Aptamer-, heparin- or cocktail-based inhibition of S1-ACE2 protein complexes.

The Spike protein (S1) from the Severe acute respiratory syndrome 2 virus binds to angiotensin converting enzyme 2 (ACE2) receptor to initiate infection. Hence, antiviral therapeutic targeting the S1-ACE2 interface is of interest. Herein, we compare the inhibition efficacy of an aptamer to heparin or their cocktail, against wild-type (WT), Omicron, Delta, and Lambda S1-ACE2 complexes. The aptamer-protein complexes had the dissociation constant KD values in the 2-13 nM range. The aptamer half-maximal inhibitory concentration against WT S1-ACE was 17 nM, with the % inhibition in the 12-35% range. Several aptamer-S1 protein complexes were also stable at low pH with 60% inhibition. Despite the similarity in S1 sequences, the extent of inhibition (2-27%) with heparin was highly dependent on the type of S1 protein. More importantly, heparin did not inhibit the WT S1-ACE2 complex but was effective with mutants. The aptamer-heparin cocktail was less effective compared to aptamer or heparin, individually. Modelling data show that either a direct or proximal binding to RBD sites by aptamer or heparin prevents the ACE2 binding. Overall, heparin was as an effective inhibitor as aptamer against certain variants, and represents the more cost-effective neutralizing agent against emerging coronaviruses.

[Introduction to the hierarchical taxonomy of psychopathology]. / Introduction à la taxonomie hiérarchique de la psychopathologie.

INTRODUCTION: In clinical practice, the usefulness of diagnosis based on the Diagnostic or Statistical Manual of Mental Disorders (DSM) or the International Classification of Diseases, 11th edition, appears essential from a clinical, research, epidemiological, administrative, economic and political level. However, such diagnostic systems have shortcomings in terms of validity, little consideration of comorbidities and strong intra-class heterogeneity. On a structural level, the operationalization of its criteria is based on a reliability which has been defined a posteriori and which does not lead to improving the validity of the diagnosis but rather to the reification of the diagnostic categories. METHODS: First published in its current form in 2017, the Hierarchical Taxonomy of Psychopathology (HiTOP) constitutes a nosological alternative based on statistics. It conceptualizes psychopathology as a set of hierarchical dimensions, i.e. in "transdiagnostic" continua. The HiTOP is structured according to super-spectra, spectra, sub-factors, syndromes, components and symptoms. This comes from the current dimensional psychology and quantitative nosology. This article describes the basic principles of the HiTOP project and its potential to integrate into clinical and psychiatric research based on its advantages and limitations. RESULTS: Unlike the DSM, which is descriptive and categorical, the HiTOP is first a dimensional classification. This dimensionality describes psychiatric phenomena on continua, each dimension providing a diagnostic continuum to situate a clinical patient. This dimensionality avoids the reification of categories and it limits the dichotomy between normal and pathological. In addition, HiTOP shows a hierarchical structure: vertical refinement of dimensions allows to circumvent the problem of comorbidities, proposes a new conception of etiopathogenic mechanisms, and improves management of care. DISCUSSION: Thus, we provide an illustration of the applications of a dimensional and hierarchical classification in current clinical practice and scientific research, compared to traditional nosology. The challenges of the HiTOP arise in terms of validity, i.e. in the relation of dimensions with physiopathological mechanisms, in clinical terms, i.e. in the potential contribution of dimensions in relation to categories. Moreover, methodological challenges will be important given the inherent limitations of the HiTOP. CONCLUSION: The HiTOP allows to examine the conceptualization of psychiatric disorders, the search for explanatory mechanisms, and treatment from another perspective for psychiatry.

[Risk factors and the issue of the boundary between the normal and the pathological: an epistemological analysis]. / Les facteurs de risque et le problème de la démarcation entre le normal et le pathologique : une analyse épistémologique.

The fact that numerous risk factors are continuous raises the problem of the boundary between the normal and the pathological states. Moreover, a risk factor approach to disease deeply calls into question the relevance of these concepts. In this paper, I propose an epistemological analysis of the notion of "continuity" as it is used in the context of "risk factor epidemiology".

[Cohort study and multivariate analysis: an epistemological and historical analysis of the Framingham heart study]. / Enquête de cohorte et analyse multivariée : une analyse épistémologique et historique du rôle fondateur de l'étude de Framingham.

BACKGROUND: Begun in 1947 and still ongoing, the epidemiological study of heart disease known as the Framingham study was one of the first prospective studies based on a large cohort and has rapidly been considered as the prototype and model for the cohort study. Nevertheless, an examination of its history reveals that the protocol does not at all correspond to today's standards for this type of study. How, then, can we account for the remarkable reputation of this study? METHODS: This paper consists in an epistemological and historical analysis of the Framingham study that provides some of the answers to this question. In my treatment of the study's methodology, I focus on the issue of how the study population was constituted, and the manner in which the multiple factor analyses were conducted, two issues that are now central to cohort studies and more generally to analytic epidemiology. RESULTS: I show how the study population of Framingham and its long-term follow-up have contributed significantly to the interpretation of the cohort as a sort of "population-laboratory". The data generated by this study, which have been very widely used by epidemiologists and other researchers, are unparalleled in terms of the amount of detailed clinical information available for such a long follow-up period. Furthermore, multivariate statistical modelling, which has become a standard statistical tool for clinical as well as epidemiological studies was introduced in the context of this study to improve the identification of significant factors in the simultaneous analysis of multiple correlations. Multivariate analysis has since proved crucial in shaping the epidemiological concept of "risk factor" and in analysing multifactorial diseases. Indeed, I suggest that the modern idea of multifactorial diseases depends on the adaptation of this statistical method. CONCLUSION: Thus, the Framingham study played a leading role not only in remodelling epidemiology after Second World War, in particular because of its contribution to the establishment of the cohort study as a standard method of investigation in etiological research, but also in constituting the "risk factor approach" to disease.

Field demonstration of pervaporation for the separation of volatile organic compounds from a surfactant-based soil remediation fluid.

As part of a Department of Defense project, the US Environmental Protection Agency was responsible for designing, building and field operating a pilot-scale pervaporation unit. The field site was an active dry cleaning facility on the grounds of Marine Corps Base Camp Lejeune in Jacksonville, NC. The overall goal of the project was to remove tetrachloroethylene (PCE) from the soil beneath the dry cleaning shop using a surfactant-based soil remediation fluid and to recycle/reuse the surfactant. In order to reinject the recovered surfactant, the pervaporation unit was required to achieve an average 95% removal of contaminants from the extracted fluid over the duration of the test period. PCE removal averaged 95.8% during peak surfactant levels and exceeded 99.9% in the absence of surfactant, thereby meeting the reinjection requirement. Removal of a group of secondary contaminants at the site, termed Varsol compounds, was monitored via concentrations of three Varsol marker compounds: decane, undecane and 1,3,5-trimethylbenzene. The pervaporation system processed 100,000 gal of groundwater and surfactant solution over a period of 70 days. In order to evaluate and validate process performance, a variety of process variables and properties were monitored over the course of the demonstration. Pervaporation costs are projected to be on the order of $20 per 1000 gal of surfactant solution treated for a moderate size system (10 gpm).

[The outpatient clinic's role : port of entrance and dispatching centre ?]. / La clinique externe : comment peut-elle prétendre aux rôles de porte d'entrée et de plaque tournante?

This article presents an overview of the plan of transformation of services adopted in 1998 at Louis-H. Lafontaine Hospital. The authors present the history, principles and models, the role played by outpatients clinics as well as the challenges related to the implementation of this new plan.

Chronic recurrent multifocal osteomyelitis causing spinal cord compression.

Chronic recurrent multifocal osteomyelitis (CRMO) is a very rare condition of unknown etiology and most commonly occurs during childhood or adolescence. The purpose of this paper is to present a case of CRMO in a vertebral location with severe kyphosis, spinal cord compression, and neurological dysfunction requiring anterior decompression and fusion. After 12 weeks, the patient was physically able to return to school. At 2-year follow-up, neurological and functional outcomes are fair. Magnetic resonance imaging shows good restoration of the sagittal spine alignment despite residual mild kyphosis, and restoration of a normal sagittal diameter of the spinal canal.

Ipsilateral omovertebral bones in the levator scapulae muscle and the rhomboid muscle in a Sprengel deformity: case report.

Our interest was stimulated by the uncommon case of a 4-year-old girl who presented a Sprengel deformity associated with two omovertebral bones on the same side. The first omovertebral bone was situated in the levator scapulae muscle and the second omovertebral bone was lying in the rhomboid muscle. The removal of these two bones was combined with a Woodward procedure to obtain a good correction.

The human glycinamide ribonucleotide transformylase domain: purification, characterization, and kinetic mechanism.

Glycinamide ribonucleotide transformylase catalyzes the third reaction of de novo purine biosynthesis, namely, the conversion of glycinamide ribonucleotide to N-formylglycinamide ribonucleotide, with concomitant conversion of 10-formyltetrahydrofolate to tetrahydrofolate. This activity has been shown to be a target for cancer chemotherapy, which has generated renewed interest in both the enzyme and the pathway. Moreover, in higher eukaryotes this activity constitutes the C-terminal domain of a monomeric protein which also catalyzes two additional reactions of de novo purine biosynthesis. In this study, the human glycinamide ribonucleotide transformylase domain has been expressed to high levels in Escherichia coli and purified to homogeneity. Our improved expression-purification system produces the desired activity exclusively in a soluble form and in higher abundance than previously achieved. The kinetic constants have been determined and the kinetic mechanism has been established as ordered-sequential, with the folate substrate binding first. The correspondence of these data to those obtained for the glycinamide ribonucleotide transformylase activity of the mammalian trifunctional enzyme indicates that the recombinant enzyme is fully functional.

Importance of the dimer-dimer interface for allosteric signal transduction and AMP cooperativity of pig kidney fructose-1,6-bisphosphatase. Site-specific mutagenesis studies of Glu-192 and Asp-187 residues on the 190's loop.

The role of the 190's loop of fructose-1,6-bisphosphatase (Fru-1, 6-P2ase) in the allosteric regulation of Fru-1,6-P2ase has been investigated through kinetic studies on three mutant enzymes, Glu-192 --> Ala, Glu-192 --> Gln, and Asp-187 --> Ala. AMP is an allosteric inhibitor, which binds to the regulatory sites and induces the R- to T-state transition; for wild-type Fru-1,6-P2ase AMP inhibition is cooperative with a Hill coefficient of 2.0. The replacement of Asp-187, which forms an interaction across the C1:C2 monomer-monomer interface, with alanine did not change the catalytic efficiency, and it had no effect on the cooperativity of AMP inhibition; however, the apparent dissociation constant for AMP increased more than 4-fold as compared to the value for the wild-type enzyme. The replacement of Glu-192, which forms interactions across the C1:C4 dimer-dimer interface, with Ala and Gln lowered kcat from 21 s-1 for wild-type enzyme to 15 s-1 and 13 s-1, respectively, for the mutant enzymes, while their respective Km values were not changed. However, these replacements did have dramatic effects on AMP inhibition; first, cooperative AMP inhibition was lost; second, the AMP inhibition was biphasic, which can be interpreted as due to AMP binding to two classes of binding sites. The high affinity class of sites corresponds to the regulatory sites, while the low affinity class of sites may be the active sites. The results reported here, combined with the structural and kinetic results from the Lys-42 --> Ala enzyme, strongly suggest that the C1:C4 dimer-dimer interface, rather than the C1:C2 monomer-monomer interface, is critical for the propagation of the allosteric signal between the AMP sites on different subunits; in addition, cooperative AMP inhibition is essential for the enzyme to be fully inhibited by the binding of AMP to the allosteric site.

[Anterior spinal fusion by thoracoscopy. A non-traumatic technique]. / La spondylodèse antérieure sous thoracoscopie: une technique atraumatique.

PURPOSE OF THE STUDY: Video assisted thoracic surgery (VATS) is a new modality which allows visualization of, and access to the intrathoracic organs without thoracotomy. Recently, this technique has been used for anterior thoracic spine approach to perform surgery which previously required standard postero-lateral thoracotomy. The authors report their initial experience of anterior spinal fusion using thoracoscopy and give a detailed description of their surgical procedure. MATERIAL AND METHODS: This technique, started on June 1993, was performed only in one level 1 in 10 patients who had thoracic spine trauma with fracture or luxation. The procedure was performed in the lateral decubitus position. The patient was prepared in the standard manner for a full thoracotomy. Surgical instruments that are needed for conversion to an open procedure must be in the operative room. Ventilation was stopped to the ipsilateral lung. Lung's collapse of the surgical side was obtained with a double lumen tube. Carbon dioxide (CO2) insufflation was used to further collapse. The first thoracoscopic portal was placed through the sixth or seventh intercostal space in the posterior axillary line, which was the safest place. All subsequent portals were placed under thoracoscopic visualization, in a triangular way as recommended by Landreneau (1992). Only open trocars were used to avoid complication of CO2 insufflation. Once the target level has been defined, a needle was placed into the disc space and roentgenographic confirmation obtained. The parietal pleura was then divided using monopolar electrocautery. Segmental vessels of the operation field lied transversely across the midportion of the vertebral body. They were mobilised and systematically ligated with endoscopic clip to simplify the procedure. Then the intervertebral space was opened and bone and disc were removed, restricted to the anterior and middle third. The graft was placed into the thoracic cavity by using a high density calcium hydroxyapatite ceramic block. Peroperative radiologic control ascertained the good position of the implant. At the end of the procedure a chest tube was placed through the lower trocar site and the lung re-expanded. A post operative CT Scan controlled good position of the graft and complete lung expansion. Contra-indications for VATS are previous surgical procedures or empyema causing extensive pleural adhesions. Procedures not appropriate for VATS approach are some that require anterior instrumentation for stabilisation, burst fracture, or fracture with posterior wall involved. RESULTS: The planned procedure was accomplished in all but one patient who required conversion to an open procedure because of segmental artery bleeding. Mean operative time was 1 h 45 mm, and mean estimated blood loss was 650 cc. There was no complication from CO2 insufflation neither postoperative complication. With an average of 2 years follow up, anterior grafting is as good as an open technique, radiologic evaluation according to Uchida (1990) showed good incorporation of each block without any radiolucent line or displacement. DISCUSSION: According to literature this technique was performed safely in 10 cases, especially without any respiratory complications and chronic pain (impairement of pulmonary function, re-expansion failure, incisional complications, rib fractures, chronic pain and malfunction of the chest wall, limitation of shoulder girdle motion) which are considered to be the main disadvantage of traditional thoracotomy. Many authors previously used VATS for multi level thoracic discectomy for correction of spinal deformities (Mack 1995), spinal reconstructive surgery (Mac Afee 1995) or removal of protrude thoracic disc (Rosenthal 1994). CONCLUSION: This original technique demonstrates that thoracoscopy for anterior thoracic surgery is better for the patients, reducing surgical trauma of the chest wall and to the lung parenchyma (in term of post operative comfort, sh

Brachialis arteria rupture following closed posterior elbow dislocation. Case report and literature review.

Although isolated posterior closed elbow dislocation occurs frequently, associated disrupted brachialis arteria is an uncommon complication which is rarely encountered. The rarity of this complication makes adequate management of this injury controversial, but arterial reconstruction remains the treatment of choice and is recommended despite reports of success following simple arterial ligation. Complications such as intermittent claudication or gangrene of the hand are possible if brachial artery flow is not restored.

Evidence for an active T-state pig kidney fructose 1,6-bisphosphatase: interface residue Lys-42 is important for allosteric inhibition and AMP cooperativity.

During the R-->T transition in the tetrameric pig kidney fructose-1,6-bisphosphatase (Fru-1,6-P2ase, EC 3.1.3.11) a major change in the quaternary structure of the enzyme occurs that is induced by the binding of the allosteric inhibitor AMP (Ke HM, Liang JY, Zhang Y, Lipscomb WN, 1991, Biochemistry 30:4412-4420). The change in quaternary structure involving the rotation of the upper dimer by 17 degrees relative to the lower dimer is coupled to a series of structural changes on the secondary and tertiary levels. The structural data indicate that Lys-42 is involved in a complex set of intersubunit interactions across the dimer-dimer interface with residues of the 190's loop, a loop located at the pivot of the allosteric rotation. In order to test the function of Lys-42, we have replaced it with alanine using site-specific mutagenesis. The kcat and K(m) values for Lys-42-->Ala Fru-1,6-P2ase were 11 s-1 and 3.3 microM, respectively, resulting in a mutant enzyme that was slightly less efficient catalytically than the normal pig kidney enzyme. Although the Lys-42-->Ala Fru-1,6-P2ase was similar kinetically in terms of K(m) and kcat, the response to inhibition by AMP was significantly different than that of the normal pig kidney enzyme. Not only was AMP inhibition no longer cooperative, but also it occurred in two stages, corresponding to high- and low-affinity binding sites. Saturation of the high-affinity sites only reduced the activity by 30%, compared to 100% for the wild-type enzyme. In order to determine in what structural state the enzyme was after saturation of the high-affinity sites, the Lys-42-->Ala enzyme was crystallized in the presence of Mn2+, fructose-6-phosphate (Fru-6-P), and 100 microM AMP and the data collected to 2.3 A resolution. The X-ray structure showed the T state with AMP binding with full occupancy to the four regulatory sites and the inhibitor Fru-6-P bound at the active sites. The results reported here suggest that, in the normal pig kidney enzyme, the interactions between Lys-42 and residues of the 190's loop, are important for propagation of AMP cooperativity to the adjacent subunit across the dimer-dimer interface as opposed to the monomer-monomer interface, and suggest that AMP cooperativity is necessary for full allosteric inhibition by AMP.

Crystal structures of the active site mutant (Arg-243-->Ala) in the T and R allosteric states of pig kidney fructose-1,6-bisphosphatase expressed in Escherichia coli.

The active site of pig kidney fructose-1,6-bisphosphatase (EC 3.1.3.11) is shared between subunits, Arg-243 of one chain interacting with fructose-1,6-bisphosphate or fructose-2,6-bisphosphate in the active site of an adjacent chain. In this study, we present the X-ray structures of the mutant version of the enzyme with Arg-243 replaced by alanine, crystallized in both T and R allosteric states. Kinetic characteristics of the altered enzyme showed the magnesium binding and inhibition by AMP differed slightly; affinity for the substrate fructose-1,6-bisphosphate was reduced 10-fold and affinity for the inhibitor fructose-2,6-bisphosphate was reduced 1,000-fold (Giroux E, Williams MK, Kantrowitz ER, 1994, J Biol Chem 269:31404-31409). The X-ray structures show no major changes in the organization of the active site compared with wild-type enzyme, and the structures confirm predictions of molecular dynamics simulations involving Lys-269 and Lys-274. Comparison of two independent models of the T form structures have revealed small but significant changes in the conformation of the bound AMP molecules and small reorganization of the active site correlated with the presence of the inhibitor. The differences in kinetic properties of the mutant enzyme indicate the key importance of Arg-243 in the function of fructose-1,6-bisphosphatase. Calculations using the X-ray structures of the Arg-243-->Ala enzyme suggest that the role of Arg-243 in the wild-type enzyme is predominantly electrostatic in nature.

Glutamic acid residue 98 is critical for catalysis in pig kidney fructose-1,6-bisphosphatase.

Site-specific mutagenesis has been used to replace Glu-98 with flutamine in pig kidney fructose-1,6-bisphosphatase (Fru-1,6-P(2)ase) in order to evaluate the role of this residue in catalysis. The combination of lower k(cat) and higher K(m) resulted in an approximately 12,000-fold reduction in the catalytic efficiency of the Glu-98-->Gln enzyme when compared to the wild-type enzyme. The affinity of the enzyme for Mg(2+) and for the allosteric inhibitor AMP was altered only slightly; however, cooperativity in the binding of both of these effectors was eliminated. In addition, AMP could not fully inhibit the Glu-98-->Gln enzyme. These data suggest a critical role for the carboxylate of Glu-98 both as a general base in the reaction, and in the mechanism of allosteric inhibition of the enzyme by AMP.

Fructose-1,6-bisphosphatase: arginine-22 is involved in stabilization of the T allosteric state.

A comparison of the X-ray crystallographic structures of the R and T allosteric states [Ke, H. M., Liang, J.-Y., Zhang, Y., & Lipscomb, W. N. (1991) Biochemistry 30, 4412-4420] of the pig kidney fructose-1,6-bisphosphatase (EC 3.1.3.11) reveals major changes in the quaternary structure of the enzyme upon the binding of the allosteric inhibitor AMP. This change in quaternary structure involves the breaking of one set of interactions that stabilize the R state and the formation of another set of interactions that stabilize the T state of the enzyme. In particular, the interactions of Arg-22 with nearby amino acid residues are quite different in the R and T states of the enzyme. Although the crystallographic data suggest that intersubunit interactions such as those involving Arg-22 are important for stabilization of the R and/or T states, the X-ray structures do not provide direct evidence concerning the functional role of specific amino acid residues. Therefore, site-specific mutagenesis has been used to probe the function of Arg-22 in pig kidney fructose-1,6-bisphosphatase. The replacement of Arg-22 by Ala results in a mutant enzyme with enhanced catalytic efficiency compared to the wild-type, as indicated by a kinetic analysis showing a slightly lower Km and increased Vmax compared to the wild-type enzyme. In addition, the substitution enhances both substrate inhibition and the affinity of the inhibitor fructose 2,6-bisphosphate. Moreover, the replacement of Arg-22 by Ala results in a more than 10-fold loss of the ability of AMP to inhibit the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Shared active sites of fructose-1,6-bisphosphatase. Arginine 243 mediates substrate binding and fructose 2,6-bisphosphate inhibition.

The active site of pig kidney fructose-1,6-bisphosphatase (EC 3.1.3.11) is shared between subunits, Arg-243 of one chain interacting with fructose-1,6-bisphosphate or fructose-2,6-bisphosphate in the active site of an adjacent chain. In this study, Arg-243 was replaced by alanine using techniques of site-specific mutagenesis and the cloned pig kidney enzyme expressed in Escherichia coli. Compared with wild-type enzyme, kinetic parameters of the altered enzyme characterizing catalytic efficiency, magnesium binding, and inhibition by AMP differed but by less than an order of magnitude; affinity for substrate fructose 1,6-bisphosphate was 10-fold poorer, and affinity for inhibitor fructose 2,6-bisphosphate was 1000-fold poorer. Molecular dynamics simulations were undertaken to determine possible alterations in active sites of the enzyme due to replacement of Arg-243 by Ala and suggested that in the mutant enzyme loss of one cationic group leads to reorganization of the active site especially involving lysine residues 269 and 274. The differences in properties of the mutant enzyme indicate the key importance of Arg-243 in the function of fructose-1,6-bisphosphatase and confirm on a functional basis the shared active site in this important metabolic enzyme.

Characterization of a dual inhibitor of angiotensin I-converting enzyme and neutral endopeptidase.

In the present study we characterize key activities of an agent designed to simultaneously inhibit angiotensin I-converting enzyme (ACE) and neutral endopeptidase (NEP). MDL 100,240 is a thioester prodrug of MDL 100,173, which is a potent competitive inhibitor of both ACE and NEP in vitro. MDL 100,240 was shown in an ex vivo study to inhibit both of these enzymes in rat kidney. When administered to anesthetized rats, MDL 100,240 enhanced the effect of infused ANP on blood pressure, diuresis and natriuresis and of infused bradykinin on blood pressure. Moreover, MDL 100,173 and MDL 100,240 inhibited the pressor response to angiotensin I. These results indicate that MDL 100,173 and its prodrug, MDL 100,240, produced effects, in vivo, consistent with inhibition of both ACE and NEP.

Inhibition of lipid peroxidation promoted by iron(III) and ascorbate.

Peroxidation of rat liver microsomes and of phospholipid isolated from them was studied using iron(III) and ascorbate initiation. One-half equivalent of citrate per iron equivalent maintained solubility of the metal ion at neutral pH. Several metal chelators, including additional citrate, blocked peroxidation, but catalase did not. These characteristics are consistent with those reported by others (D. M. Miller and S. D. Aust (1989) Arch. Biochem. Biophys. 271, 113-119). Several antioxidants, principally tocopherol analogues and nitroxides, and, as well, a nonenzymatic component of "thymol-free" catalase, potently blocked lipid peroxidation, or, equivalently, dioxygen depletion from suspensions of peroxidizing microsomes. Chromanols were the most active antioxidants. No thiol studied had significant antioxidant activity in the test system.