Rational truncation of aptamer for cross-species application to detect krait envenomation

Abstract In majority of snakebite cases, the snake responsible for the bite remains unidentified. The traditional snakebite diagnostics method relies upon clinical symptoms and blood coagulation assays that do not provide accurate diagnosis which is important for epidemiological as well as diagnostics point of view. On the other hand, high batch-to-batch variations in antibody performance limit its application for diagnostic assays. In recent years, nucleic acid aptamers have emerged as a strong chemical rival of antibodies due to several obvious advantages, including but not limited to in vitro generation, synthetic nature, ease of functionalization, high stability and adaptability to various diagnostic formats. In the current study, we have rationally truncated an aptamer developed for α-Toxin of Bungarus multicinctus and demonstrated its utility for the detection of venom of Bungarus caeruleus. The truncated aptamer α-Tox-T2 (26mer) is found to have greater affinity than its 40-mer parent counterpart α-Tox-FL. The truncated aptamers are characterized and compared with parent aptamer for their binding, selectivity, affinity, alteration in secondary structure and limit of detection. Altogether, our findings establish the cross-species application of a DNA aptamer generated for α-Toxin of Bungarus multicinctus (a snake found in Taiwan and China) for the reliable detection of venom of Bungarus caeruleus (a snake found in the Indian subcontinent)

Machine Learning Approach to Predict Stress in Ceramic/Epoxy Composites Using Micro-Mechanical Raman Spectroscopy

Micro-mechanical Raman spectroscopy is an excellent tool for direct stress measurements in the structure. The presence of mechanical stress changes the Raman frequency of each Raman modes compared to the Raman frequencies in absence of stress. This difference in Raman frequency is linearly related to stress induced and can be calibrated to stress by uniaxial or biaxial tension/compression experiments. This relationship is not generally linear for non-linear behavior of the materials which limits its use to experimentally study flow stress and plastic deformation behavior of the material. In this work strontium titanate ceramic particles dispersed inside epoxy resin matrix were used to measure stress in epoxy resin matrix with non-linear material behavior around it. The stress concentration factor between stress induced inside ceramic particles and epoxy resin matrix was obtained by non-linear constitutive finite element model. The results of finite element model were used for training a machine learning model to predict stress in epoxy resin matrix based on stress inside ceramic particles. By measuring stress inside ceramic particles using micro-mechanical Raman spectroscopy, the stress inside epoxy matrix was obtained by predetermined stress concentration factor

Simple Methods and Rational Design for Enhancing Aptamer Sensitivity and Specificity

Aptamers are structured nucleic acid molecules that can bind to their targets with high affinity and specificity. However, conventional SELEX (Systematic Evolution of Ligands by EXponential enrichment) methods may not necessarily produce aptamers of desired affinity and specificity. Thus, to address these questions, this perspective is intended to suggest some approaches and tips along with novel selection methods to enhance evolution of aptamers. This perspective covers latest novel innovations as well as a broad range of well-established approaches to improve the individual binding parameters (aptamer affinity, avidity, specificity and/or selectivity) of aptamers during and/or post-SELEX. The advantages and limitations of individual aptamer selection methods and post-SELEX optimizations, along with rational approaches to overcome these limitations are elucidated in each case. Further the impact of chosen selection milieus, linker-systems, aptamer cocktails and detection modules utilized in conjunction with target-specific aptamers, on the overall assay performance are discussed in detail, each with its own advantages and limitations. The simple variations suggested are easily available for facile implementation during and/or post-SELEX to develop ultrasensitive and specific assays. Finally, success studies of established aptamer-based assays are discussed, highlighting how they utilized some of the suggested methodologies to develop commercially successful point-of-care diagnostic assays

Local Shock Properties Measurement Using Time-Resolved Raman Spectroscopy

In this article, the dynamic response of a heterogeneous microstructure of polymer bonded composite was analyzed to a short duration shock pulse. The composite microstructure studied is a polymerbonded sugar (PBS) with single-crystal sucrose embedded inside the polydimethylsiloxane binder. The shock pulse was created by the impact of the aluminum disk at high speeds using a laser-based projectile launch system. The mechanical response on the microscale domain was measured using ultrafast time-resolved Raman spectroscopy. The in-situ analysis of the change in Raman spectra from PBS during shock compression was captured in the time domain using a streak camera. The results show a steeply rising shock front after the impact where the shock pressure rise time was estimated from the time-resolved Raman spectra. The viscoplastic behavior in the local microscale domain was characterized by quantifying effective shock viscosity measured in the vicinity of the crystal-binder interface

Mycorrhiza: An Ecofriendly Bio-Tool for Better Survival of Plants in Nature

Modern agriculture is currently enduring rapid changes in defiance of the continuing increase of the global population and the various consequent environmental challenges. Crop quality is becoming as important as crop yield and can be characterized by several parameters. Extensive use of chemical fertilizers leads to food safety concerns globally; hence, the use of mycorrhizal symbionts have proven to be beneficial for the sustainable growth of the agricultural cropping system. Microflora inhabiting the soil entails various ecological interactions which are associated with agricultural performances. Amongst these microflora, mycorrhizal fungi are the critical suppliers of nutrients, with restricted diffusion capacities of minerals such as phosphorus, nitrate, zinc, sulfur etc. Mycorrhizae are the obligatory biotrophs that depend upon their host plant for the nutritional requirements. They act as the key contributors to sustainable agro-ecological enforcement and impact globally on the eco-systemic processes. These soil inhabitants devote themselves to the continuous nutrient flow and extemporize resistance against various environmental stresses like drought, flood, metal toxicity, salinity, etc. This review briefly highlights the taxonomic co-evolution, factors affecting mycorrhizal behaviors (phytohormonal regulation), and the concise mechanistic approach (improved water status, photosystems, stomatal conductance, ionic uptake, C & N fixation) to combat various environmental stresses (biotic/abiotic). Plant growth regulators play a crucial role in this symbiotic establishment with the plant roots. Auxins, brassinosteroids, and strigolactones are responsible for the establishment of mycorrhizal association. On the other hand, ethylene, abscisic acid, and jasmonic acids can promote or downregulate this process in the plants. Whereas, gibberellic acids and salicylic acids negatively impact on mycorrhizal association. The hormonal homeostasis (in response to fungal associations) leads to the activation of transcriptional and signaling cascades which ensues various physio-morphological changes for the benefit of the plant. The role of phytohormones in the regulation of plant-fungus mutualism, and the impact of mycorrhization on the activation of molecular and transcriptional cascades, have been described along with the potential applications of agricultural produce and soil rehabilitation

High-Strain Rate Spall Strength Measurement for CoCrFeMnNi High-Entropy Alloy

In this study, we experimentally investigate the high stain rate and spall behavior of Cantor high-entropy alloy (HEA), CoCrFeMnNi. First, the Hugoniot equations of state (EOS) for the samples are determined using laser-driven CoCrFeMnNi flyers launched into known Lithium Fluoride (LiF) windows. Photon Doppler Velocimetry (PDV) recordings of the velocity profiles find the EOS coefficients using an impedance mismatch technique. Following this set of measurements, laser-driven aluminum flyer plates are accelerated to velocities of 0.5–1.0 km/s using a high-energy pulse laser. Upon impact with CoCrFeMnNi samples, the shock response is found through PDV measurements of the free surface velocities. From this second set of measurements, the spall strength of the alloy is found for pressures up to 5 GPa and strain rates in excess of 106 s−1. Further analysis of the failure mechanisms behind the spallation is conducted using fractography revealing the occurrence of ductile fracture at voids presumed to be caused by chromium oxide deposits created during the manufacturing process

High-Strain Rate Spall Strength Measurement for CoCrFeMnNi High-Entropy Alloy

In this study, we experimentally investigate the high stain rate and spall behavior of Cantor high-entropy alloy (HEA), CoCrFeMnNi. First, the Hugoniot equations of state (EOS) for the samples are determined using laser-driven CoCrFeMnNi flyers launched into known Lithium Fluoride (LiF) windows. Photon Doppler Velocimetry (PDV) recordings of the velocity profiles find the EOS coefficients using an impedance mismatch technique. Following this set of measurements, laser-driven aluminum flyer plates are accelerated to velocities of 0.5–1.0 km/s using a high-energy pulse laser. Upon impact with CoCrFeMnNi samples, the shock response is found through PDV measurements of the free surface velocities. From this second set of measurements, the spall strength of the alloy is found for pressures up to 5 GPa and strain rates in excess of 106 s−1. Further analysis of the failure mechanisms behind the spallation is conducted using fractography revealing the occurrence of ductile fracture at voids presumed to be caused by chromium oxide deposits created during the manufacturing process

DNA Aptamer Targets Mycobacterium tuberculosis DevR/DosR Response Regulator Function by Inhibiting Its Dimerization and DNA Binding Activity

Tuberculosis is recognized as one of the major public health threats worldwide. The DevR-DevS (DosR/DosS) two-component system is considered a novel drug target in Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, owing to its central role in bacterial adaptation and long-term persistence. An increase in DevR levels and the decreased permeability of the mycobacterial cell wall during hypoxia-associated dormancy pose formidable challenges to the development of anti-DevR compounds. Using an in vitro evolution approach of Systematic Evolution of Ligands by EXponential enrichment (SELEX), we developed a panel of single-stranded DNA aptamers that interacted with Mtb DevR protein in solid-phase binding assays. The best-performing aptamer, APT-6, forms a G-quadruplex structure and inhibits DevR-dependent transcription in Mycobacterium smegmatis. Mechanistic studies indicate that APT-6 functions by inhibiting the dimerization and DNA binding activity of DevR protein. In silico studies reveal that APT-6 interacts majorly with C-terminal domain residues that participate in DNA binding and formation of active dimer species of DevR. To the best of our knowledge, this is the first report of a DNA aptamer that inhibits the function of a cytosolic bacterial response regulator. By inhibiting the dimerization of DevR, APT-6 targets an essential step in the DevR activation mechanism, and therefore, it has the potential to universally block the expression of DevR-regulated genes for intercepting dormancy pathways in mycobacteria. These findings also pave the way for exploring aptamer-based approaches to design and develop potent inhibitors against intracellular proteins of various bacterial pathogens of global concern