Complete chloroplast genome and phylogenetic analysis of Amorphophallus paeoniifolius (Araceae).

Amorphophallus paeoniifolius (Dennst.) Nicolson, 1885, often known as elephant foot yam, is a tropical tuber crop that originates from south-east Asia and belongs to the Araceae family. It is known for its high production potential and popularity as a medicinal plant. However, the phylogeny and genes for this species are still unavailable. In this study, the first complete chloroplast genome of A. paeoniifolius was reported and phylogenetic analysis was conducted with Araceae species. The chloroplast genome was 176,258 bp in length with 34.80% overall GC content and includes a large single-copy (LSC) region (93,951 bp), a small single-copy (SSC) region (15,013 bp), and a pair of inverted repeat (IRs) regions (33,647 bp). The chloroplast genome has 130 genes, which include 85 protein-coding genes, 37 tRNA genes, and eight rRNA genes. A maximum-likelihood (ML) phylogenetic analysis indicated that all Amorphophallus species formed a single monophyletic clade with a high bootstrap value and A. paeoniifolius was closely related to A. konjac, A. albus, A. krausei, and A. titanum. The chloroplast genome reported in this study will be useful for further taxonomic and evolutionary studies of Amorphophallus.

Social media users' attitudes toward cyberbullying during the COVID-19 pandemic: associations with gender and verification status.

Introduction: Social media platforms such as Twitter and Weibo facilitate both positive and negative communication, including cyberbullying. Empirical evidence has revealed that cyberbullying increases when public crises occur, that such behavior is gendered, and that social media user account verification may deter it. However, the association of gender and verification status with cyberbullying is underexplored. This study aims to address this gap by examining how Weibo users' gender, verification status, and expression of affect and anger in posts influence cyberbullying attitudes. Specifically, it investigates how these factors differ between posts pro- and anti-cyberbullying of COVID-19 cases during the pandemic. Methods: This study utilized social role theory, the Barlett and Gentile Cyberbullying Model, and general strain theory as theoretical frameworks. We applied text classification techniques to identify pro-cyberbullying and anti-cyberbullying posts on Weibo. Subsequently, we used a standardized mean difference method to compare the emotional content of these posts. Our analysis focused on the prevalence of affective and anger-related expressions, particularly examining variations across gender and verification status of the users. Results: Our text classification identified distinct pro-cyberbullying and anti-cyberbullying posts. The standardized mean difference analysis revealed that pro-cyberbullying posts contained significantly more emotional content compared to anti-cyberbullying posts. Further, within the pro-cyberbullying category, posts by verified female users exhibited a higher frequency of anger-related words than those by other users. Discussion: The findings from this study can enhance researchers' algorithms for identifying cyberbullying attitudes, refine the characterization of cyberbullying behavior using real-world social media data through the integration of the mentioned theories, and help government bodies improve their cyberbullying monitoring especially in the context of public health crises.

PAS-7 is Superior to APAIS for Assessing Preoperative Anxiety in the Chinese Population.

PURPOSE: The purpose of this study was to analyze the reliability and validity of the Perioperative Anxiety Scale-7 (PAS-7), which was created by Chinese medical professionals, by using the State-Trait Anxiety Scale (STAI-S) as the standard for the diagnosis of preoperative anxiety, and to compare whether there is a difference between the PAS-7 and the Amsterdam Preoperative Anxiety and Information Scale (APAIS) in the diagnosis of preoperative anxiety in the Chinese population. DESIGN: This study was an observational study. METHODS: The PAS-7, APAIS, and STAI-S were all completed the day before surgery. The internal consistency test was used to evaluate the scale's reliability, and exploratory factor analysis and confirmatory factor analysis were used to assess the scale's construct validity. Pearson correlation was used to analyze the correlation between PAS-7 and STAI-S, and APAIS. The area under the receiver operating characteristic (ROC) curve was used to compare the diagnostic value of PAS-7 and APAIS. FINDINGS: The PAS-7 Cronbach's α coefficient was 0.804. The indicators of the overall fitting coefficient were within the acceptable range. PAS-7 scores correlated well with STAI-S and APAIS scores (P < .01). The area under the ROC curve of PAS-7 was 0.808 (0.752-0.856), and the area under the ROC curve of APAIS was 0.674 (0.611-0.733). The difference between areas was 0.133 (0.0612-0.206), P < .001, and the diagnostic value of PAS-7 was higher than that of APAIS. CONCLUSIONS: The PAS-7 scale has high reliability and validity and can be used to assess preoperative anxiety in patients undergoing elective surgery. PAS-7 is superior to APAIS for assessing preoperative anxiety in the Chinese population.

Circ_0006949 as a potential non-invasive diagnosis biomarker promotes the proliferation of NSCLC cells via miR-4673/GLUL axis.

The 5-year survival for non-small cell lung cancer (NSCLC) remains <20 %, primarily due to the early symptoms of lung cancer are inconspicuous. Prompt identification and medical intervention could serve as effective strategies for mitigating the death rate. We therefore set out to identify biomarkers to help diagnose NSCLC. CircRNA microarray and qRT-PCR reveal that sputum circ_0006949 is a potential biomarker for the early diagnosis and therapy of NSCLC, which can enhance the proliferation and clone formation, regulate the cell cycle, and accelerate the migration and invasion of NSCLC cells. Circ_0006949 and miR-4673 are predominantly co-localized in the cytoplasm of NSCLC cell lines and tissues; it upregulates GLUL by adsorption of miR-4673 through competing endogenous RNAs mechanism. The circ_0006949/miR-4673/GLUL axis exerts pro-cancer effects in vitro and in vivo. Circ_0006949 can boost GLUL catalytic activity, and they are highly expressed in NSCLC tissues and correlate with poor prognosis. In summary, circ_0006949 is a potential biomarker for the early diagnosis and therapy of NSCLC. This novel sputum circRNA is statistically more predictive than conventional serum markers for NSCLC diagnosis. Non-invasive detection of patients with early-stage NSCLC using sputum has shown good potential for routine diagnosis and possible screening.

LncRNA FOXD2-AS1 promotes the growth, invasion and migration of OSCC cells by regulating the MiR-185-5p/PLOD1/Akt/mTOR pathway.

Although lncRNAs are recognized to contribute to the development of oral squamous-cell carcinoma (OSCC), their exact function in invasion and cell migration is not clear. In this research, we explored the molecular and cellular mechanisms of FOXD2-AS1 in OSCC. Prognostic and bioinformatics analyses were used to test for the differential expression of FOXD2-AS1-PLOD1. Following FOXD2-AS1 suppression or overexpression, changes in cell viability were measured using the CCK-8 test; changes in cell migration and invasion abilities were measured using the migration and the Transwell assay. The expression of associated genes and proteins was found using Western blot and RT-qPCR. Analysis of luciferase reporter genes was done to look for regulatory connections between various molecules. The FOXD2-AS1-PLOD1 pair, which was highly expressed in OSCC, was analyzed and experimentally verified to be closely related to the prognosis of OSCC, and a nomogram model and correction curve were constructed. The inhibition of FOXD2-AS1 resulted in the reduction of cell activity, migration, invasion ability and changes in genes related to invasion and migration. In vivo validation showed that inhibition of FOXD2-AS1 expression slowed tumor growth, and related proteins changed accordingly. The experiments verified that FOXD2-AS1 negatively regulated miR-185-5 p and that miR-185-5 p negatively regulated PLOD1. In addition, it was found that the expression of PLOD1, p-Akt and p-mTOR proteins in OSCC cells was reduced by the inhibition of FOXD2-AS1, and FOXD2-AS1 and PLOD1 were closely related to the Akt/mTOR pathway. Increased expression of FOXD2-AS1 promotes OSCC growth, invasion and migration, which is important in part by targeting miR-185-5 p/PLOD1/Akt/mTOR pathway activity.

Treatment of mixed and refractory post-tuberculosis tracheobronchial stenosis with L-shaped silicone stents: case series and a literature review.

The two patients included in the study had mixed and refractory post-tuberculosis tracheobronchial stenosis (PTTS), having experienced unsuccessful interventional therapies such as balloon dilation and V-shaped stent placement before the operation. Following the secure placement of L-shaped silicone stents, examinations with a fiberbronchoscope during the first and third months post-operation revealed a significant reduction in bronchial mucosa inflammation for both patients. Additionally, the opening diameter of the upper and lower branch segments increased, and chest CT scans indicated a noticeable absorption of left pulmonary lesions. Three months post-operation, fiberbronchoscopy confirmed the stable fixation of the stent without any movement. The patients exhibited substantial improvements in pulmonary function, dyspnea index, and blood gas analysis, with no reported adverse complications. After 7 months, a follow-up fiberbronchoscope for one case revealed excellent stent fixation. Simultaneously, the chest CT scan indicated favorable re-expansion. The placement of L-shaped silicone stents proves effective in preventing displacement, alleviating airway stenosis or obstruction, and ensuring the safety and efficacy of PTTS treatment - particularly in cases where V-shaped silicone stent placement has failed. To our knowledge, this is the first study describing the L-shaped silicone stent in two patients with PTTS.

Successful treatment of severe airway narrowing due to tuberculosis using special L-shaped silicone stentsThis article tells the story of two patients who suffered from a complex lung condition called post-tuberculosis tracheobronchial stenosis (PTTS). Imagine your airways - the tubes that carry air to your lungs - getting severely scarred and narrowed due to a past bout with tuberculosis. These two patients had tried previous treatments like balloon dilation (where a small balloon is inflated inside the narrowed airway to widen it) and using V-shaped stents (flexible supports placed in the airway to keep it open), but these methods didn't provide lasting relief. In this innovative approach, doctors used L-shaped silicone stents specifically designed to fit in the affected parts of the patients' airways. After placing these stents, regular checks showed remarkable improvements. The swelling in the airway lining reduced significantly, and the openings leading to the upper and lower parts of the lungs got wider. Chest X-rays (CT scans) even showed that the patient's left lung was healing well. Three months later, the stents stayed firmly in place, and neither patient experienced any problems. Breathing became easier, lung function tests improved, and blood tests showed better oxygen levels. Seven months down the line, one patient continued to do extremely well, with the stent securely fixed and the chest scan showing good lung expansion. This groundbreaking study shows that using L-shaped silicone stents can effectively treat PTTS when other methods fail. Not only do they stay in place, preventing blockages, but they also safely and effectively alleviate narrowing of the airways. It's the first time such L-shaped stents have been used successfully in PTTS patients, offering new hope for those facing similar challenges.

Corrigendum: Phenylpropane biosynthesis and alkaloid metabolism pathways involved in resistance of Amorphophallus spp. against soft rot disease.

[This corrects the article DOI: 10.3389/fpls.2024.1334996.].

Electrochemical Restructuring Driven Catalytic Cycle of Bi-Based Heterojunctions for High-Performance Lithium-Sulfur Batteries.

Restructuring is an important phenomenon in catalytic reactions. Conversion-type materials with suitable redox potential may undergo in situ electrochemically driven restructurings and induce highly active catalytic sites in a working lithium-sulfur battery. Herein, driven by the electrochemical conversion reaction of BiVO4, a reversible catalytic cycle of Bi/amorphous Li3VO4 (a-Li3VO4) and Bi2S3/a-Li3VO4 heterojunctions is constructed, which targets the oxidation of Li2S and the conversion of polysulfide, respectively. The heterostructures and electrochemically driven size confinement provide abundant sites for shuttle restraining and sulfur conversion. Especially, the p-block Bi and Bi2S3 could dramatically reduce the conversion energy barriers of Li2S and polysulfide by virtue of the p-p orbital hybridization, promoting bidirectional reactions of the sulfur cathode. As a result, the corresponding sulfur cathode possesses a high reversible capacity of 7.5 mAh cm-2 after 120 cycles under a high sulfur loading of 10.3 mg cm-2 with a current density of 0.38 mA cm-2. This study furnishes a feasible scheme to obtain highly effective catalysts for bidirectional sulfur redox by utilizing the electrochemically induced restructuring.

The endophytic fungal community plays a crucial role in the resistance of host plants to necrotic bacterial pathogens.

Konjac species (Amorphophallus spp.) are the only plant species in the world that are rich in a large amount of konjac glucomannan (KGM). These plants are widely cultivated as cash crops in tropical and subtropical countries in Asia, including China. Pectobacterium carotovorum subsp. carotovorum (Pcc) is one of the most destructive bacterial pathogens of konjac. Here, we analyzed the interactions between Pcc and susceptible and resistant konjac species from multiple perspectives. At the transcriptional and metabolic levels, the susceptible species A. konjac and resistant species A. muelleri exhibit similar molecular responses, activating plant hormone signaling pathways and metabolizing defense compounds such as phenylpropanoids and flavonoids to resist infection. Interestingly, we found that Pcc stress can lead to rapid recombination of endophytic microbial communities within a very short period (96 h). Under conditions of bacterial pathogen infection, the relative abundance of most bacterial communities in konjac tissue decreased sharply compared with that in healthy plants, while the relative abundance of some beneficial fungal communities increased significantly. The relative abundance of Cladosporium increased significantly in both kinds of infected konjac compared to that in healthy plants, and the relative abundance in resistant A. muelleri plants was greater than that in susceptible A. konjac plants. Among the isolated cultivable microorganisms, all three strains of Cladosporium strongly inhibited Pcc growth. Our results further elucidate the potential mechanism underlying konjac resistance to Pcc infection, highlighting the important role of endophytic microbial communities in resisting bacterial pathogen infections, especially the more direct role of fungal communities in inhibiting pathogen growth.

Phenylpropane biosynthesis and alkaloid metabolism pathways involved in resistance of Amorphophallus spp. against soft rot disease.

Soft rot of konjac (Amorphophallus spp.) is a devastating disease caused by the bacterium Pectobacterium carotovorum subsp. carotovorum (Pcc) with serious adverse effects on plantation development, corm quality and crop yield due to the current lack of effective control measures. The main objective of the present study was to elucidate the mechanisms underlying plant resistance to soft rot disease. A combination of transcriptomic and metabolomic analyses demonstrated significant enrichment of differentially expressed genes (DEG) and differentially accumulated metabolites (DAM) associated with plant hormones, phenylpropanoid biosynthesis and, in particular, alkaloid metabolism, in Amorphophallus muelleri following Pcc infection compared with A. konjac, these data implicate alkaloid metabolism as the dominant mechanism underlying disease resistance of A. muelleri. Quantitative real-time polymerase chain reaction analysis further revealed involvement of PAL, CYP73A16, CCOAOMT1, RBOHD and CDPK20 genes in the response of konjac to Pcc. Analysis of the bacteriostatic activities of total alkaloid from A. muelleri validated the assumption that alkaloid metabolism positively regulates disease resistance of konjac. Our collective results provide a foundation for further research on the resistance mechanisms of konjac against soft rot disease.

An optimized approach for solar concentrating parabolic dish based on particle swarm optimization-genetic algorithm.

Parabolic dish concentrators have demonstrated the highest thermal and optical efficiencies among the available concentrator options. This paper proposes a novel design approach for fabricating large parabolic dish concentrators by employing compliant petals optimized through Particle Swarm Optimization-Genetic Algorithm (PSO-GA). The design concept involves using cables to pull the outer corners of the petals towards the center, resulting in the creation of finely formed dish mirrors. These mirrors are constructed from thin, optimal-shaped metal petals with highly reflective surfaces. In addition, an analytical model is presented to optimize the bending stiffness of the petals by strategically arranging punched holes using PSO-GA. The proposed design concept is validated through the application of Finite Element Analysis and ray tracing software, specifically LightTools, as well as laboratory experiments. Based on the demonstration with a 1m-diameter parabolic dish, it was observed that a receiver surface with a radius of 3.5 cm could achieve an impressive sunlight collection efficiency of up to 98%. This innovative design approach offers several advantages, including simplified fabrication and transportation of flat mirror elements to field sites, which can potentially lead to cost reductions and highly efficient solar energy solutions.

The complete chloroplast genome sequence of Amorphophallus konjac (Araceae) from Yunnan, China and its phylogenetic analysis in the family Araceae.

This work determined and analyzed the complete chloroplast genome sequence of Amorphophallus konjac K. Koch ex N.E.Br 1858 from Yunnan, China. The genome size was 167,470 bp, of which contains a large single-copy region (LSC 93,443 bp), a small single-copy region (SSC 21,575 bp), and a pair of inverted repeat regions (IR 26,226 bp). The chloroplast genome has 131 genes, including 86 protein-coding genes, 37 tRNAs, and eight rRNAs. A previous study reported deletion of accD, psbE, and trnG-GCC genes in the A. konjac chloroplast genome. Our study supports the conservative structure of A. konjac and does not support the gene deletion mentioned above. Phylogenetic analysis indicated that A. konjac shares a close relationship with another A. konjac (collected from Guizhou) and A. titanium by forming a clade in the genus Amorphophallus. Our results provide some useful information to the evolution of the family Araceae.

Invasion by exogenous RNA: cellular defense strategies and implications for RNA inference.

Exogenous RNA poses a continuous threat to genome stability and integrity across various organisms. Accumulating evidence reveals complex mechanisms underlying the cellular response to exogenous RNA, including endo-lysosomal degradation, RNA-dependent repression and innate immune clearance. Across a variety of mechanisms, the natural anti-sense RNA-dependent defensive strategy has been utilized both as a powerful gene manipulation tool and gene therapy strategy named RNA-interference (RNAi). To optimize the efficiency of RNAi silencing, a comprehensive understanding of the whole life cycle of exogenous RNA, from cellular entry to its decay, is vital. In this paper, we review recent progress in comprehending the recognition and elimination of foreign RNA by cells, focusing on cellular entrance, intracellular transportation, and immune-inflammatory responses. By leveraging these insights, we highlight the potential implications of these insights for advancing RNA interference efficiency, underscore the need for future studies to elucidate the pathways and fates of various exogenous RNA forms, and provide foundational information for more efficient RNA delivery methods in both genetic manipulation and therapy in different organisms.

Characteristics of Amorphophallus konjac as indicated by its genome.

Amorphophallus konjac, belonging to the genus Amorphophallus of the Araceae family, is an economically important crop widely used in health products and biomaterials. In the present work, we performed the whole-genome assembly of A. konjac based on the NovaSeq platform sequence data. The final genome assembly was 4.58 Gb with a scaffold N50 of 3212 bp. The genome includes 39,421 protein-coding genes, and 71.75% of the assemblies were repetitive sequences. Comparative genomic analysis showed 1647 gene families have expanded and 2685 contracted in the A. konjac genome. Likewise, genome evolution analysis indicated that A. konjac underwent whole-genome duplication, possibly contributing to the expansion of certain gene families. Furthermore, we identified many candidate genes involved in the tuber formation and development, cellulose and lignification synthesis. The genome of A. konjac obtained in this work provides a valuable resource for the further study of the genetics, genomics, and breeding of this economically important crop, as well as for evolutionary studies of Araceae family.

A segmentation model to detect cevical lesions based on machine learning of colposcopic images.

Background: Semantic segmentation is crucial in medical image diagnosis. Traditional deep convolutional neural networks excel in image classification and object detection but fall short in segmentation tasks. Enhancing the accuracy and efficiency of detecting high-level cervical lesions and invasive cancer poses a primary challenge in segmentation model development. Methods: Between 2018 and 2022, we retrospectively studied a total of 777 patients, comprising 339 patients with high-level cervical lesions and 313 patients with microinvasive or invasive cervical cancer. Overall, 1554 colposcopic images were put into the DeepLabv3+ model for learning. Accuracy, Precision, Specificity, and mIoU were employed to evaluate the performance of the model in the prediction of cervical high-level lesions and cancer. Results: Experiments showed that our segmentation model had better diagnosis efficiency than colposcopic experts and other artificial intelligence models, and reached Accuracy of 93.29 %, Precision of 87.2 %, Specificity of 90.1 %, and mIoU of 80.27 %, respectively. Conclution: The DeepLabv3+ model had good performance in the segmentation of cervical lesions in colposcopic post-acetic-acid images and can better assist colposcopists in improving the diagnosis.

Comparative metabolomics analysis reveals dynamic changes in carbohydrate profiles of corms during the "relay growth" of konjac (Amorphophallus muelleri).

The type and content of carbohydrates in konjac corms are an essential factors in determining the quality of konjac; however, the pattern of carbohydrate changes and the mechanism regulating the development of mother and daughter corms in the "relay growth" process of Amorphophallus muelleri remain unclear. This study aimed to investigate changes in corm carbohydrates during the growth cycle of A. muelleri and to compare the carbohydrate composition and the expression of related genes between mother and daughter corms. Integrated metabolome and RNA-seq analyses identified 37 differential metabolites as well as 8074 genes that were differentially expressed between mother and daughter corms, the majority of which were involved in starch and sucrose metabolism. More than 80% of the differential metabolites, including sucrose and starch, tended to accumulate in the mother corms; however, konjac glucomannan (KGM), as one of the most important carbohydrates and its major component of the corm, accumulated in higher amounts in the daughter corms. In addition, the expression of invertase and alpha-amylase that promote the breakdown of sucrose and starch was 351.78- and 15.63-fold higher, respectively, in the daughter corm, whereas that of the starch synthesis gene AkWAXY was only 0.096 times as high as in the mother corms. Furthermore, the level of cellulose synthase-like protein G, which promotes KGM synthesis, was 3.85 times higher in daughter corms compared to mother corms. Thus, we inferred that the daughter and mother corms had two distinct carbohydrate utilization strategies. This study provides insights into temporal changes in carbohydrates during the growth cycle of A. muelleri.

LncPCD: a manually curated database of experimentally supported associations between lncRNA-mediated programmed cell death and diseases.

Programmed cell death (PCD) refers to controlled cell death that is conducted to keep the internal environment stable. Long noncoding RNAs (lncRNAs) participate in the progression of PCD in a variety of diseases. However, no specialized online repository is available to collect and store the associations between lncRNA-mediated PCD and diseases. Here, we developed LncPCD, a comprehensive database that provides information on experimentally supported associations of lncRNA-mediated PCD with diseases. The current version of LncPCD documents 6666 associations between five common types of PCD (apoptosis, autophagy, ferroptosis, necroptosis and pyroptosis) and 1222 lncRNAs in 331 diseases. We also manually curated a wealth of information: (1) 7 important lncRNA regulatory mechanisms, (2) 310 PCD-associated cell types in three species, (3) detailed information on lncRNA subcellular locations and (4) clinical applications for lncRNA-mediated PCD in diseases. Additionally, 10 single-cell sequencing datasets were integrated into LncPCD to characterize the dynamics of lncRNAs in diseases. Overall, LncPCD is an extremely useful resource for understanding the functions and mechanisms of lncRNA-mediated PCD in diseases. Database URL:  http://spare4.hospital.studio:9000/lncPCD/Home.jsp.

Local government debt and labor income share: Evidence from China.

This study employs a CES production function to construct a theoretical model of labor income share and uses a two-way fixed effects model to test the causal effects of local government debt (LGD) on the labor income share of enterprises. Local government debt governance policies are utilized as exogenous shocks, and a DID (Difference-in-Differences) model is applied for endogeneity testing. The results have passed a series of robustness checks. The findings suggest that LGD decreases the share of firms' labor income. The mechanism analysis suggests that LGD lowers the labor remuneration of residents, the employment of labor in enterprises, and the size of bank loans mainly; while raising the cost of using funds in enterprises. Moreover, this negative effect is more apparent in non-state-owned enterprises, small and medium-sized enterprises, and enterprises with high financing constraints. This study presents new evidence on how the labor income share of enterprises is affected from the perspective by local governments in China. It has important implications for further deepening local government debt governance and achieving common prosperity.

Purification and functional validation of LtCas12a protein.

Here, we present a protocol for generating LtCas12a protein recognizing distinct TTNA (N represented A, T, C, G) protospacer adjacent motif sequence. We describe steps for transforming and harvesting bacterial cells and protein purification including nickel affinity chromatography and dialysis. We then detail procedures for verification of LtCas12a with cis- and trans-cleavage activities. For complete details on the use and execution of this protocol, please refer to Chen et al. (2023).1.

Temporospatial inhibition of Erk signaling is required for lymphatic valve formation.

Intraluminal lymphatic valves (LVs) and lymphovenous valves (LVVs) are critical to ensure the unidirectional flow of lymphatic fluid. Morphological abnormalities in these valves always cause lymph or blood reflux, and result in lymphedema. However, the underlying molecular mechanism of valve development remains poorly understood. We here report the implication of Efnb2-Ephb4-Rasa1 regulated Erk signaling axis in lymphatic valve development with identification of two new valve structures. Dynamic monitoring of phospho-Erk activity indicated that Erk signaling is spatiotemporally inhibited in some lymphatic endothelial cells (LECs) during the valve cell specification. Inhibition of Erk signaling via simultaneous depletion of zygotic erk1 and erk2 or treatment with MEK inhibitor selumetinib causes lymphatic vessel hypoplasia and lymphatic valve hyperplasia, suggesting opposite roles of Erk signaling during these two processes. ephb4b mutants, efnb2a;efnb2b or rasa1a;rasa1b double mutants all have defective LVs and LVVs and exhibit blood reflux into lymphatic vessels with an edema phenotype. Importantly, the valve defects in ephb4b or rasa1a;rasa1b mutants are mitigated with high-level gata2 expression in the presence of MEK inhibitors. Therefore, Efnb2-Ephb4 signaling acts to suppress Erk activation in valve-forming cells to promote valve specification upstream of Rasa1. Not only do our findings reveal a molecular mechanism of lymphatic valve formation, but also provide a basis for the treatment of lymphatic disorders.