Pooled multicolour tagging for visualizing subcellular protein dynamics.

Imaging-based methods are widely used for studying the subcellular localization of proteins in living cells. While routine for individual proteins, global monitoring of protein dynamics following perturbation typically relies on arrayed panels of fluorescently tagged cell lines, limiting throughput and scalability. Here, we describe a strategy that combines high-throughput microscopy, computer vision and machine learning to detect perturbation-induced changes in multicolour tagged visual proteomics cell (vpCell) pools. We use genome-wide and cancer-focused intron-targeting sgRNA libraries to generate vpCell pools and a large, arrayed collection of clones each expressing two different endogenously tagged fluorescent proteins. Individual clones can be identified in vpCell pools by image analysis using the localization patterns and expression level of the tagged proteins as visual barcodes, enabling simultaneous live-cell monitoring of large sets of proteins. To demonstrate broad applicability and scale, we test the effects of antiproliferative compounds on a pool with cancer-related proteins, on which we identify widespread protein localization changes and new inhibitors of the nuclear import/export machinery. The time-resolved characterization of changes in subcellular localization and abundance of proteins upon perturbation in a pooled format highlights the power of the vpCell approach for drug discovery and mechanism-of-action studies.

Molecular and morphological characterization of four new ancyromonad genera and proposal for an updated taxonomy of the Ancyromonadida.

Ancyromonads are small biflagellated protists with a bean-shaped morphology. They are cosmopolitan in marine, freshwater, and soil environments, where they attach to surfaces while feeding on bacteria. These poorly known grazers stand out by their uncertain phylogenetic position in the tree of eukaryotes, forming a deep-branching "orphan" lineage that is considered key to a better understanding of the early evolution of eukaryotes. Despite their ecological and evolutionary interest, only limited knowledge exists about their true diversity. Here, we aimed to characterize ancyromonads better by integrating environmental surveys with behavioral observation and description of cell morphology, for which sample isolation and culturing are indispensable. We studied 18 ancyromonad strains, including 14 new isolates and seven new species. We described three new and genetically divergent genera: Caraotamonas, Nyramonas, and Olneymonas, together encompassing four species. The remaining three new species belong to the already-known genera Fabomonas and Ancyromonas. We also raised Striomonas, formerly a subgenus of Nutomonas, to full genus status, on morphological and phylogenetic grounds. We studied the morphology of diverse ancyromonads under light and electron microscopy and carried out molecular phylogenetic analyses, also including 18S rRNA gene sequences from several environmental surveys. Based on these analyses, we have updated the taxonomy of Ancyromonadida.

Metagenome-derived virus-microbe ratios across ecosystems.

It is generally assumed that viruses outnumber cells on Earth by at least tenfold. Virus-to-microbe ratios (VMR) are largely based on counts of fluorescently labelled virus-like particles. However, these exclude intracellular viruses and potentially include false positives (DNA-containing vesicles, gene-transfer agents, unspecifically stained inert particles). Here, we develop a metagenome-based VMR estimate (mVRM) that accounts for DNA viruses across all stages of their replication cycles (virion, intracellular lytic and lysogenic) by using normalised RPKM (reads per kilobase of gene sequence per million of mapped metagenome reads) counts of the major capsid protein (MCP) genes and cellular universal single-copy genes (USCGs) as proxies for virus and cell counts, respectively. After benchmarking this strategy using mock metagenomes with increasing VMR, we inferred mVMR across different biomes. To properly estimate mVMR in aquatic ecosystems, we generated metagenomes from co-occurring cellular and viral fractions (>50 kDa-200 µm size-range) in freshwater, seawater and solar saltern ponds (10 metagenomes, 2 control metaviromes). Viruses outnumbered cells in freshwater by ~13 fold and in plankton from marine and saline waters by ~2-4 fold. However, across an additional set of 121 diverse non-aquatic metagenomes including microbial mats, microbialites, soils, freshwater and marine sediments and metazoan-associated microbiomes, viruses, on average, outnumbered cells by barely two-fold. Although viruses likely are the most diverse biological entities on Earth, their global numbers might be closer to those of cells than previously estimated.

Expanding the molecular and morphological diversity of Apusomonadida, a deep-branching group of gliding bacterivorous protists.

Apusomonads are cosmopolitan bacterivorous biflagellate protists usually gliding on freshwater and marine sediment or wet soils. These nanoflagellates form a sister lineage to opisthokonts and may have retained ancestral features helpful to understanding the early evolution of this large supergroup. Although molecular environmental analyses indicate that apusomonads are genetically diverse, few species have been described. Here, we morphologically characterize 11 new apusomonad strains. Based on molecular phylogenetic analyses of the rRNA gene operon, we describe four new strains of the known species Multimonas media, Podomonas capensis, Apusomonas proboscidea, and Apusomonas australiensis, and rename Thecamonas oxoniensis as Mylnikovia oxoniensis n. gen., n. comb. Additionally, we describe four new genera and six new species: Catacumbia lutetiensis n. gen. n. sp., Cavaliersmithia chaoae n. gen. n. sp., Singekia montserratensis n. gen. n. sp., Singekia franciliensis n. gen. n. sp., Karpovia croatica n. gen. n. sp., and Chelonemonas dolani n. sp. Our comparative analysis suggests that apusomonad ancestor was a fusiform biflagellate with a dorsal pellicle, a plastic ventral surface, and a sleeve covering the anterior flagellum, that thrived in marine, possibly oxygen-poor, environments. It likely had a complex cell cycle with dormant and multiple fission stages, and sex. Our results extend known apusomonad diversity, allow updating their taxonomy, and provide elements to understand early eukaryotic evolution.

Phylogenomics Supports the Monophyly of Aphelids and Fungi and Identifies New Molecular Synapomorphies.

The supergroup Holomycota, composed of Fungi and several related lineages of unicellular organisms (Nucleariida, Rozellida, Microsporidia, and Aphelida), represents one of the major branches in the phylogeny of eukaryotes. Nevertheless, except for the well-established position of Nucleariida as the first holomycotan branch to diverge, the relationships among the other lineages have so far remained unresolved largely owing to the lack of molecular data for some groups. This was notably the case aphelids, a poorly known group of endobiotic phagotrophic protists that feed on algae with cellulose walls. The first molecular phylogenies including aphelids supported their sister relationship with Rozellida and Microsporidia which, collectively, formed a new group called Opisthosporidia (the "Opisthosporidia hypothesis"). However, recent phylogenomic analyses including massive sequence data from two aphelid genera, Paraphelidium and Amoeboaphelidium, suggested that the aphelids are sister to fungi (the "Aphelida $+$ Fungi hypothesis"). Should this position be confirmed, aphelids would be key to understanding the early evolution of Holomycota and the origin of Fungi. Here, we carry out phylogenomic analyses with an expanded taxonomic sampling for aphelids after sequencing the transcriptomes of two species of the genus Aphelidium (Aphelidium insulamus and Aphelidium tribonematis) in order to test these competing hypotheses. Our new phylogenomic analyses including species from the three known aphelid genera strongly rejected the Opisthosporidia hypothesis. Furthermore, comparative genomic analyses further supported the Aphelida $+$ Fungi hypothesis via the identification of 19 orthologous genes exclusively shared by these two lineages. Seven of them originated from ancient horizontal gene transfer events predating the aphelid-fungal split and the remaining 12 likely evolved de novo, constituting additional molecular synapomorphies for this clade. Ancestral trait reconstruction based on our well-resolved phylogeny of Holomycota suggests that the progenitor of both fungi and rozellids, was aphelid-like, having an amoeboflagellate state and likely preying endobiotically on cellulose-containing, cell-walled organisms. Two lineages, which we propose to call Phytophagea and Opisthophagea, evolved from this ancestor. Phytophagea, grouping aphelids and classical fungi, mainly specialized in endobiotic predation of algal cells. Fungi emerged from this lineage after losing phagotrophy in favor of osmotrophy. Opisthophagea, grouping rozellids and Microsporidia, became parasites, mostly of chitin-containing hosts. This lineage entered a progressive reductive process that resulted in a unique lifestyle, especially in the highly derived Microsporidia. [Aphelida, fungi, Holomycota, horizontal gene transfer, phylogenomics, synapomorphy.].

Independent Size Expansions and Intron Proliferation in Red Algal Plastid and Mitochondrial Genomes.

Proliferation of selfish genetic elements has led to significant genome size expansion in plastid and mitochondrial genomes of various eukaryotic lineages. Within the red algae, such expansion events are only known in the plastid genomes of the Proteorhodophytina, a highly diverse group of mesophilic microalgae. By contrast, they have never been described in the much understudied red algal mitochondrial genomes. Therefore, it remains unclear how widespread such organellar genome expansion events are in this eukaryotic phylum. Here, we describe new mitochondrial and plastid genomes from 25 red algal species, thereby substantially expanding the amount of organellar sequence data available, especially for Proteorhodophytina, and show that genome expansions are common in this group. We confirm that large plastid genomes are limited to the classes Rhodellophyceae and Porphyridiophyceae, which, in part, are caused by lineage-specific expansion events. Independently expanded mitochondrial genomes-up to three times larger than typical red algal mitogenomes-occur across Proteorhodophytina classes and a large shift toward high GC content occurred in the Stylonematophyceae. Although intron proliferation is the main cause of plastid and mitochondrial genome expansion in red algae, we do not observe recent intron transfer between different organelles. Phylogenomic analyses of mitochondrial and plastid genes from our expanded taxon sampling yielded well-resolved phylogenies of red algae with strong support for the monophyly of Proteorhodophytina. Our work shows that organellar genomes followed different evolutionary dynamics across red algal lineages.

Past President's Address: Protists of the Mesopelagic and a Bit on the Long Path to the Deep Sea.

The deep sea has long been a mysterious and attractive habitat for protistologists. However, logistical difficulties severely limit sampling opportunities. Consequently, our knowledge of the protists in the deep sea, (arguably the largest habitat on earth), is relatively sparse. Here, we present a unique time-series concerning three different protist taxa that share only the characteristics of being relatively large, robust to sampling, and easily identifiable to species level using light microscopy: tintinnid ciliates, phaeogromid cercozoans (e.g. Challengerids) and amphisolenid dinoflagellates. We sampled a near-shore deep water site in the N.W. Mediterranean Sea at 250 m depth over a 2-yr period at approximately weekly intervals from January 2017 to December 2018. To our knowledge, no previous studies have employed sampling on a similar time scale. We found taxa that appear to be restricted to deep waters, distinct seasonal patterns of abundance in some taxa, and in others nonseasonal successional patterns. Based on data from sampling following a flash flood event, the Challengerid population appeared to respond positively to a pulse of terrigenous input. Some of the distinct mesopelagic tintinnid ciliates and amphisolinid dinoflagellates were also found in two samples from the North Atlantic mesopelagic gathered from near the Azores Islands in September 2018. We conclude that there are a variety of protist taxa endemic to the mesopelagic, that the populations are dynamic, and they may be widely distributed in the deep waters of the world ocean.

In-depth analyses of deep subsurface sediments using 454-pyrosequencing reveals a reservoir of buried fungal communities at record-breaking depths.

The deep subseafloor, extending from a few centimeters below the sediment surface to several hundred meters into sedimentary deposits, constitutes the deep biosphere and harbors an unexpected microbial diversity. Several studies have described the occurrence, turnover, activity and function of subseafloor prokaryotes; however, subsurface eukaryotic communities still remain largely underexplored. Ribosomal RNA surveys of superficial and near-surface marine sediments have revealed an unexpected diversity of active eukaryotic communities, but knowledge of the diversity of deep subseafloor microeukaryotes is still scarce. Here, we investigated the vertical distribution of DNA and RNA fungal signatures within subseafloor sediments of the Canterbury basin (New Zealand) by 454 pyrotag sequencing of fungal genetic markers. Different shifts between the fungal classes of Tremellomycetes, Sordariomycetes, Eurotiomycetes, Saccharomycetes, Wallemiomycetes, Dothideomycetes, Exobasidiomycetes and Microbotryomycetes were observed. These data provide direct evidence that fungal communities occur at record depths in deep sediments of the Canterbury basin and extend the depth limit of fungal presence and activity, respectively 1740 and 346 mbsf. As most of the fungal sequences retrieved have a cosmopolitan distribution, it indicates that fungi are able to adapt to the deep subseafloor conditions at record-depth and must play important ecological roles in biogeochemical cycles.

Phylogenetic and functional diversity of microbial communities associated with subsurface sediments of the Sonora Margin, Guaymas Basin.

Subsurface sediments of the Sonora Margin (Guaymas Basin), located in proximity of active cold seep sites were explored. The taxonomic and functional diversity of bacterial and archaeal communities were investigated from 1 to 10 meters below the seafloor. Microbial community structure and abundance and distribution of dominant populations were assessed using complementary molecular approaches (Ribosomal Intergenic Spacer Analysis, 16S rRNA libraries and quantitative PCR with an extensive primers set) and correlated to comprehensive geochemical data. Moreover the metabolic potentials and functional traits of the microbial community were also identified using the GeoChip functional gene microarray and metabolic rates. The active microbial community structure in the Sonora Margin sediments was related to deep subsurface ecosystems (Marine Benthic Groups B and D, Miscellaneous Crenarchaeotal Group, Chloroflexi and Candidate divisions) and remained relatively similar throughout the sediment section, despite defined biogeochemical gradients. However, relative abundances of bacterial and archaeal dominant lineages were significantly correlated with organic carbon quantity and origin. Consistently, metabolic pathways for the degradation and assimilation of this organic carbon as well as genetic potentials for the transformation of detrital organic matters, hydrocarbons and recalcitrant substrates were detected, suggesting that chemoorganotrophic microorganisms may dominate the microbial community of the Sonora Margin subsurface sediments.

Physiological features of Halomonas lionensis sp. nov., a novel bacterium isolated from a Mediterranean Sea sediment.

A novel halophilic bacterium, strain RHS90(T), was isolated from marine sediments from the Gulf of Lions, in the Mediterranean Sea. Its metabolic and physiological characteristics were examined under various cultural conditions, including exposure to stressful ones (oligotrophy, high pressure and high concentrations of metals). Based on phylogenetic analysis of the 16S rRNA gene, the strain was found to belong to the genus Halomonas in the class Gammaproteobacteria. Its closest relatives are Halomonas axialensis and Halomonas meridiana (98% similarity). DNA-DNA hybridizations indicated that the novel isolate is genotypically distinct from these species. The DNA G + C content of the strain is 54.4 mol%. The main fatty acids (C18:1ω7c, 2-OH iso-C15:0, C16:0 and/or C19:0 cyclo ω8c), main polar lipids (diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and an unidentified phosphoglycolipid) and major respiratory quinone (ubiquinone Q9) were determined. The novel isolate is heterotrophic, mesophilic, euryhaline (growth optimum ranging from 2 to 8% w/v NaCl) and is able to grow under stressful conditions. The strain accumulates poly-ß-hydroxyalkanoates granules and compatible solutes. Based on genotypic, chemotaxonomic and phenotypic distinctiveness, this isolate is likely to represent a novel species, for which the name Halomonas lionensis is proposed. The type strain of H. lionensis is RHS90(T) (DSM 25632(T) = CIP 110370(T) = UBOCC 3186(T)).

Microorganisms persist at record depths in the subseafloor of the Canterbury Basin.

The subsurface realm is colonized by microbial communities to depths of >1000 meters below the seafloor (m.b.sf.), but little is known about overall diversity and microbial distribution patterns at the most profound depths. Here we show that not only Bacteria and Archaea but also Eukarya occur at record depths in the subseafloor of the Canterbury Basin. Shifts in microbial community composition along a core of nearly 2 km reflect vertical taxa zonation influenced by sediment depth. Representatives of some microbial taxa were also cultivated using methods mimicking in situ conditions. These results suggest that diverse microorganisms persist down to 1922 m.b.sf. in the seafloor of the Canterbury Basin and extend the previously known depth limits of microbial evidence (i) from 159 to 1740 m.b.sf. for Eukarya and (ii) from 518 to 1922 m.b.sf. for Bacteria.

Phaeobacter leonis sp. nov., an alphaproteobacterium from Mediterranean Sea sediments.

A novel Gram-stain-negative, strictly aerobic, heterotrophic bacterium, designated 306(T), was isolated from near-surface (109 cm below the sea floor) sediments of the Gulf of Lions, in the Mediterranean Sea. Strain 306(T) grew at temperatures between 4 and 32 °C (optimum 17-22 °C), from pH 6.5 to 9.0 (optimum 8.0-9.0) and between 0.5 and 6.0% (w/v) NaCl (optimum 2.0%). Its DNA G+C content was 58.8 mol%. On the basis of 16S rRNA gene sequence similarity, the novel isolate belongs to the class Alphaproteobacteria and is related to the genus Phaeobacter. It shares 98.7% 16S rRNA sequence identity with Phaeobacter arcticus, its closest phylogenetic relative. It contained Q-10 as the only respiratory quinone, C(18:1)ω7c and C(16:0) as major fatty acids (>5%) and phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, diphosphatidylglycerol, two unidentified lipids and an aminolipid as polar lipids. The chemotaxonomic data are consistent with the affiliation of strain 306(T) to the genus Phaeobacter. Results of physiological experiments, biochemical tests and DNA-DNA hybridizations (with P. arcticus) indicate that strain 306(T) is genetically and phenotypically distinct from the five species of the genus Phaeobacter with validly published names. Strain 306(T) therefore represents a novel species, for which the name Phaeobacter leonis sp. nov. is proposed. The type strain is 306(T) ( =DSM 25627(T) =CIP 110369(T) =UBOCC 3187(T)).

Lymphatic microvessels density, VEGF-C, and VEGFR-3 expression in 25 cases of breast invasive lobular carcinoma.

Invasive lobular carcinoma (ILC) is the second most common type of invasive breast cancer, having distinct morphologically but also prognostic and therapeutic features. This type of breast cancer shows a higher rate of multiple metastases with a more frequent axillary-lymph-node involvement. Related to these dissemination and metastatic features, we aimed to study the immunohistochemical expression of D2-40, VEGF-C and VEGFR-3 in 25 cases of ILCs stratified according to the histopathological and molecular classification. Regardless of histopathological or molecular subtype, the statistical tests proved that for ILC, the highest D2-40 lymphatic microvessels density (LMVD) was in the peritumoral areas. In classical subtype, the LMVD values were positively correlated with the degree of tumor differentiation and pTNM clinical stages and when these cases were classified based on the molecular criteria the highest recorded values were found in the luminal B subtype. In addition, regardless of the histopathological and molecular subtypes, the D2-40 LMVD varied in the same direction for both VEGF-C and VEGFR-3 categories, with the highest LMVD values recorded in those cases with the highest VEGF-C and VEGFR-3 reactivity, especially in the peritumoral areas. Considering only the molecular luminal A and B subtypes, we have noted that VEGF-C and VEGFR-3 expression was significantly higher in luminal A subtype compared to luminal B. This immunoprofile suggests the existence of a tumor type-specific lymphangiogenesis that may have certain prognostic and therapeutic implications.

Invasive papillary carcinoma of the mammary gland: histopathologic and immunohistochemical aspects.

Papillary lesions of the breast, both being and malignant, can prove to be a very challenging diagnosis in histological preparations. This study emphasizes on the importance of immunohistochemistry and in particular, the identification of myoepithelial cells for the correct evaluation of these lesions.

Breast invasive lobular carcinoma: a retrospective clinicopathologic study of 25 cases.

Invasive lobular carcinoma (ILC) is the second most common type of invasive breast cancer, having distinct prognostic and biologic implications. As an objective of the present work, we analyzed the clinicopathologic characteristics and prognostic factor of this invasive breast cancer variant. Clinical and morphological data of 25 cases of ILC collected during 2006-2011 were reviewed. Histopathologically, 11 cases were of classic type, and the others were non-classic with solid and histiocytoid subtypes being mostly encountered. Overall the non-classic ILC type was diagnosed in more aged patients (with a median age at onset of 59 years), with a predominance for a more advanced tumor degree differentiation (78.5% as grade 2 and 3), in advanced pTNM stages (50% in stage III and IV), with 50% lymph node involvement and with over 70% ER and Her2 reactivity. Statistically, we found that for the solid variant prevailed a PR+ and Her2- status while in histiocytoid subtype the PR- and Her2+ immunoprofile was most encountered. We conclude that non-classic ILC type represents a distinct entity of invasive breast carcinoma with a worsen prognostic than the conventional ILC type.

DNA extractions from deep subseafloor sediments: novel cryogenic-mill-based procedure and comparison to existing protocols.

Extracting DNA from deep subsurface sediments is challenging given the complexity of sediments types, low biomasses, resting structures (spores, cysts) frequently encountered in deep sediments, and the potential presence of enzymatic inhibitors. Promising results for cell lysis efficiency were recently obtained by use of a cryogenic mill (Lipp et al., 2008). These findings encouraged us to devise a DNA extraction protocol using this tool. Thirteen procedures involving a combination of grinding in liquid nitrogen (for various durations and beating rates) with different chemical solutions (phenol, chloroform, SDS, sarkosyl, proteinase, GTC), or with use of DNA recovery kits (MagExtractor®) were compared. Effective DNA extraction was evaluated in terms of cell lysis efficiency, DNA extraction efficiency, DNA yield and determination of prokaryotic diversity. Results were compared to those obtained by standard protocols: the FastDNA®SPIN kit for soil and the Zhou protocol. For most sediment types grinding in a cryogenic mill at a low beating rate in combination with direct phenol-chloroform extraction resulted in much higher DNA yields than those obtained using classical procedures. In general (except for clay-rich sediments), this procedure provided high-quality crude extracts for direct downstream nested-PCR, from cell numbers as low as 1.1×10(6) cells/cm(3). This procedure is simple, rapid, low-cost, and could be used with minor modifications for large-scale DNA extractions for a variety of experimental goals.

Marinitoga litoralis sp. nov., a thermophilic, heterotrophic bacterium isolated from a coastal thermal spring on Ile Saint-Paul, Southern Indian Ocean.

A novel thermophilic, anaerobic and organotrophic bacterium, designated strain MC3T, was isolated from a coastal thermal spring on Ile Saint-Paul in the Southern Indian Ocean. Cells of strain MC3T were motile rods, 0.8-1.0 microm wide and 1.0-2.4 microm long during exponential phase and up to 7.0 microm long during stationary phase. Strain MC3T was an anaerobic organotroph able to use diverse organic compounds. It was also able to reduce sulfur to sulfide. Growth was observed at temperatures ranging from 45 to 70 degrees C (optimum at 60 degrees C), between pH 5.5 and 7.5 (optimum at pH 6) and from 8 to 46 g NaCl l(-1) (optimum at 26 g l(-1)). The total G+C content of the genomic DNA was 26.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparisons indicated that strain MC3T was affiliated with the genus Marinitoga within the order Thermotogales. It shared 94.4-95.7% 16S rRNA gene sequence similarity with strains of other Marinitoga species; Marinitoga hydrogenitolerans was found to be the most closely related organism. Based on the data from the phylogenetic analysis and the physiological properties of the novel isolate, strain MC3T should be classified as a representative of a novel species, for which the name Marinitoga litoralis sp. nov. is proposed; the type strain is MC3T (=DSM 21709T =JCM 15581T).