Deficiency of P2RY11 causes narcolepsy and attenuates the recruitment of neutrophils and macrophages in the inflammatory response in zebrafish.

Purinergic receptor P2Y11, a G protein-coupled receptor that is stimulated by extracellular ATP, has been demonstrated to be related to the chemotaxis of granulocytes, apoptosis of neutrophils, and secretion of cytokines in vitro. P2Y11 mutations were associated with narcolepsy. However, little is known about the roles of P2RY11 in the occurrence of narcolepsy and inflammatory response in vivo. In this study, we generated a zebrafish P2Y11 mutant using CRISPR/Cas9 genome editing and demonstrated that the P2Y11 mutant replicated the narcolepsy-like features including reduced HCRT expression and excessive daytime sleepiness, suggesting that P2Y11 is essential for HCRT expression. Furthermore, we accessed the cytokine expression in the mutant and revealed that the P2RY11 mutation disrupted the systemic inflammatory balance by reducing il4, il10 and tgfb, and increasing il6, tnfa, and il1b. In addition, the P2RY11-deficient larvae with caudal fin injuries exhibited significantly slower migration and less recruitment of neutrophils and macrophages at damaged site, and lower expression of anti-inflammatory cytokines during tissue damage. All these findings highlight the vital roles of P2RY11 in maintaining HCRT production and secreting anti-inflammatory cytokines in the native environment, and suggested that P2RY11-deficient zebrafish can serve as a reliable and unique model to further explore narcolepsy and inflammatory-related diseases with impaired neutrophil and macrophage responses.

Effects of diquafosol sodium in povidone iodine-induced dry eye model.

AIM: To investigate the effects of diquafosol sodium (DQS) for dry eye model induced with povidone-iodine (PI) solution. METHODS: Ten Sprague Dawley rats as the control group. Thirty Sprague Dawley rats were used to establish the dry eye model with stimulation of 10 g/L PI for 14d, then divided rats into three groups: dry eye group with no treatment (DED group, n=10); phosphate buffer saline treated group (PBS group, n=10); diquafosol treated group (DQS group, n=10). Clinical changes were observed by tear production test, fluorescein staining, tear break-up time (TBUT) test, corneal confocal microscope and ocular surface comprehensive analyzer. Eyeballs were collected on day 10 of treatment for hematoxylin-eosin (HE), periodic acid-Schiff (PAS), and alcian blue staining. TUNEL assay, polymorphonuclear (PMN) and mucin 1 (MUC1) immunofluorescence were performed and corneal ultrastructural changes were detected by electron microscopy. RESULTS: Compared with DED and PBS groups, tear production (7.26±0.440 vs 4.07±0.474 mm; 7.26±0.440 vs 3.74±0.280 mm; all P<0.01) and TBUT (7.37±0.383s vs 1.49±0.260s; 7.37±0.383s vs 1.42±0.437s; all P<0.01) were significantly increased in DQS group. HE, PAS, and alcian blue staining and MUC1 immunofluorescence showed mucins and conjunctival goblet cells density (8.45±0.718 vs 5.21±0.813 cells/0.1 mm2; 8.45±0.718 vs 5.36±0.615 cells/0.1 mm2; all P<0.01) increased in DQS group. Confocal microscopy, PMN immunofluorescence and TUNEL staining showed inflammatory infiltration and corneal epithelial cells apoptosis decreased in DQS group. The increased number of microvilli in corneal epithelial and the recovered cell junction were observed in DQS group. CONCLUSION: PI instillation can induce goblet cells and mucin loss, epithelial cell apoptosis and inflammation, which are consistent with the pathological manifestations of dry eye. Diquafosol can repair the ocular surface damage caused by PI, reduce corneal inflammation, inhibit corneal epithelial cell apoptosis, promote mucin secretion and maintain tear film stability.

Mechanically sensitive HSF1 is a key regulator of left-right symmetry breaking in zebrafish embryos.

The left-right symmetry breaking of vertebrate embryos requires nodal flow. However, the molecular mechanisms that mediate the asymmetric gene expression regulation under nodal flow remain elusive. Here, we report that heat shock factor 1 (HSF1) is asymmetrically activated in the Kupffer's vesicle of zebrafish embryos in the presence of nodal flow. Deficiency in HSF1 expression caused a significant situs inversus and disrupted gene expression asymmetry of nodal signaling proteins in zebrafish embryos. Further studies demonstrated that HSF1 is a mechanosensitive protein. The mechanical sensation ability of HSF1 is conserved in a variety of mechanical stimuli in different cell types. Moreover, cilia and Ca2+-Akt signaling axis are essential for the activation of HSF1 under mechanical stress in vitro and in vivo. Considering the conserved expression of HSF1 in organisms, these findings unveil a fundamental mechanism of gene expression regulation by mechanical clues during embryonic development and other physiological and pathological transformations.

PT-Symmetric LC Passive Wireless Sensing.

Parity-time (PT) symmetry challenges the long-held theoretical basis that only Hermitian operators correspond to observable phenomena in quantum mechanics. Non-Hermitian Hamiltonians satisfying PT symmetry also have a real-valued energy spectrum. In the field of inductor-capacitor (LC) passive wireless sensors, PT symmetry is mainly used for improving performance in terms of multi-parameter sensing, ultrahigh sensitivity, and longer interrogation distance. For example, the proposal of both higher-order PT symmetry and divergent exceptional points can utilize a more drastic bifurcation process around exceptional points (EPs) to accomplish a significantly higher sensitivity and spectral resolution. However, there are still many controversies regarding the inevitable noise and actual precision of the EP sensors. In this review, we systematically present the research status of PT-symmetric LC sensors in three working areas: exact phase, exceptional point, and broken phase, demonstrating the advantages of non-Hermitian sensing concerning classical LC sensing principles.

Melatonin attenuates 17ß-trenbolone induced insomnia-like phenotype and movement deficiency in zebrafish.

17ß-trenbolone (17ß-TBOH) is one of the dominant metabolites of trenbolone acetate, which is widely applied in beef cattle operations around the globe. The effects of environmental concentrations of 17ß-trenbolone on the early development of zebrafish embryos have received very little attention. Melatonin could regulate sleep-wake cycle and plays a protective role in various adverse conditions. Here, environmentally realistic concentrations of 17ß-trenbolone (1 ng/L, 10 ng/L, 50 ng/L) has been exposure to zebrafish embryos at 2 h postfertilization (hpf). The results showed that 10 ng/L and 50 ng/L 17ß-trenbolone disturbed the distribution of caudal primary motoneurons and downregulated expression of motoneuron development related genes along with locomotion decreasing. While melatonin could recover the detrimental effects caused by 17ß-trenbolone. Interestingly, 17ß-trenbolone exposure increased waking activity and decreased rest even in a low dose (1 ng/L). Moreover, it upregulated hypocretin/orexin (Hcrt) signaling which promotes wakefulness. Melatonin restored the insomnia-like alternation induced by 17ß-trenbolone exposure. Collectively, we conclude that 17ß-trenbolone disturbed motoneuron development and altered sleep/wake behavior, while melatonin could alleviate the deleterious influence on motoneuron development and recover the circadian rhythm.

Venlafaxine plus melatonin ameliorate reserpine-induced depression-like behavior in zebrafish.

Venlafaxine (VEN) is one of the first clinical drugs for the treatment of depression. Long-term use may cause a potentially life-threatening serotonin syndrome. Melatonin (MT) could ameliorate depression behavior. Therefore, the aim of this study was to investigate the antidepressant effects of venlafaxine in combination with melatonin on zebrafish. Reserpine was used to induce depression-like behavioral zebrafish. To explore the effects of combined use of venlafaxine and melatonin on depression-like zebrafish induced by reserpine. We tested the depressive behavior of adult zebrafish through a novel tank test, and evaluated the levels of serotonin (5-HT), dopamine (DA) and noradrenaline (NA) in zebrafish brain using enzyme-linked immunosorbent assay (ELISA), besides that the gene expression of serotonin transporters a (serta), dopamine transporters (dat) and norepinephrine transporters (net), vesicular monoamine transporter2 (vmat2) and monoamine oxidase (mao) were evaluated by qRT-PCR. The results showed that, compared with reserpine-only group, venlafaxine (VEN, 0.025 mg/L) and melatonin (MT, 1 µM) increased the parameters of exploration in the top of the tank and decreased freezing behavior significantly. Compared with reserpine-only group, the use of VEN combined with MT increased serotonin and norepinephrine levels significantly, while there was no obvious difference in dopamine content. The results of qRT-PCR showed that the use of VEN combined with MT significantly reduced the expression of serta and promoted the expression of vmat2, but had no significant effect on the expression of net, dat and mao. The results indicated that venlafaxine combined with melatonin showed more effective role to remedy the depressive symptoms in zebrafish, providing a reference for the clinical application of antidepressants.

The effects of fluorene-9-bisphenol on female zebrafish (Danio rerio) reproductive and exploratory behaviors.

Endocrine disruptor chemicals induce adverse effects to animals' development, reproduction and behavior in environment. We investigated the effects of fluorene-9-bisphenol (BHPF), one substitute of bisphenol A, on courtship behavior and exploratory behavior of adult zebrafish. Customized apparatus was used to evaluate courtship behavior. The result showed that the male spent less time with BHPF and anti-oestrogenic fulvestrant (FULV) treated female in region of approaching (ROA). Courtship index between BHPF-exposed female and male decreased. The body orientation of BHPF- and FULV-exposed female to male decreased. Furthermore, BHPF exposure downregulated the expression of genes related to estrogen receptor, steroidogenesis and upregulated oxidative stress related genes. It indicated that BHPF exposure interfered the preference of male and female in courtship, and induced detrimental effects on reproduction. BHPF treatment decreased locomotor activity and time spent in top, increased freezing bouts, and induced anxiety/depression-like behavior. The tyrosine hydroxylase in brain decreased under BHPF exposure. Here we showed the potential adverse effects of BHPF on reproduction and exploratory behaviors.

Melatonin protects embryonic development and maintains sleep/wake behaviors from the deleterious effects of fluorene-9-bisphenol in zebrafish (Danio rerio).

Environmental endocrine chemicals have various adverse effects on the development of vertebrates. Fluorene-9-bisphenol (BHPF), a substitute of bisphenol A (BPA), is widely used in commercial production. The effects of BHPF on development and behavior are unclear. Melatonin plays a protective role under many unfavorable conditions. In this study, we investigated the effects of BHPF on the development and behaviors of zebrafish and whether melatonin reverses effects induced by BHPF. Zebrafish embryos were exposed to 0.1, 10, or 1000 nmol/L BHPF with or without 1 µmol/L melatonin from 2 hours postfertilization to 6 days postfertilization. The results showed that 0.1 and 10 nmol/L BHPF had little effect on development. High-dose BHPF (1000 nmol/L) delayed the development, increased mortality and surface tension of embryonic chorions, caused aberrant expression of the key genes (ntl, shh, krox20, pax2, cmlc2) in early development detected by in situ hybridization, and damaged the CaP motor neurons, which were associated with locomotion ability detected by immunofluorescence. Melatonin addition reversed or weakened these adverse effects of BHPF on development, and melatonin alone increased surface tension as the effects of high-dose BHPF. However, all groups of BHPF exposure triggered insomnia-like behaviors, with increased waking activity and decreased rest behaviors. BHPF acted on the hypocretin (hcrt) system and upregulated the expression of sleep/wake regulators such as hcrt, hcrt receptor (hcrtr), arylalkylamine N-acetyltransferase-2 (aanat2). Melatonin recovered the alternation of sleep/wake behaviors induced by BHPF and restored abnormal gene expression to normal levels. This study showed that high-dose BHPF had adverse effects on early development and induced behavioral alternations. However, melatonin prevented BHPF-induced aberrant development and sleep/wake behaviors.

Impact of low-dose chronic exposure to Bisphenol A (BPA) on adult male zebrafish adaption to the environmental complexity: Disturbing the color preference patterns and reliving the anxiety behavior.

The extensive usage of xenobiotic endocrine disrupting chemicals (XEDCs), such as Bisphenol A (BPA), has created obvious threat to aquatic ecosystems worldwide. Although a comprehensive understanding of the adverse effect of BPA on behaviors and physiology have been proven, the potential impact of low-dose BPA on altering the basic ability of aquatic organism in adapting to the surrounded complex environment still remains elusive. In this research, we report that treatment of adult male zebrafish with chronic (7 weeks) low-dose (0.22 nM-2.2 nM) BPA, altered the ability in adapting the complex environment by disturbing the natural color preference patterns. In addition, chronic 50 ng/L (0.22 nM) BPA exposure alleviated the anxiety behavior of male zebrafish confronted with the novel environment by enhancing the preference towards light in the light/dark preference test. This phenotype was associated with less expression of serotonin (5-TH) in the hypothalamus and the down-regulation of tyrosine hydroxylase (TH) in brain tissues. As such, our results show that low-dose BPA remnant in surface waters altered zebrafish behavior that are known to have ecological and evolutionary consequences. HIGHLIGHTS: Here we reported that the impact of chronic low-dose BPA exposure on the basic capability of zebrafish to adapt to the environmental complexity. Specifically, BPA at low concentration, under the environmental safety level and 3000-fold lower than the accepted human daily exposure, interfered with the ability to discriminate color and alleviate anxiety induced by the novel environment, which finally altered the capability of male zebrafish to adapt to the environmental complexity. These findings revealed the ecological effect of low-dose BPA and regular BPA concentration standard are not necessarily safe. The result also provided the consideration of retuning the hazard concentration level of BPA.

Behavioural effect of low-dose BPA on male zebrafish: Tuning of male mating competition and female mating preference during courtship process.

The ubiquity of environmental pollution by endocrine disrupting chemicals (EDCs) such as bisphenol A (BPA) is progressively considered as a major threat to aquatic ecosystems worldwide. Numerous toxicological studies have proved that BPA are hazardous to aquatic environment, along with alterations in the development and physiology of aquatic vertebrates. However, generally, there is a paucity in knowledge of behavioural and physiological effects of BPA with low concentration, for example, 0.22 nM (50 ng/L) and 2.2 nM (500 ng/L). Here we show that treatment of adult male zebrafish (Danio rerio) with 7 weeks low-dose (0.22 nM-2.2 nM) BPA, resulted in alteration in histological structure of testis tissue and abnormality in expression levels of genes involved in testicular steroidogenesis. Furthermore, low-dose BPA treatment decreased the male locomotion during courtship; and was associated with less courtship behaviours to female but more aggressive behaviours to mating competitor. Interestingly, during the courtship test, we observed that female preferred control male to male under low-dose BPA exposure. Subsequently, we found that the ability of female to chose optimal mating male through socially mutual interaction and dynamics of male zebrafish, which was based on visual discrimination. In sum, our results shed light on the potential behavioural and physiological effect of low-dose BPA exposure on courtship behaviours of zebrafish, which could exert profound consequences on natural zebrafish populations.

The Difference between Anxiolytic and Anxiogenic Effects Induced by Acute and Chronic Alcohol Exposure and Changes in Associative Learning and Memory Based on Color Preference and the Cause of Parkinson-Like Behaviors in Zebrafish.

We describe an interdisciplinary comparison of the effects of acute and chronic alcohol exposure in terms of their disturbance of light, dark and color preferences and the occurrence of Parkinson-like behavior in zebrafish through computer visual tracking, data mining, and behavioral and physiological analyses. We found that zebrafish in anxiolytic and anxious states, which are induced by acute and chronic repeated alcohol exposure, respectively, display distinct emotional reactions in light/dark preference tests as well as distinct learning and memory abilities in color-enhanced conditional place preference (CPP) tests. Additionally, compared with the chronic alcohol (1.0%) treatment, acute alcohol exposure had a significant, dose-dependent effect on anxiety, learning and memory (color preference) as well as locomotive activities. Acute exposure doses (0.5%, 1.0%, and 1.5%) generated an "inverted V" dose-dependent pattern in all of the behavioral parameters, with 1.0% having the greatest effect, while the chronic treatment had a moderate effect. Furthermore, by measuring locomotive activity, learning and memory performance, the number of dopaminergic neurons, tyrosine hydroxylase expression, and the change in the photoreceptors in the retina, we found that acute and chronic alcohol exposure induced varying degrees of Parkinson-like symptoms in zebrafish. Taken together, these results illuminated the behavioral and physiological mechanisms underlying the changes associated with learning and memory and the cause of potential Parkinson-like behaviors in zebrafish due to acute and chronic alcohol exposure.

Protective effect of allicin against glycidamide-induced toxicity in male and female mice.

Acrylamide is known to be a neurotoxic, genotoxic, and carcinogenic compound. Glycidamide has a close relationship to the toxic mechanism of acrylamide. In order to explore the toxic mechanism of acrylamide, we further discussed the effects of oral administration of allicin on glycidamide-induced toxicity by determining the hematological parameters like AST, ALT, LDH, BUN, creatinine, ROS, and 8-OHdG, and biochemical parameters such as MDA, MPO, SOD, GST and GSH in the kidney, liver, brain and lung of male and female mice for the first time. We found that the same dose of glycidamide had more toxic effects and damage effects to the mice compared to the previous study of acrylamide. It could markedly increase the level of AST, ALT, LDH, BUN, ROS, 8-OHdG, MDA, MPO while decrease the SOD, GST and GSH. However, our data showed the oral administered allicin with a concentration of 5, 10, and 20 mg/kg b.w./day could significantly decrease the damage indexes of AST, ALT, LDH, BUN, ROS, 8-OHdG, MDA, and MPO, while increase the antioxidant indicators of SOD, GST and GSH. Thus allicin could be used as an effective dietary supplement for the chemoprevention of glycidamide genotoxicity internally, and to prevent the tissue damage and toxicity induced by glycidamide.

SiO2 nanoparticles change colour preference and cause Parkinson's-like behaviour in zebrafish.

With advances in the development of various disciplines, there is a need to decipher bio-behavioural mechanisms via interdisciplinary means. Here, we present an interdisciplinary study of the role of silica nanoparticles (SiO2-NPs) in disturbing the neural behaviours of zebrafish and a possible physiological mechanism for this phenomenon. We used adult zebrafish as an animal model to evaluate the roles of size (15-nm and 50-nm) and concentration (300 µg/mL and 1000 µg/mL) in SiO2-NP neurotoxicity via behavioural and physiological analyses. With the aid of video tracking and data mining, we detected changes in behavioural phenotypes. We found that compared with 50-nm nanosilica, 15-nm SiO2-NPs produced greater significant changes in advanced cognitive neurobehavioural patterns (colour preference) and caused potentially Parkinson's disease-like behaviour. Analyses at the tissue, cell and molecular levels corroborated the behavioural results, demonstrating that nanosilica acted on the retina and dopaminergic (DA) neurons to change colour preference and to cause potentially Parkinson's disease-like behaviour.

[The genetic screening of a dominant zebrafish mutant in long-term memory].

OBJECTIVE: To screen the learning and memory mutant from N-ethyl-N-nitrosourea (ENU) mutagenic zebrafish F1, and to get the new model animal to study the mechanism of learning and memory. METHODS: Zebrafish mutant was screened by inhibitory avoidance behavioral test and identified by the expression of gene c-fos with qRT-PCR. RESULTS: We isolated a zebrafish mutant related to learning and memory, fgt. In this fgt zebrafish mutant long-term memory was much lower than that in wild-type when tested at 24 h after training. The 24 h long-term memory in about half of fgt mutant F2 (13/30) were significantly lower than those in wild-type, and the others relatively normal. Compared with the expression in wild-type fishes, the expression of immediate-early genes (IEGs) c-fos in half of fgt mutant F2 (13/30) after exploring in a novel environment increased distinctly from the basal control levels statistically, and the others relatively normal, which were in accordance with the behavioral results. CONCLUSION: The zebrafish mutant fgt is a dominant mutant with defect in long-term memory.

Glycosyl-modified diporphyrins for in vitro and in vivo fluorescence imaging.

The application of probes for optical imaging is becoming popular as they have high safety and good biocompatibility. We prepared two kinds of glycosyl-modified diporphyrins, and their potentials as fluorescent probes were tested for the first time. After preparation of the glycosyl-modified porphyrin monomers, Ag-promoted coupling of the monomers was used to obtain glucose-modified porphyrin dimer (GPD) and lactose-modified porphyrin dimer (LPD). The strong interaction between the two porphyrin rings achieves red-shifted emission, and thus circumvents autofluorescence and light-scattering in biological samples. Although the glycosylation improves solubility, it also yielded selective attachment to cell membranes, and to chorions of early developmental-stage zebrafish. Patch-clamp experiments revealed the biocompatibility and low toxicity of GPD and LPD. Moreover, an in vivo imaging experiment provided direct evidence that zebrafish chorion contains sugar-binding proteins. The modification and derivatization make porphyrins potential bioimaging probes for specific optical imaging.

[Metaphase outcome of total hip arthroplasty with Zweymuller system in treating developmental dysplasia of the hip].

OBJECTIVE: To analyze the metaphase outcome of total hip arthroplasty with Zweymuller system and deepening acetabulum technique in treating DDH. METHODS: From Jan.1998 to Dec. 2004,56 patients (62 hips) with DDH (secondary osteoarthritis) were treated with total hip arthroplasty with Zweymuller system. There were 14 males (15 hips) and 42 females (47 hips) with an average age of 48.6 years,ranged from 30 to 67 years. All patients had pain of hip joint and functional disturbance before operation. Observation items included postoperative complications,imaging and function of hip joint. The function of hip joint was analyzed according to Harris scoring. RESULTS: All patients were followed up from 5 to 11 years with an average of 6.5 years. X-rays showed that the acetabular cup was in the position of true acetabulum, which combined tightly with the peripheral bone, the abduction angle of the acetabular cup was from 35 degrees to 45 degrees, introversion and extroversion of the femoral prosthesis was within 3 degrees, operated legs were shorter with a mean of (0.5 +/- 0.2) cm. The complications were as following:deep vein thrombosis in 20 cases,which were improved after thrombolysis;hip dislocation in 1 case,which was treated with reduction and immobilization for 3 weeks; ectopic ossification in 4 patients,all were Brook II type; no found infection or nerve injury. The Harris scoring was 87.4 +/- 3.5 postoperative,which was significant higher than that preoperative (43.2 +/- 6.7). CONCLUSION: The metaphase outcome of total hip arthroplasty with Zweymuller system and deepening acetabulum technique in treating DDH can obtain good result.

[Epidemiology of soil-transmitted nematode infections in central mountain area of Hainan province].

The epidemiology of soil-transmitted intestinal nematodes were observed in the central mountain area without anti-helminthic therapy from 1986 to 2008. The results showed that the overall prevalence decreased from 96.4% in 1986 to 35.7% in 2008. The prevalence of Ancylostoma duodenale, Ascaris lumbricoides and Trichuris trichiura decreased from 84.7%. 80.9%, 31.8% in 1986 to 32.5%, 0.3%, 4.2% in 2008, respectively. The proportion of light infection with Ancylostoma duodenale, Ascaris lumbricoides and Trichuris trichiura increased from 56.6%, 41.2%, and 66.9% in 1986 to 97.9%, 100%, and 83.7% in 2008, respectively. While that of heavy infection decreased from 6.8%, 11.9%, and 3.8% in 1986 all to zero in 2008. Water and toilet renovation, rural income increase and the improvement of sanitation and living conditions made the prevalence of soil-transmitted intestinal nematode decreased.

[Isolation, phylogenetic analysis and developmental expression parttern of AmphiRab23b in amphioxus].

The hedgehog (Hh) pathway plays an important role during the embryonic development and is related to the progression of cancers. Rab23 is a crucial functional molecule in Hh pathway. However, there is no report about amphioxus Rab23 up to now except the annotations of two isoforms in the genome of Florida lancelet (Branchiostoma floridae). Here a 2062 bp full-length cDNA sequence of the Rab23, AmphiRab23b, was isolated from Chinese amphioxus (Branchiostoma belcheri), which included the UTRs and an open reading frame of 714 bp, encoding a protein of 237 amino acids. Phylogenetic analysis suggested that AmphiRab23b falled outside the vertebrate clade. But sequence analysis indicated that this putative AmphiRab23b protein contained a specific Rab23_lke domain, which implied that Rab23 gene was functional conservative during evolution. And its developmental expression pattern showed that AmphiRab23b was expressed in the differentiating neural plate and alimentary canal, as the same as the expression pattern of the homologous vertebrate genes, which suggested that AmphiRab23b may function in the development of nervous system and alimentary canal.