Fast multiclonal clusterization of V(D)J recombinations from high-throughput sequencing

International audienceBACKGROUND: V(D)J recombinations in lymphocytes are essential for immunological diversity. They are also usefulmarkers of pathologies. In leukemia, they are used to quantify the minimal residual disease duringpatient follow-up. However, the full breadth of lymphocyte diversity is not fully understood. RESULTS: We propose new algorithms that process high-throughput sequencing (HTS) data to extract unnamedV(D)J junctions and gather them into clones for quantification. This analysis is based on a seedheuristic and is fast and scalable because in the first phase, no alignment is performed with germlinedatabase sequences. The algorithms were applied to TR HTS data from a patient with acutelymphoblastic leukemia, and also on data simulating hypermutations. Our methods identified themain clone, as well as additional clones that were not identified with standard protocols. CONCLUSIONS: The proposed algorithms provide new insight into the analysis of high-troughput sequencing data forleukemia, and also to the quantitative assessment of any immunological profile. The methodsdescribed here are implemented in a C++ open-source program called Vidjil

Multi-loci diagnosis of acute lymphoblastic leukaemia with high-throughput sequencing and bioinformatics analysis

International audienceHigh-throughput sequencing (HTS) is considered a technical revolution that has improved our knowledge of lymphoid and autoimmune diseases, changing our approach to leukaemia both at diagnosis and during follow-up. As part of an immunoglobulin/T cell receptor-based minimal residual disease (MRD) assessment of acute lymphoblastic leukaemia patients, we assessed the performance and feasibility of the replacement of the first steps of the approach based on DNA isolation and Sanger sequencing, using a HTS protocol combined with bioinformatics analysis and visualization using the Vidjil software. We prospectively analysed the diagnostic and relapse samples of 34 paediatric patients, thus identifying 125 leukaemic clones with recombinations on multiple loci (TRG, TRD, IGH and IGK), including Dd2/Dd3 and Intron/KDE rearrangements. Sequencing failures were halved (14% vs. 34%, P = 0.0007), enabling more patients to be monitored. Furthermore, more markers per patient could be monitored, reducing the probability of false negative MRD results. The whole analysis, from sample receipt to clinical validation, was shorter than our current diagnostic protocol, with equal resources. V(D)J recombination was successfully assigned by the software, even for unusual recombinations. This study emphasizes the progress that HTS with adapted bioinformatics tools can bring to the diagnosis of leukaemia patients

Site-Directed Mutagenesis of the Streptomyces R61 Dd-Peptidase. Catalytic Function of the Conserved Residues around the Active Site and a Comparison with Class-a and Class-C Beta-Lactamases

The importance of various residues in the Streptomyces R61 penicillin-sensitive DD-peptidase has been assessed by site-directed mutagenesis. The replacement of the active Ser62 by a Cys residue yielded an inactive protein which was also unable to recognize penicillin. The activity of the Lys65 → Arg mutant with the peptide and thiol ester substrates was decreased 100-200-fold and the rate of penicillin inactivation was decreased 20 000-fold or more. The mutant thus behaved as a poor, but penicillin-resistant, DD-peptidase. The other studied mutations, the mutations Phe358 → Leu, Tyr90 → Asn, Thr101 → Asn, Phe164 → Ala, Asp225 → Glu and Asp225 → Asn had little influence on the catalytic and penicillin-binding properties. The Asp225 mutants did not exhibit an increased sensitivity to cefotaxime. The Phe164 → Ala mutant was significantly more unstable than the wild-type enzyme

Fast multiclonal clusterization of V(D)J recombinations from high-throughput sequencing

International audienceBACKGROUND: V(D)J recombinations in lymphocytes are essential for immunological diversity. They are also usefulmarkers of pathologies. In leukemia, they are used to quantify the minimal residual disease duringpatient follow-up. However, the full breadth of lymphocyte diversity is not fully understood. RESULTS: We propose new algorithms that process high-throughput sequencing (HTS) data to extract unnamedV(D)J junctions and gather them into clones for quantification. This analysis is based on a seedheuristic and is fast and scalable because in the first phase, no alignment is performed with germlinedatabase sequences. The algorithms were applied to TR HTS data from a patient with acutelymphoblastic leukemia, and also on data simulating hypermutations. Our methods identified themain clone, as well as additional clones that were not identified with standard protocols. CONCLUSIONS: The proposed algorithms provide new insight into the analysis of high-troughput sequencing data forleukemia, and also to the quantitative assessment of any immunological profile. The methodsdescribed here are implemented in a C++ open-source program called Vidjil

Impact of collaborative physician-pharmacist stewardship strategies on prophylactic antibiotic practices: a quasi-experimental study.

peer reviewedBACKGROUND: An effective use of surgical antibiotic prophylaxis (SAP) appears essential to prevent the development of infections linked to surgery while inappropriate and excessive prescriptions of prophylactic antibiotics increase the risk of adverse effects, bacterial resistance and Clostridium difficile infections. In this study, we aimed to analyze SAP practices in an acute secondary hospital in Belgium during the years 2016-2021 in order to evaluate the impacts of combined stewardship interventions, implemented thanks to a physician-pharmacist collaboration. METHODS: A quasi-experimental study on SAP practices was conducted during 5 years (2016-2021) in a Belgian University Hospital. We first performed a retrospective observational transversal study on a baseline group (2016.1-2016.4). Then, we constituted a group of patients (2017.1-2017.4) to test a combined intervention strategy of stewardship which integrated the central role of a pharmacist in antibiotic stewardship team and in the pre-operative delivery of nominative kits of antibiotics adapted to patient factors. After this test, we collected patient data (2018.1-2018.4) to evaluate the sustained effects of stewardship interventions. Furthermore, we evaluated SAP practices (2019.1-2019.4) after the diffusion of a computerized decision support system. Finally, we analyzed SAP practices in the context of the COVID-19 pandemic (2020.1-2020.4 and 2021.1-2021.4). The groups were compared from year to year in terms of compliance to institutional guidelines, as evaluated from seven criteria (χ2 test). RESULTS: In total, 760 surgical interventions were recorded. The observational study within the baseline group showed that true penicillin allergy, certain types of surgery and certain practitioners were associated with non-compliance (p < 0.05). Compared with the baseline group, the compliance was significantly increased in the test group for all seven criteria assessed (p < 0.05). However, the effects were not fully sustained after discontinuation of the active interventions. Following the diffusion of the computerized decision support system, the compliance to guidelines was not significantly improved. Finally, the COVID-19 pandemic did not appear to affect the practices in terms of compliance to guidelines. CONCLUSIONS: This study shows that optimization of SAP practices is achievable within a proactive multidisciplinary approach including real-time pharmaceutical interventions in the operating area and in the care units practicing SAP

Impact of collaborative physician-pharmacist stewardship strategies on prophylactic antibiotic practices: a quasi-experimental study.

peer reviewed[en] BACKGROUND: An effective use of surgical antibiotic prophylaxis (SAP) appears essential to prevent the development of infections linked to surgery while inappropriate and excessive prescriptions of prophylactic antibiotics increase the risk of adverse effects, bacterial resistance and Clostridium difficile infections. In this study, we aimed to analyze SAP practices in an acute secondary hospital in Belgium during the years 2016-2021 in order to evaluate the impacts of combined stewardship interventions, implemented thanks to a physician-pharmacist collaboration. METHODS: A quasi-experimental study on SAP practices was conducted during 5 years (2016-2021) in a Belgian University Hospital. We first performed a retrospective observational transversal study on a baseline group (2016.1-2016.4). Then, we constituted a group of patients (2017.1-2017.4) to test a combined intervention strategy of stewardship which integrated the central role of a pharmacist in antibiotic stewardship team and in the pre-operative delivery of nominative kits of antibiotics adapted to patient factors. After this test, we collected patient data (2018.1-2018.4) to evaluate the sustained effects of stewardship interventions. Furthermore, we evaluated SAP practices (2019.1-2019.4) after the diffusion of a computerized decision support system. Finally, we analyzed SAP practices in the context of the COVID-19 pandemic (2020.1-2020.4 and 2021.1-2021.4). The groups were compared from year to year in terms of compliance to institutional guidelines, as evaluated from seven criteria (χ2 test). RESULTS: In total, 760 surgical interventions were recorded. The observational study within the baseline group showed that true penicillin allergy, certain types of surgery and certain practitioners were associated with non-compliance (p < 0.05). Compared with the baseline group, the compliance was significantly increased in the test group for all seven criteria assessed (p < 0.05). However, the effects were not fully sustained after discontinuation of the active interventions. Following the diffusion of the computerized decision support system, the compliance to guidelines was not significantly improved. Finally, the COVID-19 pandemic did not appear to affect the practices in terms of compliance to guidelines. CONCLUSIONS: This study shows that optimization of SAP practices is achievable within a proactive multidisciplinary approach including real-time pharmaceutical interventions in the operating area and in the care units practicing SAP

HPTLC Fingerprinting and Cytotoxicity of Secondary Metabolites of Equisetum Diffusum D. Don Extracts

peer reviewedThe aerial parts of Equisetum diffusum D.Don (Equisetaceae ED), a Vietnamese folk medicine used for hypertensive, anti-inflammatory, diuretic and hemostatic properties, were collected in Northern Vietnam. The secondary metabolites of the sequential n-hexane, ethyl acetate and methanol extracts were profiled by HPTLC with chromatographic conditions and derivatization reagents characteristic for flavonoids, polyphenols and terpenoids/steroids. All these metabolite classes were present in the methanol extract whereas the ethyl acetate extract comprised some polyphenols and flavonoids; no characteristic compound class could be identified in the n-hexane extract. HPLC allowed to determine the concentration of isoquercitroside, the major flavonoid of the methanol extract (1.60 ± 0.04 mg/g dry weight; n=3)

Syllabus de Travaux pratiques de toxicologie (2ème Grade en Pharmacie)

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Travaux pratiques de toxicologie analytique

7e édition 1998-1999/1Institut de Pharmacie, Service de Toxicologie, Chimie Bioanalytique et Chimie Physique Appliquée TOXI 017info:eu-repo/semantics/published

Multiclonal Diagnosis and MRD Follow-up in ALL with HTS Coupled with a Bioinformatic Analysis

International audienc