Nuclear Protein Research Articles

Antisense Oligonucleotide as a New Technology Application for CsLOB1 Gene Silencing Aiming at Citrus Canker Resistance.

Citrus canker disease, caused by Xanthomonas citri subsp. citri, poses a significant threat to global citrus production. The control of the disease in the field relies mainly on the use of conventional tools such as copper compounds, which are harmful to the environment and could lead to bacterial resistance. This scenario stresses the need for new and sustainable technologies to control phytopathogens, representing a key challenge in developing studies that translate basic into applied knowledge. During infection, X. citri subsp. citri secretes a transcriptional activator-like effector that enters the nucleus of plant cells, activating the expression of the canker susceptibility gene LATERAL ORGAN BOUNDARIES 1 (LOB1). In this study, we explored the use of antisense oligonucleotides (ASOs) with phosphorothioate modifications to transiently inhibit the gene expression of CsLOB1 in Citrus sinensis. We designed and validated three potential ASO sequences, which led to a significant reduction in disease symptoms compared with the control. The selected ASO3-CsLOB1 significantly decreased the expression level of CsLOB1 when delivered through two distinct delivery methods, and the reduction of the symptoms ranged from approximately 15 to 83%. Notably, plants treated with ASO3 did not exhibit an increase in symptom development over the evaluation period. This study highlights the efficacy of ASO technology, based on short oligonucleotide chemically modified sequences, as a promising tool for controlling phytopathogens without the need for genetic transformation or plant regeneration. Our results demonstrate the potential of ASOs as a biotechnological tool for the management of citrus canker disease.

XPO1 serves as a prognostic marker involving AKT/MAPK/TGFBR1 pathway in OSCC.

Exportin 1 (XPO1) is a nuclear export protein that facilitates the transportation of various substances. XPO1 promotes tumor development as a poor prognostic factor in a variety of tumors and is a therapeutic target for screening inhibitors. However, the role of XPO1 in oral squamous cell carcinoma (OSCC) has yet to be determined. The expression patterns of XPO1 mRNA in OSCC were investigated using bioinformatics tools, and the expression levels of XPO1 protein in OSCC specimens were confirmed by immunohistochemical assays. Survival analysis was conducted to evaluate the impact of XPO1 on prognosis. GO and KEGG enrichment analyses were utilized to uncover the signaling pathways mediated by XPO1. Additionally, we examined the association between XPO1 and AKT/MAPK/TGFBR1 and immune infiltration. XPO1 mRNA and protein expression levels were significantly enhanced in OSCC and associated with OSCC severity. Enhanced XPO1 expression was indicative of poor survival. Functional analysis showed that XPO1 mediated pathways associated with cell cycle and DNA replication and reduced immune infiltration in OSCC. Additionally, XPO1 mRNA and protein expression levels had significant positive relationships with AKT/MAPK/TGFBR1. XPO1, as a marker of poor prognosis in OSCC, can promote OSCC through AKT/MAPK/TGFBR1.

Molecular and toxicological mechanisms behind the effects of chromium (VI) on the male reproductive system of Mytilus galloprovincialis: First evidence for poly-ADP-ribosylation of protamine-like II

Studies on the molecular mechanisms of heavy metal toxicity in invertebrate reproduction are limited. Given that PARP-catalysed ADP-ribosylation is also involved in counteracting heavy metal toxicity and maintaining genomic integrity, and that PARylation is implicated in chromatin remodelling but its role in sperm chromatin remains to be elucidated, we investigated the effects of chromium(VI) at 1, 10 and 100 nM on the reproductive health of Mytilus galloprovincialis. The damage to the gonads was assessed by morphological analyses and the damage indices PARP and ɣH2A.X were measured. Changes in the binding of protamine-like (PL) to DNA and the possibility of poly(ADP-ribosyl)ation of PL proteins were also investigated. Gonadal chromium accumulation and morphological damage were found, especially when the mussels were exposed to the highest dose of chromium(VI). In addition, the maximum expression of gonadal ɣH2A.X and PARP were obtained at 100 and 10 nM Cr(VI), respectively. Interestingly, for the first time in all exposed conditions, poly(ADP)-ribosylation was detected on PL-II, which, together with PL-III and PL-IV, are the major nuclear basic proteins of Mytilus galloprovincialis sperm chromatin. Since PL-II is involved in the final high level of sperm chromatin compaction, this post-translational modification altered the binding of the PL protein to DNA, favouring the action of micrococcal nuclease on sperm chromatin. This study provides new insights into the effects of chromium(VI) on Mytilus galloprovincialis reproductive system and proposes a molecular mechanism hypothesis describing the toxic effects of this metal on PL-DNA binding, sperm chromatin and gonads.

NRBP1 promotes malignant phenotypes of glioblastoma by regulating PI3K/Akt activation.

Glioblastoma (GBM) is the most aggressive of intracranial gliomas. Despite the maximal treatment intervention, the median survival rate is still about 14-16 months. Nuclear receptor-binding protein 1 (NRBP1) has a potential growth-promoting role on biology function of cells. In this study, we investigated whether NRBP1 promotes GBM malignant phenotypes and the potential mechanisms. The correlation between NRBP1 and glioma grade, prognosis in TCGA/CGGA databases and our clinical data were analyzed. Next, we conducted knockout and overexpression of NRBP1 on GBM cells to verify that NRBP1 promoted cell proliferation, invasion, and migration invitro and invivo. Finally, we detected the impact of NRBP1 on PI3K/Akt signaling pathway and EMT. There was a correlation between elevated NRBP1 expression and advanced stage glioma, as well as decreased overall and disease-free survival. The suppression of proliferation, invasion, and migration of tumor cells was observed upon NRBP1 knockout, and invitro studies also demonstrated the induction of apoptotic cell death. Whereas, its overexpression is associated with high multiplication rate, migration, invasion, and apoptotic escape. GO enrichment and KEGG analysis revealed that NRBP1 regulated differentially expressed gene clusters are involved in PI3K/Akt signaling pathway, as well as EMT mediated by this pathway. Moreover, the effects of NRBP1 knockdown and overexpression on GBM were mitigated by MK-2206 and SC79, both of which respectively function as an inhibitor and an activator of the PI3K/Akt signaling pathway. Similarly, the suppression of NRBP1 led to a decrease in tumor growth, whereas its overexpression promoted tumor growth in a mouse model. This study shows that NRBP1 promotes malignant phenotypes in GBM by activating PI3K/Akt pathway. Hence, it can function as both a predictive indicator and a new target for therapies in GBM treatment.

Tape-assisted fabrication method for constructing PDMS membrane-containing culture devices with cyclic radial stretching stimulation.

Advanced in vitro culture systems have emerged as alternatives to animal testing and traditional cell culture methods in biomedical research. Polydimethylsiloxane (PDMS) is frequently used in creating sophisticated culture devices owing to its elastomeric properties, which allow mechanical stretching to simulate physiological movements in cell experiments. We introduce a straightforward method that uses three types of commercial tape-generic, magic and masking-to fabricate PDMS membranes with microscale thicknesses (47.2 µm for generic, 58.1 µm for magic and 89.37 µm for masking) in these devices. These membranes are shaped as the bases of culture wells and can perform cyclic radial movements controlled via a vacuum system. In experiments with A549 cells under three mechanical stimulation conditions, we analysed transcriptional regulators responsive to external mechanical stimuli. Results indicated increased nuclear yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) activity in both confluent and densely packed cells under cyclically mechanical strains (Pearson's coefficient (PC) of 0.59 in confluent and 0.24 in dense cells) compared with static (PC = 0.47 in confluent and 0.13 in dense) and stretched conditions (PC = 0.55 in confluent and 0.20 in dense). This technique offers laboratories without microfabrication capabilities a viable option for exploring cellular behaviour under dynamic mechanical stimulation using PDMS membrane-equipped devices.

Effect of Selinexor on Proliferation and Apoptosis of Acute Myeloid Leukemia Kasumi-1 Cells

To investigate the effects of selinexor, a inhibitor of nuclear export protein 1 (XPO1) on the proliferation inhibition and apoptosis of Kasumi-1 cells in acute myeloid leukemia (AML). MTS method was used to detect the inhibitory effect of different concentrations of selinexor on the proliferation of Kasumi-1 cells at different time points. The apoptosis rate and cell cycle changes after treatment with different concentration of selinexor were detected by flow cytometry. Selinexor inhibited the growth of Kasumi-1 cells at different time points in a concentration-dependent manner (r 24 h=0.7592, r 48 h=0.9456, and r 72 h=0.9425). Selinexor inhibited Kasumi-1 cells growth in a time-dependent manner (r =0.9057 in 2.5 μmol/L group, r =0.9897 in 5 μmol/L group and r =0.9994 in 10 μmol/L group). Selinexor could induce apoptosis of Kasumi-1 cells in a dose-dependent manner (r =0.9732), and the apoptosis of Kasumi-1 cells was more obvious with the increase of drug concentration. The proportion of G0/G1 phase was significantly increased and the proportion of S phase was significantly decreased after the treatment of Kasumi-1 cells by selinexor. With the increase of drug concentration, the proportion of Kasumi-1 cells cycle arrest in G0/G1 phase was increased and the cell synthesis was decreased. Selinexor can promote the death of tumor cells by inhibiting Kasumi-1 cells proliferation, inducing apoptosis and blocking cell cycle.

A multiscale modeling to determine in vitro mechanical responses of different cells at the cell-substrate interface under fluid perfusion.

Investigating the influence of different cellular mechanical and physical properties on cells in vitro is important for assessing cellular activities like differentiation, proliferation, and migration. Evaluating the mechanical response of the cells lodged on a scaffold due to variations in substrate roughness, substrate elasticity, fluid flow, and the shapes of the cells is the main goal of the study. In this comprehensive analysis, a combination of the fluid structure interaction method and the submodeled finite element technique was employed to anticipate the mechanical responses across various cells at the interface between cells and the substrate. Fluid inlet velocity, substrate roughness, and substrate material were varied in this analysis. Different cell shapes were considered along with various components such as cell membrane, cytoplasm, nucleus, and cytoskeletons. This analysis shows the effect of these individual parameters on the elastic strain and strain energy density of cells at the cell-substrate interface. The results highlight that substrate roughness has a more significant impact on the mechanical response of cells at the interface than substrate elasticity. However, effect of the substrate elasticity becomes crucial for extremely soft substrate materials. The results of this research can be applied to identify the optimal parameters for fluid flow and create a suitable condition for cell culture.

Membranous translocation of murine double minute 2 promotes the increased renal tubular immunogenicity in ischemia-reperfusion-induced acute kidney injury.

Kidneys from donors with prolonged warm and cold ischemia are prone to posttransplant T cell-mediated rejection (TCMR) due to ischemia-reperfusion injury (IRI). However, the precise mechanisms still remain obscure. Renal tubular epithelial cells (TECs) are the main target during IRI. Meanwhile, we have previously reported that murine double minute 2 (MDM2) actively participates in TEC homeostasis during IRI. In this study, we established a murine model of renal IRI and a cell model of hypoxia-reoxygenation by culturing immortalized rat renal proximal tubule cells (NRK-52E) in a hypoxic environment for different time points followed by 24 h of reoxygenation and incubating NRK-52E cells in a chemical anoxia-recovery environment. We found that during renal IRI MDM2 expression increased on the membrane of TECs and aggregated mainly on the basolateral side. This process was accompanied by a reduction of a transmembrane protein, programmed death ligand 1 (PD-L1), a coinhibitory second signal for T cells in TECs. Using mutant plasmids of MDM2 to anchor MDM2 on the cell membrane or nuclei, we found that the upregulation of membrane MDM2 could promote the ubiquitination of PD-L1 and lead to its ubiquitination-proteasome degradation. Finally, we set up a coculture system of TECs and CD4+ T cells in vitro; our results revealed that the immunogenicity of TECs was enhanced during IRI. In conclusion, our findings suggest that the increased immunogenicity of TECs during IRI may be related to ubiquitinated degradation of PD-L1 by increased MDM2 on the cell membrane, which consequently results in T-cell activation and TCMR.NEW & NOTEWORTHY Ischemic acute kidney injury (AKI) donors can effectively shorten the waiting time for kidney transplantation but increase immune rejection, especially T cell-mediated rejection (TCMR), the mechanism of which remains to be elucidated. Our study demonstrates that during ischemia-reperfusion injury (IRI), the translocation of tubular murine double minute 2 leads to basolateral programmed death ligand 1 degradation, which ultimately results in the occurrence of TCMR, which may provide a new therapeutic strategy for preventing AKI donor-associated TCMR.

Viral hijacking of hnRNPH1 unveils a G-quadruplex-driven mechanism of stress control

Viral genomes are enriched with G-quadruplexes (G4s), non-canonical structures formed in DNA or RNA upon assembly of four guanine stretches into stacked quartets. Because of their critical roles, G4s are potential antiviral targets, yet their function remains largely unknown. Here, we characterize the formation and functions of a conserved G4 within the polymerase coding region of orthoflaviviruses of the Flaviviridae family. Using yellow fever virus, we determine that this G4 promotes viral replication and suppresses host stress responses via interactions with hnRNPH1, a host nuclear protein involved in RNA processing. G4 binding to hnRNPH1 causes its cytoplasmic retention with subsequent impacts on G4-containing tRNA fragments (tiRNAs) involved in stress-mediated reductions in translation. As a result, these host stress responses and associated antiviral effects are impaired. These data reveal that the interplay between hnRNPH1 and both host and viral G4 targets controls the integrated stress response and viral replication.

GSK3β Inhibitors Inhibit TGFβ Signaling in the Human Trabecular Meshwork.

Primary open-angle glaucoma (POAG) is a leading cause of blindness, and its primary risk factor is elevated intraocular pressure (IOP) due to pathologic changes in the trabecular meshwork (TM). We previously showed that there is a cross-inhibition between TGFβ and Wnt signaling pathways in the TM. In this study, we determined if activation of the Wnt signaling pathway using small-molecule Wnt activators can inhibit TGFβ2-induced TM changes and ocular hypertension (OHT). Primary human TM (pHTM) cells and transduced SBE-GTM3 cells were treated with or without Wnt and/or TGFβ signaling activators and used for luciferase assays; for the extraction of whole-cell lysate, conditioned medium, cytosolic proteins, and nuclear proteins for Western immunoblotting (WB); or for immunofluorescent staining. Human donor eyes were perfusion cultured to study the effect of Wnt activators on IOP. We found that the small-molecule Wnt activators (GSK3β inhibitors) (BIO, SB216763, and CHIR99021) activated canonical Wnt signaling in pHTM cells without toxicity at tested concentrations. This activation inhibited TGFβ signaling as well as TGFβ2-induced extracellular matrix deposition and formation of cross-linked actin networks in pHTM cells or SBE-GTM3 cells. We also observed nuclear translocation of both Smad4 and β-catenin in pHTM cells, which suggested that the cross-inhibition between the TGFβ and Wnt signaling pathways may occur in the nucleus. Using our ex vivo model, we found that CHIR99021 inhibited TGFβ2-induced OHT in perfusion-cultured human eyes. Our results showed that small-molecule Wnt activators have the potential for treating TGFβ signaling-induced OHT in patients with POAG.

Protective Effects of Curcumin and its Analogues via the Nrf2 Pathway in Metabolic Syndrome.

Metabolic Syndrome (MetS) refers to a set of medical conditions including insulin resistance, central obesity, atherogenic dyslipidemia, and hypertension. Due to these dysregulations, if not treated, MetS could increase the risk of CVA, CVD, and diabetes. As described by WHO, CVD is the leading cause of mortality in the world which motivates researchers to investigate the management of its risk factors, especially MetS. It is reported that oxidative stress secondary to the abundant generation of free radicals oxygen species (ROS) and the ensuing altered redox status play an important role as a mediator in MetS. As a result, using new antioxidant agents with higher bioavailability has been proposed as an efficient treatment. Curcumin (a polyphenol of the diarylheptanoids class), which is used as a traditional medicine for various diseases including cardiovascular diseases and diabetes, is characterized by its antioxidant properties which, at least in part, are mediated via the activation of the Nrf2/ARE signaling pathway. Nrf2 is a transcription factor that plays a key role in regulating internal defense systems and increases antioxidant levels to decrease oxidative damage and cell apoptosis. Nrf2 expression and stability are enhanced by curcumin, leading to a higher rate of Nrf2 migration to the cell nucleus to regulate ARE gene expression, thus protecting cells against oxidative stress. In this article, we provide a comprehensive review of the molecular effect of curcumin and its derivatives via Nrf2 regulation in several conditions, such as diabetes, hypertension, dyslipidemia, and obesity.

Specific spectral sub-images for machine learning evaluation of optical differences between carbon ion and X ray radiation effects

Advances in radiotherapy, particularly the exploration of alternative radiation types such as carbon ions have updated our understanding of its effects and applicability on chondrosarcoma cells. Here we compare the optical effects produced by carbon ions (CI) and X-rays (XR) radiations on chondrosarcoma cells nuclei and set an automated method for evaluating the radiation-induced alterations without the need of chemical marking. Hyperspectral images (HSI) of SW1353 chondrosarcoma line carry detectable optical changes of the cells irradiated either with CI or XR compared to non-irradiated ones (REF). The differences between the spectral profiles of CI, XR and REF nuclei classes led to partitioning the HSIs into spectral sub-images. The changes are detected by support vector machine (SVM) classifiers whose performances are evaluated by the most used point metrics: sensitivity (SEN), accuracy (ACC), and precision (PREC), applied on spatial feature values. Specific interaction mechanisms by radiation type reveal distinct subintervals where HSIs changes are more prominent, and the classifiers perform at best. For CI the best classifiers are obtained for sub-images in the interval (424–436 nm), while for XR the best classifiers are obtained for sub-images in the interval (436–445 nm). The classifiers work better with texture features than roughness features in both cases. The classifier with the best SEN point metric in the testing phase is the most suitable to measure the irradiation efficiency irrespective of the radiation type. The altered nuclei are easier to discriminate when irradiated with CI than with XR. The study proves that SVM with optical data offers a rapid, automated, and label-free method for evaluating radiation-induced alterations in chondrosarcoma nuclei, thereby enabling effective analysis of extensive data.

Study on the Effects of Low-Intensity Pulsed Ultrasound and Iron Ions for Proliferation and Differentiation of Osteoblasts

ObjectiveThis study involved the proliferation and differentiation of osteoblasts treated with low-intensity pulsed ultrasound (LIPUS) and iron (Fe3+) ions, respectively. The biological effects of LIPUS and Fe3+ ions on the proliferation and differentiation of osteoblasts were also evaluated. MethodsMC3T3-E1 cells were seeded in six-well plates with the medium, which contained different concentrations of Fe3+ (0, 100, 200, 300, 400, 500, 600 and 700 μg L-1, respectively). LIPUS treatment was directed at the bottom of the plate for 20 min at an intensity of 80 mW cm-2 every day. ResultsViability results showed that a dose of 400 μg L-1 Fe3+ ions had the best effect at promoting osteogenic proliferation in cell culture. The results of alkaline phosphatase staining and mineralization indicated that the differentiation of osteoblasts was promoted by LIPUS and Fe3+ ions. Fluorescence staining results showed that the number of cell nuclei in the LIPUS, Fe3+ and LIPUS-Fe groups increased by 37.20%, 55.81% and 89.76%, respectively. Migration data indicated that migration and proliferation rates were increased by LIPUS and Fe3+, and the results of protein expression indicated that LIPUS and Fe3+ may increase the expression of Wnt, β-catenin, and Runx2, hence promoting normal bone regeneration and development. ConclusionThe combination of LIPUS (1.5 MHz, 80 mW cm-2) and Fe3+ accelerates the proliferation and differentiation of osteoblasts significantly compared with single-factor treatment (stimulated by LIPUS and Fe3+ ions, respectively). This study could establish a foundation for LIPUS-responsive biomaterials in the repair and regeneration of bone tissues.

Transcriptomic Identification of Potential C2H2 Zinc Finger Protein Transcription Factors in Pinus massoniana in Response to Biotic and Abiotic Stresses.

Biotic and abiotic stresses have already seriously restricted the growth and development of Pinus massoniana, thereby influencing the quality and yield of its wood and turpentine. Recent studies have shown that C2H2 zinc finger protein transcription factors play an important role in biotic and abiotic stress response. However, the members and expression patterns of C2H2 TFs in response to stresses in P. massoniana have not been performed. In this paper, 57 C2H2 zinc finger proteins of P. massoniana were identified and divided into five subgroups according to a phylogenetic analysis. In addition, six Q-type PmC2H2-ZFPs containing the plant-specific motif 'QALGGH' were selected for further study under different stresses. The findings demonstrated that PmC2H2-ZFPs exhibit responsiveness towards various abiotic stresses, including drought, NaCl, ABA, PEG, H2O2, etc., as well as biotic stress caused by the pine wood nematode. In addition, PmC2H2-4 and PmC2H2-20 were nuclear localization proteins, and PmC2H2-20 was a transcriptional activator. PmC2H2-20 was selected as a potential transcriptional regulator in response to various stresses in P. massoniana. These findings laid a foundation for further study on the role of PmC2H2-ZFPs in stress tolerance.

Nuclear Argonaute protein NRDE-3 switches small RNA binding partners during embryogenesis coincident with the formation of SIMR granules.

RNA interference (RNAi) is a conserved gene regulation mechanism that utilizes the Argonaute protein and their associated small RNAs to exert regulatory function on complementary transcripts. While the majority of germline-expressed RNAi pathway components reside in perinuclear germ granules, it is unknown whether and how RNAi pathways are spatially organized in other cell types. Here we find that the small RNA biogenesis machinery is spatially and temporally organized during embryogenesis. Specifically, the RNAi factor, SIMR-1, forms visible concentrates during mid-embryogenesis that contain an RNA-dependent RNA polymerase, a poly-UG polymerase, and the unloaded nuclear Argonaute protein, NRDE-3. Further, we observe that many other RNAi factors form foci in embryonic cells distinct from SIMR granules, including the Argonaute protein CSR-1, underscoring a potential role for cytoplasmic concentrates of RNAi factors to promote gene regulation in embryos. Curiously, coincident with the appearance of the "SIMR granules", the small RNAs bound to NRDE-3 switch from predominantly CSR-class 22G-RNAs to ERGO-dependent 22G-RNAs. Thus, our study defines two separable roles for NRDE-3, targeting germline-expressed genes during early embryogenesis and switching later in embryogenesis to repress recently duplicated genes and retrotransposons in somatic cells, highlighting the plasticity of Argonaute proteins and the need for more precise temporal characterization of Argonaute-small RNA interactions.

Segmented fluorescence correlation spectroscopy (FCS) on a commercial laser scanning microscope

Performing accurate Fluorescence Correlation Spectroscopy (FCS) measurements in cells can be challenging due to cellular motion or other intracellular processes. In this respect, it has recently been shown that analysis of FCS data in short temporal segments (segmented FCS) can be very useful to increase the accuracy of FCS measurements inside cells. Here, we demonstrate that segmented FCS can be performed on a commercial laser scanning microscope (LSM), even in the absence of the dedicated FCS module. We show how data can be acquired on a Leica SP8 confocal microscope and then exported and processed with a custom software in MATLAB. The software performs segmentation of the data to extract an average ACF and measure the diffusion coefficient in specific subcellular regions. First of all, we measure the diffusion of fluorophores of different size in solution, to show that good-quality ACFs can be obtained in a commercial LSM. Next, we validate the method by measuring the diffusion coefficient of GFP in the nucleus of HeLa cells, exploiting variations of the intensity to distinguish between nucleoplasm and nucleolus. As expected, the measured diffusion coefficient of GFP is slower in the nucleolus relative to nucleoplasm. Finally, we apply the method to HeLa cells expressing a PARP1 chromobody to measure the diffusion coefficient of PARP1 in different subcellular regions. We find that PARP1 diffusion is slower in the nucleolus compared to the nucleoplasm.

TOX2 nuclear-cytosol translocation is linked to leukemogenesis of acute T-cell leukemia by repressing TIM3 transcription.

Nuclear factors TOX and TOX2 upregulate TIM3 expression and lead to T-cell exhaustion in malignancies. Here, we demonstrate two distinct TIM3 expression patterns (high & low) with high TOX and TOX2 levels in T-cell acute lymphoblastic leukemia (T-ALL) specimens and cell lines. However, the mechanisms regulated by TOX and TIM3 signaling in leukemogenesis are unclear. We found that TOX and TOX2 proteins each directly upregulated HAVCR2 transcription, while the cellular localization of TOX2 was different in Jurkat and MOLT3 cells (nucleus) and lymphoblastic cell T2 and normal T cells (cytoplasm). Nuclear TOX and TOX2 formed a protein complex and repressed HAVCR2 promoter activity by recruiting transcriptional corepressor LCOR and deacetylase HDAC3. The nuclear-cytosol translocation of TOX2 was deacetylation-dependent and cooperatively mediated by deacetylase Sirt1 and kinase TBK1. Radiation damage induced TOX2 nuclear translocation and decreased Sirt1, TIM3, and caspase 1 expression in normal T cells. Accordingly, knockdown of TOX, TOX2 or LCOR; HDAC3 inhibition; or TIM3 overexpression induced Jurkat cell apoptosis in vitro and slow growth in vivo. Thus, our findings demonstrate a novel regulatory mechanism involving TOX-TOX2 and the TIM3 pathway in the leukemogenesis of T-ALL.

Optogenetic Strategies for Optimizing the Performance of Phospholipids Biosensors.

High-performance biosensors play a crucial role in elucidating the intricate spatiotemporal regulatory roles and dynamics of membrane phospholipids. However, enhancing the sensitivity and imaging performance remains a significant challenge. Here, optogenetic-based strategies are presented to optimize phospholipid biosensors. These strategies involves presequestering unbound biosensors in the cell nucleus and regulating their cytosolic levels with blue light to minimize background signal interference in phospholipid detection, particularly under conditions of high expression levels of biosensor. Furthermore, optically controlled phase separation and the SunTag system are employed to generate punctate probes for substrate detection, thereby amplifying biosensor signals and enhancing visualization of the detection process. These improved phospholipid biosensors hold great potential for enhancing the understanding of the spatiotemporal dynamics and regulatory roles of membrane lipids in live cells and the methodological insights in this study might be valuable for developing other high-performance biosensors.

Over Activation of IL-6/STAT3 Signaling Pathway in Juvenile Dermatomyositis.

Juvenile dermatomyositis (JDM) is characterized by persistent non-purulent inflammation in the muscle and skin. The underlying mechanisms still remain uncertain. This study aims to elucidate the mechanism of interleukin-6 (IL-6) activation of Janus kinase/signal transducer and activator of transcription 3 pathway (JAK/STAT3), contributing to the pathogenesis of JDM. Serum IL-6 levels were compared between 72 newly diagnosed patients with JDM and the same patient cohort in treatment remission. Single-cell RNA sequencing (scRNA-seq) was employed to identify differential signaling pathway expression in muscle biopsy samples from two patients with JDM and healthy controls. Immunohistochemistry was used to examine differences in STAT3 phosphorylation between JDM and control muscle tissues. In vitro, skeletal muscle cell lines were stimulated with IL-6, and the transcription levels of genes related to mitochondrial calcium channels were quantified via reverse transcription-polymerase chain reaction (RT-PCR). Reactive oxygen species (ROS) production was measured in both IL-6 treated and untreated groups. ROS levels were then compared between IL-6 receptor antagonist pre-treated skeletal muscle cells and untreated cells. IL-6 levels in newly onset patients with JDM were significantly higher compared to the same patient cohort in remission states (p < 0.0001). Serum IL-6 was significantly increased in patients with negative myositis specific antibody (MSA), positive melanoma differentiation associated protein 5 (MDA5) and positive nuclear matrix protein 2 (NXP2), yet not for JDM with positive transcriptional intermediary factor γ(TIF1γ), based on subgroup analysis. ScRNA-seq analysis of muscle biopsies from patients with MDA5-positive JDM and patients with MSA negative JDM revealed abnormal activation of the JAK/STAT3 pathway in skeletal myocytes, macrophages, and vascular endothelial cells. The phosphorylation levels of STAT3 were elevated in active JDM cases. Transcription of the calcium channel modulation gene sarcolipin (SLN) was significantly higher in JDM primary skeletal muscle cells compared to normal cells. In vitro, IL-6 enhanced SLN transcription and induced ROS production, and blocking the IL-6 receptor resulted in decreased ROS generation in skeletal muscle cells. IL-6/JAK/STAT3 signaling pathway was abnormally activated in patients with JDM. IL-6 may be involved in the pathogenesis of muscle damage by triggering the development of calcium overload and production of ROS. Blockade of the IL-6/JAK/STAT3 pathway can be a potential treatment option for JDM, especially MDA5-positive patients and those who are negative for MSA.

Mapping the nuclear landscape with multiplexed super-resolution fluorescence microscopy.

The nucleus coordinates many different processes. Visualizing how these are spatially organized requires imaging protein complexes, epigenetic marks, and DNA across scales from single molecules to the whole nucleus. To accomplish this, we developed a multiplexed imaging protocol to localize 13 different nuclear targets with nanometer precision in single cells. We show that nuclear specification into active and repressive states exists along a spectrum of length scales, emerging below one micron and becoming strengthened at the nanoscale with unique organizational principles in both heterochromatin and euchromatin. HP1-α was positively correlated with DNA at the microscale but uncorrelated at the nanoscale. RNA Polymerase II, p300, and CDK9 were positively correlated at the microscale but became partitioned below 300 nm. Perturbing histone acetylation or transcription disrupted nanoscale organization but had less effect at the microscale. We envision that our imaging and analysis pipeline will be useful to reveal the organizational principles not only of the cell nucleus but also other cellular compartments.