Educating EVs to Improve Bone Regeneration: Getting Closer to the Clinic

The incidence of bone-related disorders is continuously growing as the aging of the population in developing countries continues to increase. Although therapeutic interventions for bone regeneration exist, their effectiveness is questioned, especially under certain circumstances, such as critical size defects. This gap of curative options has led to the search for new and more effective therapeutic approaches for bone regeneration; among them, the possibility of using extracellular vesicles (EVs) is gaining ground. EVs are secreted, biocompatible, nano-sized vesicles that play a pivotal role as messengers between donor and target cells, mediated by their specific cargo. Evidence shows that bone-relevant cells secrete osteoanabolic EVs, whose functionality can be further improved by several strategies. This, together with the low immunogenicity of EVs and their storage advantages, make them attractive candidates for clinical prospects in bone regeneration. However, before EVs reach clinical translation, a number of concerns should be addressed. Unraveling the EVs’ mode of action in bone regeneration is one of them; the molecular mediators driving their osteoanabolic effects in acceptor cells are now beginning to be uncovered. Increasing the functional and bone targeting abilities of EVs are also matters of intense research. Here, we summarize the cell sources offering osteoanabolic EVs, and the current knowledge about the molecular cargos that mediate bone regeneration. Moreover, we discuss strategies under development to improve the osteoanabolic and bone-targeting potential of EVs.     

Topology of eukaryotic multispanning transmembrane proteins: use of LacZ fusions for the localization of cytoplasmic domains in COS.M6 cells

In Escherichia coli, the topology of inner membrane proteins can be studied conveniently with the alkaline phosphatase/beta-galactosidase (PhoA/LacZ) gene fusion system. PhoA is enzymatically active only when fused to external domains, LacZ when fused to cytoplasmic domains. In eukaryotic cells, only time consuming methods exist to study the topology of membrane proteins. We have extended in the first systematic study the PhoA/LacZ gene fusion system originally developed for E.coli for use in eukaryotic COS.M6 cells. We have fused PhoA and LacZ to the putative external and cytoplasmic loops of rat aquaporin 2 (AQP2), for which a model with six transmembrane domains was proposed previously. The fusion proteins were expressed in E.coli and COS.M6 cells and immunoblot analyses and enzyme activity assays were performed to localize the protein domains in both cell types. The data obtained in E.coli correlated mostly with the predictions of the six transmembrane domain model. However, two fusions were found to exhibit both high PhoA and high LacZ activity, thereby complicating the construction of a complete AQP2 model. In COS.M6 cells, the PhoA fusions were inactive. In contrast, the LacZ fusions succeeded and showed an activity pattern in complete agreement with the predictions of the six transmembrane domain model. Therefore, LacZ fusions can localize cytoplasmic loops in COS.M6 cells by means of a simple enzymatic assay with high reliability and may be used in future studies to develop topological models of other eukaryotic membrane proteins in their authentic cell systems.      

粉煤灰对混凝土拌合物泵送性能的影响

介绍了粉煤灰对掺有M17塑化剂的混凝土拌合物泵送性能和混凝土强度的影响，通过扫描电镜对粉煤灰颗粒形貌进行了观察，初步探讨了粉煤灰的作用机理。试验说明在接有M17塑化剂的泵送混凝土中加入粉煤灰，可增加坍落度，降低泌水率，泵逆性能可得到改善。     

Ag基可钢化低辐射镀膜玻璃钢化性能研究

通过对Ag基可钢化低辐射玻璃产品的钢化试验,分析比较钢化前后色差、辐射率等参数的变化,研究其性能变化规律,为深加工客户提供参考。     

涂料行业要准备迎接知识经济的挑战

阐述了知识经济的由来,知识经济的支柱产业将使涂料工业面临严峻挑战,以及涂料行业应该如何从思想上引起足够的重视,采取哪些措施准备迎接挑战。     

Binding mode of TMC-95A analogues to eukaryotic 20S proteasome

The complex thermodynamics that govern noncovalent protein-ligand interactions are still not fully understood, despite the exponential increase in experimental structural data available from X-ray crystallography and NMR spectroscopy. The eukaryotic 20S proteasome offers an ideal system for such studies as it contains in duplicate three proteolytically active sites with different substrate specificities. The natural product TMC-95A inhibits these proteolytic centers noncovalently with distinct affinities. X-ray crystallographic analysis of the complexes of the yeast proteasome core particle with this natural inhibitor and two synthetic analogues clearly revealed highly homologous hydrogen-bonding networks involving mainly the peptide backbone despite the strongly differentiated binding affinities to the three active sites of the 20S proteasome. The natural product and the two analogues are constrained in a rigid beta-type extended conformation by the endocyclic biaryl clamp, which preorganizes the peptide backbone for optimal adaptation of the ligands to the active site clefts and thus favors the binding processes entropically. However, the biaryl clamp also dictates the orientation of the P1 and P3 residues and their mode of interaction with the protein binding subsites. This limitation is optimally solved in TMC-95A with the conformationally restricted (Z)-prop-1-enyl group acting as P1 residue, at least for the chymotrypsin-like active site; however, it critically affects the inhibitory potencies of the analogues, thus suggesting the use of less-rigid endocyclic clamps in the design of proteasome inhibitors that allow for a better presentation of residues interacting with the active site clefts of the enzyme.     

Relationship of sexual maturation rate to response of oriental fruit fly strains (Diptera: Tephritidae) to methyl eugenol

Laboratory-reared and wild, fruit-reared adults of the Oriental fruit fly,Dacus dorsalis Hendel, were tested for response to methyl eugenol at various ages. Virgin laboratory (2, 4, 6, 8, and 10 days old) and wild (7, 11, 15, 19, and 23 days old) flies were released into an outdoor field cage and trapped over a two-day period. Response of males increased with age as ca. 32% and 22% of laboratory and wild males responded at 2 and 7 days of age, respectively, while ca. 93% of both strains responded at 10 and 23 days of age, respectively. These correspond approximately to the ages at which they reach sexual maturity. Female response did not increase with age and fluctuated between 15% and 29% for the laboratory strain and 10% and 45% for the wild strain. The age-related response profiles, when integrated with sexual maturation curves, indicate that one of the major reasons the male-annihilation technique is effective is because methyl eugenol is able to attract 40–50% of male flies prior to the onset of sexual maturation.     

Triacylglycerols of the red microalga Porphyridium cruentum can contribute to the biosynthesis of eukaryotic galactolipids

A mutant of the red microalga Porphyridium cruentum was selected on the basis of impaired growth at suboptimal temperatures (15 vs. 25°C). Fatty acid and lipid analyses revealed diminished proportions of eicosapentaenoic acid (from 41 to 30%) and of the eukaryotic molecular species (from 38 to 28% of monogalactosyldiacylglycerol (MGDG) and elevated proportion (10 vs. 2%) of triacylglycerols (TAG) in the mutant, as compared with the wild type. Pulse labeling of the wild type cells with radioactive fatty acid precursors indicated an initial incorporation of the fatty acids into phosphatidylcholine (PC) and TAG. Following the pulse, the label of PC and TAG decreased with time (from 25 to 5% of the total dpm in TAG) while that of chloroplastic polar lipids, mainly MGDG, continued to increase. In the mutant, however, the labeling of TAG after the pulse was higher (30% of the total dpm) than that of the wild type and decreased only slightly to 20%. This may indicate that in P. cruentum, TAG can contribute to the biosynthesis of eukaryotic species of MGDG.     

Stable expression of soluble therapeutic peptides in eukaryotic cells by multimerisation: application to the HIV-1 fusion inhibitory peptide C46

A major drawback of therapeutic peptides is their short half-life, which results in the need for multiple applications and high synthesis costs. To overcome this, we established a eukaryotic expression system that allows the stable expression of small therapeutic peptides by multimerisation. By inserting the sequence encoding the therapeutic peptide between a signal peptide and the multimerising domain of the alpha-chain from the human C4bp plasma protein, therapeutic peptides as small as 5 kDa are secreted as multimers from transfected cells; this allows easy purification. As proof of principle, we show that the T20-derived HIV-1 fusion inhibitory peptide C46 in its multimeric form: i) was efficiently secreted, ii) was more stable than the current antiviral drug T20 in vitro and in vivo, and iii) inihibited HIV-1 entry with similar efficiency in vitro. Besides the gain in stability, multimerisation also leads to increased valency and allows the combination of several therapeutic functions. Furthermore, by expressing the multimers from cells, post-translational modifications could easily be introduced.     

Spontaneous crowding of ribosomes and proteins inside vesicles: a possible mechanism for the origin of cell metabolism

One of the open questions in the origin of life is the spontaneous formation of primitive cell-like compartments from free molecules in solution and membranes. "Metabolism-first" and "replicator-first" theories claim that early catalytic cycles first evolved in solution, and became encapsulated inside lipid vesicles later on. "Compartment-first" theories suggest that metabolism progressively occurred inside compartments. Both views have some weaknesses: the low probability of co-entrapment of several compounds inside the same compartment, and the need to control nutrient uptake and waste release, respectively. By using lipid vesicles as early-cell models, we show that ribosomes, proteins and lipids spontaneously self-organise into cell-like compartments to achieve high internal concentrations, even when starting from dilute solutions. These findings suggest that the assembly of cell-like compartments, despite its low probability of occurrence, is indeed a physically realistic process. The spontaneous achievement of high local concentration might provide a rational account for the origin of primitive cellular metabolism.     

机会与做好了准备的人访陕西延长石油化建股份有限公司董事长张恺颙

<正>《石油化工建设》杂志(以下简称:PCC)感谢张董事长在百忙之中接受我们的采访!在我们杂志采访的建设企业领导中,您是我们采访的第一个上市公司的董事长。尽管延长化建上市已经     

Construction of a plasmid vector for the regulatable high level expression of eukaryotic genes in Escherichia coli: an application to overproduction of chicken lysozyme

A novel expression vector pKPl500 for synthesizing unfused proteinin Escherichia coli was constructed. pKP1500 perserves the tacpromoter, the lacZ SD sequence, unique restriction sites (EcoRI,SmaI, BamHI, SalI, PstI and HindIII) and the rrnB terminatorsof pKK223-3, but the replication origin is replaced with thatof pUC9. Construction of this plasmid is based upon the observationthat the copy number control of pUC9 is temperature dependent.At 28°C, the copy number of pKP1500 is less than 25 perchromosome, approximately the same copy number as that of pKK223-3,which contains the replication origin of pBR322, whereas at42°C, the copy number increases about 10 times and reachesup to 230 copies per chromosome. The main advantage of thissystem is that the temperature-dependent copy control and regulatableexpression of the tac promoter make cells car rying pKPl500derivatives stable against selective pressure by detrimentaloverproduction of foreign proteins at a low temperature andpermits high expression of cloned DNAs at a high temperature.When chicken lysozyme cDNA carrying the initiation codon (ATG)immediately upstream from the Lys¹ codon was inserted downstreamfrom the tac promoter and the SD sequence, the pKP1500 derivativeproduced lysozyme at about 25% of the total cellular proteins.This value is more than 10 times higher than that obtained withthe pKK223-3 derivative carrying the same lysozyine cDNA. Bycomparison, the expression of eukaryotic genes from the tacpromoter reported by others has usually been less than a few% of the total cellular protein. pKPl500 would therefore beuseful for the high level production of unfused proteins fromeukaryotic cDNAs in E. coli.     

Development of organic maturation in the thermal aureoles of sills and its relation to sediment compaction

Vitrinite reflectance profiles have been constructed for twelve borehole sequences from the Carboniferous of the eastern and central Midland Valley of Scotland. These successions are intruded by the early Stephanian, tholeiitic, (quartz-dolerite) ‘Midland Valley Sill’, or by Namurian/early Westphalian alkaline (olivine-dolerite or teschenite) sills, or by both alkaline and tholeiitic sill types. Patterns of reflectance variation in sequences invaded by the Midland Valley Sill are similar to those observed in Carboniferous successions in the north of England, where they are intruded by the ‘Great Whin Sill’, which is comagmatic with the Midland Valley Sill. The reflectance patterns all reveal extensive thermal aureoles that have modified organic matter for many tens of metres above and below the sill-sediment contacts. In contrast, no aureoles of similar scale appear to have developed, either above or below the alkaline sills. Reflectance values only rise to substantial levels in the immediate vicinity of the sediment-intrusion contacts. The most likely explanation for the radical contrast between the reflectance patterns produced by the two sill types, must be sought in the physical state of the sediments at the time of intrusion. The degree of sediment compaction, the volume of pore water in the sediments and the initial maturation level of the organic matter at the time the magma invaded the sediments will be the most significant factors, as opposed to the temperature levels of the magmas, which were probably similar (T ≈ 1000 °C), but which, because of the physical condition of the sediments, produced little rise in temperature of the organic matter even very close to the alkaline sills. The data suggest that relationships between reflectance (thermal) gradients, stratigraphic level of sill intrusion and the timing of intrusion should be carefully examined to ensure successful geothermal evaluation of sedimentary basins in which the burial history has been interrupted by intrusive igneous activity. Geochemical/petrological appraisals of sedimentary basins for hydrocarbon prospects must also be influenced by these results.     

Drying behavior and quality parameters of dried beef (biltong) subjected to different pre-treatments and maturation stages

The drying behavior of fresh, matured, and frozen beef, marinated with 0.5% salt, 1% salt, salt and vinegar, and blind samples, dried at 70°C, was investigated. Weight and color (CIELAB) were measured and images of the samples were created with a hyperspectral imaging camera. Results show that the marinade and the type of beef influences the drying behavior of beef, but not the final color. Results from the hyperspectral imaging show that it is possible to build good fitting prediction models resulting in high R² (min. 0.81, max. 0.98) and low RMSE (min. 0.08, max. 2.35) for moisture content, a^* and b^* values.     

Chemical inhibitors when timing is critical: a pharmacological concept for the maturation of T cell contacts

Cellular signal transduction proceeds through a complex network of molecular interactions and enzymatic activities. The timing of these molecular events is critical for the propagation of a signal and the generation of a specific cellular response. To define the timing of signalling events, we introduce the combination of high-resolution confocal microscopy with the application of small-molecule inhibitors at various stages of signal transduction in T cells. Inhibitors of Src-family tyrosine kinases and actin dynamics were employed to dissect the role of the lymphocyte-specific tyrosine kinase Lck in the formation and maintenance of T cell receptor/CD3-dependent contacts. Anti-CD3epsilon-coated coverslips served as a highly defined stimulus. The kinetics of the recruitment of the yellow fluorescent protein-tagged signalling protein ZAP-70 were detected by high-resolution confocal microscopy. The analysis revealed that at 5 min after receptor engagement, Lck activity was required for maintenance of contacts. In contrast, after 20 min of receptor engagement, the contacts were Lck-independent. The relevance of the timing of inhibitor application provides a pharmacological concept for the maturation of T cell-substrate contacts.