Imaging changes in membrane potential using genetically encoded fluorescent voltage indicators (GEVIs) has great potential for monitoring neuronal activity with high spatial and temporal resolution. Brightness and photostability of fluorescent proteins and rhodopsins have limited the utility of existing GEVIs. We engineered a novel GEVI, 'Voltron', that utilizes bright and photostable synthetic dyes instead of protein-based fluorophores, extending the combined duration of imaging and number of neurons imaged simultaneously by more than tenfold relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies. In mouse cortex, Voltron allowed single-trial recording of spikes and subthreshold voltage signals from dozens of neurons simultaneously, over 15 minutes of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of spike timing with behavior.Animal behavior is produced by patterns of neuronal activity that span a wide range of spatial and temporal scales. To understand how neural circuits mediate behavior thus requires high-speed recording from ensembles of neurons for long periods of time.Although the activity of large numbers of neurons can now be routinely recorded using genetically encoded calcium indicators (GECIs) (1), the slow kinetics of calcium signals complicate the measurement of action potentials, and sub-threshold voltage signals are missed entirely (1-3). Voltage imaging using genetically encoded voltage indicators (GEVIs) can overcome these challenges, enabling imaging of fast spikes and subthreshold dynamics in genetically defined neurons (4, 5). The high imaging speed and excitation intensity required for voltage imaging, combined with the smaller volume of the cellular membrane, place increased demands on voltage indicators relative to GECIs.Extant GEVIs rely on fluorescence from either microbial rhodopsins (6-8) or fluorescent proteins (FPs) (9-13). These fluorophores lack the brightness and photostability to allow in vivo voltage imaging from large fields of view over timescales of many behavioral events, precluding the millisecond-timescale interrogation of neural circuits. Recent development of improved rhodamine dyes such as the Janelia Fluor ® (JF) dyes enable their use in complex biological experiments due to their high brightness and photostability (14), compatibility with self-labeling protein tags (15, 16), and the ability to traverse the blood-brain barrier for in vivo delivery (17). Here we describe a 'chemigenetic' GEVI scaffold-termed 'Voltron'-which incorporates these synthetic fluorophore dyes. Voltron provides an increased photon yield that enables in vivo imaging of neuronal spiking and sub-threshold voltage signals in model organisms with order-of-magnitude improvement in the number of neurons imaged simultaneously over substantially longer durations.Our design for a chemigenetic voltage indicator combines a voltage-sensitive microbial rhodopsin domain (6, 7, 11) with a self-labeling protein tag domain (F...

Shikonin, a major ingredient in the Chinese traditional herb Lithospermum erythrorhixon, exhibits multiple biological functions including antimicrobial, anti-inflammatory, and antitumor effects. In this study, we delineated the molecular mechanisms of shikonin in the apoptosis of 143B osteosarcoma cells. Shikonin reduced the cell viability of 143B cells in a dose-and time-dependent manner. The IC 50 at 24 h and 48 h for 143B cells was 4.55 and 2.01 m mM, respectively. A significantly elicited hypodiploid cell population was found in cells treated with 2, 4, and 8 m mM shikonin for 24 h. Moreover, treatment with shikonin induced reactive oxygen species (ROS) generation, increased extracellular signal-regulated kinase (ERK) phosphorylation, decreased B-cell lymphoma-2 (Bcl2) expression, and was accompanied by poly(ADP-ribose) polymerase (PARP) cleavage. Pretreatment with the antioxidant agent N-acetyl cysteine (NAC) not only reversed shikonin-induced ROS generation but also significantly attenuated the cytotoxic effects of shikonin in 143B cells. Furthermore, NAC attenuated shikonin-induced ERK phosphorylation. Taken together, our results reveal that shikonin increased ROS generation and ERK activation, and reduced Bcl2, which consequently caused the cells to undergo apoptosis. Therefore, shikonin may be a promising chemotherapeutic agent for osteosarcoma treatment.

BackgroundAcetaminophen (APAP) overdose causes acute liver failure (ALF) in animals and humans via the rapid depletion of intracellular glutathione (GSH) and the generation of excess reactive oxygen species (ROS) that damage hepatocytes. Stem cell therapy is a potential treatment strategy for ALF.MethodsWe isolated mesenchymal stem cells (MSCs) from mice omentum adipose tissue-derived stem cells (ASCs) and transplanted them into a mouse model of APAP-induced ALF to explore their therapeutic potential. In addition, we performed in vitro co-culture studies with omentum-derived ASCs and primary isolated hepatocytes to demonstrate the hepatoprotective effect of omentum-derived ASCs on hepatocytes that were subjected to APAP-induced damage.ResultASC transplantation significantly improved the survival rate of mice with ALF and attenuated the severity of APAP-induced liver damage by suppressing cytochrome P450 activity to reduce the accumulation of toxic nitrotyrosine and the upregulation of NF-E2-related factor 2 (Nrf2) expression, resulting in an increase in the subsequent antioxidant activity. These effects protected the hepatocytes from APAP-induced damage through the suppression of downstream MAPK signal activation and inflammatory cytokine production.Conclusionsour results demonstrate that omentum-derived ASCs are an alternative source of ASCs that regulate the antioxidant response and may represent a beneficial therapeutic strategy for ALF.

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