Optogenetic approaches for studying neuronal functions have proven their utility in the neurosciences. However, optogenetic tools capable of inducing synaptic plasticity at the level of single ...synapses have been lacking. Here, we engineered a photoactivatable (pa)CaMKII by fusing a light-sensitive domain, LOV2, to CaMKIIα. Blue light or two-photon excitation reversibly activated paCaMKII. Activation in single spines was sufficient to induce structural long-term potentiation (sLTP) in vitro and in vivo. paCaMKII activation was also sufficient for the recruitment of AMPA receptors and functional LTP in single spines. By combining paCaMKII with protein activity imaging by 2-photon FLIM-FRET, we demonstrate that paCaMKII activation in clustered spines induces robust sLTP via a mechanism that involves the actin-regulatory small GTPase, Cdc42. This optogenetic tool for dissecting the function of CaMKII activation (i.e., the sufficiency of CaMKII rather than necessity) and for manipulating synaptic plasticity will find many applications in neuroscience and other fields.
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► LiBs and NiMH batteries currently share more than 80% of batteries market. ► Recycling of these cells is an environmental and economical necessity. ► Cost, inflexibility, and losses ...of valuables are the major recycling drawbacks. ► A flowsheet for recycling mixed stream LiBs and NiMH battery scrap was proposed. ► The economic effect of pre-treatment on battery recycling was evaluated.
Production of LiBs and NiMH batteries is expected to increase rapidly due to the soaring price of oil and gas which increases interest in renewable energy as well as the introduction of hybrid vehicles (HVs), and electric vehicles (EVs) which used secondary batteries as an effective energy storage device. Development of an efficient recycling scheme to recover the valuable parts and safely dispose the harmful one at batteries end life is a necessity. The challenge, however, is how to recover all the valuable metals without sacrificing the economics of recycling process.
Several LiBs and NiMH batteries recycling processes have been developed in recent years. A review of these processes and their development timeline was presented in this paper. It was found that the major drawback of these recycling processes is the losses of some of batteries valuable parts since these recycling processes are not originally developed for this type of batteries. Also, some of these processes are expensive and designed for specific types of batteries which ignore contamination of recycling stream with impurities and other type of batteries.
Using minerals processing operations such as grinding, sieving, magnetic, electrostatic, and gravity separations to liberate batteries electrodal materials and concentrate valuable metals is critical step in any recycling process. This may be due to the simplicity, efficiency, flexibility, and high throughput of these separation processes. The literature showed that applying these processes reduces the volume of LiBs and NiMH scrap, liberates their valuables, reduces the need for leachate purification in hydrometallurgical process, and facilitates the decomposing of battery’s electrolyte. Based on these results a flowsheet to recycle mixed stream LiBs, and NiMH battery scrap was proposed.
Fluorescence lifetime imaging microscopy (FLIM)-based Förster resonance energy transfer (FRET) measurement (FLIM-FRET) is one of the powerful methods for imaging of intracellular protein activities ...such as protein-protein interactions and conformational changes. Here, using saturation mutagenesis, we developed a dark yellow fluorescent protein named ShadowY that can serve as an acceptor for FLIM-FRET. ShadowY is spectrally similar to the previously reported dark YFP but has a much smaller quantum yield, greater extinction coefficient, and superior folding property. When ShadowY was paired with mEGFP or a Clover mutant (Clover
) and applied to a single-molecule FRET sensor to monitor a light-dependent conformational change of the light-oxygen-voltage domain 2 (LOV2) in HeLa cells, we observed a large FRET signal change with low cell-to-cell variability, allowing for precise measurement of individual cell responses. In addition, an application of ShadowY to a separate-type Ras FRET sensor revealed an EGF-dependent large FRET signal increase. Thus, ShadowY in combination with mEGFP or Clover
is a promising FLIM-FRET acceptor.
Measurement of Förster resonance energy transfer by fluorescence lifetime imaging microscopy (FLIM-FRET) is a powerful method for visualization of intracellular signaling activities such as ...protein-protein interactions and conformational changes of proteins. Here, we developed a dark green fluorescent protein (ShadowG) that can serve as an acceptor for FLIM-FRET. ShadowG is spectrally similar to monomeric enhanced green fluorescent protein (mEGFP) and has a 120-fold smaller quantum yield. When FRET from mEGFP to ShadowG was measured using an mEGFP-ShadowG tandem construct with 2-photon FLIM-FRET, we observed a strong FRET signal with low cell-to-cell variability. Furthermore, ShadowG was applied to a single-molecule FRET sensor to monitor a conformational change of CaMKII and of the light oxygen voltage (LOV) domain in HeLa cells. These sensors showed reduced cell-to-cell variability of both the basal fluorescence lifetime and response signal. In contrast to mCherry- or dark-YFP-based sensors, our sensor allowed for precise measurement of individual cell responses. When ShadowG was applied to a separate-type Ras FRET sensor, it showed a greater response signal than did the mCherry-based sensor. Furthermore, Ras activation and translocation of its effector ERK2 into the nucleus could be observed simultaneously. Thus, ShadowG is a promising FLIM-FRET acceptor.
In copper (Cu) smelters, precious metals in recycled materials are distributed into electrolytic Cu slime. During the precious metal refining process, rhodium (Rh) and ruthenium (Ru) are concentrated ...from the slime into residues containing selenium (Se) and tellurium (Te). In this study, new methods were developed for refining Rh and Ru by obtaining aqueous solutions of Rh and Ru and separating Se and Te using a hydrometallurgical process followed by a pyrometallurgical process. In the hydrometallurgical process, Se and Te are dissolved by air oxidation in a sodium hydroxide (NaOH) solution, and Cu is dissolved using diluted sulfuric acid (H2SO4). Se and Te are removed by dissolution and separated from the undissolved Rh and Ru. In the pyrometallurgical process, the residual Se and Te in the Rh and Ru concentrates obtained from the hydrometallurgical process are chlorinated and separated by volatilization. Rh, Ru, Se, and Te are chlorinated by heating to 1000–1100K in chlorine (Cl2) gas. Volatilization was used for separation of Se and Te from Rh and Ru because the vapor pressures of Se and Te chlorides are higher than those of Rh and Ru chlorides. Because the chlorides of Rh and Ru have poor solubility, they are mixed with sodium chloride (NaCl), heated, and converted to soluble complex salts containing Rh and Ru, which are later dissolved in water to obtain an aqueous solution containing concentrated Rh and Ru.
•We developed a method for refining Rh and Ru from electrolytic copper slime.•Se and Te are dissolved using a sodium hydroxide solution and air oxidation.•The residual Se and Te are volatilized by heating in chlorine gas.•The chlorides of Rh and Ru are converted into soluble sodium salts of Rh and Ru.•The salts were dissolved in water to obtain concentrated Rh and Ru solutions.
Organophosphate esters are used as additives in flame retardants and plasticizers, and they are ubiquitous in the indoor environment. Phosphorus flame retardants (PFRs) are present in residential ...dust, but few epidemiological studies have assessed their impact on human health. We measured the levels of 11 PFRs in indoor floor dust and multi‐surface dust in 182 single‐family dwellings in Japan. We evaluated their correlations with asthma and allergies of the inhabitants. Tris(2‐butoxyethyl) phosphate was detected in all samples (median value: 580 μg/g in floor dust, 111 μg/g in multi‐surface dust). Tris(2‐chloro‐iso‐propyl) phosphate (TCIPP) was detected at 8.69 μg/g in floor dust and 25.8 μg/g in multi‐surface dust. After adjustment for potential confounders, significant associations were found between the prevalence of atopic dermatitis and the presence of TCIPP and tris(1,3‐dichloro‐2‐propyl) phosphate in floor dust per log10‐unit, odds ratio (OR): 2.43 and 1.84, respectively. Tributyl phosphate was significantly associated with the prevalence of asthma (OR: 2.85 in floor dust, 5.34 in multi‐surface dust) and allergic rhinitis (OR: 2.55 in multi‐surface dust). PFR levels in Japan were high compared with values reported previously for Europe, Asia‐Pacific, and the USA. Higher levels of PFRs in house dust were related to the inhabitants' health status.
Electron tomography of the plasma membrane (PM) identified several layers of cortical actin meshwork running parallel to the PM cytoplasmic surface throughout the PM. Here, cortical actin structures ...and dynamics were examined in living cells, using super-resolution microscopy, with (x,y)- and z-resolutions of ~140 and ~400 nm, respectively, and single-molecule imaging. The super-resolution microscopy identified sub-micron-sized actin clusters that appeared identical by both phalloidin post-fixation staining and Lifeact-mGFP expression followed by fixation, and therefore, these actin clusters were named "actin-pl-clusters". In live cells, the actin-pl-clusters visualized by Lifeact-mGFP linked two or more actin filaments in the fine actin meshwork, acting as a node of the meshwork, and dynamically moved on/along the meshwork in a myosin II-dependent manner. Their formation depended on the Arp2/3 activities, suggesting that the movements could involve both the myosin motor activity and actin polymerization-depolymerization. The actin-pl-clusters differ from the actin nodes/asters found previously after latrunculin treatments, since myosin II and filamin A were not colocalized with the actin-pl-clusters, and the actin-pl-clusters were much smaller than the previously reported nodes/asters. The Lifeact linked to a fluorescently-labeled transmembrane peptide from syntaxin4 (Lifeact-TM) expressed in the PM exhibited temporary immobilization in the PM regions on which actin-pl-clusters and stress fibers were projected, showing that ≥66% of actin-pl-clusters and 89% of stress fibers were located in close proximity (within 3.5 nm) to the PM cytoplasmic surface. Podosome-associated cytoplasmic proteins, Tks4, Tks5, cortactin, and N-WASP, were transiently recruited to actin-pl-clusters, and thus, we propose that actin-pl-clusters also represent "actin podosome-like clusters".
Chromosome 5p partial monosomy (5p-syndrome) and chromosome 6p partial trisomy are chromosomal abnormalities that result in a variety of symptoms, but liver dysfunction is not normally one of them. ...Alagille syndrome (OMIM #118450) is a multisystem disorder that is defined clinically by hepatic bile duct paucity and cholestasis, in association with cardiac, skeletal, and ophthalmologic manifestations, and characteristic facial features. Alagille syndrome is caused by mutations in JAG1 on chromosome 20 or NOTCH2 on chromosome 1. Here, we report a preterm infant with karyotype 46,XX,der(5)t(5,6)(p15.2;p22.3) and hepatic dysfunction, who was diagnosed as having incomplete Alagille syndrome.
The Japanese infant was diagnosed based on the cardiac abnormalities, ocular abnormalities, characteristic facial features, and liver pathological findings. Analysis of the JAG1 and NOTCH sequences failed to detect any mutations in these genes.
These results suggest that, besides the genes that are known to be responsible for Alagille syndrome, other genetic mutations also may cause Alagille syndrome.
Elucidating temporal windows of signaling activity required for synaptic and behavioral plasticity is crucial for understanding molecular mechanisms underlying these phenomena. Here, we developed ...photoactivatable autocamtide inhibitory peptide 2 (paAIP2), a genetically encoded, light-inducible inhibitor of CaMKII activity. The photoactivation of paAIP2 in neurons for 1–2 min during the induction of LTP and structural LTP (sLTP) of dendritic spines inhibited these forms of plasticity in hippocampal slices of rodents. However, photoactivation ∼1 min after the induction did not affect them, suggesting that the initial 1 min of CaMKII activation is sufficient for inducing LTP and sLTP. Furthermore, the photoactivation of paAIP2 expressed in amygdalar neurons of mice during an inhibitory avoidance task revealed that CaMKII activity during, but not after, training is required for the memory formation. Thus, we demonstrated that paAIP2 is useful to elucidate the temporal window of CaMKII activation required for synaptic plasticity and learning.
•PaAIP2 is a genetically encoded, light-inducible inhibitor of CaMKII•PaAIP2 inhibits CaMKII with high specificity and with second-to-minute temporal resolution•PaAIP2 measures the temporal window of CaMKII required for synaptic plasticity•PaAIP2 measures the kinetics of CaMKII activity during learning and memory
Murakoshi, Shin et al. developed a light-inducible inhibitor of CaMKII and demonstrated that a brief illumination of blue light to neurons expressing this inhibitor blocks synaptic plasticity and learning.
Genetically encoded fluorescence resonance energy transfer (FRET) biosensors have been successfully used to visualize protein activity in living cells. The sensitivity and accuracy of FRET ...measurements directly depend on biosensor folding efficiency, expression pattern, sensitivity, and dynamic range. Here, to improve the folding efficiency of the Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) FRET biosensor, we amplified the association domain of the CaMKIIα gene using error-prone polymerase chain reaction (PCR) and fused it to the N-terminus of mCherry in a bacterial expression vector. We also created an Escherichia coli expression library based on a previously reported fluorescent protein folding reporter method, and found a bright red fluorescent colony that contained the association domain with four mutations (F394L, I419V, A430T, and I434T). In vitro assays using the purified mutant protein confirmed improved folding kinetics of the downstream fluorescent protein, but not of the association domain itself. Furthermore, we introduced these mutations into the previously reported CaMKIIα FRET sensor and monitored its Ca2+/calmodulin-dependent activation in HeLa cells using 2-photon fluorescence lifetime imaging microscopy (2pFLIM), and found that the expression pattern and signal reproducibility of the mutant sensor were greatly improved without affecting the autophosphorylation function and incorporation into oligomeric CaMKIIα. We believe that our improved CaMKIIα FRET sensor would be useful in various types of cells and tissues, providing data with high accuracy and reproducibility. In addition, the method described here may also be applicable for improving the performance of all currently available FRET sensors.
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