Identification of R-Spondin Gene Signature Predictive of Metastatic Progression in BRAFV600E-Positive Papillary Thyroid Cancer

Papillary thyroid carcinoma (PTC) is the most common malignancy of the thyroid gland and early stages are curable. However, a subset of PTCs shows an unusually aggressive phenotype with extensive lymph node metastasis and higher incidence of locoregional recurrence. In this study, we investigated a large cohort of PTC cases with an unusual aggressive phenotype using a high-throughput RNA sequencing (RNA-Seq) to identify differentially regulated genes associated with metastatic PTC. All metastatic PTC with mutated BRAF (V600E) but not BRAF wild-type expressed an up-regulation of R-Spondin Protein 4 (RSPO4) concomitant with an upregulation of genes involved in focal adhesion and cell-extracellular matrix signaling. Further immunohistochemistry validation confirmed the upregulation of these target genes in metastatic PTC cases. Preclinical studies using established PTC cell lines support that RSPO4 overexpression is associated with BRAF V600E mutation and is a critical upstream event that promote activation of kinases of focal adhesion signaling known to drive cancer cell locomotion and invasion. This finding opens up the potential of co-targeting B-Raf, RSPO and focal adhesion proteins as a pharmacological approach for aggressive BRAF V600E PTC

Characterization of fluorescein arsenical hairpin (FIAsH) as a probe for single-molecule fluorescence spectroscopy

Sherpa Romeo green journal. Open access article. Creative Commons Attribution 4.0 International License (CC BY 4.0) appliesIn recent years, new labelling strategies have been developed that involve the genetic insertion of small amino-acid sequences for specific attachment of small organic fluorophores. Here, we focus on the tetracysteine FCM motif (FLNCCPGCCMEP), which binds to fluorescein arsenical hairpin (FlAsH), and the ybbR motif (TVLDSLEFIASKLA) which binds fluorophores conjugated to Coenzyme A (CoA) via a phosphoryl transfer reaction. We designed a peptide containing both motifs for orthogonal labelling with FlAsH and Alexa647 (AF647). Molecular dynamics simulations showed that both motifs remain solvent-accessible for labelling reactions. Fluorescence spectra, correlation spectroscopy and anisotropy decay were used to characterize labelling and to obtain photophysical parameters of free and peptide-bound FlAsH. The data demonstrates that FlAsH is a viable probe for single-molecule studies. Single-molecule imaging confirmed dual labeling of the peptide with FlAsH and AF647. Multiparameter single-molecule Förster Resonance Energy Transfer (smFRET) measurements were performed on freely diffusing peptides in solution. The smFRET histogram showed different peaks corresponding to different backbone and dye orientations, in agreement with the molecular dynamics simulations. The tandem of fluorophores and the labelling strategy described here are a promising alternative to bulky fusion fluorescent proteins for smFRET and single-molecule tracking studies of membrane proteins.Ye

Association of fluorescent protein pairs and it's significant impact on fluorescence and energy transfer

Fluorescent proteins (FPs) are commonly used in pairs to monitor dynamic biomolecular events through changes in proximity via distance dependent processes such as Förster resonance energy transfer (FRET). The impact of FP association is assessed by predicting dimerization sites in silico and stabilizing the dimers by bio‐orthogonal covalent linkages. In each tested case dimerization changes inherent fluorescence, including FRET. GFP homodimers demonstrate synergistic behavior with the dimer being brighter than the sum of the monomers. The homodimer structure reveals the chromophores are close with favorable transition dipole alignments and a highly solvated interface. Heterodimerization (GFP with Venus) results in a complex with ≈87% FRET efficiency, significantly below the 99.7% efficiency predicted. A similar efficiency is observed when the wild‐type FPs are fused to a naturally occurring protein–protein interface system. GFP complexation with mCherry results in loss of mCherry fluorescence. Thus, simple assumptions used when monitoring interactions between proteins via FP FRET may not always hold true, especially under conditions whereby the protein–protein interactions promote FP interaction

Sequence dependent variations in RNA duplex are related to non-canonical hydrogen bond interactions in dinucleotide steps

Background: Sequence determines the three-dimensional structure of RNAs, and thereby plays an important role in carrying out various biological functions. RNA duplexes containing Watson-Crick (WC) basepairs, interspersed with non-Watson-Crick basepairs, are the dominant structural unit and form the scaffold for the 3-dimensional structure of RNA. It is therefore crucial to understand the geometric variation in the dinucleotide steps that form the helices. We have carried out a detailed analysis of the dinucleotide steps formed by AU and GC Watson-Crick basepairs in RNA structures (both free and protein bound) and compared the results to that seen in DNA. Further, the effect of protein binding on these steps was examined by comparing steps in free RNA structures with protein bound RNA structures. Results: Characteristic sequence dependent geometries are observed for the RR, RY and YR type of dinucleotide steps in RNA. Their geometric parameters show correlated variations that are different from those observed in B-DNA helices. Subtle, but statistically significant differences are seen in roll, slide and average propeller-twist values, between the dinucleotide steps of free RNA and protein bound RNA structures. Many non-canonical cross-strand and intra-strand hydrogen bonds were identified that can stabilise the RNA dinucleotide steps, among which YR steps show presence of many new unreported interactions. Conclusions: Our work provides for the first time a detailed analysis of the conformational preferences exhibited by Watson-Crick basepair containing steps in RNA double helices. Overall, the WC dinucleotide steps show considerable conformational variability. Furthermore, we have identified hydrogen bond interactions in several of the dinucleotide steps that could play a role in determining the preferred geometry, in addition to the intra-basepair hydrogen bonds and stacking interactions. Protein binding affects the conformation of the steps that are in direct contact, as well as allosterically affect the steps that are not in direct physical contact

Właściwości mechaniczne i termiczne kompozytów epoksydowych wzmocnionych tkaniną bawełniano-bambusową i włóknem szklanym

Five-layer epoxy composites consisting of two outer layers made of glass fiber and three inner layers of cotton-bamboo fabric were obtained by compression molding. The influence of cotton-bamboo fabric/glass fiber content (35, 40, 45 and 50 wt%) and the order of stacking laminate layers on the mechanical properties (tensile, flexural, compressive, impact strength), thermal properties (TGA) and structure (FTIR, SEM) of the composites was investigated. The best mechanical and thermal properties were obtained with the content of 45 wt% cotton-bamboo fabric/glass fiber.Metodą prasowania tłocznego otrzymano pięciowarstwowe kompozyty epoksydowe składające się z dwóch warstw zewnętrznych wykonanych z włókna szklanego oraz trzech wewnętrznych z tkaniny bawełniano-bambusowej. Zbadano wpływ zawartości włókna szklanego (35, 40, 45 i 50% mas) oraz kolejności układania warstw laminatu na właściwości mechaniczne (wytrzymałość na rozciąganie, zginanie i ściskanie oraz udarność), termiczne (TGA) oraz strukturę (FTIR, SEM) kompozytów. Najlepsze właściwości mechaniczne i termiczne uzyskano przy zawartości 45% mas. włókna szklanego

Insights into the Structural Dynamics of Nucleocytoplasmic Transport of tRNA by Exportin-t

Exportin-t (Xpot) transports mature 5'- and 3'-end processed tRNA from the nucleus to the cytoplasm by associating with a small G-protein Ran (RAs-related nuclear protein), in the nucleus. The release of tRNA in cytoplasm involves RanGTP hydrolysis. Despite the availability of crystal structures of nuclear and cytosolic forms of Xpot, the molecular details regarding the sequential events leading to tRNA release and subsequent conformational changes occurring in Xpot remain unknown. We have performed a combination of classical all-atom and accelerated molecular dynamics simulations on a set of complexes involving Xpot to study a range of features including conformational flexibility of free and cargo-bound Xpot and functionally critical contacts between Xpot and its cargo. The systems investigated include free Xpot and its different complexes, bound either to Ran (GTP/GDP) or tRNA or both. This approach provided a statistically reliable estimate of structural dynamics of Xpot after cargo release. The mechanistic basis for Xpot opening after cargo release has been explained in terms of dynamic structural hinges, about which neighboring region could be displaced to facilitate the nuclear to cytosolic state transition. Post-RanGTP hydrolysis, a cascade of events including local conformational change in RanGTP and loss of critical contacts at Xpot/tRNA interface suggest factors responsible for eventual release of tRNA. The level of flexibility in different Xpot complexes varied depending on the arrangement of individual HEAT repeats. Current study provides one of the most comprehensive and robust analysis carried out on this protein using molecular dynamics schemes

Stacking Interactions in RNA and DNA: Roll-Slide Energy Hyperspace for Ten Unique Dinucleotide Steps

Understanding dinucleotide sequence directed structures of nuleic acids and their variability from experimental observation remained ineffective due to unavailability of statistically meaningful data. We have attempted to understand this from energy scan along twist, roll, and slide degrees of freedom which are mostly dependent on dinucleotide sequence using ab initio density functional theory. We have carried out stacking energy analysis in these dinucleotide parameter phase space for all ten unique dinucleotide steps in DNA and RNA using DFT-D by B97X-D/6-31G(2d,2p), which appears to satisfactorily explain conformational preferences for AU/AU step in our recent study. We show that values of roll, slide, and twist of most of the dinucleotide sequences in crystal structures fall in the low energy region. The minimum energy regions with large twist values are associated with the roll and slide values of B-DNA, whereas, smaller twist values correspond to higher stability to RNA and A-DNA like conformations. Incorporation of solvent effect by CPCM method could explain the preference shown by some sequences to occur in B-DNA or A-DNA conformations. Conformational preference of BII sub-state in B-DNA is preferentially displayed mainly by pyrimidine-purine steps and partly by purine-purine steps. The purine-pyrimidine steps show largest effect of 5-methyl group of thymine in stacking energy and the introduction of solvent reduces this effect significantly. These predicted structures and variabilities can explain the effect of sequence on DNA and RNA functionality. (c) 2014 Wiley Periodicals, Inc. Biopolymers 103: 134-147, 2015

MolBridge: a program for identifying nonbonded interactions in small molecules and biomolecular structures

Identification and analysis of nonbonded interactions within a molecule and with the surrounding molecules are an essential part of structural studies, given the importance of these interactions in defining the structure and function of any supramolecular entity. MolBridge is an easy to use algorithm based purely on geometric criteria that can identify all possible nonbonded interactions, such as hydrogen bond, halogen bond, cation-pi, pi-pi and van der Waals, in small molecules as well as biomolecules. The user can either upload three-dimensional coordinate files or enter the molecular ID corresponding to the relevant database. The program is available in a standalone form and as an interactive web server with Jmol and JME incorporated into it. The program is freely downloadable and the web server version is also available at http://nucleix.mbu.iisc.ernet.in/molbridge/index.php

Energy hyperspace for stacking interaction inAU/AU dinucleotide step: dispersion-corrected density functional theory study

Double helical structures of DNA and RNA are mostly determined by base pair stacking interactions, which give them the base sequence-directed features, such as small roll values for the purine–pyrimidine steps. Earlier attempts to characterize stacking interactions were mostly restricted to calculations on fiber diffraction geometries or optimized structure using ab initio calculations lacking variation in geometry to comment on rather unusual large roll values observed in AU/AU base pair step in crystal structures of RNA double helices. We have generated stacking energy hyperspace by modeling geometries with variations along the important degrees of freedom, roll, and slide, which were chosen via statistical analysis as maximally sequence dependent. Corresponding energy contours were constructed by several quantum chemical methods including dispersion corrections. This analysis established the most suitable methods for stacked base pair systems despite the limitation imparted by number of atom in a base pair step to employ very high level of theory. All the methods predict negative roll value and near-zero slide to be most favorable for the purine–pyrimidine steps, in agreement with Calladine's steric clash based rule. Successive base pairs in RNA are always linked by sugar–phosphate backbone with C3′-endo sugars and this demands C1′–C1′ distance of about 5.4 Å along the chains. Consideration of an energy penalty term for deviation of C1′–C1′ distance from the mean value, to the recent DFT-D functionals, specifically ωB97X-D appears to predict reliable energy contour for AU/AU step. Such distance-based penalty improves energy contours for the other purine–pyrimidine sequences also