Thymidine Kinase 1 (TK1) is a pivotal enzyme in fundamental biochemistry and molecular diagnosis, but recognition and molecule detection is a challenging task. Here, we constructed a DNA-integrated hybrid nanochannel sensor for TK1 activity and inhibition assay. Single-stranded DNA containing thymidine was used as a substrate to functionalize the nanochannels, restricting the ion current through channels. With kinase, the thymidine at the termini of the substrate DNA is phosphorylated, elevating surface charge density and mitigating the pore-obstruction effect by increasing transmembrane ion current. The kinase-induced distinctness can be accurately monitored by this hybrid nanodevice, which benefits from its high sensitivity to the change of surface charge. The excellent analytical performance in both kinase enzyme activity and inhibition analysis resulted in efficient and selective evaluation in human serum. Furthermore, compared to current approaches, it greatly simplifies and offers a direct method of analysis, making it a promising sensor technology for cancer management as well as the activities of multiple types of nucleic acid kinases.

Hyperproliferative  diseases are the first step for tumor formation; thymidine kinase 1 (TK1) mRNA is closely related to cell proliferation. Therefore, the risk of malignant proliferation can be identified by sensitively detecting the variance in TK1 mRNA concentration, which can be used for tumor auxiliary diagnosis and monitoring tumor treatment. Owing to the low abundance and instability of TK1 mRNA in real samples, the development of a sensitive and fast mRNA detection method is necessary. A DNA nanosensor that can be used for detecting TK1 mRNA based on bipedal 3D DNA walker-driven proximal catalytic hairpin assembly (P-CHA) was developed. P-CHA hairpins were hybridized to a linker DNA strand coupled with magnetic nanoparticles to increase their local concentrations. The bipedal DNA walking on the surface of NPs accelerates reaction kinetics using the proximity effect. Taking advantage of the signal amplification of P-CHA as well as the rapid reaction rate of the DNA walker in 80 min, the proposed sensor detects TK1 mRNA with a low detection limit of 14 pM and may then be applied to clinical diagnosis.

Herpes simplex virus type 1 (HSV-1) is a neurotropic alphaherpesvirus that establishes a lifelong infection in sensory neurons of infected individuals, accompanied with intermittent reactivation of latent virus causing (a)symptomatic virus shedding. Whereas acyclovir (ACV) is a safe and highly effective antiviral to treat HSV-1 infections, long-term usage can lead to emergence of ACV resistant (ACVR) HSV-1 and subsequently ACV refractory disease. Here, we isolated an HSV-1 strain from a patient with reactivated herpetic eye disease that did not respond to ACV treatment. The isolate carried a novel non-synonymous F289S mutation in the viral UL23 gene encoding the thymidine kinase (TK) protein. Because ACV needs conversion by viral TK and subsequently cellular kinases to inhibit HSV-1 replication, the UL23 gene is commonly mutated in ACVR HSV-1 strains. The potential role of the F289S mutation causing ACVR was investigated using CRISPR/Cas9-mediated HSV-1 genome editing. Reverting the F289S mutation in the original clinical isolate to the wild-type sequence S289F resulted in an ACV-sensitive (ACVS) phenotype, and introduction of the F289S substitution in an ACVS HSV-1 reference strain led to an ACVR phenotype. In summary, we identified a new HSV-1 TK mutation in the eye of a patient with ACV refractory herpetic eye disease, which was identified as the causative ACVR mutation with the aid of CRISPR/Cas9-mediated genome engineering technology. Direct editing of clinical HSV-1 isolates by CRISPR/Cas9 is a powerful strategy to assess whether single residue substitutions are causative to a clinical ACVR phenotype.

OBJECTIVE: Classical serum cancer biomarkers, such as carcinoembryonic antigen (CEA) and cancer antigen 19-9 (CA 19-9), remain important tools in colorectal cancer (CRC) management for disease follow up. However, their sensitivity and specificity are low for diagnostic and prognostic evaluation. The aim of this study was to evaluate the potential of biomarkers reflecting biological activity of tumors - tissue polypeptide specific antigen (TPS), cytokeratin fragment 19 (CYFRA 21-1), thymidine kinase (TK), insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGF-BP3) - together with the CEA and CA 19-9 in CRC diagnosis and prognosis.
METHODS: This is a retrospective study including 148 CRC patients and 68 age-matched healthy subjects. Serum biomarkers were measured in pre-operative serum samples using immunoanalytical methods. The end-point for the diagnostic evaluation was the area under the receiving operating characteristic curve (AUC ROC) of the biomarkers. The end-point for the prognostic evaluation was overall survival.
RESULTS: Serum levels of CEA, CA 19-9, TPS, and TK were significantly increased in CRC early-stage patients compared with healthy controls. Each of the studied biomarkers had AUC between 0.6 and 0.7. Analysis of survival demonstrated that the patients with CEA, CA 19-9, cytokeratin, and TK above optimal cut offs had significantly shorter survival. A multivariate analysis performed on all the study biomarkers resulted in the selection of CYFRA 21-1 as the best performing biomarker with hazard ratio 10.413.
CONCLUSIONS: The combination of cytokeratins and thymidine kinase with classical cancer biomarkers enables the prediction of tumor aggressiveness and long-term prognosis.

Amplified nanoprobes based on hybridization chain reaction (HCR) have been widely developed for the detection of intracellular low abundance mRNA. However, the formed chain-like assembly decorated with fluorophore would be degraded rapidly by endogenous enzyme, resulting in failure of the long-term fluorescence imaging. To address this issue, herein, a composite signal-amplifying strategy that integrates HCR into protein-binding signal amplification (HPSA) was communicated for the in situ imaging of mRNA by avoiding signal fluctuation. Different from conventional HCR-based nanoprobes (HCR-nanoprobe), the HCR was used as the signal-triggered mode and the amplifying signal generated from in situ fluorophore-protein binding in cells, which can maintain high stability of the signal for a long time. As a proof-of-principle, a nanobeacon based on HPSA (HPSA-nanobeacon) was constructed to detect TK1 mRNA. Taking advantage of the double signal-amplifying mode, the endogenous TK1 mRNA was sensitively detected and the fluorescence signal was maintained for more than 8 h in HepG2 cells. The attempt in this work provides a new option to the current signal-amplifying strategy for sensing nucleic acid targets with high stability, significantly enhancing the acquisition of intracellular molecular information.

UNASSIGNED: The aim of this study was to develop a nomogram, using serum thymidine kinase 1 protein (STK1p) combined with ultrasonography parameters, to early predict central lymph node metastasis (CLNM) in patients with papillary thyroid carcinoma (PTC) pre-surgery.
UNASSIGNED: Patients with PTC pre-surgery in January 2021 to February 2023 were divided into three cohorts: the observation cohort (CLNM, n = 140), the control cohort (NCLNM, n = 128), and the external verification cohort (CLNM, n = 50; NCLNM, n = 50). STK1p was detected by an enzyme immunodot-blot chemiluminescence analyzer and clinical parameters were evaluated by ultrasonography.
UNASSIGNED: A suitable risk threshold value for STK1p of 1.7 pmol/L was selected for predicting CLNM risk by receiver operating characteristic (ROC) curve analysis. Multivariate analysis identified the following six independent risk factors for CLNM: maximum tumor size >1 cm [odds ratio (OR) = 2.406, 95% confidence interval (CI) (1.279-4.526), p = 0.006]; capsule invasion [OR = 2.664, 95% CI (1.324-5.360), p = 0.006]; irregular margin [OR = 2.922; 95% CI (1.397-6.111), p = 0.004]; CLN flow signal [OR = 3.618, 95% CI (1.631-8.027), p = 0.002]; tumor-foci number ≥2 [OR = 4.064, 95% CI (2.102-7.859), p < 0.001]; and STK1p ≥1.7 pmol/L [OR = 7.514, 95% CI (3.852-14.660), p < 0.001]. The constructed nomogram showed that the area under the ROC curve for the main dataset was 0.867 and that for the validation dataset was 0.830, exhibiting effectivity, and was recalculated to a total score of approximately 383. Through monitoring the response post-surgery, all patients were assessed as tumor-free at 12 months post-surgery, which was significantly associated with a reduction in STK1p to disease-free levels.
UNASSIGNED: We demonstrate for the first time that a novel nomogram including STK1p combined with ultrasonography can assist in the clinical prevention of CLNM, by facilitating timely, individualized prophylactic CLNM dissection, thereby reducing the risk of secondary surgery and the probability of recurrence.

A precisely designed dual-color biosensor has realized a visual assessment of thymidine kinase 1 (TK1) mRNA in both living cells and cell lysates. The oligonucleotide probe is constructed by hybridizing the antisense strand of the target and two recognition sequences, in which FAM serves as the donor and TAMRA as the acceptor. Once interacting with the target, two recognition strands are replaced, and then the antisense complementary sequence forms a more stable double-stranded structure. Due to the increasing spatial distance between two dyes, the FRET is attenuated, leading to a rapid recovery of FAM fluorescence and a reduction of TAMRA fluorescence. A discernible color response from orange to green could be observed by the naked eye, with a limit of detection (LOD) of 0.38 nM and 5.22 nM for spectrometer- and smartphone-based assays, respectively. The proposed ratiometric method transcends previous reports in its capacities in visualizing TK1 expression toward reliable nucleic acid biomarker analysis, which might establish a general strategy for ratiometric biosensing via strand displacement.

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Complex structures and devices, both natural and artificial, can often undergo assembly and disassembly. Assembly and disassembly allow multiple stimuli to initiate, for example, the assembly and disassembly of primary cilia under the control of E3 ubiquitin ligases and deubiquitinases. Although biology relies on such schemes, they are rarely available in materials science. Here, we demonstrate a DNA-functionalized colloidal Au response to endogenous biomarkers to trigger simultaneous assembly and disassembly techniques. Colloidal Au is initially inert because the starting DNA strands are paired and prehybridized. TK1 mRNA competes to bind one of the paired strands and release its complement. The released complement binds to the next colloidal Au to initiate assembly, and APE1 can shear the colloidal Au assembly binding site to initiate disassembly. Our strategy provides temporal and spatial logic control during colloidal Au assembly and disassembly, and this simultaneous assembly and disassembly process can be used for sequential detection and cellular imaging of two biomarkers, effectively reducing signal false-positive results and shortening detection time. This work highlights biomarker-controlled colloidal Au simultaneous assembly and disassembly in ways that are simple and versatile, with the potential to enrich the application scope of DNA nanotechnology and provide an idea for the application of precision medicine testing.

Thymidine kinase 1 (TK1) is a marker of cell proliferation that can be used for early screening, treatment monitoring, and evaluating the prognosis of patients with tumors. The main purpose of this study was to develop clinically applicable TK1 antibodies, establish an appropriate detection method, and provide material and technical support for the research and clinical application for different types of tumors. Experimental mice were immunized with the C-terminal 31 peptide of human TK1 to screen monoclonal cell lines capable of stably secreting specific antibodies. Monoclonal antibodies were then prepared, purified and screened for optimal pairing following the identification of purity and isotype. Finally, based on the principles adopted by the double-antibody sandwich detection method, we constructed a lateral flow immunochromatographic assay (LFIA) to quantify the concentration of TK1 in serum samples when using a gold nanoparticle-labeled anti-TK1 monoclonal antibody as a probe. The limit of detection for TK1 in serum was 0.31 pmol/L with a detection range of 0.31-50 pmol/L. The spiked recoveries ranged from 97.7% to 109.0% with an analytical precision of 5.7-8.2%; there was no cross-reactivity with common proteins in the serum. The established LFIA also exhibited good consistency with commercially available chemiluminescent immunoassay kits for the detection of clinical samples. The LFIA developed in this study has the advantages of high sensitivity, accuracy, reproducibility and strong specificity, and provides a new technical tool for the quantitative detection of TK1.