ECOPHAGE: Combating Antimicrobial Resistance Using Bacteriophages for Eco-Sustainable Agriculture and Food Systems.

The focus of this meeting was to discuss the suitability of using bacteriophages as alternative antimicrobials in the agrifood sector. Following a One Health approach, the workshop explored the possibilities of implementing phage application strategies in the agriculture, animal husbandry, aquaculture, and food production sectors. Therefore, the meeting had gathered phage researchers, representatives of the agrifood industry, and policymakers to debate the advantages and potential shortcomings of using bacteriophages as alternatives to traditional antimicrobials and chemical pesticides. Industry delegates showed the latest objectives and demands from consumers. Representatives of regulatory agencies (European Medicines Agency (EMA) and Spanish Agency of Medicines and Health Products (AEMPS)) presented an update of new regulatory aspects that will impact and support the approval and implementation of phage application strategies across the different sectors.

Solarplast®-An Enzymatically Treated Spinach Extract.

In the modern world we are constantly bombarded by environmental and natural stimuli that can result in oxidative stress. Antioxidant molecules and enzymes help the human body scavenge reactive oxygen species and prevent oxidative damage. Most organisms possess intrinsic antioxidant activity, but also benefit from the consumption of antioxidants from their diet. Leafy green vegetables such as spinach are a well-researched rich source of dietary antioxidant molecules. However, plant cell walls are difficult to digest for many individuals and the bio-accessibility of nutrients and antioxidants from these sources can be limited by the degree of digestion and assimilation. Through a specific enzymatic process, Solarplast® contains organic spinach protoplasts without the cell wall, which may facilitate higher yield and efficacy of beneficial antioxidant molecules. In this study, analytical techniques coupled to in vitro bioassays were used to determine the potential antioxidant activity of Solarplast® and determine its antioxidant enzymatic capabilities. Solarplast® demonstrated superior antioxidant activity when compared to frozen spinach leaves in TOC, FRAP and TEAC antioxidant assays. Several antioxidant enzymes were also increased in Solarplast®, when compared to frozen spinach. As a functional readout, Solarplast® attenuated hydrogen peroxide-, ethanol- and acetaminophen-induced increases in oxidative stress and cytotoxicity in both intestinal (HT-29) and liver (HepG2) cell lines. These findings suggest that Solarplast® may represent a non-GMO, plant-based food supplement to help reduce oxidative stress in the human body.

Bacillus megaterium Renuspore® as a potential probiotic for gut health and detoxification of unwanted dietary contaminants.

Exposure to diverse environmental pollutants and food contaminants is ever-increasing. The risks related to the bioaccumulation of such xenobiotics in the air and food chain have exerted negative effects on human health, such as inflammation, oxidative stress, DNA damage, gastrointestinal disorders, and chronic diseases. The use of probiotics is considered an economical and versatile tool for the detoxification of hazardous chemicals that are persistent in the environment and food chain, potentially for scavenging unwanted xenobiotics in the gut. In this study, Bacillus megaterium MIT411 (Renuspore®) was characterized for general probiotic properties including antimicrobial activity, dietary metabolism, and antioxidant activity, and for the capacity to detoxify several environmental contaminants that can be found in the food chain. In silico studies revealed genes associated with carbohydrate, protein and lipid metabolism, xenobiotic chelation or degradation, and antioxidant properties. Bacillus megaterium MIT411 (Renuspore®) demonstrated high levels of total antioxidant activities, in addition to antimicrobial activity against Escherichia coli, Salmonella enterica, Staphylococcus aureus, and Campylobacter jejuni in vitro. The metabolic analysis demonstrated strong enzymatic activity with a high release of amino acids and beneficial short-chain fatty acids (SCFAs). Moreover, Renuspore® effectively chelated the heavy metals, mercury and lead, without negatively impacting the beneficial minerals, iron, magnesium, or calcium, and degraded the environmental contaminants, nitrite, ammonia, and 4-Chloro-2-nitrophenol. These findings suggest that Renuspore® may play a beneficial role in supporting gut health metabolism and eliminating unwanted dietary contaminants.

Immunomodulatory and Antioxidant Properties of a Novel Potential Probiotic Bacillus clausii CSI08.

Spore-forming bacteria of the Bacillus genus have demonstrated potential as probiotics for human use. Bacillus clausii have been recognized as efficacious and safe agents for preventing and treating diarrhea in children and adults, with pronounced immunomodulatory properties during several in vitro and clinical studies. Herein, we characterize the novel strain of B. clausii CSI08 (Munispore®) for probiotic attributes including resistance to gastric acid and bile salts, the ability to suppress the growth of human pathogens, the capacity to assimilate wide range of carbohydrates and to produce potentially beneficial enzymes. Both spores and vegetative cells of this strain were able to adhere to a mucous-producing intestinal cell line and to attenuate the LPS- and Poly I:C-triggered pro-inflammatory cytokine gene expression in HT-29 intestinal cell line. Vegetative cells of B. clausii CSI08 were also able to elicit a robust immune response in U937-derived macrophages. Furthermore, B. clausii CSI08 demonstrated cytoprotective effects in in vitro cell culture and in vivo C. elegans models of oxidative stress. Taken together, these beneficial properties provide strong evidence for B. clausii CSI08 as a promising potential probiotic.

In vitro safety and functional characterization of the novel Bacillus coagulans strain CGI314.

Introduction: Bacillus coagulans species have garnered much interest in health-related functional food research owing to their desirable probiotic properties, including pathogen exclusion, antioxidant, antimicrobial, immunomodulatory and food fermentation capabilities coupled with their tolerance of extreme environments (pH, temperature, gastric and bile acid resistance) and stability due to their endosporulation ability. Methods: In this study, the novel strain Bacillus coagulans CGI314 was assessed for safety, and functional probiotic attributes including resistance to heat, gastric acid and bile salts, the ability to adhere to intestinal cells, aggregation properties, the ability to suppress the growth of human pathogens, enzymatic profile, antioxidant capacity using biochemical and cell-based methods, cholesterol assimilation, anti-inflammatory activity, and attenuation of hydrogen peroxide (H2O2)-induced disruption of the intestinal-epithelial barrier. Results: B. coagulans CGI314 spores display resistance to high temperatures (40°C, 70°C, and 90°C), and gastric and bile acids [pH 3.0 and bile salt (0.3%)], demonstrating its ability to survive and remain viable under gastrointestinal conditions. Spores and the vegetative form of this strain were able to adhere to a mucous-producing intestinal cell line, demonstrated moderate auto-aggregation properties, and could co-aggregate with potentially pathogenic bacteria. Vegetative cells attenuated LPS-induced pro-inflammatory cytokine gene expression in HT-29 intestinal cell lines and demonstrated broad antagonistic activity toward numerous urinary tract, intestinal, oral, and skin pathogens. Metabolomic profiling demonstrated its ability to synthesize several amino acids, vitamins and short-chain fatty acids from the breakdown of complex molecules or by de novo synthesis. Additionally, B. coagulans CGI314's strong antioxidant capacity was demonstrated using enzyme-based methods and was further supported by its cytoprotective and antioxidant effects in HepG2 and HT-29 cell lines. Furthermore, B. coagulans CGI314 significantly increased the expression of tight junction proteins and partially ameliorated the detrimental effects of H2O2 induced intestinal-epithelial barrier integrity. Discussion: Taken together these beneficial functional properties provide strong evidence for B. coagulans CGI314 as a promising potential probiotic candidate in food products.

In vitro and in silico assessment of probiotic and functional properties of Bacillus subtilis DE111®.

Bacillus subtilis DE111® is a safe, well-tolerated commercially available spore-forming probiotic that has been clinically shown to support a healthy gut microbiome, and to promote digestive and immune health in both adults and children. Recently it was shown that this spore-forming probiotic was capable of germinating in the gastrointestinal tract as early as 3 h after ingestion. However, a better understanding of the mechanisms involved in the efficacy of DE111® is required. Therefore, the present investigation was undertaken to elucidate the functional properties of DE111® through employing a combination of in vitro functional assays and genome analysis. DE111® genome mining revealed the presence of several genes encoding acid and stress tolerance mechanisms in addition to adhesion proteins required to survive and colonize harsh gastrointestinal environment including multi subunit ATPases, arginine deiminase (ADI) pathway genes (argBDR), stress (GroES/GroEL and DnaK/DnaJ) and extracellular polymeric substances (EPS) biosynthesis genes (pgsBCA). DE111® harbors several genes encoding enzymes involved in the metabolism of dietary molecules (protease, lipases, and carbohyrolases), antioxidant activity and genes associated with the synthesis of several B-vitamins (thiamine, riboflavin, pyridoxin, biotin, and folate), vitamin K2 (menaquinone) and seven amino acids including five essential amino acids (threonine, tryptophan, methionine, leucine, and lysine). Furthermore, a combined in silico analysis of bacteriocin producing genes with in vitro analysis highlighted a broad antagonistic activity of DE111® toward numerous urinary tract, intestinal, and skin pathogens. Enzymatic activities included proteases, peptidases, esterase's, and carbohydrate metabolism coupled with metabolomic analysis of DE111® fermented ultra-high temperature milk, revealed a high release of amino acids and beneficial short chain fatty acids (SCFAs). Together, this study demonstrates the genetic and phenotypic ability of DE111® for surviving harsh gastric transit and conferring health benefits to the host, in particular its efficacy in the metabolism of dietary molecules, and its potential to generate beneficial SCFAs, casein-derived bioactive peptides, as well as its high antioxidant and antimicrobial potential. Thus, supporting the use of DE111® as a nutrient supplement and its pottential use in the preparation of functional foods.

Presence and Germination of the Probiotic Bacillus subtilis DE111® in the Human Small Intestinal Tract: A Randomized, Crossover, Double-Blind, and Placebo-Controlled Study.

Spore-based probiotics offer important advantages over other probiotics as they can survive the harsh gastric conditions of the stomach and bile salts in the small intestine, ultimately germinating in the digestive tract. A novel clinical trial in 11 ileostomy participants was conducted to directly investigate the presence and germination of the probiotic strain Bacillus subtilis DE111® in the small intestine. Three hours following ingestion of DE111®, B. subtilis spores (6.4 × 104 ± 1.3 × 105 CFU/g effluent dry weight) and vegetative cells (4.7 × 104 ± 1.1 × 105 CFU/g effluent dry weight) began to appear in the ileum effluent. Six hours after ingestion, spore concentration increased to 9.7 × 107 ± 8.1 × 107 CFU/g and remained constant to the final time point of 8 h. Vegetative cells reached a concentration of 7.3 × 107 ± 1.4 × 108 CFU/g at 7 h following ingestion. These results reveal orally ingested B. subtilis DE111® spores are able to remain viable during transit through the stomach and germinate in the small intestine of humans within 3 h of ingestion.

Topology of the yeast Ras converting enzyme as inferred from cysteine accessibility studies.

The Ras converting enzyme (Rce1p) is an endoprotease that is involved in the post-translational processing of the Ras GTPases and other isoprenylated proteins. Its role in Ras biosynthesis marks Rce1p as an anticancer target. By assessing the chemical accessibility of cysteine residues substituted throughout the Saccharomyces cerevisiae Rce1p sequence, we have determined that yeast Rce1p has eight segments that are protected from chemical modification. Notably, the three residues that are essential for yeast Rce1p function (E156, H194, and H248) are all chemically inaccessible and associated with separate protected segments. By specifically assessing the chemical reactivity and glycosylation potential of the NH2 and COOH termini of Rce1p, we further demonstrate that Rce1p has an odd number of transmembrane spans. Substantial evidence that the most NH2-terminal segment functions as a transmembrane segment with the extreme NH2 terminus projecting into the endoplasmic reticulum (ER) lumen is presented. Because each of the remaining seven segments is too short to contain two spans and is flanked by chemically reactive positions, we infer that these segments are not transmembrane segments but rather represent compact structural features and/or hydrophobic loops that penetrate but do not fully span the bilayer (i.e., re-entrant helices). We thus propose a topological model in which yeast Rce1p contains a single transmembrane helix localized at its extreme NH2 terminus and one or more re-entrant helices and/or compact structural domains that populate the cytosolic face of the ER membrane. Lastly, we demonstrate that the natural cysteine residues of Rce1p are chemically inaccessible and fully dispensable for in vivo enzyme activity, formally eliminating the possibility of a cysteine-based enzymatic mechanism for this protease.

The effect of apparent latency on simulator sickness while using a see-through helmet-mounted display: reducing apparent latency with predictive compensation.

OBJECTIVE: The aim of this study was to determine the effect of head movement frequency and predictive compensation on (a) latency produced in a monocular see-through helmet-mounted display (HMD) test bed and (b) simulator sickness experienced by users wearing the HMD. BACKGROUND: There is conflicting research regarding latency as a significant factor in the onset of simulator sickness. Predictive compensation has been shown to mitigate the magnitude of latency, but little is known about the extent of its effect on simulator sickness. METHOD: A video camera recorded HMD and simulator imagery to accurately measure apparent latency produced at three head movement frequencies. Predictive compensation strategies were manipulated to measure the difference in apparent latency produced by the test bed in various conditions. Similar methodology was employed with human participants to measure simulator sickness experienced by users of this test bed. RESULTS: In Experiment 1, apparent latency increased significantly as head movement frequency increased. Predictive compensation strategies significantly reduced apparent latency present in the test bed. In Experiment 2, predictive compensation significantly reduced the magnitude of simulator sickness. CONCLUSION: Predictive compensation can be effectively implemented to reduce apparent latency, resulting in a lower magnitude of simulator sickness. APPLICATION: Potential applications include HMD use in which head position is tracked and visual imagery is linked to head or body movement, such as in virtual and augmented reality systems, and is thus critical to functionality and performance.

Finding the team for Mars: a psychological and human factors analysis of a Mars Desert Research Station crew.

A two-week mission in March and April of 2011 sent six team members to the Mars Desert Research Station (MDRS). MDRS, a research facility in the high Utah desert, provides an analogue for the harsh and unusual working conditions that will be faced by men and women who one day explore Mars. During the mission a selection of quantitative and qualitative psychological tests were administered to the international, multidisciplinary team. A selection of the results are presented along with discussion.

The N-terminal transmembrane domain of lambda S is required for holin but not antiholin function.

The lambda S gene encodes a holin, S105, and an antiholin, S107, which differs by its Met-Lys N-terminal extension. The model for the lysis-defective character of S107 stipulates that the additional N-terminal basic residue keeps S107 from assuming the topology of S105, which is N-out, C-in, with three transmembrane domains (TMDs). Here we show that the N terminus of S105 retains its fMet residue but that the N terminus of S107 is fully deformylated. This supports the model that in S105, TMD1 inserts into the membrane very rapidly but that in S107, it is retained in the cytoplasm. Further, it reveals that, compared to S105, S107 has two extra positively charged moieties, Lys2 and the free N-terminal amino group, to hinder its penetration into an energized membrane. Moreover, an allele, S105(DeltaTMD1), with TMD1 deleted, was found to be defective in lysis, insensitive to membrane depolarization, and dominant to the wild-type allele, indicating that the lysis-defective, antiholin character of S107 is due to the absence of TMD1 from the bilayer rather than to its ectopic localization at the inner face of the cytoplasmic membrane. Finally, the antiholin function of the deletion protein was compromised by the substitution of early-lysis missense mutations in either the deletion protein or parental S105 but restored when both S105(DeltaTMD1) and holin carried the substitution.

The holin of bacteriophage lambda forms rings with large diameter.

Holins control the length of the infection cycle of tailed phages (the Caudovirales) by oligomerizing to form lethal holes in the cytoplasmic membrane at a time dictated by their primary structure. Nothing is currently known about the physical basis of their oligomerization or the structure of the oligomers formed by any known holin. Here we use electron microscopy and single-particle analysis to characterize structures formed by the bacteriophage lambda holin (S105) in vitro. In non-ionic or mild zwitterionic detergents, purified S105, but not the lysis-defective variant S105A52V, forms rings of at least two size classes, the most common having inner and outer diameters of 8.5 and 23 nm respectively, and containing approximately 72 S105 monomers. The height of these rings, 4 nm, closely matches the thickness of the lipid bilayer. The central channel is of unprecedented size for channels formed by integral membrane proteins, consistent with the non-specific nature of holin-mediated membrane permeabilization. S105 present in detergent-solubilized rings and in inverted membrane vesicles showed similar sensitivities to proteolysis and cysteine-specific modification, suggesting that the rings are representative of the lethal holes formed by S105 to terminate the infection cycle and initiate lysis.

Topological dynamics of holins in programmed bacterial lysis.

The fate of phage-infected bacteria is determined by the holin, a small membrane protein that triggers to disrupt the membrane at a programmed time, allowing a lysozyme to attack the cell wall. S(21)68, the holin of phage 21, has two transmembrane domains (TMDs) with a predicted N-in, C-in topology. Surprisingly, TMD1 of S(21)68 was found to be dispensable for function, to behave as a SAR ("signal-anchor-release") domain in exiting the membrane to the periplasm, and to engage in homotypic interactions in the soluble phase. The departure of TMD1 from the bilayer coincides with the lethal triggering of the holin and is accelerated by membrane depolarization. Basic residues added at the N terminus of S(21)68 prevent the escape of TMD1 to the periplasm and block hole formation by TMD2. Lysis thus depends on dynamic topology, in that removal of the inhibitory TMD1 from the bilayer frees TMD2 for programmed formation of lethal membrane lesions.

+Gz acceleration loss of consciousness: time course of performance deficits with repeated experience.

OBJECTIVES: We examine the time course of performance recovery from gravity-induced loss of consciousness (GLOC) and evaluate the utility of exposing participants to repeated bouts of GLOC in promoting recovery time. BACKGROUND: A substantial number of accidents among fighter pilots have resulted from episodes of GLOC. U.S. Air Force doctrine holds that when pilots experience GLOC, impairment lasts for 24 s, in which there are 12 s of complete unconsciousness and 12 s of confusion. However, there is reason to suspect that performance efficiency associated with GLOC is degraded well before unconsciousness sets in and that more than 24 s are required for performance efficiency to return to baseline levels. Additionally, there is a belief that repeated exposure to GLOC will reduce recovery time. METHOD: Centrifuge simulators were used to induce GLOC in U.S. Air Force personnel with math and tracking tasks employed to emulate flight performance. Participants were tested once per week for 4 consecutive weeks. RESULTS: On average, performance deficits appeared 7.44 s prior to the onset of unconsciousness and persisted for 55.6 s following the GLOC event. Repeated exposure failed to moderate these results. CONCLUSION: The temporal course of performance deficits produced by GLOC far exceeds prior estimates. The problem is more serious than previously envisioned and it is not alleviated by repeated exposure to GLOC. APPLICATION: U.S. Air Force doctrine regarding the severity of GLOC and the utility of repeated exposure to this problem needs to be revised and these data incorporated into future aircraft auto recovery systems.

Asymmetric binding of membrane proteins to GroEL.

The interaction of GroEL with non-native soluble proteins has been studied intensively and structure-function relationships have been established in considerable detail. Recently, we found that GroEL is also able to bind membrane proteins in the absence of detergents and deliver them to liposomes in a biologically active state. Here, we report that three well-studied membrane proteins (bacteriorhodopsin, LacY, and the bacteriophage lambda holin) bind asymmetrically to tetradecameric GroEL. Each of the membrane proteins was visualized in one of the center cavities of GroEL using single particle analysis.

Solubilization and delivery by GroEL of megadalton complexes of the lambda holin.

GroEL can solubilize membrane proteins by binding them in its hydrophobic cavity when detergent is removed by dialysis. The best-studied example is bacteriorhodopsin, which can bind in the GroEL chaperonin at two molecules per tetradecamer. Applying this approach to the holin and antiholin proteins of phage lambda, we find that both proteins are solubilized by GroEL, in an ATP-sensitive mode, but to vastly different extents. The antiholin product, S107, saturates the chaperonin at six molecules per tetradecameric complex, whereas the holin, S105, which is missing the two N-terminal residues of S107, forms a hyper-solubilization complex with up to 350 holin molecules per GroEL, or approximately 4 MDa of protein per 0.8 MDa tetradecamer. Gel filtration chromatography and immunoprecipitation experiments confirmed the existence of complexes of the predicted masses for both S105 and S107 solubilization. For S105, negatively stained electron microscopic images show structures consistent with protein shells of the holin assembled around the chaperonin tetradecamer. Importantly, S105 can be delivered rapidly and efficiently to artificial liposomes from these complexes. In these delivery experiments, the holin exhibits efficient membrane-permeabilizing activity. The S107 antiholin can block formation of the hypersolubilization complexes, suggesting that their formation is related to an oligomerization step intrinsic to holin function.

A signal-arrest-release sequence mediates export and control of the phage P1 endolysin.

The Lyz endolysin of bacteriophage P1 was found to cause lysis of the host without a holin. Induction of a plasmid-cloned lyz resulted in lysis, and the lytic event could be triggered prematurely by treatments that dissipate the proton-motive force. Instead of requiring a holin, export was mediated by an N-terminal transmembrane domain (TMD) and required host sec function. Exported Lyz of identical SDS/PAGE mobility was found in both the membrane and periplasmic compartments, indicating that periplasmic Lyz was not generated by the proteolytic cleavage of the membrane-associated form. In gene fusion experiments, the Lyz TMD directed PhoA to both the membrane and periplasmic compartments, whereas the TMD of the integral membrane protein FtsI restricts Lyz to the membrane. Thus, the N-terminal domain of Lyz is both necessary and sufficient not only for export of this endolysin to the membrane but also for its release into the periplasm. The unusual N-terminal domain, rich in residues that are weakly hydrophobic, thus functions as a signal-arrest-release sequence, which first acts as a normal signal-arrest domain to direct the endolysin to the periplasm in membrane-tethered form and then allows it to be released as a soluble active enzyme in the periplasm. Examination of the protein sequences of related bacteriophage endolysins suggests that the presence of an N-terminal signal-arrest-release sequence is not unique to Lyz. These observations are discussed in relation to the role of holins in the control of host lysis by bacteriophage encoding a secretory endolysin.

Functional bacteriorhodopsin is efficiently solubilized and delivered to membranes by the chaperonin GroEL.

Soluble complexes between the tetradecameric chaperonin GroEL and integral membrane proteins can be efficiently formed by detergent dialysis. For example, GroEL14 was found to bind a limit of two molecules of bacteriorhodopsin (BR). The GroEL-solubilized BR molecules were rapidly ejected from the chaperonin complexes on the addition of ATP or adenosine 5'-[beta,gamma-imido]triphosphate but not AMP, indicating that conformational changes induced by nucleotide binding eliminate a binding site for the hydrophobic transmembrane domains. BR retains its native conformation in the GroEL complexes, as judged by the spectral characteristics of the bound retinal. Moreover, the chaperonin-solubilized BR could be transferred efficiently to liposomes and used to effect a light-driven proton gradient, indicating that both native conformation and vectorial insertion were accomplished. These results suggest new approaches to the study of purified integral membrane proteins in their natural membrane environment and raise the prospect that GroEL may have a role in the integration of proteins into the cytoplasmic membrane in vivo.

Sizing the holin lesion with an endolysin-beta-galactosidase fusion.

Double-stranded DNA phages require two proteins for efficient host lysis: the endolysin, a muralytic enzyme, and the holin, a small membrane protein. In an event that defines the end of the vegetative cycle, the lambda holin S acts suddenly to permeabilize the membrane. This permeabilization enables the R endolysin to attack the cell wall, after which cell lysis occurs within seconds. A C-terminal fusion of the R endolysin with full-length beta-galactosidase (beta-Gal) was tested for lytic competence in the context of the late-gene expression system of an induced lambda lysogen. Under these conditions, the hybrid R-beta-Gal product, an active tetrameric beta-Gal greater than 480 kDa in mass, was fully functional in lysis mediated by the S holin. Western blot analysis demonstrated that the lytic competence was not due to the proteolytic release of the endolysin domain of the R-beta-Gal fusion protein. The ability of this massive complex to be released by the S holin suggests that S causes a generalized membrane disruption rather than a regular oligomeric membrane pore. Similar results were obtained with an early lysis variant of the S holin and also in parallel experiments with the T4 holin, T, in an identical lambda context. However, premature holin lesions triggered by depolarization of the membrane were nonpermissive for the hybrid endolysin, indicating that these premature lesions constituted less-profound damage to the membrane. Finally, a truncated T holin functional in lysis with the endolysin is completely incompetent for lysis with the hybrid endolysin. A model for the formation of the membrane lesion within homo-oligomeric rafts of holin proteins is discussed.