Comparison of Cyanopeptolin Genes in Planktothrix, Microcystis, and Anabaena Strains: Evidence for Independent Evolution within Each Genus

The major cyclic peptide cyanopeptolin 1138, produced by Planktothrix strain NIVA CYA 116, was characterized and shown to be structurally very close to the earlier-characterized oscillapeptin E. A cyanopeptolin gene cluster likely to encode the corresponding peptide synthetase was sequenced from the same strain. The 30-kb oci gene cluster contains two novel domains previously not detected in nonribosomal peptide synthetase gene clusters (a putative glyceric acid-activating domain and a sulfotransferase domain), in addition to seven nonribosomal peptide synthetase modules. Unlike in two previously described cyanopeptolin gene clusters from Anabaena and Microcystis, a halogenase gene is not present. The three cyanopeptolin gene clusters show similar gene and domain arrangements, while the binding pocket signatures deduced from the adenylation domain sequences and the additional tailoring domains vary. This suggests loss and gain of tailoring domains within each genus, after the diversification of the three clades, as major events leading to the present diversity. The ABC transporter genes associated with the cyanopeptolin gene clusters form a monophyletic clade and accordingly are likely to have evolved as part of the functional unit. Phylogenetic analyses of adenylation and condensation domains, including domains from cyanopeptolins and microcystins, show a closer similarity between the Planktothrix and Microcystis cyanopeptolin domains than between these and the Anabaena domain. No clear evidence of recombination between cyanopeptolins and microcystins could be detected. There were no strong indications of horizontal gene transfer of cyanopeptolin gene sequences across the three genera, supporting independent evolution within each genus.     

The conformational effects of N-glycosylation on the tailpiece from serum IgM

1H-NMR spectroscopy has been used to study the conformation and dynamics of the isolated tailpiece from human serum immunoglobulin M, a 22-residue peptide containing a single asparagine glycosylation site. The peptide is isolated as a set of glycoforms, varying only in the sequence of the oligosaccharide attached at the glycosylation site. The oligosaccharides present have the general formula (Man)n(GlcNAc)2, with 45% having n = 6, 45% having n = 8 and 10% having n = 7 and/or 9. They have been identified and their NMR parameters compared to those found for the isolated oligosaccharides in free solution. The conformation and dynamics of the peptide component have also been studied, using NOE data and hydrogen-exchange experiments, and the results compared to those obtained from the aglycosyl peptide of the same sequence. The presence of the peptide is found to have no measurable effect on the conformation of the oligosaccharides. However, the presence of oligosaccharide causes a decrease in the conformational mobility of the backbone and sidechains of the peptide in the region of the glycosylation site. This is proposed to result from interactions between the oligosaccharide core and the amino acid side chains. Further, the conformation of the N-glycosidic linkage has been shown to be both rigid and planar. Thus, the conformational space available to an N-linked oligosaccharide in a glycoprotein relative to the protein may depend to a large extent upon the flexibility of the asparagine side chain. Various roles for the different glycoforms of the tail peptide are discussed.     

Gut Microbiota Dynamics during Dietary Shift in Eastern African Cichlid Fishes

The gut microbiota structure reflects both a host phylogenetic history and a signature of adaptation to the host ecological, mainly trophic niches. African cichlid fishes, with their array of closely related species that underwent a rapid dietary niche radiation, offer a particularly interesting system to explore the relative contribution of these two factors in nature. Here we surveyed the host intra- and interspecific natural variation of the gut microbiota of five cichlid species from the monophyletic tribe Perissodini of lake Tanganyika, whose members transitioned from being zooplanktivorous to feeding primarily on fish scales. The outgroup riverine species Astatotilapia burtoni, largely omnivorous, was also included in the study. Fusobacteria, Firmicutes and Proteobacteria represented the dominant components in the gut microbiota of all 30 specimens analysed according to two distinct 16S rRNA markers. All members of the Perissodini tribe showed a homogenous pattern of microbial alpha and beta diversities, with no significant qualitative differences, despite changes in diet. The recent diet shift between zooplantkon- and scale-eaters simply reflects on a significant enrichment of Clostridium taxa in scale-eaters where they might be involved in the scale metabolism. Comparison with the omnivorous species A. burtoni suggests that, with increased host phylogenetic distance and/or increasing herbivory, the gut microbiota begins differentiating also at qualitative level. The cichlids show presence of a large conserved core of taxa and a small set of core OTUs (average 13–15%), remarkably stable also in captivity, and putatively favoured by both restricted microbial transmission among related hosts (putatively enhanced by mouthbrooding behavior) and common host constraints. This study sets the basis for a future large-scale investigation of the gut microbiota of cichlids and its adaptation in the process of the host adaptive radiation.     

Discrimination of Recombinant from Natural Human Growth Hormone Using DNA Aptamers

Detection of athletes who use synthetic human growth hormone (hGH; or somatotropin) to enhance physical strength and obtain an advantage in competitive sports is a formidable problem, as rhGH is virtually identical to the natural pituitary hormone. However, some post-translational and other modifications have been documented by chromatographic separation and mass spectrometry (MS) in a small percentage of rhGH. In the present work, development of DNA aptamers against research-grade rhGH and natural hGH with adsorption of the rhGH aptamers against natural hGH was shown to produce a small family of aptamer sequences that bound consistently with greater affinity to rhGH over a low nanogram-to-microgram range in ELISA-like microplate assays. This collection of rhGH discriminatory aptamer sequences shared some short sequence segments and secondary structural features. The top rhGH discriminatory aptamers also appeared to cross-react with human myoglobin and BSA but not with bone collagen peptides and an unrelated viral envelope peptide. The cross-reactivity results suggested several strings of up to five consecutive amino acids that might serve as common epitopes for aptamer binding. SDS-PAGE revealed that the rhGH existed largely as a 45-kDa dimer, and the natural hGH was almost exclusively monomeric. The existence of the rhGH dimer suggests that a discontinuous “bridge” epitope may exist on the rhGH, which spans the subunits, thereby accounting somewhat for the difference in detection. Overall, these results suggest that aptamers might be useful for routine, presumptive laboratory screening to identify athletes who are potentially cheating by administration of rhGH.     

Setting an optimal α that minimizes errors in null hypothesis significance tests

Null hypothesis significance testing has been under attack in recent years, partly owing to the arbitrary nature of setting α (the decision-making threshold and probability of Type I error) at a constant value, usually 0.05. If the goal of null hypothesis testing is to present conclusions in which we have the highest possible confidence, then the only logical decision-making threshold is the value that minimizes the probability (or occasionally, cost) of making errors. Setting α to minimize the combination of Type I and Type II error at a critical effect size can easily be accomplished for traditional statistical tests by calculating the α associated with the minimum average of α and β at the critical effect size. This technique also has the flexibility to incorporate prior probabilities of null and alternate hypotheses and/or relative costs of Type I and Type II errors, if known. Using an optimal α results in stronger scientific inferences because it estimates and minimizes both Type I errors and relevant Type II errors for a test. It also results in greater transparency concerning assumptions about relevant effect size(s) and the relative costs of Type I and II errors. By contrast, the use of α = 0.05 results in arbitrary decisions about what effect sizes will likely be considered significant, if real, and results in arbitrary amounts of Type II error for meaningful potential effect sizes. We cannot identify a rationale for continuing to arbitrarily use α = 0.05 for null hypothesis significance tests in any field, when it is possible to determine an optimal α.