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The bluish-purple petals of Chinese bellflower, Platycodon grandiflorum (kikyo in Japanese), contain platyconin ( 1 ) as the major anthocyanin. Platyconin ( 1 ) is a polyacylated anthocyanin with two caffeoyl residues at the 7-position, and its color is stable in a diluted, weakly acidic aqueous solutions. HPLC analysis of the fresh petal extract showed the presence of several minor pigments. Photo-diode array detection of minor pigments suggested that some of these were polyacylated anthocyanins. To establish the relationship between structure and stability of the acylated anthocyanins and to obtain information on their biosynthetic pathways, minor pigments were isolated from the petals, and their structures were determined by MS and NMR analyses. Four known ( 2 - 5 ) and three new anthocyanins ( 6 - 8 ) were identified, which contained a delphinidin chromophore, and four of these ( 5 - 8 ) were diacylated anthocyanins, in which the acyl-glucosyl-acyl-glucosyl chain was attached at the 7- O -position of the delphinidin chromophore. These diacylated anthocyanins exhibited a bluish-purple color at pH 6, which was stable for more than a week.

This is the first study describing the morphological, ecological and physiological characteristics of two downstream-migrating and two non-migrating female Pacific bicolor eels, Anguilla bicolor pacifica. The total length and age of the downstream-migrating eels were 1005 mm and 10 years and 1110 mm and 11 years old, respectively, and those of the non-migrating eels were 892 mm and 8 years and 805 mm and 9 years, respectively. Silvering-related characteristics (silvering index, eye index, pectoral-fin index, gut-somatic index and swimbladder-somatic index) and reproductive physiological characteristics (gonado-somatic index, follicle diameter, oocyte stage, transcription of gonadotropins and concentration of sex steroids) of the migrating eels were more advanced than those of the non-migrating eels., (© 2020 Fisheries Society of the British Isles.)

Strigolactones (SLs) are plant hormones that suppress shoot branching through perception by their receptor protein DWARF 14 (D14). The artificial regulation of SL signaling has been considered a potent agricultural technique because plant architecture is strongly related to crop yield. In this communication, we describe the development of a small-molecule D14 inhibitor that functions at sub-micromolar levels. This potent inhibitor may be a lead compound for a first-in-class plant growth regulator.

The auxin-inducible degron (AID) system enables rapid depletion of target proteins within the cell by applying the natural auxin IAA. The AID system is useful for investigating the physiological functions of essential proteins; however, this system generally requires high dose of auxin to achieve effective depletion in vertebrate cells. Here, we describe a super-sensitive AID system that incorporates the synthetic auxin derivative 5-Ad-IAA and its high-affinity-binding partner OsTIR1F74A. The super-sensitive AID system enabled more than a 1000-fold reduction of the AID inducer concentrations in chicken DT40 cells. To apply this system to various mammalian cell lines including cancer cells containing multiple sets of chromosomes, we utilized a single-step method where CRISPR/Cas9-based gene knockout is combined with insertion of a pAID plasmid. The single-step method coupled with the super-sensitive AID system enables us to easily and rapidly generate AID-based conditional knockout cells in a wide range of vertebrate cell lines. Our improved method that incorporates the super-sensitive AID system and the single-step method provides a powerful tool for elucidating the roles of essential genes., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Autophagy and the ubiquitin-proteasome system are the major degradation processes for intracellular components in eukaryotes. Although ubiquitination acts as a signal inducing organelle-targeting autophagy, the interaction between ubiquitination and autophagy in chloroplast turnover has not been addressed. In this study, we found that two chloroplast-associated E3 enzymes, SUPPRESSOR OF PPI1 LOCUS1 and PLANT U-BOX4 (PUB4), are not necessary for the induction of either piecemeal autophagy of chloroplast stroma or chlorophagy of whole damaged chloroplasts in Arabidopsis ( Arabidopsis thaliana ). Double mutations of an autophagy gene and PUB4 caused synergistic phenotypes relative to single mutations. The double mutants developed accelerated leaf chlorosis linked to the overaccumulation of reactive oxygen species during senescence and had reduced seed production. Biochemical detection of ubiquitinated proteins indicated that both autophagy and PUB4-associated ubiquitination contributed to protein degradation in the senescing leaves. Furthermore, the double mutants had enhanced susceptibility to carbon or nitrogen starvation relative to single mutants. Together, these results indicate that autophagy and chloroplast-associated E3s cooperate for protein turnover, management of reactive oxygen species accumulation, and adaptation to starvation., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

CRY1 and CRY2 are essential components of the circadian clock controlling daily physiological rhythms. Accumulating evidences indicate distinct roles of these highly homologous proteins, in addition to redundant functions. Therefore, the development of isoform-selective compounds represents an effective approach towards understanding the similarities and differences of CRY1 and CRY2 by controlling each isoform individually. We conducted phenotypic screenings of circadian clock modulators, and identified KL101 and TH301 that selectively stabilize CRY1 and CRY2, respectively. Crystal structures of CRY-compound complexes revealed conservation of compound-binding sites between CRY1 and CRY2. We further discovered a unique mechanism underlying compound selectivity in which the disordered C-terminal region outside the pocket was required for the differential effects of KL101 and TH301 against CRY isoforms. By using these compounds, we found a new role of CRY1 and CRY2 as enhancers of brown adipocyte differentiation, providing the basis of CRY-mediated regulation of energy expenditure.

Downstream-migrating (n = 64) and non-migrating (n = 21) female Celebes eels Anguilla celebesensis were captured from the Poso Lake-River system on Sulawesi Island, Indonesia, and their reproductive physiological characteristics were examined. A histological observation of the ovaries revealed that most non-migrating eels were at the perinucleolus (43%) or oil-droplet (48%) stage, whereas most migrating eels were at the early vitellogenic (36%) or midvitellogenic (61%) stage. Transcript levels of gonadotropin genes (fshb, lhb) in the pituitary gland and concentrations of sex steroids [11-ketotestosterone (11-KT), testosterone, 17β-oestradiol (E 2 )] in blood plasma of migrating eels were significantly higher than those of non-migrating eels. The fshb messenger (m)RNA levels were lower in perinucleolus and oil-droplet stages and then significantly increased in the early vitellogenic stage. The lhb mRNA levels in vitellogenic-stage eels were significantly higher than those in perinucleolus- and oil-droplet-stage eels. The 11-KT levels of eels at the oil-droplet and vitellogenic stages were significantly higher than those of eels at the perinucleolus stage. The E 2 levels at the vitellogenic stage were significantly higher than those at the perinucleolus and oil-droplet stages. These dynamics of the reproductive hormones represented the physiological background of oogenesis in A. celebesensis that has remarkably well-developed oocytes just before downstream migration., (© 2019 The Fisheries Society of the British Isles.)

Our understanding of maternal control of development in vertebrates remains incomplete. In this study, we investigated levels of maternal transcripts in good and poor quality eggs from artificially matured Japanese eel, using RNA-Seq and quantitative polymerase chain reaction (qPCR), to identify candidate maternal transcripts related to development. De novo assembly or mapping of reads to the eel draft genome yielded 619,029 contigs and 85,906 transcripts, respectively; normalized read counts to these assemblies were calculated using reads (RPKM) or fragments (FPKM) per kilobase of transcript per million mapped reads. In silico screening identified 1,594 contigs and 150 transcripts with lower RPKM or FPKM in poor than in good quality eggs, 245 contigs, and 85 transcripts of which could be annotated by BLASTx, respectively. From selected contigs or transcripts, six genes (dnajb4, gnpat, card14, pdp1, fcgbp, ttn) had significantly lower messenger RNA levels in poor than in good quality eggs by qPCR. Multiple regression analysis showed that five genes (gnpat, b4galnt1, acsl6, rtkn, trim24) significantly correlated with hatchability. Taken together, 10 genes were identified as candidate maternal transcripts, regulating development in Japanese eel. Our results contribute to understanding the molecular basis for maternal control of development in vertebrates., (© 2019 Wiley Periodicals, Inc.)

Seven South Pacific anguillid eel species live from New Guinea to French Polynesia, but their spawning areas and life histories are mostly unknown despite previous sampling surveys. A July-October 2016 research cruise was conducted to study the spawning areas and times, and larval distributions of South Pacific anguillid eels, which included a short 155°E station-line northeast of New Guinea and five long transects (5-25°S, 160°E-140°W) crossing the South Equatorial (SEC) and other currents. This survey collected nearly 4000 anguilliform leptocephali at 179 stations using an Isaacs-Kidd Midwater Trawl accompanied by 104 CTD casts. Based on mor-phometric observations and DNA sequencing, 74 anguillid leptocephali were collected, which in the southern areas included 29 larvae of six species: Anguilla bicolor pacifica , A. marmorata , A. australis , A. reinhardtii , A. megastoma , and A. obscura (all anguillid species of the region were caught except A. dieffenbachii ). Small A. australis (9.0-16.8 mm) and A. reinhardtii (12.4, 12.5 mm) leptocephali were collected south of the Solomon Islands, other A. australis (10.8-12.0 mm) larvae were caught northwest of Fiji along with an A. obscura (20.0 mm) larva, and an A. marmorata (7.8 mm) larva was collected near Samoa. Considering collection sites, larval ages from otolith analysis, and westward SEC drift, multiple spawning locations occurred from south of the Solomon Islands and the Fiji area (16-20 days old larvae) to near Samoa (19 days old larva) during June and July in areas where high-salinity Subtropical Underwater (STUW, ~150 m depth) and the warm, low-salinity surface Fresh Pool were present. Five long hydrographic sections showed the strong Fresh Pool in the west and the STUW formation area in the east., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

In Hokkaido, the northernmost region of Japan, soybean [ Glycine max (L.) Merr.] crops are damaged by cold weather. Chilling temperatures negatively affect seed appearance by causing seed coat discoloration around the hilum region, which is called cold-induced discoloration (CD). An assay for CD tolerance using a phytotron was developed, and two quantitative trait loci (QTLs) associated with CD tolerance were identified. The major QTL was located in the proximal region of the I locus, and structural variation of this locus can serve as a useful DNA marker, called the Ic marker. To use this marker in breeding programs, the effects need to be assessed under field conditions because the Ic marker has been developed solely under phytotron conditions. The aim of this study was thus to assess the effect of the Ic marker under a cool field environment. We confirmed that the Ic allele was highly effective using 27 cultivars and breeding lines including a near-isogenic line grown in the field where severe cold-weather damage occurred. This allele had no negative influence on the agronomic traits in the near-isogenic line. Our results suggest that marker-assisted selection for the Ic allele is effective for improving CD tolerance in breeding programs., (Copyright © 2019 by JAPANESE SOCIETY OF BREEDING.)

Synthetic molecules can be powerful tools to overcome the limitations of the biological approaches. Especially redundancy, lethality, and intractability of the target genes, which often hamper the progress of plant science, could be bypassed by elaborately designed small molecules. In this review, we discuss how synthetic chemistry can contribute to increasing our understanding of plant hormone signaling. Specific focus will be on the visualization and hijacking of hormone signaling with novel synthetic chemicals, with emphasis on perception of ABA, strigolactones, and auxins., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Compounds targeting the circadian clock have been identified as potential treatments for clock-related diseases, including cancer. Our cell-based phenotypic screen revealed uncharacterized clock-modulating compounds. Through affinity-based target deconvolution, we identified GO289, which strongly lengthened circadian period, as a potent and selective inhibitor of CK2. Phosphoproteomics identified multiple phosphorylation sites inhibited by GO289 on clock proteins, including PER2 S693. Furthermore, GO289 exhibited cell type-dependent inhibition of cancer cell growth that correlated with cellular clock function. The x-ray crystal structure of the CK2α-GO289 complex revealed critical interactions between GO289 and CK2-specific residues and no direct interaction of GO289 with the hinge region that is highly conserved among kinases. The discovery of GO289 provides a direct link between the circadian clock and cancer regulation and reveals unique design principles underlying kinase selectivity.

Downstream-migrating Anguilla celebesensis eels were predominant relative to Anguilla marmorata in October, November, December, January and February (75.9-92%), while no A. celebesensis occurred and A. marmorata were predominant in May and July (96-100%), at the outlet of Poso Lake, Sulawesi Island, Indonesia. Merging these results with those from published data suggests that most A. celebesensis start downstream migration during the early to middle rainy season, and A. marmorata migrate almost year-round with a peak from the late rainy to middle dry season., (© 2018 The Fisheries Society of the British Isles.)

Contents Summary 417 I. Introduction 417 II. Auxin analogs 1: Plant growth regulators 418 III. Auxin analogs 2: Molecular genetics and chemical biology 418 IV. Auxin analogs 3: Structure-guided chemical design 418 V. Auxin analogs 4: Synthetic orthogonal auxin-TIR1 pair 420 VI. Conclusions and future perspectives 422 Acknowledgements 422 References 423 SUMMARY: Plant biologists have been fascinated by auxin - a small chemical hormone so simple in structure yet so powerful - which regulates virtually every aspect of plant growth, development and behavior. Synthetic chemistry has played a major role in unraveling the physiological effects of auxin and the application of synthetic analogs has had a dramatic effect on tissue culture, horticulture and the agriculture of economically relevant plant species. Chemical genetics of the model plant, Arabidopsis thaliana, has helped to elucidate the nuclear auxin signaling pathway mediated by the receptor, TIR1, and opened the door to structure-guided, rational designs of auxin agonists and antagonists. Further improvement and tuning of such analogs has been achieved through derivatization and screening. Finally, by harnessing synthetic chemistry and receptor engineering, an orthogonal auxin-TIR1 pair has been created and developed, enabling spatiotemporal control of auxin perception and response. This synergism of chemistry, biology and engineering sparks new ideas and directions to delineate, uncover and manipulate auxin signaling., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Background: Primary duodenal adenocarcinoma (PDC) is a rare and lethal disease, and cases with nodal or distant metastasis have a poor prognosis. There are several reports of unresectable duodenal adenocarcinoma responding to systemic chemotherapy. However, there is little data on conversion surgery for PDC with distant metastasis., Case Presentation: We report a 55-year-old man with unresectable PDC with liver and nodal metastases responding to systemic chemotherapy with capecitabine and oxaliplatin (XELOX). His metastatic lesions completely disappeared by 18-fluorodeoxyglucose positron emission tomography/computed tomography after six courses of XELOX. Then, he underwent pancreaticoduodenectomy with lymph node dissection and partial resection of the liver. Postoperatively, the histological effect was determined to be grade 3, and the patient was diagnosed as having achieved pathological complete response (pCR). He is disease-free with no evidence of metastatic lesion for 14 months after surgery. Conversion surgery allowed R0 resection for unresectable PDC, and pCR can be achieved with XELOX treatment., Conclusion: To the best of our knowledge, this case is the first report of conversion surgery for unresectable PDC with liver and para-aortic lymph node metastases.

A series of new AHX derivatives were synthesized by Pd-catalyzed C-H arylation. Their rice-growth-promoting activity was evaluated in vivo. Among the synthesized compounds, C8 phenyl-substituted AHX showed remarkable growth-promoting activity on rice. The present study shows the power and significant opportunity of C-H functionalization chemistry to rapidly transform biologically active natural products into more active compounds.

Cross-linkable 7-deaza-6-vinylguanosine (ADVP) and 7-propynyl-7-deaza-6-vinylguanosine (ADpVP) derivatives were synthesized and successfully incorporated into 2'-OMe-RNA oligonucleotides by solid-phase oligonucleotide synthesis. Analysis of their cross-link properties revealed that the 7-propynyl substituent on ADpVP induces a significant enhancement of the cross-link kinetics of the proximal 6-vinyl group to the complementary uracil base in the target RNA compared to that of ADVP. In addition, the 2'-OMe-RNA oligonucleotide containing ADpVP exhibited a higher antisense effect on luciferase production in the cell lysate than that of ADVP. These results suggested that the 7-substituted 7-deaza-6-vinylguanosine derivatives can be used as potent cross-linkers to target mRNA inside of cells.

Auxin regulates diverse aspects of plant growth and development through induction of the interaction between TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALING F-BOX proteins (TIR1/AFBs) and AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) co-receptor proteins and the subsequent transcriptional regulation. The artificial control of endogenous auxin signaling should enable the precise delineation of auxin-mediated biological events as well as the agricultural application of auxin. To this end, we previously developed a synthetic auxin-receptor pair that consists of 5-(3-methoxyphenyl)-IAA (convexIAA, cvxIAA) and the engineered TIR1 whose phenylalanine at position 79 in the auxin-binding pocket is substituted to glycine (TIR1F79G) (concaveTIR1, ccvTIR1). This synthetic auxin-receptor pair works orthogonally to natural auxin signaling in transgenic plants harboring the engineered TIR1 by exogenous application of 5-(3-methoxyphenyl)-IAA, and has potential to be utilized as novel agricultural/horticultural tools. In the present study, we report an improved version of the synthetic cvxIAA-ccvTIR1 pair such that synthetic IAA can act at lower concentrations. Using a yeast two-hybrid system, we screened various 5-substituted IAAs and identified 5-adamantyl-IAA, named pico_cvxIAA, which mediates interaction of TIR1F79G and IAA3 proteins at a 1,000-fold lower concentration than the original version, 5-(3-methoxyphenyl)-IAA. Furthermore, we found that TIR1F79A interacts with IAA3 protein in the presence of picomolar concentrations of 5-adamantyl-IAA, 10,000-fold lower than our prototype version of the cvxIAA-ccvTIR1 pair. In addition, pull-down assays confirmed that 5-adamantyl-IAA mediates in vitro interaction of TIR1F79A and IAA7-DII peptides at lower concentrations. The improved synthetic IAA-TIR1 pair with high affinity would be beneficial for basic science as well as for practical use in agriculture/horticulture.

Jun Aoyama, Sam Wouthuyzen, Michael J. Miller, Hagi Y. Sugeha, Mari Kuroki, Shun Watanabe, Augy Syahailatua, Fadly Y. Tantu, Seishi Hagihara, Triyanto, Tsuguo Otake, and Katsumi Tsukamoto (2018) Sulawesi Island of north-central Indonesia is located in a region where at least 6 species of tropical anguillid eels are present, but the reproductive ecology and biodiversity of these eels in each area of the Indonesian archipelago remains poorly understood. Some information about these species was obtained from collections of their leptocephalus larvae made during several times of the year and from year-round collections of their recruitment-stage glass eels at a few locations. A sampling survey of anguillid leptocephali was conducted in March 2010 in both the Celebes Sea and Tomini Bay of Sulawesi Island to learn about the biodiversity and reproductive ecology of the eels in the region. Twenty-eight anguillid leptocephali were collected at 13 different stations, with genetic identification indicating that 3 species of eels had spawned in the two areas. Larvae were more abundant in the Celebes Sea (N = 21; 16.0-52.1 mm TL) than in Tomini Bay (N = 7; 9.6-54.8 mm). The abundant 16-21 mm size-class of Anguilla bornensis in the Celebes Sea indicated that species had recently spawned there, and spawning had also occurred in Tomini Bay by A. celebesensis (17.4 mm). These data and previous life history information suggest that A. celebesensis may have two spawning seasons in the Celebes Sea, but only one main spawning season in Tomini Bay. Anguilla borneensis may spawn at several times of the year in the Celebes Sea. Anguilla marmorata and A. biocolor pacifica spawn outside the Indonesian Seas, with A. marmorata recruiting in large numbers in the Sulawesi Island region during much of the year. Other spawning locations of A. celebesensis and A. interioris likely exist in Indonesian waters. Therefore, further research is needed to understand the reproductive ecologies and biodiversity of the tropical anguillid eels in each region of Indonesia in relation to geographic and climatic factors.

The phytohormone auxin is the information carrier in a plethora of developmental and physiological processes in plants 1 . It has been firmly established that canonical, nuclear auxin signalling acts through regulation of gene transcription 2 . Here, we combined microfluidics, live imaging, genetic engineering and computational modelling to reanalyse the classical case of root growth inhibition 3 by auxin. We show that Arabidopsis roots react to addition and removal of auxin by extremely rapid adaptation of growth rate. This process requires intracellular auxin perception but not transcriptional reprogramming. The formation of the canonical TIR1/AFB-Aux/IAA co-receptor complex is required for the growth regulation, hinting to a novel, non-transcriptional branch of this signalling pathway. Our results challenge the current understanding of root growth regulation by auxin and suggest another, presumably non-transcriptional, signalling output of the canonical auxin pathway.

Plant-derived strigolactones have diverse functions at ecological scale, including effects upon the growth of plants themselves. The parasitic plants from the family Orobanchaceae interfere with the ecological and hormonal functions of strigolactones to generate unique germination abilities based on the sensing of host-derived strigolactones. Although the recent discovery of strigolactone receptors has enabled us to begin elucidating the mechanism of strigolactone perception, how perception relates to plant parasitism is still a mystery. In this review, we explore emerging questions by introducing recent advances in strigolactone research in parasitic plants. We also attempt to construct a conceptual framework for the unique in planta dynamics of strigolactone perception uncovered through the use of fluorescent probes for strigolactone receptors. Understanding the mechanisms of strigolactone-related processes is essential for controlling the parasitic plant Striga hermonthica, which has caused devastating damage to crop production in Africa.

The phytohormone auxin indole-3-acetic acid (IAA) regulates nearly all aspects of plant growth and development. Despite substantial progress in our understanding of auxin biology, delineating specific auxin response remains a major challenge. Auxin regulates transcriptional response via its receptors, TIR1 and AFB F-box proteins. Here we report an engineered, orthogonal auxin-TIR1 receptor pair, developed through a bump-and-hole strategy, that triggers auxin signaling without interfering with endogenous auxin or TIR1/AFBs. A synthetic, convex IAA (cvxIAA) hijacked the downstream auxin signaling in vivo both at the transcriptomic level and in specific developmental contexts, only in the presence of a complementary, concave TIR1 (ccvTIR1) receptor. Harnessing the cvxIAA-ccvTIR1 system, we provide conclusive evidence for the role of the TIR1-mediated pathway in auxin-induced seedling acid growth. The cvxIAA-ccvTIR1 system serves as a powerful tool for solving outstanding questions in auxin biology and for precise manipulation of auxin-mediated processes as a controllable switch.

DWARF14 (D14) is a strigolactone receptor that plays a central role in suppression of shoot branching, and hence is a potential target to increase crop productions and biomass. Recently, we reported a fluorescence turn-on probe, Yoshimulactone Green (YLG), which generates a strong fluorescence upon the hydrolysis by D14-type strigolactone receptors. Herein, we applied a YLG-based in vitro assay to a high-throughput chemical screening and identified a novel small molecule DL1 as a potent inhibitor of D14. DL1 competes with endogenous strigolactones, thereby increasing the number of shoot branching in a model plant Arabidopsis as well as in rice. Thus, DL1 is expected to be useful not only as a tool to understand the biological roles of D14 receptors in plant growth and development, but also as a potent agrochemical to improve the crop yield., Competing Interests: The authors declare no competing financial interest.

Englerin A, a guaiane sesquiterpene isolated from Phyllanthus engleri, showed highly potent and selective growth inhibitory activities against renal cancer cell lines. We synthesized the key tricyclic intermediate from commercially available 2,2-dimethyl-1,3-dioxan-5-one via regio- and diastereoselective [4+3] cycloaddition between the formyl enol silyl ether and the disubstituted furan, in 4.8% total yield over 10 steps.

The increasing climate changes and global warming are leading to colossal agricultural problems such as abatement of food production and quality. As stomatal development is considered to play a key role in crop plant productivity and water-use efficiency, studying stomatal development is useful for understanding the productivity of plant systems for both natural and agricultural systems. Herein, we report the first-in-class synthetic small molecules enhancing the number of stomata in Arabidopsis thaliana that have been discovered by screening of the chemical library and further optimized by the Pd-catalyzed C-H arylation reaction. The present study shows not only huge potential of small molecules to control the cellular and developmental processes of stomata without using genetically modified plants, but also the power of C-H functionalization chemistry to rapidly identify the optimized compounds.

Sturgeons (Acipenseriformes) are among the most endangered species in the world due to fragmentation and destruction of their natural habitats and to overexploitation, mainly for highly priced caviar. This has led to the development of sturgeon culture, originally for reintroduction, but more recently for caviar production. In both cases, accurate species identification is essential. We report a new tool for accurate identification of Huso huso and Acipenser ruthenus based on nuclear DNA markers. We employed ddRAD sequencing to identify species-specific nucleotide variants, which served as specific binding sites for diagnostic primers. The primers allowed identification of Huso huso and Acipenser ruthenus as well as their discrimination from A. baerii, A. schrenckii, A. gueldenstaedtii, A. stellatus, A. persicus, A. mikadoi, A. transmontanus, and H. dauricus and identification of A. ruthenus and H. huso hybrids with these species, except hybrid between A. ruthenus and A. stellatus. The species-specific primers also allowed identification of bester (H. huso × A. ruthenus), the most commercially exploited sturgeon hybrid. The tool, based on simple PCR and gel electrophoresis, is rapid, inexpensive, and reproducible. It will contribute to conservation of remaining wild populations of A. ruthenus and H. huso, as well as to traceability of their products.

Purpose: Acute kidney injury (AKI) is one of the critical complications after cardiac surgery. In the kidney, angiotensin II (Ang II) is formed by independent mechanisms, and activity of the intrarenal renin-angiotensin-aldosterone (RAAS) system contributes to the progression of kidney damage. Although atrial natriuretic peptide (ANP) exerts protective effects against renal injury by inhibiting the RAAS, the mechanisms of this effect have not been completely clarified. We investigated how human ANP (hANP) could prevent renal damage induced by cardiopulmonary bypass., Methods: Forty-eight patients undergoing cardiac surgery were divided into two groups, with and without hANP infusion. Urinary angiotensinogen, neutrophil gelatinase-associated lipocalin (NGAL) and L-type fatty acid-binding protein (L-FABP) were measured during and after surgery in both groups. Plasma renin activity, Ang II, aldosterone and serum creatinine were also measured., Results: Urinary angiotensinogen levels in the hANP group were significantly lower than in the non-hANP group after cardiopulmonary bypass surgery, at the end of surgery and 3 h after surgery. At 3 h after surgery, urinary NGAL levels in the hANP and non-hANP groups were 371.1 ± 413.6 and 761.4 ± 437.8 μg/gCr, respectively (p < 0.01). Urinary L-FABP levels at the end of surgery in the hANP and non-hANP groups were 238.8 ± 107.4 and 573.9 ± 370.1 μg/gCr, respectively (p < 0.01). Moreover, hANP seemed to significantly reduce the incidence of postoperative AKI., Conclusions: hANP demonstrated renal protective effects during cardiac surgery, and could possibly reduce the incidence of AKI after ischemia-reperfusion surgery. Moreover, this protective effect of hANP is likely induced by inhibition of the intrarenal RAAS.

The maturation-inducing hormone 17α,20β-dihydroxy-4-pregnen-3-one (DHP) was first identified in the amago salmon. Although carbonyl reductase-like 20β-hydroxysteroid dehydrogenase (CR/20β-HSD) was reported to convert 17α-hydroxyprogesterone (17OHP) to DHP in rainbow trout, we previously found that CR/20β-HSD messenger RNA (mRNA) was not upregulated in stimulated granulosa cells from masu salmon, which suggested that DHP is synthesized by a different enzyme. Accordingly, the current study aimed to identify the specific 20β-hydroxysteroid dehydrogenase (20β-HSD) responsible for DHP production by granulosa cells during final oocyte maturation in masu salmon. RNA sequencing was performed on granulosa layers that were isolated from ovarian follicles at 1 month before ovulation and incubated with or without forskolin, which was used to mimic luteinizing hormone, and ∼12 million reads were obtained, which yielded 71,062 contigs of >100 bp. tBlastx analysis identified 1 contig (#f103496) as similar to 17β-hydroxysteroid dehydrogenase type 12 (hsd17β12); however, because the full-length #f103496 sequence was different from hsd17β12, it was termed hsd17β12-like (hsd17β12l). We found that mammalian cells transfected with full-length hsd17β12l exhibited considerable 20β-HSD activity, as indicated by efficient conversion of exogenous 17OHP to DHP. In addition, we found that hsd17β12l mRNA levels were consistently low in follicles during vitellogenic growth; however, the levels increased significantly during final oocyte maturation. The levels of hsd17β12l mRNA were also considerably increased in granulosa layers in which 20β-HSD activity was induced by salmon pituitary extract. Therefore, we suggest that hsd17β12l, not CR/20β-HSD, is the 20β-HSD responsible for DHP production by granulosa cells in masu salmon during final oocyte maturation., (Copyright © 2017 by the Endocrine Society.)

Unsymmetrical cyanine dyes, such as thiazole orange, are useful for the detection of nucleic acids with fluorescence because they dramatically enhance the fluorescence upon binding to nucleic acids. Herein, we synthesized a series of unsymmetrical cyanine dyes and evaluated their fluorescence properties. A systematic structure-property relationship study has revealed that the dialkylamino group at the 2-position of quinoline in a series of unsymmetrical cyanine dyes plays a critical role in the fluorescence enhancement. Four newly designed unsymmetrical cyanine dyes showed negligible intrinsic fluorescence in the free state and strong fluorescence upon binding to double-stranded DNA (dsDNA) with a quantum yield of 0.53 to 0.90, which is 2 to 3 times higher than previous unsymmetrical cyanine dyes. A detailed analysis of the fluorescence lifetime revealed that the dialkylamino group at the 2-position of quinoline suppressed nonradiative decay in favor of increased fluorescence quantum yield. Moreover, these newly developed dyes were able to stain the nucleus specifically in fixed HeLa cells examined by using a confocal laser-scanning microscope., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Acute kidney injury (AKI) is a serious complication after cardiac surgery, being associated with a high mortality. We assessed three urinary biomarkers, L-type fatty acid-binding protein (L-FABP), neutrophil gelatinase-associated lipocalin (NGAL), and angiotensinogen, which are elevated through different mechanisms, and investigated which of these biomarkers was the earliest and most useful indicator of AKI after cardiac surgery., Methods: This study was a prospective observational study conducted at a single-institution university hospital. All patients were adults aged under 80 years who underwent cardiac surgery with cardiopulmonary bypass between November 2013 and January 2015. Perioperatively, urine samples were obtained from all patients at five points. Based on AKI criteria, patients were divided into two groups: AKI group (n = 11) and non-AKI group (n = 39), according to postoperative serum creatinine (Cr) levels., Results: Urinary L-FABP, NGAL, angiotensinogen, and Cr were measured perioperatively. L-FABP was significantly higher in the AKI group than in the non-AKI group at the end of surgery and 3 h after surgery. L-FABP levels were 601.5 ± 341.7 and 233.8 ± 127.2 μg/g Cr in the AKI and non-AKI groups, respectively. Three hours after surgery, NGAL levels were 950.5 ± 827.9 and 430.0 ± 250.6 μg/g Cr in the AKI and non-AKI groups, respectively, the level being significantly higher in the AKI group than in the non-AKI group. There were no significant differences in urinary angiotensinogen levels between the two groups at any time point., Conclusions: We demonstrated the utility of L-FABP and NGAL, but not angiotensinogen in the early recognition of AKI. The problem of the different peak points among biomarkers needs to be resolved for discovery of a panel of biomarkers.

To characterize type I and II collagen in the Amur sturgeon at the molecular level, mRNAs encoding the proα chain of both types of collagen were cloned and sequenced. Full sequences of both were obtained, and the molecular phylogeny based on the deduced amino acid sequence indicated that the correct sequences of the target genes were obtained. Analyses of primary structure of the proα chains revealed that type I and II collagen share the basic structure of the proα chain of fibril collagen, but have different characteristics, especially in residues related to thermal stability. In the triple helical domain, Gly-Pro-Pro sequence stabilizing the tripeptide unit was more frequent in type II than in type I, and Gly-Gly, which likely decline in thermal stability, was more frequent in type I than in type II. These results suggested that the denaturation temperature of type II would be remarkably higher than type I. The spatial pattern of gene expression was analyzed by quantitative real-time PCR, which showed that relatively ubiquitous type I gene and strongly skewed distribution of type II gene, which highly expressed only in vertebra, snout cartilage, and notochord. This pattern was similar to the distribution pattern of each collagen protein detected by previous biochemical analyses using Amur and Bester sturgeons. The present study is the first report of the cloning of the full-length cDNAs for both of type I and type II collagen in the Amur sturgeon, and is the first comparative analysis of type I and II collagens in a sturgeon species at the molecular level. The results provide basic and general information on collagens in sturgeons., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The dimethylamino (Me2N) group is arguably the most versatile functional group capable of highly efficient and site-selective directed aromatic functionalizations at the ortho-, meta-, and para-positions depending on reaction conditions. While the repertoire of Me2N-directed reactions is growing at a rapid pace, the lack of a general method to transform this group to other functionalities hampers its wider application in organic synthesis. Here we report nickel-catalyzed C-N borylations of aryl- and benzyl-dimethylamines that permit the conversion of a huge library of largely underutilized Me2N-containing organic molecules into various functional molecules by taking advantage of the wealth of existing C-B functionalization methods., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Sturgeons (chondrostean, acipenseridae) are ancient fish species, widely known for their caviar. Nowadays, most of them are critically endangered. The sterlet (Acipenser ruthenus) is a common Eurasian sturgeon species with a small body size and the fastest reproductive cycle among sturgeons. Such species can be used as a host for surrogate production; application is of value for recovery of critically endangered and huge sturgeon species with an extremely long reproductive cycle. One prerequisite for production of the donor's gametes only is to have a sterile host. Commonly used sterilization techniques in fishes such as triploidization or hybridization do not guarantee sterility in sturgeon. Alternatively, sterilization can be achieved by using a temporary germ cell exclusion-specific gene by a knockdown agent, the antisense morpholino oligonucleotide (MO). The targeted gene for the MO is the dead end gene (dnd) which is a vertebrate-specific gene encoding a RNA-binding protein which is crucial for migration and survival of primordial germ cells (PGCs). For this purpose, a dnd homologue of Russian sturgeon (Agdnd), resulting in the same sequence in the start codon region with isolated fragments of sterlet dnd (Ardnd), was used. Reverse transcription polymerase chain reaction confirmed tissue-specific expression of Ardnd only in the gonads of both sexes. Dnd-MO for depletion of PGCs together with fluorescein isothiocyanate (FITC)-biotin-dextran for PGCs labeling was injected into the vegetal region of one- to four-cell-stage sterlet embryos. In the control groups, only FITC was injected to validate the injection method and labeling of PGCs. After optimization of MO concentration together with volume injection, 250-μM MO was applied for sterilization of sturgeon embryos. Primordial germ cells were detected under a fluorescent stereomicroscope in the genital ridge of the FITC-labeled control group only, whereas no PGCs were present in the body cavities of morphants at 21 days after fertilization. Moreover, the body cavities of MO-treated and nontreated fish were examined by histology and in situ hybridization, showing gonads which had no germ cells in morphants at various stages (60, 150, and 210 days after fertilization). Taken together, these results report the first known and functional method of sturgeon sterilization., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Estradiol-17β (E2) and maturation-inducing hormone (MIH) are two steroid hormones produced in the teleost ovary that are required for vitellogenic growth and final oocyte maturation and ovulation. During this transition, the main steroid hormone produced in the ovary shifts from estrogens to progestogens. In the commercially important Japanese eel (Anguilla japonica), the MIH 17α,20β-dihydroxy-4-pregnen-3-one (DHP) is generated from its precursor by P450c17, which has both 17α-hydroxylase and C17-20 lyase activities. In order to elucidate the regulatory mechanism underlying the steroidogenic shift from E2 to DHP and the mechanistic basis for the failure of this shift in artificially matured eels, the cDNA for cyp17a2-which encodes P450c17-II-was isolated from the ovary of wild, mature Japanese eel and characterized, and the expression patterns of cyp17a1 and cyp17a2 during induced ovarian development were investigated in cultured eel ovaries. Five cDNAs (types I-V) encoding P450c17-II were identified that had minor sequence variations. HEK293T cells transfected with all but type II P450c17-II converted exogenous progesterone to 17α-hydroxyprogesterone (17α-P), providing evidence for 17α-hydroxylase activity; however, a failure to convert 17α-P to androstenedione indicated that C17-20 lyase activity was absent. Cyp17a2 mRNA was expressed mainly in the head kidney, ovary, and testis, and quantitative PCR analysis demonstrated that expression in the ovary increased during induced vitellogenesis and oocyte maturation/ovulation. In contrast, P450c17-I showed both 17α-hydroxylase and C17-20 lyase activities, and cyp17a1 expression increased until the mid-vitellogenic stage and remained high thereafter. Considering the high level of cyp17a2 transcript in the eel ovary at the migratory nucleus stage together with our previous report demonstrating that eel ovaries have strong 17α-P-to-DHP conversion activity, the failure of artificially maturing eels to produce the maturation-inducing DHP may be explained by a deficiency in 17α-P production due to the persistence of cyp17a1 expression after the completion of vitellogenesis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Elucidating the signaling mechanism of strigolactones has been the key to controlling the devastating problem caused by the parasitic plant Striga hermonthica. To overcome the genetic intractability that has previously interfered with identification of the strigolactone receptor, we developed a fluorescence turn-on probe, Yoshimulactone Green (YLG), which activates strigolactone signaling and illuminates signal perception by the strigolactone receptors. Here we describe how strigolactones bind to and act via ShHTLs, the diverged family of α/β hydrolase-fold proteins in Striga. Live imaging using YLGs revealed that a dynamic wavelike propagation of strigolactone perception wakes up Striga seeds. We conclude that ShHTLs function as the strigolactone receptors mediating seed germination in Striga. Our findings enable access to strigolactone receptors and observation of the regulatory dynamics for strigolactone signal transduction in Striga., (Copyright © 2015, American Association for the Advancement of Science.)

In Hokkaido, northern Japan, soybean [Glycine max (L.) Merr.] crops are damaged by cold weather. Chilling temperatures result in the appearance of cracking seeds (CS) in soybean crops, especially those grown in eastern and northern Hokkaido. Seed coats of CS are severely split on the dorsal side, and the cotyledons are exposed and frequently separated. CS occurrence causes unstable production because these seeds have no commodity value. However, little is known about the CS phenomenon. The aims of this study were to identify the cold-sensitive stage associated with CS occurrence and to develop a method to select CS-tolerant lines. First, we examined the relationship between chilling temperatures after flowering and CS occurrence in field tests. The average temperature 14 to 21 days after flowering was negatively correlated with the rate of CS. Second, we evaluated differences in CS tolerance among soybean cultivars and breeding lines in field tests. 'Toyohomare' and 'Toiku-238' were more CS-tolerant than 'Yukihomare' and 'Toyomusume'. Third, we developed a selection method in which plants were subjected to 21-day chilling-temperature treatment from 10 days after flowering in a phytotron. This enabled comparisons of CS tolerance among cultivars. This selection method will be useful for breeding CS-tolerant soybeans.

A novel crosslink-forming nucleobase, 2-amino-6-(1-ethylthiovinyl)purine (ATVP), which is triggered by the oxidation of sulfide to sulfoxide, has been developed. The oxidation of ATVP within the duplex proceeded with H2O2 and FeCl2. We have successfully developed the crosslinking reactions activated by oxidation.

Masking the miRNA binding site: Crosslink-forming oligonucleotide (CFO) was used for target gene-specific inhibition of microRNA (miRNA) functions. This method can interfere with specific miRNA-mRNA interactions by recognizing sequences unique to the 3'-UTR that are inherent in each mRNA., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Synthetic light-driven molecular motors are molecular machines capable of rotation under photo-irradiation. In this paper, we report the synthesis of peptide-conjugated molecular motors and evaluate their DNA-binding properties.

Oligodeoxynucleotides (ODNs) have been widely used for inhibiting the gene expression in antisense or antigene methods, and the interstrand cross-linking (ICL) forming ODNs have been expected to ensure the inhibition by these methods. Previously, we reported a highly efficient and selective ICL reaction toward cytosine using the 2-amino-6-vinylpurine derivative under acidic conditions. In this Letter, we report the synthesis of ODN containing 6-amino-2-vinylpurine derivatives and evaluation of the cross-linking reactivity., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The development of convenient methods for controlling the protein expression is an important challenge in the postgenomic era. We applied the crosslink forming oligonucleotide (CFO) as a terminator of the ribosomal translation. In this study, we demonstrated that the improved reactivity of our CFO under physiological conditions enabled the sequence-specific introduction of a steric block for a ribosome on mRNAs. In vitro and in cell translation experiments revealed that the crosslinked mRNA can produce the truncated proteins in which the translation terminates at the desired position., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

While the cultivated soybean, Glycine max (L.) Merr., is more recalcitrant to pod dehiscence (shattering-resistant) than wild soybean, Glycine soja Sieb. & Zucc., there is also significant genetic variation in shattering resistance among cultivated soybean cultivars. To reveal the genetic basis and develop DNA markers for pod dehiscence, several research groups have conducted quantitative trait locus (QTL) analysis using segregated populations derived from crosses between G. max accessions or between a G. max and G. soja accession. In the populations of G. max, a major QTL was repeatedly identified near SSR marker Sat&#95;366 on linkage group J (chromosome 16). Minor QTLs were also detected in several studies, although less commonality was found for the magnitudes of effect and location. In G. max × G. soja populations, only QTLs with a relatively small effect were detected. The major QTL found in G. max was further fine-mapped, leading to the development of specific markers for the shattering resistance allele at this locus. The markers were used in a breeding program, resulting in the production of near-isogenic lines with shattering resistance and genetic backgrounds of Japanese elite cultivars. The markers and lines developed will hopefully contribute to the rapid production of a variety of shattering-resistant soybean cultivars.

We previously reported that oligodeoxynucleotides containing 2-amino-6-vinylpurine (2-AVP: 1) exhibit efficient selective cross-linking to cytosine. In this study, the 2'-OMe nucleoside analogue (2) of 2-AVP was designed in order to increase its affinity to RNA and enhance metabolic stability. It has been demonstrated that 2'-OMe oligonucleotides bearing 2 achieve highly selective cross-linking to the thymine base in DNA and show higher antisense effect on luciferase production in cell lysate., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

We report the use of exciton-coupled circular dichroism (ECCD) spectroscopy in multianalyte sensing systems in complex matrices. To prepare ECCD sensors, anionic anthrylhydazides are reacted with formylphenylboronic acids to give hydrazinoanthrylboronic acids that in turn are reacted with multivalent catechols in aqueous solution. The ECCD signal between the anthracene chromophores in the resulting boronate ester products depends strongly on the structure of the boronic acid sensor and the polyphenol analyte. This dependence of the ECCD signal on analyte structure is interesting for sensing applications. Best ECCD response is found for epigallocatechin gallate (EGCG), a key polyphenol in green tea. Weakly bell-shaped pH profiles, sensitivity to ionic strength and decreasing ECCD with decreasing solvent polarity imply that the CD active product is stabilized by hydrophobic interactions between the anthracene chromophores and by formation of the conjugate bases of the boronic esters. Analyte screening reveals selectivity for divalent catechols, with effective concentrations down to EC(50) = 5 microM for EGCG. Monovalent or achiral catechols such as (+)-catechin, protocatechuate or homoprotocatechuate are not detected. However, the latter two become detectable when attached to a chiral, divalent 1,2-cyclohexylamine scaffold. Application of this simple, user-friendly ECCD system to polyphenol sensing in various green tea extracts delivers easily accessible and reproducible values in the expected range., (2009 Wiley-Liss, Inc.)

A series of glycosyl haloacetamides were synthesized as potential inhibitors of cytoplasmic peptide:N-glycanase (PNGase), an enzyme that removes N-glycans from misfolded glycoproteins. Chloro-, bromo-, and iodoacetamidyl chitobiose and chitotetraose derivatives exhibited a significant inhibitory activity. No inhibitory activity was observed with of fluoroacetamididyl derivatives. Moreover, N-acetylglucosamine derivatives, beta-chloropropionamidyl chitobiose, and chloroacetamidyl cellooligosaccharide derivatives did not show any activity. These results underscore the importance of the N-acetyl groups of chitobiose for PNGase recognition. In addition, reactivity and position of the leaving group at the reducing end are also important factors.

The Michael addition of hydrazide signal amplifiers to short-lived orthoquinone analytes is introduced as a new method to contribute toward the discrimination of polyphenols (e.g. epigallocatechin gallate, resveratrol) with synthetic pores.

Multicomponent sensing in complex matrices with synthetic pores became feasible with the introduction of amplifiers. Amplifiers are defined as molecules that can covalently capture undetectable analytes after enzymatic signal generation and drag them into the pore for transduction. Here, we introduce converters as molecules that can shift the reactivity of amplifiers in situ to capture chemoorthogonal analytes. For this purpose, a series of dialkoxynaphthalene (DAN) and dialkoxyanthracene (DAA) hydrazinoboronic acids was prepared in situ from DAN and DAA hydrazides and formylphenylboronic acids. These converted amplifiers efficiently inactivate synthetic pores with internal naphthalenediimide clamps. This pore inactivation by DAN and DAA hydrazinoboronic acids vanishes in the presence of catechols such as (+)-catechin, presumably because the obtained boronate esters are too large to bind within the synthetic beta-barrel pore or because they prefer to partition into the bilayer membrane. The resulting increase in pore activity with increasing catechol concentration at constant amplifier concentration is shown to be compatible with the sensing of polyphenols in green tea.

Synthetic ion channels and pores attract current attention as multicomponent sensors in complex matrixes. This application requires the availability of reactive signal amplifiers that covalently capture analytes and drag them into the pore. pi-Basic 1,5-dialkoxynaphthalenes (1,5-DAN) are attractive amplifiers because aromatic electron donor-acceptor (AEDA) interactions account for their recognition within pi-acidic naphthalenediimide (NDI) rich synthetic pores. Focusing on amplifier design, we report here the synthesis of a complete collection of DAN and dialkoxyanthracene amplifiers, determine their oxidation potentials by cyclic voltammetry, and calculate their quadrupole moments. Blockage experiments reveal that subtle structural changes in regioisomeric DAN amplifiers can be registered within NDI pores. Frontier orbital overlap in AEDA complexes, oxidation potentials, and, to a lesser extent, quadrupole moments are shown to contribute to isomer recognition by synthetic pores. Particularly important with regard to practical applications of synthetic pores as multianalyte sensors, we further demonstrate that application of the lessons learned with DAN regioisomers to the expansion to dialkoxyanthracenes provides access to privileged amplifiers with submicromolar activity.

Cytoplasmic peptide:N-glycanase (PNGase) is an enzyme that removes N-glycans from misfolded glycoproteins. The function of cytoplasmic PNGase plays a significant role in the degradation of misfolded glycoproteins, which is critical for cell viability. Recently, we reported that haloacetoamidyl derivatives of high-mannose-type oligosaccharides selectively modify the catalytic cysteine of cytoplasmic PNGase and serve as its specific inhibitor. Interestingly, a drastically simplified chloroacetamidyl chitobiose derivative [(GlcNAc)(2)-ClAc] was also reactive to PNGase. In our work, it was conjugated to a hydrophobic fluorophore in order to render (GlcNAc)(2)-ClAc cells permeable. We demonstrated that this compound [BODIPY-(GlcNAc)(2)-ClAc] specifically binds to cytoplasmic PNGase from budding yeast (Png1). To date, only Z-VAD-fmk is known as an inhibitor of PNGase. BODIPY-(GlcNAc)(2)-ClAc and Z-VAD-fmk share the same binding site on Png1, while BODIPY-(GlcNAc)(2)-ClAc has markedly stronger inhibitory activity. The functional analysis of PNGase using Z-VAD-fmk should be carefully interpreted because of its intrinsic property as a caspase inhibitor. In sharp contrast, chloroacetamidyl chitobiose was not reactive to caspase. In addition, BODIPY-(GlcNAc)(2)-ClAc did not bind either chitobiose-binding lectins or PNGase from other sources. Moreover, fluorescent microscopy clearly showed that BODIPY-(GlcNAc)(2)-ClAc was efficiently introduced into cells. These results suggest that this compound could be an in vivo inhibitor of cytoplasmic PNGase.