Reverse transcription as key step in RNA in vitro evolution with unnatural base pairs.

Unnatural base pairs (UBPs) augment the chemical diversity of artificial nucleic acids and can thus enable the generation of new aptamers and catalytic nucleic acids by in vitro selection. However, owing to a lack of methodologies, the reverse transcription of UBPs, a key step in RNA aptamer selection, has not been sufficiently characterized. Here, we present a series of versatile assays to investigate the reverse transcription of the TPT3:NaM base pair as a representative for hydrophobic unnatural base pairs. We determine the fidelity and retention of the UBP for four different reverse transcriptases (RT) in the context of RNA in vitro evolution. The retention of the TPT3:NaM pair during the RNA in vitro selection process was investigated using a novel click-chemistry based electromobility shift assay. Real-time monitoring of reverse transcription kinetics revealed considerable differences in polymerase activity processing the TPT3:NaM base pair. Our findings identified SuperScript IV RT as the most efficient RT for processing the TPT3:NaM pair. Our approach can be applied universally to study newly developed UBPs, not only at the reverse transcription level, but also during PCR and in vitro transcription.

Glucose micro-biosensor for scanning electrochemical microscopy characterization of cellular metabolism in hypoxic microenvironments.

Mapping of the metabolic activity of tumor tissues represents a fundamental approach to better identify the tumor type, elucidate metastatic mechanisms and support the development of targeted cancer therapies. The spatially resolved quantification of Warburg effect key metabolites, such as glucose and lactate, is essential. Miniaturized electrochemical biosensors scanned over cancer cells and tumor tissue to visualize the metabolic characteristics of a tumor is attractive but very challenging due to the limited oxygen availability in the hypoxic environments of tumors that impedes the reliable applicability of glucose oxidase-based glucose micro-biosensors. Herein, the development and application of a new glucose micro-biosensor is presented that can be reliably operated under hypoxic conditions. The micro-biosensor is fabricated in a one-step synthesis by entrapping during the electrochemically driven growth of a polymeric matrix on a platinum microelectrode glucose oxidase and a catalytically active Prussian blue type aggregate and mediator. The as-obtained functionalization improves significantly the sensitivity of the developed micro-biosensor for glucose detection under hypoxic conditions compared to normoxic conditions. By using the micro-biosensor as non-invasive sensing probe in Scanning Electrochemical Microscopy (SECM), the glucose uptake by a breast metastatic adenocarcinoma cell line, with an epithelial morphology, is measured.

Enhanced Uptake and Phototoxicity of C60@albumin Hybrids by Folate Bioconjugation.

Fullerenes are considered excellent photosensitizers, being highly suitable for photodynamic therapy (PDT). A lack of water solubility and low biocompatibility are, in many instances, still hampering the full exploitation of their potential in nanomedicine. Here, we used human serum albumin (HSA) to disperse fullerenes by binding up to five fullerene cages inside the hydrophobic cavities. Albumin was bioconjugated with folic acid to specifically address the folate receptors that are usually overexpressed in several solid tumors. Concurrently, tetramethylrhodamine isothiocyanate, TRITC, a tag for imaging, was conjugated to C60@HSA in order to build an effective phototheranostic platform. The in vitro experiments demonstrated that: (i) HSA disperses C60 molecules in a physiological environment, (ii) HSA, upon C60 binding, maintains its biological identity and biocompatibility, (iii) the C60@HSA complex shows a significant visible-light-induced production of reactive oxygen species, and (iv) folate bioconjugation improves both the internalization and the PDT-induced phototoxicity of the C60@HSA complex in HeLa cells.

Multivalent binding kinetics resolved by fluorescence proximity sensing.

Multivalent protein interactors are an attractive modality for probing protein function and exploring novel pharmaceutical strategies. The throughput and precision of state-of-the-art methodologies and workflows for the effective development of multivalent binders is currently limited by surface immobilization, fluorescent labelling and sample consumption. Using the gephyrin protein, the master regulator of the inhibitory synapse, as benchmark, we exemplify the application of Fluorescence proximity sensing (FPS) for the systematic kinetic and thermodynamic optimization of multivalent peptide architectures. High throughput synthesis of +100 peptides with varying combinatorial dimeric, tetrameric, and octameric architectures combined with direct FPS measurements resolved on-rates, off-rates, and dissociation constants with high accuracy and low sample consumption compared to three complementary technologies. The dataset and its machine learning-based analysis deciphered the relationship of specific architectural features and binding kinetics and thereby identified binders with unprecedented protein inhibition capacity; thus, highlighting the value of FPS for the rational engineering of multivalent inhibitors.

Absolute Versus Relative Success: Why Overconfidence Creates an Inefficient Equilibrium.

Overconfidence is one of the most ubiquitous biases in the social sciences, but the evidence regarding its overall costs and benefits is mixed. To test the possibility that overconfidence might yield important relative benefits that offset its absolute costs, we conducted an experiment (N = 298 university students) in which pairs of participants bargained over the unequal allocation of a prize that was earned through a joint effort. We manipulated confidence using a binary noisy signal to investigate the causal effect of negotiators' beliefs about their relative contribution to the outcome of the negotiation. Our results provide evidence that high levels of confidence lead to relative benefits (how much one earns compared with one's partner) but absolute costs (how much money one receives overall). These results suggest that overconfidence creates an inefficient equilibrium whereby overconfident negotiators benefit over their partners even as they bring about joint losses.

Human Serum Albumin-Oligothiophene Bioconjugate: A Phototheranostic Platform for Localized Killing of Cancer Cells by Precise Light Activation.

The electronic, optical, and redox properties of thiophene-based materials have made them pivotal in nanoscience and nanotechnology. However, the exploitation of oligothiophenes in photodynamic therapy is hindered by their intrinsic hydrophobicity that lowers their biocompatibility and availability in water environments. Here, we developed human serum albumin (HSA)-oligothiophene bioconjugates that afford the use of insoluble oligothiophenes in physiological environments. UV-vis and electrophoresis proved the conjugation of the oligothiophene sensitizers to the protein. The bioconjugate is water-soluble and biocompatible, does not have any "dark toxicity", and preserves HSA in the physiological monomeric form, as confirmed by dynamic light scattering and circular dichroism measurements. In contrast, upon irradiation with ultralow light doses, the bioconjugate efficiently produces reactive oxygen species (ROS) and leads to the complete eradication of cancer cells. Real-time monitoring of the photokilling activity of the HSA-oligothiophene bioconjugate shows that living cells "explode" upon irradiation. Photodependent and dose-dependent apoptosis is one of the primary mechanisms of cell death activated by bioconjugate irradiation. The bioconjugate is a novel theranostic platform able to generate ROS intracellularly and provide imaging through the fluorescence of the oligothiophene. It is also a real-time self-reporting system able to monitor the apoptotic process. The induced phototoxicity is strongly confined to the irradiated region, showing localized killing of cancer cells by precise light activation of the bioconjugate.

The EYCN and the Power of Networking.

Networking is a symbiosis-it is about establishing, building and cultivating relationships that you will maintain over a long time and which may lead to mutually beneficial exchanges in your future. Your professional contacts might also become your close friends, since you frequently share the same experiences or merely have similar scientific and general interests or nerdy humour. Chemical societies are a fantastic way to expand your network and engaging or following the activities of the European Young Chemists Network (EYCN) might be the perfect starting point for you.

Doxorubicin-Loaded Squid Pen Plaster: A Natural Drug Delivery System for Cancer Cells.

The native structure of the ß-chitin in the gladius (squid pen) of Loligo vulgaris squid can be used as a natural plaster to entrap and release a model drug, doxorubicin, in a targeted and controlled way. Local pH determines the protonation state of the doxorubicin molecules, controlling the two phenomena. Confocal microscopy shows that doxorubicin is uniformly embedded in the ß-chitin squid pen and is not simply adsorbed on its surface. Coculture with HeLa cells reveals that the ß-chitin squid pen plaster is perfectly biocompatible, while when it is loaded with doxorubicin it shows high cytotoxicity toward the cancer cells. The drug, once released, rapidly accumulates inside the cells. In conclusion, the native structure of a ß-chitin squid pen can be potentially applied as a "green" pH-responsive drug vehicle for controlled release.

Label-free metabolic imaging by mid-infrared optoacoustic microscopy in living cells.

We develop mid-infrared optoacoustic microscopy (MiROM) for label-free, bond-selective, live-cell metabolic imaging, enabling spatiotemporal monitoring of carbohydrates, lipids and proteins in cells and tissues. Using acoustic detection of optical absorption, MiROM converts mid-infrared sensing into a positive-contrast imaging modality with negligible photodamage and high sensitivity. We use MiROM to observe changes in intrinsic carbohydrate distribution from a diffusive spatial pattern to tight co-localization with lipid droplets during adipogenesis.

Stable and Biocompatible Monodispersion of C60 in Water by Peptides.

The lack of solubility in water and the formation of aggregates hamper many opportunities for technological exploitation of C60. Here, different peptides were designed and synthesized with the aim of monomolecular dispersion of C60 in water. Phenylalanines were used as recognizing moieties, able to interact with C60 through π-π stacking, while a varying number of glycines were used as spacers, to connect the two terminal phenylalanines. The best performance in the dispersion of C60 was obtained with the FGGGF peptidic nanotweezer at a pH of 12. A full characterization of this adduct was carried out. The peptides disperse C60 in water with high efficiency, and the solutions are stable for months both in pure water and in physiological environments. NMR measurements demonstrated the ability of the peptides to interact with C60. AFM measurements showed that C60 is monodispersed. Electrospray ionization mass spectrometry determined a stoichiometry of C60@(FGGGF)4. Molecular dynamics simulations showed that the peptides assemble around the C60 cage, like a candy in its paper wrapper, creating a supramolecular host able to accept C60 in the cavity. The peptide-wrapped C60 is fully biocompatible and the C60 "dark toxicity" is eliminated. C60@(FGGGF)4 shows visible light-induced reactive oxygen species (ROS) generation at physiological saline concentrations and reduction of the HeLa cell viability in response to visible light irradiation.

Proteins as supramolecular hosts for C60: a true solution of C60 in water.

Hybrid systems have great potential for a wide range of applications in chemistry, physics and materials science. Conjugation of a biosystem to a molecular material can tune the properties of the components or give rise to new properties. As a workhorse, here we take a C60@lysozyme hybrid. We show that lysozyme recognizes and disperses fullerene in water. AFM, cryo-TEM and high resolution X-ray powder diffraction show that the C60 dispersion is monomolecular. The adduct is biocompatible, stable in physiological and technologically-relevant environments, and easy to store. Hybridization with lysozyme preserves the electrochemical properties of C60. EPR spin-trapping experiments show that the C60@lysozyme hybrid produces ROS following both type I and type II mechanisms. Due to the shielding effect of proteins, the adduct generates significant amounts of 1O2 also in aqueous solution. In the case of type I mechanism, the protein residues provide electrons and the hybrid does not require addition of external electron donors. The preparation process and the properties of C60@lysozyme are general and can be expected to be similar to other C60@protein systems. It is envisaged that the properties of the C60@protein hybrids will pave the way for a host of applications in nanomedicine, nanotechnology, and photocatalysis.

Amino acid sensing in hypothalamic tanycytes via umami taste receptors.

OBJECTIVE: Hypothalamic tanycytes are glial cells that line the wall of the third ventricle and contact the cerebrospinal fluid (CSF). While they are known to detect glucose in the CSF we now show that tanycytes also detect amino acids, important nutrients that signal satiety. METHODS: Ca2+ imaging and ATP biosensing were used to detect tanycyte responses to l-amino acids. The downstream pathway of the responses was determined using ATP receptor antagonists and channel blockers. The receptors were characterized using mice lacking the Tas1r1 gene, as well as an mGluR4 receptor antagonist. RESULTS: Amino acids such as Arg, Lys, and Ala evoke Ca2+ signals in tanycytes and evoke the release of ATP via pannexin 1 and CalHM1, which amplifies the signal via a P2 receptor dependent mechanism. Tanycytes from mice lacking the Tas1r1 gene had diminished responses to lysine and arginine but not alanine. Antagonists of mGluR4 greatly reduced the responses to alanine and lysine. CONCLUSION: Two receptors previously implicated in taste cells, the Tas1r1/Tas1r3 heterodimer and mGluR4, contribute to the detection of a range of amino acids by tanycytes in CSF.

Glucose and Lactate Miniaturized Biosensors for SECM-Based High-Spatial Resolution Analysis: A Comparative Study.

With the aim of developing miniaturized enzymatic biosensors suitable for in vitro diagnostic applications, such as monitoring of metabolites at single cell level, glucose and lactate biosensors were fabricated by immobilizing enzymes (glucose oxidase and lactate oxidase, respectively) on 10 µm Pt ultramicroelectrodes. These electrodes are meant to be employed as probes for scanning electrochemical microscopy (SECM), which is a unique technique for high-spatial-resolution electrochemical-based analysis. The use of enzymatic moieties improves sensitivity, time scale response, and information content of the microprobes; however, protein immobilization is a key step in the biosensor preparation that greatly affects the overall performance. A crucial aspect is the miniaturization of the sensing, preserving their sensitivity. In this work, we investigated the most common enzyme immobilization techniques. Several fabrication routes are reported and the main figures of merit, such as sensitivity, detection limit, response time, reproducibility, spatial resolution, biosensor efficiency, permeability, selectivity, and the ability to block electro-active interfering species, are investigated and compared. With the intent of using the microprobes for in vitro functional imaging of single living cells, we carefully evaluate the spatial resolution achieved by our modified electrodes on 2D SECM imaging. Metabolic activity of single MCF10A cells were obtained by monitoring the glucose concentrations in close proximity of single living cell, using the UME-based biosensor probes prepared. A voltage-switch approach was implemented to disentangle the topographical contribution of the cells enabling quantitative measurements of cellular uptakes.

Complex Media and Enzymatic Kinetics.

Enzymatic reactions in complex environments often take place with concentrations of enzyme comparable to that of substrate molecules. Two such cases occur when an enzyme is used to detect low concentrations of substrate/analyte or inside a living cell. Such concentrations do not agree with standard in vitro conditions, aimed at satisfying one of the founding hypotheses of the Michaelis-Menten reaction scheme, MM. It would be desirable to generalize the classical approach and show its applicability to complex systems. A permeable micrometrically structured hydrogel matrix was fabricated by protein cross-linking. Glucose oxidase enzyme (GOx) was embedded in the matrix and used as a prototypical system. The concentration of H2O2 was monitored in time and fitted by an accurate solution of the enzymatic kinetic scheme, which is expressed in terms of simple functions. The approach can also find applications in digital microfluidics and in systems biology where the kinetics response in the linear regimes often employed must be replaced.

Ultrasensitive nanostructured immunosensor for stem and carcinoma cell pluripotency gatekeeper protein NANOG.

AIMS: To develop an immunosensor for ultrasensitive detection of the NANOG protein. NANOG regulates pluripotency in stem cells and some cancer cells. This article reports the first electrochemical immunosensor for ultrasensitive detection and absolute quantification of the NANOG protein. The sensor features dense capture antibody-coated gold nanoparticle layers on a pyrolytic graphite underlayer. MATERIALS & METHODS: Two separate multilabel detection strategies were used to achieve moderate and ultra-high sensitivity. RESULTS: Good sensitivity was achieved for NANOG over the concentration range 0.1-160 pg/ml. The moderate-sensitivity approach gave a detection limit of 25 pg/ml, while the ultrasensitive method achieved a 250-fold lower detection limit of 0.1 pg/ml. Amounts of NANOG detected in human embryonic stem cell lysates correlated well with qualitative western blots and mRNA expression. CONCLUSION: The electrochemical gold nanoparticle immunosensor is suitable for measuring NANOG protein expression in stem and carcinoma cell tissue lysates at very low levels.

¹H, ¹³C, and ¹5N NMR resonance assignments of reduced full length and shortened forms of the Grx domain of Mus musculus TGR.

Two forms of the glutaredoxin (Grx) domain (full length Grx domain and short Grx lacking the N-terminal region) of Mus musculus thioredoxin glutathione reductase (TGR) were isotopically labelled with (15)N and (13)C isotopes, expressed and purified to homogeneity. We report here the (1)H, (13)C and (15)N NMR assignment for both Grx forms of this mouse TGR. This investigation represents the first NMR analysis of a mammalian TGR.