Rapid SNP diagnostics using asymmetric isothermal amplification and a new mismatch-suppression technology

We developed a rapid single nucleotide polymorphism (SNP) detection system named smart amplification process version 2 (SMAP 2). Because DNA amplification only occurred with a perfect primer match, amplification alone was sufficient to identify the target allele. To achieve the requisite fidelity to support this claim, we used two new and complementary approaches to suppress exponential background DNA amplification that resulted from mispriming events. SMAP 2 is isothermal and achieved SNP detection from whole human blood in 30 min when performed with a new DNA polymerase that was cloned and isolated from Alicyclobacillus acidocaldarius (Aac pol). Furthermore, to assist the scientific community in configuring SMAP 2 assays, we developed software specific for SMAP 2 primer design. With these new tools, a high-precision and rapid DNA amplification technology becomes available to aid in pharmacogenomic research and molecular-diagnostics applications.     

HopAZ1, a type III effector of Pseudomonas amygdali pv. tabaci,induces a hypersensitive response in tobacco wildfire-resistant Nicotiana tabacum ‘N509’

Pseudomonas amygdali pv. tabaci (formerly Pseudomonas syringae pv. tabaci; Pta) is a gram-negative bacterium that causes bacterial wildfire disease in Nicotiana tabacum. The pathogen establishes infections by using a type III secretion system to inject type III effector proteins (T3Es) into cells, thereby interfering with the host__s immune system. To counteract the effectors, plants have evolved disease-resistance genes and mechanisms to induce strong resistance on effector recognition. By screening a series of Pta T3E-deficient mutants, we have identified HopAZ1 as the T3E that induces disease resistance in N. tabacum ‘N509’. Inoculation with the Pta ∆hopAZ1 mutant did not induce resistance to Pta in N509. We also found that the Pta ∆hopAZ1 mutant did not induce a hypersensitive response and promoted severe disease symptoms in N509. Furthermore, a C-terminal truncated HopAZ1 abolished HopAZ1-dependent cell death in N509. These results indicate that HopAZ1 is the avirulence factor that induces resistance to Pta by N509.     

New insights on ChlD1 function in Photosystem II from site-directed mutants of D1/T179 in Thermosynechococcus elongatus

The monomeric chlorophyll, Chl_D1, which is located between the P_D1P_D2 chlorophyll pair and the pheophytin, Pheo_D1, is the longest wavelength chlorophyll in the heart of Photosystem II and is thought to be the primary electron donor. Its central Mg²⁺ is liganded to a water molecule that is H-bonded to D1/T179. Here, two site-directed mutants, D1/T179H and D1/T179V, were made in the thermophilic cyanobacterium, Thermosynechococcus elongatus, and characterized by a range of biophysical techniques. The Mn₄CaO₅ cluster in the water-splitting site is fully active in both mutants. Changes in thermoluminescence indicate that i) radiative recombination occurs via the repopulation of *Chl_D1 itself; ii) non-radiative charge recombination reactions appeared to be faster in the T179H-PSII; and iii) the properties of P_D1P_D2 were unaffected by this mutation, and consequently iv) the immediate precursor state of the radiative excited state is the Chl_D1⁺Pheo_D1^? radical pair. Chlorophyll bleaching due to high intensity illumination correlated with the amount of ¹O₂ generated. Comparison of the bleaching spectra with the electrochromic shifts attributed to Chl_D1 upon Q_A^? formation, indicates that in the T179H-PSII and in the WT*3-PSII, the Chl_D1 itself is the chlorophyll that is first damaged by ¹O₂, whereas in the T179V-PSII a more red chlorophyll is damaged, the identity of which is discussed. Thus, Chl_D1 appears to be one of the primary damage site in recombination-mediated photoinhibition. Finally, changes in the absorption of Chl_D1 very likely contribute to the well-known electrochromic shifts observed at ~430?nm during the S-state cycle.     

Longitudinal distribution and microhabitat use of young Japanese eel Anguilla japonica in a small river flowing through paddy areas

Although the Japanese eel Anguilla japonica is a commercially important species, its habitat use is not well understood during its life stages in the river. In this study, we investigated the longitudinal distribution and microhabitat use of young Japanese eels (<200 mm in total length [TL], which correspond to elver and early yellow stages) using 180 quadrates (1 m × 1 m) in six stations in a small river (approximately 11.5 km long, 3.0–25.0 m wide) that flows through paddy areas in Fukushima Prefecture, Japan. No differences were observed in the TL of eels among the sampling stations. The analysis using generalized linear models showed that eel density increased as number of weirs decreased. The analysis using generalized additive models showed that water depth, current velocity, and substrate complexity were important factors determining microhabitat use. Eels used shallow habitats (<35 cm) with slow currents (5–40 cm/s) and high complex riverbeds (>0.35 in index of substrate complexity). These findings provide useful information to conserve and manage wild eels inhabiting small rivers flowing through paddy areas.     

Identification of triterpene biosynthetic genes from Momordica charantia using RNA-seq analysis

Cucurbitaceae plants contain characteristic triterpenoids. Momordica charantia, known as a bitter melon, contains cucurbitacins and multiflorane type triterpenes, which confer bitter tasting and exhibit pharmacological activities. Their carbon skeletons are biosynthesized from 2,3-oxidosqualene by responsible oxidosqualene cyclase (OSC). In order to identify OSCs in M. charantia, RNA-seq analysis was carried out from ten different tissues. The functional analysis of the resulting four OSC genes revealed that they were cucurbitadienol synthase (McCBS), isomultiflorenol synthase (McIMS), β-amyrin synthase (McBAS) and cycloartenol synthase (McCAS), respectively. Their distinct expression patterns based on RPKM values and quantitative RT-PCR suggested how the characteristic triterpenoids were biosynthesized in each tissue. Although cucurbitacins were finally accumulated in fruits, McCBS showed highest expression in leaves indicating that the early step of cucurbitacins biosynthesis takes place in leaves, but not in fruits.

Abbreviations: OSC: oxidosqualene cyclase; RPKM: reads perkilobase of exon per million mapped reads     

Fern gametophytes of Angiopteris lygodiifolia and Osmunda japonica harbor diverse Mucoromycotina fungi

Journal of Plant Research - Mycorrhizal symbiosis between plants and fungi is ubiquitous, and has been played key roles in plant terrestrialization and diversification. Although arbuscular...     

Competitive Chemiluminescent Immunoassay for Estradiol Using an N-Functionalized Acridinium Ester

We have compared three competitive chemiluminescent immunoassays (CLIA) for estradiol (E2) using an N-functionalized acridinium ester (AE). The assays were a standard competitive assay using immobilized antibody and directly labeled antigen (type A), an immobilized antibody and indirectly labeled antigen (type B), and an immobilized antigen and labeled antibody (type C). In an antibody-immobilized system, the assay using both AE- and E2-labeled thyroglobulin as a tracer (type B) was more sensitive than that using AE directly coupled with E2 (type A). Subsequently, a comparison of the antibody-immobilized system (type B) and an antigen-immobilized system (type C) showed that the latter was slightly more sensitive than the former. The sensitivity of the CLIA (type C) was similar or superior to commercially available CLIA or radioimmunoassays for E2. Thus, the N-functionalized AE proved to be a useful labeling reagent for a competitive CLIA with high sensitivity.     

Familial Hyperaldosteronism Type 3 with a Rapidly Growing Adrenal Tumor: An In Situ Aldosterone Imaging Study

Primary aldosteronism is most often caused by aldosterone-producing adenoma (APA) and bi-lateral adrenal hyperplasia. Most APAs are caused by somatic mutations of various ion channels and pumps, the most common being the inward-rectifying potassium channel KCNJ5. Germ line mutations of KCNJ5 cause familial hyperaldosteronism type 3 (FH3), which is associated with severe hyperaldosteronism and hypertension. We present an unusual case of FH3 in a young woman, first diagnosed with primary aldosteronism at the age of 6 years, with bilateral adrenal hyperplasia, who underwent unilateral adrenalectomy (left adrenal) to alleviate hyperaldosteronism. However, her hyperaldosteronism persisted. At the age of 26 years, tomography of the remaining adrenal revealed two different adrenal tumors, one of which grew substantially in 4 months; therefore, the adrenal gland was removed. A comprehensive histological, immunohistochemical, and molecular evaluation of various sections of the adrenal gland and in situ visualization of aldosterone, using matrix-assisted laser desorption/ionization imaging mass spectrometry, was performed. Aldosterone synthase (CYP11B2) immunoreactivity was observed in the tumors and adrenal gland. The larger tumor also harbored a somatic β-catenin activating mutation. Aldosterone visualized in situ was only found in the subcapsular regions of the adrenal and not in the tumors. Collectively, this case of FH3 presented unusual tumor development and histological/molecular findings.