The Evolution of Nucleic Acid-Based Diagnosis Methods from the (pre-)CRISPR to CRISPR era and the Associated Machine/Deep Learning Approaches in Relevant RNA Design.

Nucleic acid tests (NATs) are considered as gold standard in molecular diagnosis. To meet the demand for onsite, point-of-care, specific and sensitive, trace and genotype detection of pathogens and pathogenic variants, various types of NATs have been developed since the discovery of PCR. As alternatives to traditional NATs (e.g., PCR), isothermal nucleic acid amplification techniques (INAATs) such as LAMP, RPA, SDA, HDR, NASBA, and HCA were invented gradually. PCR and most of these techniques highly depend on efficient and optimal primer and probe design to deliver accurate and specific results. This chapter starts with a discussion of traditional NATs and INAATs in concert with the description of computational tools available to aid the process of primer/probe design for NATs and INAATs. Besides briefly covering nanoparticles-assisted NATs, a more comprehensive presentation is given on the role CRISPR-based technologies have played in molecular diagnosis. Here we provide examples of a few groundbreaking CRISPR assays that have been developed to counter epidemics and pandemics and outline CRISPR biology, highlighting the role of CRISPR guide RNA and its design in any successful CRISPR-based application. In this respect, we tabularize computational tools that are available to aid the design of guide RNAs in CRISPR-based applications. In the second part of our chapter, we discuss machine learning (ML)- and deep learning (DL)-based computational approaches that facilitate the design of efficient primer and probe for NATs/INAATs and guide RNAs for CRISPR-based applications. Given the role of microRNA (miRNAs) as potential future biomarkers of disease diagnosis, we have also discussed ML/DL-based computational approaches for miRNA-target predictions. Our chapter presents the evolution of nucleic acid-based diagnosis techniques from PCR and INAATs to more advanced CRISPR/Cas-based methodologies in concert with the evolution of deep learning (DL)- and machine learning (ml)-based computational tools in the most relevant application domains.

Overcoming microbial resuscitation using stable ultrafine gold nanosystems.

While ultrafine gold nanosystems (UGNs) are being extensively studied for their antimicrobial activities, hitherto, no report is available on their propensity towards mitigating bacterial resuscitation-a potential factor contributing to the antimicrobial resistance. The investigations herein with two categories of gold nanosystems-modulated for their stability and surface accessibility through glutathione capping-have provided insights into overcoming resuscitation. Additionally, the study cautions that even moderate resistance development in bacteria exposed to nanosystems can result in significant cross-resistance against conventional antibiotics.

Amplifying bactericidal activity: Surfactant-mediated AgBr thin film coating over two-dimensional vertically aligned ZnO nanorods for dark-light dual mode disinfection.

Thin film coatings with potent antibacterial properties find critical applications in diverse domains such as medical devices, frequently touched surfaces, and food packaging for combating microbial proliferation across diverse scenarios. Two-dimensional photocatalytic antimicrobial coatings, offering a substantial actual-to-apparent surface ratio, hold immense potential for achieving this objective. However, realizing antibacterial performance not just under light but also in dark conditions remains a challenge. To address this, we present AgBr-coated vertically aligned ZnO nanorods (NRs) thin film architecture, employing a unique surfactant-mediated solution-phase spin-coating approach for achieving uniform deposition of AgBr onto ZnO NRs. The resulting ZnO NRs/AgBr heterojunction architectures have been characterized for their microstructural, morphological, elemental, optical, and wettability attributes. The studies have ascertained the tunability of AgBr content by modulating the concentration of its surfactant-based precursor solution. Further, valence band (VB) analyses revealed an increase in the electron density near to the VB edge. The dual role of AgBr as an antimicrobial agent and a photosensitizer, effectively enhancing the visible-light photodisinfection efficacy of ZnO NRs, has been evident through the dark-light dual mode antibacterial studies. Electron paramagnetic resonance measurements have shown hydroxyl radicals being majorly responsible for the visible-light photodisinfection performance. Encouragingly, reusability assessments showcase significant promise, while artificial sweat-wiping studies on the structures unveil heightened photodisinfection efficacy. This enhancement could be attributed to components like urea and lactic acid, speculated to augment the photocatalytic efficiency by minimizing charge recombination.

Polymerizable sol-gel synthesis of dark-visible light antibacterial magnetically-recoverable AgBr-loaded iron oxide/alumina nanocomposite.

The demand for a facile approach for synthesizing multifunctional nanocomposites is increasingly vital across diverse applications. In this study, a polymerizable sol-gel synthesis has been reported to obtain nanocomposites of magnetic iron oxide deposited over alumina nanopowder. The synthesis is mediated by the deposition of a calculated amount of iron(III) methacrylate, along with ethylene glycol dimethacrylate crosslinker, over alumina nanopowder, followed by thermally-inducing free radical polymerization at 125 °C for 30 min. The powder thus obtained has been subjected to calcination at 400 °C for 150 min and the resultant nanocomposites were characterized using wide-angle x-ray scattering, attenuated total reflectance-Fourier transform infrared spectroscopy, x-ray photoelectron spectroscopy, field-emission scanning electron microscopy, ultraviolet-diffuse reflectance spectroscopy, vibrating sample magnetometer and Brunauer-Emmett-Teller surface area measurements. The nanocomposites containing 15 and 20 wt% of iron oxide have been found to exhibit a saturation magnetization (Ms) value ranging from 12 to 14 emu g-1. To the nanocomposite containing 20 wt% of iron oxide, 5 wt% of AgBr was loaded through thoroughly mixing a surfactant-based precursor, silver-tetraoctyl ammonium bromide (Ag-TOAB), followed by thermolysis. All the nanocomposites have been studied for their antibacterial activity against a representative gram-negative bacterium,Escherichia coli, under dark and visible light conditions. While a 3 mg ml-1loading of the AgBr-loaded nanocomposite has exhibited complete clearance of the bacterial growth by 90 min in the dark, a similar activity has been observed in 60 min under light. The study has revealed the multifunctionality and high potential of the AgBr-loaded iron oxide/alumina nanocomposite as a promising dual-mode antibacterial and magnetically recoverable photocatalyst material.

Probing the Effects of Antimicrobial-Lysozyme Derivatization on Enzymatic Degradation of Poly(ε-caprolactone) Film and Fiber.

Surface derivatization is essential for incorporating unique functionalities into biodegradable polymers. Nonetheless, its precise effects on enzymatic biodegradation still lack comprehensive understanding. In this study, a facile solution-based method is employed to surface derivatize poly(ε-caprolactone) films and electrospun fibers with lysozyme, aiming to impart antimicrobial properties and examine the impact on enzymatic degradation. The derivatized films and fibers have shown high antibacterial efficacy against Escherichia coli and Staphylococcus aureus. Through gravimetric analysis, it is observed that the degradation rate experiences a slight decrease upon lysozyme derivatization. However, this reduction is effectively countered by the inclusion of Tween-20, as affirmed by isothermal titration calorimetry. Comparing films and fibers, the latter undergoes degradation at a more accelerated pace, coupled with a rapid decline in molecular weight. This study provides valuable insights into the factors influencing the degradation of surface-derivatized biopolymers through electrospinning, offering a simple strategy to mitigate biomaterial-associated infections.

Direct experimental evidence for the boronic acid-mediated bridging of DNA hybrids.

The use of terminal deoxynucleotidyl transferase for the first time in a mechanistic exploration-through colorimetric sensing and isothermal titration calorimetric studies-has provided direct experimental evidence of a boronic acid moiety bridging two DNA duplexes via the 3' hydroxyl groups, offering new opportunities and insights into the domain of DNA (nano)biotechnology.

Boronic acid chemistry for fluorescence-based quantitative DNA sensing.

Contrary to the long-standing opinion of boronic acids being typically reactive with 1,2- and 1,3-diols and hence not suitable for quantitative sensing of DNA containing only a mono-ol unit, this proof-of-concept study has successfully shown the feasibility to quantitatively detect DNA in the concentration range of 5 to 50 nM plausibly through boronic acid-mediated bridging of two DNA double helices via the 3' hydroxy groups, which opens up new avenues in the realm of oligonucleotide biochemistry.

Enhanced antibacterial, antioxidant and anticancer activity of caffeic acid by simple acid-base complexation with spermine/spermidine.

Caffeic acid (CA) is a naturally occurring plant-derived polyphenol possessing diverse biological properties. However, the poor water-solubility of CA restricts its widespread applications. On the other hand, biogenic amines such as spermine and spermidine are natural constituents in eukaryotes. In this work, we present water-soluble complexes of CA with spermine and spermidine by exploiting the acid-base interaction. Four different compositions have been prepared by varying the CA to amine ratios, whose chemical structures have been probed in detail using Fourier-transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance (NMR) studies that have revealed the acid-base interaction between the constituent precursors. The obtained acid-base complexes at their native pH values have shown enhanced antibacterial and antioxidant activities than pristine CA. Further, the CA-polyamine complexes have shown high anticancer performances in the concentration range that is compatible with the normal cell lines.

Water-soluble caffeic acid-dopamine acid-base complex exhibits enhanced bactericidal, antioxidant, and anticancer properties.

Despite the highly potent biological characteristics, the poor water-solubility of caffeic acid (CA) limits its applications in various domains. Here, we present a facile approach, wherein CA has been treated with dopamine hydrochloride (Dopa.HCl) to obtain a water-soluble acid-base complex, which does not possess any covalent bond between the individual components and thus retains their nativity. Simple mixing of CA and Dopa.HCl did not provide water solubility to CA, but the complex became readily soluble in water when the mineral acid was scavenged using sodium bicarbonate. The obtained CA-Dopa complex had been characterized using FT-IR, 1H NMR, 13C NMR, 2D 1H-1H NOESY NMR, XPS, and DSC techniques. The complex was found to exhibit excellent bactericidal, antibiofilm, antioxidant, and anticancer properties in the physiologically relevant pH range of 5.5 to 7.5. The results have revealed the high potential of the simple acid-base complex of CA in diverse domains.

Lactobacillus amylovorus derived lipase-mediated silver derivatization over poly(ε-caprolactone) towards antimicrobial coatings.

Owing to the probiotic origin, lipases-derived from the Lactobacilli sp. are considered to be promising biomaterials for in vivo applications. On a different note, poly(ε-caprolactone) (PCL)-an FDA-approved polymer for implantable applications-lacks inherent antimicrobial property, because of which suitable modifications are required to render it with bactericidal activity. Here, we employ Lactobacillus amylovorous derived lipase to surface derivatize the PCL films with silver that is a highly efficient inorganic broad-spectrum antimicrobial substance. Two different surface functionalization strategies have been employed over the alkaline hydrolyzed PCL films towards this purpose: In the first strategy, lipase-capped silver nanoparticles (Ag NPs) have been synthesized in a first step, which have been covalently immobilized over the activated carboxylic groups on the PCL film surface in a subsequent step. In the second strategy, the lipase was covalently immobilized over the activated carboxylic groups of the PCL film surface in the first step, over which silver was deposited in the second step using the dip-coating method. While the characterization study using X-ray photoelectron spectroscopy (XPS) has revealed the successful derivatization of silver over the PCL film, the surface characterization using field-emission scanning electron microscopy (FE-SEM) study has shown a distinct morphological change with higher silver loading in both strategies. The antimicrobial studies employing E. coli have revealed 100 % inhibition in the bacterial growth in 4-6 h with the Ag NPs-immobilized PCL films as opposed to >8 h with those prepared through the dip-coating method. Additionally, the cytotoxicity assay using mouse fibroblast cells has shown that the PCL films immobilized with lipase-capped Ag NPs exhibit high cell compatibility, similar to that of pristine PCL film, and thereby making it suitable for in vivo applications.