Analysis of DNA Origami Nanostructures Using Capillary Electrophoresis.

DNA origami nanostructures are engineered nanomaterials (ENMs) that possess significant customizability, biocompatibility, and tunable structural and functional properties, making them potentially useful materials in fields, such as medicine, biocomputing, biomedical engineering, and measurement science. Despite the potential of DNA origami as a functional nanomaterial, a major barrier to its applicability is the difficulty associated with obtaining pure, well-folded structures. Therefore, rapid methods of analysis to ensure purity are needed to support the rapid development of this class of nanomaterials. Here, we present the development of capillary electrophoresis (CE) as an analytical tool for DNA origami. CE was investigated under both capillary zone electrophoresis (CZE) and capillary transient isotachophoresis (ctITP) modes. Optimization of both systems yielded baseline resolved separations of folded DNA origami nanostructures from excess staple strands. The ctITP separation mode demonstrated superior performance in terms of peak resolution (Rs = 2.05 ± 0.3), peak efficiency (N = 12,200 ± 230), and peak symmetry (As = 1.29 ± 0.032). The SYBR family dyes (Gold, Green I, and Green II) were investigated as highly efficient, noncovalent fluorophores for on-column labeling of DNA origami and detection using laser-induced fluorescence. Finally, ctITP analysis conditions were also applied to DNA origami nanostructures with different shapes and for the differentiation of DNA origami aggregates.

Single-Round DNA Aptamer Selection by Combined Use of Capillary Electrophoresis and Next Generation Sequencing: An Aptaomics Approach for Identifying Unique Functional Protein-Binding DNA Aptamers.

In DNA aptamer selection, existing methods do not discriminate aptamer sequences based on their binding affinity and function and the reproducibility of the selection is often poor, even for the selection of well-known aptamers like those that bind the commonly used model protein thrombin. In the present study, a novel single-round selection method (SR-CE selection) was developed by combining capillary electrophoresis (CE) with next generation sequencing. Using SR-CE selection, a successful semi-quantitative and semi-comprehensive aptamer selection for thrombin was demonstrated with high reproducibility for the first time. Selection rules based on dissociation equilibria and kinetics were devised to obtain families of analogous sequences. Selected sequences of the same family were shown to bind thrombin with high affinity. Furthermore, data acquired from SR-CE selection was mined by creating sub-libraries that were categorized by the functionality of the aptamers (e. g., pre-organized aptamers versus structure-induced aptamers). Using this approach, a novel fluorescent molecular recognition sensor for thrombin with nanomolar detection limits was discovered. Thus, in this proof-of-concept report, we have demonstrated the potential of a "DNA Aptaomics" approach to systematically design functional aptamers as well as to obtain high affinity aptamers.

Bovine Serum Albumin Enhances Silver Nanoparticle Dissolution Kinetics in a Size- and Concentration-Dependent Manner.

The dissolution of silver nanoparticles (AgNPs) to release Ag(I)(aq) is an important mechanism in potentiating AgNP cytotoxicity and imparting their antibacterial properties. However, AgNPs can undergo other simultaneous biophysicochemical transformations, such as protein adsorption, which can mediate AgNP dissolution behaviors. We report the comprehensive analysis of AgNP dissolution and protein adsorption behaviors with monolayer surface coverage of AgNPs by bovine serum albumin (BSA). AgNP dissolution rate constants, kdissolution, were quantified over several particle sizes (10, 20, and 40 nm) and BSA concentrations (0-2 nM) using linear sweep stripping voltammetry. Across all particle sizes, the dissolution rate constant increased with increasing BSA concentrations. However, protein-enhanced dissolution behaviors were most pronounced for 10 nm AgNPs, which exhibited 3.6-fold and 7.7-fold relative enhancement when compared to 20 and 40 nm AgNPs, respectively. Changes to AgNP surface properties upon interaction with BSA were monitored using dynamic light scattering and zeta potential measurements, while BSA-AgNP complex formation was evaluated using UV-vis spectroscopy and circular dichroism spectroscopy. A subtle increase in the BSA-AgNP association constant was observed with an increase in the AgNP size. Together, these results suggest that the AgNP size dependence of BSA-enhanced dissolution of AgNPs is possibly mediated through both displacement of Ag(I)(aq)-loaded BSA by excess protein in the bulk solution and minimized accessibility of the AgNP surface because of BSA adsorption.

In Situ Quantification of Silver Nanoparticle Dissolution Kinetics in Simulated Sweat Using Linear Sweep Stripping Voltammetry.

Linear sweep stripping voltammetry (LSSV) is demonstrated as a sensitive, rapid, and cost-efficient analytical technique for the quantification of silver nanoparticle (AgNP) dissolution rates in simulated sweat. LSSV does not require the extensive sample preparation (e.g., ultrafiltration or centrifugation) needed by more commonly employed techniques, such as atomic spectroscopy. The limit of detection (LOD) of Ag(I)(aq) was 14 ± 6 µg L-1, and measured dissolution rate constants, kdissolution, varied from 0.0168-0.1524 h-1, depending on solution conditions. These values are comparable and agree well with those determined by others in the literature using atomic spectroscopy. Importantly, LSSV had the necessary sensitivity to distinguish the effects of SSW solution conditions on AgNP dissolution rates. Specifically, enhanced dissolution rates were observed with decreased pH and with increased NaCl concentration. The colloidal stability of AgNPs in SSW solutions was also characterized using dynamic light scattering (DLS), ζ potential, and quantitative UV-vis spectroscopy measurements. An increase in AgNP aggregation rate was observed with increased NaCl concentration in SSW, suggesting that the enhancement in AgNP dissolution is driven by the large Cl/Ag ratio, even as the AgNPs undergo significant aggregation.

Short-chained oligo(ethylene oxide)-functionalized gold nanoparticles: realization of significant protein resistance.

Protein corona formed on nanomaterial surfaces play an important role in the bioavailability and cellular uptake of nanomaterials. Modification of surfaces with oligoethylene glycols (OEG) are a common way to improve the resistivity of nanomaterials to protein adsorption. Short-chain ethylene oxide (EO) oligomers have been shown to improve the protein resistance of planar Au surfaces. We describe the application of these EO oligomers for improved protein resistance of 30 nm spherical gold nanoparticles (AuNPs). Functionalized AuNPs were characterized using UV-Vis spectroscopy, dynamic light scattering (DLS), and zeta potential measurements. Capillary electrophoresis (CE) was used for separation and quantitation of AuNPs and AuNP-protein mixtures. Specifically, nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) was employed for the determination of equilibrium and rate constants for binding between citrate-stabilized AuNPs and two model proteins, lysozyme and fibrinogen. Semi-quantitative CE analysis was carried out for mixtures of EO-functionalized AuNPs and proteins, and results demonstrated a 2.5-fold to 10-fold increase in protein binding resistance to lysozyme depending on the AuNP surface functionalization and a 15-fold increase in protein binding resistance to fibrinogen for both EO oligomers examined in this study. Graphical abstract Using capillary electrophoresis, the addition of short-chained oligo(ethylene oxide) ligands to gold nanoparticles was shown to improve protein binding resistance up to 15-fold.

Combining capillary electrophoresis and next-generation sequencing for aptamer selection.

Next-generation sequencing (NGS) machines can sequence millions of DNA strands in a single run, such as oligonucleotide (oligo) libraries comprising millions to trillions of discrete oligo sequences. Capillary electrophoresis is an attractive technique to select tight binding oligos or "aptamers" because it requires minimal sample volumes (e.g., 100 nL) and offers a solution-phase selection environment through which enrichment of target-binding oligos can be determined quantitatively. We describe here experiments using capillary transient isotachophoresis (ctITP)-based nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) as a method for selecting aptamers from a randomized library containing a known (29mer) thrombin-binding aptamer. Our capillary electrophoresis (CE)-selected samples were sequenced by the Ion Torrent Personal Genome Machine (PGM) and analyzed for selection efficiency. We show that a single round of CE selection can enrich a randomer synthetic DNA oligo mixture for thrombin-binding activity from 0.4% aptamer content before selection to >15% aptamer content.

Quantitative separation of polystyrene nanoparticles in environmental matrices with picogram detection limits using capillary electrophoresis.

We developed a capillary electrophoresis method to separate polystyrene particles (PSPs) with different sizes or different surface functionalities. Separations were performed in buffer and 100 mg L-1 clay or 100 mg L-1 Suwanee River humic acid. In all solutions, PSPs were baseline or near-baseline resolved in less than 15 minutes.

Interventions for increasing the uptake of immunisations in healthcare workers: A systematic review.

OBJECTIVE: This systemic review aimed to evaluate the effectiveness of interventions for increasing the uptake of immunisation in healthcare workers (HCWs) compared to no or alternative interventions. METHODS: A systematic review was undertaken (until March 2022) using a search strategy established a priori to capture studies that examined the effect of interventions on vaccination levels in HCWs. We included randomised controlled trials (RCT), cluster RCTs, controlled before-after (CBA) studies and interrupted time-series (ITS) studies. We described studies descriptively and synthesized results with a fixed-effect or random-effects model meta-analysis, where appropriate. The risk of bias was assessed for each study; the quality evidence per comparison was assessed using Grading of Recommendations Assessment, Development and Evaluation (GRADE). RESULTS: We identified three RCTs, six cluster RCTs and four ITS studies. There was a diverse range of interventions; many included an educational component. Based on the evidence examined the following may be effective strategies in increasing the proportion of HCWs vaccinated: policy interventions, targeted and multicomponent strategies, tailored programs directed at management, physician delivered education with a vaccine 'champion' and individual decision analysis. Limited eligible studies restricted synthesis and interpretation of findings. No studies evaluated the effectiveness of legislation. Nor did we find studies evaluating the effectiveness of incentives on their own or studies focusing solely on improving access to vaccination. We judged all the studies as either unclear or high risk of bias. CONCLUSION: Few robust studies that evaluate interventions to increase vaccination in HCWs are available. A limitation of this systematic review is that interventions are diverse, poorly reported and few were sufficiently alike to combine in an evaluation. More research on the effects of interventions to increase vaccination in HCWs is required, this should address a variety of vaccines and not just influenza vaccination.

A Nature's Way-Our Way Pilot Project Case Assemblage: (Re)Storying Child/Physical Literacy/Land Relationships for Indigenous Preschool-Aged Children's Wholistic Wellness.

Physical literacy (PL) is gaining more attention from educational policy-makers, practitioners, and researchers as a way to improve health and wellness outcomes for children and youth. While the development of PL is important for early years children, there is limited attention in the literature that explores the political, cultural, and social discourses imbued in colonialism that implicate how PL is actualized in Indigenous early childhood education (ECE) contexts. This case assemblage explores how the culturally rooted, interdisciplinary, and community-based PL initiative, Nature's Way-Our Way (NWOW), negotiated movement with three early childhood educators in the pilot project with an early childhood education centre (ECEC) in Saskatchewan, Canada. Through postqualitative approaches to research, this case assemblage adopts new materialist methodologies to show how the natural order of knowing in movement was disrupted through moments of rupture generating stories of PL to encompass radical relationality with land. As land becomes a vital and lively part of PL storying, it can function as an important protective factor for Indigenous preschool-aged children's wholistic wellness.

Silver nanoparticle interactions with glycated and non-glycated human serum albumin mediate toxicity.

Introduction: Biomolecules bind to and transform nanoparticles, mediating their fate in biological systems. Despite over a decade of research into the protein corona, the role of protein modifications in mediating their interaction with nanomaterials remains poorly understood. In this study, we evaluated how glycation of the most abundant blood protein, human serum albumin (HSA), influences the formation of the protein corona on 40 nm silver nanoparticles (AgNPs) and the toxicity of AgNPs to the HepG2 human liver cell line. Methods: The effects of glycation on AgNP-HSA interactions were quantified using circular dichroism spectroscopy to monitor protein structural changes, dynamic light scattering to assess AgNP colloidal stability, zeta potential measurements to measure AgNP surface charge, and UV-vis spectroscopy and capillary electrophoresis (CE) to evaluate protein binding affinity and kinetics. The effect of the protein corona and HSA glycation on the toxicity of AgNPs to HepG2 cells was measured using the WST cell viability assay and AgNP dissolution was measured using linear sweep stripping voltammetry. Results and Discussion: Results from UV-vis and CE analyses suggest that glycation of HSA had little impact on the formation of the AgNP protein corona with protein-AgNP association constants of ≈2x107 M-1 for both HSA and glycated HSA (gHSA). The formation of the protein corona itself (regardless of whether it was formed from HSA or glycated HSA) caused an approximate 2-fold decrease in cell viability compared to the no protein AgNP control. While the toxicity of AgNPs to cells is often attributed to dissolved Ag(I), dissolution studies showed that the protein coated AgNPs underwent less dissolution than the no protein control, suggesting that the protein corona facilitated a nanoparticle-specific mechanism of toxicity. Overall, this study highlights the importance of protein coronas in mediating AgNP interactions with HepG2 cells and the need for future work to discern how protein coronas and protein modifications (like glycation) may alter AgNP reactivity to cellular organisms.

Comparative B cell and antibody responses induced by adenoviral vectored and mRNA vaccines against COVID-19.

Both vector and mRNA vaccines were an important part of the response to the COVID-19 pandemic and may be required in future outbreaks and pandemics. However, adenoviral vectored (AdV) vaccines may be less immunogenic than mRNA vaccines against SARS-CoV-2. We assessed anti-spike and anti-vector immunity among infection-naïve Health Care Workers (HCW) following two doses of AdV (AZD1222) versus mRNA (BNT162b2) vaccine. 183 AdV and 274 mRNA vaccinees enrolled between April and October 2021. Median ages were 42 and 39 years, respectively. Blood was collected at least once, 10-48 days after vaccine dose 2. Surrogate virus neutralization test (sVNT) and spike binding antibody titres were a median of 4.2 and 2.2 times lower, respectively, for AdV compared to mRNA vaccinees (p<0.001). Median percentages of memory B cells that recognized fluorescent-tagged spike and RBD were 2.9 and 8.3 times lower, respectively for AdV compared to mRNA vaccinees. Titres of IgG reactive with human Adenovirus type 5 hexon protein rose a median of 2.2-fold after AdV vaccination but were not correlated with anti-spike antibody titres. Together the results show that mRNA induced substantially more sVNT antibody than AdV vaccine due to greater B cell expansion and targeting of the RBD. Pre-existing AdV vector cross-reactive antibodies were boosted following AdV vaccination but had no detectable effect on immunogenicity.

Superior immunogenicity of mRNA over adenoviral vectored COVID-19 vaccines reflects B cell dynamics independent of anti-vector immunity: Implications for future pandemic vaccines.

Both vector and mRNA vaccines were an important part of the response to the COVID-19 pandemic and may be required in future outbreaks and pandemics. The aim of this study was to validate whether immunogenicity differs for adenoviral vectored (AdV) versus mRNA vaccines against SARS-CoV-2, and to investigate how anti-vector immunity and B cell dynamics modulate immunogenicity. We enrolled SARS-CoV-2 infection-naïve health care workers who had received two doses of either AdV AZD1222 (n = 184) or mRNA BNT162b2 vaccine (n = 274) between April and October 2021. Blood was collected at least once, 10-48 days after vaccine dose 2 for antibody and B cell analyses. Median ages were 42 and 39 years, for AdV and mRNA vaccinees, respectively. Surrogate virus neutralization test (sVNT) and spike binding antibody titres were a median of 4.2 and 2.2 times lower, respectively, for AdV compared to mRNA vaccinees (p < 0.001). Median percentages of memory B cells that recognized fluorescent-tagged spike and RBD were 2.9 and 8.3 times lower, respectively for AdV compared to mRNA vaccinees. Titres of IgG reactive with human adenovirus type 5 hexon protein rose a median of 2.2-fold after AdV vaccination but were not correlated with anti-spike antibody titres. Together the results show that mRNA induced substantially more sVNT antibody than AdV vaccine, which reflected greater B cell expansion and targeting of the RBD rather than an attenuating effect of anti-vector antibodies. ClinicalTrials.gov Identifier: NCT05110911.

The Primarily Undergraduate Nanomaterials Cooperative: A New Model for Supporting Collaborative Research at Small Institutions on a National Scale.

The Primarily Undergraduate Nanomaterials Cooperative (PUNC) is an organization for research-active faculty studying nanomaterials at Primarily Undergraduate Institutions (PUIs), where undergraduate teaching and research go hand-in-hand. In this perspective, we outline the differences in maintaining an active research group at a PUI compared to an R1 institution. We also discuss the work of PUNC, which focuses on community building, instrument sharing, and facilitating new collaborations. Currently consisting of 37 members from across the United States, PUNC has created an online community consisting of its Web site (nanocooperative.org), a weekly online summer group meeting program for faculty and students, and a Discord server for informal conversations. Additionally, in-person symposia at ACS conferences and PUNC-specific conferences are planned for the future. It is our hope that in the years to come PUNC will be seen as a model organization for community building and research support at primarily undergraduate institutions.

Photolichenoid dermatitis: a presenting sign of human immunodeficiency virus.

Photolichenoid dermatitis is an uncommon eruptive dermatitis that often occurs in association with a photosensitizing drug. Photodermatitis, in general, is an uncommon clinical manifestation of human immunodeficiency virus (HIV), most often affecting patients of African and Native American descent. Photolichenoid dermatitis has infrequently been reported in patients with HIV who have not been exposed to a photosensitizing drug. We report a case of an African patient with a photodistributed depigmenting eruption without exposure to a photosensitizing drug. Histologic examination revealed a patchy perivascular and bandlike lymphocytic infiltrate with melanophages, interface changes, and dyskeratotic keratinocytes, consistent with photolichenoid dermatitis. Laboratory examination was significant for a positive HIV-2 antibody. Photolichenoid dermatitis may be a presenting sign of HIV infection and may not necessarily be associated with exposure to a photosensitizing drug. Testing for HIV should be done in patients who present with photodistributed depigmenting eruptions, even in the absence of exposure to a photosensitizing drug, and particularly in patients of African and Native American descent.

High separation efficiency of gold nanomaterials of different aspect ratio and size using capillary transient isotachophoresis.

Two modes of capillary electrophoresis (CE), capillary zone electrophoresis (CZE) and capillary transient isotachophoresis (ctITP), were compared for the detection and separation of spherical gold nanoparticles (AuNPs) and gold nanorods (AuNRs). The development of ctITP using two different leading ions is described. Overall, when compared to traditional capillary zone electrophoresis (CZE), ctITP resulted in improved peak shape and peak efficiency. Specifically, the number of theoretical plates for AuNR samples increased by a factor of 2-2.5 depending on the choice of leading ion. Further, using ctITP two AuNRs differing by aspect ratio were baseline resolved, whereas the same AuNRs could not be separated using CZE or other techniques like single particle inductively coupled plasma mass spectrometry (spICP-MS) and asymmetric flow field-flow fractionation (AF4). The results of this study demonstrate that ctITP is an efficient on-line technique for the improved detection and separation of gold nanomaterials in CE.

Healthy ageing in the Nun Study: definition and neuropathologic correlates.

BACKGROUND: Although the concept of healthy ageing has stimulated considerable interest, no generally accepted definition has been developed nor has its biological basis been determined. OBJECTIVE: To develop a definition of healthy ageing and investigate its association with longevity and neuropathology. METHODS: Analyses were based on cognitive, physical, and post-mortem assessments from 1991 to 1998 in the Nun Study, a longitudinal study of ageing in participants 75+ years at baseline. We defined three mutually exclusive levels of healthy ageing (excellent, very good, and good) based on measures of global cognitive function, short-term memory, basic and instrumental activities of daily living, and self-rated function. Mortality analyses were based on 636 participants; neuropathologic analyses were restricted to 221 who had died and were autopsied. RESULTS: Only 11% of those meeting criteria for the excellent level of healthy ageing at baseline subsequently died, compared with 24% for the very good, 39% for the good, and 60% for the remaining participants. Survival curves showed significantly greater longevity with higher levels of healthy ageing. The risk of not attaining healthy ageing, adjusted for age, increased two-fold in participants with brain infarcts alone, six-fold in those with Alzheimer neuropathology alone, and more than thirteen-fold in those with both brain infarcts and Alzheimer neuropathology. CONCLUSIONS: The biological validity of our definition of healthy ageing is supported by its strong association with mortality and longevity. Avoiding Alzheimer and stroke neuropathology is critical to the maintenance of healthy ageing, and the presence of both pathologies dramatically decreases the likelihood of healthy ageing.