Aerobic rice cultivation progresses water productivity, and it can save almost 50% of irrigation water compared to lowland rice with the appropriate development of genotypes and management practices. Two field trials were conducted during 2020, and 2021 seasons to determine the validation of different rice varieties under aerobic cultivation based on their plant defense system, physio-morphological traits, stress indices, grain yield, and water productivity. The experiments were designed in a split-plot design with four replications. Two planting methods, transplanting and aerobic cultivation, were denoted as the main plots, and ten rice genotypes were distributed in the subplots. The results revealed that the planting method varied significantly in all measured parameters. The transplanting method with well watering had the highest value of all measured parameters except leaf rolling, membrane stability index, antioxidant, proline, and the number of unfilled grains. EHR1, Giza179 and GZ9399 as well as A22 genotypes a chief more antioxidant defense system that operated under aerobic conditions. Giza179, EHR1, GZ9399, and Giza178 showed high cell membrane stability and subsequently high validation under such conditions, and also showed efficiency in decreasing water consumption and improving water use efficiency. In conclusion, this study proves that Giza179, EHR1, GZ9399, Giza178, and A22 are valid genotypes for aerobic conditions. Oryza sativa L.) is an essential staple crop that secures food for almost two-thirds of the world's population1. The cultivation of rice occupies about twenty percent of the agricultural land area planted for cereals2. In Egypt, rice production faces many grand challenges such as freshwater scarcity, salt stress, the high cost of inputs, and overpopulation. It is estimated that rice's annual production deficit will increase from 400,000 t in 2016 to 800,000 by 20303. Nowadays, the insufficient available freshwater for agriculture pose a challenge for big water consumer crops production . The 79 million hectares of irrigated rice worldwide consume 34–43% of the irrigation water around the globe4.Rice  is easy to use for determining physiological status of plant under drought stress7. Also, RWC% is a vital guide to recognize the tolerant variety under various stresses such as aerobic cultivation with minimal water supply13 and salinity18. Decreasing freshwater resources are threatening agricultural production in many parts of the world19.Water resources management is one of the main factors affecting rice production globally20. Among all, RWC%, leaf rolling, grain yield, leaf drying, root/shoot ratio, and root length offer high scope for rice development for aerobic cultivation7. There is an ever-increasing need to improve water productivity to stay commercially competitive. Abiotic stresses, namely, water deficit developed by aerobic cultivation or drought, are the most critical factors negatively influencing growth, and several physiological and biochemical processes21. Plants developed many adaptation mechanisms, such as antioxidant defense systems, i.e., Superoxide dismutase (SOD), Catalase (CAT), and Peroxidase (POD). These enzymatic components play a central role in the defense system. The SOD decomposes superoxide (O2) to H2O2 by catalyzing superoxide anion radical disputation. The POD and CAT decrease H2O2 in water using various substrates as electron donors to preserve the roots and help the plants endure stress25. Aerobic rice improves water productivity with the appropriate development of genotypes and management practices7. Aerobic rice is specially developed rice, combines drought tolerance of upland rice and yield potential in lowland rice6. Therefore, it can save almost 50% of irrigation water compared to lowland rice. Aerobic rice cultivars with high yield potential and moderate drought stress tolerance have been developed through traditional upland farming with improved irrigated cultivars6. Aerobic rice cultivars achieve high yields under appropriate management practices and deep root systems, as well as water stress tolerance in the vegetative and reproductive stages26.Plants suffer from various stresses, such as drought, low and high temperatures, salinity, etc.The objectives of the current study were (1) to determine the physiological, morphological, and stress indices of some Egyptian rice varieties under aerobic cultivation; (2) the possibility to explore such type of cultivation in Egypt, and (3) to nominate valid Egyptian rice varieties to lead cultivation under aerobic conditions.Hordeum spp.). The results of the chemical soil properties are presented in Table The experiment was conducted at the experimental farm of Sakha Agricultural Research Station, Kafr El-Sheikh, Egypt, during 2020 and 2021 seasons to compare the performance of some rice varieties under normal transplanting and aerobic cultivation conditions. All experiments were preceded by a barley crop (−1) was applied in three equal doses . For the permanent field, phosphorus fertilizer at the rate of 35.5 kg P2O5 ha−1 was basally applied to the soil during the land preparation. The potassium fertilizer (57 kg K2O ha−1) was added as a basal dose and incorporated into dry soil, and zinc (Zn SO4) was applied at the rate of 23.9 kg ha−1 before continuous flood irrigation. For transplanting cultivation method, the seeds of different rice varieties were sown on May, 7th, and after 30 days, the seedlings were transplanted in the permanent field at 20 × 20 cm plant spacing in a plot area of 10 m2. As for aerobic cultivation, the 4 dry seeds/hill were sown at the space of 15 cm between each pair of hills and 30 cm between the two rows on the both sides of furrow. Origin, parentage, and variety group are presented in Table The experiment was performed in a split plot design with four replications. The main plots were devoted to two planting methods: normal transplanting and aerobic cultivation, and subplots were occupied by ten varieties. As for the aerobic cultivation pattern, the dry seeds were used in dry land on furrows. The furrow size was 70 cm from mid-bottom of each of the two furrows, with a depth of 30 cm. Rice irrigation during the first 25 days of rice growth was done as in drill seeded rice, and then watering was done each eight days to maturity by filling the bottom of furrow. The recommended dose of nitrogen fertilizer , chlorophyll content (with SPAD meter M502), stability index, RWC%, length, and root volume. Also, some enzymatic antioxidants such as catalase, peroxidase, and super oxidase dismutase, and proline activity leaf content were assessed. In the field, leaf rolling was visually estimated according to the IRRI scale. The stomatal conductance was measured at head in the field. Root length and root volume was estimated as described by Bradford2 and homogenized in 2.0 ml of extraction buffer: 100 mm potassium phosphate (pH 7.8), 0.1 mm ethylene diaminetetra acetic acid (EDTA) and 10 mm ascorbic acid. The homogenate was centrifuged at 13,000g for 15 min at 4 °C. CAT activity was assayed in the supernatant at 240 nm based on the consumption of H2O228. The activity of SOD was determined at 560 nm according to the method of Beauchamp and Fridovich29. Activity of POX was measured at 420 nm as described by Kar and Mishra30.Leaf samples  were soaked in liquid Ng. From extract, 2 mL of supernatant was added to 2 mL of ninhydrin reagent and 2 mL of glacial acetic acid. Proline was determined as mg g−1 FW using a spectrophotometer.Leaf sample (0.3 g) were placed in 3% sulphosalicylic acid and centrifuged for 20 min at 3000×31 as the following:RWC was calculated according to González and González32, the membrane stability index (MSI) was calculated as following equationYoungest leaf tissues (0.2 g) were washed by deionized water, cut into sections (1 cm length), placed in 10 mL deionized water, and heated at 40 °C in a water bath for 30 min. Then the electrical conductivity (EC1) was measured by using a conductivity meter . Subsequently, the content was boiled for 10 min in a boiling water bath (100 °C), and the conductivity (EC2) was measured. Finally, by using the formula described by Premachandra et al.−2 s−1) was measured at the heading stage via a portable photosynthesis measurement system  according to Hubbard et al.33.The stomatal conductance (gs) , peroxidase (POX) and superoxide dismutase (SOD) increase in under drought conditions. These antioxidant enzymes can protect cells from oxidative damage. Similarly, proline synthesis increased under drought conditions, showing that proline could also act as part of a survival mechanism under drought50. Moreover, drought leads to an increase in proline accumulation, which proline helps to maintain tissue water status. Accordingly, rice plants adapted to drier environments have increased the activity of antioxidant enzymes to eliminate ROS13. Continuously, the varieties; EHR1, Giza179, GZ9399, Giza178, and A22 showed apparent capacity to exert an avoidant concentration of proline under aerobic cultivation (stress conditions), which acts as an osomo-protectant and water supplier under drought. The other genotypes including Giza177, Sakha102 and Sakha106 had low efficiency to accumulate sufficient level of proline to enable them to grow healthy under aerobic conditions, indicating their drought sensitivity. Like antioxidants, all tested rice entries were at the same level of significance regarding to the proline accumulation under the transplanting method (well watering). Therefore, the ability of rice genotype to accumulate continuously reasonable concentrations of both proline and antioxidants could be recognized as valid genotype under aerobic conditions.From going findings, the tested rice genotypes were at par regarding the antioxidants plant content under the transplanting method (well watering) because gene expression less and low free radical formation. Under aerobic conditions, there are great variations among genotypes with respect to their ability to produce more antioxidant against free radicals development under such stress. Rice genotypes of EHR1, Giza179, GZ9399, Giza178 and A22 had high concentration of enzymatic antioxidants comparing others under the same conditions and particularly under aerobic condition in the terms of low water input. On contrary, Giza177, Sakha102 and Sakha106 showed weakness to release reasonable concentrations of antioxidants under aerobic conditions (stress case) even comparing to their concentration under transplanting. Oxidative damage by releasing reactive oxygen species (ROS) under abiotic stresses; high and low temperature, salt stress, water stress, and water dehydration represents a negative effect on plant growth and yield. One of the unavoidable consequences of drought stress is raising ROS production in the different cellular compartments, namely in the chloroplasts, the peroxisomes and the mitochondria. Drought caused greater inhibition of photosystem II quantum efficiency, carboxylation efficiency, and photosynthetic capacity parameters as a result of more accumulation of reactive oxygen species (ROS)51. Aerobic cultivation significantly decreased the measured physio-morphological such as leaf area index, flag leaf area, and chlorophyll content, but increased leaf rolling. The ten rice genotypes differed regarding the change rate under aerobic conditions owing to their adaptation capabilities. Like the antioxidant concept, Giza179, EHR1, GZ9399, Giza178 and A22 as one resilience category, showed higher leaf area index, flag leaf area, chlorophyll content and low leaf rolling combined with high stomata conductance under aerobic conditions irrespective of transplanting method. The high physio-morphological concept possessed by the resilience group might be reflected on raising its adaption, growth, photosynthesis, development and finally sink. On contrast, the worse category varieties including Giza177, Sakha102 and Sakha107 brought severe reduction in its physio-morphological traits along with high leaf rolling indicating its invalidation under aerobic conditions. It is important to address that high leaf roiling is a sensitivity indicator, not tolerance, which was matched with poor growth. Furthermore, the reduction in chlorophyll of sensitive varieties might affect the growth and physiological events such as photosynthesis and energy synthesis. Generally, the tolerant group keeps reasonable root length with proper volume under aerobic conditions comparing to other group which failed to give a good root system. It became clear that good root system is an essential option for validation under aerobic conditions. High leaf rolling for sensitive group might be affected the stomatal conductance and subsequently reducing gas exchange and restricting photosynthesis. One of the essential defense mechanisms in plants is avoiding drought, by adjusting some plant attributes, such as the reduction of leaf rolling or leaf size.Water deficit causes negative effects on the enzymes activities resulting in many changes in plant tissues, cell damage, nutrient uptake and photosynthesis rate52. Cell membrane stability diminished rapidly in Kentucky bluegrass exposed to drought and heat stress simultaneously53. The decrease in chlorophyll and increase in EL% may be due to the role of free radicals in damaging the membrane phospholipids of the cell wall and plant pigments. The antioxidant enzymes are the most efficient mechanisms against oxidative stress. Apart from catalase, various peroxidases and peroxiredoxins, four enzymes are involved in the ascorbate–glutathione cycle, a pathway that allows the scavenging of superoxide radicals and H2O227. The cell membrane stability was reduced under aerobic conditions, but the genotypes that possessed high concentrations of measured antioxidants under the current study kept high capability of cell membrane without damage such as GZ9399, Giza179 and EHR1. The previous varieties had low stability index, since a low index is favorable that indicating a low EL%. The low EL% provided the ability of these varieties to create satisfied defense system like antioxidants. The antioxidant could eliminate the hazard free radical developed under drought stress, which damaged the cell membrane55. The results pertaining to sensitive varieties such as Giza177, Sakha102 and Sakha106 confirmed the previous concept, which had a high stability index that matched the low concentration of antioxidants indicated apparent cell membrane damage. The damage of cell membrane induced more EL% of plant cell that was hold true with drought sensitive varieties such Giza177. Proline is acting as antioxidant and as osmo-protectant material under abiotic stresses, particularly water deficit and salt stresses11. Improved water status may be achieved through osmotic adjustment and/or changes in cell wall elasticity that results in maintaining physiological activity for extended periods of drought. Giza179, GZ9399 and EHR1 showed high proline accumulation that was completely matched with a high RWC under aerobic conditions.It is well known that the conservation of integrity and stability of membranes under water stress is a major component of drought tolerance in plants56. Again, EHR1, GZ9399 and Giza179 might have a high affinity to regulate stomata conductance under aerobic conditions with high efficiency that might be due to their ability to do balance between the K and ABA concentration in the guard cells. Furthermore, The mentioned genotypes as resilience one based on previous findings showed less leaf rolling under aerobic conditions, reflecting on optimum regulating of stomata conductance and subsequently CO2 sufficient supply needed for photosynthesis. The group including Giza177, Sakha102 and Sakha106 exhibited low stomata conductance owing to sharp leaf rolling for a long time under aerobic conditions and poor adaptation. Meanwhile, aerobic cultivation showed a reduction in such traits with various levels according to the adaptation abilities of varying genotypes. Less water input might have increased water loss through stomata, and increased respiration rates resulted in a reduction of the most of the physiological processes and photosynthetic rate57. Rice plants adapted to dry environments develop fewer stomata on the surface to avoid losing more water and growth limiting, also, decreased transpiration rate as a function enhanced the drought tolerance. The management of stomata conductance is one of the most important parameters for plant resistance under stress.The fast and first response of plants to water deficit is the closure of their stomata to minimize the transpiration of water. Recently, stomatal closure was generally accepted to be the main determinant for decreased photosynthesis under mild to moderate drought32. The previous concept was found under aerobic conditions comparing to transplanting method (well water supply). This may be attributed to the role of water and nutrients for producing new tillers and total number of grains/panicle6. Aerobic cultivation significantly reduced the number of filled grains combined with increasing unfilled grain, but the tolerant varieties; Giza79, EHR1, GZ9399, A22, and Giza178 were less affected comparing to another opposite group involving Giza177, Sakha102 and Sakha106. Low water supply under aerobic conditions might affect metabolism, development, photosynthesis assimilates and filled grains. Giza179, EHR1, GZ9399, A22 and Giza178 were found effective to developed different drought mechanisms, which reflected on their healthy growth and contentment sources that ensure proper sink along with heavy grain (panicle). Well sources and sink of tolerant rice genotype brought acceptable grain yield under aerobic conditions. The opposites was corrected with well watering under transplanting regarding plant development, physiological processes such as photosynthesis, enzyme activity, which assimilates transportation from source to sink, resulting in improved yield and yield components of rice58. Grain yield was reduced as a result of lower amounts of water supply. The reduction in grain yield might be attributed to declining in 1000 grain weight and spikelets/panicle51. Water deficit during early flowering reduced the number of fertile spikelets and resulted in decreased final grain yield production, this might also be associated with the reduction in leaf area, lower photosynthetic rates, and assimilate transportation from source to sink and dry matter partitioning59.Aerobic conditions with low water input might reduce cell division, elongation, and plant height as well as panicle length; also, low water input resulted in a low number of panicle/hillIt is concluded that water deficit stress greatly influenced physiological functions and biochemical activities that negatively affect plant growth. Superoxide dismutase and catalase as well as, proline and RWC increased under drought stress condition, particularly with tolerant varieties, indicating that the tolerance to aerobic stress is not imputable to a single component, but it is a multifactorial response. Thereby, based on the performance of growth, biochemical, yield, and yield attributes, Giza179, GZ9399, EHR1 and A22, and Giza178 is recommended to lead cultivation under aerobic conditions.Supplementary Figures.


Nanoparticles were characterized by UV–vis spectroscopy, dynamic light scattering, and total reflectance X-ray fluorescence spectroscopy, whereas the prepared substrates were characterized by scanning electron microscopy and Raman spectroscopy with a model molecule, malachite green. The relationship between Raman intensity and the loading of malachite green dye exhibited linearity, indicating the uniform spatial distribution of hotspots across the substrate. The limit of detection was determined as 2.9 μM of malachite green when 10 uL was distributed over a ca. 25 mm2 surface area. Moreover, the same substrate, after thorough washing in ethanol, was successfully employed for the detection of bovine serum albumin at a concentration level of 55 μg mL−1, demonstrating its reusability and versatility. Our findings highlight the potential of these substrates for various applications in biomedical research, clinical diagnosis, and beyond.Raman spectroscopy is a powerful analytical technique widely used for quantitative and qualitative analysis. However, the development of inexpensive, reproducible, and reusable enhancing substrates remains a challenge for material scientists and analytical chemists. In this study, we address this challenge by demonstrating the deposition of core–shell nanoparticles consisting of a gold core and a thin inert SiO Over the past century, Raman spectroscopy has developed into a powerful analytical tool in various fields, including chemistry , biology5–106 in comparison with regular Raman measurements, whereas the chemical mechanism adds another order or two to the EF due to charge transfer between either the molecules themselves [6–108. Today, the origin of Raman enhancement is described with electromagnetic (EMM) and chemical (ChM) mechanisms. The electromagnetic mechanism is associated with plasmon excitation in metallic particles rich with electrons, as well as plasmon interaction in close proximity to the particle surface. The enhancement factor (EF) for the electromagnetic mechanism is typically about 10emselves ,12. ThusIn recent years, gold nanoparticles (AuNPs) have emerged as one of the most promising materials for SERS substrates due to their unique chemical (relatively stable to oxidation and surface reactions), optical (matching with common laser wavelength used in SERS), and electronic properties . A number of methods to synthesize AuNPs with different sizes and shapes have been developed, although the classical approach known as Frens–Turkevich synthesis  prevailsThe advances in nanotechnology have enabled the development of Raman-enhancing particles covered with a thin layer of oxides such as silica or alumina, the so-called shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS). SHINERS have greatly enhanced the sensitivity of Raman spectroscopy and allowed for the detection of trace amounts of molecules, making them useful in a variety of fields, such as infectious disease diagnostics and food safety monitoring . DespiteRaman spectroscopy has been widely reported and recognized for its capability to detect a diverse range of small peptide molecules such as bovine serum albumin (BSA) ,23. MoreFinally, for successful analytical application, it is crucial that Raman-enhancing substrates exhibit not only enhanced signals but also maintain uniformity and linearity across a wide range of analyte concentrations . The relIn this study, we present a novel approach for the preparation of reusable SERS substrates based on gold nanoparticles with a silica shell deposited on a glass slide within a confined space. The present configuration was designed to minimize the undesirable “coffee-ring” effect commonly observed in conventional deposition methods, ensuring uniformity and reproducibility in the deposition process, which is crucial for the practical applications of SERS substrates. Such uniformity and reproducibility in the deposition process will directly affect the quality of the substrate and its enhancing properties. The synthesized nanoparticles were thoroughly characterized by instrumental methods. The enhancing properties of the prepared SERS substrates were investigated using Raman spectroscopy, employing malachite green as a model molecule. Furthermore, the substrates demonstrated excellent stability and reproducibility as shown by Raman mapping of the sample surface, allowing for repeated detection and washing of deposited analyte molecules without any visible damage. Finally, in a practical demonstration of their capabilities, we applied the substrates to detect bovine serum albumin (BSA), a widely studied peptide, known for its relevance in biomedical research and clinical diagnosis. The results clearly indicated that our SERS substrates were capable of detecting BSA at low concentrations, revealing their high sensitivity and potential utility in the field of bioanalysis, biomedical research, and clinical diagnosis.Reagents. Solutions of tetrachloroauric acid in HCl , (3-aminopropyl)triethoxysilane  were purchased from Aladdin, China. Sodium citrate  and silver nitrate  were received from Innochem, China. Ascorbic acid (99.9%) was bought from Meligen, Russia. Sodium silicate (Na2SiO3·9H2O) was ordered from Shanghai Lingfeng Chemical Reagent Co., Ltd., China. Malachite green  and concentrated HCl were received from Macklin, China. Bovine serum albumin  was bought from Sigma-Aldrich, Burlington, Massachusetts, USA.All chemicals were used as received without further purification. In all experiments, MilliQ water (18.2 MΩ cm) was used.Synthesis of gold nanoparticles. Nanoparticles were synthesized according to the previously published method [d method ,29 with d method . Briefly4 solution and 0.4 mL 10 mM AgNO3 were added in a round-bottom flask with 170 mL of deionized water with the subsequent addition of 15 mL of seed nanoparticles. After that, 30 mL of 5 mM solution of ascorbic acid was added dropwise at the rate of 0.5 mL min−1.Further, seed nanoparticles were grown according to the procedure described in  via a seSynthesis of core–shell particles. To cover gold nanoparticles with a thin shell of silica, the APTES-mediated procedure was applied [ applied ,21. In aPreparation of enhancing substrate. The substrates were prepared on glass slides that were thoroughly cleaned with oxygen plasma prior to the deposition process. To create a confined space with a consistent deposition surface area of approximately 25 mm2 and mitigate the coffee-ring effect to some extent, the drop-casting method was employed. Specifically, 5 to 30 μL of the gold nanoparticle solution was carefully deposited within the confined space delimited by Kapton tape.As illustrated in To clean the enhancing substrates and remove any residual dye or peptide, a thorough washing process was carried out using an ethanol solution. The substrates were subjected to multiple washing cycles to ensure efficient removal of the analyte molecules. This cleaning step was essential to maintain the substrate’s reusability and ensure that subsequent measurements would not be influenced by previous analyte deposits.UV–vis spectroscopy. Absorption spectra in the UV–visible regions were recorded using a high-quality spectrophotometer T9  with a wavelength range of 350 to 800 nm and a precision of 1 nm. The mean diameter and concentration of the particles were determined based on previously published results [ results ,30.Dynamic light scattering (DLS) and zeta potential. Dynamic light scattering (DLS) and zeta potential measurements were performed using a Litesizer 500 instrument . The instrument utilized laser irradiation at a wavelength of λ = 658 nm. DLS measurements were carried out in a glass cell, whereas a specially designed cell with incorporated electrodes were used for zeta potential measurements.Scanning electron microscopy (SEM). SEM images were acquired using an EM-6200 microscope  operating at an acceleration voltage of 20 keV.Transmission electron microscopy (TEM). TEM images were acquired using a Tecnai Spirit microscope  operating at an acceleration voltage of 120 keV.Total reflection X-ray fluorescence (TXRF). The composition of the samples was analyzed qualitatively and quantitatively using total reflection X-ray fluorescence (TXRF) with an XRF S4 T-STAR instrument from Bruker (Germany). To perform the quantitative analysis, a nanoparticle sample volume of 100 μL was mixed with a predetermined quantity of GaCl3 in an HCl solution. Subsequently, the mixture was deposited onto an acrylic substrate and dried at a temperature of 50 °C for a duration of 15 min. To ensure the accuracy and reliability of the measurements, each sample underwent 3 to 5 independent measurements.Raman spectra measurements. Raman spectra were acquired using a Raman microscope InVia  equipped with a 633 nm laser. The laser delivered a power of approximately 0.1 mW on the sample surface. The typical time to acquire the SERS spectrum was 5 s with 5 accumulations. All data were processed using the WiRE software, which provided tools for data analysis and spectral interpretation.−7 M to 10−5 M in ethanol to intensify the drying process. After the droplet had dried completely, Raman spectra were recorded. To calculate the enhancement factor (EF), the Raman spectrum of pristine MG was recorded after being deposited on a pristine glass surface of 30 μL of a 10−4 M MG solution. In the case of bovine serum albumin (BSA), a similar procedure was followed, but the ethanol was replaced with an aqueous solution.The typical procedure for measuring Raman spectra involved the deposition of 10 μL of malachite green (MG) dye solution on the surface. The dye concentrations ranged from 10To ensure statistical significance and accuracy, 3 to 5 separate points on the surface were measured for each concentration of MG. This allowed for the calculation of error bars, providing a measure of the variability within the measurements and ensuring reliable data interpretation and analysis.For mapping experiments, a grid of 100 × 100 points was scanned with a step size of 2 μm between each point. This allowed for the acquisition of spatially resolved Raman data, enabling the investigation of sample heterogeneity and the identification of localized SERS hotspots. All maps were treated and plotted with WiRE software (version 5.3).Gold nanoparticles were synthesized following the procedure described earlier. The characterization of the gold nanoparticles is presented in 2 shell on the gold core causes a further 2 nm red shift. This shift can be attributed to changes in the dielectric constant as well as an overall increase in the diameter of the nanoparticles.In To determine the concentrations of gold nanoparticles in the solution, Haiss’s approach  was utilSimilarly, DLS and zeta potential measurements were performed to evaluate the changes in the particle diameter and surface charge during the synthesis process B. The re2 surface, which contributes to the overall surface charge of the nanoparticles.Furthermore, zeta potential measurements provided insights into the surface charge of the nanoparticles. Initially, both types of gold nanoparticles exhibited a zeta potential of approximately −39 mV. The addition of a silica shell shifted the zeta potential to −43 mV. This shift toward a more negative value can be attributed to the presence of the negatively charged SiOTo validate the mean particle diameter obtained from α and Lβ lines of gold observed at 9.71 and 11.44 keV, respectively, and the Ga lines used as an internal standard, the TXRF spectrum also revealed the presence of small peaks corresponding to the Si Kα and Kβ lines at 1.71 and 1.86 keV, respectively [Further, to confirm the presence of silica in the synthesized samples, total reflection X-ray fluorescence (TXRF) analysis was performed on acrylic sample holders C. In addectively .2 particles. By using TXRF, it becomes possible to estimate the maximum thickness of the silica shell on the gold core by calculating the concentration ratio between gold and silicon (Au/Si). To that end, the concentration ratio between Au and Si should be calculated as follows:Ci—element concentration from the TXRF experiment in mg L−1; Vi—volume; ρi—crystallographic density in g cm–3; and ω—mass % of Si in SiO2. VAu and VSiO2 can be calculated from geometry as follows:r—radius of the gold core and h—thickness of the silica shell.Notably, to remove any excess unreacted silica and large silica aggregates, the samples underwent multiple washes with deionized water followed by centrifugation and redispersion. This rigorous purification process ensures that only the desired silica shell remains on the gold core and very small SiOCSi = 511 mg L−1 and CAu = 2561 mg L−1, the thickness (h) can be estimated as 9.5 nm. Additionally, the thickness of the SiO2 shell was estimated using the mentioned above UV–vis spectra and DLS particle size distributions. The shell thickness was calculated as half of the difference between the diameters of particles with and without the shell. The UV–vis analysis yielded an estimated shell thickness of 3.5 nm. On the other hand, DLS measurements provided a slightly different estimation, with a shell thickness of 4 ± 5 nm. According to TEM, shell thickness can be estimated as 4 to 5 nm –(3) along with measured  to 5 nm E. OveralTherefore, the silica shell deposited on the surface of the gold nanoparticles is relatively thin, which minimally interferes with Raman measurements. However, its presence serves as a protective barrier, shielding the gold surface from contamination and preventing unexpected reactions with analytes.After the preparation of the enhancing substrates through drop-casting in the confined space, the substrates were further characterized using malachite green (MG) dye deposition. The experimental procedure is described in detail in the Materials and Methods section, Raman spectra measurements. In summary, the MG dye was added drop-wise onto the substrate surface, followed by drying, and the subsequent Raman spectra measurements .The characterization of the prepared enhancing substrates, consisting of different types of gold nanoparticles with and without a silica shell, is presented in This improvement can be attributed to the possibility of pin-holes within the silica shell, as described in Ref. , where d−1 (in-plane C-H (ring), δ(C-H)) and 1616 cm−1 (stretching of the benzene ring). The observed MG bands and their corresponding vibrational types are summarized in Two of the most prominent bands corresponding to benzene ring vibrations in the malachite green dye were selected for further experimentation and calculation of enhancement factors. These bands are located at 1172 cm2 factor reaching 0.9842 and 0.9793 for the 1172 cm−1 and 1616 cm−1 bands, respectively. These linear dependencies are illustrated in −1 band and in −1 band. At high MG concentrations, the saturation of the surface with MG molecules can be observed, as the scattering intensity reaches a plateau with relatively high intensity and high relative errors.To investigate the loading of MG to the substrate surface, a gradual increase in MG concentration was achieved by successive deposition of MG solutions onto the enhancing substrate C. Import−1 and 1616 cm−1 bands, respectively. Furthermore, based on the presented data and observed linear dependence, the limit of detection (LOD) was calculated. The LOD values were determined to be 2.94 μM and 2.89 μM of MG for the 1172 cm−1 and 3274 counts μM−1, respectively).The linearity of the Raman scattering intensity observed in the previous experiments further confirms the high quality of the enhancing substrate. The sensitivity can be rationalized as a derivative of the intensity over the concentration range. In both cases, the observed coefficient is relatively high , while −1 band can be found in −1 across the enhancing surface. Similar to the observations made for the 1172 cm−1 band, the mapping results for the 1616 cm−1 band demonstrate the uniformity and consistent distribution of the hotspots associated with the presence of gold nanoparticles.Additional mapping information for the 1616 cmTo complete the substrate characterization, the enhancement factor of the substrates was calculated to evaluate their performance. The analytical enhancement factor (AEF) was determined using the following calculation method, as described in Refs. ,37,38:, AEF can be calculated as 3.4 × 10−1, can be easily identified. However, the application of the enhancing substrate enables the resolution of all bands in the spectrum and facilitates their assignment to specific amino acids present in BSA  and spe−1, Ref. ).2 shell (roughly 4 nm thick) in a space confined by Kapton tape using a drop-casting procedure. The characterized enhancing substrates exhibited a linear dependence between Raman intensity and the loading of MG dye, serving as a model molecule. This finding was further supported by Raman scattering mapping, which confirmed the uniform distribution of hotspots and Raman scattering intensity on the enhancing surface. The calculated enhancement factor of approximately 4 × 104 and LOD of approximately 2.9 μM demonstrated the capability of detecting various natural molecules. While this enhancement factor is lower than the anticipated values for pure electromagnetic mechanisms (which can reach up to 106), it is important to note that the present Raman substrate provides a robust and reusable scaffold for enhanced Raman spectroscopy. The combination of gold nanoparticles with a thin SiO2 shell offers an efficient platform for signal enhancement and molecular detection. Although such an EF is an order of magnitude lower than anticipated for pure electromagnetic mechanism (up to 106), the present Raman substrate nevertheless provides a scaffold for reusable substrates.To conclude, we showed a facile, cost-effective, and rapid route for preparing the enhancing Raman substrate based on gold nanoparticles with a mean diameter of 44 nm covered with a thin SiO−1 with improved peak resolution, allowing for the identification of specific amino acids.The practical application of the substrates was demonstrated by detecting and analyzing BSA as a model peptide molecule at a concentration level of 55 μg mLNotably, the prepared enhancing substrates exhibit excellent reusability, which was demonstrated by successfully reusing the same substrate through a simple washing process with an ethanol solution. This remarkable feature allows for the repeated use of the substrate without compromising its performance or effectiveness.Overall, this study introduces a novel approach for the preparation of highly efficient and reusable SERS substrates based on gold nanoparticles. These substrates hold great potential for a wide range of applications in the field of analytical chemistry and biosensing.


Sclerotinia sclerotiorum and Athelia rolfsii. Their control is often performed with fungicides which cause public concern over the environment and human health. The main aims of this study were to determine the biocontrol efficacy of a Streptomyces strain in vitro and in vivo conditions on lettuce seedlings against Athelia rolfsii and Sclerotinia sclerotiorum as severe fungal soilborne pathogens through the application of its vegetative propagules and putative bioactive crude extracts via filtrate culture. The results obtained confirm a significant effectiveness of CARA17 strain to control the severity of both fungal soilborne pathogens during two different experiments: when it is used as vegetative propagules and as a culture filtrate containing putative bioactive metabolites in vitro and in vivo conditions. These preliminary results demonstrated that the actinomycetes CARA17 strain is valid as a biocontrol agent (BCA) against both the severe phytopathogens used in this study. The biocontrol action performed from the CARA17 strain is clearly and mainly due to the putative bioactive crude extracts produced, but further studies are necessary to identify which metabolites  are produced from this Streptomyces strain.Lettuce crop is an important horticultural crop of several Mediterranean countries, including Italy. The Italian region which is a major producer of lettuce crops is Apulia, where this crop is cultivated in open fields an in greenhouses. Since several microbial pathogens are responsible for important diseases found on lettuce produced in greenhouses, in this study, the experimental activities focused on the most severe fungal soilborne pathogens, i.e.,  Lactuca sativa L.) is a common horticultural plant for countries within the Mediterranean basin, and it is one of the most economically important vegetable crops worldwide. In particular, this crop is of great economic importance in Italy, as it is the third most-consumed product after potatoes (700 thousand tons) and tomatoes (580 thousand tons). In Italy, lettuce is represented by several varieties and is produced in both greenhouse and open-field conditions, at 480 thousand tons. Apulia is the country’s major lettuce-producing region with 609 thousand hectares; yet, lettuce is produced in all Italian regions [Bremia lactucae, responsible for downy mildew, and Botrytis cinerea, responsible for grey mold, are two of the most destructive aerial pathogens of lettuce plants worldwide [Pythium and Phytophthora cryptogea; basal leaf decay, which is associated with Rhizoctonia solani, Sclerotinia sclerotiorum, and S. minor [Athelia rolfsii; and vascular diseases (wilt), associated mainly with Fusarium oxysporum f.sp. lactucae [Sclerotinia spp. and Athelia rolfsii, which result in very difficult to control problems in greenhouse and open-filled crops, leading to significant crop yield losses [Lettuce  are the most important and are more widely used against different fungal pathogens such as Fusarium oxysporum f. sp. lactucae [Sclerotinia sclerotiorum, and Chalara thielavioides [Pseudomonas spp. is used against Verticillium dahliae [Bacillus spp. against several phytopathogens [Streptomyces is the largest genus belonging to the family Streptomycetaceae [Streptomyces spp. are the most spread Gram-positive filamentous bacteria that are ubiquitous in the soil as free-living organisms and symbionts of plants and animals [Therefore, a substantial amount of research has regarded biological control as a promising alternative approach to controlling soilborne diseases in sustainable and organic agriculture. Indeed, many microbial antagonists have been demonstrated to be able to control a large number of pathogens, such as fungi and bacteria. For instance, lactucae , Sclerotavioides . Pseudom dahliae , and Bacathogens . It is kathogens ,16. Amoncetaceae , with incetaceae ,19,20. I animals . They ar animals .Streptomyces isolates were able to produce antibacterial metabolites crude extracts effective to inhibit the growth of some bacteria such as Staphylococcus aureus, Bacillus cereus, and Pseudomonas aeruginosa. Although a few studies have been carried out to assess the biocontrol efficacy and mechanism of Streptomyces spp. [Kurnianto et al.  demonstrces spp. ,17, to dStreptomyces strain in dual culture and its bioactive crude extracts against Athelia rolfsii and Sclerotinia sclerotiorum in vitro conditions; (ii) to determine the biocontrol efficacy of a Streptomyces strain on lettuce seedlings against both severe fungal soilborne pathogens in vivo conditions, by application of its propagules; and (iii) to evaluate the antagonistic efficacy of putative metabolites (bioactive compounds) produced from Streptomyces strain as metabolite crude extracts in vivo conditions.Therefore, the main aims of the present study were: (i) to assess the effectiveness of p < 0.05), and according to Levene tests, the homogeneity of variance was also significant . One-way ANOVA demonstrated that significant differences in inhibitory activity (IA) were recorded when the culture filtrates of CARA17 strain were pretreated at two temperatures (121 and 100 °C) and added to a GYMA medium with different amounts  against the fungal pathogens A. rolfsii and S. sclerotiorum. Indeed, The identification and characterization of fungal phytopathogens and CARA17 strain, as well as the antagonistic activity in dual culture were ascertained as reported in previous research . RegardiS. sclerotiorum resulted to be sensible  regarding to the presence of CARA17 strain, while A. rolfsii showed no inhibition  after 21 days  were collected when the lettuce seedlings were treated by dipping in CARA17 filtrate inoculum by S. sclerotiorum and A. rolfsii, respectively. However, the DS values collected from the roots of seedlings treated only with fungal soilborne pathogens were 3.9 for S. sclerotiorum and 5.0 for A. rolfsii. No DS values were recorded from lettuce seedlings used as control treated with both propagule and filtrate inocula of CARA17 and with the tap water. The DS values followed a similar trend regarding the symptoms observed on leaves. The percentages of the re-isolation of both fungal pathogens from symptomatic seedlings were higher than from healthy seedlings  treated with both propagule and filtrate inocula of CARA17 and with tap water. The disease severity (DS) values assessed by observations carried out on the roots of lettuce seedlings treated by dipping in CARA17 propagule inoculum were 0.6 for eedlings .Streptomyces species is known as an effective biocontrol agent against numerous plant pathogens [Streptomyces strains used as BCAs have shown various degrees of disease-suppressive activity, depending on whether they were tested in laboratories, fields, or greenhouses and the methods used [Streptomyces spp. to control several diseases occurring on cereal crops carried out in different conditions . Moreover, Law et al. [Streptomyces spp. are able to control Magnaporthe oryzae as a causal agent of rice blast; Kaur et al. [Streptomyces spp. to control Fusarium moniliforme and F. graminearum. Similar results were obtained by Carlucci et al. [Streptomyces albidoflavus CARA17 strain to control several severe fungal phytopathogens occurring on fennel plants in southern Italy in vitro and in vivo conditions, such as Athelia rolfsii, Fusarium oxysporum, Plectosphaerella ramiseptata, Sclerotina sclerotiorum, and Verticillium dahliae.It is known that the control of several severe fungal phytopathogens is very difficult, and increasing amounts of fungicides are necessary despite consumer demands for products with low or no chemical residues for the health of both humans and ecosystems. To satisfy consumers, many studies have been conducted with the aim to detect and prove the potential ability of biological control agents (BCAs) to control fungal and bacterial phytopathogens ,24,25,26athogens ,23,24,25athogens ,26,27 asathogens ,25,27,28ods used  reportedw et al.  describer et al.  and Color et al.  showed ti et al.  that disSclerotina sclerotiorum and Athelia rolfsii on lettuce plants. In particular, two different methods of inoculation were tested in this study in order to verify the ability of crude extracts from a Streptomyces CARA17 strain at inhibiting the abovementioned soilborne pathogens in vitro and in vivo conditions.The same actinomyces strain has been used in the current study in order to demonstrate that it is able to control Streptomyces before adding to a culture medium at three different concentrations  during the assessment of essays in vitro conditions. In this case, the culture filtrate was able to present significant inhibitory activity percentages against the mycelial growth of S. sclerotiorum and A. rolfsii at already lower concentrations when the culture filtrate was thermally pretreated at 100 °C. These results highlight that the higher concentrations of culture filtrates are more efficacious, and the high temperatures of pretreatment can reduce the effectiveness of putative bioactive compounds because, most likely, these are thermally sensitive. For these reasons, for setting up the greenhouse experiment, the culture filtrate of CARA17 strain was thermally treated at 100 °C. The results obtained from the greenhouse experiment demonstrated that the culture filtrate was able to significantly prevent the severity of fungal pathogens used, although the inoculations carried out with propagule inoculum showed a better prevention on the lettuce seedlings with the higher number of healthy seedlings. The better effectiveness of the propagule inoculum than filtrate inoculum is probably due to synergic actions played from crude extracts and vegetative propagules according according to Passari et al.’s work [Streptomyces albidoflavus CARA17 strain was more efficacious in protecting the lettuce seedlings against S. sclerotiorum than A. rolfsii in both inoculation kinds of the antagonist. These data confirmed the higher severity of A. rolfsii.The culture filtrate of a CARA17 strain was thermally pretreated at two different temperatures to inactivate vegetative propagules of BCA .’s work , which h.’s work , whose wConyothirium minitans [Trichoderma spp. [Pseudomonas brassicacearum [Bacillus amyloliquefaciens [Based on the results obtained from this study, we suggest that the CARA17 strain might be considered a suitable biological control agent (BCA), similarly to other antagonistic fungal and bacterial micro-organisms, as well as minitans , Trichodrma spp. ,36, Pseucacearum ,38, and The current results discussed here confirm the previous data  and encoStreptomyces albidoflavus used in the research activities described herein was isolated and characterized in early research as reported by Carlucci et al. [Athelia rolfsii and Sclerotinia sclerotiorum used in this study were isolated from fennel plants during previous surveys carried out in the northern Apulia region (2017–2020). The assessment of antagonistic activity played from the Streptomyces strain CARA17 in vitro conditions against A. rolfsii and S. sclerotiprum was performed as described by Carlucci et al. [The CARA17 strain of i et al. . The phyi et al. . All funSclerotinia sclerotiorum and Athelia rolfsii, two kinds of experiments were performed. The first consisted of preparation of dual culture as described by Carlucci et al. [6 cfu per ml of CARA17 propagules collected from 21 day-old colony, incubated at 28 ± 3 °C at 75 rpm for 21 days, and then filtered on Whatman filter, 5.5 µm in size. The filtered culture was evaluated for antifungal activity by inclusion of 15, 30, and 50% (v:v) in GYMA medium and early-sterilized at 115 °C for 10 min. After adding CARA17 culture filtrate, the GYMA  was again sterilized at 100 and 121 °C for 10 min. The Petri plates obtained were inoculated with an agar-mycelium disc (5 mm diameter) from both phytopathogens fungi (A. rolfsii and S. sclerotiorum), and incubated at 28 ± 3 °C for six days to evaluate the inhibition activity played from CARA17 filtrate. Five replicates were performed for both soilborne fungal pathogens. Plates inoculated with phytopathogens and the sterile agar discs were used as controls. The inhibitory activity (IA) was calculated as the percentage of mycelium growth inhibition compared to the control according to Carlucci et al. [p = 0.01).To assess the inhibitory activity of CARA17 strain against i et al. . The sec7 cfu/mL in sterile Tween 20 solution (0.2%). The other inoculum kind consisted of preparing a liquid culture of CARA17 strain grown into liquid medium (broth of GYM) at 28 ± 3 °C in darkness at 75 rpm. After 21 days, the antagonist culture was filtered on Whatman paper filters (size 5.5 µm) and subjected to 100 °C for 10 min to inactivate all vegetative propagules of antagonist.Inoculation preparation of biological control agent CARA17 strain: Two different inoculum kinds of CARA17 strain were prepared. One consisted of preparing the inoculum solution with the CARA17 strain propagules by collecting spores and small fragments of mycelium, scraped from surface the of 21 day-old Streptomyces colonies, grown on PDA  medium at 28 ± 3 °C in darkness until reaching a concentration of 1 × 10A. rolfsii and S. sclerotiorum: The inoculum solution with each fungal pathogen was prepared by scraping from the surface of 21 day-old colonies grown on PDA medium at 21 ± 3 °C in darkness until reaching a concentration of 1 × 107 cfu/mL in sterile Tween 20 solution (0.2%).Inoculation preparation of soilborne pathogens, A. rolfsii and S. sclerotiorum as severe fungal pathogens of Lactuca sativa var. Iceberg using 30 day-old seedlings.The experimental design was performed as two independent batches at the end of August 2022 and consisted of two different inoculum kinds of CARA17 strain as described above, against two fungi targets such as The experiment consisted of setting up of two main trials: (a) dipping twelve lettuce seedlings in the inoculum solution of propagules of CARA17 strain for 12 h (propagule inoculum); (b) dipping twelve lettuce seedlings in the CARA17 strain inoculum solution of culture filtrated for 12 h (filtrate inoculum). After dipping, the lettuce seedlings were transplanted in pots containing 1.5 kg of soil and peat (3:1)  and wetting them with 500 mL of irrigation water. Subsequently, 50 mL of inoculum solution of both fungal soilborne pathogens were poured into the soil of each pot around the collar of the lettuce seedlings.As control trials, pots containing lettuce seedlings treated with both kinds of CARA17 strain inocula (propagule and filtrate inocula), two other trials treated only with each fungal pathogen (no treatment), and another trial not treated with either the CARA17 strain or the fungal soilborne pathogens  were prepared. Each trial was replicated five times. The pots with lettuce seedlings were placed in a greenhouse with temperature and humidity not controlled. During the growth of the seedlings, no fertilizers, pesticides or fungicides were used. They were only subjected to irrigation practice when necessary, using the same water volumes for each pot. After 100 days, the lettuce plants were gently removed from the pots, the roots and collars were carefully washed, and the presence/absence of browning/rot symptoms observed on the roots and leaves were evaluated and described using an empirical scale from 0 to 5, where 0 = no symptoms observed; 1 = 1–20%; 2 = 21–40%; 3 = 41–60%; 4 = 61–80%; and 5 = 81–100%. The disease severities (DS) on the roots and leaves were determined according to the following formula:All fungi underwent re-isolation from the root of the inoculated plants to fulfil Koch’s postulates.


Lung adenocarcinoma (LUAD) is a highly heterogeneous disease that ranks first in morbidity and mortality. Abnormal arginine metabolism is associated with inflammatory lung disease and may influence alterations in the tumor immune microenvironment. However, the potential role of arginine and proline metabolic patterns and immune molecular markers in LUAD is unclear. Gene expression, somatic mutations, and clinicopathological information of LUAD were downloaded from The Cancer Genome Atlas (TCGA) database. Univariate Cox regression analysis was performed to identify metabolic genes associated with overall survival (OS). Unsupervised clustering divided the sample into two subtypes with different metabolic and immunological profiles. Gene set enrichment analysis (GESA) and gene set variation analysis (GSVA) were used to analyze the underlying biological processes of the two subtypes. Drug sensitivity between subtypes was also predicted; then prognostic features were developed by multivariate Cox regression analysis. In addition, validation was obtained in the GSE68465, and GSE50081 dataset. Then, gene expression, and clinical characterization of hub genes CPS1 and SMS were performed; finally, in vitro validation experiments for knockdown of SMS were performed in LUAD cell lines. In this study, we first identified 12 arginine and proline-related genes (APRGs) significantly associated with OS and characterized the clinicopathological features and tumor microenvironmental landscape of two different subtypes. Then, we established an arginine and proline metabolism-related scoring system and identified two hub genes highly associated with prognosis, namely CPS1, and SMS. In addition, we performed CCK8, transwell, and other functional experiments on SMS to obtain consistent results. Our comprehensive analysis revealed the potential molecular features and clinical applications of APRGs in LUAD. A model based on 2 APRGs can accurately predict survival outcomes in LUAD, improve our understanding of APRGs in LUAD, and pave a new pathway to guide risk stratification and treatment strategy development for LUAD patients. Lung cancer also exhibits molecular and genetic heterogeneity5, which presents a challenge for the diagnosis and treatment of lung cancer. In recent years, immunotherapies such as immune checkpoint inhibitors (ICIs) have brought a new direction to lung cancer treatment and have been shown to improve survival in advanced NSCLC6. However, only some lung adenocarcinoma patients respond to them, and most patients are prone to primary and secondary drug resistance8. Therefore, there is a need to find new molecular markers and construct prognostic models to stratify and tailor treatment regimens for patients with lung adenocarcinoma.Lung adenocarcinoma (LUAD) is one of the most common respiratory tumors and is classified as non-small cell lung cancer (NSCLC), accounting for approximately 40% of all lung cancers. According to the latest research data published by the World Health Organization (WHO), LUAD ranks second among all tumors worldwide and has the highest mortality rate, with an average 5-year survival rate of only 15%9. Among these, amino acid metabolism plays an important role in tumor cell growth and survival, including providing a carbon source for the TCA cycle, a nitrogen source for base synthesis, and regulating redox homeostasis. Previous studies have identified the involvement of arginase in obstructive airway diseases such as asthma and pulmonary hypertension12. The latest study shows that arginine metabolism is closely related to TME14 and is involved in T-cell activation and immunomodulatory responses15. As a multifunctional amino acid, arginine has multiple biological functions in metabolism and signaling pathways, and many tumors exhibit exogenous arginine-dependent growth16, and can maintain tumor growth through the synthesis of derived metabolic polyamines17. Proline metabolism is associated with ATP production, protein and nucleotide synthesis, and redox homeostasis in tumor cells18. The inhibition of proline biosynthesis is thought to inhibit tumor growth. For example, Myeloid-derived suppressor cells (MDSCs) that play an immunosuppressive role in TME can express high levels of ARG1, iNOS, TGF-β, ROS, IDO, etc., while depleting arginine to inhibit T cell function to promote tumor immune escape20. Meanwhile, arginine deprivation therapies targeting arginine have shown positive results in clinical practice22. In contrast, the molecular alterations and metabolic profiles of arginine and proline as non-essential amino acids in lung adenocarcinoma have not been fully investigated.In the tumor microenvironment (TME), tumor cells form a metabolic competition with immune cells. Many tumors have evolved to evade immune surveillance, using their metabolic flexibility and redirecting nutrients to their advantageCPS1) is the first rate-limiting enzyme of the urea cycle, and some LUAD patients show significant overexpression24, has been found to maintain pyrimidine and DNA synthesis in KRAS/LKB1 co-mutated non-small cell lung cancer (NSCLC) and imposes a specific metabolic vulnerability25. And the study suggests that CPS1 may be a biomarker of poor prognosis and a potential therapeutic target. Spermine synthase (SMS), a key enzyme in polyamine metabolism, plays an important role in spermine anabolism26. High expression in cancers such as hepatocellular carcinoma and head and neck squamous cell carcinoma is associated with poor prognosis28. In addition, recent studies have shown that SMS and MYC can synergistically inhibit the expression of pro-apoptotic protein Bim to maintain the growth of colorectal cancer cells29. The dysregulation of polyamine metabolism and its requirement occurs frequently in tumors, and multiple oncogenes MYC and RAS play a direct role in regulating polyamine synthesis, which represents a complementary relationship between polyamine metabolism and tumor proliferation regulation to promote cancer progression30.Carbamoyl phosphate synthase 1  database and GEO (http://www.ncbi.nlm.nih.gov/geo/) database. Construction of relevant prognostic features using 500 LUAD cases from the TCGA database, and the sample inclusion criteria for TCGA were 01A (Primary Tumor) type samples containing complete survival information. GSE68465 were used to validate the prognostic and immunological features of LUAD based on genes related to arginine and proline metabolism. To avoid the interference of irrelevant factors, most tumor samples with a follow-up time greater than 2000 days were excluded, a total of 333 samples were included in the GSE68465 dataset, and the GSE68465 dataset was used as the first validation set. The datasets GSE30219, GSE37745, and GSE50081 from the same microarray platform (Affymetrix HG-U133 Plus 2.0 Array) were integrated into a new cohort and used as the second validation set to validate the ARG-based prognostic model, and sample inclusion criteria for the GSE50081 integration cohort were samples with the histologic type of adenocarcinoma. The 54 arginine and proline-related genes were obtained from the human C2 KEGG gene set of the MsigDB database (KEGG_ARGININE_AND_PROLINE_METABOLISM)31. These genes were integrated with clinical information from 500 lung adenocarcinoma samples for univariate Cox regression analysis, and 12 APRGs associated with OS were screened according to p < 0.05 before subsequent analysis. Finally, two prognostic hub genes were identified by stepwise multivariate regression analysis. The RNA-seq data types processed for TCGA were log2(TPM + 1). The transcriptome-in-a-million (TPM) values for the LUAD cell line transcriptome were obtained from the Cancer Cell Line Encyclopedia (CCLE) (https://sites.broadinstitute.org/ccle/).Transcriptomic data and corresponding clinical data of LUAD patients were obtained from the TCGA  in LUAD, and the R package “ggplot2” was used for mapping forest plot. Prognostic genes were identified at a p < 0.05 criterion. The 12 genes associated with survival were analyzed univariately and then clustered by consensus using the R package “ConsensusClusterPlus”. The optimal number of clusters was assessed by the cumulative distribution function (CDF) plot and the consensus heat map with an optimal k-value of 2. The distribution of groups was studied using the UAMP dimensionality reduction analysis using the R package “umap”. In addition, univariate and multivariate Cox regression analyses were performed to confirm whether characteristics could be used as independent predictors of survival. The R package “survival” and “survminer” were applied, survival analysis was performed and survival curves were plotted, and log-rank tests were performed to assess the difference in survival between the two groups. p < 0.05 was considered significant.32. Single-sample gene enrichment analysis (ssGSEA) was introduced to quantify the relative infiltration of 28 immune cell types in the tumor microenvironment, with unique combinations of characteristic genes for each immune cell subtype derived from recent literature34. The results were presented using the ggplot2, ggpubr, and pheatmap R packages.We used ESTIMATE algorithm to assess the level of immune cell infiltration, stromal content, and tumor purity for each sample. The enrichment levels of 64 immune features were quantified by the Xcell algorithm, and the relative proportions of 22 immune cell types in each tumor tissue were estimated using the CIBERSORT algorithm, and samples with p > 0.05 in the results were excluded and the remaining samples were further analyzed35. The gene set “c2.cp.kegg.v7.4.symbols.gmt” was also downloaded from MSigDB as a reference gene set.We used the ssGSEA and GSVA algorithms in the R package “GSVA” to study the variation of biological processes among two clusters. Well-defined biological features were obtained from the Hallmark gene set  and the gene set constructed by Mariathasan et al. (from the IMvigor210CoreBiologies package)36 for ontology (GO) enrichment analysis, FDR < 0.05 and |logFC| > 1 were set as threshold values. Gene set enrichment analysis (GSEA) was used to explore potential functional and signaling pathway enrichment in patients with two immune escape subtypes of lung adenocarcinoma.By using the “limma” and “clusterProfiler” R packageshttps://portal.gdc.cancer.gov/). Somatic mutation data were integrated using the R package “maftools”, and mutations in five tumor driver genes were analyzed to extract tumor mutational load (TMB) values. In addition, given that T-cell inflammatory GEP and PD-L1 (CD274) expression predict immunotherapeutic response, we evaluated GEP scores and PD-L1 expression levels between the two subtypes. Predicting potential immune checkpoint blockade responses (ICB) in LUAD using tumor immune dysfunction and exclusion (TIDE)37. Higher TIDE scores indicate that cells are more likely to induce immune escape and thus have a lower response rate to ICB treatment. We also invoked an immune signature-based approach for hot and cold tumor identification and analyzed the differences in response to immunotherapy and the proportion of hot and cold tumors between the two subtypes.Somatic mutations were obtained from the TCGA database  of 198 drugs between the two subtypes. p < 0.05 was the threshold of significance.The OncoPredict R software package was developed by Maeser et al. to predict in vivo drug sensitivity in cancer patientsTo further explore the association between prognosis-related core genes and lung adenocarcinoma prognosis, we performed multivariate Cox regression analysis on 12 modeled genes and constructed a risk model based on genes related to arginine and proline metabolism, resulting in the risk score formula risk score = Ʃ (βi × Expi). Where βi coefficient represents the weight of the respective marker and Expi represents the expression value. According to the risk score formula, patients were divided into low-risk and high-risk groups, with the median risk score as the cut-off point. Subsequently, nomograms predicting the likelihood of OS at 1, 3, and 5 years were constructed by the R package “rms” based on the results of multivariate Cox regression analysis. Calibration plots were drawn to assess the predictive accuracy of the nomograms. To validate the predicted values of metabolism-related features, we used TCGA data as the training set and GSE68465, GSE50081, and other datasets as the validation cohort. The R package “survminer” was used to estimate OS by survival analysis in the high-risk and low-risk groups, and the R package “timeROC” was used to assess the prognostic value of the feature model. In addition, we analyzed the possibility of risk score as an independent prognostic feature using univariate and multivariate Cox regression.CPS1 and SMS, in LUAD normal versus LUAD by TCGA database and GEPIA2  database40, then followed by protein expression analysis in the UALCAN database. Kaplan Meier  and GEPIA online databases were used to analyze the survival curve analysis of the CPS1 and SMS genes41. In addition, we obtained mRNA expression levels of CPS1 and SMS in a variety of lung adenocarcinoma cell lines in the CCLE database. Immunohistochemical analysis of protein expression of CPS1 and SMS in normal and LUAD tissues was downloaded from the Human Protein Atlas (HPA) database (http://www.proteinatlas.org/)42. The correlation of CPS1 and SMS gene expression with clinical characteristics such as age, gender, stage, and TNM stage was also analyzed.First, we analyzed the expression levels of two modeled genes, SMS and interfering RNA control were purchased from Gima Genetics . For transient transfection, A549 and H1299 cells were transfected with siRNA for 12 h using a transfection reagent (Lipofectamine 2000), followed by functional assays.Human lung adenocarcinoma cell lines A549 and H1299 were mainly purchased from the cell bank of the Chinese Academy of Sciences . We performed in vitro culture experiments using A549 and H1299 cells cultured in DMEM medium and RPMI 1640 medium  supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin (Gibco). Small interfering RNA (siRNA) targeting SMS-Forward: TAGTGGGGATGTAATTTGGCAG; SMS-Reverse: CCACACGTTTTTCGCATGTATTT; GAPDH-Forward: GACCACAGTCCATGCCATCA; GAPDH-Reverse: GTCAAAGGTGGAGGAGTGGG. Protein blotting analysis of RIPA lysis buffer  containing PMSF  was used to collect proteins from A549 and H1299 cells. 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate protein samples, and polyvinylidene difluoride (PVDF) membranes  were used to transfer the separated proteins. The closure was performed for 15 min using Rapid Closure Solution, followed by incubation with primary antibodies: SMS  and Vinculin  overnight at 4 °C, followed by secondary antibody incubation for 2 h. Original images of the western blot are available in the Supplementary File.RNA was extracted from lung adenocarcinoma cell lines  interfering with si-SMS and NC as controls. SYBR Green qPCR mix  was used to synthesize cDNA for real-time PCR. primers were as follows: SMS siRNA, and A549 and H1299 cells were cultured in 96-well plates (3000 cells/well). The proliferation capacity of the treated cells was assayed at 4, 24, 48, and 72 h. 10% Cell Counting Kit-8 (CCK8) reagent  was added to each plate according to the kit instructions, and OD450 values were analyzed using an enzyme marker . For colony formation experiments, 2000 cells were inoculated in cell culture plates and allowed to grow until visible colonies were formed. We then fixed the clones with paraformaldehyde for 15 min, stained the clones with 1% crystalline violet for 20 min, and counted the number of clones (> 50 cells).Cell proliferation and colony formation assays were performed 24 h after transfection with SMS siRNA for 24 h and cultured in 24-well culture plates with 8 mm pore membrane inserts to measure cell migration and invasion capacity. 4 × 104 cells were inoculated in 200 μl of serum-free medium in the upper chamber of the transwell, and 800 μl of medium containing 10% FBS was added to the lower chamber. After incubation for 48 h, cells that migrated across the membrane were fixed with paraformaldehyde, stained with 1% crystal violet and counted under a light microscope (200×). In addition, A549 and H1299 cells were cultured in 24-well plates and scraped with a 200 μl pipette tip. Cells were cultured in DMEM and RPMI 1640 medium without FBS. Wound images were captured at 0 and 24 h and the wound area was quantified by ImageJ software (40×).Transwell migration and wound healing assays A549 and H1299 cells were transfected with Statistical analysis and plots were performed using R software (version 4.0.1) and GraphPad software. The Wilcoxon test was used for the test between the two paired groups, categorical variables were compared by Chi-square test or Fisher exact test, and the statistical significance of the cell line experiment was assessed by t-test in GraphPad Prism version 9 software. Differences were considered statistically significant at *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.https://www.ncbi.nlm.nih.gov/geo), and The Cancer Genome Atlas .The data used in this study were obtained from publicly available datasets, such as the GEO database  about LUAD, using univariate Cox regression analysis for 54 metabolic genes, and 12 genes were associated with OS (p < 0.05) in the TCGA cohort, namely TP53, KRAS, STK11, EGFR, and KEAP1. The results showed that cluster 2 possessed a higher overall mutation rate and higher mutations in KRAS, STK11, and KEAP1 genes compared to cluster 143  as a multi viral HAVCR1 is aberrantly expressed in tumors as a receptor for multiple viruses and is highly expressed in cluster 2, may serve as a feature of immune escape in lung adenocarcinoma45. In immunosuppressive mediator secretion, by comparing the expression of cytokines such as chemokines and receptors, interferons and receptors, and interleukins and receptors in the three subtypes most immunosuppressive mediators were lowly expressed in cluster 2, such as chemokines involved in T cell recruitment and inflammatory response: CXCL9 and CXCL10 were expressed at high levels in cluster 1, while the enrichment of CCL4 and CXCL13 may have promoted the cluster 1 establishment of the thermal tumor microenvironment46. The low expression of IL-37 in cluster 2 suggests a reduced antitumor activity and potential for tumor metastasis47  expression levels and GEP scores between the two clusters and found that cluster 1 had significantly higher PD-L1 expression and possessed a higher GEP score, which may predict that cluster 1 possesses a better immunotherapeutic effect49  + (0.48094 × SMS expression). The risk score of each patient was calculated based on this model, and LUAD patients were divided into high-risk and low-risk groups according to the median risk score  has been shown to be associated with poor prognosis in lung adenocarcinoma, and spermine synthase (SMS) has been reported to be associated with poor prognosis in hepatocellular carcinoma, head and neck squamous cell carcinoma, and colorectal cancer. First, we compared the difference in mRNA expression between normal and tumor groups in lung adenocarcinoma with CPS1 and SMS genes in the TCGA and GEPIA2 databases, and also validated them in the GEO database , disease-free survival (DFS), and recurrence-free survival (RFS), respectively. The group with high expression of CPS1 in OS and DFS had lower survival rates, while RFS showed no significant difference; SMS showed significant differences in OS, DFS, and RFS, indicating that high expression of SMS in LUAD resulted in poorer prognosis , and validated its predictive power. Finally, we performed cellular experiments to validate the role of SMS as a hub prognostic gene for APRG in LUAD progression, further validating the feasibility of the prognostic model.In this study, we reveal the landscape of arginine and proline metabolism-related genes and TME in LUAD. Screening of APRGs by univariate Cox regression analysis in the TCGA dataset yielded 12 APRGs associated with OS, revealing two different molecular isoforms based on the expression of the 12 APRGs. Cluster 1 patients had a longer survival time compared to cluster 2 patients. GSVA enrichment analysis showed that cluster 2 was significantly enriched in a variety of substance metabolisms including arginine and proline metabolism, riboflavin metabolism, and histidine metabolism. The enriched pathways in cluster 2 suggest that they play an important role in the development of LUAD and that there is a crosstalk between arginine and proline metabolism and other signaling pathways. We also identified TME features between the two clusters, with cluster 1 showing high immune cell infiltration and high stromal cell infiltration, which is close to the immune inflammatory or immune rejection type, and cluster 2 with impaired T cell activation and unique immune escape features, along with high methylation levels and reduced expression of most antitumor immune molecules, which is more closely related to the immune rejection or immune desert type. In addition, we assessed the immunotherapeutic response and drug sensitivity of both subtypes by algorithms such as TIDE, which provides a reference for clinical treatment. In addition, we developed a validated risk model for prognostic APRGs, which included 2 APRGs , spermidine synthase (SRM), and glutaminase (GLS), the upstream genes of polyamine metabolism60, its synergistic relationship with polyamine metabolism has been demonstrated in bladder cancer, neuroblastoma, and colorectal cancer63. In addition, the study by Yubin Guo et al. revealed a complex mechanism of MYC and SMS synergistically regulating CRC apoptosis.Among the two signature genes in the prognostic model of APRGs, both 64 and hypoxia-derived genes65, radiotherapy autophagy-related genes66, m6A-associated lncRNA67, etc., all of which have shown some degree of predictive power for prognosis, immune response, etc. in LUAD patients. In a recent study, circulating l-arginine was suggested to predict the survival of patients treated with ICIs68. The specific mechanism of the potential activation of rapamycin complex 1 (mTORC1) by altered arginine concentration remains to be investigated69.Recently, an increasing number of studies based on metabolism-related genes70; Pansy et al. reviewed the regulatory mechanisms of immune checkpoints, co-suppressor molecules, and cytokines in the process of immune escape from tumors71; Liu et al. also described the mechanisms by which immunosuppressive cells promote immune escape from tumors72; and Nagarsheth et al. elaborated on the roles of chemokines in cancer immunity and tumorigenesis73. Therefore, exploring the immune escape profile of lung adenocarcinoma patients may ultimately benefit clinical immunotherapy.In addition, evidence supporting tumor immune escape can be found in the following literature and related studies: in the study by Jhunjhunwala et al. we found the most critical antigen presentation and HLA deletion features consistent in this studySMS affects LUAD progression from both in vivo and in vivo experiments.In summary, this study characterized the arginine metabolism-related gene markers in LUAD and developed a prognostic model of APRGs based on two signature genes, which demonstrated strong ability to predict prognosis and evaluate immune evasion in LUAD. There remain, however, some limitations to our study. Firstly, all samples used in the survey were collected retrospectively and analyzed against publicly available database data. Thus, inherent case selection bias may influence the results and more compelling prospective studies are needed to confirm our findings. Secondly, because of the limited sample size, large cohort studies are essential to evaluate the value of the model. Thirdly, to improve the future understanding of arginine and proline metabolism, it is also crucial to validate the molecular understanding based on functional in vivo and in vitro experiments. In conclusion, some crucial clinical information including surgery, targeted therapy, and chemoradiotherapy is not available for analysis in most of the data sets, which is thought to affect the prognosis of the immune response as well as the status of arginine and proline metabolism. Future studies will continue to address the specific mechanisms by which Supplementary Information.


Increasing or decreasing ENE at the end of the embryonic period promoted an increase or a decrease in the numbers of dopamine (DA) neurons, respectively. This plasticity of dopaminergic specification occurs in the subpallium (SP) of zebrafish larvae at 6 d postfertilization (dpf), within a relatively stable population of vMAT2-positive cells. Nondopaminergic vMAT2-positive cells hence constitute an unanticipated biological marker for a reserve pool of DA neurons that can be recruited by ENE. Modulating ENE also affected larval locomotion several days after the end of the treatments. In particular, the increase of ENE from 2 to 3 dpf promoted hyperlocomotion of larvae at 6 dpf, reminiscent of zebrafish endophenotypes reported for attention deficit hyperactivity disorders (ADHDs). These results provide a convenient framework for identifying environmental factors that could disturb ENE as well as to study the molecular mechanisms linking ENE to neurotransmitter specification.During the embryonic period, neuronal communication starts before the establishment of the synapses with alternative forms of neuronal excitability, called here embryonic neural excitability (ENE). ENE has been shown to modulate the unfolding of development transcriptional programs, but the global consequences for developing organisms are not all understood. Here, we monitored calcium (Ca Using short-term pharmacological treatments by bath application that increase or decrease ENE we detected changes in the postmitotic differentiation of the dopaminergic phenotype, occuring within a reserve pool of vMAT2-positive cells. We also report changes in locomotion, with transient increase of ENE leading to hyperlocomotion, a phenotype associated with attention deficit hyperactivity disorder (ADHD) in this model. Our results provide a convenient framework to study the molecular mechanisms linking ENE to neurotransmitter specification.Spontaneous calcium (Ca2+) concentration with different spatiotemporal dynamics have also been described in differentiating neurons, either locally, in filopodia or growth cone or globally, at the level of the soma , in the Xenopus and rat brain, with consequences on several behaviors . We then analyzed the consequences of these transient pharmacological treatments a few days later, between 6 and 7 d postfertilization (dpf). We report quantifiable changes in the specification of the dopaminergic phenotype in SP-DA neurons. We also report induced changes in locomotion within the same time frame. These results suggest a contribution of ENE to the specification of the dopaminergic phenotype and the establishment of motor control in the zebrafish. They also open important perspectives for this model to decipher the molecular events leading from environmental factors to the modifications of developmental trajectories.All experiments were conducted following animal care guidelines provided by the French ethical committee and under the supervision of authorized investigators.Zebrafish were raised according to standard procedures . Brieflykca4, Tgicm06, Tgf13, Tgzf34Tg, and zf710Tg(Et.slc18a2:GFP).Wild-type zebrafish were of AB background. The following transgenic zebrafish lines were used: m), tetrodotoxin , ω conotoxin (0.08 μm), nifedipine (0.4 μm), and flunarizine (2 μm) were purchased from R&D Systems and prepared in water except for veratridine which required dimethyl sulfoxide (DMSO) for dissolution and flunarizine that requires ethanol (EtOH) for dissolution. Control exposures were performed using the same concentration of DMSO in EM without the drug. The specific period and duration of applications are indicated in the corresponding figure legends. All pharmacological treatments were performed by bath application followed by three washes in EM. Embryos were randomly distributed in wells (30 embryos per well) of a six-well plate containing 5 ml of solution (EM+DMSO or EM+drug). Embryos exposed to drugs or the control solution were observed for morphologic abnormalities every day until 5 dpf. Malformations  were considered experimental end-points, and when detected the corresponding animals were excluded from the study.Pharmacological compounds, veratridine . We used three different gal4 lines to drive UAS-dependent expression of GCaMP: a  line, in which the TATA-box binding protein (TBP) promoter drives constitutive ubiquitous expression of the transgene, a Tg line in which the expression is trigerred following a transient increase in temperature, and a  line, in which the Elavl3 promoter drives pan-neuronal expression of the transgene.For CaHsp70:GAl4;UAS:GCaMP6f) line were exposed to a 38°C temperature for 1.5 h. Upon heat shock activation, GCaMP6f is expressed in all the cells of the animals and remains detectable for several days.At 24 hpf, embryos of the , then individually embedded in low melting agarose (Life Technologies), ventral side up, for imaging.x-/y-axis were corrected using the “Stackreg” plugin . Regions of interest (ROIs) were drawn manually over individual cell bodies and the average gray level from pixels in ROIs was measured over time using the MultiMeasure plugin (Optinav). Sequential values of fluorescence were then treated in MATLAB (MathWorks). Transients were defined as an increase of fluorescence higher than 2.5 times the standard deviation of the baseline. The duration and amplitude of transients were calculated using the “peak” function. Incidence was scored as the number of cells generating transients divided by the estimated total number of cells in the imaged field and was expressed as a percentage. Frequency was calculated as the total number of transients in a given cell divided by the total acquisition time and was expressed as transients per hour.Thirty-minute time-lapse series were acquired at 1 Hz, at a single focal plane, on an Olympus BX60 microscope (Olympus Corporation) equipped with a 40 × 0.6 N water immersion objective. A nonlaser spinning disk system  was used for illumination and image acquisition. Images were processed with Fiji. Movements of the preparation in the Six- to 7-dpf zebrafish larvae were deeply anesthetized using 0.2% Ethyl3-aminobenzoate methanesulfonate  diluted in EM, then they were fixed in ice-cold 4% paraformaldehyde  in 1× PBS (Fisher Scientific) containing 0.1% Tween 20% (PBST) overnight at 4°C. Samples were dehydrated and stored in MeOH at −20°C.2O2) solution in ethanol (EtOH) 100% for 30 min, to deactivate endogenous peroxidases. They were then successively incubated in EtOH:Xylene 1:1 without agitation for 1 h and, at −20°C, in EtOH:acetone 1:2 without agitation for 20 min. The washes performed between these steps were performed in EtOH. After the final wash, samples were rehydrated in PBST.Immunofluorescence was performed in 2-ml microtubes. Unless specified otherwise in the protocol, incubations were performed at room temperature (RT), and thorough PBST washes were performed between each step. The samples were first incubated in 3% hydrogen peroxide , 1% Triton X-100, 1% Tween 20, 1% DMSO, 1% bovine serum albumin (BSA) in PBS 1×] for 3 h.To unmask the antigens, samples were incubated in PBST:Tris 150 mTwo protocols were used for primary antibody staining, adapted from published protocols . For theThe samples were then incubated in blocking buffer 2  for 1 h at RT and incubated with a first secondary antibody . Briefly, the AB mix was prepared by adding 10 μl of solution A and 10 μl of solution B in 1 ml PBST/1% Triton X-100. One hour after the preparation, the samples were incubated in the AB mix for 1 h. Samples were then incubated in Tyramide-TAMRA (1:200 in PBST) for 20 min, then 0.012% HBefore a second primary antibody incubation, the samples underwent a step of fixation in PFA 4% for 2 h at RT and were washed overnight in PBST.For the other primary antibodies . This value was used as a low threshold in subsequent analysis.We recorded the locomotion of individual larvae at 6–7 dpf, a period of relative stability for swimming parameters .Results are shown as scatterplots overlaid with box and whisker plots showing minimum (bottom whisker), maximum (top whisker), mean (cross), median (line), first quartile (bottom of the box), and third quartile (top of the box), and each value (dots). For means comparisons, we first performed the Shapiro–Wilk test for normality. If all samples passed the normality test, we then checked for equality of variance. When all samples had similar variances, we performed an ordinary ANOVA test. When differences in variances were detected, we performed Brown–Forsythe and Welch ANOVA tests (BFW ANOVA). Multiple comparisons were then performed using Dunnett’s test. If one sample or more did not pass the normality test, we used the nonparametric Kruskal–Wallis test coupled with Dunn’s test for multiple comparisons (KW). Tests and Statistical tests were performed using Prism (GraphPad), except for the equality of variance test performed with the online software Brightstat .Xenopus (m), which blocks the inactivation of voltage-dependent sodium channels. To decrease ENE, we used a cocktail containing TTX (2 μm), ω-conotoxin (0.08 μm), nifedipine (0.4 μm), and flunarizine (2 μm) targeting voltage-dependent sodium channels, N, L, and T subtypes of voltage-dependent Ca2+ channels, respectively. We refer to this cocktail as TCNF.To study the contribution of embryonic neural excitability (ENE) to zebrafish larval development, we adapted the transient pharmacological treatments previously validated in Xenopus . Bath trXenopus A. To inc2+ transients as a proxy. We followed the dynamics of intracellular Ca2+ concentration using time-lapse imaging of 2- to 3-dpf zebrafish embryos expressing the genetically encoded Ca2+ reporter GCaMP6f  line, in which the transgene is expressed on heat shock activation  promoter  corresponding to individual cell bodies. We used four parameters to analyze the results, the average frequency of transients per ROI, the incidence of active ROIs in the field of view , and the duration and amplitude of individual transients . In control conditions, we detected the presence of sporadic spontaneous transients in the telencephalon of the three transgenic backgrounds we used  neurons distributed as two neighboring subpopulations, in the subpallium (SP), and in the olfactory bulb (OB).To identify dopaminergic neurons in the telencephalon of 6- to 7-dpf larvae we used anatomic landmarks such as the position of brain ventricles and large fiber bundles combined to two markers of the catecholaminergic phenotype: the enzyme limiting the synthesis of catecholamines , tyrosine hydroxylase (TH), and the transporter accumulating monoamines inside exocytotic vesicles, vesicular monoamine transporter (vMAT). Indeed, in zebrafish, like in mammals, adrenaline and noradrenaline-containing neuronal soma, labeled by the same markers, are not detected anterior to the midbrain-hindbrain boundary, rather, they are restricted to neuronal subpopulations located in the locus coeruleus, the medulla oblongata and the area postrema , 2003. Tth; previously known as th1 and th2). th2 is mostly expressed in the caudal hypothalamus and is very rare in the nuclei studied here (+ cells in the remaining manuscript) but does not recognize Th2 , but only the latter is detected in the zebrafish brain (Tg(Et.vMAT2:eGFP) transgenic line (referred to as vMAT2+ cells in the remaining of the manuscript).In zebrafish, TH is encoded by two paralogous genes  were distributed bilaterally along the midline, and relatively close to it  were distributed at the anterior end of the forebrain and were projecting anteriorly before rapidly branching in multiple directions.In the OB, the cell bodies of vMAT2+ cell bodies and projections were similar as for vMAT2+ cells. All TH1+ cells were also vMAT2+, while some vMAT2+ cells were TH1−  subpopulations. This suggests that ENE influences the specification of monoaminergic precursors during an embryonic critical window, driving them toward the dopaminergic fate.In the OB, increasing ENE from 48 to 72 hpf did not change the number of TH11+ cells A,D. As is region A,D. The  as well E. Overal+ cells was stable following the increase or the decrease of ENE . Overall, increasing ENE increased locomotion while decreasing ENE decreased locomotion. Although the distinction between bursts and cruises may be viewed as arbitrary, the overall positive correlation between ENE and global larval activity  is robust.To exclude a direct contribution of the treatments applied in the embryonic period on the parameters measured in the larval period, we assessed the reversibility of the treatments by following the time course of the effects of acute pharmacological treatments in two sets of experiments.First, on 5-dpf larvae, we performed 10-min recording sessions and analyzed the overall distance covered before the application of the treatments, then +2 h after the application of the treatments, and lastly 24 h after washing out the drugs. Acute treatments of swimming larvae had the expected effects: veratridine application induced an increase in the distance covered, while TCNF application led to a decrease in the distance covered , which result in noncell-autonomous phenotype switching  were similar to what has been reported in Xenopus , although these two behaviors are believed to involve distinct networks, suggesting that ENE should modulate a common regulatory circuit. Determining whether direct activation of dopaminergic neurons in the subpallium could stimulate cruises or bursts using optogenetic tools is an open perspective for the future. It would be interesting to test whether other behaviors such as prepulse inhibition are also affected following alterations of ENE.Links between movement control and the neuromodulator effect of dopamine are widely described. In zebrafish, it has been involved in the initiation of movement , via dirThe developing zebrafish provide an attracting model in which to perform transcriptomic analysis to identify genes differentially expressed in vMAT2 cells following modifications of ENE. The results would help decipher the cellular and molecular mechanisms underlying the modulatory role of ENE during brain differentiation.The data presented here point to an implication of ENE in the regulation of dopaminergic differentiation in the developing brain, suggesting that ENE could act as an intermediate between environmental factors and the molecular changes leading to the alteration of dopamine-related behaviors. The zebrafish model is also adapted to test how environmental factors such as malnutrition, stress, and drug exposure could modulate ENE and potentially influence the maturation of the dopaminergic systems.