Dimer Formation Research Articles

Two Monoclonal Antibodies Targeting Distinct Subdomains of Human Norovirus P Protein.

The human norovirus (HuNov) major capsid VP1comprises an S (shell) and a P (protruding) domain; the latter is responsible for virus attachment and infection. The dimeric formation of P (containing P1 and P2 subdomains) is indispensable for forming a receptor-binding pocket, enabling HuNov to dock to attachment factor histo-blood group antigens (HBGAs) on the host cell. Thus, the P-specific antibody may hamper the engagement of P and HBGA, thereby inhibiting virus infection. In this study, we developed and characterized two HuNov P-specific murine monoclonal antibodies (MAbs), namely, 5C6 and 1H12. They can bind to P protein with high affinity, as evidenced by the results of indirect fluorescent assay, western blot, and Biolayer interferometry assay. Particularly, the MAb 1H12 recognizes the P2 subdomain, whereas the 5C6 targets the distal P1. These MAbs may contribute to the exploration of novel epitopes on HuNov VP1 and to the development of new antivirals.

MAD2L2 Dimerization Is Not Essential for Mitotic Regulation

MAD2L2 is a small HORMA domain protein that plays a crucial role in DNA repair and mitosis. In both TLS and shieldin, the dimerization of MAD2L2 via its HORMA domain is critical for the stability and function of these complexes. However, in mitosis, the dimerization state of MAD2L2 remains unknown. To assess the importance of MAD2L2’s dimerization during mitosis, we utilized CRISPR/Cas9 to generate MAD2L2 knockout cells, which were subsequently complemented with MAD2L2 species carrying different dimer-disrupting point mutations. We assessed the ability of these MAD2L2 dimer-disrupting mutants to regulate mitosis by evaluating early mitotic events and mitotic fidelity. Our findings indicate that MAD2L2 can function in its monomeric form during mitosis, suggesting that MAD2L2 homodimerization is dispensable for early mitotic regulation. Furthermore, our results suggest that the binding of CDH1 to MAD2L2 is a key regulating factor in mitosis that may actively prevent the formation of MAD2L2 dimers, thereby shifting the cellular balance toward MAD2L2-CDH1 interaction. Thus, the equilibrium between the monomeric and dimeric forms of MAD2L2 is an important cellular factor regulating the MAD2L2-containing complexes.

Synthesis, XRD structural analysis and theoretical studies of a potential inhibitor against rheumatoid arthritis (RA).

This work focused on analyzing the properties of N-(5-nitrothiazol-2-yl)furan-2-carboxamide (C8H5N3O4S, NTFC) as a possible inhibitor of the rheumatoid arthritis process. The synthesis of NTFC was carried out and good-quality crystals were obtained and studied by NMR (1H and 13C), DEPT 135, UV-Vis, IR, MS and single-crystal X-ray diffraction. The structure of NTFC consists of two rings, thiazole and furan, and a central C-N-C(=O)-C segment, which appears to be planar. This central amide segment forms angles of 2.61 (10) and 7.97 (11)° with the planes of the thiazole and furan rings, respectively. The crystal structure of NTFC exhibits N-H...N, N-H...O and C-H...O hydrogen bonds, and C-H...π and π-π interactions that facilitate self-assembly and the formation of hydrogen-bonded dimers, which implies the appearance of R22(8) graph-set motifs in this interaction. The stability of the dimeric unit is complemented by the formation of strong intramolecular C-S...O interactions of chalcogen character, with an S...O distance of 2.6040 (18) Å. Hirshfeld surface (HS) analysis revealed that O...H/H...O interactions were dominant, accounting for 36.8% of the total HS, and that N-H...N interactions were fundamental to the formation of the dimeric structure. The molecular electrostatic potential (MEP) map showed a maximum energy of 46.73 kcal mol-1 and a minimum of -36.06 kcal mol-1. The interaction energies of molecular pairs around NTFC are highest for those interactions linked by N-H hydrogen bonds. The properties of the NTFC ligand as a potential inhibitor of the DHODH (dihydroorotate dehydrogenase) enzyme were evaluated by molecular docking, showing coupling energies very close to those obtained with the control drug for rheumatoid arthritis, i.e. leflunomide.

O-GlcNAcylation of enolase 1 serves as a dual regulator of aerobic glycolysis and immune evasion in colorectal cancer

Aerobic glycolysis and immune evasion are two key hallmarks of cancer. However, how these two features are mechanistically linked to promote tumor growth is not well understood. Here, we show that the glycolytic enzyme enolase-1 (ENO1) is dynamically modified with an O-linked β-N-acetylglucosamine (O-GlcNAcylation), and simultaneously regulates aerobic glycolysis and immune evasion via differential glycosylation. Glycosylation of threonine 19 (T19) on ENO1 promotes its glycolytic activity via the formation of active dimers. On the other hand, glycosylation of serine 249 (S249) on ENO1 inhibits its interaction with PD-L1, decreases association of PD-L1 with the E3 ligase STUB1, resulting in stabilization of PD-L1. Consequently, blockade of T19 glycosylation on ENO1 inhibits glycolysis, and decreases cell proliferation and tumor growth. Blockade of S249 glycosylation on ENO1 reduces PD-L1 expression and enhances T cell-mediated immunity against tumor cells. Notably, elimination of glycosylation at both sites synergizes with PD-L1 monoclonal antibody therapy to promote antitumor immune response. Clinically, ENO1 glycosylation levels are up-regulated and show a positive correlation with PD-L1 levels in human colorectal cancers. Thus, our findings provide a mechanistic understanding of how O-GlcNAcylation bridges aerobic glycolysis and immune evasion to promote tumor growth, suggesting effective therapeutic opportunities.

Nickel Bis(dithiolene) Complexes and Tetrathiafulvalenes from Sterically Hindered Di‐ and Tetra‐substituted Dithiine‐dithiolo‐thione Precursors

Three variants of 5,6‐dihydro‐1,4‐dithiin‐2,3‐(1’,3’‐dithiole‐2’‐thione) (dddt) bearing up to four substituents on the ethylene bridge have been obtained by hetero‐Diels‐Alder reaction. The substituted dddt derivatives have the advantage of providing modulation of the steric hindrance and stereogenic centres, and represent valuable intermediates for both tetrathiafulvalene (TTF) and metal‐dithiolene complexes. Within this work, disubstituted and tetrasubstituted dddt derivatives have been used for the synthesis of Ni(II) monoanionic and neutral complexes, as well as for tetra‐ and octa‐methylated EDT‐TTF and BEDT‐TTF (EDT = ethylenedithio, BEDT = bis(ethylenedithio)) derivatives. While the influence of the bulkiness and number of the substituents is observed in the solid state packing of both types of materials (complexes and TTF), the absence of the counterion in the case of the neutral complexes translated into increased intermolecular interactions between the donors and the formation of 1D and 2D layers. The two examples of tetrasubstituted TTF radical cation salts reported in here show semiconducting behaviour due to the formation of dimers and lateral interactions through the S atoms. Our findings highlight the importance of substitution on these type of dddt precursors and their potential for electroactive molecular materials.

Structural and theoretical insights into the influence of thiocyanate ligands on divalent metal complexes with terpyridine derivatives

Four complexes with terpyridine derivative, thiocyanate anion, and selected d-electron metals were studied. The obtained complexes of the formulation [M(ftpy)(SCN)2(CH3OH)]•DMF (M = Mn (1), Co (2), Ni (3)) and [Cu(ftpy)(NCS)2]•H2O (4), where ftpy = 4′‐(furan-2-yl)-2,2′:6′,2′′‐terpyridine, were characterized using FTIR and UV–Vis spectroscopies, magnetic studies, elemental analysis, and single crystal X-ray crystallography. All compounds crystallize in a triclinic crystal system with space group P1¯ and 1–3 are isomorphous and isostructural. The effect of the modification of the terpyridine ligand and the presence of the thiocyanate ligand on the structure has been verified. Interesting dependence was found in the structure of compound 4, where the two thiocyanate ligands are differently coordinated, one via N and the other via S, and the formation of complex dimers connected through hydrogen bonds is observed. The π-stacking interactions and the resulting solid-state architectures of compounds 1–4 were inspected through a combination of DFT calculations, QTAIM, and NCIPlot analyses, and the stability of the complexes was studied by UV–Vis spectroscopy in various solvents.

Impairing protein-protein interactions in an essential tRNA modification complex: An innovative antimicrobial strategy against Pseudomonas aeruginosa.

Protein-protein interactions (PPIs) have been recognized as a promising target for the development of new drugs, as proved by the growing number of PPI modulators reaching clinical trials. In this context, peptides represent a valid alternative to small molecules, owing to their unique ability to mimic the target protein structure and interact with wider surface areas. Among the possible fields of interest, bacterial PPIs represent an attractive target to face the urgent necessity to fight antibiotic resistance. Growing attention has been paid to the YgjD/YeaZ/YjeE complex responsible for the essential t6A37 tRNA modification in bacteria. We previously identified an α-helix on the surface of Pseudomonas aeruginosa YeaZ, crucial for the YeaZ-YeaZ homodimer formation and the conserved YeaZ-YgjD interactions. Herein, we present our studies for impairing the PPIs involved in the formation of the YeaZ dimers through synthetic peptide derivatives of this helical moiety, both in vitro with purified components and on P. aeruginosa cells. Our results proved the possibility of targeting those PPIs which are usually essential for protein functioning and thus are refractory to mutational changes and antibiotic resistance development.

New Hypothesis on Enhancing β-Sheet Formation during the Tau Fragment Dimer Transition from a Flexible Monomer: Insights into Primary Nucleation Processes by Histidine Behaviors.

Slight perturbations in pH can have significant effects on the primary nucleation processes of the tau protein. The behaviors of histidine due to its pivotal role in modulating H-bonding network interactions and electrostatic interactions have garnered considerable attention, as it can influence the structural characteristics and aggregation properties. However, the nucleation mechanisms and related intermediates are still unclear. In the current study, we performed nine independent replica exchange molecular dynamics simulations to investigate dimer formation involving R3(εδ) in conjunction with the R1, R2, and R4 monomers. Our findings substantiate that, in comparison to R1-R3(εδ) and R4-R3(εδ) systems, the R2-R3(εδ) systems consistently manifest the highest averaged β-sheet content, with the fundamental feature of R3(εδ) promoting R2 rearrangement. Our comprehensive analysis reveals that high-β-sheet-rich systems exhibit a conserved three/five β-strand structure. In these β-strand-rich systems, one chain [R1/R2/R4 or R3(εδ)] with robust intrachain H-bonding interactions coordinates with another chain through interchain H-bonding interactions, contributing to the overall stability. Furthermore, we discuss distinct histidine behaviors, including backbone/side chain interactions and donor/acceptor roles. This study provides a comprehensive understanding of the aggregation propensities of soluble tau oligomers and sheds light on the primary nucleation mechanism. It contributes to a new perspective for understanding protein folding and misfolding.

Kinetics of Polyampholyte Dimerization: Influence of Charge Sequences

Polyampholytes (PAs) exhibit complex behaviors in various environments influenced by their charge distribution. This study focuses on the kinetics of dimerization of PAs, aiming to elucidate the underlying mechanisms and clarify relevant characteristics of the charge sequence. We focus on PAs with non-zero net charges, employing molecular dynamics simulations and theoretical analyses to examine how charge sequences influence the rates of dimer formation and dissociation. Our findings reveal that the charge sequence of tails and the blockiness of the minority charge group markedly influence the kinetics of dimerization: large blockiness and tails with a high number of majority-type charges slow down the dissociation of dimers. Additionally, the presence of an extended (central) block of the majority charge promotes structural diversity. Within dimer states, blocks alternate between intra- and inter-chain contacts. The duration times in the dimer states are significantly longer than the typical dwell times of block inter-contacts, with a notable extension when multiple blocks are engaged. Intrinsically disordered proteins (IDPs) play crucial roles in cellular functions, primarily due to their ability to undergo rapid conformational changes and form transient complexes. These properties largely depend on the sequence of charged residues. We provide insights into the fundamental principles governing the structural and dynamical properties of polyampholytic IDP, emphasizing the importance of sequence-specific effects on both aggregation and dissociation.

Comparative Bindings of Glycosaminoglycans with CXCL8 Monomer and Dimer: Insights from Conformational Dynamics and Kinetics of Hydrogen Bonds.

GAGs bind to both the monomeric and dimeric forms of CXCL8, helping to form a concentration gradient of the chemokine that facilitates the recruitment of neutrophils to an injury site and supports other biological functions. In this study, atomistic molecular dynamics simulations were conducted to investigate the binding behavior of two hexameric GAGs sulfated at two different positions, chondroitin sulfate (CS) and heparan sulfate (HS), with the monomer (SIL8) and dimer (DIL8) forms of the CXCL8 protein. The results support that the conformational diversity of CS and HS appeared to be more when binding with monomer SIL8 than dimer DIL8. CS gained more configurational entropy from glycosidic linkage flexibility when bound to SIL8 than DIL8, with a higher energy barrier, whereas HS exhibited a lower energy barrier for configurational entropy when bound to SIL8 and DIL8. The monomer SIL8 exhibited more favorable and preferential binding with GAGs compared to DIL8. Formation of hydrogen bonds with the basic amino acids of SIL8 and GAG was more rigid and required higher activation energy to break than the other identified hydrogen bondings. Water molecules involved in hydrogen bonding with GAGs, excluding those with basic amino acids of DIL8, showed longer lifetimes and slower relaxation compared to SIL8. This suggests that water-mediated interactions also favor binding of DIL8 with GAGs. Despite having more basic amino acids, DIL8 did not display stronger binding than SIL8, indicating the significant role of basic residues in stabilizing the GAG-protein interactions in the monomers. The reason could be that the greater number of nonbasic amino acids in dimeric CXCL8 stabilizes the complex by forming water-mediated hydrogen bonds, reducing the conformational preferences for binding with GAGs. In contrast, the monomeric form of CXCL8 exhibits a higher conformational preference for protein-GAG interactions.

Synthesis, structures and Hirshfeld surface analyses of 2-hydroxy-N′-methylacetohydrazide and 2-hydroxy-N-methylacetohydrazide

The structures of the title compounds 2-hydroxy-N′-methylacetohydrazide, 1, and 2-hydroxy-N-methylacetohydrazide, 2, both C3H8N2O2, as regioisomers differ in the position of the methyl group relative to the N atoms in 2-hydroxy-acetohydrazide. In the structure of 1, the 2-hydroxy-acetohydrazide core [OH—C—C(=O)—NH—NH] is almost planar and the methyl group is rotated relative to this plane. As opposed to 1, in the structure of 2 all non-hydrogen atoms lie in the same plane. The hydroxyl and carbonyl groups in structures 1 and 2 are in trans and cis positions, respectively. The methyl amino group and carbonyl group are in the cis position relative to the C—N bond in structure 1, while the amino group and carbonyl group are in the trans position relative to the C—N bond in stucture 2. In the crystal, molecules of 1 are linked by N—H...O and O—H...N intermolecular hydrogen bonds, forming layers parallel to the ab crystallographic plane. A Hirshfeld surface analysis showed that the H...H contacts dominate the crystal packing with a contribution of 55.3%. The contribution of the H...O/O...H interaction is somewhat smaller, amounting to 30.8%. In the crystal, as a result of the intermolecular O—H...O hydrogen bonds, molecules of 2 form dimers, which are linked by N—H...O hydrogen bonds and a three-dimensional supramolecular network The major contributors to the Hirshfeld surface are H...H (58.5%) and H...O/O...H contacts (31.7%).

Aminolysis of five-membered cyclic carbonate catalyzed by guanidines: Solvent effects on the formation of guanidine dimers

This paper presents an experimental and computational investigation on the aminolysis reactions of five-membered cyclic carbonate (5CC) catalyzed by mono- or bi-cyclic guanidines in various solvents. For both liquid and solid 5CC substrate employed, higher polar solvent favors the monomeric active guanidine responsible for the catalytic aminolysis reactions. The formation of the dimeric state of the guanidine catalyst, which is seemingly inert to catalytic activity, is prevented by the bulkier substituents connecting to the guanidine moiety. Extensive DFT calculations further confirm that the relative stabilities of dimeric guanidine is depended on both the polarities of the solvents and the bulkiness of the substituents connected to the guanidine moiety, which is consistent with the experimental kinetic results.

Preformed mincle dimers stabilized by an interchain disulfide bond in the neck region.

The sugar-binding receptor mincle stimulates macrophages when it encounters surface glycans on pathogens, such as trehalose dimycolate glycolipid in the outer membrane of mycobacteria. Binding of oligosaccharide ligands to the extracellular C-type carbohydrate-recognition domain (CRD) in mincle initiates intracellular signaling through the common Fc receptor γ (FcRγ) adapter molecule associated with mincle. One potential mechanism for initiation of signaling involves clustering of receptors, so it is important to understand the oligomeric state of mincle. Affinity purification of mincle from transfected mammalian cells has been used to show that mincle exists as a pre-formed, disulfide-linked dimer. Deletion of cysteine residues and chemical crosslinking further demonstrate that the dimers of mincle are stabilized by a disulfide bond between cysteine residues in the neck sequence that links the CRD to the membrane. In contrast, cysteine residues in the transmembrane region of mincle are not required for dimer formation or association with FcRγ. A protocol has been developed for efficient production of a disulfide-linked extracellular domain fragment of mincle in a bacterial expression system by appending synthetic dimerization domains to guide dimer formation in the absence of the membrane anchor.

A rapid, sensitive, and high-throughput pathogen detection method and application of identifying pathogens based on multiplex PCR technology combined with capillary electrophoresis

Respiratory infections are one of the leading causes of death worldwide. Rapid and accurate detection of pathogens is crucial for timely treatment. This study established a detection method based on multiplex PCR combined with capillary electrophoresis, capable of simultaneously detecting 28 common respiratory pathogens. We used specially designed homo-tag primers, significantly reducing the formation of primer dimers and improving the specificity and sensitivity of amplification. After two rounds of PCR amplification, the products were subjected to fragment analysis using the ABI 3500XL Genetic Analyzer, enabling automated result interpretation. The detection limit of this method reaches 2.77 × 102 copies/ml, with some pathogens reaching as low as 2.77 × 10 copies/ml. The detection of 147 clinical specimens showed that the overall consistency of this method with culture, colloidal gold, fluorescent quantitative PCR, and NGS was 75.5%. The multiplex PCR detection method established in this study is rapid, sensitive, and high-throughput, can be used for routine screening of common pathogens in respiratory infections, providing an important basis for clinical diagnosis and treatment.

Disruption of CAD Oligomerization by Pathogenic Variants

CAD, the multi-enzymatic protein essential for initiating the de novo biosynthesis of pyrimidine nucleotides, forms large hexamers whose structure and function are not fully understood. Defects in CAD cause a severe neurometabolic disorder that is challenging to diagnose. We developed a cellular functional assay to identify defective CAD variants, and in this study, we characterized five pathogenic missense mutations in CAD’s dihydroorotase (DHO) and aspartate transcarbamylase (ATC) domains. All mutations impaired enzymatic activities, with two notably disrupting the formation of DHO dimers and ATC trimers. Combining crystal structures and AlphaFold predictions, we modeled the hexameric CAD complex, highlighting the central role of the DHO and ATC domains in its assembly. Our findings provide insight into CAD’s stability, function, and organization, revealing that correct oligomerization of CAD into a supramolecular complex is required for its function in nucleotide synthesis and that mutations affecting this assembly are potentially pathogenic.

Synthesis and characterization of Bis(imino)phosphine [NPN] ligands and their copper(I) halide complexes

A series of four new bis(imino)phosphine [NPN] ligands PhP(C6H4-2-HC = NAr)2 (Ar = 4-OMeC6H4 (L1, 1), 4-ClC6H4 (L2, 2), 4-BrC6H4 (L3, 3), 2,6-iPr2C6H3 (L4, 4)) were synthesized and structurally characterized. Their reactions with Cu(I) halides CuX (X = Cl, Br, I) were explored for the assembly of coordination complexes LnCuX, and the effects of substituent (Ar) and anion (X) were also investigated in a comparative manner. It was found that the reaction of methoxy substituted L1 (Ar = 4-OMeC6H) with CuX gave the mononuclear Cu(I) complexes L1CuX (X = Cl (1a), Br (1b), I (1c)) with isostructural feature. In contrast, when halogen-substituted ligands L2-3 (4-ClC6H4, 4-BrC6H4) were applied, their reactions with CuCl afforded the formation of tetranuclear dimer [Ln(CuCl)2]2 (n = 2 (2a), 3 (3a)) bridged by the Cu2Cl2 square, regardless of the composition of the reactants. On the other hand, reactions of L2-3 with CuBr and CuI generated the mononuclear Cu(I) complexes LnCuX (n = 2, X = Br (2b), I (2c); n = 3, X = Br (3b), I (3c)) again. In all these cases above, L1-3 ligands played the tridentate role by biting the copper(I) centers through NPN sites. When the bulky L4 (Ar = 2,6-iPr2C6H3) was involved, the binuclear dimer (L4CuI)2 (4c) bridged by iodide anions was obtained, where each L4 ligand coordinated the copper center using the bidentate P^N mode with another imino arm pendant. In further exploration, 1,10-o-phenanthroline (denoted as phen) was introduced to produce the heteroleptic complex L1CuCl(phen) (1d), where both imino arms in L4 were pendent. The obtained ligand (L1-4) and Cu(I) complexes were characterized by melting point, elemental analysis, FT-IR, UV–Vis, NMR, and single crystal X-ray diffraction.

The role of proton-coupled electron transfer from protein to heme in dehaloperoxidase

At least two of the six methionine (Met) residues in dehaloperoxidase (DHP) are shown to act as electron donors in both autoreduction and protein-heme crosslinking. Autoreduction observed in the two isozymes, DHP-A and DHP-B, is explained by the high heme reduction potential and an endogenous source of electrons from methionine (Met) or cysteine (Cys). This study provides evidence of a connection to protein-heme crosslinking that occurs when DHP is activated by H2O2 in competition with substrate oxidation and autoreduction. The autoreduction yields of DHP-A and DHP-B are comparable and both are inversely proportional to DHP concentration. Both isoenzymes show an anti-cooperative effect on autoreduction kinetics associated with protein dimerization. Despite the presence of five tyrosine (Tyr) amino acids in DHP-A and four Tyr in DHP-B, the mass spectral evidence does not support a Tyr-heme or interprotein Tyr-Tyr crosslinking event as observed in some mammalian myoglobins. LC-MS and tandem MS/MS studies revealed three amino acids that were involved in the heme-protein crosslink, Cys73, Met63 and Met64. Cys73 facilitates dimer formation in DHP-A which also appears to slow the rate of autoreduction, but is not involved in covalent protein-heme crosslinking. Based on mutational studies, Met63 and 64 are involved in both covalent heme crosslinking and autoreduction. Proton-coupled electron transfer and crosslinking by Met to the heme may serve to regulate DHP function and protect it from uncontrolled oxidative damage.

3′-8″- Biflavones: A Review of Their Structural Diversity, Natural Occurrence, Role in Plants, Extraction and Identification

Dimeric forms of flavonoids, known as biflavonoids, are much less studied compared to monomeric forms. It is estimated that nearly 600 different natural biflavonoids have been described to date, containing various subtypes that can be subdivided according to the position of their combinations and the nature of the subunits. The group in which two monomers are linked by a 3′-8″-C atom includes the first isolated biflavonoid ginkgetin, derivatives of amentoflavone, and several other compounds. 3′-8″-biflavones recently attracted much attention as potential molecules with biological activity such as antiviral and antimicrobial activity and as effective molecules for the treatment of neurodegenerative and metabolic diseases and in cancer therapies. With the growing interest in them as pharmacologically active molecules, there is also increasing interest in finding new natural sources of 3′-8″-biflavones and optimizing methods for their extraction and identification. Herein, we have summarized the available data on the structural diversity, natural occurrence, role in plants, extraction, and identification of 3′-8″-biflavones.

Novel Anti-Trop2 Nanobodies Disrupt Receptor Dimerization and Inhibit Tumor Cell Growth.

Background: Trop2 (trophoblast cell-surface antigen 2) is overexpressed in multiple malignancies and is closely associated with poor prognosis, thus positioning it as a promising target for pan-cancer therapies. Despite the approval of Trop2-targeted antibody-drug conjugates (ADCs), challenges such as side effects, drug resistance, and limited efficacy persist. Recent studies have shown that the dimeric forms of Trop2 are crucial for its oncogenic functions, and the binding epitopes of existing Trop2-targeted drugs lie distant from the dimerization interface, potentially limiting their antitumor efficacy. Method: A well-established synthetic nanobody library was screened against Trop2-ECD. The identified nanobodies were extensively characterized, including their binding specificity and affinity, as well as their bioactivities in antigen-antibody endocytosis, cell proliferation, and the inhibition of Trop2 dimer assembly. Finally, ELISA based epitope analysis and AlphaFold 3 were employed to elucidate the binding modes of the nanobodies. Results: We identified two nanobodies, N14 and N152, which demonstrated high affinity and specificity for Trop2. Cell-based assays confirmed that N14 and N152 can facilitate receptor internalization and inhibit growth in Trop2-positive tumor cells. Epitope analysis uncovered that N14 and N152 are capable of binding with all three subdomains of Trop2-ECD and effectively disrupt Trop2 dimerization. Predictive modeling suggests that N14 and N152 likely target the epitopes at the interface of Trop2 cis-dimerization. The binding modality and mechanism of action demonstrated by N14 and N152 are unique among Trop2-targeted antibodies. Conclusions: we identified two novel nanobodies, N14 and N152, that specifically bind to Trop2. Importantly, these nanobodies exhibit significant anti-tumor efficacy and distinctive binding patterns, underscoring their potential as innovative Trop2-targeted therapeutics.

High-glucose impact on UVB responses in human epidermal keratinocytes: Insights on diabetic skin's resistance to photocarcinogenesis

Ultraviolet (UV) B-induced damage in human epidermal keratinocytes (HEKs) initiates photocarcinogenesis. However, how diabetes influences photocarcinogenesis is not well understood. To investigate the impact of high-glucose environments on responses to UVB, we cultured HEKs in normal-glucose (NG) or high-glucose (HG) conditions (G6 and G26), followed by UVB irradiation at 25 mJ/cm2 (G6UVB and G26UVB). We performed next-generation sequencing and analyzed HEKs' expression profiles bioinformatically to identify candidate genes and cellular responses involved. We found UVB induced consistent responses in both NG- and HG-cultivated HEKs, but it also triggered certain distinct processes and pathways specifically in the HG groups. The 459 differentially expressed (DE) genes in the HG groups revealed their roles in chromatin remodeling, nucleosome assembly, and interferon signaling activation. Moreover, the 29 DE genes identified in G26UVB/G6UVB comparison, including the potent tumor suppressor gene TFPI2, were considered key genes contributing to HEKs' altered response to UVB in HG environments. UVB irradiation induced significantly higher TFPI2 expression in HG-cultivated HEKs than their NG-cultivated counterpart. Finally, HG-cultivation significantly increased oxidative stress, cyclobutane pyrimidine dimer formation, and apoptosis, while reducing HEKs' viability after UVB irradiation. These changes under HG conditions probably mediate cell fate toward death and tumor regression. Overall, our findings provide evidence and associated molecular basis on how HG conditions reduce keratinocytes' photocarcinogenic potential following UVB exposure.