The Spx stress regulator confers high-level ß-lactam resistance and decreases susceptibility to last-line antibiotics in methicillin-resistant Staphylococcus aureus.

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a leading cause of mortality worldwide. MRSA has acquired resistance to next-generation ß-lactam antibiotics through the horizontal acquisition of the mecA resistance gene. Development of high resistance is, however, often associated with additional mutations in a set of chromosomal core genes, known as potentiators, which, through poorly described mechanisms, enhance resistance. The yjbH gene was recently identified as a hot spot for adaptive mutations during severe infections. Here, we show that inactivation of yjbH increased ß-lactam MICs up to 16-fold and transformed MRSA cells with low levels of resistance to being homogenously highly resistant to ß-lactams. The yjbH gene encodes an adaptor protein that targets the transcriptional stress regulator Spx for degradation by the ClpXP protease. Using CRISPR interference (CRISPRi) to knock down spx transcription, we unambiguously linked hyper-resistance to the accumulation of Spx. Spx was previously proposed to be essential; however, our data suggest that Spx is dispensable for growth at 37°C but becomes essential in the presence of antibiotics with various targets. On the other hand, high Spx levels bypassed the role of PBP4 in ß-lactam resistance and broadly decreased MRSA susceptibility to compounds targeting the cell wall or the cell membrane, including vancomycin, daptomycin, and nisin. Strikingly, Spx potentiated resistance independently of its redox-sensing switch. Collectively, our study identifies a general stress pathway that, in addition to promoting the development of high-level, broad-spectrum ß-lactam resistance, also decreases MRSA susceptibility to critical antibiotics of last resort.

Design of 3D-Photoprintable, Bio-, and Hemocompatible Nonisocyanate Polyurethane Elastomers for Biomedical Implants.

Polyurethanes (PUs) have adjustable mechanical properties, making them suitable for a wide range of applications, including in the biomedical field. Historically, these PUs have been synthesized from isocyanates, which are toxic compounds to handle. This has encouraged the search for safer and more environmentally friendly synthetic routes, leading today to the production of nonisocyanate polyurethanes (NIPUs). Among these NIPUs, polyhydroxyurethanes (PHUs) bear additional hydroxyl groups, which are particularly attractive for derivatizing and adjusting their physicochemical properties. In this paper, polyether-based NIPU elastomers with variable stiffness are designed by functionalizing the hydroxyl groups of a poly(propylene glycol)-PHU by a cyclic carbonate carrying a pendant unsaturation, enabling them to be post-photo-cross-linked with polythiols (thiol-ene). Elastomers with remarkable mechanical properties whose stiffness can be adjusted are obtained. Thanks to the unique viscous properties of these PHU derivatives and their short gel times observed by rheology experiments, formulations for light-based three-dimensional (3D) printing have been developed. Objects were 3D-printed by digital light processing with a resolution down to the micrometer scale, demonstrating their ability to target various designs of prime importance for personalized medicine. In vitro biocompatibility tests have confirmed the noncytotoxicity of these materials for human fibroblasts. In vitro hemocompatibility tests have revealed that they do not induce hemolytic effects, they do not increase platelet adhesion, nor activate coagulation, demonstrating their potential for future applications in the cardiovascular field.

A Clinically Selected Staphylococcus aureus clpP Mutant Survives Daptomycin Treatment by Reducing Binding of the Antibiotic and Adapting a Rod-Shaped Morphology.

Daptomycin is a last-resort antibiotic used for the treatment of infections caused by Gram-positive antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA). Treatment failure is commonly linked to accumulation of point mutations; however, the contribution of single mutations to resistance and the mechanisms underlying resistance remain incompletely understood. Here, we show that a single nucleotide polymorphism (SNP) selected during daptomycin therapy inactivates the highly conserved ClpP protease and is causing reduced susceptibility of MRSA to daptomycin, vancomycin, and ß-lactam antibiotics as well as decreased expression of virulence factors. Super-resolution microscopy demonstrated that inactivation of ClpP reduced binding of daptomycin to the septal site and diminished membrane damage. In both the parental strain and the clpP strain, daptomycin inhibited the inward progression of septum synthesis, eventually leading to lysis and death of the parental strain while surviving clpP cells were able to continue synthesis of the peripheral cell wall in the presence of 10× MIC daptomycin, resulting in a rod-shaped morphology. To our knowledge, this is the first demonstration that synthesis of the outer cell wall continues in the presence of daptomycin. Collectively, our data provide novel insight into the mechanisms behind bacterial killing and resistance to this important antibiotic. Also, the study emphasizes that treatment with last-line antibiotics is selective for mutations that, like the SNP in clpP, favor antibiotic resistance over virulence gene expression.

Prospective flow cytometry analysis of leucocyte subsets in critically ill patients who develop sepsis: a pilot study.

PURPOSE: Sepsis in critically ill patients with injury bears a high morbidity and mortality. Extensive phenotypic monitoring of leucocyte subsets in critically ill patients at ICU admission and during sepsis development is still scarce. The main objective of this study was to identify early changes in leukocyte phenotype which would correlate with later development of sepsis. METHODS: Patients who were admitted in a tertiary ICU for organ support after severe injury (elective cardiac surgery, trauma, necessity of prolonged ventilation or stroke) were sampled on admission (T1) and 48-72 h later (T2) for phenotyping of leukocyte subsets by flow cytometry and cytokines measurements. Those who developed secondary sepsis or septic shock were sampled again on the day of sepsis diagnosis (Tx). RESULTS: Ninety-nine patients were included in the final analysis. Nineteen (19.2%) patients developed secondary sepsis or septic shock. They presented significantly higher absolute monocyte counts and CRP at T1 compared to non-septic patients (1030/µl versus 550/µl, p = 0.013 and 5.1 mg/ml versus 2.5 mg/ml, p = 0.046, respectively). They also presented elevated levels of monocytes with low expression of L-selectin (CD62Lneg monocytes) (OR[95%CI] 4.5 (1.4-14.5), p = 0.01) and higher SOFA score (p < 0.0001) at T1 and low mHLA-DR at T2 (OR[95%CI] 0.003 (0.00-0.17), p = 0.049). Stepwise logistic regression analysis showed that both monocyte markers and high SOFA score (> 8) were independently associated with nosocomial sepsis occurrence. No other leucocyte count or surface marker nor any cytokine measurement correlated with sepsis occurrence. CONCLUSION: Monocyte counts and change of phenotype are associated with secondary sepsis occurrence in critically ill patients with injury.

Maturation of heart valve cell populations during postnatal remodeling.

Heart valve cells mediate extracellular matrix (ECM) remodeling during postnatal valve leaflet stratification, but phenotypic and transcriptional diversity of valve cells in development is largely unknown. Single cell analysis of mouse heart valve cells was used to evaluate cell heterogeneity during postnatal ECM remodeling and leaflet morphogenesis. The transcriptomic analysis of single cells from postnatal day (P)7 and P30 murine aortic (AoV) and mitral (MV) heart valves uncovered distinct subsets of melanocytes, immune and endothelial cells present at P7 and P30. By contrast, interstitial cell populations are different from P7 to P30. P7 valve leaflets exhibit two distinct collagen- and glycosaminoglycan-expressing interstitial cell clusters, and prevalent ECM gene expression. At P30, four interstitial cell clusters are apparent with leaflet specificity and differential expression of complement factors, ECM proteins and osteogenic genes. This initial transcriptomic analysis of postnatal heart valves at single cell resolution demonstrates that subpopulations of endothelial and immune cells are relatively constant throughout postnatal development, but interstitial cell subpopulations undergo changes in gene expression and cellular functions in primordial and mature valves.

P2X1: a unique platelet receptor with a key role in thromboinflammation.

Thromboinflammation involves complex interactions between actors of inflammation and immunity and components of the hemostatic system, which are elicited upon infection or tissue injury. In this context, the interplay between platelets and innate immune cells has been intensively investigated. The ATP-gated P2X1 ion channel, expressed on both platelets and neutrophils is of particular interest. On platelets, this ion channel contributes to platelet activation and thrombosis, especially under high shear stress conditions of small arteries, whereas on neutrophils, it is involved in chemotaxis and in mitigating the activation of circulating cells. In vitro studies indicate that it may also be implicated in platelet-dependent immune responses during bacterial infection. More recently, in a mouse model of intestinal epithelial barrier disruption causing systemic inflammation, it has been reported that neutrophil P2X1 ion channel could play a protective role against exaggerated inflammation-associated thrombosis. This review will focus on this unique role of the ATP-gated P2X1 ion channel in thromboinflammation, highlighting possible implications and pointing to the need for further investigation of the role of P2X1 ion channels in the interplay between platelets and neutrophils during thrombus formation under various sterile or infectious inflammatory settings and in distinct vascular beds.

AMPK-ACC signaling modulates platelet phospholipids and potentiates thrombus formation.

AMP-activated protein kinase (AMPK) α1 is activated in platelets on thrombin or collagen stimulation, and as a consequence, phosphorylates and inhibits acetyl-CoA carboxylase (ACC). Because ACC is crucial for the synthesis of fatty acids, which are essential for platelet activation, we hypothesized that this enzyme plays a central regulatory role in platelet function. To investigate this, we used a double knock-in (DKI) mouse model in which the AMPK phosphorylation sites Ser79 on ACC1 and Ser212 on ACC2 were mutated to prevent AMPK signaling to ACC. Suppression of ACC phosphorylation promoted injury-induced arterial thrombosis in vivo and enhanced thrombus growth ex vivo on collagen-coated surfaces under flow. After collagen stimulation, loss of AMPK-ACC signaling was associated with amplified thromboxane generation and dense granule secretion. ACC DKI platelets had increased arachidonic acid-containing phosphatidylethanolamine plasmalogen lipids. In conclusion, AMPK-ACC signaling is coupled to the control of thrombosis by specifically modulating thromboxane and granule release in response to collagen. It appears to achieve this by increasing platelet phospholipid content required for the generation of arachidonic acid, a key mediator of platelet activation.

Platelet-rich plasma (PRP) and tendon healing: comparison between fresh and frozen-thawed PRP.

Platelet-rich plasma (PRP) is increasingly used in the treatment of musculoskeletal diseases. Its preservation by freezing it for the realization of multiple injections in clinical use has never been discussed. Calcaneal tendons of rats were surgically sectioned. Platelet concentration of the PRP was 2.5 x 106/µl with autologous plasma of rats. Frozen-thawed PRP was prepared by performing two cycles of freezing and thawing on PRP aliquots. Both platelet preparations were injected in the lesion. Biomechanical and histological evaluations were carried out after 7, 20 or 40 days post surgery. After 7 and 40 days, no significant difference was observed between the PRP and the frozen-thawed PRP group. There is however a difference 20 days after surgery: the ultimate tensile strength (UTS) was greater in the fresh PRP group. No obvious difference with histological aspect was observed between the two groups. In conclusion, fresh PRP and frozen-thawed PRP injections can lead to similar results in the healing process of section calcaneal tendons of rats. Improvements with fresh PRP are slight. PRP could thus be frozen to be preserved if multiple injections are needed (e.g. osteoarthritis).

Dexfenfluramine and Pergolide Cause Heart Valve Disease via Valve Metabolic Reprogramming and Ongoing Matrix Remodeling.

Several clinical reports indicate that the use of amphetaminic anorectic drugs or ergot derivatives could cause valvular heart disease (VHD). We sought to investigate whether valvular lesions develop in response to long-term oral administration of these drugs and to identify drug-targeted biological processes that may lead to VHD. Treatment of New Zealand White rabbits with pergolide, dexfenfluramine, or high-dose serotonin for 16 weeks induced valvular alterations characterized by extracellular matrix remodeling. Transcriptome profiling of tricuspid valves using RNA sequencing revealed distinct patterns of differentially expressed genes (DEGs) that clustered according to the different treatments. Genes that were affected by the three treatments were functionally enriched for reduced cell metabolism processes. The two drugs yielded more changes in gene expression than serotonin and shared most of the DEGs. These DEGs were mostly enriched for decreased biosynthetic processes, increased cell-matrix interaction, and cell response to growth factors, including TGF-ß, which was associated with p38 MAPK activation. Treatment with pergolide specifically affected genes involved in homeostasis, which was corroborated by the activation of the master regulator of cell energy homeostasis, AMPK-α, as well as decreased levels of metabolism-related miR-107. Thus, both pergolide and dexfenfluramine may cause VHD through valve metabolic reprogramming and matrix remodeling.

Extracorporeal CO2 removal and regional citrate anticoagulation in an experimental model of hypercapnic acidosis.

Low flow extracorporeal veno-venous CO2 removal (ECCO2 R) therapy is used to remove CO2 while reducing ventilation intensity. However, the use of this technique is limited because efficiency of CO2 removal and potential beneficial effects on pulmonary hemodynamics are not precisely established. Moreover, this technique requires anticoagulation that may induce severe complications in critically ill patients. Therefore, our study aimed at determining precise efficiency of CO2 extraction and its effects on right ventricular (RV) afterload, and comparing regional anticoagulation with citrate to systemic heparin anticoagulation during ECCO2 R. This study was performed in an experimental model of severe hypercapnic acidosis performed in two groups of three pigs. In the first group (heparin group), pigs were anticoagulated with a standard protocol of unfractionated heparin while citrate was used for ECCO2 R device anticoagulation in the second group (citrate group). After sedation, analgesia and endotracheal intubation, pigs were connected to a volume-cycled ventilator. Severe hypercapnic acidosis was obtained by reducing tidal volume by 60%. ECCO2 R was started in both groups when arterial pH was lower than 7.2. Pump Assisted Lung Protection (PALP, Maquet, Rastatt, Germany) system was used to remove CO2 . CO2 extraction, arterial pH, PaCO2 as well as systemic and pulmonary hemodynamic were continuously followed. Mean arterial pH was normalized to 7.37 ± 1.4 at an extracorporeal blood flow of 400 mL/min, coming from 7.11 ± 1.3. RV end-systolic pressure increased by over 30% during acute hypercapnic acidosis and was normalized in parallel with CO2 removal. CO2 extraction was not significantly increased in citrate group as compared to heparin group. Mean ionized calcium and MAP were significantly lower in the citrate group than in the heparin group during ECCO2 R (1.03 ± 0.20 vs. 1.33 ± 0.19 and 57 ± 14 vs. 68 ± 15 mm Hg, respectively). ECCO2 R was highly efficient to normalize pH and PaCO2 and to reduce RV afterload resulting from hypercapnic acidosis. Regional anticoagulation with citrate solution was as effective as standard heparin anticoagulation but did not improve CO2 removal and lead to more hypocalcemia and hypotension.

Valve Disease in Heart Failure: Secondary but Not Irrelevant.

Secondary regurgitation caused by the remodeling and dysfunction of the left or right heart chamber may complicate heart failure, worsening both symptoms and prognosis. Outcome studies have shown that patients' prognosis worsened as the severity of secondary regurgitation increases. Imaging and more specifically echocardiography plays a central role for diagnosis and serial assessment of secondary regurgitation as well as for timing the intervention and guiding the procedure.

Variations of circulating cardiac biomarkers during and after anthracycline-containing chemotherapy in breast cancer patients.

BACKGROUND: Over time, the chance of cure after the diagnosis of breast cancer has been increasing, as a consequence of earlier diagnosis, improved diagnostic procedures and more effective treatment options. However, oncologists are concerned by the risk of long term treatment side effects, including congestive heart failure (CHF). METHODS: In this study, we evaluated innovative circulating cardiac biomarkers during and after anthracycline-based neoadjuvant chemotherapy (NAC) in breast cancer patients. Levels of cardiac-specific troponins T (cTnT), N-terminal natriuretic peptides (NT-proBNP), soluble ST2 (sST2) and 10 circulating microRNAs (miRNAs) were measured. RESULTS: Under chemotherapy, we observed an elevation of cTnT and NT-proBNP levels, but also the upregulation of sST2 and of 4 CHF-related miRNAs (miR-126-3p, miR-199a-3p, miR-423-5p, miR-34a-5p). The elevations of cTnT, NT-proBNP, sST2 and CHF-related miRNAs were poorly correlated, suggesting that these molecules could provide different information. CONCLUSIONS: Circulating miRNA and sST2 are potential biomarkers of the chemotherapy-related cardiac dysfunction (CRCD). Nevertheless, further studies and long-term follow-up are needed in order to evaluate if these new markers may help to predict CRCD and to identify the patients at risk to later develop CHF.

Cardiac Imaging: Multimodality Advances and Surveillance Strategies in Detection of Cardiotoxicity.

Contemporary cancer management has increased the overall number of cancer survivors, but cardiotoxicity remains a subject of concern, which is a major cause of noncancer mortality among survivors. Among the potential cardiovascular complications, left ventricular (LV) systolic dysfunction is a poor prognostic factor. The importance of its early detection is based on the principle that the likelihood of response to heart failure (HF) treatment is temporally related to the initiation of HF treatment. For these reasons, cardiac monitoring is commonly applied in general practice, based on serial measurements of LV ejection fraction (LVEF); transthoracic echocardiography (TTE) is generally used. However, the LVEF, as a diagnostic and predictive parameter, has significant limitations, which calls for more effective multimodality imaging strategies. This approach requires further study, but there is increasing available data in the literature, encouraging the combination of multimodality imaging parameters and techniques for early cancer therapeutic-related cardiac dysfunction (CTRCD) detection.

DUSP3 Genetic Deletion Confers M2-like Macrophage-Dependent Tolerance to Septic Shock.

DUSP3 is a small dual-specificity protein phosphatase with an unknown physiological function. We report that DUSP3 is strongly expressed in human and mouse monocytes and macrophages, and that its deficiency in mice promotes tolerance to LPS-induced endotoxin shock and to polymicrobial septic shock after cecal ligation and puncture. By using adoptive transfer experiments, we demonstrate that resistance to endotoxin is macrophage dependent and transferable, and that this protection is associated with a striking increase of M2-like macrophages in DUSP3(-/-) mice in both the LPS and cecal ligation and puncture models. We show that the altered response of DUSP3(-/-) mice to sepsis is reflected in decreased TNF production and impaired ERK1/2 activation. Our results demonstrate that DUSP3 plays a key and nonredundant role as a regulator of innate immune responses by mechanisms involving the control of ERK1/2 activation, TNF secretion, and macrophage polarization.

Rasa3 controls megakaryocyte Rap1 activation, integrin signaling and differentiation into proplatelet.

Rasa3 is a GTPase activating protein of the GAP1 family which targets Ras and Rap1. Ubiquitous Rasa3 catalytic inactivation in mouse results in early embryonic lethality. Here, we show that Rasa3 catalytic inactivation in mouse hematopoietic cells results in a lethal syndrome characterized by severe defects during megakaryopoiesis, thrombocytopenia and a predisposition to develop preleukemia. The main objective of this study was to define the cellular and the molecular mechanisms of terminal megakaryopoiesis alterations. We found that Rasa3 catalytic inactivation altered megakaryocyte development, adherence, migration, actin cytoskeleton organization and differentiation into proplatelet forming megakaryocytes. These megakaryocyte alterations were associated with an increased active Rap1 level and a constitutive integrin activation. Thus, these mice presented a severe thrombocytopenia, bleeding and anemia associated with an increased percentage of megakaryocytes in the bone marrow, bone marrow fibrosis, extramedular hematopoiesis, splenomegaly and premature death. Altogether, our results indicate that Rasa3 catalytic activity controls Rap1 activation and integrin signaling during megakaryocyte differentiation in mouse.

Dual-specificity phosphatase 3 deficiency or inhibition limits platelet activation and arterial thrombosis.

BACKGROUND: A limitation of current antiplatelet therapies is their inability to separate thrombotic events from bleeding occurrences. A better understanding of the molecular mechanisms leading to platelet activation is important for the development of improved therapies. Recently, protein tyrosine phosphatases have emerged as critical regulators of platelet function. METHODS AND RESULTS: This is the first report implicating the dual-specificity phosphatase 3 (DUSP3) in platelet signaling and thrombosis. This phosphatase is highly expressed in human and mouse platelets. Platelets from DUSP3-deficient mice displayed a selective impairment of aggregation and granule secretion mediated by the collagen receptor glycoprotein VI and the C-type lectin-like receptor 2. DUSP3-deficient mice were more resistant to collagen- and epinephrine-induced thromboembolism compared with wild-type mice and showed severely impaired thrombus formation on ferric chloride-induced carotid artery injury. Intriguingly, bleeding times were not altered in DUSP3-deficient mice. At the molecular level, DUSP3 deficiency impaired Syk tyrosine phosphorylation, subsequently reducing phosphorylation of phospholipase Cγ2 and calcium fluxes. To investigate DUSP3 function in human platelets, a novel small-molecule inhibitor of DUSP3 was developed. This compound specifically inhibited collagen- and C-type lectin-like receptor 2-induced human platelet aggregation, thereby phenocopying the effect of DUSP3 deficiency in murine cells. CONCLUSIONS: DUSP3 plays a selective and essential role in collagen- and C-type lectin-like receptor 2-mediated platelet activation and thrombus formation in vivo. Inhibition of DUSP3 may prove therapeutic for arterial thrombosis. This is the first time a protein tyrosine phosphatase, implicated in platelet signaling, has been targeted with a small-molecule drug.

P2X1 expressed on polymorphonuclear neutrophils and platelets is required for thrombosis in mice.

Adenosine triphosphate (ATP) and its metabolite, adenosine, are key regulators of polymorphonuclear neutrophil (PMN) functions. PMNs have recently been implicated in the initiation of thrombosis. We investigated the role of ATP and adenosine in PMN activation and recruitment at the site of endothelial injury. Following binding to the injured vessel wall, PMNs are activated and release elastase. The recruitment of PMNs and the subsequent fibrin generation and thrombus formation are strongly affected in mice deficient in the P2X1-ATP receptor and in wild-type (WT) mice treated with CGS 21680, an agonist of the A2A adenosine receptor or NF449, a P2X1 antagonist. Infusion of WT PMNs into P2X1-deficient mice increases fibrin generation but not thrombus formation. Restoration of thrombosis requires infusion of both platelets and PMNs from WT mice. In vitro, ATP activates PMNs, whereas CGS 21680 prevents their binding to activated endothelial cells. These data indicate that adenosine triphosphate (ATP) contributes to polymorphonuclear neutrophil (PMN) activation leading to their adhesion at the site of laser-induced endothelial injury, a necessary step leading to the generation of fibrin, and subsequent platelet-dependent thrombus formation. Altogether, our study identifies previously unknown mechanisms by which ATP and adenosine are key molecules involved in thrombosis by regulating the activation state of PMNs.

MicroRNAs in Valvular Heart Diseases: Potential Role as Markers and Actors of Valvular and Cardiac Remodeling.

miRNAs are a class of over 5000 noncoding RNAs that regulate more than half of the protein-encoding genes by provoking their degradation or preventing their translation. miRNAs are key regulators of complex biological processes underlying several cardiovascular disorders, including left ventricular hypertrophy, ischemic heart disease, heart failure, hypertension and arrhythmias. Moreover, circulating miRNAs herald promise as biomarkers in acute myocardial infarction and heart failure. In this context, this review gives an overview of studies that suggest that miRNAs could also play a role in valvular heart diseases. This area of research is still at its infancy, and further investigations in large patient cohorts and cellular or animal models are needed to provide strong data. Most studies focused on aortic stenosis, one of the most common valvular diseases in developed countries. Profiling and functional analyses indicate that miRNAs could contribute to activation of aortic valve interstitial cells to a myofibroblast phenotype, leading to valvular fibrosis and calcification, and to pressure overload-induced myocardial remodeling and hypertrophy. Data also indicate that specific miRNA signatures, in combination with clinical and functional imaging parameters, could represent useful biomarkers of disease progression or recovery after aortic valve replacement.