Structure of putative epidermal sensory receptors in an acoel flatworm, Praesagittifera naikaiensis.

Acoel flatworms possess epidermal sensory-receptor cells on their body surfaces and exhibit behavioral repertoires such as geotaxis and phototaxis. Acoel epidermal sensory receptors should be mechanical and/or chemical receptors; however, the mechanisms of their sensory reception have not been elucidated. We examined the three-dimensional relationship between epidermal sensory receptors and their innervation in an acoel flatworm, Praesagittifera naikaiensis. The distribution of the sensory receptors was different between the ventral and dorsal sides of worms. The nervous system was mainly composed of a peripheral nerve net, an anterior brain, and three pairs of longitudinal nerve cords. The nerve net was located closer to the body surface than the brain and the nerve cords. The sensory receptors have neural connections with the nerve net in the entire body of worms. We identified five homologs of polycystic kidney disease (PKD): PKD1-1, PKD1-2, PKD1-3, PKD1-4, and, PKD2, from the P. naikaiensis genome. All of these PKD genes were implied to be expressed in the epidermal sensory receptors of P. naikaiensis. PKD1-1 and PKD2 were dispersed across the entire body of worms. PKD1-2, PKD1-3, and PKD1-4 were expressed in the anterior region of worms. PKD1-4 was also expressed around the mouth opening. Our results indicated that P. naikaiensis possessed several types of epidermal sensory receptors to convert various environmental stimuli into electrical signals via the PKD channels and transmit the signals to afferent nerve and/or effector cells.

Reduction of oocyte shedding and cecal inflammation by 5-aminolevulinic acid daily supplementation in laying hens infected with Eimeria tenella.

The present study aimed to evaluate the effects of 5-aminolevulinic acid (5-ALA) on Eimeria tenella infection in laying hens. Oocyst shedding and histopathology were evaluated. A reduced oocyst shedding was observed 5 and 7 days post-infection (dpi) in the 5-ALA-administered group, but the total number of oocysts during the first infection period was not different between control and 5-ALA-treated groups. After E. tenella attack infection, the period of oocyst shedding in the 5-ALA-administered group lasted less long than that in controls. During the attack infection period, the total number of fecal oocysts in the 5-ALA-treated group was significantly lower than that in the control group. However, the parasite burden score in hens receiving 5-ALA was higher than that in controls after E. tenella attack infection. The lesion scores at 5 and 30 dpi in the control group were significantly lower than those in the 5-ALA-administered group. Therefore, 5-ALA administration might be beneficial against E. tenella infection in laying hens.

De novo transcriptome analysis of the centrohelid Raphidocystis contractilis to identify genes involved in microtubule-based motility.

The centrohelid heliozoan Raphidocystis contractilis has many radiating axopodia, each containing axopodial microtubules. The axopodia show rapid contraction at nearly a video rate (30 frames per second) in response to mechanical stimuli. The axopodial contraction is accompanied by cytoskeletal microtubule depolymerization, but the molecular mechanism of this phenomenon has not been elucidated. In this study, we performed de novo transcriptome sequencing of R. contractilis to identify genes involved in microtubule dynamics such as the rapid axopodial contraction. The transcriptome sequencing generated 7.15-Gbp clean reads in total, which were assembled as 31,771 unigenes. Using the obtained gene sets, we identified several microtubule-severing proteins which might be involved in the rapid axopodial contraction, and kinesin-like genes that occur in gene duplication. On the other hand, some genes for microtubule motor proteins involved in the formation and motility of flagella were not found in R. contractilis, suggesting that the gene repertoire of R. contractilis reflected the morphological features of nonflagellated protists. Our transcriptome analysis provides basic information for the analysis of the molecular mechanism underlying microtubule dynamics in R. contractilis.

Differential effects of orally administered Lactobacillus acidophilus L-55 on the gene expression of cytokines and master immune switches in the ileum and spleen of laying hen with an attenuated Newcastle disease virus vaccine.

This study aimed to evaluate the benefits of oral administration of Lactobacillus acidophilus strain L-55 (LaL-55) to chickens inoculated with a Newcastle disease virus (NDV)-based live-attenuated vaccine by examining the mRNA expression of several genes related to viral infection in the spleen and ileum by quantitative reverse transcription polymerase chain reaction. In the spleen, interferon (IFN)-α was significantly higher in the low- and middle-dose LaL-55 groups at 6 weeks than at 4 weeks. IFN regulatory factor (IRF)-3 and IRF-7 expression was significantly higher in the low-dose LaL-55 group than in the middle- and high-dose LaL-55 groups. In the ileum, melanoma differentiation-associated gene 5 showed a dose-dependent increase at 4 weeks. IFN-γ and IRF-7 showed dose-dependent increases at 6 weeks. These results suggested that LaL-55 boosts the immune response to the NDV vaccine, albeit by different mechanisms in the spleen and ileum.

Relationship between Eimeria tenella associated-early clinical signs and molecular changes in the intestinal barrier function.

The major clinical signs of coccidiosis in chickens due to Eimeria parasite are diarrhea and bloody feces. Previous studies showed that the impairment of the intestinal epithelial barrier and the elevation of the intestinal permeability are causes of clinical signs associated with coccidia challenges. Nevertheless, the information about molecular changes of the epithelial barrier at the early stage of the infection with a specific Eimeria species has not been mentioned. Hence, this study aims to elucidate the temporal relationships between epithelial barrier conditions and clinical signs in chickens infected with Eimeria tenella over the time from the earliest stages of infection. White Leghorn chickens were inoculated with 1 × 104 oocysts of E. tenella. Thereafter the chickens were monitored for their daily clinical signs through observation, and between 5 dpi to 10 dpi, feces were collected for oocysts counting. Chickens were then administrated with fluorescein isothiocyanate-dextran (FITC-d) for gastrointestinal permeability test and tissues were collected each day for histopathological observation and total RNA extraction. Finally, the mRNA expression levels of the tight and adherens junction genes and cytokine genes were evaluated using the quantitative real-time polymerase chain reaction (qRT-PCR). In this study, clinical signs such as diarrhea and bloody feces were observed concurrently from 3 to 8 dpi. Histopathology changes such as severe inflammation, hemorrhage, and epithelial desquamation were identified in the cecum specimens. The FITC-d level in the E. tenella-infected group was significantly higher than in the control group. In the infected group, the expression of claudin-2 gene was also upregulated, whereas the expressions of claudin-3 and E-cadherin genes were decreased as compared to the control group. These results implied that clinical signs of avian coccidiosis were associated with the intestinal barrier disruption via changes in expression levels of claudins and E-cadherin at the intestine.

Reduction of macrophages by carrageenan decreases oocyst output and modifies local immune reaction in chick cecum with Eimeria tenella.

This study aimed to evaluate the disease severity and local immune responses in macrophage-depleted chicks with Eimeria tenella. Macrophages were reduced by intraperitoneal injection of a carrageenan solution at 12, 13, and 16 days old, whereas the control group received intraperitoneal phosphate-buffered saline. Both chick groups were orally inoculated with E. tenella sporulated oocysts at 14 days old. Feces were collected daily, which were then quantified for oocysts. The chicks were sacrificed on day 5, and the ceca were collected for histopathological observation. The gene expression levels were measured using real-time quantitative reverse transcription-polymerase chain reaction. Macrophage-depleted chicks have been observed to shed a significantly reduced number of fecal oocysts compared to the infected control group. The parasite burden score in cecum specimens of macrophage-depleted chicks was significantly lower than those of infected control on day 5 after infection. Furthermore, macrophage reduction yielded significantly lower cecum histopathological scores and CD4 expression than those of the infected control group. The expression of interleukin (IL)-18, IL-22, interferon-γ, and inducible nitric oxide synthase was also noted to be significantly upregulated in both infected control and macrophage-depleted chicks compared to uninfected chicks. IL-4, IL-13, IL-17, and perforin expressions were also higher with macrophage depletion than in both control groups. These results suggest that macrophages serve as an invasive gate or a transporting vehicle to the site of first merogony. Furthermore, mononuclear phagocytes may play an important role in local immune responses, thus contributing to parasite development during early E. tenella infection.

Involvement of cancer-derived EMT cells in the accumulation of 18F-fluorodeoxyglucose in the hypoxic cancer microenvironment.

A high rate of glycolysis, one of the most common features of cancer, is used in positron emission tomography (PET) imaging to visualize tumor tissues using 18F-fluorodeoxyglucose (18F-FDG). Heterogeneous intratumoral distribution of 18F-FDG in tissues has been established in some types of cancer, and the maximum standardized uptake value (SUVmax) has been correlated with poor prognosis. However, the phenotype of cells that show high 18F-FDG accumulation in tumors remains unknown. Here, we combined quantitative micro-autoradiography with fluorescence immunohistochemistry to simultaneously visualize 18F-FDG distribution, the expression of multiple proteins, and hypoxic regions in the cancer microenvironment of a human A431 xenograft tumor in C.B-17/Icr-scid/scid mice. We found that the highest 18F-FDG accumulation was in cancer-derived cells undergoing epithelial-mesenchymal transition (EMT) in hypoxic regions, implicating these regions as a major contributor to increased glucose metabolism, as measured by 18F-FDG-PET.

Alteration of chemokine production in bovine endometrial epithelial and stromal cells under heat stress conditions.

After parturition, cows frequently develop uterine bacterial infections, resulting in the onset of endometritis. To eliminate the bacteria, bovine endometrial cells secrete chemokines, such as IL-6 and MCP1, which attract macrophages (MΦs) to the subepithelial stroma. These attracted MΦs are not only involved in bacterial elimination but also the orchestration of inflammation and tissue repair. These immune responses aid in the recovery from endometritis; however, the recovery from endometritis takes longer in summer than in any other season. Based on these findings, we hypothesized that heat stress (HS) affects the chemokine production in endometrial cells. To confirm this hypothesis, we compared IL-6 and MCP1 production induced by lipopolysaccharide (LPS) in bovine endometrial epithelial and stromal cells under normal (38.5°C) and HS conditions (40.5°C). In the endometrial epithelial cells, IL-6 production stimulated by LPS was significantly (p < .05) suppressed under HS conditions. MCP1 production in endometrial epithelial cells was not detected under both the control and HS conditions regardless of the presence of LPS. Moreover, LPS significantly (p < .05) stimulated IL-6 and MCP1 production in endometrial stromal cells. Moreover, HS significantly (p < .05) enhanced their production compared to that under the control conditions. In addition, HS did not affect the migration ability of MΦs; however, the supernatant of the endometrial stromal cells cultured under the HS condition significantly (p < .05) attracted the MΦs when compared to the control condition. These results suggest that HS disrupts chemokine production in two types of endometrial cells and alters the distribution of MΦs in the endometrium during the summer.

Oral administration of the probiotic bacterium Lactobacillus acidophilus strain L-55 modulates the immunological parameters of the laying hen inoculated with a Newcastle disease virus-based live attenuated vaccine.

Probiotic supplements containing living bacteria have attracted interest as a potential source of health benefits for humans and livestock. The aim of this study was to determine whether administration of Lactobacillus acidophilus strain L-55 (LaL-55) enhances the immune response among chicks exposed to a Newcastle disease virus (NDV)-based live attenuated vaccine. Oral administration of LaL-55 augmented the elevation in the total numbers of leukocytes and lymphocytes following inoculation with the NDV-based live attenuated vaccine. Monocyte counts increased after LaL-55 administration independent of inoculation with the NDV vaccine. Among chicks that were administered LaL-55, there was a dose-dependent increase in the NK cell activity measured by a 51Cr release assay at 2 weeks after the secondary NDV vaccine inoculation. Two weeks after the secondary inoculation with the NDV vaccine, interferon (IFN)-γ-mRNA expression was significantly elevated in mononuclear splenocytes from chicks that were administered LaL-55. Meanwhile, LaL-55 administration did not change the mRNA levels of IFN-α, IFN-ß, and interleukin-1ß. These results may suggest that coadministration of LaL-55 with an NDV vaccine augments the immune response against the virus. Therefore, LaL-55 may help protect against viral diseases in poultry.

Morphological and molecular identification of Eimeria spp. in breeding chicken farms of Japan.

There have been no reports of the prevalence of Eimeria spp. in poultry breeding farms in Japan unlike those of broiler farms. From 2017 to 2018, we examined the prevalence of Eimeria spp. on breeding farms in Japan by oocyst morphology and PCR analyses. A total of 143 samples was collected from 37 breeding farms in 21 prefectures of Japan. We detected oocysts of seven species at 34 of 37 breeding farms by PCR, and we identified E. brunetti at 51.5% of farms found to be positive for Eimeria. The differences in the identification of Eimeria spp. between the morphology and PCR assay methods of oocysts were pronounced for E. maxima and E. necatrix. We confirmed that molecular tools were more suitable for accurately estimating prevalence of Eimeria spp., and these findings suggest that E. brunetti could be widespread in Japan.

Daily Meal Supplemented with Astaxanthin-Enriched Yolk Has Mitigative Effects against Hypertension in Spontaneously Hypertensive Rats.

The aim of this study was to investigate the effects of egg yolk powder enriched with astaxanthin (ASX-E) on blood pressure in spontaneously hypertensive rats (SHR) and to verify the benefits of ASX-E as a functional food. To investigate the antihypertensive effect, SHR were fed with an ASX-E mixed diet before hypertension development. Blood pressures were determined periodically during the study by the tail-cuff method. At the end of the study, animals were euthanized, and their thoracic aortas were collected to determine vascular conductance. The thoracic aorta tension was measured with a force displacement transducer. Concentration-dependent response relationships were determined by cumulative addition of 10-9-10-4 M Carbamoylcholine (Cch). Blood pressures of the SHR in the ASX-E mixed diet group were ASX-dose-dependently lower than that of those in the control group. In SHR fed with an ASX-E mixed diet, Cch induced vasorelaxation in the thoracic aorta with endothelium lining but not without endothelium. However, the antihypertensive effect of ASX-E was not observed on blood pressures in SHR that were fed with ASX-E only after the development of hypertension. Results suggest that ASX-E protects endothelial function and thereby prevents the development of hypertension. Hence, the results of our research indicate that daily consumption of ASX-E has a potential benefit on human health.

Apoptosis inhibition mitigates aging effects in Drosophila melanogaster.

Aging is a natural biological process that results in progressive loss of cell, tissue, and organ function. One of the causing factors of the aging process is the decrease in muscle mass, which has not been fully verified in Drosophila. Apoptotic cell death may result in aberrant cell loss and can eventually diminish tissue function and muscle atrophy. If so, inhibition of apoptosis may prolong longevity and reduce motor function and muscle mass decline with age in Drosophila flies. Here, we used Drosophila melanogaster as study material, and induced the overexpression of Drosophila inhibitor of apoptosis protein 1 gene to inhibit apoptosis, and investigated the effect of apoptosis inhibition on the longevity and age-related declines in flight and climbing ability and muscle mass. As a result, the inhibition of apoptosis tended to mitigate the aging effects and prolonged longevity and reduced climbing ability decline with age. The current study suggests that apoptosis inhibition could mitigate the aging effects in D. melanogaster. Although such effects have already been known in mammals, the current results suggest that the apoptosis may play a similar role in insects as well.

Transitions in morphological forms and rapid development of the asexual schizonts of Eimeria tenella through serial passaging in chicks.

Attenuated strains of avian Eimeria parasites, generated by the selection of precocious lines through serial passaging in chicks, have been used widely as live vaccines. Detailed morphological transitions including their life cycle depending on the passages remain poorly understood. Here, we showed early development and acceleration of transitions in morphological forms of the asexual schizonts of E. tenella that had been attenuated for virulence by serial passaging. Our results may be helpful in understanding parasitism, facilitating further molecular analyses such as comparative genomic or transcriptomic tests.

Mulberry juice freeze-dried powder attenuates the disease severity by the maintaining of colon mucosa in mice with DSS-induced acute colitis.

This study aimed to evaluate the microbial compositions and gene expression related to inflammation in dextran sodium sulfate (DSS)-induced acute colitis and the effect of mulberry supplementation. Male BALB/c mice received a diet supplemented with mulberry juice freeze-dried powder (MFP) or not for 3 weeks. After 3 weeks, the mice received water containing 5% (w/v) DSS or not for 1 week. The disease activity index score in mice fed MFP was significantly decreased. A significant decrease in Bifidobacterium spp. and the Clostridium perfringens subgroup was observed in mice not fed MFP. The number of goblet cell and NLRP6 expression were observed in mice fed a diet supplemented with MFP compared with mice not fed MFP. These results may indicate that mulberry mitigates DSS-induced acute colitis by a changing the gut microbial flora and by improving mucosal conditions.

Antiplasmodial properties of kaempferol-3-O-rhamnoside isolated from the leaves of Schima wallichii against chloroquine-resistant Plasmodium falciparum.

Previous intervention studies have shown that the most effective agents used in the treatment of malaria were isolated from natural sources. Plants consumed by non-human primates serve as potential drug sources for human disease management due to the similarities in anatomy, physiology and disease characteristics. The present study investigated the antiplasmodial properties of the primate-consumed plant, Schima wallichii (S. wallichii) Korth. (family Theaceae), which has already been reported to have several biological activities. The ethanol extract of S. wallichii was fractionated based on polarity using n-hexane, ethyl acetate and water. The antiplasmodial activity was tested in vitro against chloroquine-resistant Plasmodium falciparum (P. falciparum) at 100 µg/ml for 72 h. The major compound of the most active ethyl acetate fraction was subsequently isolated using column chromatography and identified by nuclear magnetic resonance. The characterized compound was also tested against chloroquine-resistant P. falciparum in culture to evaluate its antiplasmodial activity. The ethanol extract of S. wallichii at 100 µg/ml exhibited a significant parasite shrinkage after 24 h of treatment. The ethyl acetate fraction at 100 µg/ml was the most active fraction against chloroquine-resistant P. falciparum. Based on the structural characterization, the major compound isolated from the ethyl acetate fraction was kaempferol-3-O-rhamnoside, which showed promising antiplasmodial activity against chloroquine-resistant P. falciparum with an IC50 of 106 µM after 24 h of treatment. The present study has provided a basis for the further investigation of kaempferol-3-O-rhamnoside as an active compound for potential antimalarial therapeutics.

Changes in estrogen receptor expression in the chick thymus during late embryonic development.

In chickens, although estrogen receptors (ER) are reported to be associated with the immunological processes, detailed information about the differences in ER expression in the tissues related to the development of lymphocytes is not fully known, especially during the developmental stage. To learn more about this immunological relationship, we used semi-quantitative polymerase chain reaction method to detect the ER expression levels in the thymus tissues of chicks during the developmental stage. Furthermore, ER-expressing cells were detected by immunohistochemistry. The results of this study show that the expression level of ER increased on embryonic day 16 and decreased on day 20. Furthermore, ER expression was significantly higher in male than in female chickens at day 16. The increased expression on day 16 and decreased level on day 20 were also reproduced in the incidence of immunoreactive cells, although there was a 1-day delay in the elevated incidence of the cells. This study revealed the changes in ER expression and the incidence of ER-positive cells in the thymus of chickens during the developmental stage.

Derivatives of Dictyostelium discoideum differentiation-inducing factor-3 suppress the activities of Trypanosoma cruzi in vitro and in vivo.

Chagas disease (human American trypanosomiasis), which is caused by the protozoan parasite Trypanosoma cruzi, is responsible for numerous deaths each year; however, established treatments for the disease are limited. Differentiation-inducing factor-1 (DIF-1) and DIF-3 are chlorinated alkylphenones originally found in the cellular slime mold Dictyostelium discoideum that have been shown to possess pharmacological activities. Here, we investigated the effects of DIF-3 derivatives on the infection rate and growth of T. cruzi by using an in vitro assay system utilizing host human fibrosarcoma HT1080 cells. Certain DIF-3 derivatives, such as butoxy-DIF-3 (Bu-DIF-3), at micro-molar levels strongly suppressed both the infection rate and growth of T. cruzi in HT1080 cells and exhibited little toxicity for HT1080 cells. For example, the IC50 of DIF-3 and Bu-DIF-3 versus the growth of T. cruzi in HT1080 cells were 3.95 and 0.72µM, respectively, and the LD50 of the two compounds versus HT1080 cells were both greater than 100µM. We also examined the effects of DIF-3 and Bu-DIF-3 on T. cruzi activity in C57BL/6 mice. Intraperitoneally administered Bu-DIF-3 (50mg/kg) significantly suppressed the number of trypomastigotes in blood with no apparent adverse effects. These results strongly suggest that DIF-3 derivatives could be new lead compounds in the development of anti-trypanosomiasis drugs.

Selenium-induced apoptosis-like cell death in Plasmodium falciparum.

Plasmodium falciparum has for some time been developing resistance against known anti-malarial drugs, and therefore a new drug is urgently needed. Selenium (Se), an essential trace element, in the form of inorganic Se, selenite (SeO32-), has been reported to have an anti-plasmodial effect, but its mechanism is still unclear. In the present study, we evaluated the anti-plasmodial effect of several Se compounds against P. falciparum in vitro. The anti-plasmodial effect of several Se compounds was analysed and their apoptosis-inducing activity was evaluated by morphological observation, DNA fragmentation assay and mitochondrial function analysis. SeO32-, methylseleninic acid, selenomethionine and selenocystine have anti-plasmodial effects with 50% inhibition concentration at 9, 10, 45, and 65 µm, respectively, while selenate and methylselenocysteine up to 100 µm have no effect on parasite growth. The effective Se compounds caused the parasites to become shrunken and pyknotic and significantly increased mitochondrial damage against P. falciparum compared to the untreated control. In conclusion, SeO32-, methylseleninic acid, selenomethionine and selenocystine have anti-plasmodial activities that induce apoptosis-like cell death in P. falciparum, and the anti-plasmodial effects of Se seem to be based on its chemical forms. The apoptosis-like cell-death mechanism in P. falciparum can be beneficial to respond to the growing problem of drug resistance.

The role of VAMP7/TI-VAMP in cell polarity and lysosomal exocytosis in vivo.

VAMP7 or tetanus neurotoxin-insensitive vesicle- associated membrane protein (TI-VAMP) has been proposed to regulate apical transport in polarized epithelial cells, axonal transport in neurons and lysosomal exocytosis. To investigate the function of VAMP7 in vivo, we generated VAMP7 knockout mice. Here, we show that VAMP7 knockout mice are indistinguishable from control mice and display a similar localization of apical proteins in the kidney and small intestine and a similar localization of axonal proteins in the nervous system. Neurite outgrowth of cultured mutant hippocampal neurons was reduced in mutant neurons. However, lysosomal exocytosis was not affected in mutant fibroblasts. Our results show that VAMP7 is required in neurons to extend axons to the full extent. However, VAMP7 does not seem to be required for epithelial cell polarity and lysosomal exocytosis.

Efficacy of mefloquine treatment and genetic profiles in uncomplicated Plasmodium falciparum malaria in southern Lao PDR.

We conducted a 28-day follow-up of 17 Laotian patients diagnosed with uncomplicated Plasmodium falciparum malaria treated with mefloquine (Mephaquine, MQ) alone to determine the efficacy. All patients were completely cured with MQ, without reappearance of asexual stage parasitemia at follow-up. Of the 7 isolates tested for genotypic analysis, one isolate was a Y86 mutant type of the pfmdr1 gene, the others were N86 wild. These findings suggest no MQ resistance in the study area possibly because the drug is rarely used in southern Lao PDR.