Brachylaima asakawai sp. nov. (Trematoda: Brachylaimidae), a rodent intestinal fluke in Hokkaido,Japan, with a finding of the first and second intermediate hosts

In the 1970’s and 1980’s, an unknown species of the genus Brachylaima (Trematoda: Brachylaimidae) had been recorded from the intestines of Rattus norvegicus and Apodemus speciosus in Hokkaido, Japan. The rodent fluke was characteristic in extending a bilateral vitellarium till the level of posterior margin of anterior testis and in keeping almost the same-sized spherical ovary and testes. In this study, the rodent fluke was rediscovered from A. speciosus, Apodemus argenteus, and Myodes rufocanus in Hokkaido. The resultant parasite collection enabled us to make a mitochondrial DNA (mtDNA) barcode for tracking its intermediate hosts. The metacercaria of the rodent fluke was detected frequently from the kidney of three species of land snails (Discus pauper, Succinea lauta, and Ainohelix editha). However, its sporocyst with cercariae was found only from the hepatopancreas of D. pauper, a fairly small snail. The wide-spectrum of the second intermediate host seems to increase the chance of transmitting the parasite to various mammals and birds. The use of indigenous land snails as the first and second intermediate hosts, the distinctiveness of the mtDNA sequence, and the characteristic morphology of all the developmental stages prompted us to propose Brachylaima asakawai sp. nov. for the rodent intestinal fluke in Hokkaido. The present field survey suggests that the life cycle of the new species is primarily dependent on a predator-prey relationship between rodents and D. pauper.     

A reproducible swine model of a surgically created saccular thoracic aortic aneurysm

A reproducible swine thoracic aortic aneurysm (TAA) model is useful for investigating new therapeutic interventions. We report a surgical method for creating a reproducible swine saccular TAA model. We used eight female swine weighing 20–25 kg (LWD; ternary species). All procedures were performed under general anesthesia and involved left thoracotomy. Following aortic cross-clamping, the thoracic aorta was surgically dissected and the media and intima were resected, and the dissection plane was extended by spreading the outer layer for aneurysmal space. Subsequently, only the adventitial layer of the aorta was sutured. At 2 weeks after these procedures, angiography and computed tomography were performed. After follow-up imaging, the model animals were euthanized. Macroscopic, histological, and immunohistological examinations were performed. All model animals survived, and a saccular TAA was confirmed by follow-up imaging in all cases. The mean length of the shorter and the longer aortic diameter after the procedure were 14.01 ± 1.0 mm and 18.35 ± 1.4 mm, respectively (P<0.001). The rate of increase in the aortic diameter was 131.7 ± 13.8%, and the mean length of aneurysmal change at thoracic aorta was 22.4 ± 1.9 mm. Histological examination revealed intimal tears and defects of elastic fibers in the media. Immunostaining revealed MMP-2 and MMP-9 expressions at the aneurysm site. We report our surgical method for creating a swine saccular TAA model. Our model animal may be useful to investigate new therapeutic interventions for aortic disease.     

Conductometric Method for Detecting Ion Efflux during a Single Action Potential of Chara

A simple device was developed to detect net ion efflux duringa single action potential of Chara as an increase in the electricconductance of the bathing solution. The device showed a sufficientlyhigh sensitivity and rapid response to the increase in the electricconductance. Net efflux of monovalent ions was estimated as65–660 pmol cm^–2 impluse^–1 (average 220 pmolcm^–2 impulse^–1). (Received June 28, 1985; Accepted November 13, 1985)     

The Vibrio parahaemolyticus effector VopC mediates Cdc42‐dependent invasion of cultured cells but is not required for pathogenicity in an animal model of infection

Vibrio parahaemolyticus is a Gram‐negative marine bacterium that causes acute gastroenteritis in humans. The virulence of V. parahaemolyticus is dependent upon a type III secretion system (T3SS2). One effector for T3SS2, VopC, is a homologue of the catalytic domain of cytotoxic necrotizing factor (CNF), and was recently reported to be a Rho family GTPase activator and to be linked to internalization of V. parahaemolyticus by non‐phagocytic cultured cells. Here, we provide direct evidence that VopC deamidates Rac1 and CDC42, but not RhoA, in vivo. Our results alsosuggest that VopC, through its activation of Rac1, contributes to formation of actin stress fibres in infected cells. Invasion of host cells, which occurs at a low frequency, does not seem linked to Rac1 activation, but instead appears to require CDC42. Finally, using an infant rabbit model of V. parahaemolyticus infection, we show that the virulence of V. parahaemolyticus is not dependent upon VopC‐mediated invasion. Genetic inactivation of VopC did not impair intestinal colonization nor reduce signs of disease, including fluid accumulation, diarrhoea and tissue destruction. Thus, although VopC can promote host cell invasion, such internalization is not a critical step of the disease process, consistent with the traditional view of V. parahaemolyticus as an extracellular pathogen.     

Risk Assessment Visualization of Rubidium Compounds: Comparison of Renal and Hepatic Toxicities,In vivo

Rubidium has been considered to be nontoxic. Its use includes thin film on glass deposition and as medical contrast medium. Recent technology innovations also involve the use of rubidium, but there is limited information about the biological effects of its various compounds. In the present risk assessment study, a series of rubidium compounds with different counter anions—acetate, bromide, carbonate, chloride, and fluoride—were orally administrated in a single dose to several groups of rats. Cumulative 24-h urine samples were obtained, and the levels of rubidium, fluoride, N-acetyl-β-D-glucosaminidase and creatinine were measured to evaluate possible acute renal effects. Daily samples of serum were also obtained to determine the levels of aspartate and alanine aminotransferases to assess possible acute hepatic effects. Urinary rubidium excretion recovery of 8.0–10.5 % shows that urine can be a useful diagnostic tool for rubidium exposure. The present results reveal that rubidium shows different biological effects depending on the counter anion. A pattern of large significant NAG leakage and elevation of ALT observed in rats treated with anhydrous rubidium fluoride indicates renal and hepatic toxicities that can be attributed to fluoride. The techniques reported in this study will be of help to assess the potential risks of toxicity of rubidium compounds with a variety of anions.     

Novel antibody assessment method for microbial compositional alteration in the oral cavity

Recently, it has been demonstrated that dysbiosis, an alteration in commensal microflora composition, is intimately involved in the onset of a variety of diseases. It is becoming increasingly evident that the composition of commensal microflora in the oral cavity is closely connected to oral diseases, such as periodontal disease, and systemic diseases, such as inflammatory bowel disease. Next-generation sequencing techniques are used as a method to examine changes in bacterial flora, but additional analytical methods to assess bacterial flora are needed to understand bacterial activity in more detail. In addition, the oral environment is unique because of the role of secretory antibodies contained in saliva in the formation of bacterial flora. The present study aimed to develop a new method for evaluating the compositional change of microbiota using flow cytometry (FCM) with specific antibodies against the bacterial surface antigen, as well as salivary antibodies. Using specific antibodies against Streptococcus mutans, a causative agent of dental caries, and human IgA, bacterial samples from human saliva were analyzed via FCM. The results showed that different profiles could be obtained depending on the oral hygiene status of the subjects. These results suggest that changes in the amount and type of antibodies that bind to oral bacteria may be an indicator for evaluating abnormalities in the oral flora. Therefore, the protocol established in this report could be applied as an evaluation method for alterations in the oral microbiota.     

High-efficiency bioaffinity separation of cells and proteins using novel thermoresponsive biotinylated magnetic nanoparticles

Thermoresponsive magnetic nanoparticles with an upper critical solution temperature (UCST) in aqueous solution were synthesized for the first time. Named Therma-Max, the material was synthesized by redox copolymerization of N-acryloyl glycinamide with a monomer form of biotin using methacrylated dextran-magnetite. While the resulting Therma-Max was completely dispersed at temperatures above the UCST (18°C) and could not be separated by a permanent magnet, it was rapidly flocculated when the temperature fell below the UCST and was easily separated by a permanent magnet. The flocculated particles dispersed completely when the temperature was raised to above the UCST. Because biotin was immobilized on the Therma-Max, avidin and antibodies were subsequently immobilized with good efficiency. Furthermore, transiently transfected Arabidopsis protoplasts, which have surface display of CD4 antigen, were efficiently captured and enriched by using a biotinylated anti-CD4 antibody in combination with avidin-conjugated Therma-Max. Also, the silkworm storage protein (SP2) was efficiently separated from the silkworm hemolymph by using biotinylated anti-IgG antibody and anti-SP2 antibody in combination with avidin-conjugated Therma-Max. In both cases, it was confirmed that specificity and adsorption capacity were markedly improved by converting the conventional micro-size fine magnetic particles to nano-size particles. These results show the potential of Therma-Max with a UCST in bioaffinity separation of cells and biomolecules.     

Group A Streptococcus modulates RAB1- and PIK3C3 complex-dependent autophagy

ABSTRACT

Autophagy selectively targets invading bacteria to defend cells, whereas bacterial pathogens counteract autophagy to survive in cells. The initiation of canonical autophagy involves the PIK3C3 complex, but autophagy targeting Group A Streptococcus (GAS) is PIK3C3-independent. We report that GAS infection elicits both PIK3C3-dependent and -independent autophagy, and that the GAS effector NAD-glycohydrolase (Nga) selectively modulates PIK3C3-dependent autophagy. GAS regulates starvation-induced (canonical) PIK3C3-dependent autophagy by secreting streptolysin O and Nga, and Nga also suppresses PIK3C3-dependent GAS-targeting-autophagosome formation during early infection and facilitates intracellular proliferation. This Nga-sensitive autophagosome formation involves the ATG14-containing PIK3C3 complex and RAB1 GTPase, which are both dispensable for Nga-insensitive RAB9A/RAB17-positive autophagosome formation. Furthermore, although MTOR inhibition and subsequent activation of ULK1, BECN1, and ATG14 occur during GAS infection, ATG14 recruitment to GAS is impaired, suggesting that Nga inhibits the recruitment of ATG14-containing PIK3C3 complexes to autophagosome-formation sites. Our findings reveal not only a previously unrecognized GAS-host interaction that modulates canonical autophagy, but also the existence of multiple autophagy pathways, using distinct regulators, targeting bacterial infection.

Abbreviations: ATG5: autophagy related 5; ATG14: autophagy related 14; ATG16L1: autophagy related 16 like 1; BECN1: beclin 1; CALCOCO2: calcium binding and coiled-coil domain 2; GAS: group A streptococcus; GcAV: GAS-containing autophagosome-like vacuole; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; Nga: NAD-glycohydrolase; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4P: phosphatidylinositol-4-phosphate; RAB: RAB, member RAS oncogene GTPases; RAB1A: RAB1A, member RAS oncogene family; RAB11A: RAB11A, member RAS oncogene family; RAB17: RAB17, member RAS oncogene family; RAB24: RAB24, member RAS oncogene family; RPS6KB1: ribosomal protein S6 kinase B1; SLO: streptolysin O; SQSTM1: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1; WIPI2: WD repeat domain, phosphoinositide interacting 2     

N-(3-oxobutyl)cytisine: A new lupin alkaloid from eChinosophora koreensis

A new lupin alkaloid, N-(3,-oxobutyl)cytisine, was isolated from the aerial parts of Echinosophora koreensis. Its structure was determined by s