UNASSIGNED: DNA polymerase gamma (POLG)-related disorders are a group of rare neurodegenerative mitochondrial diseases caused by pathogenic variants in POLG, the gene encoding POLG. Patients may experience a range of signs and symptoms, including seizures, vision loss, myopathy, neuropathy, developmental impairment or regression, and liver failure. The diseases follow a progressive, degenerative course, with most affected individuals dying within 3 months-12 years of diagnosis. At present, there are no effective treatments for POLG-related disorders.
UNASSIGNED: In this study we report the interim 6-month data from a long term open-label, single arm phase 2 trial, in which we assessed the safety and efficacy of combination therapy with deoxycytidine and deoxythymidine (dC/dT) in children with POLG-related disorders. dC/dT was given enterally in powder form, dissolved in water. The primary outcome measures included Newcastle Mitochondrial Disease Scale (NMDS) score, serum growth differentiation factor 15 (GDF-15; a biomarker of mitochondrial dysfunction), electroencephalography (EEG), seizure diary, and blood and urine tests to assess end organ and mitochondrial function. Secondary outcome measures included recording of all adverse events to evaluate the safety of the intervention. The trial is registered with ClinicalTrials.gov, NCT04802707 (https://clinicaltrials.gov/ct2/show/NCT04802707). Data were collected from 14 October, 2021 to 13 December, 2023.
UNASSIGNED: We present 6-month interim data from the first ten people with POLG-related disorders enrolled in the trial, six with Alpers-Huttenlocher syndrome, two with ataxia-neuropathy spectrum, and two who do not fit into a classical POLG-related phenotype. During the 6 months of treatment, NMDS score improved from a mean of 27.3 at baseline to 20.7 at 6 months (estimated difference 6.0; 95% CI 2.5-∞). GDF-15 values remained stable or decreased in all patients; the mean decreased from 1031 pg/ml to 729 pg/ml (estimated difference 200; 95% CI 12-∞). 8/10 patients had abnormal baseline EEG; improvement in EEG was seen in 5 of these 8. There were no significant changes in other blood and urine testing. Regarding adverse events, two patients experienced diarrhea that spontaneously resolved.
UNASSIGNED: dC/dT is a promising treatment option for people with POLG-related disorders. Further research is needed to assess the long-term safety and efficacy in POLG-related disorders, as well as safety and efficacy in other mitochondrial DNA depletion disorders.
UNASSIGNED: This study was primarily funded by the Liam Foundation, with additional funding from the Savoy Foundation, Grand Défi Pierre Lavoie Foundation, and Fonds de Recherche du Québec - Santé.

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.

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 β 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.

Three compounds with fluorescence quantum yields in the range of 10- 5 to 10- 4 and emission spectra covering the UV/Vis spectral range are suggested as new references for the determination of small fluorescence quantum yields. The compounds are thymidine (dT) in water, dibenzoylmethane (DBM) in ethanol, and malachite green chloride (MG) in water, representing the blue, green, and red regions of the spectrum, respectively. All compounds are easily handled, photostable, and commercially available. Furthermore, these compounds exhibit a mirror-image symmetry between their absorption and fluorescence spectra. This symmetry, along with closely aligned fluorescence excitation and absorption spectra, confirms that the observed emissions originate from the compounds themselves. The fluorescence quantum yields were determined via a relative approach as well as Strickler-Berg analysis in conjunction with time resolved fluorescence spectroscopy. Within the respective error margins, the two approaches yielded identical results.

OBJECTIVE: 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.
CONCLUSIONS: Chronic hepatitis B patients with TC and CC genotype in rs3826160 have high telbivudine plasma concentration are at risk of elevated creatine kinase.

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.

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.

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.

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.

OBJECTIVE: 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.
CONCLUSIONS: Baseline [ 11 C]4DST PET/CT volume-based parameters can provide important prognostic information with p16-negative oropharyngeal, hypopharyngeal, and supraglottic cancer patients.