Mapping cancer biology in space: applications and perspectives on spatial omics for oncology.

Technologies to decipher cellular biology, such as bulk sequencing technologies and single-cell sequencing technologies, have greatly assisted novel findings in tumor biology. Recent findings in tumor biology suggest that tumors construct architectures that influence the underlying cancerous mechanisms. Increasing research has reported novel techniques to map the tissue in a spatial context or targeted sampling-based characterization and has introduced such technologies to solve oncology regarding tumor heterogeneity, tumor microenvironment, and spatially located biomarkers. In this study, we address spatial technologies that can delineate the omics profile in a spatial context, novel findings discovered via spatial technologies in oncology, and suggest perspectives regarding therapeutic approaches and further technological developments.

Anti-rheumatic property and physiological safety of KMU-11342 in in vitro and in vivo models.

Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disorder characterized by joint destruction due to synovial hypertrophy and the infiltration of inflammatory cells. Despite substantial progress in RA treatment, challenges persist, including suboptimal treatment responses and adverse effects associated with current therapies. This study investigates the anti-rheumatic capabilities of the newly identified multi-protein kinase inhibitor, KMU-11342, aiming to develop innovative agents targeting RA. In this study, we synthesized the novel multi-protein kinase inhibitor KMU-11342, based on indolin-2-one. We assessed its cardiac electrophysiological safety using the Langendorff system in rat hearts and evaluated its toxicity in zebrafish in vivo. Additionally, we examined the anti-rheumatic effects of KMU-11342 on human rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS), THP-1 cells, and osteoclastogenesis in RAW264.7 cells. KMU-11342 demonstrated the ability to inhibit LPS-induced chemokine inhibition and the upregulation of pro-inflammatory cytokines, cyclooxygenase-2, inducible nitric oxide synthase, p-IKKα/ß, p-NF-κB p65, and the nuclear translocation of NF-κB p65 in RA-FLS. It effectively suppressed the upregulation of NLR family pyrin domain containing 3 (NLRP3) and caspase-1 cleavage. Furthermore, KMU-11342 hindered the activation of osteoclast differentiation factors such as RANKL-induced TRAP, cathepsin K, NFATc-1, and c-Fos in RAW264.7 cells. KMU-11342 mitigates LPS-mediated inflammatory responses in THP-1 cells by inhibiting the activation of NLRP3 inflammasome. Notably, KMU-11342 exhibited minimal cytotoxicity in vivo and electrophysiological cardiotoxicity ex vivo. Consequently, KMU-11342 holds promise for development as a therapeutic agent in RA treatment.

Efficient Haze Removal from a Single Image Using a DCP-Based Lightweight U-Net Neural Network Model.

In this paper, we propose a lightweight U-net architecture neural network model based on Dark Channel Prior (DCP) for efficient haze (fog) removal with a single input. The existing DCP requires high computational complexity in its operation. These computations are challenging to accelerate, and the problem is exacerbated when dealing with high-resolution images (videos), making it very difficult to apply to general-purpose applications. Our proposed model addresses this issue by employing a two-stage neural network structure, replacing the computationally complex operations of the conventional DCP with easily accelerated convolution operations to achieve high-quality fog removal. Furthermore, our proposed model is designed with an intuitive structure using a relatively small number of parameters (2M), utilizing resources efficiently. These features demonstrate the effectiveness and efficiency of the proposed model for fog removal. The experimental results show that the proposed neural network model achieves an average Peak Signal-to-Noise Ratio (PSNR) of 26.65 dB and a Structural Similarity Index Measure (SSIM) of 0.88, indicating an improvement in the average PSNR of 11.5 dB and in SSIM of 0.22 compared to the conventional DCP. This shows that the proposed neural network achieves comparable results to CNN-based neural networks that have achieved SOTA-class performance, despite its intuitive structure with a relatively small number of parameters.

A circulating cell-free DNA methylation signature for the detection of hepatocellular carcinoma.

To address the shortcomings of current hepatocellular carcinoma (HCC) surveillance tests, we set out to find HCC-specific methylation markers and develop a highly sensitive polymerase chain reaction (PCR)-based method to detect them in circulating cell-free DNA (cfDNA). The analysis of large methylome data revealed that Ring Finger Protein 135 (RNF135) and Lactate Dehydrogenase B (LDHB) are universally applicable HCC methylation markers with no discernible methylation level detected in any other tissue types. These markers were used to develop Methylation Sensitive High-Resolution Analysis (MS-HRM), and their diagnostic accuracy was tested using cfDNA from healthy, at-risk, and HCC patients. The combined MS-HRM RNF135 and LDHB analysis detected 57% of HCC, outperforming the alpha-fetoprotein (AFP) test's sensitivity of 45% at comparable specificity. Furthermore, when used with the AFP test, the methylation assay can detect 70% of HCC. Our findings suggest that the cfDNA methylation assay could be used for HCC liquid biopsy.

Staphylococcus aureus causes aberrant epidermal lipid composition and skin barrier dysfunction.

BACKGROUND: Staphylococcus (S) aureus colonization is known to cause skin barrier disruption in atopic dermatitis (AD) patients. However, it has not been studied how S. aureus induces aberrant epidermal lipid composition and skin barrier dysfunction. METHODS: Skin tape strips (STS) and swabs were obtained from 24 children with AD (6.0 ± 4.4 years) and 16 healthy children (7.0 ± 4.5 years). Lipidomic analysis of STS samples was performed by mass spectrometry. Skin levels of methicillin-sensitive and methicillin-resistant S. aureus (MSSA and MRSA) were evaluated. The effects of MSSA and MRSA were evaluated in primary human keratinocytes (HEKs) and organotypic skin cultures. RESULTS: AD and organotypic skin colonized with MRSA significantly increased the proportion of lipid species with nonhydroxy fatty acid sphingosine ceramide with palmitic acid ([N-16:0 NS-CER], sphingomyelins [16:0-18:0 SM]), and lysophosphatidylcholines [16:0-18:0 LPC], but significantly reduced the proportion of corresponding very long-chain fatty acids (VLCFAs) species (C22-28) compared to the skin without S. aureus colonization. Significantly increased transepidermal water loss (TEWL) was found in MRSA-colonized AD skin. S. aureus indirectly through interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, IL-6, and IL-33 inhibited expression of fatty acid elongase enzymes (ELOVL3 and ELOVL4) in HEKs. ELOVL inhibition was more pronounced by MRSA and resulted in TEWL increase in organotypic skin. CONCLUSION: Aberrant skin lipid profiles and barrier dysfunction are associated with S. aureus colonization in AD patients. These effects are attributed to the inhibition of ELOVLs by S. aureus-induced IL-1ß, TNF-α, IL-6, and IL-33 seen in keratinocyte models and are more prominent in MRSA than MSSA.

Marine Polyether Phycotoxin Palytoxin Induces Apoptotic Cell Death via Mcl-1 and Bcl-2 Downregulation.

Palytoxin is considered one of the most potent biotoxins. As palytoxin-induced cancer cell death mechanisms remain to be elucidated, we investigated this effect on various leukemia and solid tumor cell lines at low picomolar concentrations. As palytoxin did not affect the viability of peripheral blood mononuclear cells (PBMC) from healthy donors and did not create systemic toxicity in zebrafish, we confirmed excellent differential toxicity. Cell death was characterized by a multi-parametric approach involving the detection of nuclear condensation and caspase activation assays. zVAD-sensitive apoptotic cell death was concomitant with a dose-dependent downregulation of antiapoptotic Bcl-2 family proteins Mcl-1 and Bcl-xL. Proteasome inhibitor MG-132 prevented the proteolysis of Mcl-1, whereas the three major proteasomal enzymatic activities were upregulated by palytoxin. Palytoxin-induced dephosphorylation of Bcl-2 further exacerbated the proapoptotic effect of Mcl-1 and Bcl-xL degradation in a range of leukemia cell lines. As okadaic acid rescued cell death triggered by palytoxin, protein phosphatase (PP)2A was involved in Bcl-2 dephosphorylation and induction of apoptosis by palytoxin. At a translational level, palytoxin abrogated the colony formation capacity of leukemia cell types. Moreover, palytoxin abrogated tumor formation in a zebrafish xenograft assay at concentrations between 10 and 30 pM. Altogether, we provide evidence of the role of palytoxin as a very potent and promising anti-leukemic agent, acting at low picomolar concentrations in cellulo and in vivo.

Cathodic electroorganic reaction on silicon oxide dielectric electrode.

The faradaic reaction at the insulator is counterintuitive. For this reason, electroorganic reactions at the dielectric layer have been scarcely investigated despite their interesting aspects and opportunities. In particular, the cathodic reaction at a silicon oxide surface under a negative potential bias remains unexplored. In this study, we utilize defective 200-nm-thick n+-Si/SiO2 as a dielectric electrode for electrolysis in an H-type divided cell to demonstrate the cathodic electroorganic reaction of anthracene and its derivatives. Intriguingly, the oxidized products are generated at the cathode The experiments under various conditions provide consistent evidence supporting that the electrochemically generated hydrogen species, supposedly the hydrogen atom, is responsible for this phenomenon. The electrogenerated hydrogen species at the dielectric layer suggests a synthetic strategy for organic molecules.

The stem cell marker Prom1 promotes axon regeneration by down-regulating cholesterol synthesis via Smad signaling.

Axon regeneration is regulated by a neuron-intrinsic transcriptional program that is suppressed during development but that can be reactivated following peripheral nerve injury. Here we identify Prom1, which encodes the stem cell marker prominin-1, as a regulator of the axon regeneration program. Prom1 expression is developmentally down-regulated, and the genetic deletion of Prom1 in mice inhibits axon regeneration in dorsal root ganglion (DRG) cultures and in the sciatic nerve, revealing the neuronal role of Prom1 in injury-induced regeneration. Elevating prominin-1 levels in cultured DRG neurons or in mice via adeno-associated virus-mediated gene delivery enhances axon regeneration in vitro and in vivo, allowing outgrowth on an inhibitory substrate. Prom1 overexpression induces the consistent down-regulation of cholesterol metabolism-associated genes and a reduction in cellular cholesterol levels in a Smad pathway-dependent manner, which promotes axonal regrowth. We find that prominin-1 interacts with the type I TGF-ß receptor ALK4, and that they synergistically induce phosphorylation of Smad2. These results suggest that Prom1 and cholesterol metabolism pathways are possible therapeutic targets for the promotion of neural recovery after injury.

Comparison of Trends in the Incidence of Traumatic Spinal Cord Injury in Daily Life, Automobile Accidents, and Industrial Accidents: A National Multi-Insurance Study in Korea.

BACKGROUND: To examine the incidence of traumatic spinal cord injury (TSCI) from all etiologies, we measured and compared the incidence of TSCI from three national or quasi-national databases in South Korea, namely, the National Health Insurance Service (NHIS), automobile insurance (AUI), and Industrial Accident Compensation Insurance (IACI). METHODS: We reviewed patients with TSCI reported in the NHIS database between 2009 and 2018, and in the AUI and IACI databases between 2014 and 2018. TSCI patients were defined as those first admitted to the hospital with a diagnosis of TSCI according to the International Classification of Diseases (10th revision) criteria. Age-adjusted incidence was calculated using direct standardization using the 2005 South Korean population or the 2000 US population as the standard population. The annual percentage changes (APC) of TSCI incidence were calculated. The Cochrane-Armitage trend test was performed according to the injured body region. RESULTS: In the NHIS database, age-adjusted TSCI incidence using the Korean standard population increased significantly from 2009 to 2018 (from 33.73 per million in 2009 to 38.14 per million in 2018, APC = 1.2%, P = 0.014). Contrarily, age-adjusted incidence in the AUI database significantly decreased from 13.88 per million in 2014 to 11.57 per million in 2018 (APC = - 5.1%, P = 0.009). In the IACI database, the age-adjusted incidence showed no significant difference, while crude incidence showed a significant increase (from 22.02 per million in 2014 to 28.92 per million in 2018, APC = 6.1%, P = 0.038). According to the age group, all the three databases showed high incidences of TSCI in those in their 60s and 70s or older. Among those in their 70s or older, the incidence of TSCI increased dramatically in the NHIS and IACI databases, while no significant trend was found in AUI database. In 2018, the number of TSCI patients was the highest among those over 70 years of age in the NHIS, whereas among those in their 50s were the highest in both AUI and IACI. The proportion of patients with cervical spinal cord injury was the most common in all these databases. CONCLUSIONS: The differences in trends in the incidence of TSCI may be due to the different etiologies and different characteristics of subjects depending on insurance type. These results imply the need for tailored medical strategies for the different injury mechanisms represented by three national insurance services in South Korea.

Echinatin induces reactive oxygen species-mediated apoptosis via JNK/p38 MAPK signaling pathway in colorectal cancer cells.

Colorectal cancer (CRC) is a very common and deadly cancer worldwide, and oxaliplatin is used as first-line chemotherapy. However, resistance usually develops, limiting treatment. Echinatin (Ech) is the main component of licorice and exhibits various therapeutic effects on inflammation-mediated diseases and cancer, ischemia/reperfusion, and liver injuries. The present study elucidated the underlying molecular mechanism of Ech-induced apoptosis in both oxaliplatin-sensitive (HT116 and HT29) and -resistant (HCT116-OxR and HT29-OxR) CRC cells. To evaluate the antiproliferative activities of Ech, we performed MTT and soft agar assays. Ech reduced viability, colony size, and numbers of CRC cells. The underlying molecular mechanisms were explored by various flow cytometry analyses. Ech-induced annexin-V stained cells, reactive oxygen species (ROS) generation, cell cycle arrest, JNK/p38 MAPK activation, endoplasmic reticulum (ER) stress, mitochondrial membrane potential depolarization, and multi-caspase activity. In addition apoptosis-, cell cycle-, and ER stress-related protein levels were confirmed by western blotting. Moreover, we verified ROS-mediated cell death by treatment with inhibitors such as N-acetyl-L-cysteine, SP600125, and SB203580. Taken together, Ech exhibits anticancer activity in oxaliplatin-sensitive and -resistant CRCs by inducing ROS-mediated apoptosis through the JNK/p38 MAPK signaling pathway. This is the first study to show that Ech has the potential to treat drug-resistant CRC, providing new directions for therapeutic strategies targeting drug-resistant CRC.

An Implementation of Inverse Cosine Hardware for Sound Rendering Applications.

Sound rendering is the process of determining the sound propagation path from an audio source to a listener and generating 3D sound based on it. This task demands complex calculations, including trigonometric functions. This paper presents hardware-based inverse cosine function calculations using the table method and linear approximation. This approach maintains a high accuracy while limiting hardware size for suitability in sound rendering applications. Consequently, our proposed hardware-based inverse cosine calculation method is a valuable tool for achieving high efficiency and accuracy in 3D sound rendering.

Novel Multi-View RGB Sensor for Continuous Motion Analysis in Kinetic Chain Exercises: A Pilot Study for Simultaneous Validity and Intra-Test Reliability.

As the number of musculoskeletal disorders caused by smartphone usage, sedentary lifestyles, and active sports activities increases, there is a growing demand for precise and accurate measurement and evaluation of issues such as incorrect compensation patterns, asymmetrical posture, and limited joint operation range. Urgent development of new inspection equipment is necessary to address issues such as convenience, economic feasibility, and post-processing difficulties. Using 4DEYE®, a new multi-view red, green, and blue (RGB) sensor-based motion analysis equipment, and the VICON® ratio, which are infrared-based markers, we conducted a comparative analysis of the simultaneous validity of the joint angle (trajectory) and reliability. In this study, five healthy participants who could perform movements were selected for the pilot study and two movements (Y-balance and side dip) were analyzed. In addition, the ICC (Intraclass Correlation Coefficient) was analyzed using the SPSS (Statistical Package for the Social Sciences) V.18 while the number of data frames of each equipment was equalized using the MATLAB program. The results revealed that side dips, which are open kinetic chain exercises (intraclass correlation coefficient ICC(2.1), 0.895-0.996), showed very high concordance with the Y-balance test, a closed kinetic chain exercise (ICC(2.1), 0.678-0.990). The joint measurement results were similar regardless of the movement in the open or closed kinetic chain exercise, confirming the high reliability of the newly developed multiview RGB sensor. This is of great significance because we obtained important and fundamental results that can be used in various patterns of exercise movements in the future.

Vapor Pressure and Toxicity Prediction for Novichok Agent Candidates Using Machine Learning Model: Preparation for Unascertained Nerve Agents after Chemical Weapons Convention Schedule 1 Update.

The recent terrorist attacks using Novichok agents and subsequent operations have necessitated an understanding of its physicochemical properties, such as vapor pressure and toxicity, as well as unascertained nerve agent structures. To prevent continued threats from new types of nerve agents, the organization for the prohibition of chemical weapons (OPCW) updated the chemical weapons convention (CWC) schedule 1 list. However, this information is vague and may encompass more than 10 000 possible chemical structures, which makes it almost impossible to synthesize and measure their properties and toxicity. To assist this effort, we successfully developed machine learning (ML) models to predict the vapor pressure to help with escape and removal operations. The model shows robust and high-accuracy performance with promising features for predicting vapor pressure when applied to Novichok materials and accurate predictions with reasonable errors. The ML classification model was successfully built for the swallow globally harmonized system class of organophosphorus compounds (OP) for toxicity predictions. The tuned ML model was used to predict the toxicity of Novichok agents, as described in the CWC list. Although its accuracy and linearity can be improved, this ML model is expected to be a firm basis for developing more accurate models for predicting the vapor pressure and toxicity of nerve agents in the future to help handle future terror attacks with unknown nerve agents.

Two Cases of Mange Mite (Sarcoptes scabiei) Infestation in Long-Tailed Goral (Naemorhedus caudatus) in Republic of Korea.

The long-tailed goral, Naemorhedus caudatus (Mammalia: Bovidae), is one of the endangered animals in the Republic of Korea (Korea). Sarcoptic mange mites infested in diverse species of mammals, including humans, but no case has been reported in long-tailed gorals. We report 2 cases of mange mite, Sarcoptes scabiei, infestation in long-tailed gorals. Mange mites were sampled in the skin legions of the 2 long-tailed gorals, which were rescued in 2 different regions, Uljin-gun, Gyeongsangbuk-do and Cheorwon-gun, Gangwon-do, Korea. Our results showed that the ectoparasite was the itch mite that burrowed into skin and caused scabies on the morphological inspection and placed within the phylogenetic relations of the species. The present study confirmed for the first time in Korea that mange mites are pathogenic scabies of long-tailed goral. Closer surveillance of this pathogenic ectoparasite in zoonotic and infectious ecosystems is warranted.

Therapeutic potential of α,ß-thujone through metabolic reprogramming and caspase-dependent apoptosis in ovarian cancer cells.

The therapeutic potential of α,ß-thujone, a functional compound found in many medicinal plants of the Cupressaceae, Asteraceae, and Lamiaceae families, has been demonstrated, including in inflammation and cancers. However, its pharmacological functions and mechanisms of action in ovarian cancer remain unclear. We investigated the anticancer properties of α,ß-thujone in ES2 and OV90 human ovarian cancer cells and its effect on sensitization to cisplatin. α,ß-thujone inhibited cancer cell proliferation and induced cell death through caspase-dependent intrinsic apoptotic pathways. Moreover, α,ß-thujone-mediated endoplasmic reticulum stress was associated with the loss of mitochondrial functions and altered metabolic landscape of ovarian cancer cells. α,ß-Thujone attenuated blood vessel formation in transgenic zebrafish, implying it has significant antiangiogenic potential. In addition, α,ß-thujone sensitized ovarian cancer cells to cisplatin, causing synergistic pharmacological effects. Collectively, our results suggest that α,ß-thujone has therapeutic potential in human ovarian cancer and functions via regulating multiple intracellular stress-associated metabolic reprogramming and caspase-dependent apoptotic pathways.

Osthole interacts with an ER-mitochondria axis and facilitates tumor suppression in ovarian cancer.

Osthole is a natural coumarin found in a variety of plants and has been reported to have diverse biological functions, including antimicrobial, antiviral, immunomodulatory, and anticancer effects. Here, we investigated the natural derivative osthole as a promising anticancer compound against ovarian cancer and evaluated its ability to suppress and abrogate tumor progression. In addition, we found the endoplasmic reticulum-mitochondrial axis-mediated anticancer mechanisms of osthole against ES2 and OV90 ovarian cancer cells and demonstrated its calcium-dependent pharmacological potential. Mechanistically, osthole was found to target the phosphatidylinositol 3-kinase/mitogen-activated protein kinase signaling pathway to facilitate tumor suppression in ovarian cancer. Furthermore, we identified the effects of osthole in a three-dimensional tumor-formation model using the zebrafish xenograft assay, providing convincing evidence of the pharmacological effects of osthole within the anchorage-independent tumor microenvironment. These findings suggest that osthole has strong potential as a pharmacological agent for targeting ovarian cancer.

Syntrophic co-culture of a methanotroph and heterotroph for the efficient conversion of methane to mevalonate.

As the bioconversion of methane becomes increasingly important for bio-industrial and environmental applications, methanotrophs have received much attention for their ability to convert methane under ambient conditions. This includes the extensive reporting of methanotroph engineering for the conversion of methane to biochemicals. To further increase methane usability, we demonstrated a highly flexible and efficient modular approach based on a synthetic consortium of methanotrophs and heterotrophs mimicking the natural methane ecosystem to produce mevalonate (MVA) from methane. In the methane-conversion module, we used Methylococcus capsulatus Bath as a highly efficient methane biocatalyst and optimized the culture conditions for the production of high amounts of organic acids. In the MVA-synthesis module, we used Escherichia coli SBA01, an evolved strain with high organic acid tolerance and utilization ability, to convert organic acids to MVA. Using recombinant E. coli SBA01 possessing genes for the MVA pathway, 61 mg/L (0.4 mM) of MVA was successfully produced in 48 h without any addition of nutrients except methane. Our platform exhibited high stability and reproducibility with regard to cell growth and MVA production. We believe that this versatile system can be easily extended to many other value-added processes and has a variety of potential applications.

A functional analysis of Carabelli trait in Australian aboriginal dentition.

OBJECTIVES: Carabelli is a nonmetric dental trait variably expressed as a small pit to a prominent cusp in the maxillary molars of modern humans. Investigations on the occurrence and expression rates of this trait have been conducted extensively, tracing its origin to genetic sources. However, there remains a lack of understanding about its potential role in chewing. In this study, we examine molar macrowear with the aim of reconstructing Carabelli trait occlusal dynamics occurring during chewing. METHODS: We have examined 96 deciduous and permanent maxillary molars of children and young adults from Yuendumu, an Australian Aboriginal population that was at an early stage of transition from a nomadic and hunter-gatherer way of life to a more settled existence. We apply a well-established method, called Occlusal Fingerprint Analysis, which is a digital approach for analyzing dental macrowear allowing the reconstruction of jaw movements required to produce wear pattern specific to each tooth. RESULTS: Carabelli trait slightly enlarges the surface functional area, especially in those molars where this feature is expressed in its cuspal form and it is closer to the occlusal plane. Moreover, the highly steep contact planes would also indicate that Carabelli wear areas contribute to increasing the shearing abilities of the occluded teeth, which are particularly important when processing fibrous and tough foods. CONCLUSIONS: The macrowear analysis suggests that Carabelli trait in the Aboriginal people from Yuendumu slightly enhanced occlusion and probably played some functional role during mastication. Future biomechanical and microwear analyses could provide additional information on the mechanical adaptation of Carabelli trait in modern human dentition.

Picolinafen exerts developmental toxicity via the suppression of oxidative stress and angiogenesis in zebrafish embryos.

Picolinafen, a phytoene desaturase-inhibiting herbicide, has been used since 2001 to control the growth of broadleaf weeds. Picolinafen has lower solubility and volatility, and shows lower toxicity to non-target insect species than other types of herbicide. Although picolinafen has been detected in lakes near urban environments and induces chronic toxicity in the mammals, birds, and some aquatic organisms, no study has investigated the toxicity or mode of action of picolinafen in zebrafish. In this study, we demonstrated the lethality and acute LC50 value of picolinafen towards zebrafish embryos. Picolinafen hampered the development of embryos by the induction of morphological abnormalities via apoptosis. Additionally, picolinafen suppressed the generation of reactive oxygen species and angiogenesis. Also, the angiogenesis related genes, flt1 and flt4 mRNA expression was decreased in zebrafish embryos. This study provides a mechanistic understanding of the developmental toxicity of picolinafen in vertebrates.

Benfuresate induces developmental toxicity in zebrafish larvae by generating apoptosis and pathological modifications.

Benfuresate (2,3-dihydro-3,3-dimethylbenzofuran-5-yl ethanesulphonate) is a widely used pre-emergence herbicide of the benzofurane group, which works through the inhibition of lipid synthesis. During embryonic development of zebrafish, benfuresate retards growth while causing internal changes in the body, including alteration of the expression of cell cycle regulators, induction of apoptosis, and suppression of the circulatory system. Acute toxicity towards benfuresate is seen across the range of 5-15 µM in a dose-dependent manner and contributes to pathological conditions and subsequent morphological changes. For embryos 120 h post fertilization (hpf), benfuresate exposure results in an array of malformations involving eye or otolith development, pericardial edema, yolk sac edema, and abnormal curvature of the spine. Mechanistically, benfuresate exposure altered the transcription levels of the proliferative pathway genes ccnd1, ccne1, cdk2, and cdk6, all of which sensitize cells to apoptosis. Benfuresate exposure also affected vascular formation, including the formation of various vessels (DA, SIVs, CA, CV) whose functions in lymphatic-blood circulation were disrupted following decreased vegfaa, vegfc, flt1, flt4, and kdrl expression. These findings provide evidence of embryo-larval toxicity due to benfuresate and highlight the perils of herbicide exposure for non-target organisms far removed from application sites, especially in aquatic environments.