Discovery and SAR analysis of 5-chloro-4-((substituted phenyl)amino)pyrimidine bearing histone deacetylase inhibitors

Histone deacetylases (HDACs) are validated targets for the development of anticancer drugs in epigenetics. In the discovery of novel HDAC inhibitors with anticancer potency, the 5-chloro-4-((substituted phenyl)amino)pyrimidine fragment is assembled as a cap group into the structure of HDAC inhibitors. The SAR revealed that presence of small groups (such as methoxy substitution) is beneficial for the HDAC inhibitory activity. In the enzyme inhibitory selectivity test, compound L20 exhibited class I selectivity with IC₅₀ values of 0.684 µM (selectivity index of >1462), 2.548 µM (selectivity index of >392), and 0.217 µM (selectivity index of >4608) against HDAC1, HDAC2 and HDAC3 compared with potency against HDAC6 (IC₅₀ value of >1000 µM), respectively. In the antiproliferative assay, compound L20 showed both hematological and solid cancer inhibitory activities. In the flow cytometry, L20 promoted G0/G1 phase cell cycle arrest and apoptosis of K562 cells.     

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.     

Direct adenylation from 5′-OH-terminated oligonucleotides by a fusion enzyme containing Pfu RNA ligase and T4 polynucleotide kinase

5′-Adenylated oligonucleotides (AppOligos) are widely used for single-stranded DNA/RNA ligation in next-generation sequencing (NGS) applications such as microRNA (miRNA) profiling. The ligation between an AppOligo adapter and target molecules (such as miRNA) no longer requires ATP, thereby minimizing potential self-ligations and simplifying library preparation procedures. AppOligos can be produced by chemical synthesis or enzymatic modification. However, adenylation via chemical synthesis is inefficient and expensive, while enzymatic modification requires pre-phosphorylated substrate and additional purification. Here we cloned and characterized the Pfu RNA ligase encoded by the PF0353 gene in the hyperthermophilic archaea Pyrococcus furiosus. We further engineered fusion enzymes containing both Pfu RNA ligase and T4 polynucleotide kinase. One fusion enzyme, 8H-AP, was thermostable and can directly catalyze 5′-OH-terminated DNA substrates to adenylated products. The newly discovered Pfu RNA ligase and the engineered fusion enzyme may be useful tools for applications using AppOligos.     

活菌制剂活菌数急剧衰降的原因与减缓衰降的方法

市售活菌制剂口服液活菌数急剧衰降的原因是营养缺乏,特别是缺乏部分氨基酸及某些维生素。增加动物性蛋白胨及强化酵母膏,添加适量维生素可使活菌数一年后保持在１０^６／ｍｌ以上。     

Distinct basolateral amygdala excitatory inputs mediate the somatosensory and aversive-affective components of pain

Pain is a multidimensional perception that includes unpleasant somatosensory and affective experiences; however, the underlying neural circuits that mediate different components of pain remain elusive. Although hyperactivity of basolateral amygdala glutamatergic (BLA^Glu) neurons is required for the somatosensory and emotional processing of pain, the precise excitatory inputs to BLA^Glu neurons and their roles in mediating different aspects of pain are unclear. Here, we identified two discrete glutamatergic neuronal circuits in male mice: a projection from the insular cortex glutamatergic (IC^Glu) to BLA^Glu neurons, which modulates both the somatosensory and affective components of pain, and a projection from the mediodorsal thalamic nucleus (MD^Glu) to BLA^Glu neurons, which modulates only the aversive-affective component of pain. Using whole-cell recording and fiber photometry, we found that neurons within the IC→BLA and MD→BLA pathways were activated in mice upon inflammatory pain induced by injection of complete Freund’s adjuvant (CFA) into their paws. Optical inhibition of the IC^Glu→BLA pathway increased the nociceptive threshold and induced behavioral place preference in CFA mice. In contrast, optical inhibition of the MD^Glu→BLA pathway did not affect the nociceptive threshold but still induced place preference in CFA mice. In normal mice, optical activation of the IC^Glu→BLA pathway decreased the nociceptive threshold and induced place aversion, while optical activation of the MD^Glu→BLA pathway only evoked aversion. Taken together, our results demonstrate that discrete IC^Glu→BLA and MD^Glu→BLA pathways are involved in modulating different components of pain, provide insights into its circuit basis, and better our understanding of pain perception.     

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.