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.

Mechanical effects of needle texture on acupoint tissue.

OBJECTIVE: This study aims to clarify how the stimulation of acupuncture points is achieved by needles with different surface texture during acupuncture; it also seeks to lessen injury at the insertion site and increase the therapeutic efficacy of acupuncture, by simulating the mechanical effects of various needle surface patterns on Zusanli (ST36) without changing the radius of acupuncture needles. METHODS: Five acupuncture needle models with different surface patterns, including the smooth needle, the lined needle, the ringed needle, the left-hand threaded needle and the right-hand threaded needle, and a layered model of the Zusanli acupoint were used to investigate how to reduce tissue damage and increase stimulation during acupuncture treatment. Puncturing of the skin as well as lifting-inserting and twisting needle manipulations were simulated using these models, and the degree of damage and force of stimulation caused by the acupuncture needles with different surface patterns during acupuncture were compared. RESULTS: The smooth needle and the lined needle caused the least tissue damage during insertion, while the left-hand threaded and the right-hand threaded needles caused the most damage. The ringed needle, the left-hand threaded needle and the right-hand threaded needle stimulated the acupoint tissue more during lifting-inserting manipulations, while the lined needle and the smooth needle produced less stimulation. The stimulation of the lined needle on the acupoint tissue was the largest during twisting manipulation, whereas the left-hand threaded needle and the right-hand threaded needle had smaller effects. In lifting-inserting and twisting manipulations, both the left-hand threaded needle and right-hand threaded needle provided more stimulation, but the torsion direction in which they produced better stimulation was the opposite. CONCLUSION: According to the simulation results, the ringed pattern enhances stimulation best in the lifting-inserting manipulation, whereas the lined pattern enhances stimulation best in the twisting manipulation. Both the right-hand and left-hand thread patterns have certain enhancing effects in these two operations. Taking the geometric properties of the pattern into account, the left-hand thread pattern and the right-hand thread pattern have the geometric characteristics of both the lined pattern and the ringed pattern. To conclude, a pattern perpendicular to the movement direction during the acupuncture manipulation creates more stimulation. These results have significance for future needle design. Please cite this article as: Sun MZ, Wang X, Li YC, Yao W, Gu W. Mechanical effects of needle texture on acupoint tissue. J Integr Med. 2023; 21(3): 254-267.

The food preservative sodium propionate induces hyperglycaemic state and neurological disorder in zebrafish.

Propionate is an effective mould inhibitor widely used as a food preservative. In this study, we used zebrafish to explore the adverse effects of long-term exposure to low concentrations of sodium propionate and the underlying molecular mechanisms (from larvae to adult). When exposed for 3 months, we found that blood glucose, total cholesterol, and triglyceride levels increased, and zebrafish developed a hyperglycaemic state. New tank test results showed depression in zebrafish reduced 5-hydroxytryptamine levels in the brain and damaged the dopamine system. At the same time, the results of the color preference test showed that zebrafish had cognitive impairments. In addition, Hypothalamic-Pituitary-Adrenal axis analysis revealed abnormal gene expression, increased cortisol levels, and reduced glucocorticoid receptor mRNA levels, which were consistent with depressive behavior. We also observed abnormal transcription of inflammatory and apoptotic factors. Overall, we found that chronic exposure to sodium propionate induces depressive symptoms. This may be related to the activation of the HPA axis by the hyperglycaemic state, thereby inducing inflammation and disrupting the dopaminergic system. In summary, this study provides theoretical and technical support for the overlap of the emotional pathogenesis associated with diabetes.

Exposure of zebrafish embryos to sodium propionate disrupts circadian behavior and glucose metabolism-related development.

Sodium propionate is widely used as a preservative in food. The widespread use of preservatives is known to cause both environmental and public health problems. This study aimed to investigate the effects of sodium propionate on the developmental behavior and glucose metabolism of zebrafish. Our results showed that sodium propionate had no significant effect on the embryonic morphological development of zebrafish embryos but changed the head eye area. Then we found sodium propionate disturbed the thigmotaxis behavior, impaired neural development. Moreover, changes in clock gene expression disrupted the circadian rhythm of zebrafish. Circadian genes regulated insulin sensitivity and secretion in various tissues. Then our results showed that the disorder of circadian rhythm in zebrafish affected glucose metabolism and insulin resistance, which damaged the development of retina. Therefore, the safety of propionate should be further evaluated.

Wavelength of a Turing-type mechanism regulates the morphogenesis of meshwork patterns.

The meshwork pattern is a significant pattern in the development of biological tissues and organs. It is necessary to explore the mathematical mechanism of meshwork pattern formation. In this paper, we found that the meshwork pattern is formed by four kinds of stalk behaviours: stalk extension, tip bifurcation, side branching and tip fusion. The Turing-type pattern underlying the meshwork pattern is a Turing spot pattern, which indicates that the Turing instability of the spot pattern promotes activator peak formation and then guides the formation of meshwork patterns. Then, we found that the Turing wavelength decreased in turn from tip bifurcation to side branching to tip fusion via statistical evaluation. Through the functional relationship between the Turing wavelength and model parameters ([Formula: see text] and [Formula: see text]), we found that parameters [Formula: see text] and [Formula: see text] had monotonic effects on the Turing wavelength and that parameter [Formula: see text] had nonmonotonic effects. Furthermore, we performed simulations of local meshwork pattern formation under variable model parameter values. The simulation results verified the corresponding relationship between the Turing wavelength and stalk behaviours and the functional relationship between the Turing wavelength and model parameters. The simulation results showed that the Turing wavelength regulated the meshwork pattern and that the small Turing wavelength facilitated dense meshwork pattern formation. Our work provides novel insight into and understanding of the formation of meshwork patterns. We believe that studies associated with network morphogenesis can benefit from our work.

Overexpression of steroid receptor coactivators alleviates hyperglycemia-induced endothelial cell injury in rats through activating the PI3K/Akt pathway.

Hyperglycemia is a major factor in vascular endothelial injury that finally leads to a cardiovascular event. Steroid receptor coactivators (SRCs) are a group of non-DNA binding proteins that induce structural changes in steroid receptors (nuclear receptors) critical for transcriptional activation. SRCs, namely, SRC-1, SRC-2, and SRC-3, are implicated in the regulation of vascular homeostasis. In this study we investigate the role of SRCs in hyperglycemia-induced endothelial injury. Aortic endothelial cells were prepared from normal and diabetic rats, respectively. Diabetic rats were prepared by injection of streptozotocin (50 mg/kg, i.p.). The expression levels of SRC-1 and SRC-3 were significantly decreased in endothelial cells from the diabetic rats. Similar phenomenon was also observed in aortic endothelial cells from the normal rats treated with a high glucose (25 mM) for 4 h or 8 h. The expression levels of SRC-2 were little affected by hyperglycemia. Overexpression of SRC-1 and SRC-3 in high glucose-treated endothelial cells significantly increased the cell viability, suspended cell senescence, and inhibited cell apoptosis compared with the control cells. We further showed that overexpression of SRC-1 and SRC-3 markedly suppressed endothelial injury through restoring nitric oxide production, upregulating the expression of antioxidant enzymes (SOD, GPX, and CAT), and activating the PI3K/Akt pathway. The beneficial effects of SRC-1 and SRC-3 overexpression were blocked by treatment with the PI3K inhibitor LY294002 (10 mM) or with the Akt inhibitor MK-2206 (100 nM). In conclusion, hyperglycemia decreased SRC-1 and SRC-3 expression levels in rat aortic endothelial cells. SRC-1 and SRC-3 overexpression might protect against endothelial injury via inhibition of oxidative stress and activation of PI3K/Akt pathway.

Automatic multiple zebrafish tracking based on improved HOG features.

As an excellent model organism, zebrafish have been widely applied in many fields. The accurate identification and tracking of individuals are crucial for zebrafish shoaling behaviour analysis. However, multi-zebrafish tracking still faces many challenges. It is difficult to keep identified for a long time due to fish overlapping caused by the crossings. Here we proposed an improved Histogram of Oriented Gradient (HOG) algorithm to calculate the stable back texture feature map of zebrafish, then tracked multi-zebrafish in a fully automated fashion with low sample size, high tracking accuracy and wide applicability. The performance of the tracking algorithm was evaluated in 11 videos with different numbers and different sizes of zebrafish. In the Right-tailed hypothesis test of Wilcoxon, our method performed better than idTracker, with significant higher tracking accuracy. Throughout the video of 16 zebrafish, the training sample of each fish had only 200-500 image samples, one-fifth of the idTracker's sample size. Furthermore, we applied the tracking algorithm to analyse the depression and hypoactivity behaviour of zebrafish shoaling. We achieved correct identification of depressed zebrafish among the fish shoal based on the accurate tracking results that could not be identified by a human.

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.

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.

Behavioural screening of zebrafish using neuroactive traditional Chinese medicine prescriptions and biological targets.

The mechanism of the therapeutic action of antidepressants remains uncertain in traditional Chinese medicine (TCM). In this study, we selected 7 classical TCM prescriptions and utilised an automatic video-tracking system to monitor the rest/wake behaviour of larval zebrafish at 4 days post-fertilisation (dpf) for 48 hours. We found that the curative effects of the prescriptions were dose-dependent. K-means clustering was performed according to the shared behavioural phenotypes of the zebrafish. The results revealed that the rest/wake behavioural profiles induced by the same class of prescriptions were similar. A correlation analysis was conducted between the TCM prescriptions and the known compounds. The results showed that the TCM prescriptions correlated well with some well-known compounds. Therefore, we predicted that they may share a similar mechanism of action. This paper describes the first study to combine TCM research with zebrafish rest/wake behaviour in vivo and presents a powerful approach for the discovery of the mechanism of action of TCM prescriptions.

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.

Cytokeratin 8 is increased in hepatitis C virus cells and its ectopic expression induces apoptosis of SMMC7721 cells.

AIM: To investigate cytokeratin 8 (CK8) overexpression during hepatitis C virus (HCV) infection and its pathogenesis, and the effect of ectopic CK8 expression on hepatoma cell lines. METHODS: We successfully established an in vitro HCV cell culture system (HCVcc) to investigate the different expression profiles of CK8 in Huh-7-HCV and Huh-7.5-HCV cells. The expression of CK8 at the mRNA level was determined by real-time polymerase chain reaction (RT-PCR). The expression of CK8 at the protein level was evaluated by Western blotting. We then constructed a eukaryotic expression combination vector containing the coding sequence of human full length CK8 gene. CK8 cDNA was amplified by reverse transcription-PCR and inserted into pEGFP-C1 and the positive clone pEGFP-CK8 was obtained. After confirming the sequence, the recombinant plasmid was transfected into SMMC7721 cells with lipofectamine2000 and CK8 expression was detected using inverted fluorescence microscopy, RT-PCR and Western blotting. Besides, we identified biological function of CK8 on SMMC7721 cells, including cell proliferation, cell cycle and apoptosis detection. RESULTS: RT-PCR showed that the expression level of CK8 in Huh-7-HCV and Huh-7.5-HCV cells was 2.88 and 2.95 times higher than in control cells. Western blot showed that CK8 expression in Huh-7-HCV and Huh-7.5-HCV cells was 2.53 and 3.26 times higher than that in control cells, respectively. We found that CK8 at mRNA and protein levels were both significantly increased in HCVcc. CK8 was up-regulated in SMMC7721 cells. CK8 expression at the mRNA level was significantly upregulated in SMMC7721/pEGFP-CK8 cells. CK8 expression in SMMC7721/ pEGFP-CK8 cells was 2.69 times higher than in SMMC7721 cells, and was 2.64 times higher than in SMMC7721/pEGFP-C1 cells. CK8 expression at the protein level in SMMC7721/pEGFP-CK8 cells was 2.46 times higher than in SMMC7721 cells, and was 2.29 times higher than in SMMC7721/pEGFP-C1 cells. Further analysis demonstrated that forced expression of CK8 slowed cell growth and induced apoptosis of SMMC7721 cells. CONCLUSION: CK8 up-regulation might have a functional role in HCV infection and pathogenesis, and could be a promising target for the treatment of HCV infection.

Multi-bit binary decoder based on Belousov-Zhabotinsky reaction.

It is known that Belousov-Zhabotinsky (BZ) reaction can be applied to chemical computation, e.g., image processing, computational geometry, logical computation, and so on. In the field of logical computation, some basic logic gates and basic combinational logic circuits, such as adder, counter, memory cell, have already been implemented in simulations or in chemical experiments. In this paper, we focus on another important combinational logic circuit, binary decoder. Integrating AND gate and NOT gate, we first design and implement a one-bit binary decoder through numerical simulation. Then we show that one-bit decoder can be extended to design two-bit, three-bit, or even higher bit binary decoders by a cascade method. The simulation results demonstrate the effectiveness of these devices. The chemical realization of decoders can guide the construction of more sophisticated functions based on BZ reaction; meanwhile, the cascade method can facilitate the design of other combinational logic circuits.

An assessment of the impact of SiO2 nanoparticles of different sizes on the rest/wake behavior and the developmental profile of zebrafish larvae.

In this study, zebrafish larvae are introduced as an in vivo platform to examine the neurotoxicity and developmental toxicity associated with continuous exposure to a concentration gradient of different sizes of SiO2 nanoparticles (15 nm and 50 nm diameter) to determine the dose effect and size effect of SiO2 nanoparticle (NP)-induced toxicity. Bovine serum albumin (BSA-V) is utilized as a stabilizing agent to prevent coagulation of the SiO2 nanoparticles. To the best of our knowledge, this study is the first to describe locomotor activity assays linking rest/wake behavioral profiles for the purpose of investigating the neurotoxicity of NPs. In addition, developmental toxicological endpoints including mortality, LC50 , malformation, and cartilaginous deformity are assessed. The results show a concentration-dependent increase in behavioral neurotoxicity, mortality, and malformation among larvae treated with the SiO2 nanoparticles of 15 nm and 50 nm. A comparison of the 15 nm and 50 nm NPs by K-means clustering analysis demonstrates that the 15 nm NPs have a greater neurotoxic effect than the 50 nm NPs, with the 50 nm NPs exhibiting greater developmental toxicity on the zebrafish larvae than the 15 nm NPs.

Prohibitin is overexpressed in Huh-7-HCV and Huh-7.5-HCV cells harboring in vitro transcribed full-length hepatitis C virus RNA.

BACKGROUND: Currently, up-regulated proteins and apoptosis in hepatitis C is a hot topic in exploring the pathogenic mechanism of Heptitis C Virus(HCV). Some recent studies shows that prohibitin is overexpressed in cells expressing HCV core proteins, and up-regulated prohibitin is also found in human hepatoma cell line HCC-M, lung cancer, prostate cancer, and other cancers. Prohibitin is an important member of the membrane protein superfamily, and it plays a role of molecular chaperones in mitochondrial protein stability. Meanwhile, it has a permissive action on tumor growth or acts as an oncosuppressor. Based on our previously established the in vitro HCV cell-culture system (HCVcc), here we aimed to investigate the different expression profiles of prohibitin in Huh-7-HCV and Huh-7.5-HCV cells METHODS: The total cellular RNA of Huh-7, Huh-7.5, Huh-7-HCV and Huh-7.5-HCV cells were extracted, and then the first-strand cDNA was reversely transcribed. The expression of prohibitin at the mRNA level was assessed by real-time PCR with GAPDH as the control. Furthermore, the expression of prohibitin at the protein level was evaluated by western blot with GAPDH as an internal control. RESULTS: Our results of real-time PCR showed that the mRNA expression level of prohibitin in Huh-7-HCV cells was 2.09 times higher than that in Huh-7 cells, while, the mRNA level of prohibitin in Huh-7.5-HCV cells was 2.25 times higher than that in Huh-7.5 cells. The results of western blot showed that the protein expression level of prohibitin in Huh-7-HCV cells was 2.38 times higher than that in Huh-7 cells, while the protein expression of prohibitin in Huh-7.5-HCV cells was 2.29 times higher than that in Huh-7.5 cells. CONCLUSIONS: The expression of prohibitin was relatively high in Huh-7-HCV and Huh-7.5-HCV cells harboring in vitro transcribed full-length HCV RNA.

Inhibitory effect of emodin and Astragalus polysaccharide on the replication of HBV.

AIM: To evaluate the anti-viral effect of emodin plus Astragalus polysaccharide (APS) in hepatitis B virus (HBV) transgenic mice. METHODS: Sixty HBV transgenic mice (HBV TGM) whose weight varied between 18 and 24 g were randomly divided into 3 groups, with 20 mice in each group. Group A was the normal control, where the mice were treated with physiological saline; group B was the positive control where the mice were treated with lamivudine solution (100 mL/kg per day). Group C was the experimental group where the mice were treated with physiological saline containing emodin and APS (57.59 mg/kg per day and 287.95 mg/kg per day, respectively). The mice were treated daily for 3 wk. After 1 wk recovery time, the mice were sacrificed and serum as well as liver tissues were collected for ELISA and histological examination. RESULTS: After 21 d treatment, HBV DNA levels in group B and group C significantly declined when compared with group A (P < 0.05). However, a significant increase in HBV DNA content was observed in group B, whereas this phenomenon was not observed in group C. A reduction in the contents of HBsAg, HBeAg and HBcAg in the mice from group B and C was observed when compared with group A. CONCLUSION: Emodin and APS have a weak but persistent inhibitory effect on HBV replication in vivo, which may function as a supplementary modality in the treatment of hepatitis B infection.