The complex polyploid genome architecture of sugarcane.

Sugarcane, the world's most harvested crop by tonnage, has shaped global history, trade and geopolitics, and is currently responsible for 80% of sugar production worldwide1. While traditional sugarcane breeding methods have effectively generated cultivars adapted to new environments and pathogens, sugar yield improvements have recently plateaued2. The cessation of yield gains may be due to limited genetic diversity within breeding populations, long breeding cycles and the complexity of its genome, the latter preventing breeders from taking advantage of the recent explosion of whole-genome sequencing that has benefited many other crops. Thus, modern sugarcane hybrids are the last remaining major crop without a reference-quality genome. Here we take a major step towards advancing sugarcane biotechnology by generating a polyploid reference genome for R570, a typical modern cultivar derived from interspecific hybridization between the domesticated species (Saccharum officinarum) and the wild species (Saccharum spontaneum). In contrast to the existing single haplotype ('monoploid') representation of R570, our 8.7 billion base assembly contains a complete representation of unique DNA sequences across the approximately 12 chromosome copies in this polyploid genome. Using this highly contiguous genome assembly, we filled a previously unsized gap within an R570 physical genetic map to describe the likely causal genes underlying the single-copy Bru1 brown rust resistance locus. This polyploid genome assembly with fine-grain descriptions of genome architecture and molecular targets for biotechnology will help accelerate molecular and transgenic breeding and adaptation of sugarcane to future environmental conditions.

Epigenetic insights to pediatric uropathology: Celebrating the fundamental biology vision of Tony Khoury.

INTRODUCTION: Many pediatric urology conditions affect putatively normal tissues or appear too commonly to be based solely on specific DNA mutations. Understanding epigenetic mechanisms in pediatric urology, therefore, has many implications that can impact cell and tissue responses to settings, such as environmental and hormonal influences on urethral development, uropathogenic infections, obstructive stimuli, all of which originate externally or extracellularly. Indeed, the cell's response to external stimuli is often mediated epigenetically. In this commentary, we highlight work on the critical role that epigenetic machinery, such as DNA methyltransferases (DNMTs), Enhancer of Zeste Polycomb Repressive Complex 2 Subunit (EZH2), and others play in regulating gene expression and cellular functions in three urological contexts. DESIGN: Animal and cellular constructs were used to model clinical pediatric uropathology. The hypertrophy, trabeculation, and fibrosis of the chronically obstructed bladder was explored using smooth muscle cell models employing disorganised vs. normal extracellular matrix (ECM), as well as a new animal model of chronic obstructive bladder disease (COBD) which retains its pathologic features even after bladder de-obstruction. Cell models from human and murine hypospadias or genital tubercles (GT) were used to illustrate developmental responses and epigenetic dependency of key developmental genes. Finally, using bladder urothelial and organoid culture systems, we examined activity of epigenetic machinery in response to non uropathogenic vs. uropathogenic E.coli (UPEC). DNMT and EZH2 expression and function were interrogated in these model systems. RESULTS: Disordered ECM exerted a principal mitogenic and epigenetic role for on bladder smooth muscle both in vitro and in CODB in vivo. Key genes, e.g., BDNF and KCNB2 were under epigenetic regulation in actively evolving obstruction and COBD, though each condition showed distinct epigenetic responses. In models of hypospadias, estrogen strongly dysregulated WNT and Hox expression, which was normalized by epigenetic inhibition. Finally, DNA methylation machinery in the urothelium showed specific activation when challenged by uropathogenic E.coli. Similarly, UPEC induces hypermethylation and downregulation of the growth suppressor p16INK4A. Moreover, host cells exposed to UPEC produced secreted factors inducing epigenetic responses transmissible from one affected cell to another without ongoing bacterial presence. DISCUSSION: Microenvironmental influences altered epigenetic activity in the three described urologic contexts. Considering that many obstructed bladders continue to display abnormal architecture and dysfunction despite relief of obstruction similar to after resection of posterior valves or BPH, the epigenetic mechanisms described highlight novel approaches for understanding the underlying smooth muscle myopathy of this crucial clinical problem. Similarly, there is evidence for an epigenetic basis of xenoestrogen on development of hypospadias, and UTI-induced pan-urothelial alteration of epigenetic marks and propensity for subsequent (recurrent) UTI. The impact of mechanical, hormonal, infectious triggers on genitourinary epigenetic machinery activity invite novel avenues for targeting epigenetic modifications associated with these non-cancer diseases in urology. This includes the use of deactivated CRISPR-based technologies for precise epigenome targeting and editing. Overall, we underscore the importance of understanding epigenetic regulation in pediatric urology for the development of innovative therapeutic and management strategies.

Urethral compliance in hypospadias operated by tubularized incised urethral plate (TIP) with and without a dorsal inlay graft: an experimental controlled study.

OBJECTIVE: Abnormal uroflowmetries are common after tubularized incised plate urethroplasties (TIP), perhaps due to low compliance. We hypothesized that (1) abnormal uroflowmetries after TIP might be caused by segmental lower compliance; (2) by adding a graft to the raw area in the incised plate (TIPG), compliance might be improved by preventing secondary intention healing of the dorsal incision. METHODS: A standardized penectomy was performed in 27 adult male rabbits: 9 normal non-operated controls (G1), 6 weeks after TIP (G2: n = 9) or TIPG (G3: n = 9). A standardized isolated segment (including the whole urethroplasty in G1 and G2) was progressively distended with air (1, 2 and 3 ml) in the 3 groups. The respective intraluminal pressures were measured with a tensiometer. RESULTS: Pressure measurements were feasible and reproducible for this model. Mean pressures tended to be higher in the experimental groups (G1: 59.7 mmHg vs. G2: 79.6 mmHg vs. G3: 100.1 mmHg for 1 ml injections; G1: 233.1 mmHg vs. G2: 241 mmHg vs. G3: 308.4 mmHg for 2 ml injections and G1: 457.3 mmHg vs. G2: 429 mmHg vs. G3: 520 mmHg for 3 ml injections) without reaching the statistical significance. CONCLUSION: In this model, the elasticity of the TIP or TIPG neourethras tended to be reduced when compared to controls. The placement of an inlay graft on the dorsal incised area did not increase the compliance. This model allows the measurement of segmental intraluminal urethral pressures generated by controlled air distension and may be a useful tool to evaluate the experimental urethroplasty models.

Potential for Isotoxic Re-irradiation Stereotactic Ablative Body Radiotherapy in Locally Recurrent Rectal Cancer.

AIMS: The non-surgical management of locally recurrent rectal cancer (LRRC) is an area of unmet need, with no defined standard treatment and extremely poor outcomes. Patients typically receive radiotherapy during initial multimodality treatment and historically re-irradiation has been limited to conservative doses with subsequent short-term symptom control. Recently, stereotactic ablative body radiotherapy (SABR) has shown promise in re-irradiation of LRRC in England, but is limited to a relatively modest dose prescription of 30Gy in five fractions. We propose that SABR can be achieved in LRRC to higher doses using an isotoxic dose prescription with fixed 15% per annum tissue recovery for acceptable organ at risk (OAR) constraints. MATERIALS AND METHODS: Patients with LRRC at a local centre treated with SABR re-irradiation were audited. Patients were identified, the dose and time since previous radiotherapy determined, re-irradiation OAR constraints calculated and retrospective re-planning carried out. RESULTS: In patients currently receiving SABR (17 patients, 21 targets), dose escalation above 30 Gy in five fractions was achievable, with a biological effective dose of 80 Gy (alpha/beta = 10) deliverable to 80% or more of the planning target volume in eight of the 21 targets. CONCLUSIONS: Isotoxic SABR re-irradiation should be considered a potential treatment option for LRRC to maximise patient outcomes while limiting excess toxicity. Although probably conservative, clinical outcome data are needed to determine the suitability of OAR constraints using 15% per annum tissue recovery and the impact on local control rates, patient quality of life and overall survival of isotoxic SABR.

Technical Radiotherapy Advances - The Role of Magnetic Resonance Imaging-Guided Radiation in the Delivery of Hypofractionation.

Safe delivery of hypofractionated radiotherapy requires high levels of accuracy due to the high doses of radiation delivered per fraction. Magnetic resonance guided radiotherapy (MRgRT) represents a new treatment paradigm which allows improved visualisation of targets and organs at risk, alongside the capability to adapt the treatment plan in real time prior to treatment delivery. There are challenges to delivering hypofractionated radiotherapy with conventional image-guided radiotherapy (IGRT) techniques and MRgRT may help to improve accuracy in radiation delivery in a number of clinical and anatomical scenarios. Specifically, there is an emerging role of MRgRT in delivering stereotactic body radiotherapy (SBRT) for locally advanced pancreatic cancer (LAPC) due to the superior soft tissue contrast provided by Magnetic Resonance Imaging combined with the ability to accommodate variation in anatomical appearances during treatment delivery. Reported data on the use of MRgRT in LAPC and it's role in enabling dose escalation are discussed in this article. There are further potential benefits to the use of MRgRT, for example the use of functional imaging during treatment delivery and generation of synthetic computed tomography, which have previously been impractical or unachievable. The overall aim of this article is to demonstrate the utility of MRgRT in facilitating safe delivery of hypofractionated radiotherapy and to highlight ways in which it may help to overcome challenges posed by current IGRT techniques.

UK 2022 Consensus on Normal Tissue Dose-Volume Constraints for Oligometastatic, Primary Lung and Hepatocellular Carcinoma Stereotactic Ablative Radiotherapy.

The use of stereotactic ablative radiotherapy (SABR) in the UK has expanded over the past decade, in part as the result of several UK clinical trials and a recent NHS England Commissioning through Evaluation programme. A UK SABR Consortium consensus for normal tissue constraints for SABR was published in 2017, based on the existing literature at the time. The published literature regarding SABR has increased in volume over the past 5 years and multiple UK centres are currently working to develop new SABR services. A review and update of the previous consensus is therefore appropriate and timely. It is hoped that this document will provide a useful resource to facilitate safe and consistent SABR practice.

The tyrosine kinase p56lck is essential in coxsackievirus B3-mediated heart disease.

Infections are thought to be important in the pathogenesis of many heart diseases. Coxsackievirus B3 (CVB3) has been linked to chronic dilated cardiomyopathy, a common cause of progressive heart disease, heart failure and sudden death. We show here that the sarcoma (Src) family kinase Lck (p56lck) is required for efficient CVB3 replication in T-cell lines and for viral replication and persistence in vivo. Whereas infection of wild-type mice with human pathogenic CVB3 caused acute and very severe myocarditis, meningitis, hepatitis, pancreatitis and dilated cardiomyopathy, mice lacking the p56lck gene were completely protected from CVB3-induced acute pathogenicity and chronic heart disease. These data identify a previously unknown function of Src family kinases and indicate that p56lck is the essential host factor that controls the replication and pathogenicity of CVB3.

Inhibition of DNA methylation during chronic obstructive bladder disease (COBD) improves function, pathology and expression.

Partial bladder outlet obstruction due to prostate hyperplasia or posterior urethral valves, is a widespread cause of urinary dysfunction, patient discomfort and also responsible for immense health care costs. Even after removal or relief of obstruction, the functional and pathologic aspects of obstruction remain as a chronic obstructive bladder disease (COBD). Epigenetic changes, such as DNA methylation, contribute to the persistent character of many chronic diseases, and may be altered in COBD. We tested whether candidate genes and pathways and the pathophysiology of COBD were affected by a hypomethylating agent, decitabine (DAC). COBD was created in female Sprague-Dawley rats by surgical ligation of the urethra for 6 weeks, followed by removal of the suture. Sham ligations were performed by passing the suture behind the urethra. After removal of the obstruction or sham removal, animals were randomized to DAC treatment (1 mg/kg/3-times/week intraperitoneally) or vehicle (normal saline). Bladder function was non-invasively tested using metabolic cages, both one day prior to de-obstruction at 6 weeks and prior to sacrifice at 10 weeks. Residual volume and bladder mass were measured for each bladder. Bladders were examined by immunostaining as well as qPCR. The effects of DNA methyltransferase (DNMT)-3A knockout or overexpression on smooth muscle cell (SMC) function and phenotype were also examined in bladder SMC and ex vivo culture. Residual volumes of the DAC treated group were not significantly different from the NS group. Compared to COBD NS, COBD DAC treatment helped preserve micturition volume with a significant recovery of the voiding efficiency (ratio of the maximum voided volume/maximum bladder capacity) by one third (Fig. 1, p > 0.05). Brain-derived neurotrophic factor (BDNF) variants 1 and 5 were upregulated by COBD and significantly reduced by DAC treatment. Deposition of collagen in the COBD bladder was reduced by DAC, but gross hypertrophy remained. In bladder SMC, DNMT3A overexpression led to a loss of contractile function and phenotype. In bladders, persistently altered by COBD, inhibition of DNA-methylation enhances functional recovery, unlike treatment during partial obstruction, which exacerbates obstructive pathology. The underlying mechanisms may relate to the gene expression changes in BDNF and their effects on signaling in the bladder.

Image-guided Radiotherapy to Manage Respiratory Motion: Lung and Liver.

Organ motion as a result of respiratory and cardiac motion poses significant challenges for the accurate delivery of radiotherapy to both the thorax and the upper abdomen. Modern imaging techniques during radiotherapy simulation and delivery now permit better quantification of organ motion, which in turn reduces tumour and organ at risk position uncertainty. These imaging advances, coupled with respiratory correlated radiotherapy delivery techniques, have led to the development of a range of approaches to manage respiratory motion. This review summarises the key strategies of image-guided respiratory motion management with a focus on lung and liver radiotherapy.

Inhibition of calcium/calmodulin-dependent protein kinase in Drosophila disrupts behavioral plasticity.

One of the major mediators of calcium action in neurons is the multifunctional calcium/calmodulin-dependent protein kinase (CaM kinase), an enzyme with the capability of directly regulating its own activity by autophosphorylation. To assess the involvement of CaM kinase in experience-dependent behavior in an intact animal, we have designed a specific peptide inhibitor of CaM kinase and made transgenic Drosophila that express it under control of an inducible promoter. These flies fail to learn normally in two behavioral plasticity paradigms: acoustic priming, a nonassociative measure of sensitization, and courtship conditioning, a measure of associative learning. The magnitude of the learning defect in the associative paradigm appears to be proportional to the level of expression of the peptide gene in the two transgenic lines and can be increased by heat shock induction of gene expression. These results suggest that CaM kinase activity is required for plastic behaviors in an intact animal.

UK Consensus on Normal Tissue Dose Constraints for Stereotactic Radiotherapy.

Six UK studies investigating stereotactic ablative radiotherapy (SABR) are currently open. Many of these involve the treatment of oligometastatic disease at different locations in the body. Members of all the trial management groups collaborated to generate a consensus document on appropriate organ at risk dose constraints. Values from existing but older reviews were updated using data from current studies. It is hoped that this unified approach will facilitate standardised implementation of SABR across the UK and will allow meaningful toxicity comparisons between SABR studies and internationally.

Recommendations for MRI-based contouring of gross tumor volume and organs at risk for radiation therapy of pancreatic cancer.

PURPOSE: Local recurrence is a common and morbid event in patients with unresectable pancreatic adenocarcinoma. A more conformal and targeted radiation dose to the macroscopic tumor in nonmetastatic pancreatic cancer is likely to reduce acute toxicity and improve local control. Optimal soft tissue contrast is required to facilitate delineation of a target and creation of a planning target volume with margin reduction and motion management. Magnetic resonance imaging (MRI) offers considerable advantages in optimizing soft tissue delineation and is an ideal modality for imaging and delineating a gross tumor volume (GTV) within the pancreas, particularly as it relates to conformal radiation planning. Currently, no guidelines have been defined for the delineation of pancreatic tumors for radiation therapy treatment planning. Moreover, abdominal MRI sequences are complex and the anatomy relevant to the radiation oncologist can be challenging. The purpose of this study is to provide recommendations for delineation of GTV and organs at risk (OARs) using MRI and incorporating multiple MRI sequences. METHODS AND MATERIALS: Five patients with pancreatic cancer and 1 healthy subject were imaged with MRI scans either on 1.5T or on 3T magnets in 2 separate institutes. The GTV and OARs were contoured for all patients in a consensus meeting. RESULTS: An overview of MRI-based anatomy of the GTV and OARs is provided. Practical contouring instructions for the GTV and the OARs with the aid of MRI were developed and included in these recommendations. In addition, practical suggestions for implementation of MRI in pancreatic radiation treatment planning are provided. CONCLUSIONS: With this report, we attempt to provide recommendations for MRI-based contouring of pancreatic tumors and OARs. This could lead to better uniformity in defining the GTV and OARs for clinical trials and in radiation therapy treatment planning, with the ultimate goal of improving local control while minimizing morbidity.

Susceptibility to myocarditis is dependent on the response of alphabeta T lymphocytes to coxsackieviral infection.

Viral myocarditis is an important cause of heart failure and dilated cardiomyopathy. T lymphocytes are implicated in myocardial damage in murine models of coxsackievirus B3 (CVB3) myocarditis. We used knockout mice lacking CD4 (CD4(-/-)), CD8 (CD8(-/-)), both coreceptors (CD4(-/-)CD8(-/-)), or the T-cell receptor beta chain (TCRbeta(-/-)) to address the contribution of T-cell subpopulations to host susceptibility to CVB3 myocarditis. Severity of disease was magnified in CD8(-/-) mice but attenuated in CD4(-/-) mice, consistent with a pathogenic role for CD4(+) lymphocytes. Elimination of both CD4 and CD8 molecules from T lymphocytes by genetic knockout better protected mice from myocarditis, demonstrating that both CD4(+) and CD8(+) T cells contribute to host susceptibility. The same benefit occurred in TCRbeta(-/-) mice, with prolonged survival and minimal myocardial disease observed after CVB3 infection. Elevated interferon-gamma and decreased tumor necrosis factor-alpha expression are associated with attenuated myocardial damage in CD4(-/-)CD8(-/-) mice. These results show that the presence of TCRalphabeta(+) T cells enhances host susceptibility to myocarditis. The severity of myocardial damage and associated mortality are dependent on the predominant T-cell type available to respond to CVB3 infection. One mechanism by which CD4(+) and CD8(+) T-cell subsets influence the pathogenesis of myocarditis may involve specific cytokine expression patterns.

Stereotactic body radiotherapy for liver metastases.

The role for local ablative therapies in the management paradigm of oligometastatic liver disease is increasing. The evidence base supporting the use of stereotactic body radiotherapy for liver metastases has expanded rapidly over the past decade, showing high rates of local control with low associated toxicity. This review summarises the evidence base to date, discussing optimal patient selection, challenges involved with treatment delivery and optimal dose and fractionation. The reported toxicity associated with liver stereotactic body radiotherapy is presented, together with possible pitfalls in interpreting the response to treatment using standard imaging modalities. Finally, potential avenues for future research in this area are highlighted.

Initial UK Experience of Stereotactic Body Radiotherapy for Extracranial Oligometastases: Can We Change the Therapeutic Paradigm?

AIMS: To retrospectively review the toxicity and early outcome data from patients who have received stereotactic body radiotherapy (SBRT) for extracranial oligometastases at a single UK institution. MATERIALS AND METHODS: Eligible patients had ≤3 extracranial metastases and performance status ≤2. Prior systemic therapy and radical treatment of oligometastastic relapse with any standard treatment modality was permitted. Patients with synchronous metastatic disease were excluded unless they had evidence of controlled primary disease after radical therapy. Follow-up consisted of clinical examination, biochemical and radiological assessments in accordance with standard clinical care. Progression events were defined using RECIST. Toxicity was evaluated using CTCAE v4.0. Local control, progression-free survival (PFS), freedom from widespread distant metastasis (defined as disease not amenable to further radical salvage therapy) and overall survival were calculated. RESULTS: Between July 2011 and April 2014, 73 patients with 87 metastases received SBRT (range 1-3 per patient). The median follow-up was 14.5 months (range 0-26.4). The median PFS was 14.5 months (1 year PFS 57%, 2 year 28%); 1 year overall survival 96%, 2 year 79.8%; 2 year local control 88%. At 2 years, 46% of patients were free from widespread distant metastases. No ≥ grade 3 acute or late toxicity was observed. CONCLUSION: At this time point, observed toxicity is minimal with excellent local control rates. This promising treatment paradigm requires further investigation in the context of a randomised controlled trial to establish if the addition of SBRT to standard care improves survival outcomes.

Genetic control of monoterpene composition in the essential oil of Melaleuca alternifolia (Cheel).

Previous studies into the population structure of Melaleuca alternifolia by both isozyme and microsatellite analysis revealed little evidence for genetic structuring within genetic provenances. In contrast, analysis of the oil composition within these same regions showed distinct clustering of chemotypes within the provenances suggesting either that chemotype was not under genetic control, or that there is strong environmental selection for plant chemotypes. To investigate the level of genetic control of monoterpene composition in the essential oil of M. alternifolia, individuals representing the three extreme chemotypes of high terpinen-4-ol, high 1,8-cineole and high terpinolene were crossed with an individual with the commercially desirable high terpinen-4-ol oil profile. The progeny resulting from these crosses displayed oil profiles that were intermediate to that of the parent. Further analysis of the survey of oil chemotypes within the natural population also suggests that these intermediate chemotypes may arise naturally between regions containing high proportions of the extreme chemotypes. These results imply that there is a level of genetic structure for chemotype determination within the genetic provenance that is undetected by isozyme and microsatellite analysis. This information could play a vital role in the selection of appropriate genetic material to be used in future essential oil selection and breeding programs.

A retrospective epidemiological analysis of non-accidental head injury in children in Scotland over a 15 year period.

A retrospective analysis of the epidemiological characteristics of non-accidental head injury (NAHI) in children in Scotland over the last fifteen years from 1981 until March 1996 was performed. The information was provided by the Information and Statistics Division of the Scottish Health Service. The average incidence of NAHI calculated over this period was 0.04 cases per year per 1000 children under 5 years. Fifty-five per cent of all cases occurred in those children who were less than a year old. 41% of cases were inflicted by a parent but in 47% the perpetrator could not be identified. The mortality rate was found to be 2%. Non-accidental head injury cases identified using the ICD-9 coding classification system gives a surprisingly low incidence. This number is probably an underestimate and the reasons for this are discussed. A prospective epidemiological analysis of NAHI in children in Scotland is being undertaken to determine the true incidence.