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1.
Clin Chem ; 68(2): 313-321, 2022 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-34871369

RESUMEN

BACKGROUND: To date, the usage of Galaxy, an open-source bioinformatics platform, has been reported primarily in research. We report 5 years' experience (2015 to 2020) with Galaxy in our hospital, as part of the "Assistance Publique-Hôpitaux de Paris" (AP-HP), to demonstrate its suitability for high-throughput sequencing (HTS) data analysis in a clinical laboratory setting. METHODS: Our Galaxy instance has been running since July 2015 and is used daily to study inherited diseases, cancer, and microbiology. For the molecular diagnosis of hereditary diseases, 6970 patients were analyzed with Galaxy (corresponding to a total of 7029 analyses). RESULTS: Using Galaxy, the time to process a batch of 23 samples-equivalent to a targeted DNA sequencing MiSeq run-from raw data to an annotated variant call file was generally less than 2 h for panels between 1 and 500 kb. Over 5 years, we only restarted the server twice for hardware maintenance and did not experience any significant troubles, demonstrating the robustness of our Galaxy installation in conjunction with HTCondor as a job scheduler and a PostgreSQL database. The quality of our targeted exome sequencing method was externally evaluated annually by the European Molecular Genetics Quality Network (EMQN). Sensitivity was mean (SD)% 99 (2)% for single nucleotide variants and 93 (9)% for small insertion-deletions. CONCLUSION: Our experience with Galaxy demonstrates it to be a suitable platform for HTS data analysis with vast potential to benefit patient care in a clinical laboratory setting.


Asunto(s)
Biología Computacional , Laboratorios Clínicos , Biología Computacional/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Análisis de Secuencia de ADN , Programas Informáticos
2.
PLoS One ; 17(12): e0271145, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36477212

RESUMEN

Chronic hepatitis B (CHB) is a global health care challenge and a major cause of liver disease. To find new therapeutic avenues with a potential to functionally cure chronic Hepatitis B virus (HBV) infection, we performed a focused screen of epigenetic modifiers to identify potential inhibitors of replication or gene expression. From this work we identified isonicotinic acid inhibitors of the histone lysine demethylase 5 (KDM5) with potent anti-HBV activity. To enhance the cellular permeability and liver accumulation of the most potent KDM5 inhibitor identified (GS-080) an ester prodrug was developed (GS-5801) that resulted in improved bioavailability and liver exposure as well as an increased H3K4me3:H3 ratio on chromatin. GS-5801 treatment of HBV-infected primary human hepatocytes reduced the levels of HBV RNA, DNA and antigen. Evaluation of GS-5801 antiviral activity in a humanized mouse model of HBV infection, however, did not result in antiviral efficacy, despite achieving pharmacodynamic levels of H3K4me3:H3 predicted to be efficacious from the in vitro model. Here we discuss potential reasons for the disconnect between in vitro and in vivo efficacy, which highlight the translational difficulties of epigenetic targets for viral diseases.


Asunto(s)
Virus de la Hepatitis B , Hepatitis B Crónica , Humanos , Animales , Ratones , Antivirales/farmacología , Hepatitis B Crónica/tratamiento farmacológico , Epigenómica
3.
Toxicol Sci ; 183(1): 105-116, 2021 08 30.
Artículo en Inglés | MEDLINE | ID: mdl-34117767

RESUMEN

GS-9695 and GS-9822 are next-generation noncatalytic site integrase inhibitors (NCINIs) with significantly improved potency against human immunodeficiency virus compared with previous drugs such as BI-224436. Development stopped due to vacuolation of the bladder urothelium seen in cynomolgus monkey but not in rat; this lesion was absent in equivalent preclinical studies with BI-224436 (tested in dog and rat). Lesions were unlikely to be attributable to target because NCINIs specifically target viral integrase protein and no mammalian homologue is known. Secondary pharmacology studies, mitochondrial toxicity studies, immunophenotyping, and analysis of proteins implicated in cell-cell interactions and/or bladder integrity (E-cadherin, pan-cytokeratin, uroplakins) failed to offer any plausible explanation for the species specificity of the lesion. Because it was characterized by inflammation and disruption of urothelial morphology, we investigated physicochemical changes in the bladder of cynomolgus monkey (urinary pH 5.5-7.4) that might not occur in the bladder of rats (urinary pH 7.3-8.5). In measurements of surface activity, GS-9822 showed an unusual transition from a monolayer to a bilayer at the air/water interface with decreasing pH, attributed to the strong association between drug molecules in adjacent bilayer leaflets and expected to be highly disruptive to the urothelium. Structural analysis of GS-9822 and GS-9695 showed zwitterionic characteristics over the range of pH expected in cynomolgus monkey but not rat urine. This exotic surface behavior is unlikely with BI-224436 since it would transition from neutral to cationic (never zwitterionic) with decreasing pH. These data provide useful insights to guide discovery and development of NCINIs, related compounds, and zwitterions.


Asunto(s)
Inhibidores de Integrasa VIH , Urotelio , Animales , Perros , Concentración de Iones de Hidrógeno , Macaca fascicularis , Ratas , Especificidad de la Especie
4.
Radiology ; 256(2): 397-405, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20530754

RESUMEN

PURPOSE: To compare an algorithm of gradually ramped-up power to a full-power-level technique to determine which technical parameters maximized tissue coagulation by using a saline-perfused electrode. MATERIALS AND METHODS: Institutional review board approval was not necessary and animal committee approval was unnecessary because an ex vivo bovine liver model was used and the animals were not specifically killed for this study. This four-part experiment utilized multiple ablations of ex vivo bovine liver with a standard radiofrequency (RF) generator and an internally cooled needle. First, 10 RF ablations were performed at 20-60 W for 12 minutes. Second, ablation volumes obtained from an algorithm of eight ablations performed at 50 W were compared with those obtained from an algorithm of eight ablations that were gradually ramped-up to 50 W, until full impedance. Third, volumes obtained from 10 ablations performed at impedance control power levels were compared with those obtained from 10 ablations performed with a gradual ramp-up of power that started at 50 W, terminating at full impedance. Last, the third part was repeated, but with 11 ablations continuing past full impedance for 12 minutes each. RESULTS: In the first part, maximum measurements of tissue coagulation seemed to plateau from 40 to 60 W. The second part produced significantly larger measurements of tissue coagulation than did the use of a constant power level of 50 W. The third and final parts produced larger measurements of tissue coagulation than did utilizing full power for 12 minutes. Larger measurements and volumes were obtained from repeat ablations after the generator reached impedance level than were obtained from ablations stopped at maximum impedance. CONCLUSION: A gradual ramp-up of power and repeating ablations after power impedance level is reached are the two methods that increased tissue ablation in this ex vivo experiment.


Asunto(s)
Ablación por Catéter/instrumentación , Electrodos , Hepatectomía/instrumentación , Hepatectomía/métodos , Hígado/fisiología , Hígado/cirugía , Cirugía Asistida por Computador/métodos , Animales , Temperatura Corporal , Ablación por Catéter/métodos , Bovinos , Frío , Técnicas In Vitro
5.
Mol Pharm ; 7(6): 1948-58, 2010 Dec 06.
Artículo en Inglés | MEDLINE | ID: mdl-20925429

RESUMEN

Repeated administration of chemotherapeutics is typically required for the effective treatment of highly aggressive tumors and often results in systemic toxicity. We have created a copper-doxorubicin complex within the core of liposomes and applied the resulting particle in multidose therapy. Copper and doxorubicin concentrations in the blood pool were similar at 24 h (∼40% of the injected dose), indicating stable circulation of the complex. Highly quenched doxorubicin fluorescence remained in the blood pool over tens of hours, with fluorescence increasing only with the combination of liposome disruption and copper trans-chelation. At 48 h after injection, doxorubicin fluorescence within the heart and skin was one-fifth and one-half, respectively, of fluorescence observed with ammonium sulfate-loaded doxorubicin liposomes. After 28 days of twice per week doxorubicin administration of 6 mg/kg, systemic toxicity (cardiac hypertrophy and weight and hair loss) was not detected with the copper-doxorubicin liposomes but was substantial with ammonium sulfate-loaded doxorubicin liposomes. We then incorporated two strategies designed to enhance efficacy, mTOR inhibition (rapamycin) to slow proliferation and therapeutic ultrasound to enhance accumulation and local diffusion. Tumor accumulation was ∼10% ID/g and was enhanced approximately 2-fold with the addition of therapeutic ultrasound. After the 28-day course of therapy, syngeneic tumors regressed to a premalignant phenotype of ∼(1 mm)(3) or could not be detected.


Asunto(s)
Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Neoplasias de la Mama/tratamiento farmacológico , Cobre/administración & dosificación , Cobre/farmacología , Doxorrubicina/administración & dosificación , Doxorrubicina/farmacología , Nanopartículas/química , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Neoplasias de la Mama/patología , Cobre/efectos adversos , Cobre/química , Modelos Animales de Enfermedad , Doxorrubicina/efectos adversos , Doxorrubicina/química , Femenino , Liposomas/química , Ratones , Sirolimus/administración & dosificación , Sirolimus/efectos adversos , Sirolimus/química , Sirolimus/farmacología , Terapia por Ultrasonido , Ensayos Antitumor por Modelo de Xenoinjerto
6.
Nat Med ; 25(9): 1377-1384, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31501601

RESUMEN

People living with HIV (PLWH) have expressed concern about the life-long burden and stigma associated with taking pills daily and can experience medication fatigue that might lead to suboptimal treatment adherence and the emergence of drug-resistant viral variants, thereby limiting future treatment options1-3. As such, there is strong interest in long-acting antiretroviral (ARV) agents that can be administered less frequently4. Herein, we report GS-CA1, a new archetypal small-molecule HIV capsid inhibitor with exceptional potency against HIV-2 and all major HIV-1 types, including viral variants resistant to the ARVs currently in clinical use. Mechanism-of-action studies indicate that GS-CA1 binds directly to the HIV-1 capsid and interferes with capsid-mediated nuclear import of viral DNA, HIV particle production and ordered capsid assembly. GS-CA1 selects in vitro for unfit GS-CA1-resistant capsid variants that remain fully susceptible to other classes of ARVs. Its high metabolic stability and low solubility enabled sustained drug release in mice following a single subcutaneous dosing. GS-CA1 showed high antiviral efficacy as a long-acting injectable monotherapy in a humanized mouse model of HIV-1 infection, outperforming long-acting rilpivirine. Collectively, these results demonstrate the potential of ultrapotent capsid inhibitors as new long-acting agents for the treatment of HIV-1 infection.


Asunto(s)
Fármacos Anti-VIH/farmacología , Proteínas de la Cápside/antagonistas & inhibidores , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Indazoles/farmacología , Piridinas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Fármacos Anti-VIH/uso terapéutico , Cápside/efectos de los fármacos , Cápside/metabolismo , Proteínas de la Cápside/genética , ADN Viral/efectos de los fármacos , Preparaciones de Acción Retardada , Farmacorresistencia Viral/efectos de los fármacos , Infecciones por VIH/genética , Infecciones por VIH/virología , VIH-1/genética , VIH-1/patogenicidad , VIH-2/efectos de los fármacos , VIH-2/patogenicidad , Humanos , Indazoles/uso terapéutico , Cumplimiento de la Medicación , Ratones , Piridinas/uso terapéutico
7.
J Control Release ; 178: 108-17, 2014 Mar 28.
Artículo en Inglés | MEDLINE | ID: mdl-24434424

RESUMEN

Advancements in liposomal drug delivery have produced long circulating and very stable drug formulations. These formulations minimize systemic exposure; however, unfortunately, therapeutic efficacy has remained limited due to the slow diffusion of liposomal particles within the tumor and limited release or uptake of the encapsulated drug. Here, the carboxyl-terminated CRPPR peptide, with affinity for the receptor neuropilin-1 (NRP), which is expressed on both endothelial and cancer cells, was conjugated to liposomes to enhance the tumor accumulation. Using a pH sensitive probe, liposomes were optimized for specific NRP binding and subsequent cellular internalization using in vitro cellular assays. Liposomes conjugated with the carboxyl-terminated CRPPR peptide (termed C-LPP liposomes) bound to the NRP-positive primary prostatic carcinoma cell line (PPC-1) but did not bind to the NRP-negative PC-3 cell line, and binding was observed with liposomal peptide concentrations as low as 0.16mol%. Binding of the C-LPP liposomes was receptor-limited, with saturation observed at high liposome concentrations. The identical peptide sequence bearing an amide terminus did not bind specifically, accumulating only with a high (2.5mol%) peptide concentration and adhering equally to NRP positive and negative cell lines. The binding of C-LPP liposomes conjugated with 0.63mol% of the peptide was 83-fold greater than liposomes conjugated with the amide version of the peptide. Cellular internalization was also enhanced with C-LPP liposomes, with 80% internalized following 3h incubation. Additionally, fluorescence in the blood pool (~40% of the injected dose) was similar for liposomes conjugated with 0.63mol% of carboxyl-terminated peptide and non-targeted liposomes at 24h after injection, indicating stable circulation. Prior to doxorubicin treatment, in vivo tumor accumulation and vascular targeting were increased for peptide-conjugated liposomes compared to non-targeted liposomes based on confocal imaging of a fluorescent cargo, and the availability of the vascular receptor was confirmed with ultrasound molecular imaging. Finally, over a 4-week course of therapy, tumor knockdown resulting from doxorubicin-loaded, C-LPP liposomes was similar to non-targeted liposomes in syngeneic tumor-bearing FVB mice and C-LPP liposomes reduced doxorubicin accumulation in the skin and heart and eliminated skin toxicity. Taken together, our results demonstrate that a carboxyl-terminated RXXR peptide sequence, conjugated to liposomes at a concentration of 0.63mol%, retains long circulation but enhances binding and internalization, and reduces toxicity.


Asunto(s)
Antibióticos Antineoplásicos/administración & dosificación , Doxorrubicina/administración & dosificación , Neoplasias/tratamiento farmacológico , Neuropilina-1/metabolismo , Oligopéptidos/administración & dosificación , Animales , Antibióticos Antineoplásicos/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Medios de Contraste/administración & dosificación , Medios de Contraste/química , Doxorrubicina/química , Femenino , Gadolinio/administración & dosificación , Gadolinio/química , Compuestos Heterocíclicos/administración & dosificación , Compuestos Heterocíclicos/química , Humanos , Liposomas , Ratones , Neoplasias/metabolismo , Neoplasias/patología , Neuropilina-1/química , Oligopéptidos/química , Compuestos Organometálicos/administración & dosificación , Compuestos Organometálicos/química , Polietilenglicoles/química , Carga Tumoral/efectos de los fármacos
8.
IEEE Trans Biomed Eng ; 58(7): 2002-12, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21402506

RESUMEN

Gold nanoparticles (GNPs) are nontoxic, can be functionalized with ligands, and preferentially accumulate in tumors. We have developed a 13.56-MHz RF-electromagnetic field (RF-EM) delivery system capable of generating high E-field strengths required for noninvasive, noncontact heating of GNPs. The bulk heating and specific heating rates were measured as a function of NP size and concentration. It was found that heating is both size and concentration dependent, with 5 nm particles producing a 50.6 ± 0.2 °C temperature rise in 30 s for 25 µg/mL gold (125 W input). The specific heating rate was also size and concentration dependent, with 5 nm particles producing a specific heating rate of 356 ± 78 kW/g gold at 16 µg/mL (125 W input). Furthermore, we demonstrate that cancer cells incubated with GNPs are killed when exposed to 13.56 MHz RF-EM fields. Compared to cells that were not incubated with GNPs, three out of four RF-treated groups showed a significant enhancement of cell death with GNPs (p<0.05). GNP-enhanced cell killing appears to require temperatures above 50 °C for the experimental parameters used in this study. Transmission electron micrographs show extensive vacuolization with the combination of GNPs and RF treatment.


Asunto(s)
Oro/química , Hipertermia Inducida/instrumentación , Nanopartículas del Metal/química , Neoplasias/terapia , Muerte Celular/efectos de la radiación , Línea Celular Tumoral , Ácido Cítrico , Campos Electromagnéticos , Diseño de Equipo , Calor , Humanos , Hipertermia Inducida/métodos , Microscopía Electrónica de Transmisión , Nanotecnología , Tamaño de la Partícula
9.
IEEE Trans Biomed Eng ; 57(1): 155-66, 2010 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-20064754

RESUMEN

A new system is presented for generating controlled tissue heating with a clinical ultrasound scanner, and initial in vitro and in vivo results are presented that demonstrate both transient and sustained heating in the mild-hyperthermia range of 37 ( degrees )C-42 ( degrees )C. The system consists of a Siemens Antares ultrasound scanner, a custom dual-frequency three-row transducer array and an external temperature feedback control system. The transducer has two outer rows that operate at 1.5 MHz for tissue heating and a center row that operates at 5 MHz for B-mode imaging to guide the therapy. We compare the field maps obtained using a hydrophone against calculations of the ultrasound beam based on monochromatic and linear assumptions. Using the finite-difference time-domain (FDTD) method, we compare predicted time-dependent thermal profiles to measured profiles for soy tofu as a tissue-mimicking phantom. In vitro results show differential heating of 6 ( degrees )C for chicken breast and tofu. In vivo tests of the system were performed on three mice bearing Met-1 tumors, which is a model of aggressive, metastatic, and highly vascular breast cancer. In superficially implanted tumors, we demonstrate controlled heating to 42 ( degrees )C. We show that the system is able to maintain the temperature to within 0.1 ( degrees )C of the desired temperature both in vitro and in vivo.


Asunto(s)
Hipertermia Inducida/métodos , Neoplasias Experimentales/diagnóstico por imagen , Terapia por Ultrasonido/métodos , Animales , Pollos , Hipertermia Inducida/instrumentación , Carne , Ratones , Alimentos de Soja , Terapia por Ultrasonido/instrumentación , Ultrasonografía
10.
J Control Release ; 118(3): 275-84, 2007 Apr 23.
Artículo en Inglés | MEDLINE | ID: mdl-17300849

RESUMEN

A new acoustically-active delivery vehicle was developed by conjugating liposomes and microbubbles, using the high affinity interaction between avidin and biotin. Binding between microbubbles and liposomes, each containing 5% DSPE-PEG2kBiotin, was highly dependent on avidin concentration and observed above an avidin concentration of 10 nM. With an optimized avidin and liposome concentration, we measured and calculated as high as 1000 to 10,000 liposomes with average diameters of 200 and 100 nm, respectively, attached to each microbubble. Replacing avidin with neutravidin resulted in 3-fold higher binding, approaching the calculated saturation level. High-speed photography of this new drug delivery vehicle demonstrated that the liposome-bearing microbubbles oscillate in response to an acoustic pulse in a manner similar to microbubble contrast agents. Additionally, microbubbles carrying liposomes could be spatially concentrated on a monolayer of PC-3 cells at the focal point of ultrasound beam. As a result of cell-vehicle contact, the liposomes fused with the cells and internalization of NBD-cholesterol occurred shortly after incubation at 37 degrees C, with internalization of NBD-cholesterol substantially enhanced in the acoustic focus.


Asunto(s)
Acústica , Sistemas de Liberación de Medicamentos/métodos , Liposomas/administración & dosificación , Microburbujas , Biotinilación/métodos , Línea Celular Tumoral , Humanos , Masculino
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