Single RNA imaging in living cells offers precise insights into the spatiotemporal regulation of gene expression. However, achieving stable, efficient and reproducible RNA labeling remains challenging. To address this, we developed TSSOT (Tandem Split-mNeonGreen-based Signal Optimized mRNA Tracking), a robust live-cell imaging system that ensures consistent RNA labeling across multiple cell passages and provides highly reproducible, quantitative measurements at single-molecule resolution within individual cells. Leveraging TSSOT, we assessed the efficacy of various gene manipulation techniques and identified critical factors influencing Cas13d-mediated RNA cleavage. TSSOT further demonstrated that downregulation of mRNA export factors NXF1 and ALYREF leads to profound nuclear mRNA export defects. Furthermore, we employed TSSOT to spatiotemporally resolve the dynamic redistribution of mRNAs during stress granule formation. By providing precise quantification of RNA abundance, spatial organization, and temporal behavior, TSSOT is a valuable tool for live-cell RNA analysis, contributing to a better understanding of the dynamic RNA landscape.

DNA methylation is a significant component in proximal chromatin regulation and plays crucial roles in regulating gene expression and maintaining the repressive state of retrotransposon elements. However, accurate profiling of the proteomics which simultaneously identifies specific DNA sequences and their associated epigenetic modifications remains a challenge. Here, we report a strategy termed SelectID (selective profiling of epigenetic control at genome targets identified by dCas9), which introduces methylated DNA binding domain into dCas9-mediated proximity labeling system to enable in situ protein capture at repetitive elements with 5-methylcytosine (5mC) modifications. SelectID is demonstrated as feasible as dCas9-TurboID system at specific DNA methylation regions, such as the chromosome 9 satellite. Using SelectID, we successfully identify CHD4 as potential repressors of methylated long interspersed nuclear element-1 (LINE-1) retrotransposon through direct binding at the 5' untranslated region (5'UTR) of young LINE-1 elements. Overall, our SelectID approach has opened up avenues for uncovering potential regulators of specific DNA regions with DNA methylation, which will greatly facilitate future studies on epigenetic regulation.

CRISPR activation (CRISPRa) is a powerful tool for endogenous gene activation, yet the mechanisms underlying its optimal transcriptional activation remain unclear. By monitoring real-time transcriptional bursts, we find that CRISPRa modulates both burst duration and amplitude. Our quantitative imaging reveals that CRISPR-SunTag activators, with three tandem VP64-p65-Rta (VPR), form liquid-like transcriptional condensates and exhibit high activation potency. Although visible CRISPRa condensates are associated with some RNA bursts, the overall levels of phase separation do not correlate with transcriptional bursting or activation strength in individual cells. When the number of SunTag scaffolds is increased to 10 or more, solid-like condensates form, sequestering co-activators such as p300 and MED1. These condensates display low dynamicity and liquidity, resulting in ineffective gene activation. Overall, our studies characterize various phase-separated CRISPRa systems for gene activation, highlighting the foundational principles for engineering CRISPR-based programmable synthetic condensates with appropriate properties to effectively modulate gene expression.

B-cell acute lymphoblastic leukaemia (B-ALL) in adolescents and adults remains challenging due to high relapse rates and suboptimal outcomes. Although minimal residual disease (MRD) and cytogenetic risk classification are independent prognostic indicators for B-ALL in adolescents and adults, their combined utility remains under explored. This retrospective study analysed 609 adolescent and adult patients with B-ALL to evaluate the integrated prognostic value of MRD at end of induction (MRD1) and after first consolidation (MRD2), combined with cytogenetic risk stratification (standard risk [SR] vs. poor risk [PR]). Although cytogenetic risk alone was not an independent prognostic factor, MRD positivity at either time point significantly predicted inferior 5-year RFS and OS in PR patients. Allogeneic haematopoietic stem cell transplantation (allo-HSCT) improved outcomes overall; however, PR patients with MRD2 positivity remained at high risk of relapse post-transplant. Multivariate analysis identified MRD1 and MRD2 as independent predictors of both relapse (RFS HR = 2.048) and mortality (OS HR = 1.979) in PR patients (all p < 0.01). These results demonstrate that combining MRD assessment with cytogenetic risk improves risk stratification, precisely identifying poor-risk patients with persistent MRD who may benefit from treatment intensification, including novel strategies post-transplant.

Background : Secondary central nervous system lymphoma (SCNSL) characterized by the involvement of the CNS at initial diagnosis of systemic lymphoma or in the setting of relapse. Accurate diagnosis and therapeutic response of SCNSL are particular challenges, which need to be improved. We aimed to evaluate the diagnostic and the post-therapeutic prognostic value of cerebrospinal fluid (CSF) cytokine in patients with SCNSL. Methods: In this retrospective study involving 234 NHL patients and 53 other CNS patients, the NHL patients was divided into two sub-groups based on with and without CNS involvement: SCNSL (n=57) and non-SCNSL (n=177). The CSF cytokine profiles (interleukin (IL)-2, -4, -6, and -10, tumor necrosis factor-α, interferon-γ, and IL-17A) of these patients were measured by cytometric bead assay and evaluated the ability of CSF cytokine levels as diagnostic, therapeutic and prognostic biomarkers for SCNSL. Results: CSF IL-6 and IL-10 levels were significantly higher in SCNSL patients than non-SCNSL and other CNS patients. CSF levels of IL-6 and IL-10 were significantly higher in patients with an stage at Ⅲ and Ⅳ, CSF WBC counts>5/µL, CSF protein ≥0.45g/L.Using a CSF IL-6 cut-off value of 10.13 pg/ml yielded a diagnostic sensitivity and specificity was 62.34% and 87.57%, respectively (AUC, 0.7798; 95% CI, 0.7083 to 0.8514); For a CSF IL-10 cutoff value of 7.82, the sensitivity was 76.23%, and the specificity was 98.31% (AUC, 0.8164; 95% CI, 0.7466 to 0.8862). Combined CSF IL-6 and IL-10 significantly improved the diagnostic effect of SCNSL (AUC, 0.8959; 95% CI, 0.8371 to 0.9547). The levels of IL-6 and IL-10 in cerebrospinal fluid of SCNSL patients who responded to intrathecal chemotherapy were significantly decreased during follow-up, while the levels of these two cytokines showed fluctuating changes in patients with progressive SCNSL. Moreover, poor Progression free survival (PFS) for patients with SCNSL was associated with the increased CSF IL-10 level at diagnosis with CNS involvement (P=0.035), but not with the increased CSF IL-6 level. Conclusion: Our study confirmed CSF level of IL-6 and IL-10 were potentially effective diagnostic biomarkers for SCNSL, and poor progression free survival (PFS) for patients with SCNSL was associated with the increased CSF IL-10 level.

Maximizing cell survival under stress requires rapid and transient adjustments of RNA and protein synthesis. However, capturing these dynamic changes at both single-cell level and across an organism has been challenging. Here, we developed a system named MONITTR (MS2-embedded mCherry-based monitoring of transcription) for real-time simultaneous measurement of nascent transcripts and endogenous protein levels in C. elegans. Utilizing this system, we monitored the transcriptional bursting of fasting-induced genes and found that the epidermis responds to fasting by modulating the proportion of actively transcribing nuclei and transcriptional kinetics of individual alleles. Additionally, our findings revealed the essential roles of the transcription factors NHR-49 and HLH-30 in governing the transcriptional kinetics of fasting-induced genes under fasting. Furthermore, we tracked transcriptional dynamics during heat-shock response and ER unfolded protein response and observed rapid changes in the level of nascent transcripts under stress conditions. Collectively, our study provides a foundation for quantitatively investigating how animals spatiotemporally modulate transcription in various physiological and pathological conditions.

Neurons often grow highly branched and cell-type specific dendrite morphologies to receive and integrate information, which is the basis of precise neural circuit formation. Previous studies have identified numerous mechanisms that promote dendrite branching. In contrast, it is much less understood how this process is negatively regulated. Here we show that EAT-17/EVI5 acts together with the dynein adaptor protein BICD-1 and the motor protein dynein in C. elegans epidermal cells to restrict branching of PVD sensory dendrites. Loss-of-function mutants of these genes cause both ectopic branching and accumulation of the dendrite branching ligand SAX-7/L1CAM on epidermal plasma membranes. Mutants of genes regulating endo-lysosomal trafficking, including rab-5/RAB5 and dyn-1/DNM1, show similar defects. Biochemical characterization, genetic analysis, and imaging results support that EAT-17 and BICD-1 directly interact with each other and function in the endocytic degradation pathway to remove ectopically localized dendrite branching ligands to restrict abnormal branching. Fang et al. show that EAT-17 and BICD-1 function in the endocytic degradation pathway to suppress excessive dendrite branching, via down-regulating the level of SAX-7, a dendrite branching ligand, on the C. elegans epidermal plasma membranes.

Investigating gene function relies on the efficient manipulation of endogenous gene expression. Currently, a limited number of tools are available to robustly manipulate endogenous gene expression between “on” and “off” states. In this study, we insert a 63 bp coding sequence of T3H38 ribozyme into the 3’ untranslated region (UTR) of C. elegans endogenous genes using the CRISPR/Cas9 technology, which reduces the endogenous gene expression to a nearly undetectable level and generated loss-of-function phenotypes similar to that of the genetic null animals. To achieve conditional knockout, a cassette of loxP -flanked transcriptional termination signal and ribozyme is inserted into the 3’ UTR of endogenous genes, which eliminates gene expression spatially or temporally via the controllable expression of the Cre recombinase. Conditional endogenous gene turn-on can be achieved by either injecting morpholino, which blocks the ribozyme self-cleavage activity or using the Cre recombinase to remove the loxP -flanked ribozyme. Together, our results demonstrate that these ribozyme-based tools can efficiently manipulate endogenous gene expression both in space and time and expand the toolkit for studying the functions of endogenous genes.

RNA polymerase I (Pol I) synthesizes about 60% of cellular RNA by transcribing multiple copies of the ribosomal RNA gene (rDNA). The transcriptional activity of Pol I controls the level of ribosome biogenesis and cell growth. However, there is currently a lack of methods for monitoring Pol I activity in real time. Here, we develop LiveArt (live imaging-based analysis of rDNA transcription) to visualize and quantify the spatiotemporal dynamics of endogenous ribosomal RNA (rRNA) synthesis. LiveArt reveals mitotic silencing and reactivation of rDNA transcription, as well as the transcriptional kinetics of interphase rDNA. Using LiveArt, we identify SRFBP1 as a potential regulator of rRNA synthesis. We show that rDNA transcription occurs in bursts and can be altered by modulating burst duration and amplitude. Importantly, LiveArt is highly effective in the screening application for anticancer drugs targeting Pol I transcription. These approaches pave the way for a deeper understanding of the mechanisms underlying nucleolar functions.

Background: The development of diffuse large B-cell lymphoma (DLBCL), a prevalent subgroup of non-Hodgkin lymphoma (NHL), potentially involves various cytokines. We aimed to determine the correlation between deregulated serum levels of cytokines and clinical features and investigate their impact on the prognosis of patients with DLBCL. Methods: We conducted a retrospective study of 77 patients with newly diagnosed DLBCL to explore the relationships between different cytokines, adverse clinical features, and poor outcomes. The Mann-Whitney U test was used to compare the cytokine profiles between patients with DLBCL and healthy controls. The Kaplan-Meier method was used to analyze the probability of survival, and the log-rank tests were used to evaluate the differences between survival curves. The Cox proportional hazards regression model was used to performed univariate and multivariate analyses to evaluate prognostic variables for survival analyze. Results: Serum levels of interleukin-2 (IL-2), tumor necrosis factor (TNF)-& alpha;, IL-6, IL-10, and IFN-& gamma; were significantly elevated in patients with untreated DLBCL. Serum levels of IL-6 and IL-10 were significantly higher in patients with an International Prognostic Index (IPI) of 3-5, bone marrow involvement, serum levels of LDH > 250 U/L, and I32-microglobulin (I32-MG) levels > 2.3 mg/L. Patients with B symptoms only had higher serum IL10 levels, whereas patients with a partial response or no response to treatment had significantly elevated serum levels of IL-6 as well as IL-10. Significant positive correlations were observed between the levels of IL-6 and IL-10 with those of I32-MG and LDH. Patients with levels of IL-6 > 4.5 or IL-10 > 5.0 pg/mL, as well as combined elevated IL-6 and IL-10 levels, exhibited shorter progression-free survival and overall survival. Additionally, univariate and multivariate analyses revealed that serum levels of IL-6 > 4.5 pg/mL and IL-10 > 5.0 pg/mL and IPI 3-5 were independent prognostic factors for relapse and survival in patients with DLBCL. Conclusions: Pre-treatment serum IL-6 and IL-10 levels in patients with newly diagnosed DLBCL might be powerful markers for determining treatment response and predicting the prognosis of DLBCL.

The covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is the major obstacle to curing chronic hepatitis B (CHB). Current cccDNA detection methods are mostly based on biochemical extraction and bulk measurements. They nevertheless generated a general sketch of its biological features. However, an understanding of the spatiotemporal features of cccDNA is still lacking. To achieve this, we established a system combining CRISPR-Tag and recombinant HBV minicircle technology to visualize cccDNA at single-cell level in real time. Using this system, we found that the observed recombinant cccDNA (rcccDNA) correlated quantitatively with its active transcripts when a low to medium number of foci (<20) are present, but this correlation was lost in cells harboring high copy numbers (≥20) of rcccDNA. The disruption of HBx expression seems to displace cccDNA from the dCas9-accessible region, while HBx complementation restored the number of observable cccDNA foci. This indicated regulation of cccDNA accessibility by HBx. Second, observable HBV and duck HBV (DHBV) cccDNA molecules are substantially lost during cell division, and the remaining ones were distributed randomly to daughter cells. In contrast, Kaposi's sarcoma-associated herpesvirus (KSHV)-derived episomes can be retained in a LANA (latency-associated nuclear antigen)-dependent manner. Last, the dynamics of rcccDNA episomes in nuclei displayed confined diffusion at short time scales, with directional transport over longer time scales. In conclusion, this system enables the study of physiological kinetics of cccDNA at the single-cell level. The differential accessibility of rcccDNA to dCas9 under various physiological conditions may be exploited to elucidate the complex transcriptional and epigenetic regulation of the HBV minichromosome. IMPORTANCE Understanding the formation and maintenance of HBV cccDNA has always been a central issue in the study of HBV pathobiology. However, little progress has been made due to the lack of robust assay systems and its resistance to genetic modification. Here, a live-cell imaging system by grafting CRISPR-Tag into the recombinant cccDNA was established to visualize its molecular behavior in real time. We found that the accessibility of rcccDNA to dCas9-based imaging is related to HBx-regulated mechanisms. We also confirmed the substantial loss of observable rcccDNA in one-round cell division and random distribution of the remaining molecules. Molecular dynamics analysis revealed the confined movement of the rcccDNA episome, suggesting its juxtaposition to chromatin domains. Overall, this novel system offers a unique platform to investigate the intranuclear dynamics of cccDNA within live cells.

Herpes simplex virus 1 (HSV-1), a representative of the family Herpesviridae , is a ubiquitous pathogen that can establish lifelong infections and widely affects human health. Viral infection is a dynamic process that involves many steps and interactions with various cellular structures, including host chromatin.

Technologies for gene activation are valuable tools for the study of gene functions and have a wide range of potential applications in bioengineering and medicine. In contrast to existing methods based on recruiting transcriptional modulators via DNA-binding proteins, we developed a strategy termed Narta (nascent RNA-guided transcriptional activation) to achieve gene activation by recruiting artificial transcription factors (aTFs) to transcription sites through nascent RNAs of the target gene. Using Narta, we demonstrate robust activation of a broad range of exogenous and endogenous genes in various cell types, including zebrafish embryos, mouse and human cells. Importantly, the activation is reversible, tunable and specific. Moreover, Narta provides better activation potency of some expressed genes than CRISPRa and, when used in combination with CRISPRa, has an enhancing effect on gene activation. Quantitative imaging illustrated that nascent RNA-directed aTFs could induce the high-density assembly of coactivators at transcription sites, which may explain the larger transcriptional burst size induced by Narta. Overall, our work expands the gene activation toolbox for biomedical research.

Cortical actin, a thin layer of actin network underneath the plasma membranes, plays critical roles in numerous processes, such as cell morphogenesis and migration. Neurons often grow highly branched dendrite morphologies, which is crucial for neural circuit assembly. It is still poorly understood how cortical actin assembly is controlled in dendrites and whether it is critical for dendrite development, maintenance and function. In the present study, we find that knock-out of C. elegans chdp-1, which encodes a cell cortex-localized protein, causes dendrite formation defects in the larval stages and spontaneous dendrite degeneration in adults. Actin assembly in the dendritic growth cones is significantly reduced in the chdp-1 mutants. PVD neurons sense muscle contraction and act as proprioceptors. Loss of chdp-1 abolishes proprioception, which can be rescued by expressing CHDP-1 in the PVD neurons. In the high-ordered branches, loss of chdp-1 also severely affects the microtubule cytoskeleton assembly, intracellular organelle transport and neuropeptide secretion. Interestingly, knock-out of sax-1, which encodes an evolutionary conserved serine/threonine protein kinase, suppresses the defects mentioned above in chdp-1 mutants. Thus, our findings suggest that CHDP-1 and SAX-1 function in an opposing manner in the multi-dendritic neurons to modulate cortical actin assembly, which is critical for dendrite development, maintenance and function.

Background: Identifying specific risk factors associated with multiple myeloma (MM) remains a significant issue. Different cytokines take part in the pathogenesis, progression, and prognosis of MM. Therefore, this study aimed to investigate the correlations between serum cytokine levels and clinical characteristics and determine their effects on disease progression and survival of MM patients. Methods: We retrospectively analyzed the serum levels of 7 cytokines in 105 patients with newly diagnosed MM and in 20 healthy subjects. Interleukin (IL)-2, IL-4, IL-6, IL-10, and IL-17A, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma were quantitatively determined by cytometric bead assay techniques. The concentrations of each cytokine were compared between the MM patients and healthy subjects using the Mann-Whitney U test. The Kaplan-Meier method was used to analyze progression-free survival (PFS) and overall survival (OS). Results: Serum IL-2, IL-4, IL-6, IL-10, IL-17A, TNF-alpha, and IFN-gamma levels were higher in patients with newly diagnosed MM than in healthy controls. Positively significant correlations were found between IL-6, IL-10, IL-17A, and beta 2-microglobulin. Significant correlations were also observed between IL-6 and IL-10, and lactate dehydrogenase. The overall response rate of low-IL-6 and IL-17A patients was significantly higher than that of high-IL-10 and IL-17A patients (P < 0.01). Univariate and multivariate analyses revealed that serum IL-6 levels were >3 pg/mL, serum IL-17A levels were >4 pg/mL, and treatment regimens were independent prognostic factors for PFS and OS. Conclusions: Cytokine deregulation, especially that of IL-6 and IL-17A, may be a powerful predictor of clinical prognosis for MM patients.

A wealth of single-cell imaging studies have contributed novel insights into chromatin organization and gene regulation. However, a comprehensive understanding of spatiotemporal gene regulation requires developing tools to combine multiple monitoring systems in a single study. Here, we report a versatile tag, termed TriTag, which integrates the functional capabilities of CRISPR-Tag (DNA labeling), MS2 aptamer (RNA imaging) and fluorescent protein (protein tracking). Using this tag, we correlate changes in chromatin dynamics with the progression of endogenous gene expression, by recording both transcriptional bursting and protein production. This strategy allows precise measurements of gene expression at single-allele resolution across the cell cycle or in response to stress. TriTag enables capturing an integrated picture of gene expression, thus providing a powerful tool to study transcriptional heterogeneity and regulation.

CRISPR/Cas-based mRNA imaging has been developed to labeling of high-abundance mRNAs. A lack of non-genetically encoded mRNA-tagged imaging tools has limited our ability to explore the functional distributions of endogenous low-abundance mRNAs in cells. Here, we developed a CRISPR-Sunspot method based on the SunTag signal amplification system that allows efficient imaging of low-abundance mRNAs with CRISPR/Cas9. Methods: We created a stable TRE3G-dCas9-EGFP cell line and generated an Inducible dCas9-EGFP imaging system for assessment of two factors, sgRNA and dCas9, which influence imaging quality. Based on SunTag system, we established a CRISPR-Sunspot imaging system for amplifying signals from single-molecule mRNA in live cells. CRISPR-Sunspot was used to track co-localization of Camk2a mRNA with regulatory protein Xlr3b in neurons. CRISPR-Sunspot combined with CRISPRa was used to determine elevated mRNA molecules. Results: Our results showed that manipulating the expression of fluorescent proteins and sgRNA increased the efficiency of RNA imaging in cells. CRISPR-Sunspot could target endogenous mRNAs in the cytoplasm and amplified signals from single-molecule mRNA. Furthermore, CRISPR-Sunspot was also applied to visualize mRNA distributions with its regulating proteins in neurons. CRISPR-Sunspot detected the co-localization of Camk2a mRNA with overexpressed Xlr3b proteins in the neuronal dendrites. Moreover, we also manipulated CRISPR-Sunspot to detect transcriptional activation of target gene such as HBG1 in live cells. Conclusion: Our findings suggest that CRISPR-Sunspot is a novel applicable imaging tool for visualizing the distributions of low-abundance mRNAs in cells. This study provides a novel strategy to unravel the molecular mechanisms of diseases caused by aberrant mRNA molecules.

Hereditary sensory neuropathy (HSN) comprises a group of progressive peripheral neuropathies predominantly affecting the sensory nerves. To date, two different ATL3 gene mutations have been reported to be responsible for HSN type 1F (HSN1F). Here, we report a family in which the members presented numbness of the lower limbs and recurrent foot ulceration. Symptoms of foot ulcers disappeared in the years after onset, which suggests that the family members showed benign and mild symptoms compared with the affected patients reported previously. Laboratory examinations and electrophysiological data suggested axonal degeneration of the peripheral sensory nerves, while motor neurons were not involved. Exome sequencing revealed the previously reported c.C1013G (p.Pro338Arg) mutation of the ATL3 gene. This is the first report of ATL3 mutation in Chinese patients with HSN. Cells expressing mutant ATL3 exhibited disruption of the endoplasmic reticulum network, suggesting a dominant-negative effect. There was no significant difference in the expression of the endoplasmic reticulum stress marker binding immunoglobulin protein (BiP) between cells expressing wild-type or mutant ATL3. Further studies are required to ascertain the relevance of the changes in endoplasmic reticulum morphology to axonal degeneration of sensory nerves.