Establishment and validation of a 5-factor diagnostic model for obstructive and non-obstructive azoospermia based on routine clinical parameters.

Azoospermia is a serious leading male-factor cause of infertility in couples of childbearing age. The two main azoospermia types, obstructive (OA) and non-obstructive (NOA) azoospermia, differ in their treatment approaches. Therefore, their clinical diagnosis is extremely important, requiring an accurate, efficient, and easy-to-use diagnostic model. This retrospective observational study included 707 patients with azoospermia treated between 2017 and 2021, 498 with OA, and 209 with NOA. Hematological and seminal plasma parameters, hormone levels, and testicular volume were used in logistic regression analysis to evaluate and compare their diagnostic performance, results showed that the optimal diagnostic model is constructed by five variables including semen volume, semen pH, seminal plasma neutral α-glucosidase activity, follicle-stimulating hormone in the serum, and testicular volume, compared with follicle-stimulating hormone-based and testicular volume-based models. The 5-factor diagnostic model had an accuracy of 90.4%, sensitivity of 96.4%, positive predictive value of 90.6%, negative predictive value of 89.8%, and area under the curve of 0.931, all higher than in the other two models. However, its specificity (76.1%) was slightly lower than in the other models. Meantime, the internal 5-fold cross-validation results indicated that the 5-factor diagnostic model had a good clinical application value. This study established an accurate, efficient, and relatively accessible 5-factor diagnostic model for OA and NOA, providing a reference for clinical decision-making when selecting an appropriate treatment.

GOLPH3 promotes tumor malignancy via inhibition of ferroptosis by upregulating SLC7A11 in cholangiocarcinoma.

Golgi phosphoprotein 3 (GOLPH3) has been reported as an oncogene in various tumors; however, the role and function of GOLPH3 and its relevant molecular mechanism in cholangiocarcinoma (CCA) are unclear. Herein, GOLPH3 expression in CCA tissues was observed to be significantly higher than that in paired adjacent noncancerous tissues. Clinicopathological analysis showed that GOLPH3 expression correlated positively with the tumor-node-metastasis stage. In addition, GOLPH3 expression correlated inversely with the overall survival of patients with CCA. Multivariate analysis showed that GOLPH3 was an independent prognostic factor for patients with CCA. Transcriptome analysis (RNA sequencing) of GOLPH3 knockdown cells showed that the expression levels of nine ferroptosis-related genes were significantly changed, indicating the important biological function of GOLPH3 in ferroptosis in CCA cells. Furthermore, GOLPH3 knockdown could significantly promote Erastin-induced ferroptosis in vitro and suppress tumor growth in vivo. Overexpression of GOLPH3 had the opposite effect on this phenotype. Further studies revealed that GOLPH3 knockdown was significantly associated with a decrease in cysteine content, an accumulation of the lipid peroxidation product malondialdehyde, an increase in reactive oxygen species, and sensitized CCA cells to Erastin-induced ferroptosis. Moreover, changes in GOLPH3 expression were found to be consistent with the expression of light chain subunit solute carrier family 7 member 11 (SLC7A11). Thus, our study suggested that GOLPH3 functions as an oncoprotein in CCA and may suppress ferroptosis by facilitating SLC7A11 expression, suggesting that GOLPH3 could serve as a therapeutic target for CCA treatment.

Serum aspartate aminotransferase is an adverse prognostic indicator for patients with resectable pancreatic ductal adenocarcinoma.

OBJECTIVE: In this study, the association between preoperative levels of serum liver enzymes and overall survival (OS) was evaluated in patients with resectable pancreatic cancer. METHODS: Preoperative serum levels of alanine aminotransferase (ALT), aspartate aminotransferases (AST), γ-glutamyltransferase, alkaline phosphatase, and lactate dehydrogenase of 101 patients with pancreatic ductal adenocarcinoma (PDAC) were collected. Univariate and multivariate Cox hazard models were used to identify independent variables associated with OS in this cohort. RESULTS: Patients with elevated AST levels had significantly worse OS than patients with lower AST levels. A nomogram was created using TNM staging and AST levels and was shown to be more accurate in prediction than the American Joint Committee on Cancer 8th edition standard method. CONCLUSION: Preoperative AST levels could be a novel independent prognostic biomarker for patients with PDAC. The incorporation of AST levels into a nomogram with TNM staging can be an accurate predictive model for OS in patients with resectable PDAC.

Reproductive outcomes of women with moderate to severe intrauterine adhesions after transcervical resection of adhesion: A systematic review and meta-analysis.

BACKGROUND: Intrauterine adhesions (IUA) refers to the adhesion of the inner wall of the uterus, resulting in complete or partial occlusion of the uterine cavity, which causes a series of symptoms. Transcervical resection of adhesion (TCRA) is the standard surgical method for patients with IUA. However, the recurrence rate of women with moderate to severe IUA is high and it has raised a big concern about the reproductive outcomes. METHODS: We conducted a scoping review by using 4 databases, including Google Scholar, PubMed, Scopus, Embase, and web of science, to retrieve relevant literature from September 1, 2001, to February 1, 2023, and to explore the reproductive outcomes in women with moderate to severe IUA after TCRA treatment. Following defined guidelines, data extraction was carried out by 2 researchers, and the findings were examined by 2 senior academics. The papers were evaluated by 2 reviewers using the inclusion and exclusion criteria. Using a form developed especially for this study, pertinent information was retrieved, including the first author, research design, study duration, age, intervention measurement, pregnancy rate, techniques of conception, and live birth rate. Two researchers conducted a quality assessment to determine any potential bias using the Cochrane technique and the Newcastle-Ottawa scale. RevMan 5.4.1 (The Cochrane Collaboration, London, United Kingdom) was used for data analysis, while I2 was used to evaluate heterogeneity. RESULTS: In total, this study included 2099 participants. After a detailed systematic review and meta-analyses, the results showed that pregnancy and live birth rates were increased significantly after TCRA, and the risk difference of the pregnancy rate was 1.75 [1.17, 2.62]. Besides, in 2 retrospective studies, the risk difference of live birth rate was 2.26, with a 95% confidence interval of 1.99 to 2.58. Moreover, the menstrual status of women also was improved, and the risk difference of hypermenorrhoea and amenorrhea were -0.28 [-0.37, -0.19] and -0.06 [0.26, 0.13], respectively. CONCLUSIONS: Taken together, TCRA is the useful strategy for the treatment of moderate to severe IUA to enhance the reproductive outcomes in women.

Tumor malignancy by genetic transfer between cells forming cell-in-cell structures.

Cell-in-cell structures (CICs) refer to a type of unique structure with one or more cells within another one, whose biological outcomes are poorly understood. The present study aims to investigate the effects of CICs formation on tumor progression. Using genetically marked hepatocellular cancer cell lines, we explored the possibility that tumor cells might acquire genetic information and malignant phenotypes from parental cells undergoing CICs formation. The present study showed that the derivatives, isolated from CICs formed between two subpopulations by flow cytometry sorting, were found to inherit aggressive features from the parental cells, manifested with increased abilities in both proliferation and invasiveness. Consistently, the CICs clones expressed a lower level of E-cadherin and a higher level of Vimentin, ZEB-1, Fibronectin, MMP9, MMP2 and Snail as compared with the parental cells, indicating epithelial-mesenchymal transition. Remarkably, the new derivatives exhibited significantly enhanced tumorigenicity in the xenograft mouse models. Moreover, whole exome sequencing analysis identified a group of potential genes which were involved in CIC-mediated genetic transfer. These results are consistent with a role of genetic transfer by CICs formation in genomic instability and malignancy of tumor cells, which suggest that the formation of CICs may promote genetic transfer and gain of malignancy during tumor progression.

Homotypic cell-in-cell structures as an adverse prognostic predictor of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common malignant liver tumors. A homotypic cell-in-cell structure (hoCIC) refers to one or more cells internalized into the same type as their neighbors, which predominantly occurs in multiple tumors. The objective of this study was to investigate the prognostic value of hoCICs in HCC and its relationship with other clinicopathological features. By immunostaining analysis of a panel of HCC tissues, we found that hoCICs were prevalent in tumor tissues (54/90) but not in para-tumor tissues (17/90). The presence of hoCICs in tumor tissues was closely associated with E-cadherin expression. The presence of CICs was identified as significantly associated with poor survival rates of patients with HCC, comparable to traditional clinicopathological parameters, such as histological grade [hazard ratio (HR) = 0.734, p = 0.320]. Multivariate Cox regression analysis further confirmed that CICs were an independent risk factor for poor survival (HR = 1.902, p = 0.047). In addition, hoCICs were the predominant contributor in a nomogram model constructed for survival prediction at 1, 3, and 5 years [the areas under the curve (AUCs) were 0.760, 0.733, and 0.794, respectively]. Stratification analysis indicated that hoCICs tend to selectively affect patients with high-grade disease (HR = 2.477, p = 0.009) and at the early TNM stage (HR = 2.351, p = 0.05). Thus, hoCICs predict poor survival of patients with HCC, particularly those with higher grades and at an early stage.

Construction and validation of a predictive model for hepatocellular carcinoma based on serum markers.

BACKGROUND: Early hepatocellular carcinoma (HCC) detection with non-invasive biomarkers remains an unmet clinical need. We aimed to construct a predictive model based on the pre-diagnostic levels of serum markers to predict the early-stage onset of HCC. METHODS: A total of 339 HCC patients (including 157 patients from Changzhou cohort and 182 patients from Wuxi cohort) were enrolled in our retrospective study. Levels of 25 baseline serum markers were collected. Propensity score matching (PSM) analysis was conducted to balance the distributions of patients' gender, age, and the surveillance time between HCC group and control group. Then, Receiver operating characteristic (ROC) and Logistic regression analysis were performed to screen the independent predictive variables and construct a non-invasive predictive model. Subsequently, ROC curve and Kaplan-Meier (K-M) curve were used to evaluate the predictive values of the model. Clinical net benefit of the model was demonstrated by decision curve analysis (DCA) and clinical impact curve. RESULTS: Five independent predictive variables for HCC onset and two general characteristics of patients (age and gender) were incorporated into the score model. ROC and DCA curves showed that the score model had better predictive performance in discrimination and clinical net benefit compared with single variable or other score systems, with the area under the curve (AUC) of 0.890 (95% CI 0.856-0.925) in Changzhou cohort and 0.799 (95% CI 0.751-0.849) in Wuxi cohort. Meanwhile, stratification analysis indicated that the score model had good predictive values for patients with early tumor stage (AJCC stage I) or small tumors (< 2 cm). Moreover, the score of HCC patient began to increase at 30 months before clinical diagnosis and reach a peak at 6 months. CONCLUSION: Based on this model, we could optimize the current risk stratification at an early stage and consider further intensive surveillance programs for high-risk patients. It could also help clinicians to evaluate the progression and predict the prognosis of HCC patients.

Cell-in-Cell: From Cell Biology to Translational Medicine.

Cell-in-cell structures (CICs) refer to cytoplasmic internalization of a cell by another cell, which are found throughout various biological systems and have been a part of scientific dogma for a long time. However, neither the mechanisms underlying this phenomenon nor their possible roles in disease development have resulted in major breakthroughs until recent years. In view of the ubiquity of CICs in inflammatory tissue and tumors, it is tempting to think that these specific structures could be associated with clinical diagnosis and treatment and thus would become a new hotspot for translational medicine. Translational medicine is a new concept in the field of international biomedical research that appeared in the last 20 years, which transforms basic research into clinical application. With the growing interest in this field, this review addresses recent research on CICs and their potential clinical implications in cytomorphological diagnosis and the pathology of human diseases, while discussing as yet unanswered questions. We also put forward future directions to reduce the gap in our knowledge caused by our currently limited understanding of CICs.

Golgi Phosphoprotein 3 Promotes Colon Cancer Cell Metastasis Through STAT3 and Integrin α3 Pathways.

Background: Golgi phosphoprotein 3 (GOLPH3) overexpression was recently reported to be associated with a poor clinical outcome in patients with colorectal cancer (CRC). However, the underlying molecular mechanism through which GOLPH3 promotes CRC metastasis remains poorly understood. Methods: In vitro genetic ablation of GOLPH3 was performed using siRNA transfection, and a stably overexpressed GOLPH3 colon cancer cell line was constructed using the lentivirus system. Cell invasion and migration assays were conducted with or without Matrigel. Immunoblotting, qRT-PCR, immunofluorescence and immunohistochemistry were utilized to study the expression level of GOLPH3, ZEB1, integrin α3 and phosphorylation level of STAT3, AKT/mTOR and Raf/MEK/ERK pathways. Co-immunoprecipitation was used to investigate the interaction between GOLPH3 and p-STAT3 (Tyr705) or total STAT3. Results: Overexpression of GOLPH3 was found in CRC tissues and colon cancer cell lines. Knockdown of GOLPH3 using siRNAs significantly suppressed the invasion and migration of HCT116 and HCT8 cells. In contrast, the overexpression of GOLPH3 promoted the migratory and invasive ability of colon cancer cells. The phosphorylation level of STAT3 as well as the protein and mRNA levels of ZEB1 and integrin α3, were significantly decreased after GOLPH3 knockdown. Moreover, Integrin α3 expression was correlated with GOLPH3 expression in CRC tissues. Co-immunoprecipitation assay revealed that GOLPH3 interacted with pSTAT3 (Tyr705) and total STAT3. Our further experiments suggested that GOLPH3 facilitated IL-6 induced STAT3 activation and subsequently induced transcription of integrin α3 and ZEB1, which promoted the metastasis and progression of CRC. Conclusion: Our current work demonstrates that GOLPH3 facilitates STAT3 activation and regulates the expression of EMT transcription factor ZEB1 and Integrin α3 in colon cancer cells. These findings indicate that GOLPH3 plays a critical role in CRC metastasis and might be a new therapeutic target for CRC treatment.

Prediction of early recurrence and response to adjuvant Sorafenib for hepatocellular carcinoma after resection.

BACKGROUND: Early recurrence of hepatocellular carcinoma (HCC) is a major obstacle to improving the prognosis, and no widely accepted adjuvant therapy guideline for patients post-liver resection is available. Currently, all available methods and biomarkers are insufficient to accurately predict post-operation HCC patients' risk of early recurrence and their response to adjuvant therapy. METHODS: In this study, we downloaded four gene expression datasets (GSE14520, GSE54236, GSE87630, and GSE109211) from the Gene Expression Omnibus database and identified 34 common differentially expressed genes associated with HCC dysregulation and response to adjuvant sorafenib. Then, we constructed a novel 11-messenger RNA predictive model by using ROC curves analysis, univariate Cox regression analysis, and LASSO Cox regression analysis. Furthermore, we validated the predictive values of the risk model in GSE14520 and TCGA-LIHC cohorts by using Kaplan-Meier survival analysis, multivariable Cox regression analysis, and decision curve analysis, respectively. RESULTS: The risk score model could identify patients with a high risk of HCC recurrence at the early stage and could predict the response of patients to adjuvant sorafenib. Patients with a high risk score had a worse recurrence rate in training cohorts (2-year: p < 0.0001, hazard ratio (HR): 4.658, confidence interval 95% CI [2.895-7.495]; 5-year: p < 0.0001, HR: 3.251, 95% CI [2.155-4.904]) and external validation cohorts (2-year: p < 0.001, HR: 3.65, 95% CI [2.001-6.658]; 5-year: p < 0.001, HR: 3.156, 95% CI [1.78-5.596]). The AUC values of the risk score model for predicting tumor early recurrence were 0.746 and 0.618, and that of the risk score model for predicting the response to adjuvant sorafenib were 0.722 and 0.708 in the different cohort, respectively. Multivariable Cox regression analysis and decision curve analysis also showed that the risk score model was superior to and independent of other clinicopathologic characteristics. Moreover, the risk score model had excellent abilities to predict the overall survival and HCC recurrence of patients with the same tumor stage category. CONCLUSIONS: Our risk model is a reliable and superior predictive tool. With this model, we could optimize the risk stratification based on early tumor recurrence and could evaluate the response of patients to adjuvant sorafenib after liver resection.

Specific CD8+ TCR Repertoire Recognizing Conserved Antigens of SARS-CoV-2 in Unexposed Population: A Prerequisite for Broad-Spectrum CD8+ T Cell Immunity.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has developed variants escaping neutralization antibody immunity established against the original virus. An understanding of broad-spectrum adaptive immunity, including CD8+ T cell immunity to wide range of epitopes, could help translational efforts to improve coronavirus disease 2019 (COVID-19) prevention and therapy. However, there have been few direct studies in which such immunity exists in a population. METHODS: We selected SARS-CoV-2-conserved structural peptides that are not prone to mutation as antigens for broad-spectrum CD8+ T cell immunity. Peripheral blood mononuclear cells (PBMCs) from unexposed healthy donors were stimulated with these peptides in vitro and CD8+ T cell-specific response was monitored. The conserved peptide-specific CD8+ T cells were sorted for T cell receptor (TCR) repertoire sequencing. The presence of specific complementary determining region 3 (CDR3) clones was analyzed in a healthy cohort. RESULTS: For each structural protein, including S, E, M, N, the conserved peptides could potentially provide the largest number of major histocompatibility complex-I (MHC-I) epitopes in the Oriental and Caucasian populations. For conserved peptides from spike (S), envelope (E), membrane (M), nucleocapsid (N) proteins, we found that there were no cross-reactive memory T cells in the unexposed individuals. Instead, their T cells contain naïve TCR repertoire recognizing these conserved peptides. Using TCR sequencing and CDR3 clustering for the conserved peptides specific T cells, we found that the recovered patients had a higher proportion of TCR repertoire similar with that of specific CD8+ T cells in unexposed individuals. Meanwhile, CDR3 clones of the above T cells were widely present in the healthy population. CONCLUSIONS: This study provides evidence of broad-spectrum SARS-CoV-2 specific CD8+ TCR repertoire in unexposed healthy population, which is implicated in the development and implementation of broad-spectrum vaccines against COVID-19.

SARS-CoV-2 spike protein dictates syncytium-mediated lymphocyte elimination.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is highly contagious and causes lymphocytopenia, but the underlying mechanisms are poorly understood. We demonstrate here that heterotypic cell-in-cell structures with lymphocytes inside multinucleate syncytia are prevalent in the lung tissues of coronavirus disease 2019 (COVID-19) patients. These unique cellular structures are a direct result of SARS-CoV-2 infection, as the expression of the SARS-CoV-2 spike glycoprotein is sufficient to induce a rapid (~45.1 nm/s) membrane fusion to produce syncytium, which could readily internalize multiple lines of lymphocytes to form typical cell-in-cell structures, remarkably leading to the death of internalized cells. This membrane fusion is dictated by a bi-arginine motif within the polybasic S1/S2 cleavage site, which is frequently present in the surface glycoprotein of most highly contagious viruses. Moreover, candidate anti-viral drugs could efficiently inhibit spike glycoprotein processing, membrane fusion, and cell-in-cell formation. Together, we delineate a molecular and cellular rationale for SARS-CoV-2 pathogenesis and identify novel targets for COVID-19 therapy.

Anti-DFS70 Antibodies Among Patient and Healthy Population Cohorts in China: Results From a Multicenter Training Program Showing Spontaneous Abortion and Pediatric Systemic Autoimmune Rheumatic Diseases Are Common in Anti-DFS70 Positive Patients.

Objective: Anti-DFS70 antibodies correlating with the nuclear dense fine speckled (DFS) pattern in the HEp-2 indirect immunofluorescence assay (IFA) are less common in patients with systemic autoimmune rheumatic disease (SARD) than in healthy subjects and their clinical associations remain elusive. We hosted a multi-center HEp-2 IFA training program to improve the ability of clinical laboratories to recognize the DFS pattern and to investigate the prevalence and relevance of anti-DFS70 antibodies. Methods: DFS pattern sera identified by HEp-2 IFA in 29 centers in China were redirected to a central laboratory for anti-DFS70 testing by line immunoblot assay (LIA), enzyme-linked immunosorbent assay (ELISA), and IFA with HEp-2 ELITE/DFS70-KO substrate. Anti-extractable nuclear antigen antibodies were measured by LIA and the clinical relevance was examined in adult and pediatric patients. Results: HEp-2 IFA positive rate and DFS pattern in positive sera were 36.2% (34,417/95,131) and 1.7% (582/34,417) in the patient cohort, and 10.0% (423/4,234) and 7.8% (33/423) in a healthy population, respectively. Anti-DFS70 prevalence among sera presenting the DFS pattern was 96.0, 93.7, and 49.6% by ELISA, LIA, and HEp-2 ELITE, respectively. 15.5% (52/336) of adult and 50.0% (20/40) of pediatric anti-DFS70 positive patients were diagnosed with SARD. Diseases most common in anti-DFS70 positive patients were spontaneous abortion (28.0%) in adults and juvenile idiopathic arthritis (22.5%) in pediatric patients. Conclusion: Accurate DFS pattern identification increased the detection rate of anti-DFS70 antibodies by ELISA and LIA. Anti-DFS70 antibodies are remarkably high in cases of spontaneous abortion and in pediatric SARD patients, but not prevalent in adult SARD patients.

GOLPH3 Promotes Cancer Growth by Interacting With STIP1 and Regulating Telomerase Activity in Pancreatic Ductal Adenocarcinoma.

Overexpression of Golgi phosphoprotein 3 (GOLPH3) predicts poor prognosis and is a potential therapeutic target in pancreatic ductal adenocarcinoma (PDAC). However, its role and underlying molecular mechanisms in the progression of PDAC remain unknown. In the present study, using high-throughput bimolecular fluorescence complementation (BiFC) analysis, we identified that stress-inducible protein-1 (STIP1) interacts with GOLPH3 and confirmed the interaction using co-localization and co-immunoprecipitation. The levels of GOLPH3 and STIP1 in PDAC tissues and adjacent non-cancerous pancreatic tissues were determined using immunohistochemistry (IHC) and quantitative real-time reverse transcription PCR. Real-time Quantitative-telomere repeat amplification (Q-TRAP) was applied to detect relative telomerase activity, and cell proliferation was measured when small interfering RNAs targeting GOLPH3 or STIP1 were transfected into PDAC cell lines. BALB/c nude mice were used to assess tumor growth inhibition of BXPC3 cells stably transfected with GOLPH3 short hairpin RNA. In summary, GOLPH3 was found to interact with STIP1 and both proteins were overexpressed and co-localized in PDAC tissues and cell lines. Moreover, suppression of GOLPH3 expression using shRNAs in PANC1 and BXPC3 cells inhibited tumor cell proliferation both in vitro and in vivo. Mechanistically, GOLPH3 interacts with STIP1 to activate telomerase reverse transcriptase (hTERT) and telomerase activity by c-Myc, and then upregulates cell cycle-related signaling proteins, including cyclin D1, to promote tumor cell growth, suggesting that disrupting the interaction between STIP1 and GOLPH3 would be a promising new strategy to treat PDAC.

Cordycepin enhances the chemosensitivity of esophageal cancer cells to cisplatin by inducing the activation of AMPK and suppressing the AKT signaling pathway.

Although cisplatin (cDDP), is a first-line chemotherapy drug for esophageal cancer, it still has the potential to develop drug resistance and side effects. There is increasing evidence that cordycepin can work synergistically with other chemotherapy drugs. Therefore, we investigated whether combination therapy of cordycepin and cDDP may enhance the therapeutic effect of cDDP. We performed a series of functional tests to study the effect of medical treatment on esophageal cancer cells. We then used GO analysis to examine the pathways affected by treatment with cordycepin and cDDP. Next, we observed changes in the abundance of the selected pathway proteins. The in vivo animal model supported the results of the in vitro experiments. Co-treatment with cordycepin and cDDP inhibited cell growth, migration, and metastasis, as well as induced apoptosis. Cordycepin was found to effectively enhance activation of AMPK and inhibited activity of AKT. In all treatment groups, the expression levels of p-PI3K, p-Akt, p-p70S6K, Caspase-3, and Bcl-2 were significantly reduced, while the expression levels of p-AMPK, cleaved Caspase-3, and Bax increased, and the total levels of Akt, PI3K, and p70S6K levels remained unchanged. Overall, cordycepin was found to enhance the chemical sensitivity of esophageal cancer cells to cisplatin by inducing AMPK activation and inhibiting the AKT signaling pathway. Combination therapy of cordycepin and cisplatin represent a novel potential treatment of esophageal cancer.

Effect and mechanism of the CACNA2D1-CGRP pathway in osteoarthritis-induced ongoing pain.

This study built an OA model in rats by monosodium iodoacetate (MIA) injection to determine the effects and mechanism of the voltage-dependent calcium channel subunit alpha-2/delta-1 (CACNA2D1)-calcitonin gene-related protein (CGRP) pathway in osteoarthritis (OA)-induced ongoing pain. CACNA2D1 expression was measured by qPCR assay, western blotting assay, and immunofluorescence. Pain behaviors in rats were assessed with the measurement of thermal paw withdrawal latency (PWL) and mechanical paw withdrawal threshold (PWT). The expression of CACNA2D1, neuropeptide Y (NPY), activating transcription factor 3 (ATF3), CGRP, protein kinase A (PKA), phosphorylated (p)-PKA, adenylyl cyclase (AC), protein kinase C (PKC), p-PKC, phospholipase C (PLC), and mitogen-activated protein kinase (MAPK) signaling pathway proteins were measured, OA rats had higher CACNA2D1 expression than normal rats. Knockdown of CACNA2D1 led to the elevation of the pain threshold of OA rats, and CACNA2D1 over-expression decreased the pain threshold of normal rats. Moreover, CACNA2D1 over-expression inhibited the expression of CGRP, up-regulated the expressions of NPY, ATF3, p-PKA, AC, p-PKC, PLC, p-Jun N-terminal kinase (JNK), and p-p38, and had no significant effect on phosphorylated extracellular signal-regulated kinase (p-ERK) expression in vivo and in vitro. Using this model of MIA-induced OA, we demonstrated that CACNA2D1 might be involved in the process of pain by modulating the CGRP and AC-PKA/PKC/MAPK signaling pathways in the dorsal root ganglion.

HLA class I restricted epitopes prediction of common tumor antigens in white and East Asian populations: Implication on antigen selection for cancer vaccine design.

Tumor antigens processed and presented by human leukocyte antigen (HLA) Class I alleles are important targets in tumor immunotherapies. Clinical trials showed that presence of CD8+ T cells specific to tumor associated antigens (TAAs) and tumor neoantigens is one of the main factors resulting in tumor regression. Affinity prediction of tumor antigen epitopes to HLA is an important reference index for peptide selection, which is highly individualized. In this study, we selected 6 CTAs (cancer-testis antigens) commonly used in cancer immunotherapy and top 95 hot mutations from the Cancer Genome Atlas for analyzing potential epitopes with high affinities to the common HLA class I molecules in white and East Asian population, respectively. The results showed that the overall difference in CTAs epitope prediction is small between the two populations. Meanwhile, there is a linear relationship between the CTAs peptide length and the relative overall epitope occurrence. However, the difference is bigger for epitopes prediction of missense mutations between the two populations. It is worth noting that, both in the two populations, the single point mutations with the highest incidences have the lowest epitope occurrences while the mutations with the highest epitope occurrences are with low mutation incidences. This may be the result of long-term selection by the host immunosurveillance. Frameshift/inframe indel mutation neoantigens are between CTAs and spot mutation neoantigens in the relationship between peptide length and predicted epitope number. Our results help provide clues for tumor antigen and epitope selection in cancer vaccine design.

Cordycepin Reverses Cisplatin Resistance in Non-small Cell Lung Cancer by Activating AMPK and Inhibiting AKT Signaling Pathway.

Cisplatin (DDP) is the first-line chemotherapeutic agent against lung cancer. However, the therapeutic effect of DDP loses over time due to the acquired drug resistance in non-small cell lung cancer (NSCLC) cells. In recent years, the role of the traditional Chinese medicine (TCM) cordycepin (Cor) in cancer treatment has been attracting attention. However, the effects of Cor on DDP resistance in NSCLC are unclear. In the present study, we aimed to investigate the effects of Cor in combination with DDP on cell proliferation and apoptosis in NSCLC and explore possible underlying mechanisms. The cell proliferation and apoptosis were analyzed in NSCLC parental (A549) and DDP-resistant (A549DDP) cells treated with DDP alone or in combination with Cor both in vitro and in vivo. Different genes and signaling pathways were investigated between DDP-sensitive and DDP-resistant A549 cells by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The perturbations of the MAPK and PI3K-AKT signaling pathways were evaluated by Western blot analysis. Our data showed that Cor markedly enhanced DDP inhibition on cell proliferation and promotion of apoptosis compared to the DDP-alone group in both A549 and A549DDP cells. The synergic actions were associated with activation of AMPK; inhibition of AKT, mTOR, and downstream P709S6K; and S6 phosphorylation in the AKT pathway compared with DDP alone. Collectively, combination of Cor and DDP has a synergistic effect in inhibiting proliferation and promoting apoptosis of NSCLC cells in the presence or absence of DDP resistance. The antitumor activity is associated with activation of AMPK and inhibition of the AKT pathway to enhance DDP inhibition on NSCLC. Our results suggested that Cor in combination with DDP could be an additional therapeutic option for the treatment of DDP-resistant NSCLC.