Plausible Emergence of Autocatalytic Cycles under Prebiotic Conditions

The emergence of life in a prebiotic world is an enormous scientific question of paramount philosophical importance. Even when life (in any sense we can define it) can be observed and replicated in the laboratory, it is only an indication of one possible pathway for life emergence, and is by no means be a demonstration of how life really emerged. The best we can hope for is to indicate plausible chemical–physical conditions and mechanisms that might lead to self-organizing and autopoietic systems. Here we present a stochastic simulation, based on chemical reactions already observed in prebiotic environments, that might help in the design of new experiments. We will show how the definition of simple rules for the synthesis of random peptides may lead to the appearance of networks of autocatalytic cycles and the emergence of memory

A Flavone-Based Solvatochromic Probe with A Low Expected Perturbation Impact on the Membrane Physical State

The study of the cell membrane is an ambitious and arduous objective since its physical state is regulated by a series of processes that guarantee its regular functionality. Among the different methods of analysis, fluorescence spectroscopy is a technique of election, non-invasive, and easy to use. Besides, molecular dynamics analysis (MD) on model membranes provides useful information on the possibility of using a new probe, following its positioning in the membrane, and evaluating the possible perturbation of the double layer. In this work, we report the rational design and the synthesis of a new fluorescent solvatochromic probe and its characterization in model membranes. The probe consists of a fluorescent aromatic nucleus of a 3-hydroxyflavone moiety, provided with a saturated chain of 18 carbon atoms and a zwitterionic head so to facilitate the anchoring to the polar heads of the lipid bilayer and avoid the complete internalization. It was possible to study the behavior of the probe in GUV model membranes by MD analysis and fluorescence microscopy, demonstrating that the new probe can efficiently be incorporated in the lipid bilayer, and give a color response, thanks to is solvatochromic properties. Moreover, MD simulation of the probe in the membrane supports the hypothesis of a reduced perturbation of the membrane physical state

Multifaceted Analyses of Isolated Mitochondria Establish the Anticancer Drug 2-Hydroxyoleic Acid as an Inhibitor of Substrate Oxidation and an Activator of Complex IV-Dependent State 3 Respiration

The synthetic fatty acid 2-hydroxyoleic acid (2OHOA) has been extensively investigated as a cancer therapy mainly based on its regulation of membrane lipid composition and structure, activating various cell fate pathways. We discovered, additionally, that 2OHOA can uncouple oxi-dative phosphorylation, but this has never been demonstrated mechanistically. Here, we explored the effect of 2OHOA on mitochondria isolated by ultracentrifugation from U118MG glioblastoma cells. Mitochondria were analyzed by shotgun lipidomics, molecular dynamic simulations, spectrophotometric assays for determining respiratory complex activity, mass spectrometry for assessing beta oxidation and Seahorse technology for bioenergetic profiling. We showed that the main impact of 2OHOA on mitochondrial lipids is their hydroxylation, demonstrated by simulations to decrease co-enzyme Q diffusion in the liquid disordered membranes embedding respiratory complexes. This de-creased co-enzyme Q diffusion can explain the inhibition of disjointly measured complexes I-III activ-ity. However, it doesn’t explain how 2OHOA increases complex IV and state 3 respiration in intact mitochondria. This increased respiration probably allows mitochondrial oxidative phosphorylation to maintain ATP production against the 2OHOA-mediated inhibition of glycolytic ATP production. This work correlates 2OHOA function with its modulation of mitochondrial lipid composition, reflecting both 2OHOA anticancer activity and adaptation to it by enhancement of state 3 respiration. © 2022 by the authors. Li-censee MDPI, Basel, Switzerland

Characterization of Silter Cheeses Produced in Valley and Alpine Pastures by a Proteomic Approach.

Silter is an Italian hard cheese manufactured with milk produced by cows fed at different altitude, valley or alpine pasture. The chemical, rheological and sensory properties of cheeses can be affected by the modification in milk composition due to the breed which at different altitude causes the modification of protein content, κ-CN glycosylation, plasmin activity, and coagulation properties. The influence of milk plasmin activity on dairy production was investigated in seven Silter cheeses, four produced in the valley and three from alpine mountain, through alkaline urea-polyacrylamide gel electrophoresis; two-dimensional gel electrophoresis coupled to mass spectrometry and image analysis. Results demonstrated that Silter cheese obtained from cows reared in alpine pasture is characterized by a more evident proteolysis, determining high levels of β-CN and αs1-CN fragments. Therefore, the most relevant fragmentation was attributed to a more intense activity of plasmin and to a different dosage of rennet to make up for the reduced coagulation properties of alpine milk

Protein analysis by two-dimensional LC using size-exclusion and reverse phase chromatography with column switching

Over the years, two-dimensional Liquid Chromatography (2D-LC) has played a key role in the field of proteomics for the analysis and isolation of proteins from complex matrices, before their characterization by Mass Spectrometry1,2. In 2D-LC method development, a large number of experimental variables have to be extensively studied to take full advantage of the claimed features of the chromatographic separations3. In the last decade, significant improvements in LC column technology and instrumentation have been made and the introduction of superficially porous particles opened new possibilities in terms of speed and resolution. Here an automated method combining size exclusion with reverse phase chromatography and UV detection is proposed for protein separations in food samples. For the first dimension separation, a size-exclusion column, packed with 3 μm ultra-pure silica particles densely bonded with a proprietary hydrophilic surface chemistry, was used, ensuring high efficiency and resolution. A reversed phase widepore column was used for the second dimension separation, based on core-shell particle technology that provides striking increases in peak capacity and resolution at lower backpressures. Different collection trap systems were placed on a 10-port switching valve and tested to focus the proteins coming from the first dimension column, before their selective elution in the second dimension column. A protein standard mixture of bovine serum albumin, -lactoglobulin and glucose oxidase was used to optimize the gradient chromatographic separation conditions, after evaluating their retention behavior in each single column. Finally, the optimized 2D-LC method was applied to the protein analysis in eggs, infant milk powder and soy flour samples

Multifaceted Analyses of Isolated Mitochondria Establish the Anticancer Drug 2-Hydroxyoleic Acid as an Inhibitor of Substrate Oxidation and an Activator of Complex IV-Dependent State 3 Respiration

The synthetic fatty acid 2-hydroxyoleic acid (2OHOA) has been extensively investigated as a cancer therapy mainly based on its regulation of membrane lipid composition and structure, activating various cell fate pathways. We discovered, additionally, that 2OHOA can uncouple oxidative phosphorylation, but this has never been demonstrated mechanistically. Here, we explored the effect of 2OHOA on mitochondria isolated by ultracentrifugation from U118MG glioblastoma cells. Mitochondria were analyzed by shotgun lipidomics, molecular dynamic simulations, spectrophotometric assays for determining respiratory complex activity, mass spectrometry for assessing beta oxidation and Seahorse technology for bioenergetic profiling. We showed that the main impact of 2OHOA on mitochondrial lipids is their hydroxylation, demonstrated by simulations to decrease co-enzyme Q diffusion in the liquid disordered membranes embedding respiratory complexes. This decreased co-enzyme Q diffusion can explain the inhibition of disjointly measured complexes I–III activity. However, it doesn’t explain how 2OHOA increases complex IV and state 3 respiration in intact mitochondria. This increased respiration probably allows mitochondrial oxidative phosphorylation to maintain ATP production against the 2OHOA-mediated inhibition of glycolytic ATP production. This work correlates 2OHOA function with its modulation of mitochondrial lipid composition, reflecting both 2OHOA anticancer activity and adaptation to it by enhancement of state 3 respiration