Red-Shifted Voltage-Sensitive Fluorescent Proteins

SummaryElectrical signals generated by nerve cells provide the basis of brain function. Whereas single or small numbers of cells are easily accessible using microelectrode recording techniques, less invasive optogenetic methods with spectral properties optimized for in vivo imaging are required for elucidating the operation mechanisms of neuronal circuits composed of large numbers of neurons originating from heterogeneous populations. To this end, we generated and characterized a series of genetically encoded voltage-sensitive fluorescent proteins by molecular fusion of the voltage-sensing domain of Ci-VSP (Ciona intestinalis voltage sensor-containing phosphatase) to red-shifted fluorescent protein operands. We show how these indicator proteins convert voltage-dependent structural rearrangements into a modulation of fluorescence output and demonstrate their applicability for optical recording of individual or simultaneous electrical signals in cultured hippocampal neurons at single-cell resolution without temporal averaging

Engineering of a Genetically Encodable Fluorescent Voltage Sensor Exploiting Fast Ci-VSP Voltage-Sensing Movements

Ci-VSP contains a voltage-sensing domain (VSD) homologous to that of voltage-gated potassium channels. Using charge displacement (‘gating’ current) measurements we show that voltage-sensing movements of this VSD can occur within 1 ms in mammalian membranes. Our analysis lead to development of a genetically encodable fluorescent protein voltage sensor (VSFP) in which the fast, voltage-dependent conformational changes of the Ci-VSP voltage sensor are transduced to similarly fast fluorescence read-outs

Accelerated evaluation method for the SRAM cell write margin using word line voltage shift

An accelerated evaluation method for the SRAM cell write margin is proposed based on the conventional Write Noise Margin (WNM) definition. The WNM is measured under a lower word line voltage than the power supply voltage VDD. A lower word line voltage is used because the access transistor operates in the saturation mode over a wide range of threshold voltage variation. The final WNM at the VDD word line voltage, the Accelerated Write Noise Margin (AWNM), is obtained by shifting the measured WNM at the lower word line voltage. The amount of WNM shift is determined from the WNM dependence on the word line voltage. As a result, the cumulative frequency of the AWNM displays a normal distribution. A normal distribution of the AWNM drastically improves development efficiency, because the write failure probability can be estimated by a small number of samples. Effectiveness of the proposed method is verified using the Monte Carlo simulation. © 2011 IEEE

Engineering and Characterization of an Enhanced Fluorescent Protein Voltage Sensor

BACKGROUND: Fluorescent proteins have been used to generate a variety of biosensors to optically monitor biological phenomena in living cells. Among this class of genetically encoded biosensors, reporters for membrane potential have been a particular challenge. The use of presently known voltage sensor proteins is limited by incorrect subcellular localization and small or absent voltage responses in mammalian cells. RESULTS: Here we report on a fluorescent protein voltage sensor with superior targeting to the mammalian plasma membrane and high responsiveness to membrane potential signaling in excitable cells. CONCLUSIONS AND SIGNIFICANCE: This biosensor, which we termed VSFP2.1, is likely to lead to new methods of monitoring electrically active cells with cell type specificity, non-invasively and in large numbers, simultaneously

High Energy Particle Measurements during Long Discharge in LHD

The spatial resolved energy spectra can be observed during a long discharge of NBI plasma bycontinuously scanning the neutral particle analyzer. In these discharges, the plasmas are initiated by the ECH heating, after that NBI#2 (Co-injection) sustains the plasma during 40-60 seconds. The scanned pitch angle is from 44 degrees to 74 degrees. The injected neutral beam (hydrogen) energy of NBI#2 is only 130 keV because the original ion source polarity is negative. The shape of spectra is almost similar from 44 degrees to 53 degrees. However the spectra from 55 degrees are strongly varied. It reflects the injection pitch angle of the beam according to the simulation (53 degrees ot R* = 3.75 m in simulation). The beam keeps the pitch angle at incidence until the beam energy becomes to the energy, which the pitch angle scattering is occurred by the energy loss due to the electron collision. The low flux region can be observed around 10-15 keV, which is 15 times of the electron temperature. The energy region may be equal to the energy at which the pitch angle scattering is occurred. At the energy, the particle is scattered by the collision with the plasma ions and some of particles may run away from the plasma because they have a possibility to enter the loss cone. According to the simulation, the loss cone can be expected at the 10 keV with the small angular dependence. The depth of the loss cone is deep at the small pitch angle. The hollow in the spectrum may be concluded to be the loss cone as the tendency is almost agreed with the experimental result

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target

The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force

「コロナ制圧タスクフォース」COVID-19患者由来の血液細胞における遺伝子発現の網羅的解析 --重症度に応じた遺伝子発現の変化には、ヒトゲノム配列の個人差が影響する--. 京都大学プレスリリース. 2022-08-23.Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection

Generation of Flag/DYKDDDDK Epitope Tag Knock-In Mice Using i-GONAD Enables Detection of Endogenous CaMKIIα and β Proteins

Specific antibodies are necessary for cellular and tissue expression, biochemical, and functional analyses of protein complexes. However, generating a specific antibody is often time-consuming and effort-intensive. The epitope tagging of an endogenous protein at an appropriate position can overcome this problem. Here, we investigated epitope tag position using AlphaFold2 protein structure prediction and developed Flag/DYKDDDDK tag knock-in CaMKIIα and CaMKIIβ mice by combining CRISPR-Cas9 genome editing with electroporation (i-GONAD). With i-GONAD, it is possible to insert a small fragment of up to 200 bp into the genome of the target gene, enabling efficient and convenient tagging of a small epitope. Experiments with commercially available anti-Flag antibodies could readily detect endogenous CaMKIIα and β proteins by Western blotting, immunoprecipitation, and immunohistochemistry. Our data demonstrated that the generation of Flag/DYKDDDDK tag knock-in mice by i-GONAD is a useful and convenient choice, especially if specific antibodies are unavailable

Calcium Signaling in Mitral Cell Dendrites of Olfactory Bulbs of Neonatal Rats and Mice During Olfactory Nerve Stimulation and β-Adrenoceptor Activation

Synapses formed by the olfactory nerve (ON) provide the source of excitatory synaptic input onto mitral cells (MC) in the olfactory bulb. These synapses, which relay odor-specific inputs, are confined to the distally tufted single primary dendrites of MCs, the first stage of central olfactory processing. β-adrenergic modulation of electrical and chemical signaling at these synapses may be involved in early odor preference learning. To investigate this possibility, we combined electrophysiological recordings with calcium imaging in olfactory bulb slices prepared from neonatal rats and mice. Activation of ON-MC synapses induced postsynaptic potentials, which were associated with large postsynaptic calcium transients. Neither electrical nor calcium responses were affected by β-adrenergic agonists or antagonist. Immunocytochemical analysis of MCs and their tufted dendrites revealed clear immunoreactivity with antibodies against α1A (Cav2.1, P/Q-type) and α1B (Cav2.2, N-type), but not against α1C (Cav1.2, L-type) or α1D (Cav1.3, L-type) calcium channel subunits. Moreover, nimodipine, a blocker of L-type calcium channels, had no effect on either electrical or calcium signaling at ON-MC synapses. In contrast to previous evidence, we concluded that in neonatal rats and mice (P5-P8), mitral cells do not express significant amounts of L-type calcium channels, the calcium channel type that is often targeted by β-adrenergic modulation. The absence of β-adrenergic modulation on either electrical or calcium signaling at ON-MC synapses of neonatal rats and mice excludes the involvement of this mechanism in early odor preference learning