Neuroblastoma (NB) is the most common extracranial solid tumor in children. Despite treatment advances, the survival rates of high-risk NB patients remain low. This highlights the urgent need for a deeper understanding of the molecular mechanisms driving NB progression to support the development of new therapeutic strategies. In this study, we demonstrated that the reduced levels of DNAJC12, a protein involved in metabolic regulation, are associated with poor prognosis in NB patients. Our data indicate that low DNAJC12 expression activates glycolysis in NB cells, leading to increased lactic acid production and histone H4 lysine 5 lactylation (H4K5la). Elevated H4K5la upregulates the transcription of COL1A1, a gene implicated in cell metastasis. Immunohistochemistry staining of NB patient samples confirmed that high H4K5la levels correlate with poor clinical outcomes. Furthermore, we showed that inhibiting glycolysis, reducing H4K5la, or targeting COL1A1 can mitigate the invasive behavior of NB cells. These findings reveal a critical link between metabolic reprogramming and epigenetic modifications in the context of NB progression, suggesting that H4K5la could serve as a novel diagnostic and prognostic marker, and shed light on identifying new therapeutic targets within metabolic pathways for the treatment of this aggressive pediatric cancer.
Abstract Background Metagenomic next-generation sequencing (mNGS) has the potential to become a complementary, if not essential, test in some clinical settings. However, the clinical application of mNGS in a large population of children with various types of infectious diseases (IDs) has not been previously evaluated. Methods From April 2019 to April 2021, 640 samples were collected at a single pediatric hospital and classified as ID [479 (74.8%)], non-ID [NID; 156 (24.4%)], and unknown cases [5 (0.8%)], according to the final clinical diagnosis. We compared the diagnostic performance in pathogen detection between mNGS and standard reference tests. Results According to final clinical diagnosis, the sensitivity and specificity of mNGS were 75.0% (95% CI: 70.8%–79.2%) and 59.0% (95% CI: 51.3%–66.7%), respectively. For distinguishing ID from NID, the sensitivity of mNGS was approximately 45.0% higher than that of standard tests (75.0% vs 30.0%; P < 0.001). For fungal detection, mNGS showed positive results in 93.0% of cases, compared to 43.7% for standard tests (P < 0.001). Diagnostic information was increased in respiratory system samples through the addition of meta-transcriptomic sequencing. Further analysis also showed that the read counts in sequencing data were highly correlated with clinical diagnosis, regardless of whether infection was by single or multiple pathogens (Kendall’s tau b = 0.484, P < 0.001). Conclusions For pediatric patients in critical condition with suspected infection, mNGS tests can provide valuable diagnostic information to resolve negative or inconclusive routine test results, differentiate ID from NID cases, and facilitate accurate and effective clinical therapeutic decision-making.
Ischemic brain injury is not uncommon after open-heart surgery with cardiopulmonary bypass and seriously undermines the patients’ life quality. Therefore, potential protective effects of limb ischemic preconditioning (LIP) on subsequent ischemic injury of the brain were investigated by evaluating anti-inflammatory effects and apoptosis of pyramidal neurons in the CA1 hippocampus. One hundred and eight Sprague-Dawley rats were divided into the middle cerebral artery occlusion (MCAO) group (n=54) and the LIP group (n=54). A thread was used to occlude the middle cerebral artery in the MCAO group and the LIP group animals were pretreated with LIP followed by MCAO. In the two groups, nine samples were collected at each time-point of 0, 6, 12, 24, 48 and 72 h after MCAO to detect IL-6 and IL-17 and their mRNA levels. Neurological severity scores (NSS) were examined before the animals were sacrificed. Compared with the LIP group, cerebral histopathological changes in the MCAO group were most distinct and significantly more infiltrated inflammatory and apoptotic neuronal cells were observed at 24, 48 and 72 h post-surgery. IL-17 and IL-6 mRNA levels analyzed by quantitative real-time polymerase chain reaction (PCR) (qRT-PCR) were significantly reduced in the LIP group compared with the MCAO group at the 12, 24 and 48 h time-points. A significant reduction in IL-17 expression level was determined by enzyme-linked immunosorbent assay (ELISA) in the LIP group at 12, 24 and 48 h, while IL-6 was significantly reduced at the 24 and 48 h time-points. The NSSs were not significantly different between the groups. Therefore, in a MCAO rat model, we have proved that LIP pretreatment can protect the brain from infarction after ischemic injury and induce ischemic tolerance, potentially, by reducing IL-17 to provide anti-inflammatory effects and attenuate apoptosis of hippocampal neuronal cells.
Objective
To investigate the change of extracellular histone level as well as the mechanism of the cytotoxicity of extracellular histones on vascular endothelial cell in sepsis.
Methods
Septic children admitted to PICU in Shanghai Children′s Medical Center between January 2010 and December 2014 were included in the present study.According to the diagnostic criteria of sepsis, the patients were divided into the sepsis group(51 cases) and the severe sepsis group(79 cases), with healthy children as the control group(108 cases). Patients in the severe sepsis group were further divided into the survival group(45 cases) and the non-survival group(34 cases) based on 28-day mortality.The plasma concentration of extracellular histones in these children was determined and its correlation with the severity of sepsis was analyzed.Human umbilical vein endothelial cells(HUVECs) were incubated with calf thymus histone(CTH) at various concentrations(0, 50, 100, 200 and 300 μg/ml) or different time periods(200 μg/ml, 0, 5, 15, 30, 45 and 60 minutes). The treated cells were subject to flow cytometer to measure the cell survival rate and scanning/transmission electron microscopy to observe their morphological changes.Western blot was used to detect the expression of IκB and phosphor-p38/p38 in nuclear factor(NF)-κB and mitogen-activated protein kinase(MAPK)signaling pathways, while ELISA was used to determine the levels of tumor necrosis factor-α and interleukin-6.
Results
The levels of circulating histones in the septic children(2.29±1.00) and severe septic children(19.17±10.20) were significantly higher than that of healthy controls(0.23±0.26)(P<0.001), and the histone levels in the severe septic children were even higher(P<0.001). Among the children diagnosed as severe sepsis, the level of circulating histones in the non-survivors was significantly higher than that in the survivors(29.47±5.99 vs.10.94±2.68, P<0.001). The survival rate of HUVEC gradually decreased along with the increase of CTH concentration or the treatment period in vitro.Data from electron microscopy showed that CTH treatment could directly disrupt the plasma membrane of HUVEC.Histones could also activate NF-κB and MAPK pathways, leading to the release of large amount of tumor necrosis factor-α and interleukin-6.
Conclusion
The levels of extracellular histones in the septic children are correlated with the severity of sepsis.CTH can induce HUVEC death in a dose-and time-dependent manner.Extracellular histone-induced endothelial dysfunction may mediate the progression of sepsis and such cytotoxicity might be due to the destruction of endothelial cell membranes and activation of the NF-κB and MAPK signaling pathways.
Key words:
Sepsis; Extracellular histones; Endothelial cells; Multiple organ dysfunction syndrome; Inflammatory mediators
The objective of this study was to assess the perioperative changes in circulating histones and their relationships with other biomarkers and clinical outcomes after cardiac surgery with cardiopulmonary bypass (CPB) in patients.Forty-eight patients with congenital cardiac diseases undergoing corrective procedure with CPB were prospectively enrolled in this study. Circulating histones, N-terminal pro-brain natriuretic peptide (NT-proBNP), procalcitonin (PCT) and C-reactive protein (CRP) were measured preoperatively (T0) and at 0 (T1), 24 (T2), 48 (T3) and 72 (T4) h postoperatively. The relationships between biomarkers and clinical outcomes were analysed.Circulating histones, NT-proBNP, PCT and CRP increased significantly postoperatively, with histones reaching the peak value earliest at T1. Circulating histone levels were higher in patients with adverse events. Receiver operating characteristic curve analysis showed that peak histone levels had a better predictive value for adverse events postoperatively. Peak histone levels correlated with the peak level of NT-proBNP (r = 0.563, P < 0.01), PCT (r = 0.551, P < 0.01), CRP (r = 0.606, P < 0.01) and clinical parameters such as ventilation time (r = 0.601, P < 0.01) and intensive care unit time (r = 0.623, P < 0.01).Circulating histones reached peak levels faster than NT-proBNP, PCT and CRP. Furthermore, peak histone levels correlated with biomarkers and postoperative clinical outcomes. Circulating histones may be used as a prognostic indicator for patients after cardiac surgery with CPB.ClinicalTrials.gov (ID: NCT02325765).
Smad4, a key transcription factor in the transforming growth factor-β signaling pathway, is involved in a variety of cell physiologic and pathologic processes. Here, we characterized megakaryocyte/platelet-specific Smad4 deficiency in mice to elucidate its effect on platelet function. We found that megakaryocyte/platelet-specific loss of Smad4 caused mild thrombocytopenia and significantly extended first occlusion time and tail bleeding time in mice. Smad4-deficient platelets showed reduced agonist-induced platelet aggregation. Further studies showed that a severe defect was seen in integrin αIIbβ3-mediated bidirectional (inside-out and outside-in) signaling in Smad4-deficient platelets, as evidenced by reduced fibrinogen binding and α-granule secretion, suppressed platelet spreading and clot retraction. Microarray analysis showed that the expression levels of multiple genes were altered in Smad4-deficient platelets. Among these genes, spleen tyrosine kinase (Syk) and Rho-associated coiled-coil containing protein kinase 2 (ROCK2) were downregulated several times as confirmed by quantitative reverse-transcription polymerase chain reaction and immunoblotting. Further research showed that Smad4 directly regulates ROCK2 transcription but indirectly regulates Syk. Megakaryocyte/platelet-specific Smad4 deficiency caused decreased expression levels of Syk and ROCK2 in platelets. These results suggest potential links among Smad4 deficiency, attenuated Syk, and ROCK2 expression and defective platelet activation.
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