Proteomic analysis of brain metastatic lung adenocarcinoma reveals intertumoral heterogeneity and specific alterations associated with the timing of brain metastases.

BACKGROUND: Brain metastases are associated with considerable negative effects on patients' outcome in lung adenocarcinoma (LADC). Here, we investigated the proteomic landscape of primary LADCs and their corresponding brain metastases. MATERIALS AND METHODS: Proteomic profiling was conducted on 20 surgically resected primary and brain metastatic LADC samples via label-free shotgun proteomics. After sample processing, peptides were analyzed using an Ultimate 3000 pump coupled to a QExactive HF-X mass spectrometer. Raw data were searched using PD 2.4. Further data analyses were carried out using Perseus, RStudio and GraphPad Prism. Proteomic data were correlated with clinical and histopathological parameters and the timing of brain metastases. Mass spectrometry-based proteomic data are available via ProteomeXchange with identifier PXD027259. RESULTS: Out of the 6821 proteins identified and quantified, 1496 proteins were differentially expressed between primary LADCs and corresponding brain metastases. Pathways associated with the immune system, cell-cell/matrix interactions and migration were predominantly activated in the primary tumors, whereas pathways related to metabolism, translation or vesicle formation were overrepresented in the metastatic tumors. When comparing fast- versus slow-progressing patients, we found 454 and 298 differentially expressed proteins in the primary tumors and brain metastases, respectively. Metabolic reprogramming and ribosomal activity were prominently up-regulated in the fast-progressing patients (versus slow-progressing individuals), whereas expression of cell-cell interaction- and immune system-related pathways was reduced in these patients and in those with multiple brain metastases. CONCLUSIONS: This is the first comprehensive proteomic analysis of paired primary tumors and brain metastases of LADC patients. Our data suggest a malfunction of cellular attachment and an increase in ribosomal activity in LADC tissue, promoting brain metastasis. The current study provides insights into the biology of LADC brain metastases and, moreover, might contribute to the development of personalized follow-up strategies in LADC.

The antifibrotic potential of a sustained release formulation of a PDGFß-receptor targeted rho kinase inhibitor.

Rho kinase activity in hepatic stellate cells (HSCs) is associated with activation, transformation and contraction of these cells, leading to extracellular matrix production and portal hypertension in liver cirrhosis. Inhibition of rho kinase activity can reduce these activities, but may also lead to side effects, for instance systemic hypotension. This can be circumvented by liver-specific delivery of a rho kinase inhibitor to effector cells. Therefore, we targeted the rho kinase inhibitor Y27632 to the key pathogenic cells in liver fibrosis, i.e. myofibroblasts including activated HSCs that highly express the PDGFß-receptor, using the drug carrier pPB-MSA. This carrier consists of mouse serum albumin (MSA) covalently coupled to several PDGFßR-recognizing moieties (pPB). We aimed to create a prolonged release system of such a targeted construct, by encapsulating pPB-MSA-Y27632 in biodegradable polymeric microspheres, thereby reducing short-lasting peak concentrations and the need for frequent administrations. Firstly, we confirmed the vasodilating potency of PDGFß-receptor targeted Y27632 in vitro in a contraction assay using HSCs seeded on a collagen gel. We subsequently demonstrated the in vivo antifibrotic efficacy of pPB-MSA-Y27632-loaded microspheres in the Mdr2-/- mouse model of progressive biliary liver fibrosis. A single subcutaneous microsphere administration followed by organ harvest one week later clearly attenuated liver fibrosis progression and significantly suppressed the expression of fibrosis related genes, such as several collagens, profibrotic cytokines and matrix metalloproteinases. In conclusion, we demonstrate that polymeric microspheres are suitable as drug delivery system for the sustained systemic delivery of targeted protein constructs with antifibrotic potential, such as pPB-MSA-Y27632. This formulation appears suitable for the sustained treatment of liver fibrosis and possibly other chronic diseases.

Bioconjugation strategies to couple supramolecular exo-functionalized palladium cages to peptides for biomedical applications.

Supramolecular Pd2L4 cages (L = ligand) hold promise as drug delivery systems. With the idea of achieving targeted delivery of the metallacages to tumor cells, the bioconjugation of exo-functionalized self-assembled Pd2L4 cages to peptides following two different approaches is reported for the first time. The obtained bioconjugates were analyzed and identified by high-resolution mass spectrometry.

Ustekinumab in hidradenitis suppurativa: clinical results and a search for potential biomarkers in serum.

BACKGROUND: Treatment of hidradenitis suppurativa (HS) is difficult and the search for effective therapies continues. OBJECTIVES: To evaluate the efficacy of ustekinumab and to discover a potential biomarker for HS. METHODS: Seventeen patients were included in this open-label study and treated with 45 or 90 mg ustekinumab at weeks 0, 4, 16 and 28. Proteomic technology and enzyme-linked assay analysis was applied to sera. RESULTS: Twelve patients completed the protocol. Moderate-to-marked improvement of the modified Sartorius score was achieved in 82% of patients at week 40 and the Hidradenitis Suppurativa Clinical Response 50 in 47%. With regard to the expression of 54 serum proteins, at baseline, a significant difference was observed between patients and healthy controls. Involved pathways were related to inflammation, immune cell signalling and tissue morphology/development. Good responders had milder disease and lower expression of leukotriene A4-hydrolase (LTA4H). Interleukin (IL)-2R, tumour necrosis factor-α, IL-17A and IL-17F were not elevated and did not change during treatment. CONCLUSIONS: The majority of patients improved with ustekinumab. Although no biomarker was discovered, low LTA4H concentrations with mild disease severity may be predictive of the effectiveness of ustekinumab.

Investigation of potential markers of acid resistance in Lactobacillus plantarum by comparative proteomics.

AIMS: To investigate cell determinants of resistance to gastric acidity in Lactobacillus plantarum using comparative proteomics. METHODS AND RESULTS: Among ten Lact. plantarum strains that were tested for their acid resistance in vitro, three strains with different phenotypes were selected for comparative proteomic analysis: LC 804 (resistant), CIP A159 (intermediate) and CECT 4185 (sensitive). Constitutive differences between whole-cell proteomes of the three strains were studied. Among the differentially expressed proteins between strains, 17 have previously been reported to be involved in acid resistance processes. The effect of a low-pH exposure on these proteomic patterns was investigated, which led to the identification of five putative determinants of acid resistance (heat-shock protein GrpE, methionine synthase and 30S ribosomal protein S2) or sensitivity (phosphotransacetylase and adenylosuccinate synthase). Analysis also revealed three additional proteins involved in cell envelope biogenesis (3-oxoacyl-synthase II, dTDP-glucose 4,6-dehydratase and dTDP-4-dehydrorhamnose 3,5-epimerase) likely to be key factors of intrinsic acid tolerance in Lact. plantarum. CONCLUSIONS: The approach used in this study enabled the identification of potential markers of acid tolerance in Lact. plantarum, which may serve for phenotyping and screening purposes. SIGNIFICANCE AND IMPACT OF THE STUDY: The present work represents a further step towards the identification of bacterial biomarkers for each particular probiotic feature.

Toxicological potential of 2-alkylcyclobutanones--specific radiolytic products in irradiated fat-containing food--in bacteria and human cell lines.

Food irradiation has been considered as a safe processing technology to improve food safety and preservation, eliminating efficiently bacterial pathogens, parasites and insects. This study aims to characterize the toxicological potential of 2-alkylcyclobutanones (2-ACBs), radiolytic derivatives of triglycerides, formed uniquely upon irradiation of fat-containing food. In irradiated food they are generated proportionally to fat content and absorbed radiation dose. The cyto- and genotoxic potentials of various highly pure synthetic 2-ACBs were studied in bacteria and human cell lines. While pronounced cytotoxicity was evident in bacteria, no mutagenic activity has been revealed by the Ames test in Salmonella strains TA 97, TA 98 and TA 100. In mammalian cells genotoxicity was demonstrated mainly by the induction of DNA base lesions recognized by the Fpg protein as determined by both the Comet Assay and the Alkaline Unwinding procedure. Formation of DNA strand breaks was observed by the Alkaline Unwinding procedure but not by the Comet Assay. The extent of cytotoxicity and genotoxicity were dependent on chain length and degree of unsaturation of the fatty acid chain. Further studies will have to clarify mechanisms of action and potential relevance for human exposure situation.

Detection of 2-alkylcyclobutanones, markers for irradiated foods, in adipose tissues of animals fed with these substances.

Laboratory rats received a freshly prepared drinking fluid containing 0.005% 2-tetradecyl- or 2-tetradecenyl-cyclobutanones daily for 4 months. These two compounds were recovered in the adipose tissues of the animals that consumed them. Less than 1% of the 2-alkylcyclobutanones ingested daily were excreted in the feces. In addition, our data indicate that 2-alkylcyclobutanones are able to cross the intestinal barrier, to enter into the bloodstream, and to be stored in the adipose tissue of an animal. However, the amounts of these substances detected in the adipose tissues and in the feces were much smaller than the amounts ingested.

Supercritical fluid extraction of hydrocarbons and 2-alkylcyclobutanones for the detection of irradiated foodstuffs.

Supercritical carbon dioxide can be used to carry out a selective and fast extraction (30 min) of volatile hydrocarbons and 2-alkylcyclobutanones contained in irradiated foods. After elimination of the traces of triglycerides still contained in the extracts on a silica column, the compounds were analysed by gas chromatography-mass spectroscopy (2-alkylcyclobutanones) and gas chromatography-flame ionization detection (volatile hydrocarbons). The present method was applied successfully to freeze-dried samples (1 g or less) of cheese, chicken, avocados and to various ingredients (chocolate, liquid whole eggs) included in non-irradiated cookies. It was faster (4-5 h) than the reference methods EN 1784 (volatile hydrocarbons) and EN 1785 (2-alkylcyclobutanones), which take 1.5 days each. The minimal dose detectable by this method is, in addition, slightly lower than those of the reference methods.

2-Alkylcyclobutanones as markers for irradiated foodstuffs. III. Improvement of the field of application on the EN 1785 method by using silver ion chromatography.

The inclusion of a purification step by silver ion chromatography in the EN 1785 analytical protocol for 2-alkylcyclobutanones (validated by the European Committee for Standardization for the detection of ionizing radiation treatment) has considerably improved the quality of the chromatograms obtained, allowing the detection of food samples irradiated at very low doses (0.1 kGy) or irradiated ingredients included in low proportions in non irradiated foodstuffs. This analytical modification of the protocol EN 1785 ought thus to permit a very considerable extension of its current field of application.