Tertiary Structure Research Articles

Linking Protein Stability to Pathogenicity: Predicting Clinical Significance of Single-Missense Mutations in Ocular Proteins Using Machine Learning.

Understanding the effect of single-missense mutations on protein stability is crucial for clinical decision-making and therapeutic development. The impact of these mutations on protein stability and 3D structure remains underexplored. Here, we developed a program to investigate the relationship between pathogenic mutations with protein unfolding and compared seven machine learning (ML) models to predict the clinical significance of single-missense mutations with unknown impacts, based on protein stability parameters. We analyzed seven proteins associated with ocular disease-causing genes. The program revealed an R-squared value of 0.846 using Decision Tree Regression between pathogenic mutations and decreased protein stability, with 96.20% of pathogenic mutations in RPE65 leading to protein instability. Among the ML models, Random Forest achieved the highest AUC (0.922) and PR AUC (0.879) in predicting the clinical significance of mutations with unknown effects. Our findings indicate that most pathogenic mutations affecting protein stability occur in alpha-helices, beta-pleated sheets, and active sites. This study suggests that protein stability can serve as a valuable parameter for interpreting the clinical significance of single-missense mutations in ocular proteins.

Analysis of multi-disulfide bridges for insulin aspart by stepwise reduction and differentiated alkylation.

Disulfide bridge, an important post-translation modification in protein, plays a key role in stabilizing three-dimensional structure of proteins, maintaining correct folded conformation, and thus regulating the biological activities. Disulfide bridge assignment is essential to understand the essence of life process and to develop protein pharmaceutical. In this study, a novel method termed as stepwise reduction and differentiated alkylation (SRDA) was developed analyzing disulfide connectivity for proteins. As a demonstration, three disulfide bridges in insulin aspart were successfully characterized using this SRDA method combined with LC-HRMSn. Firstly, tris (2-carboxyethyl) phosphine (TCEP) was used to partially reduce disulfide bridges with N-ethylmaleimide (NEM) used to block the generated free thiol. Then, dithiothreitol (DTT) was used to reduce the rest disulfide bonds with iodoacetamide (IAM) used to block the newly generated free thiol. After that, an LC-HRMSn method was established to assign disulfide connectivity for an insulin aspart study material, based on the different mass shifts arising from differentiated alkylation. Moreover, this approach allows for the quantitative analysis of various disulfide bond pairings, which can be applied to studies on the consistency and stability among different batches of samples. The results show that SRDA is a valuable tool for reliable quality control and quality assessment of disulfide-rich proteins such as insulin analogues.

Viral RNA Interactome: The Ultimate Researcher’s Guide to RNA–Protein Interactions

RNA molecules in the cell are bound by a multitude of RNA-binding proteins (RBPs) with a variety of regulatory consequences. Often, interactions with these RNA-binding proteins are facilitated by the complex secondary and tertiary structures of RNA molecules. Viral RNAs especially are known to be heavily structured and interact with many RBPs, with roles including genome packaging, immune evasion, enhancing replication and transcription, and increasing translation efficiency. As such, the RNA–protein interactome represents a critical facet of the viral replication cycle. Characterization of these interactions is necessary for the development of novel therapeutics targeted at the disruption of essential replication cycle events. In this review, we aim to summarize the various roles of RNA structures in shaping the RNA–protein interactome, the regulatory roles of these interactions, as well as up-to-date methods developed for the characterization of the interactome and directions for novel, RNA-directed therapeutics.

Multi-frequency power ultrasound (MFPU) pretreatment of crayfish (Procambarus clarkii): Effect on the enzymatic hydrolysis process and subsequent Maillard reaction

Crayfish, as a new consumer trend, has become one of the most popular freshwater aquatic foods in China, and its processed product output has been increasing year by year. In this study, the effect of multi-frequency power ultrasound (MFPU) pretreatment in various working modes including mono-, dual- and tri-frequency on the enzymatic hydrolysis and Maillard reaction of crayfish was investigated. Additionally, the underlying mechanism was also explored. Results showed that the degree of hydrolysis (DH) of crayfish was 23.89 %, while it increased to 40.78 % after MFPU pretreatment at 20/40 kHz dual-frequency, indicating that MFPU pretreatment significantly (p < 0.05) enhanced the enzymatic hydrolysis of crayfish. The crayfish protein after MFPU pretreatment exhibited a decrease in α-helix and increase in β-fold content, and the occurrence in the unfolding of the protein structure and alteration of tertiary structure. According to scanning electron microscopy (SEM) and atomic force microscopy (AFM) analyses, the MFPU pretreatment resulted in the fragmentation of crayfish protein. During the analyses of subsequent Maillard reaction, the composition and quantity of volatiles detected by the electronic nose and GC–MS indicated that MFPU pretreatment enhanced flavor of the Maillard reaction products. In conclusion, MFPU pretreatment is a promising technology for improving the degree of hydrolysis of crayfish protein and enhancing the flavor of Maillard reaction products.

Genetic Diversity and Recombination Analysis of Canine Parvoviruses Prevalent in Central and Eastern China, from 2020 to 2023

Canine parvovirus type-2 (CPV-2), the primary causative agent of serious canine enteric diseases, is highly contagious and associated with high fatality rates worldwide. To comprehend the current emergence of CPV-2 in central and eastern China, 130 rectal swabs from domestic or stray dogs with gastroenteritis symptoms were collected during 2020–2023. A total of 118 positive samples were detected via polymerase chain reaction, and further used to amplify and sequence the VP2 gene. Sequence analysis of the deduced amino acids of VP2 protein indicated that CPV-2c was the most prevalent variant (n = 106, 89.83%), followed by the novel CPV-2a (n = 10, 8.47%) and CPV-2b (n = 2, 1.69%) variants. The VP2 protein from the obtained and reference strains showed 86.95% (AH2103 and HB2108) to 99.94% identity. Based on the nine predicted recombination events, some prevalent CPV-2c strains were highly similar to previously isolated strains, indicating their complex evolution and recombination. The predicted analysis suggested that mutations in the antigen epitope (Val219Ile, Phe267Tyr, and Asn426Glu) and other mutations (Met87Leu, Ile101Thr, and Ser297Ala) affect the tertiary structure of the VP2 protein. This research will help us understand the recent evolution and mutation of Chinese CPV-2 and provide suggestions for updating the CPV-2 vaccine.

Combination therapy with immune agonists and senescence-inducing agents delivers promise for immunotherapeutic success in pancreatic cancer

Reprogramming the PDAC tumor microenvironment via nanoparticles may help to induce tertiary lymphoid structures that support antitumor immunity.

Unravelling the Complexity of Amyloid Peptide Core Interfaces.

Amyloids, large intermolecular sandwiched β-sheet structures, underlie several protein misfolding diseases but have also been shown to have functional roles and can be a basis for designing smart and responsive nanomaterials. Short segments of proteins, called aggregation-prone regions (APRs), have been identified that nucleate amyloid formation. Here we present the database of 173 APR crystal structures currently available in the PDB, and a tool, ACW, for analyzing their topologies and the 267 inter-β-sheet interfaces of zipper regions assigned in these structures. We defined a new descriptor of zipper interfaces, the surface detail index (SDi), which quantifies the intertwining between the side chains of both β-sheets of the zipper, an important factor for the molecular recognition and self-assembly of these mesostructures. This allowed a comparative analysis of the zipper interfaces and identification of 6 clusters with different intertwining, steric fit, and size characteristics using three complementary descriptors, SDi, shape complementarity, and buried surface area. 60% of the APR structures are formed by parallel β-sheets, of which 52% belong to the topological class 1. This could be explained by the better fit and a deeper entanglement of the zipper regions of the parallel structures than of the antiparallel structures, as the analysis showed that both their shape complementarity (0.79 vs 0.70) and SDi (1.53 vs 1.32) were higher. The higher abundance of certain residues (Asn and Gln in parallel and Leu and Ala in antiparallel β-sheets) can be explained by their ability to form different ladder-like secondary interaction patterns within β-sheets. Analogous to the hierarchy of protein structure, we interpreted the primary, secondary, tertiary, and quaternary structure levels of APRs revealing different characteristics of the zipper regions for both parallel and antiparallel β-sheet structures, which may provide clues to the structural conditions of amyloid core formation and the rational design of amyloid polymorphs.

Complement activation by IgG subclasses is governed by their ability to oligomerize upon antigen binding.

Complement activation through antibody-antigen complexes is crucial in various pathophysiological processes and utilized in immunotherapies to eliminate infectious agents, regulatory immune cells, or cancer cells. The tertiary structures of the four IgG antibody subclasses are largely comparable, with the most prominent difference being the hinge regions connecting the Fab and Fc domains, providing them with unique structural flexibility. Complement recruitment and activation depend strongly on IgG subclass, which is commonly rationalized by differences in hinge flexibility and the respective affinities for C1, the first component of the classical complement pathway. However, a unifying mechanism of how these different IgG subclass properties combine to modulate C1 activation has not yet been proposed. We here demonstrate that complement activation is determined by their varying ability to form IgG oligomers on antigenic surfaces large enough to multivalently bind and activate C1. We directly visualize the resulting IgG oligomer structures and characterize their distribution by means of high-speed atomic force microscopy, quantify their complement recruitment efficiency from quartz crystal microbalance experiments, and characterize their ability to activate complement on tumor cell lines as well as in vesicle-based complement lysis assays. We present a mechanistic model of the multivalent interactions that govern C1 binding to IgG oligomers and use it to extract kinetic rate constants from real-time interaction data from which we further calculate equilibrium dissociation constants. Together, we provide a comprehensive view on the parameters that govern complement activation by the different IgG subclasses, which may inform the design of future antibody therapies.

Genome-wide identification and expression analysis of the OSC gene family in Platycodon grandiflorus

Platycodon grandiflorus stands as one of the most extensively utilized traditional Chinese medicinal herbs, with triterpenoids and their derivatives serving as its primary medicinal components. Oxido squalene cyclase (OSC), serving as a crucial enzyme in the triterpenoid synthesis pathway, has the capability to enzymatically generate significant quantities of sterols and triterpenoid intermediates. While the OSC gene family has been identified in numerous species, bioinformatics research on this family remains scant. Presently, the specific members of this gene family in Platycodon grandiflorus have yet to be definitively determined. In this study, we successfully identified a total of 15 PgOSC genes within the genome of Platycodon grandiflorus by conducting homology comparisons. These genes were discovered to be unevenly distributed across the five chromosomes of the species, organized in the form of gene clusters. Subsequently, we conducted a thorough analysis of the OSC gene family’s evolutionary relationship by constructing a phylogenetic tree. Other characteristics of PgOSC family members, including gene structure, conserved motifs, protein three-dimensional structure, subcellular localization, and cis-acting elements were thoroughly characterized. Furthermore, We analyzed the expression of PgOSC gene in different tissues of Platycodon grandiflorus by qRT-PCR, and found that the expression of PgOSC genes in root was higher than that in stem and leaf. Upon comparing the effects of salt, heat, and drought treatments, we observed a significant induction of PgOSC gene expression in Platycodon grandiflorus specifically under salt stress conditions. In summary, this study comprehensively identified and analyzed the OSC gene family, aiming to provide basic biological information for exploring the members of PgOSC gene family.

Tertiary Lymphoid Structure in Dental Pulp: The Role in Combating Bacterial Infections.

Tertiary lymphoid structure (TLS) is associated with various pathologies, including those of cancers and chronic infections. Depending on the organ, multiple factors regulate the formation of TLS. However, the role of TLS in immune response and the molecules that drive its formation remain uncertain. The dental pulp, includes a few immune cells surrounded by rigid mineralized tissue, and opens to the outside through the apical foramen. Owing to this special organization, the dental pulp generates a directional immune response to bacterial infection. Considering this aspect, the dental pulp is an ideal model for comprehensively studying the TLS. In the present study, single-cell RNA sequencing of healthy and inflamed human dental pulp reveals known markers of TLS, including C-C motif chemokine ligand 19 (CCL19), lysosome-associated membrane glycoprotein 3 (LAMP3), CC chemokine receptor 7 (CCR7), and CD86, present in inflamed dental pulp. Compared with the healthy pulp, types and proportions of immune cells increase, along with enhanced cellular communication. Multiple immunofluorescence staining reveals that typical TLS emerges in dental pulp with pulpitis, consistent with the high expression of CC chemokine ligand 3 (CCL3), which may be a key driver of TLS formation. Moreover, TLS is also observed in a mouse model of pulpitis. These findings collectively offer insights into the formation and function of TLS in response to infection.

Investigating pH-induced conformational switch in PIM-1: An integrated multi spectroscopic and MD simulation study

PIM-1 is a Ser/Thr kinase, which has been extensively studied as a potential target for cancer therapy due to its significant roles in various cancers, including prostate and breast cancers. Given its importance in cancer, researchers are investigating the structure of PIM-1 for pharmacological inhibition to discover therapeutic intervention. This study examines structural and conformational changes in PIM-1 across different pH using various spectroscopic and computational techniques. Spectroscopic results indicate that PIM-1 maintains its secondary and tertiary structure within the pH range of 7.0–9.0. However, protein aggregation occurs in the acidic pH range of 5.0–6.0. Additionally, kinase assays suggested that PIM-1 activity is optimal within the pH range of 7.0–9.0. Subsequently, we performed a 100 ns all-atom molecular dynamics (MD) simulation to see the effect of pH on PIM-1 structural stability at the molecular level. MD simulation analysis revealed that PIM-1 retains its native conformation in alkaline conditions, with some residual fluctuations in acidic conditions as well. A strong correlation was observed between our MD simulation, spectroscopic, and enzymatic activity studies. Understanding the pH-dependent structural changes of PIM-1 can provide insights into its role in disease conditions and cellular homeostasis, particularly regarding protein function under varying pH conditions.

Reduced intestinal-to-diffuse conversion and immunosuppressive responses underlie superiority of neoadjuvant immunochemotherapy in gastric adenocarcinoma.

Neoadjuvant immunochemotherapy (NAIC) achieves superior clinical benefits over neoadjuvant chemotherapy (NAC) in multiple types of human cancers, including gastric adenocarcinoma (GAC). However, it is poorly understood how the malignant epithelial cells and tumor immune microenvironment (TIME) might respond distinctly to NAIC and NAC that underlies therapeutic efficacy. Here treatment-naive and paired tumor tissues from multiple centers were subjected to pathological, immunological, and transcriptomic analysis. NAIC demonstrated significantly increased rate of pathological complete response compared to NAC (pCR: 25%vs. 4%, p<0.05). Interestingly, pretreatment intestinal subtype of Lauren's classification was predictive of pathologic regression following NAIC, but not NAC. A substantial portion of cancers underwent intestinal-to-diffuse transition, which occurred less following NAIC and correlated with treatment failure. Moreover, NAIC prevented reprogramming to an immunosuppressive TIME with less active fibroblasts and exhausted CD8+ T cells, and increased numbers of mature tertiary lymphoid structures. Mechanistically, activation of the tumor necrosis factor alpha (TNFα)/nuclear factor-kappa B (NF-κB) signaling pathway was associated with response to NAIC. Together, NAIC is superior to NAC for locally advanced GAC, likely due to reduced intestinal-to-diffuse conversion and reprogramming to an immuno-active TIME. Modulation of the histological conversion and immunosuppressive TIME could be translatable approaches to improve neoadjuvant therapeutic efficacy.

Deciphering the Multifaceted Immune Landscape of Unresectable Primary Liver Cancer to Predict Immunotherapy Response.

Immunotherapies employing PD-1/PD-L1 immune checkpoint inhibitors (ICIs) are vital for primary liver cancer (PLC), but response rates remain unsatisfying. Accurate differentiation of responders from non-responders to immunotherapy is imperative. Here, single-cell-scaled mass cytometry analysis on sequential peripheral blood mononuclear cells (PBMCs) from ICI-treated PLC patients is conducted, and tissue residence of immune subpopulations is assessed via multiplex immunohistochemistry. In the discovery cohort (n = 24), responders have lower baseline B cell and HLA-DR+CD8+T cell, and higher CD14+CD16- classical monocyte (CM) proportions. CMs decrease more in responders PBMCs, while HLA-DR+CD8+T cells conformably amplify after ICI-exposure. Responsive individuals display upregulated exhaustion and activation markers in peripheral immune lineages. In the expanded cohort of 77 patients, the augment of the B cells in non-responders is re-confirmed. Responders demonstrate much higher enrichment of B cells or tertiary lymphoid structures in tumor compared to non-responders. A prospective model that excelled in early discrimination of responders is developed using generalized linear model and achieves a satisfactory AUC over 0.9 in all three independent cohorts. Integratedly, the study unveils dynamic immune landscapes in PLC patients undergoing ICI-based therapy, aiding in PLC patient stratification for ICI-based treatment and fostering new response monitoring strategies.

Development of Cashew and Pistachio Ladders through a Food-Processing Approach.

Following successful oral immunotherapy (OIT) for peanut allergy using boiled peanuts (BOPI trial), this study investigated the potential of wet-thermal-processing-induced allergen modification, specifically soaking and boiling (1-4 h) to reduce the allergenicity of cashew and pistachio allergens. In addition, this study provides a foundation of understanding for developing safer forms of cashew/pistachio administration for application in OIT by gradual exposure to increasing doses of modified allergens with reduced potency as an "allergen ladder". An SDS-PAGE analysis and an intrinsic-fluorescence spectroscopy revealed altered tertiary structures of the allergens, leading to their denaturation and even degradation. Notably, the reduction in both allergen-specific polyclonal IgG and human-specific IgE (sIgE) binding correlated with the treatment time, with the most significant decrease observed after 4 h of boiling. In contrast, shorter soaking treatments showed negligible effects on the IgE-binding capacity of these nuts, therefore indicating a further need for optimization. These findings indicate that extended boiling effectively reduced the amounts of the highly potent allergenic component Ana o 3 in cashew and Pis v 1 in pistachio, as confirmed by ELISA using polyclonal anti-Ana o 3 antibodies, and an immunoblot showed decreased IgE epitope binding in cashew and pistachio allergens, which further modified their allergenic profiles. This approach shows promise as a viable method for offering a safer therapeutic option for cashew/pistachio allergy.

Solvation of molecules from the family of "domain of unknown function" 3494 and their ability to bind to ice.

In 2012, the molecular structure of a new, broad class of ice-binding proteins, classified as "domain of unknown function" (DUF) 3494, was described for the first time. These proteins have a common tertiary structure and are characterized by a very wide spectrum of antifreeze activity (from weakly active to hyperactive). The ice-binding surface (IBS) region of these molecules differs significantly in its structure from the IBS of previously known antifreeze proteins (AFPs), showing a complete lack of regularity and high hydrophilicity. The presence of a regular, repeating structural motif in the IBS region of hitherto known AFP molecules, combined with the hydrophobic nature of this surface, promotes the formation of an ice-like ordering of the solvation water layer and, as a result, facilitates the process of transformation of this water layer into ice. It is, therefore, surprising that the newly discovered DUF3494 class of proteins clearly breaks out of this characteristic. In this paper, using molecular dynamics simulations, we analyze the solvation water structure of the IBS region of both DUF3494 family molecules and AFPs. As we show, although the IBS of DUF3494 molecules does not form an ice-like water structure in the solvation layer, this is compensated by the formation of the equivalent of "anchored clathrate water," in the form of a relatively large number of water molecules bound to the surface of the protein molecule and providing potential binding sites for it to the ice surface.

Lymphoid Aggregates in Canine Cutaneous and Subcutaneous Sarcomas: Immunohistochemical and Gene Expression Evidence for Tertiary Lymphoid Structures.

Canine cutaneous/subcutaneous soft-tissue sarcomas (STS) are diversely derived mesenchymal neoplasms with a risk of recurrence and/or metastasis depending on the extent of surgical excision and histologic grade. Lymphoid aggregates (LAs) are often described in these tumours but not characterised. In humans, LA characterised as tertiary lymphoid structures (TLSs) improve the prognosis of many tumours, including sarcomas. We sought to determine if LA meeting a size criterion (> 700 cells) in canine sarcomas met the criteria of TLS and the overall prevalence of LA of any size. RNA expression in large LAs versus aggregate-adjacent sarcoma tissue (AAS) was measured in laser capture microdissected tissue and compared to curl-derived RNA from aggregate-free sarcomas and lymph nodes. CD3, CD20, MUM-1 and PNAd expressions were measured using immunohistochemistry. CD20 and CD3 mRNA were more highly expressed in LA versus AAS (13.8 fold, p = 0.0003 and 2.3 fold, p = 0.043). This was supported by the IHC findings. The large LAs were also enriched in chemokine RNA expression characteristic of TLS (CXCR5 5.8 fold, p < 00001, CCL19 3.68 fold, p = 0.0209, CCL21 6.87 fold, p = 0.0209 and CXCL13 2.68 fold, p = 0.0924). Plasma cells and high endothelial venules were identified in LA containing tumours but not in control tissue. Large LAs were present in 12% of tumours, and LA of any size in 30%. We conclude that large LAs in canine STS are consistent with TLS.

From Zebrafish To Humans: In Silico Comparative Study of RAD50 Sequences

DNA damage, particularly the occurrence of DNA double-strand breaks (DSBs), presents a significant hazard to the integrity and viability of cells. Improper repair of DSBs can result in chromosomal alterations, oncogenic changes, or cell demise. The MRE11-RAD50-NBS1 (MRN) complex plays a crucial role in DNA repair and signaling under the Ataxia Telangiectasia Mutated (ATM) kinase regulation. In this study, we employed comprehensive computational techniques to analyze the structure of RAD50 in Danio rerio (Zebrafish), utilized as a model organism. Additionally, we conducted in silico assessments of RAD50 from both Zebrafish and humans, comparing their characteristics. The substantial sequence resemblance between DrRAD50 and HsRAD50 suggests that DrRAD50 could potentially serve as a valuable model for HsRAD50. However, it is important to acknowledge that sequence similarity alone does not necessarily imply functional equivalence. Further functional studies are needed to confirm the extent of their functional similarities. By examining the secondary and tertiary protein structures of RAD50, we observed a notable likeness between Zebrafish and Human RAD50 proteins. In silico analysis demonstrated that the sequence of RAD50 in zebrafish shares 70% similarity with the human RAD50 protein.

Identification and validation of a prognostic model based on three TLS-Related genes in oral squamous cell carcinoma

BackgroundThe tertiary lymphoid structures (TLSs) have an immunomodulatory function and have a positive impact on the survival outcomes of patients with oral squamous cell carcinoma (OSCC). However, there is a lack of standard approaches for quantifying TLSs and prognostic models using TLS-related genes (TLSRGs). These limitations limit the widespread use of TLSs in clinical practice.MethodsA convolutional neural network was used to automatically detect and quantify TLSs in HE-stained whole slide images. By employing bioinformatics and diverse statistical methods, this research created a prognostic model using TCGA cohorts and explored the connection between this model and immune infiltration. The expression levels of three TLSRGs in clinical specimens were detected by immunohistochemistry. To facilitate the assessment of individual prognostic outcomes, we further constructed a nomogram based on the risk score and other clinical factors.ResultsTLSs were found to be an independent predictor of both overall survival (OS) and disease-free survival in OSCC patients. A larger proportion of the TLS area represented a better prognosis. After analysis, we identified 69 differentially expressed TLSRGs and selected three pivotal TLSRGs to construct the risk score model. This model emerged as a standalone predictor for OS and exhibited close associations with CD4 + T cells, CD8 + T cells, and macrophages. Immunohistochemistry revealed high expression levels of CCR7 and CXCR5 in TLS + OSCC samples, while CD86 was highly expressed in TLS- OSCC samples. The nomogram demonstrates excellent predictive ability for overall survival in OSCC patients.ConclusionsThis is the first prognostic nomogram based on TLSRGs, that can effectively predict survival outcomes and contribute to individual treatment strategies for OSCC patients.

Anlotinib plus TQB2450, a PD-L1 Antibody, in Patients with Advanced Alveolar Soft Part Sarcoma: a single-arm, phase 2 trial.

Alveolar soft part sarcoma (ASPS) is an ultra-rare soft-tissue sarcoma with a high rate of metastasis and no established treatment. This study aimed to explore the efficacy and safety of anlotinib (a tyrosine-kinase inhibitor) and TQB2450 (a PD-L1 inhibitor) in ASPS patients. This single-arm, phase 2 study evaluated the efficacy of TQB2450, an anti-programmed death ligand 1 (PD-L1) agent, combined with anlotinib, a TKI, in adults with advanced ASPS. TQB2450 was given intravenously (1,200 mg) on day 1, and anlotinib (12 mg/day) was taken orally from day 1 to day 14 every 3 weeks. The primary endpoint was overall response rate, with secondary endpoints including duration of response (DOR), progression-free survival (PFS), and overall survival (OS). Lymphocyte infiltration and tertiary lymphoid structure (TLS) were also analyzed as potential prognostic biomarkers. The study enrolled 29 patients, with 28 evaluable (one withdrew due to acute pancreatitis). An objective response was achieved in 82.1% of patients, including 4 complete and 19 partial responses. The median time to response was 2.8 months, and the DOR was not reached, with an estimated median PFS of 35.2 months. Grade 3-4 treatment-related adverse events occurred in 44.8% of patients, with no study-related deaths. Responders had a higher proportion of TLS area, density, and CD20-positive immune cells. The combination of anlotinib and TQB2450 is effective and tolerable in ASPS patients. TLS may serve as a prognostic biomarker, meriting further investigation.

Serous Ovarian Carcinoma: Detailed Analysis of Clinico-Pathological Characteristics as Prognostic Factors.

Serous ovarian carcinoma (SOC) is the most common subtype of epithelial ovarian cancer, with high-grade (HGSOC) and low-grade (LGSOC) subtypes presenting distinct clinical behaviours. This study aimed to evaluate histopathologic features in SOC, correlating these with prognostic outcomes, and explore the potential clinical implications. We analysed 51 SOC cases for lymphovascular space invasion (LVSI), tumour border configuration (TBC), microvessel density (MVD), tumour budding (TB), the tumour-stroma ratio (TSR), the stromal type, tumour-infiltrating lymphocytes (TILs), and tertiary lymphoid structures (TLSs). A validation cohort of 54 SOC cases from The Cancer Genome Atlas (TCGA) was used for comparison. In the discovery set, significant predictors of aggressive behaviour included LVSI, high MVD, high TB, and low TILs. These findings were validated in the validation set where the absence of TLSs, lower peritumoural TILs, immature stromal type, and low TSR were associated with worse survival outcomes. The stromal type was identified as an independent prognostic predictor in SOC across both datasets. Inter-observer variability analysis demonstrated substantial to almost perfect agreement for these features, ensuring the reproducibility of the findings. The histopathological evaluation of immune and stromal features, such as TILs, TLSs, TB, TSR, and stromal type, provides critical prognostic information for SOC. Incorporating these markers into routine pathological assessments could enhance risk stratification and guide treatment, offering practical utility, particularly in low-resource settings when molecular testing is not feasible.