Biology — Open Access Journal

Chlamydomonas reinhardtii is a model alga of increasing interest as a cell factory for the production of valuable compounds, including therapeutic proteins and bioactive metabolites. Expression of foreign genes in the chloroplast is particularly advantageous as: (i) accumulation of product in this sub-cellular compartment minimises potential toxicity to the rest of the cell; (ii) genes can integrate at specific loci of the chloroplast genome (plastome) by homologous recombination; (iii) the high ploidy of the plastome and the high-level expression of chloroplast genes can be exploited to achieve levels of recombinant protein as high as 5% total cell protein; (iv) the lack of any gene silencing mechanisms in the chloroplast ensures stable expression of transgenes. However, the generation of C. reinhardtii chloroplast transformants requires efficient methods of selection, and ideally methods for subsequent marker removal. Additionally, the use of reporter genes is critical to achieving a comprehensive understanding of gene expression, thereby informing experimental design for recombinant applications. This review discusses currently available selection and reporter systems for chloroplast engineering in C. reinhardtii, as well as those used for chloroplast engineering in higher plants and other microalgae, and looks to the future in terms of possible new markers and reporters that will further advance the C. reinhardtii chloroplast as an expression platform. Full article

One of the significant organ systems which decline in aging is the kidney. While the causes of age-associated decline in renal function are likely multifactorial, oxidative stress and inflammation are hypothesized to play important roles in the structural and functional changes of the kidney. During aging there is a general decline in the glomerular filtration rate (GFR), a primary measurement used to assess kidney function. Inflammation and oxidative stress have been hypothesized to have a significant detrimental effect on renal function in aging and this may be attenuated by renal protective dietary ingredients. These dietary ingredients may affect renal function directly or through a microbiome-mediated secondary product. Likewise, structural changes including renal tubular atrophy, interstitial fibrosis, and glomerulosclerosis have all been described in aging. Such detrimental changes may benefit from dietary ingredients that may delay or attenuate the occurrence of such changes. This review will describe the physiology and pathophysiology of aging in renal function with an emphasis on dogs and cats that develop a decline in kidney function naturally. In addition, the varying biomarkers of health and renal dysfunction will be discussed. Finally, we will evaluate the aid in the management of this normal decline through dietary intervention in animal models. Full article

Eukaryotic dsDNA viruses use small basic protamine-like proteins or histones, typically <15 kDa, to condense and encapsidate their genomic (g)DNAs during virogenesis. Ascoviruses are large dsDNA (~100–200 kbp) viruses that are pathogenic to lepidopteran larvae. Little is known about the molecular basis for condensation and encapsidation of their gDNAs. Previous proteomic analysis showed that Spodoptera frugiperda ascovirus (SfAV-1a) virions contain a large unique DNA-binding protein (P64; 64 kDa, pI = 12.2) with a novel architecture proposed to condense its gDNA. Here we used physical, biochemical, and transmission electron microscopy techniques to demonstrate that P64’s basic C-terminal domain condenses SfAV-1a gDNA. Moreover, we demonstrate that only P64 homologs in other ascovirus virions are unique in stably binding DNA. As similar protein families or subfamilies were not identified in extensive database searches, our collective data suggest that ascovirus P64 homologs comprise a novel family of atypical large viral gDNA condensing proteins. Full article

Invasive plant species are expected to display high dispersal capacity but low levels of genetic diversity due to the founder effect occurring at each invasion episode. Understanding the history of invasions and the levels of genetic diversity of such species is an important task for planning management and monitoring strategy for these events. Peruvian Peppertree (Schinus molle L.) is a pioneer tree species native from South America which was introduced in North America, Europe and Africa, becoming a threat to these non-native habitats. In this study, we report the discovery and characterization of 17 plastidial (ptSSR) and seven nuclear (nSSR) markers for S. molle based on low-coverage whole-genome sequencing data acquired through next-generation sequencing. The markers were tested in 56 individuals from two natural populations sampled in the Brazilian Caatinga and Pampa biomes. All loci are moderately to highly polymorphic and revealed to be suitable for genetic monitoring of new invasions, for understanding the history of old invasions, as well as for genetic studies of native populations in their natural occurrence range and of orchards established with commercial purposes. Full article

Flavoprotein monooxygenases create valuable compounds that are of high interest for the chemical, pharmaceutical, and agrochemical industries, among others. Monooxygenases that use flavin as cofactor are either single- or two-component systems. Here we summarize the current knowledge about two-component flavin adenine dinucleotide (FAD)-dependent monooxygenases and describe their biotechnological relevance. Two-component FAD-dependent monooxygenases catalyze hydroxylation, epoxidation, and halogenation reactions and are physiologically involved in amino acid metabolism, mineralization of aromatic compounds, and biosynthesis of secondary metabolites. The monooxygenase component of these enzymes is strictly dependent on reduced FAD, which is supplied by the reductase component. More and more representatives of two-component FAD-dependent monooxygenases have been discovered and characterized in recent years, which has resulted in the identification of novel physiological roles, functional properties, and a variety of biocatalytic opportunities. Full article

The self-organisation of a fertilised egg to form a blastocyst structure, which consists of three distinct cell lineages (trophoblast, epiblast and hypoblast) arranged around an off-centre cavity, is unique to mammals. While the starting point (the zygote) and endpoint (the blastocyst) are similar in all mammals, the intervening events have diverged. This review examines and compares the descriptive and functional data surrounding embryonic gene activation, symmetry-breaking, first and second lineage establishment, and fate commitment in a wide range of mammalian orders. The exquisite detail known from mouse embryogenesis, embryonic stem cell studies and the wealth of recent single cell transcriptomic experiments are used to highlight the building principles underlying early mammalian embryonic development. Full article

Animals often use public information for mate-choice decisions by observing conspecifics as they choose their mates and then copying this witnessed decision. When the copier, however, is detected by the choosing individual, the latter often alters its behavior and spends more time with the previously non-preferred mate. This behavioral change is called the audience effect. The deception hypothesis states that the choosing individual changes its behavior to distract the audience from the preferred mate. The deception hypothesis, however, only applies if the audience indeed copies the pretended mate choice of the observed individual. So far, this necessary prerequisite has never been tested. We investigated in Atlantic molly males and females whether, first, focal fish show an audience effect, i.e., alter their mate choices in the presence of an audience fish, and second, whether audience fish copy the mate choice of the focal fish they had just witnessed. We found evidence that male and female Atlantic mollies copy the pretended mate choice of same-sex focal fish. Therefore, a necessary requirement of the deception hypothesis is fulfilled. Our results show that public information use in the context of mate choice can be costly. Full article

Agriculture and intensive farming methods are the greatest cause of nitrogen pollution. In particular, nitrification (the conversion of ammonia to nitrate) plays a role in global climate changes, affecting the bio-availability of nitrogen in soil and contributing to eutrophication. In this paper, the Rhodotorula diobovata DSBCA06 was investigated for growth kinetics on nitrite, nitrate, or ammonia as the sole nitrogen sources (10 mM). Complete nitrite removal was observed in 48 h up to 10 mM initial nitrite. Nitrogen was almost completely assimilated as organic matter (up to 90% using higher nitrite concentrations). The strain tolerates and efficiently assimilates nitrite at concentrations (up to 20 mM) higher than those previously reported in literature for other yeasts. The best growth conditions (50 mM buffer potassium phosphate pH 7, 20 g/L glucose as the sole carbon source, and 10 mM nitrite) were determined. In the perspective of applications in inorganic nitrogen removal, other metabolic features relevant for process optimization were also evaluated, including renewable sources and heavy metal tolerance. Molasses, corn, and soybean oils were good substrates, and cadmium and lead were well tolerated. Scale-up tests also revealed promising features for large-scale applications. Overall, presented results suggest applicability of nitrogen assimilation by Rhodotorula diobovata DSBCA06 as an innovative tool for bioremediation and treatment of wastewater effluents. Full article

Two broad features are jointly necessary for autonomous agency: organisational closure and the embodiment of an objective-function providing a ‘goal’: so far only organisms demonstrate both. Organisational closure has been studied (mostly in abstract), especially as cell autopoiesis and the cybernetic principles of autonomy, but the role of an internalised ‘goal’ and how it is instantiated by cell signalling and the functioning of nervous systems has received less attention. Here I add some biological ‘flesh’ to the cybernetic theory and trace the evolutionary development of step-changes in autonomy: (1) homeostasis of organisationally closed systems; (2) perception-action systems; (3) action selection systems; (4) cognitive systems; (5) memory supporting a self-model able to anticipate and evaluate actions and consequences. Each stage is characterised by the number of nested goal-directed control-loops embodied by the organism, summarised as will-nestedness $\mathcal{N}$. Organism tegument, receptor/transducer system, mechanisms of cellular and whole-organism re-programming and organisational integration, all contribute to causal independence. Conclusion: organisms are cybernetic phenomena whose identity is created by the information structure of the highest level of causal closure (maximum $\mathcal{N}$), which has increased through evolution, leading to increased causal independence, which might be quantifiable by ‘Integrated Information Theory’ measures. Full article

Mitochondrial dysfunction is a hallmark of diabetes, but the metabolic alterations during early stages of the disease remain unknown. The ability of liver cells to rearrange their metabolism plays an important role in compensating the energy shortage and may provide cell survival. Moringa oleifera leaves have been studied for its health properties against diabetes, insulin resistance, and non-alcoholic liver disease. We postulated that M. oleifera executes a protective function on mitochondrial functionality in HepG2 treated with high glucose. We evaluated the effect of high glucose treatment on the mitochondrial function of HepG2 cells using a Seahorse extracellular flux analyzer (Agilent, Santa Clara, CA, USA), blue native polyacrylamide gel electrophoresis (BN-PAGE), and western blot analysis. For assessment of mitochondrial abnormalities, we measured the activity of mitochondrial Complex I and IV as well as uncoupling protein 2, and sirtuin 3 protein contents. Our results demonstrate that, under conditions mimicking the hyperglycemia, Complex I activity, UCP2, Complex III and IV subunits content, supercomplex formation, and acetylation levels are modified with respect to the control condition. However, basal oxygen consumption rate was not affected and mitochondrial reactive oxygen species production remained unchanged in all groups. Treatment of HepG2 cells with M. oleifera extract significantly increased both protein content and mitochondrial complexes activities. Nonetheless, control cells’ respiratory control ratio (RCR) was 4.37 compared to high glucose treated cells’ RCR of 15.3, and glucose plus M. oleifera treated cells’ RCR of 5.2, this indicates high-quality mitochondria and efficient oxidative phosphorylation coupling. Additionally, the state app was not altered between different treatments, suggesting no alteration in respiratory fluxes. These findings enhance understanding of the actions of M. oleifera and suggest that the known antidiabetic property of this plant, at least in part, is mediated through modulating the mitochondrial respiratory chain. Full article

The study of mating choices often focuses on correlates of traits to the overall outcome of a mating interaction. However, mating interactions can proceed through a series of stages, with opportunities for assessment at each stage. We compared whether male or female size predicted mating interaction outcome across several stages of mating in five species of North American leiobunine harvestmen (commonly known as daddy longlegs). Leiobunine harvestmen have been previously shown to exhibit incredible morphological diversity consistent with a spectrum of male–female antagonism. Across all of the species, we found a general progression of female size predicting the outcome (success and timing) of early stages of interactions, and male size or male size relative to female size predicting the outcome and timing of later stages of interactions. We also found that size was not a strong predictor of outcome in the two species on the lower end of the antagonism spectrum. The variation in how female and male size predicted outcomes across species and stages of mating suggests that multiple mechanisms may operate to shape mating dynamics within and across species. Given the close relatedness of the species studied, the patterns we uncovered suggest a rapid evolution of the traits and processes predicting the outcome of mating interactions. Full article

Pathogenesis-related proteins (PRs) are induced in plants after infection by pathogens and/or abiotic stress. Among these proteins, the family 10 (PR-10) influences the biosynthesis of secondary metabolites and shows antimicrobial ribonuclease activity. TcPR-10p (Pathogenesis-related Protein 10 of Theobroma cacao) was isolated from resistant and susceptible Moniliophthora perniciosa cacao cultivars. Cell survival with Saccharomyces cerevisiae mutant lines deficient in ATP-binding cassette (ABC) transporter proteins indicated the influence on resistance to TcPR-10p. Proteins of the ABC transport type are considered important in the process of resistance to antimicrobials and toxins. Thus, the objective of this work was to observe the sensitivity of ABC transporter yeast mutants in the presence of the TcPR-10p. Chronic exposure of S. cerevisiae mitochondrial (BYatm1Δ and BYmdl1Δ) and vacuole (BYnft1Δ, BYvmr1Δ, BYybt1Δ, BYycf1Δ and BYbpt1Δ) ABC transporter mutants to TcPR-10p (3 μg/mL, 0, 6, 12 and 24 h) was performed. Two TcPR-10p sensitive strains (BYmdl1Δ and BYnft1Δ) were submitted to a fluorescence test with the fluorogenic dihydroethidium (DHE), to visualize the presence of oxidative stress in the cells. Oxidative stress-increased sensitivity was confirmed by flow cytometry indicating induced cell death either via apoptosis or necrosis. This yeast data combined with previous data of literature (of M. perniciosa sensitivity to TcPR-10p) show that increased sensitivity to TcPR-10p in these mutants could be due to the TcPR10p-generated higher levels of intracellular reactive oxygen species (ROS), leading to increased cell death either via necrosis or apoptosis. Full article

Carbamyl phosphate (CP) is well-known as an essential intermediate of pyrimidine and arginine/urea biosynthesis. Chemically, CP can be easily synthesized from dihydrogen phosphate and cyanate. Enzymatically, CP can be synthesized using three different classes of enzymes: (1) ATP-grasp fold protein based carbamyl phosphate synthetase (CPS); (2) Amino-acid kinase fold carbamate kinase (CK)-like CPS (anabolic CK or aCK); and (3) Catabolic transcarbamylase. The first class of CPS can be further divided into three different types of CPS as CPS I, CPS II, and CPS III depending on the usage of ammonium or glutamine as its nitrogen source, and whether N-acetyl-glutamate is its essential co-factor. CP can donate its carbamyl group to the amino nitrogen of many important molecules including the most well-known ornithine and aspartate in the arginine/urea and pyrimidine biosynthetic pathways. CP can also donate its carbamyl group to the hydroxyl oxygen of a variety of molecules, particularly in many antibiotic biosynthetic pathways. Transfer of the carbamyl group to the nitrogen group is catalyzed by the anabolic transcarbamylase using a direct attack mechanism, while transfer of the carbamyl group to the oxygen group is catalyzed by a different class of enzymes, CmcH/NodU CTase, using a different mechanism involving a three-step reaction, decomposition of CP to carbamate and phosphate, transfer of the carbamyl group from carbamate to ATP to form carbamyladenylate and pyrophosphate, and transfer of the carbamyl group from carbamyladenylate to the oxygen group of the substrate. CP is also involved in transferring its phosphate group to ADP to generate ATP in the fermentation of many microorganisms. The reaction is catalyzed by carbamate kinase, which may be termed as catabolic CK (cCK) in order to distinguish it from CP generating CK. CP is a thermally labile molecule, easily decomposed into phosphate and cyanate, or phosphate and carbamate depending on the pH of the solution, or the presence of enzyme. Biological systems have developed several mechanisms including channeling between enzymes, increased affinity of CP to enzymes, and keeping CP in a specific conformation to protect CP from decomposition. CP is highly important for our health as both a lack of, or decreased, CP production and CP accumulation results in many disease conditions. Full article

Rab GTPases are essential regulators of many cellular processes and play an important role in downstream signaling vital to proper cell function. We sought to elucidate the role of novel D. discoideum GTPase RabS. Cell lines over-expressing DdRabS and expressing DdRabS N137I (dominant negative (DN)) proteins were generated, and it was determined that DdRabS localized to endosomes, ER-Golgi membranes, and the contractile vacuole system. It appeared to function in vesicular trafficking, and the secretion of lysosomal enzymes. Interestingly, microscopic analysis of GFP-tagged DdRabS (DN) cells showed differential localization to lysosomes and endosomes compared to GFP-tagged DdRabS overexpressing cells. Both cell lines over-secreted lysosomal glycosidase enzymes, especially β-glucosidase. Furthermore, DdRabS overexpressing cells were defective in aggregation due to decreased cell–cell cohesion and sensitivity to cAMP, leading to abnormal chemotactic migration, the inability to complete development, and increased induced cell death. These data support a role for DdRabS in trafficking along the vesicular and biosynthetic pathways. We hypothesize that overexpression of DdRabS may interfere with GTP activation of related proteins essential for normal development resulting in a cascade of defects throughout these processes. Full article

One particular class of Transposable Elements (TEs), called Long Terminal Repeats (LTRs), retrotransposons, comprises the most abundant mobile elements in plant genomes. Their copy number can vary from several hundreds to up to a few million copies per genome, deeply affecting genome organization and function. The detailed classification of LTR retrotransposons is an essential step to precisely understand their effect at the genome level, but remains challenging in large-sized genomes, requiring the use of optimized bioinformatics tools that can take advantage of supercomputers. Here, we propose a new tool: Inpactor, a parallel and scalable pipeline designed to classify LTR retrotransposons, to identify autonomous and non-autonomous elements, to perform RT-based phylogenetic trees and to analyze their insertion times using High Performance Computing (HPC) techniques. Inpactor was tested on the classification and annotation of LTR retrotransposons in pineapple, a recently-sequenced genome. The pineapple genome assembly comprises 44% of transposable elements, of which 23% were classified as LTR retrotransposons. Exceptionally, 16.4% of the pineapple genome assembly corresponded to only one lineage of the Gypsy superfamily: Del, suggesting that this particular lineage has undergone a significant increase in its copy numbers. As demonstrated for the pineapple genome, Inpactor provides comprehensive data of LTR retrotransposons’ classification and dynamics, allowing a fine understanding of their contribution to genome structure and evolution. Inpactor is available at https://github.com/simonorozcoarias/Inpactor. Full article

Deletions and mutations involving the Retinoic Acid Induced 1 (RAI1) gene at 17p11.2 cause Smith-Magenis syndrome (SMS). Here we report a patient with autism as the main clinical presentation, with some SMS-like features and a rare de novo RAI1 gene mutation, c.3440G > A (p.R1147Q). We functionally characterized the RAI1 p.R1147Q mutant protein. The mutation, located near the nuclear localization signal, had no effect on the subcellular localization of the mutant protein. However, similar to previously reported RAI1 missense mutations in SMS patients, the RAI1 p.R1147Q mutant protein showed a significant deficiency in activating in vivo transcription of a reporter gene driven by a BDNF (brain-derived neurotrophic factor) intronic enhancer. In addition, expression of other genes associated with neurobehavioral abnormalities and/or neurodevelopmental disorders were found to be altered in this patient. These results suggest a likely contribution of RAI1, either alone or in combination of other factors, to social behavior and reinforce the RAI1 gene as a candidate gene in patients with autistic manifestations or social behavioral abnormalities. Full article

Periplasmic ligand-binding proteins (PBPs) bind ligands with a high affinity and specificity. They undergo a large conformational change upon ligand binding, and they have a robust protein fold. These physical features have made them ideal candidates for use in protein engineering projects to develop novel biosensors and signaling molecules. The Escherichia coli MppA (murein peptide permease A) PBP binds the murein tripeptide, l-alanyl-γ-d-glutamyl-meso-diaminopimelate, (l-Ala-γ-d-Glu-meso-Dap), which contains both a D-amino acid and a gamma linkage between two of the amino acids. We have solved a high-resolution X-ray crystal structure of E. coli MppA at 1.5 Å resolution in the unliganded, open conformation. Now, structures are available for this member of the PBP protein family in both the liganded/closed form and the unliganded/open form. Full article

Chlamydomonas reinhardtii (Chlamydomonas) strains that are toxic to mosquito larvae because they express chloroplast transgenes that are based on the mosquitocidal proteins of Bacillus thuringiensis subsp. israelensis (Bti) could be very useful in mosquito control. Chlamydomonas has several advantages for this approach, including genetic controls not generally available with industrial algae. The Bti toxin is produced by sporulating bacteria and has been used for mosquito control for >30 years without creating highly resistant mosquito populations. The suite of toxins is four main proteins: three Cry proteins and the cytotoxic Cyt1Aa (27 kDa). Cyt1Aa is not very toxic to mosquitoes by itself, but it prevents the development of resistance. The production of Cyt1Aa in other microbes, however, has been challenging due to its affinity for certain membrane phospholipids. Here we report on the production of recombinant Cyt1Aa (rCyt1A) in the chloroplast of photosynthetic Chlamydomonas at levels of at least 0.3% total protein. Live cell bioassays demonstrated toxicity of the rCyt1Aa Chlamydomonas to larvae of Aedes aegypti. We also expressed the chloroplast cyt1Aa gene in a wild-type Chlamydomonas strain (21 gr) that can grow on nitrate. These results have implications for developing a Chlamydomonas strain that will be toxic to mosquito larvae but will not induce strongly resistant populations. Full article

Life is based on two aspects: matter and a non-material, electrical component. In a dynamic system of reciprocal causality, matter and the so-called bioelectricity interact with one another, forming a functional unity. The aim of this essay is to summarize evidence for bioelectricity, for the sensitivity of biosystems to external physical factors and for the interactions of internal bioelectricity with internal biochemical structures. I propose non-material information of bioelectrical states to be just as inheritable from generation to generation as is the material genetic code. Full article