Toxicity evaluation of neonicotinoids to earthworm (Eisenia fetida) behaviors by a novel locomotion tracking assay.

Pesticides are substances used for controlling, preventing, and repelling pests in agriculture. Among them, neonicotinoids have become the fastest-growing class of insecticides because of their efficiency in targeting pests. They work by strongly binding to nicotinic acetylcholine receptors (nAChRs) in the central nervous system of insects, leading to receptor blockage, paralysis, and death. Despite their selectivity for insects, these substances may be hazardous to non-target creatures, including earthworms. Although earthworms may be invasive in some regions like north America, they contribute to the development of soil structure, water management, nutrient cycling, pollution remediation, and cultural services, positively impacting the environment, particularly in the soil ecosystem. Thus, this study aimed to develop a novel earthworm behavior assay since behavior is a sensitive marker for toxicity assay, and demonstrated its application in evaluating the toxicity of various neonicotinoids. Here, we exposed Eisenia fetida to 1 and 10 ppb of eight neonicotinoids (acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram pestanal, thiacloprid, thiametoxam, and sulfoxaflor) for 3 days to observe their behavior toxicities. Overall, all of the neonicotinoids decreased their locomotion, showed by a reduction of average speed by 24.94-68.63% and increment in freezing time movement ratio by 1.51-4.25 times, and altered their movement orientation and complexity, indicated by the decrement in the fractal dimension value by 24-70%. Moreover, some of the neonicotinoids, which were acetamiprid, dinotefuran, imidacloprid, nitenpyram, and sulfoxaflor, could even alter their exploratory behaviors, which was shown by the increment in the time spent in the center area value by 6.94-12.99 times. Furthermore, based on the PCA and heatmap clustering results, thiametoxam was found as the neonicotinoid that possessed the least pronounced behavior toxicity effects among the tested pesticides since these neonicotinoid-treated groups in both concentrations were grouped in the same major cluster with the control group. Finally, molecular docking was also conducted to examine neonicotinoids' possible binding mechanism to Acetylcholine Binding Protein (AChBP), which is responsible for neurotransmission. The molecular docking result confirmed that each of the neonicotinoids has a relatively high binding energy with AChBP, with the lowest binding energy was possessed by thiametoxam, which consistent with its relatively low behavior toxicities. Thus, these molecular docking results might hint at the possible mechanism behind the observed behavior alterations. To sum up, the present study demonstrated that all of the neonicotinoids altered the earthworm behaviors which might be due to their ability to bind with some specific neurotransmitters and the current findings give insights into the toxicities of neonicotinoids to the environment, especially animals in a soil ecosystem.

Assessments of carbon nanotubes toxicities in zebrafish larvae using multiple physiological and molecular endpoints.

In recent years, carbon nanotubes (CNTs) have become one of the most promising materials for the technology industry. However, due to the extensive usage of these materials, they may be released into the environment, and cause toxicities to the organism. Here, their acute toxicities in zebrafish embryos and larvae were evaluated by using various assessments that may provide us with a novel perspective on their effects on aquatic animals. Before conducting the toxicity assessments, the CNTs were characterized as multiwall carbon nanotubes (MWCNTs) functionalized with hydroxyl and carboxyl groups, which improved their solubility and dispersibility. Based on the results, abnormalities in zebrafish behaviors were observed in the exposed groups, indicated by a reduction in tail coiling frequency and alterations in the locomotion as the response toward photo and vibration stimuli that might be due to the disruption in the neuromodulatory system and the formation of reactive oxygen species (ROS) by MWCNTs. Next, based on the respiratory rate assay, exposed larvae consumed more oxygen, which may be due to the injuries in the larval gill by the MWCNTs. Finally, even though no irregularity was observed in the exposed larval cardiac rhythm, abnormalities were shown in their cardiac physiology and blood flow with significant downregulation in several cardiac development-related gene expressions. To sum up, although the following studies are necessary to understand the exact mechanism of their toxicity, the current study demonstrated the environmental implications of MWCNTs in particularly low concentrations and short-term exposure, especially to aquatic organisms.

Evaluation of Tacrolimus' Adverse Effects on Zebrafish in Larval and Adult Stages by Using Multiple Physiological and Behavioral Endpoints.

Tacrolimus (FK506) is a common immunosuppressant that is used in organ transplantation. However, despite its importance in medical applications, it is prone to adverse side effects. While some studies have demonstrated its toxicities to humans and various animal models, very few studies have addressed this issue in aquatic organisms, especially zebrafish. Here, we assessed the adverse effects of acute and chronic exposure to tacrolimus in relatively low doses in zebrafish in both larval and adult stages, respectively. Based on the results, although tacrolimus did not cause any cardiotoxicity and respiratory toxicity toward zebrafish larvae, it affected their locomotor activity performance in light-dark locomotion tests. Meanwhile, tacrolimus was also found to slightly affect the behavior performance, shoaling formation, circadian rhythm locomotor activity, and color preference of adult zebrafish in a dose-dependent manner. In addition, alterations in the cognitive performance of the fish were also displayed by the treated fish, indicated by a loss of short-term memory. To help elucidate the toxicity mechanism of tacrolimus, molecular docking was conducted to calculate the strength of the binding interaction between tacrolimus to human FKBP12. The results showed a relatively normal binding affinity, indicating that this interaction might only partly contribute to the observed alterations. Nevertheless, the current research could help clinicians and researchers to further understand the toxicology of tacrolimus, especially to zebrafish, thus highlighting the importance of considering the toxicity of tacrolimus prior to its usage.

A comprehensive painkillers screening by assessing zebrafish behaviors after caudal fin amputation.

Recently, the usage of zebrafish for pain studies has increased in the past years, especially due to its robust pain-stimulated behaviors. Fin amputation has been demonstrated to induce a noxious response in zebrafish. However, based on the prior study, although lidocaine, the most used painkiller in zebrafish, has been shown to ameliorate amputated zebrafish behaviors, it still causes some prolonged effects. Therefore, alternative painkillers are always needed to improve the treatment quality of fin-amputated zebrafish. Here, the effects of several analgesics in recovering zebrafish behaviors post-fin amputation were evaluated. From the results, five painkillers were found to have potentially beneficial effects on amputated fish behaviors. Overall, these results aligned with their binding energy level to target proteins of COX-1 and COX-2. Later, based on their sub-chronic effects on zebrafish survivability, indomethacin, and diclofenac were further studied. This combination showed a prominent effect in recovering zebrafish behaviors when administered orally or through waterborne exposure, even with lower concentrations. Next, based on the ELISA in zebrafish brain tissue, although some changes were found in the treated group, no statistical differences were observed in most of the tested biomarkers. However, since heatmap clustering showed a similar pattern between biochemical and behavior endpoints, the minor changes in each biomarker may be sufficient in changing the fish behaviors.

Using DeepLabCut for markerless cardiac physiology and toxicity estimation in water fleas (Daphnia magna).

Daphnia magna is one species of water flea that has been used for a long time for ecotoxicity studies. In addition, Daphnia has a myogenic heart that is very useful for cardiotoxicity studies. Previous attempts to calculate the cardiac parameter endpoints in Daphnia suffer from the drawback of tedious operation and high variation due to manual counting errors. Even the previous method that utilized deep learning to help the process suffer from either overestimation of parameters or the need for specialized equipment to perform the analysis. In this study, we utilized DeepLabCut software previously used for animal pose tracking and demonstrated that ResNet_152 was the best fit for training the network. The trained network also showed comparable results with ImageJ and Kymograph, which was mostly done manually. In addition to that, several macro scripts in either Excel or Python format were developed to help summarize the data for faster analysis. The trained network was then challenged to analyze the potential cardiotoxicity of imidacloprid and pendimethalin in D. magna, and it showed that both pesticides cause alteration in their cardiac performance. Overall, this method provides a simple and automatic method to analyze the cardiac performance of Daphnia by utilizing DeepLabCut. The method proposed in this paper can contribute greatly to scientists conducting fast and accurate cardiotoxicity measurements when using Daphnia as a model.

Using crayfish behavior assay as a simple and sensitive model to evaluate potential adverse effects of water pollution: Emphasis on antidepressants.

The freshwater crayfish, Procambarus clarkii is an excellent aquatic animal model that is highly adaptable and tolerant. P. clarkii is widely used as a toxicity model to study various pharmaceutical exposure. This animal model has complex behavioral traits and is considered sensitive to environmental changes, making it an excellent candidate to study psychoactive drugs based on a behavioral approach. However, up to now, most behavioral studies on crayfish use manual observation and scoring that require panelists. In this study, we aim to develop an automation pipeline to analyze crayfish behavior automatically. We use a deep-learning approach to label body parts in multiple crayfish, and based on the trajectory results, the intra- or inter-individual crayfish were calculated. Reliable and fast results of several behavior endpoints in multiple crayfish were retrieved. We then validated the detection performance of numerous crayfish in specific gender groups (male-male and female-female). Based on the result, the male crayfish displayed significantly higher aggression than females. We also tested the antidepressant exposure on this animal model to evaluate the psychoactive effects of this drug. As male crayfish display more distinct agonistic behavior than females, we exposed them to sertraline (SRT) 1 ppb for 7 and 14 days. It was revealed that sertraline was able to alter several behavioral endpoints in crayfish. Significant increases in extend claw ratio, total distance moved, average speed, and rapid movement were displayed in sertraline-exposed crayfish but decreased interaction time and longest interaction time. In addition, SRT 14 days exposure could atler the aggressiveness and bold behavior In the present method, DeepLabCut (DLC) has been utilized to analyze the locomotion behavior of multiple crayfish. This established method provides rapid and accurate ecotoxicity measurements using freshwater crayfish, which beneficient and applicable for environmental research.

The Effect of the Pyrethroid Pesticide Fenpropathrin on the Cardiac Performance of Zebrafish and the Potential Mechanism of Toxicity.

Fenpropathrin, a pyrethroid insecticide, has been widely used for many years in agricultural fields. It works by disturbing the voltage-gated sodium channel, leading to paralysis and the death of the target animal. While past studies have focused on neurodegeneration following fenpropathrin poisoning in humans, relatively few pieces of research have examined its effect on other peripheral organs. This study successfully investigated the potential toxicity of fenpropathrin on the cardiovascular system using zebrafish as an animal model. Zebrafish larvae exposed to varying doses of fenpropathrin underwent an evaluation of cardiac physiology by measuring the heart rate, stroke volume, cardiac output, and shortening fraction. The blood flow velocity and the dorsal aorta diameter were also measured to assess the impact of fenpropathrin exposure on the vascular system. Furthermore, molecular docking was performed to evaluate the pesticide binding affinity to various proteins associated with the cardiovascular system, revealing the potential mechanism of the fenpropathrin cardiotoxic effect. The findings demonstrated a significant dose-dependent increase in the heart rate stroke volume, cardiac output, shortening fraction, and ejection fraction of zebrafish larvae after 24 h of acute treatment with fenpropathrin. Additionally, zebrafish treated at a concentration of 1 ppm exhibited significantly larger blood vessels in diameter and an increased blood flow velocity compared to the control group. According to molecular docking, fenpropathrin showed a high affinity for various voltage-gated sodium channels like scn1lab, cacna1sb, and clcn3. Finally, from the results, we found that fenpropathrin caused cardiomegaly, which may have been induced by the voltage-gated sodium channel disruption. This study highlights the significant disruption of fenpropathrin in the cardiovascular system and emphasizes the need for further research on the health implications of this pesticide.

Investigating Potential Cardiovascular Toxicity of Two Anti-Leukemia Drugs of Asciminib and Ponatinib in Zebrafish Embryos.

BCR-ABL, a fusion protein kinase, is a druggable target exclusively expressed in patients with chronic myeloid leukemia (CML). Several anti-leukemia medicines targeting this protein have been developed in recent years. However, therapeutic options are limited for CML patients bearing multiple BCR-ABL1 mutations. Ponatinib (PON), a potent tyrosinase inhibitor, was one of the approved drugs for managing BCR-ABL1 T315I mutant disease. However, treatment of patients with PON reported severe side effects related to cardiovascular events. Asciminib (ASC) was the first allosteric inhibitor approved to target the myristoyl pocket of BCR-ABL protein to inhibit protein activity. The different mechanism of inhibition opens the possibility of co-exposure with both medicines. Reports on cardiovascular side effects due to the combination use of PON + ASC in pre-clinical and clinical studies are minimal. Thus, this study aimed to observe the potential cardiovascular-related side effect after co-exposure to ASC and PON using zebrafish as an animal model. In this study, zebrafish were acutely exposed to both compounds. The cardiovascular physiology parameters and gene expression related to cardiovascular development were evaluated. We demonstrate that combining ASC with PON at no observed effect concentration (NOEC) did not cause any significant change in the cardiac performance parameter in zebrafish. However, a significant increase in nkx2.5 expression level and a substantial decrease in blood flow velocity were recorded, suggesting that combining these compounds at NOEC can cause mild cardiovascular-related side effects.

OpenBloodFlow: A User-Friendly OpenCV-Based Software Package for Blood Flow Velocity and Blood Cell Count Measurement for Fish Embryos.

The transparent appearance of fish embryos provides an excellent assessment feature for observing cardiovascular function in vivo. Previously, methods to conduct vascular function assessment were based on measuring blood-flow velocity using third-party software. In this study, we reported a simple software, free of costs and skills, called OpenBloodFlow, which can measure blood flow velocity and count blood cells in fish embryos for the first time. First, videos captured by high-speed CCD were processed for better image stabilization and contrast. Next, the optical flow of moving objects was extracted from the non-moving background in a frame-by-frame manner. Finally, blood flow velocity was calculated by the Gunner Farneback algorithm in Python. Data validation with zebrafish and medaka embryos in OpenBloodFlow was consistent with our previously published ImageJ-based method. We demonstrated consistent blood flow alterations by either OpenBloodFlow or ImageJ in the dorsal aorta of zebrafish embryos when exposed to either phenylhydrazine or ractopamine. In addition, we validated that OpenBloodFlow was able to conduct precise blood cell counting. In this study, we provide an easy and fully automatic programming for blood flow velocity calculation and blood cell counting that is useful for toxicology and pharmacology studies in fish.

Ractopamine at the Center of Decades-Long Scientific and Legal Disputes: A Lesson on Benefits, Safety Issues, and Conflicts.

Ractopamine (RAC) is a synthetic phenethanolamine, ß-adrenergic agonist used as a feed additive to develop leanness and increase feed conversion efficiency in different farm animals. While RAC has been authorized as a feed additive for pigs and cattle in a limited number of countries, a great majority of jurisdictions, including the European Union (EU), China, Russia, and Taiwan, have banned its use on safety grounds. RAC has been under long scientific and political discussion as a controversial antibiotic as a feed additive. Here, we will present significant information on RAC regarding its application, detection methods, conflicts, and legal divisions that play a major role in controversial deadlock and why this issue warrants the attention of scientists, agriculturists, environmentalists, and health advocates. In this review, we highlight the potential toxicities of RAC on aquatic animals to emphasize scientific evidence and reports on the potentially harmful effects of RAC on the aquatic environment and human health.

Acute and Chronic Effects of Fin Amputation on Behavior Performance of Adult Zebrafish in 3D Locomotion Test Assessed with Fractal Dimension and Entropy Analyses and Their Relationship to Fin Regeneration.

The fin is known to play an important role in swimming for many adult fish, including zebrafish. Zebrafish fins consist of paired pectoral and pelvic with unpaired dorsal, anal, and caudal tail fins with specific functions in fish locomotion. However, there was no study comparing the behavior effects caused by the absence of each fin. We amputated each fin of zebrafish and evaluated their behavior performance in the 3D locomotion test using fractal dimension and entropy analyses. Afterward, the behavior recovery after the tail fin amputation was also evaluated, together with the fin regeneration process to study their relationship. Finally, we conducted a further study to confirm whether the observed behavior alterations were from pain elicited by fin amputation procedure or not by using lidocaine, a pain-relieving drug. Amputation in the caudal fin resulted in the most pronounced behavior alterations, especially in their movement complexity. Furthermore, we also found that their behavior was fully recovered before the caudal fin was fully regenerated, indicating that these behavioral changes were not majorly due to a mechanical change in tail length; instead, they may come from pain elicited from the fin amputation, since treatment with lidocaine could ameliorate the behavioral effects after the amputation procedure. However, lidocaine did not accelerate the behavior recovery process; instead, it caused the fishes to display some slight side effects. This study highlights the potential moderate severity of fin amputation in zebrafish and the importance of analgesia usage. However, side effects may occur and need to be considered since fin amputation is routinely conducted for various research, especially genomic screening.

Alpinumisoflavone against cancer pro-angiogenic targets: In silico, In vitro, and In ovo evaluation.

BACKGROUND: Breast cancer is currently the world's most predominant malignancy. In cancer progression, angiogenesis is a requirement for tumor growth and metastasis.Alpinumisoflavone (AIF), a bioactive isoflavonoid, exhibited good binding affinity with the angiogenesis pathway's druggable target through molecular docking. OBJECTIVES: To confirm AIF's angiogenesis inhibitory activity, cytotoxic potential toward breast cancer cells, and druggability. METHODS: Antiangiogenic activity was evaluated in six pro-angiogenic proteins in vitro, duck chorioallantoic membrane (CAM) in ovo, molecular docking and druggability in silico. RESULTS: Findings showed that AIF significantly inhibited (p = < 0.001) the HER2(IC50 = 2.96 µM), VEGFR-2(IC50 = 4.80 µM), MMP-9(IC50 = 23.00 µM), FGFR4(IC50 = 57.65 µM), EGFR(IC50 = 92.06 µM) and RET(IC50 = > 200 µM) activity in vitro.AIF at 25 µM-200 µM significantly inhibited (p = < 0.001) the total number of branch points (IC50 = 14.25 µM) and mean length of tubule complexes (IC50 = 3.52 µM) of duck CAM comparable (p = > 0.001) with the positive control 200 µM celecoxib on both parameters.AIF inhibited the growth of the estrogen-receptor-positive (ER +) human breast cancer cells (MCF-7) by 44.92 ± 1.79% at 100 µM while presenting less toxicity to human dermal fibroblast neonatal (HDFn) normal cells.The positive control 100 µM doxorubicin showed 86.66 ± 0.93% and 92.97 ± 1.27% inhibition with MCF-7 (IC50 = 3.62 µM) and HDFn, (IC50 = 27.16 µM) respectively.In docking, AIF has the greatest in silico binding affinity on HER2 (-10.9 kcal/mol) among the key angiogenic molecules tested. In silico rat oral LD50 calculation indicates that AIF is moderate to slightly toxic at 146.4 mg/kg with 1.1 g/kg and 20.1 mg/kg upper and lower 95% confidence limits. Lastly, it sufficiently complies with Lipinski's, Veber's, Egan's, Ghose's, and Muegge's Rule, supporting its oral drug-like property. CONCLUSION: This study revealed that AIF possesses characteristics of a phytoestrogen compound with significant binding affinity, inhibitory activity against pro-angiogenic proteins, and cytotoxic potential against ER + breast cancer cells.The acceptable and considerable safety and drug-likeness profiles of AIF are worthy of further confirmation in vivo and advanced pre-clinical studies so that AIF can be elevated as a promising molecule for breast cancer therapy.

Toxicity Assessment of an Anti-Cancer Drug of p-Toluene Sulfonamide in Zebrafish Larvae Based on Cardiovascular and Locomotion Activities.

p-Toluene sulfonamide (p-TSA), a small molecular drug with antineoplastic activity is widely gaining interest from researchers because of its pharmacological activities. In this study, we explored the potential cardio and neural toxicity of p-TSA in sublethal concentrations by using zebrafish as an in vivo animal model. Based on the acute toxicity assay, the 96hr LC50 was estimated as 204.3 ppm, suggesting the overall toxicity of p-TSA is relatively low in zebrafish larvae. For the cardiotoxicity test, we found that p-TSA caused only a minor alteration in treated larvae after no overall significant alterations were observed in cardiac rhythm and cardiac physiology parameters, as supported by the results from expression level measurements of several cardiac development marker genes. On the other hand, we found that acute p-TSA exposure significantly increased the larval locomotion activity during the photomotor test while prolonged exposure (4 days) reduced the locomotor startle reflex activities in zebrafish. In addition, a higher respiratory rate and blood flow velocity was also observed in the acutely treated fish groups compared to the untreated group. Finally, by molecular docking, we found that p-TSA has a moderate binding affinity to skeletal muscle myosin II subfragment 1 (S1), ATPase activity, actin- and Ca2+-stimulated myosin S1 ATPase, and v-type proton ATPase. These binding interactions between p-TSA and proteins offer insights into the potential molecular mechanism of action of p-TSA on observed altered responses toward photo and vibration stimuli and minor altered vascular performance in the zebrafish larvae.

Using DeepLabCut as a Real-Time and Markerless Tool for Cardiac Physiology Assessment in Zebrafish.

DeepLabCut (DLC) is a deep learning-based tool initially invented for markerless pose estimation in mammals. In this study, we explored the possibility of adopting this tool for conducting markerless cardiac physiology assessment in an important aquatic toxicology model of zebrafish (Danio rerio). Initially, high-definition videography was applied to capture heartbeat information at a frame rate of 30 frames per second (fps). Next, 20 videos from different individuals were used to perform convolutional neural network training by labeling the heart chamber (ventricle) with eight landmarks. Using Residual Network (ResNet) 152, a neural network with 152 convolutional neural network layers with 500,000 iterations, we successfully obtained a trained model that can track the heart chamber in a real-time manner. Later, we validated DLC performance with the previously published ImageJ Time Series Analysis (TSA) and Kymograph (KYM) methods. We also evaluated DLC performance by challenging experimental animals with ethanol and ponatinib to induce cardiac abnormality and heartbeat irregularity. The results showed that DLC is more accurate than the TSA method in several parameters tested. The DLC-trained model also detected the ventricle of zebrafish embryos even in the occurrence of heart abnormalities, such as pericardial edema. We believe that this tool is beneficial for research studies, especially for cardiac physiology assessment in zebrafish embryos.

Evaluation of Locomotion Complexity in Zebrafish after Exposure to Twenty Antibiotics by Fractal Dimension and Entropy Analysis.

Antibiotics are extensively used in aquaculture to prevent bacterial infection and the spread of diseases. Some antibiotics have a relatively longer half-life in water and may induce some adverse effects on the targeted fish species. This study analyzed the potential adverse effects of antibiotics in zebrafish at the behavioral level by a phenomic approach. We conducted three-dimensional (3D) locomotion tracking for adult zebrafish after acute exposure to twenty different antibiotics at a concentration of 100 ppb for 10 days. Their locomotor complexity was analyzed and compared by fractal dimension and permutation entropy analysis. The dimensionality reduction method was performed by combining the data gathered from behavioral endpoints alteration. Principal component and hierarchical analysis conclude that three antibiotics: amoxicillin, trimethoprim, and tylosin, displayed unique characteristics. The effects of these three antibiotics at lower concentrations (1 and 10 ppb) were observed in a follow-up study. Based on the results, these antibiotics can trigger several behavioral alterations in adult zebrafish, even in low doses. Significant changes in locomotor behavioral activity, such as total distance activity, average speed, rapid movement time, angular velocity, time in top/bottom duration, and meandering movement are highly related to neurological motor impairments, anxiety levels, and stress responses were observed. This study provides evidence based on an in vivo experiment to support the idea that the usage of some antibiotics should be carefully addressed since they can induce a significant effect of behavioral alterations in fish.

Establishing a High-Throughput Locomotion Tracking Method for Multiple Biological Assessments in Tetrahymena.

Protozoa are eukaryotic, unicellular microorganisms that have an important ecological role, are easy to handle, and grow rapidly, which makes them suitable for ecotoxicity assessment. Previous methods for locomotion tracking in protozoa are largely based on software with the drawback of high cost and/or low operation throughput. This study aimed to develop an automated pipeline to measure the locomotion activity of the ciliated protozoan Tetrahymena thermophila using a machine learning-based software, TRex, to conduct tracking. Behavioral endpoints, including the total distance, velocity, burst movement, angular velocity, meandering, and rotation movement, were derived from the coordinates of individual cells. To validate the utility, we measured the locomotor activity in either the knockout mutant of the dynein subunit DYH7 or under starvation. Significant reduction of locomotion and alteration of behavior was detected in either the dynein mutant or in the starvation condition. We also analyzed how Tetrahymena locomotion was affected by the exposure to copper sulfate and showed that our method indeed can be used to conduct a toxicity assessment in a high-throughput manner. Finally, we performed a principal component analysis and hierarchy clustering to demonstrate that our analysis could potentially differentiate altered behaviors affected by different factors. Taken together, this study offers a robust methodology for Tetrahymena locomotion tracking in a high-throughput manner for the first time.

Automated Cardiac Chamber Size and Cardiac Physiology Measurement in Water Fleas by U-Net and Mask RCNN Convolutional Networks.

Water fleas are an important lower invertebrate model that are usually used for ecotoxicity studies. Contrary to mammals, the heart of a water flea has a single chamber, which is relatively big in size and with fast-beating properties. Previous cardiac chamber volume measurement methods are primarily based on ImageJ manual counting at systolic and diastolic phases which suffer from low efficiency, high variation, and tedious operation. This study provides an automated and robust pipeline for cardiac chamber size estimation by a deep learning approach. Image segmentation analysis was performed using U-Net and Mask RCNN convolutional networks on several different species of water fleas such as Moina sp., Daphnia magna, and Daphnia pulex. The results show that Mask RCNN performs better than U-Net at the segmentation of water fleas' heart chamber in every parameter tested. The predictive model generated by Mask RCNN was further analyzed with the Cv2.fitEllipse function in OpenCV to perform a cardiac physiology assessment of Daphnia magna after challenging with the herbicide of Roundup. Significant increase in normalized stroke volume, cardiac output, and the shortening fraction was observed after Roundup exposure which suggests the possibility of heart chamber alteration after roundup exposure. Overall, the predictive Mask RCNN model established in this study provides a convenient and robust approach for cardiac chamber size and cardiac physiology measurement in water fleas for the first time. This innovative tool can offer many benefits to other research using water fleas for ecotoxicity studies.

Performance Comparison of Five Methods for Tetrahymena Number Counting on the ImageJ Platform: Assessing the Built-in Tool and Machine-Learning-Based Extension.

Previous methods to measure protozoan numbers mostly rely on manual counting, which suffers from high variation and poor efficiency. Although advanced counting devices are available, the specialized and usually expensive machinery precludes their prevalent utilization in the regular laboratory routine. In this study, we established the ImageJ-based workflow to quantify ciliate numbers in a high-throughput manner. We conducted Tetrahymena number measurement using five different methods: particle analyzer method (PAM), find maxima method (FMM), trainable WEKA segmentation method (TWS), watershed segmentation method (WSM) and StarDist method (SDM), and compared their results with the data obtained from the manual counting. Among the five methods tested, all of them could yield decent results, but the deep-learning-based SDM displayed the best performance for Tetrahymena cell counting. The optimized methods reported in this paper provide scientists with a convenient tool to perform cell counting for Tetrahymena ecotoxicity assessment.

Inhibitory Effect of Sulfated Polysaccharide from Codium edule P.C. Silva Against 2,4-Dinitrofluorobenzene (DNFB)- Induced Allergic Contact Dermatitis on Female BALB/c Mice.

Purpose: Sulfated polysaccharide from Codium species has been reported for its antiinflammatoryactivities. However, the effect of sulfated polysaccharide from Codium edule on allergic responses has not been studied. The study was conducted to determine the effect ofsulfated polysaccharide (F1) from C. edule on allergic contact dermatitis (ACD) induced by2,4-dinitrofluorobenzene (DNFB) in female BALB/c mice. Methods: F1 was isolated using DEAE sepharose gel chromatography and chemically identifiedby LC-MS analyses. The effects of F1 on changes in ear thickness, allergic responses, andhistology were evaluated. The effects of F1 on the production of inflammatory cytokinesinterferon gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-ɑ) in serum were also quantifiedand compared with standard prednisolone therapy. Results: F1 was identified as a heteropolysaccharide with ß-D-galactans and ß-L-arabinans units.F1 was non-toxic at 2000 mg/kg. Administration of F1 in DNFB-challenged mice significantlysuppressed the increase in ear thickness, erythema, desquamation, and proliferation ofinflammatory cells. F1 significantly decreased the production of inflammatory markers, IFN-γand TNF-α in a dose-dependent manner when compared to the untreated group (P < 0.05). Conclusion: Results suggest that F1 from C. edule is a bioactive sulfated heteropolysaccharidewith anti-inflammatory activity and might be a valuable candidate molecule for the treatmentof allergic diseases such as ACD.

In Ovo and In Silico Evaluation of the Anti-Angiogenic Potential of Syringin.

INTRODUCTION: Cancer is considered as one of the deadliest human diseases today. Angiogenesis, the propagation of new blood vessels from pre-existing vasculature, is a critical step in the progression of cancer as it is essential in the growth and metastasis of tumors. Hence, suppression of angiogenesis is a promising approach in cancer therapy. Syringin, a phenylpropanoid glycoside with a molecular formula of C17H24O9, has been found to exhibit chemopreventive effects. However, its anti-angiogenic activity and the underlying mechanism of action are still unknown. METHODS: In this work, in ovo chorioallantoic membrane (CAM) assay has been conducted to evaluate the effect of syringin on neovascularization. Additionally, reverse molecular docking studies have been performed in order to identify the probable enzyme targets in the angiogenesis pathway. RESULTS: Treatment with syringin showed significant dose-dependent inhibition of blood vessel length and junctions in the CAM of duck eggs; the anti-angiogenic activity of syringin at 100 µM and 200 µM is comparable with 200 µM of the positive control celecoxib. The results of reverse docking studies indicate that syringin binds the strongest to dihydrofolate reductase (DHFR) and, to some extent, with transforming growth factor-beta receptor type 1 (TGF-ßR1), vascular endothelial growth factor receptor 2 (VEGFR2), and matrix metalloproteinase-2 (MMP-2). Furthermore, ADMET models revealed that syringin potentially possesses excellent pharmacokinetic and toxicity profiles. CONCLUSION: This study demonstrates the potential of syringin as an anti-angiogenic agent and elicits further investigations to establish its application in cancer suppression.