Development of PCR primers enabling the design of flexible sticky ends for efficient concatenation of long DNA fragments.

We developed chemically modified PCR primers that allow the design of flexible sticky ends by introducing a photo-cleavable group at the phosphate moiety. Nucleic acid derivatives containing o-nitrobenzyl photo-cleavable groups with a tert-butyl group at the benzyl position were stable during strong base treatment for oligonucleotide synthesis and thermal cycling in PCR reactions. PCR using primers incorporating these nucleic acid derivatives confirmed that chain extension reactions completely stopped at position 1 before and after the site of the photo-cleavable group was introduced. DNA fragments of 2 and 3 kbp, with sticky ends of 50 bases, were successfully concatenated with a high yield of 77%. A plasmid was constructed using this method. Finally, we applied this approach to construct a 48.5 kbp lambda phage DNA, which is difficult to achieve using restriction enzyme-based methods. After 7 days, we were able to confirm the generation of DNA of the desired length. Although the efficiency is yet to be improved, the chemically modified PCR primer offers potential to complement enzymatic methods and serve as a DNA concatenation technique.

Creation of single molecular conjugates of metal-organic cages and DNA.

Here we report the development of an equimolar conjugate of a metal-organic cage (MOC) and DNA (MOC-DNA). Several MOC-DNA conjugates were assembled into a programmed structure by coordinating with a template DNA having a complementary base sequence. Moreover, conjugation with the MOC drastically enhanced the permeability of DNA through the lipid bilayer, presenting great potential as a drug delivery system.

A 2'-modified uridine analog, 2'-O-(methylthiomethoxy)methyl uridine, for siRNA applications.

The medicinal applications of siRNAs have been intensively examined but are still hindered by their low molecular stability under biological conditions and off-target effects, etc. The introduction of chemical modifications to the nucleoside is a promising strategy for solving these limitations. Herein, we describe the development of a new uridine analog, U*, that has a (methylthiomethoxy)methoxy group at the 2' position. The phosphoramidite reagent corresponding to U* was easily synthesized and the RNA oligonucleotides containing U* were stably prepared using a standard protocol for oligonucleotide synthesis. The introduction of U* into the siRNA resulted in positive or negative effects on the targeted gene silencing in a position-dependent manner, and the positive effects were attributed to the improved stability under biological conditions. The thermodynamic analysis of the U*-modified RNAs revealed a slight destabilization of the dsRNA, based depending on which U was strategically utilized to restrain the off-target effects of the siRNA. This study describes a rare example of nucleoside analogs with a large substitution at the 2'-position in the context of an siRNA application and is informative for the development of other analogs to further improve the molecular properties of siRNAs for medicinal applications.

Diagnosis and Treatment for Acute Focal Bacterial Nephritis With Renal Abscess Based on Magnetic Resonance Imaging Evaluation.

A 5-year-old boy was diagnosed with left acute focal bacterial nephritis (AFBN) complicated with renal abscess (RA) on magnetic resonance imaging (MRI). MRI is useful for diagnosing AFBN and RA complications. He was administered antibiotics for 3 weeks on evaluation of MRI findings. Evaluation of apparent diffusion coefficient values over time may be useful as an index of treatment of RA.

Protein-losing enteropathy in an infant with severe atopic dermatitis.

Severe atopic dermatitis (AD) may lead to various complications such as hypoproteinaemia. We describe the case of a 7-month-old male infant with severe AD complicated with protein-losing enteropathy (PLE). He was diagnosed with AD at 2 months of age; however, because of familial steroid phobia, topical corticosteroids were not administered. At 7 months of age, he was admitted to our hospital for decreased feeding, diarrhoea, reduced urine volume and recurrent vomiting. Class 3 topical corticosteroid treatment was initiated. On day 3, eczema had almost resolved. However, serum protein levels had not improved; oliguria persisted and oedema worsened. Serum albumin scintigraphy revealed radioisotopes in the distal duodenum, leading to PLE diagnosis. Systemic prednisolone and albumin were administered, with no PLE relapse after discontinuation. To our knowledge, only two infant PLE cases associated with AD were reported to date. PLE should be considered in patients with severe AD and persistent hypoproteinaemia.

Berberine stimulates fibroblast growth factor 21 by modulating the molecular clock component brain and muscle Arnt-like 1 in brown adipose tissue.

Fibroblast growth factor 21 (FGF21), a member of the FGF subfamily that acts through the FGF receptor 1 with the co-receptor ß-Klotho, functions as an important metabolic regulator of peripheral glucose tolerance and lipid homeostasis in an endocrine or autocrine and/or paracrine manner. Previous studies showed that FGF21 ameliorated and prevented the development of metabolic disorders, such as obesity and diabetes mellitus. In the present study, we demonstrated that berberine, a naturally occurring compound, stimulated FGF21 expression in brown adipose tissue (BAT). Furthermore, the up-regulated expression of FGF21 in brown adipocytes in response to berberine was due, at least in part, to the activation of the AMP-activated protein kinase pathway. We also found that berberine reversed high-fat diet-induced obesity concomitant with its regulation of the expression of Fgf21 and the core clock component brain and muscle Arnt-like 1 (Bmal1) in BAT. Berberine significantly up-regulated the gene expression and production of FGF21 in a dose-dependent manner in C3H10T1/2 brown adipocytes. Furthermore, the knockdown of Bmal1 prevented the up-regulated expression of FGF21 in response to berberine in C3H10T1/2 brown adipocytes, suggesting that Bmal1 links the regulatory mechanisms of FGF21 in response to berberine. The present results suggest that berberine stimulates the expression of FGF21 by modulating molecular clock Bmal1 in BAT, which may, in turn, attenuate diet-induced obesity. They also indicate the potential of berberine as a therapeutic agent for obesity and obesity-associated metabolic disorders related to circadian misalignments.

Baicalein stimulates fibroblast growth factor 21 expression by up-regulating retinoic acid receptor-related orphan receptor α in C2C12 myotubes.

Retinoic acid receptor-related orphan receptor (ROR) α has been implicated in various physiological functions, including the immune system, inflammation, and circadian rhythms. In the present study, the synthetic RORα/γ agonist SR1078 stimulated the production and gene expression of fibroblast growth factor 21 (FGF21) in C2C12 myotubes. FGF21, a member of the FGF family, plays important roles in the regulation of peripheral glucose tolerance and lipid metabolism and improves metabolic health. The mRNA expression and secretion of FGF21 was significantly weaker in Rora-silenced cells than in cells transfected with non-targeting control siRNA. SR1078 significantly up-regulated C/EBP homologous protein (CHOP), an established marker of ER stress, in a dose-dependent manner in C2C12 myotubes, while CHOP expression was decreased in Rora-silenced C2C12 cells, suggesting that RORα is involved in the regulation of FGF21 expression and stimulates ER stress in C2C12 myotubes. The naturally occurring compound baicalein up-regulated FGF21 expression and secretion in C2C12 myotubes. Additionally, the up-regulation of CHOP mRNA and protein expression was observed in C2C12 myotubes after the baicalein treatment. Furthermore, the knockdown of RORα prevented the augmentation of FGF21 and up-regulation of CHOP in response to baicalein in C2C12 cells. Collectively, these results suggest that baicalein stimulates the ER stress response and FGF21 expression through an RORα-dependent mechanism in C2C12 myotubes, and indicate the potential of baicalein as an effective anti-obesity therapy via its ability to enhance FGF21 production.

Empathy with fictional stories: reconsideration of the fantasy scale of the interpersonal reactivity index.

The present research compared empathy for real people with empathy for fictional characters. 95 university students (53 men, 42 women) ages 18-22 years (M = 19.5, SD = 1.9) completed the Interpersonal Reactivity Index (IRI) and the Fictional IRI. The IRI is a widely used measure of human empathy, and the Fictional IRI contains items from the original IRI that have been modified to investigate empathy for fictional characters. Empathy for characters in fictional stories was found to correlate statistically significantly with empathy for real people on all but a few factors. The results of the present study indicate that empathy for real people and empathy for fictional characters are similar, suggesting that the Fantasy subscale of the IRI, which is limited to fictional stories, should be reconsidered.

Enantioselective borodeuteride reduction of aldimines catalyzed by cobalt complexes: preparation of optically active deuterated primary amines.

[reaction: see text] The enantioselective borodeuteride reduction catalyzed by optically active beta-ketoiminato cobalt complexes was applied to N-(di(o-tolyl)phosphinyl)aldimines to afford the corresponding optically active deuterated primary amines in high yields with high enantiomeric excesses after simple deprotection. The present deuteride reduction of aldimines is in the opposite sense of the enantioselective for the previously reported borohydride reduction of ketones or diphenylphosphinyl aldimines. The stereochemical course in these enantioselective reductions is discussed.