Design of Deinococcus radiodurans thioredoxin reductase with altered thioredoxin specificity using computational alanine mutagenesis

In this study, the X-ray crystal structure of the complex between Escherichia coli thioredoxin reductase (EC TrxR) and its substrate thioredoxin (Trx) was used as a guide to design a Deinococcus radiodurans TrxR (DR TrxR) mutant with altered Trx specificity. Previous studies have shown that TrxRs have higher affinity for cognate Trxs (same species) than that for Trxs from different species. Computational alanine scanning mutagenesis and visual inspection of the EC TrxR-Trx interface suggested that only four residues (F81, R130, F141, and F142) account for the majority of the EC TrxR-Trx interface stability. Individual replacement of equivalent residues in DR TrxR (M84, K137, F148, and F149) with alanine resulted in drastic changes in binding affinity, confirming that the four residues account for most of TrxR-Trx interface stability. When M84 and K137 were changed to match equivalent EC TrxR residues (K137R and M84F), the DR TrxR substrate specificity was altered from its own Trx to that of EC Trx. The results suggest that a small subset of the TrxR-Trx interface residues is responsible for the majority of Trx binding affinity and species-specific recognition.     

Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

We developed an IgG1 domain-tethering approach to guide the correct assembly of 2 light and 2 heavy chains, derived from 2 different antibodies, to form bispecific monovalent antibodies in IgG1 format. We show here that assembling 2 different light and heavy chains by sequentially connecting them with protease-cleavable polypeptide linkers results in the generation of monovalent bispecific antibodies that have IgG1 sequence, structure and functional properties. This approach was used to generate a bispecific monovalent antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor that: 1) can be produced and purified using standard IgG1 techniques; 2) exhibits stability and structural features comparable to IgG1; 3) binds both targets simultaneously; and 4) has potent anti-tumor activity. Our strategy provides new engineering opportunities for bispecific antibody applications, and, most importantly, overcomes some of the limitations (e.g., half-antibody and homodimer formation, light chains mispairing, multi-step purification), inherent with some of the previously described IgG1-based bispecific monovalent antibodies.     

微生物细胞工厂中多基因表达的控制策略

微生物代谢工程和合成生物学是当今微生物技术领域研究的热点,微生物的生长速度快、容易进行大规模培养;遗传背景清楚、遗传操作简便可靠等性质使其在与人类生活相关的多个领域中起到重要的作用。微生物细胞工厂是指人工设计的能够进行物质生产的微生物代谢体系。许多微生物细胞工厂的构建由于引入多个基因或整条代谢途径,而可能导致代谢失衡、部分代谢中间产物积累等问题,需要使用一定的调控策略加以控制。以下对涉及多个基因作用的微生物细胞工厂中所使用的调控策略,分为若干层次进行了总结和探讨,并对今后多基因控制策略的发展方向进行了预测与展望。     

对比分析IPS e.max Press铸瓷高嵌体与普兰梅卡CADCAM系统制作的高嵌体修复后牙牙体缺损的效果

摘要 目的：本研究旨在探讨对比分析IPS e.max Press铸瓷高嵌体与普兰梅卡CADCAM系统制作的高嵌体修复后牙牙体缺损的效果。方法：以2021年8月-2022年8月我院诊治的60例（86颗）后牙牙体缺损患者为研究对象,采用随机数字表法将其分为研究组（给予普兰梅卡CADCAM系统制作的高嵌体修复）和对照组（给予IPS e.max Press铸瓷高嵌体修复）各30例,对比两组修复前及修复1年后咀嚼功能、牙龈情况、RANK、CXCL16水平、IL-6、IL-8水平,统计修复结果及美牙效果满意度。结果：（1）修复前,两组咀嚼功能比较无差异（P＞0.05）;修复1年后,研究组咀嚼功能优于对照组（P＜0.05）;（2）修复前,两组牙龈指数、菌斑指数评分比较无差异（P＞0.05）;修复1年后,研究组牙龈指数、菌斑指数评分优于对照组（P＜0.05）;（3）修复前,两组RANK、CXCL16水平比较无差异（P＞0.05）;修复1年后,研究组RANK、CXCL16水平低于对照组（P＜0.05）;（4）修复前,两组IL-6、IL-8水平比较无差异（P＞0.05）;修复1年后,研究组IL-6、IL-8水平与对照组比较（P＞0.05）;（5）修复1年后,研究组修复体边缘适合性、修复体邻面解剖形态优于对照组（P＜0.05）;两组修复体表面及边缘着色、修复体折裂与固位比较,差异不显著（P＞0.05）;（6）修复1年后,研究组美牙效果满意度优于对照组（P＜0.05）。结论：与IPS e.max Press铸瓷高嵌体相比,普兰梅卡CADCAM系统制作的高嵌体修复后牙牙体缺损的效果更佳,能够提高咀嚼功能,改善牙龈指数,提高美牙效果满意度。     

Functional analysis of phosphorylation of the mitotic centromere-associated kinesin by Aurora B kinase in human tumor cells

Mitotic centromere-associated kinesin (MCAK) is the best characterized member of the kinesin-13 family and plays important roles in microtubule dynamics during mitosis. Its activity and subcellular localization is tightly regulated by an orchestra of mitotic kinases, such as Aurora B. It is well known that serine 196 of MCAK is the major phosphorylation site of Aurora B in Xenopus leavis extracts and that this phosphorylation regulates its catalytic activity and subcellular localization. In the current study, we have addressed the conserved phosphorylation site serine 192 in human MCAK to characterize its function in more depth in human cancer cells. Our data confirm that S192 is the major phosphorylation site of Aurora B in human MCAK and that this phosphorylation has crucial roles in regulating its catalytic activity and localization at the kinetochore/centromere region in mitosis. Interfering with this phosphorylation leads to a delayed progression through prometa- and metaphase associated with mitotic defects in chromosome alignment and segregation. We show further that MCAK is involved in directional migration and invasion of tumor cells, and interestingly, interference with the S192 phosphorylation affects this capability of MCAK. These data provide the first molecular explanation for clinical observation, where an overexpression of MCAK was associated with lymphatic invasion and lymph node metastasis in gastric and colorectal cancer patients.     

Electrostatic fields at the active site of ribulose-1,5-bisphosphate carboxylase.

A macroscopic approach has been employed to calculate the electrostatic potential field of nonactivated ribulose-1,5-bisphosphate carboxylase and of some complexes of the enzyme with activator and substrate. The overall electrostatic field of the L2-type enzyme from the photosynthetic bacterium Rhodospirillum rubrum shows that the core of the dimer, consisting of the two C-terminal domains, has a predominantly positive potential. These domains provide the binding sites for the negatively charged phosphate groups of the substrate. The two N-terminal domains have mainly negative potential. At the active site situated between the C-terminal domain of one subunit and the N-terminal domain of the second subunit, a large potential gradient at the substrate binding site is found. This might be important for polarization of chemical bonds of the substrate and the movement of protons during catalysis. The immediate surroundings of the activator lysine, K191, provide a positive potential area which might cause the pK value for this residue to be lowered. This observation suggests that the electrostatic field at the active site is responsible for the specific carbamylation of the epsilon-amino group of this lysine side chain during activation. Activation causes a shift in the electrostatic potential at the position of K166 to more positive values, which is reflected in the unusually low pK of K166 in the activated enzyme species. The overall shape of the electrostatic potential field in the L2 building block of the L8S8-type Rubisco from spinach is, despite only 30% amino acid homology for the L-chains, strikingly similar to that of the L2-type Rubisco from Rhodospirillum rubrum. A significant difference between the two species is that the potential is in general more positive in the higher plant Rubisco. In particular, the second phosphate binding site has a considerably more positive potential, which might be responsible for the higher affinity for the substrate of L8S8-type enzymes. The higher potential at this site might be due to two remote histidine residues, which are conserved in the plant enzymes.     

Potential applications of natural origin polymer-based systems in soft tissue regeneration

Despite the many advances in tissue engineering approaches, scientists still face significant challenges in trying to repair and replace soft tissues. Nature-inspired routes involving the creation of polymer-based systems of natural origins constitute an interesting alternative route to produce novel materials. The interest in these materials comes from the possibility of constructing multi-component systems that can be manipulated by composition allowing one to mimic the tissue environment required for the cellular regeneration of soft tissues. For this purpose, factors such as the design, choice, and compatibility of the polymers are considered to be key factors for successful strategies in soft tissue regeneration. More recently, polysaccharide-protein based systems have being increasingly studied and proposed for the treatment of soft tissues. The characteristics, properties, and compatibility of the resulting materials investigated in the last 10 years, as well as commercially available matrices or those currently under investigation are the subject matter of this review.