Evaluation of the Influence of Halogenation on the Binding of Bisphenol A to the Estrogen-Related Receptor γ.

Halogenation of organic compounds is one the most important transformations in chemical synthesis and is used for the production of various industrial products. A variety of halogenated bisphenol analogs have recently been developed and are used as alternatives to bisphenol A (BPA), which is a raw material of polycarbonate that has adverse effects in animals. However, limited information is available on the potential toxicity of the halogenated BPA analogs. In the present study, to assess the latent toxicity of halogenated BPA analogs, we evaluated the binding and transcriptional activities of halogenated BPA analogs to the estrogen-related receptor γ (ERRγ), a nuclear receptor that contributes to the growth of nerves and sexual glands. Fluorinated BPA analogs demonstrated strong ERRγ binding potency, and inverse antagonistic activity, similar to BPA. X-ray crystallography and fragment molecular orbital (FMO) calculation revealed that a fluorine-substituted BPA analog could interact with several amino acid residues of ERRγ-LBD, strengthening the binding affinity of the analogs. The ERRγ binding affinity and transcriptional activity of the halogenated BPAs decreased with the increase in the size and number of halogen atom(s). The IC50 values, determined by the competitive binding assay, correlated well with the binding energy obtained from the docking calculation, suggesting that the docking calculation could correctly estimate the ERRγ binding potency of the BPA analogs. These results confirmed that ERRγ has a ligand binding pocket that fits very well to BPA. Furthermore, this study showed that the binding affinity of the BPA analogs can be predicted by the docking calculation, indicating the importance of the calculation method in the risk assessment of halogenated compounds.

Crystal Structure and Band-Gap Engineering of a Semiconducting Coordination Polymer Consisting of Copper(I) Bromide and a Bridging Acceptor Ligand.

A new semiconducting 3D coordination polymer, [Cu2Br2(ttz)] n (1), with an acceptor bridging ligand, 1,2,4,5-tetrazine (ttz), was synthesized. The complex shows large absorption bands extending to the near-IR region, indicating a small band gap in the coordination polymer. This complex shows higher conductivity than those of [CuBr(pyz)] n (2), including pyrazine (pyz) with a higher lowest unoccupied molecular orbital level. We performed density functional theory band calculations using the VASP program to understand the electronic states and conducting paths of the coordination polymer.

Bacterial clade with the ribosomal RNA operon on a small plasmid rather than the chromosome.

rRNA is essential for life because of its functional importance in protein synthesis. The rRNA (rrn) operon encoding 16S, 23S, and 5S rRNAs is located on the "main" chromosome in all bacteria documented to date and is frequently used as a marker of chromosomes. Here, our genome analysis of a plant-associated alphaproteobacterium, Aureimonas sp. AU20, indicates that this strain has its sole rrn operon on a small (9.4 kb), high-copy-number replicon. We designated this unusual replicon carrying the rrn operon on the background of an rrn-lacking chromosome (RLC) as the rrn-plasmid. Four of 12 strains close to AU20 also had this RLC/rrn-plasmid organization. Phylogenetic analysis showed that those strains having the RLC/rrn-plasmid organization represented one clade within the genus Aureimonas. Our finding introduces a previously unaddressed viewpoint into studies of genetics, genomics, and evolution in microbiology and biology in general.

Rapid Removal and Mineralization of Bisphenol A by Heterosupramolecular Plasmonic Photocatalyst Consisting of Gold Nanoparticle-Loaded Titanium(IV) Oxide and Surfactant Admicelle.

The establishment of technology for rapid and complete removal and mineralization of harmful phenolic compounds from water is of great importance for environmental conservation. Visible-light irradiation (λ > 430 nm, light intensity integrated from 420 to 485 nm = 6.0 mW cm-2) of Au nanoparticle (NP)-loaded TiO2 (Au/TiO2) in dilute aqueous solutions of bisphenol A (BPA) and p-cresol (PC) causes degradation of the phenols. The addition of trimethylstearylammonium chloride (C18TAC) enhances the adsorption of BPA on Au/TiO2 to greatly increase the rate of reaction. Consequently, 10 µM phenols are completely removed from the solutions within 2.5 h irradiation, and prolonging irradiation time to 24 h quantitatively oxidizes BPA to CO2. Dynamic light scattering ζ-potential measurements indicate that a C18TAC bilayer or admicelle is formed on the Au/TiO2 particle surface at C18TAC concentration >50 µM. The action spectrum for reaction shows that this reaction is driven by the Au NP localized surface plasmon resonance excitation-induced interfacial electron transfer from Au to TiO2. We propose a possible reaction scheme on the basis of the experimental results including intermediate analysis.

Management of surgical instruments with radio frequency identification tags.

PURPOSE: To prevent malpractices, medical staff has adopted inventory time-outs and/or checklists. Accurate inventory and maintenance of surgical instruments decreases the risk of operating room miscounting and malfunction. In our previous study, an individual management of surgical instruments was accomplished using Radio Frequency Identification (RFID) tags. The purpose of this paper is to evaluate a new management method of RFID-tagged instruments. DESIGN/METHODOLOGY/APPROACH: The management system of RFID-tagged surgical instruments was used for 27 months in clinical areas. In total, 13 study participants assembled surgical trays in the central sterile supply department. FINDINGS: While using the management system, trays were assembled 94 times. During this period, no assembly errors occurred. An instrument malfunction had occurred after the 19th, 56th, and 73 th uses, no malfunction caused by the RFID tags, and usage history had been recorded. Additionally, the time it took to assemble surgical trays was recorded, and the long-term usability of the management system was evaluated. ORIGINALITY/VALUE: The system could record the number of uses and the defective history of each surgical instrument. In addition, the history of the frequency of instruments being transferred from one tray to another was recorded. The results suggest that our system can be used to manage instruments safely. Additionally, the management system was acquired of the learning effect and the usability on daily maintenance. This finding suggests that the management system examined here ensures surgical instrument and tray assembly quality.

Symbiosis island shuffling with abundant insertion sequences in the genomes of extra-slow-growing strains of soybean bradyrhizobia.

Extra-slow-growing bradyrhizobia from root nodules of field-grown soybeans harbor abundant insertion sequences (ISs) and are termed highly reiterated sequence-possessing (HRS) strains. We analyzed the genome organization of HRS strains with the focus on IS distribution and symbiosis island structure. Using pulsed-field gel electrophoresis, we consistently detected several plasmids (0.07 to 0.4 Mb) in the HRS strains (NK5, NK6, USDA135, 2281, USDA123, and T2), whereas no plasmids were detected in the non-HRS strain USDA110. The chromosomes of the six HRS strains (9.7 to 10.7 Mb) were larger than that of USDA110 (9.1 Mb). Using MiSeq sequences of 6 HRS and 17 non-HRS strains mapped to the USDA110 genome, we found that the copy numbers of ISRj1, ISRj2, ISFK1, IS1632, ISB27, ISBj8, and IS1631 were markedly higher in HRS strains. Whole-genome sequencing showed that the HRS strain NK6 had four small plasmids (136 to 212 kb) and a large chromosome (9,780 kb). Strong colinearity was found between 7.4-Mb core regions of the NK6 and USDA110 chromosomes. USDA110 symbiosis islands corresponded mainly to five small regions (S1 to S5) within two variable regions, V1 (0.8 Mb) and V2 (1.6 Mb), of the NK6 chromosome. The USDA110 nif gene cluster (nifDKENXSBZHQW-fixBCX) was split into two regions, S2 and S3, where ISRj1-mediated rearrangement occurred between nifS and nifB. ISs were also scattered in NK6 core regions, and ISRj1 insertion often disrupted some genes important for survival and environmental responses. These results suggest that HRS strains of soybean bradyrhizobia were subjected to IS-mediated symbiosis island shuffling and core genome degradation.

Preferential association of endophytic bradyrhizobia with different rice cultivars and its implications for rice endophyte evolution.

Plant colonization by bradyrhizobia is found not only in leguminous plants but also in nonleguminous species such as rice. To understand the evolution of the endophytic symbiosis of bradyrhizobia, the effect of the ecosystems of rice plantations on their associations was investigated. Samples were collected from various rice (Oryza sativa) tissues and crop rotational systems. The rice endophytic bradyrhizobia were isolated on the basis of oligotrophic properties, selective medium, and nodulation on siratro (Macroptilium atropurpureum). Six bradyrhizobial strains were obtained exclusively from rice grown in a crop rotational system. The isolates were separated into photosynthetic bradyrhizobia (PB) and nonphotosynthetic bradyrhizobia (non-PB). Thai bradyrhizobial strains promoted rice growth of Thai rice cultivars better than the Japanese bradyrhizobial strains. This implies that the rice cultivars possess characteristics that govern rice-bacterium associations. To examine whether leguminous plants in a rice plantation system support the persistence of rice endophytic bradyrhizobia, isolates were tested for legume nodulation. All PB strains formed symbioses with Aeschynomene indica and Aeschynomene evenia. On the other hand, non-PB strains were able to nodulate Aeschynomene americana, Vigna radiata, and M. atropurpureum but unable to nodulate either A. indica or A. evenia. Interestingly, the nodABC genes of all of these bradyrhizobial strains seem to exhibit low levels of similarity to those of Bradyrhizobium diazoefficiens USDA110 and Bradyrhizobium sp. strain ORS285. From these results, we discuss the evolution of the plant-bradyrhizobium association, including nonlegumes, in terms of photosynthetic lifestyle and nod-independent interactions.

Efficacy and safety of cell-free and concentrated ascites reinfusion therapy (CART) in gynecologic cancer patients with a large volume of ascites.

AIM: The aim of this study was to evaluate the efficacy and safety of cell-free concentrated ascites reinfusion therapy (CART) on a large amount of ascites. MATERIAL AND METHODS: Fifty-eight CART procedures were performed in nine patients with ovarian, endometrial, or cervical cancer from February 2013 to September 2014. The medical records were retrospectively reviewed for the amount of collected ascites, vital signs, and laboratory results before and after CART. RESULTS: No obvious change in the plasma protein and plasma albumin concentration was found after CART for < 5 L of ascites; however, obvious increases in both were observed in CART for ≥ 5 L of ascites (P < 0.001). The optimum cut-off value for obtaining a positive variant of plasma protein and plasma albumin after CART was 7.9 L. CART for ≥ 5 L of ascites did not increase the risk of transient water retention in the body (odds ratio = 2.2; 95% confidence interval: 0.35-13.83; P = 0.38); however, CART for ≥ 7.9 L of ascites increased the risk of water retention (odds ratio = 8.4; 95% confidence interval: 1.91-44.09; P = 0.004). The optimal cut-off value of ascites for predicting water retention due to CART was 9.2 L. CONCLUSION: Massive ascites collection in CART < 9.2 L appears to be a safe and effective treatment for improving general condition, plasma protein, and electrolytes in gynecologic cancer patients.

The nitrate-sensing NasST system regulates nitrous oxide reductase and periplasmic nitrate reductase in Bradyrhizobium japonicum.

The soybean endosymbiont Bradyrhizobium japonicum is able to scavenge the greenhouse gas N2O through the N2O reductase (Nos). In previous research, N2O emission from soybean rhizosphere was mitigated by B. japonicum Nos(++) strains (mutants with increased Nos activity). Here, we report the mechanism underlying the Nos(++) phenotype. Comparative analysis of Nos(++) mutant genomes showed that mutation of bll4572 resulted in Nos(++) phenotype. bll4572 encodes NasS, the nitrate (NO3(-))-sensor of the two-component NasST regulatory system. Transcriptional analyses of nosZ (encoding Nos) and other genes from the denitrification process in nasS and nasST mutants showed that, in the absence of NO3(-) , nasS mutation induces nosZ and nap (periplasmic nitrate reductase) via nasT. NO3(-) addition dissociated the NasS-NasT complex in vitro, suggesting the release of the activator NasT. Disruption of nasT led to a marked decrease in nosZ and nap transcription in cells incubated in the presence of NO3(-). Thus, although NasST is known to regulate the NO3(-)-mediated response of NO3(-) assimilation genes in bacteria, our results show that NasST regulates the NO3(-) -mediated response of nosZ and napE genes, from the dissimilatory denitrification pathway, in B. japonicum.

Metaproteomic identification of diazotrophic methanotrophs and their localization in root tissues of field-grown rice plants.

In a previous study by our group, CH4 oxidation and N2 fixation were simultaneously activated in the roots of wild-type rice plants in a paddy field with no N input; both processes are likely controlled by a rice gene for microbial symbiosis. The present study examined which microorganisms in rice roots were responsible for CH4 oxidation and N2 fixation under the field conditions. Metaproteomic analysis of root-associated bacteria from field-grown rice (Oryza sativa Nipponbare) revealed that nitrogenase complex-containing nitrogenase reductase (NifH) and the alpha subunit (NifD) and beta subunit (NifK) of dinitrogenase were mainly derived from type II methanotrophic bacteria of the family Methylocystaceae, including Methylosinus spp. Minor nitrogenase proteins such as Methylocella, Bradyrhizobium, Rhodopseudomonas, and Anaeromyxobacter were also detected. Methane monooxygenase proteins (PmoCBA and MmoXYZCBG) were detected in the same bacterial group of the Methylocystaceae. Because these results indicated that Methylocystaceae members mediate both CH4 oxidation and N2 fixation, we examined their localization in rice tissues by using catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). The methanotrophs were localized around the epidermal cells and vascular cylinder in the root tissues of the field-grown rice plants. Our metaproteomics and CARD-FISH results suggest that CH4 oxidation and N2 fixation are performed mainly by type II methanotrophs of the Methylocystaceae, including Methylosinus spp., inhabiting the vascular bundles and epidermal cells of rice roots.

A rice gene for microbial symbiosis, Oryza sativa CCaMK, reduces CH4 flux in a paddy field with low nitrogen input.

Plants have mutualistic symbiotic relationships with rhizobia and fungi by the common symbiosis pathway, of which Ca(2+)/calmodulin-dependent protein kinase (encoded by CCaMK) is a central component. Although Oryza sativa CCaMK (OsCCaMK) is required for fungal accommodation in rice roots, little is known about the role of OsCCaMK in rice symbiosis with bacteria. Here, we report the effect of a Tos17-induced OsCCaMK mutant (NE1115) on CH4 flux in low-nitrogen (LN) and standard-nitrogen (SN) paddy fields compared with wild-type (WT) Nipponbare. The growth of NE1115 was significantly decreased compared with that of the WT, especially in the LN field. The CH4 flux of NE1115 in the LN field was significantly greater (156 to 407% in 2011 and 170 to 816% in 2012) than that of the WT, although no difference was observed in the SN field. The copy number of pmoA (encodes methane monooxygenase in methanotrophs) was significantly higher in the roots and rhizosphere soil of the WT than in those of NE1115. However, the mcrA (encodes methyl coenzyme M reductase in methanogens) copy number did not differ between the WT and NE1115. These results were supported by a (13)C-labeled CH4-feeding experiment. In addition, the natural abundance of (15)N in WT shoots (3.05‰) was significantly lower than in NE1115 shoots (3.45‰), suggesting greater N2 fixation in the WT because of dilution with atmospheric N2 (0.00‰). Thus, CH4 oxidation and N2 fixation were simultaneously activated in the root zone of WT rice in the LN field and both processes are likely controlled by OsCCaMK.

Separation of the diastereomers of phosphorothioated siRNAs by anion-exchange chromatography under non-denaturing conditions.

In recent years, several small interfering RNA (siRNA) therapeutics have been approved, and most of them are phosphorothioate (PS)-modified for improving nuclease resistance. This chemical modification induces chirality in the phosphorus atom, leading to the formation of diastereomers. Recent studies have revealed that Sp and Rp configurations of PS modifications of siRNAs have different biological properties, such as nuclease resistance and RNA-induced silencing complex (RISC) loading. These results highlight the importance of determining diastereomeric distribution in quality control. Although various analytical approaches have been used to separate diastereomers (mainly single-stranded oligonucleotides), it becomes more difficult to separate all of them as the number of PS modifications increases. Despite siRNA exhibits efficacy in the double-stranded form, few reports have examined the separation of diastereomers in the double-stranded form. In this study, we investigated the applicability of non-denaturing anion-exchange chromatography (AEX) for the separation of PS-modified siRNA diastereomers. Separation of the four isomers of the two PS bonds tended to improve in the double-stranded form compared to the single-stranded form. In addition, the effects of the analytical conditions and PS-modified position on the separation were evaluated. Moreover, the elution order of the Sp and Rp configurations was confirmed, and the steric difference between them, i.e., the direction of the anionic sulfur atom, appeared to be important for the separation mechanism in non-denaturing AEX. Consequently, all 16 peak tops of the four PS modifications were detected in one sequence, and approximately 30 peak tops were detected out of 64 isomers of six PS bonds, indicating that non-denaturing AEX is a useful technique for the quality control of PS-modified siRNA therapeutics.

A copper(I) thiocyanate-based photoresponsive semiconducting 2D coordination polymer.

A coordination polymer, [Cu(SCN)(iqi)]n (iqi = isoquinoline), containing copper(I) thiocyanate and a nitrogen-containing π-conjugated ligand, iqi, has been synthesized and its physical properties were evaluated. This coordination polymer has a two-dimensional (2D) sheet structure consisting of copper(I) thiocyanate and shows photoluminescence derived from 3MLCT and photoconductive properties.

Genome analysis suggests that the soil oligotrophic bacterium Agromonas oligotrophica (Bradyrhizobium oligotrophicum) is a nitrogen-fixing symbiont of Aeschynomene indica.

Agromonas oligotrophica (Bradyrhizobium oligotrophicum) S58(T) is a nitrogen-fixing oligotrophic bacterium isolated from paddy field soil that is able to grow in extra-low-nutrient environments. Here, the complete genome sequence of S58 was determined. The S58 genome was found to comprise a circular chromosome of 8,264,165 bp with an average GC content of 65.1% lacking nodABC genes and the typical symbiosis island. The genome showed a high level of similarity to the genomes of Bradyrhizobium sp. ORS278 and Bradyrhizobium sp. BTAi1, including nitrogen fixation and photosynthesis gene clusters, which nodulate an aquatic legume plant, Aeschynomene indica, in a Nod factor-independent manner. Although nonsymbiotic (brady)rhizobia are significant components of rhizobial populations in soil, we found that most genes important for nodule development (ndv) and symbiotic nitrogen fixation (nif and fix) with A. indica were well conserved between the ORS278 and S58 genomes. Therefore, we performed inoculation experiments with five A. oligotrophica strains (S58, S42, S55, S72, and S80). Surprisingly, all five strains of A. oligotrophica formed effective nitrogen-fixing nodules on the roots and/or stems of A. indica, with differentiated bacteroids. Nonsymbiotic (brady)rhizobia are known to be significant components of rhizobial populations without a symbiosis island or symbiotic plasmids in soil, but the present results indicate that soil-dwelling A. oligotrophica generally possesses the ability to establish symbiosis with A. indica. Phylogenetic analyses suggest that Nod factor-independent symbiosis with A. indica is a common trait of nodABC- and symbiosis island-lacking strains within the members of the photosynthetic Bradyrhizobium clade, including A. oligotrophica.

Tris-(µ4-azepane-1-carbodi-thio-ato)bis-(µ3-azepane-1-carbodi-thio-ato)-µ9-bromido-tetra-µ2-bromido-octa-copper(I)-copper(II).

The reaction of Cu(Hm-dtc)2 (H2m-dtc is azepane-1-carbodi-thioic acid), CuBr2 and methyl iso-thio-cyanate yielded the title mixed-valence nona-nuclear Cu(I)/Cu(II) compound, [Cu9Br5(C7H12NS2)5] or [Cu(I) 8Cu(II)Br5(Hm-dtc)5], encapsulating a bromide anion in the center of the Cu9Br4S10 cluster cage. The cage consists of a mononuclear Cu(II) unit [Cu(Hm-dtc)2], three µ4-bridging Hm-dtc(-) ligands, eight Cu(I) ions with distorted tetra-hedral or trigonal pyramidal coordination geometries and four µ2-bridging bromide anions. The incorporated central bromide anion inter-acts with nine Cu ions with shorter Cu-Br separations than the sum of the van der Waals radii for Cu and Br.

Retention behavior of short double-stranded oligonucleotide and its potential impurities by anion-exchange chromatography under non-denaturing conditions.

Small interfering RNA (siRNA), consisting of two complementary single-stranded RNAs with overhanging bases, is being adopted as a potent and specific inhibitor of target gene expression. However, non-duplexed single strands and undesired double strands composed of impurities (e.g., n-1 mer) could be produced in addition to the target double strand in the siRNA manufacturing process. Compared to the liquid chromatography analysis of single strands, the analysis of the duplexes under non-denaturing conditions is challenging, since restricted chromatographic conditions are required to maintain the Watson-Crick hydrogen bonds. This study reports the analysis of double-stranded oligomers having approximately 20 base pairs with some overhanging bases as non-denatured forms by anion-exchange chromatography (AEX). Optimization of the chromatographic conditions could potentially achieve the adequate separation of excess single strands from the double strand. Non-optimal duplexes, such as duplexes with long overhangs or bulge structures, were prepared by intentionally deleting terminal or middle nucleotide(s) of either the sense or the antisense strand, and these non-optimal duplexes were eluted at different retention times in most of the cases. Interestingly, under alkaline chromatographic conditions (pH 9.0), non-optimal duplexes containing a shortmer tended to exhibit a stronger retention than their parent duplexes, although they possessed a less negative charge. This study demonstrated some retention behavior of double strands with overhangs by AEX under non-denaturing conditions.

Structural diversity of copper(I)-ethylene complexes with 2,4-bis(2-pyridyl)pyrimidine directed by anions.

The 3 : 1 reaction of [Cu(C2H4)n]ClO4 with 2,4-bis(2-pyridyl)pyrimidine (bpprd) in Me2CO under C2H4 afforded yellow prism crystals of the dinuclear Cu(I)-C2H4 complex [Cu2(bpprd)(η2-C2H4)2(ClO4)2] (1). The 3 : 1 reaction of [Cu(C2H4)n]NO3 with bpprd in Me2CO under C2H4 afforded yellow plate crystals of the tetranuclear Cu(I)-C2H4 complex [Cu4(bpprd)2(η2-C2H4)4(µ-NO3)2](NO3)2 (2). The 10 : 1 reaction of [Cu(C2H4)n]BF4 with bpprd in Me2CO under C2H4 afforded yellow plate crystals of the dinuclear Cu(I)-C2H4 complex [Cu2(bpprd)(η2-C2H4)2(BF4)]BF4 (3). The 3 : 1 reaction of [Cu(C2H4)n]BF4 with bpprd in Me2CO under C2H4 afforded red prism crystals of the polymeric Cu(I)-C2H4 complex {[Cu6(bpprd)4(η2-C2H4)2(µ-η2:η2-C2H4)(µ-BF4)2](BF4)4}n (4). The X-ray crystal structures of complexes 1-4 have been determined. The structural diversity of Cu(I)-C2H4 complexes bridged by bpprd with different anions was demonstrated. The 1D Cu(I)-bpprd/C2H4 coordination polymer 4 bridged by unusual µ-η2:η2-C2H4 and the µ-BF4- anion is of particular significance. Complex 1 exhibited relatively well-resolved 1H NMR signals of bpprd and C2H4 (δ = 4.97 ppm) in (CD3)2CO at 23 °C.

High cytotoxicity of a degraded TBBPA, dibromobisphenol A, through apoptotic and necrosis pathways.

Halogenated flame retardants comprising bisphenol A (BPA) derivatives, such as tetrabromobisphenol A (TBBPA), have been studied their adverse effects on human health. However, despite the fact that these halogenated BPAs are easily degraded in the environment, the risks to living organisms due to these degraded products have mostly been overlooked. To evaluate the potential toxicity of degraded TBBPAs and related compounds, we examined the cytotoxicity of halogenated bisphenol A derivatives possessing one to four halogen atoms in vitro. The results indicated that the degraded TBBPA derivatives exhibited strong cytotoxicity against HeLa cells than TBBPA. Interestingly, the di-halogenated BPA derivatives possessing two halogen atoms exhibited the strongest cytotoxicity among tested compounds. In addition, a lactate dehydrogenase release assay, fluorescence spectroscopy and flow cytometry results indicated that dibromo-BPA and diiodo-BPA induced both apoptotic and necrotic cell death by damaging the cell membranes of HeLa cells. Moreover, Escherichia coli growth was inhibited in the presence of dehalogenated TBBPA and related compounds. These findings suggest that halogenated BPA derivatives that leak from various flame-retardant-containing products require strict monitoring, as not only TBBPA but also its degraded products in environment can exert adverse effects to human health.

Evolution of Bradyrhizobium-Aeschynomene mutualism: living testimony of the ancient world or highly evolved state?

Until recently it had been well established that the initial step in legume-rhizobia symbioses was flavonoid and Nod factor (NF) signaling. However, NF-independent symbiosis is now known to occur between Bradyrhizobium and some species of Aeschynomene. Since its discovery, this unusual symbiotic system has attracted attention, and efforts have been devoted to revealing the NF-independent symbiotic mechanism, although the molecular mechanisms of nodule initiation still remain to be elucidated. NF-independent symbiosis is also interesting from the perspective of the evolution of legume-rhizobia symbiosis. In this mini-review, we discuss the current literature on the NF-independent symbiotic system in terms of phylogeny of the partners, infection, bacteroid differentiation, nodule structure, photosynthesis, endophytic features and model host plant. We also discuss NF-independent symbiosis, which is generally regarded to be more primitive than NF-dependent symbiosis, because the bacteria invade host plants via 'crack entry'. We propose three possible scenarios concerning the evolution of NF-independent symbiosis, which do not exclude the possibility that the NF-independent system evolved from NF-dependent interactions. Finally, we examine an interesting question on Bradyrhizobium-Aeschynomene mutualism, which is how do they initiate symbiosis without NF. Phylogenetic and genomic analyses of symbiotic and non-symbiotic bradyrhizobia with A. indica may be crucial to address the question, because of the very narrow phylogeny of natural endosymbionts without nod genes compared with other legume-rhizobia symbioses.

The genotype of the calcium/calmodulin-dependent protein kinase gene (CCaMK) determines bacterial community diversity in rice roots under paddy and upland field conditions.

The effects of the Oryza sativa calcium/calmodulin-dependent protein kinase OsCCaMK genotype (dominant homozygous [D], heterozygous [H], recessive homozygous [R]) on rice root-associated bacteria, including endophytes and epiphytes, were examined by using a Tos17 rice mutant line under paddy and upland field conditions. Roots were sampled at the flowering stage and were subjected to clone library analyses. The relative abundance of Alphaproteobacteria was noticeably decreased in R plants under both paddy and upland conditions (0.8% and 3.0%, respectively) relative to those in D plants (10.3% and 17.4%, respectively). Population shifts of the Sphingomonadales and Rhizobiales were mainly responsible for this low abundance in R plants. The abundance of Anaerolineae (Chloroflexi) and Clostridia (Firmicutes) was increased in R plants under paddy conditions. The abundance of a subpopulation of Actinobacteria (Saccharothrix spp. and unclassified Actinosynnemataceae) was increased in R plants under upland conditions. Principal coordinate analysis revealed unidirectional community shifts in relation to OsCCaMK gene dosage under both conditions. In addition, shoot length, tiller number, and plant weight decreased as the OsCCaMK gene dosage decreased under upland conditions. These results suggest significant impacts of OsCCaMK on both the diversity of root-associated bacteria and rice plant growth under both paddy and upland field conditions.