The structure of Lactobacillus brevis surface layer reassembled on liposomes differs from native structure as revealed by SAXS

AbstractThe reassembly of the S-layer protein SlpA of Lactobacillus brevis ATCC 8287 on positively charged liposomes was studied by small angle X-ray scattering (SAXS) and zeta potential measurements. SlpA was reassembled on unilamellar liposomes consisting of 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-3-trimethylammonium-propane, prepared by extrusion through membranes with pore sizes of 50nm and 100nm. Similarly extruded samples without SlpA were used as a reference. The SlpA-containing samples showed clear diffraction peaks in their SAXS intensities. The lattice constants were calculated from the diffraction pattern and compared to those determined for SlpA on native cell wall fragments. Lattice constants for SlpA reassembled on liposomes (a=9.29nm, b=8.03nm, and γ=84.9°) showed a marked change in the lattice constants b and γ when compared to those determined for SlpA on native cell wall fragments (a=9.41nm, b=6.48nm, and γ=77.0°). The latter are in good agreement with values previously determined by electron microscopy. This indicates that the structure formed by SlpA is stable on the bacterial cell wall, but SlpA reassembles into a different structure on cationic liposomes. From the (10) reflection, the lower limit of crystallite size of SlpA on liposomes was determined to be 92nm, corresponding to approximately ten aligned lattice planes

Nanosilver-Silica Composite : Prolonged Antibacterial Effects and Bacterial Interaction Mechanisms for Wound Dressings

Infected superficial wounds were traditionally controlled by topical antibiotics until the emergence of antibiotic-resistant bacteria. Silver (Ag) is a kernel for alternative antibacterial agents to fight this resistance quandary. The present study demonstrates a method for immobilizing small-sized (similar to 5 nm) silver nanoparticles on silica matrix to form a nanosilver-silica (Ag-SiO2) composite and shows the prolonged antibacterial effects of the composite in vitro. The composite exhibited a rapid initial Ag release after 24 h and a slower leaching after 48 and 72 h and was effective against both methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli). Ultraviolet (UV)-irradiation was superior to filter-sterilization in retaining the antibacterial effects of the composite, through the higher remaining Ag concentration. A gauze, impregnated with the Ag-SiO2 composite, showed higher antibacterial effects against MRSA and E. coli than a commercial Ag-containing dressing, indicating a potential for the management and infection control of superficial wounds. Transmission and scanning transmission electron microscope analyses of the composite-treated MRSA revealed an interaction of the released silver ions with the bacterial cytoplasmic constituents, causing ultimately the loss of bacterial membranes. The present results indicate that the Ag-SiO2 composite, with prolonged antibacterial effects, is a promising candidate for wound dressing applications.Peer reviewe

Silica-gentamicin nanohybrids : combating antibiotic resistance, bacterial biofilms, and in vivo toxicity

Introduction: Antibiotic resistance is a growing concern in health care. Methicillin-resistant Staphylococcus aureus (MRSA), forming biofilms, is a common cause of resistant orthopedic implant infections. Gentamicin is a crucial antibiotic preventing orthopedic infections. Silica-gentamicin (SiO2-G) delivery systems have attracted significant interest in preventing the formation of biofilms. However, compelling scientific evidence addressing their efficacy against planktonic MRSA and MRSA biofilms is still lacking, and their safety has not extensively been studied. Materials and methods: In this work, we have investigated the effects of SiO2-G nanohybrids against planktonic MRSA as well as MRSA and Escherichia coli biofilms and then evaluated their toxicity in zebrafish embryos, which are an excellent model for assessing the toxicity of nanotherapeutics. Results: SiO2-G nanohybrids inhibited the growth and killed planktonic MRSA at a minimum concentration of 500 mu g/mL. SiO2-G nanohybrids entirely eradicated E. coli cells in biofilms at a minimum concentration of 250 mu g/mL and utterly deformed their ultrastructure through the deterioration of bacterial shapes and wrinkling of their cell walls. Zebrafish embryos exposed to SiO2-G nanohybrids (500 and 1,000 mu g/mL) showed a nonsignificant increase in mortality rates, 13.4 +/- 9.4 and 15%+/- 7.1%, respectively, mainly detected 24 hours post fertilization (hpf). Frequencies of malformations were significantly different from the control group only 24 hpf at the higher exposure concentration. Conclusion: Collectively, this work provides the first comprehensive in vivo assessment of SiO2-G nanohybrids as a biocompatible drug delivery system and describes the efficacy of SiO2-G nanohybrids in combating planktonic MRSA cells and eradicating E. coli biofilms.Peer reviewe

Characterization of the SpaCBA Pilus Fibers in the Probiotic Lactobacillus rhamnosus GG

Lactobacillus rhamnosus GG is a human intestinal isolate that has been studied intensively because of its probiotic properties. We have previously shown that L. rhamnosus GG produces proteinaceous pili that earlier had been observed only in Gram-positive pathogens (M. Kankainen et al., Proc. Natl. Acad. Sci. U. S. A. 106:17193-17198, 2009). These pili were found to be encoded by the spaCBA gene cluster, and the pilus-associated SpaC pilin was shown to confer on the cells a mucus-binding ability. In addition to the spaCBA cluster, another putative pilus cluster, spaFED, was predicted from the L. rhamnosus GG genome sequence. Herein, we show that only SpaCBA pili are produced by L. rhamnosus, and we describe a detailed analysis of cell wall-associated and affinity-purified SpaCBA pili by Western blotting and immunogold electron microscopy. Our results indicate that SpaCBA pili are heterotrimeric protrusions with a SpaA subunit as the shaft-forming major pilin. Only a few SpaB subunits could be observed in pilus fibers. Instead, SpaB pilins were found at pilus bases, as assessed by immunogold double labeling of thin sections of cells, suggesting that SpaB is involved in the termination of pilus assembly. The SpaC adhesin was present along the whole pilus length at numbers nearly equaling those of SpaA. The relative amount and uniform distribution of SpaC within pili not only makes it possible to exert both long-distance and intimate contact with host tissue but also provides mucus-binding strength, which explains the prolonged intestinal residency times observed for L. rhamnosus GG compared to that of nonpiliated lactobacill

Comparative genome analysis of Lactobacillus casei strains isolated from Actimel and Yakult products reveals marked similarities and points to a common origin

The members of the Lactobacillus genus are widely used in the food and feed industry and show a remarkable ecological adaptability. Several Lactobacillus strains have been marketed as probiotics as they possess health-promoting properties for the host. In the present study, we used two complementary next-generation sequencing technologies to deduce the genome sequences of two Lactobacillus casei strains LcA and LcY, which were isolated from the products Actimel and Yakult, commercialized as probiotics. The LcA and LcY draft genomes have, respectively, an estimated size of 3067 and 3082 Mb and a G+ C content of 46.3%. Both strains are close to identical to each other and differ by no more than minor chromosomal re-arrangements, substitutions, insertions and deletions, as evident from the verified presence of one insertion-deletion (InDel) and only 29 single-nucleotide polymorphisms (SNPs). In terms of coding capacity, LcA and LcY are predicted to encode a comparable exoproteome, indicating that LcA and LcY are likely to establish similar interactions with human intestinal cells. Moreover, both L. casei LcA and LcY harboured a 59.6 kb plasmid that shared high similarities with plasmids found in other L. casei strains, such as W56 and BD-II. Further analysis revealed that the L. casei plasmids constitute a good evolution marker within the L. casei species. The plasmids of the LcA and LcY strains are almost identical, as testified by the presence of only three verified SNPs, and share a 3.5 kb region encoding a remnant of a lactose PTS system that is absent from the plasmids of W56 and BD-II but conserved in another smaller L. casei plasmid (pLC2W). Our observations imply that the results obtained in animal and human experiments performed with the Actimel and Yakult strains can be compared with each other as these strains share a very recent common ancestor

A computational study on altered theta-gamma coupling during learning and phase coding

There is considerable interest in the role of coupling between theta and gamma oscillations in the brain in the context of learning and memory. Here we have used a neural network model which is capable of producing coupling of theta phase to gamma amplitude firstly to explore its ability to reproduce reported learning changes and secondly to memory-span and phase coding effects. The spiking neural network incorporates two kinetically different GABAA receptor-mediated currents to generate both theta and gamma rhythms and we have found that by selective alteration of both NMDA receptors and GABAA,slow receptors it can reproduce learning-related changes in the strength of coupling between theta and gamma either with or without coincident changes in theta amplitude. When the model was used to explore the relationship between theta and gamma oscillations, working memory capacity and phase coding it showed that the potential storage capacity of short term memories, in terms of nested gamma-subcycles, coincides with the maximal theta power. Increasing theta power is also related to the precision of theta phase which functions as a potential timing clock for neuronal firing in the cortex or hippocampus

BopA does not have a major role in the adhesion of bifidobacterium bifidum to intestinal epithelial cells, extracellular matrix proteins, and mucus

he ability of bifidobacteria to adhere to the intestine of the human host is considered to be important for efficient colonization and achieving probiotic effects. Bifidobacterium bifidum strains DSM20456 and MIMBb75 adhere well to the human intestinal cell lines Caco-2 and HT-29. The surface lipoprotein BopA was previously described to be involved in mediating adherence of B. bifidum to epithelial cells, but thioacylated, purified BopA inhibited the adhesion of B. bifidum to epithelial cells in competitive adhesion assays only at very high concentrations, indicating an unspecific effect. In this study, the role of BopA in the adhesion of B. bifidum was readdressed. The gene encoding BopA was cloned and expressed without its lipobox and hydrophobic signal peptide in Escherichia coli, and an antiserum against the recombinant BopA was produced. The antiserum was used to demonstrate the abundant localization of BopA on the cell surface of B. bifidum. However, blocking of B. bifidum BopA with specific antiserum did not reduce adhesion of bacteria to epithelial cell lines, arguing that BopA is not an adhesin. Also, adhesion of B. bifidum to human colonic mucin and fibronectin was found to be BopA independent. The recombinant BopA bound only moderately to human epithelial cells and colonic mucus, and it failed to bind to fibronectin. Thus, our results contrast the earlier findings on the major role of BopA in adhesion, indicating that the strong adhesion of B. bifidum to epithelial cell lines is BopA independent

Representation of Time-Varying Stimuli by a Network Exhibiting Oscillations on a Faster Time Scale

Sensory processing is associated with gamma frequency oscillations (30–80 Hz) in sensory cortices. This raises the question whether gamma oscillations can be directly involved in the representation of time-varying stimuli, including stimuli whose time scale is longer than a gamma cycle. We are interested in the ability of the system to reliably distinguish different stimuli while being robust to stimulus variations such as uniform time-warp. We address this issue with a dynamical model of spiking neurons and study the response to an asymmetric sawtooth input current over a range of shape parameters. These parameters describe how fast the input current rises and falls in time. Our network consists of inhibitory and excitatory populations that are sufficient for generating oscillations in the gamma range. The oscillations period is about one-third of the stimulus duration. Embedded in this network is a subpopulation of excitatory cells that respond to the sawtooth stimulus and a subpopulation of cells that respond to an onset cue. The intrinsic gamma oscillations generate a temporally sparse code for the external stimuli. In this code, an excitatory cell may fire a single spike during a gamma cycle, depending on its tuning properties and on the temporal structure of the specific input; the identity of the stimulus is coded by the list of excitatory cells that fire during each cycle. We quantify the properties of this representation in a series of simulations and show that the sparseness of the code makes it robust to uniform warping of the time scale. We find that resetting of the oscillation phase at stimulus onset is important for a reliable representation of the stimulus and that there is a tradeoff between the resolution of the neural representation of the stimulus and robustness to time-warp. Author Summary Sensory processing of time-varying stimuli, such as speech, is associated with high-frequency oscillatory cortical activity, the functional significance of which is still unknown. One possibility is that the oscillations are part of a stimulus-encoding mechanism. Here, we investigate a computational model of such a mechanism, a spiking neuronal network whose intrinsic oscillations interact with external input (waveforms simulating short speech segments in a single acoustic frequency band) to encode stimuli that extend over a time interval longer than the oscillation's period. The network implements a temporally sparse encoding, whose robustness to time warping and neuronal noise we quantify. To our knowledge, this study is the first to demonstrate that a biophysically plausible model of oscillations occurring in the processing of auditory input may generate a representation of signals that span multiple oscillation cycles.National Science Foundation (DMS-0211505); Burroughs Wellcome Fund; U.S. Air Force Office of Scientific Researc

Low-cost irradiance sensors for irradiation assessments inside tree canopies.

The solar irradiation that a crop receives is directly related to the physical and biological processes that affect the crop. However, the assessment of solar irradiation poses certain problems when it must be measured through fruit inside the canopy of a tree. In such cases, it is necessary to check many test points, which usually requires an expensive data acquisition system. The use of conventional irradiance sensors increases the cost of the experiment, making them unsuitable. Nevertheless, it is still possible to perform a precise irradiance test with a reduced price by using low-cost sensors based on the photovoltaic effect. The aim of this work is to develop a low-cost sensor that permits the measurement of the irradiance inside the tree canopy. Two different technologies of solar cells were analyzed for their use in the measurement of solar irradiation levels inside tree canopies. Two data acquisition system setups were also tested and compared. Experiments were performed in Ademuz (Valencia, Spain) in September 2011 and September 2012 to check the validity of low-cost sensors based on solar cells and their associated data acquisition systems. The observed difference between solar irradiation at high and low positions was of 18.5% ± 2.58% at a 95% confidence interval. Large differences were observed between the operations of the two tested sensors. In the case of a-Si cells based mini-modules, an effect of partial shadowing was detected due to the larger size of the devices, the use of individual c-Si cells is recommended over a-Si cells based mini-modules