Directed evolution of human dihydrofolate reductase: towards a better understanding of binding at the active site

La dihydrofolate réductase humaine (DHFRh) est une enzyme essentielle à la prolifération cellulaire, ce qui en fait une cible de choix pour le traitement de différents cancers. À cet effet, plusieurs inhibiteurs spécifiques de la DHFRh, les antifolates, ont été mis au point : le méthotrexate (MTX) et le pemetrexed (PMTX) en sont de bons exemples. Malgré l’efficacité clinique certaine de ces antifolates, le développement de nouveaux traitements s’avère nécessaire afin de réduire les effets secondaires liés à leur utilisation. Enfin, dans l’optique d’orienter la synthèse de nouveaux composés inhibiteurs des DHFRh, une meilleure connaissance des interactions entre les antifolates et leur enzyme cible est primordiale. À l’aide de l’évolution dirigée, il a été possible d’identifier des mutants de la DHFRh pour lesquels l’affinité envers des antifolates cliniquement actifs se voyait modifiée. La mutagenèse dite ¬¬de saturation a été utilisée afin de générer des banques de mutants présentant une diversité génétique au niveau des résidus du site actif de l’enzyme d’intérêt. De plus, une nouvelle méthode de criblage a été mise au point, laquelle s’est avérée efficace pour départager les mutations ayant entrainé une résistance aux antifolates et/ou un maintient de l’activité enzymatique envers son substrat natif, soient les phénotypes d’activité. La méthode de criblage consiste dans un premier temps en une sélection bactérienne à haut débit, puis dans un second temps en un criblage sur plaques permettant d’identifier les meilleurs candidats. Plusieurs mutants actifs de la DHFRh, résistants aux antifolates, ont ainsi pu être identifiés et caractérisés lors d’études de cinétique enzymatique (kcat et IC50). Sur la base de ces résultats cinétiques, de la modélisation moléculaire et des données structurales de la littérature, une étude structure-activité a été effectuée. En regardant quelles mutations ont les effets les plus significatif sur la liaison, nous avons commencé à construire un carte moléculaire des contacts impliqués dans la liaison des ligands. Enfin, des connaissances supplémentaires sur les propriétés spécifiques de liaison ont put être acquises en variant l’inhibiteur testé, permettant ainsi une meilleure compréhension du phénomène de discrimination du ligand.Human dihydrofolate reductase (hDHFR) is an essential enzyme for cellular proliferation and it has long been the target of antifolate drugs for the treatment of various types of cancer. Despite the clinical effectiveness of current antifolate treatments, new drugs are required to reduce the side-effects associated with their use. An essential requirement for design of new antifolates is a better understanding of how these drugs interact with their targets. We applied directed evolution to identify mutant hDHFR variants with modified binding to some clinically relevant antifolates. A saturation mutagenesis approach was used to create genetic diversity at active-site residues of hDHFR and a new, efficient screening strategy was developed to identify the amino acids that preserved native activity and/or conferred antifolate resistance. The screening method consists in a high-throughput first-tier bacterial selection coupled with a second-tier in vitro assay that allows for rapid detection of the best variants among the leads, according to user-defined parameters. Many active, antifolate-resistant mutants of hDHFR were identified. Moreover, the approach has proven efficient in rapidly assessing kinetic (kcat) and inhibition parameters of the hDHFR variants (IC50). Structure-function relationship analysis based on kinetic investigation, available structural and functional data as well as modeling were performed. By monitoring which mutations have the greatest effect on binding, we have begun to build a molecular picture of the contacts involved in drug binding. By varying the drugs we test against, we gain a better understanding of the specific binding properties that determine ligand discrimination

Kinetic and continuum modeling of high-temperature oxygen and nitrogen binary mixtures

The present paper provides a comprehensive comparative analysis of thermochemistry models of various fidelity levels developed in leading research groups around the world. Fully kinetic, hybrid kinetic-continuum, and fully continuum approaches are applied to analyze parameters of hypersonic flows starting from the revision of single-temperature rate constants up to the application in 1-D postshock conditions. Comparison of state-specific and two-temperature approaches shows there are very significant and often qualitative differences in the time-dependent nonequilibrium reaction rates and their ratio to the corresponding single-temperature rates. A major impact of the vibration-dissociation coupling on the temporal relaxation of gas properties is shown. For instance, the legacy Park's model has a strongly nonlinear behavior of nonequilibrium reaction rate with vibrational temperature, while a nearly linear shape exists for all state-specific approaches. Analysis of vibrational level populations in the nonequilibrium region shows a profound impact of the numerical approach and the model on the population ratios, and thus vibrational temperatures inferred from such ratios. The difference in the ultraviolet absorption coefficients, calculated by a temperature-based spectral code using vibrational populations from state-specific and kinetic approaches, is found to exceed an order of magnitude

Kinetic and continuum modeling of high-temperature air relaxation

Fully kinetic, vibrationally kinetic, and continuum solvers with varying model fidelity are used in this work to model the high-temperature relaxation of air in 7230 and 15,000 K adiabatic heat baths and a 6 km/s hypersonic flow over a cylinder. The results show significant impact of uncertainties in vibrational relaxation times and reaction rate constants on thermal and chemical relaxation, in particular, on gas temperature and species mole fractions. Most notably, these uncertainties need to be reduced for collisions that include nitric oxide. Order-of-magnitude differences in the nitric oxide dissociation and recombination rates have a large impact on the peak NO mole fraction immediately behind the shock and surface-distributed heat flux, respectively. High-fidelity kinetic and continuum approaches are found to have different reaction channels having the largest effect on species mole fractions and gas temperature: N-2+O exchange and O-2+O dissociation in the former, and NO+O and O-2+N-2 dissociation in the latter

Hemophagocytic inflammatory syndrome in ADA-SCID: report of two cases and literature review

Hemophagocytic inflammatory syndrome (HIS) is a rare form of secondary hemophagocytic lymphohistiocytosis caused by an impaired equilibrium between natural killer and cytotoxic T-cell activity, evolving in hypercytokinemia and multiorgan failure. In the context of inborn errors of immunity, HIS occurrence has been reported in severe combined immunodeficiency (SCID) patients, including two cases of adenosine deaminase deficient-SCID (ADA-SCID). Here we describe two additional pediatric cases of ADA-SCID patients who developed HIS. In the first case, HIS was triggered by infectious complications while the patient was on enzyme replacement therapy; the patient was treated with high-dose corticosteroids and intravenous immunoglobulins with HIS remission. However, the patient required HLA-identical sibling donor hematopoietic stem cell transplantation (HSCT) for a definitive cure of ADA-SCID, without HIS relapse up to 13 years after HSCT. The second patient presented HIS 2 years after hematopoietic stem cell gene therapy (GT), secondarily to Varicella-Zoster vaccination and despite CD4+ and CD8+ lymphocytes’ reconstitution in line with other ADA SCID patients treated with GT. The child responded to trilinear immunosuppressive therapy (corticosteroids, Cyclosporine A, Anakinra). We observed the persistence of gene-corrected cells up to 5 years post-GT, without HIS relapse. These new cases of children with HIS, together with those reported in the literature, support the hypothesis that a major dysregulation in the immune system can occur in ADA-SCID patients. Our cases show that early identification of the disease is imperative and that a variable degree of immunosuppression could be an effective treatment while allogeneic HSCT is required only in cases of refractoriness. A deeper knowledge of immunologic patterns contributing to HIS pathogenesis in ADA-SCID patients is desirable, to identify new targeted treatments and ensure patients’ long-term recovery

Management of patients with advanced prostate cancer—metastatic and/or castration-resistant prostate cancer: report of the Advanced Prostate Cancer Consensus Conference (APCCC) 2022

Background: Innovations in imaging and molecular characterisation together with novel treatment options have improved outcomes in advanced prostate cancer. However, we still lack high-level evidence in many areas relevant to making management decisions in daily clinical practise. The 2022 Advanced Prostate Cancer Consensus Conference (APCCC 2022) addressed some questions in these areas to supplement guidelines that mostly are based on level 1 evidence. Objective: To present the voting results of the APCCC 2022. Design, setting, and participants: The experts voted on controversial questions where high- level evidence is mostly lacking: locally advanced prostate cancer; biochemical recurrence after local treatment; metastatic hormone-sensitive, non-metastatic, and metastatic castration- resistant prostate cancer; oligometastatic prostate cancer; and managing side effects of hormonal therapy. A panel of 105 international prostate cancer experts voted on the consensus questions. Outcome measurements and statistical analysis: The panel voted on 198 pre-defined questions, which were developed by 117 voting and non-voting panel members prior to the conference following a modified Delphi process. A total of 116 questions on metastatic and/or castration- resistant prostate cancer are discussed in this manuscript. In 2022, the voting was done by a web-based survey because of COVID-19 restrictions. Results and limitations: The voting reflects the expert opinion of these panellists and did not incorporate a standard literature review or formal meta-analysis. The answer options for the consensus questions received varying degrees of support from panellists, as reflected in this article and the detailed voting results are reported in the supplementary material. We report here on topics in metastatic, hormone-sensitive prostate cancer (mHSPC), non-metastatic, castration-resistant prostate cancer (nmCRPC), metastatic castration-resistant prostate cancer (mCRPC), and oligometastatic and oligoprogressive prostate cancer. Conclusions: These voting results in four specific areas from a panel of experts in advanced prostate cancer can help clinicians and patients navigate controversial areas of management for which high-level evidence is scant or conflicting and can help research funders and policy makers identify information gaps and consider what areas to explore further. However, diagnostic and treatment decisions always have to be individualised based on patient characteristics, including the extent and location of disease, prior treatment(s), co-morbidities, patient preferences, and treatment recommendations and should also incorporate current and emerging clinical evidence and logistic and economic factors. Enrolment in clinical trials is strongly encouraged. Importantly, APCCC 2022 once again identified important gaps where there is non-consensus and that merit evaluation in specifically designed trials. Patient summary: The Advanced Prostate Cancer Consensus Conference (APCCC) provides a forum to discuss and debate current diagnostic and treatment options for patients with advanced prostate cancer. The conference aims to share the knowledge of international experts in prostate cancer with healthcare providers worldwide. At each APCCC, an expert panel votes on pre-defined questions that target the most clinically relevant areas of advanced prostate cancer treatment for which there are gaps in knowledge. The results of the voting provide a practical guide to help clinicians discuss therapeutic options with patients and their relatives as part of shared and multidisciplinary decision-making. This report focuses on the advanced setting, covering metastatic hormone-sensitive prostate cancer and both non-metastatic and metastatic castration-resistant prostate cancer. Twitter summary: Report of the results of APCCC 2022 for the following topics: mHSPC, nmCRPC, mCRPC, and oligometastatic prostate cancer. Take-home message: At APCCC 2022, clinically important questions in the management of advanced prostate cancer management were identified and discussed, and experts voted on pre-defined consensus questions. The report of the results for metastatic and/or castration- resistant prostate cancer is summarised here

The wide-field, multiplexed, spectroscopic facility WEAVE : survey design, overview, and simulated implementation

Funding for the WEAVE facility has been provided by UKRI STFC, the University of Oxford, NOVA, NWO, Instituto de Astrofísica de Canarias (IAC), the Isaac Newton Group partners (STFC, NWO, and Spain, led by the IAC), INAF, CNRS-INSU, the Observatoire de Paris, Région Île-de-France, CONCYT through INAOE, Konkoly Observatory (CSFK), Max-Planck-Institut für Astronomie (MPIA Heidelberg), Lund University, the Leibniz Institute for Astrophysics Potsdam (AIP), the Swedish Research Council, the European Commission, and the University of Pennsylvania.WEAVE, the new wide-field, massively multiplexed spectroscopic survey facility for the William Herschel Telescope, will see first light in late 2022. WEAVE comprises a new 2-degree field-of-view prime-focus corrector system, a nearly 1000-multiplex fibre positioner, 20 individually deployable 'mini' integral field units (IFUs), and a single large IFU. These fibre systems feed a dual-beam spectrograph covering the wavelength range 366-959 nm at R ∼ 5000, or two shorter ranges at R ∼ 20,000. After summarising the design and implementation of WEAVE and its data systems, we present the organisation, science drivers and design of a five- to seven-year programme of eight individual surveys to: (i) study our Galaxy's origins by completing Gaia's phase-space information, providing metallicities to its limiting magnitude for ∼ 3 million stars and detailed abundances for ∼ 1.5 million brighter field and open-cluster stars; (ii) survey ∼ 0.4 million Galactic-plane OBA stars, young stellar objects and nearby gas to understand the evolution of young stars and their environments; (iii) perform an extensive spectral survey of white dwarfs; (iv) survey  ∼ 400 neutral-hydrogen-selected galaxies with the IFUs; (v) study properties and kinematics of stellar populations and ionised gas in z 1 million spectra of LOFAR-selected radio sources; (viii) trace structures using intergalactic/circumgalactic gas at z > 2. Finally, we describe the WEAVE Operational Rehearsals using the WEAVE Simulator.PostprintPeer reviewe

Case Reports1. A Late Presentation of Loeys-Dietz Syndrome: Beware of TGFβ Receptor Mutations in Benign Joint Hypermobility

Background: Thoracic aortic aneurysms (TAA) and dissections are not uncommon causes of sudden death in young adults. Loeys-Dietz syndrome (LDS) is a rare, recently described, autosomal dominant, connective tissue disease characterized by aggressive arterial aneurysms, resulting from mutations in the transforming growth factor beta (TGFβ) receptor genes TGFBR1 and TGFBR2. Mean age at death is 26.1 years, most often due to aortic dissection. We report an unusually late presentation of LDS, diagnosed following elective surgery in a female with a long history of joint hypermobility. Methods: A 51-year-old Caucasian lady complained of chest pain and headache following a dural leak from spinal anaesthesia for an elective ankle arthroscopy. CT scan and echocardiography demonstrated a dilated aortic root and significant aortic regurgitation. MRA demonstrated aortic tortuosity, an infrarenal aortic aneurysm and aneurysms in the left renal and right internal mammary arteries. She underwent aortic root repair and aortic valve replacement. She had a background of long-standing joint pains secondary to hypermobility, easy bruising, unusual fracture susceptibility and mild bronchiectasis. She had one healthy child age 32, after which she suffered a uterine prolapse. Examination revealed mild Marfanoid features. Uvula, skin and ophthalmological examination was normal. Results: Fibrillin-1 testing for Marfan syndrome (MFS) was negative. Detection of a c.1270G > C (p.Gly424Arg) TGFBR2 mutation confirmed the diagnosis of LDS. Losartan was started for vascular protection. Conclusions: LDS is a severe inherited vasculopathy that usually presents in childhood. It is characterized by aortic root dilatation and ascending aneurysms. There is a higher risk of aortic dissection compared with MFS. Clinical features overlap with MFS and Ehlers Danlos syndrome Type IV, but differentiating dysmorphogenic features include ocular hypertelorism, bifid uvula and cleft palate. Echocardiography and MRA or CT scanning from head to pelvis is recommended to establish the extent of vascular involvement. Management involves early surgical intervention, including early valve-sparing aortic root replacement, genetic counselling and close monitoring in pregnancy. Despite being caused by loss of function mutations in either TGFβ receptor, paradoxical activation of TGFβ signalling is seen, suggesting that TGFβ antagonism may confer disease modifying effects similar to those observed in MFS. TGFβ antagonism can be achieved with angiotensin antagonists, such as Losartan, which is able to delay aortic aneurysm development in preclinical models and in patients with MFS. Our case emphasizes the importance of timely recognition of vasculopathy syndromes in patients with hypermobility and the need for early surgical intervention. It also highlights their heterogeneity and the potential for late presentation. Disclosures: The authors have declared no conflicts of interes

The LifeCycle Project-EU Child Cohort Network : a federated analysis infrastructure and harmonized data of more than 250,000 children and parents

Early life is an important window of opportunity to improve health across the full lifecycle. An accumulating body of evidence suggests that exposure to adverse stressors during early life leads to developmental adaptations, which subsequently affect disease risk in later life. Also, geographical, socio-economic, and ethnic differences are related to health inequalities from early life onwards. To address these important public health challenges, many European pregnancy and childhood cohorts have been established over the last 30 years. The enormous wealth of data of these cohorts has led to important new biological insights and important impact for health from early life onwards. The impact of these cohorts and their data could be further increased by combining data from different cohorts. Combining data will lead to the possibility of identifying smaller effect estimates, and the opportunity to better identify risk groups and risk factors leading to disease across the lifecycle across countries. Also, it enables research on better causal understanding and modelling of life course health trajectories. The EU Child Cohort Network, established by the Horizon2020-funded LifeCycle Project, brings together nineteen pregnancy and childhood cohorts, together including more than 250,000 children and their parents. A large set of variables has been harmonised and standardized across these cohorts. The harmonized data are kept within each institution and can be accessed by external researchers through a shared federated data analysis platform using the R-based platform DataSHIELD, which takes relevant national and international data regulations into account. The EU Child Cohort Network has an open character. All protocols for data harmonization and setting up the data analysis platform are available online. The EU Child Cohort Network creates great opportunities for researchers to use data from different cohorts, during and beyond the LifeCycle Project duration. It also provides a novel model for collaborative research in large research infrastructures with individual-level data. The LifeCycle Project will translate results from research using the EU Child Cohort Network into recommendations for targeted prevention strategies to improve health trajectories for current and future generations by optimizing their earliest phases of life.Peer reviewe

Management of patients with advanced prostate cancer. Part I: Intermediate-/high-risk and locally advanced disease, biochemical relapse, and side effects of hormonal treatment: Report of the Advanced Prostate Cancer Consensus Conference 2022

© 2023 The Authors. Published by Elsevier on behalf of European Association of Urology. This is an open access article available under a Creative Commons licence. The published version can be accessed at the following link on the publisher’s website: https://doi.org/10.1016/j.eururo.2022.11.002Background: Innovations in imaging and molecular characterisation and the evolution of new therapies have improved outcomes in advanced prostate cancer. Nonetheless, we continue to lack high-level evidence on a variety of clinical topics that greatly impact daily practice. To supplement evidence-based guidelines, the 2022 Advanced Prostate Cancer Consensus Conference (APCCC 2022) surveyed experts about key dilemmas in clinical management. Objective: To present consensus voting results for select questions from APCCC 2022. Design, setting, and participants: Before the conference, a panel of 117 international prostate cancer experts used a modified Delphi process to develop 198 multiple-choice consensus questions on (1) intermediate- and high-risk and locally advanced prostate cancer, (2) biochemical recurrence after local treatment, (3) side effects from hormonal therapies, (4) metastatic hormone-sensitive prostate cancer, (5) nonmetastatic castration-resistant prostate cancer, (6) metastatic castration-resistant prostate cancer, and (7) oligometastatic and oligoprogressive prostate cancer. Before the conference, these questions were administered via a web-based survey to the 105 physician panel members (“panellists”) who directly engage in prostate cancer treatment decision-making. Herein, we present results for the 82 questions on topics 1–3. Outcome measurements and statistical analysis: Consensus was defined as ≥75% agreement, with strong consensus defined as ≥90% agreement. Results and limitations: The voting results reveal varying degrees of consensus, as is discussed in this article and shown in the detailed results in the Supplementary material. The findings reflect the opinions of an international panel of experts and did not incorporate a formal literature review and meta-analysis. Conclusions: These voting results by a panel of international experts in advanced prostate cancer can help physicians and patients navigate controversial areas of clinical management for which high-level evidence is scant or conflicting. The findings can also help funders and policymakers prioritise areas for future research. Diagnostic and treatment decisions should always be individualised based on patient and cancer characteristics (disease extent and location, treatment history, comorbidities, and patient preferences) and should incorporate current and emerging clinical evidence, therapeutic guidelines, and logistic and economic factors. Enrolment in clinical trials is always strongly encouraged. Importantly, APCCC 2022 once again identified important gaps (areas of nonconsensus) that merit evaluation in specifically designed trials. Patient summary: The Advanced Prostate Cancer Consensus Conference (APCCC) provides a forum to discuss and debate current diagnostic and treatment options for patients with advanced prostate cancer. The conference aims to share the knowledge of international experts in prostate cancer with health care providers and patients worldwide. At each APCCC, a panel of physician experts vote in response to multiple-choice questions about their clinical opinions and approaches to managing advanced prostate cancer. This report presents voting results for the subset of questions pertaining to intermediate- and high-risk and locally advanced prostate cancer, biochemical relapse after definitive treatment, advanced (next-generation) imaging, and management of side effects caused by hormonal therapies. The results provide a practical guide to help clinicians and patients discuss treatment options as part of shared multidisciplinary decision-making. The findings may be especially useful when there is little or no high-level evidence to guide treatment decisions.We gratefully acknowledge the following organisations for providing financial support for the APCCC 2022: The City of Lugano and Movember Foundation. Ros Eeles is supported by a National Institute of Health Research grant to the Biomedical Research Centre at The Institute of Cancer Research and Royal Marsden NHS Foundation Trust. We also acknowledge sponsorship from several for-profit organisations for APCCC 2022, including Advanced Accelerator Applications, Amgen, Astellas, AstraZeneca, Bayer Health Care, Debiopharm, MSD, Janssen Oncology, Myovant Sciences, Orion Pharma, Pfizer Oncology, Roche, Telix Innovations SA, Ferring Pharmaceuticals, Lantheus, and Tolmar. These for-profit organisations supported the conference financially but had no input on the scientific content or the final publication.Accepted versio