Geometric morphometric analyses of sexual dimorphism and allometry in two sympatric snakes: Natrix helvetica (Natricidae) and Vipera berus (Viperidae)

The non-venomous grass snake (Natrix helvetica) and the venomous adder (Vipera berus) are two native species that are often found in sympatry in Great Britain and Europe. They occupy partially overlapping ecological niches and prey on small vertebrates, but use different feeding strategies. Here, we investigated the morphologies of grass snakes and adders from Dorset (UK) using twodimensional geometric morphometrics to assess the degree of sexual dimorphism in size and shape together with the relative impact of allometry and general body dimensions on head shape. Both species showed significant sexual dimorphism in head size, but not in head shape. We found a clear allometric pattern in N. helvetica, whereas allometry in V. berus was generally less pronounced. Body dimensions were strongly correlated with head shape in the grass snake, but not in the adder. The fact that V. berus is venomous appears to explain the lack of allometric patterns and the lack of an association between body dimensions and head shape. The high degree of size dimorphism identified in both species could originate from the advantages of reduced intraspecific competition that are conveyed by a partial differentiation in feeding morphology

Macroevolutionary ecomorphology of the Carnivora skull: adaptations and constraints in the extant species

The mammalian order Carnivora is characterized by a broad taxonomic and ecological diversity. By using a large sample of extant species, we tested the impact of ecological factors on carnivoran skull (cranium and mandible) morphology, taking advantage of a combined geometric morphometrics and comparative method approach. We implemented several evolutionary models to account for different tempo and mode of evolution in size and shape data. These models validated the association between skull morphology and diet at the interspecific scale. The functional distinction between pinniped (aquatic) and fissiped (mostly terrestrial) taxa was found valid only in mandible shape and cranial size. High levels of morphological disparity and evolutionary rates were identified in specialized dietary groups, and positive association between rates and disparity was found for skull size. Cranium and mandible showed consistent patterns of covariation that reflect constrained functional processes, which stabilize the ecomorphological evolution of Carnivora. Aquatic adaptations allowed carnivorans to invade and persist within novel regions of the mandibular morphospace. This ecological shift did not increase morphological disparity but occurred at a faster rate than in terrestrial species. Those species exhibit a stronger level of cranio-mandibular covariation due to constraints imposed by more demanding masticatory adaptations

Comparison of alternative integration sites in the chromosome and the native plasmids of the cyanobacterium Synechocystis sp. PCC 6803 in respect to expression efficiency and copy number

Background: Synechocystis sp. PCC 6803 provides a well-established reference point to cyanobacterial metabolic engineering as part of basic photosynthesis research, as well as in the development of next-generation biotechnological production systems. This study focused on expanding the current knowledge on genomic integration of expression constructs in Synechocystis, targeting a range of novel sites in the chromosome and in the native plasmids, together with established loci used in literature. The key objective was to obtain quantitative information on site-specific expression in reference to replicon copy numbers, which has been speculated but never compared side by side in this host. Results: An optimized sYFP2 expression cassette was successfully integrated in two novel sites in Synechocystis chromosome (slr0944; sll0058) and in all four endogenous megaplasmids (pSYSM/slr5037-slr5038; pSYSX/slr6037; pSYSA/slr7023; pSYSG/slr8030) that have not been previously evaluated for the purpose. Fluorescent analysis of the segregated strains revealed that the expression levels between the megaplasmids and chromosomal constructs were very similar, and reinforced the view that highest expression in Synechocystis can be obtained using RSF1010-derived replicative vectors or the native small plasmid pCA2.4 evaluated in comparison. Parallel replicon copy number analysis by RT-qPCR showed that the expression from the alternative loci is largely determined by the gene dosage in Synechocystis, thereby confirming the dependence formerly proposed based on literature. Conclusions: This study brings together nine different integrative loci in the genome of Synechocystis to demonstrate quantitative differences between target sites in the chromosome, the native plasmids, and a RSF1010-based replicative expression vector. To date, this is the most comprehensive comparison of alternative integrative sites in Synechocystis, and provides the first direct reference between expression efficiency and replicon gene dosage in the context. In the light of existing literature, the findings support the view that the small native plasmids can be notably more difficult to target than the chromosome or the megaplasmids, and that the RSF1010-derived vectors may be surprisingly well maintained under non-selective culture conditions in this cyanobacterial host. Altogether, the work broadens our views on genomic integration and the rational use of different integrative loci versus replicative plasmids, when aiming at expressing heterologous genes in Synechocystis.The research was financially supported by the Academy of Finland Centre of Excellence (#307335), NordForsk Nordic Centre of Excellence (#82845) and Jane and Aatos Erkko Foundation (#4605–26422). The work also received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Action—Innovative Training Network 2017 (#764920), and Fundação para a Ciência e a Tecnologia (CEECIND/00259/2017 to CCP)

Expanding the toolbox for Synechocystis sp. PCC 6803 : validation of replicative vectors and characterization of a novel set of promoters

Cyanobacteria are promising ‘low-cost’ cell factories since they have minimal nutritional requirements, high metabolic plasticity and can use sunlight and CO2 as energy and carbon sources. The unicellular Synechocystis sp. PCC 6803, already considered the ‘green’ Escherichia coli, is the best studied cyanobacterium but to be used as an efficient and robust photoautotrophic chassis it requires a customized and well-characterized toolbox. In this context, we evaluated the possibility of using three self-replicative vectors from the Standard European Vector Architecture (SEVA) repository to transform Synechocystis. Our results demonstrated that the presence of the plasmid does not lead to an evident phenotype or hindered Synechocystis growth, being the vast majority of the cells able to retain the replicative plasmid even in the absence of selective pressure. In addition, a set of heterologous and redesigned promoters were characterized exhibiting a wide range of activities compared to the reference PrnpB, three of which could be efficiently repressed. As a proof-of-concept, from the expanded toolbox, one promoter was selected and assembled with the ggpS gene [encoding one of the proteins involved in the synthesis of the native compatible solute glucosylglycerol (GG)] and the synthetic device was introduced into Synechocystis using one of the SEVA plasmids. The presence of this device restored the production of the GG in a ggpS deficient mutant validating the functionality of the tools/device developed in this study

Molecular characterization of Bifidobacterium longum biovar longum NAL8 plasmids and construction of a novel replicon screening system

In this study, we performed molecular characterization and sequence analysis of three plasmids from the human intestinal isolate Bifidobacterium longum biovar longum NAL8 and developed a novel vector screening system. Plasmids pNAL8H (10 kb) and pNAL8M (4.9 kb) show close sequence similarity to and the same gene organization as the already characterized B. longum plasmids. The B. longum plasmid pNAC1 was identified as being most closely related to pNAL8L (3.5 kb). However, DNA sequence analysis suggested that direct repeat-rich sites could have promoted several recombination events to diversify the two plasmid molecules. We verified the likely rolling circle replication of plasmid pNAL8L and studied the phylogenetic relationship in all the Bifidobacterium plasmids fully sequenced to date based on in silico comparative sequence analysis of their replication proteins and iteron regions. Our transformation experiments confirmed that the ColE1 replication origin from high-copy-number pUC vectors could interfere with the replication apparatus of Bifidobacterium plasmids and give rise to false positive clones. As a result, we developed a system suitable for avoiding possible interference by other functional replication modules on the vector and for screening functional replicons from wild-type plasmids

Severe hereditary spherocytosis and distal renal tubular acidosis associated with the total absence of band 3

Absence of band 3, associated with the mutation Coimbra (V488M) in the homozygous state, caused severe hereditary spherocytosis in a young child. Although prenatal testing was made available to the parents, it was declined. Because the fetus stopped moving near term, an emergency cesarean section was performed and a severely anemic, hydropic female baby was delivered. She was resuscitated and initially kept alive with respiratory assistance and hypertransfusion therapy. Cord blood smears revealed erythroblastosis, poikilocytosis, and red cells with stalk-like elongations. Band 3 and protein 4.2 were absent; spectrin, ankyrin, and glycophorin A were significantly reduced. Renal tubular acidosis was detected by the age of 3 months. Nephrocalcinosis appeared soon thereafter. After 3 years of follow-up the child is doing reasonably well on a regimen that includes regular blood transfusions and daily bicarbonate supplements. The long-term prognosis remains uncertain given the potential for hematologic and renal complications