Electron tomography of prolamellar bodies and their transformation into grana thylakoids in cryofixed Arabidopsis cotyledons.

The para-crystalline structures of prolamellar bodies (PLBs) and light-induced etioplast-to-chloroplast transformation have been investigated via electron microscopy. However, such studies suffer from chemical fixation artifacts and limited volumes of 3D reconstruction. Here, we examined Arabidopsis thaliana cotyledon cells by electron tomography (ET) to visualize etioplasts and their conversion into chloroplasts. We employed scanning transmission ET to image large volumes and high-pressure freezing to improve sample preservation. PLB tubules were arranged in a zinc blende-type lattice-like carbon atoms in diamonds. Within 2 h after illumination, the lattice collapsed from the PLB exterior and the disorganized tubules merged to form thylakoid sheets (pre-granal thylakoids), which folded and overlapped with each other to create grana stacks. Since the nascent pre-granal thylakoids contained curved membranes in their tips, we examined the expression and localization of CURT1 (CURVATURE THYLAKOID1) proteins. CURT1A transcripts were most abundant in de-etiolating cotyledon samples, and CURT1A was concentrated at the PLB periphery. In curt1a etioplasts, PLB-associated thylakoids were swollen and failed to form grana stacks. In contrast, PLBs had cracks in their lattices in curt1c etioplasts. Our data provide evidence that CURT1A is required for pre-granal thylakoid assembly from PLB tubules during de-etiolation, while CURT1C contributes to cubic crystal growth in the dark.

High performance production process development and scale-up of an anti-TSLP nanobody.

Nanobodies (Nbs) represent a class of single-domain antibodies with great potential application value across diverse biotechnology fields, including therapy and diagnostics. Thymic Stromal Lymphopoietin (TSLP) is an epithelial cell-derived cytokine, playing a crucial role in the regulation of type 2 immune responses at barrier surfaces such as skin and the respiratory/gastrointestinal tract. In this study, a method for the expression and purification of anti-TSLP nanobody (Nb3341) was established at 7 L scale and subsequently scaled up to 100 L scale. Key parameters, including induction temperature, methanol feed and induction pH were identified as key factors by Plackett-Burman design (PBD) and were optimized in 7 L bioreactor, yielding optimal values of 24 °C, 8.5 mL/L/h and 6.5, respectively. Furthermore, Diamond Mix-A and Diamond MMC were demonstrated to be the optimal capture and polishing resins. The expression and purification process of Nb3341 at 100L scale resulted in 22.97 g/L titer, 98.7% SEC-HPLC purity, 95.7% AEX-HPLC purity, 4 ppm of HCP content and 1 pg/mg of HCD residue. The parameters of the scaling-up process were consistent with the results of the optimized process, further demonstrating the feasibility and stability of this method. This study provides a highly promising and competitive approach for transitioning from laboratory-scale to commercial production-scale of nanobodies.

Engineering of the function of diamond-like carbon binding peptides through structural design.

The use of phage display to select material-specific peptides provides a general route towards modification and functionalization of surfaces and interfaces. However, a rational structural engineering of the peptides for optimal affinity is typically not feasible because of insufficient structure-function understanding. Here, we investigate the influence of multivalency of diamond-like carbon (DLC) binding peptides on binding characteristics. We show that facile linking of peptides together using different lengths of spacers and multivalency leads to a tuning of affinity and kinetics. Notably, increased length of spacers in divalent systems led to significantly increased affinities. Making multimers influenced also kinetic aspects of surface competition. Additionally, the multivalent peptides were applied as surface functionalization components for a colloidal form of DLC. The work suggests the use of a set of linking systems to screen parameters for functional optimization of selected material-specific peptides.

In vitro effects of nanosized diamond particles on macrophages.

The effects of synthetic diamond nanoparticles (4-6 nm) on mouse macrophage biotropism and biocompatibility and the modulation of the macrophage functions (expression of IL-1α, TNF-α, GM-CSF, bFGF, and TGF-ß) by nanoparticles in different concentrations were studied in vitro during exposure of different duration. Macrophage endocytosis of nanodiamonds increased with increasing the concentration of nanoparticles in culture and incubation time. Nanodiamonds exhibited high biotropism and biocompatibility towards macrophages; in doses of 10-20 µg/ml, they induced expression of GM-CSF and TGF-ß, inhibited expression of bFGF, and did not stimulate IL-1α and TNF-α. These data indicate that nanodiamond capture by macrophages in the studied experimental model led to modulation of the functional status of macrophages that determine their capacity to stimulate reparative processes without increasing proinflammatory and profibrogenic status.

Molecular recognition of HER-1 in whole-blood samples.

Multimode sensing was proposed for molecular screening and recognition of HER-1 in whole blood. The tools used for molecular recognition were platforms based on nanostructured materials such as the complex of Mn(III) with meso-tetra (4-carboxyphenyl) porphyrin, and maltodextrin (dextrose equivalence between 4 and 7), immobilized in diamond paste, graphite paste or C60 fullerene paste. The identification of HER-1 in whole-blood samples, at molecular level, is performed using stochastic mode and is followed by the quantification of it using stochastic and differential pulse voltammetry modes. HER-1 can be identified in the concentration range between 280 fg/ml and 4.86 ng/ml using stochastic mode, this making possible the early detection of cancers such as gastrointestinal, pancreatic and lung cancers. The recovery tests performed using whole-blood samples proved that the platforms can be used for identification and quantification of HER-1 with high sensitivity and reliability in such samples, these making them good molecular screening tools.

Ultrasensitive Diamond Microelectrode Application in the Detection of Ca2+ Transport by AnnexinA5-Containing Nanostructured Liposomes.

This report describes the innovative application of high sensitivity Boron-doped nanocrystalline diamond microelectrodes for tracking small changes in Ca2+ concentration due to binding to Annexin-A5 inserted into the lipid bilayer of liposomes (proteoliposomes), which could not be assessed using common Ca2+ selective electrodes. Dispensing proteoliposomes to an electrolyte containing 1 mM Ca2+ resulted in a potential jump that decreased with time, reaching the baseline level after ~300 s, suggesting that Ca2+ ions were incorporated into the vesicle compartment and were no longer detected by the microelectrode. This behavior was not observed when liposomes (vesicles without AnxA5) were dispensed in the presence of Ca2+. The ion transport appears Ca2+-selective, since dispensing proteoliposomes in the presence of Mg2+ did not result in potential drop. The experimental conditions were adjusted to ensure an excess of Ca2+, thus confirming that the potential reduction was not only due to the binding of Ca2+ to AnxA5 but to the transfer of ions to the lumen of the proteoliposomes. Ca2+ uptake stopped immediately after the addition of EDTA. Therefore, our data provide evidence of selective Ca2+ transport into the proteoliposomes and support the possible function of AnxA5 as a hydrophilic pore once incorporated into lipid membrane, mediating the mineralization initiation process occurring in matrix vesicles.

The aminopeptidase B (Ap-B) is phosphorylated in HEK293 cells.

Proteolysis is a post-translational modification (PTM) that affects the whole proteome. First regarded as only destructive, it is more precise than expected. It is finely regulated by other PTMs like phosphorylation. Aminopeptidase B (Ap-B), a M1 metallopeptidase, hydrolyses the peptide bond on the carbonyl side of basic residues at the NH2-terminus of peptides. 2D electrophoresis (2DE) was used to show that Ap-B is modified by phosphorylation. Detection of Ap-B by western blot after 2DE reveals several isoforms with different isoelectric points. Using alkaline phosphatase, Pro-Q Diamond phosphorylation-specific dye and kinase-specific inhibitors, we confirmed that Ap-B is phosphorylated. Phosphorylation can alter the structure of proteins leading to changes in their activity, localization, stability and association with other interacting molecules. We showed that Ap-B phosphorylation might delay its turnover. Our study illustrates the central role of the crosstalk between kinases and proteases in the regulation of many biological processes.

Diamonds in the rough: identification of individual naphthenic acids in oil sands process water.

Expansion of the oil sands industry of Canada has seen a concomitant increase in the amount of process water produced and stored in large lagoons known as tailings ponds. Concerns have been raised, particularly about the toxic complex mixtures of water-soluble naphthenic acids (NA) in the process water. To date, no individual NA have been identified, despite numerous attempts, and while the toxicity of broad classes of acids is of interest, toxicity is often structure-specific, so identification of individual acids may also be very important. Here we describe the chromatographic resolution and mass spectral identification of some individual NA from oil sands process water. We conclude that the presence of tricyclic diamondoid acids, never before even considered as NA, suggests an unprecedented degree of biodegradation of some of the oil in the oil sands. The identifications reported should now be followed by quantitative studies, and these used to direct toxicity assays of relevant NA and the method used to identify further NA to establish which, or whether all NA, are toxic. The two-dimensional comprehensive gas chromatography-mass spectrometry method described may also be important for helping to better focus reclamation/remediation strategies for NA as well as in facilitating the identification of the sources of NA in contaminated surface waters.

Osteoblast Cell Response on Polycrystalline Diamond-Coated Additively Manufactured Scaffolds.

Additive manufacturing of metals using selective laser melting can create customized parts with various degrees of complexity and geometry for medical implants. However, challenges remain in accepting orthopedic implants due to the bio-inert surface of metal scaffolds, resulting in a lack of osseointegration. Here, we show that polycrystalline diamond (PCD) coatings on selective laser melted titanium (SLM-Ti) scaffolds can improve the cell-to-material interaction of osteoblasts. The results show that by controlling the uniformity of the diamond coatings, we can mediate the biological response of osteoblasts, such as cell adhesion, proliferation, and spreading. The osteoblasts show favorable cell adhesion and spreading on non-planar PCD-coated scaffolds compared to the un-coated SLM-Ti scaffold. This study plays an important role in understanding the key physicochemical behavior of bone cell growth on customized orthopedic implant materials.

Simultaneous multisite detection of quantal release from PC12 cells using micro graphitic-diamond multi electrode arrays.

Micro graphitic - diamond - multi electrode arrays (µG-D-MEAs) are suitable for measuring multisite quantal dopamine (DA) release from PC12 cells. Following cell stimulation with high extracellular KCl and electrode polarization at +650 mV, amperometric spikes are detected with a mean frequency of 0.60 ±â€¯0.16 Hz. In each recording, simultaneous detection of secretory events is occurred in approximately 50% of the electrodes. Kinetic spike parameters and background noise are preserved among the different electrodes. Comparing the amperometric spikes recorder under control conditions with those recorders from PC12 cells previously incubated for 30 min with the dopamine precursor Levodopa (L-DOPA, 20 µM) it appears that the quantal size of amperometric spikes is increased by 250% and the half-time width (t1/2) by over 120%. On the contrary, L-DOPA has no effect on the frequency of secretory events. Overall, these data demonstrate that the µG-D-MEAs represent a reliable bio-sensor to simultaneously monitor quantal exocytotic events from different cells and in perspective can be exploited as a drug-screening tool.

Label-free DNA sensor by boron-doped diamond electrode using an ac impedimetric approach.

An electrochemical biosensor using a boron-doped diamond (BDD) electrode is described for differentiating between gene sequences according to DNA hybridization events using an ac impedimetric approach. BDD electrodes were dipped into a 1% solution of polyethylenimine (PEI) to adsorb a thin layer of positively charged PEI on the surface of BDD, then PEI-modified BDD electrodes were used to immobilize negatively charged single-stranded PCR fragments from Exon 7 of human p53 gene. Alternating current impedimetric measurements were first performed on these systems in phosphate buffered saline (PBS) and then upon exposure to single-stranded DNA (ssDNA). When the ssDNA-immobilized BDD electrode and solution ssDNA were completely complementary, a large drop in impedance was measured. Complementary DNA could be clearly detected at concentrations down to 10 (-19) g mL (-1) at a fixed frequency (10 Hz). Higher concentrations of DNA gave faster hybridization with saturation occurring at levels above 1.0 pg mL (-1.) Responses were much lower upon exposure to noncDNA, even at higher concentrations. The results show it is possible to directly detect target DNA at a fixed frequency and without additional labeling.

Peptide-grafted nanodiamonds: preparation, cytotoxicity and uptake in cells.

Nanodiamonds that were prepared by high pressure/high temperature were functionalized with biomolecules for biological applications. Nanodiamonds (NDs, < or =35 nm) that were coated by silanization or with polyelectrolyte layers were grafted with a fluorescent thiolated peptide via a maleimido function; this led to an aqueous colloidal suspension that was stable for months. These substituted NDs were not cytotoxic for CHO cells. Their capacity to enter mammalian cells, and their localisation inside were ascertained after labelling the nucleus and actin, by examining the cells by confocal, reflected light and fluorescence microscopy.

Mesenchymal stem cell interaction with ultra-smooth nanostructured diamond for wear-resistant orthopaedic implants.

Ultra-smooth nanostructured diamond (USND) can be applied to greatly increase the wear resistance of orthopaedic implants over conventional designs. Herein we describe surface modification techniques and cytocompatibility studies performed on this new material. We report that hydrogen (H)-terminated USND surfaces supported robust mesenchymal stem cell (MSC) adhesion and survival, while oxygen- (O) and fluorine (F)-terminated surfaces resisted cell adhesion, indicating that USND can be modified to either promote or prevent cell/biomaterial interactions. Given the favorable cell response to H-terminated USND, this material was further compared with two commonly used biocompatible metals, titanium alloy (Ti-6Al-4V) and cobalt chrome (CoCrMo). MSC adhesion and proliferation were significantly improved on USND compared with CoCrMo, although cell adhesion was greatest on Ti-6Al-4V. Comparable amounts of the pro-adhesive protein, fibronectin, were deposited from serum on the three substrates. Finally, MSCs were induced to undergo osteoblastic differentiation on the three materials, and deposition of a mineralized matrix was quantified. Similar amounts of mineral were deposited onto USND and CoCrMo, whereas mineral deposition was slightly higher on Ti-6Al-4V. When coupled with recently published wear studies, these in vitro results suggest that USND has the potential to reduce debris particle release from orthopaedic implants without compromising osseointegration.

Reduction of bacterial adhesion on modified DLC coatings.

The high incidence of infections caused by the use of implanted biomedical devices, including catheters, bone fracture fixation pins and heart valves, etc. has a severe impact on human health and health care costs. Diamond-like carbon (DLC) films as biomaterial for medical devices have been attracting great interest due to their excellent properties such as low friction and chemical inertness. It has been demonstrated that the properties of DLC films can be further improved by the addition of selective elements into DLC films. In this paper Si- and N-doped DLC coatings with various silicon and nitrogen contents on 316 stainless steel substrates were prepared by a magnetron sputtering technique. Bacterial adhesion to the modified DLC coatings was evaluated with Pseudomonas aeruginosa (ATCC 33347) which frequently cause medical device infections. The results showed that the addition of N or Si in DLC coatings had a significant influence on bacterial adhesion. In general the modified DLC coatings with N or Si performed better than the pure DLC coatings in inhibiting bacterial adhesion. The bacterial adhesion mechanism on the modified DLC coatings was explained with thermodynamic theory.

Biomedical applications of diamond-like carbon coatings: a review.

Owing to its superior tribological and mechanical properties with corrosion resistance, biocompatibility, and hemocompatibility, diamond-like carbon (DLC) has emerged as a promising material for biomedical applications. DLC films with various atomic bond structures and compositions are finding places in orthopedic, cardiovascular, and dental applications. Cells grew on to DLC coating without any cytotoxity and inflammation. DLC coatings in orthopedic applications reduced wear, corrosion, and debris formation. DLC coating also reduced thrombogenicity by minimizing the platelet adhesion and activation. However, some contradictory results (Airoldi et al., Am J Cardiol 2004;93:474-477, Taeger et al., Mat-wiss u Werkstofftech 2003;34:1094-1100) were also reported that no significant improvement was observed in the performance of DLC-coated stainless stent or DLC-coated femoral head. This controversy should be discussed based on the detailed information of the coating such as atomic bond structure, composition, and/or electronic structure. In addition, instability of the DLC coating caused by its high level of residual stress and poor adhesion in aqueous environment should be carefully considered. Further in vitro and in vivo studies are thus required to confirm its use for medical devices.

Benign response to particles of diamond and SiC: bone chamber studies of new joint replacement coating materials in rabbits.

Wear particles from total joint replacements are thought to accelerate prosthetic loosening. Diamond coating may improve the smoothness and wear characteristics of the femoral head component of total hip replacements, and thus increase their longevity. The brittleness of a thin diamond coat may be overcome by using an SiC-whisker diamond composite. This study describes the reactions of regenerating bone tissue to phagocytosable particles of diamond and SiC, using implanted bone harvest chambers in rabbits. The particles were dispersed in hyaluronan and introduced into a canal transversing the implant. The tissue that entered the canal during the following 3 weeks was then harvested. In previous studies using this model, particles of high density polyethylene, bone cement and chromium-cobalt all caused an inflammatory reaction and a marked decrease in the amount of ingrown bone. In the present study, neither the diamond nor the SiC particles caused any decrease in bone formation. It appears that particles of diamond and SiC are comparatively harmless.

Biocompatibility of chemical-vapour-deposited diamond.

The biocompatibility of chemical-vapour-deposited (CVD) diamond surfaces has been assessed. Our results indicate that CVD diamond is as biocompatible as titanium (Ti) and 316 stainless steel (SS). First, the amount of adsorbed and 'denatured' fibrinogen on CVD diamond was very close to that of Ti and SS. Second, both in vitro and in vivo there appears to be less cellular adhesion and activation on the surface of CVD diamond surfaces compared to Ti and SS. This evident biocompatibility, coupled with the corrosion resistance and notable mechanical integrity of CVD diamond, suggests that diamond-coated surfaces may be highly desirable in a number of biomedical applications.

Concurrent improvement in biocompatibility and bioinertness of diamond-like carbon films with nitrogen doping.

The surfaces of implantable biomaterials improving biocompatibility and bioinertness are critical for new application of bioimplantable devices. Diamond-like carbon (DLC) film is a promising biomaterial with use for coating bioimplantable devices because of its good biocompatibility, bioinertness, and mechanical properties. In this study, concurrent improvement in biocompatibility and bioinertness of DLC films has been achieved using N-incorporation technique. The N doping degree was found to play an important role in affecting the biocompatibility and bioinertness of N-doped DLC films. The results indicated that the N-doped DLC films deposited at N(2) concentration of 5% could help to create suitable condition of surface/structure/adhesion combination of DLC films in the both affinity of the L929 mouse fibroblasts and electrochemical inertness in the Hank's balanced salt solutions (simulating human body fluids). N doping supports the attachment and proliferation of cells and prevents the permeation of electrolyte solutions, thereby simultaneity improved the biocompatibility and bioinertness of DLC films. This finding is useful for the fabrication and encapsulation of in vivo devices without induced immune response in the human body.

The creation of a biomimetic interface between boron-doped diamond and immobilized proteins.

Immobilization of proteins on a solid electrode is to date done by chemical cross-linking or by addition of an adjustable intermediate. In this paper we introduce a concept where a solid with variable surface properties is optimized to mediate binding of the electron-transfer protein Cytochrome c (Cyt c) by mimicking the natural binding environment. It is shown that, as a carbon-based material, boron-doped diamond can be adjusted by simple electrochemical surface treatments to the specific biochemical requirements of Cyt c. The structure and functionality of passively adsorbed Cyt c on variously terminated diamond surfaces were characterized in detail using a combination of electrochemical techniques and atomic force microscopy with single-molecule resolution. Partially oxidized diamond allowed stable immobilization of Cyt c together with high electron transfer activity, driven by a combination of electrostatic and hydrophobic interactions. This surface mimics the natural binding partner, where coarse orientation is governed by electrostatic interaction of the protein's dipole and hydrophobic interactions assist in formation of the electron transfer complex. The optimized surface mediated electron transfer kinetics around 100 times faster than those reported for other solids and even faster kinetics than on self-assembled monolayers of alkanethiols.

Biocompatibility and mechanical properties of diamond-like coatings on cobalt-chromium-molybdenum steel and titanium-aluminum-vanadium biomedical alloys.

Diamond-like carbon (DLC) films are favored for wear components because of diamond-like hardness, low friction, low wear, and high corrosion resistance (Schultz et al., Mat-wiss u Werkstofftech 2004;35:924-928; Lappalainen et al., J Biomed Mater Res B Appl Biomater 2003;66B:410-413; Tiainen, Diam Relat Mater 2001;10:153-160). Several studies have demonstrated their inertness, nontoxicity, and the biocompatibility, which has led to interest among manufacturers of surgical implants (Allen et al., J Biomed Mater Res B Appl Biomater 2001;58:319-328; Uzumaki et al., Diam Relat Mater 2006;15:982-988; Hauert, Diam Relat Mater 2003;12:583-589; Grill, Diam Relat Mater 2003;12:166-170). In this study, hydrogen-free amorphous, tetrahedrally bonded DLC films (ta-C) were deposited at low temperatures by physical vapor deposition on medical grade Co28Cr6Mo steel and the titanium alloy Ti6Al4V (Scheibe et al., Surf Coat Tech 1996;85:209-214). The mechanical performance of the ta-C was characterized by measuring its surface roughness, contact angle, adhesion, and wear behavior, whereas the biocompatibility was assessed by osteoblast (OB) attachment and cell viability via Live/Dead assay. There was no statistical difference found in the wettability as measured by contact angle measurements for the ta-C coated and the uncoated samples of either Co28Cr6Mo or Ti6Al4V. Rockwell C indentation and dynamic scratch testing on 2-10 µm thick ta-C films on Co28Cr6Mo substrates showed excellent adhesion with HF1 grade and up to 48 N for the critical load L(C2) during scratch testing. The ta-C coating reduced the wear from 3.5 × 10(-5) mm(3)/Nm for an uncoated control sample (uncoated Co28Cr6Mo against uncoated stainless steel) to 1.1 × 10(-7) mm(3)/Nm (coated Co28Cr6Mo against uncoated stainless steel) in reciprocating pin-on-disk testing. The lowest wear factor of 3.9 × 10(-10) mm(3)/Nm was measured using a ta-C coated steel ball running against a ta-C coated and polished Co28Cr6Mo disk. Student's t-test found that the ta-C coating had no statistically significant (p < 0.05) effect on OB attachment, when compared with the uncoated control samples. There was no significant difference (p < 0.05) in the Live/Dead assay results in cell death between the ta-C coated Co28Cr6Mo and Ti6Al4V samples and the uncoated controls. Therefore, these ta-C coatings show improved wear and corrosion (Dorner-Reisel et al., Diam Relat Mater 2003;11:823-827; Affato et al., J Biomed Mater Res B Appl Biomater 2000;53:221-226; Dorner-Reisel et al., Surf Coat Tech 2004;177-178:830-837; Kim et al., Diam Relat Mater 2004;14:35-41) performance and excellent in vitro cyto-compatibility, when compared with currently used uncoated Co28Cr6Mo and Ti6Al4V implant materials.