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In this study, Tartary buckwheat was fermented by Monascus purpureus, and the fermented Tartary buckwheat oil (FTBO) was extracted under supercritical CO2 conditions. γ-Oryzanol, fatty acids, antioxidant capacities, and non-target metabolomics were investigated, and the lipid-lowering and immune effects of FTBO on high-fat mice were evaluated. The results showed that γ-oryzanol content reached 27.09 mg/g; oleic acid content increased by 12.56%; FTBO improved the antioxidant capacities of DPPH, ABTS, and Hydroxyl compared with raw Tartary buckwheat oil (TBO); and 207 metabolites were identified, including carboxylic acids and its derivatives and fatty acyls. FTBO gavage in high-fat mice had a dose effect. Three dose groups reduced TC, TG, and LDL-C levels in the liver and plasma and increased HDL-C level in the liver. Among them, the high-dose group exhibited the most significant effect on reducing inflammatory factors of ET, TNF-α, IL-1β, and IL-6 in the liver, which were 18.85, 570.12, 50.47, and 26.22 pg/mL, respectively (p ˂ 0.05). Liver damage in mice was also effectively ameliorated. Therefore, fermented Tartary buckwheat by M. purpureus is an effective method to enhance its biological activity. FTBO can be used as a functional product to lower blood lipids and modulate immune activity.

Background Immune checkpoint blockade resistance narrows the efficacy of cancer immunotherapies, but the underlying mechanism remains elusive. Delineating the inherent mechanisms of anti-PD1 resistance is important to improve outcome of patients with advanced HCC. Method The level of cricTMEM181 was measured in HCC patients with anti-PD1 therapy by RNA sequencing and then confirmed by qPCR and Sanger sequencing. Immune status in tumor microenvironment of HCC patients or mice models was evaluated by flow cytometry and IHC. Exosomes from HCC cell lines were isolated by ultracentrifugation, and their internalization by macrophage was confirmed by immunofluorescence. The underlying mechanism of HCC-derived exosomal circTMEM181 to macrophage was confirmed by SILAC, RNA FISH and RNA immunoprecipitation. The ATP–ADO pathway amplified by HCC–macrophage interaction was evaluated through ATP, AMP and ADO measurement and macrophage-specific CD39 knockout mice. The role of circTMEM181 in anti-PD1 therapy and its clinical significance were also determined in our retrospective HCC cohorts. Results Here, we found that circTMEM181 was elevated in hepatocellular carcinoma (HCC) patients responding poorly to anti-PD1 therapy and in HCC patients with a poor prognosis after operation. Moreover, we also found that high exosomal circTMEM181 favored the immunosuppressive microenvironment and endowed anti-PD1 resistance in HCC. Mechanistically, exosomal circTMEM181 sponged miR-488-3p and upregulated CD39 expression in macrophages. Using macrophage-specific CD39 knockout mice and pharmacologic approaches, we revealed a novel mode of anti-PD1 resistance in HCC. We discovered that cell-specific CD39 expression in macrophages and CD73 expression in HCC cells synergistically activated the eATP–adenosine pathway and produced more adenosine, thereby impairing CD8+ T cell function and driving anti-PD1 resistance. Conclusion In summary, HCC-derived exosomal circTMEM181 contributes to immunosuppression and anti-PD1 resistance by elevating CD39 expression, and inhibiting the ATP–adenosine pathway by targeting CD39 on macrophages can rescue anti-PD1 therapy resistance in HCC. Graphical Abstract

To investigate the relationship between imaging findings and S100A4 overexpression in pancreatic ductal adenocarcinoma (PDAC) and to determine imaging biomarkers of S100A4 overexpression from whole-tumor evaluation with MRI and texture analysis. A total of 60 patients with pathologically confirmed PDAC were included in the study. All patients underwent preoperative abdominal contrast-enhanced MRI examination with Magnetom Aera (Siemens Healthcare, Germany, 1.5 T) at our institute. Whole-tumor evaluation including texture analysis was performed. Sections of specimens were reviewed, and the S100A4 expression status was quantitatively evaluated. Univariate and multivariate logistic regression analyses were conducted to find imaging biomarkers that could predict S100A4 overexpression. Twenty-four tumors (40.0%) had negative results for S100A4 overexpression, and 36 tumors (60.0%) exhibited overexpression. After univariate and multivariate analysis, distal pancreatic duct dilatation, T1WI_10th percentile and the enhancement rate difference between delayed phase (DP) and portal venous phase (PVP) were identified to predict S100A4 overexpression in PDAC independently (p = 0.009, 0.012 and 0.044), with odds ratios (ORs) of 0.102, 0.139 and 4.645, respectively. The area under the ROC curve (AUC) values were 0.715, 0.707 and 0.691. The AUC value of the proposed model was 0.877 with a sensitivity of 80.6% and specificity of 75.0%. A model including distal pancreatic duct dilatation, T1WI_10th percentile and the enhancement rate difference between the DP and PVP could predict S100A4 overexpression in PDAC as imaging biomarkers.

Adlay, as a traditional Chinese medicine, has been used in nourishing foods, which are rich in a variety of nutrients (special biological compounds). The study was designed to optimize the fermentation parameters of dehulled, polished and broken adlay fermented by Bacillus subtilis BJ3-2 with regard to tetramethylpyrazine (TMP) yield and fibrinolytic enzyme activity. Then the proximate and bioactive components of B. subtilis-fermented adlay were evaluated. Box–Behnken design results showed that the TMP yield was 6.93 mg/g DW (dried weight) of B. subtilis-fermented polished adlay, which was about 136 times higher than that of B. subtilis-fermented soybean (BSB). The fibrinolytic enzyme activity was 2236.17 U/g in B. subtilis-fermented dehulled adlay, and slightly less than in BSB. B. subtilis-fermented adlay contained higher fat, free amino acids and fatty acids contents but lower protein and starch contents than raw adlay. Except for coixol and coixan, the levels of γ-aminobutyric acid, triterpenes, phenolics, flavonoids and coixenolide in B. subtilis-fermented adlay increased by 14.05, 2.02, 2.31 and 1.36 times, respectively. The contents of phenolic acids including caffeic, gallic, catechinic and chlonogenic acids in the free phenolic extracts significantly increased (p

The physicochemical properties and antioxidant activity of B. subtilis‐fermented polished adlay (BPA) subjected to different drying methods (hot‐air drying, HAD; infrared‐radiation drying, IRD; vacuum drying, VD; microwave‐vacuum drying, MVD; and freeze‐vacuum drying, FVD) were evaluated in this study. Results showed FVD was ideal for maintaining the natural appearance and higher contents of proximate compositions, free fatty acids, tetramethylpyrazine (6.91 mg/g DW), coixol (0.62 mg/g DW), coixenolide (4.21% DW), coixan (35.10% DW), and triterpenoids (17.41 mg/g DW). The higher contents of total phenolics and flavonoids, stronger antioxidant activity, and higher color differences were observed in HAD and IRD samples. MVD displayed the shorter drying time, higher γ‐aminobutyric acid content, and higher retention ratios of tetramethylpyrazine (75.54%), coixol (87.10%), coixenolide (98.57%), and coixan (99.11%). Pearson's correlation coefficient exhibited that the positive correlation between the contents of phenolics and flavonoids and the antioxidant activities of all dried BPA samples was observed (R 2 > 0.881, p, The physicochemical properties and antioxidant activity of B. subtilis‐fermented polished adlay (BPA) treated by different drying methods were evaluated in this study. Through analysing comprehensive quality, microwave‐vacuum drying should be a potential preservation method to obtain high‐quality dried BPA for short drying time and high comprehensive quality.

Dehulled adlay was fermented with Bacillus subtilis BJ3-2, the anti-proliferative activities of the extracts from fermented dehulled adlay were investigated with six types of tumor cells, and then the bioactive components and the anti-proliferative mechanism were primarily explored. Results showed that all the extracts of B. subtilis-fermented dehulled adlay (BDA) and dehulled adlay (DA) had no inhibition effect on human embryonic kidney 239T cells. The anti-proliferative activities of the extracts from BDA against six types of tumor cells were almost always significantly higher than DA. Compared with others, the n-butanol extract of BDA (BDA-Nb) exhibited stronger anti-proliferative activities against human leukemia K562 cells and human non-small cell lung cancer A549 cells. Importantly, the anti-proliferative activity of fermented dehulled adlay against K562 cells was firstly discovered. Meanwhile, BDA-Nb was rich in tetramethylpyrazine, γ-aminobutyric acid, protocatechuic, 2,3,4-trihydroxybenzoic, chlorogenic, p-hydroxybenzoic, caffeic, trans-cinnamic, ferulic acids, and rutin. BDA-Nb induced the proliferative inhibition of K562 and A549 cells due to abnormal cell morphology, the increased cell population in G1 phase and apoptosis rate, the downregulation of Bcl-2, and the upregulation of Bax and caspase-3/8/9. These results indicate that dehulled adlay fermented with B. subtilis could be a potential therapeutic agent for leukemia and lung cancer.

What is already known about this topic? The Co-Administration of Multiple Vaccines were implemented in many countries and have been shown to significantly reduce many times of visiting the vaccination clinic. What is added by this report? It is the first time to calculate the cost of visiting vaccination clinic from transportation and work-absence for children’s families in Guangdong. What are the implications for public health practice? We demonstrated the importance of Co-Administration of Multiple Vaccines that reduce the vaccination cost of children’s families. The policy should be promoted as soon as possible.

The effects of five drying methods on kinetics, biocompounds and antioxidant activity of Bacillus subtilis-fermented dehulled adlay (BDA) were evaluated in this study. The Page model (MR = Ae−ktB) ...

Background: Our previous studies showed that tetraspanin CD151 was implicated in the progression of hepatocellular carcinoma (HCC), mainly depending on the formation of functional complexes with molecular partners, including Mortalin. In this study, we investigate the role of mortalin in CD151-depedent progression of HCCs. Methods: Immunofluorescent staining, western blot and quantitative real-time polymerase chain reaction (qRT-PCR) were used to investigate the expression and location of CD151 and Mortalin in four HCC cell lines with different metastatic ability. The relationship between Mortalin and CD151 was investigated in HCCLM3 cells using co-immunoprecipitation. CD151 or Mortalin expression in HCC cells were modified by transfection technology. Wound-healing assay and Transwell assay were used to assay the role of CD151 and Mortalin in cell migration and invasion. The expression and prognostic implication of CD151 and Mortalin in 187 cases of HCCs were analyzed. Results: Expression of Mortalin in HCC cells was positive related to their metastatic ability and its tendency was in line with the expression of CD151. Immunofluorescent staining showed that Mortalin was located in cytoplasm, while positive staining for CD151 was observed in cytoplasm and membrane of HCC cells. co-IP revealed that Mortalin formed a complex with CD151. Down-regulation of Mortalin induced a moderate decreased CD151 protein, but not CD151 mRNA, while inhibition of CD151 did not influence the expression of Mortalin at the level of both protein and mRNA. Interference of Mortalin significantly inhibited the invasion and migration of HCC cells with high CD151 expression and partially restored the invasion and migration of HCC cells induced by CD151 over-expression. Clinically, high Mortalin expression correlated with malignant phenotype of HCC, such as microvascular invasion (p=0.017) and tumor diameter (p=0.001). HCC patients expressing high Mortalin were tend to have higher expression of CD151. HCC patients expressing high level of CD151 showed the poorer prognosis in a Mortalin-dependent manner. Conclusions: Mortalin maybe stabilize of the structure of CD151-dependent tetraspanin-enriched microdomains and implicate in the progression of HCC.

Rationale: PD1/PD-L1 immune checkpoint inhibitors have shown promising results for several malignancies. However, PD1/PD-L1 signaling and its therapeutic significance remains largely unknown in intrahepatic cholangiocarcinoma (ICC) cases with complex etiology. Methods: We investigated the expression and clinical significance of CD3 and PD1/PD-L1 in 320 ICC patients with different risk factors. In addition, we retrospectively analyzed 7 advanced ICC patients who were treated with PD1 inhibitor. Results: The cohort comprised 233 patients with HBV infection, 18 patients with hepatolithiasis, and 76 patients with undetermined risk factors. PD-L1 was mainly expressed in tumor cells, while CD3 and PD1 were expressed in infiltrating lymphocytes of tumor tissues. PD1/PD-L1 signals were activated in tumor tissues, and expression was positively correlated with HBV infection and lymph node invasion. More PD1+ T cells and higher PD-L1 expression were observed in tumor tissues of ICC patients with HBV infection compared to patients with hepatolithiasis or undetermined risk factors. More PD1+ T cells and/or high PD-L1 expression negatively impacted the prognosis of patients with HBV infection but not those with hepatolithiasis. Multivariate analysis showed PD1/PD-L1 expression was an independent indicator of ICC patient prognosis. Advanced ICC patients with HBV infection and less PD1+ T cells tended to have good response to anti-PD1 therapy. Conclusion: Hyperactivated PD1/PD-L1 signals in tumor tissues are a negative prognostic marker for ICCs after resection. HBV infection- and hepatolithiasis-related ICCs have distinct PD1/PD-L1 profiles. Further, PD1+ T cells could be used as a biomarker to predict prognosis and assay the efficiency of anti-PD1 immunotherapy in ICC patients with HBV infection.

Lipophilic tocols, γ-oryzanol, and coixenolide in coix seed before and after fermentation by Monascus purpureus were determined. Antioxidant and anticancer activities of raw and fermented coix seed were evaluated using free-radical-scavenging assays and polyunsaturated fatty acid oxidation model, and human laryngeal carcinoma cell HEp2, respectively. Compared to the raw seed, the tocols, γ-oryzanol, and coixenolide contents increased approximately 4, 25, and 2 times, respectively, in the fermented coix seed. Especially, γ-tocotrienol and γ-oryzanol reached 72.5 and 655.0 μg/g in the fermented coix seed. The lipophilic extract from fermented coix seed exhibited higher antioxidant activity in scavenging free radicals and inhibiting lipid oxidation. The inhibitory concentrations for 50% cell survival (IC50) of lipophilic extract from fermented coix seed in inhibiting HEp2 cells decreased by 42%. This study showed that coix seed fermented by M. purpureus increased free and readily bioavailable lipophilic antioxidants and anticancer activity. Therefore, fermentation could enhance the efficacy of the health promoting function of coix seeds.

Objective Differentiation‐inducing therapy for tumors is a strategy that aims to induce the differentiation and maturation of cancer stem cells (CSCs). The differentiation‐inducing capacity of arsenic trioxide (ATO) in hepatocellular carcinoma (HCC) and the underlying mechanism were previously unknown. Methods In the present study, we explored the ATO‐induced differentiation of CSCs in HCC by detecting the expression of CSC‐related markers and tumorigenicity variation in vivo and in vitro. We developed a combined chemotherapeutic approach to HCC by characterizing the effects of combinatorial treatment with 5‐fluorouracil (5‐FU)/cisplatin and ATO in vitro and in patient‐derived xenograft models. Changes in gene expression patterns were investigated by gene microarray analysis. Results ATO effectively induced differentiation of CSCs by downregulation of CSC‐related genes and suppression of tumorigenicity capability. Combinatorial treatment with ATO and 5‐FU/cisplatin significantly enhanced therapeutic effects in HCC cells compared with the treatment with 5‐FU/cisplatin alone. Synergistic inhibition of the LIF/JAK1/STAT3 and NF‐kB signaling pathways by ATO and 5‐FU/cisplatin is a potential molecular mechanism underlying the differentiation effect. Conclusions ATO induced the differentiation of HCC CSCs and potentiated the cytotoxic effects of 5‐FU/cisplatin through synergistic inhibition of the LIF/JAK1/STAT3 and NF‐kB signaling pathways. These results offer new insights for the clinical treatment of HCC., Arsenic trioxide induces differentiation of cancer stem cells in hepatocellular carcinoma through inhibition of LIF/JAK1/STAT3 and NF‐kB signaling pathways synergistically

An intensive short-term chemotherapy regimen has substantially prolonged the overall survival of Burkitt’s lymphoma (BL) patients, which has been further improved by addition of rituximab. However, the inevitable development of resistance to rituximab and the toxicity of chemotherapy remain obstacles. We first prepared two BL cell lines resistant to rituximab-mediated CDC. Using a phosphorylation antibody microarray, we revealed that PI3K/AKT pathway contained the most phosphorylated proteins/hits, while apoptosis pathway that may be regulated by PKC displayed the greatest fold enrichment in the resistant cells. The PI3K/AKT inhibitor IPI-145 failed to reverse the resistance. In contrast, the pan-PKC inhibitor midostaurin exhibited potent antitumor activity in both original and resistant cells, alone or in combination with rituximab. Notably, midostaurin promoted apoptosis by reducing the phosphorylation of PKC and consequently of downstream Bad, Bcl-2 and NF-κB. Therefore, midostaurin improved rituximab activity by supplementing pro-apoptotic effects. In vivo, midostaurin alone powerfully prolonged the survival of mice bearing the resistant BL cells compared to rituximab alone treatments. Addition of midostaurin to rituximab led to dramatically improved survival compared to rituximab but not midostaurin monotherapy. Our findings call for further evaluation of midostaurin alone or in combination with rituximab in treating resistant BL in particular.

Aim To investigate the volatile flavour components (VFCs), microbiota and their correlations of three categories of sufu, a Chinese fermented soybean food. Methods and results The VFCs were analyzed by headspace solid-phase micro-extraction (HS-SPME) combined with gas chromatography and mass spectrometry. A total of 141 VFCs were tentatively identified, in which 12 esters, eight aldehydes, five alcohols and nine miscellaneous compounds were recognized as the characteristic VFCs through the relative odour activity value. The complex microbial community was revealed by Illumina MiSeq sequencing. Among the total 202 bacteria and 125 fungi species identified, 16 bacteria and 7 fungi species were revealed as dominant community members. Furthermore, the VFC-microbiota correlation was characterized by Spearman's rank correlation coefficients (ρ). Based on the positive VFC-microbiota correlation, two bacterial species and three fungal species were selected as potential flavour-producing microbiota. Conclusions A variety of VFCs and complicated microbiota were observed in the three categories of sufu. Lactococcus lactis, Sphingobacterium sp., Pichia fermentans, Kodamaea ohmeri and Saccharomyces rouxii were the potential flavour-producing microbiota. Significance and impact of the study The preliminary results would be useful for designing starter cultures to produce sufu with desirable sensory properties, consistent flavour and shorter ripening time. Furthermore, these results will also provide a new insight to improve the flavour quality of traditional fermented soybean food.

In this study, the physicochemical, taste, and functional changes in low-salt Sufu paste were investigated during fermentation as enhanced by a mixed starter (Pichia fermentans, Kodamaea ohmeri, and Lactococcus lactis subsp. lactis) and α-ketoglutarate. The total free amino acids increased from 0.02 mg/g dry matter to 12.63 mg/g dry matter, and monosodium glutamate-like was the major free amino acid group (3.89 mg/g dry matter), followed by bitter according to the taste characteristics. Functional components such as γ-aminobutyric acid, riboflavin (VB2), and puerarin significantly increased to 30.96, 4.97, and 12.03 mg/g dry matter, respectively, at the end of post-ripening (p

Amplification of the fibroblast growth factor receptor 1 (FGFR1) is believed to predict response to FGFR inhibitors. The aim of this study was to investigate the frequency and the prognostic impact of FGFR1 amplification in patients with resected esophageal squamous cell carcinoma (ESCC) by using fluorescent in situ hybridization. Microarrayed paraffin embedded blocks were constructed, and the cohort of tissues came from 506 patients with ESCC. FGFR1 high amplification (FGFR1high ) was defined by an FGFR1/centromere 8 ratio of ≥ 2.0, or average number of FGFR1 signals/tumor cell nucleus ≥ 6.0, or percentage of tumor cells containing ≥ 15 FGFR1 signals, or large cluster in ≥ 10% of cancer cells. FGFR1 low amplification was defined by ≥ 5 FGFR1 signals in ≥ 50% of cancer cells. Kaplan-Meier curves with log-rank tests and Cox proportional hazards model were used to analyze patients' survival. Among 506 patients, high amplification, low amplification, and disomy were detected in 8.7%, 3.6% and 87.7%, respectively. In general, the FGFR1high group trended towards worse disease-free survival (DFS) and overall survival (OS) compared to the FGFR1 low amplification/disomy (FGFR1low/disomy ) group (DFS, P=0.108; OS, P=0.112), but this trend was amplified for patients with DFS ≥ 30 months (DFS, P=0.009; OS, P=0.007). Furthermore, when patients were stratified into stage I-II and stage III-IV, the FGFR1high group directly presented with adverse DFS and OS than the FGFR1low/disomy group in stage I-II patients (DFS, P=0.019; OS, P=0.034), especially with DFS ≥ 30 months (DFS, P=0.002; OS, P=0.001). However, for patients in stage III-IV, FGFR1high had no effect on prognosis regardless of DFS time. FGFR1high occurs in a minority of ESCC, and it predicts delayed poor prognosis in stage I and II ESCC patients.

The nutritional, functional and tasty components of three categories of Sufu, divided by strains in the culture phase, were comprehensively investigated in this study. The levels of isoflavones, γ-aminobutyric acid (GABA), phytosterols and soyasaponin were 0.58-2.20, 7.46-57.95, 0.73-2.72 and 10.89-23.35 mg/g dry matter (DM), respectively. Glu was the most abundant of the 17 detected free amino acids (FAAs), followed by Phe, Leu, Val and Asp. Additionally, potential tasty peptide profiles were typed by the segment of molecular weight (MW) < 300 Da, ranging from 79.22% ± 4.12% to 95.24% ± 2.93%. The complex taste impression based on the electronic tongue showed that the bitterness intensity was the highest, which was followed by saltiness and the umami intensity. To some extent, different Sufu categories can be distinguished according to the electronic tongue. The results provide a theoretical basis for improving the quality control and standardization of the manufacturing process.

Adlay, as a traditional Chinese medicine, has been used in nourishing foods, which are rich in a variety of nutrients (special biological compounds). The study was designed to optimize the fermentation parameters of dehulled, polished and broken adlay fermented by Bacillus subtilis BJ3-2 with regard to tetramethylpyrazine (TMP) yield and fibrinolytic enzyme activity. Then the proximate and bioactive components of B. subtilis-fermented adlay were evaluated. Box–Behnken design results showed that the TMP yield was 6.93 mg/g DW (dried weight) of B. subtilis-fermented polished adlay, which was about 136 times higher than that of B. subtilis-fermented soybean (BSB). The fibrinolytic enzyme activity was 2236.17 U/g in B. subtilis-fermented dehulled adlay, and slightly less than in BSB. B. subtilis-fermented adlay contained higher fat, free amino acids and fatty acids contents but lower protein and starch contents than raw adlay. Except for coixol and coixan, the levels of γ-aminobutyric acid, triterpenes, phenolics, flavonoids and coixenolide in B. subtilis-fermented adlay increased by 14.05, 2.02, 2.31 and 1.36 times, respectively. The contents of phenolic acids including caffeic, gallic, catechinic and chlonogenic acids in the free phenolic extracts significantly increased (p

To retrospectively investigate the clinicopathological features and prognosis of early esophageal squamous cell neoplasm (ESCN) treated with endoscopic resection (ER), especially, to compare the prognosis in patients with sm2 cancer and non-sm2 cancer. From 2007 to 2013, 368 patients were included in our analysis. The patients were 252 (68.5 %) men and 116 (31.5 %) women with a median age of 61 (range 16–84 years) years. Hyperplasia, mild dysplasia, moderate dysplasia, severe dysplasia, m1, m2, m3, sm1, and sm2 were diagnosed in 47 (12.8 %), 27 (7.3 %), 34 (9.2 %), 61 (16.6 %), 54 (14.7 %), 38 (10.3 %), 63 (17.1 %), 12 (3.3 %), and 32 (8.7 %) cases. The mean (range) follow-up time was 29 (0–84) months. The cumulative overall 1-, 3-, and 5-year metachronous esophageal lesion rates were 4.1, 12.9, and 32.6 %. The incidence of lymph node or distant metastasis was 1.54 % in m3, 6.25 % in sm2, and 0 in other subgroups. The overall 1-, 3-, and 5-year survival rates were 99.5, 97.3, and 87.5 %. There was significant difference between sm2 and non-sm2 patients in metastatic rate (P = 0.021); however, no difference existed between m3 patients and sm2 patients (P = 0.252). The difference of metachronous esophageal lesion (P = 0.401) and survival (P = 0.634) between sm2 and non-sm2 patients was not obvious. Our study showed that ER was an effective and relatively safe treatment for superficial ESCN. ER is still appropriate in select sm2 patients. To monitor the second primary cancer in sm2 is necessary during the follow-up.

Phenolic, fatty acid, and phytosterol contents in sweet potato (SP) fermented by Lactobacillus acidophilus were evaluated and compared with those of raw and boiled SPs. The differences in the profiles and levels of phenolics between the raw and boiled SPs were not as significant as the differences between those and the fermented SP. The levels of caffeic acid and 3,5-dicaffeoylquinic acid in fermented SP were more than 4 times higher than those in raw and boiled SPs. Two phenolics, p-coumaric acid and ferulic acid, which were not detected in either raw or boiled SP, were found in fermented SP. The level of each fatty acid or phytosterol increased in fermented SP and decreased in boiled SP. Among the hydrophilic and lipophilic extracts obtained from raw and fermented SPs, the hydrophilic extract of fermented SP exhibited the highest capability of inhibiting cancer-cell PC-12 proliferation. However, each of the extracts had very low cytotoxicities to normal-monkey-kidney-cell growth. The results indicated that SP fermented by L. acidophilus significantly increased free antioxidant-rich phenolics and inhibited cancer-cell-proliferation activity without cytotoxicity to normal cells.

In the present study, we aimed to determine the prognostic impact and clinicopathological feature of FGF4 amplification in patients with esophageal squamous cell carcinoma (ESCC). Fluorescence in situ hybridization with FGF4 probe was analyzed using tissue microarray consisting of representative cores of 267 ESCC cases. FGF4 amplification was observed in 52.8% (141/267) of patients. Patients with FGF4 amplification showed a significantly shorter disease-free survival (DFS) or disease-specific overall survival (OS) compared with those without FGF4 amplification (both P < .05). Moreover, FGF4 amplification was an independent prognostic factor (DFS, P = .036; OS, P = .021) along with clinical stage and lymph node metastasis in multivariate analysis. Among stage I-II or III patients whose DFS was greater than or equal to 24 months (n = 125 or 32), patients with FGF4 amplification showed a significantly worse prognosis (OS, P = .027 or P = .010). Moreover, the survival curve of stage I-II patients with FGF4 amplification was identical to stage III patients without FGF4 amplification (DFS, P = .643; OS, P = .707). Taken together, FGF4 amplification was an independent prognostic factor in ESCC patients, and ESCC might have potentially been upstaged by FGF4 amplification. Therefore, FGF4 amplification in combination with clinical stage could be used as a relatively accurate predictor for the 5-year probability of death and recurrence for ESCC patients.

One paraffin block is routinely used for human epidermal growth factor receptor 2 (Her2/neu) immunohistochemistry (IHC) assessment. Here, we investigated if picking 2 paraffin blocks for Her2/neu evaluation on 1 slide is an economical, efficient, and practical method, which may reduce false negativity of Her2/neu IHC assessment due to intratumoral heterogeneity. A total of 251 gastric cancer (GC) patients were divided into a cohort using 1 tumor tissue paraffin block (single-block group, n = 132) and a cohort using dual tumor tissue paraffin blocks (dual-block group, n = 119) when evaluating Her2/neu expression status by IHC. In dual-block group, we combined the results from 2 different paraffin blocks and used the higher one as the final score. The number of IHC 1+, 2+, and 3+ specimens in the single-block group was 31 (23.5%), 40 (30.3%), and 19 (14.4%), respectively. The combined final IHC score in the dual-block group of 1+, 2+, and 3+ was 26 (21.8%), 34 (28.6%), and 23 (19.3%), respectively. Inconsistent Her2/neu expression between blocks was found in 36 (30.3%) cases in the dual-block group. The pooled data in the single-block group and the dual-block group indicated that, when using dual blocks, the Her2/neu-positive (3+) rate of GC was higher compared to that in the single-block group. Our results implied that using dual paraffin blocks to assess Her2/neu expression of GC may help identify more patients with Her2/neu-positive GC who could benefit from targeted therapy, by reducing false-negative rate of Her2 status assessment. This is an efficient, economical, and practical method for Her2/neu evaluation of GC.

The risk of developing hepatocellular carcinoma (HCC) is strongly associated with hepatitis B virus infection. Hepatic angiomyolipoma (AML), a rare benign tumor, is composed of a heterogeneous mixture of adipose cells, smooth muscle cells and blood vessels. Here, we report the case of a 44-year-old man who developed HCC with a concomitant hepatic AML and a cavernous hemangioma, in the absence of cirrhosis. To our knowledge, based on an extensive literature search using the www.pubmed.gov website, this is the first report of an HCC case with both concomitant AML and cavernous hemangioma at the same position in the liver. The presence of the hepatitis B surface antigen was detected, but the liver function was normal. Clinical and pathological data were collected before and during the treatment. Hepatic AML was diagnosed based on the typical histological characteristics and immunohistochemical staining, which revealed, a positive staining with a melanocytic cell-specific monoclonal antibody. There was no evidence of tuberous sclerosis complex in this patient. Although the HCC was poor- to moderately-differentiated, the characteristics of the AML and the cavernous hemangioma in this patient did not match any criteria for malignancy. Hepatectomy followed by transarterial chemoembolization treatment were effective therapeutic methods for the adjacent lesions in this patient. This case is an interesting coincidence.

Background: Excessive complement activation plays an important role in the pathogenesis of atherosclerosis (AS). We therefore wanted to investigate whether complement is activated in areas of AS by detecting the deposition of C3b/iC3b and membrane attack complex (MAC). We also analyzed the relationships between C3b/iC3b and MAC levels and the clinicopathological features of patients with AS. Methods: The sample comprised 79 patients who had been diagnosed with AS. Their levels of C3b/iC3b and MAC deposition were evaluated by immunohistochemistry (IHC). The results were translated into scores, and the patients’ clinical features were recorded. Results: Compared with normal arteries, significantly greater deposits of C3b/iC3b and MAC were found in AS arteries. In the group with more C3b/iC3b deposition, the ratio of patients with hypertension was higher. Moreover, in the group with more MAC deposition, the ratio of patients with hypertriglyceridemia was higher. Conclusions: The finding of C3b/iC3b and MAC deposition in atherosclerotic arteries points to the activation of complement. Greater amounts of C3b/iC3b and MAC deposition imply excessive complement activation, which can lead to the development of AS. Hypertension and hypertriglyceridemia may, respectively, contribute to the activation of complement C3 or the formation of MAC.

We undertook a study of tumour infiltrating lymphocytes (TILs) in a large and relatively homogeneous group of patients with completely resected esophageal squamous cell carcinoma (ESCC). Hematoxylin and eosin–stained sections of 235 ESCC tumours were evaluated for density of TILs in intratumoural (iTIL) and stromal compartments (sTIL). Foxp3+, CD4+, and CD8+ T cells in tumoural and stromal areas were evaluated by immunohistochemistry. Of the 235 tumours, high sTIL (>10%), and iTIL (>10%) were observed in 101 (43.0%) and 98 (41.7%), respectively. The median follow-up period was 36.0 months (95% CI 29.929–42.071). Univariate analysis revealed that sTIL (>10%), iTIL (>20%), vessels involvement, lymph node metastasis, and clinical stage were significantly associated with postoperative outcome. In multivariate analysis, high sTIL (HR: 0.664, P = 0.019 for Disease free survival; HR: 0.608, P = 0.005 for Overall survival) was identified as independent better prognostic factor. Further analysis, sTIL was identified as independently prognostic factor in Stage III-IVa disease, which was not found in Stage I-II disease. Our study demonstrated that sTIL was associated with better ESCC patients’ survival, especially in Stage III-IVa disease. Assessment of sTIL could be useful to discriminate biological behavior for ESCC patients.

Aims Cell microarray (CMA) is a high-throughput scientific research tool, which has greatly accelerated many analyses based at the cellular level. However, there are few described methods for constructing CMAs. Here, we introduce a new, simple, high-output CMA method that is applicable to a broad range of cellular samples. Methods In this method, a recipient block (length, 3.6 cm; width, 2.7 cm; depth, 2 cm) with 40 dot markers was moulded using a transparent plastic box. Adenocarcinoma cells were collected from malignant pleural effusions, cell cylinders were moulded with plastic piping and the cylinders were manually arrayed one by one into the corresponding location of the 60 °C pre-softened recipient block using the guide holes drilled with a steel needle. We constructed a 40-cylinder CMA to prove this method. The expression of cytokeratin 7 (CK7) in the CMA was examined to confirm antigen preservation and epidermal growth factor receptor (EGFR) gene mutation was screened for in five samples. Results The CMA prepared by this method had well-defined array configurations, good cellular morphology and well-preserved proteins and DNA. A total of 1000 sections could be easily gained from this CMA block. Conclusions This simple and low-cost method provides a novel way of preparing a high-output CMA.

Three extracts from Zijuan (Camellia sinensis), Kudingcha (Ilex kudingcha) and Mingri (Angelica keiskei) leaves were used to fortify the health promoting function of traditional buckwheat tea. Three fortified teas with similar sensory property to the original tea and higher level of total flavonoids and catechin were chosen to evaluate their capability in lowering hyperglycemic and hyperlipidemic stress using mouse models. The blood glucose level in the hyperglycemic mice was significantly reduced by 10.84 to 34.12% after oral gavage of the three fortified teas (0.4 mL of 20% in solution) each day for 7 days, while it only lowered 4.56% in the original buckwheat tea treatment (P

The biology underlying bone-specific metastasis (BM) of hepatocellular carcinoma (HCC) is poorly understood. The goal of the present study is to further elucidate the molecular and cellular mechanisms underlying HCC with BM. The expression of connective tissue growth factor (CTGF) and interleukin-11 (IL-11) in RNA extracted from 127 formalin-fixed, paraffin-embedded HCC specimens was examined by quantitative real-time polymerase chain reaction. A cellular hypoxic model was established in vitro to investigate CTGF and osteoprotegerin (OPG) expression and roles in hypoxia-induced tumor aggressiveness. The mean CTGF expression in BM versus non-metastatic samples was 3.63-times higher, and IL-11 expression was detected in 62.5 % (10/16) of BM samples versus only in 18.9 % (21/111) of the non-metastatic ones. Highly metastatic HCC cell lines tended to show strong expression of CTGF and IL-11, but low expression of OPG. Hypoxic stimulation of HCC 97L cells increased the level of CTGF mRNA by 2.80-fold within 1.5 h, and hypoxia-inducible factor-1α mRNA levels in these cells could be increased by stimulation with recombinant CTGF protein. Furthermore, OPG and matrix metalloproteinase-2 and -9 levels were also induced under hypoxic conditions. Expression levels of intratumoral CTGF or IL-11 were independent prognostic factors for the development of BM in HCC patients. Tumor hypoxia enhanced the expression of CTGF, which initiates the invasive angiogenesis cascade and enhances expression of many hypoxia-associated genes. Cellular release of OPG may play a role in tumor cell survival. The hypoxia-induced cascade in HCC cells may contribute to invasion and metastasis in vivo.

Aims HER2 is currently the only biomarker used to select eligible patients with advanced gastric cancer (GC) for targeted therapy. The aims of this study were to verify the value of dual-block HER2 assessment and to explore whether increasing the block number is more beneficial by carrying out a randomized prospective cohort study in which dual-block and all-block HER2 assessment were compared in resected specimens of GC. Methods and results Five hundred and forty-nine resected GC specimens were randomly enrolled into two cohorts: a dual-block group (n = 274) with two primary tumour blocks tested, and an all-block group (n = 275) with all primary tumour blocks tested. Immunohistochemical staining of HER2 was performed. For HER2-equivocal (2+) cases, fluorescence in-situ hybridization (FISH) was performed. As compared with single-block assessment, dual-block assessment increased the HER2 immunohistochemistry (IHC)-positive (3+) rate. The rate with dual-block assessment (11.3%) was significantly higher than that with block 1 assessment (8.8%) (P = 0.016) and block 2 assessment (9.1%) (P = 0.031). Similarly, all-block assessment demonstrated a higher HER2 3+ rate (12.4%) than single-block assessment (block 1, 6.5%; block 2, 6.2%; block 3, 7.2%; block 4, 8.7%) (P < 0.05). HER2 3+ rates of all-block and dual-block assessments showed no significant difference (P = 0.703). After IHC and FISH results had been combined, the HER2-positive rate with all-block assessment (13.5%) was slightly higher than that with dual-block assessment (12.0%), although the difference was not statistically significant (P = 0.62). Conclusions Dual-block immunohistochemical assessment is an effective, practical and economic approach that is suitable for the preliminary screening of HER2. We recommend that dual-block HER2 assessment be routinely performed on resected specimens of GC. All-block assessment can be a supplement to dual-block assessment if necessary.

// Dongxian Jiang 1, * , Qi Song 1, * , Haixing Wang 1 , Jie Huang 1 , Hao Wang 2 , Jun Hou 1 , Xiaojing Li 1 , Yifan Xu 1 , Akesu Sujie 1 , Haiying Zeng 1 , Lijie Tan 2 , Yingyong Hou 1, 3 1 Department of Pathology, Zhongshan Hospital, Fudan University, Shanghai 200032, P. R. China 2 Department of thoracic surgery, Zhongshan Hospital, Fudan University, Shanghai 200032, P. R. China 3 Department of Pathology, School of Basic Medical Sciences & Zhongshan Hospital, Fudan University, Shanghai 200032, P. R. China * These authors have contributed equally to this work Correspondence to: Yingyong Hou, email: houyingyong@aliyun.com Keywords: PD-L1 expression, clinical stage, lymph node metastasis, prognostic marker, ESCC Received: August 15, 2016 Accepted: November 22, 2016 Published: December 26, 2016 ABSTRACT Accumulating evidence has shown that PD-L1 expression is associated with clinicopathological features in various human malignancies. We searched for correlations between PD-L1 expression and clinicopathological data in esophageal squamous cell carcinoma (ESCC) patients. PD-L1 expression in primary tumors from 278 patients was evaluated using immunohistochemistry (IHC) in ESCC tissue microarray. Survival curves were constructed by using the Kaplan-Meier method. Univariate and multivariate Cox proportional hazard regression models were performed to identify associations with outcome variables. Overall, tumoral PD-L1 expression (≥10%, 20% or 30% as cut-off value) was associated with favorable DFS and OS upon multivariate analysis. When the patients stratified into stage I-II (168, 60.4%) and stage III-IV (110, 39.6%), or with lymph node metastasis (133, 47.8%), the prognostic role was not consistent. In patients with stage I-II disease, tumoral PD-L1 expression (≥5%, 10%, 20% or 30%) was associated with better DFS and OS upon multivariate analysis. In patients without lymph node metastasis, tumoral PD-L1 expression (≥1%, 5%, 10%, 20%, or 30%) was associated with improved DFS and OS in univariate or multivariate analysis. However, PD-L1 expression was not correlated with prognosis in patients with stage III-IV disease or with lymph node metastasis. Our results for the first time showed the prognostic role of tumoral PD-L1 expression was variable in different stages and lymph node status of ESCC. Tumoral PD-L1 expression was independent favorable predictor in ESCC patients with Stage I-II disease or without lymph node metastasis, not in stage III-IV or lymph node metastasis.

To study the clinicopathological characteristics of hepatic angiomyolipoma (HAML) and to evaluate the correlation between clinicopathological parameters and tumor subtypes.Retrospective analysis of clinicopathological features was conducted in 182 cases of HAML.HAML patients were predominantly female (M:F=1:4) and most commonly presented with non-specific symptoms. The median age at diagnosis was 46 years, ranged from 17 to 77 years. Tumor diameter was ranged from 0.3 to 32.0 cm with an average of 5.0 cm. Majority of the tumor was epithelioid type (112/165, 67.9%). Extramedullary hematopoiesis, multinucleated giant cells, intranuclear inclusions, nucleolus, cellular atypia, invasive growth pattern, multiple masses, hyperpigmentation and purpura-like changes mostly occurred in the epithelioid type (P0.05). Extramedullary hematopoiesis was commonly seen in HAML, the significance of which was still uncertain.Most of HAML are epithelioid type, characterized by a proliferation of predominantly epithelioid cells, in which extramedullary hematopoiesis is commonly seen. Some morphologic features that may predict malignant such as necrosis, mitotic figures, and tumor emboli are only found in the epithelioid HAML. Mitotic activity, tumor necrosis, tumor thrombus, giant cells, periportal invasion, multiple lesions and tumors size over 10 cm are closely related with tumor recurrence and metastasis.

Purpose: To investigate the expression of metadherin (MTDH) for its prognostic value in hepatocellular carcinoma (HCC) and its role in promoting HCC metastasis. Experimental Design: This study employed a tissue microarray containing samples from 323 HCC patients to examine the expression of MTDH and its correlation with other clinicopathologic characteristics. The role of MTDH in the regulation of HCC metastasis was investigated both in vitro and in vivo using short hairpin RNA (shRNA)–mediated downregulation of MTDH in HCC cell lines with various metastatic potentials. Results: The expression of MTDH was markedly higher in HCC tumors than in normal liver tissue. Particularly high MTDH expression was observed in tumors with microvascular invasion, pathologic satellites, poor differentiation, or tumor-node-metastasis stages II to III. Furthermore, the clinical outcome was consistently poorer for the MTDHhigh group than for the MTDHlow group in the 1-, 3-, and 5-year overall survival (OS) rates and in the 1-, 3-, 5-year cumulative recurrence rates. In a nude mice model, the shRNA-mediated downregulation of MTDH resulted in a reduced migratory capacity in HCC cell lines, as well as a reduction in pulmonary and abdominal metastasis. Furthermore, we found that the expression level of MTDH correlated with four epithelial–mesenchymal transition (EMT) markers. Knockdown of MTDH expression in HCC cell lines resulted in downregulation of N-cadherin and snail, upregulation of E-cadherin, and translocation of β-catenin. Conclusions: MTDH may promote HCC metastasis through the induction of EMT process and may be a candidate biomarker for prognosis as well as a target for therapy. Clin Cancer Res; 17(23); 7294–302. ©2011 AACR.

We aimed to develop a clinicopathological model that would predict the risk of bone metastasis (BM) in hepatocellular carcinoma (HCC). We first evaluated a training cohort of 201 HCC patients who had undergone hepatectomy and found that the following factors independently predicted BM development: vascular invasion, tumor-node-metastasis stage, CXCR4, connective tissue growth factor, and interleukin-11. These variables were used to construct a clinicopathological prediction model that may be scored from 0 to 19. The predictive value of the model was demonstrated in a validation cohort of 179 post-hepatectomy HCC patients. During a median follow-up of 54.3 months for the training cohort and 52.5 months for the validation cohort, 23 patients (11.4%) in the former and 19 patients (10.6%) in the latter developed BM. A cutoff value of 9.4 best discriminated BM risk and was able to exclude future BM development with high accuracy in the validation cohort. The sensitivity and specificity of the method were 73.7 and 78.7%, respectively, the positive predictive value was 29.2%, and the negative predictive value 96.2%. The 1- and 2-year cumulative BM rates were (respectively) 10.8% and 27.4% in the high-risk group and 2.4 and 4.3% in the low-risk group. The hazard ratio for BM of the high- versus low-risk group was 9.240 (95% CI: 3.319–25.722). The simple prediction model constructed from clinicopathological parameters is accurate in predicting BM development in HCC patients.