Nanosilver inhibits the progression of pancreatic cancer by inducing a paraptosis-like mixed type of cell death.

Silver has been in clinical use since ancient times and silver nanoparticles (AgNPs) have attracted attention in cancer therapy. We investigated the mechanisms by which AgNPs inhibit pancreatic ductal adenocarcinoma (PDAC). AgNPs were synthesized and 3 human PDAC and 2 nonmalignant primary cell lines were treated with AgNPs. MTT, MAPK, colony, spheroid and scratch assays, Western blotting, TEM, annexin V, 7-AAD, and H2DCFDA staining, FACS analysis, mRNA array and bioinformatics analyses, tumor xenograft transplantation, and immunohistochemistry of the treated cells were performed. We found that minimal AgNPs amounts selectively eradicated PDAC cells within a few hours. AgNPs inhibited cell migration and spheroid and colony formation, damaged mitochondria, and induced paraptosis-like cell death with the presence of cytoplasmic vacuoles, dilation of the ER and mitochondria, ROS formation, MAPK activity, and p62 and LC3b expression, whereas effects on the nucleus, DNA fragmentation, or caspases were not detectable. AgNPs strongly decreased tumor xenograft growth without side effects and reduced the expression of markers for proliferation and DNA repair, but upregulated paraptosis markers. The results highlight nanosilver as complementary agent to improve the therapeutic efficacy in pancreatic cancer.

A complementary study approach unravels novel players in the pathoetiology of Hirschsprung disease.

Hirschsprung disease (HSCR, OMIM 142623) involves congenital intestinal obstruction caused by dysfunction of neural crest cells and their progeny during enteric nervous system (ENS) development. HSCR is a multifactorial disorder; pathogenetic variants accounting for disease phenotype are identified only in a minority of cases, and the identification of novel disease-relevant genes remains challenging. In order to identify and to validate a potential disease-causing relevance of novel HSCR candidate genes, we established a complementary study approach, combining whole exome sequencing (WES) with transcriptome analysis of murine embryonic ENS-related tissues, literature and database searches, in silico network analyses, and functional readouts using candidate gene-specific genome-edited cell clones. WES datasets of two patients with HSCR and their non-affected parents were analysed, and four novel HSCR candidate genes could be identified: ATP7A, SREBF1, ABCD1 and PIAS2. Further rare variants in these genes were identified in additional HSCR patients, suggesting disease relevance. Transcriptomics revealed that these genes are expressed in embryonic and fetal gastrointestinal tissues. Knockout of these genes in neuronal cells demonstrated impaired cell differentiation, proliferation and/or survival. Our approach identified and validated candidate HSCR genes and provided further insight into the underlying pathomechanisms of HSCR.

Alterations of the erythrocyte membrane proteome and cytoskeleton network during storage--a possible tool to identify autologous blood transfusion.

Mature red blood cells (RBCs) are the end-stage of a development process that starts in the bone marrow and continues to differentiate, through reticulocyte stage, entering into the circulation with a four-month lifespan. While stored, RBCs undergo different changes. The aim of this study was to evaluate changes occurring in RBC membranes during storage that could be used as possible markers to detect the misuse of blood transfusion in sports. Whole blood was collected from two volunteers in blood bags and stored for 42 days at 4°C. At different times (1, 7, 21, and 42 days of storage) whole blood was extracted under sterile conditions and submitted to RBC membrane ghost preparation and further analysis. Proteomic methods were applied using two strategies: protein oriented using 2-DE gels and peptide oriented using isobaric tags for relative and absolute quantitation (iTRAQ). In both approaches, the goal was to compare detectable changes in RBC membrane proteome before and after standard storage at different times. Some of the changes were confirmed with both methodologies employed, while with others only with one of them. Complementarities of the methods in this case showed to be an advantage. Changes were observed in two different protein complexes. In one of them, changes consisted of proteins decreasing, while increasing in the other during storage of RBCs. They are mostly located in cytoskeleton--spectrin ß, band 4.2, ankyrin-1, tropomodulin-1, ß adducin, band 4.9 (dematin), tropomyosin, while some changes were also observed in transmembrane proteins (glycophorin C, aquaporin-1, band 3).

Determination of several psychiatric drugs in whole blood using capillary gas-liquid chromatography with nitrogen phosphorus detection: comparison of two solid phase extraction procedures.

A simple and reliable gas chromatographic method with nitrogen-phosphorus detection without derivatization was developed for the detection of several psychiatric drugs in whole blood as part of systematic toxicological analyses (STA). Drugs included mirtazapine, chlorpromazine, methotrimeprazine (levomepromazine), clothiapine, olanzapine, clozapine, haloperidol, and thioridazine. All drugs were studied at concentrations of 100-2,000 microg/L, except haloperidol that was studied at concentrations of 400-8,000 microg/L. In order to select the best blood purification procedure and therefore increase the signal to noise ratio we have compared two solid-phase extraction (SPE) columns, Chem Elut and Bond Elut Certify, for their recovery, precision, sensitivity and matrix purification efficiency. Recoveries for these drugs using Chem Elut columns at 500 and 2,000 microg/L (2,000 and 8,000 microg/L for haloperidol) were in the range 21-65%, with intra-assay and inter-assay precisions of less than 17% and 19%, respectively. Limits of detection (LODs) and limits of quantitation (LOQs) for mirtazapine, chlorpromazine, methotrimeprazine, clothiapine, olanzapine, clozapine, and thioridazine ranged from 62 to 161 microg/L and from 205 to 531 microg/L, respectively. LOD and LOQ for haloperidol were 442 and 1,458 microg/L, respectively. Recoveries of these compounds using Bond Elut Certify columns at 500 and 2,000 microg/L (2,000 and 8,000 microg/L for haloperidol) were in the range 44-97%, with intra-assay and inter-assay precisions of less than 7% and 14%, respectively. LODs and LOQs for mirtazapine, chlorpromazine, methotrimeprazine, clothiapine, olanzapine, clozapine, and thioridazine ranged from 37 to 66 microg/L and from 122 to 218 microg/L, respectively. LOD and LOQ for haloperidol were 156 and 515 microg/L, respectively. Linearity was observed in the studied range for all compounds with r(2) values of >0.999. The use of the mixed-mode bonded-silica Bond Elut Certify columns showed advantages comparing with Chem Elut columns for the screening of these psychotropic agents such as higher recoveries, cleaner extracts, better sensitivity, better precision and less solvent consumption and subsequent disposal.

A comparative solid-phase extraction study for the simultaneous determination of fluvoxamine, mianserin, doxepin, citalopram, paroxetine, and etoperidone in whole blood by capillary gas-liquid chromatography with nitrogen-phosphorus detection.

This paper reports a simple and reliable gas chromatographic method with nitrogen-phosphorus detection without derivatization for the simultaneous detection of fluvoxamine, mianserin, doxepin, citalopram, paroxetine, and etoperidone in whole blood as part of a systematic toxicological analysis (STA). All drugs were studied at concentration levels of 100-2000 ng/mL, except paroxetine for which it was necessary to study at concentration levels of 400-8000 ng/mL. A comparative and validation study using two solid-phase extraction (SPE) columns, Chem Elut and Bond Elut Certify, was developed regarding their recovery, precision, sensitivity, and matrix purification efficiency. The Chem Elut columns, diatomaceous earth, are closely related to conventional liquid-liquid extraction. The Bond Elut Certify columns, more recently developed in the market, are mixed SPE (reversed-phase and cation exchange sorbent). Recoveries for the antidepressants using Chem Elut columns at 500 ng/mL (2000 ng/mL for paroxetine) were in the range 43-72% with intra- and interassay precisions of less than 10% and 16%, respectively. Limits of detection (LODs) and quantitation (LOQs) for fluvoxamine, mianserin, doxepin, citalopram, and etoperidone ranged from 18 to 236 ng/mL and 60 to 786 ng/mL, respectively. LOD and LOQ for paroxetine were 303 and 1009 ng/mL, respectively. Recoveries of these compounds using Bond Elut Certify columns at 500 ng/mL (2000 ng/mL for paroxetine) were in the range 52-83% with intra- and interassay precisions of less than 6% and 8%, respectively. LODs and LOQs for fluvoxamine, mianserin, doxepin, citalopram, and etoperidone ranged from 7 to 28 ng/mL and 23 to 93 ng/mL, respectively. LOD and LOQ for paroxetine were 113 and 376 ng/mL, respectively. An excellent linearity was observed with both procedures from the LOQs up to the upper studied concentration level. In general, higher recoveries, cleaner extracts, better sensitivity, better precision, and reduced solvent consumption and disposal were achieved for the screening of these antidepressants with the use of the mixed SPE Bond Elut Certify compared with Chem Elut columns. The application of these methods on a forensic case study is also presented.