Chloramphenicol Derivatives with Antibacterial Activity Identified by Functional Metagenomics.

A functional metagenomic approach identified novel and diverse soil-derived DNAs encoding inhibitors to methicillin-resistant Staphylococcus aureus (MRSA). A metagenomic DNA soil library containing 19 200 recombinant Escherichia coli BAC clones with 100 Kb average insert size was screened for antibiotic activity. Twenty-seven clones inhibited MRSA, seven of which were found by LC-MS to possess modified chloramphenicol ( Cm) derivatives, including three new compounds whose structures were established as 1-acetyl-3-propanoylchloramphenicol, 1-acetyl-3-butanoylchloramphenicol, and 3-butanoyl-1-propanoylchloramphenicol. Cm was used as the selectable antibiotic for cloning, suggesting that heterologously expressed enzymes resulted in derivatization of Cm into new chemical entities with biological activity. An esterase was found to be responsible for the enzymatic regeneration of Cm, and the gene trfA responsible for plasmid copy induction was found to be responsible for inducing antibacterial activity in some clones. Six additional acylchloramphenicols were synthesized for structure and antibacterial activity relationship studies, with 1- p-nitrobenzoylchloramphenicol the most active against Mycobacterium intracellulare and Mycobacterium tuberculosis, with MICs of 12.5 and 50.0 µg/mL, respectively.

Enantioselective Cascade Reaction for Synthesis of Quinolinones through Synergistic Catalysis Using Cu-Pybox and Chiral Benzotetramisole as Catalysts.

In contrast to the well-studied asymmetric catalyzed synthesis of tetrahydroquinolines, the asymmetric methodologies toward 3,4-dihydroquinolin-2-ones are quite rare. Herein, the first asymmetric cascade reaction is reported between ethynyl benzoxazinanones and mixed-anhydrides generated from aryl acetic acids and pivaloyl chloride, based on synergistic catalysis. This allowed the formation of attractive 3,4-dihydroquinolin-2-ones bearing two vicinal chiral centers at C3 and C4 in high yields with excellent diastereo- and enantioselectivities. A plausible chiral induction model for this reaction was proposed. The utility of this methodology was exemplified by further elaboration of the cyclization products by removal of the N-protecting groups.

Amide-Phosphonium Salt as Bifunctional Phase Transfer Catalyst for Asymmetric 1,6-Addition of Malonate Esters to para-Quinone Methides.

Asymmetric 1,6-addition of malonates to para-quinone methides has been developed by using amide-phosphonium salts derived from easily available chiral α-amino acids as bifunctional phase transfer catalysts. Stabilized para-quinone methides with various substituents on the phenyl ring were reacted with diphenyl malonates to give functionalized diaryl methines in excellent yields and high to excellent ee's. Furthermore, to show the utility of this methodology, a gram scale synthesis of an 1,6-addition adduct and its further elaboration into the key intermediate for synthesis of GPR40 agonists were also described.

Efficient de novo assembly of large and complex genomes by massively parallel sequencing of Fosmid pools.

BACKGROUND: Sampling genomes with Fosmid vectors and sequencing of pooled Fosmid libraries on the Illumina platform for massive parallel sequencing is a novel and promising approach to optimizing the trade-off between sequencing costs and assembly quality. RESULTS: In order to sequence the genome of Norway spruce, which is of great size and complexity, we developed and applied a new technology based on the massive production, sequencing, and assembly of Fosmid pools (FP). The spruce chromosomes were sampled with ~40,000 bp Fosmid inserts to obtain around two-fold genome coverage, in parallel with traditional whole genome shotgun sequencing (WGS) of haploid and diploid genomes. Compared to the WGS results, the contiguity and quality of the FP assemblies were high, and they allowed us to fill WGS gaps resulting from repeats, low coverage, and allelic differences. The FP contig sets were further merged with WGS data using a novel software package GAM-NGS. CONCLUSIONS: By exploiting FP technology, the first published assembly of a conifer genome was sequenced entirely with massively parallel sequencing. Here we provide a comprehensive report on the different features of the approach and the optimization of the process.We have made public the input data (FASTQ format) for the set of pools used in this study:ftp://congenie.org/congenie/Nystedt_2013/Assembly/ProcessedData/FosmidPools/.(alternatively accessible via http://congenie.org/downloads).The software used for running the assembly process is available at http://research.scilifelab.se/andrej_alexeyenko/downloads/fpools/.

[Changes of non-structural carbohydrates and nitrogen contents of needles and twigs in Pinus sylvestris var. mongolica plantations along an aridity gradient]. / 沿干旱梯度樟子松人工林针叶和枝条非结构性碳水化合物及氮含量的变化.

With six Pinus sylvestris var. mongolica plantations (Huinan, Xifeng, Fujia, Zhanggutai, Naiman and Wulanaodu) along an aridity gradient in the Horqin sandy land, we examined the changes in non-structural carbohydrates (NSCs) and nitrogen (N) contents of current and one-year-old needles and twigs, to explore the carbon supply and demand status as well as the nutrient accumulation strategies of P. sylvestris var. mongolica under drought. The results showed that the contents of NSCs and soluble sugars in needles and twigs of P. sylvestris var. mongolica plantations significantly decreased with increasing aridity. From the most humid site (Huinan) to the most aridity site (Wulanaodu), the soluble sugar contents in current and one-year old needles of P. sylvestris var. mongolica decreased from 12.8% and 12.5% to 9.0% and 9.5%, respectively. The soluble sugar contents in current-year old twigs decreased from 15.6% to 9.2%. With increasing aridity, the starch contents in needles and twigs remained relatively stable, soluble sugars/starch ratio in current and one-year old needles decreased, the N contents in current and one-year old twigs significantly increased. The P. sylvestris var. mongolica plantations in the Horqin sandy land consumed soluble sugar storage under drought, resulting in a risk of mortality from 'carbon starvation'. P. sylvestris var. mongolica tended to maintain stable starch storage and accumulate N in twigs to cope with long-term drought stress.

BAC library resources for map-based cloning and physical map construction in barley (Hordeum vulgare L.).

BACKGROUND: Although second generation sequencing (2GS) technologies allow re-sequencing of previously gold-standard-sequenced genomes, whole genome shotgun sequencing and de novo assembly of large and complex eukaryotic genomes is still difficult. Availability of a genome-wide physical map is therefore still a prerequisite for whole genome sequencing for genomes like barley. To start such an endeavor, large insert genomic libraries, i.e. Bacterial Artificial Chromosome (BAC) libraries, which are unbiased and representing deep haploid genome coverage, need to be ready in place. RESULT: Five new BAC libraries were constructed for barley (Hordeum vulgare L.) cultivar Morex. These libraries were constructed in different cloning sites (HindIII, EcoRI, MboI and BstXI) of the respective vectors. In order to enhance unbiased genome representation and to minimize the number of gaps between BAC contigs, which are often due to uneven distribution of restriction sites, a mechanically sheared library was also generated. The new BAC libraries were fully characterized in depth by scrutinizing the major quality parameters such as average insert size, degree of contamination (plate wide, neighboring, and chloroplast), empty wells and off-scale clones (clones with <30 or >250 fragments). Additionally a set of gene-based probes were hybridized to high density BAC filters and showed that genome coverage of each library is between 2.4 and 6.6 X. CONCLUSION: BAC libraries representing >20 haploid genomes are available as a new resource to the barley research community. Systematic utilization of these libraries in high-throughput BAC fingerprinting should allow developing a genome-wide physical map for the barley genome, which will be instrumental for map-based gene isolation and genome sequencing.

Effects of tongmai huoxue yin (see text) on tumor necrosis factor-alpha in the acute cerebral ischemia model rat.

OBJECTIVE: To observe the interfering action of Tongmai Huoxue Yin (see text) on the acute cerebral ischemia model rat. METHODS: Total 60 SD rats, 30 females and 30 males, were randomly divided into 4 groups, sham-operation group, model group, Nimodipine group and Tongmai Huoxue Yin group, 15 rats in each group. The acute cerebral ischemia rat model was duplicated, the middle cerebral artery (MCA) were ligated and the thread was inserted for the rats in the model group, Nimodipine group and Tongmai Huoxue Yin group, for the rats in the sham-operation group, the arteries were separated without ligature and the thread was not inserted. After the modeling has succeed, the water-decocted concentrated solution of 20-fold Tongmai Huoxue Yin clinical dosage was intragastrically administrated in a dose of 3 mL/100 g d divided into twice, 1.5 mL/100 g once. Distilled water 3 mL/100 g x d was intragastrically administrated, 1.5 mL/100 g once, for the rat in the model group, Nimodipne suspension 3 mL/100 g x d (0.6 mg / 100 g) for the Nimodipine group and 3 mL/100 g x d (5.4 g/100 g) for the Tongmai Huoxue Yin group, no drugs for the sham-operation group. And changes of tumor necrosis factor-alpha (TNF-alpha) contents in the serum and brain tissue were investigated. RESULTS: Compared with the model group, compared with the sham-operation group, serum TNF-alpha content at 5 h of focal cerebral ischemic ischemia in the model group started to increase and reached to the high peak at 12 h, but in both the Tongmai Huoxue Yin group and the Nimodipine group decreased in varying degrees at the same time; compared with the sham-operation group, brain TNF-alpha content at 6 h of focal cerebral ischemic ischemia in the model group started to increase and reached to the high peak at 12 h, but in both the Tongmai Huoxue Yin group and the Nimodipine group decreased in varying degrees, with the most obviously decreased at 24 h of ischemia.Tongmai Huoxue Yin could significantly decrease TNF-alpha content in the brain tissue. CONCLUSION: Tongmai Huoxue Yin has a protective action on acute cerebral ischemia injury in the rat.

Palladium-Catalyzed anti-Carbosilylation of Alkynes to Access Isoquinolinone-Containing Exocyclic Vinylsilanes.

A Pd-catalyzed trans-selective carbosilylation reaction of alkynes has been developed. The trans-vinylpalladium species, generated through intramolecular syn-carbopalladation of alkynes and subsequent cis-trans isomerization, were captured by hexamethyldisilane to form multisubstituted vinylsilanes. This reaction provides a useful strategy for the stereoselective synthesis of isoquinolinone-containing exocyclic tetrasubstituted vinylsilanes.

Intermolecular C-H silylation through cascade carbopalladation and vinylic to aryl 1,4-palladium migration.

A palladium-catalyzed remote C-H silylation reaction has been developed through vinylic to aryl 1,4-palladium migration. By using alkyne-tethered aryl iodides as the starting materials and hexamethyldisilane as the silylating reagent, the reaction involves cascade intramolecular carbopalladation, 1,4-palladium migration, and silylation with hexamethyldisilane, and leads to the formation of exocyclic alkene-containing 5-silylisoquinolines as the final products.

Palladium-catalyzed diastereoselective cross-coupling of two aryl halides via C-H activation: synthesis of chiral eight-membered nitrogen heterocycles.

A method for the synthesis of enantiopure eight-membered nitrogen heterocycles has been developed through diastereoselective cross-coupling of 2-iodobiphenyls with 2-bromobenzylamines. The products represent a novel type of chiral scaffold, which feature easy modification and high configurative stability and have the potential to be applied in asymmetric synthesis. Palladacycles that were formed via the C-H activation of 2-iodobiphenyls should act as the intermediates. The reaction provides a new strategy for the synthesis of medium-sized ring compounds.

Diastereoselective Construction of Eight-Membered Carbocycles through Palladium-Catalyzed C(sp3)-H Functionalization.

A palladium-catalyzed cross-coupling reaction of 2-alkylphenyl bromides with biphenylene has been developed. The reactions formed eight-membered carbocycles through C(sp3)-H activation and the formation of two C-C bonds, and the chiral products were obtained with excellent diastereoselectivity. The reaction provides a new strategy for the construction of eight-membered carbocycles, and the products represent a novel type of chiral scaffold.

Palladium-Catalyzed Dual Coupling Reaction of 2-Iodobiphenyls with o-Bromoanilines through C-H Activation: An Approach for the Synthesis of Tribenzo[b,d,f]azepines.

A novel and straightforward approach for the synthesis of tribenzo[b,d,f]azepines starting from 2-iodobiphenyls and 2-bromoanilines has been developed. A wide range of tribenzo[b,d,f]azepines were obtained in good to excellent yields via a cascade intermolecular palladium-catalyzed C-H activation/dual coupling reaction. C,C-palladacycles, which are generated by C-H activation of 2-iodobiphenyls, should be the reaction intermediates.

Palladium-catalyzed intermolecular C-H silylation initiated by aminopalladation.

A Pd(ii)-catalyzed intermolecular C-H silylation reaction initiated by aminopalladation has been developed. The C-H bonds were activated by an alkyl Pd(ii) species generated through aminopalladation and then disilylated with hexamethyldisilane to form disilylated indolines as the final products. The reaction provides a new method for the introduction of silyl groups into complex organic molecules.

Synthesis of 3,4-Fused Tricyclic Indoles through Cascade Carbopalladation and C-H Amination: Development and Total Synthesis of Rucaparib.

3,4-Fused tricyclic indole scaffolds are ubiquitous in bioactive natural products and pharmaceuticals. A new protocol for the synthesis of 3,4-fused tricyclic indoles has been developed through cascade carbopalladation and C-H amination with N,N-di-tert-butyldiaziridinone. The protocol allows access to a range of 3,4-fused tricyclic indoles, including those containing various linkers and fused with medium-sized rings. Rucaparib can be synthesized via this reaction, providing an advantageous synthetic method for the FDA-approved cancer medicine.

Enantioselective synthesis of quaternary 3,4-dihydroisoquinolinones via Heck carbonylation reactions: development and application to the synthesis of Minalrestat analogues.

Minalrestat and its analogues represent structurally novel aldose reductase inhibitors, and the asymmetric synthesis of such pharmaceutically privileged molecules has not been reported yet. We have developed a palladium-catalyzed enantioselective intramolecular carbonylative Heck reaction by using formate esters as the source of CO, which represents the first enantioselective synthesis of quaternary 3,4-dihydroisoquinolines. The reaction provides a facile and efficient method for the synthesis of enantiopure nitrogen-containing heterocyclic compounds bearing an all-carbon quaternary stereocenter. The reaction has been successfully applied to the first asymmetric synthesis of Minalrestat analogues.

Synthesis of Indolines by Palladium-Catalyzed Intermolecular Amination of Unactivated C(sp3)-H Bonds.

A new palladium-catalyzed C(sp3)-H amination reaction has been developed. 1-(tert-Butyl)-2-iodobenzene and its derivatives undergo palladium-catalyzed C-H activation to form palladacycles. The palladacycles are aminated with diaziridinone to form indolines as the final products. The reaction represents a new method for the synthesis of 3,3-disubstituted indolines, which are essential structural motifs in many bioactive compounds and pharmaceutical targets.

Use of Multiple Sequencing Technologies To Produce a High-Quality Genome of the Fungus Pseudogymnoascus destructans, the Causative Agent of Bat White-Nose Syndrome.

White-nose syndrome has recently emerged as one of the most devastating wildlife diseases recorded, causing widespread mortality in numerous bat species throughout eastern North America. Here, we present an improved reference genome of the fungal pathogen Pseudogymnoascus destructans for use in comparative genomic studies.

Trialkyl Methanetricarboxylate as Dialkyl Malonate Surrogate in Copper-Catalyzed Enantioselective Propargylic Substitution.

The first copper-catalyzed enantioselective propargylation of trialkyl methantricarboxylate with propargylic alcohol derivatives was developed. The tricarboxylate unit in the obtained adducts could be easily transformed into a malonate moiety by treating with in situ generated NaOEt in excellent yield without racemization.

Changes in spatial patterns of Caragana stenophylla along a climatic drought gradient on the Inner Mongolian Plateau.

Few studies have investigated the influence of water availability on plant population spatial patterns. We studied changes in the spatial patterns of Caragana stenophylla along a climatic drought gradient within the Inner Mongolian Plateau, China. We examined spatial patterns, seed density, "nurse effects" of shrubs on seedlings, transpiration rates and water use efficiency (WUE) of C. stenophylla across semi-arid, arid, and intensively arid zones. Our results showed that patches of C. stenophylla populations shifted from a random to a clumped spatial pattern towards drier environments. Seed density and seedling survival rate of C. stenophylla decreased from the semi-arid zone to the intensively arid zone. Across the three zones, there were more C. stenophylla seeds and seedlings underneath shrub canopies than outside shrub canopies; and in the intensively arid zone, there were almost no seeds or seedlings outside shrub canopies. Transpiration rates of outer-canopy leaves and WUE of both outer-canopy and inner-canopy leaves increased from the semi-arid zone to the intensively arid zone. In the intensively arid zone, transpiration rates and WUE of inner-canopy leaves were significantly lower and higher, respectively, than those of outer-canopy leaves. We conclude that, as drought stress increased, seed density decreased, seed proportions inside shrubs increased, and "nurse effects" of shrubs on seedlings became more important. These factors, combined with water-saving characteristics associated with clumped spatial patterns, are likely driving the changes in C. stenophylla spatial patterns.

Meeting report: 1st international functional metagenomics workshop may 7-8, 2012, st. Jacobs, ontario, Canada.

This report summarizes the events of the 1(st) International Functional Metagenomics Workshop. The workshop was held on May 7 and 8, 2012, in St. Jacobs, Ontario, Canada and was focused on building an international functional metagenomics community, exploring strategic research areas, and identifying opportunities for future collaboration and funding. The workshop was initiated by researchers at the University of Waterloo with support from the Ontario Genomics Institute (OGI), Natural Sciences and Engineering Research Council of Canada (NSERC) and the University of Waterloo.