SERS as a Probe of Surface Chemistry Enabled by Surface-Accessible Plasmonic Nanomaterials.

ConspectusWhen the size of materials is reduced, their volume decreases much faster than their surface area, which in the most extreme case leads to 2D nanomaterials which are "all surface". Since atoms at the surface have free energies, electronic states, and mobility which are very different from bulk atoms, nanomaterials that have large surface-to-volume ratios can display remarkable new properties compared to their bulk counterparts. More generally, the surface is where nanomaterials interact with their environment, which in turn places surface chemistry at the heart of catalysis, nanotechnology, and sensing applications. Understanding and utilizing nanosurfaces are not possible without appropriate spectroscopic and microscopic characterization techniques. An emerging technique in this area is surface-enhanced Raman spectroscopy (SERS), which utilizes the interaction between plasmonic nanoparticles and light to enhance the Raman signals of molecules near the nanoparticles' surfaces. SERS has the great advantage that it can provide detailed in situ information on surface orientation and binding between molecules and the nanosurface. A long-standing dilemma that has limited the applications of SERS in surface chemistry studies is the choice between surface-accessibility and plasmonic activity. More specifically, the synthesis of metal nanomaterials with strong plasmonic and SERS-enhancing properties typically involves the use of strongly adsorbing modifier molecules, but these modifiers also passivate the surface of the product material, which prevents the general application of SERS in the analysis of weaker molecule-metal interactions.In this Account, we discuss our efforts in the development of modifier-free synthetic approaches to synthesize surface-accessible, plasmonic nanomaterials for SERS. We start by discussing the definition of "modifiers" and "surface-accessibility", especially in the context of surface chemistry studies in SERS. As a general rule of thumb, the chemical ligands on surface-accessible nanomaterials should be easily displaceable by a wide range of target molecules relevant to potential applications. We then introduce modifier-free approaches for the bottom-up synthesis of colloidal nanoparticles, which are the basic building blocks for nanotechnology. Following this, we introduce modifier-free interfacial self-assembly approaches developed by our group that allow the creation of multidimensional plasmonic nanoparticle arrays from different types of nanoparticle-building blocks. These multidimensional arrays can be further combined with different types of functional materials to form surface-accessible multifunctional hybrid plasmonic materials. Finally, we demonstrate applications for surface-accessible nanomaterials as plasmonic substrates for SERS studies of surface chemistry. Importantly, our studies revealed that the removal of modifiers led to not only significantly enhanced properties but also the observation of new surface chemistry phenomena that had been previously overlooked or misunderstood in the literature. Realizing the current limitations of modifier-based approaches provides new perspectives in manipulating molecule-metal interactions in nanotechnology and can have significant implications in the design and synthesis of the next generation of nanomaterials.

Identification of two novel QTL for Fusarium head blight resistance in German wheat cultivar Centrum.

Fusarium head blight (FHB) is a devastating disease that occurs in warm and humid environments. The German wheat Centrum has displayed moderate to high levels of FHB resistance in the field for many years. In this study, an F6:8 recombinant inbred line (RIL) population derived from cross Centrum × Xinong 979 was evaluated for FHB response following point inoculation in five environments. The population and parents were genotyped using the GenoBaits Wheat 16 K Panel. Stable quantitative trait loci (QTL) associated with FHB resistance in Centrum were mapped on chromosome arms 2DS and 5BS. The most effective QTL, located in 2DS, was identified as a new chromosome region represented by a 1.4 Mb interval containing 17 candidate genes. Another novel QTL was mapped in chromosome arm 5BS of a 5BS-7BS translocation chromosome. In addition, two environmentally-sensitive QTL were mapped on chromosome arms 2BL from Centrum and 5AS from Xinong 979. Polymorphisms of flanking allele-specifc quantitative PCR (AQP) markers AQP-6 for QFhb.nwafu-2DS and 16K-13073 for QFhb.nwafu-5BS were validated in a panel of 217 cultivars and breeding lines. These markers could be useful for marker-assisted selection of FHB resistance and also provide a starting point for fine mapping and marker-based cloning of the resistance genes.

A comparative study of the prevalence of myopia and behavioral changes in primary school students.

OBJECTIVE: To analyze the changes in the prevalence of myopia and its relation to ocular biological parameters, and behaviors among primary school students in China, and understand the prevention and control of myopia. METHODS: Cross-sectional surveys were performed on 7-9-year-old children in the yrs. 2012 and 2019. In addition, spherical equivalent refraction (SER), axial length (AL), and AL/corneal radius ratio (AL/CR ratio) were collected without cycloplegia. Participants completed detailed questionnaires on behavior related to myopia. RESULTS: Data was collected on 623 children (8.02 ± 0.57 years old) in 2012 and 536 students in 2019 (8.09 ± 0.65 years old). The prevalence of myopia was 37.7% in 2012 and 39.9% in 2019. The SER was -0.25 (0.92) D in 2012 and -0.25 (1.25) in 2019. There was no statistical difference in the prevalence of myopia and SER over the 7 years (all P > 0.05). In 2019, the prevalence of myopia among girls demonstrated an increasing trend (33.8% vs. 37.8%), but there was no statistical difference (P > 0.05). The mean AL and AL/CR ratio of boys were decreasing (all P < 0.05). The proportion of children reading more than 2 h and using digital devices for more than 2 h per day after their classes in the 2019 group both decreased (all P < 0.05). However, the proportion of activities performed outdoors for more than 2 h./day decreased significantly (P = 0.001). CONCLUSION: Compared with 2012, the prevalence of myopia in primary school students in 2019 was under control, which may be related to the improvement of children's near-work behavior, but there was the problem of insufficient outdoor activity time. In terms of ocular biological parameters, the risk of myopia for boys in 2019 was lower.

Transient receptor potential canonical 6 knockdown ameliorated diabetic kidney disease by inhibiting nuclear factor of activated T cells 2 expression in glomerular mesangial cells.

PURPOSE: Glomerular mesangial cell (GMC) dysfunction plays a vital role in the pathogenesis of diabetic kidney disease (DKD). Transient receptor potential canonical 6 (TRPC6) has been demonstrated to be involved in the development of DKD. However, the underlying mechanism remains unclear. The present study investigated the role of TRPC6 in GMC dysfunction and the related mechanism. METHODS: Diabetic rats and cultured GMCs were used in the experiment. The diabetic rat model was created by intraperitoneal injection of streptozotocin. Protein and mRNA levels were assessed by Western blotting and quantitative RT-PCR, respectively. Histological changes in the kidneys were observed by immunochemistry and hematoxylin and eosin. TRPC6 knockdown was achieved by adenovirus-mediated TRPC6 shRNA delivery in vivo and TRPC6 siRNA transfection in vitro. RESULTS: TRPC6 expression was increased in diabetic rat kidneys. Knockdown of TRPC6 attenuated diabetes-induced kidney functional deterioration. In addition, the increases in extracellular matrix components, including collagen IV, collagen I, and fibronectin production, as well as NFAT2 expression were also suppressed. In cultured GMCs, high glucose (25 mM, HG) treatment increased the expression of TRPC6. Knockdown of TRPC6 alleviated HG-induced increases in collagen IV, fibronectin, and NFAT2 expression. Knockdown of NFAT2 also inhibited the upregulation of proteins, including collagen IV and fibronectin, in HG-treated GMCs. CONCLUSION: These results demonstrate that inhibition of TRPC6/NFAT2 signaling attenuates GMC dysfunction and the development of DKD and suggest that pharmacological targeting of TRPC6/NFAT2 in GMCs may provide beneficial effects for DKD.

Advanced Raman Spectroscopy Detection of Oxidative Damage in Nucleic Acid Bases: Probing Chemical Changes and Intermolecular Interactions in Guanosine at Ultralow Concentration.

DNA/RNA synthesis precursors are especially vulnerable to damage induced by reactive oxygen species occurring following oxidative stress. Guanosine triphosphates are the prevalent oxidized nucleotides, which can be misincorporated during replication, leading to mutations and cell death. Here, we present a novel method based on micro-Raman spectroscopy, combined with ab initio calculations, for the identification, detection, and quantification of oxidized nucleotides at low concentration. We also show that the Raman signature in the terahertz spectral range (<100 cm-1) contains information on the intermolecular assembly of guanine in tetrads, which allows us to further boost the oxidative damage detection limit. Eventually, we provide evidence that similar analyses can be carried out on samples in very small volumes at very low concentrations by exploiting the high sensitivity of surface-enhanced Raman scattering combined with properly designed superhydrophobic substrates. These results pave the way for employing such advanced spectroscopic methods for quantitatively sensing the oxidative damage of nucleotides in the cell.

Longitudinal association between myopia and parental myopia and outdoor time among students in Wenzhou: a 2.5-year longitudinal cohort study.

BACKGROUND: To evaluate the impact of parental myopia and outdoor time on myopia among students in Wenzhou. METHODS: We examined 1388 primary students from first grade to third grade in Wenzhou from September 2012 to March 2015. We performed noncycloplegic refractometry on each student every six months and axial length (AL) measurements every year. At the commencement of our study, children were asked to complete a questionnaire regarding near work activity and outdoor activity, whereas parents were asked to complete a self-administered questionnaire regarding their background circumstances and their history of myopia. RESULTS: A total of 1294 students (93.2%) returned for follow-up examinations. Children with initial and final no myopia spent more time on outdoor activities than those with new onset myopia (1.92 vs. 1.81 h/d, p = 0.022), and elongation of AL in children with a high level (> 2.5 h/day) of outdoor time (0.22 ± 0.13 mm/Y) was less than those with a low level (≤ 1.5 h/day) of outdoor time (0.24 ± 0.14 mm/Y, p = 0.045). The proportion of rapid myopia progression (≤-0.5D/Y) was 16.7%, 20.2% and 31.5% among the children with no myopic parent, one myopic parent and two myopic parents, respectively (X2 = 28.076, p < 0.001), and the elongation of AL in children among different numbers of myopic parents was significantly different (p < 0.001). A high level of outdoor time was a protective factor for children with one myopic parent (HR 0.49, 95% CI 0.27-0.88; p = 0.018). CONCLUSIONS: In this sample, parental myopia and outdoor time were associated with myopia in children. A high level of outdoor time was a protective factor for children with one myopic parent.

Involvement of BIG1 and BIG2 in regulating VEGF expression and angiogenesis.

VEGF stimulates the formation of new blood vessels by inducing endothelial cell (EC) proliferation and migration. Brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein (BIG)1 and 2 accelerate the replacement of bound GDP with GTP to activate ADP-ribosylation factor (Arf)1, which regulates vesicular transport between the Golgi and plasma membrane. Although it has been reported that treating cells with BFA interferes with Arf1 activation to inhibit VEGF secretion, the role of BIG1 and BIG2 in VEGF trafficking and expression, EC migration and proliferation, and vascular development remains unknown. Here, we found that inactivation of Arf1 reduced VEGF secretion but did not affect the levels of VEGF protein. Interestingly, however, BIG1 and BIG2 knockdown significantly decreased the levels of VEGF mRNA and protein in glioblastoma U251 cells and HUVECs. Furthermore, depletion of BIG1 and BIG2 inhibited HUVEC angiogenesis by diminishing cell migration. Angioblast migration and intersegmental vessel sprouting were also impaired when the BIG2 homolog, Arf guanine nucleotide exchange factor (arfgef)2, was knocked down in zebrafish with endothelial expression of green fluorescent protein (GFP). Depletion of arfgef2 by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) also caused defects in vascular development of zebrafish embryos. Taken together, these data reveal that BIG1 and BIG2 participate in endothelial cell angiogenesis.-Lu, F.-I., Wang, Y.-T., Wang, Y.-S., Wu, C.-Y., Li, C.-C. Involvement of BIG1 and BIG2 in regulating VEGF expression and angiogenesis.

An overview of therapeutic anticancer drug monitoring based on surface enhanced (resonance) Raman spectroscopy (SE(R)RS).

Therapeutic drug monitoring (TDM) is important for many therapeutic regimens and has particular relevance for anticancer drugs which often have serious effects and whose optimum dosage can vary significantly between different patients. Many of the features of surface enhanced (resonance) Raman spectroscopy (SE(R)RS) suggest it should be very suitable for TDM of anticancer drugs and some initial studies which explore the potential of SE(R)RS for TDM of anticancer drugs have been published. This review brings this work together in an attempt to draw some general observations about key aspects of the approach, including the nature of the substrate used, matrix interference effects and factors governing adsorption of the target molecules onto the enhancing surface. There is now sufficient evidence to suggest that none of these pose real difficulties in the context of TDM. However, some issues, particularly the need to carry out multiplex measurements for TDM of combination therapies, have yet to be addressed.

Ultrahigh piezoelectricity in ferroelectric ceramics by design.

Piezoelectric materials, which respond mechanically to applied electric field and vice versa, are essential for electromechanical transducers. Previous theoretical analyses have shown that high piezoelectricity in perovskite oxides is associated with a flat thermodynamic energy landscape connecting two or more ferroelectric phases. Here, guided by phenomenological theories and phase-field simulations, we propose an alternative design strategy to commonly used morphotropic phase boundaries to further flatten the energy landscape, by judiciously introducing local structural heterogeneity to manipulate interfacial energies (that is, extra interaction energies, such as electrostatic and elastic energies associated with the interfaces). To validate this, we synthesize rare-earth-doped Pb(Mg1/3Nb2/3)O3-PbTiO3 (PMN-PT), as rare-earth dopants tend to change the local structure of Pb-based perovskite ferroelectrics. We achieve ultrahigh piezoelectric coefficients d33 of up to 1,500 pC N-1 and dielectric permittivity ε33/ε0 above 13,000 in a Sm-doped PMN-PT ceramic with a Curie temperature of 89 °C. Our research provides a new paradigm for designing material properties through engineering local structural heterogeneity, expected to benefit a wide range of functional materials.

Exploiting the chemical differences between Ag and Au colloids allows dramatically improved SERS detection of "non-adsorbing" molecules.

In colloidal SERS only analytes that can spontaneously adsorb onto nanoparticles are detected. Therefore, considerable effort has been placed on modifying the surface properties of colloidal particles, particularly Ag particles, to promote the absorption of "difficult" analytes which do not spontaneously adsorb to as-prepared nanoparticles. In contrast, much less attention has been paid to the role which the identity of the underlying metal plays in the absorption since it is widely believed that the chemical properties of Ag and Au are very similar. This leads to the assumption that molecules which do not adsorb to Ag, such as hydrocarbons, will also not adsorb to aggregated Au colloids for SERS measurements. Here, we challenge this common perception by showing that SERS detection of "difficult" aromatic targets such as naphthalene, trinitrotoluene and 3,4-methylenedioxymethamphetamine which cannot be achieved even at >10-3 M concentrations with bare aggregated Ag colloids is possible at ≥10-8 M with unmodified aggregated Au colloids. For naphthalene and 3,4-methylenedioxymethamphetamine the detection limit obtained in this work with bare citrate-capped Au particles exceeds the previous best limit of detection obtained with surface-modified nanoparticles by an order of magnitude.

Enhancement of ß-catenin activity by BIG1 plus BIG2 via Arf activation and cAMP signals.

Multifunctional ß-catenin, with critical roles in both cell-cell adhesion and Wnt-signaling pathways, was among HeLa cell proteins coimmunoprecipitated by antibodies against brefeldin A-inhibited guanine nucleotide-exchange factors 1 and 2 (BIG1 or BIG2) that activate ADP-ribosylation factors (Arfs) by accelerating the replacement of bound GDP with GTP. BIG proteins also contain A-kinase anchoring protein (AKAP) sequences that can act as scaffolds for multimolecular assemblies that facilitate and limit cAMP signaling temporally and spatially. Direct interaction of BIG1 N-terminal sequence with ß-catenin was confirmed using yeast two-hybrid assays and in vitro synthesized proteins. Depletion of BIG1 and/or BIG2 or overexpression of guanine nucleotide-exchange factor inactive mutant, but not wild-type, proteins interfered with ß-catenin trafficking, leading to accumulation at perinuclear Golgi structures. Both phospholipase D activity and vesicular trafficking were required for effects of BIG1 and BIG2 on ß-catenin activation. Levels of PKA-phosphorylated ß-catenin S675 and ß-catenin association with PKA, BIG1, and BIG2 were also diminished after BIG1/BIG2 depletion. Inferring a requirement for BIG1 and/or BIG2 AKAP sequence in PKA modification of ß-catenin and its effect on transcription activation, we confirmed dependence of S675 phosphorylation and transcription coactivator function on BIG2 AKAP-C sequence.

Ultra-Stable Plasmonic Colloidal Aggregates for Accurate and Reproducible Quantitative SE(R)RS in Protein-Rich Biomedia.

Au/Ag colloids aggregated with simple salts are amongst the most commonly used substrates in surface-enhanced (resonance) Raman spectroscopy (SE(R)RS). However, salt-induced aggregation is a dynamic process, which means that SE(R)RS enhancements vary with time and that measurements therefore need to be taken at a fixed time point, normally within a short time-window of a few minutes. Here, we present an emulsion templated method which allows formation of densely-packed quasi-spherical Au/Ag colloidal aggregates. Since the particles in the product aggregates retain their weakly adsorbed charged ligands and the ionic strength remains low these charged aggregates resist further aggregation while still providing intense SE(R)RS enhancement which remains stable for days. This eliminates a major source of irreproducibility in conventional colloidal SE(R)RS measurements and paves the way for SE(R)RS analysis in complex systems, such as protein-rich bio-solutions where conventional aggregated colloids fail.

STIM1 Knockout Enhances PDGF-Mediated Ca2+ Signaling through Upregulation of the PDGFR⁻PLCγ⁻STIM2 Cascade.

Platelet-derived growth factor (PDGF) has mitogenic and chemotactic effects on fibroblasts. An increase in intracellular Ca2+ is one of the first events that occurs following the stimulation of PDGF receptors (PDGFRs). PDGF activates Ca2+ elevation by activating the phospholipase C gamma (PLCγ)-signaling pathway, resulting in ER Ca2+ release. Store-operated Ca2+ entry (SOCE) is the major form of extracellular Ca2+ influx following depletion of ER Ca2+ stores and stromal interaction molecule 1 (STIM1) is a key molecule in the regulation of SOCE. In this study, wild-type and STIM1 knockout mouse embryonic fibroblasts (MEF) cells were used to investigate the role of STIM1 in PDGF-induced Ca2+ oscillation and its functions in MEF cells. The unexpected findings suggest that STIM1 knockout enhances PDGFR⁻PLCγ­STIM2 signaling, which in turn increases PDGF-BB-induced Ca2+ elevation. Enhanced expressions of PDGFRs and PLCγ in STIM1 knockout cells induce Ca2+ release from the ER store through PLCγ­IP3 signaling. Moreover, STIM2 replaces STIM1 to act as the major ER Ca2+ sensor in activating SOCE. However, activation of PDGFRs also activate Akt, ERK, and JNK to regulate cellular functions, such as cell migration. These results suggest that alternative switchable pathways can be observed in cells, which act downstream of the growth factors that regulate Ca2+ signaling.

Revealing the three dimensional architecture of focal adhesion components to explain Ca2+-mediated turnover of focal adhesions.

BACKGROUND: Focal adhesions (FAs) are large, dynamic protein complexes located close to the plasma membrane, which serve as the mechanical linkages and a biochemical signaling hub of cells. The coordinated and dynamic regulation of focal adhesion is required for cell migration. Degradation, or turnover, of FAs is a major event at the trailing edge of a migratory cell, and is mediated by Ca2+/calpain-dependent proteolysis and disassembly. Here, we investigated how Ca2+ influx induces cascades of FA turnover in living cells. METHODS: Images obtained with a total internal reflection fluorescence microscope (TIRFM) showed that Ca2+ ions induce different processes in the FA molecules focal adhesion kinase (FAK), paxillin, vinculin, and talin. Three mutated calpain-resistant FA molecules, FAK-V744G, paxillin-S95G, and talin-L432G, were used to clarify the role of each FA molecule in FA turnover. RESULTS: Vinculin was resistant to degradation and was not significantly affected by the presence of mutated calpain-resistant FA molecules. In contrast, talin was more sensitive to calpain-mediated turnover than the other molecules. Three-dimensional (3D) fluorescence imaging and immunoblotting demonstrated that outer FA molecules were more sensitive to calpain-mediated proteolysis than internal FA molecules. Furthermore, cell contraction is not involved in degradation of FA. CONCLUSIONS: These results suggest that Ca2+-mediated degradation of FAs was mediated by both proteolysis and disassembly. The 3D architecture of FAs is related to the different dynamics of FA molecule degradation during Ca2+-mediated FA turnover. GENERAL SIGNIFICANCE: This study will help us to clearly understand the underlying mechanism of focal adhesion turnover by Ca2+.

[Effect of low-dose exposure to sodium arsenite on proliferation of HBE and HaCaT cells].

OBJECTIVE: To investigate the effect of chronic exposure to sodium arsenite at a dose of 1. 0 µmol / L on proliferation of human bronchial epithelial cells( HBE) and human keratinocytes( HaCaT) and discuss the mechanism of arsenic carcinogenesis. METHODS: Malignant transformation model of HBE and HaCaT cells cultured in vitro were used in this study. MTT assay was used to detect the capacity of proliferation. Flow cytometry was used to detect cell cycle. The expression of cell cycle related protein like cyclin E, cyclin D1 and cyclin A protein were inspected by Western blot. RESULTS: The treated cells, including passage 36 and 43 of HBE cells and passage 28 and 35 of HaCaT cells grow faster than the control group( P < 0. 01 and P < 0. 05). The treated cells in the G1 phase were decreased( P < 0. 05), however cells in the S phase were increased( P <0. 05). In addition, the expression of cyclin E displayed a trend of up-regulation( P <0. 05), and it was maintained at a high level in advanced period. CONCLUSION: By increasing the expression of cyclin E in HBE and HaCaT cells, low dose of sodium arsenite made cells escaping from the G1 phase to S phase, accelerating cell cycle progression and proliferation, a way that may lead to malignant transformation.

Aggregation-induced emission and intermolecular charge transfer effect in triphenylamine fluorophores containing diphenylhydrazone structures.

Three new chromophores incorporating acceptor-π-donor-π-acceptor structural motifs and mono-, di- and tri-branched diphenylsulfone base linked to triphenylamine through a hydrazone π-bridge were synthesized, and the photoluminescence properties of the three chromophores were studied in solutions as well as in aggregated states. All the fluorophores emitted strong blue fluorescence in THF. Mono- and di-branched triphenylamine both exhibited increasing blue fluorescence and displayed an AIEE effect in the aggregated state. Tri-branched triphenylamine emitted green fluorescence and presented the AIE effect in the aggregated state. These interesting phenomena have been interpreted by a molecular stacking mode with molecular dynamics (MD) and DFT calculations. The unique propeller shaped molecular configuration of triphenylamine prevented face to face π-π stacking and induced the hindered rotation, which resulted in the AIEE or AIE effect in the aggregated state. The enlarged coplanarity of diphenylhydrazone chains increased the conjugation of tri-branched triphenylamine, which was beneficial to the formation of ICT and AIE and resulted in emitting green ICT fluorescence in the aggregated state. Fluorescent microscope imaging and the fluorescent pictures of the powder states certified the strong AIEE effect or AIE effect in the solid.

Arf guanine nucleotide-exchange factors BIG1 and BIG2 regulate nonmuscle myosin IIA activity by anchoring myosin phosphatase complex.

Brefeldin A-inhibited guanine nucleotide-exchange factors BIG1 and BIG2 activate, through their Sec7 domains, ADP ribosylation factors (Arfs) by accelerating the replacement of Arf-bound GDP with GTP for initiation of vesicular transport or activation of specific enzymes that modify important phospholipids. They are also implicated in regulation of cell polarization and actin dynamics for directed migration. Reciprocal coimmunoprecipitation of endogenous HeLa cell BIG1 and BIG2 with myosin IIA was demonstrably independent of Arf guanine nucleotide-exchange factor activity, because effects of BIG1 and BIG2 depletion were reversed by overexpression of the cognate BIG molecule C-terminal sequence that follows the Arf activation site. Selective depletion of BIG1 or BIG2 enhanced specific phosphorylation of myosin regulatory light chain (T18/S19) and F-actin content, which impaired cell migration in Transwell assays. Our data are clear evidence of these newly recognized functions for BIG1 and BIG2 in transduction or integration of mechanical signals from integrin adhesions and myosin IIA-dependent actin dynamics. Thus, by anchoring or scaffolding the assembly, organization, and efficient operation of multimolecular myosin phosphatase complexes that include myosin IIA, protein phosphatase 1δ, and myosin phosphatase-targeting subunit 1, BIG1 and BIG2 serve to integrate diverse biophysical and biochemical events in cells.

Continuous activation of Nrf2 and its target antioxidant enzymes leads to arsenite-induced malignant transformation of human bronchial epithelial cells.

Long-term exposure to arsenite leads to human lung cancer, but the underlying mechanisms of carcinogenesis remain obscure. The transcription factor of nuclear factor-erythroid-2 p45-related factor (Nrf2)-mediated antioxidant response represents a critical cellular defense mechanism and protection against various diseases. Paradoxically, emerging data suggest that the constitutive activation of Nrf2 is associated with cancer development, progression and chemotherapy resistance. However, the role of Nrf2 in the occurrence of cancer induced by long-term arsenite exposure remains to be fully understood. By establishing transformed human bronchial epithelial (HBE) cells via chronic low-dose arsenite treatment, we showed that, in acquiring this malignant phenotype, continuous low level of ROS and sustained enhancement of Nrf2 and its target antioxidant enzyme levels were observed in the later-stage of arsenite-induced cell transformation. The downregulation of Keap1 level may be responsible for the over-activation of Nrf2 and its target enzymes. To validate these observations, Nrf2 was knocked down in arsenite-transformed HBE cells by SiRNA transfection, and the levels of Nrf2 and its target antioxidant enzymes, ROS, cell proliferation, migration, and colony formation were determined following these treatments. Results showed that blocked Nrf2 expression significantly reduced Nrf2 and its target antioxidant enzyme levels, restored ROS levels, and eventually suppressed cell proliferation, migration, and colony formation of the transformed cells. In summary, the results of the study strongly suggested that the continuous activation of Nrf2 and its target antioxidant enzymes led to the over-depletion of intracellular ROS levels, which contributed to arsenite-induced HBE cell transformation.

DNA Polymerases as targets for gene therapy of hepatocellular carcinoma.

BACKGROUND: Hepatocyte carcinoma (HCC) is one of the most common malignancies worldwide. Despite many achievements in diagnosis and treatment, HCC mortality remains high due to the malignant nature of the disease. Novel approaches, especially for targeted therapy, are being extensively explored. Gene therapy is ideal for such purpose for its specific expression of exogenous genes in HCC cells driven by tissue-specific promoter. However strategies based on correction of mutations or altered expressions of genes responsible for the development/progression of HCC have limitations because these aberrant molecules are not presented in all cancerous cells. In the current work, we adopted a novel strategy by targeting the DNA replication step which is essential for proliferation of every cancer cell. METHODS: A recombinant adenovirus with alpha fetoprotein (AFP) promoter-controlled expressions of artificial microRNAs targeting DNA polymerases α, δ, ε and recombinant active Caspase 3, namely Ad/AFP-Casp-AFP-amiR, was constructed. RESULTS: The artificial microRNAs could efficiently inhibit the expression of the target polymerases in AFP-positive HCC cells at both RNA and protein levels, and HCC cells treated with the recombinant virus Ad/AFP-Casp-AFP-amiR exhibited significant G0/1 phase arrest. The proliferation of HCC cells were significantly inhibited by Ad/AFP-Casp-AFP-amiR with increased apoptosis. On the contrary, the recombinant adenovirus Ad/AFP-Casp-AFP-amiR did not inhibit the expression of DNA polymerases α, δ or ε in AFP-negative human normal liver cell HL7702, and showed no effect on the cell cycle progression, proliferation or apoptosis. CONCLUSIONS: Inhibition of DNA polymerases α, δ and ε by AFP promoter-driven artificial microRNAs may lead to effective growth arrest of AFP-positive HCC cells, which may represent a novel strategy for gene therapy by targeting the genes that are essential for the growth/proliferation of cancer cells, avoiding the limitations set by any of the individually altered gene.

Colchicine derivative as a potential anti-glioma compound.

Colchicine, an anti-microtubule and antimitotic drug, is a common therapeutically agent for gout, which is thought to have potential anti-tumor effects. Owing to concerns of colchicines poisoning, the development of derivatives with low dose efficacy and less side effects is of obvious interest. In this study, we characterized the inhibitory effects of a colchicine derivative named AD1 on the cell proliferation of human malignant glioblastoma (MG) cell lines, U87MG and U373MG. We found that 50 % of U87MG and U373MG cells were reduced in the cultures after exposure to AD1 for 24 h at 10 and 50 nM, respectively. Moreover, α-tubulin immunostaining indicated that AD1 induced the disruption of the microtubule polymerization in glioma cells with apoptotic features including membrane budding/blebbing or fragmented nuclei. Increased levels of reactive oxygen species (ROS) were also detected in AD1-treated U87MG and U373MG cells compared to that observed in the control culture. Moreover, examination of microtubule-associated protein 1A/1B-light chain 3 (LC3I)/LC3II conversion and acridine orange staining for autophagic vesicles, combined with flow cytometry, showed that treatment with AD1 induced the autophagic pathway in U87MG and U373MG cells. Furthermore, we found that the intermittent intravenous administration of AD1 suppressed glioma growth in rat brain receiving intracerebral injection with rat C6 glioma cells. Taken together, our findings reveal that treatment with AD1 at nanomolar scales can reduce glioma cell viability effectively, with the occurrence of a rise in ROS and cellular autophagy. In conjunction with the observations from in vivo study, the colchicine derivative AD1 has chemotherapeutic potential to suppress glioma progression.