Integrated analysis of mRNA and miRNA expression in HeLa cells expressing low levels of Nucleolin.

Nucleolin is an essential protein that plays important roles in the regulation of cell cycle and cell proliferation. Its expression is up regulated in many cancer cells but its molecular functions are not well characterized. Nucleolin is present in the nucleus where it regulates gene expression at the transcriptional and post-transcriptional levels. Using HeLa cells depleted in nucleolin we performed an mRNA and miRNA transcriptomics analysis to identify biological pathways involving nucleolin. Bioinformatic analysis strongly points to a role of nucleolin in lipid metabolism, and in many signaling pathways. Down regulation of nucleolin is associated with lower level of cholesterol while the amount of fatty acids is increased. This could be explained by the decreased and mis-localized expression of the transcription factor SREBP1 and the down-regulation of enzymes involved in the beta-oxidation and degradation of fatty acids. Functional classification of the miRNA-mRNA target genes revealed that deregulated miRNAs target genes involved in apoptosis, proliferation and signaling pathways. Several of these deregulated miRNAs have been shown to control lipid metabolism. This integrated transcriptomic analysis uncovers new unexpected roles for nucleolin in metabolic regulation and signaling pathways paving the way to better understand the global function of nucleolin within the cell.

Net-Zero-Energy Model for Sustainable Wastewater Treatment.

A large external energy input prevents wastewater treatment from being environmentally sustainable. A net-zero-energy (NZE) wastewater treatment concept based on biomass energy recycling was proposed to avoid wasting resources and to promote energy recycling in wastewater treatment plants (WWTPs). Simultaneously, a theoretical model and boundary condition based on energy balance were established to evaluate the feasibility of achieving NZE in WWTPs; the model and condition were employed to analyze data from 20 conventional WWTPs in China. A total of six WWTPs can currently export excess energy, eight WWTPs can achieve 100% energy self-sufficiency by adjusting the metabolic material allocation, and six municipal WWTPs cannot achieve net-zero energy consumption based on the evaluation of the theoretical model. The NZE model offset 79.5% of the electricity and sludge disposal cost compared with conventional wastewater treatment. The NZE model provides a theoretical basis for the optimization of material regulation for the effective utilization of organic energy from wastewater and promotes engineering applications of the NZE concept in WWTPs.

Photoresponsive microvalve for remote actuation and flow control in microfluidic devices.

Microvalves with different actuation methods offer great integrability and flexibility in operation of lab-on-chip devices. In this work, we demonstrate a hydrogel-based and optically controlled modular microvalve that can be easily integrated within a microfluidic device and actuated by an off-chip laser source. The microvalve is based on in-channel trapping of microgel particles, which are composed of poly(N-isopropylacrylamide) and polypyrrole nanoparticles. Upon irradiation by a near-infrared (NIR) laser, the microgel undergoes volumetric change and enables precisely localized fluid on/off switching. The response rate and the "open" duration of the microvalve can be simply controlled by adjusting the laser power and exposure time. We showed that the trapped microgel can be triggered to shrink sufficiently to open a channel within as low as ∼1-2 s; while the microgel swells to re-seal the channel within ∼6-8 s. This is so far one of the fastest optically controlled and hydrogel-based microvalves, thus permitting speedy fluidic switching applications. In this study, we successfully employed this technique to control fluidic interface between laminar flow streams within a Y-junction device. The optically triggered microvalve permits flexible and remote fluidic handling, and enables pulsatile in situ chemical treatment to cell culture in an automatic and programmed manner, which is exemplified by studies of chemotherapeutic drug induced cell apoptosis under different drug treatment strategies. We find that cisplatin induced apoptosis is significantly higher in cancer cells treated with a pulsed dose, as compared to continuous flow with a sustained dose. It is expected that our NIR-controlled valving strategy will provide a simple, versatile, and powerful alternative for liquid handling in microfluidic devices.

Near-infrared light triggerable deformation-free polysaccharide double network hydrogels.

To prepare a hydrogel with robust mechanical properties and programmable remotely-controlled releasing ability, we synthesized an agarose/alginate double network hydrogel incorporating polypyrrole (PPy) nanoparticles as a near-infrared (NIR) laser responsive releasing system. This hydrogel exhibited pulsatile releasing behaviours according to the laser switching while maintaining its morphology and mechanical strength.

Near-infrared photothermal activation of microgels incorporating polypyrrole nanotransducers through droplet microfluidics.

Poly(N-isopropylacrylamide) (pNIPAM) composite microgels incorporating polypyrrole (PPy) nanoparticles were produced using droplet microfluidics. The composite microgels exhibited site-specific de-swelling-swelling properties that were activated by near-infrared light. Their applications for programmable drug release by pulsed-light control were also demonstrated.

Effects of oligo(3-hydroxyalkanoates) on the viability and insulin secretion of murine beta cells.

Biopolyesters of polyhydroxyalkanoates (PHAs), including poly-3-hydroxybutyrate (PHB), co-polyester of 3-hydroxybutyrate and 4-hydroxybutyrate (P3HB4HB), and co-polyester of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) have been well investigated for their biocompatibility. For in vivo application, it is very important that the degradation products of PHAs, especially the oligomers, are not harmful to the cells and surrounding tissues. In this study, in vitro effects of oligo(3-hydroxybutyrate) (OHB), oligo(3-hydroxybutyrate-co-4-hydroxybutyrate) (O3HB4HB) and oligo(3-hydroxybutyrate-co-3-hydroxyhexanoate) (OHBHHx) on growth and differentiation of the murine beta cell line NIT-1 were investigated. Among the three oligo-hydroxyalkanoates (Oligo-HAs), cells treated with OHBHHx displayed higher viability, as measured by CCK-8 assay. Flow cytometric analysis of NIT-1 cells indicated that Oligo-HAs had an inhibitory effect on cell apoptosis. The cytosolic Ca(2+) transient of NIT-1 cells increased when fed with 0.04 g/l Oligo-HAs. For gap junction intercellular communication of cells, the effect of OHBHHx was the best among all materials tested. More importantly, extracellular insulin secretion was up-regulated after growing in OHBHHx for 48 h. The results demonstrated that the degradation products of PHAs, especially OHBHHx from PHBHHx, were not harmful to the beta cells. Therefore, PHBHHx warrant further study for application as a pancreatic tissue engineering material.

Production of polyhydroxyalkanoates with high 3-hydroxydodecanoate monomer content by fadB and fadA knockout mutant of Pseudomonas putida KT2442.

Pseudomonas putida KT2442 produces medium-chain-length (MCL) polyhydroxyalkanoates (PHA) consisting of 3-hydroxyhexanoate (HHx), 3-hydroxyoctanoate (HO), 3-hydroxydecanoate (HD), and 3-hydroxydodecanoate (HDD) from a wide-range of carbon sources. In this study, fadA and fadB genes encoding 3-ketoacyl-CoA thiolase and 3-hydroxyacyl-CoA dehydrogenase in P. putida KT2442 were knocked out to weaken the beta-oxidation pathway. Two-step culture was proven as the optimal method for PHA production in the mutant termed P. putida KTOY06. In a shake-flask culture, when dodecanoate was used as a carbon source, P. putida KTOY06 accumulated 84 wt % PHA, much higher than 50 wt % PHA in its wild type KT2442. The PHA monomer composition was completely different: the HDD fraction in PHA produced by KTOY06 was 41 mol %, much higher compared with 7.5 mol % only in KT2442. The fermentor-scale culture indicated the HDD fraction in PHA decreased during the culture time from 35 to 25 mol % in a one-step fermentation process or from 75 to 49 mol % in a two-step fermentation process. It is for the first time that PHA with a dominant HDD fraction was produced. Thermal and mechanical properties assays indicated that this new type PHA with a high HDD fraction had higher crystallinity and tensile strength than PHA with a low HDD fraction did, demonstrating an improved application property.

Overexpression of QM induces cell differentiation and mineralization in MC3T3-E1.

It has been reported that QM was highly expressed by cells isolated from epiphyseal cartilage as opposed to proliferative chondrocytes. In vitro investigation of the expression of QM revealed higher QM expression in nonmineralizing osteoblast and pericyte cultures as compared with mineralizing cultures. These evidences suggest that QM may play an essential role in cell differentiation before mineralization. However, our research results showed that QM overexpression in MC3T3-E1 enhanced cell differentiation and mineralization. In this study, alkaline phosphatase (ALP) activity and nodule mineralization were increased in MC3T3-E1 from QM overexpression cultures relative to normal expression QM cultures. RT-PCR revealed upregulation of the marker genes type I collagen, ALP, osteocalcin, osterix and BMP-2 and a slight decrease of a negative regulator osteopontin. These results suggest that the increasing of QM expression could stimulate osteoblast differentiation and mineralization in MC3T3-E1.

Dual effects of cycloheximide on U937 apoptosis induced by its combination with VP-16.

In this study, cycloheximide (CHX) and VP-16 alone and in combination (C&V) have been used to strongly trigger apoptosis in U937 cells. The presence of CHX markedly prevented VP-16-induced apoptosis, suggesting that in this process de novo protein synthesis is required. But interestingly, C&V had shown more similarities with CHX but not VP-16 alone, including the effects on cell cycle distribution and induction of apoptosis, which occurred more quickly and was steadily enhanced by increasing concentrations of CHX or by N-alpha-tosyl-L-lysyl-chloromethyl ketone (TLCK), a serine protease inhibitor. These results indicate that CHX, not VP-16, is indeed the dominant inducer of U937 apoptosis, when they are coadministered. In particular, VP-16 even promoted CHX-induced apoptosis, but did not alter its selection of cell types. In T-cells resistant to CHX (Molt-4), we have detected no apoptotic response to their combination. These findings may well explain why the inhibitory effects of CHX on apoptosis induced by the same stimuli are usually different according to the cell type used, and also suggest that CHX may have the potential to lower side effects and drug resistance of cancer therapy.

Effects of chebulinic acid on differentiation of human leukemia K562 cells.

AIM: To study effects of chebulinic acid on erythroid and megakaryocytic differentiation in K562 cells. METHODS: The benzidine staining method was used to evaluate hemoglobin synthesis; the expression of erythroid specific glycophorin A (GPA) protein and megakaryocytic surface marker CD61 was determined by flow cytometry using fluorescence labeled antibodies; erythroid and megakaryocytic mRNA expression was analyzed by RT-PCR. RESULTS: During erythroid differentiation induced by butyric acid (BA) or hemin, chebulinic acid not only inhibited the hemoglobin synthesis of BA- and hemin-treated K562 cells in concentration-dependent manner with IC50 of 4 micromol/L and 40 micromol/L respectively, but also inhibited another erythroid differentiation marker acetylcholinesterase at the concentration of 50 micromol/L in the cells either treated or untreated with each erythroid differentiation inducers, whereas chebulinic acid 50 micromol/L did not change GPA protein expression in these cells significantly. When K562 cells were treated with TPA 50 microg/L for 72 h to induce megakaryocytic differentiation, the presence of chebulinic acid 50 micromol/L slightly provoked the decrease of GPA protein expression induced by TPA. Chebulinic acid did not change the TPA-induced CD61 expression at the same concentration. Chebulinic acid also reduced the mRNA levels of erythroid relative genes including gamma-globin, PBGD, NF-E2, and GATA-1 genes in K562 cells either treated or untreated with BA, whereas chebulinic acid upregulated the mRNA levels of GATA-2 transcription factor in these cells. CONCLUSION: Chebulinic acid had inhibitory effect on erythroid differentiation likely through changing transcriptional activation of differentiation relative genes, which suggests that chebulinic acid or other tannins might influence the efficiency of some anti-tumor drugs-induced differentiation or the hematopoiesis processes.

Mitochondrial dysfunction as an early event in the process of apoptosis induced by woodfordin I in human leukemia K562 cells.

Tannins are a group of widely distributed plant polyphenols, some of which are beneficial to health because of their chemopreventive activities. In the present study, we investigated the effects and action mechanisms of woodfordin I, a macrocyclic ellagitannin dimer, on human chronic myelogenous leukemia (CML) K562 cells. The results showed that woodfordin I was able to suppress the proliferation and induce apoptosis in K562 cells. Apoptosis was evaluated by cytomorphology, internucleosomal DNA fragmentation, and externalization of phosphatidylserine. Woodfordin I treatment caused a rapid and sustained loss of mitochondrial transmembrane potential (MMP), transient generation of reactive oxygen species (ROS), transient elevation of intracellular Ca2+ concentration, and cytosolic accumulation of cytochrome c. The activation of caspase-9 and 3, but not caspase-8, was also demonstrated, indicating that the apoptotic signaling triggered by woodfordin I was mediated through the intrinsic mitochondria-dependent pathway. Western blot and immunofluorescence analysis revealed that the anti-apoptotic Bcl-2 and Bcl-xL levels were downregulated, together with the pro-apoptotic Bax protein. Significantly, woodfordin I-induced apoptosis was associated with a decline in the levels of c-Abl, Bcr-Abl, and cellular protein tyrosine phosphorylation. Considering the consequence of all the events in the process of woodfordin I-induced apoptosis, the mitochondrial dysfunction is directly responsible for the pro-apoptotic effects on K562 cells. Furthermore, because CML is a malignancy of pleuripotent hematopoietic cells caused by the dysregulated tyrosine kinase activity of Bcr-Abl, these findings suggest that woodfordin I may be a potential lead compound against CML.