A Genetically Encoded BRET-based SARS-CoV-2 Mpro Protease Activity Sensor

The SARS-CoV-2 main protease, Mpro, is critical for its replication and is an appealing target for designing anti-SARS-CoV-2 agents. In this regard, a number of assays have been developed based on its cleavage sequence preferences to monitor its activity. These include the usage of Fluorescence Resonance Energy Transfer (FRET)-based substrates in vitro and a FlipGFP reporter, one which fluoresces after Mpro-mediated cleavage, in live cells. Here, we have engineered a pair of genetically encoded, Bioluminescence Resonance Energy Transfer (BRET)-based sensors for detecting SARS-CoV-2 Mpro proteolytic activity in living host cells as well as in vitro assays. The sensors were generated by sandwiching Mpro N-terminal autocleavage sites, either AVLQSGFR (short) or KTSAVLQSGFRKME (long), in between the mNeonGreen and nanoLuc proteins. Co-expression of the sensor with the Mpro in live cells resulted in its cleavage in a dose- and time-dependent manner while mutation of the critical C145 residue (C145A) in Mpro completely abrogated the sensor cleavage. Importantly, the BRET-based sensors displayed increased sensitivities and specificities as compared to the recently developed FlipGFP-based Mpro sensor. Additionally, the sensors recapitulated the inhibition of Mpro by the well-characterized pharmacological agent GC376. Further, in vitro assays with the BRET-based Mpro sensors revealed a molecular crowding-mediated increase in the rate of Mpro activity and a decrease in the inhibitory potential of GC376. The sensor developed here will find direct utility in studies related to drug discovery targeting the SARS-CoV-2 Mpro and functional genomics application to determine the effect of sequence variation in Mpro.

[Profile of the victims of sexual violence in child psychiatry consultation]. / Enfants victimes de violences sexuelles en consultation de pédopsychiatrie.

INTRODUCTION: Child sexual abuse is a major health problem that remains under-declared and under-reported worldwide. In this paper we have taken an interest in establishing the profile of the victims in a clinical population of children consulting for sexual abuse. METHOD: We conducted a descriptive retrospective study in the child and teenager psychiatry department of the Mongi-Slim hospital (Tunis, Tunisia). We included all patients referred for treatment or medical expertise during the period from January 2013 to June 2019 and recorded sociodemographic data and clinical characteristics on pre-established charts from patient files. RESULTS: The total number of consultants was 150 patients with 61,33 % of female subjects (n=92). The average age was 9,9 years old with extremes ranging from 2 to 18 years old. The assault was unique in 62,7 % and happened in a place known by the victim in 47,33 %. Sexual abuse with non-penetrating contact was the most frequent (48 %). The average age at which the first sexual abuse happened was 9 years old. In the vast majority of cases, the abuser was a male. Concerning the identity of the abuser, he was someone of the family in 37,3 % of the cases and in half of these cases, he was the father. In the other cases where the abuser wasn't a family member, the identity was known by the child and/or his family in 48,66 %. A psychiatric diagnosis was made in 58 % of the cases with a percentage of 52,1 % among girls and 60,3 % among boys. Main diagnoses were acute stress disorder in 10,6 %, post trauma stress disorder in 19,3 %, adjustment disorder in14,6 % and a major depressive disorder in 8 %. CONCLUSION: Knowing the profile of victims of child sexual abuse and taking into consideration the social and psychiatric impact can help in adapting the means to intervene properly in order to take care of the victims and prevent such abuse.

Traumatic right diaphragmatic hernia in children: Diagnostic difficulties.

Traumatic right diaphragmatic hernia is rare in children. Its diagnosis can be difficult in the acute phase of trauma because its signs are not specific, especially in a poly trauma context. We report two cases of traumatic right diaphragmatic hernia following a blunt thoraco-abdominal trauma, highlighting some difficulties in establishing an early diagnosis and the need for a high index of suspicion.

Validation of two-dimensional methods for left atrial volume measurement: a comparison of echocardiography with cardiac computed tomography.

BACKGROUND: Left atrial volume (LAVol) is an important predictor of cardiovascular outcomes. Different formulas are applied to calculate LAVol using two-dimensional transthoracic echocardiography (2DTTE) with variable reference values. The objective of the study was to evaluate the accuracy of methods to calculate LAVol by 2DTTE or cardiac computed tomography (CT). METHODS AND RESULTS: Overall 177 consecutive patients who underwent both a 2DTTE and retrospective electrocardiogram (ECG)-gated coronary CT angiography (CTA) within 15 days were included for this study. LA volume measurements were calculated by 2DTTE and 2DCT using the biplane area-length, biplane Simpson's, prolate-ellipsoid-1 and prolate-ellipsoid-2 methods. These results were compared with those measured by CT using a volumetric method. There was very good correlation between the CT and echocardiographic measures for LAVol, but significant underestimation of the echocardiographic methods when compared to the reference standard (33.5%, 39.1%, 48.1%, and 53.2% for the biplane area-length, biplane Simpson's, prolate-ellipsoid-1, and prolate-ellipsoid-2 methods, respectively). The biplane area-length method using 2DTTE had the closest volume estimation of all echocardiographic methods to the reference standard (67.6 ± 25.5 mL vs. 106 ± 35.5 mL, r = 0.712). Similarly, the biplane area-length method using CT most accurately predicted LAVol (103.3 ± 36.0 mL, r = 0.965). CONCLUSIONS: Compared to CT, 2DTTE provides reasonable assessment of LAVol, although all measurement methods underestimate LAVol. For both 2DTTE and CT, the biplane area-length method appears to provide the most accurate 2D estimate of LAVol.

Determining patient prognosis using computed tomography coronary angiography.

In addition to demonstrating luminal narrowings, cardiac computed tomography angiography (CTA) has the ability to detect nonstenotic plaque, vessel wall calcification and can assess left ventricular function. CTA prognostic studies have considered these components individually and in combination to produce novel risk factor scores to help predict clinical outcomes. In this article, we will consider the utility of CTA to predict clinical risk by considering the evidence for luminal stenosis, plaque scores, plaque descriptors and models combining these elements. We will also discuss some of the emerging applications of CTA that will likely provide future prognostic data in coronary artery disease patients. Although initially described as an anatomical investigation to determine the presence of coronary disease, CTA is being explored as a tool for functional imaging and may soon provide a noninvasive technique of anatomical and functional assessment previously only possible by invasive methods.

Changes in the activity of a CpG neuron after the reinforcement of an operantly conditioned behavior in Lymnaea.

We have previously shown that the aerial respiratory behavior of the mollusk Lymnaea stagnalis can be operantly conditioned, and the central pattern generating (CPG) neurons underlying this behavior have been identified. As neural correlates of operant conditioning remain poorly defined in both vertebrates and invertebrates, we have used the Lymnaea respiratory CPG to investigate neuronal changes associated with the change in behavior after conditioning. After operant conditioning of the intact animals, semi-intact preparations were dissected, so that changes in the respiratory behavior (pneumostome openings) and underlying activity of the identified CPG neuron, right pedal dorsal 1 (RPeD1), could be monitored simultaneously. RPeD1 was studied because it initiates the rhythmic activity of the CPG and receives chemo-sensory input from the pneumostome area. Pneumostome openings and RPeD1 activity were monitored both before and after a reinforcing training stimulus applied to the open pneumostome of operantly conditioned and yoked control preparations. After presentation of the reinforcing stimulus, there was a significant reduction in both breathing behavior and RPeD1 activity in operant preparations but not in yoked and naïve controls. Furthermore these changes were only significant in the subgroup of operantly conditioned animals described as good learners and not in poor learners. These data strongly suggest that changes in RPeD1 activity may underlie the behavioral changes associated with the reinforcement of operant conditioning of the respiratory behavior.

[Isotope angiocardiographic study of 30 cases of ventricular tachycardia with Fourier phase analysis]. / Etude de 30 tachycardies ventriculaires par angiocardiographie isotopique avec analyse de phase de Fourier.

Twenty-seven patients (15 men, 12 women; mean age 48.9 years) suffering from ventricular tachycardia (VT) (n = 30) were studied by radionuclide angiocardiography with Fourier phase analysis, both in sinus rhythm and during tachycardia. VT was spontaneous, electrically inducible, sustained, haemodynamically stable and monomorphous, with a mean rate of 174 beats/min (range: 115-260 beats). Heart diseases responsible for VT were: non-obstructive cardiomyopathy (n = 7), hypertrophic cardiomyopathy (n = 1), ischaemic heart disease (n = 5), probable right ventricular arrhythmogenic dysplasia (n = 4), congenital left ventricular aneurysm (n = 2), sequela of myocarditis (n = 2) and aortic valve regurgitation (n = 1); no heart disease was detectable in 5 patients. On surface electrocardiogram there was good concordance between the initial radionuclide site of VT activation and the configuration and electrical axis of QRS. At Fourier phase analysis all 17 VT of the right lag type originated in the left ventricle, arising from the apical septum (n = 7) or lateral segment (n = 2) in case of left axis, from the basal segment (n = 6) or the lateral segment (n = 1) in case of vertical or right axis, and from the middle left septum (n = 1) in case of normal axis. Nine VT of the left lag type originated in the right ventricle, arising from the basal septum or the latero-basal region in case of vertical or right axis (n = 6), from the apical septum or the inferior-apical region in case of left axis (n = 2) and from the middle septum in case of normal axis (n = 1). Four of our patients (3 with coronary disease and 1 with congenital left ventricular aneurysm) had VT of the left lag type and an initial radionuclide site of activation in the middle part of the left septum in case of left axis (n = 2) and in the basal part of that septum in case of right axis (n = 2). Seven patients were operated upon for recurrent VT: 4 had intra-operative mapping which in every case confirmed the results of radionuclide angiocardiography, a method which in the other 3 patients was the only surgeon's guide. Correlations between the site of origin of VT at radionuclide mapping and kinetic abnormalities visualized at radiological angiography and gamma-ray angiocardiography were common in our study. In one of our patients the same lesion gave birth to 2 VT of different morphologies.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of capsicum fruit on theophylline absorption and bioavailability in rabbits.

Absorption and bioavailability of theophylline from a sustained-release gelatin capsule were investigated in 10 male rabbits after oral administration (20 mg/kg), with and without a ground capsicum fruit suspension. Comparison of pharmacokinetic parameters showed that the concomitant absorption of capsicum increases areas under plasma curves (from 86.06 +/- 9.78 mg H/liter to 138.32 +/- 17.27 mg H/liter, P less than 0.001), peak plasma levels (from 6.65 +/- 0.76 to 8.78 +/- 0.98 mg/liter, P less than 0.01), and mean residence times (from 14.94 +/- 2.97 to 20.98 +/- 5.75 H, P less than 0.001). A second administration of the capsicum suspension, 11 hours after dosing, produced a new rise of theophylline plasma levels in every rabbit. The variations in pharmacokinetic and bioavailability parameters are discussed in accordance with the mechanisms of action of capsaicin, an active compound present in capsicum fruit.