In Vitro Study of Tumor-Homing Peptide-Modified Magnetic Nanoparticles for Magnetic Hyperthermia.

Cancer cells have higher heat sensitivity compared to normal cells; therefore, hyperthermia is a promising approach for cancer therapy because of its ability to selectively kill cancer cells by heating them. However, the specific and rapid heating of tumor tissues remains challenging. This study investigated the potential of magnetic nanoparticles (MNPs) modified with tumor-homing peptides (THPs), specifically PL1 and PL3, for tumor-specific magnetic hyperthermia therapy. The synthesis of THP-modified MNPs involved the attachment of PL1 and PL3 peptides to the surface of the MNPs, which facilitated enhanced tumor cell binding and internalization. Cell specificity studies revealed an increased uptake of PL1- and PL3-MNPs by tumor cells compared to unmodified MNPs, indicating their potential for targeted delivery. In vitro hyperthermia experiments demonstrated the efficacy of PL3-MNPs in inducing tumor cell death when exposed to an alternating magnetic field (AMF). Even without exposure to an AMF, an additional ferroptotic pathway was suggested to be mediated by the nanoparticles. Thus, this study suggests that THP-modified MNPs, particularly PL3-MNPs, hold promise as a targeted approach for tumor-specific magnetic hyperthermia therapy.

Preparation of Functional Nanoparticles-Loaded Magnetic Carbon Nanohorn Nanocomposites towards Composite Treatment.

Combination therapy for cancer is expected for the synergetic effect of different treatments, and the development of promising carrier materials is demanded for new therapeutics. In this study, nanocomposites including functional nanoparticles (NPs) such as samarium oxide NP for radiotherapy and gadolinium oxide NP as a magnetic resonance imaging agent were synthesized and chemically combined with iron oxide NP-embedded or carbon dot-coating iron oxide NP-embedded carbon nanohorn carriers, where iron oxide NP is a hyperthermia reagent and carbon dot exerts effects on photodynamic/photothermal treatments. These nanocomposites exerted potential for delivery of anticancer drugs (doxorubicin, gemcitabine, and camptothecin) even after being coated with poly(ethylene glycol). The co-delivery of these anticancer drugs played better drug-release efficacy than the independent drug delivery, and the thermal and photothermal procedures enlarged the drug release. Thus, the prepared nanocomposites can be expected as materials to develop advanced medication for combination treatment.

Role of HIKESHI on Hyperthermia for Castration-Resistant Prostate Cancer and Application of a Novel Magnetic Nanoparticle with Carbon Nanohorn for Magnetic Hyperthermia.

The prognosis of castration-resistant prostate cancer (CRPC) is technically scarce; therefore, a novel treatment for CRPC remains warranted. To this end, hyperthermia (HT) was investigated as an alternative therapy. In this study, the analysis focused on the association between CRPC and heat shock protein nuclear import factor "hikeshi (HIKESHI)", a factor of heat tolerance. Silencing the HIKESHI expression of 22Rv1 cells (human CRPC cell line) treated with siRNAs inhibited the translocation of heat shock protein 70 from the cytoplasm to the nucleus under heat shock and enhanced the effect of hyperthermia. Moreover, a novel magnetic nanoparticle was developed via binding carbon nanohorn (CNH) and iron oxide nanoparticle (IONP) with 3-aminopropylsilyl (APS). Tumor-bearing model mice implanted with 22 Rv1 cells were examined to determine the effect of magnetic HT (mHT). We locally injected CNH-APS-IONP into the tumor, which was set under an alternative magnetic field and showed that tumor growth in the treatment group was significantly suppressed compared with other groups. This study suggests that HIKESHI silencing enhances the sensitivity of 22Rv1 cells to HT, and CNH-APTES-IONP deserves consideration for mHT.

Direct chiral separation of abscisic acid by high-performance liquid chromatography with a phenyl column and a mobile phase containing γ-cyclodextrin.

Abscisic acid (2-cis,4-trans-abscisic acid) is a plant hormone that has an asymmetric carbon atom. We tried to separate the enantiomers of native abscisic acid by HPLC using a phenyl column and a chiral mobile phase containing γ-cyclodextrin. The optimum mobile phase conditions were found to be 0.8% (w/v) γ-cyclodextrin, 4% (v/v) acetonitrile, and 20 mM phosphate buffer (pH 6.0). It was found that (R)-abscisic acid was earlier detected than (S)-abscisic acid. Since γ-cyclodextrin is hardly retained on a phenyl column, it was suggested that (R)-abscisic acid formed a more stable complex with γ-cyclodextrin than the (S)-abscisic acid. Abscisic acid in an acacia honey sample was successfully enantioseparated with the proposed method and only (S)-abscisic acid was detected. A biologically inactive 2-trans,4-trans-abscisic acid, which was prepared by irradiation of abscisic acid with a light-emitting diode lamp at 365 nm, was partially enantioseparated by the proposed method. Since the irradiation of (S)-abscisic acid-induced cis-to-trans isomerization to produce one 2-trans,4-trans-abscisic acid enantiomer, it is reasonable that racemization did not proceed during the cis-to-trans isomerization. (S)-Abscisic acid and probably (S)-2-trans,4-trans-abscisic acid were detected in a honey sample, where the peak area of (S)-abscisic acid was 7 times larger than that of (S)-2-trans,4-trans-abscisic acid.

LC-MS/MS assay for the investigation of acetylated Alpha-synuclein in serum from postmortem Alzheimer's disease pathology.

Alpha-synuclein (α-Syn), a neuronal protein, has been linked to the inflammation and development of neurodegenerative diseases. In a number of neurodegenerations, α-Syn has been investigated in the central nervous system and cerebrospinal fluid. However, there are few studies concerning the variations in peripheral α-Syn in postmortem Alzheimer's disease (AD) pathology. In this study, the quantitative procedure for the determination of peripheral acetylated α-Syn regarding N-terminal amino acid's site (α-Syn1-6; MDVFMK and Ac-α-Syn1-6; Ac-MDVFMK) was developed using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and tryptic digestion without antibody. Serum samples were selected from postmortem specimens based on autopsy pathological examination of AD remark. The LC-MS/MS assay with ACQUITY UPLC BEH C18 column was applied on the basis of electrospray positive ionization. When subjected to N-terminal α-Syn peptides using MonoSpin Typsin HP preparation, doubly- and singly-charged α-Syn1-6 and Ac-α-Syn1-6 ions were observed at m/z 386 > 104 and m/z 813 > 72, respectively, which correspond to quantitative profiling with internal standards. In the calibration, the range of 10-1000 nmol/L showed r2 = 0.999 and recovery from 86.0% to 115.0% (RSD < 9.0%). Using this procedure, peripheral α-Syn1-6 from serum samples could not be detected. On the other hand, Ac-α-Syn1-6 levels were measured from 106.9 to 319.8 nmol/L (AD; n = 10) and 147.1-292.0 nmol/L (control; n = 10) with an insignificant difference. From these preliminary results, individual Ac-α-Syn levels in serum were inferred with nonspecific biomarker regarding to AD pathology.

Molecular Structure of Gardenia Blue Pigments by Reaction of Genipin with Benzylamine and Amino Acids.

Genipin was reacted with benzylamine and several amino acids to prepare gardenia blue (GB). The time-course of GB formation with benzylamine was monitored by high-performance liquid chromatography (HPLC), liquid chromatography time-of-flight mass spectrometry (LC-TOFMS), and 1H and 13C NMR measurements. In this experiment, we determined the molecular structures of some intermediates using accurate masses and additional NMR techniques such as heteronuclear multiple bond correlation (HMBC). GBs with amino acids (GB-AAs) were characterized by both liquid and solid-state NMR measurements. Interestingly, many significant peaks appeared in the solid-state NMR spectra, although the 13C NMR spectra from solution samples did not show any distinct peaks. Therefore, we determined that GB-AAs had an alternating copolymer structure composed of methyne and 5H-2-pyrindine, which was substituted by amino acids at N atom and linked with methyne at 5 and 7 positions. To confirm this molecular structure, the pyrolysis gas chromatography-mass spectrometry (GC-MS) measurement of GB-AAs was carried out, and 5H-2-pyrindine and its methyl derivatives were formed as main pyrolysis products from the polymer chains.

Simultaneous Determination of Trigonelline, Caffeine, Chlorogenic Acid and Their Related Compounds in Instant Coffee Samples by HPLC Using an Acidic Mobile Phase Containing Octanesulfonate.

In order to analyze trigonelline, caffeine, chlorogenic acid, and their related compounds simultaneously, an HPLC method using an InertSustain C18 column and a mobile phase containing octanesulfonate as an ion-pairing reagent under an acidic condition was developed. The optimum mobile phase conditions were determined to be 0.1% phosphoric acid, 4 mM octanesulfonate, and 15% methanol at 35°C. Using the proposed method, trigonelline, nicotinic acid, caffeine, theophylline, chlorogenic acid, and caffeic acid in ten instant coffee samples were analyzed. These analytes except for theophylline were detected in all samples. An increase in the caffeine content in instant coffee samples tended to decrease in both trigonelline and chlorogenic acid contents, and the trigonelline content was found to be correlated well with the chlorogenic acid content (R(2) = 0.887).

Simultaneous determination of post-translational racemization and isomerization of N-terminal amyloid-ß in Alzheimer's brain tissues by covalent chiral derivatized ultraperformance liquid chromatography tandem mass spectrometry.

Typical markers of protein aging are spontaneous post-translational modifications such as amino acid racemization (AAR) and amino acid isomerization (AAI) during the degradation of peptides. The post-translational AAR and AAI could significantly induce the density and localization of plaque deposition in brain tissues. Alzheimer's disease (AD) is reliably related to the formation and aggregation of amyloid-ß peptide (Aß) plaques in the human brain. No current analytical methods can simultaneously determine AAR and AAI during the degradation of Aß from AD patients. We now report a covalent chiral derivatized ultraperformance liquid chromatography tandem mass spectrometry (CCD-UPLC-MS/MS) method for the determination of post-translational AAR and AAI of N-terminal Aß (N-Aß1-5) in human brain tissues. When subjected to tryptic N-Aß1-5 from post-translationally modified natural Aß in focal brain tissues by the CCD procedure, it was monitored at m/z 989.6→637.0/678.9 during electrospray collision-induced dissociation. These N-Aß1-5 fragments with l-aspartic acid (l-Asp), d-Asp, l-isoAsp, and d-isoAsp could be separated using the UPLC system with a conventional reversed-phase column and mobile phase. The quantification of these peptides was determined using a stable isotope [(15)N]-labeled Aß1-40 internal standard. The CCD-UPLC-MS/MS assay of potential N-Aß1-5 allowed for the discovery of the present and ratio levels of these N-Aß1-5 sequences with l-Asp, d-Asp, l-isoAsp, and d-isoAsp.

Macrophage migration inhibitory factor and stearoyl-CoA desaturase 1: potential prognostic markers for soft tissue sarcomas based on bioinformatics analyses.

The diagnosis and treatment of soft tissue sarcomas (STSs) has been particularly difficult, because STSs are a group of highly heterogeneous tumors in terms of histopathology, histological grade, and primary site. Recent advances in genome technologies have provided an excellent opportunity to determine the complete biological characteristics of neoplastic tissues, resulting in improved diagnosis, treatment selection, and investigation of therapeutic targets. We had previously developed a novel bioinformatics method for marker gene selection and applied this method to gene expression data from STS patients. This previous analysis revealed that the extracted gene combination of macrophage migration inhibitory factor (MIF) and stearoyl-CoA desaturase 1 (SCD1) is an effective diagnostic marker to discriminate between subtypes of STSs with highly different outcomes. In the present study, we hypothesize that the combination of MIF and SCD1 is also a prognostic marker for the overall outcome of STSs. To prove this hypothesis, we first analyzed microarray data from 88 STS patients and their outcomes. Our results show that the survival rates for MIF- and SCD1-positive groups were lower than those for negative groups, and the p values of the log-rank test are 0.0146 and 0.00606, respectively. In addition, survival rates are more significantly different (p = 0.000116) between groups that are double-positive and double-negative for MIF and SCD1. Furthermore, in vitro cell growth inhibition experiments by MIF and SCD1 inhibitors support the hypothesis. These results suggest that the gene set is useful as a prognostic marker associated with tumor progression.

On-line solid-phase extraction LC-MS/MS for the determination of Ac-SDKP peptide in human plasma from hemodialysis patients.

We developed a high-throughput method based on on-line solid-phase extraction liquid chromatography tandem mass spectrometry (SPE-LC-MS/MS) to determine N-terminal thymosin-ß fragment peptide (N-acetyl-seryl-aspartyl-lysyl-proline, Ac-SDKP) in human plasma samples. Quantification of Ac-SDKP was performed using direct injection for on-line SPE based on C(18), reversed-phase LC separation and stable isotope dilution electrospray ionization-MS/MS in multiple reaction-monitoring (MRM) mode. The Ac-SDKP-(13)C(6), (15)N(2) (m/z 496 → 137) was synthesized for the internal standard. The MRM ion for Ac-SDKP was m/z 488 → 129 (quantitative ion)/226. The limit of detection and lower limit of quantitation were 0.05 and 0.1 ng/mL in standard solution, respectively. Recovery values were 98.3-100.4% with inter-day (relative standard deviation, RSD, 0.4-14.1%) and intra-day (RSD, 0.8-19.7%) assays. This method was applied to the measurement of Ac-SDKP levels in plasma from hemodialyzed subjects. Concentrations were 0.59 ± 0.23 ng/mL (pre-hemodialyzed subjects, n = 9) and 0.44 ± 0.19 ng/mL (post-hemodialyzed subjects, n = 9). All plasma Ac-SDKP levels were decreased by dialysis. Thus, plasma Ac-SDKP was decreased through dialysis in chronic kidney disease. The findings in this study will be useful for the treatment of anemia in chronic kidney disease with dialysis.

Formation of acrylamide from glucans and asparagine.

Model foods consisting of carbohydrates, asparagine (Asn), albumin, and sodium chloride were heated at 180°C for various times, and the levels of acrylamide (AA) in these foods were determined by LC/MS/MS. When glucans such as ß-cyclodextrin (ß-CD), starch and cellulose were used as carbohydrates in the above model, the levels of AA formed were approximately the same as or much higher than those observed in the glucose model. Glucans were heated in the absence of Asn for one hour, and their degradation products were analyzed for sugar components by HPAEC-PAD and for volatile compounds by GC/MS. The amounts of glucose detected in the glucan models, however, were too low to consider that AA was formed from the glucans in these model foods via the intermediate production of glucose. By contrast, several carbonyl compounds such as acetaldehyde and acetone were detected in the glucan degradation products. Furthermore, AA was formed when acetaldehyde and Asn were heated together in sealed vials at 180°C. These results showed that AA was formed from glucans and Asn, not via glucose produced by glucan hydrolysis, but via volatile carbonyl compounds such as acetaldehyde produced by glucan pyrolysis.

Quantification of N-acetyl-seryl-aspartyl-lysyl-proline in hemodialysis patients administered angiotensin-converting enzyme inhibitors by stable isotope dilution liquid chromatography-tandem mass spectrometry.

We developed a sensitive, selective and accurate method based on liquid chromatography with tandem mass spectrometry (LC-MS/MS) to determine N-terminal thymosin-ß peptides of Ac-SDKP and Ac-ADKP in human plasma samples. Quantification of Ac-SDKP and Ac-ADKP was performed using solid phase extraction (SPE) based on C(18), reversed phase LC separation, and stable isotope dilution electrospray ionization-MS/MS in multiple reaction-monitoring (MRM) mode. The Ac-SDKP-(13)C(6), (15)N(2) and Ac-ADKP-d(7) were synthesized for the internal standards. These MRM monitoring ions were m/z 488→129 (quantitative ion)/226 for Ac-SDKP, m/z 496→137 for Ac-SDKP-(13)C(6), (15)N(2), m/z 472→129 (quantitative ion)/226 for Ac-ADKP, and m/z 479→129 for Ac-ADKP-d(7), respectively. Lower limit of quantitation (LLOQ) of Ac-SDKP and Ac-ADKP was 0.1ng/mL in human plasma. Recovery values were ranged from 94.7% to 106.3% for inter- (RSD: 0.6-3.5%) and intra- (RSD: 0.4-4.9%) day assays. Plasma Ac-SDKP levels were significantly higher in hemodialyzed subjects treated with angiotensin-converting enzyme inhibitors of enalapril (27.3±24.6ng/mL, n=10) and trandolapril (12.3±16.9ng/mL, n=18) than healthy (0.4±0.2ng/mL, n=7) and hemodialyzed subjects (0.6±0.2ng/mL, n=34). This analytical method would be useful to measure N-terminal thymosin-ß peptides in human plasma for the clinical study.

Allyl isothiocyanate that induces GST and UGT expression confers oxidative stress resistance on C. elegans, as demonstrated by nematode biosensor.

BACKGROUND: Electrophilic xenobiotics and endogenous products from oxidative stresses induce the glutathione S-transferases (GSTs), which form a large family within the phase II enzymes over both animal and plant kingdoms. The GSTs thus induced in turn detoxify these external as well as internal stresses. Because these stresses are often linked to ageing and damage to health, the induction of phase II enzymes without causing adverse effects would be beneficial in slowing down ageing and keeping healthy conditions. METHODOLOGY/PRINCIPAL FINDINGS: We have tested this hypothesis by choosing allyl isothiocyanate (AITC), a functional ingredient in wasabi, as a candidate food ingredient that induces GSTs without causing adverse effects on animals' lives. To monitor the GST induction, we constructed a gst::gfp fusion gene and used it to transform Caenorhabditis elegans for use as a nematode biosensor. With the nematode biosensor, we found that AITC induced GST expression and conferred tolerance on the nematode against various oxidative stresses. We also present evidence that the transcription factor SKN-1 is involved in regulating the GST expression induced by AITC. CONCLUSIONS/SIGNIFICANCE: We show the applicability of the nematode biosensor for discovering and evaluating functional food substances and chemicals that would provide anti-ageing or healthful benefits.

Determination of avoparcin in animal tissues and milk using LC-ESI-MS/MS and tandem-SPE.

A highly sensitive and selective method using LC-ESI-MS/MS and tandem-SPE was developed to detect trace amounts of avoparcin (AV) antibiotics in animal tissues and milk. Data acquisition using MS/MS was achieved by applying multiple reaction monitoring of the product ions of [M + 3H](3+) and the major product ions of AV-alpha and -beta at m/z 637 --> 86/113/130 and m/z 649 --> 86/113/130 in ESI(+) mode. The calculated instrumental LODs were 3 ng/mL. The sample preparation was described that the extraction using 5% TFA and the tandem-SPE with an ion-exchange (SAX) and InertSep C18-A cartridge clean-up enable us to determine AV in samples. Ion suppression was decreased by concentration rates of each sample solution. These SPE concentration levels could be used to detect quantities of 5 ppb (milk), 10 ppb (beef), and 25 ppb (chicken muscle and liver). The matrix matching calibration graphs obtained for both AV-alpha (r >0.996) and -beta (r >0.998) from animal tissues and milk were linear over the calibration ranges. AV recovery from samples was higher than 73.3% and the RSD was less than 12.0% (n = 5).

Acrylamide-responsive genes in the nematode Caenorhabditis elegans.

As acrylamide is a known neurotoxin for many animals and potential carcinogen for humans, it came as a surprise when the Swedish National Food Agency and Stockholm University reported in 2002 that it is formed during the frying or baking of foods. We report here genomic and proteomic analyses on genes and proteins of Caenorhabditis elegans exposed to 500 mg/l acrylamide. Of the 21,120 genes profiled, 409 genes were more than twofold upregulated and 111 genes were downregulated. Upregulated genes included many that encode detoxification enzymes such as glutathione S-transferases (GSTs), uridine diphosphate-glucuronosyl/glucosyl transferases, and short-chain type dehydrogenases but only one cytochrome P450. Subsequent proteomic analysis confirmed the heavy involvement of GSTs. Because of their high expression levels and central roles in acrylamide metabolism, we analyzed the in vivo expression patterns of eight gst genes. Although all encoded GST and were more than twofold upregulated by acrylamide treatment, their expression patterns were varied, and their regulation involved the transcription factor SKN-1 (a C. elegans homolog of Nuclear factor E2-related factors 1 and 2). We then selected the gst-4::gfp-transformed C. elegans to study the detoxification rate of acrylamide and its metabolite glycidimide in living animals. This animal detects acrylamide as a green fluorescence protein (GFP) expression signal in a dose- and time-dependent manner and may prove to be a useful tool not only for rapidly and inexpensively detecting acrylamide, a harmful substance in food, but also for analyzing mechanisms of GST induction by acrylamide and other inducers like oxidative stresses.

A rapid and inexpensive method to screen for common foods that reduce the action of acrylamide, a harmful substance in food.

By DNA microarray and protein 2-DE screens for Caenorhabditis elegans genes up-regulated by acrylamide, we selected the gst-4 gene and constructed a gst::gfp fusion gene, which was used to transform C. elegans into a biosensor for acrylamide. This biosensor detects acrylamide as a GFP-expression signal in a dose- and time-dependent manner. When the biosensor was exposed to acrylamide together with commercially available powdered green tea, GFP levels decreased to the control level, suggestive of acrylamide detoxification or prevention of GST induction. The present methodology should be applicable for screening of not only harmful substances but also substances that reduce or counteract their harmfulness or action, with appropriately constructed visible biosensors.

Extremely low dose of acrylamide decreases lifespan in Caenorhabditis elegans.

The neurotoxic, genotoxic and carcinogenic effects of industrial exposure to acrylamide have been studied in animals and humans for more than 30 years. A recent search for the cause of high background levels of acrylamide in industrially unexposed people revealed that it is formed during the frying or baking of foods by means of the Maillard reaction. To evaluate the biological consequences of continuous exposure to acrylamide at levels found in common foodstuffs, we studied the effects of acrylamide on the three parameters of (1) growth, (2) fecundity and (3) lifespan in the nematode Caenorhabditis elegans. As for growth and fecundity, no deleterious consequences were observed when the animals were raised in the acrylamide concentrations of 0.5 microg/L to 5mg/L, which are commonly found in daily consumed foodstuffs. Conversely, in 500 mg/L of acrylamide, they showed retarded growth with reduced body and brood sizes. Unlike the first two parameters, however, lifespan decreased significantly even in 0.5 microg/L of acrylamide, the maximum theoretical concentration allowed in drinking water by the WHO and US EPA. Very interestingly, the acrylamide effect on lifespan is biphasic as lifespan increased to near normalcy in 5 mg/L and decreased again in 500 mg/L.

Application of ion-trap LC/MS/MS for determination of acrylamide in processed foods.

Ion-trap LC/MS/MS was evaluated for use in the determination of acrylamide (AA) in processed foods. Multiple reaction monitoring (MRM) analysis of a series of AA standard solutions containing deuterium-labeled acrylamide (AA-d3) as an internal standard was performed. A linear relationship between the concentration of AA and the ratio of peak area (AA/AA-d3) in the extracted ion chromatogram (m/z 55, 58 derived from m/z 72, 75, respectively) was obtained over a wide range of 2-20,000 ng/mL. The quantification limit of AA was 2 ng/mL. In analyses of 37 commercial foods, AA was detected in a potato snack at the maximum value of 3,570 ng/g and found in 23 foods prepared or cooked at high temperature. The samples were analyzed in triplicate and the relative standard deviations (RSD) were less than 15% in many processed foods.