In vitro immunomodulatory activity of Lactobacillus fermentum CECT5716 and Lactobacillus salivarius CECT5713: two probiotic strains isolated from human breast milk

Commensal bacteria, including some species of lactobacilli commonly present in human breast milk, appear to colonize the neonatal gut and contribute to protection against infant infections, suggesting that lactobacilli could potentially modulate immunity. In this study, we evaluated the potential of two Lactobacillus strains isolated from human milk to modulate the activation and cytokine profile of peripheral blood mononuclear cell (PBMC) subsets in vitro. Moreover, these effects were compared to the same probiotic species of non-milk origin. Lactobacillus salivarius CECT5713 and Lactobacillus fermentum CECT5716 at 105, 106 and 107 bacteria/mL were co-cultured with PBMC (106/mL) from 8 healthy donors for 24 h. Activation status (CD69 and CD25 expressions) of natural killer (NK) cells (CD56+), total T cells (CD3+), cytotoxic T cells (CD8+) and CD4+ T cells was determined by flow cytometry. Regulatory T cells (Treg) were also quantified by intracellular Foxp3 evaluation. Regarding innate immunity, NK cells were activated by addition of both Lactobacillus strains, and in particular, the CD8+ NK subset was preferentially induced to highly express CD69 (90%, p<0.05). With respect to acquired immunity, approximately 9% of CD8+ T cells became activated after co-cultivation with L. fermentum or L salivarius. Although CD4+ T cells demonstrated a weaker response, there was a preferential activation of Treg cells (CD4+CD25+Foxp3+) after exposure to both milk probiotic bacteria (p<0.05). Both strains significantly induced the production of a number of cytokines and chemokines, including TNFα, IL-1β, IL-8, MIP-1α, MIP-1β, and GM-CSF, but some strain-specific effects were apparent. This work demonstrates that L salivarius CECT5713 and L. fermentum CECT5716 enhanced both natural and acquired immune responses, as evidenced by the activation of NK and T cell subsets and the expansion of Treg cells, as well as the induction of a broad array of cytokines.

Changes in race-specific virulence in pseudomonas syringae pv. phaseolicola are Associated with a chimeric transposable element and rare deletion events in a plasmid-borne pathogenicity island

Virulence for bean and soybean is determined by effector genes in a plasmid-borne pathogenicity island (PAI) in race 7 strain 1449B of Pseudomonas syringae pv. phaseolicola. One of the effector genes, avrPphF, confers either pathogenicity, virulence, or avirulence depending on the plant host and is absent from races 2, 3, 4, 6, and 8 of this pathogen. Analysis of cosmid clones and comparison of DNA sequences showed that the absence of avrPphF from strain 1448A is due to deletion of a continuous 9.5-kb fragment. The remainder of the PAI is well conserved in strains 1448A and 1449B. The left junction of the deleted region consists of a chimeric transposable element generated from the fusion of homologs of IS1492 from Pseudomonas putida and IS1090 from Ralstonia eutropha. The borders of the deletion were conserved in 66 P. syringae pv. phaseolicola strains isolated in different countries and representing the five races lacking avrPphF. However, six strains isolated in Spain had a 10.5-kb deletion that extended 1 kb further from the right junction. The perfect conservation of the 28-nucleotide right repeat of the IS1090 homolog in the two deletion types and in the other 47 insertions of the IS1090 homolog in the 1448A genome strongly suggests that the avrPphF deletions were mediated by the activity of the chimeric mobile element. Our data strongly support a clonal origin for the races of P. syringae pv. phaseolicola lacking avrPphF.

In vitro evaluation of the antimicrobial activity of a range of probiotics against pathogens: Evidence for the effects of organic acids

The aim of this study was to investigate the antimicrobial properties of fifteen selected strains belonging to the Lactobacillus, Bifidobacterium, Lactococcus, Streptococcus and Bacillus genera against Gram-positive and Gram-negative pathogenic bacteria. In vitro antibacterial activity was initially investigated by an agar spot method. Results from the agar spot test showed that most of the selected strains were able to produce active compounds on solid media with antagonistic properties against Salmonella Typhimurium, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and Clostridium difficile. These results were also confirmed when cell-free culture supernatants (CFCS) from the putative probiotics were used in an agar well diffusion assay. Neutralization of the culture supernatants with alkali reduced the antagonistic effects. These experiments are able to confirm the capacity of potential probiotics to inhibit selected pathogens. One of the main inhibitory mechanisms may result from the production of organic acids from glucose fermentation and consequent lowering of culture pH. This observation was confirmed when the profile of organic acids was analysed demonstrating that lactic and acetic acid were the principal end products of probiotic metabolism. Furthermore, the assessment of the haemolytic activity and the susceptibility of the strains to the most commonly used antimicrobials, considered as basic safety aspects, were also studied. The observed antimicrobial activity was mainly genus-specific, additionally significant differences could be observed among species.

Epoch-specific functional networks involved in working memory

Working memory (WM) is not a unitary construct. There are distinct processes involved in encoding information, maintaining it on-line, and using it to guide responses. The anatomical configurations of these processes are more accurately analyzed as functionally connected networks than collections of individual regions. In the current study we analyzed event-related functional magnetic resonance imaging (fMRI) data from a Sternberg Item Recognition Paradigm WM task using a multivariate analysis method that allowed the linking of functional networks to temporally-separated WM epochs. The length of the delay epochs was varied to optimize isolation of the hemodynamic response (HDR) for each task epoch. All extracted functional networks displayed statistically significant sensitivity to delay length. Novel information extracted from these networks that was not apparent in the univariate analysis of these data included involvement of the hippocampus in encoding/probe, and decreases in BOLD signal in the superior temporal gyrus (STG), along with default-mode regions, during encoding/delay. The bilateral hippocampal activity during encoding/delay fits with theoretical models of WM in which memoranda held across the short term are activated long-term memory representations. The BOLD signal decreases in the STG were unexpected, and may reflect repetition suppression effects invoked by internal repetition of letter stimuli. Thus, analysis methods focusing on how network dynamics relate to experimental conditions allowed extraction of novel information not apparent in univariate analyses, and are particularly recommended for WM experiments for which task epochs cannot be randomized.

Gut microbial activity, implications for health and disease: the potential role of metabolite analysis

Microbial metabolism of proteins and amino acids by human gut bacteria generates a variety of compounds including phenol, indole, and sulfur compounds and branched chain fatty acids, many of which have been shown to elicit a toxic effect on the lumen. Bacterial fermentation of amino acids and proteins occurs mainly in the distal colon, a site that is often fraught with symptoms from disorders including ulcerative colitis (UC) and colorectal cancer (CRC). In contrast to carbohydrate metabolism by the gut microbiota, proteolysis is less extensively researched. Many metabolites are low molecular weight, volatile compounds. This review will summarize the use of analytical methods to detect and identify compounds in order to elucidate the relationship between specific dietary proteinaceous substrates, their corresponding metabolites, and implications for gastrointestinal health.

Connecting mean field models of neural activity to EEG and fMRI data

Progress in functional neuroimaging of the brain increasingly relies on the integration of data from complementary imaging modalities in order to improve spatiotemporal resolution and interpretability. However, the usefulness of merely statistical combinations is limited, since neural signal sources differ between modalities and are related non-trivially. We demonstrate here that a mean field model of brain activity can simultaneously predict EEG and fMRI BOLD with proper signal generation and expression. Simulations are shown using a realistic head model based on structural MRI, which includes both dense short-range background connectivity and long-range specific connectivity between brain regions. The distribution of modeled neural masses is comparable to the spatial resolution of fMRI BOLD, and the temporal resolution of the modeled dynamics, importantly including activity conduction, matches the fastest known EEG phenomena. The creation of a cortical mean field model with anatomically sound geometry, extensive connectivity, and proper signal expression is an important first step towards the model-based integration of multimodal neuroimages.

Understanding the transition to seizure by modeling the epileptiform activity of general anesthetic agents

A central difficulty in modeling epileptogenesis using biologically plausible computational and mathematical models is not the production of activity characteristic of a seizure, but rather producing it in response to specific and quantifiable physiologic change or pathologic abnormality. This is particularly problematic when it is considered that the pathophysiological genesis of most epilepsies is largely unknown. However, several volatile general anesthetic agents, whose principle targets of action are quantifiably well characterized, are also known to be proconvulsant. The authors describe recent approaches to theoretically describing the electroencephalographic effects of volatile general anesthetic agents that may be able to provide important insights into the physiologic mechanisms that underpin seizure initiation.

Are greenhouse gas signals of Northern Hemisphere winter extra-tropical cyclone activity dependent on the identification and tracking algorithm?

For Northern Hemisphere extra-tropical cyclone activity, the dependency of a potential anthropogenic climate change signal on the identification method applied is analysed. This study investigates the impact of the used algorithm on the changing signal, not the robustness of the climate change signal itself. Using one single transient AOGCM simulation as standard input for eleven state-of-the-art identification methods, the patterns of model simulated present day climatologies are found to be close to those computed from re-analysis, independent of the method applied. Although differences in the total number of cyclones identified exist, the climate change signals (IPCC SRES A1B) in the model run considered are largely similar between methods for all cyclones. Taking into account all tracks, decreasing numbers are found in the Mediterranean, the Arctic in the Barents and Greenland Seas, the mid-latitude Pacific and North America. Changing patterns are even more similar, if only the most severe systems are considered: the methods reveal a coherent statistically significant increase in frequency over the eastern North Atlantic and North Pacific. We found that the differences between the methods considered are largely due to the different role of weaker systems in the specific methods.

The resting-state neurovascular coupling relationship: rapid changes in spontaneous neural activity in the somatosensory cortex are associated with haemodynamic fluctuations that resemble stimulus-evoked haemodynamics

Although promise exists for patterns of resting-state blood oxygen level-dependent (BOLD) functional magnetic resonance imaging (fMRI) brain connectivity to be used as biomarkers of early brain pathology, a full understanding of the nature of the relationship between neural activity and spontaneous fMRI BOLD fluctuations is required before such data can be correctly interpreted. To investigate this issue, we combined electrophysiological recordings of rapid changes in multi-laminar local field potentials from the somatosensory cortex of anaesthetized rats with concurrent two-dimensional optical imaging spectroscopy measurements of resting-state haemodynamics that underlie fluctuations in the BOLD fMRI signal. After neural ‘events’ were identified, their time points served to indicate the start of an epoch in the accompanying haemodynamic fluctuations. Multiple epochs for both neural ‘events’ and the accompanying haemodynamic fluctuations were averaged. We found that the averaged epochs of resting-state haemodynamic fluctuations taken after neural ‘events’ closely resembled the temporal profile of stimulus-evoked cortical haemodynamics. Furthermore, we were able to demonstrate that averaged epochs of resting-state haemodynamic fluctuations resembling the temporal profile of stimulus-evoked haemodynamics could also be found after peaks in neural activity filtered into specific electroencephalographic frequency bands (theta, alpha, beta, and gamma). This technique allows investigation of resting-state neurovascular coupling using methodologies that are directly comparable to that developed for investigating stimulus-evoked neurovascular responses.

Inhibition of LRRK2 kinase activity stimulates macroautophagy

Leucine Rich Repeat Kinase 2 (LRRK2) is one of the most important genetic contributors to Parkinson's disease. LRRK2 has been implicated in a number of cellular processes, including macroautophagy. To test whether LRRK2 has a role in regulating autophagy, a specific inhibitor of the kinase activity of LRRK2 was applied to human neuroglioma cells and downstream readouts of autophagy examined. The resulting data demonstrate that inhibition of LRRK2 kinase activity stimulates macroautophagy in the absence of any alteration in the translational targets of mTORC1, suggesting that LRRK2 regulates autophagic vesicle formation independent of canonical mTORC1 signaling. This study represents the first pharmacological dissection of the role LRRK2 plays in the autophagy/lysosomal pathway, emphasizing the importance of this pathway as a marker for LRRK2 physiological function. Moreover it highlights the need to dissect autophagy and lysosomal activities in the context of LRRK2 related pathologies with the final aim of understanding their aetiology and identifying specific target for disease modifying therapies in patients.

Response to zinc deficiency of two rice lines with contrasting tolerance is determined by root growth maintenance and organic acid exudation rates, and not by zinc-transporter activity

Zinc (Zn)-deficient soils constrain rice (Oryza sativa) production and cause Zn malnutrition. The identification of Zn-deficiency-tolerant rice lines indicates that breeding might overcome these constraints. Here, we seek to identify processes underlying Zn-deficiency tolerance in rice at the physiological and transcriptional levels. A Zn-deficiency-tolerant line RIL46 acquires Zn more efficiently and produces more biomass than its nontolerant maternal line (IR74) at low Zn(ext) under field conditions. We tested if this was the result of increased expression of Zn(2+) transporters; increased root exudation of deoxymugineic acid (DMA) or low-molecular-weight organic acids (LMWOAs); and/or increased root production. Experiments were performed in field and controlled environment conditions. There was little genotypic variation in transcript abundance of Zn-responsive root Zn(2+)-transporters between the RIL46 and IR74. However, root exudation of DMA and LMWOA was greater in RIL46, coinciding with increased root expression of putative ligand-efflux genes. Adventitious root production was maintained in RIL46 at low Zn(ext), correlating with altered expression of root-specific auxin-responsive genes. Zinc-deficiency tolerance in RIL46 is most likely the result of maintenance of root growth, increased efflux of Zn ligands, and increased uptake of Zn-ligand complexes at low Zn(ext); these traits are potential breeding targets.

Applications of micromorphology to understanding activity areas and site formation processes in experimental hut floors

Experimental buildings at Butser Ancient Farm and St. Fagans (UK) and Lejre (Denmark) were sampled to investigate micromorphology of known activity areas, to contribute to our understanding of the internal use of space in excavated buildings and formation processes of house floor deposits. The experimental buildings provided important information relating to activity residues and sediments over the 16 years that the buildings were in use. Specifically, these results contribute to our understanding of the routes and cycles for transportation of materials in occupation contexts, which can be used to inform archaeological studies. It has been possible to identify internal ‘hot spots’ within the buildings for the deposition of activity residues and for the formation of specific deposit types. Analysis also highlighted postdepositional alterations occurring in internal occupation deposits, which has provided a means of identifying roofed and unroofed spaces in the archaeological record.

Impact of the enhanced permeability and retention (EPR) effect and cathepsins levels on the activity of polymer-drug conjugates

Polymer-drug conjugates have demonstrated clinical potential in the context of anticancer therapy. However, such promising results have, to date, failed to translate into a marketed product. Polymer-drug conjugates rely on two factors for activity: (i) the presence of a defective vasculature, for passive accumulation of this technology into the tumour tissue (enhanced permeability and retention (EPR) effect) and (ii) the presence of a specific trigger at the tumour site, for selective drug release (e.g., the enzyme cathepsin B). Here, we retrospectively analyse literature data to investigate which tumour types have proved more responsive to polymer-drug conjugates and to determine correlations between the magnitude of the EPR effect and/or expression of cathepsin B. Lung, breast and ovarian cancers showed the highest response rate (30%, 47% and 41%, respectively for cathepsin-activated conjugates and 31%, 43%, 40%, across all conjugates). An analysis of literature data on cathepsin content in various tumour types showed that these tumour types had high cathepsin content (up to 3835 ng/mg for lung cancer), although marked heterogeneity was observed across different studies. In addition, these tumour types were also reported as having a high EPR effect. Our results suggest that a pre-screening of patient population could bring a more marked clinical benefit.

Potential anti-obesogenic properties of non-digestible carbohydrates: specific focus on resistant dextrin

Alterations in the composition and metabolic activity of the gut microbiota appear to contribute to the development of obesity and associated metabolic diseases. However, the extent of this relationship remains unknown. Modulating the gut microbiota with non-digestible carbohydrates (NDC) may exert anti-obesogenic effects through various metabolic pathways including changes to appetite regulation, glucose and lipid metabolism and inflammation. The NDC vary in physicochemical structure and this may govern their physical properties and fermentation by specific gut bacterial populations. Much research in this area has focused on established prebiotics, especially fructans (i.e. inulin and fructo-oligosaccharides); however, there is increasing interest in the metabolic effects of other NDC, such as resistant dextrin. Data presented in this review provide evidence from mechanistic and intervention studies that certain fermentable NDC, including resistant dextrin, are able to modulate the gut microbiota and may alter metabolic process associated with obesity, including appetite regulation, energy and lipid metabolism and inflammation. To confirm these effects and elucidate the responsible mechanisms, further well-controlled human intervention studies are required to investigate the impact of NDC on the composition and function of the gut microbiota and at the same time determine concomitant effects on host metabolism and physiology.

Minimal regulation of platelet activity by PECAM-1.

PECAM-1 is a member of the superfamily of immunoglobulins (Ig) and is expressed on platelets at moderate level. PECAM-1 has been reported to have contrasting effects on platelet activation by the collagen receptor GPVI and the integrin, alphaIIbbeta3, even though both receptors signal through Src-kinase regulation of PLCgamma2. The present study compares the role of PECAM-1 on platelet activation by these two receptors and by the lectin receptor, CLEC-2, which also signals via PLCgamma2. Studies using PECAM-1 knockout-mice and cross-linking of PECAM-1 using specific antibodies demonstrated a minor inhibitory role on platelet responses to the above three receptors and also under some conditions to the G-protein agonist thrombin. The degree of inhibition was considerably less than that produced by PGI2, which elevates cAMP. There was no significant difference in thrombus formation on collagen in PECAM-1-/- platelets relative to litter-matched controls. The very weak inhibitory effect of PECAM-1 on platelet activation relative to that of PGI2 indicate that the Ig-receptor is not a major regulator of platelet activation. PECAM-1 has been reported to have contrasting effects on platelet activation. The present study demonstrates a very mild or negligible effect on platelet activation in response to stimulation by a variety of agonists, thereby questioning the physiological role of the immunoglobulin receptor as a major regulator of platelet activation.