Exploring the Structure-Function Loop Adaptability of a (β/α)(8)-Barrel Enzyme through Loop Swapping and Hinge Variability

Cellulosomes are large extracellular multi-enzyme complexes that exhibit elevated activity on plant cell-wall polysaccharides. In the present study, the relationships between the conformational flexibility and efficacy of cellulosomes, and the inter-modules linkers of their scaffold protein were investigated. For this purpose, the length of the intrinsically disordered Ser/Thr-rich 50-residue linker connecting a Clostridium thermocellum and a Clostridium cellulolyticum cohesin in a hybrid scaffoldin (Scaf4) was changed by sequences ranging from 4 to 128 residues. The composition was also modified and new linkers composed of series of N, S or repeats of the EPPV motif were generated. Two model cellulases (Cel48F and Cel9G) appended with appropriate dockerins were subsequently bound to the engineered scaffoldins. All the resulting minicomplexes displayed the same activity on crystalline cellulose as the complex based on the initial Scaf4, and were found to be 2-fold more active than Cel48F and Cel9G bound to separate cohesins. Small-angle X-ray scattering assays of the engineered scaffoldins confirmed, however, that the size and the conformational flexibility of some of the new inter-cohesins linkers differed significantly from that of the initial 50 residue linker displayed by the parental Scaf4. Our data suggest that the synergy induced by proximity does not require a specific inter-cohesins sequence or distance. The present study reveals that complexation onto the hybrid scaffoldins modifies the type of soluble sugars released from crystalline cellulose by the selected cellulases, compared to the free enzyme system.     

Comments on duck circovirus (DuCV) genotype definition

A standardised methodology has been used to define genotypes based on pairwise sequence comparisons (PASC). PASC is a widely accepted method in virus taxonomy, which is based on the histogram of pairwised differences among sequences. Recently, Zhang et al. (2013) concluded that the average p-distance of duck circovirus (DuCV) between genotypes 1 and 2 was 0.170, and subtype distance thresholds were 0.032 in DuCV-1 and 0.018 in DuCV-2, respectively. However, there might be some concerns on the methodology application to define the genotype of DuCV. Taking into account the concerns mentioned above, our authors conducted the PASC analyses of 54 capsid gene (ORF2) and genomic sequences including all the sequences from Zhang et al. (2013). Our results confirmed the existence of two DuCV genotypes (1 and 2) and, we suggest that DuCV ORF2 and genomic distance genotype thresholds were 0.061 and 0.038, respectively.     

Multiple early factors anticipate post-acute COVID-19 sequelae

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Identification,genotyping, and molecular evolution analysis of duck circovirus

Duck circovirus (DuCV) is a contagious immunosuppressive virus affecting many duck species, which is responsible for multiple outbreaks in poultry industries worldwide. In this study, the first DuCV isolate GH01 was identified in Sichuan by PCR, which shared a high level of nucleotide identity (81.8–99.4%) with sequences of other DuCV isolates available in GenBank. Comparative phylogenetic and pairwise sequence comparison analyses indicated that DuCV could be divided into two genotypes (DuCV-1 and DuCV-2) and six subtypes (1a, 1b, 1c, 2a, 2b and 2c) based on the complete genome sequence. The results revealed that both DuCV-1 and DuCV-2 had evolved from the same ancestor but undergone divergent evolution. Interestingly, phylogenetic analyses indicated that three isolates were classified into a cluster DuCV-2a using complete DuCV genome sequence and cap gene, except rep gene. Recombination analyses revealed that DuCV-2a arose from recombination between DuCV-1a and DuCV-2b isolates within the rep genes, and the recombination events mainly occur both in non-structural protein coding region and structural protein coding region. In addition, the mechanisms of recombination supporting the genetic variability in DuCV isolates were investigated. Likewise, selective pressure indicated that purifying selection had been a major driving force in maintaining diversity among the DuCV isolates. Because eradicating the virus from commercial ducks is impossible, it is necessary to take effective control measures and implement them throughout the world.     

Cloning,expression, and characterization of a cellobiose dehydrogenase from Thielavia terrestris induced under cellulose growth conditions

The enzyme cellobiose dehydrogenase (CDH) is of considerable interest, not only for its biotechnological applications, but also its potential biological role in lignocellulosic biomass breakdown. The enzyme catalyzes the oxidation of cellobiose and other cellodextrins, utilizing a variety of one- and two-electron acceptors, although the electron acceptor employed in nature is still unknown. In this study we show that a CDH is present in the secretome of the thermophilic ascomycete Thielavia terrestris when grown with cellulose, along with a mixture of cellulases and hemicellulases capable of breaking down lignocellulosic biomass. We report the cloning of this T. terrestris CDH gene (cbdA), its recombinant expression in Aspergillus oryzae, and purification and characterization of the T. terrestris CDH protein (TtCDH). The TtCDH shows spectral properties and enzyme activity similar to other characterized CDH enzymes. Substrate specificity was determined for a number of carbohydrate electron donors in the presence of the two-electron acceptor 2,6-dichlorophenol-indophenol. The TtCDH also shows dramatic synergy with Thermoascus aurantiacus glycoside hydrolase family 61A protein in the presence of a β-glucosidase for the cleavage of cellulose.     

Epigenetic memory of coronavirus infection in innate immune cells and their progenitors

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