Na+/K+-ATPase: More than an Electrogenic Pump.

The sodium pump, or Na+/K+-ATPase (NKA), is an essential enzyme found in the plasma membrane of all animal cells. Its primary role is to transport sodium (Na+) and potassium (K+) ions across the cell membrane, using energy from ATP hydrolysis. This transport creates and maintains an electrochemical gradient, which is crucial for various cellular processes, including cell volume regulation, electrical excitability, and secondary active transport. Although the role of NKA as a pump was discovered and demonstrated several decades ago, it remains the subject of intense research. Current studies aim to delve deeper into several aspects of this molecular entity, such as describing its structure and mode of operation in atomic detail, understanding its molecular and functional diversity, and examining the consequences of its malfunction due to structural alterations. Additionally, researchers are investigating the effects of various substances that amplify or decrease its pumping activity. Beyond its role as a pump, growing evidence indicates that in various cell types, NKA also functions as a receptor for cardiac glycosides like ouabain. This receptor activity triggers the activation of various signaling pathways, producing significant morphological and physiological effects. In this report, we present the results of a comprehensive review of the most outstanding studies of the past five years. We highlight the progress made regarding this new concept of NKA and the various cardiac glycosides that influence it. Furthermore, we emphasize NKA's role in epithelial physiology, particularly its function as a receptor for cardiac glycosides that trigger intracellular signals regulating cell-cell contacts, proliferation, differentiation, and adhesion. We also analyze the role of NKA ß-subunits as cell adhesion molecules in glia and epithelial cells.

Ouabain Induces Transcript Changes and Activation of RhoA/ROCK Signaling in Cultured Epithelial Cells (MDCK).

Ouabain, an organic compound with the ability to strengthen the contraction of the heart muscle, was originally derived from plants. It has been observed that certain mammalian species, including humans, naturally produce ouabain, leading to its classification as a new type of hormone. When ouabain binds to Na+/K+-ATPase, it elicits various physiological effects, although these effects are not well characterized. Previous studies have demonstrated that ouabain, within the concentration range found naturally in the body (10 nmol/L), affects the polarity of epithelial cells and their intercellular contacts, such as tight junctions, adherens junctions, and gap junctional communication. This is achieved by activating signaling pathways involving cSrc and Erk1/2. To further investigate the effects of ouabain within the hormonally relevant concentration range (10 nmol/L), mRNA-seq, a high-throughput sequencing technique, was employed to identify differentially expressed transcripts. The discovery that the transcript encoding MYO9A was among the genes affected prompted an exploration of whether RhoA and its downstream effector ROCK were involved in the signaling pathways through which ouabain influences cell-to-cell contacts in epithelial cells. Supporting this hypothesis, this study reveals the following: (1) Ouabain increases the activation of RhoA. (2) Treatment with inhibitors of RhoA activation (Y27) and ROCK (C3) eliminates the enhancing effect of ouabain on the tight junction seal and intercellular communication via gap junctions. These findings further support the notion that ouabain acts as a hormone to emphasize the epithelial phenotype.

Ouabain's Influence on TRPV4 Channels of Epithelial Cells: An Exploration of TRPV4 Activity, Expression, and Signaling Pathways.

Ouabain, a substance originally obtained from plants, is now classified as a hormone because it is produced endogenously in certain animals, including humans. However, its precise effects on the body remain largely unknown. Previous studies have shown that ouabain can influence the phenotype of epithelial cells by affecting the expression of cell-cell molecular components and voltage-gated potassium channels. In this study, we conducted whole-cell clamp assays to determine whether ouabain affects the activity and/or expression of TRPV4 channels. Our findings indicate that ouabain has a statistically significant effect on the density of TRPV4 currents (dITRPV4), with an EC50 of 1.89 nM. Regarding treatment duration, dITRPV4 reaches its peak at around 1 h, followed by a subsequent decline and then a resurgence after 6 h, suggesting a short-term modulatory effect related to on TRPV4 channel activity and a long-term effect related to the promotion of synthesis of new TRPV4 channel units. The enhancement of dITRPV4 induced by ouabain was significantly lower in cells seeded at low density than in cells in a confluent monolayer, indicating that the action of ouabain depends on intercellular contacts. Furthermore, the fact that U73122 and neomycin suppress the effect caused by ouabain in the short term suggests that the short-term induced enhancement of dITRPV4 is due to the depletion of PIP2 stores. In contrast, the fact that the long-term effect is inhibited by PP2, wortmannin, PD, FR18, and IKK16 suggests that cSrc, PI3K, Erk1/2, and NF-kB are among the components included in the signaling pathways.

Trichoderma as a biological control agent: mechanisms of action, benefits for crops and development of formulations.

Currently, the food and economic losses generated by the attack of phytopathogens on the agricultural sector constitute a severe problem. Conventional crop protection techniques based on the application of synthetic pesticides to combat these undesirable microorganisms have also begun to represent an inconvenience since the excessive use of these substances is associated with contamination problems and severe damage to the health of farmers, consumers, and communities surrounding the fields, as well as the generation of resistance by the phytopathogens to be combated. Using biocontrol agents such as Trichoderma to mitigate the attack of phytopathogens represents an alternative to synthetic pesticides, safe for health and the environment. This work explains the mechanisms of action through which Trichoderma exerts biological control, some of the beneficial aspects that it confers to the development of crops through its symbiotic interaction with plants, and the bioremedial effects that it presents in fields contaminated by synthetic pesticides. Also, detail the production of spore-based biopesticides through fermentation processes and formulation development.

On-Demand Chemomagnetic Modulation of Striatal Neurons Facilitated by Hybrid Magnetic Nanoparticles.

Minimally invasive manipulation of cell signaling is critical in basic neuroscience research and in developing therapies for neurological disorders. Here, we describe a wireless chemomagnetic neuromodulation platform for the on-demand control of primary striatal neurons that relies on nanoscale heating events. Iron oxide magnetic nanoparticles (MNPs) are functionally coated with thermoresponsive poly (oligo (ethylene glycol) methyl ether methacrylate) (POEGMA) brushes loaded with dopamine. Dopamine loaded MNPs-POEGMA are co-cultured with primary striatal neurons. When alternating magnetinec fields (AMF) are applied, MNPs undergo hysteresis power loss and dissipate heat. The local heat produced by MNPs initiates a thermodynamic phase transition on POEGMA brushes resulting in polymer collapse and dopamine release. AMF-triggered dopamine release enhances the response of dopamine ion channels expressed on the cell membranes enhancing the activity of ~50% of striatal neurons subjected to the treatment. Chemomagnetic actuation on dopamine receptors is confirmed by blocking D1 and D2 receptors. The reversible thermodynamic phase transition of POEGMA brushes allow the on-demand release of dopamine in multiple microdoses. AMF-triggered dopamine release from MNPs-POEGMA causes no cell cytotoxicity nor promotes cell ROS production. This research represents a fundamental step forward for the chemomagnetic control of neural activity using hybrid magnetic nanomaterials with tailored physical properties.

Ouabain-Induced Changes in the Expression of Voltage-Gated Potassium Channels in Epithelial Cells Depend on Cell-Cell Contacts.

Ouabain is a cardiac glycoside, initially isolated from plants, and currently thought to be a hormone since some mammals synthesize it endogenously. It has been shown that in epithelial cells, it induces changes in properties and components related to apical-basolateral polarity and cell-cell contacts. In this work, we used a whole-cell patch clamp to test whether ouabain affects the properties of the voltage-gated potassium currents (Ik) of epithelial cells (MDCK). We found that: (1) in cells arranged as mature monolayers, ouabain induced changes in the properties of Ik; (2) it also accelerated the recovery of Ik in cells previously trypsinized and re-seeded at confluence; (3) in cell-cell contact-lacking cells, ouabain did not produce a significant change; (4) Na+/K+ ATPase might be the receptor that mediates the effect of ouabain on Ik; (5) the ouabain-induced changes in Ik required the synthesis of new nucleotides and proteins, as well as Golgi processing and exocytosis, as evidenced by treatment with drugs inhibiting those processes; and (5) the signaling cascade included the participation of cSrC, PI3K, Erk1/2, NF-κB and ß-catenin. These results reveal a new role for ouabain as a modulator of the expression of voltage-gated potassium channels, which require cells to be in contact with themselves.

Ouabain Enhances Gap Junctional Intercellular Communication by Inducing Paracrine Secretion of Prostaglandin E2.

Ouabain is a cardiac glycoside that has been described as a hormone, with interesting effects on epithelial physiology. We have shown previously that ouabain induces gap junctional intercellular communication (GJIC) in wild, sensitive cells (MDCK-S), but not in cells that have become insensitive (MDCK-I) by modifying their Na+-K+-ATPase. We have also demonstrated that prostaglandin E2 (PGE2) is able to induce increased GJIC by a mechanism other than ouabain, that does not depend on Na+-K+-ATPase. In this work we show, by dye transfer assays, that when MDCK-S and MDCK-I are randomly mixed, to form monolayers, the latter stablish GJIC, because of stimulation by a compound released to the extracellular media, by MDCK-S cells, after treatment with ouabain, as evidenced by the fact that monolayers of only MDCK-I cells, treated with a conditioned medium (CM) that is obtained after incubation of MDCK-S monolayers with ouabain, significantly increase their GJIC. The further finding that either (1) pre-treatment with COX-2 inhibitors or (2) addition to CM of antagonists of EP2 receptor abolish CM's ability to induce GJIC in MDCK-I monolayers indicate that PGE2 is the GJIC-inducing compound. Therefore, these results indicate that, in addition to direct stimulation, mediated by Na+-K+-ATPase, ouabain enhances GJIC indirectly through the paracrine production of PGE2.

Prostaglandin E2 Enhances Gap Junctional Intercellular Communication in Clonal Epithelial Cells.

Prostaglandins are a group of lipids that produce diverse physiological and pathological effects. Among them, prostaglandin E2 (PGE2) stands out for the wide variety of functions in which it participates. To date, there is little information about the influence of PGE2 on gap junctional intercellular communication (GJIC) in any type of tissue, including epithelia. In this work, we set out to determine whether PGE2 influences GJIC in epithelial cells (MDCK cells). To this end, we performed dye (Lucifer yellow) transfer assays to compare GJIC of MDCK cells treated with PGE2 and untreated cells. Our results indicated that (1) PGE2 induces a statistically significant increase in GJIC from 100 nM and from 15 min after its addition to the medium, (2) such effect does not require the synthesis of new mRNA or proteins subunits but rather trafficking of subunits already synthesized, and (3) such effect is mediated by the E2 receptor, which, in turn, triggers a signaling pathway that includes activation of adenylyl cyclase and protein kinase A (PKA). These results widen the knowledge regarding modulation of gap junctional intercellular communication by prostaglandins.

Fabrication of Microbolometer Arrays Based on Polymorphous Silicon-Germanium.

This work reports the development of arrays of infrared sensors (microbolometers) using a hydrogenated polymorphous silicon-germanium alloy (pm-SixGe1-x:H). Basically, polymorphous semiconductors consist of an amorphous semiconductor matrix with embedded nanocrystals of about 2-3 nm. The pm-SixGe1-x:H alloy studied has a high temperature coefficient of resistance (TCR) of 4.08%/K and conductivity of 1.5 × 10-5 S∙cm-1. Deposition of thermosensing film was made by plasma-enhanced chemical vapor deposition (PECVD) at 200 °C, while the area of the devices is 50 × 50 µm2 with a fill factor of 81%. Finally, an array of 19 × 20 microbolometers was packaged for electrical characterization. Voltage responsivity values were obtained in the range of 4 × 104 V/W and detectivity around 2 × 107 cm∙Hz1/2/W with a polarization current of 70 µA at a chopper frequency of 30 Hz. A minimum value of 2 × 10-10 W/Hz1/2 noise equivalent power was obtained at room temperature. In addition, it was found that all the tested devices responded to incident infrared radiation, proving that the structure and mechanical stability are excellent.

Ouabain Promotes Gap Junctional Intercellular Communication in Cancer Cells.

Gap junctions are molecular structures that allow communication between neighboring cells. It has been shown that gap junctional intercellular communication (GJIC) is notoriously reduced in cancer cells compared to their normal counterparts. Ouabain, a plant derived substance, widely known for its therapeutic properties on the heart, has been shown to play a role in several types of cancer, although its mechanism of action is not yet fully understood. Since we have previously shown that ouabain enhances GJIC in epithelial cells (MDCK), here we probed whether ouabain affects GJIC in a variety of cancer cell lines, including cervico-uterine (CasKi, SiHa and Hela), breast (MDA-MB-321 and MCF7), lung (A549), colon (SW480) and pancreas (HPAF-II). For this purpose, we conducted dye transfer assays to measure and compare GJIC in monolayers of cells with and without treatment with ouabain (0.1, 1, 10, 50 and 500 nM). We found that ouabain induces a statistically significant enhancement of GJIC in all of these cancer cell lines, albeit with distinct sensitivity. Additionally, we show that synthesis of new nucleotides or protein subunits is not required, and that Csrc, ErK1/2 and ROCK-Rho mediate the signaling mechanisms. These results may contribute to explaining how ouabain influences cancer.

Ouabain Modulates the Adherens Junction in Renal Epithelial Cells.

BACKGROUND/AIMS: Ouabain, a well-known plant-derived toxin, is also a hormone found in mammals at nanomolar levels that binds to a site located in the a-subunit of Na⁺,K⁺-ATPase. Our main goal was to understand the physiological roles of ouabain. Previously, we found that ouabain increases the degree of tight junction sealing, GAP junction-mediated communication and ciliogenesis. Considering our previous results, we investigated the effect of ouabain on adherens junctions. METHODS: We used immunofluorescence and immunoblot methods to measure the effect of 10 nM ouabain on the cellular and nuclear content of E-cadherin, ß-catenin and γ-catenin in cultured monolayers of Marin Darby canine renal cells (MDCK). We also studied the effect of ouabain on adherens junction biogenesis through sequential Ca²âº removal and replenishment. Then, we investigated whether c-Src and ERK1/2 kinases are involved in these responses. RESULTS: Ouabain enhanced the cellular content of the adherens junction proteins E-cadherin, ß-catenin and γ-catenin and displaced ß-catenin and γ-catenin from the plasma membrane into the nucleus. Ouabain also increased the expression levels of E-cadherin and ß-catenin in the plasma membrane after Ca²âº replenishment. These effects on adherens junctions were sensitive to PP2 and PD98059, suggesting that they depend on c-Src and ERK1/2 signaling. The translocation of ß-catenin and γ-catenin into the nucleus was specific because ouabain did not change the localization of the tight junction proteins ZO-1 and ZO-2. Moreover, in ouabain-resistant MDCK cells, which express a Na⁺,K⁺-ATPase α1-subunit with low affinity for ouabain, this hormone was unable to regulate adherens junctions, indicating that the ouabain receptor that regulates adherens junctions is Na⁺,K⁺-ATPase. CONCLUSION: Ouabain (10 nM) upregulated adherens junctions. This novel result supports the proposition that one of the physiological roles of this hormone is the modulation of cell contacts.

Toward Smaller Aqueous-Phase Plasmonic Gold Nanoparticles: High-Stability Thiolate-Protected ∼4.5 nm Cores.

Most applications of aqueous plasmonic gold nanoparticles benefit from control of the core size and shape, control of the nature of the ligand shell, and a simple and widely applicable preparation method. Surface functionalization of such nanoparticles is readily achievable but is restricted to water-soluble ligands. Here we have obtained highly monodisperse and stable smaller aqueous gold nanoparticles (core diameter ∼4.5 nm), prepared from citrate-tannate precursors via ligand exchange with each of three distinct thiolates: 11-mercaptoundecanoic acid, α-R-lipoic acid, and para-mercaptobenzoic acid. These are characterized by UV-vis spectroscopy for plasmonic properties; Fourier transform infrared (FTIR) spectroscopy for ligand-exchange confirmation; X-ray diffractometry for structural analysis; and high-resolution transmission electron microscopy for structure and size determination. Chemical reduction induces a blueshift, maximally +0.02 eV, in the localized surface plasmon resonance band; this is interpreted as an electronic (-) charging of the monolayer-protected cluster (MPC) gold core, corresponding to a -0.5 V change in electrochemical potential.

Single nanowire measurements of room temperature ferromagnetism in FeSi nanowires and the effects of Mn-doping.

Semiconductors with magnetic response at room temperature are sought for spintronics in solid-state devices. Among possible materials for this applications, the magnetic response of FeSi and doped FeSi have produced contradictory results at the nanoscale and more precise measurements and deeper studies are needed to clarify its potential capabilities. For that reason, in this work, single nanowire measurements of ferromagnetic semiconducting FeSi and Mn-doped FeSi nanostructures have been performed using magnetic force microscopy and electron holography. Results obtained confirm the presence of magnetic domains at room temperature with a magnetic moment per Fe atom of [Formula: see text] Spin polarized density functional calculations confirm a net magnetic moment between [Formula: see text] in Fe surface atoms with an estimated Curie temperature of 417 K by means of the molecular field approximation. The nanowires present a crystalline B20 cubic structure as confirmed by x-ray diffraction and high-resolution electron microscopy. Their electrical transport measurements confirm p-type nature and thermal activation. A remanent magnetization of 1.5 × 10-5 emu and 0.5 × 10-5 emu was measured at room temperature for FeSi and Mn-doped FeSi respectively, with spin freezing behavior around 30 K for the Mn-doped nanowires.

Ouabain Modulates the Distribution of Connexin 43 in Epithelial Cells.

BACKGROUND/AIMS: The fact that ouabain has been identified as an endogenous substance, led us to inquire its physiological role in epithelial cells. Based on previous observations, we hypothesized that it influences processes related to cell contacts. Previously we have shown that nanomolar concentrations of ouabain up-regulate tight junctions, accelerate ciliogenesis, and increase gap junctional intercellular communication (GJIC). Given that silencing assays indicated that connexin 43 (Cnx43) is involved in the GJIC response, in the present work we study whether ouabain affects Cnx43 expression and distribution. METHODS: We seeded confluent monolayers of epithelial renal MDCK cells and incubated them with 10 nM ouabain during 1 h. Then we measured, by densitometric analysis of Western blot assays, the amount of Cnx43 in cells and in fractions enriched of plasma membrane. We also studied its localization with immunofluorescence and confocal microscopy. RESULTS: Cnx43 is remarkably displayed, outlining the borders of cells gathered in clusters, randomly scattered throughout the monolayer. Ouabain increases the density of such clusters, as well as the average number of cells per cluster, without inducing the synthesis of new Cnx43. It also promotes relocation towards the membrane, of subunits already available. The fact that such changes are inhibited by PP2 and PD98059 indicates that a signaling pathway, that includes c-Src and ERK1/2, is involved in this response. CONCLUSION: Ouabain induces the translocation of Cnx43 from the cytoplasm to the plasma membrane. These findings support our hypothesis that one of the physiological roles of ouabain is the modulation of physiological processes that depend on cell to cell contacts.

High-Concentration Aqueous Dispersions of Nanoscale 2D Materials Using Nonionic, Biocompatible Block Copolymers.

Conditions for the dispersion of molybdenum disulfide (MoS2) in aqueous solution at concentrations up to 0.12 mg mL(-1) using a range of nonionic, biocompatible block copolymers (i.e., Pluronics and Tetronics) are identified. Furthermore, the optimal Pluronic dispersant for MoS2 is found to be effective for a range of other 2D materials such as molybdenum diselenide, tungsten diselenide, tungsten disulfide, tin selenide, and boron nitride.

Resonance properties of Ag-ZnO nanostructures at terahertz frequencies.

Nanoantennas have been fabricated by scaling down traditional antenna designs using nanolithographic techniques and testing them at different optical wavelengths, these particular nanoantennas have shown responses in a broad range of frequencies going from visible wavelengths to the range of the terahertz. Some self-assembled nanostructures exist that exhibit similar shapes and properties to those of traditional antenna structures. In this work the emission and absorption properties of self-assembled nanostructures made of zinc oxide nanorods on silver nanowires, which resemble traditional dipole antennas, were measured and simulated in order to test their antenna performance. These structures show resonant properties in the 10-120 THz range, with the main resonance at 60 THz. The radiation pattern of these nanostructures was also obtained by numerical simulations, and it is shown that it can be tailored to increase or decrease its directivity as a function of the location of the energy source of excitation. Experimental measurements were performed by Raman spectroscopy and Fourier Transform Infrared Spectroscopy (FTIR) in order to show existing vibrational frequencies at the resonant frequencies of the nanostructures, measurements were made from ~9 to 103 THz and the results were in agreement with the simulations. These characteristics make these metal-semiconductor Ag/ZnO nanostructures useful as self-assembled nanoantennas in applications such as terahertz spectroscopy and sensing at terahertz frequencies.

Ouabain increases gap junctional communication in epithelial cells.

BACKGROUND/AIMS: The finding that endogenous ouabain acts as a hormone prompted efforts to elucidate its physiological function. In previous studies, we have shown that 10 nM ouabain (i.e., a concentration within the physiological range) modulates cell-cell contacts such as tight junctions and apical/basolateral polarity. In this study, we examined whether 10 nM ouabain affects another important cell-cell feature: gap junction communication (GJC). METHODS: We employed two different approaches: 1) analysis of the cell-to-cell diffusion of neurobiotin injected into a particular MDCK cell (epithelial cells from dog kidneys) in a confluent monolayer by counting the number of neighboring cells reached by the probe and 2) measurement of the electrical capacitance. RESULTS: We found that 10 nM ouabain increase GJC by 475% within 1 hour. The Na+-K+-ATPase acts as a receptor of ouabain. In previous works we have shown that ouabain activates c-Src and ERK1/2 in 1 hour; in the present study we show that the inhibition of these proteins block the effect of ouabain on GJC. This increase in GJC does not require synthesis of new protein components, because the inhibitors cycloheximide and actinomycin D did not affect this phenomenon. Using silencing assays we also demonstrate that this ouabain-induced enhancement of GJC involves connexins 32 and 43. CONCLUSION: Ouabain 10 nM increases GJC in MDCK cells.

Structural order in ultrathin films of the monolayer protected clusters based upon 4 nm gold nanocrystals: an experimental and theoretical study.

The structural order in ultrathin films of monolayer protected clusters (MPCs) is important in a number of application areas but can be difficult to demonstrate by conventional methods, particularly when the metallic core dimension, d, is in the intermediate size-range, 1.5 < d < 5.0 nm. Here, improved techniques for the synthesis of monodisperse thiolate-protected gold nanoparticles have made possible the production of dodecane-thiolate saturated ∼4 ± 0.5 nm Au clusters with single-crystal core structure and morphology. An ultrathin ordered film or superlattice of these nanocrystal-core MPCs is prepared and investigated using aberration corrected scanning/transmission electron microscopy (STEM) which allowed imaging of long-range hexagonally ordered superlattices of the nanocrystals, separated by the thiolate groups. The lattice constants determined by direct imaging are in good agreement with those determined by small-angle electron diffraction. The STEM image revealed the characteristic grain boundary (GB) with sigma (Σ) 13 in the interface between two crystals. The formation and structures found are interpreted on the basis of theoretical calculations employing molecular dynamics (MD) simulations and coarse-grained (CG) approach.

New insights into the properties and interactions of carbon chains as revealed by HRTEM and DFT analysis.

Atomic carbon chains have raised interest for their possible applications as graphene interconnectors as the thinnest nanowires; however, they are hard to synthesize and subsequently to study. We present here a reproducible method to synthesize carbon chains in situ TEM. Moreover, we present a direct observation of the bond length alternation in a pure carbon chain by aberration corrected TEM. Also, cross bonding between two carbon chains, 5 nm long, is observed experimentally and confirmed by DFT calculations. Finally, while free standing carbon chains were observed to be straight due to tensile loading, a carbon chain inside the walls of a carbon nanotube showed high flexibility.

High precision orientation mapping from 4D-STEM precession electron diffraction data through quantitative analysis of diffracted intensities.

The association of scanning transmission electron microscopy (STEM) and detection of a diffraction pattern at each probe position (so-called 4D-STEM) represents one of the most promising approaches to analyze structural properties of materials with nanometric resolution and low irradiation levels. This is widely used for texture analysis of materials using automated crystal orientation mapping (ACOM). Herein, we perform orientation mapping in InP nanowires exploiting precession electron diffraction (PED) patterns acquired by an axial CMOS camera. Crystal orientation is determined at each probe position by the quantitative analysis of diffracted intensities minimizing a residue comparing experiments and simulations in analogy to x-ray structural refinement. Our simulations are based on the two-beam dynamical diffraction approximation and yield a high angular precision (∼0.03°), much lower than the traditional ACOM based on pattern matching algorithms (∼1°). We anticipate that simultaneous exploration of both spot positions and high precision crystal misorientation will allow the exploration of the whole potentiality provided by PED-based 4D-STEM for the characterization of deformation fields in nanomaterials.