Search results
Found 12436 matches for
The Nuffield Department of Surgical Sciences is the academic department of surgery at the University of Oxford, and hosts a multidisciplinary team of senior clinical academic surgeons, senior scientists, junior clinicians and scientists in training.
A hierarchical model of transcriptional dynamics allows robust estimation of transcription rates in populations of single cells with variable gene copy number.
MOTIVATION: cis-regulatory DNA sequence elements, such as enhancers and silencers, function to control the spatial and temporal expression of their target genes. Although the overall levels of gene expression in large cell populations seem to be precisely controlled, transcription of individual genes in single cells is extremely variable in real time. It is, therefore, important to understand how these cis-regulatory elements function to dynamically control transcription at single-cell resolution. Recently, statistical methods have been proposed to back calculate the rates involved in mRNA transcription using parameter estimation of a mathematical model of transcription and translation. However, a major complication in these approaches is that some of the parameters, particularly those corresponding to the gene copy number and transcription rate, cannot be distinguished; therefore, these methods cannot be used when the copy number is unknown. RESULTS: Here, we develop a hierarchical Bayesian model to estimate biokinetic parameters from live cell enhancer-promoter reporter measurements performed on a population of single cells. This allows us to investigate transcriptional dynamics when the copy number is variable across the population. We validate our method using synthetic data and then apply it to quantify the function of two known developmental enhancers in real time and in single cells. AVAILABILITY: Supporting information is submitted with the article.
Evidence for phenotypic plasticity among multihost Campylobacter jejuni and C. coli lineages, obtained using ribosomal multilocus sequence typing and Raman spectroscopy.
Closely related bacterial isolates can display divergent phenotypes. This can limit the usefulness of phylogenetic studies for understanding bacterial ecology and evolution. Here, we compare phenotyping based on Raman spectrometric analysis of cellular composition to phylogenetic classification by ribosomal multilocus sequence typing (rMLST) in 108 isolates of the zoonotic pathogens Campylobacter jejuni and C. coli. Automatic relevance determination (ARD) was used to identify informative peaks in the Raman spectra that could be used to distinguish strains in taxonomic and host source groups (species, clade, clonal complex, and isolate source/host). Phenotypic characterization based on Raman spectra showed a degree of agreement with genotypic classification using rMLST, with segregation accuracy between species (83.95%), clade (in C. coli, 98.41%), and, to some extent, clonal complex (86.89% C. jejuni ST-21 and ST-45 complexes) being achieved. This confirmed the utility of Raman spectroscopy for lineage classification and the correlation between genotypic and phenotypic classification. In parallel analysis, relatively distantly related isolates (different clonal complexes) were assigned the correct host origin irrespective of the clonal origin (74.07 to 96.97% accuracy) based upon different Raman peaks. This suggests that the phenotypic characteristics, from which the phenotypic signal is derived, are not fixed by clonal descent but are influenced by the host environment and change as strains move between hosts.
Dynamic analysis of stochastic transcription cycles.
In individual mammalian cells the expression of some genes such as prolactin is highly variable over time and has been suggested to occur in stochastic pulses. To investigate the origins of this behavior and to understand its functional relevance, we quantitatively analyzed this variability using new mathematical tools that allowed us to reconstruct dynamic transcription rates of different reporter genes controlled by identical promoters in the same living cell. Quantitative microscopic analysis of two reporter genes, firefly luciferase and destabilized EGFP, was used to analyze the dynamics of prolactin promoter-directed gene expression in living individual clonal and primary pituitary cells over periods of up to 25 h. We quantified the time-dependence and cyclicity of the transcription pulses and estimated the length and variation of active and inactive transcription phases. We showed an average cycle period of approximately 11 h and demonstrated that while the measured time distribution of active phases agreed with commonly accepted models of transcription, the inactive phases were differently distributed and showed strong memory, with a refractory period of transcriptional inactivation close to 3 h. Cycles in transcription occurred at two distinct prolactin-promoter controlled reporter genes in the same individual clonal or primary cells. However, the timing of the cycles was independent and out-of-phase. For the first time, we have analyzed transcription dynamics from two equivalent loci in real-time in single cells. In unstimulated conditions, cells showed independent transcription dynamics at each locus. A key result from these analyses was the evidence for a minimum refractory period in the inactive-phase of transcription. The response to acute signals and the result of manipulation of histone acetylation was consistent with the hypothesis that this refractory period corresponded to a phase of chromatin remodeling which significantly increased the cyclicity. Stochastically timed bursts of transcription in an apparently random subset of cells in a tissue may thus produce an overall coordinated but heterogeneous phenotype capable of acute responses to stimuli.
Physiological levels of TNFalpha stimulation induce stochastic dynamics of NF-kappaB responses in single living cells.
Nuclear factor kappa B (NF-kappaB) signalling is activated by cellular stress and inflammation and regulates cytokine expression. We applied single-cell imaging to investigate dynamic responses to different doses of tumour necrosis factor alpha (TNFalpha). Lower doses activated fewer cells and those responding showed an increasingly variable delay in the initial NF-kappaB nuclear translocation and associated IkappaBalpha degradation. Robust 100 minute nuclear:cytoplasmic NF-kappaB oscillations were observed over a wide range of TNFalpha concentrations. The result is supported by computational analyses, which identified a limit cycle in the system with a stable 100 minute period over a range of stimuli, and indicated no co-operativity in the pathway activation. These results suggest that a stochastic threshold controls functional all-or-nothing responses in individual cells. Deterministic and stochastic models simulated the experimentally observed activation threshold and gave rise to new predictions about the structure of the system and open the way for better mechanistic understanding of physiological TNFalpha activation of inflammatory responses in cells and tissues.
Spatially resolved clonal copy number alterations in benign and malignant tissue.
Defining the transition from benign to malignant tissue is fundamental to improving early diagnosis of cancer1. Here we use a systematic approach to study spatial genome integrity in situ and describe previously unidentified clonal relationships. We used spatially resolved transcriptomics2 to infer spatial copy number variations in >120,000 regions across multiple organs, in benign and malignant tissues. We demonstrate that genome-wide copy number variation reveals distinct clonal patterns within tumours and in nearby benign tissue using an organ-wide approach focused on the prostate. Our results suggest a model for how genomic instability arises in histologically benign tissue that may represent early events in cancer evolution. We highlight the power of capturing the molecular and spatial continuums in a tissue context and challenge the rationale for treatment paradigms, including focal therapy.
Genomic evolution shapes prostate cancer disease type.
The development of cancer is an evolutionary process involving the sequential acquisition of genetic alterations that disrupt normal biological processes, enabling tumor cells to rapidly proliferate and eventually invade and metastasize to other tissues. We investigated the genomic evolution of prostate cancer through the application of three separate classification methods, each designed to investigate a different aspect of tumor evolution. Integrating the results revealed the existence of two distinct types of prostate cancer that arise from divergent evolutionary trajectories, designated as the Canonical and Alternative evolutionary disease types. We therefore propose the evotype model for prostate cancer evolution wherein Alternative-evotype tumors diverge from those of the Canonical-evotype through the stochastic accumulation of genetic alterations associated with disruptions to androgen receptor DNA binding. Our model unifies many previous molecular observations, providing a powerful new framework to investigate prostate cancer disease progression.
Genomic copy number predicts oesophageal cancer years before transformation
SummaryCancer arises through a process of somatic evolution and recent studies have shown that aneuploidies and driver gene mutations precede cancer diagnosis by several years to decades1–4Here, we address the question whether such genomic signals can be used for early detection and pre-emptive cancer treatment. To this end we study Barrett’s oesophagus, a genomic copy number driven neoplastic precursor lesion to oesophageal adenocarcinoma5. We use shallow whole genome sequencing of 777 biopsies sampled from 88 patients in surveillance for Barrett’s oesophagus over a period of up to 15 years. These data show that genomic signals exist that distinguish progressive from stable disease with an AUC of 0.87 and a sensitivity of 50% even ten years prior to histopathological disease transformation. These finding are validated on two independent cohorts of 75 and 248 patients. Compared against current patient management guidelines genomic risk classification enables earlier treatment for high risk patients as well as reduction of unnecessary treatment and monitoring for patients who are unlikely to develop cancer.
Prostate cancer evolution from multilineage primary to single lineage metastases with implications for liquid biopsy.
The evolutionary progression from primary to metastatic prostate cancer is largely uncharted, and the implications for liquid biopsy are unexplored. We infer detailed reconstructions of tumor phylogenies in ten prostate cancer patients with fatal disease, and investigate them in conjunction with histopathology and tumor DNA extracted from blood and cerebrospinal fluid. Substantial evolution occurs within the prostate, resulting in branching into multiple spatially intermixed lineages. One dominant lineage emerges that initiates and drives systemic metastasis, where polyclonal seeding between sites is common. Routes to metastasis differ between patients, and likely genetic drivers of metastasis distinguish the metastatic lineage from the lineage that remains confined to the prostate within each patient. Body fluids capture features of the dominant lineage, and subclonal expansions that occur in the metastatic phase are non-uniformly represented. Cerebrospinal fluid analysis reveals lineages not detected in blood-borne DNA, suggesting possible clinical utility.
Sequencing of prostate cancers identifies new cancer genes, routes of progression and drug targets.
Prostate cancer represents a substantial clinical challenge because it is difficult to predict outcome and advanced disease is often fatal. We sequenced the whole genomes of 112 primary and metastatic prostate cancer samples. From joint analysis of these cancers with those from previous studies (930 cancers in total), we found evidence for 22 previously unidentified putative driver genes harboring coding mutations, as well as evidence for NEAT1 and FOXA1 acting as drivers through noncoding mutations. Through the temporal dissection of aberrations, we identified driver mutations specifically associated with steps in the progression of prostate cancer, establishing, for example, loss of CHD1 and BRCA2 as early events in cancer development of ETS fusion-negative cancers. Computational chemogenomic (canSAR) analysis of prostate cancer mutations identified 11 targets of approved drugs, 7 targets of investigational drugs, and 62 targets of compounds that may be active and should be considered candidates for future clinical trials.
Rare disease gene association discovery in the 100,000 Genomes Project.
Up to 80% of rare disease patients remain undiagnosed after genomic sequencing1, with many probably involving pathogenic variants in yet to be discovered disease-gene associations. To search for such associations, we developed a rare variant gene burden analytical framework for Mendelian diseases, and applied it to protein-coding variants from whole-genome sequencing of 34,851 cases and their family members recruited to the 100,000 Genomes Project2. A total of 141 new associations were identified, including five for which independent disease-gene evidence was recently published. Following in silico triaging and clinical expert review, 69 associations were prioritized, of which 30 could be linked to existing experimental evidence. The five associations with strongest overall genetic and experimental evidence were monogenic diabetes with the known β cell regulator3,4 UNC13A, schizophrenia with GPR17, epilepsy with RBFOX3, Charcot-Marie-Tooth disease with ARPC3 and anterior segment ocular abnormalities with POMK. Further confirmation of these and other associations could lead to numerous diagnoses, highlighting the clinical impact of large-scale statistical approaches to rare disease-gene association discovery.
Time to return of bowel function following perioperative probiotics in colorectal cancer surgery (PICCS-1): study protocol for a randomized controlled trial.
BACKGROUND: Postoperative ileus occurs in up to 30% of patients following major oncologic surgery for colorectal cancer, leading to significant morbidity, patient distress, as well as increased utilization of healthcare resources. Various modalities to reduce postoperative ileus rates have been explored. One such modality is the perioperative administration of probiotics which have hitherto achieved inconsistent success. Here, we design a trial to determine whether the perioperative administration with probiotics given together with nutritional supplementation can help to reduce postoperative ileus rates. METHODS: We propose a parallel three-arm randomized controlled trial. In Arm 1, no nutritional supplementation is provided to the patient. In Arm 2, Nestle Isocal is provided to the participant. Nestle Isocal provides nutritional supplementation but without any probiotic. In Arm 3, Nestle Boost Optimum is provided to the patient. Nestle Boost Optimum contains a similar nutritional profile to Isocal, but with the addition of Lactobacillus paracasei. The primary outcome is the time to first bowel movement in days from the day of surgery. Secondary outcomes are time to first flatus, infective complications, and adverse events related to the administration of nutritional supplementation. Statistical analysis will be conducted in an intention-to-treat approach. ANOVA with the Tukey test will be used to compare continuous variables, while the χ2 test will be used for categorical variables. DISCUSSION: Nutritional supplementation with probiotics is a convenient, non-pill alternative for patients. Furthermore, the interventions are commonly found in the formulary of many hospitals worldwide. If successful, probiotics in nutritional supplementation could be a cost-effective and simple way to reduce postoperative ileus. TRIAL REGISTRATION: ClinicalTrials.gov NCT06456229. This trial was registered on 11 June 2024. Thai Clinical Trials Registry TCTR20240706003. This trial was registered on 6 July 2024.
Safety of Nonsteroidal Anti-inflammatory Drugs in Major Gastrointestinal Surgery: A Prospective, Multicenter Cohort Study.
BACKGROUND: Significant safety concerns remain surrounding the use of nonsteroidal anti-inflammatory drugs (NSAIDs) following gastrointestinal surgery, leading to wide variation in their use. This study aimed to determine the safety profile of NSAIDs after major gastrointestinal surgery. METHODS: Consecutive patients undergoing elective or emergency abdominal surgery with a minimum one-night stay during a 3-month study period were eligible for inclusion. The administration of any NSAID within 3 days following surgery was the main independent variable. The primary outcome measure was the 30-day postoperative major complication rate, as defined by the Clavien-Dindo classification (Clavien-Dindo III-V). Propensity matching with multivariable logistic regression was used to produce odds ratios (OR) and 95 % confidence intervals. RESULTS: From 9264 patients, 23.9 % (n = 2212) received postoperative NSAIDs. The overall major complication rate was 11.5 % (n = 1067). Following propensity matching and adjustment, use of NSAIDs were not significantly associated with any increase in major complications (OR 0.90, 0.60-1.34, p = 0.560). CONCLUSIONS: Early use of postoperative NSAIDs was not associated with an increase in major complications following gastrointestinal surgery.
Multicentre prospective cohort study of body mass index and postoperative complications following gastrointestinal surgery.
BACKGROUND: There is currently conflicting evidence surrounding the effects of obesity on postoperative outcomes. Previous studies have found obesity to be associated with adverse events, but others have found no association. The aim of this study was to determine whether increasing body mass index (BMI) is an independent risk factor for development of major postoperative complications. METHODS: This was a multicentre prospective cohort study across the UK and Republic of Ireland. Consecutive patients undergoing elective or emergency gastrointestinal surgery over a 4-month interval (October-December 2014) were eligible for inclusion. The primary outcome was the 30-day major complication rate (Clavien-Dindo grade III-V). BMI was grouped according to the World Health Organization classification. Multilevel logistic regression models were used to adjust for patient, operative and hospital-level effects, creating odds ratios (ORs) and 95 per cent confidence intervals (c.i.). RESULTS: Of 7965 patients, 2545 (32·0 per cent) were of normal weight, 2673 (33·6 per cent) were overweight and 2747 (34·5 per cent) were obese. Overall, 4925 (61·8 per cent) underwent elective and 3038 (38·1 per cent) emergency operations. The 30-day major complication rate was 11·4 per cent (908 of 7965). In adjusted models, a significant interaction was found between BMI and diagnosis, with an association seen between BMI and major complications for patients with malignancy (overweight: OR 1·59, 95 per cent c.i. 1·12 to 2·29, P = 0·008; obese: OR 1·91, 1·31 to 2·83, P = 0·002; compared with normal weight) but not benign disease (overweight: OR 0·89, 0·71 to 1·12, P = 0·329; obese: OR 0·84, 0·66 to 1·06, P = 0·147). CONCLUSION: Overweight and obese patients undergoing surgery for gastrointestinal malignancy are at increased risk of major postoperative complications compared with those of normal weight.
High-resolution spatio-temporal bioactivity of a novel peptide revealed by optical imaging in rat orbitofrontal cortex in vitro: possible implications for neurodegenerative diseases.
Acetylcholinesterase (AChE) is now well known to have a secondary, non-enzymatic function independent of cholinergic transmission. In the last decade, the part of the molecule responsible for this action has been identified, i.e. a 14 amino acid peptide fragment ('T14'), deriving from the C-terminus of AChE: this peptide has been shown to be bioactive in a range of preparations and to act at an allosteric site on α₇ nicotinic acetylcholine receptors (α₇-nAChRs). Of particular significance is the finding that AChE-related peptides trigger calcium-induced neurotoxicity that may be pivotal in the process of neurodegenerative diseases, such as Alzheimer's. However to date all studies have been performed on isolated cell preparations. The aim of this study was therefore to characterise the bioactivity of T14 on meso-scale in vitro cortical networks ('neuronal assemblies') from rat brain slices containing orbitofrontal cortex. Local field potential (LFP) recordings showed that the T14 peptide has a selective, holistic action on cortical networks in a modulatory biphasic manner i.e. predisposing excitation at concentrations of up to 1 μM, after which the trend is reversed in favour of inhibition at higher doses (>1 μM). By contrast, a scrambled variant of the T14 peptide sequence (S14), showed no significant changes in neuronal activation. Optical imaging using voltage-sensitive dyes (VSDI) corroborated the electrophysiological findings and also provided further insight into the spatial dynamics of the effects of the peptide: T14 application had a facilitatory effect i.e. increased the time-course of activation at sub-micromolar concentrations only (700 nM) without significantly affecting the spread of evoked assemblies. Moreover: co-applying T14 with the α₇-nAChR competitive antagonist methyllycaconitine (MLA) produced inhibition in activation synchrony not seen with either agent on their own, suggesting an additive inhibitory effect. In conclusion, the T14 peptide derived from AChE produced a dose-dependent biphasic modulation of cortical networks activity dependent on the α₇-nAChR: these findings should thus provide a more comprehensive insight into the immediate actions of a novel bioactive agent of high potential relevance to neurodegenerative disorders such as Alzheimer's disease.
Deep Brain Stimulation of the Nucleus Accumbens in Severe Enduring Anorexia Nervosa: A Pilot Study.
INTRODUCTION: Anorexia nervosa (AN) is one of the most debilitating psychiatric disorders, becoming severe and enduring in a third of cases; with few effective treatments. Deep brain stimulation is a reversible, adjustable neurosurgical procedure that has been gaining ground in psychiatry as a treatment for depression and obsessive-compulsive disorder, yet few studies have investigated AN. Abnormal eating behavior and the compulsive pursuit of thinness in AN is, in part, a consequence of dysfunction in reward circuitry and the nucleus accumbens (NAcc) is central to reward processing. METHODS: Phase 1 prospective open-label pilot study of seven individuals with severe enduring AN. Electrodes were implanted bilaterally into the NAcc with stimulation at the anterior limb of the internal capsule using rechargeable implantable pulse generators. The protocol of 15 months included 12 months of deep brain stimulation incorporating two consecutive, randomized blind on-off fortnights 9 months after stimulation onset. The primary objectives were to investigate safety and feasibility, together with changes in eating disorder psychopathology. RESULTS: Feasibility and safety was demonstrated with no serious adverse events due to deep brain stimulation. Three patients responded to treatment [defined as > 35% reduction in Eating Disorders Examination (EDE) score at 12 months] and four patients were non-responders. Responders had a statistically significant mean reduction in EDE scores (50.3% reduction; 95% CI 2.6-98.2%), Clinical Impairment Assessment (45.6% reduction; 95% CI 7.4-83.7%). Responders also had a statistically significant mean reduction in Hamilton Depression Scale, Hamilton Anxiety Scale and Snaith-Hamilton pleasure scale. There were no statistically significant changes in Body Mass Index, Yale-Brown-Cornell Eating Disorder Scale, Yale-Brown Obsessive-Compulsive Scale and World Health Organization Quality of Life Psychological subscale. CONCLUSION: This study provides some preliminary indication that deep brain stimulation to the NAcc. Might potentially improve some key features of enduring AN. In this small study, the three responders had comorbid obsessive-compulsive disorder which predated AN diagnosis. Future studies should aim to further elucidate predictors of outcome. CLINICAL TRIAL REGISTRATION: [www.ClinicalTrials.gov], identifier [Project ID 128658].
Deep Brain Stimulation (DBS) and Motor Cortex Stimulation (MCS) for Central Post-Stroke Pain: A Systematic Review And Meta-Analysis.
INTRODUCTION: Deep Brain Stimulation (DBS) and Motor Cortex stimulation (MCS) are invasive interventions in order to treat various neuropathic pain syndromes such as Central Post-Stroke Pain. While each treatment has varying degree of success, comparative analysis has not yet been performed, and the success rates of these techniques using validated, objective pain scores have not been synthesised. METHODS: A systematic review and meta-analysis was conducted in accordance with PRISMA guidelines. Three databases were searched, and articles published from January 2000 October 2024 were included (last search date 25 October 2024). Meta-Analysis was performed using random effects models. We evaluated the performance of DBS or MCS by assessing studies that reported pain relief using Visual Analogue Scale (VAS) or Numerical Rating Scale (NRS) scores. RESULTS: Of the 478 articles identified, 32 were included in the analysis (330 patients- 139 DBS, & 191 MCS). The improvement in mean VAS score for patients that underwent DBS post-surgery was 48.6% compared to a score of 53.1% for patients that had MCS. The pooled number of patients who improved after DBS was 0.62 (95% CI, 0.51-0.71, I2=16%). The pooled number of patients who improved after MCS was 0.64 (95% CI, 0.53-0.74, I2=40%). CONCLUSION: The use of neurosurgical interventions such as DBS and MCS are last-resort treatments for Central Post-Stroke Pain, with limited studies exploring and comparing these two techniques. While our study shows that MCS might be a slightly better treatment option, further research would need to be done to determine the appropriate surgical intervention in the treatment of Central Post-Stroke Pain.