Comparative analysis of RNA secondary structure accuracy on predicted RNA 3D models.

RNA structure is conformationally dynamic, and accurate all-atom tertiary (3D) structure modeling of RNA remains challenging with the prevailing tools. Secondary structure (2D) information is the standard prerequisite for most RNA 3D modeling. Despite several 2D and 3D structure prediction tools proposed in recent years, one of the challenges is to choose the best combination for accurate RNA 3D structure prediction. Here, we benchmarked seven small RNA PDB structures (40 to 90 nucleotides) with different topologies to understand the effects of different 2D structure predictions on the accuracy of 3D modeling. The current study explores the blind challenge of 2D to 3D conversions and highlights the performances of de novo RNA 3D modeling from their predicted 2D structure constraints. Our results show that conformational sampling-based methods such as SimRNA and IsRNA1 depend less on 2D accuracy, whereas motif-based methods account for 2D evidence. Our observations illustrate the disparities in available 3D and 2D prediction methods and may further offer insights into developing topology-specific or family-specific RNA structure prediction pipelines.

Free-Energy Landscape and Rate Estimation of the Aromatic Ring Flips in Basic Pancreatic Trypsin Inhibitors Using Metadynamics.

Aromatic side chains (phenylalanine and tyrosine) of a protein flip by 180° around the Cß-Cγ axis (χ2 dihedral of the side chain), producing two symmetry-equivalent states. The study of ring flip dynamics with nuclear magnetic resonance (NMR) experiments helps to understand local conformational fluctuations. Ring flips are categorized as slow (milliseconds and onward) or fast (nanoseconds to near milliseconds) based on timescales accessible to NMR experiments. In this study, we investigated the ability of the infrequent metadynamics approach to estimate the flip rate and discriminate between slow and fast ring flips for eight individual aromatic side chains (F4, Y10, Y21, F22, Y23, F33, Y35, and F45) of the basic pancreatic trypsin inhibitor. Well-tempered metadynamics simulations were performed to estimate the ring-flipping free-energy surfaces for all eight aromatic residues. The results indicate that χ2 as a standalone collective variable (CV) is not sufficient to obtain computationally consistent results. Inclusion of a complementary CV, such as χ1(Cα-Cß), solved the problem for most residues and enabled us to classify fast and slow ring flips. This indicates the importance of librational motions in ring flips. Multiple pathways and mechanisms were observed for residues F4, Y10, and F22. Recrossing events were observed for residues F22 and F33, indicating a possible role of friction effects in ring flipping. The results demonstrate the successful application of infrequent metadynamics to estimate ring flip rates and identify certain limitations of the approach.

Advances in Animal Models and Cutting-Edge Research in Alternatives: Proceedings of the Third International Conference on 3Rs Research and Progress, Vishakhapatnam, 2022.

Animal experimentation has been integral to drug discovery and development and safety assessment for many years, since it provides insights into the mechanisms of drug efficacy and toxicity (e.g. pharmacology, pharmacokinetics and pharmacodynamics). However, due to species differences in physiology, metabolism and sensitivity to drugs, the animal models can often fail to replicate the effects of drugs and chemicals in human patients, workers and consumers. Researchers across the globe are increasingly applying the Three Rs principles by employing innovative methods in research and testing. The Three Rs concept focuses on: the replacement of animal models (e.g. with in vitro and in silico models or human studies), on the reduction of the number of animals required to achieve research objectives, and on the refinement of existing experimental practices (e.g. eliminating distress and enhancing animal wellbeing). For the last two years, Oncoseek Bio-Acasta Health, a 3-D cell culture-based cutting-edge translational biotechnology company, has organised an annual International Conference on 3Rs Research and Progress. This series of global conferences aims to bring together researchers with diverse expertise and interests, and provides a platform where they can share and discuss their research to promote practices according to the Three Rs principles. In November 2022, the 3rd international conference, Advances in Animal Models and Cutting-Edge Research in Alternatives, took place at the GITAM University in Vishakhapatnam (AP, India) in a hybrid format (i.e. online and in-person). These conference proceedings provide details of the presentations, which were categorised under five different topic sessions. It also describes a special interactive session on in silico strategies for preclinical research in oncology, which was held at the end of the first day.

Epitaxial growth of 1D GaN-based heterostructures on various substrates for photonic and energy applications.

GaN is an important III-V semiconductor for a variety of applications owing to its large direct band gap. GaN nanowires (NWs) have demonstrated significant potential as critical building blocks for nanoelectronics and nanophotonic devices, as well as integrated nanosystems. We present a comprehensive analysis of the vapor-liquid-solid (VLS) as a general synthesis technique for NWs on a variety of substrates, the morphological and structural characterization, and applications of GaN NWs in piezoelectric nanogenerators, light-emitting diodes, and solar-driven water splitting. We begin by summarizing the overall VLS growth process of GaN NWs, followed by the growth of NWs on several substrates. Subsequently, we review the various uses of GaN NWs in depth.

High Performance, Stable, and Flexible Piezoelectric Nanogenerator Based on GaN:Mg Nanowires Directly Grown on Tungsten Foil.

Rapid development of micro-electromechanical systems increases the need for flexible and durable piezoelectric nanogenerators (f-PNG) with high output power density. In this study, a high-performance, flexible, and highly stable f-PNG is prepared by directly growing the Mg-doped semi-insulating GaN nanowires (NWs) on a 30-µm-thick tungsten foil using vapor-liquid-solid growth mechanism. The direct growth of NWs on metal foil extends the overall lifetime of the f-PNG. The semi-insulating GaN NWs significantly enhance the piezoelectric performance of the f-PNG by reducing free electron density. Additionally, the direct integration of NWs on the tungsten foil improves the conductivity, resulting in current enhancement (2.5 mA) with an output power density of 13 mW cm-2 . The piezoelectric performance of the f-PNG is investigated under several bending angles, actuation frequencies, continuous vibrations, and airflow velocities. The maximum output voltage exhibited by the f-PNG is 20 V at a bending angle of 155°. The f-PNG is connected to the backside of an index finger to monitor finger bending behavior by changing the current density. Depending on its flexibility and sensitivity, the f-PNG can be used as a health-monitoring sensor to be mounted on joints (fingers, hands, elbows, and knees) to monitor their repeated bending and relaxation.

Advances in Animal Models and Cutting-Edge Research in Alternatives: Proceedings of the Second International Conference on 3Rs Research and Progress, Hyderabad, 2021.

The fact that animal models fail to replicate human disease faithfully is now being widely accepted by researchers across the globe. As a result, they are exploring the use of alternatives to animal models. The time has come to refine our experimental practices, reduce the numbers and eventually replace the animals used in research with human-derived and human-relevant 3-D disease models. Oncoseek Bio-Acasta Health, which is an innovative biotechnology start-up company based in Hyderabad and Vishakhapatnam, India, organises an annual International Conference on 3Rs Research and Progress. In 2021, this conference was on 'Advances in Research Animal Models and Cutting-Edge Research in Alternatives'. This annual conference is a platform that brings together eminent scientists and researchers from various parts of the world, to share recent advances from their research in the field of alternatives to animals including new approach methodologies, and to promote practices to help refine animal experiments where alternatives are not available. This report presents the proceedings of the conference, which was held in hybrid mode (i.e. virtual and in-person) in November 2021.

Accurate prediction of B-form/A-form DNA conformation propensity from primary sequence: A machine learning and free energy handshake.

DNA carries the genetic code of life, with different conformations associated with different biological functions. Predicting the conformation of DNA from its primary sequence, although desirable, is a challenging problem owing to the polymorphic nature of DNA. We have deployed a host of machine learning algorithms, including the popular state-of-the-art LightGBM (a gradient boosting model), for building prediction models. We used the nested cross-validation strategy to address the issues of "overfitting" and selection bias. This simultaneously provides an unbiased estimate of the generalization performance of a machine learning algorithm and allows us to tune the hyperparameters optimally. Furthermore, we built a secondary model based on SHAP (SHapley Additive exPlanations) that offers crucial insight into model interpretability. Our detailed model-building strategy and robust statistical validation protocols tackle the formidable challenge of working on small datasets, which is often the case in biological and medical data.

Interleaflet Decoupling in a Lipid Bilayer at Excess Cholesterol Probed by Spectroscopic Ellipsometry and Simulations.

Artificial lipid membranes are often investigated as a replica of the cell membrane in the form of supported lipid bilayers (SLBs). In SLBs, the phase state of a lipid bilayer strongly depends on the presence of molecules such as cholesterol, ceramide, and physical parameters such as temperature. Cholesterol is a key molecule of biological membranes and it exerts condensing effect on lipid bilayers. In this paper, we demonstrate the influence of excess cholesterol content on a supported lipid bilayer of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (fluid-phase) using spectroscopic ellipsometry (SE) and coarse-grained (CG) molecular dynamics (MD) simulations. The results show the condensation effect due to cholesterol addition up to 30% and interleaflet decoupling at excess cholesterol beyond 30%. SE results show the separation of individual leaflets of the bilayer and influence of cholesterol on the biophysical properties such as thickness and optical index. CG simulations were performed at different ratios of DOPC:cholesterol mixtures to explore cholesterol-driven bilayer properties and stability. The simulations displayed the accumulation of cholesterol molecules at the interface of the lower and upper leaflets of the bilayer, thus leading to undulations in the bilayer. This work reports the successful application of SE technique to study lipid-cholesterol interactions for the first time.

The Effects of Furosemide on Oxygenation in Mechanically Ventilated Children with Bronchiolitis.

Fluid balance management, including diuretic administration, may influence outcomes among mechanically ventilated children. We retrospectively compared oxygenation saturation index (OSI) before and after the initial furosemide bolus among 65 mechanically ventilated children. Furosemide was not associated with a significant change in median OSI (6.25 [interquartile range: 5.01-7.92] vs. 6.06 [4.73-7.54], p = 0.48), but was associated with expected changes in fluid balance and urine output. Secondary analysis suggested more favorable effects of furosemide in children with worse baseline OSI. The reported common use of furosemide by pediatric intensivists obligates further study to better establish its efficacy, or lack thereof, in mechanically ventilated children.

Spectroscopic Ellipsometry of fluid and gel phase lipid bilayers in hydrated conditions.

The biological membranes play a crucial role in the various biological processes due to their characteristic physical properties. The parameters such as membrane composition, thickness undulations, and the influence of external stimuli play a crucial role in the phase state behavior of biological membranes. The supported lipid bilayer (SLBs) systems closely represent cell membranes and are often studied to understand their behavior. In the current study, we have implemented spectroscopic Ellipsometry (SE) technique to explore the optical properties of various single component lipid bilayers having different phase state (fluid and gel) under hydrated or native conditions. The measurement of ellipsometric angles ψ and Δ were obtained from SE and were utilized to realize an explicit phase state dependence. Further, we obtained the thickness (t) and refractive index (n) by modeling of these SE results. These results were found to be inter-related to the phase state and lateral organization. The bilayer thickness is further verified against values obtained from coarse-grained molecular dynamics simulations. The proposed method was tested on various lipid bilayers under their native phase state at room temperature and was found to have a correlation with AFM measurements.

Insilico direct folding of thrombin-binding aptamer G-quadruplex at all-atom level.

The reversible folding of the thrombin-binding DNA aptamer G-quadruplexes (GQs) (TBA-15) starting from fully unfolded states was demonstrated using a prolonged time scale (10-12 µs) parallel tempering metadynamics (PTMetaD) simulation method in conjunction with a modified version of the AMBER bsc1 force field. For unbiased descriptions of the folding free energy landscape of TBA-15, this force field was minimally modified. From this direct folding simulation using the modified bsc1 force field, reasonably converged free energy landscapes were obtained in K+-rich aqueous solution (150 mM), providing detailed atomistic pictures of GQ folding mechanisms for TBA-15. This study found that the TBA folding occurred via multiple folding pathways with two major free energy barriers of 13 and 15 kcal/mol in the presence of several intermediate states of G-triplex variants. The early formation of these intermediates was associated with a single K+ ion capturing. Interestingly, these intermediate states appear to undergo facile transitions among themselves through relatively small energy barriers.

Understanding B-DNA to A-DNA transition in the right-handed DNA helix: Perspective from a local to global transition.

The right-handed DNA helix exhibits two major conformations, A-DNA and B-DNA, depending on the environmental conditions. The B-DNA to A-DNA (B→A) transition is sequence specific, cooperative, and reversible. The reduced water activity due to the addition of solvents like ethanol or the presence of protein or drug molecules causes B→A transition. In several biological cases, B→A transition occurs at a local level where small fragments of a long DNA sequence undergoes B→A transition. In this review, we have discussed various aspects of B→A transition such as the role of water, sequence specificity, mechanism of B→A transition, etc. The review primarily focuses on the B→A mechanism involved at a local level, and finally its connection to the global transition in theoretical and experimental studies.

Water modulates the ultraslow dynamics of hydrated ionic liquids near CG rich DNA: consequences for DNA stability.

Ionic liquids are known to stabilize DNA for much longer than water can. While the source of this stability has commonly been attributed to thermodynamic aspects, we probe the dynamical aspects of the ionic liquids near DNA to further our understanding of this stability. Using molecular dynamics simulation, we calculated the mean residence time (MRT) of the cations of five different ionic liquids (ILs) in the grooves and around phosphate groups of AT and CG rich DNA segments. We find the residence time of different cations next to CG rich DNA to be much higher compared to that next to AT rich DNA, with a negligible difference with the variation of anions. The interaction energy between cations and DNA, however, shows exactly the opposite trend; it is much lower (indicating a stronger interaction) for AT than for CG. Investigation of DNA parameters reveals an insignificant difference for the DNA sequences under consideration. Analysis of water behavior provides a rationale for the long MRTs of cations; water molecules have been found to be denser and to possess higher MRT when next to CG-rich DNA, thus resulting in a crowded environment. Our results indicate that the dynamics influence the binding of ILs to different DNA sequences, possibly by modulating the entropy of the binding process.

Ionic liquid induced G-quadruplex formation and stabilization: spectroscopic and simulation studies.

Among different polymorphs of DNA, G-quadruplex (GQ) formation in guanine rich sequences has received special attention due to its direct relevance to cellular aging and abnormal cell growths. To date, smaller ions like Na+, K+, Li+, and NH4+ are the best possible selective GQ stabilizing materials. Herein, we report that an ionic liquid (IL), i.e. guanidinium tris(pentafluoroethyl)trifluorophosphate, can not only instigate the GQ formation in the absence of conventional GQ forming ions (like Na+, K+, NH4+, etc.), but also stabilizes the GQ structure. This conformational transition has been confirmed through different spectroscopic tools and molecular dynamics (MD) simulation studies. MD simulation shows that one of the guanidinium cations resides in the G-tetrad core, while bulky anions prefer to stay near the GQ surface resulting in GQ formation and stabilization. This study thus brings out a special type of ionic liquid that acts as a GQ stabilizer. The origin of GQ stabilization by IL presented here may also help in the future design of IL for GQ formation and stabilization.

Computational Approach to Explore the B/A Junction Free Energy in DNA.

Protein-DNA interactions induce conformational changes in DNA such as B- to A-form transitions at a local level. Such transitions are associated with a junction free energy cost at the boundary of two different conformations in a DNA molecule. In this study, we performed umbrella sampling simulations to find the free energy values of the B-A transition at the dinucleotide and trinucleotide level of DNA. Using a combination of dinucleotide and trinucleotide free energy costs obtained from simulations, we calculated the B/A junction free energy. Our study shows that the B/A junction free energy is 0.52 kcal mol(-1) for the A-philic GG step and 1.59 kcal mol(-1) for the B-philic AA step. This observation is in agreement with experimentally derived values. After excluding junction effects, we obtained an absolute free energy cost for the B- to A-form conversion for all the dinucleotide steps. These absolute free energies may be used for predicting the propensity of structural transitions in DNA.

Avian scavengers and the threat from veterinary pharmaceuticals.

Veterinary use of the non-steroidal anti-inflammatory drug diclofenac on domesticated ungulates caused populations of resident Gyps vultures in the Indian sub-continent to collapse. The birds died when they fed on carrion from treated animals. Veterinary diclofenac was banned in 2006 and meloxicam was advocated as a 'vulture-safe' alternative. We examine the effectiveness of the 2006 ban, whether meloxicam has replaced diclofenac, and the impact of these changes on vultures. Drug residue data from liver samples collected from ungulate carcasses in India since 2004 demonstrate that the prevalence of diclofenac in carcasses in 2009 was half of that before the ban and meloxicam prevalence increased by 44%. The expected vulture death rate from diclofenac per meal in 2009 was one-third of that before the ban. Surveys at veterinary clinics show that diclofenac use in India began in 1994, coinciding with the onset of rapid Gyps declines ascertained from measured rates of declines. Our study shows that one pharmaceutical product has had a devastating impact on Asia's vultures. Large-scale research and survey were needed to detect, diagnose and quantify the problem and measure the response to remedial actions. Given these difficulties, other effects of pharmaceuticals in the environment may remain undetected.

Depth inpainting by tensor voting.

Depth maps captured by range scanning devices or by using optical cameras often suffer from missing regions due to occlusions, reflectivity, limited scanning area, sensor imperfections, etc. In this paper, we propose a fast and reliable algorithm for depth map inpainting using the tensor voting (TV) framework. For less complex missing regions, local edge and depth information is utilized for synthesizing missing values. The depth variations are modeled by local planes using 3D TV, and missing values are estimated using plane equations. For large and complex missing regions, we collect and evaluate depth estimates from self-similar (training) datasets. We align the depth maps of the training set with the target (defective) depth map and evaluate the goodness of depth estimates among candidate values using 3D TV. We demonstrate the effectiveness of the proposed approaches on real as well as synthetic data.

Sequence dependent free energy profiles of localized B- to A-form transition of DNA in water.

DNA carries an inherent polymorphism, which surfaces under various external conditions. While B-form remains predominant under normal physiological conditions for most of the DNA sequences, low humidity and increased ion concentration cause B- to A-form transition. Certain proteins and molecules also sometimes cause local deformation of the DNA to the specific A-form. Previous experimental and computational studies focused on the overall B- to A-form transition. Here for the first time we investigated thermodynamics and mechanism of B- to A-form transition in water for various DNA sequences at a local dinucleotide base pair level. We introduced a new reaction coordinate Zp', based on the unique order parameter Zp, to drive B- to A-form transition locally and thereby calculate free energy profiles for the same for all the ten different dinucleotide steps embedded in a twelve base pair DNA. Results show that the trend of "A" and "B" philicity observed in experiment is preserved even at this local dinucleotide level, indicating its localized origin. Higher free energy cost obtained here is attributed to the cost of creating B∕A junctions along with formation of B->A transition at dimer level. We find that while water energetically stabilizes A-form for all the ten different dinucleotide steps to various extents, entropy acts against it. Therefore, we find that the stability of B-form DNA in water is entropic in origin. Mechanism of the conversion appears to be triggered by Slide; however, backbone parameters change concertedly.