Search Results (324)

Uterine corpus endometrial carcinoma (UCEC) is the most common gynecologic malignancy, and aberrant canonical NF-κB signaling is implicated in uterine corpus endometrial carcinoma (UCEC), yet the expression, epigenetic, and prognostic profile of IκB kinase (IKK) complex subunits CHUK (IKKα), IKBKB (IKKβ), and IKBKG (NEMO) remains undefined. This study assessed expression, methylation, miRNA regulation, and clinical relevance of IKK-complex genes in UCEC using public datasets. RNA-seq expression (TCGA-UCEC; GEPIA2) showed significant downregulation of IKBKB in tumors versus normal endometrium (unpaired Wilcoxon), whereas CHUK and IKBKG showed non-significant increases; immunohistochemistry (Human Protein Atlas) illustrated heterogeneity and suggested possible mRNA–protein discordance for IKBKB. Promoter methylation analysis (UALCAN; Illumina 450K) identified CHUK hypomethylation and IKBKG hypermethylation in tumors, with no significant change for IKBKB. Stratification showed IKBKB suppression across clinicopathological strata and TP53 mutant/wild-type tumors; IKBKG decreased across subtypes and stages. Pan-cancer profiling (TIMER2.0) highlighted IKBKB as the broadly dysregulated IKK member across malignancies. STRING networks indicated connectivity with NF-κB mediators (e.g., RELA, NFKB1, TRAF6). miRNA predictions (miRDB/TargetScan) revealed shared regulation (CHUK–IKBKB: 14 miRNAs; CHUK–IKBKG: 2; IKBKB–IKBKG: 0). Kaplan–Meier analysis indicated that elevated IKBKG expression correlated with reduced overall survival (HR = 1.85, p = 0.0037), while CHUK expression did not correlate with survival, and IKBKB exhibited a non-significant trend. In a multivariable Cox regression analysis, high IKBKG expression continued to show a significant association with reduced overall survival, even after adjusting for age, histological type, histological grade, and TCGA molecular subtype (adjusted HR = 1.58, 95% CI = 1.00–2.47, p = 0.048). These findings collectively suggest the potential prognostic significance of IKBKG in UCEC, although independent validation in larger, comprehensively annotated cohorts is still required. Collectively, these findings suggest IKBKG as a candidate negative prognostic marker and indicator in UCEC, warranting experimental validation. Full article

Background: Telomerase is a ribonucleic multimeric reverse transcriptase complex protecting the chromosomal ends from erosion and thereby from cellular senescence. The prognostic value of the components of this complex and their interrelationships with the immune system are not well understood. Objectives: We aimed to examine 15 telomerase-related genes across 7489 tumor samples from the TCGA database. Methods: The mRNA expression of these genes was analyzed using Kaplan–Meier statistics and hierarchical clustering analyses, alone or in combination with tumor infiltration counts for 11 immune cell types. As an additional analysis, univariable and multivariable Cox regression analyses have been performed. Results: Thirteen of 21 tumor types showed significant associations between gene expression in tumors and survival times of patients. Most gene correlations were found in hepatocellular carcinoma and renal clear cell carcinoma. In hepatocellular carcinoma, a high expression of DKC1, NHP2, GAR1, WRAP53, and ACD was associated with shorter survival. In renal clear cell carcinoma, TERT, DKC1, and PARN correlated with shorter survival, and NAF1, TERF2, POT1, and TINF2 with longer survival. DKC1 was the only gene significantly associated with poor prognosis in both tumor types. The telomerase-related genes correlated with patterns of immune cell infiltration, which influenced the survival of patients. The associations of mutation burden and neoantigen load with survival varied depending on the gene and patient groups. In renal clear cell carcinoma, TERT, DKC1, and PARN showed strong interactions with immune cell infiltration and neoantigen load. Conclusions: The combination of telomerase-related gene expression and immune-cell infiltration was associated with overall survival in hepatocellular carcinoma and renal clear cell carcinoma and warrants further evaluation as prognostic markers. Full article

Background: Trimeric envelope-proximal domains in viral class I fusion proteins are conserved targets of broadly neutralizing antibodies (bNAbs), but it has proven difficult to develop vaccines against those targets. The HIV-1 gp41 membrane-proximal external region (MPER) is one such target. Induction of a neutralizing response likely depends on the immunogen having a close-to-native structure. Methods: Native sequence MPER was displayed on genome-reduced bacteria as a coiled-coil homotrimer using a Haemophilus influenzae Hia trimeric autotransporter. Vaccine designs incorporated additional features, including trimerization domains to stabilize MPER, tandem MPER repeats to increase antigen valency, and immunomodulatory elements. Antigen exposure was assessed by flow cytometry, antibody responses were evaluated by ELISA, and functional activity was measured using HIV-1 pseudovirus neutralization assays. Results: Trimer stabilization improved MPER exposure, but immunogen visibility alone did not predict neutralization. After three immunizations, neutralizing activity was detected only in the most extensively engineered vaccine, which neutralized tier 2 virus CNE55. After five immunizations, the same vaccine also neutralized the tier 2 virus 25710-2.43. A further design modification that included an extended Hia-derived spacer increased MPER exposure and antibody binding, with neutralization detected against MN.3, X1632_S2_B10, and 25710-2.43 viruses in subsets of animals. Conclusions: As a proof-of-concept, native-sequence MPER can induce detectable, though modest and virus-dependent, HIV-1 neutralizing activity when displayed in a carefully controlled trimeric bacterial surface-display platform. The results show that MPER vaccine performance depends not only on antigen exposure, but also on multimeric organization, immunomodulatory context, and antigen-scaffold geometry. Analogous coiled-coil trimeric bacterial surface display immunogens may inform vaccine development for stem/stalk regions of other Class I fusion protein viruses. Full article

Galectin-3-mediated processes are important during many aspects of cancer progression, but they are not well understood. Galectin-3 is present extracellularly, and because of its carbohydrate recognition domain (CRD) and unstructured N-terminal domain (NTD), galectin-3 undergoes multimerization, which influences events such as carbohydrate-controlled cell–cell interactions. Investigations reported herein using an in vitro wound-healing assay show that exogenous galectin-3 inhibits cancer cellular migration. Since the addition of the galectin-3 CRD without the NTD does not arrest cellular migration, we attribute the effect of full-length galectin-3 on migration to the extracellular interactions between multimeric, full-length galectin-3 and extracellular receptors. In this publication, lactose-functionalized dendrimers serve as multivalent binding partners for galectin-3 and are used to mitigate extracellular multivalent galectin/carbohydrate interactions. The addition of lactose-functionalized dendrimers provides significant restoration of cellular migration in the presence of exogenous galectin-3 without increasing cellular viability. Thus, although galectin-3/protein interactions within the cell are known to increase both cellular migration and viability, cell surface galectin-3/carbohydrate interactions have the opposite impact and decrease cellular migration. Full article

Background/Objectives: ADAMTS13 is a plasma metalloprotease that cleaves von Willebrand Factor (vWF), a multimeric glycoprotein involved in platelet recruitment during primary hemostasis. Inhibition of ADAMTS13 activity by autoantibodies causes immune thrombotic thrombocytopenic purpura (iTTP). Growing evidence has established allostery as a key contributor to iTTP pathophysiology. This review summarizes the current understanding of how ADAMTS13 structure contributes to its function and regulation and examines anti-ADAMTS13 antibodies as pathological allosteric modulators in iTTP and their associated allosteric mechanisms. Methods: We searched the published literature investigating the role of allostery in iTTP, with special emphasis on studies that satisfy the functional definition of allostery, which addresses how a ligand binding to a protein influences a second ligand-binding event at a distinct site. Results: Anti-ADAMTS13 antibodies primarily affect catalytic turnover as opposed to substrate binding affinity, consistent with their characterization as V-type allosteric ligands. Biophysical studies have revealed extensive and distal structural changes upon antibody binding, including near the enzyme’s active site. However, more recent work utilizing full-length Immunoglobulin G (IgG) molecules and polyclonal iTTP patient plasma suggests that the mechanistic complexity is greater than initially appreciated, with multiple mechanisms likely coexisting. Conclusions: While significant progress has been made to understand allostery in ADAMTS13 and iTTP, many questions remain unresolved. Further elucidation of these underlying mechanisms could help inform the development of diagnostic strategies and targeted therapies for this potentially fatal disorder. Full article

Von Willebrand disease (VWD) is the most common inherited bleeding disorder, yet the contribution of specific VWF domains to its pathogenesis remains incompletely understood. In particular, the role of the D4 domain in VWF secretion, intracellular maturation, and multimer formation has not been fully elucidated. Here, we investigated the functional impact of a heterozygous p.Cys2163Tyr variant located in the D4 domain, identified in a patient with a severe bleeding phenotype, using clinical evaluation, genetic analysis, family studies, and in vitro expression assays. Laboratory testing revealed markedly reduced VWF:Ag (6.8 IU/dL), VWF:GPIbR (0.1 IU/dL), and FVIII:C levels, indicating a severe VWD phenotype in the proband. Plasma VWF multimer analysis showed a markedly reduced overall VWF signal with an almost complete absence of high-molecular-weight multimers, supporting classification of the phenotype as severe type 2A VWD. The heterozygous c.6488G>A (p.Cys2163Tyr) variant was also present in asymptomatic family members, indicating incomplete segregation with the severe phenotype and suggesting that this variant alone is insufficient to explain the proband’s disease severity. Notably, the proband’s mother exhibited mildly reduced VWF levels in the absence of this variant, suggesting the possible contribution of an additional unidentified defect or modifier affecting the maternal allele. In vitro expression demonstrated preserved intracellular VWF antigen, markedly reduced secretion of mutant VWF, and loss of high-molecular-weight VWF multimers. Together, these findings indicate that VWF p.Cys2163Tyr is a functionally deleterious variant that markedly impairs VWF secretion and high-molecular-weight multimer formation in vitro. However, the incomplete segregation observed in the family suggests that this heterozygous variant alone may not fully account for the proband’s severe type 2A VWD phenotype. Full article

The continuous evolution of the SARS-CoV-2 virus, marked by the emergence of new variants, poses a significant threat to the efficacy of existing vaccines. However, a promising approach to addressing vaccine failure caused by viral mutations (particularly in the spike protein) is the development of a variant-proof (conserved), non-spike, multiepitope universal nanostructure vaccine with multifunctionality, biocompatibility, self-adjuvanticity, and structural similarity to pathogens in terms of size and shape. This study aimed to design a self-assembled nanostructure vaccine (SANV) featuring pentameric and trimeric coiled-coil peptide motifs, as well as other functional motifs, including epitopes, TAT, PADRE, and adjuvant. The cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL), and B lymphocyte (BL) epitopes of SANV were screened from the IEDB with more than 50% individual predicted population coverage (PPC) and fused using linkers to enable self-assembly. The multimerization of the 24 SANV monomers was modeled using the GalaxyHomomer and AlphaFold web servers. Subsequently, the leading SANV constructs with (SANVa9) and without (SANVb6) adjuvant were analyzed for their physicochemical profiles and assessed for antigenicity, allergenicity, solubility, and antioxidant potential. Furthermore, the molecular interactions, specificity, and stability of SANVa9 and SANVb6 with the broadly neutralizing sarbecovirus antibody 5817 and toll-like receptors (TLR2, TLR3, and TLR7) were analyzed using molecular docking and simulation over a 100-nanosecond time scale. Finally, the comparative immune simulation profiles of SANVa9 and SANVb6 with controls indicated stronger, broad-spectrum immune responses that could be translated into in vitro and in vivo studies and warrant further evaluation before clinical use. Full article

KCTD proteins exhibit significant structural complexity, arising from their modular organization, oligomerization, and intricate biological partnerships. Although their biological importance has been assessed for two decades, the biochemical basis of their activities is only partially understood. This is certainly due to the limited structural information that was available until a few years ago. Fortunately, some recent insightful structural studies and the advent of machine-learning-based approaches are rapidly changing the scenario. By surveying the literature and structural databases and integrating this information with ad hoc 3D-structure predictions, we provide a detailed view of the structural biology of these proteins at different levels: individual domains, full-length oligomers, functional hetero-oligomers formed by different family members, and complexes with functional partners. Collectively, these surveys and analyses provide insights into the family’s evolutionary history and its structure–function relationships. The family-wide coverage of structural information also indicates the extent to which structural similarities are reflected in functional analogies. Finally, the potential functional implications of the intricate architecture of these multimeric proteins and the tendency of their members to hetero-oligomerize are discussed from a functional perspective. Full article

High-dose intravenous immunoglobulin (IVIG) is used to treat autoimmune and inflammatory diseases, and several studies demonstrate that the therapeutic effects of IVIG can be recapitulated with the fragment crystallizable (Fc) portion. Further, recent data indicate that recombinant multimeric Fc molecules exhibit potent anti-inflammatory properties. In this study, we investigated the biochemical and biological properties of different recombinant human IgG1 Fc molecules with increasing valency and avidity, combined with mutations to increase binding affinity to complement protein C1q. These molecules were investigated for their potential dual antagonism: to antagonize Fcγ receptor (FcγR) effector functions (Ab-dependent cellular phagocytosis) in vitro, and to inhibit the activation of the classical complement pathway. C1q-binding mutants demonstrated an exponential increase in potency to inhibit the classical pathway in correlation with increasing multimerization. Importantly, in contrast to other multimeric Fc constructs such as Fc hexamers, no generation of complement C4a was observed. Reducing the binding affinity to FcγRIIB resulted in a half-life extension of the trivalent hIgG1-Fc molecules in human neonatal Fc receptor transgenic (hFcRn Tg) mice. Our data demonstrate a potent anti-inflammatory effect of recombinant human IgG1-Fc C1q-binding mutants in vitro and in vivo, mediated by blockade of FcγRs and inhibition of complement activation. Full article

Lactoferrin (LF)-derived peptides (LDPs) are short cationic and amphipathic fragments generated primarily from the N-terminal lobe of LF through pepsin-mediated proteolytic processes. The best-characterized LDPs include lactoferricin (LFcin), lactoferrampin (LFampin), and LF1-11. In addition to these native peptides, a growing range of engineered LDPs has been developed by modifying the LFcin-derived RRWQWR motif through the incorporation of non-natural amino acids, cyclization, multimerization, and conjugation with chemotherapeutic agents. LDPs have garnered significant interest as potential anticancer peptides due to their ability to preferentially engage with the surfaces of malignant cells and initiate various tumor-suppressive mechanisms. This review article provides an overview of the principal classes of LDPs and elucidates how structural features influence membrane interaction, selectivity, intracellular targeting, apoptotic pathways, and immune modulation. It also discusses current mechanistic insights and examines the major challenges and opportunities for translating innovative LDPs into clinically useful cancer therapeutics. Full article

Fibroblast activation protein (FAP) has emerged as a promising target for the development of cancer radiotheranostics due to its selective overexpression in cancer-associated fibroblasts (CAFs) within the tumor stroma. Affinity and selectivity refer to the binding affinities of FAP inhibitors toward FAP and related family members, whereas the accumulation of radiolabeled-FAP inhibitors varies by tumor type. Although monomeric FAP inhibitors (FAPIs) have shown extraordinary utility in diagnostic imaging, their clinical application in radiotherapy has been limited by short tumor retention times and heterogeneous uptake. To address these challenges, homomultimeric FAPI ligands—featuring two or more identical FAP-targeting motifs—have been developed with the aim of enhancing binding avidity and prolonging tumor residence. This review comprehensively examines the evolution of homomultimeric FAPI ligands, from molecular design and preclinical validation to early clinical implementation. We highlight how dimeric and higher-order multimeric constructs improve tumor retention and therapeutic efficacy compared to monomers, while also discussing the impact of linker chemistry, valency, and scaffold architecture on pharmacokinetics and targeting efficiency. Preclinical studies demonstrate that optimized dimers and trimers achieve superior tumor-to-background ratios and sustained tumor uptake, whereas excessive multimerization can lead to steric hindrance and reduced efficacy. Clinical data from pioneering studies using agents such as [¹⁷⁷Lu]Lu-DOTAGA.(SA.FAPi)₂ and [¹⁷⁷Lu]Lu-DOTAGA.Glu.(FAPi)₂ confirm prolonged tumor retention, encouraging therapeutic responses and a favorable safety profile in advanced cancers. However, translational challenges remain, including the need for better preclinical models that reflect stromal FAP heterogeneity, optimized radiometal–chelator pairs, and standardized dosing protocols for comparative clinical trials. Overall, homomultimeric FAPI ligands represent a significant advance in FAP-targeted theranostics, offering a robust platform for personalized cancer management. Full article

Background/Objectives: In the first months of their life, infants rely on maternal antibodies for immune protection. Breastmilk is a major source of these defenses, supplying secretory IgA, IgG, and IgM that help guard mucosal surfaces against pathogens such as SARS-CoV-2. Most studies on breastmilk immunity in the context of COVID-19 have emphasized circulating monomeric IgA, rather than the multimeric secretory IgA (sIgA) that is active at mucosal barriers. This study assessed in-depth the contribution of breastmilk antibody subtypes to SARS-CoV-2 neutralization capacity and how these profiles differ following maternal COVID-19 infection versus vaccination during pregnancy or postpartum. Methods: In this prospective cohort study, breastmilk samples were collected longitudinally from individuals who had COVID-19 during pregnancy or received COVID-19 mRNA vaccination during pregnancy or postpartum. Serological assays measured IgG, IgM, systemic IgA, and secretory IgA against SARS-CoV-2 spike and nucleocapsid antigens. Results: COVID-19 infection during pregnancy resulted in significantly higher systemic and secretory IgA levels compared to vaccination. Secretory IgA demonstrated a strong correlation with neutralization capacity. Principal component analysis revealed distinct antibody profiles in COVID-19-exposed individuals versus vaccinated cohorts, with significant overlap between pregnancy and postpartum vaccination groups. Conclusions: Although both COVID-19 vaccination and disease elicit sustained COVID-19-related antibodies in breastmilk, COVID-19 infection elicits a broader and more diverse antibody response in breastmilk, specifically with a greater secretory IgA generation. These findings support the value of maternal vaccination to safely confer mucosal immunity to neonates and the need for optimized vaccine formulations for mucosal immunity. Full article

Single-domain antibodies (sdAbs) are the smallest antigen-binding antibody (Ab) fragments (12–15 kDa) and have emerged as a versatile therapeutic platform. Their compact size, high solubility, stability, and ability to access cryptic epitopes distinguish them from conventional monoclonal Abs (mAbs) and larger Ab fragments. These properties are particularly attractive in ophthalmology, where molecular size, tissue penetration, and formulation constraints critically influence therapeutic performance. This narrative review summarizes the structural features, engineering strategies, immunogenicity considerations, and production platforms of sdAbs, with a focus on ocular applications. Preclinical studies demonstrate promising efficacy in retinal vascular diseases through targeting of VEGFA, ANG2, TNFα, and complement components, as well as in inflammatory and anterior segment disorders. SdAbs can be formatted as multimeric or Fc-fused constructs to extend intraocular half-life or delivered via gene therapy vectors as a sustained intraocular “biofactory” approach. Notably, recent work demonstrates the feasibility of vector-encoded sdAbs targeting complement C3 in vivo. While challenges remain regarding immunogenicity, pharmacokinetics, and regulatory pathways, the approval of several sdAb-based drugs in other fields underscores their clinical potential. SdAbs represent a promising next-generation modality for ocular therapeutics, enabling innovative strategies beyond conventional antibody formats. Full article

Background: Effective vaccines are essential to overcome the limitations of livestock immunisation, particularly in low- and middle-income countries (LMICs), where scalable, thermostable, and easy-to-administer solutions are needed. Nanoparticle-based delivery systems, such as the Spontaneous Nanoliposome Antigen Particle (SNAP) technology using CoPoP liposomes, offer a promising alternative for subunit vaccine development, although their performance in large animal species remains poorly characterised. CoPoP enables the rapid non-covalent multimeric display of His-tagged protein antigens combined with immunomodulators on liposomes incorporating cobalt porphyrin–phospholipid (CoPoP). Objective: To evaluate the immunogenicity of CoPoP-based liposomes delivering the Theileria parva p67C antigen in cattle and compare their performance in murine models. Methods: Cattle and mice were immunised with p67C formulated in CoPoP liposomes incorporating QS-21 and/or PHAD immunomodulators. Humoral and cellular responses were assessed. Parallel in vitro stimulation of bovine PBMC with Quil-A was used to investigate the mechanistic effects of saponins on bovine cells. Results: CoPoP liposome formulations did not improve p67C immunogenicity in cattle, with antibody responses at least two-fold lower than previously reported results and no detectable cellular responses. In contrast, the same platform induced up to 2000-fold higher antibody titres in mice. This disparity is likely driven by differences in antigen dose relative to body mass, tissue architecture, lymphatic accessibility, and innate immune signalling differences. PHAD-mediated TLR4 activation appeared less effective in cattle, whereas QS-21 induced a broader immune activation, likely through conserved inflammasome pathways. Despite limited immunogenicity, antigen presentation by CoPoP liposomes was preserved. Conclusions: SNAP-based CoPoP liposomes showed strong immunogenicity in mice but limited efficacy in cattle, highlighting the challenges of cross-species translation. Optimisation of antigen dose and adjuvant selection for the targeted species is required, with QS-21 representing a more promising candidate than the TLR4 agonist. The scalability and versatility of SNAP technology support its continued development for multivalent livestock vaccines. Full article

Protein–protein interactions (PPIs) are fundamental to viral replication, regulating processes such as assembly, genome packaging, and virion maturation. Despite their biological importance, these interactions remain challenging to study and are relatively underexploited as therapeutic targets. Split reporter systems, based on protein-fragment complementation, provide quantitative platforms to measure PPIs by reconstituting reporter activity when interacting protein partners are brought into proximity. These systems can be applied in vitro and in live cells which enables detection of dynamic and multimeric interactions in physiologically relevant contexts. Major classes of split reporter systems include β-lactamase, alkaline phosphatase, luciferase-based platforms, green fluorescent protein, and horseradish peroxidase. Assay performance depends on factors such as fusion protein stability, expression levels, and reporter kinetics, which influence sensitivity, dynamic range, and reliability. These approaches have been applied to study viral protein interactions across diverse systems, including HIV-1 matrix and nucleocapsid proteins, flaviviral capsid proteins, hepatitis B virus core protein, and chikungunya virus capsid. Split reporter assays also enable high-throughput screening for small-molecule inhibitors that disrupt viral PPIs and multimerization. This provides a functional readout linked to viral replication. Despite the challenges that exist in assay optimization and protein stability, the sensitivity and versatility of these systems provide a framework to interrogate viral protein interactions and support the development of antiviral therapeutics.: Full article