Optimization of a GCaMP calcium indicator for neural activity imaging

© The Author(s), 2012. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Journal of Neuroscience 32 (2012): 13819-13840, doi:10.1523/JNEUROSCI.2601-12.2012.Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of “GCaMP5” sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.A.F. has been supported by a European Molecular Biology Organization long-term fellowship. Work in H.B.’s laboratory was funded by the National Institutes of Health (NIH) Nanomedicine Development Center “Optical Control of Biological Function,” and work in S.S.-H.W.’s laboratory was funded by NIH R01 NS045193

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

Effect of the Characteristic Properties of Membrane on Long-Term Stability in the Vacuum Membrane Distillation Process

Membrane distillation (MD) is a technology that can treat feed solutions with higher osmotic pressure, as well as produce high-purity water. However, the water production cost of the MD process is expensive. In this study, to decrease the water production cost, we attempted to evaluate the effect of membrane characteristics on the long-term stability of a vacuum MD (VMD) system. We fabricated four different types of polyvinylidene difluoride hollow fiber membranes, and operated a VMD system with 3.5 wt% NaCl aqueous solution at 65 °C as a feed under 11 kPa of air gap pressure. Consequently, in the proposed VMD system, it is found that the liquid entry pressure (LEP) is the most important factor. When LEP was higher than 0.37 MPa, the pilot-scale module was very stable for long-term operations, and the vapor flux was approximately 19.3 kg/m2·h with a total salt retention factor of over 99.9% during the 300-h operation

Studies on the Pinctada fucata BMP-2 Gene: Structural Similarity and Functional Conservation of Its Osteogenic Potential within the Animal Kingdom

Bone morphogenetic protein (BMP)-2 plays an important role in morphogenesis in both vertebrates and invertebrates. BMP-2 is one of the most powerful bioactive substances known to induce the osteogenic differentiation of mesenchymal cells. We examined the structural and functional conservation of Pinctada fucata BMP-2 in inducing osteogenesis in the murine mesenchymal stem cells, C3H10T1/2. Exposure of C3H10T1/2 cells to the recombinant mature fragment of Pinctada fucata BMP-2 resulted in osteoblastic differentiation. The sequence, SVPKPCCVPTELSSL, within the C-terminal portion of Pinctada fucata BMP-2, is homologous to the knuckle epitope of human BMP-2. This synthetic polypeptide was able to induce differentiation of C3H10T1/2 along the osteoblastic lineage, as confirmed by an increase in alkaline phosphatase activity, and the accumulation of calcium, as determined by von Kossa staining. Furthermore, using immunohistochemical staining, we observed an increased expression of collagen type I, osteopontin, and osteocalcin, which are known markers of osteogenesis. These results show that BMP-2 is conserved, not only in terms of its homology at the amino acid sequence, but also in terms of driving the formation of hard tissues in vertebrates and invertebrates