Functional characterization and target discovery of glycoside hydrolases from the digestome of the lower termite Coptotermes gestroi

Background

Lignocellulosic materials have been moved towards the forefront of the biofuel industry as a sustainable resource. However, saccharification and the production of bioproducts derived from plant cell wall biomass are complex and lengthy processes. The understanding of termite gut biology and feeding strategies may improve the current state of biomass conversion technology and bioproduct production.

Results

The study herein shows comprehensive functional characterization of crude body extracts from Coptotermes gestroi along with global proteomic analysis of the termite's digestome, targeting the identification of glycoside hydrolases and accessory proteins responsible for plant biomass conversion. The crude protein extract from C. gestroi was enzymatically efficient over a broad pH range on a series of natural polysaccharides, formed by glucose-, xylose-, mannan- and/or arabinose-containing polymers, linked by various types of glycosidic bonds, as well as ramification types. Our proteomic approach successfully identified a large number of relevant polypeptides in the C. gestroi digestome. A total of 55 different proteins were identified and classified into 29 CAZy families. Based on the total number of peptides identified, the majority of components found in the C. gestroi digestome were cellulose-degrading enzymes. Xylanolytic enzymes, mannan- hydrolytic enzymes, pectinases and starch-degrading and debranching enzymes were also identified. Our strategy enabled validation of liquid chromatography with tandem mass spectrometry recognized proteins, by enzymatic functional assays and by following the degradation products of specific 8-amino-1,3,6-pyrenetrisulfonic acid labeled oligosaccharides through capillary zone electrophoresis.

Conclusions

Here we describe the first global study on the enzymatic repertoire involved in plant polysaccharide degradation by the lower termite C. gestroi. The biochemical characterization of whole body termite extracts evidenced their ability to cleave all types of glycosidic bonds present in plant polysaccharides. The comprehensive proteomic analysis, revealed a complete collection of hydrolytic enzymes including cellulases (GH1, GH3, GH5, GH7, GH9 and CBM 6), hemicellulases (GH2, GH10, GH11, GH16, GH43 and CBM 27) and pectinases (GH28 and GH29).     

Cell-to-cell and phloem-mediated transport of potato virus X. The role of virions

Movement-deficient potato virus X (PVX) mutants tagged with the green fluorescent protein were used to investigate the role of the coat protein (CP) and triple gene block (TGB) proteins in virus movement. Mutants lacking either a functional CP or TGB were restricted to single epidermal cells. Microinjection of dextran probes into cells infected with the mutants showed that an increase in the plasmodesmal size exclusion limit was dependent on one or more of the TGB proteins and was independent of CP. Fluorescently labeled CP that was injected into epidermal cells was confined to the injected cells, showing that the CP lacks an intrinsic transport function. In additional experiments, transgenic plants expressing the PVX CP were used as rootstocks and grafted with nontransformed scions. Inoculation of the PVX CP mutants to the transgenic rootstocks resulted in cell-to-cell and systemic movement within the transgenic tissue. Translocation of the CP mutants into sink leaves of the nontransgenic scions was also observed, but infection was restricted to cells close to major veins. These results indicate that the PVX CP is transported through the phloem, unloads into the vascular tissue, and subsequently is transported between cells during the course of infection. Evidence is presented that PVX uses a novel strategy for cell-to-cell movement involving the transport of filamentous virions through plasmodesmata.     

Model of cytosine-, thymine-containing oligodeoxyribonucleotide protonation in solution

In the present study, the experimental data on the pH-induced formation of the i-motif structure in the nucleotide sequence 5′-CCTTTCCTTTTCCTTTCC-3′ (25°C, pH 3.3-8.9) obtained by spectroscopic techniques, such as UV molecular absorption and circular dichroism, has been analyzed using the chemometric soft modeling-based MCR-ALS approach and the hard modeling-based matrix method. Soft modeling using 2 or 3 spectral species correctly reproduced the spectral variations observed experimentally. The use of hard chemical modeling has allowed proposing an equilibrium model, which describes spectral changes as functions of solution acidity. Additionally, the intrinsic protonation constant K _in and the cooperativity parameter ω have been calculated from fitting of the circular dichroism as well as the molecular absorption spectra. The results indicated that folding was accompanied by a cooperative process, i.e. the enhancement of protonated structure stability upon protonation.     

Trafficking and localization studies of recombinant alpha1, 3- fucosyltransferase VI stably expressed in CHO cells

Peripheral alpha1,3-fucosylation of glycans occurs by the action of either one of five different alpha1,3-fucosyltransferases (Fuc-Ts) cloned to date. Fuc-TVI is one of the alpha1,3-fucosyltransferases which is capable to synthesize selectin ligands. The major alpha1, 3- fucosyltransferase activity in human plasma is encoded by the gene for fucosyltransferase VI, which presumably originates from liver cells. While the sequence, chromosomal localization, and kinetic properties of Fuc-TVI are known, immunocytochemical localization and trafficking studies have been impossible because of the lack of specific antibodies. Here we report on the development and characterization of a peptide-specific polyclonal antiserum monospecific to Fuc-TVI and an antiserum to purified soluble recombinant Fuc-TVI crossreactive with Fuc-TIII and Fuc-TV. Both antisera were applied for immunodetection in stably transfected CHO cells expressing the full-length form of this enzyme (CHO clone 61/11). Fuc-TVI was found to be a resident protein of the Golgi apparatus. In addition, more than 30% of cell-associated and released enzyme activity was found in the medium. Maturation and release of Fuc-TVI was analyzed in metabolically labeled CHO 61/11 cells followed by immunoprecipitation. Fuc-TVI occurred in two forms of 47 kDa and 43 kDa bands, while the secreted form was detected as a 43 kDa. These two different intracellular forms arose by posttranslational modification, as shown by pulse-chase experiments. Fuc-TVI was released to the supernatant by proteolytic cleavage as a partially endo-H resistant glycoform.      

ABC: software for interactive browsing of genomic multiple sequence alignment data

Background  

Alignment and comparison of related genome sequences is a powerful method to identify regions likely to contain functional elements. Such analyses are data intensive, requiring the inclusion of genomic multiple sequence alignments, sequence annotations, and scores describing regional attributes of columns in the alignment. Visualization and browsing of results can be difficult, and there are currently limited software options for performing this task.     

Procathepsin and acid phosphatase are stored in Musca domestica yolk spheres

Yolk spheres present in mature invertebrate oocytes are composed of yolk proteins and proteolytic enzymes. In the fly Musca domestica, yolk proteins are degraded during embryogenesis by a cathepsin-like proteinase that is stored as a zymogen. An acid phosphatase is also active in the yolk spheres during Musca embryogenesis. In this paper we show that procathepsin and acid phosphatase are initially stored by a different pathway from the one followed by yolk protein precursors. Both enzymes are taken up by the oocytes and transitorily stored into small vesicles (lysosomes) surrounding the early yolk spheres. Fusion of both structures, the early yolk spheres and lysosomes, creates the mature yolk spheres.     

7th SOSORT consensus paper: conservative treatment of idiopathic & Scheuermann's kyphosis

Abstract

Thoracic hyperkyphosis is a frequent problem and can impact greatly on patient's quality of life during adolescence. This condition can be idiopathic or secondary to Scheuermann disease, a disease disturbing vertebral growth. To date, there is no sound scientific data available on the management of this condition. Some studies discuss the effects of bracing, however no guidelines, protocols or indication's of treatment for this condition were found. The aim of this paper was to develop and verify the consensus on managing thoracic hyperkyphosis patients treated with braces and/or physiotherapy.

Methods

The Delphi process was utilised in four steps gradually modified according to the results of a set of recommendations: we involved the SOSORT Board twice, then all SOSORT members twice, with a Pre-Meeting Questionnaire (PMQ), and during a Consensus Session at the SOSORT Lyon Meeting with a Meeting Questionnaire (MQ).

Results

There was an unanimous agreement on the general efficacy of bracing and physiotherapy for this condition. Most experts suggested the use of 4-5 point bracing systems, however there was some controversy with regards to physiotherapeutic aims and modalities.

Conclusion

The SOSORT panel of experts suggest the use of rigid braces and physiotherapy to correct thoracic hyperkyphosis during adolescence. The evaluation of specific braces and physiotherapy techniques has been recommended.     

Chorioallantoic placentation in Galea spixii (Rodentia,Caviomorpha, Caviidae)

Background  

Placentas of guinea pig-related rodents are appropriate animal models for human placentation because of their striking similarities to those of humans. To optimize the pool of potential models in this context, it is essential to identify the occurrence of characters in close relatives.