ABCB1 overexpression through locus amplification represents an actionable target to combat paclitaxel resistance in pancreatic cancer cells.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest types of cancer and the chemotherapies such as gemcitabine/nab-paclitaxel are confronted with intrinsic or acquired resistance. The aim of this study was to investigate mechanisms underlying paclitaxel resistance in PDAC and explore strategies to overcome it. METHODS: Three paclitaxel (PR) and gemcitabine resistant (GR) PDAC models were established. Transcriptomics and proteomics were used to identify conserved mechanisms of drug resistance. Genetic and pharmacological approaches were used to overcome paclitaxel resistance. RESULTS: Upregulation of ABCB1 through locus amplification was identified as a conserved feature unique to PR cells. ABCB1 was not affected in any of the GR models and no cross resistance was observed. The ABCB1 inhibitor verapamil or siRNA-mediated ABCB1 depletion sensitized PR cells to paclitaxel and prevented efflux of ABCB1 substrates in all models. ABCB1 expression was associated with a trend towards shorter survival in patients who had received gemcitabine/nab-paclitaxel treatment. A pharmacological screen identified known and novel kinase inhibitors that attenuate efflux of ABCB1 substrates and sensitize PR PDAC cells to paclitaxel. CONCLUSION: Upregulation of ABCB1 through locus amplification represents a novel, conserved mechanism of PDAC paclitaxel resistance. Kinase inhibitors identified in this study can be further (pre) clinically explored as therapeutic strategies to overcome paclitaxel resistance in PDAC.

Low-melting point agarose as embedding medium for MALDI mass spectrometry imaging and laser-capture microdissection-based proteomics.

The combination of MALDI mass spectrometry imaging, laser-capture microdissection, and quantitative proteomics allows the identification and characterization of molecularly distinct tissue compartments. Such workflows are typically performed using consecutive tissue sections, and so reliable sectioning and mounting of high-quality tissue sections is a prerequisite of such investigations. Embedding media facilitate the sectioning process but can introduce contaminants which may adversely affect either the mass spectrometry imaging or proteomics analyses. Seven low-temperature embedding media were tested in terms of embedding temperature and cutting performance. The two media that provided the best results (5% gelatin and 2% low-melting point agarose) were compared with non-embedded tissue by both MALDI mass spectrometry imaging of lipids and laser-capture microdissection followed by bottom-up proteomics. Two out of the seven tested media (5% gelatin and 2% low-melting point agarose) provided the best performances on terms of mechanical properties. These media allowed for low-temperature embedding and for the collection of high-quality consecutive sections. Comparisons with non-embedded tissues revealed that both embedding media had no discernable effect on proteomics analysis; 5% gelatin showed a light ion suppression effect in the MALDI mass spectrometry imaging experiments, 2% agarose performed similarly to the non-embedded tissue. 2% low-melting point agarose is proposed for tissue embedding in experiments involving MALDI mass spectrometry imaging of lipids and laser-capture microdissection, proteomics of consecutive tissue sections.

Proteomics analysis of circulating small extracellular vesicles: Focus on the contribution of EVs to tumor metabolism.

The term small extracellular vesicle (sEV) is a comprehensive term that includes any type of cell-derived, membrane-delimited particle that has a diameter < 200 nm, and which includes exosomes and smaller microvesicles. sEVs transfer bioactive molecules between cells and are crucial for cellular homeostasis and particularly during tumor development, where sEVs provide important contributions to the formation of the premetastic niche and to their altered metabolism. sEVs are thus legitimate targets for intervention and have also gained increasing interest as an easily accessible source of biomarkers because they can be rapidly isolated from serum/plasma and their molecular cargo provides information on their cell-of origin. To target sEVs that are specific for a given cell/disease it is essential to identify EV surface proteins that are characteristic of that cell/disease. Mass-spectrometry based proteomics is widely used for the identification and quantification of sEV proteins. The methods used for isolating the sEVs, preparing the sEV sample for proteomics analysis, and mass spectrometry analysis, can have a strong influence on the results and requires careful consideration. This review provides an overview of the approaches used for sEV proteomics and discusses the inherent compromises regarding EV purity versus depth of coverage. Additionally, it discusses the practical applications of the methods to unravel the involvement of sEVs in regulating the metabolism of pancreatic ductal adenocarcinoma (PDAC). The metabolic reprogramming in PDAC includes enhanced glycolysis, elevated glutamine metabolism, alterations in lipid metabolism, mitochondrial dysfunction and hypoxia, all of which are crucial in promoting tumor cell growth. A thorough understanding of these metabolic adaptations is imperative for the development of targeted therapies to exploit PDAC's vulnerabilities.

Determination of Spectroscopic Parameters of Ag(I) and Ag(II) Emission Lines Using Time-Independent Extended C-Sigma Method.

The knowledge of the spectroscopic parameters of the elemental emission lines is important for diagnostics of laser-induced plasmas and the application of calibration-free/fundamental parameters analytical methods. In this paper, we used the recently proposed time-independent extended C-sigma method for determining, for the first time, the transition probabilities and Stark broadening coefficients of several neutral (TIECS) and ionic silver emission lines. The method allows for a compensation of self-absorption in the plasma, thus providing a measure of the spectroscopic parameters which is not affected by the optical thickness of the plasma.

Flaxseed Cake as a Tool for the Improvement of Nutraceutical and Sensorial Features of Sourdough Bread.

Flaxseed has been recently studied for the formulation of healthy functional foods that are also useful for the prevention of chronic diseases. In this context, the production of sourdough bread fortified with different percentages of flaxseed cake was performed and the interactions among the bioactive compounds derived from both sourdough and flaxseed cake were investigated. The organoleptic properties as well as nutraceutical and chemical characteristics regarding pH, ethanol, lactic and acetic acid content, fatty acids profile, the concentration of total polyphenols, antioxidant capacity, and aroma volatile organic compounds were determined to evaluate the efficacy of leavening in the different matrices in comparison with the traditional bread. The results obtained demonstrated that flaxseed cake-enriched sourdough bread can represent a potential vehicle for bioactive compounds with the possibility of obtaining high-quality products with improved nutritional profiles and desired health attributes. Furthermore, the bread obtained with the addition of 7.5% of flaxseed cake was individuated as the best formulation to produce sourdough bread fortified with flaxseed cake by the overlap between three series of information coming from physical-chemical, nutritional, and sensorial analyses. In conclusion, in the operating conditions adopted, the use of flaxseed cake could represent a viable alternative for the production of fortified bread based on sourdough technology.

Analytical and mathematical methods for revealing hidden details in ancient manuscripts and paintings: A review.

In this work, a critical review of the current nondestructive probing and image analysis approaches is presented, to revealing otherwise invisible or hardly discernible details in manuscripts and paintings relevant to cultural heritage and archaeology. Multispectral imaging, X-ray fluorescence, Laser-Induced Breakdown Spectroscopy, Raman spectroscopy and Thermography are considered, as techniques for acquiring images and spectral image sets; statistical methods for the analysis of these images are then discussed, including blind separation and false colour techniques. Several case studies are presented, with particular attention dedicated to the approaches that appear most promising for future applications. Some of the techniques described herein are likely to replace, in the near future, classical digital photography in the study of ancient manuscripts and paintings.