Prospectively Isolated Tetraspanin+ Neoblasts Are Adult Pluripotent Stem Cells Underlying Planaria Regeneration.

Proliferating cells known as neoblasts include pluripotent stem cells (PSCs) that sustain tissue homeostasis and regeneration of lost body parts in planarians. However, the lack of markers to prospectively identify and isolate these adult PSCs has significantly hampered their characterization. We used single-cell RNA sequencing (scRNA-seq) and single-cell transplantation to address this long-standing issue. Large-scale scRNA-seq of sorted neoblasts unveiled a novel subtype of neoblast (Nb2) characterized by high levels of PIWI-1 mRNA and protein and marked by a conserved cell-surface protein-coding gene, tetraspanin 1 (tspan-1). tspan-1-positive cells survived sub-lethal irradiation, underwent clonal expansion to repopulate whole animals, and when purified with an anti-TSPAN-1 antibody, rescued the viability of lethally irradiated animals after single-cell transplantation. The first prospective isolation of an adult PSC bridges a conceptual dichotomy between functionally and molecularly defined neoblasts, shedding light on mechanisms governing in vivo pluripotency and a source of regeneration in animals. VIDEO ABSTRACT.

Clathrin-mediated endocytosis facilitates the internalization of Magnaporthe oryzae effectors into rice cells.

Fungi and oomycetes deliver effectors into living plant cells to suppress defenses and control plant processes needed for infection. Little is known about the mechanism by which these pathogens translocate effector proteins across the plasma membrane into the plant cytoplasm. The blast fungus Magnaporthe oryzae secretes cytoplasmic effectors into a specialized biotrophic interfacial complex (BIC) before translocation. Here, we show that cytoplasmic effectors within BICs are packaged into punctate membranous effector compartments that are occasionally observed in the host cytoplasm. Live cell imaging with fluorescently labeled proteins in rice (Oryza sativa) showed that these effector puncta colocalize with the plant plasma membrane and with CLATHRIN LIGHT CHAIN 1, a component of clathrin-mediated endocytosis (CME). Inhibiting CME using virus-induced gene silencing and chemical treatments resulted in cytoplasmic effectors in swollen BICs lacking effector puncta. By contrast, fluorescent marker colocalization, gene silencing, and chemical inhibitor studies failed to support a major role for clathrin-independent endocytosis in effector translocation. Effector localization patterns indicated that cytoplasmic effector translocation occurs underneath appressoria before invasive hyphal growth. Taken together, this study provides evidence that cytoplasmic effector translocation is mediated by CME in BICs and suggests a role for M. oryzae effectors in coopting plant endocytosis.

Specific inhibition of an anticancer target, polo-like kinase 1, by allosterically dismantling its mechanism of substrate recognition.

Polo-like kinase 1 (Plk1) is considered an attractive target for anticancer therapy. Over the years, studies on the noncatalytic polo-box domain (PBD) of Plk1 have raised the expectation of generating highly specific protein-protein interaction inhibitors. However, the molecular nature of the canonical PBD-dependent interaction, which requires extensive water network-mediated interactions with its phospholigands, has hampered efforts to identify small molecules suitable for Plk1 PBD drug discovery. Here, we report the identification of the first allosteric inhibitor of Plk1 PBD, called Allopole, a prodrug that can disrupt intracellular interactions between PBD and its cognate phospholigands, delocalize Plk1 from centrosomes and kinetochores, and induce mitotic block and cancer cell killing. At the structural level, its unmasked active form, Allopole-A, bound to a deep Trp-Phe-lined pocket occluded by a latch-like loop, whose adjoining region was required for securely retaining a ligand anchored to the phospho-binding cleft. Allopole-A binding completely dislodged the L2 loop, an event that appeared sufficient to trigger the dissociation of a phospholigand and inhibit PBD-dependent Plk1 function during mitosis. Given Allopole's high specificity and antiproliferative potency, this study is expected to open an unexplored avenue for developing Plk1 PBD-specific anticancer therapeutic agents.

A multiplex real-time RT-qPCR assay for simultaneous detection of porcine epidemic diarrhea virus, porcine deltacoronavirus, and swine acute diarrhea syndrome coronavirus.

Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV) cause intestinal diseases with similar manifestations in suckling piglets. In this study, we developed a multiplex real-time PCR for differential diagnosis of PEDV, PDCoV, and SADS-CoV. The assay demonstrated high specificity with a detection limit of 5 copies/µl for each virus. The assay specifically detected PEDV, PDCoV, and SADS-CoV and excluded all other swine pathogens circulating in pigs. Furthermore, the assay exhibited satisfactory performance in analyzing clinical samples. The data indicate that the newly developed multiplex real-time PCR method can be applied for differential diagnosis of porcine enteric coronaviruses.

Prevalence of Parainfluenza Viruses in Seoul, 2021 - 2022.

BACKGROUND: Parainfluenza virus (PIV) is a significant etiological agent of acute lower respiratory tract infections (ALRIs) in infants and young children. The present study has been conducted to investigate the prevalence of recently identified respiratory viruses. METHODS: In total, 543 oropharyngeal or nasopharyngeal swab samples collected from hospitalized patients with acute respiratory symptoms (ARS) between January and December 2021 (5,653 females and 4,950 males) were tested for respiratory viruses using RT-PCR. RESULTS: At least one respiratory virus was detected by RT-PCR in 119 out of 175 samples (68%). The most frequently detected virus was human rhinovirus (HRV) (34, 6.5%), followed by human parainfluenza viruses (HPIVs) (19, 3.6%), human bocavirus (HBoV) (8, 1.5%), human adenovirus (HAdV) (7, 1.3%), and human respiratory syncytial virus (HRSV) (4, 0.8%). HPIV-3 accounted for 3.6% (19/175) of all viral pathogens and was the second most frequently detected viral pathogen in our study. HPIV-3 infections peaked in the fall (November) of 2021. Phylogenetic analysis of the coding region of the viral protein HA revealed that all 35 (100%) of 35 HPIV-infected patients were infected with HPIV-3. CONCLUSIONS: HPIV was an important causative pathogen associated with ALRI in children hospitalized in Korea in the late fall of 2021, as the social distancing rules for COVID-19 were relaxed. These findings highlight the im-portance of HPIV as a cause of ALRI.

Relation Between Masticatory Performance and Skeletal Properties in Patients With Skeletal Class III Malocclusion.

This study aimed to measure masticatory performance (MP) using ß-carotene gummy jelly to investigate its relationship with skeletal properties in decompensated patients diagnosed with skeletal class III malocclusion. The study included 78 patients (38 men and 40 women) diagnosed with skeletal class III malocclusion without temporomandibular joint disorder and periodontal disease. MP was measured using a new masticatory measuring device and ß-carotene in the gummy jelly. Lateral and posteroanterior cephalograms were obtained, and skeletal properties (Me deviation, ANB, SNB, APDI, Wits, ODI, facial axis, body length, ramus length, SN-GoGn, anterior facial height, posterior facial height, saddle angle, articular angle, and gonial angle) were evaluated. MP differences according to age and sex and the effect of skeletal properties on MP were analyzed using multiple linear regression analysis. The MP of all patients was 3690.55±1428.77 mm², MP of the male group was 4043.05±1498.09 mm², and MP of the female group was 3355.68±1272.19 mm². Among the items investigated, the variable that affected MP was posterior facial height. Posterior facial height showed a positive correlation (P=0.022). There was no significant difference between MP and other skeletal properties (P>0.05). The severity of the hypodivergency in skeletal class III could affect MP. The relationship between facial asymmetry or skeletal relation and MP could not be explained in this study.

Cost-effectiveness of the anti-vascular endothelial growth factor intravitreal injection and panretinal photocoagulation for patients with proliferative diabetic retinopathy in South Korea.

BACKGROUND: We determined the cost-effectiveness of the anti-vascular endothelial growth factor (VEGF) intravitreal injection versus panretinal photocoagulation (PRP) for patients with proliferative diabetic retinopathy (PDR) in South Korea. METHODS: We simulated four treatment strategies using PRP and the anti-VEGF injection by constructing a Markov model for a hypothetical cohort of 50-year-old PDR patients: (1) PRP only; (2) anti-VEGF injection only; (3) PRP first; and (4) anti-VEGF injection first. RESULTS: In this cost-effectiveness analysis, compared with only-PRP, the incremental cost-effectiveness ratio was $95,456 per quality-adjusted life-year (QALY) for PRP first, $34,375 per QALY for anti-VEGF injection first, and $33,405 per QALY for anti-VEGF injection only from a healthcare perspective. From the societal and payer perspective, strategy (2) was more cost-saving and effective than (1). In the probabilistic sensitivity analysis, only-PRP was cost-effective up to the willingness-to-pay (WTP) of about $42,000, while anti-VEGF injection only was cost-effective from a healthcare perspective. From the societal and payer perspectives, regardless of the value of WTP, anti-VEGF injection only was the most cost-effective strategy. CONCLUSION: In our study, the anti-VEGF injection for PDR was cost-effective from the payer and societal perspectives.

Developmental toxicity of chlorpyrifos-methyl and its primary metabolite, 3,5,6-trichloro-2-pyridinol to early life stages of zebrafish (Danio rerio).

Chlorpyrifos-methyl (CPM) is one of the thiophosphate insecticides, and it is mainly metabolized to 3,5,6-trichloro-2-pyridinol (TCP) in the environment. As CPM is a strongly toxic and TCP is persistent in the environment, CPM and TCP need to be evaluate their toxicities using animal model organisms. With this regard, CPM and TCP were treated on zebrafish (Danio rerio) embryos and LC50 values were determined as over 2000 µg/L and 612.5 µg/L, respectively. For the hatchability, CPM did not exhibit any interference, while TCP showed weak inhibition. In the CPM-treated embryos, pericardial edema and bleeding were observed at 48 hpf, but recovered afterwards. The pericardial edema and yolk sac edema were observed in TCP-treated zebrafish embryos at the concentration of 500 µg/L after 72 hpf. TCP induced abnormal heart development and the heartbeat was dramatically decreased in Tg(cmlc2:EGFP) embryos at the level of 500 µg/L. The expression level of heart development-related genes such as gata, myl7, and cacna1c was significantly decreased in the TCP 500 µg/L-treated embryos at the 96 hpf. Taken together, TCP appears to be more toxic than the parent compound towards the zebrafish embryos. It is highly requested that TCP needs to be monitored with a strong public concern because it affects presumably heart development in early-stage aquatic vertebrates.

Substantial Lipid Increases During Menopausal Transition in Korean Middle-Aged Women.

BACKGROUND: Adverse lipid profiles are observed in postmenopausal women. However, there is insufficient evidence of the association between lipids and reproductive aging in Korean women. We aimed to characterize lipid changes with respect to timing relative to menopause in Korean middle-aged women. METHODS: This study included 1,436 premenopausal women who had a natural menopause during the follow-up period (median = 15.76 years) from the Korean Genome and Epidemiology Study (KoGES) Ansan and Anseong cohort. Lipid levels were measured every 2 years, and the magnitudes of annual lipid changes and differences in the changes by premenopausal body mass index were estimated using piecewise linear mixed-effects models. RESULTS: All lipid levels increased greatly from 3 or 5 years before menopause to 1 year after menopause in all women, regardless of their premenopausal body mass index. During the period, high-density lipoprotein cholesterol (HDL-C) levels increased at 0.42 mg/dL per year (95% confidence interval [CI], 0.29 to 0.55 mg/dL). Nevertheless, non-HDL-C levels simultaneously increased at 3.42 mg/dL per year (95% CI, 3.11 to 3.72 mg/dL), and an annual change in the non-HDL-C to HDL-C ratio was 0.05 (95% CI, 0.04 to 0.06). One year after menopause, changes in all lipid parameters significantly slowed down, except for the non-HDL-C to HDL-C ratio (P < 0.001 for all). The ratio continued to increase until 3 years after menopause, but thereafter, the change leveled off. CONCLUSION: Women experienced remarkable increases in lipid levels during menopausal transition, highlighting the need for early intervention strategies for cardiovascular disease prevention in women.

Two Years of Experience and Methodology of Korean COVID-19 Living Clinical Practice Guideline Development.

BACKGROUND: In Korea, during the early phase of the coronavirus disease 2019 (COVID-19) pandemic, we responded to the uncertainty of treatments under various conditions, consistently playing catch up with the speed of evidence updates. Therefore, there was high demand for national-level evidence-based clinical practice guidelines for clinicians in a timely manner. We developed evidence-based and updated living recommendations for clinicians through a transparent development process and multidisciplinary expert collaboration. METHODS: The National Evidence-based Healthcare Collaborating Agency (NECA) and the Korean Academy of Medical Sciences (KAMS) collaborated to develop trustworthy Korean living guidelines. The NECA-supported methodological sections and 8 professional medical societies of the KAMS worked with clinical experts, and 31 clinicians were involved annually. We developed a total of 35 clinical questions, including medications, respiratory/critical care, pediatric care, emergency care, diagnostic tests, and radiological examinations. RESULTS: An evidence-based search for treatments began in March 2021 and monthly updates were performed. It was expanded to other areas, and the search interval was organized by a steering committee owing to priority changes. Evidence synthesis and recommendation review was performed by researchers, and living recommendations were updated within 3-4 months. CONCLUSION: We provided timely recommendations on living schemes and disseminated them to the public, policymakers and various stakeholders using webpages and social media. Although the output was successful, there were some limitations. The rigor of development issues, urgent timelines for public dissemination, education for new developers, and spread of several new COVID-19 variants have worked as barriers. Therefore, we must prepare systematic processes and funding for future pandemics.

Redox-engineering enhances maize thermotolerance and grain yield in the field.

Increasing populations and temperatures are expected to escalate food demands beyond production capacities, and the development of maize lines with better performance under heat stress is desirable. Here, we report that constitutive ectopic expression of a heterologous glutaredoxin S17 from Arabidopsis thaliana (AtGRXS17) can provide thermotolerance in maize through enhanced chaperone activity and modulation of heat stress-associated gene expression. The thermotolerant maize lines had increased protection against protein damage and yielded a sixfold increase in grain production in comparison to the non-transgenic counterparts under heat stress field conditions. The maize lines also displayed thermotolerance in the reproductive stages, resulting in improved pollen germination and the higher fidelity of fertilized ovules under heat stress conditions. Our results present a robust and simple strategy for meeting rising yield demands in maize and, possibly, other crop species in a warming global environment.

A chimeric MERS-CoV virus-like particle vaccine protects mice against MERS-CoV challenge.

BACKGROUND: Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory disease in humans, with a case fatality rate of approximately 35%, thus posing a considerable threat to public health. The lack of approved vaccines or antivirals currently constitutes a barrier in controlling disease outbreaks and spread. METHODS: In this study, using a mammalian expression system, which is advantageous for maintaining correct protein glycosylation patterns, we constructed chimeric MERS-CoV virus-like particles (VLPs) and determined their immunogenicity and protective efficacy in mice. RESULTS: Western blot and cryo-electron microscopy analyses demonstrated that MERS-CoV VLPs were efficiently produced in cells co-transfected with MERS-CoV spike (S), envelope, membrane and murine hepatitis virus nucleocapsid genes. We examined their ability as a vaccine in a human dipeptidyl peptidase 4 knock-in C57BL/6 congenic mouse model. Mice immunized with MERS VLPs produced S-specific antibodies with virus neutralization activity. Furthermore, MERS-CoV VLP immunization provided complete protection against a lethal challenge with mouse-adapted MERS-CoV and improved virus clearance in the lung. CONCLUSIONS: Overall, these data demonstrate that MERS-CoV VLPs have excellent immunogenicity and represent a promising vaccine candidate.

Quantitative differential analysis of norovirus outbreak samples using RT-ddPCR.

Noroviruses cause acute gastroenteritis with symptoms of diarrhoea and vomiting, and their high infectivity allows outbreaks to readily occur. Quickly identifying and isolating potential contaminants is an effective method to prevent the spread of outbreaks. A total of 376 samples collected from nine outbreaks were categorized as either patient, asymptomatic individual, cook or environmental samples, according to the source of contamination. Using real-time PCR and sequencing analysis, norovirus GII genotypes were detected in 34·9% of samples from patients, 19·2% from asymptomatic individuals, 2·4% from the environment and 1·4% from cooks. Our findings showed contrasting results in samples categories quantified based on the limit of blank and detection limit by reverse transcription droplet digital PCR, which is a more sensitive testing method than real-time-PCR.

Anti-Inflammatory and Neuroprotective Mechanisms of GTS-21, an α7 Nicotinic Acetylcholine Receptor Agonist, in Neuroinflammation and Parkinson's Disease Mouse Models.

Neuroinflammation is crucial in the progression of neurodegenerative diseases. Thus, controlling neuroinflammation has been proposed as an important therapeutic strategy for neurodegenerative disease. In the present study, we examined the anti-inflammatory and neuroprotective effects of GTS-21, a selective α7 nicotinic acetylcholine receptor (α7 nAChR) agonist, in neuroinflammation and Parkinson's disease (PD) mouse models. GTS-21 inhibited the expression of inducible nitric oxide synthase (iNOS) and proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated BV2 microglial cells and primary microglia. Further research revealed that GTS-21 has anti-inflammatory properties by inhibiting PI3K/Akt, NF-κB, and upregulating AMPK, Nrf2, CREB, and PPARγ signals. The effects of GTS-21 on these pro-/anti-inflammatory signaling molecules were reversed by treatment with an α7 nAChR antagonist, suggesting that the anti-inflammatory effects of GTS-21 are mediated through α7 nAChR activation. The anti-inflammatory and neuroprotective properties of GTS-21 were then confirmed in LPS-induced systemic inflammation and MPTP-induced PD model mice. In LPS-injected mouse brains, GTS-21 reduced microglial activation and production of proinflammatory markers. Furthermore, in the brains of MPTP-injected mice, GTS-21 restored locomotor activity and dopaminergic neuronal cell death while inhibiting microglial activation and pro-inflammatory gene expression. These findings suggest that GTS-21 has therapeutic potential in neuroinflammatory and neurodegenerative diseases such as PD.

Severe Fever with Thrombocytopenia Syndrome Virus in Ticks in the Republic of Korea.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a zoonotic, tick-borne RNA virus of the genus Bandavirus (Family Phenuiviridae), mainly reported in China, Japan, and the Republic of Korea (Korea). For the purpose of this study, a total of 3,898 adult and nymphal ticks of species Haemaphysalis longicornis (94.2%), Haemaphysalis flava (5.0%), Ixodes nipponensis (0.8%), and 1 specimen of Ixodes ovatus, were collected from the Deogyusan National Park, Korea, between April 2016 and June 2018. A single-step reverse transcriptase-nested PCR was performed, targeting the S segment of the SFTSV RNA. Total infection rate (IR) of SFTSV in individual ticks was found to be 6.0%. Based on developmental stages, IR was 5.3% in adults and 6.0% in nymphs. The S segment sequences obtained from PCR were divided into 17 haplotypes. All haplotypes were phylogenetically clustered into clades B-2 and B-3, with 92.7% sequences in B-2 and 7.3% in B-3. These observations indicate that the Korean SFTSV strains were closer to the Japanese than the Chinese strains. Further epidemiological studies are necessary to better understand the characteristics of the Korean SFTSV and its transmission cycle in the ecosystem.

Heat shock protein 70 could enhance porcine epidemic diarrhoea virus replication by interacting with membrane proteins.

In this study, we investigated the role of heat shock protein 70 (HSP70) in porcine epidemic diarrhoea virus (PEDV) replication. We found that PEDV infection induced strong HSP70 overexpression in the very early stage of infection. We also confirmed that HSP70 overexpression increased the speed of PEDV replication, resulting in the generation of more virions. In contrast, knockout of HSP70 in cells significantly downregulated PEDV protein expression, resulting in a significant reduction in PEDV replication. Most importantly, we confirmed that among the structural proteins of PEDV, membrane (M) proteins have this important role. We found that membrane proteins control cellular HSP70 expression in PEDV-infected cells. We confirmed HSP70/M complex formation by both immunoprecipitation and immunofluorescence assays. Additionally, PEDV M overexpression induced strong HSP70 expression. All our results clearly confirmed that in PEDV-infected cells, the M protein plays a very important role in PEDV replication in collaboration with HSP70.

CGFS-type glutaredoxin mutations reduce tolerance to multiple abiotic stresses in tomato.

Sessile organisms such as plants have adopted diverse reactive oxygen species (ROS) scavenging mechanisms to mitigate damage under abiotic stress conditions. Though CGFS-type glutaredoxin (GRX) genes are important regulators of ROS homeostasis, each of their functions in crop plants have not yet been well understood. We performed a targeted mutagenesis analysis of four CGFS-type GRXs (SlGRXS14, SlGRXS15, SlGRXS16, and SlGRXS17) in tomato plants (Solanum lycopersicum) using a multiplex clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system and found that Slgrxs mutants were more sensitive to various abiotic stresses compared with the wild-type tomatoes. Slgrxs15 mutants were embryonic lethal. Single, double, and triple combinations of Slgrxs14, 16, and 17 mutants were examined under heat, chilling, drought, heavy metal toxicity, nutrient deficiency, and short photoperiod stresses. Slgrxs14 and 17 mutants showed hypersensitivity to almost all stresses while Slgrxs16 mutants were affected by chilling stress and showed milder sensitivity to other stresses. Additionally, Slgrxs14 and 17 mutants showed delayed flowering time. Our results indicate that the CGFS-type SlGRXs have specific roles against abiotic stresses, providing valuable resources to develop tomato and, possibly, other crop species that are tolerant to multiple abiotic stresses by genetic engineering.

Utility of the Patient-Reported Outcomes Measurement Information System (PROMIS) to measure primary health outcomes in cancer patients: a systematic review.

OBJECTIVE: To systematically review the literature on how the Patient-Reported Outcomes Measurement Information System (PROMIS) measure system is used to assess patient-reported outcomes (PROs) in cancer patients. METHODS: We conducted a systematic review following the PRISMA guidelines. Articles were identified through searches of PubMed, EMBASE, and additional manual review of the publications listed on the PROMIS website. We included studies measuring outcomes, including physical function, fatigue, pain, anxiety, and depression in cancer patients. Eligible articles included interventional and observational studies published in English between 2009 and 2019. RESULTS: A total of 1789 records were identified and screened by three reviewers, 118 articles were reviewed in full text, and 42 articles met the inclusion criteria. The majority of studies used the PROMIS measure system to prospectively assess longitudinal changes in PROs; the number of measurements ranges from 2 to 4 with the time points of follow-up set at 3, 6, and 12 months after the baseline assessment. Depression and fatigue were the most frequently measured outcomes. Fixed-length short forms with four items were the most common measure types. A transition toward utilizing a web- or smartphone-based electronic tool was observed to limit the burden of the conventional paper-based survey to collect and store PROs. CONCLUSION: The PROMIS measure system is increasingly popular to measure PROs in cancer patients with acceptance of its various short forms and electronic-based systems to administer data electronically. Findings from this review highlight various aspects of PROMIS and may help health professionals in their choice of PRO tools for optimizing care and support for cancer patients.

Developmental toxicity of 3-phenoxybenzoic acid (3-PBA) and endosulfan sulfate derived from insecticidal active ingredients: Abnormal heart formation by 3-PBA in zebrafish embryos.

Pyrethroid and organochlorine insecticides are enormously used to control agricultural and indoor insect pests. The metabolites of pyrethroid and endosulfan were used to evaluate environmental toxicities using a representative animal model, zebrafish (Danio rerio) embryos in this study. The LC50 values in 3-phenoxy benzoic acid (3-PBA) and endosulfan sulfate (ES) were 1461 µg/L and 1459 µg/L, respectively. At the concentration of 2000 µg/L, spine curvature was observed in the ES-treated embryos. ES showed seizure-like events with an EC50 value of 354 µg/L. At the concentration of 1000 µg/L, the pericardial edema was observed in 3-PBA-treated embryos. The inhibition of heart development and the reduction of beating rates were observed in Tg(cmlc2:EGFP) embryos after the exposure to 3-PBA. Down-regulation of the vmhc gene coding ventricular myosin during heart development was significantly found in 3-PBA-treated embryos at 48 hpf, but recovered afterward. It indicates that ventricular malformation occurred at the initial stage of 3-PBA exposure. Considered together, both 3-PBA and ES need public concerns with periodic monitoring of these metabolites in households and agricultural areas to prevent humans and environmental organisms from their unexpected attacks.

Acute toxicity of the insecticide EPN upon zebrafish (Danio rerio) embryos and its related adverse effects: Verification of abnormal cardiac development and seizure-like events.

Toxicological studies of O-ethyl-O-(4-nitrophenyl) phenylphosphonothioate (EPN) to aquatic vertebrates have been reported, but no reports on toxic mechanism was reported. As zebrafish (Danio rerio) embryos were exposed to EPN, no changes in their survival and hatching rates were observed until 96 h post fertilization (hpf), even at the highest treated concentration of 500 µg/L. In both 250 µg/L and 500 µg/L, edemas were observed in the heart and yolk sac, and a blood pool was also found. Acridine orange staining confirmed apoptotic phynotype, which was the strongest in embryos at 48 hpf. No noticeable difference in the formation and the shape of blood vessels of Tg(fli1a:EGFP) was observed. However, the total body length and number of somite were decreased. Heart formation in Tg(cmlc2:EGFP) were not properly proceeded, and the ventricle did not beat normally at 500 µg/L level. Cardiac development-related genes, myl7 and nppa, were significantly down- and up-regulated in a concentration-dependent manner. The slowed heartbeat was confirmed using Tg(gata1:EGFP), showing stagnant blood flow and seizure-like events were observed. Altogether, EPN can be the cause for the abnormal heart development accompanied by blood stagnation in embryos, interfering normal development with their inner circulatory system.