Two distinct binding modes provide the RNA-binding protein RbFox with extraordinary sequence specificity.

Specificity of RNA-binding proteins for target sequences varies considerably. Yet, it is not understood how certain few proteins achieve markedly higher sequence specificity than most others. Here we show that the RNA Recognition Motif of RbFox accomplishes extraordinary sequence specificity by employing functionally and structurally distinct binding modes. Affinity measurements of RbFox for all binding site variants reveal the existence of two distinct binding modes. The first exclusively accommodates cognate and closely related RNAs with high affinity. The second mode accommodates all other RNAs with reduced affinity by imposing large thermodynamic penalties on non-cognate sequences. NMR studies indicate marked structural differences between the two binding modes, including large conformational rearrangements distant from the RNA-binding site. Distinct binding modes by a single RNA-binding module explain extraordinary sequence selectivity and reveal an unknown layer of functional diversity, cross talk and regulation in RNA-protein interactions.

Drug-Like Small Molecules That Inhibit Expression of the Oncogenic MicroRNA-21.

We report the discovery of drug-like small molecules that bind specifically to the precursor of the oncogenic and pro-inflammatory microRNA-21 with mid-nanomolar affinity. The small molecules target a local structure at the Dicer cleavage site and induce distinctive structural changes in the RNA, which correlate with specific inhibition of miRNA processing. Structurally conservative single nucleotide substitutions eliminate the conformational change induced by the small molecules, which is also not observed in other miRNA precursors. The most potent of these compounds reduces cellular proliferation and miR-21 levels in cancer cell lines without inhibiting kinases or classical receptors, while closely related compounds without this specific binding activity are inactive in cells. These molecules are highly ligand-efficient (MW < 330) and display specific biochemical and cellular activity by suppressing the maturation of miR-21, thereby providing an avenue toward therapeutic development in multiple diseases where miR-21 is abnormally expressed.

A functional SNP regulates E-cadherin expression by dynamically remodeling the 3D structure of a promoter-associated non-coding RNA transcript.

Transcription of E-cadherin, a tumor suppressor that plays critical roles in cell adhesion and the epithelial-mesenchymal transition, is regulated by a promoter-associated non-coding RNA (paRNA). The sense-oriented paRNA (S-paRNA) includes a functional C/A single nucleotide polymorphism (SNP rs16260). The A-allele leads to decreased transcriptional activity and increased prostate cancer risk. The polymorphic site is known to affect binding of a microRNA-guided Argonaute 1 (AGO1) complex and recruitment of chromatin-modifying enzymes to silence the promoter. Yet the SNP is distant from the microRNA-AGO1 binding domain in both primary sequence and secondary structure, raising the question of how regulation occurs. Here we report the 3D NMR structure of the 104-nucleotide domain of the S-paRNA that encompasses the SNP and the microRNA-binding site. We show that the A to C change alters the locally dynamic and metastable structure of the S-paRNA, revealing how the single nucleotide mutation regulates the E-cadherin promoter through its effect on the non-coding RNA structure.

A Slow Dynamic RNA Switch Regulates Processing of microRNA-21.

The microRNAs are non-coding RNAs which post-transcriptionally regulate the expression of many eukaryotic genes, and whose dysregulation is a driver of human disease. Here we report the discovery of a very slow (0.1 s-1) conformational rearrangement at the Dicer cleavage site of pre-miR-21, which regulates the relative concentration of readily- and inefficiently-processed RNA structural states. We show that this dynamic switch is affected by single nucleotide mutations and can be biased by small molecule and peptide ligands, which can direct the microRNA to occupy the inefficiently processed state and reduce processing efficiency. This result reveals a new mechanism of RNA regulation and suggests a chemical approach to suppressing or activating pathogenic microRNAs by selective stabilization of their unprocessed or processed states.

Structure of the dengue virus RNA promoter.

Dengue virus, a single-stranded positive sense RNA virus, is the most prevalent mosquito-borne pathogen in the world. Like all RNA viruses, it uses conserved structural elements within its genome to control essential replicative steps. A 70 nt stem-loop RNA structure (called SLA), found at the 5'-end of the genome of all flaviviruses, functions as the promoter for viral replication. This highly conserved structure interacts with the viral polymerase NS5 to initiate RNA synthesis. Here, we report the NMR structure of a monomeric SLA from dengue virus serotype 1, assembled to high-resolution from independently folded structural elements. The DENV1 SLA has an L-shaped structure, where the top and side helices are coaxially stacked, and the bottom helix is roughly perpendicular to them. Because the sequence is highly conserved among different flavivirus genomes, it is very likely that the three-dimensional fold and local structure of SLA are also conserved among flaviviruses and required for efficient replication. This work provides structural insight into the dengue promoter and provides the foundation for the discovery of new antiviral drugs that target this essential replicative step.

Innovative Eco-Friendly Conductive Ink Based on Carbonized Lignin for the Production of Flexible and Stretchable Bio-Sensors.

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.

Genome-wide association studies reveal the role of polymorphisms affecting factor H binding protein expression in host invasion by Neisseria meningitidis.

Many invasive bacterial diseases are caused by organisms that are ordinarily harmless components of the human microbiome. Effective interventions against these microbes require an understanding of the processes whereby symbiotic or commensal relationships transition into pathology. Here, we describe bacterial genome-wide association studies (GWAS) of Neisseria meningitidis, a common commensal of the human respiratory tract that is nevertheless a leading cause of meningitis and sepsis. An initial GWAS discovered bacterial genetic variants, including single nucleotide polymorphisms (SNPs), associated with invasive meningococcal disease (IMD) versus carriage in several loci across the meningococcal genome, encoding antigens and other extracellular components, confirming the polygenic nature of the invasive phenotype. In particular, there was a significant peak of association around the fHbp locus, encoding factor H binding protein (fHbp), which promotes bacterial immune evasion of human complement by recruiting complement factor H (CFH) to the meningococcal surface. The association around fHbp with IMD was confirmed by a validation GWAS, and we found that the SNPs identified in the validation affected the 5' region of fHbp mRNA, altering secondary RNA structures, thereby increasing fHbp expression and enhancing bacterial escape from complement-mediated killing. This finding is consistent with the known link between complement deficiencies and CFH variation with human susceptibility to IMD. These observations demonstrate the importance of human and bacterial genetic variation across the fHbp:CFH interface in determining IMD susceptibility, the transition from carriage to disease.

Decision making based on hybrid modeling approach applied to cellulose acetate based historical films conservation.

Preserving culture heritage cellulose acetate-based historical films is a challenge due to the long-term instability of these complex materials and a lack of prediction models that can guide conservation strategies for each particular film. In this work, a cellulose acetate degradation model is proposed as the basis for the selection of appropriate strategies for storage and conservation for each specimen, considering its specific information. Due to the formulation complexity and diversity of cellulose acetate-based films produced over the decades, we hereby propose a hybrid modeling approach to describe the films degradation process. The problem is addressed by a hybrid model that uses as a backbone a first-principles based model to describe the degradation kinetics of the pure cellulose diacetate polymer. The mechanistic model was successfully adapted to fit experimental data from accelerated aging of plasticized films. The hybrid model considers then the specificity of each historical film via the development of two chemometric models. These models resource on gas release data, namely acetic acid, and descriptors of the films (manufacturing date, AD-strip value and film type) to assess the current polymer degradation state and estimate the increase in the degradation rate. These estimations are then conjugated with storage conditions (e.g., temperature and relative humidity, presence of adsorbent in the film's box) and used to feed the mechanistic model to provide the required time degradation simulations. The developed chemometric models provided predictions with accuracy more than 87%. We have found that the storage archive as well as the manufacturing company are not determining factors for conservation but rather the manufacturing date, off gas data as well as the film type. In summary, this hybrid modeling was able to develop a practical tool for conservators to assess films conservation state and to design storage and conservation policies that are best suited for each cultural heritage film.

Efficient NMR Screening Approach to Discover Small Molecule Fragments Binding Structured RNA.

We describe a scalable nuclear magnetic resonance (NMR) screening approach to identify and prioritize small molecule fragments that bind to structured RNAs. This approach is target agnostic and, therefore, amenable to many RNA structures and libraries, and it provides initial hits for further synthetic elaboration and structure-based drug discovery efforts. We demonstrate the approach on the pre-miR-21 stem-loop, which is of significant interest in oncology and metabolic diseases. We screened the pre-miR-21 hairpin using a small (420 compounds) commercially available fragment library and identified 18 hits in the first round of triage screening. This was further refined to four fragments which passed all screening cascade filters. Among these four hits, a thiadiazole fragment was demonstrated to bind the Dicer cleavage site of pre-miR-21 by target-detected NMR experiments and through the observation of clear intermolecular NOEs.

High blood flow shear stress values are associated with circulating tumor cells cluster disaggregation in a multi-channel microfluidic device.

Metastasis represents a dynamic succession of events involving tumor cells which disseminate through the organism via the bloodstream. Circulating tumor cells (CTCs) can flow the bloodstream as single cells or as multicellular aggregates (clusters), which present a different potential to metastasize. The effects of the bloodstream-related physical constraints, such as hemodynamic wall shear stress (WSS), on CTC clusters are still unclear. Therefore, we developed, upon theoretical and CFD modeling, a new multichannel microfluidic device able to simultaneously reproduce different WSS characterizing the human circulatory system, where to analyze the correlation between SS and CTC clusters behavior. Three physiological WSS levels (i.e. 2, 5, 20 dyn/cm2) were generated, reproducing values typical of capillaries, veins and arteries. As first validation, triple-negative breast cancer cells (MDA-MB-231) were injected as single CTCs showing that higher values of WSS are correlated with a decreased viability. Next, the SS-mediated disaggregation of CTC clusters was computationally investigated in a vessels-mimicking domain. Finally, CTC clusters were injected within the three different circuits and subjected to the three different WSS, revealing that increasing WSS levels are associated with a raising clusters disaggregation after 6 hours of circulation. These results suggest that our device may represent a valid in vitro tool to carry out systematic studies on the biological significance of blood flow mechanical forces and eventually to promote new strategies for anticancer therapy.

A microbial sensor platform based on bacterial bioluminescence (luxAB) and green fluorescent protein (gfp) reporters for in situ monitoring of toxicity of wastewater nitrification process dynamics.

To avoid the upset of nitrification process in wastewater treatment plants, monitoring of influent toxic chemicals is essential for stable operation. Toxic chemical compounds can interfere with the biological nitrogen removal, thus affecting plant efficiency and effluent water quality. Here we report the development of fluorescence and bioluminescence bioassays, based on E. coli engineered to contain the promoter region of ammonia oxidation pathway (AmoA1) of Nitrosomonas europaea and a reporter gene (lux or gfp). The fluorescence or bioluminescence signal was measured with newly designed optical devices. The microbial sensors were tested and validated at different concentrations of nitrification-inhibiting compounds such as allylthiourea, phenol, and mercury. The signal decrease was immediate and proportional to inhibitor concentration. The developed bacterial bioassays could detect the inhibition of the nitrification process in wastewater for allylthiourea concentrations of 1 µg/L for E.coli pMosaico-Pamo-gfp and 0.5 µg/L for E.coli pMosaico-Pamo-luxAB. The results were confirmed using water from a wastewater plant, containing nitrification-inhibiting compounds.

3D fluid-dynamic ovarian cancer model resembling systemic drug administration for efficacy assay.

Recently, 3D in vitro cancer models have become important alternatives to animal tests for establishing the efficacy of anticancer treatments. In this work, 3D SKOV-3 cell-laden alginate hydrogels were established as ovarian tumor models and cultured within a fluid-dynamic bioreactor (MIVO®) device able to mimic the capillary flow dynamics feeding the tumor. Cisplatin efficacy tests were performed within the device over time and compared with (i) the in vitro culture under static conditions and (ii) a xenograft mouse model with SKOV-3 cells, by monitoring and measuring cell proliferation or tumor regression, respectively, over time. After one week of treatment with 10 µM cisplatin, viability of cells within the 3D hydrogels cultured under static conditions remained above 80%. In contrast, the viability of cells within the 3D hydrogels cultured within dynamic MIVO® decreased by up to 50%, and very few proliferating Ki67-positive cells were observed through immunostaining. Analysis of drug diffusion, confirmed by computational analysis, explained that these results are due to different cisplatin diffusion mechanisms in the two culture conditions. Interestingly, the outcome of the drug efficacy test in the xenograft model was about 44% of tumor regression after 5 weeks, as predicted in a shorter time in the fluid-dynamic in vitro tests carried out in the MIVO® device. These results indicate that the in vivo-like dynamic environment provided by the MIVO® device allows to better model the 3D tumor environment and predict in vivo drug efficacy than a static in vitro model.

Quantum dots functionalised artificial peptides bioinspired to the D1 protein from the Photosystem II of Chlamydomonas reinhardtii for endocrine disruptor optosensing.

Herein we describe the design and synthesis of novel artificial peptides mimicking the plastoquinone binding niche of the D1 protein from the green photosynthetic alga Chlamydomonas reinhardtii, also able to bind herbicides. In particular, molecular dynamics (MD) simulations were performed to model in silico the behaviour of three peptides, D1Pep70-H, D1Pep70-S264K and D1Pep70-S268C, as genetic variants with different affinity towards the photosynthetic herbicide atrazine. Then the photosynthetic peptides were functionalised with quantum dots for the development of a hybrid optosensor for the detection of atrazine, one of the most employed herbicides for weed control in agriculture as well as considered as a putative endocrine disruptor case study. The excellent agreement between computational and experimental results self consistently shows resistance or super-sensitivity toward the atrazine target, with detection limits in the µg/L concentration range, meeting the requirements of E.U. legislation.

High-Tech and Nature-Made Nanocomposites and Their Applications in the Field of Sensors and Biosensors for Gas Detection.

Gas sensors have been object of increasing attention by the scientific community in recent years. For the development of the sensing element, two major trends seem to have appeared. On one hand, the possibility of creating complex structures at the nanoscale level has given rise to ever more sensitive sensors based on metal oxides and metal-polymer combinations. On the other hand, gas biosensors have started to be developed, thanks to their intrinsic ability to be selective for the target analyte. In this review, we analyze the recent progress in both areas and underline their strength, current problems, and future perspectives.

3D Perfusable Hydrogel Recapitulating the Cancer Dynamic Environment to in Vitro Investigate Metastatic Colonization.

Metastasis is a dynamic process involving the dissemination of circulating tumor cells (CTCs) through blood flow to distant tissues within the body. Nevertheless, the development of an in vitro platform that dissects the crucial steps of metastatic cascade still remains a challenge. We here developed an in vitro model of extravasation composed of (i) a single channel-based 3D cell laden hydrogel representative of the metastatic site, (ii) a circulation system recapitulating the bloodstream where CTCs can flow. Two polymers (i.e., fibrin and alginate) were tested and compared in terms of mechanical and biochemical proprieties. Computational fluid-dynamic (CFD) simulations were also performed to predict the fluid dynamics within the polymeric matrix and, consequently, the optimal culture conditions. Next, once the platform was validated through perfusion tests by fluidically connecting the hydrogels with the external circuit, highly metastatic breast cancer cells (MDA-MB-231) were injected and exposed to physiological wall shear stress (WSS) conditions (5 Dyn/cm2) to assess their migration toward the hydrogel. Results indicated that CTCs arrested and colonized the polymeric matrix, showing that this platform can be an effective fluidic system to model the first steps occurring during the metastatic cascade as well as a potential tool to in vitro elucidate the contribution of hemodynamics on cancer dissemination to a secondary site.

The Long Noncoding RNA CCAT2 Induces Chromosomal Instability Through BOP1-AURKB Signaling.

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, γ-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.

NMR structure of Dengue West Nile viruses stem-loop B: A key cis-acting element for flavivirus replication.

Flaviviruses are major emerging human pathogenic viruses that pose a persistent and growing menace to global health. They are enveloped single-stranded RNA viruses with positive polarity transmitted by arthropod vectors like mosquitoes or ticks, responsible for a significant and growing number of human deaths and illnesses. The 5'- and 3'-untranslated regions (UTRs) are highly structured and contain conserved cis-acting RNA elements that participate in viral translation, replication and host adaptation. Despite their role in fiaviviruses replication, few high-resolution structural studies have investigated the RNA elements required for viral replication. Here we report the NMR structures of stem-loop B from WNV and DENV4 viruses. Because this element is required for cyclization of the genome and the activity of the replicative viral enzymes, viral replication rates are sensitive to even small changes in these RNAs. Therefore, this work provides structural insight into a new drug target to reduce flavivirus replication rates.

Primordial emergence of a nucleic acid-binding protein via phase separation and statistical ornithine-to-arginine conversion.

De novo emergence demands a transition from disordered polypeptides into structured proteins with well-defined functions. However, can polypeptides confer functions of evolutionary relevance, and how might such polypeptides evolve into modern proteins? The earliest proteins present an even greater challenge, as they were likely based on abiotic, spontaneously synthesized amino acids. Here we asked whether a primordial function, such as nucleic acid binding, could emerge with ornithine, a basic amino acid that forms abiotically yet is absent in modern-day proteins. We combined ancestral sequence reconstruction and empiric deconstruction to unravel a gradual evolutionary trajectory leading from a polypeptide to a ubiquitous nucleic acid-binding protein. Intermediates along this trajectory comprise sequence-duplicated functional proteins built from 10 amino acid types, with ornithine as the only basic amino acid. Ornithine side chains were further modified into arginine by an abiotic chemical reaction, improving both structure and function. Along this trajectory, function evolved from phase separation with RNA (coacervates) to avid and specific double-stranded DNA binding. Our results suggest that phase-separating polypeptides may have been an evolutionary resource for the emergence of early proteins, and that ornithine, together with its postsynthesis modification to arginine, could have been the earliest basic amino acids.

An evolutionarily conserved RNA structure in the functional core of the lincRNA Cyrano.

The wide prevalence and regulated expression of long noncoding RNAs (lncRNAs) highlight their functional roles, but the molecular basis for their activities and structure-function relationships remains to be investigated, with few exceptions. Among the relatively few lncRNAs conserved over significant evolutionary distances is the long intergenic noncoding RNA (lincRNA) Cyrano (orthologous to human OIP5-AS1), which contains a region of 300 highly conserved nucleotides within tetrapods, which in turn contains a functional stretch of 26 nt of deep conservation. This region binds to and facilitates the degradation of the microRNA miR-7, a short ncRNA with multiple cellular functions, including modulation of oncogenic expression. We probed the secondary structure of Cyrano in vitro and in cells using chemical and enzymatic probing, and validated the results using comparative sequence analysis. At the center of the functional core of Cyrano is a cloverleaf structure maintained over the >400 million years of divergent evolution that separates fish and primates. This strikingly conserved motif provides interaction sites for several RNA-binding proteins and masks a conserved recognition site for miR-7. Conservation in this region strongly suggests that the function of Cyrano depends on the formation of this RNA structure, which could modulate the rate and efficiency of degradation of miR-7.

Comparative structure, dynamics and evolution of acyl-carrier proteins from Borrelia burgdorferi, Brucella melitensis and Rickettsia prowazekii.

Acyl carrier proteins (ACPs) are small helical proteins found in all kingdoms of life, primarily involved in fatty acid and polyketide biosynthesis. In eukaryotes, ACPs are part of the fatty acid synthase (FAS) complex, where they act as flexible tethers for the growing lipid chain, enabling access to the distinct active sites in FAS. In the type II synthesis systems found in bacteria and plastids, these proteins exist as monomers and perform various processes, from being a donor for synthesis of various products such as endotoxins, to supplying acyl chains for lipid A and lipoic acid FAS (quorum sensing), but also as signaling molecules, in bioluminescence and activation of toxins. The essential and diverse nature of their functions makes ACP an attractive target for antimicrobial drug discovery. Here, we report the structure, dynamics and evolution of ACPs from three human pathogens: Borrelia burgdorferi, Brucella melitensis and Rickettsia prowazekii, which could facilitate the discovery of new inhibitors of ACP function in pathogenic bacteria.