Sanger Gap Sequencing for Genetic Alphabet Expansion of DNA

Genetic alphabet expansion technology, creating new replicable and functional DNA molecules with unnatural base pairs (UBPs), is the novel promising research area of xenobiology. Recently, this technology has rapidly advanced, resulting in the need for a sequencing method for DNA molecules containing UBPs. However, all of the conventional sequencing methods, such as Sanger methods, are for four-letter DNA molecules. Here, we present an improved Sanger sequencing method (Sanger gap sequencing) for DNAs containing our UBP, Ds-Px, which appears as gaps in the sequencing peak patterns. By improving the sequencing reaction for efficient Ds-Px pairing and using modified Px substrates, we have developed a sequencing method with increased processivity and clear gap patterns for multiple Ds-Px pairs in various sequence contexts. This method is useful for UBP applications such as high-affinity DNA aptamer generation and semisynthetic organism creation involving UBPs. In addition, through this research, we found that the side chains of UBs greatly affect the efficiency of UB pairings in replication, thus suggesting further development of UBPs.     

Dolichol: A Component of the Cellular Antioxidant Machinery

Dolichol, an end product of the mevalonate pathway, has been proposed as a biomarker of aging, but its biological role, not to mention its catabolism, has not been fully understood. UV‐B radiation was used to induce oxidative stress in isolated rat hepatocytes by the collagenase method. Effects on dolichol, phospholipid‐bound polyunsaturated fatty acids (PL‐PUFA) and known lipid soluble antioxidants [coenzyme Q (CoQ) and α‐tocopherol] were studied. The increase in oxidative stress was detected by a probe sensitive to reactive oxygen species (ROS). Peroxidation of lipids was assessed by measuring the release of thiobarbituric acid reactive substances (TBARS). Dolichol, CoQ, and α‐tocopherol were assessed by high‐pressure liquid chromatography (HPLC), PL‐PUFA by gas–liquid chromatography (GC). UV‐B radiation caused an immediate increase in ROS as well as lipid peroxidation and a simultaneous decrease in the levels of dolichol and lipid soluble antioxidants. Decrease in dolichol paralleled changes in CoQ levels and was smaller to that in α‐tocopherol. The addition of mevinolin, a competitive inhibitor of the enzyme 3‐hydroxy‐3‐methylglutaryl CoA reductase (HMG‐CoAR), magnified the loss of dolichol and was associated with an increase in TBARS production. Changes in PL‐PUFA were minor. These findings highlight that oxidative stress has very early and similar effects on dolichol and lipid soluble antioxidants. Lower levels of dolichol are associated with enhanced peroxidation of lipids, which suggest that dolichol may have a protective role in the antioxidant machinery of cell membranes and perhaps be a key to understanding some adverse effects of statin therapy.     

Agomelatine beyond Borders: Current Evidences of Its Efficacy in Disorders Other than Major Depression

Agomelatine, a melatonergic antidepressant with a rapid onset of action, is one of the most recent drugs in the antidepressant category. Agomelatine’s antidepressant actions are attributed to its sleep-promoting and chronobiotic actions mediated by MT1 and MT2 receptors present in the suprachiasmatic nucleus, as well as to its effects on the blockade of 5-HT2c receptors. Blockade of 5-HT2c receptors causes release of both noradrenaline and dopamine at the fronto-cortical dopaminergic and noradrenergic pathways. The combined actions of agomelatine on MT1/MT2 and 5-HT2c receptors facilitate the resynchronization of altered circadian rhythms and abnormal sleep patterns. Agomelatine appeared to be effective in treating major depression. Moreover, evidence exists that points out a possible efficacy of such drug in the treatment of bipolar depression, anxiety disorders, alcohol dependence, migraines etc. Thus, the aim of this narrative review was to elucidate current evidences on the role of agomelatine in disorders other than major depression.