Objective To explore the relationship between circadian rhythm genes and the occurrence, development, prognosis, and tumor microenvironment (TME) of lung adenocarcinoma (LUAD). Methods The Cancer Genome Atlas data were used to evaluate the expression, copy number variation, and somatic mutation frequency of circadian gene sets in LUAD. Gene ontology, Kyoto encyclopedia of genes and genomes, and gene set enrichment analysis were used to explore the potential mechanisms by which circadian rhythm genes affected LUAD progression. Cox regression, least absolute shrinkage and selection operator regression, support vector machine recursive feature elimination, and random forest screened circadian genes and established prognostic models, and on this basis constructed nomogram to predict patients’ 1-, 3-, and 5-year survival rates. Kaplan-Meier survival curves, receiver operating characteristic (ROC) curves, and time-dependent ROC curves were drawn to evaluate the predictive ability of the model, and the external dataset of GEO further verified the prognostic value of the prediction model. In addition, we evaluated the association of the prognostic model with immune cells and immune checkpoint genes. Single cell RNA sequencing (scRNA-seq) analysis was used to explore the molecular characteristics between prognostically relevant circadian genes and different immune cell populations in TME. Results Differentially expressed circadian rhythm genes were mainly enriched in biological processes related to cGMP-PKG signaling pathway, lipid and atherosclerosis, and JAK-STAT signaling pathway. Seven circadian rhythm genes: LGR4, CDK1, KLF10, ARNTL2, RORA, NPAS2, PTGDS were screened out, and a RiskScore model was established. According to the median RiskScore, samples were divided into a high-risk group and a low-risk group. Compared with patients in the low-risk group, patients in the high-risk group showed a poorer prognosis (P<0.001). Immunological characterization analysis showed that there were differences in the infiltration of multiple immune cells between the low-risk group and high-risk group. Most immune checkpoint genes had higher expression levels in the high-risk group than those in the low-risk group, and RiskScore was positively correlated with the expression of CD276, TNFSF4, PDCD1LG2, CD274, and TNFRSF9, and negatively correlated with the expression of CD40LG and TNFSF15. The scRNA-seq analysis showed that RORA and KLF10 were mainly expressed in natural killer cells. Conclusion The prognostic model based on seven feature circadian rhythm genes has certain predictive value for predicting survival of LUAD patients. Dysregulated expression of circadian genes may regulate the occurrence, progression as well as prognosis of LUAD through affecting TME, which provides a possible direction for finding potential strategies for treating LUAD from the perspective of mechanism by which circadian disorder affects immune cells.

Objective To determine the prognostic biomarkers and new therapeutic targets of the lung adenocarcinoma (LUAD), based on which to establish a prediction model for the survival of LUAD patients. Methods An integrative analysis was conducted on gene expression and clinicopathologic data of LUAD, which were obtained from the UCSC database. Subsequently, various methods, including screening of differentially expressed genes (DEGs), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and Gene Set Enrichment Analysis (GSEA), were employed to analyze the data. Cox regression and least absolute shrinkage and selection operator (LASSO) regression were used to establish an assessment model. Based on this model, we constructed a nomogram to predict the probable survival of LUAD patients at different time points (1-year, 2-year, 3-year, 5-year, and 10-year). Finally, we evaluated the predictive ability of our model using Kaplan-Meier survival curves, receiver operating characteristic (ROC) curves, and time-dependent ROC curves. The validation group further verified the prognostic value of the model. Results The different-grade pathological subtypes' DEGs were mainly enriched in biological processes such as metabolism of xenobiotics by cytochrome P450, natural killer cell-mediated cytotoxicity, antigen processing and presentation, and regulation of enzyme activity, which were closely related to tumor development. Through Cox regression and LASSO regression, we constructed a reliable prediction model consisting of a five-gene panel (MELTF, MAGEA1, FGF19, DKK4, C14ORF105). The model demonstrated excellent specificity and sensitivity in ROC curves, with an area under the curve (AUC) of 0.675. The time-dependent ROC analysis revealed AUC values of 0.893, 0.713, and 0.632 for 1-year, 3-year, and 5-year survival, respectively. The advantage of the model was also verified in the validation group. Additionally, we developed a nomogram that accurately predicted survival, as demonstrated by calibration curves and C-index. Conclusion We have developed a prognostic prediction model for LUAD consisting of five genes. This novel approach offers clinical practitioners a personalized tool for making informed decisions regarding the prognosis of their patients.

Perineural invasion (PNI) by tumor cells is a key phenotype of highly-invasive oral squamous cell carcinoma (OSCC). Since Schwann cells (SCs) and fibroblasts maintain the physiological homeostasis of the peripheral nervous system, and we have focused on cancer-associated fibroblasts (CAFs) for decades, it's imperative to elucidate the impact of CAFs on SCs in PNI⁺ OSCCs. We describe a disease progression-driven shift of PNI^- towards PNI⁺ during the progression of early-stage OSCC (31%, n = 125) to late-stage OSCC (53%, n = 97), characterized by abundant CAFs and nerve demyelination. CAFs inhibited SC proliferation/migration and reduced neurotrophic factors and myelin in vitro, and this involved up-regulated ER stress and decreased MAPK signals. Moreover, CAFs also aggravated the paralysis of the hind limb and PNI in vivo. Unexpectedly, leukemia inhibitory factor (LIF) was exclusively expressed on CAFs and up-regulated in metastatic OSCC. The LIF inhibitor EC330 restored CAF-induced SC inactivation. Thus, OSCC-derived CAFs inactivate SCs to aggravate nerve injury and PNI development.
Schwann Cells/metabolism* ; Mouth Neoplasms/metabolism* ; Humans ; Cancer-Associated Fibroblasts/metabolism* ; Animals ; Carcinoma, Squamous Cell/metabolism* ; Neoplasm Invasiveness/pathology* ; Male ; Female ; Mice ; Cell Movement/physiology* ; Cell Proliferation/physiology* ; Cell Line, Tumor ; Leukemia Inhibitory Factor/metabolism* ; Middle Aged

Objective:To retrospectively analyze the changes in the proportion of refined lymphocyte subsets in peripheral blood of patients with Graves disease (GD), and their correlation with the clinical characteristics and efficacy of GD, and to explore the immunological pathogenesis of Graves disease for seeking new therapeutic targets.Methods:A total of 97 newly diagnosed GD patients (GD group), 27 patients after treatment (treatment group), and 31 healthy individuals (control group) who visited the Fifth Medical Center of the PLA General Hospital from 2018 to 2021 were included in this study. The data of refined lymphocyte subsets, thyroid function, blood routine and clinical treatment of the three groups were compared and analyzed. The t-test and rank sum test were used to compare the proportions of lymphocyte subsets among different groups, and Pearson correlation analysis was used to analyze the correlation between the proportions of lymphocyte subsets and thyroid function indicators.Results:The proportion of B cells in GD group was higher than that in the control group [16.2%(11.8%, 21.8%) vs 10.2%(8.1%,13.6%)], while the proportion of natural killer (NK) cells was lower [9.4%(4.9%, 13.6%) vs 14.6%(12.1%,18.8%)], and the differences were statistically significant ( P<0.05). Abnormal T cell differentiation: the proportions of functional cells, including activated T cells, memory T cells, clustering antigen(CD)4+memory T cells, Th1 cells, and Tc1 cells, were lower than that in the control group [3.2%(2.1%, 5.7%) vs 5.8%(3.0%, 9.3%), P<0.05; 36.7% (29.9%, 48.1%) vs 48.0%(39.2%,57.7%), P<0.05; 23.1%(17.4%, 30.1%) vs 28.9%(23.3%,34.6%), P<0.05; 16.4% (11.8%, 23.6%) vs 24.3%(16.9%,28.5%), P<0.05; 28.5% (14.7%, 39.2%) vs 46.3%(21.6%,69.2%), P<0.05]. The proportion of activated T cells in the treatment group was higher than that in the GD group [6.5% (4.6%, 13.6%) vs 3.2% (2.1%, 5.7%), P<0.05]. The total triiodothyronine results showed positive correlations with B cells ( r=0.356, P<0.01) and negative correlations with NK cells ( r=?0.416, P<0.01), while the total thyroxine values showed negative correlations with NK cells and activated T cells ( r=?0.318,?0.335; P<0.01). Thyroid stimulating hormone and CD8+initial T cells were positively correlated ( r=0.382, P<0.01). The proportion of B cells, cytotoxic T cells and suppressor T cells in CD8+cells of patients with complications [such as Graves orbitopathy (GO), thyroid toxic cardiomyopathy, etc.] was significantly different from that of the simple GD patients [18.3% (14.1%, 27.1%) vs 14.6% (10.8%, 21.4%), Z=2.54, P<0.05; 73.4%(65.6%,83.6%)vs 65.0%(50.3%,79.3%), Z=2.93, P<0.05; 26.6%(16.4%, 37.5%)vs 35.0%(20.7%,49.7%), Z=?2.74, P<0.05]. The proportion of suppressor T cells in GO patients was lower than that in non-GO patients [6.1% (3.4%, 8.1%) vs 8.5% (4.9%, 13.6%), Z=?3.20 P<0.05]. Conclusion:There are significant alterations in the circulating immune cells of GD patients, suggesting that immunological abnormalities play a crucial role in the onset and progression of the disease.

Objective:To retrospectively analyze the changes in the proportion of refined lymphocyte subsets in peripheral blood of patients with Graves disease (GD), and their correlation with the clinical characteristics and efficacy of GD, and to explore the immunological pathogenesis of Graves disease for seeking new therapeutic targets.Methods:A total of 97 newly diagnosed GD patients (GD group), 27 patients after treatment (treatment group), and 31 healthy individuals (control group) who visited the Fifth Medical Center of the PLA General Hospital from 2018 to 2021 were included in this study. The data of refined lymphocyte subsets, thyroid function, blood routine and clinical treatment of the three groups were compared and analyzed. The t-test and rank sum test were used to compare the proportions of lymphocyte subsets among different groups, and Pearson correlation analysis was used to analyze the correlation between the proportions of lymphocyte subsets and thyroid function indicators.Results:The proportion of B cells in GD group was higher than that in the control group [16.2%(11.8%, 21.8%) vs 10.2%(8.1%,13.6%)], while the proportion of natural killer (NK) cells was lower [9.4%(4.9%, 13.6%) vs 14.6%(12.1%,18.8%)], and the differences were statistically significant ( P<0.05). Abnormal T cell differentiation: the proportions of functional cells, including activated T cells, memory T cells, clustering antigen(CD)4+memory T cells, Th1 cells, and Tc1 cells, were lower than that in the control group [3.2%(2.1%, 5.7%) vs 5.8%(3.0%, 9.3%), P<0.05; 36.7% (29.9%, 48.1%) vs 48.0%(39.2%,57.7%), P<0.05; 23.1%(17.4%, 30.1%) vs 28.9%(23.3%,34.6%), P<0.05; 16.4% (11.8%, 23.6%) vs 24.3%(16.9%,28.5%), P<0.05; 28.5% (14.7%, 39.2%) vs 46.3%(21.6%,69.2%), P<0.05]. The proportion of activated T cells in the treatment group was higher than that in the GD group [6.5% (4.6%, 13.6%) vs 3.2% (2.1%, 5.7%), P<0.05]. The total triiodothyronine results showed positive correlations with B cells ( r=0.356, P<0.01) and negative correlations with NK cells ( r=?0.416, P<0.01), while the total thyroxine values showed negative correlations with NK cells and activated T cells ( r=?0.318,?0.335; P<0.01). Thyroid stimulating hormone and CD8+initial T cells were positively correlated ( r=0.382, P<0.01). The proportion of B cells, cytotoxic T cells and suppressor T cells in CD8+cells of patients with complications [such as Graves orbitopathy (GO), thyroid toxic cardiomyopathy, etc.] was significantly different from that of the simple GD patients [18.3% (14.1%, 27.1%) vs 14.6% (10.8%, 21.4%), Z=2.54, P<0.05; 73.4%(65.6%,83.6%)vs 65.0%(50.3%,79.3%), Z=2.93, P<0.05; 26.6%(16.4%, 37.5%)vs 35.0%(20.7%,49.7%), Z=?2.74, P<0.05]. The proportion of suppressor T cells in GO patients was lower than that in non-GO patients [6.1% (3.4%, 8.1%) vs 8.5% (4.9%, 13.6%), Z=?3.20 P<0.05]. Conclusion:There are significant alterations in the circulating immune cells of GD patients, suggesting that immunological abnormalities play a crucial role in the onset and progression of the disease.

As the research work went further and more detailed, a variety of new treatments compete to come out.However, it remains unclear that how the antigen works to distinguish cancer cells and normal cells.Neoantigen, which is located in the tumor cell surface of a specific antigen, its presence makes human immunotherapy into new areas which may make personalized treatment possible in the near future.Emerging data suggest that the identification of such newantigens is a major factor in clinical immunotherapy.They can form a biomarker in cancer immunotherapy to provide targets for a variety of therapeutic approaches to attack, which allows T cells to selectively enhance the immune response against this class of antigens.

American medical colleges attach great importance to the humanistic education.The rich humanities curriculum is fully integrated into the teaching practice thus the mutual penetration of medicine and the humanities is achieved.They have specific and detailed examination evaluation system and the teaching method was more practical and participatory.By contrast,nost of the medical colleges in our country neglect the humanities construction and the cultivation of medical students' humanistic spirit.The coverage of the humanities curriculum is relatively narrow and the faculty is insufficient.We should promote the development of humanistic cducation,attach importance to the construction of medical humanities,set up appropriate curriculum system,increase the investment in faculty and reform the teaching method and examination method.

Objective To assess the correlation of serum 25-hydroxyvitamin D (25-OHD) levels with vitamin D-binding protein (the group-specific component,GC) gene polymorphism in chronic obstructive pulmonary disease (COPD).Methods In a cross-sectional case-control study,250participants,including 116 COPD patients with smoking history and 134 healthy smokers,were investigated.A questionnaire about smoking history,vitamin D intake and comorbidities was collected.General pulmonary function was done by routine.Serum 25-OHD levels were detected by ELISA.The genetic variants (rs4588and rs7041) were genotyped by real time fluorescence polymerase chain reaction (RT-PCR) with TaqMan probe technology.Results The COPD patients had lower serum vitamin D level than the smoker subjects (36.58 nmol/L vs 43.80 nmol/L,P ＜0.001).In the COPD patients,vitamin D level was 39.43 nmol/L in those with percentage of predicted forced expiratory volume in 1 second (FEV1 ％ pred) greater than or equal to 80％.In other groups with FEV1 ％ pred 50％-80％,30％-50％ and lower than 30％,vitamin D levels were 35.32 nmol/L,32.21 nmol/L,26.25 nmol/L respectively (P ＜ 0.01).Moreover,there was a significant relevance of 25-OHD levels with FEV1 ％ pred in both COPD patients and healthy smokers (r2 =1.911; P ＜0.000 1).The mean 25-OHD concentration had a negative correlation with Global Initiative for Obstructive Lung Disease (GOLD) stages.Homozygous carriers of vitamin D-binding protein gene rs7041 T allele were independently related to 25-OHD levels and susceptibility of COPD (P ＜ 0.01 ; OR =2.140,95％ CI 1.157-3.959,P =0.015 respectively).Conclusions Patients with COPD are at high risk of vitamin D deficiency and the severity of COPD is inversely correlated with vitamin D levels.Furthermore,homozygous carrier of rs7041 T allele influences 25-OHD serum levels and is related to susceptibility of COPD,which may be a potential candidate gene for screening COPD.

Objective To investigate the expression of macrophage migration inhibition factor (MIF) in pulmonary tissues of the smokers with and without chronic obstructive pulmonary disease (COPD).Methods The subjects were assigned into three groups:non-smokers without COPD (control group,n =12),smokers without COPD (smoker group,n =13) and smokers with COPD (COPD group,n =16).The specimens were obtained from lung tissues as far away from cancer focus as possible (＞ 5cm).Real-time quantitative PCR and immunohistochemistry were used to investigate the expression and distribution of MIF in pulmonary tissues.The relationship between the severity of airflow obstruction and the differential expressions of MIF in lung tissues of the smokers with or without COPD was analyzed.Results (1) MIF mRNA expression in COPD group (4.87 ± 1.79) was higher than that in the smoker group (2.16 ±0.72;P＜0.01),which was higher than that in the control group (1.09 ±0.48;P ＜0.01).(2)Immunohistochemistry analysis showed that MIF protein expression in lung tissues of the COPD group (0.277±0.025) was higher than that in the smokers group (0.199 ±0.034;P ＜0.01),which was significantly higher than that in control group (0.130 ±0.021 ;P ＜0.01).(3) Correlation analysis of MIF mRNA expression in the lung tissues and pulmonary function parameters of forced expired volume in one second (FEV1) percentage of predicted (FEV1 pred) and ratio of FEV1 to forced vital capacity (FEV1/FVC) suggested that MIF mRNA expression in the lung tissues was negatively related with FEV1 pred (r=-0.578,P ＜ 0.01) and FEV1/FVC (r =-0.607,P ＜ 0.01).Conclusions MIF expression significantly increases in the smokers with COPD,and MIF level in the lung is positively correlated with airflow limitation.The results suggest that MIF may play an important role in the pathogenesis of smokinginduced COPD.

In order to investigate the effects of puerarin on pulmonary vascular remodeling and protein kinase C-alpha (PKC-alpha) in chronic exposure smoke rats, 54 male Wistar rats were randomly divided into 7 groups: control group (C group), smoke exposure groups (S(4w) group, S(8w) group), puerarin groups (P(4w) group, P(8w) group), propylene glycol control groups (PC(4w) group, PC(8w) group). Rats were exposed to cigarette smoke or air for 4 to 8 weeks. Rats in puerarin groups also received puerarin. To evaluate vascular remodeling, alpha-smooth muscle actin (alpha-SM-actin) staining was used to count the percentage of completely muscularised vessels to intraacinar pulmonary arteries (CMA/IAPA) which was determined by morphometric analysis of histological sections. Pulmonary artery smooth muscle cell (PASMC) apoptosis was detected by in situ end labeling technique (TUNEL), and proliferation by proliferating cell nuclear antigen (PCNA) staining. Reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence staining and Western blot analysis were done to detect the PKC-alpha mRNA and protein expression in pulmonary arteries. The results showed that in cigarette smoke-exposed rats the percentage of CMA/IAPA and alpha-SM-actin expression were increased greatly, PASMC apoptosis was increased and proliferation was markedly increased; Apoptosis indices (AI) and proliferation indices (PI) were higher than in C group; AI and PI were correlated with vascular remodeling indices; The expression of PKC-alpha mRNA and protein in pulmonary arteries was significantly higher than in C group. In rats treated with puerarin, the percentage of CMA/IAPA and cell proliferation was reduced, whereas PASMC apoptosis was increased; The expression levels of PKC-alpha mRNA and protein were lower than in smoke exposure rats. There was no difference among all these data between S groups and PC groups. These findings suggested that cigarette smoke-induced pulmonary vascular remodeling was most likely an effect of the imbalance of PASMC proliferation and apoptosis. Puerarin appears to be able to reduce cell proliferation and vascular remodeling possibly through PKC signaling transduction pathway.
Apoptosis ; Cell Proliferation ; Endothelium, Vascular/*drug effects ; Isoflavones/*pharmacology ; Lung/*drug effects ; Myocytes, Smooth Muscle/cytology ; Myocytes, Smooth Muscle/*drug effects ; Protein Kinase C-alpha/*metabolism ; Pulmonary Artery/cytology ; Pulmonary Artery/*drug effects ; Rats, Wistar ; Smoking ; Tobacco Smoke Pollution ; Vasodilator Agents/pharmacology