Crystal structure of a tetrameric RNA G-quadruplex formed by hexanucleotide repeat expansions of C9orf72 in ALS/FTD.

The abnormal GGGGCC hexanucleotide repeat expansions (HREs) in C9orf72 cause the fatal neurodegenerative diseases including amyotrophic lateral sclerosis and frontotemporal dementia. The transcribed RNA HREs, short for r(G4C2)n, can form toxic RNA foci which sequestrate RNA binding proteins and impair RNA processing, ultimately leading to neurodegeneration. Here, we determined the crystal structure of r(G4C2)2, which folds into a parallel tetrameric G-quadruplex composed of two four-layer dimeric G-quadruplex via 5'-to-5' stacking in coordination with a K+ ion. Notably, the two C bases locate at 3'- end stack on the outer G-tetrad with the assistance of two additional K+ ions. The high-resolution structure reported here lays a foundation in understanding the mechanism of neurological toxicity of RNA HREs. Furthermore, the atomic details provide a structural basis for the development of potential therapeutic agents against the fatal neurodegenerative diseases ALS/FTD.

Time domain turbo equalization based on vector approximate message passing for multiple-input multiple-output underwater acoustic communications.

This paper proposes a high-performance receiver for underwater acoustic communications based on time reversal processing for multiple-input multiple-output (MIMO) systems. The receiver employs the vector approximate message passing (VAMP) algorithm as a soft equalizer in turbo equalization. By performing self-iteration between the inner soft slicer and the inner soft equalizer, the VAMP algorithm achieves near-optimal performance. Furthermore, an iterative channel-estimation-based soft successive interference cancellation method is incorporated to suppress co-channel interference in the MIMO system. Additionally, the introduction of passive time reversal technology can combine multiple channels into a single channel, which greatly reduces the computational complexity of the MIMO system, especially for large MIMO systems. The effectiveness of the proposed receiver is verified using experimental data collected in Songhua Lake, China in 2019. The results demonstrate that the proposed receiver significantly reduces the complexity of the traditional parallel-VAMP receiver without sacrificing performance and outperforms other receivers of the same type. Moreover, our experimental results also verify that the VAMP-turbo outperforms the generalized approximate message passing (GAMP)-turbo in terms of bit error rate and convergence performance.

Broadening the Voltage Window of 3D-Printed MXene Micro-Supercapacitors with a Hybridized Electrolyte.

The burgeoning demand for miniaturized energy storage devices compatible with the miniaturization trend of electronic technologies necessitates advancements in micro-supercapacitors (MSCs) that promise safety, cost efficiency, and high-speed charging capabilities. However, conventional aqueous MSCs face a significant limitation due to their inherently narrow electrochemical potential window, which restricts their operational voltage and energy density compared to their organic and ionic liquid counterparts. In this study, we introduce an innovative aqueous NaCl/H2O/EG hybrid gel electrolyte (comprising common salt (NaCl), H2O, ethylene glycol (EG), and SiO2) for Ti3C2Tx MXene MSCs that substantially widens the voltage window to 1.6 V, a notable improvement over traditional aqueous system. By integrating the hybrid electrolyte with 3D-printed MXene electrodes, we realized MSCs with remarkable areal capacitance (1.51 F cm-2) and energy density (675 µWh cm-2), significantly surpassing existing benchmarks for aqueous MSCs. The strategic formulation of the hybrid electrolyte-a low-concentration NaCl solution with EG-ensures both economic and environmental viability while enabling enhanced electrochemical performance. Furthermore, the MSCs fabricated via 3D printing technology exhibit exceptional flexibility and are suitable for modular device integration, offering a promising avenue for the development of high-performance, sustainable energy storage devices. This advancement not only provides a tangible solution to the challenge of limited voltage windows in aqueous MXene MSCs but also sets a new precedent for the design of next-generation MSCs that align with the needs of an increasingly microdevice-centric world.

Discovery of the covalent SARS-CoV-2 Mpro inhibitors from antiviral herbs via integrating target-based high-throughput screening and chemoproteomic approaches.

The main proteases (Mpro ) are highly conserved cysteine-rich proteins that can be covalently modified by numerous natural and synthetic compounds. Herein, we constructed an integrative approach to efficiently discover covalent inhibitors of Mpro from complex herbal matrices. This work begins with biological screening of 60 clinically used antiviral herbal medicines, among which Lonicera japonica Flos (LJF) demonstrated the strongest anti-Mpro effect (IC50 = 37.82 µg/mL). Mass spectrometry (MS)-based chemical analysis and chemoproteomic profiling revealed that LJF extract contains at least 50 constituents, of which 22 exhibited the capability to covalently modify Mpro . We subsequently verified the anti-Mpro effects of these covalent binders. Gallic acid and quercetin were found to potently inhibit severe acute respiratory syndrome coronavirus 2 Mpro in dose- and time- dependent manners, with the IC50 values below 10 µM. The inactivation kinetics, binding affinity and binding mode of gallic acid and quercetin were further characterized by fluorescence resonance energy transfer, surface plasmon resonance, and covalent docking simulations. Overall, this study established a practical approach for efficiently discovering the covalent inhibitors of Mpro from herbal medicines by integrating target-based high-throughput screening and MS-based assays, which would greatly facilitate the discovery of key antiviral constituents from medicinal plants.

Multiple paternally inherited chloroplast capture events associated with Taxus speciation in the Hengduan Mountains.

Mountainous regions provide a multitude of habitats and opportunities for complex speciation scenarios. Hybridization leading to chloroplast capture, which can be revealed by incongruent phylogenetic trees, is one possible outcome. Four allopatric Taxus lineages (three species and an undescribed lineage) from the Hengduan Mountains, southwest China, exhibit conflicting phylogenetic relationships between nuclear and chloroplast phylogenies. Here, we use multi-omic data at the population level to investigate their historical speciation processes. Population genomic analysis based on ddRAD-seq data revealed limited contemporary inter-specific gene flow involving only populations located close to another species. In a historical context, chloroplast and nuclear data (transcriptome) consistently showed conflicting phylogenetic relationships for T. florinii and the Emei type lineage. ILS and chloroplast recombination were excluded as possible causes, and transcriptome and ddRAD-seq data revealed an absence of the mosaic nuclear genomes that characterize hybrid origin scenarios. Therefore, T. florinii appears to have originated when a lineage of T. florinii captured the T. chinensis plastid type, whereas plastid introgression in the opposite direction generated the Emei Type. All four species have distinct ecological niche based on community investigations and ecological niche analyses. We propose that the origins of both species represent very rare examples of chloroplast capture events despite the paternal cpDNA inheritance of gymnosperms. Specifically, allopatrically and/or ecologically diverged parental species experienced a rare secondary contact, subsequent hybridization and reciprocal chloroplast capture, generating two new lineages, each of which acquired a unique ecological niche. These events might have been triggered by orogenic activities of the Hengduan Mountains and an intensification of the Asian monsoon in the late Miocene, and may represent a scenario more common in these mountains than presently known.

Rheology of graphene oxide stabilized Pickering emulsions.

Pickering emulgels stabilized by graphene oxide (GO) with didodecyldimethylammonium bromide (DDAB) as an auxiliary surfactant and liquid paraffin as the oil phase have proved to be an excellent 3D printable ink. This paper elucidates the structure of such emulgels by a combination of microscopy before and after intensive shear as well as broadband dielectric spectroscopy and rheology in the linear and nonlinear regime. An increase of the DDAB surfactant and GO-contents leads to a systematic increase of modulus and viscosity, a reduction of the limits of the nonlinear regime and a more complicated variation of the normal forces, with negative normal forces at high shear rate  for low GO-contents and positive normal forces at high GO-contents. The interfacial jamming behavior studied by morphology, rheology and dielectric spectroscopy is explained based on droplet deformation, jamming and recovery phenomena.

A rationally engineered specific near-infrared fluorogenic substrate of human pancreatic lipase for functional imaging and inhibitor screening.

Obesity, now widespread all over the world, is frequently associated with several chronic diseases. Human pancreatic lipase (hPL) is a crucial digestive enzyme responsible for the digestion of dietary lipids in humans, and the inhibition of hPL is effective in reducing triglyceride intake and thus preventing and treating obesity. In this work, a practical sequential screening strategy was developed to construct a highly selective near-infrared fluorogenic substrate 7-STCFC for hPL. Under physiological conditions, 7-STCFC can be rapidly hydrolyzed by hPL to form 7-HTCFC, which triggers 254-fold NIR signal enhancement at 670 nm. 7-STCFC was successfully applied for the sensing and imaging of endogenous PL in living systems (including living cells, tissues and organs) with low cytotoxicity and high imaging resolution. Moreover, a high-throughput screening platform was established using 7-STCFC, and the inhibitory effects of 94 kinds of herbs toward hPL were evaluated. Among them, Pu-erh tea stood out with outstanding hPL inhibitory effects, and the inhibitory ingredients and involved inhibitory mechanism were further revealed, which strongly facilitates the discovery of novel anti-obesity agents targeting hPL. Collectively, these findings suggested that our strategy was practical to develop an isoform-specific fluorogenic substrate for a target enzyme, and 7-STCFC was a powerful tool for monitoring PL activity in complex biological systems with value for exploring physiological functions and rapid screening of inhibitors.

Crystal structure of parallel G-quadruplex formed by the two-repeat ALS- and FTD-related GGGGCC sequence.

The hexanucleotide repeat expansion, GGGGCC (G4C2), within the first intron of the C9orf72 gene is known to be the most common genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The G4C2 repeat expansions, either DNA or RNA, are able to form G-quadruplexes which induce toxicity leading to ALS/FTD. Herein, we report a novel crystal structure of d(G4C2)2 that self-associates to form an eight-layer parallel tetrameric G-quadruplex. Two d(G4C2)2 associate together as a parallel dimeric G-quadruplex which folds into a tetramer via 5'-to-5' arrangements. Each dimer consists of four G-tetrads connected by two CC propeller loops. Especially, the 3'-end cytosines protrude out and form C·C+•C·C+/ C·C•C·C+ quadruple base pair or C•C·C+ triple base pair stacking on the dimeric block. Our work sheds light on the G-quadruplexes adopted by d(G4C2) and yields the invaluable structural details for the development of small molecules to tackle neurodegenerative diseases, ALS and FTD.

Aldh inhibitor restores auditory function in a mouse model of human deafness.

Genetic hearing loss is a common health problem with no effective therapy currently available. DFNA15, caused by mutations of the transcription factor POU4F3, is one of the most common forms of autosomal dominant non-syndromic deafness. In this study, we established a novel mouse model of the human DFNA15 deafness, with a Pou4f3 gene mutation (Pou4f3Δ) identical to that found in a familial case of DFNA15. The Pou4f3(Δ/+) mice suffered progressive deafness in a similar manner to the DFNA15 patients. Hair cells in the Pou4f3(Δ/+) cochlea displayed significant stereociliary and mitochondrial pathologies, with apparent loss of outer hair cells. Progression of hearing and outer hair cell loss of the Pou4f3(Δ/+) mice was significantly modified by other genetic and environmental factors. Using Pou4f3(-/+) heterozygous knockout mice, we also showed that DFNA15 is likely caused by haploinsufficiency of the Pou4f3 gene. Importantly, inhibition of retinoic acid signaling by the aldehyde dehydrogenase (Aldh) and retinoic acid receptor inhibitors promoted Pou4f3 expression in the cochlear tissue and suppressed the progression of hearing loss in the mutant mice. These data demonstrate Pou4f3 haploinsufficiency as the main underlying cause of human DFNA15 deafness and highlight the therapeutic potential of Aldh inhibitors for treatment of progressive hearing loss.

N-Doped Porous Carbon-Nanofiber-Supported Fe3C/Fe2O3 Nanoparticles as Anode for High-Performance Supercapacitors.

Exploring anode materials with an excellent electrochemical performance is of great significance for supercapacitor applications. In this work, a N-doped-carbon-nanofiber (NCNF)-supported Fe3C/Fe2O3 nanoparticle (NCFCO) composite was synthesized via the facile carbonizing and subsequent annealing of electrospinning nanofibers containing an Fe source. In the hybrid structure, the porous carbon nanofibers used as a substrate could provide fast electron and ion transport for the Faradic reactions of Fe3C/Fe2O3 during charge-discharge cycling. The as-obtained NCFCO yields a high specific capacitance of 590.1 F g-1 at 2 A g-1, superior to that of NCNF-supported Fe3C nanoparticles (NCFC, 261.7 F g-1), and NCNFs/Fe2O3 (NCFO, 398.3 F g-1). The asymmetric supercapacitor, which was assembled using the NCFCO anode and activated carbon cathode, delivered a large energy density of 14.2 Wh kg-1 at 800 W kg-1. Additionally, it demonstrated an impressive capacitance retention of 96.7%, even after 10,000 cycles. The superior electrochemical performance can be ascribed to the synergistic contributions of NCNF and Fe3C/Fe2O3.

Visualizing the Surface Photocurrent Distribution in Perovskite Photovoltaics.

Defect states play an important role in the photovoltaic performance of metal halide perovskites. Particularly, the passivation of surface defects has made great contributions to high-performance perovskite photovoltaics. This highlights the importance of understanding the surface defects from a fundamental level by developing more accurate and operando characterization techniques. Herein, a strategy to enable the surface carriers and photocurrent distributions on perovskite films to be visualized in the horizontal direction is put forward. The visual image of photocurrent distribution is realized by combining the static local distribution of carriers provided by scanning near-field optical microscopy with the dynamic transporting of carriers achieved via a scanning photocurrent measurement system. Taking a surface passivated molecule as an example, a comprehensive defect scene including static and dynamic as well as local and entire conditions is obtained using this strategy. The comprehensive analysis of the trap states in perovskite films is pioneered vertically and horizontally, which will powerfully promote the deep understanding of defect mechanisms and carrier behavior for the goal of fabricating high-performance perovskite optoelectronic devices.

Impact of genetic variation in CYP2C19, CYP2D6, and CYP3A4 on oxycodone and its metabolites in a large database of clinical urine drug tests.

Urine drug testing (UDT) is a tool for monitoring drug use, including oxycodone. While variation in cytochrome P450 (CYP) genes is known to alter oxycodone metabolism, its impact on UDT results of oxycodone and its metabolites has not been well-studied. Here, multivariate analysis was performed on retrospective UDT results of 90,379 specimens collected from 14,684 genotyped patients prescribed oxycodone. Genetic variation in CYP2D6 and CYP2C19 had a significant impact on oxymorphone/oxycodone ratios, with a 6.9-fold difference between CYP2D6 ultrarapid metabolizers (UMs) and poor metabolizers (PMs; p < 10-300) and a 1.6-fold difference between CYP2C19 UMs and PMs (p = 1.50 × 10-4). CYP2D6 variation also significantly impacted noroxycodone/oxycodone ratios (p = 6.95 × 10-38). Oxycodone-positive specimens from CYP2D6 PMs were ~5-fold more likely to be oxymorphone-negative compared to normal metabolizers. These findings indicate that multivariate analysis of UDT data may be used to reveal the real-world impact of genetic and non-genetic factors on drug metabolism.

Causal relationship between genetically predicted depression and cancer risk: a two-sample bi-directional mendelian randomization.

BACKGROUND: Depression has been reported to be associated with some types of cancer in observational studies. However, the direction and magnitude of the causal relationships between depression and different types of cancer remain unclear. METHODS: We performed the two-sample bi-directional mendelian randomization with the publicly available GWAS summary statistics to investigate the causal relationship between the genetically predicted depression and the risk of multiple types of cancers, including ovarian cancer, breast cancer, lung cancer, glioma, pancreatic cancer, lymphoma, colorectal cancer, thyroid cancer, bladder cancer, and kidney cancer. The total sample size varies from 504,034 to 729,150. Causal estimate was calculated by inverse variance weighted method. We also performed additional sensitivity tests to evaluate the validity of the causal relationship. RESULTS: After correction for heterogeneity and horizontal pleiotropy, we only detected suggestive evidence for the causality of genetically predicted depression on breast cancer (OR = 1.09, 95% CI: 1.03-1.15, P = 0.0022). The causal effect of depression on breast cancer was consistent in direction and magnitude in the sensitivity analysis. No evidence of causal effects of depression on other types of cancer and reverse causality was detected. CONCLUSIONS: The result of this study suggests a causative effect of genetically predicted depression on specific type of cancer. Our findings emphasize the importance of depression in the prevention and treatment of breast cancer.

Genetic spectrum of CAKUT and risk factors for kidney failure: a pediatric multicenter cohort study.

BACKGROUND: Congenital anomalies of the kidney and urinary tracts (CAKUT) are the leading cause of kidney failure in children with phenotypic and genotypic heterogeneity. Our objective was to describe the genetic spectrum and identify the risk factors for kidney failure in children with CAKUT. METHODS: Clinical and genetic data were derived from a multicenter network (Chinese Children Genetic Kidney Disease Database, CCGKDD) and the Chigene database. A total of 925 children with CAKUT who underwent genetic testing from 2014 to 2020 across China were studied. Data for a total of 584 children wereobtained from the CCGKDD, including longitudinal data regarding kidney function. The risk factors for kidney failure were determined by the Kaplan-Meier method and Cox proportional hazards models. RESULTS: A genetic diagnosis was established in 96 out of 925 (10.3%) children, including 72 (8%) with monogenic variants, 20 (2%) with copy number variants (CNVs), and 4 (0.4%)with major chromosomal anomalies. Patients with skeletal abnormalities were more likely to have large CNVs or abnormal karyotypes than monogenic variants. Eighty-two patients from the CCGKDD progressed to kidney failure at a median age of 13.0 (95% confidence interval, 12.4-13.6) years, and twenty-four were genetically diagnosed with variants of PAX2, TNXB, EYA1, HNF1B and GATA3 or the 48, XXYY karyotype. The multivariate analysis indicated that solitary kidney, posterior urethral valves, bilateral hypodysplasia, the presence of certain variants and premature birth were independent prognostic factors. CONCLUSIONS: The genetic spectrum of CAKUT varies among different subphenotypes. The identified factors indicate areas that require special attention.

CircPVT1 promotes the tumorigenesis and metastasis of osteosarcoma via mediation of miR-26b-5p/CCNB1 axis.

INTRODUCTION: Osteosarcoma (OS) is the most aggressive malignancy among the bone tumors in the world. Circular RNAs (circRNAs) have been reported to be participated in multiple cancers, including OS. Meanwhile, circPVT1 has been proved to be upregulated in OS. However, the mechanism by which circPVT1 mediates the tumorigenesis of OS remains to be further explored. MATERIALS AND METHODS: Protein and gene expressions in OS cells were measured by western blot and RT-qPCR, respectively. Cell growth was assessed by flow cytometry and colony formation, respectively. In addition, cell migration was assessed by wound healing, and invasion was evaluated by Transwell assay. Meanwhile, the correlation among circPVT1, miR-26b-5p and CCNB1 was explored by RNA pull-down and dual luciferase assay. Finally, in vivo model was established to explore the role of circPVT1 in OS in vivo. RESULTS: CircPVT1 and CCNB1 were significantly upregulated in OS cells, while miR-26b-5p was downregulated. Knockdown of circPVT1 notably inhibited proliferation and induced apoptosis of OS cells. CircPVT1 shRNA significantly suppressed the OS cell invasion and migration. Meanwhile, circPVT1 sponged miR-26b-5p and CCNB1 was found to be the direct target of miR-26b-5p. Furthermore, silencing of circPVT1 inhibited the growth and metastasis of OS in vivo. CONCLUSION: Silencing of circPVT1 notably suppressed the tumorigenesis and metastasis of OS via miR-26b-5p/CCNB1 axis. Therefore, circPVT1 might be used as a target for OS treatment.

Cu-based MOF-derived architecture with Cu/Cu2O nanospheres anchored on porous carbon nanosheets for efficient capacitive deionization.

The design of high-performance electrode materials with excellent desalination ability has always been a research goal for efficient capacitive deionization (CDI). Herein, a hybrid architecture with Cu/Cu2O nanospheres anchored on porous carbon nanosheets (Cu/Cu2O/C) was first synthesized by pyrolyzing a two-dimensional (2D) Cu-based metal-organic framework and then evaluated as a cathode for hybrid CDI. The as-prepared Cu/Cu2O/C exhibits a hierarchically porous structure with a high specific surface area of 305 m2 g-1 and large pore volume of 0.55 cm3 g-1, which is favorable to accelerating ion migration and diffusion. The porous carbon nanosheet matrix with enhanced conductivity will facilitate the Faradaic reactions of Cu/Cu2O nanospheres during the desalination process. The Cu/Cu2O/C hybrid architecture displays a high specific capacitance of 142.5 F g-1 at a scan rate of 2 mV s-1 in 1 M NaCl solution. The hybrid CDI constructed using the Cu/Cu2O/C cathode and a commercial activated carbon anode exhibits a high desalination capacity of 16.4 mg g-1 at an operation voltage of 1.2 V in 500 mg L-1 NaCl solution. Additionally, the hybrid CDI exhibits a good cycling stability with 18.3% decay in the desalination capacity after 20 electrosorption-desorption cycles. Thus, the Cu/Cu2O/C composite is expected to be a promising cathode material for hybrid CDI.

Polyaniline coated MOF-derived Mn2O3 nanorods for efficient hybrid capacitive deionization.

Mn-based oxides are efficient pseudocapacitive electrode materials and have been investigated for capacitive deionization (CDI). However, their poor conductivity seriously affects their desalination performance. In this work, polyaniline coated Mn2O3 nanorods (PANI/Mn2O3) are synthesized by oxidizing a Mn-based metal organic framework (MOF) and subsequent in-situ chemical polymerization. The polyaniline not only acts as a conductive network for faradaic reactions of Mn2O3, but also enhances the desalination rate. PANI/Mn2O3 has a specific capacitance of 87 F g-1 (at 1 A g-1), superior to that of Mn2O3 nanorod (52 F g-1 at 1 A g-1). The hybrid CDI cell constructed with a PANI/Mn2O3 cathode and an active carbon anode shows a high desalination capacity of 21.6 mg g-1, superior recyclability with only 11.3% desalination capacity decay after 30 desalination cycles and fast desalination rate of 2.2 mg g-1 min-1. PANI/Mn2O3 is a potential candidate for high performance CDI applications.

Structural basis of DNA replication origin recognition by human Orc6 protein binding with DNA.

The six-subunit origin recognition complex (ORC), a DNA replication initiator, defines the localization of the origins of replication in eukaryotes. The Orc6 subunit is the smallest and the least conserved among ORC subunits. It is required for DNA replication and essential for viability in all species. Orc6 in metazoans carries a structural homology with transcription factor TFIIB and can bind DNA on its own. Here, we report a solution structure of the full-length human Orc6 (HsOrc6) alone and in a complex with DNA. We further showed that human Orc6 is composed of three independent domains: N-terminal, middle and C-terminal (HsOrc6-N, HsOrc6-M and HsOrc6-C). We also identified a distinct DNA-binding domain of human Orc6, named as HsOrc6-DBD. The detailed analysis of the structure revealed novel amino acid clusters important for the interaction with DNA. Alterations of these amino acids abolish DNA-binding ability of Orc6 and result in reduced levels of DNA replication. We propose that Orc6 is a DNA-binding subunit of human/metazoan ORC and may play roles in targeting, positioning and assembling the functional ORC at the origins.

High S100A7 expression is associated with early muscle invasion and poor survival in bladder carcinoma.

Muscle-invasive bladder carcinoma (MIBC) accounts for 25% of newly diagnosed bladder carcinomas (BCs) and presents a high risk of progression and metastasis. This study aimed to identify reliable biomarkers associated with muscle invasion and prognosis to identify potential therapeutic targets for MIBC. Four gene datasets were downloaded from the Gene Expression Omnibus, and the integrated differentially expressed genes (DEGs) were then subjected to gene ontology (GO) terms and pathway enrichment analyses. Correlation analysis between the expression of the top-ranking DEGs and pathological T stages was performed to identify the genes associated with early muscle invasion. The corresponding prognostic values were evaluated, and co-expressed genes mined in the cBioPortal database were loaded into ClueGo in Cytoscape for pathway enrichment analysis. Using data mining from the STRING and TCGA databases, protein-protein interaction and competitive endogenous RNA networks were constructed. In total, 645 integrated DEGs were identified and these were mainly enriched in 26 pathways, including cell cycle, bladder cancer, DNA replication, and PPAR signaling pathway. S100A7 expression was significantly increased from the T2 stage and showed significantly worse overall survival and disease-specific survival in patients with BC. In total, 144 genes co-expressed with S100A7 in BC were significantly enriched in the IL-17 pathway. S100A7 was predicted to directly interact with LYZ, which potentially shows competitive binding with hsa-mir-140 to affect the expression of six lncRNAs in MIBC. In conclusion, high S100A7 expression was predicted to be associated with early muscle invasion and poor survival in patients with BC.

Simultaneous Real-Time Three-Dimensional Localization and FRET Measurement of Two Distinct Particles.

Many biological processes employ mechanisms involving the locations and interactions of multiple components. Given that most biological processes occur in three dimensions, the simultaneous measurement of three-dimensional locations and interactions is necessary. However, the simultaneous three-dimensional precise localization and measurement of interactions in real time remains challenging. Here, we report a new microscopy technique to localize two spectrally distinct particles in three dimensions with an accuracy (2.35σ) of tens of nanometers with an exposure time of 100 ms and to measure their real-time interactions using fluorescence resonance energy transfer (FRET) simultaneously. Using this microscope, we tracked two distinct vesicles containing t-SNAREs or v-SNARE in three dimensions and observed FRET simultaneously during single-vesicle fusion in real time, revealing the nanoscale motion and interactions of single vesicles in vesicle fusion. Thus, this study demonstrates that our microscope can provide detailed information about real-time three-dimensional nanoscale locations, motion, and interactions in biological processes.