Characterizing the Behavior of Water Interacting With a Nano-Pore Material: A Structural Investigation in Native Environment Using Magnetic Resonance Approaches.

The study of fluid absorption, particularly that of water, into nanoporous materials has garnered increasing attention in the last decades across a broad range of disciplines. However, most investigation approaches to probe such behaviors are limited by characterization conditions. In this study, a combined MRI and MAS NMR method was used to study a nanoporous silica glass to acquire information about its structural framework and interactions with confined water. Specifically, MRI was used for a quantitative analysis of water extent. While MAS NMR techniques provided structural information of silicate materials, including interactive surface area and framework packing. Analysis of water spin-spin relaxation times (T2) suggested differences in water confinement within the characterized framework. Subsequent unsuccessful delivery of paramagnetic molecule into the pores enabled a quantitative assessment of the dimensions that "bottleneck" the pores. Finally, pore sizes were derived from the molecular size, density function theory (DFT) simulation and characterizations on standard samples. Our result matches with Brunauer-Emmett-Teller (BET) analysis.. The use of a paramagnetic probe for pore size determination introduces a new approach of characterization in the liquid phase, offering an alternative to the conventional BET analysis that uses gas molecule as probes.

Immunotherapy targeting isoDGR-protein damage extends lifespan in a mouse model of protein deamidation.

Aging results from the accumulation of molecular damage that impairs normal biochemical processes. We previously reported that age-linked damage to amino acid sequence NGR (Asn-Gly-Arg) results in "gain-of-function" conformational switching to isoDGR (isoAsp-Gly-Arg). This integrin-binding motif activates leukocytes and promotes chronic inflammation, which are characteristic features of age-linked cardiovascular disorders. We now report that anti-isoDGR immunotherapy mitigates lifespan reduction of Pcmt1-/- mouse. We observed extensive accumulation of isoDGR and inflammatory cytokine expression in multiple tissues from Pcmt1-/- and naturally aged WT animals, which could also be induced via injection of isoDGR-modified plasma proteins or synthetic peptides into young WT animals. However, weekly injection of anti-isoDGR mAb (1 mg/kg) was sufficient to significantly reduce isoDGR-protein levels in body tissues, decreased pro-inflammatory cytokine concentrations in blood plasma, improved cognition/coordination metrics, and extended the average lifespan of Pcmt1-/- mice. Mechanistically, isoDGR-mAb mediated immune clearance of damaged isoDGR-proteins via antibody-dependent cellular phagocytosis (ADCP). These results indicate that immunotherapy targeting age-linked protein damage may represent an effective intervention strategy in a range of human degenerative disorders.

Protein Cage Relaxivity Measurement for Magnetic Resonance Imaging Contrast Agents.

Contrast agents are employed to enhance the differentiation of diseased cells or lesions from normal tissues in magnetic resonance imaging (MRI). Protein cages have been explored as templates to synthesize superparamagnetic MRI contrast agents for decades. The biological origin imparts natural precision in forming confined nano-sized reaction vessels. With natural capacity to bind divalent metal ions, ferritin protein cages have been used for the synthesis of nanoparticles containing MRI contrast agents inside their core. Furthermore, ferritin is known to bind transferrin receptor 1 (TfR1) which is overexpressed on specific cancer cell types and could be used for targeted cellular imaging. In addition to iron, other metal ions such as manganese and gadolinium have been encapsulated within the core of ferritin cages. To compare the magnetic properties of ferritin loaded with contrast agents, a protocol for calculating the contrast enhancement power of protein nanocage is required. The contrast enhancement power is demonstrated as relaxivity and can be measured using MRI and solution nuclear magnetic resonance (NMR) methods. In this chapter, we present methods for measuring and calculating the relaxivity of ferritin nanocages loaded with paramagnetic ions in solution (in tube) with NMR and MRI.

Complete Sequences of the Velvet Worm Slime Proteins Reveal that Slime Formation is Enabled by Disulfide Bonds and Intrinsically Disordered Regions.

The slime of velvet worms (Onychophora) is a strong and fully biodegradable protein material, which upon ejection undergoes a fast liquid-to-solid transition to ensnare prey. However, the molecular mechanisms of slime self-assembly are still not well understood, notably because the primary structures of slime proteins are yet unknown. Combining transcriptomic and proteomic studies, the authors have obtained the complete primary sequences of slime proteins and identified key features for slime self-assembly. The high molecular weight slime proteins contain cysteine residues at the N- and C-termini that mediate the formation of multi-protein complexes via disulfide bonding. Low complexity domains in the N-termini are also identified and their propensity for liquid-liquid phase separation is established, which may play a central role in slime biofabrication. Using solid-state nuclear magnetic resonance, rigid and flexible domains of the slime proteins are mapped to specific peptide domains. The complete sequencing of major slime proteins is an important step toward sustainable fabrication of polymers inspired by the velvet worm slime.

Magnetoferritin enhances T2 contrast in magnetic resonance imaging of macrophages.

Imaging of immune cells has wide implications in understanding disease progression and staging. While optical imaging is limited in penetration depth due to light properties, magnetic resonance (MR) imaging provides a more powerful tool for the imaging of deep tissues where immune cells reside. Due to poor MR signal to noise ratio, tracking of such cells typically requires contrast agents. This report presents an in-depth physical characterization and application of archaeal magnetoferritin for MR imaging of macrophages - an important component of the innate immune system that is the first line of defense and first responder in acute inflammation. Magnetoferritin is synthesized by loading iron in apoferritin in anaerobic condition at 65 °C. The loading method results in one order of magnitude enhancement of r1 and r2 relaxivities compared to standard ferritin synthesized by aerobic loading of iron at room temperature. Detailed characterizations of the magnetoferritin revealed a crystalline core structure that is distinct from previously reported ones indicating magnetite form. The magnetite core is more stable in the presence of reducing agents and has higher peroxidase-like activities compared to the core in standard loading. Co-incubation of macrophage cells with magnetoferritin in-vitro shows significantly higher enhancement in T2-MRI contrast of the immune cells compared to standard ferritin.

Abundant neuroprotective chaperone Lipocalin-type prostaglandin D synthase (L-PGDS) disassembles the Amyloid-ß fibrils.

Misfolding of Amyloid ß (Aß) peptides leads to the formation of extracellular amyloid plaques. Molecular chaperones can facilitate the refolding or degradation of such misfolded proteins. Here, for the first time, we report the unique ability of Lipocalin-type Prostaglandin D synthase (L-PGDS) protein to act as a disaggregase on the pre-formed fibrils of Aß(1-40), abbreviated as Aß40, and Aß(25-35) peptides, in addition to inhibiting the aggregation of Aß monomers. Furthermore, our proteomics results indicate that L-PGDS can facilitate extraction of several other proteins from the insoluble aggregates extracted from the brain of an Alzheimer's disease patient. In this study, we have established the mode of binding of L-PGDS with monomeric and fibrillar Aß using Nuclear Magnetic Resonance (NMR) Spectroscopy, Small Angle X-ray Scattering (SAXS), and Transmission Electron Microscopy (TEM). Our results confirm a direct interaction between L-PGDS and monomeric Aß40 and Aß(25-35), thereby inhibiting their spontaneous aggregation. The monomeric unstructured Aß40 binds to L-PGDS via its C-terminus, while the N-terminus remains free which is observed as a new domain in the L-PGDS-Aß40 complex model.

Cerebral Ischemic Preconditioning: the Road So Far .

Cerebral preconditioning constitutes the brain's adaptation to lethal ischemia when first exposed to mild doses of a subtoxic stressor. The phenomenon of preconditioning has been largely studied in the heart, and data from in vivo and in vitro models from past 2-3 decades have provided sufficient evidence that similar machinery exists in the brain as well. Since preconditioning results in a transient protective phenotype labeled as ischemic tolerance, it can open many doors in the medical warfare against stroke, a debilitating cerebrovascular disorder that kills or cripples thousands of people worldwide every year. Preconditioning can be induced by a variety of stimuli from hypoxia to pharmacological anesthetics, and each, in turn, induces tolerance by activating a multitude of proteins, enzymes, receptors, transcription factors, and other biomolecules eventually leading to genomic reprogramming. The intracellular signaling pathways and molecular cascades behind preconditioning are extensively being investigated, and several first-rate papers have come out in the last few years centered on the topic of cerebral ischemic tolerance. However, translating the experimental knowledge into the clinical scaffold still evades practicality and faces several challenges. Of the various preconditioning strategies, remote ischemic preconditioning and pharmacological preconditioning appears to be more clinically relevant for the management of ischemic stroke. In this review, we discuss current developments in the field of cerebral preconditioning and then examine the potential of various preconditioning agents to confer neuroprotection in the brain.