A genetically related cluster of Salmonella Typhimurium cases in humans associated with ruminant livestock and related food chains, United Kingdom, August 2021-December 2022.

Following an outbreak of Salmonella Typhimurium in Wales in July 2021 associated with sheep meat and offal, further genetically related cases were detected across the UK. Cases were UK residents with laboratory-confirmed Salmonella Typhimurium in the same 5-single-nucleotide polymorphism (SNP) single-linkage cluster with specimen date between 01/08/2021-2031/12/2022. We described cases using routine (UK) and enhanced (Wales only) surveillance data. Exposures in cases in Wales were compared with non-Typhimurium Salmonella case-controls. Environmental Health Practitioners and the Food Standards Agency investigated supply chains of food premises reported by ≥2 cases. Animal, carcass, and environmental samples taken for diagnostic or monitoring purposes for gastrointestinal pathogens were included in microbiological investigations. We identified 142 cases: 75% in England, 23% in Wales and 3% in Scotland. Median age was 32 years, and 59% were male. Direct contact with sheep was associated with becoming a case (aOR: 14, 95%CI: 1.4-145) but reported by few (6/32 cases). No single food item, premises, or supplier linked all cases. Multi-agency collaboration enabled the identification of isolates in the same 5-SNP single-linkage cluster from a sheep carcass at an English abattoir and in ruminant, wildlife, poultry, and environmental samples, suggesting multiple vehicles and pathways of infection.

De Novo Resistance to Arg10-Teixobactin Occurs Slowly and Is Costly.

Bacterial pathogens are rapidly evolving resistance to all clinically available antibiotics. One part of the solution to this complex issue is to better understand the resistance mechanisms to new and existing antibiotics. Here, we focus on two antibiotics. Teixobactin is a recently discovered promising antibiotic that is claimed to "kill pathogens without detectable resistance" (L. L. Ling, T. Schneider, A. J. Peoples, A. L. Spoering, et al., Nature 517:455-459, 2015, https://doi.org/10.1038/nature14098). Moenomycin A has been extensively used in animal husbandry for over 50 years with no meaningful antibiotic resistance arising. However, the nature, mechanisms, and consequences of the evolution of resistance to these "resistance-proof" compounds have not been investigated. Through a fusion of experimental evolution, whole-genome sequencing, and structural biology, we show that Staphylococcus aureus can develop significant resistance to both antibiotics in clinically meaningful timescales. The magnitude of evolved resistance to Arg10-teixobactin is 300-fold less than to moenomycin A over 45 days, and these are 2,500-fold and 8-fold less than evolved resistance to rifampicin (control), respectively. We have identified a core suite of key mutations, which correlate with the evolution of resistance, that are in genes involved in cell wall modulation, lipid synthesis, and energy metabolism. We show the evolution of resistance to these antimicrobials translates into significant cross-resistance against other clinically relevant antibiotics for moenomycin A but not Arg10-teixobactin. Lastly, we show that resistance is rapidly lost in the absence of antibiotic selection, especially for Arg10-teixobactin. These findings indicate that teixobactin is worth pursuing for clinical applications and provide evidence to inform strategies for future compound development and clinical management.

Radiobiological effects of hypoxia-dependent uptake of 64Cu-ATSM: enhanced DNA damage and cytotoxicity in hypoxic cells.

PURPOSE: Hypoxia occurs frequently in cancers and can lead to therapeutic resistance due to poor perfusion and loss of the oxygen enhancement effect. (64)Cu-ATSM has shown promise as a hypoxia diagnostic agent due to its selective uptake and retention in hypoxic cells and its emission of positrons for PET imaging. (64)Cu also emits radiotoxic Auger electrons and beta(-) particles and may therefore exhibit therapeutic potential when concentrated in hypoxic tissue. METHODS: MCF-7 cells were treated with 0-10 MBq/ml (64)Cu-ATSM under differing oxygen conditions ranging from normoxia to severe hypoxia. Intracellular response to hypoxia was measured using Western blotting for expression of HIF-1alpha, while cellular accumulation of (64)Cu was measured by gamma counting. DNA damage and cytotoxicity were measured with, respectively, the Comet assay and clonogenic survival. RESULTS: (64)Cu-ATSM uptake in MCF-7 cells increased as atmospheric oxygen decreased (up to 5.6 Bq/cell at 20.9% oxygen, 10.4 Bq/cell at 0.1% oxygen and 26.0 Bq/cell at anoxia). Toxicity of (64)Cu-ATSM in MCF-7 cells also increased as atmospheric oxygen decreased, with survival of 9.8, 1.5 and 0% in cells exposed to 10 MBq/ml at 20.9, 0.1 and 0% oxygen. The Comet assay revealed a statistically significant increase in (64)Cu-ATSM-induced DNA damage under hypoxic conditions. CONCLUSION: The results support a model in which hypoxia-enhanced uptake of radiotoxic (64)Cu induces sufficient DNA damage and toxicity to overcome the documented radioresistance in hypoxic MCF-7 cells. This suggests that (64)Cu-ATSM and related complexes have potential for targeted radionuclide therapy of hypoxic tumours.

The Face of Clostriodes Difficile Infections in the Outpatient Setting.

BACKGROUND: It has long been well-established that Clostridiodes difficile infections (CDI) can cause severe morbidity and mortality. However, most of the literature to date has focused on hospital-diagnosed infections with less emphasis on clinic-based CDI cases. Guidelines from the 2010 IDSA/SHEA for CDI advocate for metronidazole as first-line therapy for mild to moderate CDI cases. However, the 2017 guidelines recommend oral vancomycin or fidaxomicin as first-line therapy due to their superior efficacy. Objective: The purpose of this study was to compare Clostriodes difficile infections in convenience samples of clinic vs. hospital patients. METHODS: In 2019, a retrospective, case-controlled study was performed by the first six authors between 2015-2017 (i.e., prior to the 2017 IDSA/SHEA CDI guidelines) to compare ambulatory and hospital CDI treatment prescriptions. Analytic data included frequency of White blood cells (WBC) and creatinine collection, frequency of severe CDI cases, compliance with the 2010 guidelines, CDI recurrence, and mortality. RESULTS: An eligible subgroup of N = 92 hospital patients at Spectrum Health Lakeland were more likely to have WBC (98.4% vs 32.6%, p<0.001) and creatinine (97.8 vs. 39.4, P < 0.001) drawn than 184 patients receiving clinic-based care. Hospital sampled patients were more likely to have severe CDI (46.7% vs 6.7%, p < 0.001). Mortality was less common in hospital patients (1.1% vs. 7.6%, p = 0.017) and the recurrence rates were similar. (21.2% inpatient vs. 28.3% outpatient, (p = 0.224). CONCLUSIONS: Based on these results, assessment of CDI severity remains limited in the ambulatory population due to the lack of severity markers. It is unclear if this is due to lack of available laboratory resources or difference in clinical presentation. Of those sample patients who have available markers of severity, patients receiving clinic-based diagnoses were less likely assessed to have severe CDI. Keywords: Cloistriodes difficile infection, ambulatory, severity markers.

Comparison of the Video Game Functional Assessment-Revised (VGFA-R) and Internet Gaming Disorder Test (IGD-20).

Initially labeled as internet addiction in the mid-1990s (e.g., Griffiths, 1996; Young, 1996), researchers have since focused on how specific online activities result in negative consequences for those who overuse and have problems with online applications such as online gambling and online sex (Griffiths, 2000; Potenza, 2017). More recently, this has been applied to online problematic video game play, often used synonymously with terms such as online video game addiction, online gaming addiction, and Internet gaming disorder (IGD). With the publication of the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5; American Psychiatric Association [APA], 2013), IGD was identified by the APA as warranting further study. The current proposed diagnostic criterion in the DSM-5 requires the presence of five of nine symptoms over a 12-month period. These include: (a) preoccupation or obsession with Internet games, (b) withdrawal symptoms when not playing Internet games, (c) an increasing need over time to spend more and more time playing video games, (d) failed attempts to stop or curb Internet gaming, (e) loss of interest in other activities such as hobbies, (f) continued overuse of Internet games even with knowledge of the impact of overuse on their life, (g) lying about extent of Internet game usage, (h) uses Internet games to relieve anxiety or guilt, and (i) has lost or put at risk an opportunity or relationship because of Internet games (American Psychiatric Association [APA], 2013). However, it is unclear if the disorder represents addiction to the internet or if IGD evaluates specific behaviors occurring within the context of the video gaming (Starcevic and Billieux, 2017; Young and Brand, 2017).

A randomized clinical trial of the Recovery Line among methadone treatment patients with ongoing illicit drug use.

BACKGROUND: Relapse, drug use, and treatment dropout are common challenges facing patients receiving methadone. Though effective, multiple barriers to face-to-face counseling exist. The Recovery Line (RL), an automated, self-management system based on Cognitive Behavioral Therapy, is a phone-based adjunctive treatment that provides low cost, consistent delivery and immediate therapeutic availability 24 h a day. METHODS: The current study was a 12-week randomized clinical efficacy trial of treatment-as-usual (TAU) only or RL + TAU for methadone treatment patients with continued illicit drug use (N = 82). Previous small trial phases evaluated methods to increase participant engagement and use of the RL and were incorporated into the current RL version. Primary outcomes were days of self-reported illicit drug abstinence and urine screens negative for illicit drugs. RESULTS: Days of self-reported illicit drug abstinence improved for patients in RL + TAU but not in TAU. Percent of urine screens negative for illicit drugs, coping skills efficacy, and retention in methadone treatment did not differ by condition. Patients in RL + TAU attended more substance use disorder treatment and self-help group sessions during treatment than those in TAU. RL system use was generally low and more system use was correlated with abstinence outcomes. CONCLUSIONS: Although the RL did not impact urine screen outcomes, it increases self-reported abstinence. Additional methods to increase patient engagement with automated, self-management systems for substance use disorder are needed.

p53-dependent global nucleotide excision repair of cisplatin-induced intrastrand cross links in human cells.

Cisplatin is an extremely effective chemotherapeutic agent used for the treatment of testicular and other solid tumours. It induces a variety of structural modifications in DNA, the most abundant being the GpG- and ApG-1,2-intrastrand cross links formed between adjacent purine bases. These cross links account for approximately 90% of cisplatin-induced DNA damage and are thought to be responsible for the cytotoxic activity of the drug. In human cells, the nucleotide excision repair (NER) process removes the intrastrand cross links from the genome, the efficiency of which is likely to be an important determinant of cisplatin cytotoxicity. We have investigated whether the p53 tumour suppressor status affects global NER of cisplatin-induced intrastrand cross links in human cells. We have used a (32)P-postlabelling method to monitor the removal of GpG- and ApG-intrastrand cross links from two human cell models (the 041TR system, in which p53 is regulated by a tetracycline-inducible promoter, together with WI38 fibroblasts and the SV40-transformed derivative VA13) that each differ in p53 status. We demonstrate that the absence of functional p53 leads to persistence of both cisplatin-induced intrastrand cross links in the genome, suggesting that p53 regulates NER of these DNA lesions. This observation extends the role of p53 in NER beyond enhancing the removal of environmentally induced DNA lesions to include those of clinical origin. Given the frequency of p53 mutations in human tumours, these results may have implications for the use of cisplatin in cancer chemotherapy.

Cysteines and Disulfide-Bridged Macrocyclic Mimics of Teixobactin Analogues and Their Antibacterial Activity Evaluation against Methicillin-Resistant Staphylococcus Aureus (MRSA).

Teixobactin is a highly potent cyclic depsipeptide which kills a broad range of multi-drug resistant, Gram-positive bacteria, such as Methicillin-resistant Staphylococcus aureus (MRSA) without detectable resistance. In this work, we describe the design and rapid synthesis of novel teixobactin analogues containing two cysteine moieties, and the corresponding disulfide-bridged cyclic analogues. These analogues differ from previously reported analogues, such as an Arg10-teixobactin, in terms of their macrocyclic ring size, and feature a disulfide bridge instead of an ester linkage. The new teixobactin analogues were screened against Methicillin-resistant Staphylococcus aureus and Methicillin-sensitive Staphylococcus aureus. Interestingly, one teixobactin analogue containing all l-amino acid building blocks showed antibacterial activity against MRSA for the first time. Our data indicates that macrocyclisation of teixobactin analogues with disulfide bridging is important for improved antibacterial activity. In our work, we have demonstrated the unprecedented use of a disulfide bridge in constructing the macrocyclic ring of teixobactin analogues.

Design and Syntheses of Highly Potent Teixobactin Analogues against Staphylococcus aureus, Methicillin-Resistant Staphylococcus aureus (MRSA), and Vancomycin-Resistant Enterococci (VRE) in Vitro and in Vivo.

The cyclic depsipeptide, teixobactin, kills a number of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), and Mycobacterium tuberculosis without detectable resistance. To date, teixobactin is the only molecule in its class that has shown in vivo antibacterial efficacy. In this work, we designed and synthesized 10 new in vivo ready teixobactin analogues. These analogues showed highly potent antibacterial activities against Staphylococcus aureus, MRSA, and vancomycin-resistant enterococci (VRE) in vitro. One analogue, d-Arg4-Leu10-teixobactin, 2, was found to be noncytotoxic in vitro and in vivo. Moreover, topical instillation of peptide 2 in a mouse model of S. aureus keratitis decreased the bacterial bioburden (>99.0% reduction) and corneal edema significantly as compared to untreated mouse corneas. Collectively, our results have established the high therapeutic potential of a teixobactin analogue in attenuating bacterial infections and associated severities in vivo.

The Myxobacterium Myxococcus xanthus Can Sense and Respond to the Quorum Signals Secreted by Potential Prey Organisms.

The myxobacterium Myxococcus xanthus is a predatory member of the soil microfauna, able to consume bacteria (Gram-negative, Gram-positive), archaea, and fungi. Many potential prey of M. xanthus communicate amongst themselves using acyl homoserine lactones (AHLs) as quorum signals. M. xanthus cannot itself produce AHLs, but could potentially benefit by responding to exogenous AHLs produced during signaling between proximal prey. Four AHLs of different side chain length were tested and all found to delay sporulation of M. xanthus vegetative cells, and to stimulate germination of myxospores, increasing the proportion of predatory vegetative cells in the population. The predatory activity and expansion rates of M. xanthus colonies were also found to be stimulated by AHLs. Thermally inactivated AHLs had no effect on M. xanthus cells, and the response to AHLs depended (non-linearly) on the length of AHL side chain, suggesting that the effect of AHLs was mediated by specific signaling within M. xanthus, rather than being a consequence of the chemical or physical properties of AHLs. Therefore, it seems that the presence of xenic quorum signaling molecules enhances the predatory activity of M. xanthus. AHLs increase the proportion of the population capable of predation, and stimulate the motility and predatory activity of vegetative cells. We therefore propose that in the wild, M. xanthus uses AHLs as markers of nearby prey, potentially eavesdropping on the conversations between prey organisms.

Measures of behavioral function predict duration of video game play: Utilization of the Video Game Functional Assessment - Revised.

Background Internet gaming disorder (IGD) was introduced in the DSM-5 as a way of identifying and diagnosing problematic video game play. However, the use of the diagnosis is constrained, as it shares criteria with other addictive orders (e.g., pathological gambling). Aims Further work is required to better understand IGD. One potential avenue of investigation is IGD's relationship to the primary reinforcing behavioral functions. This study explores the relationship between duration of video game play and the reinforcing behavioral functions that may motivate or maintain video gaming. Methods A total of 499 video game players began the online survey, with complete data from 453 participants (85% white and 28% female), were analyzed. Individuals were placed into five groups based on self-reported hours of video gaming per week, and completed the Video Game Functional Assessment - Revised (VGFA-R). Results The results demonstrated the escape and social attention function were significant in predicting duration of video game play, whereas sensory and tangible were not significant. Conclusion Future implications of the VGFA-R and behaviorally based research are discussed.

Delay Discounting of Video Game Players: Comparison of Time Duration Among Gamers.

Video game addiction or Internet game disorder, as proposed by the DSM-5 (Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition), has similar criterion characteristics to other impulse control disorders. There is limited research examining video game addiction within a behavioral economic framework using delay discounting. The current study evaluated delay-discounting patterns of money and video game play by usual weekly hours of video game play. A total of 104 participants were split into 1 of 3 groups of video game players (low, medium, and high) and were asked to complete a monetary and video game discounting procedure through an online survey. Results showed significant differences between groups within both the monetary (p = 0.003) and video game discounting procedures (p = 0.004). Additionally, a positive linear relationship was noted between the groups across both procedures. The results of the current article reinforce previous findings that more hours of video game use are associated with greater impulsivity and provide implications for future research.

Syntheses of potent teixobactin analogues against methicillin-resistant Staphylococcus aureus (MRSA) through the replacement of l-allo-enduracididine with its isosteres.

The recently discovered cyclic depsipeptide, teixobactin, is a highly potent antibiotic against multi-drug resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and Mycobaterium tuberculosis. It comprises of 4 D amino acids and a rare l-allo-enduracididine amino acid. The synthesis of a properly protected l-allo-enduracididine amino acid and its incorporation into teixobactin is time consuming, synthetically challenging and low yielding and is therefore a major bottleneck in the development of potent analogues of teixobactin. In this article, we have synthesised 8 analogues of teixobactin using commercially available building blocks by replacing the l-allo-enduracididine amino acid with its isosteres. Furthermore, we have tested all the compounds against a panel of Gram positive bacteria including MRSA and explained the observed trend in biological activity. Although all the analogues were active, three analogues from this work, showed very promising activity against MRSA (MIC 1 µg mL-1). We can conclude that amino acids which are the closest isosteres of l-allo-enduracididine are the key to synthesising simplified potent analogues of teixobactin using rapid syntheses and improved yields.

Customized recommendations and reminder text messages for automated, computer-based treatment during methadone.

The Recovery Line is an automated, computer-based intervention based on cognitive behavioral therapy (CBT) designed to provide real-time assistance by phone for patients in methadone maintenance. Preliminary efficacy findings were promising, however, as with other computer-based systems for substance use disorder, patient system use was less than recommended. Development and evaluation of system functions to increase patient engagement and use is needed. Thus, we conducted two randomized trials to evaluate system functions designed to increase patient use of the Recovery Line among methadone-maintained patients with continued illicit drug use. In Trial 1 (n = 60), patients received customized, system use recommendations or no recommendations on each Recovery Line call. Ratings of system usability were higher for customized recommendations (CR), but number of calls and total call time did not differ by condition. Trial 2 evaluated characteristics of reminder messages (message frame and reminder latency). Participants (N = 67) received gain- and loss-frame reminder messages, and were randomly assigned to immediate, short, or long term message latency. Although message framing had no effect, gender interacted with latency condition such that females did not differ by message latency, while males had significantly greater total contact time in the short latency conditions. Number of calls differed by condition over time such that the shorter latencies led to greater calls initially, but dissipated over time. Overall the study indicates that computer-based self-management systems can be adapted to increase patient engagement and use. (PsycINFO Database Record

Teixobactin analogues reveal enduracididine to be non-essential for highly potent antibacterial activity and lipid II binding.

Teixobactin is a highly promising antibacterial depsipeptide consisting of four d-amino acids and a rare l-allo-enduracididine amino acid. l-allo-Enduracididine is reported to be important for the highly potent antibacterial activity of teixobactin. However, it is also a key limiting factor in the development of potent teixobactin analogues due to several synthetic challenges such as it is not commercially available, requires a multistep synthesis, long and repetitive couplings (16-30 hours). Due to all these challenges, the total synthesis of teixobactin is laborious and low yielding (3.3%). In this work, we have identified a unique design and developed a rapid synthesis (10 min µwave assisted coupling per amino acid, 30 min cyclisation) of several highly potent analogues of teixobactin with yields of 10-24% by replacing the l-allo-enduracididine with commercially available non-polar residues such as leucine and isoleucine. Most importantly, the Leu10-teixobactin and Ile10-teixobactin analogues have shown highly potent antibacterial activity against a broader panel of MRSA and Enterococcus faecalis (VRE). Furthermore, these synthetic analogues displayed identical antibacterial activity to natural teixobactin (MIC 0.25 µg mL-1) against MRSA ATCC 33591 despite their simpler design and ease of synthesis. We have confirmed lipid II binding and measured the binding affinities of individual amino acid residues of Ala10-teixobactin towards geranyl pyrophosphate by NMR to understand the nature and strength of binding interactions. Contrary to current understanding, we have shown that a cationic amino acid at position 10 is not essential for target (lipid II) binding and potent antibacterial activity of teixobactin. We thus provide strong evidence contrary to the many assumptions made about the mechanism of action of this exciting new antibiotic. Introduction of a non-cationic residue at position 10 allows for tremendous diversification in the design and synthesis of highly potent teixobactin analogues and lays the foundations for the development of teixobactin analogues as new drug-like molecules to target MRSA and Mycobacterium tuberculosis.

Acquired resistance to oxaliplatin is not directly associated with increased resistance to DNA damage in SK-N-ASrOXALI4000, a newly established oxaliplatin-resistant sub-line of the neuroblastoma cell line SK-N-AS.

The formation of acquired drug resistance is a major reason for the failure of anti-cancer therapies after initial response. Here, we introduce a novel model of acquired oxaliplatin resistance, a sub-line of the non-MYCN-amplified neuroblastoma cell line SK-N-AS that was adapted to growth in the presence of 4000 ng/mL oxaliplatin (SK-N-ASrOXALI4000). SK-N-ASrOXALI4000 cells displayed enhanced chromosomal aberrations compared to SK-N-AS, as indicated by 24-chromosome fluorescence in situ hybridisation. Moreover, SK-N-ASrOXALI4000 cells were resistant not only to oxaliplatin but also to the two other commonly used anti-cancer platinum agents cisplatin and carboplatin. SK-N-ASrOXALI4000 cells exhibited a stable resistance phenotype that was not affected by culturing the cells for 10 weeks in the absence of oxaliplatin. Interestingly, SK-N-ASrOXALI4000 cells showed no cross resistance to gemcitabine and increased sensitivity to doxorubicin and UVC radiation, alternative treatments that like platinum drugs target DNA integrity. Notably, UVC-induced DNA damage is thought to be predominantly repaired by nucleotide excision repair and nucleotide excision repair has been described as the main oxaliplatin-induced DNA damage repair system. SK-N-ASrOXALI4000 cells were also more sensitive to lysis by influenza A virus, a candidate for oncolytic therapy, than SK-N-AS cells. In conclusion, we introduce a novel oxaliplatin resistance model. The oxaliplatin resistance mechanisms in SK-N-ASrOXALI4000 cells appear to be complex and not to directly depend on enhanced DNA repair capacity. Models of oxaliplatin resistance are of particular relevance since research on platinum drugs has so far predominantly focused on cisplatin and carboplatin.

p53 controls global nucleotide excision repair of low levels of structurally diverse benzo(g)chrysene-DNA adducts in human fibroblasts.

Benzo(g)chrysene is a widespread environmental contaminant and potent carcinogen. We have measured the formation and nucleotide excision repair of covalent DNA adducts formed by the DNA-reactive metabolite of this compound in human fibroblasts, in which expression of the p53 tumor suppressor gene could be controlled by a tetracycline-inducible promoter. Cells were exposed for 1 h to 0.01, 0.1, or 1.2 microM (+/-)-anti-benzo(g)chrysene diol-epoxide, and DNA adducts were assessed at various post-treatment times by subjecting isolated DNA to (32)P-postlabeling analysis. Four major DNA adducts were detected, corresponding to the reaction of either the (+)- or (-)-anti-benzo(g)chrysene diol-epoxide stereoisomer with adenine or guanine. Treatment with 1.2 microM resulted in a level of 1100 total adducts/10(8) nucleotides for both p53-proficient and -deficient cells; removal of adducts was not observed in either case. In cells treated with 0.1 microM, the maximum level of total adducts at 24 h was 150/10(8) nucleotides in p53-proficient cells and 210 adducts/10(8) nucleotides in p53-deficient cells. A concentration of 0.01 microM resulted in a maximum of 20 adducts/10(8) nucleotides in p53-proficient cells at 4 h, but 40 adducts/10(8) nucleotides persisted in p53-deficient cells at 24 h. Whereas there were clear differences in the time course of adduct levels in p53-proficient compared with p53-deficient cells treated with 0.1 microM or 0.01 microM, these levels did not decrease extensively over 3 days. This is likely because of the stabilization of the diol-epoxide in cells, and consequent exposure and formation of adducts for many hours after the initial treatment. Furthermore, despite minor quantitative differences, all 4 of the adducts behaved similarly with respect to the effect of p53 expression on their removal. p53 appears to minimize the appearance of benzo(g)chrysene adducts in human cells by up-regulating global nucleotide excision repair and reducing the maximum adduct levels achieved. The fact that this p53-dependent effect is noted at levels of DNA adducts that are commonly found in human tissues (i.e., <100 adducts/10(8) nucleotides) because of environmental factors such as smoking is particularly significant with respect to human carcinogenesis related to environmental exposure.

Functional characterization of global genomic DNA repair and its implications for cancer.

The most versatile cellular pathway for dealing with a large variety of structurally-unrelated DNA alterations is nucleotide excision repair (NER). Most genomic damage, if not repaired, may contribute to mutagenesis and carcinogenesis, as well as to cellular lethality. There are two subpathways of NER, termed global genomic repair (GGR) and transcription-coupled repair (TCR); While GGR deals with all repairable lesions throughout the genome, TCR is selective for the transcribed DNA strand in expressed genes. Proteins involved in the initial recognition of lesions for GGR as well as for TCR (i.e. RNA polymerase) may sometimes initiate gratuitous repair events in undamaged DNA. However, the damage recognition enzymes for GGR are normally maintained at very low levels unless the cells are genomically stressed. Following UV irradiation in human fibroblasts the efficiency of GGR is upregulated through activation of the p53 tumor suppressor gene. The transactivation role of p53 includes control of expression of the genes, XPC and XPE, which are implicated in GGR but not TCR. These inducible responses are essential for the efficient repair of the most prominent lesion produced by UV, the cyclobutane pyrimidine dimer (CPD). They are also clinically relevant, as we have shown them to operate upon chemical carcinogen DNA damage at levels to which humans are environmentally exposed (e.g. through smoking). Thus, for benzo(a)pyrene (at 10-50 adducts per 10(8) nucleotides) repair was essentially complete within 1 day in p53(+/+) human fibroblasts while no repair was detected within 3 days in p53(-/-) cells. The levels of all four DNA adducts formed by benzo(g)chrysene, also exhibited p53-dependent control in human fibroblasts. However, unlike humans most rodent tissues are deficient in the p53-dependent GGR pathway. Since rodents are used as surrogates for humans in environmental cancer risk assessment it is very important that we determine how they differ from humans with respect to DNA repair and oncogenic responses to environmental genotoxins.

p53-dependent radiobiological responses to internalised indium-111 in human cells.

INTRODUCTION: The p53 tumour suppressor protein plays a pivotal role in the response of mammalian cells to DNA damage. It regulates cell cycle progression, apoptosis and DNA repair mechanisms and is therefore likely to influence response to targeted radionuclide therapy. This study investigated the role of p53 in the cellular response to the Auger-emitting radionuclide indium-111. METHODS: Two stable clones of a HT1080 fibrosarcoma cell line, differing only in p53 status due to RNAi-mediated knockdown of p53 expression, were incubated for 1 h with [¹¹¹In]-oxinate (0-10 MBq/ml). Radiopharmaceutical uptake into HT1080 cells was measured in situ using a non-contact phosphorimager method. Cellular sensitivity and DNA damage were measured by, respectively, clonogenic survival analysis and the single cell gel electrophoresis (Comet) assay. RESULTS: Mean uptake of [¹¹¹In]-oxinate in HT1080 cells was unaffected by p53 status, reaching a maximum of 9Bq/cell. [¹¹¹In]-oxinate-induced cytotoxicity was also identical in both clones, as measured by IC50 (0.68 MBq/ml). However the formation of DNA damage, measured immediately after treatment with [¹¹¹In]-oxinate, was found to be up to 2.5-fold higher in the p53-deficient HT1080 clone. CONCLUSIONS: The increased DNA damage induced in p53-deficient HT1080 cells suggests an early deficiency in the repair of DNA damage during the treatment period. However, the similarity in cellular sensitivity, irrespective of p53 status, suggests that reduced p53 leads to a concomitant reduction in p53-dependent cytotoxicity despite the persistence of DNA damage. The results may provide insight into how tumours that differ in p53 status respond to therapeutic radionuclides.