Acellular embryoid body and hydroxybutyl chitosan composite hydrogels promote M2 macrophage polarization and accelerate diabetic cutaneous wound healing.

Diabetic wound healing is delayed due to persistent inflammation, and macrophage-immunomodulating biomaterials can control the inflammatory phase and shorten the healing time. In this study, acellular embryoid bodies (aEBs) were prepared and mixed with thermosensitive hydroxybutyl chitosan (HBC) hydrogels to produce aEB/HBC composite hydrogels. The aEB/HBC composite hydrogels exhibited reversible temperature-sensitive phase transition behavior and a hybrid porous network. In vitro analysis showed that the aEB/HBC composite hydrogels exhibited better antimicrobial activity than the PBS control, aEBs or HBC hydrogels and promoted M0 to M2 polarization but not M1 to M2 macrophage repolarization in culture. The in vivo results showed that the aEB/HBC composite hydrogels accelerated cutaneous wound closure, re-epithelialization, ingrowth of new blood vessels, and collagen deposition and reduced the scar width during wound healing in diabetic mice over time. Macrophage phenotype analysis showed that the aEB/HBC composite hydrogels induce M2 macrophage reactions continually, upregulate M2-related mRNA and protein expression and downregulate M1-related mRNA and protein expression. Therefore, the aEB/HBC composite hydrogels have excellent antimicrobial activity, promote M2 macrophage polarization and accelerate the functional and structural healing of diabetic cutaneous wounds.

Visible-Light-Induced, Palladium-Mediated Desaturation/Sulfonation Cascade To Access 4-Sulfonyltetrahydropyridine Scaffolds.

Herein, we report a visible-light-induced, palladium-catalyzed desaturation/sulfonation cascade, offering a concise route to a series of highly valuable 4-sulfonyltetrahydropyridine scaffolds from inexpensive and readily available piperidine derivatives with sodium sulfinates. The key to the success of this transformation is the well-designed sequence of palladium-mediated 1,5-hydrogen atom transfer/ß-hydride elimination/allylic sulfonation process, which demonstrates the synthetic potentials for orchestrating synthetic events by rationally taking advantage of varied catalytic modes.

Diastereoselective Access to Seven-Membered Benzosultams via Palladium-Catalyzed Ring-Opening [3 + 2]-Annulation.

A palladium-catalyzed ring-opening [3 + 2]-annulation of spirovinylcyclopropanyl oxindoles with seven-membered cyclic N-sulfonylimines has been developed. A wide range of seven-membered benzosultams featuring both a quaternary center and axially chiral biaryl scaffolds have been afforded in an average yield of 87% with moderate to excellent diastereoselectivities. The enantioenriched benzosultams were also accessed successfully in good yields with excellent atropoenantioselectivities enabled by the Pd2(dba)3/(S,S,S)-SKP ligand. The practical utility of this protocol was further demonstrated by the gram-scale reaction and diversified synthetic transformations of the desired seven-membered benzosultam.

[Discussion on "sugar needle - comfortable acupuncture and moxibustion"].

The cases of feeling comfort during acupuncture and moxibustion treatment in literature were summarized and its biological basis was explored. A simple classification of comfort was made, and the importance of obtaining comfort in acupuncture treatment was pointed out. Considering the pursuit of less pain and harmlessness in modern clinical treatment, sugar needle should be advocated and popularized in current clinical practice of acupuncture and moxibustion.

Role of carnitine in adaptation of Chromohalobacter salexigens DSM 3043 and its mutants to osmotic and temperature stress in defined medium.

L-Carnitine is widespread in nature, but little information is available on its metabolism and physiological functions in moderate halophiles. In this study, we found that Chromohalobacter salexigens DSM 3043 could utilize carnitine not only as a nutrient, but also as an osmolyte. When grown at 37 °C under salt-stress conditions, the strain utilized carnitine as an osmoprotectant by enzymatically converting it into GB. When grown at low and high temperature, both carnitine and its metabolic intermediate GB were simultaneously accumulated intracellularly, serving as cryoprotectants and thermoprotectants. The genes (csal_3172, csal_3173, and csal_3174) which were predicted to participate in L-carnitine degradation to GB were deleted to construct the corresponding mutants. The effects of salinity and temperature on the growth rates and cytoplasmic solute pools of the C. salexigens wild-type and mutant strains were investigated. 13C-NMR analysis revealed that GB was still detected in the Δcsal_3172Δcsal_3173Δcsal_3174 mutant grown in a defined medium with added DL-carnitine, but not with L-carnitine, indicating that an unidentified D-carnitine degradation pathway exists in C. salexigens. Taken together, the data presented in this study expand our knowledge on carnitine metabolism and its physiological functions in C. salexigens exposed to single or multiple environmental abiotic stress.

Effect of naturally derived surgical hemostatic materials on the proliferation of A549 human lung adenocarcinoma cells.

Hemostatic materials are generally applied in surgical operations for cancer, but their effects on the growth and recurrence of tumors are unclear. Herein, three commonly used naturally derived hemostatic materials, gelatin sponge, Surgicel (oxidized regenerated cellulose), and biopaper (mixture of sodium hyaluronate and carboxymethyl chitosan), were cocultured with A549 human lung adenocarcinoma cells in vitro. Furthermore, the performance of hemostatic materials and the tumorigenicity of the materials with A549 â€‹cells were observed after subcutaneous implantation into BALB/c mice. The in vitro results showed that biopaper was dissolved quickly, with the highest cell numbers at 2 and 4 days of culture. Gelatin sponges retained their structure and elicited the least cell infiltration during the 2- to 10-day culture. Surgicel partially dissolved and supported cell growth over time. The in vivo results showed that biopaper degraded rapidly and elicited an acute Th1 lymphocyte reaction at 3 days after implantation, which was decreased at 7 days after implantation. The gelatin sponge resisted degradation and evoked a hybrid M1/M2 macrophage reaction at 7-21 days after implantation, and a protumor M2d subset was confirmed. Surgicel resisted early degradation and caused obvious antitumor M2a macrophage reactions. Mice subjected to subcutaneous implantation of A549 â€‹cells and hemostatic materials in the gelatin sponge group had the largest tumor volumes and the shortest overall survival (OS), while the Surgicel and the biopaper group had the smallest volumes and the longest OS. Therefore, although gelatin sponges exhibited cytotoxicity to A549 â€‹cells in vitro, they promoted the growth of A549 â€‹cells in vivo, which was related to chronic M2d macrophage reaction. Surgicel and biopaper inhibited A549 â€‹cell growth in vivo, which is associated with chronic M2a macrophage reaction or acute Th1 lymphocyte reaction.

Photoredox-Catalyzed Cascade of o-Hydroxyarylenaminones to Access 3-Aminated Chromones.

Reported herein is a photoredox-catalyzed amination of o-hydroxyarylenaminones with tert-butyl ((perfluoropyridin-4-yl)oxy)carbamate, a versatile amidyl-radical precursor developed in our laboratory. This work establishes a new cascade pathway for the assembly of a range of 3-aminochromones under mild conditions. Downstream transformations of the obtained 3-aminochromones to construct diverse amino pyrimidines greatly broaden the applications of this photocatalyzed protocol.

Exosome-based rare earth nanoparticles for targeted in situ and metastatic tumor imaging with chemo-assisted immunotherapy.

In this research, a tumor exosome system DOX/2DG@E-RENPs with good biocompatibility, low immunogenicity, and a high targeting effect was proposed for theranostics with high chemo-/starvation/immunotherapy efficiency. DOX and 2-deoxy-D-glucose (DOX/2DG) together with rare earth nanoparticles (RENPs) can be simultaneously carried on the exosome by endocytosis of tumor cells and then exocytosis in vitro. This platform has a good monodispersity with an average size of 70 nm, and the system can emit upconversion luminescence and NIR II luminescence under a single NIR laser. In particular, this exosome can target homing cancer cells and kill the origin tumor cells. The strong targeting effect was proved by different cell lines with exosomes from different orthogonal cells (normal/cancer cells and human/mouse sources, respectively), and the in vivo NIR II imaging guided targeted cancer imaging and liver metastases can be realized by intravenous injection of E-RENPs. Furthermore, the good targeted therapeutic effect and in vivo NIR II imaging and metastases of this platform can be proved. The chemotherapy, starvation therapy, and immunotherapy (immune checkpoint inhibitors of an anti-PD-L1 antibody) could achieve effective synergistic therapy for lung adenocarcinoma, and the immunotherapy can be further proved by the clinical data. This will provide a new strategy for the precise targeting and treatment of tumors.

Targeted Luminescent Probes for Precise Upconversion/NIR II Luminescence Diagnosis of Lung Adenocarcinoma.

In this research, the antibody of the searched hub genes has been proposed to combine with a rare-earth composite for an upconversion luminescence (UCL) and downconversion (DCL) NIR-II imaging strategy for the diagnosis of lung adenocarcinoma (LUAD). Weighted gene co-expression network analysis is used to search the most relevant hub genes, and the required top genes that contribute to tumorigenesis (negative: CLEC3B, MFAP4, PECAM1, and FHL1; positive: CCNB2, CDCA5, HMMR, and TOP2A) are identified and validated by survival analysis and transcriptional and translational results. Meanwhile, fluorescence imaging probes (NaYF4:Yb,Er,Eu@NaYF4:Nd, denoted as NYF:Eu NPs) with multimodal optical imaging properties of downconversion and upconversion luminescence in the visible region and luminescence in the near infrared II region are designed with various uniform sizes and enhanced penetration and sensitivity. Finally, when the NYF:Eu NP probe is combined with antibodies of these chosen positive hub genes (such as, TOP2A and CCNB2), the in vitro and in vivo animal experiments (flow cytometry, cell counting kit-8 assay using A549 cells, and in vivo immunohistochemistry IHC microscopy images of LUAD from patient cases) indicate that the designed nanoprobes can be excellently used as a targeted optical probe for future accurate diagnosis and surgery navigation of LUAD in contrast with other cancer cells and normal cells. This strategy of antibodies combined with optical probes provides a dual-modal luminescence imaging method for precise medicine.

Association of drinking behavior and self injury behavior in adolescents / 中国学校卫生

Objective@#To explore the association between drinking behavior and self injury behavior in adolescents.@*Methods@#A total of 9 247 students from 4 middle schools were investigated. Drinking behavior and self injury behavior were collected from questionnaire survey. Univariate and multivariate Logistic regression analysis were used to analyze the relationship between drinking behavior and self injury behavior.@*Results@#Among the 9 247 middle school students, 52.8% reported ever drinking, 24.9% reported drinking behavior in the past 30 days, and 14.6% reported been drunk in the past year. The average age of drinking for the first time was 12.47±3.05. About 47.2% of the participants had self injury behavior. Male with younger drinking age ( OR =1.52), had been drunken ( OR =1.35) and frequent drinking ( OR =1.54) increased the incidence of self injury. Female reported drinking at younger age ( OR =1.69), had been drunk ( OR =1.82) and lived in cities and towns ( OR =1.20) had a higher risk of self injury.@*Conclusion@#Drinking at younger age, heavy and frequent drinking are associated with higher risk of self injury in adolescents in sex specific fashion.

[Distribution of Antibiotic Concentration in Domestic Wastewater Treatment Facilities in Villages and Towns].

Antibiotics discharged into the environment cause increased environmental resistance. Four types of antibiotics (quinolones, tetracyclines, macrolides, and ß-lactams) were selected for this study. In a comparison with the municipal wastewater plant, the concentration and removal of antibiotics in influent and effluent of domestic wastewater treatment facilities of different scales in villages and towns was investigated using high-performance liquid chromatography (HPLC) and tandem mass spectrometry (MS). The results showed that the highest amount of ofloxacin in rural wastewater treatment facilities reached 32663.5 ng·L-1. Due to the different situations of influent fluctuation, discharge requirements, and management between urban and rural wastewater plants, only 33% of the rural domestic wastewater facilities detected an antibiotics removal rate of more than 60%. The effective removal of some antibiotics can be achieved when the rural domestic wastewater treatment facilities maintain the standard discharge of conventional pollutants.

Role of N,N-Dimethylglycine and Its Catabolism to Sarcosine in Chromohalobacter salexigens DSM 3043.

Chromohalobacter salexigens DSM 3043 can grow on N,N-dimethylglycine (DMG) as the sole C, N, and energy source and utilize sarcosine as the sole N source under aerobic conditions. However, little is known about the genes and enzymes involved in the conversion of DMG to sarcosine in this strain. In the present study, gene disruption and complementation assays indicated that the csal_0990, csal_0991, csal_0992, and csal_0993 genes are responsible for DMG degradation to sarcosine. The csal_0990 gene heterologously expressed in Escherichia coli was proven to encode an unusual DMG dehydrogenase (DMGDH). The enzyme, existing as a monomer of 79 kDa with a noncovalently bound flavin adenine dinucleotide, utilized both DMG and sarcosine as substrates and exhibited dual coenzyme specificity, preferring NAD+ to NADP+ The optimum pH and temperature of enzyme activity were determined to be 7.0 and 60°C, respectively. Kinetic parameters of the enzyme toward its substrates were determined accordingly. Under high-salinity conditions, the presence of DMG inhibited growth of the wild type and induced the production and accumulation of trehalose and glucosylglycerate intracellularly. Moreover, exogenous addition of DMG significantly improved the growth rates of the four DMG- mutants (Δcsal_0990, Δcsal_0991, Δcsal_0992, and Δcsal_0993) incubated at 37°C in S-M63 synthetic medium with sarcosine as the sole N source. 13C nuclear magnetic resonance (13C-NMR) experiments revealed that not only ectoine, glutamate, and N-acetyl-2,4-diaminobutyrate but also glycine betaine (GB), DMG, sarcosine, trehalose, and glucosylglycerate are accumulated intracellularly in the four mutants.IMPORTANCE Although N,N-dimethylglycine (DMG) dehydrogenase (DMGDH) activity was detected in cell extracts of microorganisms, the genes encoding microbial DMGDHs have not been determined until now. In addition, to our knowledge, the physiological role of DMG in moderate halophiles has never been investigated. In this study, we identified the genes involved in DMG degradation to sarcosine, characterized an unusual DMGDH, and investigated the role of DMG in Chromohalobacter salexigens DSM 3043 and its mutants. Our results suggested that the conversion of DMG to sarcosine is accompanied by intramolecular delivery of electrons in DMGDH and intermolecular electron transfer between DMGDH and other electron acceptors. Moreover, an unidentified methyltransferase catalyzing the production of glycine betaine (GB) from DMG but sharing no homology with the reported sarcosine DMG methyltransferases was predicted to be present in the cells. The results of this study expand our understanding of the physiological role of DMG and its catabolism to sarcosine in C. salexigens.

An optimized lanthanide-chlorophyll nanocomposite for dual-modal imaging-guided surgery navigation and anti-cancer theranostics.

In this research, an optimized phosphor combined with naturally green chlorophyll (Phosphor-Chlorophyll, denoted as Ph-chl) was designed for dual-modal luminescence-guided anti-tumor surgery and photodynamic therapy (PDT). A genetic algorithm (GA) is used to solve the "low up-conversion luminescence (UCL) and short-wavelength infrared (SWIR) intensity" problem by coding the proportions of elements in the phosphor in order to find the optimal phosphor with enhanced red UCL emission and SWIR luminescence using Yb/Er in the core and Yb/Nd in the shell. Moreover, when phosphors with different emission light colors (blue and green) are combined with chlorophyll as the control, the results indicate that phosphors with red emission as the energy donor have high PDT efficiency to activate the chlorophyll for reactive oxygen species (ROS) production. Additionally, due to the enhanced penetration and retention (EPR) effect, the as-synthesized Ph-chl could be used for surgery navigation with a higher SWIR imaging effect focusing on cancer rather than normal tissues and paracancerous tissue. Thus, the high dual-modal luminescence guided surgery properties of the final optimized phosphor will promote its further use in minimally-invasive endoscopic clinical surgery navigation.

Glycine Betaine Monooxygenase, an Unusual Rieske-Type Oxygenase System, Catalyzes the Oxidative N-Demethylation of Glycine Betaine in Chromohalobacter salexigens DSM 3043.

Although some bacteria, including Chromohalobacter salexigens DSM 3043, can use glycine betaine (GB) as a sole source of carbon and energy, little information is available about the genes and their encoded proteins involved in the initial step of the GB degradation pathway. In the present study, the results of conserved domain analysis, construction of in-frame deletion mutants, and an in vivo functional complementation assay suggested that the open reading frames Csal_1004 and Csal_1005, designated bmoA and bmoB, respectively, may act as the terminal oxygenase and the ferredoxin reductase genes in a novel Rieske-type oxygenase system to convert GB to dimethylglycine in C. salexigens DSM 3043. To further verify their function, BmoA and BmoB were heterologously overexpressed in Escherichia coli, and 13C nuclear magnetic resonance analysis revealed that dimethylglycine was accumulated in E. coli BL21(DE3) expressing BmoAB or BmoA. In addition, His-tagged BmoA and BmoB were individually purified to electrophoretic homogeneity and estimated to be a homotrimer and a monomer, respectively. In vitro biochemical analysis indicated that BmoB is an NADH-dependent flavin reductase with one noncovalently bound flavin adenine dinucleotide (FAD) as its prosthetic group. In the presence of BmoB, NADH, and flavin, BmoA could aerobically degrade GB to dimethylglycine with the concomitant production of formaldehyde. BmoA exhibited strict substrate specificity for GB, and its demethylation activity was stimulated by Fe2+ Phylogenetic analysis showed that BmoA belongs to group V of the Rieske nonheme iron oxygenase (RO) family, and all the members in this group were able to use quaternary ammonium compounds as substrates.IMPORTANCE GB is widely distributed in nature. In addition to being accumulated intracellularly as a compatible solute to deal with osmotic stress, it can be utilized by many bacteria as a source of carbon and energy. However, very limited knowledge is presently available about the molecular and biochemical mechanisms for the initial step of the aerobic GB degradation pathway in bacteria. Here, we report the molecular and biochemical characterization of a novel two-component Rieske-type monooxygenase system, GB monooxygenase (BMO), which is responsible for oxidative demethylation of GB to dimethylglycine in C. salexigens DSM 3043. The results gained in this study extend our knowledge on the catalytic reaction of microbial GB degradation to dimethylglycine.

Photooxidatively crosslinked acellular tumor extracellular matrices as potential tumor engineering scaffolds.

Acellular tumor extracellular matrices (ECMs) have limitations when employed as three-dimensional (3D) scaffolds for tumor engineering. In this work, methylene blue-mediated photooxidation was used to crosslink acellular tumor ECMs. Photooxidative crosslinking greatly increased the stiffness of acellular tumor ECM scaffolds but barely altered the Amide III band of the secondary structure of polypeptides and proteins. MCF-7, HepG2 and A549 cells cultured on photooxidatively crosslinked acellular tumor ECM scaffolds exhibited greater cell number per scaffold, more IL-8 and VEGF secretion, and increase migration and invasion abilities than cells cultured on uncrosslinked acellular tumor ECM scaffolds. The three tumor cell lines cultured on the stiffer photooxidatively crosslinked acellular matrices acquire mesenchymal properties (mesenchymal shift) and dedifferentiated phenotypes. Furthermore, the malignant phenotypes induced in vitro when cultured on the crosslinked scaffold promoted the in vivo tumor growth of BALB/c nude mice. Finally, the dedifferentiated cancer cells, including MCF-7, HepG2 and A549 cells, were less sensitive to chemotherapeutics. Thus, photooxidatively crosslinked acellular tumor ECMs have potentials as 3D tumor engineering scaffolds for cancer research. STATEMENT OF SIGNIFICANCE: Natural material scaffolds have been successfully used as 3D matrices to study the in vitro tumor cell growth and mimic the in vivo tumor microenvironment. Acellular tumor ECMs are developed as 3D scaffolds for tumor engineering but have limitations in terms of elastic modulus and cell spheroid formation. Here we use methylene blue-mediated photooxidation to crosslink acellular tumor ECMs and investigate the influence of photooxidative crosslinking on structural, mechanical and biological characteristics of acellular tumor ECM scaffolds. It is the first study to evaluate the feasibility of photooxidatively crosslinked acellular tumor ECMs as 3D scaffolds for cancer research and the results are encouraging. Moreover, this study provides new research areas in regard to photodynamic therapy (PDT) for Cancer.

Establishment of a markerless gene deletion system in Chromohalobacter salexigens DSM 3043.

Chromohalobacter salexigens DSM 3043 can grow over a wide range of salinity, which makes it as an excellent model organism for understanding the mechanism of prokaryotic osmoregulation. Functional analysis of C. salexigens genes is an essential way to reveal their roles in cellular osmoregulation. However, the lack of an effective markerless gene deletion system has prevented construction of multiple gene deletion mutants for the members in the genus. Here, we report the development of a markerless gene deletion system in C. salexigens using allelic exchange method. In this system, the in vitro mutant allele of target gene was inserted into a pK18mobsacB-based integrative vector pMDC21, which contained a chloramphenicol resistance cassette as the positive selection marker and a sacB gene from Bacillus subtilis as the counterselectable marker. To validate this system, two single-gene deletion mutants and a double-gene deletion mutant were constructed. In addition, our results showed that growth of the merodiploids and sucrose screening at 25 °C were more effective to decrease the occurrence of spontaneous sucrose resistance colonies than at higher temperature (30 or 37 °C), and growth of the merodiploids in mineral salt medium instead of the complex medium was critical to increase the recovery rate of deletion mutants.

MiR-19a promotes epithelial-mesenchymal transition through PI3K/AKT pathway in gastric cancer.

AIM: To investigate the mechanism by which miR-19a is up-regulated in gastric cancer (GC), which plays an oncogenic role. METHODS: In the present study, we investigated the role of miR-19a in gastric tissues as well as two GC cell lines. In vivo, we detected the basal expression level of miR-19a using real-time reverse transcription-PCR (RT-PCR), and the relevance between expression of miR-19a and clinicopathological information was analyzed. In vitro, miR-19a was ectopically expressed using overexpression and knock-down strategies. RESULTS: Overexpression of miR-19a was significantly associated with metastasis of GC and inferior overall prognosis. However, no significant correlation was found between miR-19a expression and other characteristics such as age, gender, tobacco, alcohol or tumor size. Cell proliferation, migration and invasion assays showed that overexpression of miR-19a promoted the proliferation, migration and invasion, and that overexpression of miR-19a promoted the epithelial-mesenchymal transition through activating the PI3K/AKT pathway. Blocking the PI3K/AKT pathway could cancel the effect of miR-19a. CONCLUSION: All together, our results suggest that miR-19a could be used as a promising therapeutic target in the treatment of GC.

Decreased expression of sestrin 2 predicts unfavorable outcome in colorectal cancer.

Sestrin 2 is a conserved antioxidant protein that is involved in p53­dependent antioxidant defenses and protects cells against oxidative stresses. The present study was conducted to examine the expression of sestrin 2 in colorectal cancer (CRC) and investigate a possible relationship between sestrin 2 expression and prognosis in CRC. The expression of sestrin 2 in human CRC tissues and cell lines was evaluated by immunohistochemical or immunofluorescent staining and western blot analysis. The correlations between sestrin 2 expression in human CRC tissues and clinicopathological variables, including overall survival (OS) and disease­free survival (DFS), were analyzed. Both human CRC tissues and cell lines showed a decreased expression of sestrin 2. Furthermore, a low expression of sestrin 2 was significantly correlated with advanced tumor stage, lymphatic invasion, lymph node metastasis, vascular invasion and liver metastasis. Survival analysis showed that patients with low sestrin 2 staining had a significantly worse DFS and OS. Additionally, early or advanced stage CRC patients with a low expression of sestrin 2 had a shorter survival. In univariate analysis, the patients with low sestrin 2 expression, advanced tumor stage, lymphatic invasion, lymphatic node metastasis, vascular invasion, liver metastasis and peritoneal metastasis had shorter OS and DFS. In multivariate analysis, only low sestrin 2 expression, advanced tumor stage, lymphatic node metastasis, vascular invasion and liver metastasis remained as independent prognostic factors of poor OS and DFS. The findings suggested that a decreased expression of sestrin 2 is associated with an unfavorable prognosis, which suggests that it is a novel and crucial predictor for CRC metastasis.

Development of an acellular tumor extracellular matrix as a three-dimensional scaffold for tumor engineering.

Tumor engineering is defined as the construction of three-dimensional (3D) tumors in vitro with tissue engineering approaches. The present 3D scaffolds for tumor engineering have several limitations in terms of structure and function. To get an ideal 3D scaffold for tumor culture, A549 human pulmonary adenocarcinoma cells were implanted into immunodeficient mice to establish xenotransplatation models. Tumors were retrieved at 30-day implantation and sliced into sheets. They were subsequently decellularized by four procedures. Two decellularization methods, Tris-Trypsin-Triton multi-step treatment and sodium dodecyl sulfate (SDS) treatment, achieved complete cellular removal and thus were chosen for evaluation of histological and biochemical properties. Native tumor tissues were used as controls. Human breast cancer MCF-7 cells were cultured onto the two 3D scaffolds for further cell growth and growth factor secretion investigations, with the two-dimensional (2D) culture and cells cultured onto the Matrigel scaffolds used as controls. Results showed that Tris-Trypsin-Triton multi-step treated tumor sheets had well-preserved extracellular matrix structures and components. Their porosity was increased but elastic modulus was decreased compared with the native tumor samples. They supported MCF-7 cell repopulation and proliferation, as well as expression of growth factors. When cultured within the Tris-Trypsin-Triton treated scaffold, A549 cells and human colorectal adenocarcinoma cells (SW-480) had similar behaviors to MCF-7 cells, but human esophageal squamous cell carcinoma cells (KYSE-510) had a relatively slow cell repopulation rate. This study provides evidence that Tris-Trypsin-Triton treated acellular tumor extracellular matrices are promising 3D scaffolds with ideal spatial arrangement, biomechanical properties and biocompatibility for improved modeling of 3D tumor microenvironments.

High expression of CASK correlates with progression and poor prognosis of colorectal cancer.

Calcium/calmodulin-dependent serine protein kinase (CASK), which localizes at cell-cell adhesion sites and binds to the heparan sulfate proteoglycan syndecan-2, is involved in cell proliferation, cytoskeletal remodeling, and cell migration. To demonstrate the role of CASK in colorectal cancer (CRC) carcinogenesis, we examined the expression of CASK and its binding protein syndecan-2 in human CRC tissues. The expression of CASK was measured in CRC specimens and the controls from adenomas and normal mucosae by immunohistochemical staining and Western blot analysis. Syndecan-2 protein level was tested in CRC samples and the controls by Western blot analysis. The correlations between CASK expression and clinicopathological variables, including disease-free survival (DFS) and overall survival (OS), were analyzed. Compared to the controls, both CASK and syndecan-2 expression were enhanced in CRC tissues. Furthermore, high expression of CASK and syndecan-2 was significantly correlated with advanced tumor stage, lymphatic invasion, lymph node metastasis, vascular invasion, liver metastasis, and unresectable metastatic CRC. Survival analysis showed that patients with low CASK staining had a significantly better survival compared to patients with high CASK staining. In multivariate analysis, CASK overexpression, advanced tumor stage, lymph node metastasis, vasvular invasion, and liver metastasis were independent prognostic factors of poor DFS and OS. Our present study indicates that CASK overexpression is associated with an unfavorable prognosis. CASK is an independent prognostic factor for CRC, which suggests that it is a novel and crucial predictor for CRC metastasis.