International Journal of Molecular Sciences

Switchable β-barrel-type fluorescent proteins are essential genetically encoded probes for super-resolution imaging. The space required for chromophore cis–trans isomerisation can also provide an opportunity to introduce bulkier chemistry at the 3-position of the phenolic ring. Here, we report, to our knowledge, the first successful genetic encoding of 3-cyano-L-tyrosine (3CNY) into a protein. Using genetic code expansion, the cyano-containing tyrosine derivative is incorporated directly into the chromophore of mKate, a pH-dependent switchable red fluorescent protein. While mKate adopts a fluorescent phenolate cis-state chromophore at physiological pH, substituting the native tyrosine with 3CNY yields a functional protein exhibiting hypsochromically shifted spectral properties. Time-dependent density functional theory (TD-DFT) calculations indicate that 3CNY incorporation results in a trans state at pH 8 but, unlike mKate, is fluorescent. The electron-withdrawing cyano group potentially perturbs conjugation across the chromophore, thus lowering the barrier to cis–trans isomerisation. The trans form may also be stabilised by hydrogen bonds from the cyano group to the rest of the protein. Overall, the introduction of a genetically encoded 3-CNY tyrosine analogue into a fluorescent protein chromophore expands our mechanistic understanding and enables the incorporation of a new chemical tag directly into the chromophore.

Calcitonin gene-related peptide (CGRP), encoded by the CALCA gene, plays a central role in migraine pathophysiology; however, it remains unclear whether migraine is associated with persistent systemic molecular alterations affecting the CGRP pathway during the interictal phase. Transient receptor potential cation channel subfamily A member 1 (TRPA1) stimulates the release of CGRP from nerve endings. We investigated gene-specific and global epigenetic regulatory mechanisms associated with CALCA and TRPA1 expression in the peripheral blood of 56 patients with episodic migraine in the interictal phase and 36 headache-free controls. Expression of CALCA, TRPA1, and genes encoding epigenetic regulators, histone deacetylases 1 and 6 (HDAC1 and HDAC6), was assessed by quantitative real-time PCR. DNA methylation of CALCA and TRPA1 was evaluated using methylation-specific PCR. Expression of other epigenetic regulators miRNA-375, miRNA-382, and miRNA-34a was determined using TaqMan MicroRNA assays. No significant differences were observed between migraine patients and controls in CALCA or TRPA1 expression, nor in DNA methylation of these genes. Similarly, no differences were found in the expression of miRNA-375 and miRNA-382. In contrast, miRNA-34a expression was significantly reduced in migraine patients. Moreover, the expression of HDAC1 and HDAC6 was significantly decreased in migraine. Despite the established role of CGRP in migraine, no evidence of persistent systemic dysregulation of CALCA or TRPA1 was detected in peripheral blood during the interictal phase. However, altered expression of miRNA-34a, HDAC1, and HDAC6 suggests the presence of broader epigenetic and post-transcriptional regulatory disturbances. These findings support a model in which migraine is associated with dysregulation of regulatory mechanisms rather than stable alterations of individual target genes.

Vernal keratoconjunctivitis (VKC) is a chronic pediatric ocular allergy that can progress from seasonal to persistent inflammation with vision-threatening complications. Although Th2-associated mechanisms have been implicated, the immune correlates of disease severity are not well defined. Tear samples from VKC patients (moderate intermittent and moderate persistent) and healthy controls were collected using Schirmer’s strips. Cytokine profiling was performed using the OLINK^® Target 48 Cytokine Panel. Differential expression, correlation with clinical features, and pathway enrichment analyses were performed. Compared to control, IL-15, CXCL11, CXCL9, MMP12, and CCL13 were significantly elevated in VKC, with higher levels in the persistent phenotype. These cytokines correlated with symptom duration, limbal involvement, and papillary hypertrophy. Pathway analysis revealed enrichment of IL-17, JAK–STAT, and chemokine signaling pathways. VKC severity is associated with distinct tear cytokine signatures, with the persistent phenotype showing enhanced chronic inflammatory signaling. These findings identify candidate tear-based markers of disease severity that require validation in larger, independent, and longitudinal cohorts.