Mechanisms restraining break‐induced replication at two‐ended DNA double‐strand breaks

DNA synthesis during homologous recombination is highly mutagenic and prone to template switches. Two‐ended DNA double‐strand breaks (DSBs) are usually repaired by gene conversion with a short patch of DNA synthesis, thus limiting the mutation load to the vicinity of the DSB. Single‐ended DSBs are repaired by break‐induced replication (BIR), which involves extensive and mutagenic DNA synthesis spanning up to hundreds of kilobases. It remains unknown how mutagenic BIR is suppressed at two‐ended DSBs. Here, we demonstrate that BIR is suppressed at two‐ended DSBs by proteins coordinating the usage of two ends of a DSB: (i) ssDNA annealing proteins Rad52 and Rad59 that promote second end capture, (ii) D‐loop unwinding helicase Mph1, and (iii) Mre11‐Rad50‐Xrs2 complex that promotes synchronous resection of two ends of a DSB. Finally, BIR is also suppressed when Sir2 silences a normally heterochromatic repair template. All of these proteins are particularly important for limiting BIR when recombination occurs between short repetitive sequences, emphasizing the significance of these mechanisms for species carrying many repetitive elements such as humans.     

LncRNA SNHG1 promotes sepsis‐induced myocardial injury by inhibiting Bcl‐2 expression via DNMT1

Myocardial injury is a frequently occurring complication of sepsis. This study aims to investigate the molecular mechanism of long noncoding RNA (lncRNA) small nucleolar RNA host gene 1 (SNHG1)‐mediated DNA methyltransferase 1/B‐cell lymphoma‐2 (DNMT1/Bcl‐2) axis in sepsis‐induced myocardial injury. Mice and HL‐1 cells were treated with lipopolysaccharide (LPS) to establish animal and cellular models simulating sepsis and inflammation. LncRNA SNHG1 was screened out as a differentially expressed lncRNA in sepsis samples through microarray profiling, and the upregulated expression of lncRNA SNHG1 was confirmed in myocardial tissues of LPS‐induced septic mice and HL‐1 cells. Further experiments suggested that silencing of lncRNA SNHG1 reduced the inflammation and apoptotic rate of LPS‐induced HL‐1 cells. LncRNA SNHG1 inhibited Bcl‐2 expression by recruiting DNMT1 to Bcl‐2 promoter region to cause methylation. Inhibition of Bcl‐2 promoter methylation reduced the inflammation and apoptotic rate of LPS‐induced HL‐1 cells. In vivo experiments substantiated that lncRNA SNHG1 silencing alleviated sepsis‐induced myocardial injury in mice. Taken together, lncRNA SNHG1 promotes LPS‐induced myocardial injury in septic mice by downregulating Bcl‐2 through DNMT1‐mediated Bcl‐2 methylation.     

MicroRNA‐495‐3p diminishes doxorubicin‐induced cardiotoxicity through activating AKT

Doxorubicin (Dox) is a broad‐spectrum antitumour agent; however, its clinical application is impeded due to the cumulative cardiotoxicity. The present study aims to investigate the role and underlying mechanisms of microRNA‐495‐3p (miR‐495‐3p) in Dox‐induced cardiotoxicity. Herein, we found that cardiac miR‐495‐3p expression was significantly decreased in Dox‐treated hearts, and that the miR‐495‐3p agomir could prevent oxidative stress, cell apoptosis, cardiac mass loss, fibrosis and cardiac dysfunction upon Dox stimulation. In contrast, the miR‐495‐3p antagomir dramatically aggravated Dox‐induced cardiotoxicity in mice. Besides, we found that the miR‐495‐3p agomir attenuated, while the miR‐495‐3p antagomir exacerbated Dox‐induced oxidative stress and cellular injury in vitro. Mechanistically, we demonstrated that miR‐495‐3p directly bound to the 3′‐untranslational region of phosphate and tension homology deleted on chromosome ten (PTEN), downregulated PTEN expression and subsequently activated protein kinase B (PKB/AKT) pathway, and that PTEN overexpression or AKT inhibition completely abolished the cardioprotective effects of the miR‐495‐3p agomir. Our study for the first time identify miR‐495‐3p as an endogenous protectant against Dox‐induced cardiotoxicity through activating AKT pathway in vivo and in vitro.     

Histone chaperone CAF‐1 promotes HIV‐1 latency by leading the formation of phase‐separated suppressive nuclear bodies

HIV‐1 latency is a major obstacle to achieving a functional cure for AIDS. Reactivation of HIV‐1‐infected cells followed by their elimination via immune surveillance is one proposed strategy for eradicating the viral reservoir. However, current latency‐reversing agents (LRAs) show high toxicity and low efficiency, and new targets are needed to develop more promising LRAs. Here, we found that the histone chaperone CAF‐1 (chromatin assembly factor 1) is enriched on the HIV‐1 long terminal repeat (LTR) and forms nuclear bodies with liquid–liquid phase separation (LLPS) properties. CAF‐1 recruits epigenetic modifiers and histone chaperones to the nuclear bodies to establish and maintain HIV‐1 latency in different latency models and primary CD4⁺ T cells. Three disordered regions of the CHAF1A subunit are important for phase‐separated CAF‐1 nuclear body formation and play a key role in maintaining HIV‐1 latency. Disruption of phase‐separated CAF‐1 bodies could be a potential strategy to reactivate latent HIV‐1.     

IDH2 stabilizes HIF‐1α‐induced metabolic reprogramming and promotes chemoresistance in urothelial cancer

Drug resistance contributes to poor therapeutic response in urothelial carcinoma (UC). Metabolomic analysis suggested metabolic reprogramming in gemcitabine‐resistant urothelial carcinoma cells, whereby increased aerobic glycolysis and metabolic stimulation of the pentose phosphate pathway (PPP) promoted pyrimidine biosynthesis to increase the production of the gemcitabine competitor deoxycytidine triphosphate (dCTP) that diminishes its therapeutic effect. Furthermore, we observed that gain‐of‐function of isocitrate dehydrogenase 2 (IDH2) induced reductive glutamine metabolism to stabilize Hif‐1α expression and consequently stimulate aerobic glycolysis and PPP bypass in gemcitabine‐resistant UC cells. Interestingly, IDH2‐mediated metabolic reprogramming also caused cross resistance to CDDP, by elevating the antioxidant defense via increased NADPH and glutathione production. Downregulation or pharmacological suppression of IDH2 restored chemosensitivity. Since the expression of key metabolic enzymes, such as TIGAR, TKT, and CTPS1, were affected by IDH2‐mediated metabolic reprogramming and related to poor prognosis in patients, IDH2 might become a new therapeutic target for restoring chemosensitivity in chemo‐resistant urothelial carcinoma.     

Overexpression of c‐Jun inhibits erastin‐induced ferroptosis in Schwann cells and promotes repair of facial nerve function

Myelin undergoes various changes after nerve injury, and c‐Jun has a close relationship with Schwann cells (SCs). However, it remains unclear whether c‐Jun can be involved in nerve repair by regulating ferroptosis. To explore this, we first set up a facial nerve injury model and detected the changes of ferroptosis‐related proteins and c‐Jun by immunofluorescence and Western blot. Then, we cultured RSC 96 and pSCs, and studied the potential regulatory relationships by a combination of experimental methods such as CCK‐8, ELISA, immunofluorescence, qRT‐PCR, Western blot and viral transfection. Finally, we corroborated the role of c‐Jun through animal experiments. Our experiments revealed that ferroptosis occurs after facial nerve injury. Erastin decreased GPX4, c‐Jun proteins and GSH content, while PTGS2, NRF2, HO‐1 proteins, MDA, Fe²⁺ and ROS contents increased. This effect was inhibited after c‐Jun overexpression but was reversed after the addition of c‐Jun siRNA. Besides, we proved in vivo that c‐Jun could inhibit ferroptosis and promote the recovery of facial nerve function. In conclusion, our study identified the relationship between c‐Jun and ferroptosis during peripheral nerve injury repair, which provides new ideas for studying peripheral nerve injury and repair.     

N‐glycosylation of PD‐1 promotes binding of camrelizumab

PD‐1 is a highly glycosylated inhibitory receptor expressed mainly on T cells. Targeting of PD‐1 with monoclonal antibodies (MAbs) to block the interaction with its ligand PD‐L1 has been successful for the treatment of multiple tumors. However, polymorphisms at N‐glycosylation sites of PD‐1 exist in the human population that might affect antibody binding, and dysregulated glycosylation has been observed in the tumor microenvironment. Here, we demonstrate varied N‐glycan composition in PD‐1, and show that the binding affinity of camrelizumab, a recently approved PD‐1‐specific MAb, to non‐glycosylated PD‐1 proteins from E. coli is substantially decreased compared with glycosylated PD‐1. The structure of the camrelizumab/PD‐1 complex reveals that camrelizumab mainly utilizes its heavy chain to bind to PD‐1, while the light chain sterically inhibits the binding of PD‐L1 to PD‐1. Glycosylation of asparagine 58 (N58) promotes the interaction with camrelizumab, while the efficiency of camrelizumab to inhibit the binding of PD‐L1 is substantially reduced for glycosylation‐deficient PD‐1. These results increase our understanding of how glycosylation affects the activity of PD‐1‐specific MAbs during immune checkpoint therapy.     

Requirements for human‐induced pluripotent stem cells

‘Requirements for Human‐Induced Pluripotent Stem Cells’ is the first set of guidelines on human‐induced pluripotent stem cells in China, jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research. This standard specifies the technical requirements, test methods, and instructions for use, labeling, packaging, storage, transportation, and waste handling for human‐induced pluripotent stem cells, which apply to the production and quality control of human‐induced pluripotent stem cells. It was released by the Chinese Society for Cell Biology on 9 January 2021 and came into effect on 9 April 2021. We hope that the publication of these guidelines will promote institutional establishment, acceptance, and execution of proper protocols and accelerate the international standardization of human‐induced pluripotent stem cells for applications.     

TIMELESS‐TIPIN and UBXN‐3 promote replisome disassembly during DNA replication termination in Caenorhabditis elegans

The eukaryotic replisome is rapidly disassembled during DNA replication termination. In metazoa, the cullin‐RING ubiquitin ligase CUL‐2^LRR‐1 drives ubiquitylation of the CMG helicase, leading to replisome disassembly by the p97/CDC‐48 “unfoldase”. Here, we combine in vitro reconstitution with in vivo studies in Caenorhabditis elegans embryos, to show that the replisome‐associated TIMELESS‐TIPIN complex is required for CUL‐2^LRR‐1 recruitment and efficient CMG helicase ubiquitylation. Aided by TIMELESS‐TIPIN, CUL‐2^LRR‐1 directs a suite of ubiquitylation enzymes to ubiquitylate the MCM‐7 subunit of CMG. Subsequently, the UBXN‐3 adaptor protein directly stimulates the disassembly of ubiquitylated CMG by CDC‐48_UFD‐1_NPL‐4. We show that UBXN‐3 is important in vivo for replisome disassembly in the absence of TIMELESS‐TIPIN. Correspondingly, co‐depletion of UBXN‐3 and TIMELESS causes profound synthetic lethality. Since the human orthologue of UBXN‐3, FAF1, is a candidate tumour suppressor, these findings suggest that manipulation of CMG disassembly might be applicable to future strategies for treating human cancer.     

LRG‐1 promotes fat graft survival through the RAB31‐mediated inhibition of hypoxia‐induced apoptosis

Autologous adipose tissue is an ideal soft tissue filling material, and its biocompatibility is better than that of artificial tissue substitutes, foreign bodies and heterogeneous materials. Although autologous fat transplantation has many advantages, the low retention rate of adipose tissue limits its clinical application. Here, we identified a secretory glycoprotein, leucine‐rich‐alpha‐2‐glycoprotein 1 (LRG‐1), that could promote fat graft survival through RAB31‐mediated inhibition of hypoxia‐induced apoptosis. We showed that LRG‐1 injection significantly increased the maintenance of fat volume and weight compared with the control. In addition, higher fat integrity, more viable adipocytes and fewer apoptotic cells were observed in the LRG‐1‐treated groups. Furthermore, we discovered that LRG‐1 could reduce the ADSC apoptosis induced by hypoxic conditions. The mechanism underlying the LRG‐1‐mediated suppression of the ADSC apoptosis induced by hypoxia was mediated by the upregulation of RAB31 expression. Using LRG‐1 for fat grafts may prove to be clinically successful for increasing the retention rate of transplanted fat.     

Knockdown of circular RNA VANGL1 inhibits TGF‐β‐induced epithelial‐mesenchymal transition in melanoma cells by sponging miR‐150‐5p

Melanoma is one of the most aggressive and life‐threatening skin cancers, and in this research, we aimed to explore the functional role of circular RNA VANGL1 (circVANGL1) in melanoma progression. The expression levels of circVANGL1 were observed to be significantly increased in clinical melanoma tissues and cell lines. Moreover, circVANGL1 knockdown suppressed, while circVANGL1 overexpression promoted the proliferation, migration and invasion abilities of melanoma cells. Further investigations confirmed the direct binding relation between circVANGL1 and miR‐150‐5p in melanoma, and restoration of miR‐150‐5p blocked the effects of circVANGL1 overexpression in melanoma cells. We further found that circVANGL1 was up‐regulated by TGF‐β treatment, and the enhanced EMT of TGF‐β‐treated melanoma cells was blocked by circVANGL1 knockdown. In conclusion, these results indicated that circVANGL1 might serve as a promising therapeutic target for melanoma.     

Overexpression of PD‐L1 causes germ cells to slough from mouse seminiferous tubules via the PD‐L1/PD‐L1 interaction

Spermatogenesis is a cyclical process in which different generations of spermatids undergo a series of developmental steps at a fixed time and finally produce spermatids. Here, we report that overexpression of PD‐L1 (B7 homolog1) in the testis causes sperm developmental disorders and infertility in male mice, with severe malformation and sloughing during spermatid development, characterized by disorganized and collapsed seminiferous epithelium structure. PD‐L1 needs to be simultaneously expressed on Sertoli cells and spermatogonia to cause spermatogenesis failure. After that, we excluded the influence of factors such as the PD‐L1 receptor and humoral regulation, confirming that PD‐L1 has an intrinsic function to interact with PD‐L1. Studies have shown that PD‐L1 not only serves as a ligand but also plays a receptor‐like role in signal transduction. PD‐L1 interacts with PD‐L1 to affect the adhesive function of germ cells, causing malformation and spermatid sloughing. Taken together, these results indicate that PD‐L1 can interact with PD‐L1 to cause germ cell detachment and male infertility.     

Macrophage‐derived exosomal miR‐4532 promotes endothelial cells injury by targeting SP1 and NF‐κB P65 signalling activation

Atherosclerosis is a complex pathological process involving macrophages, endothelial cells and vascular smooth muscle cells that can lead to ischemic heart disease; however, the mechanisms underlying cell‐to‐cell communication in atherosclerosis are poorly understood. In this study, we focused on the role of exosomal miRNAs in crosstalk between macrophages and endothelial cells and explored the rarely studied molecular mechanisms involved. Our in vitro result showed that macrophage‐derived exosomal miR‐4532 significantly disrupted human umbilical vein endothelial cells (HUVECs) function by targeting SP1 and downstream NF‐κB P65 activation. In turn, increased endothelin‐1 (ET‐1), intercellular cell adhesion molecule‐1 (ICAM‐1) and vascular cell adhesion molecule‐1 (VCAM‐1) and decreased endothelial nitric oxide synthase (eNOS) expression in HUVECs increased attraction of macrophages, exacerbating foam cell formation and transfer of exosomal miR‐4532 to HUVECs. MiR‐4532 overexpression significantly promoted endothelial injury and pretreatment with an inhibitor of miR‐4532 or GW4869 (exosome inhibitor) could reverse this injury. In conclusion, our data reveal that exosomes have a critical role in crosstalk between HUVECs and macrophages. Further, exosomal miR‐4532 transferred from macrophages to HUVECs and targeting specificity protein 1 (SP1) may be a novel therapeutic target in patients with atherosclerosis.     

Deficiency of Ninjurin1 attenuates LPS/D‐galactosamine‐induced acute liver failure by reducing TNF‐α‐induced apoptosis in hepatocytes

Nerve injury‐induced protein 1 (Ninjurin1, Ninj1) is a membrane protein that mediates cell adhesion. The role of Ninj1 during inflammatory response has been widely investigated in macrophages and endothelial cells. Ninj1 is expressed in various tissues, and the liver also expresses high levels of Ninj1. Although the hepatic upregulation of Ninj1 has been reported in human hepatocellular carcinoma and septic mice, little is known of its function during the pathogenesis of liver diseases. In the present study, the role of Ninj1 in liver inflammation was explored using lipopolysaccharide (LPS)/D‐galactosamine (D‐gal)‐induced acute liver failure (ALF) model. When treated with LPS/D‐gal, conventional Ninj1 knock‐out (KO) mice exhibited a mild inflammatory phenotype as compared with wild‐type (WT) mice. Unexpectedly, myeloid‐specific Ninj1 KO mice showed no attenuation of LPS/D‐gal‐induced liver injury. Whereas, Ninj1 KO primary hepatocytes were relatively insensitive to TNF‐α‐induced caspase activation as compared with WT primary hepatocytes. Also, Ninj1 knock‐down in L929 and AML12 cells and Ninj1 KO in HepG2 cells ameliorated TNF‐α‐mediated apoptosis. Consistent with in vitro results, hepatocyte‐specific ablation of Ninj1 in mice alleviated LPS/D‐gal‐induced ALF. Summarizing, our in vivo and in vitro studies show that lack of Ninj1 in hepatocytes diminishes LPS/D‐gal‐induced ALF by alleviating TNF‐α/TNFR1‐induced cell death.