Nanoparticle Surface-Enhanced Raman Scattering of Bacteriorhodopsin Stabilized by Amphipol A8-35

Surface-enhanced Raman spectroscopy (SERS) has developed dramatically since its discovery in the 1970s, because of its power as an analytical tool for selective sensing of molecules adsorbed onto noble metal nanoparticles (NPs) and nanostructures, including at the single-molecule (SM) level. Despite the high importance of membrane proteins (MPs), SERS application to MPs has not really been studied, due to the great handling difficulties resulting from the amphiphilic nature of MPs. The ability of amphipols (APols) to trap MPs and keep them soluble, stable, and functional opens up onto highly interesting applications for SERS studies, possibly at the SM level. This seems to be feasible since single APol-trapped MPs can fit into gaps between noble metal NPs, or in other gap-containing SERS substrates, whereby the enhancement of Raman scattering signal may be sufficient for SM sensitivity. The goal of the present study is to give a proof of concept of SERS with APol-stabilized MPs, using bacteriorhodopsin (BR) as a model. BR trapped by APol A8-35 remains functional even after partial drying at a low humidity. A dried mixture of silver Lee–Meisel colloid NPs and BR/A8-35 complexes give rise to SERS with an average enhancement factor in excess of 10². SERS spectra resemble non-SERS spectra of a dried sample of BR/APol complexes.     

Numerical study of nanoparticle sensors based on the detection of the two-photon-induced luminescence of gold nanorod antennas

We investigate numerically the modification of the nonlinear optical properties of a nanoantenna in the trapping of nanoparticles (NPs) by using both the discrete dipole approximation method and the finite-difference time-domain technique. The nanoantenna, which is formed by two gold nanorods (GNRs) aligned end to end and separated by a small gap, can emit strong two-photon-induced luminescence (TPL) under the excitation of a femtosecond laser light which is resonant with its longitudinal surface plasmon resonance. In addition, the excited antenna can stably trap small NPs which in turn induce modifications in the emitted TPL. These two features make it a promising candidate for building highly sensitive detectors for NPs of different materials and sizes. It is demonstrated that sensors built with antennas possess higher sensitivities than those built with single GNRs and nanorod-based antennas are more sensitive than nanoprism-based antennas. In addition, it is found that the trapping probability for a second NP is significantly reduced for the antenna with a trapped NP, implying that trapping of NPs may occur sequentially. A relationship between the TPL of the system (antenna?+?NP) and the optical potential energy of the NP is established, enabling the extraction of the information on the optical potential energy and optical force by recording the TPL of the system. It is shown that the sequential trapping and releasing of NPs flowing in a microfluid channel can be realized by designing two different antennas arranged closely.     

Development of uniform density control with self-assembled colloidal gold nanoparticles on a modified silicon substrate

Here, we present a simple method for controlling the density of Au nanoparticles (Au NPs) on a modified silicon substrate, by destabilizing the colloidal Au NPs with 3-mercaptopropyltrimethoxylsilane (3-MPTMS) for microelectromechanical-system-based applications to reduce tribological issues. A silicon surface was pretreated with a 3-MPTMS solution, immediately after which thiolated Au NPs were added to it, resulting in their uniform deposition on the silicon substrate. Without any material property change of the colloidal Au NPs, we observed the formation of large clusters Au NPs on the modified silicon surface. Analysis by scanning electron microscopy with energy dispersive X-ray spectroscopy indicated that the addition of 3-MPTMS resulted in an alternation of the chemical characteristics of the solution. Atomic force microscopy imaging supported the notion that silicon surface modification is the most important factor on tribological properties of materials along with ligand-modified Au NPs. The density of Au NPs on a silicon surface was significantly dependent on several factors, including the concentration of colloidal Au NPs, deposition time, and concentration of 3-MPTMS solution, while temperature range which was used throughout experiment was determined to have no significant effect. A relatively high density of Au NPs forms on the silicon surface as the concentrations of Au NPs and 3-MPTMS are increased. In addition, the maximum deposition of Au NPs on silicon wafer was observed at 3 h, while the effects of temperature variation were minimal.     

Effect of TiO2 nanoparticles on oxidative damage and antioxidant defense systems in chickpea seedlings during cold stress

The effects of TiO₂ nanoparticles (NPs) on physiologo-biochemical responses were studied in two chickpea (Cicer arietimun L.) genotypes differing in cold sensitivity (tolerant Sel11439 and sensitive ILC533) during cold stress (CS). The results showed that hydrogen peroxide and MDA contents and electrolyte leakage index (ELI) increased under CS conditions in both genotypes and that these damage indices were higher in ILC533 than in Sel11439 plants. In plants treated with TiO₂ NPs, a decreased H₂O₂ level was accompanied by a decrease in the MDA content and ELI compared to control plants, and these changes occurred more effectively in Sel11439 than in ILC533 plants. The antioxidant enzymes were more effective in cell protection against CS in Sel11439 plants compared to ILC533 plants, as well as in plants treated with TiO₂ NPs compared to control plants. The lipoxygenase activity was induced efficiently only in Sel11439 plants treated with TiO₂ NPs during CS, probably indicating its role in stress response (which was confirmed by measuring allen oxide synthase activity). TiO₂ NPs caused stability of chlorophyll and carotenoid contents during CS. Results suggest that TiO₂ NPs confer an increased tolerance of chickpea plants to CS, decreasing the level of injuries and increasing the capacity of defense systems.     

Impact of TiO2 nanoparticles on Vicia narbonensis L.: potential toxicity effects

This work was aimed to provide further information about toxicology of TiO₂ nanoparticles (NPs) on Vicia narbonensis L., considering different endpoints. After exposure to TiO₂ nanoparticle suspension (mixture of rutile and anatase, size <100 nm) at four different concentrations (0.2, 1.0, 2.0 and 4.0 ‰), the seeds of V. narbonensis were let to germinate in controlled environmental conditions. After 72 h, the extent of the success of the whole process (seed germination plus root elongation) was recorded as the vigour index, an indicator of possible phytotoxicity. After the characterisation of the hydric state of different materials, oxidative stress and enzymatic and nonenzymatic antioxidant responses were considered as indicators of possible cytotoxicity and to assess if damage induced by TiO₂ NPs was oxidative stress-dependent. Cytohistochemical detection of in situ DNA fragmentation as genotoxicity endpoint was monitored by TUNEL reaction. The treatments with TiO₂ NPs in our system induced phytotoxic effects, ROS production and DNA fragmentation. The nonenzymatic and enzymatic antioxidant responses were gradually and differentially activated and were able to maintain the oxidative damage to levels not significantly different from the control. On the other hand, the results of DNA fragmentation suggested that the mechanisms of DNA repair were not effective enough to eliminate early genotoxicity effects.     

Apparent Reduction of ADAM10 in Scrapie-Infected Cultured Cells and in the Brains of Scrapie-Infected Rodents

It has been described that A disintegrin and metalloproteinase (ADAM10) may involve in the physiopathology of prion diseases, but the direct molecular basis still remains unsolved. In this study, we confirmed that ADAM10 was able to cleave recombinant human prion protein in vitro. Using immunoprecipitation tests (IP) and immunofluorescent assays (IFA), reliable molecular interaction between the native cellular form of PrP (PrP^C) and ADAM10 was observed not only in various cultured neuronal cell lines but also in brain homogenates of healthy hamsters and mice. Only mature ADAM10 (after removal of its prodomain) molecules showed the binding activity with the native PrP^C. Remarkably more prion protein (PrP)-ADAM10 complexes were detected in the membrane fraction of cultured cells. In the scrapie-infected SMB cell model, the endogenous ADAM10 levels, especially the mature ADAM10, were significantly decreased in the fraction of cell membrane. IP and IFA tests of prion-infected SMB-S15 cells confirmed no detectable PrP-ADAM10 complex in the cellular lysates and PrP-ADAM10 co-localization on the cell surface. Furthermore, we demonstrated that the levels of ADAM10 in the brain homogenates of scrapie agent 263K-infected hamsters and agent ME7-infected mice were also almost diminished at the terminal stage, showing time-dependent decreases during the incubation period. Our data here provide the solid molecular basis for the endoproteolysis of ADAM10 on PrP molecules and interaction between ADAM10 and PrP^C. Obvious loss of ADAM10 during prion infection in vitro and in vivo highlights that ADAM10 may play essential pathophysiological roles in prion replication and accumulation.     

Polymorphism of DNA Repair Gene xrcc1 and Lung Cancer Risk

The current large-scale meta-analysis was performed to reach a reliable conclusion on the association between X-ray repair cross-complementing 1 (xrcc1) rs1799782 and the development of lung cancer. Studies that investigated the association between rs1799782 and lung cancer risk were identified by searching PubMed. We calculated odds ratio (OR) with corresponding 95 % confidence interval (CI) for Trp/Trp vs Arg/Arg, Trp/Trp + Arg/Trp vs Arg/Arg, and Trp/Trp vs Arg/Trp + Arg/Arg contrast models. Combining all 25 studies, we yielded three summary ORs: 1.07 (95 % CI 0.92–1.23) for Trp/Trp vs Arg/Arg, 0.93 (95 % CI 0.87–1.00) for Trp/Trp + Arg/Trp vs Arg/Arg, and 1.08 (95 % CI 0.94–1.25) for Trp/Trp vs Arg/Trp + Arg/Arg, suggesting rs1799782 was not associated with overall risk of lung cancer. Strikingly, a significantly deceased risk was found among Caucasian populations (Trp/Trp + Arg/Trp vs Arg/Arg, OR = 0.86, 95 % CI 0.76–0.97). This study confirms that xrcc1 rs1799782 may lower the risk of lung cancer among Caucasians.     

Mechanism of bFGF-binding Peptide Reversing Adriamycin Resistance in Human Gastric Cancer Cells

Development of drug resistance is a challenging problem in cancer chemotherapy. It has been shown that basic fibroblast growth factor (bFGF) plays an important role in an epigenetic mechanism of drug resistance. We have isolated a bFGF binding peptide P7 with inhibitory activity against bFGF-induced proliferation of human gastric cancer cells by screening a phage display library. In this study, we found that P7 peptide also has efficacy of reversing bFGF-induced resistance to Adriamycin (ADM) in human gastric cancer cells. Further investigations with SGC-7901 cells revealed that inhibition of Akt activation triggered by bFGF, and reversal of bFGF-induced up-regulation of Bcl-2 and XIAP and down-regulation of Bax, contribute to P7 peptide counteracting the anti-apoptotic effect of bFGF, and further reversing bFGF-induced resistance to ADM. The results suggested that the bFGF-binding peptide may have therapeutic potential of drug resistance in gastric cancer.     

Ag and Sn Nanoparticles to Enhance the Near-Infrared Absorbance of a-Si:H Thin Films

Silver (Ag) and tin (Sn) nanoparticles (NPs) were deposited by thermal evaporation onto heated glass substrates with a good control of size, shape and surface coverage. This process has the advantage of allowing the fabrication of thin-film solar cells with incorporated NPs without vacuum break, since it does not require chemical processes or post-deposition annealing. The X-ray diffraction, TEM and SEM properties are correlated with optical measurements and amorphous silicon hydrogenated (a-Si:H) films deposited on top of both types of NPs show enhanced absorbance in the near-infrared. The results are interpreted with electromagnetic modelling performed with Mie theory. A broad emission in the near-infrared region is considerably increased after covering the Ag nanoparticles with an a-Si:H layer. Such effect may be of interest for possible down-conversion mechanisms in novel photovoltaic devices.     

The amino-terminal sequence ofStreptomyces griseus carboxypeptidase

The amino-terminal sequence of carboxypeptidase fromStreptomyces griseus was determined using a new protocol for automatic Edman degradation that reduced background noise. The sequence of the first 48 residues is: Asp-Phe-Pro-Pro-Ala-Asp-Ser-Arg-Tyr-His-Asn-Tyr-Ala-Glu-Met-Asn-Ala-Ala-Ile-Asp-Ala-Arg-Ile-Ala-Ala-Asn-Pro-Ser-Ile-Met-Ser-Lys-Arg-Val-Ile-Gly-Lys-Thr-Tyr-Gln-Gly-(Arg)-Asp-Val-Ile-Ala-Val-Lys, which is homologous to that of other zinc-containing carboxypeptidase from vertebrate and invertebrate sources.     

On the existence of two GABA pools associated with newly synthesized GABA and with newly taken up GABA in nerve terminals

[¹⁴C]Glutamic acid and [³H]GABA were injected into the lateral ventricle of mouse and then [¹⁴C]GABA and [³H]GABA in synaptosomes isolated from the animals were analysed. The [¹⁴C]GABA was interpreted to be newly synthesized GABA from [¹⁴C]glutamic acid while the [³H]GABA to be newly taken up GABA. We have obtained the following results: (1) when the animals were pretreated with aminooxyacetic acid and thus the GABA content in synaptosomes increased to about 2 times of the control level, only the [³H]GABA was enhanced to 3 times of the control level without any change of [¹⁴C]GABA, (2) the release of [¹⁴C]GABA from synaptosomes by high K⁺ depolarization was 1.5 times greater than that of [³H]GABA, (3) the releases of both [¹⁴C]GABA and [³H]GABA were increased in the presence of cold GABA,l-2,4-diaminobutyric acid or γ-amino-β-hydroxybutyric acid, but only slightly increased in the presence of β-alanine. These results would suggest that newly synthesized GABA and newly taken up GABA localized individually in different pools, which might localize either in different nerve terminals or separately in the same nerve terminal.     

EGFR-AKT-mTOR activation mediates epiregulin-induced pleiotropic functions in cultured osteoblasts

Epidermal growth factor (EGF) receptor (EGFR) emerges as an essential molecule for the regulating of osteoblast cellular functions. In the current study, we explored the effect of epiregulin, a new EGFR ligand, on osteoblast functions in vitro, and studied the underlying mechanisms. We found that epiregulin-induced EGFR activation in both primary osteoblasts and osteoblast-like MC3T3-E1 cells. Meanwhile, epiregulin activated AKT-mammalian target of rapamycin (mTOR) and Erk-mitogen-activated protein kinase (MAPK) signalings in cultured osteoblasts, which were blocked by EGFR inhibitor AG1478 or monoclonal antibody against EGFR (anti-EGFR). Further, in primary and MC3T3-E1 osteoblasts, epiregulin promoted cell proliferation and increased alkaline phosphatase activity, while inhibiting dexamethasone (Dex)-induced cell death. Such effects by epiregulin were largely inhibited by AG1478 or anti-EGFR. Notably, AKT-mTOR inhibitors, but not Erk inhibitors, alleviated epiregulin-induced above pleiotropic functions in osteoblasts. Meanwhile, siRNA depletion of Sin1, a key component of mTOR complex 2 (mTORC2), also suppressed epiregulin-exerted effects in MC3T3-E1 cells. Together, these results suggest that epiregulin-induced pleiotropic functions in cultured osteoblasts are mediated through EGFR-AKT-mTOR signalings.     

Expression of TNF-α, IFN-γ, TGF-β, and IL-4 in the Spinal Tuberculous Focus and Its Impact on the Disease

To investigate the expression of TNF-α, IFN-γ, TGF-β, and IL-4 in the spinal tuberculous focus and its relationship with the lesions type, severity, and bone destruction. The pathological samples of patients with spinal tuberculosis (TB) were divided into hyperplasia group and necrosis group according to their intra-operative and post-operative pathological findings. Normal bone tissues were taken as the control group. Pathology and expression of TNF-α, IFN-γ, TGF-β, and IL-4 in different tissues were compared among these three groups using immunohistochemical staining, quantitative image analysis, and measurement of bone tissue. 286 granulomas observed in the 14 samples in the hyperplasia group, which included 84 necrotizing and 202 non-necrotizing granulomas. As for the 20 samples in the necrosis group, there were 356 necrotizing and 186 non-necrotizing granulomas among all the 542 granulomas. The proportion of necrotizing granulomas in the necrosis group was significantly higher than that of the hyperplasia group. By inter-group comparison, expression of TNF-α, IFN-γ of granulomas in the hyperplasia group was significantly higher than that of the necrosis group, while the expression of TGF-β, IL-4 of granulomas in the necrosis group was significantly higher than that of the hyperplasia group. Also, expression of IFN-γ of non-necrotizing granulomas was significantly higher than that of necrotizing granulomas in the hyperplasia group, and expression of TGF-β in necrotizing granulomas was significantly higher than that of non-necrotizing granulomas in the necrosis group. The lesions were mainly bone resorption in the hyperplasia group, whereas mostly necrotic bones accompanied by local fibrosis in the necrosis group. Expression levels of TNF-α, IFN-γ in the hyperplasia group have a positive correlation to bone loss, whereas expression levels of TGF-β, IL-4 in the necrosis group have a positive correlation to the bone formation. The high expressions of TNF-α, IFN-γ in the spinal tuberculous focus were associated with protective immune cells. TGF-β and IL-4 were related to allergic lesions, fibrosis and osteogenesis. Expression imbalance of TNF-α, IFN-γ, TGF-β, and IL-4 might aggravate the allergy of TB.     

Immune Response of the VEGF/bFGF Complex Peptide Vaccine and Function of Immune Antibodies in Inhibiting Migration of HUVEC Cells and Proliferation of Cancer Cells

Overexpression of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) plays a key role in tumor angiogenesis and metastasis in tumors. VEGF/bFGF complex peptide (VBP3) was designed to elicit the body to produce both high titer anti-VEGF and anti-bFGF antibodies to inhibit tumor angiogenesis and tumor growth. BALB/c mice were immunized with the VEGF/bFGF complex peptide, and the immune responses were assayed. Splenocytes were separated from the immunized mice and the CD4, CD8 T cells and IFN-γ were assayed by Flow cytometry. The results showed that the VBP3 could effectively stimulate immune response in mice and resulted in the increase of CD4 and CD8 T cells. CD4+ T cells and CD8+ T cells were increased from 10.78 to 15.13 and 6.82 to 11.58 % respectively. Polyclonal antibodies purified from the VBP3 immunized mice showed good anti-proliferation function to lung cancer cells, and resulted in the decrease of phosphroylation level of Akt and Erk assayed by the Western-blot. Transwell assays showed that the migration of HUVEC cells was inhibited by the antibodies. The results revealed that the VBP3 have good immunogenicity and may be used as a vaccine for tumor therapy.