Evaluation of a D-Octaarginine-linked polymer as a transfection tool for transient and stable transgene expression in human and murine cell lines.

Poly(N-vinylacetamide-co-acrylic acid) coupled with d-octaarginine (VP-R8) promotes the cellular uptake of peptides/proteins in vitro; however, details of the transfection efficacy of VP-R8, such as the cell types possessing high gene transfer, are not known. Herein, we compared the ability of VP-R8 to induce the cellular uptake of plasmid DNA in mouse and human cell lines from different tissues and organs. A green fluorescent protein (GFP)-expression plasmid was used as model genetic material, and fluorescence as an indicator of uptake and plasmid-derived protein expression. Three mouse and three human cell lines were incubated with a mixture of plasmid and VP-R8, and fluorescence analysis were performed two days after transfection. To confirm stable transgene expression, we performed drug selection three days after transfection. A commercially available polymer-based DNA transfection reagent (PTR) was used as the transfection control and standard for comparing transgene expression efficiency. In the case of transient transgene expression, slight-to-moderate GFP expression was observed in all cell lines transfected with plasmid via VP-R8; however, transfection efficiency was lower than using the PTR for gene delivery. In the case of stable transgene expression, VP-R8 promoted drug-resistance acquisition more efficiently than the PTR did. Cells that developed drug resistance after VP-R8-mediated gene transfection expressed GFP more efficiently than cells that developed drug resistance after transfection with the PTR. Thus, VP-R8 shows potential as an in vitro or ex vivo nonviral transfection tool for generating cell lines with stable transgene expression.

Polypod-like structured guanine-rich oligonucleotide aptamer as a selective and cytotoxic nanostructured DNA to cancer cells.

Guanine-rich oligonucleotide (GRO) can be developed as an effective anticancer agent owing to its high selectivity, affinity and antiproliferative activity in cancer cells. In this study, to increase the potency of GRO29A, a 29-mer GRO aptamer against nucleolin, an overexpressed protein in cancer cells, GRO29A was incorporated into three or six pods of polypod-like structured DNA (polypodna), tripodna or hexapodna, respectively. The polypod-like structured GROs, tri-G3, consisting of one tripodna and three GRO29A, or hexa-G1, hexa-G3 or hexa-G6, each of which comprises one hexapodna and one, three or six GRO29A, respectively, were designed. Tri-G3, hexa-G1 and hexa-G3 were prepared in high yield, except for hexa-G6. Polypod-like structured GROs had quadruplex structures under physiological salt conditions, and degraded at a slower rate in buffer containing serum. Cellular interaction experiments using fluorescently labelled DNA samples showed that the uptake of hexa-G3 by nucleolin-positive MCF-7 cells was more than 2-fold higher than GRO29A, and the interaction was increasingly dependent on the number of GRO29A in the structures. Hexa-G3 inhibited the proliferation of MCF-7 cells in more than 40%, but not of CHO cells. These results indicate that polypod-like structured GROs are useful DNA aptamers with high selectivity and cytotoxicity against nucleolin-positive cancer cells.

The effects of a participatory structured group educational program on the development of CKD: a population-based study.

BACKGROUND: The type of lifestyle guidance that is effective for preventing development of chronic kidney disease (CKD) is unknown. Here, we aim to investigate the effects of a participatory structured group education (SGE) program on the development of CKD in a population-based study. METHODS: We retrospectively analyzed 1060 adult special health check-up examinees with CKD. Examinees with an estimated glomerular filtration rate (eGFR) from 50 to 60 mL/min/1.73 m2 and/or proteinuria 1+ were encouraged to attend an SGE program. The SGE program included participatory small group discussions on the attendees' remaining risk factors. The primary outcome of this study was the change in eGFR per year. RESULTS: The changes in eGFR in examinees who attended the SGE program (n = 209, + 2.9 mL/min/1.73 m2 [95% confidence interval (CI) + 1.9 to + 3.9]) significantly improved compared with control (n = 383, + 1.2 mL/min/1.73 m2 [95% CI + 0.5 to + 1.9], p = 0.006). Attending an SGE program was independently and positively related to the changes in eGFR at 1 year after attendance, after adjusting for classical covariates (ß = 1.55 [95% CI 0.37-2.73], p = 0.01). Attending an SGE program was effective for the examinees with a lower eGFR compared with those with only proteinuria. CONCLUSIONS: Our SGE program showed the beneficial effects of preventing the development of CKD, independent of classical factors. The type of SGE program that is more effective for preventing development of CKD should be investigated in a long-term analysis.

Comparison of c-Fos expression in brain regions involved in maternal behavior of virgin and lactating female mice.

Maternal care is indispensable for the survival of mammalian offspring. Although virgin female mice avoid pups, they actively display maternal behavior after parturition. To determine which brain regions are involved in the qualitative differences observed in the responses of virgin and lactating females to pups, we compared the expression of c-Fos, which is a marker of neuronal activation, in brain regions involved in regulating maternal behavior. Pup presentation increased the number of c-Fos-positive cells in both the ventrotegmental area (VTA) and nucleus accumbens to a greater extent in lactating females than in virgin females. The bed nucleus of striaterminalis (BNST), which innervates VTA neurons to regulate both aversive and rewarding responses, showed increased number of c-Fos-positive cells following pup presentation in virgin females, butnotin lactating females. On the other hand, the number of c-Fos-positive cells in the medial preoptic area (MPOA) increased in both virgin and lactating females. The number of c-Fos-positive cells in lactating females not presented with pups was high and similar to that in virgin females presented with pups. Moreover, c-Fos-positive GABAergicneurons projecting from the MPOA to the BNST was confirmed using a retrograde tracer Fluorogold in lactating females. Our results indicate that constitutive GABAergic modulation projecting from the MPOA may suppress the activity of BNST neurons and prevent avoidance responses to pups in lactating females.

Enhanced water dispersibility of coenzyme Q10 by complexation with albumin hydrolysate.

The biologically important coenzyme Q10 (CoQ10) is widely used as a drug for chronic heart failure, as a nutritional supplement, and in cosmetics. However, the oral bioavailability of CoQ10 is poor due to its extremely low solubility in aqueous media. In this study, complexation of CoQ10 with albumin hydrolysate as a peptide mixture (Pep) was shown to enhance the water dispersibility of CoQ10. An aqueous solution of Pep and an acetone solution of CoQ10 were mixed and lyophilized to obtain a white-yellow powder containing peptides and CoQ10 complex (Q10-Pep). The water dispersibility of Q10-Pep was much higher than that of CoQ10 alone and increased with the quantity of Pep. The particle size of Q10-Pep in aqueous media was 170-280 nm, suggesting that Q10-Pep was present as a hydrocolloidal material. Characterization of Q10-Pep using differential scanning calorimetry showed that CoQ10 was incorporated in the hydrocolloid in an amorphous state.