IR780-dye loaded gold nanoparticles as new near infrared activatable nanotheranostic agents for simultaneous photodynamic and photothermal therapy and intracellular tracking by surface enhanced resonant Raman scattering imaging.

Due to the good transparency of the human tissue in the biological spectral window, near-infrared (NIR)-dye loaded nanosystems enable more effective light-activated therapy and better contrast imaging with major impact on nanomedicine. Herein, we prepare Pluronic coated gold nanoparticles incorporating the hydrophobic NIR dye, IR780 iodide (GNP-Plu-IR780) to provide water-solubility and stability and demonstrate the proficiency of combining photodynamic and photothermal therapeutic activity with surface-enhanced resonance Raman scattering (SERRS) imaging facility. The potential of GNP-Plu-IR780 to operate as NIR-activatable agents was first assessed in aqueous solution by singlet oxygen generation measurements and monitoring the temperature increase of the nanoparticles. Subsequent in vitro uptake studies by dark field and differential interference contrast (DIC) microscopy reveal massive internalization of GNP-Plu-IR780 by murine colon carcinoma cells (C-26). Moreover, by exploiting the SERRS effect under 785 nm laser excitation we were able to perform intracellular tracking of GNP-Plu-IR780. Finally, NIR irradiation experiments conducted in vitro against C-26 cells show efficient phototherapeutic activity induced by GNP-Plu-IR780 with no dark cytotoxicity. Moreover, when compared to the administration of free drug or non-loaded GNP-Plu, the higher phototherapeutic activity of GNP-Plu-IR780 indicates the occurrence of cooperative synergistic effects by simultaneous photodynamic and photothermal activity.

Flexible and Tunable 3D Gold Nanocups Platform as Plasmonic Biosensor for Specific Dual LSPR-SERS Immuno-Detection.

Early medical diagnostic in nanomedicine requires the implementation of innovative nanosensors with highly sensitive, selective, and reliable biomarker detection abilities. In this paper, a dual Localized Surface Plasmon Resonance - Surface Enhanced Raman Scattering (LSPR- SERS) immunosensor based on a flexible three-dimensional (3D) gold (Au) nanocups platform has been implemented for the first time to operate as a relevant "proof-of-concept" for the specific detection of antigen-antibody binding events, using the human IgG - anti-human IgG recognition interaction as a model. Specifically, polydimethylsilane (PDMS) elastomer mold coated with a thin Au film employed for pattern replication of hexagonally close-packed monolayer of polystyrene nanospheres configuration has been employed as plasmonic nanoplatform to convey both SERS and LSPR readout signals, exhibiting both well-defined LSPR response and enhanced 3D electromagnetic field. Synergistic LSPR and SERS sensing use the same reproducible and large-area plasmonic nanoplatform providing complimentary information not only on the presence of anti-human IgG (by LSPR) but also to identify its specific molecular signature by SERS. The development of such smart flexible healthcare nanosensor platforms holds promise for mass production, opening thereby the doors for the next generation of portable point-of-care devices.

Revealing the structure and functionality of graphene oxide and reduced graphene oxide/pyrene carboxylic acid interfaces by correlative spectral and imaging analysis.

A high-end correlated spectral and imaging multianalysis, adapted for bidimensional systems, is presented here to analyze graphene oxide (GO) and reduced GO (rGO) modified with pyrene carboxylic acid (PCA). Confocal Raman mapping was used next to two-photon excited Fluorescence Lifetime Imaging Microscopy (FLIM) to characterize the distribution of PCA on GO and rGO and compared to UV-vis and X-ray Photoelectron Spectroscopy (XPS) analysis of the materials. Raman imaging clearly highlights the difference in the spatial distribution of PCA molecules on GO and rGO. Two-photon excited FLIM helped in gaining insight into the elusive phenomena and effects occurring at the GO-PCA interface level. Apart from the charge transfer effects from PCA molecules to GO, the GO structure depends on the molecular orientation and the spatial distribution of PCA molecules identified by different sp2 network domains in Raman mapping. Heating of GO-PCA results in an enhancement of the sp2 network presumably as the PCA aromatic core becomes fused into the GO nanosheets whilst enriching the resulting rGO nanosheets with carboxyl functionalities. This "healing" effect observed in rGO-PCA might be of high importance for applications using rGO-PCA matrices and interfaces in particular for electrical devices.

Bone metabolism regulators and arterial stiffness in postmenopausal women.

BACKGROUND: Osteoprotegerin (OPG), osteopontin (OPN) and matrix Gla protein (MGP) are markers of bone metabolism but they are also involved in vascular calcification. However, their precise role is not completely understood. Arterial stiffness is considered an independent predictor of cardiovascular events and it may be one of the causes of the increased cardiovascular risk associated with postmenopausal status. Medial and intimal calcification may increase arterial stiffness. The aim of our study was to assess the relationship of OPG, OPN and MGP with aortic pulse wave velocity (aPWV) as a marker of arterial stiffness in postmenopausal women. MATERIALS AND METHODS: Circulating OPG, OPN and serum total MGP were measured in 144 postmenopausal women using the enzyme-linked immunosorbent assay method. Aortic PWV was determined by an oscillometric method. RESULTS: Osteoprotegerin correlated with age (p<0.001, r=0.27), aPWV (p<0.001, r=0.32) and hypersensitive C reactive protein (hsCRP) (p<0.001, r=0.37), OPN correlated directly with hsCRP (p<0.001, r=0.39) and inversely with high density lipoprotein cholesterol (p=0.02, r=-0.02). No significant association was found between total MGP and clinical, biochemical and vascular parameters. The correlation between OPG and aPWV persisted even after the adjustment for various potential confounders (p=0.02, r=0.19). In multiple regression analysis in the whole study population the most important predictors of aPWV were OPG (ß=0.230, p=0.006), hsCRP (ß=0.212, p=0.01) and systolic blood pressure (ß=0.163, p=0.04). After exclusion of patients treated with statins the independent predictors were hsCRP (ß=0.275, p=0.005) and OPG (ß=0.199, p=0.04). CONCLUSION: Circulating OPG, but not OPN and total MGP, is associated with aPWV and may be a marker of the increased arterial stiffness and cardiovascular risk in postmenopausal women.

Lack of association between ACE ID polymorphism and colorectal cancer in Romanian patients.

BACKGROUND: The insertion/deletion polymorphism of the angiotensin I-converting enzyme (ACE) gene has recently been linked to the pathogenesis of human cancers. The goal of this study was to analyze the possible association between ACE gene I/D polymorphism and colorectal cancer in Romanian patients. METHODS: Blood samples were obtained, after informed consent, from individuals with colorectal cancer (n=108, M:W = 64:44), and healthy persons (n=150, M:W = 84:66). Genomic DNA was extracted from peripheral blood leucocytes using commercial kits and the insertion (I) / deletion (D) polymorphism was assessed by PCR. Statistical analysis was done using the chi2 test. We determined the odds ratio using the genotype II as risk factor. A p value < 0.05 was considered statistically significant. RESULTS: The distribution of ACE II: ID: DD genotypes was 23.1%: 46.3%: 30.6% in patients and respectively 20%: 48.7%: 31.3% in controls. The distribution of genotype (chi2 0.37, p = 0.54) and alleles (chi2 0.19, p = 0.65) did not differ significantly between cancer patients and control. CONCLUSIONS: Study results do not demonstrate an association between ACE ID polymorphism and colorectal cancer in our patients.

Evaluation of a bead-based enzyme immunoassay for the rapid detection of osteocalcin in human serum.

BACKGROUND: Circulating osteocalcin is a well-known marker for bone formation, but none of the commercial kits currently available can be used in automated systems. Here we present the first semiautomated assay for human serum osteocalcin. METHODS: Polystyrene beads were coated with antibodies against the COOH terminus of osteocalcin and used in the COBAS((R)) EIA System. Osteocalcin was detected with peroxidase-conjugated antibodies against the osteocalcin NH(2) terminus. RESULTS: The time required to analyze an unknown sample was 60 min, with a lower detection limit of 4.5 microg/L and a linear dose-response curve between 4.5 and 100 microg/L. The intraassay imprecision (CV) was 5-8% (n = 21); the interassay variation was 6-9% (n = 14). In samples from human volunteers and patients, data generated with the newly developed assay were comparable to those obtained with standard microtiter plate-based assays. CONCLUSIONS: The coated beads assay may be implemented on fully automated analyzers, which not only may further reduce imprecision but may also substantially increase the applicability of osteocalcin as a marker for bone metabolism in the routine clinical setting.

Improved bone metabolism in female elite athletes after vitamin K supplementation.

In female elite athletes strenuous exercise may result in hypoestrogenism and amenorrhoea. As a consequence a low peak bone mass and rapid bone loss are often seen in relatively young athletes. In postmenopausal women, increased intake of vitamin K may result in an increase of serum markers for bone formation, a decrease of urinary markers for bone resorption, and a decrease in urinary calcium loss. In the present paper we report an intervention study among eight female athletes, four of whom had been amenorrhoeic for more than one year, whereas the others had been using oral contraceptives. All participants received vitamin K supplementation (10 mg/day) during one month, and various bone markers were measured before and after treatment. At baseline the athletes not using oral contraceptives were biochemically vitamin K-deficient as deduced from the calcium binding capacity of the circulating bone protein osteocalcin. In all subjects increased vitamin K was associated with an increased calcium-binding capacity of osteocalcin. In the low-estrogen group vitamin K supplementation induced a 15-20% increase of bone formation markers and a parallel 20-25% decrease of bone resorption markers. This shift is suggestive for an improved balance between bone formation and resorption.

Induction of prothrombin synthesis by K-vitamins compared in vitamin K-deficient and in brodifacoum-treated rats.

Vitamin K is a group name for a number of prenylated 2-methyl-1,4-naphtoquinones, which may differ in their ability to function as a cofactor for prothrombin biosynthesis. To quantify the bioactivity of different forms of vitamin K, two experimental animal systems are frequently used: vitamin K-deficient rats and anticoagulated rats. In this paper both models are compared, and it is shown that the results obtained depend on the model used. The main reason for this discrepancy is the difference in recycling of vitamin K-epoxide, which results in a 500 times higher vitamin K requirement in anticoagulated rats. Absorption and hepatic accumulation of long chain menaquinones seem to be restricted to a maximum, whereas also the lipophilic nature of long chain menaquinones may hamper the quinone-quinol reduction in anticoagulated animals. If these data may be extrapolated to patients, food items rich in K1 and MK-4 would be expected to influence the stability of oral anticoagulation to a much larger extent than food items primarily containing higher menaquinones.

Bone markers during a 6-month space flight: effects of vitamin K supplementation.

Rapid bone loss is a serious health problem for astronauts during long lasting missions in space. We have recorded the changes of biochemical markers for bone metabolism in one of the astronauts during the 6-month space flight of the EUROMIR-95 mission. Immediately after launch both bone resorption markers and urinary calcium excretion increased about two fold, whereas bone formation markers remained unchanged. After 12 1/2 weeks the astronaut received vitamin K1 (10 mg/day for 6 weeks). Vitamin K is known to be involved in the formation of gamma-carboxyglutamate (Gla) in proteins, such as the calcium-binding bone Gla-proteins osteocalcin and matrix Gla-protein. Concomitant with the start of vitamin K treatment, the calcium-binding capacity of osteocalcin increased, and so did the urinary excretion of free Gla. This is suggestive for a subclinical vitamin K-deficiency in the astronaut before vitamin K-supplementation. During periods of high vitamin K status markers for bone formation (osteocalcin and bone alkaline phosphatase) had increased as compared to the first part of the flight. The mean increases were 14 and 23%, respectively. Our data suggest that increased intake of vitamin K may contribute to counteracting microgravity-induced loss of bone mass during long lasting space missions, but need confirmation in more astronauts.

Nutritional vitamin K-intake and urinary gamma-carboxyglutamate excretion in the rat.

Using the rat as an experimental animal model we have found that prothrombin synthesis reaches its maximal level at a relatively low dietary vitamin K intake. At still higher vitamin K intakes, however, the urinary Gla-excretion was substantially increased, showing a different vitamin K requirement for liver and extrahepatic tissues. The increased urinary Gla-excretion was found for both phylloquinone and menaquinone-4, but not for menaquinone-8, which questions the bioavailability of higher menaquinones for extrahepatic tissues. A discrepancy was found between effects of nutritional vitamin K-deficiency and treatment with a vitamin K-antagonist (brodifacoum). With both regimens plasma prothrombin rapidly decreased to well below 10% of the starting values, but in case of K-deficiency urinary Gla had hardly decreased in 7 days, whereas after 3 days of brodifacoum treatment Gla-excretion had decreased to 17% of the starting values. An explanation for this observation is that prothrombin procoagulant activity does not decrease proportional to the prothrombin Gla-content, but that a wide range of undercarboxylated prothrombins have lost nearly all activity. During vitamin K-deficiency the remaining low levels of vitamin K would mainly give rise to undercarboxylated prothrombin, whereas during brodifacoum treatment only non-carboxylated prothrombin is formed. It seems plausible that in the latter case the urinary Gla originates from proteins with long half-life times, such as the bone Gla-proteins.

Effects of vitamin K on bone mass and bone metabolism.

Vitamin K is involved in blood coagulation and in bone metabolism via the carboxylation of glutamate residues in (hepatic) blood coagulation factors and (osteoblastic) bone proteins. The bioavailability of nutritional vitamin K depends on the type of food, the dietary fat content, the length of the aliphatic side chain in the K-vitamer and probably also the genetically determined polymorphism of apolipoprotein E. Although undercarboxylation of blood coagulation factors is very rare, undercarboxylated osteocalcin (bone Gla-protein) is frequently found in postmenopausal women. Supplementation of these women with extra vitamin K causes the markers for bone formation to increase. In parallel, a decrease of the markers for bone resorption is frequently seen. Insufficient data are available to conclude that the regular administration of vitamin K concentrates will reduce the loss of bone mass in white women at risk for developing postmenopausal osteoporosis.