Implications of surface charge and curvature for the binding orientation of Thermomyces lanuginosus lipase on negatively charged or zwitterionic phospholipid vesicles as studied by ESR spectroscopy

The triglyceride lipase (EC 3.1.1.3) Thermomyces lanuginosus lipase (TLL) binds with high affinity to unilamellar phospholipid vesicles that serve as a diluent interface for both lipase and substrate, but it displays interfacial activation on only small and negatively charged such vesicles [Cajal, Y., et al. (2000) Biochemistry 39, 413-423]. The productive-mode binding orientation of TLL at the lipid-water interface of small unilamellar vesicles (SUV) consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) was previously determined using electron spin resonance (ESR) spectroscopy in combination with site-directed spin-labeling [Hedin, E. M. K., et al. (2002) Biochemistry 41, 14185-14196]. In our investigation, we have studied the interfacial orientation of TLL when bound to large unilamellar vesicles (LUV) consisting of POPG, and bound to SUV consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC). Eleven single-cysteine TLL mutants were spin-labeled as previously described, and studied upon membrane binding using the water soluble spin-relaxation agent chromium(III) oxalate (Crox). Furthermore, dansyl-labeled vesicles revealed the intermolecular fluorescence quenching efficiency between each spin-label positioned on TLL, and the lipid membrane. ESR exposure and fluorescence quenching data show that TLL associates closer to the negatively charged PG surface than the zwitterionic PC surface, and binds to both POPG LUV and POPC SUV predominantly through the concave backside of TLL opposite the active site, as revealed by the contact residues K74C-SL, R209C-SL, and T192C-SL. This orientation is significantly different compared to that on the POPG SUV, and might explain the differences in activation of the lipase. Evidently, both the charge and accessibility (curvature) of the vesicle surface determine the TLL orientation at the phospholipid interface.     

The ligamentum infundibulo-cornuale in the pig: morphological and physiological studies of the smooth muscle component

The ligamentum infundibulo-cornuale (LIC) in the pig runs along the anterolateral side of the tubal isthmus, connecting the uterotubal junction and the edge of the infundibulum and has a comparatively well-developed muscular component running under the mesothelium. The well-vascularized smooth muscle cells held close cell-to-cell contacts and received innervation by adrenergic and cholinergic-like nerve terminals. Isolated LIC preparations, collected during oestrus showed a rhythmic spontaneous motility in vitro, the frequency and the relative amplitude of the contractions being highest during the preovulatory period. In vitro, noradrenaline and adrenaline elicited contractile (alpha) and relaxatory (beta) responses, while isoprenaline induced only beta-responses, as demonstrated by pretreatment with selective blockers. Oxytocin, PGF2 alpha and PGE2 always increased the muscular activity of the LIC. Indomethacin inhibited, in a concentration-dependent and reversible manner, the spontaneous motility of the porcine LIC, which could be fully restored by PGF2 alpha, indicating an endogenous local synthesis of prostaglandins in the tissue. The present results suggest that, in the pig, the LIC consists of a well-arranged, richly innervated bulk of smooth muscle which shows rhythmic spontaneous activity at the time of ovulation that could assist ova pick-up.     

Enzyme-Mediated Protecting Group Chemistry on the Hydroxyl Groups of Nucleosides

Abstract

Enzymes are well known catalysts for carrying out regio- and stereoselective reactions in organic synthesis. Results from enzyme-mediated protections and deprotections of the hydroxyl groups of pentofuranose nucleosides are discussed in this review.     

Inferring Adaptive Introgression Using Hidden Markov Models

Adaptive introgression—the flow of adaptive genetic variation between species or populations—has attracted significant interest in recent years and it has been implicated in a number of cases of adaptation, from pesticide resistance and immunity, to local adaptation. Despite this, methods for identification of adaptive introgression from population genomic data are lacking. Here, we present Ancestry_HMM-S, a hidden Markov model-based method for identifying genes undergoing adaptive introgression and quantifying the strength of selection acting on them. Through extensive validation, we show that this method performs well on moderately sized data sets for realistic population and selection parameters. We apply Ancestry_HMM-S to a data set of an admixed Drosophila melanogaster population from South Africa and we identify 17 loci which show signatures of adaptive introgression, four of which have previously been shown to confer resistance to insecticides. Ancestry_HMM-S provides a powerful method for inferring adaptive introgression in data sets that are typically collected when studying admixed populations. This method will enable powerful insights into the genetic consequences of admixture across diverse populations. Ancestry_HMM-S can be downloaded from https://github.com/jesvedberg/Ancestry_HMM-S/.     

Genet age in marginal populations of two clonal Carex species in the Siberian Arctic

During a Swedish-Russian expedition to northern Siberia 1994, we sampled two marginal populations of two Carex species at two high arctic sites ( C, stans Drej. on Faddeyevsky Island and C. ensifolia V. Krecz ssp. arctisibirica Jurtz. at north-eastern Taymyr Peninsula), both north of previously documented localities in that areas for the two species. These populations were composed of a few distinct patches of ramet colonies, some of them shaped like fairy rings with dead centres. We measured the size of all colonies and collected samples for detailed morphometric analyses of rhizome growth. By using RAPD (random amplified polymorphic DNA) analysis we established that the largest colony at each site consisted of a single genet, based on 41 polymorphic bands amplified with three primers. Pooled samples from each of two additional colonies of C. stans on Faddeyevsky Island were analysed and showed that clones of the same species at the same site were relatively dissimilar (Dice's similarity index 0.26 0,43), We then assumed that each ramet colony represented a single genet. Based on the morphometric data, we developed a deterministic growth model that simulates the clonal growth of these species and enabled estimates of the time since establishment of the genets. The estimated age of the five C. Stans clones varied from 17 to 154 yr and the age of the two C. ensifolia ssp, arctisibirica clones was well over 3000 yr.     

Synthesis of Novel 3′-C-Hydroxymethyl Nucleosides by a Convergent Strategy

Abstract

Synthesis of 1-(2-deoxy-3-C-hydroxymethyl-α/β-D-elythro-pentofuranosyl)thymine and -cytosine by a convergent strategy using the precursors 7 and 8 have been accomplished.     

Differential ultracentrifugation enables deep plasma proteomics through enrichment of extracellular vesicles

Human plasma is a rich source of biomedical information and biomarkers. However, the enormous dynamic range of plasma proteins limits its accessibility to mass spectrometric (MS) analysis. Here, we show that enrichment of extracellular vesicles (EVs) by ultracentrifugation increases plasma proteome depth by an order of magnitude. With this approach, more than two thousand proteins are routinely and reproducibly quantified by label-free quantification and data independent acquisition (DIA) in single-shot liquid chromatography tandem mass spectrometry runs of less than one hour. We present an optimized plasma proteomics workflow that enables high-throughput with very short chromatographic gradients analyzing hundred samples per day with deep proteome coverage, especially when including a study-specific spectral library generated by repeated injection and gas-phase fractionation of pooled samples. Finally, we test the workflow on clinical biobank samples from malignant melanoma patients in immunotherapy to demonstrate the improved proteome coverage supporting the potential for future biomarker discovery.