RNA demethylase ALKBH5 promotes colorectal cancer progression by posttranscriptional activation of RAB5A in an m6A-YTHDF2-dependent manner.

BACKGROUND: N6-methyladenosine (m6A) modification is an emerging epigenetic regulatory mechanism in tumourigenesis. Considering that AlkB homolog 5 (ALKBH5) is a well-described m6A demethylase in previous enzyme assays, we aimed to investigate the role of m6A methylation alteration conferred by disturbed ALKBH5 in colorectal cancer (CRC) development. METHODS: Expression of ALKBH5 and its correlation with clinicopathological characteristics of CRC were evaluated using the prospectively maintained institutional database. The molecular role and underlying mechanism of ALKBH5 in CRC were explored using in vitro and in vivo experiments with methylated RNA immunoprecipitation sequencing (MeRIP-seq), RNA-seq, MeRIP-qPCR, RIP-qPCR and luciferase reporter assays. RESULTS: ALKBH5 expression was significantly upregulated in CRC tissues compared to the paired adjacent normal tissues, and higher expression of ALKBH5 was independently associated with worse overall survival in CRC patients. Functionally, ALKBH5 promoted the proliferative, migrative and invasive abilities of CRC cells in vitro and enhanced subcutaneous tumour growth in vivo. Mechanistically, RAB5A was identified as the downstream target of ALKBH5 in CRC development, and ALKBH5 posttranscriptionally activated RAB5A by m6A demethylation, which impeded the YTHDF2-mediated degradation of RAB5A mRNA. In addition, we demonstrated that dysregulation of the ALKBH5-RAB5A axis could affect the tumourigenicity of CRC. CONCLUSIONS: ALKBH5 facilitates the progression of CRC by augmenting the expression of RAB5A via an m6A-YTHDF2-dependent manner. Our findings suggested that ALKBH5-RAB5A axis might serve as valuable biomarkers and effective therapeutic targets for CRC.

Fasting-mimicking diet synergizes with ferroptosis against quiescent, chemotherapy-resistant cells.

BACKGROUND: More than ten randomized clinical trials are being tested to evaluate the efficacy, effectiveness and safety of a fasting-mimicking diet (FMD) combined with different antitumor agents. METHODS: UMI-mRNA sequencing, Cell-cycle analysis, Label retention, metabolomics, Multilabeling et al. were used to explore mechanisms. A tandem mRFP-GFP-tagged LC3B, Annexin-V-FITC Apoptosis, TUNEL, H&E, Ki-67 and animal model was used to search for synergistic drugs. FINDINGS: Here we showed that fasting or FMD retards tumor growth more effectively but does not increase 5-fluorouracil/oxaliplatin (5-FU/OXA) sensitivity to apoptosis in vitro and in vivo. Mechanistically, we demonstrated that CRC cells would switch from an active proliferative to a slow-cycling state during fasting. Furthermore, metabolomics shows cell proliferation was decreased to survive nutrient stress in vivo, as evidenced by a low level of adenosine and deoxyadenosine monophosphate. CRC cells would decrease proliferation to achieve increased survival and relapse after chemotherapy. In addition, these fasting-induced quiescent cells were more prone to develop drug-tolerant persister (DTP) tumor cells postulated to be responsible for cancer relapse and metastasis. Then, UMI-mRNA sequencing uncovered the ferroptosis pathway as the pathway most influenced by fasting. Combining fasting with ferroptosis inducer treatment leads to tumor inhibition and eradication of quiescent cells by boosting autophagy. INTERPRETATION: Our results suggest that ferroptosis could improve the antitumor activity of FMD + chemotherapy and highlight a potential therapeutic opportunity to avoid DTP cells-driven tumor relapse and therapy failure. FUNDING: A full list of funding bodies can be found in the Acknowledgements section.

Small dataset for hot cracking susceptibility of Al alloys and Ni alloys using dynamic X-ray radiography (DXR) technique.

Hot cracking as the major concern in the manufacturing process of metal alloys is detrimental to part performance and can lead to catastrophic failure. However, current research in this field is restricted to the scarcity of the relevant hot cracking susceptibility data. Here, using the DXR technique provided at 32-ID-B beamline of Advanced Photon Source (APS) at Argonne National Laboratory, we characterized the hot cracking formation in Laser Powder Bed Fusion (L-PBF) process for ten commercial alloys (Al7075, Al6061, Al2024, Al5052, Haynes 230, Haynes 160, Haynes X, Haynes 120, Haynes 214, and Haynes 718). The extracted DXR images captured the post-solidification hot cracking distribution and allow the quantification of the hot cracking susceptibility of those alloys. We further exploited this in our recent effort on hot cracking susceptibility prediction [1] and established a hot cracking susceptibility dataset posted on Mendeley Data for the purpose of facilitating the relevant research in this field.

DNA methylation profile in CpG-depleted regions uncovers a high-risk subtype of early-stage colorectal cancer.

BACKGROUND: The current risk stratification system defined by clinicopathological features does not identify the risk of recurrence in early-stage (stage I-II) colorectal cancer (CRC) with sufficient accuracy. We aimed to investigate whether DNA methylation could serve as a novel biomarker for predicting prognosis in early-stage CRC patients. METHODS: We analyzed the genome-wide methylation status of CpG loci using Infinium MethylationEPIC array run on primary tumor tissues and normal mucosa of early-stage CRC patients to identify potential methylation markers for prognosis. The machine-learning approach was applied to construct a DNA methylation-based prognostic classifier for early-stage CRC (MePEC) using the 4 gene methylation markers FAT3, KAZN, TLE4, and DUSP3. The prognostic value of the classifier was evaluated in 2 independent cohorts (n = 438 and 359, respectively). RESULTS: The comprehensive analysis identified an epigenetic subtype with high risk of recurrence based on a group of CpG loci in the CpG-depleted region. In multivariable analysis, the MePEC classifier was independently and statistically significantly associated with time to recurrence in validation cohort 1 (hazard ratio = 2.35, 95% confidence interval = 1.47 to 3.76, P < .001) and cohort 2 (hazard ratio = 3.20, 95% confidence interval = 1.92 to 5.33, P < .001). All results were further confirmed after each cohort was stratified by clinicopathological variables and molecular subtypes. CONCLUSIONS: We demonstrated the prognostic statistical significance of a DNA methylation profile in the CpG-depleted region, which may serve as a valuable source for tumor biomarkers. MePEC could identify an epigenetic subtype with high risk of recurrence and improve the prognostic accuracy of current clinical variables in early-stage CRC.

Metabolomics reveals that CAF-derived lipids promote colorectal cancer peritoneal metastasis by enhancing membrane fluidity.

Patients with peritoneal metastasis (PM) of colorectal cancer (CRC) have poorer overall survival outcomes than those without PM. Cancer-associated fibroblasts (CAFs) are a major component of the tumor microenvironment and mediate CRC progression and PM. It is imperative to identify and develop novel therapeutic targets for PM-CRC driven by CAFs. Using lipidomics, we reveal that the abundance of phosphatidylcholine (PC) with unsaturated acyl chains was increased in clinical PM-CRC specimens. Additionally, we found that CAFs were present at a higher relative abundance in primary PM-CRC tumors and that membrane fluidity in CRC cells was increased after incubation with CAF-conditioned medium (CM) through three independent methods: lipidomics, fluorescence recovery after photobleaching (FRAP), and generalized polarization. Then, we found that increased membrane fluidity can enhance glucose uptake and metabolism, as supported by real-time bioenergetics analysis and U-13C glucose labeling. Interestingly, stearoyl-CoA desaturase 1 (SCD), the rate-limiting enzyme in the biosynthesis of unsaturated fatty acids (uS-FAs), was expressed at low levels in PM and associated with poor prognosis in CRC patients. Importantly, by untargeted metabolomics analysis and fatty acid ([U-13C]-stearic acid) tracing analyses, we found that CRC cells take up lipids and lipid-like metabolites secreted from CAFs, which may compensate for low SCD expression. Both in vitro and in vivo experiments demonstrated that sodium palmitate (C16:0) treatment could decrease the CAF-induced change in cell membrane fluidity, limit glucose metabolism, suppress cell invasiveness, and impair tumor growth and intraperitoneal dissemination. An increased C16:0 concentration was shown to induce apoptosis linked to lipotoxicity. Furthermore, C16:0 effectively enhanced the antitumor activity of 5-fluorouracil (5-FU) in vitro and was well tolerated in vivo. Taken together, these findings suggest that adding the saturated fatty acid (S-FA) C16:0 to neoadjuvant chemotherapy may open new opportunities for treating PM-CRC in the future.

DNA methylation-based signature of CD8+ tumor-infiltrating lymphocytes enables evaluation of immune response and prognosis in colorectal cancer.

BACKGROUND: Tumor-infiltrating lymphocytes (TILs), especially CD8+ TILs, can be used for predicting immunotherapy responsiveness and survival outcome. However, the evaluation of CD8+ TILs currently relies on histopathological methodology with high variability. We therefore aimed to develop a DNA methylation signature for CD8+ TILs (CD8+ MeTIL) that could evaluate immune response and prognosis in colorectal cancer (CRC). METHODS: A CD8+ MeTIL signature score was constructed by using CD8+ T cell-specific differentially methylated positions (DMPs) that were identified from Illumina EPIC methylation arrays. Immune cells, colon epithelial cells, and two CRC cohorts (n=282 and 335) were used to develop a PCR-based assay for quantitative analysis of DNA methylation at single-base resolution (QASM) to determine CD8 + MeTIL signature score. RESULTS: Three CD8+ T cell-specific DMPs were identified to construct the CD8+ MeTIL signature score, which showed a dramatic discriminability between CD8+ T cells and other cells. The QASM assay we developed for CD8+ MeTIL markers could measure CD8+ TILs distributions in a fully quantitative, accurate, and simple manner. The CD8+ MeTIL score determined by QASM assay showed a strong association with histopathology-based CD8+ TIL counts and a gene expression-based immune marker. Furthermore, the low CD8+ MeTIL score (enriched CD8+ TILs) was associated with MSI-H tumors and predicted better survival in CRC cohorts. CONCLUSIONS: This study developed a quantitative DNA methylation-based signature that was reliable to evaluate CD8+ TILs and prognosis in CRC. This approach has the potential to be a tool for investigations on CD8+ TILs and a biomarker for therapeutic approaches, including immunotherapy.

The Addition of Preoperative Radiation Is Insufficient for Lateral Pelvic Control in a Subgroup of Patients With Low Locally Advanced Rectal Cancer: A Post Hoc Study of a Randomized Controlled Trial.

BACKGROUND: The local recurrence of rectal cancer has been improved by total mesorectal excision following neoadjuvant chemoradiotherapy. However, in patients with low locally advanced rectal cancer, lateral pelvic recurrence remains to be addressed. OBJECTIVE: This study aimed to determine the efficiency of neoadjuvant radiotherapy in addressing lateral pelvic recurrence and which subgroup of patients might be optimal to receive lateral lymph node dissection. DESIGN: The MRI/CT images were reassessed for lateral lymph node status. The lateral lymph nodes with short axis ≥5 mm and ≥4 mm were considered positive in pretreatment and restaging MRI/CT. SETTING: This was a post hoc analysis of a prospective randomized controlled trial (FOWARC, NCT01211210). PATIENTS: A total of 495 patients with stage II or III rectal adenocarcinoma were included in the original trial. According to the excluding criteria, the finally included population consists of 253 patients; of these, 195 patients received neoadjuvant chemoradiotherapy and 94 received chemotherapy alone. MAIN OUTCOMES AND MEASURES: The primary outcome was the 5-year lateral pelvic recurrence rate. RESULTS: Compared with patients receiving chemotherapy alone, patients receiving additional radiotherapy had a marginal significance of lower lateral pelvic recurrence rate (6.6% vs 13.0%; p = 0.051). In the subset with pretreatment positive lateral lymph nodes, patients had a lateral pelvic recurrence rate of 22.6% and 45.1% after neoadjuvant chemoradiotherapy and chemotherapy alone. Of note, 34.9% of the pretreatment positive lateral lymph nodes were persistent after neoadjuvant chemoradiotherapy, culminating in a lateral pelvic recurrence rate of 63.3%. LIMITATIONS: This is a post hoc analysis, and only the patients from the leading center were included, which limited the sample size. In addition, the lateral lymph node dissection was not performed in this cohort. CONCLUSIONS: The addition of radiotherapy in neoadjuvant regimens could not address lateral pelvic recurrence adequately. Some subgroups of patients might need additional dissection. See Video Abstract at http://links.lww.com/DCR/B613. LA INCLUSION DE LA RADIOTERAPIA PREOPERATORIA ES INSUFICIIENTE EN EL CONTROL PLVICO LATERAL EN UN SUBGRUPO DE PACIENTES CON CNCER DE RECTO INFERIOR LOCALMENTE AVANZADO UN ESTUDIO POSTHOC CONTROLADO Y RANDOMIZADO: ANTECEDENTES:La recurrencia local del cancer de recto ha disminuido al efectuar una excision mesorrectal total seguida de quimioradioterapia neoadyuvante. No obstante, en pacientes con cancer de tercio inferior de recto avanzado localmente, aún está por controlarse la recurrencia pélvicaOBJETIVOS:Determinar la eficacia de la radioterapia neoadyuvante en el control de la recurrencia pélvica lateral y en que subgrupo de pacientes sería conveniente efecutar una excisión lateral de las cadenas ganglionares.DISEÑO:Se reevaluaron las imágenes tomográficas y de resonancia magnética del status de las cadenas ganglionares linfáticas laterales. Los ganglios linfáticos laterales con un eje-corto > 5 mm y ≥ 4 mm se consideraron como positivos previo al tratamiento y reestadificados con RM y TAC respectivamente.ESCENARIO:Es un análisis post hoc de un studio prospectivo randomizado controlado (FOWARC, NCT01211210).PACIENTESSe incluyeron un total de 495 pacientes en estdio II o III con adenomcarcinoma rectal en el estudio original. De acuerdo a los criterios de exclusión, la población final incluida consistió en 253 pacientes; de estos, 195 recibieron quimioradioterapia neoadyuvante y 94 quimioterapia sola.EVALUACION DE LOS RESULTADOS PRINCIPALES:El parámetro mas importante fue la tasa de recurrencia pélvica lateral a cinco años.RESULTADOS:En comparación con los pacientes que recibieron quimioterapia sola, aquellos que además fueron sometidos a radioterapia adicional presentaron un margen significativo de menor tasa de recurrencia pélvica lateral (6.6% vs. 13.0%; p=0.051). En el grupo de pacientes con ganglios linfáticos laterales positivos, los enfermos presentaron una tasa de recurrencia pélvica lateral de 22.6% y 45.1% después de quimioradiaterapia neoadyuvante en comparación con quimioterapia sola respectivamente. Cabe mencionar que el 34.9% de los pacientes con ganglios linfáticos laterales positivos antes del tratamiento persistieron después de la quimioradioterapia neoadyuvante, reportándose finalmente una recurrencia pélvica lateral de un 63.3%.LIMITACIONES:Se trata de un análisis posthoc y solo los pacientes del hospital fueron incluidos, lo que limita el tamaño de la muestra. Además, no se efectuó la disección de los ganglios linfáticos laterales en este grupo.CONCLUSIONES:La radioterapia en los esquemas de neoadyuvancia no logran controlar la recurrencia pélvica lateral en forma adecuada. Algunos subgrupos de pacientes podría requerir de disección adicional. Consulte Video Resumen en http://links.lww.com/DCR/B613.

Genome-wide analysis identifies critical DNA methylations within NTRKs genes in colorectal cancer.

BACKGROUND: Neurotrophic tropomyosin receptor kinases (NTRKs) are a gene family function as oncogene or tumor suppressor gene in distinct cancers. We aimed to investigate the methylation and expression profiles and prognostic value of NTRKs gene in colorectal cancer (CRC). METHODS: An analysis of DNA methylation and expression profiles in CRC patients was performed to explore the critical methylations within NTRKs genes. The methylation marker was validated in a retrospectively collected cohort of 229 CRC patients and tested in other tumor types from TCGA. DNA methylation status was determined by quantitative methylation-specific PCR (QMSP). RESULTS: The profiles in six CRC cohorts showed that NTRKs gene promoter was more frequently methylated in CRC compared to normal mucosa, which was associated with suppressed gene expression. We identified a specific methylated region within NTRK3 promoter targeted by cg27034819 and cg11525479 that best predicted survival outcome in CRC. NTRK3 promoter methylation showed independently predictive value for survival outcome in the validation cohort (P = 0.004, HR 2.688, 95% CI [1.355, 5.333]). Based on this, a nomogram predicting survival outcome was developed with a C-index of 0.705. Furthermore, the addition of NTRK3 promoter methylation improved the performance of currently-used prognostic model (AIC: 516.49 vs 513.91; LR: 39.06 vs 43.64, P = 0.032). Finally, NTRK3 promoter methylation also predicted survival in other tumors, including pancreatic cancer, glioblastoma and stomach adenocarcinoma. CONCLUSIONS: This study highlights the essential value of NTRK3 methylation in prognostic evaluation and the potential to improve current prognostic models in CRC and other tumors.

Improved Survival Outcome and Access to Cancer Screening from Hemorrhoid in Patients with Rectal Cancer.

BACKGROUND: The interventions for hemorrhoid increase access to rectal cancer screening and thus might reduce cancer death. We aimed to examine the impact of hemorrhoid on survival outcomes in rectal cancer. METHODS: We identified 510 patients with stage I to III rectal cancer from a prospectively collected database. Patients were divided into hemorrhoid and non-hemorrhoid group. The primary endpoints were disease-free survival (DFS) and overall survival (OS). RESULTS: Hemorrhoid group had significantly more stage I-II diseases in comparison to nonhemorrhoid group (71.1% vs. 55.9%, P = 0.049). The hemorrhoid group had significantly better DFS and OS compared to nonhemorrhoid group, the hazard ratios (HRs) of which were 0.39 (95% CI 0.17-0.88, P = 0.018) and 0.33 (95% CI 0.12-0.92, P = 0.034), respectively. Multivariate analysis revealed that hemorrhoid was independently associated with DFS [adjusted HR 0.43 (95% CI 0.17-0.95, P = 0.045)]. A nomogram for predicting DFS outcome was generated based on hemorrhoid history, with a concordance index of 0.71 (95% CI 0.66-0.75, P < 0.001). CONCLUSIONS: There may exist a screening effect and survival benefit from hemorrhoid in rectal cancer, which supports the significance of rectal cancer screening in lowering its mortality.

Nomograms for Prediction of Molecular Phenotypes in Colorectal Cancer.

BACKGROUND: Colorectal cancer (CRC) patients with different molecular phenotypes, including microsatellite instability (MSI), CpG island methylator phenotype (CIMP), and somatic mutations in BRAF and KRAS gene, vary in treatment response and prognosis. However, molecular phenotyping under adequate quality control in a community-based setting may be difficult. We aimed to build the nomograms based on easily accessible clinicopathological characteristics to predict molecular phenotypes. METHODS: Three hundred and six patients with pathologically confirmed stage I-IV CRC were included in the cohort. The assays for MSI, CIMP, and mutations in BRAF and KRAS gene were performed using resected tumor samples. The candidate predictors were identified from clinicopathological variables using multivariate Logistic regression analyses to construct the nomograms that could predict each molecular phenotype. RESULTS: The incidences of MSI, CIMP, BRAF mutation and KRAS mutation were 25.3% (72/285), 2.5% (7/270), 3.4% (10/293), and 34.8% (96/276) respectively. In the multivariate Logistic analysis, poor differentiation and high neutrophil/lymphocyte ratio (NLR) were independently associated with MSI; poor differentiation, high NLR and high carcinoembryonic antigen/tumor size ratio (CSR) were independently associated with CIMP; poor differentiation, lymphovascular invasion and high CSR were independently associated with BRAF mutation; poor differentiation, proximal tumor, mucinous tumor and high NLR were independently associated with KRAS mutation. Four nomograms for MSI, CIMP, BRAF mutation and KRAS mutation were developed based on these independent predictors, the C-indexes of which were 61.22% (95% CI: 60.28-62.16%), 95.57% (95% CI: 95.20-95.94%), 83.56% (95% CI: 81.54-85.58%), and 69.12% (95% CI: 68.30-69.94%) respectively. CONCLUSION: We established four nomograms using easily accessible variables that could well predict the presence of MSI, CIMP, BRAF mutation and KRAS mutation in CRC patients.

High platelet-to-lymphocyte ratio predicts improved survival outcome for perioperative NSAID use in patients with rectal cancer.

PURPOSE: Nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to block tumor-associated inflammation in rectal cancer. However, the perioperative use of NSAIDs remains controversial. This study was designed to investigate whether the perioperative use of NSAIDs influences outcomes and to provide a predictive marker to identify patients who would benefit from NSAIDs. METHODS: We enrolled 515 patients with stage I to III rectal cancer in this retrospective study. Patients were classified into the NSAID and non-NSAID groups according to their perioperative use of NSAIDs. The whole cohort was stratified by platelet-to-lymphocyte ratio (PLR). The primary endpoints were disease-free survival (DFS) and overall survival (OS). RESULTS: The NSAID group had a 12.6% lower risk of recurrence than the non-NSAID group (P = 0.015), while the association with survival was nonsignificant. In the high-PLR subset, the NSAID group had a 17.3% lower risk of recurrence (P = 0.003) and a better DFS (P = 0.033) outcome than the non-NSAID group. Multivariate analysis confirmed this independent significant association with DFS (P = 0.023). In the low-PLR subset, the association of NSAID use with survival was nonsignificant. CONCLUSION: Perioperative use of NSAIDs was associated with improved survival outcomes in rectal cancer patients with high PLR.

Prognostic value of preoperative carcinoembryonic antigen/tumor size in rectal cancer.

BACKGROUND: Carcinoembryonic antigen (CEA) is a commonly used biomarker in colorectal cancer. However, controversy exists regarding the insufficient prognostic value of preoperative serum CEA alone in rectal cancer. Here, we combined preoperative serum CEA and the maximum tumor diameter to correct the CEA level, which may better reflect the malignancy of rectal cancer. AIM: To assess the prognostic impact of preoperative CEA/tumor size in rectal cancer. METHODS: We retrospectively reviewed 696 stage I to III rectal cancer patients who underwent curative tumor resection from 2007 to 2012. These patients were randomly divided into two cohorts for cross-validation: training cohort and validation cohort. The training cohort was used to generate an optimal cutoff point and the validation cohort was used to further validate the model. Maximally selected rank statistics were used to identify the optimum cutoff for CEA/tumor size. The Kaplan-Meier method and log-rank test were used to plot the survival curve and to compare the survival data. Univariate and multivariate Cox regression analyses were used to determine the prognostic value of CEA/tumor size. The primary and secondary outcomes were overall survival (OS) and disease-free survival (DFS), respectively. RESULTS: In all, 556 patients who satisfied both the inclusion and exclusion criteria were included and randomly divided into the training cohort (2/3 of 556, n = 371) and the validation cohort (1/3 of 556, n = 185). The cutoff was 2.429 ng/mL per cm. Comparison of the baseline data showed that high CEA/tumor size was correlated with older age, high TNM stage, the presence of perineural invasion, high CEA, and high carbohydrate antigen 19-9 (CA 19-9). Kaplan-Meier curves showed a manifest reduction in 5-year OS (training cohort: 56.7% vs 81.1%, P < 0.001; validation cohort: 58.8% vs 85.6%, P < 0.001) and DFS (training cohort: 52.5% vs 71.9%, P = 0.02; validation cohort: 50.3% vs 79.3%, P = 0.002) in the high CEA/tumor size group compared with the low CEA/tumor size group. Univariate and multivariate analyses identified CEA/tumor size as an independent prognostic factor for OS (training cohort: hazard ratio (HR) = 2.18, 95% confidence interval (CI): 1.28-3.73, P = 0.004; validation cohort: HR = 4.83, 95%CI: 2.21-10.52, P < 0.001) as well as DFS (training cohort: HR = 1.47, 95%CI: 0.93-2.33, P = 0.096; validation cohort: HR = 2.61, 95%CI: 1.38-4.95, P = 0.003). CONCLUSION: Preoperative CEA/tumor size is an independent prognostic factor for patients with stage I-III rectal cancer. Higher CEA/tumor size is associated with worse OS and DFS.

Novel Assay for Quantitative Analysis of DNA Methylation at Single-Base Resolution.

BACKGROUND: The DNA methylation profile provides valuable biological information with potential clinical utility. Several methods, such as quantitative methylation-specific PCR (qMSP), have been developed to examine methylation of specific CpG sites. Existing qMSP-based techniques fail to examine the genomic methylation at a single-base resolution, particularly for loci in gene bodies or extensive CpG open seas lacking flanking CpGs. Therefore, we established a novel assay for quantitative analysis of single-base methylation. METHODS: To achieve a robust single-base specificity, we developed a PCR-based method using paired probes following bisulfite treatment. The 6-carboxyfluorescein- and 2'-chloro-7'phenyl-1,4-dichloro-6-carboxy-fluorescein-labeled probes conjugated with minor groove binder were designed to specifically bind to the methylated and unmethylated allele of targeted single CpGs at their 3' half regions, respectively. The methylation percentage was calculated by values of methylation / (methylation + unmethylation). RESULTS: In the detection of single CpGs within promoters or bodies of 4 human genes, the quantitative analysis of the single-base methylation assay showed a detection capability in the 1 to 1:10000 dilution experiments with linearity over 4 orders of magnitude (R 2 = 0.989-0.994; all P < 0.001). In a cohort of 10 colorectal cancer samples, the assay showed a comparable detection performance with bisulfite pyrosequencing (R 2 = 0.875-0.990; all P < 0.001), which was better than conventional qMSP methods normalized by input control reaction (R 2 = 0.841 vs 0.769; P = 0.002 vs 0.009). CONCLUSIONS: This assay is highly specific and sensitive for determining single-base methylation and, thus, is potentially useful for methylation-based panels in diagnostic and prognostic applications.

Texture-Dependent Adhesion in Polydopamine Nanomembranes.

The conformal nature of in situ polymerization of adhesive dopamine molecules permits the strong underwater adhesion between polydopamine (PDA) nanomembranes and the target substrates. However, the adhesive interaction between the postdeposit PDA nanomembranes and other macrobodies is strongly influenced by the texture of PDA nanomembranes. Here we report the texture-dependent adhesion of PDA nanomembranes both in air and aqueous environments. Despite the nanometer-scale roughness of PDA nanomembranes, interfacial adhesion between PDA nanomembranes and elastomeric bodies are the strong function of the root-mean-square roughness of PDA nanomembranes, root-mean-square gradient of PDA nanomembranes, and the elasticity of the bulk materials. Reduced adhesion due to increased texture is intensified in hydrated conditions, possibly hinting that the conventional explanation of the negative effect of water to adhesion from a molecular level needs to be revisited. These findings can inform the role of adhesive interaction in conformal coatings and provide an explanation for the differential adhesion observed in freestanding PDA nanomembranes.

MicroRNAs Involved in Asthma After Mesenchymal Stem Cells Treatment.

Administration of human bone marrow-derived mesenchymal stem cells (BM-MSCs) significantly alleviates allergic airway inflammation. There are no studies that refer to the role of microRNAs (miRNAs) after the BM-MSCs treatment in airway allergic inflammation. We induced a mouse model of asthma and performed the transplantation of BM-MSCs. We analyzed aberrant miRNAs and key immune regulators using both miRNA and messenger RNA (mRNA) polymerase chain reaction (PCR) arrays. We identified that 296 miRNAs were differently expressed after the induction of asthma and/or the treatment of BM-MSCs, in which 14 miRNAs presented the reverse variation tendency between asthma induction and BM-MSCs transplantation. Mmu-miR-21a-3p, mmu-miR-449c-5p, and mmu-miR-496a-3p were further confirmed to be differently expressed with additional samples and quantitative real-time PCR. With an mRNA PCR array, we identified 19 genes to be involved in the allergy induction and the administration of BM-MSCs. Further target genes analysis revealed that mmu-miR-21a-3p was significantly correlated with the immune regulator activin A receptor, Type IIA (Acvr2a). Mmu-miR-21a-3p had opposite expression with Acvr2a after asthma and BM-MSCs treatment. Acvr2a had binding sites for miR-21a for both mice and human, suggesting that miR-21/Acvr2a axis is conserved between human and mice. Dual-luciferase reporter assay showed that mmu-miR-21a-3p negatively regulated the transcript of Acvr2a. In addition, has-miR-21a inhibitor significantly increased the expression of Acvr2a mRNA in BEAS-2B cells under lipopolysaccharide stimulation. Our results suggest that there were different miRNA and mRNA profiles after asthma induction and BM-MSCs treatment, and the miR-21/Acvr2a axis is an important mechanism for the induction of asthmatic inflammation.

One-step fabrication of inorganic/organic hybrid microspheres with tunable surface texture for controlled drug release application.

In this paper, we report one-step fabrication of poly(lactide-co-glycolic acid)/titanium oxide (PLGA/TiO2) hybrid microspheres with tunable surface texture via droplet-based microfluidics. Surface texture of microspheres can be continuously tuned by changing the mass ratio between titanium tetraisopropoxide (TTIP) and PLGA in the dispersed phase. The fast hydrolysis of TTIP on the droplet surface can generate a thin shell membrane, resulting in a wrinkled surface after extraction of organic solvent. In vitro drug release monitoring of tanshinone IIA-loaded PLGA/TiO2 hybrid microsphere reveals that surface texture can affect the drug release rate to a large extent without sacrificing the drug encapsulation efficiency. Our finding might benefit the sustained drug delivery where variable drug release rate and high drug encapsulation efficiency are both required.

Connexin 43 Upregulation in Mouse Lungs during Ovalbumin-Induced Asthma.

BACKGROUND: Connexin (Cx)-based gap junction channels play important roles in the inflammatory response. Cx43 is involved in the pathogenesis of some lung diseases such as acute lung injury. However, the Cx43 expression in asthma is unclear. In the present study, we used a murine model of ovalbumin (OVA)-induced allergic airway disease to examine the levels of Cx43 and analyze the relationship between Cx43 and airway inflammation in allergic airway disease. METHODS: Asthma was induced in mice via sensitization and challenge with OVA. Cx43 mRNA and protein expression levels were investigated via QT-PCR, western blot, and immunohistochemistry 0 h, 8 h, 1 d, 2 d and 4 d after the first challenge. The relationship between Cx43 protein levels and inflammatory cell infiltration, cytokine levels was analyzed. RESULTS: The OVA-induced mice exhibited typical pathological features of asthma, including airway hyper-responsiveness; strong inflammatory cell infiltration surrounding the bronchia and vessels; many inflammatory cells in the bronchoalveolar lavage fluid (BALF); higher IL-4, IL-5 and IL-13 levels; and high OVA specific IgE levels. Low Cx43 expression was detected in the lungs of control (PBS) mice. A dramatic increase in the Cx43 mRNA and protein levels was found in the asthmatic mice. Cx43 mRNA and protein expression levels increased in a time-dependent manner in asthma mice, and Cx43 was mostly localized in the alveolar and bronchial epithelial layers. Moreover, lung Cx43 protein levels showed a significant positive correlation with inflammatory cell infiltration in the airway and IL-4 and IL-5 levels in the BALF at different time points after challenge. Interestingly, the increase in Cx43 mRNA and protein levels occurred prior to the appearance of the inflammatory cell infiltration. CONCLUSION: Our data suggest that there is a strong upregulation of Cx43 mRNA and protein levels in the lungs in asthma. Cx43 levels also exhibited a positive correlation with allergic airway inflammation. Cx43 may represent a target to treat allergic airway diseases in the future.

Characterization of the genotype and integration patterns of hepatitis B virus in early- and late-onset hepatocellular carcinoma.

UNLABELLED: Early-onset hepatocellular carcinoma (HCC) accounts for 15%-20% of total HCC cases in Asia, and the incidence is increasing. The low frequency of cirrhosis and poor prognosis of early-onset HCC suggests that its mechanisms may differ from late-onset HCC. Although hepatitis B virus (HBV) infection is epidemiologically associated with HCC, the role of HBV in early-onset HCC remains poorly understood. Here, we report a comparative study of HBV subgenotypes and integration in early- (≤30) and late-onset (≥70) HBV-associated HCC using a novel high-throughput viral integration detection method. We report that HBV B2 is predominantly present in early-onset HCC. HBV integration is a common phenomenon, both in early- and late-onset HCC, which favors integrating into human repeat regions. Moreover, we found a breakpoint in 8q24 located between c-Myc and plasmocytoma variant translocation 1 (PVT1), which was detected in 12.4% (14 of 113) of early-onset HCCs, but only 1.4% (2 of 145) in late-onset HCCs. HBV integrating this site results in c-MYC, PVT1, and microRNA-1204 overexpression in tumors, thereby potentially contributing to the development of early-onset HCC. CONCLUSION: HBV genotype and integration patterns may be distinct in early-onset HCC. Our results may shed light on HCC risk factors in young HBV carriers. Further studies are needed to elucidate at which time in tumor development this integration event occurs and whether it plays an important, causative role in HCC development or progression.

Low CADM2 expression predicts high recurrence risk of hepatocellular carcinoma patients after hepatectomy.

PURPOSE: To investigate the expression and clinical significance of CADM2 in hepatocellular carcinomas (HCC). METHODS: The level of expression of CADM2 mRNA was assessed in frozen tumor specimens and adjacent noncancerous tissues from 30 HCC patients by real-time PCR. The protein level was determined by immunohistochemistry on a tissue microarray containing tumor and adjacent noncancerous tissues from 234 HCC patients. Clinicopathological characteristics associated analysis was performed through SPSS18 . RESULTS: CADM2 was strikingly down regulated in HCC. CADM2 expression was associated with differentiation (P = 0.000), serum alpha-fetoprotein (P = 0.003), vascular invasion (P = 0.001), and hepatitis B surface antigen (HBsAg, P = 0.038). Furthermore, patients with low CADM2 expression had significantly poorer recurrence-free survival (RFS) (40.8 and 34.2 % vs. 56.3 and 50.1 % in 3- and 5-year RFS, respectively, P = 0.005). Subgroup analysis revealed that the difference in RFS between groups with low- and high-CADM2 expression still existed among patients belonging to stage 0 or A of BCLC staging system (P = 0.008), patients with tumor ≤5 cm in size (P = 0.013), and alpha-fetoprotein-negative patients (P = 0.003). Moreover, low expression was more frequently observed in the early recurrence group (within 2 years, P = 0.007). Further multivariate Cox regression analysis indicated that CADM2 expression level, tumor size, tumor number, vascular invasion, HBsAg were independent risk factors for HCC recurrence. CONCLUSION: CADM2 serves as a novel predictor of RFS in HCC patients after curative resection.

Hepatitis B virus X protein (HBx)-related long noncoding RNA (lncRNA) down-regulated expression by HBx (Dreh) inhibits hepatocellular carcinoma metastasis by targeting the intermediate filament protein vimentin.

UNLABELLED: The hepatitis B virus X protein (HBx) has been implicated as an oncogene in both epigenetic modifications and genetic regulation during hepatocarcinogenesis, but the underlying mechanisms are not entirely clear. Long noncoding RNAs (lncRNAs), which regulate gene expression with little or no protein-coding capacity, are involved in diverse biological processes and in carcinogenesis. We asked whether HBx could promote hepatocellular carcinoma (HCC) by regulating the expression of lncRNAs. In this study we investigated the alteration in expression of lncRNAs induced by HBx using microarrays and real-time quantitative polymerase chain reaction (PCR). Our results indicate that HBx transgenic mice have a specific profile of liver lncRNAs compared with wildtype mice. We identified an lncRNA, down-regulated expression by HBx (termed lncRNA-Dreh), which can inhibit HCC growth and metastasis in vitro and in vivo, act as a tumor suppressor in the development of hepatitis B virus (HBV)-HCC. LncRNA-Dreh could combine with the intermediate filament protein vimentin and repress its expression, and thus further change the normal cytoskeleton structure to inhibit tumor metastasis. We also identified a human ortholog RNA of Dreh (hDREH) and found that its expression level was frequently down-regulated in HBV-related HCC tissues in comparison with the adjacent noncancerous hepatic tissues, and its decrement significantly correlated with poor survival of HCC patients. CONCLUSION: These findings support a role of lncRNA-Dreh in tumor suppression and survival prediction in HCC patients. This discovery contributes to a better understanding of the importance of the deregulated lncRNAs by HBx in HCC and provides a rationale for the potential development of lncRNA-based targeted approaches for the treatment of HBV-related HCC.