Thrombomodulin Regulates PTEN/AKT Signaling Axis in Endothelial Cells.

BACKGROUND: We recently demonstrated that deletion of thrombomodulin gene from endothelial cells results in upregulation of proinflammatory phenotype. In this study, we investigated the molecular basis for the altered phenotype in thrombomodulin-deficient (TM-/-) cells. METHODS: Different constructs containing deletions or mutations in the cytoplasmic domain of thrombomodulin were prepared and introduced to TM-/- cells. The phenotype of cells expressing different derivatives of thrombomodulin and tissue samples of thrombomodulin-knockout mice were analyzed for expression of distinct regulatory genes in established signaling assays. RESULTS: The phosphatase and tensin homolog were phosphorylated and its recruitment to the plasma membrane was impaired in TM-/- cells, leading to hyperactivation of AKT (protein kinase B) and phosphorylation-dependent nuclear exclusion of the transcription factor, forkhead box O1. The proliferative/migratory properties of TM-/- cells were enhanced, and cells exhibited hypersensitivity to stimulation by angiopoietin 1 and vascular endothelial growth factor. Reexpression of wild-type thrombomodulin in TM-/- cells normalized the cellular phenotype; however, thrombomodulin lacking its cytoplasmic domain failed to restore the normal phenotype in TM-/- cells. Increased basal permeability and loss of VE-cadherin were restored to normal levels by reexpression of wild-type thrombomodulin but not by a thrombomodulin construct lacking its cytoplasmic domain. A thrombomodulin cytoplasmic domain deletion mutant containing 3-membrane-proximal Arg-Lys-Lys residues restored the barrier-permeability function of TM-/- cells. Enhanced phosphatase and tensin homolog phosphorylation and activation of AKT and mTORC1 (mammalian target of rapamycin complex 1) were also observed in the liver of thrombomodulin-KO mice. CONCLUSIONS: These results suggest that the cytoplasmic domain of thrombomodulin interacts with the actin cytoskeleton and plays a crucial role in regulation of phosphatase and tensin homolog/AKT signaling in endothelial cells.

Clp ATPases differentially affect natural competence development in Streptococcus mutans.

In naturally competent bacteria, DNA transformation through horizontal gene transfer is an evolutionary mechanism to receive extracellular DNA. Bacteria need to maintain a state of competence to accept foreign DNA, and this is an energy-driven phenomenon that is tightly controlled. In Streptococcus, competence development is a complex process that is not fully understood. In this study, we used Streptococcus mutans, an oral bacterium, to determine how cell density affects competence development. We found that in S. mutans the transformation efficiency is maximum when the transforming DNA was added at low cell density and incubated for 2.5 h before selecting for transformants. We also found that S. mutans cells remain competent until the mid-logarithmic phase, after which the competence decreases drastically. Surprisingly, we observed that individual components of Clp proteolytic complexes differentially regulate competence. If the transformation is carried out at the early growth phase, both ClpP protease and ClpX ATPase are needed for competence. In contrast, we found that both ClpC and ClpE negatively affect competence. We also found that if the transformation is carried out at the mid-logarithmic growth phase ClpX is still required for competence, but ClpP negatively affects competence. While the exact reason for this differential effect of ClpP and ClpX on transformation is currently unknown, we found that both ClpC and ClpE have a negative effect on transformation, which was not reported before.

Antibody deposition on vascular endothelial cells contributes to localized inflammation in salivary glands.

BACKGROUND: Oral and ocular dryness due to reduced saliva and tear production, exocrine gland inflammation, and autoantibodies to multiple cellular proteins are the cardinal features of Sjögren's Disease. Among the autoantibody specificities, anti-Ro52 is linked with higher disease severity. We have previously reported that mice immunized with recombinant Ro52 developed IgG deposits in salivary and lacrimal glands and showed reduced saliva and tear production. Furthermore, passive transfer of sera from Ro52-immunized mice rapidly induced glandular dysfunction without immune cell infiltration in recipient mice. METHODS: To identify mechanisms driving antibody-mediated salivary gland dysfunction, hyperimmune rabbit antiserum to mouse Ro52 was passively transferred into NZM2758 female mice, pretreated with alum adjuvant. Alum-pretreated mice given hyperimmune rabbit antiserum to maltose-binding protein served as controls. Antibody deposition and its distribution in the salivary glands were studied by immunofluorescence staining for rabbit IgG, nerve fibers, and endothelial cells. The nCounter inflammation panel was used to determine differentially expressed genes in the salivary gland. RESULTS: Rabbit IgG deposits were detected in salivary glands of anti-Ro52 immune sera recipients. The rabbit IgG was present on the endothelial cells in small blood vessels, and it did not co-localize with nerve fibers. Ingenuity pathway analysis of the gene expression dataset predicted the canonical vascular endothelial growth factor (VEGF) pathway as the most activated and Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) as the most inhibited pathway in the salivary glands of anti-Ro52 sera recipients. CONCLUSION: Our study suggests that autoantibody deposition on salivary gland endothelial cells might play a critical role in the pathogenesis of Sjögren's Disease.

ClpX/P-Dependent Degradation of Novel Substrates in Streptococcus mutans.

Regulated proteolysis is where AAA+ ATPases (ClpX, ClpC, and ClpE) are coupled to a protease subunit (ClpP) to facilitate degradation of misfolded and native regulatory proteins in the cell. The process is intricately linked to protein quality control and homeostasis and modulates several biological processes. In streptococci, regulated proteolysis is vital to various functions, including virulence expression, competence development, bacteriocin production, biofilm formation, and stress responses. Among the various Clp ATPases, ClpX is the major one that recognizes specific amino acid residues in its substrates and delivers them to the ClpP proteolytic chamber for degradation. While multiple ClpX substrates have been identified in Escherichia coli and other bacteria, little is known about the identity of these substrates in streptococci. Here, we used a preliminary proteomic analysis to identify putative ClpX substrates using Streptococcus mutans as a model organism. SMU.961 is one such putative substrate where we identified the Glu-Lue-Gln (ELQ) motif at the C terminus that is recognized by ClpX/P. We identified several other proteins, including MecA, which also harbor ELQ and are degraded by ClpX/P. This is surprising since MecA is known to be degraded by ClpC/P in Bacillus subtilis; however, ClpX/P-mediated MecA degradation is unknown. We also identified Glu and Gln as the crucial residues for ClpX recognition. Our data indicate a species and perhaps strain-specific recognition of ELQ by streptococcal ClpX/P. At present, we do not know whether this species-dependent degradation by ClpX/P is unique to S. mutans, and we are currently examining the phenomenon in other pathogenic streptococci. IMPORTANCE ClpX/P is a major intracellular proteolytic complex that is responsible for protein quality control in the cell. ClpX, an AAA+ ATPase, distinguishes the potential substrates by recognizing short motifs at the C-terminal end of proteins and delivers the substrates for degradation by ClpP protease. The identity of these ClpX substrates, which varies greatly among bacteria, is known only for a few well-studied species. Here, we used Streptococcus mutans as a model organism to identify ClpX substrates. We found that a short motif of three residues is successfully recognized by ClpX/P. Interestingly, the motif is not present at the ultimate C-terminal end; rather it is present close to the end. This result suggests that streptococcal ClpX ATPase can recognize internal motifs.

Involvement of ClpE ATPase in Physiology of Streptococcus mutans.

Streptococcus mutans, a dental pathogen, harbors at least three Clp ATPases (ClpC, ClpE, and ClpX) that form complexes with ClpP protease and participate in regulated proteolysis. Among these, the function of ClpE ATPase is poorly understood. We have utilized an isogenic clpE-deficient strain derived from S. mutans UA159 and evaluated the role of ClpE in cellular physiology. We found that loss of ClpE leads to increased susceptibility against thiol stress but not to oxidative and thermal stress. Furthermore, we found that the mutant displays altered tolerance against some antibiotics and altered biofilm formation. We performed a label-free proteomic analysis by comparing the mutant with the wild-type UA159 strain under nonstressed conditions and found that ClpE modulates a relatively limited proteome in the cell compared to the proteomes modulated by ClpX and ClpP. Nevertheless, we found that ClpE deficiency leads to an overabundance of some cell wall synthesis enzymes, ribosomal proteins, and an unknown protease encoded by SMU.2153. Our proteomic data strongly support some of the stress-related phenotypes that we observed. Our study emphasizes the significance of ClpE in the physiology of S. mutans. IMPORTANCE When bacteria encounter environmental stresses, the expression of various proteins collectively known as heat shock proteins is induced. These heat shock proteins are necessary for cell survival specifically under conditions that induce protein denaturation. A subset of heat shock proteins known as the Clp proteolytic complex is required for the degradation of the misfolded proteins in the cell. The Clp proteolytic complex contains an ATPase and a protease. A specific Clp ATPase, ClpE, is uniquely present in Gram-positive bacteria, including streptococci. Here, we have studied the functional role of the ClpE protein in Streptococcus mutans, a dental pathogen. Our results suggest that ClpE is required for survival under certain antibiotic exposure and stress conditions but not others. Our results demonstrate that loss of ClpE leads to a significantly altered cellular proteome, and the analysis of those changes suggests that ClpE's functions in S. mutans are different from its functions in other Gram-positive bacteria.

Redox Sensing Modulates the Activity of the ComE Response Regulator of Streptococcus mutans.

Streptococcus mutans, a dental pathogen, encodes the ComDE two-component system comprised of a histidine kinase (ComD) and a response regulator (ComE). This system is necessary for production of bacteriocins and development of genetic competence. ComE interacts with its cognate promoters to activate the transcription of bacteriocin and competence-related genes. Previous transcriptomic studies indicated that expressions of bacteriocin genes were upregulated in the presence of oxygen. To understand the relationship between the aerobic condition and bacteriocin expression, we analyzed the S. mutans ComE sequence and its close homologs. Surprisingly, we noticed the presence of cysteine (Cys) residues located at positions 200 and 229, which are highly conserved among the ComE homologs. Here, we investigated the role of Cys residues of S. mutans ComE in the activation of bacteriocin transcription using the PnlmA promoter that expresses bacteriocin NlmA. We constructed both single mutants and double mutants by replacing the Cys residues with serine and performed complementation assays. We observed that the presence of Cys residues is essential for PnlmA activation. With purified ComE mutant proteins, we found that ComE double mutants displayed a nearly 2-fold lower association rate than wild-type ComE. Furthermore, 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence studies indicated that the double mutants displayed wider conformation changes than wild-type ComE. Finally, we demonstrated that close streptococcal ComE homologs successfully activate the PnlmA expression in vivo. This is the first report suggesting that S. mutans ComE and its homologs can sense the oxidation status of the cell, a phenomenon similar to the AgrA system of Staphylococcus aureus but with different outcomes. IMPORTANCE Streptococci are an important species that prefer to grow under anaerobic or microaerophilic environments. Studies have shown that streptococci growth in an aerobic environment generates oxidative stress responses by activating various defense systems, including production of antimicrobial peptides called bacteriocins. This study highlights the importance of a two-component response regulator (ComE) that senses the aerobic environment and induces bacteriocin production in Streptococcus mutans, a dental pathogen. We believe increased bacteriocin secretion under aerobic conditions is necessary for survival and colonization of S. mutans in the oral cavity by inhibiting other competing organisms. Redox sensing by response regulator might be a widespread phenomenon since two other ComE homologs from pathogenic streptococci that inhabit diverse environmental niches also perform a similar function.

Unusual "cardiac" cause of hemoptysis: Accessory cardiac bronchus.

Hemoptysis is a common presenting feature of tuberculosis, pulmonary parenchymal malignancy, bronchiectasis, or a cardiac pathology as mitral stenosis. Relevant clinical history, physical examination, laboratory investigations, and radiology usually identify the cause of hemoptysis in the majority of the cases. We report a case of a 50-year-old male with intermittent hemoptysis which was the presenting feature of accessory cardiac bronchus.

Sub-pulmonic stenosis caused by a right ventricular outflow tract vegetation in a children with restricted ventricular septal defect.

Infective endocarditis involving the right heart is rarely observed in the pediatric population. Echocardiography plays an important role in its diagnosis, and surgery is indicated in patients with heart failure and persistent sepsis not responding to medical treatment. Here, we report a rare case of restricted ventricular septal defect complicated by a vegetation developed in the right ventricular outflow tract and causing sub-pulmonic stenosis in a 3-year-old male child.

Protective Role of Activated Protein C against Viral Mimetic Poly(I:C)-Induced Inflammation.

Activated protein C (APC) is an anticoagulant plasma serine protease which exhibits potent cytoprotective and anti-inflammatory activities. Here, we studied protective effects of APC on the proinflammatory function of polyinosinic:polycytidylic acid [poly(I:C)], a synthetic analog of viral double-stranded RNA, in cellular and animal models. Poly(I:C) induced histone H3 extranuclear translocation via interaction with toll-like receptor 3 in two established endothelial cell lines. Furthermore, poly(I:C) induced histone H3 extranuclear translocation in J774A.1 macrophages and human neutrophils and formation of macrophage and neutrophil extracellular traps (ETs). Mechanistically, poly(I:C) was found to upregulate expression of peptidylarginine deiminase 4 and enhance its interaction with histone H3, thereby leading to increased histone citrullination and neutrophil ET formation. Poly(I:C) elicited proinflammatory signaling responses by inducing nuclear factor kappa B activation and disrupting endothelial cell permeability. In vivo, poly(I:C) enhanced cell surface expression of Mac-1 on neutrophils in mice and facilitated their infiltration to lung tissues. Poly(I:C) also downregulated thrombomodulin expression in mouse tissues and reduced its circulating soluble level in plasma. We demonstrate in this study that APC and a signaling-selective mutant of APC effectively inhibit proinflammatory signaling effects of poly(I:C) in both cellular and animal models. We further demonstrate that unlike the requirement for endothelial protein C receptor on endothelial cells, the integrin Mac-1 is involved in the protease-activated receptor 1-dependent APC inhibition of macrophage ET formation in J774A.1 cells. Taken together, these results support a key role for APC signaling in inhibiting the viral mimetic-induced proinflammatory signaling responses and histone translocation-associated formation of ETs by innate immune cells.

Significance of Individual Domains of ClpL: A Novel Chaperone from Streptococcus mutans.

ClpL is a member of the HSP100 family of AAA+ chaperones that is widely present in Gram-positive but surprisingly absent in Gram-negative bacteria. ClpL is involved in various cellular processes, including stress tolerance response, long-term survival, virulence, and antibiotic resistance. ClpL is poorly characterized, and its molecular mechanisms of chaperone activity are largely unclear. Here, we biochemically characterized the ClpL protein from Streptococcus mutans, a dental pathogen, to understand its biological functions. ClpL harbors five domains: N-domain, two nucleotide binding domains (NBD-1 and NBD-2), M-domain, and C-domain. NBD-1 and NBD-2 contain distinct Walker A and B motifs for ATP binding and hydrolysis, respectively. We found that ClpL predominantly exists as a trimer in solution; however, upon ATP binding, it rapidly forms a hexameric structure. To study structure-function activity, we constructed several substitution and deletion mutants. We found that mutations in the Walker A and B motifs interfered with ATP hydrolysis and oligomerization. Similarly, deletions of N-, M-, and C-domains abolished both ATPase activity and oligomerization. Because we previously found that ClpL acts as a chaperone, we analyzed the chaperone activity. Surprisingly, we found that the NBD-2 mutants did not display any chaperone activity, indicating that ATP binding and hydrolysis by NBD-2 are essential for the chaperone. However, NBD-1 mutants showed chaperone activities, but the activities were variable depending on the nature of the mutations. Our results indicate that unlike other HSP100 family chaperones, ClpL is a novel chaperone that does not require any additional secondary chaperones for its activity.

Left Atrial Dissection and Rupture Following Excision of Left Atrial Myxoma: Role of Transesophageal Echocardiography.

Left atrial mass after excision of a left atrial myxoma may occur due to residual or additional masses, such as biatrial or multicentric myxomas and inverted left atrial appendage. In this E-challenge, the authors present a case where intraoperative transesophageal echocardiography allowed visualization of a left atrial mass after excision of a left atrial myxoma. Detailed examination demonstrated that the mass was due to left atrial dissection that progressed to rupture, allowing its early detection and repair. A high index of suspicion, as well as coordination between the surgeon and the perioperative echocardiographer, played a crucial role in the detection and management of this complication.

Plasmodium falciparum histidine rich protein HRPII inhibits the anti-inflammatory function of antithrombin.

BACKGROUND: It has been reported that histidine-rich protein II (HRPII), secreted by the malaria parasite, Plasmodium falciparum (Pf), inhibits the heparin-dependent anticoagulant activity of antithrombin (AT) in vitro and in plasma-based assay systems. OBJECTIVE: The objective of this study was to test the hypothesis that HRPII may also interact with the AT-binding vascular glycosaminoglycans (GAGs), thereby inhibiting the anti-inflammatory signaling function of the serpin. METHODS: We expressed HRPII in bacteria, purified it to homogeneity and studied its effect on endothelial cell signaling in the absence and presence of AT employing established signaling assays. RESULTS: We demonstrate that a low concentration of HRPII potently disrupts the barrier permeability function of endothelial cells. Moreover, HRPII competitively inhibits the protective effect of AT by a concentration-dependent manner. Similarly, AT inhibits the pro-inflammatory activity of HRPII by a concentration-dependent manner. The siRNA knockdown of 3-O-sulfotransferase 1 (3-OST-1), the enzyme responsible for the essential 3-O-sulfation of the AT-binding GAGs, downregulates the pro-inflammatory function of HRPII in endothelial cells, supporting the hypothesis that HRPII competitively inhibits the interaction of AT with 3-OS containing vascular GAGs. Histidine-rich protein II elicits its barrier-disruptive effect by the Src-dependent phosphorylation of vascular endothelial (VE)-cadherin and AT counteracts this effect. We further demonstrate that inorganic polyphosphates bind HRPII with a high affinity to amplify the pro-inflammatory signaling function of HRPII in both cellular and in vivo permeability models. CONCLUSION: We postulate that Pf-derived HRPII and polyphosphate can contribute to the pathogenesis of malaria infection by downregulating the AT-dependent anti-inflammatory and anticoagulant pathways.

Thrombomodulin Regulation of Mitogen-Activated Protein Kinases.

The multifaceted role of mitogen-activated protein kinases (MAPKs) in modulating signal transduction pathways in inflammatory conditions such as infection, cardiovascular disease, and cancer has been well established. Recently, coagulation factors have also emerged as key players in regulating intracellular signaling pathways during inflammation. Among coagulation factors, thrombomodulin, as a high affinity receptor for thrombin on vascular endothelial cells, has been discovered to be a potent anti-inflammatory and anti-tumorigenic signaling molecule. The protective signaling function of thrombomodulin is separate from its well-recognized role in the clotting cascade, which is to function as an anti-coagulant receptor in order to switch the specificity of thrombin from a procoagulant to an anti-coagulant protease. The underlying protective signaling mechanism of thrombomodulin remains largely unknown, though a few published reports link the receptor to the regulation of MAPKs under different (patho)physiological conditions. The goal of this review is to summarize what is known about the regulatory relationship between thrombomodulin and MAPKs.

Activated protein C inhibits lipopolysaccharide-mediated acetylation and secretion of high-mobility group box 1 in endothelial cells.

Essentials APC elicits cytoprotective responses in endothelial cells via EPCR-dependent cleavage of PAR1. APC inhibits LPS-mediated translocation and extracellular secretion of HMGB1 in endothelial cells. Signaling activity of APC inhibits LPS-mediated acetylation of HMGB1 by epigenetic mechanisms. APC inhibits LPS-mediated HMGB1 expression in CD31-positive endothelial cells in cremaster muscle. SUMMARY: Background Activated protein C (APC) inhibits high-mobility group box 1 (HMGB1) signaling and its lipopolysaccharide (LPS)-mediated release by endothelial protein C receptor (EPCR)-dependent activation of protease-activated receptor 1 (PAR1) in endothelial cells. Post-translational acetylation is known to modulate the subcellular localization of HMGB1, and its hyperacetylated form is translocated to the cytoplasm of innate immune cells before being secreted into the extracellular space. Objective To determine whether APC inhibits LPS-mediated HMGB1 secretion from endothelial cells by modulating its acetylation status. Methods The subcellular localization of HMGB1 in LPS-treated endothelial cells was monitored in the absence and presence of APC by western blot analysis of fractionated cell lysates and confocal immunofluorescence microscopy. Results Both western blot and immunofluorescence data indicated that APC effectively inhibits LPS-mediated translocation of HMGB1 from the nucleus to the cytoplasm by EPCR-dependent and PAR1-dependent mechanisms. When EPCR was ligated by the Gla-domain of protein C/APC, thrombin also inhibited LPS-mediated HMGB1 translocation. Further studies revealed that APC inhibits the translocation of HMGB1 from the nucleus to the cytoplasm by inhibiting LPS-mediated hyperacetylation of HMGB1 by (de)acetylating enzymes. Furthermore, the translocated HMGB1 was found to be associated with lysosome-associated membrane protein 1 in LPS-treated endothelial cells. The in vivo relevance of these findings was investigated in the mouse cremaster muscle, and this demonstrated that both wild-type APC and a signaling-selective mutant of APC inhibit LPS-mediated HMGB1 expression and translocation in CD31-positive endothelial cells. Conclusion These results suggest that APC inhibits LPS-mediated cytoplasmic translocation and secretion of HMGB1 in endothelial cells by epigenetic mechanisms.

Inorganic Polyphosphate Amplifies High Mobility Group Box 1-Mediated Von Willebrand Factor Release and Platelet String Formation on Endothelial Cells.

Objective- Inorganic polyphosphate (polyP) is known to modulate coagulation, inflammation, and metabolic pathways. It also amplifies inflammatory responses of HMGB1 (high mobility group box 1) in endothelial cells. The objective of this study was to evaluate the effect of polyP on von Willebrand factor (VWF) release from endothelial cells with or without HMGB1. Approach and Results- EA.hy926 endothelial cells were treated with different concentrations of polyP70 alone or in combination with different concentrations of HMGB1. VWF release was measured by an ELISA assay in the absence or presence of pharmacological inhibitors of the receptor for advanced glycation end products, P2Y1, and Ca2+. A flow chamber assay was used to monitor polyP70-mediated platelet recruitment and VWF-platelet string formation. PolyP70 and HMGB1 induced VWF release from endothelial cells by a concentration-dependent manner. PolyP70 amplified HMGB1-mediated VWF release from endothelial cells. This was also true if boiled platelet releasate was used as the source of polyP. Gene silencing or pharmacological inhibitors of receptor for advanced glycation end products, P2Y1, and Ca2+ significantly inhibited VWF release. PolyP70 and HMGB1 synergistically promoted VWF-platelet string formation in the flow chamber assay, which was inhibited by the anti-GPIbα (glycoprotein Ib alpha) antibody. VWF release by polyP70-HMGB1 complex required phosphorylation of Src and phospholipase C because inhibitors of Src, phospholipase C, and Ca2+ signaling significantly decreased VWF secretion. The polyP70-HMGB1 complex also increased angiopoietin-2 release, indicating that Weibel-Palade body exocytosis is involved in the VWF release. Conclusions- PolyP70 can promote thrombotic and inflammatory pathways by inducing VWF release and platelet string formation on endothelial cells.

A Method for the Measurement of Salivary Gland Function in Mice.

Patients with Sjögren's syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Polymyxin susceptibility testing, interpretative breakpoints and resistance mechanisms: An update.

Emerging multidrug-resistant (MDR) nosocomial pathogens are a great threat. Polymyxins, an old class of cationic polypeptide antibiotic, are considered as last-resort drugs in treating infections caused by MDR Gram-negative bacteria. Increased use of polymyxins in treating critically ill patients necessitates routine polymyxin susceptibility testing. However, susceptibility testing both of colistin and polymyxin B (PMB) is challenging. In this review, currently available susceptibility testing methods are briefly discussed. The multicomponent composition of colistin and PMB significantly influences susceptibility testing. In addition, poor diffusion in the agar medium, adsorption to microtitre plates and the synergistic effect of the surfactant polysorbate 80 with polymyxins have a great impact on the performance of susceptibility testing methods This review also describes recently identified chromosomal resistance mechanisms, including modification of lipopolysaccharide (LPS) with 4-amino-4-deoxy-l-arabinose (L-Ara4-N) and phosphoethanolamine (pEtN) resulting in alteration of the negative charge, as well as the plasmid-mediated colistin resistance determinants mcr-1, mcr-1.2, mcr-2 and mcr-3.

Intraoperative assessment of transient and persistent regional left ventricular wall motion abnormalities in patients undergoing coronary revascularization surgery using real time three-dimensional transesophageal echocardiography: A prospective observational study.

OBJECTIVE: To evaluate the feasibility of intraoperative real time three-dimensional echocardiography (RT3DE) for identification and quantification of transient and persistent regional wall motion abnormalities (RWMAs) in patients with ischemic cardiomyopathy undergoing coronary artery bypass grafting (CABG). DESIGN: A prospective observational study SETTING: Single-center study in an academic tertiary care hospital PARTICIPANTS: A series of 42 patients undergoing elective CABG over a 2-year period were included. INTERVENTION: After induction of anesthesia, a comprehensive transesophageal echocardiography (TEE) examination was performed to evaluate regional wall motion using two-dimensional wall motion score index (WMSI) and RT3D echocardiographic parameters at three specific time points during the operative phase. MEASUREMENTS AND MAIN RESULTS: The 3D assessment of LV function was based on the quantification of change in LV chamber volume over time from each segment excursion. Patients were divided into two groups and subgroups based on TEE findings. There was significant mechanical dyssynchrony in patients with RWMAs (WMSI > 1; systolic dyssynchrony index [SDI] = 7.0 ± 3.66) as compared to the patients having normal wall motion (WMSI = 1; SDI = 2.0 ± 0.95; P = .001). Patients with contractile dysfunction were found to have low values of segmental excursion and high values of negative excursion on parametric imaging. Persistent RWMAs due to hibernating myocardium showed significant resolution of mechanical dyssynchrony after revascularization. Parametric imaging could detect transient RWMAs due to stunning and graft dysfunction. Early activating segments (EAS) on "timing bull's-eye" may represent hypercontractile segments and may influence inotrope administration. CONCLUSION: The RT3DE is a valuable modality for precise quantification of regional wall motion during revascularization procedure.

A Multicenter Evaluation of a Closed-Loop Anesthesia Delivery System: A Randomized Controlled Trial.

BACKGROUND: Closed-loop systems for anesthesia delivery have been shown to outperform traditional manual control in different clinical settings. The present trial was aimed at evaluating the feasibility and efficacy of Bispectral Index (BIS)-guided closed-loop anesthesia delivery system (CLADS) in comparison with manual control across multiple centers in India. METHODS: Adult patients scheduled for major surgical procedures of an expected duration of 1 to 3 hours were randomized across 6 sites into 2 groups: a CLADS group and a manual group. In the manual control group, propofol infusion was titrated manually by the attending anesthesiologist to a BIS of 50 during induction and maintenance. Analgesia was maintained with fentanyl infusion and nitrous oxide in both groups. In the CLADS group, both induction and maintenance of anesthesia were performed automatically using CLADS. The primary outcome measure was the performance of the system as assessed by the percentage of total anesthesia time BIS remained ±10 of target BIS. The secondary outcome measures were a percentage of anesthesia-time heart rate and mean arterial pressure within 25% of the baseline, median absolute performance error, wobble, and global score. Wobble indicates intraindividual variability in the control of BIS, and global score reflects the overall performance; lower values indicate superior performance for both parameters. The performance parameters of the system also were compared among the participating sites. RESULTS: Two hundred forty-two patients were randomized. BIS was maintained within ±10 of target for significantly longer time in the CLADS group (81.4% ± 8.9 % of anesthesia duration) than in the manual group (55.34% ± 25%, P < 0.0001). The indices that assess performance were significantly better in the CLADS group than the manual group as follows: median absolute performance error was 10 (10, 12) (median [interquartile range]) in the CLADS group versus 18 (14, 24) in the manual group, P < 0.0001; wobble was 9 (8, 10) in CLADS group versus 10 (8, 14) in the manual group, P = 0.0009; and Global score, which reflects overall performance, was 24 (19, 30) in the CLADS group versus 51 (31, 99) in the manual group, P < 0.0001. The percentage of time heart rate was within 25% of the baseline was significantly greater in the CLADS group (heart rate of 95 [87, 99], median [interquartile range], in the CLADS group versus 90 [75, 98] in the manual group P = 0.0031). On comparison of data between the centers, the performance parameters did not differ significantly among the centers in the CLADS group (P = 0.94), but the parameters differed significantly among the centers in the manual group (P < 0.001). CONCLUSIONS: Our study in a multicenter setting proves the consistently better performance of automated anesthesia drug delivery compared with conventional manual control. This highlights an important advantage of an automated system for delivering standardized anesthesia, thereby overcoming differences in practices among anesthesiologists.