Avanti lipid tools: Connecting lipids,technology, and cell biology

Lipid research is challenging owing to the complexity and diversity of the lipidome. Here we review a set of experimental tools developed for the seasoned lipid researcher, as well as, those who are new to the field of lipid research. Novel tools for probing protein–lipid interactions, applications for lipid binding antibodies, enhanced systems for the cellular delivery of lipids, improved visualization of lipid membranes using gold-labeled lipids, and advances in mass spectrometric analysis techniques will be discussed. Because lipid mediators are known to participate in a host of signal transduction and trafficking pathways within the cell, a comprehensive lipid toolbox that aids the science of lipidomics research is essential to better understand the molecular mechanisms of interactions between cellular components. This article is part of a Special Issue entitled Tools to study lipid functions.     

Light-harvesting chlorophyll protein (LHCII) drives electron transfer in semiconductor nanocrystals

Type-II quantum dots (QDs) are capable of light-driven charge separation between their core and the shell structures; however, their light absorption is limited in the longer-wavelength range. Biological light-harvesting complex II (LHCII) efficiently absorbs in the blue and red spectral domains. Therefore, hybrid complexes of these two structures may be promising candidates for photovoltaic applications. Previous measurements had shown that LHCII bound to QD can transfer its excitation energy to the latter, as indicated by the fluorescence emissions of LHCII and QD being quenched and sensitized, respectively. In the presence of methyl viologen (MV), both fluorescence emissions are quenched, indicating an additional electron transfer process from QDs to MV. Transient absorption spectroscopy confirmed this notion and showed that electron transfer from QDs to MV is much faster than fluorescence energy transfer between LHCII and QD. The action spectrum of MV reduction by LHCII-QD complexes reflected the LHCII absorption spectrum, showing that light absorbed by LHCII and transferred to QDs increased the efficiency of MV reduction by QDs. Under continuous illumination, at least 28 turnovers were observed for the MV reduction. Presumably, the holes in QD cores were filled by a reducing agent in the reaction solution or by the dihydrolipoic-acid coating of the QDs. The LHCII-QD construct can be viewed as a simple model of a photosystem with the QD component acting as reaction center.     

Characterization of Protease-Activated Receptor (PAR) ligands: Parmodulins are reversible allosteric inhibitors of PAR1-driven calcium mobilization in endothelial cells

Several classes of ligands for Protease-Activated Receptors (PARs) have shown impressive anti-inflammatory and cytoprotective activities, including PAR2 antagonists and the PAR1-targeting parmodulins. In order to support medicinal chemistry studies with hundreds of compounds and to perform detailed mode-of-action studies, it became important to develop a reliable PAR assay that is operational with endothelial cells, which mediate the cytoprotective effects of interest. We report a detailed protocol for an intracellular calcium mobilization assay with adherent endothelial cells in multiwell plates that was used to study a number of known and new PAR1 and PAR2 ligands, including an alkynylated version of the PAR1 antagonist RWJ-58259 that is suitable for the preparation of tagged or conjugate compounds. Using the cell line EA.hy926, it was necessary to perform media exchanges with automated liquid handling equipment in order to obtain optimal and reproducible antagonist concentration-response curves. The assay is also suitable for study of PAR2 ligands; a peptide antagonist reported by Fairlie was synthesized and found to inhibit PAR2 in a manner consistent with reports using epithelial cells. The assay was used to confirm that vorapaxar acts as an irreversible antagonist of PAR1 in endothelium, and parmodulin 2 (ML161) and the related parmodulin RR-90 were found to inhibit PAR1 reversibly, in a manner consistent with negative allosteric modulation.     

Phylogenetic analyses and evolutionary timescale of Coleoptera based on mitochondrial sequence

Coleopteran phylogeny was analysed using mitochondrial genome (mitogenome) sequence. The optimal tree topology was given by the dataset consisted of all coding genes except for the exclusion of the 3rd codon sites (mtDNA12) using Bayesian Inference Method. This topology supports the monophyly of four suborders, and the sister group relationship between Adephaga and Myxophaga and between Polyphaga and Archostemata. In Polyphaga, Cucujiformia and Elateroidea formed independent node respectively, the remaining species grouped together except for Cyphon sp, among which, only Cucujiformia and Scarabaeiformia were supported as monophyletic group, respectively. Within Cucujiformia, the monophyly of Chrysomeloidea, Curculionoidea and Tenebrionoidea were supported respectively, among which Tenebrionoidea occupied the basal position of Cucujiformia. Cleroidea grouped together with Bothrideridae and Coccinellidae, and formed an independent node, which lead to the paraphyly of Cucujoidea. The monophyly of Elateriformia was not supported because of the division of Scirtoidea and Buprestoidea. Furthermore, using a Bayesian relaxed clock calibrated with fossil data, we estimated that most superfamilies within Polyphaga originated in the Jurassic period.     

Contribution of arginine (HC3) 141 alpha to the Bohr effect of the fourth binding step in the reaction of ligand with human hemoglobin

A few years ago we reported that histidine (HC3) 146 beta plays a major role in the pH-dependent properties of the R-state of human hemoglobin, accounting for close to 50% of the R-state Bohr effect. We have extended these studies by examining the role of arginine 141 alpha, another group known to affect the overall Bohr effect. We have compared the pH dependencies of the rate constants for the dissociation and combination of the fourth carbon monoxide molecule, l4 and l'4, respectively, for native hemoglobin A (HbA) and a control reconstituted HbA, and des-(Arg 141 alpha) HbA, the hemoglobin molecule resulting from the enzymatic removal of the C-terminal arginine of the alpha-chain of human Hb. From these kinetic constants the pH dependence of L4, the affinity constant for the fourth carbon monoxide molecule, has been estimated. We find that the removal of arginine 141 alpha reduces the pH dependence of log L4 by about 80% between pH 6 and 8, where the alkaline Bohr effect normally occurs. The sum of the effects of the removal of His 146 beta and of Arg 141 alpha is greater than 100%. This suggests that at least one of these modifications alters the contributions of other residues of this Bohr effect.     

Synthesis and structure–activity relationships of a novel and selective bone morphogenetic protein receptor (BMP) inhibitor derived from the pyrazolo[1.5-a]pyrimidine scaffold of Dorsomorphin: The discovery of ML347 as an ALK2 versus ALK3 selective MLPCN probe

A structure–activity relationship of the 3- and 6-positions of the pyrazolo[1,5-a]pyrimidine scaffold of the known BMP inhibitors dorsomorphin, 1, LDN-193189, 2, and DMH1, 3, led to the identification of a potent and selective compound for ALK2 versus ALK3. The potency contributions of several 3-position substituents were evaluated with subtle structural changes leading to significant changes in potency. From these studies, a novel 5-quinoline molecule was identified and designated an MLPCN probe molecule, ML347, which shows >300-fold selectivity for ALK2 and presents the community with a selective molecular probe for further biological evaluation.     

Computational analysis and functional expression of ancestral copepod luciferase

We recently reported the cDNA sequences of 11 copepod luciferases from the superfamily Augaptiloidea in the order Calanoida. They were classified into two groups, Metridinidae and Heterorhabdidae/Lucicutiidae families, by phylogenetic analyses. To elucidate the evolutionary processes, we have now further isolated 12 copepod luciferases from Augaptiloidea species (Metridia asymmetrica, Metridia curticauda, Pleuromamma scutullata, Pleuromamma xiphias, Lucicutia ovaliformis and Heterorhabdus tanneri). Codon-based synonymous/nonsynonymous tests of positive selection for 25 identified copepod luciferases suggested that positive Darwinian selection operated in the evolution of Heterorhabdidae luciferases, whereas two types of Metridinidae luciferases had diversified via neutral mechanism. By in silico analysis of the decoded amino acid sequences of 25 copepod luciferases, we inferred two protein sequences as ancestral copepod luciferases. They were expressed in HEK293 cells where they exhibited notable luciferase activity both in intracellular lysates and cultured media, indicating that the luciferase activity was established before evolutionary diversification of these copepod species.     

妊娠期糖尿病肾功能损害患者血清miR-135b、miR-141表达水平的变化及意义

摘要 目的：探讨血清miR-135b、miR-141在妊娠期糖尿病（GDM）肾功能损害患者中的表达及作用。方法：选取2019年3月至2020年12月在我院就诊的GDM患者200例作为GDM组,根据有无肾功能损伤分为GDM组（n=83）和GDM肾功能损伤组（n=117）,再将肾病患者根据尿白蛋白/肌酐比值（UACR）分为轻度65例、中度37例和重度15例。另选择同期在我院产检的正常妊娠孕妇100例为对照组。测定孕妇血清中miR-135b、miR-141水平及肾功能指标,Pearson法分析GDM肾病患者血清miR-135b、miR-141与肾功能指标的相关性。采用非条件Logistic 多元回归分析GDM肾功能损伤的危险因素。结果：GDM肾功能损伤组孕妇血清miR-135b、miR-141表达水平高于GDM组和对照组,GDM组明显高于对照组（P<0.05）。GDM肾功能损伤组肌酐（Scr）、β₂-微球蛋白（β₂-MG）、UCAR表达水平高于GDM组和对照组,肾小球滤过率（eGFR）低于GDM组和对照组,差异有统计学意义（P<0.05）。血清miR-135b、miR-141表达水平与Scr、β₂-MG、UCAR呈正相关（P<0.05）,与eGFR呈负相关（P<0.05）。肾功能重度损伤孕妇血清miR-135b、miR-141表达水平明显高于肾功能中度损伤和轻度损伤孕妇,且肾功能中度损伤明显高于轻度损伤（P<0.05）。非条件 Logistic 多元回归分析结果显示,miR-135b、miR-141表达水平是孕妇GDM发生的危险因素（P<0.05）。结论：GDM肾功能损伤患者血清miR-135b、miR-141水平异常升高,且随着肾功能损害的加重其水平逐渐增加,可为GDM肾功能损伤的预测和治疗提供一定参考。