Significant metal enhanced fluorescence of Ag2S quantum dots in the second near-infrared window.

The amplification of light in NIR-II from Ag2S QDs via metal enhanced fluorescence (MEF) is reported for the first time. Significant fluorescence enhancement of over 100 times for Ag2S QDs deposited on Au-nanostructured arrays, paves the way for novel sensing and imaging applications based on Ag2S QDs, with improved detection sensitivity and contrast enhancement.

Inhibition of Leptin-ObR Interaction Does not Prevent Leptin Translocation Across a Human Blood-Brain Barrier Model.

The adipocyte-derived hormone leptin regulates appetite and energy homeostasis through the activation of leptin receptors (ObR) on hypothalamic neurones; hence, leptin must be transported through the blood-brain barrier (BBB) to reach its target sites in the central nervous system. During obesity, however, leptin BBB transport is decreased, in part precluding leptin as a viable clinical therapy against obesity. Although the short isoform of the ObR (ObRa) has been implicated in the transport of leptin across the BBB as a result of its elevated expression in cerebral microvessels, accumulating evidence indicates that leptin BBB transport is independent of ObRa. In the present study, we employed an ObR-neutralising antibody (9F8) to directly examine the involvement of endothelial ObR in leptin transport across an in vitro human BBB model composed of the human endothelial cell line hCMEC/D3. Our results indicate that, although leptin transport across the endothelial monolayer was nonparacellular, and energy- and endocytosis-dependent, it was not inhibited by pre-treatment with 9F8, despite the ability of the latter to recognise hCMEC/D3-expressed ObR, prevent leptin-ObR binding and inhibit leptin-induced signal transducer and activator of transcription 3 (STAT-3) phosphorylation in hCMEC/D3 cells. Furthermore, hCMEC/D3 cells expressed the transporter protein low-density lipoprotein receptor-related protein-2 (LRP-2), which is capable of binding and endocytosing leptin. In conclusion, our results demonstrate that leptin binding to and signalling through ObR is not required for efficient transport across human endothelial monolayers, indicating that ObR is not the primary leptin transporter at the human BBB, a role which may fall upon LRP-2. A deeper understanding of leptin BBB transport will help clarify the exact causes for leptin resistance seen in obesity and aid in the development of more efficient BBB-penetrating leptin analogues.

Direct in situ observation of ZnO nucleation and growth via transmission X-ray microscopy.

The nucleation and growth of a nanostructure controls its size and morphology, and ultimately its functional properties. Hence it is crucial to investigate growth mechanisms under relevant growth conditions at the nanometer length scale. Here we image the nucleation and growth of electrodeposited ZnO nanostructures in situ, using a transmission X-ray microscope and specially designed electrochemical cell. We show that this imaging technique leads to new insights into the nucleation and growth mechanisms in electrodeposited ZnO including direct, in situ observations of instantaneous versus delayed nucleation.

Avoiding artefacts during electron microscopy of silver nanomaterials exposed to biological environments.

Electron microscopy has been applied widely to study the interaction of nanomaterials with proteins, cells and tissues at nanometre scale. Biological material is most commonly embedded in thermoset resins to make it compatible with the high vacuum in the electron microscope. Room temperature sample preparation protocols developed over decades provide contrast by staining cell organelles, and aim to preserve the native cell structure. However, the effect of these complex protocols on the nanomaterials in the system is seldom considered. Any artefacts generated during sample preparation may ultimately interfere with the accurate prediction of the stability and reactivity of the nanomaterials. As a case study, we review steps in the room temperature preparation of cells exposed to silver nanomaterials (AgNMs) for transmission electron microscopy imaging and analysis. In particular, embedding and staining protocols, which can alter the physicochemical properties of AgNMs and introduce artefacts thereby leading to a misinterpretation of silver bioreactivity, are scrutinized. Recommendations are given for the application of cryogenic sample preparation protocols, which simultaneously fix both particles and diffusible ions. By being aware of the advantages and limitations of different sample preparation methods, compromises or selection of different correlative techniques can be made to draw more accurate conclusions about the data.

Mapping functional groups on oxidised multi-walled carbon nanotubes at the nanometre scale.

Despite voluminous research on the acid oxidation of carbon nanotubes (CNTs), there is a distinct lack of experimental results showing distributions of functional groups at the nanometre length scale. Here, functional peaks have been mapped across individual multi-walled CNTs with low-dose, monochromated electron energy-loss spectroscopy (EELS) in the scanning transmission electron microscope (STEM). Density functional theory simulations show that the EELS features are consistent with oxygenated functional groups, most likely carboxyl moieties.

InAs(1-x)P(x) nanowires grown by catalyst-free molecular-beam epitaxy.

We report on the self-catalysed growth of vertical InAs(1-x)P(x) nanowires on Si(111) substrates by solid-source molecular-beam epitaxy. High-resolution transmission electron microscopy revealed the mixed wurtzite and zincblende structure of the nanowires. Energy dispersive x-ray spectroscopy and x-ray diffraction measurements were used to study the phosphorus content x in the InAs(1-x)P(x) nanowires, which was shown to be in the range 0-10 %. The dependence of phosphorus incorporation in the nanowires on the phosphorus flux in the growth chamber was investigated. The incorporation rate coefficients of As and P in InAs(1x)P(x) nanowires were found to be in the ratio 10 ± 5 to 1.

Reduced size-independent mechanical properties of cortical bone in high-fat diet-induced obesity.

Overweight and obesity are rapidly expanding health problems in children and adolescents. Obesity is associated with greater bone mineral content that might be expected to protect against fracture, which has been observed in adults. Paradoxically, however, the incidence of bone fractures has been found to increase in overweight and obese children and adolescents. Prior studies have shown some reduced mechanical properties as a result of high-fat diet (HFD) but do not fully address size-independent measures of mechanical properties, which are important to understand material behavior. To clarify the effects of HFD on the mechanical properties and microstructure of bone, femora from C57BL/6 mice fed either a HFD or standard laboratory chow (Chow) were evaluated for structural changes and tested for bending strength, bending stiffness and fracture toughness. Here, we find that in young, obese, high-fat fed mice, all geometric parameters of the femoral bone, except length, are increased, but strength, bending stiffness, and fracture toughness are all reduced. This increased bone size and reduced size-independent mechanical properties suggests that obesity leads to a general reduction in bone quality despite an increase in bone quantity; yield and maximum loads, however, remained unchanged, suggesting compensatory mechanisms. We conclude that diet-induced obesity increases bone size and reduces size-independent mechanical properties of cortical bone in mice. This study indicates that bone quantity and bone quality play important compensatory roles in determining fracture risk.

Hydroxyapatite nano and microparticles: correlation of particle properties with cytotoxicity and biostability.

Synthetic colloid and gel hydroxyapatite (HA) nanoparticles (NPs) were spray dried to form microparticles (MPs). These are intended for use as slow release vaccine vectors. The physico-chemical properties of gel and colloid NPs and MPs were compared to those of HA obtained commercially. Their cytotoxicity to human monocytes'-derived macrophages (HMMs) was assessed in vitro using a range of techniques. These included the MTT assay, LDH leakage and a confocal based live-dead cell assay. Cytotoxicity differed significantly between preparations, with the suspended gel preparation being the most toxic (31-500 microg/ml). Other preparations were also toxic but only at higher concentrations (>250 microg/ml). Transmission electron microscopy (TEM) and stereology showed variable cellular uptake and subsequent dissolution of the various forms of HA. We have demonstrated that HA particle toxicity varied considerably and that it was related to their physico-chemical properties. Cell death correlated strongly with particle load. The intracellular dissolution of particles as a function of time in HMM suggests that increased cytoplasmic calcium load is likely to be the cause of cell death. Some HA NPs eluded the phagocytic pathway and a few were even seen to enter the nuclei through nuclear pores.

Ultrastructural examination of dentin using focused ion-beam cross-sectioning and transmission electron microscopy.

Focused ion-beam (FIB) milling is a commonly used technique for transmission electron microscopy (TEM) sample preparation of inorganic materials. In this study, we seek to evaluate the FIB as a TEM preparation tool for human dentin. Two particular problems involving dentin, a structural analog of bone that makes up the bulk of the human tooth, are examined. Firstly, the process of aging is studied through an investigation of the mineralization in 'transparent' dentin, which is formed naturally due to the filling up of dentinal tubules with large mineral crystals. Next, the process of fracture is examined to evaluate incipient events that occur at the collagen fiber level. For both these cases, FIB-milling was able to generate high-quality specimens that could be used for subsequent TEM examination. The changes in the mineralization suggested a simple mechanism of mineral 'dissolution and reprecipitation', while examination of the collagen revealed incipient damage in the form of voids within the collagen fibers. These studies help shed light on the process of aging and fracture of mineralized tissues and are useful steps in developing a framework for understanding such processes.

Composition-controlled nanocomposites of apatite and collagen incorporating silicon as an osseopromotive agent.

The development of a novel biocomposite of apatite (Ap) and collagen incorporating low-level additions of silicon (Si) as an osseopromotive agent is detailed. Designed to mimic the structural and compositional characteristics of developing bone, this composite is produced via a coprecipitation method, through which the weight percentage of Ap (i.e., the Ap/collagen ratio) can be varied. Coprecipitates produced at Ap contents of 80 wt % (Ap/collagen=4:1), 60 wt % (Ap/collagen=3:2), and 40 wt % (Ap/collagen=2:3) Ap showed markedly different morphologies, ranging from ceramic-like particulates to rope-like macro-fibrils; at all three Ap contents, however, the nanostructural features of the composites remained qualitatively indistinguishable, with equiaxed Ap nanocrystals distributed randomly throughout a matrix of amorphous collagen. Si incorporation was observed to occur preferentially in the collagenous phase-a result with potential impact on local controlled release of Si.

A "healthy apatite" for bone repair.

Bone is unique in its ability to adapt structure to functional requirements, but as is all too obvious in an ever-ageing population it is susceptible to a number of degenerative diseases. Therefore there is an increasing need for materials for bone replacement. Clearly, the ideal material with which to replace bone, would be bone itself, but the major problem now facing us is that there is an insufficient supply of the natural bone to satisfy the clinical requirements. Hence, there is a need for the development of chemically synthesised bone graft substitutes

Comparison of in vivo dissolution processes in hydroxyapatite and silicon-substituted hydroxyapatite bioceramics.

The incorporation of silicate into hydroxyapatite (HA) has been shown to significantly increase the rate of bone apposition to HA bioceramic implants. However, uncertainty remains about the mechanism by which silicate increases the in vivo bioactivity of HA. In this study, high-resolution transmission electron microscopy was used to observe dissolution from HA, 0.8 wt% Si-HA and 1.5 wt% Si-HA implants after 6 and 12 weeks in vivo. Our observations confirmed that defects, in particular those involving grain boundaries, were the starting point of dissolution in vivo. Dissolution was observed to follow the order 1.5 wt% Si-HA>0.8 wt% Si-HA>pure HA and it was found to be particularly prevalent at grain boundaries and triple-junctions. These observations may help to explain the mechanism by which silicate ions increase the in vivo bioactivity of pure HA, and highlight the enhanced potential of these ceramics for biomedical applications.

Pyloric ceca are significant sites of newly synthesized 22:6n-3 in rainbow trout (Oncorhynchus mykiss).

In this pulse-chase study, rainbow trout fed a diet containing deuterated (D5) (17,17,18,18,18)-18:3n-3 ethyl ester accumulated D5-22:6n-3 in pyloric ceca to a greater extent than in liver 2 d post-dose. The ratio of newly synthesized D5-22:6n-3 in ceca to that in liver 2 d after feeding D5-18:3n-3 was 4.7 +/- 1.2 when expressed as per mg tissue and 5.2 +/- 2.4 when expressed as per mg protein. The amount of D5-22:6n-3 in ceca then declined whereas that in liver and blood increased, with the ratio of ceca to liver falling to 1.7 and 1.4, respectively, by day 5 and approaching unity by day 9. A crude cecal mucosa fraction contained 123 +/- 50 ng D5-22:6n-3/mg protein/mg D5-18:3n-3 eaten 2 d after feeding the tracer, compared with 35 +/- 21 ng D5-22:6n-3/mg protein/mg D5-18:3n-3 eaten in liver. Three days later the amount in cecal mucosa had fallen by one-third and that in liver had increased threefold. Most of the D5-18:3n-3 was catabolized very rapidly. The ratio of D5-18:3n-3 to 21:4n-6 (a relatively inert FA marker) in the diet was 4.0, but this fell to 0.30 in ceca and ca. 0.8 in liver, blood, and whole carcass one day after feeding. These results indicate that ceca are active in the synthesis of 22:6n-3 and the oxidation of 18:3n-3.

The ultrastructure of the plasma-sprayed hydroxyapatite-bone interface predisposing to bone bonding.

The deposition of biological apatite and subsequent formation of bone on hydroxyapatite implants depends on the partial dissolution of the implant surface and the reprecipitation of carbonated apatite from the biological milieu. Previous investigations in vitro have shown that the degree of dissolution and reprecipitation decreases as the coating crystallinity increases. These findings prompted the current study of the effects of coating crystallinity on the mechanism of bone bonding. The process of mineralization of bone associated with a hydroxyapatite coating was compared to the normal process of ossification. Plasma-sprayed hydroxyapatite (PSHA) coated titanium alloy (6% Al-4% V) rods as received and annealed for 0.7 h at 600 degrees C in air to increase the coating crystallinity were implanted in the proximal and distal femora and proximal tibiae of adult mongrel dogs for 3 h, 3 and 10 days. Bony sites containing the implant were prepared for ultramicrotomy and transmission electron microscopy using an anhydrous embedding procedure: fixation in ethylene glycol and embedment in Spurr's resin. The results demonstrated the precipitation of biological apatite crystallites on non-annealed PSHA coatings in vivo within 3 h of implantation. After 3 and 10 days there were differences in the ultrastructure of the mineral phase on the surfaces of non-annealed and annealed surfaces. Observations showed that there was little difference in the mechanism of mineralization of bone associated with HA-coated prostheses and the normal process of ossification.

Optimization of cytokine stability in stored amniotic fluid.

OBJECTIVE: Many studies use stored amniotic fluid samples to assay cytokines and other proteins for outcome-based research; however, there is little information on the optimal methods of storage. The objective of our study was to evaluate cytokine stability in amniotic fluid stored at different temperatures both with and without a proteolytic enzyme inhibitor. STUDY DESIGN: Patients undergoing midtrimester genetic amniocentesis for routine indications gave consent for the study. After the sample was centrifuged, the acellular portion of the sample was mixed to homogeneity and aliquoted in 0.5-mL increments and stored for 1 year at 4 degrees C, -20 degrees C, and -80 degrees C with and without the protease inhibitor aprotinin. Enzyme-linked immunoassays for angiogenin, interleukin-6, and vascular endothelial growth factor were performed simultaneously on each aliquot. RESULTS: Thirty samples were assayed for each storage condition. Results were calculated as the percentage of its own sister aliquot stored at -80 degrees C without aprotinin. In all samples, there was a significant relation between storage temperature and cytokine levels, with the lowest levels found at 4 degrees C and the highest at -80 degrees C (angiogenin, P =.004; interleukin-6, P <.001; vascular endothelial growth factor, P =.02). The addition of aprotinin improved stability only for angiogenin at all temperatures (all P <.05). CONCLUSIONS: Degradation of cytokines occurs when amniotic fluid samples are stored for prolonged periods at temperatures greater than -80 degrees C. The addition of a protease inhibitor helps stem the degradation of some cytokines.

Management of isoimmunization in the presence of multiple maternal antibodies.

OBJECTIVE: Evaluation and management of patients with multiple maternal antibody isoimmunization is unclear. The presence of > or = 1 maternal antibody may suggest a worse scenario. The objective of this study was 2-fold: first, to determine whether the presence of multiple antibodies predicts a more severe course than single antibodies and second, to determine the utility of the Queenan curves/protocol in evaluating multiple-antibody isoimmunization. STUDY DESIGN: Amniotic fluid DeltaOD(450) measurements were obtained from the antenatal testing logbook and confirmed by chart review. Cases were categorized by antibody type and clinical outcomes obtained by chart review. RESULTS: Twenty-four pregnancies with isoimmunization and multiple maternal antibodies were identified; of these, 17 had 2 antibodies (anti-D and -C in 13; anti-D and -E in 1; anti-D and -Jka in 1; anti-c and -E in 1; and anti-c and -Jka in 1), and 7 had > 2 antibodies (anti-D, -C, and -E in 4; anti-D, -C, and -N in 1; anti-c, -E, and -FYA in 1; and anti-E, -K, -Fya, -S, and -C in 1). Eleven patients (46%) required at least 1 intrauterine fetal transfusion (mean initial fetal hematocrit, 15%; range, 4.9%-24%). In those not transfused, no DeltaOD(450) measurements occurred in the Queenan "fetal death risk" zone. Poorest outcomes (multiple transfusions/hydrops/fetal demise) were in patients with anti-D and anti-C, with or without anti-E. The absence of anti-D was associated with no need for fetal transfusions. The overall transfusion rate was significantly higher compared with a group of 57 isoimmunization patients with only anti-D (46% vs. 25%, P < or =.05). CONCLUSIONS: The presence of anti-D appears to be the most significant factor guiding the course of isoimmunization with multiple antibodies. The presence of another antibody with anti-D appears to significantly increase the need for intrauterine fetal transfusions. The Queenan protocol can successfully treat patients with multiple maternal red blood cell antibodies.

Cultured fish cells metabolize octadecapentaenoic acid (all-cis delta3,6,9,12,15-18:5) to octadecatetraenoic acid (all-cis delta6,9,12,15-18:4) via its 2-trans intermediate (trans delta2, all-cis delta6,9,12,15-18:5).

Octadecapentaenoic acid (all-cis delta3,6,9,12,15-18:5; 18:5n-3) is an unusual fatty acid found in marine dinophytes, haptophytes, and prasinophytes. It is not present at higher trophic levels in the marine food web, but its metabolism by animals ingesting algae is unknown. Here we studied the metabolism of 18:5n-3 in cell lines derived from turbot (Scophthalmus maximus), gilthead sea bream (Sparus aurata), and Atlantic salmon (Salmo salar). Cells were incubated in the presence of approximately 1 microM [U-14C]18:5n-3 methyl ester or [U-14C]18:4n-3 (octadecatetraenoic acid; all-cis delta6,9,12,15-18:4) methyl ester, both derived from the alga Isochrysis galbana grown in H14CO3-, and also with 25 microM unlabeled 18:5n-3 or 18:4n-3. Cells were also incubated with 25 microM trans delta2, all-cis delta6,9,12,15-18:5 (2-trans 18:5n-3) produced by alkaline isomerization of 18:5n-3 chemically synthesized from docosahexaenoic acid (all-cis delta4,7,10,13,16,19-22:6). Radioisotope and mass analyses of total fatty acids extracted from cells incubated with 18:5n-3 were consistent with this fatty acid being rapidly metabolized to 18:4n-3 which was then elongated and further desaturated to eicosatetraenoic acid (all-cis delta8,11,14,17,19-20:4) and eicosapentaenoic acid (all-cis delta5,8,11,14,17-20:5). Similar mass increases of 18:4n-3 and its elongation and further desaturation products occurred in cells incubated with 18:5n-3 or 2-trans 18:5n-3. We conclude that 18:5n-3 is readily converted biochemically to 18:4n-3 via a 2-trans 18:5n-3 intermediate generated by a delta3,delta2-enoyl-CoA-isomerase acting on 18:5n-3. Thus, 2-trans 18:5n-3 is implicated as a common intermediate in the beta-oxidation of both 18:5n-3 and 18:4n-3.

Biosynthesis and tissue deposition of docosahexaenoic acid (22:6n-3) in rainbow trout (Oncorhynchus mykiss).

Rainbow trout (Oncorhynchus mykiss) weighing ca. 5 g and previously acclimated for 8 wk on a diet comprising vegetable oil (11%), fish meal (5%), and casein (48%) as the major constituents were fed a pulse of diet containing deuterated (D5) (17,17,18,18,18)-18:3n-3 ethyl ester. The synthesis and tissue distribution of D5-22:6n-3 was determined 3, 7, 14, 24, and 35 d after the pulse. The whole-body accumulation of D5-22:6n-3 was linear over the first 7 d, corresponding to a rate of 0.54 +/- 0.12 microg D5-22:6n-3/g fish/mg D5-18:3n-3 eaten/d. Maximal accretion of D5-22:6n-3 was 4.3 +/- 1.2 microg/g fish/mg of D5-18:3n-3 eaten after 14 d. The amount of D5-22:6n-3 peaked in liver at day 7, in brain and eyes at day 24, and plateaued after day 14 in visceral and eye socket adipose tissue and in the whole fish. The majority of D5-22:6n-3 was found in the carcass (remaining tissues minus the above tissues analyzed separately) at all times. On a per milligram lipid basis, liver and eyes had the highest concentration of D5-22:6n-3. The experimental diet also contained 21:4n-6 ethyl ester as a marker to estimate the amount of food eaten by individual fish. From such estimates it was calculated that the great majority of the D5-tracer was catabolized, with the combined recovery of D5-18:3n-3 plus D5-22:6n-3 being 2.6%. The recovery of 21:4n-6 was 57.6%. The concentration of 22:6n-3 in the fish decreased during the 13-wk period, and the amount of 22:6n-3 synthesized from 18:3n-3 was only about 5% of that obtained directly from the fish meal in the diet.

Modification of odd-chain length unsaturated fatty acids by hepatocytes of rainbow trout (Oncorhynchus mykiss) fed diets containing fish oil or olive oil.

Hepatocytes isolated from rainbow trout fed on diets containing either fish oil or olive oil were incubated with individual odd-chain length unsaturated fatty acids (19:1n-9, 19:2n-6, 19:3n-3, 21:2n-6, 21:3n-6, 21:4n-6, 21:3n-3, and 21:5n-3) to examine whether these fatty acids were substrates for modification by desaturation and elongation. All odd-chain length fatty acids were readily assimilated into the lipids of hepatocytes from both dietary groups of fish, but their conversion to longer-chain, more unsaturated derivatives was more pronounced with cells from trout fed olive oil. Thus, the conversion of 19:2n-6 and 21:2n-6 to 21:3n-6 and 21:4n-6, and of 19:3n-3 to 21:4n-3 and 21:5n-3, was most obvious in cells from the olive oil group, as was the conversion of 21:3n-6 and 21:3n-3 to 21:4n-6 and 21:4n-3, respectively. Elongation of 19:1n-9 to 21:1n-9 and 23:1n-9 occurred in cells from both groups. No 23:6n-3 was detectable as a product of 19:3n-3 or 21:3n-3. However, this fatty acid was a major product formed by cells from fish fed olive oil presented with 21:5n-3. Cells from both groups of fish incorporated 21:4n-6 and 21:5n-3 into their lipids largely without modification but chain-shortened around 40, 23, and 19% of the incorporated 21:2n-6, 21:3n-3, and 19:1n-9, respectively. The results demonstrate that odd-chain length unsaturated fatty acids can act as substrates for the desaturation, elongation, and chain-shortening systems of trout hepatocytes.