Monoclonal C5-1 antibody produced in transgenic alfalfa plants exhibits a N-glycosylation that is homogenous and suitable for glyco-engineering into human-compatible structures

Structural analysis of the N-glycosylation of alfalfa proteins was investigated in order to evaluate the capacity of this plant to perform this biologically important post-translational modification. We show that, in alfalfa, N-linked glycans are processed into a large variety of mature oligosaccharides having core-xylose and core alpha(1,3)-fucose, as well as terminal Lewis(a) epitopes. In contrast, expression of the C5-1 monoclonal antibody in alfalfa plants results in the production of plant-derived IgG1 which is N-glycosylated by a predominant glycan having a alpha(1,3)-fucose and a beta(1,2)-xylose attached to a GlcNAc2Man3GlcNAc2 core. Since this core is common to plant and mammal N-linked glycans, it therefore appears that alfalfa plants have the ability to produce recombinant IgG1 having a N-glycosylation that is suitable for in vitro or in vivo glycan remodelling into a human-compatible plantibody. For instance, as proof of concept, in vitro galactosylation of the alfalfa-derived C5-1 mAb resulted in a homogenous plantibody harbouring terminal beta(1,4)-galactose residues as observed in the mammalian IgG.     

Dynamics of α-Hb chain binding to its chaperone AHSP depends on heme coordination and redox state

Background

AHSP is an erythroid molecular chaperone of the α-hemoglobin chains (α-Hb). Upon AHSP binding, native ferric α-Hb undergoes an unprecedented structural rearrangement at the heme site giving rise to a 6th coordination bond with His(E7).

Methods

Recombinant AHSP, WT α-Hb:AHSP and α-Hb^HE7Q:AHSP complexes were expressed in Escherichia coli. Thermal denaturation curves were measured by circular dichroism for the isolated α-Hb and bound to AHSP. Kinetics of ligand binding and redox reactions of α-Hb bound to AHSP as well as α-Hb release from the α-Hb:AHSP complex were measured by time-resolved absorption spectroscopy.

Results

AHSP binding to α-Hb is kinetically controlled to prevail over direct binding with β-chains and is also thermodynamically controlled by the α-Hb redox state and not the liganded state of the ferrous α-Hb. The dramatic instability of isolated ferric α-Hb is greatly decreased upon AHSP binding. Removing the bis-histidyl hexacoordination in α-HbH58(E7)Q:AHSP complex reduces the stabilizing effect of AHSP binding. Once the ferric α-Hb is bound to AHSP, the globin can be more easily reduced by several chemical and enzymatic systems compared to α-Hb within the Hb-tetramer.

Conclusion

α-Hb reduction could trigger its release from AHSP toward its final Hb β-chain partner producing functional ferrous Hb-tetramers. This work indicates a preferred kinetic pathway for Hb-synthesis.

General significance

The cellular redox balance in Hb-synthesis should be considered as important as the relative proportional synthesis of both Hb-subunits and their heme cofactor. The in vivo role of AHSP is discussed in the context of the molecular disorders observed in thalassemia.     

Relationship between Spectral Characteristics of Spontaneous Postural Sway and Motion Sickness Susceptibility

Motion sickness (MS) usually occurs for a narrow band of frequencies of the imposed oscillation. It happens that this frequency band is close to that which are spontaneously produced by postural sway during natural stance. This study examined the relationship between reported susceptibility to motion sickness and postural control. The hypothesis is that the level of MS can be inferred from the shape of the Power Spectral Density (PSD) profile of spontaneous sway, as measured by the displacement of the center of mass during stationary, upright stance. In Experiment 1, postural fluctuations while standing quietly were related to MS history for inertial motion. In Experiment 2, postural stability measures registered before the onset of a visual roll movement were related to MS symptoms following the visual stimulation. Study of spectral characteristics in postural control showed differences in the distribution of energy along the power spectrum of the antero-posterior sway signal. Participants with MS history provoked by exposure to inertial motion showed a stronger contribution of the high frequency components of the sway signal. When MS was visually triggered, sick participants showed more postural sway in the low frequency range. The results suggest that subject-specific PSD details may be a predictor of the MS level. Furthermore, the analysis of the sway frequency spectrum provided insight into the intersubject differences in the use of postural control subsystems. The relationship observed between MS susceptibility and spontaneous posture is discussed in terms of postural sensory weighting and in relation to the nature of the provocative stimulus.     

Massive Diversification in Aging Colonies of Escherichia coli

The evolutionary success of bacteria depends greatly on their capacity to continually generate phenotypic diversity. Structured environments are particularly favorable for diversification because of attenuated clonal interference, which renders selective sweeps nearly impossible and enhances opportunities for adaptive radiation. We examined at the microscale level the emergence and the spatial and temporal dynamics of phenotypic diversity and their underlying causes in Escherichia coli colonies. An important dynamic heterogeneity in the growth, metabolic activity, morphology, gene expression patterns, stress response induction, and death patterns among cells within colonies was observed. Genetic analysis indicated that the phenotypic variation resulted mostly from mutations and that indole production, oxidative stress, and the RpoS-regulated general stress response played an important role in the generation of diversity. We observed the emergence and persistence of phenotypic variants within single colonies that exhibited variable fitness compared to the parental strain. Some variants showed improved capacity to produce biofilms, whereas others were able to use different nutrients or to tolerate antibiotics or oxidative stress. Taken together, our data show that bacterial colonies provide an ecological opportunity for the generation and maintenance of vast phenotypic diversity, which may increase the probability of population survival in unpredictable environments.     

Studying sources of incongruence in arthropod molecular phylogenies: Sea spiders (Pycnogonida) as a case study

In this report, we analyze the phylogeny of Pycnogonida using the three nuclear and three mitochondrial markers currently sequenced for studying inter- and intrafamilial relationships within Arthropoda: 18S and 28S rRNA genes, Histone H3, cytochrome c oxidase subunit 1 (CO1), 12S and 16S rRNA genes. We identify several problems in previous studies, due to the use of inappropriate sequences (taxonomic misidentification, DNA contamination, sequencing errors, missing data) or taxa (outgroup choice). Our analyses show that most markers are not powerful to study the phylogeny of sea spiders. The results suggest however a recent diversification of the group (Mesozoic rather than Paleozoic) and the early divergence of Austrodecidae, followed by Colossendeidae, Pycnogonidae and Rhynchothoracidae. Except Ammotheidae and Callipallenidae, all other families were recovered as monophyletic. Analyses of synonymous sites in CO1 sequences reveal an extreme heterogeneity of nucleotide composition within sea spiders, as six unrelated species show a reverse strand-specific bias. We therefore suggest that several independent reversals of asymmetric mutational constraints occurred during the evolution of Pycnogonida, as a consequence of genomic inversions involving either the control region or a fragment containing the CO1 gene. These hypotheses are supported by the comparison of two complete mitochondrial genomes of sea spiders (Achelia bituberculata and Nymphon gracile) with that of Limulus.     

The phylogenetic position of the 'living fossils' Neoglyphea and Laurentaeglyphea (Decapoda: Glypheidea)

The Glypheidea is a group of lobster-like decapods that appeared in the Triassic and that was thought to be extinct until 1975, when a specimen of the species Neoglyphea inopinata was caught off the Philippines. More recently, in 2005, a specimen of another glypheid species, Laurentaeglyphea neocaledonica, was discovered near New Caledonia. Here, we construct a decapod molecular data set including the two extant glypheid species sequenced from eight nuclear and mitochondrial genes. Our study strongly shows that the two extant genera of glypheids cluster together, and further confirms the status of Glypheidea as a separate infraorder. Moreover the reptantian decapods are divided into two major groups, one including Brachyura, Anomura, and Axiidea, and the other including Astacidea, Polychelida, Achelata, and Glypheidea. Although commonly nicknamed 'Jurassic shrimps' and considered as 'living fossils', glypheids are therefore a derived decapod lineage.     

Blindsnake evolutionary tree reveals long history on Gondwana

Worm-like snakes (scolecophidians) are small, burrowing species with reduced vision. Although largely neglected in vertebrate research, knowledge of their biogeographical history is crucial for evaluating hypotheses of snake origins. We constructed a molecular dataset for scolecophidians with detailed sampling within the largest family, Typhlopidae (blindsnakes). Our results demonstrate that scolecophidians have had a long Gondwanan history, and that their initial diversification followed a vicariant event: the separation of East and West Gondwana approximately 150 Ma. We find that the earliest blindsnake lineages, representing two new families described here, were distributed on the palaeolandmass of India+Madagascar named here as Indigascar. Their later evolution out of Indigascar involved vicariance and several oceanic dispersal events, including a westward transatlantic one, unexpected for burrowing animals. The exceptional diversification of scolecophidians in the Cenozoic was probably linked to a parallel radiation of prey (ants and termites) as well as increased isolation of populations facilitated by their fossorial habits.     

The Extracellular A-loop of Dual Oxidases Affects the Specificity of Reactive Oxygen Species Release

NADPH oxidase (Nox) family proteins produce superoxide (O₂^⨪) directly by transferring an electron to molecular oxygen. Dual oxidases (Duoxes) also produce an O₂^⨪ intermediate, although the final species secreted by mature Duoxes is H₂O₂, suggesting that intramolecular O₂^⨪ dismutation or other mechanisms contribute to H₂O₂ release. We explored the structural determinants affecting reactive oxygen species formation by Duox enzymes. Duox2 showed O₂^⨪ leakage when mismatched with Duox activator 1 (DuoxA1). Duox2 released O₂^⨪ even in correctly matched combinations, including Duox2 + DuoxA2 and Duox2 + N-terminally tagged DuoxA2 regardless of the type or number of tags. Conversely, Duox1 did not release O₂^⨪ in any combination. Chimeric Duox2 possessing the A-loop of Duox1 showed no O₂^⨪ leakage; chimeric Duox1 possessing the A-loop of Duox2 released O₂^⨪. Moreover, Duox2 proteins possessing the A-loops of Nox1 or Nox5 co-expressed with DuoxA2 showed enhanced O₂^⨪ release, and Duox1 proteins possessing the A-loops of Nox1 or Nox5 co-expressed with DuoxA1 acquired O₂^⨪ leakage. Although we identified Duox1 A-loop residues (His¹⁰⁷¹, His¹⁰⁷², and Gly¹⁰⁷⁴) important for reducing O₂^⨪ release, mutations of these residues to those of Duox2 failed to convert Duox1 to an O₂^⨪-releasing enzyme. Using immunoprecipitation and endoglycosidase H sensitivity assays, we found that the A-loop of Duoxes binds to DuoxA N termini, creating more stable, mature Duox-DuoxA complexes. In conclusion, the A-loops of both Duoxes support H₂O₂ production through interaction with corresponding activators, but complex formation between the Duox1 A-loop and DuoxA1 results in tighter control of H₂O₂ release by the enzyme complex.     

The plant endosphere world – bacterial life within plants

The plant endosphere is colonized by complex microbial communities and microorganisms, which colonize the plant interior at least part of their lifetime and are termed endophytes. Their functions range from mutualism to pathogenicity. All plant organs and tissues are generally colonized by bacterial endophytes and their diversity and composition depend on the plant, the plant organ and its physiological conditions, the plant growth stage as well as on the environment. Plant-associated microorganisms, and in particular endophytes, have lately received high attention, because of the increasing awareness of the importance of host-associated microbiota for the functioning and performance of their host. Some endophyte functions are known from mostly lab assays, genome prediction and few metagenome analyses; however, we have limited understanding on in planta activities, particularly considering the diversity of micro-environments and the dynamics of conditions. In our review, we present recent findings on endosphere environments, their physiological conditions and endophyte colonization. Furthermore, we discuss microbial functions, the interaction between endophytes and plants as well as methodological limitations of endophyte research. We also provide an outlook on needs of future research to improve our understanding on the role of microbiota colonizing the endosphere on plant traits and ecosystem functioning.     

FreshOmics: A manually curated and standardized –omics database for investigating freshwater microbiomes

Freshwater is a critical resource for human survival but severely threatened by anthropogenic activities and climate change. These changes strongly impact the abundance and diversity of the microbial communities which are key players in the functioning of these aquatic ecosystems. Although widely documented since the emergence of high-throughput sequencing approaches, the information on these natural microbial communities is scattered among thousands of publications and it is therefore difficult to investigate the temporal dynamics and the spatial distribution of microbial taxa within or across ecosystems. To fill this gap and in the FAIR principles context we built a manually curated and standardized microbial freshwater –omics database (FreshOmics). Based on recognized ontologies (ENVO, MIMICS, GO, ISO), FreshOmics describes 29 different types of freshwater ecosystems and uses standardized attributes to depict biological samples, sequencing protocols and article attributes for more than 2487 geographical locations across 71 countries around the world. The database contains 24,808 sequence identifiers (i.e., Run_Id / Exp_ID, mainly from SRA/DDBJ SRA/ENA, GSA and MG-RAST repositories) covering all sequence-based -omics approaches used to investigate bacteria, archaea, microbial eukaryotes, and viruses. Therefore, FreshOmics allows accurate and comprehensive analyses of microbial communities to answer questions related to their roles in freshwater ecosystems functioning and resilience, especially through meta-analysis studies. This collection also highlights different sort of errors in published works (e.g., wrong coordinates, sample type, material, spelling).