On the phylogeny and evolution of Mesozoic and extant lineages of Adephaga (Coleoptera,Insecta)

The relationships of extant and extinct lineages of Adephaga were analysed formally for the first time. Emphasis is placed on the aquatic and semiaquatic groups and their evolution in the Mesozoic. ?Triadogyrus and ?Mesodineutus belong to Gyrinidae, the sister group of the remaining families. ?Triaplidae are the sister group of the following groups (Haliplidae, Geadephaga, Dytiscoidea incl. ?Liadytidae, ?Parahygrobiidae and ?Coptoclavidae [major part]). The lack of a ventral procoxal joint and a very short prosternal process are plesiomorphies of ?Triaplidae. ?Coptoclavidae and ?Timarchopsinae are paraphyletic. ?Timarchopsis is placed in a geadephagan clade. In contrast to other coptoclavids, its metathorax is close to the condition found in Haliplidae, with a complete transverse ridge and coxae with large plates and free mesal walls. ?Coptoclavidae s.str., i.e. excl. ?Timarchopsis, is a dytiscoid subgroup. The mesal metacoxal walls are fused, the coxal plates are reduced, and the transverse ridge is absent. ?Stygeonectes belongs to this dytiscoid coptoclavid unit and is therefore misplaced in ?Timarchopsinae. ?Liadytidae belongs to a dytiscoid subgroup, which also comprises the extant families Aspidytidae, Amphizoidae, Hygrobiidae and Dytiscidae. ?Parahygrobia is the sister group of Hygrobiidae. The larvae are characterized by a broad gula, the absence of the lacinia, retractile maxillary bases and very long urogomphi set with long setae. ?Liadytiscinae is the sister group of extant Dytiscidae. There is no support for a clade ?Eodromeinae and for Trachypachidae incl. ?Eodromeinae. ?Fortiseode is nested within Carabidae. The exclusion of fossil taxa has no effect on the branching pattern. The evolution of Adephaga in the Mesozoic is discussed. Possible reasons for the extinction of ?Coptoclavidae are the rise of teleost fish and the competition of Gyrinidae and Dytiscidae, which possess efficient defensive glands and larval mandibular sucking channels.     

Metabolic engineering of Escherichia coli for enhanced biosynthesis of poly(3-hydroxybutyrate) based on proteome analysis

We have previously analyzed the proteome of recombinant Escherichia coli producing poly(3-hydroxybutyrate) [P(3HB)] and revealed that the expression level of several enzymes in central metabolism are proportional to the amount of P(3HB) accumulated in the cells. Based on these results, the amplification effects of triosephosphate isomerase (TpiA) and fructose-bisphosphate aldolase (FbaA) on P(3HB) synthesis were examined in recombinant E. coli W3110, XL1-Blue, and W lacI mutant strains using glucose, sucrose and xylose as carbon sources. Amplification of TpiA and FbaA significantly increased the P(3HB) contents and concentrations in the three E. coli strains. TpiA amplification in E. coli XL1-Blue lacI increased P(3HB) from 0.4 to 1.6 to g/l from glucose. Thus amplification of glycolytic pathway enzymes is a good strategy for efficient production of P(3HB) by allowing increased glycolytic pathway flux to make more acetyl-CoA available for P(3HB) biosynthesis.     

Expression of a lipase on the cell-surface of Escherichia coli using the OmpW anchoring motif and its application to enantioselective reactions

Microbial-surface display is the expression of proteins or peptides on the surface of cells by fusing an appropriate protein as an anchoring motif. Here, the outer membrane protein W (OmpW) was selected as a fusion partner for functional expression of Pseudomonas fluorescence SIK W1 lipase (TliA) on the cell-surface of Escherichia coli. Localization of the truncated OmpW-TliA fusion protein on the cell-surface was confirmed by immunoblotting and functional assay of lipase activity. Enantioselective hydrolysis of rac-phenylethyl butanoate by the displayed lipase resulted in optically active (R)-phenyl ethanol with 96 % enantiomeric excess and 44 % of conversion in 5 days. Thus, a small outer membrane protein OmpW, is a useful anchoring motif for displaying an active enzyme of ~50 kDa on the cell-surface and the surface-displayed lipase can be employed as an enantioselective biocatalyst in organic synthesis.     

Altitudinal Distribution of Ammonia-Oxidizing Archaea and Bacteria in Alpine Grassland Soils Along the South-Facing Slope of Nyqentangula Mountains,Central Tibetan Plateau

Nitrogen is a major limiting nutrient for the net primary production of terrestrial ecosystems, especially on sentinel alpine ecosystem. Ammonia oxidation is the first and rate-limiting step on nitrification process and is thus crucial to nitrogen cycle. To decipher climatic influence on ammonia oxidizers, their communities were characterized by qPCR and clone sequencing by targeting amoA genes (encoding the alpha subunit of ammonia mono-oxygenase) in soils from 7 sites over an 800 m elevation transect (4400–5200 m a.s.l.), based on “space-to-time substitution” strategy, on a steppe-meadow ecosystem located on the central Tibetan Plateau (TP). Archaeal amoA abundance outnumbered bacterial amoA abundance at lower altitude (<4800 m a.s.l.), but bacterial amoA abundance was greater in surface soils at higher altitude (≥4800 m a.s.l.). Archaeal amoA abundance decreased with altitude in surface soil, while its abundance stayed relatively stable and was mostly greater than bacterial amoA abundance in subsurface soils. Conversely, bacterial amoA abundance gradually increased with altitude at all three soil depths. Statistical analysis indicated that altitude-dependent factors, in particular pH and precipitation, had a profound effect on the abundance and community of ammonia-oxidizing bacteria, but only on the community composition of ammonia-oxidizing archaea along the altitudinal gradient. These findings imply that the shifts in the relative abundance and/or community structure of ammonia-oxidizing bacteria and archaea may result from the precipitation variation along the altitudinal gradient. Thus, we speculate that altitude-related factors (mainly precipitation variation combing changed pH), would play a vital role in affecting nitrification process on this alpine grassland ecosystem located at semi-arid area on TP.     

Three-Dimensional Structure of CAP-Gly Domain of Mammalian Dynactin Determined by Magic Angle Spinning NMR Spectroscopy: Conformational Plasticity and Interactions with End-Binding Protein EB1

Microtubules and their associated proteins play important roles in vesicle and organelle transport, cell motility and cell division. Perturbation of these processes by mutation typically gives rise to severe pathological conditions. In our efforts to obtain atomic information on microtubule-associated protein/microtubule interactions with the goal to understand mechanisms that might potentially assist in the development of treatments for these diseases, we have determined the three-dimensional structure of CAP-Gly (cytoskeleton-associated protein, glycine-rich) domain of mammalian dynactin by magic angle spinning NMR spectroscopy. We observe two conformations in the β2 strand encompassing residues T43-V44-A45, residues that are adjacent to the disease-associated mutation, G59S. Upon binding of CAP-Gly to microtubule plus-end tracking protein EB1, the CAP-Gly shifts to a single conformer. We find extensive chemical shift perturbations in several stretches of residues of CAP-Gly upon binding to EB1, from which we define accurately the CAP-Gly/EB1 binding interface. We also observe that the loop regions may exhibit unique flexibility, especially in the GKNDG motif, which participates in the microtubule binding. This study in conjunction with our previous reports suggests that conformational plasticity is an intrinsic property of CAP-Gly likely due to its unusually high loop content and may be required for its biological functions.     

Ssp DnaE split-intein mediated split-Cre reconstitution in tobacco

The Cre/loxP system is increasingly exploited for spatial and temporal gene activation or inactivation. In this study, a novel approach for gene activation using a Cre/loxp system in tobacco is described. As the DnaE intein in Synechocystis sp. strain PCC6803 is capable of catalyzing a protein trans-splicing reaction to assemble a mature protein from two separate precursors, the N- and C-terminal ends of the Cre enzyme, split between Gly190 and Gly191, were fused to N- and C-terminals of the Ssp DnaE split intein,respectively. Subsequently, in-frame fusions of NCre/NInt and CInt/CCre are assembled into the pCAMBIA1300 cloning vector, and used for co-expression, along with the BAR selectable marker gene for BASTA herbicide resistance in tobacco. A Cre-dependent excision recombination event is monitored when tobacco leaf explants are screened for resistance to Basta, but along with absence of beta-glucuronidase activity. Based on herbicide resistance, an efficient recombination event is observed, in vivo Bar activation following co-expression of NCre/NInt and CInt/CCre fusion genes in pCAGUS/BAR transgenic lines. Moreover, the recombination efficiency is comparable to that of intact Cre gene expression. However, no Cre recombination event is observed when only the NCre and CCre genes or the NCre/NInt fusion gene and the CCre genes are co-expressed. Thus, the Ssp DnaE split intein-mediated Cre activity reconstitution observed in this study provides an alternative approach for the traditional Cre/loxP system, and this may aid in achieving dynamic regulation of gene expression in transgenic plants.     

Interactions between soybean ABA receptors and type 2C protein phosphatases

The plant hormone abscisic acid (ABA) plays important roles in regulating plant growth, development, and responses to environmental stresses. Proteins in the PYR/PYL/RCAR family (hereafter referred to as PYLs) are known as ABA receptors. Since most studies thus far have focused on Arabidopsis PYLs, little is known about PYL homologs in crop plants. We report here the characterization of 21 PYL homologs (GmPYLs) in soybean. Twenty-three putative GmPYLs can be found from soybean genome sequence and categorized into three subgroups. GmPYLs interact with AtABI1 and two GmPP2Cs in diverse manners. A lot of the subgroup I GmPYLs interact with PP2Cs in an ABA-dependent manner, whereas most of the subgroup II and III GmPYLs bind to PP2Cs in an ABA-independent manner. The subgroup III GmPYL23, which cannot interact with any of the tested PP2Cs, differs from other GmPYLs. The CL2/gate domain is crucial for GmPYLs-PP2Cs interaction, and a mutation in the conserved proline (P109S) abolishes the interaction between GmPYL1 and AtABI1. Furthermore, the ABA dependence of GmPYLs-PP2Cs interactions are partially correlated with two amino acid residues preceding the CL2/gate domain of GmPYLs. We also show that GmPYL1 interacts with AtABI1 in an ABA-dependent manner in plant cells. Three GmPYLs differentially inhibit AtABI1 and GmPP2C1 in an ABA-dependent or -enhanced manner in vitro. In addition, ectopically expressing GmPYL1 partially restores ABA sensitivity of the Arabidopsis triple mutant pyr1/pyl1/pyl4. Taken together, our results suggest that soybean GmPYLs are ABA receptors that function by interacting and inhibiting PP2Cs.     

Autoimmune regulator, Aire, is a novel regulator of chondrocyte differentiation

Chondrocyte differentiation is controlled by various regulators, such as Sox9 and Runx2, but the process is complex. To further understand the precise underlying molecular mechanisms of chondrocyte differentiation, we aimed to identify a novel regulatory factor of chondrocyte differentiation using gene expression profiles of micromass-cultured chondrocytes at different differentiation stages. From the results of microarray analysis, the autoimmune regulator, Aire, was identified as a novel regulator. Aire stable knockdown cells, and primary cultured chondrocytes obtained from Aire^−/− mice, showed reduced mRNA expression levels of chondrocyte-related genes. Over-expression of Aire induced the early stages of chondrocyte differentiation by facilitating expression of Bmp2. A ChIP assay revealed that Aire was recruited on an Airebinding site (T box) in the Bmp2 promoter region in the early stages of chondrocyte differentiation and histone methylation was modified. These results suggest that Aire can facilitate early chondrocyte differentiation by expression of Bmp2 through altering the histone modification status of the promoter region of Bmp2.     

Characterization of zebrafish mutants with defects in bone calcification during development

Using the fluorescent dyes calcein and alcian blue, we stained the F3 generation of chemically (ENU) mutagenized zebrafish embryos and larvae, and screened for mutants with defects in bone development. We identified a mutant line, bone calcification slow (bcs), which showed delayed axial vertebra calcification during development. Before 4–5 days post-fertilization (dpf), the bcs embryos did not display obvious abnormalities in bone development (i.e., normal number, size and shape of cartilage and vertebrae). At 5–6 dpf, when vertebrae calcification starts, bcs embryos began to show defects. At 7 dpf, for example, in most of the bcs embryos examined, calcein staining revealed no signals of vertebrae mineralization, whereas during the same developmental stages, 2–14 mineralized vertebrae were observed in wild-type animals. Decreases in the number of calcified vertebrae were also observed in bcs mutants when examined at 9 and 11 dpf, respectively. Interestingly, by 13 dpf the defects in bcs mutants were no longer evident. There were no significant differences in the number of calcified vertebrae between wild-type and mutant animals. We examined the expression of bone development marker genes (e.g., Sox9b, Bmp2b, and Cyp26b1, which play important roles in bone formation and calcification). In mutant fish, we observed slight increases in Sox9b expression, no alterations in Bmp2b expression, but significant increases in Cyp26b1 expression. Together, the data suggest that bcs delays axial skeletal calcification, but does not affect bone formation and maturation.