Marked Strain Research Articles

Cuticle deposition ceases during strawberry fruit development

BackgroundIdeally, the barrier properties of a fruit’s cuticle persist throughout its development. This presents a challenge for strawberry fruit, with their rapid development and thin cuticles. The objective was to establish the developmental time course of cuticle deposition in strawberry fruit.ResultsFruit mass and surface area increase rapidly, with peak growth rate coinciding with the onset of ripening. On a whole-fruit basis, the masses of cutin and wax increase but on a unit surface-area basis, they decrease. The decrease is associated with marked increases in elastic strain.The expressions of cuticle-associated genes involved in transcriptional regulation (FaSHN1, FaSHN2, FaSHN3), synthesis of cutin (FaLACS2, FaGPAT3) and wax (FaCER1, FaKCS10, FaKCR1), and those involved in transport of cutin monomers and wax constituents (FaABCG11, FaABCG32) decreased until maturity. The only exceptions were FaLACS6 and FaGPAT6 that are presumably involved in cutin synthesis, and FaCER1 involved in wax synthesis. This result was consistent across five strawberry cultivars.Strawberry cutin consists mainly of C16 and C18 monomers, plus minor amounts of C19, C20, C22 and C24 monomers, ω-hydroxy acids, dihydroxy acids, epoxy acids, primary alcohols, carboxylic acids and dicarboxylic acids. The most abundant monomer is 10,16-dihydroxyhexadecanoic acid. Waxes comprise mainly long-chain fatty acids C29 to C46, with smaller amounts of C16 to C28. Wax constituents are carboxylic acids, primary alcohols, alkanes, aldehydes, sterols and esters.ConclusionThe downregulation of cuticle deposition during development accounts for the marked cuticular strain, for the associated microcracking, and for their high susceptibility to the disorders of water soaking and cracking.

Confinement mechanism and modeling of basalt TRM confined concrete: Effect of mortar matrix grade

Textile reinforced mortar (TRM) confinement significantly enhances the compressive strength and ultimate axial strain of concrete. This study mainly investigated the influence of mortar strength on the stress-strain behavior of TRM-confined concrete to advance understanding of its confinement mechanism. We conducted 48 compression tests on concrete columns, comparing those without jackets to those confined in basalt TRM (BTRM). Variables included the number of textile layers (0 to 4) and mortar matrix strengths (from low-grade ‘M1′ to high-grade ‘M3′). Results indicated that a critical confinement stiffness ratio of 0.024 is essential for strain hardening behavior with marked strain capacity enhancement. Low-grade mortar diminished the effective confinement stiffness of BTRM jackets, manifesting as either a steeper descending plateau in the strain-softening stress-strain curve or a more gradual ascending plateau in the strain-hardening curve. High-strength mortar significantly reduced both the hoop rupture strain of BTRM jackets and the ultimate axial strain of the confined concrete. This study integrated the identified BTRM confinement mechanism into an existing stress-strain model for FRP-confined concrete, enhancing its predictive accuracy for BTRM-confined concrete. Specially, a coefficient, termed km, was introduced to quantify the mortar grade's impact on confinement stiffness in the confining pressure equation. Predictive results closely matched experimental data, especially in replicating plateau characteristics of the stress-strain curve.

High-resolution reconstruction-based investigation of multi-scale lamellar microstructures by coupled crystal plasticity and in-situ experiment

This study aims to establish a novel approach to better understand and predict material behavior in multi-scale lamellar microstructures. High-resolution reconstruction is built on collaborative characterization at both grain and sub-grain scale. A framework for microstructural characterization integrates, secondary electron imaging (SEI), electron backscatter diffraction (EBSD), and image recognition, to ensure that the reconstructed microstructure adequately represents the equivalent grain size, lamellar spacing, and lamellar geometric orientation. Importantly, a high-resolution reconstruction algorithm was developed combining the Laguerre-Voronoi tessellation and minimum bounding sphere (MBS), which allows the reconstructed model retain multi-scale microstructures. Finally, the mechanical properties of pearlite were investigated by combining crystal plasticity simulation with in-situ scanning electron microscopy (SEM) tensile testing based on high-resolution reconstruction. Stress-strain field analysis revealed that cross-lamellae strain bands originate from ferrite large deformation-induced shear transmission, with marked strain distribution heterogeneity within identical lamellae. This concurrently validated the excellent reliability of the novel strategy.

Chromosomal engineering of inducible isopropanol- butanol-ethanol production in Clostridium acetobutylicum.

The use of environmentally damaging petrochemical feedstocks can be displaced by fermentation processes based on engineered microbial chassis that recycle biomass-derived carbon into chemicals and fuels. The stable retention of introduced genes, designed to extend product range and/or increase productivity, is essential. Accordingly, we have created multiply marked auxotrophic strains of Clostridium acetobutylicum that provide distinct loci (pyrE, argH, purD, pheA) at which heterologous genes can be rapidly integrated using allele-coupled exchange (ACE). For each locus, ACE-mediated insertion is conveniently selected on the basis of the restoration of prototrophy on minimal media. The Clostridioides difficile gene (tcdR) encoding an orthogonal sigma factor (TcdR) was integrated at the pyrE locus under the control of the lactose-inducible, bgaR::PbgaL promoter to allow the simultaneous control of genes/operons inserted at other disparate loci (purD and pheA) that had been placed under the control of the PtcdB promoter. In control experiments, dose-dependent expression of a catP reporter gene was observed with increasing lactose concentration. At the highest doses tested (10mM) the level of expression was over 10-fold higher than if catP was placed directly under the control of bgaR::PbgaL and over 2-fold greater than achieved using the strong Pfdx promoter of the Clostridium sporogenes ferredoxin gene. The utility of the system was demonstrated in the production of isopropanol by the C. acetobutylicum strain carrying an integrated copy of tcdR following the insertion of a synthetic acetone operon (ctfA/B, adc) at the purD locus and a gene (sadh) encoding a secondary dehydrogenase at pheA. Lactose induction (10mM) resulted in the production of 4.4g/L isopropanol and 19.8g/L Isopropanol-Butanol-Ethanol mixture.

Giant electric field-induced strain and structure evolution of NaTaO3-modified 0.94(Bi0.5Na0.5)TiO3-0.06BaTiO3 Pb-free ceramics

To acquire giant electric field-induced strain in non-Pb materials is attracting a great deal of attention in the past decade. In the current investigation, the crystal and domain structures as well as the electrical performances of (1-x) (0.94Bi0.5Na0.5TiO3-0.06BaTiO3)-xNaTaO3 (BNBT-xNT) specimens were systematically studied to achieve enhanced strain. The introduction of NT makes the phase structure transit from rhombohedral-tetragonal to pseudo-cubic structure. The original domain structure of BNBT is destroyed, and the disorder degree of the local structure increases. Simultaneously, the remnant polarization, coercive field, and piezoelectric coefficients were significantly decreased. The transition from ferroelectric to ergodic relaxation can be effectively modified, thus lowering the transition zone to room temperature. Finally, the BNBT-3NT ceramics achieve marked strain coefficients at room temperature, with a maximum strain of 0.394% under 65 kV/cm and a d33* of 606 pm/V.

Application of prophage sequence analysis to investigate a disease outbreak involving Salmonella Adjame, a rare serovar and implications for the population structure.

Outbreak investigation of foodborne salmonellosis is hindered when the food source is contaminated by multiple strains of Salmonella, creating difficulties matching an incriminated organism recovered from patients with the specific strain in the suspect food. An outbreak of the rare Salmonella Adjame was caused by multiple strains of the organism as revealed by single-nucleotide polymorphism (SNP) variation. The use of highly discriminatory prophage analysis to characterize strains of Salmonella should enable a more precise strain characterization and aid the investigation of foodborne salmonellosis. We have carried out genomic analysis of S. Adjame strains recovered during the course of a recent outbreak and compared them with other strains of the organism (n = 38 strains), using SNPs to evaluate strain differences present in the core genome, and prophage sequence typing (PST) to evaluate the accessory genome. Phylogenetic analyses were performed using both total prophage content and conserved prophages. The PST analysis of the S. Adjame isolates showed a high degree of strain heterogeneity. We observed small clusters made up of 2-6 isolates (n = 27) and singletons (n = 11) in stark contrast with the three clusters observed by SNP analysis. In total, we detected 24 prophages of which only four were highly prevalent, namely: Entero_p88 (36/38 strains), Salmon_SEN34 (35/38 strains), Burkho_phiE255 (33/38 strains) and Edward_GF (28/38 strains). Despite the marked strain diversity seen with prophage analysis, the distribution of the four most common prophages matched the clustering observed using core genome. Mutations in the core and accessory genomes of S. Adjame have shed light on the evolutionary relationships among the Adjame strains and demonstrated a convergence of the variations observed in both fractions of the genome. We conclude that core and accessory genomes analyses should be adopted in foodborne bacteria outbreak investigations to provide a more accurate strain description and facilitate reliable matching of isolates from patients and incriminated food sources. The outcomes should translate to a better understanding of the microbial population structure and an 46 improved source attribution in foodborne illnesses.

High-strain rate compressive behavior of concrete with two different substituted recycled plastic aggregates: Experimental characterization and probabilistic modeling

This paper presents a pilot study on the characterization of the high-strain rate compressive behavior of a novel concrete with two different substituted recycled plastic aggregates. A reference mix with an average compressive strength of about 55MPa is considered. Recycled PolyEthylene Terephthalate (PET) powder and recycled mixed plastic (composed of PolyPropylene (PP) and PolyEthylene (PE)) granules were adopted to substitute fine and coarse aggregates. Two different substitution strategies are employed. In the first one, the PET powder is used to substitute the fine sand by volume. In the second one, the PET powder is used to substitute the fine sand while the recycled mixed plastic granules are used to substitute the coarse sand and fine coarse aggregates by volume (50% for PET powder and 50% for recycled mixed plastic granules). Four total replacement levels (5%, 10%, 20%, and 30%) by volume were considered. The fresh concrete properties (slump and density) and quasi-static compressive behavior, are investigated. The micro-scale characterization of the material using SEM scans provided a complete understanding of the observed macro-scale behavior. Tests were performed using conventional quasi-static loading with a compressive testing machine and high-strain rate tests with a Φ80-mm Split-Hopkinson Pressure Bar (SHPB) for strain rates up to 200s−1. The dynamic tests revealed the marked strain rate dependency, although specimens with large plastic volume substitution were more sensitive to the strain rate effect. A probabilistic data-driven model for the Dynamic Increase Factor (DIF) is also proposed based on the test data. Ultimately, this study indicates that the proposed material has a good high-strain rate compressive behavior and can be a promising material to be employed for protective techniques against impact and blast loads.

Fate of Listeria monocytogenes and Shiga Toxin-Producing Escherichia coli on Bresaola Slices During Storage

The viability of multistrain cocktails of genetically marked strains of Listeria monocytogenes and Shiga toxin-producing Escherichia coli (STEC) were separately monitored on slices of one brand of a commercially produced bresaola (ca. pH 6.7 and aw 0.899) during extended storage at refrigeration and abusive temperatures. Two slices (ca. 8 g each; ca.10.2 cm wide, ca. 11 cm long) of bresaola were layered horizontally within a nylon-polyethylene bag. The outer surface of each slice was inoculated (50μL total; ca. 3.5 log colony-forming units [CFU]/package) with a rifampicin-resistant (100μg/mL) cocktail of either L. monocytogenes (5 strains) or STEC (8 strains). Bags were vacuum-sealed and then stored at 4°C or 10°C for 180 or 90 d, respectively. In each of 5 trials, 3 bags were analyzed for pathogen presence at each sampling interval via the US Department of Agriculture–Agricultural Research Service package rinse method. In general, levels of L. monocytogenes and STEC decreased by 3.0 and 2.4 log CFU/package, respectively, after 180 d when bresaola was stored at 4°C. When bresaola was stored at 10°C for 90 d, levels of L. monocytogenes and STEC decreased by 2.4 and 3.1 log CFU/package, respectively. Thus, the sliced bresaola evaluated herein did not provide a favorable environment for either persistence or outgrowth of surface-inoculated cells of L. monocytogenes or STEC.

High-strain rate tension behavior of Fiber-Reinforced Rubberized Concrete

This paper investigates for the first time the high-strain rate splitting tension behavior of Fiber-Reinforced Rubberized Concrete (FRRC). Splitting tension tests were performed on Φ100-mm specimens. Quasi-static tests were carried out using conventional quasi-static loading with a compressive testing machine and dynamic high-strain rate tests with a Φ80-mm Split Hopkinson Pressure Bar (SHPB) for strain rates up to 10s−1. The experimental program comprises the characterization of Plain Concrete (PC), Fiber Reinforced Concrete (FRC), Rubberized Concrete (RuC), and FRRC with a full test matrix (a total of 240 specimens) covering fiber contents up to 1.5% and rubber volume substitution ratios up to 30%. A high-speed camera was used to capture the cracking development processes together with Digital Image Correlation (DIC). The addition of fibers enhances the deformation capacity with a marked increase in the tensile strength both for quasi-static and dynamic conditions while the substitution with rubber aggregates significantly reduces the tensile strength with a similar enhancement of the deformation capacity. For FRRC, a combination of the two effects was observed. FRC, RuC, and FRRC are characterized by marked strain rate dependency within the considered test range. Furthermore, data-driven models for compression and tension strength reduction factor (SRFc and SRFt) and tension Dynamic Increase Factor (DIF) are proposed for different types of FRRC. Finally, a discussion on the image-based damage development processes is provided based on the high-speed camera videos. This study indicates that FRRC has an excellent high-strain rate tension behavior reducing and controlling the crack propagation.

High-strain rate compressive behavior of Fiber-Reinforced Rubberized Concrete

The compressive mechanical properties of concrete-based materials are strongly affected by the presence of additions in the mix and load application velocity. Fiber-Reinforced Rubberized Concrete (FRRC) was recently proposed in the literature as a concrete-based material with the aim of obtaining a ductile material with reduced environmental burden and characterized by excellent energy absorption performances during impact loading. FRRC joins and balances the advantages and drawbacks of Fiber Reinforce Concrete (FRC) and Rubberized Concrete (RuC). However, its behavior has rarely been investigated under dynamic loadings, although gaining sufficient knowledge on their high strain rate behavior is crucial for their full-scale application. This study aims to investigate the high-strain rate compressive behavior of FRRC with fine rubber aggregates and micro-straight steel fibers. Tests were performed using conventional quasi-static loading with a compressive testing machine and high-strain rate tests with a Φ80-mm Split Hopkinson Pressure Bar (SHPB) for strain rates up to 200s−1. The experimental program comprises the characterization of Plain Concrete (PC), FRC, RuC, and FRRC with a full test matrix (a total of 192 specimens) covering fiber contents up to 1.5% and rubber volume substitution ratios up to 30%. The micro-scale characterization of the material using SEM scans provided a complete understanding of the observed macro-scale behavior. A high-speed camera was used to capture the cracking development processes. The measurements both for quasi-static and dynamic tests indicate that the addition of fibers improves the ductility or toughness with a slight increase in the compressive strength and the substitution with rubber aggregates significantly reduces the compressive strength with improved ductility or toughness. The combination of fiber and rubber (FRRC) leads to a combination of the previously described single marginal effects. The dynamic tests reveal the marked strain rate dependency, although specimens with large rubber volume substitution were more sensitive to the strain rate effect within the considered test range. Based on the test data, data-driven models for strength reduction factor (SRF) and dynamic increase factor (DIF) are proposed for different types of FRRC. The image-based waveform discussion provides evidence of the “double-peak” phenomenon highlighting the different features of the observed signals and damage evolution. Ultimately, this study indicates that FRRC has an excellent high-strain rate compressive behavior and can be a promising material to be employed to protect structures against impact and blast loads.

Phage-delivered CRISPR-Cas9 for strain-specific depletion and genomic deletions in the gut microbiome.

SUMMARYMechanistic insights into the role of the human microbiome in the predisposition to and treatment of disease are limited by the lack of methods to precisely add or remove microbial strains or genes from complex communities. Here, we demonstrate that engineered bacteriophage M13 can be used to deliver DNA to Escherichia coli within the mouse gastrointestinal (GI) tract. Delivery of a programmable exogenous CRISPR-Cas9 system enables the strain-specific depletion of fluorescently marked isogenic strains during competitive colonization and genomic deletions that encompass the target gene in mice colonized with a single strain. Multiple mechanisms allow E. coli to escape targeting, including loss of the CRISPR array or even the entire CRISPR-Cas9 system. These results provide a robust and experimentally tractable platform for microbiome editing, a foundation for the refinement of this approach to increase targeting efficiency, and a proof of concept for the extension to other phage-bacterial pairs of interest.

Phage-Delivered CRISPR-Cas9 for Strain-Specific Depletion and Genomic Deletions in the Gut Microbiome

Mechanistic insights into the role of the human microbiome in the predisposition to and treatment of disease are limited by the lack of methods to precisely add or remove microbial strains or genes from complex communities. Here, we demonstrate that engineered bacteriophage M13 can be used to deliver DNA to Escherichia coli within the mouse gastrointestinal (GI) tract. Delivery of a programmable exogenous CRISPR-Cas9 system enabled the strain-specific depletion of fluorescently marked isogenic strains during competitive colonization and genomic deletions that encompass the target gene in mice colonized with a single strain. Multiple mechanisms enabled E. coli to escape targeting, including loss of the CRISPR array or even the entire CRISPR-Cas9 system. These results provide a robust and experimentally tractable platform for microbiome editing, a foundation for the refinement of this approach to increase targeting efficiency, and a proof-of-concept for the extension to other phage-bacterial pairs of interest.

Inducing cellular immune responses with a marked Mycobacterium avium subsp. paratuberculosis strain in dairy calves

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne’s disease, a chronic granulomatous enteritis with a high global prevalence in dairy cattle. This disease causes significant economic loss in the dairy industry and has been challenging to control, as current diagnostic assays are low in sensitivity and specificity, and previously developed vaccines do not prevent infection and face regulatory concerns due to interference with bovine tuberculosis diagnostics. To remediate this issue, positive and negative immune markers were created in a MAP strain as a step towards a vaccine capable of differentiating infected from vaccinated animals (DIVA). A gene coding for an immunogenic protein (MAP1693c) in the MAP genome was replaced with a library of epitope-tagged immunogenic genes (pepA) via a stable allelic exchange method. These markers were evaluated in a calf infection trial, where Holstein-Friesian dairy calves were inoculated at two weeks of age with either the marked strain or the parent strain, or remained uninfected controls. Cellular immune responses to the markers were measured using an interferon gamma release assay (IGRA). There were no MAP1693c marker-specific differences in cellular immune responses between infection groups. A scrambled version of the HA (human influenza hemagglutinin) epitope, but not the actual HA epitope, induced a significant IFN-γ response in marker-infected calves compared to WT-infected and uninfected groups at 4.5 months post-inoculation. This scrambled HA epitope thus holds potential as a diagnostic tool as part of a DIVA vaccine for Johne’s disease.

Marked Neurospora crassa Strains for Competition Experiments and Bayesian Methods for Fitness Estimates.

The filamentous fungus Neurospora crassa, a model microbial eukaryote, has a life cycle with many features that make it suitable for studying experimental evolution. However, it has lacked a general tool for estimating relative fitness of different strains in competition experiments. To remedy this need, we constructed N. crassa strains that contain a modified csr-1 locus and developed an assay for detecting the proportion of the marked strain using a post PCR high resolution melting assay. DNA extraction from spore samples can be performed on 96-well plates, followed by a PCR step, which allows many samples to be processed with ease. Furthermore, we suggest a Bayesian approach for estimating relative fitness from competition experiments that takes into account the uncertainty in measured strain proportions. We show that there is a fitness effect of the mating type locus, as mating type mat a has a higher competitive fitness than mat A. The csr-1* marker also has a small fitness effect, but is still a suitable marker for competition experiments. As a proof of concept, we estimate the fitness effect of the qde-2 mutation, a gene in the RNA interference pathway, and show that its competitive fitness is lower than what would be expected from its mycelial growth rate alone.

Surface modification of poly(tetrafluoroethylene) and poly(ethylene terephthalate) films via environmental crazing

AbstractThis work addresses the phenomenon of the development of a patterned surface relief on polymer films via different modes of environmental crazing. Commercial films of semicrystalline poly(tetrafluoroethylene) (PTFE) and amorphous glassy poly(ethylene terephthalate) (PET) were subjected to tensile drawing in the presence of physically active liquid environments (carbon tetrachloride or aliphatic alcohols). The structure parameters and wettability of the modified films were studied by AFM, SEM, profilometer measurements and contact angle measurements. Environmental intercrystallite crazing of PTFE is accompanied by the development of an unstable structure with a high free surface, which experiences marked strain recovery upon unloading. As a result of the relief formation, PTFE hydrophobicity is enhanced (the water contact angle increases by 25°). Classical environmental crazing of PET films is accompanied by the formation of an anisotropic surface relief which is an assembly of crazes oriented perpendicular to the direction of tensile drawing, thus leading to the phenomenon of anisotropic wetting. The proposed approach for structural surface modification makes it possible to use the advantages of surface instability and spontaneous self‐organization of the polymer towards the development of a unique surface microrelief. © 2020 Society of Chemical Industry

Strain and sex-based glucocentric & behavioral differences between KK/HlJ and C57BL/6J mice

IntroductionSmall-animal models are the most widely used preclinical model for studying the etiology, pathology and treatment of diabetes, prediabetes and diabetic comorbidities. Diabetic patients are burdened with higher rates of depression, anxiety and cognitive decline due to inadequate control of blood glucose levels, vascular damage and aberrant CNS insulin signaling. The C57BL/6J model is amongst the most widely used mouse model due to its susceptibility to diet-induced obesity (DIO). This strain has also been well-characterized in behavioral research studies. However the C57BL/6J model has a number of limitations: [1] overt fasting hyperglycemia can only be induced by dietary manipulation and/or chemical ablation of the pancreatic beta cells. [2] There is heterogeneity in the obesogenic response to hypercaloric feeding as well as sex-dependent differences, with males being more responsive. The KK inbred strain has been used to study aspects of the metabolic syndrome and prediabetes due to inherent glucose intolerance, hyperinsulinemia and insulin resistance. However KK/HlJ mice are less well-characterized and there have been fewer behavioral studies reported. The aim of this study was to examine differences in male and female glucocentric parameters between KK/HlJ and C57BL/6J mice, and to compare their performance in a variety of standard behavioral tests relating to general, anxiogenic and cognitive paradigms. MethodsStrain differences in male and female KK/HlJ and C57BL/6J mouse adiposity, glucose and insulin parameters were studied together with group differences in standard Open Field, Object Recognition, Elevated Plus Maze, Light-Dark Transition, Porsolt test, Marble Burying, Social Recognition and Morris Water Maze tests. Correlations between behavioral variables were analyzed. Results and conclusionIn addition to being uniformly larger, hyperinsulinemic and more insulin intolerant than C57BL/6J mice, we observed marked strain and sex-differences in KK/HlJ behavior. KK/HlJ mice exhibited less locomotor and vertical exploratory behavior in comparison to C57BL/6J, whereas object exploration and novel object discrimination were superior in KK/HlJ mice. Female KK/HlJ mice were faster swimmers, whereas the males exhibited greater spatial cognition and place-learning during the MWM test.

Effect of cyclic deformation on xenogeneic heart valve biomaterials.

Glutaraldehyde-fixed bovine pericardium is currently the most popular biomaterial utilized in the creation of bioprosthetic heart valves. However, recent studies indicate that glutaraldehyde fixation results in calcification and structural valve deterioration, limiting the longevity of bioprosthetic heart valves. Additionally, glutaraldehyde fixation renders the tissue incompatible with constructive recipient cellular repopulation, remodeling and growth. Use of unfixed xenogeneic biomaterials devoid of antigenic burden has potential to overcome the limitations of current glutaraldehyde-fixed biomaterials. Heart valves undergo billion cycles of opening and closing throughout the patient’s lifetime. Therefore, understanding the response of unfixed tissues to cyclic loading is crucial to these in a heart valve leaflet configuration. In this manuscript we quantify the effect of cyclic deformation on cycle dependent strain, structural, compositional and mechanical properties of fixed and unfixed tissues. Glutaraldehyde-fixed bovine pericardium underwent marked cyclic dependent strain, resulting from significant changes in structure, composition and mechanical function of the material. Conversely, unfixed bovine pericardium underwent minimal strain and maintained its structure, composition and mechanical integrity. This manuscript demonstrates that unfixed bovine pericardium can withstand cyclic deformations equivalent to 6 months of in vivo heart valve leaflet performance.

Binding activity to intestinal cells and transient colonization in mice of two Lactobacillus paracasei subsp. paracasei strains with high aggregation potential.

Surface properties like hydrophobicity, aggregation ability, adhesion to mucosal surfaces and epithelial cells and transit time are key features for the characterization of probiotic strains. In this study, we used two Lactobacillus paracasei subsp. paracasei strains (BGNJ1-64 and BGSJ2-8) strains which were previously described with very strong aggregation capacity. The aggregation promoting factor (AggLb) expressed in these strains showed high level of binding to collagen and fibronectin, components of extracellular matrix. The working hypothesis was that strains able to aggregate have an advantage to resist in intestinal tract. So, we assessed whether these strains and their derivatives (without aggLb gene) are able to bind or not to intestinal components and we compared the transit time of each strains in mice. In that purpose parental strains (BGNJ1-64 and BGSJ2-8) and their aggregation negative derivatives (BGNJ1-641 and BGSJ2-83) were marked with double antibiotic resistance in order to be tracked in in vivo experiments in mice. Comparative analysis of binding ability of WT and aggregation negative strains to different human intestinal cell lines and mucin revealed no significant difference among them, excluding involvement of AggLb in interaction with surface of intestinal cells and mucin. In vivo experiments showed that surviving and transit time of marked strains in mice did not drastically depend on the presence of the AggLb aggregation factor.

Potentiating α2 subunit containing perisomatic GABAA receptors protects against seizures in a mouse model of Dravet syndrome.

Dravet syndrome mice (Scn1a+/- ) demonstrate a marked strain dependence for the severity of seizures which is correlated with GABAA receptor α2 subunit expression. The α2 /α3 subunit selective positive allosteric modulator (PAM) AZD7325 potentiates inhibitory postsynaptic currents (IPSCs) specifically in perisomatic synapses. AZD7325 demonstrates stronger effects on IPSCs in the seizure resistant mouse strain, consistent with higher α2 subunit expression. AZD7325 demonstrates seizure protective effects in Scn1a+/- mice without apparent sedative effects in vivo. GABAA receptor potentiators are commonly used for the treatment of epilepsy, but it is not clear whether targeting distinct GABAA receptor subtypes will have disproportionate benefits over adverse effects. Here we demonstrate that the α2 /α3 selective positive allosteric modulator (PAM) AZD7325 preferentially potentiates hippocampal inhibitory responses at synapses proximal to the soma of CA1 neurons. The effect of AZD7325 on synaptic responses was more prominent in mice on the 129S6/SvEvTac background strain, which have been demonstrated to be seizure resistant in the model of Dravet syndrome (Scn1a+/- ), and in which the α2 GABAA receptor subunits are expressed at higher levels relative to in the seizure prone C57BL/6J background strain. Consistent with this, treatment of Scn1a+/- mice with AZD7325 elevated the temperature threshold for hyperthermia-induced seizures without apparent sedative effects. Our results in a model system indicate that selectively targeting α2 is a potential therapeutic option for Dravet syndrome.

Investigating the role of nitric oxide in plant root colonisation by Streptomyces spp.

Streptomyces is a genus of soil dwelling actinomycetes that play an important role in plant health through association with plant roots. They provide an array of benefits to the plant host such as infectious disease prevention and plant growth promotion. We investigate the role of nitric oxide (NO), a ubiquitous signalling molecule used by plants and bacteria alike, in root colonisation by Streptomyces coelicolor. Plating studies were conducted for Arabidopsis thaliana and Triticum aestivum. Relative colonisation was determined by comparing selective recovery of marked mutant strains, alongside a marked control. The effect of increased endogenous NO was interrogated with a deletion mutant of nsrR-hmpA – genes responsible for NO detoxification in Streptomyces. Strains were also engineered to express recombinant NO synthase genes, to investigate the impact of NO production by the bacteria. We show that S. coelicolor ΔnsrR-hmpA is significantly more competent at colonising T. aestivum rhizosphere compared to the control (P<0.005). Endosphere colonisation is sporadic for both mutant and control, this observation is supported by fCLSM imaging. Preliminary data indicates that the Streptomyces strains engineered to express NO synthase at high levels, colonise poorly. This suggests that NO is a dynamic and finely tuned signalling molecule. We are excited to present promising new evidence to support an as yet undescribed link between NO and plant root colonisation by Streptomyces coelicolor. Understanding the mechanisms that underpin this process is the first step in exploiting these interactions for agricultural technology.