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Supplementary Figure 1. Validation of the lncRNA microarray data using qRT-PCR. Supplementary Figure 2. Expression levels of DZnep-induced lncRNAs in a panel of HCC cell lines and non-tumor cell lines. Supplementary Figure 3. Expression levels of DZnep-induced lncRNAs in paired human HCC tissues and adjacent non-tumor tissues. Supplementary Figure 4. High specificity of ChIP assay in measuring true interaction between EZH2 and the promoters of EZH2 targeted genes. Supplementary Figure 5. Inhibition of TCAM1P-004 and RP11-598D14.1 with siRNAs promoted cell proliferation. Supplementary Figure 6. Knockdown of TCAM1P-004 and RP11-598D14.1 with shRNAs inhibited cell apoptosis, promoted colony formation ability and transformation ability, and stimulated tumor growth in xenograft mice model. Supplementary Figure 7. Overexpression of TCAM1P-004 and RP11-598D14.1 repressed cell colony formation and transformation abilities, and tumor growth in xenograft mice model. Supplementary Figure 8. Overexpression of TCAM1P-004 or RP11-598D14.1 promoted apoptosis in subcutaneous tumors derived from Hep3B cells. Supplementary Figure 9. Inhibition of TCAM1P-004 or RP11-598D14.1 could induce tumorigenesis of non-tumor MIHA cells. Supplementary Figure 10. KEGG pathway enrichment analysis of TCAM1P-004 and RP11-598D14.1 regulated genes. Supplementary Figure 11. qRT-PCR validated signaling pathways related to TCAM1P-004 and RP11-598D14.1. Supplementary Figure 12. Expression of DDIT and PFKFB4 in HCC cell lines and HCC tumor samples. Supplementary Figure 13. Expression of PFKFB4 in HCC tumor samples. Supplementary Figure 14. Alignment analysis between lncRNA TCAM1P-004 and RP11-598D14.1. Supplementary Figure 15. EZH1 showed no significant role in HCC.

Long noncoding RNAs (lncRNA) play critical roles in the development of cancer, including hepatocellular carcinoma (HCC). However, the mechanisms underlying their deregulation remain largely unexplored. In this study, we report that two lncRNAs frequently downregulated in HCC function as tumor suppressors and are epigenetically silenced by histone methyltransferase EZH2. lncRNAs TCAM1P-004 and RP11-598D14.1 were inhibited by EZH-mediated trimethylation of H3K27me3 at their promoters. Downregulation of TCAM1P-004 and RP11-598D14.1 was frequently observed in HCC tumors compared with adjacent normal tissues. Both lncRNAs inhibited cell growth, cell survival, and transformation in HCC cells in vitro as well as tumor formation in vivo. Using RNA pull-down and mass spectrometry, we demonstrated that TCAM1P-004 bound IGF2BP1 and HIST1H1C, whereas RP11-598D14.1 bound IGF2BP1 and STAU1. These lncRNA–protein interactions were critical in regulating p53, MAPK, and HIF1α pathways that promoted cell proliferation in HCC. Overexpression of EZH2 was critical in repressing TCAM1P-004 and RP11-598D14.1, and EZH2-TCAM1P-004/RP11-598D14.1–regulated pathways were prevalent in human HCC. Aberrant suppression of TCAM1P-004 and RP11-598D14.1 led to loss of their tumor-suppressive effects by disrupting the interaction with IGF2BP1, HIST1H1C, and STAU1, which in turn promoted HCC development and progression. Collectively, these findings demonstrate the role of TCAMP1P-004 and RP11-598D14.1 in suppressing tumor growth and suggest that EZH2 may serve as a therapeutic target in HCC.Significance:EZH2-mediated loss of lncRNAs TCAM1P-004 and RP11-598D14.1 hinders the formation of tumor suppressor lncRNA–protein complexes and subsequently promotes HCC growth.

Supplementary Table S1. Primer sequences used for qRT-PCR.

Supplementary Figure Legends. Legend for Supplementary Figures S1-S5.

Supplementary Figures S1-S5. The established miR-34a reporter was responsive to the endogeneous miR-34a level (S1); Rubone activated the expression of miR-34a (S2); Rubone inhibited Huh7 xenografted tumor growth (S3); Rubone activated pri-miR-34a expressions and inhibited angiogenesis in HepG2 xenografts (S4); Knockdown of p53 reversed pri-miR-34a expression and cell growth inhibition induced by Rubone in HepG2 and Bel-7404 cells (S5).

Supplementary Materials and Methods. Description of additional methods and procedures used in the study.

Long noncoding RNAs (lncRNA) play critical roles in the development of cancer, including hepatocellular carcinoma (HCC). However, the mechanisms underlying their deregulation remain largely unexplored. In this study, we report that two lncRNAs frequently downregulated in HCC function as tumor suppressors and are epigenetically silenced by histone methyltransferase EZH2. lncRNAs TCAM1P-004 and RP11-598D14.1 were inhibited by EZH-mediated trimethylation of H3K27me3 at their promoters. Downregulation of TCAM1P-004 and RP11-598D14.1 was frequently observed in HCC tumors compared with adjacent normal tissues. Both lncRNAs inhibited cell growth, cell survival, and transformation in HCC cells in vitro as well as tumor formation in vivo. Using RNA pull-down and mass spectrometry, we demonstrated that TCAM1P-004 bound IGF2BP1 and HIST1H1C, whereas RP11-598D14.1 bound IGF2BP1 and STAU1. These lncRNA–protein interactions were critical in regulating p53, MAPK, and HIF1α pathways that promoted cell proliferation in HCC. Overexpression of EZH2 was critical in repressing TCAM1P-004 and RP11-598D14.1, and EZH2-TCAM1P-004/RP11-598D14.1–regulated pathways were prevalent in human HCC. Aberrant suppression of TCAM1P-004 and RP11-598D14.1 led to loss of their tumor-suppressive effects by disrupting the interaction with IGF2BP1, HIST1H1C, and STAU1, which in turn promoted HCC development and progression. Collectively, these findings demonstrate the role of TCAMP1P-004 and RP11-598D14.1 in suppressing tumor growth and suggest that EZH2 may serve as a therapeutic target in HCC. Significance: EZH2-mediated loss of lncRNAs TCAM1P-004 and RP11-598D14.1 hinders the formation of tumor suppressor lncRNA–protein complexes and subsequently promotes HCC growth.

In order to effectively solve the problem in over-turn of a bedridden person with the assistance of external force, a double bed face- three embedded leave over-turn nursing robot with the flexible compensation was put forward, with the abstraction of the bedridden person as an organism. This robot, on the basis of concept gesture of the person in bed and the state of the robot supporting and proving the gesture with the actions and combination of the two bed faces, held the complete function of over-turn nursing with 7 states corresponding to 5 gestures of the bedridden person obeying the fundamental requirements of safety, rapidity, and comport. The design method of “PS-MM-KD” was proposed for multi-mechanism coalescent system with related specific tasks induced from the original problems with Systems Engineering. Mechanics and Mechanisms, then applied in the concrete sub-system design followed by analysis and verification of both the scheme and the sub-systems in the design, using the Kinematics and Dynamics, implementing the gears, chain wheel, slewing mechanism, screw nut and mortise and tenon joint type clutch mechanism design successfully. Based on those above, a “two-bed face/three-leaf embedded flexible compensation nursing robot” was designed adopting to all ages, people of various kinds of body geometry. PLC, sensor and logic algorithm were used to carry out the control and operation of 7 state-5 posture sequences for realization of the automation and intelligent over-turning in safety, comfort, and convenience.

As is expressed in Part I, Functions and coalescence design of the over-turn nursing robot, the performance and requirements have been put forward with systematic design of several mechanisms. Here, in order to control and function well the over-turn nursing robot, the three-dimensional and five-dimensional Euclidean space with the real number were adopted in terms of sets for gesture of the bedridden person and the corresponding state of the robot, respectively. The matrix method was employed to define and describe the gestures-robot performance and its transition path. The gesture-state sequence matrix not only accurately and clearly expressed the gesture series, state sequence and their corresponding relations, but also laid a theoretical and technical foundation for the path planning from the current gesture to the target one. The control and operation of 7 states and 5 gestures were done to realize the automation and intelligent over-turning safely, comfortably and conveniently.

A simple, rapid, and green method was developed for the simultaneous analysis of polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and polybrominated biphenyl ethers in aquatic products using subcritical 1,1,1,2-tetrafluoroethane extraction coupled with gas chromatography and mass spectrometry. Effects of the extraction temperature, pressure, and cosolvent volume on the extraction efficiency were investigated by extracting spiked oyster samples. The results show that the maximum extraction efficiency was obtained at 40°C, 12 MPa, and a cosolvent (dichloromethane) volume of 5.0 mL. Under these conditions, the calibration curves had good linearity with square of the correlation larger than 0.998 in the concentration range of 5-800 ng/mL; limit of detection and limit of quantitation were 0.16-2.83 and 0.55-9.43 ng/g, respectively. At spiked levels of 10, 30, and 50 ng/g, the average recoveries were 70.4-80.4% for polycyclic aromatic hydrocarbons, 74.0-83.6% for polychlorinated biphenyls, and 66.9-78.0% for polybrominated biphenyl ethers, with average relative standard deviations of less than 16.3%. The established method has no significant differences in recovery compared to traditional methods and is suitable for the analysis of real samples.

The effects of supercritical carbon dioxide (SC-CO2) treatments on Vibrio parahaemolyticus cells were determined using viable plate count method at different treatment times (10 and 40min), pressures (10-25MPa), and temperature (40°C). Using the changes in the physical (absorbance, transmission electron microscope and contents of fatty acids) and chemical indexes (pH value, activity of Na+K+-ATPase, SDS-PAGE) were for further understand the mechanisms of bacterial inactivation under SC-CO2. The result showed that 25MPa treatment for 40min in 40°C could significantly (P

Objectives Cellular aggregates are readily applicable in cell‐based therapy. The effects of agitation and inoculation density on the aggregation of cells in spinner flask and the molecular mechanism of aggregation were investigated. Materials and methods The aggregation kinetics of cells in spinner flask was evaluated with bovine articular chondrocytes (bACs), rabbit bone marrow‐derived mesenchymal stem cells (rMSCs) and their mixture. The morphology of cellular aggregates was studied with scanning electron microscopy and gene expression of cell adhesion‐related molecules was analysed. Results It was shown that suspension culture in spinner flask induced the aggregation of bACs and rMSCs. Both cells exhibited increased aggregation rate and aggregate size with decreasing agitation rate and increasing cell inoculation density. Additionally, aggregate size increased with extended culture time. By analysing gene expression of integrin β1 and cadherin, it was indicated that these molecules were potentially involved in the aggregation process of bACs and rMSCs, respectively. Aggregates composed of both bACs and rMSCs were also prepared, showing rMSCs in the core and bACs in the periphery. Conclusions Cellular aggregates were prepared in dynamic suspension culture using spinner flask, the key parameters to the aggregation process were identified, and the molecular mechanism of aggregation was revealed. This would lay a solid foundation for the large‐scale production of cellular aggregates for cell‐based therapy, such as cartilage regeneration.

Coculture has emerged as a strategy to improve chondrogenesisof mesenchymal stem cells (MSCs) for cartilage regeneration. In the present study, a new system integrating cell encapsulation, dynamic stimulation and coculture with articular chondrocytes (ACs), termed as three-dimensional (3D) dynamic coculture, was developed for chondrogenic differentiation of MSCs. In addition, a novel mechanism was exploited to enable facile separation of cocultured cells. Rabbit bone-marrow derived MSCs (rMSCs) and rabbit ACs (rACs) were encapsualted in alginate gel beads, respectively. In rACs-laden beads, magnetic Fe 3 O 4 nanoparticles were incorporated. Dynamic coculture was performed in a spinner flask. Fe 3 O 4 nanoparticles had no negative effects on growth of rACs, although a slight inhibition on production of glycosaminoglycans (GAG) by rACs was noticed. An alginate concentration of above 1.8 w/v% was favorable for dynamic culture of rMSCs and rACs. Coculturing rMSCs with rACs in chondrogenic medium in this system demonstrated an improved chondrogenesis than monocultured rMSCs according to GAG quantification, histological staining, and gene expression analysis of collagen I, II and X. However, supplementation of 2% FBS did not show posive effects on chondrogenesis of cocultured rMSCs. This proof-of-concept study demonstrates that such a separable 3D dynamic coculture system holds great potential in cartilage tissue regeneration.

BACKGROUNDEarly detection of lung cancer to allow curative treatment remains challenging. Cell-free circulating tumor DNA (ctDNA) analysis may aid in malignancy assessment and early cancer diagnosis of lung nodules found in screening imagery.METHODSThe multi-center clinical study enrolled 192 patients with operable occupying lung diseases. Plasma ctDNA, white blood cell genomic DNA (gDNA) and tumor tissue gDNA of each patient were analyzed by ultra-deep sequencing to an average of 35,000X of the coding regions of 65 lung cancer-related genes.RESULTSThe cohort consists of a quarter of benign lung diseases and three quarters of cancer patients with all histopathology subtypes. 64% of the cancer patients is at Stage I. Gene mutations detection in tissue gDNA and plasma ctDNA results in a sensitivity of 91% and specificity of 88%. When ctDNA assay was used as the test, the sensitivity was 69% and specificity 96%. As for the lung cancer patients, the assay detected 63%, 83%, 94% and 100%, for Stage I, II, III and IV, respectively. In a linear discriminant analysis, combination of ctDNA, patient age and a panel of serum biomarkers boosted the overall sensitivity to 80% at a specificity of 99%. 29 out of the 65 genes harbored mutations in the lung cancer patients with the largest number found in TP53 (30% plasma and 62% tumor tissue samples) and EGFR (20% and 40%, respectively).CONCLUSIONPlasma ctDNA was analyzed in lung nodule assessment and early cancer detection while an algorithm combining clinical information enhanced the test performance.

BackgroundEarly detection of lung cancer to allow curative treatment remains challenging. Cell-free circulating tumour (ct) DNA (ctDNA) analysis may aid in malignancy assessment and early cancer diagnosis of lung nodules found in screening imagery.MethodsThe multicentre clinical study enrolled 192 patients with operable occupying lung diseases. Plasma ctDNA, white cell count genomic DNA (gDNA) and tumour tissue gDNA of each patient were analysed by ultra-deep sequencing to an average of 35 000× of the coding regions of 65 lung cancer-related genes.ResultsThe cohort consists of a quarter of benign lung diseases and three quarters of cancer patients with all histopathology subtypes. 64% of the cancer patients are at stage I. Gene mutations detection in tissue gDNA and plasma ctDNA results in a sensitivity of 91% and specificity of 88%. When ctDNA assay was used as the test, the sensitivity was 69% and specificity 96%. As for the lung cancer patients, the assay detected 63%, 83%, 94% and 100%, for stages I, II, III and IV, respectively. In a linear discriminant analysis, combination of ctDNA, patient age and a panel of serum biomarkers boosted the overall sensitivity to 80% at a specificity of 99%. 29 out of the 65 genes harboured mutations in the patients with lung cancer with the largest number found in TP53 (30% plasma and 62% tumour tissue samples) and EGFR (20% and 40%, respectively).ConclusionPlasma ctDNA was analysed in lung nodule assessment and early cancer detection, while an algorithm combining clinical information enhanced the test performance.Trial registration numberNCT03081741.

Scaffolds capable of providing dual neurotrophic factor (NTF) delivery with different release kinetics, spatial delivery of NTFs at different loci and topographical guidance are promising for enhanced peripheral nerve regeneration. In this study, we have designed and fabricated multi-layered aligned-fiber scaffolds through combining emulsion electrospinning, sequential electrospinning and high-speed electrospinning (HS-ES) to modulate the release behavior of glial cell line-derived growth factor(GDNF) and nerve growth factor (NGF). GDNF and NGF were incorporated into poly(lactic-co-glycolic acid) (PLGA) fibers and poly(D,L-lactic acid) (PDLLA) fibers, respectively. Aligned fibers were obtained in each layer of multi-layered scaffolds and relatively thick tri-layered and tetra-layered scaffolds with controlled layer thickness were obtained. Their morphology, structure, properties, and the in vitro release of growth factors were examined. Dual and spatio-temporal release of GDNF and NGF with different release kinetics from multi-layered scaffolds was successfully demonstrated. High separation efficiency by PDLLA fibrous barrier layer for spatial neurotrophic factor delivery from both tri-layered scaffolds and tetra-layered scaffolds was achieved.

Electrospun fibrous scaffolds have been extensively used as cell-supporting matrices or delivery vehicles for various biomolecules in tissue engineering. Biodegradable scaffolds with tunable degradation behaviors are favorable for various resorbable tissue replacements. In nerve tissue engineering, delivery of growth factors (GFs) such as nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) from scaffolds can be used to promote peripheral nerve repair. In this study, using the established dual-source dual-power electrospinning technique, bicomponent scaffolds incorporated with NGF and GDNF were designed and demonstrated as a strategy to develop scaffolds providing dual GF delivery. NGF and GDNF were encapsulated in poly(D, L-lactic acid) (PDLLA) and poly(lactic-co-glycolic acid) (PLGA) nanofibers, respectively, via emulsion electrospinning. Bicomponent scaffolds with various mass ratios of GDNF/PLGA fibers to NGF/PDLLA fibers were fabricated. Their morphology, structure, properties, and the in vitro degradation were examined. Both types of core?shell structured fibers were evenly distributed in bicomponent scaffolds. Robust scaffolds with varying component ratios were fabricated with average fiber diameter ranging from 307???100 nm to 688???129 nm. The ultimate tensile stress and elastic modulus could be tuned ranging from 0.23???0.07 MPa to 1.41???0.23 MPa, 11.1???3.0 MPa to 75.9???3.3 MPa, respectively. Adjustable degradation was achieved and the weight loss of scaffolds ranged from 9.2% to 44.0% after 42 day degradation test. GDNF and NGF were incorporated with satisfactory encapsulation efficiency and their bioactivity were well preserved. Sustained release of both types of GFs was also achieved.

BackgroundRemarkable advances for clinical diagnosis and treatment in cancers including lung cancer involve cell-free circulating tumor DNA (ctDNA) detection through next generation sequencing. However, before the sensitivity and specificity of ctDNA detection can be widely recognized, the consistency of mutations in tumor tissue and ctDNA should be evaluated. The urgency of this consistency is extremely obvious in lung cancer to which great attention has been paid to in liquid biopsy field.MethodsWe have developed an approach named systematic error correction sequencing (Sec-Seq) to improve the evaluation of sequence alterations in circulating cell-free DNA. Averagely 10 ml preoperative blood samples were collected from 30 patients containing pulmonary space occupying pathological changes by traditional clinic diagnosis. cfDNA from plasma, genomic DNA from white blood cells, and genomic DNA from solid tumor of above patients were extracted and constructed as libraries for each sample before subjected to sequencing by a panel contains 50 cancer-associated genes encompassing 29 kb by custom probe hybridization capture with average depth >40000, 7000, or 6300 folds respectively.ResultsDetection limit for mutant allele frequency in our study was 0.1%. The sequencing results were analyzed by bioinformatic expertise based on our previous studies on the baseline mutation profiling of circulating cell-free DNA and the clinicopathological data of these patients. Among all the lung cancer patients, 78% patients were predicted as positive by ctDNA sequencing when the shreshold was defined as at least one of the hotspot mutations detected in the blood (ctDNA) was also detected in tumor tissue. Pneumonia and pulmonary tuberculosis were detected as negative according to the above standard. When evaluating all hotspots in driver genes in the panel, 24% mutations detected in tumor tissue (tDNA) were also detected in patients blood (ctDNA). When evaluating all genetic variations in the panel, including all the driver genes and passenger genes, 28% detected in tumor tissue (tDNA) were also detected in patients blood (ctDNA). Positive detection rates of plasma ctDNA in stage I lung cancer patients is 85%, compared with 17% of tumor biomarkers.ConclusionWe demonstrated the importance of sequencing both circulating cell-free DNA and genomic DNA in tumor tissue for ctDNA detection in lung cancer currently. We also determined and confirmed the consistency of ctDNA and tumor tissue through NGS according to the criteria explored in our studies. Our strategy can initially distinguish the lung cancer from benign lesions of lung. Our work shows that the consistency will be benefited from the optimization in sensitivity and specificity in ctDNA detection.

Cell-free DNA (cfDNA) in plasma has emerged as a potential important biomarker in clinical diagnostics, particularly in cancer. However, somatic mutations are also commonly found in healthy individuals, which interfere with the effectiveness for cancer diagnostics. This study examined the background somatic mutations in white blood cells (WBC) and cfDNA in healthy controls based on sequencing data from 821 non-cancer individuals and several cancer samples with the aim of understanding the patterns of mutations detected in cfDNA. We determined the mutation allele frequencies in both WBC and cfDNA using a panel of 50 cancer-associated genes that covers 20 K-nucleotide region and ultra-deep sequencing with average depth >40000-fold. Our results showed that most of the mutations in cfDNA were highly correlated to WBC. We also observed that the NPM1 gene was the most frequently mutated gene in both WBC and cfDNA. Our study highlighted the importance of sequencing both cfDNA and WBC to improve the sensitivity and accuracy for calling cancer-related mutations from circulating tumour DNA, and shedded light on developing a strategy for early cancer diagnosis by cfDNA sequencing.

Small molecules that restore the expression of growth-inhibitory microRNAs (miRNA) downregulated in tumors may have potential as anticancer agents. miR34a functions as a tumor suppressor and is downregulated or silenced commonly in a variety of human cancers, including hepatocellular carcinoma (HCC). In this study, we used an HCC cell–based miR34a luciferase reporter system to screen for miR34a modulators that could exert anticancer activity. One compound identified as a lead candidate, termed Rubone, was identified through its ability to specifically upregulate miR34a in HCC cells. Rubone activated miR34a expression in HCC cells with wild-type or mutated p53 but not in cells with p53 deletions. Notably, Rubone lacked growth-inhibitory effects on nontumorigenic human hepatocytes. In a mouse xenograft model of HCC, Rubone dramatically inhibited tumor growth, exhibiting stronger anti-HCC activity than sorafenib both in vitro and in vivo. Mechanistic investigations showed that Rubone decreased expression of cyclin D1, Bcl-2, and other miR34a target genes and that it enhanced the occupancy of p53 on the miR34a promoter. Taken together, our results offer a preclinical proof of concept for Rubone as a lead candidate for further investigation as a new class of HCC therapeutic based on restoration of miR34a tumor-suppressor function. Cancer Res; 74(21); 6236–47. ©2014 AACR.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and chronic hepatitis B virus (HBV) infection is the major risk factor of HCC. The virus encodes HBV X (HBx) protein that plays a critical role in the development of HCC. Studies have revealed numerous HBx-altered genes and signalling pathways that heavily contribute to tumourigenesis of non-tumour hepatocytes. However, the role of HBx in regulating other critical gene regulators such as microRNAs is poorly understood, which impedes the exploration of a complete HBx-associated carcinogenic network. Besides, critical microRNAs that drive the transformation of non-tumour hepatocytes are yet to be identified. Here, we overexpressed C-terminal truncated HBx protein in a non-tumour hepatocyte cell line MIHA, and measured a panel of cancer-associated miRNAs. We observed that oncogenic miR-21 was upregulated upon ectopic expression of this viral protein variant. HBx-miR-21 pathway was prevalent in HCC cells as inhibition of HBx in Hep3B and PLC/PRF/5 cells significantly suppressed miR-21 expression. Subsequently, we showed that the upregulation of miR-21 was mediated by HBx-induced interleukin-6 pathway followed by activation of STAT3 transcriptional factor. The high dependency of miR-21 expression to HBx protein suggested a unique viral oncogenic pathway that could aberrantly affect a network of gene expression. Importantly, miR-21 was essential in the HBx-induced transformation of non-tumour hepatocytes. Inhibition of miR-21 effectively attenuated anchorage-independent colony formation and subcutaneous tumour growth of MIHA cells. Our study suggested that overexpression of miR-21 was critical to promote early carcinogenesis of hepatocytes upon HBV infection.

Cell-based therapy using chondrocytes for cartilage repair suffers from chondrocyte dedifferentiation. In the present study, the effects of an integrated three-dimensional and dynamic culture on rabbit articular chondrocytes were investigated. Cells (passages 1 and 4) were encapsulated in alginate gel beads and cultured in spinner flasks in chondrogenic and chondrocyte growth media. Subcutaneous implantation of the cell-laden beads was performed to evaluate the ectopic chondrogenesis. It was found that cells remained viable after 35 days in the three-dimensional dynamic culture. Passage 1 cells demonstrated a proliferative growth in both media. Passage 4 cells showed a gradual reduction in DNA content in growth medium, which was attenuated in chondrogenic medium. Deposition of glycosaminoglycans (GAG) was found in all cultures. While passage 1 cells generally produced higher amounts of GAG than passage 4 cells, GAG/DNA became similar on day 35 for both cells in growth media. Interestingly, GAG/DNA in growth medium was greater than that in chondrogenic medium for both cells. Based on GAG quantification and gene expression analysis, encapsulated passage 1 cells cultured in growth medium displayed the best ectopic chondrogenesis. Taken together, the three-dimensional and dynamic culture for chondrocytes holds great potential in cartilage regeneration.

e24036Background: Cell-free circulating tumor DNA (ctDNA) for cancer detection drew significant clinical attention. Concordance of mutations in tumor tissue and ctDNA should be evaluated first. Met...

e23057 Background: Cell-free circulating tumor DNA (ctDNA) has emerged as an effective blood-based biomarker for clinical diagnostics in cancers, improving the accuracy and efficiency of cancer treatment for clinic. However, the somatic mutations originated from inheritance or normal biological metabolism in circulating cell-free DNA (cfDNA) in plasma can largely interfere with the detection of ctDNA. The baseline for ctDNA evaluation is urgently needed for accuracy identification of ctDNAs in patients. Methods: Averagely 20 ml blood samples were collected from more than 1200 individuals including healthy volunteers, lung cancer, colorectal cancer, pancreatic cancer, gastric cancer patients, etc. Genomic DNA from white blood cells and cfDNA from plasma were extracted and constructed as sequencing libraries using a panel contains 50 cancer-associated genes, respectively for each sample. Then they are subjected to ultra-deep sequencing with average depth > 40000 folds covering ~21K nucleotide regions. Results: The background somatic mutation frequencies were detected in genomic DNA and cfDNA in healthy controls. The results showed that most of mutations in cfDNA were consistent with those in genomic DNA. Using data from 1200 individuals, we generated the baseline mutation profiling of cfDNA, which was referred in the ctDNA determination in cancer samples, which significantly improved the accuracy of ctDNA detection compared with tissue biopsy. Conclusions: Our studies demonstrated the importance of sequencing both cfDNA and genomic DNA for ctDNA detection in cancers. We also determined the baseline mutation profiling of circulating cfDNA from more than 1200 healthy individuals and confirmed the value of it by comparing with DNA sequencing data in cancer tissue. Our work offers clue on how to improve the detection accuracy of circulating tumor DNA in early cancer diagnosis.

While chondrogenesis of mesenchymal stem cells (MSCs) in vitro has been extensively studied, their participation in cartilage tissue repair remains unresolved. This study was designed to elucidate if MSCs affect the phenotype of articular chondrocytes (ACs). A combination of noncontact coculture modes was developed. Human or rabbit MSCs and rabbit ACs (rACs) were encapsulated in alginate hydrogel beads [three-dimensional (3D)] or cultured in a monolayer [two-dimensional (2D)] and subsequently cocultured in the Transwell(®) system. After coculture, cell morphology, growth, deposition of the cartilaginous extracellular matrix (ECM), and gene expression of rACs were investigated. It was found that upon coculture without a cell-cell contact, both 2D and 3D cultured MSCs dramatically induced the morphological transformation of 2D cultured rACs from round to a spindle-like shape, and however inhibited the generation of cellular aggregates of 3D cultured rACs. Most strikingly, a coculture resulted in a significantly less deposition of the cartilaginous ECM, including glycosaminoglycans and collagen type II by both 2D and 3D cultured rACs. Importantly, similar observations were achieved for rACs cultured in an MSC-conditioned medium, confirming the definite paracrine interactions between MSCs and rACs. Based on the analysis of gene expression, this phenotypic change of rACs was not identical as the dedifferentiation. To the best of our knowledge, this is a first study demonstrating that MSCs could downregulate chondrocytic differentiation of ACs and warrants considerations in cartilage tissue repair.