Inhibitor design to target a unique feature in the folate pocket of Staphylococcus aureus dihydrofolate reductase.

Staphylococcus aureus (Sa) is a serious concern due to increasing resistance to antibiotics. The bacterial dihydrofolate reductase enzyme is effectively inhibited by trimethoprim, a compound with antibacterial activity. Previously, we reported a trimethoprim derivative containing an acryloyl linker and a dihydophthalazine moiety demonstrating increased potency against S. aureus. We have expanded this series and assessed in vitro enzyme inhibition (Ki) and whole cell growth inhibition properties (MIC). Modifications were focused at a chiral carbon within the phthalazine heterocycle, as well as simultaneous modification at positions on the dihydrophthalazine. MIC values increased from 0.0626-0.5 µg/mL into the 0.5-1 µg/mL range when the edge positions were modified with either methyl or methoxy groups. Changes at the chiral carbon affected Ki measurements but with little impact on MIC values. Our structural data revealed accommodation of predominantly the S-enantiomer of the inhibitors within the folate-binding pocket. Longer modifications at the chiral carbon, such as p-methylbenzyl, protrude from the pocket into solvent and result in poorer Ki values, as do modifications with greater torsional freedom, such as 1-ethylpropyl. The most efficacious Ki was 0.7 ± 0.3 nM, obtained with a cyclopropyl derivative containing dimethoxy modifications at the dihydrophthalazine edge. The co-crystal structure revealed an alternative placement of the phthalazine moiety into a shallow surface at the edge of the site that can accommodate either enantiomer of the inhibitor. The current design, therefore, highlights how to engineer specific placement of the inhibitor within this alternative pocket, which in turn maximizes the enzyme inhibitory properties of racemic mixtures.

The toxin from a ParDE toxin-antitoxin system found in Pseudomonas aeruginosa offers protection to cells challenged with anti-gyrase antibiotics.

Toxin-antitoxin systems are mediators of diverse activities in bacterial physiology. For the ParE-type toxins, their reported role of gyrase inhibition utilized during plasmid-segregation killing indicates they are toxic. However, their location throughout chromosomes leads to questions about function, including potential non-toxic outcomes. The current study has characterized a ParDE system from the opportunistic human pathogen Pseudomonas aeruginosa (Pa). We identified a protective function for this ParE toxin, PaParE, against effects of quinolone and other antibiotics. However, higher concentrations of PaParE are themselves toxic to cells, indicating the phenotypic outcome can vary based on its concentration. Our assays confirmed PaParE inhibition of gyrase-mediated supercoiling of DNA with an IC50 value in the low micromolar range, a species-specificity that resulted in more efficacious inhibition of Escherichia coli derived gyrase versus Pa gyrase, and overexpression in the absence of antitoxin yielded an expected filamentous morphology with multi-foci nucleic acid material. Additional data revealed that the PaParE toxin is monomeric and interacts with dimeric PaParD antitoxin with a KD in the lower picomolar range, yielding a heterotetramer. This work provides novel insights into chromosome-encoded ParE function, whereby its expression can impart partial protection to cultures from selected antibiotics.

Shared decision-making for biologic treatment of autoimmune disease: influence on adherence, persistence, satisfaction, and health care costs.

BACKGROUND: Shared decision-making (SDM), a process whereby physicians and patients collaborate to select interventions, is not well understood for biologic treatment of autoimmune conditions. METHODS: This was a cross-sectional survey of adults initiating treatment for Crohn's disease or ulcerative colitis (inflammatory bowel disease, IBD) or psoriatic arthritis or rheumatoid arthritis (RA/PA). Survey data were linked to administrative claims for 6 months before (baseline) and after (follow-up) therapy initiation. Measures included the Shared Decision Making Questionnaire, Patient Activation Measure (PAM), Morisky Medication Adherence Scale (MMAS), general health, and treatment satisfaction. Claims-based Quan-Charlson comorbidity scores, persistence, medication possession ratio (MPR), and health care costs were examined. Patients were compared by participation (SDM) and nonparticipation (non-SDM) in SDM. RESULTS: Among 453 respondents, 357 were eligible, and 306 patients (204 RA/PA and 102 IBD) were included in all analyses. Overall (n=357), SDM participants (n=120) were more often females (75.0% vs 62.5%, P=0.018), had lower health status (48.0 vs 55.4, P=0.005), and higher Quan-Charlson scores (1.0 vs 0.7, P=0.035) than non-SDM (n=237) participants. Lower MMAS scores (SDM 0.17 vs non-SDM 0.41; P<0.05) indicated greater likelihood of adherence; SDM participants also reported higher satisfaction with medication and had greater activation (PAM: SDM vs non-SDM: 66.9 vs 61.6; P<0.001). Mean MPR did not differ, but persistence was longer among SDM participants (111.2 days vs 102.2 days for non-SDM; P=0.029). Costs did not differ by SDM status overall, or among patients with RA/PA. The patients with IBD, however, experienced lower (P=0.003) total costs ($9,404 for SDM vs $25,071 for non-SDM) during follow-up. CONCLUSION: This study showed greater likelihood of adherence and satisfaction for patients who engaged in SDM and reduced health care costs among patients with IBD who engaged in SDM. This study provides a basis for defining SDM participation and detecting differences by SDM participation for biologic treatment selection for autoimmune conditions.

Toxin-Antitoxin Modules Are Pliable Switches Activated by Multiple Protease Pathways.

Toxin-antitoxin (TA) modules are bacterial regulatory switches that facilitate conflicting outcomes for cells by promoting a pro-survival phenotypic adaptation and/or by directly mediating cell death, all through the toxin activity upon degradation of antitoxin. Intensive study has revealed specific details of TA module functions, but significant gaps remain about the molecular details of activation via antitoxin degradation used by different bacteria and in different environments. This review summarizes the current state of knowledge about the interaction of antitoxins with cellular proteases Lon and ClpP to mediate TA module activation. An understanding of these processes can answer long-standing questions regarding stochastic versus specific activation of TA modules and provide insight into the potential for manipulation of TA modules to alter bacterial growth.

Association Between Corporate Wellness Program Participation and Changes in Health Risks.

OBJECTIVE: To assess the relationship between wellness program participation and employee health risks. METHODS: Data from 173,901 health-risk appraisals and wellness program participation records were used to assess changes in seven health risks (blood pressure, body weight, cholesterol level, nutrition, physical inactivity, stress, and tobacco use). RESULTS: Controlling for baseline covariates, high-risk members who completed only a coaching program were significantly more likely to lower five out of seven health risks than were high-risk members in the comparison group. Participation in multiple wellness activities (eg, biometric screening) increased the odds that risks would be reduced.In addition, the number of risk levels that improved was greater than the total that worsened. CONCLUSIONS: This study provides evidence that wellness program participation was associated with significant risk reduction, particularly among individuals who participated in more than one program.

A novel method for collecting vaginal pool for fetal lung maturity studies.

OBJECTIVE: Fetal lung maturity (FLM) studies using vaginal pool can guide management in near-term gestations with rupture of membranes. Because an adequate sample may be difficult to collect using a syringe, we tested collection using a sterile sponge. STUDY DESIGN: In this prospective study, vaginal pool was collected via both a syringe and a sponge from each gravida. Study patients experienced rupture of membranes between 34-41 weeks of gestation. Each sample was analyzed using the TDx-FLM II assay and phosphatidylglycerol (PG) testing. RESULTS: Fifty patients were enrolled; 44 demonstrated concordant syringe/sponge results. There was 1 instance of mature TDx-FLM testing using a sponge but not a syringe; PG testing in this case was absent in both samples. Using the kappa statistic, agreement between the methods was "substantial" for TDx-FLM and "almost perfect" for PG. CONCLUSION: Collecting vaginal pool with a sponge may provide clinically useful information, particularly when PG is present.

Preventive foot-care practices among adults with diabetes in North Carolina, 1997 to 2001.

Preventive foot-care practices, such as annual foot examinations by a health-care provider, can substantially reduce the risk of lower-extremity amputations. We examined the level of preventive foot-care practices (reported rates of having at least one foot examination by a physician) among patients with diabetes mellitus in North Carolina and determined the factors associated with these practices. Of 1,245 adult respondents to the 1997 to 2001 North Carolina Behavioral Risk Factor Surveillance System, 71.6% reported that they had had their feet examined within the past year, a rate that is much higher than that previously reported by Bell and colleagues in the same population for 1994 to 1995 (61.7%). Foot care was more common among insulin users than nonusers, those having diabetes for 20 years or longer than those having diabetes for less than 10 years, blacks than whites, and those who self-monitored their blood glucose level daily than those who did not. The results of this study indicate that diabetes educational services can be directed at populations at high risk of ignoring the recommended foot-care practices indicated in these analyses, thereby reducing diabetes-related lower-extremity complications.

Phenylpropanolamine appears not to promote weight loss in patients with schizophrenia who have gained weight during clozapine treatment.

BACKGROUND: Weight gain is a common side effect of clozapine treatment and may expose patients to obesity-associated health risks. We proposed that concomitant treatment with an appetite suppressant such as phenylpropanolamine (PPA) would lead to a decrease in appetite and therefore loss of weight. METHOD: This was a 12-week, double-blind, randomized, placebo-controlled trial of PPA, 75 mg/day, in outpatients with treatment-refractory schizophrenia (DSM-IV) who were stable on clozapine treatment for at least 4 months and had gained > 10% of their baseline body weight since starting clozapine. Patients were evaluated for adverse effects and weighed weekly. A Positive and Negative Syndrome Scale (PANSS) assessment, a short dietary quiz, and blood indices were completed monthly. RESULTS: Sixteen patients were equally randomly assigned to receive PPA or placebo. The groups did not differ in mean age, baseline weight, dose of clozapine, baseline PANSS scores, or the percent of weight gained since the start of clozapine. There was no significant effect of treatment on weight (t = 0.219, df = 10, p = .831). There was no significant change in either the total PANSS scores (t = -0.755, df = 10, p = .468), the positive or negative symptom cluster scores, or any of the remaining variables. CONCLUSION: Phenylpropanolamine 75 mg/day was well tolerated but was not effective in reversing established weight gain associated with clozapine treatment in stable outpatients with schizophrenia.