Subcritical low temperature extraction of bioactive ingredients from foods and food by-products and its applications in the agro-food industry.

Bioactive ingredients are part of the food chain and are responsible for numerous health benefits. Subcritical low temperature extraction has been employed to acquire bioactive ingredients because of its excellent properties, such as energy conservation, low temperature, elimination of residual solvent, and high extraction yield and quality. This review aims to provide a clear picture of the basics of subcritical-temperature extraction, its bioactive ingredient extraction efficiency, and possible applications in the agro-food industry. This review suggested that the extraction temperature, time, co-solvents, solid-fluid ratio, and pressure impacted the extraction efficiency of bioactive ingredients from foods and food by-products. Subcritical solvents are appropriate for extracting low polar ingredients, while the inclusion of co-solvents could extract medium and high polar substances. Bioactive ingredients from foods and food by-products can be used as antioxidants, colorants, and nutritional supplements. Additionally, this technology could remove pesticide residues in tea, concentrate edible proteins, and reduce cigarette tar. A new trend toward using subcritical low temperature extraction in extracting bioactive ingredients will acquire momentum.

MicroRNAs 15A and 16-1 Activate Signaling Pathways That Mediate Chemotaxis of Immune Regulatory B cells to Colorectal Tumors.

BACKGROUND & AIMS: B cells infiltrate tumors, but little is known about how they affect tumor growth and progression. microRNA15A (MIR15A or miRNA15A) and microRNA16-1 (MIR16-1 or miRNA16-1) regulate cell proliferation, apoptosis, and drug resistance. We investigated their involvement in B-cell-mediated immune suppression by colorectal tumors. METHODS: Mice with disruptions of the gene cluster that encodes MIR15A and MIR16-1 (knockout mice), and control (C57BL/B6) mice were given azoxymethane with dextran sodium sulfate (AD) to induce formation of colorectal tumors. Mice were given anti-CD20 to delete B cells, or injections of agomir to increase MIR15A and MIR16-1. Proliferation of CD8+T cells was measured by carboxyfluorescein-succinimidyl-ester analysis. Colon tissues were collected from mice and analyzed by flow cytometry, microRNA (miRNA) sequencing, and for cytokine production. Intestinal epithelial cells (IECs) were isolated and transfected with miRNA mimics, to identify their targets. We analyzed miRNA expression patterns and quantified B cells in colorectal cancer tissue microarrays derived from 90 patients who underwent surgical resection, from July 2006 through April 2008, in Shanghai, China; expression data were compared with clinical outcomes. RESULTS: Tumors that developed in knockout mice following administration of AD were larger and contained greater numbers of B cells than tumors that grew in control mice. Most of the B cells in the tumors were positive for immunoglobulin A (IgA+). IgA+ B cells expressed high levels of immune regulatory molecules (programmed death ligand 1, interleukin 10, and transforming growth factor beta), and repressed the proliferation and activation of CD8+ T cells. Levels of MIR15A and MIR16-1 were reduced in colon tumors from mice, compared with nontumor colon tissue. Incubation of IECs with IL17A reduced expression of MIR15A and MIR16-1. Transgenic expression of MIR15A and MIR16-1 in IECs decreased activation of NF-κB and STAT1 by reducing expression of I-kappaB kinases; this resulted in reduced production of chemokine (C-X-C motif) ligands 9 and 10 and decreased chemotaxis of IgA+ B cells. Tumors in mice injected with AD and agomir grew more slowly than tumors in mice not given in agomir and contained fewer IgA+ B cells. We found a negative correlation between levels of MIR15A and MIR16-1 and numbers of IgA+B cells in human colorectal tumor tissues; high levels of MIR15A and MIR16-1 and low numbers of IgA+B cells were associated with longer survival times of patients. CONCLUSIONS: We found increased levels of MIR15A and MIR16-1 to reduce numbers of IgA+ B cells in colorectal tumor tissues and correlate with increased survival time of patients. In mice that lack MIR15A and MIR16-1, colon tumors grow more rapidly and contain increased numbers of IgA+ B cells. MIR15A and MIR16-1 appear to activate signaling pathways required for B-cell-mediated immune suppression.

Molecular profiling of the biphasic components of hepatic carcinosarcoma by the use of targeted next-generation sequencing.

AIMS: To better understand the tumourogenesis and molecular features of hepatic carcinosarcoma (HCS). METHODS AND RESULTS: We selected 13 cases of HCS, including the clinicopathological and immunohistochemical features, and analysed the molecular alterations in separately microdissected carcinomatous and sarcomatous components in eight cases by using targeted next-generation sequencing with a panel of 329 cancer-related genes. As a result, transitional areas were observed between the two components of HCS in all cases. Concordance and overlap in genetic alterations were identified in the two histological components of the eight HCS patients, indicating the clonal relatedness of the two tumour components. The most common gene alterations found in both components were TP53 (75%, 6/8) and NF1/2 (38%, 3/8) mutations and VEGFA amplification (25%, 2/8), which may be strongly associated with HCS tumorigenesis. Unique mutations and amplifications found only in one component were also identified. Amplifications involving MET (38%, n = 3/8) and PDGFRA (25%, n = 2/8) were present only in the sarcomatous components, whereas mutation affecting ERBB4 (25%, n = 2/8) and amplifications of CCND1 and FGF3/4/19 (38%, n = 3/8) were present only in the carcinomatous components, indicating their involvement in the clonal evolution of HCS. Furthermore, multiple potential therapeutic targets were identified for HCS. CONCLUSIONS: Our findings indicate that HCS could have been of monoclonal origin, and that the diverse clonal evolution might be driven by special molecular alterations in each tumour component. Our results also identify multiple therapeutic targets of HCS, which are valuable for the personalised treatment of HCS.

MicroRNA-194 protects against chronic hepatitis B-related liver damage by promoting hepatocyte growth via ACVR2B.

Persistent infection with the hepatitis B virus leads to liver cirrhosis and hepatocellular carcinoma. MicroRNAs (miRNAs) play an important role in a variety of biological processes; however, the role of miRNAs in chronic hepatitis B (CHB)-induced liver damage remains poorly understood. Here, we investigated the role of miRNAs in CHB-related liver damage. Microarray analysis of the expression of miRNAs in 22 CHB patients and 33 healthy individuals identified miR-194 as one of six differentially expressed miRNAs. miR-194 was up-regulated in correlation with increased liver damage in the plasma or liver tissues of CHB patients. In mice subjected to 2/3 partial hepatectomy, miR-194 was up-regulated in liver tissues in correlation with hepatocyte growth and in parallel with the down-regulation of the activin receptor ACVR2B. Overexpression of miR-194 in human liver HL7702 cells down-regulated ACVR2B mRNA and protein expression, promoted cell proliferation, acceleratedG1 to S cell cycle transition, and inhibited apoptosis, whereas knockdown of miR-194 had the opposite effects. Luciferase reporter assays confirmed that ACVR2B is a direct target of miR-194, and overexpression of ACVR2B significantly repressed cell proliferation and G1 to S phase transition and induced cell apoptosis. ACVR2B overexpression abolished the effect of miR-194, indicating that miR-194 promotes hepatocyte proliferation and inhibits apoptosis by down-regulating ACVR2B. Taken together, these results indicate that miR-194 plays a crucial role in hepatocyte proliferation and liver regeneration by targeting ACVR2B and may represent a novel therapeutic target for the treatment of CHB-related liver damage.

Identification and Functional Evaluation of miR-4633-5p as a Biomarker and Tumor Suppressor in Metastatic Melanoma.

BACKGROUND/AIMS: Sinonasal mucosal melanoma (SMM) is a rare but extremely aggressive disease. Interestingly, however, as lethal as SMM, a few patients could survive for over 5 years without metastasis. However, biomarkers for metastatic SMM are lacking. METHODS: Laser-capture microdissection combined with microRNA microarray and RT-qPCR was performed in formalin-fixed paraffin-embedded tissue samples from SMM patients whose follow-up studies were carried out in parallel. In vitro cell proliferation and invasion assays, gelatin zymography, western blot analysis and RT-qPCR were performed in melanoma cell lines. RESULTS: In the discovery stage, miR-4633-5p expressed differentially in sinonasal mucosal melanoma patients with short and long disease-specific survival. Subsequent large-sample validation revealed that expression of miR-4633-5p was lower in metastatic SMM than in non-metastatic patients (P< 0.001). Moreover, miR-4633-5plow was able to identify metastatic SMM with specificity of 100% (5/5) and sensitivity of 87.5% (21/24). Multivariate analysis further pinpointed miR-4633-5p as an independent marker for metastasis (relative risk: 54.22, P< 0.001). In vitro, overexpression of miR-4633-5p suppressed the growth and invasiveness of melanoma cells through inhibiting activation of Akt pathway and secretion of MMP2, while knockdown of miR-4633-5p reversed the inhibitory effects. CONCLUSION: Our findings underpin miR-4633-5p as a predictive biomarker in metastatic SMM and a pivotal tumor suppressor that negatively regulates the invasive growth of melanoma cells. Quantitative detection of miR-4633-5p can diagnostically predict the risk of metastasis in SMM patients, which, in turn, may lead to more personalized treatment with better prognosis.

B Subunit of Human Chorionic Gonadotropin Promotes Tumor Invasion and Predicts Poor Prognosis of Early-Stage Colorectal Cancer.

BACKGROUND/AIMS: It is well established that many non-trophoblastic tumors secrete HCG (human chorionic gonadotropin) and that such secretion is correlated with the poor prognosis of tumor patients. This study aims to analyze the correlation between ß-HCG expression and outcome of colorectal cancer (CRC) and understand its role in CRC pathology Methods: We detected the mRNA and protein expression of ß-HCG in human CRC tissues with RT-qPCR and immunohistochemistry, and we compared the clinical-pathological characteristics, prognosis and progression between the ß-HCG positive and negative groups. We also generated CRC cell lines with ß-HCG over-expression as well as ß-HCG stable knockout, and evaluated cell function and mechanism in vitro and in vivo. RESULTS: Fifty out of 136 CRC patients (37%) expressed ß-HCG at the invasive front. Clinical-pathological data showed that ß-HCG was positively correlated with Dukes staging (P=0.031) and lymph node metastasis (P=0.012). Survival analysis suggested that the patients with high expression of ß-HCG had poorer prognosis than those with low ß-HCG expression (P=0.0289). ß-HCG expression level was also positively correlated with tumor invasion in early-stage CRC patient tissues (P=0.0227). Additionally ß-HCG promoted the migration and invasion of CRC in vitro and in vivo but had no effect on the proliferation of tumor cells. CONCLUSION: Our study demonstrated that ß-HCG was ectopically expressed in the CRC patients and its high expression correlated with poor prognosis of early-stage CRC. Additionally it worked as an oncogene that promotes the migration and invasion of CRC by epithelial-mesenchymal transition (EMT).

Selective versus non-selective removal for dental caries: a systematic review and meta-analysis.

OBJECTIVE: Selective and non-selective methods for caries removal were controversial so far, thus we aimed to compare the efficacy of selective and non-selective caries removal by conducting meta-analysis of randomized controlled trials (RCTs). MATERIALS AND METHODS: Eligible RCTs studies comparing selective caries removal with non-selective caries removal were retrieved by searching PubMed, EMBASE and Cochrane Library till 15 July 2017. The pooled odds ratios (ORs) with 95% confidence intervals (CIs) were calculated for outcome indictors, including pulpal exposure, pulpal symptoms and failure using Inverse variance-random effects or Mantel-Haenszel-fixed effects models. RESULTS: Totally, seven studies were eligible for the meta-analysis. Compared with the non-selective caries removal group, the risk of pulpal exposure was significantly reduced in the selective caries removal group (OR = 0.11, 95% CI: 0.04-0.30). No significant difference was observed in pulpal symptoms (OR = 0.79, 95% CI: 0.30-2.12) and failure (OR = 1.40, 95% CI: 0.69-2.84) between the groups. CONCLUSIONS: The efficacy of selective caries removal appears comparable to that of non-selective caries removal in children, with similar pulpal symptoms and failure, but selective caries removal may result in a low incidence of pulpal exposure. However, larger-scale RCTs with long-term follow-up are required to confirm this conclusion.

Association of Mutations in Toll-like Receptor 2 Signaling Genes With Fulminant Form of Hepatitis B-Related Acute Liver Failure.

Background: The fulminant form of hepatitis B-related acute liver failure (FHB-ALF) is a rare but highly fatal outcome of acute hepatitis B virus (HBV) infection. Its related host factors have not been studied to our knowledge. Methods: To identify functionally relevant biological pathway(8) in FHB-ALF pathogenesis, pathway enrichment analysis was conducted on a data set of rare case-specific variants derived from exomic sequencing of 10 unrelated cases. Key variants in identified pathways were validated using 312 controls with HBV disease. Mechanistic studies of a recurrent Toll-like receptor (TLR) 2 gene (TLR2) variant were performed in vitro and in vivo. Results: The TLR signaling pathway was highly enriched, with associated variants found in 9 of the 10 cases. Notably, a rare heterozygous single-nucleotide variation causing F679I mutation in TLR2 was identified in 2 unrelated cases. In vitro analysis demonstrated F679I to cause loss of function. In both heterozygous and homozygous TLR2 knockout mice, injection of HBV replicon plasmid resulted in more prominent alanine aminotransferase elevations and hepatic necroinflammation than in wild-type mice. Mechanistic analyses demonstrated reduced regulatory T-cell percentages in postexposure TLR2 knockout mice. Conclusions: TLR2 signaling is very likely impaired in patients with FHB-ALF. The recurrence of rare case-specific TLR2 variant strongly suggests mechanistic contribution to fulminancy in HBV infection.

Stiffness Value and Serum Biomarkers in Liver Fibrosis Staging: Study in Large Surgical Specimens in Patients with Chronic Hepatitis B.

Purpose To investigate the capabilities of stiffness value and serum biomarkers in the staging of liver fibrosis in patients with chronic hepatitis B (CHB), with pathologic findings in large surgical specimens serving as the reference standard. Materials and Methods This study was approved by the institutional review board, and informed consent was obtained from all patients. Liver stiffness (determined by means of ultrasonography-based elastography point quantification), aspartate aminotransferase-platelet ratio index (APRI), and fibrosis index (based on the four-factor Fibrosis-4 [FIB-4] calculation) were obtained in 386 patients with CHB. With pathologic fibrosis stages in large surgical specimens as the reference standard, capabilities and cutoffs of stiffness and serum biomarkers were first investigated in a cohort of 284 patients and then validated in an independent cohort of 102 patients by means of area under the receiver operating characteristic curve (AUC) analysis. Results Liver stiffness demonstrated significantly stronger correlation with fibrosis stages than did APRI and FIB-4 (r = 0.738 vs r = 0.477 vs r = 0.427, respectively; P < .05 for all). In the development cohort, liver stiffness had significantly higher AUCs in identifying fibrosis of stage 1 or higher, stage 2 or higher, stage 3 or higher, and stage 4 or higher (0.97, 0.96, 0.91, and 0.87, respectively) than APRI (0.89, 0.84, 0.73, and 0.74, respectively) and FIB-4 (0.82, 0.79, 0.70, and 0.72, respectively). In the validation cohort, liver stiffness was validated as showing significantly higher AUCs in identifying fibrosis of stage 1 or higher, stage 2 or higher, stage 3 or higher, and stage 4 or higher (0.99, 0.95, 0.89, and 0.88, respectively) than APRI (0.83, 0.76, 0.78, and 0.68, respectively) and FIB-4 (0.76, 0.69, 0.75, and 0.67, respectively). Conclusion Liver stiffness demonstrated considerable capability in identifying each stage of liver fibrosis in patients with CHB, whereas serum biomarkers showed limited capabilities. (©) RSNA, 2016 Online supplemental material is available for this article.

A plasma microRNA panel for early detection of colorectal cancer.

Colonoscopy remains the standard screening method for detecting colorectal cancer (CRC) at an early stage. However, many people avoid having a colonoscopy because of the fear for its potential complications. Our study aimed to identify plasma microRNAs for preliminarily screening CRC in general population, so that some unnecessary colonoscopies can be avoided. We investigated plasma microRNA expression in three independent cohorts including the discovery (n = 80), training (n = 112), and validation (n = 49) phases recruited at two medical centers. Microarrays were used for screening 723 microRNAs in 80 plasma samples to identify candidate microRNAs. Quantitative reverse-transcriptase PCR was performed on the 161 training and validation plasma samples to evaluate the candidate microRNAs discovered from microarrays. A logistic regression model was constructed based on the training cohort and then verified by using the validation dataset. Area under the receiver operating characteristic curve (AUC) was used to evaluate the diagnostic accuracy. We identified a panel of miR-409-3p, miR-7, and miR-93 that yielded high diagnostic accuracy in discriminating CRC from healthy group (AUC: 0.866 and 0.897 for training and validation dataset, respectively). Moreover, the diagnostic performance of the microRNA panel persisted in nonmetastasis CRC stages (Dukes' A-B, AUC: 0.809 and 0.892 for training and validation dataset, respectively) and in metastasis CRC stages (Dukes' C-D, AUC: 0.917 and 0.865 for training and validation dataset, respectively). In conclusion, our study reveals a plasma microRNA panel that has potential clinical value in early CRC detection and would play a critical role on preliminarily screening CRC in general population.

Hepatocellular carcinoma: stiffness value and ratio to discriminate malignant from benign focal liver lesions.

PURPOSE: To investigate the use of stiffness value and stiffness ratio (ratio of lesion to background liver parenchyma values) to discriminate malignant from benign focal liver lesions by using histologic results as the reference standard. MATERIALS AND METHODS: This study was approved by the institutional review board, and written informed consent was obtained. Three hundred seventy-three patients with focal liver lesions proven at histologic examination underwent measurement of liver stiffness with elastography point quantification. First, stiffness values in two regions of the background liver parenchyma (at 0.5-2 cm and >2 cm from the lesion periphery) near 163 hepatocellular carcinomas were analyzed to determine a reference background liver for calculating the stiffness ratio. Second, the use of the lesion stiffness value and the stiffness ratio for prediction of liver malignancy was investigated in a cohort of patients with 58 benign and 201 malignant lesions. Results were validated in another independent cohort of patients with 25 benign and 89 malignant lesions by using analysis of the area under the receiver operating characteristic (AUC) curve. RESULTS: The coefficient of variation for the background liver at 0.5-2 cm from the lesion was higher (196%) than that at greater than 2 cm from the lesion (66%). In the development phase, diagnostic accuracy with use of the stiffness value was significantly higher than that with use of the stiffness ratio for discrimination of malignant from benign lesions (AUC, 0.86 vs 0.66, respectively; P < .001). Diagnostic performance with the stiffness value was lower than that with the stiffness ratio (AUC, 0.53 vs 0.86, respectively; P < .001) for discrimination of cirrhotic nodules from other benign lesions. Diagnostic performance with the stiffness value was significantly lower than that with the stiffness ratio (AUC, 0.58 vs 0.71 respectively; P = .007) for discrimination of metastasis from primary liver cancers. In the validation phase, similar findings were revealed for the discrimination of malignant from benign lesions (AUC, 0.87 vs 0.67; P < .001) and discrimination between metastasis and primary liver cancers (AUC, 0.49 vs 0.73; P < .001). CONCLUSION: Use of stiffness values measured in the liver parenchyma at more than 2 cm from the lesion allowed better diagnostic performance than did values measured in a region closer to the tumor. Stiffness value was more accurate than stiffness ratio for differentiation of malignant from benign focal liver lesions, but the stiffness ratio might be useful for subclassification of benign and malignant lesions. Online supplemental material is available for this article.

Hepatitis B virus X protein induces the histone H3 lysine 9 trimethylation on the promoter of p16 gene in hepatocarcinogenesis.

Our previous study showed hepatitis B virus X protein (HBx) suppresses the p16 expression in hepatocarcinogenesis. In this study we explored the relationship between HBx and trimethylation of H3K9 (H3K9me3), and elucidated the underlying mechanisms in HBx inducing the tumor suppressor p16 gene silence. SMMC-7721 and HepG2 hepatoma cell lines were transfected with HBx-expressing plasmid. Immunohistochemistry, Western blotting and real-time polymerase chain reaction, were performed to detect the expressions of HBx, H3K9me3, and jumonji domain-containing protein 2B (JMJd2B). H3K9me3 enrichment on the p16 promoter was measured by immunoprecipitation-PCR (ChIP-PCR) analyses, and 39 cases of hepatitis B virus (HBV) associated-hepatocellular carcinoma (HCC) and corresponding noncancerous liver tissues were also examined. We demonstrated that HBx was able to upregulate H3K9me3 and suppress JMJd2B mRNA and protein levels in SMMC-7721 and HepG2 hepatoma cell lines. JMJd2B, as a specific target of H3K9me3 for demethylation, was inversely correlated with the levels of H3K9me3 in SMMC-7721 (r=-0.666, P<0.05) and HepG2 cells (r=-0.625, P<0.05). The ChIP-PCR data indicated that HBx remarkably increased H3K9me3 on the p16 promoter region. Immunohistochemistry analysis showed that H3K9me3 expression in HBx positive HCC samples were significantly higher than that in HBx negative HCC tissues and were associated with decreased levels of JMJd2B expression. JMJd2B immunoreactivity was also remarkably inversed to that of HBx in HCC tissues (r=-0.630, P<0.05). Our results provide evidence that HBx is able to induce H3K9me3 on the p16 promoter via the decrease of demethylase JMJd2B expression and thus promote the repression of p16 gene expression to enhance hepatocarcinogenesis.

Human miR-1228 as a stable endogenous control for the quantification of circulating microRNAs in cancer patients.

Circulating microRNAs are promising biomarkers for non-invasive testing and dynamic monitoring in cancer patients. However, no consensus exists regarding the normalization of circulating microRNAs in the quantification, making the results incomparable. We investigated global circulating microRNA profiles to identify a stable endogenous control for quantifying circulating microRNAs using three cohorts (n = 544), including 168 control individuals (healthy subjects and those with chronic hepatitis B and cirrhosis) and 376 cancer patients (hepatocellular, colorectal, lung, esophageal, gastric, renal, prostate, and breast cancer patients). GeNorm, NormFinder, and coefficient of variability (CV) were used to select the most stable endogenous control, whereas Ingenuity Pathway Analysis (IPA) was adopted to explore its signaling pathways. Seven candidates (miR-1225-3p, miR-1228, miR-30d, miR-939, miR-940, miR-188-5p, and miR-134) from microarray analysis and four commonly used controls (miR-16, miR-223, let-7a, and RNU6B) from literature were subjected to real-time quantitative reverse transcription-polymerase chain reaction validation using independent cohorts. MiR-1228 (CV = 5.4%) with minimum M value and S value presented as the most stable endogenous control across eight cancer types and three controls. IPA showed miR-1228 to be involved extensively in metabolism-related signal pathways and organ morphology, implying that miR-1228 functions as a housekeeping gene. Functional network analysis found that "hematological system development" was on the list of the top networks that associate with miR-1228, implying that miR-1228 plays an important role in the hematological system. The results explained the steady expression of miR-1228 in the blood. In conclusion, miR-1228 is a promising stable endogenous control for quantifying circulating microRNAs in cancer patients.

A microRNA panel to discriminate carcinomas from high-grade intraepithelial neoplasms in colonoscopy biopsy tissue.

OBJECTIVE: It is a challenge to differentiate invasive carcinomas from high-grade intraepithelial neoplasms in colonoscopy biopsy tissues. In this study, microRNA profiles were evaluated in the transformation of colorectal carcinogenesis to discover new molecular markers for identifying a carcinoma in colonoscopy biopsy tissues where the presence of stromal invasion cells is not detectable by microscopic analysis. METHODS: The expression of 723 human microRNAs was measured in laser capture microdissected epithelial tumours from 133 snap-frozen surgical colorectal specimens. Three well-known classification algorithms were used to derive candidate biomarkers for discriminating carcinomas from adenomas. Quantitative reverse-transcriptase PCR was then used to validate the candidates in an independent cohort of macrodissected formalin-fixed paraffin-embedded colorectal tissue samples from 91 surgical resections. The biomarkers were applied to differentiate carcinomas from high-grade intraepithelial neoplasms in 58 colonoscopy biopsy tissue samples with stromal invasion cells undetectable by microscopy. RESULTS: One classifier of 14 microRNAs was identified with a prediction accuracy of 94.1% for discriminating carcinomas from adenomas. In formalin-fixed paraffin-embedded surgical tissue samples, a combination of miR-375, miR-424 and miR-92a yielded an accuracy of 94% (AUC=0.968) in discriminating carcinomas from adenomas. This combination has been applied to differentiate carcinomas from high-grade intraepithelial neoplasms in colonoscopy biopsy tissues with an accuracy of 89% (AUC=0.918). CONCLUSIONS: This study has found a microRNA panel that accurately discriminates carcinomas from high-grade intraepithelial neoplasms in colonoscopy biopsy tissues. This microRNA panel has considerable clinical value in the early diagnosis and optimal surgical decision-making of colorectal cancer.

Limited dependence on soil nitrogen fixation as subtropical forests develop.

Soil nitrogen (N) fixation, driven by microbial reactions, is critical to support the entrance of nitrogen in nutrient poor and pioneer ecosystems. However, how and why N fixation and soil diazotrophs evolve as forests develop remain poorly understood. Here, we used a 60-year forest rewilding chronosequence and found that soil N fixation activity gradually decreased with increasing forest age, experiencing dramatic drops of 64.8% in intermediate stages and 93.0% in the oldest forests. Further analyses revealed loses in diazotrophic diversity and a significant reduction in the abundance of important diazotrophs (e.g., Desulfovibrio and Pseudomonas) as forest develops. This reduction in N fixation, and associated shifts in soil microbes, was driven by acidification and increases in N content during forest succession. Our results provide new insights on the life history of one of the most important groups of soil organisms in terrestrial ecosystems, with consequences for understanding the buildup of nutrients as forest soil develops.

Targeting the chromatin structural changes of antitumor immunity.

Epigenomic imbalance drives abnormal transcriptional processes, promoting the onset and progression of cancer. Although defective gene regulation generally affects carcinogenesis and tumor suppression networks, tumor immunogenicity and immune cells involved in antitumor responses may also be affected by epigenomic changes, which may have significant implications for the development and application of epigenetic therapy, cancer immunotherapy, and their combinations. Herein, we focus on the impact of epigenetic regulation on tumor immune cell function and the role of key abnormal epigenetic processes, DNA methylation, histone post-translational modification, and chromatin structure in tumor immunogenicity, and introduce these epigenetic research methods. We emphasize the value of small-molecule inhibitors of epigenetic modulators in enhancing antitumor immune responses and discuss the challenges of developing treatment plans that combine epigenetic therapy and immunotherapy through the complex interaction between cancer epigenetics and cancer immunology.

Optimized pregelatinized starch technique for cell block preparation in cell cultures.

The aim of the present study was to optimize the pregelatinized starch technique for cell block preparation and apply this approach in cultured cells of all types of growing forms, suspension and adherent. In order to evenly mix the starch powder and the cell suspension, we crafted a special plastic dropper. To prove the effectiveness of this optimized technique we used different cell lines, NCI-H69, NCI-H345, HCT-116, SKBR3 and MDA-MB-231. The morphology features, immunocytochemistry (ICC) and fluorescent/chromogenic in-situ hybridization (FISH/CISH) on the cell block sections were evaluated. The morphology features, the ICC and ISH results of cell block sections prepared by the new method were satisfactory comparing with the results obtained in biopsies, the gold standard test for this kind of analysis. The most attractive advantage of our optimized pregelatinized starch technique is that this new method is based on cell suspensions instead of cell sediment, so with our technique every section will contain cells due to the even distribution of the starch powder and the cells forming a homogeneous cell block. To the authors' knowledge, this is the first description on cell block preparation based on cell suspension.

Inhibition of the inflammatory cytokine tumor necrosis factor-alpha with etanercept provides protection against lethal H1N1 influenza infection in mice.

INTRODUCTION: Factors implicated in influenza-mediated morbidity and mortality include robust cytokine production (cytokine storm), excessive inflammatory infiltrates, and virus-induced tissue destruction. Tumor necrosis factor-alpha (TNF-α) is an important pro-inflammatory cytokine present during influenza infection, but it is unclear whether direct inhibition of TNF-α can elicit protection against influenza infection. METHODS: In this study, the commercially available TNF-α inhibitor etanercept was used to inhibit TNF-α induced by lethal A/FM/1/47 (H1N1) influenza virus infection of mice. The effects of TNF-α inhibition on mouse survival, pathologic changes, immune cell infiltration, inflammatory cytokine secretion, Toll-like receptor expression, and activation of the NF-κB (nuclear factor kappa B) signaling pathway were evaluated. RESULTS: The intranasal delivery of etanercept provided significant protection against mortality (30% of mice survived up to 14 days after infection) in mice treated with etanercept. In contrast, no survivors were found beyond 6 days in mice treated with saline after lethal challenge with H1N1 influenza virus. It was observed that etanercept significantly reduced inflammatory cell infiltration (for example, macrophages and neutrophils), inflammatory cytokine secretion (for example, interleukin-6, TNF-α, and interferon gamma), and expression of Toll-like receptors (TLR-3, TLR-4, and TLR-7). Etanercept also downregulated and inhibited the cascade proteins of the NF-κB signaling pathway (for example, MyD88, TRIF, NF-κB, and p65), as well as enhanced host control of virus replication. CONCLUSIONS: These findings indicate that etanercept, by blocking TNF-α, can significantly downregulate excessive inflammatory immune responses and provide protection against lethal influenza infection, making its use a novel strategy for controlling severe influenza-induced viral pneumonia.

Lipopolysaccharide-induced toll-like receptor 4 signaling in cancer cells promotes cell survival and proliferation in hepatocellular carcinoma.

BACKGROUND: Recent studies have shown that toll-like receptor 4 (TLR4) is involved in hepatocarcinogenesis. However, the significance of TLR4 signaling in cancer development and progression remains unclear. AIM: The purpose of this study was to investigate the role of TLR4 in cancer cell survival and proliferation in hepatocellular carcinoma (HCC). METHODS: Fifty-three HCC and ten normal liver specimens were analyzed by immunohistochemistry, and three cell lines (HL-7702, PLC/PRF/5 and HepG2) were used for in vitro studies. Lipopolysaccharide (LPS), a specific ligand of TLR4, was used to activate TLR4 signaling. The effects of LPS-TLR4 signaling on cell survival, proliferation and invasion were examined. Specific inhibitors of NF-κB and MAPK (JNK, ERK and p38) signaling pathways were used to explore the role of each pathway in LPS-TLR4 signaling. RESULTS: TLR4 was overexpressed in HCC cell lines and in human HCC tissues, where it correlated with Ki-67 expression. LPS-induced activation of TLR4 signaling promoted cancer cell survival and proliferation. LPS-TLR4 signaling was associated with regulation on the activation of NF-κB and MAPK signaling pathways. LPS-TLR4-induced activation of ERK and JNK signaling promotes cell proliferation through regulating Bax translocation to mitochondria. Activation of NF-κB and p38 mediates cytotoxicity of LPS, and inhibition on these two pathways promotes cell proliferation in HCC cells. CONCLUSION: Our results indicate that TLR4 signaling in cancer cells promotes cell survival and proliferation in HCC.

PEGylated human catalase elicits potent therapeutic effects on H1N1 influenza-induced pneumonia in mice.

Therapeutic recombinant human catalase (rhCAT) can quench infection-induced reactive oxygen species (ROS), thereby alleviating the associated tissue damage. Although the intranasal route is efficient to deliver native rhCAT to the lung, the therapeutic effect is limited by rapid elimination from the blood. In this study, we modified rhCAT with the active polymer, polyethylene glycol monomethyl ether (PEG)-5000, and analyzed the pharmacokinetics of PEGylated rhCAT in mice. The high tetra-PEGylation ratio was about 60%, and PEGylation prolonged the half-life of rhCAT in serum (75 vs. 13.5 min for native rhCAT). The protective effects of PEG-rhCAT were investigated in a mouse model of influenza virus A (H1N1)-associated pneumonia. PEG-rhCAT was more effectively delivered than native rhCAT and was associated with higher survival ratio, less extensive lung injuries, reduced ROS levels, and lower viral replication. Collectively, these findings indicate that PEGylation can enhance the therapeutic efficacy of native rhCAT and suggest that PEGylated rhCAT may represent a novel complement therapy for H1N1 influenza-induced pneumonia.