Exploring molecular profiles of calcification in aortic vascular smooth muscle cells and aortic valvular interstitial cells.

Cardiovascular calcification can occur in vascular and valvular structures and is commonly associated with calcium deposition and tissue mineralization leading to stiffness and dysfunction. Patients with chronic kidney disease and associated hyperphosphatemia have an elevated risk for coronary artery calcification (CAC) and calcific aortic valve disease (CAVD). However, there is mounting evidence to suggest that the susceptibility and pathobiology of calcification in these two cardiovascular structures may be different, yet clinically they are similarly treated. To better understand diversity in molecular and cellular processes that underlie hyperphosphatemia-induced calcification in vascular and valvular structures, we exposed aortic vascular smooth muscle cells (AVSMCs) and aortic valve interstitial cells (AVICs) to high (2.5 mM) phosphate (Ph) conditions in vitro, and examined cell-specific responses. To further identify hyperphosphatemic-specific responses, parallel studies were performed using osteogenic media (OM) as an alternative calcific stimulus. Consistent with clinical observations made by others, we show that AVSMCs are more susceptible to calcification than AVICs. In addition, bulk RNA-sequencing reveals that AVSMCs and AVICs activate robust ossification-programs in response to high phosphate or OM treatments, however, the signaling pathways, cellular processes and osteogenic-associated markers involved are cell- and treatment-specific. For example, compared to VSMCs, VIC-mediated calcification involves biological processes related to osteo-chondro differentiation and down regulation of 'actin cytoskeleton'-related genes, that are not observed in VSMCs. Furthermore, hyperphosphatemic-induced calcification in AVICs and AVSMCs is independent of P13K signaling, which plays a role in OM-treated cells. Together, this study provides a wealth of information suggesting that the pathogenesis of cardiovascular calcifications is significantly more diverse than previously appreciated.

Identification of amino acid domains of Borrelia burgdorferi P66 that are surface exposed and important for localization, oligomerization, and porin function of the protein.

P66, a bifunctional integral outer membrane protein, is necessary for Borrelia burgdorferi to establish initial infection and to disseminate in mice. The integrin binding function of P66 facilitates extravasation and dissemination, but the role of its porin function during murine infection has not been investigated. A limitation to studying P66 porin function during mammalian infection has been the lack of structural information for P66. In this study, we experimentally characterized specific domains of P66 with regard to structure and function. First, we aligned the amino acid sequences of P66 from Lyme disease-causing Borrelia and relapsing fever-causing Borrelia to identify conserved and unique domains between these disease-causing clades. Then, we examined whether specific domains of P66 are exposed on the surface of the bacteria by introducing c-Myc epitope tags into each domain of interest. The c-Myc epitope tag inserted C-terminally to E33 (highly conserved domain), to T187 (integrin binding region domain and a non-conserved domain), and to E334 (non-conserved domain) were all detected on the surface of Borrelia burgdorferi. The c-Myc epitope tag inserted C-terminally to E33 and D303 in conserved domains disrupted P66 oligomerization and porin function. In a murine model of infection, the E33 and D303 mutants exhibited decreased infectivity and dissemination. Taken together, these results suggest the importance of these conserved domains, and potentially P66 porin function, in vivo.

Increasing Trends of Association of 16S rRNA Methylases and Carbapenemases in Enterobacterales Clinical Isolates from Switzerland, 2017-2020.

Aminoglycosides (AGs) in combination with ß-lactams play an important role in antimicrobial therapy in severe infections. Pan-resistance to clinically relevant AGs increasingly arises from the production of 16S rRNA methylases (RMTases) that are mostly encoded by plasmids in Gram-negative bacteria. The recent emergence and spread of isolates encoding RMTases is worrisome, considering that they often co-produce extended-spectrum ß-lactamases (ESBLs) or carbapenemases. Our study aimed to retrospectively analyze and characterize the association of carbapenem- and aminoglycoside-resistant clinical isolates in Switzerland during a 3.5-year period between January 2017 and June 2020. A total of 103 pan-aminoglycoside- and carbapenem-resistant clinical isolates were recovered at the NARA (Swiss National Reference Center for Emerging Antibiotic Resistance) during the 2017-2020 period. Carbapenemase and RMTase determinants were identified by PCR and sequencing. The characterization of plasmids bearing resistance determinants was performed by a mating-out assay followed by PCR-based replicon typing (PBRT). Clonality of the isolates was investigated by multilocus sequence typing (MLST). Over the 991 Enterobacterales collected at the NARA during this period, 103 (10.4%) of them were resistant to both carbapenems and all aminoglycosides. Among these 103 isolates, 35 isolates produced NDM-like carbapenemases, followed by OXA-48-like (n = 23), KPC-like (n = 21), or no carbapenemase (n = 13), OXA-48-like and NDM-like co-production (n = 7), and VIM-like enzymes (n = 4). The RMTases ArmA, RmtB, RmtC, RmtF, RmtG, and RmtB + RmtF were identified among 51.4%, 13.6%, 4.9%, 24.3%, 1%, and 1%, respectively. Plasmid co-localization of the carbapenemase and the RMTase encoding genes was found among ca. 20% of the isolates. A high diversity was identified in terms of the nature of associations between RMTase and carbapenemase-encoding genes, of incompatibility groups of the corresponding plasmids, and of strain genetic backgrounds, highlighting heterogeneous importations rather than clonal dissemination.

New Delhi Metallo-ß-Lactamase-Producing Enterobacterales Bacteria, Switzerland, 2019-2020.

Carbapenemase-producing Enterobacterales (CPE) bacteria are a critical global health concern; New Delhi metallo-ß-lactamase (NDM) enzymes account for >25% of all CPE found in Switzerland. We characterized NDM-positive CPE submitted to the Swiss National Reference Center for Emerging Antibiotic Resistance during a 2-year period (January 2019-December 2020) phenotypically and by using whole-genome sequencing. Most isolates were either Klebsiella pneumoniae (59/141) or Escherichia coli (52/141), and >50% were obtained from screening swabs. Among the 108 sequenced isolates, NDM-1 was the most prevalent variant, occurring in 56 isolates, mostly K. pneumoniae (34/56); the next most prevalent was NDM-5, which occurred in 49 isolates, mostly E. coli (40/49). Fourteen isolates coproduced a second carbapenemase, predominantly an OXA-48-like enzyme, and almost one third of isolates produced a 16S rRNA methylase conferring panresistance to aminoglycosides. We identified successful plasmids and global lineages as major factors contributing to the increasing prevalence of NDMs in Switzerland.

Animal models to study pathogenesis and treatments of cardiac disorders in rheumatoid arthritis: Advances and challenges for clinical translation.

Although cardiac diseases such as acute myocardial infarction, heart failure and arrhythmias are the leading cause of cardiovascular complications in rheumatoid arthritis (RA), their pathogenesis is far from being understood and optimal therapeutic options to treat specifically these disorders in RA are lacking. Preclinical studies on animal models of arthritis can help to decipher the complex link between arthritis and the heart, and to identify critical pathways and novel therapeutic targets. This review presented the available data on cardiac disorders in animal models of RA, as well as the current knowledge on pathophysiology and pharmacology of these disorders. Future directions for translational studies in a cardiorheumatic perspective are proposed.

Psoriatic arthritis and physical activity: a systematic review.

INTRODUCTION: The positive effects of physical activity in both rheumatoid arthritis and ankylosing spondylitis have been proven, but no clear data is yet published regarding psoriatic arthritis (PsA). The aims of this study were (i) to assess the level of physical activity (PA) in these patients and (ii) to review the effects of PA on articular disease, extra articular symptoms, and overall well-being. METHODS: The research strategy was performed on Pubmed, Cochrane, PEDro databases using the following keywords: "psoriatic arthritis AND physical activity" without restriction. The PRISMA methodology was used to select and analyze articles. We searched for all studies published online and in English before January 2021. RESULTS: A total of 319 studies were retrieved by our search but only 13 could be included. Two reports showed that 17 and 68% of patients reported practicing regularly physical activity. Exercise improved the BASDAI (Bath Ankylosing Spondylitis Disease Activity Index), the general symptoms (pain and fatigue), and the quality of life. Muscle strength and some of cardiovascular comorbidities were also improved. While the studies concerning the risk of enthesitis or flare induced by physical activity are conflicting, recent clinical trials did not mention any adverse event. Finally, rehabilitation programs were associated with a reduction mainly of pain and fatigue. CONCLUSION: Studies show clear beneficial effects of exercise in PsA on disease activity, on well-being, and on comorbidities, and they seem to outweigh the risk of enthesitis induced by mechanical stress. Further investigations are necessary to confirm these results and to precise the modalities of exercise. Key Points • Psoriatic arthritis patients have a sedentary lifestyle • Physical activity has beneficial effects on disease activity, well-being and reduced some cardiovascular risk factor in psoriatic arthritis • Risk of enthesitis and flares is low with exercise in psoriatic arthritis.

Biomarker variation in patients successfully treated with tocilizumab for severe coronavirus disease 2019 (COVID-19): results of a multidisciplinary collaboration.

OBJECTIVES: Acute respiratory distress syndrome (ARDS) related to SARS-CoV-2 is likely due to a cytokine storm characterised by a major release of pro-inflammatory cytokines, including interleukin-6 (IL-6). Blocking excessive IL-6 production might be the key to the COVID-19-ARDS treatment. Beneficial effects of IL-6 blockade using a humanised anti-IL-6 receptor antibody, tocilizumab (TCZ) were previously reported in patients with COVID-19 related ARDS. The aim of the study was to study the variation over time of several biomarkers, demonstrated to be predictors of poor prognostic, in subjects successfully treated with TCZ for severe COVID-19. METHODS: Retrospective analysis of a case series of patients with COVID-19-ARDS, evidenced by RT-PCR and lung CT-scan. Patients with increasing O2 requirements, within the window of opportunity for TCZ treatment (Day 7 to Day 17 after onset of symptoms) were treated with TCZ (2 infusions of 8 mg/kg). Demographic, biological and clinical data were collected from the patients' files. Serum levels of CRP, ferritin, fibrinogen, lymphocytes, platelets, creatinine, D-dimer and liver enzymes were assayed at the time of the first TCZ administration, then every two days for 8 days. RESULTS: 40 patients were treated with TCZ. Most of them had several comorbidities, and all had multiple biological abnormalities (lymphopenia, increased CRP, ferritin, fibrinogen, D-dimer, liver enzymes). 30 patients (75%) benefited from TCZ and 10 patients died. In the survivors, following TCZ administration CRP decreased dramatically as early as day 4 (-86.7%, p<0.0001) and returned to normal at day 6. Fibrinogen and lymphocyte count returned to normal values at day 6. Ferritin also decreased significantly. No significant change was observed for D-dimer (p=0.68) and other studied biomarkers (haemoglobin, leucocyte count, AST). CONCLUSIONS: In patients with COVID-19 acute respiratory distress syndrome, treatment with TCZ resulted in favourable evolution in 75% of the cases. Biomarkers of inflammation (CRP, ferritin, fibrinogen) decreased dramatically as early as the 4th day after TCZ injection. Lymphopenia, a predictor of poor prognostic, was reversed 6 days after TCZ injection.

Ongoing dissemination of OXA-244 carbapenemase-producing Escherichia coli in Switzerland and their detection.

OXA-244 is a derivative of OXA-48 showing weaker carbapenemase activity, compromising the detection of corresponding producers in clinical laboratories. Since 2017, the Swiss National Reference Center for Emerging Antibiotic Resistance noticed an increased identification of OXA-244-producing Escherichia coli (n=15) within the country. Different methods (biochemical and immunoassay tests, screening culture media) were tested for the detection of OXA-244 producers. Whole genome sequencing was used to investigate the genetic relatedness between the isolates and the genetic structures at the origin of the acquisition of the blaOXA-244 gene. The mSuperCARBA® medium and the NG-Test CARBA5 assay were found to be suitable tools for detecting all OXA-244-producing isolates. Other selective media did not perform optimally. Among the fifteen strains, five sequence types were identified, with ST38 being predominant. The blaOXA-244 gene was located on the chromosome for all isolates. Overall, detection of OXA-244 producers is challenging and specific guidelines must be followed.

Stringent Response Regulators Contribute to Recovery from Glucose Phosphate Stress in Escherichia coli.

In enteric bacteria such as Escherichia coli, the transcription factor SgrR and the small RNA SgrS regulate the response to glucose phosphate stress, a metabolic dysfunction that results in growth inhibition and stems from the intracellular accumulation of sugar phosphates. SgrR activates the transcription of sgrS, and SgrS helps to rescue cells from stress in part by inhibiting the uptake of stressor sugar phosphates. While the regulatory targets of this stress response are well described, less is known about how the SgrR-SgrS response itself is regulated. To further characterize the regulation of the glucose phosphate stress response, we screened global regulator gene mutants for growth changes during glucose phosphate stress. We found that deleting dksA, which encodes a regulator of the stringent response to nutrient starvation, decreases growth under glucose phosphate stress conditions. The stringent response alarmone regulator ppGpp (synthesized by RelA and SpoT) also contributes to recovery from glucose phosphate stress: as with dksA, mutating relA and spoT worsens the growth defect of an sgrS mutant during stress, although the sgrS relA spoT mutant defect was only detectable under lower stress levels. In addition, mutating dksA or relA and spoT lowers sgrS expression (as measured with a P sgrS -lacZ fusion), suggesting that the observed growth defects may be due to decreased induction of the glucose phosphate stress response or related targets. This regulatory effect could occur through altered sgrR transcription, as dksA and relA spoT mutants also exhibit decreased expression of a P sgrR -lacZ fusion. Taken together, this work supports a role for stringent response regulators in aiding the recovery from glucose phosphate stress.IMPORTANCE Glucose phosphate stress leads to growth inhibition in bacteria such as Escherichia coli when certain sugar phosphates accumulate in the cell. The transcription factor SgrR and the small RNA SgrS alleviate this stress in part by preventing further sugar phosphate transport. While the regulatory mechanisms of this response have been characterized, the regulation of the SgrR-SgrS response itself is not as well understood. Here, we describe a role for stringent response regulators DksA and ppGpp in the response to glucose phosphate stress. sgrS dksA and sgrS relA spoT mutants exhibit growth defects under glucose phosphate stress conditions. These defects may be due to a decrease in stress response induction, as deleting dksA or relA and spoT also results in decreased expression of sgrS and sgrR This research presents one of the first regulatory effects on the glucose phosphate stress response outside SgrR and SgrS and depicts a novel connection between these two metabolic stress responses.

NI(ii) phosphine and phosphide complexes supported by a PNP-pyrrole pincer ligand.

The reaction between [(PNpyrP)NiCl] (1, PNpyrP = 2,5-bis((di-iso-propylphosphino)-methyl)-1H-pyrrolide) and TlPF6 in the presence of a monodentate phosphine ligand led to cationic nickel phosphine and phosphite complexes, [(PNpyrP)Ni(PHPh2)][PF6] (2), [(PNpyrP)Ni(PMe3)][PF6] (3), and [(PNpyrP)Ni{P(OMe)3}][PF6] (4). Compound 2 can be deprotonated resulting in the generation of a terminal phosphido complex, [(PNpyrP)Ni(PPh2)] (5). When 3 is subjected to a base, a methyl proton of PMe3 is abstracted to afford [(PNpyrP)Ni(CH2PMe2)] (6), containing a methylene bridge between Ni and the external phosphine. Compounds 2-6 were characterized by single crystal X-ray diffraction in addition to multi-nuclear NMR spectroscopy and elemental analysis.

Borrelia burgdorferi outer surface protein C (OspC) binds complement component C4b and confers bloodstream survival.

Borrelia burgdorferi (Bb) is the causative agent of Lyme disease in the United States, a disease that can result in carditis, and chronic and debilitating arthritis and/or neurologic symptoms if left untreated. Bb survives in the midgut of the Ixodes scapularis tick, or within tissues of immunocompetent hosts. In the early stages of infection, the bacteria are present in the bloodstream where they must resist clearance by the innate immune system of the host. We have found a novel role for outer surface protein C (OspC) from B. burgdorferi and B. garinii in interactions with the complement component C4b and bloodstream survival in vivo. Our data show that OspC inhibits the classical and lectin complement pathways and competes with complement protein C2 for C4b binding. Resistance to complement is important for maintenance of the lifecycle of Bb, enabling survival of the pathogen within the host as well as in the midgut of a feeding tick when ospC expression is induced.

Ag(I) and Tl(I) precursors as transfer agents of a pyrrole-based pincer ligand to late transition metals.

A PNP ligand, PN(pyr)P ((PN(pyr)P)H = 2,5-bis((di-iso-propylphosphino)methyl)pyrrole), which employs a pyrrole unit as a central anionic nitrogen donor, was designed. The corresponding group 10 metal chlorides as well as iridium and ruthenium compounds were isolated. In order to conduct this work, [(PN(pyr)P)Tl] and [(PN(pyr)P)Ag]2 were synthesized and characterized. The thallium and silver species were paramount in the formation of the iridium and ruthenium complexes, which could not be isolated using (PN(pyr)P)H or the corresponding lithium pyrrolide salt. Interestingly, the solid state molecular structure of [(PN(pyr)P)Tl] indicates that the metal center engages in an η(2) intermolecular interaction with the backbone of a neighboring pyrrole molecule instead of the expected bonding to the phosphine arms.