Cryo-Electron Microscopy Reveals Cardiac Myosin Binding Protein-C M-Domain Interactions with the Thin Filament

Cardiac myosin binding protein C (cMyBP-C) modulates cardiac contraction via direct interactions with cardiac thick (myosin) and thin (actin) filaments (cTFs). While its C-terminal domains (e.g. C8-C10) anchor cMyBP-C to the backbone of the thick filament, its N-terminal domains (NTDs) (e.g. C0, C1, M, and C2) bind to both myosin and actin to accomplish its dual roles of inhibiting thick filaments and activating cTFs. While the positions of C0, C1 and C2 on cTF have been reported, the binding site of the M-domain on the surface of the cTF is unknown. Here, we used cryo-EM to reveal that the M-domain interacts with actin via helix 3 of its ordered tri-helix bundle region, while the unstructured part of the M-domain does not maintain extensive interactions with actin. We combined the recently obtained structure of the cTF with the positions of all the four NTDs on its surface to propose a complete model of the NTD binding to the cTF. The model predicts that the interactions of the NTDs with the cTF depend on the activation state of the cTF. At the peak of systole, when bound to the extensively activated cTF, NTDs would inhibit actomyosin interactions. In contrast, at falling Ca²⁺ levels, NTDs would not compete with the myosin heads for binding to the cTF, but would rather promote formation of active cross-bridges at the adjacent regulatory units located at the opposite cTF strand. Our structural data provides a testable model of the cTF regulation by the cMyBP-C.     

Penetration by Botryosphaeriaceae species in avocado,guava and persimmon fruit during postharvest

Botryosphaeriaceae species have a wide host range and a worldwide distribution. These fungal species can colonize several plant organs, such as the trunk, leaves and fruit. Some Botryosphaeriaceae species cause important diseases on persimmon, avocado and guava fruit. However, there is a lack of information regarding the mechanisms of penetration by Botryosphaeriaceae species on these tropical and subtropical fruits. This study aimed to better understand the mechanisms involved in fungal penetration, host specificity and aggressiveness of Botryosphaeria dothidea, Lasiodiplodia pseudotheobromae and Neofusicoccum parvum on avocado (Persea americana), guava (Psidium guajava) and persimmon (Diospyros kaki) fruit. Scanning electron microscopy (SEM) image analysis showed that in avocado fruit, the three studied Botryosphaeriaceae species penetrated through lenticels. In guava fruit, penetration through stomata was verified for Botryosphaeria dothidea and Neofusicoccum parvum. In persimmon fruit, an appressoria-like structure was observed for B. dothidea, which suggests direct penetration. Disease incidence in wounded fruit was 24% higher than in non-wounded fruit. L. pseudotheobromae and N. parvum showed differences in aggressiveness in guava fruit. The longest incubation period was observed for N. parvum inoculated on guava, with an average of 4.5 days, and the shortest incubation period was verified for B. dothidea inoculated on avocado, with an average of 2.8 days. The area under the disease progress curve (AUDPC) did not differ between Botryosphaeriaceae species on avocado, whereas on guava and persimmon fruit, the AUDPC was lower for B. dothidea. The information regarding penetration mechanisms and aggressiveness is important to improve postharvest disease control strategies.     

TFG clusters COPII‐coated transport carriers and promotes early secretory pathway organization

In mammalian cells, cargo‐laden secretory vesicles leave the endoplasmic reticulum (ER) en route to ER‐Golgi intermediate compartments (ERGIC) in a manner dependent on the COPII coat complex. We report here that COPII‐coated transport carriers traverse a submicron, TFG (Trk‐fused gene)‐enriched zone at the ER/ERGIC interface. The architecture of TFG complexes as determined by three‐dimensional electron microscopy reveals the formation of flexible, octameric cup‐like structures, which are able to self‐associate to generate larger polymers in vitro. In cells, loss of TFG function dramatically slows protein export from the ER and results in the accumulation of COPII‐coated carriers throughout the cytoplasm. Additionally, the tight association between ER and ERGIC membranes is lost in the absence of TFG. We propose that TFG functions at the ER/ERGIC interface to locally concentrate COPII‐coated transport carriers and link exit sites on the ER to ERGIC membranes. Our findings provide a new mechanism by which COPII‐coated carriers are retained near their site of formation to facilitate rapid fusion with neighboring ERGIC membranes upon uncoating, thereby promoting interorganellar cargo transport.     

Why is intracellular ice lethal? A microscopical study showing evidence of programmed cell death in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum

Background and Aims Conservation of the genetic diversity afforded by recalcitrant seeds is achieved by cryopreservation, in which excised embryonic axes (or, where possible, embryos) are treated and stored at temperatures lower than −180 °C using liquid nitrogen. It has previously been shown that intracellular ice forms in rapidly cooled embryonic axes of Acer saccharinum (silver maple) but this is not necessarily lethal when ice crystals are small. This study seeks to understand the nature and extent of damage from intracellular ice, and the course of recovery and regrowth in surviving tissues.Methods Embryonic axes of A. saccharinum, not subjected to dehydration or cryoprotection treatments (water content was 1·9 g H₂O g⁻¹ dry mass), were cooled to liquid nitrogen temperatures using two methods: plunging into nitrogen slush to achieve a cooling rate of 97 °C s⁻¹ or programmed cooling at 3·3 °C s⁻¹. Samples were thawed rapidly (177 °C s⁻¹) and cell structure was examined microscopically immediately, and at intervals up to 72 h in vitro. Survival was assessed after 4 weeks in vitro. Axes were processed conventionally for optical microscopy and ultrastructural examination.Key Results Immediately following thaw after cryogenic exposure, cells from axes did not show signs of damage at an ultrastructural level. Signs that cells had been damaged were apparent after several hours of in vitro culture and appeared as autophagic decomposition. In surviving tissues, dead cells were sloughed off and pockets of living cells were the origin of regrowth. In roots, regrowth occurred from the ground meristem and procambium, not the distal meristem, which became lethally damaged. Regrowth of shoots occurred from isolated pockets of surviving cells of peripheral and pith meristems. The size of these pockets may determine the possibility for, the extent of and the vigour of regrowth.Conclusions Autophagic degradation and ultimately autolysis of cells following cryo-exposure and formation of small (0·2–0·4 µm) intracellular ice crystals challenges current ideas that ice causes immediate physical damage to cells. Instead, freezing stress may induce a signal for programmed cell death (PCD). Cells that form more ice crystals during cooling have faster PCD responses.     

Role of cyclooxygenase-2 in Trypanosoma cruzi survival in the early stages of parasite host-cell interaction

Chagas disease, caused by the intracellular protozoan Trypanosoma cruzi, is a serious health problem in Latin America. During this parasitic infection, the heart is one of the major organs affected. The pathogenesis of tissue remodelling, particularly regarding cardiomyocyte behaviour after parasite infection and the molecular mechanisms that occur immediately following parasite entry into host cells are not yet completely understood. When cells are infected with T. cruzi, they develop an inflammatory response, in which cyclooxygenase-2 (COX-2) catalyses rate-limiting steps in the arachidonic acid pathway. However, how the parasite interaction modulates COX-2 activity is poorly understood. In this study, the H9c2 cell line was used as our model and we investigated cellular and biochemical aspects during the initial 48 h of parasitic infection. Oscillatory activity of COX-2 was observed, which correlated with the control of the pro-inflammatory environment in infected cells. Interestingly, subcellular trafficking was also verified, correlated with the control of Cox-2 mRNA or the activated COX-2 protein in cells, which is directly connected with the assemble of stress granules structures. Our collective findings suggest that in the very early stage of the T. cruzi-host cell interaction, the parasite is able to modulate the cellular metabolism in order to survives.     

伦敦白胶和茶：一种既快又安全的用于透射电子显微镜的细菌样品制备方法

【目的】检测乌龙茶提取物是否可作为电子染色剂取代醋酸双氧铀用于细菌细胞染色,使其能在透射电子显微镜下进行观察。【方法】利用伦敦白胶对细菌样品(大肠杆菌和金黄色葡萄球菌)进行胶块的制备,再在复染铅与不复染铅这两种情况下对超薄切片样品进行3种不同染色剂的电子染色,之后在透射电子显微镜下观察比较其不同之处。这3种不同的染色剂分别是醋酸双氧铀、0.05%乌龙茶提取物以及0.1%乌龙茶提取物。首先将带有超薄切片样品的铜网悬浮于不同的待比较染液中10?15 min,若需进一步用柠檬酸铅复染,则将经3次蒸馏水冲洗过后的铜网再次悬浮于柠檬酸铅染液中8?10 min。【结果】复染铅的情况下,在透射电子显微镜下无论是大肠杆菌还是金黄色葡萄球菌,利用3种电子染色剂进行染色的结果均非常相似。【结论】实验结果表明,在观察细菌结构中,乌龙茶提取物可以替代醋酸双氧铀进行透射电子显微镜样品的电子染色。     

Molecular architecture of the ribosome‐bound Hepatitis C Virus internal ribosomal entry site RNA

Internal ribosomal entry sites (IRESs) are structured cis‐acting RNAs that drive an alternative, cap‐independent translation initiation pathway. They are used by many viruses to hijack the translational machinery of the host cell. IRESs facilitate translation initiation by recruiting and actively manipulating the eukaryotic ribosome using only a subset of canonical initiation factor and IRES transacting factors. Here we present cryo‐EM reconstructions of the ribosome 80S‐ and 40S‐bound Hepatitis C Virus (HCV) IRES. The presence of four subpopulations for the 80S•HCV IRES complex reveals dynamic conformational modes of the complex. At a global resolution of 3.9 Å for the most stable complex, a derived atomic model reveals a complex fold of the IRES RNA and molecular details of its interaction with the ribosome. The comparison of obtained structures explains how a modular architecture facilitates mRNA loading and tRNA binding to the P‐site. This information provides the structural foundation for understanding the mechanism of HCV IRES RNA‐driven translation initiation.     

Microscopic study on resorption of β-tricalcium phosphate materials by osteoclasts

Sintered compounds prepared with β-tricalcium phosphate (β-TCP) are commonly used as biocompatible materials for bone regenerative medicine. Although implanted β-TCP is gradually replaced with new bone after resorption by osteoclasts, exactly how osteoclasts resorb β-TCP is not well understood. To elucidate this mechanism, we analyzed the structure of β-TCP discs on which mouse mature osteoclasts were cultured using scanning electron microscopy. We found that β-TCP was resorbed by mature osteoclasts on one side of each disc, as evidenced by the formation of multiple spine-like crystals at the exposed areas. Because osteoclasts secrete acid to resorb bone minerals, we mimicked this acidification by dipping β-TCP slices into HCl solution (pH 2.0). However, no spine-like crystals appeared even though the size of each β-TCP particle was reduced. On dentin slices, osteoclasts formed clear actin rings, which are cytoskeletal structures characteristic of bone-resorbing osteoclasts. No clear actin rings were observed in osteoclasts cultured on β-TCP slices, although small actin dots were observed. Analysis by transmission electron microscopy showed that osteoclasts attached to β-TCP particles. These results suggest that osteoclasts resorb β-TCP particles independently of clear actin ring formation.     

A surface acoustic wave sensor functionalized with a polypyrrole molecularly imprinted polymer for selective dopamine detection

A surface acoustic wave sensor operating at 104 MHz and functionalized with a polypyrrole molecularly imprinted polymer has been designed for selective detection of dopamine (DA). Optimization of pyrrole/DA ratio, polymerization and immersion times permitted to obtain a highly selective sensor, which has a sensitivity of 0.55°/mM (≈550 Hz/mM) and a detection limit of ≈ 10 nM. Morphology and related roughness parameters of molecularly imprinted polymer surfaces, before and after extraction of DA, as well as that of the non imprinted polymer were characterized by atomic force microscopy. The developed chemosensor selectively recognized dopamine over the structurally similar compound 4‐hydroxyphenethylamine (referred as tyramine), or ascorbic acid,which co‐exists with DA in body fluids at a much higher concentration. Selectivity tests were also carried out with dihydroxybenzene, for which an unexpected phase variation of order of 75% of the DA one was observed. Quantum chemical calculations, based on the density functional theory, were carried out to determine the nature of interactions between each analyte and the PPy matrix and the DA imprinted PPy polypyrrole sensing layer in order to account for the important phase variation observed during dihydroxybenzene injection. Copyright © 2015 John Wiley & Sons, Ltd.     

中国淫羊藿属药用植物花粉特征及其分类学意义

淫羊藿属(Epimedium L.)植物多为常用药材,分类和药材鉴定较为困难。该文采用扫描电镜对中国产淫羊藿属31种药用植物花粉的形态、大小和表面纹饰等进行了比较观察。结果显示:(1)淫羊藿属植物花粉形态多为长球形,少数为球形,大小为(15.5~25.0)×(27.3~48.1)μm,极面观多为三裂圆形,具三孔沟。(2)花粉粒外壁表面纹饰有网状、条网状、条纹状等类型。(3)花粉粒外壁纹饰如网眼的形状、大小和网脊表面特征等在各物种间有一定差异。研究结果为淫羊藿属药材鉴定与植物分类提供了重要的参考。