AlkB is a Fe(II)/α-ketoglutarate-dependent dioxygenase that repairs some alkylated bases of DNA and RNA in Escherichia coli. In the course of catalysis, oxidation of a co-substrate (α-ketoglutarate, αKG) leads to the formation of a highly reactive \'oxyferryl\' enzyme-bound intermediate, Fe(IV) = O, ensuring hydroxylation of the alkyl nucleobase adducts. Previous studies have revealed that AlkB is a flexible protein and can adopt different conformations during interactions with cofactors and DNA. To assess the conformational dynamics of the enzyme in complex with single- or double-stranded DNA in real-time mode, we employed the stopped-flow fluorescence method. N1-Methyladenine (m1A) introduced into a sequence of 15-mer oligonucleotides was chosen as the specific damage. Single-turnover kinetics were monitored by means of intrinsic fluorescence of the protein\'s Trp residues, fluorescent base analogue 2-aminopurine (2aPu), and a dye-quencher pair (FAM/BHQ1). For all the fluorescent labels, the fluorescent traces showed several phases of consistent conformational changes, which were assigned to specific steps of the enzymatic process. These data offer an overall picture of the structural dynamics of AlkB and DNA during their interaction.

Accumulating evidence has revealed that aberrant abundance of 5-hydroxymethylcytosine (5hmC) is critically involved in tumorigenesis. The aim of the present study was to investigate the level of 5hmC in primary oral squamous cell carcinoma (OSCC) and determine its clinical significance as well as prognostic value in predicting patients\' outcomes. The expression levels of 5hmC in 95 human OSCC samples and 24 normal oral mucosa were evaluated by immunohistochemical staining. Moreover, the associations between the expression status of 5hmC and several clinicopathological parameters as well as patients\' survival were further statistically assessed. Our immunohistochemical results revealed that 5hmC was significantly downregulated in a significant fraction of OSCC as compared their normal counterparts. However, elevated 5hmC level was found to be significantly associated with pathological grade and cervical node metastasis with P-values of 0.0239 and 0.0041, respectively. Results from Kaplan-Meier cumulative survival analyses indicated that high expression of 5hmC in OSCC was significantly associated with decreased overall survival, disease-free and disease-specific survival as compared to those with low 5hmC (Log-rank, P=0.0210, 0.0313, 0.0415, respectively). Furthermore, the univariate and multivariate survival analyses further identified the expression status of 5hmC as an independent prognostic factor affecting patients\' survival. Taken together, our results reveal a significant decrease of 5hmC level in a large subset of OSCC. However, high level of 5hmC associates with tumor aggressive features and unfavorable prognosis in a fraction of OSCC patients.