are interpretable in ecological terms. However, computational difficulties in the determination of the β‐function parameters initiated controversial debates on the applicability and usefulness of this function in vegetation modelling and gradient analysis. We propose a simple algorithm for fitting the β‐function to observed data. Analytic properties of the algorithm (its ability to recover the known species responses along gradients) are tested using a series of simulated data. In most cases the algorithm correctly estimated parameters of the simulated responses.     

A simple method to improve probe set estimates from oligonucleotide arrays

A popular commercially available oligonucleotide microarray technology employs sets of 25 base pair oligonucleotide probes for measurement of gene expression levels. A mathematical algorithm is required to compute an estimate of gene expression from the multiple probes. Previously proposed methods for summarizing gene expression data have either been substantially ad hoc or have relied on model assumptions that may be easily violated. Here we present a new algorithm for calculating gene expression from probe sets. Our approach is functionally related to leave-one-out cross-validation, a non-parametric statistical technique that is often applied in limited data situations. We illustrate this approach using data from our study seeking a molecular fingerprint of STAT3 regulated genes for early detection of human cancer.     

The community structure of human cellular signaling network

Living cell is highly responsive to specific chemicals in its environment, such as hormones and molecules in food or aromas. The reason is ascribed to the existence of widespread and diverse signal transduction pathways, between which crosstalks usually exist, thus constitute a complex signaling network. Evidently, knowledge of topology characteristic of this network could contribute a lot to the understanding of diverse cellular behaviors and life phenomena thus come into being. In this presentation, signal transduction data is extracted from KEGG to construct a cellular signaling network of Homo sapiens, which has 931 nodes and 6798 links in total. Computing the degree distribution, we find it is not a random network, but a scale-free network following a power-law of P(K) approximately K(-gamma), with gamma approximately equal to 2.2. Among three graph partition algorithms, the Guimera's simulated annealing method is chosen to study the details of topology structure and other properties of this cellular signaling network, as it shows the best performance. To reveal the underlying biological implications, further investigation is conducted on ad hoc community and sketch map of individual community is drawn accordingly. The involved experiment data can be found in the supplementary material.     

Dynamic Interaction of the Sec Translocon with the Chaperone PpiD

The Sec translocon constitutes a ubiquitous protein transport channel that consists in bacteria of the three core components: SecY, SecE, and SecG. Additional proteins interact with SecYEG during different stages of protein transport. During targeting, SecYEG interacts with SecA, the SRP receptor, or the ribosome. Protein transport into or across the membrane is then facilitated by the interaction of SecYEG with YidC and the SecDFYajC complex. During protein transport, SecYEG is likely to interact also with the protein quality control machinery, but details about this interaction are missing. By in vivo and in vitro site-directed cross-linking, we show here that the periplasmic chaperone PpiD is located in front of the lateral gate of SecY, through which transmembrane domains exit the SecY channel. The strongest contacts were found to helix 2b of SecY. Blue native PAGE analyses verify the presence of a SecYEG-PpiD complex in native Escherichia coli membranes. The PpiD-SecY interaction was not influenced by the addition of SecA and only weakly influenced by binding of nontranslating ribosomes to SecYEG. In contrast, PpiD lost contact to the lateral gate of SecY during membrane protein insertion. These data identify PpiD as an additional and transient subunit of the bacterial SecYEG translocon. The data furthermore demonstrate the highly modular and versatile composition of the Sec translocon, which is probably essential for its ability to transport a wide range of substrates across membranes in bacteria and eukaryotes.     

生态样带边界分析的改进算法与网络计算

确定生态样带的边界,其目的是找出样带中的一系列不连续点,判定生物物种扩散的自然屏障,分析生物分布及其景观多样性．生态样带边界分析对有害生物的扩散、生物物种分布研究以及生物多样性保护具有一定的意义．本文在前人研究的基础上,对生态样带边界分析算法进行了改进,在算法中加入了不同的距离测度,由方差／均值比率确定窗口宽度数,用置信区间及其统计检验来确定样带边界．改进算法减少了主观性,具有较高的灵敏度,可检测出更细致的边界点．研制了改进算法的Java网络软件,可在多种兼容Java的网络洲览器,如Internet Explorer,HotJava,Nescape Navigator上调用和运行．以稻田生态环境的节肢动物样带对算法进行了检验和实例分析．结果显示,低营养级的植食性节肢动物,其分布受可见边界的显著影响．高营养级的捕食性节肢动物和寄生性节肢动物的分布也受到可见边界的一定影响,但不很显著．另外,也检测到植食性节肢动物在样带中的扩散方向．相对于植食性节肢动物,捕食性节肢动物和寄生性节肢动物的扩散无明显规律性．     

Data mining the protein data bank: automatic detection and assignment of carbohydrate structures

Knowledge of the 3D structure of glycans is a prerequisite for a complete understanding of the biological processes glycoproteins are involved in. However, due to a lack of standardised nomenclature, carbohydrate compounds are difficult to locate within the Protein Data Bank (PDB). Using an algorithm that detects carbohydrate structures only requiring element types and atom coordinates, we were able to detect 1663 entries containing a total of 5647 carbohydrate chains. The majority of chains are found to be N-glycosidically bound. Noncovalently bound ligands are also frequent, while O-glycans form a minority. About 30% of all carbohydrate containing PDB entries comprise one or several errors. The automatic assignment of carbohydrate structures in PDB entries will improve the cross-linking of glycobiology resources with genomic and proteomic data collections, which will be an important issue of the upcoming glycomics projects. By aiding in detection of erroneous annotations and structures, the algorithm might also help to increase database quality.     

Phospholipid Flippases Lem3p-Dnf1p and Lem3p-Dnf2p Are Involved in the Sorting of the Tryptophan Permease Tat2p in Yeast

The type 4 P-type ATPases are flippases that generate phospholipid asymmetry in membranes. In budding yeast, heteromeric flippases, including Lem3p-Dnf1p and Lem3p-Dnf2p, translocate phospholipids to the cytoplasmic leaflet of membranes. Here, we report that Lem3p-Dnf1/2p are involved in transport of the tryptophan permease Tat2p to the plasma membrane. The lem3Δ mutant exhibited a tryptophan requirement due to the mislocalization of Tat2p to intracellular membranes. Tat2p was relocalized to the plasma membrane when trans-Golgi network (TGN)-to-endosome transport was inhibited. Inhibition of ubiquitination by mutations in ubiquitination machinery also rerouted Tat2p to the plasma membrane. Lem3p-Dnf1/2p are localized to endosomal/TGN membranes in addition to the plasma membrane. Endocytosis mutants, in which Lem3p-Dnf1/2p are sequestered to the plasma membrane, also exhibited the ubiquitination-dependent missorting of Tat2p. These results suggest that Tat2p is ubiquitinated at the TGN and missorted to the vacuolar pathway in the lem3Δ mutant. The NH₂-terminal cytoplasmic region of Tat2p containing ubiquitination acceptor lysines interacted with liposomes containing acidic phospholipids, including phosphatidylserine. This interaction was abrogated by alanine substitution mutations in the basic amino acids downstream of the ubiquitination sites. Interestingly, a mutant Tat2p containing these substitutions was missorted in a ubiquitination-dependent manner. We propose the following model based on these results; Tat2p is not ubiquitinated when the NH₂-terminal region is bound to membrane phospholipids, but if it dissociates from the membrane due to a low level of phosphatidylserine caused by perturbation of phospholipid asymmetry in the lem3Δ mutant, Tat2p is ubiquitinated and then transported from the TGN to the vacuole.     

Identification of amino acids important for substrate specificity in sucrose transporters using gene shuffling

Plant sucrose transporters (SUTs) are H(+)-coupled uptake transporters. Type I and II (SUTs) are phylogenetically related but have different substrate specificities. Type I SUTs transport sucrose, maltose, and a wide range of natural and synthetic α- and β-glucosides. Type II SUTs are more selective for sucrose and maltose. Here, we investigated the structural basis for this difference in substrate specificity. We used a novel gene shuffling method called synthetic template shuffling to introduce 62 differentially conserved amino acid residues from type I SUTs into OsSUT1, a type II SUT from rice. The OsSUT1 variants were tested for their ability to transport the fluorescent coumarin β-glucoside esculin when expressed in yeast. Fluorescent yeast cells were selected using fluorescence-activated cell sorting (FACS). Substitution of five amino acids present in type I SUTs in OsSUT1 was found to be sufficient to confer esculin uptake activity. The changes clustered in two areas of the OsSUT1 protein: in the first loop and the top of TMS2 (T80L and A86K) and in TMS5 (S220A, S221A, and T224Y). The substrate specificity of this OsSUT1 variant was almost identical to that of type I SUTs. Corresponding changes in the sugarcane type II transporter ShSUT1 also changed substrate specificity, indicating that these residues contribute to substrate specificity in type II SUTs in general.