The distribution of N6-methyladenosine (m6A) controls its substrate RNA fate, playing key roles in various biological processes. However, the mechanism underlying site-selective m6A deposition of RNAs, especially in the start codon regions, and the role in epigenetic information transduction connecting tumorigenesis remain largely unknown. Here, we identified RBM15B mainly modulates m6A modifications in the 5'untranslated regions (UTRs) and around the start codons of mRNAs transcribed. This process is guided by H3K79me2 histone methylation, a critical epigenetic modification in mixed lineage leukemia. We show that the H47 of RBM15B is a key residue for the recognition of H3K79me2. The selective m6A modification orchestrated by the H3K79me2-RBM15B axis enhances translation efficiency of oncogenic transcripts, and promotes self-renewal of leukemic stem cells and leukemia maintenance. We further demonstrate that blockade of the H3K79me2-RBM15B-m6A axis inhibits the survival of leukemia cells and promotes cell differentiation, and impairs hematological malignancies. This study uncovers a novel selective m6A deposition mechanism mediated by H3K79me2 and RBM15B, highlighting promising therapeutic targets for hematological malignancies.

CARD8 (caspase activation and recruitment domain-containing 8) senses intracellular danger signals, leading to inflammasome formation and pyroptosis. Recently studies have shown that CARD8 was highly expressed in hematopoietic cells, however, the mechanism by which the released C- terminal domain of CARD8 (CARD8-CT) oligomerizes and nucleates, and its role in acute myeloid leukemia (AML) treatment has not been fully understood. Here, we identified a long noncoding RNA IL10RB-AS1 as a novel regulator that can enhance the CARD8-CT oligomer assembly and promote pro-caspase-1-dependent pyroptosis in AML cells. Mechanically, IL10RB-AS1 recruits DEAD-box helicase 3, X-linked (DDX3X) to CARD8 and enhances the DDX3X-CARD8 interaction to promote CARD8-CT nucleation. This process activates the CARD8 inflammasome and triggers an ASC-independent pyroptosis response to perturbations in cellular homeostasis. We found that the C-terminal domain of DDX3X is essential for its interaction with both IL10RB-AS1 and CARD8-CT, facilitating inflammasome activation during AML cell pyroptosis. Importantly, IL10RB-AS1 is highly expressed across AML and correlates with poor outcomes. This elevated expression of IL10RB-AS1 promotes DPP8/9 inhibitor induced pro-caspase-1-dependent pyroptosis in vivo, suggesting that high IL10RB-AS1 levels may predict cellular sensitivity to these inhibitors. Our findings not only reveal a novel mechanism by which a lncRNA orchestrates CARD8-CT nucleation but also highlight new therapeutic opportunities for DPP8/9 inhibitors in hematopoietic malignancies with high IL10RB-AS1 expression.

Membrane proteins play a critical role in cellular processes such as chemical transport, signal transduction, and enzymatic catalysis. A comprehensive understanding of membrane protein types and accurately measuring their expression levels are essential for advancing cell biology. However, current methods often fail to characterize the types and amounts of multiple membrane proteins in specific cell types due to inherent measurement limitations. Here, we have developed a single-cell image analysis technique based on a cross-reactive imaging matrix using membrane protein tags. This technique allows quantitative and qualitative analysis of multiple membrane protein types and their expression levels within single cells. As a proof of concept, we used four specific DNA aptamers to tag four different membrane proteins in seven different cell types. The four proteins are MUC1, EGFR, HeR2, and TLR4. The seven cell lines are 3t3, 4T1, A549, CT26, HeLa, Hepa1-6, Raw, respectively. We captured a comprehensive image matrix using wide-field fluorescence imaging, allowing precise quantification of protein expression at the single-cell level. Our approach introduces a multi-labeling analysis method for membrane proteins and reveals the influence of membrane protein diversity on cell heterogeneity analysis. We found that the accuracy of cell heterogeneity recognition can be affected by adjusting the number of membrane proteins. As the diversity of membrane protein species increases, the accuracy of cell type recognition increases. Our approach facilitates the evaluation of heterogeneity in diverse cells, providing new insights into cellular diversity and a powerful tool for studying single-cell biology.

Protein concentration is an important factor regulating liquid-to-solid phase transition of proteins with prion-like domains (PLDs). Here, we present a protocol for investigating the effect of prion-like protein concentration on phase transition of fibrillarin (FBL) condensates in vitro using phase separation assays. We describe steps for construction of fluorescent-protein-tagged FBL plasmids, protein expression and purification in vitro, and detection of condensed states of FBL. This protocol has potential applications in phase separation assays for studying phase transitions in any prion-like protein. For complete details on the use and execution of this protocol, please refer to Sun et al.1.

DNA damage response (DDR) is a complicated network that responds to DNA lesions to prevent their accumulation; a defective DDR is one hallmark of cancer. Although targeting DDR pathways has been considered as a therapeutic approach, DDR inhibitors have also been reported ineffective for treating some low mutation burden cancers, such as Mixed-lineage leukemia (MLL)-rearranged (MLL-r) leukemia, a clinically fatal and refractory malignancy. Exploring the roles and mechanisms of DDR pathways in these low mutation burden cancers may help understand the chromatin biology and develop therapeutic strategies. Here, we identified a set of DDR-related chromatin-associated circular RNAs (cacircRNAs) that regulate DNA repair via the non-homologous end joining (NHEJ) pathway, which is vital for meeting the high DNA repair demands during the progression of MLL-r leukemia. Among these cacircRNAs, we identified ciCRLF3(2) as a previously unknown component of the NHEJ complex. We showed that ciCRLF3(2) recruits NHEJ regulators to DNA lesions, supporting abundant DNA repair in leukemia cells. ciCRLF3(2) abundance is abnormally upregulated in MLL-r leukemia and indicates a poor prognosis. Targeting ciCRLF3(2) suppressed NHEJ-mediated DNA repair, leading to DNA damage and broad anti-cancer effects in vitro and in vivo. A patient-derived xenograft model of MLL-r leukemia further indicated that ciCRLF3(2) depletion can decrease the leukemic burden. These findings demonstrate the function of cacircRNAs in DDR and chromatin biology and reveal a new avenue for developing strategies to treat low mutation burden cancers, such as MLL-r leukemia.

Rice bacterial blight is a devastating disease worldwide, causing significant yield losses. Understanding how plants defend against microbial infection is critical for sustainable crop production. In this study, we show that ALEX1, a previously identified pathogen-induced long noncoding RNA, localizes to the nucleus and directly binds AUXIN RESPONSE FACTOR 3 (ARF3). We showed that ARF3 forms the condensates in the nucleus via its intrinsically disordered middle region (MR), and that these ARF3 condensates display solid-like properties. We further revealed that ALEX1 directly binds the MR of ARF3 to regulate ARF3 condensate dynamics and promote ARF3 homodimerization. The dispersed, dimeric form of ARF3, referred to as its functional phase state, enhances its ability to transcriptionally repress the expression of downstream target genes such as JAZ13, thereby modulating the jasmonic acid signaling pathway and enhancing pathogen resistance in rice. Collectively, this study reveals the role of a long noncoding RNA in regulating protein condensation and complex assembly, thus contributing to plant pathogen resistance.

The factors predicting the development of heterotopic ossification (HO) of the elbow in children with untreated chronic Monteggia fractures (UCMFs) remained unclear. This multicentre study was designed to evaluate the radiographic data from paediatric patients with UCMFs and to identify the risk factors for HO formation and their radiographic characteristics. We retrospectively reviewed 274 patients (mean age at injury: 5.82 ± 2.62 years) with UCMFs with all types of anterior (group A) and non-anterior (group B) radial head (RH) dislocations. Radiographs were used to assess the presence, size and bone density of HO. The risk factors evaluated included age at injury, sex, laterality, interval from injury to diagnosis, presence of radial or median nerve injury, immobilization of the fractured ulna after injury, direction of RH dislocation and distance of RH dislocation (DD-RH). The results were compared with 76 patient demographics-matched paediatric acute Monteggia fractures (PAMFs) undergoing surgery within 48 h after injury. The HO rate (13.1

PURPOSE:Tyrosine kinase inhibitors combined with immune checkpoint blockades produce enhanced antitumor activity in the treatment of advanced hepatocellular carcinoma (HCC). Sitravatinib is a novel multitarget tyrosine kinase inhibitor that targets TYRO3, AXL, and MERTK receptors, c-MET, etc. This study aimed to investigate the antitumor efficacy and immunomodulatory activity of sitravatinib in HCC. EXPERIMENTAL DESIGN:Human HCC cell lines and xenograft models were used to explore the antitumor activity of sitravatinib. Subcutaneous and orthotopic immunocompetent murine HCC models were used to assess the therapeutic efficacy of sitravatinib and PD-1 blockade combination therapy. Cocultures for tumor cells and T cells were performed to verify the immunomodulatory effect of sitravatinib in tumors. RESULTS:Sitravatinib showed potent antitumor activity and immunomodulatory capabilities both invitro and in vivo. Sitravatinib treatment synergized with PD-1 blockade to generate an increased antitumor efficacy, leading to significant enrichment of cytotoxic CD8+ T cells and a reduction in the infiltration of regulatory T cells in tumors. Mechanically, on the one hand, sitravatinib reinforced MHC-I expression by blocking the TYRO3-STAT1 axis, thereby sensitizing tumor cells to T-cell killing. On the other hand, sitravatinib suppressed tumor-secreted IL33 by inhibiting TYRO3 activity in HCC cells, resulting in reduced regulatory T-cell differentiation and consequently liberating CD8+ T-cell cytotoxic capacity. In the clinic, one patient with advanced HCC treated with sitravatinib plus PD-1 blockade achieved near-complete response and remained disease progression free for >2 years. CONCLUSIONS:Collectively, we demonstrated a rationale for combining sitravatinib with PD-1 blockade in the treatment for HCC.

Abstract Background The risk factors for proximal radial abnormalities (PRA) in paediatric patients with untreated chronic Monteggia fractures (CMFs) are unclear. This multicentre study aimed to evaluate the risk factors for PRA in children with untreated CMFs. Materials and methods The clinical data of 142 patients (mean age at the time of injury: 5.73 years) with untreated unilateral CMFs were retrospectively reviewed. The radial neck-shaft angle (RNSAP) and radial head size (RHL) were measured on anteroposterior (AP) and lateral (L) radiographs, respectively. The RHL size was the ratio of the widest width of the proximal radial metaphysis to the narrowest radial neck width. The En-RNSAP and En-RHL were the ratios of the enlargement (En) of the RNSAP angle and RHL size of the injured elbow to those of the uninjured elbow, respectively. Paired-sample t-tests, single-factor analyses and multiple linear regression analyses were performed to evaluate the correlation between the differences in these parameters between the injured and uninjured elbows and the assessed risk factors. These risk factors included institution, sex, laterality, age at injury, time from injury to diagnosis, direction of RH dislocation, distance of RH dislocation (DD-RH), presence of radial or median nerve injury, heterotopic ossification and immobilization of the elbow after injury. Results In children with untreated CMFs (mean time from injury to diagnosis: 14.6 months), Student’s t-test revealed a significant difference in the RHL size (P < 0.001) but not in the RNSAP angle (P = 0.075) between the injured and uninjured elbows. Pearson correlation analysis revealed a potential correlation between En-RHL and age at the time of injury (P = 0.069), time from injury to diagnosis (P < 0.001) and DD-RH (P < 0.001), excluding other risk factors (P > 0.05). Multiple linear regression analysis revealed that age at the time of injury (P = 0.047), time from injury to diagnosis (P = 0.007) and DD-RH (P = 0.001) were risk factors for an increased En-RHL in patients with untreated CMFs; the variability in En-RHL among the other three risk factors was 21.4%. Conclusions In paediatric patients with unilateral untreated CMFs, PRA of the injured elbow consisted mainly of RH enlargement or radial neck narrowing rather than valgus deformities of the proximal radius. Older age at injury, increased time from injury to diagnosis and DD-RH were risk factors for more severe PRA. Level of evidence III.

Aims To investigate the risk factors for unsuccessful radial head reduction (RHR) in children with chronic Monteggia fractures (CMFs) treated surgically. Methods A total of 209 children (mean age 6.84 years (SD 2.87)), who underwent surgical treatment for CMFs between March 2015 and March 2023 at six institutions, were retrospectively reviewed. Assessed risk factors included age, sex, laterality, dislocation direction and distance, preoperative proximal radial metaphysis width, time from injury to surgery, reduction method, annular ligament reconstruction, radiocapitellar joint fixation, ulnar osteotomy, site of ulnar osteotomy, preoperative and postoperative ulnar angulation, ulnar fixation method, progressive ulnar distraction, and postoperative cast immobilization. Independent-samples t-test, chi-squared test, and logistic regression analysis were used to identify the risk factors associated with unsuccessful RHR. Results Redislocation occurred during surgery in 48 patients (23%), and during follow-up in 44 (21.1%). The mean follow-up of patients with successful RHR was 13.25 months (6 to 78). According to the univariable analysis, time from injury to surgery (p = 0.002) and preoperative dislocation distance (p = 0.042) were identified as potential risk factors for unsuccessful RHR. However, only time from injury to surgery (p = 0.007) was confirmed as a risk factor by logistic regression analysis. Receiver operating characteristic curve analysis and chi-squared test confirmed that a time from injury to surgery greater than 1.75 months increased the rate of unsuccessful RHR above the cutoff (p = 0.002). Conclusion Time from injury to surgery is the primary independent risk factor for unsuccessful RHR in surgically treated children with CMFs, particularly in those with a time from injury to surgery of more than 1.75 months. No other factors were found to influence the incidence of unsuccessful RHR. Surgical reduction of paediatric CMFs should be performed within the first two months of injury whenever possible.

Introduction Inflammatory diseases, such as diabetes mellitus, rheumatoid arthritis, and inflammatory bowel disease, lead to systemic immune microenvironment disturbances, contributing to bone loss, yet the mechanisms by which specific receptors regulate this process in inflammatory bone loss remain poorly understood. As a G-protein-coupled receptor, the Apelin receptor plays a crucial role in the regulation of inflammation and immune microenvironment. However, the precise mechanisms governing its role in inflammatory bone loss remain incompletely understood. Objective This study aims to investigate how APJ regulates macrophage polarization to mitigate inflammatory bone loss. Methods Lipopolysaccharide induced systemic inflammatory bone loss model in mice was used to explore the relationship between bone loss and osteoclast activation, macrophage polarization and APJ. In vitro studies, Bone marrow derived macrophages and siRNA were used to elucidate the regulatory influence of APJ on the immune microenvironment and osteoclast differentiation, while high-throughput sequencing is leveraged to uncover the underlying mechanisms through which APJ modulates macrophage polarization. Results Our study established a link between APJ and macrophage M1 polarization in systemic inflammatory bone loss mice. The activation of APJ effectively mitigated M1 polarization in macrophages, suppressed excessive osteoclast activation, and alleviated systemic inflammatory bone loss. In vitro high-throughput sequencing analysis revealed that APJ modulates macrophage polarization, linking to mitochondrial autophagy and the NOD-like receptor signaling pathway and the involvement of the AMPK and MAPK signaling pathways in signal transduction after APJ activation was also suggested. Subsequent experiments substantiated that APJ predominantly enhances mitophagy and diminishes the accumulation of reactive oxygen species by regulating the AMPK/BNIP3/PINK1/PARKIN axis, thereby suppressing the activation of macrophage M1 polarization and osteoclastogenesis. Conclusion This study elucidated the underlying mechanism by which APJ modulates macrophage polarization, thereby proposing a new therapeutic target for addressing inflammatory bone loss.

Purpose Although mRNA vaccines have shown certain clinical benefits in multiple malignancies, their therapeutic efficacies against hepatocellular carcinoma (HCC) remains uncertain. This study focused on establishing a novel risk score system based on immune subtypes so as to identify optimal HCC mRNA vaccination population. Methods GEPIA, cBioPortal and TIMER databases were utilized to identify candidate genes for mRNA vaccination in HCC. Subsequently, immune subtypes were constructed based on the candidate genes. According to the differential expressed genes among various immune subtypes, a risk score system was established using machine learning algorithm. Besides, multi-color immunofluorescence of tumor tissues from 72 HCC patients were applied to validate the feasibility and efficiency of the risk score system. Results Twelve overexpressed and mutated genes associated with poor survival and APCs infiltration were identified as potential candidate targets for mRNA vaccination. Three immune subtypes (e.g. IS1, IS2 and IS3) with distinct clinicopathological and molecular profiles were constructed according to the 12 candidate genes. Based on the immune subtype, a risk score system was developed, and according to the risk score from low to high, HCC patients were classified into four subgroups on average (e.g. RS1, RS2, RS3 and RS4). RS4 mainly overlapped with IS3, RS1 with IS2, and RS2+RS3 with IS1. ROC analysis also suggested the significant capacity of the risk score to distinguish between the three immune subtypes. Higher risk score exhibited robustly predictive ability for worse survival, which was further independently proved by multi-color immunofluorescence of HCC samples. Notably, RS4 tumors exhibited an increased immunosuppressive phenotype, higher expression of the twelve potential candidate targets and increased genome altered fraction, and therefore might benefit more from vaccination. Conclusions This novel risk score system based on immune subtypes enabled the identification of RS4 tumor that, due to its highly immunosuppressive microenvironment, may benefit from HCC mRNA vaccination.

Circular RNAs (circRNAs) have been implicated in tumorigenesis and progression of various cancers. However, the underlying mechanisms of circRNAs in hepatocellular carcinoma (HCC) have not been fully elucidated. Herein, a new oncogenic circRNA, hsa_circ_0070039 (circNUP54), was identified to be significantly upregulated in HCC through circRNA sequencing. As verified in 68 HCC samples, circNUP54 overexpression was correlated with aggressive cancerous behaviors and poor outcomes. Moreover, the function experiments showed that knockdown of circNUP54 inhibited the malignant progression of HCC in vitro and in vivo, whereas overexpression of circNUP54 had the opposite role. Mechanistic investigations carried out by RNA pull-down, RNA immunoprecipitation, and immunofluorescence revealed that circNUP54 interacted with the RNA-binding protein Hu-antigen R (HuR) and promoted its cytoplasmic export. The cytoplasmic accumulation of HuR stabilized the downstream BIRC3 mRNA through its binding to the 3′ UTR region. Consequently, the encoded protein of BIRC3, cellular inhibitor of apoptosis 2 (cIAP2), proceeded to activate the NF-κB signal pathway and ultimately contributed to HCC progression. In addition, depletion of BIRC3 rescued the pro-tumorigenic effect of circNUP54 on HCC cells. Overall, this study demonstrated that circNUP54 facilitates HCC progression via regulating the HuR/BIRC3/NF-κB axis, which may serve as a promising therapeutic target for HCC treatment.

FMS-like tyrosine kinase-3 internal tandem duplication (FLT3-ITD) acute myeloid leukemia (AML) is an adverse subtype of leukemia associated with a poor patient prognosis. FLT3 inhibitors exhibit promising clinical activity, however, their effectiveness is limited by drug resistance. Therefore, overcoming resistance to FLT3 inhibitors remains an important clinical problem, necessitating the development of new strategies. In this study, we identified a lncRNA, AL713998.1, which was upregulated in both FLT3-ITD AML and quizartinib-resistant cells (LFQR). In vitro experiments showed that LFQR silencing significantly inhibited cell proliferation, impaired self-renewal capacity, caused cell cycle arrest and induced cell differentiation, specifically in FLT3-ITD AML. In addition, the interaction of LFQR with SFPQ was verified using RNA pull-down assays, mass spectrometry and RNA immunoprecipitation. Mechanistically, LFQR promotes SFPQ binding to the promoter region of FLT3 and facilitates transcriptional regulation, thereby increasing the expression of kinase and oncoproteins in FLT3-ITD AML cells. Notably, LFQR inhibition also impaired the cytoactivity of quizartinib cells and reversed their resistance to quizartinib. Overall, our study highlights that LFQR may serve as a potential therapeutic target in FLT3-ITD AML, and its knockdown can significantly improve the efficacy of quizartinib, thus providing a promising strategy for overcoming resistance to FLT3 inhibitors.

The upregulation of programmed death ligand 1 (PD-L1) plays a crucial role in facilitating cancer cells to evade immune surveillance through immunosuppression. However, the precise regulatory mechanisms of PD-L1 in hepatocellular carcinoma (HCC) remain undefined. The correlation between PD-L1 and ubiquitin-like molecules (UBLs) was studied using sequencing data from 20 HCC patients in our center, combined with TCGA data. Specifically, the association between FAT10 and PD-L1 was further validated at both the protein and mRNA levels in HCC tissues from our center. Subsequently, the effect of FAT10 on tumor progression and immune suppression was examined through both in vivo and in vitro experiments. Utilizing sequencing data, qPCR, and Western blotting assays, we confirmed that FAT10 was highly expressed in HCC tissues and positively correlated with PD-L1 expression. Additionally, in vitro experiments demonstrated that the overexpression of FAT10 fostered the proliferation, migration, and invasion of HCC cells. Furthermore, the overexpression of FAT10 in HCC cells led to an increase in PD-L1 expression, resulting in the inhibition of T cell proliferation and the enhancement of HCC cell resistance to T cell-mediated cytotoxicity. Moreover, in vivo experiments utilizing the C57BL/6 mouse model revealed that overexpression of FAT10 effectively suppressed the infiltration of CD8 + GZMB + and CD8 + Ki67 + T cells, as well as reduced serum levels of TNF-α and IFN-γ. Mechanistically, we further identified that FAT10 upregulates PD-L1 expression via activating the PI3K/AKT/mTOR pathway, but not in a ubiquitin-like modification. In conclusion, our findings indicate that FAT10 promotes immune evasion of HCC via upregulating PD-L1 expression, suggesting its potential as a novel target to enhance the efficiency of immunotherapy in HCC.

Hepatocellular carcinoma (HCC) has a high incidence and dismal prognosis, making it a significant global health burden. To change this, the development of new therapeutic strategies is imminent. The claudin (CLDN) family, as key components of tight junctions (TJs), plays an important role in the initiation and development of cancer. Dysregulated expression of CLDNs leads to loss of intercellular adhesion and aberrant cell signaling, which are closely related to cancer cell invasion, migration, and epithelial–mesenchymal transition (EMT). CLDN1, CLDN3, CLDN4, CLDN5, CLDN6, CLDN7, CLDN9, CLDN10, CLDN11, CLDN14, and CLDN17 are aberrantly expressed in HCC, which drives the progression of the disease. Consequently, they have tremendous potential as prognostic indicators and therapeutic targets. This article summarizes the aberrant expression, molecular mechanisms, and clinical application studies of different subtypes of CLDNs in HCC, with a particular emphasis on CLDN1.

Liquid-to-solid phase transition of proteins with prion-like domains (PLDs) has been associated with neurodegenerative diseases and aging. High protein concentration is one important aspect triggering the transition; however, several prion-like proteins, including fibrillarin (FBL), an important phase-separated protein in the nucleolus for pre-rRNA processing, show relatively high expression levels in certain cells, especially cancer cells, without obvious phase transitions and growth arrest. How cells maintain prion-like protein proteostasis is still unknown. Here, we attempt to answer the question, with FBL as an example. We find that lncRNA DNAJC3-AS1 can buffer the behavior of FBL condensation and maintain the state and function of fibrillar component/dense fibrillar component (FC/DFC) units in human cell lines through two mechanisms, not only facilitating FBL condensation but also inhibiting excessive aggregation by binding multiple PLDs and partially blocking their interactions. We propose that lncRNAs could supply buffered systems to sustain functional phase states of prion-like proteins.

Background: The incidence of aggravation or occurrence of avascular necrosis (AVN) following hardware removal in surgically treated pediatric femoral neck fractures who achieved radiologic consolidation is unknown. This study aimed to investigate the risk factors for this complication. Methods: Seventy-one pediatric (mean age: 9.8±3.9 y) were retrospectively analyzed. Risk factors (age, sex, laterality, severity of initial displacement, type of fracture, time from trauma to reduction, reduction and fixation method, quality of reduction, time required to achieve radiologic union, duration of hardware retention, presence of AVN before hardware removal and follow-up time) were recorded. The severity of AVN was assessed based on radiographs with Ratliff’s classification. Results: Following hardware removal, the aggravation/occurrence of AVN was detected in 11 hips (15.5%). Among the 5 hips (7%) with aggravation of AVN, 1 (1.4%) with type II AVN and 3 (4.2%) with type III AVN exhibited aggravation of type I AVN, while the remaining hip (1.4%; type I) showed enlargement of the involved AVN area. Six hips (8.5%) developed AVN following hardware removal: 2 (2.8%) were classified as type I and 4 (5.6%) as type III. Receiver operating characteristic curve analysis indicated that hardware retention >7 months after union is associated with a decreased rate of aggravation or occurrence of AVN of the femoral neck or head following hardware removal. Conclusions: The incidence of aggravation or occurrence of AVN following hardware removal in surgically treated pediatric femoral neck fractures is 15.5%; hardware retention >7 months after radiologic union may reduce the risk of aggravation or occurrence of AVN of the femoral neck or head postimplant removal. Level of evidence: Level III.

Background Mixed-lineage leukemia ( MLL ) fusion gene caused by chromosomal rearrangement is a dominant oncogenic driver in leukemia. Due to having diverse MLL rearrangements and complex characteristics, MLL leukemia treated by currently available strategies is frequently associated with a poor outcome. Therefore, there is an urgent need to identify novel therapeutic targets for hematological malignancies with MLL rearrangements. Methods qRT-PCR, western blot, and spearman correction analysis were used to validate the regulation of LAMP5-AS1 on LAMP5 expression. In vitro and in vivo experiments were conducted to assess the functional relevance of LAMP5-AS1 in MLL leukemia cell survival. We utilized chromatin isolation by RNA purification (ChIRP) assay, RNA pull-down assay, chromatin immunoprecipitation (ChIP), RNA fluorescence in situ hybridization (FISH), and immunofluorescence to elucidate the relationship among LAMP5-AS1, DOT1L, and the LAMP5 locus. Autophagy regulation by LAMP5-AS1 was evaluated through LC3B puncta, autolysosome observation via transmission electron microscopy (TEM), and mRFP-GFP-LC3 puncta in autophagic flux. Results The study shows the crucial role of LAMP5-AS1 in promoting MLL leukemia cell survival. LAMP5-AS1 acts as a novel autophagic suppressor, safeguarding MLL fusion proteins from autophagic degradation. Knocking down LAMP5-AS1 significantly induced apoptosis in MLL leukemia cell lines and primary cells and extended the survival of mice in vivo. Mechanistically, LAMP5-AS1 recruits the H3K79 histone methyltransferase DOT1L to LAMP5 locus, directly activating LAMP5 expression. Importantly, blockade of LAMP5-AS1-LAMP5 axis can represses MLL fusion proteins by enhancing their degradation. Conclusions The findings underscore the significance of LAMP5-AS1 in MLL leukemia progression through the regulation of the autophagy pathway. Additionally, this study unveils the novel lncRNA-DOT1L-LAMP5 axis as promising therapeutic targets for degrading MLL fusion proteins.

Cell imaging technology is undoubtedly a powerful tool for studying single-cell heterogeneity due to its non-invasive and visual advantages. It covers microscope hardware, software, and image analysis techniques, which are hindered by low throughput owing to abundant hands-on time and expertise. Herein, a cellular nucleus image-based smarter microscope system for single-cell analysis is reported to achieve high-throughput analysis and high-content detection of cells. By combining the hardware of an automatic fluorescence microscope and multi-object recognition/acquisition software, we have achieved more advanced process automation with the assistance of Robotic Process Automation (RPA), which realizes a high-throughput collection of single-cell images. Automated acquisition of single-cell images has benefits beyond ease and throughout and can lead to uniform standard and higher quality images. We further constructed a single-cell image database-based convolutional neural network (Efficient Convolutional Neural Network, E-CNN) exceeding 20618 single-cell nucleus images. Computational analysis of large and complex data sets enhances the content and efficiency of single-cell analysis with the assistance of Artificial Intelligence (AI), which breaks through the super-resolution microscope's hardware limitation, such as specialized light sources with specific wavelengths, advanced optical components, and high-performance graphics cards. Our system can identify single-cell nucleus images that cannot be artificially distinguished with an accuracy of 95.3%. Overall, we build an ordinary microscope into a high-throughput analysis and high-content smarter microscope system, making it a candidate tool for Imaging cytology.