Directed Evolution and In Silico Analysis of Reaction Centre Proteins Reveal Molecular Signatures of Photosynthesis Adaptation to Radiation Pressure

Evolutionary mechanisms adopted by the photosynthetic apparatus to modifications in the Earth's atmosphere on a geological time-scale remain a focus of intense research. The photosynthetic machinery has had to cope with continuously changing environmental conditions and particularly with the complex ionizing radiation emitted by solar flares. The photosynthetic D1 protein, being the site of electron tunneling-mediated charge separation and solar energy transduction, is a hot spot for the generation of radiation-induced radical injuries. We explored the possibility to produce D1 variants tolerant to ionizing radiation in Chlamydomonas reinhardtii and clarified the effect of radiation-induced oxidative damage on the photosynthetic proteins evolution. In vitro directed evolution strategies targeted at the D1 protein were adopted to create libraries of chlamydomonas random mutants, subsequently selected by exposures to radical-generating proton or neutron sources. The common trend observed in the D1 aminoacidic substitutions was the replacement of less polar by more polar amino acids. The applied selection pressure forced replacement of residues more sensitive to oxidative damage with less sensitive ones, suggesting that ionizing radiation may have been one of the driving forces in the evolution of the eukaryotic photosynthetic apparatus. A set of the identified aminoacidic substitutions, close to the secondary plastoquinone binding niche and oxygen evolving complex, were introduced by site-directed mutagenesis in un-transformed strains, and their sensitivity to free radicals attack analyzed. Mutants displayed reduced electron transport efficiency in physiological conditions, and increased photosynthetic performance stability and oxygen evolution capacity in stressful high-light conditions. Finally, comparative in silico analyses of D1 aminoacidic sequences of organisms differently located in the evolution chain, revealed a higher ratio of residues more sensitive to oxidative damage in the eukaryotic/cyanobacterial proteins compared to their bacterial orthologs. These results led us to hypothesize an archaean atmosphere less challenging in terms of ionizing radiation than the present one

Personalize, participate, predict, and prevent: 4Ps in inflammatory bowel disease

Inflammatory bowel disease (IBD), which includes Crohn's disease (CD) and ulcerative colitis (UC), is a complex, immune-mediated, disorder which leads to several gastrointestinal and systemic manifestations determining a poor quality of life, disability, and other negative health outcomes. Our knowledge of this condition has greatly improved over the last few decades, and a comprehensive management should take into account both biological (i.e., disease-related, patient-related) and non-biological (i.e., socioeconomic, cultural, environmental, behavioral) factors which contribute to the disease phenotype. From this point of view, the so called 4P medicine framework, including personalization, prediction, prevention, and participation could be useful for tailoring ad hoc interventions in IBD patients. In this review, we discuss the cutting-edge issues regarding personalization in special settings (i.e., pregnancy, oncology, infectious diseases), patient participation (i.e., how to communicate, disability, tackling stigma and resilience, quality of care), disease prediction (i.e., faecal markers, response to treatments), and prevention (i.e., dysplasia through endoscopy, infections through vaccinations, and post-surgical recurrence). Finally, we provide an outlook discussing the unmet needs for implementing this conceptual framework in clinical practice

Chapter Computational Biology, Protein Engineering, and Biosensor Technology: a Close Cooperation for Herbicides Monitoring

Pest contro

Chapter Computational Biology, Protein Engineering, and Biosensor Technology: a Close Cooperation for Herbicides Monitoring

Pest contro

The Arabidopsis BEL1-LIKE HOMEODOMAIN Proteins SAW1 and SAW2 Act Redundantly to Regulate KNOX Expression Spatially in Leaf Margins[W]

In Arabidopsis thaliana, the BEL1-like TALE homeodomain protein family consists of 13 members that form heterodimeric complexes with the Class 1 KNOX TALE homeodomain proteins, including SHOOTMERISTEMLESS (STM) and BREVIPEDICELLUS (BP). The BEL1-like protein BELLRINGER (BLR) functions together with STM and BP in the shoot apex to regulate meristem identity and function and to promote correct shoot architecture. We have characterized two additional BEL1-LIKE HOMEODOMAIN (BLH) proteins, SAWTOOTH1 (BLH2/SAW1) and SAWTOOTH2 (BLH4/SAW2) that, in contrast with BLR, are expressed in lateral organs and negatively regulate BP expression. saw1 and saw2 single mutants have no obvious phenotype, but the saw1 saw2 double mutant has increased leaf serrations and revolute margins, indicating that SAW1 and SAW2 act redundantly to limit leaf margin growth. Consistent with this hypothesis, overexpression of SAW1 suppresses overall growth of the plant shoot. BP is ectopically expressed in the leaf serrations of saw1 saw2 double mutants. Ectopic expression of Class 1 KNOX genes in leaves has been observed previously in loss-of-function mutants of ASYMMETRIC LEAVES (AS1). Overexpression of SAW1 in an as1 mutant suppresses the as1 leaf phenotype and reduces ectopic BP leaf expression. Taken together, our data suggest that BLH2/SAW1 and BLH4/SAW2 establish leaf shape by repressing growth in specific subdomains of the leaf at least in part by repressing expression of one or more of the KNOX genes

Mutations of Photosystem II D1 Protein That Empower Efficient Phenotypes of <i>Chlamydomonas reinhardtii</i> under Extreme Environment in Space

<div><p>Space missions have enabled testing how microorganisms, animals and plants respond to extra-terrestrial, complex and hazardous environment in space. Photosynthetic organisms are thought to be relatively more prone to microgravity, weak magnetic field and cosmic radiation because oxygenic photosynthesis is intimately associated with capture and conversion of light energy into chemical energy, a process that has adapted to relatively less complex and contained environment on Earth. To study the direct effect of the space environment on the fundamental process of photosynthesis, we sent into low Earth orbit space engineered and mutated strains of the unicellular green alga, <i>Chlamydomonas reinhardtii,</i> which has been widely used as a model of photosynthetic organisms. The algal mutants contained specific amino acid substitutions in the functionally important regions of the pivotal Photosystem II (PSII) reaction centre D1 protein near the Q_B binding pocket and in the environment surrounding Tyr-161 (Y_Z) electron acceptor of the oxygen-evolving complex. Using real-time measurements of PSII photochemistry, here we show that during the space flight while the control strain and two D1 mutants (A250L and V160A) were inefficient in carrying out PSII activity, two other D1 mutants, I163N and A251C, performed efficient photosynthesis, and actively re-grew upon return to Earth. Mimicking the neutron irradiation component of cosmic rays on Earth yielded similar results. Experiments with I163N and A251C D1 mutants performed on ground showed that they are better able to modulate PSII excitation pressure and have higher capacity to reoxidize the Q_A⁻ state of the primary electron acceptor. These results highlight the contribution of D1 conformation in relation to photosynthesis and oxygen production in space.</p></div