Development of an all-in-one inducible lentiviral vector for gene specific analysis of reprogramming

Fair comparison of reprogramming efficiencies and in vitro differentiation capabilities among induced pluripotent stem cell (iPSC) lines has been hampered by the cellular and genetic heterogeneity of de novo infected somatic cells. In order to address this problem, we constructed a single cassette all-in-one inducible lentiviral vector (Ai-LV) for the expression of three reprogramming factors (Oct3/4, Klf4 and Sox2). To obtain multiple types of somatic cells having the same genetic background, we generated reprogrammable chimeric mice using iPSCs derived from Ai-LV infected somatic cells. Then, hepatic cells, hematopoietic cells and fibroblasts were isolated at different developmental stages from the chimeric mice, and reprogrammed again to generate 2nd iPSCs. The results revealed that somatic cells, especially fetal hepatoblasts were reprogrammed 1200 times more efficiently than adult hepatocytes with maximum reprogramming efficiency reaching 12.5%. However, we found that forced expression of c-Myc compensated for the reduced reprogramming efficiency in aged somatic cells without affecting cell proliferation. All these findings suggest that the Ai-LV system enables us to generate a panel of iPSC clones derived from various tissues with the same genetic background, and thus provides an invaluable tool for iPSC research.     

Targeted delivery to macrophages and dendritic cells by chemically modified mannose ligand-conjugated siRNA

Extrahepatic delivery of small interfering RNAs (siRNAs) may have applications in the development of novel therapeutic approaches. However, reports on such approaches are limited, and the scarcity of reports concerning the systemically targeted delivery of siRNAs with effective gene silencing activity presents a challenge. We herein report for the first time the targeted delivery of CD206-targetable chemically modified mannose–siRNA (CMM–siRNA) conjugates to macrophages and dendritic cells (DCs). CMM–siRNA exhibited a strong binding ability to CD206 and selectively delivered contents to CD206-expressing macrophages and DCs. Furthermore, the conjugates demonstrated strong gene silencing ability with long-lasting effects and protein downregulation in CD206-expressing cells in vivo. These findings could broaden the use of siRNA technology, provide additional therapeutic opportunities, and establish a basis for further innovative approaches for the targeted delivery of siRNAs to not only macrophages and DCs but also other cell types.     

Studies on the interaction between polyion and counterions by sound velocity (II)

Sound velocities in polyacrylate solutions neutralized by tetraalkylammonium hydroxide were measured at various concentrations of added NACl. From the results, the degree of counterion binding to polyion and the extent of the changes in hydration volume due to ion binding were determined as a function of the degree of neutralization, alpha. The ion binding accompanying the volume changes appeared above about alpha = 0.6 and the ion binding process depended on the charge density of the polyion. The effect of the size of the tetraalkylammonium ion on ion binding was negligibly small.     

Soprano singing in gibbons

Diversifications in primate vocalization, including human speech, are believed to reflect evolutionary modifications in vocal anatomy and physiology. Gibbon song is acoustically unique, comprising loud, melodious, penetrating pure tone‐like calls. In a white‐handed gibbon, Hylobates lar, the fundamental frequency (f₀) of song sounds is amplified distinctively from the higher harmonics in normal air. In a helium‐enriched atmosphere, f₀ does not shift, but it is significantly suppressed and 2f₀ is emphasized. This implies that the source is independent of the resonance filter of the supralaryngeal vocal tract (SVT) in gibbons, in contrast to musical wind instruments, in which the filter primarily determines f₀. Acoustic simulation further supported that gibbons' singing is produced analogously to professional human soprano singing, in which a precise tuning of the first formant (F₁) of the SVT to f₀ amplifies exclusively the f₀ component of the source. Thus, in gibbons, as in humans, dynamic control over the vocal tract configuration, rather than anatomical modifications, has been a dominant factor in determining call structure. The varied dynamic movements were adopted in response to unique social and ecological pressures in gibbons, allowing monogamous gibbons to produce pure‐tonal melodious songs in the dense tropical forests with poor visibility. Am J Phys Anthropol, 2012. © 2012 Wiley Periodicals, Inc.     

Wound-induced Accumulation of Jasmonic Acid in Tissues of Potato Tubers

The level of jasmonic acid (JA) in medullary tissues of intactpotato tubers was found to be very low [ca. 4 ng (g fr wt)^–1].When the tissues were excised and incubated on moistened filterpaper, the level of JA began to increase rapidly, reached amaximum 4 h after excision and decreased thereafter. The maximumlevel of JA observed after 4 h was 450 ng (g fr wt)^–1.The level of JA-related compounds, as estimated by a bioassayfor potato tuber-inducing activity, showed similar fluctuationsto those in the level of JA, and neither methyl jasmonate nortuberonic acid could be detected in the tissues at any time.These results suggest that the increase in the level of JA isdue to the synthesis of JA and the decrease is due to the degradationof JA. The accumulation of JA was found only in tissues of dormanttubers that had been stored for less than 5 months and not inthose of sprouting tubers. Two inhibitors of animal phospholipaseA₂, namely, manoalide and quinacrine, did not inhibit the accumulationof JA, a result that suggests that activation of phospholipaseA₂ is not involved in the synthesis of JA. Actinomycin D andchloramphenicol also had no effect on the accumulation of JAbut cycloheximide had a considerable inhibitory effect. Theresults suggest that a newly synthesized protein(s) is the ratelimitingfactor in the biosynthesis of JA. (Received January 17, 1994; Accepted April 14, 1994)     

5′ Untranslated region of the Hsp12 gene contributes to efficient translation in Aspergillus oryzae

We describe a 5′ untranslated region (5′UTR) that dramatically increases the expression level of an exogenous gene in Aspergillus oryzae. Using a series of 5′UTR::GUS (uidA) fusion constructs, we analyzed the translation efficiency of chimeric mRNAs with different 5′UTRs at different temperatures. We found that the 5′UTR of a heat-shock protein gene, Hsp12, greatly enhanced the translation efficiency of the chimeric GUS mRNA at normal temperature (30°C). Moreover, at high temperature (37°C), the translation efficiency of the mRNA containing the Hsp12 5′UTR was far superior to that of mRNAs containing nonheat-shock 5′UTRs, resulting in much more efficient expression of GUS protein (about 20-fold higher GUS activity compared to the control construct). This 5′UTR can be used in combination with various strong promoters to enhance the expression of foreign proteins in A. oryzae.