Surface Electromyography and Gait Features in Patients after Anterior Cruciate Ligament Reconstruction.

OBJECTIVE: An important reason for the poor recovery of anterior cruciate ligament (ACL) injuries is the poor recovery of muscle function. Therefore, we used surface electromyography (sEMG) and gait analysis to explore the muscle activation patterns and gait characteristics between lower limbs under different exercise states in patients, following anterior cruciate ligament reconstruction (ACLR). METHODS: Forty-one adults with unilateral ACL injuries in Binzhou Medical University Hospital from October 2022 to June 2023 were allocated to three groups according to the time after ACL reconstruction: group A (≤3 months, 16), group B (3 months-1 year, 13), and group C (>1 year, 12). Patients were tested by sEMG and gait, while straight leg raising (SLR), walking at normal speed, fast walking, and walking up and down the stairs. Two related sample tests were performed for the normalized root mean square (RMS) values and gait parameters. RESULTS: Muscle function changes varied in different training tasks. The RMS value of the involved side was more than the uninvolved side in biceps femoris and semitendinosus of group A (p < 0.010), and for the bilateral rectus femoris (RS), vastus medialis (VM), and vastus lateralis in group B, only the comparison of the RS was significant in group C during fast walking and going up and down the stairs. The ground impact (0.90 [0.63, 1.33] vs. 0.71 [0.43, 1.02], p = 0.035) of the uninvolved side was significantly decreased compared to those of the involved side in patients with ACLR when going down the stairs. CONCLUSION: Different muscles need to be focused on at different stages of the postoperative period. sEMG and gait analysis can guide the development of a rehabilitation program.

Slow-replicating leukemia cells represent a leukemia stem cell population with high cell-surface CD74 expression.

Persistence of quiescent leukemia stem cells (LSCs) after treatment most likely contributes to chemotherapy resistance and poor prognosis of leukemia patients. Identification of this quiescent cell population would facilitate eradicating LSCs. Here, using a cell-tracing PKH26 (PKH) dye that can be equally distributed to daughter cells following cell division in vivo, we identify a label-retaining slow-cycling leukemia cell population from AML1-ETO9a (AE9a) leukemic mice. We find that, compared with cells not maintaining PKH-staining, a higher proportion of PKH-retaining cells are in G0 phase, and PKH-retaining cells exhibit increased colony formation ability and leukemia initiation potential. In addition, PKH-retaining cells possess high chemo-resistance and are more likely to be localized to the endosteal bone marrow region. Based on the transcriptional signature, HLA class II histocompatibility antigen gamma chain (Cd74) is highly expressed in PKH-retaining leukemia cells. Furthermore, cell surface CD74 was identified to be highly expressed in LSCs of AE9a mice and CD34+ human leukemia cells. Compared to Lin-CD74- leukemia cells, Lin-CD74+ leukemia cells of AE9a mice exhibit higher stemness properties. Collectively, our findings reveal that the identified slow-cycling leukemia cell population represents an LSC population, and CD74+ leukemia cells possess stemness properties, suggesting that CD74 is a candidate LSC surface marker.

Pediococcus pentosaceus MIANGUAN Enhances the Immune Response to Vaccination in Mice.

Increasing evidence shows that some probiotics can improve vaccine responses as adjuvants. This study aimed to evaluate the effect of Pediococcus pentosaceus MIANGUAN (PPM) on SARS-CoV-2 vaccine-elicited immune response in mice. Six-week-old female ICR mice were primed and boosted with SARS-CoV-2 vaccine intramuscularly at weeks 0 and 4, respectively. Mice were gavaged with PPM (5 × 109 CFU/mouse) or PBS (control) for 3 days immediately after boosting vaccination. Compared to the control, oral PPM administration resulted in significantly higher levels of RBD-specific IgG binding antibodies (> 2.3-fold) and RBD-specific IgG1 binding antibodies (> 4-fold) in the serum. Additionally, PPM-treated mice had higher titers of RBD-specific IgG binding antibodies (> 2.29-fold) and neutralization antibodies (> 1.6-fold) in the lung compared to the control mice. The transcriptional analyses showed that the B cell receptor (BCR) signaling pathway was upregulated in both splenocytes and BAL cells in the PPM group vs. the control group. In addition, the number of IFN-γ-producing splenocytes (mainly in CD4 + T cells as determined by flow cytometry) in response to restimulation of RBD peptides was significantly increased in the PPM group. RNA sequencing showed that the genes associated with T cell activation and maturation and MHC class II pathway (CD4, H2-DMa, H2-DMb1, H2-Oa, Ctss) were upregulated, suggesting that oral administration of PPM may enhance CD4 + T cell responses through MHC class II pathway. Furthermore, PPM administration could downregulate the expression level of proinflammatory genes. To conclude, oral administration of PPM could boost SARS-CoV-2 vaccine efficacy through enhancing the specific humoral and cellular immunity response and decrease the expression of inflammation pathways.

Spatial-linked alignment tool (SLAT) for aligning heterogenous slices.

Spatially resolved omics technologies reveal the spatial organization of cells in various biological systems. Here we propose SLAT (Spatially-Linked Alignment Tool), a graph-based algorithm for efficient and effective alignment of spatial slices. Adopting a graph adversarial matching strategy, SLAT is the first algorithm capable of aligning heterogenous spatial data across distinct technologies and modalities. Systematic benchmarks demonstrate SLAT's superior precision, robustness, and speed over existing state-of-the-arts. Applications to multiple real-world datasets further show SLAT's utility in enhancing cell-typing resolution, integrating multiple modalities for regulatory inference, and mapping fine-scale spatial-temporal changes during development. The full SLAT package is available at https://github.com/gao-lab/SLAT .

Elucidating the cell metabolic heterogeneity during hematopoietic lineage differentiation based on Met-Flow.

Cell metabolism is critically involved in the differentiation of the hematopoietic lineage and, therefore, has attracted the attention of researchers, however, in-depth studies on cellular metabolic activity of hematopoietic cells (HCs) require attention. This investigation compared the metabolic activity of HCs at critical lineage differentiation stages, including hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs), and differentiated blood cells, via multiple methods and basic reference values. Primary metabolic processes of HCs, including anabolism, catabolism, phosphate, and glucose metabolism, were analyzed, and their maps were drawn. The data revealed that GLUT1 expression in HSCs was substantially higher than in all progenitor cells and mature myeloid blood cells, indicating their strong glucose uptake capacity. In myeloid differentiation, the ACAC expression of HPC2 was markedly higher than in neutrophils and monocytes. The ACAC, ASS1, ATP5A, and PRDX2 of HPC2 expression in lymphoid differentiation was substantially greater than in B and Natural-killer cells. CLP, CMP, GMP, MEP, and HPC1 inherit increased glucose uptake stem cell properties. In lymphocyte subsets, the expression of ACAC, ASS1, ATP5A, CPT1A, and PRDX2 in CD4+ T subgroups (naive and memory CD4+ T and nTreg) were elevated than in B subgroups (pro-, pre-, immature and mature Bs) and CD8+ T subgroups. Furthermore, leukemia stem cells (LSCs) had increased levels of ACAC, CPT1A, G6PD, IDH2, and PRDX2 than leukemia cells, indicating a stronger metabolic capacity of LSCs than differentiated leukemia cells.

Bioinformatics and Raman spectroscopy-based identification of key pathways and genes enabling differentiation between acute myeloid leukemia and T cell acute lymphoblastic leukemia.

Acute myeloid leukemia (AML) and T cell acute lymphoblastic leukemia (T-ALL) are two of the most prevalent hematological malignancies diagnosed among adult leukemia patients, with both being difficult to treat and associated with high rates of recurrence and mortality. In the present study, bioinformatics approaches were used to analyze both of these types of leukemia in an effort to identify characteristic gene expression patterns that were subsequently validated via Raman spectroscopy. For these analyses, four Gene Expression Omnibus datasets (GSE13204, GSE51082, GSE89565, and GSE131184) pertaining to acute leukemia were downloaded, and differentially expressed genes (DEGs) were then identified through comparisons of AML and T-ALL patient samples using the R Bioconductor package. Shared DEGs were then subjected to Gene Ontology (GO) enrichment analyses and were used to establish a protein-protein interaction (PPI) network analysis. In total, 43 and 129 upregulated and downregulated DEGs were respectively identified. Enrichment analyses indicated that these DEGs were closely tied to immune function, collagen synthesis and decomposition, inflammation, the synthesis and decomposition of lipopolysaccharide, and antigen presentation. PPI network module clustering analyses further led to the identification of the top 10 significantly upregulated and downregulated genes associated with disease incidence. These key genes were then validated in patient samples via Raman spectroscopy, ultimately confirming the value of these genes as tools that may aid the differential diagnosis and treatment of AML and T-ALL. Overall, these results thus highlight a range of novel pathways and genes that are linked to the incidence and progression of AML and T-ALL, providing a list of important diagnostic and prognostic molecular markers that have the potential to aid in the clinical diagnosis and treatment of these devastating malignancies.

Coupled deglycosylation-ubiquitination cascade in regulating PD-1 degradation by MDM2.

Posttranslational modifications represent a key step in modulating programmed death-1 (PD-1) functions, but the underlying mechanisms remain incompletely defined. Here, we report crosstalk between deglycosylation and ubiquitination in regulating PD-1 stability. We show that the removal of N-linked glycosylation is a prerequisite for efficient PD-1 ubiquitination and degradation. Murine double minute 2 (MDM2) is identified as an E3 ligase of deglycosylated PD-1. In addition, the presence of MDM2 facilitates glycosylated PD-1 interaction with glycosidase NGLY1 and promotes subsequent NGLY1-catalyzed PD-1 deglycosylation. Functionally, we demonstrate that the absence of T cell-specific MDM2 accelerates tumor growth by primarily upregulating PD-1. By stimulating the p53-MDM2 axis, interferon-α (IFN-α) reduces PD-1 levels in T cells, which, in turn, exhibit a synergistic effect on tumor suppression by sensitizing anti-PD-1 immunotherapy. Our study reveals that MDM2 directs PD-1 degradation via a deglycosylation-ubiquitination coupled mechanism and sheds light on a promising strategy to boost cancer immunotherapy by targeting the T cell-specific MDM2-PD-1 regulatory axis.

Transfrontal External Ventricular Drainage Combined with OMMAYA Sac Implantation under Laser Navigation were Performed: A Technical Note.

BACKGROUND: Hydrocephalus caused by spontaneous intracerebral hemorrhage (ICH) is an independent risk factor with adverse effects on the progression of the disease. Until now, the choice of intraventricular catheter placement and intraventricular fibrinolysis (IVF) has been mainly based on the personal experience of the neurosurgeon. OBJECTIVE: We will introduce the clinical effect of the new external ventricular drainage (EVD), an independent innovation of our medical center, on ICH patients, hoping to inspire more neurosurgeons to apply our method. METHODS: In this open retrospective study, We analyzed the clinical data, radiological manifestations, and prognostic scores of 10 patients with the spontaneous intracerebral hemorrhage who received transfrontal lateral ventricle puncture and drainage under laser navigation in our hospital. RESULTS: A total of 10 patients with an average age of 58.10±9.97 years were enrolled for emergency surgery. All operations were completed according to the consensus specifications. It took 11.25±3.81 days for the intracranial pressure to return to normal. On admission, patients had a median GCS of 10. The median preoperative GCS was 8. The median GCS at discharge score was 15. At discharge, the median NIHSS score was 4. After 6 months of follow-up, patients had a median NIHSS score of 4. At discharge, the median ADL score of patients was 85. After 6 months of follow-up, the median ADL score of the patients was 95. CONCLUSION: In treating patients with ICH, the emergency treatment of transfrontal external ventricular drainage combined with OMMAYA sac implantation under laser navigation is a surgical method worthy of further study.

Surface electromyography characteristics of patients with anterior cruciate ligament injury in different rehabilitation phases.

Background: Anterior cruciate ligament reconstruction (ACLR) is a common treatment for anterior cruciate ligament (ACL) injury. However, after ACLR, a significant proportion of patients do not return to pre-injury levels. Research on muscle function during movement has important implications in rehabilitation. Methods: Sixty patients with unilateral ACL injury were recruited for this study and assigned into three groups: group A, individuals with an ACL injury before 6 months; group B, individuals with ACLR from 6 months to 1 year; and group C, individuals with ACLR 1 year later. Surface electromyography (SEMG) signals were collected from the bilateral rectus femoris (RF), vastus medialis (VM), vastus lateralis (VL), biceps femoris (BF), and semitendinosus (ST). The tasks performed during the experiment included straight leg raising (SLR) training at 30°, SLR training at 60°, ankle dorsiflexion, walking, and fast walking. Results: In the maximum muscle strength test, the affected side of the BF in group A (199.4 ± 177.12) was significantly larger than in group B (53.91 ± 36.61, p = 0.02) and group C (75.08 ± 59.7, p = 0.023). In the walking test, the contralateral side of the RF in group B (347.53 ± 518.88) was significantly greater than that in group C (139.28 ± 173.78, p = 0.029). In the SLR training (60°) test, the contralateral side of the RF in group C (165.37 ± 183.06) was significantly larger than that in group A (115.09 ± 62.47, p = 0.023) and smaller than that in group B (226.21 ± 237.17, p = 0.046); In the ankle dorsiflexion training test, the contralateral side of the RF in group B (80.37 ± 87.9) was significantly larger than that in group C (45.61 ± 37.93, p = 0.046). Conclusion: This study showed the EMG characteristics of patients with ACL injury helped to determine which muscle requires more training and which exercise model would be best suited for intervention.

Comparative Evaluation of Standard RT-PCR Assays and Commercial Real-Time RT-PCR Kits for Detection of Lassa Virus.

Lassa virus (LASV) is a causative agent of hemorrhagic fever epidemic in West Africa. In recent years, it has been transmitted several times to North America, Europe, and Asia. Standard reverse transcription (RT)-PCR and real-time RT-PCR are extensively used for early detection of LASV. However, the high nucleotide diversity of LASV strains complicates the development of appropriate diagnostic assays. Here, we analyzed LASV diversity clustered with geographic location and evaluated the specificity and sensitivity of two standard RT-PCR methods (GPC RT-PCR/1994 and 2007) and four commercial real-time RT-PCR kits (namely, Da an, Mabsky, Bioperfectus, and ZJ) to detect six representative LASV lineages using in vitro synthesized RNA templates. The results showed that the GPC RT-PCR/2007 assay had better sensitivity compared to the GPC RT-PCR/1994 assay. The Mabsky and ZJ kits were able to detect all RNA templates of six LASV lineages. Contrastingly, the Bioperfectus and Da an kits failed to detect lineages IV and V/VI. The limit of detection for lineage I with the Da an, Bioperfectus, and ZJ kits were significantly higher than that of the Mabsky kit at an RNA concentration of 1 × 1010 to 1 × 1011 copies/mL. The Bioperfectus and Da an kits detected lineages II and III at an RNA concentration of 1 × 109 copies/mL, higher than that of the other kits. In conclusion, the GPC RT-PCR/2007 assay and the Mabsky kit were suitable assays for the detection of LASV strains based on good analytical sensitivity and specificity. IMPORTANCE Lassa virus (LASV) is a significant human pathogen causing hemorrhagic fever in West Africa. Increased traveling around the world raises the risk of imported cases to other countries. The high nucleotide diversity of LASV strains clustered with geographic location complicates the development of appropriate diagnostic assays. In this study, we showed that the GPC reverse transcription (RT)-PCR/2007 assay and the Mabsky kit are suitable for detecting most LASV strains. Future assays for molecular detection of LASV should be based on specific countries/regions along with new variants.

The repression of oncoprotein SET by the tumor suppressor p53 reveals a p53-SET-PP2A feedback loop for cancer therapy.

The oncoprotein SET is frequently overexpressed in many types of tumors and contributes to malignant initiation and progression through multiple mechanisms, including the hijacking of the tumor suppressors p53 and PP2A. Targeting aberrant SET represents a promising strategy for cancer intervention. However, the mechanism by which endogenous SET is regulated in cancer cells remains largely unknown. Here, we identified the tumor suppressor p53 as a key regulator that transcriptionally repressed the expression of SET in both normal and cancer cells. In addition, p53 stimulated PP2A phosphatase activity via p53-mediated transcriptional repression of SET, whereby SET-mediated inhibition of PP2A was alleviated. Moreover, targeting the interaction between SET and PP2A catalytic subunit (PP2Ac) with FTY720 enhanced stress-induced p53 activation via PP2A-mediated dephosphorylation of p53 on threonine 55 (Thr55). Therefore, our findings uncovered a previously unknown p53-SET-PP2A regulatory feedback loop. To functionally potentiate this feedback loop, we designed a combined therapeutic strategy by simultaneously administrating a p53 activator and SET antagonist in cancer cells and observed a dramatic synergistic effect on tumor suppression. Our study reveals mechanistic insight into the regulation of the oncoprotein SET and raises a potential strategy for cancer therapy by stimulating the p53-SET-PP2A feedback loop.

Alternatively mechanistic insights into acetylation in p53-mediated transcriptional regulation of cancer cell-intrinsic PD-1.

Since the recent discovery of cancer cell-intrinsic programmed cell death protein-1 (PD-1), the mechanisms that manipulate PD-1 functions in tumor development beyond its immune checkpoint roles have become attractive research topics in oncology. Our previous study validated that PD-1 exists in lung cancer cells and is directly transactivated by p53 in a DNA-binding domain (DBD) acetylation-dependent manner. Here, we report that the carboxyl-terminal domain (CTD) of p53 likewise participates in PD-1 transcriptional regulation in cancer cells under different regulatory mechanisms. By mutating the lysine residues within the CTD to mimic either acetylation-deficient or fully acetylated status, we proved that acetylated CTD dramatically impeded p53-mediated transactivation of PD-1. Furthermore, we identified bromodomain-containing protein 4 (BRD4) as a transcriptional coactivator of p53 that facilitates p53-mediated PD-1 transcription. Mechanistically, BRD4 specifically bound to the unacetylated CTD of p53, while CTD acetylation almost completely destroyed the BRD4-p53 interaction and thus led to compromised PD-1 expression. Collectively, this study unveils an alternative mechanism of p53 acetylation-directed PD-1 transcriptional regulation, which would broaden our current understanding of the molecular regulatory network of cancer cell-intrinsic PD-1.

Elucidating the mitochondrial function of murine lymphocyte subsets and the heterogeneity of the mitophagy pathway inherited from hematopoietic stem cells.

Background: Mitochondria are mainly involved in ATP production to meet the energy demands of cells. Researchers are increasingly recognizing the important role of mitochondria in the differentiation and activation of hematopoietic cells, but research on how mitochondrial metabolism influence different subsets of lymphocyte at different stages of differentiation and activation are yet to be carried out. In this work, the mitochondrial functions of lymphocytes were compared at different differentiation and activation stages and included CD8+ T lymphocytes, CD4+ T lymphocytes, B lymphocytes, NK cells as well as their subsets. For this purpose, a complete set of methods was used to comprehensively analyze mitophagy levels, mitochondrial reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and the mitochondrial mass (MM) of subsets of lymphocytes. It is expected that this will provide a complete set of standards, and drawing the mitochondrial metabolic map of lymphocyte subsets at different stages of differentiation and activation. Results and discussion: Of all lymphocytes, B cells had a relatively high mitochondrial metabolic activity which was evident from the higher levels of mitophagy, ROS, MMP and MM, and this reflected the highly heterogeneous nature of the mitochondrial metabolism in lymphocytes. Among the B cell subsets, pro-B cells had relatively higher levels of MM and MMP, while the mitochondrial metabolism level of mature B cells was relatively low. Similarly, among the subsets of CD4+ T cell, a relatively higher level of mitochondrial metabolism was noted for naive CD4+ T cells. Finally, from the CD8+ T cell subsets, CD8+ Tcm had relatively high levels of MM and MMP but relatively low ones for mitophagy, with effector T cells displaying the opposite characteristics. Meanwhile, the autophagy-related genes of lymphoid hematopoietic cells including hematopoietic stem cells, hematopoietic progenitor cells and lymphocyte subsets were analyzed, which preliminarily showed that these cells were heterogeneous in the selection of mitophagy related Pink1/Park2, BNIP3/NIX and FUNDC1 pathways. The results showed that compared with CD4+ T, CD8+ T and NK cells, B cells were more similar to long-term hematopoietic stem cell (LT-HSC) and short-term hematopoietic stem cell (ST-HSC) in terms of their participation in the Pink1/Park2 pathway, as well as the degree to which the characteristics of autophagy pathway were inherited from HSC. Compared with CLP and B cells, HSC are less involved in BNIP3/NIX pathway. Among the B cell subsets, pro-B cells inherited the least characteristics of HSC in participating in Pink1/Park2 pathway compared with pre-B, immature B and immature B cells. Among CD4+ T cell subsets, nTreg cells inherited the least characteristics of HSC in participating in Pink1/Park2 pathway compared with naive CD4+ T and memory CD4+ T cells. Among the CD8+ T cell subsets, compared with CLP and effector CD8+ T cells, CD8+ Tcm inherit the least characteristics of HSC in participating in Pink1/Park2 pathway. Meanwhile, CLP, naive CD4+ T and effector CD8+ T were more involved in BNIP3/NIX pathway than other lymphoid hematopoietic cells. Conclusion: This study is expected to provide a complete set of methods and basic reference values for future studies on the mitochondrial functions of lymphocyte subsets at different stages of differentiation and activation in physiological state, and also provides a standard and reference for the study of infection and immunity based on mitochondrial metabolism.

[Bone Marrow Osteoblasts Promotes the Proliferation Leukemia Stem Cell by Up-regulating Interleukin-1].

OBJECTIVE: To explore the extrinsic regulation mechanism of bone marrow microenvironment in leukemia cells, and investigate the promoting effect of osteoblast niche on the proliferation and self-renewal of leukemia stem cell by up-regulating the expression of interleukin-1 (IL-1) in leukemia cell. METHODS: The gene expression profiles on leukemia cells derived from AE9a mouse bone marrow endosteum and central bone marrow were determined by RNA sequencing and gene set enrichment analysis (GSEA). Quantitative real-time PCR (qRT-PCR) was used to detect the expression of IL-1 in AE9a mouse leukemia cells co-cultured with or without osteoblasts in vitro. In addition, qRT-PCR was also used to determine the expression of IL-1 in bone marrow mononuclear cell (BMMNC) from 43 patients with acute myeloid leukemia (AML). For leukemia cells co-cultured with osteoblasts or treated with IL-1ß, colony forming ability of AE9a leukemia cells was determined by colony formation assay. RESULTS: In AE9a leukemia mouse, RNA-seq data and GSEA showed that the enrichment of the upregulated genes in leukemia cells located in endosteum fell into inflammatory response gene set, among them, IL-1α and IL-1ß were significantly higher expressed in AE9a leukemia cells that located osteoblast niche (IL-1α: P<0.001, IL-1ß:P<0.001). After AE9a leukemia cells were co-cultured with osteoblasts in vitro, the expression of IL-1α and IL-1ß in leukemia cells were increased by 2.5 and 3.5 times respectively. In colony formation assay, the number of colonies was increased significantly after leukemia cells were co-cultured with osteoblasts (P<0.001). In addition, when AE9a leukemia cells were treated with IL-1ß, the number of colonies was also increased significantly (P<0.01). In AML patients, BMMNC with high percentage of CD34 positive cells exhibited higher level of IL-1 expression. CONCLUSION: Osteoblast niche can promote leukemia cell proliferation and self-renewal through up-regulating the expression of IL-1 in leukemia cells. In AML patients, the expression level of IL-1 was correlated to the percentage of CD34 positive cells in BMMNC.

Multi-omics single-cell data integration and regulatory inference with graph-linked embedding.

Despite the emergence of experimental methods for simultaneous measurement of multiple omics modalities in single cells, most single-cell datasets include only one modality. A major obstacle in integrating omics data from multiple modalities is that different omics layers typically have distinct feature spaces. Here, we propose a computational framework called GLUE (graph-linked unified embedding), which bridges the gap by modeling regulatory interactions across omics layers explicitly. Systematic benchmarking demonstrated that GLUE is more accurate, robust and scalable than state-of-the-art tools for heterogeneous single-cell multi-omics data. We applied GLUE to various challenging tasks, including triple-omics integration, integrative regulatory inference and multi-omics human cell atlas construction over millions of cells, where GLUE was able to correct previous annotations. GLUE features a modular design that can be flexibly extended and enhanced for new analysis tasks. The full package is available online at https://github.com/gao-lab/GLUE .

Mutant U2AF1-induced differential alternative splicing causes an oxidative stress in bone marrow stromal cells.

Alternative splicing (AS) is a critical regulatory process of gene expression. In bone marrow microenvironment, AS plays a critical role in mesenchymal stem cells fate determination by forming distinct isoforms of important regulators. As a spliceosome factor, U2AF1 is essential for the catalysis of pre-mRNA splicing, and its mutation can cause differential AS events. In the present study, by forced expression of mutant U2AF1 (U2AF1S34F) in the mouse bone marrow stroma OP9 cells, we determine AS changes in U2AF1S34F transduced OP9 cells and investigate their role in stroma cell biological functions. We find that abundant differential RNA splicing events are induced by U2AF1S34F in OP9 cells. U2AF1S34F causes increased generation of hydrogen peroxide, promotes production of cytokines and chemokines. U2AF1S34F transduced OP9 cells also exhibit dysfunction of mitochondria. RNA-seq data, gene ontology (GO), and gene set enrichment analysis reveal that differentially expressed genes downregulated in response to U2AF1S34F are enriched in peroxisome component and function. U2AF1S34F can also cause release of hydrogen peroxide from OP9 cells. Furthermore, we investigate the influence of U2AF1S34F-induced oxidative stress in stromal cells on hematopoietic cells. When co-culturing mouse bone marrow mononuclear cells with OP9 cells, the U2AF1S34F expressing OP9 cells induce phosphorylation of histone H2AX in hematopoietic cells. Collectively, our results reveal that mutant U2AF1-induced differential AS events cause oxidative stress in bone marrow stromal cells and can further lead to DNA damage and genomic instability in hematopoietic cells.

An unexpected role for p53 in regulating cancer cell-intrinsic PD-1 by acetylation.

Cancer cell-intrinsic programmed cell death protein-1 (PD-1) has emerged as a tumor regulator in an immunity-independent manner, but its precise role in modulating tumor behaviors is complex, and how PD-1 is regulated in cancer cells is largely unknown. Here, we identified PD-1 as a direct target of tumor suppressor p53. Notably, p53 acetylation at K120/164 played a critical role in p53-mediated PD-1 transcription. Acetylated p53 preferentially recruited acetyltransferase cofactors onto PD-1 promoter, selectively facilitating PD-1 transcription by enhancing local chromatin acetylation. Reexpression of PD-1 in cancer cells inhibited tumor growth, whereas depletion of cancer cell-intrinsic PD-1 compromised p53-dependent tumor suppression. Moreover, histone deacetylase inhibitor (HDACi) activated PD-1 in an acetylated p53-dependent manner, supporting a synergistic effect by HDACi and p53 on tumor suppression via stimulating cancer cell-intrinsic PD-1. Our study reveals a mechanism for activating cancer cell-intrinsic PD-1 and indicates that p53-mediated PD-1 activation is critically involved in tumor suppression in an immunity-independent manner.