Structural basis of broad-spectrum ß-lactam resistance in Staphylococcus aureus.

Broad-spectrum ß-lactam antibiotic resistance in Staphylococcus aureus is a global healthcare burden1,2. In clinical strains, resistance is largely controlled by BlaR13, a receptor that senses ß-lactams through the acylation of its sensor domain, inducing transmembrane signalling and activation of the cytoplasmic-facing metalloprotease domain4. The metalloprotease domain has a role in BlaI derepression, inducing blaZ (ß-lactamase PC1) and mecA (ß-lactam-resistant cell-wall transpeptidase PBP2a) expression3-7. Here, overcoming hurdles in isolation, we show that BlaR1 cleaves BlaI directly, as necessary for inactivation, with no requirement for additional components as suggested previously8. Cryo-electron microscopy structures of BlaR1-the wild type and an autocleavage-deficient F284A mutant, with or without ß-lactam-reveal a domain-swapped dimer that we suggest is critical to the stabilization of the signalling loops within. BlaR1 undergoes spontaneous autocleavage in cis between Ser283 and Phe284 and we describe the catalytic mechanism and specificity underlying the self and BlaI cleavage. The structures suggest that allosteric signalling emanates from ß-lactam-induced exclusion of the prominent extracellular loop bound competitively in the sensor-domain active site, driving subsequent dynamic motions, including a shift in the sensor towards the membrane and accompanying changes in the zinc metalloprotease domain. We propose that this enhances the expulsion of autocleaved products from the active site, shifting the equilibrium to a state that is permissive of efficient BlaI cleavage. Collectively, this study provides a structure of a two-component signalling receptor that mediates action-in this case, antibiotic resistance-through the direct cleavage of a repressor.

The incidence, impact, and risk factors for moderate to severe persistent pain after breast cancer surgery: a prospective cohort study.

BACKGROUND: Few Australasian studies have evaluated persistent pain after breast cancer surgery. OBJECTIVE: To evaluate the incidence, impact, and risk factors of moderate to severe persistent pain after breast cancer surgery in a New Zealand cohort. DESIGN: Prospective cohort study. METHODS: Consented patients were reviewed at 3 timepoints (preoperative, 2 weeks and 6 months postoperative). Pain incidence and interference, psychological distress and upper limb disability were assessed perioperatively. Clinical, demographic, psychological, cancer treatment-related variables, quantitative sensory testing, and patient genotype (COMT, OPRM1, GCH1, ESR1, and KCNJ6) were assessed as risk factors using multiple logistic regression. RESULTS: Of the 173 patients recruited, 140 completed the 6-month follow-up. Overall, 15.0% (n = 21, 95% CI: 9.5%-22.0%) of patients reported moderate to severe persistent pain after breast cancer surgery with 42.9% (n = 9, 95% CI: 21.9%-66.0%) reporting likely neuropathic pain. Pain interference, upper limb dysfunction and psychological distress were significantly higher in patients with moderate to severe pain (P < .004). Moderate to severe preoperative pain (OR= 3.60, 95% CI: 1.13-11.44, P = .03), COMT rs6269 GA genotype (OR = 5.03, 95% CI: 1.49-17.04, P = .009) and psychological distress at postoperative day 14 (OR= 1.08, 95% CI: 1.02-1.16, P = .02) were identified as risk factors. Total intravenous anesthesia (OR= 0.31, 95% CI: 0.10 - 0.99, P = .048) was identified as protective. CONCLUSION: The incidence of moderate to severe persistent pain after breast cancer surgery is high with associated pain interference, physical disability, and psychological distress. Important modifiable risk factors were identified to reduce this important condition.

Assessment of ED triage of anaphylaxis patients based on the Emergency Severity Index.

OBJECTIVES: To describe the emergency department (ED) triage of anaphylaxis patients based on the Emergency Severity Index (ESI), assess the association between ESI triage level and ED epinephrine administration, and determine characteristics associated with lower acuity triage ESI assignment (levels 3 and 4). METHODS: We conducted a cohort study of adult and pediatric anaphylaxis patients between September 2010 and September 2018 at an academic ED. Patient characteristics and management were compared between Emergency Severity Index (ESI) triage level 1 or 2 versus levels 3 or 4 using logistic regression analysis. We adhered to STROBE reporting guidelines. RESULTS: A total of 1090 patient visits were included. There were 26 (2%), 515 (47%), 489 (45%), and 60 (6%) visits that were assigned an ESI triage level of 1, 2, 3, and 4, respectively. Epinephrine was administered in the ED to 53% of patients triaged ESI level 1 or 2 and to 40% of patients triaged ESI level 3 or 4. Patients who were assigned a lower acuity ESI level of 3 or 4 had a longer median time from ED arrival to epinephrine administration compared to those with a higher acuity ESI level of 1 or 2 (28 min compared to 13 min, p < .001). A lower acuity ESI level was more likely to be assigned to visits with a chief concern of hives, rash, or pruritus (OR 2.33 [95% CI, 1.20-4.53]) and less likely to be assigned to visits among adults (OR, 0.43 [0.31-0.60]), patients who received epinephrine from emergency medical services (OR 0.56 [0.38-0.82]), presented with posterior pharyngeal or uvular angioedema (OR, 0.56 [0.38-0.82]), hypoxemia (OR, 0.34 [0.18-0.64]), or increased heart (OR 0.83 [0.73-0.95]) or respiratory (OR 0.70 [0.60-0.82]) rates. CONCLUSION: Patients triaged to lower acuity ESI levels experienced delays in ED epinephrine administration. Adult and pediatric patients with skin-related chief concerns were more likely to be to be assigned lower acuity ESI levels. Further studies are needed to identify interventions that will improve ED anaphylaxis triage.

The Prevalence, Impact, and Risk Factors for Persistent Pain After Breast Cancer Surgery in a New Zealand Population.

OBJECTIVE: Few Australasian studies have assessed persistent pain after breast cancer surgery. This study aims to evaluate the prevalence, impact, and risk factors of moderate to severe persistent pain after breast cancer surgery in a New Zealand population. METHODS: Retrospective cross-sectional study of patients who underwent breast cancer surgery between six and 48 months previously. Validated questionnaires were used to assess pain prevalence and impact, psychological distress, and upper limb function. Patients' clinical records were assessed for potential risk factors. RESULTS: Of the 375 patients who were sent questionnaires, 201 were included in the study. More than half of the patients (N = 111, 55%) reported breast surgery related-persistent pain, with 46 (23%) rating the pain as moderate to severe. Neuropathic pain was reported by 21 (46%) patients with moderate to severe pain. Pain interference, upper limb dysfunction, and psychological distress were significantly higher in patients with moderate to severe pain (P < 0.001). Non-European ethnicity (odds ratio [OR] = 5.02, 95% confidence interval [CI] = 2.05-12.25, P < 0.001), reconstruction surgery (OR = 4.10, 95% CI = 1.30-13.00, P = 0.02), and axillary node dissection (OR = 4.33, 95% CI = 1.19-15.73, P < 0.03) were identified as risk factors for moderate to severe pain by multivariate logistic regression analysis. CONCLUSIONS: Moderate to severe persistent pain after breast cancer surgery affects many New Zealand patients, and is associated with impaired daily life activities, physical disability, and psychological distress. Large numbers of patients undergo breast cancer surgery annually. This study emphasizes the importance of identification and management of these patients perioperatively.

25-Hydroxyvitamin D and blood pressure: a plateau effect in adults with African ancestry living at different latitudes.

OBJECTIVE: This study examined the association of 25-hydroxyvitamin D [25(OH)D] levels and blood pressure above and below 25(OH)D levels of 20 ng/ml in young adults with African ancestry. METHODS: This cross-sectional analysis utilized data from a pooled sample of 2242 adults with African ancestry from five different latitudes (403 in the United States, 474 in South Africa, 479 in Ghana, 448 in Jamaica, and 438 in Seychelles). Piecewise linear regression models with a single knot were fitted to determine above and below a 25(OH)D level of 20 ng/ml the slope of SBP and DBP while adjusting for covariates including calcium intake and site. RESULTS: The mean age was 34.4 (6.1) years, and 46.3% were men. Mean SBP and DBP were 118.1 (15.6) and 73.2 (12.2) mmHg, respectively, and were significantly higher among the United States vs Ghana, Jamaica, and Seychelles groups (P < 0.001 for all comparisons). 25(OH)D levels were significantly lower in the United States vs all other sites (P < 0.001 for all comparisons). When 25(OH)D levels were less than 20 ng/ml, slopes of SBP [-0.33 (95% confidence interval (CI) -0.57, -0.07)] and DBP [-0.21 (95% CI -0.40, -0.02)] were negative and significantly different from zero after adjustment for covariates. In contrast, with 25(OH)D levels above 20 ng/ml, the slopes of SBP [-0.03 (95% CI -0.13, 0.06)] and DBP [-0.04 (-0.11, 0.03)] did not differ significantly from zero. CONCLUSION: The cross-sectional association of 25(OH)D with blood pressure is strongest when 25(OH)D levels are less than 20 ng/ml in young adults with African ancestry.

High-throughput screening of potential inhibitors for the metabolism of the investigational anti-cancer drug 5,6-dimethylxanthenone-4-acetic acid.

By screening potential inhibitors of drug metabolism using the in vitro models, potential drug-drug interactions in vivo may be predicted with the use of appropriate pharmacokinetic principles. This study aimed to develop a rapid screening system using human liver microsomes to efficiently identify the potential inhibitors of DMXAA metabolism. Initial IC50 was estimated by using a two-point method, and then Ki values were determined if required and compared with those initial IC50 values. More than 100 compounds including known substrates and inhibitors of human uridine diphosphate glucuronosyltransferases (UGTs) and cytochrome P450 (CYP), anti-cancer drugs and xanthenone analogues were screened for their inhibitory effect on DMXAA glucuronidation and 6-methylhydroxylation in human liver microsomes. Both metabolites of DMXAA, DMXAA acyl glucuronide (DMXAA-G) and 6-hydroxymethyl-5-methylxanthenone-4-acetic acid (6-OH-MXAA), formed in human liver microsomes were quantitated by validated HPLC methods. The results indicated that there was a significant relationship (r2 = 0.966, P < 0.001) between the two-point IC50 values and the apparent Ki values for 20 compounds showing significant inhibitory effects on DMXAA metabolism, suggesting the usefulness of the two-point determination for the initial screening of compounds. This study has been completed using a strategy for rapid HPLC analysis and thus provided early access to detailed information for potential inhibitors of DMXAA metabolism and allows for further DMXAA-drug interaction studies.

Inhibition of human CYP1A2 oxidation of 5,6-dimethyl-xanthenone-4-acetic acid by acridines: a molecular modelling study.

1. The aim of the present study was to investigate the structural requirements for the inhibition of 6-methyl-hydroxylation of the antitumour agent 5,6-dimethyl-xanthenone-4-acetic acid (DMXAA) by acridine analogues and use a CYP1A2 homology model to provide some insight into this interaction. 2. Concentrations causing 50% inhibition (IC50) of the 6-methylhydroxylation of DMXAA were determined in human liver microsomes in the presence of various acridines. Some of the acridines were also tested for their ability to inhibit the CYP1A2-mediated 7-ethoxyresorufin O-de-ethylation. The molecular modelling studies of human CYP1A2 used the crystal structure of rabbit CYP2C5 as a template based on protein sequence homology and an interactive docking procedure using a dynamic hydrogen bond feature. 3. The in vitro IC50 studies for the inhibition of 6-methylhydroxylation of DMXAA indicated: (i) the importance of the position of the carboxamide side-chain on the acridine nucleus (and, to a lesser extent, its composition); (ii) the addition of hydroxyl groups to the 5-, 6- and 7-position of the acridine nucleus diminished the inhibitory potency; and (iii) amsacrine (acridine nucleus with methansulphonanilide side-chain at the 9-position) had no significant inhibitory effect. Similar structural trends were observed for the inhibition of O-de-ethylation of 7-ethoxyresorufin by acridines, supporting the involvement of CYP1A2 in DMXAA 6-methyl hydroxylation. 4. The molecular modelling studies indicated: (i) both DMXAA and N-[2-(dimethylamino)-ethyl]acridine-4-carboxamide (DACA) form two hydrogen bonds plus putative pi-pi stacking interactions with the CYP1A2-binding domain, typical of CYP1A2 substrates and inhibitors; (ii) the DMXAA 6-methyl group is 4.0 A from the central iron atom of the heme moiety and ideal for oxidation; (iii) the known oxidation sites for DACA are orientated away from the heme iron, supporting the non-involvement of CYP1A2; and (iv) amsacrine did not fit the putative CYP1A2 site owing to the steric hindrance of the bulky methanesulphonanilide side-chain. 5. These results suggest that docking studies with this homology model may be useful in the design of further acridine anticancer agents, in particular to identify agents that do not interact either as substrates or inhibitors with the CYP1A2-binding domain.