Analysis of Renal Cell Carcinoma as a First Step for Developing Mass Spectrometry-Based Diagnostics

Immediate diagnosis of human specimen is an essential prerequisites in medical routines. This study aimed to establish a novel cancer diagnostics system based on probe electrospray ionization-mass spectrometry (PESI-MS) combined with statistical data processing. PESI-MS uses a very fine acupuncture needle as a probe for sampling as well as for ionization. To demonstrate the applicability of PESI-MS for cancer diagnosis, we analyzed nine cases of clear cell renal cell carcinoma (ccRCC) by PESI-MS and processed the data by principal components analysis (PCA). Our system successfully delineated the differences in lipid composition between non-cancerous and cancerous regions. In this case, triacylglycerol (TAG) was reproducibly detected in the cancerous tissue of nine different individuals, the result being consistent with well-known profiles of ccRCC. Moreover, this system enabled us to detect the boundaries of cancerous regions based on the expression of TAG. These results strongly suggest that PESI-MS will be applicable to cancer diagnosis, especially when the number of data is augmented.     

Direct profiling of phytochemicals in tulip tissues and in vivo monitoring of the change of carbohydrate content in tulip bulbs by probe electrospray ionization mass spectrometry

Probe electrospray ionization (PESI) is a recently developed ESI-based ionization technique which generates electrospray from the tip of a solid needle. In this study, we have applied PESI interfaced with a time of flight mass spectrometer (TOF-MS) for direct profiling of phytochemicals in a section of a tulip bulb in different regions, including basal plate, outer and inner rims of scale, flower bud and foliage leaves. Different parts of tulip petals and leaves have also been investigated. Carbohydrates, amino acids and other phytochemicals were detected. A series of in vivo PESI-MS experiments were carried out on the second outermost scales of four living tulip bulbs to monitoring the change of carbohydrate content during the first week of initial growth. The breakdown of carbohydrates was observed which was in accordance with previous reports achieved by other techniques. This study has indicated that PESI-MS can be used for rapid and direct analysis of phytochemicals in living biological systems with advantages of low sample consumption and little sample preparation. Therefore, PESI-MS can be a new choice for direct analysis/profiling of bioactive compounds or monitoring metabolic changes in living biological systems.     

Metabonomic profiling of renal cell carcinoma: high-resolution proton nuclear magnetic resonance spectroscopy of human serum with multivariate data analysis

Metabonomic profiling using proton nuclear magnetic resonance (¹H NMR) spectroscopy and multivariate data analysis of human serum samples was used to characterize metabolic profiles in renal cell carcinoma (RCC). We found distinct, easily detectable differences between (a) RCC patients and healthy humans, (b) RCC patients with metastases and without metastases, and (c) RCC patients before and after nephrectomy. Compared to healthy human serum, RCC serum had higher levels of lipid (mainly very low-density lipoproteins), isoleucine, leucine, lactate, alanine, N-acetylglycoproteins, pyruvate, glycerol, and unsaturated lipid, together with lower levels of acetoacetate, glutamine, phosphatidylcholine/choline, trimethylamine-N-oxide, and glucose. This pattern was somewhat reversed after nephrectomy. Altered metabolite concentrations are most likely the result of the cells switching to glycolysis to maintain energy homeostasis following the loss of ATP caused by impaired TCA cycle in RCC. Serum NMR spectra combined with principal component analysis techniques offer an efficient, convenient way of depicting tumour biochemistry and stratifying tumours under different pathophysiological conditions. It may be able to assist early diagnosis and postoperative surveillance of human malignant diseases using single blood samples.     

Remobilization of pentavalent antimony and vanadium from a granular iron hydroxide material--a comparative study of different leaching systems

The remobilization of antimony and vanadium from previously loaded commercial granular ferric-hydroxide GEH material (intended for water treatment) was examined by using a sequential extraction procedure and three different leaching systems to evaluate their physicochemical mobility and potential availability under different simulated environmental conditions. A classical batch extraction, an extraction cell (EC) and rotating-coiled columns (RCC) were used as extraction systems.For each system it could be shown that the content of ion-exchangeable antimony and vanadium in previously loaded material is negligible (<1.5%). The oxyanions were sorbed strongly and could be predominantly remobilized through reducing agents, which means through dissolution of the iron (hydr)oxide matrix.The major advantages of dynamic systems in comparison to batchwise fractionation technique are the drastically reduced extraction time and the possibility of generating information to the leaching kinetics. It is shown that the efficiency of the three leaching systems is quite different employing Wenzel's sequential fractionation protocol. Only by working with RCC, the iron (hydr)oxide matrix was completely dissolved within four steps resulting in the total mobilization of antimony and vanadium. EC seems to be less suitable for leaching studies of Sb and V sorbed on iron(hydr)oxide. The remobilizable proportion of the several fractions was lower in comparison to batch and RCC and seems to be a result of an agglomeration of the GEH in the EC device.     

Effect of acid concentration on closed-vessel microwave-assisted digestion of plant materials

The efficiency of microwave-assisted acid digestion of plants using different concentrations of nitric acid (2.0, 3.0, 5.0, 7.0 and 14 mol l⁻¹) with hydrogen peroxide (30% v/v) was evaluated by measuring the residual carbon content (RCC) using inductively coupled plasma optical emission spectrometry (ICP-OES) with axial viewing. Certified reference materials were used for evaluating the accuracy attained when 2 mol l⁻¹ HNO₃ was employed for digestion. Under all experimental conditions RCC values were always lower than 13% w/v, and even the highest concentration did not cause any interference with element recovery. It seems that the high pressure reached for closed-vessel operation improved the oxidative action of nitric acid due to consequent temperature increase, even when this reagent was not used at high concentrations. According to acid–base titration data, residual acid in the digestates varied from 1.2 to 4.0 mol l⁻¹, depending on the acid concentration initially added. It can be concluded that for plant materials, microwave-assisted acid digestion can be carried out under mild conditions, which implies that digestates do not need extensive dilution before introduction by pneumatic nebulization to ICP-OES. An additional advantage is the lower amount of residue generated when working with less concentrated acid solutions.     

Online Electrospray Ionization Mass Spectrometric Monitoring of Protease-Catalyzed Reactions in Real Time

Although there are a lot of well established methods for monitoring enzyme-catalyzed reactions, most of them are based on changes in spectroscopic properties during the conversion of substrates to products. However, reactions without optical changes are common, which are inapplicable to these spectroscopic methods. As an alternative technique for enzymologic research, mass spectrometry (MS) is favored due to its specificity, sensitivity, and the ability to obtain stoichiometric information. In this work, probe electrospray ionization (PESI) source coupled with a time of flight mass spectrometer was employed to monitor some typical protease-catalyzed reactions, including pepsinolysis and trypsinolysis of cytochrome c in real time. Due to the high electrical conductivity of each reaction system, corona discharges are likely to occur, which would decrease intensities of mass spectrometric signals. An ultra-fine sampling probe and an auxiliary vapor spray were adopted to prevent corona discharges. Experimental results from peptic and tryptic digestions of cytochrome c showed different and characteristic catalytic pathways. With the data presented in this study, PESI-MS can be considered as a potential tool for real-time monitoring of enzymatic reactions because of its simplicity in instrumental configuration, wide applicability under harsh conditions, and flexibility in combination with other techniques.     

Dynamic fractionation of trace metals in soil and sediment samples using rotating coiled column extraction and sequential injection microcolumn extraction: A comparative study

Dynamic fractionation has been recognized as an appealing alternative to conventional equilibrium-based sequential extraction procedures (SEPs) for partitioning of trace elements (TE) in environmental solid samples. This paper reports the first attempt for harmonization of flow-through dynamic fractionation using two novel methods, the so-called sequential injection microcolumn (SIMC) extraction and rotating coiled column (RCC) extraction. In SIMC extraction, a column packed with the solid sample is clustered in a sequential injection system, while in RCC, the particulate matter is retained under the action of centrifugal forces. In both methods, the leachants are continuously pumped through the solid substrates by the use of either peristaltic or syringe pumps.A five-step SEP was selected for partitioning of Cu, Pb and Zn in water soluble/exchangeable, acid-soluble, easily reducible, easily oxidizable and moderately reducible fractions from 0.2 to 0.5 g samples at an extractant flow rate of 1.0 mL min⁻¹ prior to leachate analysis by inductively coupled plasma-atomic emission spectrometry.Similarities and discrepancies between both dynamic approaches were ascertained by fractionation of TE in certified reference materials, namely, SRM 2711 Montana Soil and GBW 07311 sediment, and two real soil samples as well. Notwithstanding the different extraction conditions set by both methods, similar trends of metal distribution were in generally found. The most critical parameters for reliable assessment of mobilisable pools of TE in worse-case scenarios are the size-distribution of sample particles, the density of particles, the content of organic matter and the concentration of major elements. For reference materials and a soil rich in organic matter, the extraction in RCC results in slightly higher recoveries of environmentally relevant fractions of TE, whereas SIMC leaching is more effective for calcareous soils.     

Zinc oxide nanoparticles promotes ferroptosis to repress cancer cell survival and inhibits invasion and migration by targeting miR-27a-3p/YAP axis in renal cell carcinoma

BackgroundRenal cell carcinoma (RCC) is a prevalent malignancy with growing mortality and high metastasis. Ferroptosis has been identified as an essential process in cancer development, but the regulatory mechanism underlying the RCC progression remains obscure. The nanomaterial zinc oxide nanoparticles (ZONs) have presented anti-cancer function. Here, we identified the critical role of ZONs in promoting ferroptosis of RCC cells by regulating miR-27a-3p/YAP axis.MethodsThe effect of ZONs on RCC was analyzed by qPCR, Western blot, MTT assays, colony formation assays, Flow cytometry analysis, transwell assays, wound healing assays, iron assays, lipid ROS detection, luciferase reporter gene assays, and tumor xenograft.ResultsThe treatment of ZONs repressed expression of GPX4 and SLC7A11 and enhanced ROS accumulation and iron/Fe²⁺ levels in RCC cells. Ferroptosis activator erastin repressed RCC cell viabilities and ZONs further repressed this effect. ZONs inhibited invasion and migration of RCC cells and treatment of ZONs represses RCC cell survival in vitro. ZONs suppressed RCC cell growth in tumorigenicity mouse model. Mechanically, ZONs down-regulated YAP expression by inducing miR-27a-3p, in which YAP overexpression and miR-27a-3p inhibition reverse ZONs -inhibited RCC cell survival in vitro.DiscussionThus, we concluded that ZONs induced RCC cell ferroptosis to suppress RCC cell survival by targeting miR-27a-3p/YAP axis. The clinical significance of ZONs for the treatment of RCC is required to further study and may benefit the targeted therapy of RCC.     

14-3-3 protein beta/alpha as a urinary biomarker for renal cell carcinoma: proteomic analysis of cyst fluid

Although various samples, including tissue, cells, serum, and urine, from patients with renal cell carcinoma (RCC) have been analyzed, biomarkers with diagnostic value have yet to be identified. We used a proteomics approach to analyze cyst fluid in cases of cyst-associated RCC to identify accessible and abundant proteins that are overexpressed and/or secreted by RCC cells. Proteins in the cyst fluid were separated by reverse-phase high-performance liquid chromatography and agarose two-dimensional gel electrophoresis and were identified by tandem mass spectrometry. We conducted a National Center for Biotechnology Information search and a MEDLINE search to predict the function of these identified proteins and to select a tumor-marker candidate protein. Our search resulted in the identification and selection of the differentially regulated protein known as 14-3-3 protein beta/alpha, which was overexpressed in cyst fluid from cyst-associated RCC but has not been previously associated with RCC. We then measured its incidence through Western blotting of various normal and RCC samples (serum, urine, tissue, and cyst fluid). The expression levels of 14-3-3 protein beta/alpha were higher in urine samples from patients with RCC than in samples from healthy volunteers. Receiver operating characteristic (ROC) curve analyses were performed to assess this potential biomarker; these data (area under the ROC curve value was 0.8813) indicate a high degree of accuracy for this screening method. 14-3-3 Protein beta/alpha may be a diagnostically useful biomarker for early diagnosis of RCC.     

Mapping and expression pattern analysis of key components of the major histocompatibility complex class I antigen processing and presentation pathway in a representative human renal cell carcinoma cell line.

Renal cell carcinoma (RCC) represent approximately 5% of all cancer deaths. At the time of presentation, over 50% of the patients have already developed locally advanced or metastatic disease with five-year survival rates of less than 20%. Although relative resistant to conventional regimens, RCC are partially susceptible to T cell-based immunotherapy. To further develop this treatment modality, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was applied for both the mapping of the key components of the major histocompatibility complex (MHC) class I antigen processing and presentation machinery (APM) and the characterization of the constitutive and cytokine-regulated protein expression profiles in a representative human RCC cell line. The latter aspect is based on the fact, that the expression level of some of the APM components can be altered in response to interferon (IFN)-gamma treatment. Total cell lysates from untreated and IFN-gamma-treated tumor cells were separated on 2-D PAGE gels using broad range immobilized pH gradient (IPG) strips. Serial Western blot analyses using sets of APM-specific antibodies were performed to target the relevant protein spots. Protein verification was mostly accomplished via peptide mass finger-printing using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). To date, the majority of the APM-related components have been identified and mapped. In addition, the different protein expression profiles of untreated and IFN-gamma-treated RCC cells are under investigation.     

Multivariate statistical differentiation of renal cell carcinomas based on lipidomic analysis by ambient ionization imaging mass spectrometry

Desorption electrospray ionization (DESI) mass spectrometry (MS) was used in an imaging mode to interrogate the lipid profiles of thin tissue sections of 11 sample pairs of human papillary renal cell carcinoma (RCC) and adjacent normal tissue and nine sample pairs of clear cell RCC and adjacent normal tissue. DESI-MS images showing the spatial distributions of particular glycerophospholipids (GPs) and free fatty acids in the negative ion mode were compared to serial tissue sections stained with hematoxylin and eosin (H&E). Increased absolute intensities as well as changes in relative abundance were seen for particular compounds in the tumor regions of the samples. Multivariate statistical analysis using orthogonal projection to latent structures treated partial least square discriminate analysis (PLS-DA) was used for visualization and classification of the tissue pairs using the full mass spectra as predictors. PLS-DA successfully distinguished tumor from normal tissue for both papillary and clear cell RCC with misclassification rates obtained from the validation set of 14.3% and 7.8%, respectively. It was also used to distinguish papillary and clear cell RCC from each other and from the combined normal tissues with a reasonable misclassification rate of 23%, as determined from the validation set. Overall DESI-MS imaging combined with multivariate statistical analysis shows promise as a molecular pathology technique for diagnosing cancerous and normal tissue on the basis of GP profiles.     

A hyphenated microLC-Q-TOF-MS platform for exosomal lipidomics investigations: application to RCC urinary exosomes

Urinary exosomes are released from every renal epithelial cell type facing the urinary space and therefore, they may carry molecular markers of renal dysfunction and structural injury. Here, we present a hyphenated microLC-Q-TOF-MS platform for lipidomics studies applied to investigate the urinary exosome lipid repertoire. Lipids were separated by reversed-phase chromatography using a linear gradient of formic acid 0.2% and tetrahydrofuran, in 40 min of analysis. Features (m/z with associated own retention time) were extracted by MarkerLynx(TM) (Waters) and processed, demonstrating good analytical performance in terms of repeatability and mass accuracy of the microLC Q-TOF MS platform. In particular, a stable retention time (RSD less than 4%) and relative intensity (RSD from 2.9% to 11%) were observed. Moreover, the method takes advantages by the use of a lock spray interface (Waters) that allows readjusting the m/z data after acquisition, obtaining inaccuracy below 6 ppm in measuring the m/z value of the reference compound during chromatographic run. The method was employed in a preliminary application to perform comparative analysis from healthy control subjects and renal cell carcinoma (RCC) patients, in order to possibly highlight differences in lipid composition to be exploited as potential tumor biomarker. Differential lipid composition in RCC urinary exosomes was achieved and tentatively identified by accurate mass, providing a preliminary indication of a relationship between lipid composition of urinary exosomes and RCC disease. Among the total features significantly different in RCC exosomes, the ion at m/z 502.3 was taken as an example for molecular confirmation by MS/MS fragmentation analysis.     

Use of rotating-coiled columns for sample preparation in the analysis of petropolluted soils

It is shown for the first time that rotating-coiled columns (RCC) can be used as sample preparation tools in the analysis of petropolluted soils. The use of RCC allows for the extraction of petroleum products from soils in the dynamic mode without using additional means of sample preparation. A test sample of soil is retained in an RCC as a stationary phase. Petroleum products are extracted from soil in 15–25 mL of an organic solvent (n-hexane, acetonitrile). Sample preparation lasts no more than 60 min. Examples are presented for the extraction of hydrocarbons, constituents of diesel fuel, and phthalates in the analysis of petropolluted soil samples taken from the territory of oil-extracting and oil-refining enterprises of the Krasnoyarsk region and the Khanty-Mansiisk autonomous area.     

Fractionation of Sb and As in soil and sludge samples using different continuous-flow extraction techniques

The fractionation of Sb and As in soil and sludge samples had been comparably studied using two continuous-flow systems: a microcolumn (MC) and a rotating coiled column (RCC). The leachants were applied in correspondence with a five-step sequential extraction scheme addressing water-soluble, non-specifically sorbed, specifically sorbed, and bound to amorphous and crystalline Fe/Al oxide fractions of Sb and As. Inductively coupled plasma atomic emission spectroscopy was applied to determine antimony, arsenic, and major elements in the effluent and in the residual fractions after their digestion. Resemblances and discrepancies of the two methods were evaluated by the fractionation of Sb and As in forest soil, river sludge, and dumped waste (soil) samples. For the forest soil sample, which is very poor in organic matter, RCC and MC extractions yielded similar quantitative values of As and Sb contents in individual leachable fractions. However, for the river sludge sample with a moderate concentration of C _org (3.3 %), the results obtained by both continuous-flow methods are in satisfactory agreement. RCC extraction enabled water-soluble and non-specifically sorbed As fractions to be recovered, whereas after MC leaching, these environmentally relevant forms of As were not detected. For the soil rich in organic matter (C _org = 11.5 %), the discrepancy between the data of RCC and MC fractionations is significant. RCC extraction provides about six times higher recoveries of As and Sb bound to amorphous Fe/Al oxides. More efficient leaching of As and Sb in RCC may be attributed to the migration of organic-rich particles with low density inside the column that might enhance the mixing of the solid and liquid phases.     

Fractionation of nano- and microparticles in a rotating conoidal coiled column

A method for the continuous-flow fractionation of particles in a transverse centrifugal field in a rotating conoidal coiled (RCC) column has been developed. A model of a planetary centrifuge with a conoidal drum of a special construction has been tested. The effects of the rotation and revolution speed of the conoidal RCC, as well as the direction and pumping rate of the mobile phase on the behavior of particles smaller than 1 μm have been studied. The conditions have been selected and optimized for the retention and elution of spherical particles of the ‘150 nm’, ‘400 nm’, and ‘900 nm’ standard samples of silica gel (Polyscience Inc.). The possibility of using RCC for the analysis and production of monodisperse standard particle samples has been demonstrated. In particular, the ‘900 nm’ particles have been separated from admixtures of small (100–200 nm) particles and nonspherical 1–2 μm particles present in the sample. The separated fractions have been characterized by electron microscopy.     

Low C24‐OH and C22‐OH sulfatides in human renal cell carcinoma

Histopathologic diagnosis of renal cell carcinoma (RCC) may sometimes be difficult with small biopsy samples. We applied histology‐directed matrix‐assisted laser desorption/ionization mass spectrometry to RCC samples to evaluate whether and how lipid profiles are different between RCC and normal tissue. We evaluated 59 RCC samples and 24 adjacent normal tissue samples collected from patients who underwent surgery. Five peaks were significantly differently expressed (p < 10^?7) between RCCs and adjacent normal tissue samples. C24‐OH sulfatide (ST‐OH {18:1/24:0}[M‐H]^?; m/z 906.7 in the negative ion mode) and C22‐OH sulfatide (ST‐OH {18:1/22:0}[M‐H]^?; m/z 878.6 in the negative ion mode) were most significantly underexpressed in RCC samples, compared with adjacent normal tissue samples. With 100 random training‐to‐test partitions within these samples, the median prediction accuracy (RCC vs. normal) ranged from 96.3% to 100% at p cutoff values for feature selection ranging from 0.001 to 10^?7. Two oncocytoma samples were predicted as normal tissue by five lipids that were differentially expressed between RCC and normal tissue at p < 10^?7. Clear‐cell, papillary, and chromophobe RCCs were different in lipid profiles. Permutation p‐ values for 0.632+ bootstrap cross‐validated misclassification rates were less than 0.05 for all the classifiers. Thus, lipid profiles differentiate RCC from normal tissue and may possibly classify the histology of RCC. © 2014 The Authors. Journal of Mass Spectrometry published by John Wiley & Sons, Ltd.     

Alkynylxenon(II) fluorides

Alkynylxenon(II) fluorides, RCC(triple bond)XeF, have been prepared from the reactions of the corresponding trimethyl(alkynyl)silanes, Me3(-)SiC(triple bond)CR, and XeF2 in the presence of [NMe4F in common organic solvents at low temperature. The existence of the linear unit C(triple bond)C-Xe-F was proved for PhC(triple bond)CXeF by the 19F-13C NMR correlation method using the HMBC pulse sequence.     

Photosensitizing and radiosensitizing effects of hypericin on human renal carcinoma cells in vitro

The renal cell carcinoma (RCC) is extremely resistant to chemotherapy and radiotherapy. The prognosis of patients with metastatic RCC still remains poor, the median survival is less than 12 months. Therefore, new therapeutic options are desirable. The aim of this study was to investigate the photosensitizing and radiosensitizing effects of hypericin on human RCC cells in vitro. First the RCC-derived cell lines A498 and ACHN were incubated with different concentrations of hypericin. In vitro uptake and intracellular distribution of hypericin were confirmed by fluorescence microscopy. Subsequently cells were illuminated and irradiated with a dose of 2-8 Gy, respectively. Finally, metabolic activity, apoptosis and clonogenic survival were investigated. Uptake of hypericin was observed for almost all cells. Hypericin treatment combined with illumination led to a 94-97% decrease in metabolic activity and caused apoptosis in nearly 100% of RCC cells. Hypericin enhanced the radiosensitivity of A498 cells in vitro. The clonogenic survival after irradiation was significantly reduced by hypericin treatment. Taken together, the photosensitizing and radiosensitizing effects of hypericin on human RCC cells we found in this investigation could be of clinical relevance, e.g. for radiotherapy and intraoperative photodynamic therapy, respectively.     

Possibilities for the harmonization of methods of the dynamic fractionation of elements in soils and bottom sediments

The paper presents the first comparative study of the fractionation of heavy metals, arsenic, antimony, and selenium in soils, sludge and bottom sediments using two systems for dynamic extraction, rotating coiled columns (RCC) and cylindrical microcolumns (MC). It has been revealed that the results of the fractionation of the forms of elements are related with the physicochemical properties of soils, sludge, and bottom sediments and the special features of mixing the sample and eluent in RCC and MC. In particular, it has been demonstrated that, as compared to MC, RCC provides more efficient recovery of the most mobile (ecologically relevant) forms of arsenic, antimony, and selenium from samples rich with organic matter. However, in the majority of cases, despite the different efficiency of the recovery of individual fractions, RCC and MC provide comparable diagrams of the distribution of the extracted forms of elements. The influence of the rate of the eluent flow on the processes of the dynamic extraction of heavy metals, arsenic and antimony in RCC and MC has been estimated. The directions of research for the further harmonization of the methods of dynamic fractionation of forms of elements in soils, sludge, and bottom sediments with different physicochemical mobilities and potential biological availabilities are outlined.