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RNAs contain post-transcriptional modifications, which fulfill a variety of functions in translation, secondary structure stabilization and cellular stress survival. Here, 2-methylthiocytidine (ms2C) is identified in tRNA of E. coli and P. aeruginosa using NAIL-MS (nucleic acid isotope labeling coupled mass spectrometry) in combination with genetic screening experiments. ms2C is only found in 2-thiocytidine (s2C) containing tRNAs, namely tRNAArgCCG, tRNAArgICG, tRNAArgUCU and tRNASerGCU at low abundances. ms2C is not formed by commonly known tRNA methyltransferases. Instead, we observe its formation in vitro and in vivo during exposure to methylating agents. More than half of the s2C containing tRNA can be methylated to carry ms2C. With a pulse-chase NAIL-MS experiment, the repair mechanism by AlkB dependent sulfur demethylation is demonstrated in vivo. Overall, we describe ms2C as a bacterial tRNA modification and damage product. Its repair by AlkB and other pathways is demonstrated in vivo by our powerful NAIL-MS approach., Bacterial tRNA is modified by thiolation of nucleosides. Here the authors identify 2-methylthiocytidine in bacterial tRNA using nucleic acid isotope labeling coupled mass spectrometry. Exposure to methylating agents converts 2-thiocytidine to 2-methylthiocytidine, which is repaired by demethylase AlkB in vivo.

Protein-protein interactions (PPIs) play a key role in many biological processes and are intriguing targets for drug discovery campaigns. Advancements in experimental and computational techniques are leading to a growth of data accessibility, and, with it, an increased need for the analysis of PPIs. In this respect, visualization tools are essential instruments to represent and analyze biomolecular interactions. In this chapter, we reviewed some of the available tools, highlighting their features, and describing their functions with practical information on their usage.

Background & Aims Promoted by pancreatitis, oncogenic KrasG12D triggers acinar cells' neoplastic transformation through acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia. Anterior gradient 2 (Agr2), a known inhibitor of p53, is detected at early stage of pancreatic ductal adenocarcinoma (PDAC) development. RNA polymerase II (RNAPII) is a key nuclear enzyme; regulation of its nuclear localization in mammalian cells represents a potential therapeutic target. Methods A mouse model of inflammation-accelerated KrasG12D-driven ADM and pancreatic intraepithelial neoplasia development was used. Pancreas-specific Agr2 ablation was performed to access its role in pancreatic carcinogenesis. Hydrophobic hexapeptides loaded in liposomes were developed to disrupt Agr2–RNAPII complex. Results We found that Agr2 is up-regulated in ADM-to-pancreatic intraepithelial neoplasia transition in inflammation and KrasG12D-driven early pancreatic carcinogenesis. Genetic ablation of Agr2 specifically blocks this metaplastic-to-neoplastic process. Mechanistically, Agr2 directs the nuclear import of RNAPII via its C-terminal nuclear localization signal, undermining the ATR-dependent p53 activation in ADM lesions. Because Agr2 binds to the largest subunit of RNAPII in a peptide motif-dependent manner, we developed a hexapeptide to interfere with the nuclear import of RNAPII by competitively disrupting the Agr2-RNAPII complex. This novel hexapeptide leads to dysfunction of RNAPII with concomitant activation of DNA damage response in early neoplastic lesions; hence, it dramatically compromises PDAC initiation in vivo. Moreover, the hexapeptide sensitizes PDAC cells and patient-derived organoids harboring wild-type p53 to RNAPII inhibitors and first-line chemotherapeutic agents in vivo. Of note, this therapeutic effect is efficient across various cancer types. Conclusions Agr2 is identified as a novel adaptor protein for nuclear import of RNAPII in mammalian cells. Also, we provide genetic evidence defining Agr2-dependent nuclear import of RNAPII as a pharmaceutically accessible target for prevention and treatment in PDAC in the context of wild-type p53.

Protein-protein interactions (PPIs) play a key role in many biological processes and are intriguing targets for drug discovery campaigns. Advancements in experimental and computational techniques are leading to a growth of data accessibility, and, with it, an increased need for the analysis of PPIs. In this respect, visualization tools are essential instruments to represent and analyze biomolecular interactions. In this chapter, we reviewed some of the available tools, highlighting their features, and describing their functions with practical information on their usage.

When a NLU model is updated, new utter- ances must be annotated to be included for training. However, manual annotation is very costly. We evaluate a semi-supervised learning workflow with a human in the loop in a produc- tion environment. The previous NLU model predicts the annotation of the new utterances, a human then reviews the predicted annotation. Only when the NLU prediction is assessed as incorrect the utterance is sent for human anno- tation. Experimental results show that the pro- posed workflow boosts the performance of the NLU model while significantly reducing the annotation volume. Specifically, in our setup, we see improvements of up to 14.16% for a recall-based metric and up to 9.57% for a F1- score based metric, while reducing the annota- tion volume by 97% and overall cost by 60% for each iteration.

The accuracy of an online shopping system via voice commands is particularly important and may have a great impact on customer trust. This paper focuses on the problem of detecting if an utterance contains actual and purchasable products, thus referring to a shopping-related intent in a typical Spoken Language Understanding architecture consist- ing of an intent classifier and a slot detec- tor. Searching through billions of products to check if a detected slot is a purchasable item is prohibitively expensive. To overcome this problem, we present a framework that (1) uses a retrieval module that returns the most rele- vant products with respect to the detected slot, and (2) combines it with a twin network that decides if the detected slot is indeed a pur- chasable item or not. Through various exper- iments, we show that this architecture outper- forms a typical slot detector approach, with a gain of +81% in accuracy and +41% in F1 score.

In all domains of life, the nucleobases of tRNA can be methylated. These methylations are introduced either by enzymes or by the reaction of methylating agents with the nucleophilic centers of the nucleobases. Herein, we present a systematic approach to identify the methylation sites within RNA in vitro and in vivo. For discrimination between enzymatic tRNA methylation and tRNA methylation damage in bacteria, we used nucleic acid isotope labeling coupled mass spectrometry (NAIL‐MS). With NAIL‐MS, we clearly observed the formation of 7‐methylguanosine, 3‐methyluridine, and 6‐methyladenosine during exposure of bacteria to the alkylating agent methyl methanesulfonate (MMS) in vivo. These damage products were not reported to form in tRNA in vivo, as they were masked by the enzymatically formed modified nucleosides in previous studies. In addition, we found formation of the known damage products 1‐methyladenosine and 3‐methylcytidine in vivo. With a dynamic NAIL‐MS setup, we observed tRNA repair by demethylation of these two RNA modifications in vivo. Furthermore, we saw the potential repair of 6‐methyladenosine but not 7‐methylguanosine in bacterial tRNA.

Due to the increasingly widespread diffusion of lasers in many scientific and technological fields, the engineering and the fabrication of systems able to protect either human eyes or delicate equipment from laser radiation damage is nowadays attracting lots of interest in the scientific community. In this work, the optical limiting properties of fulleropyrrolidine, gold nanoparticles and hybrid systems in solution and in a polycarbonate matrix are investigated using a continuous wave laser at 514 nm, by optical limiting, Z-scan and temporal response measurements. The comparison of the results, obtained with different techniques, has allowed us to show that thermal effects account for most of the nonlinear response in gold nanoparticles and in the hybrid system; moreover, the latter exhibits a lower nonlinear threshold and a faster response compared to the former. This paper provides a contribution to the engineering of efficient protection devices in the continuous wave regime.

La competencia es un factor esencial para el buen funcionamiento de los mercados. Fomenta la innovacion, la productividad y el crecimiento, lo cual, a su vez, genera riqueza y ayuda a reducir la pobreza. En los ultimos anos, Mexico ha puesto en marcha ambiciosas reformas para liberalizar y fortalecer su economia. La competencia se incremento en muchos sectores, sobre todo en el energetico —electricidad, petroleo y gas— y en los servicios de telecomunicaciones. La funcion de los reguladores se fortalecio y se faculto a las autoridades en materia de competencia para desalentar la colusion y otras practicas anticompetitivas.

Competition is essential to the functioning of markets. It fosters innovation, productivity and growth, which, in turn, create wealth and help to reduce poverty. Over the past years, Mexico has implemented ambitious reforms to free up its economy. Competition was enhanced in many sectors, most notably in the energy sector – electricity, oil and gas – and in telecommunication services. The role of regulators has been strengthened and the competition authorities have been empowered to deter collusion and other anticompetitive practices. Building on these achievements, Mexico now needs to ensure that entrepreneurship thrives and opportunities are spread evenly across the country. Removing existing barriers in services trade would benefit manufacturing and further facilitate the country’s integration into global value chains.

Kommunitarismus und Tugendethik schopfen in weiten Teilen aus denselben Quellen. Schlaglichtartig wird die historische Entwicklung des Tugendbegriffs bzw. der Tugendethik skizziert und tugendethische Gedanken bei kommunitarischen Philosophen, vor allem Alasdair MacIntyre, Michael Sandel, Charles Taylor und Martha Nussbaum, vorgestellt. Die Tragfahigkeit des auf Aristoteles zuruckgehenden Tugendmodells in Zusammenhangen zeitgenossischer Gesellschaften wird diskutiert, Gemeinsamkeiten und Unterschiede von Kommunitarismus und Tugendethik beleuchtet und ein Blick auf die stets diskutierte Frage der Universalisierbarkeit geworfen.

Screening nanoparticle toxicity directly on cell culture can be a fast and cheap technique. Nevertheless, to obtain results in accordance with those observed in live animals, the conditions in which cells are cultivated should resemble the one encountered in live systems. Microfluidic devices offer the possibility to satisfy this requirement, in particular with endothelial cell lines, because they are capable to reproduce the flowing media and shear stress experienced by these cell lines in vivo. In this work, we exploit a microfluidic device to observe how human umbilical vein endothelial cells (HUVEC) viability changes when subject to a continuous flow of culture medium, in which spherical citrate-stabilized gold nanoparticles of different sizes and at varying doses are investigated. For comparison, the same experiments are also run in multiwells where the cells do not experience the shear stress induced by the flowing medium. We discuss the results considering the influence of mode of exposure and nanoparticle size (24 and 13 nm). We observed that gold nanoparticles show a lower toxicity under flow conditions with respect to static and the HUVEC viability decreases as the nanoparticle surface area per unit volume increases, regardless of size. Electronic supplementary material The online version of this article (10.1007/s11051-017-3993-5) contains supplementary material, which is available to authorized users.

Toxicity of gold nanoparticles towards human endothelial cells is observed under static and flow conditions by means of a microfluidic device which mimics the in vivo environmental conditions for this type of cells.

In this work time- and frequency-resolved spectroscopic techniques are employed for the investigation of protein adsorption (protein corona formation) on gold nanoparticles. We will follow the interaction of ∼ 20 nm citrate-capped gold nanoparticles with the model protein bovine serum albumin under both static and flow conditions, to monitor in details the binding process in the early stages. In parallel to standard techniques (UV-Visible absorption, circular dichroism and fluorescence spectroscopy), an advanced technique such as fluorescence correlation spectroscopy is employed for the characterization of the kinetic and thermodynamic constants involved in protein corona formation. The dissociation constant characterizing adsorption of bovine serum albumin on gold nanoparticles is measured in solutions under static conditions as a function of protein concentration. A preliminary characterization of the adsorption dynamic, instead, is achieved by fabrication of a simple Y shaped microfluidic device in polydimethylsiloxane and glass. Water solutions of nanoparticles and protein are injected in the two inlets and mixing of the two species occurs only in the central part of the main channel. Following the evolution of the fluorescence correlation spectroscopy signal along the main channel of the microfluidic device provides adsorption kinetic data with a time resolution defined only by the flow rate applied at the inlets.

In the attempt to improve optical limiting of cw lasers by exploiting the thermo-optic effect exhibited by gold nanostructures, we investigated two coupled systems consisting of either gold nanoparticles (AuNPs) or gold-silica core-shell (AuNSs), both functionalized with a thiolated-fulleropyrrolidine (C 60 Py). We measured the optical limiting behavior under cw illumination at 514 and 647 nm, resonant with the surface plasmon resonance at around 520 of AuNPs and at 650 nm of AuNSs, respectively. Temporal response analysis shows the variation of transmitted irradiance in a 300 milliseconds time interval, corresponding to the blinking time of the human eye. Comparing the present results we those previously obtained for AuNPs1 we demonstrate an improvement of the response of functionalized nanoparticles (AuNPs- C 60 Py) with respect to bare AuNPs.

A new in vitro model system, adding advection and shear stress associated with a flowing medium, is proposed for the investigation of nanoparticles uptake and toxicity towards endothelial cells, since these processes are normally present when nanoparticles formulations are intravenously administered. In this model system, mechanical forces normally present in vivo, such as advection and shear stress were applied and carefully controlled by growing human umbilical vein endothelial cells inside a microfluidic device and continuously infusing gold nanoparticle (Au NPs) solution in the device. The tests performed in the microfluidic device were also run in multiwells, where no flow is present, so as to compare the two model systems and evaluate if gold nanoparticles toxicity differs under static and flow culture conditions. Full characterization of Au NPs in water and in culture medium was accomplished by standard methods. Two-photon fluorescence correlation spectroscopy was also employed to map the flow speed of Au NPs in the microfluidic device and characterize Au NPs before and after interactions with the cells. Au NPs uptake in both in vitro systems was investigated through electron and fluorescence microscopy and ICP-AES, and NPs toxicity measured through standard bio-analytical tests. Comparison between experiments run in multiwells and in microfluidic device plays a pivotal role for the investigation of nanoparticle-cell interaction and toxicity assessment: our work showed that administration of equal concentrations of Au NPs under flow conditions resulted in a reduced sedimentation of nanoparticle aggregates onto the cells and lower cytotoxicity with respect to experiments run in ordinary static conditions (multiwells).

In this work we report two-photon fluorescence correlation spectroscopy experiments run on gold nanoparticles in aqueous solution. We compare the photophysical properties of nanoparticles obtained by the Turkevich method and by laser ablation as a function of increasing laser power. Fluorescence correlation spectroscopy curves for all nanoparticles show the rotational diffusion contribution at short lag times, even if the residual rotation contrast coefficient at high excitation laser power is lower for laser ablation nanoparticles with respect to Turkevich syntheses. Moreover, in contrast to Turkevich nanoparticles, laser ablation ones do not show an increase of the number of bright tracers in the excitation volume under higher excitation power. Nanoparticles from Turkevich synthesis are also tested as possible flow tracers inside a very simple microfluidic device featuring a single straight channel. Our findings confirm that gold nanoparticles can indeed be used as extremely sensitive probes to observe local flow inside micrometric size channels, for example, such as mammalian capillaries, since it can distinguish subtle changes in flow speed, as the ones imparted by a syringe pump.

Gold nanoshells, with a silica core and different core and shell dimensions, have been extensively investigated. Optical far-field properties, namely extinction and absorption, have been separately determined by means of spectrophotometry and photoacoustic spectroscopy, respectively, in the 440-900 nm range. The enhancement factor for surface-enhanced Raman scattering, which is related to near-field effects, has been measured from 568 to 920 nm. The absorption contribution to extinction decreases as the overall diameter increases. Moreover, absorption and scattering display different spectral distributions, the latter being red shifted. The Surface Enhanced Raman Scattering enhancement profile, measured using thiobenzoic acid as a Raman probe, is further shifted to the red. The latter result suggests that the enhancement is dominated by the presence of hot spots, which are possibly related to the surface roughness of gold nanoshell particles.

The goal of this work is to study the optical limiting properties of gold nanoparticles colloidal suspensions in organic solvent and to make a comparison with nanoparticles embedded in a polymeric matrix. The idea is to realize a solid state protection device based on gold nanoparticles (AuNPs) against cw lasers. This is desirable because polymers are usually cheap, flexible, and mechanically robust and temporally stable. In particular we use polycarbonate, a common polymer used to produce high quality optical lenses with superior mechanical properties. Thermally induced nonlinear optical properties of AuNPs, characterized by a surface plasmon resonance with a peak at about 520 nm where the human eye is most sensitive, have been reported for solution samples. The z-scan analysis performed on AuNPs in organic solvent confirms the presence of a nonlinear absorption coefficient comparable with literature for water colloids. We have investigated the optical limiting behavior of AuNPs doped thin film under cw illumination at 488 nm and their temporal response. A critical parameter in assessing the effectiveness of the optical limiting action is the time response of the material at varying input powers. It gives us information on the transmitted irradiance in a 300 milliseconds time interval, corresponding to the blinking time of the human eye, therefore on the total fluence reaching the retina. Finally we also report a characterization of the optical limiting action of film at 488, 514 and 647 nm.

Many small protein reactive organic and inorganic chemicals can cause allergic contact dermatitis, a T cell mediated inflammatory skin disease. In vitro alternatives to animal testing are needed for the identification of chemicals that pose such risks to human health. We here publish the standard operation procedure for a human T cell priming assay developed primarily for the identification of contact allergens within the integrated EU project Sens-it-iv. This multiparametric flow cytometry based assay identifies chemical specific T cells based on their frequency and antigen-specific production of the cytokines IFN-γ and TNF-α at the single cell level. Using sorted naive T cells and monocyte-derived dendritic cells pulsed with the test chemical or with chemical-protein conjugates, the successful priming of an antigen-specific T cell response is controlled after antigen-specific restimulation by cytokine readout. As the most specific response of the immune system to contact allergens the analysis of the chemical-specific T cell response may be a useful in vitro assay for hazard identification in immunotoxicology. This assay may be a valuable, highly specific element of an integrated testing strategy for the identification of chemicals and drugs that cause T cell mediated respiratory or gastrointestinal tract hypersensitivities or adverse drug reactions.

Arginase 1, an enzyme induced by Th2 cytokines, is a hallmark of alternatively activated macrophages and is responsible for the hydrolysis of L-arginine into ornithine, the building block for the production of polyamines. Upregulation of arginase 1 has been observed in a variety of diseases, but the mechanisms by which arginase contributes to pathology are not well understood. We reveal here a unique role for arginase 1 in the pathogenesis of nonhealing leishmaniasis, a prototype Th2 disease, and demonstrate that the activity of this enzyme promotes pathology and uncontrolled growth of Leishmania parasites in vivo. Inhibition of arginase activity during the course of infection has a clear therapeutic effect, as evidenced by markedly reduced pathology and efficient control of parasite replication. Despite the clear amelioration of the disease, this treatment does not alter the Th2 response. To address the underlying mechanisms, the arginase-induced L-arginine catabolism was investigated and the results demonstrate that arginase regulates parasite growth directly by affecting the polyamine synthesis in macrophages.