The regulation of peptidoglycan hydrolysis in Escherichia coli

PhD ThesisThe bacterial cell envelope heteropolymer, peptidoglycan (PG), is essential for maintaining the osmotic stability and shape of most bacteria. PG biosynthesis is the target of our most successful antibiotics, the β-lactams and glycopeptides. However, the spread of antibiotic resistant strains highlights the need for novel antibiotic targets. Gram-negative bacteria possess a mainly single layered PG, which is enlarged in growing and dividing bacteria by the coordinated action of PG synthases and hydrolases. PG synthesis in Gram-negative bacteria is regulated from the cytoplasmic membrane (CM), by prokaryotic cytoskeletal elements, and from the outer membrane (OM) by the lipoproteins, LpoA and LpoB. LpoA/B interact with, and are essential for the in vivo activity of, the major PG synthases PBP1A and PBP1B, respectively. While the regulation of PG synthesis has been well studied in recent years, the mechanisms of PG hydrolysis regulation in E. coli remain poorly understood. E. coli possesses ~30 PG hydrolases with relatively few known regulators. In this work, we have structurally characterised LpoA from E. coli using nuclear magnetic resonance (NMR) spectroscopy of the N-terminal domain and use this to further the understanding of the in vitro and in vivo interaction of LpoA/PBP1A. We also studied PBP1A and LpoA in Haemophilus influenzae; in this species LpoA is essential. In a search for novel LpoA interaction partners we discovered the in vitro and in vivo interaction with the PG hydrolase, PBP4 and show that PBP4 also interacts with PBP1A. Subsequently, we optimised a process for the rapid identification of in vitro interactions and identified >20 interactions between PG synthases, PG hydrolases and other cell envelope proteins. We therefore present a putative PG hydrolysing complex with direct associations to the PG synthesis machinery. Through direct functional interactions with at least five PG hydrolases, we present the characterisation of the OM-anchored lipoprotein NlpI, of currently unknown cellular function, as a regulator of hydrolase activity. We show the in vitro regulation of activity by NlpI and the in vivo relevance of these interactions using a β-lactamase induction assay. This work significantly enhances our understanding of how PG synthesis and hydrolysis are coordinated as multi-enzyme complexes and presents the characterisation of a novel regulator of hydrolase activity, NlpI

Priorities for synthesis research in ecology and environmental science

ACKNOWLEDGMENTS We thank the National Science Foundation grant #1940692 for financial support for this workshop, and the National Center for Ecological Analysis and Synthesis (NCEAS) and its staff for logistical support.Peer reviewedPublisher PD

Priorities for synthesis research in ecology and environmental science

ACKNOWLEDGMENTS We thank the National Science Foundation grant #1940692 for financial support for this workshop, and the National Center for Ecological Analysis and Synthesis (NCEAS) and its staff for logistical support.Peer reviewedPublisher PD

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same regio

Characteristics, Trends, and Outcomes of Liver Transplantation for Primary Sclerosing Cholangitis in Female Versus Male Patients: An Analysis From the European Liver Transplant Registry.

BACKGROUND The influence of sex on primary sclerosing cholangitis (PSC), pre- and postliver transplantation (LT) is unclear. Aims are to assess whether there have been changes in incidence, profile, and outcome in LT-PSC patients in Europe with specific emphasis on sex. METHODS Analysis of the European Liver Transplant Registry database (PSC patients registered before 2018), including baseline demographics, donor, biochemical, and clinical data at LT, immunosuppression, and outcome. RESULTS European Liver Transplant Registry analysis (n = 6463, 32% female individuals) demonstrated an increasing number by cohort (1980-1989, n = 159; 1990-1999, n = 1282; 2000-2009, n = 2316; 2010-2017, n = 2549) representing on average 4% of all transplant indications. This increase was more pronounced in women (from 1.8% in the first cohort to 4.3% in the last cohort). Graft survival rate at 1, 5, 10, 15, 20, and 30 y was 83.6%, 70.8%, 57.7%, 44.9%, 30.8%, and 11.6%, respectively. Variables independently associated with worse survival were male sex, donor and recipient age, cholangiocarcinoma at LT, nondonation after brain death donor, and reduced size of the graft. These findings were confirmed using a more recent LT population closer to the current standard of care (LT after the y 2000). CONCLUSIONS An increasing number of PSC patients, particularly women, are being transplanted in European countries with better graft outcomes in female recipients. Other variables impacting outcome include donor and recipient age, cholangiocarcinoma, nondonation after brain death donor, and reduced graft size

The Novel, Nicotinic Alpha7 Receptor Partial Agonist, BMS-933043, Improves Cognition and Sensory Processing in Preclinical Models of Schizophrenia.

The development of alpha7 nicotinic acetylcholine receptor agonists is considered a promising approach for the treatment of cognitive symptoms in schizophrenia patients. In the present studies we characterized the novel agent, (2R)-N-(6-(1H-imidazol-1-yl)-4-pyrimidinyl)-4'H-spiro[4-azabicyclo[2.2.2]octane-2,5'-[1,3]oxazol]-2'-amine (BMS-933043), in vitro and in rodent models of schizophrenia-like deficits in cognition and sensory processing. BMS-933043 showed potent binding affinity to native rat (Ki = 3.3 nM) and recombinant human alpha7 nicotinic acetylcholine receptors (Ki = 8.1 nM) and agonist activity in a calcium fluorescence assay (EC50 = 23.4 nM) and whole cell voltage clamp electrophysiology (EC50 = 0.14 micromolar (rat) and 0.29 micromolar (human)). BMS-933043 exhibited a partial agonist profile relative to acetylcholine; the relative efficacy for net charge crossing the cell membrane was 67% and 78% at rat and human alpha7 nicotinic acetylcholine receptors respectively. BMS-933043 showed no agonist or antagonist activity at other nicotinic acetylcholine receptor subtypes and was at least 300 fold weaker at binding to and antagonizing human 5-HT3A receptors (Ki = 2,451 nM; IC50 = 8,066 nM). BMS-933043 treatment i) improved 24 hour novel object recognition memory in mice (0.1-10 mg/kg, sc), ii) reversed MK-801-induced deficits in Y maze performance in mice (1-10 mg/kg, sc) and set shift performance in rats (1-10 mg/kg, po) and iii) reduced the number of trials required to complete the extradimensional shift discrimination in neonatal PCP treated rats performing the intra-dimensional/extradimensional set shifting task (0.1-3 mg/kg, po). BMS-933043 also improved auditory gating (0.56-3 mg/kg, sc) and mismatch negativity (0.03-3 mg/kg, sc) in rats treated with S(+)ketamine or neonatal phencyclidine respectively. Given this favorable preclinical profile BMS-933043 was selected for further development to support clinical evaluation in humans

BMS-933043 improves MMN in neonatal PCP-treated rats.

<p>Results from 3 independent studies are presented as the mean ± SEM AUC determined from the averaged difference wave for each subject after treatment with either vehicle or BMS-933043 (n = 12–13/group). For reference, the dashed line shows the average AUC in untreated adult rats determined in separate studies. Each treatment was analyzed by one sample, two tailed t test; ** p<0.01 compared to a hypothetical zero.</p

BMS-933043 improves EDS performance in neonatal PCP-treated rats.

<p>Results are presented as the mean ± SEM number of trials required to reach the performance criterion of 6 successive correct entries into the baited pot for each discrimination (n = 9–10/treatment). Results were analyzed by 2 way repeated measures ANOVA followed by Dunnett’s post hoc analysis; **** p = 0.0001 compared to neonatal PCP/Vehicle treated rats.</p