Engineered fluorescent protein (FP) chimeras that modulate their fluorescence in response to changes in calcium ion (Ca²⁺) concentration are powerful tools for visualizing intracellular signaling ...activity. However, despite a decade of availability, the palette of single FP-based Ca²⁺ indicators has remained limited to a single green hue. We have expanded this palette by developing blue, improved green, and red intensiometric indicators, as well as an emission ratiometric indicator with an 11,000% ratio change. This series enables improved single-color Ca²⁺ imaging in neurons and transgenic Caenorhabditis elegans. In HeLa cells, Ca²⁺ was imaged in three subcellular compartments, and, in conjunction with a cyan FP—yellow FP—based indicator, Ca²⁺ and adenosine 5′-triphosphate were simultaneously imaged. This palette of indicators paints the way to a colorful new era of Ca²⁺ imaging.
We have used protein engineering to expand the palette of genetically encoded calcium ion (Ca2+) indicators to include orange and improved red fluorescent variants, and validated the latter for ...combined use with optogenetic activation by channelrhodopsin-2 (ChR2). These indicators feature intensiometric signal changes that are 1.7- to 9.7-fold improved relatively to the progenitor Ca2+ indicator, R-GECO1. In the course of this work, we discovered a photoactivation phenomenon in red fluorescent Ca2+ indicators that, if not appreciated and accounted for, can cause false-positive artifacts in Ca2+ imaging traces during optogenetic activation with ChR2. We demonstrate, in both a beta cell line and slice culture of developing mouse neocortex, that these artifacts can be avoided by using an appropriately low intensity of blue light for ChR2 activation.
Semi-dwarf traits have been widely introgressed into cereal crops to improve lodging resistance. In sorghum (Sorghum bicolor L. Moench), four major unlinked dwarfing genes, Dw1-Dw4, have been ...introduced to reduce plant height, and among them, Dw3 and Dw1 have been cloned. Dw3 encodes a gene involved in auxin transport, whereas, Dw1 was recently isolated and identified as a gene encoding a protein of unknown function. In this study, we show that DW1 is a novel component of brassinosteroid (BR) signaling. Sorghum possessing the mutated allele of Dw1 (dw1), showed similar phenotypes to rice BR-deficient mutants, such as reduced lamina joint bending, attenuated skotomorphogenesis, and insensitivity against feedback regulation of BR-related genes. Furthermore, DW1 interacted with a negative regulator of BR signaling, BRASSINOSTEROID INSENSITIVE 2 (BIN2), and inhibited its nuclear localization, indicating that DW1 positively regulates BR signaling by inhibiting the function of BIN2. In contrast to rice and wheat breeding which used gibberellin (GA) deficiency to reduce plant height, sorghum breeding modified auxin and BR signaling. This difference may result from GA deficiency in rice and wheat does not cause deleterious side effects on plant morphology, whereas in sorghum it leads to abnormal culm bending.
Sorghum is the fifth most important cereal crop. Here we performed molecular genetic analyses of the 'SUGARY FETERITA' (SUF) variety, which shows typical sugary endosperm traits (e.g., wrinkled ...seeds, accumulation of soluble sugars, and distorted starch). Positional mapping indicated that the corresponding gene was located on the long arm of chromosome 7. Within the candidate region of 3.4 Mb, a sorghum ortholog for maize
(
) encoding a starch debranching enzyme ISA1 was found. Sequencing analysis of
in SUF uncovered nonsynonymous single nucleotide polymorphisms (SNPs) in the coding region, containing substitutions of highly conserved amino acids. Complementation of the rice
(
) mutant line with the
gene recovered the sugary endosperm phenotype. Additionally, analyzing mutants obtained from an EMS-induced mutant panel revealed novel alleles with phenotypes showing less severe wrinkles and higher Brix scores. These results suggested that
was the corresponding gene for the sugary endosperm. Expression profiles of starch synthesis genes during the grain-filling stage demonstrated that a loss-of-function of
affects the expression of most starch synthesis genes and revealed the fine-tuned gene regulation in the starch synthetic pathway in sorghum. Haplotype analysis using 187 diverse accessions from a sorghum panel revealed the haplotype of SUF showing severe phenotype had not been used among the landraces and modern varieties. Thus, weak alleles (showing sweet and less severe wrinkles), such as in the abovementioned EMS-induced mutants, are more valuable for grain sorghum breeding. Our study suggests that more moderate alleles (
produced by genome editing) should be beneficial for improving grain sorghum.
Introduction: This study aimed to describe the quantitative features of the microvasculature in the cystic lesions of branch retinal vein occlusion (BRVO). Methods: A total of 43 eyes with BRVO, ...treated with anti-vascular endothelial growth factor therapy, were analyzed. Using wide-field swept-source optical coherence tomography angiography (OCTA), en face OCT images were obtained by depth-integrated reflectivity of the retina, and vascular density (VD), vascular length (VL), vascular lacunarity, and fractal dimension (FD) were evaluated in a 12 × 12-mm area of retinal nonperfusion. Results: The mean area of affected lesions was 38.7 ± 19.8 mm2, and cystic lesions were 8.5 ± 10.1 mm2. VD, VL, and FD were significantly decreased in the cystic lesions compared to other affected lesions in the same eyes (p = 0.0010, p = 0.0001, and p = 0.0003, respectively) and in all eyes (p = 0.0281, p = 0.0050, and p < 0.0001, respectively). VD in cystic lesions within the vascular arcade (25 eyes) correlated with best-corrected visual acuity on OCTA (r = −0.433, and p = 0.0492). Conclusions: Vascular structure in the cystic lesions was unpreserved compared to the other lesions in BRVO. These findings may help in understanding the pathophysiology of retinal edema in BRVO.
Semi-dwarfing genes have contributed to enhanced lodging resistance, resulting in increased crop productivity. In the history of grain sorghum breeding, the spontaneous mutation, dw1 found in Memphis ...in 1905, was the first widely used semi-dwarfing gene. Here, we report the identification and characterization of Dw1. We performed quantitative trait locus (QTL) analysis and cloning, and revealed that Dw1 encodes a novel uncharacterized protein. Knockdown or T-DNA insertion lines of orthologous genes in rice and Arabidopsis also showed semi-dwarfism similar to that of a nearly isogenic line (NIL) carrying dw1 (NIL-dw1) of sorghum. A histological analysis of the NIL-dw1 revealed that the longitudinal parenchymal cell lengths of the internode were almost the same between NIL-dw1 and wildtype, while the number of cells per internode was significantly reduced in NIL-dw1. NIL-dw1dw3, carrying both dw1 and dw3 (involved in auxin transport), showed a synergistic phenotype. These observations demonstrate that the dw1 reduced the cell proliferation activity in the internodes, and the synergistic effect of dw1 and dw3 contributes to improved lodging resistance and mechanical harvesting.
Heterosis helps increase the biomass of many crops; however, while models for its mechanisms have been proposed, it is not yet fully understood. Here, we use a QTL analysis of the progeny of a ...high-biomass sorghum F
hybrid to examine heterosis. Five QTLs were identified for culm length and were explained using the dominance model. Five resultant homozygous dominant alleles were used to develop pyramided lines, which produced biomasses like the original F
line. Cloning of one of the uncharacterised genes (Dw7a) revealed that it encoded a MYB transcription factor, that was not yet proactively used in modern breeding, suggesting that combining classic dw1or dw3, and new (dw7a) genes is an important breeding strategy. In conclusion, heterosis is explained in this situation by the dominance model and a combination of genes that balance the shortness and early flowering of the parents, to produce F
seed yields.
To investigate the chronological corneal changes associated with long-term rigid gas-permeable contact lens (RGP-CL) wear in patients with keratoconus (KC).
Clinical records of 405 patients with KC ...or with KC suspect were retrospectively reviewed. Patients with mild-to-moderate KC and uneventful follow-up were classified into the CL (RGP-CL wear) and non-CL (without CL wear) groups. Inclusion criteria were (1) at least 3-year follow-up and (2) Scheimpflug-based corneal imaging examination at each visit. The anterior (ARC) and posterior (PRC) radius of curvature obtained in a 3.0 mm optical zone, the thinnest pachymetry reading of the corneal thickness (Tmin), and maximum keratometry values (Kmax) were investigated as tomographic parameters.
Twenty-two and 15 patients who met the inclusion criteria were included in the CL and non-CL groups, respectively (31 and 20 eyes, respectively). The mean observation periods were 75 (CL group) and 63 (non-CL group) months. A multivariable non-linear regression analysis to assess the change in tomographic parameters over the follow-up period and difference of the trend between the two groups demonstrated no significant differences in the chronological change in ARC, PRC and Tmin between the CL and non-CL groups (p=0.318, p=0.280 and p=0.874, respectively).
Based on corneal tomographic evaluation over 5-6 years, the effects of long-term RGP-CL wear had no effect on KC progression.
Directed protein evolution provides a series of fluorescent protein-based indicators for multicolor Ca2
+
imaging.
Engineered fluorescent protein (FP) chimeras that modulate their fluorescence in ...response to changes in calcium ion (Ca
2+
) concentration are powerful tools for visualizing intracellular signaling activity. However, despite a decade of availability, the palette of single FP-based Ca
2+
indicators has remained limited to a single green hue. We have expanded this palette by developing blue, improved green, and red intensiometric indicators, as well as an emission ratiometric indicator with an 11,000% ratio change. This series enables improved single-color Ca
2+
imaging in neurons and transgenic
Caenorhabditis elegans
. In HeLa cells, Ca
2+
was imaged in three subcellular compartments, and, in conjunction with a cyan FP–yellow FP–based indicator, Ca
2+
and adenosine 5′-triphosphate were simultaneously imaged. This palette of indicators paints the way to a colorful new era of Ca
2+
imaging.
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