Mycobacterium avium Infections of Acanthamoeba Strains: Host Strain Variability,Grazing-Acquired Infections,and Altered Dynamics of Inactivation with Monochloramine

Stable Mycobacterium avium infections of several Acanthamoeba strains were characterized by increased infection resistance of recent environmental isolates and reduced infectivity in the presence of other bacteria. Exposure of M. avium in coculture with Acanthamoeba castellanii to monochloramine yielded inactivation kinetics markedly similar to those observed for A. castellanii alone.Acanthamoebae are widely distributed in the environment (20) and generally function ecologically as predators of bacteria (23), although numerous types of bacteria resist predation (22). Acanthamoebae are very resistant to a range of disinfectants (5, 6, 8, 28), and bacteria within acanthamoebae are generally afforded extra protection (16). A notable example is the opportunistic pathogen Mycobacterium avium (10), which can survive within Acanthamoeba species trophozoites and cysts (4, 26), resulting in increased resistance to several antimicrobials (22). It has been demonstrated that many Mycobacterium spp. are able to infect the laboratory strain Acanthamoeba polyphaga (1). Acanthamoeba cultures undergo many physiological changes after several passages in the laboratory (15, 17, 21), although it is not known if prolonged cultivation of Acanthamoeba alters their capacity to be infected by M. avium. This knowledge is important for assessing the environmental relevance of associations between Acanthamoeba and M. avium. Therefore, we studied the infectivity and infection stability of M. avium with several laboratory and environmental Acanthamoeba strains for 28 days under high-nutrient (peptone-yeast extract-glucose [PYG] medium) and low-nutrient (Page''s amoeba saline [PAS]) conditions.     

Quantitative Proteomics and Metabolomics Analysis of Normal Human Cerebrospinal Fluid Samples*

The analysis of cerebrospinal fluid (CSF) is used in biomarker discovery studies for various neurodegenerative central nervous system (CNS) disorders. However, little is known about variation of CSF proteins and metabolites between patients without neurological disorders. A baseline for a large number of CSF compounds appears to be lacking. To analyze the variation in CSF protein and metabolite abundances in a number of well-defined individual samples of patients undergoing routine, non-neurological surgical procedures, we determined the variation of various proteins and metabolites by multiple analytical platforms. A total of 126 common proteins were assessed for biological variations between individuals by ESI-Orbitrap. A large spread in inter-individual variation was observed (relative standard deviations [RSDs] ranged from 18 to 148%) for proteins with both high abundance and low abundance. Technical variation was between 15 and 30% for all 126 proteins. Metabolomics analysis was performed by means of GC-MS and nuclear magnetic resonance (NMR) imaging and amino acids were specifically analyzed by LC-MS/MS, resulting in the detection of more than 100 metabolites. The variation in the metabolome appears to be much more limited compared with the proteome: the observed RSDs ranged from 12 to 70%. Technical variation was less than 20% for almost all metabolites. Consequently, an understanding of the biological variation of proteins and metabolites in CSF of neurologically normal individuals appears to be essential for reliable interpretation of biomarker discovery studies for CNS disorders because such results may be influenced by natural inter-individual variations. Therefore, proteins and metabolites with high variation between individuals ought to be assessed with caution as candidate biomarkers because at least part of the difference observed between the diseased individuals and the controls will not be caused by the disease, but rather by the natural biological variation between individuals.The analysis of CSF¹ is indispensable in the diagnosis and understanding of various neurodegenerative CNS disorders (1–3). CSF is a fluid that has different functions, such as the protection of the brain from outside forces, transport of biological substances, and excretion of toxic and waste substances. It is in close contact with the extracellular fluid of the brain. Therefore, the composition of CSF can reflect biological processes of the brain (4). By discovering the characterization of the proteome and metabolome of CSF we may gain better insight on the pathogenesis of CNS disorders. This would be significant because, for many of these disorders, the etiology is still unclear.CSF is produced in the ventricles of the brain and in the subarachnoidal spaces. Humans normally produce around 500 mL of CSF each day, and the total volume of CSF at a given time is approximately 150 mL. CSF reflects the composition of blood plasma, although the concentrations of most proteins and metabolites in CSF are lower. However, individual proteins and metabolites can act differently. Active transport from blood and secretion from the brain contribute to the specific composition of CSF. This composition can be disturbed in neurological disorders (5–6). Since CNS-specific proteins and metabolites are typically low in abundance compared with their levels in blood, this change in composition is more likely to be found in CSF because in blood the more abundant plasma proteins can completely mask the signal of the less abundant proteins. Also, if the disease markers do not cross the blood-brain-barrier, then the CSF is the only viable biofluid source. Therefore, CSF might be an excellent source for biomarker discovery for CNS disorders if we follow the hypothesis that neurological diseases induce alterations in CSF protein and metabolite levels.Analysis of metabolites in CSF has been common practice in clinical chemistry for decades to analyze biomarkers for inborn errors of metabolism. The approaches used are either metabolite profiling of CSF using NMR (7), or targeted analysis of one or a few metabolites using specific analytical methods (8). Metabolomics includes the analysis of metabolites in biofluids by NMR or MS-based approaches, i.e. LC-MS or GC-MS. Several metabolite profiling studies were performed on CSF using NMR, some of which were published only recently (9,10). Surprisingly, very few metabolomics studies using MS-based methods have been performed on CSF to date (11,12). One of the reasons is the fact that the human CSF metabolome has not yet been characterized very well. Many CSF metabolites remain unidentified, and for those that have been identified there is not much known about normal concentration ranges. A systematic categorization of the CSF metabolome is necessary and expected to be beneficial for future biomarker discoveries. Recently, Wishart et al. made a good start in exploring the human CSF metabolome with their computer-aided literature survey that resulted in 308 detectable metabolites in human CSF (13).The CSF proteome has been characterized to a much larger extent than the CSF metabolome and is currently the topic of investigations in several research groups worldwide. Recently, studies have been published with numerous identities and quantities of CSF proteins. Pan and co-workers were able to identify 2,594 proteins in well-characterized pooled human CSF samples using strict proteomics criteria with a combination of linear trap quadrupole LTQ-FT (Thermo Fisher Scientific, Bremen, Germany) and MALDI TOF/TOF equipment (14). They were also able to quantify several proteins using a targeted LC MALDI TOF/TOF approach (15). Hu et al. have studied the intra- and inter-individual variation in human CSF and found large variations in protein concentrations in six patients by means of two dimensional–gel electrophoresis (16), focusing mainly on the variations within individuals at two different time-points. Although only a limited number of proteins was analyzed, the variation between the time-points was profound, exceeding 200% for seven proteins.Unique CSF biomarkers may contribute to a deeper understanding of the mechanisms of CNS disorders. However, for this assumption to come true, there are still challenges ahead. Although CSF is not as complex as blood (almost missing the cellular part and the clotting system present in blood), it is expected to consist of thousands of organic- and non-organic salts, sugars, lipids, and proteins. A large part of the CSF consists of a few highly abundant metabolites and proteins, which hamper, if no precautions are undertaken, the identification and quantification of metabolites and proteins that occur in lower amounts. The analysis of the CSF metabolome is complicated because of the diverse chemical nature of metabolites and the lower concentration of metabolites compared with blood. Analytical method development is still required because it is not possible to identify the entire range of CSF metabolites with one single analytical method. Although in proteome research efforts have been made to quantify proteins, metabolomics studies up to now either do not provide quantitative information or they only give information for the most abundant metabolites.Another challenge is the sample amount obtained by lumbar puncture to collect CSF. Lumbar puncture is an invasive method that is not performed as frequently as blood sampling. However, often after the analysis of various clinical parameters, only a limited amount of CSF sample is available for biomarker discovery. Metabolomics studies are hampered by limited CSF sample amount. Therefore, analytical methods are required that are suitable to handle relatively small sample volumes.The main objectives of this study were (1) to analyze the variation in CSF protein and metabolite abundances in a number of well-defined individual samples by multiple analytical platforms; and (2) to integrate metabolomics and proteomics to present biological variations in metabolite and protein abundances and compare these with technical variations with the currently used analytical methods. The results will facilitate and increase the application of CSF for future biomarker discovery studies in the field of neurodegenerative diseases and neuro-oncology.     

Regulated intramembrane proteolysis of amyloid precursor protein and regulation of expression of putative target genes

gamma-Secretase-dependent regulated intramembrane proteolysis of amyloid precursor protein (APP) releases the APP intracellular domain (AICD). The question of whether this domain, like the Notch intracellular domain, is involved in nuclear signalling is highly controversial. Although some reports suggest that AICD regulates the expression of KAI1, glycogen synthase kinase-3beta, Neprilysin and APP, we found no consistent effects of gamma-secretase inhibitors or of genetic deficiencies in the gamma-secretase complex or the APP family on the expression levels of these genes in cells and tissues. Finally, we demonstrate that Fe65, an important AICD-binding protein, transactivates a wide variety of different promoters, including the viral simian virus 40 promoter, independent of AICD coexpression. Overall, the four currently proposed target genes are at best indirectly and weakly influenced by APP processing. Therefore, inhibition of APP processing to decrease Abeta generation in Alzheimer's disease will not interfere significantly with the function of these genes.     

Development and plasticity-related changes in protein expression patterns in cat visual cortex: a fluorescent two-dimensional difference gel electrophoresis approach

During early postnatal brain development, changes in visual input can lead to specific alteration of function and connectivity in mammalian visual cortex. In cat, this so-called critical period exhibits maximal sensory-driven adaptations around postnatal day 30 (P30), and ceases toward adulthood. We examined the molecular framework that directs age- and experience-dependent plasticity in cat visual cortex, by comparing protein expression profiles at eye opening (postnatal day 10 (P10), when experience-dependent plasticity starts), the peak of the critical period (P30), and in adulthood. Using 2-D DIGE, we performed comparisons of P10-P30 and P30-adult brain protein samples. Sixty protein spots showed statistically significant intensity changes in at least one comparison. Fifty-one spots were identified using quadrupole-TOF MS/MS or LC-MS/MS, containing 37 different proteins. The progressive increase or decrease in protein expression levels could be correlated to age-dependent postnatal brain development. Four spots containing transferrin, 14-3-3 alpha/beta and cypin, showed maximal protein expression levels at P30, thereby showing a positive correlation to critical period plasticity. Western analysis indeed revealed a clear effect of visual deprivation on cypin expression in cat visual cortex. Our results therefore demonstrate the power of 2-D DIGE as a tool toward understanding the molecular basis of nervous system development and plasticity.     

Proteomic analysis of global protein expression changes in the endothelin-1 rat model for cerebral ischemia: Rescue effect of mild hypothermia

Mild hypothermia is a promising neuroprotective therapy in stroke management. However, little is known about its effects on the global protein expression patterns in brain regions affected by ischemic stroke. We investigated protein expression changes associated with the neuroprotective effects of hypothermia via a functional proteomics approach through the analysis of the core (striatum) and the penumbra (cortex) after an ischemic insult in rats induced by endothelin-1 (Et-1). Functional outcome, infarct volume and related global protein expression changes were assessed 24 h after the insult using two-dimensional difference gel electrophoresis. Mild hypothermia, induced 20 min after endothelin-1 infusion, improved the neurological outcome, reflected by a 36% reduction in infarct volume and a significantly better neurological deficit score. Hypothermia was typically associated with opposite protein expression changes inthe cortex to those induced by stroke under normothermic conditions, but not in the striatum. The main cellular processes rescued by hypothermia and potentially involved in the protection of the cortex are cellular assembly and organization, followed by cell signaling, thereby confirming that hypothermia is neuroprotective through multiple molecular and cellular pathways.     

Heritability and Clinical Determinants of Serum Indoxyl Sulfate and p-Cresyl Sulfate,Candidate Biomarkers of the Human Microbiome Enterotype

Background

Indoxyl sulfate and p-cresyl sulfate are unique microbial co-metabolites. Both co-metabolites have been involved in the pathogenesis of accelerated cardiovascular disease and renal disease progression. Available evidence suggests that indoxyl sulfate and p-cresyl sulfate may be considered candidate biomarkers of the human enterotype and may help to explain the link between diet and cardiovascular disease burden.

Objective and Design

Information on clinical determinants and heritability of indoxyl sulfate and p-cresyl sulfate serum is non-existing. To clarify this issue, the authors determined serum levels of indoxyl sulfate and p-cresyl sulfate in 773 individuals, recruited in the frame of the Flemish Study on Environment, Genes and Health Outcomes (FLEMENGHO study).

Results

Serum levels of indoxyl sulfate and p-cresyl sulfate amounted to 3.1 (2.4–4.3) and 13.0 (7.4–21.5) μM, respectively. Regression analysis identified renal function, age and sex as independent determinants of both co-metabolites. Both serum indoxyl sulfate (h² = 0.17) and p-cresyl sulfate (h² = 0.18) concentrations showed moderate but significant heritability after adjustment for covariables, with significant genetic and environmental correlations for both co-metabolites.

Limitations

Family studies cannot provide conclusive evidence for a genetic contribution, as confounding by shared environmental effects can never be excluded.

Conclusions

The heritability of indoxyl sulfate and p-cresyl sulfate is moderate. Besides genetic host factors and environmental factors, also renal function, sex and age influence the serum levels of these co-metabolites.     

Randomised Double-Blind Comparison of Placebo and Active Drugs for Effects on Risks Associated with Blood Pressure Variability in the Systolic Hypertension in Europe Trial

Background

In the Systolic Hypertension in Europe trial ({"type":"clinical-trial","attrs":{"text":"NCT02088450","term_id":"NCT02088450"}}NCT02088450), we investigated whether systolic blood pressure variability determines prognosis over and beyond level.

Methods

Using a computerised random function and a double-blind design, we randomly allocated 4695 patients (≥60 years) with isolated systolic hypertension (160–219/<95 mm Hg) to active treatment or matching placebo. Active treatment consisted of nitrendipine (10–40 mg/day) with possible addition of enalapril (5–20 mg/day) and/or hydrochlorothiazide (12.5–25.0 mg/day). We assessed whether on-treatment systolic blood pressure level (SBP), visit-to-visit variability independent of the mean (VIM) or within-visit variability (WVV) predicted total (n = 286) or cardiovascular (n = 150) mortality or cardiovascular (n = 347), cerebrovascular (n = 133) or cardiac (n = 217) endpoints.

Findings

At 2 years, mean between-group differences were 10.5 mm Hg (p<0.0001) for SBP, 0.29 units (p = 0.20) for VIM, and 0.07 mm Hg (p = 0.47) for WVV. Active treatment reduced (p≤0.048) cardiovascular (−28%), cerebrovascular (−40%) and cardiac (−24%) endpoints. In analyses dichotomised by the median, patients with low vs. high VIM had similar event rates (p≥0.14). Low vs. high WVV was not associated with event rates (p≥0.095), except for total and cardiovascular mortality on active treatment, which were higher with low WVV (p≤0.0003). In multivariable-adjusted Cox models, SBP predicted all endpoints (p≤0.0043), whereas VIM did not predict any (p≥0.058). Except for an inverse association with total mortality (p = 0.042), WVV was not predictive (p≥0.15). Sensitivity analyses, from which we excluded blood pressure readings within 6 months after randomisation, 6 months prior to an event or both were confirmatory.

Conclusions

The double-blind placebo-controlled Syst-Eur trial demonstrated that blood-pressure lowering treatment reduces cardiovascular complications by decreasing level but not variability of SBP. Higher blood pressure level, but not higher variability, predicted risk.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02088450","term_id":"NCT02088450"}}NCT02088450     

NMR and pattern recognition can distinguish neuroinflammation and peripheral inflammation

Multiple Sclerosis (MScl) is a neurodegenerative disease of the CNS, associated with chronic neuroinflammation. Cerebrospinal fluid (CSF), being in closest interaction with CNS, was used to profile neuroinflammation to discover disease-specific markers. We used the commonly accepted animal model for the neuroinflammatory aspect of MScl: the experimental autoimmune/allergic encephalomyelitis (EAE). A combination of advanced (1)H NMR spectroscopy and pattern recognition methods was used to establish the metabolic profile of CSF of EAE-affected rats (representing neuroinflammation) and of two control groups (healthy and peripherally inflamed) to detect specific markers for early neuroinflammation. We found that the CSF metabolic profile for neuroinflammation is distinct from healthy and peripheral inflammation and characterized by changes in concentrations of metabolites such as creatine, arginine, and lysine. Using these disease-specific markers, we were able to detect early stage neuroinflammation, with high accuracy in a second independent set of animals. This confirms the predictive value of these markers. These findings from the EAE model may help to develop a molecular diagnosis for the early stage MScl in humans.     

Variation in volatile leaf oils of five Eucalyptus species harvested from Jbel Abderrahman arboreta (Tunisia)

Hydrodistillation of the dried leaves of five species of the genus Eucalyptus L' Hér ., viz., E. dundasii Maiden , E. globulus Labill ., E. kitsoniana Maiden , E. leucoxylon F. Muell ., and E. populifolia Hook ., harvested from Jbel Abderrahman arboreta (region of Nabeul, northeast of Tunisia) in April 2006, afforded essential oils in yields varying from 0.9±0.3 to 3.8±0.6%, dependent on the species. E. globulus and E. Kitsoniana provided the highest and the lowest percentage of essential oil amongst the species examined, respectively. Analysis by GC (RI) and GC/MS allowed the identification of 127 compounds, representing 93.8 to 98.7% of the total oil composition. The contents of the different samples varied according to the species. The main components were 1,8‐cineole ( 2 ; 4.7–59.2%), followed by α‐pinene ( 1 ; 1.9–23.6%), trans‐pinocarveol ( 6 ; 3.5–21.6%), globulol ( 8 ; 4.3–12.8%), p‐cymene ( 3 ; 0.5–6.7%), α‐terpineol (1.5–4.5%), borneol (0.2–4.4%), pinocarvone (1.1–3.8%), aromadendrene (1.4–3.4%), isospathulenol (0.0–1.9%), fenchol ( 4 ; 0.1–2.5%), limonene (1.0–2.4%), epiglobulol (0.6–2.1%), viridiflorol ( 9 ; 0.8–1.8%), and spathulenol (0.1–1.6%). E. leucoxylon was the richest species in 2 . Principal component analysis (PCA) and hierarchical cluster analysis (HCA) separated the five Eucalyptus leaf essential oils into four groups, each constituting a chemotype.     

Backwash intensity and frequency impact the microbial community structure and function in a fixed-bed biofilm reactor

Linkages among bioreactor operation and performance and microbial community structure were investigated for a fixed-bed biofilm system designed to remove perchlorate from drinking water. Perchlorate removal was monitored to evaluate reactor performance during and after the frequency and intensity of the backwash procedure were changed, while the microbial community structure was studied using clone libraries and quantitative PCR targeting the 16S rRNA gene. When backwash frequency was increased from once per month to once per day, perchlorate removal initially deteriorated and then recovered, and the relative abundance of perchlorate-reducing bacteria (PRB) initially increased and then decreased. This apparent discrepancy suggested that bacterial populations other than PRB played an indirect role in perchlorate removal, likely by consuming dissolved oxygen, a competing electron acceptor. When backwash intensity was increased, the reactor gradually lost its ability to remove perchlorate, and concurrently the relative abundance of PRB decreased. The results indicated that changes in reactor operation had a profound impact on reactor performance through altering the microbial community structure. Backwashing is an important yet poorly characterized procedure when operating fixed-bed biofilm reactors. Compared to backwash intensity, changes in backwash frequency exerted less disturbance on the microbial community in the current study. If this finding can be confirmed in future work, backwash frequency may serve as the primary parameter when optimizing backwash procedures.