Low temperature inhibits anthocyanin accumulation in strawberry fruit by activating FvMAPK3-induced phosphorylation of FvMYB10 and degradation of Chalcone Synthase 1.

Low temperature causes poor coloration of strawberry (Fragaria sp.) fruits, thus greatly reducing their commercial value. Strawberry fruits accumulate anthocyanins during ripening, but how low temperature modulates anthocyanin accumulation in plants remains largely unknown. We identified MITOGEN-ACTIVATED PROTEIN KINASE3 (FvMAPK3) as an important negative regulator of anthocyanin accumulation that mediates the poor coloration of strawberry fruits in response to low temperature. FvMAPK3 activity was itself induced by low temperature, leading to the repression of anthocyanin accumulation via two mechanisms. Activated FvMAPK3 acted as the downstream target of MAPK KINASE4 (FvMKK4) and SUCROSE NONFERMENTING1-RELATED KINASE2.6 (FvSnRK2.6) to phosphorylate the transcription factor FvMYB10 and reduce its transcriptional activity. In parallel, FvMAPK3 phosphorylated CHALCONE SYNTHASE1 (FvCHS1) to enhance its proteasome-mediated degradation. These results not only provide an important reference to elucidate the molecular mechanisms underlying low-temperature-mediated repression of anthocyanin accumulation in plants, but also offer valuable candidate genes for generating strawberry varieties with high tolerance to low temperature and good fruit quality.

Role of the PARP1/NF-κB Pathway in DNA Damage and Apoptosis of TK6 Cells Induced by Hydroquinone.

Hydroquinone(HQ) is a widely used industrial raw material and is a topical lightening product found in over-the-counter products. However, inappropriate exposure to HQ can pose certain health hazards. This study aims to explore the mechanisms of DNA damage and cell apoptosis caused by HQ, with a focus on whether HQ activates the nuclear factor-κB (NF-κB) pathway to participate in this process and to investigate the correlation between the NF-κB pathway activation and poly(ADP-ribose) polymerase 1(PARP1). Through various experimental techniques, such as DNA damage detection, cell apoptosis assessment, cell survival rate analysis, immunofluorescence, and nuclear-cytoplasmic separation, the cytotoxic effects of HQ were verified, and the activation of the NF-κB pathway was observed. Simultaneously, the relationship between the NF-κB pathway and PARP1 was verified by shRNA interference experiments. The results showed that HQ could significantly activate the NF-κB pathway, leading to a decreased cell survival rate, increased DNA damage, and cell apoptosis. Inhibiting the NF-κB pathway could significantly reduce HQ-induced DNA damage and cell apoptosis and restore cell proliferation and survival rate. shRNA interference experiments further indicated that the activation of the NF-κB pathway was regulated by PARP1. This study confirmed the important role of the NF-κB pathway in HQ-induced DNA damage and cell apoptosis and revealed that the activation of the NF-κB pathway was mediated by PARP1. This research provides important clues for a deeper understanding of the toxic mechanism of HQ.

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.

Fluorene-9-bisphenol acts on the gut-brain axis by regulating oxytocin signaling to disturb social behaviors in zebrafish.

Previous studies have identified the exposure to ubiquitous environmental endocrine disruptors may be a risk factor of neurological disorders. However, the effects of fluorene-9-bisphenol (BHPF) in environmental exposure concentrations associated with these disorders are poorly understood. In this study, classic light-dark and social behavior tests were performed on zebrafish larvae and adults exposed BHPF exposure to evaluate social behavioral disorders and the microbiota-gut-brain axis was assessed to reveal the potential mechanisms underlying the behavioral abnormalities observed. Our results demonstrated that zebrafish larvae exposed to an environmentally relevant concentration (0.1 nM) of BHPF for 7 days showed a diminished response to external environmental factors (light or dark). Zebrafish larvae exposed to BHPF for 7 days or adults exposed to BHPF for 30 days at 1 µM displayed significant behavioral inhibition and altered social behaviors, including social recognition, social preference, and social fear contagion, indicating autism-like behaviors were induced by the exposure. BHPF exposure reduced the distribution of Nissl bodies in midbrain neurons and significantly reduced 5-hydroxytryptamine signaling. Oxytocin (OXT) levels and expression of its receptor oxtra in the gut and brain were down-regulated by BHPF exposure. In addition, the expression levels of genes related to the excitation-inhibitory balance of synaptic transmission changed. Microbiomics revealed increased community diversity and altered abundance of some microflora, such as an elevation in Bacillota and Bacteroidota and a decline in Mycoplasmatota in zebrafish guts, which might contribute to the abnormal neural circuits and autism-like behaviors induced by BHPF. Finally, the rescue effect of exogenous OXT on social behavioral defects induced by BHPF exposure was verified in zebrafish, highlighting the crucial role of OXT signaling through gut-brain axis in the regulatory mechanisms of social behaviors affected by BHPF. This study contributes to understanding the effects of environmental BHPF exposure on neuropsychiatric disorders and attracts public attention to the health risks posed by chemicals in aquatic organisms. The potential mental disorders should be considered in the safety assessments of environmental pollutants.

Sensors for Robots.

Currently, robots are playing significant roles in industry [...].

Liquid-Driven Microinjection System for Precise Fundus Injection.

Microinjection is usually applied to the treatment of some retinal disorders, such as retinal vein cannulation and displaced submacular hemorrhage. Currently, the microinjection procedure is usually performed by using the viscous fluid control of a standard vitrectomy system, which applies a fixed air pressure through foot pedal activation. The injection process with the fixed pressure is uncontrollable and lacks feedback, the high flow rate of the injected drug may cause damage to the fundus tissue. In this paper, a liquid-driven microinjection system with a flow sensor is designed and developed specifically for fundus injection. In addition, a PID sliding mode control (SMC) method is proposed to achieve precise injection in the injection system. The experimental results of fundus simulation injection demonstrate that the microinjection system meets the requirements of fundus injection and reduces the impact of the injection process on the fundus tissue.

Melatonin protects zebrafish pancreatic development and physiological rhythms from sodium propionate-induced disturbances via the hypothalamic-pituitary-thyroid axis.

BACKGROUND: The widespread use of sodium propionate as a preservative in food may affect public health. We aimed to assess the effects of sodium propionate on circadian rhythms and pancreatic development in zebrafish and the possible underlying mechanisms. RESULTS: In this experiment, we analyzed the relationship between circadian rhythms and pancreatic development and then revealed the role of the thyroid endocrine system in zebrafish. The results showed that sodium propionate interfered with the rhythmic behavior of zebrafish, and altered the expression of important rhythmic genes. Experimental data revealed that pancreatic morphology and developmental genes were altered after sodium propionate exposure. Additionally, thyroid hormone levels and key gene expression associated with the hypothalamic-pituitary-thyroid axis were significantly altered. Melatonin at a concentration of 1 µmol L-1, with a mild effect on zebrafish, observably alleviated sodium propionate-induced disturbances in circadian rhythms and pancreatic development, as well as regulating the thyroid system. CONCLUSION: Melatonin, while modulating the thyroid system, significantly alleviates sodium propionate-induced circadian rhythm disturbances and pancreatic developmental disorders. We further revealed the deleterious effects of sodium propionate as well as the potential therapeutic effects of melatonin on circadian rhythm, pancreatic development and the thyroid system. © 2024 Society of Chemical Industry.

The deleterious effects and potential therapeutic strategy of fluorene-9-bisphenol on circadian activity and liver diseases in zebrafish and mice.

Increasing evidence indicates that disturbance of the clock genes, which leads to systemic endocrine perturbation, plays a crucial role in the pathogenesis of metabolic and liver diseases. Fluorene-9-bisphenol (BHPF) is utilized in the manufacturing of plastic materials but its biological effects on liver homeostasis remain unknown. The impacts and involved mechanisms of BHPF on the liver diseases, metabolism, and circadian clock were comprehensively studied by zebrafish and mouse models. The therapeutic effect of melatonin (MT) was also verified. Zebrafish and mouse models with either acute exposure (0.5 and 1 µmol/L, 1-4 days post-fertilization) or chronic oral exposure (0.5 and 50 mg/(kg·2 days), 30 days) were established with various BHPF concentrations. Herein, we identified a crucial role for estrogenic regulation in liver development and circadian locomotor rhythms damaged by BHPF in a zebrafish model. BHPF mice showed chaos in circadian activity through the imbalance of circadian clock component Brain and Muscle Aryl hydrocarbon receptor nuclear translocator-like 1 in the liver and brain. The liver sexual dimorphic alteration along with reduced growth hormone and estrogens played a critical role in damaged glucose metabolism, hepatic inflammation, and fibrosis induced by BHPF. Besides, sleep improvement by exogenous MT alleviated BHPF-related glucose metabolism and liver injury in mice. We proposed the pathogenesis of metabolic and liver disease resulting from BHPF and promising targeted therapy for liver metabolism disorders associated with endocrine perturbation chemicals. These results might play a warning role in the administration of endocrine-disrupting chemicals in everyday life and various industry applications.

Applicability of Scoring Calyculin A-Induced Premature Chromosome Condensation Objects for Dose Assessment Including for Radiotherapy Patients.

As an extension to a previous study, a linear calibration curve covering doses from 0 to 10 Gy was constructed and evaluated in the present study using calyculin A-induced premature chromosome condensation (PCC) by scoring excess PCC objects. The main aim of this study was to assess the applicability of this PCC assay for doses below 2 Gy that are critical for triage categorization. Two separate blind tests involving a total of 6 doses were carried out; 4 out of 6 dose estimates were within the 95% confidence limits (95% CL) with the other 2 just outside. In addition, blood samples from five cancer patients undergoing external beam radiotherapy (RT) were also analyzed, and the results showed whole-body dose estimates statistically comparable to the dicentric chromosome assay (DCA) results. This is the first time that calyculin A-induced PCC was used to analyze clinical samples by scoring excess objects. Although dose estimates for the pre-RT patient samples were found to be significantly higher than the mean value for the healthy donors and were also significantly higher than those obtained using DCA, all these pre-treatment patients fell into the same category as those who may have received a low dose (<1 Gy) and do not require immediate medical care during emergency triage. Additionally, for radiological accidents with unknown exposure scenario, PCC objects and rings can be scored in parallel for the assessment of both low- and high-dose exposures. In conclusion, scoring excess objects using calyculin A-induced PCC is confirmed to be another potential biodosimetry tool in radiological emergency particularly in mass casualty scenarios, even though the data need to be interpreted with caution when cancer patients are among the casualties.

International Comparison Exercise for Biological Dosimetry after Exposures with Neutrons Performed at Two Irradiation Facilities as Part of the BALANCE Project.

In the case of a radiological or nuclear event, biological dosimetry can be an important tool to support clinical decision-making. During a nuclear event, individuals might be exposed to a mixed field of neutrons and photons. The composition of the field and the neutron energy spectrum influence the degree of damage to the chromosomes. During the transatlantic BALANCE project, an exposure similar to a Hiroshima-like device at a distance of 1.5 km from the epicenter was simulated, and biological dosimetry based on dicentric chromosomes was performed to evaluate the participants ability to discover unknown doses and to test the influence of differences in neutron spectra. In a first step, calibration curves were established by irradiating blood samples with 5 doses in the range of 0-4 Gy at two different facilities in Germany (Physikalisch-Technische Bundesanstalt [PTB]) and the USA (the Columbia IND Neutron Facility [CINF]). The samples were sent to eight participating laboratories from the RENEB network and dicentric chromosomes were scored by each participant. Next, blood samples were irradiated with 4 blind doses in each of the two facilities and sent to the participants to provide dose estimates based on the established calibration curves. Manual and semiautomatic scoring of dicentric chromosomes were evaluated for their applicability to neutron exposures. Moreover, the biological effectiveness of the neutrons from the two irradiation facilities was compared. The calibration curves from samples irradiated at CINF showed a 1.4 times higher biological effectiveness compared to samples irradiated at PTB. For manual scoring of dicentric chromosomes, the doses of the test samples were mostly successfully resolved based on the calibration curves established during the project. For semiautomatic scoring, the dose estimation for the test samples was less successful. Doses >2 Gy in the calibration curves revealed nonlinear associations between dose and dispersion index of the dicentric counts, especially for manual scoring. The differences in the biological effectiveness between the irradiation facilities suggested that the neutron energy spectrum can have a strong impact on the dicentric counts.

Decabromodiphenyl ethane affects embryonic development by interfering with nuclear F-actin in zygotes and leads to cognitive and social disorders in offspring mice.

Decabromodiphenyl ethane (DBDPE) is a novel retardant. DBDPE is used in various flammable consumer products such as electronics, building materials, textiles, and children's toys. The presence of DBDPE in humans makes it extremely urgent to assess the health effects of DBDPE exposure. Here, we used female mice as an animal model to investigate the effects of DBDPE on embryonic development and offspring health. The results showed that 50 µg/kg bw/day of DBDPE exposure did not affect spindle rotation in oocytes after fertilization, but led to a decrease of pronuclei (PN) in zygotes. Further investigation found that DBDPE interferes with the self-assembly of F-actin in PN, resulting in PN reduction, DNA damage, and reduced expression of zygotic genome activating genes, and finally leading to abnormal embryonic development. More importantly, we found that maternal DBDPE exposure did not affect the growth and development of the first generation of offspring (F1) mice, but resulted in behavioral defects in F1 mice. Female F1 mice from DBDPE-exposed mothers exhibited increased motor activity and deficits in social behavior. Both female and male F1 mice from DBDPE-exposed mothers exhibited cognitive memory impairment. These results suggest that DBDPE has developmental toxicity on embryos and has a cross-generational interference effect. It is suggested that people should pay attention to the reproductive toxicity of DBDPE. In addition, it also provides a reference for studying the origin of neurological diseases and indicates that adult diseases caused by environmental pollutants may have begun in the embryonic stage.

Interaction of elastic waves in solids with quadratic and cubic nonlinearity.

This article investigates the interactions of two-plane waves in weakly nonlinear elastic solids containing quadratic and cubic nonlinearity. The analytical solutions for generated combined harmonic waves are derived using the Green's function approach applied to a generated system of quasi-linear equations of motion. Wave mixing solutions are obtained and include shape functions that permit closed-form solutions for a variety of interaction geometries. An explicit example is highlighted for a spherical interaction volume assuming isotropic elastic constants. Several parameters of the generated field after mixing are analyzed including resonant and nonresonant mixing, the role of interaction angle, and the frequencies of the two incident waves. Wave mixing offers the potential for sensing localized elastic nonlinearity and the present model can be used to help design experimental configurations.

Neuron Contact Detection Based on Pipette Precise Positioning for Robotic Brain-Slice Patch Clamps.

A patch clamp is the "gold standard" method for studying ion-channel biophysics and pharmacology. Due to the complexity of the operation and the heavy reliance on experimenter experience, more and more researchers are focusing on patch-clamp automation. The existing automated patch-clamp system focuses on the process of completing the experiment; the detection method in each step is relatively simple, and the robustness of the complex brain film environment is lacking, which will increase the detection error in the microscopic environment, affecting the success rate of the automated patch clamp. To address these problems, we propose a method that is suitable for the contact between pipette tips and neuronal cells in automated patch-clamp systems. It mainly includes two key steps: precise positioning of pipettes and contact judgment. First, to obtain the precise coordinates of the tip of the pipette, we use the Mixture of Gaussian (MOG) algorithm for motion detection to focus on the tip area under the microscope. We use the object detection model to eliminate the encirclement frame of the pipette tip to reduce the influence of different shaped tips, and then use the sweeping line algorithm to accurately locate the pipette tip. We also use the object detection model to obtain a three-dimensional bounding frame of neuronal cells. When the microscope focuses on the maximum plane of the cell, which is the height in the middle of the enclosing frame, we detect the focus of the tip of the pipette to determine whether the contact between the tip and the cell is successful, because the cell and the pipette will be at the same height at this time. We propose a multitasking network CU-net that can judge the focus of pipette tips in complex contexts. Finally, we design an automated contact sensing process in combination with resistance constraints and apply it to our automated patch-clamp system. The experimental results show that our method can increase the success rate of pipette contact with cells in patch-clamp experiments.

Robotic Intracellular Pressure Measurement Using Micropipette Electrode.

Intracellular pressure, a key physical parameter of the intracellular environment, has been found to regulate multiple cell physiological activities and impact cell micromanipulation results. The intracellular pressure may reveal the mechanism of these cells' physiological activities or improve the micro-manipulation accuracy for cells. The involvement of specialized and expensive devices and the significant damage to cell viability that the current intracellular pressure measurement methods cause significantly limit their wide applications. This paper proposes a robotic intracellular pressure measurement method using a traditional micropipette electrode system setup. First, the measured resistance of the micropipette inside the culture medium is modeled to analyze its variation trend when the pressure inside the micropipette increases. Then, the concentration of KCl solution filled inside the micropipette electrode that is suitable for intracellular pressure measurement is determined according to the tested electrode resistance-pressure relationship; 1 mol/L KCl solution is our final choice. Further, the measurement resistance of the micropipette electrode inside the cell is modeled to measure the intracellular pressure through the difference in key pressure before and after the release of the intracellular pressure. Based on the above work, a robotic measurement procedure of the intracellular pressure is established based on a traditional micropipette electrode system. The experimental results on porcine oocytes demonstrate that the proposed method can operate on cells at an average speed of 20~40 cells/day with measurement efficiency comparable to the related work. The average repeated error of the relationship between the measured electrode resistance and the pressure inside the micropipette electrode is less than 5%, and no observable intracellular pressure leakage was found during the measurement process, both guaranteeing the measurement accuracy of intracellular pressure. The measured results of the porcine oocytes are in accordance with those reported in related work. Moreover, a 90% survival rate of operated oocytes was obtained after measurement, proving limited damage to cell viability. Our method does not rely on expensive instruments and is conducive to promotion in daily laboratories.

Integrated analysis of transcriptome profiling of lncRNAs and mRNAs in livers of type 2 diabetes mellitus.

Long noncoding RNAs (lncRNAs) influence the progression of almost all human diseases, but the participation of lncRNAs in type 2 diabetes mellitus (T2DM) has not been fully elucidated. This study aimed to systematically compare the transcriptome profiling of lncRNAs and mRNAs in livers between patients with T2DM and controls, to identify key genes associated with T2DM pathogenesis, and to predict the underlying molecular mechanisms. As a result, a total of 1,512 differentially expressed (DE) lncRNAs and 1,923 DE mRNAs were identified through microarray analysis. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that multiple metabolic processes were dysregulated such as small molecule, organic acid, lipid, and branched chain amino acid metabolism. Protein-protein interaction network was constructed, and 10 hub mRNAs were identified, including EHHADH, ATM, ACOX1, PIK3R1, EGFR, UQCRFS1, HMGCL, UQCRC2, NDUFS3, and F2. RT-qPCR was conducted to verify the validity of microarray results. Then, coding-noncoding co-expression network and competing endogenous RNA (ceRNA) network were analyzed to predict the lncRNA-mRNA and lncRNA-miRNA-mRNA regulatory patterns. Subsequently, 10 key intermediating miRNAs in ceRNA networks with a node degree >80 were identified, including hsa-miR-5692a, hsa-miR-12136, hsa-miR-5680, hsa-miR-1305, hsa-miR-6833-5p, hsa-miR-7159-5p, hsa-miR-548as-3p, hsa-miR-6873-3p, hsa-miR-1290, and hsa-miR-4768-5p. In conclusion, this study evaluated the transcriptome profiling of lncRNAs and mRNAs in livers from patients with T2DM, with a value for understanding the molecular mechanism of disease pathogenesis and identifying effective biomarkers in clinical diagnosis.

The masculinization steroid milieu caused by fluorene-9-bisphenol disrupts sex-typical courtship behavior in female zebrafish (Danio rerio).

In vertebrates, the behavior of congenital sex differences between males and females is highly dependent on steroid signals and hormonal milieu. The presence of endocrine disrupting chemicals (EDCs) in the environment generally plays a similar role to sex hormones, so its interference with aquatic organism population stability can not be ignored and is worth studying. Fluorene-9-bisphenol (BHPF) has been clarified as an endocrine disruptor on organisms by several studies but its mechanism in perturbation of courtship behavior of female zebrafish is not clear. Here, we proposed an automated multi-zebrafish tracking method quantifying the courtship process and reported that zebrafish females exposed to BHPF, are not receptive to males but rather court females, and lose normal ovarian function with an altered sex steroid milieu. Our results showed that BHPF damaged 17ß-estradiol synthesis by down-regulation of sox3 and cyp19a1a, linking apoptosis with ovary development and female fecundity. The down-regulated expression of estrogen signaling through an estrogen receptor, esr2b, caused the induction of masculinization of female courtship behavior and sexual preference in zebrafish females after BHPF treatment. This process might be mediated by inhibiting the transcription of a neuropeptide B (npb) in the brain. Our study reveals that the estrogen signaling pathway may play an important role in classical courtship behavior and sexual preference of zebrafish. This study provided evidence that anti-estrogenic chemical exposure caused adverse effects on the regulation of the brain-gonad-estrogen axis of aquatic organisms, which should be of concern and highlighted the importance of controlling environmental contamination.

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.

A Machine Learning Method for Automated In Vivo Transparent Vessel Segmentation and Identification Based on Blood Flow Characteristics.

In vivo transparent vessel segmentation is important to life science research. However, this task remains very challenging because of the fuzzy edges and the barely noticeable tubular characteristics of vessels under a light microscope. In this paper, we present a new machine learning method based on blood flow characteristics to segment the global vascular structure in vivo. Specifically, the videos of blood flow in transparent vessels are used as input. We use the machine learning classifier to classify the vessel pixels through the motion features extracted from moving red blood cells and achieve vessel segmentation based on a region-growing algorithm. Moreover, we utilize the moving characteristics of blood flow to distinguish between the types of vessels, including arteries, veins, and capillaries. In the experiments, we evaluate the performance of our method on videos of zebrafish embryos. The experimental results indicate the high accuracy of vessel segmentation, with an average accuracy of 97.98%, which is much more superior than other segmentation or motion-detection algorithms. Our method has good robustness when applied to input videos with various time resolutions, with a minimum of 3.125 fps.

Cardiotoxicity of Zebrafish Induced by 6-Benzylaminopurine Exposure and Its Mechanism.

6-BA is a common plant growth regulator, but its safety has not been conclusive. The heart is one of the most important organs of living organisms, and the cardiogenesis process of zebrafish is similar to that of humans. Therefore, based on wild-type and transgenic zebrafish, we explored the development of zebrafish heart under 6-BA exposure and its mechanism. We found that 6-BA affected larval cardiogenesis, inducing defective expression of key genes for cardiac development (myl7, vmhc, and myh6) and AVC differentiation (bmp4, tbx2b, and notch1b), ultimately leading to weakened cardiac function (heart rate, diastolic speed, systolic speed). Acridine orange staining showed that the degree of apoptosis in zebrafish hearts was significantly increased under 6-BA, and the expression of cell-cycle-related genes was also changed. In addition, HPA axis assays revealed abnormally expressed mRNA levels of genes and significantly increased cortisol contents, which was also consistent with the observed anxiety behavior in zebrafish at 3 dpf. Transcriptional abnormalities of pro- and anti-inflammatory factors in immune signaling pathways were also detected in qPCR experiments. Collectively, we found that 6-BA induced cardiotoxicity in zebrafish, which may be related to altered HPA axis activity and the onset of inflammatory responses under 6-BA treatment.

Regulation of apoptosis by Pacific oyster Crassostrea gigas reveals acclimation strategy to CO2 driven acidification.

Ocean acidification (OA) has posed formidable threats to marine calcifiers. In response to elevated CO2 levels, marine calcifiers have developed multiple strategies to survive, such as taking advantage of apoptosis, but its regulation mechanism remains largely unknown. Here, we used the Pacific oyster Crassostrea gigas as model to understand the apoptotic responses and regulation mechanism at short- (7 d) to long-term (56 d) CO2 exposure (pH = 7.50). The apoptosis of hemocytes was significantly induced after short-term treatment (7-21 d) but was suppressed under long-term CO2 exposure (42-56 d). Similarly, caspase-3 and caspase-9 were also increased post short-term exposure and fell back to normal levels after long-term exposure. These data together indicated diverse regulation mechanisms of apoptosis through different exposure periods. Through analysis of the B-cell lymphoma 2 (Bcl-2) family mitochondrial apoptosis regulators, we showed that only CgBcl-XL's expression kept at high levels after 42- and 56-day CO2 exposure. CgBcl-XL shared sequence, and structural similarity with its mammalian counterpart, and knockdown of CgBcl-XL in hemocytes via RNA interference promoted apoptosis. The protein level of CgBcl-XL was significantly increased after long-term CO2 exposure (28-56 d), and its distribution in hemocytes became more concentrated and dense. Therefore, CgBcl-XL serves as an essential anti-apoptotic protein for tipping the balance of cell apoptosis, which may play a key role in survival under long-term CO2 exposure. These results reveal a potential adaptation strategy of oysters towards OA and the variable environment changes through the modulation of apoptosis.