An Alternative and Conserved Cell Wall Enzyme That Can Substitute for the Lipid II Synthase MurG.

The cell wall is a stress-bearing structure and a unifying trait in bacteria. Without exception, synthesis of the cell wall involves formation of the precursor molecule lipid II by the activity of the essential biosynthetic enzyme MurG, which is encoded in the division and cell wall synthesis (dcw) gene cluster. Here, we present the discovery of a cell wall enzyme that can substitute for MurG. A mutant of Kitasatospora viridifaciens lacking a significant part of the dcw cluster, including murG, surprisingly produced lipid II and wild-type peptidoglycan. Genomic analysis identified a distant murG homologue, which encodes a putative enzyme that shares only around 31% amino acid sequence identity with MurG. We show that this enzyme can replace the canonical MurG, and we therefore designated it MglA. Orthologues of mglA are present in 38% of all genomes of Kitasatospora and members of the sister genus Streptomyces CRISPR interference experiments showed that K. viridifaciens mglA can also functionally replace murG in Streptomyces coelicolor, thus validating its bioactivity and demonstrating that it is active in multiple genera. All together, these results identify MglA as a bona fide lipid II synthase, thus demonstrating plasticity in cell wall synthesis.IMPORTANCE Almost all bacteria are surrounded by a cell wall, which protects cells from environmental harm. Formation of the cell wall requires the precursor molecule lipid II, which in bacteria is universally synthesized by the conserved and essential lipid II synthase MurG. We here exploit the unique ability of an actinobacterial strain capable of growing with or without its cell wall to discover an alternative lipid II synthase, MglA. Although this enzyme bears only weak sequence similarity to MurG, it can functionally replace MurG and can even do so in organisms that naturally have only a canonical MurG. The observation that MglA proteins are found in many actinobacteria highlights the plasticity in cell wall synthesis in these bacteria and demonstrates that important new cell wall biosynthetic enzymes remain to be discovered.

Ethylene oligomerisation chromium catalysts with unsymmetrical PCNP ligands.

Chromium(iii) complexes of chelating diphosphines, with PNP or PCNCP backbones, are excellent catalysts for ethylene tetra- and/or trimerisations. A missing link within this ligand series are unsymmetric chelating diphosphines based on a PCNP scaffold. New bidentate PCNP ligands of the type Ph2PCH2N(R)PPh2 (R = 1-naphthyl or 5-quinoline groups, 2a-d) have been synthesised and shown to be extremely effective ligands for ethylene tri-/tetramerisations. Three representative tetracarbonyl Cr0 complexes bearing a single PN(R)P (5), PCN(R)P (6), or PCN(R)CP (7) diphosphine (R = 1-naphthyl) have been prepared from Cr(CO)4(η4-nbd) (nbd = norbornadiene). Furthermore we report a single crystal X-ray diffraction study of these compounds and discuss their structural parameters.

Ethylene Tetramerisation: A Structure-Selectivity Correlation.

The effect of ethylene tetramerisation ligand structures on 1-octene selectivity is well studied. However, by-product formation is less understood. In this work, a range of PNP ligand structures are correlated with the full product selectivity and with catalyst activity. As steric bulk on the N-substituent increases, the product selectivity shifts from >10 % to < 3% of both C6 cyclics and C16+ by-products. 1-Octene peaks at ca. 70%. Thereafter, only 1-hexene increases. Similar selectivity changes were observed for ortho-Ph-substituted PNP ligands. The C10-14 selectivity was less affected by the ligand structure. The ligand effect on the changes in selectivity is explained mechanistically. Lastly, an increase in ligand steric bulk was found to improve catalyst activity and reduce polymer formation by an order of magnitude. It is proposed that steric bulk promotes formation of cationic catalytic species which are responsible for selective ethylene oligomerisation.

Strategies for achieving viral hepatitis C micro-elimination in the Netherlands.

The Netherlands is striving to achieve national elimination of the hepatitis C virus (HCV) as one of the first countries worldwide. The favorable HCV epidemiology with both low prevalence and incidence, together with access to care and treatment, present excellent conditions to further build on towards this objective. The Dutch national plan on viral hepatitis, introduced in 2016, defines targets in the HCV healthcare cascade and provides a structural framework for the development of elimination activities. Since many different stakeholders are involved in HCV care in the Netherlands, focus has been placed on micro-elimination initiatives as a pragmatic and efficient approach. These numerous micro-eliminations projects have brought the Netherlands closer to HCV elimination. In the near future, efforts specifically have to be made in order to optimize case-finding strategies and to successfully accomplish the nationwide implementation of the registration and monitoring system of viral hepatitis mono-infections, before this final goal can be reached. The upcoming years will then elucidate if the Dutch' hands on approach has resulted in sufficient progress against HCV and if the Netherlands will lead the way towards nationwide HCV elimination.

Targeted next-generation sequencing using a multigene panel in myeloid neoplasms: Implementation in clinical diagnostics.

INTRODUCTION: Detection of mutations in patients with myeloid neoplasms (MNs) has shown great potential for diagnostic and prognostic purposes. Next-generation sequencing (NGS) is currently implemented for the diagnostic profiling of the four major MN subgroups. METHODS: First, we validated the targeted NGS approach using the TruSight Myeloid panel. Next, we screened 287 patients with a clinical suspicion of MN and 61 follow-up patients with documented MN. RESULTS: Validation of the NGS workflow resulted in maximal precision, accuracy, sensitivity, and specificity for gene variants with an allele frequency of at least 5% and a minimal read depth of 300. In our diagnostic screen, we identified at least one somatic mutation in 89% of patients with proven MN. Of the 155 newly diagnosed MN cases, 126 (81%) showed at least one mutation, confirming clonality. Moreover, the co-occurrence of mutated genes in the different MN subentities facilitates their classification and justifies the diagnostic use of a pan-myeloid panel. Furthermore, several of these mutations provide additional prognostic information independently of traditional prognostic scoring systems. CONCLUSION: Pan-myeloid targeted NGS fits elegantly in the routine diagnostic approach of MNs allowing for an improved diagnosis, subclassification, and prognosis.

Detection of Alpha-Toxin and Other Virulence Factors in Biofilms of Staphylococcus aureus on Polystyrene and a Human Epidermal Model.

BACKGROUND & AIM: The ability of Staphylococcus aureus to successfully colonize (a)biotic surfaces may be explained by biofilm formation and the actions of virulence factors. The aim of the present study was to establish the presence of 52 proteins, including virulence factors such as alpha-toxin, during biofilm formation of five different (methicillin resistant) S. aureus strains on Leiden human epidermal models (LEMs) and polystyrene surfaces (PS) using a competitive Luminex-based assay. RESULTS: All five S. aureus strains formed biofilms on PS, whereas only three out of five strains formed biofilms on LEMs. Out of the 52 tested proteins, six functionally diverse proteins (ClfB, glucosaminidase, IsdA, IsaA, SACOL0688 and nuclease) were detected in biofilms of all strains on both PS and LEMs. At the same time, four toxins (alpha-toxin, gamma-hemolysin B and leukocidins D and E), two immune modulators (formyl peptide receptor-like inhibitory protein and Staphylococcal superantigen-like protein 1), and two other proteins (lipase and LytM) were detectable in biofilms by all five S. aureus strains on LEMs, but not on PS. In contrast, fibronectin-binding protein B (FnbpB) was detectable in biofilms by all S. aureus biofilms on PS, but not on LEMs. These data were largely confirmed by the results from proteomic and transcriptomic analyses and in case of alpha-toxin additionally by GFP-reporter technology. CONCLUSION: Functionally diverse virulence factors of (methicillin-resistant) S. aureus are present during biofilm formation on LEMs and PS. These results could aid in identifying novel targets for future treatment strategies against biofilm-associated infections.

The decrease in procarboxypeptidase U (TAFI) concentration in acute ischemic stroke correlates with stroke severity, evolution and outcome.

BACKGROUND AND OBJECTIVES: Procarboxypeptidase U (proCPU, TAFI) concentration in plasma is potentially related to thrombotic tendency, and elevated proCPU levels have been reported in ischemic stroke patients. Improved insight into the role of proCPU in acute ischemic stroke is essential for the development of more adequate therapeutics that may include carboxypeptidase inhibitors. In this study we investigated whether the plasma concentration of proCPU and the proCPU kinetic profile in acute ischemic stroke are related to initial stroke severity, stroke evolution in the subacute phase and long-term stroke outcome. METHODS: Plasma concentration of proCPU was assessed in 136 stroke patients at admission (7.5 h after stroke onset), at 24 h, at 72 h and at day 7 after stroke onset. We evaluated the relation between change in proCPU concentrations and (a) stroke severity (patients with TIA vs. stroke patients, NIHSS score at admission), (b) stroke evolution (stroke progression, infarct volume at 72 h), and (c) stroke outcome (mRS score at month 3). RESULTS: ProCPU concentration decreased significantly in the first 72 h after stroke onset and thereafter returned to baseline. This biphasic time course, with its nadir at 72 h, was more pronounced in patients with severe stroke, unfavourable stroke evolution in the first 72 h and poor long-term outcome. CONCLUSIONS: The decrease in proCPU concentration in the first 72 h after stroke onset correlates with more severe stroke, unfavourable stroke evolution, and poor long-term stroke outcome.

Carboxypeptidase U (TAFIa): a new drug target for fibrinolytic therapy?

Procarboxypeptidase U (TAFI) is a recently discovered plasma procarboxypeptidase that upon activation by thrombin or thrombin-thrombomodulin turns into a potent antifibrinolytic enzyme. Its prominent bridging function between coagulation and fibrinolysis raised the interest of many research groups and of the pharmaceutical industry. The development of carboxypeptidase U (CPU) inhibitors as profibrinolytic agents is an attractive concept and possibilities for rational drug design will become more readily available in the near future as a result of the recently published crystal structure. Numerous studies have been performed and many of them show beneficial effects of CPU inhibitors for the improvement of endogenous fibrinolysis in different animal sepsis and thrombosis models. CPU inhibitors combined with tissue-type plasminogen activator (t-PA) seem to increase the efficiency of pharmacological thrombolysis allowing lower dosing of t-PA and subsequently fewer bleeding complications. This review will focus on recently obtained in vivo data and the benefits/risks of targeting CPU for the treatment of thrombotic disorders.

Mortality and causes of death in patients with hemophilia, 1992-2001: a prospective cohort study.

BACKGROUND: Clotting factor products have been safe for HIV since 1985, and for hepatitis C since 1992. Few studies have reported on mortality in the total population of hemophilia patients after the period of risk of viral infection transmission. OBJECTIVES: We studied the mortality, causes of death, and life expectancy of hemophilia patients between 1992 and 2001. We compared these findings with those of previous cohorts, together spanning the periods before, during, and after the use of potentially contaminated clotting products. PATIENTS AND METHODS: We performed a prospective cohort study among 967 patients with hemophilia A and B. Death rates, overall and cause-specific, were compared with national mortality figures for males adjusted for age and calendar period as standardized mortality ratio (SMRs). RESULTS: Between 1992 and 2001, 94 (9.7%) patients had died and two patients were lost to follow-up (0.2%). Mortality was 2.3-times higher in hemophilia patients than in the general male population (SMR 2.3 95% confidence interval 1.9-2.8). In patients with severe hemophilia, life expectancy decreased from 63 (1972-1985) to 59 years (1992-2001). Exclusion of virus-related deaths resulted in a life expectancy at birth of 72 years. CONCLUSIONS: AIDS was the main cause of death (26%) and 22% of deaths were because of hepatitis C. In patients not affected by viral infections, there still appeared to be a trend toward a moderately increased mortality compared with the Dutch male population. Thus, mortality of patients with hemophilia is still increased; this is largely because of the consequences of viral infections.