Removal and detoxification of iprodione in water using didodecyldimethylammonium bromide-montmorillonite organoclay and manganese dioxide.

Combination of organoclay sorption with manganese(IV) oxide (MnO2) catalyzed catechol oxidation was studied for the removal of a dicarboximide fungicide, iprodione, from water. Iprodion in water was sorbed on didodecyldimethylammonium bromide (DDAB)-modified montmorillonite (MT) organoclay and converted into the degraded product, 3,5-dichloroaniline (DCA). The degree of sorption increased by the modification with DDAB, because of the formation of a hydrophobic region for the incorporation of iprodione and negligibly interfered by coexisting MnO2. The half-life for the degradation of irodione in water at 25 °C was 7 days, whreas it reduced to 15 min in the organoclay. The activation energy, 65.4 ± 4.8 kJ mol-1, for the first-order reaction in the aqueous solution (pH 7.0) decreased to 43.9 ± 1.8 kJ mol-1 in the organoclay, indicating the catalytic activity of the organoclay that accelerates the hydrolysis reaction of iprodione. In the coexistence of appropriate amounts of MnO2 and catechol, the degraded product, DCA, reacted with oxidized products of catechol to form a water-insoluble precipitate and was successfully eliminated from water. The results obtained in the present study strongly suggest the applicability of the combined method of organoclay sorption method and MnO2-catalyzed oxidation for the diffusion control of toxic agrochemicals.

Quantitative analysis of powder samples using screen-printing techniques as sample preparation methods for LA-ICP-MS.

The combination of laser ablation and inductively coupled plasma mass spectrometry (LA-ICP-MS) offers a powerful tool for directly analyzing solid samples. However, LA-ICP-MS has a limitation in quantitative analyses owing to a requirement for matrix-matched standard materials. In this study, we have developed a sample preparation method that facilitates quantitative analyses by LA-ICP-MS. The sample powder is dispersed in a liquid resin and film-like samples are prepared from the resulting paste by a screen-printing technique. The sample includes the analyte spiked with internal standards and is prepared by mixing standard solutions in the sample paste. Because all reagents except for the sample powder are liquids, homogeneous samples can be easily obtained. The internal standard and concentration of the spiked analyte can be tailored for each sample, which is a requirement for accurate quantitative analyses. The amount of sample and concentration of the spiked analyte are controlled against an internal standard, enabling internal standardization without the need to have an element of known concentration in the sample. The accuracy of this method was evaluated by analyzing impurity elements in TiO2 powder; however, it is expected that other materials could also be analyzed. The versatility and flexibility of this method suggest great potential for quantitative analyses by LA-ICP-MS, for which reliable matrix-matched standard materials are required.

Interim analysis of post-marketing surveillance of ravulizumab for paroxysmal nocturnal hemoglobinuria in Japan.

Ravulizumab is a long-acting C5 inhibitor available for treating paroxysmal nocturnal hemoglobinuria (PNH). Post-marketing surveillance (PMS) was implemented following its approval in September 2019 in Japan. We report safety data obtained through to December 2021 for 218 patients and effectiveness data for 194 patients (182 switched from eculizumab and 12 complement inhibitor-naïve). Over a median follow-up of 74.4 weeks, 193 adverse events (AEs) were reported in 66/218 patients (30.3%; incidence 72.73/100 patient-years). The two most frequent AEs were anemia and pyrexia (each 3.01/100 patient-years). The incidence of serious AEs was 36.93/100 patient-years. In patients who switched from eculizumab, lactate dehydrogenase (LDH) and hemoglobin (Hb) levels were maintained over 26 weeks of ravulizumab treatment. In complement inhibitor-naïve patients, LDH decreased significantly and Hb increased significantly over 26 weeks of ravulizumab treatment. These data for Japanese patients with PNH who were naïve to complement inhibitors and patients who switched from eculizumab show that the safety and effectiveness of ravulizumab are consistent with the published clinical trial data. However, transfusion independence was less likely in patients with bone marrow failure. Further follow-up data from this PMS will help to elucidate the long-term clinical safety and effectiveness of ravulizumab for treating PNH.

Real-world safety profile of eculizumab in patients with paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, or generalized myasthenia gravis: an integrated analysis of post-marketing surveillance in Japan.

Eculizumab is a C5 inhibitor approved for the treatment of paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), and anti-acetylcholine receptor antibody-positive generalized myasthenia gravis (AChR + gMG) in Japan. We report integrated safety data from post-marketing surveillance in these three indications, focusing on commonly occurring adverse events (AEs) and infection-related AEs. Of 1219 patients registered, 1055 (PNH: 780; aHUS: 192; AChR + gMG: 83) had available safety data. Total eculizumab exposure was 3977.361 patient-years. AEs were reported in 74.03% of patients. AEs with an incidence of  ≥ 1.0 per 100 patient-years included hemolysis, headache, nasopharyngitis, renal impairment, anemia, pneumonia, upper respiratory tract inflammation, influenza, condition aggravated, and infection. The incidence of infection-related AEs was 21.30 per 100 patient-years, the most frequent types (≥ 1.0 per 100 patient-years) being nasopharyngitis, pneumonia, influenza, and infection. Meningococcal infections were reported in four patients (0.10 per 100 patient-years). Two patients died from meningococcal sepsis, with a mortality rate of 0.05 per 100 patient-years. This is the largest safety dataset on eculizumab in Japan derived from more than 10 years of clinical experience. No new safety signals were observed and the safety profile of eculizumab was consistent with that in previous clinical trials and international real-world safety analyses.

Determinants of Subtype-Selectivity of the Anthelmintic Paraherquamide A on Caenorhabditis elegans Nicotinic Acetylcholine Receptors.

The anthelmintic paraherquamide A acts selectively on the nematode L-type nicotinic acetylcholine receptors (nAChRs), but the mechanism of its selectivity is unknown. This study targeted the basis of paraherquamide A selectivity by determining an X-ray crystal structure of the acetylcholine binding protein (AChBP), a surrogate nAChR ligand-binding domain, complexed with the compound and by measuring its actions on wild-type and mutant Caenorhabditis elegans nematodes and functionally expressed C. elegans nAChRs. Paraherquamide A showed a higher efficacy for the levamisole-sensitive [L-type (UNC-38/UNC-29/UNC-63/LEV-1/LEV-8)] nAChR than the nicotine-sensitive [N-type (ACR-16)] nAChR, a result consistent with in vivo studies on wild-type worms and worms with mutations in subunits of these two classes of receptors. The X-ray crystal structure of the Ls-AChBP-paraherquamide A complex and site-directed amino acid mutation studies showed for the first time that loop C, loop E, and loop F of the orthosteric receptor binding site play critical roles in the observed L-type nAChR selective actions of paraherquamide A. SIGNIFICANCE STATEMENT: Paraherquamide A, an oxindole alkaloid, has been shown to act selectively on the L-type over N-type nAChRs in nematodes, but the mechanism of selectivity is unknown. We have co-crystallized paraherquamide A with the acetylcholine binding protein, a surrogate of nAChRs, and found that structural features of loop C, loop E, and loop F contribute to the L-type nAChR selectivity of the alkaloid. The results create a new platform for the design of anthelmintic drugs targeting cholinergic neurotransmission in parasitic nematodes.

Competitive chrodrimanin B interactions with rat brain GABAA receptors revealed by radioligand binding assays.

Meroterpenoid compounds chrodrimanins produced by Talaromyces sp. YO-2 have been shown to act as competitive antagonists of silkworm larval GABAA receptors using electrophysiology, yet no further evidence has been provided to support such an action. We have investigated the actions of chrodrimanin B on rat brain GABAA receptors by binding assays with non-competitive ligand of GABAA receptors [3H]EBOB and competitive ligands [3H]gabazine and [3H]muscimol. Chrodrimanin B did not significantly affect the binding of [3H]EBOB while reducing the binding of [3H]gabazine and [3H]muscimol to the rat membrane preparations. Chrodrimanin B increased the dissociation constant Kd of [3H]gabazine and [3H]muscimol without significantly affecting the maximum binding, pointing to competitive interactions of chrodrimanin B with rat GABAA receptors in support of our previous observation that the compound acts as a competitive antagonist on the silkworm larval GABA receptor.

Stimulation of asymbiotic sporulation in arbuscular mycorrhizal fungi by fatty acids.

Arbuscular mycorrhizal (AM) fungi are obligate symbionts that depend on living host plants to complete their life cycle1,2. This feature, which leads to their unculturability in the absence of plants, strongly hinders basic research and agricultural application of AM fungi. However, at least one AM fungus can grow and develop fertile spores independently of a host plant in co-culture with the bacterium Paenibacillus validus3. The bacteria-derived substances are thought to act as stimulants or nutrients for fungal sporulation, but these molecules have not been identified. Here, we show that (S)-12-methyltetradecanoic acid4,5, a methyl branched-chain fatty acid isolated from bacterial cultures, stimulates the branching of hyphae germinated from mother spores and the formation of secondary spores in axenic culture of the AM fungus Rhizophagus irregularis. Extensive testing of fatty acids revealed that palmitoleic acid induces more secondary spores than the bacterial fatty acid in R. irregularis. These induced spores have the ability to infect host plant roots and to generate daughter spores. Our work shows that, in addition to a major source of organic carbon6-9, fatty acids act as stimulants to induce infection-competent secondary spores in the asymbiotic stage and could provide the key to developing the axenic production of AM inoculum.

Anti-plant viral activity of peptaibols, trichorzins HA II, HA V, and HA VI, isolated from Trichoderma harzianum HK-61.

Three peptaibols, trichorzins HA II (1), HA V (2), and HA VI (3), were isolated from okara fermented with Trichoderma harzianum HK-61 as anti-plant viral agents. Their structures were confirmed by spectroscopic and chemical methods. At micro molar concentrations, the trichorzins inhibited infections by Cucumber mosaic virus in the cowpea plant Vigna sesquipedalis.

Biosynthesis and Structure-Activity Relationship Studies of Okaramines That Target Insect Glutamate-Gated Chloride Channels.

Prenylated indole alkaloid okaramines selectively target insect glutamate-gated chloride channels (GluCls). Because of their highly complex structures, including azocine and azetidine rings, total synthesis of okaramine A or B has not been achieved, preventing evaluation of the biological activities of okaramines. Biosynthetic approaches provide alternatives to accessing structurally diverse derivatives and enabling the elucidation of structure-activity relationships. To explore the biosynthetic potential of okaramines, gene knockout experiments of an okaramine-producer fungus were performed. The deletion mutants of the oxygenase genes okaB, okaD, okaE, and okaG provided analogues that were unlikely to be accumulated in the normal biosynthetic process of the wild-type strain. Analysis of the structure-activity relationships of okaramines collected from the fungal cultures revealed that 1,4-dihydroazocine and N-aliphatic group attached to the indole were crucial for GluCl-activating activity. This provided insights into further derivatization of the complex structure of okaramines in order to facilitate the development of new insecticides.

An L319F mutation in transmembrane region 3 (TM3) selectively reduces sensitivity to okaramine B of the Bombyx mori l-glutamate-gated chloride channel.

Okaramines produced by Penicillium simplicissimum AK-40 activate l-glutamate-gated chloride channels (GluCls) and thus paralyze insects. However, the okaramine binding site on insect GluCls is poorly understood. Sequence alignment shows that the equivalent of residue Leucine319 of the okaramine B sensitive Bombyx mori (B. mori) GluCl is a phenylalanine in the okaramine B insensitive B. mori γ-aminobutyric acid-gated chloride channel of the same species. This residue is located in the third transmembrane (TM3) region, a location which in a nematode GluCl is close to the ivermectin binding site. The B. mori GluCl containing the L319F mutation retained its sensitivity to l-glutamate, but responses to ivermectin were reduced and those to okaramine B were completely blocked.

Comparison of salivary hemoglobin measurements for periodontitis screening.

Salivary hemoglobin (Hb) for screening of periodontitis is approved under the pharmaceutical affairs law of Japan. Two reagents are commercially available for the modified fecal occult blood test: Saliva Hemo Plus and OC-AUTO S Latex Reagent. We simultaneously measured split specimens from 561 samples by using these two methods and compared the differences and agreement between both methods. Moreover, saliva samples were collected from 10 subjects at five time points during the day for analysis of circadian variations and fluctuation. The Pearson's correlation coefficient for these two reagents was 0.794. The Bland-Altman plot of differences in salivary Hb levels measured by the two reagents indicated that the difference included fixed errors (0.55 µg/mL). On analysis of circadian variations, no statistically significant differences were observed using the Friedman test. However, fixed errors were observed between wake-up time and before dinner and before lunch and before dinner, and no random errors were observed by Bland-Altman analysis. In conclusion, the salivary Hb levels measured using OC-AUTO S Latex Reagent were lower than those measured using Saliva Hemo Plus, along with a tendency for higher levels in the morning. Thus, when performing salivary tests these observations must be considered.

Cytototoxic constituents from the bark of Chisocheton cumingianus (Meliaceae).

A new lanostane-type triterpenoid, 3ß-hydroxy-25-ethyl-lanost-9(11),24(24')-diene (1), along with 3ß-hydroxy-lanost-7-ene (2) and ß-sitosterol-3-O-acetate (3) was isolated from the stem bark of C. cumingianus. The chemical structure of the new compound was elucidated on the basis of spectroscopic data. All of the compounds were evaluated for their cytotoxic effects against P-388 murine leukemia cells. Compounds 1-3 showed cytotoxicity against P-388 murine leukemia cells with IC50 values of 28.8 ± 0.10, 4.29 ± 0.03, and 100.18 ± 0.16 µg/ml, respectively.

Okaramine insecticidal alkaloids show similar activity on both exon 3c and exon 3b variants of glutamate-gated chloride channels of the larval silkworm, Bombyx mori.

The okaramine indole alkaloids were recently shown to be more selective than ivermectin in activating the glutamate-gated chloride channels of the silkworm larvae of Bombyx mori (BmGluCls). Those studies were carried out using the exon 3b variant as a representative of BmGluCls. However, it remains unknown whether okaramines are similarly effective on other silkworm GluCl variants and whether they share the same binding site as ivermectin on GluCls. To begin to address these questions, we examined the potency of four okaramines on the exon 3c variant of BmGluCls by two-electrode voltage clamp voltage recordings of glutamate-induced chloride currents. The potency of okaramines in activating the exon 3c BmGluCl agreed well with findings on the exon 3b BmGluCl and insecticidal potency. Okaramine B (10µM) reduced the maximum binding (Bmax) but not the dissociation constant (KD) of [3H]ivermectin in studies on plasma membrane fractions of HEK293 cells expressing the exon 3c variant. These findings indicate that activation of GluCls is important in the insecticidal actions of okaramines.

Carlactone-type strigolactones and their synthetic analogues as inducers of hyphal branching in arbuscular mycorrhizal fungi.

Hyphal branching in the vicinity of host roots is a host recognition response of arbuscular mycorrhizal fungi. This morphological event is elicited by strigolactones. Strigolactones are carotenoid-derived terpenoids that are synthesized from carlactone and its oxidized derivatives. To test the possibility that carlactone and its oxidized derivatives might act as host-derived precolonization signals in arbuscular mycorrhizal symbiosis, carlactone, carlactonoic acid, and methyl carlactonoate as well as monohydroxycarlactones, 4-, 18-, and 19-hydroxycarlactones, were synthesized chemically and evaluated for hyphal branching-inducing activity in germinating spores of the arbuscular mycorrhizal fungus Gigaspora margarita. Hyphal branching activity was found to correlate with the degree of oxidation at C-19 methyl. Carlactone was only weakly active (100 ng/disc), whereas carlactonoic acid showed comparable activity to the natural canonical strigolactones such as strigol and sorgomol (100 pg/disc). Hydroxylation at either C-4 or C-18 did not significantly affect the activity. A series of carlactone analogues, named AD ester and AA'D diester, was synthesized by reacting formyl Meldrum's acid with benzyl, cyclohexylmethyl, and cyclogeranyl alcohols (the A-ring part), followed by coupling of the potassium enolates of the resulting formylacetic esters with the D-ring butenolide. AD ester analogues exhibited moderate activity (1 ng-100 pg/disc), while AA'D diester analogues having cyclohexylmethyl and cyclogeranyl groups were highly active on the AM fungus (10 pg/disc). These results indicate that the oxidation of methyl to carboxyl at C-19 in carlactone is a prerequisite but BC-ring formation is not essential to show hyphal branching activity comparable to that of canonical strigolactones.

Meroterpenoid Chrodrimanins Are Selective and Potent Blockers of Insect GABA-Gated Chloride Channels.

Meroterpenoid chrodrimanins, produced from Talaromyces sp. YO-2, are known to paralyze silkworm (Bombyx mori) larvae, but their target is unknown. We have investigated the actions of chrodrimanin B on ligand-gated ion channels of silkworm larval neurons using patch-clamp electrophysiology. Chrodrimanin B had no effect on membrane currents when tested alone at 1 µM. However, it completely blocked the γ-aminobutyric acid (GABA)-induced current and showed less pronounced actions on acetylcholine- and L-glutamate-induced currents, when delivered at 1 µM for 1 min prior to co-application with transmitter GABA. Thus, chrodrimanins were also tested on a wild-type isoform of the B. mori GABA receptor (GABAR) RDL using two-electrode voltage-clamp electrophysiology. Chrodrimanin B attenuated the peak current amplitude of the GABA response of RDL with an IC50 of 1.66 nM. The order of the GABAR-blocking potency of chrodrimanins B > D > A was in accordance with their reported insecticidal potency. Chrodrimanin B had no open channel blocking action when tested at 3 nM on the GABA response of RDL. Co-application with 3 nM chrodrimanin B shifted the GABA concentration response curve to a higher concentration and further increase of chrodrimanin B concentration to 10 nM; it reduced maximum current amplitude of the GABA response, pointing to a high-affinity competitive action and a lower affinity non-competitive action. The A282S;T286V double mutation of RDL, which impairs the actions of fipronil, hardly affected the blocking action of chrodrimanin B, indicating a binding site of chrodrimanin B distinct from that of fipronil. Chrodrimanin B showed approximately 1,000-fold lower blocking action on human α1ß2γ2 GABAR compared to RDL and thus is a selective blocker of insect GABARs.

Frontier studies on highly selective bio-regulators useful for environmentally benign agricultural production.

Fungal metabolites active for insects were obtained from fermentation products using okara media. The mechanisms of action of these compounds against insects were clarified using voltage clamp electrophysiology. The branching factor inducing hyphal branching in arbuscular mycorrhizal (AM) fungi was isolated from the root exudates of Lotus japonicus and identified as 5-deoxystrigol. Strigolactones were originally identified as seed germination stimulants of parasitic weeds; therefore, synthetic strigolactones were developed to exhibit the inducing activity of hyphal branching in AM fungi and diminish the stimulating activity of seed germination of parasitic weeds. Signaling molecules, acylhomoserine lactones (AHLs), in quorum sensing were identified in the fungal strain Mortierella alpina A-178, and the true producer of AHLs was clarified as symbiotic bacteria in the fungus. Since acyl-(S)-adenosylmethionine analogs may be good candidates for competitive inhibitors of AHL synthases, intermediate mimics in the biosynthesis of AHLs have been synthesized.

Medicaol, a strigolactone identified as a putative didehydro-orobanchol isomer, from Medicago truncatula.

A major strigolactone produced by the model legume Medicago truncatula (barrel medic) has been tentatively identified as a didehydro-orobanchol isomer. In this study, a putative didehydro-orobanchol isomer was isolated from root exudates collected from barrel medic grown hydroponically under phosphate-starved conditions. The structure and absolute configurations of this strigolactone, named medicaol, were determined by a combination of spectroscopic analysis and spectral comparison with 4-deoxymedicaol which was synthesized using solvolysis and rearrangement of hydroxymethylhexahydroindenone to tetrahydroazulenone as a key step. Medicaol has a seven-membered cycloheptadiene in the A ring instead of a typical six-membered cyclohexene. Medicaol and 4-deoxymedicaol showed activity comparable to their corresponding six-membered A ring relatives, orobanchol and 4-deoxyorobanchol (ent-2'-epi-5-deoxystrigol), in inducing hyphal branching of germinating spores of an arbuscular mycorrhizal fungus Gigaspora margarita. Plausible biosynthetic pathways from 4-deoxyorobanchol to medicaol are also proposed.

Synthetic and structure-activity relationship of insecticidal bufadienolides.

A new synthetic analog of bufadienolide, methyl isobryophyllinate A (1), and a known synthetic analog, methyl isobersaldegenate-1,3,5-orthoacetate (2), were obtained by methanolysis of bryophyllin A (3) and bersaldegenin-1,3,5-orthoacetate (5) in basic solution. Structure-insecticidal activity relationship studies revealed both orthoacetate and alpha-pyrone moieties seemed to be essential structural elements for exhibiting insecticidal activity, whereas oxygenated substituents in the C ring enhanced the insecticidal activity against the third instar larvae of silkworm (Bombyx mori).

GluCl a target of indole alkaloid okaramines: a 25 year enigma solved.

In 1989, indole alkaloid okaramines isolated from the fermentation products of Penicillium simplicissimum were shown to be insecticidal, yet the mechanism of their toxicity to insects remains unknown. We therefore examined the action of okaramine B on silkworm larval neurons using patch-clamp electrophysiology. Okaramine B induced inward currents which reversed close to the chloride equilibrium potential and were blocked by fipronil. Thus it was tested on the silkworm RDL (resistant-to-dieldrin) γ-aminobutyric-acid-gated chloride channel (GABACl) and a silkworm L-glutamate-gated chloride channel (GluCl) expressed in Xenopus laevis oocytes. Okaramine B activated GluCl, but not RDL. GluCl activation by okaramines correlated with their insecticidal activity, offering a solution to a long-standing enigma concerning their insecticidal actions. Also, unlike ivermectin, okaramine B was inactive at 10 µM on human α1ß2γ2 GABACl and α1ß glycine-gated chloride channels and provides a new lead for the development of safe insect control chemicals.

An acyl-SAM analog as an affinity ligand for identifying quorum sensing signal synthases.

N-Acylhomoserine lactones (AHLs) are quorum sensing signals produced by Gram-negative bacteria. We here report the affinity purification of AHL synthases using beads conjugated with an enzyme inhibitor, which was designed based on the catalytic intermediate acyl-SAM.