Phylogenomic position of eupelagonemids, abundant, and diverse deep-ocean heterotrophs.

Eupelagonemids, formerly known as Deep Sea Pelagic Diplonemids I (DSPD I), are among the most abundant and diverse heterotrophic protists in the deep ocean, but little else is known about their ecology, evolution, or biology in general. Originally recognized solely as a large clade of environmental ribosomal subunit RNA gene sequences (SSU rRNA), branching with a smaller sister group DSPD II, they were postulated to be diplonemids, a poorly studied branch of Euglenozoa. Although new diplonemids have been cultivated and studied in depth in recent years, the lack of cultured eupelagonemids has limited data to a handful of light micrographs, partial SSU rRNA gene sequences, a small number of genes from single amplified genomes, and only a single formal described species, Eupelagonema oceanica. To determine exactly where this clade goes in the tree of eukaryotes and begin to address the overall absence of biological information about this apparently ecologically important group, we conducted single-cell transcriptomics from two eupelagonemid cells. A SSU rRNA gene phylogeny shows that these two cells represent distinct subclades within eupelagonemids, each different from E. oceanica. Phylogenomic analysis based on a 125-gene matrix contrasts with the findings based on ecological survey data and shows eupelagonemids branch sister to the diplonemid subgroup Hemistasiidae.

Multiple parallel origins of parasitic Marine Alveolates.

Microbial eukaryotes are important components of marine ecosystems, and the Marine Alveolates (MALVs) are consistently both abundant and diverse in global environmental sequencing surveys. MALVs are dinoflagellates that are thought to be parasites of other protists and animals, but the lack of data beyond ribosomal RNA gene sequences from all but a few described species means much of their biology and evolution remain unknown. Using single-cell transcriptomes from several MALVs and their free-living relatives, we show that MALVs evolved independently from two distinct, free-living ancestors and that their parasitism evolved in parallel. Phylogenomics shows one subgroup (MALV-II and -IV, or Syndiniales) is related to a novel lineage of free-living, eukaryovorous predators, the eleftherids, while the other (MALV-I, or Ichthyodinida) is related to the free-living predator Oxyrrhis and retains proteins targeted to a non-photosynthetic plastid. Reconstructing the evolution of photosynthesis, plastids, and parasitism in early-diverging dinoflagellates shows a number of parallels with the evolution of their apicomplexan sisters. In both groups, similar forms of parasitism evolved multiple times and photosynthesis was lost many times. By contrast, complete loss of the plastid organelle is infrequent and, when this does happen, leaves no residual genes.

Gene Transfer Agents in Bacterial Endosymbionts of Microbial Eukaryotes.

Gene transfer agents (GTAs) are virus-like structures that package and transfer prokaryotic DNA from donor to recipient prokaryotic cells. Here, we describe widespread GTA gene clusters in the highly reduced genomes of bacterial endosymbionts from microbial eukaryotes (protists). Homologs of the GTA capsid and portal complexes were initially found to be present in several highly reduced alphaproteobacterial endosymbionts of diplonemid protists (Rickettsiales and Rhodospirillales). Evidence of GTA expression was found in polyA-enriched metatranscriptomes of the diplonemid hosts and their endosymbionts, but due to biases in the polyA-enrichment methods, levels of GTA expression could not be determined. Examining the genomes of closely related bacteria revealed that the pattern of retained GTA head/capsid complexes with missing tail components was common across Rickettsiales and Holosporaceae (Rhodospirillales), all obligate symbionts with a wide variety of eukaryotic hosts. A dN/dS analysis of Rickettsiales and Holosporaceae symbionts revealed that purifying selection is likely the main driver of GTA evolution in symbionts, suggesting they remain functional, but the ecological function of GTAs in bacterial symbionts is unknown. In particular, it is unclear how increasing horizontal gene transfer in small, largely clonal endosymbiont populations can explain GTA retention, and, therefore, the structures may have been repurposed in endosymbionts for host interactions. Either way, their widespread retention and conservation in endosymbionts of diverse eukaryotes suggests an important role in symbiosis.

Microbial communities in sandy beaches from the three domains of life differ by microhabitat and intertidal location.

The microbial communities of sandy beaches are poorly described despite the biogeochemical importance and ubiquity of these ecosystems. Using metabarcoding of the 16S and 18S rRNA genes, we investigated the diversity, microhabitats (with or between sand grains) and intertidal distributions of microorganisms (including meiofauna) from pristine sandy beaches in British Columbia, Canada, and hypothesized that abiotic variations due to microhabitat or intertidal gradients influence the distribution of microorganisms on local scales. Bacterial, archaeal and protistan communities of the sand were clearly distinct from interstitial communities, and from planktonic communities of the overlying seawater, which correlated with differences in function and lifestyle (e.g., sulphur reduction and gliding motility). In contrast, meiofaunal communities could not be distinguished by sample type, suggesting that they are more frequently mobilized between these microhabitats. Across intertidal zones, high intertidal, mid intertidal and low intertidal/swash communities were distinct and correlated with moisture, organic carbon and phosphate content, implying that the distribution of microorganisms is influenced by intertidal abiotic gradients. However, few taxa at the genus or species level individually contributed to this zonation pattern; rather, a unique combination of multiple microbial taxa was probably responsible. Although significant differences in microbial community composition on sandy beaches can be attributed to microhabitat and intertidal gradients, further investigations are needed to assess community assembly processes, the consistency of these distributions, and the functions of the majority of the microorganisms observed in the sand and their effects on the biogeochemistry and ecology of sandy beaches.

Factors Associated with Treatment Satisfaction Among People Living with HIV in Japan and Other Selected Countries: Examination of the Intertwined Roles of Medication, Patient, and Provider Characteristics.

We examined satisfaction and perceived challenges with antiretroviral therapy (ART) among people living with HIV (PLHIV) in Japan vs three other Asian countries (China, Taiwan, South Korea), and 21 non-Asian countries, using data from the 2019 Positive Perspectives Study (pooled sample size from all 25 countries = 2389). Participants in other Asian countries were more likely than those in Japan to report they missed ART ≥ 1 time in the past month because they were depressed/overwhelmed (57.4%[89/155] vs 32.0%[24/75]), had privacy concerns (56.8%[88/155] vs 30.7%[23/75]), were concerned about the potential long-term negative impacts of ART (46.5%[72/155] vs 26.7%[20/75]), or just wanted to forget about HIV (45.8%[71/155] vs 22.7%[17/75]). ART satisfaction however did not differ significantly between surveyed PLHIV in Japan (54.7%[41/75]) vs those in other Asian countries (47.7%[74/155]). The percentage who felt that daily ART dosing limited their lives was 36.0%[27/75] among participants from Japan, 48.4%[75/155] among participants from other Asian countries, and 27.3%[589/2159] among those from non-Asian countries. Within a structural equation model using pooled data from all 25 countries, positive correlations were seen between ART satisfaction and "provider engagement" (ß = 0.35), high perceived control over ART dosing schedule (ß = 0.28), and the belief that ART prevents HIV transmission (ß = 0.16). Conversely, negative correlations were seen between ART satisfaction and experience of ART side-effects (ß = - 0.24), high "ART anxiety" (ß = - 0.20); and being on multi-tablet regimens (ß = - 0.13). Those ART-satisfied reported higher self-rated health and greater ART adherence. These findings underscore the need for patient-centered care to enhance treatment satisfaction and improve ART adherence.

Characterization of new cristamonad species from kalotermitid termites including a novel genus, Runanympha.

Cristamonadea is a large class of parabasalian protists that reside in the hindguts of wood-feeding insects, where they play an essential role in the digestion of lignocellulose. This group of symbionts boasts an impressive array of complex morphological characteristics, many of which have evolved multiple times independently. However, their diversity is understudied and molecular data remain scarce. Here we describe seven new species of cristamonad symbionts from Comatermes, Calcaritermes, and Rugitermes termites from Peru and Ecuador. To classify these new species, we examined cells by light and scanning electron microscopy, sequenced the symbiont small subunit ribosomal RNA (rRNA) genes, and carried out barcoding of the mitochondrial large subunit rRNA gene of the hosts to confirm host identification. Based on these data, five of the symbionts characterized here represent new species within described genera: Devescovina sapara n. sp., Devescovina aymara n. sp., Macrotrichomonas ashaninka n. sp., Macrotrichomonas secoya n. sp., and Macrotrichomonas yanesha n. sp. Additionally, two symbionts with overall morphological characteristics similar to the poorly-studied and probably polyphyletic 'joeniid' Parabasalia are classified in a new genus Runanympha n. gen.: Runanympha illapa n. sp., and Runanympha pacha n. sp.

Taxonomy of the Apicomplexan Symbionts of Coral, including Corallicolida ord. nov., Reassignment of the Genus Gemmocystis, and Description of New Species Corallicola aquarius gen. nov. sp. nov. and Anthozoaphila gnarlus gen. nov. sp. nov.

Corals (Metazoa; Cnidaria; Anthozoa) have recently been shown to play host to a widespread and diverse group of intracellular symbionts of the phylum Apicomplexa. These symbionts, colloquially called "corallicolids", are mostly known through molecular analyses, and no formal taxonomy has been proposed. Another apicomplexan, Gemmocystis cylindrus (described from the coral Dendrogyra cylindrus), may be related to corallicolids, but lacks molecular data. Here, we isolate and describe motile trophozoite (feeding) corallicolids cells using microscopic (light, SEM, and TEM) and molecular phylogenetic analysis to provide the basis for a formal description. Phylogenetic analyses using nuclear and plastid rRNA operons, and three mitochondrial protein sequences derived from single cell transcriptomes, all confirm that these organisms fall into a discrete deep-branching clade within the Apicomplexa not closely related to any known species or major subgroup. As a result, we assign this clade to a new order, Corallicolida ord. nov., and family, Corallicolidae fam. nov. We describe a type species, Corallicola aquarius gen. nov. sp. nov. from its Rhodactis sp. host, and also describe a second species, Anthozoaphila gnarlus gen. nov. sp. nov., from the coral host Madracis mirabilis. Finally, we propose reassigning the incertae sedis taxon G. cylindrus from the order Agamococcidiorida to the Corallicolida, based on similarities in morphology and host localization to that of the corallicolids.

[Relations of alcohol consumption and sleep among community-dwelling elderly living in cold region of Russia: a cross-sectional study].

AIM: Alcohol consumption is high in the colder regions of Russia, and it is related to poor sleep quality, mental and physical health problems. Little known on the actual situation, and no appropriate amount of drinking has been shown as a health guidance. The purpose of this study is to examine the relationship between alcohol consumption (in pure alcohol) and sleep among older people living in the Russian Siberian region, and the factors related to alcohol consumption. METHODS: A self-reported questionnaire survey was administered to 422 elderly over the age of 60 living in Novosibirsk, the central city of Siberia. Question items were basic attributes, health status, drinking habits, Short Form-8 Health Survey, Geriatric Depression Scale, and Pittsburgh Sleep Quality Index. For drinking elderly, daily amount of alcohol converted in pure alcohol was calculated, and logistic regression analysis among the two groups was compared based on the median value (32 g). RESULTS: The valid responses from the survey was 416 (98.9%). Of these, 293 with drinking habits were subjected to logistic regression analysis using pure alcohol (≥32 g/day) as the dependent variable. Significant relationships were found with gender (OR=0.586; 95%CI: 0.345-0.995), years of education (OR=1.538; 95%CI: 1.239-1.910), insomnia (OR=2.442; 95%CI: 1.185-5.032), alcohol intake, due to better sleep (OR=4.120; 95%CI: 1.044-16.258), effects of drinking, arousal during the night (OR=2.586; 95%CI: 1.317-5.077), effects of drinking, from family (OR=26.938; 95%CI: 3.368-215.431). CONCLUSIONS: Among the elderly people in colder regions of Russia, high alcohol consumption reduces sleep quality, suggesting the need for appropriate standards for pure alcohol and health education.

Single-Cell Transcriptomics of Abedinium Reveals a New Early-Branching Dinoflagellate Lineage.

Dinoflagellates possess many cellular characteristics with unresolved evolutionary histories. These include nuclei with greatly expanded genomes and chromatin packaged using histone-like proteins and dinoflagellate-viral nucleoproteins instead of histones, highly reduced mitochondrial genomes with extensive RNA editing, a mix of photosynthetic and cryptic secondary plastids, and tertiary plastids. Resolving the evolutionary origin of these traits requires understanding their ancestral states and early intermediates. Several early-branching dinoflagellate lineages are good candidates for such reconstruction, however these cells tend to be delicate and environmentally sparse, complicating such analyses. Here, we employ transcriptome sequencing from manually isolated and microscopically documented cells to resolve the placement of two cells of one such genus, Abedinium, collected by remotely operated vehicle in deep waters off the coast of Monterey Bay, CA. One cell corresponds to the only described species, Abedinium dasypus, whereas the second cell is distinct and formally described as Abedinium folium, sp. nov. Abedinium has classically been assigned to the early-branching dinoflagellate subgroup Noctilucales, which is weakly supported by phylogenetic analyses of small subunit ribosomal RNA, the single characterized gene from any member of the order. However, an analysis based on 221 proteins from the transcriptome places Abedinium as a distinct lineage, separate from and basal to Noctilucales and the rest of the core dinoflagellates. The transcriptome also contains evidence of a cryptic plastid functioning in the biosynthesis of isoprenoids, iron-sulfur clusters, and heme, a mitochondrial genome with all three expected protein-coding genes (cob, cox1, and cox3), and the presence of some but not all dinoflagellate-specific chromatin packaging proteins.

Inter- and Intramolecular Alkyne-Carbonyl Metathesis/1,6-Addition/Oxidative Aromatization Tandem Reactions between 1,6-Diynes and Aldehydes.

We report an efficient synthetic route to 9-arylfluorenes and biaryl compounds from 1,6-diynes and aldehydes via inter- and intramolecular alkyne-carbonyl metathesis/1,6-addition/oxidative aromatization reactions. These tandem reactions are initiated by a BF3·Et2O-promoted tandem inter- and intramolecular alkyne-carbonyl metathesis of 1,6-diynes with carbonyl compounds followed by an In(OTf)3-catalyzed 1,6-addition/oxidative aromatization with iodobenzene diacetate.

A Revised Taxonomy of Diplonemids Including the Eupelagonemidae n. fam. and a Type Species, Eupelagonema oceanica n. gen. & sp.

Recent surveys of marine microbial diversity have identified a previously unrecognized lineage of diplonemid protists as being among the most diverse heterotrophic eukaryotes in global oceans. Despite their monophyly (and assumed importance), they lack a formal taxonomic description, and are informally known as deep-sea pelagic diplonemids (DSPDs) or marine diplonemids. Recently, we documented morphology and molecular sequences from several DSPDs, one of which is particularly widespread and abundant in environmental sequence data. To simplify the communication of future work on this important group, here we formally propose to erect the family Eupelagonemidae to encompass this clade, as well as a formal genus and species description for the apparently most abundant phylotype, Eupelagonema oceanica, for which morphological information and single-cell amplified genome data are currently available.

Phylogeny, Evidence for a Cryptic Plastid, and Distribution of Chytriodinium Parasites (Dinophyceae) Infecting Copepods.

Spores of the dinoflagellate Chytriodinium are known to infest copepod eggs causing their lethality. Despite the potential to control the population of such an ecologically important host, knowledge about Chytriodinium parasites is limited: we know little about phylogeny, parasitism, abundance, or geographical distribution. We carried out genome sequence surveys on four manually isolated sporocytes from the same sporangium, which seemed to be attached to a copepod nauplius, to analyze the phylogenetic position of Chytriodinium based on SSU and concatenated SSU/LSU rRNA gene sequences, and also characterize two genes related to the plastidial heme pathway, hemL and hemY. The results suggest the presence of a cryptic plastid in Chytriodinium and a photosynthetic ancestral state of the parasitic Chytriodinium/Dissodinium clade. Finally, by mapping Tara Oceans V9 SSU amplicon data to the recovered SSU rRNA gene sequences from the sporocytes, we show that globally, Chytriodinium parasites are most abundant within the pico/nano- and mesoplankton of the surface ocean and almost absent within microplankton, a distribution indicating that they generally exist either as free-living spores or host-associated sporangia.

Single cell genomics of uncultured marine alveolates shows paraphyly of basal dinoflagellates.

Marine alveolates (MALVs) are diverse and widespread early-branching dinoflagellates, but most knowledge of the group comes from a few cultured species that are generally not abundant in natural samples, or from diversity analyses of PCR-based environmental SSU rRNA gene sequences. To more broadly examine MALV genomes, we generated single cell genome sequences from seven individually isolated cells. Genes expected of heterotrophic eukaryotes were found, with interesting exceptions like presence of proteorhodopsin and vacuolar H+-pyrophosphatase. Phylogenetic analysis of concatenated SSU and LSU rRNA gene sequences provided strong support for the paraphyly of MALV lineages. Dinoflagellate viral nucleoproteins were found only in MALV groups that branched as sister to dinokaryotes. Our findings indicate that multiple independent origins of several characteristics early in dinoflagellate evolution, such as a parasitic life style, underlie the environmental diversity of MALVs, and suggest they have more varied trophic modes than previously thought.

Mast cell hyperactivity underpins the development of oxygen-induced retinopathy.

Mast cells are classically thought to play an important role in protection against helminth infections and in the induction of allergic diseases; however, recent studies indicate that these cells also contribute to neovascularization, which is critical for tissue remodeling, chronic inflammation, and carcinogenesis. Here, we demonstrate that mast cells are essential for sprouting angiogenesis in a murine model of oxygen-induced retinopathy (OIR). Although mouse strains lacking mast cells did not exhibit retinal neovascularization following hypoxia, these mice developed OIR following infusion of mast cells or after injection of mast cell tryptase (MCT). Relative hypoxia stimulated mast cell degranulation via transient receptor potential ankyrin 1. Subsequent surges in MCT stimulated retinal endothelial cells to produce monocyte chemotactic protein-1 (MCP1) and angiogenic factors, leading to sprouting angiogenesis. Mast cell stabilizers as well as specific tryptase and MCP1 inhibitors prevented the development of OIR in WT mice. Preterm infants with early retinopathy of prematurity had markedly higher plasma MCT levels than age-matched infants without disease, suggesting mast cells contribute to human disease. Together, these results suggest therapies that suppress mast cell activity should be further explored as a potential option for preventing eye diseases and subsequent blindness induced by neovascularization.

Morphological Identification and Single-Cell Genomics of Marine Diplonemids.

Recent global surveys of marine biodiversity have revealed that a group of organisms known as "marine diplonemids" constitutes one of the most abundant and diverse planktonic lineages [1]. Though discovered over a decade ago [2, 3], their potential importance was unrecognized, and our knowledge remains restricted to a single gene amplified from environmental DNA, the 18S rRNA gene (small subunit [SSU]). Here, we use single-cell genomics (SCG) and microscopy to characterize ten marine diplonemids, isolated from a range of depths in the eastern North Pacific Ocean. Phylogenetic analysis confirms that the isolates reflect the entire range of marine diplonemid diversity, and comparisons to environmental SSU surveys show that sequences from the isolates range from rare to superabundant, including the single most common marine diplonemid known. SCG generated a total of ∼915 Mbp of assembled sequence across all ten cells and ∼4,000 protein-coding genes with homologs in the Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology database, distributed across categories expected for heterotrophic protists. Models of highly conserved genes indicate a high density of non-canonical introns, lacking conventional GT-AG splice sites. Mapping metagenomic datasets [4] to SCG assemblies reveals virtually no overlap, suggesting that nuclear genomic diversity is too great for representative SCG data to provide meaningful phylogenetic context to metagenomic datasets. This work provides an entry point to the future identification, isolation, and cultivation of these elusive yet ecologically important cells. The high density of nonconventional introns, however, also portends difficulty in generating accurate gene models and highlights the need for the establishment of stable cultures and transcriptomic analyses.

Tandem Reaction of Enynyl Acetate: Precursor of Allenyl Ketones.

Deacetylation of enynyl acetates under basic conditions allows convenient access to reactive allenyl ketones, which can then undergo 1,4-addition of nucleophiles to furnish ß,γ-unsaturated ketones. Benzofuran and indole derivatives have also been obtained from enynyl acetates with an o-hetero-atom-substituted aryl group via intramolecular 1,4-addition.

Copper-Catalyzed Tandem Decyclization-Cyclization Reaction of N-Alkynyl-3-hydroxyisoindolin-1-ones Generated from N-Alkynyl Phthalimides: Selective Synthesis of ortho-(2-Oxazolyl)phenyl Ketones.

Selective carbophilic monoaddition on N-alkynyl phthalimides was performed with organometallic reagents to afford 3-substituted N-alkynyl-3-hydroxyisoindolin-1-ones (α-hydroxy ynamides) as a new subgroup of ynamides. Owing to the alkynyl motif on the nitrogen atom, α-hydroxy ynamides were easily isomerized to the corresponding ortho-(2-oxazolyl)phenyl ketones in a CuCl-catalyzed tandem decyclization-cyclization reaction under mild conditions.

Enantioselective γ-Alkylation of α,ß-Unsaturated Malonates and Ketoesters by a Sequential Ir-Catalyzed Asymmetric Allylic Alkylation/Cope Rearrangement.

A catalytic, enantioselective γ-alkylation of α,ß-unsaturated malonates and ketoesters is reported. This strategy entails a highly regio- and enantioselective iridium-catalyzed α-alkylation of an extended enolate, and a subsequent translocation of chirality to the γ-position via a Cope rearrangement.

Phospholipid methylation controls Atg32-mediated mitophagy and Atg8 recycling.

Degradation of mitochondria via selective autophagy, termed mitophagy, contributes to mitochondrial quality and quantity control whose defects have been implicated in oxidative phosphorylation deficiency, aberrant cell differentiation, and neurodegeneration. How mitophagy is regulated in response to cellular physiology remains obscure. Here, we show that mitophagy in yeast is linked to the phospholipid biosynthesis pathway for conversion of phosphatidylethanolamine to phosphatidylcholine by the two methyltransferases Cho2 and Opi3. Under mitophagy-inducing conditions, cells lacking Opi3 exhibit retardation of Cho2 repression that causes an anomalous increase in glutathione levels, leading to suppression of Atg32, a mitochondria-anchored protein essential for mitophagy. In addition, loss of Opi3 results in accumulation of phosphatidylmonomethylethanolamine (PMME) and, surprisingly, generation of Atg8-PMME, a mitophagy-incompetent lipid conjugate of the autophagy-related ubiquitin-like modifier. Amelioration of Atg32 expression and attenuation of Atg8-PMME conjugation markedly rescue mitophagy in opi3-null cells. We propose that proper regulation of phospholipid methylation is crucial for Atg32-mediated mitophagy.

Construction of Tertiary Chiral Centers by Pd-catalyzed Asymmetric Allylic Alkylation of Prochiral Enolate Equivalents.

The palladium-catalyzed decarboxylative allylic alkylation of enol carbonates derived from lactams and ketones is described. Employing these substrates with an electronically tuned Pd catalyst system trisubstituted chiral centers are produced. These stereocenters have been previously challenging to achieve using Pd complex/chiral P-N ligand systems.