Specific Modifications Research Articles

Holistic in silico developability assessment of novel classes of small proteins using publicly available sequence-based predictors.

The development of novel therapeutic proteins is a lengthy and costly process, with an average attrition rate of 91% (Thomas et al. Clinical Development Success Rates and Contributing Factors 2011-2020, 2021). To increase the probability of success and ensure robust drug supply beyond approval, it is essential to assess the developability profile of new potential drug candidates as early and broadly as possible in development (Jain et al. MAbs, 2023. https://doi.org/10.1016/j.copbio.2011.06.002 ). Predicting these properties in silico is expected to be the next leap in innovation as it would enable significantly reduced development timelines combined with broader screens at lower costs. However, developing predictive algorithms typically requires substantial datasets generated under very defined conditions, a limiting factor especially for new classes of therapeutic proteins that hold immense clinical promise. Here we describe a strategy for assessing the developability of a novel class of small therapeutic Anticalin® proteins using machine learning in conjunction with a knowledge-driven approach. The knowledge-driven approach considers developability attributes such as aggregation propensity, charge variants, immunogenicity, specificity, thermal stability, hydrophobicity, and potential post-translational modifications, to calculate a holistic developability score. Based on sequence-derived descriptors as input parameters we established novel statistical models designed to predict the developability scores for Anticalin proteins. The best models yielded low root mean square errors across the entire dataset and were further validated by removing input data from individual screening campaigns and predicting developability scores for those drug candidates. The adoption of the described workflow will enable significantly streamlined preclinical development of Anticalin drug candidates and could potentially be applied to other therapeutic protein scaffolds.

Evaluation and prediction of bending properties of glulam beams made from young plantation-grown Eucalyptus nitens

ABSTRACT Interest in making glulam from plantation-grown Eucalyptus nitens has increased, but current structural timber standards were not designed for species grown in plantations. Additionally, the applicability of classical composite beam theory remains uncertain due to the unique features of this species. This study was designed to assess the hypothesis that the bending performance of glulam made from young plantation-grown E. nitens is reliable and predictable. We tested the hypothesis through experimental procedures and theoretical and statistical analyses. First, we assessed the bending performance of 20 glulam beams, which were manufactured in five configurations. Second, we compared our experimental results with predictive values to evaluate the accuracy of methods in current standards. Third, in our pursuit of improved predictive tools, we developed theoretical and parametric models using mechanical properties of the board elements. Our improved models showed a decreased deviation in both stiffness and strength values. A Monte Carlo simulation was used to determine the distribution for both stiffness and strength. The results validated that this product can be allocated a GL grade using current standards and the bending performance is predictable. The study enhances confidence in incorporating E. nitens into glulam products and offers valuable data for future specification modifications.

Motion Coordination of Multiple Autonomous Mobile Robots under Hard and Soft Constraints

This paper presents a distributed approach to the motion control problem for a platoon of unicycle robots moving through an unknown environment filled with static obstacles under multiple hard and soft operational constraints. Each robot has an onboard camera to determine its relative position in relation to its predecessor and proximity sensors to detect and avoid nearby obstascles. Moreover, no robot apart from the leader can independently localize itself within the given workspace. To overcome this limitation, we propose a novel distributed control protocol for each robot of the fleet, utilizing the Adaptive Performance Control (APC) methodology. By utilizing the APC approach to address input constraints via the on-line modification of the error specifications, we ensure that each follower effectively tracks its predecessor without encountering collisions with obstacles, while simultaneously maintaining visual contact with its preceding robot, thus ensuring the inter-robot visual connectivity. Finally, extensive simulation results are presented to demonstrate the effectiveness of the presented control system along with a real-time experiment conducted on an actual robotic system to validate the feasibility of the proposed approach in real-world scenarios.

Imaging plant cell walls using fluorescent stains: The beauty is in the details.

Plants continuously face various environmental stressors throughout their lifetime. To be able to grow and adapt in different environments, they developed specialized tissues that allowed them to maintain a protected yet interconnected body. These tissues undergo specific primary and secondary cell wall modifications that are essential to ensure normal plant growth, adaptation and successful land colonization. The composition of cell walls can vary among different plant species, organs and tissues. The ability to remodel their cell walls is fundamental for plants to be able to cope with multiple biotic and abiotic stressors. A better understanding of the changes taking place in plant cell walls may help identify and develop new strategies as well as tools to enhance plants' survival under environmental stresses or prevent pathogen attack. Since the invention of microscopy, numerous imaging techniques have been developed to determine the composition and dynamics of plant cell walls during normal growth and in response to environmental stimuli. In this review, we discuss the main advances in imaging plant cell walls, with a particular focus on fluorescent stains for different cell wall components and their compatibility with tissue clearing techniques. Lay Description: Plants are continuously subjected to various environmental stresses during their lifespan. They evolved specialized tissues that thrive in different environments, enabling them to maintain a protected yet interconnected body. Such tissues undergo distinct primary and secondary cell wall alterations essential to normal plant growth, their adaptability and successful land colonization. Cell wall composition may differ among various plant species, organs and even tissues. To deal with various biotic and abiotic stresses, plants must have the capacity to remodel their cell walls. Gaining insight into changes that take place in plant cell walls will help identify and create novel tools and strategies to improve plants' ability to withstand environmental challenges. Multiple imaging techniques have been developed since the introduction of microscopy to analyse the composition and dynamics of plant cell walls during growth and in response to environmental changes. Advancements in plant tissue cleaning procedures and their compatibility with cell wall stains have significantly enhanced our ability to perform high-resolution cell wall imaging. At the same time, several factors influence the effectiveness of cleaning and staining plant specimens, as well as the time necessary for the process, including the specimen's size, thickness, tissue complexity and the presence of autofluorescence. In this review, we will discuss the major advances in imaging plant cell walls, with a particular emphasis on fluorescent stains for diverse cell wall components and their compatibility with tissue clearing techniques. We hope that this review will assist readers in selecting the most appropriate stain or combination of stains to highlight specific cell wall components of interest.

Computationally inferred cell-type specific epigenome-wide DNA methylation analysis unveils distinct methylation patterns among immune cells for HIV infection in three cohorts.

Epigenome-wide association studies (EWAS) have identified CpG sites associated with HIV infection in blood cells in bulk, which offer limited knowledge of cell-type specific methylation patterns associated with HIV infection. In this study, we aim to identify differentially methylated CpG sites for HIV infection in immune cell types: CD4+ T-cells, CD8+ T-cells, B cells, Natural Killer (NK) cells, and monocytes. Applying a computational deconvolution method, we performed a cell-type based EWAS for HIV infection in three independent cohorts (Ntotal = 1,382). DNA methylation in blood or in peripheral blood mononuclear cells (PBMCs) was profiled by an array-based method and then deconvoluted by Tensor Composition Analysis (TCA). The TCA-computed CpG methylation in each cell type was first benchmarked by bisulfite DNA methylation capture sequencing in a subset of the samples. Cell-type EWAS of HIV infection was performed in each cohort separately and a meta-EWAS was conducted followed by gene set enrichment analysis. The meta-analysis unveiled a total of 2,021 cell-type unique significant CpG sites for five inferred cell types. Among these inferred cell-type unique CpG sites, the concordance rate in the three cohorts ranged from 96% to 100% in each cell type. Cell-type level meta-EWAS unveiled distinct patterns of HIV-associated differential CpG methylation, where 74% of CpG sites were unique to individual cell types (false discovery rate, FDR <0.05). CD4+ T-cells had the largest number of unique HIV-associated CpG sites (N = 1,624) compared to any other cell type. Genes harboring significant CpG sites are involved in immunity and HIV pathogenesis (e.g. CD4+ T-cells: NLRC5, CX3CR1, B cells: IFI44L, NK cells: IL12R, monocytes: IRF7), and in oncogenesis (e.g. CD4+ T-cells: BCL family, PRDM16, monocytes: PRDM16, PDCD1LG2). HIV-associated CpG sites were enriched among genes involved in HIV pathogenesis and oncogenesis that were enriched among interferon-α and -γ, TNF-α, inflammatory response, and apoptotic pathways. Our findings uncovered computationally inferred cell-type specific modifications in the host epigenome for people with HIV that contribute to the growing body of evidence regarding HIV pathogenesis.

The Mutagenic Plasticity of the Cholera Toxin B-Subunit Surface Residues: Stability and Affinity.

Mastering selective molecule trafficking across human cell membranes poses a formidable challenge in healthcare biotechnology while offering the prospect of breakthroughs in drug delivery, gene therapy, and diagnostic imaging. The cholera toxin B-subunit (CTB) has the potential to be a useful cargo transporter for these applications. CTB is a robust protein that is amenable to reengineering for diverse applications; however, protein redesign has mostly focused on modifications of the N- and C-termini of the protein. Exploiting the full power of rational redesign requires a detailed understanding of the contributions of the surface residues to protein stability and binding activity. Here, we employed Rosetta-based computational saturation scans on 58 surface residues of CTB, including the GM1 binding site, to analyze both ligand-bound and ligand-free structures to decipher mutational effects on protein stability and GM1 affinity. Complimentary experimental results from differential scanning fluorimetry and isothermal titration calorimetry provided melting temperatures and GM1 binding affinities for 40 alanine mutants among these positions. The results showed that CTB can accommodate diverse mutations while maintaining its stability and ligand binding affinity. These mutations could potentially allow modification of the oligosaccharide binding specificity to change its cellular targeting, alter the B-subunit intracellular routing, or impact its shelf-life and in vivo half-life through changes to protein stability. We anticipate that the mutational space maps presented here will serve as a cornerstone for future CTB redesigns, paving the way for the development of innovative biotechnological tools.

Quantitative phosphoproteomics explain cryopreservation-induced reductions in ram sperm motility

Sperm cryopreservation decreases motility, probably due to changes in protein phosphorylation. Our objective was to use quantitative phosphoproteomics for systematic comparative analyses of fresh versus frozen-thawed sperm to identify factors causing cryo-injury. Ejaculates were collected (artificial vagina) from six Dorper rams, pooled, extended, and frozen over liquid nitrogen. Overall, 915, 3382, and 6875 phosphorylated proteins, phosphorylated peptides, and phosphorylation sites, respectively, were identified. At least two modified sites were present in 57.94% of the 6875 phosphosites identified, of which AKAP4 protein contained up to 331 modified sites. There were 732 phosphorylated peptides significantly up-regulated and 909 significantly down-regulated in frozen-thawed versus fresh sperm. Moreover, the conserved motif [RxxS] was significantly down-regulated in frozen-thawed sperm. Phosphorylation of sperm-specific proteins, e.g., AKAP3/4, CABYR, FSIP2, GSK3A/B, GPI, and ODF1/2 make them potential biomarkers to assess the quality of frozen-thawed ram sperm. Furthermore, these differentially phosphorylated proteins and modification sites were implicated in cryopreservation-induced changes in sperm energy production, fiber sheath composition, and various biological processes. We concluded that abnormal protein phosphorylation modifications are key regulators of reduced sperm motility. These novel findings implicated specific protein phosphorylation modifications in sperm cryo-injury. SignificanceThis study used phosphorylated TMT quantitative proteomics to explore regulation of epigenetic modifications in frozen-thawed ram sperm. This experiment demonstrated that ram sperm freezing affects phosphorylation site modifications of proteins, especially those related to functions such as sperm motility and energy production. Furthermore, it is important to link functions of phosphorylated proteins with changes in sperm quality after freezing and thawing, and to clarify intrinsic reasons for sperm quality changes, which is of great importance for elucidating mechanisms of sperm freezing damage. Based on these protein markers and combined with cryoprotectant design theory, it provides a theoretical basis and data reference to study sperm cryoprotectants.

Modification Strategies of High-Energy Li-Rich Mn-Based Cathodes for Li-Ion Batteries: A Review.

Li-rich manganese-based oxide (LRMO) cathode materials are considered to be one of the most promising candidates for next-generation lithium-ion batteries (LIBs) because of their high specific capacity (250 mAh g-1) and low cost. However, the inevitable irreversible structural transformation during cycling leads to large irreversible capacity loss, poor rate performance, energy decay, voltage decay, etc. Based on the recent research into LRMO for LIBs, this review highlights the research progress of LRMO in terms of crystal structure, charging/discharging mechanism investigations, and the prospects of the solution of current key problems. Meanwhile, this review summarizes the specific modification strategies and their merits and demerits, i.e., surface coating, elemental doping, micro/nano structural design, introduction of high entropy, etc. Further, the future development trend and business prospect of LRMO are presented and discussed, which may inspire researchers to create more opportunities and new ideas for the future development of LRMO for LIBs with high energy density and an extended lifespan.

The lncRNA Snhg11, a new candidate contributing to neurogenesis, plasticity, and memory deficits in Down syndrome.

Down syndrome (DS) stands as the prevalent genetic cause of intellectual disability, yet comprehensive understanding of its cellular and molecular underpinnings remains limited. In this study, we explore the cellular landscape of the hippocampus in a DS mouse model, the Ts65Dn, through single-nuclei transcriptional profiling. Our findings demonstrate that trisomy manifests as a highly specific modification of the transcriptome within distinct cell types. Remarkably, we observed a significant shift in the transcriptomic profile of granule cells in the dentate gyrus (DG) associated with trisomy. We identified the downregulation of a specific small nucleolar RNA host gene, Snhg11, as the primary driver behind this observed shift in the trisomic DG. Notably, reduced levels of Snhg11 in this region were also observed in a distinct DS mouse model, the Dp(16)1Yey, as well as in human postmortem brain tissue, indicating its relevance in Down syndrome. To elucidate the function of this long non-coding RNA (lncRNA), we knocked down Snhg11 in the DG of wild-type mice. Intriguingly, this intervention alone was sufficient to impair synaptic plasticity and adult neurogenesis, resembling the cognitive phenotypes associated with trisomy in the hippocampus. Our study uncovers the functional role of Snhg11 in the DG and underscores the significance of this lncRNA in intellectual disability. Furthermore, our findings highlight the importance of DG in the memory deficits observed in Down syndrome.

Microbiological and metabolic pathways analysing the mechanisms of alfalfa polysaccharide and sulfated alfalfa polysaccharide in alleviating obesity

Alfalfa polysaccharide (AP) and sulfated alfalfa polysaccharide (SAP) exhibit potential for alleviating obesity. This study aimed to analyze the mechanism of action of AP and SAP in alleviating obesity through combined microbiomics and metabolomics. The research selected validated optimal AP and SAP concentration for experiment. The results showed that AP and SAP down-regulated colonic inflammatory gene expression, regulated intestinal pH to normal, and restored intestinal growth. Microbial sequencing showed that AP and SAP altered the microbial composition ratio. AP increased the relative abundance of Muribaculaceae and Romboutsia. SAP increased the relative abundance of Dubosiella, Fecalibaculum and Desulfovibrionaceae. Metabolomic analysis showed that AP regulated steroid hormone biosynthesis, neuroactive ligand-receptor interactions and bile secretion pathways. SAP focuses more on pathways related to amino acid metabolism. Meanwhile, AP and SAP down-regulated the mRNA expression of colonic COX-2, PepT-1 and HK2 and up-regulated the mRNA expression of TPH1. Correlation analysis showed a strong correlation between metabolites and gut bacteria. Dubosiella, Faecalibaculum may be the critical marker flora for polysaccharides to alleviate obesity. This study indicates that AP and SAP alleviate obesity through different pathways and that specific polysaccharide modifications affect characteristic microbial and metabolic pathways, providing new insights into polysaccharide modifications.

Surface Modification of Polystyrene with Boronic Acid for Immunoaffinity-Based Cell Enrichment.

Obtaining an enriched and phenotypically pure cell population from heterogeneous cell mixtures is important for diagnostics and biosensing. Existing techniques such as fluorescent-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) require preincubation with antibodies (Ab) and specialized equipment. Cell immunopanning removes the need for preincubation and can be done with no specialized equipment. The majority of the available antibody-mediated analyte capture techniques require a modification to the Abs for binding. In this work, no antibody modification is used because we take advantage of the carbohydrate chain in the Fc region of Ab. We use boronic acid as a cross-linker to bind the Ab to a modified surface. The process allows for functional orientation and cleavable binding of the Ab. In this study, we created an immunoaffinity matrix on polystyrene (PS), an inexpensive and ubiquitous plastic. We observed a 37% increase in Ab binding compared with that of a passive adsorption approach. The method also displayed a more consistent antibody binding with 17 times less variation in Ab loading among replicates than did the passive adsorption approach. Surface topography analysis revealed that a dextran coating reduced nonspecific antibody binding. Elemental analysis (XPS) was used to characterize the surface at different stages and showed that APBA molecules can bind upside-down on the surface. While upside-down antibodies likely remain functional, their elution behavior might differ from those bound in the desired way. Cell capture experiments show that the new surface has 43% better selectivity and 2.4-fold higher capture efficiency compared to a control surface of passively adsorbed Abs. This specific surface chemistry modification will allow the targeted capture of cells or analytes with the option of chemical detachment for further research and characterization.

Engineering circular bacteriocins: structural and functional effects of α-helix exchanges and disulfide introductions in circularin A.

Circular bacteriocins form a distinct group of antimicrobial peptides (AMPs) characterized by their unique head-to-tail ligated circular structure and functional properties. They belong to the ribosomally synthesized and post-translationally modified peptide (RiPP) family. The ribosomal origin of these peptides facilitates rapid diversification through mutations in the precursor genes combined with specific modification enzymes. In this study, we primarily explored the bacteriocin engineering potential of circularin A, a circular bacteriocin produced by Clostridium beijerinckii ATCC 25752. Specifically, we employed strategies involving α-helix replacements and disulfide bond introductions to investigate their effects on both biosynthesis and bioactivity of the bacteriocin. The results show the feasibility of peptide engineering to introduce certain structural properties into circularin A through carefully designed approaches. The introduction of cysteines for potential disulfide bonds resulted in a substantial reduction in bacteriocin biosynthesis and/or bioactivity, indicating the importance of maintaining dynamic flexibility of α-helices in circularin A, while reduction of the potential disulfide in one case increased the activity. The 5 α-helices of circularin A were respectively replaced by corresponding helices from another circular peptide, enterocin AS-48, and modestly active peptides were obtained in a few cases. Overall, this study provides valuable insights into the engineering potential of circular bacteriocins as antimicrobial agents, including their structural and functional restrictions and their suitability as peptide engineering scaffolds. This helps to pave the way for the development of novel antimicrobial peptides with tailored properties based on circular bacteriocins.

Glycosylation-related genes mediated prognostic signature contribute to prognostic prediction and treatment options in ovarian cancer: based on bulk and single‑cell RNA sequencing data

BackgroundOvarian cancer (OC) is a complex disease with significant tumor heterogeneity with the worst prognosis and highest mortality among all gynecological cancers. Glycosylation is a specific post-translational modification that plays an important role in tumor progression, immune escape and metastatic spread. The aim of this work was to identify the major glycosylation-related genes (GRGs) in OC and construct an effective GRGs signature to predict prognosis and immunotherapy.MethodsAUCell algorithm was used to identify glycosylation-related genes (GRGs) based on the scRNA-seq and bulk RNA-seq data. An effective GRGs signature was conducted using COX and LASSO regression algorithm. The texting dataset and clinical sample data were used to assessed the accuracy of GRGs signature. We evaluated the differences in immune cell infiltration, enrichment of immune checkpoints, immunotherapy response, and gene mutation status among different risk groups. Finally, RT-qPCR, Wound-healing assay, Transwell assay were performed to verify the effect of the CYBRD1 on OC.ResultsA total of 1187 GRGs were obtained and a GRGs signature including 16 genes was established. The OC patients were divided into high- and low- risk group based on the median riskscore and the patients in high-risk group have poor outcome. We also found that the patients in low-risk group have higher immune cell infiltration, enrichment of immune checkpoints and immunotherapy response. The results of laboratory test showed that CYBRD1 can promote the invasion, and migration of OC and is closely related to the poor prognosis of OC patients.ConclusionsOur study established a GRGs signature consisting of 16 genes based on the scRNA-seq and bulk RNA-seq data, which provides a new perspective on the prognosis prediction and treatment strategy for OC.

A42 A HIGH SALT DIET INCREASES ELASTOLYTIC ACTIVITY AND WORSENS COLITIS IN GNOTOBIOTIC MICE

Abstract Background Recent evidence has linked ultra-processed foods, which are high in salt, to increased risk of inflammatory bowel disease (IBD). A high salt diet (HSD) was also shown to be colitogenic in specific pathogen-free mice, in part, through modulation of the microbiota. We recently described high microbial proteolytic activity (PA) in feces from IBD patients, that when transferred to germ-free mice, increased acute colitis severity. However, the drivers of high microbial PA are unknown. Aims To determine whether HSD increases proteolytic activity, thereby exacerbating colitis. Methods Adult germ-free (GF) C57BL/6 mice were colonized with feces from a patient with UC in flare (n=13). Mice were fed either a control diet (CD; 7004, Teklad) or a HSD (7004 supplemented with 4% NaCl) plus 1% NaCl in drinking water. Three weeks following colonization, chronic colitis was induced in half of the mice by three cycles (five days each; 2.0%, 1.5% and 1.5%, respectively) of DSS in drinking water with a five-day wash-out period between cycles. Fecal PA and predicted microbial protease profiles were measured before colitis induction. Weight loss and disease activity (e.g., occult blood in feces and stool consistency) was evaluated for the duration of colitis and microscopic colitis was evaluated at endpoint. Results In mice colonized with UC microbiota, a HSD increased elastolytic activity (p=.03 versus CD) and induced a unique predicted protease and peptidase profile. After colitis induction, mice consuming a HSD lost more weight than mice consuming the CD (p=.0001) and had higher disease activity (p=.0004). Additionally, HSD induced more severe microscopic colitis scores compared with the CD (p=.0001). Conclusions These results raise the hypothesis that a HSD drives microbial proteolytic activity of IBD microbiota. However, whether this shift is causally associated with the worsening of colitis remains unclear. Mechanistic insight underlying this HSD-microbial proteolytic function could lead to specific dietary modifications or anti-proteolytic therapies for IBD in patients with proteolytic-driven inflammation. Funding Agencies CCC, CIHR

A numerical study on the aerodynamic effects of dynamic twisting on forward flight flapping wings

To better understand the secret of natural flying vertebrates such as how humming-birds twist their wings to achieve superb flight ability, we presented a numerical investigation of dynamic twisting based on a hummingbird-like flapping wing model. Computational fluid dynamic simulations were performed to examine the effects of dynamic twisting on the unsteady flow field, the generation of instantaneous aerodynamic forces, and the time-averaged aerodynamic performance. This research reveals the details of leading-edge vortices (LEVs) and the underlying mechanisms behind the positive effects of wing torsion. The results demonstrated that wing torsion can effectively maintain the favorable distribution of effective angle of attack along the wing spanwise, resulting in a higher time-averaged thrust and vertical force. Further, the proper parameters of dynamic twisting can also improve the propulsive efficiency in forward flight. Dynamic twisting also showed a superior ability in controlling the airflow separation over the wing surface and maintaining the stability of the LEV. The amplitudes of effective angle of attack associated with the highest peak thrust and the maximum thrust-to-power at different advanced ratios were also explored, and it was found that the amplitudes decrease with increasing advanced ratio. To improve the efficiency during larger advanced ratio, specific modifications to the pitching of the wing were proposed in this work. The research in this paper has promising implications for the bio-inspired flapping wing.

Recent Advance in the Study on 5‐Formylcytosine (f5C) RNA Modification

AbstractThe widespread involvement of 5‐formylcytosine f5C RNA in gene function regulation and its impact on crucial life processes like cell differentiation, embryonic development, and disease development underscores the significance of detecting this specific base modification. This detection holds great importance for basic epigenetics research and the early diagnosis and pathogenesis research of various diseases. This review aims to summarize recent research progress in f5C detection methods using selective chemical labeling, with the hope of aiding future research endeavors.

Temperature distribution in CrMnNi steel-Mg-PSZ functionally graded material during FAST/SPS

Within the scope of this study, the macroscopic temperature distribution in the steady-state temperature condition during Field Assisted Sintering Technique/Spark Plasma Sintering (FAST/SPS) in functionally graded materials (FGM) was investigated. The sample material exhibited a diameter of 50 mm and a thickness of 9 mm. The FGM samples were composed of steel X2CrMnNi 16-7-6 and Mg-PSZ ceramic (MgO partly stabilized ZrO2). By varying the sintering tool setup, a specific modification of the macroscopic temperature distribution within the sample material and the sintering tool was achieved. The aim of the modifications was to optimize the temperature gradients in order to allow for the simultaneous compaction of all FGM layers under ideal circumstances. This involves achieving enhanced ceramic densification without inducing steel melting. Finite element method (FEM) simulations were carried out to get information about the temperature distribution within the sample material and the sintering tool. Furthermore, for this propose, thermocouple temperature measurements were conducted at various measurement points on pre-sintered FGM within the specific sintering tool setup. Additionally, all findings regarding the temperature distribution within the sample material and the sintering tool were compared with results concerning the temperature distribution from the microstructural and mechanical characterization of the center and edge regions of the FGM samples. The FGM samples exhibited a significant increase in temperature from the center to the edge in the radial direction depending on the used sintering tool setup. While the FGM samples showed only minor vertical temperature gradients in the center, the highest vertical temperature gradients were determined at the edge of the FGM sample using an asymmetric sintering tool setup.

Developing Cheap but Useful Machine Learning-Based Models for Investigating High-Entropy Alloy Catalysts.

This work aims to address the challenge of developing interpretable ML-based models when access to large-scale computational resources is limited. Using CoMoFeNiCu high-entropy alloy catalysts as an example, we present a cost-effective workflow that synergistically combines descriptor-based approaches, machine learning-based force fields, and low-cost density functional theory (DFT) calculations to predict high-quality adsorption energies for H, N, and NHx (x = 1, 2, and 3) adsorbates. This is achieved using three specific modifications to typical DFT workflows including: (1) using a sequential optimization protocol, (2) developing a new geometry-based descriptor, and (3) repurposing the already-available low-cost DFT optimization trajectories to develop a ML-FF. Taken together, this study illustrates how cost-effective DFT calculations and appropriately designed descriptors can be used to develop cheap but useful models for predicting high-quality adsorption energies at significantly lower computational costs. We anticipate that this resource-efficient philosophy may be broadly relevant to the larger surface catalysis community.

The impact of genomic distance on enhancer-promoter interactions at the CFTR locus.

We identified and characterized multiple cell-type selective enhancers of the CFTR gene promoter in previous work and demonstrated active looping of these elements to the promoter. Here we address the impact of genomic spacing on these enhancer:promoter interactions and on CFTR gene expression. Using CRISPR/Cas9, we generated clonal cell lines with deletions between the -35 kb airway enhancer and the CFTR promoter in the 16HBE14o- airway cell line, or between the intron 1 (185 + 10 kb) intestinal enhancer and the promoter in the Caco2 intestinal cell line. The effect of these deletions on CFTR transcript abundance, as well as the 3D looping structure of the locus was investigated in triplicate clones of each modification. Our results indicate that both small and larger deletions upstream of the promoter can perturb CFTR expression and -35 kb enhancer:promoter interactions in the airway cells, though the larger deletions are more impactful. In contrast, the small intronic deletions have no effect on CFTR expression and intron 1 enhancer:promoter interactions in the intestinal cells, whereas larger deletions do. Clonal variation following a specific CFTR modification is a confounding factor particularly in 16HBE14o- cells.

Abstract B018: Triple CDK2/4/6 inhibition via p27 modulation: A novel approach to targeting cell cycle dysregulation

Abstract The dysregulation of cell cycle control is a hallmark of cancer and targeting cyclin-dependent kinases (CDKs) has emerged as a promising strategy in advanced breast cancer. The G1 CDKs are the recipients of the oncogenic signals that travel from the cell surface to the nucleus, and as such, selective inhibition of CDK2, CDK4 and CDK6 has been a goal in oncology for several decades. We present a novel approach involving modulation of the CDK interacting protein p27Kip1, to simultaneously inhibit CDK2/4/6. p27 is responsible for turning the G1 CDKs ON and OFF during periods of proliferation and quiescence, respectively, and this transition is mediated by a specific modification to p27 itself, by the tyrosine kinase BRK (Breast tumor Related Kinase). A naturally occurring ALTernatively spliced form of BRK, named ALT, can bind to p27, blocking BRK's association with and phosphorylation of p27. The binding of ALT to p27 locks both CDK4/6 and CDK2 into the OFF conformation, inhibiting activity of both kinases. p27’s association with CDK2 also prevents p27’s degradation. In in vitro kinase assays, inducible ALT expression in ER+ breast cancer cells, inhibited CDK4/6 and CDK2 activities simultaneously, resulting in durable growth arrest. As p27 associates preferentially with the G1 CDKs, the specificity of ALT for these kinases is achieved by p27 itself, thus avoiding off-target kinase interactions with the rest of the CDK kinome. We engineered IpY.20, a smaller, more stable variant of ALT, formulated within a liposome to increase its serum residence time and facilitate delivery into tumor cells. IpY.20 inhibits cell proliferation of HR+ and triple negative (TN) breast cancer, ovarian, uterine and NSCLC, preferentially over non-tumor cells, supporting its clinical utility. Unlike CDK4/6i and CDK2i which only cause cytostasis, IpY.20 arrests proliferation and induces cell death, demonstrating that p27 inhibition is a viable therapeutic approach to combat CDK4/6i-dependent drug resistance. In vivo, IpY.20 reduces tumor volume in palbociclib-resistant ER+ xenografts, TN xenografts and the MMTV-ErbB2 spontaneous breast cancer model. IpY.20 does not cause the adverse effects seen in the presence of CDK4/6i or CDK2i: IpY.20 does not cause neutropenia, and in combination with palbociclib, IpY.20 rescues the CDK4/6i-dependent decrease in neutrophils, suggesting better tolerability in patients. IpY.20 is detected in tumors within 15 min. of single dose I.V. administration and continues to be detectable for 24 h. IpY.20 is currently completing CMC process optimization, batch-to-batch consistency, and stability as it moves into the IND program. These findings underscore the significance of targeting CDK2, CDK4, and CDK6 simultaneously via p27 modulation as a potential therapy. Given the difficulties in inhibiting the CDK2/4/6 hub with small molecule CDKi (off-target kinase selectivity, adverse effects, lack of tumor cell killing), targeting p27 may provide advantages to the post-CDK4/6i setting. Citation Format: Grace Chen, Natalia Zisman, Krishna Allamneni, Stacy W Blain. Triple CDK2/4/6 inhibition via p27 modulation: A novel approach to targeting cell cycle dysregulation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B018.