Step-specific investigation of SNARE-mediated membrane fusion

Cholesterol is a major component of biological membranes and is known to affect vesicle fusion. However, the mechanism by which cholesterol modulates SNARE-dependent intracellular fusion is not well understood. First, using the fluorescence assay and employing dye-labeled SNAREs and the fluorescent lipids on yeast post golgi trafficking SNARE-mediated model membrane fusion, we dissected cholesterol effects on individual fusion steps including SNARE complex formation, hemifusion, pore formation, and pore dilation. At physiological high concentrations, cholesterol stimulated hemifusion as much as 30-fold, but its stimulatory effect diminished to 10- and 3-folds for subsequent pore formation and pore expansion at 40 mole%, respectively. Therefore, the results show that cholesterol serves as a strong stimulator for hemifusion but acts as mild stimulators for pore opening and expansion. Strong stimulation of hemifusion and mild stimulation of pore formation are consistent with the fusion model based on the intrinsic negative curvature of cholesterol. However, even a milder effect of cholesterol on pore expansion is contradictory to such a simple curvature-based prediction. Thus, we speculate that cholesterol also affects the conformation of the transmembrane domains of SNAREs, which modulates the fusion kinetics. Furthermore, we investigated the effect of cholesterol in the specific steps of fusion: docking, lipid mixing, spontaneous fusion, and Ca2+-triggered fusion using single vesicular study. Cholesterol enhances the kinetics of all these steps. Cholesterol on both sides of the vesicles is required for content mixing. In addition, cholesterol helped Syntaxins assemble to form higher oligomers meanwhile the higher oligomer ratio of Vamp was not affected by cholesterol content. α-Synuclein (α-Syn), a major component of Lewy bodies that are considered as a hallmark of Parkinson\u27s disease (PD), has been implicated in neuroexocytosis. Overexpression of α-Syn decreases the neurotransmitter release. However, the mechanism by which α-Syn inhibits the neurotransmitter release is still unclear. Here, we investigated the effect of α-Syn on SNARE assembly and SNARE-dependent liposome fusion using fluorescence methods. The results show that α-Syn inhibits bulk lipid mixing. Furthermore, mutants linked to familial PD A30P, A53T, and E46K are less effective than, similar to, more than the wild-type in fusion-inhibition, respectively, correlating well with the rank order of their individual membrane affinities. A negatively-charged lipid that strongly favors α-Syn\u27s membrane binding is also required for the fusion-inhibiting function. Finally, the single-vesicle fusion assay reveals that α-Syn specifically inhibits vesicle docking, without interfering with lipid mixing and pore opening. Thus, α-Syn may inhibit SNARE-dependent vesicle docking through membrane binding

Fabrication of three-dimensional suspended, interlayered and hierarchical nanostructures by accuracy-improved electron beam lithography overlay

Nanofabrication techniques are essential for exploring nanoscience and many closely related research fields such as materials, electronics, optics and photonics. Recently, three-dimensional (3D) nanofabrication techniques have been actively investigated through many different ways, however, it is still challenging to make elaborate and complex 3D nanostructures that many researchers want to realize for further interesting physics studies and device applications. Electron beam lithography, one of the two-dimensional (2D) nanofabrication techniques, is also feasible to realize elaborate 3D nanostructures by stacking each 2D nanostructures. However, alignment errors among the individual 2D nanostructures have been difficult to control due to some practical issues. In this work, we introduce a straightforward approach to drastically increase the overlay accuracy of sub-20 nm based on carefully designed alignmarks and calibrators. Three different types of 3D nanostructures whose designs are motivated from metamaterials and plasmonic structures have been demonstrated to verify the feasibility of the method, and the desired result has been achieved. We believe our work can provide a useful approach for building more advanced and complex 3D nanostructures.114sciescopu

Deep sub-wavelength nanofocusing of UV-visible light by hyperbolic metamaterials

Confining light into a sub-wavelength area has been challenging due to the natural phenomenon of diffraction. In this paper, we report deep sub-wavelength focusing via dispersion engineering based on hyperbolic metamaterials. Hyperbolic metamaterials, which can be realized by alternating layers of metal and dielectric, are materials showing opposite signs of effective permittivity along the radial and the tangential direction. They can be designed to exhibit a nearly-flat open isofrequency curve originated from the large-negative permittivity in the radial direction and small-positive one in the tangential direction. Thanks to the ultraflat dispersion relation and curved geometry of the multilayer stack, hyperlens can magnify or demagnify an incident beam without diffraction depending on the incident direction. We numerically show that hyperlens-based nanofocusing device can compress a Gaussian beam down to tens-of-nanometers of spot size in the ultraviolet (UV) and visible frequency range. We also report four types of hyperlenses using different material combinations to span the entire range of visible frequencies. The nanofocusing device based on the hyperlens, unlike conventional lithography, works under ordinary light source without complex optics system, giving rise to practical applications including truly nanoscale lithography and deep sub-wavelength scale confinement.1165Nsciescopu

12-and 24-Month-Old Infants' Search Behavior Under Informational Uncertainty

Infants register and react to informational uncertainty in the environment. They also form expectations about the probability of future events as well as update the expectation according to changes in the environment. A novel line of research has started to investigate infants' and toddlers' behavior under uncertainty. By combining these research areas, the present research investigated 12- and 24-month-old infants' searching behaviors under varying degree of informational uncertainty. An object was hidden in one of three possible locations and probabilistic information about the hiding location was manipulated across trials. Infants' time delay in search initiation for a hidden object linearly increased across the level of informational uncertainty. Infants' successful searching also varied according to probabilistic information. The findings suggest that infants modulate their behaviors based on probabilistic information. We discuss the possibility that infants' behavioral reaction to the environmental uncertainty constitutes the basis for the development of subjective uncertainty

Metacognitive variety, from Inner Mongolian Buddhism to Post-Truth

In this chapter I present a case study based on ethnographic research carried out in Inner Mongolia, northern China. A Buddhist teacher and his students have subtly different metacognitive relationships to Buddhism and their practice and knowledge are dramatically different as a result. I offer this case study as an example of metacognitive variety, and argue that a similar approach is required to understand other cases in which people reflect, and attempt to act, on their own cognition and cognitive experience, including the transformations that have been described as 'post-truth'. In conclusion I make some methodological remarks about the study of metacognition through ethnography

Young Children's Sensitivity to Their Own Ignorance in Informing Others

Prior research suggests that young children selectively inform others depending on others' knowledge states. Yet, little is known whether children selectively inform others depending on their own knowledge states. To explore this issue, we manipulated 3-to 4-year-old children's knowledge about the content of a box and assessed the impact on their decisions to inform another person. Moreover, we assessed the presence of uncertainty gestures while they inform another person in light of the suggestions that children's gestures reflect early developing, perhaps transient, epistemic sensitivity. Finally, we compared children's performance in the informing context to their explicit verbal judgment of their knowledge states to further confirm the existence of a performance gap between the two tasks. In their decisions to inform, children tend to accurately assess their ignorance, whereas they tend to overestimate their own knowledge states when asked to explicitly report them. Moreover, children display different levels of uncertainty gestures depending on the varying degrees of their informational access. These findings suggest that children's implicit awareness of their own ignorance may be facilitated in a social, communicative context

Knowing minds: Linking early perspective taking and later metacognitive insight

Recent metacognitive research using a partial knowledge task indicates that a firm understanding of ‘knowing about knowing’ develops surprisingly late, at around 6 years of age. To reveal the mechanisms subserving this development, the partial knowledge task was used in a longitudinal study with 67 children (33 girls) as an outcome measure at 5;9 (years;months). In addition, first- and second-order false belief was assessed at 4;2, 5;0, and 5;9. At 2;6, perspective taking and executive abilities were evaluated. Metacognition at 5;9 was correlated with earlier theory of mind and perspective taking – even when verbal intelligence and executive abilities were partialled out. This highlights the importance of perspective taking for the development of an understanding of one’s own mind

Metacognition of curiosity: people underestimate the seductive lure of non-instrumental information

Curiosity - the desire to seek information - is fundamental for learning and performance. Studies on curiosity have shown that people are intrinsically motivated to seek information even if it does not bring an immediate tangible benefit (i.e., non-instrumental information), but little is known as to whether people have the metacognitive capability to accurately monitor their motivation for seeking information. We examined whether people can accurately predict their own non-instrumental information-seeking behavior. Across six experiments (Experiments 1A-1E and 2, total N = 579), participants predicted that they would engage in information-seeking behavior less frequently than they actually did, suggesting that people tend to underestimate the motivational lure of curiosity. Overall, there was no consistent statistical evidence that this underestimation was altered by contextual factors (e.g., the cost to seek information). These results were consistent with the theoretical account that it is difficult for people to make sense of the internally rewarding value of information in advance

Thermally robust ring-shaped chromium perfect absorber of visible light

A number of light-absorbing devices based on plasmonic materials have been reported, and their device efficiencies (or absorption) are high enough to be used in real-life applications. Many light-absorbing applications such as thermophotovoltaics and energy-harvesting and energy-sensing devices usually require high-temperature durability; unfortunately, noble metals used for plasmonics are vulnerable to heat. As an alternative, refractory plasmonics has been introduced using refractory metals such as tungsten (3422 degrees C) and transition metal nitrides such as titanium nitride (2930 degrees C). However, some of these materials are not easy to handle for device fabrications owing to their ultra-high melting point. Here, we propose a light absorber based on chromium (Cr), which is heat tolerant due to its high melting temperature (1907 degrees C) and is compatible with fabrication using conventional semiconductor manufacturing processes. The fabricated device has >95% average absorption of visible light (500-800 nm) independent of polarization states. To verify its tolerance of heat, the absorber was also characterized after annealing at 600 degrees C. Because of its compactness, broadband operational wavelength, and heat tolerance, this Cr perfect absorber will have applications in high-temperature photonic devices such as solar thermophotovoltaics.111Ysciescopu