Synthesis and evaluation of new N6-substituted adenosine-5′-N-methylcarboxamides as A3 adenosine receptor agonists

A number of N⁶-substituted adenosine-5′-N-methylcarboxamides were synthesised and their pharmacology, in terms of their receptor affinity, selectivity and cardioprotective effects, were explored. The first series of compounds, 4a–4f and 5a–5f, showed modest receptor affinity for the A₃AR with K_i values in the low to mid μM range. However, the incorporation of a 4-(2-aminoethyl)-2,6-di-tert-butylphenol group in the N⁶-position (in compounds 4g and 5g) significantly improved the affinity with K_i values of 30 and 9 nM, respectively. Improvements in affinity, as well as selectivity were seen when a functionalised linker was introduced. The N⁶-phenyl series, compounds 7a–7d, demonstrated low to mid nanomolar receptor affinities (K_i = 2.3–45.0 nM), with 7b displaying 109-fold selectivity for the A₃AR (vs A₁). The N⁶-benzyl series 9a–9c also proved to be potent and selective A₃AR agonists and the longer chain length linker 13 was tolerated at the A₃AR without abrogation of affinity or selectivity. Cardioprotection was demonstrated by a simulated ischaemia cell culture assay, whereby 7b, 7c, 9a, 9b and 9c all showed cardioprotective effects at 100 nM comparable or better than the benchmark A₃AR agonist IB-MECA, but which were indistinguishable by statistical analysis. For example, compound 9c reduced cell death by 68.0 ± 3.6%.     

Three novel missense mutations in the antithrombin III (AT3) gene causing recurrent venous thrombosis

The polymerase chain reaction and direct sequencing were used to determine the nature of the mutations in the antithrombin III (AT3) gene in seven unrelated patients with familial antithrombin III (ATIII) deficiency and recurrent venous thrombosis. Three novel mutations were found, two associated with a type I deficiency state (Pro80Thr and His120Tyr) manifesting reduced synthesis of ATIII. The other novel lesion (Met251Ile) was associated with a dysfunctional ATIII protein (type II ATIII deficiency) and is predicted to interfere either with a heparin-induced conformational change in the ATIII molecule or with docking to thrombin. A novel polymorphism (Tyr158Cys) was also found to occur in several individuals of Scandinavian origin.     

Statistical characterization of protein ensembles

When accounting for structural fluctuations or measurement errors, a single rigid structure may not be sufficient to represent a protein. One approach to solve this problem is to represent the possible conformations as a discrete set of observed conformations, an ensemble. In this work, we follow a different richer approach, and introduce a framework for estimating probability density functions in very high dimensions, and then apply it to represent ensembles of folded proteins. This proposed approach combines techniques such as kernel density estimation, maximum likelihood, cross-validation, and bootstrapping. We present the underlying theoretical and computational framework and apply it to artificial data and protein ensembles obtained from molecular dynamics simulations. We compare the results with those obtained experimentally, illustrating the potential and advantages of this representation.     

TREHALOSE PHOSPHATE SYNTHASE11-dependent trehalose metabolism promotes Arabidopsis thaliana defense against the phloem-feeding insect Myzus persicae

Agricultural productivity is limited by the removal of sap, alterations in source-sink patterns, and viral diseases vectored by aphids, which are phloem-feeding pests. Here we show that TREHALOSE PHOSPHATE SYNTHASE11 (TPS11) gene-dependent trehalose metabolism regulates Arabidopsis thaliana defense against Myzus persicae (Sülzer), commonly known as the green peach aphid (GPA). GPA infestation of Arabidopsis resulted in a transient increase in trehalose and expression of the TPS11 gene, which encodes a trehalose-6-phosphate synthase/phosphatase. Knockout of TPS11 function abolished trehalose increases in GPA-infested leaves of the tps11 mutant plant and attenuated defense against GPA. Trehalose application restored resistance in the tps11 mutant, confirming that the lack of trehalose accumulation is associated with the inability of the tps11 mutant to control GPA infestation. Resistance against GPA was also higher in the trehalose hyper-accumulating tre1 mutant and in bacterial otsB gene-expressing plants, further supporting the conclusion that trehalose plays a role in Arabidopsis defense against GPA. Evidence presented here indicates that TPS11-dependent trehalose regulates expression of the PHYTOALEXIN DEFICIENT4 gene, which is a key modulator of defenses against GPA. TPS11 also promotes the re-allocation of carbon into starch at the expense of sucrose, the primary plant-derived carbon and energy source for the insect. Our results provide a framework for the signaling function of TPS11-dependent trehalose in plant stress responses, and also reveal an important contribution of starch in controlling the severity of aphid infestation.     

Inhibition of mycolic acid transport across the Mycobacterium tuberculosis plasma membrane

New chemotherapeutics active against multidrug-resistant Mycobacterium tuberculosis are urgently needed. We report on the identification of an adamantyl urea compound that shows potent bactericidal activity against M. tuberculosis and a unique mode of action, namely the abolition of the translocation of mycolic acids from the cytoplasm, where they are synthesized to the periplasmic side of the plasma membrane and are in turn transferred onto cell wall arabinogalactan or used in the formation of virulence-associated, outer membrane, trehalose-containing glycolipids. Whole-genome sequencing of spontaneous-resistant mutants of M. tuberculosis selected in vitro followed by genetic validation experiments revealed that our prototype inhibitor targets the inner membrane transporter MmpL3. Conditional gene expression of mmpL3 in mycobacteria and analysis of inhibitor-treated cells validate MmpL3 as essential for mycobacterial growth and support the involvement of this transporter in the translocation of trehalose monomycolate across the plasma membrane.     

Depletion of M. tuberculosis GlmU from Infected Murine Lungs Effects the Clearance of the Pathogen

M. tuberculosis N-acetyl-glucosamine-1-phosphate uridyltransferase (GlmU_Mtb) is a bi-functional enzyme engaged in the synthesis of two metabolic intermediates N-acetylglucosamine-1-phosphate (GlcNAc-1-P) and UDP-GlcNAc, catalyzed by the C- and N-terminal domains respectively. UDP-GlcNAc is a key metabolite essential for the synthesis of peptidoglycan, disaccharide linker, arabinogalactan and mycothiols. While glmU _Mtb was predicted to be an essential gene, till date the role of GlmU_Mtb in modulating the in vitro growth of Mtb or its role in survival of pathogen ex vivo / in vivo have not been deciphered. Here we present the results of a comprehensive study dissecting the role of GlmU_Mtb in arbitrating the survival of the pathogen both in vitro and in vivo. We find that absence of GlmU_Mtb leads to extensive perturbation of bacterial morphology and substantial reduction in cell wall thickness under normoxic as well as hypoxic conditions. Complementation studies show that the acetyl- and uridyl- transferase activities of GlmU_Mtb are independently essential for bacterial survival in vitro, and GlmU_Mtb is also found to be essential for mycobacterial survival in THP-1 cells as well as in guinea pigs. Depletion of GlmU_Mtb from infected murine lungs, four weeks post infection, led to significant reduction in the bacillary load. The administration of Oxa33, a novel oxazolidine derivative that specifically inhibits GlmU_Mtb, to infected mice resulted in significant decrease in the bacillary load. Thus our study establishes GlmU_Mtb as a strong candidate for intervention measures against established tuberculosis infections.     

Complete genome sequence of Meiothermus ruber type strain (21)

Meiothermus ruber (Loginova et al. 1984) Nobre et al. 1996 is the type species of the genus Meiothermus. This thermophilic genus is of special interest, as its members share relatively low degrees of 16S rRNA gene sequence similarity and constitute a separate evolutionary lineage from members of the genus Thermus, from which they can generally be distinguished by their slightly lower temperature optima. The temperature related split is in accordance with the chemotaxonomic feature of the polar lipids. M. ruber is a representative of the low-temperature group. This is the first completed genome sequence of the genus Meiothermus and only the third genome sequence to be published from a member of the family Thermaceae. The 3,097,457 bp long genome with its 3,052 protein-coding and 53 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

A whole‐genome,radiation hybrid mapping resource of hexaploid wheat

Generating a contiguous, ordered reference sequence of a complex genome such as hexaploid wheat (2n = 6x = 42; approximately 17 GB) is a challenging task due to its large, highly repetitive, and allopolyploid genome. In wheat, ordering of whole‐genome or hierarchical shotgun sequencing contigs is primarily based on recombination and comparative genomics‐based approaches. However, comparative genomics approaches are limited to syntenic inference and recombination is suppressed within the pericentromeric regions of wheat chromosomes, thus, precise ordering of physical maps and sequenced contigs across the whole‐genome using these approaches is nearly impossible. We developed a whole‐genome radiation hybrid (WGRH) resource and tested it by genotyping a set of 115 randomly selected lines on a high‐density single nucleotide polymorphism (SNP) array. At the whole‐genome level, 26 299 SNP markers were mapped on the RH panel and provided an average mapping resolution of approximately 248 Kb/cR₁₅₀₀ with a total map length of 6866 cR₁₅₀₀. The 7296 unique mapping bins provided a five‐ to eight‐fold higher resolution than genetic maps used in similar studies. Most strikingly, the RH map had uniform bin resolution across the entire chromosome(s), including pericentromeric regions. Our research provides a valuable and low‐cost resource for anchoring and ordering sequenced BAC and next generation sequencing (NGS) contigs. The WGRH developed for reference wheat line Chinese Spring (CS‐WGRH), will be useful for anchoring and ordering sequenced BAC and NGS based contigs for assembling a high‐quality, reference sequence of hexaploid wheat. Additionally, this study provides an excellent model for developing similar resources for other polyploid species.