Background/Objective(s) Evidence suggests ocrelizumab (OCR) breastmilk transfer is minimal, although little is known on the pharmacodynamics (PD) in breastfed infants. SOPRANINO (NCT04998851) is the first prospective study measuring both PD and pharmacokinetic breastmilk parameters alongside infant humoral response to vaccines, safety, growth and development. Material(s) and Method(s) Women with multiple sclerosis (MS) who gave birth to one healthy-term infant and decided to start/ resume OCR at 2–24 weeks postpartum while partially or exclusively breastfeeding were enrolled. Key exclusion criteria were potential OCR foetal exposure and anomalies interfering with breastfeeding. Co-primary outcomes were proportion of infants with B-cell levels below the lower limit of normal at 30 days post-infusion and OCR average daily infant dose (ADID) over 60 days post-infusion. Result(s) Thirteen women with MS and their infants were enrolled (median [range] infant age at maternal infusion [months]: 2.0 [0.5–5.0]). OCR levels in breastmilk were negligible (median ADID: 45.09 µg; median relative infant dose: 0.27%) and OCR was undetectable in infant serum (median [range]: 0.0 µg/ml [0.0–0.0]). All infant B-cell levels were within age-specific normal ranges (figure). Eleven infants (84.6%) had ≥1 adverse event (AE), of which 10 experienced ≥1 infancy infection. Most infant AEs were mild to moderate (Grade 1 or 2), with one severe (Grade 3) bronchiolitis reported that resolved. No serious AEs occurred. Conclusion(s) Our results show negligible transfer of OCR into breastmilk, undetectable OCR levels in infant serum as well as normal infant B-cell levels and expected infant health, indicating that breastfeeding is compatible with OCR.

Background & AimRecombinant full-length human laminins, Biolaminin, can improve the hPSC culture at different scales, from microtiter plates for gene editing, to upscaling with hollow-fibers and microcarriers. Due to high cellular yield and attachment, 34% savings in matrix and material costs could be achievedThe cell culture environment must be carefully designed to mimic the natural cell niche, including relevant medium and substrate, to achieve standardized, reproducible, and biorelevant cells. The development of recombinant and scalable protein matrices for efficient expansion and differentiation of hPSC has been a milestone for emerging cell therapies. Laminins are an extracellular matrix (ECM) protein family compassed of 16 different isoforms and essential component of the basement membranes through the human body, vital for tissue development and homeostasis.The extracellular matrix (ECM) protein, laminin-521 is the major ECM protein expressed in the inner cell mass of the pre-implanted embryo and, therefore the most biologically relevant matrix for hiPSCs and hESCs, directly involved in their survival and self-renewal, independent of platform size or material.Methods, Results & ConclusionIn microtiter plates, Biolaminin 521 has been proven to be an excellent substrate for gene editing, automated workflows, and imaging analysis. High-content image analysis system assessed the best hiPSC confluence and single-cell cloning efficiency on LN521, compared to other ECMs. For CRISPR/Cas9, Biolaminin 521 coating supported single hPSC seeding at a very low density. For imaging on glass surfaces, the purity and adhesive properties of Biolaminin 521 proved to be a major advantage.In large-scale expansion systems for PSCs, Biolaminin 521 coating significantly increased the cell numbers and population doublings, on both porous hollow fibers and microcarriers. By providing survival, proliferation, and migration signals, Biolaminin 521 coating resulted in a 7.5-fold increase in cell expansion with 90% viability for PSCs. This had a direct effect on improved attachment (87%) and surface area usage (85% spreading). Thus cutting the overall matrix and material costs by 34% due to the increased culture efficiency.In conclusion, by re-creating the natural cell niche, with recombinant full-length human laminins in vitro, human ESC and iPSC culture can be supported from clonal stages to stable long-time expansion, independent of the platform or level of automation.

Origin of earliest land plants from ancestral algae dramatically accelerated the evolution of Earth's terrestrial ecosystems, in which microbial symbioses have played key roles. Recent molecular diversification analyses identify the rare, geographically-limited moss Takakia as Earth's most archaic modern land plant. Despite occupying a phylogenetic position pivotal for understanding earliest plants, Takakia microbial associations are poorly known. Here, we describe symbiosis-related structural features and contig-based metagenomic data that illuminate the evolutionary transition from streptophyte algae to early embryophytes. We observed that T. lepidozioides shares with streptophyte algae secretion of microbe-harboring mucilage and bacterial taxa such as Rhizobium and genes indicating nitrogen fixation. We find that Takakia root-analogs produce lateral mucilage organs that are more complex than generally understood, having structural analogies to angiosperm lateral roots adapted for N-fixation symbioses, including presence of intracellular microbes. We also find structural and metagenomic evidence for mycorrhiza-like species of glomalean fungi (including Rhizophagus irregularis) not previously known for mosses, as well as ascomycete fungi (e.g. Rhizoscyphus ericae) that associate with other early-diverging plants. Because Takakia is the oldest known modern plant genus, this study of plants of a remote locale not strongly influenced by human activities may indicate microbiome features of early land plants.

Invasive pollinators can disrupt native pollination mutualisms. We investigated the impact of the invasion of the European bumble bee Bombus terrestris in NW Patagonia, Argentina, on the pollination mutualism between the native legume Vicia nigricans and its main pollinator, the native bumble bee B. dahlbomii, and its consequences on plant reproduction. We assessed visitation frequency by both bumble bees and reproductive output of V. nigricans across space (in 12 sites) and time (over five seasons, from 1999 to 2012), before and after invasion in 2006. In 2012, we studied the behavior, visitation frequency, and nectar consumption by both bumble bees, the nectar consumed, the quantity and quality of pollen deposited and the reproductive output of V. nigricans in a subset of five sites across a gradient of B. terrestris invasion. Six years after invasion, B. terrestris became the most frequent visitor and nectar robber of V. nigricans flowers, while visits by B. dahlbomii dropped by 50%, fruit set by 43% and seeds per fruit by 32%. This detrimental effect on plant reproduction resulted from the high frequency of nectar robbing by B. terrestris, the nectar depletion, and the concomitant reduction of legitimate visits by the native bumble bee, which in turn lead to a decreased deposition of high quality pollen. Our study demonstrates that biological invasions can alter ecological interactions by replacing a plant-pollinator mutualism by a plant-nectar robber antagonism, hence disrupting plant reproduction and undermining long-term population viability. This conclusion raises the urgent need to regulate the international trade of non-native pollinators.

The modern pteridophyte genus Equisetum is the only survivor of Sphenopsida, an ancient clade known from the Devonian. This genus, of nearly worldwide distribution, comprises approximately 15 extant species. However, genomic information is limited. In this study, we assembled the complete chloroplast genome of the giant species Equisetum xylochaetum from a metagenomic sequence and compared the plastid genome structure and protein-coding regions with information available for two other Equisetum species using network analysis. Equisetum chloroplast genomes showed conserved traits of quadripartite structure, gene content, and gene order. Phylogenetic analysis based on plastome protein-coding regions corroborated previous reports that Equisetum is monophyletic, and that E. xylochaetum is more closely related to E. hyemale than to E. arvense. Single-gene phylogenetic estimation and haplotype analysis showed that E. xylochaetum belonged to the subgenus Hippochaete. Single-gene haplotype analysis revealed that E. arvense, E. hyemale, E. myriochaetum, and E. variegatum resolved more than one haplotype per species, suggesting the presence of a high diversity or a high mutation rate of the corresponding nucleotide sequence. Sequences from E. bogotense appeared as a distinct group of haplotypes representing the subgenus Paramochaete that diverged from Hippochaete and Equisetum. In addition, the taxa that were frequently located at the joint region of the map were E. scirpoides and E. pratense, suggesting the presence of some plastome characters among the Equiseum subgenera.

Abstract Background and Aims Canyon stream beds in the hyperarid Atacama Desert surprisingly harbour magnificent groves of endemic giant horsetail wetland plants, Equisetum xylochaetum. Our previous metagenomic study of eukaryotes closely associated with this plant indicated that the microbiome included prokaryotes that might likewise influence host success and environment. We explored this possibility by using the metagenomic sequence to characterize prokaryote taxa and functional genes present in the microbiome of E. xylochaetum sampled from remote sites differing in the degree of anthropogenic disturbance. We focused on biogeochemical functions known to be important in wetland ecosystems. Methods To ensure that analyses were conducted on microbes most closely associated with plants, we extracted DNA from well-washed plant organs whose microbial biofilms were revealed with scanning electron microscopy. To assess the benefits of longer sequences for taxonomic and gene classifications, results of analyses performed using contigs were compared with those obtained with unassembled reads. We employed methods widely used to estimate genomic coverage of single taxa for genomic analysis to infer relative abundances of taxa and functional genes. Key Results Key functional bacterial genera (e.g. Hydrogenophaga, Sulfuritalea and Rhodoferax) inferred from taxonomic and functional gene analysis of contigs – but not unassembled reads – to occur on surfaces of (or within) plants at relatively high abundance (>50× genomic coverage) indicated roles in nitrogen, sulfur and other mineral cycling processes. Comparison between sites revealed impacts on biogeochemical functions, e.g. reduced levels of the nifH gene marker under disturbance. Vanadium nitrogenases were more important than molybdenum nitrogenases, indicated by both functional genes and taxa such as Rhodomicrobium and Phaeospirillum inferred from contigs but not unassembled reads. Conclusions Our contig-based metagenomic analyses revealed that microbes performing key wetland biogeochemical functions occur as tightly adherent biofilms on the plant body, not just in water or sediments, and that disturbance reduces such functions, providing arguments for conservation efforts.

Abstract. Understanding features that fostered the persistence of Equisetum–Earth's oldest extant vascular plant genus–since Mesozoic times and through episodes of significant global environmental change, is of current interest in view of modern challenges to plant survival. In addition to known structural and physiological adaptations, we hypothesized that microscopy and shotgun metagenomic sequencing might reveal eukaryotic microorganisms such as fungi that may aid Equisetum survival. Here, we report evidence for several lineages of eukaryotic microbes associated with giant Equisetum xylochaetum, which dominates vegetation in saline streambeds of remote valleys in the hyper-arid Atacama Desert, Chile. Plant material was collected and field-preserved at two comparatively low-disturbance sites; DNA extracted in Chile using low-shear methods was later sequenced, 18S and 28S rDNA taxonomic marker sequences were selected for SILVAngs classification, allowing comparisons to eukaryotic microorganisms previously inferred for earlier-diverging plant lineages. SEM, fluorescence microscopy, and/or LM of toluidine blue-stained sections of roots indicated protists, epiphytic and endophytic fungi, and cortical nematodes. Eukaryotic genera inferred from 18S rDNA at >100X mean sequencing depth included the ciliate Engelmanniella, hyphal chytrid Monoblepharella, predatory ascomycete Cephaliophora, a salpingoecid choanoflagellate, and an annelid worm. 23S rDNA sequences indicated ascomycete Capnodiales fungi at one site and four types of Pezizomycotina fungi at the other. No evidence for vesicular-arbuscular mycorrhizal fungi was found, but we hypothesized that Equisetum may benefit from other types of fungal associations, some possibly inherited from ancestral plant lineages.

Cladophora is an algal genus known to be ecologically important. It provides habitats for microorganisms known to provide ecological services such as biosynthesis of cobalamin (vitamin B12) and nutrient cycling. Most knowledge of microbiomes was obtained from studies of lacustrine Cladophora species. However, whether lotic freshwater Cladophora microbiomes are as complex as the lentic ones or provide similar ecological services is not known. To illuminate these issues, we used amplicons of 16S rDNA, 18S rDNA, and ITS to investigate the taxonomy and diversity of the microorganisms associated with replicate Cladophora samples from three sites along the Nan River, Thailand. Results showed that the diversity of prokaryotic and eukaryotic members of Cladophora microbiomes collected from different sampling sites was statistically different. Fifty percent of the identifiable taxa were shared across sampling sites: these included organisms belonging to different trophic levels, decomposers, and heterotrophic bacteria. These heterogeneous assemblages of bacteria, by functional inference, have the potential to perform various ecological functions, i.e., cellulose degradation, cobalamin biosynthesis, fermentative hydrogen production, ammonium oxidation, amino acid fermentation, dissimilatory reduction of nitrate to ammonium, nitrite reduction, nitrate reduction, sulfur reduction, polyphosphate accumulation, denitrifying phosphorus-accumulation, and degradation of aromatic compounds. Results suggested that river populations of Cladophora provide ecologically important habitat for microorganisms that are key to nutrient cycling in lotic ecosystems.

We report the first New Zealand case of Anncaliia algerae myositis in a 55-year-old man with a history of psoriatic arthritis, treated with long-term immunosuppressive therapy. He resided in the city of Rotorua, which is famous for geothermal hot springs. A vastus lateralis muscle biopsy was performed to investigate the cause of an unexplained myositis. Light microscopy demonstrated a necrotizing myositis with scattered clusters of ovoid spores within the myocyte cytoplasm resembling microsporidia. DNA analysis by PCR and electron microscopy confirmed microsporidial myositis with features characteristic of A. algerae. Immunosuppressive drugs were stopped and the patient was treated with cholestyramine wash and albendazole. The patient deteriorated with involvement of bulbar and respiratory muscles requiring intensive care and ventilation. He died 3 weeks after diagnosis. Post-mortem examination of skeletal muscle from tongue and intercostal muscles also revealed numerous organisms confirming disseminated disease. (c) 2021 Elsevier B.V. All rights reserved.

Palynological investigations of the upper Emsian rocks from the Bukowa Góra Quarry (Holy Cross Mountains; Poland) revealed an organic fraction classified as remains of filamentous algae. Individual cells were arranged linearly and contacted each other. Specimens occurred as short filaments having no branches. Cell walls appeared single-layered, very thin and translucent. Cell surfaces were sometimes covered with very delicate striations, possibly generated by compressional folding. A comparison of the main features of the described taxa with extant algae indicated closest relationship with the chlorophycean green algal orders Ulotrichales and Cladophorales. Representatives of these orders occupy mainly freshwater environments. Palynofacies investigation revealed high content of land origin palynomorphs with complete lack of marine components. The presence of such delicate organisms as algae in sediments might indicate very fast deposition in a proximal, very near-shore, possibly shallow water/lagoonal or alluvial environment.

Prokaryotic Nostoc, one of the world's most conspicuous and widespread algal genera (similar to eukaryotic algae, plants, and animals) is known to support a microbiome that influences host ecological roles. Past taxonomic characterizations of surface microbiota (epimicrobiota) of free‐living Nostoc sampled from freshwater systems employed 16S rRNA genes, typically amplicons. We compared taxa identified from 16S, 18S, 23S, and 28S rRNA gene sequences filtered from shotgun metagenomic sequence and used microscopy to illuminate epimicrobiota diversity for Nostoc sampled from a wetland in the northern Chilean Altiplano. Phylogenetic analysis and rRNA gene sequence abundance estimates indicated that the host was related to Nostoc punctiforme PCC 73102. Epimicrobiota were inferred to include 18 epicyanobacterial genera or uncultured taxa, six epieukaryotic algal genera, and 66 anoxygenic bacterial genera, all having average genomic coverage ≥90X. The epicyanobacteria Geitlerinemia, Oscillatoria, Phormidium, and an uncultured taxon were detected only by 16S rRNA gene; Gloeobacter and Pseudanabaena were detected using 16S and 23S; and Phormididesmis, Neosynechococcus, Symphothece, Aphanizomenon, Nodularia, Spirulina, Nodosilinea, Synechococcus, Cyanobium, and Anabaena (the latter corroborated by microscopy), plus two uncultured cyanobacterial taxa (JSC12, O77) were detected only by 23S rRNA gene sequences. Three chlamydomonad and two heterotrophic stramenopiles genera were inferred from 18S; the streptophyte green alga Chaetosphaeridium globosum was detected by microscopy and 28S rRNA genes, but not 18S rRNA genes. Overall, >60% of epimicrobial taxa were detected by markers other than 16S rRNA genes. Some algal taxa observed microscopically were not detected from sequence data. Results indicate that multiple taxonomic markers derived from metagenomic sequence data and microscopy increase epimicrobiota detection.

Cultivation of the filamentous chlorophyte Oedogonium in municipal wastewater effluent is known to improve water quality and yield lipid- and protein-rich biomass for industrial applications. Chlorophyte celluloses, whose molecular organization and physical traits differ from those of plants, represent yet another valuable extractive, and algal oxygen production is of economic value in wastewater treatment. Consequently, we explored cellulose and oxygen production from Oedogonium biomass batch-cultivated in treated secondary municipal wastewater effluent. We compared biomass, cellulose, and oxygen production outside and within an adjacent greenhouse, under differing dissolved CO2 and pH conditions, and during temperate-zone seasonal change from summer through fall. Overall production did not differ within or outside the greenhouse, but outside production was higher in summer and lower in fall as air temperatures declined. Batch cultivation offered advantages, but high levels of mixing and CO2 were essential to maintain neutral pH for optimal algal growth and oxygen production.

Local isolates of the species-rich, fast-growing chlorophyte genusOedogoniumhave been employed in outdoor biomass cultivation for industrial applications such as wastewater remediation, but such isolates may not express structural traits needed for definitive species identification by microscopy alone. We used maximum likelihood and Bayesian phylogenetic methods to evaluate molecular marker gene sequences derived from genomic sequences to characterise an industrial isolate,Oedogoniumsp. Lake Mendota strain, which could not be reliably classified at species level using only morphology. Molecular markers evaluated wererbcL, 23S rDNA, 28S rDNA, 18S rDNA, and a sequence that included ITS1+5.8S rDNA+ITS2. All five markers distinguished the Lake Mendota strain from otherOedogoniumaccessions in GenBank. Using full genome sequences allowed the efficient acquisition of multiple types of marker sequences whose sequencing depth was known. In the absence of a classical species determination, these markers provided a taxonomic reference background for future genomic, biochemical, and other investigations of the local isolate. Local isolates are advantageous for outdoor industrial biomass cultivation applications because such isolates are already adapted to climatic conditions prevailing at cultivation sites. The results should encourage industrial use of local isolates ofOedogoniumbecause these can be characterised by molecular markers even if structural features needed for accurate species determinations are not expressed.

A relatively neglected element of the biota of the Lower Devonian Rhynie chert Lagerstätte are filamentous green algae exceptionally preserved by silicification. Palynological processing of sediments associated with the cherts has yielded palynomorphs that we also interpret as the remains of filamentous green algae and one such taxon is described herein. Cells occur individually, in masses or joined end-to-end as an unbranched filament. The cells are characterised by end walls that form a ‘collar’ structure and inner bodies interpreted as reproductive structures. Because of a lack of preserved characters taxonomic precision is limited, although we suggest the fossils are most likely either zygnematalean or oedogonialean algae that inhabited ponds or lakes and were either attached to substrates and/or free-floating.

Premise of research.Recent high-throughput molecular analyses indicate that lichen consortia include bacterial communities, but these are not always characterized at the generic taxonomic level, a process that can indicate microbial functions. Further, the eukaryotic components of lichens and spatial relationships among lichen microbiota are incompletely understood. To provide such information, we acquired new shotgun metagenomic sequence data for use with microscopy to focus on prokaryotic and eukaryotic genera of a Peltigera ponojensis microbiome.Methodology.16S, 23S, 18S, and 28S rDNA sequences filtered from deep shotgun metagenomic Illumina and long-read Roche 454 metagenomic sequence data were employed with the SILVAngs analytic pipeline to infer generic diversity, and lichen microbiota were imaged with light and scanning electron microscopy.Pivotal results.Correlative microscopy indicated the presence of morphologically diverse microbes on upper and lower lichen surfaces, and putative bacteria were observed within the photobiont zone. Sphingomonas, Methylobacterium, and Nostoc were the most abundant of similar to 50 relatively abundant bacterial genera, those inferred to occur in 100 16S and/or 23S rDNA reads. More than 140 additional prokaryotic genera were detected in 10 reads. Sequences classifying with eukaryotic representatives of Rhizaria, Amoebozoa, Alveolata, Metazoa, and Viridiplantae were detected in 10 18S and/or 28S rDNA reads. The most abundant protist was the rhizarian Protaspa (=Protaspis), indicated by >100 28S rDNA reads. More than 9000 18S rDNA reads classified with Peltigera and >1000 with Solorina; 10 additional ascomycete genera and two basidomycete genera were detected at >10 reads.Conclusions.Molecular inference of a core of nonoxygenic bacterial genera common to P. ponojensis and free-living Nostoc microbiomes suggests that some functionally significant cyanolichen microbiota may derive from those of ancestral free-living cyanobacteria, possibly indicating an early step in the evolution of cyanolichen microbiomes. A surprising diversity of microbiome eukaryotes suggested that lichens may serve as a habitat for such organisms.

Since rooted vascular plants rose to dominance, associated microbiota have powerfully influenced global biogeochemistry by mobilizing N and P and otherwise aiding plant health, thereby fostering sequestration of CO2 into coal and soil organics. Less well understood are evolutionary history and biogeochemical roles of microbiomes of ecologically significant nonvascular plants and related algae, whose lineages penetrate more deeply into time. Because analyses of diverse host-microbiome systems indicate that evolutionary history commonly influences microbiome composition and function, we mapped onto a geological time frame biogeochemical features inferred in our previous metagenomic studies of a phylogenetic spectrum of multicellular, freshwater-terrestrial, nonvascular autotroph-microbiome systems: Nostoc commune, representing microbialite-forming cyanobacteria; the Nostoc lichen Peltigera ponojensis; chlorophyte Cladophora glomerata; streptophyte alga Coleochaete pulvinata; moss Sphagnum fimbriatum; and liverwort Conocephalum conicum. Widespread and often abundant today, these species represent ancient lineages of minimal ages ranging from 1450 to 380 Ma, information we used to estimate present global annual magnitudes of organic/inorganic C sequestration, N fixation, and methane oxidation and to calculate biogeochemical impacts during periods from lineage origin to the rise of vascular plants and from lineage origins to present. Functional comparisons indicated that (1) functionally diverse bacterial associates have likely enhanced global C- and N-cycle roles of cyanobacteria for ∼1.5 billion years; (2) by 700–800 Ma, chlorophyte microbiomes retaining ancient functional features (e.g., vitamin B12 biosynthesis and N fixation) had added diverse methane-oxidizing bacteria and eukaryotes, including early metazoans; (3) by ∼500 Ma, streptophyte algal microbiomes possessing ancient functions had incorporated early-diverging fungi; (4) by 450 Ma, microbiomes of early plants included diverse prokaryotes, protists, fungi, and early terrestrial animals; and (5) by 385 Ma, plant microbiomes included P-mobilizing fungi. Autotroph-microbiome systems appear to have accreted functions of global significance over deep time, a new concept that illuminates the early evolution of terrestrial life and that aids modeling past, present, and future biogeochemical impacts.

Known Proterozoic algal fossils raise compelling questions about the origin and diversification of cyanobacteria and eukaryotic algae, and their ecological influence in deep time. This Perspectives article describes particular examples of persistent evolutionary and biogeochemical issues whose resolution would be aided by additional algal fossil evidence from Proterozoic deposits, which have been the subjects of recent intensive study. New Proterozoic geosciences literature relevant to the early diversification of algae is surveyed. Previously underappreciated algal traits that might improve taxonomic attributions of fossil remains are highlighted. Processes that phycologists could use to improve detection of algal fossils are recommended. Potential geological sources of new Proterozoic fossils are suggested.