Fungi at the scene of the crime: innocent bystanders or accomplices in oral infections?

Purpose of Review: Over the last decade, microbiome studies have enhanced our knowledge and understanding of the polymicrobial nature of oral infections. Recently, profiling of the fungal microbiome has expanded our conventional understanding of oral ecology, revealing the critical importance of yeasts within this complex microbiome. This review aims to explore our current appreciation of interkingdom interactions in oral disease. Recent Findings: There is a growing evidence base of interactions and pathogenic synergy and antagonism with bacterial species within oral disease. Recent studies have helped to develop our knowledge of how Candida albicans, alongside bacteria such as Porphyromonas gingivalis, Streptococcus mutans, Staphylococcus aureus, Enterococcus faecalis, and Lactobacillus species, influence overall pathogenicity. Summary: Clinical and experimental evidence makes a compelling case for a role for C. albicans in a number of oral infections, though whether its role is an active accomplice or passive bystander remains to be determined

Investigation of advanced counterrotation blade configuration concepts for high speed turboprop systems. Task 5: Unsteady counterrotation ducted propfan analysis. Computer program user's manual

The primary objective of this study was the development of a time-marching three-dimensional Euler/Navier-Stokes aerodynamic analysis to predict steady and unsteady compressible transonic flows about ducted and unducted propfan propulsion systems employing multiple blade rows. The computer codes resulting from this study are referred to as ADPAC-AOACR (Advanced Ducted Propfan Analysis Codes-Angle of Attack Coupled Row). This report is intended to serve as a computer program user's manual for the ADPAC-AOACR codes developed under Task 5 of NASA Contract NAS3-25270, Unsteady Counterrotating Ducted Propfan Analysis. The ADPAC-AOACR program is based on a flexible multiple blocked grid discretization scheme permitting coupled 2-D/3-D mesh block solutions with application to a wide variety of geometries. For convenience, several standard mesh block structures are described for turbomachinery applications. Aerodynamic calculations are based on a four-stage Runge-Kutta time-marching finite volume solution technique with added numerical dissipation. Steady flow predictions are accelerated by a multigrid procedure. Numerical calculations are compared with experimental data for several test cases to demonstrate the utility of this approach for predicting the aerodynamics of modern turbomachinery configurations employing multiple blade rows

FREQ-Seq: A Rapid, Cost-Effective, Sequencing-Based Method to Determine Allele Frequencies Directly from Mixed Populations

Understanding evolutionary dynamics within microbial populations requires the ability to accurately follow allele frequencies through time. Here we present a rapid, cost-effective method (FREQ-Seq) that leverages Illumina next-generation sequencing for localized, quantitative allele frequency detection. Analogous to RNA-Seq, FREQ-Seq relies upon counts from the >105 reads generated per locus per time-point to determine allele frequencies. Loci of interest are directly amplified from a mixed population via two rounds of PCR using inexpensive, user-designed oligonucleotides and a bar-coded bridging primer system that can be regenerated in-house. The resulting bar-coded PCR products contain the adapters needed for Illumina sequencing, eliminating further library preparation. We demonstrate the utility of FREQ-Seq by determining the order and dynamics of beneficial alleles that arose as a microbial population, founded with an engineered strain of Methylobacterium, evolved to grow on methanol. Quantifying allele frequencies with minimal bias down to 1% abundance allowed effective analysis of SNPs, small in-dels and insertions of transposable elements. Our data reveal large-scale clonal interference during the early stages of adaptation and illustrate the utility of FREQ-Seq as a cost-effective tool for tracking allele frequencies in populations.Organismic and Evolutionary Biolog

Gaining insights from Candida biofilm heterogeneity: one size does not fit all

Despite their clinical significance and substantial human health burden, fungal infections remain relatively under-appreciated. The widespread overuse of antibiotics and the increasing requirement for indwelling medical devices provides an opportunistic potential for the overgrowth and colonization of pathogenic Candida species on both biological and inert substrates. Indeed, it is now widely recognized that biofilms are a highly important part of their virulence repertoire. Candida albicans is regarded as the primary fungal biofilm forming species, yet there is also increasing interest and growing body of evidence for non-Candida albicans species (NCAS) biofilms, and interkingdom biofilm interactions. C. albicans biofilms are heterogeneous structures by definition, existing as three-dimensional populations of yeast, pseudo-hyphae, and hyphae, embedded within a self-produced extracellular matrix. Classical molecular approaches, driven by extensive studies of laboratory strains and mutants, have enhanced our knowledge and understanding of how these complex communities develop, thrive, and cause host-mediated damage. Yet our clinical observations tell a different story, with differential patient responses potentially due to inherent biological heterogeneity from specific clinical isolates associated with their infections. This review explores some of the recent advances made in an attempt to explore the importance of working with clinical isolates, and what this has taught us

A Collaborative Web-Based Approach to Planning Research, Integration, and Testing Using a Wiki

The National Aeronautics and Space Administration Integrated Vehicle Health Management program touches on many different research areas while striving to enable the automated detection, diagnosis, prognosis, and mitigation of adverse events at the aircraft and system level. At the system level, the research focus is on the evaluation of multidisciplinary integrated methods, tools, and technologies for achieving the program goal. The participating program members form a diverse group of government, industry, and academic researchers. The program team developed the Research and Test Integration Plan in order to track significant test and evaluation activities, which are important for understanding, demonstrating, and communicating the overall project state and project direction. The Plan is a living document, which allows the project team the flexibility to construct conceptual test scenarios and to track project resources. The Plan also incorporates several desirable feature requirements for Plan users and maintainers. A wiki has proven to be the most efficient and effective means of implementing the feature requirements for the Plan. The wiki has proven very valuable as a research project management tool, and there are plans to expand its scope

The Ability of Flux Balance Analysis to Predict Evolution of Central Metabolism Scales with the Initial Distance to the Optimum

The most powerful genome-scale framework to model metabolism, flux balance analysis (FBA), is an evolutionary optimality model. It hypothesizes selection upon a proposed optimality criterion in order to predict the set of internal fluxes that would maximize fitness. Here we present a direct test of the optimality assumption underlying FBA by comparing the central metabolic fluxes predicted by multiple criteria to changes measurable by a 13C-labeling method for experimentally-evolved strains. We considered datasets for three Escherichia coli evolution experiments that varied in their length, consistency of environment, and initial optimality. For ten populations that were evolved for 50,000 generations in glucose minimal medium, we observed modest changes in relative fluxes that led to small, but significant decreases in optimality and increased the distance to the predicted optimal flux distribution. In contrast, seven populations evolved on the poor substrate lactate for 900 generations collectively became more optimal and had flux distributions that moved toward predictions. For three pairs of central metabolic knockouts evolved on glucose for 600–800 generations, there was a balance between cases where optimality and flux patterns moved toward or away from FBA predictions. Despite this variation in predictability of changes in central metabolism, two generalities emerged. First, improved growth largely derived from evolved increases in the rate of substrate use. Second, FBA predictions bore out well for the two experiments initiated with ancestors with relatively sub-optimal yield, whereas those begun already quite optimal tended to move somewhat away from predictions. These findings suggest that the tradeoff between rate and yield is surprisingly modest. The observed positive correlation between rate and yield when adaptation initiated further from the optimum resulted in the ability of FBA to use stoichiometric constraints to predict the evolution of metabolism despite selection for rate

A 3-year follow-up study of inpatients with lower limb ulcers: evidence of an obesity paradox?

Objectives: To determine whether body composition is related to long-term outcomes amongst vascular inpatients with lower limb ulcers. Design: Prospective study with 3 years follow-up. Materials and methods: Body mass index (BMI), fat, and fat-free mass were measured and associations with readmission to hospital (number, cause, length of stay) and all-cause mortality were explored. Results: Thirty patients (22 men, 8 women) participated in the study. Ten patients (33%) had a BMI ≥ 30 kg/m2. 18/20 (90%) patients with a BMI < 30 kg/m2 and 9/10 (90%) patients with a BMI ≥ 30 kg/m2 were admitted to hospital in the 3 years of follow-up. Patients with a BMI < 30 kg/m2 were admitted more frequently, earlier and for longer compared to those with BMI ≥ 30 kg/m2 but these did not reach statistical significance. The 3 year mortality rate for patients with BMI ≥ 30 kg/m2 was 20% (n = 2/10) compared to 70% (n = 14/20) with a BMI <30 kg/m2, P = 0.019. Conclusion: This preliminary study suggests that higher BMI may have a protective effect against mortality in vascular patients with lower limb ulcers. These findings contradict the universal acceptance that obesity leads to poor health outcomes. Further work is required to confirm these findings and explore some of the potential mechanisms for this effect

Development of an Optimized Medium, Strain and High-Throughput Culturing Methods for Methylobacterium extorquens

Methylobacterium extorquens strains are the best-studied methylotrophic model system, and their metabolism of single carbon compounds has been studied for over 50 years. Here we develop a new system for high-throughput batch culture of M. extorquens in microtiter plates by jointly optimizing the properties of the organism, the growth media and the culturing system. After removing cellulose synthase genes in M. extorquens strains AM1 and PA1 to prevent biofilm formation, we found that currently available lab automation equipment, integrated and managed by open source software, makes possible reliable estimates of the exponential growth rate. Using this system, we developed an optimized growth medium for M. extorquens using response surface methodologies. We found that media that used EDTA as a metal chelator inhibited growth and led to inconsistent culture conditions. In contrast, the new medium we developed with a PIPES buffer and metals chelated by citrate allowed for fast and more consistent growth rates. This new Methylobacterium PIPES (‘MP’) medium was also robust to large deviations in its component ingredients which avoided batch effects from experiments that used media prepared at different times. MP medium allows for faster and more consistent growth than other media used for M. extorquens.Organismic and Evolutionary Biolog

Urinary ATP and visualization of intracellular bacteria: a superior diagnostic marker for recurrent UTI in renal transplant recipients?

Renal transplant recipients (RTR) are highly susceptible to urinary tract infections (UTIs) with over 50% of patients having at least one UTI within the first year. Yet it is generally acknowledged that there is considerable insensitivity and inaccuracy in routine urinalysis when screening for UTIs. Thus a large number of transplant patients with genuine urine infections may go undiagnosed and develop chronic recalcitrant infections, which can be associated with graft loss and morbidity. Given a recent study demonstrating ATP is released by urothelial cells in response to bacteria exposure, possibly acting at metabotropic P2Y receptors mediating a proinflammatory response, we have investigated alternative, and possibly more appropriate, urinalysis techniques in a cohort of RTRs.Mid-stream urine (MSU) samples were collected from 53 outpatient RTRs. Conventional leukocyte esterase and nitrite dipstick tests, and microscopic pyuria counts (in 1 ?l), ATP concentration measurements, and identification of intracellular bacteria in shed urothelial cells, were performed on fresh unspun samples and compared to ‘gold-standard’ bacterial culture results.Of the 53 RTRs, 22% were deemed to have a UTI by ‘gold-standard’ conventional bacteria culture, whereas 87%, 8% and 4% showed evidence of UTIs according to leukocyte esterase dipstick, nitrite dipstick, and a combination of both dipsticks, respectively. Intracellular bacteria were visualized in shed urothelial cells of 44% of RTRs, however only 1 of the 23 RTRs (44%) was deemed to have a UTI by conventional bacteria culture. A significant association of the ‘gold-standard’ test with urinary ATP concentration combined with visualization of intracellular bacteria in shed urothelial cells was determined using the Fisher’s exact test.It is apparent that standard bedside tests for UTIs give variable results and that seemingly quiescent bacteria in urothelial cells are very common in RTRs and may represent a focus of subclinical infection. Furthermore, our results suggest urinary ATP concentration combined with detection of intracellular bacteria in shed urinary epithelial cells may be a sensitive means by which to detect ‘occult’ infection in RTRs

Chemical diffusion of fluorine in melts in the system Na2OAl2O3SiO2

The volatilization of fluorine from three melts in the system Na2OAl2O3SiO2 has been investigated at 1 atm pressure and 1200–1400°C. The melts chosen have base compositions corresponding to albite, jadeite and a peraluminous melt with 75 mole % SiO2. Melt spheres were suspended from platinum loops in a vertical tube furnace in a flow of oxygen gas, then quenched, sectioned and analysed by electron microprobe. The microprobe scans indicate that transport of fluorine to the melt-vapor interface is by binary, concentration-independent interdiffusion of fluorine and oxygen. FO interdiffusivity increases in the order albite < peraluminous < jadeite. There is no simple reciprocal relationship between FO interdiffusivity and melt viscosity. Comparison with data on high-pressure interdiffusivity of fluorine and oxygen in jadeite melt indicates that FO interdiffusivity increases with pressure from 0.001 to 10 kbar while the activation energy remains unchanged. Fluorine chemical diffusivity in albite melt is substantially lower than H2O chemical diffusivity in obsidian melts suggesting that different diffusive mechanisms are responsible for the transport of F and H2O in igneous melts. Fluorine diffuses in albite melt via an anionic exchange with oxygen whereas water probably diffuses in obsidian melt via an alkali exchange mechanism