Structure-Based Site-Directed Mutagenesis of Hydroxynitrile Lyase from Cyanogenic Millipede, Oxidus gracilis for Hydrocyanation and Henry Reactions.

Hydroxynitrile lyase (HNL) from the cyanogenic millipede Oxidus gracillis (OgraHNL) is a crucial enzyme in the cyanogenesis pathway. Here, the crystal structures of OgraHNL complexed with sulfate, benzaldehyde (BA), (R)-mandelonitrile ((R)-Man), (R)-2-chloromandelonitrile ((R)-2-Cl-Man), and acetone cyanohydrin (ACN) were solved at 1.6, 1.7, 2.3, 2.1, and 2.0 Šresolutions, respectively. The structure of OgraHNL revealed that it belonged to the lipocalin superfamily. Based on this structure, positive variants were designed to further improve the catalytic activity and enantioselectivity of the enzyme for asymmetric hydrocyanation and Henry reactions.

Transcriptionally linked simultaneous overexpression of P450 genes for broad-spectrum herbicide resistance.

Broad-spectrum herbicide resistance (BSHR), often linked to weeds with metabolism-based herbicide resistance, poses a threat to food production. Past studies have revealed that overexpression of catalytically promiscuous enzymes explains BSHR in some weeds; however, the mechanism of BSHR expression remains poorly understood. Here, we investigated the molecular basis of high-level resistance to diclofop-methyl in BSHR late watergrass (Echinochloa phyllopogon) found in the United States, which cannot be solely explained by the overexpression of promiscuous cytochrome P450 monooxygenases CYP81A12/21. The BSHR late watergrass line rapidly produced 2 distinct hydroxylated diclofop acids, only 1 of which was the major metabolite produced by CYP81A12/21. RNA-seq and subsequent reverse transcription quantitative PCR (RT-qPCR)-based segregation screening identified the transcriptionally linked overexpression of a gene, CYP709C69, with CYP81A12/21 in the BSHR line. The gene conferred diclofop-methyl resistance in plants and produced another hydroxylated diclofop acid in yeast (Saccharomyces cerevisiae). Unlike CYP81A12/21, CYP709C69 showed no other herbicide-metabolizing function except for a presumed clomazone-activating function. The overexpression of the 3 herbicide-metabolizing genes was also identified in another BSHR late watergrass in Japan, suggesting a convergence of BSHR evolution at the molecular level. Synteny analysis of the P450 genes implied that they are located at mutually independent loci, which supports the idea that a single trans-element regulates the 3 genes. We propose that transcriptionally linked simultaneous overexpression of herbicide-metabolizing genes enhances and broadens the metabolic resistance in weeds. The convergence of the complex mechanism in BSHR late watergrass from 2 countries suggests that BSHR evolved through co-opting a conserved gene regulatory system in late watergrass.

Subgenomic T cell receptor alpha and delta (TRA/TRD) loci in common carp.

In jawed vertebrates, the T cell receptor alpha (TRA) and delta (TRD) genes, which encode the TRα and TRδ chains, respectively, are located as a nested structure on a single chromosome. To date, no animal has been reported to harbor multiple TRA/TRD loci on different chromosomes. Therefore, herein, we describe the first full annotation of the TRA/TRD genomic regions of common carp, an allo-tetraploid fish species that experiences cyprinid-specific whole-genome duplication (WGD) in evolution. Fine genomic maps of TRA/TRD genomic regions 1 and 2, on LG30 and LG22, respectively, were constructed using the annotations of complete sets of TRA and TRD genes, including TRA/TRD variable (V), TRA junction (J), and constant (C), TRD diversity (D), and the J and C genes. The structure and synteny of the TRA/TRD genomic regions were highly conserved in zebrafish, indicating that these regions are on individual chromosomes. Furthermore, analysis of the variable regions of the TRA and TRD genes in a monoclonal T cell line revealed that both subgenomic regions 1 and 2 were indeed rearranged. Although carp TRAV and TRDV genes were phylogenetically divided into different lineages, they were mixed and organized into the TRA/TRD V gene clusters on the genome, similar to that in other vertebrates. Notably, 285 potential TRA/TRD V genes were detected in the TRA/TRD genomic regions, which is the most abundant number of genes in vertebrates and approximately two-fold that in zebrafish. The recombination signal sequences (RSSs) at the end of each V gene differed between TRAV and TRDV, suggesting that RSS variations might separate each V gene into a TRα or TRδ chain. This study is the first to describe subgenomic TRA/TRD loci in animals. Our findings provide fundamental insights to elucidate the impact of WGD on the evolution of immune repertoire.

Exploration and utilization of novel aldoxime, nitrile, and nitro compounds metabolizing enzymes from plants and arthropods.

Aldoxime (R1R2C=NOH) and nitrile (R-C≡N) are nitrogen-containing compounds that are found in species representing all kingdoms of life. The enzymes discovered from the microbial "aldoxime-nitrile" pathway (aldoxime dehydratase, nitrile hydratase, amidase, and nitrilase) have been thoroughly studied because of their industrial importance. Although plants utilize cytochrome P450 monooxygenases to produce aldoxime and nitrile, many biosynthetic pathways are yet to be studied. Cyanogenic millipedes accumulate various nitrile compounds, such as mandelonitrile. However, no such aldoxime- and nitrile-metabolizing enzymes have been identified in millipedes. Here, I review the exploration of novel enzymes from plants and millipedes with characteristics distinct from those of microbial enzymes, the catalysis of industrially useful reactions, and applications of these enzymes for nitrile compound production.

Identification and characterization of cytochrome P450 CYP77A59 of loquat (Rhaphiolepis bibas) responsible for biosynthesis of phenylacetonitrile, a floral nitrile compound.

MAIN CONCLUSION: Cytochrome P450 CYP77A59 is responsible for the biosynthesis of phenylacetonitrile in loquat flowers. Flowers of some plants emit volatile nitrile compounds, but the biosynthesis of these compounds is unclear. Loquat (Rhaphiolepis bibas) flowers emit characteristic N-containing volatiles, such as phenylacetonitrile (PAN), (E/Z)-phenylacetaldoxime (PAOx), and (2-nitroethyl)benzene (NEB). These volatiles likely play a defense role against pathogens and insects. PAN and NEB are commonly biosynthesized from L-phenylalanine via (E/Z)-PAOx. Two cytochrome P450s-CYP79D80 and "promiscuous fatty acid ω-hydroxylase" CYP94A90, which catalyze the formation of (E/Z)-PAOx from L-phenylalanine and NEB from (E/Z)-PAOx, respectively-are involved in NEB biosynthesis. However, the enzymes catalyzing the formation of PAN from (E/Z)-PAOx in loquat have not been identified. In this study, we aimed to identify candidate cytochrome P450s catalyzing PAN formation in loquat flowers. Yeast whole-cell biocatalyst assays showed that among nine candidate cytochrome P450s, CYP77A58 and CYP77A59 produced PAN from (E/Z)-PAOx. CYP77As catalyzed the dehydration of aldoximes, which is atypical of cytochrome P450; the reaction was NADPH-dependent, with an optimum temperature and pH of 40 °C and 8.0, respectively. CYP77As acted on (E/Z)-PAOx, (E/Z)-4-hydroxyphenylacetaldoxime, and (E/Z)-indole-3-acetaldoxime. Previously characterized CYP77As are known to hydroxylate fatty acids; loquat CYP77As did not act on tested fatty acids. We observed higher expression of CYP77A59 in flowers than in buds; expression of CYP77A58 was remarkably reduced in the flowers. Because the flowers, but not buds, emit PAN, CYP77A59 is likely responsible for the biosynthesis of PAN in loquat flowers. This study will help us understand the biosynthesis of floral nitrile compounds.

A promiscuous fatty acid ω-hydroxylase CYP94A90 is likely to be involved in biosynthesis of a floral nitro compound in loquat (Eriobotrya japonica).

Nitro groups are often associated with synthetically manufactured compounds such as medicines and explosives, and rarely with natural products. Loquat emits a nitro compound, (2-nitroethyl)benzene, as a flower scent. The nitro compound exhibits fungistatic activity and is biosynthesised from l-phenylalanine via (E/Z)-phenylacetaldoxime. Although aldoxime-producing CYP79s have been intensively studied, it is unclear what enzymes form nitro groups from aldoximes either in plants or in other organisms. Here, we report the identification of two cytochrome P450s that are likely to be involved in (2-nitroethyl)benzene biosynthesis in loquat through differential gene expression analysis using RNA-seq and functional identification using yeast and tobacco. CYP79D80 and CYP94A90 catalysed the formation of (E/Z)-phenylacetaldoxime from l-phenylalanine and (2-nitroethyl)benzene from the aldoxime, respectively. Expression profiles of CYP79D80 and CYP94A90 were correlated with the emission of (2-nitroethyl)benzene from loquat flowers. CYP94A90 also functioned as a fatty acid ω-hydroxylase as do other CYP94A fatty acid ω-hydroxylases. The CYP94As tested from other plants were all found to catalyse the formation of (2-nitroethyl)benzene from (E/Z)-phenylacetaldoxime. CYP79D80 and CYP94A90 are likely to operate in concert to biosynthesise (2-nitroethyl)benzene in loquat. CYP94A90 and other CYP94As are 'promiscuous fatty acid ω-hydroxylases', catalysing the formation of nitro groups from aldoximes, and are widely distributed in dicot plants.

Transmission properties of microwaves at an optical Weyl point in a three-dimensional chiral photonic crystal.

Microwave transmission measurements were performed for a three-dimensional (3D) layer-by-layer chiral photonic crystal (PhC), whose photonic band structure contains 3D singular points, Weyl points. For the frequency and wavevector in the vicinity of a Weyl point, the transmitted intensity was found to be inversely proportional to the square of the propagation length. In addition, the transmitted wave was well-collimated in the plane parallel to the PhC layers, even for point-source incidence. When a plane wave was incident on the PhC containing metal scatters, the planar wavefront was reconstructed after the transmission, indicating a cloaking effect.

[Disseminated aspergillosis due to Aspergillus udagawae during immunosuppressive treatment for myelodysplastic syndrome].

An 80 year old male who had received immunosuppressive therapy for myelodysplastic syndrome presented with fever, fatigue, and elevated serum Aspergillus antigen. Computed tomography revealed infiltrative shadows in the left lower lung and subcutaneous nodules. A polymerase chain reaction assay from lung and subcutaneous nodule samples identified the presence Aspergillus udagawae. A. udagawae is a cryptic species that shares similar morphological characteristics with A. fumigatus but genetically differs from the latter in its susceptibility to antifungal drugs. When immunosuppressed patients with hematological malignancies develop disseminated aspergillosis, biopsy and fungal tests are crucial to identify the causative fungus, including cryptic species, for deciding the appropriate therapeutic intervention.

Functional characterization of cytochrome P450 CYP81A subfamily to disclose the pattern of cross-resistance in Echinochloa phyllopogon.

KEY MESSAGE: CYP81A P450s armor Echinochloa phyllopogon against diverse and several herbicide chemistries. CYP81A substrate preferences can be a basis for cross-resistance prediction and management in E. phyllopogon and other related species. Metabolism-based herbicide resistance is a major threat to agriculture, as it is unpredictable and could extend resistance to different chemical groups and modes of action, encompassing existing, novel and to-be-discovered herbicides. Limited information on the enzymes involved in herbicide metabolism has hindered the prediction of cross-resistance in weeds. Members of CYP81A subfamily in multiple herbicide resistant (MHR) Echinochloa phyllopogon were previously identified for conferring cross-resistance to six unrelated herbicide classes. This suggests a critical role of CYP81As in endowing unpredictable cross-resistances in E. phyllopogon, thus the functions of all its nine putative functional CYP81A genes to 33 herbicides from 24 chemical groups were characterized. Ectopic expression in Arabidopsis thaliana identified the CYP81As that can confer resistance to multiple and diverse herbicides. The CYP81As were further characterized for their enzymatic functions in Escherichia coli. CYP81A expression in E. coli was optimized via modification of the N-terminus, co-expression with HemA gene and culture at optimal temperature. CYP81As metabolized its herbicide substrates into hydroxylated, N-/O-demethylated or both products. The cross-resistance pattern conferred by CYP81As is geared towards all chemical groups of acetolactate synthase inhibitors and is expanded to herbicides inhibiting photosystem II, phytoene desaturase, protoporphyrinogen oxidase, 4-hydroxyphenylpyruvate dioxygenase, and 1-deoxy-D-xylulose 5-phosphate synthase. Cross-resistance to herbicides pyrimisulfan, propyrisulfuron, and mesotrione was predicted and confirmed in MHR E. phyllopogon. This study demonstrated that the functional characterization of the key enzymes for herbicide metabolism could disclose the cross-resistance pattern and identify appropriate chemical options to manage the existing and unexpected cross-resistances in E. phyllopogon.

Robotic CT-guided out-of-plane needle insertion: comparison of angle accuracy with manual insertion in phantom and measurement of distance accuracy in animals.

OBJECTIVES: To evaluate the accuracy of robotic CT-guided out-of-plane needle insertion in phantom and animal experiments. METHODS: A robotic system (Zerobot), developed at our institution, was used for needle insertion. In the phantom experiment, 12 robotic needle insertions into a phantom at various angles in the XY and YZ planes were performed, and the same insertions were manually performed freehand, as well as guided by a smartphone application (SmartPuncture). Angle errors were compared between the robotic and smartphone-guided manual insertions using Student's t test. In the animal experiment, 6 robotic out-of-plane needle insertions toward targets of 1.0 mm in diameter placed in the kidneys and hip muscles of swine were performed, each with and without adjustment of needle orientation based on reconstructed CT images during insertion. Distance accuracy was calculated as the distance between the needle tip and the target center. RESULTS: In the phantom experiment, the mean angle errors of the robotic, freehand manual, and smartphone-guided manual insertions were 0.4°, 7.0°, and 3.7° in the XY plane and 0.6°, 6.3°, and 0.6° in the YZ plane, respectively. Robotic insertions in the XY plane were significantly (p < 0.001) more accurate than smartphone-guided insertions. In the animal experiment, the overall mean distance accuracy of robotic insertions with and without adjustment of needle orientation was 2.5 mm and 5.0 mm, respectively. CONCLUSION: Robotic CT-guided out-of-plane needle insertions were more accurate than smartphone-guided manual insertions in the phantom and were also accurate in the in vivo procedure, particularly with adjustment during insertion. KEY POINTS: • Out-of-plane needle insertions performed using our robot were more accurate than smartphone-guided manual insertions in the phantom experiment and were also accurate in the in vivo procedure. • In the phantom experiment, the mean angle errors of the robotic and smartphone-guided manual out-of-plane needle insertions were 0.4° and 3.7° in the XY plane (p < 0.001) and 0.6° and 0.6° in the YZ plane (p = 0.65), respectively. • In the animal experiment, the overall mean distance accuracies of the robotic out-of-plane needle insertions with and without adjustments of needle orientation during insertion were 2.5 mm and 5.0 mm, respectively.

Robotic needle insertion during computed tomography fluoroscopy-guided biopsy: prospective first-in-human feasibility trial.

INTRODUCTION: This was a prospective, first-in-human trial to evaluate the feasibility and safety of insertion of biopsy introducer needles with our robot during CT fluoroscopy-guided biopsy in humans. MATERIALS AND METHODS: Eligible patients were adults with a lesion ≥ 10 mm in an extremity or the trunk requiring pathological diagnosis with CT fluoroscopy-guided biopsy. Patients in whom at-risk structures were located within 10 mm of the scheduled needle tract were excluded. Ten patients (4 females and 6 males; mean [range] age, 72 [52-87] years) with lesions (mean [range] maximum diameter, 28 [14-52] mm) in the kidney (n = 4), lung (n = 3), mediastinum (n = 1), adrenal gland (n = 1), and muscle (n = 1) were enrolled. The biopsy procedure involved robotic insertion of a biopsy introducer needle followed by manual acquisition of specimens using a biopsy needle. The patients were followed up for 14 days. Feasibility was defined as the distance of ≤ 10 mm between needle tip after insertion and the nearest lesion edge on the CT fluoroscopic images. The safety of robotic insertion was evaluated on the basis of machine-related troubles and adverse events according to the Clavien-Dindo classification. RESULTS: Robotic insertion of the introducer needle was feasible in all patients, enabling pathological diagnosis. There was no machine-related trouble. A total of 11 adverse events occurred in 8 patients, including 10 grade I events and 1 grade IIIa event. CONCLUSION: Insertion of biopsy introducer needles with our robot was feasible at several locations in the human body. KEY POINTS: • Insertion of biopsy introducer needles with our robot during CT fluoroscopy-guided biopsy was feasible at several locations in the human body.

Medial meniscus posterior root tear causes swelling of the medial meniscus and expansion of the extruded meniscus: a comparative analysis between 2D and 3D MRI.

PURPOSE: This study aimed to clarify the advantages of three-dimensional (3D) magnetic resonance imaging (MRI) over two-dimensional (2D) MRI in measuring the size of the medial meniscus (MM) and to analyse the volumes of MM and the extruded meniscus in patients with MM posterior root tear (MMPRT), at 10° and 90° knee flexion. METHODS: This study included 17 patients with MMPRTs and 15 volunteers with uninjured knees. The MMs were manually segmented for 3D reconstruction; thereafter, the extruded part separated from the tibial edge was determined. The length, width, height, and extrusion of MM were measured by the 2D and 3D methods, and compared. The MM volume, extruded meniscus volume, and their ratio were also calculated using 3D analysis software in the two groups. RESULTS: The estimated length and posterior height of MM were larger with 3D MRI than with 2D MRI measurements. The MM volume was significantly greater in MMPRT knees than in normal knees, with increasing MM height. In MMPRT knees, the mean volume of the extruded meniscus and its ratio significantly increased by 304 mm3 (p = 0.02) and 9.1% (p < 0.01), respectively, during knee flexion. CONCLUSIONS: This study demonstrated that 3D MRI could estimate the precise MM size and that MMPRT caused swelling of the meniscus due to the increased thickness in the posteromedial part. The clinical significance of this study lies in its 3D evaluation of MM volume, which should help the surgeon understand the biomechanical failure of MM function and improve MMPRT repair technique. LEVEL OF EVIDENCE: III.

Ancient features of the MHC class II presentation pathway, and a model for the possible origin of MHC molecules.

Major histocompatibility complex (MHC) molecules are only found in jawed vertebrates and not in more primitive species. MHC class II type structures likely represent the ancestral structure of MHC molecules. Efficient MHC class II transport to endosomal compartments depends on association with a specialized chaperone, the MHC class II invariant chain (aliases Ii or CD74). The present study identifies conserved motifs in the CLIP region of CD74 molecules, used for binding in the MHC class II binding groove, throughout jawed vertebrates. Peculiarly, in CD74a molecules of Ostariophysi, a fish clade including for example Mexican tetra and zebrafish, the CLIP region has duplicated. In mammals, in endosomal compartments, the peptide-free form of classical MHC class II is stabilized by binding to nonclassical MHC class II "DM," a process that participates in "peptide editing" (selection for high affinity peptides). Hitherto, DM-lineage genes had only been reported from the level of amphibians, but the present study reveals the existence of DMA and DMB genes in lungfish. This is the first study which details how classical and DM lineage molecules have distinguishing glycine-rich motifs in their transmembrane regions. In addition, based on extant MHC class II structures and functions, the present study proposes a model for early MHC evolution, in which, in an ancestral jawed vertebrate, the ancestral MHC molecule derived from a heavy-chain-only antibody type molecule that cycled between the cell surface and endosomal compartments.

CYP81A P450s are involved in concomitant cross-resistance to acetolactate synthase and acetyl-CoA carboxylase herbicides in Echinochloa phyllopogon.

Californian populations of Echinochloa phyllopogon have evolved multiple-herbicide resistance (MHR), posing a threat to rice production in California. Previously, we identified two CYP81A cytochrome P450 genes whose overexpression is associated with resistance to acetolactate synthase (ALS) inhibitors from two chemical groups. Resistance mechanisms to other herbicides remain unknown. We analyzed the sensitivity of an MHR line to acetyl-CoA carboxylase (ACCase) inhibitors from three chemical groups, followed by an analysis of herbicide metabolism and segregation of resistance of the progenies in sensitive (S) and MHR lines. ACCase herbicide metabolizing function was investigated in the two previously identified P450s. MHR plants exhibited resistance to all the ACCase inhibitors by enhanced herbicide metabolism. Resistance to the ACCase inhibitors segregated in a 3 : 1 ratio in the F2 generation and completely co-segregated with ALS inhibitor resistance in F6 lines. Expression of the respective P450 genes conferred resistance to the three herbicides in rice, which is in line with the detection of hydroxylated herbicide metabolites in vivo in transformed yeast. CYP81As are super P450s that metabolize multiple herbicides from five chemical classes, and concurrent overexpression of the P450s induces metabolism-based resistance to the three ACCase inhibitors in MHR E. phyllopogon, as it does to ALS inhibitors.

Effectiveness and Safety of Ultrasound Guided Lower Extremity Nerve Blockade in Infragenicular Bypass Grafting for High Risk Patients With Chronic Limb Threatening Ischaemia.

OBJECTIVES: Surgical revascularisation to accomplish limb salvage remains preferable in some patients with chronic limb threatening ischaemia (CLTI). The aim of this study was to evaluate the effectiveness and safety of ultrasound guided lower extremity nerve blockade (UGNB) in infragenicular bypass surgery (IGBS). METHODS: This was a single centre, retrospective clinical study. Fifty-nine patients with CLTI (67 limbs) who underwent IGBS under UGNB (femoral and sciatic nerve blockade) at Asahikawa Medical University between January 2012 and December 2017 were compared with patients with CLTI (137 limbs) who underwent IGBS under general anaesthesia (GA) over the same period. Propensity score matching based on pre-operative comorbidities was used to minimise background differences of the two groups. RESULTS: Fifty-six pairs of CLTIs were matched and analysed (55% dialysis dependent). Procedure duration was similar between the two groups, but intraoperative catecholamine index and intravenous fluid volume were lower with UGNB compared with GA (2.9 ± 4.6 vs. 5.9 ± 6.5; p < .01 and 1831 ± 990 vs. 2335 ± 931 mL; p < .01, respectively). The mean arterial blood pressure during induction of anaesthesia was significantly decreased with GA. Post-operatively, the time period to resume a clear liquid and solid food diet was significantly shorter with UGNB (P<0.01 for both outcome measures). Intravenous fluid volume was significanlty lower, while cardiac complications and delirium, based on the NEECHAM confusion scale, occurred significantly less often with UGNB than GA. These significant differences show advantages of UGNB compared to GA. No mortality or major amputations were observed in either group. Early graft thrombosis was observed in five limbs (8.9%) with UGNB and in four limbs with GA (7.1%) (p = .73). CONCLUSIONS: UGNB has advantages for intra- and post-operative management and could be a useful method to prevent peri-operative complications for high risk patients with CLTI. To ensure the effectiveness of UGNB for IGBS for future indications, a randomised study is required.

What could be the mechanisms of immunological memory in fish?

Vaccination is the most effective strategy to control infectious diseases in species with adaptive immunity. In human and in mouse, vaccination typically induces specific memory cells, which can mediate a fast anamnestic response upon infection by the targeted pathogen. In these species, successful vaccination induces a long-lasting protection, long after the titres of specific antibodies and the frequency of specific T cells have returned to steady state. Vaccination is also an important challenge in aquaculture, since alternative treatments are either too costly, or, in the case of antibiotics, are harmful for the environment or may result in dangerous resistances. However, the mechanisms of the long-term protection elicited by vaccines in fish remain poorly understood. Although fish possess typical B- and T-cells expressing diverse repertoires of immunoglobulins and T-cell receptors, many features of antigen specific responses are different from what is known in mouse and in human. Memory is one of the most elusive properties of fish adaptive immunity, and its basis is widely unknown. In this opinion article, we discuss the concept of immune memory in the context of the fish immunity. We illustrate the complexity of this question by discussing the results of experiments showing that protection can be passed through adoptive transfer of leukocytes from vaccinated donor fish to naive histocompatible recipients. Combined with tools developed in Targetfish and in previous projects, such as monoclonal antibodies against B- and T-cell markers, we propose that such models of protection transfer provide excellent systems to dissect the mechanisms of B- and T-cell memory in the future.

Teleost cytotoxic T cells.

Cell-mediated cytotoxicity is one of the major mechanisms by which vertebrates control intracellular pathogens. Two cell types are the main players in this immune response, natural killer (NK) cells and cytotoxic T lymphocytes (CTL). While NK cells recognize altered target cells in a relatively unspecific manner CTLs use their T cell receptor to identify pathogen-specific peptides that are presented by major histocompatibility (MHC) class I molecules on the surface of infected cells. However, several other signals are needed to regulate cell-mediated cytotoxicity involving a complex network of cytokine- and ligand-receptor interactions. Since the first description of MHC class I molecules in teleosts during the early 90s of the last century a remarkable amount of information on teleost immune responses has been published. The corresponding studies describe teleost cells and molecules that are involved in CTL responses of higher vertebrates. These studies are backed by functional investigations on the killing activity of CTLs in a few teleost species. The present knowledge on teleost CTLs still leaves considerable room for further investigations on the mechanisms by which CTLs act. Nevertheless the information on teleost CTLs and their regulation might already be useful for the control of fish diseases by designing efficient vaccines against such diseases where CTL responses are known to be decisive for the elimination of the corresponding pathogen. This review summarizes the present knowledge on CTL regulation and functions in teleosts. In a special chapter, the role of CTLs in vaccination is discussed.

Proton transport properties of proton-conducting phosphate glasses at their glass transition temperatures.

The proton transport properties of 32 kinds of proton-conducting phosphate glasses with broad ranges of glass transition temperature, proton conductivity, and the proton carrier concentration were studied. Almost constant proton mobility of around 2 × 10-8 cm2 V-1 s-1 at the glass transition temperature, corresponding to a diffusion coefficient of approximately 4 × 10-10 cm2 s-1, was found for the glasses. The reason why the diffusion coefficient of protons is almost constant in various proton-conducting phosphate glasses was discussed based on the role of the protons as a cross-linker within the phosphate framework via hydrogen bonding. We evaluated the highest proton conductivity of the phosphate glasses and melts based on the almost constant mobility at their glass transition temperatures and obtained a highest expected proton conductivity of 7.5 × 10-3 S cm-1 at 300 °C. The potential of proton-conducting phosphate glasses as electrolytes in intermediate temperature fuel cells was also discussed.

New cell lines for efficient propagation of koi herpesvirus and infectious salmon anaemia virus.

The production of piscine viruses, in particular of koi herpesvirus (KHV, CyHV-3) and infectious salmon anaemia virus (ISAV), is still challenging due to the limited susceptibility of available cell lines to these viruses. A number of cell lines from different fish species were compared to standard diagnostic cell lines for KHV and ISAV regarding their capability to exhibit a cytopathic effect (CPE) and to accumulate virus. Two cell lines, so far undescribed, appeared to be useful for diagnostic purposes. Fr994, a cell line derived from ovaries of rainbow trout (Oncorhynchus mykiss), produced constantly high ISA virus (ISAV) titres and developed a pronounced CPE even at high cell passage numbers, while standard cell lines are reported to gradually loose these properties upon propagation. Another cell line isolated from the head kidney of common carp (Cyprinus carpio), KoK, showed a KHV induced CPE earlier than the standard cell line used for diagnostics. A third cell line, named Fin-4, established from the fin epithelium of rainbow trout did not promote efficient replication of tested viruses, but showed antigen sampling properties and might be useful as an in vitro model for virus uptake or phagocytosis.

Post-depositional changes in elemental leaching from recovered soils separated from disaster waste and tsunami deposits generated by the Great East Japan Earthquake and tsunami.

The Great East Japan Earthquake and subsequent tsunami in 2011 generated massive amounts of disaster waste and tsunami deposits, one-third of which comprised soil and sediment, which are expected to be re-used; however, there has been no previous experience or knowledge of recovered soil. In this study, up-flow column leaching tests were conducted to investigate the elemental leaching behavior in this soil and sediment following its separation and treatment (hereafter termed "recovered soil") to assess whether it can be safely re-used without posing any environmental risk. The pH of the leaching water was slightly alkaline throughout the test period, regardless of the source of the recovered soil. Concentrations of calcium and sulfate ions in the leaching water varied in a similar way to the electrical conductivity (EC), with a stable state being observed initially followed by a rapid decrease until typical concentrations were reached, whereas sodium and chloride ions derived from seawater made a relatively small contribution to the EC. In terms of toxic elements, zinc and copper concentrations decreased as the volume of leaching water increased, whereas lead, fluoride, and arsenic concentrations increased as the concentration of calcium and sulfate ions decreased, indicating that the dissolution of large quantities of compounds such as calcium sulfate controlled the toxic element behavior in the recovered soils. Consequently, there is a need for continuous and careful monitoring of areas where recovered soil is re-used or treatment of the recovered soil prior to its re-use.