Susceptibility of Pseudomonas aeruginosa Biofilm to Alpha-Helical Peptides: D-enantiomer of LL-37

Pseudomonas aeruginosa is a highly versatile opportunistic pathogen and its ability to produce biofilms is a direct impediment to the healing of wounds and recovery from infection. Interest in anti-microbial peptides (AMPs) has grown due to their potential therapeutic applications and their possible use against antibiotic resistant bacteria. LL-37 is the only cathelicidin expressed by humans. In this study, we tested LL-37 and the effect of a protease-resistant LL-37 peptide mimetic, the peptide enantiomer D-LL-37, for anti-microbial and anti-biofilm activity against P. aeruginosa. Both forms of the peptide were equally effective as AMPs with similar killing kinetics. Circular dichroism spectra were obtained to demonstrate the chirality of D- and L-LL-37, and the trypsin resistance of D-LL-37 was confirmed. The helical cathelicidin from the cobra Naja atra (NA-CATH), and synthetic peptide variations (ATRA-1, ATRA-2, NA-CATH:ATRA1-ATRA1) were also tested. Although the cobra cathelicidin and related peptides had strong anti-microbial activity, those tested did not inhibit Pseudomonas biofilm formation, neither did control peptides. Both D- and L-LL-37 inhibited the attachment of Pseudomonas to a 96-well plate and decreased the amount of pre-formed (established) biofilm. D-LL-37 is able to promote Pseudomonas motility and decrease biofilm formation by altering the rate of twitching as well as by downregulating the expression of the biofilm-related genes, rhlA and rhlB, similar to L-LL-37. Both L- and D-LL-37 protected Galleria mellonella in vivo against Pseudomonas infection, while NA-CATH:ATRA1-ATRA1 peptide did not. This study demonstrates the ability and equivalence of D-LL-37 compared to L-LL-37 to promote bacterial twitching motility and inhibit biofilm formation, and protect against in vivo infection, and suggests that this peptide could be a critical advancement in the development of new treatments for P. aeruginosa infection

Cigarette smoke extract induces differential expression levels of beta-defensin peptides in human alveolar epithelial cells

BACKGROUND: The damaging effects of cigarette smoke on the lungs are well known in terms of cancer risks. Additional molecular changes within the lung tissue can also occur as a result of exposure to cigarette smoke. The human β-defensin (hBD) class of antimicrobial peptides is the focus of our research. In addition to antimicrobial activity, β-defensins also have immunomodulatory functions. Over 30 previously unrecognized β-defensin genes have recently been identified in the human genome, many with yet to be determined functions. We postulated that altered β-defensin production may play a role in the pathogenesis observed in the lungs of smokers. Our hypothesis is that cigarette smoke exposure will affect the expression of β-defensins in human lung alveolar epithelial cells (A549). METHODS: We exposed A549 cells to cigarette smoke extract (CSE) and measured the changes in mRNA levels of several antimicrobial peptides by quantitative real-time PCR, and directly observed peptide expression in cells by immunofluorescence (IF) microscopy. RESULTS: We found that hBD3, hBD5, and hBD9 gene expression was upregulated in A549 cells exposed to CSE. HBD1, hBD8, hBD18 and LL-37 gene expression did not significantly change upon exposure to CSE. Expression of hBD3 and hBD4 peptides was visualized by IF. CONCLUSIONS: This differential expression suggests that hBD3, hBD5, and hBD9 may play a role in the changes to the lung tissue observed in smokers. Establishing differential β-defensin expression following CSE treatment will add to our understanding of the molecular response of the lung alveolar epithelium to cigarette smoke exposure

Azithromycin effectiveness against intracellular infections of Francisella

<p>Abstract</p> <p>Background</p> <p>Macrolide antibiotics are commonly administered for bacterial respiratory illnesses. Azithromycin (Az) is especially noted for extremely high intracellular concentrations achieved within macrophages which is far greater than the serum concentration. Clinical strains of Type B <it>Francisella </it>(<it>F.</it>) <it>tularensis </it>have been reported to be resistant to Az, however our laboratory <it>Francisella </it>strains were found to be sensitive. We hypothesized that different strains/species of <it>Francisella </it>(including Type A) may have different susceptibilities to Az, a widely used and well-tolerated antibiotic.</p> <p>Results</p> <p><it>In vitro </it>susceptibility testing of Az confirmed that <it>F. tularensis subsp. holarctica </it>Live Vaccine Strain (LVS) (Type B) was not sensitive while <it>F. philomiragia, F. novicida</it>, and Type A <it>F. tularensis </it>(NIH B38 and Schu S4 strain) were susceptible. In J774A.1 mouse macrophage cells infected with <it>F. philomiragia, F. novicida</it>, and <it>F. tularensis </it>LVS, 5 μg/ml Az applied extracellularly eliminated intracellular <it>Francisella </it>infections. A concentration of 25 μg/ml Az was required for <it>Francisella-</it>infected A549 human lung epithelial cells, suggesting that macrophages are more effective at concentrating Az than epithelial cells. Mutants of RND efflux components (<it>tolC </it>and <it>ftlC</it>) in <it>F. novicida </it>demonstrated less sensitivity to Az by MIC than the parental strain, but the <it>tolC </it>disc-inhibition assay demonstrated increased sensitivity, indicating a complex role for the outer-membrane transporter. Mutants of <it>acrA </it>and <it>acrB </it>mutants were less sensitive to Az than the parental strain, suggesting that AcrAB is not critical for the efflux of Az in <it>F. novicida</it>. In contrast, <it>F. tularensis </it>Schu S4 mutants Δ<it>acrB </it>and Δ<it>acrA </it>were more sensitive than the parental strain, indicating that the AcrAB may be important for Az efflux in <it>F. tularensis </it>Schu S4. <it>F. novicida </it>LPS O-antigen mutants (<it>wbtN, wbtE, wbtQ </it>and <it>wbtA</it>) were found to be less sensitive <it>in vitro </it>to Az compared to the wild-type. Az treatment prolonged the survival of <it>Galleria </it>(<it>G</it>.) <it>mellonella </it>infected with <it>Francisella</it>.</p> <p>Conclusion</p> <p>These studies demonstrate that Type A <it>Francisella </it>strains, as well as <it>F. novicida </it>and <it>F. philomiragia</it>, are sensitive to Az <it>in vitro. Francisella </it>LPS and the RND efflux pump may play a role in Az sensitivity. Az also has antimicrobial activity against intracellular <it>Francisella</it>, suggesting that the intracellular concentration of Az is high enough to be effective against multiple strains/species of <it>Francisella</it>, especially in macrophages. Az treatment prolonged survival an <it>in vivo </it>model of <it>Francisella-</it>infection.</p

Risk governance of potential emerging risks to drinking water quality: Analysing current practices

The presence of emerging contaminants in the aquatic environment may affect human health via exposure to drinking water. And, even if some of these emerging contaminants are not a threat to human health, their presence might still influence the public perception of drinking water quality. Over the last decades, much research has been done on emerging contaminants in the aquatic environment, most of which has focused on the identification of emerging contaminants and the characterisation of their toxic potential. However, only limited information is available on if, and how, scientific information is implemented in current policy approaches. The opportunities for science to contribute to the policy of emerging contaminants in drinking water have, therefore, not yet been identified. A comparative analysis was performed of current approaches to the risk governance of emerging chemical contaminants in drinking water (resources) to identify any areas for improvement. The policy approaches used in the Netherlands, Germany, Switzerland and the state of Minnesota were analysed using the International Risk Governance Council framework as a normative concept. Quality indicators for the analysis were selected based on recent literature. Information sources used were scientific literature, policy documents, and newspaper articles. Subsequently, suggestions for future research for proactive risk governance are given. Suggestions include the development of systematic analytical approaches to various information sources so that potential emerging contaminants to drinking water quality can be identified quickly. In addition, an investigation into the possibility and benefit of including the public concern about emerging contaminants into the risk governance process was encouraged.</p

Kinetic Characterization and Phosphoregulation of the Francisella tularensis 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (MEP Synthase)

Deliberate and natural outbreaks of infectious disease underscore the necessity of effective vaccines and antimicrobial/antiviral therapeutics. The prevalence of antibiotic resistant strains and the ease by which antibiotic resistant bacteria can be intentionally engineered further highlights the need for continued development of novel antibiotics against new bacterial targets. Isoprenes are a class of molecules fundamentally involved in a variety of crucial biological functions. Mammalian cells utilize the mevalonic acid pathway for isoprene biosynthesis, whereas many bacteria utilize the methylerythritol phosphate (MEP) pathway, making the latter an attractive target for antibiotic development. In this report we describe the cloning and characterization of Francisella tularensis MEP synthase, a MEP pathway enzyme and potential target for antibiotic development. In vitro growth-inhibition assays using fosmidomycin, an inhibitor of MEP synthase, illustrates the effectiveness of MEP pathway inhibition with F. tularensis. To facilitate drug development, F. tularensis MEP synthase was cloned, expressed, purified, and characterized. Enzyme assays produced apparent kinetic constants (KMDXP = 104 µM, KMNADPH = 13 µM, kcatDXP = 2 s−1, kcatNADPH = 1.3 s−1), an IC50 for fosmidomycin of 247 nM, and a Ki for fosmidomycin of 99 nM. The enzyme exhibits a preference for Mg+2 as a divalent cation. Titanium dioxide chromatography-tandem mass spectrometry identified Ser177 as a site of phosphorylation. S177D and S177E site-directed mutants are inactive, suggesting a mechanism for post-translational control of metabolic flux through the F. tularensis MEP pathway. Overall, our study suggests that MEP synthase is an excellent target for the development of novel antibiotics against F. tularensis

Novel Insights into Autism Knowledge and Stigmatizing Attitudes Toward Mental Illness in Dutch Youth and Family Center Physicians

Education and Child Studie

Screening for abnormal glycosylation in a cohort of adult liver disease patients

Congenital Disorders of Glycosylation (CDG) are a rapidly expanding group of rare genetic defects in glycosylation. In a novel CDG subgroup of Vacuolar-ATPase assembly defects various degrees of hepatic injury have been described, including end stage liver disease. However, the CDG diagnostic workflow can be complex as liver disease per se may be associated with abnormal glycosylation. Therefore, we collected serum samples of patients with a wide range of liver pathology to study the performance and yield of two CDG screening methods. Our aim was to identify glycosylation patterns that could help to differentiate between primary and secondary glycosylation defects in liver disease. To this end, we analyzed serum samples of 1042 adult liver disease patients. This cohort consisted of 567 liver transplant candidates and 475 chronic liver disease patients. Our workflow consisted of screening for abnormal glycosylation by transferrin isoelectric focusing (tIEF), followed by in-depth analysis of the abnormal samples with quadruple time-of-flight mass spectrometry (QTOF-MS). Screening with tIEF resulted in identification of 247 (26%) abnormal samples. QTOF-MS analysis of 110 of those did not reveal glycosylation abnormalities comparable with those seen in V-ATPase assembly factor deficiencies. However, two patients presented with isolated sialylation deficiency. Fucosylation was significantly increased in liver transplant candidates compared to healthy controls and patients with chronic liver disease. In conclusion, a significant percentage of patients with liver disease presented with abnormal CDG screening results, however, not indicative for a V-ATPase assembly factor defect. Advanced glycoanalytical techniques assist in the dissection of secondary and primary glycosylation defects. This article is protected by copyright. All rights reserved

Parotid Gland Stem Cell Sparing Radiation Therapy for Patients With Head and Neck Cancer:A Double-Blind Randomized Controlled Trial

BACKGROUND: Radiotherapy for head and neck cancer (HNC) frequently leads to salivary gland damage and subsequent xerostomia. The radiation response of parotid glands of rats, mice, and patients critically depends on dose to its stem cells, mainly located in the gland's main ducts (stem cell rich (SCR) region). Therefore, this double-blind randomized controlled trial aimed to test the hypothesis that parotid gland stem cell sparing radiotherapy preserves parotid gland function better than currently-used whole parotid gland sparing radiotherapy. METHODS: HNC patients (n=102) treated with definitive radiotherapy were randomized between standard parotid sparing and stem cell sparing (SCS) techniques. The primary endpoint was >75% reduction in parotid gland saliva production compared to pretreatment production (FLOW12M). Secondary endpoints were several aspects of xerostomia 12 months after treatment. RESULTS: Fifty-four patients were assigned to the standard arm and 48 to the SCS arm. Only dose to the SCR regions (contralateral 16 and 11 Gy (p=0.004) and ipsilateral 26 and 16 Gy (p=0.001), standard and SCS arm respectively) and pretreatment patient-rated daytime xerostomia (35% and 13% (p=0.01), standard and SCS arm respectively) differed significantly between the arms. In the SCS arm, 1 patient (2.8%) experienced FLOW12M compared to 2 (4.9%) in the standard arm (p=1.00). However, a trend towards better relative parotid gland salivary function in favor of SCS radiotherapy was shown. Moreover, multivariable analysis showed that mean contralateral SCR region dose was the strongest dosimetric predictor for moderate-to-severe patient-rated daytime xerostomia and grade ≥2 physician-rated xerostomia, the latter including complaints of alteration in diet. CONCLUSIONS: No significant better parotid function was observed in SCS radiotherapy. However, additional multivariable analysis showed that dose to the SCR region was more predictive for development of parotid gland function related xerostomia endpoints, than dose to the entire parotid gland

Lipoprotein(a) plasma levels are not associated with incident microvascular complications in type 2 diabetes mellitus

Aims/hypothesis: Microvascular disease in type 2 diabetes is a significant cause of end-stage renal disease, blindness and peripheral neuropathy. The strict control of known risk factors, e.g. lifestyle, hyperglycaemia, hypertension and dyslipidaemia, reduces the incidence of microvascular complications, but a residual risk remains. Lipoprotein (a) [Lp(a)] is a strong risk factor for macrovascular disease in the general population. We hypothesised that plasma Lp(a) levels and the LPA gene SNPs rs10455872 and rs3798220 are associated with the incident development of microvascular complications in type 2 diabetes. Methods: Analyses were performed of data from the DiaGene study, a prospective study for complications of type 2 diabetes, collected in the city of Eindhoven, the Netherlands (n = 1886 individuals with type 2 diabetes, mean follow-up time = 6.97 years). To assess the relationship between plasma Lp(a) levels and the LPA SNPs with each newly developed microvascular complication (retinopathy n = 223, nephropathy n = 246, neuropathy n = 236), Cox proportional hazards models were applied and adjusted for risk factors for microvascular complications (age, sex, mean arterial pressure, non-HDL-cholesterol, HDL-cholesterol, BMI, duration of type 2 diabetes, HbA1c and smoking). Results: No significant associations of Lp(a) plasma levels and the LPA SNPs rs10455872 and rs3798220 with prevalent or incident microvascular complications in type 2 diabetes were found. In line with previous observations the LPA SNPs rs10455872 and rs3798220 did influence the plasma Lp(a) levels. Conclusions/interpretation: Our data show no association between Lp(a) plasma levels and the LPA SNPs with known effect on Lp(a) plasma levels with the development of microvascular complications in type 2 diabetes. This indicates that Lp(a) does not play a major role in the development of microvascular complications. However, larger studies are needed to exclude minimal effects of Lp(a) on the development of microvascular complications