Association of GAL-8 promoter methylation levels with coronary plaque inflammation.

BACKGROUND AND AIMS: Coronary heart disease (CHD) is a condition that carries a high risk of mortality and is associated with aging. CHD is characterized by the chronic inflammatory response of the coronary intima. Recent studies have shown that the methylation level of blood mononuclear cell DNA is closely associated with adverse events in CHD, but the roles and mechanisms of DNA methylation in CHD remain elusive. METHODS AND RESULTS: In this study, the DNA methylation status within the epigenome of human coronary tissue in the sudden coronary death (SCD) group and control (CON) group of coronary heart disease was analyzed using the Illumina® Infinium Methylation EPIC BeadChip (850 K chip), resulting in the identification of a total of 2553 differentially methylated genes (DMGs). The differentially methylated genes were then subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and significant differential DNA methylation was found. Among the differentially hypomethylated genes were GAL-8, LTF, and RFPL3, while the highly methylated genes were TMEM9B, ANK3, and C6orF48. These genes were mainly enriched in 10 significantly enriched pathways, such as cell adhesion junctions, among which the differentially methylated gene GAL-8 was involved in inflammatory pathway signaling. For functional analysis of GAL-8, we first examined the differences in GAL-8 promoter methylation levels among different subgroups of human coronary tissue in the CON, CHD, and SCD groups using pyrophosphate sequencing. The results revealed reduced GAL-8 promoter methylation levels in the SCD group, while the difference between the CHD and CON groups was not statistically significant (P > 0.05). The reduced GAL-8 promoter methylation level was associated with upregulated GAL-8 expression, which led to increased expression of the inflammatory markers TNF-α, IL-1ß, MCP-1, MIP-2, MMP-2, and MMP-9. This enhanced inflammatory response contributed to the accumulation of foam cells, thickening of the intima of human coronary arteries, and increased luminal stenosis, which promoted the occurrence of sudden coronary death. Next, we found that GAL-8 promoter methylation levels in PBMC were consistent with human coronary tissue. The unstable angina group (UAP) had significantly lower GAL-8 promoter methylation levels than stable angina (SAP) and healthy controls (CON) (P < 0.05), and there was a significant correlation between reduced GAL-8 promoter methylation levels and risk factors for coronary heart disease. These findings highlight the association between decreased GAL-8 promoter methylation and the presence of coronary heart disease risk factors. ROC curve analysis suggests that methylation of the GAL 8 promoter region is an independent risk factor for CHD. In conclusion, our study confirmed differential expression of GAL-8, LTF, MUC4D, TMEM9B, MYOM2, and ANK3 genes due to DNA methylation in the SCD group. We also established the consistency of GAL-8 promoter methylation alterations between human coronary tissue and patient peripheral blood monocytes. The decreased methylation level of the GAL-8 promoter may be related to the increased expression of GAL-8 and the coronary risk factors. CONCLUSIONS: Accordingly, we hypothesized that reduced levels of GAL-8 promoter methylation may be an independent risk factor for adverse events in coronary heart disease.

A diagnostic strategy for pulmonary fat embolism based on routine H&E staining using computational pathology.

Pulmonary fat embolism (PFE) as a cause of death often occurs in trauma cases such as fractures and soft tissue contusions. Traditional PFE diagnosis relies on subjective methods and special stains like oil red O. This study utilizes computational pathology, combining digital pathology and deep learning algorithms, to precisely quantify fat emboli in whole slide images using conventional hematoxylin-eosin (H&E) staining. The results demonstrate deep learning's ability to identify fat droplet morphology in lung microvessels, achieving an area under the receiver operating characteristic (ROC) curve (AUC) of 0.98. The AI-quantified fat globules generally matched the Falzi scoring system with oil red O staining. The relative quantity of fat emboli against lung area was calculated by the algorithm, determining a diagnostic threshold of 8.275% for fatal PFE. A diagnostic strategy based on this threshold achieved a high AUC of 0.984, similar to manual identification with special stains but surpassing H&E staining. This demonstrates computational pathology's potential as an affordable, rapid, and precise method for fatal PFE diagnosis in forensic practice.

Age estimation by modified Suchey-Brooks method using three-dimensional reconstructed CT images of Chinese Han population.

The method proposed by Suchey-Brooks for adult age estimation based on the surface morphology of the pubic symphysis has been widely accepted. The applicability of the method varies considerably in different populations. The present study established a virtual reference sample and aimed to develop population-specific criteria that can be used for age estimation in different skeletal samples. First, The dry bone specimens from 100 individuals were compared with their corresponding three-dimensional (3D) reconstruction model and showed high inter-method agreement (k = 0.743-0.811), suggesting that the virtual bone model and physical bone specimens have comparable performances in describing the surface morphology of the pubic symphysis. We retrospectively collected clinical computed tomography (CT) data from 895 Chinese patients to create a virtual reference sample of the pubic symphysis. Based on the original Suchey-Brooks method, each of the 895 reference samples was assigned a phase, for each sex and phase, data on the mean age, standard deviation, and 95% age range of the corresponding sample were obtained, which was then used as the "method modified for Chinese" (modified method) and compared to the "SB method". Compared to the SB method, modified method had a lower inaccuracy in dry bones for males over 35 years and females over 45 years, in dry bone CT test sample for males over 55 years and females over 45 years, and in postmortem CT test sample for males over 35 years and females over 55 years. The modified method can improve the accuracy of age estimation for older samples over 40 years. It has shown considerable reliability when applied as a population-specific criterion, but its accuracy is still not sufficient, and caution is needed when using it.

Blockade of CXCR4 promotes macrophage autophagy through the PI3K/AKT/mTOR pathway to alleviate coronary heart disease.

OBJECTIVE: Autophagy is important in regulating inflammation and cholesterol efflux, suggesting that targeting autophagy may slow down atherosclerosis (AS). Since the pathological basis of coronary artery disease (CAD) is atherosclerosis, it is crucial to investigate the role of autophagy in atherosclerosis. This study aimed to investigate the role of the chemokine CXC chemokine receptor 4 (CXCR4) in promoting macrophage autophagy through the phosphoinositide-3 kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway to alleviate coronary artery disease. METHODS: The human left coronary artery and myocardium were collected to detect CXCR4, MAP1LC3(LC3) and SQSTM1(p62) expression. ApoE-/- mice were used to establish an atherosclerosis mice model, while human monocytes (THP-1) were used to establish a foam cell model and co-cultured with foam cells using siRNACXCR4. Western blotting was conducted to quantify CXCR4, PI3K/AKT/mTOR pathway protein, LC3, Beclin1 and p62 protein levels. The left coronary artery from humans and mouse aorta and myocardium were stained with Hematoxylin and Eosin (H&E), macrophages with Oil Red O staining and foam cells were assessed by Movat's staining. CXCR4 levels, PI3K/AKT/mTOR pathway protein, LC3 and p62 were detected by immunohistochemistry (IHC) and immunofluorescence assays. Detection of autophagosomes in macrophages using transmission electron microscopy. We further assessed whether the effect of CXCR4-mediated macrophage autophagy on the formation of atherosclerosis and structural changes in the myocardium was mediated via the PI3K/AKT/mTOR signaling pathway. RESULTS: CXCR4 and p62 proteins were upregulated in human coronary lesions, mouse aorta, myocardial tissue, and foam cells, while LC3II/LC3I was downregulated. p85 (P-PI3K), Ser473 (P-AKT), and Ser2448 (P-mTOR) phosphorylated proteins associated with the PI3K/AKT/mTOR pathway were detected in AS and foam cell models. Upregulated CXCR4 inhibited autophagy of macrophages and increased the severity of atherosclerotic lesions. After specific knockdown of CXCR4 by adeno-associated virus (AAV9-CXCR4-RNAi) and siRNACXCR4, the above indicators were reversed, macrophage autophagy was promoted, the severity of atherosclerotic lesions was reduced, and the disorganized arrangement of myocardial architecture was improved. CONCLUSION: Knockdown of CXCR4 reduces the extent of coronary artery disease by promoting macrophage autophagy through the PI3K/AKT/mTOR pathway to attenuate atherosclerosis.

DEL-1 suppression attenuates atherosclerosis by modulating macrophagic GSK-3ß/CEBP-ß signaling pathway.

OBJECTIVE: The study aims to investigate the effect of developmental endothelial locus-1(DEL-1) expression in atherosclerotic plaque formation and its mechanism. METHODS: Human left coronary arteries were collected to detect the DEL-1 expression. The ApoE-/- mice were used to establish the atherosclerosis mice model. The left coronary artery and mouse aorta were stained with HE, Oil Red O, and Movat staining. The DEL-1 levels, chemokines CXC chemokine receptor 4 (CXCR4) and its ligand stromal cell-derived factor-1alpha (SDF-1α), pathway protein glycogen synthase kinase-3ß (GSK-3ß), CCAAT enhanced binding protein ß (C/EBPß), and downstream inflammatory factors (C-X-C motif chemokine 2 (MIP-2or CXCL2), macrophage inflammatory protein-1alpha (MIP-1α or CCL3),Tumor Necrosis Factor alpha (TNF-α) were detected by immunoblotting and immunohistochemistry. Pearson correlation coefficient was used to analyze the correlation between DEL-1 gene expression and inflammatory factors in the lesion group and the correlation between DEL-1 gene expression and structure-related indexes. RESULTS: Compared with Control group(CON), the intravascular plaque area was widened, accompanied by narrowed lumens. The number of plaque foam cells was significantly increased in the high fat and high cholesterol (AS group) or AAV9-eGFP group (P < 0.05). Compared to CON, the enhanced fluorescence intensity of DEL-1 with CD68 in the AS or AAV9-eGFP groups. Diminished fluorescence of DEL-1 with CD68 expression in AAV9-CXCR4 group compared to AS group or AAV9-eGFP group. The DEL-1 and its downstream proteins in AS group or AAV9-eGFP group were mainly accumulated in the macrophage cytoplasm. The DEL-1 expression level was significantly and positively correlated with plaque area, lumen stenosis, plaque foam cell count, TNFα, CXCL2, and CCL3 levels. CONCLUSION: DEL-1 inhibition decreases macrophagic inflammatory factors involved in atherosclerotic plaque formation.

Application of nanoindentation technology in testing the mechanical properties of skull materials.

Three-point bending test, compression test and tensile test can detect the mechanical properties of the whole layer of skull, but cannot detect the mechanical properties of the inner plate, the diploe and the outer plate of the skull. In this study, nanoindentation technology was applied to detect mechanical properties of micro-materials of the skull, and differences in micro-mechanical properties of the inner, diploe and outer plates of the skull and cranial suture of human carcasses at different ages were analyzed. The differences in hardness (HIT) and modulus of elasticity (E) were statistically significant among different age groups (P < 0.01). In terms of structure, the E of diploe was higher than that of other structures, while HIT had no significant statistical difference. In terms of location, both HIT and E showed that left frontal (LF) was significantly higher than coronal suture (CS). The above results were consistent with the multi-factor ANOVAs. In addition, the multi-factor ANOVAs further explained the interaction of HIT and E with age, location and structure. It was believed that the nanoindentation technique could be used to analyze laws of micromechanical properties of different structures of human cadaveric skull and cranial suture.

Label-Free Diagnosis of Pulmonary Fat Embolism Using Fourier Transform Infrared (FT-IR) Spectroscopic Imaging.

The diagnosis of pulmonary fat embolism (PFE) is of great significance in the field of forensic medicine because it can be considered a major cause of death or a vital reaction. Conventional histological analysis of lung tissue specimens is a widely used method for PFE diagnosis. However, variable and labor-intensive tissue staining procedures impede the validity and informativeness of histological image analysis. To obtain complete information from tissues, a method based on infrared imaging of unlabeled tissue sections was developed to identify pulmonary fat emboli in the present study. We selected 15 PFE-positive lung samples and 15 PFE-negative samples from real cases. Oil red O (ORO) staining and infrared spectral imaging collection were both performed on all lung tissue samples. And the fatty tissue of the abdominal wall and the embolized lipid droplets in the lungs were taken for comparison. The results of the blind, evaluation by pathologists, showed good agreement between the infrared spectral imaging of the lung tissue and the standard histological stained images. Fourier transform infrared (FT-IR) spectroscopic imaging significantly simplifies the typical painstakingly laborious histological staining procedure. And we found a difference between lipid droplets embolized in abdominal wall fat and lung tissue.

Identification of myocardial fibrosis by ATR-FTIR spectroscopy combined with chemometrics.

Different degrees of myocardial fibrosis can often be observed in sudden cardiac death cases, so that the identification of myocardial fibrosis is an important step in forensics to identify cardiac death. Previous methods are restricted by complex algorithms, high cost, low sensitivity and high requirements. Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy is an efficient and rapid method to identify tissue types, which has been used increasingly in forensics. This study aims to identify novel biophysical biomarkers of myocardial fibrosis and establish a prediction model by using ATR-FTIR analysis combined with chemometrics. A total of 129 tissue blocks taken from human hearts were cut into slices, and then ATR-FTIR spectroscopy and hematoxylin and eosin (HE) staining were performed. By using HE staining, the samples were divided into the experimental group (with myocardial fibrosis) and the control group (without myocardial fibrosis). The chemometrics classification results showed that the sensitivity and specificity of the training dataset were 0.91 and 1.0 respectively, and the sensitivity and specificity of the predictive dataset were 0.862 and 0.900. This study demonstrated that ATR-FTIR spectroscopy combined with chemometrics is a novel method for identifying myocardial fibrosis.

Cathepsin C Is Involved in Macrophage M1 Polarization via p38/MAPK Pathway in Sudden Cardiac Death.

This study was aimed at identifying molecular markers associated with the pathogenesis of sudden cardiac death (SCD). It provides a proteomic analysis of human left anterior descending coronary artery from subjects diagnosed with SCD through histological examination and cases of nondisease accidental deaths through autopsy. A total of 2784 proteins were obtained from label-free quantitative proteomic analysis. This included a total of 265 differential proteins which were involved in SCD-related processes, such as inflammation, muscle system process regulation, metal ion transport, and lysosomal pathway. Western blotting was carried out to measure the expressions of cathepsin C (CTSC), focal adhesion kinase (FAK), p-FAK, and proteins related to the p38/MAPK signaling pathway, whereas immunohistochemistry was performed to determine the localization and expression of CTSC, TNF-α, and CD206 in arterial tissues. It was found that CTSC were the most expressed proteins with a significant upward trend in SCD cases. Besides, CTSC regulated macrophage polarization to M1 through the FAK-induced p38/MAPK signaling pathway. This promoted the release of inflammatory factors and eventually increased the inflammatory response. In conclusion, this study implies that CTSC may be one of the key molecular targets for promoting macrophage M1 polarization in SCD, which may provide new therapeutic insights into the treatment of inflammatory diseases.

Fatal postoperative malignant hyperthermia following rhinoplasty in a patient with one variant of the ryanodine receptor 1 (RYR1) gene.

Malignant hyperthermia (MH) is characterized by a rapid rise in body temperature after using inhalational anesthetics and depolarizing muscle relaxants. A 19-year-old female had a rapidly developing fever up to 43.0 °C, after rhinoplasty surgery. Inhalational anesthetics and depolarizing muscle relaxants were used in general anesthesia. It was suspected that the patient died of MH. The medico-legal autopsy findings showed classical MH histopathological changes in the skeletal muscles, cardiac muscles, as well as lungs. Additionally, postmortem blood biochemical results indicated rhabdomyolysis. A combination of clinical records and autopsy revealed that MH might have caused the death. A diagnostic genetic testing was performed to confirm the existence of MH, and an MH diagnostic variant RYR1 c.7048G >A (p. A2350T) was detected. Eventually, the cause of death was determined as MH based on clinical records, autopsy, and genetic analysis. This case highlights that diagnostic genetic analysis plays a vital role in postmortem diagnosis of MH in routine medico-legal contexts.

Significances of viable synergistic autophagy-associated cathepsin B and cathepsin D (CTSB/CTSD) as potential biomarkers for sudden cardiac death.

BACKGROUND: The Cathepsins family, including cathepsin B and cathepsin D, potentially affects the entire processes involved in atherosclerosis. Although coronary heart disease (CHD) has been widely studied as the basis of Sudden Cardiac Death (SCD), the relationship between CHD and CTSB/D remains unclear. METHODS: We screened for differentially expressed proteins (DEPs) associated with autophagy by limma package in R. For the genes corresponding to the DEPs after screening, we used various databases to carry out functional enrichment of related DEGs to explore their possible influence on a specific aspect of the disease. Functional enrichment analysis of DEGs was performed by DAVID, Metascape and GSEA. STRING and Cytoscape were obtained the hub genes, the analysis of interaction networks through the GENMANIA and Networkanalyst. Western Blot was used to validate the protein expression level of target genes. TF and miRNA prediction were performed using Networkanalyst and visualized using Cytoscape. RESULTS: The expression levels of members of the cathepsin family were up regulated in CHD tissues compared with the control. GO and KEGG revealed that cathepsin was markedly enriched in endopeptidase activities, immune responses, lysosome pathways, et al. The correlation analysis showed that in patients with CHD, the CTSB/CTSD expression were negatively correlated with ATG4D and BNIP3, but positively with BCL2L1, CAPNS1, and TP53. In the TF-mRNA-miRNA network, has-miR-24-3p and has-miR-128-3p had higher degrees, CTSB/CTSD could be targeted by them. CONCLUSIONS: Our findings elucidated the expression and regulatory role of cathepsins in coronary heart disease induced SCD and might further explore the potential mechanisms of autophagy in CHD.

The role of OX40L and ICAM-1 in the stability of coronary atherosclerotic plaques and their relationship with sudden coronary death.

BACKGROUND: Coronary heart disease is related to sudden death caused by multi-factors and a major threat to human health.This study explores the role of OX40L and ICAM-1 in the stability of coronary plaques and their relationship with sudden coronary death. METHODS: A total of 118 human coronary arteries with different degrees of atherosclerosis and/or sudden coronary death comprised the experimental group and 28 healthy subjects constituted the control group were isolated from patients. The experimental group was subdivided based on whether the cause of death was sudden coronary death and whether it was accompanied by thrombosis, plaque rupture, plaque outflow and other secondary changes: group I: patients with coronary atherosclerosis but not sudden coronary death, group II: sudden coronary death without any of the secondary changes mentioned above, group III: sudden coronary death with coronary artery atherosclerotic lesions accompanied by either of the above secondary changes. The histological structure of the coronary artery was observed under a light microscope after routine HE staining, and the related indexes of atherosclerotic plaque lesions were assessed by image analysis software. The expressions of OX40L and ICAM-1 were detected by real-time quantitative PCR (RT-PCR), immunohistochemistry (IHC) and Western blotting, and the correlations between the expressions and the stability of coronary atherosclerotic plaque and sudden coronary death were analyzed. RESULTS: (1) The expression of OX40L protein in the control group and the three experimental groups showed an increasing trend, and the difference between groups was statistically significant (P < 0.05). (2) The expression of the ICAM-1 protein in the control group and the three experimental groups showed a statistically significant (P < 0.05) increasing trend. (3) The expression of OX40L and ICAM-1 mRNAs increased in the control and the three experimental groups and the difference was statistically significant (P < 0.05). CONCLUSION: The expression of OX40L and ICAM-1 proteins and mRNAs is positively correlated with the stability of coronary atherosclerotic plaque and sudden coronary death.

Differential Expression of miRNAs in the Hippocampi of Offspring Rats Exposed to Fluorine Combined with Aluminum during the Embryonic Stage and into Adulthood.

A previous study from our team found that continuous exposure to fluorine combined with aluminum (FA) impaired the neurobehavioral reflexes, spatial learning, and memory of offspring rats. To date, the specific mechanisms for these changes are unclear. Here, high-throughput sequencing was utilized to analyze the microRNA (miRNA) profile of the hippocampi in the offspring of rats exposed to FA during the embryonic stage and into adulthood through tap water supplemented with NaF and AlCl3 at concentrations of (0, 0); (60, 600); (120, 600); and (240, 600) mg/L, respectively. qRT-PCR was performed to validate the reliability of the sequence data. Twenty differentially expressed miRNAs were selected for further analysis using bioinformatics tools. Several genes related to neuromodulation were found to be regulated by miR-10a-5p, miR-34b-5p, and miR-182, which might be harmful to normal nerve function. The protein levels of brain-derived neurotrophic factor (BDNF) and tyrosine receptor kinase B (TrkB) in hippocampus were markedly downregulated. These data suggest that miR-10a-5p, miR-34b-5p, and miR-182 and BDNF-TrkB signaling pathway are involved in mechanisms of hippocampal damage in the offspring of rats exposed to FA. HIGHLIGHTS: • Multiple miRNAs were significantly differentially expressed in offspring rat hippocampus after fluorine combined with aluminum (FA) exposure. • Twenty differentially expressed miRNAs might mediate FA-induced developmental neurotoxicity. • MiR-10a-5p, miR-34b-5p, and miR-182 were closely related to neurotoxic signaling of FA. • The BDNF-TrkB learning and memory-associated pathway was downregulated in the hippocampus after FA exposure.

Clinicopathological Characteristics of Traumatic Head Injury in Juvenile, Middle-Aged and Elderly Individuals.

BACKGROUND Traumatic head injury is a leading cause of death and disability worldwide. How clinicopathological features differ by age remains unclear. This epidemiological study analyzed the clinicopathological features of patients with head injury belonging to 3 age groups. MATERIAL AND METHODS Data of patients with traumatic head injury were obtained from the Department of Cerebral Surgery of the Affiliated Hospital of Guizhou Medical University and the Guizhou Provincial People's Hospital in 2011-2015. Their clinicopathological parameters were assessed. The patients were divided into 3 age groups: elderly (≥65 years), middle-aged (18-64 years), and juvenile (≤17 years) individuals. RESULTS Among 3356 hospitalizations for traumatic head injury (2573 males and 783 females, 654 died (19.49%), the highest and lowest mortality rates were in the elderly and juvenile groups, respectively. Fall was the most common cause in juvenile and elderly individuals (32.79% and 43.95%, respectively), while traffic injury was most common in the elderly group (35.08%). The manners of injury differed considerably among the 3 age groups. Scalp injury, skull fracture, intracranial hematoma, and cerebral injury were the most common mechanisms in juvenile (67.32%), middle-aged (63.50%), elderly (69.56%) and middle-aged (90.44%) individuals, respectively. Scalp injury and skull fracture types differed among the groups. Epidural, subdural, and intracerebral hematomas were most common in juvenile, middle-aged, and elderly individuals, respectively. Cerebral contusion showed the highest frequency in the 3 groups, and concussion the lowest. CONCLUSIONS Patients with traumatic HI show remarkable differences in clinicopathological features among juvenile, middle-aged, and elderly individuals.