Microparticles in marine mussels at regional and localized scales across the Salish Sea, Washington.

Microparticles (MP; particles <5 mm) are ubiquitous in marine environments. Understanding MP concentrations at different spatial scales in the Salish Sea, Washington, USA, can provide insight into how ecologically and economically important species may be affected. We collected mussels across the Salish Sea at regional and localized scales, chemically processed tissue to assess MP contamination, and used visual and chemical analyses for particle identification. Throughout the Salish Sea, mussel MP concentrations averaged 0.75 ± 0.09 MP g-1 wet tissue. At a regional scale, we identified slight differences in concentrations and morphotypes of MP while at a localized scale these metrics were not significant and did not differ from controls. In a subset of particles, 20 % were identified as synthetic materials, which include polyethylene terephthalate (PET), polypropylene (PP), polystyrene (PS), and nylon. Differences in MP sources, heterogeneous transport of MP, and distinct shellfish feeding mechanisms may contribute to plastic contamination patterns in the Salish Sea.

Exploiting weak supervision to facilitate segmentation, classification, and analysis of microplastics (<100 µm) using Raman microspectroscopy images.

Reliable quantification and characterization of microplastics are necessary for large-scale and long-term monitoring of their behaviors and evolution in the environment. This is especially true in recent times because of the increase in the production and use of plastics during the pandemic. However, because of the myriad of microplastic morphologies, dynamic environmental forces, and time-consuming and expensive methods to characterize microplastics, it is challenging to understand microplastic transport in the environment. This paper describes a novel approach that compares unsupervised, weakly-supervised, and supervised approaches to facilitate segmentation, classification, and the analysis of <100 µm-sized microplastics without the use of pixel-wise human-labeled data. The secondary aim of this work is to provide insight into what can be accomplished when no human annotations are available, using the segmentation and classification tasks as use cases. In particular, the weakly-supervised segmentation performance surpasses the baseline performance set by the unsupervised approach. Consequently, feature extraction (derived from the segmentation results) provides objective parameters describing microplastic morphologies that will result in better standardization and comparisons of microplastic morphology across future studies. The weakly-supervised performance for microplastic morphology classification (e.g., fiber, spheroid, shard/fragment, irregular) also exceeds the performance of the supervised analogue. Moreover, in contrast to the supervised method, our weakly-supervised approach provides the benefit of pixel-wise detection of microplastic morphology. Pixel-wise detection is used further to improve shape classifications. We also demonstrate a proof-of-concept for distinguishing microplastic particles from non-microplastic particles using verification data from Raman microspectroscopy. As the automation of microplastic monitoring progresses, robust and scalable identification of microplastics based on their morphology may be achievable.

High-throughput profiling of antibody self-association in multiple formulation conditions by PEG stabilized self-interaction nanoparticle spectroscopy.

Affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS) is an assay developed to monitor the propensity of antibody self-association, hence assessing its colloidal stability. It has been widely used by pharmaceutical companies to screen antibodies at the early discovery stages, aiming to flag potential issues with high concentration formulation. However, the original assay format is not suitable for certain formulation conditions, in particular histidine buffer. In addition, the previous data extrapolation method is suboptimal and cumbersome for processing large amounts of data (100s of molecules) in a high-throughput fashion. To address these limitations, we developed an assay workflow with two major improvements: 1) use of a stabilizing reagent to enable screening of a broader range of formulation conditions beyond phosphate-buffered saline, pH 7.4; and 2) inclusion of a novel algorithm and robust data processing schema that empowers streamlined data analysis. The optimized assay format expands the screening applicability to a wider range of formulation conditions critical for downstream development. Such capability is enhanced by a custom data management workflow for optimal data extraction, analysis, and automation. Our protocol and the R/Shiny application for analysis are publicly available and open-source to benefit the broader scientific community.

Low incidence of microplastic contaminants in Pacific oysters (Crassostrea gigas Thunberg) from the Salish Sea, USA.

Plastic pollution is a threat to marine life with long term impacts to ecosystems and organisms in the sea. In this study, we quantified the presence of microparticles in wild populations of Pacific oysters (Crassostrea gigas) from the Salish Sea, Washington State. Examination under a dissecting microscope revealed 63% of oysters contained microparticles (~1.75 microparticles per oyster) and microfibers were the dominant type of particles. Using Raman microspectroscopy (RMS) and Fourier transform infrared microspectroscopy (µ-FTIR) we found that only ~2% of these microparticles were synthetic and included polymers such as polystyrene, polyethylene, polypropylene, poly(bisphenol A carbonate), rayon, and polyacrylate. It is important to note that of the 447 microparticles analyzed with RMS, 41% showed fluorescence interference, impeding the determination of their identification. The remaining microparticles were cellulose derivatives, shell fragments, biological or proteinaceous material, salts, minerals, and gypsum. Fourier transform infrared spectroscopy equipped with a diamond attenuated total reflectance accessory (ATR-FTIR) showed the presence of sorbitan derivatives in all samples examined (n = 213). These findings provide the first baseline for microplastic and other particles in oysters from the west coast of the United States integrating results from ATR-FTIR, µ-FTIR, and RMS, in addition to visual sorting. These results suggest there is low retention of plastic particles in Pacific oysters from the Salish Sea, but further research is needed to determine the composition of microparticles with fluorescence interference.

Development of a high-throughput solubility screening assay for use in antibody discovery.

Poor solubility is a common challenge encountered during the development of high concentration monoclonal antibody (mAb) formulations, but there are currently no methods that can provide predictive information on high-concentration behavior of mAbs in early discovery. We explored the utility of methodologies used for determining extrapolated solubility as a way to rank-order mAbs based on their relative solubility properties. We devised two approaches to accomplish this: 1) vapor diffusion technique utilized in traditional protein crystallization practice, and 2) polyethylene glycol (PEG)-induced precipitation and quantitation by turbidity. Using a variety of in-house mAbs with known high-concentration behavior, we demonstrated that both approaches exhibited reliable predictability of the relative solubility properties of these mAbs. Optimizing the latter approach, we developed a format that is capable of screening a large panel of mAbs in multiple pH and buffer conditions. This simple, material-saving, high-throughput approach enables the selection of superior molecules and optimal formulation conditions much earlier in the antibody discovery process, prior to time-consuming and material intensive high-concentration studies.

Self-Sterilizing, Self-Cleaning Mixed Polymeric Multifunctional Antimicrobial Surfaces.

Mitigation of bacterial adhesion and subsequent biofilm formation is quickly becoming a strategy for the prevention of hospital-acquired infections. We demonstrate a basic strategy for surface modification that combines the ability to control attachment by microbes with the ability to inactivate microbes. The surface consists of two active materials: poly(p-phenylene ethynylene)-based polymers, which can inactivate a wide range of microbes and pathogens, and poly(N-isopropylacrylamide)-based polymers, which can switch between an hydrophobic "capture" state and a hydrophilic "release" state. The combination of these materials creates a surface that can both bind microbes in a switchable way and kill surface-bound microbes efficiently. Considerable earlier work with cationic poly(p-phenylene ethynylene) polyelectrolytes has demonstrated and characterized their antimicrobial properties, including the ability to efficiently destroy or deactivate Gram-negative and Gram-positive bacteria, fungi, and viruses. Similarly, much work has shown (1) that surface-polymerized films of poly(N-isopropylacrylamide) are able to switch their surface thermodynamic properties from a swollen, relatively hydrophilic state at low temperature to a condensed, relatively hydrophobic state at higher temperature, and (2) that this switch can control the binding and/or release of microbes to poly(N-isopropylacrylamide) surfaces. The active surfaces described herein were fabricated by first creating a film of biocidal poly(p-phenylene ethynylene) using layer-by-layer methods, and then conferring switchable adhesion by growing poly(N-isopropylacrylamide) through the poly(p-phenylene ethynylene) layer, using surface-attached polymerization initiators. The resulting multifunctional, complex films were then characterized both physically and functionally. We demonstrate that such films kill and subsequently induce widespread release of Gram-negative and Gram-positive bacteria.

Anti-tumor activity of stability-engineered IgG-like bispecific antibodies targeting TRAIL-R2 and LTbetaR.

Bispecific antibodies (BsAbs) represent an emerging class of biologics that achieve dual targeting with a single agent. Recombinant DNA technologies have facilitated a variety of creative bispecific designs with many promising therapeutic applications; however, practical methods for producing high quality BsAbs that have good product stability, long serum half-life, straightforward purification, and scalable production have largely been limiting. Here we describe a protein-engineering approach for producing stable, scalable tetravalent IgG-like BsAbs. The stability-engineered IgG-like BsAb was envisioned to target and crosslink two TNF family member receptors, TRAIL-R2 (TNF-Related Apoptosis Inducing Ligand Receptor-2) and LTbetaR (Lymphotoxin-beta Receptor), expressed on the surface of epithelial tumor cells with the goal of triggering an enhanced anti-tumor effect. Our IgG-like BsAbs consists of a stability-engineered anti-LTbetaR single chain Fv (scFv) genetically fused to either the N- or C-terminus of the heavy chain of a fulllength anti-TRAIL-R2 IgG1 monoclonal antibody. Both N- or C-terminal BsAbs were active in inhibiting tumor cell growth in vitro, and with some cell lines demonstrated enhanced activity relative to the combination of parental Abs. Pharmacokinetic studies in mice revealed long serum half-lives for the BsAbs. In murine tumor xenograft models, therapeutic treatment with the BsAbs resulted in reduction in tumor volume either comparable to or greater than the combination of parental antibodies, indicating that simultaneously targeting and cross-linking receptor pairs is an effective strategy for treating tumor cells. These studies support that stability-engineering is an enabling step for producing scalable IgG-like BsAbs with properties desirable for biopharmaceutical development.