Reverse transcription-quantitative polymerase chain reaction: description of a RIN-based algorithm for accurate data normalization

<p>Abstract</p> <p>Background</p> <p>Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the gold standard technique for mRNA quantification, but appropriate normalization is required to obtain reliable data. Normalization to accurately quantitated RNA has been proposed as the most reliable method for in vivo biopsies. However, this approach does not correct differences in RNA integrity.</p> <p>Results</p> <p>In this study, we evaluated the effect of RNA degradation on the quantification of the relative expression of nine genes (<it>18S</it>, <it>ACTB</it>, <it>ATUB</it>, <it>B2M</it>, <it>GAPDH</it>, <it>HPRT</it>, <it>POLR2L</it>, <it>PSMB6</it> and <it>RPLP0</it>) that cover a wide expression spectrum. Our results show that RNA degradation could introduce up to 100% error in gene expression measurements when RT-qPCR data were normalized to total RNA. To achieve greater resolution of small differences in transcript levels in degraded samples, we improved this normalization method by developing a corrective algorithm that compensates for the loss of RNA integrity. This approach allowed us to achieve higher accuracy, since the average error for quantitative measurements was reduced to 8%. Finally, we applied our normalization strategy to the quantification of <it>EGFR</it>, <it>HER2 </it>and <it>HER3 </it>in 104 rectal cancer biopsies. Taken together, our data show that normalization of gene expression measurements by taking into account also RNA degradation allows much more reliable sample comparison.</p> <p>Conclusion</p> <p>We developed a new normalization method of RT-qPCR data that compensates for loss of RNA integrity and therefore allows accurate gene expression quantification in human biopsies.</p

Higher risk for influenza-associated pulmonary aspergillosis (IAPA) in asthmatic patients: A Swiss multicenter cohort study on IAPA in critically ill influenza patients

Background: Influenza-associated pulmonary aspergillosis (IAPA) is an important complication of severe influenza with high morbidity and mortality. Methods: We conducted a retrospective multicenter study in tertiary hospitals in Switzerland during 2017/2018 and 2019/2020 influenza seasons. All adults with PCR-confirmed influenza infection and treatment on intensive-care unit (ICU) for >24 h were included. IAPA was diagnosed according to previously published clinical, radiological, and microbiological criteria. We assessed risk factors for IAPA and predictors for poor outcome, which was a composite of in-hospital mortality, ICU length of stay ≥7 days, mechanical ventilation ≥7 days, or extracorporeal membrane oxygenation. Results: One hundred fifty-eight patients (median age 64 years, 45% females) with influenza were included, of which 17 (10.8%) had IAPA. Asthma was more common in IAPA patients (17% vs. 4% in non-IAPA, P = 0.05). Asthma (OR 12.0 [95% CI 2.1-67.2]) and days of mechanical ventilation (OR 1.1 [1.1-1.2]) were associated with IAPA. IAPA patients frequently required organ supportive therapies including mechanical ventilation (88% in IAPA vs. 53% in non-IAPA, P = 0.001) and vasoactive support (75% vs. 45%, P = 0.03) and had more complications including ARDS (53% vs. 26%, P = 0.04), respiratory bacterial infections (65% vs. 37%, P = 0.04), and higher ICU-mortality (35% vs. 16.4%, P = 0.05). IAPA (OR 28.8 [3.3-253.4]), influenza A (OR 3.3 [1.4-7.8]), and higher SAPS II score (OR 1.07 [1.05-1.10]) were independent predictors of poor outcome. Interpretation: High clinical suspicion, early diagnostics, and therapy are indicated in IAPA because of high morbidity and mortality. Asthma is likely an underappreciated risk factor for IAPA. Keywords: asthma; influenza; influenza-associated aspergillosis; intensive care medicine; invasive aspergillosis

Higher risk for influenza-associated pulmonary aspergillosis (IAPA) in asthmatic patients: A Swiss multicenter cohort study on IAPA in critically ill influenza patients.

BACKGROUND Influenza-associated pulmonary aspergillosis (IAPA) is an important complication of severe influenza with high morbidity and mortality. METHODS We conducted a retrospective multicenter study in tertiary hospitals in Switzerland during 2017/2018 and 2019/2020 influenza seasons. All adults with PCR-confirmed influenza infection and treatment on intensive-care unit (ICU) for >24 h were included. IAPA was diagnosed according to previously published clinical, radiological, and microbiological criteria. We assessed risk factors for IAPA and predictors for poor outcome, which was a composite of in-hospital mortality, ICU length of stay ≥7 days, mechanical ventilation ≥7 days, or extracorporeal membrane oxygenation. RESULTS One hundred fifty-eight patients (median age 64 years, 45% females) with influenza were included, of which 17 (10.8%) had IAPA. Asthma was more common in IAPA patients (17% vs. 4% in non-IAPA, P = 0.05). Asthma (OR 12.0 [95% CI 2.1-67.2]) and days of mechanical ventilation (OR 1.1 [1.1-1.2]) were associated with IAPA. IAPA patients frequently required organ supportive therapies including mechanical ventilation (88% in IAPA vs. 53% in non-IAPA, P = 0.001) and vasoactive support (75% vs. 45%, P = 0.03) and had more complications including ARDS (53% vs. 26%, P = 0.04), respiratory bacterial infections (65% vs. 37%, P = 0.04), and higher ICU-mortality (35% vs. 16.4%, P = 0.05). IAPA (OR 28.8 [3.3-253.4]), influenza A (OR 3.3 [1.4-7.8]), and higher SAPS II score (OR 1.07 [1.05-1.10]) were independent predictors of poor outcome. INTERPRETATION High clinical suspicion, early diagnostics, and therapy are indicated in IAPA because of high morbidity and mortality. Asthma is likely an underappreciated risk factor for IAPA

Assessment of epidermal growth factor receptor (EGFR) expression in primary colorectal carcinomas and their related metastases on tissue sections and tissue microarray

Metastatic colorectal carcinomas (CRC) resistant to chemotherapy may benefit from targeting monoclonal therapy cetuximab when they express the epidermal growth factor receptor (EGFR). Because of its clinical implications, we studied EGFR expression by immunohistochemistry on tissue sections of primary CRC (n=32) and their related metastases (n=53). A tissue microarray (TMA) was generated from the same paraffin blocks to determine whether this technique could be used for EGFR screening in CRC. On tissue sections, 84% of the primary CRC and 94% of the metastases were EGFR-positive. When matched, they showed a concordant EGFR-positive status in 78% of the cases. Moreover, staining intensity and extent of EGFR-positive cells in the primary CRC correlated with those observed in the synchronous metastases. On TMA, 65% of the primary CRC, 66% of the metastases, and 43% of the matched primary CRC metastases were EGFR-positive. There was no concordant EGFR status between the primary and the metastatic sites. A strong discrepancy of EGFR status was noted between TMA and tissue sections. In conclusion, EGFR expression measured in tissue sections from primary CRC and their related metastases was found to be similar and frequent, but it was significantly underestimated by the TMA technique

BMJ Med

OBJECTIVE: To evaluate the efficacy of covid-19 convalescent plasma to treat patients admitted to hospital for moderate covid-19 disease with or without underlying immunodeficiency (CORIPLASM trial). DESIGN: Open label, randomised clinical trial. SETTING: CORIMUNO-19 cohort (publicly supported platform of open label, randomised controlled trials of immune modulatory drugs in patients admitted to hospital with moderate or severe covid-19 disease) based on 19 university and general hospitals across France, from 16 April 2020 to 21 April 2021. PARTICIPANTS: 120 adults (n=60 in the covid-19 convalescent plasma group, n=60 in the usual care group) admitted to hospital with a positive SARS-CoV2 test result, duration of symptoms 40. MAIN OUTCOME MEASURES: Primary outcomes were proportion of patients with a WHO Clinical Progression Scale score of ≥6 on the 10 point scale on day 4 (higher values indicate a worse outcome), and survival without assisted ventilation or additional immunomodulatory treatment by day 14. Secondary outcomes were changes in WHO Clinical Progression Scale scores, overall survival, time to discharge, and time to end of dependence on oxygen supply. Predefined subgroups analyses included immunosuppression status, duration of symptoms before randomisation, and use of steroids. RESULTS: 120 patients were recruited and assigned to covid-19 convalescent plasma (n=60) or usual care (n=60), including 22 (covid-19 convalescent plasma) and 27 (usual care) patients who were immunocompromised. 13 (22%) patients who received convalescent plasma had a WHO Clinical Progression Scale score of ≥6 at day 4 versus eight (13%) patients who received usual care (adjusted odds ratio 1.88, 95% credible interval 0.71 to 5.24). By day 14, 19 (31.6%) patients in the convalescent plasma group and 20 (33.3%) patients in the usual care group needed ventilation, additional immunomodulatory treatment, or had died. For cumulative incidence of death, three (5%) patients in the convalescent plasma group and eight (13%) in the usual care group died by day 14 (adjusted hazard ratio 0.40, 95% confidence interval 0.10 to 1.53), and seven (12%) patients in the convalescent plasma group and 12 (20%) in the usual care group by day 28 (adjusted hazard ratio 0.51, 0.20 to 1.32). In a subgroup analysis performed in patients who were immunocompromised, transfusion of covid-19 convalescent plasma was associated with mortality (hazard ratio 0.39, 95% confidence interval 0.14 to 1.10). CONCLUSIONS: In this study, covid-19 convalescent plasma did not improve early outcomes in patients with moderate covid-19 disease. The efficacy of convalescent plasma in patients who are immunocompromised should be investigated further. TRIAL REGISTRATION: ClinicalTrials.gov NCT04345991

Brockett\u27s necessary conditions and the stabilization of nonlinear control systems

Brockett\u27s theorem states the three necessary conditions for the existence of a continuously differentiable closed loop control that asymptotically stabilizes the nonlinear control system to an equilibrium point. Kinematic systems are shown to fail to meet Brockett\u27s third necessary condition. A normal form is introduced so that nonholonomic control systems are defined directly over a reduced constraint distribution. In normal form, nonholonomic control systems can then easily be shown to fail to be stabilizable to a point via a $C^1$ control. The conditions for the smooth stabilization of the nonholonomic systems to an equilibrium submanifold are then presented. For a particular case of the reduced form of mechanical control systems (Chaplygin systems), stabilization to a point can be achieved by applying the concept of geometric phase and using piecewise differentiable state controls

Brockett's necessary conditions and the stabilization of nonlinear control systems

Brockett's theorem states the three necessary conditions for the existence of a continuously differentiable closed loop control that asymptotically stabilizes the nonlinear control system to an equilibrium point. Kinematic systems are shown to fail to meet Brockett's third necessary condition. A normal form is introduced so that nonholonomic control systems are defined directly over a reduced constraint distribution. In normal form, nonholonomic control systems can then easily be shown to fail to be stabilizable to a point via a C1 control. The conditions for the smooth stabilization of the nonholonomic systems to an equilibrium submanifold are then presented. For a particular case of the reduced form of mechanical control systems (Chaplygin systems), stabilization to a point can be achieved by applying the concept of geometric phase and using piecewise differentiable state controls

Recherche linéaire et fusion de données par ajustement de faisceaux (application à la localisation par vision)

Les travaux présentés dans ce manuscrit concernent le domaine de la localisation et la reconstruction 3D par vision artificielle. Dans ce contexte, la trajectoire d une caméra et la structure3D de la scène filmée sont initialement estimées par des algorithmes linéaires puis optimisées par un algorithme non-linéaire, l ajustement de faisceaux. Cette thèse présente tout d abord une technique de recherche de l amplitude de déplacement (recherche linéaire), ou line search pour les algorithmes de minimisation itérative. La technique proposée est non itérative et peut être rapidement implantée dans un ajustement de faisceaux traditionnel. Cette technique appelée recherche linéaire algébrique globale (G-ALS), ainsi que sa variante à deux dimensions (Two way-ALS), accélèrent la convergence de l algorithme d ajustement de faisceaux. L approximation de l erreur de reprojection par une distance algébrique rend possible le calcul analytique d une amplitude de déplacement efficace (ou de deux pour la variante Two way-ALS), par la résolution d un polynôme de degré 3 (G-ALS) ou 5 (Two way-ALS). Nos expérimentations sur des données simulées et réelles montrent que cette amplitude, optimale en distance algébrique, est performante en distance euclidienne, et permet de réduire le temps de convergence des minimisations. Une difficulté des algorithmes de localisation en temps réel par la vision (SLAM monoculaire) est que la trajectoire estimée est souvent affectée par des dérives : dérives d orientation, de position et d échelle. Puisque ces algorithmes sont incrémentaux, les erreurs et approximations sont cumulées tout au long de la trajectoire, et une dérive se forme sur la localisation globale. De plus, un système de localisation par vision peut toujours être ébloui ou utilisé dans des conditions qui ne permettent plus temporairement de calculer la localisation du système. Pour résoudre ces problèmes, nous proposons d utiliser un capteur supplémentaire mesurant les déplacements de la caméra. Le type de capteur utilisé varie suivant l application ciblée (un odomètre pour la localisation d un véhicule, une centrale inertielle légère ou un système de navigation à guidage inertiel pour localiser une personne). Notre approche consiste à intégrer ces informations complémentaires directement dans l ajustement de faisceaux, en ajoutant un terme de contrainte pondéré dans la fonction de coût. Nous évaluons trois méthodes permettant de sélectionner dynamiquement le coefficient de pondération et montrons que ces méthodes peuvent être employées dans un SLAM multi-capteur temps réel, avec différents types de contrainte, sur l orientation ou sur la norme du déplacement de la caméra. La méthode est applicable pour tout autre terme de moindres carrés. Les expérimentations menées sur des séquences vidéo réelles montrent que cette technique d ajustement de faisceaux contraint réduit les dérives observées avec les algorithmes de vision classiques. Ils améliorent ainsi la précision de la localisation globale du système.The works presented in this manuscript are in the field of computer vision, and tackle the problem of real-time vision based localization and 3D reconstruction. In this context, the trajectory of a camera and the 3D structure of the filmed scene are initially estimated by linear algorithms and then optimized by a nonlinear algorithm, bundle adjustment. The thesis first presents a new technique of line search, dedicated to the nonlinear minimization algorithms used in Structure-from-Motion. The proposed technique is not iterative and can be quickly installed in traditional bundle adjustment frameworks. This technique, called Global Algebraic Line Search (G-ALS), and its two-dimensional variant (Two way-ALS), accelerate the convergence of the bundle adjustment algorithm. The approximation of the reprojection error by an algebraic distance enables the analytical calculation of an effective displacement amplitude (or two amplitudes for the Two way-ALS variant) by solving a degree 3 (G-ALS) or 5 (Two way-ALS) polynomial. Our experiments, conducted on simulated and real data, show that this amplitude, which is optimal for the algebraic distance, is also efficient for the Euclidean distance and reduces the convergence time of minimizations. One difficulty of real-time tracking algorithms (monocular SLAM) is that the estimated trajectory is often affected by drifts : on the absolute orientation, position and scale. Since these algorithms are incremental, errors and approximations are accumulated throughout the trajectory and cause global drifts. In addition, a tracking vision system can always be dazzled or used under conditions which prevented temporarily to calculate the location of the system. To solve these problems, we propose to use an additional sensor measuring the displacement of the camera. The type of sensor used will vary depending on the targeted application (an odometer for a vehicle, a lightweight inertial navigation system for a person). We propose to integrate this additional information directly into an extended bundle adjustment, by adding a constraint term in the weighted cost function. We evaluate three methods (based on machine learning or regularization) that dynamically select the weight associated to the constraint and show that these methods can be used in a real time multi-sensor SLAM, and validate them with different types of constraint on the orientation or on the scale. Experiments conducted on real video sequences show that this technique of constrained bundle adjustment reduces the drifts observed with the classical vision algorithms and improves the global accuracy of the positioning system.CLERMONT FD-Bib.électronique (631139902) / SudocSudocFranceF