Nanochannel-based biosensor for ultrasensitive and label-free detection of thymidine kinase activity.

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.

[Metabolic engineering of Escherichia coli for thymidine production].

Thymidine, as a crucial precursor of anti-AIDS drugs (e.g., zidovudine and stavudine), has wide application potential in the pharmaceutical industry. In this study, we introduced the thymidine biosynthesis pathway into the wild-type Escherichia coli MG1655 by systems metabolic engineering to improve the thymidine production in E. coli. Firstly, deoA, tdk, udp, rihA, rihB, and rihC were successively deleted to block the thymidine degradation pathway and salvage pathway in the wild-type E. coli MG1655. Then, the pyrimidine nucleoside operons from Bacillus subtilis F126 were introduced to enlarge the metabolic flux of the uridylic acid synthesis pathway. Finally, the expression of uridylate kinase, ribonucleoside diphosphate reductase, thymidine synthase, and 5'-nucleotidase in the thymidine biosynthesis pathway was optimized to enhance the metabolic flux from uridylic acid to thymidine. The engineered THY6-2 strain produced 11.10 g/L thymidine in a 5 L bioreactor with a yield of 0.04 g/g glucose and productivity of 0.23 g/(L·h). In this study, we constructed a strain that used glucose as the only carbon source for efficient production of thymidine and did not harbor plasmids, which provided a reference for the research on other pyrimidine nucleosides.

Thymidine-dependent Staphylococcus aureus and lung function in patients with cystic fibrosis: a 10-year retrospective case-control study.

OBJECTIVE: Thymidine-dependent small-colony variants (TD-SCVs) of Staphylococcus aureus are being isolated with increasing frequency from patients with cystic fibrosis (CF). The aim of this study was to evaluate the relationship between TD-SCV isolation and pulmonary function in patients with CF, as well as to determine whether the emergence of TD-SCVs was associated with trimethoprim-sulfamethoxazole (TMP-SMX) use and with coinfection with other microorganisms. METHODS: This was a retrospective case-control study including patients with CF who visited the Clinical Hospital Complex of the Federal University of Paraná, in Curitiba, Brazil, between 2013 and 2022. Demographic, clinical, and spirometric data, as well as information on TD-SCVs and other isolated microorganisms, were collected from the medical records of patients with CF and TD-SCVs (TD-SCV group; n = 32) and compared with those of a matched group of patients with CF without TD-SCVs (control group; n = 64). RESULTS: Isolation of TD-SCVs was positively associated with TMP-SMX use (p = 0.009), hospitalization (p < 0.001), and impaired pulmonary function (p = 0.04). CONCLUSIONS: The use of TMP-SMX seems to contribute to the emergence of TD-SCVs, the isolation of which was directly associated with worse pulmonary function in our sample.

Distinguishing Isomeric Cyclobutane Thymidine Dimers by Ion Mobility and Tandem Mass Spectrometry.

Irradiation of the major conformation of duplex DNA found in cells (B form) produces cyclobutane pyrimidine dimers (CPDs) from adjacent pyrimidines in a head-to-head orientation (syn) with the C5 substituents in a cis stereochemistry. These CPDs have crucial implications in skin cancer. Irradiation of G-quadruplexes and other non-B DNA conformations in vitro produces, however, CPDs between nonadjacent pyrimidines in nearby loops with syn and head-to-tail orientations (anti) with both cis and trans stereochemistry to yield a mixture of six possible isomers of the T=T dimer. This outcome is further complicated by formation of mixtures of nonadjacent CPDs of C=T, T=C, and C=C, and successful analysis depends on development of specific and sensitive methods. Toward meeting this need, we investigated whether ion mobility mass spectrometry (IMMS) and MS/MS can distinguish the cis,syn and trans,anti T=T CPDs. Ion mobility can afford baseline separation and give relative mobilities that are in accord with predicted cross sections. Complementing this ability to distinguish isomers is MS/MS collisional activation where fragmentation also distinguishes the two isomers and confirms conclusions drawn from ion mobility analysis. The observations offer early support that ion mobility and MS/MS can enable the distinction of DNA photoproduct isomers.

Thymidylate synthase disruption to limit cell proliferation in cell therapies.

Stem and progenitor cells hold great promise for regenerative medicine and gene therapy approaches. However, transplantation of living cells entails a fundamental risk of unwanted growth, potentially exacerbated by CRISPR-Cas9 or other genetic manipulations. Here, we describe a safety system to control cell proliferation while allowing robust and efficient cell manufacture, without any added genetic elements. Inactivating TYMS, a key nucleotide metabolism enzyme, in several cell lines resulted in cells that proliferate only when supplemented with exogenous thymidine. Under supplementation, TYMS-/--pluripotent stem cells proliferate, produce teratomas, and successfully differentiate into potentially therapeutic cell types such as pancreatic ß cells. Our results suggest that supplementation with exogenous thymidine affects stem cell proliferation, but not the function of stem cell-derived cells. After differentiation, postmitotic cells do not require thymidine in vitro or in vivo, as shown by the production of functional human insulin in mice up to 5 months after implantation of stem cell-derived pancreatic tissue.

Creatine kinase elevation in chronic hepatitis B patients with telbivudine therapy: influence of telbivudine plasma concentration and single nucleotide polymorphisms of TK2, RRM2B, and NME4.

PURPOSE: To study the correlations of genetic variants of telbivudine phosphorylase kinases and telbivudine plasma concentration with creatine kinase elevation in chronic hepatitis B patients who received telbivudine. METHODS: An observational study was performed in China chronic hepatitis B patients receiving telbivudine therapy at 600 mg once daily. Plasma concentration was measured 12 h after taking telbivudine using ultra-performance liquid chromatography-tandem mass spectrometry and SNPs located in RRM2B, TK2, and NME4 was detected by MALDI-TOF mass spectrometry. All statistical analyses were performed with R 4.3.1 and all graphs were drawn by Origin 2023b and P value < 0.05 was considered statistically significant. RESULTS: A total of 140 patients receiving telbivudine therapy were recruited with a median plasma concentration of 952.49 (781.07-1238.98) ng/mL. The value of plasma concentration was proportional to the grade of creatine kinase elevation and the best telbivudine plasma concentration threshold to discriminate the grade 3/4 CK elevation was 1336.61 ng/mL. Multivariate analysis revealed that plasma concentration and rs3826160 were the independent risk factor of telbivudine-induced creatine kinase elevation. Patients with TC and CC genotype in rs3826160 not only had a higher incidence of creatine kinase elevation but also a higher plasma concentration than TT genotype carriers. CONCLUSION: Chronic hepatitis B patients with TC and CC genotype in rs3826160 have high telbivudine plasma concentration are at risk of elevated creatine kinase.

Cas9-assisted biological containment of a genetically engineered human commensal bacterium and genetic elements.

Sophisticated gene circuits built by synthetic biology can enable bacteria to sense their environment and respond predictably. Engineered biosensing bacteria outfitted with such circuits can potentially probe the human gut microbiome to prevent, diagnose, or treat disease. To provide robust biocontainment for engineered bacteria, we devised a Cas9-assisted auxotrophic biocontainment system combining thymidine auxotrophy, an Engineered Riboregulator (ER) for controlled gene expression, and a CRISPR Device (CD). The CD prevents the engineered bacteria from acquiring thyA via horizontal gene transfer, which would disrupt the biocontainment system, and inhibits the spread of genetic elements by killing bacteria harboring the gene cassette. This system tunably controlled gene expression in the human gut commensal bacterium Bacteroides thetaiotaomicron, prevented escape from thymidine auxotrophy, and blocked transgene dissemination. These capabilities were validated in vitro and in vivo. This biocontainment system exemplifies a powerful strategy for bringing genetically engineered microorganisms safely into biomedicine.

Fragmentation of 5-fluorouridine induced by low energy (< 12 eV) electrons: insights into the radiosensitization of DNA.

5-Fluorouracil is now routinely used in chemo- and radiotherapy. Incorporated within DNA, the molecule is bound to the sugar backbone, forming the 5-fluorouridine sub-unit investigated in the present work. For the clinical usage of the latter, no information exists on the mechanisms that control the radiosensitizing effect at the molecular level. As low energy (< 12 eV) electrons are abundantly produced along the radiation tracks during cancer treatment using beams of high energy particles, we study how these ballistic secondary electrons damage the sensitizing molecule. The salient result from our study shows that the N-glycosidic bonds are principally affected with a cross-section of approximately two orders of magnitude higher than the canonical thymidine, reflecting to some degree the surviving factor of radiation-treated carcinoma cells with and without 5-fluorouracil incorporation. This result may help in the comprehension of the radiosensitizing effect of the fluoro-substituted thymidine in DNA.

A synergistic two-drug therapy specifically targets a DNA repair dysregulation that occurs in p53-deficient colorectal and pancreatic cancers.

The tumor-suppressor p53 is commonly inactivated in colorectal cancer and pancreatic ductal adenocarcinoma, but existing treatment options for p53-mutant (p53Mut) cancer are largely ineffective. Here, we report a therapeutic strategy for p53Mut tumors based on abnormalities in the DNA repair response. Investigation of DNA repair upon challenge with thymidine analogs reveals a dysregulation in DNA repair response in p53Mut cells that leads to accumulation of DNA breaks. Thymidine analogs do not interrupt DNA synthesis but induce DNA repair that involves a p53-dependent checkpoint. Inhibitors of poly(ADP-ribose) polymerase (PARPis) markedly enhance DNA double-strand breaks and cell death induced by thymidine analogs in p53Mut cells, whereas p53 wild-type cells respond with p53-dependent inhibition of the cell cycle. Combinations of trifluorothymidine and PARPi agents demonstrate superior anti-neoplastic activity in p53Mut cancer models. These findings support a two-drug combination strategy to improve outcomes for patients with p53Mut cancer.

4'-C-Acetamidomethyl-2'-O-methoxyethyl Nucleic Acid Modifications Improve Thermal Stability, Nuclease Resistance, Potency, and hAgo2 Binding of Small Interfering RNAs.

In this study, we designed the 4'-C-acetamidomethyl-2'-O-methoxyethyl (4'-C-ACM-2'-O-MOE) uridine and thymidine modifications, aiming to test them into small interfering RNAs. Thermal melting studies revealed that incorporating a single 4'-C-ACM-2'-O-MOE modification in the DNA duplex reduced thermal stability. In contrast, an increase in thermal stability was observed when the modification was introduced in DNA:RNA hybrid and in siRNAs. Thermal destabilization in DNA duplex was attributed to unfavorable entropy, which was mainly compensated by the enthalpy factor to some extent. A single 4'-C-ACM-2'-O-MOE thymidine modification at the penultimate position of the 3'-end of dT20 oligonucleotides in the presence of 3'-specific exonucleases, snake venom phosphodiesterase (SVPD), demonstrated significant stability as compared to monomer modifications including 2'-O-Me, 2'-O-MOE, and 2'-F. In gene silencing studies, we found that the 4'-C-ACM-2'-O-MOE uridine or thymidine modifications at the 3'-overhang in the passenger strand in combination with two 2'-F modifications exhibited superior RNAi activity. The results suggest that the dual modification is well tolerated at the 3'-end of the passenger strand, which reflects better siRNA stability and silencing activity. Interestingly, 4'-C-ACM-2'-O-MOE-modified siRNAs showed considerable gene silencing even after 96 h posttransfection; it showed that our modification could induce prolonged gene silencing due to improved metabolic stability. Molecular modeling studies revealed that the introduction of the 4'-C-ACM-2'-O-MOE modification at the 3'-end of the siRNA guide strand helps to anchor the strand within the PAZ domain of the hAgo2 protein. The overall results indicate that the 4'-C-ACM-2'-O-MOE uridine and thymidine modifications are promising modifications to improve the stability, potency, and hAgo2 binding of siRNAs.

Predictive value of local control by 4'-[methyl-11C]-thiotymidine PET volume parameters in p16-negative oropharyngeal, hypopharyngeal, and supraglottic squamous cell carcinoma.

PURPOSE: We investigated the potential of baseline 4'-[methyl- 11 C]-thiothymidine ([ 11 C]4DST) PET for predicting loco-regional control of head and neck squamous cell carcinoma (HNSCC). METHODS: A retrospective analysis was performed using volumetric parameters, such as SUVmax, proliferative tumor volume (PTV), and total lesion proliferation (TLP), of pretreatment [ 11 C]4DST PET for 91 patients with HNSCC with primary lesions in the oral cavity, hypopharynx, supraglottis, and oropharynx, which included p16-negative patients. PTV and TLP were calculated for primary lesions and metastatic lymph nodes combined. We examined the association among the parameters and relapse-free survival and whether case selection focused on biological characteristics improved the accuracy of prognosis prediction. RESULTS: The area under the curves (AUCs) using PTV and TLP were high for the oropharyngeal/hypopharyngeal/supraglottis groups (0.91 and 0.87, respectively), whereas that of SUVmax was 0.66 ( P  < 0.01). On the other hand, the oral group had lower AUCs for PTV and TLP (0.72 and 0.77, respectively). When all cases were examined, the AUCs using PTV and TLP were 0.84 and 0.83, respectively. CONCLUSION: Baseline [ 11 C]4DST PET/CT volume-based parameters can provide important prognostic information with p16-negative oropharyngeal, hypopharyngeal, and supraglottic cancer patients.

Understanding the Multi-Functional Role of TCTP in the Regeneration Process of Earthworm, Perionyx excavatus.

BACKGROUND: Regeneration is a highly complex process that requires the coordination of numerous molecular events, and identifying the key ruler that governs is important to investigate. While it has been shown that TCTP is a multi-functional protein that regulates cell proliferation, differentiation, apoptosis, anti-apoptosis, stem cell maintenance, and immune responses, but only a few studies associated to regeneration have been reported. To investigate the multi-functional role of TCTP in regeneration, the earthworm Perionyx excavatus was chosen. METHODS: Through pharmacological suppression of TCTP, amputation, histology, molecular docking, and western blotting, the multi-function role of TCTP involved in regeneration is revealed. RESULTS: Amputational studies show that P. excavatus is a clitellum-independent regenerating earthworm resulting in two functional worms upon amputation. Arresting cell cycle at the G1/S boundary using 2 mM Thymidine confirms that P. excavatus execute both epimorphosis and morphallaxis regeneration mode. The pharmacological suppression of TCTP using buclizine results in regeneration suppression. Following the combinatorial injection of 2 mM Thymidine and buclizine, the earthworm regeneration is completely blocked, which suggests a critical functional role of TCTP in morphallaxis. The pharmacological inhibition of TCTP also suppresses the key proteins involved in regeneration: Wnt3a (stem cell marker), PCNA (cell proliferation) and YAP1 (Hippo signalling) but augments the expression of cellular stress protein p53. CONCLUSION: The collective results indicate that TCTP synchronously is involved in the process of stem cell activation, cell proliferation, morphallaxis, and organ development in the regeneration event.

DFT investigation of the regioselective allylation of pyrimidine 2'-deoxynucleosides.

To understand the regioselectivity observed in the allylation of pyrimidine nucleosides and to identify the factors directing the reaction, a theoretical study of the regioselective allylation was carried out. Several key points were considered such as: the structure of the deprotonated nucleobase in the presence of Na+; the effect of the solvent on the dissociation and aggregation reactions of thymidine/Na+ ion pair; and the likely allylation reaction mechanisms involved. The results showed that the regioselectivity observed experimentally can be attributed to a greater stability of a dimeric form coupled to an increase of the reaction barrier in THF due to larger Na+ binding to the nucleobase.

Thymidine Kinase-Independent Click Chemistry DNADetect Probes for DNA Proliferation Assessment in Malaria Parasites.

Metabolic chemical probes are small-molecule reagents that utilize naturally occurring biosynthetic enzymes for in situ incorporation into biomolecules of interest. These reagents can be used to label, detect, and track important biological processes within living cells including protein synthesis, protein glycosylation, and nucleic acid proliferation. A limitation of current chemical probes, which have largely focused on mammalian cells, is that they often cannot be applied to other organisms due to metabolic differences. For example, the thymidine derivative 5-ethynyl-2'-deoxyuridine (EdU) is a gold standard metabolic chemical probe for assessing DNA proliferation in mammalian cells; however, it is unsuitable for the study of malaria parasites due to Plasmodium species lacking the thymidine kinase enzyme that is essential for metabolism of EdU. Herein, we report the design and synthesis of new thymidine-based probes that sidestep the requirement for a thymidine kinase enzyme in Plasmodium. Two of these DNADetect probes exhibit robust labeling of replicating asexual intraerythrocytic Plasmodium falciparum parasites, as determined by flow cytometry and fluorescence microscopy using copper-catalyzed azide-alkyne cycloaddition to a fluorescent azide. The DNADetect chemical probes are synthetically accessible and thus can be made widely available to researchers as tools to further understand the biology of different Plasmodium species, including laboratory lines and clinical isolates.

Telbivudine-induced rhabdomyolysis in a patient undergoing haemodialysis: A case report and review of literature.

Herein, we describe a case of acute rhabdomyolysis in a man in his early 50s undergoing haemodialysis and receiving the antiviral drug, telbivudine, for chronic hepatitis B virus (HBV) infection. Following diagnosis by electromyography (EMG), magnetic resonance image (MRI) scans and laboratory data (i.e., elevated serum creatinine kinase (CK) and myoglobin) telbivudine was discontinued and the patient was treated with methylprednisolone. While his CK and myoglobin levels decreased rapidly, his muscle weakness and pain improved slowly. Learning points include: patients undergoing haemodialysis and concomitantly receiving antiviral treatment for HBV, should have their serum levels of CK and myoglobin monitored regularly; treatment with corticosteroids maybe required; relief from rhabdomyolysis-induced muscle weakness and pain may be slow due to nerve fibre damage.

Tritiated thymidine induces developmental delay, oxidative stress and gene overexpression in developing zebrafish (Danio rerio).

Tritium is a betta emitter radionuclide. Being an isotope of hydrogen, it is easily transferred to different environmental compartments, and to human and non-human biota. Considering that tritium levels are expected to rise in the upcoming decades with the development of nuclear facilities producing tritium using fission processes, investigating the potential toxicity of tritium to human and non-human biota is necessary. Tritiated thymidine, an organic form of tritium, has been used in this study to assess its toxicity on fish embryo development. Zebrafish embryos (3.5 hpf; hours post fertilization) have been exposed to tritiated thymidine at three different activity concentrations (7.5; 40; 110 kBq/mL) for four days. These experiments highlighted that zebrafish development was affected by the exposure to organic tritium, with smaller larvae at 3 dpf after exposure to the two lowest dose rates (22 and 170 µGy/h), a delayed hatching after exposure to the two highest dose rates (170 and 470 µGy/h), an increase in the spontaneous tail movement (1 dpf) and a decrease in the heartbeat (3 dpf) after exposure to the highest dose rate. The results also highlighted an increase in ROS production in larvae exposed to the intermediate dose rate. A dysregulation of many genes, involved in apoptosis, DNA repair or oxidative stress, was also found after 1 day of exposure to the lowest tritium dose rate. Our results thus suggest that exposure to tritiated thymidine from a dose rate as low as 22 µGy/h can lead to sublethal effects, with an effect on the development, dysregulation of many genes and increase of the ROS production. This paper provides valuable information on toxic effects arising from the exposure of fish to an organic form of tritium, which was the main objective of this study.

Screening the NCI diversity set V for anti-MRSA activity: cefoxitin synergy and LC-MS/MS confirmation of folate/thymidine biosynthesis inhibition.

IMPORTANCE: New antibacterial agents are urgently needed to counter increasingly resistant bacteria. One approach to this problem is library screening for new antibacterial agents. Library screening efforts can be improved by increasing the information content of the screening effort. In this study, we screened the National Cancer Institute diversity set V against methicillin-resistant Staphylococcus aureus (MRSA) with several enhancements. One of these is to screen the library before and after microsomal metabolism as means to identify potential active metabolites. A second enhancement is to screen the library in the absence and presence of sub-minimum inhibitory concentration levels of another antibiotic, such as cefoxitin in this study. This identified four agents with synergistic activity with cefoxitin out of 16 agents with good MRSA activity alone. Finally, active agents from this effort were counter-screened in the presence of thymidine, which quickly identified three folate/thymidine biosynthesis inhibitors, and also screened for bactericidal vs bacteriostatic activity.

Hybrids of 1,4-Naphthoquinone with Thymidine Derivatives: Synthesis, Anticancer Activity, and Molecular Docking Study.

One of the most essential health problems is cancer, the first or second cause of death worldwide. Head and neck cancers are hard to detect due to non-specific symptoms. The treatment often relies on a combination of radio and chemotherapy. For this reason, the research of new anticancer compounds is fundamental. The natural and synthetic compounds with 1,4-naphthoquinone scaffold is characterized by high anticancer activity. The study aimed to evaluate the synthesis and anticancer activity of hybrids 1,4-naphthoquinone with thymidine derivatives. The series of compounds allows us to check the influence of the substituent in the C3' position of the thymidine moiety on the cytotoxicity against squamous cancer cell lines (SCC-9 and SCC-25) and submandibular gland cancer (A-253). An annexin V/propidium iodide (PI) co-staining assay shows that derivatives cause the apoptotic in SCC-25 and A-253 cell lines. The molecular docking study examined the interaction between the active site of the BCL-2 protein and the hybrids.

Evaluation of deoxythymidine-based cationic amphiphiles as antimicrobial, antibiofilm, and anti-inflammatory agents.

OBJECTIVES: We recently designed a series of cationic deoxythymidine-based amphiphiles that mimic the cationic amphipathic structure of antimicrobial peptides (AMPs). Among these amphiphiles, ADG-2e and ADL-3e displayed the highest selectivity against bacterial cells. In this study, ADG-2e and ADL-3e were evaluated for their potential as novel classes of antimicrobial, antibiofilm, and anti-inflammatory agents. METHODS: Minimum inhibitory concentrations of ADG-2e and ADL-3e against bacteria were determined using the broth microdilution method. Proteolytic resistance against pepsin, trypsin, α-chymotrypsin, and proteinase K was determined by radial diffusion and HPLC analysis. Biofilm activity was investigated using the broth microdilution and confocal microscopy. The antimicrobial mechanism was investigated by membrane depolarization, cell membrane integrity analysis, scanning electron microscopy (SEM), genomic DNA influence and genomic DNA binding assay. Synergistic activity was evaluated using checkerboard method. Anti-inflammatory activity was investigated using ELISA and RT-PCR. RESULTS: ADG-2e and ADL-3e showed good resistance to physiological salts and human serum, and a low incidence of drug resistance. Moreover, they exhibit proteolytic resistance against pepsin, trypsin, α-chymotrypsin, and proteinase K. ADG-2e and ADL-3e were found to kill bacteria by an intracellular target mechanism and bacterial cell membrane-disrupting mechanism, respectively. Furthermore, ADG-2e and ADL-3e showed effective synergistic effects when combined with several conventional antibiotics against methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Pseudomonas aeruginosa (MDRPA). Importantly, ADG-2e and ADL-3e not only suppressed MDRPA biofilm formation but also effectively eradicated mature MDRPA biofilms. Furthermore, ADG-2e and ADL-3e drastically decreased tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) gene expression and protein secretion in lipopolysaccharide (LPS)-stimulated macrophages, implying potent anti-inflammatory activity in LPS-induced inflammation. CONCLUSION: Our findings suggest that ADG-2e and ADL-3e could be further developed as novel antimicrobial, antibiofilm, and anti-inflammatory agents to combat bacterial infections.