Bioactive Compounds from Agrifood Byproducts: Their Use in Medicine and Biology.

Agrifood produces a high amount of waste, millions of tons per year worldwide, the disposal of which is a significant environmental, organizational, logistical, economic and ethic problem and in the last decades the scientific interest about this argument has increased significantly [...].

Use of ancient grains for the management of diabetes mellitus: A systematic review and meta-analysis.

AIMS: A systematic review and meta-analysis of published randomized controlled trials was conducted to collate evidence from studies implementing ancient grains and investigate the impact of ancient grain consumption on health outcomes of patients with Diabetes Mellitus (DM). DATA SYNTHESIS: Twenty-nine randomized controlled trials were included, and 13 were meta-analyzed. Interventions ranged from 1 day to 24 weeks; most samples were affected by DM type 2 (n = 28 studies) and the ancient grains used were oats (n = 10 studies), brown rice (n = 6 studies), buckwheat (n = 4 studies), chia (n = 3 studies), Job's Tears (n = 2 studies), and barley, Khorasan and millet (n = 1 study). Thirteen studies that used oats, brown rice, and chia provided data for a quantitative synthesis. Four studies using oats showed a small to moderate beneficial effect on health outcomes including LDL-c (n = 717, MD: 0.30 mmol/l, 95% CI: 0.42 to -0.17, Z = 4.61, p < 0.05, I2 = 0%), and TC (n = 717, MD: 0.44 mmol/l, 95% CI: 0.63 to -0.24, Z = 4.40, p < 0.05, I2 = 0%). Pooled analyses of studies using chia and millet did not show significant effects on selected outcomes. CONCLUSIONS: For adults affected by DM type 2, the use of oats may improve lipidic profile. Further experimental designs are needed in interventional research to better understand the effects of ancient grains on diabetes health outcomes. PROSPERO REGISTRATION: CRD42023422386.

AGS Gastric Cells: Antioxidant Activity and Metabolic Effects of Phenolic Extracts from Different Monocultivar Virgin Olive Oils.

The effects of the phenolic compounds of extra virgin olive oil (EVOO) on AGS cells have never been studied so far, which is the aim of this study. The profiles of the main phenolic components in EVOOs, mainly secoiridoid compounds derived from the transformation of oleuropein during the olive milling process, were evaluated and compared. Oils of different origins were evaluated aiming at verifying whether chemical differences in the phenolic composition of the dry extracts played a role in the metabolism and in maintaining the cellular redox state of AGS cells. The following key enzymes of some metabolic pathways were studied: lactate dehydrogenase, enolase, pyruvate kinase, glucose 6-phosphate dehydrogenase, citrate synthase, 3-Hydroxyacyl-CoA dehydrogenase and hexokinase. As confirmed through PCA analysis, pretreatments with the dry extracts of EVOOs at different concentrations appeared to be able to counteract the enzymatic activity alterations due to oxidative stress induced by H2O2 1 mM and 2 mM. The studied phytocomplexes showed the ability to protect AGS cells from oxidative damage and the secoiridoid derivatives from both oleuropein and ligstroside contributed to the observed effects. The results suggested that EVOOs with medium to high concentrations of phenols can exert this protection.

Virgin Olive Oil By-Products: Biological Activity of Phenolic Extract of Pâté on AGS Gastric Cells.

Pâté is a by-product of olive oil production which represents an abundant source of phenolic compounds and can be used for food formulation, reducing its environmental impact and promoting a circular economy. In this context, the effects of a hydroalcoholic extract of pâté were evaluated for the first time in an AGS human cell line commonly used as model of gastric mucosa. Pâté was obtained from Tuscan olives; the total phenolic content was 16.6 mg/g dried extract, with verbascoside and secoiridoid derivatives as the most abundant phenols. The phenolic pâté extract did not alter viability, distribution of cell cycle phases or proliferation and migration of AGS cells at the tested concentrations. Seven enzymes were chosen to investigate the metabolic effect of the pâté extract in the context of oxidative stress. Pâté produced a statistically significant increase in the activity of key enzymes of some metabolic pathways: Lactate dehydrogenase, Enolase, Pyruvate kinase, Glucose 6-phosphate dehydrogenase, Citrate synthase, 3-Hydroxyacyl-CoA dehydrogenase and Hexokinase. Pre-treatments with the extract of pâté at 100 µg/mL or 200 µg/mL, as observed through PCA analysis, appeared able to counteract the enzymatic activity alterations due to oxidative stress induced by H2O2 1 mM and 2 mM. The results indicate that dried pâté, due to its phenolic components, can be proposed as a new functional food ingredient.

Ingestible light source for intragastric antibacterial phototherapy: a device safety study on a minipig model.

Helicobacter pylori gastric infections are among the most diffused worldwide, suffering from a rising rate of antibiotic resistance. In this context, some of the authors have previously designed an ingestible device in the form of a luminous capsule to perform antibacterial photodynamic inactivation in the stomach. In this study, the light-emitting capsules were tested to verify the safety of use prior to perform clinical efficacy studies. First, laboratory tests measured the capsule temperature while in function and verified its chemical resistance in conditions mimicking the gastric and gut environments. Second, safety tests in a healthy minipig model were designed and completed, to verify both the capsule integrity and the absence of side effects, associated with its illumination and transit throughout the gastrointestinal tract. To this aim, a capsule administration protocol was defined considering a total of 6 animals with n = 2 treated with 8 capsules, n = 2 treated with 16 capsules and n = 2 controls with no capsule administration. Endoscopies were performed in sedated conditions before-after every capsule administration. Biopsies were taken from the corpus and antrum regions, while the gastric cavity temperature was monitored during illumination. The bench tests confirmed a very good chemical resistance and a moderate (about 3 °C) heating of the capsules. The animal trials showed no significant effects on the gastric wall tissues, both visually and histologically, accompanied with overall good animal tolerance to the treatment. The integrity of the administered capsules was verified as well. These encouraging results pose the basis for the definition of successive trials at the clinical level.

Scratch assay microscopy: A reaction-diffusion equation approach for common instruments and data.

Scratch assay is an easy and widely used "in vitro" technique to study cell migration and proliferation. In this work we focus on its modelling and on the capability to distinguish between these two phenomena that the simpler and common models are not able to disentangle. We adapted a model based on reaction-diffusion equation for being used with common microscopy instruments/data and therefore taking place in the gap between simpler modelling approaches and complex ones. An optimized image analysis pipeline and numerical least-squares fit provide estimates of the scratch proliferation and diffusion coefficients l and D. This work is intended as a first of a series in which the model is tested and its robustness and reproducibility are evaluated. Test samples were NIH3T3 cells scratch assays with proliferation and migration stimulated by varying the foetal bovine serum amount in the culture medium (10%, 7.5%, 5% and 2.5%). Results demonstrate, notwithstanding an expected l-D anticorrelation, the model capability to disentangle them. The 7.5% serum treatment can be identified as the model sensitivity limit. Treat-control l and D variations showed an intra-experiment reproducibility (∼±0.05∕h and ∼±200µm2∕h respectively) consistent with single fit typical uncertainties (∼±0.02∕h and ∼±300µm2∕h respectively).

Synergistic effect of photodynamic therapy at 400 nm and doxycycline against Helicobacter pylori.

Aim: The objective of this study was to investigate the possible synergy between doxycycline and photodynamic therapy against Helicobacter pylori and to evaluate the possible side effects on adenocarcinoma gastric cells with and without protoporphyrin IX. Materials & methods: Three H. pylori strains (ATCC 700392, 43504 and 49503) were grown on solid medium either with, or without, doxycycline at subinhibitory concentrations, and irradiated for 10, 20 and 30 minutes with a 400 nm-peaked light source. The phototoxicity tests on AGS cells were evaluated by MTT assay. Results: The photodynamic therapy and doxycycline combination showed an antibacterial synergistic effect with no significant toxicities. Conclusion: The synergistic treatment could be considered as an interesting therapeutic option.

Glyoxylate cycle activity in Pinus pinea seeds during germination in altered gravity conditions.

This work inserts in the research field regarding the effects of altered gravity conditions on biological plant processes. Pinus pinea seeds germination was studied in simulated microgravity (2x10-3g) and hypergravity (20g) conditions. The effects of simulated gravity were evaluated monitoring the levels of the key enzymes, involved in the main metabolic pathway during germination process of lipid-rich seeds (oilseeds): isocitrate lyase and malate synthase for glyoxylate cycle, 3-hydroxyacyl-CoA dehydrogenase for beta-oxidation, isocitrate dehydrogenase for Krebs cycle, pyruvate kinase for glycolysis and glucose 6 phosphate dehydrogenase for pentose phosphate shunt. The simulated micro and hypergravity conditions were obtained by a Random Position Machine and a Hyperfuge, respectively. Results show that the levels of some tested enzymes, at different lag times of the germination process, have the same trend of controls (g = 1), but with significant differences from quantitative point of view. They are higher in microgravity conditions and lower in hypergravity ones, suggesting that, from a biochemical point of view, the germination process results accelerated in microgravity conditions and delayed in hypergravity ones. These biochemical results show a good correlation with morphological ones, obtained with the measurement of the length of the seeds sprouting radicle. These results give promising indications regarding the possibility to grow plant with lipid-rich seeds in spatial environment, to obtain food sources for astronauts during long term space missions and to reconstitute new atmosphere.

Unstained cell imaging: Morphological insights from coupled fixation and darkfield microscopy.

Cell staining techniques are well established in cell biology and associated with a broad range of dedicated dyes; however, they are accompanied by non-negligible costs, preparation time and unavoidable alterations of the sample with foreign molecules. In this context, we point out and propose the use of darkfield microscopy (DM) combined with different fixation protocols (to be used anyway) to enhance the different cell structures and districts as a timesaving and inexpensive support to the techniques that need staining or immuno-staining protocols and products. In a first step, we have analysed the effect of different fixation protocols on DM images for various human cellular lines. The presented imaging study shows that cell morphology actually changes with the fixation protocols that enhance, through contrast and luminosity variations, different shapes and patterns and thus structures of the cells. The different chemical action of various fixations, in fact, modifies the local scattering coefficient, thus affecting in a different way the morphology shown by DM images. As a second step we have compared the observed DM morphologies to those of selective fluorescent staining being therefore able to associate them to specific cell districts (e.g. nucleus, membrane or cytoskeleton). The obtained results indicate that this common microscopy technique can give images with particular cellular structures or districts enhanced more than others depending on the choice of fixation protocol. Therefore Darkfield Microscopy can be considered as a simpler, cheaper and faster method to provide morphological indications, respect to staining techniques, even at low and medium magnifications.

Side effects of intra-gastric photodynamic therapy: an in vitro study.

Since many years it has been acknowledged that some bacterial species, among which H. pylori, P. aeruginosa, P. acnes accumulate endogenous photosensitizers (PS) in the form of porphyrins. This makes antibacterial photodynamic therapy (PDT) easier to perform due to the possible avoidance of external PS. In this study, we focus on gastric infections associated with the presence of Helicobacter pylori (H. pylori), known to accumulate and release both protoporphyrin IX (PPIX) and coproporphyrins. PDT versus H. pylori can be carried out by modified endoscopes or by new ingestible luminous devices under development. In both cases of in vitro and in vivo applications, either for therapy (PDT) or diagnosis, scientific literature lacks studies on the possible side-effects of light treatments on the surrounding tissues. To this aim we evaluated in vitro side-effects due to a possible intrinsic photosensitivity of gastric mucosa or to a photosensitization by the PS released from the bacterium itself. Photo-toxicity studies were conducted on the AGS cell line (ATCC® CRL-1739™), commonly used as a model for the stomach mucosa tissue, considering PPIX as the photosensitizing agent. After first evaluations of PPIX dark toxicity, its uptake and accumulation sites, photo-toxicity tests were conducted using a LED light source peaked at 400 nm, by varying both PPIX concentration (50 nM - 2 µM) and light dose in the range 0.6-13 J/cm2, representing different treatment procedures found in literature. The oxidative stress consequent to irradiation was investigated both in terms of ROS production and assessment of the activity of enzymes involved in ROS-related biological mechanisms. A significant phototoxic effect was found only for PPIX concentration > 100 nM for all tested light doses. This indicates that the evaluated photo-treatments do not cause side effects even with the sensitization due to PPIX released by the bacteria.

EGFR-Targeted Magnetic Nanovectors Recognize, in Vivo, Head and Neck Squamous Cells Carcinoma-Derived Tumors.

Head and neck squamous cell carcinomas (HNSCC) are a diverse group of tumors with high morbidity and mortality that have remained mostly unchanged over the past decades. The epidermal growth factor receptor (EGFR) is often overexpressed and activated in these tumors and strongly contributes to their pathogenesis. Still, EGFR-targeted therapies such as monoclonal antibodies and kinase inhibitors have demonstrated only limited improvements in the clinical outcome of this disease. Here, we take advantage of the extraordinary affinity of EGF for its cognate receptor to specifically target magnetite-containing nanoparticles to HNSCC cells and mediate, in vitro, their cellular upload. On the basis of this, we show efficient accumulation, in vivo, of such nanoparticles in subcutaneous xenograft tumor tissues in sufficient amounts to be able to mediate visualization by magnetic resonance imaging. Overall, our EGF-coated nanosystem may warrant, in the near future, novel and very efficient theranostic approaches to HNSCC.

B-cell lymphoma 2 and ß-catenin expression in colorectal cancer and their prognostic role following surgery.

The prognosis of colorectal cancer depends on the stage of the disease. However, even within the same stage there may be different outcomes in terms of recurrence and survival. Therefore, it is clear that as well as pathological stage, novel biomarkers that are capable of improving risk stratification and therapeutic decision-making are required. The present study aimed to evaluate the potential roles of two previously proposed biomarkers of tumour status: B-cell lymphoma 2 (Bcl-2) and ß-catenin. A total of 412 patients undergoing surgery for primary colorectal cancer were studied. Tumour specimens of the patients were collected, fixed and processed for immunohistochemical detection of Bcl-2 and ß-catenin. The data were then analyzed in relation to disease-free survival and overall survival. Pathological stage was the only variable that was significantly correlated with both disease-free and overall survival. The expression levels of neither Bcl-2 nor ß-catenin were able to accurately predict prognosis. However, there was a clear association between nuclear ß-catenin expression levels and disease-free survival in the three tumour stages. There was an increased hazard ratio in stage I and II nuclear ß-catenin positive tumours, whereas there was a marked decrease in risk in stage III positive tumours. A similar effect was also observed with regards to overall survival, however this finding was not significant. The results of the present study suggest that conventional pathological tumour staging is the only accurate prognostic method. Neither Bcl-2 or ß-catenin were shown to be useful biomarkers for the prognosis of colorectal cancer. However, the heterogeneous behaviour of nuclear ß-catenin expression in the various tumour stages may indicate a possible role in predicting the response of patients to chemotherapy. Therefore, nuclear ß-catenin expression may be a biomarker for the prediction of improved responses to chemotherapy.