Engineering of enzymes using non-natural amino acids

In enzyme engineering, the main targets for enhancing properties are enzyme activity, stereoselective specificity, stability, substrate range, and the development of unique functions. With the advent of genetic code extension technology, non-natural amino acids (nnAAs) are able to be incorporated into proteins in a site-specific or residue-specific manner, which breaks the limit of 20 natural amino acids for protein engineering. Benefitting from this approach, numerous enzymes have been engineered with nnAAs for improved properties or extended functionality. In the present review, we focus on applications and strategies for using nnAAs in enzyme engineering. Notably, approaches to computational modelling of enzymes with nnAAs are also addressed. Finally, we discuss the bottlenecks that currently need to be addressed in order to realise the broader prospects of this genetic code extension technique.     

LGR5 regulates osteogenic differentiation of human thoracic ligamentum flavum cells by Wnt signalling pathway

Thoracic ossification of the ligamentum flavum (TOLF) is ectopic ossification of the spinal ligaments. Histologically, the development of TOLF can be described as the process of endochondral ossification. However, the underlying aetiology has not been completely clarified. In this investigation, the gene expression profile associated with leucine‐rich repeat‐containing G‐protein‐coupled receptors (LGR) and Wnt signalling pathway in the thoracic ligamentum flavum cells (TLFCs) of different ossification stages was analysed via RNA sequencing. We further confirmed the significant differences in the related gene expression profile by Gene Ontology (GO) enrichment analysis. LGR5 was first identified in primary human TLFCs during osteogenic differentiation. To evaluate the effect of LGR5 on osteogenic differentiation, LGR5 has been knocked down and overexpressed in human TLFCs. We observed that the knockdown of LGR5 inhibited the activity of Wnt signalling and attenuated the potential osteogenic differentiation of TLFCs, while overexpression of LGR5 activated the Wnt signalling pathway and increased osteogenic differentiation. Our results provide important evidence for the potent positive mediatory effects of LGR5 on osteogenesis by enhancing the Wnt signalling pathway in TOLF.     

Age‐related memory vulnerability to interfering stimuli is caused by gradual loss of MAPK‐dependent protection in Drosophila

Age‐related memory impairment (AMI) is a common phenomenon across species. Vulnerability to interfering stimuli has been proposed to be an important cause of AMI. However, the molecular mechanisms underlying this vulnerability‐related AMI remain unknown. Here we show that learning‐activated MAPK signals are gradually lost with age, leading to vulnerability‐related AMI in Drosophila. Young flies (2‐ or 3‐day‐old) exhibited a significant increase in phosphorylated MAPK levels within 15 min after learning, whereas aged flies (25‐day‐old) did not. Compared to 3‐day‐old flies, significant 1 h memory impairments were observed in 15‐, 20‐, and 30‐day‐old flies, but not in 10‐day‐old flies. However, with post‐learning interfering stimuli such as cooling or electric stimuli, 10‐day‐old flies had worse memory performance at 1 h than 3‐day‐old flies, showing a premature AMI phenomenon. Increasing learning‐activated MAPK signals through acute transgene expression in mushroom body (MB) neurons restored physiological trace of 1 h memory in a pair of MB output neurons in aged flies. Decreasing such signals in young flies mimicked the impairment of 1 h memory trace in aged flies. Restoring learning‐activated MAPK signals in MB neurons in aged flies significantly suppressed AMI even with interfering stimuli. Thus, our data suggest that age‐related loss of learning‐activated neuronal MAPK signals causes memory vulnerability to interfering stimuli, thereby leading to AMI.     

Rhabdomyolysis happened after the start of dabigatran etexilate treatment: A case report

There are few reports of rhabdomyolysis caused by anticoagulants, and it is extremely rare for it to be caused by dabigatran etexilate. An 86-year-old female experienced sudden muscle weakness and pain, a significant increase in Creatine kinase, and renal impairment after oral administration of dabigatran etexilate for 3 weeks. The enhanced thigh MRI showed abnormal signal in multiple thigh muscle groups, indicating that the lesions should be considered inflammatory diseases. In conclusion, the possibility of rhabdomyolysis should be ruled out when muscle weakness and myalgia occur at the beginning of dabigatran etexilate treatment.