Tertiary Structure Research Articles

Phytochemical inhibitors from Leucas aspera against the target proteins induced by Trichophyton mentagrophytes using computational techniques.

Dermatophytosis is a cutaneous infection that is able to degrade the keratinized tissues of the animal/human body, like skin, nails, and hair, causing chronic or subacute infection with the contact of some specific fungal strains. Trichophyton mentagrophytes are the most potential fungal pathogen causing dermatophytoses. The present study focuses on computationally based in silico antifungal activity of selected phytocompounds of Leucas aspera (Willd.) Link. against dermatophytic fungus, T. mentagrophytes. Validation and screening of derived phytocompounds is performed using Lipinski rule of five and toxicity test through Protox-II. Five target genes involved in dermatophytosis, induced by T. mentagrophytes are retrieved from the UniProt Database, and the corresponding proteins such as glucan 1,3-beta-glucosidase ARB_02797, Probable class II chitinase ARB_00204, squalene monooxygenase, actin, and ubiquitin are selected for in silico study. Three-dimensional structures of the target protein were computationally determined and validated through modeling tools and techniques due to the lack of validated protein structures in the database. Then, these proteins are used for in silico molecular docking through the AutoDock Vina tool to find out the promising phytocompounds. This study could be utilized in designing more effective drugs against T. mentagrophytes. Based on this work, a plant-based natural alternative can be added to the treatment of dermatophytosis rather than synthetic supplements.

Traditional cooking methods decreased the allergenicity of egg proteins.

Egg allergy is one of the most common food allergies globally. This study aimed to assess the impact of four traditional cooking methods on the allergenicity of egg proteins using a comprehensive strategy, including simulated gastrointestinal digestion in vitro, serology experiments, a rat basophilic leukemia (RBL)-2H3 cell degranulation model, and a passive cutaneous anaphylaxis (PCA) mice model, and the structure changes were detected by circular dichroism (CD)spectra and ultraviolet (UV) spectra. The results showed that the processed egg proteins were more readily digested compared to raw egg proteins. The serological experiments revealed a significant reduction in immunoglobulin E binding of egg proteins after thermal treatments (p<0.05), particularly after frying. Subsequently, the RBL-2H3 cell degranulation experiment demonstrated a marked decrease in the level of egg allergens-induced β-hexosaminidase release after cooking (p<0.05). Moreover, the results from the PCA mice model indicated that the increase in vascular permeability was effectively relieved in the treated groups, especially in frying group (p<0.05). Additionally, the α-helix and β-turn contents of processed egg proteins were significantly decreased (p<0.05) compared with native egg proteins. The UV spectra findings showed that all cooking treatments caused significant alterations in the tertiary structure, and fluorescence analysis indicated that cooking decreased the surface hydrophobicity of egg proteins. In conclusion, four traditional cooking methods reduced the allergenicity of egg proteins, particularly frying, and this reduction was associated with structural changes that could contribute to the destruction or masking of epitopes of egg allergens. PRACTICAL APPLICATION: Egg allergy has a serious impact on public health, and there is no ideal treatment method at present. This study demonstrated that four traditional cooking methods (boiling, steaming, baking, and frying) reduced the allergenicity of egg proteins, especially frying, and the results will provide a basis for the development of hypoallergenic egg products.

Tertiary lymphoid structures as a potential prognostic biomarker for combined hepatocellular-cholangiocarcinoma.

Combined hepatocellular-cholangiocarcinoma (cHCC-CCA), as a rare primary hepatic tumor, is challenging to accurately assess in terms of the clinical outcomes and prognostic risk factors in patients. This study aimed to clarify the function of tertiary lymphoid structure (TLS) status in predicting the outcome of cHCC-CCA and to preliminarily explore the possible mechanism of TLS formation. The TLSs, with different spatial distributions and densities, of 137 cHCC-CCA were quantified, and their association with prognosis was assessed by Cox regression and Kaplan-Meier analyses. We further validated TLS possible efficacy in predicting immunotherapy responsiveness in two cHCC-CCA case reports. TLS composition and its relationship to CXCL12 expression were analysed by fluorescent multiplex immunohistochemistry. A high intratumoural TLS score was correlated with prolonged survival, whereas a high TLS density in adjacent tissue indicated a worse prognosis in cHCC-CCA. Mature TLSs were related to favorable outcomes and showed more CD8 + T cells infiltrating tumor tissues. We further divided the cHCC-CCA patients into four immune grades by combining the peri-TLS and intra-TLS, and these grades were an independent prognostic factor. In addition, our reported cases suggested a potential value of TLS in predicting immunotherapy response in cHCC-CCA patients. Our findings suggested that CXCL12 expression in cHCC-CCA tissue was significantly correlated with TLS presence. The spatial distribution and density of TLSs revealing the characteristics of the cHCC-CCA immune microenvironment, significantly correlated with prognosis and provided a potential immunotherapy response biomarker for cHCC-CCA.

TCL1A-expressing B cells are critical for tertiary lymphoid structure formation and the prognosis of oral squamous cell carcinoma

BackgroundOral squamous cell carcinoma (OSCC) is a malignant tumor with a poor prognosis. Traditional treatments have limited effectiveness. Regulation of the immune response represents a promising new approach for OSCC treatment. B cells are among the most abundant immune cells in OSCC. However, the role of B cells in OSCC treatment has not been fully elucidated.MethodsSingle-cell RNA sequencing analysis of 13 tissues and 8 adjacent normal tissues from OSCC patients was performed to explore differences in B-cell gene expression between OSCC tissues and normal tissues. We further investigated the relationship between differentially expressed genes and the immune response to OSCC. We utilized tissue microarray data for 146 OSCC clinical samples and RNA sequencing data of 359 OSCC samples from The Cancer Genome Atlas (TCGA) to investigate the role of T-cell leukemia 1 A (TCL1A) in OSCC prognosis. Multiplex immunohistochemistry (mIHC) was employed to investigate the spatial distribution of TCL1A in OSCC tissues. We then investigated the effect of TCL1A on B-cell proliferation and trogocytosis. Finally, lentiviral transduction was performed to induce TCL1A overexpression in B lymphoblastoid cell lines (BLCLs) to verify the function of TCL1A.ResultsOur findings revealed that TCL1A was predominantly expressed in B cells and was associated with a better prognosis in OSCC patients. Additionally, we found that TCL1A-expressing B cells are located at the periphery of lymphatic follicles and are associated with tertiary lymphoid structures (TLS) formation in OSCC. Mechanistically, upregulation of TCL1A promoted the trogocytosis of B cells on dendritic cells by mediating the upregulation of CR2, thereby improving antigen-presenting ability. Moreover, the upregulation of TCL1A expression promoted the proliferation of B cells.ConclusionThis study revealed the role of B-cell TCL1A expression in TLS formation and its effect on OSCC prognosis. These findings highlight TCL1A as a novel target for OSCC immunotherapy.

HLA-A+ tertiary lymphoid structures with reactivated tumor infiltrating lymphocytes are associated with a positive immunotherapy response in esophageal squamous cell carcinoma.

Immune checkpoint blockade (ICB) therapy provides remarkable clinical benefits for multiple cancer types. However, the overall response rate to ICB therapy remains low in esophageal squamous cell carcinoma (ESCC). This study aimed to identify biomarkers of ICB therapy for ESCC and interrogate its potential clinical relevance. We investigated gene expression in 42 treatment-naïve ESCC tumor tissues and identified differentially expressed genes, tumor-infiltrating lymphocytes and immune-related genes signatures associated with differential immunotherapy responses. We systematically assessed the tumor microenvironment using the NanoString GeoMx digital spatial profiler, single-cell RNA-seq and multiplex immunohistochemistry in ESCC. Finally, we evaluated the associations between HLA-A-positive tertiary lymphoid structures (TLSs) and patients' responses to ICB in 60 ESCC patients. Tumor infiltrating B lymphocytes and several immune-related gene signatures, such as the antigen presenting machinery (APM) signature, are significantly elevated in ICB treatment responders. Multiplex immunohistochemistry identified the presence of HLA-A+ TLSs and showed that TLS-resident cells increasingly express HLA-A as TLSs mature. Most TLS-resident HLA-A+ cells are tumor-infiltrating T (TIL-T) or tumor-infiltrating B (TIL-B) lymphocytes. Digital spatial profiling of spatially distinct TIL-T lymphocytes and single-cell RNA-seq data from 60 ESCC tumor tissues revealed that CXCL13-expressing exhausted TIL-Ts inside TLSs are reactivated with elevated expression of the APM signature as TLSs mature. Finally, we demonstrated that HLA-A+ TLSs and their major cellular components, TIL-Ts and TIL-Bs, are associated with a clinical benefit from ICB treatment for ESCC. HLA-A+ TLSs are present in ESCC tumor tissues. TLS-resident TIL-Ts with elevated expression of the APM signature may be reactivated. HLA-A+ TLSs and their major cellular components, TIL-Ts and TIL-Bs, may serve as biomarkers for ICB-treated ESCC patients.

Comprehensive analysis of the bZIP gene family in ‘Duli’ pear (Pyrus betulifolia Bunge): Insights for research on abiotic stress, especially drought, in pear

Plants are unavoidably subjected to various abiotic stresses, such as drought, salt, and cold, during their growth and development. Drought, accompanied by water shortages in North China, has become an environmental factor seriously affecting agriculture production. Basic leucine zipper (bZIP) is one of the most diverse transcription factor families in plants. It participates in the regulation of many physiological processes and plays an important role in plants’ response to abiotic stress. In this study, for the first time, 88 bZIP family members (PbbZIPs) in ‘Duli’ pear (Pyrus betulifolia Bunge) were identified using both hidden Markov model (HMM) and basic local alignment search tool (BLAST) methods. A phylogenetic tree of all the PbbZIPs members was constructed. The physicochemical properties, subcellular localisation, chromosomal location, gene structure, conserved motifs and domains, secondary and tertiary structures, and cis-acting promoter elements of the 88 family members were comprehensively analysed. The results indicated that PbbZIPs may play relevant roles in stress response in ‘Duli’. Therefore, we determined the expressions of all PbbZIPs in ‘Duli’ seedlings under drought, salt, and cold conditions, and in seedlings treated with stress-related hormones such as abscisic acid (ABA), salicylic acid (SA), methyl jasmonate (MeJA), and melatonin (MT). PbbZIPs responsive to various abiotic stresses and hormones were screened. In addition, we determined the expression patterns of 12 PbbZIP genes that strongly responded to both drought and ABA in ‘Duli’ tissue culture plants treated with different concentrations of PEG4000 at different time points. Finally, we selected three key genes (PbbZIP65, PbbZIP77 and PbbZIP81) that may play a major role in drought resistance in ‘Duli’, and preliminarily identified their function through the VIGS method, finding that PbbZIP77 could positively regulate the drought resistance of ‘Duli’ seedlings, while PbbZIP65/81 could negatively regulate the drought resistance of ‘Duli’ seedlings. This study not only broadens our knowledge about the bZIP gene family in ‘Duli’ pear, but also provides notable guidance for further functional research on PbbZIPs in abiotic stress, especially drought resistance, in the pear plant.

Abstract PR010: Exploratory analysis of tumor tertiary lymphoid structures using a novel artificial intelligence–based approach in patients with muscle-invasive urothelial carcinoma from the CheckMate 274 trial

Abstract Introduction: The phase 3 randomized CheckMate 274 trial in patients (pts) with high-risk muscle-invasive urothelial carcinoma (MIUC) demonstrated improved disease-free survival (DFS) with nivolumab (NIVO) vs placebo (PBO) in intent-to-treat (ITT) and tumor programmed death ligand 1 expression ≥1% pts (primary endpoints). With extended follow-up, continued efficacy was observed, and immature overall survival favored NIVO in both populations. Extending from previous exploratory tumor biomarker analyses (Necchi et al. ESMO 2022), we conducted a post hoc analysis of tertiary lymphoid structures (TLS), which are ectopic lymphoid structures often found within the solid tumor microenvironment. A subset of TLS has been associated with improved outcomes to neoadjuvant immunotherapy in UC. Using artificial intelligence (AI)–based algorithms, we explored the biomarker utility of TLS from CheckMate 274. Methods: We used a reproducible AI-based algorithm to identify and classify TLS in situ using hematoxylin and eosin (H&amp;E) images from pretreatment tumor tissue specimens (n=272/699 treated pts). Images were scored for the presence or absence of lymphoid aggregate (LA), immature TLS (iTLS), and mature TLS (mTLS) and scored as either positive (+) or negative (−) for the respective TLS category. Relationships between TLS categories and DFS were explored (minimum follow-up, 5.9 months). Results: Most tumors exhibited some form of LA/TLS (245/272; 90.1%), with a higher incidence of LA (237/272; 87.1%) compared with mTLS (89/272; 32.7%). There was heterogeneity of TLS within the tumors, with most tumors demonstrating more than 1 form of TLS (198/272; 72.8%). In the PBO arm, median DFS was modest for most TLS categories (5.7–8.3 months) but was substantially improved for mTLS+ tumors (22.1 months; 95% CI, 5.4–not estimable [NE]); n=41), representing a 2.7- to 3.9-fold increase relative to any other category. In contrast, treatment with NIVO resulted in 2.1- to 4.0-fold improvements in median DFS relative to PBO across all but the mTLS+ category (22.9 months; 95% CI,11.9–NE; n=48), which was similar to the PBO arm. The largest increases in median DFS (95% CI) for NIVO vs PBO were observed for LA+ (27.6 [16.1–NE; n=114] vs 8.3 [5.6–11.9; n=123] months; HR, 0.56; 95% CI, 0.39–0.80; P=0.0015), iTLS+ (27.0 [16.1–NE; n=91] vs 8.2 [5.4–21.2; n=105] months; HR, 0.61; 95% CI, 0.41–0.91; P=0.0143), and mTLS− tumors (24.6 [11.4–NE; n=81] vs 6.1 [5.2–8.5; n=102] months; HR, 0.53; 95% CI, 0.36–0.78; P=0.0012]). Conclusion: These results suggest that a novel AI-based TLS maturity classification using H&amp;E images is feasible to investigate the tumor microenvironment in MIUC, with potential broad application in other studies. Citation Format: Johannes Alfred Witjes, Matthew D. Galsky, Jürgen E. Gschwend, Dean F. Bajorin, Matthew I. Milowsky, Gautam Jha, Roger Li, Jun Li, Vanessa Matos-Cruz, Scott Ely, George Lee, Vipul Baxi, Joshua Zhang, Saurabh Gupta, Justin M. David. Exploratory analysis of tumor tertiary lymphoid structures using a novel artificial intelligence–based approach in patients with muscle-invasive urothelial carcinoma from the CheckMate 274 trial [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr PR010.

E3 ubiquitin ligase UBR5 promotes gemcitabine resistance in pancreatic cancer by inducing O-GlcNAcylation-mediated EMT via destabilization of OGA.

Pancreatic cancer (PC) is among the deadliest malignancies, with an extremely poor diagnosis and prognosis. Gemcitabine (GEM) remains the first-line drug for treating PC; however, only a small percentage of patients benefit from current immunotherapies or targeted therapies. Resistance to GEM is prevalent and affects long-term survival. We found that ubiquitin-protein ligase E3 module N-recognition 5 (UBR5) is a therapeutic target against GEM resistance. UBR5 was markedly upregulated in clinical GEM-resistant PC samples and GEM-resistant PC cells. UBR5 knockdown markedly increased GEM sensitivity in GEM-resistant PC cell lines. UBR5-mediated GEM resistance was accompanied by activation of epithelial-mesenchymal transition (EMT) and could be mitigated by inhibiting EMT. Further analysis revealed that UBR5 promoted GEM resistance in PC cells by enhancing O-GlcNAcylation-mediated EMT. In addition, UBR5 knockdown resulted in increased O-GlcNAase (OGA) levels, an essential negatively regulated enzyme in the O-GlcNAcylation process. We identified a negative association between OGA and UBR5 levels, which further supported the hypothesis that O-GlcNAcylation-mediated GEM resistance induced by UBR5 is OGA-dependent in PC cells. Mechanistic studies revealed that UBR5 acts as an E3 ubiquitin ligase of OGA and regulates O-GlcNAcylation by binding and modulating OGA, facilitating its degradation and ubiquitination. Additionally, high-throughput compound library screening using three-dimensional protein structure analysis and drug screening identified a Food and Drug Administration drug, Y-39983 dihydrochloride, as a potent GEM sensitiser and UBR5 inhibitor. The combination of Y-39983 dihydrochloride and GEM attenuated tumour growth in a mouse xenograft tumour model. Collectively, these data demonstrated that UBR5 plays a pivotal role in the sensitisation of PC to GEM and provides a potential therapeutic strategy to overcome GEM resistance.

Backbone Modification in a Protein Hydrophobic Core.

Targeted protein backbone modification can recreate tertiary structures reminiscent of folds found in nature on artificial scaffolds with improved biostability. Incorporation of altered monomers in such entities is typically limited to sites distant from the hydrophobic core to avoid potential disruptions to folding. This is limiting, as it is advantageous in some applications to incorporate artificial connectivity at buried sites. Here, we report an examination of protein backbone modification targeted specifically to hydrophobic core positions and its impacts on tertiary folded structure and fold stability. Different artificial monomer types are placed at core, core-flanking, or solvent-exposed positions in a compact three-helix protein. Effects on structure and folding energetics are assessed by NMR spectroscopy and biophysical methods. Results show that artificial residues can be well accommodated in the hydrophobic core of a defined tertiary fold, with effects on stability only modestly larger than identical changes at solvent-exposed sites. Collectively, these results provide new insights into folding behavior of protein-like artificial chains as well as strategies for the design of such molecules.

The Rapid Evolution of De Novo Proteins in Structure and Complex.

Recent studies in the rice genome-wide have established that de novo genes, evolving from non-coding sequences, enhance protein diversity through a stepwise process. However, the pattern and rate of their evolution in protein structure over time remain unclear. Here, we addressed these issues within a surprisingly short evolutionary timescale (<1 million years for 97% of Oryza de novo genes) with comparative approaches to gene duplicates. We found that de novo genes evolve faster than gene duplicates in the intrinsic disordered regions (IDRs, such as random coils), secondary structure elements (such as α-helix and β-strand), hydrophobicity, and molecular recognition features (MoRFs). In de novo proteins, specifically, we observed an 8-14% decay in random coils and IDR lengths and a 2.3-6.5% increase in structured elements, hydrophobicity, and MoRFs, per million years on average. These patterns of structural evolution align with changes in amino acid composition over time as well. We also revealed higher positive charges but smaller molecular weights for de novo proteins than duplicates. Tertiary structure predictions showed that most de novo proteins, though not typically well-folded on their own, readily form low-energy and compact complexes with other proteins facilitated by extensive residue contacts and conformational flexibility, suggesting a faster-binding scenario in de novo proteins to promote interaction. These analyses illuminate a rapid evolution of protein structure in de novo genes in rice genomes, originating from non-coding sequences, highlighting their quick transformation into active, protein complex-forming components within a remarkably short evolutionary timeframe.

AlphaFind: discover structure similarity across the proteome in AlphaFold DB.

AlphaFind is a web-based search engine that provides fast structure-based retrieval in the entire set of AlphaFold DB structures. Unlike other protein processing tools, AlphaFind is focused entirely on tertiary structure, automatically extracting the main 3D features of each protein chain and using a machine learning model to find the most similar structures. This indexing approach and the 3D feature extraction method used by AlphaFind have both demonstrated remarkable scalability to large datasets as well as to large protein structures. The web application itself has been designed with a focus on clarity and ease of use. The searcher accepts any valid UniProt ID, Protein Data Bank ID or gene symbol as input, and returns a set of similar protein chains from AlphaFold DB, including various similarity metrics between the query and each of the retrieved results. In addition to the main search functionality, the application provides 3D visualizations of protein structure superpositions in order to allow researchers to instantly analyze the structural similarity of the retrieved results. The AlphaFind web application is available online for free and without any registration at https://alphafind.fi.muni.cz.

4-nitroquinoline 1-oxide-induced oral epithelial lesions exhibit time- and stage-dependent changes in the tumor immune microenvironment

Oral tongue squamous cell carcinoma (OTSCC) is the most common cancer of the oral cavity and is associated with high morbidity due to local invasion and lymph node metastasis. Tumor infiltrating lymphocytes (TILs) are associated with good prognosis in oral cancer patients and dictate response to treatment. Ectopic sites for immune activation in tumors, known as tertiary lymphoid structures (TLS), and tumor-associated high-endothelial venules (TA-HEVs), which are specialized lymphocyte recruiting vessels, are associated with a favorable prognosis in OSCC. Why only some tumors support the development of TLS and HEVs is poorly understood. In the current study we explored the infiltration of lymphocyte subsets and the development of TLS and HEVs in oral epithelial lesions using the 4-nitroquinoline 1-oxide (4NQO)-induced mouse model of oral carcinogenesis. We found that the immune response to 4NQO-induced oral epithelial lesions was dominated by T cell subsets. The number of T cells (CD4+, FoxP3+, and CD8+), B cells (B220+) and PNAd+ HEVs increased from the earliest to the latest endpoints. All the immune markers increased with the severity of the dysplasia, while the number of HEVs and B cells further increased in SCCs. HEVs were present already in early-stage lesions, while TLS did not develop at any timepoint. This suggests that the 4NQO model is applicable to study the dynamics of the tumor immune microenvironment at early phases of oral cancer development, including the regulation of TA-HEVs in OTSCC.

A unique Ca2+-inhibited C-type lectin in shrimp Fenneropenaeuschinensis

C-type lectins (CTLs) are glycan-binding pattern recognition receptors (PRRs) that can bind to carbohydrates on pathogen surfaces, triggering immune responses in shrimp innate immunity. In this study, a unique Ca2+-inhibited CTL named FcLec was identified and characterized in Chinese shrimp Fenneropenaeus chinensis. The full-length cDNA sequence of FcLec was 976 bp (GenBank accession number KU361826), with a 615 bp open reading frame (ORF) encoding 204 amino acids. FcLec possesses a C-type lectin-like domain (CTLD) containing four conserved cysteines (Cys105, Cys174, Cys192, and Cys200) and two sugar-binding site structures (QPD and LNP). The tertiary structure of FcLec deduced revealed three α-helices and eight β-pleated sheets. The mRNA expression levels of FcLec in hemocytes and the hepatopancreas were markedly elevated after stimulation with Vibrio anguillarum and white spot syndrome virus (WSSV). The recombinant FcLec protein exhibited Ca2+-independent hemagglutination and bacterial agglutination, but these activities were observed only in the presence of EDTA to chelate metal ions. These findings suggest that FcLec plays important and functionally distinct roles in the shrimp's innate immune response to bacteria and viruses, enriching the current understanding of the relationship between CTL activity and Ca2+ in invertebrates.

Novel polymorphisms and genetic studies of the shadow of prion protein gene (SPRN) in pheasants

BackgroundPrion diseases in mammals are caused by the structural conversion of the natural prion protein (PrPC) to a pathogenic isoform, the “scrapie form of prion protein (PrPSc).” Several studies reported that the shadow of prion protein (Sho), encoded by the shadow of prion protein gene (SPRN), is involved in prion disease development by accelerating the conformational conversion of PrPC to PrPSc. Until now, genetic polymorphisms of the SPRN gene and the protein structure of Sho related to fragility to prion disease have not been investigated in pheasants, which are a species of poultry.MethodsHere, we identified the SPRN gene sequence by polymerase chain reaction (PCR) and compared the SPRN gene and Sho protein sequences among various prion disease-susceptible and -resistant species to identify the distinctive genetic features of pheasant Sho using Clustal Omega. In addition, we investigated genetic polymorphisms of the SPRN gene in pheasants and analyzed genotype, allele, and haplotype frequencies, as well as linkage disequilibrium among the genetic polymorphisms. Furthermore, we used in silico programs, namely Mutpred2, MUpro and AMYCO, to investigate the effect of non-synonymous single nucleotide polymorphisms (SNPs). Finally, the predicted secondary and tertiary structures of Sho proteins from various species were analyzed by Alphafold2.ResultsIn the present study, we reported pheasant SPRN gene sequences for the first time and identified a total of 14 novel SNPs, including 7 non-synonymous and 4 synonymous SNPs. In addition, the pheasant Sho protein sequence showed 100% identity with the chicken Sho protein sequence. Furthermore, amino acid substitutions were predicted to affect the hydrogen bond distribution in the 3D structure of the pheasant Sho protein.ConclusionTo the best of our knowledge, this is the first report of the genetic and structural features of the pheasant SPRN gene.

Systematic dissection of tumor-normal single-cell ecosystems across a thousand tumors of 30 cancer types

The complexity of the tumor microenvironment poses significant challenges in cancer therapy. Here, to comprehensively investigate the tumor-normal ecosystems, we perform an integrative analysis of 4.9 million single-cell transcriptomes from 1070 tumor and 493 normal samples in combination with pan-cancer 137 spatial transcriptomics, 8887 TCGA, and 1261 checkpoint inhibitor-treated bulk tumors. We define a myriad of cell states constituting the tumor-normal ecosystems and also identify hallmark gene signatures across different cell types and organs. Our atlas characterizes distinctions between inflammatory fibroblasts marked by AKR1C1 or WNT5A in terms of cellular interactions and spatial co-localization patterns. Co-occurrence analysis reveals interferon-enriched community states including tertiary lymphoid structure (TLS) components, which exhibit differential rewiring between tumor, adjacent normal, and healthy normal tissues. The favorable response of interferon-enriched community states to immunotherapy is validated using immunotherapy-treated cancers (n = 1261) including our lung cancer cohort (n = 497). Deconvolution of spatial transcriptomes discriminates TLS-enriched from non-enriched cell types among immunotherapy-favorable components. Our systematic dissection of tumor-normal ecosystems provides a deeper understanding of inter- and intra-tumoral heterogeneity.

Filled Elastomers: Mechanistic and Physics-Driven Modeling and Applications as Smart Materials

Elastomers are made of chain-like molecules to form networks that can sustain large deformation. Rubbers are thermosetting elastomers that are obtained from irreversible curing reactions. Curing reactions create permanent bonds between the molecular chains. On the other hand, thermoplastic elastomers do not need curing reactions. Incorporation of appropriated filler particles, as has been practiced for decades, can significantly enhance mechanical properties of elastomers. However, there are fundamental questions about polymer matrix composites (PMCs) that still elude complete understanding. This is because the macroscopic properties of PMCs depend not only on the overall volume fraction (ϕ) of the filler particles, but also on their spatial distribution (i.e., primary, secondary, and tertiary structure). This work aims at reviewing how the mechanical properties of PMCs are related to the microstructure of filler particles and to the interaction between filler particles and polymer matrices. Overall, soft rubbery matrices dictate the elasticity/hyperelasticity of the PMCs while the reinforcement involves polymer–particle interactions that can significantly influence the mechanical properties of the polymer matrix interface. For ϕ values higher than a threshold, percolation of the filler particles can lead to significant reinforcement. While viscoelastic behavior may be attributed to the soft rubbery component, inelastic behaviors like the Mullins and Payne effects are highly correlated to the microstructures of the polymer matrix and the filler particles, as well as that of the polymer–particle interface. Additionally, the incorporation of specific filler particles within intelligently designed polymer systems has been shown to yield a variety of functional and responsive materials, commonly termed smart materials. We review three types of smart PMCs, i.e., magnetoelastic (M-), shape-memory (SM-), and self-healing (SH-) PMCs, and discuss the constitutive models for these smart materials.

Predicting Antimicrobial Peptides Using ESMFold-Predicted Structures and ESM-2-Based Amino Acid Features with Graph Deep Learning.

Currently, antimicrobial resistance constitutes a serious threat to human health. Drugs based on antimicrobial peptides (AMPs) constitute one of the alternatives to address it. Shallow and deep learning (DL)-based models have mainly been built from amino acid sequences to predict AMPs. Recent advances in tertiary (3D) structure prediction have opened new opportunities in this field. In this sense, models based on graphs derived from predicted peptide structures have recently been proposed. However, these models are not in correspondence with state-of-the-art approaches to codify evolutionary information, and, in addition, they are memory- and time-consuming because depend on multiple sequence alignment. Herein, we presented a framework to create alignment-free models based on graph representations generated from ESMFold-predicted peptide structures, whose nodes are characterized with amino acid-level evolutionary information derived from the Evolutionary Scale Modeling (ESM-2) models. A graph attention network (GAT) was implemented to assess the usefulness of the framework in the AMP classification. To this end, a set comprised of 67,058 peptides was used. It was demonstrated that the proposed methodology allowed to build GAT models with generalization abilities consistently better than 20 state-of-the-art non-DL-based and DL-based models. The best GAT models were developed using evolutionary information derived from the 36- and 33-layer ESM-2 models. Similarity studies showed that the best-built GAT models codified different chemical spaces, and thus they were fused to significantly improve the classification. In general, the results suggest that esm-AxP-GDL is a promissory tool to develop good, structure-dependent, and alignment-free models that can be successfully applied in the screening of large data sets. This framework should not only be useful to classify AMPs but also for modeling other peptide and protein activities.

Insertion-Deletion Events Are Depleted in Protein Regions with Predicted Secondary Structure.

A fundamental goal in evolutionary biology and population genetics is to understand how selection shapes the fate of new mutations. Here, we test the null hypothesis that insertion-deletion (indel) events in protein-coding regions occur randomly with respect to secondary structures. We identified indels across 11,444 sequence alignments in mouse, rat, human, chimp, and dog genomes and then quantified their overlap with four different types of secondary structure-alpha helices, beta strands, protein bends, and protein turns-predicted by deep-learning methods of AlphaFold2. Indels overlapped secondary structures 54% as much as expected and were especially underrepresented over beta strands, which tend to form internal, stable regions of proteins. In contrast, indels were enriched by 155% over regions without any predicted secondary structures. These skews were stronger in the rodent lineages compared to the primate lineages, consistent with population genetic theory predicting that natural selection will be more efficient in species with larger effective population sizes. Nonsynonymous substitutions were also less common in regions of protein secondary structure, although not as strongly reduced as in indels. In a complementary analysis of thousands of human genomes, we showed that indels overlapping secondary structure segregated at significantly lower frequency than indels outside of secondary structure. Taken together, our study shows that indels are selected against if they overlap secondary structure, presumably because they disrupt the tertiary structure and function of a protein.

A Genome-Wide Analysis of the BAM Gene Family and Identification of the Cold-Responsive Genes in Pomegranate (Punica granatum L.)

Beta-amylases (BAMs, EC 3.2.1.2), belonging to a multigene family, play a pivotal role in starch breakdown and are also involved in hormonal and stress responses, notably to cold stress. Pomegranate trees (Punica granatum L.) are adapted to warm climates and are sensitive to cold temperatures. In this study, we analyzed eight PgBAM genes from the pomegranate genome dataset. These members unevenly distributed across chromosomes and were categorized into four groups based on their orthologous members. The motif composition was highly consistent among most members. In contrast, exon numbers and arrangements were conserved within groups or subgroups, whereas significant diversity was observed between different groups. A syntenic analysis revealed that three PgBAM members (PgBAM1/4/5) showed a total of 11 syntenic relationships with the BAM members from Arabidopsis, kiwifruit, and Chinese white pear, respectively. Promoter binding motif prediction suggested potential roles for PgBAMs’ genes in light, stress, hormones, and development signaling. Gene expression indicated that PgBAM4 was predominantly expressed in leaves, PgBAM7 in flowers, and PgBAM8 in roots and leaves and during fruit ripening, particularly in pericarp development. A transcriptome analysis identified the starch and sucrose metabolism pathway (map00500) as a key factor in the cold stress response of cold-sensitive cultivar ‘Tunisia’ seedlings. PgBAM4 exhibited remarkable expression and was closely associated with the cold-responsive BAM genes, characterized by a closer phylogenetic relationship, conserved catalytic residues, and similar secondary and tertiary structures. Moreover, the differences in soluble sugar levels and PgBAM4 expression were closely associated with the varying cold stress resistance observed between ‘Tunisia’ and ‘Sanbai’ seedlings. Furthermore, yeast one-hybrid assays confirmed that PgCBF7, a critical transcription factor for enhancing freezing tolerance, binds to the promoter region of PgBAM4. Our findings provide a systematic overview of the PgBAM gene family and shed new light on the regulatory mechanisms underlying cold stress tolerance in pomegranate.

Effect of cold plasma treatment of sunflower seed protein modification on its structural and functional properties and its mechanism

Sunflower seed protein is a protein rich in amino acids, but its structure is more difficult to be applied due to its poor functional properties. The purpose of this study is to investigate the effect of cold plasma treatment on the structural and functional properties of sunflower seed protein. The results showed that the energetic particles and active substances generated by cold plasma treatment could improve the spatial structure of the protein by bombardment/etching and oxidation, respectively, which led to the change of its secondary structure: α-helices and random coil were transformed to β-sheet and β-turns, and the tertiary structure: the fluorescence intensity was increased, the roughness was decreased, which finally led to the increase of the zeta-potential, the solubility increased to 7.341, and the emulsion increased to 7.341, which led to the increase of the solubility and the fluorescence intensity. to 7.341, and the emulsion activity index and emulsion stability index increased from 1.842 m2/g, 107.5 min to 3.3887 m2/g, 171.4 min. These changes were attributed to the fact that the cold plasma treatment altered the structure of sunflower proteins from tightly hydrophobic to loosely hydrophilic, with the exposure of the internal hydrophobic groups and the protein aggregation from large to small. This provides a theoretical basis for its application in food.