Binding site selection analysis of protein-DNA interactions via solid phase sequencing of oligonucleotide mixtures.

By combining the concept of degenerate oligonucleotide mutagenesis (1,2,3,4) and the convenience of solid phase chemical DNA sequencing (5), we have developed a rapid procedure for determining the specificity of DNA-binding proteins in vitro. Starting with a degenerate oligonucleotide mixture, the technique assays for alternative nucleotides in fractions that are bound or non-bound to the protein of interest. In contrast to previous approaches using degenerate oligonucleotides, it does not involve cloning but rather employs direct sequencing of the oligonucleotide mixtures after attachment to a solid support. Solid state processing obviates the need for both DNA extractions from polyacrylamide gels and time-consuming ethanol precipitations. Because of its convenience and sensitivity, this binding site selection analysis is well suited to determining rapidly the sequence preference of DNA-binding proteins that are available in small amounts, and complements well established approaches like methylation interference or missing contact assays. The solid phase reaction protocol we propose can also improve these latter approaches.     

A major difference between the divergence patterns within the lines-1 families in mice and voles

L1 retroposons are represented in mice by subfamilies of interspersed sequences of varied abundance. Previous analyses have indicated that subfamilies are generated by duplicative transposition of a small number of members of the L1 family, the progeny of which then become a major component of the murine L1 population, and are not due to any active processes generating homology within preexisting groups of elements in a particular species. In mice, more than a third of the L1 elements belong to a clade that became active approximately 5 Mya and whose elements are > or = 95% identical. We have collected sequence information from 13 L1 elements isolated from two species of voles (Rodentia: Microtinae: Microtus and Arvicola) and have found that divergence within the vole L1 population is quite different from that in mice, in that there is no abundant subfamily of homologous elements. Individual L1 elements from voles are very divergent from one another and belong to a clade that began a period of elevated duplicative transposition approximately 13 Mya. Sequence analyses of portions of these divergent L1 elements (approximately 250 bp each) gave no evidence for concerted evolution having acted on the vole L1 elements since the split of the two vole lineages approximately 3.5 Mya; that is, the observed interspecific divergence (6.7%-24.7%) is not larger than the intraspecific divergence (7.9%-27.2%), and phylogenetic analyses showed no clustering into Arvicola and Microtus clades.      

Molecular phylogeny and divergence times of drosophilid species

The phylogenetic relationships and divergence times of 39 drosophilid species were studied by using the coding region of the Adh gene. Four genera--Scaptodrosophila, Zaprionus, Drosophila, and Scaptomyza (from Hawaii)--and three Drosophila subgenera--Drosophila, Engiscaptomyza, and Sophophora--were included. After conducting statistical analyses of the nucleotide sequences of the Adh, Adhr (Adh-related gene), and nuclear rRNA genes and a 905-bp segment of mitochondrial DNA, we used Scaptodrosophila as the outgroup. The phylogenetic tree obtained showed that the first major division of drosophilid species occurs between subgenus Sophophora (genus Drosophila) and the group including subgenera Drosophila and Engiscaptomyza plus the genera Zaprionus and Scaptomyza. Subgenus Sophophora is then divided into D. willistoni and the clade of D. obscura and D. melanogaster species groups. In the other major drosophilid group, Zaprionus first separates from the other species, and then D. immigrans leaves the remaining group of species. This remaining group then splits into the D. repleta group and the Hawaiian drosophilid cluster (Hawaiian Drosophila, Engiscaptomyza, and Scaptomyza). Engiscaptomyza and Scaptomyza are tightly clustered. Each of the D. repleta, D. obscura, and D. melanogaster groups is monophyletic. The splitting of subgenera Drosophila and Sophophora apparently occurred about 40 Mya, whereas the D. repleta group and the Hawaiian drosophilid cluster separated about 32 Mya. By contrast, the splitting of Engiscaptomyza and Scaptomyza occurred only about 11 Mya, suggesting that Scaptomyza experienced a rapid morphological evolution. The D. obscura and D. melanogaster groups apparently diverged about 25 Mya. Many of the D. repleta group species studied here have two functional Adh genes (Adh-1 and Adh-2), and these duplicated genes can be explained by two duplication events.      

Sulfate reduction and S-oxidation in a moorland pool sediment

In an oligotrophic moorland pool in The Netherlands, S cycling near the sediment/water boundary was investigated by measuring (1) SO₄ ^2– reduction rates in the sediment, (2) depletion of SO₄ ^2– in the overlying water column and (3) release of³⁵S from the sediment into the water column. Two locations differing in sediment type (highly organic and sandy) were compared, with respect to reduction rates and depletion of SO₄ ^2– in the overlying water.Sulfate reduction rates in sediments of an oligotrophic moorland pool were estimated by diagenetic modelling and whole core³⁵SO₄ ^2– injection. Rates of SO₄ ^2– consumption in the overlying water were estimated by changes in SO₄ ^2– concentration over time in in situ enclosures. Reduction rates ranged from 0.27–11.2 mmol m^–2 d^–1. Rates of SO₄ ^2– uptake from the enclosed water column varied from –0.5, –0.3 mmol m^–2 d^–1 (November) to 0.43–1.81 mmol m^–2 d^–1 (July, August and April). Maximum rates of oxidation to SO₄ ^2– in July 1990 estimated by combination of SO₄ ^2– reduction rates and rates of in situ SO₄ ^2– uptake in the enclosed water column were 10.3 and 10.5 mmol m^–2 d^–1 at an organic rich and at a sandy site respectively.Experiments with³⁵S^2– and³⁵SO₄ ^2– tracer suggested (1) a rapid formation of organically bound S from dissimilatory reduced SO₄ ^2– and (2) the presence of mainly non SO₄ ^2–-S derived from reduced S transported from the sediment into the overlying water. A³⁵S^2– tracer experiment showed that about 7% of³⁵S^2– injected at 1 cm depth in a sediment core was recovered in the overlying water column.Sulfate reduction rates in sediments with higher volumetric mass fraction of organic matter did not significantly differ from those in sediments with a lower mass fraction of organic matter.Corresponding author     

Ant versus bird exclusion effects on the arthropod assemblage of an organic citrus grove

1. Predation‐exclusion experiments have highlighted that top‐down control is pervasive in terrestrial communities, but most of these experiments are simplistic in that they only excluded a single group of predators and the effect of removal was evaluated on a few species from the community. The main goal of our study was to experimentally establish the relative effects of ants and birds on the same arthropod assemblage of canopy trees. 2. We conducted 1‐year long manipulative experiments in an organic citrus grove intended to quantify the independent effects of bird and ant predators on the abundance of arthropods. Birds were excluded with plastic nets whereas ants were excluded with sticky barriers on the trunks. The sticky barrier also excluded other ground dwelling insects, like the European earwig Forficula auricularia L. 3. Both the exclusion of ants and birds affected the arthropod community of the citrus canopies, but the exclusion of ants was far more important than the exclusion of birds. Indeed, almost all groups of arthropods had higher abundance in ant‐excluded than in control trees, whereas only dermapterans were more abundant in bird‐excluded than in control trees. A more detailed analysis conducted on spiders also showed that the effect of ant exclusion was limited to a few families rather than being widespread over the entire diverse spectrum of spiders. 4. Our results suggest that the relative importance of vertebrate and invertebrate predators in regulating arthropod populations largely depends on the nature of the predator–prey system.     

早期液体复苏对感染性休克患者血流动力学的影响

目的：早期液体复苏对感染性休克患者血流动力学的影响。方法：选取2012年2月-2013年2月我院ICU收治的26例感染性休克患者作为研究对象,随机分为对照组和试验组,各13例。两组患者均采用PICCO监测,并根据早期复苏目标导向（Earlygoaldirectedtherapy,EGDT）进行早期液体复苏治疗。对照组和试验组复苏液分别为林格液和6％羟乙基淀粉130／0．4氯化钠溶液。分别于复苏开始时（Oh）、8h和24h收集患者的血流动力学参数。结果：两组患者CO及PAWP水平均随着时间的延长下降,而CI、CVP及SVR水平均随着时间的增加上升。除对照组CI外,与开始复苏（oh）相比较试验组和对照组的C0、CI、CVP、SVR及PAWP与开始复苏（O小时）相比较均有显著差异（P值均〈0．05）。经重复测量资料的．方差分析进行比较发现,与对照组相比较,试验组CVP和SVR上升水平及PAWP下降水平明显,差异具有统计学意义（P值均〈0．05）。结论：感染性休克患者使用6％羟乙基淀粉130／0．4氯化钠溶液进行复苏,能更好的改善患者的血流动力学指标。     

Sex‐dependent alterations in BDNF‐TrkB signaling in the hippocampus of reelin heterozygous mice: a role for sex steroid hormones

Neurodevelopmental psychiatric disorders such as schizophrenia may be caused by a combination of gene × environment, gene × gene, and/or gene × sex interactions. Reduced expression of both Reelin and Brain‐Derived Neurotrophic factor (BDNF) has been associated with schizophrenia in human post‐mortem studies. However, it remains unclear how Reelin and BDNF interact (gene × gene) and whether this is sex‐specific (gene × sex). This study investigated BDNF‐TrkB signaling in the hippocampus of male and female Reelin heterozygous (Rln^+/?) mice. We found significantly increased levels of BDNF in the ventral hippocampus (VHP) of female, but not male Rln^+/? compared to wild‐type (WT) controls. While levels of TrkB were not significantly altered, phosphorylated TrkB (pTrkB) levels were significantly lower, again only in female Rln^+/? compared to WT. This translated to downstream effects with a significant decrease in phosphorylated ERK1 (pERK1). No changes in BDNF, TrkB, pTrkB or pERK1/2 were observed in the dorsal hippocampus of Rln^+/? mice. Ovariectomy (OVX) had no effect in WT controls, but caused a significant decrease in BDNF expression in the VHP of Rln^+/? mice to the levels of intact WT controls. The high expression of BDNF was restored in OVX Rln^+/? mice by 17β‐estradiol treatment, suggesting that Rln^+/? mice respond differently to an altered estradiol state than WT controls. In addition, while OVX had no significant effect on TrkB or ERK expression/phosphorylation, OVX + estradiol treatment markedly increased TrkB and ERK1 phosphorylation in Rln^+/? and, to a lesser extent in WT controls, compared to intact genotype‐matched controls. These data may provide a better understanding of the interaction of Reelin and BDNF in the hippocampus, which may be involved in schizophrenia.     

Spontaneous neural activity is required for the establishment and maintenance of the olfactory sensory map

We have developed a genetic approach to examine the role of spontaneous activity and synaptic release in the establishment and maintenance of an olfactory sensory map. Conditional expression of tetanus toxin light chain, a molecule that inhibits synaptic release, does not perturb targeting during development, but neurons that express this molecule in a competitive environment fail to maintain appropriate synaptic connections and disappear. Overexpression of the inward rectifying potassium channel, Kir2.1, diminishes the excitability of sensory neurons and more severely disrupts the formation of an olfactory map. These studies suggest that spontaneous neural activity is required for the establishment and maintenance of the precise connectivity inherent in an olfactory sensory map.     

A data management system for structural genomics

Background  

Structural genomics (SG) projects aim to determine thousands of protein structures by the development of high-throughput techniques for all steps of the experimental structure determination pipeline. Crucial to the success of such endeavours is the careful tracking and archiving of experimental and external data on protein targets.