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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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cells regularly repair numerous mutations. However, the effect of CRISPR/Cas9-induced dsDNA breaks on the repair processes of naturally occurring genome-wide mutations is unclear. In this study, we used TSCE5 cells with the heterozygous thymidine kinase genotype (TK +/- ) to examine these effects. We strategically inserted the target sites for guide RNA (gRNA)/Cas9 and I-SceI into the functional allele and designed the experiment such that deletions of > 81 bp or base substitutions within exon five disrupted the TK gene, resulting in a TK -/- genotype. TSCE5 cells in the resting state exhibited 16 genome-wide mutations that affected cellular functions. After gRNA/Cas9 editing, these cells produced 859 mutations, including 67 high-impact variants that severely affected cellular functions under standard culture conditions. Mutation profile analysis indicated a significant accumulation of C to A substitutions, underscoring the widespread induction of characteristic mutations by gRNA/Cas9. In contrast, gRNA/Cas9-edited cells under conditions of S∼G2/M arrest and cyclin-dependent kinase 1 inhibition showed only five mutations. Transcriptomic analysis revealed the downregulation of DNA replication genes and upregulation of alternative DNA repair genes, such as zinc finger protein 384 (ZNF384) and dual specificity phosphatase, under S∼G2/M conditions. Additionally, activation of nucleotide and base excision repair gene, including O-6-methylguanine-DNA methyltransferase and xeroderma pigmentosum complementation group C, was observed. This study highlights the profound impact of CRISPR/Cas9 editing on genome-wide mutation processes and underscores the emergence of novel DNA repair pathways. Finally, our findings provide significant insights into the maintenance of genome integrity during genome editing., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The monkeypox spread has been announced a public health emergency of international concern (PHEIC) by the World Health Organization (WHO). Both monkeypox and smallpox viruses are placed in the genus Orthopoxvirus. Despite recommendations for the administration of smallpox drugs versus monkeypox, no specific drug for monkeypox has yet been introduced. A reliable and effective method against this outbreak can be the use of natural products. This study aimed for identification of natural flavonoid derivatives as potential thymidine kinase inhibitors, the main drug target of monkeypox virus. Thymidine kinase protein structure was predicted by homology modeling and the quality of generated model was evaluated. Then, the interaction between natural flavonoids and the modeled thymidine kinase was explored by molecular docking. Based on docking results, more than half of the flavonoids with higher docking scores compared to reference drug (ganciclovir) were exhibited better binding affinities toward the protein. In addition, stability of the top flavonoids including eupatorin, fisetin, rhamnetin and scutellarein, was confirmed by MD simulations and binding free energy calculations using MM/PBSA analysis. These selected compounds were also shown acceptable results for drug likeness and ADMET analysis. Therefore, the results of the study showed that these flavonoids could be considered as potential thymidine kinase inhibitors for use against monkeypox virus., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Neural stem cells (NSCs) are considered to be valuable candidates for delivering a variety of anti-cancer agents, including oncolytic viruses, to brain tumors. However, owing to the previously reported tumorigenic potential of NSC cell lines after intranasal administration (INA), here we identified the human hepatic stellate cell line LX-2 as a cell type capable of longer resistance to replication of oncolytic adenoviruses (OAVs) as a therapeutic cargo, and that is non-tumorigenic after INA. Our data show that LX-2 cells can longer withstand the OAV XVir-N-31 replication and oncolysis than NSCs. By selecting the highly migratory cell population out of LX-2, an offspring cell line with a higher and more stable capability to migrate was generated. Additionally, as a safety backup, we applied genomic herpes simplex virus thymidine kinase (HSV-TK) integration into LX-2, leading to high vulnerability to ganciclovir (GCV). Histopathological analyses confirmed the absence of neoplasia in the respiratory tracts and brains of immuno-compromised mice 3 months after INA of LX-2 cells. Our data suggest that LX-2 is a novel, robust, and safe cell line for delivering anti-cancer and other therapeutic agents to the brain., (© 2023 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Malignant tumors have become a significant risk factor for human mortality. Although there have been notable advancements in the treatment of tumors, patient prognosis remains poor. In recent years, gene diagnosis and gene therapy have brought great benefits to patients. Deoxythymidine kinase (DTYMK) is a highly promising biomarker, has been studied by many scholars, and plays a crucial role in the occurrence and development of various types of cancer. The abnormal expression of DTYMK is involved in tumor occurrence and development, and may also serve as a biomarker for tumor diagnosis, treatment, and prognosis. Several experimental studies have shown that DTYMK can impact tumor progression by regulating mechanisms such as cell cycle, tumor microenvironment, immune infiltration, and signaling pathways. Therefore, this article focuses on clarifying the mechanism of DTYMK in tumors and exploring its clinical application value to help patients prolong their survival cycle and improve their quality of life., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Glioblastoma multiforme (GBM) is the most invasive form of primary brain astrocytoma, resulting in poor clinical outcomes. Herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapy is considered a promising strategy for GBM treatment. Since Connexin43 (Cx43) expression is reduced in GBM cells, increasing Cx43 levels could enhance the effectiveness of gene therapy. The present study aims to examine the impact of fluoxetine on HSV-TK/GCV gene therapy in human GBM cells using human olfactory ensheathing cells (OECs) as vectors. The effect of fluoxetine on Cx43 levels was assessed using the western blot technique. GBM-derived astrocytes and OECs-TK were Cocultured, and the effect of fluoxetine on the Antitumor effect of OEC-TK/GCV gene therapy was evaluated using MTT assay and flow cytometry. Our results showed that fluoxetine increased Cx43 levels in OECs and GBM cells and augmented the killing effect of OECs-TK on GBM cells. Western blot data revealed that fluoxetine enhanced the Bax/Bcl2 ratio and the levels of cleaved caspase-3 in the coculture of OECs-TK and GBM cells. Moreover, flow cytometry data indicated that fluoxetine increased the percentage of apoptotic cells in the coculture system. This study suggests that fluoxetine, by upregulating Cx43 levels, could strengthen the Antitumor effect of OEC-TK/GCV gene therapy on GBM cells., (© 2023 Wiley Periodicals LLC.)

Breast cancer is the most common cancer diagnosis worldwide accounting for 1 out of every 8 cancer diagnoses. The elevated expression of Thymidine Kinase 1 (TK1) is associated with more aggressive tumor grades, including breast cancer. Recent studies indicate that TK1 may be involved in cancer pathogenesis; however, its direct involvement in breast cancer has not been identified. Here, we evaluate potential pathogenic effects of elevated TK1 expression by comparing HCC 1806 to HCC 1806 TK1-knockdown cancer cells (L133). Transcriptomic profiles of HCC 1806 and L133 cells showed cell cycle progression, apoptosis, and invasion as potential pathogenic pathways affected by TK1 expression. Subsequent in-vitro studies confirmed differences between HCC 1806 and L133 cells in cell cycle phase progression, cell survival, and cell migration. Expression comparison of several factors involved in these pathogenic pathways between HCC 1806 and L133 cells identified p21 and AKT3 transcripts were significantly affected by TK1 expression. Creation of a protein-protein interaction map of TK1 and the pathogenic factors we evaluated predict that the majority of factors evaluated either directly or indirectly interact with TK1. Our findings argue that TK1 elevation directly increases HCC 1806 cell pathogenicity and is likely occurring by p21- and AKT3-mediated mechanisms to promote cell cycle arrest, cellular migration, and cellular survival., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Bitter et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Immune cells can recognize tumor-associated antigens released from dead tumor cells, which elicit immune responses, potentially resulting in tumor regression. Tumor cell death induced by chemotherapy has also been reported to activate immunity. However, various studies have reported drug-induced immunosuppression or suppression of inflammation by apoptotic cells. Thus, this study aimed to investigate whether apoptotic tumor cells trigger antitumor immunity independent of anticancer treatment. Local immune responses were evaluated after direct induction of tumor cell apoptosis using a Herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system. The inflammatory response was significantly altered at the tumor site after apoptosis induction. The expression of cytokines and molecules that activate and suppress inflammation simultaneously increased. The HSV-tk/GCV-induced tumor cell apoptosis resulted in tumor growth suppression and promoted T lymphocyte infiltration into tumors. Therefore, the role of T cells after inducing tumor cell death was explored. CD8 T cell depletion abrogated the antitumor efficacy of apoptosis induction, indicating that tumor regression was mainly dependent on CD8 T cells. Furthermore, CD4 T cell depletion inhibited tumor growth, suggesting the potential role of CD4 T cells in suppressive tumor immunity. Tumor tissues were evaluated after tumor cell apoptosis and CD4 T cell depletion to elucidate this immunological mechanism. Foxp3 and CTLA4, regulatory T-cell markers, decreased. Furthermore, arginase 1, an immune-suppressive mediator induced by myeloid cells, was significantly downregulated. These findings indicate that tumors accelerate CD8 T cell-dependent antitumor immunity and CD4 T cell-mediated suppressive immunity. These findings could be a therapeutic target for immunotherapy in combination with cytotoxic chemotherapy., (© 2023 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Metabolic reprogramming plays a crucial role in the development of hepatocellular carcinoma (HCC). However, the key drivers of metabolic reprogramming underlying HCC progression remain unclear. Using a large-scale transcriptomic database and survival correlation screening, we identify thymidine kinase 1 (TK1) as a key driver. The progression of HCC is robustly mitigated by TK1 knockdown and significantly aggravated by its overexpression. Furthermore, TK1 promotes the oncogenic phenotypes of HCC not only through its enzymatic activity and production of deoxythymidine monophosphate (dTMP) but also by promoting glycolysis via binding with protein arginine methyltransferase 1 (PRMT1). Mechanistically, TK1 directly binds PRMT1 and stabilizes it by interrupting its interactions with tripartite-motif-containing 48 (TRIM48), which inhibits its ubiquitination-mediated degradation. Subsequently, we validate the therapeutic capacity of hepatic TK1 knockdown in a chemically induced HCC mouse model. Therefore, targeting both the enzyme-dependent and -independent activity of TK1 may be therapeutically promising for HCC treatment., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: The viral thymidine kinase (TK) phosphorylates the antiviral medication famciclovir (FCV), which treats herpes simplex virus (HSV-TK). The phosphorylated FCV destroys the infected cells by preventing cellular DNA synthesis., Objective: We hypothesize that FCV impurity, which is a related substance to FCV, should be efficient in killing cells independent of HSV-TK and is currently the most widely used suicide agent for gene therapy of cancer., Methods: This study proposes the binding affinity of these derivatives for the active site of TK through molecular docking to a protein (PDB ID: 1W4R). The derivatives' reliability was ensured through the in-silico preliminary drug designing model by screening their Lipinski rule of five violations, if any, ADMET prediction for their profile using online tools. Using MOE 2009.10 computational software, we performed molecular docking of approximately 22 famciclovir derivatives alongside the famciclovir drug., Results: Our results suggest that these derivatives are indicative of possible chemical stability irrespective of all the parameters used to evaluate the selected derivatives as a possible drug candidates for their cytotoxicity. FC20 (i.e., 2-(2-(2-((1-(9-(4-Acetoxy-3-(acetoxymethyl)butyl)-2-amino-9Hpurin- 8-yl)ethyl)amino)-9H-purin-9-yl)ethyl)propane-1,3-diyl diacetate) and FC21 (i.e., 2-Amino-1,9- dihydro-9-(4-hydroxybutyl)-6H-purin-6-one), showed maximum and minimum scores of -26.95 and - 7.21 kcal/mol, respectively when compared to famciclovir (-15.4122 kcal/mol)., Conclusion: Considering that there might be a cytotoxicity effect due to competition between protein TK and the suicidal gene of famciclovir derivatives. The outcome of the study proved that the FCV impurity could successfully modify an HSV-TK-dependent antiviral drug into an anti-tumor drug. Further, it can be used for the design and development of novel compounds of FCV impurity that could be cytotoxic agents if properly delivered to cancer cells., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Brivudin, (( E )-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) can be considered the gold standard for the treatment of varicella-zoster virus (VZV) infections, such as herpes zoster (shingles). It is available for clinical use in most European countries (except for the UK) and over the whole world (except for the US and Canada). Besides VZV its activity spectrum also includes various other herpesviruses, such as herpes simplex virus type 1 (HSV-1). Its activity against VZV and HSV-1 depends on phosphorylation by the virus-encoded thymidine kinase (TK). In its active form (BVDU TP or BVDU 5'-triphosphate), it can act as both substrate and inhibitor of the viral (i.e., HSV-1) DNA polymerase. It has proven to be effective against herpes zoster, including post-herpetic neuralgia (PHN). It is contra-indicated in patients concomitantly treated by 5-fluorouracil (FU), since its degradation product, ( E )-5-(2-bromovinyl)uracil, is inhibitory to the catabolism of FU, which may enhance the toxicity of the latter. A new compound, the bicyclic nucleoside analogue (BCNA) Cf-1743, has been described, which is a more potent inhibitor of VZV replication than BVDU and which does not interfere with the catabolism of FU. It is applicable orally, as its 5'-valine ester FV-100 (Fermavir), but has not (yet) been marketed for clinical use.

Chimeric fusion transcription factors are oncogenic hallmarks of several devastating cancer entities including pediatric sarcomas, such as Ewing sarcoma (EwS) and alveolar rhabdomyosarcoma (ARMS). Despite their exquisite specificity, these driver oncogenes have been considered largely undruggable due to their lack of enzymatic activity.Here, we show in the EwS model that - capitalizing on neomorphic DNA-binding preferences - the addiction to the respective fusion transcription factor EWSR1-FLI1 can be leveraged to express therapeutic genes.We genetically engineered a de novo enhancer-based, synthetic and highly potent expression cassette that can elicit EWSR1-FLI1-dependent expression of a therapeutic payload as evidenced by episomal and CRISPR-edited genomic reporter assays. Combining in silico screens and immunohistochemistry, we identified GPR64 as a highly specific cell surface antigen for targeted transduction strategies in EwS. Functional experiments demonstrated that anti-GPR64-pseudotyped lentivirus harboring our expression cassette can specifically transduce EwS cells to promote the expression of viral thymidine kinase sensitizing EwS for treatment to otherwise relatively non-toxic (Val)ganciclovir and leading to strong anti-tumorigenic, but no adverse effects in vivo. Further, we prove that similar vector designs can be applied in PAX3-FOXO1-driven ARMS, and to express immunomodulatory cytokines, such as IL-15 and XCL1, in tumor entities typically considered to be immunologically 'cold'.Collectively, these results generated in pediatric sarcomas indicate that exploiting, rather than suppressing, the neomorphic functions of chimeric transcription factors may open inroads to innovative and personalized therapies, and that our highly versatile approach may be translatable to other cancers addicted to oncogenic transcription factors with unique DNA-binding properties., (© 2022. The Author(s).)

Pseudorabies (also called Aujeszky's disease) is a highly infectious viral disease caused by the pseudorabies virus (PRV, or Suid herpesvirus 1). Although the disease has been controlled by immunization with the PRV-attenuated vaccine, the emerging PRV variants can escape the immune surveillance in the vaccinated pig, resulting in recent outbreaks. Furthermore, the virus has been detected in other animals and humans, indicating cross-transmission of PRV. However, the mechanism of PRV cross-species transmission needs further study. In this study, we compared the amino acid sequences of glycoproteins (gD), gL, and thymidine kinase (TK) of PRV strains, human PRV hSD-1 2019 strain, and the attenuated strain Bartha-K61, followed by predication of their spatial conformation. In addition, the interactions between the viral gD protein and host nectin-1, nectin-2, and HS were also evaluated via molecular docking. The results showed that the amino acid sequence homology of the gD, gL, and TK proteins of hSD-1 2019 and JL-CC was 97.5%, 94.4%, and 99.1%, respectively. Moreover, there were mutations in the amino acid sequences of gD, gL, and TK proteins of hSD-1 2019 and JL-CC compared with the corresponding reference sequences of the Bartha strain. The mutations of gD, gL, and TK might not affect the spatial conformation of the protein domain but may affect the recognition of antibodies and antigen epitopes. Moreover, the gD protein of JL-CC, isolated previously, can bind to human nectin-1, nectin-2, and HS, suggesting the virus may be highly infectious and pathogenic to human beings.

Thymidylates are generated by several partially overlapping metabolic pathways in different subcellular locations. This interconnectedness complicates an understanding of how thymidylates are formed in vivo. Analyzing a comprehensive collection of mutants and double mutants on the phenotypic and metabolic level, we report the effect of de novo thymidylate synthesis, salvage of thymidine, and conversion of cytidylates to thymidylates on thymidylate homeostasis during seed germination and seedling establishment in Arabidopsis (Arabidopsis thaliana). During germination, the salvage of thymidine in organelles contributes predominantly to the thymidylate pools and a mutant lacking organellar (mitochondrial and plastidic) thymidine kinase has severely altered deoxyribonucleotide levels, less chloroplast DNA, and chlorotic cotyledons. This phenotype is aggravated when mitochondrial thymidylate de novo synthesis is additionally compromised. We also discovered an organellar deoxyuridine-triphosphate pyrophosphatase and show that its main function is not thymidylate synthesis but probably the removal of noncanonical nucleotide triphosphates. Interestingly, cytosolic thymidylate synthesis can only compensate defective organellar thymidine salvage in seedlings but not during germination. This study provides a comprehensive insight into the nucleotide metabolome of germinating seeds and demonstrates the unique role of enzymes that seem redundant at first glance., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Effective experimental prophylactic vaccines against viral pathogens such as herpes simplex virus type 1 (HSV-1) have been shown to protect the host through T and/or B lymphocyte-driven responses. Previously, we found a live-attenuated HSV-1 mutant, 0ΔNLS used as a prophylactic vaccine, provided significant protection against subsequent ocular HSV-1 challenge aligned with a robust neutralizing antibody response. Yet, how the virus mutant elicited the humoral immune response relative to parental virus was unknown. Herein, we present the characterization of B cell subsets in vaccinated mice at times after primary vaccination and following boost compared to the parental virus, termed GFP105. We found that 0∆NLS-vaccinated mice possessed more CD4 + follicular helper T (T FH ) cells, germinal B cells and class-switched B cells within the first 7 days post-vaccination. Moreover, 0∆NLS vaccination resulted in an increase in plasmablasts and plasma cells expressing amino-acid transporter CD98 along with an elevated titer of HSV-1-specific antibody compared to GFP105-vaccinated animals. Furthermore, O∆NLS-vaccine-induced CD4 + (T FH ) cells produced significantly more IL-21 compared to mice immunized with the parental HSV-1 strain. In contrast, there were no differences in the number of regulatory B cells comparing the two groups of immunized mice. In comparing sera recognition of HSV-1-encoded proteins, it was noted antiserum from GFP105-vaccinated mice immunoprecipitated HSV-1 thymidine kinase (TK) and glycoprotein M (gM) whereas sera from 0∆NLS-immunized mice did not even though both groups of vaccinated mice displayed similar neutralizing antibody titers to HSV-1 and were highly resistant to ocular HSV-1 challenge. Collectively, the results suggest (1) the live-attenuated HSV-1 mutant 0∆NLS elicits a robust B cell response that drives select B cell responses greater than the parental HSV-1 and (2) HSV-1 TK and gM are likely expendable components in efficacy of a humoral response to ocular HSV-1 infection., (© 2022. The Author(s).)

Thymidine kinase (TK2) deficiency causes mitochondrial DNA depletion syndrome. We aimed to report the clinical, biochemical, genetic, histopathological, and ultrastructural features of a cohort of paediatric patients with TK2 deficiency. Mitochondrial DNA was isolated from muscle biopsies to assess depletions and deletions. The TK2 genes were sequenced using Sanger sequencing from genomic DNA. All muscle biopsies presented ragged red fibres (RRFs), and the prevalence was greater in younger ages, along with an increase in succinate dehydrogenase (SDH) activity and cytochrome c oxidase (COX)-negative fibres. An endomysial inflammatory infiltrate was observed in younger patients and was accompanied by an overexpression of major histocompatibility complex type I (MHC I). The immunofluorescence study for complex I and IV showed a greater number of fibres than those that were visualized by COX staining. In the ultrastructural analysis, we found three major types of mitochondrial alterations, consisting of concentrically arranged lamellar cristae, electrodense granules, and intramitochondrial vacuoles. The pathological features in the muscle showed substantial differences in the youngest patients when compared with those that had a later onset of the disease. Additional ultrastructural features are described in the muscle biopsy, such as sarcomeric de-structuration in the youngest patients with a more severe phenotype.

Thymidine kinase 1 (TK1), involved in DNA precursor synthesis, is used as a serum biomarker in cancer diagnostics in both human and veterinary medicine. We investigated the utility of serum TK1 protein (TK1p) and TK1 activity (TK1a) determinations for prognosis and monitoring of canine hematological malignancies. The combination of TK1p or TK1a with canine C-reactive protein (CRP) determinations was also investigated. Serum samples from 51 client-owned dogs with naive hematological malignancies and from 149 healthy subjects were included. Serum TK1p levels were determined using a prototype TK1-ELISA, TK1a using the [ 3 H]-dThd phosphorylation assay, and CRP using an immunoturbidimetric assay. Mean TK1p in sera from dogs with tumors was significantly higher than from healthy dogs (mean ± SD = 3.9 ± 5.9 vs. 0.45 ± 0.15 ng/mL). Similarly, TK1a in hematological malignancies was significantly higher than in healthy dogs (mean ± SD = 15.1 ± 31.3 vs. 0.96 ± 0.33 pmol/min/mL). The receiver-operating characteristic indicated that a combination of TK1p or TK1a with CRP gave higher sensitivity than either biomarker alone for the prognosis of hematological malignancies. Median pretreatment TK1p and TK1a levels were significantly higher than in dogs in remission and correlated with clinical outcome. Kaplan-Meier curve analysis showed that naive dogs with high TK1p, TK1a, and CRP had significantly shorter survival. This study present two new polyclonal antibodies used in an ELISA system to determine TK1p. The study also show that combining TK1p or TK1a with CRP gave higher sensitivity than either biomarker alone. Monitoring patients in the study while undergoing chemotherapy, suggests that the TK1 + CRP combination could be useful in a biomarker panel, possibly aiding the prognosis and therapy monitoring of hematological malignancies in dogs., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Many cancers carry recurrent, change-of-function mutations affecting RNA splicing factors. Here, we describe a method to harness this abnormal splicing activity to drive splicing factor mutation-dependent gene expression to selectively eliminate tumor cells. We engineered synthetic introns that were efficiently spliced in cancer cells bearing SF3B1 mutations, but unspliced in otherwise isogenic wild-type cells, to yield mutation-dependent protein production. A massively parallel screen of 8,878 introns delineated ideal intronic size and mapped elements underlying mutation-dependent splicing. Synthetic introns enabled mutation-dependent expression of herpes simplex virus-thymidine kinase (HSV-TK) and subsequent ganciclovir (GCV)-mediated killing of SF3B1-mutant leukemia, breast cancer, uveal melanoma and pancreatic cancer cells in vitro, while leaving wild-type cells unaffected. Delivery of synthetic intron-containing HSV-TK constructs to leukemia, breast cancer and uveal melanoma cells and GCV treatment in vivo significantly suppressed the growth of these otherwise lethal xenografts and improved mouse host survival. Synthetic introns provide a means to exploit tumor-specific changes in RNA splicing for cancer gene therapy., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Dioscin (Dio), steroid saponin, exists in several medicinal herbs with potent anticancer efficacy. This study aimed to explore the effect of Dio on the immune-related modulation and synergistic therapeutic effects of the herpes simplex virus thymidine kinase/ganciclovir (HSV-Tk/GCV) suicide gene therapy system in murine melanoma, thereby providing a research basis to improve the potential immunomodulatory mechanism underlying combination therapy. Using both in vitro and in vivo experiments, we confirmed the immunocidal effect of Dio-potentiated suicide gene therapy on melanoma. The results showed that Dio upregulated connexin 43 (Cx43) expression and improved gap junction intercellular communication (GJIC) in B16 cells while increasing the cross-presentation of antigens by dendritic cells (DCs), eventually promoting the activation and antitumor immune killing effects of CD8 + T lymphocytes. In contrast, inhibition or blockade of the GJIC function (overexpression of mutant Cx43 tumor cells/Gap26) partially reversed the potentiating effect. The significant synergistic effect of Dio on HSV-Tk/GCV suicide gene therapy was further investigated in a B16 xenograft mouse model. The increased number and activation ratio of CD8 + T lymphocytes and the levels of Gzms-B, IFN-γ, and TNF-α in mice reconfirmed the potential modulatory effects of Dio on the immune system. Taken together, Dio targets Cx43 to enhance GJIC function, improve the antigens cross-presentation of DCs, and activate the antitumor immune effect of CD8 + T lymphocytes, thereby providing insights into the potential immunomodulatory mechanism underlying combination therapy., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Objective: Gastric cancer is one of the most lethal malignancies in the world. However, the current research on the diagnosis and treatment of nano-ultrasound contrast agents in the field of tumor is mostly focused on breast cancer, ovarian cancer, prostate cancer, liver cancer, etc. Due to the interference of gas in the stomach, there is no report on the treatment of gastric cancer. Herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) therapy system is the most mature tumor suicide gene in cancer treatment. At the same time, in order to improve its safety and efficiency, we designed a gastric tumor targeted ultrasound-triggered phase-transition nano ultrasound contrast agent PFH/AGM-CBA/HSV-TK/Liposome (PAHL)-Affibody complex., Methods: In our study, guanidinylated SS-PAAs polymer poly(agmatine/N, N'-cystamine-bis-acrylamide) (AGM-CBA) was used as a nuclear localization vector of suicide gene to form a polyplex, perfluorohexane (PFH) was used as ultrasound contrast agent, liposomes were used to encapsulate perfluorohexane droplets and the polyplexes of AGM-CBA/HSV-TK, and affibody molecules were conjugated to the prepared PAHL in order to obtain a specific targeting affinity to human epidermal growth factor receptor type 2 (ErbB2) at gastric cancer cells. With the aid of ultrasound targeted microbubble destruction technology and the nuclear localization effect of AGM-CBA vector, the transfection efficiency of the suicide gene in gastric cancer cells was significantly increased, leading to significant apoptosis of gastric cancer cells., Results: It was shown that PAHL-Affibody complex was nearly spherical with an average diameter of 560 ± 28.9 nm, having higher and specific affinity to ErbB2 (+) gastric cells. In vitro experiments further confirmed that PAHL could target gastric cancer cells expressing ErbB2. In a contrast-enhanced ultrasound scanning study, the prepared ultrasound-triggered phase-change nano-ultrasound contrast agent, PAHL, showed improved ultrasound enhancement effects. With the application of the low-frequency ultrasound, the gene transfection efficiency of PAHL was significantly improved,   thereby inducing significant apoptosis in gastric cancer cells., Conclusion: This study constructs PFH/AGM-CBA/HSV-TK/Liposome-Affibody nano ultrasound contrast agent, which provides new ideas for the treatment strategy of ErbB2-positive gastric cancer and provides some preliminary experimental basis for its inhibitory effect., Competing Interests: The authors declare no conflicts of interest., (© 2022 Zhou et al.)

Background: Deoxythymidine triphosphate (dTTP) is an essential building block of DNA, and defects in enzymes involved in dTTP synthesis cause neurodegenerative disorders. For instance, mutations in DTYMK, the gene coding for thymidylate kinase (TMPK), cause severe microcephaly in human. However, the mechanism behind this is not well-understood. Here we used the zebrafish model and studied (i) TMPK, an enzyme required for both the de novo and the salvage pathways of dTTP synthesis, and (ii) thymidine kinases (TK) of the salvage pathway in order to understand their role in neuropathology., Results: Our findings reveal that maternal-stored dNTPs are only sufficient for 6 cell division cycles, and the levels of dNTPs are inversely correlated to cell cycle length during early embryogenesis. TMPK and TK activities are prominent in the cytosol of embryos, larvae and adult fish and brain contains the highest TMPK activity. During early development, TMPK activity increased gradually from 6 hpf and a profound increase was observed at 72 hpf, and TMPK activity reached its maximal level at 96 hpf, and remained at high level until 144 hpf. The expression of dtymk encoded Dtymk protein correlated to its mRNA expression and neuronal development but not to the TMPK activity detected. However, despite the high TMPK activity detected at later stages of development, the Dtymk protein was undetectable. Furthermore, the TMPK enzyme detected at later stages showed similar biochemical properties as the Dtymk enzyme but was not recognized by the Dtymk specific antibody., Conclusions: Our results suggest that active dNTP synthesis in early embryogenesis is vital and that Dtymk is essential for neurodevelopment, which is supported by a recent study of dtymk knockout zebrafish with neurological disorder and lethal outcomes. Furthermore, there is a novel TMPK-like enzyme expressed at later stages of development., (© 2022. The Author(s).)

The role played by certain domestic species such as dogs as a translational model in comparative oncology shows great interest to develop new therapeutic strategies in brain tumors. Gliomas are a therapeutic challenge that represents the most common form of malignant primary brain tumors in humans and the second most common form in dogs. Gene-directed enzyme/prodrug therapy using adipose mesenchymal stem cells (Ad-MSCs) expressing the herpes simplex virus thymidine kinase (TK) has proven to be a promising alternative in glioblastoma therapy, through its capacity to migrate and home to the tumor and delivering local cytotoxicity avoiding other systemic administration. In this study, we demonstrate the possibility for canine Ad-MSCs (cAd-MSCs) to be genetically engineered efficiently with a lentiviral vector to express TK (TK-cAd-MSCs) and in combination with ganciclovir (GCV) prodrug demonstrated its potential antitumor efficacy in vitro and in vivo in a mice model with the human glioblastoma cell line U87. TK-cAd-MSCs maintained cell proliferation, karyotype stability, and MSCs phenotype. Genetic modification significantly affects its secretory profile, both the analyzed soluble factors and exosomes. TK-cAd-MSCs showed a high secretory profile of some active antitumor immune response cytokines and a threefold increase in the amount of secreted exosomes, with changes in their protein cargo. We also found that the prodrug protein is not released directly into the culture medium by TK-cAd-MSCs. We believe that our work provides new perspectives for glioblastoma gene therapy in dogs and a better understanding of this therapy in view of its possible implantation in humans., Competing Interests: The authors have declared that no competing interests exist.

Cell therapies based on pluripotent stem cells (PSC), have opened new therapeutic strategies for neurodegenerative diseases. However, insufficiently differentiated PSC can lead to tumor formation. Ideally, safety switch therapies should selectively kill proliferative transplant cells while preserving post-mitotic neurons. In this study, we evaluated the potential of nucleoside analogs and thymidine kinase-based suicide genes. Among tested thymidine kinase variants, the humanized SR39 (SR39h) variant rendered cells most sensitive to suicide induction. Unexpectedly, post-mitotic neurons with ubiquitous SR39h expression were killed by ganciclovir, but were spared when SR39h was expressed under the control of the cell cycle-dependent Ki67 promoter. The efficacy of six different nucleoside analogs to induce cell death was then evaluated. Penciclovir (PCV) showed the most interesting properties with an efficiency comparable to ganciclovir (GCV), but low toxicity. We tested three nucleoside analogs in vivo: at concentrations of 40 mg/kg/day, PCV and GCV prevented tumor formation, while acyclovir (ACV) did not. In summary, SR39h under the control of a cell cycle-dependent promoter appears most efficient and selective as safety switch for neural transplants. In this setting, PCV and GCV are efficient inducers of cell death. Because of its low toxicity, PCV might become a preferred alternative to GCV.

Despite its importance in the nucleoside (and nucleoside prodrug) metabolism, the structure of the active conformation of human thymidine kinase 1 (hTK1) remains elusive. We perform microsecond molecular dynamics simulations of the inactive enzyme form bound to a bisubstrate inhibitor that was shown experimentally to activate another TK1-like kinase, Thermotoga maritima TK ( Tm TK). Our results are in excellent agreement with the experimental findings for the Tm TK closed-to-open state transition. We show that the inhibitor induces an increase of the enzyme radius of gyration due to the expansion on one of the dimer interfaces; the structural changes observed, including the active site pocket volume increase and the decrease in the monomer-monomer buried surface area and of the number of hydrogen bonds (as compared to the inactive enzyme control simulation), indicate that the catalytically competent (open) conformation of hTK1 can be assumed in the presence of an activating ligand.

Mitochondrial thymidine kinase 2 (TK2) is an essential enzyme for mitochondrial dNTP synthesis in many tissues. Deficiency in TK2 activity causes devastating mitochondrial diseases. Here we investigated several residues involved in substrate binding and catalysis. We showed that mutations of Gln-110 and Glu-133 affected Mg 2+ and ATP binding, and thus are crucial for TK2 function. Furthermore, mutations of Gln-110 and Tyr-141 altered the kinetic behavior, suggesting their involvement in substrate binding through conformational changes. Since the 3 D structure of TK2 is still unknown, and thus, the identification of key amino acids for TK2 function may help to explain how TK2 mutations cause mitochondrial diseases.

Defects in the replication, maintenance, and repair of mitochondrial DNA (mtDNA) constitute a growing and genetically heterogeneous group of mitochondrial disorders. Multiple genes participate in these processes, including thymidine kinase 2 (TK2) encoding the mitochondrial matrix protein TK2, a critical component of the mitochondrial nucleotide salvage pathway. TK2 deficiency (TK2d) causes mtDNA depletion, multiple deletions, or both, which manifest predominantly as mitochondrial myopathy. A wide clinical spectrum phenotype includes a severe, rapidly progressive, early onset form (median survival: < 2 years); a less severe childhood-onset form; and a late-onset form with a variably slower rate of progression. Clinical presentation typically includes progressive weakness of limb, neck, facial, oropharyngeal, and respiratory muscle, whereas limb myopathy with ptosis, ophthalmoparesis, and respiratory involvement is more common in the late-onset form. Deoxynucleoside monophosphates and deoxynucleosides that can bypass the TK2 enzyme defect have been assessed in a mouse model, as well as under open-label compassionate use (expanded access) in TK2d patients, indicating clinical efficacy with a favorable side-effect profile. This treatment is currently undergoing testing in clinical trials intended to support approval in the US and European Union (EU). In the early expanded access program, growth differentiation factor 15 (GDF-15) appears to be a useful biomarker that correlates with therapeutic response. With the advent of a specific treatment and given the high morbidity and mortality associated with TK2d, clinicians need to know how to recognize and diagnose this disorder. Here, we summarize translational research about this rare condition emphasizing clinical aspects.

T-cell therapy with T cells that are re-directed to hepatitis B virus (HBV)-infected cells by virus-specific receptors is a promising therapeutic approach for treatment of chronic hepatitis B and HBV-associated cancer. Due to the high number of target cells, however, side effects such as cytokine release syndrome or hepatotoxicity may limit safety. A safeguard mechanism, which allows depletion of transferred T cells on demand, would thus be an interesting means to increase confidence in this approach. In this study, T cells were generated by retroviral transduction to express either an HBV-specific chimeric antigen receptor (S-CAR) or T-cell receptor (TCR), and in addition either inducible caspase 9 (iC9) or herpes simplex virus thymidine kinase (HSV-TK) as a safety switch. Real-time cytotoxicity assays using HBV-replicating hepatoma cells as targets revealed that activation of both safety switches stopped cytotoxicity of S-CAR- or TCR-transduced T cells within less than one hour. In vivo , induction of iC9 led to a strong and rapid reduction of transferred S-CAR T cells adoptively transferred into AAV-HBV-infected immune incompetent mice. One to six hours after injection of the iC9 dimerizer, over 90% reduction of S-CAR T cells in the blood and the spleen and of over 99% in the liver was observed, thereby limiting hepatotoxicity and stopping cytokine secretion. Simultaneously, however, the antiviral effect of S-CAR T cells was diminished because remaining S-CAR T cells were mostly non-functional and could not be restimulated with HBsAg. A second induction of iC9 was only able to deplete T cells in the liver. In conclusion, T cells co-expressing iC9 and HBV-specific receptors efficiently recognize and kill HBV-replicating cells. Induction of T-cell death via iC9 proved to be an efficient means to deplete transferred T cells in vitro and in vivo containing unwanted hepatotoxicity., Competing Interests: KW and UP are co-founders and shareholders of SCG Cell Therapy Pte. Ltd. KW is partially employed by SCG Cell Therapy GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Klopp, Schreiber, Kosinska, Pulé, Protzer and Wisskirchen.)

Stochastic fluctuations in gene expression ("noise") are often considered detrimental, but fluctuations can also be exploited for benefit (e.g., dither). We show here that DNA base excision repair amplifies transcriptional noise to facilitate cellular reprogramming. Specifically, the DNA repair protein Apex1, which recognizes both naturally occurring and unnatural base modifications, amplifies expression noise while homeostatically maintaining mean expression levels. This amplified expression noise originates from shorter-duration, higher-intensity transcriptional bursts generated by Apex1-mediated DNA supercoiling. The remodeling of DNA topology first impedes and then accelerates transcription to maintain mean levels. This mechanism, which we refer to as "discordant transcription through repair" ("DiThR," which is pronounced "dither"), potentiates cellular reprogramming and differentiation. Our study reveals a potential functional role for transcriptional fluctuations mediated by DNA base modifications in embryonic development and disease., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Our goal was to analyze post mortem tissues of an adult patient with late-onset thymidine kinase 2 (TK2) deficiency who died of respiratory failure. Compared with control tissues, we found a low mtDNA content in the patient's skeletal muscle, liver, kidney, small intestine, and particularly in the diaphragm, whereas heart and brain tissue showed normal mtDNA levels. mtDNA deletions were present in skeletal muscle and diaphragm. All tissues showed a low content of OXPHOS subunits, and this was especially evident in diaphragm, which also exhibited an abnormal protein profile, expression of non-muscular β-actin and loss of GAPDH and α-actin. MALDI-TOF/TOF mass spectrometry analysis demonstrated the loss of the enzyme fructose-bisphosphate aldolase, and enrichment for serum albumin in the patient's diaphragm tissue. The TK2-deficient patient's diaphragm showed a more profound loss of OXPHOS proteins, with lower levels of catalase, peroxiredoxin 6, cytosolic superoxide dismutase, p62 and the catalytic subunits of proteasome than diaphragms of ventilated controls. Strong overexpression of TK1 was observed in all tissues of the patient with diaphragm showing the highest levels. TK2 deficiency induces a more profound dysfunction of the diaphragm than of other tissues, which manifests as loss of OXPHOS and glycolytic proteins, sarcomeric components, antioxidants and overactivation of the TK1 salvage pathway that is not attributed to mechanical ventilation.

Pretreatment D-dimer levels have been reported to predict survival in several types of malignancies in human patients. The objective of this study was to evaluate the prognostic value of pretreatment D-dimer level in dogs with intermediate to high-grade non-Hodgkin lymphoma (NHL). In a prospective, randomized, double-blind study of F14512 vs etoposide phosphate, we assessed the prognostic value of pretreatment plasma D-dimer level in 48 client-owned dogs diagnosed with intermediate to high-grade NHL. The correlation between pretreatment plasma D-dimer level and various clinical features, progression-free survival (PFS) and overall survival (OS) was analysed. The median value of pretreatment plasma D-dimer level was 0.4 μg/mL (range: 0.1-14.3 μg/mL). High pretreatment plasma D-dimer level (>0.5 μg/mL) was detected in 44% (21/48) of dogs. High D-dimer levels were not correlated with naive vs relapsed lymphoma, clinical stage, substage, immunophenotype or treatment group. D-dimer levels >0.5 μg/mL were significantly associated with inferior median PFS (54 vs 104 days, P = .011) and OS (93 vs 169 days, P = .003). In the multivariate analysis, high D-dimer levels remained an independent predictor for worse PFS (HR: 3.21, 95% CI: 1.57-6.56, P = .001) and OS (HR: 3.87, 95% CI: 1.88-7.98; P < .001). This study suggests that pretreatment plasma D-dimer level can serve as a predictor of prognosis in dogs with intermediate to high-grade NHL. Further studies are warranted to confirm these findings., (© 2020 The Authors. Veterinary and Comparative Oncology published by John Wiley & Sons Ltd.)

The pathological hallmark of Parkinson's disease (PD) is the loss of neuromelanin-containing dopaminergic neurons within the substantia nigra pars compacta (SNpc). Additionally, numerous studies indicate an altered synaptic function during disease progression. To gain new insights into the molecular processes underlying the alteration of synaptic function in PD, a proteomic study was performed. Therefore, synaptosomes were isolated by density gradient centrifugation from SNpc tissue of individuals at advanced PD stages (N = 5) as well as control subjects free of pathology (N = 5) followed by mass spectrometry-based analysis. In total, 362 proteins were identified and assigned to the synaptosomal core proteome. This core proteome comprised all proteins expressed within the synapses without regard to data analysis software, gender, age, or disease. The differential analysis between control subjects and PD cases revealed that CD9 antigen was overrepresented and fourteen proteins, among them Thymidine kinase 2 (TK2), mitochondrial, 39S ribosomal protein L37, neurolysin, and Methionine-tRNA ligase (MARS2) were underrepresented in PD suggesting an alteration in mitochondrial translation within synaptosomes.

Acquisition of resistance to gemcitabine is a challenging clinical and biological hallmark property of refractory pancreatic cancer. Here, we investigated whether glycogen synthase kinase (GSK)-3β, an emerging therapeutic target in various cancer types, is mechanistically involved in acquired resistance to gemcitabine in human pancreatic cancer. This study included 3 gemcitabine-sensitive BxPC-3 cell-derived clones (BxG30, BxG140, BxG400) that acquired stepwise resistance to gemcitabine and overexpressed ribonucleotide reductase (RR)M1. Treatment with GSK3β-specific inhibitor alone attenuated the viability and proliferation of the gemcitabine-resistant clones, while synergistically enhancing the efficacy of gemcitabine against these clones and their xenograft tumors in rodents. The gemcitabine-resensitizing effect of GSK3β inhibition was associated with decreased expression of RRM1, reduced phosphorylation of Rb protein, and restored binding of Rb to the E2 transcription factor (E2F)1. This was followed by decreased E2F1 transcriptional activity, which ultimately suppressed the expression of E2F1 transcriptional targets including RRM1, CCND1 encoding cyclin D1, thymidylate synthase, and thymidine kinase 1. These results suggested that GSK3β participates in the acquisition of gemcitabine resistance by pancreatic cancer cells via impairment of the functional interaction between Rb tumor suppressor protein and E2F1 pro-oncogenic transcription factor, thereby highlighting GSK3β as a promising target in refractory pancreatic cancer. By providing insight into the molecular mechanism of gemcitabine resistance, this study identified a potentially novel strategy for pancreatic cancer chemotherapy., (© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

A variety of positive/negative selection systems have been exploited as genome engineering tools and screening platforms for genetic switches. While numerous positive-selection systems are available, only a handful of negative-selection systems are useful for such applications. We previously reported a powerful negative-selection system using herpes simplex virus thymidine kinase (HsvTK) and the mutagenic nucleoside analog 6-(β-d-2-deoxyribofuranosyl)-3,4-dihydro-8H-pyrimido [4,5-c][1,2] oxazin-7-one (dP). Upon addition of 1000 nM dP, cells expressing HsvTK quickly die, with unprecedented efficacy. However, this selection procedure elevates the spontaneous mutation rate of the host cells by 10-fold due to the mutagenic nature of dP. To decrease the operative concentration of dP required for negative selection, we systematically created the strains of Escherichia coli either by removing or overexpressing genes involved in DNA/RNA metabolism. We found that over-expression of NupC and NupG (nucleoside uptake-related inner membrane transporters), Tsx (outer membrane transporter), NdK (nucleotide kinase) sensitized E. coli cells to dP. Simultaneous overexpression of these three genes (ndk-nupC-tsx) significantly improved the dP-sensitivity of E. coli, lowering the necessary operative concentration of dP for negative selection by 10-fold. This enabled robust and selective elimination of strains harboring chromosomally-encoded hsvtk simply by adding as low as 100 nM dP, which causes only a modest increase in the spontaneous mutation frequency as compared to the cells without hsvtk., (Copyright © 2020 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

To investigate the expression level and clinical significance of fucosidase (AFU), glutamyltranspeptidase (GGT), and thymidine kinase 1 (TK1) in the serum of patients with primary liver cancer (PHC). A total of 135 PHC patients in Baoji Central Hospital from September 2014 to February 2018 were selected as a research group (RG), while 127 healthy subjects were collected as a control group (CG). Enzyme-linked immunosorbent assay (ELISA) was used to detect the AFU, GGT, and TK1 concentrations in serum of the two groups, and the diagnostic value of combined detection of the three for PHC was analyzed. AFU, GGT, and TK1 concentrations in serum of the RG were dramatically higher than those of the CG (P&lt; 0.050). ROC curve analysis showed that the sensitivity of AFU, GGT, and TK1 in the single diagnosis of PHC was 88.00, 94.00, and 66.00% respectively, and the specificity was 68.00, 54.00, and 66.00% respectively. The sensitivity and specificity of the combined diagnosis of PHC were 76.00 and 90.00%, respectively. AFU, GGT, and TK1 concentrations were different in the presence or absence of liver cirrhosis, TNM stage, and tissue type (P&lt; 0.050). AFU, GGT, and TK1 concentrations in PHC patients were dramatically higher than those in healthy people. Combined detection of the three has good diagnostic value for PHC.

Aim: We investigated the effect of the combination of heat shock protein 90α (HSP90α), alpha-fetoprotein (AFP) and thymidine kinase 1 (TK1) in the diagnosis of hepatocellular carcinoma (HCC). Methods & results: A total of 409 patients with HCC, 101 patients with benign liver disorder and 78 healthy individuals were retrospectively analyzed. HSP90α level was higher in HCC patients than in controls. The expression of HSP90α showed a positive correlation with tumor stage, differentiation, lymph node metastasis and tumor thrombus formation. The combination of HSP90α, AFP and TK1 improved the diagnostic sensitivity (89.24%) and the area under the receiver operating characteristic curve (0.919). Conclusion: The detection of HSP90α, AFP and TK1 is more efficient than a single tumor marker for the diagnosis of HCC.

GDF-15 is a biomarker for mitochondrial diseases. We investigated the application of GDF-15 as biomarker of disease severity and response to deoxynucleoside treatment in patients with thymidine kinase 2 (TK2) deficiency and compared it to FGF-21. GDF-15 and FGF-21 were measured in serum from 24 patients with TK2 deficiency treated 1-49 months with oral deoxynucleosides. Patients were grouped according to age at treatment and biomarkers were analyzed at baseline and various time points after treatment initiation. GDF-15 was elevated on average 30-fold in children and 6-fold in adults before the start of treatment. There was a significant correlation between basal GDF-15 and severity based on pretreatment distance walked (6MWT) and weight (BMI). During treatment, GDF-15 significantly declined, and the decrease was accompanied by relevant clinical improvements. The decline was greater in the paediatric group, which included the most severe patients and showed the greatest clinical benefit, than in the adult patients. The decline of FGF-21 was less prominent and consistent. GDF-15 is a potential biomarker of severity and of therapeutic response for patients with TK2 deficiency. In addition, we show evidence of clinical benefit of deoxynucleoside treatment, especially when treatment is initiated at an early age.

Glioblastoma, the most common and aggressive brain tumor with low survival rate, is difficult to be cured by neurosurgery or radiotherapy. Mounting evidence has reported the anti-inflammatory and anticancer effects of curcumin on several types of cancer in preclinical studies and clinical trials. To our knowledge, there is no platform or system that could be used to effectively and real-timely evaluate the therapeutic efficacy of curcumin for glioblastoma multiforme (GBM). In this study, we constructed a lentivirus vector with triple-reporter genes ( Fluc / GFP / tk ) and transduced into rat F98 glioblastoma cells to establish an orthotopic F98/ FGT glioma-bearing rat model. In the model, the therapeutic efficacies for curcumin alone, radiation alone, and their combination were evaluated via noninvasive bioluminescent imaging and overall survival measurements. At the cell level, curcumin is capable of causing a G2/M cell cycle arrest and sensitizing the F98 cells to radiation. In animal model, curcumin synergistically enhances the effects of radiotherapy on suppressing the growth of both transplanted glioma cells and in situ brain tumors, and extending the overall survival periods longer than those of curcumin alone and radiation alone treatments. In conclusion, we have demonstrated that curcumin may serve as a novel radiosensitizer to combine with radiotherapy using the triple-reporter F98/ FGT animal model for effective and simultaneous evaluation of therapeutic efficacy.

Two non-animal test methods, KeratinoSens™ and LuSens, have been approved by the Organization of Economic Cooperation and Development (OECD) test guidelines for evaluating the sensitization potential of chemicals, and been positioned as a method for appraising key event (KE)-2, namely, the keratinocyte response component of the Adverse Outcome Pathway (AOP) in sensitization process. However, these two methods require separate cytotoxicity tests to determine the concentrations to be tested in the main test. Therefore, we developed a simple and highly accurate KE-2 test method named α-Sens that uses the dual luciferase assay system and attempted a further application of luciferase-based determination of cell viability to calculate the normalized Antioxidant response element (ARE)-mediated transcriptional activity, named normalized ARE Activity (nAA), to evaluate the sensitizing potential of chemicals. A cell line carrying the ARE-inducible Firefly luciferase reporter gene and Thymidine kinase (TK) promoter-driven Renilla luciferase gene was established and used for the α-Sens. A total of 28 chemicals, consisting of 19 skin sensitizers and nine non-skin sensitizers were tested by this assay system. The α-Sens yielded an accuracy (%), sensitivity (%), and specificity (%) against corresponding values for local lymph node assay of 96.4 %, 95.0 %, and 100 %, respectively, and for human data of 100 % for all. The α-Sens gave clear positive results for phenyl benzoate and eugenol, chemicals for which KeratinoSens™ or LuSens yielded false-negative results, using a new parameter. Our results suggest that better prediction capacity could be achieved by using nAA as a classifier compared to other existing KE-2 test methods. In conclusion, the α-Sens is promising as a simple and highly accurate in vitro skin sensitization test method for evaluation of KE-2., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In a retrospective study design, we explored the relationship between serum thymidine kinase 1 (TK1) concentration before radiotherapy and clinical parameters and evaluated the prognostic value of serum TK1 concentration before radiotherapy in breast cancer patients with type 2 diabetes mellitus. The present study finally consisted of 428 breast cancer patients with a mean age of 53.0 years. Compared with low TK1 group, the high TK1 group tended to have larger tumor size (P=0.011) and had more lymph node number (P=0.021). Significant differences were also observed in clinical stages I, II and III (P=0.000). There was no significant difference between TK1 and other clinical parameters. For disease-free survival (DFS), the univariate analysis indicated that the high TK1 increased the risk of poor prognosis (HR = 2.38, 95% CI: 1.64-4.23, P=0.000). The Kaplan-Meier curve indicated the high TK1 group was poorer than that in the low TK1 group (P=0.002). For the overall survival (OS), similar results were found that the high TK1 was related to poor OS (HR = 1.89, 95% CI: 1.34-3.67, P=0.000). The multivariate Cox regression indicated that the TK1 was still associated with DFS (HR = 1.83, 95% CI: 1.22-3.17, P=0.001) and OS (HR = 1.63, 95% CI: 1.19-2.08, P=0.006). The high pretreatment serum TK1 levels in breast cancer patients were associated with poor OS and DFS. TK1 could be a potential predictive factor in differential diagnosis of poor prognosis from all patients., (© 2020 The Author(s).)

Background: Deficiency in thymidine kinase 2 (TK2) or p53 inducible ribonucleotide reductase small subunit (p53R2) is associated with tissue specific mitochondrial DNA (mtDNA) depletion. To understand the mechanisms of the tissue specific mtDNA depletion we systematically studied key enzymes in dTMP synthesis in mitochondrial and cytosolic extracts prepared from adult rat tissues., Results: In addition to mitochondrial TK2 a cytosolic isoform of TK2 was characterized, which showed similar substrate specificity to the mitochondrial TK2. Total TK activity was highest in spleen and lowest in skeletal muscle. Thymidylate synthase (TS) was detected in cytosols and its activity was high in spleen but low in other tissues. TS protein levels were high in heart, brain and skeletal muscle, which deviated from TS activity levels. The p53R2 proteins were at similar levels in all tissues except liver where it was ~ 6-fold lower. Our results strongly indicate that mitochondria in most tissues are capable of producing enough dTTP for mtDNA replication via mitochondrial TK2, but skeletal muscle mitochondria do not and are most likely dependent on both the salvage and de novo synthesis pathways., Conclusion: These results provide important information concerning mechanisms for the tissue dependent variation of dTTP synthesis and explained why deficiency in TK2 or p53R2 leads to skeletal muscle dysfunctions. Furthermore, the presence of a putative cytosolic TK2-like enzyme may provide basic knowledge for the understanding of deoxynucleoside-based therapy for mitochondrial disorders.

Pre-clinical and clinical studies have shown that engineered tumoricidal neural stem cells (tNSCs) are a promising treatment strategy for the aggressive brain cancer glioblastoma (GBM). Yet, stabilizing human tNSCs within the surgical cavity following GBM resection is a significant challenge. As a critical step toward advancing engineered human NSC therapy for GBM, we used a preclinical variant of the clinically utilized NSC line HB1.F3.CD and mouse models of human GBM resection/recurrence to identify a polymeric scaffold capable of maximizing the transplant, persistence, and tumor kill of NSC therapy for post-surgical GBM. Using kinetic bioluminescence imaging, we found that tNSCs delivered into the mouse surgical cavity wall by direct injection persisted only 3 days. We found that delivery of tNSCs into the cavity on nanofibrous electrospun poly-l-lactic acid scaffolds extended tNSC persistence to 8 days. Modifications to fiber surface coating, diameter, and morphology of the scaffold failed to significantly extend tNSC persistence in the cavity. In contrast, tNSCs delivered into the post-operative cavity on gelatin matrices (GEMs) persisted 8-fold longer as compared to direct injection. GEMs remained permissive to tumor-tropic homing, as tNSCs migrated off the scaffolds and into invasive tumor foci both in vitro and in vivo. To mirror envisioned human brain tumor trials, we engineered tNSCs to express the prodrug/enzyme thymidine kinase (tNSCs tk ) and transplanted the therapeutic cells in the post-operative cavity of mice bearing resected orthotopic patient-derived GBM xenografts. Following administration of the prodrug ganciclovir, residual tumor volumes in mice receiving GEM/tNSCs were reduced by 10-fold at day 35, and median survival was extended from 31 to 46 days. Taken together, these data begin to define design parameters for effective scaffold/tNSC composites and suggest a new approach to maximizing the efficacy of tNSC therapy in human patient trials., (Published by Elsevier Inc.)

Gene-directed enzyme-prodrug therapy (GDEPT) is a promising approach for cancer therapy, but it suffers from poor targeted delivery in vivo. Polyethylenimine (PEI) is a cationic polymer efficient in delivering negatively charged nucleic acids across cell membranes; however, it is highly toxic in vivo. Hence, we efficiently reduced PEI toxicity without compromising its transfection efficiency by conjugating it with poly(d,l-lactic- co -glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) as triblock copolymers through a multistep synthetic process. The synthesized nanoparticles showed efficient delivery of loaded nucleic acids to tumor cells in vitro and in vivo in mice. We used this nanoparticle to deliver a rationally engineered thymidine kinase (TK)-p53-nitroreductase (NTR) triple therapeutic gene against hepatocellular carcinoma (HCC), where p53 tumor suppressor gene is mutated in more than 85% of cancers. TK-p53-NTR triple gene therapy restores p53 function and potentiates cancer cell response to delivered prodrugs (ganciclovir (GCV) and CB1954). We used SP94 peptide-functionalized PLGA-PEG-PEI nanoparticles for the optimal delivery of TK-p53-NTR therapeutic gene in vivo. The nanoparticles prepared from the conjugated polymer showed high loading efficiency for the DNA and markedly enhanced TK-NTR-mediated gene therapy upon the simultaneous coexpression of p53 by the concurrent rescue of the endogenous apoptotic pathway in HCC cells of both p53-mutant and wild-type phenotypes in vitro. In vivo delivery of TK-p53-NTR genes by SP94-targeted PLGA-PEG-PEI NP in mice resulted in a strong expression of suicide genes selectively in tumors, and subsequent administration of GCV and CB1954 led to a decline in tumor growth, and established a superior therapeutic outcome against HCC. We demonstrate a highly efficient approach that exogenously supplements p53 to enable synergy with the outcome of TK-NTR suicide gene therapy against HCC.

Direct loading of fluorophore-labeled DNA molecules (named as flares) on gold nanoparticles (AuNPs) is a controllable and straightforward approach for intracellular imaging of target DNA molecules. However, the modification of AuNPs with flares requires a tedious and time-consuming procedure, additional reagents, or adenosine-rich DNA molecules. Here, we developed a rapid, simple, reagentless, and room-temperature approach for the modification of monolayer molybdenum disulfide nanosheets (M-MoS2 NSs) with dual-fluorophore-labeled flares, which were implemented for the ratiometric imaging of TK1 mRNA in living cells. The duplexes were prepared by hybridizing thiolated single-stranded DNA (ssDNA) to 6-carboxyfluorescein (FAM)- and 5-carboxytetramethylrhodamine (TAMRA)-labeled flares. The nanoflares were fabricated by conjugating the formed duplexes to the surface sulfur vacancy sites of the M-MoS2 NSs. The time required for preparing the nanoflares was found to be within 1 h. In the nanoflares, FAM stays away from TAMRA, leading to inefficient fluorescence resonance energy transfer (FRET). The presence of perfectly matched DNA (DNApm) molecules induces the liberation of the flares from the nanoflares. The liberated flares fold into hairpin-shaped structures, causing high FRET efficiency from FAM to TAMRA and efficient FAM-TAMRA static quenching. Following this mechanism, the nanoflares provided an effective platform for the ratiometric sensing of DNApm molecules with a limit of detection (at a signal-to-noise ratio of 3) of 8 nM and the linear range of 25-500 nM. Confocal microscopy experiments demonstrated that the nanoflares can be used to ratiometrically image TK1 mRA in HeLa and MCF-7 cells.

Resistance testing of antivirals to herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) can be done by phenotypic and genotypic methods. The determination of a resistant phenotype is based on the calculation of inhibitory concentrations for the antiviral drug, which should be tested. The main advantage of this resistance test is a clear interpretation of laboratory findings, but the method is time-consuming and a considerable experience is required by handling infectious virus. Genotypic resistance testing is based on the detection of resistance-related mutations in viral genes encoding the thymidine kinase and DNA polymerase, which need to be amplified and sequenced. This approach has the advantage of being faster, but only frameshift mutations, stops of translation, and amino acid substitutions described in the literature can be interpreted without doubt. By contrast, numerous novel amino acid substitutions are diagnostically less conclusive.

In vitro genetic toxicology assays are used to assess the genotoxic potential of chemicals or mixtures. They measure chromosome damage (e.g., micronucleus [MN] formation) or gene mutation, and different combinations of data generated from such assays are evaluated in concert in order to identify genotoxic hazards. Mode-of-action (MoA) information is also fundamental to understanding any apparent genotoxic response. In view of the importance of these types of data for full characterization of genotoxic potential, we leveraged relevant endpoints already established in the human TK6 cell line to develop a single integrated assay that measures MN formation, gene mutation (at the thymidine kinase locus), and MoA (DNA damage response biomarkers). Several prototypical direct-acting genotoxins (methyl methanesulfonate, mitomycin C, and 4-nitroquinoline 1-oxide), pro-genotoxins (benzo[a]pyrene and cyclophosphamide monohydrate), and one non-DNA reactive genotoxin (vinblastine sulfate) were assessed in the approach and found to elicit genotoxic profiles that were generally consistent with their MoA. In contrast, the non-genotoxic agents D-mannitol and (2-chloroethyl) trimethyl-ammonium chloride induced negligible effects on all endpoints up to a top concentration of 10 mM. Sodium diclofenac, presumed to be non-genotoxic, provoked an induction in the phosphoserine 10 -H3-positive cell population within a small window of concentrations (0.157-0.314 mM), as well as increases in γH2AX, nuclear p53, and MN at higher concentrations, although it had no effect on the mutation frequency endpoint. G 2 M cell cycle arrest was also largely observed in cells that exhibited genotoxicity in the in vitro MN assay. The TK6 cell-based integrated assay represents an in vitro approach that permits comprehensive genotoxicity analysis in a human-relevant test system. Moreover, its vis-à-vis nature may facilitate further comprehension of the range of effects that can manifest in human cells in response to DNA-damaging agents., Competing Interests: Declaration of Competing Interest All authors are employees of Philip Morris International., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.)

Thymidine kinase 1 (TK1) and deoxycytidine kinase (dCK) are required for the activation of thymidine and deoxycytidine analogs used in antiviral and anticancer therapies. Many anticancer drugs cause oxidative stress, and the rise of GSSG and other reactive oxygen species may lead to alteration in gene expression, protein, nucleic acids and lipid modifications. Here, we investigated the effects of oxidative stress and nucleoside analog on the expression and activity of TK1 and dCK. Treatment with GSSG resulted in glutathionylation of dCK and dGK but not TK1 and Dm-dNK, and glutathionylation led to increased dCK activity but decreased dGK activity. Treatment with hydrogen peroxide resulted in induction of TK1, however, the TK1 activity did not correlate with TK1 protein levels, indicating that TK1 protein was inactive. The cellular expression of dCK, however, was reduced but dCK activity was not affected at concentration ≤ 4 mM. Treatment with TFT or 5FdU resulted in downregulation of both TK1 and dCK. However, araC and dFdC treatment led to increased dCK protein but decreased dCK activity. In contrast, both TK1 protein and activity were upregulated after araC and dFdC treatment. Doxorubicin treatment led to upregulation of the TK1 but downregulation of dCK. In conclusion TK1 and dCK expression and activity are apparently affected by oxidative stress and treatment by nucleoside analogs. These results demonstrate the pharmacokinetic importance of characterizing the expression and activity of TK1 and dCK during chemotherapy with thymidine and deoxycytidine analogs in order to optimize their efficacy.

Determining mutant frequencies in endogenous reporter genes is a tool for identifying potentially genotoxic environmental agents, and discovering phenotypes prone to genomic instability and diseases, such as cancer. Here, we describe a high-throughput method for identifying mouse spleen lymphocytes with mutations in the endogenous X-linked hypoxanthine guanine phosphoribosyl transferase (Hprt) gene and the endogenous autosomal thymidine kinase (Tk) gene. The selective clonal expansion of mutant lymphocytes is based upon the phenotypic properties of HPRT- and TK-deficient cells. The same procedure can be utilized for quantifying Hprt mutations in most strains of mice (and, with minor changes, in other mammalian species), while mutations in the Tk gene can be determined only in transgenic mice that are heterozygous for inactivation of this gene. Expanded mutant clones can be further analyzed to classify the types of mutations in the Tk gene (small intragenic mutations vs. large chromosomal mutations) and to determine the nature of intragenic mutation at both the Hprt and Tk genes.

A physiological decrease in extracellular pH (pHe) alters the efficiency of DNA repair and increases formation of DNA double-strand breaks (DSBs). Whether this could translate into genetic instability and variations, was investigated using the TK6 cell model, in which positive selection of the TK1 gene loss-of-function mutations can be achieved from resistance to trifluorothymidine. Cell exposure to suboptimal pH (down to 6.9) for 3 weeks resulted in the 100 % frequency of a stronger frameshift mutation that has spread to both TK1 alleles, whereas weaker frameshift mutations within the 3'exon were eliminated during the selection. Suboptimal pHe values were also found to alter the proportion of the TK1 splicing variant expressed as percent spliced in index values and promote selection of truncated exons as well as intron retention. Although recovery at pH 7.4 did not reverse the selected frameshift mutation, reversal of splice variants and exon truncation towards control values were observed. Hence, suboptimal pHe can induce a combination of mutational events and splicing alterations within the same gene in the resistant clones. This model of positive selection for loss-of-function clearly demonstrates that suboptimal pHe may confer a similar growth advantage when such instability occurs within tumor suppressor genes., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2019 Elsevier B.V. All rights reserved.)