Nucleic Acid Nanotechnology: Trends, Opportunities and Challenges.

Nucleic acids (DNA and RNA) hold great potential for the advancement of future medicine but suffer from unsatisfactory clinical success due to the challenges accompanied with their delivery. Nucleic acid-mediated nanomaterials have riveted the researchers from the past two decades and exhilarating tasks have prevailed. Nucleic acid nanotechnology offers unique control over the shape, size, time, mechanics and anisotropy. It can transfect numerous types of tissues and cells without any toxic effect, minimize the induced immune response, and penetrate most of the biological barriers and hence it reveals itself as a versatile tool for multidisciplinary research field and for various therapeutic purposes. Nucleic acid combines with other nanoscale objects also by altering the chemical functional groups and reproducing the varied array of nanomaterials. Interestingly, nucleic acidderived nanomaterials are characterized easily at atomic level accuracy. However, this advent of nanoscience has vital issues which must be addressed, such as the high cost of nucleic acids, their self-assembly nature, etc. Hence, the aim of this review is to highlight the systematic advances and methodology of nucleic acid-mediated synthesis of nanomaterials and their therapeutic applications.

Serine 408 phosphorylation is a molecular switch that regulates structure and function of the occludin α-helical bundle.

Occludin is a tetramembrane-spanning tight junction protein. The long C-terminal cytoplasmic domain, which represents nearly half of occludin sequence, includes a distal bundle of three α-helices that mediates interactions with other tight junction components. A short unstructured region just proximal to the α-helical bundle is a phosphorylation hotspot within which S408 phosphorylation acts as molecular switch that modifies tight junction protein interactions and barrier function. Here, we used NMR to define the effects of S408 phosphorylation on intramolecular interactions between the unstructured region and the α-helical bundle. S408 pseudophosphorylation affected conformation at hinge sites between the three α-helices. Further studies using paramagnetic relaxation enhancement and microscale thermophoresis indicated that the unstructured region interacts with the α-helical bundle. These interactions between the unstructured domain are enhanced by S408 phosphorylation and allow the unstructured region to obstruct the binding site, thereby reducing affinity of the occludin tail for zonula occludens-1 (ZO-1). Conversely, S408 dephosphorylation attenuates intramolecular interactions, exposes the binding site, and increases the affinity of occludin binding to ZO-1. Consistent with an increase in binding to ZO-1, intravital imaging and fluorescence recovery after photobleaching (FRAP) analyses of transgenic mice demonstrated increased tight junction anchoring of enhanced green fluorescent protein (EGFP)-tagged nonphosphorylatable occludin relative to wild-type EGFP-occludin. Overall, these data define the mechanisms by which S408 phosphorylation modifies occludin tail conformation to regulate tight junction protein interactions and paracellular permeability.

Nanodroplet Oligomers (NanDOs) of Aß40.

Using atomic force microscopy (AFM) and nuclear magnetic resonance (NMR), we describe small Aß40 oligomers, termed nanodroplet oligomers (NanDOs), which form rapidly and at Aß40 concentrations too low for fibril formation. NanDOs were observed in putatively monomeric solutions of Aß40 (e.g., by size exclusion chromatography). Video-rate scanning AFM shows rapid fusion and dissolution of small oligomer-sized particles, of which the median size increases with peptide concentration. In NMR (13C HSQC), a small number of chemical shifts changed with a change in peptide concentration. Paramagnetic relaxation enhancement NMR experiments also support the formation of NanDOs and suggest prominent interactions in hydrophobic domains of Aß40. Addition of Zn2+ to Aß40 solutions caused flocculation of NanDO-containing solutions, and selective loss of signal intensity in NMR spectra from residues in the N-terminal domain of Aß40. NanDOs may represent the earliest aggregated form of Aß40 in the aggregation pathway and are akin to premicelles in solutions of amphiphilies.

Improved air quality during COVID-19 at an urban megacity over the Indo-Gangetic Basin: From stringent to relaxed lockdown phases.

The enforced lockdown amid COVID-19 pandemic eased anthropogenic activities across India. The satellite-derived aerosol optical depth (AOD) and absorption AOD showed a significant reduction of ~30% over the Indo-Gangetic Basin (IGB) in north India during the lockdown period in 2020 with respect to the previous year 2019, when no such lockdown was in effect. Further, near-surface air pollutants were investigated at an urban megacity Delhi during 01 March to 31 May 2020. Except O3, a drastic reduction in PM10, PM2.5, NO, NO2 and CO concentrations were observed by ~58%, 47%, 76%, 68% and 58%, respectively during the lockdown period of 2020 as compared to 2019. While, O3 was low in the initial phase and gradually increased with progression of lockdown phases, the mean O3 during the entire lockdown period was nearly similar in both the years. Though, all the measured pollutants showed significant reduction during the entire lockdown, a phase-wise enhancement, associated with the conditional relaxations was observed in their concentrations. Thus, the present results may help, not only to assess the impact of outbreak on air quality, but also in designing the mitigation policies in urban megacities in more efficient ways to combat the air pollution problems.

Atomic-level differences between brain parenchymal- and cerebrovascular-seeded Aß fibrils.

Alzheimer's disease is characterized by neuritic plaques, the main protein components of which are ß-amyloid (Aß) peptides deposited as ß-sheet-rich amyloid fibrils. Cerebral Amyloid Angiopathy (CAA) consists of cerebrovascular deposits of Aß peptides; it usually accompanies Alzheimer's disease, though it sometimes occurs in the absence of neuritic plaques, as AD also occurs without accompanying CAA. Although neuritic plaques and vascular deposits have similar protein compositions, one of the characteristic features of amyloids is polymorphism, i.e., the ability of a single pure peptide to adopt multiple conformations in fibrils, depending on fibrillization conditions. For this reason, we asked whether the Aß fibrils in neuritic plaques differed structurally from those in cerebral blood vessels. To address this question, we used seeding techniques, starting with amyloid-enriched material from either brain parenchyma or cerebral blood vessels (using meninges as the source). These amyloid-enriched preparations were then added to fresh, disaggregated solutions of Aß to make replicate fibrils, as described elsewhere. Such fibrils were then studied by solid-state NMR, fiber X-ray diffraction, and other biophysical techniques. We observed chemical shift differences between parenchymal vs. vascular-seeded replicate fibrils in select sites (in particular, Ala2, Phe4, Val12, and Gln15 side chains) in two-dimensional 13C-13C correlation solid-state NMR spectra, strongly indicating structural differences at these sites. X-ray diffraction studies also indicated that vascular-seeded fibrils displayed greater order than parenchyma-seeded fibrils in the "side-chain dimension" (~ 10 Å reflection), though the "hydrogen-bond dimensions" (~ 5 Å reflection) were alike. These results indicate that the different nucleation conditions at two sites in the brain, parenchyma and blood vessels, affect the fibril products that get formed at each site, possibly leading to distinct pathophysiological outcomes.

Biogenesis and Application of Nickel Nanoparticles: A Review.

Biogenic synthesis of Nanoparticles (NPs) is attractive due to their ecological benefits and cheap, rapid, and sustainable nature. Among them, Nickel Oxide NPs (NiO-NPs) are acquired for their varied catalytic and clinical applications, as they have antibacterial, antifungal, cytotoxic, anticancer, antioxidant, remediation, and enzyme inhibition properties. Though several chemical-dependent methods were applied for the fabrication of nanoparticles, due to their substantial disadvantages, mainly toxicity and higher cost synthesis methods, the more secure, greener, eco-friendly, cost-effective, and synthetic methods are in demand. Greener approaches can take away the arduousness and complications of physicochemical methods. The present review is aimed at displaying the recent advancement related to the catalytic activity, antimicrobial activity, cytotoxicity, and antioxidant application of green synthesized Nickle. In this study, nickle oxide nanoparticles have been highlighted along with their sustainable synthesis options.

Chemical characterization of PM2.5 and source apportionment of organic aerosol in New Delhi, India.

Delhi is one of the most polluted cities worldwide and a comprehensive understanding and deeper insight into the air pollution and its sources is of high importance. We report 5 months of highly time-resolved measurements of non-refractory PM2.5 and black carbon (BC). Additionally, source apportionment based on positive matrix factorization (PMF) of the organic aerosol (OA) fraction is presented. The highest pollution levels are observed during winter in December/January. During that time, also uniquely high chloride concentrations are measured, which are sometimes even the most dominant NR-species in the morning hours. With increasing temperature, the total PM2.5 concentration decreases steadily, whereas the chloride concentrations decrease sharply. The concentrations measured in May are roughly 6 times lower than in December/January. PMF analysis resolves two primary factors, namely hydrocarbon-like (traffic-related) OA (HOA) and solid fuel combustion OA (SFC-OA), and one or two secondary factors depending on the season. The uncertainties of the PMF analysis are assessed by combining the random a-value approach and the bootstrap resampling technique of the PMF input. The uncertainties for the resolved factors range from ±18% to ±19% for HOA, ±7% to ±19% for SFC-OA and ±6 % to ±11% for the OOAs. The average correlation of HOA with equivalent black carbon from traffic (eBCtr) is R2 = 0.40, while SFC-OA has a correlation of R2 = 0.78 with equivalent black carbon from solid fuel combustion (eBCsf). Anthracene (m/z 178) and pyrene (m/z 202) (PAHs) are mostly explained by SFC-OA and follow its diurnal trend (R2 = 0.98 and R2 = 0.97). The secondary oxygenated aerosols are dominant during daytime. The average contribution during the afternoon hours (1 pm-5 pm) is 59% to the total OA mass, with contributions up to 96% in May. In contrast, the primary sources are more important during nighttime: the mean nightly contribution (22 pm-3 am) to the total OA mass is 48%, with contributions up to 88% during some episodes in April.

ß-amyloid model core peptides: Effects of hydrophobes and disulfides.

The mechanism by which a disordered peptide nucleates and forms amyloid is incompletely understood. A central domain of ß-amyloid (Aß21-30) has been proposed to have intrinsic structural propensities that guide the limited formation of structure in the process of fibrillization. In order to test this hypothesis, we examine several internal fragments of Aß, and variants of these either cyclized or with an N-terminal Cys. While Aß21-30 and variants were always monomeric and unstructured (circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMRS)), we found that the addition of flanking hydrophobic residues in Aß16-34 led to formation of typical amyloid fibrils. NMR showed no long-range nuclear overhauser effect (nOes) in Aß21-30, Aß16-34, or their variants, however. Serial 1 H-15 N-heteronuclear single quantum coherence spectroscopy, 1 H-1 H nuclear overhauser effect spectroscopy, and 1 H-1 H total correlational spectroscopy spectra were used to follow aggregation of Aß16-34 and Cys-Aß16-34 at a site-specific level. The addition of an N-terminal Cys residue (in Cys-Aß16-34) increased the rate of fibrillization which was attributable to disulfide bond formation. We propose a scheme comparing the aggregation pathways for Aß16-34 and Cys-Aß16-34, according to which Cys-Aß16-34 dimerizes, which accelerates fibril formation. In this context, cysteine residues form a focal point that guides fibrillization, a role which, in native peptides, can be assumed by heterogeneous nucleators of aggregation.

ß-Amyloid aggregation and heterogeneous nucleation.

In this article, we consider the role of heterogeneous nucleation in ß-amyloid aggregation. Heterogeneous nucleation is more common and occurs at lower levels of supersaturation than homogeneous nucleation. The nucleation period is also the stage at which most of the polymorphism of amyloids arises, this being one of the defining features of amyloids. We focus on several well-known heterogeneous nucleators of ß-amyloid, including lipid surfaces, especially those enriched in gangliosides and cholesterol, and divalent metal ions. These two broad classes of nucleators affect ß-amyloid particularly in light of the amphiphilicity of these peptides: the N-terminal region, which is largely polar and charged, contains the metal binding site, whereas the C-terminal region is aliphatic and is important in lipid binding. Notably, these two classes of nucleators can interact cooperatively, aggregation begetting greater aggregation.

A versatile method for producing labeled or unlabeled Aß55, Aß40, and other ß-amyloid family peptides.

We present a straightforward, versatile method for expressing and purifying ß-amyloid (Aß40) and transmembrane peptides derived from ß-amyloid precursor protein (Aß55). In principle, these methods should be applicable to other types of strongly aggregating peptides. We start with a DNA plasmid encoding a HexaHis tag with a flexible, hydrophilic linker sequence, followed by a cleavage site, and then Aß peptides. The HexaHis tag rather than a protein fusion partner (e.g., GST) obviates the need for a folded protein in affinity purification. Second, we present two cleavage methods, using either Factor Xa or BNPS-Skatole. Although the latter procedure requires subsequent reduction of the product, we describe methods for minimizing side reactions. Because the use of BNPS-Skatole obviates the need for a folded protein in the cleavage reaction, it is compatible with harsh conditions (e.g., inclusion of detergents and denaturants) needed to solubilize the fusion proteins; such conditions tend to inactivate Factor Xa. Finally, we also describe purification strategies for Aß40 and Aß55 using FPLC and/or reverse phase HPLC. Yields of peptide after these BNPS-Skatole cleavage and peptide reduction, though subquantitative, greatly exceed those obtained using Factor Xa cleavage, as the reaction of BNPS-Skatole is insensitive to the presence of detergents and denaturants, and therefore can be used to produce highly aggregative and low solubility peptides such as Aß55. Trp is a low abundance amino acid in proteins generally, and for peptides like Aß55, and other transmembane peptides lacking Trp in relevant positions, this cleavage method remains a useful option.

High affinity interactions of Pb2+ with synaptotagmin I.

Lead (Pb) is a potent neurotoxin that disrupts synaptic neurotransmission. We report that Synaptotagmin I (SytI), a key regulator of Ca2+-evoked neurotransmitter release, has two high-affinity Pb2+ binding sites that belong to its cytosolic C2A and C2B domains. The crystal structures of Pb2+-complexed C2 domains revealed that protein-bound Pb2+ ions have holodirected coordination geometries and all-oxygen coordination spheres. The on-rate constants of Pb2+ binding to the C2 domains of SytI are comparable to those of Ca2+ and are diffusion-limited. In contrast, the off-rate constants are at least two orders of magnitude smaller, indicating that Pb2+ can serve as both a thermodynamic and kinetic trap for the C2 domains. We demonstrate, using NMR spectroscopy, that population of these sites by Pb2+ ions inhibits further Ca2+ binding despite the existing coordination vacancies. Our work offers a unique insight into the bioinorganic chemistry of Pb(ii) and suggests a mechanism by which low concentrations of Pb2+ ions can interfere with the Ca2+-dependent function of SytI in the cell.

Non-Native Metal Ion Reveals the Role of Electrostatics in Synaptotagmin 1-Membrane Interactions.

C2 domains are independently folded modules that often target their host proteins to anionic membranes in a Ca2+-dependent manner. In these cases, membrane association is triggered by Ca2+ binding to the negatively charged loop region of the C2 domain. Here, we used a non-native metal ion, Cd2+, in lieu of Ca2+ to gain insight into the contributions made by long-range Coulombic interactions and direct metal ion-lipid bridging to membrane binding. Using X-ray crystallography, NMR, Förster resonance energy transfer, and vesicle cosedimentation assays, we demonstrate that, although Cd2+ binds to the loop region of C2A/B domains of synaptotagmin 1 with high affinity, long-range Coulombic interactions are too weak to support membrane binding of individual domains. We attribute this behavior to two factors: the stoichiometry of Cd2+ binding to the loop regions of the C2A and C2B domains and the impaired ability of Cd2+ to directly coordinate the lipids. In contrast, electron paramagnetic resonance experiments revealed that Cd2+ does support membrane binding of the C2 domains in full-length synaptotagmin 1, where the high local lipid concentrations that result from membrane tethering can partially compensate for lack of a full complement of divalent metal ions and specific lipid coordination in Cd2+-complexed C2A/B domains. Our data suggest that long-range Coulombic interactions alone can drive the initial association of C2A/B with anionic membranes and that Ca2+ further augments membrane binding by the formation of metal ion-lipid coordination bonds and additional Ca2+ ion binding to the C2 domain loop regions.

Mapping the Hydrogen Bond Networks in the Catalytic Subunit of Protein Kinase A Using H/D Fractionation Factors.

Protein kinase A is a prototypical phosphoryl transferase, sharing its catalytic core (PKA-C) with the entire kinase family. PKA-C substrate recognition, active site organization, and product release depend on the enzyme's conformational transitions from the open to the closed state, which regulate its allosteric cooperativity. Here, we used equilibrium nuclear magnetic resonance hydrogen/deuterium (H/D) fractionation factors (φ) to probe the changes in the strength of hydrogen bonds within the kinase upon binding the nucleotide and a pseudosubstrate peptide (PKI5-24). We found that the φ values decrease upon binding both ligands, suggesting that the overall hydrogen bond networks in both the small and large lobes of PKA-C become stronger. However, we observed several important exceptions, with residues displaying higher φ values upon ligand binding. Notably, the changes in φ values are not localized near the ligand binding pockets; rather, they are radiated throughout the entire enzyme. We conclude that, upon ligand and pseudosubstrate binding, the hydrogen bond networks undergo extensive reorganization, revealing that the open-to-closed transitions require global rearrangements of the internal forces that stabilize the enzyme's fold.

Synchronous opening and closing motions are essential for cAMP-dependent protein kinase A signaling.

Conformational fluctuations play a central role in enzymatic catalysis. However, it is not clear how the rates and the coordination of the motions affect the different catalytic steps. Here, we used NMR spectroscopy to analyze the conformational fluctuations of the catalytic subunit of the cAMP-dependent protein kinase (PKA-C), a ubiquitous enzyme involved in a myriad of cell signaling events. We found that the wild-type enzyme undergoes synchronous motions involving several structural elements located in the small lobe of the kinase, which is responsible for nucleotide binding and release. In contrast, a mutation (Y204A) located far from the active site desynchronizes the opening and closing of the active cleft without changing the enzyme's structure, rendering it catalytically inefficient. Since the opening and closing motions govern the rate-determining product release, we conclude that optimal and coherent conformational fluctuations are necessary for efficient turnover of protein kinases.

Conformational propensities and dynamics of a ßγ-crystallin, an intrinsically disordered protein.

The three-dimensional folded structure of a protein has been considered essential for its function. However, recently many proteins have been identified to function without having a definite structure and they have been classified as intrinsically disordered proteins (IDPs). Recently, we have identified a ßγ-crystallin domain in the genome of a marine bacterium called Hahella chejuensis on the basis of known sequence signatures. This protein, called Hahellin, was characterized by NMR spectroscopy as an IDP, which upon Ca(2+)-binding was shown to undergo a large conformational transformation and acquires a typical ßγ-crystallin fold. In this paper, we have characterized this IDP by a combined use of NMR and Replica Exchange Molecular Dynamics simulation and found it to be in a highly dynamic, inter-converting population having a molten globular state with the C-terminal Greek key motif relatively more flexible as compared to its N-terminal counterpart. Network analysis and clustering on the observed conformational ensemble showed a heterogeneous mixture of eleven distinct clusters, classified into near-native and far-native populations, which are not in equilibrium. Several conformational clusters display an increased propensity for helical conformation(s) and a decreased ß-strand propensity, which is consistent with the NMR observations made on this protein. The negatively charged Ca(2+)-coordinating residues form parts of the highly flexible polypeptide stretches, and thus act as seeds for the origin of different conformational clusters observed. This study thus helps us to understand the relationship between the role of conformational dynamics and the structural propensities of the intrinsically disordered state of apo-Hahellin.

Backbone ¹H, ¹³C and ¹5N resonance assignments of an intrinsically unstructured ßγ-crystallin from Hahella chejuensis.

The sequence specific backbone (1)H, (13)C and (15)N resonance assignments of an intrinsically unstructured ßγ-crystallin from Hahella chejuensis are reported. The secondary structure chracterization of the unstructured protein reveals that large fraction of residues exhibits ß-strand propensity, as in the case of the Ca(2+)-bound structured protein.

Contribution of anthropogenic aerosols in direct radiative forcing and atmospheric heating rate over Delhi in the Indo-Gangetic Basin.

INTRODUCTION: The present work is aimed to understand direct radiation effects due to aerosols over Delhi in the Indo-Gangetic Basin (IGB) region, using detailed chemical analysis of surface measured aerosols during the year 2007. METHODS: An optically equivalent aerosol model was formulated on the basis of measured aerosol chemical compositions along with the ambient meteorological parameters to derive radiatively important aerosol optical parameters. The derived aerosol parameters were then used to estimate the aerosol direct radiative forcing at the top of the atmosphere, surface, and in the atmosphere. RESULTS: The anthropogenic components measured at Delhi were found to be contributing ∼ 72% to the composite aerosol optical depth (AOD(0.5) ∼ 0.84). The estimated mean surface and atmospheric forcing for composite aerosols over Delhi were found to be about -69, -85, and -78 W m(-2) and about +78, +98, and +79 W m(-2) during the winter, summer, and post-monsoon periods, respectively. The anthropogenic aerosols contribute ∼ 90%, 53%, and 84% to the total aerosol surface forcing and ∼ 93%, 54%, and 88% to the total aerosol atmospheric forcing during the above respective periods. The mean (± SD) surface and atmospheric forcing for composite aerosols was about -79 (± 15) and +87 (± 26) W m(-2) over Delhi with respective anthropogenic contributions of ∼ 71% and 75% during the overall period of observation. CONCLUSIONS: Aerosol induced large surface cooling, which was relatively higher during summer as compared to the winter suggesting an increase in dust loading over the station. The total atmospheric heating rate at Delhi averaged during the observation was found to be 2.42 ± 0.72 K day(-1), of which the anthropogenic fraction contributed as much as ∼ 73%.

Conformational heterogeneity and dynamics in a ßγ-crystallin from Hahella chejuensis.

Most of the ßγ-crystallins are structural proteins with high intrinsic stability, which gets enhanced by Ca(2+)-binding in microbial members. Functions of most of these proteins are yet to be known. However, a few of them were reported to be involved in Ca(2+)-dependent and stress-related functions. Hahellin, a microbial homolog, is a natively unfolded protein that acquires a well-folded structure upon Ca(2+) binding. Although the structure of ßγ-crystallin domains is well understood, the dynamical features are yet to be explored. We have investigated for the first time the equilibrium dynamics, conformational heterogeneity and associated low-lying free-energy states of hahellin in its Ca(2+)-bound form using NMR spectroscopy to understand the dynamics of a ßγ-crystallin domain. Hahellin shows large conformational heterogeneity with nearly 40% of the residues, some of which are part of Ca(2+)-binding loops, accessing alternative states. Further, out of the two Greek key motifs, which together constitute the ßγ-crystallin domain, the second Greek key motif is floppy as compared to its relatively rigid counterpart. Taken together, we believe that these characteristics might be of importance to understand the stability and functions of ßγ-crystallin domains.

A natively unfolded ßγ-crystallin domain from Hahella chejuensis.

To date, very few ßγ-crystallins have been identified and structurally characterized. Several of them have been shown to bind Ca(2+) and thereby enhance their stability without any significant change in structure. Although Ca(2+)-induced conformational changes have been reported in two putative ßγ-crystallins from Caulobacter crescentus and Yersinia pestis, they are shown to be partially unstructured, and whether they acquire a ßγ-crystallin fold is not known. We describe here a ßγ-crystallin domain, hahellin, its Ca(2+) binding properties and NMR structure. Unlike any other ßγ-crystallin, hahellin is characterized as a pre-molten globule (PMG) type of natively unfolded protein domain. It undergoes drastic conformational change and acquires a typical ßγ-crystallin fold upon Ca(2+) binding and hence acts as a Ca(2+)-regulated conformational switch. However, it does not bind Mg(2+). The intrinsically disordered Ca(2+)-free state and the close structural similarity of Ca(2+)-bound hahellin to a microbial ßγ-crystallin homologue, Protein S, which shows Ca(2+)-dependent stress response, make it a potential candidate for the cellular functions. This study indicates the presence of a new class of natively unfolded ßγ-crystallins and therefore the commencement of the possible functional roles of such proteins in this superfamily.

Sequence specific 1H, 13C and 15N resonance assignments of hahellin in 8 M urea.

The sequence specific (1)H, (13)C and (15)N resonance assignments of hahellin in 8 M urea-denatured state have been accomplished by NMR spectroscopy. Secondary chemical shift analysis reveals the native-like propensities for ß-rich conformation in the denatured state.