Biology subjects

Li, G.

Publications and source records attributed to Li, G..

At least 343 records · Page 19Linked to original sources

bioRxiv2022

Integrated Multi-Omics Analysis of Brain Aging in Female Nonhuman Primates Reveals Altered Signaling Pathways Relevant to Age-Related Disorders

The prefrontal cortex (PFC) has been implicated as a key brain region responsible for age-related cognitive decline. Little is known about aging-related molecular changes in PFC that may mediate these effects. To date, no studies have used untargeted discovery methods with integrated analyses to determine PFC molecular changes in healthy female primates. We quantified PFC changes associated with healthy aging in female baboons by integrating multiple omics data types (transcriptomics, proteomics, metabolomics) from samples across the adult age span. Our integrated omics approach using unbiased weighted gene co-expression network analysis (WGCNA) to integrate data and treat age as a continuous variable, revealed highly interconnected known and novel pathways associated with PFC aging. We found GABA tissue content associated with these signaling pathways, providing one potential biomarker to assess PFC changes with age. These highly coordinated pathway changes during aging may represent early steps for aging-related decline in PFC functions, such as learning and memory, and provide potential biomarkers to assess cognitive status in humans.

systems biology↗

bioRxiv2022

Gap-free nuclear and mitochondrial genomes of Ustilaginoidea virens strain JS60-2, a fungal pathogen causing rice false smut

Rice false smut (RFS), caused by Ustilaginoidea virens, has become a major disease in recent years, and mycotoxins produced by U. virens often threaten food safety. To study fungal pathogenesis and identify potential targets for developing new fungicides, gap-free nuclear and complete mitochondrial genomes of U. virens JS60-2 were sequenced and assembled. Using the second and third generation sequencing data, we assembled a 38.02-Mb genome that consists of seven contigs with the contig N50 being 6.32-Mb. In total, 8,486 protein-coding genes were annotated in the genome, including 21 secondary metabolism gene clusters. We also assembled the complete mitochondrial genome, which is 102,498 bp, with 28% GC content. The JS60-2 genomes assembled in this study will facilitate research on U. virens and contribute to RFS control.

genomics↗

bioRxiv2022

Smaller total and subregional cerebellar volumes in posttraumatic stress disorder: a mega-analysis by the ENIGMA-PGC PTSD workgroup

BackgroundThe cerebellum critically contributes to higher-order cognitive and emotional functions such fear learning and memory. Prior research on cerebellar volume in PTSD is scant and has neglected neuroanatomical subdivisions of the cerebellum that differentially map on to motor, cognitive, and affective functions. MethodsWe quantified cerebellar lobule volumes using structural magnetic resonance imaging in 4,215 adults (PTSD n= 1640; Control n=2575) across 40 sites from the from the ENIGMA-PGC PTSD working group. Using a new state-of-the-art deep-learning based approach for automatic cerebellar parcellation, we obtained volumetric estimates for the total cerebellum and 28 subregions. Linear mixed effects models controlling for age, gender, intracranial volume, and site were used to compare cerebellum total and subregional volume in PTSD compared to healthy controls. The Benjamini-Hochberg procedure was used to control the false discovery rate (p-FDR < .05). ResultsPTSD was associated with significant grey and white matter reductions of the cerebellum. Compared to controls, people with PTSD demonstrated smaller total cerebellum volume. In addition, people with PTSD showed reduced volume in subregions primarily within the posterior lobe (lobule VIIB, crus II), but also the vermis (VI, VIII), flocculonodular lobe (lobule X), and cerebellar white matter (all p-FDR < 0.05). Effects of PTSD on volume were consistent, and generally more robust, when examining symptom severity rather than diagnostic status. ConclusionsThese findings implicate regionally specific cerebellar volumetric differences in the pathophysiology of PTSD. The cerebellum appears to play an important role in high-order cognitive and emotional processes, far beyond its historical association with vestibulomotor function. Further examination of the cerebellum in trauma-related psychopathology will help to clarify how cerebellar structure and function may disrupt cognitive and affective processes at the center of translational models for PTSD.

neuroscience↗

bioRxiv2022

Gaq-PKD/PKCμ regulates the IkB transcription to limit the NF-kB mediated inflammatory response essential for early pregnancy

Decidualization, denoting the transformation of endometrial stromal cells into specialized decidual cells, is a prerequisite for normal embryo implantation and a successful pregnancy in human. Here we demonstrated that knockout of Gaq lead to an aberrantly enhanced inflammatory state during decidualization. Furthermore, we showed that deficiency of Gaq resulted in over-activation of nuclear factor (NF)-{kappa}B signaling, due to the decreased expression of NF{kappa}BIA, which encode the I{kappa}B protein and is the negative regulator for NF{kappa}B. Mechanistically, Gaq deficiency decreased the PKD/PKC phosphorylation levels, so leading to attenuated HDAC5 phosphorylation and thus its nuclear export. Aberrantly high level of nuclear HADC5 retarded histone acetylation to inhibit NF{kappa}BIA transcription during decidualization. Consistently, pharmacological activation of the PKD/PKC or inhibition of the HDAC5 signaling restored the inflammatory state and proper decidual response. Finally, we disclosed that over-active inflammatory state in Gaq deficient decidua deferred the blastocyst hatching and adhesion in vitro, and the decidual expression of Gq was significantly lower in women with recurrent pregnancy loss compared with normal pregnancy. In brief, we showed here that Gq as a key regulator of the inflammatory cytokines expression and decidual homeostasis in response to differentiation cues, which is required for successful implantation and early pregnancy.

cell biology↗

bioRxiv2022

A meta-analysis of genome-wide association studies identifies new genetic loci associated with all-cause and vascular dementia

Dementia is multifactorial with Alzheimer (AD) and vascular (VaD) pathologies making the largest contributions. Genome-wide association studies (GWAS) have identified over 70 genetic risk loci for AD but the genomic determinants of other dementias, including VaD remain understudied. We hypothesize that common forms of dementia will share genetic risk factors and conducted the largest GWAS to date of "all-cause dementia" (ACD) and examined the genetic overlap with VaD. Our dataset includes 809,299 individuals from European, African, Asian, and Hispanic ancestries with 46,902 and 8,702 cases of ACD and VaD, respectively. We replicated known AD loci at genome-wide significance for both ACD and VaD and conducted bioinformatic analyses to prioritize genes that are likely functionally relevant, and shared with closely related traits and risk factors. For ACD, novel loci identified were associated with energy transport (SEMA4D), neuronal excitability (ANO3), amyloid deposition in the brain (RBFOX1), and MRI markers of small vessel disease (HBEGF). Novel VaD loci were associated with hypertension, diabetes, and neuron maintenance (SPRY2, FOXA2, AJAP1, and PSMA3). Our study identified genetic risks underlying all-cause dementia, demonstrating overlap with neurodegenerative processes, vascular risk factors (Type-II diabetes, blood pressure, lipid) and cerebral small vessel disease. These novel insights could lead to new prevention and treatment strategies for all dementias.

genetics↗

bioRxiv2022

RNAcontacts, a pipeline for predicting contacts from RNA proximity ligation assays

BackgroundHigh-throughput RNA proximity ligation assays are molecular methods that simultaneously analyze spatial proximity of many RNAs in living cells. Their principle is based on cross-linking, fragmentation, and consequent religation of RNAs followed by high-throughput sequencing. The generated fragments have two distinct types of splits, one resulting from pre-mRNA splicing, and the other resulting from ligating spatially close RNA strands. FindingsHere, we present RNAcontacts, a universal pipeline for detecting RNA-RNA contacts in high-throughput RNA proximity ligation assays. It circumvents the inherent problem of mapping sequences with two distinct split types using a two-pass alignment, in which splice junctions are inferred from a control RNA-seq experiment on the first pass and then provided to the aligner on the second pass as bona fide introns. This approach allows for a more sensitive detection of RNA contacts and has higher specificity with respect to splice junctions that are present in the biological sample in comparison to previously developed methods. RNAcontacts extracts contacts, clusters their ligation points, computes the read support, and generates tracks for the visualization through the UCSC Genome Browser. It is implemented in a reproducible and scalable workflow management system Snakemake that allows fast and uniform processing of multiple datasets. ConclusionsRNAcontacts represents a generic pipeline for the detection of RNA contacts that can be used with any proximity ligation method as long as one of the interacting partners is RNA. RNAcontacts is available via github at https://github.com/smargasyuk/RNAcontacts/

bioinformatics↗

bioRxiv2022

Exosome-derived lncRNA HOXA-AS3 promotes castration resistance and progression of prostate cancer via the miR-29b-3p/Mcl-1/STAT3 axis

Prostate cancer is one of the leading causes of cancer-related deaths in men. While endocrine therapy is effective in the early stage of metastasis, significantly inhibits the progression of metastatic prostate cancer, most patients eventually develop CRPC. Tumor microenvironment are involved in the progression of prostate cancer as an "accomplice", cancer cell-secreted exosomes were identified as crucial messengers can carry lncRNAs to participate in intercellular communication. we revealed PC-3-derived exosomes promote androgen resistance in LNCaP cells. HOXA-AS3 as a ceRNA of miRNA-29b-3p affects the proliferation and invasion ability of prostate cancer cells. A series of molecular experiments, cell experiments and clinical tissue verification experiments confirmed that HOXA-AS3 participates in the castration resistance and progression of prostate cancer through regulating the miR-29b-3p/Mcl-1/STAT3/Cytochrome C/caspases-9 pathway. Dysregulation of HOXA-AS3 is observed in many cancer types, and this study shows the importance of this lncRNA in controlling prostate cancer cell progression, thus highlighting it as a potential biomarker for inhibiting prostate cancer progression and a target for immunotherapy.

genomics↗

bioRxiv2022

PF-D-Trimer, a broadly protective SARS-CoV-2 subunit vaccine: immunogenicity and application

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has had and still has a considerable impact on global public health. One of the characteristics of SARS-CoV-2 is a surface homotrimeric spike protein, the primary responsible for the host immune response upon infection. Here we show the preclinical studies of a broad protective SARS-CoV-2 subunit vaccine developed from our Trimer Domain platform using the Delta spike protein, from antigen design to purification, vaccine evaluation and manufacturability. The prefusion trimerized Delta spike protein, PF-D-Trimer, was highly expressed in Chinese hamster ovary (CHO) cells, purified by a rapid one-step anti-Trimer Domain monoclonal antibody immunoaffinity process and prepared as a vaccine formulation with an adjuvant. The immunogenicity studies demonstrated that this vaccine candidate induces robust immune responses in mouse, rat and Syrian hamster models. It also protects K18-hACE2 transgenic mice in a homologous virus challenge. The neutralizing antibodies induced by this vaccine display a cross-reactive capacity against the ancestral WA1 and Delta variants as well as different Omicron, including BA.5.2. The Trimer Domain platform was proven to be a key technology in the rapid production of the PF-D-Trimer vaccine and may be crucial to accelerate the development of updated versions of SARS-CoV-2 vaccines.

immunology↗

bioRxiv2022

The chromosome-level genome of Water-Wisteria establishes an ecological plant model and provides insight into the molecular mechanisms underlying heterophylly in amphibious plants

Heterophylly is a phenomenon in which an individual plant dramatically changes its leaf shape in response to the surrounding environment. Hygrophila difformis (Acanthaceae), also known as water wisteria, has recently emerged as a model plant to study heterophylly because of its striking leaf shape variation in response to various ecological factors. Under submerged conditions, H. difformis develops complex leaves and in terrestrial conditions it develops simple leaves. Here, we sequenced and assembled the chromosome-level genome of triploid H. difformis (scaffold N50: 60.43 Mb, genome size: 871.92 Mb), which reveals 36,099 predicted protein-coding genes distributed over 15 pseudochromosomes. H. difformis diverged from its relatives during the Oligocene climate-change period and expanded the gene families related to its amphibious lifestyle. Genes involved in environmental stimuli, leaf development, and other pathways are differentially expressed in submerged and terrestrial conditions, possibly modulating morphological and physiological acclimation to changing environments. We confirmed that auxin plays a role in the heterophylly of H. difformis. Finally, we discovered candidate genes that respond to different environmental conditions and elucidated the role of LATE MERISTEM IDENTITY 1 (LMI1) in heterophylly. Our study establishes H. difformis as a model for studying the interconnections between ecological adaptation and plant morphological features.

plant biology↗

bioRxiv2022

Biologically relevant integration of transcriptomics profiles from cancer cell lines, patient-derived xenografts and clinical tumors using deep neural networks

Cell lines and patient-derived xenografts are essential to cancer research, however, the results derived from such models often lack clinical translatability, as these models do not fully recapitulate the complex cancer biology. It is critically important to better understand the systematic differences between cell lines, xenografts and clinical tumors, and to be able to identify pre-clinical models that sufficiently resemble the biological characteristics of clinical tumors across different cancers. On another side, direct comparison of transcriptional profiles from pre-clinical models and clinical tumors is infeasible due to the mixture of technical artifacts and inherent biological signals. To address these challenges, we developed MOBER, Multi-Origin Batch Effect Remover method, to simultaneously extract biologically meaningful embeddings and remove batch effects from transcriptomic datasets of different origin. MOBER consists of two neural networks: conditional variational autoencoder and source discriminator neural network that is trained in adversarial fashion. We applied MOBER on transcriptional profiles from 932 cancer cell lines, 434 patient-derived tumor xenografts and 11159 clinical tumors and identified pre-clinical models with greatest transcriptional fidelity to clinical tumors, and models that are transcriptionally unrepresentative of their respective clinical tumors. MOBER can conserve the biological signals from the original datasets, while generating embeddings that do not encode confounder information. In addition, it allows for transformation of transcriptional profiles of pre-clinical models to resemble the ones of clinical tumors, and therefore can be used to improve the clinical translation of insights gained from pre-clinical models. As a batch effect removal method, MOBER can be applied widely to transcriptomics datasets of different origin, allowing for integration of multiple datasets simultaneously.

bioinformatics↗

bioRxiv2022

Genetically encoded sensors for measuring histamine release both in vitro and in vivo

Histamine (HA) is a key biogenic monoamine involved in a wide range of physiological and pathological processes in both the central nervous system and the periphery. Because the ability to directly measure extracellular HA in real-time will provide important insights into the functional role of HA in complex circuits under a variety of conditions, we developed a series of genetically encoded G protein-coupled receptor activation-based (GRAB) HA (GRABHA) sensors. These sensors produce a robust increase in fluorescence upon HA application, with good photostability, sub-second kinetics, nanomolar affinity, and high specificity. Using these GRABHA sensors, we measured electrical stimulation-evoked HA release in acute brain slices with high spatiotemporal resolution. Moreover, we recorded HA release in the preoptic area of the hypothalamus and in the medial prefrontal cortex during the sleep-wake cycle in freely moving mice, finding distinct patterns of HA release in these specific brain regions. Together, these in vitro and in vivo results show that our GRABHA sensors have high sensitivity and specificity for measuring extracellular HA, thus providing a robust new set of tools for examining the role of HA signaling in both health and disease.

neuroscience↗

bioRxiv2022

Stability of SARS-CoV-2 in cold-chain transportation environments and the efficacy of disinfection measures

Cold-chain environment could extend the survival duration of SARS-CoV-2 and increases the risk of transmission. However, the effect of clod-chain environmental factors and packaging materials on SARS-CoV-2 stability and the efficacy of intervention measures to inactivate SARS-CoV-2 under cold-chain environment remains uncertain. This study aimed to unravel cold-chain environmental factors that preserved the stability of SARS-CoV-2 and disinfection measures against SARS-CoV-2 under the cold-chain environment. The spike gene of SARS-CoV-2 isolated from Wuhan hu-1 was used to construct the SARS-CoV-2 pseudovirus and used as model of the SARS-CoV-2 virus. The decay rate of SARS-CoV-2 pseudovirus in the cold-chain environment, various types of packaging material surfaces i.e., PE plastic, stainless steel, Teflon and cardboard, and in frozen seawater was investigated. The influence of LED visible light(wavelength 450 nm-780 nm) and airflow movement on the stability of SARS-CoV-2 pseudovirus at -18{degrees} C were subsequently assessed. The results show that SARS-CoV-2 pseudovirus decayed more rapidly on porous cardboard surface compared with the non-porous surfaces including PE plastic, stainless steel and Teflon. Compared with 25{degrees} C, the decay rate of SARS-CoV-2 pseudovirus was significantly lower at low temperature. Seawater preserved viral stability both at -18{degrees} C and repeated freeze-thawing cycles compared with deionized water. LED visible light illumination and airflow movement environment at -18{degrees} C reduced the SARS-CoV-2 pseudovirus stability. In conclusion, our results indicate cold-chain temperature and seawater as risk factors for SARS-CoV-2 transmission and LED visible light illumination and airflow movement as possible disinfection measures of SARS-CoV-2 under the cold-chain environment. ImportanceIt is widely recognized that low temperature is a condition for maintaining virus vitality, and cold-chain transportation spreads the events of the SARS-CoV-2 were reported. This study provides that the decay rate of the SARS-CoV-2 pseudovirus at low temperatures varies on different packaging materials, and salt ions present in frozen foods such as seafood may protect virus survival. These results provide evidence for the possibility of SARS-CoV-2 transmission through cold-chain transport and also suggest the importance for disinfection of items. However, the commonly used disinfection methods of ultraviolet radiation and chemical reagents are generally not suitable for the disinfection of frozen food. Our study shows LED visible light illumination and airflow movement as possible disinfection measures of SARS-CoV-2 under the cold-chain environment. This has implications for reducing the long-distance transmission of the virus through cold-chain transportation.

microbiology↗

bioRxiv2022

Multifunctional requirements for ERK1/2 signaling in the development of ganglionic eminence derived glia and cortical inhibitory neurons

The RAS/RAF/MEK/ERK1/2 intracellular signaling pathway is activated by numerous cues during brain development and dysregulated in neurodevelopmental syndromes, particularly the RASopathies and certain forms of autism. Cortical excitatory/inhibitory imbalance is thought to be critical in the neuropathogenesis of these conditions. However, the developmental functions of ERK1/2 signaling in cortical inhibitory neurons (CINs) and other medial ganglionic eminence (MGE)-derived non-neuronal cells are poorly understood. Here, we genetically modulated ERK1/2 signaling in mouse MGE neural progenitors or GABAergic neurons in vivo. We find that MEK-ERK1/2 signaling is essential for regulating MGE-derived oligodendrocyte number in the anterior commissure. While Erk1/2 inactivation does not alter CIN number, we discovered a significant and persistent reduction in somatostatin, but not parvalbumin, expression in a subset of CINs. ERK1/2 signaling is also necessary for chemogenetic activity-dependent FOSB expression in CINs in vivo. Interestingly, one week of chronic chemogenetic stimulation in juvenile or adult animals partially rescues the decrease in somatostatin expression in Erk1/2 mutant CINs. Our data demonstrate ERK1/2 signaling is required for the establishment of MGE-derived glia, whereas in CINs, ERK1/2 drives activity dependent-responses and the expression of somatostatin in a subset of neurons.

neuroscience↗

bioRxiv2022

Genome-wide investigation of multiplexed CRISPR-Cas12a-mediated editing in rice

We previously reported highly specific genome editing in rice by CRISPR-Cas9 and Cas12a with single DNA double strand break (DSB) (Tang et al., 2018). Two concurrent DSBs by CRISPR-Cas9 could generate defined deletions (Zhou et al., 2014), inversions (Schmidt et al., 2020), and translocations (Beying et al., 2020). Off-target effects of many simultaneous DSBs are unknown in plants. Here, we used whole-genome sequencing (WGS) to investigate off-target effects in rice plants edited by highly multiplexable CRISPR-Cas12a systems (Zhang et al., 2021). Our WGS study revealed highly specific multiplexed genome editing by CRISPR-Cas12a. We found low-frequency large chromosomal deletion and duplication events in plants that endured many (e.g., >50) simultaneous DSBs, but not in plants that endured a lower order DSBs (e.g., <10). While our short reads sequencing may not capture all chromosomal rearrangements, our results nevertheless shed important light on the analysis and regulation of engineered crops derived from multiplexed genome editing.

genomics↗

bioRxiv2022

Analysis of the mechanism of castor tolerance to Cd metal based on proteomics and metabolomics

The pollution of soil with heavy metals is an increasingly serious worldwide problem, and cadmium (Cd) has attracted attention because of its high toxicity to almost all plants. Since castor tolerates the accumulation of heavy metals, it has the potential for heavy metal soil remediation. We studied the mechanism of the tolerance of castor to Cd stress treatments at three doses: 300 mg/L, 700 mg/L, and 1,000 mg/L. Our research provides new ideas for revealing the defense and detoxification mechanisms of Cd-stressed castor. By combining the results of physiology, differential proteomics and comparative metabolomics, we conducted a comprehensive analysis of the networks that regulate the response of castor to Cd stress. The physiological results mainly emphasize the super-sensitive responses of castor plant roots to Cd stress and the effects of Cd stress on plants antioxidant system, ATP synthesis and ion homeostasis. We confirmed these results at the protein and metabolite levels. At the same time, proteomics and metabolomics show that castor plants mainly block the root systems absorption of Cd2+ by enhancing the strength of the cell wall, and inducing programmed cell death in response to the three different doses of Cd stress. In addition, the plasma membrane ATPase encoding gene (RcHA4), which was significantly upregulated in our differential proteomics and RT-qPCR studies, was transgenically overexpressed in wild type Arabidopsis thaliana for functional verification. The results indicated that this gene plays an important role in improving plant Cd tolerance.

molecular biology↗

bioRxiv2022

Prior-guided Individualized Thalamic Parcellation based on Local Diffusion Characteristics using Deep Learning

As a gateway for projections entering and exiting the cerebral cortex, the human thalamus processes information from sensory to cognition relevant to various neuropsychiatric disorders. It is composed of dozens of nuclei, which have been difficult to identify with clinical MR sequences. However, delineating thalamic nuclei accurately at an individual level is essential for precise neuromodulation treatment. Here, we not only identified the fine-grained thalamic nuclei using local diffusion properties in vivo but also employed a deep learning strategy to achieve highly reproducible individual-level parcellation. Using High-quality diffusion MRI (dMRI), we first constructed a fine-grained group thalamus atlas based on thalamic local diffusion features. Then, the high-probability core area of the group thalamus atlas was wrapped into the native space as prior guidance for individualized thalamus construction. Finally, we trained the semi-supervised multiple classification models to accurately construct the individualized thalamus atlas with single-subject local diffusion characteristics. Compared to group atlas registration and single-subject clustering strategies, our individualized thalamus atlas combines population commonality and individual specificity and is superior in depicting the individual thalamic nuclei boundaries. Besides, our atlas provides a more conspicuous capacity to capture the individual specificity of thalamic nuclei. Through the evaluation by 3.0T\7.0T and test-retest dMRI datasets, the proposed high-probability group prior guided individualized thalamus construction pipeline is robust and repeatable in different magnetic field strengths and scanning batches. In addition, the individual parcellation of the thalamic nuclei has a good correspondence with the histological atlas and captured both higher group consistency and inter-subjects variations, which could be a valuable solution for precision clinical treatment.

neuroscience↗

bioRxiv2022

Foxm1 drives cardiomyocyte proliferation in adult zebrafish after cardiac injury

The regenerative capacity of the mammalian heart is poor with one potential reason being that adult cardiomyocytes cannot proliferate at sufficient levels to replace lost tissue. During development and early neonatal stages, cardiomyocytes can successfully divide under injury conditions; however, as these cells mature their ability to proliferate rapidly decreases. Therefore, understanding which regulatory programs are required to induce post-mitotic, mature cardiomyocytes into a proliferative state is essential in order to enhance cardiac regeneration. Unlike mammals, adult zebrafish cardiomyocytes in the injury border zone do proliferate. This model provides an opportunity to elucidate how these border zone cells respond to different stimuli post-injury and to study which regulatory programs are required for adult cardiomyocyte proliferation. Here we report the forkhead transcription factor, foxm1, is required for cardiomyocyte proliferation after cardiac injury through transcriptional regulation of cell cycle genes. Transcriptomic analysis of injured adult zebrafish hearts revealed that foxm1 expression is increased after injury in border zone cardiomyocytes. foxm1 mutants showed decreased cardiomyocyte proliferation after ventricular resection, resulting in larger fibrotic scars. Moreover, decreased expression of cell cycle progression genes suggests that Foxm1 is required for different cell cycle checkpoints during cardiomyocyte division. Subsequent analyses of Foxm1 targets revealed the microtubule and kinetochore binding protein, cenpf, is required for cardiac regeneration as cenpf mutants failed to regenerate due to increased cardiomyocyte binucleation. Thus, foxm1 and cenpf are required for cardiomyocytes to complete mitosis during zebrafish cardiac regeneration.

developmental biology↗

bioRxiv2022

DNA packaging via hierarchical chromatin structures revealed by live-cell 3D imaging

Single-molecule localization microscopy is a powerful superresolution imaging technique to study biological questions by visualizing subcellular fine structures with nanometer-scale precision. However, its application in live-cell imaging studies has been impeded by the paucity of self-blinking organic fluorophores that enable high spatiotemporal resolution and labeling/localization density at a moderate laser intensity. Herein, we report a self-blinking Si-rhodamine dye 6-HESiR with a suitably increased "ON" fraction and a fluorogenic self-blinking dsDNA probe 6-HoeHESiR as a powerful tool for 3D superresolution imaging of native chromatin in eukaryotes without the use of photoswitching buffer and high laser intensity. With the probe 6-HoeHESiR, 3D superresolution imaging of in vitro reconstituted nucleosomal arrays and chromatin fibers yielded results consistent with EM analysis. Similar euchromatin and heterochromatin structures were visualized in fixed and live cells with high spatiotemporal resolution and labeling density, providing the first live-cell evidence for a hierarchical model of chromatin organization. 3D imaging results obtained in the presence of selective inhibitors of histone deacetylases also corroborate chromatin fiber decompaction upon hyperacetylation of histones.

cell biology↗