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.

Progesterone and ß-hCG Determination Using an Electrochemical Combo-Strip for Pregnancy Monitoring.

The development of analytical devices that can allow an easy, rapid and cost-effective measurement of multiple markers, such as progesterone and ß-hCG, could have a role in decreasing the burden associated with pregnancy-related complications, such as ectopic pregnancies. Indeed, ectopic pregnancies are a significant contributor to maternal morbidity and mortality in both high-income and low-income countries. In this work, an effective and highly performing electrochemical strip for a combo determination of progesterone and ß-hCG was developed. Two immunosensing approaches were optimized for the determination of these two hormones on the same strip. The immunosensors were realized using cost-effective disposable electrode arrays and reagent-saving procedures. Each working electrode of the array was modified with both the IgG anti-ß-hCG and anti-progesterone, respectively. By adding the specific reagents, progesterone or ß-hCG can then be determined. Fast quantitative detection was achieved, with the analysis duration being around 1 h. Sensitivity and selectivity were assessed with a limit of detection of 1.5 × 10-2 ng/mL and 2.45 IU/L for progesterone and ß-hCG, respectively. The proposed electrochemical combo-strip offers great promise for rapid, simple, cost-effective, and on-site analysis of these hormones and, thus, for the development of a point-of-care diagnostic tool for early detection of pregnancy-related complications.

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.

Lscß and lscγ, two novel levansucrases of Pseudomonas syringae pv. actinidiae biovar 3, the causal agent of bacterial canker of kiwifruit, show different enzymatic properties.

Bacterial canker disease caused by Pseudomonas syringae pv. actinidiae (Psa) biovar 3 involved all global interest since 2008. We have found that in Psa3 genome, similarly to other P. syringae, there are three putative genes, lscα, lscß and lscγ, coding for levansucrases. These enzymes, breaking the sucrose moiety and releasing glucose can synthetize the fructose polymer levan, a hexopolysaccharide that is well known to be part of the survival strategies of many different bacteria. Considering lscα non-coding because of a premature stop codon, in the present work we cloned and expressed the two putatively functional levansucrases of Psa3, lscß and lscγ, in E. coli and characterized their biochemical properties such as optimum of pH, temperature and ionic strength. Interestingly, we found completely different behaviour for both sucrose splitting activity and levan synthesis between the two proteins; lscγ polymerizes levan quickly at pH 5.0 while lscß has great sucrose hydrolysis activity at pH 7.0. Moreover, we demonstrated that at least in vitro conditions, they are differentially expressed suggesting two distinct roles in the physiology of the bacterium.

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).

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.

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.

Cancer associated fibroblasts transfer lipids and proteins to cancer cells through cargo vesicles supporting tumor growth.

Fibroblasts are the most abundant cells in connective tissue and, with fibrillar extracellular matrix, form the structural scaffolding of organs. In solid tumors, interaction with cancer cells induces fibroblasts transdifferentiation into an activated form, which become a fundamental part of the tumor stroma. Within tumor microenvironment stromal and cancer cells engage a crosstalk that is mediated by soluble factors, cellcell contacts and extracellular vesicles trafficlking. Here we report that fibroblasts have the ability to transfer a remarkable amount of proteins and lipids to neighboring cells, in an ectosome-dependent fashion, identifying a novel and native property of these cells. Cancer-associated fibroblasts show an enhanced production and delivering of ectc:Jsomes to cancer cells compared to normal fibroblasts. As a consequence of this phenomenon, tumor cells increase their proliferation rate, indicating that ectosome-mediated trafficking could be a relevant mechanism mediating the trophic function of activated connective tissue on tumor cells.

Effect of IR laser on myoblasts: a proteomic study.

Laser therapy is used in physical medicine and rehabilitation to accelerate muscle recovery and in sports medicine to prevent damages produced by metabolic disturbances and inflammatory reactions after heavy exercise. The aim of this research was to get insight into possible benefits deriving from the application of an advanced IR laser system to counteract deficits of muscle energy metabolism and stimulate the recovery of hypotrophic tissue. We studied the effect of IR laser treatment on proliferation, differentiation, cytoskeleton organization and global protein expression in C2C12 myoblasts. We found that laser treatment induced a decrease in the cell proliferation rate without affecting cell viability, while leading to cytoskeletal rearrangement and expression of the early differentiation marker MyoD. The differential proteome analysis revealed the up-regulation and/or modulation of many proteins known to be involved in cell cycle regulation, cytoskeleton organization and differentiation.

The insulin-mimetic effect of Morin: a promising molecule in diabetes treatment.

BACKGROUND: Type-2 diabetes is a worldwidely diffuse disease characterized by insulin resistance that arises from alterations of receptor and/or post-receptor events of insulin signalling. Studies performed with PTP1B-deficent mice demonstrated that PTP1B is the main negative regulator of insulin signalling. Inhibition or down regulation of this enzyme causes enhanced insulin sensitivity. Hence this enzyme represents the most attractive target for development of innovative anti-diabetic drugs. METHODS: Selection of new PTP1B inhibitors among an in house library of polyphenolic compounds was carried out screening their activity. The inhibition mechanism of Morin was determined by kinetic analyses. The cellular action of Morin was assayed on HepG2 cells. Analyses of the insulin signalling pathways was carried out by Western blot methods, glycogen synthesis was estimated by measuring the incorporation of [(3)H]-glucose, gluconeogenesis rate was assayed by measuring the glucose release in the cell medium. Cell growth was estimated by cell count. Docking analysis was conducted with SwissDock program. RESULTS: We demonstrated that Morin: i) is a non-competitive inhibitor of PTP1B displaying a Ki in the µM range; ii) increases the phosphorylation of the insulin receptor and Akt; iii) inhibits gluconeogenesis and enhances glycogen synthesis. Morin does not enhance cell growth. CONCLUSIONS: We have identified Morin as a new small molecular non-competitive inhibitor of PTP1B, which behaves as an activator and sensitizer of the insulin receptor stimulating the metabolic pathways only. GENERAL SIGNIFICANCE: Our study suggests that Morin is a useful lead for development of new low Mr compounds potentially active as antidiabetic drugs.

Evaluation of SCO1 deletion on Saccharomyces cerevisiae metabolism through a proteomic approach.

The Saccharomyces cerevisiae gene SCO1 has been shown to play an essential role in copper delivery to cytochrome c oxidase. Biochemical studies demonstrated specific transfer of copper from Cox17p to Sco1p, and physical interactions between the Sco1p and Cox2p. Deletion of SCO1 yeast gene results in a respiratory deficient phenotype. This study aims to gain a more detailed insight on the effects of SCO1 deletion on S. cerevisiae metabolism. We compared, using a proteomic approach, the protein pattern of SCO1 null mutant strain and wild-type BY4741 strain grown on fermentable and on nonfermentable carbon sources. The analysis showed that on nonfermentable medium, the SCO1 mutant displayed a protein profile similar to that of actively fermenting yeast cells. Indeed, on 3% glycerol, this mutant displayed an increase of some glycolytic and fermentative enzymes such as glyceraldehyde-3-phosphate dehydrogenase 1, enolase 2, pyruvate decarboxylase 1, and alcohol dehydrogenase 1. These data were supported by immunoblotting and enzyme activity assay. Moreover, the ethanol assay and the oxygen consumption measurement demonstrated a fermentative activity in SCO1 mutant on respiratory medium. Our results suggest that on nonfermentable carbon source, the lack of Sco1p causes a metabolic shift from respiration to fermentation.

Plasma protein carbonylation and physical exercise.

Regular physical activity is associated with a reduced risk of coronary heart disease, as it probably modifies the balance between free-radical generation and antioxidant activity. On the other hand, however, acute physical activity increases oxygen uptake and leads to a temporary imbalance between the production of reactive oxygen and nitrogen species (RONS) and their disposal: this phenomenon is called oxidative stress. Proteins are one of the most important oxidation targets during physical exercise and carbonylation is one of the most common oxidative protein modifications. In cells there is a physiological level of oxidized proteins that doesn't interfere with cell function; however, an increase in oxidized protein levels may cause a series of cellular malfunctions that could lead to a disease state. For this reason the quantification of protein oxidation is important to distinguish a healthy state from a disease state. Several studies have demonstrated an increase of carbonylated plasma proteins in athletes after exercise, but none have identified targets of this oxidation. Recently a process of protein decarbonylation has been discovered, this may indicate that carbonylation could be involved in signal transduction. The aim of our research was to characterize plasma protein carbonylation in response to physical exercise in trained male endurance athletes. We analyzed by proteomic approach their plasma proteins at resting condition and after two different kinds of physical exercise (PE). We used 2D-GE followed by western blot with specific antibodies against carbonylated proteins. The 2D analysis identified Haptoglobin as potential protein target of carbonylation after PE. We also identified Serotransferrin and Fibrinogen whose carbonylation is reduced after exercise. These methods have allowed us to obtain an overview of plasma protein oxidation after physical exercise.

Reciprocal control of cell proliferation and migration.

In adult tissue the quiescent state of a single cell is maintained by the steady state conditions of its own microenvironment for what concern both cell-cell as well as cell-ECM interaction and soluble factors concentration. Physiological or pathological conditions can alter this quiescent state through an imbalance of both soluble and insoluble factors that can trigger a cellular phenotypic response. The kind of cellular response depends by many factors but one of the most important is the concentration of soluble cytokines sensed by the target cell. In addition, due to the intrinsic plasticity of many cellular types, every single cell is able, in response to the same stimulus, to rapidly switch phenotype supporting minimal changes of microenviromental cytokines concentration. Wound healing is a typical condition in which epithelial, endothelial as well as mesenchymal cells are firstly subjected to activation of their motility in order to repopulate the damaged region and then they show a strong proliferative response in order to successfully complete the wound repair process. This schema constitute the leitmotif of many other physiological or pathological conditions such as development vasculogenesis/angiogenesis as well as cancer outgrowth and metastasis.Our review focuses on the molecular mechanisms that control the starting and, eventually, the switching of cellular phenotypic outcome in response to changes in the symmetry of the extracellular environment.

Kinetic mechanism of the Zn-dependent aryl-phosphatase activity of myo-inositol-1-phosphatase.

Myo-inositol-1-phosphatase (EC 3.1.3.25) is able to hydrolyze myo-inositol-1-phosphate in the presence of Mg(2+) ions at neutral pH, and also p-nitrophenyl phosphate in the presence of Zn(2+)-ions at acidic pH. This enzyme plays a role in phosphatidylinositol cell signalling and is a putative target of lithium therapy in manic depression. We elucidate here the kinetic mechanism of the Zn-dependent activity of myo-inositol-1-phosphatase. As part of this analysis it was necessary to determine the basicity constants of p-nitrophenyl phosphate and the stability constant of its metal-complex in the presence of zinc chloride. We find that the Zn-dependent reaction may be described either by a rapid-equilibrium random mechanism or an ordered steady-state mechanism in which the substrate binds to the free enzyme prior to the metal ion. In both models the Zn-substrate complex acts as a high affinity inhibitor, yielding a dead-end species through its binding to the enzyme-Zn-substrate in rapid-equilibrium or to the enzyme-phosphate complexes in a steady-state model. Phosphate is a competitive inhibitor of the enzyme with respect to the substrate and an uncompetitive inhibitor with respect to zinc ions.

Gravitational stress on germinating Pinus pinea seeds.

In the germination of lipid-rich seeds, the glyoxylate cycle plays a control role in that, bypassing the two decarboxylative steps of the Krebs cycle; it allows the net synthesis of carbohydrates from lipids. The activity of isocitrate lyase, the key enzyme of the glyoxylate cycle, is an indicator of the state of seed germination: stage of germination, growth of embryo, activation and progress of protein synthesis, depletion of lipidic supplies. In order to investigate the effects of gravity on seed germination, we carried out a study on the time pattern of germination of Pinus pinea seeds that were subjected to a hypergravitational stress (1000 g for 64 h at 4 degrees C), either in a dry or in a wet environment, before to be placed in germination plates. During the whole time of germination, we monitored the state of embryo growth and the most representative enzymes of the main metabolic pathways. In treated wet seeds, we observed an average germination of only 20% with a slowdown of the enzyme activities assayed and a noticeable degradation of lipidic reserves with respect to the controls. These differences in germination are not found for dry seeds.

Enzyme catalysis in microgravity: steady-state kinetic analysis of the isocitrate lyase reaction.

Two decades of research in microgravity have shown that certain biochemical processes can be altered by weightlessness. Approximately 10 years ago, our team, supported by the European Space Agency (ESA) and the Agenzia Spaziale Italiana, started the Effect of Microgravity on Enzyme Catalysis project to test the possibility that the microgravity effect observed at cellular level could be mediated by enzyme reactions. An experiment to study the cleavage reaction catalyzed by isocitrate lyase was flown on the sounding rocket MASER 7, and we found that the kinetic parameters were not altered by microgravity. During the 28th ESA parabolic flight campaign, we had the opportunity to replicate the MASER 7 experiment and to perform a complete steady-state analysis of the isocitrate lyase reaction. This study showed that both in microgravity and in standard g controls the enzyme reaction obeyed the same kinetic mechanism and none of the kinetic parameters, nor the equilibrium constant of the overall reaction were altered. Our results contrast with those of a similar experiment, which was performed during the same parabolic flight campaign, and showed that microgravity increased the affinity of lipoxygenase-1 for linoleic acid. The hypotheses suggested to explain this change effect of the latter were here tested by computer simulation, and appeared to be inconsistent with the experimental outcome.