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Nucleic Acid Labeling, Ligation, and Modification

In recent years the CuI-catalyzed [3+2] azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne [3+2] cycloaddition (SPAAC) reactions have been used to great effect in the nucleic acid field for DNA and RNA labeling, strand ligation, and the generation of artificial nucleic acid backbones. This chapter focuses on the most recent examples of DNA and RNA functionalization and ligation in which the CuAAC and SPAAC reactions have been applied. Chemical ligation of DNA and RNA strands has certain advantages over enzymatic ligation; it can be carried out on a large scale, and it is compatible with a wide range of chemical modifications in the DNA strands as well as being compatible with nucleic acid analogs. To achieve chemical ligation, alkyne- and azide-labeled DNA strands can be joined together efficiently by the CuAAC reaction to give an artificial triazole backbone at the point of ligation.

CMV serostatus is associated with improved survival and delayed toxicity onset following anti-PD-1 checkpoint blockade.

Cytomegalovirus (CMV) is a globally endemic latent herpes virus that profoundly impacts T cell immunity. We investigated the oncological consequences of CMV infection across 341 prospectively recruited patients receiving immune checkpoint blockade (ICB) for melanoma. CMV+ patients with metastatic melanoma (MM) have higher lymphocyte counts, reduced neutrophil to lymphocyte ratio and divergent CD8+ T cell gene expression. Combination anti-CTLA-4/anti-PD-1 ICB, but not single-agent anti-PD-1 ICB, induces cytotoxicity and CMV-associated gene expression in CD8+ T cells from CMV- patients. Correspondingly, overall survival was independent of CMV serostatus in combination anti-CTLA-4/anti-PD-1 ICB recipients (CMV+ hazard ratio for death: 1.02, P = 0.92), whereas CMV+ single-agent anti-PD-1 ICB recipients had improved overall survival (CMV+ hazard ratio for death: 0.37, P 

Irradiation at Ultra-High (FLASH) Dose Rates Reduces Acute Normal Tissue Toxicity in the Mouse Gastrointestinal System.

PURPOSE: Preclinical studies using ultra-high dose rate (FLASH) irradiation have demonstrated reduced normal tissue toxicity compared with conventional dose rate (CONV) irradiation, although this finding is not universal. We investigated the effect of temporal pulse structure and average dose rate of FLASH compared with CONV irradiation on acute intestinal toxicity. MATERIALS AND METHODS: Whole abdomens of C3H mice were irradiated with a single fraction to various doses, using a 6 MeV electron linear accelerator with single pulse FLASH (dose rate = 2-6 × 106 Gy/s) or conventional (CONV; 0.25 Gy/s) irradiation. At 3.75 days postirradiation, fresh feces were collected for 16S rRNA sequencing to assess changes in the gut microbiota. A Swiss roll-based crypt assay was used to quantify acute damage to the intestinal crypts to determine how tissue toxicity was affected by the different temporal pulse structures of FLASH delivery. RESULTS: We found statistically significant improvements in crypt survival for mice irradiated with FLASH at doses between 7.5 and 12.5 Gy, with a dose modifying factor of 1.1 for FLASH (7.5 Gy, P < .01; 10 Gy, P < .05; 12.5 Gy, P < .01). This sparing effect was lost when the delivery time was increased, either by increasing the number of irradiation pulses or by prolonging the time between 2 successive pulses. Sparing was observed for average dose rates of ≥280 Gy/s. Fecal microbiome analysis showed that FLASH irradiation caused fewer changes to the microbiota than CONV irradiation. CONCLUSIONS: This study demonstrates that FLASH irradiation can spare mouse small intestinal crypts and reduce changes in gut microbiome composition compared with CONV irradiation. The higher the average dose rate, the larger the FLASH effect, which is also influenced by temporal pulse structure of the delivery.

Use of intraoperative fluorescence to enhance robot-assisted radical prostatectomy.

Robot-assisted radical prostatectomy has become the standard of care for the removal of localized prostate cancer. Positive outcomes depend upon the precise removal of the prostate and associated tissue without damage to nearby structures. This process can be aided by fluorescence-guided surgery to enhance the visual contrast between different structures. Here the authors have conducted a systematic review using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines to identify ten investigations into the use of fluorescence-guided surgery in robot-assisted radical prostatectomy. These studies used fluorescent tracers to identify structures, including the prostate, neurovascular bundle and lymph nodes. These studies demonstrate the safe and effective use of fluorescence-guided surgery in robot-assisted radical prostatectomy and pave the way for further developments in this field.

Binuclear lanthanide complexes as magnetic resonance and optical imaging probes for redox sensing.

We report a family of lanthanide(III) complexes that act as redox probes via both magnetic resonance and luminescence outputs. The ligands are functionalized with nitro, azobenzene and azide groups which are reduced to a common aniline product, and each responds to both chemical and biocatalytic reductive conditions at different cathodic onset potentials. By judicious choice of complexed Ln(III), the probes can be optimized either for use in magnetic resonance imaging (Ln = Gd), or as highly efficient turn-on luminescence probes (Ln = Tb). The Tb(III) analogues are essentially non-emissive, until reductive generation of the aniline affords a complex which when excited by visible light (488 nm) emits green light with a quantum yield of 45% and millisecond long luminescent lifetimes (ms). The tunable redox response and imaging modalities of these versatile complexes have the potential to open up new approaches to imaging hypoxia.

Characterization of Effects of mTOR Inhibitors on Aging in Caenorhabditis elegans

Abstract Pharmacological inhibition of the mechanistic target of rapamycin (mTOR) signaling pathway with rapamycin can extend lifespan in several organisms. Although this includes the nematode Caenorhabditis elegans, effects in this species are relatively weak and sometimes difficult to reproduce. Here we test effects of drug dosage and timing of delivery to establish the upper limits of its capacity to extend life, and investigate drug effects on age-related pathology and causes of mortality. Liposome-mediated rapamycin treatment throughout adulthood showed a dose-dependent effect, causing a maximal 21.9% increase in mean lifespan, but shortening of lifespan at the highest dose, suggesting drug toxicity. Rapamycin treatment of larvae delayed development, weakly reduced fertility and modestly extended lifespan. By contrast, treatment initiated later in life robustly increased lifespan, even from Day 16 (or ~70 years in human terms). The rapalog temsirolimus extended lifespan similarly to rapamycin, but effects of everolimus were weaker. As in mouse, rapamycin had mixed effects on age-related pathologies, inhibiting one (uterine tumor growth) but not several others, suggesting a segmental antigeroid effect. These findings should usefully inform future experimental studies with rapamycin and rapalogs in C. elegans.

Machine learning predicts lifespan and underlying causes of death in aging C. elegans

Unraveling effects of anti-aging drugs on C. elegans using liposomes.

Liposome-mediated delivery is a possible means to overcome several shortcomings with C. elegans as a model for identifying and testing drugs that retard aging. These include confounding interactions between drugs and the nematodes' bacterial food source and failure of drugs to be taken up into nematode tissues. To explore this, we have tested liposome-mediated delivery of a range of fluorescent dyes and drugs in C. elegans. Liposome encapsulation led to enhanced effects on lifespan, requiring smaller quantities of compounds, and enhanced uptake of several dyes into the gut lumen. However, one dye (Texas red) did not cross into nematode tissues, showing that liposomes cannot ensure the uptake of all compounds. Of six compounds previously reported to extend lifespan (vitamin C, N-acetylcysteine, glutathione (GSH), trimethadione, thioflavin T (ThT), and rapamycin), this effect was reproduced for the latter four in a condition-dependent manner. For GSH and ThT, antibiotics abrogated life extension, implying a bacterially mediated effect. With GSH, this was attributable to reduced early death from pharyngeal infection and associated with alterations of mitochondrial morphology in a manner suggesting a possible innate immune training effect. By contrast, ThT itself exhibited antibiotic effects. For rapamycin, significant increases in lifespan were only seen when bacterial proliferation was prevented. These results document the utility and limitations of liposome-mediated drug delivery for C. elegans. They also illustrate how nematode-bacteria interactions can determine the effects of compounds on C. elegans lifespan in a variety of ways.

Highly effective inhibition of mild steel corrosion in HCl solution by using pyrido[1,2-a]benzimidazoles

Pyrido[1,2-a]benzimidazoles are reported herein for the first time as effective inhibitors of mild steel corrosion in hydrochloric acid.

A survey of dosimetry quality assurance practice at UK small animal radiation research platform (SARRP) facilities.

Improvements in preclinical radiation research have been made to better mimic the equipment and techniques implemented in the clinic. The development of dedicated small animal radiation units facilitates such advances by combining treatment planning, image guidance and conformal delivery. One area significantly behind its clinical equivalent are standardised dosimetry quality assurance (QA) protocols, hampering the translatability of results into the development of clinical interventions. Approach: The aim of the study described herein was to summarise the current QA procedures implemented at several institutions on Small Animal Radiation Research Platforms (SARRPs), the system used by the six institutions surveyed, and to determine the barriers to implementing a standard dosimetry protocol. Participants at UK research institutions were invited to complete a questionnaire to ascertain their current preclinical QA practice. Main results: All participants involved undertake regular dose output measurements and most perform image guidance QA measurements. Consistency in QA procedures differed when more complex plan verification or end-to-end testing was discussed. Significance: This survey demonstrates that, although improvements are being made in the awareness of the importance of regular dosimetry tests, there is still a way to go to standardise the procedures with regards to more complex verifications. Incorporating robust QA procedures and strict dose constraints would ensure the reliability and ethical integrity of experiments involving small animals. This approach not only protects the welfare of the animals but also enhances the quality and reproducibility of the preclinical results.

Performance analysis of an artificial intelligence algorithm for detection and localisation of tracheal tube position by chest radiography.

A qualitative exploration of the barriers to and facilitators of clozapine monitoring in a secure psychiatric setting.

AIMS AND METHOD: To explore the beliefs and understanding of staff and patients at a secure mental health unit regarding clozapine monitoring, and to identify barriers to and facilitators of monitoring. Qualitative semi-structured interviews and focus groups were conducted with 17 staff members and six patients. RESULTS: Six key themes were identified. The key facilitator of effective monitoring was the motivation of staff to help patients to become independent and facilitate recovery. An important barrier was a lack of clarity around the roles of different staff groups in monitoring. Staff and patients widely supported the establishment of an in-patient clozapine clinic and perceived that it would prepare patients for discharge. CLINICAL IMPLICATIONS: An in-patient clozapine clinic is a robust mechanism for clozapine monitoring in secure settings. The barriers and facilitators identified here could be applied to other secure units to guide their systems of clozapine monitoring.

Surgical and Medical Management of Epithelial Ovarian Cancer

Epithelial cancer of the ovary, fallopian tube or primary peritoneum, collectively described as ‘ovarian cancer’ or EOC, is relatively uncommon. It represents 2% of total cancer cases in the UK (data from 2013) but is the most lethal of all gynaecological cancers. This is partly due to its insidious presentation but also because of its intrinsic histological and molecular heterogeneity. EOC comprises at least five distinct histological subtypes (high-grade serous, endometrioid, clear cell, mucinous, seromucinous and low-grade serous), the most common and well-studied being high-grade serous ovarian cancer (HGSOC). For the majority of patients after successful initial treatment with debulking surgery and chemotherapy, the disease will relapse and become increasingly chemotherapy resistant with each episode of recurrence. Future treatment strategies, as well as improving response to front-line therapy, are focusing on ways to overcome chemotherapy resistance in the relapsed setting, with the judicious use of novel cytotoxic and/or targeted therapies. These options are realized with improvements in our understanding of the molecular behaviour of the disease. In this chapter, we summarize the current status quo of the surgical and medical management of ovarian cancer and present results from a number of key studies that have explored genetic, molecular and histological-targeted strategies in the treatment of this disease.

Analysis of sagittal plane cine magnetic resonance imaging for measurement of pancreatic tumor residual motion during breath hold and evaluation of gating margins used in radiotherapy treatment.

BACKGROUND AND PURPOSE: In pancreatic radiotherapy, residual tumor motion during treatment increases the risk of toxicity. Cine imaging acquired during magnetic resonance guided radiotherapy (MRgRT) enables real-time treatment gating in response to anatomical motion, which can reduce this risk; however, treatment gating can negatively impact the efficiency of treatment. This study aimed to quantify the extent of residual tumor motion during breath hold and evaluate the appropriateness of the treatment gating margins used in current clinical practice. MATERIALS AND METHODS: Cine imaging acquired during pancreatic MRgRT of 11 patients on the ViewRay MRIdian was analyzed. The total duration of treatment analyzed was 12 h 13 min. Improved methods for processing and analyzing cine imaging were developed: breath holds were systematically separated with frequency analysis, residual motion was measured with consideration of both the tracking structure contour and centroid, and residual motion measurements were supported by phantom measurements of image scaling, resolution, and noise. Residual motion was measured at angles 0°, 45°, 90°, and 135° to the superior-inferior (SI) direction. Total residual motion was measured by combining directional measurements. RESULTS: The minimum tracking structure displacement resolvable through cine imaging was found to be 1.5 mm; therefore, residual motion analysis was limited to 1.5 mm spatial resolution. Total residual motion was contained within margins Δ = $\Delta =\, $ ±1.5, ±3, and ±4.5mm with mean percentage frequencies of 97.0%, 91.1%, and 67.8%. Most residual motion was observed in the SI direction, and significantly more residual motion was measured for the tracking structure contour than the centroid. CONCLUSION: The results demonstrate that patients are largely able to maintain breath hold positions to within a 3 mm margin, thus provide evidence that supports the use of a 3mm gating margin in clinical practice. Residual motion frequently exceeded 1.5 mm so a reduction in gating margin would have an undesirable impact on treatment efficiency.

Regional patterns of early-onset colorectal cancer from the GEOCODE (Global Early-Onset COlorectal Cancer DatabasE)-European consortium: Retrospective cohort study

Background: The incidence of early-onset colorectal cancer is increasing, but in Europe this growth shows a heterogeneous pattern in different countries and regions. Methods: Patients from six countries who participated in the Global Early-Onset COlorectal Cancer DatabasE (GEOCODE)-Europe group were included. The inclusion criteria were patients with colorectal adenocarcinoma diagnosed between 18 and 49 years of age, between January 2010 and December 2017, with at least 3 years of follow-up. Patients with inherited colorectal cancer syndromes were excluded. Results: A total of 851 patients were included with almost equal sex distribution, most were diagnosed at age 39 years or older and 42% of patients were overweight or obese. Diagnoses were predominantly at later stages (62.5% stage III-IV) and tumours were predominantly located in the distal colon (76.9% left colon and rectum). Comparative analysis between countries demonstrated that the UK had a younger age at diagnosis and the Italian cohort had a higher prevalence of being overweight or obese. Patients from Luxembourg had more advanced stage diagnoses and those from The Netherlands had more polyps. Patients from the UK had a greater family history of colorectal cancer. Comparison of Mediterranean versus non-Mediterranean countries showed significant differences in the age at diagnosis and body mass index. The prevalence of early-onset colorectal cancer over the age of 40 years in Mediterranean versus non-Mediterranean countries was 71.4% versus 62.1% (P = 0.002), and early-onset colorectal cancer was diagnosed at a more advanced stage in Mediterranean countries versus non-Mediterranean countries (65.3% versus 54.7%; P = 0.033). Family history of colorectal cancer in a first-degree relative was more common in non-Mediterranean versus Mediterranean countries (19.1% versus 11.4%; P < 0.001). Conclusion: This study highlights significant geographical disparities in the clinical, pathological and familial features of early-onset colorectal cancer across European countries.

Neoadjuvant Treatment of Rectal Cancer: A Repeat UK-wide Survey After Implementation of National Intensity Modulated Radiotherapy Guidance.

AIMS: Rectal cancer management has changed significantly in the last decade with the introduction of total neoadjuvant therapy (TNT), minimally invasive surgery, brachytherapy, and organ preservation. A national survey of intensity modulated radiotherapy (IMRT) was carried out in 2020 to support the development of national Royal College of Radiologists (RCR) guidance, published in 2021. We performed a repeat survey in collaboration with the RCR, to inform iterations of the RCR Guidance and establish treatment patterns across the UK to facilitate future research and development. MATERIALS AND METHODS: A web-based survey was developed and tested by the authors prior to dissemination by the RCR to all UK radiotherapy centres. The repeat survey requested details and strategies of current radiotherapy techniques, including details on setup, doses, organs at risk, peer review, and verification, and asked for the standard management of 5 clinical cases within each multidisciplinary team (MDT) serving that radiotherapy centre. Descriptive statistical analysis was carried out. RESULTS: In total, 42 of 60 (70%) of the NHS centres across the UK answered the repeat IMRT rectal survey, which reflected 70 MDTs answering the clinical scenarios questions. 100% of centres that responded are routinely using IMRT, with 95% of centres using it in all patients. Variation in treatment delivery has reduced since the previous survey. The greatest difference is still in the use of simultaneous integrated boost and definition of organs at risk. The management for the clinical cases was widely different, with answers generally equally distributed between 2-4 options. The highest-scoring treatment strategies ranged from 24% to 57%. CONCLUSION: RCR guidance has helped standardise the delivery of radiotherapy to treat rectal cancer in the UK. The variation in neoadjuvant treatment represents an exciting, evolving time in rectal cancer management. Clinical trials are needed to further homogenise treatment, but a degree of national variation is likely to continue.

Supplementary Figure S2 from Combined Oxygen-Enhanced MRI and Perfusion Imaging Detect Hypoxia Modification from Banoxantrone and Atovaquone and Track Their Differential Mechanisms of Action

<p>Supplementary Figure S2: Confirmation that oxygen increases tumor R1.</p>

Supplementary Figure S5 from Combined Oxygen-Enhanced MRI and Perfusion Imaging Detect Hypoxia Modification from Banoxantrone and Atovaquone and Track Their Differential Mechanisms of Action

<p>Supplementary Figure S5 compares the natural history growth of Calu6 and U87 xenografts.</p>

Supplementary Figure S6 from Combined Oxygen-Enhanced MRI and Perfusion Imaging Detect Hypoxia Modification from Banoxantrone and Atovaquone and Track Their Differential Mechanisms of Action

<p>Supplementary Figure S6 shows that banoxantrone induces tumor necrosis.</p>

Supplementary Figure S4 from Combined Oxygen-Enhanced MRI and Perfusion Imaging Detect Hypoxia Modification from Banoxantrone and Atovaquone and Track Their Differential Mechanisms of Action

<p>Supplementary Figure S4 confirms that there was no drug effect on cell number or viability.</p>