Endophytic fungi from the medicinal plant <Emphasis Type="Italic">Lippia sidoides</Emphasis> Cham. and their antimicrobial activity

Endophytic fungi were isolated from healthy tissues of Lippia sidoides Cham. (Verbenaceae), a medicinal plant used as an antiseptic in the northeast of Brazil. From 480 fragments of leaves (240) and stems (240), a total of 203 endophytic fungi were isolated, representing 14 species belonging to the groups Ascomycota, Coelomycetes and Hyphomycetes. Endophytic colonization was greater in leaves (50.4%) than in stems (35.4%). Colletotrichum gloeosporiodes had the maximum colonization frequency (12.3%), followed by Alternaria alternata (7.08%), Guignardia bidwelli (6.87%) and Phomopsis archeri (5.41%). Some species showed specificity for the host tissue: Curvularia pallescens, Dreschlera dematioidea, G. bidwellii, Microascus desmosporum, Peacilomyces variotti, Periconia byssoides and Ulocladium oudemansii were only isolated from leaves while Fusarium lateritium and Phoma tracheiphila were only isolated from stems. Through a preliminary screening and fermentation assay, 16 isolates where found to produce antimicrobial metabolites against bacterial and fungi. The diversity and role of endophytes in medicinal plants is briefly discussed. In conclusion, endophytic fungi from L. sidoides have pharmaceutical potential and can be seen as an attractive source of biologically active compounds.     

Dendritic cells and innate defense against tumor cells

Tumor growth results from a delicate balance between intrinsic dysregulation of oncogenes, tumor suppressor and stability genes counteracted by extrinsic defenses composed of immune cells shaping tumor immunogenicity. Although immune subversion might be the ultimate outcome of this process, a complex network of cellular interactions take place eventually leading to tumor specific cognate immune responses. The links between innate and cognate antitumor immunity eliciting protective T cell responses are instigated by cytokines, chemokines and damage associated molecular patterns. The intricate differentiation pathway whereby dendritic cells could undergo an efficient maturation program in the tumor microenvironment appears crucial. We will discuss the role of innate effectors and cancer therapies in the process of defense against tumor cells.     

How to make siRNA lipoplexes efficient? Add a DNA cargo

Background

We recently reported an efficient formulation of siRNA targeting TNF-α, that was able to restore immunological balance in a mouse arthritis model following intravenous injection.

Method

Since this efficient formulation included the pre association of siRNA with a DNA cargo, we decided to extensively characterise siRNA lipoplexes with or without DNA cargo, in order to better understand the DNA cargo enhancing effect.

Results

We showed that addition of DNA cargo to siRNA lipoplexes led to specific gene extinction in vitro, using reduced siRNA concentration. This procedure is also applicable to other lipid vectors, like Lipofectamine or DMRIE-C. No structural modification could be observed in siRNA lipoplexes upon addition of DNA cargo using dynamic light scattering or transmission electronic microscopy. Nevertheless, we observed some slight differences, in the amount of lipid required to obtain neutrality of the complex and in stability of the complex towards incubation with heparan sulfate.

Conclusions

These results suggest that the addition of DNA cargo to siRNA complexes is an easy procedure that leads to more efficient complexes to transfer siRNA at low concentration and in the presence of serum.     

A preliminary study of the effects of handling type on horses’ emotional reactivity and the human–horse relationship

Handling is a crucial component of the human–horse relationship. Here, we report data from an experiment conducted to assess and compare the effect of two training methods. Two groups of six Welsh mares were trained during four sessions of 50 min, one handled with traditional exercises (halter leading, grooming/brushing, lifting feet, lunging and pseudo-saddling (using only girth and saddle pad) and the second group with natural horsemanship exercises (desensitization, yielding to body pressure, lunging and free-lunging). Emotional reactivity (ER) and the human–horse relationship (HHR) were assessed both prior to and following handling. A social isolation test, a neophobia test and a bridge test were used to assess ER. HHR was assessed through test of spontaneous approach to, and forced approach by, an unknown human.Horses’ ER decreased after both types of handling as indicated by decreases in the occurrence of whinnying during stressful situations. Head movement (jerk/shake) was the most sensitive variable to handling type. In the spontaneous approach tests, horses in the traditional handling group showed higher latencies to approach a motionless person after handling than did the natural horsemanship group. Our study suggests that natural horsemanship exercises could be more efficient than traditional exercises for improving horses’ HHR.     

The Deinococcus radiodurans SMC protein is dispensable for cell viability yet plays a role in DNA folding

Deinococcus radiodurans contains a highly condensed nucleoid that remains to be unaltered following the exposure to high doses of γ-irradiation. Proteins belonging to the structural maintenance of chromosome protein (SMC) family are present in all organisms and were shown to be involved in chromosome condensation, pairing, and/or segregation. Here, we have inactivated the smc gene in the radioresistant bacterium D. radiodurans, and, unexpectedly, found that smc null mutants showed no discernible phenotype except an increased sensitivity to gyrase inhibitors suggesting a role of SMC in DNA folding. A defect in the SMC-like SbcC protein exacerbated the sensitivity to gyrase inhibitors of cells devoid of SMC. We also showed that the D. radiodurans SMC protein forms discrete foci at the periphery of the nucleoid suggesting that SMC could locally condense DNA. The phenotype of smc null mutant leads us to speculate that other, not yet identified, proteins drive the compact organization of the D. radiodurans nucleoid.     

Analysis of the glucagon receptor first extracellular loop by the substituted cysteine accessibility method

Glucagon is an important hormone for the prevention of hypoglycemia, and contributes to the hyperglycemia observed in diabetic patients, yet very little is known about its receptor structure and the receptor-glucagon interaction. In related receptors, the first extracellular loop, ECL1, is highly variable in length and sequence, suggesting that it might participate in ligand recognition. We applied a variant of the SCAM (Substituted Cysteine Accessibility Method) to the glucagon receptor ECL1 and sequentially mutated positions 197 to 223 to cysteine. Most of the mutations (15/27) affected the glucagon potency, due either to a modification of the glucagon binding site, or to the destabilization of the active receptor conformation. We reasoned that side chains accessible to glucagon must also be accessible to large, hydrophilic cysteine reagents. We therefore evaluated the accessibility of the introduced cysteines to maleimide-PEO₂-biotin ((+)-biotinyl-3-maleimido-propionamidyl-3,6-dioxa-octanediamine), and tested the effect of pretreatment of intact cells with a large cationic cysteine reagent, MTSET ([2-(trimethylammonium)ethyl]methanethiosulfonate bromide), on glucagon potency. Our results suggest that the second and third transmembrane helices (TM2 and TM3) are extended to position 202 and from position 215, respectively, and separated by a short β stretch (positions 203-209). Glucagon binding induced a conformational change close to TM2: L198C was accessible to the biotin reagent only in the presence of glucagon. Most other mutations affected the receptor activation rather than glucagon recognition, but S217 and D218 (at the top of TM3) were good candidates for glucagon recognition and V221 was very close to the binding site.     

Dual TCR expression biases lung inflammation in DO11.10 transgenic mice and promotes neutrophilia via microbiota-induced Th17 differentiation

A commonly used mouse model of asthma is based on i.p. sensitization to OVA together with aluminum hydroxide (alum). In wild-type BALB/c mice, subsequent aerosol challenge using this protein generates an eosinophilic inflammation associated with Th2 cytokine expression. By constrast, in DO11.10 mice, which are transgenic for an OVA-specific TCR, the same treatment fails to induce eosinophilia, but instead promotes lung neutrophilia. In this study, we show that this neutrophilic infiltration results from increased IL-17A and IL-17F production, whereas the eosinophilic response could be restored upon blockade of IFN-γ, independently of the Th17 response. In addition, we identified a CD4(+) cell population specifically present in DO11.10 mice that mediates the same inflammatory response upon transfer into RAG2(-/-) mice. This population contained a significant proportion of cells expressing an additional endogenous TCR α-chain and was not present in RAG2(-/-) DO11.10 mice, suggesting dual antigenic specificities. This particular cell population expressed markers of memory cells, secreted high levels of IL-17A, and other cytokines after short-term restimulation in vitro, and triggered a neutrophilic response in vivo upon OVA aerosol challenge. The relative numbers of these dual TCR lymphocytes increased with the age of the animals, and IL-17 production was abolished if mice were treated with large-spectrum antibiotics, suggesting that their differentiation depends on foreign Ags provided by gut microflora. Taken together, our data indicate that dual TCR expression biases the OVA-specific response in DO11.10 mice by inhibiting eosinophilic responses via IFN-γ and promoting a neutrophilic inflammation via microbiota-induced Th17 differentiation.