Characterization of DNA-protein complexes by nanoparticle tracking analysis and their association with systemic lupus erythematosus.

Nanotechnology enables investigations of single biomacromolecules, but technical challenges have limited the application in liquid biopsies, for example, blood plasma. Nonetheless, tools to characterize single molecular species in such samples represent a significant unmet need with the increasing appreciation of the physiological importance of protein structural changes at nanometer scale. Mannose-binding lectin (MBL) is an oligomeric plasma protein and part of the innate immune system through its ability to activate complement. MBL also serves a role as a scavenger for cellular debris, especially DNA. This may link functions of MBL with several inflammatory diseases in which cell-free DNA now appears to play a role, but mechanistic insight has been lacking. By making nanoparticle tracking analysis possible in human plasma, we now show that superoligomeric structures of MBL form nanoparticles with DNA. These oligomers correlate with disease activity in systemic lupus erythematosus patients. With the direct quantification of the hydrodynamic radius, calculations following the principles of Taylor dispersion in the blood stream connect the size of these complexes to endothelial inflammation, which is among the most important morbidities in lupus. Mechanistic insight from an animal model of lupus supported that DNA-stabilized superoligomers stimulate the formation of germinal center B cells and drive loss of immunological tolerance. The formation involves an inverse relationship between the concentration of MBL superoligomers and antibodies to double-stranded DNA. Our approach implicates the structure of DNA-protein nanoparticulates in the pathobiology of autoimmune diseases.

Gut reactions: Eosinophils add another string to their bow.

Eosinophils are found in abundance in the gut. In this issue of Immunity, Chu et al. (2014) report that eosinophil-deficient mice have impaired intestinal immunoglobulin A production, accompanied by a disrupted mucosal layer and alterations in microbiota density and composition.

Advanced bioengineering of female germ cells to preserve fertility.

Oogenesis and folliculogenesis are considered as complex and species-specific cellular differentiation processes, which depend on the in vivo ovarian follicular environment and endocrine cues. Considerable efforts have been devoted to driving the differentiation of female primordial germ cells toward mature oocytes outside of the body. The recent experimental attempts have laid stress on offering a suitable microenvironment to assist the in vitro folliculogenesis and oogenesis. Despite developing a variety of bioengineering techniques and generating functional mature gametes through in vitro oogenesis in earlier studies, we still lack knowledge of appropriate microenvironment conditions for building biomimetic culture systems for female fertility preservation. Therefore, this review paper can provide a source for a large body of scientists developing cutting-edge in vitro culture systems for female germ cells or setting up the next generation of reproductive medicine as feasible options for female infertility treatment. The focal point of this review outlines advanced bioengineering technologies such as 3D biofabricated hydrogels/scaffolds and microfluidic systems utilized with female germlines for fertility preservation through in vitro folliculogenesis and oogenesis.

Managing ecosystems in a sea of uncertainty: invasive species management and assisted colonizations.

Managing ecosystems in the face of complex species interactions, and the associated uncertainty, presents a considerable ecological challenge. Altering those interactions via actions such as invasive species management or conservation translocations can result in unintended consequences, supporting the need to be able to make more informed decisions in the face of this uncertainty. We demonstrate the utility of ecosystem models to reduce uncertainty and inform future ecosystem management. We use Phillip Island, Australia, as a case study to investigate the impacts of two invasive species management options and consider whether a critically endangered mammal is likely to establish a population in the presence of invasive species. Qualitative models are used to determine the effects of apex predator removal (feral cats) and invasive prey removal (rabbits, rats, and mice). We extend this approach using Ensemble Ecosystem Models to consider how suppression, rather than eradication influences the species community; and consider whether an introduction of the critically endangered eastern barred bandicoot is likely to be successful in the presence of invasive species. Our analysis revealed the potential for unintended outcomes associated with feral cat control operations, with rats and rabbits expected to increase in abundance. A strategy based on managing prey species appeared to have the most ecosystem-wide benefits, with rodent control showing more favorable responses than a rabbit control strategy. Eastern barred bandicoots were predicted to persist under all feral cat control levels (including no control). Managing ecosystems is a complex and imprecise process. However, qualitative modeling and ensemble ecosystem modeling address uncertainty and are capable of improving and optimizing management practices. Our analysis shows that the best conservation outcomes may not always be associated with the top-down control of apex predators, and land managers should think more broadly in relation to managing bottom-up processes as well. Challenges faced in continuing to conserve biodiversity mean new, bolder, conservation actions are needed. We suggest that endangered species are capable of surviving in the presence of feral cats, potentially opening the door for more conservation translocations.

It's a small world after all: A Canadian resident's perspective on COVID-19.

COVID-19 has infected millions of people, with an estimated total dead in the hundreds of thousands. This has significantly impacted health care, including who is delivering it, how it is delivered, and how it is taught. This article describes challenges of the COVID-19 pandemic from the perspective of a Canadian nuclear medicine resident, including new risks with nuclear imaging, navigating new and sometimes challenging guidelines, as well as working and living within the confines of social distancing.

Fostering of advanced mutualism with gut microbiota by Immunoglobulin A.

Immunoglobulin A (IgA), the most abundantly secreted antibody isotype in mammals, not only provides direct immune protection to neonates via maternal milk but also helps program the infant immune system by regulating the microbiota. IgA continues to maintain dynamic interactions with the gut microbiota throughout life and this influences immune system homeostasis as well as other physiological processes. The secretory IgA produced independently of T-cell selection are commonly referred to as natural or innate antibodies. Our studies have shown that innate-IgA, while effective at excluding microorganisms from the gut, does not promote mutualism with the microbiota in the same way as adaptive-IgA that is selected in T cell-dependent germinal center reactions. Adaptive-IgA fosters more advanced mutualism with the microbiota than innate-IgA by selecting and diversifying beneficial microbial communities. In this review, we suggest that the diversified microbiota resulting from adaptive-IgA pressure was pivotal in promoting ecological adaptability and speciation potential of mammals.

Kinetics of the Topochemical Transformation of (PbSe) m(TiSe2) n(SnSe2) m(TiSe2) n to (Pb0.5Sn0.5Se) m(TiSe2) n.

Solid-state reaction kinetics on atomic length scales have not been heavily investigated due to the long times, high reaction temperatures, and small reaction volumes at interfaces in solid-state reactions. All of these conditions present significant analytical challenges in following reaction pathways. Herein we use in situ and ex situ X-ray diffraction, in situ X-ray reflectivity, high-angle annular dark field scanning transmission electron microscopy, and energy-dispersive X-ray spectroscopy to investigate the mechanistic pathways for the formation of a layered (Pb0.5Sn0.5Se)1+δ(TiSe2) m heterostructure, where m is the varying number of TiSe2 layers in the repeating structure. Thin film precursors were vapor deposited as elemental-modulated layers into an artificial superlattice with Pb and Sn in independent layers, creating a repeating unit with twice the size of the final structure. At low temperatures, the precursor undergoes only a crystallization event to form an intermediate (SnSe2)1+γ(TiSe2) m(PbSe)1+δ(TiSe2) m superstructure. At higher temperatures, this superstructure transforms into a (Pb0.5Sn0.5Se)1+δ(TiSe2) m alloyed structure. The rate of decay of superlattice reflections of the (SnSe2)1+γ(TiSe2) m(PbSe)1+δ(TiSe2) m superstructure was used as the indicator of the progress of the reaction. We show that increasing the number of TiSe2 layers does not decrease the rate at which the SnSe2 and PbSe layers alloy, suggesting that at these temperatures it is reduction of the SnSe2 to SnSe and Se that is rate limiting in the formation of the alloy and not the associated diffusion of Sn and Pb through the TiSe2 layers.

Nanoparticle Adsorption on Antifouling Polymer Brushes.

Polymer brushes have been widely used to functionalize surfaces and provide antifouling capabilities against proteins and cells. Many efforts have focused on methods for functionalization of antifouling polymer brush surfaces for interactions with specific cells, proteins, and bacteria, but none have focused on immobilizing nanoparticles (NPs) on these surfaces. This article demonstrates that both pristine NPs and protein-coated NPs can adsorb onto well-functioning antifouling polymer brush coatings formed from poly-l-lysine-graft-poly(ethylene glycol) (PLL-g-PEG) and methoxy PEG-thiol. The role of ionic strength in solution, substrate surface material, and NP surface charge in the interaction was investigated to explore the forces behind the interaction. The adsorption of different types of NPs onto the surface was studied, determining that polystyrene, gold, carbon black, and silica particles can adsorb onto PLL-g-PEG. We show that the approach can be applied in, and studied by, both surface plasmon resonance and fluorescence imaging and suggest its application as a means to study NP-protein interactions, such as the protein corona. NPs self-assembled at antifouling polymer brush surfaces provide a novel platform for both scientific studies and applications in biotechnology.

Chiral plasmonic nanocrescents: large-area fabrication and optical properties.

Large-area arrays of substrate-supported plasmonic gold crescents are fabricated by using the new colloidal lithography technique, which is based on an in situ-deposited silica resistance layer. The method provides the means to control the particles' asymmetry just by changing the mutual deposition angle of gold and silica. Asymmetric crescent structures exhibit a pronounced circular dichroism in near-infrared region, with the chiral asymmetry factor reaching 0.2. According to the simulation, the optical chirality enhancement reaches between one and two orders of magnitude and is localized near the crescents' tips.

Immune antibodies and helminth products drive CXCR2-dependent macrophage-myofibroblast crosstalk to promote intestinal repair.

Helminth parasites can cause considerable damage when migrating through host tissues, thus making rapid tissue repair imperative to prevent bleeding and bacterial dissemination particularly during enteric infection. However, how protective type 2 responses targeted against these tissue-disruptive multicellular parasites might contribute to homeostatic wound healing in the intestine has remained unclear. Here, we observed that mice lacking antibodies (Aid-/-) or activating Fc receptors (Fcrg-/-) displayed impaired intestinal repair following infection with the murine helminth Heligmosomoides polygyrus bakeri (Hpb), whilst transfer of immune serum could partially restore chemokine production and rescue wound healing in Aid-/- mice. Impaired healing was associated with a reduced expression of CXCR2 ligands (CXCL2/3) by macrophages (MΦ) and myofibroblasts (MF) within intestinal lesions. Whilst antibodies and helminths together triggered CXCL2 production by MΦ in vitro via surface FcR engagement, chemokine secretion by intestinal MF was elicited by helminths directly via Fcrg-chain/dectin2 signaling. Blockade of CXCR2 during Hpb challenge infection reproduced the delayed wound repair observed in helminth infected Aid-/- and Fcrg-/- mice. Finally, conditioned media from human MΦ stimulated with infective larvae of the helminth Ascaris suum together with immune serum, promoted CXCR2-dependent scratch wound closure by human MF in vitro. Collectively our findings suggest that helminths and antibodies instruct a chemokine driven MΦ-MF crosstalk to promote intestinal repair, a capacity that may be harnessed in clinical settings of impaired wound healing.

Antibody-mediated trapping of helminth larvae requires CD11b and Fcγ receptor I.

Infections with intestinal helminths severely impact on human and veterinary health, particularly through the damage that these large parasites inflict when migrating through host tissues. Host immunity often targets the motility of tissue-migrating helminth larvae, which ideally should be mimicked by anti-helminth vaccines. However, the mechanisms of larval trapping are still poorly defined. We have recently reported an important role for Abs in the rapid trapping of tissue-migrating larvae of the murine parasite Heligmosomoides polygyrus bakeri. Trapping was mediated by macrophages (MΦ) and involved complement, activating FcRs, and Arginase-1 (Arg1) activity. However, the receptors and Ab isotypes responsible for MΦ adherence and Arg1 induction remained unclear. Using an in vitro coculture assay of H. polygyrus bakeri larvae and bone marrow-derived MΦ, we now identify CD11b as the major complement receptor mediating MΦ adherence to the larval surface. However, larval immobilization was largely independent of CD11b and instead required the activating IgG receptor FcγRI (CD64) both in vitro and during challenge H. polygyrus bakeri infection in vivo. FcγRI signaling also contributed to the upregulation of MΦ Arg1 expression in vitro and in vivo. Finally, IgG2a/c was the major IgG subtype from early immune serum bound by FcγRI on the MΦ surface, and purified IgG2c could trigger larval immobilization and Arg1 expression in MΦ in vitro. Our findings reveal a novel role for IgG2a/c-FcγRI-driven MΦ activation in the efficient trapping of tissue-migrating helminth larvae and thus provide important mechanistic insights vital for anti-helminth vaccine development.

Podosome Formation and Development in Monocytes Restricted by the Nanoscale Spatial Distribution of ICAM1.

We studied podosome formation and development in activated monocytes (THP1) at ICAM1 (intercellular adhesion molecule 1) nanopatterns of circular and ring-shaped domains and show that cellular binding to a preclustered ICAM1 nanopattern requires ligand patches of at least 200 nm (corresponding to 14 or more integrins). Podosome-like adhesion formation depends on the structure of the ligand pattern under the developing podosome with larger single domains promoting adhesion in a single patch and multiple smaller domains allowing podosome formation by integration of at least 2 smaller domains on either side of the podosome core. Maturation to rosette structures and recruitment of proteases were only observed with macroscopic ICAM1 presentation.

Calcium-phosphate-osteopontin particles for caries control.

Caries is caused by acid production in biofilms on dental surfaces. Preventing caries therefore involves control of microorganisms and/or the acid produced. Here, calcium-phosphate-osteopontin particles are presented as a new approach to caries control. The particles are made by co-precipitation and designed to bind to bacteria in biofilms, impede biofilm build-up without killing the microflora, and release phosphate ions to buffer bacterial acid production if the pH decreases below 6. Analysis of biofilm formation and pH in a five-species biofilm model for dental caries showed that treatment with particles or pure osteopontin led to less biofilm formation compared to untreated controls or biofilms treated with osteopontin-free particles. The anti-biofilm effect can thus be ascribed to osteopontin. The particles also led to a slower acidification of the biofilm after exposure to glucose, and the pH always remained above 5.5. Hence, calcium-phosphate-osteopontin particles show potential for applications in caries control.

Hierarchical protein patterning by meso to molecular scale self-assembly.

Numerous protein patterning methodologies are used extensively for biomedical research and development. We have developed a novel bottom-up protein patterning method using a combination of self-assembly processes in the meso to molecular scale range to allow hierarchical protein patterns to be straightforwardly fabricated with low cost over large areas. As a proof of principle, we patterned vitronectin in various dimensional hierarchies using meso to nanoscale colloids and self-assembled monolayers.

Spatial mapping and quantification of soft and hard protein coronas at silver nanocubes.

Protein coronas around silver nanocubes were quantified in serum-containing media using localized surface plasmon resonances. Both soft and hard coronas showed exposure-time and concentration-dependent changes in protein surface density with time-dependent hardening. We observed spatially dependent kinetics of the corona-formation at cube edges/corners versus facets at short incubation times, where the polymer stabilization agent delayed corona hardening. The soft corona contained more protein than the hard corona at all time-points (8-fold difference with 10% serum conditions).

The nanoscale geometrical maturation of focal adhesions controls stem cell differentiation and mechanotransduction.

We show that the nanoscale adhesion geometry controls the spreading and differentiation of epidermal stem cells. We find that cells respond to such hard nanopatterns similarly to their behavior on soft hydrogels. Cellular responses were seen to stem from local changes in diffusion dynamics of the adapter protein vinculin and associated impaired mechanotransduction rather than impaired recruitment of proteins involved in focal adhesion formation.

From rings to crescents: a novel fabrication technique uncovers the transition details.

A novel fabrication route is reported for the generation of substrate-supported symmetric and asymmetric metal nanostructures. We combine a colloidal template and angled evaporation to deposit in situ mask materials for subsequent lithographic pattern transfer. The technique is demonstrated for the fabrication of concentric and nonconcentric gold rings and crescents. Optical properties of localized plasmon resonances in such structures are studied by UV-vis-NIR spectroscopy and finite-difference time domain simulations during the transition from rings to crescents revealing the development of strong quadrupolar modes.

Onset of bonding plasmon hybridization preceded by gap modes in dielectric splitting of metal disks.

Dielectric splitting of nanoscale disks was studied experimentally and via finite-difference time-domain (FDTD) simulations through systematic introduction of multiple ultrathin dielectric layers. Tunable, hybridized dark bonding modes were seen with first-order gap modes preceding the appearance of bonding dipole-dipole disk modes. The observed bright dipolar mode did not show the energy shift expected from plasmon hybridization but activated dark higher order gap modes. Introducing lateral asymmetry was shown to remodel the field distribution resulting in 3D asymmetry that reoriented the dipole orientation away from the dipole of the elementary disk modes.

Smart Biointerfaces via Click Chemistry-Enabled Nanopatterning of Multiple Bioligands and DNA Force Sensors.

Nanoscale biomolecular placement is crucial for advancing cellular signaling, sensor technology, and molecular interaction studies. Despite this, current methods fall short in enabling large-area nanopatterning of multiple biomolecules while minimizing nonspecific interactions. Using bioorthogonal tags at a submicron scale, we introduce a novel hole-mask colloidal lithography method for arranging up to three distinct proteins, DNA, or peptides on large, fully passivated surfaces. The surfaces are compatible with single-molecule fluorescence microscopy and microplate formats, facilitating versatile applications in cellular and single-molecule assays. We utilize fully passivated and transparent substrates devoid of metals and nanotopographical features to ensure accurate patterning and minimize nonspecific interactions. Surface patterning is achieved using bioorthogonal TCO-tetrazine (inverse electron-demand Diels-Alder, IEDDA) ligation, DBCO-azide (strain-promoted azide-alkyne cycloaddition, SPAAC) click chemistry, and biotin-avidin interactions. These are arranged on surfaces passivated with dense poly(ethylene glycol) PEG brushes crafted through the selective and stepwise removal of sacrificial metallic and polymeric layers, enabling the directed attachment of biospecific tags with nanometric precision. In a proof-of-concept experiment, DNA tension gauge tether (TGT) force sensors, conjugated to cRGD (arginylglycylaspartic acid) in nanoclusters, measured fibroblast integrin tension. This novel application enables the quantification of forces in the piconewton range, which is restricted within the nanopatterned clusters. A second demonstration of the platform to study integrin and epidermal growth factor (EGF) proximal signaling reveals clear mechanotransduction and changes in the cellular morphology. The findings illustrate the platform's potential as a powerful tool for probing complex biochemical pathways involving several molecules arranged with nanometer precision and cellular interactions at the nanoscale.

Building better habitats: Spatiotemporal signaling cues in 3D biointerfaces for tailored cellular functionality.

A promising research direction in the field of biological engineering is the design and functional programming of three-dimensional (3D) biointerfaces designed to support living cell functionality and growth in vitro, offering a route to precisely regulate cellular behaviors and phenotypes for addressing therapeutic challenges. While traditional two-dimensional (2D) biointerfaces have provided valuable insights, incorporating specific signaling cues into a 3D biointeractive microenvironment at the right locations and time is now recognized as crucial for accurately programming cellular decision-making and communication processes. This approach aims to engineer cell-centric microenvironments with the potential to recapitulate complex biological functions into a finite set of growing cellular organizations. Additionally, they provide insights into the hierarchical logic governing the relationship between molecular components and higher-order multicellular functionality. The functional live cell-based microenvironment engineered through such innovative biointerfaces has the potential to be used as an in vitro model system for expanding our understanding of cellular behaviors or as a therapeutic habitat where cellular functions can be reprogrammed.