Blood leukocytes as a non-invasive diagnostic tool for thyroid nodules: a prospective cohort study.

BACKGROUND: Thyroid nodule (TN) patients in China are subject to overdiagnosis and overtreatment. The implementation of existing technologies such as thyroid ultrasonography has indeed contributed to the improved diagnostic accuracy of TNs. However, a significant issue persists, where many patients undergo unnecessary biopsies, and patients with malignant thyroid nodules (MTNs) are advised to undergo surgery therapy. METHODS: This study included a total of 293 patients diagnosed with TNs. Differential methylation haplotype blocks (MHBs) in blood leukocytes between MTNs and benign thyroid nodules (BTNs) were detected using reduced representation bisulfite sequencing (RRBS). Subsequently, an artificial intelligence blood leukocyte DNA methylation (BLDM) model was designed to optimize the management and treatment of patients with TNs for more effective outcomes. RESULTS: The DNA methylation profiles of peripheral blood leukocytes exhibited distinctions between MTNs and BTNs. The BLDM model we developed for diagnosing TNs achieved an area under the curve (AUC) of 0.858 in the validation cohort and 0.863 in the independent test cohort. Its specificity reached 90.91% and 88.68% in the validation and independent test cohorts, respectively, outperforming the specificity of ultrasonography (43.64% in the validation cohort and 47.17% in the independent test cohort), albeit with a slightly lower sensitivity (83.33% in the validation cohort and 82.86% in the independent test cohort) compared to ultrasonography (97.62% in the validation cohort and 100.00% in the independent test cohort). The BLDM model could correctly identify 89.83% patients whose nodules were suspected malignant by ultrasonography but finally histological benign. In micronodules, the model displayed higher specificity (93.33% in the validation cohort and 92.00% in the independent test cohort) and accuracy (88.24% in the validation cohort and 87.50% in the independent test cohort) for diagnosing TNs. This performance surpassed the specificity and accuracy observed with ultrasonography. A TN diagnostic and treatment framework that prioritizes patients is provided, with fine-needle aspiration (FNA) biopsy performed only on patients with indications of MTNs in both BLDM and ultrasonography results, thus avoiding unnecessary biopsies. CONCLUSIONS: This is the first study to demonstrate the potential of non-invasive blood leukocytes in diagnosing TNs, thereby making TN diagnosis and treatment more efficient in China.

Effects of calcitonin on lumbar spinal stenosis.

STUDY DESIGN: Systematic review and meta-analysis. OBJECTIVE: There is some controversy about the effects of calcitonin (CT) on lumbar spinal stenosis (LSS). This systematic review and meta-analysis is to assess the strength of the evidence supporting the use of CT in the treatment of patients with LSS. MATERIAL AND METHOD: We performed an electronic search depicting randomized controlled trials (RCTs) through 4 databases from the date of database creation to January 2023. 3 different researchers conducted independent literature screening, data extractions, and quality assessments. The outcome measures included visual analogue scale (VAS), walking distance, and oswestry disability index (ODI). Meta-analysis and trial sequence analysis (TSA) were carried out using RevMan 5.4, Stata 16.0, and TSA 0.9. GRADE 3.6 was used to evaluate the evidence quality. RESULTS: We accepted 9 studies with 496 participants. The meta-analysis revealed that CT offered no significant improvement in VAS, walking distance, or ODI in patients with LSS. CONCLUSION: There is no evidence that CT has a benefit in patients with LSS, either alone or in combination with other treatments, or depending on the route of administration, according to the systematic review and meta-analysis of relevant RCTs.

MeIS: DNA methylation-based immune response signatures for thyroid nodule diagnostics.

CONTEXT: Accurately distinguishing between benign thyroid nodules (BTNs) and papillary thyroid cancers (PTCs) with current conventional methods poses a significant challenge. OBJECTIVE: We identify DNA methylation markers of immune response-related genes for distinguishing BTNs and PTCs. METHODS: In this study, we analyzed a public reduced representative bisulfite sequencing (RRBS) dataset and revealed distinct methylation patterns associated with immune signals in PTCs and BTNs. Based on these findings, we developed a diagnostic classifier named as the Methylation-based Immune Response Signature (MeIS), which was composed of fifteen DNA methylation markers associated with immune response-related genes. We validated the MeIS's performance in two independent cohorts: ZS's retrospective cohort (50 PTC and 18 BTN surgery-leftover samples) and ZS's preoperative cohort (31 PTC and 30 BTN fine-needle aspiration (FNA) samples). RESULTS: The MeIS classifier demonstrated significant clinical promise, achieving AUCs of 0.96, 0.98, 0.89 and 0.90 in the training set, validation set, ZS's retrospective cohort, and ZS's preoperative cohort, respectively. For the cytologically indeterminate thyroid nodules, in the ZS's retrospective cohort, MeIS exhibited a sensitivity of 91% and a specificity of 82%; in the ZS's preoperative cohort, MeIS achieved a sensitivity of 84% and a specificity of 74%. Additionally, combining MeIS and BRAFV600E detection improved the detecting performance of cytologically indeterminate thyroid nodules, yielding sensitivities of 98% and 87%, and specificities of 82% and 74% in the ZS's retrospective cohort and ZS's preoperative cohort, respectively. CONCLUSIONS: The fifteen markers we identified can be employed to improve the diagnostic of cytologically indeterminate thyroid nodules.

Tough, high conductivity pectin polysaccharide-based hydrogel for strain sensing and real-time information transmission.

Hydrogels from natural polymers are eco-friendly, biocompatible and adjustable for manufacturing wearable sensors. However, it is still challenging to prepare natural polymer hydrogel sensors with excellent properties (e.g., high conductivity). Here, we developed a physically cross-linked, highly conductive and multifunctional hydrogel (named PPTP) to address this challenge. The natural renewable pectin-based PPTP hydrogel is synthesized by introducing tannic acid (TA), calcium chloride (CaCl2), and sodium chloride (NaCl) into the pectin/polyvinyl alcohol (PVA) dual network structure. The hydrogel exhibits excellent characteristics, including unique tensile strength (2.6155 MPa), high electrical conductivity (7 S m-1), and high sensitivity (GF = 3.75). It is also recyclable, further enhancing its eco-friendly nature. The PPTP hydrogel can be used for monitoring human joint activities, as flexible electrodes for monitoring electrocardiogram (ECG) signals, and touchable screen pen for electronic skin. Moreover, when combined with Morse code and wireless Bluetooth technology, PPTP hydrogels can be used for underwater and land information encryption, and decryption. Our unique PPTP hydrogel offers promising opportunities for medical monitoring, information transfer, and human-computer interaction.

A diagnostic model based on DNA methylation haplotype block characteristics for identifying papillary thyroid carcinoma from thyroid adenoma.

Papillary thyroid carcinoma (PTC) is the most prevalent form of thyroid cancer. Methylation of some genes plays a crucial role in the tendency to malignancy as well as poor prognosis of thyroid cancer, suggesting that methylation features can serve as complementary markers for molecular diagnosis. In this study, we aimed to develop and validate a diagnostic model for PTC based on DNA methylation markers. A total of 142 thyroid nodule tissue samples containing 84 cases of PTC and 58 cases of thyroid adenoma (TA) were collected for reduced representation bisulfite sequencing (RRBS) and subsequent analysis. The diagnostic model was constructed by the logistic regression (LR) method followed by 5-cross validation and based on 94 tissue methylation haplotype block (MHB) markers. The model achieved an area under the receiver operating characteristic curve (AUROC) of 0.974 (95% CI, 0.964-0.981) on 108 training samples and 0.917 (95% CI, 0.864-0.973) on 27 independent testing samples. The diagnostic model scores showed significantly high in males (P = 0.0016), age ≤ 45 years (P = 0.026), high body mass index (BMI) (P = 0.040), lymph node metastasis (P = 0.00052) and larger nodules (P = 0.0017) in the PTC group, and the risk score of this diagnostic model showed significantly high in recurrent PTC group (P = 0.0005). These results suggest that the diagnostic model can be expected to be a powerful tool for PTC diagnosis and there are more potential clinical applications of methylation markers to be excavated.

Breast tumor-on-chip: from the tumor microenvironment to medical applications.

With the development of microfluidic technology, tumor-on-chip models have gradually become a new tool for the study of breast cancer because they can simulate more key factors of the tumor microenvironment compared with traditional models in vitro. Here, we review up-to-date advancements in breast tumor-on-chip models. We summarize and analyze the breast tumor microenvironment (TME), preclinical breast cancer models for TME simulation, fabrication methods of tumor-on-chip models, tumor-on-chip models for TME reconstruction, and applications of breast tumor-on-chip models and provide a perspective on breast tumor-on-chip models. This review will contribute to the construction and design of microenvironments for breast tumor-on-chip models, even the development of the pharmaceutical field, personalized/precision therapy, and clinical medicine.

Circular RNA hsa_circ_0084054 promotes the progression of periodontitis with diabetes via the miR-508-3p/PTEN axis.

BACKGROUND AND OBJECTIVE: Diabetes is an important risk factor for periodontitis, and circular RNA (circRNA) may play an important role in aggravating inflammation and accelerating disease progression by regulating miRNA/mRNA. This study aimed to investigate the role and mechanism of the hsa_circ_0084054/miR-508-3p/PTEN axis in the progression of periodontitis with diabetes. METHODS: First, circRNA sequencing was used to screen the differentially expressed circRNAs of periodontal ligament cells (PDLCs) treated with high glucose and/or Porphyromonas gingivalis lipopolysaccharide (LPS) in vitro, and the overtly differentially expressed hsa_circ_0084054 was selected and was also verified in periodontal ligament (PDL) tissue from periodontitis patients with diabetes. Then, its ring structure was tested by Sanger sequencing, RNase R, and actinomycin D assays. The bioinformatics analysis, dual luciferase reporter assay, and RIP assay were used to explore the interaction of hsa_circ_0084054/miR-508-3p/PTEN axis, whose effects on inflammation, oxidative stress, and apoptosis of PDLCs were evaluated through the measurement of inflammatory factors, reactive oxygen species (ROS), total superoxide dismutase (SOD), malondialdehyde (MDA), and Annexin V/PI assay. RESULTS: By high-throughput sequencing, it was found that hsa_circ_0084054 was significantly increased in HG + LPS group compared with control group and LPS group, which was also verified in periodontal ligament (PDL) tissue from periodontitis patients with diabetes. Silencing hsa_circ_0084054 in PDLCs decreased the expression of inflammatory factors (IL-1ß, IL-6, TNF-α), the levels of ROS and MDA, and the proportion of apoptotic cells; conversely, SOD activity was enhanced. In addition, we found that hsa_circ_0084054 could up-regulate the expression of PTEN through sponge miR-508-3p to inhibit AKT phosphorylation, finally trigger the aggravation of oxidative stress and inflammation in periodontitis patients with diabetes. CONCLUSION: hsa_circ_0084054 can aggravate inflammation and promote the progression of periodontitis with diabetes by regulating miR-508-3p/PTEN signaling axis, which may serve as a new target for the intervention of periodontitis with diabetes.

Six macrophage-associated genes in synovium constitute a novel diagnostic signature for osteoarthritis.

Background: Synovial macrophages play important roles in the formation and progression of osteoarthritis (OA). This study aimed to explore the biological and clinical significance of macrophage-associated genes (MAGs) in OA. Methods: The OA synovial gene expression profiles GSE89408 and GSE82107 were obtained from the GEO database. Single-sample gene set enrichment analysis (ssGSEA) and GSEA were employed to decipher differences in immune infiltration and macrophage-associated biological pathways, respectively. Protein-protein interaction (PPI) network analysis and machine learning were utilized to establish a macrophage-associated gene diagnostic signature (MAGDS). RT-qPCR was performed to test the expression of key MAGs in murine models. Results: OA synovium presented high levels of immune infiltration and activation of macrophage-associated biological pathways. A total of 55 differentially expressed MAGs were identified. Using PPI analysis and machine learning, a MAGDS consisting of IL1B, C5AR1, FCGR2B, IL10, IL6, and TYROBP was established for OA diagnosis (AUC = 0.910) and molecular pathological evaluation. Patients with high MAGDS scores may possess higher levels of immune infiltration and expression of matrix metalloproteinases (MMPs), implying poor biological alterations. The diagnostic value of MAGDS was also validated in an external cohort (AUC = 0.886). The expression of key MAGs was validated in a murine model using RT-qPCR. Additionally, a competitive endogenous RNA network was constructed to reveal the potential posttranscriptional regulatory mechanisms. Conclusions: We developed and validated a MAGDS model with the ability to accurately diagnose and characterize biological alterations in OA. The six key MAGs may also be latent targets for immunoregulatory therapy.

Augmented Production of Platelets From Cord Blood With Euchromatic Histone Lysine Methyltransferase Inhibition.

Cord blood hematopoietic stem/progenitor cells (CB-HSPCs) have emerged as a promising supply for functional platelets to potentially alleviate the increasing demand for platelet transfusions, but the clinical application has been limited by the undefined molecular mechanism and insufficient platelet production. Here, we performed single-cell profiling of more than 16 160 cells to construct a dynamic molecular landscape of human megakaryopoiesis from CB-HSPCs, enabling us to uncover, for the first time, cellular heterogeneity and unique features of neonatal megakaryocytes (MKs) and to also offer unique resources for the scientific community. By using this model, we defined the genetic programs underlying the differentiation process from megakaryocyte-erythroid progenitors (MEPs) to MKs via megakaryocyte progenitors (MKPs) and identified inhibitors of euchromatic histone lysine methyltransferase (EHMT), which, when applied at the early stage of differentiation, significantly increase the final platelet production. At the mechanistic level, we found that EHMT inhibitors act to selectively induce the expansion of MEPs and MKPs. Together, we uncover new mechanistic insights into human megakaryopoiesis and provide a novel chemical strategy for future large-scale generation and clinical applications of platelets.

Effect of Self-Efficacy Intervention Combined with Humanistic Nursing on Self-Care Ability and Quality of Life in Patients Receiving Chemotherapy for Malignant Tumors.

Background: We aimed to explore the effect of self-efficacy intervention combined with humanistic nursing on self-care ability and quality of life in patients receiving chemotherapy for malignant tumors. Methods: A total of 410 patients were enrolled, who received chemotherapy for malignant tumors in Fuyang People's Hospital from June 2019 to June 2021. They were equally divided into the experimental group and the control group by a random number table. The former was given routine nursing, while self-efficacy intervention combined with humanistic nursing on the bases of routine care was introduced for the latter. Baseline information was collected from all patients. The psychological status of patients before and after intervention was assessed by self-rating anxiety scale (SAS), self-rating depression scale (SDS) and Visual analogue scale (VAS), while self-efficacy score and self-care ability scale for evaluating self-care ability of patients. Additionally, there was an evaluation of quality of life and nursing satisfaction in each group. Results: Before intervention, no significant difference was identified in psychological status, self-care ability and quality of life between the two groups. After the intervention, the above three indexes in the experimental group were significantly better than those of the control group were. The experimental group had higher nursing satisfaction than the control group. Conclusion: In patients with malignant tumor undergoing chemotherapy, self-efficacy intervention combined with humanistic nursing can significantly improve the self-care ability, quality of life and nursing satisfaction of patients, which is therefore worthy of promotion in clinical.

Expression and purification of recombinant human CCL5 and its biological characterization.

C-C motif chemokine ligand 5 (CCL5) is crucial in the tumor microenvironment. It has been previously reported to act as a key role in tumor invasion and metastasis. However, the function of exogenous CCL5 in ovarian cancer has not been well-characterized. The present study attempted to express and purify recombinant CCL5 protein and investigate the exogenous CCL5 in ovarian cancer cell proliferation. The human CCL5 was amplified and inserted into the pET-30a vectors for prokaryotic expression in Escherichia coli BL21. Soluble His-CCL5 was successfully expressed with 0.1 mmol/L of isopropyl-ß-D-1-tiogalactopiranoside at 25 ℃ and purified by affinity chromatography. Additionally, methyl thiazolyl tetrazolium (MTT) assay demonstrated that CCL5 promotes ovarian cancer cell proliferation; increases the phosphorylation levels of extracellular-signal-regulated kinase and mitogen-activated protein kinase/ERK kinase, and increases the mRNA levels of Jun, NF-κB2, Nras, Relb, and Traf2. Furthermore, treatment with the MEK inhibitor reduced the Jun, NF-κB2, and Traf2 mRNA levels, indicating that exogenous CCL5 increased ovarian cancer cell proliferation, through MEK/ERK pathway activation, and Jun, NF-κB2, and Traf2 expression. The present study provided primary data for further studies to discover more CCL5 functions in ovarian cancer.

Exogenous interleukin-1 beta promotes the proliferation and migration of HeLa cells via the MEK/ERK signaling pathway.

OBJECTIVE: Interleukin-1 beta (IL-1ß) is a crucial cytokine that has been implicated in cancer and metastasis development. However, its possible mechanistic role in cervical cancer remains unclear. This study aimed to investigate the functions of exogenous IL-1ß in cervical cancer cell proliferation and migration. METHODS: HeLa cell proliferation and migration were measured using MTT and Transwell assays. A lentivirus-mediated packaging system was used to construct an IL-1ß overexpressing cell line. MEK/ERK signal transduction was inhibited by pretreatment with the MEK inhibitor PD98059. qRT-PCR and Western blotting were used to test the expression of relevant genes. RESULTS: Exogenous IL-1ß promoted the proliferation and migration of HeLa cells. In addition, overexpression of IL-1ß in HeLa cells promoted cell proliferation. Mechanistically, exogenous IL-1ß increased the phosphorylated MEK and ERK levels in HeLa cells and the expression of JUN, RELB, and NF-κB2. Alternatively, blockade of MEK inhibited the promoting proliferation effects of IL-1ß and the expression of JUN, RELB, and NF-κB2. CONCLUSIONS: Our data suggest that exogenous IL-1ß regulates HeLa cell functions by regulating the MEK/ERK signaling pathway and by targeting JUN, RELB, and NF-κB2. Our study uncovered a potential association across IL-1ß, cervical tumor development, and cancer progression.

Association between Interleukin-1ß Polymorphism at Rs16944 and Glucose Metabolism: A Cohort Study.

BACKGROUND: This study explored the correlation between the interleukin-1ß gene rs16944 polymorphism and diabetes through epidemiological and follow-up investigations. METHODS: The study was conducted on 600 subjects with normal glucose metabolism recruited from participants of the Risk Evaluation of cAncers in Chinese type 2 diabeTic Individuals: A lONgitudinal (REACTION) study in Luzhou, China in 2011. All subjects received a unified standardized questionnaire, physical examination, laboratory examination, and follow-up in 2016. Subjects were divided into normal glucose metabolism (NC), pre-diabetes (PDM), and type 2 diabetes mellitus (T2DM) groups according to their glucose metabolism after follow-up. The IL-1ß gene rs16944 polymorphism was analyzed using the polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) technique. RESULTS: After follow-up, 386, 156, and 58 cases were observed in the NC, PDM, and T2DM groups, respectively. Serum IL-1ß levels were compared to baselines at follow-up in the 3 groups; the difference in the T2DM group was statistically significant. The frequency distributions of the IL-1ß gene rs16944 genotypes, i.e., CC, CT, and TT, were significantly different in the 3 groups, and the distributions in the T2DM and NC groups were significantly different. The frequency distributions of the C and T alleles of IL-1ß rs16944 were not significantly different. Logistic regression analysis identified the CC+CT genotype as an independent risk factor for the development of diabetes in patients with normal glucose metabolism (OR = 2.457, 95% CI: 1.238-4.877). CONCLUSIONS: The IL-1ß gene rs16944 C/T polymorphism may cause genetic susceptibility to T2DM in the Luzhou population. The CC+CT genotypes may increase T2DM risk.

Antioxidant interventions in autism spectrum disorders: A meta-analysis.

BACKGROUND: Autism spectrum disorder (ASD) might be associated with oxidative stress, and antioxidants are commonly used in the treatment of young people with ASD. However, the evidence about the effectiveness of these interventions remains debatable. We performed a meta-analysis to evaluate the effect of antioxidants on the symptoms of patients with autism. METHODS: Data sources: PubMed and Web of Science databases. STUDY SELECTION: We selected placebo-controlled, double-blind, randomized clinical trials published until February 2021 to evaluate the efficacy of antioxidant interventions on ASD. DATA ANALYSIS: Aberrant Behavior Checklist (ABC), Repetitive Behavior Scale-Revised (RBS), Social Responsiveness Scale (SRS), Developmental Behavior Checklist (DBC) and Clinical Global Impressions Severity scale (CGIS) were used to evaluate the 22 different symptom outcomes. The Hedges-adjusted g value was used to estimate the effect of each dietary intervention relative to the placebo. RESULTS: In this meta-analysis, we examined 13 double-blind randomized clinical trials, comprising a total of 570 patients with ASD: 293 in the intervention group and 277 in the placebo group. Antioxidants (N-acetylcysteine (NAC), other antioxidants) are more effective than placebos in improving the irritability among symptoms in the ABC and communication disturbance symptoms in the DBC. There was a good trend of improvement in the stereotypic behavior symptoms in the ABC. Treatment with NAC antioxidants showed a good trend of improvement in irritability in the ABC and symptoms of hyperactivity. The effect size was small, and there was a low risk of statistical heterogeneity and publication bias. LIMITATIONS: The number of studies in this meta-analysis was small and the sample size was small. CONCLUSION: This meta-analysis suggests that antioxidant intervention has a potential role in the management of some symptoms in patients with ASD, and indicates the feasibility of using antioxidants to treat autism in the future.

Deciphering cell lineage specification of human lung adenocarcinoma with single-cell RNA sequencing.

Lung adenocarcinomas (LUAD) arise from precancerous lesions such as atypical adenomatous hyperplasia, which progress into adenocarcinoma in situ and minimally invasive adenocarcinoma, then finally into invasive adenocarcinoma. The cellular heterogeneity and molecular events underlying this stepwise progression remain unclear. In this study, we perform single-cell RNA sequencing of 268,471 cells collected from 25 patients in four histologic stages of LUAD and compare them to normal cell types. We detect a group of cells closely resembling alveolar type 2 cells (AT2) that emerged during atypical adenomatous hyperplasia and whose transcriptional profile began to diverge from that of AT2 cells as LUAD progressed, taking on feature characteristic of stem-like cells. We identify genes related to energy metabolism and ribosome synthesis that are upregulated in early stages of LUAD and may promote progression. MDK and TIMP1 could be potential biomarkers for understanding LUAD pathogenesis. Our work shed light on the underlying transcriptional signatures of distinct histologic stages of LUAD progression and our findings may facilitate early diagnosis.

Hyaline fibromatosis syndrome: a case presenting with gingival enlargement as the only clinical manifestation and a report of two new mutations in the ANTXR2 gene.

BACKGROUND: Hyaline fibromatosis syndrome (HFS) is a rare autosomal recessive disorder caused by mutations in the gene for anthrax toxin receptor-2 (ANTXR2). The clinical features of HFS include skin thickening with nodules, papules and plaques, gingival enlargement, joint stiffness and contractures, and systemic manifestations. Notably, in all patients with HFS reported in the literature, gingival enlargement has never occurred alone. CASE PRESENTATION: A case of a child with gingival enlargement as the only clinical manifestation, who was later diagnosed with HFS, is described. In this case, the absence of skin and joint lesions and other characteristic clinical presentations gave rise to a diagnostic problem. This uncommon condition was clinically indistinguishable from other diseases or conditions that presented with diffuse gingival enlargement. A definitive diagnosis of HFS was reached through genetic analysis. Trio whole exome sequencing revealed compound heterozygous mutations of ANTXR2 in this patient and two new mutations were reported. CONCLUSIONS: The findings of this case serve as an important reminder to clinicians. When dental practitioners encounter gingival manifestations of HFS without accompanied skin or joint involvement, there is a need to pay attention to the differential diagnosis and increase awareness of HFS.

DNA methylation patterns at and beyond the histological margin of early-stage invasive lung adenocarcinoma radiologically manifested as pure ground-glass opacity.

BACKGROUND: Early-stage lung cancers radiologically manifested as ground-glass opacities (GGOs) have been increasingly identified, among which pure GGO (pGGO) has a good prognosis after local resection. However, the optimal surgical margin is still under debate. Precancerous lesions exist in tumor-adjacent tissues beyond the histological margin. However, potential precancerous epigenetic variation patterns beyond the histological margin of pGGO are yet to be discovered and described. RESULTS: A genome-wide high-resolution DNA methylation analysis was performed on samples collected from 15 pGGO at tumor core (TC), tumor edge (TE), para-tumor tissues at the 5 mm, 10 mm, 15 mm, 20 mm beyond the tumor, and peripheral normal (PN) tissue. TC and TE were tested with the same genetic alterations, which were also observed in histologically normal tissue at 5 mm in two patients with lower mutation allele frequency. According to the difference of methylation profiles between PN samples, 2284 methylation haplotype blocks (MHBs), 1657 differentially methylated CpG sites (DMCs), and 713 differentially methylated regions (DMRs) were identified using reduced representation bisulfite sequencing (RRBS). Two different patterns of methylation markers were observed: Steep (S) markers sharply changed at 5 mm beyond the histological margin, and Gradual (G) markers changed gradually from TC to PN. S markers composed 86.2% of the tumor-related methylation markers, and G markers composed the other 13.8%. S-marker-associated genes enriched in GO terms that were related to the hallmarks of cancer, and G-markers-associated genes enriched in pathways of stem cell pluripotency and transcriptional misregulation in cancer. Significant difference in DNA methylation score was observed between peripheral normal tissue and tumor-adjacent tissues 5 mm further from the histological margin (p < 0.001 in MHB markers). DNA methylation score at and beyond 10 mm from histological margin is not significantly different from peripheral normal tissues (p > 0.05 in all markers). CONCLUSIONS: According to the methylation pattern observed in our study, it was implied that methylation alterations were not significantly different between tissues at or beyond P10 and distal normal tissues. This finding explained for the excellent prognosis from radical resections with surgical margins of more than 15 mm. The inclusion of epigenetic characteristics into surgical margin analysis may yield a more sensitive and accurate assessment of remnant cancerous and precancerous cells in the surgical margins.

Study on the Effect of Antiplatelet and Gastric Mucosal Protection of Traditional Chinese Medicine Invigorating Qi and Hemostasis.

BACKGROUND: To investigate the effects of Chinese herbal medicine in tonifying qi and attaining hemostasis caused by the metabolism of the drug clopidogrel and as a result of platelet and gastric mucosa injury in an ischemia-reperfusion rat model. METHODS: A pharmacokinetic model was established to record the drug metabolism parameters of clopidogrel metabolites. Then, absorption of the drug was compared with approaches using the traditional Chinese medicine (TCM) approach of tonifying qi and establishing hemostasis, to using the drug pantoprazole and applying these approaches in combination with clopidogrel. Intragastric administration was performed, and all indicators were tested. RESULTS: The area under the curve (AUC; 0-T, 300.342 ± 35.832 mg/L* h; AUC 0-∞, 320.462 ± 40.213 mg/L* h), the plasma peak concentration (30.622 ± 9.917 mg/L*), and the peak time and half-life (7.954 ± 1.121 h) in the clopidogrel and the TCM groups were higher than those in the clopidogrel and pantoprazole groups. In terms of antiplatelet aggregation, compared with model group, the platelet aggregation rate induced by arachidonic acid (AA) and adenosine diphosphate (ADP) was significantly decreased by the TCM approach of tonifying qi and stopping bleeding (p < 0.05). The ADP, thromboxane A2, GPII B/Pa-A, CD62P and platelet factor 4 content in the TCM yiqi decoction and hemostasis approach were significantly decreased (p < 0.01). Compared with the clopidogrel group, the gastrin and motilin in the serum, the cyclooxygenase (COX)-1 and prostaglandin E2 in gastric tissue, and expression of vascular endothelial growth factor messenger ribonucleic acid in the serum were all significantly increased using TCM approach to protect against gastric mucosal injury (p < 0.05). CONCLUSION: TCM invigorating qi and hemostasis has an inhibitory effect on platelet activation. It can reduce the local inflammatory reaction at the same time as protecting gastric mucosa.

Characterization of Cellular Heterogeneity and an Immune Subpopulation of Human Megakaryocytes.

Megakaryocytes (MKs) and their progeny platelets function in a variety of biological processes including coagulation, hemostasis, inflammation, angiogenesis, and innate immunity. However, the divergent developmental and cellular landscape of adult MKs remains mysterious. Here, by deriving the single-cell transcriptomic profiling of MKs from human adult bone marrow (BM), cellular heterogeneity within MKs is unveiled and an MK subpopulation with high enrichment of immune-associated genes is identified. By performing the dynamic single-cell transcriptomic landscape of human megakaryopoiesis in vitro, it is found that the immune signatures of MKs can be traced back to the progenitor stage. Furthermore, two surface markers, CD148 and CD48, are identified for mature MKs with immune characteristics. At the functional level, these CD148+ CD48+ MKs can respond rapidly to immune stimuli both in vitro and in vivo, exhibit high-level expression of immune receptors and mediators, and may function as immune-surveillance cells. The findings uncover the cellular heterogeneity and a novel immune subset of human adult MKs and should greatly facilitate the understanding of the divergent functions of MKs under physiological and pathological conditions.

PKD3 promotes metastasis and growth of oral squamous cell carcinoma through positive feedback regulation with PD-L1 and activation of ERK-STAT1/3-EMT signalling.

Oral squamous cell carcinoma (OSCC) has a high incidence of metastasis. Tumour immunotherapy targeting PD-L1 or PD-1 has been revolutionary; however, only a few patients with OSCC respond to this treatment. Therefore, it is essential to gain insights into the molecular mechanisms underlying the growth and metastasis of OSCC. In this study, we analysed the expression levels of protein kinase D3 (PKD3) and PD-L1 and their correlation with the expression of mesenchymal and epithelial markers. We found that the expression of PKD3 and PD-L1 in OSCC cells and tissues was significantly increased, which correlated positively with that of mesenchymal markers but negatively with that of epithelial markers. Silencing PKD3 significantly inhibited the growth, metastasis and invasion of OSCC cells, while its overexpression promoted these processes. Our further analyses revealed that there was positive feedback regulation between PKD3 and PD-L1, which could drive EMT of OSCC cells via the ERK/STAT1/3 pathway, thereby promoting tumour growth and metastasis. Furthermore, silencing PKD3 significantly inhibited the expression of PD-L1, and lymph node metastasis of OSCC was investigated with a mouse footpad xenograft model. Thus, our findings provide a theoretical basis for targeting PKD3 as an alternative method to block EMT for regulating PD-L1 expression and inhibiting OSCC growth and metastasis.