Efficacy and safety of XELOX combined with anlotinib and penpulimab vs XELOX as an adjuvant therapy for ctDNA-positive gastric and gastroesophageal junction adenocarcinoma: a protocol for a randomized, controlled, multicenter phase II clinical trial (EXPLORING study).

Background: The efficacy of current adjuvant chemotherapy for gastric adenocarcinoma/gastroesophageal junction adenocarcinoma (GA/GEJA) leaves much to be desired. ctDNA could serve as a potential marker to identify patients who are at higher risk of recurrence. Reinforcing standard adjuvant chemotherapy with immunotherapy has already been indicated to significantly improve clinical outcome, albeit such evidence is rare in GA/GEJA. Here, we intend to explore the clinical benefit of the reinforcement of adjuvant immunotherapy and antiangiogenics alongside with chemotherapy in patients who are deemed in high risk of recurrence by ctDNA analysis, which might shed light on further improvements in adjuvant therapy for GA/GEJA. Methods/Design: This study is designed as a prospective, multicenter, randomized, controlled phase II study in patients histologically or cytologically diagnosed with GA/GEJA who underwent D2 gastrectomy and achieved R0 or R1 resection. From February 2022, a total of 300 stage III patients will be enrolled and subjected according to ctDNA sequencing results, and those with positive results will subsequently be randomized 1:1 to arm A or B. Patients in arm A will receive anlotinib, penpulimab and XELOX for 6-8 cycles, maintained with anlotinib and penpulimab for up to 1 year, while patients in arm B will receive XELOX alone for 6-8 cycles. ctDNA-negative patients will be assigned to arm C, and patients who are ctDNA positive but failed in randomization will be assigned to arm D. Patients in arms C and D will receive the investigator's choice of therapy. The primary endpoint is the median disease-free survival (DFS) of arm A versus arm B determined via CT/MRI imaging. Secondary endpoints include the DFS of ctDNA positive patients versus ctDNA negative patients, the 2- and 3-year DFS rates, overall survival (OS), the impact of hallmark molecules on the treatment response, adverse events (AEs), and the impact of nutrition status or exercise on recurrence. Discussion: We expect that ctDNA would be a strong prognostic factor and ctDNA-positive patients are at higher risk of relapse than ctDNA-negative patients. The addition of anlotinib and penpulimab to XELOX, may contribute to delaying relapse in ctDNA-positive patients. Trial registration: https://www.clinicaltrials.gov, identifier NCT05494060.

Immuno-genomic-radiomics to predict response of biliary tract cancer to camrelizumab plus GEMOX in a single-arm phase II trial.

Background & Aims: Immunotherapy is an option for the treatment of advanced biliary tract cancer (BTC), although it has a low response rate. In this post hoc analysis, we investigated the predictive value of an immuno-genomic-radiomics (IGR) analysis for patients with BTC treated with camrelizumab plus gemcitabine and oxaliplatin (GEMOX) therapy. Methods: Thirty-two patients with BTC treated with camrelizumab plus GEMOX were prospectively enrolled. The relationship between high-throughput computed tomography (CT) radiomics features with immuno-genomic expression was tested and scaled with a full correlation matrix analysis. Odds ratio (OR) of IGR expression for objective response to camrelizumab plus GEMOX was tested with logistic regression analysis. Association of IGR expression with progression-free survival (PFS) and overall survival (OS) was analysed with a Cox proportional hazard regression. Results: CT radiomics correlated with CD8+ T cells (r = -0.72-0.71, p = 0.004-0.047), tumour mutation burden (TMB) (r = 0.59, p = 0.039), and ARID1A mutation (r = -0.58-0.57, p = 0.020-0.034). There was no significant correlation between radiomics and programmed cell death protein ligand 1 expression (p >0.96). Among all IGR biomarkers, only four radiomics features were independent predictors of objective response (OR = 0.09-3.81; p = 0.011-0.044). Combining independent radiomics features into an objective response prediction model achieved an area under the curve of 0.869. In a Cox analysis, radiomics signature [hazard ratio (HR) = 6.90, p <0.001], ARID1A (HR = 3.31, p = 0.013), and blood TMB (HR = 1.13, p = 0.023) were independent predictors of PFS. Radiomics signature (HR = 6.58, p <0.001) and CD8+ T cells (HR = 0.22, p = 0.004) were independent predictors of OS. Prognostic models integrating these features achieved concordance indexes of 0.677 and 0.681 for PFS and OS, respectively. Conclusions: Radiomics could act as a non-invasive immuno-genomic surrogate of BTC, which could further aid in response prediction for patients with BTC treated with immunotherapy. However, multicenter and larger sample studies are required to validate these results. Impact and implications: Immunotherapy is an alternative for the treatment of advanced BTC, whereas tumour response is heterogeneous. In a post hoc analysis of the single-arm phase II clinical trial (NCT03486678), we found that CT radiomics features were associated with the tumour microenvironment and that IGR expression was a promising marker for tumour response and long-term survival. Clinical trial number: Post hoc analysis of NCT03486678.

Development of a classification model and an immune-related network based on ferroptosis in periodontitis.

BACKGROUND AND OBJECTIVES: Periodontitis is an immunoinflammatory disease characterized by irreversible periodontal attachment loss and bone destruction. Ferroptosis is a kind of immunogenic cell death that depends on the participation of iron ions and is involved in various inflammatory and immune processes. However, information regarding the relationship between ferroptosis and immunomodulation processes in periodontitis is extremely limited. The purpose of this study was to investigate the correlation between ferroptosis and immune responses in periodontitis. METHODS: Gene expression profiles of gingivae were collected from the Gene Expression Omnibus data portal. After detecting differentially expressed ferroptosis-related genes (FRGs), we used univariate logistic regression analysis followed by logistic least absolute shrinkage and selection operator (LASSO) regression to establish a ferroptosis-related classification model in an attempt to accurately distinguish periodontitis gingival tissues from healthy samples. The infiltration level of immunocytes in periodontitis was then assessed through single-sample gene-set enrichment analysis. Subsequently, we screened out immune-related genes by weighted correlation network analysis and protein-protein interaction (PPI) analysis and constructed an immune-related network based on FRGs and immune-related genes. RESULTS: A total of 24 differentially expressed FRGs were detected, and an 8-FRG combined signature constituted the classification model. The established model showed outstanding discriminating ability according to the results of receiver operating characteristic (ROC) curve analysis. In addition, the periodontitis samples had a higher degree of immunocyte infiltration. Activated B cells had the strongest positive correlation while macrophages had a strong negative correlation with certain FRGs, and we found that XBP1, ALOX5 and their interacting genes might be crucial genes in the immune-related network. CONCLUSIONS: The FRG-based classification model had a satisfactory determination ability, which could bring new insights into the pathogenesis of periodontitis. Those genes in the immune-related network, especially hub genes along with XBP1 and ALOX5, would have the potential to serve as promising targets of immunomodulatory treatments for periodontitis.

Analysis of the Cytokine Expression in the Aqueous Humor of Individuals with BRVO-Associated Macular Edema.

Purpose: This study aimed to determine the expression levels of vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) in the aqueous humor of patients with macular edema (ME) caused by branch retinal vein occlusion (BRVO), as well as to investigate the relationship between the cytokines as mentioned earlier and best-corrected visual acuity (BCVA), ME, and the degree of ME from the molecular level. Methods: In a prospective observational study, fluorescein fundus angiography (FFA) and optical coherence tomography (OCT) were used to classify 58 patients with non-ischemic BRVO-ME into three groups according to the degree of ME: 14-mild, 17-moderate, and 27-severe. The specific concentration of IL-6, VEGF, ICAM-1, and VCAM-1 in the aqueous humor was detected using the BD CSCanto™ II Flow Cytometer (US). Spearman or Pearson correlation analysis was used to test the correlation between the levels of BCVA and severity of ME and the expression levels of IL-6, VEGF, ICAM-1, and VCAM-1 in the aqueous humor. Results: According to the obtained data, BCVA did not correlate with the severity of ME, and these four cytokines expression levels in patients' aqueous humor (P > 0.05). Moreover, BCVA did not correlate with mild, moderate, or severe ME as well (P > 0.05). However, the levels of these four cytokines were correlated with the severity of the ME. These underlined cytokines were linked to the mild, moderate, and severe degrees of ME. VEGF was also significantly correlated (r > 0.8, P < 0.0001) with the severity of ME. Conclusions: This study suggests that the severity of ME in BRVO-ME patients is significantly correlated with the expression levels of IL-6, VEGF, ICAM-1, and VCAM-1 in the aqueous humor. Lowering the level of disease-associated cytokines may potentially reduce the degree of ME. Therefore, an in-depth study of the levels and the relationship may provide some evidence for the pathogenesis, treatment, and prevention of BRVO-ME.

Sensitive and rapid determination of heat shock protein 70 using lateral flow immunostrips and upconversion nanoparticle fluorescence probes.

Heat shock protein 70 (Hsp70), belonging to the heat shock protein (HSP) family, is reported to be a potential diagnostic biomarker. In this work, a lateral flow immunostrip was fabricated for the sensitive and rapid determination of Hsp70 by the incorporation of fluorescence and upconversion nanoparticle probes. The upconversion nanoparticles (UCNPs, size ∼39 nm, λex = 980 nm; λem = 540 nm) consisting of a NaYF4:Yb/Er core and polyacrylic acid-modified shell were covalently coupled with Hsp70 antibodies to form the signal probe, which was characterized by dynamic light scattering and zeta potential analyses. The lateral flow assay (LFA) was constructed based on the sandwich-type immunoassay using a sample pad, a test pad, and an adsorption pad on a PVP backing. Hsp70 antibody, IgG antibody and the signal probe were separately dropped on the test zone, the control zone of the test pad, and the sample pad, respectively. In the sandwich LFA, since two antibodies bind to Hsp70 antigenic epitopes, i.e. specific binding, it provided superior specificity and high sensitivity, making it an ideal sensing platform for complex samples like serum Hsp70 samples. The important parameters for the preparation of the lateral flow immunostrips were optimized. Under the optimized conditions, Hsp70 can be detected using the increased fluorescence intensity of UCNPs with a wide linear range from 0.11 to 12 ng mL-1, low detection limit of 0.06 ng mL-1, small sample volume (120 µL), short assay time (15 min) and good reproducibility. The fluorescence method was successfully applied in the determination of Hsp70 in serum samples with good recovery. By combining the accessibility of the lateral flow immunostrips and upconversion nanoparticles, the fluorescence method can serve as a point-of-care testing method for protein assays with high sensitivity and fast detection.

Case fatality rate of the adult in-patients with COVID-19 and digestive system tumors: A systematic review and meta-analysis.

BACKGROUND: During the coronavirus disease 2019 (COVID-19) pandemic, endoscopic screening for gastrointestinal tumors was suspended or delayed in most countries. Thus, our study aimed to quantify the impact of COVID-19 on the clinical outcomes of patients with digestive system tumors through a systematic review and meta-analysis. METHODS: We systematically searched the PubMed, Web of Science, Cochrane Library, and Embase databases as of March 7, 2021 to identify the case fatality rate (CFR) of COVID-19 patients diagnosed with digestive system tumors. A random-effects model was used for meta-analysis, I2 was used to assess heterogeneity, and funnel plot was used to assess publication bias. RESULTS: A total of 13 studies were included, involving 2943 tumor patients with COVID-19, of which 871 were digestive system tumors, and the CFR was 24% (95% CI, 18%-30%; I2 = 55.7%). The mortality rate of colorectal cancer was 21% (95% CI, 14%-27%; I2 = 0.0%), gastric cancer was 25% (95% CI, 6%-45%; I2 = 0.0%), and hepatobiliary cancer was 29%. In general, there was no significant difference in the CFR of digestive system tumors. CONCLUSION: The combined CFR of digestive system tumors and COVID-19 patients was 24%, which is much higher than that of the general population. Under the premise of fully complying with the international guidelines to limit the spread of COVID-19, we call for the resumption of endoscopic screening programs and selective surgery as soon as possible. REGISTRATION INFORMATION: PROSPERO registration no. CRD42021248194.

Development of a lncRNA-based prognostic signature for oral squamous cell carcinoma.

BACKGROUND: We aimed to establish a long noncoding RNA (lncRNA)-based signature for accurately predicting prognosis and guiding the personalized clinical management of oral squamous cell carcinoma (OSCC). METHODS: OSCC RNA sequencing profiles were acquired from The Cancer Genome Atlas and Gene Expression Omnibus. Univariate Cox regression, least absolute shrinkage and selection operator (LASSO) and multivariate Cox regression analyses were performed to construct a lncRNA-based prognostic signature. Kaplan-Meier survival analysis, receiver operating characteristic (ROC) curves and calibration curves were used to assess the effectiveness and accuracy of the signature. Additionally, we conducted single-sample gene-set enrichment analysis to infer the different degrees of immunocyte infiltration. Weighted correlation network analysis, enrichment analysis and Spearman's correlation analysis were implemented to screen immune-related genes that interact with the lncRNA signature. RESULTS: In total, 14 lncRNAs were defined as potential prognostic biomarkers. Based on these lncRNAs, patients were divided into low- and high-risk subgroups with different survival times (p < 0.001). In addition, the reliability of the prognostic signature was verified by Kaplan-Meier analysis, ROC analysis and calibration curves. Patients in the low-risk group exhibited more significant immune cell infiltration. Simultaneously, a potential regulatory network consisting of eight lncRNAs and 159 protein-coding genes in the top 10 immune-related biological process terms was constructed. CONCLUSIONS: Our findings suggested that the 14-lncRNA signature has satisfactory performance in predicting the prognosis of OSCC, thereby providing new insights to the pathogenesis, clinical patient management and therapeutic intervention. The different immune cell infiltration statuses of OSCC patients may encourage immunotherapy.

Low-dose apatinib combined with camrelizumab and the SOX regimen in the neoadjuvant treatment of locally advanced gastric/gastroesophageal junction adenocarcinoma (SPACE-neo): a protocol for an open-label, single-arm, clinical trial.

Background: Neoadjuvant chemotherapy with S-1 plus oxaliplatin (SOX regimen) has shown promising results in pathological response rate and survival rate in patients with locally advanced resectable gastric cancer (LAGC). We previously carried out the SPACE study to assess efficacy and safety of low-dose apatinib combined with camrelizumab and the SOX regimen as a first-line treatment of advanced gastric/gastroesophageal junction adenocarcinoma (AGC/GEJC). The preliminary results demonstrated a high objective response rate. However, the SPACE study was conducted in patients with AGC, but the efficacy of LAGC patients is not yet known. The SPACE-neo study is designed to investigate whether this combination could improve outcomes in patients with locally advanced gastric/gastroesophageal junction cancer (LAGC/GEJC) as neoadjuvant therapy. Methods: SPACE-neo is a prospective, open-label, single-arm study conducted in China at the First Affiliated Hospital of Nanjing Medical University (Jiangsu Province Hospital). Thirty-two patients with human epidermal growth factor receptor 2 (HER2)-negative or HER2-unknown LAGC/GEJC confirmed by histopathology or cytology will be recruited. Included patients shall be clinically staged as M0 and either T3 to T4 or N+ assessed by ultrasound endoscopy and thoracoabdominal-enhanced computed tomography or magnetic resonance imaging. The patients will receive three cycles of this combined regimen as a neoadjuvant treatment. Each patient will receive screening visits within 2 weeks before the first cycle and planned visits before every cycle of treatment. Key monitoring data include imaging data, pathological findings, and adverse events associated with neoadjuvant and surgical treatment. The primary endpoints are major pathological response (MPR) and safety. MPR is the proportion of patients whose residual tumor cells make up less than 10% of the primary tumor from among the total cohort. Clopper-Pearson method will be used to estimate the 95% confidence interval of MPR and safety data will be reported as descriptive statistical analysis. Discussion: The SPACE-neo trial aims to evaluate the safety and preliminary efficacy of this regimen in the neoadjuvant treatment of LAGC/GEJC. It is hoped that the study can achieve a higher pathological response rate and longer survival rate. Trial Registration: ChiCTR.gov.cn: ChiCTR2100049305.

Genomic alterations in biliary tract cancer predict prognosis and immunotherapy outcomes.

BACKGROUND: Recently, immunotherapy with immune checkpoint inhibitors (ICIs) has shown promising efficacy in biliary tract cancer (BTC), which includes gallbladder cancer (GBC) and cholangiocarcinoma (CHOL). Understanding the association between immunotherapy outcomes and the genomic profile of advanced BTC may further improve the clinical benefits from immunotherapy. METHODS: Genomic tumor DNA was isolated from 98 Chinese patients with advanced BTC and used for targeted next-generation sequencing of 416 cancer-related genes to identify the genomic alterations common to advanced BTC. Thirty-four patients had received ICI camrelizumab plus gemcitabine and oxaliplatin (from the NCT03486678 trial) as a first-line treatment. Tumor-infiltrating immune cells were evaluated using immunofluorescence staining. RESULTS: KRAS and TP53 mutations were much more frequent in the advanced-stage BTC cohort than in other cohorts with mostly early stage disease. Specifically, KRAS-TP53 co-mutations were favored in advanced CHOL, with a favorable response to immunotherapy, while single KRAS mutations predicted poor prognosis and immunotherapy outcomes for CHOL. Compared with GBC, CHOL had more mutations in genes involved in KRAS signaling; a high mutation load in these genes correlated with poor immunotherapy outcomes and may subsequently cause inferior immunotherapy outcomes for CHOL relative to GBC. Furthermore, a genomic signature including 11 genes was developed; their mutated subtype was associated with poor prognosis and immunotherapy outcomes in both CHOL and GBC. Transcriptome analyses suggested immune dysfunction in the signature mutated subtype, which was validated by tumor microenvironment (TME) evaluation based on detection of immune cell infiltration. Importantly, the signature wild-type subtype with favorable TME may be an advantageous population of immunotherapy. CONCLUSIONS: Genomic alterations in advanced BTC were associated with specific prognosis and immunotherapy outcomes. Combining genomic classification with TME evaluation further improved the stratification of immunotherapy outcomes.

Agarose-based spheroid culture enhanced stemness and promoted odontogenic differentiation potential of human dental follicle cells in vitro.

Human dental follicle cells (HDFCs) are an ideal cell source of stem cells for dental tissue repair and regeneration and they have great potential for regenerative medicine applications. However, the conventional monolayer culture usually reduces cell proliferation and differentiation potential due to the continuous passage during in vitro expansion. In this study, primary HDFC spheroids were generated on 1% agarose, and the HDFCs spontaneously formed cell spheroids in the agarose-coated dishes. Compared with monolayer culture, the spheroid-derived HDFCs exhibited increased proliferative ability for later passage HDFCs as analysed by Cell Counting Kit-8 (CCK-8). The transcription-quantitative polymerase chain reaction (qRT-PCR), western blot and immunofluorescence assay showed that the expression of stemness marker genes Sox2, Oct4 and Nanog was increased significantly in the HDFC spheroids. Furthermore, we found that the odontogenic differentiation capability of HDFCs was significantly improved by spheroid culture in the agarose-coated dishes. On the other hand, the osteogenic differentiation capability was weakened compared with monolayer culture. Our results suggest that spheroid formation of HDFCs in agarose-coated dishes partially restores the proliferative ability of HDFCs at later passages, enhances their stemness and improves odontogenic differentiation capability in vitro. Therefore, spheroid formation of HDFCs has great therapeutic potential for stem cell clinical therapy.

Current Progress and Future Perspectives of Immune Checkpoint Inhibitors in Biliary Tract Cancer.

Biliary tract cancer (BTC) is an uncommon and aggressive neoplasm, with most patients presenting in an advanced stage. Systemic chemotherapy is the limited treatment available but is unsatisfactory, while targeted therapy is still awaiting validation from clinical trials. Given the potential effect of immune checkpoint inhibitors (ICIs) in the treatment of BTC, this review aims to summarize the evidence-based benefits and predictive biomarkers for using inhibitors of cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) ligand, or programmed cell death protein-1 and its ligand (PD-1 and PD-L1) as monotherapy or combined with other anti-tumor therapies, while also pointing out certain pitfalls with the use of ICIs which need to be addressed.

Lateral flow immunostrips for the sensitive and rapid determination of 8-hydroxy-2'-deoxyguanosine using upconversion nanoparticles.

Lateral flow immunostrips were newly designed and a sensitive and rapid fluorometric method for the determination of 8-hydroxy-2'-deoxyguanosine (8-OHdG) as a model target of small biomarker molecules was developed. The upconversion nanoparticles (UCNPs, NaYF4:Yb/Er core, and polyacrylic acid (PAA)-modified shell, size ~ 39 nm, excitation wavelength = 980 nm; emission wavelength = 540 nm) were employed as fluorescence signal material. The 8-OHdG antibody (Ab) was taken as the recognition probe while UCNP-labeled Ab was taken as the signal probe. Bovine serum albumin (BSA) was designed as carrier protein for 8-OHdG to form 8-OHdG-BSA conjugate as the capture probe. The lateral flow immunostrips were prepared by laminating a sample pad (glass fiber membrane), a test pad (nitrocellulose membrane), and adsorption pad (filter paper) on PVP backing. The capture probe was immobilized on the test zone while an IgG antibody taken as the control probe was immobilized on the control zone. When the signal probe and the sample were in sequence loaded on the sample pad, 8-OHdG analyte bound with the signal probe, and then the excess of the signal probe move along the strip and is collected by the capture probe on the test zone while the remnant signal probe is collected by the control probe on the control zone. The signal probe and capture probe were synthesized and characterized. The fluorescence intensity on the test zone was inversely proportional to the concentration of 8-OHdG for the quantitative determination while the fluorescence emission on the control zone was observed to validate the assay. The developed method showed a wide linear range from 0.10 to 10 nM, a quite low detection limit of 0.05 nM, small sample volume requirement (100 µL), short assay time (15 min), and good method reproducibility (RSD = 4.4%, nine immunostrips). Graphical abstract Schematic illustration of the configuration and measurement principle of lateral flow fluorescence immunostrip for 8-OHdG: (a) configuration; (b) preparation: load of capture probe (BSA-8-OHdG, 2 µL) on test zone; load of control probe (IgG Ab, 2 µL) on control zone; load of signal probe (UCNP-Ab, 16 µL) on sample pad; (c) measurement: load of sample (8-OHdG, 100 µL) on sample pad, collection, and measurement.

Deoxyelephantopin decreases the release of inflammatory cytokines in macrophage associated with attenuation of aerobic glycolysis via modulation of PKM2.

Growing evidence suggests that activated immune cells undergo metabolic reprogramming in the regulation of the innate inflammatory response. Remarkably, macrophages activated by lipopolysaccharide (LPS) induce a switch from oxidative phosphorylation to aerobic glycolysis, and consequently results in release of proinflammatory cytokines. Pyruvate Kinase M2 (PKM2) plays a vital role in the process of macrophage activation, promoting the inflammatory response in sepsis and septic shock. Deoxyelephantopin (DET), a naturally occurring sesquiterpene lactone from Elephantopus scaber, has been shown to counteracts inflammation during fulminant hepatitis progression, but the underlying mechanism remains unclear. Here, we studied the function of the DET on macrophage activation and investigated the anti-inflammatory effects of DET associated with interfering with glycolysis in macrophage. Our results first demonstrated that DET attenuates LPS-induced interleukin-1ß (IL-1ß) and high-mobility group box 1 (HMGB1) release in vitro and in vivo and protected mice against lethal endotoxemia. Furthermore, DET decreased the expression of pyruvate dehydrogenase kinase 1 (PDK1), glucose transporter 1(GLUT1), lactate dehydrogenase A (LDHA), and reduced lactate production dose-dependently in macrophages. Moreover, we further revealed that DET attenuates aerobic glycolysis in macrophages associated with regulating the nuclear localization of PKM2. Our results provided a novel mechanism for DET suppression of macrophages activation implicated in anti-inflammatory therapy.

A Defined Combination of Four Active Principles From the Danhong Injection Is Necessary and Sufficient to Accelerate EPC-Mediated Vascular Repair and Local Angiogenesis.

Many compounds in Chinese medicine formulae, including Danhong injection (DHI) formulae, are capable of stimulating angiogenesis and promoting vascular repair, but their chemical basis and action mechanisms remain poorly defined. The aim of this study is to determine the minimal native chemical composition of DHI for the pro-angiogenesis activity and to evaluate its contribution from local endothelial cells (ECs) and bone marrow-derived endothelial progenitor cells (EPCs). Our study demonstrated that the action of DHI in accelerating the recovery of hindlimb blood flow in a ischemic rat model was attributable to its local CXCR4-mediated pro-angiogenesis activity in mature endothelial cells, as well as to its ability to promote the proliferation, migration, adhesion, and angiogenesis of EPCs via integrated activation of SDF-1α/CXCR4, VEGF/KDR, and eNOS/MMP-9 signal pathways. Combination experiments narrowed down the angiogenic activity into a few components in DHI. Reconstitution experiment defined that a combination of tanshinol, protocatechuic aldehyde, salvianolic acid B, and salvianolic acid C in their native proportion was necessary and sufficient for DHI's angiogenic activity. Compared with the full DHI, the minimal reconstituted four active principles had the same effects in promoting tube formation in vitro, improving perfusion and recovery of ischemic limb, and enhancing angiogenesis in ischemic mice post-hindlimb ischemia in vivo.

Quercetin Inhibits the Proliferation of Glycolysis-Addicted HCC Cells by Reducing Hexokinase 2 and Akt-mTOR Pathway.

Increased glycolysis in tumor cells is associated with increased risk of tumor progression and mortality. Therefore, disruption of glycolysis, one of the main sources of cellular energy supply, can serve as a target for suppressing tumor growth and progression. Of note, hexokinase-2 (HK2) plays vital roles in glucose metabolism. Moreover, the expression of HK2 alters the metabolic phenotype and supports the continuous growth of tumor cells, making it an attractive target for cancer therapy. Quercetin (QUE), a bioactive flavonoid, has a profound anti-tumor effect on hepatocellular carcinoma (HCC), but the precise underlying mechanism of this effect is unclear. In the present study, we reported that QUE inhibited the proliferation of HCC cells that relied on aerobic glycolysis. We further found that QUE could decrease the protein levels of HK2 and suppress the AKT/mTOR pathway in HCC cells. In addition, QUE significantly restrained the growth of HCC xenografts and decreased HK-2 expression in vivo. Taken together, we have revealed that QUE suppresses the progression of HCC by inhibiting HK2-dependentglycolysis, which may have a promising potential to be an effective treatments for HCC, especially for those patients with high HK2 expression.

Identification of Key Genes and Pathways in Triple-Negative Breast Cancer by Integrated Bioinformatics Analysis.

PURPOSE: Triple-negative breast cancer refers to breast cancer that does not express estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor receptor 2 (Her2). This study aimed to identify the key pathways and genes and find the potential initiation and progression mechanism of triple-negative breast cancer (TNBC). METHODS: We downloaded the gene expression profiles of GSE76275 from Gene Expression Omnibus (GEO) datasets. This microarray Super-Series sets are composed of gene expression data from 265 samples which included 67 non-TNBC and 198 TNBC. Next, all the differentially expressed genes (DEGs) with p<0.01 and fold change ≥1.5 or ≤-1.5 were identified. RESULT: 56 upregulated and 151 downregulated genes were listed and the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analysis was performed. These significantly changed genes were mainly involved in the biological process termed prostate gland morphogenesis, inner ear morphogenesis, cell maturation, digestive tract morphogenesis, autonomic nervous system development, monovalent inorganic anion homeostasis, neural crest cell development, regulation of dendrite extension and glial cell proliferation, immune system process termed T cell differentiation, regulation of immune response, and macrophage activation. Genes are mainly involved in the KEGG pathway termed Oocyte meiosis. All DEGs underwent survival analysis using datasets from The Cancer Genome Atlas (TCGA) integrated by cBioPortal, of which amplification of SRY-related HMG-box 8 (SOX8), androgen receptor (AR), and Chromosome 9 Open Reading Frame 152 (C9orf152) were significantly negative while Nik Related Kinase (NRK) and RAS oncogene family 30 (RAB30) were positively correlated to the life expectancy (p<0.05). CONCLUSIONS: In conclusion, these pathways and genes identified could help understanding the mechanism of development of TNBC. Besides, SOX8, AR, C9orf152, NRK and RAB30, and other key genes and pathways might be promising targets for the TNBC treatment.

The effect of pre-process and transport strategies on survival, microbiologic, and physiologic of Patinopecten yessoensis.

The supply chain of shellfish is complex, with animals being subjected to several stressors during the depuration, temporary keeping, and waterless-low-temp transportation processing. In this paper, the recycled water system for depuration and temporary keeping was used to realize both depuration and temporary keeping of Patinopecten yessoensis. The samples were divided into three groups based on three different pre-process involved: samples in group 1 were depurated for 48 hr straight, whereas those in group 2 were first depured for 24 hr and then cooled for 24 hr; samples in group 3 was directly kept in a polyethylene insulation box. Then group 1 and group 2 were transported in a 3L polyethylene insulation box with ice packs (250 ml) to study the quality of transport based on the different pre-process. As a result, in group 1 (depuration for 48 hr), the first death occurred after 56 hr, and all shellfishes died after 102 hr with total bacterial density of 2,630 CFU/ml. In group 2 (depuration for 24 hr and temporary keeping for 24 hr), the first death occurred after 104 hr and the total number of bacteria was increasing steadily within 0-104 hr. After 120 hr, all shellfishes died with total bacterial density of 1,090 CFU/ml. In group 3 (directly transport), all shellfishes died in 64 hr. The total number of bacteria in groups 1 and 2 declined significantly in the depuration process. The bacteria number (p < 0.05) in group 3 was significantly different from that in groups 1 and 2. The crude protein, crude fat, and glycogen of all groups declined. However, compared to groups 1 and 3, the consumption of glycogen in group 2 (p < 0.05) was delayed by the gradual cooling procedure. Those results prove that the depuration and temporary keeping procedures can improve the sterilization of the bacteria. The survival rate is less sensitive to the temperature change. The results provide satisfactory references for the P. yessoensis' quality studies with depuration, temporary keeping, and waterless-low-temp transportation technologies.

Selected Phytoestrogens Distinguish Roles of ERα Transactivation and Ligand Binding for Anti-Inflammatory Activity.

Estrogen receptor α (ERα) is a ligand-activated transcriptional activator that is also involved vascular inflammation and atherosclerosis. Whether different ligands may affect this activity has not been explored. We screened a panel of phytoestrogens for their role in ERα binding and transcriptional transcription, and correlated the findings to anti-inflammatory activities in vascular endothelial cells stably expressing either a wild-type or mutant form of ERα deficient in its membrane association. Taxifolin and silymarin were "high binders" for ERα ligand binding; quercetin and curcumin were "high activators" for ERα transactivation. Using these phytoestrogens as functional probes, we found, in endothelial cells expressing wild-type ERα, the ERα high activator, but not the ERα high binder, promoted ERα nuclear translocation, estrogen response element (ERE) reporter activity, and the downstream gene expression. In endothelial cells expressing membrane association-deficient mutant ERα, the ERα nuclear translocation was significantly enhanced by taxifolin and silymarin, which still failed to activate ERα. Inflammation response was examined using the systemic or vascular inflammation inducers lipopolysaccharide or oxidized low-density lipoprotein. In both cases, only the ERα high activator inhibited nuclear translocation of nuclear factor κB, JNK, and p38, and the production of inflammatory cytokines IL-1ß and TNFα. We confirm a threshold nuclear accumulation of ERα is necessary for its transactivation. The anti-inflammatory activity of phytoestrogens is highly dependent on ERα transactivation, less so on the ligand binding, and independent of its membrane association. A pre-examination of phytoestrogens for their mode of ERα interaction could facilitate their development as better targeted receptor modifiers.

Homogeneous Immunosorbent Assay Based on Single-Particle Enumeration Using Upconversion Nanoparticles for the Sensitive Detection of Cancer Biomarkers.

Prostate-specific antigen (PSA) is an intercellular glycoprotein produced primarily by the prostate gland, which is commonly chosen as the initial target for the early diagnosis of prostate cancer. In this work, we demonstrate a simple yet sensitive sandwich-type single-particle enumeration (SPE) immunoassay for the quantitative detection of PSA in a flow chamber. The design is based on the luminescence resonance energy transfer (LRET) between upconversion nanoparticles (UCNPs) and gold nanoparticles (GNPs). The carboxyl group-functionalized UCNPs are conjugated with anti-PSA detection antibodies (Ab1) and serve as the luminescence energy donor, while GNPs are modified with anti-PSA capture antibodies (Ab2) and act as the energy acceptor. In the presence of target antigen (i.e., PSA), the specific immnuoreaction brings the donor and acceptor into close proximity, resulting in quenched luminescence. Through statistical counting of the target-dependent fluorescent particles on the glass slide surface, the quantity of antigens in the solution is accurately determined. The dynamic range for PSA detection in Tris-buffered saline (TBS) is 0-500 pM and the limit-of-detection (LOD) is 1.0 pM, which is much lower than the cutoff level in patients' serum samples. In the serum sample assay, comparable LOD was also achieved (i.e., 2.3 pM). As a consequence, this method will find promising applications for the selective detection of cancer biomarkers in clinical diagnosis.

[Effect of low-intensity pulsed ultrasound combined with triamcinolone acetonide on oral mucosal ulcer healing in syrian hamster].

PURPOSE: To evaluate the effect of low-intensity pulsed ultrasound（LIPUS） combined with triamcinolone acetonide on oral mucosal ulcer in syrian hamster in several ways, including healing time, contents of superoxide dismutase（SOD）and malondialdehyde (MDA). METHODS: Sixty syrian hamsters were randomly divided into 5 groups, including a baseline group (containing a normal baseline group and a model baseline group, n=6) and 4 experimental groups (LIPUS processing and drug use group, LIPUS group, drug group and a normal control group without any processing, n=12). Four experimental groups and model baseline group were given oxygen free radicals to model the oral mucosal ulcer. At 24 h after the last treatment, the healing time of ulcer, content of SOD and MDA were compared between each group. SPSS 20.0 software package was used for statistical analysis. RESULTS: Compared with LIPUS group,drug group and control group, the healing time of oral mucosal ulcer in LIPUS and drug combined group was shortened. At 24 h after the last treatment, the activity of SOD showed that the LIPUS and drug combined group[(2.32±0.30) U/mgprot] were significantly higher than the model baseline group[(1.48±0.29) U/mgprot], the LIPUS group[(1.83±0.15) U/mgprot], the drug group[(1.76±0.25) U/mgprot] and control group[(1.71±0.18) U/mgprot] (P<0.05). The results of MDA content showed that the LIPUS and drug combined group [(8.17±0.21) nmol/mgprot] were significantly lower than the model baseline group[(9.41±0.22) nmol/mgprot], the LIPUS group[(9.00±0.44) nmol/mgprot], the drug group [(9.04±0.43) nmol/mgprot] and control group[(9.03±0.46) nmol/mgprot] (P<0.05). After oral mucosal ulcer healing, the activity of SOD and MDA showed that the LIPUS and drug combined group, the LIPUS group, the drug group and control group were not significantly different from the normal baseline group (P>0.05). CONCLUSIONS: Low-intensity pulsed ultrasound combined with triamcinolone acetonide can effectively improve the activity of SOD and reduce the contents of MDA in ulcerated tissues, and therefore accelerate the process of ulcer healing．.