Binding of an antimicrobial peptide to bacterial cells: interaction with different species, strains and cellular components

Antimicrobial peptides (AMPs) selectively kill bacteria by disrupting their cell membranes, and are promising compounds to fight drug-resistant microbes. Biophysical studies on model membranes have characterized AMP/membrane interactions and the mechanism of bilayer perturbation, showing that accumulation of cationic peptide molecules in the external leaflet leads to the formation of pores ("carpet" mechanism). However, similar quantitative studies on real cells are extremely limited. Here, we investigated the interaction of the dansylated PMAP23 peptide (DNS-PMAP23) with a Gram-positive bacterium, showing that 10(7) bound peptide molecules per cell are needed to kill it. This result is consistent with our previous finding for Gram-negative strains, where a similar high threshold for killing was determined, demonstrating the general relevance of the carpet model for real bacteria. However, in the case of the Gram-positive strain, this number of molecules even exceeds the total surface available on the bacterial membrane. The high affinity of DNS-PMAP23 for the anionic teichoic acids of the Gram-positive cell wall, but not for the lipopolysaccharides of Gram-negative bacteria, provides a rationale for this finding. To better define the role of anionic lipids in peptide/cell association, we studied DNS-PMAP23 interaction with E. coli mutant strains lacking phosphatidylglycerol and/or cardiolipin. Surprisingly, these strains showed a peptide affinity similar to that of the wild type. This finding was rationalized by observing that these bacteria have an increased content of other anionic lipids, thus maintaining the total membrane charge essentially constant. Finally, studies of DNS-PMAP23 association to dead bacteria showed an affinity an order of magnitude higher compared to that of live cells, suggesting strong peptide binding to intracellular components that become accessible after membrane perturbation. This effect could play a role in population resistance to AMP action, since dead bacteria could protect the surviving cells by sequestering significant amounts of peptide molecules. Overall, our data indicate that quantitative studies of peptide association to bacteria can lead to a better understanding of the mechanism of action of AMPs

Endodontic Re-treatment of a Tooth with a Floor Perforation in a Patient in Treatment with Oral Bisphosphonate

Prevention of bisphosphonate related osteonecrosis of the jaws (BRONJ) represents an important challenge for clinicians. Dent alveolar surgery is usually performed to reduce the risk local and systemic complications, In fact, removing the tooth that is the source of the infectious outbreak, brings to the removal of the infected tissue and determines a correct healing of the disease. However, in patients are undergoing bisphosphonate this procedure expose patients to an increased risk of BRONJ. However, in patients who are in treatment with systemic bisphosphonates, this procedure exposes them to an increased risk of BRONJ. When possible, avoid the surgical interventions in favors of endodontic treatment could be an important clinical options in reducing the possibility of BRONJ occurrence. In this paper, we report a case of endodontic re-treatment in a tooth with a floor perforation in a patient was assuming oral alendronate. Limits and advantages of performing endodontic retreatment for the prevention of BRONJ development have still not well elucidated. Due to the very low power of this study, no conclusions should be made to perform an endodontic retreatment instead of performing a tooth extraction. However, this case report support the execution of future studies related to the calculation of BRONJ incidence after surgical extraction vs endodontic retreatment

Lower cardiorespiratory fitness contributes to increased insulin resistance and fasting glycaemia in middle-aged South Asian compared with European men living in the UK

AIMS/HYPOTHESIS: This study aimed to determine the extent to which increased insulin resistance and fasting glycaemia in South Asian men, compared with white European men, living in the UK, was due to lower cardiorespiratory fitness (maximal oxygen uptake [[Formula: see text]]) and physical activity. METHODS: One hundred South Asian and 100 age- and BMI-matched European men without diagnosed diabetes, aged 40–70 years, had fasted blood taken for measurement of glucose concentration, HOMA-estimated insulin resistance (HOMA(IR)), plus other risk factors, and underwent assessment of physical activity (using accelerometry), [Formula: see text], body size and composition, and demographic and other lifestyle factors. For 13 South Asian and one European man, HbA(1c) levels were >6.5% (>48 mmol/mol), indicating potential undiagnosed diabetes; these men were excluded from the analyses. Linear regression models were used to determine the extent to which body size and composition, fitness and physical activity variables explained differences in HOMA(IR) and fasting glucose between South Asian and European men. RESULTS: HOMA(IR) and fasting glucose were 67% (p < 0.001) and 3% (p < 0.018) higher, respectively, in South Asians than Europeans. Lower [Formula: see text], lower physical activity and greater total adiposity in South Asians individually explained 68% (95% CI 45%, 91%), 29% (11%, 46%) and 52% (30%, 80%), respectively, and together explained 83% (50%, 119%) (all p < 0.001) of the ethnic difference in HOMA(IR). Lower [Formula: see text] and greater total adiposity, respectively, explained 61% (9%, 111%) and 39% (9%, 76%) (combined effect 63% [8%, 115%]; all p < 0.05) of the ethnic difference in fasting glucose. CONCLUSIONS/INTERPRETATION: Lower cardiorespiratory fitness is a key factor associated with the excess insulin resistance and fasting glycaemia in middle-aged South Asian, compared with European, men living in the UK. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00125-013-2969-y) contains peer-reviewed but unedited supplementary material, which is available to authorised users

Measurement of the angular correlation between the two gamma rays emitted in the radioactive decays of a 60^{60}Co source with two NaI(Tl) scintillator

We implemented a didactic experiment to study the angular correlation between the two gamma rays emitted in typical $^{60}$Co radioactive decays. We used two NaI(Tl) scintillators, already available in our laboratory, and a low-activity $^{60}$Co source. The detectors were mounted on two rails, with the source at their center. The first rail was fixed, while the second could be rotated around the source. We performed several measurements by changing the angle between the two scintillators in the range from $90^\circ$ to $180^\circ$. Dedicated background runs were also performed, removing the source from the experimental setup. We found that the signal rate increases with the angular separation between the two scintillators, with small discrepancies from the theoretical expectations.Comment: 15 pages, 12 figure

Oral High-Dose Atorvastatin Treatment in Relapsing-Remitting Multiple Sclerosis

BACKGROUND:Recent data from animal models of multiple sclerosis (MS) and from a pilot study indicated a possible beneficial impact of statins on MS. METHODOLOGY/PRINCIPAL FINDINGS:Safety, tolerability and effects on disease activity of atorvastatin given alone or in combination with interferon-beta (IFN-beta) were assessed in a phase II open-label baseline-to-treatment trial in relapsing-remitting MS (RRMS). Patients with at least one gadolinium-enhancing lesion (CEL) at screening by magnetic resonance imaging (MRI) were eligible for the study. After a baseline period of 3 monthly MRI scans (months -2 to 0), patients followed a 9-month treatment period on 80 mg atorvastatin daily. The number of CEL in treatment months 6 to 9 compared to baseline served as the primary endpoint. Other MRI-based parameters as well as changes in clinical scores and immune responses served as secondary endpoints. Of 80 RRMS patients screened, 41 were included, among them 16 with IFN-beta comedication. The high dose of 80 mg atorvastatin was well tolerated in the majority of patients, regardless of IFN-beta comedication. Atorvastatin treatment led to a substantial reduction in the number and volume of CEL in two-sided multivariate analysis (p = 0.003 and p = 0.008). A trend towards a significant decrease in number and volume of CEL was also detected in patients with IFN-beta comedication (p = 0.060 and p = 0.062), in contrast to patients without IFN-beta comedication (p = 0.170 and p = 0.140). Immunological investigations showed no suppression in T cell response but a significant increase in IL-10 production. CONCLUSIONS/SIGNIFICANCE:Our data suggest that high-dose atorvastatin treatment in RRMS is safe and well tolerated. Moreover, MRI analysis indicates a possible beneficial effect of atorvastatin, alone or in combination with IFN-beta, on the development of new CEL. Thus, our findings provide a rationale for phase II/III trials, including combination of atorvastatin with already approved immunomodulatory therapy regimens. TRIAL REGISTRATION:ClinicalTrials.gov NCT00616187

Development of a questionnaire to assess sedentary time in older persons -- a comparative study using accelerometry

Background: There is currently no validated questionnaire available to assess total sedentary time in older adults. Most studies only used TV viewing time as an indicator of sedentary time. The first aim of our study was to investigate the self-reported time spent by older persons on a set of sedentary activities, and to compare this with objective sedentary time measured by accelerometry. The second aim was to determine what set of self-reported sedentary activities should be used to validly rank people's total sedentary time. Finally we tested the reliability of our newly developed questionnaire using the best performing set of sedentary activities. Methods. The study sample included 83 men and women aged 65-92 y, a random sample of Longitudinal Aging Study Amsterdam participants, who completed a questionnaire including ten sedentary activities and wore an Actigraph GT3X accelerometer for 8 days. Spearman correlation coefficients were calculated to examine the association between self-reported time and objective sedentary time. The test-retest reliability was calculated using the intraclass correlation coefficient (ICC). Results: Mean total self-reported sedentary time was 10.4 (SD 3.5) h/d and was not significantly different from mean total objective sedentary time (10.2 (1.2) h/d, p = 0.63). Total self-reported sedentary time on an average day (sum of ten activities) correlated moderately (Spearman's r = 0.35, p < 0.01) with total objective sedentary time. The correlation improved when using the sum of six activities (r = 0.46, p < 0.01), and was much higher than when using TV watching only (r = 0.22, p = 0.05). The test-retest reliability of the sum of six sedentary activities was 0.71 (95% CI 0.57-0.81). Conclusions: A questionnaire including six sedentary activities was moderately associated with accelerometry-derived sedentary time and can be used to reliably rank sedentary time in older persons. © 2013 Visser and Koster; licensee BioMed Central Ltd

To treat or not to treat: comparison of different criteria used to determine whether weight loss is to be recommended

Background: Excess body fat is a major risk factor for disease primarily due to its endocrine activity. In recent years several criteria have been introduced to evaluate this factor. Nevertheless, treatment need is currently assessed only on the basis of an individual's Body Mass Index (BMI), calculated as body weight (in kg) divided by height in m2. The aim of our study was to determine whether application of the BMI, compared to adiposity-based criteria, results in underestimation of the number of subjects needing lifestyle intervention. Methods: We compared treatment need based on BMI classification with four adiposity-based criteria: percentage body fat (%BF), considered both alone and in relation to metabolic syndrome risk (MS), waist circumference (WC), as an index of abdominal fat, and Body Fat Mass Index (BFMI, calculated as fat mass in kg divided by height in m2) in 63 volunteers (23 men and 40 women, aged 20 – 65 years). Results: According to the classification based on BMI, 6.3% of subjects were underweight, 52.4% were normal weight, 30.2% were overweight, and 11.1% were obese. Agreement between the BMI categories and the other classification criteria categories varied; the most notable discrepancy emerged in the underweight and overweight categories. BMI compared to almost all of the other adiposity-based criteria, identified a lower percentage of subjects for whom treatment would be recommended. In particular, the proportion of subjects for whom clinicians would strongly recommend weight loss on the basis of their BMI (11.1%) was significantly lower than those identified according to WC (25.4%, p = 0.004), %BF (28.6%, p = 0.003), and MS (33.9%, p = 0.002). Conclusion: The use of the BMI alone, as opposed to an assessment based on body composition, to identify individuals needing lifestyle intervention may lead to unfortunate misclassifications. Population-specific data on the relationships between body composition, morbidity, and mortality are needed to improve the diagnosis and treatment of at-risk individual

Preliminary analysis of immune activation in early onset type 2 diabetes

Introduction. First Nations and other Aboriginal children are disproportionately affected by cardiometabolic diseases, including type 2 diabetes (T2D). In T2D, the disruption of insulin signalling can be driven by pro-inflammatory immunity. Pro-inflammatory responses can be fueled by toll-like receptors (TLR) on immune cells such as peripheral blood mononuclear cells (PBMC, a white blood cell population). TLR4 can bind to lipids from bacteria and food sources activating PBMC to produce cytokines tumour necrosis factor (TNF)-&#x03B1; and interleukin (IL)-1&#x03B2;. These cytokines can interfere with insulin signalling. Here, we seek to understand how TLR4 activation may be involved in early onset T2D. We hypothesized that immune cells from youth with T2D (n=8) would be more reactive upon TLR4 stimulation relative to cells from age and body mass index (BMI)-matched controls without T2D (n=8). Methods. Serum samples were assayed for adipokines (adiponectin and leptin), as well as cytokines. Freshly isolated PBMC were examined for immune reactivity upon culture with TLR4 ligands bacterial lipopolysaccharide (LPS, 2 and 0.2 ng/ml) and the fatty acid palmitate (200 &#x00B5;M). Culture supernatants were evaluated for the amount of TNF-&#x03B1; and IL-1&#x03B2; produced by PBMC. Results. Youth with T2D displayed lower median serum adiponectin levels compared to controls (395 vs. 904 ng/ml, p&#x003C;0.05). PBMC isolated from youth with and without T2D produced similar levels of TNF-&#x03B1; and IL-1&#x03B2; after exposure to the higher LPS concentration. However, at the low LPS dose the T2D cohort exhibited enhanced IL-1&#x03B2; synthesis relative to the control cohort. Additionally, exposure to palmitate resulted in greater IL-1&#x03B2; synthesis in PBMCs isolated from youth with T2D versus controls (p&#x003C;0.05). These differences in cytokine production corresponded to greater monocyte activation in the T2D cohort. Conclusion. These preliminary results suggest that cellular immune responses are exaggerated in T2D, particularly with respect to IL-1&#x03B2; activity. These studies aim to improve the understanding of the biology behind early onset T2D and its vascular complications that burden First Nations people