Dual role of Ovol2 on the germ cell lineage segregation during gastrulation in mouse embryogenesis.

In mammals, primordial germ cells (PGCs), the origin of the germ line, are specified from the epiblast at the posterior region where gastrulation simultaneously occurs, yet the functional relationship between PGC specification and gastrulation remains unclear. Here, we show that OVOL2, a transcription factor conserved across the animal kingdom, balances these major developmental processes by repressing the epithelial-to-mesenchymal transition (EMT) that drives gastrulation and the upregulation of genes associated with PGC specification. Ovol2a, a splice variant encoding a repressor domain, directly regulates EMT-related genes and, consequently, induces re-acquisition of potential pluripotency during PGC specification, whereas Ovol2b, another splice variant missing the repressor domain, directly upregulates genes associated with PGC specification. Taken together, these results elucidate the molecular mechanism underlying allocation of the germ line among epiblast cells differentiating into somatic cells through gastrulation. This article has an associated 'The people behind the papers' interview.

The 33rd International Conference on Arabidopsis Research (ICAR2023).

The 33rd International Conference on Arabidopsis Research (ICAR2023) was held at Makuhari Messe International Conference Hall in Chiba prefecture from June 5 to 9, 2023. This annual conference, which rotates among hosts in North America, Asia-Oceania, and Europe, covers the full range of plant biology research involving Arabidopsis and other plant species. The conference hosted more than 1200 participants, including approximately 800 international attendees from 42 countries (or regions), and featured about 900 oral and poster presentations. Reflecting the conference theme, "Arabidopsis for Sustainable Development Goals (SDGs)," there were numerous exemplary presentations regarding basic plant science using Arabidopsis and translational research conducted to achieve SDGs by exploiting the knowledge gained from Arabidopsis to improve crop production. The conference concluded on a high note, with more than 99% of survey respondents expressing their general satisfaction with ICAR2023. This report aims to summarize the organization, objectives, and outcomes of the conference.

A decision tree model for predicting high mono-N-desethylamiodarone concentrations and reducing tissue toxicity in patients with low-dose amiodarone therapy: A multicentral retrospective cohort study.

OBJECTIVE: High plasma levels of mono-N-desethylamiodarone (MDEA), an active amiodarone metabolite, may be associated with tissue toxicity in heart failure (patients with heart rhythm disturbances); therefore, a tool that can identify patients for whom therapeutic drug monitoring (TDM) of MDEA is required. This multicenter study aimed to develop a decision tree (DT) model that can identify patients with heart rhythm disturbances at high MDEA concentrations. MATERIALS AND METHODS: A multicenter retrospective cohort study was conducted, including 157 adult patients with heart failure who received oral amiodarone treatment. A χ2 automatic interaction-detection algorithm was used to construct a DT model. In the DT analysis, the dependent variable was set as an MDEA trough plasma concentration of ≥ 0.6 µg/mL during the steady-state period. Explanatory variables were selected as factors with p < 0.05 in multivariate logistic regression analysis. RESULTS: The adjusted odds ratios for the daily dose of amiodarone and body mass index were 1.01 (95% coefficient interval: 1.008 - 1.021, p < 0.001) and 0.91 (95% confidence interval: 0.834 - 0.988, p = 0.025), respectively. For DT analysis, the risk of reaching plasma MDEA concentrations ≥ 0.6 µg/mL was relatively high, combined with a daily dose of amiodarone > 100 mg and body mass index ≤ 22.3 kg/m2 at 69.0% (20/29), and its trend was also detected in the sensitivity analysis. CONCLUSION: Patients taking a daily amiodarone dose > 100 mg and with a body mass index ≤ 22.3 kg/m2 warrant TDM implementation for MDEA to minimize the risk of MDEA-induced tissue toxicity.

Widespread and transgenerational retrotransposon activation in inter- and intraspecies recombinant inbred populations of Lotus japonicus.

Transposable elements (TEs) constitute a large proportion of genomes of multicellular eukaryotes, including flowering plants. TEs are normally maintained in a silenced state and their transpositions rarely occur. Hybridization between distant species has been regarded as a 'shock' that stimulates genome reorganization, including TE mobilization. However, whether crosses between genetically close parents that result in viable and fertile offspring can induce TE transpositions has remained unclear. Here, we investigated the activation of long terminal repeat (LTR) retrotransposons in three Lotus japonicus recombinant inbred line (RIL) populations. We found that at least six LTR retrotransposon families were activated and transposed in 78% of the RILs investigated. LORE1a, one of the transposed LTR retrotransposons, showed transgenerational epigenetic activation, indicating the long-term effects of epigenetic instability induced by hybridization. Our study highlights TE activation as an unexpectedly common event in plant reproduction.

Identification of Chemicals That Abrogate Folate-Dependent Inhibition of Starch Accumulation in Non-Photosynthetic Plastids of Arabidopsis.

Folate, also known as vitamin B9, is an essential cofactor for a variety of enzymes and plays a crucial role in many biological processes. We previously reported that plastidial folate prevents starch biosynthesis triggered by the influx of sugar into non-starch-accumulating plastids, such as etioplasts, and chloroplasts under darkness; hence the loss of plastidial folate induces the accumulation of starch in plastids. To understand the molecular mechanism underlying this phenomenon, we screened our in-house chemical library and searched their derivatives to identify chemicals capable of inducing starch accumulation in etioplasts. The results revealed four chemicals, compounds #120 and #375 and their derivatives, compounds #120d and #375d, respectively. The derivative compounds induced starch accumulation in etioplasts and suppressed hypocotyl elongation in dark-grown Arabidopsis seedlings. They also inhibited the post-germinative growth of seedlings under illumination. All four chemicals contained the sulfonamide group as a consensus structure. The sulfonamide group is also found in sulfa drugs, which exhibit antifolate activity, and in sulfonylurea herbicides. Further analyses revealed that compound #375d induces starch accumulation by inhibiting folate biosynthesis. By contrast, compound #120d neither inhibited folate biosynthesis nor exhibited the herbicide activity. Protein and metabolite analyses suggest that compound #120d abrogates folate-dependent inhibition of starch accumulation in etioplasts by enhancing starch biosynthesis.

Cefcapene pivoxil-induced hypocarnitinemic hypoglycemia in elderly man with subclinical ACTH deficiency: a case report.

BACKGROUND: Drug-induced hypocarnitinemia has been noted as a cause of hypoglycemia in children. However, adult cases are extremely rare and pre-existing conditions (including endocrine disorders and frailty) have been suggested to be involved. Hypoglycemia due to drug-induced hypocarnitinemia is quite rare, and there were few reports of pivoxil-containing cephalosporin (PCC)-induced hypocarnitinemia in adults. CASE PRESENTATION: We present a case of an 87-year-old man with malnutrition, and frailty. He developed severe hypoglycemia with unconsciousness after taking cefcapene pivoxil hydrochloride, one of PCC, and hypocarnitinemia was diagnosed. Despite levocarnitine administration, asymptomatic mild hypoglycemia had persisted. Subsequent investigation revealed subclinical ACTH deficiency due to empty sella, which played a key role to maintain mild hypoglycemia as underlying disorder, and PCC-induced hypocarnitinemia triggered severe hypoglycemia. The patient responded to hydrocortisone therapy. CONCLUSIONS: We need to be aware of the facts that PCC can induce severe hypocarnitinemic hypoglycemia in elderly adults associated with frailty, malnutrition, and subclinical ACTH syndrome.

Machine Learning-Based Prediction of Digoxin Toxicity in Heart Failure: A Multicenter Retrospective Study.

Digoxin toxicity (plasma digoxin concentration ≥0.9 ng/mL) is associated with worsening heart failure (HF). Decision tree (DT) analysis, a machine learning method, has a flowchart-like model where users can easily predict the risk of adverse drug reactions. The present study aimed to construct a flowchart using DT analysis that can be used by medical staff to predict digoxin toxicity. We conducted a multicenter retrospective study involving 333 adult patients with HF who received oral digoxin treatment. In this study, we employed a chi-squared automatic interaction detection algorithm to construct DT models. The dependent variable was set as the plasma digoxin concentration (≥ 0.9 ng/mL) in the trough during the steady state, and factors with p < 0.2 in the univariate analysis were set as the explanatory variables. Multivariate logistic regression analysis was conducted to validate the DT model. The accuracy and misclassification rates of the model were evaluated. In the DT analysis, patients with creatinine clearance <32 mL/min, daily digoxin dose ≥1.6 µg/kg, and left ventricular ejection fraction ≥50% showed a high incidence of digoxin toxicity (91.8%; 45/49). Multivariate logistic regression analysis revealed that creatinine clearance <32 mL/min and daily digoxin dose ≥1.6 µg/kg were independent risk factors. The accuracy and misclassification rates of the DT model were 88.2 and 46.2 ± 2.7%, respectively. Although the flowchart created in this study needs further validation, it is straightforward and potentially useful for medical staff in determining the initial dose of digoxin in patients with HF.

Sleep-related factors and circulating levels of sex hormones in premenopausal Japanese women.

Sleep disruption and circadian disruption have been proposed to be risk factors of breast cancer. The present study examined the associations of sleep-related factors, referring to night shift work, sleep habits, and sleep disturbances, with the plasma levels of sex hormones in premenopausal Japanese women. Study participants were 432 women who had regular menstrual cycles less than 40 days long. Information on their history of night shift work and sleep disturbances was obtained using a self-administered questionnaire. Information on their sleep habits, such as usual wake-up times, bedtimes, and ambient light level while sleeping, was obtained in an interview. The participants' height and weight were measured. Plasma concentrations of estradiol, testosterone, dehydroepiandrosterone sulfate, sex hormone-binding globulin (SHBG), FSH, and LH were also measured. After controlling for the phase of the menstrual cycle and other covariates, more years of night shift work ≥ once a week during the past 10 years was significantly associated with a lower SHBG and a higher free estradiol level. Shorter sleep duration was significantly associated with the higher total, bioavailable, and free testosterone levels. Sleep disturbance by awaking after sleep onset was significantly associated with a high free estradiol level. The data suggest that long-term night shift work, short sleep duration, and arousal during sleep are associated with higher estradiol or testosterone levels in premenopausal women.

The rhizobial autotransporter determines the symbiotic nitrogen fixation activity of Lotus japonicus in a host-specific manner.

Leguminous plants establish endosymbiotic associations with rhizobia and form root nodules in which the rhizobia fix atmospheric nitrogen. The host plant and intracellular rhizobia strictly control this symbiotic nitrogen fixation. We recently reported a Lotus japonicus Fix- mutant, apn1 (aspartic peptidase nodule-induced 1), that impairs symbiotic nitrogen fixation. APN1 encodes a nodule-specific aspartic peptidase involved in the Fix- phenotype in a rhizobial strain-specific manner. This host-strain specificity implies that some molecular interactions between host plant APN1 and rhizobial factors are required, although the biological function of APN1 in nodules and the mechanisms governing the interactions are unknown. To clarify how rhizobial factors are involved in strain-specific nitrogen fixation, we explored transposon mutants of Mesorhizobium loti strain TONO, which normally form Fix- nodules on apn1 roots, and identified TONO mutants that formed Fix+ nodules on apn1 The identified causal gene encodes an autotransporter, part of a protein secretion system of Gram-negative bacteria. Expression of the autotransporter gene in M. loti strain MAFF3030399, which normally forms Fix+ nodules on apn1 roots, resulted in Fix- nodules. The autotransporter of TONO functions to secrete a part of its own protein (a passenger domain) into extracellular spaces, and the recombinant APN1 protein cleaved the passenger protein in vitro. The M. loti autotransporter showed the activity to induce the genes involved in nodule senescence in a dose-dependent manner. Therefore, we conclude that the nodule-specific aspartic peptidase, APN1, suppresses negative effects of the rhizobial autotransporter in order to maintain effective symbiotic nitrogen fixation in root nodules.

Multi-omics analysis on an agroecosystem reveals the significant role of organic nitrogen to increase agricultural crop yield.

Both inorganic fertilizer inputs and crop yields have increased globally, with the concurrent increase in the pollution of water bodies due to nitrogen leaching from soils. Designing agroecosystems that are environmentally friendly is urgently required. Since agroecosystems are highly complex and consist of entangled webs of interactions between plants, microbes, and soils, identifying critical components in crop production remain elusive. To understand the network structure in agroecosystems engineered by several farming methods, including environmentally friendly soil solarization, we utilized a multiomics approach on a field planted with Brassica rapa We found that the soil solarization increased plant shoot biomass irrespective of the type of fertilizer applied. Our multiomics and integrated informatics revealed complex interactions in the agroecosystem showing multiple network modules represented by plant traits heterogeneously associated with soil metabolites, minerals, and microbes. Unexpectedly, we identified soil organic nitrogen induced by soil solarization as one of the key components to increase crop yield. A germ-free plant in vitro assay and a pot experiment using arable soils confirmed that specific organic nitrogen, namely alanine and choline, directly increased plant biomass by acting as a nitrogen source and a biologically active compound. Thus, our study provides evidence at the agroecosystem level that organic nitrogen plays a key role in plant growth.

NDR1/HIN1-Like Protein 13 Interacts with Symbiotic Receptor Kinases and Regulates Nodulation in Lotus japonicus.

Lysin-motif receptor-like kinases (LysM-RLKs) are involved in the recognition of microbe-associated molecular patterns to initiate pattern-triggered immunity (PTI). LysM-RLKs are also required for recognition of microbe-derived symbiotic signal molecules upon establishing mutualistic interactions between plants and microsymbionts. A LysM-RLK CHITIN ELICITOR RECEPTOR KINASE1 (CERK1) plays central roles both in chitin-mediated PTI and in arbuscular mycorrhizal symbiosis, suggesting the overlap between immunity and symbiosis, at least in the signal perception and the activation of downstream signal cascades. In this study, we screened for the interacting proteins of Nod factor Receptor1 (NFR1), a CERK1 homolog in the model legume Lotus japonicus, and obtained a protein orthologous to NONRACE-SPECIFIC DISEASE RESISTANCE1/HARPIN-INDUCED1-LIKE13 (NHL13), a protein involved in the activation of innate immunity in Arabidopsis thaliana, which we named LjNHL13a. LjNHL13a interacted with NFR1 and with the symbiosis receptor kinase SymRK. LjNHL13a also displayed positive effects in nodulation. Our results suggest that NHL13 plays a role both in plant immunity and symbiosis, possibly where they overlap. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

GFS9 Affects Piecemeal Autophagy of Plastids in Young Seedlings of Arabidopsis thaliana.

Chloroplasts, and plastids in general, contain abundant protein pools that can be major sources of carbon and nitrogen for recycling. We have previously shown that chloroplasts are partially and sequentially degraded by piecemeal autophagy via the Rubisco-containing body. This degradation occurs during plant development and in response to the environment; however, little is known about the fundamental underlying mechanisms. To discover the mechanisms of piecemeal autophagy of chloroplasts/plastids, we conducted a forward-genetics screen following ethyl-methanesulfonate mutagenesis of an Arabidopsis (Arabidopsis thaliana) transgenic line expressing chloroplast-targeted green fluorescent protein (CT-GFP). This screen allowed us to isolate a mutant, gfs9-5, which hyperaccumulated cytoplasmic bodies labeled with CT-GFP of up to 1.0 µm in diameter in the young seedlings. We termed these structures plastid bodies (PBs). The mutant was defective in a membrane-trafficking factor, green fluorescent seed 9 (GFS9), and PB accumulation in gfs9-5 was promoted by darkness and nutrient deficiency. Transmission electron microscopy indicated that gfs9-5 hyperaccumulated structures corresponding to autophagosomes and PBs. gfs9-5 hyperaccumulated membrane-bound endogenous ATG8 proteins, transgenic yellow fluorescent protein (YFP)-ATG8e proteins and autophagosome-like structures labeled with YFP-ATG8e. The YFP-ATG8e signal was associated with the surface of plastids and their protrusions in gfs9-5. Double mutants of gfs9 and autophagy-defective 5 did not accumulate PBs. In gfs9-5, the YFP-ATG8e proteins and PBs could be delivered to the vacuole and autophagic flux was increased. We discuss a possible connection between GFS9 and autophagy and propose a potential use of gfs9-5 as a new tool to study piecemeal plastid autophagy.

Growth and Nitrate Reductase Activity Are Impaired in Rice Osnlp4 Mutants Supplied with Nitrate.

Nitrate is an important nutrient and signaling molecule in plants, which modulates the expression of many genes and regulates plant growth. In paddy-grown rice (Oryza sativa), nitrogen is mostly supplied in the form of ammonium but can also be supplied in the form of nitrate. Several nitrogen transporters and nitrate assimilation enzymes have been identified and functionally characterized in rice. However, little is known regarding the nitrate sensing system in rice, and the regulatory mechanisms of nitrate-related genes remain to be elucidated. In recent years, NIN-like proteins (NLPs) have been described as key transcription factors of nitrogen responses in Arabidopsis thaliana, which implies that OsNLP4 is involved in the regulation of nitrate assimilation and nitrogen use efficiency in rice. Here, we show that OsNLP4 can influence plant growth by affecting nitrate reductase (NR) activity. The growth of OsNLP4 knockdown mutants was reduced when nitrate was supplied, but not when ammonium was supplied. The nitrate concentration was significantly reduced in osnlp4 mutants. Furthermore, the concentrations of iron and molybdenum, essential elements for NR activity, were reduced in OsNLP4 knockdown mutants. We propose that, in addition to the regulation of gene expression within the nitrate signaling pathway, OsNLP4 can affect the NR activity and nitrate-dependent growth of rice. Our results support a working model for the role of OsNLP4 in the nitrate signaling pathway.

Cell death during crisis is mediated by mitotic telomere deprotection.

Tumour formation is blocked by two barriers: replicative senescence and crisis. Senescence is triggered by short telomeres and is bypassed by disruption of tumour-suppressive pathways. After senescence bypass, cells undergo crisis, during which almost all of the cells in the population die. Cells that escape crisis harbour unstable genomes and other parameters of transformation. The mechanism of cell death during crisis remains unexplained. Here we show that human cells in crisis undergo spontaneous mitotic arrest, resulting in death during mitosis or in the following cell cycle. This phenotype is induced by loss of p53 function, and is suppressed by telomerase overexpression. Telomere fusions triggered mitotic arrest in p53-compromised non-crisis cells, indicating that such fusions are the underlying cause of cell death. Exacerbation of mitotic telomere deprotection by partial TRF2 (also known as TERF2) knockdown increased the ratio of cells that died during mitotic arrest and sensitized cancer cells to mitotic poisons. We propose a crisis pathway wherein chromosome fusions induce mitotic arrest, resulting in mitotic telomere deprotection and cell death, thereby eliminating precancerous cells from the population.

The telomere deprotection response is functionally distinct from the genomic DNA damage response.

Loss of chromosome end protection through telomere erosion is a hallmark of aging and senescence. Here we developed an experimental system that mimics physiological telomere deprotection in human cells and discovered that the telomere deprotection response is functionally distinct from the genomic DNA damage response. We found that, unlike genomic breaks, deprotected telomeres that are recognized as DNA damage but remain in the fusion-resistant intermediate state activate differential ataxia telangiectasia mutated (ATM) signaling where CHK2 is not phosphorylated. Also unlike genomic breaks, we found that deprotected telomeres do not contribute to the G2/M checkpoint and are instead passed through cell division to induce p53-dependent G1 arrest in the daughter cells. Telomere deprotection is therefore an epigenetic signal passed between cell generations to ensure that replication-associated telomere-dependent growth arrest occurs in stable diploid G1 phase cells before genome instability can occur.

Role of macrophage-mediated Toll-like receptor 4-interleukin-1R signaling in ectopic tongue pain associated with tooth pulp inflammation.

BACKGROUND: The existence of referred pain and ectopic paresthesia caused by tooth pulp inflammation may make definitive diagnosis difficult and cause misdiagnosis or mistreatment; thus, elucidation of that molecular mechanism is urgent. In the present study, we investigated the mechanisms underlying ectopic pain, especially tongue hyperalgesia, after tooth pulp inflammation. METHODS: A rat model with mandibular first molar tooth pulp exposure was employed. Tooth pulp exposure-induced heat and mechanical-evoked tongue hypersensitivity was measured, and immunohistochemical staining for Iba1, a marker of active macrophages, IL-1ß, IL-1 type I receptor (IL-1RΙ), and toll-like receptor 4 in the trigeminal ganglion was performed. In addition, we investigated the effects of injections of liposomal clodronate Clophosome-A (LCCA), a selective macrophage depletion agent, lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS, a toll-like receptor 4 antagonist), IL-1ß, or heat shock protein 70 (Hsp70, a selective agonist of toll-like receptor 4), to examine changes in tongue hypersensitivity and in the regulation of IL-1RΙ, toll-like receptor 4, and transient receptor potential vanilloid 1 (TRPV1) biosynthesis. RESULTS: At day 1 after tooth pulp exposure, obvious tooth pulp inflammation was observed. Tooth pulp exposure-induced heat and mechanical tongue hypersensitivity was observed from days 1 to 3 after tooth pulp exposure. The production of IL-1ß in activated macrophages and toll-like receptor 4 and IL-1RΙ expression were significantly increased in trigeminal ganglion neurons innervating the tongue following tooth pulp exposure. Intra-trigeminal ganglion injection of LCCA significantly suppressed tongue hypersensitivity; however, toll-like receptor 4 and IL-1RΙ expression in trigeminal ganglion neurons innervating the tongue was not significantly altered. Intra-trigeminal ganglion injection of LPS-RS significantly suppressed tongue hypersensitivity and reduced IL-1RΙ expression in the trigeminal ganglion neurons innervating the tongue following tooth pulp exposure. Intra-trigeminal ganglion injection of recombinant Hsp70 significantly promoted tongue hypersensitivity and increased IL-1RI expression in trigeminal ganglion neurons innervating the tongue in naive rats. Furthermore, intra-trigeminal ganglion injection of recombinant IL-1ß led to tongue hypersensitivity and enhanced TRPV1 expression in trigeminal ganglion neurons innervating the tongue in naive rats. CONCLUSIONS: The present findings suggest that the neuron-macrophage interaction mediated by toll-like receptor 4 and IL-1RI activation in trigeminal ganglion neurons affects the pathogenesis of abnormal tongue pain following tooth pulp inflammation via IL-1RI and TRPV1 signaling in the trigeminal ganglion. Further research may contribute to the establishment of new therapeutic and diagnostic methods.

Target-specific micronucleus induction by colon carcinogens: 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 1,2-dimethylhydrazine.

Genotoxicity occurring at the target organs of carcinogenesis is important for understanding the mechanisms of chemical carcinogenicity and also for setting of threshold estimation. In vivo gene mutations have been evaluated by transgenic animal models in which any organ can be targeted; however, the methodologies that have been applied to assess chromosomal aberrations including micronucleus induction, are organ restricted, (often to bone marrow hematopoietic cells, as a common example). For food and food-related chemicals, the digestive tract is the important target organ as it is the organ of first contact. In the present study, we used 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 1,2-dimethylhydrazine (DMH) as model chemicals of carcinogens primarily targeting the colon. We evaluated the applicability of colon cells and hepatocytes, together with bone marrow cells, in the micronucleus assay. Both model chemicals induced micronuclei in the colon, which is the target organ of these carcinogens, after short- and long-term treatment(s). The results demonstrate the target specificity of micronucleus induction and the assay using organs other than bone marrow will play an important role in understanding the mechanism of carcinogenicity and predicting new carcinogenic agents.

Loss-of-function of ASPARTIC PEPTIDASE NODULE-INDUCED 1 (APN1) in Lotus japonicus restricts efficient nitrogen-fixing symbiosis with specific Mesorhizobium loti strains.

The nitrogen-fixing symbiosis of legumes and Rhizobium bacteria is established by complex interactions between the two symbiotic partners. Legume Fix- mutants form apparently normal nodules with endosymbiotic rhizobia but fail to induce rhizobial nitrogen fixation. These mutants are useful for identifying the legume genes involved in the interactions essential for symbiotic nitrogen fixation. We describe here a Fix- mutant of Lotus japonicus, apn1, which showed a very specific symbiotic phenotype. It formed ineffective nodules when inoculated with the Mesorhizobium loti strain TONO. In these nodules, infected cells disintegrated and successively became necrotic, indicating premature senescence typical of Fix- mutants. However, it formed effective nodules when inoculated with the M. loti strain MAFF303099. Among nine different M. loti strains tested, four formed ineffective nodules and five formed effective nodules on apn1 roots. The identified causal gene, ASPARTIC PEPTIDASE NODULE-INDUCED 1 (LjAPN1), encodes a nepenthesin-type aspartic peptidase. The well characterized Arabidopsis aspartic peptidase CDR1 could complement the strain-specific Fix- phenotype of apn1. LjAPN1 is a typical late nodulin; its gene expression was exclusively induced during nodule development. LjAPN1 was most abundantly expressed in the infected cells in the nodules. Our findings indicate that LjAPN1 is required for the development and persistence of functional (nitrogen-fixing) symbiosis in a rhizobial strain-dependent manner, and thus determines compatibility between M. loti and L. japonicus at the level of nitrogen fixation.

Establishment of novel monoclonal antibodies for identification of type A spermatogonia in teleosts†.

We recently established a germ cell transplantation system in salmonids. Donor germ cells transplanted into the body cavity of recipient embryos migrate toward and are incorporated into the recipient gonad, where they undergo gametogenesis. Among the various types of testicular germ cells, only type A spermatogonia (A-SG) can be incorporated into the recipient gonads. Enriching for A-SG is therefore important for improving the efficiency of germ cell transplantation. To enrich for A-SG, an antibody against a cell surface marker is a convenient and powerful approach used in mammals; however, little is known about cell surface markers for A-SG in fish. To that end, we have produced novel monoclonal antibodies (mAbs) against cell-surface molecules of rainbow trout (Oncorhynchus mykiss) A-SG. We inoculated mice with living A-SG isolated from pvasa-GFP transgenic rainbow trout using GFP-dependent flow cytometry. By fusing lymph node cells of the inoculated mice with myeloma cells, we generated 576 hybridomas. To identify hybridomas that produce mAbs capable of labeling A-SG preferentially and effectively, we screened them using cell ELISA, fluorescence microscopy, and flow cytometry. We thereby identified two mAbs that can label A-SG. By using flow cytometry with these two antibodies, we could enrich for A-SG with transplantability to recipient gonads from amongst total testicular cells. Furthermore, one of these mAbs could also label zebrafish (Danio rerio) spermatogonia. Thus, we expect these monoclonal antibodies to be powerful tools for germ cell biology and biotechnology.

Specific visualization of live type A spermatogonia of Pacific bluefin tuna using fluorescent dye-conjugated antibodies†.

During our previous work toward establishing surrogate broodstock that can produce donor-derived gametes by germ cell transplantation, we found that only type A spermatogonia (ASGs) have the potency to colonize recipient gonads. Therefore, the ability to visualize ASGs specifically would allow the sequential analysis of donor cell behavior in the recipient gonads. Here we produced monoclonal antibodies that could recognize the cell surface antigens of ASGs in Pacific bluefin tuna (Thunnus orientalis), with the aim of visualizing live ASGs. We generated monoclonal antibodies by inoculating Pacific bluefin tuna testicular cells containing ASGs into mice and then screened them using cell-based enzyme-linked immunosorbent assay (ELISA), immunocytochemistry, flow cytometry (FCM), and immunohistochemistry, which resulted in the selection of two antibodies (Nos. 152 and 180) from a pool of 1152 antibodies. We directly labeled these antibodies with fluorescent dye, which allowed ASG-like cells to be visualized in a one-step procedure using immunocytochemistry. Molecular marker analyses against the FCM-sorted fluorescent cells confirmed that ASGs were highly enriched in the antibody-positive fraction. To evaluate the migratory capability of the ASGs, we transplanted visualized cells into the peritoneal cavity of nibe croaker (Nibea mitsukurii) larvae. This resulted in incorporated fluorescent cells labeled with antibody No. 152 being detected in the recipient gonads, suggesting that the visualized ASGs possessed migratory and incorporation capabilities. Thus, the donor germ cell visualization method that was developed in this study will facilitate and simplify Pacific bluefin tuna germ cell transplantation.