Diversely substituted sulfamides for fragment-based drug discovery of carbonic anhydrase inhibitors: synthesis and inhibitory profile

A series of sulfamide fragments has been synthesised and investigated for human carbonic anhydrase inhibition. One of the fragments showing greater selectivity for cancer-related isoforms hCA IX and XII was co-crystalized with hCA II showing significant potential for fragment periphery evolution via fragment growth and linking. These opportunities will be identified in the future via the screening of this fragment structure for co-operative carbonic anhydrase binding with other structurally diverse fragments.Open in a separate window     

Effect of hydrogen concentration on the community structure of hydrogenotrophic methanogens studied by T-RELP analysis of 16S rRNA gene amplicons

Terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes was used to monitor the changes in the composition of the population of methanogens in enrichment cultures under high and low hydrogen concentrations. Hydrogen concentration was shown to determine the structure of a methanogenic community. High hydrogen concentration probably favors the hydrogen-and acetate-utilizing representatives of Methanosarcinaceae, while a more diverse methanogenic community is favored by low hydrogen concentrations.     

Sculpting therapeutic monoclonal antibody N-glycans using endoglycosidases

Immunoglobulin G (IgG) monoclonal antibodies are a prominent and expanding class of therapeutics used for the treatment of diverse human disorders. The chemical composition of the N-glycan on the fragment crystallizable (Fc) region determines the effector functions through interaction with the Fc gamma receptors and complement proteins. The chemoenzymatic synthesis using endo-β-N-acetylglucosaminidases (ENGases) emerged as a strategy to obtain antibodies with customized glycoforms that modulate their therapeutic activity. We discuss the molecular mechanism by which ENGases recognize different N-glycans and protein substrates, especially those that are specific for IgG antibodies, in order to rationalize the glycoengineering of immunotherapeutic antibodies, which increase the impact on the treatment of myriad diseases.     

Chloroplast DNA variation in pearl millet and related species

The evolution of specific regions of the chloroplast genome was studied in five grass species in the genus Pennisetum, including pearl millet, and one species from a related genus (Cenchrus). Three different regions of the chloroplast DNA were investigated. The first region included a 12-kilobase pair (kbp) EcoRI fragment containing the 23S, 16S and 5S ribosomal RNA genes, which is part of a larger duplicated region of reverse orientation. The second region was contained in a 21-kbp Sa/I fragment, which spans the short single-copy sequence separating the two reverse repeat structures and which overlaps the duplicated copies of the 12-kbp Eco RI fragment. The third region was a 6-kbp EcoRI fragment located in the large single-copy region of the chloroplast genome. Together these regions account for slightly less than 25% of the chloroplast genome. Each of these DNA fragments was cloned and used as hybridization probes to determine the distribution of homologous DNA fragments generated by various restriction endonuclease digests.—A survey of 12 geographically diverse collections of pearl millet showed no indication of chloroplast DNA sequence polymorphism, despite moderate levels of nuclear-encoded enzyme polymorphism. Interspecific and intergeneric differences were found for restriction endonuclease sites in both the small and the large single-copy regions of the chloroplast genome. The reverse repeat structure showed identical restriction site distributions in all materials surveyed. These results suggest that the reverse repeat region is differentially conserved during the evolution of the chloroplast genome.     

Synthesis and evaluation of the performance of a small molecule library based on diverse tropane-related scaffolds

A unified synthetic approach was developed that enabled the synthesis of diverse tropane-related scaffolds. The key intermediates that were exploited were cycloadducts formed by reaction between 3-hydroxy-pyridinium salts and vinyl sulfones or sulfonamides. The diverse tropane-related scaffolds were formed by addition of substituents to, cyclisation reactions of, and fusion of additional ring(s) to the key bicyclic intermediates. A set of 53 screening compounds was designed, synthesised and evaluated in order to determine the biological relevance of the scaffolds accessible using the synthetic approach. Two inhibitors of Hedgehog signalling, and four compounds with weak activity against the parasite P. falciparum, were discovered. Three of the active compounds may be considered to be indotropane or pyrrotropane pseudo natural products in which a tropane is fused with a fragment from another natural product class. It was concluded that the unified synthetic approach had yielded diverse scaffolds suitable for the design of performance-diverse screening libraries.     

Amplified Fragment Length Polymorphism and Pulsed Field Gel Electrophoresis for Subspecies Differentiation ofSerpulina pilosicoli

Pulsed field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP) were compared for their ability to differentiate between 50 porcine Serpulina pilosicoli isolates. Both techniques were highly sensitive, dividing the isolates into 36 and 38 groups, respectively. Due to the low workload and high capacity and sensitivity of AFLP, this technique is recommended for future studies of S. pilosicoli. S. pilosicoli isolates were genetically diverse, and generally, identical subtypes were only identified in cultures from the same herd.     

Roseobacter clade bacteria are abundant in coastal sediments and encode a novel combination of sulfur oxidation genes

Roseobacter clade bacteria (RCB) are abundant in marine bacterioplankton worldwide and central to pelagic sulfur cycling. Very little is known about their abundance and function in marine sediments. We investigated the abundance, diversity and sulfur oxidation potential of RCB in surface sediments of two tidal flats. Here, RCB accounted for up to 9.6% of all cells and exceeded abundances commonly known for pelagic RCB by 1000-fold as revealed by fluorescence in situ hybridization (FISH). Phylogenetic analysis of 16S rRNA and sulfate thiohydrolase (SoxB) genes indicated diverse, possibly sulfur-oxidizing RCB related to sequences known from bacterioplankton and marine biofilms. To investigate the sulfur oxidation potential of RCB in sediments in more detail, we analyzed a metagenomic fragment from a RCB. This fragment encoded the reverse dissimilatory sulfite reductase (rDSR) pathway, which was not yet found in RCB, a novel type of sulfite dehydrogenase (SoeABC) and the Sox multi-enzyme complex including the SoxCD subunits. This was unexpected as soxCD and dsr genes were presumed to be mutually exclusive in sulfur-oxidizing prokaryotes. This unique gene arrangement would allow a metabolic flexibility beyond known sulfur-oxidizing pathways. We confirmed the presence of dsrA by geneFISH in closely related RCB from an enrichment culture. Our results show that RCB are an integral part of the microbial community in marine sediments, where they possibly oxidize inorganic and organic sulfur compounds in oxic and suboxic sediment layers.     

Comparative studies to assess bacterial communities on the clover phylloplane using MLST, DGGE and T-RFLP

Denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) were used to characterise the changes that occurred in Bacillus cereus group strains present in the phylloplane of clover Trifolium hybridum over 4 months. These strains had previously been analysed by multiple locus sequence typing (MLST). DGGE displayed many equally intense bands which indicated many equally abundant ribotypes. The bacterial community composition was variable and the leaves sampled as little as a week apart were found to have some temporal variability, indicating that diverse phylloplane bacterial communities follow sequential patterns from time to time. The B. cereus group community clearly clustered into early, mid and late branches, possibly due to multiple successional sequences occurring during growing seasons. The functionally and phylogenetically diverse microbial communities appeared to exhibit predictable successional patterns over shorter time scales. DGGE analysis with the molecular marker rpoB gave better resolution than 16S rRNA amplicons. There were no strong similarities between the dendrograms produced by DGGE, MLST and T-RFLP and the clustering produced by the automated T-RFLP method was variable even between the three restriction enzymes used. The DGGE–MLST method emerged as a superior method to T-RFLP–MLST for rapid typing of bacterial communities.     

Identification and Molecular Epidemiology of Campylobacter coli Isolates from Human Gastroenteritis, Food, and Animal Sources by Amplified Fragment Length Polymorphism Analysis and Penner Serotyping

Campylobacter coli is an infrequently studied but important food-borne pathogen with a wide natural distribution. We investigated its molecular epidemiology by use of amplified fragment length polymorphism (AFLP)-based genotyping and Penner serotyping. Serotype reference strains and 177 Danish isolates of diverse origin identified by routine phenotyping as C. coli were examined. Molecular tools identified some 12% of field isolates as Campylobacter jejuni, emphasizing the need for improved identification methods in routine laboratories. Cluster analysis of AFLP profiles of 174 confirmed C. coli isolates revealed a difference in the distribution of isolates from pig and poultry (chicken, duck, turkey, and ostrich) species and indicated the various poultry species, but not pigs, to be likely sources of human C. coli infection. A poor correlation was observed between serotyping and AFLP profiling, suggesting that the former method has limited value in epidemiological studies of this species.     

Micro-spatial distribution of two sibling periwinkle species across the intertidal indicates hybrdization

Populations of periwinkles Littorina saxatilis (Olivi 1792) and L. arcana Hannaford Ellis, 1978 are well suited for microevolutionary studies, being at the same time closely related and intraspecifically diverse. The divergence between these two sibling species, sympatric over large parts of their distribution areas, is small, the only morphological difference being the pallial gland complex structure in females. Molecular identification is possible with the use of a RAPD nuclear marker (cloned A2.8 DNA fragment) typical for L. arcana. However, in some individuals from sympatric populations molecular and morphological criteria suggest conflicting species affiliation, which may be explained either by hybridization or by shared ancestral polymorphism. We tested the hybridization hypotheses examining the micro-spatial distribution of these two species across the intertidal zone in two distant sites at the Barents Sea. We found that (a) the frequency of putative hybrids in sympatric populations was proportional to the frequency of L. arcana; (b) L. saxatilis bearing A2.8 DNA fragment were almost absent in the lower part of the intertidal zone, where L. arcana was absent too; (c) there was a close positive correlation between the distribution of potential parent molluscs and putative hybrids. Moreover, logistic regression models showed a good agreement between the distribution of putative hybrid frequencies and that of parental species frequencies. All our observations taken together support the hypothesis of hybridization between L. saxatilis and L. arcana. Elucidating the mechanisms that support the species status of these sympatric populations is necessary.     

Mitochondrial DNA Restriction site polymorphism in Drosophila montana and drosophila virilis

Mitochondrial DNA fragment patterns produced by ten restriction endonucleases have been studied for three geographically diverse Drosophila montana lines and two Drosophila virilis lines. The two species are estimated to differ at about 6% of mitochondrial DNA nucleotide sites. In both cases intraspecific genetic variation is relatively small (pairs of lines are estimated to differ at about 0.6% of mitochondrial DNA nucleotide sites). It appears that Japanese and North American D. montana populations have not been separated for an extended period. In D. virilis there is evidence for intraspecific variation in the size of the mitochondrial genome.     

Bacterial epibionts and endolichenic actinobacteria and fungi in the Lower Devonian lichen Chlorolichenomycites salopensis

The charcoalified fragment of the dorsiventrally organized, internally stratified presumed green algal lichen Chlorolichenomycites salopensis from the Lower Devonian Lochkovian strata in the Welsh Borderland carries bacterial colonies on the upper surface, i.e. the cortex, and actinobacterial filaments in the medulla underneath the photobiont layer. Moreover relatively thin hyphae of presumed endolichenic fungi were found. As in extant lichens, which are best regarded as consortia with an unknown number of participants, this internally stratified, fossil thallus fragment of a presumed green algal lichen harbours a diverse microbial community.     

New Molecular Screening Tools for Analysis of Free-Living Diazotrophs in Soil

Free-living nitrogen-fixing prokaryotes (diazotrophs) are ubiquitous in soil and are phylogenetically and physiologically highly diverse. Molecular methods based on universal PCR detection of the nifH marker gene have been successfully applied to describe diazotroph populations in the environment. However, the use of highly degenerate primers and low-stringency amplification conditions render these methods prone to amplification bias, while less degenerate primer sets will not amplify all nifH genes. We have developed a fixed-primer-site approach with six PCR protocols using less degenerate to nondegenerate primer sets that all amplify the same nifH fragment as a previously published PCR protocol for universal amplification. These protocols target different groups of diazotrophs and allowed for direct comparison of the PCR products by use of restriction fragment length polymorphism fingerprinting. The new protocols were optimized on DNA from 14 reference strains and were subsequently tested with bulk DNA extracts from six soils. These analyses revealed that the new PCR primer sets amplified nifH sequences that were not detected by the universal primer set. Furthermore, they were better suited to distinguish between diazotroph populations in the different soils. Because the novel primer sets were not specific for monophyletic groups of diazotrophs, they do not serve as an identification tool; however, they proved powerful as fingerprinting tools for subsets of soil diazotroph communities.     

Thermophilic Lifestyle for an Uncultured Archaeon from Hydrothermal Vents: Evidence from Environmental Genomics

We present a comparative analysis of two genome fragments isolated from a diverse and widely distributed group of uncultured euryarchaea from deep-sea hydrothermal vents. The optimal activity and thermostability of a DNA polymerase predicted in one fragment were close to that of the thermophilic archaeon Thermoplasma acidophilum, providing evidence for a thermophilic way of life of this group of uncultured archaea.     

High throughput sequencing enables discovery of microsatellites from the puff-throated bulbul (Alophoixus pallidus) and assessment of genetic diversity in Khao Yai National Park,Thailand

Bulbuls (family Pycnonotidae) are a diverse family of songbirds that carry out a number of ecologically important functions associated with seed dispersal. Since, 2003, a puff-throated bulbul (Alophoixus pallidus) population in the Mo-Singto Long-term Biodiversity Research Plot in Khao Yai National Park, Thailand has served as a model system for examining how bulbul behavior, movement, and demographics affect Southeast Asian forests. In this study, we used 454 pyrosequencing to discover microsatellites from A. pallidus that will enable the long-term mark-recapture work conducted at Mo-Singto to be complemented by molecular ecology and population genetic studies. In addition, we conducted fragment analysis to examine the level of genetic diversity exhibited by the Mo-Singto population. In total, we identified 103 DNA fragments containing microsatellite repeats and 66 fragments with sufficient flanking sequences to allow for primer design. Upon screening 26 loci via PCR-based genotyping assays, we identified nine polymorphic loci and used eight of these to examine genetic diversity in the Mo-Singto population. The results of these analyses suggest that the Mo-Singto population is moderately diverse (mean number of effective alleles across eight loci = 3.36, standard deviation = 1.78), is more-or-less in Hardy–Weinberg equilibrium, and has not recently been subject to severe population reduction.     

Analysis of Facultative Lithotroph Distribution and Diversity on Volcanic Deposits by Use of the Large Subunit of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase

A 492- to 495-bp fragment of the gene coding for the large subunit of the form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) (rbcL) was amplified by PCR from facultatively lithotrophic aerobic CO-oxidizing bacteria, colorless and purple sulfide-oxidizing microbial mats, and genomic DNA extracts from tephra and ash deposits from Kilauea volcano, for which atmospheric CO and hydrogen have been previously documented as important substrates. PCR products from the mats and volcanic sites were used to construct rbcL clone libraries. Phylogenetic analyses showed that the rbcL sequences from all isolates clustered with form IC rbcL sequences derived from facultative lithotrophs. In contrast, the microbial mat clone sequences clustered with sequences from obligate lithotrophs representative of form IA rbcL. Clone sequences from volcanic sites fell within the form IC clade, suggesting that these sites were dominated by facultative lithotrophs, an observation consistent with biogeochemical patterns at the sites. Based on phylogenetic and statistical analyses, clone libraries differed significantly among volcanic sites, indicating that they support distinct lithotrophic assemblages. Although some of the clone sequences were similar to known rbcL sequences, most were novel. Based on nucleotide diversity and average pairwise difference, a forested site and an 1894 lava flow were found to support the most diverse and least diverse lithotrophic populations, respectively. These indices of diversity were not correlated with rates of atmospheric CO and hydrogen uptake but were correlated with estimates of respiration and microbial biomass.     

Methane- and Sulfur-Metabolizing Microbial Communities Dominate the Lost City Hydrothermal Field Ecosystem

Hydrothermal venting and the formation of carbonate chimneys in the Lost City hydrothermal field (LCHF) are driven predominantly by serpentinization reactions and cooling of mantle rocks, resulting in a highly reducing, high-pH environment with abundant dissolved hydrogen and methane. Phylogenetic and terminal restriction fragment length polymorphism analyses of 16S rRNA genes in fluids and carbonate material from this site indicate the presence of organisms similar to sulfur-oxidizing, sulfate-reducing, and methane-oxidizing Bacteria as well as methanogenic and anaerobic methane-oxidizing Archaea. The presence of these metabolic groups indicates that microbial cycling of sulfur and methane may be the dominant biogeochemical processes active within this ultramafic rock-hosted environment. 16S rRNA gene sequences grouping within the Methylobacter and Thiomicrospira clades were recovered from a chemically diverse suite of carbonate chimney and fluid samples. In contrast, 16S rRNA genes corresponding to the Lost City Methanosarcinales phylotype were found exclusively in high-temperature chimneys, while a phylotype of anaerobic methanotrophic Archaea (ANME-1) was restricted to lower-temperature, less vigorously venting sites. A hyperthermophilic habitat beneath the LCHF may be reflected by 16S rRNA gene sequences belonging to Thermococcales and uncultured Crenarchaeota identified in vent fluids. The finding of a diverse microbial ecosystem supported by the interaction of high-temperature, high-pH fluids resulting from serpentinization reactions in the subsurface provides insight into the biogeochemistry of what may be a pervasive process in ultramafic subseafloor environments.     

Genetic Diversity and Antibiotic Resistance Patterns in a Campylobacter Population Isolated from Poultry Farms in Switzerland

The diversity and genetic interrelation of Campylobacter jejuni and C. coli isolated from Swiss poultry were assessed by three independent typing methods. Samples were derived prior to slaughter from 100 randomly selected flocks (five birds per flock) raised on three different farm types. The observed flock prevalence was 54% in total, with 50% for conventional and 69% for free-range farms. Birds held on farms with a confined roaming area had the lowest prevalence of 37%. Campylobacter isolates were characterized by amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism of flaA PCR fragments (flaA-RFLP), and disk diffusion testing for eight antimicrobial agents that are commonly used in veterinary or human medicine in Switzerland. Analysis of the genotypic results indicates that the Campylobacter population in Swiss poultry is genetically highly diverse. Nevertheless, occasionally, isolates with identical or nearly identical characteristics were isolated from different farms or farm types in different locations. Genetic typing by AFLP and flaA-RFLP was found to be complementary. The majority of isolates (67%) were susceptible to all tested antibiotics; however, single, double, and triple resistances were observed in 7%, 23%, and 2% of the strains, respectively. There was no correlation between genotype and antibiotic resistance. Surprisingly, sulfonamide resistance was frequently found together with streptomycin resistance. Our findings illustrate the results of common genetic exchange in the studied bacterial population.     

Genotypic Characterization of Bradyrhizobium Strains Nodulating Small Senegalese Legumes by 16S-23S rRNA Intergenic Gene Spacers and Amplified Fragment Length Polymorphism Fingerprint Analyses

We examined the genotypic diversity of 64 Bradyrhizobium strains isolated from nodules from 27 native leguminous plant species in Senegal (West Africa) belonging to the genera Abrus, Alysicarpus, Bryaspis, Chamaecrista, Cassia, Crotalaria, Desmodium, Eriosema, Indigofera, Moghania, Rhynchosia, Sesbania, Tephrosia, and Zornia, which play an ecological role and have agronomic potential in arid regions. The strains were characterized by intergenic spacer (between 16S and 23S rRNA genes) PCR and restriction fragment length polymorphism (IGS PCR-RFLP) and amplified fragment length polymorphism (AFLP) fingerprinting analyses. Fifty-three reference strains of the different Bradyrhizobium species and described groups were included for comparison. The strains were diverse and formed 27 groups by AFLP and 16 groups by IGS PCR-RFLP. The sizes of the IGS PCR products from the Bradyrhizobium strains that were studied varied from 780 to 1,038 bp and were correlated with the IGS PCR-RFLP results. The grouping of strains was consistent by the three methods AFLP, IGS PCR-RFLP, and previously reported 16S amplified ribosomal DNA restriction analysis. For investigating the whole genome, AFLP was the most discriminative technique, thus being of particular interest for future taxonomic studies in Bradyrhizobium, for which DNA is difficult to obtain in quantity and quality to perform extensive DNA:DNA hybridizations.     

Habitat degradation impacts black howler monkey (Alouatta pigra) gastrointestinal microbiomes

The gastrointestinal (GI) microbiome contributes significantly to host nutrition and health. However, relationships involving GI microbes, their hosts and host macrohabitats remain to be established. Here, we define clear patterns of variation in the GI microbiomes of six groups of Mexican black howler monkeys (Alouatta pigra) occupying a gradation of habitats including a continuous evergreen rainforest, an evergreen rainforest fragment, a continuous semi-deciduous forest and captivity. High throughput microbial 16S ribosomal RNA gene sequencing indicated that diversity, richness and composition of howler GI microbiomes varied with host habitat in relation to diet. Howlers occupying suboptimal habitats consumed less diverse diets and correspondingly had less diverse gut microbiomes. Quantitative real-time PCR also revealed a reduction in the number of genes related to butyrate production and hydrogen metabolism in the microbiomes of howlers occupying suboptimal habitats, which may impact host health.