Phosphonopeptides Revisited, in an Era of Increasing Antimicrobial Resistance

Given the increase in resistance to antibacterial agents, there is an urgent need for the development of new agents with novel modes of action. As an interim solution, it is also prudent to reinvestigate old or abandoned antibacterial compounds to assess their efficacy in the context of widespread resistance to conventional agents. In the 1970s, much work was performed on the development of peptide mimetics, exemplified by the phosphonopeptide, alafosfalin. We investigated the activity of alafosfalin, di-alanyl fosfalin and β-chloro-L-alanyl-β-chloro-L-alanine against 297 bacterial isolates, including carbapenemase-producing Enterobacterales (CPE) (n = 128), methicillin-resistant Staphylococcus aureus (MRSA) (n = 37) and glycopeptide-resistant enterococci (GRE) (n = 43). The interaction of alafosfalin with meropenem was also examined against 20 isolates of CPE. The MIC50 and MIC90 of alafosfalin for CPE were 1 mg/L and 4 mg/L, respectively and alafosfalin acted synergistically when combined with meropenem against 16 of 20 isolates of CPE. Di-alanyl fosfalin showed potent activity against glycopeptide-resistant isolates of Enterococcus faecalis (MIC90; 0.5 mg/L) and Enterococcus faecium (MIC90; 2 mg/L). Alafosfalin was only moderately active against MRSA (MIC90; 8 mg/L), whereas β-chloro-L-alanyl-β-chloro-L-alanine was slightly more active (MIC90; 4 mg/L). This study shows that phosphonopeptides, including alafosfalin, may have a therapeutic role to play in an era of increasing antibacterial resistance

Swift Observations of GRB 050603: An afterglow with a steep late time decay slope

We report the results of Swift observations of the Gamma Ray Burst GRB 050603. With a V magnitude V=18.2 about 10 hours after the burst the optical afterglow was the brightest so far detected by Swift and one of the brightest optical afterglows ever seen. The Burst Alert Telescope (BAT) light curves show three fast-rise-exponential-decay spikes with $T_{90}$=12s and a fluence of 7.6$\times 10^{-6}$ ergs cm$^{-2}$ in the 15-150 keV band. With an $E_{\rm \gamma, iso} = 1.26 \times 10^{54}$ ergs it was also one of the most energetic bursts of all times. The Swift spacecraft began observing of the afterglow with the narrow-field instruments about 10 hours after the detection of the burst. The burst was bright enough to be detected by the Swift UV/Optical telescope (UVOT) for almost 3 days and by the X-ray Telescope (XRT) for a week after the burst. The X-ray light curve shows a rapidly fading afterglow with a decay index $\alpha$=1.76$^{+0.15}_{-0.07}$. The X-ray energy spectral index was $\beta_{\rm X}$=0.71\plm0.10 with the column density in agreement with the Galactic value. The spectral analysis does not show an obvious change in the X-ray spectral slope over time. The optical UVOT light curve decays with a slope of $\alpha$=1.8\plm0.2. The steepness and the similarity of the optical and X-ray decay rates suggest that the afterglow was observed after the jet break. We estimate a jet opening angle of about 1-2$^{\circ}$Comment: 14 pages, accepted for publication in Ap

Nonthermal Hard X-ray Emission and Iron Kalpha Emission from a Superflare on II Pegasi

We report on an X-ray flare detected on the active binary system II~Pegasi with the Swift telescope. The trigger had a 10-200 keV luminosity of 2.2$\times10^{32}$ erg s$^{-1}$-- a superflare, by comparison with energies of typical stellar flares on active binary systems. The trigger spectrum indicates a hot thermal plasma with T$\sim$180 $\times10^{6}$K. X-ray spectral analysis from 0.8--200 keV with the X-Ray Telescope and BAT in the next two orbits reveals evidence for a thermal component (T$>$80 $\times10^{6}$K) and Fe K 6.4 keV emission. A tail of emission out to 200 keV can be fit with either an extremely high temperature thermal plasma (T$\sim3\times10^{8}$K) or power-law emission. Based on analogies with solar flares, we attribute the excess continuum emission to nonthermal thick-target bremsstrahlung emission from a population of accelerated electrons. We estimate the radiated energy from 0.01--200 keV to be $\sim6\times10^{36}$ erg, the total radiated energy over all wavelengths $\sim10^{38}$ erg, the energy in nonthermal electrons above 20 keV $\sim3\times10^{40}$ erg, and conducted energy $<5\times10^{43}$ erg. The nonthermal interpretation gives a reasonable value for the total energy in electrons $>$ 20 keV when compared to the upper and lower bounds on the thermal energy content of the flare. This marks the first occasion in which evidence exists for nonthermal hard X-ray emission from a stellar flare. We investigate the emission mechanism responsible for producing the 6.4 keV feature, and find that collisional ionization from nonthermal electrons appears to be more plausible than the photoionization mechanism usually invoked on the Sun and pre-main sequence stars.Comment: 41 pages, 7 figures, accepted for publication in the Astrophysical Journa

Excess Mucin Impairs Subglottic Epithelial Host Defense in Mechanically Ventilated Patients

Rationale: Aspiration of infective subglottic secretions causes ventilator-associated pneumonia (VAP) in mechanically ventilated patients. Mechanisms underlying subglottic colonization in critical illness have not been defined, limiting strategies for targeted prevention of VAP. Objectives: To characterize subglottic host defense dysfunction in mechanically ventilated patients in the intensive care unit (ICU). To determine whether subglottic mucin contributes to neutrophil phagocytic impairment and bacterial growth. Methods: Prospective subglottic sampling in mechanically ventilated patients (intubated for four or more days), and newly intubated control patients (intubated for less than 30 minutes). Isolation and culture of primary subglottic epithelial cells from controls. Laboratory analysis of host innate immune defenses. Measurements and Main Results: Twenty-four patients in the ICU and 27 newly intubated control patients were studied. Subglottic ICU samples had significantly reduced microbiological diversity and contained potential respiratory pathogens. The subglottic microenvironment in ICU was characterized by neutrophilic inflammation, significantly increased pro-inflammatory cytokines and neutrophil proteases, and altered physical properties of subglottic secretions, including accumulation of mucins. Subglottic mucin from ICU patients impaired the capacity of neutrophils to phagocytose and kill bacteria. Phagocytic function was reversible upon treatment with a mucolytic agent. Subglottic mucus promoted growth and invasion of bacterial pathogens in a novel air-liquid interface model of primary human subglottic epithelium. Conclusions: Mechanical ventilation in ICU is characterized by substantial mucin secretion and neutrophilic inflammation. Mucin impairs neutrophil dysfunction and promotes bacterial growth. Mucolytic agents reverse mucin-mediated neutrophil dysfunction. Enhanced mucus disruption and removal has potential to augment preventive benefits of subglottic drainage

Design of cohort studies in chronic diseases using routinely collected databases when a prescription is used as surrogate outcome

BACKGROUND: There has been little research on design of studies based on routinely collected data when the clinical endpoint of interest is not recorded, but can be inferred from a prescription. This often happens when exploring the effect of a drug on chronic diseases. Using the LifeLink claims database in studying the possible anti-inflammatory effects of statins in rheumatoid arthritis (RA), oral steroids (OS) were treated as surrogate of inflammatory flare-ups. We compared two cohort study designs, the first using time to event outcomes and the second using quantitative amount of the surrogate. METHODS: RA patients were extracted from the LifeLink database. In the first study, patients were split into two sub-cohorts based on whether they were using OS within a specified time window of the RA index date (first record of RA). Using Cox models we evaluated the association between time-varying exposure to statins and (i) initiation of OS therapy in the non-users of OS at RA index date and (ii) cessation of OS therapy in the users of OS at RA index date. In the second study, we matched new statin users to non users on age and sex. Zero inflated negative binomial models were used to contrast the number of days' prescriptions of OS in the year following date of statin initiation for the two exposure groups. RESULTS: In the unmatched study, the statin exposure hazard ratio (HR) of initiating OS in the 31451 non-users of OS at RA index date was 0.96(95% CI 0.9,1.1) and the statin exposure HR of cessation of OS therapy in the 6026 users of OS therapy at RA index date was 0.95 (0.87,1.05). In the matched cohort of 6288 RA patients the statin exposure rate ratio for duration on OS therapy was 0.88(0.76,1.02). There was digit preference for outcomes in multiples of 7 and 30 days. CONCLUSIONS: The 'time to event' study design was preferable because it better exploits information on all available patients and provides a degree of robustness toward confounding. We found no convincing evidence that statins reduce inflammation in RA patients

A long-lasting measles epidemic in Maroua, Cameroon 2008-2009: mass vaccination as response to the epidemic.

A measles outbreak occurred in Maroua, Cameroon, from January 2008 to April 2009. In accordance with recent World Health Organization guidelines, an outbreak-response immunization (ORI) was conducted in January 2009. The aim of this study was to investigate the causes of the epidemic in order to guide vaccination strategies

Viridot: An automated virus plaque (immunofocus) counter for the measurement of serological neutralizing responses with application to dengue virus.

The gold-standard method for quantifying neutralizing antibody responses to many viruses, including dengue virus (DENV), is the plaque reduction neutralization test (PRNT, also called the immunofocus reduction neutralization test). The PRNT conducted on 96-well plates is high-throughput and requires a smaller volume of antiserum than on 6- or 24-well plates, but manual plaque counting is challenging and existing automated plaque counters are expensive or difficult to optimize. We have developed Viridot (Viridot package), a program for R with a user interface in shiny, that counts viral plaques of a variety of phenotypes, estimates neutralizing antibody titers, and performs other calculations of use to virologists. The Viridot plaque counter includes an automatic parameter identification mode (misses <10 plaques/well for 87% of diverse DENV strains [n = 1521]) and a mode that allows the user to fine-tune the parameters used for counting plaques. We compared standardized manual and Viridot plaque counting methods applied to the same wells by two analyses and found that Viridot plaque counts were as similar to the same analyst's manual count (Lin's concordance correlation coefficient, ρc = 0.99 [95% confidence interval: 0.99-1.00]) as manual counts between analysts (ρc = 0.99 [95% CI: 0.98-0.99]). The average ratio of neutralizing antibody titers based on manual counted plaques to Viridot counted plaques was 1.05 (95% CI: 0.98-1.14), similar to the average ratio of antibody titers based on manual plaque counts by the two analysts (1.06 [95% CI: 0.84-1.34]). Across diverse DENV and ZIKV strains (n = 14), manual and Viridot plaque counts were mostly consistent (range of ρc = 0.74 to 1.00) and the average ratio of antibody titers based on manual and Viridot counted plaques was close to 1 (0.94 [0.86-1.02]). Thus, Viridot can be used for plaque counting and neutralizing antibody titer estimation of diverse DENV strains and potentially other viruses on 96-well plates as well as for formalization of plaque-counting rules for standardization across experiments and analysts

Development of the preterm gut microbiome in twins at risk of necrotising enterocolitis and sepsis

The preterm gut microbiome is a complex dynamic community influenced by genetic and environmental factors and is implicated in the pathogenesis of necrotising enterocolitis (NEC) and sepsis. We aimed to explore the longitudinal development of the gut microbiome in preterm twins to determine how shared environmental and genetic factors may influence temporal changes and compared this to the expressed breast milk (EBM) microbiome. Stool samples (n = 173) from 27 infants (12 twin pairs and 1 triplet set) and EBM (n = 18) from 4 mothers were collected longitudinally. All samples underwent PCR-DGGE (denaturing gradient gel electrophoresis) analysis and a selected subset underwent 454 pyrosequencing. Stool and EBM shared a core microbiome dominated by Enterobacteriaceae, Enterococcaceae, and Staphylococcaceae. The gut microbiome showed greater similarity between siblings compared to unrelated individuals. Pyrosequencing revealed a reduction in diversity and increasing dominance of Escherichia sp. preceding NEC that was not observed in the healthy twin. Antibiotic treatment had a substantial effect on the gut microbiome, reducing Escherichia sp. and increasing other Enterobacteriaceae. This study demonstrates related preterm twins share similar gut microbiome development, even within the complex environment of neonatal intensive care. This is likely a result of shared genetic and immunomodulatory factors as well as exposure to the same maternal microbiome during birth, skin contact and exposure to EBM. Environmental factors including antibiotic exposure and feeding are additional significant determinants of community structure, regardless of host genetics

Impacts of organic and conventional crop management on diversity and activity of free-living nitrogen fixing bacteria and total bacteria are subsidiary to temporal effects

A three year field study (2007-2009) of the diversity and numbers of the total and metabolically active free-living diazotophic bacteria and total bacterial communities in organic and conventionally managed agricultural soil was conducted at the Nafferton Factorial Systems Comparison (NFSC) study, in northeast England. The result demonstrated that there was no consistent effect of either organic or conventional soil management across the three years on the diversity or quantity of either diazotrophic or total bacterial communities. However, ordination analyses carried out on data from each individual year showed that factors associated with the different fertility management measures including availability of nitrogen species, organic carbon and pH, did exert significant effects on the structure of both diazotrophic and total bacterial communities. It appeared that the dominant drivers of qualitative and quantitative changes in both communities were annual and seasonal effects. Moreover, regression analyses showed activity of both communities was significantly affected by soil temperature and climatic conditions. The diazotrophic community showed no significant change in diversity across the three years, however, the total bacterial community significantly increased in diversity year on year. Diversity was always greatest during March for both diazotrophic and total bacterial communities. Quantitative analyses using qPCR of each community indicated that metabolically active diazotrophs were highest in year 1 but the population significantly declined in year 2 before recovering somewhat in the final year. The total bacterial population in contrast increased significantly each year. Seasonal effects were less consistent in this quantitative study

Active vitamin D (1,25-dihydroxyvitamin D) and bone health in middle-aged and elderly men: the European male aging study (EMAS)

&lt;p&gt;Context: There is little information on the potential impact of serum 1,25-dihydroxyvitamin D [1,25(OH)2D] on bone health including turnover.&lt;/p&gt; &lt;p&gt;Objective: The objective of the study was to determine the influence of 1,25(OH)2D and 25-hydroxyvitamin D [25(OH)D] on bone health in middle-aged and older European men.&lt;/p&gt; &lt;p&gt;Design, Setting, and Participants: Men aged 40–79 years were recruited from population registers in 8 European centers. Subjects completed questionnaires that included questions concerning lifestyle and were invited to attend for quantitative ultrasound (QUS) of the heel, assessment of height and weight, and a fasting blood sample from which 1,25(OH)2D, 25(OH)D, and PTH were measured. 1,25(OH)2D was measured using liquid chromatography tandem mass spectrometry. Bone markers serum N-terminal propeptide of type 1 procollagen (P1NP) and crosslinks (β-cTX) were also measured. Dual-energy x-ray absorptiometry (DXA) of the hip and lumbar spine was performed in 2 centers.&lt;/p&gt; &lt;p&gt;Main Outcome Measure(s): QUS of the heel, bone markers P1NP and β-cTX, and DXA of the hip and lumbar spine were measured.&lt;/p&gt; &lt;p&gt;Results: A total of 2783 men, mean age 60.0 years (SD 11.0) were included in the analysis. After adjustment for age and center, 1,25(OH)2D was positively associated with 25(OH)D but not with PTH. 25(OH)D was negatively associated with PTH. After adjustment for age, center, height, weight, lifestyle factors, and season, 1,25(OH)2D was associated negatively with QUS and DXA parameters and associated positively with β-cTX. 1,25(OH)2D was not correlated with P1NP. 25(OH)D was positively associated with the QUS and DXA parameters but not related to either bone turnover marker. Subjects with both high 1,25(OH)2D (upper tertile) and low 25(OH)D (lower tertile) had the lowest QUS and DXA parameters and the highest β-cTX levels.&lt;/p&gt; &lt;p&gt;Conclusions: Serum 1,25(OH)2D is associated with higher bone turnover and poorer bone health despite being positively related to 25(OH)D. A combination of high 1,25(OH)2D and low 25(OH)D is associated with the poorest bone health.&lt;/p&gt