Detection of α-Thrombin with Platelet Glycoprotein Ibα (GP1bα) for the Development of a Coagulation Marker.

The detection of prothrombotic markers is crucial for understanding thromboembolism and assessing the effectiveness of anticoagulant drugs. α-Thrombin is a marker that plays a critical role in the coagulation cascade process. However, the detection of this enzymatic molecule was hindered by the absence of an efficient modality in the clinical environment. Previously, we reported that one α-thrombin interacts with two α-chains of glycoprotein Ib (GPIbα), i.e., multivalent protein binding (MPB), using bioresponsive hydrogel nanoparticles (nanogels) and optical microscopy. In this study, we demonstrated that GPIbα-mediated platforms led to the highly sensitive and quantitative detection of α-thrombin in various diagnostic systems. Initially, a bioresponsive nanogel-based surface plasmon resonance (nSPR) assay was developed that responds to the MPB of α-thrombin to GPIbα. The use of GPIbα for the detection of α-thrombin was further validated using the enzyme-linked immunosorbent assay, which is a gold-standard protein detection technique. Additionally, GPIbα-functionalized latex beads were developed to perform latex agglutination (LA) assays, which are widely used with hospital diagnostic instruments. Notably, the nSPR and LA assays exhibited a nearly 1000-fold improvement in sensitivity for α-thrombin detection compared to our previous optical microscopy method. The superiority of our GPIbα-mediated platforms lies in their stability for α-thrombin detection through protein-protein interactions. By contrast, assays relying on α-thrombin enzymatic activity using substrates face the challenge of a rapid decrease in postsample collection. These results suggested that the MPB of α-thrombin to GPIbα is an ideal mode for clinical α-thrombin detection, particularly in outpatient settings.

Efficient production of natural sunscreens shinorine, porphyra-334, and mycosporine-2-glycine in Saccharomyces cerevisiae.

Mycosporine-like amino acids (MAAs) are promising natural sunscreens mainly produced in marine organisms. Until now, metabolic engineering efforts to produce MAAs in heterologous hosts have mainly focused on shinorine production, and the low production levels are still not suitable for industrial applications. In this study, we successfully developed Saccharomyces cerevisiae strains that can efficiently produce various disubstituted MAAs, including shinorine, porphyra-334, and mycosporine-2-glycine (M2G), which are formed by conjugating serine, threonine, and glycine to mycosporine-glycine (MG), respectively. We first generated an MG-producing strain by multiple integration of the biosynthetic genes from cyanobacteria and applying metabolic engineering strategies to increase sedoheptulose-7-phosphate pool, a substrate for MG production. Next, five mysD genes from cyanobacteria, which encode D-Ala-D-Ala ligase homologues that conjugate an amino acid to MG, were introduced into the MG-producing strain to determine the substrate preference of each MysD enzyme. MysDs from Lyngbya sp., Nostoclinckia, and Euhalothece sp. showed high specificity toward serine, threonine, and glycine, resulting in efficient production of shinorine, porphyra-334, and M2G, respectively. This is the first report on the production of porphyra-334 and M2G in S. cerevisiae. Furthermore, we identified that the substrate specificity of MysD was determined by the omega loop region of 43-45 amino acids predicted based on its structural homology to a D-Ala-D-Ala ligase from Thermus thermophilus involved in peptidoglycan biosynthesis. The substrate specificities of two MysD enzymes were interchangeable by swapping the omega loop region. Using the engineered strain expressing mysD from Lyngbya sp. or N. linckia, up to 1.53 g/L shinorine or 1.21 g/L porphyra-334 was produced by fed-batch fermentation in a 5-L bioreactor, the highest titer reported so far. These results suggest that S. cerevisiae is a promising host for industrial production of different types of MAAs, providing a sustainable and eco-friendly alternative for the development of natural sunscreens.

Tuning Surface Plasmon Resonance Responses through Size and Crosslinking Control of Multivalent Protein Binding-Capable Nanoscale Hydrogels.

Surface plasmon resonance (SPR) phenomena have been widely studied to detect biomolecules because of their high sensitivity and ability to determine biomolecular interactions with kinetic information. However, highly selective detection in specific concentration ranges relevant to target biomolecules is still a challenging task. Recently, we developed bioresponsive nanoscale hydrogels to selectively intensify SPR signals through multivalent protein binding (MPB) events with target biomolecules, including IL-2, where we were able to demonstrate exceptional selectivity for target biomolecules with minimal responses to nonspecific and monovalent binding events. In this work, we systematically explored the relationship between the physical properties of MPB-capable nanoscale hydrogels and their SPR response induced in the presence of the programmed cell death protein 1 antibody (PD-1Ab) as a model target biomolecule. First, we developed a synthetic protocol by controlling various reaction parameters to construct a library of nanoscale poly(N-isopropylacrylamide-co-acrylic acid) hydrogels (NHs) with different sizes (from 400 nm to 1 µm) and degrees of crosslinking (from 2 to 8%). Then, by incorporating MPB-capable PD-1 receptors onto the surface of NHs to form PD-1-responsive nanoscale hydrogels (PNHs), the hydrogel size and crosslinking dependency of their SPR responses were investigated. Our results reveal the appropriate hydrogel size regime and degree of crosslinking for effective PD-1Ab detection at specific concentrations range between a few nM and 1 µM. Overall, our study demonstrates that by tuning the physical properties of the nanoscale hydrogel matrix, the sensitivity and detection range of MPB-based SPR sensors can be modulated to potentially benefit clinical applications such as monitoring diverse therapeutic biomolecules.

Simple Host-Guest Assembly for High-Resolution Magnetic Resonance Imaging of Microvasculature.

Magnetic resonance angiography (MRA) is an important imaging technique that can be used to identify and characterize various types of vascular diseases. However, currently used molecular contrast agents are unsuitable for MRA due to the short intravascular retention time, the whole-body distribution, and the relatively low contrast effect. In this study, we developed a vascular analysis contrast agent (i.e., VasCA) for MRA, which is a simple and biocompatible 1:1 host-guest assembly of PEGylated ß-cyclodextrin and gadolinium chelate with renal clearable size and high relaxivity (r1 = 9.27 mM-1 s-1). Its biocompatibility was confirmed by in vivo animal studies as well as in vitro 3D cell culture. In a tumor-bearing rat model, VasCA circulated in the blood vessels much longer (4.3-fold increase) than gadoterate meglumine (Dotarem) and was mainly excreted by the renal system after intravenous injection. This feature of VasCA allows characterization of tumor microvasculature (e.g., feeding and draining vessels) as well as visualization of small vessels in the brain and body organs. Furthermore, after treatment with an angiogenesis inhibitor (i.e., sorafenib), VasCA revealed the vessel normalization process and allowed the assessment of viable and necrotic tumor regions. Our study provides a useful tool for diverse MRA applications, including tumor characterization, early-stage evaluation of drug efficacy, and treatment planning, as well as diagnosis of cardiovascular diseases.

Label-Free Analysis of Multivalent Protein Binding Using Bioresponsive Nanogels and Surface Plasmon Resonance (SPR).

Precise identification of protein-protein interactions is required to improve our understanding of biochemical pathways for biology and medicine. In physiology, how proteins interact with other proteins or small molecules is crucial for maintaining biological functions. For instance, multivalent protein binding (MPB), in which a ligand concurrently interacts with two or more receptors, plays a key role in regulating complex but accurate biological functions, and its interference is related to many diseases. Therefore, determining MPB and its kinetics has long been sought, which currently requires complicated procedures and instruments to distinguish multivalent binding from monovalent binding. Here, we show a method for quickly evaluating the MPB over monovalent binding and its kinetic parameters in a label-free manner. Engaging pNIPAm-co-AAc nanogels with MPB-capable moieties (e.g., PD-1 antigen and biocytin) permits a surface plasmon resonance (SPR) instrument to evaluate the MPB events by amplifying signals from the specific target molecules. Using our MPB-based method, PD-1 antibody that forms a type of MPB by complexing with two PD-1 proteins, which are currently used for cancer immunotherapy, is detectable down to a level of 10 nM. In addition, small multivalent cations (e.g., Ca2+, Fe2+, and Fe3+) are distinguishably measurable over monovalent cations (e.g., Na+ and K+) with the pNIPAm-co-AAc nanogels.