Characterization of TioF, a tryptophan 2,3-dioxygenase involved in 3-hydroxyquinaldic acid formation during thiocoraline biosynthesis

Thiocoraline is a thiodepsipeptide antitumor agent that belongs to the family of bisintercalator natural products that bind duplex DNA through their two planar intercalating moieties. In thiocoraline, the 3-hydroxyquinaldic acid (3HQA) chromophores required for intercalation are derived from L-Trp. We have expressed the Micromonospora sp. ML1 tryptophan 2,3-dioxygenase(TDO) TioF, purified it from E. coli, and confirmed its role in the irreversible oxidation of L-Trp to N-formylkynurenine, the proposed first step during 3HQA biosynthesis. We have established that TioF is a catalyst with broader specificity than other TDOs, but that is less promiscuous than indoleamine 2,3-dioxygenases. TioF was found to display activity with various L-Trp analogs (serotonin, D-Trp, and indole). The TioF reaction products generated during this study will be used as substrates for subsequent analysis of the other enzymes involved in 3HQA biosynthesis.     

Validation of a sensitive assay for thiocoraline in mouse plasma using liquid chromatography-tandem mass spectrometry

A sensitive high-performance liquid chromatography-tandem mass spectrometry assay for thiocoraline, an anti-tumor depsipeptide, in mouse plasma is described. Echinomycin, a quinoxaline peptide, was used as an internal standard. Thiocoraline was recovered from the mouse plasma using protein precipitation with acetonitrile and followed by solid-phase extraction of the supernatant. The mobile phase consisted of methanol (0.1% formic acid)-water (0.1% formic acid) (90:10, v/v). The analytical column was a YMC C(18). The standard curve was linear from 0.1 to 50 ng/ml (R(2)>0.99). The lower limit of quantitation was 0.1 ng/ml. The assay was specific based on the multiple reaction monitoring transitions at m/z 1157-->215 and m/z 1101-->243 for thiocoraline and the internal standard, echinomycin, respectively. The mean intra- and inter-day assay accuracies remained below 5 and 12%, respectively, for all calibration standards and quality control (QC) samples. The intra- and inter-day assay precisions were less than 11.4 and 9.5% for all QC levels, respectively. The utility of the assay was demonstrated by a pharmacokinetic study of i.v. (bolus) thiocoraline on CD-1 mice. Thiocoraline was stable in mouse plasma in an ice-water bath for 6 h and for three freeze-thaw cycles. The reconstituted thiocoraline after extraction and drying sample process was stable in the autosampler for over 24 h. The assay was able to quantify thiocoraline in plasma up to 48 h following dose. Pharmacokinetic analysis showed that thiocoraline has distinct pharmacokinetic profiling when dosed in different formulation solutions. The assay is currently used to measure thiocoraline plasma concentrations in support of a project to develop a suitable formulation with a desirable pharmacokinetic profile.     

A New Scaffold of an Old Protein Fold Ensures Binding to the Bisintercalator Thiocoraline

Thiocoraline is a thiodepsipeptide with potent antitumor activity. TioX, a protein with an unidentified function, is encoded by a gene of the thiocoraline biosynthetic gene cluster. The crystal structure of the full-length TioX protein at 2.15 Å resolution reveals that TioX protomer shares an ancient βαβββ fold motif with glyoxalase I and bleomycin resistance protein families, despite a very low sequence homology. Intriguingly, four TioX monomers form a unique 2-fold symmetric tetrameric assembly that is stabilized by four intermolecular disulfide bonds formed cyclically between Cys60 and Cys66 of adjacent monomers. The arrangement of two of the four monomers in the TioX tetramer is analogous to that in dimeric bleomycin resistance proteins. This analogy indicates that this novel higher-order structural scaffold of TioX may have evolved to bind thiocoraline. Our equilibrium titration studies demonstrate the binding of a thiocoraline chromophore analog, quinaldic acid, to TioX, thereby substantiating this model. Furthermore, a strain of Streptomyces albus containing an exogenous thiocoraline gene cluster devoid of functional tioX maintains thiocoraline production, albeit with a lower yield. Taken together, these observations rule out a direct enzymatic function of TioX and suggest that TioX is involved in thiocoraline resistance or secretion.     

Paramagnetic relaxation enhancement of membrane proteins by incorporation of the metal-chelating unnatural amino acid 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid (HQA)

The use of paramagnetic constraints in protein NMR is an active area of research because of the benefits of long-range distance measurements (>10 Å). One of the main issues in successful execution is the incorporation of a paramagnetic metal ion into diamagnetic proteins. The most common metal ion tags are relatively long aliphatic chains attached to the side chain of a selected cysteine residue with a chelating group at the end where it can undergo substantial internal motions, decreasing the accuracy of the method. An attractive alternative approach is to incorporate an unnatural amino acid that binds metal ions at a specific site on the protein using the methods of molecular biology. Here we describe the successful incorporation of the unnatural amino acid 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid (HQA) into two different membrane proteins by heterologous expression in E. coli. Fluorescence and NMR experiments demonstrate complete replacement of the natural amino acid with HQA and stable metal chelation by the mutated proteins. Evidence of site-specific intra- and inter-molecular PREs by NMR in micelle solutions sets the stage for the use of HQA incorporation in solid-state NMR structure determinations of membrane proteins in phospholipid bilayers.     

N,N,N',N'-tetramethylchloroformamidinium hexafluorophosphate (TCFH), a powerful coupling reagent for bioconjugation

Prodrugs are increasingly used as delivery vehicles for pharmaceutical agents that present solubility and/or pharmacokinetic/metabolic issues. In the course of the development of prodrugs for the antitumoral agent thiocoraline, standard coupling reagents and procedures failed to provide the desired target derivatives because of the lack of reactivity of its quinolinic alcohol. In contrast, the use of N,N,N',N'-tetramethylchloroformamidinium hexafluorophosphate (TCFH) resulted in good yields of ester prodrugs of thiocoraline and could be applicable to other drugs with similar limitations.     

水电梯级开发对河流生境质量及纵向连通性影响评价——以五布河和藻渡河为例

河流物理生境是维持河流生物多样性及生态功能的关键因素。生境质量的好坏能反应河流健康的程度。以我国西南地区的五布河和藻渡河为例,采用河流生境调查方法(RHS)和树状水系连通性指数(DCI)定量评估水电梯级开发和水坝建设对河流物理生境质量和河流纵向连通性的影响。结果表明,水电梯级开发后,五布河干流未受水坝明显影响河段、库区河段、减水河段分别为20.48、43.34、18.09 km,占总长度的25.0%、52.9%、22.1%。藻渡河干流河口至双河口段未受水坝明显影响河段、库区河段、减水河段分别为58.61、8.28、18.99 km,占总长度的68.2%、9.6%、占22.1%。水电梯级开发后,五布河干流河流片段由26个增至29个,藻渡河干流河流片段由2个增至5个。两条河流纵向连通性分别降低了7.8%和38.0%。五布河坝下减水河段生境质量降低14.1%,库区河段生境质量变化不明显。藻渡河减水河段生境质量与近自然河段无显著差异;两座坝后式电站库区河段生境质量明显低于近自然河段。水电梯级开发对两条河流物理生境的影响与水坝位置选择、建坝前的自然阻隔数量与分布、河流地貌特征、水电资源开发方式等密切相关。     

<Emphasis Type="Italic">Ascidiaceibacter salegens</Emphasis> gen. nov., sp. nov., isolated from an ascidian

An aerobic, Gram-stain negative, rod-shaped, non-motile bacterium, designated as strain HQA918^T, was isolated from an ascidian, Botryllus schlosseri, which was collected from the coast of Weihai in the north of the Yellow Sea, in China. The strain grew optimally at 28–30 °C, at pH values 7.0–8.0, and in the presence of 1.0–3.0% (w/v) sodium chloride (NaCl). A phylogenetic analysis based on 16S rRNA gene sequences showed that strain HQA918^T can be affiliated with the family Flavobacteriaceae in the phylum Bacteroidetes, with 92.7% similarity to its close relatives. The major fatty acids identified were iso-C_15:0, iso-C_15:0 3-OH, and summed feature 3 (iso-C_15:0 2-OH and/or C_16:1ω7c). The major polar lipids were phosphatidylethanolamine, one unidentified aminolipid, and five unidentified polar lipids. The G+C content of the genomic DNA was 44.1 mol%. On the basis of the phylogenetic, genotypic, phenotypic, and chemotaxonomic data, this organism should be classified as a representative of a novel genus, for which the name Ascidiaceibacter gen. nov. is proposed. The type species is Ascidiaceibacter salegens sp. nov. (type strain HQA918^T?=?KCTC 52719^T?=?MCCC 1K03259^T).     

Hospital Performance,the Local Economy,and the Local Workforce: Findings from a US National Longitudinal Study

Background

Pay-for-performance is an increasingly popular approach to improving health care quality, and the US government will soon implement pay-for-performance in hospitals nationwide. Yet hospital capacity to perform (and improve performance) likely depends on local resources. In this study, we quantify the association between hospital performance and local economic and human resources, and describe possible implications of pay-for-performance for socioeconomic equity.

Methods and Findings

We applied county-level measures of local economic and workforce resources to a national sample of US hospitals (n = 2,705), during the period 2004–2007. We analyzed performance for two common cardiac conditions (acute myocardial infarction [AMI] and heart failure [HF]), using process-of-care measures from the Hospital Quality Alliance [HQA], and isolated temporal trends and the contributions of individual resource dimensions on performance, using multivariable mixed models. Performance scores were translated into net scores for hospitals using the Performance Assessment Model, which has been suggested as a basis for reimbursement under Medicare''s “Value-Based Purchasing” program. Our analyses showed that hospital performance is substantially associated with local economic and workforce resources. For example, for HF in 2004, hospitals located in counties with longstanding poverty had mean HQA composite scores of 73.0, compared with a mean of 84.1 for hospitals in counties without longstanding poverty (p<0.001). Hospitals located in counties in the lowest quartile with respect to college graduates in the workforce had mean HQA composite scores of 76.7, compared with a mean of 86.2 for hospitals in the highest quartile (p<0.001). Performance on AMI measures showed similar patterns. Performance improved generally over the study period. Nevertheless, by 2007—4 years after public reporting began—hospitals in locationally disadvantaged areas still lagged behind their locationally advantaged counterparts. This lag translated into substantially lower net scores under the Performance Assessment Model for hospital reimbursement.

Conclusions

Hospital performance on clinical process measures is associated with the quantity and quality of local economic and human resources. Medicare''s hospital pay-for-performance program may exacerbate inequalities across regions, if implemented as currently proposed. Policymakers in the US and beyond may need to take into consideration the balance between greater efficiency through pay-for-performance and socioeconomic equity. Please see later in the article for the Editors'' Summary     

Noncovalent liposome linkage and miniaturization of capsosomes for drug delivery

We report the synthesis of poly(methacrylic acid)-co-(oleyl methacrylate) with three different amounts of oleyl methacrylate and compare the ability of these polymers with that of poly(methacrylic acid)-co-(cholesteryl methacrylate) (PMA(c)) to noncovalently anchor liposomes to polymer layers. We subsequently assembled ～1 μm diameter PMA(c)-based capsosomes, polymer hydrogel capsules that contain up to ～2000 liposomal subcompartments, and investigate the potential of these carriers to deliver water-insoluble drugs by encapsulating two different antitumor compounds, thiocoraline or paclitaxel, into the liposomes. The viability of lung cancer cells is used to substantiate the cargo concentration-dependent activity of the capsosomes. These findings cover several crucial aspects for the application of capsosomes as potential drug delivery vehicles.     

Occurrence and variability of River Habitat Survey features across Europe and the consequences for data collection and evaluation

River Habitat Survey (RHS) data collected for the EU-funded STAR project was used to identify hydromorphological characteristic features of rivers in four European regions namely: lowlands; mountain; the Alps; and the Mediterranean. Using RHS attributes, Habitat Quality Assessment (HQA) – a measure of natural habitat diversity, and Habitat Modification Score (HMS) – a measure of anthropogenic modification, we identified considerable differences in frequency, diversity and evenness of features between the regions. A relatively small subset of features clearly distinguish the hydromorphological characters of lowland, Alpine and southern European rivers. It was more difficult to distinguish mountain rivers from Alpine rivers. The highest statistical differences are observed between Lowland and Mountain region. Within the four regions studied the RHS attributes that most strongly influence the HQA and HMS indices were identified. We conclude that specific effort should be made to ensure these are recorded properly as part of the quality control of RHS data. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Biological indices applied to benthic macroinvertebrates at reference conditions of mountain streams in two ecoregions (Poland, the Slovak Republic)

The study was carried out from 2007 to 2010 in two ecoregions: the Carpathians and the Central Highlands. The objectives of our survey were to test the existing biological index metric based on benthic macroinvertebrates at reference conditions in the high- and mid-altitude mountain streams of two ecoregions according to the requirements of the EU WFD and to determine which environmental factors influence the distribution of benthic macroinvertebrates. Our results revealed statistically significant differences in the values of the physical and chemical parameters of water as well as the mean values of metrics between the types of streams at the sampling sites. RDA analysis showed that the temperature of the water, pH, conductivity, the stream gradient, values of the HQA index, and altitude were the parameters most associated with the distribution of benthic macroinvertebrate taxa and the values of the metrics. The values of biological indices should be considered according to the stream typology including altitude and geology. At the reference conditions, the suggested border values of biological indices are very harsh. The values of the biological indices of most sampling sites did not correspond to the requirements of the high status in rivers. The streams at altitudes above 1,200 m a.s.l. should be treated as another river type and new reference values should be established.     

Preliminary testing of River Habitat Survey features for the aims of the WFD hydro-morphological assessment: an overview from the STAR Project

The UK River Habitat Survey (RHS) method for the assessment of hydro-morphological features was applied within the EU STAR project simultaneously with the collection of biological data. A subset of data from 79 sites affected by hydro-morphological alteration and belonging to 7 different stream types was analysed. The different features recorded within RHS were evaluated separately considering the characteristics associated with banks, channel and riparian zone. Different scores were assigned to selected features representing hydro-morphological alteration and naturalness of habitat. The ability of the different compartments to represent the quality gradient of sites was investigated. In addition, the link between macroinvertebrate community and hydro-morphological data was investigated, directly relating indices and metrics calculated from taxa list collected in a site to scores assigned to the RHS features. The sections highly affected by morphological alteration were channel geometry and bank profile. Metrics showing the best correlation with the features selected were EPT taxa, ASPT and ICMi (Inter-calibration Common Metric index). Among the indices studied, the HQA score (Habitat Quality Assessment) apparently played the most important role in structuring biological communities and the lentic-lotic character of rivers was also important. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

In Vitro Characterization of Echinomycin Biosynthesis: Formation and Hydroxylation of L-Tryptophanyl-S-Enzyme and Oxidation of (2S,3S) β-Hydroxytryptophan

Quinoxaline-2-carboxylic acid (QXC) and 3-hydroxyquinaldic acid (HQA) feature in quinomycin family and confer anticancer activity. In light of the significant potency against cancer, the biosynthetic gene clusters have been reported from many different Streptomyces strains, and the biosynthetic pathway were proposed mainly based on the in vivo feeding experiment with isotope labeled putative intermediates. Herein we report another gene cluster from Streptomyces griseovariabilis subsp. bandungensis subsp. nov responsible for the biosynthesis of echinomycin (a member of quinomycin family, also named quinomycin A) and presented in vitro evidence to corroborate the previous hypothesis on QXC biosynthesis, showing that only with the assistance of a MbtH-like protein Qui5, did the didomain NRPS protein (Qui18) perform the loading of a L-tryptophan onto its own PCP domain. Particularly, it was found that Qui5 and Qui18 subunits form a functional tetramer through size exclusion chromatography. The subsequent hydroxylation on β-carbon of the loaded L-tryptophan proved in vitro to be completed by cytochrome P450-dependent hydroxylase Qui15. Importantly, only the Qui18 loaded L-tryptophan can be hydroxylated by Qui15 and the enzyme was inactive on free L-tryptophan. Additionally, the chemically synthesized (2S,3S) β-hydroxytryptophan was detected to be converted by the tryptophan 2,3-dioxygenase Qui17 through LC-MS, which enriched our previous knowledge that tryptophan 2,3-dioxygenase nearly exclusively acted on L-tryptophan and 6-fluoro-tryptophan.     

River morphology of mountain streams influenced by an extreme windstorm in the High Tatra Mountains (northern Slovakia)

In this study we describe how the river morphology of seven High Tatras streams has been influenced in different ways by a severe windstorm that occurred in November 2004. A control site situated in an undisturbed area is compared with six sites in windstorm damaged areas. In 2009–2010 River Habitat Survey (RHS) was used to assess the character of 500 m lengths of stream and to derive associated morphological indices (HQA, HMS sensu Raven 1998, RHS indices sensu Vaughan 2010). Large amounts of large woody debris in the channel influenced the streams despite most of it having been removed after the windstorm. Streams situated in deforested valley slopes were probably more affected by erosion; they had higher amounts of transported inorganic matter and also depositional bar features. Some changes in land-use had occurred with streams situated on the south-east side of the High Tatras having a more uniform banktop vegetation structure than the other streams. Distinct secondary succession of bank top vegetation was observed along windstorm influenced streams. The highest percentage of shrubs occurred where impacted streams remained untouched. Morphologically, the streams most affected by windstorm are those situated in deforested steep valley slopes that are affected by erosion and siltation.     

Caddisflies (Trichoptera) as good indicators of environmental stress in mountain lotic ecosystems

We analysed the influence of environmental stress (mainly deforestation of catchment due to windstorm) on taxonomic structure as well as composition of functional groups (FG) based on zonation preferences and feeding types of caddisfly species in 11 streams (12 sites) with various environmental conditions situated in the High Tatra Mts, Slovakia. Using Spearman correlation, we confirmed an expected positive association between taxonomic and functional groups richness (Ri), diversity (Shannon — Wiener Index) (Di) and equitability (Eq), but not with habitat diversity (HQA indices). By testing measured physiographical, physico-chemical and hydromorphological factors using CCA analysis we found that stream order and temperature best explained the functional and taxonomic structure of caddisfly assemblages. The occurrence of Rhyacophila glareosa decreased with increasing daily mean water temperature positively correlated with deforestation, whereas R. tristis (dominating at sites most affected by erosion) showed the opposite pattern. Composition of functional groups based on zonation preferences and feeding types distinguished well near natural sites from impacted ones, dominated by crenal/rhithral predators, and explained more of the overall variance of the species-environment relationships than taxonomic composition of caddisfly assemblages.     

Effects of flow regime alteration on fluvial habitats and riparian quality in a semiarid Mediterranean basin

The Segura River Basin is one of the most arid and regulated zones in the Mediterranean as well as Europe that includes four hydrologic river types, according to their natural flow regime: main stem rivers, stable streams, seasonal streams and temporary streams. The relationships between flow regime and fluvial and riparian habitats were studied at reference and hydrologically altered sites for each of the four types. Flow regime alteration was assessed using two procedures: (1) an indirect index, derived from variables associated with the main hydrologic pressures in the basin, and (2) reference and altered flow series analyses using the Indicators of Hydrologic Alteration (IHA) and the Indicators of Hydrologic Alteration in Rivers (IAHRIS). Habitats were characterized using the River Habitat Survey (RHS) and its derived Habitat Quality Assessment (HQA) score, whereas riparian condition was assessed using the Riparian Quality Index (RQI) and an inventory of riparian native/exotic species. Flow stability and magnitude were identified as the main hydrologic drivers of the stream habitats in the Segura Basin. Hydrologic alterations were similar to those described in other Mediterranean arid and semiarid areas where dams have reduced flow magnitude and variability and produced the inversion of seasonal patterns. Additionally, the Segura Basin presented two general trends: an increase in flow torrentiality in main stems and an increase in temporality in seasonal and temporary streams. With the indirect alteration index, main stems presented the highest degree of hydrologic alteration, which resulted in larger channel dimensions and less macrophytes and mesohabitats. However, according to the hydrologic analyses, the seasonal streams presented the greatest alteration, which was supported by the numerous changes in habitat features. These changes were associated with a larger proportion of uniform banktop vegetation as well as reduced riparian native plant richness and mesohabitat density. Both stream types presented consequent reductions in habitat and riparian quality as the degree of alteration increased. However, stable streams, those least impacted in the basin, and temporary streams, which are subject to great hydrologic stress in reference conditions, showed fewer changes in physical habitat due to hydrologic alteration. This study clarifies the relationships between hydrologic regime and physical habitat in Mediterranean basins. The hydrologic and habitat indicators that respond to human pressures and the thresholds that imply relevant changes in habitat and riparian quality presented here will play a fundamental role in the use of holistic frameworks when developing environmental flows on a regional scale.     

Interactions between metal ions and carbohydrates: the coordination behavior of neutral erythritol to zinc and europium nitrate

The single crystals of coordinated complexes of neutral erythritol (C4H10O4) with zinc nitrate and europium nitrate were synthesized and studied using FT-IR and single crystal X-ray diffraction analysis. In the structure of Zn(NO3)2.C4H10O4, ZnEN (E denotes erythritol, N represents nitrate), Zn2+ is coordinated to four hydroxyl groups from two erythritol molecules and two oxygen atoms from two nitrates. Two Zn2+ are connected by one erythritol molecule to form Zn(C4H10O4)(NO3)2 chain, and layers formed by above chain pile to produce 3D structures. In the structure of Eu(NO3)3.C4H10O4.C2H5OH, EuEN, Eu3+ is 10-coordinated by six oxygen atoms from three nitrate ions, three hydroxyl groups from one erythritol molecule and one hydroxyl group from ethanol. In the above erythritol complexes, two hydroxyl groups of erythritol coordinate to one metal ion and the other two to another metal ion or erythritol acts as three-hydroxyl groups donor. The OH groups of erythritol act as ligand to coordinate to metal ions on one hand, one the other hand, OH groups form hydrogen bonds network to build three-dimensional structures.     

Purification to homogeneity of GD3 synthase and partial purification of GM3 synthase from rat brain

A CMP-sialic acid: GM3 sialyltransferase (GD3 synthase) and a CMP-sialic acid: LacCer sialyltransferase (GM3 synthase) have been purified 10,000- and 3,000-fold, respectively, from the Triton X-100 extract of rat brain. The two enzymes were purified and resolved by affinity chromatography on two successive CDP-Sepharose columns by NaCl gradient elution. Final purification of GD3 synthase was achieved by specific elution from a 'GM3 acid'-Sepharose column with buffer containing GM3. Sodium dodecylsulfate-gel electrophoresis of GD3 synthase revealed a single major protein band with an apparent molecular weight of 55,000.     

Microdissection and PCR Amplification of Single Soybean Chromosome

Glass needles were successfully used to dissect the soybean (Glycine max L. ) single chromosome under the micromanipulator in this research. Two dissected soybean chromosomes were digested by Sau3A in two 0.5 mL Eppendorf tubes respectively. The two ends of chromosomal fragments were ligated with Sau3A linker adoptor. After two rounds of PCR amplification, smear DNA fragments ranged from 0.3 to 3 kb were acquired. Southern hybridization result showed the PCR products from the two single soybean chromosomes were homogeneous with the soybean genomic DNA, indicating that DNAs from the two single chromosomes have been successfully amplified. At the same time, the amplified products from the two of the distinguished single chromosome appeared somewhat different. The authors dissected the small chromosomes only by a traditional inverted microscope. Therefore, this research provides a plausible chance for amplification and microcloning of single small chromosomes.