Gene Expression Profiles of the Small Intestinal Mucosa of Dogs Repeatedly Infected with the Cestode \u3ci\u3eEchinococcus multilocularis\u3c/i\u3e

The data set presented in this article is related to a previous research article entitled, The timing of worm exclusion in dogs repeatedly infected with the cestode Echinococcus multilocularis (Kouguchi et al., 2016). This article describes the genes \u3e2 fold up- or down-regulated in the first- and repeated-infection groups compared to the healthy controls group. The gene expression profiles were generated using the Agilent-021193 Canine (V2) Gene Expression Microarray (GPL15379). The raw and normalized microarray data have been deposited with the Gene Expression Omnibus (GEO) database under accession number GSE105098

In Vitro Reconstitution of the Clostridium botulinum Type D Progenitor Toxin

Clostiridium botulinum D型4947株は蛋白分解作用を受けない完全型として、異なったサイズの2種類のプロジェニター毒素（MとL）を産生する。M毒素は神経毒素（NT）と非毒素系ー非血球凝集素（NTNHA）で構成されているのに対し、L毒素はM毒素と血球凝集素（HA-70、HA-17、HA-33）によって構成されている。HA-70サブコンポーネントとHA-33/17混合体は変成剤の存在下でクロマトグラフィーによりL毒素からほぼ単一の形で得られた。著者らは、純化したM毒素、HA-70とHA-33/17を混合してL毒素を再構成することに初めて成功した。再構成したL毒素とネイティブのL毒素はゲル濾過、PAGEプロファイル、血球凝集活性、赤血球への吸着活性、マウスへの経口毒力などの諸症状が全く同じであった。トリプシン処理によりニックを持つNTNHAで構成されたM毒素はHAサブコンポーネントと一緒にしてもL毒素を再構成することが出来なかったのに対し、プロテアーゼ処理したL毒素はM毒素とHAサブコンポーネントに隔離することが出来なかった。これらの結果から、著者らはM毒素が最初にNTとNTNHAの会合により形成され、その後HA-70とHA-33/17の会合によってL毒素へ変換されると結論ずけた

Adult Worm Exclusion and Histological Data of Dogs Repeatedly Infected with the Cestode \u3ci\u3eEchinococcus multilocularis\u3c/i\u3e

The data presented in this article are related to a previously published research article titled The timing of worm exclusion in dogs repeatedly infected with the cestode Echinococcus multilocularis (Kouguchi et al. 2016). This data describe a comparison of worm exclusion in the early stage of infection (1 day and 6 days post-infection) between dogs infected for the first time (control group) and dogs repeatedly infected with the parasite 4 times (repeated infection groups). We observed that 6 days post reinfection, the number of adult worms in repeated-infection groups decreased by 88.7% compared with the control group. Histological analysis comparison of the small intestinal mucosa from healthy, first infected, and repeatedly infected dogs are also reported. We observed no clear pathological abnormality, except the shortening of microvillus in reinfected dogs. However, eosinophil accumulation and eosinophilic ulcers were observed in some reinfected dogs. This data could be useful as preliminary data to develop a final host vaccine for this parasite

Effect of the anti-parasitic compounds pyrvinium pamoate and artemisinin in enzymatic and culture assays: Data on the search for new anti-echinococcal drugs

The dataset presented herein is related to a previous research article titled “Mitochondrial Complex III in Larval Stage of Echinococcus multilocularis as a Potential Chemotherapeutic Target and in vivo Efficacy of Atovaquone Against Primary Hydatid Cysts” [1]. In this report, data were collected by screening drugs for echinococcosis. We investigated the inhibitory activities of artemisinin and pyrvinium pamoate against the mitochondrial respiratory enzymes in E. multilocularis protoscoleces. Artemisinin did not inhibit mitochondrial complexes I, II, and III. However, pyrvinium pamoate inhibited complex I at 11 μM,although complexes II and III were not inhibited. In the culture assay, E. multilocularis protoscoleces were treated with atovaquone (ATV), rotenone, praziquantel, artemisinin, and pyrvinium pamoate at a final concentration of 50 µM in different culture media. The viability of protoscoleces was compared under aerobic and anaerobic conditions via culture experiments. The survival days of E. multilocularis protoscoleces were evaluated in the drug-treated group compared with those in the non-treated group. The results of these culture assays revealed that praziquantel and artemisinin did not eliminate the protoscoleces under both aerobic and anaerobic conditions. However, a stronger elimination ability was observed with the co-administration of praziquantel or artemisinin with ATV than with ATV alone under aerobic conditions. Pyrvinium pamoate completely killed protoscoleces at 5 and 7 days under aerobic and anaerobic conditions, respectively. Pyrvinium pamoate behaved identically to rotenone, the complex I inhibitor, in the culture treatment assay.The data serve as a reference for the development of novel anti-echinococcal drugs

Spontaneous Nicking in the Nontoxic-Nonhemagglutinin Component of the costridium botulinum Toxin Conplex

Clostiridium botulinum D型4947株によって産生された毒素複合体から非毒素ー非血球凝集素（NTNHA）標品を蛋白分解酵素フリーの条件下で、単独の形と神経毒(NT)/NTNHA複合体の形で調製できた。両調製物質のNTNHAはスポンテニアスにニックの入ったNTNHAとなり、15-と115-kDaフラグメント及び特異的部分でいくつかのアミノ酸の削除を生じることが長時間培養のSDS-PAGEで認められた。しかし、NT/NTNHA/血球凝集素(HA)複合体は同じ条件下でニックの入らない一本鎖のペプチドのままであった。NTNHA調製物に少量のニック型の物が含まれていることから、NTNHAのニック型は精製の残物と/あるいはNTNHAがインタクトNTNHAをそれ自身との切断酵素活性によってニックを入れたことが考えられる

Role of C-Terminal Region of HA-33 Component of Botulinum Toxin in Hemagglutination

Clostridium botulinum D型1873株とC型Yoichi株の産生するプロジェニター毒素から得られた1種類のサブコンポーネントである血球凝集素（HA-33）は、C及びD型参照株のそれより少し小さい分子サイズであるが、他のコンポーネントのの大きさに違いがないことが、SDS-PAGEを用いて見いだされた。すなわち、HA-33のN-及びC-末端シーケンス分析に基つき、両株のHA-33淡白において特異的なサイトでC-末端から33アミノ酸残基の欠落があることが見いだされた。両株のプロジェニター毒素は、参照株毒素の血球凝集力価2かそれ以下の弱い血球凝集活性を示すが、赤血球に吸着することはできない。これらの結果はHA-33の短いC-末端部分が、ボツリヌスプロジェンター毒素の血球凝集活性において重要な役割を果たしていることを示す。さらに、シーケンスモチーフの探索により、HA-33のC-末端部分は炭化水素認識サブドメインを持っていることが推測された

Medical Treatment of Echinococcus multilocularis and New Horizons for Drug Discovery: Characterization of Mitochondrial Complex II as a Potential Drug Target

As an efficient drug for alveolar echinococcosis (AE) is still not available, new chemotherapy targets are necessary. The mitochondrial respiratory chain may be a good drug candidate because parasite respiratory chains are quite different from those of mammalian hosts. For example, Ascaris suum possesses an NADH‐fumarate reductase system (fumarate respiration) that is highly adapted to anaerobic environments such as the small intestine. It is composed of mitochondrial complex I (NADH‐ubiquinone reductase), complex II (succinate‐ubiquinone reductase), and rhodoquinone. We previously demonstrated that fumarate respiration is also essential in E. multilocularis. Quinazoline, a complex I inhibitor, inhibited growth of E. multilocularis larvae in vitro. These results indicate that fumarate respiration could be a target for E. multilocularis therapy. In the current chapter, we focused on complex II, which is another component of this system, because quinazoline exhibited strong toxicity to mammalian mitochondria. We evaluated the molecular and biochemical characterization of E. multilocularis complex II as a potential drug target. In addition, we found that ascofuranone, a trypanosome cyanide‐insensitive alternative oxidase inhibitor, inhibited E. multilocularis complex II at the nanomolar order. Our findings demonstrate the potential development of targeted therapy against Echinococcus complex II

Killing Two Birds with One Stone: Discovery of Dual Inhibitors of Oxygen and Fumarate Respiration in Zoonotic Parasite, Echinococcus multilocularis

Ascofuranone (AF), a meroterpenoid isolated from various filamentous fungi, including Acremonium egyptiacum, has been reported as a potential lead candidate for drug development against parasites and cancer. In this study, we demonstrated that AF and its derivatives are potent anthelminthic agents, particularly against Echinococcus multilocularis, which is the causative agent of alveolar echinococcosis. We measured the inhibitory activities of AF and its derivatives on the mitochondrial aerobic and anaerobic respiratory systems of E. multilocularis larvae. Several derivatives inhibited complex II (succinate:quinone reductase [SQR]; IC50 = 0.037 to 0.135 mM) and also complex I to III (NADH:cytochrome c reductase; IC50 = 0.008 to 0.401 mM), but not complex I (NADH:quinone reductase), indicating that mitochondrial complexes II and III are the targets. In particular, complex II inhibition in the anaerobic pathway was notable because E. multilocularis employs NADH:fumarate reductase (fumarate respiration), in addition to NADH oxidase (oxygen respiration), resulting in complete shutdown of ATP synthesis by oxidative phosphorylation. A structure-activity relationship study of E. multilocularis complex II revealed that the functional groups of AF are essential for inhibition. Binding mode prediction of AF derivatives to complex II indicated potential hydrophobic and hydrogen bond interactions between AF derivatives and amino acid residues within the quinone binding site. Ex vivo culture assays revealed that AF derivatives progressively reduced the viability of protoscoleces under both aerobic and anaerobic conditions. These findings confirm that AF and its derivatives are the first dual inhibitors of fumarate and oxygen respiration in E. multilocularis and are potential lead compounds in the development of anti-echinococcal drugs

A Pilot Study on Developing Mucosal Vaccine against Alveolar Echinococcosis (AE) Using Recombinant Tetraspanin 3: Vaccine Efficacy and Immunology

Humans and rodents become infected with E. multilocularis by oral ingesting of the eggs, which then develop into cysts in the liver and progress an endless proliferation. Untreated AE has a fatality rate of >90% in humans. Tetraspanins have been identified in Schistosoma and showed potential as the prospective vaccine candidates. In our recent study, we first identified seven tetraspanins in E. multilocularis and evaluated their protective efficacies as vaccines against AE when subcutaneously administered to BALB/c mice. Mucosal immunization of protective proteins is able to induce strong local and systemic immune responses, which might play a crucial role in protecting humans against E. multilocularis infection via the intestine, blood and liver. We focused on Em-TSP3, which achieved significant vaccine efficacy via both s.c. and i.n. routes. The adjuvanticity of nontoxic CpG OND as i.n. vaccine adjuvant was evaluated. The widespread expression of Em-TSP3 in all the developmental stages of E. multilocularis, and the strong local and systemic immune responses evoked by i.n. administration of rEm-TSP3 with CpG OND adjuvant suggest that this study might open the way for developing efficient, nontoxic human mucosal vaccines against AE

Characterization of microRNAs expressed in the cystic legion of the liver of Mus musculus perorally infected with Echinococcus multilocularis Nemuro strain

Alveolar echinococcosis (AE) is a zoonosis caused by the metacestode of Echinococcus multilocularis. The published genome of E. multilocularis showed that approximately 86% of its genome is non-coding. Micro RNAs (miRNAs) are small non-coding regulatory RNAs, and recent studies on parasitic helminths expect miRNAs as a promising target for drug development and diagnostic markers. Prior to this study, only a few studies reported the E. multilocularis miRNA profiles in the intermediate host. The primary objective of this study was to characterize miRNA profiles via small RNA-seq in E. multilocularis Nemuro strain, a laboratory strain of Asian genotype, using mice perorally infected with the parasite eggs. The data were then compared with two previously published small RNA-seq data. We identified 44 mature miRNAs as E. multilocularis origin out of the 68 mature miRNA sequences registered in the miRNA database miRbase. The highest quantities of miRNAs detected were miR-10-5p, followed by bantam-3p, let-7-5p, miR-61-3p, and miR-71-5p. The top two most abundant miRNAs (miR-10-5p and bantam-3p) accounted for approximately 80.9% of the total parasite miRNAs. The highly expressed miRNA repertoire is mostly comparable to that obtained from the previous experiment using secondary echinococcosis created by an intraperitoneal administration of metacestodes. A detailed characterization and functional annotations of these shared miRNAs will lead to a better understanding of parasitic dynamics, which could provide a basis for the development of novel diagnostic and treatment methods for AE. Superscript/Subscript Available ABSTRACT Alveolar echinococcosis (AE) is a zoonosis caused by the metacestode of Echinococcus multilocularis. The published genome of E. multilocularis showed that approximately 86% of its genome is non-coding. Micro RNAs (miRNAs) are small non-coding regulatory RNAs, and recent studies on parasitic helminths expect miRNAs as a promising target for drug development and diagnostic markers. Prior to this study, only a few studies reported the E. multilocularis miRNA profiles in the intermediate host. The primary objective of this study was to characterize miRNA profiles via small RNA-seq in E. multilocularis Nemuro strain, a laboratory strain of Asian genotype, using mice perorally infected with the parasite eggs. The data were then compared with two previously published small RNA-seq data. We identified 44 mature miRNAs as E. multilocularis origin out of the 68 mature miRNA sequences registered in the miRNA database miRbase. The highest quantities of miRNAs detected were miR-10-5p, followed by bantam-3p, let-7-5p, miR-61-3p, and miR-71-5p. The top two most abundant miRNAs (miR-10-5p and bantam-3p) accounted for approximately 80.9% of the total parasite miRNAs. The highly expressed miRNA repertoire is mostly comparable to that obtained from the previous experiment using secondary echinococcosis created by an intraperitoneal administration of metacestodes. A detailed characterization and functional annotations of these shared miRNAs will lead to a better understanding of parasitic dynamics, which could provide a basis for the development of novel diagnostic and treatment methods for AE