Nosocomial outbreak of monoclonal VIM carbapenemase-producing Enterobacter cloacae complex in an intensive care unit during the COVID-19 pandemic: an integrated approach.

BACKGROUND: An outbreak of VIM carbapenemase-expressing Enterobacter cloacae complex occurred between March and October 2020 in an intensive care unit (ICU) of a tertiary care and teaching hospital in France. At the same time, the hospital was facing the COVID-19 first wave. AIM: To describe the management of an outbreak caused by a VIM-producing Enterobacter cloacae complex strain during the COVID-19 pandemic in an ICU and to show the importance of an integrated approach. METHODS: A multi-focal investigation was conducted including descriptive and molecular epidemiology, environmental screening, and assessment of infection prevention and control measures. FINDINGS: A total of 14 cases were identified in this outbreak with a high attributable mortality rate (85.7%). The outbreak management was coordinated by a crisis cell, and involved the implementation of multi-disciplinary actions such as: enhanced hygiene measures, microbiological and molecular analysis of patients and environmental E. cloacae complex strains, and simulation-based teaching. All 23 E. cloacae complex strains isolated from patients and environment samples belonged to multi-locus sequence type ST78 and carried bla-VIM4 gene. Using Fourier transform infrared spectroscopy, all but two isolates were also found to belong to a single cluster. Although the source of this outbreak could not be pinpointed, the spread of the strain was controlled thanks to this multi-focal approach and multi-disciplinary implementation. CONCLUSION: This investigation highlighted the usefulness of Fourier transform infra-red spectroscopy in the rapid typing of outbreak strains as well as the importance of an integrated approach to successfully fight against multidrug-resistant micro-organism dissemination and healthcare-associated infections.

Myogenic Disease and Metabolic Acidosis: Consider Multiple Acyl-Coenzyme A Dehydrogenase Deficiency.

BACKGROUND: Multiple acyl-coA dehydrogenase deficiency (MADD) is a rare, inherited, autosomal-recessive disorder leading to the accumulation of acylcarnitine of all chain lengths. Acute decompensation with cardiac, respiratory or hepatic failure and metabolic abnormalities may be life-threatening. CASE PRESENTATION: A 29-year-old woman presented with severe lactic acidosis associated with intense myalgia and muscle weakness. The clinical examination revealed symmetric upper and lower limb motor impairment (rated at 2 or 3 out of 5 on the Medical Research Council scale) and clear amyotrophy. Laboratory tests had revealed severe rhabdomyolysis, with a serum creatine phosphokinase level of 8,700 IU/L and asymptomatic hypoglycemia in the absence of ketosis. Electromyography revealed myotonic bursts in all four limbs. The absence of myositis-specific autoantibodies ruled out a diagnosis of autoimmune myositis. Finally, Acylcarnitine profile and gas chromatography-mass spectrometry analysis of organic acids led to the diagnosis of MADD. A treatment based on the intravenous infusion of glucose solutes, administration of riboflavin, and supplementation with coenzyme Q10 and carnitine was effective. Lipid consumption was strictly prohibited in the early stages of treatment. The clinical and biochemical parameters rapidly improved and we noticed a complete disappearance of the motor deficit, without sequelae. CONCLUSION: A diagnosis of MADD must be considered whenever acute or chronic muscle involvement is associated with metabolic disorders. Acute heart, respiratory or hepatic failure and metabolic abnormalities caused by MADD may be life-threatening, and will require intensive care.

The onset of left ventricular diastolic dysfunction in SHR rats is not related to hypertrophy or hypertension.

Left ventricular (LV) diastolic dysfunction, particularly relaxation abnormalities, are known to be associated with the development of LV hypertrophy (LVH). Preliminary human and animal studies suggested that early LV diastolic dysfunction may be revealed independently of LVH. However, whether LV diastolic dysfunction is compromised before the onset of hypertension and LVH remains unknown. We therefore evaluated LV diastolic function in spontaneously hypertensive rats (SHR) at different ages and tested whether LV diastolic dysfunction is associated with abnormal intracellular calcium homeostasis. LV systolic and diastolic functions were evaluated by invasive and echocardiographic methods in 3-week-old (without hypertension) and 5-week-old (with hypertension) SHR and Wistar-Kyoto control rats. Basal intracytoplasmic calcium and sarcoplasmic reticulum (SR) Ca(2+) contents were measured in cardiomyocytes using fura-2 AM. Sarco(endo)plasmic Ca(2+)-ATPase isoform 2a (SERCA 2a) and phospholamban (PLB) expressions were quantified by Western blot and quantitative RT-PCR techniques. LV relaxation dysfunction was observed in 3-week-old SHR rats before onset of hypertension and LVH. An increase in basal intracytoplasmic Ca(2+) and a decrease in SR Ca(2+) release were demonstrated in SHR. Decreased expression of SERCA 2a and Ser16 PLB (p16-PLB) protein levels was also observed in SHR rats, whereas mRNA expression was not decreased. For the first time, we have shown that LV myocardial dysfunction precedes hypertension in 3-week-old SHR rats. This LV myocardial dysfunction was associated with high diastolic [Ca(2+)](i) possibly due to decreased SERCA 2a and p16-PLB protein levels. Diastolic dysfunction may be a potential predictive marker of arterial hypertension in genetic hypertension syndromes.

Effect of intra-abdominal hypertension on left ventricular relaxation: a preliminary animal study.

BACKGROUND: In the intensive care unit, intra-abdominal hypertension (IAH) is a frequently encountered, life-threatening condition. The aim of this animal study was to evaluate the effect of IAH on left ventricular (LV) relaxation (i.e. the active phase of diastole). METHODS: Seven male rabbits were anaesthetized before mechanical ventilation. A 20 mm Hg increase in intra-abdominal pressure (IAP) was then induced by intraperitoneal infusion of 1.5% glycine solution. Haemodynamic parameters were recorded and the relaxation time constant tau (considered to be the best index of left ventricle relaxation) was calculated. All haemodynamic measurements were recorded at baseline and then after induction of IAH. RESULTS: A 20 mm Hg increase in IAP was not followed by a significant change in arterial pressure, but was associated with increases in central venous pressure (from 2 [-2 to 6] to 7 [-2 to 12] mm Hg, P= 0.03), LV end-diastolic pressure (from 7 [6-8] to 15 [11-19] mm Hg, P= 0.04) and the relaxation time constant tau (from 16 [14-18] to 43 [34-52] ms, P= 0.048). CONCLUSIONS: In this animal study, a 20 mm Hg increase in IAP impaired LV relaxation. Further studies are necessary to identify the causes of this impairment.

Pathophysiological mechanisms and consequences of cardiovascular calcifications: role of uremic toxicity.

SUMMARY: Chronic kidney disease (CKD) represents an accelerated model of the active cardiovascular calcification process. Recent data from our laboratory indicate the presence of a possible vascular remodeling leading to vascular calcification similar to that observed in bone tissue, and emphasize the role of uremic toxicity. Uremic serum not only induces differentiation of smooth muscle cells into an osteoblast-like phenotype but also inhibits the differentiation of monocyte-macrophages cells into osteoclasts. The imbalance between the two processes in vascular walls in favor of osteoblast-like formation could lead to calcification. Cardiovascular calcification may contribute to the high rate of cardiovascular disease in patients with CKD. However, uremic toxicity, which participates in the pathogenesis of cardiovascular calcification, seems to have independent effects on vascular walls, at least in the early stages of CKD. We recently reported that functional (i.e. endothelial dysfunction) rather than structural changes, including vascular calcification, may contribute to the aortic hemodynamic changes observed during early CKD. Uremic toxicity also appears to be associated with calcification of intracranial arteries. Knowledge concerning the pathogenesis and consequences of cardiovascular calcification derived from the uremic model therefore opens up new perspectives for pharmacologic treatments that may also help to prevent and/or treat cardiovascular calcification, and consequently cardiovascular mortality and morbidity, not only in CKD patients but also in the general population.

Data mining of imperfect double-stranded RNA in 3' untranslated regions of eukaryotic mRNAs.

Recent developments in the study of RNA silencing indicate that double-stranded RNA (dsRNA) can be used in eukaryotes to block expression of a corresponding cellular gene. There is also a large class of small non-coding RNAs having potential to form a distinct, stable stem-loop in numbers of eukaryotic genomes. We had reported that a large imperfect dsRNA structure with hundreds of base-pairs (bp) in the 3' untranslated region (3' UTR) of cytotoxic ribonuclease was correlated with the translation suppression. In this study, we search for such dsRNAs in a 3' UTR database. The occurrence rate of large dsRNA in 3' UTRs ranges from 0.01% in plant to 0.30% in vertebrate mRNAs. However, small imperfect dsRNAs of ~ 30 bp are much more prevalent than large ones. The small dsRNAs are statistically very significant and uniquely well-ordered. Most of them have the conserved structural features of pre-miRNAs. Our data mining of the dsRNAs in the 3' UTR database can be used to explore RNA-based regulation of gene expression.

Pleiotropic effects of the non-calcium phosphate binder sevelamer.

The number of chronic kidney disease (CKD) patients and related adverse outcomes has dramatically increased worldwide in the past decade. Therefore, numerous experimental and clinical studies have recently addressed the underlying mechanisms, in particular the marked increase in cardiovascular mortality. Hyperphosphatemia is a major problem in these patients with advanced stage of CKD. Its control by calcium-containing phosphate binders is effective, but at the price of potentially noxious calcium overload. Sevelamer hydrochloride is a phosphate binder that offers an effective control of hyperphosphatemia as calcium-rich binders but without increase of calcium load. Beyond the control of phosphate, sevelamer seems to exert pleiotropic effects which include the correction of lipid abnormalities and the clearance of some uremic toxins.

Short-strong hydrogen bonds and a low barrier transition state for the proton transfer reaction in RNase A catalysis: a quantum chemical study.

There is growing evidence that some enzymes catalyze reactions through the formation of short-strong hydrogen bonds as first suggested by Gerlt and Gassman. Support comes from several experimental and quantum chemical studies that include correlation energies on model systems. In the present study, the process of proton transfer between hydroxyl and imidazole groups, a model of the crucial step in the hydrolysis of RNA by the enzymes of the RNase A family, is investigated at the quantum mechanical level of density functional theory and perturbation theory at the MP2 level. The model focuses on the nature of the formation of a complex between the important residues of the protein and the hydroxyl group of the substrate. We have also investigated different configurations of the ground state that are important in the proton transfer reaction. The nature of bonding between the catalytic unit of the enzyme and the substrate in the model is investigated by Bader's atoms in molecule theory. The contributions of solvation and vibrational energies corresponding to the reactant, the transition state and the product configurations are also evaluated. Furthermore, the effect of protein environment is investigated by considering the catalytic unit surrounded by complete proteins--RNase A and Angiogenin. The results, in general, indicate the formation of a short-strong hydrogen bond and the formation of a low barrier transition state for the proton transfer model of the enzyme.

Anatomy of protein structures: visualizing how a one-dimensional protein chain folds into a three-dimensional shape.

Here, we depict the anatomy of protein structures in terms of the protein folding process. Via an iterative, top-down dissecting procedure, tertiary structures are spliced down to reveal their anatomy: first, to produce domains (defined by visual three-dimensional inspection criteria); then, hydrophobic folding units (HFU); and, at the end of a multilevel process, a set of building blocks. The resulting anatomy tree organization not only clearly depicts the organization of a one-dimensional polypeptide chain in three-dimensional space but also straightforwardly describes the most likely folding pathway(s). Comparison of the tree with the formation of the hydrophobic folding units through combinatorial assembly of the building blocks illustrates how the chain folds in a sequential or a complex folding pathway. Further, the tree points to the kinetics of the folding, whether the chain is a fast or a slow folder, and the probability of misfolding. Our ability to successfully dissect the protein into an anatomy tree illustrates that protein folding is a hierarchical process and further validates a building blocks protein folding model.

A gender-specific mRNA encoding a cytotoxic ribonuclease contains a 3' UTR of unusual length and structure.

A cDNA (2855 nt) encoding a putative cytotoxic ribonuclease (rapLR1) related to the antitumor protein onconase was cloned from a library derived from the liver of gravid female amphibian Rana pipiens. The cDNA was mainly comprised (83%) of 3' untranslated region (UTR). Secondary structure analysis predicted two unusual folding regions (UFRs) in the RNA 3' UTR. Two of these regions (711-1442 and 1877-2130 nt) contained remarkable, stalk-like, stem-loop structures greater than 38 and 12 standard deviations more stable than by chance, respectively. Secondary structure modeling demonstrated similar structures in the 3' UTRs of other species at low frequencies (0.01-0.3%). The size of the rapLR1 cDNA corresponded to the major hybridizing RNA cross-reactive with a genomic clone encoding onconase (3.6 kb). The transcript was found only in liver mRNA from female frogs. In contrast, immunoreactive onconase protein was detected only in oocytes. Deletion of the 3' UTR facilitated the in vitro translation of the rapLR1 cDNA. Taken together these results suggest that these unusual UFRs may affect mRNA metabolism and/or translation.

Prediction of common secondary structures of RNAs: a genetic algorithm approach.

In this study we apply a genetic algorithm to a set of RNA sequences to find common RNA secondary structures. Our method is a three-step procedure. At the first stage of the procedure for each sequence, a genetic algorithm is used to optimize the structures in a population to a certain degree of stability. In this step, the free energy of a structure is the fitness criterion for the algorithm. Next, for each structure, we define a measure of structural conservation with respect to those in other sequences. We use this measure in a genetic algorithm to improve the structural similarity among sequences for the structures in the population of a sequence. Finally, we select those structures satisfying certain conditions of structural stability and similarity as predicted common structures for a set of RNA sequences. We have obtained satisfactory results from a set of tRNA, 5S rRNA, rev response elements (RRE) of HIV-1 and RRE of HIV-2/SIV, respectively.

Distinguishing between sequential and nonsequentially folded proteins: implications for folding and misfolding.

We describe here an algorithm for distinguishing sequential from nonsequentially folding proteins. Several experiments have recently suggested that most of the proteins that are synthesized in the eukaryotic cell may fold sequentially. This proposed folding mechanism in vivo is particularly advantageous to the organism. In the absence of chaperones, the probability that a sequentially folding protein will misfold is reduced significantly. The problem we address here is devising a procedure that would differentiate between the two types of folding patterns. Footprints of sequential folding may be found in structures where consecutive fragments of the chain interact with each other. In such cases, the folding complexity may be viewed as being lower. On the other hand, higher folding complexity suggests that at least a portion of the polypeptide backbone folds back upon itself to form three-dimensional (3D) interactions with noncontiguous portion(s) of the chain. Hence, we look at the mechanism of folding of the molecule via analysis of its complexity, that is, through the 3D interactions formed by contiguous segments on the polypeptide chain. To computationally splice the structure into consecutively interacting fragments, we either cut it into compact hydrophobic folding units or into a set of hypothetical, transient, highly populated, contiguous fragments ("building blocks" of the structure). In sequential folding, successive building blocks interact with each other from the amino to the carboxy terminus of the polypeptide chain. Consequently, the results of the parsing differentiate between sequentially vs. nonsequentially folded chains. The automated assessment of the folding complexity provides insight into both the likelihood of misfolding and the kinetic folding rate of the given protein. In terms of the funnel free energy landscape theory, a protein that truly follows the mechanism of sequential folding, in principle, encounters smoother free energy barriers. A simple sequentially folded protein should, therefore, be less error prone and fold faster than a protein with a complex folding pattern.

Ion-RNA interactions in the RNA pseudoknot of a ribosomal frameshifting site: molecular modeling studies.

The three-dimensional (3-D) structure of a RNA pseudoknot that causes the efficient ribosomal frameshifting in the gag-pro region of mouse mammary tumor virus (MMTV) has been determined recently by nuclear magnetic resonance (NMR) studies. But since the structure refinement in the studies did not use metal ions and waters, it is not clear how metal ions participate in the stabilization of the pseudoknot, and what kind of ion-RNA interactions dominate in the tertiary contacts for the RNA pseudoknotting. Based on the reported structure data of the pseudoknot VPK of MMTV, we gradually refined the structure by restrained molecular dynamics (MD) using NMR distance restraints. Restrained MD simulation of the RNA pseudoknot was performed with sodium ions and water molecules. Our results are in good agreement with known NMR data and delineate the importance of the metal ion coordination in the stability of the pseudoknot. In the non-coaxially stacking pseudoknot, stem 1 (S1), stem 2 (S2), and the intervening A14 involves unconventional stacking of base pairs coordinated by Na+ and/or bridging water molecules. A6 and G7 of loop L1 make a perfect base stacking in the major groove and are further stabilized by coordinated Na+ ions and water molecules. The first 4-nucleotide (nt) ACUC of loop L2 form a sharp turn and the following 4-nt AAAA cross the minor groove of S1 and are steadied by interactions with the nucleotides of S , bridging water molecules and coordinated Na+ ions. Our studies suggest that the metal ion plays a crucial role in the RNA pseudoknotting of VPK. In the stacking interior of S1 and S2, the Na+ ion is positioned in the major groove and interacts directly with the carbonyl group O6 of G28 and carbonyl group O4 of U13 in the wobble base pair U13:G28. The ion-RNA interactions in MMTV VPK not only stabilize the RNA pseudoknot but also modify the electrostatic properties of the nucleotides at the critical parts of the pseudoknot VPK.

Evolution of a common structural core in the internal ribosome entry sites of picornavirus.

The translational control involving internal ribosome binding occurs in poliovirus (PV), human rhinoviruses (HRV), encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV), and hepatitis A virus (HAV). Internal ribosome binding utilizes cis-acting genetic elements of approximately 450 nucleotides (nt) termed the internal ribosome entry sites (IRES) found in these picornaviral 5'-untranslated region (5'UTR). Although these IRES elements are quite different in their primary sequence, a similar folding structure with a conserved 3' structural core exists in the IRES. Phylogenetic analysis and RNA folding of the 5' UTR of picornaviruses, including PV types 1-3, coxsackievirus types A and B, swine vesicular disease virus, echoviruses, enteroviruses (human and bovine), HRV, HAV, EMCV, mengovirus, Theiler's murine encephalomyelitis viruses, FMDV, and equine rhinoviruses, indicates that the predicted conserved structural core is indeed a general structural feature for all members of the picornavirus family. The evolution of a common structural core likely occurred by the gradual addition or deletion of structural domains and elements to preserve a similar tertiary structure that facilitates the utilization of the IRES in specific host-cell environments.

Phylogenetic evidence for the improved RNA higher-order structure in internal ribosome entry sequences of HCV and pestiviruses.

The strong requirement for a small segment of the 5'-proximal coding sequence of hepatitis C virus (HCV) is one of the most remarkable features in the internal initiation of HCV mRNA translation. Phylogenetic analysis and RNA folding indicate a common RNA structure of the 5' untranslated region (UTR) of HCV and the animal pestiviruses, including HCV types 1-11, bovine viral diarrhea (BVDV), border disease virus (BDV) and hog cholera (HoCV). Although the common RNA structure shares similar features to that proposed for the internal ribosome entry sequence (IRES) of picornavirus, phylogenetic evidence suggests four new tertiary interactions between conserved terminal hairpin loops and between the terminal hairpin loop of F2b and the short coding sequence for HCV and pestiviruses. We suggest that the higher-order structures of IRES cis-acting elements for HCV and animal pestivirus are composed of stem-loop structures B-C, domains E-H, stem-loop structure J and four additional tertiary interactions. The common structure of IRES elements for these viruses forms a compact structure by these tertiary interactions and stem stacking. The active structural core is centered in the junction domain of E-H that is also conserved in all members of picornaviruses. Our model suggests that the requirement for a small segment of the 5' coding sequence is to form the distinct tertiary structure that facilitates the cis-acting function of the HCV IRES in the internal initiation of the translational control.

Analysis of the structure of HIV-1 protease complexed with a hexapeptide inhibitor. Part II: Molecular dynamic studies of the active site region.

Six models of the catalytic site of HIV-1 protease complexed with a reduced peptide inhibitor, MVT-101, were investigated. These studies focused on the details of protonation of the active site, its total net charge and hydrogen bonding pattern, which was consistent with both the observed coplanar configuration of the acidic groups of the catalytic aspartates (Asp-25 and Asp-125) and the observed binding mode of the inhibitor. Molecular dynamic simulations using AMBER 4.0 indicated that the active site should be neutral. The planarity of the aspartate dyad may be due to the formation of two hydrogen bonds: one between the inner O delta 1 oxygen atoms of the two catalytic aspartates and another between the O delta 2 atom of Asp-125 and the nitrogen atom of the reduced peptide bond of the bound inhibitor. This would require two additional protonations, either of both aspartates, or of one Asp and the amido nitrogen atom of Nle-204. Our results favor the Asp-inhibitor protonation but the other one is not excluded. Implications of these findings for the mechanism of enzymatic catalysis are discussed. Dynamic properties of the hydrogen bond network in the active site and an analysis of the interaction energy between the inhibitor and the protease are presented.

A common RNA structural motif involved in the internal initiation of translation of cellular mRNAs.

The 5'-non-translated regions (5'NTR) of human immunoglobulin heavy chain binding protein (BiP), Antennapedia (Antp) ofDrosophilaand human fibroblast growth factor 2 (FGF-2) mRNAs are reported to mediate translation initiation by an internal ribosome binding mechanism. In this study, we investigate predicted features of the higher order structures folded in these 5'NTR sequences. Statistical analyses of RNA folding detected a 92 nt unusual folding region (UFR) from 129 to 220, close to the initiator AUG in the BiP mRNA. Details of the structural analyses show that the UFR forms a Y-type stem-loop structure with an additional stem-loop in the 3'-end resembling the common structure core found in the internal ribosome entry site (IRES) elements of picornavirus. The Y-type structural motif is also conserved among a number of divergent BiP mRNAs. We also find two RNA elements in the 5'-leader sequence of human FGF-2. The first RNA element (96 nt) is 2 nt upstream of the first CUG start codon located in the reported IRES element of human FGF-2. The second (107 nt) is immediately upstream of the authentic initiator AUG of the main open reading frame. Intriguingly, the folded RNA structural motif in the two RNA elements is conserved in other members of FGF family and shares the same structural features as that found in the 5'NTR of divergent BiP mRNAs. We suggest that the common RNA structural motif conserved in the diverse BiP and FGF-2 mRNAs has a general function in the internal ribosome binding mechanism of cellular mRNAs.

A common structural core in the internal ribosome entry sites of picornavirus, hepatitis C virus, and pestivirus.

Cap-independent translations of viral RNAs of enteroviruses and rhinoviruses, cardioviruses and aphthoviruses, hepatitis A and C viruses (HAV and HCV), and pestivirus are initiated by the direct binding of 40S ribosomal subunits to a cis-acting genetic element termed the internal ribosome entry site (IRES) or ribosome landing pad (RLP) in the 5' noncoding region (5'NCR). RNA higher ordered structure models for these IRES elements were derived by a combined approach using thermodynamic RNA folding, Monte Carlo simulation, and phylogenetic comparative analysis. The structural differences among the three groups of picornaviruses arise not only from point mutations, but also from the addition or deletion of structural domains. However, a common core can be identified in the proposed structural models of these IRES elements from enteroviruses and rhinoviruses, cardioviruses and aphthoviruses, and HAV. The common structural core identified within the picornavirus IRES is also conserved in the 5'NCR of the divergent viruses, HCV, and pestiviruses. Furthermore, the proposed structural motif shares a structural feature similar to that observed in the catalytic core of the group 1 intron. The conserved structural motif from these divergent sequences that looks like the common core region of group 1 introns is probably a crucial element involved in the IRES-dependent translation.

Identification of ribosome binding sites in Escherichia coli using neural network models.

This study investigated the use of neural networks in the identification of Escherichia coli ribosome binding sites. The recognition of these sites based on primary sequence data is difficult due to the multiple determinants that define them. Additionally, secondary structure plays a significant role in the determination of the site and this information is difficult to include in the models. Efforts to solve this problem have so far yielded poor results. A new compilation of E. coli ribosome binding sites was generated for this study. Feedforward backpropagation networks were applied to their identification. Perceptrons were also applied, since they have been the previous best method since 1982. Evaluation of performance for all the neural networks and perceptrons was determined by ROC analysis. The neural network provided significant improvement in the recognition of these sites when compared with the previous best method, finding less than half the number of false positives when both models were adjusted to find an equal number of actual sites. The best neural network used an input window of 101 nucleotides and a single hidden layer of 9 units. Both the neural network and the perceptron trained on the new compilation performed better than the original perceptron published by Stormo et al. in 1982.