Here, we investigated the relationship between the attenuation of lung cancer growth due to oral administration of Euglena gracilis water extract (EWE) and T cell stimulation. Orally administered EWE was revealed to increase PD-1 and PD-L1 mRNA and proteins primarily in tumor-infiltrating lymphocytes (TILs), which was correlated with a significant decrease in the tumor weights in mice. A combination treatment with EWE and anti-PD-1 antibody significantly decreased the growth of murine lung tumors more than treatment with either alone by increasing the number of TILs and attenuating T cell exhaustion. Short-chain fatty acids, which were previously shown to be increased in intestines of mice treated with oral EWE, increased both PD-1 and PD-L1 expression in splenocytes, but not in lung cancer cells in cell culture. These results suggest there is a close relationship between the EWE-induced increase of short-chain fatty acids, the increase of PD-1 expression in TILs, and the attenuation of lung tumor growth. Furthermore, EWE enhances the efficacy of anti-PD-1 antibody-based immune checkpoint blockade therapy against non-small cell lung cancer.

Metal flux methods are excellent for synthesizing high-quality hexagonal boron nitride (hBN) crystals, but the atomic mechanisms of hBN nucleation and growth in these systems are poorly understood and difficult to probe experimentally. Here, we harness classical reactive molecular dynamics (ReaxFF) to unravel the mechanisms of hBN synthesis from liquid nickel solvent over time scales up to 30 ns. These simulations mimic experimental conditions by including relatively large liquid nickel slabs containing dissolved boron and a molecular nitrogen gas phase. Overall, the reaction takes place almost exclusively on the surface of the liquid nickel, owing to the low solubility of nitrogen in bulk nickel and the intermediate species' preference for the metal-gas interface. The formation of hBN invariably begins by reaction of dinitrogen with nickel-solvated boron atoms at the surface, forming intermediate N-N-B species, which typically evolve into B-N-B units through a short-lived intermediate where a single nitrogen atom is coordinated by one nitrogen and two boron atoms. The resulting B-N-B units, in turn, coalesce with growing hBN nuclei and carry nitrogen between hBN nanocrystals in an Ostwald ripening process. The amount of hBN produced on the tens of nanosecond time scale depends critically on the boron concentration, while having a much weaker dependence on the N2 pressure for the regime considered (N2 pressures of 2.5-10 MPa, Ni-B solutions with 6-12% boron by atom fraction). The highest rate of hBN formation occurs at the lowest temperature considered (1750 K, just above the melting point of nickel), while no hBN sheets are formed at 2000 K or above. An analysis of the transition pathways for nitrogen atoms shows that the final step, incorporation of small B-N motifs into larger hBN sheets, is the rate-limiting step in the regimes considered. While raising the temperature from 1750 to 2000 K has little effect on the formation of intermediates (N-N-B, B-N-B, etc.), the lack of large hBN sheets at temperatures >1900 K is explained by decreased probability of the final step and increased probability of breakup of hBN into B-N motifs.

Icosahedral boron materials, which include regular icosahedra of 12 boron atoms have gained increasing attention due to their potential applications as superhard materials, semiconductors, and energy storage media. However, the synthesis of high quality crystals of these materials has been a major barrier to the development of these applications. To enable computational prediction of synthesis conditions yielding high-quality icosahedral boron crystals, herein we tested and refined a set of ReaxFF parameters for the nucleation and growth of such crystals. We focused on matching the relative energies of small boron clusters obtained by density functional theory since such small clusters and similar motifs are likely present in crystal nuclei and at the interface of growing crystals. Using a training set of B80 clusters, including a low-energy core-shell structure containing a B12 icosahedron core and a high-energy single-shell structure produced in preliminary ReaxFF simulations, the ReaxFF parameter set was refined to better reproduce energies calculated by density functional theory (DFT). Among existing ReaxFF parameter sets and the machine-learning interatomic potentials MACE-MP-0, MACE-MP-0b3, MACE-MPA-0, PFP v7.0.0, and SevenNet-MF-ompa, only our new parameter set and PFP v7.0.0 correctly ranked these B80 clusters. This refinement led to improved agreement with DFT for a test set of 58 clusters consisting of 8-103 boron atoms. Furthermore, our refined parameter set yielded greater local icosahedral structure than the previously existing ReaxFF parameter set for larger scale simulations of crystallization from supercooled liquid boron. Additionally, simulations of solid boron in contact with molten nickel using our refined ReaxFF parameters yielded a boron solubility value that agrees moderately well with experimental expectations, while the previous boron parameters gave a value that was much too low.

Self-assembled nanostructures such as those formed by peptide amphiphiles (PAs) are of great interest in biological and pharmacological applications. Herein, a simple and widely applicable chemical modification, a urea motif, was included in the PA's molecular structure to stabilize the nanostructures by virtue of intermolecular hydrogen bonds. Since the amino acid residue nearest to the lipid tail is the most relevant for stability, we decided to include the urea modification at that position. We prepared four groups of molecules (13 PAs in all), with varying levels of intermolecular cohesion, using amino acids with distinct β-sheet promoting potential and/or containing hydrophobic tails of distinct lengths. Each subset contained one urea-modified PA and nonmodified PAs, all with the same peptide sequence. The varied responses of these PAs to variations in pH, temperature, counterions, and biologically related proteins were examined using microscopic, X-ray, spectrometric techniques, and molecular simulations. We found that the urea group contributes to the stabilization of the morphology and internal arrangement of the assemblies against environmental stimuli for all peptide sequences. In addition, microbiological and biological studies were performed with the cationic PAs. These assays reveal that the addition of urea linkages affects the PA-cell membrane interaction, showing the potential to increase the selectivity toward bacteria. Our data indicate that the urea motif can be used to tune the stability of a wide range of PA nanostructures, allowing flexibility on the biomaterial's design and opening a myriad of options for clinical therapies.

The interaction of liquid water with hydrophobic surfaces is ubiquitous in life and technology. Yet, the molecular structure of interfacial liquid water on these surfaces is not known. By using a 3D atomic force microscope, we characterize with angstrom resolution the structure of interfacial liquid water on hydrophobic and hydrophilic silica surfaces. The combination of 3D AFM images and molecular dynamics simulations reveals that next to a hydrophobic silica surface, there is a 1.2 nm region characterized by a very low density of water. In contrast, the 3D AFM images obtained of a hydrophilic silica surface reveal the presence of hydration layers next to the surface. The gap observed on hydrophobic silica surfaces is filled with two-to-three layers of straight-chain alkanes. We developed a 2D Ising model that explains the formation of a continuous hydrocarbon layer on hydrophobic silica surfaces.

Abstract Proteins involved in immune checkpoint pathways, such as CTLA4, PD-1, and PD-L1, have become important targets for cancer immunotherapy; however, the development of small molecule drugs targeting these pathways has proven difficult due to the nature of their protein-protein interfaces. Here, using a hierarchy of computational techniques, we designed a cyclic peptide that binds CTLA4 and follow this with experimental verification of binding and biological activity, using bio-layer interferometry, cell culture, and a lung cancer mouse model in immunocompetent mice. Beginning from a template excised from the x-ray structure of the CTLA4:B7-2 complex, we generate several peptide sequences using Rosetta, a protein modelling program. These peptides are cyclized head-to-tail to improve structural and proteolytic stability and screened using molecular dynamics simulation and MM-GBSA calculation. The standard binding free energies for shortlisted peptides are then calculated in explicit-solvent simulation using a rigorous multistep Binding Free Energy Estimator (BFEE). The most promising peptide, cyc-EIDTVLTPTGWVAKRYS (hereafter CTLA4-ip), yields the standard free energy -6.6 ± 3.5 kcal/mol, which corresponds to a dissociation constant of 15 μmol/L. The binding affinity of this peptide for CTLA4 is measured experimentally (31 ± 4 μmol/L) using bio-layer interferometry. Pharmacokinetics of CTLA4-ip in a cell culture with Lewis lung carcinoma (LLC) cells and in mice revealed that this peptide is significantly more stable in both cell culture and in mice as compared to a control non-cyclic scrambled-sequence. Treatment with CTLA4-ip inhibited cancer cell growth in a co-culture of LLC cells and LLC cell-antigen primed murine T cells. Intraperitoneal administration of the CTLA4-ip (10mg/kg/day, every other day totaling 4 doses) markedly inhibited lung tumor growth in mice with an orthotropic LLC allograft model. Efficacy of the tumor growth inhibition by the CTLA4-ip was similar to that by an anti-PD-L1 antibody (10mg/kg/day, every two days totaling 3 doses). These results strongly suggest that this novel CTLA4-ip works as an immune checkpoint inhibitor to CTLA4 and is usable for lung cancer treatment. This research was supported by Kansas State University Johnson Cancer Research center (MT), Midwest Biomedical Accelerator Consortium (MT, JC), National Cancer Institute MT, JC) and the National Science Foundation (JC). Citation Format: Ravindra Thakkar, Deepa Upreti, Susumu Ishiguro, Geraldine Magnin, Keshari Sudasinghe, Anabelle Hall, Sarah DeVader, Masaaki Tamura, Jeffrey Comer. Computational design of a cyclic peptide that inhibits the CTLA4 in T cells and the growth of lung carcinoma in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1352.

Extracts from Euglena gracilis have been shown to prevent cancer growth in mouse models. However, the molecular mechanism of this anti-cancer activity has not been determined nor has the effect of Euglena extracts on tobacco smoke carcinogen-induced carcinogenesis. Here, we investigate the hypothesis that this anti-cancer activity is a result of changes in the intestinal microbiota induced by oral administration of the extract. We found that a Euglena gracilis water extract prevents lung tumorigenesis induced by a tobacco smoke-specific carcinogen (NNK) in mice treated either 2 weeks before or 10 weeks after NNK injection. Both of these treatment regimens are associated with significant increases in 27 microbiota metabolites found in the mouse feces, including large increases in triethanolamine, salicylate, desaminotyrosine, N-acetylserine, glycolate, and aspartate. Increases in the short-chain fatty acids (SCFAs) including acetate, propionate and butyrate are also observed. We also detected a significant attenuation of lung carcinoma cell growth through the induction of cell cycle arrest and apoptosis caused by low levels of SCFAs. This study provides strong evidence of anti-cancer activity in Euglena gracilis extracts against tobacco smoke carcinogen-induced tumorigenesis and demonstrates that this activity is linked to increased production of specific gut microbiota metabolites and the resultant induction of cell cycle arrest and apoptosis of lung carcinoma cells.

Nanoscale silver particles have growing applications in biomedical and other technologies due to their unique antibacterial, optical, and electrical properties. The preparation of metal nanoparticles requires the action of a capping agent, such as thiol-containing compounds, to provide colloidal stability, prevent agglomeration, stop uncontrolled growth, and attenuate oxidative damage. However, despite the extensive use of these thiol-based capping agents, the structure of the capping agent layers on the metal surface and the thermodynamics of the formation of these layers remains poorly understood. Here, we leverage molecular dynamics simulations and free energy calculation techniques, to study the behavior of citrate and four thiol-containing capping agents commonly used to protect silver nanoparticles from oxidation. We have studied the single-molecule adsorption of these capping agents to the metal-water interface, their coalescence into clusters, and the formation of complete monolayers covering the metal nanoparticle. At sufficiently high concentrations, we find that allylmercaptan, lipoic acid, and mercaptohexanol spontaneously self-assemble into ordered layers with the thiol group in contact with the metal surface. The high density and ordered structure is presumably responsible for their improved protective characteristics relative to the other compounds studied.

A cyclic peptide targeted to the immune checkpoint protein CTLA4 is designed using a hierarchy of computational methods, demonstrated to bind by a physical measurement, and shown to inhibit tumor growth in cell culture and in mice.

ABSTRACT A high-dimensional neural network interatomic potential was developed and used in molecular dynamics simulations of condensed phase Ni and Ni systems with liquid–solid phase coexistence. The reference data set was generated by sampling the potential energy surface over a broad temperature-pressure domain using ab initio MD simulations to train a unified potential. Excellent agreement was achieved between bulk face-centred cubic nickel thermal expansion simulations and relevant experimental data. The same potential also yields accurate structures and diffusivities in the liquid state. The phase transition between liquid and solid phases was simulated using the two-phase interface method. The predicted melting point temperature is within a few kelvins of the literature value. The general methodology could be applied to describe crystals with much more complex phase behaviours.

In the United States, colorectal cancer (CRC) is the second leading cause of cancer-related death in both sexes. Immune checkpoint blockade therapy (ICBT) has emerged as a powerful new tool for cancer therapy. However, only CRCs associated with microsatellite instability (MSI) or DNA mismatch repair gene defects (dMMR, ~15% of all CRCs) are sensitive to this therapy. This poor sensitivity to ICBT is either due to poor tumor infiltration of functional T cells or T cell exhaustion. To overcome this, we investigated a combination treatment with an oncolytic virus and an immune checkpoint inhibitor (ICI). Because tumor cell oncolysis generates neoantigens and increases tumor immunogenicity, T cell infiltration into the tumor tissue will be increased and the efficacy of ICBT will be enhanced. Accordingly, local immune checkpoint inhibition coupled with oncolysis may be an ideal ICBT. In the present study, a novel peptide that interferes with the PD-1/PD-L1 immune checkpoint pathway, termed PD-L1 inhibitory peptide 3 (PD-L1ip3), was computationally designed, experimentally validated for its specific binding to PD-L1, and evaluated for its antitumor effects in cell culture and in a mouse colon carcinoma syngeneic murine model. Fourteen candidate peptide sequences were generated using the PinaColada algorithm and the x-ray structure of the PD-1:PD-L1 complex and screened in molecular dynamics simulations on the microsecond timescale. Two were chosen for experimental testing, wherein the peptide denoted PD-L1ip3 showed a binding affinity for PD-L1 in the micromolar range. In cell culture studies, treatment with PD-L1ip3, but not a similar peptide with a scrambled sequence, substantially increased death of CT26 colon carcinoma cells when co-cultured with murine CD8+ T cells primed by antigens from CT26 cells. In immunocompetent mice, growth of CT26 tumor cells transduced with the PD-L1ip3 gene by an adenovirus vector was significantly slower than that of un-transduced CT26 cells. This tumor growth attenuation was further enhanced by cotreatment with the peptide form of PD-L1ip3. The present study suggests that this peptide can stimulate host antitumor immunity via a blockade of the PD-1/PD-L1 pathway, thereby increasing CD8+ T cell-induced death of colon carcinoma cells. The tumor site-specific inhibition of PD-L1 by an adenovirus carrying the PD-L1ip3 gene, together with direct peptide treatment, may be used as a local immune checkpoint blockade therapy to inhibit colon carcinoma growth. This research was supported by Kansas State University Johnson Cancer Research center (MT), K-INBRE Scholar Award (MB), National Cancer Institute (MT, JC) and the National Science Foundation (JC). Citation Format: Susumu Ishiguro, Deepa Upreti, Molly Bassette, E. R. Azhagiya Singam, Ravindra Thakkar, Mayme Loyd, Makoto Inui, Jeffrey Comer, Masaaki Tamura. A gene therapy with a novel PD-L1 inhibitory peptide secretory gene inhibits the growth of colon carcinoma in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2737.

Food-derived bioactive peptides (FBPs) are gaining interest due to their great potential in agricultural byproduct valorization and high-activity peptide screening. The introduction of bioinformatics into FBP studies further enhances the prospects of this field. This review provides a comprehensive overview and critical insight into the latest advances in bioinformatics-driven FBPs studies. The roles of databases, proteolysis simulation, bioactivity potency evaluation, quantitative structure-activity relationships (QSAR) models, molecular docking, molecular dynamics simulation, and free energy calculation in FBP studies are covered. Furthermore, critical issues related to QSAR model development, molecular docking, and integrated bioinformatics strategies are highlighted. By leveraging these bioinformatics approaches, researchers can fully utilize existing knowledge about identified peptides for checking novelty, evaluating bioactivity potency as well as rational peptide and protein hydrolysate design. QSAR models and molecular docking enable efficient screening of thousands of peptide candidates and generate new insights into bioactivity mechanisms. Directions for future research and challenges in current studies are also discussed. The employment of bioinformatics will significantly accelerate the process from the identification of high-potential FBPs to product development, assist in wet chemistry experiment design for targeted protein hydrolysates preparation, and ultimately enhance the long-term development of nutraceutical, pharmaceutical, and cosmeceutical industries.

Although effective vaccines have been developed against SARS-CoV-2, many regions in the world still have low rates of vaccination and new variants with mutations in the viral spike protein have reduced the effectiveness of most available vaccines and treatments. There is an urgent need for a drug to cure this disease and prevent infection. The SARS-CoV-2 virus enters the host cell through protein-protein interaction between the virus's spike protein and the host's angiotensin converting enzyme (ACE2). Using protein design software and molecular dynamics simulations, we have designed a 17-residue peptide (pep39), that binds to the spike protein receptor-binding domain (RBD) and blocks interaction of spike protein with ACE2. We have confirmed the binding activity of the designed peptide for the original spike protein and the delta variant spike protein using micro-cantilever and bio-layer interferometry (BLI) based methods. We also confirmed that pep39 strongly inhibits SARS-CoV-2 virus replication in Vero E6 cells. Taken together these data suggest that a newly designed spike protein RBD blocking peptide pep39 has a potential as a SARS-CoV-2 virus inhibitor.

Lung cancer is the leading cause of cancer-related mortality in the world. Our previous studies have shown that the oral administration of Euglena water extract significantly inhibits the growth of grafted lung carcinoma tumors in mice. Euglena gracilis, a single-celled alga used as a nutritional dietary supplement, possesses a broad range of medicinal properties including anticancer activity against a few types of cancers. Most studies describing this anticancer activity have used xenograft cancer mouse models, but not carcinogen-induced cancer models. Since tobacco smoke carcinogen-induced lung carcinoma in mice mimics human lung cancer development, in this study, we investigated the anti-cancer properties and the underlying mechanism of the E. gracilis water extract against lung tumorigenesis induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). A/J mice (n=10 per treatment group) were used and the treatment regimen consisted of: (i) PBS control, (ii) Euglena water extract 2 weeks before NNK injection (pre-NNK) and (iii) this extract 10 weeks after NNK injection (post-NNK). We examined the metabolite landscape of the gut microbiota by a high-throughput metabolomics approach. The Euglena water extract treatment greatly attenuated NNK-induced tumorigenesis in lungs of A/J mice. A Partial Least-Squares Discriminant Analysis demonstrated a separation of the fecal metabolites between the control and treatment groups. Using the untargeted metabolomics, we identified 20 potential metabolites that were differentially expressed in the Euglena extract (pre- and post-NNK) treatment groups as compared to the control group. Specifically, succinate, malate, triethanolamine, acetylserine were increased with the Euglena water extract treatment in comparison to the control. These metabolites are involved in the biosynthesis of short-chain fatty acids (SCFA). Furthermore, using the targeted analysis of SCFA, we observed a significant increase in acetic acid in the feces of mice treated with Euglena water extract both before and after NNK exposure; however, butyric and propionic acids were significantly higher in only post-treated mice. Moreover, in vitro treatment of both human and murine lung cancer cells with SCFA significantly suppressed their proliferation. The present study indicated that treating with the Euglena water extract both pre- and post-NNK exposure greatly inhibits NNK-induced lung tumorigenesis in mice even after early tumorigenesis has been established. This study also found that Euglena water extract increases the levels of SCFA and their precursors in the feces of the treated mice indicating a role of gut microbiota in suppressing the NNK-induced tumorigenesis. This study was supported by 2017 EUGLENA-RC2 (MT and JC), Kansas State University Johnson Cancer Research Center (MT and JC), and NIH grant P20 GM103418 (MT). Citation Format: Deepa Upreti, Susumu Ishiguro, Ayaka Nakashima, Kengo Suzuki, Jeffrey Comer, Masaaki Tamura. Euglena water extract attenuates lung tumorigenesis induced by tobacco-specific carcinogen through modulation of gut microbiota metabolites [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5944.

The antitumor effects of a partially purified water extract from Euglena gracilis (EWE) and EWE treated by boiling (bEWE) were evaluated using orthotopic lung cancer syngeneic mouse models with Lewis lung carcinoma (LLC) cells. Daily oral administration of either EWE or bEWE started three weeks prior to the inoculation of LLC cells significantly attenuated tumor growth as compared to the phosphate buffered saline (PBS) control, and the attenuation was further enhanced by bEWE. The intestinal microbiota compositions in both extract-treated groups were more diverse than that in the PBS group. Particularly, a decrease in the ratio of Firmicutes to Bacteroidetes and significant increases in Akkermansia and Muribaculum were observed in two types of EWE-treated groups. Fecal microbiota transplantation (FMT) using bEWE-treated mouse feces attenuated tumor growth to an extent equivalent to bEWE treatment, while tumor growth attenuation by bEWE was abolished by treatment with an antibiotic cocktail. These studies strongly suggest that daily oral administration of partially purified water extracts from Euglena gracilis attenuates lung carcinoma growth via the alteration of the intestinal microbiota.

A novel peptide that interferes with the PD-1/PD-L1 immune checkpoint pathway, termed PD-L1 inhibitory peptide 3 (PD-L1ip3), was computationally designed, experimentally validated for its specific binding to PD-L1, and evaluated for its antitumor effects in cell culture and in a mouse colon carcinoma syngeneic murine model. In several cell culture studies, direct treatment with PD-L1ip3, but not a similar peptide with a scrambled sequence, substantially increased death of CT26 colon carcinoma cells when co-cultured with murine CD8+ T cells primed by CT26 cell antigens. In a syngeneic mouse tumor model, the growth of CT26 tumor cells transduced with the PD-L1ip3 gene by an adenovirus vector was significantly slower than that of un-transduced CT26 cells in immunocompetent mice. This tumor growth attenuation was further enhanced by the coadministration of the peptide form of PD-L1ip3 (10 mg/kg/day). The current study suggests that this peptide can stimulate host antitumor immunity via blockade of the PD-1/PD-L1 pathway, thereby increasing CD8+ T cell-induced death of colon carcinoma cells. The tumor site-specific inhibition of PD-L1 by an adenovirus carrying the PD-L1ip3 gene, together with direct peptide treatment, may be used as a local immune checkpoint blockade therapy to inhibit colon carcinoma growth.

The graphite-water interface provides a unique environment for polypeptides that generally favors ordered structures more than in solution. Therefore, systems consisting of designed peptides and graphitic carbon might serve as a convenient medium for controlled self-assembly of functional materials. Here, we computationally designed cyclic peptides that spontaneously fold into a β-sheet-like conformation at the graphite-water interface and self-assemble, and we subsequently observed evidence of such assembly by atomic force microscopy. Using a novel protocol, we screened nearly 2000 sequences, optimizing for formation of a unique folded conformation while discouraging unfolded or misfolded conformations. A head-to-tail cyclic peptide with the sequence GTGSGTGGPGGGCGTGTGSGPG showed the greatest apparent propensity to fold spontaneously, and this optimized sequence was selected for larger scale molecular dynamics simulations, rigorous free-energy calculations, and experimental validation. In simulations ranging from hundreds of nanoseconds to a few microseconds, we observed spontaneous folding of this peptide at the graphite-water interface under many different conditions, including multiple temperatures (295 and 370 K), with different initial orientations relative to the graphite surface, and using different molecular dynamics force fields (CHARMM and Amber). The thermodynamic stability of the folded conformation on graphite over a range of temperatures was verified by replica-exchange simulations and free-energy calculations. On the other hand, in free solution, the folded conformation was found to be unstable, unfolding in tens of picoseconds. Intermolecular hydrogen bonds promoted self-assembly of the folded peptides into linear arrangements where the peptide backbone exhibited a tendency to align along one of the six zigzag directions of the graphite basal plane. For the optimized peptide, atomic force microscopy revealed growth of single-molecule-thick linear patterns of 6-fold symmetry, consistent with the simulations, while no such patterns were observed for a control peptide with the same amino acid composition but a scrambled sequence.