Bioconjugatable porphyrins bearing a compact swallowtail motif for water solubility

A broad range of applications requires access to water-soluble, bioconjugatable porphyrins. Branched alkyl groups attached at the branching site to the porphyrin meso position are known to impart high organic solubility. Such "swallowtail" motifs bearing a polar group (hydroxy, dihydroxyphosphoryl, dihydroxyphosphoryloxy) at the terminus of each branch have now been incorporated at a meso site in trans-AB-porphyrins. The incorporation of the swallowtail motif relies on rational synthetic methods whereby a 1,9-bis(N-propylimino)dipyrromethane (bearing a bioconjugatable tether at the 5-position) is condensed with a dipyrromethane (bearing a protected 1,5-dihydroxypent-3-yl unit at the 5-position). The two hydroxy groups in the swallowtail motif of each of the resulting zinc porphyrins can be transformed to the corresponding diphosphate or diphosphonate product. A 4-(carboxymethyloxy)phenyl group provides the bioconjugatable tether. The six such porphyrins reported here are highly water-soluble (> or =20 mM at room temperature in water at pH 7) as determined by visual inspection, UV-vis absorption spectroscopy, or 1H NMR spectroscopy. Covalent attachment was carried out in aqueous solution with the unprotected porphyrin diphosphonate and a monoclonal antibody against the T-cell receptor CD3epsilon. The resulting conjugate performed comparably to a commercially available fluorescein isothiocyanate-labeled antibody with Jurkat cells in flow cytometry and fluorescence microscopy assays. Taken together, this work enables preparation of useful quantities of water-soluble, bioconjugatable porphyrins in a compact architecture for applications in the life sciences.     

Enzyme-based formulations for decontamination: current state and perspectives

Development of noncorrosive, cost-effective, environmentally benign, and broad-spectrum antimicrobial formulations is necessary for clinical, industrial, and domestic purposes. Many current decontaminating formulations are effective, but they require the use of strong oxidizing agents or organic solvents that have deleterious effects on human health and the surrounding environment. The emergence of antibiotic-resistant pathogens has motivated researchers to develop enzyme-based self-decontaminating formulations as alternatives to such chemical decontamination approaches. Hydrolytic and oxidative enzymes can be used to deactivate pathogens, including bacteria, spores, viruses, and fungi. Laccases, haloperoxidases, and perhydrolases catalyze the generation of biocidal oxidants, such as iodine, bromine, hypohalous acid (e.g., HOCl or HOBr), and peracetic acid. These oxidants have broad-spectrum antimicrobial activity. Due to the multi-pathway action of these oxidants, it has proven extremely difficult for microbes to gain resistance. Thus far, few examples have been reported on enzyme-based antimicrobial formulations. For these reasons, various enzyme-containing antimicrobial formulations are highlighted in this review.     

Improving microbial fitness in the mammalian gut by in vivo temporal functional metagenomics

Elucidating functions of commensal microbial genes in the mammalian gut is challenging because many commensals are recalcitrant to laboratory cultivation and genetic manipulation. We present Temporal FUnctional Metagenomics sequencing (TFUMseq), a platform to functionally mine bacterial genomes for genes that contribute to fitness of commensal bacteria in vivo. Our approach uses metagenomic DNA to construct large‐scale heterologous expression libraries that are tracked over time in vivo by deep sequencing and computational methods. To demonstrate our approach, we built a TFUMseq plasmid library using the gut commensal Bacteroides thetaiotaomicron (Bt) and introduced Escherichia coli carrying this library into germfree mice. Population dynamics of library clones revealed Bt genes conferring significant fitness advantages in E. coli over time, including carbohydrate utilization genes, with a Bt galactokinase central to early colonization, and subsequent dominance by a Bt glycoside hydrolase enabling sucrose metabolism coupled with co‐evolution of the plasmid library and E. coli genome driving increased galactose utilization. Our findings highlight the utility of functional metagenomics for engineering commensal bacteria with improved properties, including expanded colonization capabilities in vivo.     

A novel approach for multi-domain and multi-gene family identification provides insights into evolutionary dynamics of disease resistance genes in core eudicot plants

Background

Recent advances in DNA sequencing techniques resulted in more than forty sequenced plant genomes representing a diverse set of taxa of agricultural, energy, medicinal and ecological importance. However, gene family curation is often only inferred from DNA sequence homology and lacks insights into evolutionary processes contributing to gene family dynamics. In a comparative genomics framework, we integrated multiple lines of evidence provided by gene synteny, sequence homology and protein-based Hidden Markov Modelling to extract homologous super-clusters composed of multi-domain resistance (R)-proteins of the NB-LRR type (for NUCLEOTIDE BINDING/LEUCINE-RICH REPEATS), that are involved in plant innate immunity.

Results

To assess the diversity of R-proteins within and between species, we screened twelve eudicot plant genomes including six major crops and found a total of 2,363 NB-LRR genes. Our curated R-proteins set shows a 50% average for tandem duplicates and a 22% fraction of gene copies retained from ancient polyploidy events (ohnologs). We provide evidence for strong positive selection and show significant differences in molecular evolution rates (Ka/Ks-ratio) among tandem- (mean = 1.59), ohnolog (mean = 1.36) and singleton (mean = 1.22) R-gene duplicates. To foster the process of gene-edited plant breeding, we report species-specific presence/absence of all 140 NB-LRR genes present in the model plant Arabidopsis and describe four distinct clusters of NB-LRR “gatekeeper” loci sharing syntenic orthologs across all analyzed genomes.

Conclusion

By curating a near-complete set of multi-domain R-protein clusters in an eudicot-wide scale, our analysis offers significant insight into evolutionary dynamics underlying diversification of the plant innate immune system. Furthermore, our methods provide a blueprint for future efforts to identify and more rapidly clone functional NB-LRR genes from any plant species.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-966) contains supplementary material, which is available to authorized users.     

Role of nitric oxide in vasodilation in upstream muscle during intermittent pneumatic compression.

This study investigated the dosage effects of nitric oxide synthase (NOS) inhibitor N(G)-monomethyl-L-arginine (L-NMMA) on intermittent pneumatic compression (IPC)-induced vasodilation in uncompressed upstream muscle and the effects of IPC on endothelial NOS (eNOS) expression in upstream muscle. After L-NMMA infusion, mean arterial pressure increased by 5% from baseline (99.5 +/- 18.7 mmHg; P < 0.05). Heart rate and respiratory rate were not significantly affected. One-hour IPC application on legs induced a 10% dilation from baseline in 10- to 20-microm arterioles and a 10-20% dilation in 21- to 40 microm arterioles and 41- to 70-microm arteries in uncompressed cremaster muscle. IPC-induced vasodilation was dose dependently reduced, abolished, or even reversed by concurrently infused L-NMMA. Moreover, expression of eNOS mRNA in uncompressed cremaster muscle was upregulated to 2 and 2.5 times normal at the end of 1- and 5-h IPC on legs, respectively, and the expression of eNOS protein was upregulated to 1.8 times normal. These increases returned to baseline level after cessation of IPC. The results suggest that eNOS plays an important role in regulating the microcirculation in upstream muscle during IPC.     

Turing bifurcations with a temporally varying diffusion coefficient

This paper is concerned with the possibility of Turing bifurcations in a reaction-diffusion system in which the diffusion coefficient of one species varies periodically in time. This problem was introduced and investigated numerically by Timm and Okubo (J. Math. Biol. 30, 307, 1992) in the context of predator-prey interactions in plankton populations. Here, I consider the simple case in which the temporal variation in diffusivity has a square-tooth form, alternating between two constant values, with a period that is long compared with the time scale of the kinetics. The analysis is valid for any set of reaction kinetics. I derive explicit expressions for the Floquet multipliers that determine the stability of the steady state, and thereby obtain the conditions for diffusion driven instability to occur. These conditions imply that, depending on the kinetics, the homogeneous equilibrium may be either more or less stable than when the diffusion coefficient is a constant equal to the mean of the variable diffusivity. I go on to consider the form of the solution when diffusion driven instability does occur, and I use perturbation theory to determine the effect of a small temporal variation in the diffusion coefficient on the spatial wavelength of the pattern that results from diffusion driven instability.     

The genetic basis of developmental abnormalities in interpopulation hybrids of the moss Ceratodon purpureus

Divergent populations are intrinsically reproductively isolated when hybrids between them either fail to develop properly or do not produce viable offspring. Intrinsic isolation may result from Dobzhansky-Muller (DM) incompatibilities, in which deleterious interactions among genes or gene products lead to developmental problems or underdominant chromosome structure differences between the parents. These mechanisms can be tested by studying marker segregation patterns in a hybrid mapping population. Here we examine the genetic basis of abnormal development in hybrids between two geographically distant populations of the moss Ceratodon purpureus. Approximately half of the hybrid progeny exhibited a severely reduced growth rate in early gametophyte development. We identified four unlinked quantitative trait loci (QTL) that interacted asymmetrically to cause the abnormal development phenotype. This pattern is consistent with DM interactions. We also found an excess of recombination between three marker pairs in the abnormally developing progeny, relative to that estimated in the normal progeny. This suggests that structural differences in these regions contribute to hybrid breakdown. Two QTL coincided with inferred structural differences, consistent with recent theory suggesting that rearrangements may harbor population divergence alleles. These observations suggest that multiple complex genetic factors contribute to divergence among populations of C. purpureus.