Spatially resolved clonal copy number alterations in benign and malignant tissue

Publisher Copyright: © 2022, The Author(s).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.Peer reviewe

Spatially resolved clonal copy number alterations in benign and malignant tissue

Publisher Copyright: © 2022, The Author(s).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.Peer reviewe

The Genome Sequence of the Leaf-Cutter Ant Atta cephalotes Reveals Insights into Its Obligate Symbiotic Lifestyle

Leaf-cutter ants are one of the most important herbivorous insects in the Neotropics, harvesting vast quantities of fresh leaf material. The ants use leaves to cultivate a fungus that serves as the colony's primary food source. This obligate ant-fungus mutualism is one of the few occurrences of farming by non-humans and likely facilitated the formation of their massive colonies. Mature leaf-cutter ant colonies contain millions of workers ranging in size from small garden tenders to large soldiers, resulting in one of the most complex polymorphic caste systems within ants. To begin uncovering the genomic underpinnings of this system, we sequenced the genome of Atta cephalotes using 454 pyrosequencing. One prediction from this ant's lifestyle is that it has undergone genetic modifications that reflect its obligate dependence on the fungus for nutrients. Analysis of this genome sequence is consistent with this hypothesis, as we find evidence for reductions in genes related to nutrient acquisition. These include extensive reductions in serine proteases (which are likely unnecessary because proteolysis is not a primary mechanism used to process nutrients obtained from the fungus), a loss of genes involved in arginine biosynthesis (suggesting that this amino acid is obtained from the fungus), and the absence of a hexamerin (which sequesters amino acids during larval development in other insects). Following recent reports of genome sequences from other insects that engage in symbioses with beneficial microbes, the A. cephalotes genome provides new insights into the symbiotic lifestyle of this ant and advances our understanding of host–microbe symbioses

Factors Associated with Revision Surgery after Internal Fixation of Hip Fractures

Background: Femoral neck fractures are associated with high rates of revision surgery after management with internal fixation. Using data from the Fixation using Alternative Implants for the Treatment of Hip fractures (FAITH) trial evaluating methods of internal fixation in patients with femoral neck fractures, we investigated associations between baseline and surgical factors and the need for revision surgery to promote healing, relieve pain, treat infection or improve function over 24 months postsurgery. Additionally, we investigated factors associated with (1) hardware removal and (2) implant exchange from cancellous screws (CS) or sliding hip screw (SHS) to total hip arthroplasty, hemiarthroplasty, or another internal fixation device. Methods: We identified 15 potential factors a priori that may be associated with revision surgery, 7 with hardware removal, and 14 with implant exchange. We used multivariable Cox proportional hazards analyses in our investigation. Results: Factors associated with increased risk of revision surgery included: female sex, [hazard ratio (HR) 1.79, 95% confidence interval (CI) 1.25-2.50; P = 0.001], higher body mass index (fo

Constructing RNA based genetic circuits for the detection of endogenous microRNAs

Synthetic biology is a rapidly evolving field enabling the rational design of new biological functionality. RNA is particularly amenable to this rational design processes due to relatively straightforward structural modelling principles and its ability to perform diverse roles, from that of simple messenger RNAs to complex catalytic ribozymes. Consequently, the synthetic design and assembly of RNA building blocks into functional circuits and networks can far exceed the complexity of what could be achieved using protein components. MicroRNAs (miRNAs) are fascinating short non-coding RNA molecules (~22nt long) which have the capacity to bind and tune the expression of thousands of mRNAs, functioning as molecular rheostats during development, physiology and disease. The ability to detect miRNAs, via synthetic biosensor circuits would therefore offer a unique insight into cellular behavior in addition to the ability to link miRNA expression signatures to an output expression programme of choice. In this thesis, I have developed novel synthetic circuit architectures for the detection of miRNA activity. Firstly, protein and RNA modules were used in concert to develop a biosensor circuit consisting of two serially linked NOT gates that could activate output expression in response to a specific input miRNA. Secondly, methods were devised for the construction of a single molecule biosensor device, built entirely using RNA. At the heart of this device were novel miRNA responsive allosteric ribozymes which have been created through two independent in vitro selection methodologies devised for this study. Such engineered modules now constitute part of the synthetic biology toolbox, increasing the versatility with which miRNAs can serve as inputs to artificial genetic circuits.</p

Constructing RNA based genetic circuits for the detection of endogenous microRNAs

Synthetic biology is a rapidly evolving field enabling the rational design of new biological functionality. RNA is particularly amenable to this rational design processes due to relatively straightforward structural modelling principles and its ability to perform diverse roles, from that of simple messenger RNAs to complex catalytic ribozymes. Consequently, the synthetic design and assembly of RNA building blocks into functional circuits and networks can far exceed the complexity of what could be achieved using protein components. MicroRNAs (miRNAs) are fascinating short non-coding RNA molecules (~22nt long) which have the capacity to bind and tune the expression of thousands of mRNAs, functioning as molecular rheostats during development, physiology and disease. The ability to detect miRNAs, via synthetic biosensor circuits would therefore offer a unique insight into cellular behavior in addition to the ability to link miRNA expression signatures to an output expression programme of choice. In this thesis, I have developed novel synthetic circuit architectures for the detection of miRNA activity. Firstly, protein and RNA modules were used in concert to develop a biosensor circuit consisting of two serially linked NOT gates that could activate output expression in response to a specific input miRNA. Secondly, methods were devised for the construction of a single molecule biosensor device, built entirely using RNA. At the heart of this device were novel miRNA responsive allosteric ribozymes which have been created through two independent in vitro selection methodologies devised for this study. Such engineered modules now constitute part of the synthetic biology toolbox, increasing the versatility with which miRNAs can serve as inputs to artificial genetic circuits

Evaluation of the role of miR-31-dependent reduction in dystrophin and nNOS on atrial-fibrillation-induced electrical remodelling in man

The management of atrial fibrillation remains a challenge. This condition remodels atrial electrical properties, which promote resistance to treatment. Although remodelling has long been a therapeutic target in atrial fibrillation, its causes remain incompletely understood. We aimed to evaluate the role of miR-31-dependent reduction in dystrophin and neuronal nitric oxide synthase (nNOS, also known as NOS1) on atrial electrical properties and atrial fibrillation inducibility.We recruited 258 patients (209 patients in sinus rhythm and 49 with permanent atrial fibrillation) from the John Radcliffe Hospital, Oxford, UK; written informed consent was obtained from each participant. We also used a goat model of pacing-induced atrial fibrillation (24 with atrial fibrillation vs 20 controls in normal sinus rythm) and nNos-knock-out mice (n=28 compared with 27 wild-type littermates). Gene expression of miR-31, dystrophin, and nNOS was assessed by quantitative RT-PCR; protein content was measured by immunoblotting; NOS activity was evaluated with high-performance liquid chromatography; action potential duration (APD) and rate dependent adaptation were assessed by single-cell patch-clamping, and atrial fibrillation inducibility was evaluated by transoesophageal atrial burst stimulation.We found that atrial-specific upregulation of miR-31 in human atrial fibrillation caused dystrophin (DYS) translational repression and accelerated mRNA degradation of nNOS leading to a profound reduction in atrial DYS and nNOS protein content and in nitric oxide availability. In human atrial myocytes obtained from patients in sinus rhythm, nNOS inhibition was sufficient to recapitulate hallmark features of remodelling induced by atrial fibrillation, such as shortening of APD and loss of APD rate-dependency, but had no effect in patients with atrial fibrillation. In mice, nNos gene deletion or inhibition shortened atrial APD and increased atrial fibrillation inducibility in vivo. Inhibition of miR-31 in human atrial fibrillation recovered DYS and nNOS, and normalised APD and APD rate-dependency. Prevention of miR-31 binding to nNOS 3'UTR recovered both nNOS protein and gene expression but had no effect on the DYS protein or mRNA level (consistent with the mRNA degradation of nNOS by miR-31). Prevention of miR-31 binding to DYS 3'UTR increased DYS protein but not mRNA is consistent with translation repression of DYS by miR-31; recovery of DYS protein increased nNOS protein but not mRNA in keeping with a stabilising effect of DYS on nNOS protein. In goats, a reduction in dystrophin and nNOS protein content was associated with upregulation of miR-31 in the atria but not in the ventricles.The findings suggest that atrial-specific upregulation of miR-31 in human atrial fibrillation is a key mechanism causing atrial loss of dystrophin and nNOS; this loss leads to the electrical phenotype induced by atrial fibrillation.British Heart Foundation (BHF) Programme grant (for BC and XL), BHF Centre of Excellence in Oxford (SR), Leducq Foundation (in part for BC and SR), the European Union's seventh Framework Programme Grant Agree

Human Placental Hypoxia-Inducible Factor-1α Expression Correlates with Clinical Outcomes in Chronic Hypoxia in Vivo

Placental hypoxia is causally implicated in fetal growth restriction and preeclampsia, with both occurring more frequently at high altitude (>2700 m; HA). The nuclear transcription factor hypoxia-inducible factor (HIF) may facilitate placental oxygen transport at HA by increasing erythropoiesis and placental angiogenesis. We therefore investigated HIF expression and its regulatory mechanisms in placentas from normal pregnancies at high (3100 m), moderate (1600 m), and sea level (75 m) altitudes. Moderate-altitude and sea level placentas did not differ, but HIF-1α and the von Hippel-Lindau tumor suppressor protein were overexpressed in HA placentas. The ability of von Hippel-Lindau tumor suppressor protein to form the E3 ubiquitin protein ligase complex, required for HIF-1α degradation, was unaltered in HA placentas. mRNA for factor-inhibiting HIF, a negative modulator of HIF-1α transactivation, was increased, but protein levels were diminished. Elevated HIF-1α likely contributed to the significant increase we report in HIF-1α downstream target proteins, transforming growth factor β3 in the placenta, and vascular endothelial growth factor and erythropoietin in the maternal circulation. These circulating markers and lowered birth to placental weight ratios correlated with increased HIF-1α, thereby linking molecular and systemic physiological data. The HA response to chronic hypoxia resembles preeclampsia in several aspects, illustrating the utility of the HA model in understanding placental pathologies