Preliminary insight into parental contributions to Japanese eel (Anguilla japonica) preleptocephali spawned on different nights.

Parentage sibship-inference analyses were conducted using mtDNA sequencing and six microsatellite genotypes of 182 Japanese eel preleptocephali that were collected from one net-tow near the West Mariana Ridge in May 2014. At least 328 parents were involved in producing the 182 preleptocephali, and several parents may have spawned a few times during 3 days of a spawning period. Half-sibs suggested that a few parents mated with 1-3 partners, indicating that the Japanese eel can form spawning aggregations in which several parents mate with each other in the ocean.

Development of a novel heterologous gene expression system using earthworms.

In recent years, animals and plants have received increasing attention as potential next-generation protein production systems, especially for biopharmaceuticals and animal proteins. The aim of the present study was to develop the earthworms Eisenia fetida Waki and Eisenia andrei Sagami as next-generation animal protein production hosts. These earthworms have been approved as model animals for acute toxicity tests by the Organization for Economic Co-operation and Development, and they have post-translational modification systems. However, so far, none of the studies have used earthworm transfection techniques. Thus, we developed a transfection method for E. fetida and E. andrei using microinjection and electroporation systems. The maximum survival rates and transfection efficiencies were 79.2% and 29.2% for E. fetida, and 95.8% and 50.0% for E. andrei, respectively. Furthermore, human erythropoietin was detected in the transformed earthworm tail fragments using an enzyme-linked immunosorbent assay. These results contribute to the development of a potential earthworm-based novel animal protein production system.

High-Spatial-Resolution Multimodal Imaging by Tapping-Mode Scanning Probe Electrospray Ionization with Feedback Control.

Direct extraction and ionization techniques using minute amounts of solvent can be employed for the rapid analysis of chemical components in a sample without any sample preparation steps. This type of approach is important for mass spectrometry imaging of samples with multiple chemical components that have different spatial distributions (i.e., biological tissues). To improve the spatial resolution of such imaging, it is necessary to reduce the solvent volume for extraction and deliver it to the sample surface. This report describes a feedback control system applied to tapping-mode scanning probe electrospray ionization. By combining the measurement technique of capillary probe vibration with the dynamic distance control system between the probe and the sample, the vibration amplitude of the probe is maintained while the probe scans over uneven samples. This method allows simultaneous high-resolution imaging of molecular distribution, surface topography, and amplitude/phase changes in the probe vibration. Such multimodal imaging is demonstrated on rhodamine B thin films in microwells and on a mouse brain tissue section. This technique can generally be applied to examine the multidimensional molecular distribution and the surface profiles of various objects.

Distribution of anguillid leptocephali and possible spawning areas in the South Pacific Ocean.

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.

New PCR primers for metabarcoding environmental DNA from freshwater eels, genus Anguilla.

Freshwater eels of the genus Anguilla comprise 16 species that include three subspecies and are characterized by their unique catadromous life cycles. Their life histories and nocturnal life styles make it difficult to observe them in freshwater and marine habitats. To investigate their distribution and ecology in aquatic environments, we developed new PCR primers for metabarcoding environmental DNA (eDNA) from Anguilla. The new primers (MiEel) were designed for two conserved regions of the mitochondrial ATP6 gene, which amplify a variable region with sufficient interspecific variations ranging from five to 22 nucleotide differences (one to three nucleotide differences between three subspecies pairs). We confirmed the versatility of the MiEel primers for all freshwater eels using tissue DNA extracts when analyzed separately. The metabarcoding combined with the MiEel primers using mock communities enabled simultaneous detection of Anguilla at the species level. Analysis of eDNA samples from aquarium tanks, a controlled pond and natural rivers demonstrated that the MiEel metabarcoding could successfully detect the correct Anguilla species from water samples. These results suggested that eDNA metabarcoding with MiEel primers would be useful for non-invasively monitoring the presence of the endangered anguillid eels in aquatic environments where sampling surveys are difficult.

Release of eDNA by different life history stages and during spawning activities of laboratory-reared Japanese eels for interpretation of oceanic survey data.

To assist in detection of offshore spawning activities of the Japanese eel Anguilla japonica and facilitate interpretation of results of environmental DNA (eDNA) analysis in their spawning area, we examined the eDNA concentration released by each life history stage of artificially reared Japanese eels in the laboratory using quantitative real-time PCR (qPCR). We also compared eDNA concentrations between before and after artificially induced spawning activities. eDNA was not detected from three 30 L seawater tanks containing each single fertilized egg, but eDNA was found from other tanks each containing single individuals of larval stages (preleptocephalus and leptocephalus), juvenile stages (glass eel, elver and yellow eel) or adult stage (silver eel). The eDNA concentrations increased in the life history stages, showed a significant difference among all stages, and were positively correlated with the total length and wet weight. Moreover, the eDNA concentration after spawning was 10-200 times higher than that before spawning, which indicated that the spawning events in the ocean would produce relatively high eDNA concentration. These results in the laboratory suggested that eDNA analysis appears to be an effective method for assisting oceanic surveys to estimate the presence and spawning events of the Japanese eel in the spawning area.

Visualization of Sampling and Ionization Processes in Scanning Probe Electrospray Ionization Mass Spectrometry.

Ambient sampling and ionization techniques based on direct liquid extraction and electrospray ionization are of great value for rapid analysis and mass spectrometry imaging. Scanning probe electrospray ionization (SPESI) enables the sampling and ionization of analyte molecules in a solid material using a liquid bridge and electrospray, respectively, from a single capillary probe. To further improve SPESI, it is essential to understand the dynamic behavior of nanoliter volumes of liquids during sampling and ionization. In this study, the dynamic formation and breakage of the liquid bridge and the subsequent electrospray ionization were investigated by measuring the displacement of the capillary probe using a new optical technique. Measurements revealed that both the time from the formation of the liquid bridge to its breakage and the time from the breakage of the liquid bridge to the detection of analyte ions were correlated with the physical properties of the solvent. It was also found that both of these times were positively correlated with the flow rate. These results will not only lead to the improvement of sampling and ionization efficiencies but also afford a greater understanding of the physicochemical properties of charged nanoliter volumes of liquids.

Structure-activity relationship of indoloquinoline analogs anti-MRSA.

Indolo[3,2-b]quinoline analogs (3a-3s), 4-(acridin-9-ylamino) phenol hydrochloride (4), benzofuro[3,2-b]quinoline (3t), indeno[1,2-b]quinolines (3u and 3v) have been synthesized. Those compounds were found to exhibit anti-bacterial activity towards Methicillin-resistant Staphylococcus aureus (anti-MRSA activity). Structure-activity relationship studies were conducted that indoloquinoline ring, benzofuroquinoline ring and 4-aminophenol group are essential structure for anti-MRSA activity.

The LNK1 night light-inducible and clock-regulated gene is induced also in response to warm-night through the circadian clock nighttime repressor in Arabidopsis thaliana.

Ambient temperature has two fundamental impacts on the Arabidopsis circadian clock system in the processes referred to as temperature compensation and entrainment, respectively. These temperature-related longstanding problems have not yet been fully clarified. Recently, we provided evidence that temperature signals feed into the clock transcriptional circuitry through the evening complex (EC) nighttime repressor composed of LUX-ELF3-ELF4, and that the transcription of PRR9, PRR7, GI and LUX is commonly regulated through the nighttime repressor in response to both moderate changes in temperature (∆6 °C) and differences in steady-state growth-compatible temperature (16 °C to 28 °C). These temperature-associated characteristics of the core clock genes might be relevant to the fundamental oscillator functions. Here, we further show that the recently identified LNK1 night light-inducible and clock-controlled gene, which actually has a robust peak at daytime, is induced also by warm-night through the EC nighttime repressor in a manner very similar to PRR7, which is also night light-inducible daytime gene. Based on these findings, a hypothetical view is proposed with regard to the temperature entrainment of the central oscillator.

Ambient temperature signal feeds into the circadian clock transcriptional circuitry through the EC night-time repressor in Arabidopsis thaliana.

An interlocking multiloop model has been generally accepted to describe the transcriptional circuitry of core clock genes, through which robust circadian rhythms are generated in Arabidopsis thaliana. The circadian clock must have the ability to integrate ambient temperature signals into the clock transcriptional circuitry to regulate clock function properly. Clarification of the underlying mechanism is a longstanding subject in the field. Here, we provide evidence that temperature signals feed into the clock transcriptional circuitry through the evening complex (EC) night-time repressor consisting of EARLY FLOWERING 3 (ELF3, ELF4) and LUX ARRHYTHMO (LUX; also known as PCL1). Chromatin immunoprecipitation assays showed that PSEUDO-RESPONSE REGULATOR7 (PRR7), GIGANTEA (GI) and LUX are direct targets of the night-time repressor. Consequently, transcription of PRR9/PRR7, GI and LUX is commonly regulated through the night-time repressor in response to both moderate changes in temperature (Δ6°C) and differences in the steady-state growth-compatible temperature (16-28°C). A warmer temperature inhibits EC function more, whereas a cooler temperature stimulates it more. Consequently, the expression of these target genes is up-regulated in response to a warm temperature specifically during the dark period, whereas they are reversibly down-regulated in response to a cool temperature. Transcription of another EC target, the PIF4 (PHYTOCHROME-INTERACTING FACTOR 4) gene, is modulated through the same thermoregulatory mechanism. The last finding revealed the sophisticated physiological mechanism underlying the clock-controlled output pathway, which leads to the PIF4-mediated temperature-adaptive regulation of hypocotyl elongation.

Enteral Nutrition Related Complications Relevant to Alteration of Formulas in Two Critically Ill Pediatric Patients.

The early institution of enteral nutrition is associated with beneficial outcomes and intestinal growth in pediatric patients. However, the number, frequency, and types of unfavorable events occurring with particular formulas are undefined. We experienced unexpected complications in two cases following a change in formula. One case diagnosed with myotubular myopathy experienced highly-increased gastric residuals and watery diarrhea leading to decreased calorie intake and weight loss. The second case with campomelic dysplasia suffered liver dysfunction and fever. In both cases, symptoms developed soon after of the change in formula and improved after resumption of the previous formula. Both cases had undergone tracheostomy and artificial ventilation, and had a history of feeding the same formula for an extended period of time. In chronic care patients such as ours, a change in formula may cause unexpected adverse events; therefore, caution is warranted.

Redox-sensitive structural change in the A-domain of HMGB1 and its implication for the binding to cisplatin modified DNA.

HMGB1 (high-mobility group B1) is a ubiquitously expressed bifunctional protein that acts as a nuclear protein in cells and also as an inflammatory mediator in the extracellular space. HMGB1 changes its functions according to the redox states in both intra- and extra-cellular environments. Two cysteines, Cys23 and Cys45, in the A-domain of HMGB1 form a disulfide bond under oxidative conditions. The A-domain with the disulfide bond shows reduced affinity to cisplatin modified DNA. We have solved the oxidized A-domain structure by NMR. In the structure, Phe38 has a flipped ring orientation from that found in the reduced form; the phenyl ring in the reduced form intercalates into the platinated lesion in DNA. The phenyl ring orientation in the oxidized form is stabilized through intramolecular hydrophobic contacts. The reorientation of the Phe38 ring by the disulfide bond in the A-domain may explain the reduced HMGB1 binding affinity towards cisplatinated DNA.

Expression and function of inducible costimulator on peripheral blood CD4+ T cells in Behçet's patients with uveitis: a new activity marker?

PURPOSE: Inducible costimulator (ICOS) is an important costimulatory molecule involved in T-cell activation. In this study, the role of ICOS in the pathogenesis of uveitis in Behçet's disease (BD) was investigated. METHODS: Peripheral blood mononuclear cells (PBMCs) were obtained from BD patients with uveitis in the active or remission phase and in healthy subjects. Total RNA was isolated from PMBCs, and mRNA expression was analyzed on an oligonucleotide microarray. ICOS expression on CD4(+) T cells was determined by flow cytometry, and the functional costimulatory effect of ICOS/B7RP-1 interaction was assessed on stimulation with concanavalin A (conA) or IRBP in the presence or absence of anti-ICOS mAb. RESULTS: As the result of microarray analysis, ICOS in PBMCs showed the greatest difference in expression in BD patients with uveitis compared with healthy control subjects. ICOS expression on CD4(+) T cells in BD patients with uveitis was significantly higher than that in healthy individuals, both before and after conA stimulation. Among the BD patients, ICOS expression on CD4(+) T cells was significantly higher in those with active uveitis than in those with remitted uveitis. Blockade of ICOS/B7-related protein-1 (B7RP-1) interaction by anti-ICOS mAb significantly decreased IFN-γ, IL-17, and TNF-α production by PBMCs when stimulated with conA or IRBP in BD with active uveitis. CONCLUSIONS: High ICOS expression in BD patients with uveitis contributed to the upregulation of IFN-γ, IL-17, and TNF-α production, suggesting that abnormal ICOS costimulation may play an immunopathologic role in the pathogenesis of uveitis in BD.

Effects of dioxin on vascular endothelial growth factor (VEGF) production in the retina associated with choroidal neovascularization.

PURPOSE: Cigarette smoking is the most consistent risk factor for age-related macular degeneration (AMD), especially the choroidal neovascularization (CNV)-mediated exudative type. Dioxins and dioxin-like compounds have various effects on living organisms and are also contained in cigarette smoke. However, the effects of dioxins on the eye remain elusive. In this study, the authors examined the association between dioxins and neovascularization in the eye. METHODS: C57BL/6 mice were injected intraperitoneally with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) every other day for 14 days. Messenger RNA expression of cytochrome P450 (CYP)1A1, CYP1B1, vascular endothelial growth factor (VEGF)-A and VEGF-B, and VEGF production were examined in the eyes of TCDD-treated mice and in human retinal pigment epithelial cell lines (ARPE-19) exposed to TCDD. In addition, CNV was induced by photocoagulation in mice injected with TCDD, and the volume of CNV was compared by fluorescence-labeled choroidal flat mount. RESULTS: TCDD injected intraperitoneally increased CYP1A1 mRNA expression in the iris/ciliary body and retina, indicating that TCDD acts directly on ocular tissues through the aryl hydrocarbon receptor (AhR) to promote the transcription of target genes. TCDD also promoted VEGF-A mRNA expression in the retina and the retinal pigment epithelium. TCDD-induced VEGF production at the molecular level was also observed in vivo by immunohistochemistry and in vitro using ARPE-19. Moreover, the injection of TCDD significantly exacerbated photocoagulation-induced CNV in mice. CONCLUSIONS: The authors demonstrate that dioxins are among the factors inducing abnormal vascularization in the eye through VEGF production mediated by AhR signaling.

The role of the ICOS/B7RP-1 T cell costimulatory pathway in murine experimental autoimmune uveoretinitis.

ICOS/B7RP-1 is a new member of the CD28/B7 family of costimulatory molecules and plays differential roles in autoimmune diseases. In this study, we examined the role of ICOS/B7RP-1 pathway in the pathogenesis of mouse experimental autoimmune uveoretinitis (EAU), an animal model of human autoimmune uveitis. ICOS expression was found on infiltrating CD4+ T cells in the region of the retina in EAU-induced mice. The anti-B7RP-1 monoclonal antibody (mAb)-treated or ICOS-deficient mice showed a substantial reduction of disease scores. Blockade of ICOS/B7RP-1 interaction during the effector phase ameliorated the disease, whereas its blockade during the induction phase exhibited no significant effect. Moreover, administration of anti-B7RP-1 mAb effectively ameliorated the disease induced by adoptive transfer of pathogenic T cells. The anti-B7RP-1 mAb treatment inhibited the expansion and/or effector function of pathogenic T cells, given that proliferative response and IFN-gamma production by lymph node cells were reduced upon restimulation with the antigen peptide in vitro. These results suggest that the ICOS/B7RP-1 interaction plays a critical role in the pathogenesis of uveitis. We also indicated that ICOS-mediated costimulation plays differential roles in EAU and experimental autoimmune encephalomyelitis, which is also a Th1 disease induced in the same manner as EAU.

Possibility of inducing anterior chamber-associated immune deviation by TGF-beta2 treatment of monocytes isolated from Behcet's patients.

Murine macrophages treated with TGF-beta2 are capable of inducing anterior chamber-associated immune deviation (ACAID), and these macrophages are characterized by impaired IL-12 production and CD40 expression, consequently failing to promote Th1 cell differentiation. In this study, we investigated whether human monocytes can also acquire the specific functions by TGF-beta2 treatment, even when the monocytes are isolated from patients with Behcet's disease (BD). Adherent monocytes isolated from peripheral blood mononuclear cells (PBMC) of 16 BD patients and 16 healthy controls, were cultured overnight with or without 5 ng/ml of TGF-beta2. Then, TGF-beta2-treated or untreated adherent cells were co-cultured with allogeneic CD4(+) T cells obtained from healthy subjects. TGF-beta2 treatment inhibited the abilities of adherent monocytes obtained from BD patients to stimulate the proliferation and IFN-gamma production of allogeneic CD4(+) T cells. The reduced IFN-gamma production was also confirmed by IFN-gamma mRNA expression in the co-cultured T cells. IL-12 production and CD40 molecule expression by adherent monocytes obtained from BD patients were strikingly reduced by TGF-beta2 treatment. These results suggest a possibility that adherent monocytes isolated from BD patients may acquire a property to induce ACAID by treatment with TGF-beta2.

Elevated serum levels of CXCL9/monokine induced by interferon-gamma and CXCL10/interferon-gamma-inducible protein-10 in ocular sarcoidosis.

PURPOSE: Sarcoidosis is a chronic multisystem granulomatous disorder characterized by an accumulation of activated CD4+ T cells and monocytes/macrophages in involved organs. Chemokines are required for the extravasation of leukocytes to the inflammation site. This study was undertaken to determine which chemokines are augmented in the serum of patients with active ocular sarcoidosis. METHODS: Seventeen patients with diagnosed ocular sarcoidosis, 28 with suspected ocular sarcoidosis, 16 with Behçet's disease, 17 with Vogt-Koyanagi-Harada disease, and 18 healthy subjects were studied. Serum levels of CCL2, CCL5, CXCL8, CXCL9, and CXCL10 were simultaneously measured by cytometric bead array using flow cytometer. In addition, serum CXCL9 and CXCL10 levels in the patients with diagnosed or suspected ocular sarcoidosis were compared with respect to ocular disease activity, the presence of bilateral hilar lymphadenopathy (BHL), and laboratory data. RESULTS: Serum levels of both CXCL9 and CXCL10 were markedly elevated in the patients with diagnosed or suspected ocular sarcoidosis compared with patients with other types of uveitis and healthy subjects. Although CCL2 and CXCL8 were detected in the serum of all subjects, the levels were extremely low with no significant differences between groups. Elevation of serum CXCL9 and CXCL10 in ocular sarcoidosis correlated significantly with ocular disease activity and ACE (angiotensin converting enzyme) levels and was unrelated to the presence of BHL, erythrocyte sedimentation rate, white blood cell count, serum IgG, or serum lysozyme. CONCLUSIONS: The results demonstrated that serum levels of CXCL9 and CXCL10 were elevated markedly in the patients with ocular sarcoidosis and correlated with ocular disease activity and ACE level.

CCR5-deficient mice develop experimental autoimmune uveoretinitis in the context of a deviant effector response.

PURPOSE: Experimental autoimmune uveoretinitis (EAU) is an organ-specific, Th1-cell-mediated disease that targets the neural retina. CCR5 is a chemokine receptor expressed on Th1 cells that promotes their migration. In CCR5-deficient mice, we examined the role of CCR5 in the development of EAU induced by immunization with interphotoreceptor retinoid-binding protein (IRBP) peptide. METHODS: Wild-type or CCR5-deficient B6 mice were immunized with human IRBP peptide 1-20 (hIRBP-p), and the severity of EAU was assessed clinically and histologically. Splenocytes and cells of regional lymph nodes near the eye were collected and their proliferation and production of IL-6, IL-10, IFN-gamma, and CCL2 (MCP-1) in response to hIRBP-p stimulation were measured. Moreover, the intraocular levels of these cytokines were analyzed. RESULTS: Immunization with hIRBP-p induced EAU in CCR5-deficient mice with a severity comparable to that in wild-type mice. Histologically, T-cell infiltration of the eye was reduced, but granulocyte infiltration was augmented in CCR5-deficient mice. Although splenic T cells from CCR5-deficient mice produced IFN-gamma but not IL-10 on stimulation by hIRBP-p, T cells from the regional lymph nodes failed to produce both cytokines. IL-6 production in the eye and IL-6 and CCL2 production by splenic T cells were predominantly augmented in CCR5-deficient mice. CONCLUSIONS: The development of EAU is not prevented in CCR5-deficient mice. Although T-cell infiltration into the eye is apparently reduced in CCR5-deficient mice, the defect is compensated for by granulocyte infiltration, supposedly mediated by augmented intraocular production of IL-6.

Effects of 3-methylcholanthrene on the transcriptional activity and mRNA accumulation of the oncogene hWAPL.

hWAPL is a human oncogene associated with uterine cervical cancer. Here, we demonstrate that hWAPL transcription is induced by 3-methylcholanthrene (3-MC) in the cervical carcinoma-derived cell line SiHa. hWAPL transcription was analyzed with evaluation of the mRNA and heterogeneous nuclear RNA (hnRNA) levels by quantitative real time PCR analysis. Flow cytometric analysis suggested that the alteration of hWAPL mRNA levels is independent of cell cycle profile. We also found that DMSO and some components of FBS affect hWAPL transcription. Interestingly, when the aryl hydrocarbon receptor (AhR) function was inhibited by alpha-naphthoflavone (ANF), the induction of hWAPL transcription by 3-MC was greater than that in AhR-functioning normal cells. These observations suggest that there are complex mechanisms regulating the transcription of hWAPL. Furthermore, mRNA level of a mouse homolog of hWAPL in mouse uterus was induced by 3-MC injection into the abdominal cavity. Thus, some effects from 3-MC exposure on uterus may be mediated by the unscheduled overexpression of hWAPL.