Anchorless surface associated glycolytic enzymes from Lactobacillus plantarum 299v bind to epithelial cells and extracellular matrix proteins

An important criterion for the selection of a probiotic bacterial strain is its ability to adhere to the mucosal surface. Adhesion is usually mediated by proteins or other components located on the outer cell surface of the bacterium. In the present study we characterized the adhesive properties of two classical intracellular enzymes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and enolase (ENO) isolated from the outer cell surface of the probiotic bacterium Lactobacillus plantarum 299v. None of the genes encoded signal peptides or cell surface anchoring motifs that could explain their extracellular location on the bacterial surface. The presence of the glycolytic enzymes on the outer surface was verified by western blotting using polyclonal antibodies raised against the specific enzymes. GAPDH and ENO showed a highly specific binding to plasminogen and fibronectin whereas GAPDH but not ENO showed weak binding to mucin. Furthermore, a pH dependent and specific binding of GAPDH and ENO to intestinal epithelial Caco-2 cells at pH 5 but not at pH 7 was demonstrated. The results showed that these glycolytic enzymes could play a role in the adhesion of the probiotic bacterium L. plantarum 299v to the gastrointestinal tract of the host. Finally, a number of probiotic as well non-probiotic Lactobacillus strains were analyzed for the presence of GAPDH and ENO on the outer surface, but no correlation between the extracellular location of these enzymes and the probiotic status of the applied strains was demonstrated.     

Neighboring Deschampsia flexuosa and Trientalis europaea harbor contrasting root fungal endophytic communities

Fungal endophytic communities and potential host preference of root-inhabiting fungi of boreal forest understory plants are poorly known. The objective of this study was to find out whether two neighboring plant species, Deschampsia flexuosa (Poaceae) and Trientalis europaea (Primulaceae), share similar root fungal endophytic communities and whether the communities differ between two sites. The study was carried out by analysis of pure culture isolates and root fungal colonization percentages. A total of 84 isolates from D. flexuosa and 27 isolates from T. europaea were obtained. The roots of D. flexuosa harbored 16 different isolate types based on macromorphological characteristics, whereas only 4 isolate types were found in T. europaea. The root colonization by dark septate and hyaline septate hyphae correlated with isolate numbers being higher in D. flexuosa compared to T. europaea. The different isolate types were further identified on the basis of internal transcribed spacer sequence and phylogenetic analysis. An isolate type identified as dark septate endophyte Phialocephala fortinii colonized 50 % of the T. europaea and 21 % of the D. flexuosa specimens. In addition, Meliniomyces variabilis, Phialocephala sphaeroides, and Umbelopsis isabellina were found colonizing the grass, D. flexuosa, for the first time and Mycena sp. was confirmed as an endophyte of D. flexuosa. Site-specific differences were observed in the abundance and diversity of endophytic fungi in the roots of both study plants, but the differences were not as predominant as those between plant species. It is concluded that D. flexuosa harbors both higher amount and more diverse community of endophytic fungi in its roots compared to T. europaea.     

Indicator selection in life cycle assessment to enable decision making: issues and solutions

Purpose

With an ever increasing list of indicators available, life cycle assessment (LCA) practitioners face the challenge of effectively communicating results to decision makers. Simplification of LCA is often limited to an arbitrary selection of indicators, use of single scores by using weighted values or single attribute indicators. These solutions are less attractive to decision makers, since value judgments are introduced or multi-indicator information is lost. Normalization could be a means to narrow the list of indicators by ranking indicators vs. a reference system. This paper shows three different normalization approaches that produce very different ranking of indicators. It is explained how normalization helps maintain a multi-indicator approach while keeping the most relevant indicators, allowing effective decision making.

Methods

The approaches are illustrated on a hand dishwashing case study, using ReCiPe as the impact assessment method and taking the European population (year 2000) as the reference situation. Indicators are ranked using midpoint normalization factors, and compared to the ranking from endpoint normalization broken down by midpoint contribution.

Results and discussion

Endpoint normalization shows Resources as the most relevant area of protection for this case, closely followed by Human Health and Ecosystem. Broken down by their key driving midpoints, fossil depletion, climate change and, to a lesser extent, particulate matter formation and metal depletion, are most relevant. Midpoint normalization, however, indicates Freshwater Eutrophication, Natural Land Transformation and Toxicity indicators (marine and freshwater ecotoxicity and human toxicity) are most relevant.

Conclusions

A three-step approach based on endpoint normalization is recommended to present only the most relevant indicators, allowing more effective decision making instead of communicating all LCA indicators. The selection process breaks out the normalized endpoint results into the most contributing midpoints (relevant indicators) and reports results with midpoint level units. Bias due to lack of data completeness is less of an issue in the endpoint normalization process (compared to midpoint normalization), while midpoint results are less subject to uncertainty (compared to endpoint results). Focusing on the relevant indicators and key contributing unit processes has proven to be effective for non-LCA expert decision makers to understand, use, and communicate complex LCA results.     

Tripartite efflux pumps: energy is required for dissociation,but not assembly or opening of the outer membrane channel of the pump

The MtrCDE multidrug pump, from Neisseria gonorrhoeae, is assembled from the inner and outer membrane proteins MtrD and MtrE, which are connected by the periplasmic membrane fusion protein MtrC. Although it is clear that MtrD delivers drugs to the channel of MtrE, it remains unclear how drug delivery and channel opening are connected. We used a vancomycin sensitivity assay to test for opening of the MtrE channel. Cells expressing MtrE or MtrE‐E434K were insensitive to vancomycin; but became moderately and highly sensitive to vancomycin respectively, when coexpressed with MtrC, suggesting that the MtrE channel opening requires MtrC binding and is energy‐independent. Cells expressing wild‐type MtrD, in an MtrCE background, were vancomycin‐insensitive, but moderately sensitive in an MtrCE‐E434K background. The mutation of residues involved in proton translocation inactivated MtrD and abolished drug efflux, rendered both MtrE and MtrE‐E434K vancomycin‐insensitive; imply that the pump–component interactions are preserved, and that the complex is stable in the absence of proton flux, thus sealing the open end of MtrE. Following the energy‐dependent dissociation of the tripartite complex, the MtrE channel is able to reseal, while MtrE‐E434K is unable to do so, resulting in the vancomycin‐sensitive phenotype. Thus, our findings suggest that opening of the OMP via interaction with the MFP is energy‐independent, while both drug export and complex dissociation require active proton flux.     

Soybean lecithin-based extender as an alternative for goat sperm cryopreservation

The aim of this study was to evaluate the effect of different concentrations of soybean lecithin (SL) in extenders for sperm goat cryopreservation. Sexually mature male Saanen goats (n = 4) were used, and the ejaculates were obtained using an artificial vagina method. The semen samples were pooled and diluted in a skim milk-based extender (control group; CG) or Tris extender supplemented with SL at different concentrations (G1 = 0.04%, SL G2 = 0.08% SL and G3 = 0.16%) for a final concentration of 240 × 10⁶ spermatozoa/mL. The semen samples were packed in straws (0.25 mL), frozen using an automated system and stored in liquid nitrogen (?196 °C). After thawing (37 °C/30 s), the samples were evaluated for sperm quality parameters, including sperm motility, membrane integrity, acrosome integrity and mitochondrial activity. No significant difference was observed among the experimental and control groups for all of the parameters (P > 0.05). However, even though the control group presented a significantly lower mitochondrial membrane potential compared to fresh semen (P < 0.05), the same did not occur for the extender supplemented with soybean lecithin, that is, it did not differ from fresh sperm (P > 0.05). The extender containing soybean lecithin at different concentrations preserved the sperm quality parameters in a manner similar to the conventional skim milk-based extender. Thus, it is concluded that an extender containing soybean lecithin as the lipoprotein source can be used for freezing goat semen.     

Dynamics of exocyclic groups in the Escherichia coli O91 O-antigen polysaccharide in solution studied by carbon-13 NMR relaxation

Carbon-13 relaxation data are reported for exocyclic groups of hexopyranosyl sugar residues in the repeating unit within the Escherichia coli O91 O-antigen polysaccharide in a dilute D₂O solution. The measurements of T ₁, T ₂ and heteronuclear nuclear Overhauser enhancements were carried out at 310 K at two magnetic fields (16.4 T, 21.1 T). The data were analyzed using the standard and extended Lipari–Szabo models, as well as a conformational jump model. The extended version of the Lipari–Szabo and the two-site jump models were most successful for the hydroxymethyl groups of Gal and GlcNAc sugar residues. Different dynamics was found for the hydroxymethyl groups associated with different configurations (d-gluco, d-galacto) of the sugar residues, the latter being faster than the former.     

Hepatic production of transthyretin L12P leads to intracellular lysosomal aggregates in a new somatic transgenic mouse model

Transthyretin (TTR) is a plasma and cerebrospinal fluid (CSF)-circulating homotetrameric protein. More than 100 point mutations have been identified in the TTR gene and several are related with amyloid diseases. Here we focused our attention in the TTR L12P variant associated with severe peripheral neuropathy and leptomeningeal amyloidosis. By using different cell lines derived from tissues specialized on TTR synthesis, such as the hepatocyte and the choroid plexus expressing WT, V30M, or L12P TTR variants we analyzed secretion, intracellular aggregation and degradation patterns. Also, we used liver-specific AAV gene transfer to assess expression of the L12P variant in vivo. We found the following: (i) decreased secretion with intracellular aggregation of TTR L12P in hepatoma cells relative to WT and V30M variant; this differential property of TTR L12P variant was also observed in mice injected with L12P AAV vector; (ii) differential N-glycosylation pattern of L12P variant in hepatoma cell lysates, conditioned media and mouse sera, which might represent an escape mechanism from ERAD degradation; (iii) intracellular L12P TTR aggregates mainly localized to lysosomes in cultured cells and liver; and (iv) none of the above findings were present in choroid plexus derived cells, suggesting particular secretion/quality control mechanisms that might contribute to leptomeningeal amyloidosis associated with the L12P variant. These observations open new avenues for the treatment of TTR associated leptomeningeal amyloidosis.     

MitoQ,a mitochondria-targeted antioxidant,delays disease progression and alleviates pathogenesis in an experimental autoimmune encephalomyelitis mouse model of multiple sclerosis

Oxidative stress and mitochondrial dysfunction are involved in the progression and pathogenesis of multiple sclerosis (MS). MitoQ is a mitochondria-targeted antioxidant that has a neuroprotective role in several mitochondrial and neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Here we sought to determine the possible effects of a systematic administration of MitoQ as a therapy, using an experimental autoimmune encephalomyelitis (EAE) mouse model. We studied the beneficial effects of MitoQ in EAE mice that mimic MS like symptoms by treating EAE mice with MitoQ and pretreated C57BL6 mice with MitoQ plus EAE induction. We found that pretreatment and treatment of EAE mice with MitoQ reduced neurological disabilities associated with EAE. We also found that both pretreatment and treatment of the EAE mice with MitoQ significantly suppressed inflammatory markers of EAE, including the inhibition of inflammatory cytokines and chemokines. MitoQ treatments reduced neuronal cell loss in the spinal cord, a factor underlying motor disability in EAE mice. The neuroprotective role of MitoQ was confirmed by a neuron-glia co-culture system designed to mimic the mechanism of MS and EAE in vitro. We found that axonal inflammation and oxidative stress are associated with impaired behavioral functions in the EAE mouse model and that treatment with MitoQ can exert protective effects on neurons and reduce axonal inflammation and oxidative stress. These protective effects are likely via multiple mechanisms, including the attenuation of the robust immune response. These results suggest that MitoQ may be a new candidate for the treatment of MS.     

Identification of the infectious source of an unusual outbreak of histoplasmosis,in a hotel in Acapulco,state of Guerrero,Mexico

Three isolates of Histoplasma capsulatum were identified from mice lung, liver, and spleen inoculated with soil samples of the X hotel’s ornamental potted plants that had been fertilized with organic material known as compost. The presence of H. capsulatum in the original compost was detected using the dot-enzyme-linked immunosorbent assay. Nested-PCR, using a specific protein Hcp100 coding gene sequence, confirmed the fungal identification associated with an unusual histoplasmosis outbreak in Acapulco. Although, diversity between the H. capsulatum isolate from the hotel and some clinical isolates from Guerrero (positive controls) was observed using random amplification of polymorphic DNA based-PCR, sequence analyses of H-anti and ole fragment genes revealed a high homology (92–99%) between them.