The role of outer membrane protein(S) harboring slh/oprb-domains in extracellular vesicles’ production in synechocystis sp. pcc 6803

Cyanobacteria are a group of photosynthetic prokaryotes that contribute to primary production on a global scale. These microorganisms release vesicles to the extracellular environment, spherical nanosized structures, derived essentially from the outer membrane. Even though earlier works in model Gram-negative bacteria have hypothesized that outer membrane stability is crucial in vesicle formation, the mechanisms determining vesicle biogenesis in cyanobacteria remain unknown. Here, we report on the identification of six candidate genes encoding outer membrane proteins harboring SLH/OprB-domains in the genome of the model cyanobacterium Synechocystis sp. PCC 6803. Using a genetics-based approach, one gene was found to encode an essential protein (Slr1841), while the remaining five are not essential for growth under standard conditions. Vesicle production was monitored, and it was found that a mutant in the gene encoding the second most abundant SLH/OprB protein in Synechocystis sp. PCC 6803 outer membrane (Slr1908) produces more vesicles than any of the other tested strains. Moreover, the Slr1908-protein was also found to be important for iron uptake. Altogether, our results suggest that proteins containing the SLH/OprB-domains may have dual biological role, related to micronutrient uptake and to outer membrane stability, which, together or alone, seem to be involved in cyanobacterial vesicle biogenesis.This work was financed by Fundo Europeu de Desenvolvimento Regional (FEDER) funds through the COMPETE 2020 Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal, 2020, and by Portuguese funds through Fundação para a Ciência e a Tecnolo-gia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project POCI-01-0145-FEDER-029540 (PTDC/BIA-OUT/29540/2017). Fundação para a Ciência e a Tecnologia is also greatly acknowledged for the PhD fellowship SFRH/BD/130478/2017 (SL) and FCT Investigator grant IF/00256/2015 (PO)

Strategies to obtain designer polymers based on cyanobacterial extracellular polymeric substances (EPS)

Biopolymers derived from polysaccharides are a sustainable and environmentally friendly alternative to the synthetic counterparts available in the market. Due to their distinctive properties, the cyanobacterial extracellular polymeric substances (EPS), mainly composed of heteropolysaccharides, emerge as a valid alternative to address several biotechnological and biomedical challenges. Nevertheless, biotechnological/biomedical applications based on cyanobacterial EPS have only recently started to emerge. For the successful exploitation of cyanobacterial EPS, it is important to strategically design the polymers, either by genetic engineering of the producing strains or by chemical modification of the polymers. This requires a better understanding of the EPS biosynthetic pathways and their relationship with central metabolism, as well as to exploit the available polymer functionalization chemistries. Considering all this, we provide an overview of the characteristics and biological activities of cyanobacterial EPS, discuss the challenges and opportunities to improve the amount and/or characteristics of the polymers, and report the most relevant advances on the use of cyanobacterial EPS as scaffolds, coatings, and vehicles for drug delivery.This work was financed by FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT—Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project POCI-01-0145-FEDER-028779, contract DL57/2016/CP1327/CT0007 and fellowship SFRH/BD/119920/2016

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)

Biocompatibility of the biopolymer cyanoflan for applications in skin wound healing

There is a great demand for the development of novel wound dressings to overcome the time and costs of wound care performed by a vast number of clinicians, especially in the current overburdened healthcare systems. In this study, Cyanoflan, a biopolymer secreted by a marine unicellular cyanobacterium, was evaluated as a potential biomaterial for wound healing. Cyanoflan effects on cell viability, apoptosis, and migration were assessed in vitro, while the effect on tissue regeneration and biosafety was evaluated in healthy Wistar rats. The cell viability and apoptosis of fibroblasts and endothelial cells was not influenced by the treatment with different concentrations of Cyanoflan, as observed by flow cytometry. Moreover, the presence of Cyanoflan did not affect cell motility and migratory capacity, nor did it induce reactive oxygen species production, even revealing an antioxidant behavior regarding the endothelial cells. Furthermore, the skin wound healing in vivo assay demonstrated that Cyanoflan perfectly adapted to the wound bed without inducing systemic or local oxidative or inflammatory reaction. Altogether, these results suggest that Cyanoflan is a promising biopolymer for the development of innovative applications to overcome the many challenges that still exist in skin wound healing.This work was supported by FEDER—Fundo Europeu de Desenvolvimento Regional— through the COMPETE 2020—Operational Programme for Competitiveness and Internationalization (POCI), Portugal 2020—and by Portuguese funds through FCT—Fundação para a Ciência e a Tec-nologia/Ministério da Ciência, Tecnologia e Ensino Superior—in the framework of the project POCI-01-0145-FEDER-028779 (PTDC/BIA-MIC/28779/2017), by NORTE-01-0145-FEDER-000012, Structured Programme on Bioengineering Therapies for Infectious Diseases and Tissue Regeneration, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and in the framework of the project “Institute for Research and Innovation in Health Sciences” (UID/BIM/04293/2020)

Substance P-Botulinum Mediates Long-term Silencing of Pain Pathways that can be Re-instated with a Second Injection of the Construct in Mice

Chronic pain presents an enormous personal and economic burden and there is an urgent need for effective treatments. In a mouse model of chronic neuropathic pain, selective silencing of key neurons in spinal pain signalling networks with botulinum constructs resulted in a reduction of pain behaviours associated with the peripheral nerve. However, to establish clinical relevance it was important to know how long this silencing period lasted. Now, we show that neuronal silencing and the concomitant reduction of neuropathic mechanical and thermal hypersensitivity lasts for up to 120d following a single injection of botulinum construct. Crucially, we show that silencing and analgesia can then be reinstated with a second injection of the botulinum conjugate. Here we demonstrate that single doses of botulinum-toxin conjugates are a powerful new way of providing long-term neuronal silencing and pain relief

New Avenues for Old Travellers: Phenotypic Evolutionary Trends Meet Morphodynamics, and Both Enter the Global Change Biology Era

Evolutionary trends (ETs) are traditionally defined as substantial changes in the state of traits through time produced by a persistent condition of directional evolution. ETs might also include directional responses to ecological, climatic or biological gradients and represent the primary evolutionary pattern at high taxonomic levels and over long-time scales. The absence of a well-supported operative definition of ETs blurred the definition of conceptual differences between ETs and other key concepts in evolution such as convergence, parallel evolution, and divergence. Also, it prevented the formulation of modern guidelines for studying ETs and evolutionary dynamics related to them. In phenotypic evolution, the theory of morphodynamics states that the interplay between evolutionary factors such as phylogeny, evo-devo constraints, environment, and biological function determines morphological evolution. After introducing a new operative definition, here we provide a morphodynamics-based framework for studying phenotypic ETs, discussing how understanding the impact of these factors on ETs improves the explanation of links between biological patterns and processes underpinning directional evolution. We envisage that adopting a quantitative, pattern-based, and multifactorial approach will pave the way to new potential applications for this field of evolutionary biology. In this framework, by exploiting the catalysing effect of climate change on evolution, research on ETs induced by global change might represent an ideal arena for validating hypotheses about the predictability of evolution

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

The role of the tyrosine kinase Wzc (Sll0923) and the phosphatase Wzb (Slr0328) in the production of extracellular polymeric substances (EPS) by Synechocystis PCC 6803

Many cyanobacteria produce extracellular polymeric substances (EPS) mainly composed of heteropolysaccharides with unique characteristics that make them suitable for biotechnological applications. However, manipulation/optimization of EPS biosynthesis/characteristics is hindered by a poor understanding of the production pathways and the differences between bacterial species. In this work, genes putatively related to different pathways of cyanobacterial EPS polymerization, assembly, and export were targeted for deletion or truncation in the unicellular Synechocystis sp. PCC 6803. No evident phenotypic changes were observed for some mutants in genes occurring in multiple copies in Synechocystis genome, namely ¿wzy (¿sll0737), ¿wzx (¿sll5049), ¿kpsM (¿slr2107), and ¿kpsM¿wzy (¿slr2107¿sll0737), strongly suggesting functional redundancy. In contrast, ¿wzc (¿sll0923) and ¿wzb (¿slr0328) influenced both the amount and composition of the EPS, establishing that Wzc participates in the production of capsular (CPS) and released (RPS) polysaccharides, and Wzb affects RPS production. The structure of Wzb was solved (2.28 Å), revealing structural differences relative to other phosphatases involved in EPS production and suggesting a different substrate recognition mechanism. In addition, Wzc showed the ATPase and autokinase activities typical of bacterial tyrosine kinases. Most importantly, Wzb was able to dephosphorylate Wzc in vitro, suggesting that tyrosine phosphorylation/dephosphorylation plays a role in cyanobacterial EPS production.Norte Portugal Regional Operational Programme (NORTE 2020), Grant/Award Number: NORTE‐01‐0145‐FE?ER‐000008 and NORTE‐01‐0145‐FE?ER‐000012; FCT ‐ Fundação para a Ciência e a Tecnologia/ Ministério da Ciência, Tecnologia e Ensino Superior, Grant/Award Number: PTDC/BIA‐ MIC/28779/2017, SFRH/BD /119920/2016, SFRH/B?/84914/2012 and SFRH/BD/99715/ 2014; FEDER ‐ Fundo Europeu de Desen‐ volvimento Regional funds through the COMPETE 2020 ‐ Operational Programme for Competitiveness and Internationalisation (POCI), Grant/Award Number: POCI‐01‐0145‐ FE?ER‐007274 ACKNOWLEDGMENTS: This work was financed by FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020— Operational Programme for Competitiveness and Internationalisation (POCI); projects NORTE‐01‐0145‐FEDER‐000012—Structured Programme on Bioengineering Therapies for Infectious ?iseases and Tissue Regeneration and NORTE‐01‐0145‐FEDER‐000008— Porto Neurosciences and Neurologic Disease Research Initiative at i3S, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement; and by Portuguese funds through FCT—Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project “Institute for Research and Innovation in Health Sciences” (POCI‐01‐0145‐FEDER‐007274 and PTDC/BIA‐ MIC/28779/2017) and grants SFRH/BD /119920/2016 (MS), SFRH/ BD /99715/2014 (CF), and SFRH/BD /129921/2017 (JPL). The au‐ thors thank F. Chauvat and the Commissariat à l’Energie Atomique (CEA), Direction des Sciences du Vivant, for providing the cas‐ sette for the deletion of the Synechocystis sll0923, the staff of the European Synchrotron Radiation Facility (Grenoble, France) and SOLEIL (Essonne, France) synchrotrons, Filipe Pinto, Frederico Silva, Hugo Osório, and Joana Furtado for their technical assistance

A method for mapping morphological convergence on three-dimensional digital models: the case of the mammalian sabre-tooth

Morphological convergence can be assessed using a variety of statistical methods. None of the methods proposed to date enable the visualization of convergence. All are based on the assumption that the phenotypes either converge, or do not. However, between species, morphologically similar regions of a larger structure may behave differently. Previous approaches do not identify these regions within the larger structures or quantify the degree to which they may contribute to overall convergence. Here, we introduce a new method to chart patterns of convergence on three-dimensional models using the R function conv.map. The convergence between pairs of models is mapped onto them to visualize and quantify the morphological convergence. We applied conv.map to a well-known case study, the sabre-tooth morphotype, which has evolved independently among distinct mammalian clades from placentals to metatherians. Although previous authors have concluded that sabre-tooths kill using a stabbing ‘bite’ to the neck, others have presented different interpretations for specific taxa, including the iconic Smilodon and Thylacosmilus. Our objective was to identify any shared morphological features among the sabre-tooths that may underpin similar killing behaviours. From a sample of 49 placental and metatherian carnivores, we found stronger convergence among sabre-tooths than for any other taxa. The morphological convergence is most apparent in the rostral and posterior parts of the cranium. The extent of this convergence suggests similarity in function among these phylogenetically distant species. In our view, this function is most likely to be the killing of relatively large prey using a stabbing bite. © 2021 The Authors. Palaeontology published by John Wiley & Sons Ltd on behalf of The Palaeontological Association