BackgroundVascular injury is a major contributor to the development of cardiovascular diseases. Following vascular damage, macrophages migrate to the injury site and, during the later stages of vascular repair, secrete cytokines such as interleukin-10 (IL-10) and transforming growth factor-β1a (TGFB1A), thereby promoting vascular regeneration. Previous studies have demonstrated that macrophage recruitment to sites of tissue injury is mediated by the CXCR4A-CXCL12B signaling axis. In a screening of traditional Chinese medicinal herbs for cardiovascular therapeutic potential, Salvia miltiorrhiza root was identified as a promising source of bioactive compounds capable of enhancing vascular repair through modulation of the CXCR4A-CXCL12B axis.MethodsEstablishing a vascular injury model in transgenic zebrafish lines Tg (flk1:eGFP; gata1:dsRed) using a two-photon microscopy laser system. Dynamic monitoring of vascular repair via two-photon microscopy. Evaluate macrophage migration capacity in a Tg (mpeg1:eGFP) zebrafish vascular injury model using confocal microscopy. Detection of il-10 and tgfb1a expression released by macrophages via qPCR experiments. Detect CXCR4A-CXCL12B expression at the site of zebrafish vascular injury via fluorescence in situ hybridization coupled with antibody staining.ResultsWe confirm that compounds from the selected extract promote macrophage migration to vascular injury sites by upregulating the CXCR4A-CXCR12B signaling axis. This process accelerates repair of damaged blood vessels in zebrafish by inducing the release of cytokines such as il-10 and tgfb1a.ConclusionsThis study confirms that Salvia miltiorrhiza, a traditional Chinese medicinal plant, is a valuable source of bioactive compounds with pro-angiogenic properties. Our findings provide scientific support for the traditional use of Salvia miltiorrhiza active components in treating vascular injuries.

Table S2. KRAS mutations in leukemia

Table S3. HRAS mutations in leukemia

Homeostasis is essential for hematopoiesis, and its dysregulation can lead to severe pathological conditions. Retinoic acid (RA) is a key regulator that exerts concentration-dependent effects on both embryonic and adult hematopoiesis. However, the mechanisms that modulate RA signaling in hematopoietic processes remain poorly understood. Using zebrafish as a model, we identified angiopoietin-like protein 5 (Angptl5) as a critical regulator of hematopoietic homeostasis. Loss of Angptl5 function resulted in myeloid hyperplasia in the anterior lateral plate mesoderm (ALPM) and anterior expansion of erythroid progenitors in the posterior lateral plate mesoderm (PLPM)— phenotypes consistent with attenuated RA signaling. Molecular analyses confirmed impaired RA signaling in angptl5Δ10/Δ10 mutants, and exogenous RA supplementation fully rescued the hematopoietic defects. Mechanistically, we found that Angptl5 transcriptionally activates retinol dehydrogenase dhrs9 through its interaction with Integrin α6lβ5. Our findings establish Angptl5 as a novel and essential regulator of embryonic hematopoiesis and reveal a previously unrecognized mechanism controlling hematopoietic homeostasis. These insights position Angptl5 as a potential therapeutic target for hematological disorders.

N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD), a widespread tire-derived contaminant, has drawn increasing concern for its environmental persistence and toxicity. However, its specific effects on early liver development remain poorly understood. In this study, we investigated the hepatotoxicity of 6PPD using zebrafish larvae and human L02 hepatocyte models. A novel exposure strategy was employed, initiating 6PPD treatment at 48 h post-fertilization, after liver budding, to minimize interference from early-stage developmental defects. 6PPD exposure led to a marked reduction in liver size without obvious morphological abnormalities, alongside downregulation of hepatocyte marker genes. Importantly, liver growth gradually recovered after 6PPD removal, suggesting acute and reversible toxicity. Mechanistically, 6PPD induced DNA damage and apoptosis in hepatocytes, as evidenced by elevated γ-H2AX, baxa, and casp3a expression, while hepatocyte proliferation remained unaffected. Transcriptomic and qPCR analyses revealed activation of inflammatory pathways and increased macrophage infiltration. Co-treatment with the anti-inflammatory agent dexamethasone rescued liver size and reduced DNA damage, indicating inflammation as a key mediator of 6PPD-induced toxicity. Similarly, 6PPD exposure in human hepatocytes reduced viability and increased apoptotic markers, which were alleviated by dexamethasone. These results demonstrate that 6PPD causes acute, inflammation-mediated liver toxicity during embryogenesis, with conserved mechanisms across species.

Table S1. NRAS mutations in leukemia

Acute myeloid leukemia (AML) cells rely heavily on mitochondrial oxidative phosphorylation (OXPHOS) for energy, making mitochondrial function a promising therapeutic target. TCF12, a transcription factor belonging to the basic helix-loop-helix (bHLH) family, has been implicated in various cancers and is highly expressed in AML, where it may contribute to disease progression. However, the effects of TCF12 on AML progression and the underlying mechanisms remain unclear. This study aimed to elucidate the role of TCF12 in promoting AML cell survival and uncover the underlying mechanism. We found that TCF12 was highly expressed in AML and was associated with poor patient prognosis. Knockdown of TCF12 significantly inhibited AML cell growth, suppressed glycolysis, increased ROS accumulation, and induced mitochondrial dysfunction. Additionally, TCF12 was shown to promote EZH2 transcription, whereas its knockdown increased TXNIP expression, thereby inhibiting AML progression. In summary, TCF12 promotes AML progression by regulating EZH2-mediated suppression of TXNIP, thereby enhancing glycolysis, maintaining mitochondrial function, regulating ROS levels, and promoting cell survival, which underscores its potential as a therapeutic target.

Abstract RAS mutations are prevalent in leukemia, including mutations at G12, G13, T58, Q61, K117, and A146. These mutations are often crucial for tumor initiation, maintenance, and recurrence. Although much is known about RAS function in the last 40 years, a substantial knowledge gap remains in understanding the mutation-specific biological activities of RAS in cancer and the approaches needed to target specific RAS mutants effectively. The recent approval of KRASG12C inhibitors, adagrasib and sotorasib, has validated KRAS as a direct therapeutic target and demonstrated the feasibility of selectively targeting specific RAS mutants. Nevertheless, KRASG12C remains the only RAS mutant successfully targeted with FDA-approved inhibitors for cancer treatment in patients, limiting its applicability for other oncogenic RAS mutants, such as G12D, in leukemia. Despite these challenges, new approaches have generated optimism about targeting specific RAS mutations in an allele-dependent manner for cancer therapy, supported by compelling biochemical and structural evidence, which inspires further exploration of RAS allele-specific vulnerabilities. This review will discuss the recent advances and challenges in the development of therapies targeting RAS signaling, highlight emerging therapeutic strategies, and emphasize the importance of allele-specific approaches for leukemia treatment.

Cholesterol (CH) plays a crucial role in enhancing the membrane stability of drug delivery systems (DDS). However, its association with conditions such as hyperlipidemia often leads to criticism, overshadowing its influence on the biological effects of formulations. In this study, we reevaluated the delivery effect of CH using widely applied lipid microspheres (LM) as a model DDS. We conducted comprehensive investigations into the impact of CH on the distribution, cell uptake, and protein corona (PC) of LM at sites of cardiovascular inflammatory injury. The results demonstrated that moderate CH promoted the accumulation of LM at inflamed cardiac and vascular sites without exacerbating damage while partially mitigating pathological damage. Then, the slow cellular uptake rate observed for CH@LM contributed to a prolonged duration of drug efficacy. Network pharmacology and molecular docking analyses revealed that CH depended on LM and exerted its biological effects by modulating peroxisome proliferator-activated receptor gamma (PPAR-γ) expression in vascular endothelial cells and estrogen receptor alpha (ERα) protein levels in myocardial cells, thereby enhancing LM uptake at cardiovascular inflammation sites. Proteomics analysis unveiled a serum adsorption pattern for CH@LM under inflammatory conditions showing significant adsorption with CH metabolism-related apolipoprotein family members such as apolipoprotein A-V (Apoa5); this may be a major contributing factor to their prolonged circulation in vivo and explains why CH enhances the distribution of LM at cardiovascular inflammatory injury sites. It should be noted that changes in cell types and physiological environments can also influence the biological behavior of formulations. The findings enhance the conceptualization of CH and LM delivery, providing novel strategies for investigating prescription factors' bioactivity.

BACKGROUND:Mycn, a MYC gene family member, is implicated in both carcinogenesis through amplification and Feingold syndrome through its deficiency. Previous studies have indicated that increased Mycn expression enhances vascularization in human neuroblastomas, yet its precise role in vascular development remains elusive. RESULTS:In this study, we utilized single-cell RNA-seq and live imaging analyses to confirm that mycn is expressed during zebrafish vasculogenesis. We investigated vascular development in zebrafish using a genetically engineered mycn mutation. Our findings reveal that mycn-deficient zebrafish exhibit reduced intersegmental vessels and malformed subintestinal vessels, primarily due to decreased cell proliferation in vascular endothelial cells. Importantly, we discovered that activation of PI3K signaling significantly ameliorates these vascular abnormalities. CONCLUSIONS:Our study establishes Mycn as a key regulator of vascular development in zebrafish, acting through the PI3K signaling pathway.

IntroductionCDK2 (Cyclin-dependent kinase 2) is an oncogenic cyclin-dependent kinase with potent mitogenic and immunosuppressive functions. Despite extensive research on CDK2 inhibitors, the lack of selectivity has made it unclear whether CDK2 inhibition specifically facilitate immunogenic cell death.MethodsWe used CRISPR-Cas9 system to generate Cdk2-/- MCA205 cells. Tumor cells were inoculated subcutaneously into mice while administering MTX (mitoxantrone) or anti-PD-1 antibodies treatment to observe tumor growth curves. Next, immune cell infiltration in tumor microenvironment was detected by immunofluorescence. Furthermore, apoptosis pathway was evaluated by flow cytometry and western blot. The hallmarks of immunogenic cell death were detected by flow cytometry, ELISA or qRT-PCR.ResultsWe found that mice bearing Cdk2-/- cancer cells exhibit slower tumor growth than WT cells after anthracycline analogue MTX treatment, and this phenomenon is dependent on the immune system. Furthermore, our data exhibits that Cdk2-/- cancer cells treated with MTX trigger a more robust immunostimulatory responses than WT cells, including apoptosis stress response, surface calreticulin expression, endoplasmic reticulum stress response, HMGB1 (High Mobility Group Box 1) release, and type-1 interferon response. DiscussionThis study not only suggests that CDK2 inhibition improves the outcome of chemotherapy by enhancing the type-1 interferon response but also investigates the synergistic effects of CDK2 inhibition with MTX or anti-PD-1 antibodies in immunocompetent mice.

OBJECTIVE:In this study, we aimed to create a diabetic zebrafish model and investigate the effects of high glucose levels on both inflammation and tissue regeneration. METHODS:We established the Tübingen strain of zebrafish (wild type, WT) and Tg (mpo: EGFP), and Tg (coro1a: EGFP) zebrafish models of caudal fin amputation to study tissue regeneration and assess inflammation. RESULTS:Our results revealed that elevated glucose levels led to the increased recruitment of neutrophils and macrophages following amputation. Zebrafish larvae exposed to 222 mM glucose for 14 days exhibited severely impaired limb regeneration postamputation. In addition, the levels of key inflammatory factors, including interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), vascular cell adhesion molecule-1 (VCAM-1), and monocyte chemoattractant protein-1 (MCP-1), which are closely associated with the development of diabetic wounds, were significantly increased after caudal fin amputation upon glucose exposure. Most notably, the p38 inhibitor SB203580 effectively reduced the expression of these key inflammatory factors and promoted tissue regeneration under high-glucose conditions. According to high-throughput transcriptome sequencing, caudal fin amputation following glucose exposure resulted in the enrichment of genes in several pathways involved in amino acid metabolism, lipid metabolism, and inflammation. CONCLUSIONS:Our study provides a comprehensive genetic and molecular profile that closely recapitulates human diabetes. This system offers a valuable avenue for advancing basic research in the field of inflammation and regeneration within a diabetic context and promotes translational research for diabetes therapy.

Proprotein convertase subtilisin/kexin type 5 (PCSK5) is a member of the proprotein convertase (PC) family, which processes immature proteins into functional proteins and plays an important role in the process of cell migration and transformation. Andrographolide is a non-peptide compound with PC inhibition and antitumor activity. Our research aimed to investigate the functional role of PCSK5 downregulation combined with Andro on GBM progression. Results from the cancer genome atlas (TCGA) and clinical samples revealed a significant upregulation of PCSK5 in GBM tissues than in non-tumor brain tissues. Higher expression of PCSK5 was correlated with advanced GBM stages and worse patient prognosis. PCSK5 knockdown attenuated the epithelial-mesenchymal transition (EMT)-like properties of GBM cells induced by IL-6. PCSK5 knockdown in combination with Andro treatment significantly inhibited the proliferation and invasion of GBM cells in vitro, as well as tumor growth in vivo. Mechanistically, PCSK5 downregulation reduced the expression of p-STAT3 and Matrix metalloproteinases (MMPs), which could be rescued by the p-STAT3 agonist. STAT3 silencing downregulated the expression of MMPs without affecting PCSK5. Furthermore, Andro in combination with PCSK5 silencing significantly inhibited STAT3/MMPs axis. These observations provided evidence that PCSK5 functioned as a potential tumor promoter by regulating p-STAT3/MMPs and the combination of Andro with PCSK5 silencing might be a good strategy to prevent GBM progression.

N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD), which is widely used as an antiozonant in rubber tires, has recently got much attention for its acute aquatic toxicity. However, the developmental toxicity of 6PPD in cerebrovascular network remains unknown. Here, we investigated the effects of 6PPD exposure in cerebral vascular using zebrafish. 6PPD would not affect the body length and shape of zebrafish larvae at the concentrations ranging from 20 μg/L to 1000 μg/L. 6PPD induced developmental defects in the brain in a concentration-dependent manner. The trunk vascular development would not be affected while the cerebrovascular network was disrupted upon 6PPD exposure. 6PPD would trigger excessive Reactive Oxygen Species (ROS) in the brain, indicating abnormal oxidative stress. Mechanistically, brain-specific transcriptome analysis showed that 6PPD could potentially cause the blockage of arachidonic acid (AA) metabolism-related genes and the upregulation of ferroptosis-related genes. Besides, treatment with ferroptosis inhibitor N-Acetyl-L-cysteine (NAC) attenuated oxidative damage and improved the construction of cerebrovascular network upon 6PPD exposure. Moreover, using a human vascular endothelial cell line, we further confirmed that 6PPD could trigger abnormal oxidative stress and defective expansion capacity, implying the conserved toxicity cross species. These findings are useful for the elucidation of toxicity underlying 6PPD in cerebrovascular systems of both zebrafish and humans.

背景:随着年龄的增长,脾脏的结构和功能发生改变,胸腺在青春期之后逐渐萎缩退化,导致机体发生免疫功能障碍.目的:探讨骨髓间充质干细胞对衰老猕猴胸腺及脾脏结构和功能的作用.方法:筛选出平均年龄25岁老年猕猴6只,随机分为老年组和老年治疗组各3只;老年治疗组猕猴经股静脉输注超顺磁性铁纳米颗粒标记的第4代骨髓间充质干细胞(1×107个/kg),1次/d,连续输注3 d,老年组猕猴在同一时间输注等体积的生理盐水;猕猴最后一次输注骨髓间充质干细胞5个月后给予安乐死处理,取出胸腺以及脾脏组织;苏木精-伊红染色观察胸腺组织和脾脏组织的结构变化;免疫荧光染色分析胸腺组织中各种T细胞亚群以及衰老相关基因蛋白P21的表达量变化;免疫组织化学染色分析脾脏组织中各种T细胞亚群以及衰老相关基因蛋白P21的表达量变化;安乐死处理猕猴之前抽取各组猕猴股静脉血5 mL,运用酶联免疫分析外周血血清中衰老相关分泌表型肿瘤坏死因子α和白细胞介素1α的分泌水平.结果 与结论:①超顺磁性铁纳米颗粒标记的骨髓间充质干细胞经过股静脉输注入猕猴体内能够成功定植在衰老胸腺和脾脏组织中发挥作用;②骨髓间充质干细胞移植治疗后,衰老猕猴中胸腺部分组织出现清晰的皮质与髓质交界,脂肪细胞减少,出现胸腺小体,向正常的胸腺组织结构转变;衰老猕猴中脾脏组织出现清晰的红髓与白髓分界,脾小体的边缘多完整,且巨噬细胞减少,向正常的脾脏组织结构转变;③与老年组相比,老年治疗组衰老胸腺和脾脏组织中CD3+、CD4+以及CD8+T细胞的表达量呈现增高的趋势,老年治疗组衰老胸腺和脾脏组织中P21蛋白的表达量呈现显著下降趋势;④与老年组相比,老年治疗组外周血中衰老相关分泌表型肿瘤坏死因子α以及白细胞介素1α的分泌水平呈现显著下降趋势;⑤上述结果表明,通过骨髓间充质干细胞的移植治疗,能够改善老年猕猴胸腺及脾脏组织的结构和功能.

目的 探索Toll样受体4(TLR4)在小鼠皮肤缺损创面愈合及早期瘢痕形成的作用.方法 将36只购自南京大学动物模式研究所的小鼠分为野生型组(WT)和TLR4基因敲除组(TLR4KO)切除部分背部全层皮肤建立模型,每组18只,观察小鼠创面愈合及瘢痕形成变化.实时定量聚合酶链反应(Real-time PCR)检测造模后7、14、21、28 d TLR4,11 d和33 d白细胞介素(IL)-6、IL-10 mRNA水平,苏木精-伊红(HE)和天狼星红染色评估33 d创面及变化.组间比较采用t 检验.结果 在创面愈合过程中,TLR4KO创面闭合时间高于WT[(25.91±2.81)d比(19.55±2.42)d,t=5.690,P＜0.01],TLR4KO IL-10 mRNA 指标低于 WT(0.005 比 1.000,t=37.070,P＜0.01),TLR4KOIL-6mRNA 指标高于 WT(2.650 比 1.000,t=19.290,P＜0.01).在瘢痕形成早期,TLR4KO 表皮厚度高于 WT[(45.83±1.61)μm 比(22.79±2.07)μm,t=7.645,P＜0.01],TLR4KO Ⅰ 型/Ⅲ型胶原比值高于 WT(24.54±1.27 比 7.93±1.76,t=10.820,P＜0.01),TLR4KO IL-6 mRNA 指标高于 WT(3.190 比 1.000,t=9.865,P＜0.01),TLR4KO 与 WT IL-10 mRNA 指标差异无统计学意义(1.108比1.000,t=0.960,P＞0.05).结论 TLR4功能完全丧失导致皮肤创面延迟愈合,进而导致TLR4基因敲除小鼠早期瘢痕明显,可能与炎性反应失调有关.

ABSTRACT:Aging is accompanied by significant inhibition of hematopoietic and immune system function and disruption of bone marrow structure. Aging-related alterations in the inflammatory response, immunity, and stem cell niches are at the root of hematopoietic aging. Understanding the molecular mechanisms underlying hematopoietic and bone marrow aging can aid the clinical treatment of aging-related diseases. In particular, it is unknown how the niche reprograms hematopoietic stem cells (HSCs) in an age-dependent manner to maintain normal hematopoiesis in elderly individuals. Recently, specific inhibitors and blood exchange methods have been shown to reshape the hematopoietic niche and reverse hematopoietic aging. Here, we present the latest scientific discoveries related to hematopoietic aging and hematopoietic system rejuvenation, discuss the relationships between hematopoietic niche aging and HSC aging, and describe related studies on stem cell-mediated regulation of hematopoietic aging, aiming to provide new ideas for further study.

Myelin and lymphocyte protein 2 (MAL2) is mainly involved in endocytosis under physiological conditions and mediates the transport of materials across the membranes of cell and organelle. It has been reported that MAL2 is significantly upregulated in diverse cancers. This study aimed to investigate the role of MAL2 in breast cancer (BC). Bioinformatics analysis and Immunohistochemical assay were applied to detect the correlation between MAL2 expression in breast cancer tissues and the prognosis of breast cancer patients. Functional experiments were carried out to investigate the role of MAL2 in vitro and in vivo. The molecular mechanisms involved in MAL2-induced β-catenin and c-Myc expression and β-catenin/c-Myc-mediated enhancement of BC progression were confirmed by western blot, β-catenin inhibitor and agonist, Co-IP and immunofluorescence colocalization assays. Results from the cancer genome atlas (TCGA) and clinical samples confirmed a significant upregulation of MAL2 in BC tissues than in adjacent non-tumor tissues. High expression of MAL2 was associated with worse prognosis. Functional experiments demonstrated that MAL2 knockdown reduced the migration and invasion associating with EMT, increased the apoptosis of BC cells in vitro and reduced the metastatic capacity in vivo. Mechanistically, MAL2 interacts with β-catenin in BC cells. MAL2 silencing reduced the expression of β-catenin and c-Myc, while the β-catenin agonist SKL2001 partially rescued the downregulation of c-Myc and inhibition of migration and invasion caused by MAL2 knockdown in BC cells. These observations provided evidence that MAL2 acted as a potential tumor promoter by regulating EMT and β-catenin/c-Myc axis, suggesting potential implications for anti-metastatic therapy for BC.

Much progress has been made toward generating analogs of early embryos, such as gastruloids and embry-oids, in vitro. However, methods for how to fully mimic the cell movements of gastrulation and coordinate germ-layer patterning to induce head formation are still lacking. Here, we show that a regional Nodal gradient applied to zebrafish animal pole explant can generate a structure that recapitulates the key cell movements of gastrulation. Using single-cell transcriptome and in situ hybridization analysis, we assess the dynamics of the cell fates and patterning of this structure. The mesendoderm differentiates into the anterior endoderm, pre -chordal plate, notochord, and tailbud-like cells along an anterior-posterior axis, and an anterior-posterior -patterned head-like structure (HLS) progressively forms during late gastrulation. Among 105 immediate Nodal targets, 14 genes contain axis-induction ability, and 5 of them induce a complete or partial head struc-ture when overexpressed in the ventral side of zebrafish embryos.