Fungal Endophytes of Populus trichocarpa Alter Host Phenotype, Gene Expression, and Rhizobiome Composition.

Mortierella and Ilyonectria genera include common species of soil fungi that are frequently detected as root endophytes in many plants, including Populus spp. However, the ecological roles of these and other endophytic fungi with respect to plant growth and function are still not well understood. The functional ecology of two key taxa from the P. trichocarpa rhizobiome, M. elongata PMI93 and I. europaea PMI82, was studied by coupling forest soil bioassays with environmental metatranscriptomics. Using soil bioassay experiments amended with fungal inoculants, M. elongata was observed to promote the growth of P. trichocarpa. This response was cultivar independent. In contrast, I. europaea had no visible effect on P. trichocarpa growth. Metatranscriptomic studies revealed that these fungi impacted rhizophytic and endophytic activities in P. trichocarpa and induced shifts in soil and root microbial communities. Differential expression of core genes in P. trichocarpa roots was observed in response to both fungal species. Expression of P. trichocarpa genes for lipid signaling and nutrient uptake were upregulated, and expression of genes associated with gibberellin signaling were altered in plants inoculated with M. elongata, but not I. europaea. Upregulation of genes for growth promotion, downregulation of genes for several leucine-rich repeat receptor kinases, and alteration of expression of genes associated with plant defense responses (e.g., jasmonic acid, salicylic acid, and ethylene signal pathways) also suggest that M. elongata manipulates plant defenses while promoting plant growth

Signal-3L 2.0: A Hierarchical Mixture Model for Enhancing Protein Signal Peptide Prediction by Incorporating Residue-Domain Cross-Level Features

Signal peptides play key roles in targeting and translocation of integral membrane proteins and secretory proteins. However, signal peptides present several challenges for automatic prediction methods. One challenge is that it is difficult to discriminate signal peptides from transmembrane helices, as both the H-region of the peptides and the transmembrane helices are hydrophobic. Another is that it is difficult to identify the cleavage site between signal peptides and mature proteins, as cleavage motifs or patterns are still unclear for most proteins. To solve these problems and further enhance automatic signal peptide recognition, we report a new Signal-3L 2.0 predictor. Our new model is constructed with a hierarchical protocol, where it first determines the existence of a signal peptide. For this, we propose a new residue-domain cross-level feature-driven approach, and we demonstrate that protein functional domain information is particularly useful for discriminating between the transmembrane helices and signal peptides as they perform different functions. Next, in order to accurately identify the unique signal peptide cleavage sites along the sequence, we designed a top-down approach where a subset of potential cleavage sites are screened using statistical learning rules, and then a final unique site is selected according to its evolution conservation score. Because this mixed approach utilizes both statistical learning and evolution analysis, it shows a strong capacity for recognizing cleavage sites. Signal-3L 2.0 has been benchmarked on multiple data sets, and the experimental results have demonstrated its accuracy. The online server is available at www.csbio.sjtu.edu.cn/bioinf/Signal-3L/

Identification and Molecular Characterization of Superoxide Dismutases Isolated From A Scuticociliate Parasite: Physiological Role in Oxidative Stress

Philasterides dicentrarchi is a free-living microaerophilic scuticociliate that can become a facultative parasite and cause a serious parasitic disease in farmed fsh. Both the free-living and parasitic forms of this scuticociliate are exposed to oxidative stress associated with environmental factors and the host immune system. The reactive oxygen species (ROS) generated by the host are neutralized by the ciliate by means of antioxidant defences. In this study we aimed to identify metalloenzymes with superoxide dismutase (SOD) activity apable of inactivating the superoxide anion (•O2−) generated during induction of oxidative stress. P. dicentrarchi possesses the three characteristic types of SOD isoenzymes in eukaryotes: copper/zinc-SOD, manganese-SOD and iron-SOD. The Cu/Zn-SOD isoenzymes comprise three types of homodimeric proteins (CSD1-3) of molecular weight (MW) 34–44kDa and with very diferent AA sequences. All Cu/Zn-SODs are sensitive to NaCN, located in the cytosol and in the alveolar sacs, and one of them (CSD2) is extracellular. Mn- and Fe-SOD transcripts encode homodimeric proteins (MSD and FSD, respectively) in their native state: a) MSD (MW 50kDa) is insensitive to H2O2 and NaN3 and is located in the mitochondria; and b) FSD (MW 60kDa) is sensitive to H2O2, NaN3 and the polyphenol trans-resveratrol and is located extracellularly. Expression of SOD isoenzymes increases when •O2 − is induced by ultraviolet (UV) irradiation, and the increase is proportional to the dose of energy applied, indicating that these enzymes are actively involved in cellular protection against oxidative stressThis study was financially supported by grant AGL2017-83577-R awarded by the Ministerio de Economía y Competitividad (Spain) and Fondo Europeo de Desarrollo Regional -FEDER- (European Union), by grant ED431C2017/31 from the Xunta de Galicia (Spain), and by PARAFISHCONTROL project, which received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 634429S

A multi-method and structure-based in silico vaccine designing against Echinococcus granulosus through investigating enolase protein

Introduction: Hydatid disease is a ubiquitous parasitic zoonotic disease, which causes different medical, economic and serious public health problems in some parts of the world. The causal organism is a multi-stage parasite named Echinococcus granulosus whose life cycle is dependent on two types of mammalian hosts viz definitive and intermediate hosts. Methods: In this study, enolase, as a key functional enzyme in the metabolism of E. granulosus (EgEnolase), was targeted through a comprehensive in silico modeling analysis and designing a host-specific multi-epitope vaccine. Three-dimensional (3D) structure of enolase was modeled using MODELLER v9.18 software. The B-cell epitopes (BEs) were predicted based on the multi-method approach and via some authentic online predictors. ClusPro v2.0 server was used for docking-based T-helper epitope prediction. The 3D structure of the vaccine was modeled using the RaptorX server. The designed vaccine was evaluated for its immunogenicity, physicochemical properties, and allergenicity. The codon optimization of the vaccine sequence was performed based on the codon usage table of E. coli K12. Finally, the energy minimization and molecular docking were implemented for simulating the vaccine binding affinity to the TLR-2 and TLR-4 and the complex stability. Results: The designed multi-epitope vaccine was found to induce anti-EgEnolase immunity which may have the potential to prevent the survival and proliferation of E. granulosus into the definitive host. Conclusion: Based on the results, this step-by-step immunoinformatics approach could be considered as a rational platform for designing vaccines against such multi-stage parasites. Furthermore, it is proposed that this multi-epitope vaccine is served as a promising preventive anti-echinococcosis agent

Characterization of the Isoforms of the Multiple Sclerosis Risk Protein, IL-22 Binding Protein (IL-22BP)

262 p.The human IL22RA2 gene co-produces three protein isoforms in dendritic cells (IL-22 binding protein isoform-1 [IL-22BPi1], -2 [IL-22BPi2], and -3 [IL-22BPi3]). Two of these, namely, IL-22BPi2 and IL-22BPi3, are capable of neutralizing the biological activity of IL-22. The function of IL-22BPi1, which differs from IL-22BPi2 through an in-frame, 32-amino-acid insertion provided by an alternatively spliced exon, remains unknown.This thesis focuses on the biochemical characterization of the three isoforms (in silico and in vitro), as well as the potential pharmacological targeting thereof. Additionally, it addresses the functionality of the non-synonymous single nucleotide polymorphism (SNP), rs28385692, which is located in IL22RA2 and has been found to be associated with multiple sclerosis (MS).El gen humano IL22RA2 coproduce tres isoformas en las células dendríticas (la isoforma-1 de la proteína de unión a la IL-22 [IL-22BPi1], la -2 [IL-22BPi2] y la -3 [IL-22BPi3]). Dos de ellas, la IL-22BPi2 y la IL-22BPi3, son capaces de neutralizar la actividad biológica de la IL-22. La función de la IL-22BPi1, que difiere de la IL-22BPi2 por una inserción de 32 aminoácidos mediante splicing alternativo, sigue siendo desconocida. El objetivo general de esta tesis es comprender la función y el destino de la IL-22BP mediante la caracterización bioquímica de sus isoformas. Los obteivos específicos son: estudiar la expresión de los transcritos de IL22RA2 y relacionarlos con la secreción de sus isoformas mediante su caracterización bioquímica, así como identificar los factores clave implicados en su plegamiento y secreción e investigar su secreción y función dentro de la célula; evaluar si las chaperonas identificadas pudieran ser utilizadas como posibles dianas terapéuticas que tuvieran efectos diferenciales en la secreción de las isoformas de IL-22BP; y determinar si la variante de riesgo de la EM, rs28385692, tiene efectos funcionales sobre las isoformas de la IL-22BP y en qué medida

Regulation mechanisms of human D-amino acid oxidase

The human peroxisomal FAD-dependent enzyme D-amino acid oxidase (hDAAO, EC 1.4.3.3) plays a key role in important physiological processes by catalyzing the stereospecific degradation of several D-amino acids (D-AAs). A number of studies demonstrated that a dysregulation in processes regulating D-AAs concentration is related to the mechanism(s) predisposing to several pathologies. The important role played by hDAAO in modulating D-AAs levels increased the interest for this flavoenzyme: while structural and biochemical properties have been extensively investigated, several aspects in the modulation of its functionality remain elusive. Furthermore, it has been recently suggested that DAAO could be mistargeted to the nucleus or secreted in the (mouse) intestinal lumen, where it could select the composition of gut microbiota by generating H2O2. Here, some biochemical properties of the recombinant enzyme were investigated. Moreover, we focused on mistargeting of DAAO by studying a variant lacking the N-terminal signal peptide (thus shedding light on the mechanism of microbiota selection) and two variants at position 120 (a residue belonging to a putative nuclear translocation signal): the cellular targeting of the flavoenzyme seems a way to modulate hDAAO functionality. This modulation allows hDAAO to fulfil different physiological functions, such as the control of the level of D-Ser in the brain and of other D-AAs in different tissues or the selection of microbiota in the gut

Escuticociliatosis: mecanismos de evasión de la respuesta inmunitaria del rodaballo y desarrollo de vacunas

La escuticociliatosis producida por Philasterides dicentrarchi genera mortalidades en rodaballos de cultivo provocando pérdidas económicas. Para evitarlo, es necesario desarrollar medidas profilácticas basadas en la utilización de vacunas, a partir de la búsqueda de antígenos que generen protección, y/o fármacos que inhiban mecanismos vitales para la supervivencia del ciliado. Por tanto, la identificación de antígenos protectores y de moléculas implicadas en el metabolismo, las cuales permiten al escuticociliado sobrevivir en el hospedador, han sido los objetivos principales del presente estudio. Hemos descubierto que el parásito posee dos familias de proteínas (leishmanolisinas y proteínas variables de superficie (VSPs)) asociadas a la membrana celular e involucradas en la evasión de la respuesta inmunitaria del hospedador. También hemos identificado y caracterizado proteínas de secreción producidas por los extrusomas, capaces de generar cápsulas de mucopolisacáridos que protegen del ataque de los factores humorales citotóxicos y superóxido dismutasas, que permiten al ciliado inactivar radicales libres de oxígeno tóxicos asociados al estrés oxidativo generado durante la infección. Para solventar los problemas actuales relacionados con el uso de vacunas inactivadas formuladas en adyuvantes oleosos, también hemos desarrollado un adyuvante biodegradable (hidrogel de quitosano) que presenta capacidad inmunoestimulante similar a los oleosos y resulta menos dañino para el pez. La producción de proteínas recombinantes en levadura, como potenciales antígenos para generar vacunas recombinantes, nos permite incluir los cambios aminoacídicos que se producen entre las diferentes cepas, obteniendo así un antígeno inmunogénico universal que genere respuesta frente a todas ellas. Para conseguir una mayor protección es necesario incluir en la vacuna varios antígenos empleando proteínas quiméricas recombinantes a partir de péptidos antigénicos procedentes de varias proteínas. Estas formulaciones vacunales de quitosano han generado elevados niveles de protección en ensayos realizados en rodaballo, indicando que esta estrategia podría ser útil para obtener una vacuna universal eficaz frente a la escuticociliatosis