Chimeric antigen receptor (CAR) T cell therapy has emerged as a promising approach for hematological malignancies, yet its efficacy in solid tumors is hindered by limited persistence. To address this, immune checkpoint inhibitors (ICIs) and cytokines have been explored as potential solutions. In this study, we developed a novel monoclonal antibody (mAb), m8A8, which exhibits high specificity for human PD-1 and effectively disrupts its ligand interactions. Furthermore, we engineered CAR-T cells to express human IL-7, resulting in enhanced anti-tumor efficacy in xenograft models. Additionally, the human-mouse chimeric antibody C8A8, derived from m8A8, was found to significantly amplify the anti-tumor activity of IL-7-engineered CAR-T cells. Our findings provide compelling evidence and a robust rationale for the synergistic integration of ICIs, cytokines, and CAR-T cell therapy in the treatment of solid tumors.

The spleen is a highly organized lymphoid organ with a critical role in antimicrobial immune responses. Pseudorabies virus (PRV) is widely recognized for its ability to induce immunosuppression, with the spleen being one of the primary parenchymal organs of target. Viral infections often disrupt endoplasmic reticulum (ER) homeostasis, leading to ER stress and subsequent apoptosis. This study aimed to investigate the relationship between PRV-induced spleen damage and ER stress. Both classical (Min-A) and variant (SX-2018) PRV strains caused significant histopathological damage in the mouse spleen, including marked reductions in CD8+ T cell populations and increased lymphocyte apoptosis. Further analyses revealed that PRV infection triggered ER stress and activated the PERK-eIF2α-ATF4-CHOP signaling pathway in the spleen. Notably, treatment with the ER stress inhibitor 4-phenylbutyric acid(4-PBA) mitigated lymphocyte depletion and improved survival rates in PRV-infected mice. PRV infection leads to lymphocyte depletion in mouse spleens, closely associated with ER stress and apoptosis.

Prion diseases are a group of neurodegenerative diseases characterized by mitochondrial dysfunction and neuronal death. Mitophagy is a selective form of macroautophagy that clears injured mitochondria. Prohibitin 2 (PHB2) has been identified as a novel inner membrane mitophagy receptor that mediates mitophagy. However, the role of PHB2 in prion diseases remains unclear. In this study, we isolated primary cortical neurons from rats and used the neurotoxic prion peptide PrP106–126 as a cell model for prion diseases. We examined the role of PHB2 in PrP106–126-induced mitophagy using Western blotting and immunofluorescence microscopy and assessed the function of PHB2 in PrP106–126-induced neuronal death using the cell viability assay and the TUNEL assay. The results showed that PrP106–126 induced mitochondrial morphological abnormalities and mitophagy in primary cortical neurons. PHB2 was found to be indispensable for PrP106–126-induced mitophagy and was involved in the accumulation of PINK1 and recruitment of Parkin to mitochondria in primary neurons. Additionally, PHB2 depletion exacerbated neuronal cell death induced by PrP106–126, whereas the overexpression of PHB2 alleviated PrP106–126 neuronal toxicity. Taken together, this study demonstrated that PHB2 is indispensable for PINK1/Parkin-mediated mitophagy in PrP106–126-treated neurons and protects neurons against the neurotoxicity of the prion peptide.

ABSTRACTThe roles of mitochondrial respiration in pluripotency remain largely unknown. We show here that mouse ESC mitochondria possess superior respiration capacity compared to somatic cell mitochondria, and oxidative phosphorylation (OXPHOS) generates the majority of cellular ATP in ESCs. Inhibition of OXPHOS results in extensive pluripotency and metabolic gene expression reprogram, leading to disruption of self-renewal and pluripotency. Metabolomics profiling identifies UDP-N-acetylglucosamine (UDP-GlcNAc) as one of the most significantly decreased metabolites in response to OXPHOS inhibition. The loss of ESC identity induced by OXPHOS inhibition can be ameliorated by directly adding GlcNAc both in vitro and in vivo. This work demonstrates that mitochondrial respiration, but not glycolysis, produces the majority of ATP in ESCs, and uncovers a novel mechanism whereby mitochondrial respiration is coupled with the hexosamine biosynthesis pathway to generate UDP-GlcNAc for ESC identity maintenance.SIGNIFICANCEOxidative phosphorylation (OXPHOS) and glycolysis are the two major pathways for generating ATP in mammalian cells. It is widely assumed that somatic cells utilize OXPHOS, whereas embryonic stem cells (ESCs) utilize glycolysis with low mitochondrial respiration rates even under aerobic conditions. However, the relative contribution of OXPHOS and glycolysis to ATP generation in ESCs, and the role of mitochondrial respiration in regulating ESC identity, have remained unclear. In this study, Cao et al demonstrate that mouse ESC mitochondria have a significantly higher respiration capacity than somatic cell mitochondria. Oxidative phosphorylation produces the majority of cellular ATP in mESCs and is coupled with the hexosamine biosynthesis pathway to generate UDP-GlcNAc for pluripotency maintenance. These findings define the function and mechanism of OXPHOS in regulating pluripotency, and challenge the traditional concept that mESCs rely on glycolysis over OXPHOS for their major supply of energy.HIGHLIGHTSESC mitochondria have a significantly higher respiration capability than somatic cell mitochondriaOXPHOS, but not glycolysis, produces the majority of cellular ATP in ESCsOXPHOS inhibition induces a decrease in O-GlcNAcylation and the expression of pluripotency genes in blastocysts that can be partially rescued by adding GlcNAcOXPHOS is coupled with the hexosamine biosynthesis pathway for UDP-GlcNAc biosynthesis to maintain ESC identity

Embryonic stem cells (ESCs), which are characterized by the capacity for self-renewal and pluripotency, hold great promise for regenerative medicine. Increasing evidence points to the essential role of mitophagy in pluripotency regulation. Our recent work showed that PINK1/OPTN take part in guarding ESC mitochondrial homeostasis and pluripotency. Evaluating mitophagy in ESCs is important for exploring the relationships between mitochondrial homeostasis and pluripotency. ESCs are smaller in size than adult somatic cells and the mitophagosomes in ESCs are difficult to observe. Many methods have been employed-for example, detecting colocalization of LC3-II and mitochondria-to evaluate mitophagy in ESCs. However, it is important to define an objective way to detect mitophagy in ESCs. Here, we evaluated two commonly used fluorescence-based imaging methods to detect mitophagy in ESCs. By using autophagy- or mitophagy-defective ESC lines, we showed that the mito-Keima (mt-Keima) system is a suitable and effective way for detecting and quantifying mitophagy in ESCs. Our study provides evidence that mt-Keima is an effective tool to study mitophagy function in ESCs.

Polydopamine (PDA) is a biocompatible cell-adhesive polymer with versatile applications in biomedical devices. Previous studies have shown that PDA coating could improve cell adhesion and differentiation of human mesenchymal stem cells (hMSCs). However, there is still a knowledge gap in the effect of PDA-mediated protein adsorption on the epigenetic status of MSCs. This work used gelatin-coated cell culture surfaces with and without PDA underlayer (Gel and PDA-Gel) to culture and differentiate primary human adipose-derived stem cells (hASCs). The properties of these two substrates were significantly different, which, in combination with a variation in extracellular matrix (ECM) protein bioactivity, regulated cell adhesion and migration. hASCs reduced focal adhesions by downregulating the expression of integrins such as αV, α1, α2, and β1 on the PDA-Gel compared to the Gel substrate. Interestingly, the ratio of H3K27me3 to H3K27me3+H3K4me3 was decreased, but this only occurred for upregulation of AGG and BMP4 genes during chondrogenic differentiation. This result implies that the PDA-Gel surface positively affects the chondrogenic, but not adipogenic and osteogenic, differentiation. In conclusion, for the first time, this study demonstrates the sequential effects of PDA coating on the biophysical property of adsorbed protein and then focal adhesions and differentiation of hMSCs through epigenetic regulation. This study sheds light on PDA-mediated mechanotransduction.

Autophagy-mediated mitochondrial degradation plays pivotal roles in both the acquisition and maintenance of pluripotency, but the molecular mechanisms that link autophagy-mediated mitochondrial homeostasis to pluripotency regulation are unclear. Here, we identified that the mitophagy receptor BNIP3 regulates pluripotency. In mouse ESCs, depletion of BNIP3 caused accumulation of aberrant mitochondria accompanied by decreased mitochondrial membrane potential, increased production of reactive oxygen species (ROS), and reduced ATP generation, which led to compromised self-renewal and differentiation. Impairment of mitophagy by knockdown of BNIP3 inhibited mitochondrial clearance during pluripotency induction, resulting in decreased reprogramming efficiency. These defects were rescued by reacquisition of wild-type but not LIR-deficient BNIP3 expression. Taken together, our findings highlight a critical role of BNIP3-mediated mitophagy in the induction and maintenance of pluripotency.

Embryonic stem cells (ESCs) have a significantly lower mutation load compared to somatic cells, but the mechanisms that guard genomic integrity in ESCs remain largely unknown. Here we show that BNIP3-dependent mitophagy protects genomic integrity in mouse ESCs. Deletion of Bnip3 increases cellular reactive oxygen species (ROS) and decreases ATP generation. Increased ROS in Bnip3 −/− ESCs compromised self-renewal and were partially rescued by either NAC treatment or p53 depletion. The decreased cellular ATP in Bnip3 −/− ESCs induced AMPK activation and deteriorated homologous recombination, leading to elevated mutation load during long-term propagation. Whereas activation of AMPK in X-ray-treated Bnip3 +/+ ESCs dramatically ascended mutation rates, inactivation of AMPK in Bnip3 −/− ESCs under X-ray stress remarkably decreased the mutation load. In addition, enhancement of BNIP3-dependent mitophagy during reprogramming markedly decreased mutation accumulation in established iPSCs. In conclusion, we demonstrated a novel pathway in which BNIP3-dependent mitophagy safeguards ESC genomic stability, and that could potentially be targeted to improve pluripotent stem cell genomic integrity for regenerative medicine.

Emerging evidence indicates that cellular bioenergetics is critical in determining the self-renewal and differentiation of stem cells. Human platelet lysate (hPL) contains abundant proteins, which has been shown to improve self-renewal and osteogenic differentiation of mesenchymal stem cells (MSCs). However, the detailed modulating effect of hPL on MSCs energy metabolism remains unexplored. This study showed that MSCs cultured in hPL displayed a reduced cell size and cell spreading, but an improved proliferation and osteogenic capability compared with cells maintained in fetal bovine serum (FBS). RNA sequencing revealed widespread transcriptome differences between hPL-and FBS-MSCs where the differential expressed genes (DEGs) were enriched mainly in the PI3K-Akt and metabolic signal pathways. We found a significant downregulation of HIF1A (hypoxia-inducible factor 1 alpha) and altered mitochondrial features in hPL-MSCs, indicating a metabolism switch of the hPL-treated cells from glycolysis towards mitochondrial oxidative phosphorylation (OxPhos). It was also demonstrated that hPL-MSCs tend to differentiate towards the aerobic metabolism-demanded osteocytes or adipocytes rather than the anaerobic metabolism-demanded chondrocytes using a differentiation medium. Finally, hPL-MSCs showed an impaired paracrine function where the secreted factors cannot stimulate M2 polarization of THP1 cells and angiogenesis of HUVECs. We concluded that the PI3K-Akt/HIF1A-mediated metabolic state dominated the physiological property and lineage commitment of MSCs in hPL. For the first time, this study demonstrates the molecular mechanism of hPL in the regulation of metabolism and functions of MSCs, which implies the potential of hPL as an efficient biological material for stem cell engineering and regenerative medicine. (c) 2021 Elsevier Ltd. All rights reserved.

OBJECTIVES:Dysfunction of autophagy results in accumulation of depolarized mitochondria and breakdown of self-renewal and pluripotency in ESCs. However, the regulators that control how mitochondria are degraded by autophagy for pluripotency regulation remains largely unknown. This study aims to dissect the molecular mechanisms that regulate mitochondrial homeostasis for pluripotency regulation in mouse ESCs.MATERIALS AND METHODS:Parkin+/+ and parkin-/- ESCs were established from E3.5 blastocysts of parkin+/- x parkin+/- mating mice. The pink1-/- , optn-/- and ndp52-/- ESCs were generated by CRISPR-Cas9. shRNAs were used for function loss assay of target genes. Mito-Keima, ROS and ATP detection were used to investigate the mitophagy and mitochondrial function. Western blot, Q-PCR, AP staining and teratoma formation assay were performed to evaluate the PSC stemness.RESULTS:PINK1 or OPTN depletion impairs the degradation of dysfunctional mitochondria during reprogramming, and reduces the reprogramming efficiency and quality. In ESCs, PINK1 or OPTN deficiency leads to accumulation of dysfunctional mitochondria and compromised pluripotency. The defective mitochondrial homeostasis and pluripotency in pink1-/- ESCs can be compensated by gain expression of phosphomimetic Ubiquitin (Ub-S65D) together with WT or a constitutively active phosphomimetic OPTN mutant (S187D, S476D, S517D), rather than constitutively inactive OPTN (S187A, S476A, S517A) or a Ub-binding dead OPTN mutant (D477N).CONCLUSIONS:The mitophagy receptor OPTN guards ESC mitochondrial homeostasis and pluripotency by scavenging damaged mitochondria through TBK1-activated OPTN binding of PINK1-phosphorylated Ubiquitin.

Liu, Kun Wu, Yunkun Wei, Wenting Wang, Zhonghui Zhu, Jiaqi Wang, HonganHow to prevent internal risks to the information system, especially for undefined risks, is a great challenge. A reasonable approach is to mine the behavior rules of internal staff on historical data through various data mining algorithms and then use the behavior rules to detect abnormal behaviors. However, in practice, risk control officers are often not familiar with data mining technologies, so it is hard to make them effectively choose and adapt these algorithms to find internal risks. In this paper, we propose an interactive approach for behavior rule mining and anomaly detection. Firstly, we express behavior rules and abnormal behaviors as complex events uniformly to accommodate different mining algorithms. Then, the internal staff’s history behavior logs generated during production are used for mining behavior rules. Next, mined behavior rules are applied to new logs for anomaly detection. Finally, the detected abnormal behavior will be reported to the risk control officer for evaluation, and the feedback will be used for improving mining and detection settings to form a gradual and interactive process. The experiments on the real production data show that the approach is effective and efficient to detect abnormal behavior and can be used to prevent internal risks of the information system of big corporations such as banks.

Glioma is one of the most common brain tumors. Steroid receptor coactivator3 (SRC3) has been reported to be amplified and overexpressed in breast cancer, prostate cancer, lung cancer, and other types of tumors. However, the role of SRC3 in glioma remains unclear. Objective: Our study aimed to explore the role of SRC3 in glioma proliferation and invasion. Methods: The protein and mRNA expressions of SRC3 in glioma tissues and normal brain tissues were analyzed by western blot and the TCGA database, respectively. Then, the U251 and U87 cell lines were transfected with siRNA-SRC3 and a negative control. After that, an MTS assay and a colony formation assay were performed to investigate the effect of SRC3 in glioma cell proliferation. A Transwell assay was performed to evaluate the role of SRC3 in glioma cell invasion. Moreover, SRC3 mRNA expression in glioma cell lines was determined by RNA extraction and real time-PCR analysis. SRC3 protein expression and the relevant signaling molecules’ protein expressions in glioma cell lines were detected by western blot. Results: We firstly found that SRC3 was overexpressed in glioma tissue samples and glioma cell lines (grade II and IV). Further experiments showed that SRC3 knockdown inhibited cell proliferation, colony formation, and invasion capacity. Moreover, we found that knocking down SRC3 expression reduced PCNA, MMP9, p-Akt, CyclinD1 and CDK2 expressions significantly, while p21 expression was increased dramatically. Conclusion: SRC3 may function as a cancerogenic factor in the malignant progression of glioma and may be a potential target for glioma therapy.

Internal threat is an important issue for the information systems of an organization. To deal with this problem, organizations often formulate regulations and rules to regulate the behavior of employees and prevent them from causing production risks. However, how to effectively detect violations of the rules in the production process is challenging. In this paper, we propose an event based internal threat detection method. Firstly, we establish a detection model for regulation violation by representing rules and regulations as complex events and design a rule engine to detect if these complex events occur and discover the violations of rules. Then the logs generated during product are used for activating the rule reasoning. Finally, the rule violation will be reported to the supervisor for further investigation. The experiment on the real production processes shows the method is effective and efficient to detect internal threats and can be used at major production sites.

Although much is known about transcriptional networks that control embryonic stem cell (ESC) self-renewal and differentiation, the metabolic regulation of ESC is less clear. Autophagy is a catabolic process that is activated under both stress and normal conditions to degrade damaged organelles and aggregated proteins, and thus plays pivotal roles in somatic and adult stem cell function. However, if and how ESCs harness autophagy to regulate stemness remains largely unknown. Recently, we have defined that autophagy is essential for mitochondrial homeostasis regulation in pluripotency acquirement and maintenance. Here we identified high autophagic flux as an essential mechanism to maintain ESC identity. We show that mouse ESCs exhibit a high autophagic flux that is maintained by coordinating expression of autophagy core molecular machinery genes through FOXO1, a forkhead family transcription factor. Tapering autophagic flux by manipulating either Atg3 or Foxo1 expression compromised ESC self-renewal, pluripotency, and differentiation that could be restored by gain of wild-type but not function-deficient Atg3 or Foxo1 mutants, respectively. Our results define a newly recognized role of autophagic flux in mouse ESC identity maintenance that links cellular catabolism to ESC fate regulation.

Pluripotent stem cells, including induced pluripotent and embryonic stem cells (ESCs), have less developed mitochondria than somatic cells and, therefore, rely more heavily on glycolysis for energy production.(1-3) However, how mitochondrial homeostasis matches the demands of nuclear reprogramming and regulates pluripotency in ESCs is largely unknown. Here, we identified ATG3-dependent autophagy as an executor for both mitochondrial remodeling during somatic cell reprogramming and mitochondrial homeostasis regulation in ESCs. Dysfunctional autophagy by Atg3 deletion inhibited mitochondrial removal during pluripotency induction, resulting in decreased reprogramming efficiency and accumulation of abnormal mitochondria in established iPSCs. In Atg3 null mouse ESCs, accumulation of aberrant mitochondria was accompanied by enhanced ROS generation, defective ATP production and attenuated pluripotency gene expression, leading to abnormal self-renewal and differentiation. These defects were rescued by reacquisition of wild-type but not lipidation-deficient Atg3 expression. Taken together, our findings highlight a critical role of ATG3-dependent autophagy for mitochondrial homeostasis regulation in both pluripotency acquirement and maintenance.

Pluripotent stem cells (PSCs), including embryonic and induced pluripotent stem cells (iPSCs), show atypical cell cycle regulation characterized by a high proliferation rate and a shorter G1 phase compared with somatic cells. The mechanisms by which somatic cells remodel their cell cycle to achieve the high proliferation rate of PSCs during reprogramming are unclear. Here we identify that the Ink4 protein p18, which is expressed at high levels in somatic cells but at low levels in PSCs, is a roadblock to successful reprogramming. Mild inhibition of p18 expression enhances reprogramming efficiency, while ectopic expression of p18 completely blocks reprogramming. Mechanistic studies show that expression of wild-type p18, but not a p18 D68N mutant which cannot inhibit Cdk4/6, down-regulates expression of Cdk4/6 target genes involved in DNA synthesis (TK, TS, DHFR, PCNA) and cell cycle regulation (CDK1 and CCNA2) and thus inhibits reprogramming. These results indicate that p18 blocks reprogramming by targeting Cdk4/6-mediated cell cycle regulation. Taken together, our results define a novel pathway that inhibits somatic cell reprogramming and provide a new target to enhance reprogramming efficiency.