Reactive species in RF plasma sputtering deposition of nanocrystalline calcium phosphate bio-active films

Optical emission of reactive plasma species during the synthesis of functionally graded calcium phosphate-based bioactive films has been investigated. The coatings have been deposited on Ti-6Al-4V orthopedic alloy by co-sputtering of hydroxyapatite (HA) and titanium targets in reactive plasmas of Ar + H2O gas mixtures. The species, responsible for the Ca-P-Ti film growth have been non-intrusively monitored in situ by a high-resolution optical emission spectroscopy (OES). It is revealed that the optical emission originating from CaO species dominates throughout the deposition process. The intensities of CaO, PO and CaPO species are strongly affected by variations of the operating pressure, applied RF power, and DC substrate bias. The optical emission intensity (OEI) of reaction species can efficiently be controlled by addition of H2O reactant.

Ultra-trace detection of diagnostically important biomarkers using functionalised-Surface Enhanced Raman Spectroscopy (SERS)

Here we report an ultrasensitive method for detecting bio-active compounds in biological samples by means of functionalised nanoparticles interrogated by surface enhanced Raman spectroscopy (SERS). This method is applicable to the recovery and detection of many diagnostically important peptidyl analytes such as insulin, human growth hormone, growth factors (IGFs) and erythropoietin (EPO), as well as many small molecule analytes and metabolites. Our method, developed to detect EPO, demonstrates its utility in a complex yet well defined biological system. Recombinant human EPO (rhEPO) and EPO analogues have successfully been used to treat anaemia in end-stage renal failure, chronic disorders and infections, cancer and AIDS. Current methods for EPO testing are lengthy, laborious and relatively insensitive to low concentrations. In our rapid screening methodology, gold nanoparticles were functionalised with anti-EPO antibodies to provide very high selectivity towards the EPO protein in urine. These “smart sensor” nanoparticles interact with and trap EPO. Subsequent SERS screening allows for the detection and quantisation of ultra trace amounts (<<10-15 M) of EPO in urine samples with minimal sample preparation. We present data showing that the SERS spectrum differentiates between human endogenous EPO and rhEPO in unpurified urine, and potentially distinguishes between purified EPO isoforms. The elimination of sample preparation and direct screening in biological fluids significantly reduces the time required by current methods. Antibody recognition against a variety of biological targets and the availability of portable commercial SERS analysers for rapid onsite testing suggest broad diagnostic applicability in a flexible analytical platform.

Ultra sensitive label free surface enhanced Raman spectroscopy method for the detection of biomolecules

We present a proof of concept for a novel nanosensor for the detection of ultra-trace amounts of bio-active molecules in complex matrices. The nanosensor is comprised of gold nanoparticles with an ultra-thin silica shell and antibody surface attachment, which allows for the immobilization and direct detection of bio-active molecules by surface enhanced Raman spectroscopy (SERS) without requiring a Raman label. The ultra-thin passive layer (~1.3 nm thickness) prevents competing molecules from binding non-selectively to the gold surface without compromising the signal enhancement. The antibodies attached on the surface of the nanoparticles selectively bind to the target molecule with high affinity. The interaction between the nanosensor and the target analyte result in conformational rearrangements of the antibody binding sites, leading to significant changes in the surface enhanced Raman spectra of the nanoparticles when compared to the spectra of the un-reacted nanoparticles. Nanosensors of this design targeting the bio-active compounds erythropoietin and caffeine were able to detect ultra-trace amounts the analyte to the lower quantification limits of 3.5×10−13 M and 1×10−9 M, respectively.

Structure, bonding state and in-vitro study of Ca–P–Ti film deposited on Ti6Al4V by RF magnetron sputtering

Experimental investigation of functionally graded calcium phosphate-based bio-active films on Ti-6A1-4V orthopaedic alloy prepared in an RF magnetron sputtering plasma reactor is reported. The technique involves concurrent sputtering of Hydroxyapatite (HA) and Ti targets, which results in remarkably enhanced adhesion of the film to the substrate and stability of the interface. The films have been characterized using X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS). The XPS data show that the films are composed of O, Ca, P and Ti, and reveal the formation of O=P groups and hybridization of O-Ca-P. The XRD pattern shows that the Ca-P thin films are of crystalline calcium oxide phosphate (4CaO·P2O5) with preferred orientation varying with processing parameters. High-resolution optical emission spectra show that the emission of CaO is dominant. The CaO, PO and CaPO species are strongly influenced by deposition conditions. The introduction of Ti element during deposition provides a stable interface between bio-inert substrates Ti-6A1-4V and bioactive HA coating. In-vitro cell culturing tests suggest excellent biocompatibility of the Ca-P-Ti films.

Activation of matrix metalloproteinase-2 (MMP-2) by membrane type 1 matrix metalloproteinase through an artificial receptor for ProMMP-2 generates active MMP-2

The suggested model for pro-matrix metalloproteinase-2 (proMMP-2) activation by membrane type 1 MMP (MT1-MMP) implicates the complex between MT1-MMP and tissue inhibitor of MMP-2 (TIMP-2) as a receptor for proMMP-2. To dissect this model and assess the pathologic significance of MMP-2 activation, an artificial receptor for proMMP-2 was created by replacing the signal sequence of TIMP-2 with cytoplasmic/transmembrane domain of type II transmembrane mosaic serine protease (MSP-T2). Unlike TIMP-2, MSP-T2 served as a receptor for proMMP-2 without inhibiting MT1-MMP, and generated TIMP-2-free active MMP-2 even at a low level of MT1-MMP. Thus, MSP-T2 did not affect direct cleavage of the substrate testican-1 by MT1-MMP, whereas TIMP-2 inhibited it even at the level that stimulates proMMP-2 processing. Expression of MSP-T2 in HT1080 cells enhanced MMP-2 activation by endogenous MT1-MMP and caused intensive hydrolysis of collagen gel. Expression of MSP-T2 in U87 glioma cells, which express a trace level of endogenous MT1-MMP, induced MMP-2 activation and enhanced cell-associated protease activity, activation of extracellular signal-regulated kinase, and metastatic ability into chick embryonic liver and lung. MT1-MMP can exert both maximum MMP-2 activation and direct cleavage of substrates with MSP-T2, which cannot be achieved with TIMP-2. These results suggest that MMP-2 activation by MT1-MMP potentially amplifies protease activity, and combination with direct cleavage of substrate causes effective tissue degradation and enhances tumor invasion and metastasis, which highlights the complex role of TIMP-2. MSP-T2 is a unique tool to analyze physiologic and pathologic roles of MMP-2 and MT1-MMP in comparison with TIMP-2.

An SECM study on the influence of cationic, membrane-active peptides on a gold-supported self-assembled monolayer

The influence of the membrane active peptides, Tat44–57 (activator in HIV-1) and melittin (active content of bee venom), on self-assembled monolayers of 6-mercaptohexanoic acid (MHA) on gold electrodes has been studied with scanning electrochemical microscopy (SECM). It was found that MHA, when deprotonated at physiological pH, significantly affected the relative rates of electron transfer between the [Fe(CN)6]4− solution based mediator and the underlying gold electrode, predominantly by the electrostatic interaction between the mediator and MHA. Upon the introduction of Tat44–57 ormelittin to the electrolyte, the relative rate of electron transfer through the MHA layer could be increased or decreased depending on the mediator used. However, in all cases it was found that these peptides have the ability to be incorporated into synthetic SAMs, which has implications for future electrochemical studies carried out using cell mimicking membranes immobilised on such layers.

Expression of Potato virus Y cytoplasmic inclusion protein in tobacco results in disorganization of parenchyma cells, distortion of epidermal cells, and induces mitochondrial and chloroplast abnormalities, formation of membrane whorls and atypical lipid accumulation

Infection of plant cells by potyviruses induces the formation of cytoplasmic inclusions ranging in size from 200 to 1000 nm. To determine if the ability to form these ordered, insoluble structures is intrinsic to the potyviral cytoplasmic inclusion protein, we have expressed the cytoplasmic inclusion protein from Potato virus Y in tobacco under the control of the chrysanthemum ribulose-1,5-bisphosphate carboxylase small subunit promoter, a highly active, green tissue promoter. No cytoplasmic inclusions were observed in the leaves of transgenic tobacco using transmission electron microscopy, despite being able to clearly visualize these inclusions in Potato virus Y infected tobacco leaves under the same conditions. However, we did observe a wide range of tissue and sub-cellular abnormalities associated with the expression of the Potato virus Y cytoplasmic inclusion protein. These changes included the disruption of normal cell morphology and organization in leaves, mitochondrial and chloroplast internal reorganization, and the formation of atypical lipid accumulations. Despite these significant structural changes, however, transgenic tobacco plants were viable and the results are discussed in the context of potyviral cytoplasmic inclusion protein function.

Active regulation of the epidermal calcium profile

A distinct calcium profile is strongly implicated in regulating the multi-layered structure of the epidermis. However, the mechanisms that govern the regulation of this calcium profile are currently unclear. It clearly depends on the relatively impermeable barrier of the stratum corneum (passive regulation) but may also depend on calcium exchanges between keratinocytes and extracellular fluid (active regulation). Using a mathematical model that treats the viable sublayers of unwounded human and murine epidermis as porous media and assumes that their calcium profiles are passively regulated, we demonstrate that these profiles are also actively regulated. To obtain this result, we found that diffusion governs extracellular calcium motion in the viable epidermis and hence intracellular calcium is the main source of the epidermal calcium profile. Then, by comparison with experimental calcium profiles and combination with a hypothesised cell velocity distribution in the viable epidermis, we found that the net influx of calcium ions into keratinocytes from extracellular fluid may be constant and positive throughout the stratum basale and stratum spinosum, and that there is a net outflux of these ions in the stratum granulosum. Hence the calcium exchange between keratinocytes and extracellular fluid differs distinctly between the stratum granulosum and the underlying sublayers, and these differences actively regulate the epidermal calcium profile. Our results also indicate that plasma membrane dysfunction may be an early event during keratinocyte disintegration in the stratum granulosum.

Binding of small mono- and oligomeric integrin ligands to membrane-embedded integrins monitored by surface plasmon-enhanced fluorescence spectroscopy

We recently developed a binding assay format by incorporating native transmembrane receptors into artificial phospholipid bilayers on biosensor devices for surface plasmon resonance spectroscopy. By extending the method to surface plasmon-enhanced fluorescence spectroscopy (SPFS), sensitive recording of the association of even very small ligands is enabled. Herewith, we monitored binding of synthetic mono- and oligomeric RGD-based peptides and peptidomimetics to integrins alphavbeta3 and alphavbeta5, after having confirmed correct orientation and functionality of membrane-embedded integrins. We evaluated integrin binding of RGD multimers linked together via aminohexanoic acid (Ahx) spacers and showed that the dimer revealed higher binding activity than the tetramer, followed by the RGD monomers. The peptidomimetic was also found to be highly active with a slightly higher selectivity toward alphavbeta3. The different compounds were also evaluated in in vitro cell adhesion tests for their capacity to interfere with alphavbeta3-mediated cell attachment to vitronectin. We hereby demonstrated that the different RGD monomers were similarly effective; the RGD dimer and tetramer showed comparable IC50 values, which were, however, significantly higher than those of the monomers. Best cell detachment from vitronectin was achieved by the peptidomimetic. The novel SPFS-binding assay platform proves to be a suitable, reliable, and sensitive method to monitor the binding capacity of small ligands to native transmembrane receptors, here demonstrated for integrins.

Association of increased basement membrane invasiveness with absence of estrogen receptor and expression of vimentin in human breast cancer cell lines

Lack of estrogen receptor (ER) and presence of vimentin (VIM) associate with poor prognosis in human breast cancer. We have explored the relationships between ER, VIM, and invasiveness in human breast cancer cell lines. In the matrigel outgrowth assay, ER+/VIM- (MCF-7, T47D, ZR-75-1), and ER-/VIM- (MDA-MB-468, SK-Br-3) cell lines were uninvasive, while ER-/VIM+ (BT549, MDA-MB-231, MDA-MB-435, MDA-MB-436, Hs578T) lines formed invasive, penetrating colonies. Similarly, ER-/VIM+ cell lines were significantly more invasive than either the ER+/VIM- or ER-/VIM- cell lines in the Boyden chamber chemoinvasion assay. Invasive activity in nude mice was only seen with ER-/ VIM+ cell lines MDA-MB-231, MDA-MB-435 and MDA-MB-436. Hs578T cells (ER-/VIM+) showed hematogenous dissemination to the lungs in one of five mice, but lacked local invasion. The ER-/VIM+ MCF-7ADR subline was significantly more active than the MCF-7 cells in vitro, but resembled the wild-type MCF-7 parent in in vivo activity. Data from these cell lines suggest that human breast cancer progression results first in the loss of ER, and subsequently in VIM acquisition, the latter being associated with increased metastatic potential through enhanced invasiveness. The MCF-7ADR data provide evidence that this transition can occur in human breast cancer cells. Vimentin expression may provide useful insights into mechanisms of invasion and/or breast cancer cell progression.

Lipid-modified azurin of Neisseria meningitidis is a copper protein localized on the outer membrane surface and not regulated by FNR

The laz gene of Neisseria meningitidis is predicted to encode a lipid-modified azurin (Laz). Laz is very similar to azurin, a periplasmic protein, which belongs to the copper-containing proteins in the cupredoxin superfamily. In other bacteria, azurin is an electron donor to nitrite reductase, an important enzyme in the denitrifying process. It is not known whether Laz could function as an electron transfer protein in this important pathogen. Laz protein was heterologously expressed in Escherichia coli and purified. Electrospray mass spectrometry indicated that the Laz protein contains one copper ion. Laz was shown to be redox-active in the presence of its redox center copper ion. When oxidized, Laz exhibits an intense blue colour and absorbs visible light around 626 nm. The absorption is lost when exposed to diethyldithiocarbamate, a copper chelating agent. Polyclonal antibodies were raised against purified Laz for detecting expression of Laz under different growth conditions and to determine the orientation of Laz on the outer membrane. The expression of Laz under microaerobic and microaerobic denitrifying conditions was slightly higher than that under aerobic conditions. However, the expression of Laz was similar between the wild type strain and an fnr mutant, suggesting that Fumarate/Nitrate reduction regulator (FNR) does not regulate the expression of Laz despite the presence of a partial FNR box upstream of the laz gene. We propose that some Laz protein is exposed on the outer membrane surface of N. meningitidis as the αLaz antibodies can increase killing by complement in a capsule deficient N. meningitidis strain, in a dose-dependent fashion.