Bariatric surgery, also known as metabolic surgery, is an effective treatment for morbid obesity, which also offers pronounced metabolic effects including the resolution of type 2 diabetes and a decrease in cardiovascular disease and long-term cancer risk. However, the mechanisms of surgical weight loss and the long-term consequences of bariatric surgery remain unclear. Bariatric surgery has been demonstrated to alter the composition of both the microbiome and the metabolic phenotype. We observed a marked shift toward Gammaproteobacteria, particularly Enterobacter hormaechei, following Roux-en-Y gastric bypass (RYGB) surgery in a rat model compared with sham-operated controls. Fecal water from RYGB surgery rats was highly cytotoxic to rodent cells (mouse lymphoma cell line). In contrast, fecal water from sham-operated animals showed no/very low cytotoxicity. This shift in the gross structure of the microbiome correlated with greatly increased cytotoxicity. Urinary phenylacetylglycine and indoxyl sulfate and fecal gamma-aminobutyric acid, putrescine, tyramine, and uracil were found to be inversely correlated with cell survival rate. This profound co-dependent response of mammalian and microbial metabolism to RYGB surgery and the impact on the cytotoxicity of the gut luminal environment suggests that RYGB exerts local and global metabolic effects which may have an influence on long-term cancer risk and cytotoxic load.

The European Medicines Agency (EMA) has expressed concern regarding (i) the potential for antisense oligonucleotide (ASO) therapeutics to induce sequence specific mutation at genomic DNA and (ii) the capability of ASO degradation products (nucleotide analogues) to incorporate into newly synthesised genomic DNA via DNA polymerase and cause mutation if base-pairing occurs with reduced fidelity. Treating human lymphoblastoid cells with a biologically active antisense molecule induced sequence specific mutation within genomic DNA over four fold, in a system where RAD51 protein expression was induced. This finding has implications for ASO therapeutics with individuals with an induced DNA damage response, such as cancer patients. Furthermore, a phosphorothioate nucleotide analogue potently induced mutation at genomic DNA two orders of magnitude above control. This study shows that a biologically active ASO molecule can induce heritable sequence alterations, and if degraded, its respective analogue may incorporate into genomic DNA with mutagenic consequences.

Purine tracts in duplex DNA can bind oligonucleotide strands in a sequence specific manner to form triple-helix structures. Triple-helix forming oligonucleotides (TFOs) targeting supFG1 constructs have previously been shown to be mutagenic raising safety concerns for oligonucleotide-based pharmaceuticals. We have engineered a TFO, TFO27, to target the genomic Hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus to define the mutagenic potential of such structures at genomic DNA. We report that TFO27 was resistant to nuclease degradation and readily binds to its target motif in a cell free system. Contrary to previous studies using the supFG1 reporter construct, TFO27 failed to induce mutation within the genomic HPRT locus. We suggest that it is possible that previous reports of triplex-mediated mutation using the supFG1 reporter construct could be confounded by DNA quadruplex formation. Although the present study indicates that a TFO targeting a genomic locus lacks mutagenic activity, it is unclear if this finding can be generalised to all TFOs and their targets. For the present, we suggest that it is prudent to avoid large purine stretches in oligonucleotide pharmaceutical design to minimise concern regarding off-target genotoxicity.

Background/Aims  Giant cell arteritis (GCA) is a systemic vasculitis affecting the temporal arteries and large vessels, including the aorta, in 25% of cases. Incidence of vertebral artery involvement is unknown likely owing to the reliance on conventional diagnostics including temporal artery biopsy (TAB) and temporal/axillary ultrasound which evade the vertebral arteries. These tests lose sensitivity soon after steroid initiation. Early specialist assessment and diagnostics, as part of a fast track pathway (FTP), can overcome this challenge. We present the first 12 months data from our FTP. To our knowledge this is the first GCA-FTP in the UK to utilise positron emission tomography - computed tomography (PET-CT) as an early diagnostic tool so we present a unique cohort of patients. Methods  Cohort 1: 40 patients, diagnosed between 2015-2019, before FTP implementation. Time from steroid initiation to temporal artery ultrasound (TAUS) and TAB was recorded. Cohort 2: 35 patients referred after pathway implementation. 21 had a positive diagnosis. Time from steroid initiation to diagnostic test was recorded. Results  Cohort 1: 26 (65%) patients had TAB; 3 (7.5%) had TAUS. The average time from starting steroid to investigation was 6 and 2 days respectively. Cohort 2: 17 (48%) had TAB, 30 (86%) had TAUS, 28 (80%) had PET-CT. In confirmed diagnoses, time from steroid initiation to investigations was 6.6 days, 1 day and 2.5 days respectively. In negative diagnoses time frames were 11.5, 1.5 and 2.3 days respectively. Table 1 shows sensitivity and specificity of these tests. Table 1. Sensitivity and specificity comparison between diagnostic tests Conclusion  Combining TAUS and PET-CT allows for high diagnostic accuracy without the need for invasive biopsy. 24% of patients had negative or inconclusive ultrasound and/ or negative biopsy but confirmed vasculitis on PET-CT. 50% of this cohort had vertebral involvement only. PET-CT has an important role in detecting extracranial particularly vertebral arteritis, where biopsy and ultrasound are unreliable. Our pathway design with the inclusion of PET-CT, enables us to capture all patients with GCA and satisfy NHS England criteria for tocilizumab use in refractory GCA. Continued evaluation of PET-CT and its role in predicting vascular complications is required. Disclosure  D.R. Ludwig: None. V. Morris: None. A. Shashidhara: None. S. Voo: None. R. Reshat: None.

It has been reported that DNA triplex formation induces mutagenesis as determined using plasmid-based reporter constructs (Wang et al 1996, Science, 271, p802). Triplex mediated mutagenesis has been shown to include point mutations, deletions, small insertions and homologous recombination. To the best of our knowledge, no study has successfully examined the mutagenic potential of a non-conjugated triplex-forming oligonucleotide (TFO) targeting a genomic sequence. In this study, we have designed a TFO that targets the hemizygous hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus, in the human lymphoblastoid TK6 cell line, and assessed mutagenicity through resistance to 6-thioguanine. Our TFO, TFO27, has been designed to form a triplex with a purine tract in exon 3 of the HPRT gene. Triplex formation at the target motif was shown to occur at nanomolar concentrations, confirmed by Electrophoretic Mobility Shift Assays. A scrambled oligonucleotide, SCR27, failed to form a triplex at the target motif. A range of transfection reagents were evaluated for facilitated delivery of TFO27, and resulted in variable cellular toxicity. Transfection of high concentrations of oligonucleotide, with acceptable levels of cytotoxicity, resulted in HPRT mutation with TFO27 but not SCR27. Similar experiments failed to result in mutation at the non-targeted thymidine kinase (TK) locus, suggesting locus specificity for the mode of action of TFO27. The target specificity and sequence context of these mutagenic events is being determined to establish the mechanism of mutation. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 6.