Diagnosis of oncological diseases remains at the forefront of current medical research. Carbonic Anhydrase IX (CA IX) is a cell surface hypoxia-inducible enzyme functionally involved in adaptation to acidosis that is expressed in aggressive tumors; hence, it can be used as a tumor biomarker. Herein, we propose a nanoscale graphene oxide (GO) platform functionalized with magnetic nanoparticles and a monoclonal antibody specific to the CA IX marker. The GO platforms were prepared by a modified Hummers and Offeman method from exfoliated graphite after several centrifugation and ultrasonication cycles. The magnetic nanoparticles were prepared by a chemical precipitation method and subsequently modified. Basic characterization of GO, such as the degree of oxidation, nanoparticle size and exfoliation, were determined by physical and chemical analysis, including X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM), energy dispersive X-ray analysis (EDX), and atomic force microscopy (AFM). In addition, the size and properties of the poly-L-lysine-modified magnetic nanoparticles were characterized. The antibody specific to CA IX was linked via an amidic bond to the poly-L-lysine modified magnetic nanoparticles, which were conjugated to GO platform again via an amidic bond. The prepared GO-based platform with magnetic nanoparticles combined with a biosensing antibody element was used for a hypoxic cancer cell targeting study based on immunofluorescence.

Human carbonic anhydrase IX (CAIX), a unique member of the α carbonic anhydrase family, is a transmembrane glycoprotein with high enzymatic activity by which CAIX contributes to tumorigenesis through pH regulation. Due to its aberrant expression, CAIX is considered to be a marker of tumor hypoxia and a poor prognostic factor of several human cancers. Hypoxia-activated catalytic function of CAIX is dependent on posttranslational modification of its short intracellular domain. In this work, we have identified that C-terminal Ala459 residue, which is common across CAIX of various species as well as additional transmembrane isoforms, plays an important role in CAIX activation and in pH regulation. Moreover, structure prediction I-TASSER analysis revealed involvement of Ala459 in potential ligand binding. Using tandem mass spectrometry, Protein-L-isoaspartyl methyltransferase (PIMT) was identified as a novel interacting partner, further confirmed by an in vitro pulldown assay and an in situ proximity ligation assay. Indeed, suppression of PIMT led to increased alkalinization of culture media of C33a cells constitutively expressing CAIX in hypoxia. We suggest that binding of PIMT represents a novel intracellular signal required for enzymatic activity of CAIX with a potential unidentified downstream function.

Understanding the long-term dynamics of SARS-CoV-2 neutralizing antibodies is critical for evaluating vaccine-induced protection and informing booster strategies. In this longitudinal study, we analyzed 114 serum samples from 19 individuals across six time points over a three-year period following mRNA vaccination (Comirnaty) and natural SARS-CoV-2 infection. Using pseudotype-based neutralization assays against nine SARS-CoV-2 variants, including major Omicron subvariants (BA.1–BA.5, BQ.1.1, XBB), and anti-S1 IgG ELISA, we observed that antibody levels peaked after the third vaccine dose and remained relatively stable two years later. Neutralization titers rose markedly after the second and third doses, with the highest neutralization observed at two years post-booster. Strong correlations were found between anti-S1 IgG levels and mean neutralization titers for pre-Omicron variants (r = 0.79–0.93; p < 0.05), but only moderate for Omicron subvariants (r ≈ 0.50–0.64). Notably, hybrid immunity (vaccination plus infection) resulted in higher neutralization titers at the final time point compared to vaccine-only participants. The lowest neutralization was observed against XBB, underscoring the immune evasiveness of emerging variants. These findings support the importance of booster vaccination and highlight the added durability of hybrid immunity in long-term protection.

Understanding the long-term dynamics of SARS-CoV-2 neutralizing antibodies is critical for evaluating vaccine-induced protection and informing booster strategies. In this longitudinal study, we analyzed 114 serum samples from 19 individuals across six time points over a three-year period following mRNA vaccination (Comirnaty) and natural SARS-CoV-2 infection. Using pseudotype-based neutralization assays against nine SARS-CoV-2 variants, including major Omicron subvariants (BA.1–BA.5, BQ.1.1, XBB), and anti-S1 IgG ELISA, we observed that antibody levels peaked after the third vaccine dose (mean ELISA ratio ± SD: 8.89 ± 1.65 S/C) and remained relatively stable two years later (mean: 7.53± 1.17 S/C). Neutralization titers rose markedly after the second and third doses, with the highest neutralization observed at two years post-booster. Strong correlations were found between anti-S1 IgG levels and mean neutralization titers for pre-Omicron variants (r = 0.79–0.93; p<0.05), but only moderate for Omicron subvariants (r ≈ 0.50–0.64). Notably, hybrid immunity (vaccination plus infection) resulted in higher neutralization titers at the final time point compared to vaccine-only participants. The lowest neutralization was observed against XBB, underscoring the immune evasiveness of emerging variants. These findings support the importance of booster vaccination and highlight the added durability of hybrid immunity in long-term protection.