Broadly Applicable Research Articles

A New Version of a Broadly Applicable, Cross-lingual Meaning Representation Formalism and Its Significance for Biomedical Sciences

A New Version of a Broadly Applicable, Cross-lingual Meaning Representation Formalism and Its Significance for Biomedical Sciences

Accessing a Diverse Set of Functional Red-Light Photoswitches by Selective Copper-Catalyzed Indigo N-Arylation.

The ability to correlate the structure of a molecule with its properties is the key to the rational and accelerated design of new functional compounds and materials. Taking photoswitches as an example, the thermal stability of the metastable state is a crucial property that dictates their application in molecular systems. Indigos have recently emerged as an attractive motif for designing photoswitchable molecules due to their red-light addressability, which can be advantageous in biomedical and material applications. The lack of synthetic techniques to derivatize the abundant parent dye and a thorough understanding of the impact of structural factors on the photochemical and thermal properties hinder broad applications of this emerging photoswitch class. Herein, we report an efficient copper-catalyzed indigo N-arylation that enables the installation of a wide variety of aryl moieties carrying useful functional groups. The exclusive selectivity for monoarylation likely originates from a bimetallic cooperative mechanism through a binuclear copper-indigo intermediate. Functional N-aryl-N'-alkylindigos were prepared and shown to photoisomerize efficiently under red light. Moreover, this design allows for the modulation of thermal half-lives through N-aryl substituents, while the N'-alkyl groups enable the independent attachment of functional moieties without affecting the photochromic properties. A strong correlation between the structure of the N-aryl moiety and the thermal stability of the photogenerated Z-isomers was achieved by multivariate linear regression models obtained through a data-science workflow. This work thus builds an avenue leading to versatile red-light photoswitches and a general method for structure-property correlation that is expected to be broadly applicable to the design of photoresponsive molecules.

Photo‐Arbuzov Reactions as a Broadly Applicable Surface Modification Strategy

AbstractChemical vapor deposition (CVD) polymerization is a commonly used approach in surface chemistry, providing a substrate‐independent platform for bioactive surface functionalization strategies. This work investigates the Arbuzov reaction of halogenated polymer coatings readily available via CVD polymerization, using poly(4‐chloro‐para‐xylylene) (Parylene C) as a model substance. Postpolymerization modification of these coatings via catalyst‐free and UV‐induced Arbuzov reaction using phosphites results in phosphonate‐functionalized polymers. The combination of infrared reflection‐absorption spectroscopy (IRRAS), X‐ray photoelectron spectroscopy (XPS), and time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) provides detailed insights into the reaction progress. Time‐dependent studies suggest that the non‐polar phosphites penetrate deep into the CVD films and react with the polymer film. In addition, ToF‐SIMS, scanning electron microscopy (SEM), and atomic force microscopy (AFM) confirm spatial control of the reaction, resulting in localized chemical and topographical surface modification, recognizable by changes in interference color, fluorescence, and wettability. Preliminary 3D fluorescence spectroscopy investigations indicate tunable near‐infrared emission of these polymer films. This work is the first step toward generating multifunctional polymer coatings based on chemically modifiable, CVD polymers with potential applications in biomaterials, sensors, or optoelectronics.

Abstract 6324: Antagonism-enforced braking system to enhance CAR T cell therapeutic specificity

Abstract Chimeric Antigen Receptor (CAR) T cell immunotherapy represents a breakthrough in the treatment of hematological malignancies. However, the rarity of cell surface protein targets that are specific to cancerous but not vital healthy tissue has hindered its broad application to solid tumor treatment. While new logic-gated CAR designs have shown reduced toxicity against healthy tissues, the generalizability of such approaches across tumors remains unclear. Here, we harness a universal characteristic of endogenous T cell receptors (TCRs), their ability to discriminate between self and non-self ligands through inhibition of response against self (weak) antigens, to develop a broadly applicable method of enhancing immunotherapeutic precision. We hypothesized that this discriminatory mechanism, known as antagonism, would apply across receptors, allowing for a transfer of specificity from TCRs onto CARs. We therefore systematically mapped out the responses of CAR T cells to joint TCR and CAR stimulations. We first engineered murine T cells with an ovalbumin-specific TCR to express a CAR targeting murine CD19 and discovered that the expression of a strong TCR antigen on CD19+ leukemia enhanced CAR T killing. Importantly though, the presence of a weak TCR antigen antagonized CAR T responses, assessed by in vitro multiplexed dynamic profiling as well as in vivo cytotoxicity. We developed a mathematical model based on cross-receptor inhibitory coupling that accurately predicted the extent of TCR/CAR antagonism across a wide range of immunological settings. This model was validated in a CD19+ B16 mouse melanoma model showing that TCR/CAR antagonism decreased the infiltration of a tumor-reactive T cell cluster, while TCR/CAR agonism enhanced infiltration of this T cell cluster. We then applied our quantitative knowledge of TCR/CAR crosstalk to design an Antagonism-Enforced Braking System (AEBS) for CAR T cell therapy. This was assessed in a model system using a CAR targeting the tyrosine-protein kinase erbB-2 (HER2) together with a hedgehog acyltransferase (HHAT) peptide-specific TCR that binds strongly to mutated tumor neoantigen while retaining weak affinity for the wild-type self-antigen on healthy tissue. We established a humanized in vivo model of CAR T function and found that AEBS CAR T cells maintained high anti-tumor activity against a human lung adenocarcinoma (PC9) but notably, their anti-tissue cytotoxicity against human bronchial epithelial cells (BEAS-2B) was minimized. AEBS CAR T cells therefore sharpen the discriminatory power of synthetic anti-tumor lymphocytes. Our work highlights a novel mechanism by which TCRs can enforce CAR T cell specificity, with practical implications for the rational design of future anti-leukemia immunotherapies. Citation Format: Taisuke Kondo, François X. Bourassa, Sooraj Achar, Justyn DuSold, Pablo Cespedes, Madison Wahlsten, Audun Kvalvaag, Guillaume Gaud, Paul Love, Michael Dustin, Gregoire Altan-Bonnet, Paul François, Naomi Taylor. Antagonism-enforced braking system to enhance CAR T cell therapeutic specificity [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 6324.

Abstract 5177: Ex vivo assessment of cellular mass response predicts clinical efficacy in solid tumors

Abstract Introduction: Identifying personalized, efficacious therapies for cancer remains an urgent unmet need in oncology. While genomics-based biomarkers have demonstrated significant progress, they are not universally applicable, with most patients still treated using clinical guidelines. Experimental procedures: We have previously demonstrated broad clinical applicability of a functional precision medicine (FPM) workflow that incorporates high-resolution measurements of single-cell mass changes in response to drugs. As a highly integrative biomarker of cellular state, mass change serves as a rapid and mechanism agnostic readout of drug response, demonstrating an ability to detect signal from over 100 different cytotoxic and targeted therapies within 24 hours of treatment ex vivo. As a single-cell assay that requires as few as 10,000 cells to assess drug response, this method does not require any ex vivo culture for cell expansion and is compatible with a range of clinically relevant tissue specimen formats. Together, these features have enabled this mass response framework to be implemented in a CLIA-certified laboratory with a reporting turnaround time of just two days. Summary of the new, unpublished data: Having previously shown that mass response testing is compatible with various solid tumor specimen formats such as malignant fluids, core biopsies and fine needle aspirates, here we present preliminary results that demonstrate compatibility with surgically resected tissue specimens. Specifically, we demonstrate that mass response testing results correlate with clinical response across of broad array of solid tumors. In our cohort of 24 patients, we found that mass response measurements correlated with clinical response to therapy with an accuracy of 83% (OR=19.66, P=0.003). Within this group, we also found clear examples of how mass response testing can serve as a valuable complement to existing genomic approaches to guide therapy. Importantly, we demonstrate both concordant and discrepant results using small molecule inhibitors in the setting of known biomarkers of response, highlighting the need for functional testing in precision cancer medicine. Conclusions: These results support that mass response testing is feasible and broadly applicable to solid tumor malignancies and may serve as a valuable complementary readout for informing therapeutic selection for cancer patients. Citation Format: Robert Kimmerling, Mark Stevens, Selim Olcum, Madeleine Vacha, Rachel LaBella, Katie Katsis, Reginald Aikens, Cathleen Hannah, Audrey Guo, Juanita Fujii, Zayna Shaheen, Srividya Sundaresan, Ashley Rainey, Andrew M. Blakely, Jonathan Hernandez, Anobel Tamrazi, Clifford Reid. Ex vivo assessment of cellular mass response predicts clinical efficacy in solid tumors [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 5177.

A Broadly Applicable Coarse Alignment Framework for the Point Cloud of Hull Blocks

_ The terrestrial laser scanning (TLS) technology, which has been widely used in recent years, provides a new approach to control the construction quality of hull blocks in shipyards. As the cloud-model registration problem plays the most significant role in the data processing stage of utilizing the TLS devices, this paper concentrates on developing a broadly applicable coarse alignment framework for the point cloud of hull blocks. This framework involves two steps: the recognition of the connection regions of the point clouds and the coarse registration method according to the classification results. To detect the connection regions automatically, a hand-crafted simple model of the connection regions of hull blocks is built and its supporting detection method is proposed, besides, considering the recent overwhelming success of deep learning methods, a deep learning network suitable for large hull block datasets is constructed according to the Point-Net, and convolutional neural networks paradigms. Then, the coarse alignment method based on the detected connection regions is proposed. Experimental results illustrate the good performance of the proposed framework. Keywords computers in construction; shipbuilding; automation

Design and Application of New Pyridine-Derived Chiral Ligands in Asymmetric Catalysis.

ConspectusThe innovation of chiral ligands has been crucial for the asymmetric synthesis of functional molecules, as demonstrated by several types of widely applied "privileged" ligands. In this context, chiral pyridine-derived ligands, by far some of the oldest and most widely utilized ligands in catalysis, have attracted considerable research interest in the past half-century. However, the development of broadly applicable chiral pyridine units (CPUs) has been plagued by several intertwining challenges, thus delaying advancements in many asymmetric reactions.This Account aims to summarize the recent progress in new CPU-containing ligands, focusing on a rationally designed, modular, and tunable CPU developed in our laboratory. A significant problem thwarting conventional designs is the paradox between broad reactivity and stereoselectivity; that is, while enhanced stereoselectivity may be achieved by introducing chiral elements close to the N atom, the concomitant increase in local steric hindrance often limits catalytic activity and scope. Our newly developed CPU features a rigid [6-5-3] fused-ring framework and a tunable spirocyclic ketal side wall. The well-defined three-dimensional structure minimizes local (inner layer) steric hindrance and tunes the peripheral environment (outer layer) by remote substituents, thus securing reactivity and stereoselectivity. Different chelating ligands were readily assembled using this chiral structural module, with applications in mechanistically diverse transition-metal-catalyzed reactions. Thus, a series of chiral 2,2'-bipyridine ligands were successfully employed in the development of a general, efficient, and highly enantioselective nickel-catalyzed intermolecular reductive addition, Ullmann coupling of ortho-chlorinated aryl aldehydes, and carboxylation of benzylic (pseudo)halides with CO2. Notably, these chiral 2,2'-bipyridine ligands exhibited superior catalytic activity in the reactions compared to common N-based ligands. In addition, highly enantioselective iridium-catalyzed C-H borylation was developed using a CPU-containing N,B-bidentate ligand. Furthermore, mechanistically challenging, additive-free, and broad-scope transfer hydrogenative direct asymmetric reductive amination was achieved using a half-sandwich iridium catalyst supported by a chiral N,C-bidentate ligand. The new ligands demonstrated excellent performance in securing high catalytic activity and stereoselectivity, which, when combined with experimental and computational mechanistic investigations, supported the "double-layer control" design concept.Considering the broad applications of pyridine-derived ligands, the research progress described herein should inspire the creation of novel chiral catalysts and drive the development of many catalytic asymmetric reactions.

How far can I extrapolate my species distribution model? Exploring shape, a novel method

Species distribution and ecological niche models (hereafter SDMs) are popular tools with broad applications in ecology, biodiversity conservation, and environmental science. Many SDM applications require projecting models in environmental conditions non‐analog to those used for model training (extrapolation), giving predictions that may be statistically unsupported and biologically meaningless. We introduce a novel method, Shape, a model‐agnostic approach that calculates the extrapolation degree for a given projection data point by its multivariate distance to the nearest training data point. Such distances are relativized by a factor that reflects the dispersion of the training data in environmental space. Distinct from other approaches, Shape incorporates an adjustable threshold to control the binary discrimination between acceptable and unacceptable extrapolation degrees. We compared Shape's performance to five extrapolation metrics based on their ability to detect analog environmental conditions in environmental space and improve SDMs suitability predictions. To do so, we used 760 virtual species to define different modeling conditions determined by species niche tolerance, distribution equilibrium condition, sample size, and algorithm. All algorithms had trouble predicting species niches. However, we found a substantial improvement in model predictions when model projections were truncated independently of extrapolation metrics. Shape's performance was dependent on extrapolation threshold used to truncate models. Because of this versatility, our approach showed similar or better performance than the previous approaches and could better deal with all modeling conditions and algorithms. Our extrapolation metric is simple to interpret, captures the complex shapes of the data in environmental space, and can use any extrapolation threshold to define whether model predictions are retained based on the extrapolation degrees. These properties make this approach more broadly applicable than existing methods for creating and applying SDMs. We hope this method and accompanying tools support modelers to explore, detect, and reduce extrapolation errors to achieve more reliable models.Keywords: environmental novelty, extrapolation, Mahalanobis distance, model prediction, non‐analog environmental data, transferability

Harnessing TCR/CAR Antagonism to Enhance Immunotherapeutic Precision

Chimeric Antigen Receptor (CAR) T cell immunotherapy represents a conceptual breakthrough in the treatment of hematological malignancies. However, the rarity of cell surface protein targets specific to cancerous but not vital tissue has hindered its broad application to solid tumor treatment. While new logic-gated CAR designs have shown reduced toxicity against healthy tissues, the generalizability of such approaches across tumors remains unclear. Here, we harness a universal characteristic of endogenous T cell receptors (TCRs), their ability to discriminate between self and non-self ligands through inhibition of response against self (weak) antigens, to develop a broadly applicable method of enhancing immunotherapeutic precision. We hypothesized that this discriminatory mechanism, known as antagonism, would apply across receptors, allowing for a transfer of specificityfrom TCRs onto CARs. We therefore systematically mapped out the responses of CAR T cells to joint TCR and CAR stimulations. We transduced ovalbumin-specific TCR T cells with mouse CD19 CAR to produce T cells expressing both TCR and CAR and evaluated the response of TCR/CAR T cells using in vivo and in vitro leukemia models ( Figure 1A). We discovered that strong TCR antigen enhanced CAR T killing of CD19 + leukemia, while weak TCR antigen antagonized CAR T responses as assessed in vivo cytotoxicity and in vitro multiplexed dynamic profiling ( Figure 1B). We developed a mathematical model based on cross-receptor inhibitory coupling that accurately predicted the extent of TCR/CAR antagonism across a wide range of immunological settings. This model was validated in a CD19 + B16 mouse melanoma model showing that TCR/CAR antagonism decreased the infiltration of a tumor-reactive T cell cluster (cluster 1), while TCR/CAR agonism enhanced infiltration of this cluster1 ( Figure 1C). We then applied our quantitative knowledge of TCR/CAR crosstalk to design an Antagonism-Enforced Braking System (AEBS) for CAR T cell therapy. This was assessed in a model system using a CAR targeting the tyrosine-protein kinase erbB-2 (HER2), expressed on a subset of patients with both B-ALL and AML together with a hedgehog acyltransferase (HHAT) specific TCR, which responds strongly to mutated peptides presented on tumor cells and weakly to wild-type peptides presented on healthy tissue. Consistent with our discovery of the TCR/CAR antagonism, TCR signals against healthy cells expressing wild-type HHAT peptide antigen antagonized HER2 CAR T cell responses, minimizing on-target/off-tumor cytotoxicity against healthy cells. Notably though, AEBS-CAR T cells exhibited high anti-tumor cytotoxicity against tumor cells expressing HER2 and mutated HHAT peptides ( Figure 1D). AEBS CAR T cells sharpen the discriminatory power of synthetic anti-tumor lymphocytes, laying the groundwork for future studies to engineer complex logic into cells with minimal numbers of receptors ( Figure 1E). Our work highlights a novel mechanism by which TCRs can enforce CAR T cell specificity, with practical implications for the rational design of future anti-leukemia immunotherapies.

Multiplex Prime Editing and PASSIGE TM for Non-Viral Generation of an Allogeneic CAR-T Cell Product

While CAR-T cell therapy represents a major advance in personalized immunotherapy, the autologous nature of commercially available CAR-T products have delayed its broad application beyond a subset of hematological malignancies. In particular, manufacturing autologous CAR-T therapies is costly and time-consuming, and success relies on the fitness of a patient's cells. An allogeneic off-the-shelf CAR-T product could overcome these cell quality and quantity issues and could be accessed on-demand. However, a successful allogeneic CAR-T product will require multiplex gene knockout in addition to stable CAR integration to prevent graft-versus-host disease (GvHD) and graft rejection by the patient's immune system. Strategies for the delivery and expression of CAR transgenes include semi-random integration using lentivirus or transposons, which risk unintended gene disruption or activation. Targeted integration can be achieved using nucleases in combination with a template for homology-directed repair (HDR), but efficiency of this approach is generally low and there are risks associated with the induction of double-strand breaks (DSBs), including p53 activation and chromothripsis. This is further complicated in a situation where multiplex editing is required, as multiple DSBs can lead to gross chromosomal rearrangements. Alternatively, base editors have been used for targeted gene disruption without the induction of DSBs via single nucleotide conversion to disrupt a splice site or introduce a single premature stop (pmSTOP) codon. However, this approach is limited to transition mutations and cannot be used to integrate large genetic cargo, which is required to generate CAR-T cells. Prime Editing (PE) can be used for precise and programmable gene disruption via multiple strategies, including introduction of multiple pmSTOP codons, splice site disruption, frameshift mutations, or deletion of large segments of regulatory or coding sequence. Further, Prime-Assisted Site-Specific Integrase Gene Editing (PASSIGE) can be used to precisely and flexibly integrate large genetic cargo at a specific locus. Together, PE-mediated gene knock out and PASSIGE can be tailored to generate a more broadly applicable, potentially safer, and more effective CAR-T cell product. To evaluate the efficacy of an all-PE non-viral approach to allogeneic CAR-T cell generation, PASSIGE and PE mediated gene knockout were used to generate CD19-CAR-T cells. Multiplex PE precisely disrupted expression of the endogenous T cell receptor alpha constant ( TRAC) and beta-2-microglobulin ( B2M) loci in over 90% of human T cells. Co-delivery of a non-viral DNA donor template with PASSIGE editing components resulted in targeted integration of a 3.5 kb CD19-CAR transgene expression cassette at the TRAC locus in over 60% of the T cells, with no observed impact on T cell viability, phenotype, or functionality. CD19 CAR-T cells generated using PASSIGE show potent anti-tumor activity and cytokine production in response to CD19 + tumor cell lines in vitro and in vivo. The PASSIGE-generated CD19 CAR-T cells reduce tumor burden and prolong survival of mice bearing CD19 + tumors. These results show that a PE platform can be used to generate multiplex edited CAR-T cells without the need for viral vectors and without causing DSBs. This modular, one-step, non-viral delivery Prime Editing platform expands the applicability of T cell therapies for the treatment of tumors and immune diseases.

Design of prime-editing guide RNAs with deep transfer learning

Prime editors (PEs) are promising genome-editing tools, but effective optimization of prime-editing guide RNA (pegRNA) design remains a challenge owing to the lack of accurate and broadly applicable approaches. Here we develop Optimized Prime Editing Design (OPED), an interpretable nucleotide language model that leverages transfer learning to improve its accuracy and generalizability for the efficiency prediction and design optimization of pegRNAs. Comprehensive validations on various published datasets demonstrate its broad applicability in efficiency prediction across diverse scenarios. Notably, pegRNAs with high OPED scores consistently show significantly increased editing efficiencies. Furthermore, the versatility and efficacy of OPED in design optimization are confirmed by efficiently installing various ClinVar pathogenic variants using optimized pegRNAs in the PE2, PE3/PE3b and ePE editing systems. OPED consistently outperforms existing state-of-the-art approaches. We construct the OPEDVar database of optimized designs from over two billion candidates for all pathogenic variants and provide a user-friendly web application of OPED for any desired edit.

A Broadly Applicable Diels-Alder-Based Synthesis of Ketamine-Related Arylcyclohexylamines.

Herein, we report the synthesis of aryl derivatives of ketamine and of ketamine's major metabolites hydroxynorketamine (HNK), norketamine (NK), and dehydronorketamine (DHNK) via a microwave-assisted Diels-Alder reaction to form the substituted cyclohexane core structure. Starting with aryl acrylic esters as dienophiles and siloxybutadienes as diene counterparts, a wide range of substituted arylcyclohexylamines was obtained after several modification steps of the initial Diels-Alder products [El Sheikh, S.; Weber, H.; Kortenbrede, L.; Drouvé, N. A broadly applicable Diels-Alder based Synthesis of Ketamine related Arylcyclohexylamines. ChemRxiv 2022, 10.26434/chemrxiv- 2022-xf1l9].

Study on Light Pollution Based on A Broadly Applicable Metric

Nowadays, with the development of the economy, light pollution is becoming more and more serious, affecting our health, safety and environment. This paper mainly studies the problem of light pollution. First, a light pollution index measurement model is established, and a widely applicable indicator is developed to determine the risk level of light pollution in an area. Secondly, we selected several nature reserves as specific application sites, and used the model to solve the specific index values of light pollution, and the calculation showed that the calculation results of the model were basically consistent with the actual situation. Finally, based on the classification of light pollution, three intervention strategies are given, and specific implementation measures are given.

High content cellulomics by correlative microscopy

The exponential increase in anthropogenic chemicals in our environment necessitates methods to assess impact prior to their release to predict potential environmental and health impacts. Atomic force microscopy (AFM) and laser scanning confocal microscopy (LSCM) each provide abundant information on cell behaviour. In addition to determining cell morphology and surface ultrastructure, AFM in quantitative imaging (QI) mode probes cellular mechanics and surface biochemistry with minimal applied force, and LSCM offers a window into the cell for imaging fluorescently tagged macromolecules. Data from correlative AFM-LSCM is thus complimentary, providing a comprehensive picture of cellular physiology. I will present a correlative AFM-QI-LSCM assay for the simultaneous real-time imaging of living cells in situ that generates real-time multiplexed data, including cell morphology and mechanics, surface adhesion and ultrastructure, along with the localization of intracellular macromolecules. The broad applicability of the assay is demonstrated using disparate cell types, showing altered surface properties, internal molecular arrangement and oxidative stress in model bacterial, fungal and human cells exposed to 2,4-dichlorophenoxyacetic acid. This correlative microscopy assay is broadly applicable to a variety of cell types and can be used to assess the impact of any multitude of contaminants, alone or in combination.

Au(Np#)Cl]: Highly Reactive and Broadly Applicable Au(I)─NHC Catalysts for Alkyne π-Activation Reactions.

Cationic Au(I)─NHC (NHC = N-heterocyclic carbene) complexes have become an important class of catalysts for alkyne π-activation reactions in organic synthesis. In particular, these complexes are characterized by high stability of catalytic species engendered by strong σ-donation and metal backbonding. Herein, we report the synthesis and characterization of well-defined [Au(NHC)Cl] complexes featuring recently discovered IPr# family of ligands that hinge upon modular peralkylation of aniline. These ligands have been commercialized in collaboration with MilliporeSigma (IPr#: 915653; Np#: 915912; BIAN-IPr#: 916420). Evaluation of the [Au(NHC)Cl] complexes in a series of Au(I)─NHC-catalyzed π-functionalizations of alkynes, such as hydrocarboxylation, hydroamination and hydration, resulted in the identification of wingtip-flexible [Au(Np#)Cl] as a highly reactive and broadly applicable catalyst with the re-activity outperforming the classical [Au(IPr)Cl] and [Au(IPr*)Cl] complexes. The utility of this catalyst has been demonstrated in the direct late-stage derivatization of complex pharmaceuticals. Structural and computational studies were conducted to determine steric effects, frontier molecular orbitals and bond orders of this class of catalysts. Considering the attractive features of well-defined Au(I)─NHC complexes, we anticipate that this class of bulky and wingtip-flexible Au(I)─NHCs based on the modular peralkylated naphthylamine scaffold will find broad application in π-functionalization of alkynes in various areas of organic synthesis and catalysis.

An extremely efficiency method to achieve stable superhydrophobicity on the surface of additive manufactured NiTi Alloys: “Ultrasonic Fluorination”

The realization of superhydrophobic helps to enhance the anti-corrosion, self-cleaning and other properties of the material surface. Compared with polymer materials, metallic have superior strength, hardness and stability in superhydrophobicity. For most metals, the standard superhydrophobic treatment method is a combination of micro-nano hierarchical structure and low surface energy modification Herein, SLM-NiTi with broad application prospects in biomedical and engineering fields is selected for the first time. The nanosecond laser processing method was used to construct the surface with the micro-nano hierarchical structure, and a new low surface energy modification method (ultrasonic fluorination) was innovatively proposed. Ultimately, a stable superhydrophobic surface of SLM-NiTi was successfully achieved. The results show that ultrasonic fluorination treatment can achieve surface modification in only 10 min, which is far more efficient than other low surface energy treatment methods. Simultaneously, it is found that the realization of superhydrophobicity of metallic surfaces depends on the nanosecond laser process parameters. Moreover, the frictional stability and durability of the surfaces are also demonstrated, which inspired new applications in extreme environments. We envision this work not just for SLM-NiTi alloys but for broadly applicable in various metals, dramatically improving the period for realizing superhydrophobic functions on metallic surfaces.

Hydrosilane Organocatalyst Permits Broadly Applicable Amidation under Mild Conditions

Key words amidation - peptides - hydrosilanes - peptide coupling

Identification of Broadly Applicable Adeno-Associated Virus Vectors by Systematic Comparison of Commonly Used Capsid Variants In Vitro.

Adeno-associated viruses (AAVs) represent highly attractive gene therapy vectors and potent research tools for the modulation of gene expression in animal models or difficult-to-transfect cell cultures. Engineered variants, comprising chimeric, mutated, or peptide-inserted capsids, have strongly broadened the utility of AAVs by altering cellular tropism, enabling immune evasion, or increasing transduction efficiency. In this work, the performance of 50 of the most used, predominantly published, AAVs was compared on several primary cells, cell lines, and induced pluripotent stem cell-derived models from different organs, including the adipose tissue, liver, lung, brain, and eyes. To identify the most efficient capsids for each cell type, self-complementary AAVs were standardized by digital polymerase chain reaction, arrayed on 96-well plates, and screened using high-content imaging. To enable best use of the data, all results are also provided in a web app. The utility of one selected AAV variant is further exemplified in a liver fibrosis assay based on primary hepatic stellate cells, where it successfully reversed a small interfering RNA (siRNA)-induced phenotype. Most importantly, our comparative analysis revealed that a subselection of only five AAV variants (AAV2.NN, AAV9-SLRSPPS, AAV6.2, AAV6TM, and AAV1P5) enabled efficient transduction of all tested cell types and markedly outperformed other well-established capsids, such as AAV2-7m8. These findings suggest that a core panel comprising these five capsid variants is a universally applicable and sufficient tool to identify potent AAVs for gene expression modulation in cellular systems.

Dissecting protein tyrosine phosphatase signaling by engineered chemogenetic control of its activity.

Protein tyrosine phosphatases (PTPases) are critical mediators of dynamic cell signaling. A tool capable of identifying transient signaling events downstream of PTPases is essential to understand phosphatase function on a physiological time scale. We report a broadly applicable protein engineering method for allosteric regulation of PTPases. This method enables dissection of transient events and reconstruction of individual signaling pathways. Implementation of this approach for Shp2 phosphatase revealed parallel MAPK and ROCK II dependent pathways downstream of Shp2, mediating transient cell spreading and migration. Furthermore, we show that the N-SH2 domain of Shp2 regulates MAPK-independent, ROCK II-dependent cell migration. Engineered targeting of Shp2 activity to different protein complexes revealed that Shp2-FAK signaling induces cell spreading whereas Shp2-Gab1 or Shp2-Gab2 mediates cell migration. We identified specific transient morphodynamic processes induced by Shp2 and determined the role of individual signaling pathways downstream of Shp2 in regulating these events. Broad application of this approach is demonstrated by regulating PTP1B and PTP-PEST phosphatases.

Abstract 3574: A Phase I trial of personalized multi-peptide vaccination in combination with the TLR1/2 ligand XS15 in CLL patients under Bruton-Tyrosin-Kinase-inhibitor based regimes - Preclinical development and study design

Abstract Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. Despite the therapeutic success of novel small molecules (e.g. ibrutinib, idealisib and venetoclax), patients frequently experience disease relapse due to the persistence of minimal residual disease (MRD). Targeting the residual CLL cells by immunotherapy could further improve the depth and persistence of CLL remission. Here, we report on the preclinical development and clinical implementation of a phase I trial evaluating safety, immunogenicity and efficacy in terms of MRD response of a personalized multi-peptide vaccine adjuvanted with the novel toll-like receptor (TLR) 1/2 agonist XS15 emulsified in Montanide࣪ ISA51 VG (Rammensee et al. JIC 2019) in CLL patient receiving Bruton-Tyrosin-Kinase (BTK)-inhibitor based regimes (NCT04688385). So far, broad application of peptide vaccines in cancer patients is hampered by time- and cost-intensive design and the lack of neoepitopes from tumor-specific mutations, especially in low-mutational burden malignancies. To improve on these drawbacks, we developed an immunopeptidome-guided workflow for the design of tumor-associated off-the-shelf peptide warehouses for broadly applicable personalized therapeutics (Nelde et al., Front Immunol, 2021). For the trial reported here, comparative mass spectrometry-based immunopeptidome analyses of 61 CLL patient samples and a dataset of benign tissues samples (n = 351) enabled the identification of high-frequent non-mutated CLL-associated antigens for the most common HLA allotypes (HLA-A*02, -A*24, and -B*07). These antigens were proven to be recognized as T cell epitopes by pre-existing and de novo induced T cells in CLL patients and healthy volunteers, thereby enabling the selection of a 12-peptides panel comprising 9 HLA-class I (3 for each HLA allotype) and 3 HLA class II restricted high frequent CLL-exclusive T cell epitopes as warehouse for the personalized composition of study vaccine cocktails. Our meanwhile ongoing phase I trial recruits CLL patients eligible for treatment with BTK inhibitors. For those with matching HLA allotypes, a personalized peptide-cocktail is formulated comprising 6 CLL-associated peptides (3 HLA class I and 3 HLA class II peptides), selected from our warehouse based on individual immunopeptidome analysis, and 2 highly immunogenic HLA class II control peptides, one viral (human adenovirus C) and one tumor associated (Baculoviral IAP repeat-containing protein 5). Three doses of the vaccine are administered in a 4-weekly interval, after reduction of CLL-load by BTK inhibitors. So far, 10 CLL patients were included in the trial. The first vaccinations will be applied in November and December 2021 with first immunogenicity data expected in March 2022 and to be reported at the meeting. Citation Format: Maddalena Marconato, Annika Nelde, Yacine Maringer, Marcel Wacker, Marion Richter, Monika Denk, Helmut Salih, Jonas Heitmann, Juliane Sarah Walz. A Phase I trial of personalized multi-peptide vaccination in combination with the TLR1/2 ligand XS15 in CLL patients under Bruton-Tyrosin-Kinase-inhibitor based regimes - Preclinical development and study design [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 3574.