T-DNA integration patterns in transgenic maize lines mediated by Agrobacterium tumefaciens

To explore transfer deoxyribonucleic acid (T-DNA) integration patterns in the maize genome, we improved the protocol of thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR), and amplified the flanking sequences around T-DNA integration sites from 70 independent transgenic maize lines mediated by Agrobacterium tumefaciens. Out of 64 specific amplified fragments, 32 and 9 are homologous to the sequences of the maize genome and the expression plasmid, respectively. For 26 of them, a filler sequence was found flanking the cleavage sites. These results demonstrate that cleavage occurs not only during the T-DNA borders but also inside or outside the borders. The border sequences and some inside sequences can be deleted, and filler sequences can be inserted. Illegitimate recombination is a major pattern of T-DNA integration, while some hot spots and preference are present on maize chromosomes.Key words: Agrobacterium tumefaciens, maize, thermal asymmetric interlaced PCR, transfer DNA,transgenics

Interdependent network reciprocity in evolutionary games

Besides the structure of interactions within networks, also the interactions between networks are of the outmost importance. We therefore study the outcome of the public goods game on two interdependent networks that are connected by means of a utility function, which determines how payoffs on both networks jointly influence the success of players in each individual network. We show that an unbiased coupling allows the spontaneous emergence of interdependent network reciprocity, which is capable to maintain healthy levels of public cooperation even in extremely adverse conditions. The mechanism, however, requires simultaneous formation of correlated cooperator clusters on both networks. If this does not emerge or if the coordination process is disturbed, network reciprocity fails, resulting in the total collapse of cooperation. Network interdependence can thus be exploited effectively to promote cooperation past the limits imposed by isolated networks, but only if the coordination between the interdependent networks is not disturbe

Towards personalised allele-specific CRISPR gene editing to treat autosomal dominant disorders

Abstract CRISPR/Cas9 holds immense potential to treat a range of genetic disorders. Allele-specific gene disruption induced by non-homologous end-joining (NHEJ) DNA repair offers a potential treatment option for autosomal dominant disease. Here, we successfully delivered a plasmid encoding S. pyogenes Cas9 and sgRNA to the corneal epithelium by intrastromal injection and acheived long-term knockdown of a corneal epithelial reporter gene, demonstrating gene disruption via NHEJ in vivo. In addition, we used TGFBI corneal dystrophies as a model of autosomal dominant disease to assess the use of CRISPR/Cas9 in two allele-specific systems, comparing cleavage using a SNP-derived PAM to a guide specific approach. In vitro, cleavage via a SNP-derived PAM was found to confer stringent allele-specific cleavage, while a guide-specific approach lacked the ability to distinguish between the wild-type and mutant alleles. The failings of the guide-specific approach highlights the necessity for meticulous guide design and assessment, as various degrees of allele-specificity are achieved depending on the guide sequence employed. A major concern for the use of CRISPR/Cas9 is its tendency to cleave DNA non-specifically at “off-target” sites. Confirmation that S. pyogenes Cas9 lacks the specificity to discriminate between alleles differing by a single base-pair regardless of the position in the guide is demonstrated

Hsp21potentiates antifungal drug tolerance in Candida albicans

Peer reviewedPublisher PD

The contribution of virtual enterprises to competence-based learning: an assessment from the students’ perspective: Case study

The use of virtual enterprises has evolved from secondary, vocational and professional education to the university level, becoming in recent times a relevant experiential e-learning tool based on the simulation of the functioning of a company. The purpose of this study is to analyze the perspective of students taking part in virtual enterprises about their acquisition of generic and specific managerial skills fostered by this e-learning methodology. We analyzed data of 76 students from Faculties of Business and Economics, who had participated during the academic year 2014-2015 in a virtual company during the development of their degrees at two different Italian universities, University of Bologna and Parma. Our results show that the most valued generic skills were related to the capacity of learning and adaptation, problem solving and teamwork, whilst the least valued ones were related to the skills of communication and interaction with people of other countries and cultures. In the case of specific managerial skills, the most valued ones were about understanding managerial concepts and the role and functions of economic agents, and the least valued were skills related to providing managerial advice, dealing with risks and analyzing financial statements. Besides, the analyses conducted to determine the existence of a learning profile in the students reveal that the skills acquired were quite similar, not being affected by the students’ gender or the economic activity developed by the virtual enterprise. This study makes a contribution in terms of the effectiveness of virtual enterprises for competence-based learning

A fresh look at the evolution and diversification of photochemical reaction centers

In this review, I reexamine the origin and diversification of photochemical reaction centers based on the known phylogenetic relations of the core subunits, and with the aid of sequence and structural alignments. I show, for example, that the protein folds at the C-terminus of the D1 and D2 subunits of Photosystem II, which are essential for the coordination of the water-oxidizing complex, were already in place in the most ancestral Type II reaction center subunit. I then evaluate the evolution of reaction centers in the context of the rise and expansion of the different groups of bacteria based on recent large-scale phylogenetic analyses. I find that the Heliobacteriaceae family of Firmicutes appears to be the earliest branching of the known groups of phototrophic bacteria; however, the origin of photochemical reaction centers and chlorophyll synthesis cannot be placed in this group. Moreover, it becomes evident that the Acidobacteria and the Proteobacteria shared a more recent common phototrophic ancestor, and this is also likely for the Chloroflexi and the Cyanobacteria. Finally, I argue that the discrepancies among the phylogenies of the reaction center proteins, chlorophyll synthesis enzymes, and the species tree of bacteria are best explained if both types of photochemical reaction centers evolved before the diversification of the known phyla of phototrophic bacteria. The primordial phototrophic ancestor must have had both Type I and Type II reaction centers

Repair of the TGFBI gene in human corneal keratocytes derived from a granular corneal dystrophy patient via CRISPR/Cas9-induced homology-directed repair

Abstract Granular corneal dystrophy (GCD) is an autosomal dominant hereditary disease in which multiple discrete and irregularly shaped granular opacities are deposited in the corneal stroma. GCD is caused by a point mutation in the transforming growth factor-β-induced (TGFBI) gene, located on chromosome 5q31. Here, we report the first successful application of CRISPR-Cas9-mediated genome editing for the correction of a TGFBI mutation in GCD patient-derived primary corneal keratocytes via homology-directed repair (HDR). To correct genetic defects in GCD patient cells, we designed a disease-specific guide RNA (gRNA) targeting the R124H mutation of TGFBI, which causes GCD type 2 (GCD2). An R124H mutation in primary human corneal keratocytes derived from a GCD2 patient was corrected by delivering a CRISPR plasmid expressing Cas9/gRNA and a single-stranded oligodeoxynucleotide HDR donor template in vitro. The gene correction efficiency was 20.6% in heterozygous cells and 41.3% in homozygous cells. No off-target effects were detected. These results reveal a new therapeutic strategy for GCD2; this method may also be applicable to other heredity corneal diseases