Precision Oncology via NMR-Based Metabolomics: A Review on Breast Cancer

Precision oncology is an emerging approach in cancer care. It aims at selecting the optimal therapy for the right patient by considering each patient’s unique disease and individual health status. In the last years, it has become evident that breast cancer is an extremely heterogeneous disease, and therefore, patients need to be appropriately stratified to maximize survival and quality of life. Gene-expression tools have already positively assisted clinical decision making by estimating the risk of recurrence and the potential benefit from adjuvant chemotherapy. However, these approaches need refinement to further reduce the proportion of patients potentially exposed to unnecessary chemotherapy. Nuclear magnetic resonance (NMR) metabolomics has demonstrated to be an optimal approach for cancer research and has provided significant results in BC, in particular for prognostic and stratification purposes. In this review, we give an update on the status of NMR-based metabolomic studies for the biochemical characterization and stratification of breast cancer patients using different biospecimens (breast tissue, blood serum/plasma, and urine).     

Novel KCND3 Variant Underlying Nonprogressive Congenital Ataxia or SCA19/22 Disrupt KV4.3 Protein Expression and K+ Currents with Variable Effects on Channel Properties

KCND3 encodes the voltage-gated potassium channel K_V4.3 that is highly expressed in the cerebellum, where it regulates dendritic excitability and calcium influx. Loss-of-function K_V4.3 mutations have been associated with dominant spinocerebellar ataxia (SCA19/22). By targeted NGS sequencing, we identified two novel KCND3 missense variants of the K_V4.3 channel: p.S347W identified in a patient with adult-onset pure cerebellar syndrome and p.W359G detected in a child with congenital nonprogressive ataxia. Neuroimaging showed mild cerebellar atrophy in both patients. We performed a two-electrode voltage-clamp recording of K_V4.3 currents in Xenopus oocytes: both the p.G345V (previously reported in a SCA19/22 family) and p.S347W mutants exhibited reduced peak currents by 50%, while no K+ current was detectable for the p.W359G mutant. We assessed the effect of the mutations on channel gating by measuring steady-state voltage-dependent activation and inactivation properties: no significant alterations were detected in p.G345V and p.S347W disease-associated variants, compared to controls. K_V4.3 expression studies in HEK293T cells showed 53% (p.G345V), 45% (p.S347W) and 75% (p.W359G) reductions in mutant protein levels compared with the wildtype. The present study broadens the spectrum of the known phenotypes and identifies additional variants for KCND3-related disorders, outlining the importance of SCA gene screening in early-onset and congenital ataxia.     

A Phenylacetamide Resveratrol Derivative Exerts Inhibitory Effects on Breast Cancer Cell Growth

Resveratrol (RSV) is a natural compound that displays several pharmacological properties, including anti-cancer actions. However, its clinical application is limited because of its low solubility and bioavailability. Here, the antiproliferative and anti-inflammatory activity of a series of phenylacetamide RSV derivatives has been evaluated in several cancer cell lines. These derivatives contain a monosubstituted aromatic ring that could mimic the RSV phenolic nucleus and a longer flexible chain that could confer a better stability and bioavailability than RSV. Using MTT assay, we demonstrated that most derivatives exerted antiproliferative effects in almost all of the cancer cell lines tested. Among them, derivative 2, that showed greater bioavailability than RSV, was the most active, particularly against estrogen receptor positive (ER+) MCF7 and estrogen receptor negative (ER-) MDA-MB231 breast cancer cell lines. Moreover, we demonstrated that these derivatives, particularly derivative 2, were able to inhibit NO and ROS synthesis and PGE2 secretion in lipopolysaccharide (LPS)-activated U937 human monocytic cells (derived from a histiocytoma). In order to define the molecular mechanisms underlying the antiproliferative effects of derivative 2, we found that it determined cell cycle arrest at the G1 phase, modified the expression of cell cycle regulatory proteins, and ultimately triggered apoptotic cell death in both breast cancer cell lines. Taken together, these results highlight the studied RSV derivatives, particularly derivative 2, as promising tools for the development of new and more bioavailable derivatives useful in the treatment of breast cancer.     

Proinflammatory Interleukin-33 Induces Dichotomic Effects on Cell Proliferation in Normal Gastric Epithelium and Gastric Cancer

Interleukin (IL)-33 is a member of the interleukin (IL)-1 family of cytokines linked to the development of inflammatory conditions and cancer in the gastrointestinal tract. This study is designed to investigate whether IL-33 has a direct effect on human gastric epithelial cells (GES-1), the human gastric adenocarcinoma cell line (AGS), and the gastric carcinoma cell line (NCI-N87) by assessing its role in the regulation of cell proliferation, migration, cell cycle, and apoptosis. Cell cycle regulation was also determined in ex vivo gastric cancer samples obtained during endoscopy and surgical procedures. Cell lines and tissue samples underwent stimulation with rhIL-33. Proliferation was assessed by XTT and CFSE assays, migration by wound healing assay, and apoptosis by caspase 3/7 activity assay and annexin V assay. Cell cycle was analyzed by means of propidium iodine assay, and gene expression regulation was assessed by RT-PCR profiling. We found that IL-33 has an antiproliferative and proapoptotic effect on cancer cell lines, and it can stimulate proliferation and reduce apoptosis in normal epithelial cell lines. These effects were also confirmed by the analysis of cell cycle gene expression, which showed a reduced expression of pro-proliferative genes in cancer cells, particularly in genes involved in G0/G1 and G2/M checkpoints. These results were confirmed by gene expression analysis on bioptic and surgical specimens. The aforementioned results indicate that IL-33 may be involved in cell proliferation in an environment- and cell-type-dependent manner.     

Structural Investigation and Molecular Modeling Studies of Strobilurin-Based Fungicides Active against the Rice Blast Pathogen Pyricularia oryzae

The increasing emergence of fungicide-resistant pathogens requires urgent solutions for crop disease management. Here, we describe a structural investigation of new fungicides obtained by combining strobilurin and succinate dehydrogenase inhibitor pharmacophores. We identified compounds endowed with very good activity against wild-type Pyricularia oryzae, combined in some cases with promising activity against strobilurin-resistant strains. The first three-dimensional model of P. oryzae cytochrome bc1 complex containing azoxystrobin as a ligand was developed. The model was validated with a set of commercially available strobilurins, and it well explains both the resistance mechanism to strobilurins mediated by the mutation G143A and the activity of metyltetraprole against strobilurin-resistant strains. The obtained results shed light on the key recognition determinants of strobilurin-like derivatives in the cytochrome bc1 active site and will guide the further rational design of new fungicides able to overcome resistance caused by G143A mutation in the rice blast pathogen.     

Percutaneous Coronary Intervention (PCI) Reprograms Circulating Extracellular Vesicles from ACS Patients Impairing Their Cardio-Protective Properties

Extracellular vesicles (EVs) are promising therapeutic tools in the treatment of cardiovascular disorders. We have recently shown that EVs from patients with Acute Coronary Syndrome (ACS) undergoing sham pre-conditioning, before percutaneous coronary intervention (PCI) were cardio-protective, while EVs from patients experiencing remote ischemic pre-conditioning (RIPC) failed to induce protection against ischemia/reperfusion Injury (IRI). No data on EVs from ACS patients recovered after PCI are currently available. Therefore, we herein investigated the cardio-protective properties of EVs, collected after PCI from the same patients. EVs recovered from 30 patients randomly assigned (1:1) to RIPC (EV-RIPC) or sham procedures (EV-naive) ({"type":"clinical-trial","attrs":{"text":"NCT02195726","term_id":"NCT02195726"}}NCT02195726) were characterized by TEM, FACS and Western blot analysis and evaluated for their mRNA content. The impact of EVs on hypoxia/reoxygenation damage and IRI, as well as the cardio-protective signaling pathways, were investigated in vitro (HMEC-1 + H9c2 co-culture) and ex vivo (isolated rat heart). Both EV-naive and EV-RIPC failed to drive cardio-protection both in vitro and ex vivo. Consistently, EV treatment failed to activate the canonical cardio-protective pathways. Specifically, PCI reduced the EV-naive Dusp6 mRNA content, found to be crucial for their cardio-protective action, and upregulated some stress- and cell-cycle-related genes in EV-RIPC. We provide the first evidence that in ACS patients, PCI reprograms the EV cargo, impairing EV-naive cardio-protective properties without improving EV-RIPC functional capability.     

Maternal dietary habits and mycotoxin occurrence in human mature milk

During 2006, 82 samples of human mature milk were collected at Italian hospitals and checked for aflatoxin M1 (AFM1) and ochratoxin A (OTA) by immunoaffinity column extraction and HPLC. AFM1 was detected in four (5%) of milk samples (ranging from < 7 ng/L to 140 ng/L; mean level: 55.35 ng/L); OTA was detected in 61 (74%) of milk samples (ranging from < 5 ng/L to 405 ng/L; mean level: 30.43 ng/L. OTA levels were significantly higher (p less, not double equals 0.05) in milk of habitual consumers of bread, bakery products and cured pork meat. No other statistically significant differences were observed although habitual consumers of pasta (p = 0.059), cookies (p = 0.061) and juices (p = 0.063) had mean contamination values of OTA higher than the moderate consumer. The very few AFB1 positive samples did not allow statistical comparisons. The present study confirms that the occurrence of OTA in human milk is related to maternal dietary habits. The findings support the possibility of dietary recommendations to woman, during pregnancy and lactation, aimed to tentatively reduce the OTA contamination of human milk.     

Assessment of grape skin hardness by a puncture test

BACKGROUND: The release of grape components during wine making might be related to the mechanical properties of the skin, in particular its hardness. Samples from three varieties collected during the 2005 vintage season in Piedmont, Italy, were tested for their skin hardness using a texture analyser. The goal was to understand the statistical interactions between three factors—variety, cluster position and puncture point—and their influence on the grape skin hardness. A discussion on the relationship between the size of the sample used and the confidence level is also provided. RESULTS: Results of the ANOVA test showed that there is an interaction between the variety and the puncture point when measuring the skin hardness with the break energy. The position of the berry on the cluster does not affect the berry skin break energy. We also show that a sample size depend on the variety tested. CONCLUSION: The break energy is more useful in understanding the effect of the three factors on the skin hardness. Other factors that might affect the puncture test applied to grapes need to be studied in the future and the usefulness of the test in winemaking will need to be further developed. Copyright © 2008 Society of Chemical Industry     

Detection, quantification and vitality of Listeria monocytogenes in food as determined by quantitative PCR

In this paper we describe the development of a quantitative PCR (qPCR) technique to detect, quantify and determine the vitality of Listeria monocytogenes in foods. The method was based on the amplification of the intergenic region spacer (IGS) between the 16S and 23S rRNA genes. A panel of more than 100 strains of Listeria spp. and non-Listeria was used in order to verify the specificity of the primers and Taqman probe and amplification signals were obtained only when L. monocytogenes DNA and RNA were loaded in the qPCR mix. Standard curves were constructed in several food matrices (milk, meat, soft cheese, fermented sausage, cured ham and ready-to-eat salad). The quantification limit was of 10(3)-10(4) cfu/g or ml, while for the determination of vitality it was 10(4)-10(5) cfu/g or ml. After an overnight enrichment in BHI at 37 degrees C also 10 cfu/g or ml could be detected in all the matrices used in this study. When we applied the protocol to food samples collected from the market or from small food processing plants, on a total number of 66 samples, 4 fresh cheeses from raw milk gave positive results prior to the overnight incubation, while 9 samples, of which only one represented by fresh meat and the others by cheeses from raw milk, were positive after the enrichment. Out of the 4 positive samples, only one could be quantified and it was determined to contain 4x10(3) cfu/g.     

Accumulation parameters and seasonal trends for PCBs in temperate and boreal forest plant species

The concentration of polychlorinated biphenyls (PCBs) in the air and vegetation was measured periodically in two alpine forests, during the growing season. Foliage samples from nine plant species typical of the temperate and boreal environment were collected and analyzed. Leaf concentrations of tri- and tetra-CBs showed fast response times with changing temperature and gas-phase concentrations, suggesting that a partitioning equilibrium is approached relatively rapidly (few days) in the field. Heavier compounds showed kinetically limited accumulation trends, not reaching equilibrium during the growing season. Results were used to estimate the bioconcentration factors or equilibrium plant/air partition coefficient (KPA) for each species. Values of log KPA (calculated on a mass/volume basis) ranged between 0.78 and 1.96 and were correlated to the log KOA. Uptake trends of the higher chlorinated compounds showed intraspecific differences which were partially explained by the specific leaf area (SLA).