Domain swapping reveals that the C- and N-terminal domains of DnaG and DnaB, respectively, are functional homologues

The bacterial primase (DnaG)-helicase (DnaB) interaction is mediated by the C-terminal domain of DnaG (p16) and a linker that joins the N- and C-terminal domains (p17 and p33 respectively) of DnaB. The crystal and nuclear magnetic resonance structures of p16 from Escherichia coli and Bacillus stearothermophilus DnaG proteins revealed a unique structural homology with p17, despite the lack of amino acid sequence similarity. The functional significance of this is not clear. Here, we have employed a 'domain swapping' approach to replace p17 with its structural homologue p16 to create chimeras. p33 alone hydrolyses ATP but exhibits no helicase activity. Fusing p16 (p16-p33) or DnaG (G-p33) to the N-terminus of p33 produced chimeras with partially restored helicase activities. Neither chimera interacted with DnaG. The p16-p33 chimera formed hexamers while G-p33 assembled into tetramers. Furthermore, G-p33 and DnaB formed mixed oligomers with ATPase activity better than that of the DnaB/DnaG complex and helicase activity better than the sum of the individual DnaB and G-p33 activities but worse than that of the DnaB/DnaG complex. Our combined data provide direct evidence that p16 and p17 are not only structural but also functional homologues, albeit their amino acid composition differences are likely to influence their precise roles.     

Estrus behavior and fecal steroid profiles in the Asiatic lion (Panthera leo persica) during natural and gonadotropin-induced estrus

In this paper the behavior of the Asiatic lion was studied during natural and gonadotropin-induced estrus in relation to fecal estradiol and progesterone concentration. The average length of estrus was 5.4 days and no significant difference was observed between natural and gonadotropin-induced estrus. Vocalization and rolling were the major estrus behavioral activities of Asiatic lions and the frequency of these activities were similar in both natural and gonadotropin-induced estrus and treatment with exogenous gonadotropin did not alter estrus behavioral activities. A significant positive correlation was observed between fecal estradiol and frequency of estrus behavior during natural and gonadotropin-induced estrus. Following gonadotropin treatment estrus could be induced in 69% of animals and these induced animals ovulated following hCG treatment. This study reports for the first time the successful use of the non-invasive fecal steroid assay for monitoring the induction of estrus and ovulation in the Asiatic lion.     

Towards a mechanistic understanding of reciprocal drug–microbiome interactions

Broad‐spectrum antibiotics target multiple gram‐positive and gram‐negative bacteria, and can collaterally damage the gut microbiota. Yet, our knowledge of the extent of damage, the antibiotic activity spectra, and the resistance mechanisms of gut microbes is sparse. This limits our ability to mitigate microbiome‐facilitated spread of antibiotic resistance. In addition to antibiotics, non‐antibiotic drugs affect the human microbiome, as shown by metagenomics as well as in vitro studies. Microbiome–drug interactions are bidirectional, as microbes can also modulate drugs. Chemical modifications of antibiotics mostly function as antimicrobial resistance mechanisms, while metabolism of non‐antibiotics can also change the drugs’ pharmacodynamic, pharmacokinetic, and toxic properties. Recent studies have started to unravel the extensive capacity of gut microbes to metabolize drugs, the mechanisms, and the relevance of such events for drug treatment. These findings raise the question whether and to which degree these reciprocal drug–microbiome interactions will differ across individuals, and how to take them into account in drug discovery and precision medicine. This review describes recent developments in the field and discusses future study areas that will benefit from systems biology approaches to better understand the mechanistic role of the human gut microbiota in drug actions.     

Competition between Diachasmimorpha kraussii and Fopius arisanus in Bactrocera tryoni: does native parasitoid-host association matter?

In Australia Fopius arisanus (Sonan) is an established, but exotic fruit fly egg-larval-pupal parasitoid which co-occurs with Diachasmimorpha kraussii (Fullaway), a native larval-pupal fruit fly parasitoid: both attack the native fruit fly Bactrocera tryoni (Froggatt). In interactions involving evolutionary novel host-parasitoid associations, F. arisanus consistently out-competes other parasitoid species, including D. kraussii. However, in fruit fly-parasitoid systems where there is co-evolutionary history between parasitoids and their hosts, competitive hierarchies can vary. In this study we investigated the outcome of competition between F. arisanus and D. kraussii within B. tryoni, to test whether the close evolutionary relationship between D. kraussii and B. tryoni might circumvent the competitive advantage of F. arisanus. Consistent with previous research, and despite the evolutionary relationship, dissection of multiparasitized B. tryoni larvae showed that D. kraussii was invariably suppressed by F. arisanus. A total of 47% and 74% of the eggs of D. kraussii in presence of F. arisanus were killed within a span of 24 h and 48–72 h, respectively. However, parasitoid emergence from fruit fly hosts exposed sequentially to F. arsianus and D. kraussii suggest that D. kraussii females are able to discriminate hosts already parasitized by F. arisanus. Results show that the co-evolutionary relationship between B. tryoni and D. kraussii does not help overcome the early-acting advantage of the egg parasitoid F. arisanus. Though F. arisanus may not have completely displaced D. kraussii in its native habitat, simultaneous inundative releases of these two parasitoid species (currently under consideration) might not help increase B. tryoni parasitism levels.

     

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.     

Annotated Chemical Patent Corpus: A Gold Standard for Text Mining

Exploring the chemical and biological space covered by patent applications is crucial in early-stage medicinal chemistry activities. Patent analysis can provide understanding of compound prior art, novelty checking, validation of biological assays, and identification of new starting points for chemical exploration. Extracting chemical and biological entities from patents through manual extraction by expert curators can take substantial amount of time and resources. Text mining methods can help to ease this process. To validate the performance of such methods, a manually annotated patent corpus is essential. In this study we have produced a large gold standard chemical patent corpus. We developed annotation guidelines and selected 200 full patents from the World Intellectual Property Organization, United States Patent and Trademark Office, and European Patent Office. The patents were pre-annotated automatically and made available to four independent annotator groups each consisting of two to ten annotators. The annotators marked chemicals in different subclasses, diseases, targets, and modes of action. Spelling mistakes and spurious line break due to optical character recognition errors were also annotated. A subset of 47 patents was annotated by at least three annotator groups, from which harmonized annotations and inter-annotator agreement scores were derived. One group annotated the full set. The patent corpus includes 400,125 annotations for the full set and 36,537 annotations for the harmonized set. All patents and annotated entities are publicly available at www.biosemantics.org.     

Micelle bound structure and DNA interaction of brevinin‐2‐related peptide,an antimicrobial peptide derived from frog skin

Brevinin‐2‐related peptide (BR‐II), a novel antimicrobial peptide isolated from the skin of frog, Rana septentrionalis, shows a broad spectrum of antimicrobial activity with low haemolytic activity. It has also been shown to have antiviral activity, specifically to protect cells from infection by HIV‐1. To understand the active conformation of the BR‐II peptide in membranes, we have investigated the interaction of BR‐II with the prokaryotic and eukaryotic membrane‐mimetic micelles such as sodium dodecylsulfate (SDS) and dodecylphosphocholine (DPC), respectively. The interactions were studied using fluorescence and circular dichroism (CD) spectroscopy. Fluorescence experiments revealed that the N‐terminus tryptophan residue of BR‐II interacts with the hydrophobic core of the membrane mimicking micelles. The CD results suggest that interactions with membrane‐mimetic micelles induce an α‐helix conformation in BR‐II. We have also determined the solution structures of BR‐II in DPC and SDS micelles using NMR spectroscopy. The structural comparison of BR‐II in the presence of SDS and DPC micelles showed significant conformational changes in the residues connecting the N‐terminus and C‐terminus helices. The ability of BR‐II to bind DNA was elucidated by agarose gel retardation and fluorescence experiments. The structural differences of BR‐II in zwitterionic versus anionic membrane mimics and the DNA binding ability of BR‐II collectively contribute to the general understanding of the pharmacological specificity of this peptide towards prokaryotic and eukaryotic membranes and provide insights into its overall antimicrobial mechanism. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.