Mating Pheromone (Gamone 1) in Blepharisma: A Glycoprotein Responsible for Species Diversity in Unicellular Organisms (Alveolata, Ciliophora).

The genus Blepharisma (Alveolata, Ciliophora) is a unicellular organism distributed worldwide, even in extreme environments, and comprises numerous species. While usually proliferating through cell division, Blepharisma undergoes sexual reproduction (conjugation) when cells are moderately starved. Conjugation is initiated by mating pheromones (gamone 1 and gamone 2) secreted by complementary mating-type cells. Gamone 1, a glycoprotein, functions in a species-specific manner, while gamone 2, an amino acid derivative, is a common molecule across species. The specific function of gamone 1 suggests the possibility that mutations in gamone 1 might have led to reproductive isolation during the evolutionary process, triggering species diversification. In this study, by comparing the amino acid sequences of gamone 1 homologs from 15 strains (representing five species), we found that mutations resulting in distinct amino acid properties occur across species boundaries and are mainly concentrated at two specific regions within gamone 1. These mutations potentially alter the binding affinity of gamone 1 to its receptors, suggesting their effect in causing reproductive isolation. The interspecies artificial conjugation conducted previously and the molecular phylogenetic tree constructed using the gamone 1 homolog sequences in this study provide insights into the speciation process within the genus Blepharisma.

Survey of Japanese nephrologists' use of clinical practice guidelines for rapidly progressive glomerulonephritis.

BACKGROUND: The guidelines in Japan for the treatment of rapidly progressive glomerulonephritis (RPGN) have been revised; the latest update was released in 2020. We investigated the actual usage of the new guidelines in Japan. METHODS: We distributed a survey electronically to board-certified nephrologists throughout Japan from December 15, 2021 to January 31, 2022. The survey focused on anti-neutrophil cytoplasmic antibody (ANCA)-associated RPGN and anti-glomerular basement membrane (GBM)-antibody RPGN, plus the treatment strategies and infection-prevention measures used. RESULTS: The survey was completed by 155 certified nephrologists from medical facilities across Japan. Their responses regarding treatment procedures revealed that ANCA-associated RPGN was treated with immunosuppressants and/or biologics by 58.1% of the survey respondents, and with plasma exchange (PE) in combination with corticosteroids by 21.3%. Regarding anti-GBM-antibody RPGN, 78.1% of the respondents used corticosteroids in combination with PE (63.2%), cyclophosphamide (CY) (23.9%), or rituximab (RTX) (8.4%), suggesting a discrepancy between clinical practice and the actual use of the guidelines. Trimethoprim-sulfamethoxazole was prescribed as prophylaxis by 94.8% of the respondents, reflecting the widespread recognition of the need to prevent infectious disease in patients with RPGN. CONCLUSIONS: The survey responses revealed how Japan's new RPGN guidelines are used in actual clinical practice. Our findings will contribute to the guidelines' dissemination and implementation.

Possible Third Step Preventing Conjugation between Different Species of Blepharisma.

In the genus Blepharisma, reproductive isolation between different species appears to be established at least by two barriers: (1) a mating pheromone, i.e., gamone 1, and (2) a factor involved in pair formation. Using four species, we experimentally investigated other potential barriers to interspecific conjugation in Blepharisma, as well as the first and second barriers. Cell-free fluid from type I cells (CFF1) of B. americanum had no effect on B. undulans, B. japonicum, or B. stoltei. Type II cells of B. americanum responded to CFF1 from B. americanum but not to CFF1 from B. undulans, B. japonicum, or B. stoltei. Gamone 1, therefore, seems to be the first reproductive barrier (with the inclusion of B. americanum species [megakaryotype 3]) as reported previously. In pretreated cells with complementary gamones in B. undulans and B. americanum, inter-species pair formation was rare, but pair formation between B. americanum and B. japonicum and between B. americanum and B. stoltei occurred at relatively high frequency. Most of the inter-species B. americanum−B. stoltei pairs underwent nuclear changes specific to conjugation. No significant difference was observed between the intra- and inter-species pairs over the time course of the nuclear changes, but the percentage of abnormal cells was higher in inter-species pairs than in intra-species pairs, and no progenies were produced by inter-species pairs. These results suggest a third barrier or step, in addition to the first and second ones, in nuclear changes after pair formation that prevents interspecific conjugation in Blepharisma.

Recall of extubation after remimazolam anesthesia with flumazenil antagonism during emergence: a retrospective clinical study.

PURPOSE: This study was performed to examine and compare the incidence of extubation recall in surgical patients who underwent remimazolam anesthesia with flumazenil antagonism during emergence and in those who underwent propofol anesthesia. METHODS: One hundred sixty-three patients who underwent surgery using general endotracheal or supraglottic airway anesthesia with propofol (n = 97) or remimazolam (n = 66) were retrospectively analyzed. Remimazolam was antagonized by flumazenil after discontinuation of remimazolam at the end of surgery. The endotracheal tube or supraglottic airway was removed after surgery was complete, and consciousness and adequate spontaneous breathing were confirmed. The incidence of extubation recall was compared between the remimazolam and propofol anesthesia groups using propensity score matching. RESULTS: Extubation recall was observed in 28 patients (17%). After propensity score matching, the incidence of extubation recall did not significantly differ between the remimazolam and propofol anesthesia groups (15.6% vs. 18.8%; p = 1.000). CONCLUSION: The incidence of extubation recall after remimazolam anesthesia with flumazenil antagonism during emergence did not significantly differ from that after propofol anesthesia.

Temporal dynamics of Campylobacter and Arcobacter in a freshwater lake that receives fecal inputs from migratory geese.

Migratory geese could influence the microbiological water quality; however, their impacts on pathogen dynamics remain largely unknown. In this study, we analyzed the population dynamics of Campylobacter and Arcobacter group bacteria (AGB) in a freshwater lake in Japan over two years. The bacteria were quantified by using both culture-dependent and -independent methods. The potential sources of these bacteria were examined by a high-throughput flaA sequencing approach. Campylobacter was abundantly detected both by culture-dependent and -independent methods in the lake, especially when migratory geese were present in the lake. High-throughput flaA sequencing suggests that geese were the likely source of Campylobacter in the lake. The viable population of Campylobacter exceeds the concentrations that can potentially cause 10-4 infections per person per year when water is used to grow fresh vegetables. The occurrence of AGB, on the other hand, was not directly related to the population of migratory geese. AGB were not detected in geese fecal samples. Diverse AGB flaA genotypes occurred in the lake over multiple seasons. Our results suggest that AGB likely comprise a part of the indigenous microbial population of the lake and grow in response to high nutrient, warm temperature, and low dissolved oxygen concentrations in the lake. Geese therefore can indirectly impact the AGB population by providing nutrients to cause eutrophication and lower the dissolved oxygen concentration. Since geese travel long-distance and disperse their fecal microbiota and nutrients to wide areas, they may have significant impacts on water quality and public health.

An Enigmatic Stramenopile Sheds Light on Early Evolution in Ochrophyta Plastid Organellogenesis.

Ochrophyta is an algal group belonging to the Stramenopiles and comprises diverse lineages of algae which contribute significantly to the oceanic ecosystems as primary producers. However, early evolution of the plastid organelle in Ochrophyta is not fully understood. In this study, we provide a well-supported tree of the Stramenopiles inferred by the large-scale phylogenomic analysis that unveils the eukaryvorous (nonphotosynthetic) protist Actinophrys sol (Actinophryidae) is closely related to Ochrophyta. We used genomic and transcriptomic data generated from A. sol to detect molecular traits of its plastid and we found no evidence of plastid genome and plastid-mediated biosynthesis, consistent with previous ultrastructural studies that did not identify any plastids in Actinophryidae. Moreover, our phylogenetic analyses of particular biosynthetic pathways provide no evidence of a current and past plastid in A. sol. However, we found more than a dozen organellar aminoacyl-tRNA synthases (aaRSs) that are of algal origin. Close relationships between aaRS from A. sol and their ochrophyte homologs document gene transfer of algal genes that happened before the divergence of Actinophryidae and Ochrophyta lineages. We further showed experimentally that organellar aaRSs of A. sol are targeted exclusively to mitochondria, although organellar aaRSs in Ochrophyta are dually targeted to mitochondria and plastids. Together, our findings suggested that the last common ancestor of Actinophryidae and Ochrophyta had not yet completed the establishment of host-plastid partnership as seen in the current Ochrophyta species, but acquired at least certain nuclear-encoded genes for the plastid functions.

Cytosolic Glutamine Synthetase GS1;3 Is Involved in Rice Grain Ripening and Germination.

Ammonium is combined with glutamate to form glutamine. This reaction is catalyzed by glutamine synthetase (GS or GLN). Plants harbor several isoforms of cytosolic GS (GS1). Rice GS1;3 is highly expressed in seeds during grain filling and germination, suggesting a unique role in these processes. This study aimed to investigate the role of GS1;3 for rice growth and yield. Tos17 insertion lines for GS1;3 were isolated, and the nitrogen (N), amino acid, and ammonium contents of GS1;3 mutant grains were compared to wild-type grains. The spatiotemporal expression of GS1;3 and the growth and yield of rice plants were evaluated in hydroponic culture and the paddy field. Additionally, the stable isotope of N was used to trace the foliar N flux during grain filling. Results showed that the loss of GS1;3 retarded seed germination. Seeds of GS1;3 mutants accumulated glutamate but did not show a marked change in the level of phytohormones. The expression of GS1;3 was detected at the beginning of germination, with limited promoter activity in seeds. GS1;3 mutants showed a considerably decreased ripening ratio and decreased N efflux in the 12th leaf blade under N deficient conditions. The ß-glucuronidase gene expression under control of the GS1;3 promoter was detected in the vascular tissue and aleurone cell layer of developing grains. These data suggest unique physiological roles of GS1;3 in the early stage of seed germination and grain filling under N deficient conditions in rice.

Mitochondrial DNA deletion-dependent podocyte injuries in Mito-miceΔ, a murine model of mitochondrial disease.

Focal segmental glomerulosclerosis (FSGS) is a major renal complication of human mitochondrial disease. However, its pathogenesis has not been fully explained. In this study, we focused on the glomerular injury of mito-miceΔ and investigated the pathogenesis of their renal involvement. We analyzed biochemical data and histology in mito-miceΔ. The proteinuria began to show in some mito-miceΔ with around 80% of mitochondrial DNA deletion, then proteinuria developed dependent with higher mitochondrial DNA deletion, more than 90% deletion. Mito-miceΔ with proteinuria histologically revealed FSGS. Immunohistochemistry demonstrated extensive distal tubular casts due to abundant glomerular proteinuria. Additionally, the loss of podocyte-related protein and podocyte's number were found. Therefore, the podocyte injuries and its depletion had a temporal relationship with the development of proteinuria. This study suggested mitochondrial DNA deletion-dependent podocyte injuries as the pathogenesis of renal involvement in mito-miceΔ. The podocytes are the main target of mitochondrial dysfunction originated from the accumulation of mitochondrial DNA abnormality in the kidney.

Immunohistological score of transcription factor 21 had a positive correlation with its urinary excretion and proteinuria in immunoglobulin A nephropathy.

Transcription factor 21 (TCF21) contributes to mammalian nephrogenesis, and especially to glomerular maturation. Our previous study suggested its influence on glomerular injury, showing that TCF21 expression in podocytes had a positive correlation with the urinary protein value and also with the urinary TCF21 concentration. We now focus on its influence on the clinical course of immunoglobulin A nephropathy (IgAN), as patients with IgAN constitute the largest population of individuals with primary chronic glomerulonephritis in the world. Twenty cases of IgAN were divided into two groups according to the immunohistological score (IHS) of glomerular TCF21 expression: group IHS1 (n=7) and group IHS2+3 (n=13). Sixteen of the 20 cases were followed up for 2 years. Group IHS2+3 had heavier urinary protein (p=0.03) and a greater urinary TCF21 level (p<0.001) compared to group IHS1 at baseline. None of the other factors including hematuria, estimated glomerular filtration rate (eGFR), or the Oxford classification showed a statistically significant difference between these two groups. At the 2-year follow-up, even though the rate of remission in urinary protein, hematuria and the eGFR decline were not statistically correlated to IHS, the IHS2+3 group had a slight tendency toward a steeper eGFR decline compared to IHS1 (p=0.31). The present study suggested that the higher IHS of TCF21 corresponded to heavier proteinuria and a higher urinary TCF21 level in IgAN. This could be the first step in determining the TCF21 value for predicting the prognosis for IgAN.

Literature review of allograft adenovirus nephritis and a case presenting as mass lesions in a transplanted kidney without symptoms of urinary tract infection or acute kidney injury.

Adenovirus (AdV) infection is a common complication in bone marrow/hematopoietic stem cell transplant and solid organ transplant recipients. AdV infection usually presents as hemorrhagic cystitis, but sometimes it can progress to acute kidney injury showing AdV nephritis (AdVN). We present the case of a 52-year-old Japanese female who had received a living kidney transplantation (KT) from her husband. At 21 months post-KT, the patient presented with a fever, but no renal dysfunction and no abnormal urine findings. A contrast-enhanced computed tomography (CT) scan revealed a few mass lesions with hypoperfusion in the transplanted kidney. An enhanced CT-guided biopsy targeting one of these lesions revealed a necrotizing tubulointerstitial nephritis suggesting AdVN. The polymerase chain reaction tests for ADV were negative in a urine sample but positive in the sera and the frozen kidney biopsy samples. AdVN can manifest as an unusual pattern of acute lobar nephritis/acute focal bacterial nephritis-like localization without symptoms of acute kidney injury or urinary tract infection. Enhanced CT can provide clues for clinical diagnosis.

Anti-glomerular Basement Membrane Glomerulonephritis During the First Trimester of Pregnancy.

A 28-year-old woman was admitted during the eighth week of her pregnancy because her clinical course was consistent with rapid progressive glomerulonephritis (RPGN). Anti-glomerular basement membrane antibody (anti-GBM Ab) and myeloperoxidase anti-neutrophil cytoplasmic antibody (MPO-ANCA) were positive, and the anti-GBM Ab titer being extremely high. She was treated with hemodialysis, plasma exchange and prednisolone. She survived the illness; however, neither the fetus nor her kidney function could be rescued. She had human leukocyte antigen (HLA)-DRB1*1502:01, which differs from the DRB1*1501 associated with anti-GBM GN. When patients have particular symptoms, we should check the urine and serum creatinine to exclude RPGN, even in cases of pregnancy.

Transcription factor 21 expression in injured podocytes of glomerular diseases.

Transcription factor 21 (TCF21) is one of the essential transcription factors in kidney development. To elucidate its influence on glomerular disease, we have investigated TCF21 expression in human and rat kidney tissue, and its urinary concentration. Immunohistological analysis suggested the highest TCF21 expression in nephrotic syndrome along with the urinary protein level. Urinary TCF21 concentration in human showed a positive correlation with its podocyte expression level. In nephrotic rat models, TCF21 expression in podocytes increased along with the severity of nephrotic syndrome. Next, in vitro experiments using Tcf21-expressing murine podocyte cell line, we could observe some Tcf21-dependent effects, related with actin cytoskeleton dysregulation and apoptosis. Our study illustrated TCF21 expression changes in vivo and its in vitro-functional significance injured podocytes.

Age-dependent survival in rapidly progressive glomerulonephritis: A nationwide questionnaire survey from children to the elderly.

BACKGROUND: Rapidly progressive glomerulonephritis (RPGN) has been known to have a poor prognosis. Although evidence across adult RPGN cases has accumulated over many years, the number of case series in adolescents and young adults has been limited, requiring further studies. METHODS: A total of 1,766 cases from 1989 to 2007 were included in this nationwide questionnaire survey, led by Intractable (former name, Progressive) Renal Diseases Research, Research on intractable disease, from the Ministry of Health, Labour and Welfare of Japan. To elucidate age-related differences in 2-year patient and renal survival rates, the cases were divided into the following four groups: children (0-18 years), young adults (19-39 years), the middle-aged (40-64 years), and the elderly (over 65 years). RESULTS: Of the 1,766 total RPGN cases, antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis comprised 1,128 cases (63.9% of all RPGN cases), showing a tendency to increase with age. Two-year patient survival for RPGN was 93.9% among children, 92.6% in young adults, 83.2% in the middle-aged, and 68.8% in the elderly. The younger group (children plus young adults) showed a clearly higher survival rate compared to the older group (middle-aged plus elderly) (p<0.05). ANCA-associated glomerulonephritis also showed similar age-related results with all RPGN cases. The comparison of renal prognosis showed no statistically significant differences both in RPGN and in ANCA-associated GN. CONCLUSION: The present study described the age-dependent characteristics of the classification of RPGN, especially focusing on a better prognosis of the younger group in patient survival both in RPGN and in ANCA-associated GN.

Microsatellite DNA markers applicable to paternity inference in the androdioecious gooseneck barnacle Octolasmis warwickii (Lepadiformes: Poecilasmatidae).

The gooseneck barnacle Octolasmis warwickii has a rare sexual system called androdioecy, in which hermaphrodites and dwarf males co-occur. It has been hypothesized that dwarf males can coexist with conspecific hermaphrodites when dwarf males are capable of leaving more offspring than hermaphrodites via male reproduction. This hypothesis of reproductive superiority of dwarf males can be validated by comparing the reproductive success between dwarf males and hermaphrodites through DNA marker-based parentage testing. In the present study, we developed microsatellite DNA markers for O. warwickii, and evaluated the power of these markers to infer parentage based on simulation analysis. Using next generation sequencing, we obtained 344 microsatellite sequences suitable for designing primer sets for amplification in polymerase chain reaction (PCR). Of these, we examined the PCR amplification efficiency of 54 primer sets, of which 11 passed our primer screening in a population sample (n = 35). The developed markers exhibited moderate to high levels of polymorphisms, and met Hardy-Weinberg equilibrium with little evidence of significant allelic association to each other. Our simulated paternity inference suggested that the combinational use of the markers allows a high resolution of parentage (success rate of > 99.9%) if all candidate fathers are available.

An international validation study of the IL-2 Luc assay for evaluating the potential immunotoxic effects of chemicals on T cells and a proposal for reference data for immunotoxic chemicals.

To evaluate the immunotoxic effects of xenobiotics, we have established the Multi-ImmunoTox assay, in which three stable reporter cell lines are used to evaluate the effects of chemicals on the IL-2, IFN-γ, IL-1ß and IL-8 promoters. Here, we report the official validation study of the IL-2 luciferase assay (IL-2 Luc assay). In the Phase I study that evaluated five coded chemicals in three sets of experiments, the average within-laboratory reproducibility was 86.7%. In the Phase II study, 20 coded chemicals were evaluated at multiple laboratories. In the combined results of the Phase I and II studies, the between-laboratory reproducibility was 80.0%. These results suggested that the IL-2 Luc assay was reproducible both between and within laboratories. To determine the predictivity, we collected immunotoxicological information and constructed the reference data by classifying the chemical into immunotoxic compounds targeting T cells or others according to previously reported criteria. When compared with the reference data, the average predictivity of the Phase I and II studies was 75.0%, while that of additional 60 chemicals examined by the lead laboratory was 82.5%. Although the IL-2 Luc assay alone is not sufficient to predict immunotoxicity, it will be a useful tool when combined with other immune tests.

Cytosolic GLUTAMINE SYNTHETASE1;1 Modulates Metabolism and Chloroplast Development in Roots.

Nitrogen (N) is an essential macronutrient, and the final form of endogenous inorganic N is ammonium, which is assimilated by Gln synthetase (GS) into Gln. However, how the multiple isoforms of cytosolic GSs contribute to metabolic systems via the regulation of ammonium assimilation remains unclear. In this study, we compared the effects of two rice (Oryza sativa) cytosolic GSs, namely OsGS1;1 and OsGS1;2, on central metabolism in roots using reverse genetics, metabolomic and transcriptomic profiling, and network analyses. We observed (1) abnormal sugar and organic N accumulation and (2) significant up-regulation of genes associated with photosynthesis and chlorophyll biosynthesis in the roots of Osgs1;1 but not Osgs1;2 knockout mutants. Network analysis of the Osgs1;1 mutant suggested that metabolism of Gln was coordinated with the metabolic modules of sugar metabolism, tricarboxylic acid cycle, and carbon fixation. Transcript profiling of Osgs1;1 mutant roots revealed that expression of the rice sigma-factor (OsSIG) genes in the mutants were transiently upregulated. GOLDEN2-LIKE transcription factor-encoding genes, which are involved in chloroplast biogenesis in rice, could not compensate for the lack of OsSIGs in the Osgs1;1 mutant. Microscopic analysis revealed mature chloroplast development in Osgs1;1 roots but not in the roots of Osgs1;2, Osgs1;2-complemented lines, or the wild type. Thus, organic N assimilated by OsGS1;1 affects a broad range of metabolites and transcripts involved in maintaining metabolic homeostasis and plastid development in rice roots, whereas OsGS1;2 has a more specific role, affecting mainly amino acid homeostasis but not carbon metabolism.

Pediludiella daitoensis gen. et sp. nov. (Scenedesmaceae, Chlorophyceae), a large coccoid green alga isolated from a Loxodes ciliate.

Freshwater protists often harbor unicellular green algae within their cells. In ciliates, possibly because of large host cell sizes and the small size of algal coccoids, a single host cell typically contains more than a hundred algal cells. While surveying such algae-bearing protists on Minami Daito Jima Island in Japan, we found a green Loxodes ciliate (Loxodida, Karyorelictea) that contained one or two dozens of very large coccoid algae. We isolated one of these algae and analyzed its characteristics in detail. A small subunit (SSU) rDNA phylogeny indicated Pseudodidymocystis species (Scenedesmaceae, Chlorophyceae) to be the taxon closest to the alga, although it was clearly separated from this by 39 or more different sites (inclusive of gaps). SSU rRNA structure analyses indicated that these displacements included eight compensatory base changes (CBCs) and seven hemi-CBCs. We therefore concluded that this alga belongs to a separate genus, and described it as Pediludiella daitoensis gen. et sp. nov. The shape of the isolated and cultured P. daitoensis was nearly spherical and reached up to 30 µm in diameter. Chloroplasts were arranged peripherally and often split and elongated. Cells were often vacuolated and possessed a net-like cytoplasm that resembled a football (soccer ball) in appearance, which was reflected in the genus name.

Kidney transplant patient with immunoglobulin A nephropathy subsequently diagnosed as concurrent autosomal dominant polycystic kidney disease during 17-year follow-up.

A 14-year-old Japanese boy was diagnosed with immunoglobulin A nephropathy resulting in end-stage kidney disease (ESKD). He underwent ABO-compatible living kidney transplantation from his father at the age of 27. In the process of selecting a donor before the transplantation, it turned out that his mother had polycystic kidneys and that her family had a history of hypertension and cerebrovascular diseases. The patient himself also had bilateral multiple kidney cysts, with a normal-sized kidney, confusing us to make the diagnosis of acquired cystic kidney disease (ACKD) or ADPKD difficult at that point. Seventeen years later, his native kidneys showed bilateral swelling with multiple cysts. This, along with the histories of his mother and her relatives and with the existence of multiple liver cysts, led us to confirm the diagnosis of autosomal dominant polycystic kidney disease, not of ACKD. Contrary to previous studies that have suggested the size of cysts both in ADPKD and ACKD reduced with time, the present case showed an increase of 3.0% per year in total kidney volume (TKV) by computed tomography. It suggested the possibility that TKV, after decreasing in the relatively early stage after transplantation, may later increase in the long term after ESKD due to another kidney injury.