Substrate Affinity and Specificity of the ScSth1p Bromodomain Are Fine-Tuned for Versatile Histone Recognition

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Rapid development of a restored oyster reef facilitates habitat provision for estuarine fauna

Oyster reef restoration has become a principal strategy for ameliorating the loss of natural Crassostrea virginica populations and increasing habitat provision. In 2014, a large‐scale, high‐relief, 23‐ha subtidal C. virginica reef was restored at the historically productive Half Moon Reef in Matagorda Bay, TX, using concrete and limestone substrates. Encrusting and motile fauna were sampled seasonally until 17 months postrestoration at the restored reef and at adjacent unrestored sites. Restored oysters developed rapidly and were most abundant 3 months postrestoration, with subsequent declines possibly due to interacting effects of larval settlement success on new substrate versus post‐settlement mortality due to competitors and predators. Oyster densities were 2× higher than in a restored oyster population in Chesapeake Bay that was reported to be the largest reestablished metapopulation of native oysters in the world. Resident fauna on the restored reef were 62% more diverse, had 433% greater biomass, and comprised a distinct faunal community compared to unrestored sites. The presence of three‐dimensional habitat was the most important factor determining resident faunal community composition, indicating that substrate limitation is a major hindrance for oyster reef community success in Texas and other parts of the Gulf of Mexico. There were only minor differences in density, biomass, and diversity of associated fauna located adjacent (13 m) versus distant (150 m) to the restored reef. The two substrate types compared had little influence on oyster recruitment or faunal habitat provision. Results support the use of reef restoration as a productive means to rebuild habitat and facilitate faunal enhancement.     

Resistance outside the substrate envelope: hepatitis C NS3/4A protease inhibitors

Direct acting antivirals have dramatically increased the efficacy and tolerability of hepatitis C treatment, but drug resistance has emerged with some of these inhibitors, including nonstructural protein 3/4?A protease inhibitors (PIs). Although many co-crystal structures of PIs with the NS3/4A protease have been reported, a systematic review of these crystal structures in the context of the rapidly emerging drug resistance especially for early PIs has not been performed. To provide a framework for designing better inhibitors with higher barriers to resistance, we performed a quantitative structural analysis using co-crystal structures and models of HCV NS3/4A protease in complex with natural substrates and inhibitors. By comparing substrate structural motifs and active site interactions with inhibitor recognition, we observed that the selection of drug resistance mutations correlates with how inhibitors deviate from viral substrates in molecular recognition. Based on this observation, we conclude that guiding the design process with native substrate recognition features is likely to lead to more robust small molecule inhibitors with decreased susceptibility to resistance.     

Effects of substrate patterning on cellular spheroid growth and dynamics measured by gradient light interference microscopy (GLIM)

The development of three‐dimensional (3D) cellular architectures during development and pathological processes involves intricate migratory patterns that are modulated by genetics and the surrounding microenvironment. The substrate composition of cell cultures has been demonstrated to influence growth, proliferation and migration in 2D. Here, we study the growth and dynamics of mouse embryonic fibroblast cultures patterned in a tissue sheet which then exhibits 3D growth. Using gradient light interference microscopy (GLIM), a label‐free quantitative phase imaging approach, we explored the influence of geometry on cell growth patterns and rotational dynamics. We apply, for the first time to our knowledge, dispersion‐relation phase spectroscopy (DPS) in polar coordinates to generate the radial and rotational cell mass‐transport. Our data show that cells cultured on engineered substrates undergo rotational transport in a radially independent manner and exhibit faster vertical growth than the control, unpatterned cells. The use of GLIM and polar DPS provides a novel quantitative approach to studying the effects of spatially patterned substrates on cell motility and growth.     

Substrate availability regulates the suppressive effects of Canada goldenrod invasion on soil respiration

基质有效性调节加拿大一枝黄花入侵对土壤呼吸的抑制作用 外来植物入侵不仅会降低河边近岸湿地生态系统植被多样性,而且会改变湿地生态系统的地下碳过程。外来入侵植物加拿大一枝黄花(Solidago canadensis L.)已广泛入侵我国东南部地区,但加拿大一枝黄花入侵对入侵地生态系统地下土壤碳循环过程的影响却知之甚少。本研究通过野外原位观测实验和温室模拟入侵实验,探究外来植物加拿大一枝黄花入侵对入侵地土壤呼吸的影响规律及其驱动因素。野 外原位观测实验开展于2018年7月21日至12月15日,期间每周测定样地土壤呼吸。温室模拟入侵实验开展于2019年7月15日至12月15日,期间每月1日与15日上午测定土壤呼吸、自养呼吸和异养呼吸。土壤呼吸、自养呼吸和异养呼吸通过静态箱结合深埋根系隔离法测定。野外原位观测实验和温室模拟入侵实验结果均显示,加拿大一枝黄花的入侵降低了土壤二氧化碳的排放通量。加拿大一枝黄花入侵对土壤呼吸的抑制作用可能归因于其入侵引起的土壤可利用底物质量与数量的变化,表明外来入侵植物加拿大一枝黄花可通过改变植物释放基质以及与本地植物和/或土壤微生物争夺土壤有效基质而影响土壤碳循环。这些研究结果对于评估外来入侵植物对入侵地地下碳动态的影响以及对全球变暖的贡献具有重要意义。     

Stabilization and scale-up of photosynthesis-driven ω-hydroxylation of nonanoic acid methyl ester by two-liquid phase whole-cell biocatalysis

Photoautotrophic organisms are promising hosts for biocatalytic oxyfunctionalizations because they supply reduction equivalents as well as O₂ via photosynthetic water oxidation. Thus far, research on photosynthesis-driven bioprocesses mainly focuses on strain development and the proof of principle in small-scale biocatalytic reaction setups. This study investigates the long-term applicability of the previously developed cyanobacterial strain Synechocystis sp. PCC 6803_BGT harboring the alkane monooxygenase system AlkBGT catalyzing terminal alkyl group oxyfunctionalization. For the regiospecific ω-hydroxylation of nonanoic acid methyl ester (NAME), this biocatalyst showed light intensity-independent hydroxylation activity and substantial hydrolysis of NAME to nonanoic acid. Substrate mass transfer limitation, substrate hydrolysis, as well as reactant toxicity were overcome via in situ substrate supply by means of a two-liquid phase system. The application of diisononyl phthalate as organic carrier solvent enabled 1.7-fold increased initial specific activities (5.6 ± 0.1 U/g_CDW) and 7.6-fold increased specific yields on biomass (3.8 ± 0.1 mmol_H-NAME/g_CDW) as compared with single aqueous phase biotransformations. Finally, the whole-cell biotransformation system was successfully scaled from glass tubes to a stirred-tank photobioreactor. This is the first study reporting the application of the two-liquid phase concept for efficient phototrophic whole-cell biocatalysis.     

Sucrose nonfermenting AMPK-related kinase (SNARK) regulates exercise-stimulated and ischemia-stimulated glucose transport in the heart

The signaling mechanisms mediating myocardial glucose transport are not fully understood. Sucrose nonfermenting AMP-activated protein kinase (AMPK)-related kinase (SNARK) is an AMPK-related protein kinase that is expressed in the heart and has been implicated in contraction-stimulated glucose transport in mouse skeletal muscle. We first determined if SNARK is phosphorylated on Thr²⁰⁸, a site critical for SNARK activity. Mice were treated with exercise, ischemia, submaximal insulin, or maximal insulin. Treadmill exercise slightly, but significantly increased SNARK Thr²⁰⁸ phosphorylation. Ischemia also increased SNARK Thr²⁰⁸ phosphorylation, but there was no effect of submaximal or maximal insulin. HL1 cardiomyocytes were used to overexpress wild-type (WT) SNARK and to knockdown endogenous SNARK. Overexpression of WT SNARK had no effect on ischemia-stimulated glucose transport; however, SNARK knockdown significantly decreased ischemia-stimulated glucose transport. SNARK overexpression or knockdown did not alter insulin-stimulated glucose transport or glycogen concentrations. To study SNARK function in vivo, SNARK heterozygous knockout mice (SNARK^+/−) and WT littermates performed treadmill exercise. Exercise-stimulated glucose transport was decreased by ~50% in hearts from SNARK^+/− mice. In summary, exercise and ischemia increase SNARK Thr²⁰⁸ phosphorylation in the heart and SNARK regulates exercise-stimulated and ischemia-stimulated glucose transport. SNARK is a novel mediator of insulin-independent glucose transport in the heart.     

Structural analysis of P450 AmphL from Streptomyces nodosus provides insights into substrate selectivity of polyene macrolide antibiotic biosynthetic P450s

AmphL is a cytochrome P450 enzyme that catalyzes the C8 oxidation of 8-deoxyamphotericin B to the polyene macrolide antibiotic, amphotericin B. To understand this substrate selectivity, we solved the crystal structure of AmphL to a resolution of 2.0 Å in complex with amphotericin B and performed molecular dynamics (MD) simulations. A detailed comparison with the closely related P450, PimD, which catalyzes the epoxidation of 4,5-desepoxypimaricin to the macrolide antibiotic, pimaricin, reveals key catalytic structural features responsible for stereo- and regio-selective oxidation. Both P450s have a similar access channel that runs parallel to the active site I helix over the surface of the heme. Molecular dynamics simulations of substrate binding reveal PimD can “pull” substrates further into the P450 access channel owing to additional electrostatic interactions between the protein and the carboxyl group attached to the hemiketal ring of 4,5-desepoxypimaricin. This substrate interaction is absent in AmphL although the additional substrate -OH groups in 8-deoxyamphotericin B help to correctly position the substrate for C8 oxidation. Simulations of the oxy-complex indicates that these -OH groups may also participate in a proton relay network required for O₂ activation as has been suggested for two other macrolide P450s, PimD and P450eryF. These findings provide experimentally testable models that can potentially contribute to a new generation of novel macrolide antibiotics with enhanced antifungal and/or antiprotozoal efficacy.