Using machine learning for chemical-free histological tissue staining.

Hematoxylin and eosin staining can be hazardous, expensive, and prone to error and variability. To circumvent these issues, artificial intelligence/machine learning models such as generative adversarial networks (GANs), are being used to 'virtually' stain unstained tissue images indistinguishable from chemically stained tissue. Frameworks such as deep convolutional GANs (DCGAN) and conditional GANs (CGANs) have successfully generated highly reproducible 'stained' images. However, their utility may be limited by requiring registered, paired images which can be difficult to obtain. To avoid these dataset requirements, we attempted to use an unsupervised CycleGAN pix2pix model(5,6) to turn unpaired, unstained bright-field images into pathologist-approved digitally 'stained' images. Using formalin-fixed-paraffin-embedded liver samples, 5µm section images (20x) were obtained before and after staining to create "stained" an "unstained" datasets. Model implementation was conducted using Ubuntu 20.04.4 LTS, 32 GB RAM, Intel Core i7-9750 CPU @2.6 GHz, Nvidia GeForce RTX 2070 Mobile, Python 3.7.11 and Tensorflow 2.9.1. The CycleGAN framework utilized a u-net-based generator and discriminator from pix2pix, a CGAN. The CycleGAN used a modified loss function, cycle consistent loss that assumed unpaired images, so loss was measured twice. To our knowledge, this is the first documented application of this architecture using unpaired bright-field images. Results and suggested improvements are discussed.

Investigation of Rare Earth Element Binding to a Surface-Bound Affinity Peptide Derived from EF-Hand Loop I of Lanmodulin.

Bioinspired strategies have been given extensive attention for the recovery of rare earth elements (REEs) from waste streams because of their high selectivity, regeneration potential, and sustainability as well as low cost. Lanmodulin protein is an emerging biotechnology that is highly selective for REE binding. Mimicking lanmodulin with shorter peptides is advantageous because they are simpler and potentially easier to manipulate and optimize. Lanmodulin-derived peptides have been found to bind REEs, but their properties have not been explored when immobilized on solid substrates, which is required for many advanced separation technologies. Here, two peptides, LanM1 and scrambled LanM1, are designed from the EF-hand loop 1 of lanmodulin and investigated for their binding affinity toward different REEs when surface-bound. First, the ability of LanM1 to bind REEs was confirmed and characterized in solution using circular dichroism (CD), nuclear magnetic resonance (NMR), and molecular dynamics (MD) simulations for Ce(III) ions. Isothermal titration calorimetry (ITC) was used to further analyze the binding of the LanM1 to Ce(III), Nd(III), Eu(III), and Y(III) ions and in low-pH conditions. The performance of the immobilized peptides on a model gold surface was examined using a quartz crystal microbalance with dissipation (QCM-D). The studies show that the LanM1 peptide has a stronger REE binding affinity than that of scrambled LanM1 when in solution and when immobilized on a gold surface. QCM-D data were fit to the Langmuir adsorption model to estimate the surface-bound dissociation constant (Kd) of LanM1 with Ce(III) and Nd(III). The results indicate that LanM1 peptides maintain a high affinity for REEs when immobilized, and surface-bound LanM1 has no affinity for potential competitor calcium and copper ions. The utility of surface-bound LanM1 peptides was further demonstrated by immobilizing them to gold nanoparticles (GNPs) and capturing REEs from solution in experiments utilizing an Arsenazo III-based colorimetric dye displacement assay and ultraviolet-visible (UV-vis) spectrophotometry. The saturated adsorption capacity of GNPs was estimated to be around 3.5 µmol REE/g for Ce(III), Nd(III), Eu(III), and Y(III) ions, with no binding of non-REE Ca(II) ions observed.

Scalable Production of Peptides for Enhanced Struvite Formation via Expression on the Surface of Genetically Engineered Microbes.

A promising method for recycling phosphate from wastewater is through precipitation of struvite (MgNH4PO4·6H2O), a slow-release fertilizer. Peptides have been shown to increase the yield of struvite formation, but producing peptides via solid phase synthesis is cost prohibitive. This work investigates the effects of peptide-expressing bacteria on struvite precipitation to provide a sustainable and cost-efficient means to enhance struvite precipitation. A peptide known for increased struvite yield was expressed on a membrane protein in Escherichia coli(E. coli), and then 5 mL precipitation reactions were performed in 50 mL culture tubes for at least 15 min. The yield of struvite crystals was examined, with the presence of peptide-expressing E. coli inducing significantly higher yields than nonpeptide-expressing E. coli when normalized to the amount of bacteria. The precipitate was identified as struvite through Fourier transform infrared spectroscopy and energy dispersive spectroscopy, while the morphology and size of the crystals were analyzed through optical microscopy and scanning electron microscopy. Crystals were found to have a larger area when precipitated with the peptide-expressing bacteria. Additionally, bacteria-struvite samples were thermogravimetrically analyzed to quantify their purity and determine their thermal decomposition behavior. Overall, this study presents the benefits of a novel, microbe-driven method of struvite precipitation, offering a means for scalable implementation.

Clickable polymer scaffolds enable Ce recovery with peptide ligands.

Rare earth elements (REEs) are a vital part of many technologies with particular importance to the renewable energy sector and there is a pressing need for environmentally friendly and sustainable processes to recover and recycle them from waste streams. Functionalized polymer scaffolds are a promising means to recover REEs due to the ability to engineer both transport properties of the porous material and specificity for target ions. In this work, REE adsorbing polymer scaffolds were synthesized by first introducing poly(glycidyl methacrylate) (GMA) brushes onto porous polyvinylidene fluoride (PVDF) surface through activator generated electron transfer atom transfer radical polymerization (AGET ATRP). Azide moieties were then introduced through a ring opening reaction of GMA. Subsequently, REE-binding peptides were conjugated to the polymer surface through copper catalyzed azide alkyne cycloaddition (CuAAC) click chemistry. The presence of GMA, azide, and peptide was confirmed through Fourier transform infrared spectroscopy. Polymer scaffolds functionalized with the REE-binding peptide bound cerium, while polymer scaffolds functionalized with a scrambled control peptide bound significantly less cerium. Importantly, this study shows that the REE binding peptide retains its functionality when bound to a polymer surface. The conjugation strategy employed in this work can be used to introduce peptides onto other polymeric surfaces and tailor surface specificity for a wide variety of ions and small molecules.

Grafting of short elastin-like peptides using an electric field.

Surface-grafted elastin has found a wide range of uses such as sensing, tissue engineering and capture/release applications because of its ability to undergo stimuli-responsive phase transition. While various methods exist to control surface grafting in general, it is still difficult to control orientation as attachment occurs. This study investigates using an electric field as a new approach to control the surface-grafting of short elastin-like polypeptide (ELP). Characterization of ELP grafting to gold via quartz crystal microbalance with dissipation, atomic force microscopy and temperature ramping experiments revealed that the charge/hydrophobicity of the peptides, rearrangement kinetics and an applied electric field impacted the grafted morphology of ELP. Specifically, an ELP with a negative charge on the opposite end of the surface-binding moiety assembled in a more upright orientation, and a sufficient electric field pushed the charge away from the surface compared to when the same peptide was assembled in no electric field. In addition, this study demonstrated that assembling charged ELP in an applied electric field impacts transition behavior. Overall, this study reveals new strategies for achieving desirable and predictable surface properties of surface-bound ELP.

Proteomic, Metabolomic, and Lipidomic Analyses of Lung Tissue Exposed to Mustard Gas.

Sulfur mustard (HD) poses a serious threat due to its relatively simple production process. Exposure to HD in the short-term causes an inflammatory response, while long-term exposure results in DNA and RNA damage. Respiratory tract tissue models were exposed to relatively low concentrations of HD and collected at 3 and 24 h post exposure. Histology, cytokine ELISAs, and mass spectrometric-based analyses were performed. Histology and ELISA data confirmed previously seen lung damage and inflammatory markers from HD exposure. The multi-omic mass spectrometry data showed variation in proteins and metabolites associated with increased inflammation, as well as DNA and RNA damage. HD exposure causes DNA and RNA damage that results in variation of proteins and metabolites that are associated with transcription, translation and cellular energy.

Self-Assembly and Rearrangement of a Polyproline II Helix Peptide on Gold.

Polyproline peptide sequences have gained popularity as anchors for peptide-based self-assembled monolayers (SAMs) due to their attractive properties. In this work, peptides containing the polyproline II helix (PPII) conformation were designed and assembled on gold (Au). A quartz crystal microbalance with dissipation was used to characterize SAM formation kinetics and related properties. Peptides were designed with the sequence (GPPPPPG)2C. It was discovered that a biexponential adsorption and rearrangement model describes the binding kinetics of the PPII-containing peptide on Au. In this model, an initial reversible binding step is followed by an irreversible rearrangement step, given by parameter kt. This study found kt to be approximately 0.00064 s-1 for the PPII-containing peptides. Similarly, we found that the adsorption of the PPII-containing peptide on Au, given by ΔGads, was thermodynamically favorable (-7.8 kcal mol-1) and comparable to other common thiol terminated SAMs on Au. Furthermore, we characterized SAM properties via QCM-D, Fourier-transform infrared (FTIR) spectroscopy, and electrochemical techniques to reveal high molecular density SAMs consisting of PPII helices. In addition, these SAMs were found to have high antifouling properties. Overall, this study characterizes the fundamental assembly mechanisms, particularly, rearrangement of PPII-containing peptides for the first time, which will be useful in the designing of future peptide-based SAMs with high surface coverage and antifouling properties.

Discovery of treatment for nerve agents targeting a new metabolic pathway.

The inhibition of acetylcholinesterase is regarded as the primary toxic mechanism of action for chemical warfare agents. Recently, there have been numerous reports suggesting that metabolic processes could significantly contribute to toxicity. As such, we applied a multi-omics pipeline to generate a detailed cascade of molecular events temporally occurring in guinea pigs exposed to VX. Proteomic and metabolomic profiling resulted in the identification of several enzymes and metabolic precursors involved in glycolysis and the TCA cycle. All lines of experimental evidence indicated that there was a blockade of the TCA cycle at isocitrate dehydrogenase 2, which converts isocitrate to α-ketoglutarate. Using a primary beating cardiomyocyte cell model, we were able to determine that the supplementation of α-ketoglutarate subsequently rescued cells from the acute effects of VX poisoning. This study highlights the broad impacts that VX has and how understanding these mechanisms could result in new therapeutics such as α-ketoglutarate.

A Lysine-Modified Polyethersulfone (PES) Membrane for the Recovery of Lanthanides.

Rare-earth elements (which include all lanthanides, scandium, and yttrium) play a key role in many fields including oil refining, metallurgy, electronics manufacturing, and other high-technology applications. Although the available lanthanide resources are enough for current levels of manufacturing, increased future demand for lanthanides will require new, efficient recovery methods to provide a sustainable supply. Membrane adsorbers are promising separation materials to recover lanthanides from high volumes of wastewater due to their tailorable surface chemistry, high binding capacity and high throughput. In this work, membrane adsorbers were synthesized by first using ultraviolet-initiated free radical polymerization to graft a poly(glycidyl methacrylate) (p-GMA) layer to the surface of polyethersulfone membranes. Then, the reactive epoxy groups of the grafted p-GMA were used for the covalent attachment of lysine molecules via a zinc perchlorate-catalyzed, epoxide ring-opening reaction at 35°C. Changes in membrane surface chemistry throughout the functionalization process were monitored with Fourier Transform Infrared Spectroscopy. The degree of grafting for the p-GMA film was quantified gravimetrically and increased with increasing polymerization time. Equilibrium adsorption experiments were performed for single specie solutions of La3+, Ce3+, Nd3+, Na+, Ca2+, and Mg2+ at pH 5.25 ± 0.25. Lysine-modified membranes showed negligible uptake of Na+, Ca2+, and Mg2+. The maximum capacities modeled by the Langmuir isotherm for La3+ and Ce3+ were 6.11 ± 0.58 and 6.45 ± 1.29 mg/g adsorbent, respectively. Nd3+ adsorbed to the membrane; however, the fit of the Langmuir model was not significant and it adsorbed to a lower extent than La3+ and Ce3+. Lower adsorption of the higher charge density species indicates that the primary binding mode is through the amine moieties of lysine and not the carboxylic acid. Dynamic adsorption experiments were conducted with 1 ppm La3+ feed solutions at different flow rates using either a single membrane or three membranes in series. The dynamic binding capacity at 50% breakthrough was independent of flowrate within the tested range. The low-temperature membrane functionalization methodology presented in this work can be used to immobilize biomolecules with even higher specificity, like engineered peptides or proteins, on membrane surfaces.

Exploring the effect of a peptide additive on struvite formation and morphology: a high-throughput method.

Precipitation of struvite (MgNH4PO4·6H2O), a slow-release fertilizer, provides a means of recycling phosphate from wastewater streams. In this work, a high-throughput struvite precipitation method is developed to investigate the effects of a peptide additive. The reactions occurred in small volumes (300 µL or less) in a 96-well plate for 45 minutes. The formation of struvite was monitored by fitting absorbance at 600 nm over time to a first order model with induction time, with the addition of peptide inducing significant changes to the yield parameter and formation constant in that model. The impact of struvite seed dosing was also investigated, highlighting the importance of optimization when peptide is present. The composition of the precipitate was confirmed through Fourier-transform infrared spectroscopy, while morphology and crystal size were analyzed through optical microscopy. Crystals had a higher aspect ratio when precipitated with the peptide. Finally, the utility of the high-throughput platform was demonstrated with a 25 full factorial design to capture the effects and interactions of: magnesium dose, mixing time, seed dose, pH, and temperature. Overall, this study quantifies novel effects of a sequence-defined peptide on struvite formation and morphology via a newly developed high throughput platform.

Controlling the Distribution of Perfluorinated Sulfonic Acid Ionomer with Elastin-like Polypeptide.

Proton-exchange-membrane (PEM)-based devices are promising technologies for hydrogen production and electricity generation. Currently, the amount of expensive platinum catalyst used in these devices must be reduced to be cost-competitive with other technologies. These devices typically contain Nafion ionomer thin films in the catalyst layers, which are responsible for transporting protons and gaseous species to and from electrochemically active sites. The morphology of the Nafion ionomer thin films in the catalyst layers with reduced platinum loading is impacted by interactions with the catalyst and the confinement to nanometer thicknesses, which leads to performance losses in PEM-based devices. In this study, an elastin-like polypeptide (ELP) is designed to modulate the morphology of Nafion ionomer on platinum surfaces. The ELP shows an ability to assemble into a monolayer on platinum and change the ionomer interaction with platinum, thereby modifying its thin-film structure and improving the Nafion ionomer coverage. As a proof of concept, an ELP-modified catalyst ink was prepared and morphological differences were observed. Overall, we discovered an engineered ELP that can modulate the ionomer-catalyst interface in the electrodes of PEM-based devices.

Catalyst-free, highly selective synthesis of ammonia from nitrogen and water by a plasma electrolytic system.

There is a growing need for scalable ammonia synthesis at ambient conditions that relies on renewable sources of energy and feedstocks to replace the Haber-Bosch process. Electrically driven approaches are an ideal strategy for the reduction of nitrogen to ammonia but, to date, have suffered from low selectivity associated with the catalyst. Here, we present a hybrid electrolytic system characterized by a gaseous plasma electrode that facilitates the study of ammonia formation in the absence of any material surface. We find record-high faradaic efficiency (up to 100%) for ammonia from nitrogen and water at atmospheric pressure and temperature with this system. Ammonia measurements under varying reaction conditions in combination with scavengers reveal that the unprecedented selectivity is achieved by solvated electrons produced at the plasma-water interface, which react favorably with protons to produce the key hydrogen radical intermediate. Our results demonstrate that limitations in selectivity can be circumvented by using catalyst-free solvated electron chemistry. In the absence of adsorption steps, the importance of controlling proton concentration and transport is also revealed.

Engineered interaction between short elastin-like peptides and perfluorinated sulfonic-acid ionomer.

Control of ionomer thin films on metal surfaces is important for a range of electrodes used in electrochemical applications. Engineered peptides have emerged as powerful tools in electrode assembly because binding sites and peptide structures can be modulated by changing the amino acid sequence. However, no studies have been conducted showing peptides can be engineered to interact with ionomers and metals simultaneously. In this study, we design a single-repeat elastin-like peptide to bind to gold using a cysteine residue, and bind to a perfluorinated sulfonic-acid ionomer called Nafion® using a lysine guest residue. Quartz crystal microbalance with dissipation monitoring and atomic force microscopy are used to show that an elastin-like peptide monolayer attached to gold facilitates the formation of a thin, phase-separated ionomer layer. Dynamic light scattering confirms that the interaction between the peptide with the lysine residue and the ionomer also happens in solution, and circular dichroism shows that the peptides maintain their secondary structures in the presence of ionomer. These results demonstrate that elastin-like peptides are promising tools for ionomer control in electrode engineering.

Rare case of an extraosseous cervical chordoma with both intradural and extensive extraspinal involvement.

BACKGROUND: Chordomas must be considered among the differential diagnoses for extradural spinal tumors, especially involving the clival or sacrococcygeal regions. They are often locally invasive and destructive to the osseous structures from which they arise, but rarely extend intradurally. Here, we report a unique chordoma that was intradural and spanned nearly four subaxial cervical vertebral levels. CASE DESCRIPTION: We report the case of an atypical intradural chordoma that spanned four subaxial levels of the cervical spine in an 81-year-old female. It also extended through multiple neural foramina but did not invade or destroy the bony elements of the cervical vertebrae. Notably, it demonstrated sizable extension into the deep carotid triangle abutting the internal jugular vein. CONCLUSION: This case involved an extraosseous, intradural, four-level subaxial cervical chordoma that demonstrated significant extraspinal extension into the anterior soft tissues of the neck.

B-cell lymphoma presenting as multiple nerve sheath tumors.

BACKGROUND: Diffuse Large B-cell Lymphoma (DLBCL) is the most common form of Non-Hodgkin lymphoma (NHL), accounting for 25-30 percent of cases in the United States.1 Extranodal sites are involved in approximately 40% of cases of DLBCL. CASE DESCRIPTION: In this report, we discuss the case of a patient with extranodal DLBCL within the cervical nerve roots that underwent surgical intervention due to the presence of cervical radiculopathy. CONCLUSION: The diagnosis of DLBCL was surprising given the appearance of the masses on MRI being similar to that of a neurofibroma or schwannoma. Surgical decompression provided a tissue sample for biopsy as well as an opportunity for decompression of the nerve roots and restoration of function of the patient's left upper extremity.

The use of engineered protein materials in electrochemical devices.

Bioelectrochemical technologies have an important and growing role in healthcare, with applications in sensing and diagnostics, as well as the potential to be used as implantable power sources and be integrated with automated drug delivery systems. Challenges associated with enzyme-based electrodes include low current density and short functional lifetimes. Protein engineering is emerging as a powerful tool to overcome these issues. By taking advantage of the ability to precisely define protein sequences, electrodes can be organized into high performing structures, and enable the next generation of medical devices.

Incorporating the BMP-2 peptide in genetically-engineered biomaterials accelerates osteogenic differentiation.

Protein-based biomaterials have emerged as powerful tools for tissue engineering applications. Recombinant DNA techniques can be used to precisely tune material properties at the molecular level, and multiple peptide modules can be incorporated into a single material. Here, we genetically engineered biomaterials that incorporate a peptide derived from bone morphogenetic protein-2 (BMP-2) and investigated whether the BMP-2 peptide, within the context of these materials, can promote stem cells to produce bone matrix and synergize with a cell-binding sequence (RGD). Our study is the first to demonstrate that when the BMP-2 peptide is incorporated within the backbone of protein-based biomaterials, it is active and accelerates osteogenic differentiation of mesenchymal stem cells. In particular, cells seeded on proteins containing the BMP-2 peptide had increased levels of alkaline phosphatase (AP) activity, calcium deposition, and expression of bone-related genes. However, the BMP-2 peptide did not synergize with the RGD cell-binding domain within the context of these protein-based materials. Overall, these results suggest that incorporation of the BMP-2 peptide within the context of modular proteins is a successful strategy for engineering biomaterials for applications in bone tissue engineering.