Rhabdamoeba marina is a heterotrophic relative of chlorarachnid algae.

Rhabdamoeba marina is a unique and poorly reported amoeba with an uncertain phylogenetic position. We successfully cultured R. marina from coastal seawater in Japan and performed a molecular phylogenetic analysis using the small subunit ribosomal RNA (SSU rRNA) gene sequence. Our phylogenetic analysis showed that R. marina branched as a basal lineage of Chlorarachnea, a group of marine photosynthetic algae belonging to the phylum Cercozoa within the supergroup Rhizaria. By comparing the ecological and morphological characteristics of R. marina with those of photosynthetic chlorarachneans and other cercozoans, we gained insight into the evolution and acquisition of plastids in Chlorarachnida.

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.

Timing of computed tomography imaging in adult patients with severe trauma: A nationwide cohort study in Japan.

PURPOSE: Computed tomography (CT) has become essential for the management of trauma patients. However, appropriate timing of CT acquisition remains undetermined. The purpose of this study was to assess the relationship between time to CT acquisition and mortality among adult patients with severe trauma. METHODS: We conducted a retrospective cohort study using data from the Japan Trauma Data Bank, which had 256 participating institutions from all over Japan between 2004 and 2018. Patients were categorized upon arrival as either severe trunk trauma with signs of shock or severe head trauma with coma and separately analyzed. Cases were further divided into three groups based on time elapsed between arrival at hospital and CT acquisition as immediate (0-29 min), intermediate (30-59 min), or late (≥60 min). Primary outcome was mortality on discharge, and multivariate logistic regression with adjusting for confounders was used for evaluation. RESULTS: A total of 8467 (3640 in immediate group, 3441 in intermediate group, 1386 in late group) with trunk trauma patients and 6762 (4367 in immediate group, 2031 in intermediate group, 364 in late group) with head trauma patients were eligible for analysis included in the trunk and head trauma groups, respectively. The trunk trauma patients with shock on hospital arrival was 56.4% (4773/8467), and the head trauma patients with deep coma upon EMS arrival was 44.2% (2988/6762). Mortality rate gradually increased from 5.7% to 15.8% with prolonged time to CT imaging among trunk trauma patients. Multivariate logistic regression for death on discharge among trunk trauma patients yielded an adjusted odds ratio of 1.79 (95% confidence interval: 1.42-2.27) for the late group compared to the immediate group. In contrast, among head trauma patients, an adjusted odds ratio was 0.93 (95% confidence interval: 0.71-1.20) for the late group compared to the immediate group. CONCLUSION: CT scan at or after 60 min was associated with increased death on discharge among patients with severe trunk trauma but not in those with severe head trauma.

Dinoflagellates with relic endosymbiont nuclei as models for elucidating organellogenesis.

Nucleomorphs are relic endosymbiont nuclei so far found only in two algal groups, cryptophytes and chlorarachniophytes, which have been studied to model the evolutionary process of integrating an endosymbiont alga into a host-governed plastid (organellogenesis). However, past studies suggest that DNA transfer from the endosymbiont to host nuclei had already ceased in both cryptophytes and chlorarachniophytes, implying that the organellogenesis at the genetic level has been completed in the two systems. Moreover, we have yet to pinpoint the closest free-living relative of the endosymbiotic alga engulfed by the ancestral chlorarachniophyte or cryptophyte, making it difficult to infer how organellogenesis altered the endosymbiont genome. To counter the above issues, we need novel nucleomorph-bearing algae, in which endosymbiont-to-host DNA transfer is on-going and for which endosymbiont/plastid origins can be inferred at a fine taxonomic scale. Here, we report two previously undescribed dinoflagellates, strains MGD and TGD, with green algal endosymbionts enclosing plastids as well as relic nuclei (nucleomorphs). We provide evidence for the presence of DNA in the two nucleomorphs and the transfer of endosymbiont genes to the host (dinoflagellate) genomes. Furthermore, DNA transfer between the host and endosymbiont nuclei was found to be in progress in both the MGD and TGD systems. Phylogenetic analyses successfully resolved the origins of the endosymbionts at the genus level. With the combined evidence, we conclude that the host-endosymbiont integration in MGD/TGD is less advanced than that in cryptophytes/chrorarachniophytes, and propose the two dinoflagellates as models for elucidating organellogenesis.

[A Case of Penile Strangulation Due to Metal Rings Released with the Cooperation of a Rescue Team].

In this report, we describe a case of penile strangulation via metal rings. A 65-year-old Japanese man was transferred to the emergency room of our hospital for, dysuria and penile pain following penile incarceration with metal rings. Five metal rings approximately 30 mm in diameter were incarcerated to the penile root. Physical examination, revealed marked penile swelling distal to the rings. Various methods including the use of a ring cutter, were attempted to relieve the penial strangulation. However, these techniques failed, prompting referral to a rescue team. We started cutting the rings with an air cutter. After, 90 minutes, the rings were successfully removed. This study highlights the benefit of early cooperation with the rescue team in managing patients with mechanical penile strangulation.

Relationship between the number of pediatric patients with rotavirus and telephone triage for associated symptoms.

BACKGROUND: Earlier syndromic surveillance may be effective in preventing the spread of infectious disease. However, there has been no research on syndromic surveillance for rotavirus. The study aimed to assess the relationship between the incidence of rotavirus infections and the number of telephone triages for associated symptoms in pediatric patients under 4 years old in Osaka prefecture, Japan. METHODS: This was a retrospective observational study for which the study period was the 3 years between January 2015 and December 2017. We analyzed data on children under 4 years old who were triaged by telephone triage nurses using software. The primary endpoint was the number of rotavirus patients under 4 years triaged old per week. Using a linear regression model, we calculated the R square value of the regression model to assess the relationship between the number of patients with rotavirus and the number of telephone triages made for associated symptoms. Covariates in the linear regression model were the week number indicating seasonality and the weekly number of telephone triages related to rotavirus symptoms such as stomachache and vomiting. RESULTS: During the study period, there were 102,336 patients with rotavirus, and the number of people triaged by telephone was 123,720. The highest correlation coefficient was 0.921 in the regression model with the number of telephone triages for "stomachache + nausea/vomiting" and "stomachache + diarrhea + nausea/vomiting". CONCLUSION: The number of telephone triage symptoms was positively related to the incidence of pediatric patients with rotavirus in a large metropolitan area of Japan.

Barthelonids represent a deep-branching metamonad clade with mitochondrion-related organelles predicted to generate no ATP.

We here report the phylogenetic position of barthelonids, small anaerobic flagellates previously examined using light microscopy alone. Barthelona spp. were isolated from geographically distinct regions and we established five laboratory strains. Transcriptomic data generated from one Barthelona strain (PAP020) were used for large-scale, multi-gene phylogenetic (phylogenomic) analyses. Our analyses robustly placed strain PAP020 at the base of the Fornicata clade, indicating that barthelonids represent a deep-branching metamonad clade. Considering the anaerobic/microaerophilic nature of barthelonids and preliminary electron microscopy observations on strain PAP020, we suspected that barthelonids possess functionally and structurally reduced mitochondria (i.e. mitochondrion-related organelles or MROs). The metabolic pathways localized in the MRO of strain PAP020 were predicted based on its transcriptomic data and compared with those in the MROs of fornicates. We here propose that strain PAP020 is incapable of generating ATP in the MRO, as no mitochondrial/MRO enzymes involved in substrate-level phosphorylation were detected. Instead, we detected a putative cytosolic ATP-generating enzyme (acetyl-CoA synthetase), suggesting that strain PAP020 depends on ATP generated in the cytosol. We propose two separate losses of substrate-level phosphorylation from the MRO in the clade containing barthelonids and (other) fornicates.

[A Case of Ammonium Acid Urate Staghorn Calculi Lost Only in the Drug Therapy].

We report a case of a staghorn stone containing ammonium acid urate that was effectively treated with drug therapy alone. A 46-year-old man had recurring urinary tract stones. He had no previous episode of urinary tract stones that required hospitalization and operation. He received only drug therapy for hyperuricemia in another hospital. Ultrasonography and computed tomography revealed a left staghorn stone measuring 37×34 mm. The kidney-ureter-bladder radiograph did not show any stones. His urine was acidic, and we estimated that the left staghorn stone consisted of urate. Oral administration of sodium hydrogen carbonate was initiated to alkalize the urine, and treatment with transurethral lithotripsy (TUL) was scheduled. Before the TUL, analysis of an excreted stone sample revealed that it consisted of ammonium acid urate. The staghorn stone was completely removed in 10 months after the first medical examination. At present, the patient is free of urinary tract stones.

A Non-photosynthetic Diatom Reveals Early Steps of Reductive Evolution in Plastids.

Nonphotosynthetic plastids retain important biological functions and are indispensable for cell viability. However, the detailed processes underlying the loss of plastidal functions other than photosynthesis remain to be fully understood. In this study, we used transcriptomics, subcellular localization, and phylogenetic analyses to characterize the biochemical complexity of the nonphotosynthetic plastids of the apochlorotic diatom Nitzschia sp. NIES-3581. We found that these plastids have lost isopentenyl pyrophosphate biosynthesis and ribulose-1,5-bisphosphate carboxylase/oxygenase-based carbon fixation but have retained various proteins for other metabolic pathways, including amino acid biosynthesis, and a portion of the Calvin-Benson cycle comprised only of glycolysis/gluconeogenesis and the reductive pentose phosphate pathway (rPPP). While most genes for plastid proteins involved in these reactions appear to be phylogenetically related to plastid-targeted proteins found in photosynthetic relatives, we also identified a gene that most likely originated from a cytosolic protein gene. Based on organellar metabolic reconstructions of Nitzschia sp. NIES-3581 and the presence/absence of plastid sugar phosphate transporters, we propose that plastid proteins for glycolysis, gluconeogenesis, and rPPP are retained even after the loss of photosynthesis because they feed indispensable substrates to the amino acid biosynthesis pathways of the plastid. Given the correlated retention of the enzymes for plastid glycolysis, gluconeogenesis, and rPPP and those for plastid amino acid biosynthesis pathways in distantly related nonphotosynthetic plastids and cyanobacteria, we suggest that this substrate-level link with plastid amino acid biosynthesis is a key constraint against loss of the plastid glycolysis/gluconeogenesis and rPPP proteins in multiple independent lineages of nonphotosynthetic algae/plants.

An Orphan Protist Quadricilia rotundata Finally Finds Its Phylogenetic Home in Cercozoa.

Quadricilia rotundata is a heterotrophic flagellate with four flagella. However, because this species has no clear morphological characteristics or molecular data affiliating it with any known group, Q. rotundata has been treated as a protist incertae sedis, for a long time. Here, we established a clonal culture of Q. rotundata and sequenced its 18S rDNA sequence. Molecular phylogenetic analysis successfully placed Q. rotundata in an environmental clade within Cercozoa, which contributes to expand the morphological and species diversity within Cercozoa. We also discuss morphological evolution within Cercozoa based on this finding.

Algal genomes reveal evolutionary mosaicism and the fate of nucleomorphs.

Cryptophyte and chlorarachniophyte algae are transitional forms in the widespread secondary endosymbiotic acquisition of photosynthesis by engulfment of eukaryotic algae. Unlike most secondary plastid-bearing algae, miniaturized versions of the endosymbiont nuclei (nucleomorphs) persist in cryptophytes and chlorarachniophytes. To determine why, and to address other fundamental questions about eukaryote-eukaryote endosymbiosis, we sequenced the nuclear genomes of the cryptophyte Guillardia theta and the chlorarachniophyte Bigelowiella natans. Both genomes have >21,000 protein genes and are intron rich, and B. natans exhibits unprecedented alternative splicing for a single-celled organism. Phylogenomic analyses and subcellular targeting predictions reveal extensive genetic and biochemical mosaicism, with both host- and endosymbiont-derived genes servicing the mitochondrion, the host cell cytosol, the plastid and the remnant endosymbiont cytosol of both algae. Mitochondrion-to-nucleus gene transfer still occurs in both organisms but plastid-to-nucleus and nucleomorph-to-nucleus transfers do not, which explains why a small residue of essential genes remains locked in each nucleomorph.

Viridiuvalis adhaerens gen. et sp. nov., a novel colony-forming chlorarachniophyte.

A new chlorarachniophyte, Viridiuvalis adhaerens gen. et sp. nov. was isolated from the mucus on a coral reef from Zanpa Beach, Okinawa, Japan. The main vegetative stage of V. adhaerens consisted of unicellular coccoid cells with cell walls, although sarcinoid colonies and uniflagellate zoospores were also observed. V. adhaerens had chloroplasts with nucleomorphs and pyrenoids that were completely embedded in the chloroplast. A deep plate-like invagination of the periplastidal compartment (PPC) almost partitioned the pyrenoid and chloroplast components, which were surrounded by two membranes. The nucleomorph was positioned in the base of the invagination of the PPC. Molecular phylogenetic analyses using rRNA genes showed that V. adhaerens branched as a sister lineage of the Amorphochlora clade. The sarcinoid colony, pyrenoid embedded in the chloroplast, and nucleomorph located at the base of the deep invagination of the PPC have not been reported in other chlorarachniophytes. Based on these morphological and ultrastructural characteristics and the results of the molecular phylogenetic analyses, we propose V. adhaerens as a new genus and species of chlorarachniophyte.

Fewer REBOA complications with smaller devices and partial occlusion: evidence from a multicentre registry in Japan.

BACKGROUND: Resuscitative endovascular balloon occlusion of the aorta (REBOA) performed by emergency physicians has been gaining acceptance as a less invasive technique than resuscitative thoracotomy. OBJECTIVE: To evaluate access-related complications and duration of occlusions during REBOA. METHODS: Patients with haemorrhagic shock requiring REBOA, from 18 hospitals in Japan, included in the DIRECT-IABO Registry were studied. REBOA-related characteristics were compared between non-survivors and survivors at 24 hours. 24-Hour survivors were categorised into groups with small (≤8 Fr), large (≥9 Fr) or unusual sheaths (oversized or multiple) to assess the relationship between the sheath size and complications. Haemodynamic response, occlusion duration and outcomes were compared between groups with partial and complete REBOA. RESULTS: Between August 2011 and December 2015, 142 adults undergoing REBOA were analysed. REBOA procedures were predominantly (94%) performed by emergency medicine (EM) physicians. The median duration of the small sheath (n=53) was 19 hours compared with 7.5 hours for the larger sheaths (P=0.025). Smaller sheaths were more likely to be removed using external manual compression (96% vs 45%, P<0.001). One case of a common femoral artery thrombus (large group) and two cases of amputation (unusual group) were identified. Partial REBOA was carried out in more cases (n=78) and resulted in a better haemodynamic response than complete REBOA (improvement in haemodynamics, 92% vs 70%, P=0.004; achievement of stability, 78% vs 51%, P=0.007) and allowed longer occlusion duration (median 58 vs 33 min, P=0.041). No statistically significant difference in 24-hour or 30-day survival was found between partial and complete REBOA. CONCLUSION: In Japan, EM physicians undertake the majority of REBOA procedures. Smaller sheaths appear to have fewer complications despite relatively prolonged placement and require external compression on removal. Although REBOA is a rarely performed procedure, partial REBOA, which may extend the occlusion duration without a reduction in survival, is used more commonly in Japan.

[Port-Site Metastasis of an Urothelial Carcinoma after Laparoscopic Nephroureterectomy : A Case Report].

We report a case of ureteral carcinoma in which port site metastasis was found after a laparoscopic nephroureterectomy. The patient was a 77-year-old woman with a chiefcomplaint ofgross hematuria. A tumor was found in her left ureter by computed tomography (CT). The patient was diagnosed with a left ureter carcinoma with T2N0M0 or less. She underwent retroperitoneoscopic radical nephroureterectomy. The pathological diagnosis was an urothelial carcinoma, Grade 2, pT2Nx. She was carefully followed up without any adjuvant therapy. At 26 months postoperatively, a subcutaneous tumor was found at a port site without any disseminated disease or distant metastasis by CT and positron emission tomography-CT (PETCT). She underwent surgical resection ofthe subcutaneous tumor. Pathological diagnosis was port site metastatic urothelial carcinoma. She had no recurrence or metastasis at 24 months after the surgical resection without any adjuvant therapy.

Proposal of a Twin Arginine Translocator System-Mediated Constraint against Loss of ATP Synthase Genes from Nonphotosynthetic Plastid Genomes. [Corrected].

Organisms with nonphotosynthetic plastids often retain genomes; their gene contents provide clues as to the functions of these organelles. Yet the functional roles of some retained genes-such as those coding for ATP synthase-remain mysterious. In this study, we report the complete plastid genome and transcriptome data of a nonphotosynthetic diatom and propose that its ATP synthase genes may function in ATP hydrolysis to maintain a proton gradient between thylakoids and stroma, required by the twin arginine translocator (Tat) system for translocation of particular proteins into thylakoids. Given the correlated retention of ATP synthase genes and genes for the Tat system in distantly related nonphotosynthetic plastids, we suggest that this Tat-related role for ATP synthase was a key constraint during parallel loss of photosynthesis in multiple independent lineages of algae/plants.

Entamoeba marina n. sp.; a New Species of Entamoeba Isolated from Tidal Flat Sediment of Iriomote Island, Okinawa, Japan.

The genus Entamoeba includes anaerobic lobose amoebae, most of which are parasites of various vertebrates and invertebrates. We report a new Entamoeba species, E. marina n. sp. that was isolated from a sample of tidal flat sediment collected at Iriomote Island, Okinawa, Japan. Trophozoites of E. marina were 12.8-32.1 µm in length and 6.8-15.9 µm in width, whereas the cysts were 8.9-15.8 µm in diam. and contained four nuclei. The E. marina cells contained a rounded nucleus with a small centric karyosome and uniformly arranged peripheral chromatin. Although E. marina is morphologically indistinguishable from other tetranucleated cyst-forming Entamoeba species, E. marina can be distinguished from them based on the combination of molecular phylogenetic analyses using SSU rDNA gene and the difference of collection sites. Therefore, we propose E. marina as a new species of the genus Entamoeba.

A New Heterotrophic Cryptomonad: Hemiarma marina n. g., n. sp.

We report a new heterotrophic cryptomonad Hemiarma marina n. g., n. sp. that was collected from a seaweed sample from the Republic of Palau. In our molecular phylogenetic analyses using the small subunit ribosomal RNA gene, H. marina formed a clade with two marine environmental sequences, and the clade was placed as a sister lineage of the freshwater cryptomonad environmental clade CRY1. Alternatively, in the concatenated large and small subunit ribosomal RNA gene phylogeny, H. marina was placed as a sister lineage of Goniomonas. Light and electron microscopic observations showed that H. marina shares several ultrastructural features with cryptomonads, such as flattened mitochondrial cristae, a periplast cell covering, and ejectisomes that consist of two coiled ribbon structures. On the other hand, H. marina exhibited unique behaviors, such as attaching to substrates with its posterior flagellum and displaying a jumping motion. H. marina also had unique periplast arrangement and flagellar transitional region. On the basis of both molecular and morphological information, we concluded that H. marina should be treated as new genus and species of cryptomonads.

Trachyrhizium urniformis n. g., n. sp., a Novel Marine Filose Thecate Amoeba Related to a Cercozoan Environmental Clade (Novel Clade 4).

A novel cercozoan filose thecate amoeba, Trachyrhizium urniformis n. g., n. sp., was isolated from a marine sediment sample collected at Agenashiku Island, Okinawa, Japan. We performed light and electron microscopic observations, and a molecular phylogenetic analysis using the small subunit ribosomal RNA gene of the isolate. Cells of T. urniformis are spherical in shape and are covered by a thin theca possessing a wide rounded aperture. Branching and occasionally anastomosing filopodia with small granules emerge from the aperture. The granules are transported in the filopodia bidirectionally. Transmission electron microscopy showed that cells of T. urniformis possess nucleus with permanently condensed chromatin, Golgi apparatuses, microbodies, mitochondria with tubular cristae, and extrusomes. Several morphological and ultrastructural features of T. urniformis (the presence of thecae and nucleus with permanently condensed chromatin) show similarities with those of Thecofilosea. In a phylogenetic analysis, T. urniformis included in Thecofilosea with weak statistical supports and formed a clade with two sequences that constitutes a cercozoan environmental clade, novel clade 4. On the basis of morphological and ultrastructural information and the results of the phylogenetic analysis, we propose T. urniformis as a new member of class Thecofilosea.

Plastid genome sequences of Gymnochlora stellata, Lotharella vacuolata, and Partenskyella glossopodia reveal remarkable structural conservation among chlorarachniophyte species.

Chlorarachniophyte algae have complex plastids acquired by the uptake of a green algal endosymbiont, and this event is called secondary endosymbiosis. Interestingly, the plastids possess a relict endosymbiont nucleus, referred to as the nucleomorph, in the intermembrane space, and the nucleomorphs contain an extremely reduced and compacted genome in comparison with green algal nuclear genomes. Therefore, chlorarachniophyte plastids consist of two endosymbiotically derived genomes, i.e., the plastid and nucleomorph genomes. To date, complete nucleomorph genomes have been sequenced in four different species, whereas plastid genomes have been reported in only two species in chlorarachniophytes. To gain further insight into the evolution of endosymbiotic genomes in chlorarachniophytes, we newly sequenced the plastid genomes of three species, Gymnochlora stellata, Lotharella vacuolata, and Partenskyella glossopodia. Our findings reveal that chlorarachniophyte plastid genomes are highly conserved in size, gene content, and gene order among species, but their nucleomorph genomes are divergent in such features. Accordingly, the current architecture of the plastid genomes of chlorarachniophytes evolved in a common ancestor, and changed very little during their subsequent diversification. Furthermore, our phylogenetic analyses using multiple plastid genes suggest that chlorarachniophyte plastids are derived from a green algal lineage that is closely related to Bryopsidales in the Ulvophyceae group.

Overexpression of molecular chaperone genes in nucleomorph genomes.

Chlorarachniophytes and cryptophytes possess complex plastids that were acquired by the ingestion of a green and red algal endosymbiont, respectively. The plastids are surrounded by four membranes, and a relict nucleus, called the nucleomorph, remains in the periplastidal compartment, which corresponds to the remnant cytoplasm of the endosymbiont. Nucleomorphs contain a greatly reduced genome that possesses only several hundred genes with high evolutionary rates. We examined the relative transcription levels of the genes of all proteins encoded by the nucleomorph genomes of two chlorarachniophytes and three cryptophytes using an RNA-seq transcriptomic approach. The genes of two heat shock proteins, Hsp70 and Hsp90, were highly expressed under normal conditions. It has been shown that molecular chaperone overexpression allows an accumulation of genetic mutations in bacteria. Our results suggest that overexpression of heat shock proteins in nucleomorph genomes may play a role in buffering the mutational destabilization of proteins, which might allow the high evolutionary rates of nucleomorph-encoded proteins.