Spectroscopic and Mechanistic Studies of Heterodimetallic Forms of Metallo-β-lactamase NDM-1

In an effort to characterize the roles of each metal ion in metallo-β-lactamase NDM-1, heterodimetallic analogues (CoCo-, ZnCo-, and CoCd-) of the enzyme were generated and characterized. UV–vis, 1H NMR, EPR, and EXAFS spectroscopies were used to confirm the fidelity of the metal substitutions, including the presence of a homogeneous, heterodimetallic cluster, with a single-atom bridge. This marks the first preparation of a metallo-β-lactamase selectively substituted with a paramagnetic metal ion, Co(II), either in the Zn1 (CoCd-NDM-1) or in the Zn2 site (ZnCo-NDM-1), as well as both (CoCo-NDM-1). We then used these metal-substituted forms of the enzyme to probe the reaction mechanism, using steady-state and stopped-flow kinetics, stopped-flow fluorescence, and rapid-freeze-quench EPR. Both metal sites show significant effects on the kinetic constants, and both paramagnetic variants (CoCd- and ZnCo-NDM-1) showed significant structural changes on reaction with substrate. These changes are discussed in terms of a minimal kinetic mechanism that incorporates all of the data

Molecular dissection of the pea shoot apical meristem*

The shoot apical meristem (SAM) is responsible for the development of all the above-ground parts of a plant. Our understanding of the SAM at the molecular level is incomplete. This study investigates the gene expression repertoire of SAMs in the garden pea (Pisum sativum). To this end, 10 346 EST sequences representing 7610 unique genes were generated from SAM cDNA libraries. These sequences, together with previously reported pea ESTs, were used to construct a 12K oligonucleotide array to identify genes with differential SAM expression, as compared to axillary meristems, root apical meristems, or non-meristematic tissues. A number of genes were identified, predominantly expressed in specific cell layers or domains of the SAM and thus are likely components of the gene networks involved in stem cell maintenance or the initiation of lateral organs. Further in situ hybridization analysis confirmed the spatial localization of some of these genes within the SAM. Our data also indicate the diversification of some gene expression patterns and hence functions in legume crop plants. A number of transcripts highly expressed in all three meristems have also been uncovered and these candidates may provide valuable insight into molecular networks that underpin the maintenance of meristematic functionality

Subcutaneous Implantable Cardioverter Defibrillator and LifeVest Saving Lives in Challenging Scenarios

Introduction: According to guidelines, ICD should be implanted in selected patients for primary or secondary prevention. The increasing number of procedures leads to a growth of infective complications. Case Summary: We report two cases of ICD implantation complicated with severe infections. The first procedure was complicated by endocarditis and septic shock, the second one by septic shock. After antibiotics and ICD explantation, a wearable cardioverter defibrillator (WCD) was used as a bridge to subcutaneous ICD (S-ICD). Conclusion: These two cases demonstrate the benefits of the S-ICD, but also the chance to treat high-risk patients with WCD as bridge-to-procedure, allowing full recovery before a new implantation

Saprochaete capitata aortitis in an immunocomopetent patient after myocardial revascularization

Saprochaete species infection is a rare fungal disease reported so far only in immunocompromised patients. We describe the first case of aortitis caused by Saprochaete capitata, presenting as ascending aorta aneurysm, with secondary endophthalmitis in an immunocompetent patient. Infection by Saprochaete capitata is potentially fatal, with a mortality ranging from 50% to 90% of cases. In the present case aortic aneurysm caused by Saprochaete capitata aortitis was successfully treated by the combination of accurate diagnosis with surgical and specific antifungal therapy

Spontaneous coronary artery dissection: Role of prognostic markers and relationship with genetic analysis

Spontaneous coronary artery dissection (SCAD) is increasingly recognized as an important cause of myocardial infarction (MI). Currently there is little knowledge about prognostic factors for unfavorable outcome at long term follow-up; furthermore, there is also little knowledge about the genetics of these patients. Aims: This observational and retrospective study describes long-term cardiovascular outcomes of a population affected by SCAD and assesses predictors of recurrent de novo SCAD and major adverse cardiovascular events (MACE). Furthermore, a correlation between genotype and adverse events at follow-up was sought. Methods: Baseline characteristics, angiographic features, use of medication and long-term cardiovascular events were systematically ascertained between 2000 and 2019. Next generation sequencing was performed with a panel consisting of twenty genes of interest. Variants found were filtered based on their frequency and only frequencies <1% in the general population were considered as \u201cpositive\u201d. Results: Seventy patients were enrolled and followed for a median time of 39.1 months. Median age was 52 years and the majority were women (86%). Use of hormone therapy (HT) (OR 3.64, p = 0.041) and presence of malignant ventricular arrhythmias (VAs) at onset (OR 7.03, p = 0.0073) were associated with a greater risk of recurrent de novo SCAD. Proximal type SCAD (OR 8.47, p < 0.0001) and presence of VAs at onset (OR 9.97, p = 0.047) were associated with a greater risk of MACE. A potential SCAD-associated mutation was detected in 27 patients (44%); 6 patients (22%) defined as genetically \u201cpositive\u201d developed MACE vs. 2 patients (6%) defined as \u201cnegative\u201d (p = 0.06 at univariate analysis). MACE at follow-up is reached earlier in genetically positive patients (7.9 vs. 42.5 months). Conclusion: use of HT and VAs at SCAD onset are prognostic factors for recurrent de novo SCAD. Proximal SCAD site and VAs at SCAD onset were prognostic factors for MACE. Analysis by molecular genetics seems to be a promising tool for the possible additional role it could play in MACE prediction

Identification and characterization of an unusual metallo-beta-lactamase from Serratia proteamaculans

Metallo-beta-lactamases (MBLs) are a family of metalloenzymes that are capable of hydrolyzing beta-lactam antibiotics and are an important means by which bacterial pathogens use to inactivate antibiotics. A database search of the available amino acid sequences from Serratia proteamaculans indicates the presence of an unusual MBL. A full length amino acid sequence alignment indicates overall homology to B3-type MBLs, but also suggests considerable variations in the active site, notably among residues that are relevant to metal ion binding. Steady-state kinetic measurements further indicate functional differences and identify two relevant pK (a) values for catalysis (3.8 for the enzyme-substrate complex and 7.8 for the free enzyme) and a preference for penams with modest reactivity towards some cephalosporins. An analysis of the metal ion content indicates the presence of only one zinc ion per active site in the resting enzyme. In contrast, kinetic data suggest that the enzyme may operate as a binuclear enzyme, and it is thus proposed that a catalytically active di-Zn2+ center is formed only once the substrate is present