Probing the structure of the Ff bacteriophage major coat protein transmembrane helix dimer by solution NMR

The transmembrane (TM) segment of the major coat protein from Ff bacteriophage has been extensively studied as an example of dimerization in detergent and lipid bilayer systems. However, almost all the information regarding this interaction has been gained through mutagenesis studies, with little direct structural information being available. To this end solution NMR has the potential to provide new insights into structure of the dimer. In order to evaluate the utility of this approach we have studied a selectively ¹⁵N-labeled peptide containing the TM segment of MCP (MCP_TM) by solution NMR. This peptide was found to give rise to detergent concentration-dependent spectra that were assigned to monomeric and dimeric forms. The standard free energy of this interaction in SDS was estimated from these spectra and found to be consistent with weak but specific dimerization. In addition, similar spectra could be obtained in β-octyl glucoside with intermolecular paramagnetic relaxation experiments demonstrating a parallel arrangement of TM helices in the dimer. In both detergents backbone chemical shift differences between monomeric and dimeric forms of MCP_TM showed that the largest changes occur around its GXXXG motif. The resulting structural model is consistent with observations made for MCP mutants previously characterized in biological membranes, opening the door to detailed structural characterization of this form of MCP. These results also have general implications for the study of weakly interacting TM segments by solution NMR since the use of similar sample conditions should allow structural data to be accessed for oligomeric states from a wide range systems that undergo biologically relevant but weak associations in the membrane.     

Recent advances in plasmid-based tools for establishing novel microbial chassis

A key challenge for domesticating alternative cultivable microorganisms with biotechnological potential lies in the development of innovative technologies. Within this framework, a myriad of genetic tools has flourished, allowing the design and manipulation of complex synthetic circuits and genomes to become the general rule in many laboratories rather than the exception. More recently, with the development of novel technologies such as DNA automated synthesis/sequencing and powerful computational tools, molecular biology has entered the synthetic biology era. In the beginning, most of these technologies were established in traditional microbial models (known as chassis in the synthetic biology framework) such as Escherichia coli and Saccharomyces cerevisiae, enabling fast advances in the field and the validation of fundamental proofs of concept. However, it soon became clear that these organisms, although extremely useful for prototyping many genetic tools, were not ideal for a wide range of biotechnological tasks due to intrinsic limitations in their molecular/physiological properties. Over the last decade, researchers have been facing the great challenge of shifting from these model systems to non-conventional chassis with endogenous capacities for dealing with specific tasks. The key to address these issues includes the generation of narrow and broad host plasmid-based molecular tools and the development of novel methods for engineering genomes through homologous recombination systems, CRISPR/Cas9 and other alternative methods. Here, we address the most recent advances in plasmid-based tools for the construction of novel cell factories, including a guide for helping with “build-your-own” microbial host.     

The choice of the femoral center of rotation affects material loss in total knee replacement wear testing – A parametric finite element study of ISO 14243-3

A leading cause of long-term failure of total knee replacements (TKRs) is osteolysis caused by polyethylene wear particles. The current gold standard for preclinical wear testing of TKRs is mechanical knee simulators. The definition of the femoral center of flexion-extension rotation (CoR) has been identified as one possible source of variability within TKR wear tests, since the femoral curvature varies from distal to posterior. The magnitude of the influence on wear due to changes in location of femoral CoR has not been investigated in depth. During this study, a computational framework utilizing finite element analysis for modelling wear of TKRs was developed and used to investigate the influence of the location of femoral CoR on TKR polyethylene wear during standardized displacement controlled testing (ISO 14243-3:2014). The study was carried out using a 40-point Latin Hypercube Design of Experiments approach. Volumetric wear was highly correlated to femoral CoR in both the superior/inferior and anterior/posterior directions, with a stronger relationship in the superior/inferior direction. In addition, wear scars showing linear penetration were examined, with large differences in simulations at the extreme ends of the sampling region. In this study, it was found that variations in the location of the femoral center of rotation can represent a large source of variability in the preclinical testing and evaluation of the wear performance of total knee replacements. This study represents the first attempt at quantifying the effect on wear of different femoral center of rotations across a large sampling space.     

以单细胞免疫荧光技术观察烟草细胞融合过程中微管骨架的变化

本文建立了单细胞免疫荧光标记技术并以此结合单对细胞融合技术对细胞融合过程中微管骨架组织形式的动态变化进行了追踪观察。发现在聚乙二醇(PEG)诱导条件下,一旦细胞开始粘连,细胞内微管骨架便开始解聚。在细胞融合的整个过程中一直维持着这种解聚的状态,直到融合完成,在后续的培养中微管骨架才重新出现。在微管骨架呈解聚状态时融合产物不能完成与另外的细胞融合。实验揭示了细胞的再融合能力可能受细胞本身微管骨架状态的影响。该结果为解释高等植物如何避免多精入卵提供了新的可能性。     

重组L-门冬酰胺酶工程菌的表达和PEG的化学修饰

目的提高重组L-门冬酰胺酶(rL-ASP)工程菌的表达量,分离纯化rL-ASP并对之进行PEG化学修饰。方法将带有编码rL-ASP的基因的质粒(pKA)导入不同的宿主菌中,挑出高表达菌株,同时优化发酵培养基,分离纯化获得的高纯度rL-ASP再用PEG进行化学修饰,SDS-PAGE检测修饰效果。结果在pH7.0的条件下,宿主菌为JMl09的工程菌pKA/JMl09酶活力最高,三角瓶振摇培养的酶活力可达216×103IU/L;发酵罐发酵培养,酶活力达312×103IU/L。纯化后的rL-ASP比活力为220IU/mg,rL-ASP经过PEG化学修饰生成rL-ASP-PEG,分子量发生改变。结论改变目标蛋白表达的宿主菌和优化发酵工艺,提高了rL-ASP的表达量,纯化的rL-ASP经过PEG化学修饰后分子量增大。     

Physiology of diapause in the adult bark beetle, Ips acuminatus Gyll., studied in relation to cold hardiness

The ovarioles of the bark beetle, Ips acuminatus are telotrophic. Ovarian development is suppressed at an immature stage with primary germ cells present in the germaria. Lower oxygen consumption is found in beetles during autumn and early winter, and a substantial rise in mean respiration rates occurs in the beginning of January paralleled by a resumption of pre-vitellogenesis in all females maintained either at 3°C or out of doors after return to 21°C for 2 weeks. It is concluded that I. acuminatus enters faculative diapause soon after enclosure to the adult, and that diapause is terminated by mid-winter in beetles kept for 18 weeks at either 3°C or out of doors. The specimens remain thereafter in reproductive quiescence until ovarian development can proceed.Photic cues are neither involved in the elevation of mean respiration rates, nor needed to abolish the inhibition of ovarian maturation in beetles kept at 3°C or in those returned to 21°C. However, follicle formation in ovarioles is only seen in positive phototactic females reared during “long-day” conditions, suggesting a photoperiodic regulation of the later stages of vitellogenesis.Detectable amounts of ethylene glycol are found at the beginning of November in freezing-susceptible I. acuminatus hibernating in its galleries underneath bark of Scots pine (Pinus silvestris) at 3°C. The gradual catabolism of the cryoprotective solute at 3°C through December occurred at a time when individuals achieved the competence to resume ovarian maturation during 2 weeks at 21°C, but prior to the substantial rise in their mean respiration rates. However, resumption of ovarian development in spring had no effect on the capability of outdoor beetles to enhance their supercooling capacity when subjected to sub-zero temperatures. Since the ability to respond to temperature changes occurred in post-diapause I. acuminatus as well, the maintenance of prolonged cold hardiness in specimens could not be related to diapause itself. Apparently, the ability of beetles to resynthesize ethylene glycol when a detectable level is present in the organism remains unaltered during overwintering.     

Extractive fermentation for enhanced gellan-hydrolysing enzyme production by Bacillus thuringiensis H14

A new extractive fermentation process using PEG and potassium phosphate aqueous two-phase system (ATPS) was developed for enhanced production of gellan-hydrolysing enzyme by Bacillus thuringiensis H14. Five different Bacillus sp. were tested for their ability to synthesize gellan-hydrolysing enzyme. Bacillus thuringiensis H14 was found to be the best organism for gellan-hydrolysing enzyme production. The enzyme showed maximum activity at pH 7.5 and 40 °C. The partition studies of gellan-hydrolysing enzyme in the system using PEG X (X = 9000, 6000, 4000) and potassium phosphate–water and PEG–sodium citrate–water system indicated at PEG (4000)– potassium phosphate–water is the best system for partitioning of gellan-hydrolysing enzyme into the PEG phase (K = 4.99). Gellan-hydrolysing enzyme production by Bacillus thuringiensis H14 was studied in ATPSs composed of PEG X (X = 9000, 6000, 4000) and potassium phosphate. The top phase is continuous and rich in PEG while the bottom phase is dispersed and is rich in phosphate, microbial cells being mainly retained in the bottom phase. The gellan-hydrolysing enzyme produced during fermentation partitioned into the upper PEG phase and total gellan-hydrolysing enzyme produced was 2.12, 2.29 and 2.40 times higher than that of homogeneous fermentation when the fermentations were carried out using PEG 9000–potassium phosphate–water, PEG 6000–potassium phosphate–water, PEG 4000–potassium phosphate–water systems respectively.     

聚乙二醇对平邑甜茶叶片气孔器超微结构的影响

 采用透射电镜技术对聚乙二醇（PEG）模拟干旱处理的平邑甜茶(Malus hupehensis Rehd (pingyitiancha))叶片气孔器超微结构进行了观察,并对叶片气孔器超微结构制片方法进行了改进。所观察结果如下：1）PEG胁迫后保卫细胞中叶绿体数量增加,而叶绿体中淀粉粒数量呈下降趋势;2）PEG胁迫降低保卫细胞中线粒体的数量及破坏线粒体的超微结构;3）PEG胁迫使平邑甜茶保卫细胞中的液泡变小以致不明显。     

Molecular dissection of the response of a rice leucine-rich repeat receptor-like kinase (LRR-RLK) gene to abiotic stresses

Leucine-rich repeat (LRR) receptor-like kinase (RLK) proteins play key roles in a variety of biological pathways. In a previous study, we analyzed the members of the rice LRR-RLK gene family using in silico analysis. A total of 23 LRR-RLK genes were selected based on the expression patterns of a genome-wide dataset of microarrays. The Oryza sativa gamma-ray induced LRR-RLK1 (OsGIRL1) gene was highly induced by gamma irradiation. Therefore, we studied its expression pattern in response to various different abiotic and phytohormone treatments. OsGIRL1 was induced on exposure to abiotic stresses such as salt, osmotic, and heat, salicylic acid (SA), and abscisic acid (ABA), but exhibited downregulation in response to jasmonic acid (JA) treatment. The OsGIRL1 protein was clearly localized at the plasma membrane. The truncated proteins harboring juxtamembrane and kinase domains (or only harboring a kinase domain) exhibited strong autophosphorylation. The biological function of OsGIRL1 was investigated via heterologous overexpression of this gene in Arabidopsis plants subjected to gamma-ray irradiation, salt stress, osmotic stress, and heat stress. A hypersensitive response was observed in response to salt stress and heat stress, whereas a hyposensitive response was observed in response to gamma-ray treatment and osmotic stress. These results provide critical insights into the molecular functions of the rice LRR-RLK genes as receptors of external signals.