Combined Secondary Ion Mass Spectrometry Depth Profiling and Focused Ion Beam Analysis of Cu Films Electrodeposited under Oscillatory Conditions

The recrystallization behavior of Cu films electrodeposited under oscillatory conditions in the presence of plating additives was studied by means of secondary ion mass spectrometry (SIMS) and focused ion beam analysis. When combined with bis-(sodium-sulfopropyl)-disulfide (SPS), Imep levelers (polymerizates of imidazole and epichlorohydrin) show characteristic oscillations in the galvanostatic potential/time transient measurements. These are related to the periodic degradation and restoration of the active leveler ensemble at the interface. The leveler action relies on adduct formation between the Imep and MPS (mercaptopropane sulfonic acid)-stabilized CuI complexes that appear as intermediates of the copper deposition when SPS is present in the electrolyte. SIMS depth profiling proves that additives are incorporated into the growing film preferentially under transient conditions during the structural breakdown of the leveler ensemble and its subsequent restoration. In contrast, Cu films electrodeposited in the presence of a structurally intact Imep–CuI–MPS ensemble remain largely contamination free.

Quantitative depth profiling of Ce 3+ in Pt/CeO 2 by in situ high-energy XPS in a hydrogen atmosphere

The redox property of ceria is a key factor in the catalytic activity of ceria-based catalysts. The oxidation state of well-defined ceria nanocubes in gas environments was analysed in situ by a novel combination of near-ambient pressure X-ray Photoelectron Spectroscopy (XPS) and high-energy XPS at a synchrotron X-ray source. In situ high-energy XPS is a promising new tool to determine the electronic structure of matter under defined conditions. The aim was to quantitatively determine the degree of cerium reduction in a nano-structured ceria-supported platinum catalyst as a function of the gas environment. To obtain a non-destructive depth profile at near-ambient pressure, in situ high-energy XPS analysis was performed by varying the kinetic energy of photoelectrons from 1 to 5 keV, and, thus, the probing depth. In ceria nanocubes doped with platinum, oxygen vacancies formed only in the uppermost layers of ceria in an atmosphere of 1 mbar hydrogen and 403 K. For pristine ceria nanocubes, no change in the cerium oxidation state in various hydrogen or oxygen atmospheres was observed as a function of probing depth. In the absence of platinum, hydrogen does not dissociate and, thus, does not lead to reduction of ceria.

High-Resolution Chemical Depth Profiling of Solid Material Using a Miniature Laser Ablation/Ionization Mass Spectrometer

High-resolution chemical depth profiling measurements of copper films are presented. The 10 μm thick copper test samples were electrodeposited on a Si-supported Cu seed under galvanostatic conditions in the presence of particular plating additives (SPS, Imep, PEI, and PAG) used in the semiconductor industry for the on-chip metallization of interconnects. To probe the trend of these plating additives toward inclusion into the deposit upon growth, quantitative elemental mass spectrometric measurements at trace level concentration were conducted by using a sensitive miniature laser ablation ionization mass spectrometer (LIMS), originally designed and developed for in situ space exploration. An ultrashort pulsed laser system (τ ∼ 190 fs, λ = 775 nm) was used for ablation and ionization of sample material. We show that with our LIMS system, quantitative chemical mass spectrometric analysis with an ablation rate at the subnanometer level per single laser shot can be conducted. The measurement capabilities of our instrument, including the high vertical depth resolution coupled with high detection sensitivity of ∼10 ppb, high dynamic range ≥10(8), measurement accuracy and precision, is of considerable interest in various fields of application, where investigations with high lateral and vertical resolution of the chemical composition of solid materials are required, these include, e.g., wafers from semiconductor industry or studies on space weathered samples in space research.

Metabolic profiling of praziquantel enantiomers

Praziquantel (PZQ), prescribed as a racemic mixture, is the most readily available drug to treat schistosomiasis. In the present study, ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS) based metabolomics was employed to decipher the metabolic pathways and enantioselective metabolic differences of PZQ. Many phase I and four new phase II metabolites were found in urine and feces samples of mice 24h after dosing, indicating that the major metabolic reactions encompassed oxidation, dehydrogenation, and glucuronidation. Differences in the formation of all these metabolites were observed between (R)-PZQ and (S)-PZQ. In an in vitro phase I incubation system, the major involvement of CYP3A, CYP2C9, and CYP2C19 in the metabolism of PZQ, and CYP3A, CYP2C9, and CYP2C19 exhibited different catalytic activity toward the PZQ enantiomers. Apparent Km and Vmax differences were observed in the catalytic formation of three mono-oxidized metabolites by CYP2C9 and CYP3A4 further supporting the metabolic differences for PZQ enantiomers. Molecular docking showed that chirality resulted in differences in substrate location and conformation, which likely accounts for the metabolic differences. In conclusion, in silico, in vitro, and in vivo methods revealed the enantioselective metabolic profile of praziquantel.

Noninvasive Urinary Metabolomic Profiling Identifies Diagnostic and Prognostic Markers in Lung Cancer

Lung cancer remains the most common cause of cancer deaths worldwide, yet there is currently a lack of diagnostic noninvasive biomarkers that could guide treatment decisions. Small molecules (<1,500 Da) were measured in urine collected from 469 patients with lung cancer and 536 population controls using unbiased liquid chromatography/mass spectrometry. Clinical putative diagnostic and prognostic biomarkers were validated by quantitation and normalized to creatinine levels at two different time points and further confirmed in an independent sample set, which comprises 80 cases and 78 population controls, with similar demographic and clinical characteristics when compared with the training set. Creatine riboside (IUPAC name: 2-{2-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)-oxolan-2-yl]-1-methylcarbamimidamido}acetic acid), a novel molecule identified in this study, and N-acetylneuraminic acid (NANA) were each significantly (P < 0.00001) elevated in non-small cell lung cancer and associated with worse prognosis [HR = 1.81 (P = 0.0002), and 1.54 (P = 0.025), respectively]. Creatine riboside was the strongest classifier of lung cancer status in all and stage I-II cases, important for early detection, and also associated with worse prognosis in stage I-II lung cancer (HR = 1.71, P = 0.048). All measurements were highly reproducible with intraclass correlation coefficients ranging from 0.82 to 0.99. Both metabolites were significantly (P < 0.03) enriched in tumor tissue compared with adjacent nontumor tissue (N = 48), thus revealing their direct association with tumor metabolism. Creatine riboside and NANA may be robust urinary clinical metabolomic markers that are elevated in tumor tissue and associated with early lung cancer diagnosis and worse prognosis.

Mutations upstream of fabI in triclosan resistant Staphylococcus aureus strains are associated with elevated fabI gene expression

Background The enoyl-acyl carrier protein (ACP) reductase enzyme (FabI) is the target for a series of antimicrobial agents including novel compounds in clinical trial and the biocide triclosan. Mutations in fabI and heterodiploidy for fabI have been shown to confer resistance in S. aureus strains in a previous study. Here we further determined the fabI upstream sequence of a selection of these strains and the gene expression levels in strains with promoter region mutations. Results Mutations in the fabI promoter were found in 18% of triclosan resistant clinical isolates, regardless the previously identified molecular mechanism conferring resistance. Although not significant, a higher rate of promoter mutations were found in strains without previously described mechanisms of resistance. Some of the mutations identified in the clinical isolates were also detected in a series of laboratory mutants. Microarray analysis of selected laboratory mutants with fabI promoter region mutations, grown in the absence of triclosan, revealed increased fabI expression in three out of four tested strains. In two of these strains, only few genes other than fabI were upregulated. Consistently with these data, whole genome sequencing of in vitro selected mutants identified only few mutations except the upstream and coding regions of fabI, with the promoter mutation as the most probable cause of fabI overexpression. Importantly the gene expression profiling of clinical isolates containing similar mutations in the fabI promoter also showed, when compared to unrelated non-mutated isolates, a significant up-regulation of fabI. Conclusions In conclusion, we have demonstrated the presence of C34T, T109G, and A101C mutations in the fabI promoter region of strains with fabI up-regulation, both in clinical isolates and/or laboratory mutants. These data provide further observations linking mutations upstream fabI with up-regulated expression of the fabI gene.

1H HR-MAS NMR Based Metabolic Profiling of Cells in Response to Treatment with a Hexacationic Ruthenium Metallaprism as Potential Anticancer Drug.

1H high resolution magic angle spinning (HR-MAS) NMR spectroscopy was applied in combination with multivariate statistical analyses to study the metabolic response of whole cells to the treatment with a hexacationic ruthenium metallaprism [1]6+ as potential anticancer drug. Human ovarian cancer cells (A2780), the corresponding cisplatin resistant cells (A2780cisR), and human embryonic kidney cells (HEK-293) were each incubated for 24 h and 72 h with [1]6+ and compared to untreated cells. Different responses were obtained depending on the cell type and incubation time. Most pronounced changes were found for lipids, choline containing compounds, glutamate and glutathione, nucleotide sugars, lactate, and some amino acids. Possible contributions of these metabolites to physiologic processes are discussed. The time-dependent metabolic response patterns suggest that A2780 cells on one hand and HEK-293 cells and A2780cisR cells on the other hand may follow different cell death pathways and exist in different temporal stages thereof.

CD47 protein expression in acute myeloid leukemia: A tissue microarray-based analysis

Binding of CD47 to signal regulatory protein alpha (SIRPα), an inhibitory receptor, negatively regulates phagocytosis. In acute myeloid leukemia (AML), CD47 is overexpressed on peripheral blasts and leukemia stem cells and inversely correlates with survival. Aim of the study was to investigate the correlation between CD47 protein expression by immunohistochemistry (IHC) in a bone marrow (BM) tissue microarray (TMA) and clinical outcome in AML patients. CD47 staining on BM leukemia blasts was scored semi-quantitatively and correlated with clinical parameters and known prognostic factors in AML. Low (scores 0-2) and high (score 3) CD47 protein expression were observed in 75% and 25% of AML patients. CD47 expression significantly correlated with percentage BM blast infiltration and peripheral blood blasts. Moreover, high CD47 expression was associated with nucleophosmin (NPM1) gene mutations. In contrast, CD47 expression did not significantly correlate with overall or progression free survival or response to therapy. In summary, a BM TMA permits rapid and reproducible semi-quantitative analysis of CD47 protein expression by IHC. While CD47 expression on circulating AML blasts has been shown to be a negative prognostic marker for a very defined population of AML patients with NK AML, CD47 expression on AML BM blasts is not.

Genome profiling of sterol synthesis shows convergent evolution in parasites and guides chemotherapeutic attack

Sterols are an essential class of lipids in eukaryotes, where they serve as structural components of membranes and play important roles as signaling molecules. Sterols are also of high pharmacological significance: cholesterol-lowering drugs are blockbusters in human health, and inhibitors of ergosterol biosynthesis are widely used as antifungals. Inhibitors of ergosterol synthesis are also being developed for Chagas's disease, caused by Trypanosoma cruzi. Here we develop an in silico pipeline to globally evaluate sterol metabolism and perform comparative genomics. We generate a library of hidden Markov model-based profiles for 42 sterol biosynthetic enzymes, which allows expressing the genomic makeup of a given species as a numerical vector. Hierarchical clustering of these vectors functionally groups eukaryote proteomes and reveals convergent evolution, in particular metabolic reduction in obligate endoparasites. We experimentally explore sterol metabolism by testing a set of sterol biosynthesis inhibitors against trypanosomatids, Plasmodium falciparum, Giardia, and mammalian cells, and by quantifying the expression levels of sterol biosynthetic genes during the different life stages of T. cruzi and Trypanosoma brucei. The phenotypic data correlate with genomic makeup for simvastatin, which showed activity against trypanosomatids. Other findings, such as the activity of terbinafine against Giardia, are not in agreement with the genotypic profile.

Common target genes of palatal and gingival fibroblasts for EMD: the microarray approach.

BACKGROUND AND OBJECTIVE Connective tissue grafts are frequently applied, together with Emdogain(®) , for root coverage. However, it is unknown whether fibroblasts from the gingiva and from the palate respond similarly to Emdogain. The aim of this study was therefore to evaluate the effect of Emdogain(®) on fibroblasts from palatal and gingival connective tissue using a genome-wide microarray approach. MATERIAL AND METHODS Human palatal and gingival fibroblasts were exposed to Emdogain(®) and RNA was subjected to microarray analysis followed by gene ontology screening with Database for Annotation, Visualization and Integrated Discovery functional annotation clustering, Kyoto Encyclopedia of Genes and Genomes pathway analysis and the Search Tool for the Retrieval of Interacting Genes/Proteins functional protein association network. Microarray results were confirmed by quantitative RT-PCR analysis. RESULTS The transcription levels of 106 genes were up-/down-regulated by at least five-fold in both gingival and palatal fibroblasts upon exposure to Emdogain(®) . Gene ontology screening assigned the respective genes into 118 biological processes, six cellular components, eight molecular functions and five pathways. Among the striking patterns observed were the changing expression of ligands targeting the transforming growth factor-beta and gp130 receptor family as well as the transition of mesenchymal epithelial cells. Moreover, Emdogain(®) caused changes in expression of receptors for chemokines, lipids and hormones, and for transcription factors such as SMAD3, peroxisome proliferator-activated receptor gamma and those of the ETS family. CONCLUSION The present data suggest that Emdogain(®) causes substantial alterations in gene expression, with similar patterns observed in palatal and gingival fibroblasts.

Salivary Pellicle vs Whole Saliva: The Response of Oral Fibroblasts Based on a Genome-Wide Microarray.

PURPOSE Whole saliva comprises components of the salivary pellicle that spontaneously forms on surfaces of implants and teeth. However, there are no studies that functionally link the salivary pellicle with a possible change in gene expression. MATERIALS AND METHODS This study examined the genetic response of oral fibroblasts exposed to the salivary pellicle and whole saliva. Oral fibroblasts were seeded onto a salivary pellicle and the respective untreated surface. Oral fibroblasts were also exposed to freshly harvested sterile-filtered whole saliva. A genome-wide microarray of oral fibroblasts was performed, followed by gene ontology screening with DAVID functional annotation clustering, KEGG pathway analysis, and the STRING functional protein association network. RESULTS Exposure of oral fibroblasts to saliva caused 61 genes to be differentially expressed (P < .05). Gene ontology screening assigned the respective genes into 262 biologic processes, 3 cellular components, 13 molecular functions, and 7 pathways. Most remarkable was the enrichment in the inflammatory response. None of the genes regulated by whole saliva was significantly changed when cells were placed onto a salivary pellicle. CONCLUSION The salivary pellicle per se does not provoke a significant inflammatory response of oral fibroblasts in vitro, whereas sterile-filtered whole saliva does produce a strong inflammatory response.

Possible involvement of plant ABC transporters in cadmium detoxification: a cDNA sub-microarray approach

As a nontolerant plant to a large number of toxic compounds, Arabidopsis thaliana is a suitable model to study regulation of genes involved in response to heavy metals. Using a cDNA-microarray approach, we identified some ABC transporters that are differentially regulated after cadmium treatments, making them putative candidates for being involved in Cd sequestration and redistribution in plants. Regarding yeast and fission yeast, in which Cd is able to form complexes either with glutathione (GSH) or phytochelatins (PC) subsequently transported into vacuoles via ABC transporters, it is also very likely that some plant ABC transporters are able to transport GS2–Cd or PC–Cd complexes into subcellular compartments or outside of the cell. The characterization of such transporters is of great interest for developing molecular biology approaches in phytoremediation.

Molecular profiling of lung adenosquamous carcinoma: hybrid or genuine type?

Lung adenosquamous carcinoma is a particular subtype of non-small cell lung carcinoma that is defined by the coexistence of adenocarcinoma and squamous cell carcinoma components. The aim of this study was to assess the mutational profile in each component of 16 adenosquamous carcinoma samples from a Caucasian population by a combination of next generation sequencing using the cancer hotspot panel as well as the colon and lung cancer panel and FISH. Identified mutations were confirmed by Sanger sequencing of DNA from cancer cells of each component collected by Laser Capture microdissection. Mutations typical for adenocarcinoma as well as squamous cell carcinoma were identified. Driver mutations were predominantly in the trunk suggesting a monoclonal origin of adenosquamous carcinoma. Most remarkably, EGFR mutations and mutations in the PI3K signaling pathway, which accounted for 30% and 25% of tumors respectively, were more prevalent while KRAS mutations were less prevalent than expected for a Caucasian population. Surprisingly, expression of classifier miR-205 was intermediate between that of classical adenocarcinoma and squamous cell carcinoma suggesting that adenosquamous carcinoma is a transitional stage between these tumor types. The high prevalence of therapy-relevant targets opens new options of therapeutic intervention for adenosquamous carcinoma patients.