The tautomer-specific excited state dynamics of 2,6-diaminopurine using resonance-enhanced multiphoton ionization and quantum chemical calculations.

2,6-Diaminopurine (2,6-dAP) is an alternative nucleobase that potentially played a role in prebiotic chemistry. We studied its excited state dynamics in the gas phase by REMPI, IR-UV hole burning, and ps pump-probe spectroscopy and performed quantum chemical calculations at the SCS-ADC(2) level of theory to interpret the experimental results. We found the 9H tautomer to have a small barrier to ultrafast relaxation via puckering of its 6-membered ring. The 7H tautomer has a larger barrier to reach a conical intersection and also has a sizable triplet yield. These results are discussed relative to other purines, for which 9H tautomerization appears to be more photostable than 7H and homosubstituted purines appear to be less photostable than heterosubstituted or singly substituted purines.

Aspirin reprogrammes colorectal cancer cell metabolism and sensitises to glutaminase inhibition.

BACKGROUND: To support proliferation and survival within a challenging microenvironment, cancer cells must reprogramme their metabolism. As such, targeting cancer cell metabolism is a promising therapeutic avenue. However, identifying tractable nodes of metabolic vulnerability in cancer cells is challenging due to their metabolic plasticity. Identification of effective treatment combinations to counter this is an active area of research. Aspirin has a well-established role in cancer prevention, particularly in colorectal cancer (CRC), although the mechanisms are not fully understood. METHODS: We generated a model to investigate the impact of long-term (52 weeks) aspirin exposure on CRC cells, which has allowed us comprehensively characterise the metabolic impact of long-term aspirin exposure (2-4mM for 52 weeks) using proteomics, Seahorse Extracellular Flux Analysis and Stable Isotope Labelling (SIL). Using this information, we were able to identify nodes of metabolic vulnerability for further targeting, investigating the impact of combining aspirin with metabolic inhibitors in vitro and in vivo. RESULTS: We show that aspirin regulates several enzymes and transporters of central carbon metabolism and results in a reduction in glutaminolysis and a concomitant increase in glucose metabolism, demonstrating reprogramming of nutrient utilisation. We show that aspirin causes likely compensatory changes that render the cells sensitive to the glutaminase 1 (GLS1) inhibitor-CB-839. Of note given the clinical interest, treatment with CB-839 alone had little effect on CRC cell growth or survival. However, in combination with aspirin, CB-839 inhibited CRC cell proliferation and induced apoptosis in vitro and, importantly, reduced crypt proliferation in Apcfl/fl mice in vivo. CONCLUSIONS: Together, these results show that aspirin leads to significant metabolic reprogramming in colorectal cancer cells and raises the possibility that aspirin could significantly increase the efficacy of metabolic cancer therapies in CRC.

Spore-FP1 tuberculosis mucosal vaccine candidate is highly protective in guinea pigs but fails to improve on BCG-conferred protection in non-human primates.

Tuberculosis remains a major health threat globally and a more effective vaccine than the current Bacillus Calmette Guerin (BCG) is required, either to replace or boost it. The Spore-FP1 mucosal vaccine candidate is based on the fusion protein of Ag85B-Acr-HBHA/heparin-binding domain, adsorbed on the surface of inactivated Bacillus subtilis spores. The candidate conferred significant protection against Mycobacterium. tuberculosis challenge in naïve guinea pigs and markedly improved protection in the lungs and spleens of animals primed with BCG. We then immunized rhesus macaques with BCG intradermally, and subsequently boosted with one intradermal and one aerosol dose of Spore-FP1, prior to challenge with low dose aerosolized M. tuberculosis Erdman strain. Following vaccination, animals did not show any adverse reactions and displayed higher antigen specific cellular and antibody immune responses compared to BCG alone but this did not translate into significant improvement in disease pathology or bacterial burden in the organs.

Aspirin and the metabolic hallmark of cancer: novel therapeutic opportunities for colorectal cancer.

Aspirin is a well-known nonsteroidal anti-inflammatory drug (NSAID) that has a recognized role in cancer prevention as well as evidence to support its use as an adjuvant for cancer treatment. Importantly there has been an increasing number of studies contributing to the mechanistic understanding of aspirins' anti-tumour effects and these studies continue to inform the potential clinical use of aspirin for both the prevention and treatment of cancer. This review focuses on the emerging role of aspirin as a regulator of metabolic reprogramming, an essential "hallmark of cancer" required to support the increased demand for biosynthetic intermediates needed for sustained proliferation. Cancer cells frequently undergo metabolic rewiring driven by oncogenic pathways such as hypoxia-inducible factor (HIF), wingless-related integration site (Wnt), mammalian target of rapamycin (mTOR), and nuclear factor kappa light chain enhancer of activated B cells (NF-κB), which supports the increased proliferative rate as tumours develop and progress. Reviewed here, cellular metabolic reprogramming has been identified as a key mechanism of action of aspirin and include the regulation of key metabolic drivers, the regulation of enzymes involved in glycolysis and glutaminolysis, and altered nutrient utilisation upon aspirin exposure. Importantly, as aspirin treatment exposes metabolic vulnerabilities in tumour cells, there is an opportunity for the use of aspirin in combination with specific metabolic inhibitors in particular, glutaminase (GLS) inhibitors currently in clinical trials such as telaglenastat (CB-839) and IACS-6274 for the treatment of colorectal and potentially other cancers. The increasing evidence that aspirin impacts metabolism in cancer cells suggests that aspirin could provide a simple, relatively safe, and cost-effective way to target this important hallmark of cancer. Excitingly, this review highlights a potential new role for aspirin in improving the efficacy of a new generation of metabolic inhibitors currently undergoing clinical investigation.

In-silico and in-vitro morphometric analysis of intestinal organoids.

Organoids offer a powerful model to study cellular self-organisation, the growth of specific tissue morphologies in-vitro, and to assess potential medical therapies. However, the intrinsic mechanisms of these systems are not entirely understood yet, which can result in variability of organoids due to differences in culture conditions and basement membrane extracts used. Improving the standardisation of organoid cultures is essential for their implementation in clinical protocols. Developing tools to assess and predict the behaviour of these systems may produce a more robust and standardised biological model to perform accurate clinical studies. Here, we developed an algorithm to automate crypt-like structure counting on intestinal organoids in both in-vitro and in-silico images. In addition, we modified an existing two-dimensional agent-based mathematical model of intestinal organoids to better describe the system physiology, and evaluated its ability to replicate budding structures compared to new experimental data we generated. The crypt-counting algorithm proved useful in approximating the average number of budding structures found in our in-vitro intestinal organoid culture images on days 3 and 7 after seeding. Our changes to the in-silico model maintain the potential to produce simulations that replicate the number of budding structures found on days 5 and 7 of in-vitro data. The present study aims to aid in quantifying key morphological structures and provide a method to compare both in-vitro and in-silico experiments. Our results could be extended later to 3D in-silico models.

The association between genetically elevated polyunsaturated fatty acids and risk of cancer.

BACKGROUND: The causal relevance of polyunsaturated fatty acids (PUFAs) for risk of site-specific cancers remains uncertain. METHODS: Using a Mendelian randomization (MR) framework, we assessed the causal relevance of PUFAs for risk of cancer in European and East Asian ancestry individuals. We defined the primary exposure as PUFA desaturase activity, proxied by rs174546 at the FADS locus. Secondary exposures were defined as omega 3 and omega 6 PUFAs that could be proxied by genetic polymorphisms outside the FADS region. Our study used summary genetic data on 10 PUFAs and 67 cancers, corresponding to 562,871 cases and 1,619,465 controls, collected by the Fatty Acids in Cancer Mendelian Randomization Collaboration. We estimated odds ratios (ORs) for cancer per standard deviation increase in genetically proxied PUFA exposures. FINDINGS: Genetically elevated PUFA desaturase activity was associated (P < 0.0007) with higher risk (OR [95% confidence interval]) of colorectal cancer (1.09 [1.07-1.11]), esophageal squamous cell carcinoma (1.16 [1.06-1.26]), lung cancer (1.06 [1.03-1.08]) and basal cell carcinoma (1.05 [1.02-1.07]). There was little evidence for associations with reproductive cancers (OR = 1.00 [95% CI: 0.99-1.01]; Pheterogeneity = 0.25), urinary system cancers (1.03 [0.99-1.06], Pheterogeneity = 0.51), nervous system cancers (0.99 [0.95-1.03], Pheterogeneity = 0.92) or blood cancers (1.01 [0.98-1.04], Pheterogeneity = 0.09). Findings for colorectal cancer and esophageal squamous cell carcinoma remained compatible with causality in sensitivity analyses for violations of assumptions. Secondary MR analyses highlighted higher omega 6 PUFAs (arachidonic acid, gamma-linolenic acid and dihomo-gamma-linolenic acid) as potential mediators. PUFA biosynthesis is known to interact with aspirin, which increases risk of bleeding and inflammatory bowel disease. In a phenome-wide MR study of non-neoplastic diseases, we found that genetic lowering of PUFA desaturase activity, mimicking a hypothetical intervention to reduce cancer risk, was associated (P < 0.0006) with increased risk of inflammatory bowel disease but not bleeding. INTERPRETATION: The PUFA biosynthesis pathway may be an intervention target for prevention of colorectal cancer and esophageal squamous cell carcinoma but with potential for increased risk of inflammatory bowel disease. FUNDING: Cancer Resesrch UK (C52724/A20138, C18281/A19169). UK Medical Research Council (MR/P014054/1). National Institute for Health Research (NIHR202411). UK Medical Research Council (MC_UU_00011/1, MC_UU_00011/3, MC_UU_00011/6, and MC_UU_00011/4). National Cancer Institute (R00 CA215360). National Institutes of Health (U01 CA164973, R01 CA60987, R01 CA72520, U01 CA74806, R01 CA55874, U01 CA164973 and U01 CA164973).

Determination of the Pharmacokinetics and Pharmacodynamics of Isoniazid, Rifampicin, Pyrazinamide and Ethambutol in a Cross-Over Cynomolgus Macaque Model of Mycobacterium tuberculosis Infection.

Innovative cross-over study designs were explored in non-human primate (NHP) studies to determine the value of this approach for the evaluation of drug efficacy against tuberculosis (TB). Firstly, the pharmacokinetics (PK) of each of the drugs Isoniazid (H), Rifampicin (R), Pyrazinamide (Z) and Ethambutol (E), that are standardly used for the treatment of tuberculosis, was established in the blood of macaques after oral dosing as a monotherapy or in combination. Two studies were conducted to evaluate the pharmacokinetics and pharmacodynamics of different drug combinations using cross-over designs. The first employed a balanced, three-period Pigeon design with an extra period; this ensured that treatment by period interactions and carry-over could be detected comparing the treatments HR, HZ and HRZ using H37Rv as the challenge strain of Mycobacterium tuberculosis (M. tb). Although the design accounted for considerable variability between animals, the three regimens evaluated could not be distinguished using any of the alternative endpoints assessed. However, the degree of pathology achieved using H37Rv in the model during this study was less than expected. Based on these findings, a second experiment using a classical AB/BA design comparing HE with HRZ was conducted using the M. tb Erdman strain. More extensive pathology was observed, and differences in computerized tomography (CT) scores and bacteriology counts in the lungs were detected, although due to the small group sizes, clearer differences were not distinguished. Type 1 T helper (Th1) cell response profiles were characterized using the IFN-γ ELISPOT assay and revealed differences between drug treatments that corresponded to decreases in disease burden. Therefore, the studies performed support the utility of the NHP model for the determination of PK/PD of TB drugs, although further work is required to optimize the use of cross-over study designs.

A rapid high throughput bioprinted colorectal cancer spheroid platform forin vitrodrug- and radiation-response.

We describe the development of a high-throughput bioprinted colorectal cancer (CRC) spheroid platform with high levels of automation, information content, and low cell number requirement. This is achieved via the formulation of a hydrogel bioink with a compressive Young's modulus that is commensurate with that of colonic tissue (1-3 kPa), which supports exponential growth of spheroids from a wide range of CRC cell lines. The resulting spheroids display tight cell-cell junctions, bioink matrix-cell interactions and necrotic hypoxic cores. By combining high content light microscopy imaging and processing with rapid multiwell plate bioprinting, dose-response profiles are generated from CRC spheroids challenged with oxaliplatin (OX) and fluorouracil (5FU), as well as radiotherapy. Bioprinted CRC spheroids are shown to exhibit high levels of chemoresistance relative to cell monolayers, and OX was found to be significantly less effective against tumour spheroids than in monolayer culture, when compared to 5FU.

New light on the use of Theobroma cacao by Late Classic Maya.

Cacao seeds, Theobroma cacao, provide the basis for a ceremonially important Mesoamerican food. Past efforts to identify cacao in ceramics focused on highly decorative vessel forms associated with elite ceremonial contexts, creating assumptions as to how cacao was distributed and who could access it. This study examines 54 archaeological ceramic sherds from El Pilar (Belize/Guatemala) of Late Classic (600 to 900 CE) residential and civic contexts representing a cross-section of ancient Maya inhabitants. Identification of cacao in ancient sherds has depended on the general presence of theobromine; we used the discrete presence of theophylline, a unique key biomarker for cacao in the region. Analysis was done by grinding off all outside surfaces to reduce contamination, pulverizing the inner clay matrix, extracting absorbed molecules, and concentrating the extractions. In order to obtain especially high selectivity and low limits of detection, our study utilized the technique of resonance-enhanced multiphoton ionization coupled with laser-desorption jet-cooling mass spectrometry. This technique isolates molecules in the cold gas phase where they can be selectively ionized through a resonant two-photon process. Of the sherds analyzed, 30 samples (56%) were found to contain significant amounts of theophylline and thus test positive for cacao. Importantly, cacao is present in all contexts, common to all Maya residents near and far from centers.

BCL-3 loss sensitises colorectal cancer cells to DNA damage by targeting homologous recombination.

The proto-oncogene BCL-3 is upregulated in a subset of colorectal cancers (CRC), where it has been shown to enhance tumour cell survival. However, although increased expression correlates with poor patient prognosis, the role of BCL-3 in determining therapeutic response remains largely unknown. In this study, we use combined approaches in multiple cell lines and pre-clinical mouse models to investigate the function of BCL-3 in the DNA damage response. We show that suppression of BCL-3 increases γH2AX foci formation and decreases homologous recombination in CRC cells, resulting in reduced RAD51 foci number and increased sensitivity to PARP inhibition. Importantly, a similar phenotype is seen in Bcl3-/- mice, where Bcl3-/- mouse crypts also exhibit sensitivity to DNA damage with increased γH2AX foci compared to wild type mice. Additionally, Apc.Kras-mutant x Bcl3-/- mice are more sensitive to cisplatin chemotherapy compared to wild type mice. Taken together, our results identify BCL-3 as a regulator of the cellular response to DNA damage and suggests that elevated BCL-3 expression, as observed in CRC, could increase resistance of tumour cells to DNA damaging agents including radiotherapy. These findings offer a rationale for targeting BCL-3 in CRC as an adjunct to conventional therapies and suggest that BCL-3 expression in tumours could be a useful biomarker in stratification of rectal cancer patients for neo-adjuvant chemoradiotherapy.

Culture-Free Enumeration of Mycobacterium tuberculosis in Mouse Tissues Using the Molecular Bacterial Load Assay for Preclinical Drug Development.

BACKGROUND: The turnaround times for phenotypic tests used to monitor the bacterial load of Mycobacterium tuberculosis, in both clinical and preclinical studies, are delayed by the organism's slow growth in culture media. The existence of differentially culturable populations of M.tuberculosis may result in an underestimate of the true number. Moreover, culture methods are susceptible to contamination resulting in loss of critical data points. OBJECTIVES: We report the adaptation of our robust, culture-free assay utilising 16S ribosomal RNA, developed for sputum, to enumerate the number of bacteria present in animal tissues as a tool to improve the read-outs in preclinical drug efficacy studies. METHODS: Initial assay adaptation was performed using naïve mouse lungs spiked with known quantities of M. tuberculosis and an internal RNA control. Tissues were homogenised, total RNA extracted, and enumeration performed using RT-qPCR. We then evaluated the utility of the assay, in comparison to bacterial counts estimated using growth assays on solid and liquid media, to accurately inform bacterial load in tissues from M. tuberculosis-infected mice before and during treatment with a panel of drug combinations. RESULTS: When tested on lung tissues derived from infected mice, the MBL assay produced comparable results to the bacterial counts in solid culture (colony forming units: CFU). Notably, under specific drug treatments, the MBL assay was able to detect a significantly higher number of M. tuberculosis compared to CFU, likely indicating the presence of bacteria that were unable to produce colonies in solid-based culture. Additionally, growth recovery in liquid media using the most probable number (MPN) assay was able to account for the discrepancy between the MBL assay and CFU number, suggesting that the MBL assay detects differentially culturable sub-populations of M. tuberculosis. CONCLUSIONS: The MBL assay can enumerate the bacterial load in animal tissues in real time without the need to wait for extended periods for cultures to grow. The readout correlates well with CFUs. Importantly, we have shown that the MBL is able to measure specific populations of bacteria not cultured on solid agar. The adaptation of this assay for preclinical studies has the potential to decrease the readout time of data acquisition from animal experiments and could represent a valuable tool for tuberculosis drug discovery and development.

Symptom burden, validity, and cognitive performance in Iraq and Afghanistan veterans.

INTRODUCTION: The present study evaluates the complex relationships between symptom burden, validity, and cognition in a sample of Iraq and Afghanistan veterans to identify key characteristic symptoms and validity measures driving cognitive performance. We hypothesized that symptom and performance validity would account for poorer outcomes on cognitive performance beyond psychological symptoms. METHODS: Veterans (n = 226) completed a cognitive test battery, Personality Assessment Inventory (PAI), Word Memory Test (WMT), and Miller Forensic Assessment Symptom Test (M-FAST). Partial least squares structural equation modeling (PLS-SEM) modeled the fully-adjusted relationships among PAI subscales, validity, and cognitive performance. RESULTS: 23.45% of participants failed validity indices (19.9% WMT; 7.1% M-FAST). PLS-SEM indicated PAI subscales were not directly associated with performance or symptom validity measures, and there were no direct effects between validity performance and cognitive performance. Several PAI subscales were directly associated with measures of verbal abstraction, visual processing, and verbal learning and memory. CONCLUSION: Contrary to hypotheses, symptom and performance validity did not account for poorer outcomes on cognitive performance beyond symptom burden in the PLS-SEM model. Results highlight the association between psychiatric symptoms and cognitive performance beyond validity status.

Evaluating the Efficacy of a Screening Protocol for Severe Acute Respiratory Syndrome Coronavirus 2 Virus in Asymptomatic Preoperative/Preprocedural Patients at a Military Hospital.

INTRODUCTION: Facing the COVID-19 pandemic, many hospitals implemented severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) screening protocols before aerosol-generating procedures (AGPs) in an effort to protect patients and health care workers. Given the limited prior evidence on the effectiveness of such protocols, we report the process improvement experience at a military treatment facility. MATERIALS AND METHODS: We evaluated the outcomes of patients undergoing AGPs from March to September 2020, divided into three cohorts: a preprotocol (PP) cohort who did not receive screening, an early testing (ET) cohort representing the early months of the screening protocol, and a late testing (LT) cohort managed under adaptive modifications to the screening protocol. We recorded identifiable post-procedure COVID-19 diagnoses. The study was approved as a process improvement protocol and was determined not to meet criteria for human subject research through an institutional approval process. RESULTS: Across the three cohorts, 4520 procedures were performed: 422 PP, 1297 ET, and 2801 LT. Among 4098 procedures in the ET and LT cohorts, 12 asymptomatic patients tested positive for SARS-CoV-2 (0.29% positivity rate). One left the health system before completing the procedure and another proceeded urgently under COVID precautions, while 10 were rescheduled and completed at a later date; 7 were cleared using a test-based strategy, while 3 were cleared using a time-based strategy. Of 445 patients who had SARS-CoV-2 tests performed within 30 days following their procedures, three patients with negative preoperative tests had a positive test within 30 days, all in the LT cohort but had evidence of acquiring the infection after the procedure or had a false-positive test. CONCLUSIONS: Our strategy of preprocedural SARS-CoV-2 testing successfully identified asymptomatic infected patients before surgery. Care was delayed for most of these patients without apparent detriment. Adaptation to a time-based strategy for clearance might reduce such delays, but other considerations may still influence how soon procedures should be completed after a positive test.

Evaluation of a Three-Dimensional Primary Human Hepatocyte Spheroid Model: Adoption and Industrialization for the Enhanced Detection of Drug-Induced Liver Injury.

Drug-induced liver injury is a leading cause of compound attrition during both preclinical and clinical drug development, and early strategies are in place to tackle this recurring problem. Human-relevant in vitro models that are more predictive of hepatotoxicity hazard identification, and that could be employed earlier in the drug discovery process, would improve the quality of drug candidate selection and help reduce attrition. We present an evaluation of four human hepatocyte in vitro models of increasing culture complexity (i.e., two-dimensional (2D) HepG2 monolayers, hepatocyte sandwich cultures, three-dimensional (3D) hepatocyte spheroids, and precision-cut liver slices), using the same tool compounds, viability end points, and culture time points. Having established the improved prediction potential of the 3D hepatocyte spheroid model, we describe implementing this model into an industrial screening setting, where the challenge was matching the complexity of the culture system with the scale and throughput required. Following further qualification and miniaturization into a 384-well, high-throughput screening format, data was generated on 199 compounds. This clearly demonstrated the ability to capture a greater number of severe hepatotoxins versus the current routine 2D HepG2 monolayer assay while continuing to flag no false-positive compounds. The industrialization and miniaturization of the 3D hepatocyte spheroid complex in vitro model demonstrates a significant step toward reducing drug attrition and improving the quality and safety of drugs, while retaining the flexibility for future improvements, and has replaced the routine use of the 2D HepG2 monolayer assay at GlaxoSmithKline.

Hospital ethical climate associated with the professional quality of life among nurses during the early stage of COVID-19 pandemic in Wuhan, China: A cross-sectional study.

OBJECTIVES: To describe the professional quality of life and explore its associated factors among nurses coming from other areas of China to assist with the anti-epidemic fight in Wuhan and especially examine whether the hospital ethical climate was independently associated with nurses' professional quality of life. METHODS: A cross-sectional online survey was conducted from March 2020 to April 2020. The nurses working in Wuhan from the other parts of China were the target population. The Professional Quality of Life Scale version 5, the Hospital Ethical Climate Survey, and a basic information sheet were used to collect data. Descriptive statistics, t-test, ANOVA, Pearson correlation, and multiple linear regression analysis were used to analyze the data. RESULTS: In total, 236 nurses participated in this study, and 219 valid questionnaires were analyzed. The average age of the participants was 31.2 ± 5.0 years. Most nurses were female (176/219; 80.4%) and married (145/219; 66.2%). In term of professional quality of life, nurses reported moderate (129/219; 58.9%) to high (90/219; 41.1%) levels of compassion satisfaction, low (119/219; 54.3%) to moderate (100/219; 45.7%) levels of burnout, and low (67/219; 36.0%) to high (10/219; 4.6%) levels of secondary traumatic stress. Regarding hospital ethical climate, nurses reported moderately high hospital ethical climates with an average score of 4.46. After controlling for socio-demographic characteristics, the multiple linear regression models showed that the hospital ethical climate subscale of "relationship with physicians" was independently associated with the compassion satisfaction (ß = 0.533, P < 0.01) and burnout (ß = -0.237, P < 0.05); the hospital ethical climate subscale of "relationship with peers" (ß = -0.191, P < 0.01) was independently associated with the secondary traumatic stress. CONCLUSIONS: During the early stage of the pandemic, nurses demonstrated moderate to high level of compassion satisfaction, low to moderate level of burnout, and all nurses experienced secondary traumatic stress. Nurses perceived a high level of hospital ethical climate, and the perceived hospital ethical climate played an important role in promoting nurses' professional quality of life during a life-threatening infectious disease pandemic.

High-dose Mycobacterium tuberculosis aerosol challenge cannot overcome BCG-induced protection in Chinese origin cynomolgus macaques; implications of natural resistance for vaccine evaluation.

This study describes the use of cynomolgus macaques of Chinese origin (CCM) to evaluate the efficacy and immunogenicity of the BCG vaccine against high dose aerosol Mycobacterium tuberculosis challenge. Progressive disease developed in three of the unvaccinated animals within 10 weeks of challenge, whereas all six vaccinated animals controlled disease for 26 weeks. Three unvaccinated animals limited disease progression, highlighting the intrinsic ability of this macaque species to control disease in comparison to macaques of other species and genotypes. Low levels of IFNγ were induced by BCG vaccination in CCM suggesting that IFNγ alone does not provide a sufficiently sensitive biomarker of vaccination in this model. An early response after challenge, together with the natural bias towards terminal effector memory T-cell populations and the contribution of monocytes appears to enhance the ability of CCM to naturally control infection. The high dose aerosol challenge model of CCM has value for examination of the host immune system to characterise control of infection which would influence future vaccine design. Although it may not be the preferred platform for the assessment of prophylactic vaccine candidates, the model could be well suited for testing post-exposure vaccination strategies and drug evaluation studies.

NOTUM from Apc-mutant cells biases clonal competition to initiate cancer.

The tumour suppressor APC is the most commonly mutated gene in colorectal cancer. Loss of Apc in intestinal stem cells drives the formation of adenomas in mice via increased WNT signalling1, but reduced secretion of WNT ligands increases the ability of Apc-mutant intestinal stem cells to colonize a crypt (known as fixation)2. Here we investigated how Apc-mutant cells gain a clonal advantage over wild-type counterparts to achieve fixation. We found that Apc-mutant cells are enriched for transcripts that encode several secreted WNT antagonists, with Notum being the most highly expressed. Conditioned medium from Apc-mutant cells suppressed the growth of wild-type organoids in a NOTUM-dependent manner. Furthermore, NOTUM-secreting Apc-mutant clones actively inhibited the proliferation of surrounding wild-type crypt cells and drove their differentiation, thereby outcompeting crypt cells from the niche. Genetic or pharmacological inhibition of NOTUM abrogated the ability of Apc-mutant cells to expand and form intestinal adenomas. We identify NOTUM as a key mediator during the early stages of mutation fixation that can be targeted to restore wild-type cell competitiveness and provide preventative strategies for people at a high risk of developing colorectal cancer.

5-Aminosalicylic acid inhibits stem cell function in human adenoma-derived cells: implications for chemoprophylaxis in colorectal tumorigenesis.

BACKGROUND: Most colorectal cancers (CRC) arise sporadically from precursor lesions: colonic polyps. Polyp resection prevents progression to CRC. Risk of future polyps is proportional to the number and size of polyps detected at screening, allowing identification of high-risk individuals who may benefit from effective chemoprophylaxis. We aimed to investigate the potential of 5-aminosalicylic acid (5-ASA), a medication used in the treatment of ulcerative colitis, as a possible preventative agent for sporadic CRC. METHODS: Human colorectal adenoma (PC/AA/C1, S/AN/C1 and S/RG/C2), transformed adenoma PC/AA/C1/SB10 and carcinoma cell lines (LS174T and SW620) were treated with 5-ASA. The effect on growth in two- and three-dimensional (3D) culture, ß-catenin transcriptional activity and on cancer stemness properties of the cells were investigated. RESULTS: 5-ASA was shown, in vitro, to inhibit the growth of adenoma cells and suppress ß-catenin transcriptional activity. Downregulation of ß-catenin was found to repress expression of stem cell marker LGR5 (leucine-rich G protein-coupled receptor-5) and functionally suppress stemness in human adenoma and carcinoma cells using 3D models of tumorigenesis. CONCLUSIONS: 5-ASA can suppress the cancer stem phenotype in adenoma-derived cells. Affordable and well-tolerated, 5-ASA is an outstanding candidate as a chemoprophylactic medication to reduce the risk of colorectal polyps and CRC in those at high risk.

Pathogenomic analyses of Mycobacterium microti, an ESX-1-deleted member of the Mycobacterium tuberculosis complex causing disease in various hosts.

Mycobacterium microti is an animal-adapted member of the Mycobacterium tuberculosis complex (MTBC), which was originally isolated from voles, but has more recently also been isolated from other selected mammalian hosts, including occasionally from humans. Here, we have generated and analysed the complete genome sequences of five representative vole and clinical M. microti isolates using PacBio- and Illumina-based technologies, and have tested their virulence and vaccine potential in SCID (severe combined immune deficient) mouse and/or guinea pig infection models. We show that the clinical isolates studied here cluster separately in the phylogenetic tree from vole isolates and other clades from publicly available M. microti genome sequences. These data also confirm that the vole and clinical M. microti isolates were all lacking the specific RD1mic region, which in other tubercle bacilli encodes the ESX-1 type VII secretion system. Biochemical analysis further revealed marked phenotypic differences between isolates in type VII-mediated secretion of selected PE and PPE proteins, which in part were attributed to specific genetic polymorphisms. Infection experiments in the highly susceptible SCID mouse model showed that the clinical isolates were significantly more virulent than the tested vole isolates, but still much less virulent than the M. tuberculosis H37Rv control strain. The strong attenuation of the ATCC 35872 vole isolate in immunocompromised mice, even compared to the attenuated BCG (bacillus Calmette-Guérin) vaccine, and its historic use in human vaccine trials encouraged us to test this strain's vaccine potential in a guinea pig model, where it demonstrated similar protective efficacy as a BCG control, making it a strong candidate for vaccination of immunocompromised individuals in whom BCG vaccination is contra-indicated. Overall, we provide new insights into the genomic and phenotypic variabilities and particularities of members of an understudied clade of the MTBC, which all share a recent common ancestor that is characterized by the deletion of the RD1mic region.

MTBVAC vaccination protects rhesus macaques against aerosol challenge with M. tuberculosis and induces immune signatures analogous to those observed in clinical studies.

A single intradermal vaccination with MTBVAC given to adult rhesus macaques was well tolerated and conferred a significant improvement in outcome following aerosol exposure to M. tuberculosis compared to that provided by a single BCG vaccination. Vaccination with MTBVAC resulted in a significant reduction in M. tuberculosis infection-induced disease pathology measured using in vivo medical imaging, in gross pathology lesion counts and pathology scores recorded at necropsy, the frequency and severity of pulmonary granulomas and the frequency of recovery of viable M. tuberculosis from extrapulmonary tissues following challenge. The immune profiles induced following immunisation with MTBVAC reflect those identified in human clinical trials of MTBVAC. Evaluation of MTBVAC- and TB peptide-pool-specific T-cell cytokine production revealed a predominantly Th1 response from poly- (IFN-γ+TNF-α+IL2+) and multi-(IFN-γ+TNF-α+) functional CD4 T cells, while only low levels of Th22, Th17 and cytokine-producing CD8 T-cell populations were detected together with low-level, but significant, increases in CFP10-specific IFN-γ secreting cells. In this report, we describe concordance between immune profiles measured in clinical trials and a macaque pre-clinical study demonstrating significantly improved outcome after M. tuberculosis challenge as evidence to support the continued development of MTBVAC as an effective prophylactic vaccine for TB vaccination campaigns.