Quantification and cytotoxicity of degradation products (chloropropanols) in sucralose containing e-liquids with propylene glycol and glycerol as base.

Electronic cigarettes (e-cigarettes) have gained increasing popularity in recent years, mostly because they are supposed to be less harmful than regular cigarettes. Therefore, it is highly imperative to investigate possible noxious effects to protect the consumers. E-liquids consist of propylene glycol, glycerol, aroma compounds and sweeteners. One of these sweeteners is a chlorinated version of sucrose, namely sucralose. The aim of this work was to investigate degradation products of sucralose in the presence of propylene glycol and glycerol at different temperatures of commercially available e-cigarettes. Chemical analysis and biological tests were simultaneously performed on e-liquid aerosol condensates. The results of the chemical analysis, which was executed by employing GC-MS/GC-FID, demonstrated high amounts of various chloropropanols. The most abundant one is extremely toxic, namely 3-chloropropane-1,2-diol, which can be detected at concentrations ranging up to 10,000 mg/kg. Furthermore, a cytotoxicity investigation of the condensates was performed on HUVEC/Tert2 cells in which metabolic activity was determined by means of resazurin assay. The cellular metabolic activity significantly decreased by treatment with e-liquid aerosol condensate. Due to the results of this study, we advise against the use of sucralose as sweetener in e-liquids.

Fully programmable spectrum sliced chirped microwave photonic filter.

A novel chirped microwave photonic filter (MPF) capable of achieving a large radio frequency (RF) group delay slope and a single passband response free from high frequency fading is presented. The design is based upon a Fourier domain optical processor (FD-OP) and a single sideband modulator. The FD-OP is utilized to generate both constant time delay to tune the filter and first order dispersion to induce the RF chirp, enabling full software control of the MPF without the need for manual adjustment. An optimized optical parameter region based on a large optical bandwidth >750 GHz and low slicing dispersion < ± 1 ps/nm is introduced, with this technique greatly improving the RF properties including the group delay slope magnitude and passband noise. Experimental results confirm that the structure simultaneously achieves a large in-band RF chirp of -4.2 ns/GHz, centre frequency invariant tuning and independent reconfiguration of the RF amplitude and phase response. Finally, a stochastic study of the device passband noise performance under tuning and reconfiguration is presented, indicating a low passband noise <-120 dB/Hz.

Multiplex protein analysis to determine fibrosis stage and progression in patients with chronic hepatitis C.

BACKGROUND & AIMS: Noninvasive tests cannot differentiate between adjacent stages of fibrosis, which limits assessment of disease progression and regression during therapy. We investigated whether levels of cytokines and extracellular matrix proteins in serum and biopsy samples can be used to determine actual stage of liver fibrosis in patients with chronic hepatitis C (CHC) and in prognosis. METHODS: We collected data from 383 treatment-naive patients with CHC from the Duke Hepatology Clinical Research Database and Biorepository, from 2006 through 2009, for use in the training set. Serum samples were obtained from 100 individuals without CHC (controls). We selected 37 serum biomarkers for customized array analysis by using the SearchLight multiplex sandwich enzyme-linked immunosorbent assay. Data from 434 treatment-naive patients with CHC, which were obtained from the Trent HCV cohort, were used in the validation analysis. Multivariable modeling, marker selection, and validation included randomForest and Obuchowski measures, with independent comparison with FibroSURE. RESULTS: Four serum markers (levels of hyaluronic acid, vascular cell adhesion molecule 1, alpha-2 macroglobulin, and retinol-binding protein 4) and age associated with fibrosis stage (F0-1, F2-3, or F4); these had Obuchowski measures of 0.85-0.89, with misclassification rates of 38% and 29% in training and validation sets, compared with 50% for the FibroSURE test. In the training set, area under the curve values for the multiplex markers were similar to those from the FibroSURE test: stages F0 vs F1 (0.51 vs 0.53), F1 vs F2 (0.60 vs 0.59), F2 vs F3 (0.69 vs 0.72), and F3 vs F4 (0.51 vs 0.52). Area under the curve values were similar in the validation cohort. In longitudinal analyses of 133 paired biopsies, 9 markers (level of alanine aminotransferase, γ-glutamyltransferase, hyaluronic acid, intracellular adhesion molecule 1, interleukin 4, CXCL10, CXCL9, and vascular cell adhesion molecule 1) were associated with change in the histologic activity index (P values ranging from .000 to .049), and 4 (granulocyte-macrophage colony-stimulating factor, interleukin 12, interleukin 2, and matrix metalloproteinase 13) were associated with a change in fibrosis stage (P values ranging from .001 to .042). CONCLUSIONS: We identified serum biomarkers that can be measured by multiplex enzyme-linked immunosorbent assay to determine levels of fibrosis in patients with CHC, although misclassification is frequent and results are comparable with those from the FibroSURE test. Changes in protein levels in biopsy samples were associated with progression of fibrosis in patients.

Anti-Inflammatory Extract from Soil Algae Chromochloris zofingiensis Targeting TNFR/NF-κB Signaling at Different Levels.

Inflammatory skin diseases, including atopic dermatitis (AD) and psoriasis, are increasing in populations worldwide. The treatment of patients with AD and other forms of skin inflammation is mainly based on the use of topical corticosteroids or calcineurin inhibitors, which can cause significant side effects with long-term use. Therefore, there is a great need for the development of more effective and less toxic anti-inflammatory agents suitable for the treatment of chronic skin lesions. Here, we screened a number of strains from the ASIB 505 terrestrial algae collection and identified a green algae Chromochloris zofingiensis with pronounced anti-inflammatory properties. We found that a crude nonpolar extract of C. zofingiensis (ID name NAE_2022C), grown upon nitrogen deprivation, acts as a bioactive substance by inhibiting TNFR/NF-κB responses in human skin keratinocyte HaCaT cells. We also found that NAE_2022C suppressed the secretion of pro-inflammatory cytokine tumor necrosis factor α (TNFα) and several Th1- and Th2-related chemokines in a reconstituted human epidermis. The TNFR/NF-κB pathway analysis showed multiple inhibitory effects at different levels and disclosed a direct targeting of IKKß by the extract. Bioassay-guided fractionation followed by high-resolution mass spectrometry detected diacylglyceryl-trimethylhomoserine (DGTS), Lyso-DGTS (LDGTS), 5-phenylvaleric acid, theophylline and oleamide as leading metabolites in the active fraction of NAE_2022C. Further analysis identified betaine lipid DGTS (32:0) as one of the active compounds responsible for the NAE_2022C-mediated NF-κB suppression. Overall, this study presents an approach for the isolation, screening, and identification of anti-inflammatory secondary metabolites produced by soil algae.

Fluorescent thermal shift-based method for detection of NF-κB binding to double-stranded DNA.

The nuclear factor kappa B (NF-κB) family of dimeric transcription factors regulates a wide range of genes by binding to their specific DNA regulatory sequences. NF-κB is an important therapeutic target linked to a number of cancers as well as autoimmune and inflammatory diseases. Therefore, effective high-throughput methods for the detection of NF-κB DNA binding are essential for studying its transcriptional activity and for inhibitory drug screening. We describe here a novel fluorescence-based assay for quantitative detection of κB consensus double-stranded (ds) DNA binding by measuring the thermal stability of the NF-κB proteins. Specifically, DNA binding proficient NF-κB probes, consisting of the N-terminal p65/RelA (aa 1-306) and p50 (aa 1-367) regions, were designed using bioinformatic analysis of protein hydrophobicity, folding and sequence similarities. By measuring the SYPRO Orange fluorescence during thermal denaturation of the probes, we detected and quantified a shift in the melting temperatures (ΔTm) of p65/RelA and p50 produced by the dsDNA binding. The increase in Tm was proportional to the concentration of dsDNA with apparent dissociation constants (KD) of 2.228 × 10-6 M and 0.794 × 10-6 M, respectively. The use of withaferin A (WFA), dimethyl fumarate (DMF) and p-xyleneselenocyanate (p-XSC) verified the suitability of this assay for measuring dose-dependent antagonistic effects on DNA binding. In addition, the assay can be used to analyse the direct binding of inhibitors and their effects on structural stability of the protein probe. This may facilitate the identification and rational design of new drug candidates interfering with NF-κB functions.

Olaparib is effective in combination with, and as maintenance therapy after, first-line endocrine therapy in prostate cancer cells.

A number of prostate cancer (PCa)-specific genomic aberrations (denominated BRCAness genes) have been discovered implicating sensitivity to PARP inhibition within the concept of synthetic lethality. Recent clinical studies show favorable results for the PARP inhibitor olaparib used as single agent for treatment of metastatic castration-resistant PCa. Using 2D and 3D cell culture models mimicking the different treatment and progression stages of PCa, we evaluated a potential use for olaparib in combination with first-line endocrine treatments, androgen deprivation, and complete androgen blockade, and as a maintenance therapy following on from endocrine therapy. We demonstrate that the LNCaP cell line, possessing multiple aberrations in BRCAness genes, is sensitive to olaparib. Additive effects of olaparib combined with endocrine treatments in LNCaP are noted. In contrast, we find that the TMPRSS2:ERG fusion-positive cell lines VCaP and DuCaP do not show signs of synthetic lethality, but are sensitive to cytotoxic effects caused by olaparib. In consequence, additive effects of olaparib with endocrine therapy were not observable in these cell lines, showing the need for synthetic lethality in combination treatment regimens. Additionally, we show that PCa cells remain sensitive to olaparib treatment after initial androgen deprivation implicating a possible use of olaparib as maintenance therapy. In sum, our preclinical data recommend olaparib as a synthetic lethal treatment option in combination or sequenced to first-line endocrine therapy for PCa patients with diagnosed BRCAness.

The spatial organization of lipid synthesis in the yeast Saccharomyces cerevisiae derived from large scale green fluorescent protein tagging and high resolution microscopy.

The localization pattern of proteins involved in lipid metabolism in the yeast Saccharomyces cerevisiae was determined using C-terminal green fluorescent protein tagging and high resolution confocal laser scanning microscopy. A list of 493 candidate proteins ( approximately 9% of the yeast proteome) was assembled based on proteins of known function in lipid metabolism, their interacting proteins, proteins defined by genetic interactions, and regulatory factors acting on selected genes or proteins. Overall 400 (81%) transformants yielded a positive green fluorescent protein signal, and of these, 248 (62% of the 400) displayed a localization pattern that was not cytosolic. Observations for many proteins with known localization patterns were consistent with published data derived from cell fractionation or large scale localization approaches. However, in many cases, high resolution microscopy provided additional information that indicated that proteins distributed to multiple subcellular locations. The majority of tagged enzymes localized to the endoplasmic reticulum (91), but others localized to mitochondria (27), peroxisomes (17), lipid droplets (23), and vesicles (53). We assembled enzyme localization patterns for phospholipid, sterol, and sphingolipid biosynthetic pathways and propose a model, based on enzyme localization, for concerted regulation of sterol and sphingolipid metabolism that involves shuttling of key enzymes between endoplasmic reticulum, lipid droplets, vesicles, and Golgi.