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Using dendron chemistry, we developed stability-enhanced carboxylate surface-modified (negatively charged dendron) AuNPs (Au-NCD). Since the carboxylate surface of Au-NCD is optimal for complexation with cisplatin (Pt) moieties, we further synthesized Pt-loaded Au-NCD (Au-NCD/Pt) to serve as potential therapeutic anticancer agents. The size distribution, zeta potential, and surface plasmon resonance of both Au-NCDs and Au-NCD/Pt were characterized via dynamic light scattering, scanning transmission electron microscopy, and ultraviolet–visible spectrophotometry. Surface chemistry, Pt uptake, and Pt release were evaluated using inductively coupled plasma-mass spectrometry and X-ray photoelectron spectroscopy. Colloidal stability in physiological media over a wide pH range (1–13) and shelf-life stability (up to 6 months) were also assessed. Finally, the cytotoxicity of both Au-NCD and Au-NCD/Pt to Chinese hamster ovary cells (CHO K1; as a normal cell line) and to human lung epithelial cells (A549; as a cancer cell line) was evaluated. The results of these physicochemical and functional cytotoxicity studies with Au-NCD/Pt demonstrated that the particles exhibited superlative colloidal stability, cisplatin uptake, and in vitro anticancer activity despite low amounts of Pt release from the conjugate. [ABSTRACT FROM AUTHOR]

Aim: To quantitatively evaluate the inhibition of human DNA repair proteins APE1 and MTH1 by dextran-coated γ-Fe2O3 ultrasmall superparamagnetic iron oxide nanoparticles (dUSPIONs). Materials & methods: Liquid chromatography–tandem mass spectrometry with isotope-dilution was used to measure the expression levels of APE1 and MTH1 in MCL-5 cells exposed to increasing doses of dUSPIONs. The expression levels of APE1 and MTH1 were measured in cytoplasmic and nuclear fractions of cell extracts. Results: APE1 and MTH1 expression was significantly inhibited in both cell fractions at the highest dUSPION dose. The expression of MTH1 was linearly inhibited across the full dUSPION dose range in both fractions. Conclusion: These findings warrant further studies to characterize the capacity of dUSPIONs to inhibit other DNA repair proteins in vitro and in vivo. Inhibitors of DNA repair proteins are increasingly being utilized as potential anticancer agents to supplement traditional chemotherapy and radiation-based approaches. The present study was focused on investigating the use of iron oxide nanoparticles to inhibit the expression of relevant human DNA repair proteins in a cellular model (MCL-5 cells). The authors utilized liquid chromatography–tandem mass spectrometry with isotope dilution to measure the expression levels of two different DNA repair proteins (MTH1 and APE1) in cells after the cells were exposed to increasing levels of the iron oxide nanoparticles. The authors observed significant decreases in DNA repair protein levels that were associated with increasing doses of the iron oxide nanoparticles. The authors' findings warrant more comprehensive studies using other cellular models and suitable animal models. [ABSTRACT FROM AUTHOR]

The gene Rv1885c from the genome of Mycobacterium tuberculosis [H.sub.37][R.sub.v] encodes a monofunctional and secreted chorismate mutase (*MtCM) with a 33-amino-acid cleavable signal sequence; hence, it belongs to the [sup.*]AroQ class of chorismate mutases. Consistent with the heterologously expressed [sup.*]MtCM having periplasmic destination in Escherichia coli and the absence of a discrete periplasmic compartment in M. tuberculosis, we show here that [sup.*]MtCM secretes into the culture filtrate of M. tuberculosis. [sup.*]MtCM functions as a homodimer and exhibits a dimeric state of the protein at a concentration as low as 5 nM. [sup.*]MtCM exhibits simple Michaelis-Menten kinetics with a [K.sub.m] of 0.5 [+ or -] 0.05 mM and a [k.sub.cat] of 60 [s.sup.-1] per active site (at 37[degrees]C and pH 7.5). The crystal structure of [sup.*]MtCM has been determined at 1.7 [Angstrom] resolution (Protein Data Bank identifier 2F6L). The protein has an all alpha-helical structure, and the active site is formed within a single chain without any contribution from the second chain in the dimer. Analysis of the structure shows a novel fold topology for the protein with a topologically rearranged helix containing [Arg.sub.134]. We provide evidence by site-directed mutagenesis that the residues [Arg.sub.49], [Lys.sub.60], [Arg.sub.72], [Thr.sub.105], [Glu.sub.109], and [Arg.sub.134] constitute the catalytic site; the numbering of the residues includes the signal sequence. Our investigation on the effect of phenylalanine, tyrosine, and tryptophan on [sup.*]MtCM shows that [sup.*]MtCM is not regulated by the aromatic amino acids. Consistent with this observation, the X-ray structure of [sup.*]MtCM does not have an allosteric regulatory site.

A unified extraction and quantification procedure based on stable isotope-dilution liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed for the simultaneous determination of total homocysteine and folate (5-methyltetrahydrofolic acid and folic acid) levels in human serum and plasma. This is the first report documenting the simultaneous extraction and quantification of these structurally dissimilar analytes. Analytes are quantitatively isolated from samples (500 [micro]L) prior to LC/ MS/MS analysis using a two-step stabilization process combined with [C.sub.18] solid-phase extraction. The method exhibits excellent linearity over 4 orders of magnitude for each analyte. Measurement repeatability (RSD, N = 2) ranged from 0.3% to 3% for all analytes over 1 day of analysis. Total method variability (RSD, N = 6) ranged from 0.7% to 10% for all analytes over three independent days of analysis. The accuracy and practical applicability of the method were demonstrated by applying the method to the quantitative determination of each analyte in a new NIST serum Standard Reference Material (NIST SRM 1955 Homocysteine and Folate in Frozen Human Serum) and in a small subset of normal donor plasma samples.

Two independent methods have been critically evaluated and applied to the measurement of total homocysteine in serum and plasma: solid-phase anion extraction (SPAE) gas chromatography/mass spectrometry (GC/MS) and protein precipitation liquid chromatography/tandem mass spectrometry (LC/MS/MS). In addition, analysis of samples prepared by SPAE was accomplished by liquid chromatography/mass spectrometry (LC/MS) and LC/MS/MS. These methods have been used to determine total homocysteine levels in several existing serum-based Standard Reference Materials (SRMs) from the National Institute of Standards and Technology and in patient plasma samples provided by the Centers for Disease Control and Prevention. The precision of the homocysteine measurements in serum and plasma was critically evaluated, and method comparisons were carried out using Bland--Altman plots and bias analysis. On the basis of the excellent precision and close agreement of the mass spectrometric (MS) methods, the MS-based methods will be used for certification of a serum-based SRM for homocysteine and folates.

Elevated plasma homocysteine has been identified as a strong and independent risk factor for cardiovascular diseases, and recently, it has been associated with the development of dementia in older adults. Selected ion-monitoring isotope-dilution LC/MS (electrospray) has been developed and evaluated as a reference method for the accurate determination of total homocysteine in human plasma. Homocysteine is quantitatively isolated from plasma via the use of anion-exchange resins and then detected and quantified in stabilized plasma extracts with selected ion-monitoring LC/MS. This method is shown to be highly comparable to LC/MS/MS determinations in terms of its analytical accuracy and precision, yet this alternative measurement approach does not necessitate the enhanced instrumentation or added expense required of tandem MS/MS determinations. LC/MS detection of homocysteine was linear (standard error of the estimate for the regression line was 0.0323) over 3 orders of magnitude, and the calculated limits of detection and quantification were 0.06 [micro]mol/L (0.12 ng on column) and 0.6 [micro]mol/L (1.2 ng on column), respectively. Independent calibration curves showed excellent linearity ([r.sup.2] [greater than or equal to] 0.996) between 0 and 25 [micro]mol/L homocysteine over a 3-day period. The accuracy and precision of total homocysteine measurements for patient samples and quality control pools using LC/MS were compared to total homocysteine measurements using LC/MS/MS, GC/MS, FPIA, and LC-FD. LC/MS performed well in relation to the other homocysteine methods in terms of its capability to accurately quantify plasma homocysteine over the normal range (5-15 [micro]mol/L).

Engineered nanomaterials (ENMs) come in a wide array of shapes, sizes, surface coatings, and compositions, and often possess novel or enhanced properties compared to larger sized particles of the same elemental composition. To ensure the safe commercialization of products containing ENMs, it is important to thoroughly understand their potential risks. Given that ENMs can be created in an almost infinite number of variations, it is not feasible to conduct in vivo testing on each type of ENM. Instead, new approach methodologies (NAMs) such as in vitro or in chemico test methods may be needed, given their capacity for higher throughput testing, lower cost, and ability to provide information on toxicological mechanisms. However, the different behaviors of ENMs compared to dissolved chemicals may challenge safety testing of ENMs using NAMs. In this study, member agencies within the Interagency Coordinating Committee on the Validation of Alternative Methods were queried about what types of ENMs are of agency interest and whether there is agency-specific guidance for ENM toxicity testing. To support the ability of NAMs to provide robust results in ENM testing, two key issues in the usage of NAMs, namely dosimetry and interference/bias controls, are thoroughly discussed. [ABSTRACT FROM AUTHOR]