Effect of In Vitro Micropropagation on the Chemical, Antioxidant, and Biological Characteristics of Senecio nutans Sch. Bip., an Endemic Plant of the Atacama Desert Andean Region.

The species Senecio nutans Sch. Bip., commonly called "chachacoma", is widely used as a medicinal plant by the Andean communities of Northern Chile. Ethanolic extracts of S. nutans and the main compound, 4-hydroxy-3-(3-methyl-2-butenyl) acetophenone, have shown interesting biological activity. However, due to the high-altitude areas where this species is found, access to S. nutans is very limited. Due to the latter, in this work, we carried out micropropagation in vitro and ex vitro adaptation techniques as an alternative for the massive multiplication, conservation, and in vitro production of high-value metabolites from this plant. The micropropagation and ex vitro adaptation techniques were successfully employed, and UHPLC-DAD analysis revealed no significant changes in the phenolic profile, with acetophenone 4 being the most abundant metabolite, whose antioxidant and antibacterial activity was studied. Independently of the applied culture condition, the ethanolic extracts of S. nutans presented high activity against both Gram-positive and Gram-negative bacteria, demonstrating their antimicrobial capacity. This successful initiation of in vitro and ex vitro cultures provides a biotechnological approach for the conservation of S. nutans and ensures a reliable and consistent source of acetophenone 4 as a potential raw material for pharmacological applications.

Size Matters: Free-Energy Calculations of Amino Acid Adsorption over Pristine Graphene.

There is still growing interest in graphene interactions with proteins, both for its possible biological applications and due to concerns over detrimental effects at the cellular level. As with any process involving proteins, an understanding of amino acid composition is desirable. In this work, we systematically studied the adsorption process of amino acids onto pristine graphene via rigorous free-energy calculations. We characterized the free energy, potential energy, and entropy of the adsorption of all proteinogenic amino acids. The energetic components were further separated into pair interaction contributions. A linear correlation was found between the free energy and the solvent accessible surface area change during adsorption (ΔSASAads) over pristine graphene and uncharged amino acids. Free energies over pristine graphene were compared with adsorption onto graphene oxide, finding an almost complete loss of the favorability of amino acid adsorption onto graphene. Finally, the correlation with ΔSASAads was used to successfully predict the free energy of adsorption of several penta-l-peptides in different structural states and sequences. Due to the relative ease of calculating the ΔSASAads compared to free-energy calculations, it could prove to be a cost-effective predictor of the free energy of adsorption for proteins onto nonpolar surfaces.

Protein-Mediated Electroporation in a Cardiac Voltage-Sensing Domain Due to an nsPEF Stimulus.

This study takes a step in understanding the physiological implications of the nanosecond pulsed electric field (nsPEF) by integrating molecular dynamics simulations and machine learning techniques. nsPEF, a state-of-the-art technology, uses high-voltage electric field pulses with a nanosecond duration to modulate cellular activity. This investigation reveals a relatively new and underexplored phenomenon: protein-mediated electroporation. Our research focused on the voltage-sensing domain (VSD) of the NaV1.5 sodium cardiac channel in response to nsPEF stimulation. We scrutinized the VSD structures that form pores and thereby contribute to the physical chemistry that governs the defibrillation effect of nsPEF. To do so, we conducted a comprehensive analysis involving the clustering of 142 replicas simulated for 50 ns under nsPEF stimuli. We subsequently pinpointed the representative structures of each cluster and computed the free energy between them. We find that the selected VSD of NaV1.5 forms pores under nsPEF stimulation, but in a way that significant differs from the traditional VSD opening. This study not only extends our understanding of nsPEF and its interaction with protein channels but also adds a new effect to further study.

Nanosecond Pulsed Electric Field (nsPEF): Opening the Biotechnological Pandora's Box.

Nanosecond Pulsed Electric Field (nsPEF) is an electrostimulation technique first developed in 1995; nsPEF requires the delivery of a series of pulses of high electric fields in the order of nanoseconds into biological tissues or cells. They primary effects in cells is the formation of membrane nanopores and the activation of ionic channels, leading to an incremental increase in cytoplasmic Ca2+ concentration, which triggers a signaling cascade producing a variety of effects: from apoptosis up to cell differentiation and proliferation. Further, nsPEF may affect organelles, making nsPEF a unique tool to manipulate and study cells. This technique is exploited in a broad spectrum of applications, such as: sterilization in the food industry, seed germination, anti-parasitic effects, wound healing, increased immune response, activation of neurons and myocites, cell proliferation, cellular phenotype manipulation, modulation of gene expression, and as a novel cancer treatment. This review thoroughly explores both nsPEF's history and applications, with emphasis on the cellular effects from a biophysics perspective, highlighting the role of ionic channels as a mechanistic driver of the increase in cytoplasmic Ca2+ concentration.

Exploring the Conformational Changes Induced by Nanosecond Pulsed Electric Fields on the Voltage Sensing Domain of a Ca2+ Channel.

Nanosecond Pulsed Electric Field (nsPEF or Nano Pulsed Stimulation, NPS) is a technology that delivers a series of pulses of high-voltage electric fields during a short period of time, in the order of nanoseconds. The main consequence of nsPEF upon cells is the formation of nanopores, which is followed by the gating of ionic channels. Literature is conclusive in that the physiological mechanisms governing ion channel gating occur in the order of milliseconds. Hence, understanding how these channels can be activated by a nsPEF would be an important step in order to conciliate fundamental biophysical knowledge with improved nsPEF applications. To get insights on both the kinetics and thermodynamics of ion channel gating induced by nsPEF, in this work, we simulated the Voltage Sensing Domain (VSD) of a voltage-gated Ca2+ channel, inserted in phospholipidic membranes with different concentrations of cholesterol. We studied the conformational changes of the VSD under a nsPEF mimicked by the application of a continuous electric field lasting 50 ns with different intensities as an approach to reveal novel mechanisms leading to ion channel gating in such short timescales. Our results show that using a membrane with high cholesterol content, under an nsPEF of 50 ns and E→ = 0.2 V/nm, the VSD undergoes major conformational changes. As a whole, our work supports the notion that membrane composition may act as an allosteric regulator, specifically cholesterol content, which is fundamental for the response of the VSD to an external electric field. Moreover, changes on the VSD structure suggest that the gating of voltage-gated Ca2+ channels by a nsPEF may be due to major conformational changes elicited in response to the external electric field. Finally, the VSD/cholesterol-bilayer under an nsPEF of 50 ns and E→ = 0.2 V/nm elicits a pore formation across the VSD suggesting a new non-reported effect of nsPEF into cells, which can be called a "protein mediated electroporation".

l-DOPA modulates the kinetics but not the thermodynamic equilibrium of TTA+ amphiphiles forming lyotropic nematic liquid crystals.

Lyotropic liquid crystals (LLCs) are mixtures of amphiphile molecules usually studied as mimetic of biological membrane. The equilibrium dynamics of tetradecyltrimethyl ammonium cation (TTA+) molecules forming nematic LLCs (LNLCs) is guided by a dive-in mechanism where TTA+ molecules spontaneously leave and re-enter the bicelle. Of note, this dynamic behavior could be exploited to produce drug nano-delivery systems based on LNLCs. Therefore, the understanding of the effect of pharmaceutically interesting molecules in the dynamics of the dive-in mechanism should be crucial for drug delivery applications. In this work, we studied the effects of l-DOPA in the equilibrium dynamics of TTA+ bicelles forming LNLCs, employing a transdisciplinary approach based on 2H-NMR together with molecular modeling and molecular dynamics simulations. Our data suggest that l-DOPA perturbs the kinetic of the dive-in mechanism but not the thermodynamics of this process. As whole, our results provide fundamental insights on the mechanisms by which l-DOPA govern the equilibrium of LNLCs bicelles.