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In this work, a system consisting of two resistive sensors working in tandem to detect and quantify the acetic acid released during the degradation of cellulose acetate-based ancient cinematographic and photographic films is presented. Acetic acid must be constantly monitored to prevent reaching concentrations at which autocatalytic degradation processes begin. The sensors are constituted by a thin layer of metal oxide (tungsten oxide and tin oxide) deposited over an interdigitated electrode capable of being heated, chosen to maximize the array response towards acetic acid vapors. The signals obtained from the sensor array are mathematically processed to reduce the background signal due to interferent gases produced during degradation of ancient cinematographic films. The sensor array reported a LOD of 30 ppb for acetic acid, with a linearity range up to 30 ppm. Finally, the sensor array was tested with different cinematographic and photographic film samples made of cellulose acetate, whose degradation state and acetic acid production was validated using the conventional technique (A-D strips). The presented array is suitable for remote monitoring large number of films in collections since, compared to the official technique, it has a lower detection limit (30 ppb vs. 500 ppb) and is much quicker in providing accurate acetic acid concentration in the film boxes (15 min vs. 24 h).

In this study, we report a novel way to produce carbon-based conductive inks for electronic and sensor technology applications. Carbonized lignin, obtained from the waste products of the Eucalyptus globulus tree paper industry, was used to produce a stable conductive ink. To this end, liquid-phase compositions were tested with different amounts of carbonized lignin powder to obtain an ink with optimal conductivity and rheological properties for different possible uses. The combination that showed the best properties, both regarding electrochemical properties and green compatibility of the materials employed, was cyclohexanone/cellulose acetate/carbonized lignin 5% (w/w), which was used to produce screen-printed electrodes. The electrodes were characterized from a structural and electrochemical point of view, resulting in an electrochemically active area of 0.1813 cm2, compared to the electrochemically active area of 0.1420 cm2 obtained by employing geometrically similar petroleum-based screen-printed electrodes and, finally, their performance was demonstrated for the quantification of uric acid, with a limit of detection of 0.3 μM, and their biocompatibility was assessed by testing it with the laccase enzyme and achieving a limit of detection of 2.01 μM for catechol as the substrate. The results suggest that the developed ink could be of great use in both sensor and electronic industries, reducing the overall ecological impact of traditionally used petroleum-based inks.

The RNA recognition motif (RRM), which is the most abundant RNA-binding motif in eukaryotes, is a well-structured domain of about 90 amino acids, yet the β2β3 hairpin, corresponding to strands 2 and 3 of the β-sheet, and the intervening loop make essential interactions with RNA in many RRM complexes. A series of small cyclic peptide mimics of the β2β3 hairpin of Rbfox2 protein that recognize the terminal loop of precursor miR-20b have been designed to investigate whether the full RNA-binding protein can be mimicked with a minimal structurally preorganized peptide. Within a small library of seven cyclic peptides, a peptide with low-micromolar affinity for the miR-20b precursor was found. NMR spectroscopy titration data suggest that this peptide specifically targets the apical loop of pre-miR-20b. This work shows that it is possible to mimic RNA-binding proteins with designed stable peptides, which provide a starting point for designing or evolving small peptide mimetics of RRM proteins.

This report documents the program and the outcomes of Dagstuhl Seminar 19342 ``Advances and Challenges in Protein-RNA Recognition, Regulation and Prediction''.

The RNA recognition motif (RRM) is the most common RNA binding domain across eukaryotic proteins. It is therefore of great value to engineer its specificity to target RNAs of arbitrary sequence. This was recently achieved for the RRM in Rbfox protein, where four mutations R118D, E147R, N151S, and E152T were designed to target the precursor to the oncogenic miRNA 21. Here, we used a variety of molecular dynamics-based approaches to predict specific interactions at the binding interface. Overall, we have run approximately 50 microseconds of enhanced sampling and plain molecular dynamics simulations on the engineered complex as well as on the wild-type Rbfox·pre-miRNA 20b from which the mutated systems were designed. Comparison with the available NMR data on the wild type molecules (protein, RNA, and their complex) served to establish the accuracy of the calculations. Free energy calculations suggest that further improvements in affinity and selectivity are achieved by the S151T replacement., Author summary RNA is an outstanding target for oncological intervention. Engineering the most common RNA binding motif in human proteins (called RRM) so as to bind to a specific RNA has an enormous pharmacological potential. Yet, it is highly non trivial to design RRM-bearing protein variants with RNA selectivity and affinity sufficiently high for clinical applications. Here we present an extensive molecular simulation study which shed light on the exquisite molecular recognition of the empirically-engineered complex between the RRM-bearing protein Rbfox and its RNA target pre-miR21. The simulations allow predicting a variant, the S151T, which may lead to further enhancement of selectivity and affinity for pre-miR21.

This report documents the program and the outcomes of Dagstuhl Seminar 17252 "Computational Challenges in RNA-Based Gene Regulation: Protein-RNA Recognition, Regulation and Prediction".

Solution-state nuclear magnetic resonance (NMR) is a versatile tool for the study of binding interactions between small molecules and macromolecular targets. We applied ligand-based NMR techniques to the study of human thymidylate synthase (hTS) using known nanomolar inhibitors and a library of small molecule fragments. Screening by NMR led to the rapid identification of ligand pairs that bind in proximal sites within the cofactor-binding pocket of hTS. Screening hits were used as search criteria within commercially available sources, and a subset of catalog analogs were tested for potency by in vitro assay and binding affinity by quantitative saturation transfer difference (STD)-NMR titration. Two compounds identified by this approach possess low micromolar affinity and potency, as well as excellent binding efficiency against hTS. Relative binding orientations for both leads were modeled using AutoDock, and the most likely bound conformations were validated using experimentally derived STD-NMR binding epitope data. These ligands represent novel starting points for fragment-based drug design of non-canonical TS inhibitors, and their binding epitopes highlight important and previously unexploited interactions with conserved residues in the cofactor-binding site.

The functions of most RNA molecules are critically dependent on the distinct local dynamics that characterize secondary structure and tertiary interactions and on structural changes that occur upon binding by proteins and small molecule ligands. Measurements of RNA dynamics at nucleotide resolution set the foundation for understanding the roles of individual residues in folding, catalysis, and ligand recognition. In favorable cases, local order in small RNAs can be quantitatively analyzed by NMR in terms of a generalized order parameter, S2. Alternatively, SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) chemistry measures local nucleotide flexibility in RNAs of any size using structure-sensitive reagents that acylate the 2'-hydroxyl position. In this work, we compare per-residue RNA dynamics, analyzed by both S2 and SHAPE, for three RNAs: the HIV-1 TAR element, the U1A protein binding site, and the Tetrahymena telomerase stem loop 4. We find a very strong correlation between the two measurements: nucleotides with high SHAPE reactivities consistently have low S2 values. We conclude that SHAPE chemistry quantitatively reports local nucleotide dynamics and can be used with confidence to analyze dynamics in large RNAs, RNA-protein complexes, and RNAs in vivo.

BACKGROUND & AIMS: Chromosomal instability (CIN) is a carcinogenesis event that promotes metastasis and resistance to therapy by unclear mechanisms. Expression of the colon cancer-associated transcript 2 gene (CCAT2), which encodes a long noncoding RNA (lncRNA), associates with CIN, but little is known about how CCAT2 lncRNA regulates this cancer enabling characteristic.METHODS: We performed cytogenetic analysis of colorectal cancer (CRC) cell lines (HCT116, KM12C/SM, and HT29) overexpressing CCAT2 and colon organoids from C57BL/6N mice with the CCAT2 transgene and without (controls). CRC cells were also analyzed by immunofluorescence microscopy, gamma-H2AX, and senescence assays. CCAT2 transgene and control mice were given azoxymethane and dextran sulfate sodium to induce colon tumors. We performed gene expression array and mass spectrometry to detect downstream targets of CCAT2 lncRNA. We characterized interactions between CCAT2 with downstream proteins using MS2 pull-down, RNA immunoprecipitation, and selective 2'-hydroxyl acylation analyzed by primer extension analyses. Downstream proteins were overexpressed in CRC cells and analyzed for CIN. Gene expression levels were measured in CRC and non-tumor tissues from 5 cohorts, comprising more than 900 patients.RESULTS: High expression of CCAT2 induced CIN in CRC cell lines and increased resistance to 5-fluorouracil and oxaliplatin. Mice that expressed the CCAT2 transgene developed chromosome abnormalities, and colon organoids derived from crypt cells of these mice had a higher percentage of chromosome abnormalities compared with organoids from control mice. The transgenic mice given azoxymethane and dextran sulfate sodium developed more and larger colon polyps than control mice given these agents. Microarray analysis and mass spectrometry indicated that expression of CCAT2 increased expression of genes involved in ribosome biogenesis and protein synthesis. CCAT2 lncRNA interacted directly with and stabilized BOP1 ribosomal biogenesis factor (BOP1). CCAT2 also increased expression of MYC, which activated expression of BOP1. Overexpression of BOP1 in CRC cell lines resulted in chromosomal missegregation errors, and increased colony formation, and invasiveness, whereas BOP1 knockdown reduced viability. BOP1 promoted CIN by increasing the active form of aurora kinase B, which regulates chromosomal segregation. BOP1 was overexpressed in polyp tissues from CCAT2 transgenic mice compared with healthy tissue. CCAT2 lncRNA and BOP1 mRNA or protein were all increased in microsatellite stable tumors (characterized by CIN), but not in tumors with microsatellite instability compared with nontumor tissues. Increased levels of CCAT2 lncRNA and BOP1 mRNA correlated with each other and with shorter survival times of patients.CONCLUSIONS: We found that overexpression of CCAT2 in colon cells promotes CIN and carcinogenesis by stabilizing and inducing expression of BOP1 an activator of aurora kinase B. Strategies to target this pathway might be developed for treatment of patients with microsatellite stable colorectal tumors.

The HIV-1 Tat protein and several small molecules bind to HIV-1 transactivation responsive RNA (TAR) by selecting sparsely populated but pre-existing conformations. Thus, a complete characterization of TAR conformational ensemble and dynamics is crucial to understand this paradigmatic system and could facilitate the discovery of new antivirals targeting this essential regulatory element. We show here that molecular dynamics simulations can be effectively used toward this goal by bridging the gap between functionally relevant time scales that are inaccessible to current experimental techniques. Specifically, we have performed several independent microsecond long molecular simulations of TAR based on one of the most advanced force fields available for RNA, the parmbsc0 AMBER. Our simulations are first validated against available experimental data, yielding an excellent agreement with measured residual dipolar couplings and order parameter S2. This contrast with previous molecular dynamics simulations (Salmon et al., J. Am. Chem. Soc. 2013 135, 5457–5466) based on the CHARMM36 force field, which could achieve only modest accord with the experimental RDC values. Next, we direct the computation toward characterizing the internal dynamics of TAR over the microsecond time scale. We show that the conformational fluctuations observed over this previously elusive time scale have a strong functionally oriented character in that they are primed to sustain and assist ligand binding. Fil: Musiani, Francesco. Scuola Internazionale Superiore di Studi Avanzati; Italia. Università di Bologna; Italia. Helmholtz Gemeinschaft. Forschungszentrum Jülich; Alemania Fil: Rossetti, Giulia. Helmholtz Gemeinschaft. Forschungszentrum Jülich; Alemania Fil: Capece, Luciana. International Centre for Genetic Engineering and Biotechnology; Italia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Gerger, Thomas Martin. Helmholtz Gemeinschaft. Forschungszentrum Jülich; Alemania Fil: Micheletti, Cristian. Scuola Internazionale Superiore di Studi Avanzati; Italia Fil: Varani, Gabriele. University of Washington; Estados Unidos Fil: Carloni, Paolo. Helmholtz Gemeinschaft. Forschungszentrum Jülich; Alemania