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Far-red and near-infrared (NIR) genetically encoded calcium ion (Ca2+ ) indicators (GECIs) are powerful tools for in vivo and multiplexed imaging of neural activity and cell signaling. Inspired by a previous report to engineer a far-red fluorescent protein (FP) from a biliverdin (BV)-binding NIR FP, we have developed a far-red fluorescent GECI, designated iBB-GECO1, from a previously reported NIR GECI. iBB-GECO1 exhibits a relatively high molecular brightness, an inverse response to Ca2+ with ΔF/Fmin  = -13, and a near-optimal dissociation constant (Kd ) for Ca2+ of 105 nM. We demonstrate the utility of iBB-GECO1 for four-color multiplexed imaging in MIN6 cells and five-color imaging in HEK293T cells. Like other BV-binding GECIs, iBB-GECO1 did not give robust signals during in vivo imaging of neural activity in mice, but did provide promising results that will guide future engineering efforts. SIGNIFICANCE: Genetically encoded calcium ion (Ca2+ ) indicators (GECIs) compatible with common far-red laser lines (~630-640 nm) on commercial microscopes are of critical importance for their widespread application to deep-tissue multiplexed imaging of neural activity. In this study, we engineered a far-red excitable fluorescent GECI, designated iBB-GECO1, that exhibits a range of preferable specifications such as high brightness, large fluorescence response to Ca2+ , and compatibility with multiplexed imaging in mammalian cells.

Alzheimer's disease is an age-dependent progressive neurodegenerative disorder that results in impairments of memory and cognitive function. It is hypothesized that oligonol has ameliorative effects on memory impairment and reduced cognitive functions in mice with Alzheimer's disease induced by amyloid β(25-35) (Aβ(25-35)) injection. The protective effect of an oligonol against Aβ(25-35)-induced memory impairment was investigated in an in vivo Alzheimer's mouse model. The aggregation of Aβ25-35 was induced by incubation at 37°C for 3 days before injection into mice brains (5 nmol/mouse), and then oligonol was orally administered at 100 and 200 mg/kg of body weight for 2 weeks. Memory and cognition were observed in T-maze, object recognition, and Morris water maze tests. The group injected with Aβ(25-35) showed impairments in both recognition and memory. However, novel object recognition and new route awareness abilities were dose dependently improved by the oral administration of oligonol. In addition, the results of the Morris water maze test indicated that oligonol exerted protective activity against cognitive impairment induced by Aβ(25-35). Furthermore, nitric oxide formation and lipid peroxidation were significantly elevated by Aβ(25-35), whereas oligonol treatment significantly decreased nitric oxide formation and lipid peroxidation in the brain, liver, and kidneys. The present results suggest that oligonol improves Aβ(25-35)-induced memory deficit and cognition impairment.

Photosystem II (PSII) catalyses the oxidation of water through a four-step cycle of Si states (i = 0–4) at the Mn4CaO5 cluster1–3, during which an extra oxygen (O6) is incorporated at the S3 state to form a possible dioxygen4–7. Structural changes of the metal cluster and its environment during the S-state transitions have been studied on the microsecond timescale. Here we use pump-probe serial femtosecond crystallography to reveal the structural dynamics of PSII from nanoseconds to milliseconds after illumination with one flash (1F) or two flashes (2F). YZ, a tyrosine residue that connects the reaction centre P680 and the Mn4CaO5 cluster, showed structural changes on a nanosecond timescale, as did its surrounding amino acid residues and water molecules, reflecting the fast transfer of electrons and protons after flash illumination. Notably, one water molecule emerged in the vicinity of Glu189 of the D1 subunit of PSII (D1-E189), and was bound to the Ca2+ ion on a sub-microsecond timescale after 2F illumination. This water molecule disappeared later with the concomitant increase of O6, suggesting that it is the origin of O6. We also observed concerted movements of water molecules in the O1, O4 and Cl-1 channels and their surrounding amino acid residues to complete the sequence of electron transfer, proton release and substrate water delivery. These results provide crucial insights into the structural dynamics of PSII during S-state transitions as well as O–O bond formation.Serial femtosecond crystallography reveals the structural dynamics of photosystem II during the S-state transitions that produce dioxygen, providing insight into electron transfer, water insertion, proton release and O–O bond formation on sub-microsecond timescales. [ABSTRACT FROM AUTHOR]

West Nile virus (WNV) poses a serious threat to public health, especially to the elderly and the immuno-compromised. Neither vaccines nor treatments ate available for humans. Active hexose correlated compound (AHCC) is ah extract of Lentinula edodes of the Basidiomycetefamily of fungi rich in [alpha]-glucans. In this study, we evaluated the effect of AHCC on host susceptibility in the murine model of WNV infection. Mice orally administered with AHCC (600 mg/kg) every other day for 1 wk before and at d 1 and 3 postinfection were assessed using viremia levels, survival rate, and protective immunity. AHCC administration in young (6- to 8-wk-old) mice attenuated viremia and mortality following lethal WNV infection. WNV-specific IgM and IgG production and [gamma][delta] T cell expansion were also enhanced in these mice. Aged (21- to 22-mo-old) mice were more susceptible to WNV infection than young mice, partially due to the dysfunction of [gamma][delta] T cell subsets. AHCC administration in aged mice enhanced the protective V[gamma][1.sup.+] T cell response as well as WNV-specific IgG but not IgM antibodies production. AHCC administration in aged mice attenuated viremia levels but led to no difference in mortality rate. Overall, our data suggests that AHCC enhances protective host immune responses against WNV infection in young and aged mice. Dietary supplementation with AHCC may be potentially immunotherapeutic for WNV-susceptible populations.