Robustness and stability of the gene regulatory network involved in DV boundary formation in the Drosophila wing

Gene regulatory networks have been conserved during evolution. The Drosophila wing and the vertebrate hindbrain share the gene network involved in the establishment of the boundary between dorsal and ventral compartments in the wing and adjacent rhombomeres in the hindbrain. A positive feedback-loop between boundary and non-boundary cells and mediated by the activities of Notch and Wingless/Wnt-1 leads to the establishment of a Notch dependent organizer at the boundary. By means of a Systems Biology approach that combines mathematical modeling and both in silico and in vivo experiments in the Drosophila wing primordium, we modeled and tested this regulatory network and present evidence that a novel property, namely refractoriness to the Wingless signaling molecule, is required in boundary cells for the formation of a stable dorsal-ventral boundary. This new property has been validated in vivo, promotes mutually exclusive domains of Notch and Wingless activities and confers stability to the dorsal-ventral boundary. A robustness analysis of the regulatory network complements our results and ensures its biological plausibility.     

A cytoplasm to vacuole targeting pathway in P. pastoris

The cytoplasm-to-vacuole targeting (Cvt) pathway of Saccharomyces cerevisiae delivers aminopeptidase I (Ape1) from the cytosol to the vacuole, bypassing the normal secretory route. The Cvt pathway, although well-studied, was known only in S. cerevisiae. We demonstrate its existence in the methylotrophic yeast, Pichia pastoris, where it also delivers P. pastoris Ape1 (PpApe1) to the vacuole. Most proteins known to be required for the Cvt pathway in S. cerevisiae were, to the extent we found orthologs, also required in P. pastoris. The P. pastoris Cvt pathway differs, however, from that in S. cerevisiae, in that new proteins, such as PpAtg28 and PpAtg26, are involved. The discovery of a Cvt pathway in P. pastoris makes it an excellent model system for the dissection of autophagy-related pathways in a single organism and for the discovery of new Cvt pathway components.     

Dominant marine heterotrophic flagellates are adapted to natural planktonic bacterial abundances

Grazing controls bacterial abundances and composition in many ecosystems. In marine systems, heterotrophic flagellates (HFs) are important predators. Assemblages of HFs are primarily formed by species still uncultured; therefore, many aspects of their trophic behaviour are poorly known. Here, we assessed the functional response of the whole assemblage and of four taxa grown in an unamended seawater incubation. We used fluorescently labelled bacteria to create a prey gradient of two orders of magnitude in abundance and estimated ingestion rates. Natural HFs had a half-saturation constant of 6.7 × 10⁵ prey ml⁻¹, a value lower than that of cultured flagellates and within the range of marine planktonic bacterial abundances. Minorisa minuta was well adapted to low prey abundances and very efficient in ingesting bacteria. MAST-4 and MAST-7 were also well adapted to the typical marine abundances but less voracious. In contrast, Paraphysomonas imperforata, a typical cultured species, did not achieve ingestion rate saturation even at the highest prey concentration assayed. Our study, beside to set the basis for the fundamental differences between cultured and uncultured bacterial grazers, indicate that the examined predator taxa have different functional responses, suggesting that they occupy distinct ecological niches according to their grazing strategies and prey preferences.     

Eustress application trough-controlled elicitation strategies as an effective agrobiotechnology tool for capsaicinoids increase: a review

Phytochemistry Reviews - Capsaicinoids (CAPs) are compounds synthesized and accumulated exclusively in pepper fruits. They are known as health-promoting compounds, and upbringing approaches to...     

GCAT|Panel,a comprehensive structural variant haplotype map of the Iberian population from high-coverage whole-genome sequencing

The combined analysis of haplotype panels with phenotype clinical cohorts is a common approach to explore the genetic architecture of human diseases. However, genetic studies are mainly based on single nucleotide variants (SNVs) and small insertions and deletions (indels). Here, we contribute to fill this gap by generating a dense haplotype map focused on the identification, characterization, and phasing of structural variants (SVs). By integrating multiple variant identification methods and Logistic Regression Models (LRMs), we present a catalogue of 35 431 441 variants, including 89 178 SVs (≥50 bp), 30 325 064 SNVs and 5 017 199 indels, across 785 Illumina high coverage (30x) whole-genomes from the Iberian GCAT Cohort, containing a median of 3.52M SNVs, 606 336 indels and 6393 SVs per individual. The haplotype panel is able to impute up to 14 360 728 SNVs/indels and 23 179 SVs, showing a 2.7-fold increase for SVs compared with available genetic variation panels. The value of this panel for SVs analysis is shown through an imputed rare Alu element located in a new locus associated with Mononeuritis of lower limb, a rare neuromuscular disease. This study represents the first deep characterization of genetic variation within the Iberian population and the first operational haplotype panel to systematically include the SVs into genome-wide genetic studies.     

Monitoring the colonization of sugarcane and rice plants by the endophytic diazotrophic bacterium Gluconacetobacter diazotrophicus marked with gfp and gusA reporter genes

Aims: To evaluate the colonization process of sugarcane plantlets and hydroponically grown rice seedlings by Gluconacetobacter diazotrophicus strain PAL5 marked with the gusA and gfp reporter genes. Methods and Results: Sugarcane plantlets inoculated in vitro with PAL5 carrying the gfp::gusA plasmid pHRGFPGUS did not present green fluorescence, but β‐glucuronidase (GUS)‐stained bacteria could be observed inside sugarcane roots. To complement this existing inoculation methodology for micropropagated sugarcane with a more rapid colonization assay, we employed hydroponically grown gnotobiotic rice seedlings to study PAL5–plant interaction. PAL5 could be isolated from the root surface (10⁸ CFU g^?1) and from surface‐disinfected root and stem tissues (10⁴ CFU g^?1) of inoculated plants, suggesting that PAL5 colonized the internal plant tissues. Light microscopy confirmed the presence of bacteria inside the root tissue. After inoculation of rice plantlets with PAL5 marked with the gfp plasmid pHRGFPTC, bright green fluorescent bacteria could be seen colonizing the rice root surface, mainly at the sites of lateral root emergence, at root caps and on root hairs. Conclusion: The plasmids pHRGFPGUS and pHRGFPTC are valid tools to mark PAL5 and monitor the colonization of micropropagated sugarcane and hydroponic rice seedlings. Significance and Impact of the Study: These tools are of use to: (i) study PAL5 mutants affected in bacteria–plant interactions, (ii) monitor plant colonization in real time and (iii) distinguish PAL5 from other bacteria during the study of mixed inoculants.     

Effect of crown ethers on structure, stability, activity, and enantioselectivity of subtilisin Carlsberg in organic solvents.

Colyophilization or codrying of subtilisin Carlsberg with the crown ethers 18-crown-6, 15-crown-5, and 12-crown-4 substantially improved enzyme activity in THF, acetonitrile, and 1,4-dioxane in the transesterification reactions of N-acetyl-L-phenylalanine ethylester and 1-propanol and that of (+/-)-1-phenylethanol and vinylbutyrate. The acceleration of the initial rate, V(0), ranged from less than 10-fold to more than 100-fold. All crown ethers activated subtilisin substantially, which excludes a specific macrocyclic effect from being responsible. The secondary structure of subtilisin was studied by Fourier-transform infrared (FTIR) spectroscopy. 18-Crown-6 and 15-crown-5 led to a more nativelike structure of subtilisin in the organic solvents employed when compared with that of the dehydrated enzyme obtained from buffer alone. However, the high level of activation with 12-crown-4 where this effect was not observed excluded overall structural preservation from being the primary cause of the observed enzyme activation. The conformational mobility of subtilisin was investigated by performing thermal denaturation experiments in 1,4-dioxane. Although only a small effect of temperature on subtilisin structure was observed for the samples prepared with or without 12-crown-4, both 18-crown-6 and 15-crown-5 caused the enzyme to denature at quite low temperatures (38 degrees C and 56 degrees C, respectively). No relationship between this property and V(0) was evident, but increased conformational mobility of the protein decreased its storage stability. The possibility of a "molecular imprinting" effect was also tested by removing 18-crown-6 from the subtilisin-18-crown-6 colyophilizate by washing. V(0) was only halved as a result of this procedure, an effect insignificant compared with the ca. 80-fold rate enhancement observed prior to washing in THF. This suggests that molecular imprinting is likely the primary cause of subtilisin activation by crown ethers, as recently suggested.     

Characteristics of the interaction between Hsc70 and the transferrin receptor in exosomes released during reticulocyte maturation

The transferrin receptor (TfR) of reticulocytes is released in vesicular form (exosomes) during their maturation to erythrocytes. The heat shock cognate 70-kDa protein (Hsc70) has been demonstrated to interact with the cytosolic domain of the TfR and could thus trigger the receptor toward this secretion pathway. We investigated the characteristics of the interaction between Hsc70 and the TfR in exosomes with an in vitro binding assay using TfR immobilized on Sepharose beads and purified Hsc70. The results show that Hsc70 binds to exosomal TfR with characteristics expected of a chaperone/peptide interaction. We demonstrated that heat-denatured luciferase competed for in vitro binding, dependent on the nucleotide bound to Hsc70, and that this interaction activates the ATPase activity of Hsc70. Moreover, we used immunosuppressive agents that interact with Hsc70, thus decreasing Hsc70 binding to TfR in our in vitro binding assay and enabling us to assess the role of this interaction in vivo during reticulocyte maturation.