The Effect of Output Processing on Subsequent Input Processing: A Free Recall Study

There is now growing evidence that output promotes second language acquisition. Recently, interest has been shown in examining (a) the effect of output processing on subsequent input processing, and (b) what factors mediate that effect. An experiment was conducted for two proficiency levels of Japanese learners of English under two conditions (output and non-output). First, participants in the output condition wrote a story in English based on four cartoon pictures (output task processing). Second, participants in both conditions read a model story describing the four cartoon pictures (subsequent input processing). Third, both sets of participants were asked, without any advance notice, to recall what they had read (written free recall test). Performance on the written free recall test suggests that (a) output tasks facilitated subsequent input processing, and (b) complex relationships existed among L2 proficiency levels, experiment conditions, and linguistic domains during subsequent input processing. These findings are discussed with reference to second language acquisition research. Implications for pedagogical practice are also considered

Imaging of the fluorescence spectrum of a single fluorescent molecule by prism-based spectroscopy

AbstractWe have devised a novel method to visualize the fluorescence spectrum of a single fluorescent molecule using prism-based spectroscopy. Equiping a total internal reflection microscope with a newly designed wedge prism, we obtained a spectral image of a single rhodamine red molecule attached to an essential light chain of myosin. We also obtained a spectral image of single-pair fluorescence resonance energy transfer between rhodamine red and Cy5 in a double-labeled myosin motor domain. This method could become a useful tool to investigate the dynamic processes of biomolecules at the single-molecule level

Visualization and Measurement of ATP Levels in Living Cells Replicating Hepatitis C Virus Genome RNA

Adenosine 5′-triphosphate (ATP) is the primary energy currency of all living organisms and participates in a variety of cellular processes. Although ATP requirements during viral lifecycles have been examined in a number of studies, a method by which ATP production can be monitored in real-time, and by which ATP can be quantified in individual cells and subcellular compartments, is lacking, thereby hindering studies aimed at elucidating the precise mechanisms by which viral replication energized by ATP is controlled. In this study, we investigated the fluctuation and distribution of ATP in cells during RNA replication of the hepatitis C virus (HCV), a member of the Flaviviridae family. We demonstrated that cells involved in viral RNA replication actively consumed ATP, thereby reducing cytoplasmic ATP levels. Subsequently, a method to measure ATP levels at putative subcellular sites of HCV RNA replication in living cells was developed by introducing a recently-established Förster resonance energy transfer (FRET)-based ATP indicator, called ATeam, into the NS5A coding region of the HCV replicon. Using this method, we were able to observe the formation of ATP-enriched dot-like structures, which co-localize with non-structural viral proteins, within the cytoplasm of HCV-replicating cells but not in non-replicating cells. The obtained FRET signals allowed us to estimate ATP concentrations within HCV replicating cells as ∼5 mM at possible replicating sites and ∼1 mM at peripheral sites that did not appear to be involved in HCV replication. In contrast, cytoplasmic ATP levels in non-replicating Huh-7 cells were estimated as ∼2 mM. To our knowledge, this is the first study to demonstrate changes in ATP concentration within cells during replication of the HCV genome and increased ATP levels at distinct sites within replicating cells. ATeam may be a powerful tool for the study of energy metabolism during replication of the viral genome

Increase of Pro-opiomelanocortin mRNA Prior to Tyrosinase, Tyrosinase-Related Protein 1, Dopachrome Tautomerase, Pmel-17/gp100, and P-Protein mRNA in Human Skin After Ultraviolet B Irradiation

In ultraviolet-induced tanning, the protein levels of various gene products critical for pigmentation (including tyrosinase and tyrosinase-related protein-1) are increased in response to ultraviolet B irradiation, but changes in mRNA levels of these factors have not been investigated in vivo. We have established an in situ hybridization technique to investigate mRNA levels of pro-opiomelanocortin, tyrosinase, tyrosinase-related protein-1, dopachrome tautomerase, P-protein, Pmel-17/gp100, and microphthalmia-associated transcription factor, and have analyzed the changes in mRNA levels in the ultraviolet B-exposed skin in vivo. The right or left forearm of each volunteer was irradiated with ultraviolet B, and skin biopsies were obtained at 2 and 5 d postirradiation. mRNA level of pro- opiomelanocortin was increased 2 d after ultraviolet B irradiation, and returned to a near-basal level after 5 d, whereas the mRNA levels of tyrosinase, tyrosinase-related protein-1, dopachrome tautomerase, P-protein, and Pmel-17/gp100 showed some or no increase at 2 d, but were significantly increased 5 d after ultraviolet B irradiation. Microphthalmia-associated transcription factor mRNA was slightly increased on days 2 and 5 after ultraviolet B irradiation. Our results suggest that the mechanism of the tanning response of human skin may involve the transcriptional regulation of certain pigmentary genes, and that pro-opiomelanocortin-derived melanocortins such as α-melanocyte-stimulating hormone and adrenocorticotropic hormone may play a part in regulating these genes in vivo

Plasma Nitriding-Assisted 3D Printing for Die Technology in Digital Micro-Manufacturing

A plasma nitriding-assisted 3D printing method was developed to build up the micro-punch and micro-die systems. Two dimensional punch head and core-die cavity geometries were ink-jet printed or screen-printed onto the AISI316 and SKD11 tool substrate surfaces in following their two-dimensional computer-aided design (CAD) data. The low-temperature plasma nitriding process was utilized to make nitrogen supersaturation only into the unprinted substrates. The sand-blasting and chemical etching were utilized to mechanically or chemically remove the printed parts from punch and die substrate. As sand-blasted and chemically etched AISI316 and SKD11 punches and core-dies were simply finished and used as a die set for micro-embossing, micro-piercing and micro-punching processes. In particular, a micro-pump was selected as a miniature mechanical element. Its 3D CAD geometry was sliced to 2D CAD data for each functional AISI304 stainless steel sheet. A pair of punch and die for each 2D CAD geometry for constituent sheet was prepared by the plasma nitriding-assisted 3D printing. Each sheet was punched out by using this set of punch and die to functionalize each sheet unit in correspondence to the sliced CAD data. These constituent sheets were assembled and joined to a structural unit of micro-pump

Mutations in N-terminal flanking region of blue light-sensing light-oxygen and voltage 2 (LOV2) domain disrupt its repressive activity on kinase domain in the Chlamydomonas phototropin.

Phototropin is a light-regulated kinase that mediates a variety of photoresponses such as phototropism, chloroplast positioning, and stomata opening in plants to increase the photosynthetic efficiency. Blue light stimulus first induces local conformational changes in the chromophore-bearing light-oxygen and voltage 2 (LOV2) domain of phototropin, which in turn activates the serine/threonine (Ser/Thr) kinase domain in the C terminus. To examine the kinase activity of full-length phototropin conventionally, we employed the budding yeast Saccharomyces cerevisiae. In this organism, Ser/Thr kinases (Fpk1p and Fpk2p) that show high sequence similarity to the kinase domain of phototropins exist. First, we demonstrated that the phototropin from Chlamydomonas reinhardtii (CrPHOT) could complement loss of Fpk1p and Fpk2p to allow cell growth in yeast. Furthermore, this reaction was blue light-dependent, indicating that CrPHOT was indeed light-activated in yeast cells. We applied this system to a large scale screening for amino acid substitutions in CrPHOT that elevated the kinase activity in darkness. Consequently, we identified a cluster of mutations located in the N-terminal flanking region of LOV2 (R199C, L202L, D203N/G/V, L204P, T207I, and R210H). An in vitro phosphorylation assay confirmed that these mutations substantially reduced the repressive activity of LOV2 on the kinase domain in darkness. Furthermore, biochemical analyses of the representative T207I mutant demonstrated that the mutation affected neither spectral nor multimerization properties of CrPHOT. Hence, the N-terminal flanking region of LOV2, as is the case with the C-terminal flanking Jα region, appears to play a crucial role in the regulation of kinase activity in phototropin