Electrogenetics: Bridging synthetic biology and electronics to remotely control the behavior of mammalian designer cells.

Electrogenetics, the combination of electronics and genetics, is an emerging field of mammalian synthetic biology in which electrostimulation is used to remotely program user-designed genetic elements within designer cells to generate desired outputs. Here, we describe recent advances in electro-induced therapeutic gene expression and therapeutic protein secretion in engineered mammalian cells. We also review available tools and strategies to engineer electro-sensitive therapeutic designer cells that are able to sense electrical pulses and produce appropriate clinically relevant outputs in response. We highlight current limitations facing mammalian electrogenetics and suggest potential future directions for research.

Role of Herbal Teas in Regulating Cellular Homeostasis and Autophagy and Their Implications in Regulating Overall Health.

Tea is one of the most popular and widely consumed beverages worldwide, and possesses numerous potential health benefits. Herbal teas are well-known to contain an abundance of polyphenol antioxidants and other ingredients, thereby implicating protection and treatment against various ailments, and maintaining overall health in humans, although their mechanisms of action have not yet been fully identified. Autophagy is a conserved mechanism present in organisms that maintains basal cellular homeostasis and is essential in mediating the pathogenesis of several diseases, including cancer, type II diabetes, obesity, and Alzheimer's disease. The increasing prevalence of these diseases, which could be attributed to the imbalance in the level of autophagy, presents a considerable challenge in the healthcare industry. Natural medicine stands as an effective, safe, and economical alternative in balancing autophagy and maintaining homeostasis. Tea is a part of the diet for many people, and it could mediate autophagy as well. Here, we aim to provide an updated overview of popular herbal teas' health-promoting and disease healing properties and in-depth information on their relation to autophagy and its related signaling molecules. The present review sheds more light on the significance of herbal teas in regulating autophagy, thereby improving overall health.

Applications of Solution NMR in Drug Discovery.

During the past decades, solution nuclear magnetic resonance (NMR) spectroscopy has demonstrated itself as a promising tool in drug discovery. Especially, fragment-based drug discovery (FBDD) has benefited a lot from the NMR development. Multiple candidate compounds and FDA-approved drugs derived from FBDD have been developed with the assistance of NMR techniques. NMR has broad applications in different stages of the FBDD process, which includes fragment library construction, hit generation and validation, hit-to-lead optimization and working mechanism elucidation, etc. In this manuscript, we reviewed the current progresses of NMR applications in fragment-based drug discovery, which were illustrated by multiple reported cases. Moreover, the NMR applications in protein-protein interaction (PPI) modulators development and the progress of in-cell NMR for drug discovery were also briefly summarized.

The yin and yang of mitochondrial Ca2+ signaling in cell physiology and pathology.

Mitochondria are autonomous and dynamic cellular organelles orchestrating a diverse range of cellular activities. Numerous cell-signaling pathways target these organelles and Ca2+ is one of the most significant. Mitochondria are able to rapidly and transiently take up Ca2+, thanks to the mitochondrial Ca2+ uniporter complex, as well as to extrude it through the Na+/Ca2+ and H+/Ca2+ exchangers. The transient accumulation of Ca2+ in the mitochondrial matrix impacts on mitochondrial functions and cell pathophysiology. Here we summarize the role of mitochondrial Ca2+ signaling in both physiological (yang) and pathological (yin) processes and the methods that can be used to investigate mitochondrial Ca2+ homeostasis. As an example of the pivotal role of mitochondria in pathology, we described the state of the art of mitochondrial Ca2+ alterations in different pathological conditions, with a special focus on Alzheimer's disease.

AMPK and TOR: The Yin and Yang of Cellular Nutrient Sensing and Growth Control.

The AMPK (AMP-activated protein kinase) and TOR (target-of-rapamycin) pathways are interlinked, opposing signaling pathways involved in sensing availability of nutrients and energy and regulation of cell growth. AMPK (Yin, or the "dark side") is switched on by lack of energy or nutrients and inhibits cell growth, while TOR (Yang, or the "bright side") is switched on by nutrient availability and promotes cell growth. Genes encoding the AMPK and TOR complexes are found in almost all eukaryotes, suggesting that these pathways arose very early during eukaryotic evolution. During the development of multicellularity, an additional tier of cell-extrinsic growth control arose that is mediated by growth factors, but these often act by modulating nutrient uptake so that AMPK and TOR remain the underlying regulators of cellular growth control. In this review, we discuss the evolution, structure, and regulation of the AMPK and TOR pathways and the complex mechanisms by which they interact.

Murburn concept: a paradigm shift in cellular metabolism and physiology.

Two decades of evidence-based exploratory pursuits in heme-flavin enzymology led to the formulation of a new biological electron/moiety transfer paradigm, called murburn concept. Murburn is a novel literary abstraction from "mured burning" or "mild unrestricted burning". This concept was invoked to explain the longstanding conundrum of maverick physiological dose responses and also applied to remodel the prevailing understanding of drug metabolism and cellular respiration. A conglomeration of simple ideas grounded in the known principles of thermodynamics and reaction chemistry, murburn concept invokes catalytic/functional roles for diffusible reactive species or radicals. Hitherto, diffusible reactive species were primarily seen as toxic agents of chaos, non-conducible to the maintenance of life-order. Since the murburn paradigm offers a distinctly different perspective for several biological phenomena, researchers holding conventional views of cellular metabolism pose a direct conflict of interests to the advancement of murburn concept. Murburn schemes are poised to integrate numerous metabolic motifs with holistic physiological outcomes; redefining pursuits in biology and medicine. To advance this agenda, I present a brief account of murburn concept and point out how redundant ideas are still advocated in some prestigious journals.

Analysis of Fluorescence Spectra of Citrus Polymethoxylated Flavones and Their Incorporation into Mammalian Cells.

Citrus polymethoxylated flavones (PMFs) influence biochemical cascades in human diseases, yet little is known about how these compounds interact with cells and how these associations influence the actions of these compounds. An innate attribute of PMFs is their ultraviolet-light-induced fluorescence, and the fluorescence spectra of 14 PMFs and 7 PMF metabolites were measured in methanol. These spectra were shown to be strongly influenced by the compounds' hydroxy and methoxy substituents. For a subset of these compounds, the fluorescence spectra were measured when bound to human carcinoma Huh7.5 cells. Emission-wavelength maxima of PMF metabolites with free hydroxyl substituents exhibited 70-80 nm red shifts when bound to the Huh7.5 cells. Notable solvent effects of water were observed for nearly all these compounds, and these influences likely reflect the effects of localized microenvironments on the resonance structures of these compounds when bound to human cells.

Water extract of Semecarpus parvifolia Thw. leaves inhibits cell proliferation and induces apoptosis on HEp-2 cells.

BACKGROUND: Semecarpus parvifolia Thw is used as an ingredient of poly herbal decoctions to treat cancer in traditional medicine. The present study aims to investigate the antiproliferative activity on HEp 2 cells by the water extract of S. parvifolia leaves and to evaluate potential mechanisms. METHODS: The plant extract was exposed to S. parvifolia for 24 hours and antiproliferative activity was quantified by Sulforhodamine B (SRB), 3-(4, 5-dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) and Lactate dehydrogenase (LDH) assays. Morphological changes were observed after staining cells with ethidium bromide/acridine orange (EB/AO) and Giemsa dye. Comet assay was performed to evaluate the DNA damage. The toxicity of the plant extract was determined by brine shrimp lethality assay. RESULTS: S. parvifolia leaves reduced the cell proliferation in a dose and time dependent manner. A two fold increase in NO level was observed at higher concentrations. Morphological changes characteristic to apoptosis were observed in light microscopy, Giemsa and EB/AO stained cells. Fragmented DNA further confirmed its capacity to induce apoptosis. No lethality was observed with brine shrimps. CONCLUSION: The results suggest that Semecarpus parvifolia Thw induces apoptosis in HEp-2 cells through a NO dependent pathway.

Inhibitory effects of selenium on cadmium-induced cytotoxicity in PC12 cells via regulating oxidative stress and apoptosis.

Purpose of this study is to investigate mechanism/s of cyto-protection by selenium (Na2SeO3; Se4+) against cadmium (CdCl2; Cd2+)-induced cytotoxicity using PC12 cells. In addition, Se (5, 10, 20 and 40 µM) and Cd (2.5, 5 and 10 µM)-induced cytotoxicity is determined. Cytotoxicity assays and western blot analyses confirmed that Se (≥10 µM) promotes autophagic cell death via inhibition of mTOR activation and p62 accumulation due to increase of cellular oxidative stress. On the other hand, co-presence of non-toxic Se (5 µM) and toxic Cd (5 µM) showed to increase cell viability, glutathione and glutathione peroxidase 1 (GPx1) levels, and to decrease DNA fragmentation and lactate dehydrogenase (LDH) activity compared to Cd-treated (5 µM) cells alone. Furthermore, western blot analyses of cytochrome c and ERK1 indicated that Cd-induced apoptotic cell death in PC12 cells. However, the co-exposure of Se with Cd significantly decreases the release of cytochrome c into cytosol from mitochondria, and up-regulates ERK1 protein to inhibit Cd-induced apoptosis. In conclusion, Se (≥10 µM) possess cytotoxicity in PC12 cells; however, co-presence of Se (5 µM) with Cd (5 µM) protects against Cd-induced apoptosis in PC12 cells due to inhibition of Cd-induced oxidative stress and subsequently suppression of mitochondrial apoptosis pathway.

What Do Reactive Fragments Actually Do in Cells?

Covalently binding molecules are frequently regarded as being generally promiscuous. In a recent study, binding selectivities and cellular target proteins of a wide variety of reactive fragments are examined in a proteome-wide context.

Evidences of the static magnetic field influence on cellular systems.

Efforts to elucidate the doubtful character of the static magnetic field (SMF) influence on living cells have been made, although the topic still faces controversies because confusing reports in the scientific literature. This study intended to collect the most relevant issues separated by different topics (relating the SMF to its action on cellular systems) and analyze how the many field intensities, cell types and exposure time would affect the cell or intracellular structures. The analysis was based in the search in online databases aiming to give a general view of how the data can show conformity. It is proposed that scientists have been searching for linearity in what is actually a well characterized nonlinear system and two outputs are considered: the high sensitivity of parameters in which specific cell responses are generated and also the complexity and particularity of each cellular system. It is possible to trigger effects from a SMF, however in a stochastic way and depending on the cell system.

Protective effects of five allium derived organosulfur compounds against mutation and oxidation.

In this study, we examined the ability of five allium-derived organosulfur compounds to protect cells against mutation and oxidation. The compounds tested were 1-propylmercaptan (PM), dimethyl disulfide (DMDS), diallyl disulfide (DADS), propyl disulfide (PDS), and 2,5-dimethylthiophene (DMT). Our results showed that when used at concentrations of 100-400 µmol/l, the five compounds inhibited the mutagenicity of 4-nitroquinoline-N-oxide, a direct mutagen, and benzo[a]pyrene, an indirect mutagen, toward Salmonella typhimurium TA 98 and TA 100. Furthermore, at these concentrations, all five of the compounds protected HepG2 cells against tert-butyl hydroperoxide-induced oxidative cytotoxicity. The compounds likely enhanced cell viability by suppressing the formation of reactive oxygen species and the depletion of glutathione depletion in cells. DMT and PM inhibited mutation and oxidation to a greater extent than DMDS, DADS, and PDS. These results demonstrate for the first time that DMT and PM can contribute to the antimutagenic and the antioxidative property of Allium vegetables.

Activation of Nrf2 target enzymes conferring protection against oxidative stress in PC12 cells by ginger principal constituent 6-shogaol.

Natural compounds containing phenoxyl groups and/or Michael acceptor units appear to possess antioxidant and cytoprotective properties. The ginger principal constituent 6-shogaol (6-S) represents one of such compounds. In this study, we reported that 6-S efficiently scavenges various free radicals in vitro, and displays remarkable cytoprotection against oxidative stress-induced cell damage in the neuron-like rat pheochromocytoma cell line, PC12 cells. Pretreatment of PC12 cells with 6-S significantly upregulates a series of phase II antioxidant molecules, such as glutathione, heme oxygenase 1, NAD(P)H: quinone oxidoreductase 1, thioredoxin reductase 1, and thioredoxin 1. A mechanistic study revealed that 6-S enhanced the translocation of Nrf2 from the cytosol to the nucleus and knockdown of Nrf2 abolished such protection, indicating that this cytoprotection is mediated by the activation of the transcription factor Nrf2. Another ginger constituent 6-gingerol (6-G), having a similar structure of 6-S but lacking the alpha,beta-unsaturated ketone structure (Michael acceptor moiety), failed to shelter PC12 cells from oxidative stress. Our results demonstrate that 6-S is a novel small molecule activator of Nrf2 in PC12 cells, and suggest that 6-S might be a potential candidate for the prevention of oxidative stress-mediated neurodegenerative disorders.

The effect of five Taraxacum species on in vitro and in vivo antioxidant and antiproliferative activity.

Plants belonging to the genus Taraxacum are considered a nutritious food, being consumed raw or cooked. Additionally, these plants have long been used in folk medicine due to their choleretic, diuretic, antitumor, antioxidant, antiinflammatory, and hepatoprotective properties. This genus, with its complex taxonomy, includes several species that are difficult to distinguish. Its traditional use must be related not only to T. officinale F.H. Wigg., the most studied species, but also to others. The aim of this work is to compare five different common South European species of Taraxacum (T. obovatum (Willd.) DC., T. marginellum H. Lindb., T. hispanicum H. Lindb., T. lambinonii Soest and T. lacistrum Sahlin), in order to find differences between antioxidant and cytotoxic activities among them. Dissimilarities between species in LC/MS patterns, in in vitro and intracellular antioxidant activity and also in the cytotoxicity assay were found. T. marginellum was the most efficient extract reducing intracellular ROS levels although in in vitro assays, T. obovatum was the best free radical scavenger. A relevant cytotoxic effect was found in T. lacistrum extract over HeLa and HepG2 cell lines.

Cell-based peptide screening to access the undruggable target space.

Only 20-30% of drug target proteins can be accessed by common drug classes, like small molecules or therapeutic antibodies. The vast majority of the remaining proteins are considered "undruggable" and include drug target proteins, like transcription factors, scaffold or adapter proteins, which play important roles in disease. However over the last years innovative compound classes including nucleotide derived drugs (e.g. siRNA, antisense), macrocyclic compounds and cell-permeable peptides matured significantly and hold now the potential to modulate these hard to access target proteins for therapeutic use. This article will focus on the discovery of cell-permeable peptides and discuss intracellular screening systems for peptides, which yield highly relevant peptides, because peptide selection takes place in eukaryotic cells, under conditions, which are very similar to the later therapeutic use.

A simple and high-throughput in-cell Western assay using HepG2 cell line for investigating the potential hypocholesterolemic effects of food components and nutraceutics.

Since saving time and money are critical issues while developing innovative functional foods and nutraceutics, the use of specific and high-throughput assays for the fast screening of potentially bioactive ingredients is crucial. In this context, the aim of the present investigation was the development of an in-cell Western (ICW) assay, a quantitative colorimetric cell-based technique, at the HepG2 cell line for screening and evaluating the effects of potentially bioactive compounds on the low density lipoprotein (LDL) receptor (LDLR). It is known that LDLR plays a pivotal role in the binding and endocytosis of circulating LDL, increasing its plasma clearance. The ICW was optimised and validated using monacolin K, the main hypocholesterolemic component of red yeast rice. This provided a robust and reproducible assay useful for characterising the cholesterol-lowering properties of bioactive food components. To our knowledge, this is the first application of the ICW technique in the field of functional foods and nutraceutics.

Antioxidant potential of Sutherlandia frutescens and its protective effects against oxidative stress in various cell cultures.

BACKGROUND: Sutherlandia frutescens (L.) R.Br. (SF) is a South African plant that is widely used to treat stress, infections, cancer, and chronic diseases, many of which involve oxidative stress. The aim of the study was to quantitatively assess the antioxidant potential of SF extracts in cell-free system as well as in cell lines. METHODS: Dried SF vegetative parts were extracted using six different solvents, and the extracts were assessed for total phenolic and flavonoid contents, total reducing power, iron chelating capacity, and free radical scavenging power, including, scavenging of hydroxyl radicals, superoxide anions, nitric oxide, and hydrogen peroxide. We further investigated the freeze-dried hot water extract of SF (SFE) to assess its effect against oxidative stress induced by tert-butyl hydroperoxide (t-BHP), an organic peroxide. Three different cell lines: Chinese hamster ovary (CHO), human hepatoma (HepaRG), and human pulmonary alveolar carcinoma (A549) cells, were employed to determine cell viability, intracellular reactive oxygen species (ROS) levels, and reduced to oxidized glutathione levels (GSH/GSSG). RESULTS: The results indicated that: (1) SF extracts have significant antioxidant potential that is dependent upon the nature of the extraction solvent and (2) SFE protects against tBHP-induced oxidative stress in cells by scavenging ROS and preserving intracellular GSH/GSSG. CONCLUSION: Oxidative stress is implicated in a number of disorders, and due to the public's concerns about synthetic antioxidants, various natural antioxidants are being explored for their therapeutic potential. Our findings support claims for S. frutescens being a promising adjunctive therapeutic for oxidative stress-related health problems.

SLC22, SLC44, and SLC47 transporters--organic anion and cation transporters: molecular and cellular properties.

Transporters within the SLC22, SLC44, and SLC47 families of solute carriers mediate transport of a structurally diverse array of organic electrolytes, that is, molecules that are generally charged (cationic, anionic, or zwitterionic) at physiological pH. Transporters in the SLC22 family--all of which are members of the major facilitator superfamily (MFS) of transporters--represent a mechanistically diverse set of processes, including the organic anion transporters (OATs and URAT1) that physiologically operate as organic anion (OA) exchangers, the organic cation transporters (OCTs) that operate as electrogenic uniporters of organic cations (OCs), and the so-called "novel" organic cation transporters (OCTNs) that support Na-cotransport of selected zwitterions. Whereas the OCTNs display a high degree of substrate selectivity, the physiological hallmark of the OATs and OCTs is their multiselectivity--consistent with a principal role in renal and hepatic clearance of a wide array of both endogenous and xenobiotic compounds. SLC47 consists of members of the multidrug and toxin extruder (MATE) family, which are carriers that are obligatory exchangers and that physiologically support electroneutral H⁺ exchange. The MATEs also display a characteristic multiselectivity and are frequently paired with OCTs to mediate transepithelial OC secretion, with the OCTs typically supporting basolateral OC entry and the MATEs supporting apical OC efflux. The SLC44 family contains the choline transporter-like (CTL) transporters. Largely restricted to choline and a limited set of structural congeners, the CTLs appear to support the Na-independent, electrogenic uniport of choline, thereby providing choline for membrane biogenesis. The solution of X-ray crystal structures of representative prokaryotic MFS and MATE transporters has led to the development of homology models of mammalian OAT, OCT, and MATE transporters that, in turn, have supplemented studies of the molecular basis of the complex interactions of ligands with these multiselective proteins.

Cytoprotective effect of white tea against H2O2-induced oxidative stress in vitro.

The protective effect of water extracts of white tea (WEWT) on oxidative stress in vitro is investigated. WEWT, like water extracts of green tea (WEGT) and water extracts of Pu-erh tea (WEPT), demonstrates a marked inhibition of the oxidation of liposome, albumin and LDLmodel systems. WEWT protects against H2O2-induced cytotoxicity, in a dose-dependent manner. The inhibition of ROS generation and MDA formation by WEWT in H2O2-induced Clone 9 cells parallels the effects on cell viability. Moreover, GSH and antioxidant enzymes may play an important role in the protective effect that is associated with H2O2-induced oxidative stress. The HPLC-DAD and HPLC-MS/MS analysis, shows that sixteen bioactive compounds are present in WEWT, which may partially account for its protective effect against oxidative insult. These results suggest that the mechanism of the protective actions of WEWT is related to its antioxidant potential and the maintenance of the normal redox status of the cell.