Ultrasensitive amplification-free detection of circulating miRNA via droplet-based processing of SERS tag-miRNA-magnetic nanoparticle sandwich nanocomplexes on a paper-based electrowetting-on-dielectric platform.

MicroRNAs (miRNAs) have emerged as a promising class of biomarkers for early detection of various cancers, including ovarian cancer. However, quantifying miRNAs in human blood samples is challenging owing to the issues of sensitivity and specificity. In this study, hsa-miR-200a-3p of the miR-200a sub-family, which is a biomarker of ovarian cancer, was used as the analyte to demonstrate the analytical capability of an integrated biosensing platform using an extremely sensitive surface-enhanced Raman scattering (SERS) nanotag-nanoaggregate-embedded beads (NAEBs), magnetic nanoparticles (MNPs), a pair of highly specific locked nucleic acid (LNA) probes, and a semi-automated paper-based electrowetting-on-dielectric (pEWOD) device to provide labor-less and thorough sample cleanup and recovery. A sandwich approach where NAEBs are modified by one LNA-1 probe and MNPs are modified by another LNA-2 probe was applied. Then, the target analyte miRNA-200a-3p was introduced to form a sandwich nanocomplex through hybridization with the pair of LNA probes. The pEWOD device was used to achieve short cleanup time and good recovery of the nanocomplex, bringing the total analysis time to less than 30 min. The detection limit of this approach can reach 0.26 fM through SERS detection. The versatility of this method without the need for RNA extraction from clinical samples is expected to have good potential in detecting other miRNAs.

Exosomal long noncoding RNA MLETA1 promotes tumor progression and metastasis by regulating the miR-186-5p/EGFR and miR-497-5p/IGF1R axes in non-small cell lung cancer.

BACKGROUND: Lung cancer is the most common and deadliest cancer worldwide, and approximately 90% of all lung cancer deaths are caused by tumor metastasis. Tumor-derived exosomes could potentially promote tumor metastasis through the delivery of metastasis-related molecules. However, the function and underlying mechanism of exosomal long noncoding RNA (lncRNA) in lung cancer metastasis remain largely unclear. METHODS: Cell exosomes were purified from conditioned media by differential ultracentrifugation and observed using transmission electron microscopy, and the size distributions were determined by nanoparticle tracking analysis. Exosomal lncRNA sequencing (lncRNA-seq) was used to identify long noncoding RNAs. Cell migration and invasion were determined by wound-healing assays, two-chamber transwell invasion assays and cell mobility tracking. Mice orthotopically and subcutaneously xenografted with human cancer cells were used to evaluate tumor metastasis in vivo. Western blot, qRT‒PCR, RNA-seq, and dual-luciferase reporter assays were performed to investigate the potential mechanism. The level of exosomal lncRNA in plasma was examined by qRT‒PCR. MS2-tagged RNA affinity purification (MS2-TRAP) assays were performed to verify lncRNA-bound miRNAs. RESULTS: Exosomes derived from highly metastatic lung cancer cells promoted the migration and invasion of lung cancer cells with low metastatic potential. Using lncRNA-seq, we found that a novel lncRNA, lnc-MLETA1, was upregulated in highly metastatic cells and their secreted exosomes. Overexpression of lnc-MLETA1 augmented cell migration and invasion of lung cancer. Conversely, knockdown of lnc-MLETA1 attenuated the motility and metastasis of lung cancer cells. Interestingly, exosome-transmitted lnc-MLETA1 promoted cell motility and metastasis of lung cancer. Reciprocally, targeting lnc-MLETA1 with an LNA suppressed exosome-induced lung cancer cell motility. Mechanistically, lnc-MLETA1 regulated the expression of EGFR and IGF1R by sponging miR-186-5p and miR-497-5p to facilitate cell motility. The clinical datasets revealed that lnc-MLETA1 is upregulated in tumor tissues and predicts survival in lung cancer patients. Importantly, the levels of exosomal lnc-MLETA1 in plasma were positively correlated with metastasis in lung cancer patients. CONCLUSIONS: This study identifies lnc-MLETA1 as a critical exosomal lncRNA that mediates crosstalk in lung cancer cells to promote cancer metastasis and may serve as a prognostic biomarker and potential therapeutic target for lung cancer diagnosis and treatment.

Ubiquitin-specific peptidase 5 facilitates cancer stem cell-like properties in lung cancer by deubiquitinating ß-catenin.

BACKGROUND: Lung cancer has the highest mortality rate in the world, and mounting evidence suggests that cancer stem cells (CSCs) are associated with poor prognosis, recurrence, and metastasis of lung cancer. It is urgent to identify new biomarkers and therapeutic targets for targeting lung CSCs. METHODS: We computed the single-sample gene set enrichment analysis (ssGSEA) of 1554 Reactome gene sets to identify the mRNA expression-based stemness index (mRNAsi)-associated pathways using the genome-wide RNA sequencing data of 509 patients from The Cancer Genome Atlas (TCGA) cohort of lung adenocarcinoma (LUAD). Phenotypic effects of ubiquitin-specific peptidase 5 (USP5) on the CSC-like properties and metastasis were examined by in vitro sphere formation assay, migration assay, invasion assay, and in vivo xenografted animal models. Cycloheximide chase assay, co-immunoprecipitation assay, and deubiquitination assay were performed to confirm the effect of USP5 on the deubiquitination of ß-catenin. RESULTS: We demonstrated that USP5 expression were positively correlated with the stemness-associated signatures and poor outcomes in lung cancer specimens. Silencing of endogenous USP5 reduced CSC-like characteristics, epithelial-mesenchymal transition (EMT), and metastasis in vitro and in vivo. Furthermore, USP5 interacted with ß-catenin, which resulted in deubiquitination, stabilization of ß-catenin, and activation of Wnt/ß-catenin pathway. Accordingly, expression of USP5 was positively correlated with the enrichment score of the Wnt/TCF pathway signature in human lung cancer. Silencing of ß-catenin expression suppressed USP5-enhancing sphere formation. Targeting USP5 with the small molecule WP1130 promoted the degradation of ß-catenin, and showed great inhibitory effects on sphere formation, migration, and invasion. Finally, we identified a poor-prognosis subset of tumors characterized by high levels of USP5, Wnt signaling score, and Stemness score in both TCGA-LUAD and Rousseaux_2013 datasets. CONCLUSIONS: These findings reveal a clinical evidence for USP5-enhanced Wnt/ß-catenin signaling in promoting lung cancer stemness and metastasis, implying that targeting USP5 could provide beneficial effects to improve lung cancer therapeutics.

MiR-455-5p suppresses PDZK1IP1 to promote the motility of oral squamous cell carcinoma and accelerate clinical cancer invasion by regulating partial epithelial-to-mesenchymal transition.

BACKGROUND: Lymph node and distant metastasis contribute to poor outcomes in patients with oral squamous cell carcinoma (OSCC). The mechanisms regulating cancer migration and invasion play a key role in OSCC. METHODS: We determined migration and invasion ability of OSCC by wound-healing assay, two-chamber transwell invasion assay and cell mobility tracking and evaluated tumor metastasis in vivo. Western blot (WB), qRT-PCR, RNA-seq, dual-luciferase reporter assays and nuclear/cytoplasmic fractionation were performed to investigate the potential mechanism. Immunohistochimical (IHC) staining determined vimentin and PDZK1IP1 expression in OSCC tissues. RESULTS AND CONCLUSION: In this study, we determined that miR-455-5p was associated with lymph node metastasis and clinical invasion, leading to poor outcomes in patients with OSCC. MiR-455-5p promoted oral cancer cell migration and invasion and induced epithelial-to-mesenchymal transition (EMT). We also identified a new biomarker, PDZK1IP1 (MAP17), that was targeted by miR-455-5p. PDZK1IP1 knockdown led to migration, metastasis, EMT, and increased transforming growth factor-ß signaling in OSCC. In addition, miR-455-5p overexpression and PDZK1IP1 inhibition promoted collective OSCC cell migration. According to data from the Cancer Genome Atlas database and the NCKU-OrCA-40TN data set, miR-455-5p and PDZK1IP1 are positively and negatively correlated, respectively, with partial EMT score. High miR-455-5p expression was associated with high vimentin levels and low MAP17 H-scores. The patients with low MAP17 expression had higher rates of disease recurrence than did patients with high MAP17 expression, especially for patients with clinical invasion risk factors and low MAP17 expression. These results suggest that miR-455-5p suppresses PDZK1IP1 expression and mediates OSCC progression. MiR-455-5p and PDZK1IP1 may therefore serve as key biomarkers and be involved in regulating partial EMT in OSCC cells. PDZK1IP1 expression may also serve as an independent factor that impacts outcomes in patients with clinical risk factors for recurrence.

CASZ1 promotes migration, invasion, and metastasis of lung cancer cells by controlling expression of ITGAV.

CASZ1, a zinc finger transcription factor with two isoforms, is known to play important roles in cardiac and neural development. The abnormal expression of CASZ1 is also frequently found in a variety of tumors but has different effects on different tumors; for example, it acts as a tumor suppressor in neuroblastoma but promotes cancer metastasis in ovarian cancer. However, the effect of CASZ1 in lung cancer, the most lethal cancer, remains unclear. Here, we found that the expression of CASZ1 in lung cancer is positively associated with cancer metastasis and poor prognosis. The overexpression of CASZ1b promotes lung cancer cell migration, invasion, and epithelial-mesenchymal transition and is associated with poor prognosis in lung cancer patients. The knockdown of CASZ1 resulted in the suppression of epithelial-mesenchymal transition, migration, and invasion of lung cancer cells and reduced metastasis in vivo. The results of an RNA-sequencing analysis of CASZ1-silenced cells showed that CASZ1 considerably affected the integrin-mediated pathways. CASZ1 bound to the ITGAV promoter and transcriptionally regulated ITGAV expression. Our findings demonstrate that CASZ1 plays an oncogenic role in lung cancer and that CASZ1 promotes lung cancer migration, invasion and metastasis is mediated by ITGAV.

Phocaeicola oris sp. nov., an anaerobic bacterium isolated from the saliva of a patient with oral squamous cell carcinoma.

A Gram-stain-negative or -positive, strictly anaerobic, non-spore-forming and pleomorphic bacterium (designated 14-104T) was isolated from the saliva sample of a patient with oral squamous cell carcinoma. It was an acid-tolerant neutralophilic mesophile, growing at between 20 and 40 °C (with optimum growth at 30 °C) and pH between pH 3.0 and 7.0 (with optimum growth at pH 6.0-7.0). It contained anteiso-C15 : 0 and C15 : 0 as the major fatty acids. The genome size of strain 14-104T was 2.98 Mbp, and the G+C content was 39.6 mol%. It shared <87 % 16S rRNA sequence similarity, <71 % orthologous average nucleotide identity, <76 % average amino acid identity and <68 %% of conserved proteins with its closest relative, Phocaeicola abscessus CCUG 55929T. Reconstruction of phylogenetic and phylogenomic trees revealed that strain 14-104T and P. abscessus CCUG 55929T were clustered as a distinct clade without any other terminal node. The phylogenetic and phylogenomic analyses along with physiological and chemotaxonomic data indicated that strain 14-104T represents a novel species in the genus Phocaeicola, for which the name Phocaeicola oris sp. nov. is proposed. The type strain is 14-104T (=BCRC 81305T= NBRC 115041T).

Microbiome dysbiosis inhibits carcinogen-induced murine oral tumorigenesis.

Oral cancer is one of the most common cancers worldwide and ranks fourth for the mortality rate of cancers in males in Taiwan. The oral microbiota is the microbial community in the oral cavity, which is essential for maintaining oral health, but the relationship between oral tumorigenesis and the oral microbiota remains to be clarified. This study evaluated the effect of microbiome dysbiosis on oral carcinogenesis in mice, and the impact of the microbiome and its metabolic pathways on regulating oral carcinogenesis. We found that antibiotics treatment decreases carcinogen-induced oral epithelial malignant transformation. Microbiome analysis based on 16S rRNA gene sequencing revealed that the species richness of fecal specimens was significantly reduced in antibiotic-treated mice, while that in the salivary specimens was not decreased accordingly. Differences in bacterial composition, including Lactobacillus animalis abundance, in the salivary samples of cancer-bearing mice was dramatically decreased. L. animalis was the bacterial species that increased the most in the saliva of antibiotic-treated mice, suggesting that L. animalis may be negatively associated with oral carcinogenesis. In functional analysis, the microbiome in the saliva of the tumor-bearing group showed greater potential for polyamine biosynthesis. Immunochemical staining proved that spermine oxidase, an effective polyamine oxidase, was upregulated in mouse oral cancer lesions. In conclusion, oral microbiome dysbiosis may alter polyamine metabolic pathways and reduce carcinogen-induced malignant transformation of the oral epithelium.

Galectin-1 orchestrates an inflammatory tumor-stroma crosstalk in hepatoma by enhancing TNFR1 protein stability and signaling in carcinoma-associated fibroblasts.

Most cases of hepatocellular carcinoma (HCC) arise with the fibrotic microenvironment where hepatic stellate cells (HSCs) and carcinoma-associated fibroblasts (CAFs) are critical components in HCC progression. Therefore, CAF normalization could be a feasible therapy for HCC. Galectin-1 (Gal-1), a ß-galactoside-binding lectin, is critical for HSC activation and liver fibrosis. However, few studies has evaluated the pathological role of Gal-1 in HCC stroma and its role in hepatic CAF is unclear. Here we showed that Gal-1 mainly expressed in HCC stroma, but not cancer cells. High expression of Gal-1 is correlated with CAF markers and poor prognoses of HCC patients. In co-culture systems, targeting Gal-1 in CAFs or HSCs, using small hairpin (sh)RNAs or an therapeutic inhibitor (LLS30), downregulated plasminogen activator inhibitor-2 (PAI-2) production which suppressed cancer stem-like cell properties and invasion ability of HCC in a paracrine manner. The Gal-1-targeting effect was mediated by increased a disintegrin and metalloprotease 17 (ADAM17)-dependent TNF-receptor 1 (TNFR1) shedding/cleavage which inhibited the TNF-α → JNK → c-Jun/ATF2 signaling axis of pro-inflammatory gene transcription. Silencing Gal-1 in CAFs inhibited CAF-augmented HCC progression and reprogrammed the CAF-mediated inflammatory responses in a co-injection xenograft model. Taken together, the findings uncover a crucial role of Gal-1 in CAFs that orchestrates an inflammatory CSC niche supporting HCC progression and demonstrate that targeting Gal-1 could be a potential therapy for fibrosis-related HCC.

A novel DNA aptamer targeting lung cancer stem cells exerts a therapeutic effect by binding and neutralizing Annexin A2.

Cancer remains one of the leading causes of death worldwide. Cancer stem cells (CSCs) are the underlying reason for tumor recurrence, progression, and therapeutic resistance. Aptamers are synthetic single-stranded oligonucleotides that can specifically bind to various molecular targets. Here, we aim to develop an effective aptamer-based biomarker and therapeutic tool that targets CSCs for cancer therapy. We perform whole-cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX) to screen DNA aptamers that specifically bound to lung CSCs, modeled by E-cadherin-silenced A549 cells. We develop a CSC-specific aptamer (AP-9R) specifically recognizing lung CSCs with high affinity and identify Annexin A2, a Ca2+-dependent membrane-binding protein, as its target. Annexin A2 expression was upregulated in lung CSCs and involved in cancer stemness. The expression of Annexin A2 was associated with signatures of stemness and metastasis, as well as poor clinical outcomes, in lung cancer in silico. Moreover, AP-9R decreased Annexin A2 expression and suppressed CSC properties in CSCs in vitro and in vivo. The present findings suggest that Annexin A2 is a CSC marker and regulator, and the CSC-specific aptamer AP-9R has potential theranostic applications for lung cancer.

α-Catulin promotes cancer stemness by antagonizing WWP1-mediated KLF5 degradation in lung cancer.

Background: The cytoskeletal linker protein α-Catulin has been shown to be important for tumor progression in various cancers. However, its role in the regulation of cancer stemness remains unclear. Methods: Phenotypic effects of α-Catulin on the cancer stem cell (CSC)-like properties and metastasis were examined by in vitro sphere formation assay, migration assay, invasion assay, and in vivo xenografted animal models. Yeast two-hybrid assay, co-immunoprecipitation assay, and cycloheximide chase assay were performed to confirm the effect of α-Catulin on the WWP1-mediated degradation of KLF5. CPTAC and TCGA database were analyzed to determine the clinical association of α-Catulin, KLF5, and stemness-associated signatures in lung adenocarcinoma. Results: We report that α-Catulin increases cancer stem-like properties in non-small cell lung cancer (NSCLC). The expression of α-Catulin is elevated in tumor spheres compared to sphere-derived adherent cells and promotes the acquisition of cancer stemness characteristics in vitro and in vivo. Mechanistically, the interaction of α-Catulin and the C-terminal region of Kruppel-like transcription factor KLF5 results in the inhibition of WWP1-mediated degradation of KLF5. Accordingly, increased protein expression of KLF5 is observed in clinical specimens of lung adenocarcinoma with high expression of α-Catulin compared to specimens with low α-Catulin-expression. Knockdown of KLF5 abrogates α-Catulin-driven cancer stemness. α-Catulin is known to interact with integrin-linked kinase (ILK). Notably, an ILK inhibitor disrupts the α-Catulin-KLF5 interaction, promotes the degradation of KLF5, and decreases α-Catulin-driven cancer stemness. Importantly, we identify a CTNNAL1/ILK/KLF5 three-gene signature for predicting poor overall survival in patients with lung adenocarcinoma. Conclusions: These findings reveal a molecular basis of α-Catulin-enhanced KLF5 signaling and highlight a role for α-Catulin in promoting cancer stemness.

Taxonomic and Functional Dysregulation in Salivary Microbiomes During Oral Carcinogenesis.

Exploring microbial community compositions in humans with healthy versus diseased states is crucial to understand the microbe-host interplay associated with the disease progression. Although the relationship between oral cancer and microbiome was previously established, it remained controversial, and yet the ecological characteristics and their responses to oral carcinogenesis have not been well studied. Here, using the bacterial 16S rRNA gene amplicon sequencing along with the in silico function analysis by PICRUSt2 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States 2), we systematically characterized the compositions and the ecological drivers of saliva microbiome in the cohorts of orally healthy, non-recurrent oral verrucous hyperplasia (a pre-cancer lesion), and oral verrucous hyperplasia-associated oral cancer at taxonomic and function levels, and compared them with the re-analysis of publicly available datasets. Diversity analyses showed that microbiome dysbiosis in saliva was significantly linked to oral health status. As oral health deteriorated, the number of core species declined, and metabolic pathways predicted by PICRUSt2 were dysregulated. Partitioned beta-diversity revealed an extremely high species turnover but low function turnover. Functional beta-diversity in saliva microbiome shifted from turnover to nestedness during oral carcinogenesis, which was not observed at taxonomic levels. Correspondingly, the quantitative analysis of stochasticity ratios showed that drivers of microbial composition and functional gene content of saliva microbiomes were primarily governed by the stochastic processes, yet the driver of functional gene content shifted toward deterministic processes as oral cancer developed. Re-analysis of publicly accessible datasets supported not only the distinctive family taxa of Veillonellaceae and Actinomycetaceae present in normal cohorts but also that Flavobacteriaceae and Peptostreptococcaceae as well as the dysregulated metabolic pathways of nucleotides, amino acids, fatty acids, and cell structure were related to oral cancer. Using predicted functional profiles to elucidate the correlations to the oral health status shows superior performance than using taxonomic data among different studies. These findings advance our understanding of the oral ecosystem in relation to oral carcinogenesis and provide a new direction to the development of microbiome-based tools to study the interplay of the oral microbiome, metabolites, and host health.

Autophagy Drives Galectin-1 Secretion From Tumor-Associated Macrophages Facilitating Hepatocellular Carcinoma Progression.

Galectin-1 (Gal-1) is a secretory lectin with pro-tumor activities and is associated strongly with hepatocellular carcinoma (HCC) development. Although Gal-1 is a well-known soluble pro-tumor factor in the tumor microenvironment (TME), the secretion mode of Gal-1 is not clearly defined. On the other hand, in addition to cancer cells, Gal-1 is widely expressed in tumor stromal cells, including tumor-associated macrophages (TAMs). TAMs are a significant component of stromal cells in TME; however, their contributions in producing Gal-1 to TME are still not explored. Here we reveal that TAMs can actively secrete Gal-1 in response to stimuli of HCC cells. Gal-1 produced by TAMs leads to an increase of the systemic level of Gal-1 and HCC tumor growth in mice. Mechanistically, TLR2-dependent secretory autophagy is found to be responsible for Gal-1 secretion from TAMs. Gal-1 acts as a cargo of autophagosomes to fuse with multivesicular bodies via Rab11 and VAMP7-mediated vesicle trafficking before being secreted. This autophagy-regulated Gal-1 secretion in TAMs correlates to poor overall survival and progression-free survival rates of HCC patients. Our findings uncover the secretion mode of Gal-1 via secretory autophagy and highlight the pathological role of TAM-produced Gal-1 in HCC progression.

Malignant transformation of oral potentially malignant disorders in Taiwan: An observational nationwide population database study.

ABSTRACT: Oral cancer is one of the leading causes of cancer death, which are mostly preceded by oral potentially malignant disorders (OPMDs). Taiwanese government launched a free oral cancer screening program. The aim of this study was to analyze the malignant transformation rate of OPMDs.This study was based on national-wide oral screening databases. 3,362,232 people were enrolled. Patients clinically diagnosed with leukoplakia, erythroplakia, oral submucosal fibrosis (OSF), oral verrucous hyperplasia (OVH), and oral lichen planus (OLP), from 2010 to 2013, were identified. We followed up OPMD patients in cancer registry databases to analyze the malignant transformation rate.The malignant transformation rates from the highest to the lowest were: OVH > OSF > erythroplakia > OLP > leukoplakia. The malignant transformation rate was 24.55, 12.76, 9.75, 4.23, and 0.60 per 1000 person-years in the OVH, OSF, erythroplakia, leukoplakia, and comparison cohort. The hazard ratio was 8.19 times higher in the OPMD group compared with comparison cohort group, after age and habit adjustment. Female patients with OPMDs had a high risk of malignant transformation.Nationwide screening is very important for early diagnosis. OVH had the highest malignant transformation possibility. Female OPMD patients are a rare but have a relatively high malignant transformation rate.

The polyhydroxyalkanoate (PHA) synthase 1 of Pseudomonas sp. H9 synthesized a 3-hydroxybutyrate-dominant hybrid of short- and medium-chain-length PHA.

Pseudomonas sp. H9 (H9), an environmental strain isolated from a riverbank soil sample collected in southern Taiwan, is Gram-negative and shares a 99 % sequence identity to Pseudomonas putida KT2440 based on 16S rRNA gene analysis. H9 produced novel polyhydroxyalkanoates (PHA) including a hybrid PHA, comprised of 3-hydroxyvalerate (37 mol%) and medium-chain-length (MCL) monomers from valerate, as well as a 3-hydroxyhexanoate-dominant (93 mol%) MCL-PHA from hexanoate. Next-generation sequencing analysis showed H9 had a typical class II PHA operon, consisted of phaC1H9-phaZH9-phaC2H9, in which phaC1H9 was the sole active PHA synthase in H9. Deletion of phaC1H9 gene led to a complete loss of its PHA accumulation capability. Knockout of phaC2H9 gene, in contrast, affected neither bacterial growth nor PHA accumulation. When co-expressed with the phaAB genes of Ralstonia eutropha H16 in the PHA mutant strain Pseudomonas sp. H9ΔC1, phaC1H9 synthesized a hybrid PHA consisted of 3-hydroxybutyrate (3HB) (75 mol%) and MCL-monomers, confirmed in analyses using hot-acetone fractionation and 13C-NMR spectroscopy. As a novel PHA synthase, PhaC1H9 possesses a broad substrate specificity to synthesize a hybrid of SCL- and MCL-PHA, known to have many mechanical properties for potential applications.

DNA methylation maintains the CLDN1-EPHB6-SLUG axis to enhance chemotherapeutic efficacy and inhibit lung cancer progression.

The loss of cancer-cell junctions and escape from the primary-tumor microenvironment are hallmarks of metastasis. A tight-junction protein, Claudin 1 (CLDN1), is a metastasis suppressor in lung adenocarcinoma. However, as a metastasis suppressor, the underlying molecular mechanisms of CLDN1 has not been well studied. Methods: The signaling pathway regulated by CLDN1 was analyzed by Metacore software and validated by immunoblots. The effect of the CLDN1-EPHB6-ERK-SLUG axis on the formation of cancer stem-like cells, drug resistance and metastasis were evaluated by sphere assay, aldefluor assay, flow cytometry, migration assay, cytotoxicity, soft agar assay, immunoprecipitation assay and xenograft experiments. Furthermore, the methylation-specific PCR, pyrosequencing assay, chromatin immunoprecipitation and reporter assay were used to study the epigenetic and RUNX3-mediated CLDN1 transcription. Finally, the molecular signatures of RUNX3/CLDN1/SLUG were used to evaluate the correlation with overall survival by using gene expression omnibus (GEO) data. Results: We demonstrated that CLDN1 repressed cancer progression via a feedback loop of the CLDN1-EPHB6-ERK1/2-SLUG axis, which repressed metastasis, drug resistance, and cancer stemness, indicating that CLDN1 acts as a metastasis suppressor. CLDN1 upregulated the cellular level of EPHB6 and enhanced its activation, resulting in suppression of ERK1/2 signaling. Interestingly, DNA hypermethylation of the CLDN1 promoter abrogated SLUG-mediated suppression of CLDN1 in low-metastatic cancer cells. In contrast, the histone deacetylase inhibitor trichostatin A or vorinostat facilitated CLDN1 expression in high-metastatic cancer cells and thus increased the efficacy of chemotherapy. Combined treatment with cisplatin and trichostatin A or vorinostat had a synergistic effect on cancer-cell death. Conclusions: This study revealed that DNA methylation maintains CLDN1 expression and then represses lung cancer progression via the CLDN1-EPHB6-ERK1/2-SLUG axis. Because CLDN1 enhances the efficacy of chemotherapy, CLDN1 is not only a prognostic marker but a predictive marker for lung adenocarcinoma patients who are good candidates for chemotherapy. Forced CLDN1 expression in low CLDN1-expressing lung adenocarcinoma will increase the chemotherapy response, providing a novel therapeutic strategy.

A fiber optic nanoplasmonic biosensor for the sensitive detection of ampicillin and its analogs.

A novel optical immunosensor for the screening of ampicillin (Amp) residues has been developed. The biosensor is based on fiber optic particle plasmon resonance detection and uses an enhancement method called as fiber optic nanogold-linked immunosorbent assay (FONLISA) for the sensitive detection of antibiotics. A commercial antibody which had a higher affinity for ampicillin than for other ß-lactam antibiotics was chosen. A surface competitive binding assay was used in which a fixed concentration of antibiotic-conjugated gold nanoparticles (AuNPs) competes with free unlabeled antibiotic molecules to measure the amount of binding with antibody molecules immobilized on an optical fiber. The synthesis of the 11-mercaptoundecanoic acid (MUA)-ampicillin conjugate facilitates the attachment of the Amp molecules to AuNPs via MUA which acts as a linker between them. This AuNP-Amp conjugate was then used for the detection of ß-lactam antibiotics. The practical limit of detection obtained for Amp was 0.74 ppb (7.4 × 10-10 g/mL) which is lower than the recommended maximum residue limit (MRL) for ß-lactams. The method also shows a wide linear range of 4 orders. Its applicability to the determination of ampicillin in spiked milk samples has been demonstrated with good recovery and reproducibility. Graphical abstract.

Development of microfluidic concentrator using ion concentration polarization mechanism to assist trapping magnetic nanoparticle-bound miRNA to detect with Raman tags.

MicroRNAs (miRNAs) are small noncoding single-stranded ribonucleic acid molecules. This type of endogenous oligonucleotide could be secreted into the circulation and exist stably. The detection of specific miRNAs released by cancer cells potentially provides a noninvasive means to achieve early diagnosis and prognosis of cancers. However, the typical concentration of miRNAs in blood is below the ultratrace level. This study uses a simple thermoplastic microfluidic concentration device based on an ion concentration polarization mechanism to perform enrichment and cleanup and Raman sensing beads to determine miRNA quantitatively. One sample solution containing target miRNA molecules having been hybridized with two nucleotide probes, where one probe is on a Raman tag of a nanoaggregate embedded bead (NAEB) and the other probe is on a magnetic nanoparticle (MNP), is first filled into the device. When an external field is applied across a cation exchange membrane stationed in the middle conduit of the device, the MNP-miRNA-NAEB complexed particles are enriched near the membrane edge of the cathode side. The concentrated complexed particles are further trapped using an external magnet to perform washing steps to remove excess noncomplexed NAEBs. When cleanup steps are accomplished, the remaining complexed particles are loaded into one detection capillary to acquire Raman signals from the sensing beads. Compared with that using a conventional magnetic trapping device, the cleanup time is shortened from nearly an hour to less than 10 min. Sample loss during the washing steps becomes more controllable, resulting in adequate standard curve linearity (R > 0.99) ranging from 1 to 100 pM.

MicroRNA-150-5p promotes cell motility by inhibiting c-Myb-mediated Slug suppression and is a prognostic biomarker for recurrent ovarian cancer.

Treatment of ovarian cancer (OvCa) remains challenging owing to its high recurrence rates. Detachment of cancer cells into the peritoneal fluid plays a key role in OvCa relapse, but how this occurs remains incompletely understood. Here we examined global miRNA expression profiles of paired primary/recurrent OvCa specimens and identified a novel biomarker, microRNA-150-5p (miR-150-5p), that was significantly upregulated in 16 recurrent OvCa tissues compared with their matched primary specimens. Analyses of cohorts from two other groups confirmed that expression of miR-150-5p was associated with early relapse and poor survival of OvCa patients. Inhibition of miR-150-5p significantly inhibited the migration and invasion of OvCa cells and induced a mesenchymal-epithelial transition (MET) phenotype. We demonstrated that the proto-oncogene, MYB, is an miR-150-5p target in OvCa cells and that the miR-150-5p/c-Myb/Slug axis plays important roles in regulating epithelial-mesenchymal transition (EMT) in OvCa cells. Expression of MYB was significantly correlated with good clinical outcome in OvCa and was negatively correlated with Slug expression in late-stage clinical specimens. These results suggest that miR-150-5p upregulation mediates the progression of recurrent OvCa by targeting the c-Myb/Slug pathway. Inhibition of miR-150-5p may serve as a new therapeutic strategy for preventing recurrence of OvCa.

A DNA Aptamer Targeting Galectin-1 as a Novel Immunotherapeutic Strategy for Lung Cancer.

Galectin-1 (Gal-1) is a pleiotropic homodimeric ß-galactoside-binding protein with a single carbohydrate recognition domain. It has been implicated in several biological processes that are important during tumor progression. Several lines of evidence have indicated that Gal-1 is involved in cancer immune escape and induces T cell apoptosis. These observations all emphasized Gal-1 as a novel target for cancer immunotherapy. Here, we developed a novel Gal-1-targeting DNA aptamer (AP-74 M-545) and demonstrated its antitumor effect by restoring immune function. AP-74 M-545 binds to Gal-1 with high affinity. AP-74 M-545 targets tumors in murine tumor models but suppresses tumor growth only in immunocompetent C57BL/6 mice, not in immunocompromised non-obese diabetic (NOD)/severe combined immunodeficiency (SCID) mice. Immunohistochemistry revealed increased CD4+ and CD8+ T cells in AP-74 M-545-treated tumor tissues. AP-74 M-545 suppresses T cell apoptosis by blocking the binding of Gal-1 to CD45, the main receptor and apoptosis mediator of Gal-1 on T cells. Collectively, our data suggest that the Gal-1 aptamer suppresses tumor growth by blocking the interaction between Gal-1 and CD45 to rescue T cells from apoptosis and restores T cell-mediated immunity. These results indicate that AP-74 M-545 may be a potential strategy for cancer immunotherapy.

Biogeochemical controls of surface ocean phosphate.

Surface ocean phosphate is commonly below the standard analytical detection limits, leading to an incomplete picture of the global variation and biogeochemical role of phosphate. A global compilation of phosphate measured using high-sensitivity methods revealed several previously unrecognized low-phosphate areas and clear regional differences. Both observational climatologies and Earth system models (ESMs) systematically overestimated surface phosphate. Furthermore, ESMs misrepresented the relationships between phosphate, phytoplankton biomass, and primary productivity. Atmospheric iron input and nitrogen fixation are known important controls on surface phosphate, but model simulations showed that differences in the iron-to-macronutrient ratio in the vertical nutrient supply and surface lateral transport are additional drivers of phosphate concentrations. Our study demonstrates the importance of accurately quantifying nutrients for understanding the regulation of ocean ecosystems and biogeochemistry now and under future climate conditions.