3D collagen microchamber arrays for combined chemotherapy effect evaluation on cancer cell numbers and migration.

Breast cancer metastasis involves complex mechanisms, particularly when patients are undergoing chemotherapy. In tissues, tumor cells encounter cell-cell interactions, cell-microenvironment interactions, complex nutrient, and drug gradients. Currently, two-dimensional cell culture systems and animal models are challenging to observe and analyze cell responses to microenvironments with various physical and bio-chemical conditions, and microfluidic technology has been systematically developed to address this dilemma. In this study, we have constructed a combined chemotherapy evaluation chip (CCEC) based on microfluidic technology. The chip possesses 192 diamond-shaped microchambers containing MDA-MB-231-RFP cells, and each microchamber is composed of collagen to mimic breast cancer and its surrounding microenvironment. In addition, by adding medium containing different drugs to the medium channels of CCEC, composite drug (paclitaxel+gemcitabine+7rh and paclitaxel+fluorouracil+PP2) concentration gradients, and single drug (paclitaxel, gemcitabine, 7rh, fluorouracil, PP2) concentration gradients have been established in the five collagen regions, respectively, so that each localized microchamber in the regions has a unique drug microenvironment. In this way, we evaluated the composite and single chemotherapy efficacy on the same chip by statistically analyzing their effects on the numbers and migration of the cell. The quantitative results in CCECs reveal that the inhibition effects on the numbers and migration of MDA-MB-231-RFP cell under the composite drug gradients are more optimal than those of the single drugs. Besides, the cancer cell inhibition effect between the groups composed of two drugs has also been compared, that is the paclitaxel+gemcitabine, paclitaxel+fluorouracil, and paclitaxel+PP2 have better cell numbers and migration inhibition effects than paclitaxel+7rh. The results indicate that the bio-mimetic and high-throughput combined chemotherapy evaluation platform can serve as a more efficient and accurate tool for preclinical drug development and screening.

Temporospatial nano-heterogeneity of self-assembly of extracellular polymeric substances on microplastics and water environmental implications.

Environmental behavior and ecotoxicity of microplastics (MPs) are significantly influenced by the omnipresent self-assembly of microbial extracellular polymeric substances (EPS) on them. However, mechanisms of EPS self-assembly onto MPs at nanoscale resolution and effects of aging are unclear. For the first time, temporospatial nano-heterogeneity of self-assembly of EPS onto fresh and one-year aged polypropylene (PP) MPs were investigated by atomic-force-microscopy-infrared-spectroscopy (AFM-IR). Natural aging caused high degree nanoscale fragmentation of MPs physically and chemically. Self-assembly of EPS on MPs was aging-dependent. Polysaccharides were assembled on MP surface faster than proteins. Initially, regardless of the fresh or aged MPs, polysaccharides and proteins, with the former being predominant, were successively and separately assembled to different nanospaces because of their competition for binding sites. More and more proteins and polysaccharides were superimposed on each other with assembly time due to intermolecular forces. The nanochemical textural analysis showed that the nano-heterogeneity of EPS assembly to MPs was clearly correlated with the aging-induced nanochemical and nanomechanical heterogeneity of MP surface. The spontaneous self-assembly of EPS with temporospatial nano-heterogeneity on MPs have multiple impacts on behavior, ecotoxicity and fate of MPs and their associated pollutants as well as other key ecological processes in aquatic environment.

Integrated DNA triangular prism nanomachines for two-stage dynamic recognizing and bio-imaging from surface to the inside of living cells.

The identification and detection of biomarkers in cancer cells play an essential role in the early detection of diseases, especially the detection of dual-biomarker. However, one of the most important limiting factors is how to realize the identification and labeling of biomarkers dynamically from the plasma membrane to the cytoplasm in living cells. In this study, integrated DNA triangular prism nanomachines (IDTPNs), a two-stage identification and dynamic bio-imaging strategy, recognize biomarkers from the plasma membrane to the cytoplasm have been designed. DNA triangular prism (DTP) was selected to act as a delivery platform with the aptamer Sgc8c and P53 modified on the side as the recognition molecules. Through the specific recognition of aptamers and the superior internalization of DTP, the IDTPNs realize the dynamic responses to PTK7 and p53 from the membrane to the cytoplasm in living cells. It is proved that the IDTPNs can be used for dynamic dual-biomarker recognition and bio-image from the surface to the inside of tumor cells automatically. Therefore, the strategy we developed provides a reliable platform for tumor diagnosis and biomarker research.

Chemically synthesized (Ag, Mn2O3)-codecorated ZnO nanoparticles for achieving superior visible light-induced photodegradation and enhanced gas sensing activity.

Heterostructural engineering and noble metal coupling are effective strategies to optimize semiconductor photocatalytic materials. In this work, (Ag, Mn2O3)-codecorated ZnO nanoparticles with different Mn2O3 contents (0-10 mol%) were synthesized by integrating the two strategies by a facile two-step polymer network-gel process. The photocatalytic activity of Ag/ZnO (AZM0) was significantly enhanced with the optimum Mn2O3 molar ratio of 3 mol%. The degradation efficiency of AZM3 is ∼3 times and ∼4.8 times higher than that of AZM0 for the degradation of methylene blue (MB) upon exposure to simulated sunlight and visible light, respectively. Also, this ternary nanocomposite exhibits enhanced gas sensing performance towards NO2 under ultraviolet/visible light irradiation at room temperature. The analysis of its microstructural, optical and photoelectrical characteristics suggests the synergistic coupling effects of Ag and Mn2O3, in which the significantly enhanced visible light response and hetero-interface charge carrier migration are the critical factors for the improvement of photocatalytic efficiency and gas sensing activity. Furthermore, the effects of recycling ability, the influence of the initial solution pH, the catalyst dosage and the main active species during the catalysis process on photocatalytic activity were explored. This study develops a feasible pathway to consciously construct multiheterostructures for enhancing the photocatalytic activity with great potential applications in toxic pollution abatement and noxious gas detection.

Porcine epidemic diarrhea virus E protein suppresses RIG-I signaling-mediated interferon-ß production.

Porcine epidemic diarrhea virus (PEDV) encodes many multifunctional proteins that inhibit host innate immune response during virus infection. As one of important structural proteins, PEDV E protein has been found to block the production of type I interferon (IFN) in virus life cycle, but little is known about this process that E protein subverts host innate immune. Thus, in this present study, we initiated the construction of eukaryotic expression vectors to express PEDV E protein. Subsequently, cellular localization analysis was performed and the results showed that the majority of PEDV E protein distributed at cytoplasm and localized in endoplasmic reticulum (ER). Over-expression of PEDV E protein significantly inhibited poly(I:C)-induced IFN-ß and IFN-stimulated genes (ISGs) productions. We also found that PEDV E protein remarkably suppressed the protein expression of RIG-I signaling-associated molecules, but all their corresponding mRNA levels remained unaffected and unchanged. Furthermore, PEDV E protein obviously interfered with the translocation of IRF3 from cytoplasm to nucleus through direct interaction with IRF3, which is crucial for the IFN-ß production induced by poly(I:C). Taken together, our results suggested that PEDV E protein acts as an IFN-ß antagonist through suppression of the RIG-I-mediated signaling. This study will pave the way for the further investigation into the molecular mechanisms by which PEDV E protein evades host innate immune response.

Lipid raft-associated PI3K/Akt/SREBP1 signaling regulates coxsackievirus A16 (CA16) replication.

Coxsackievirus A16 (CA16) is one of predominant Enterovirus that possesses high pathogenicity. Lipid rafts, as cholesterol - and sphingolipid - enriched membrane nanodomains, are involved into many aspects of the virus life cycle. Our previous study found that lipid rafts integrity was essential for CA16 replication, but how lipid rafts regulate CA16 replication through activating downstream signaling remains largely unknown. Thus, in this study, we revealed that lipid rafts were required for activation of PI3K/Akt signaling at early stage of CA16 infection. Treatment with wortmannin significantly reduced the expression of virus protein, indicating PI3K/Akt signaling was beneficial for early stage of virus infection. In addition, lipid rafts integrity was also indispensable for PI3K/Akt activation during the late stage of CA16 infection, which played critical functions in mediating sterol regulatory element-binding proteins 1 (SREBP1) maturation. Whereas, over-expression of SREBP1 exhibited inhibition on virus replication, suggesting that PI3K/Akt signaling and SREBP1 might positively and negatively influence virus replication in two different stages of infection, respectively. Taken together, our study demonstrates an important role of the lipid raft-associated PI3K/Akt/SREBP1 signaling in modulating CA16 replication, which will deepen our understanding mechanism of CA16 infection.

The accessory protein ORF3 of porcine epidemic diarrhea virus inhibits cellular interleukin-6 and interleukin-8 productions by blocking the nuclear factor-κB p65 activation.

Porcine epidemic diarrhea virus (PEDV) is an enveloped, single-stranded positive-sense RNA virus that belongs to a porcine entero-pathogenic alphacoronavirus, causing lethal watery diarrhea in piglets. Despite existing study reports the sole accessory protein ORF3 identified as NF-κB antagonist, the contribution of PEDV ORF3 to production of the pro-inflammatory cytokines mediated by NF-κB signaling remains largely unknown. Thus in this present study, we showed that PEDV ORF3 protein significantly inhibited the productions of pro-inflammatory cytokines interleukin-6 (IL-6) and IL-8. The phosphorylation of IκBα was inhibited by ORF3 protein, but no degradation of IκBα was induced in ORF3-expressing cells. Furthermore, PEDV ORF3 inhibited NF-κB activation through preventing nuclear factor p65 phosphorylation and down-regulating p65 expression level, as well as interfering nuclear translocation of p65, eventually resulting into the inhibition of IL-6 and IL-8 production. Our study definitely links PEDV ORF3 to inhibition of pro-inflammatory cytokines production, which will provide new insight into the molecular mechanisms of NF-κB activity inhibited by PEDV proteins to facilitate virus evasion of host innate immune.

Enterovirus 71 inhibits cytoplasmic stress granule formation during the late stage of infection.

Stress granules (SGs) are host translationally silent ribonucleo-proteins formed in cells in response to multiple types of environmental stress, including viral infection. We previously showed that the nuclear protein, 68-kDa Src-associated in mitosis protein (Sam68), is recruited to cytoplasm and form the Sam68-positive SGs at 6 hpi, but the Sam68-positive SGs disassembled beyond 12 hpi, suggesting that the SGs might be inhibited during the late stage of Enterovirus 71 (EV71) infection. However, the mechanism and function of this process remains poorly understood. Thus in this study, we demonstrated that EV71 initially induced SGs formation at the early stage of EV71 infection, and confirmed that 2Apro of EV71 was the key viral component that triggered SG formation. In contrast, SGs were diminished as EV71 infection proceeding. At the same time, arsenite-induced SGs were also blocked at the late stage of EV71 infection. This disruption of SGs was caused by viral protease 3Cpro-mediated G3BP1 cleavage. Furthermore, we demonstrated that over-expression of G3BP1-SGs negatively impacted viral replication at the cytopathic effect (CPE), protein, RNA, and viral titer levels. Our novel finding may not only help us to better understand the mechanism how EV71 interacts with the SG response, but also provide mechanistic linkage between cellular stress responses and innate immune activation during EV71 infection.

Caveolin-1 regulates the expression of tight junction proteins during hyperoxia-induced pulmonary epithelial barrier breakdown.

BACKGROUND: Bronchopulmonary dysplasia (BPD) is a common complication in preterm infants that involves the downregulation of tight junction (TJ) proteins. However, the mechanism underlying downregulation of the expression of TJ proteins during at the early stages of hyperoxia-induced BPD remains to be understood. Here, we aimed to identify the role of caveolin-1 (Cav-1) in hyperoxia-induced pulmonary epithelial barrier breakdown. METHODS: First, we established an in vitro pulmonary epithelial barrier models using primary type II alveolar epithelial cells (AEC-II) from newborn rats. AEC-II was assigned to the hyperoxic (85 % O2/5 % CO2) or normoxic (21 % O2/5 % CO2) groups. Second, AEC-II was transfected with Cav-1-siRNA to downregulate Cav-1 under normoxic exposure. Third, AEC-II was transfected with a cDNA encoding Cav-1 to upregulate Cav-1 expression under hyperoxic exposure. Then, expression levels of Cav-1 and TJ proteins were examined by immunofluorescence staining, reverse transcription-polymerase chain reaction, and Western blotting. The TJ structures visualized using a transmission electron microscope, and transepithelial resistance and apparent permeability coefficient of fluorescein isothiocyanate-dextran, which are indicators of barrier function, were measured. RESULTS: Our data showed that exposure to hyperoxia disrupted the structure and function of the pulmonary epithelial barrier and decreased the ZO-1, occludin, claudin-4, and Cav-1 expression levels. Moreover, Cav-1 knockdown attenuated the expression of the other three genes and disrupted pulmonary epithelial barrier structure and function under normoxic exposure. However, Cav-1 upregulation markedly antagonized the hyperoxia-induced pulmonary epithelial barrier destruction and TJ protein loss. CONCLUSIONS: This is the first study to present evidence illustrating the novel role of Cav-1 downregulation-mediated TJ protein loss in pulmonary epithelial barrier destruction during BPD.

Radiation-induced epigenetic bystander effects demonstrated in Arabidopsis thaliana.

Radiation-induced bystander effects (RIBE) in vivo in the higher plant Arabidopsis thaliana ( A. thaliana ) have been well demonstrated in terms of effects on development and genetics. However, there is not yet robust evidence regarding RIBE-mediated epigenetic changes in plants. To address this, in the current work the roots of A. thaliana seedlings were locally irradiated with 10 Gy of α particles, after which DNA methylation in bystander aerial plants were detected using the methylation-sensitive amplification polymorphism (MSAP) and bisulfite sequencing PCR (BSP). Results showed that irradiation of the roots led to long-distance changes in DNA methylation patterns at some CCGG sites over the whole genome, specifically from hemi-methylation to non-methylation, and the methylation ratios, mainly at CG sites, strongly indicating the existence of RIBE-mediated epigenetic changes in higher plants. Root irradiation also influenced expressions of DNA methylation-related MET1, DRM2 and SUVH4 genes and demethylation-related DML3 gene in bystander aerial plants, suggesting a modulation of RIBE to the methylation machinery in plants. In addition, the multicopy P35S:GUS in A. thaliana line L5-1, which is silenced epigenetically by DNA methylation and histone modification, was transcriptionally activated through the RIBE. The transcriptional activation could be significantly inhibited by the treatment with reactive oxygen species (ROS) scavenger dimethyl sulfoxide (DMSO), indicative of a pivotal role of ROS in RIBE-mediated epigenetic changes. Time course analyses showed that the bystander signaling molecule(s) for transcriptional activation of multicopy P35S:GUS, although of unknown chemical nature, were generated in the root cells within 24 h postirradiation.

Hyperoxia stimulates the transdifferentiation of type II alveolar epithelial cells in newborn rats.

Supplemental oxygen treatment in preterm infants may cause bronchopulmonary dysplasia (BPD), which is characterized by alveolar simplification and vascular disorganization. Despite type II alveolar epithelial cell (AEC II) damage being reported previously, we found no decrease in the AEC II-specific marker, surfactant protein C (SP-C), in the BPD model in our previous study. We thus speculated that AEC II injury is not a unique mechanism of BPD-related pulmonary epithelial repair dysfunction and that abnormal transdifferentiation can exist. Newborn rats were randomly assigned to model (85% oxygen inhalation) and control groups (room air inhalation). Expressions of AEC I (aquaporin 5, T1α) and AEC II markers (SP-C, SP-B) were detected at three levels: 1) in intact lung tissue, 2) in AEC II isolated from rats in the two groups, and 3) in AEC II isolated from newborn rats, which were further cultured under either hyperoxic or normoxic conditions. In the model group, increased AEC I was observed at both the tissue and cell level, and markedly increased transdifferentiation was observed by immunofluorescent double staining. Transmission electron microscopy revealed morphological changes in alveolar epithelium such as damaged AECs, a fused air-blood barrier structure, and opened tight junctions in the model group. These findings indicate that transdifferentiation of AECs is not suppressed but rather is increased under hyperoxic treatment by compensation; however, such repair during injury cannot offset pulmonary epithelial air exchange and barrier dysfunction caused by structural damage to AECs.

The time course of long-distance signaling in radiation-induced bystander effect in vivo in Arabidopsis thaliana demonstrated using root micro-grafting.

The radiation-induced bystander effect has been demonstrated in whole organisms as well as in multicellular tissues in vitro and single-cell culture systems in vitro. However, the time course of bystander signaling, especially in whole organisms, is not clear. Long-distance bystander/abscopal effects in vivo in plants have been demonstrated by our group. Plant grafting is a useful experimental tool for studying the root-shoot signaling of plants. In the present study, we developed a root micro-grafting technique with young seedlings of Arabidopsis thaliana in which the bystander signaling communication of root-to-shoot could easily be stopped or started at specific times after root irradiation. Using this methodology, we demonstrated the time course of long-distance signaling in radiation-induced bystander effects at the level of the organism using the expression level of the AtRAD54 gene as a biological end point. Briefly, an 8-h accumulation of damage signals in bystander parts after irradiation was essential for eliciting a bystander response. The protraction of signal accumulation was not related to the transmission speed of signaling molecules in plants and did not result from the delayed initiation of bystander signals in targeted root cells. It was suggested that the bystander effect might be induced jointly by multiple bystander signals initiated at different stages after irradiation. Moreover, reactive oxygen species (ROS) were shown to be implicated in the response process of bystander cells to radiation damage signals rather than in the generation of bystander signals in targeted cells.