Imported fire ants are significant pests of urban, agricultural, and medical importance, causing USD billions of annual losses in the United States. Synthetic insecticides are commonly used in their management. The potential adverse effects of synthetic insecticides highlight the need to develop natural-product-based alternatives for fire ant control. Repellants are useful in managing fire ants; for example, repellants can be used to prevent fire ants from invading sensitive areas, such as electrical equipment, nursing homes, and hospitals. In particular, plant-derived natural repellants may provide a safer and more environmentally friendly alternative. This study demonstrates the repellent effects of the plant-essential-oil-derived compounds carvacrol, thymol, and their acetate derivatives against imported fire ants. Carvacrol, a GRAS compound  was the most potent repellent against red, black, and imported fire ants with minimum repellent effective doses of 0.98 µg/g, 7.80 µg/g, and 0.98 µg/g, respectively, followed by thymol, carvacrol acetate, and thymol acetate. Thymol red essential oil containing carvacrol and thymol also showed repellency. These results indicated that carvacrol and thymol as well as essential oils with high contents of carvacrol and/or thymol are potentially useful in managing imported fire ants.Solenopsis invicta Buren), black imported fire ants (S. richteri Forel), and hybrid imported fire ants (S. invicta × S. richteri). They are significant pests, and their control heavily relies on synthetic insecticides. The extensive use of insecticides has led to public concern about their potential negative effects on human health and the well-being of wildlife and the environment. As an alternative, plant-derived natural compounds, particularly essential oils (EOs) and their main constituents, show promise as safe and environmentally friendly products for controlling fire ants. Repellants are useful in managing fire ants, and plant-derived natural repellants may serve as a safer and more environmentally friendly option. This study investigates the repellency of EO-derived compounds carvacrol, thymol, and their acetates against imported fire ant workers. The results revealed that carvacrol, a GRAS compound , was the most potent repellent against S. invicta, S. richteri, and their hybrid, with minimum repellent effective doses (MREDs) of 0.98 µg/g, 7.80 µg/g, and 0.98 µg/g, respectively. Thymol also exhibited strong repellency, with MREDs of 31.25 µg/g, 31.25 µg/g, and 7.8 µg/g, respectively. Furthermore, thyme-red essential oil, characterized by a thymol chemotype containing 48.8% thymol and 5.1% carvacrol, was found to effectively repel the hybrid ants with an MRED of 15.6 µg/g. In contrast, thyme essential oil, characterized by a linalool chemotype lacking thymol and carvacrol, did not exhibit any repellent effect, even at the highest tested dose of 125 µg/g. This study provides the first evidence of the potent repellency of carvacrol and thymol against imported fire ant workers, indicating their potential as promising repellents for fire ant control.In the United States, imported fire ants are commonly referred to as red imported fire ants ( Solenopsis invicta Buren) (Hymenoptera: Formicidae), black imported fire ants (Solenopsis richteri Forel), and hybrid imported fire ants (S. invicta × S. richteri), are major invasive pest ants in the United States [Imported fire ants, including red imported fire ants , often referred to as “Green Pesticides,” have gained popularity in modern organic agriculture as a safe and environmentally friendly pest control option that is compatible with biological control methods . EOs havarvacrol . Thymol arvacrol , has exharvacrol . To the Thymus vulgaris  and thyme from Thymus vulgaris , were purchased from Edens Garden .Carvacrol (purity ˃ 98%) was purchased from Tokyo Chemical Industry—TCI , and thymol (purity ˃ 99%) from Sigma Aldrich . Carvacrol acetate and thymol acetate were synthesized (see below in p-cymene (16.3%), γ-terpinene (6.4%), carvacrol (5.1%), caryophyllene (3.1%), linalool (2.4%), eucalyptol (1.4%), α-humulene (1.1%), and (-)-terpinen-4-ol (1.0%), while the major compounds in thyme EO were linalool (71.9%), linalyl acetate (13.4%), and caryophyllene (3.4%). The percentage compositions of other constituents in these EOs are shown in Thyme-red EO and thyme EO were analyzed by GC-MS using an Agilent 7890 B GC system equipped with a 5977A quadrupole mass spectrometer and a 7693 autosampler . The sample was prepared in GC-MS grade dichloromethane (Sigma-Aldrich) at a concentration of 10 mg/mL. Helium was used as the carrier gas at a flow rate of 1.5 mL/min. In split injection mode, the inlet temperature was set to 280 °C and the split ratio was set to 30:1. The oven temperature program was initially set to 60 °C, held for 2 min, then ramped up to 280 °C at a rate of 6 °C/min, and heated isothermally at 280 °C for 10 min, for a total run time of 77 min. Data acquisition was performed using Agilent MassHunter software (version B.07.06). Compound identification was based on an NIST library search and comparison with reference standards. The main constituents present in thyme-red EO were thymol (48.8%), 2Cl2 (15 mL) containing 555 µL of triethylamine and a catalytic amount of 4-dimethylaminopyridine  was stirred at room temperature for 1 h under N2. The reaction mixture was washed with HPLC-grade water (20 mL × 3). The organic phase was dried with anhydrous Na2SO4 and evaporated to dryness to obtain carvacrol acetate (530 mg). Identification of the product was confirmed by 1H and 13C NMR spectra (A mixture of carvacrol (500 mg) and acetic anhydride  in CH spectra . Its pur2Cl2 (30 mL) containing 1.11 mL of triethylamine and a catalytic amount of DMAP (70 mg) was stirred at room temperature for 2 h under N2. The solution was washed with HPLC-grade water (40 mL × 3). The organic phase was dried with anhydrous Na2SO4 and evaporated to dryness to obtain thymol acetate (1.21 g). Identification of the product was confirmed by 1H and 13C NMR spectra  and acetic anhydride  in CH spectra . Its purColonies of red imported fire ants, black imported fire ants, and hybrid ants were collected from Tunica County, MS-713; Hernando, DeSotos County, MS 38632 (34°49′56.5″ N 90°12′55.6″ W); Washington County, MS 38748 (33°09′31.2″ N 90°54′56.4″ W); and the University Field Station , respectively. The ant colonies were kept in the laboratory under conditions of 25 ± 2 °C and 50% ± 10% relative humidity and fed with crickets and a 25% honey/water solution. The ants were maintained under laboratory conditions for one month before starting the bioassays. The ant species were identified based on the venom alkaloid and hydrocarbon profiles of the collected individuals ,24. The The digging bioassay used in this study for testing repellency has been described in our previous paper , which ip ≤ 0.05 (SAS 9.4 (2012)).Data were analyzed by using an analysis of variance (ANOVA)  followed by a Ryan–Einot–Gabriel–Welsch multiple range test for mean separation at As shown in Two chemotypes of thyme oils, thyme-red EO containing thymol (48.8%) and carvacrol (5.1%) and thyme EO containing linalool (71.9%) and linalool acetate (13.4%), were also tested against the hybrid imported fire ants. The MRED of thyme-red EO was 15.6 µg/g. For thyme EO, in which thymol and carvacrol are not present, no repellent effect was observed at the highest test dose of 125 µg/g.Given the high impact of imported fire ants on agriculture, human health, and the environment, interest in developing novel repellents is gaining attention. Previously, synthetic insecticides such as bifenthrin and tefluthrin were used to repel fire ants from potting soil  and permOur study showed that carvacrol and thymol, two structurally close monoterpene isomers, had remarkable repellent activity against imported fire ant workers. Unlike the superior repellent effect of carvacrol acetate compared to carvacrol in ticks , carvacrAedes albopictus [Dermanyssus gallinae) showed that carvacrol was superior to thymol [A. albopictus [Pochazia shantungensis) [Cryptolestes ferrugineus) [The repellency of carvacrol against imported fire ant workers is approximately 32-, 8-, and 7-fold higher than thymol against red imported fire ants, black imported fire ants, and the hybrid ants, respectively. This seems to be consistent with a previous report describing mosquito repellency of the two compounds against bopictus , where co thymol . Carvacrbopictus , insectingensis) , and fumugineus) . Taken tugineus) .Drosophila melanogaster) [Thymol was initially registered as a pesticide in the United States in 1964 for use as a repellent (EPA-738-F-93-010). It repels vertebrate pests by a non-toxic mode of action, which should not result in unreasonable adverse effects on human health or the environment. In recent years, thymol’s repellent and insecticidal activities against mosquitoes have been studied extensively ,39,40. Iogaster)  has alsoOur study has also shown that thymol-red EO containing carvacrol and thymol exhibited potent repellency against hybrid imported fire ants, and these two compounds are likely the active compounds responsible for the observed repellency. The thymol-chemotype of thyme EOs or many other EOs with high contents of carvacrol and/or thymol  may exerIt is worth noting that carvacrol and thymol are listed as food additives by the US Food and Drug Administration, and they are “Generally Recognized As Safe” (GRAS) in food when used in the minimum quantity to produce their intended effect. In 2021, the Expert Panel of the Flavor and Extract Manufacturers Association further affirmed the GRAS status of carvacrol and thymol and several EOs containing these compounds under their conditions of intended use as flavor ingredients . This inThis study has demonstrated the repellency of carvacrol and thymol against imported fire ant workers. This is the first report of the repellency of the two compounds against imported fire ants. In particular, carvacrol is a more promising candidate due to its remarkable repellent effects towards all the three fire ant species tested. In the future, more structural analogues of carvacrol and thymol may be synthesized for structure–activity relationship studies. In addition, formulation studies, e.g., microencapsulation for long-lasting repellency, may be conducted to develop carvacrol- and thymol-based products for fire ant control.


Introduction: We aimed to evaluate the influence of 1,25-dihydroxyvitamin D 2D) on metabolic dysfunction and elucidate its underlying mechanism using a rat model of polycystic ovary syndrome (PCOS).Methods: Twenty-four Sprague-Dawley rats were randomly divided into four groups: control group , 1,25VD group 2D), PCOS group , PCOS+1,25VD group 2D). The treatments were administered for 8 weeks. Body weight, estrus cycle, insulin tolerance, and oral glucose tolerance of the rats in the different groups were assessed. The rats were euthanized at the 8th weeks, and plasma, ovarian, and liver samples were collected and analyzed. The hepatic lipid profile was characterized using HPLC/MRM.Results: Letrozole-induced PCOS rats exhibited increased weight, insulin resistance, postprandial glucose abnormalities, and dyslipidemia. Compared with the PCOS group rats, the PCOS+1,25VD group rats showed reduced body weight, increased sensitivity to insulin, decreased postprandial glucose, and elevated levels of high-density lipoprotein cholesterol. Moreover, abnormally increased liver concentrations of total diacylglycerol (DG) and DG species in the PCOS rats were reversed by treatment with 1,25(OH)2D. Additionally, hepatic DG and insulin sensitivity were correlated.Conclusion: 1,25(OH)2D inhibited hepatic DG accumulation and ameliorated metabolic dysfunction in PCOS rat models. Polycystic ovary syndrome (PCOS) is a commonly diagnosed endocrine disease, with a prevalence of PCOS approximately 4%–20% worldwide . HyperanVitamin D deficiency is a global health concern . Vitamin2D], an active form of vitamin D, exerts its biological function by interacting with vitamin D receptors  and triacylglycerol (TG) using a lipidomics strategy. The results of the current study may provide novel evidence for the application of vitamin D supplements as a cost-effective therapeutic strategy in PCOS management.Exposure to letrozole, an aromatase inhibitor, reportedly prevents the conversion of testosterone to estradiol, which further elevates the serum androgen levels and leads to polycystic ovarian morphology and other metabolic disorders in a rat model . 1,25-dieceptors . Herein,2D supplemental group  where rats received 0.5% CMC gavage daily and an intraperitoneal (i.p.) injection of 2.5ug/kg of 1,25(OH)2D  dissolved in a 0.5% ethanol and 95.5% culture medium solution every 2 days  where rats received 1 mg/kg of letrozole gavage and 2.5 ug/kg of i.p. 1,25(OH) 2D injection every 2 days.Twenty-four (4-5-week-old) rats, weighing 78–97 g, were purchased from the Shanghai Family Planning Research Institute, Shanghai, China. The rats (six per cage) were housed under the following conditions: 12 h light/dark cycle, 21°C–22°C, 50%–60% humidity, and free access to food and distilled water. The Soochow University Animal Care and Use Committee  approved this study . The rats were divided into the following groups: 1) control group  where rats were intragastrically administered 0.5% carboxymethyl cellulose (CMC) at a daily dose of 2 ml/kg; 2) 1,25(OH)y 2 days ; 3) LetrThe treatments were administered for 8 weeks. Rats were weighed twice per week. Vaginal smears were obtained daily during the 8th weeks to validate the induction of PCOS models. Insulin tolerance and oral glucose tolerance tests were completed on separate days of the 8th weeks. After the 8th weeks, the animals kept fasting overnight. Subsequently, they were anesthetized with chloral hydrate . The blood samples were obtained from the abdominal aorta and centrifuged at 4500 rpm for 10 min at 4°C to collect the serum. Bilateral ovary and liver samples were dissected and weighed. One ovary was treated with formalin (10% solution) to determine morphological changes; the other ovary was stored at −80°C for the lipidomics analysis  Figure . The preThe rats were kept fasting overnight. Thereafter, the rats were intraperitoneally administered insulin (1 IU/kg). Blood samples were collected from the tail vein. Blood glucose levels at 0, 15, 30, 60, and 90 min after insulin administration were measured using a blood glucometer (ASCENSIA Diabetes Care).The rats were intragastrically administered glucose (2 g/kg) after overnight fasting. Blood samples were collected from the tail vein. Blood glucose levels at 0, 15, 30, 60, 90, and 120 min after glucose administration were measured using a blood glucometer (ASCENSIA diabetes care).Serum samples were collected and enzyme-linked immunosorbent assay (ELISA) kits (Sangon Biotech) were used to analyze testosterone, luteinizing hormone, and follicle-stimulating hormone levels. Serum 25-hydroxyvitamin D [25(OH)D] levels were measured using radioimmunoassay (Roche Diagnostics). Serum triglyceride, total cholesterol, high-density lipoprotein cholesterol (HDL-c), and low-density lipoprotein cholesterol (LDL-c) levels were analyzed using an automatic biochemical analyzer (Beckman Coulter AU5800).Histological studies were performed as previously described . Ovarian2O (350 µl) and chloroform (250 µl). Subsequently, the samples were centrifuged for 5 min (12000 rpm at 4°C). After centrifugation, the lower phase was transferred to a new tube. Chloroform (500 µl) was added to the aqueous phase to extract the remaining lipids. Finally, the lipids were collected, dried using SpeedVac, and stored at −80°C until needed.The lipids were isolated from 30 mg of rat liver using the modified Bligh and Dyer’s method . Liver sAll lipidomic analyses were performed at LipidALL Technologies  using an Agilent 1290 II UPLC coupled with a Sciex QTRAP 6500 PLUS . Individpost hoc tests. Correlation analysis was performed using Pearson’s correlation coefficient. Statistical significance was set as p < 0.05.All data are expressed as mean ± standard deviation. Statistical analyses were performed using SPSS . Comparisons between two groups were performed using Student’s t-test. Comparisons among multiple groups were performed using analysis of variance (ANOVA) with Tukey’s 2D treatment had no significant effect on testosterone and the LH/FSH ratio in the PCOS rats , phosphatidylethanolamines (PE), and DGs was remarkably higher in the PCOS group than in the CON group. In contrast, the contents of phosphatidylglycerols (PG) and lysophosphatidylserines (LPS) decreased in the PCOS group. Supplementation with 1,25(OH)2D in PCOS rats decreased the DG content , DG 32:1 (16:1/16:0), DG 34:3 (18:2/16:1), DG 34:2 (16:0/18:2), DG 34:1 (18:0/18:1), DG 36:4 (18:2/18:2), DG 36:3 (18:2/18:1), DG 36:2 (18:1/18:1), DG 38:6 (18:1/20:5) and DG 38:4 (18:1/20:3)] (4 The contents of six DGs  were markedly increased in the PCOS group compared with the control group. 1,25(OH)1/20:3)] .2D3 in PCOS rats and the potential regulatory effects of 1,25(OH)2D3 on hepatic DG content.In this study, we found a significant decrease in vitamin D concentration in letrozole-induced PCOS rats compared to that in control rats. The key findings of the present study were the metabolic improvement of 1,25(OH)PCOS is a heterogeneous disease accompanied by metabolic abnormalities, such as obesity, insulin resistance, dyslipidemia, and endothelial dysfunction. Improvement of metabolic dysfunction is of paramount importance to reduce the risk of metabolic and cardiovascular disorders in patients with PCOS later in life. Additionally, improvement of metabolic dysfunction is beneficial for reproductive function and pregnancy outcomes . In the 2D3 was injected into PCOS rat models in the present study, which reduced the weight, increased insulin sensitivity, and ameliorated lipid disturbances in PCOS rats. Previous data suggested that approximately 50% of women with PCOS are overweight or obese, and excess weight aggravates metabolic and reproductive disorders in PCOS  in PCOS , and our in PCOS . In curr in PCOS . MoreoveCOS 2D3 has regulatory effects remain poorly understood. A previous study conducted using obese rat models showed that insulin resistance is associated with increased DG concentrations 2D3 could decrease the level of DG in PCOS and also confirmed the positive correlation between DG and ITT-AUC. In summary, changes in hepatic DG might be associated with the anti-obesity and insulin-sensitizing effects of 1,25(OH)2D3. Further investigation is required to assess possible underlying mechanisms.Insulin resistance plays a key role in the onset and progression of PCOS and DG is reportedly associated with insulin-related signaling pathways and is a predictor of insulin resistance . Previoutrations . Assessmtrations . Multipltrations . In the trations ; The acttrations . Furtherhe liver . Overall2D levels in rats were not measured. Serum 25(OH)D commonly reflects the vitamin D nutrient status 2D and insulin sensitivity did not prove causality, and the mechanism of DG variation that could affect insulin signaling in PCOS was not investigated in the current study.The current study had several limitations. First, the serum 1,25(OH)t status ; however2D3 on hepatic DG levels, as indicated by the lipidomic analysis. This phenomenon may explain the protective effects of vitamin D on PCOS. Further studies are required to assess the possible mechanisms underlying the DG-modulating effects of vitamin D. The results of the current study may provide novel evidence for the clinical application of vitamin D as a dietary supplement for long-term management of PCOS.In conclusion, vitamin D supplementation could prevent obesity, insulin resistance, and dyslipidemia in a letrozole-induced PCOS rat model. Moreover, this study sheds light on the regulatory effects of 1,25(OH)


Previous epidemiological evidence has demonstrated a preferential association of iTFAs, rather than rTFAs, with various diseases including cardiovascular diseases. However, it is still unknown how iTFAs exert their specific toxicity and what effective treatments are available to mitigate their toxicity. Here, we performed a comprehensive toxicological assessment of TFAs based on the toxicity mechanism that we established previously. We found that iTFAs including elaidic acid (EA), but not other types of fatty acids including rTFAs, had a strong pro-apoptotic effect upon treatment of extracellular ATP, a damage-associated molecular pattern that induces apoptosis through the apoptosis signal-regulating kinase 1 (ASK1)-p38 MAP kinase pathway. We also found that polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA), potently suppressed EA-dependent increase in ASK1 activation and apoptosis. These results demonstrate that iTFAs specifically exert toxicity by targeting ASK1, and that PUFAs serve as their effective suppressor. Our study provides a molecular basis for risk assessment of foods, and for new prevention and treatment strategies for TFA-related diseases. In the human body, fatty acid desaturating enzymes introduce only cis double bonds into fatty acids. Hence, whereas enzymatically synthesized UFAs contain all cis double bonds, hereafter referred to as cis-fatty acids (CFAs), TFAs are exclusively obtained from dietary sources1. According to the types of sources, TFAs are divided into two groups: industrial and ruminant. Industrial TFAs (iTFAs), such as elaidic acid  and linoelaidic acid , are generated as byproducts during food production processes, such as partial hydrogenation of edible oils containing CFAs, and are abundant in processed foods, including snacks and fast food products2. On the other hand, ruminant TFAs (rTFAs), such as trans-vaccenic acid , rumenic acid , and palmitelaidic acid , are mainly produced during bacterial biohydrogeneation of CFAs in the rumen of ruminants (e.g. cows and sheep), and are present in meat and dairy products , and cardiovascular diseases (CVDs)7, which has been supported by mouse model studies10. However, structure–toxicity relationships of TFAs have yet to be determined, since the molecular mechanism underlying TFA toxicity has remained elusive.12. eATP induces apoptosis through activation of the apoptosis signal-regulating kinase 1 (ASK1)-p38 mitogen activated protein (MAP) kinase pathway, initiated by reactive oxygen species (ROS) that are generated from NADPH oxidases downstream of the P2X purinoceptor 7 (P2X7)13. We showed that TFAs including EA, but not their corresponding cis isomers, potentiate eATP-induced activation of the ASK1-p38 pathway, and thereby promote apoptosis in macrophages and microglial cells12, which is closely associated with the pathogenesis of CVDs and ND, respectively. Besides these cell types, P2X7 receptor is expressed in diverse types of cells, such as epithelial cells and neuronal cells, and is involved in various diseases, including diabetes, obesity, NDs, and CVDs, through enhancing inflammatory responses and cell death15. Thus, our findings provided mechanistic insight into the etiology of TFA-related disorders.We have recently shown a novel toxicity mechanism of TFAs; TFAs promote inflammatory signaling and cell death in response to extracellular ATP (eATP), one of the damage-associated molecular patterns (DAMPs) that are known as potent pro-inflammatory factors, derived from injured cells16. We showed that EA and LEA potently promote eATP-induced activation of ASK1 and p38, and ultimately cell death, whereas TVA, RA and PEA scarcely did, in consistent with the previous epidemiological studies showing a particular association of the iTFAs with various disorders including CVDs6. Moreover, by focusing on the ameliorating effect of all-cis poly-unsaturated fatty acids (PUFAs), such as eicosapentaenoic acid  and docosahexaenoic acid , on various pathological conditions17, we found that co-treatment with PUFAs strongly blocked the pro-apoptotic action of iTFAs by suppressing ASK1 hyperactivation. These results demonstrate that iTFAs have a unique pro-apoptotic function during eATP stimulation, and that PUFAs serve as a potent suppressor for iTFA toxicity. Our study provides insight into developing new strategies for the prevention and treatment of TFA-related disorders.In this study, based on our recently established toxicity mechanism of TFAs, we performed a comprehensive toxicity assessment of five major food-derived TFAs: EA and LEA as iTFAs, and TVA, RA, and PEA as rTFAs Fig. 16. We sh11, we confirmed that EA pretreatment strongly decreased cell viability upon eATP stimulation  apparently increased ATP-induced phosphorylation of ASK1 and p38 , EPA or DHA drastically increased cell viability, suggesting that PUFAs, particularly those possessing four or more double bonds, have a strong protective effect against EA toxicity upon eATP stimulation. Since DHA had the highest inhibitory effect among PUFAs , an upstream kinase of ASK1, are essential for EA-mediated hyperactivation of ASK1 in response to eATP11. To determine the target of DHA to exert its protective action against EA toxicity, we examined the effect of DHA on eATP-induced ROS generation and CaMKII phosphorylation. Using a green fluorescent ROS indicator, DCFH-DA, we observed no significant difference in eATP-induced ROS generation with or without DHA pretreatment  is much higher than that of LEA (~ 2 µM)22. Therefore, EA likely plays a major causative role in TFA-related diseases among TFA species. Notably, although there is no reliable evidence of the actual TFA amount in human tissues, EA concentration in the liver of rodents fed with a normal diet is estimated to be 1–6 mM24, and that in rat brain is estimated to be 200 µM25. Therefore, the concentration of TFAs at which significant toxicity was observed (50–200 µM) in this study would be physiologically relevant.In this study, we demonstrated that EA and LEA, the predominant TFAs in industrially produced foods (iTFAs), but not TVA, RA, and PEA, major TFAs contained in ruminant foods (rTFAs), efficiently facilitated eATP-induced apoptosis signaling Figs. e,f and 3ly Figs. g,h and 3 20 Fig. . Based o26. Thus, it would be possible that PUFAs directly bind to PP5, elevate the phosphatase activity of PP5, and thereby prevent ASK1 hyperactivation in response to EA. Nevertheless, it is noteworthy that while DHA completely reversed the pro-apoptotic effect of EA, it did not increase cell viability when stimulated with eATP without EA . LPS was purchased from Invivogen . Aspirin was purchased from Tokyo Kasei . 1-Aminobenzotriazole, HET0016, Zileuton, PD146176, Celecoxib, MS-PPOH, ML355, and Triacsin C were purchased from Cayman .ATP and H2 at 37 °C. ASK1 KO RAW264.7 cells were established in our previous study11. HEK293A cells obtained from Thermo Fisher Scientific  were cultured in Dulbecco’s Modified Eagle Medium  containing 5% heat-inactivated fetal bovine serum  and 1% penicillin–streptomycin solution  in 5% CO2 at 37 °C.RAW264.7 and BV2 cells, obtained from ATCC, were cultured in RPMI 1640 and Dulbecco’s Modified Eagle Medium , respectively, containing 10% heat-inactivated fetal bovine serum  and 1% penicillin–streptomycin solution  in 5% CO7 and pCAGGS-rat CaMKIIα were constructed in previous studies35. Plasmid transfection was performed using Polyethylenimine “Max” , according to the manufacturer’s instructions.pcDNA3.2 HA-mouse P2X33. Briefly, fatty acids were dissolved in 0.1 N NaOH at 70 °C, and then conjugated with fatty acid-free BSA  at 55 °C for 10 min to make 5 mM BSA-conjugated fatty acid stock solutions containing 10% BSA. Cells were treated with various concentrations of BSA-conjugated fatty acids by diluting stock solutions in a medium without fetal bovine serum .OA , EA (Sigma), LA, LEA, AA, EPA, DHA (Cayman), TVA , and RA and PEA  were prepared as described previously36. The antibodies used for immunoblotting were against phospho-ASK1 (Thr-845), phospho-p38, p38 , β-actin , ASK1 , phospho-CaMKII (Thr-286), and CaMKII 37. The blots were developed with ECL , and detected with ChemiDoc Touch Imaging System .Cells were lysed in ice-cold lysis buffer containing 20 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% Triton-X100, 10% Glycerol, and 1% protease and phosphatase inhibitor cocktail . After centrifugation, the cell extracts were resolved by SDS-PAGE, and were analyzed as described previously38. RAW264.7 and BV2 cells were seeded on 96-well plates. After any stimulation or treatment, cell viability was determined using Cell Titer 96 Cell Proliferation Assay , according to the manufacturer’s protocol. The absorbance was read at 490 nm using a microplate reader . Data are normalized to control without stimulus, unless noted otherwise.Cell viability was assayed as described previously39. Briefly, after stimulation, cells seeded on a glass plate were treated with 10 μM DCFH-DA (Sigma) for 30 min, and intracellular ROS generation was observed using a Zeiss LSM800 laser confocal microscope  and the images were processed with Zen software. After background subtraction, ROS level was calculated by dividing total green fluorescence by cell numbers using Image J. Data are shown as mean ± SD of relative fluorescence intensity from three different fields of view.Intracellular ROS was detected and quantified as described previously40. Isolated lipids were methylated with 2.5% H2SO4 in methanol. The resulting fatty acid methyl esters were then extracted with hexane and subjected to gas chromatography tandem mass spectrometry (GC–MS/MS) analysis. GC–MS/MS analysis was performed by using a GCMS-QP2010 Plus  equipped with Zebron ZB-FAME 60 m × 0.25 mm × 0.20 µm . The oven temperature program was as follows: the initial temperature was 100 °C for 2 min, then raised to 280 °C at 15 °C/min, and held for 5 min. The injector and detector temperatures were set at 240 °C and 260 °C, respectively. The amounts of incorporated TFAs were calculated based on standard curves created from serial dilution of the respective fatty acids, which was normalized with the amount of extracted protein measured by Bradford method using protein assay CBB solution .Lipids were extracted by the method of Bligh and DyerAll the values are expressed as means ± SD, and statistical analyses were performed using GraphPad Prism software (v.9.3.0). All experiments were repeated at least three independent times. Two groups were compared using two-tailed Student’s t-test. Multiple-group comparisons were conducted using either the one-way ANOVA analysis or two-way ANOVA analysis followed by Tukey–Kramer or Dunnett’s test.Supplementary Figures.


Under 250P conditions, root elongation was significantly inhibited, and the density of lateral roots was dramatically increased. This suggests that 250P may inhibit the elongation of tea plant roots. Moreover, the P concentration in roots was about 4.58 times higher than that under 0P, indicating that 250P may cause P toxicity in tea plant roots. Contrary to common plants, the expression of CsPT1/2 in tea plant roots was significantly increased by four times at 250P, which indicated that tea plant roots suffering from P toxicity might be due to the excessive expression of phosphate uptake-responsible genes under 250P conditions. Additionally, 94.80% of P-containing metabolites accumulated due to 250P stimulation, most of which were energy-associated metabolites, including lipids, nucleotides, and sugars. Especially the ratio of AMP/ATP and the expression of energy sensor CsSnRKs were inhibited by P application. Therefore, under 250P conditions, P over-accumulation due to the excessive expression of CsPT1/2 may inhibit energy metabolism and thus the growth of tea plant roots.Low phosphorus (P) is a major limiting factor for plant growth in acid soils, which are preferred by tea plants. This study aims to investigate the unique mechanisms of tea plant roots adaptation to low-P conditions. Tea plant roots were harvested for multi-omics analysis after being treated with 0 µmol·L As an essential macronutrient for plant growth and development, P serves not only as a structural element of cell membranes and genetic materials but also as a constituent element of energy materials like ATP and phosphate esters . HoweverSPX to coordinate intracellular P homeostasis [SPX, a key transcription factor in the P signaling network, is usually upregulated by low P to enhance its expression [PvSPX1 in Phaseolus vulgaris significantly upregulates the expression of downstream P transport key genes, including PvPHT2 and PvPT1, thereby increasing P concentration in transgenic hairy roots [PHO2, which encodes the E2 ubiquitin ligase, is usually inhibited by low P and negatively regulates the P signaling network genes [OsSPX1 is increased in the rice pho2 mutant, indicating that upstream OsPHO2 may repress the transcriptional expression of OsSPX1 [PT1;6 are significantly induced by low P in roots [Pht1;4 gene, one of the key high-affinity phosphate transporters in Arabidopsis, is mutated [In order to adapt to low-P conditions, plants have developed various strategies to improve the efficiency of P absorption and utilization. These strategies include changing root architecture or morphology and the expression of P transporters in roots to promote P uptake, secreting organic acids or phosphatases to increase P availability in soils, and regulating key genes such as eostasis ,5. Amongpression . Overexpry roots . Howeverrk genes . The traf OsSPX1 . Downstrf OsSPX1 ,11,12. Iin roots . However mutated .Camellia sinensis) is widely consumed due to its abundant health metabolites, and it can only be cultivated in acidic soils, where Aluminum (Al) toxicity and P deficiency are major factors limiting plant growth [Phaseolus vulgaris control plants adapted to a low P environment [Tea  activity, as demonstrated by Kim et al. [Snf in Mucor circinelloides can significantly increase lipid content in cells [CsSnRKs in tea plants led to a substantial accumulation of lipid metabolites under the 250P condition 2SO4, 125.0 µmol·L−1 K2SO4, 100.0 µmol·L−1 MgSO4, 62.5 µmol·L−1 Ca(NO3)2, 37.5 µmol·L−1 CaCl2, 25.3 µmol·L−1 KH2PO4, 16.4 µmol·L−1 Fe(III)-EDTA, and slight amounts of MnSO4, H3BO3, ZnSO4, CuSO4, and (NH4)6Mo7O24 [−2 s−1, and a temperature of 27 °C.The tea plants utilized in this study were )6Mo7O24 . There wAfter P treatments, the roots were photographed using a digital camera . The newly grown roots were then scanned and analyzed using the WinRHIZO LA2400 root system  for total root length. The main root length was measured via Image J software (version 1.52a).3 for complete digestion. After cooling, the solution was added to a volume of 20 mL. Furthermore, 1 mL of digestive solution was diluted about four-fold using ultrapure water. Finally, the nutrient element quantification was performed using an inductively coupled plasma mass spectrometer, the ICP-MS7900 . Sample powder (0.010 g) was digested in nitric acid and heated gradually to 120 °C, and nutrient quantification was performed using an inductively coupled plasma mass spectrometer 7900 .After treatments, the newly grown roots were harvested, rinsed with distilled water three times, and oven-dried at 105 °C for 30 min, followed by further drying at 70 °C until a constant weight was achieved. After being ground into powder, 0.010 g of the sample was weighed and heated gradually to 120 °C in concentrated HNO−1. The column temperature is about 40 °C. The mass spectrometer is equipped with an ESI Turbo Ion-Spray interface, which allows operation in both positive and negative ion modes. The collected information was qualitatively analyzed on the basis of the Metware database, and the peak area of each chromatographic peak represented the relative content of the substance. Principal component analysis and cluster analysis were conducted on all samples, and differentially accumulated metabolites were screened based on the criteria of p < 0.05 and log2FC (fold change) > 1.The root samples were collected and detected for metabolomes by Metware Biotechnology Co., Ltd. . For metabolomic analysis, the freeze-dried sample was crushed and extracted with methanol, and the supernatant was retained for analysis. The supernatant was detected with the SB-C1 1.8 µm, 2.1 mm × 100 mm liquid chromatographic column  and analyzed by the AB4500 Q TRAP UPLC-ESI-MS/MS system  in the ESI-triple quadrupole linear ion trap (QTRAP) mass spectrometer. The mobile phase is composed of solvent A (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). The sample measurement was conducted with a gradient program. The flow rate is 0.35 mL/minhttps://ngdc.cncb.ac.cn/gwh/Assembly/12834/show accessed on 1 October 2022). The gene expression levels were calculated based on the FPKM value, and bioinformatics analyses were performed to screen for differential gene expression and conduct functional annotation based on the expression levels of genes in different samples. Differential gene screening was conducted using a threshold of padj < 0.05 and an absolute log2FC (fold change) > 1.The root samples were also analyzed by RNA-seq by Metware Biotechnology Co., Ltd. . Total RNA was extracted from the roots using the TRIzol reagent , and the RNAseq library was prepared using the NEBNext Ultra RNA library kit . The sequencing data were generated on the Illumina platform, and the raw data were filtered and aligned to the Tieguanyin genome , Phytozome (https://phytozome-next.jgi.doe.gov/ accessed on 1 November 2022), and Tair (https://www.arabidopsis.org/ accessed on 13 November 2022). The amino acid sequences were aligned, and an evolutionary tree was constructed using the Neighbor Joining (NJ) method and Poisson model in MEGA software (version 10.2.6).The amino acid sequences of homologous genes in rice, soybean, and Arabidopsis were obtained from online databases, including MSU-RGAP (t-tests with SPSS software (version 19.0).The data were analyzed by The tea plants adapt well to acid soil with limited available phosphate. Our study revealed that the tea plant roots remained healthy under 0P, while they suffered under 250P, likely due to their high P utilization efficiency under 0P and over-uptake of phosphate under high-P conditions.CsPT1/2). Moreover, P over-accumulation under 250P conditions regulates energy metabolism and thus root growth. Under low-P conditions in acid soils, tea plants regulate the synthesis and accumulation of energy-related metabolites such as lipids, carbohydrates, and adenosine by controlling the expression and activation of the energy-sensing kinase CsSnRKs. In the aspect of tea quality, high P could also inhibit the esterification of catechins in the tea plant roots, which might be carried out by decreasing the expression of esterification genes, such as CsTGY09G0002412b, and finally reducing the accumulation of EGCG. These findings suggest that tea plants have developed a delicate balance in P uptake and utilization during the evolution of adaptation to low-P conditions. In the management of tea plantations, excessive P fertilizer application should be avoided to ensure the promotion of healthy root growth and tea quality.Tea plants have unique mechanisms to adapt to low-P conditions in acid soils. However, excessive P application can inhibit the growth of tea plant roots and cause P toxicity. In this study, we found that high P (250P) inhibited the elongation of tea plant roots and caused P toxicity, which was possibly due to the excessive expression of phosphate uptake-responsible genes (


Aphidoletes aphidimyza is a predator that is an important biological agent used to control agricultural and forestry aphids. Although many studies have investigated its biological and ecological characteristics, few molecular studies have been reported. The current study was performed to identify suitable reference genes to facilitate future gene expression and function analyses via quantitative reverse transcription PCR. Eight reference genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RPS13, RPL8, RPS3, α-Tub, β-actin, RPL32, and elongation factor 1 alpha (EF1-α) were selected. Their expression levels were determined under four different experimental conditions  using qRT-PCR technology. The stability was evaluated with five methods . The results showed that GAPDH, RPL32, and EF1-α were ranked as the best reference gene combinations for measuring gene expression levels among different developing stages and in various starvation treatments. RPL8 and RPS3 were recommended to normalize the gene expression levels among different adult tissues. RPL32, β-actin, and EF1-α were recommended sugar-feeding conditions. To validate the utility of the selected reference pair, RPL8, and RPS3, we estimated the tissue-biased expression level of a chemosensory protein gene (AaphCSP1). As expected, AaphCSP1 is highly expressed in the antennae and lowly expressed in the abdomen. These findings will lay the foundation for future research on the molecular physiology and biochemistry of A. aphidimyza. Aphidoletes aphidimyza Rondani (Diptera: Cecidomyiidae) is widely utilized as an efficient predator of aphids in various agricultural systems, and its larvae can prey voraciously on more than 80 aphid species , β-actin, and elongation factor 1 alpha (EF1-α) (Quantitative reverse transcription PCR (qRT-PCR) has become one of the most popular methods for studying quantitative gene expression because of its high sensitivity, specificity, and reproducibility . The acc (EF1-α) . However (EF1-α) . Each ca (EF1-α) .A. aphidimyza reference genes were evaluated and selected based on stability under a wide range of conditions. Eight potential reference genes of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RPS13, RPL8, RPS3, α-Tub, β-actin, RPL32, and elongation factor 1 alpha (EF1-α) were assessed for expression stability by ΔCt, geNorm, NormFinder, and BestKeeper during different developmental stages, in different adult tissues, under sugar treatment conditions, and under starvation treatment conditions. Based on rankings produced by the aforementioned four statistical algorithms, an overall ranking for each experimental condition was then generated using RefFinder. The results will function as a foundation for subsequent studies investigating A. aphidimyza gene expression and gene function.In the current study A. aphidimyza strain used in this study was collected at the tobacco station in Leshan Town, Zunyi City, Guizhou Province, China, in May 2017. They were raised in an artificial climate chamber at 24°C ± 1°C, a 16:8 light-dark cycle, and 70% relative humidity. Megara japonica Matsumura on broad bean plants was utilized to feed the larvae, while honey was fed to adults.The A. aphidimyza individuals at different development stages, including 10–20 first–third larvae, 10 pupae, and 8 adults  were collected representing different developmental stages. Each experimental treatment included three biological duplicates. All experiments were performed with three independent biological replicates. Individuals of each sample were collected and kept in 1.5 mL centrifuge tubes and then were rapidly frozen in liquid nitrogen, and stored at −80°C before the total RNA extraction.aphidimyza adults were stunned by keeping them in a −20°C refrigerator for 3 min. Multiple tissue samples, including antennae (500 adults each), head , thorax , abdomen (100 adults), and legs (300 adults) were dissected on ice using a sterilized scalpel and forceps.A.A. aphidimyza adults emerging 12 h after eclosion were divided into four feeding treatments: no other food was provided, aphid honeydew, 10% sugar solution, and purified water. Each treatment was conducted for 24 h. A total of 15 adults consisting of males and females were collected.A. aphidimyza were collected and starved in 7.5-cm Petri dishes for 0, 1, 3, and 5 days (n = 10 per group).The third larvae of Total RNA was extracted from each sample using Trizol Reagent  in accordance with the manufacturer’s instructions. NanoDrop 2000  was used to assess the concentration and and purity of RNA in each sample, and the RNA samples with absorbance ratios of A260/A280 around 2.0 were selected for further analysis. RNA integrity was verified via 1.2% agarose gel electrophoresis. Lastly, the extracted RNA was digested by DNase I  to remove genomic DNA contamination. 1 μg of total RNA was used to synthesize cDNA using the Prime Script TMRT Reagent kit . The cDNA from each sample was stored at −20°C prior to the use of both PCR and RT-qPCR.GAPDH, RPL32, RPS13, β-actin, RPS3, EF1-α, Tub, and RPL8 were selected from the literature. The primers of GAPDH, RPL32, RPS13, β-actin, RPS3, EF1-α, Tub, and RPL8 were designed based on the transcriptome data of A. aphidimyza (unpublished) with NCBI Primer-BLAST to design the primers  was used to purify. The FastPure® Gel DNA Extraction Mini Kit  was used to purify and were amplified using the 2 × Phanta Max Master Mixes. The pEASY®-Blunt Zero Cloning Kit  was used to subclone purified DNA. Subcloning products were sequenced in both directions . Sequences resulting from these experiments were submitted to GenBank  and the QuantStudio™ 7Pro Real-Time PCR System . The reaction mixture consisted of a 20 μL mixture containing 10 µL of 2 × SYBR Green qPCR Master Mix, 0.4 µL of forwarding primer (10 Mmol L−1), 0.4 µL of reverse primer (10 Mmol L−1), 0.4 µL of ROX Reference Dye II (50X), 1 µL of cDNA template, and 7.8 µL of double-distilled water. The qRT-PCR reaction conditions were as follows: initial denaturation at 95°C for 30 s, followed by 40 cycles at 95°C for 3 s, and ended with an annealing step at 60°C for 30 s. A melting curve analysis was conducted in the 60°C–95°C temperature range to ensure the specificity of the primers. Three independent biological replicates were set. A standard curve was generated from the five-fold dilution series of cDNA, the slopes were analyzed, and the corresponding amplification efficiencies were calculated. RT-qPCR efficiency (E) was determined via the following equation:Primer sequences were designed using NCBI Primer-BLAST in n+1 = 0.15) Vn/n + 1 < 0.15 indicates that the optimal number of reference genes is n, and Vn/n+1 > 0.15 indicates that the optimal number is n + 1 . The primer sequence of the target gene was as follows:The chemosensory protein (CSP) of Forward: AACGCTTTTGTTGGACAGCTAC.Reverse: CAATGAATCGAAGCACACGA.RPL8 and RPS13) and instability (β-actin) of primary reference genes The average relative expressions of AaphCSP1 in different female tissues were computed based on the 2−ΔΔCT method (Based on the stability (T method  and threA. aphidimyza with a complete open reading frame (ORF) were identified by RT-PCR. The GAPDH, RPL32, RPS13, β-actin, RPS3, EF1-α, Tub, and RPL8 genes had a 936, 387, 375, 1,081, 681, 1,260, 1,194, and 669 base pair (bp), separately. The obtained sequences were submitted to the GenBank database and the accession numbers are shown in R2) were calculated using the slope of the standard curve established for each primer pair. The E-values ranged from 95.90% (Tub) to 108.79% (GAPDH), which was within the required range of 80.0%–120.0% (EF1-α) to 1.000 (RPS13) (Eight candidate genes of %–120.0% . The reg (RPS13) .β-actin) −30.75 (RPL32)  reflecting their expression under different conditions.The gene expression analysis of the eight candidate reference genes in all samples under four conditions showed a range of Ct means of 13.34 ( (RPL32) . At difffference . Among vce genes . Adults ariation . Larvae ariation .α-Tub and EF1-α were the better reference genes during different developmental stages, with M-values below 1  and pairwise variations (Vn/Vn+1). The expression stability values revealed that  below 1 . Pair-wi below 1 , indicatnd RPS13 . Accordiof 0.314 . Based oble gene . The sta β-actin . Therefog stages .EF1-α and RPL32 were below 0.4  are recommended to be used to test the target gene expression levels in sugar treatment.In this study, the geNorm algorithm results showed that the comprehensive reference gene rankings from the best to the least stable were and RPL8 . Except reatment . The Nortability . The Besectively . The staH > RPL8 . Thus, tGAPDH, EF1-α, and RPL32 were below 0.4 , and β-actin (the least stable reference gene). The highest accumulated mRNA level of AaphCSP1 was found in the antenna, followed by those in the head, the lowest level was detected in the leg, thorax, and abdomen. However, β-actin was used as a reference gene, and AaphCSP1 was highly expressed in the thorax exceeded (p < 0.05)  .Bombyx mori (Podoptera frugiperda (Anastatus japonicus (Quantitative real-time PCR is a crucial technique for studying gene expression analysis. This technique uses a quantitative method to detect gene expression, with the internal reference gene serving as a standard to calibrate the expression of the target gene. It is highly sensitive and has good repeatability, making it a standard technique for detecting or comparing mRNA levels in gene expression studies. The expression level of any single reference gene does not remain constant under all experimental treatments however, so appropriate reference genes must be chosen before RT-qPCR is performed . Referenbyx mori , Podopteugiperda , and Anaaponicus .A. aphidimyza adults, and the most stable reference genes and their rankings were not entirely consistent. For example, BestKeeper and ΔCt determined that GAPDH was the most stable gene during the various development stages of A. aphidimyza development, whereas GeNorm determined that EF1-α = α-Tub was the most stable gene, and NormFinder determined that GAPDH was the most stable gene (The stability evaluation of candidate reference genes is often performed by multiple methods based on a comprehensive analysis of relevant parameters, and the candidate reference genes are ranked according to their stability. In the present study five methods were used individually to rank stability at various developmental stages and in different tissues of ble gene . Similarble gene . Similarble gene .RPS3 and RPL8 (RPS18 had the highest expression stability across tissues in Tetranychus cinnabarinus (RPS11 had the highest expression stability in Nilaparvata lugens (RPS13 had the highest expression stability in Sesamia inferens (RPS15 and RPL13 were also reported as a stable pair of reference genes in different tissues of larvae of the cotton bollworm Helicoverpa armigera (Mythimna separata larvae RPL12 was the most stable gene in different tissues (RPS13 had the highest expression stability in different adult tissues of Tuta absoluta (RPL13 exhibited higher stability in specific larval tissues of Phthorimaea operculella (In different adult tissues, the reliable reference genes were and RPL8 . Ribosomabarinus , RPS11 ha lugens , and RPSinferens . RPS15 aarmigera . In Myth tissues . RPS13 habsoluta , whereasrculella .GAPDH remained stable during developmental stages and under sugar treatment conditions in adult A. aphidimyza (GAPDH enzyme regulates glycolysis, gluconeogenesis, and other mechanisms of energy metabolism. For example, GAPDH was the most stable reference gene during various developmental stages in Schistocerca gregaria (Spodoptera litura (Spodoptera exigua (Sesamia inferens (GAPDH expression can occasionally be erratic, such as in Harmonia axyridis (Tetranychus cinnabarinus (Sogatella furcifera (Bombus terrestris, and Bombus lucorum (EF1-α has been identified as a highly stable reference gene in various insect species. In both Thitarodes armoricanus larvae (Danaus plexippus (EF1-α is evidently insensitive to changes in tissue type or developmental stage. EF1-α has exhibited good expression stability in Phthorimaea operculella during several developmental stages and at different temperatures (EF1-α was the most reliable reference gene in adult A. aphidimyza (hidimyza . The GAPgregaria , Spodopta litura , Spodopta exigua , and Sesinferens . Howeveraxyridis , Tetranyabarinus , Sogatelurcifera , Bombus  lucorum . EF1-α hs larvae  and Danalexippus  EF1-α iseratures . Notablyhidimyza .β-actin is a highly conserved cytoskeletal protein that plays a crucial role in various eukaryotic physiological processes such as cell division, chromosome movement, organelle movement, and cytoplasmic flow. It has been identified as one of the most stable reference genes in several insect species, including Liriomyza trifolii (Drosophila melanogaster (A. aphidimyza (β-actin was less stable across temperature and photoperiod treatments in Helicoverpa armigera (Coccinella septempunctata (Henosepilachna vigintioctomaculata (Phaedon brassicae (Phthorimaea operculella (α-Tub is a gene that encodes microtubule proteins involved in a number of physiological processes such as the construction and maintenance of cell morphology, intracellular transport, chromosome movement, and cell division. It is widely considered to be a reliable reference gene in several insect species, including Chilo suppressalis larvae (Lymantria dispar (A. aphidimyza, α-Tub was the worst reference gene (trifolii , and Dronogaster . Howeverhidimyza . This obarmigera , and in punctata , Henosepmaculata , Phaedonrassicae , and Phtrculella . α-Tub is larvae  and diffa dispar . In the nce gene .RPL8 and RPS3 inaccurate gene expression analysis in A. aphidimyza, we evaluated the relative gene expression level of AaphCSP1 in the antenna, head, leg, thorax, and abdomen. Our results showed that AaphCSP1 was abundantly expressed in the antenna, moderately transcribed in the head, and lowly expressed in the thorax and abdomen (AaphCSP1 demonstrates that RPL8 and RPS3 can be used as endogenous controls to assess gene expression in A. aphidimyza.In order to demonstrate the utility of  abdomen . Our exp abdomen . Includi abdomen , reprodu abdomen , and hos abdomen . Thus, t


E. coli (ETEC) vaccine relative to a formalin-inactivated counterpart. Exposure of ETEC H10407 to PUVA using the psoralen drug 4′-Aminomethyltrioxsalen hydrochloride (AMT) yielded replication-incompetent bacteria that retained their metabolic activity. CFA/I-mediated mannose-resistant hemagglutination (MRHA) was equivalent for PUVA-inactivated and live ETEC, but was severely reduced for formalin–ETEC, indicating that PUVA preserved fimbrial protein functional integrity. The immunogenicity of PUVA–ETEC and formalin–ETEC was compared in mice ± double mutant heat-labile enterotoxin (dmLT) adjuvant. Two weeks after an intramuscular prime/boost, serum anti-ETEC IgG titers were similar for the two vaccines and were increased by dmLT. However, the IgG responses raised against several conserved ETEC proteins were greater after vaccination with PUVA–ETEC. In addition, PUVA–ETEC generated IgG specific for heat-labile toxin (LT) in the absence of dmLT, which was not a property of formalin–ETEC. These data are consistent with PUVA preserving ETEC protein antigens in their native-like form and justify the further testing of PUVA as a vaccine platform for ETEC using murine challenge models.Inactivated whole-cell vaccines present a full repertoire of antigens to the immune system. Formalin treatment, a standard method for microbial inactivation, can modify or destroy protein antigenic epitopes. We tested the hypothesis that photochemical inactivation with psoralen and UVA light (PUVA), which targets nucleic acid, would improve the immunogenicity of an Enterotoxigenic  New vaccine approaches that are safe and effective for widespread use, including in individuals with compromised immune systems, are needed. Killed whole-cell vaccines have advantages over protein subunit, nucleic acid-based, and viral vectored vaccines in that they present a broad repertoire of strain-specific and conserved protein and polysaccharide antigens to the immune system and are generally safe for use in vulnerable individuals. Killed whole-cell vaccines are agile in that they require minimal or no molecular engineering or preexisting knowledge of the role of specific proteins in inducing protective immune responses. They are relatively inexpensive to produce and are compatible with multivalency.Shigella [Pseudomonas aeruginosa [Bordetella pertussis [Salmonella [Formalin treatment has served as a standard method for killing microorganisms for the purpose of producing vaccines. Formalin modifies amino acids by the addition of reactive carbonyl groups, promoting protein crosslinks. Although formalin inactivation has produced effective vaccines, for example, the Hepatitis A vaccine (HAV) , detrimeShigella , Pseudomruginosa , Bordeteertussis , Salmonelmonella  poliovirlmonella ,7, respilmonella , and othlmonella . BecauseTreatment with psoralen drugs followed by irradiation with long-wavelength UV light UVA, 315–400 nm) disables the replication of viruses ,11 and b–400 nm dSalmonella typhimurium [Pseudomonas aeruginosa [Listeria monocytogenes [Bacillus anthracis [In the context of bacterial vaccines, psoralen + UVA has yielded promising results in preclinical studies for multiple pathogens . The trehimurium  Pseudomoruginosa ,22, Listytogenes , and Bacnthracis . In thatnthracis . DespiteE. coli (ETEC), for which psoralen inactivation for the purpose of vaccine development has not previously been reported. ETEC is a significant cause of diarrheal illness in the developing world, predominantly affecting children under the age of 5 [E. coli that overexpress CFA/I, CS3, CS5, and CS6. ETVAX is delivered with LCTBA toxoid and the adjuvant dmLT [The current study was undertaken to compare psoralen inactivation to formalin inactivation using Enterotoxigenic age of 5 ,25. In aage of 5 . To dateage of 5 . Adhesioage of 5 . Consideant dmLT . DmLT , which has previously been used to inactivate viruses ,15. The Bacterial strains and growth conditions. The ETEC strain H10407 (O78:H11), expressing LT, ST, and CFA/I , was acq600 1.5), were treated with AMT psoralen . For dose–response experiments, the range was 0.1–100 µg/mL; 0.34–340 µM. After an additional 1 h at 37 °C with shaking, 1.0 mL cultures were transferred to individual wells of a 6-well tissue culture dish and exposed (lid on to maintain sterility) to the indicated dose of UVA light at 365 nm (dose range 1.0–4.0 J/cm2) using an Analytik Jena Crosslinker . The treated bacteria were recovered from the wells and washed 3 times before plating the dilutions onto LB agar to enumerate colony forming units (CFU). Bacteria treated with AMT only and UVA light only served as controls. To detect residual CFU, the entire 1.0 mL culture was plated on LB agar. As an additional read-out of residual replication-competent bacteria, 1.0 mL of inactivated or live bacteria was inoculated into 50 mL of fresh broth and monitored for growth at 37 °C over a period of 72 h. Psoralen inactivation. Overnight CFA broth cultures (37 °C with shaking) were diluted 1:500 into 1.0 mL of fresh media and, after 5 h of culture bacteria  for 2 h with shaking at 37 °C. The bacteria were then moved to 4 °C and incubated static for 3 days. The treated bacteria were washed 3 times and killing was confirmed by plating on LB agar. Formalin inactivation. ETEC were formalin-inactivated using the method of Tobias et al. . The bacueous nonradioactive Cell Proliferation Assay ), which measures the activity of cellular dehydrogenase enzymes. After PUVA or formalin inactivation, the bacteria were washed, resuspended in LB broth, and added to 96-well plates (25 µL of bacteria in 75 µL of LB) + 10 µL/well MTS reagent. The controls were live and heat-killed bacteria and growth media only. The cultures were incubated for 1 h at 37 °C, after which, the absorbance of the soluble formazan product was measured at 490 nm using a BioTek EPOH2NS microplate reader with Gen5 software, version 3.1. Killed but metabolically active (KBMA) assay. Metabolic activity was assessed by MTS assay using a CellTiter 96 AQ600 = 1.0. The MRHA assay was performed in 96-well round bottom plates and on glass slides. For the 96-well plate assay, 100 µL of bacteria was mixed with 50 µL of PBS + 2% mannose or PBS only. Then, 50 µL/well of washed, Type A human RBC  adjusted to 1.5% in PBS was added. For the slide assay, 25 µL of bacteria suspended in 2% mannose was mixed with 25 µL of 1.5% RBC. Agglutination was visible as a ring of aggregated cells around a pellet of settled RBC in the 96-well assay or as clumping in the slide assay. Digital images of partial wells or 2 different slide fields were captured by photomicroscopy using the 4× lens of an EVOS M5000 Imaging System  after 24 h at 4 °C (96-well assay) or 1 h at 23 °C (slide assay). The controls included RBC only and H10407-P  [MRHA assay. The capacity of CFA/I fimbriae to mediate the mannose-resistant agglutination of red blood cells  was perf strain)  grown in9 CFU equivalents/mL (OD600 2.0) or 2 × 108 CFU equivalents/mL (OD600 0.2). An equal volume of PBS or dmLT adjuvant , to achieve a final concentration of 2.5 µg/mL, was added prior to the intramuscular (IM) delivery of 0.1 mL per mouse. This preparation yielded a dose of 1 × 108 (high) or 1 × 107 (low) inactivated ETEC ± 0.25 µg dmLT. Preparation of ETEC vaccines. The psoralen- and formalin-killed vaccines  were prepared as described and stored in 25% glycerol-LB media at −80 °C. Vaccine aliquots were thawed, washed three times, and adjusted to either 2 × 10Vaccination of mice with ETEC vaccines. Mouse vaccination experiments were conducted according to a protocol approved by the Wake Forest Institutional Animal Care and Use Committee (#A19-163). Female BALB/c mice (6–8 weeks old) were purchased from Charles River. Mice were housed in ventilated cages with unlimited water and commercially produced mouse chow. Mice (7 or 8 per group) were immunized with vaccines containing no adjuvant or combined with dmLT adjuvant at the indicated doses. The vaccination groups were: (1). PUVA–ETEC; (2). PUVA–ETEC + dmLT; (3). formalin–ETEC; and (4). formalin–ETEC + dmLT. The vaccines were administered intramuscularly in the hind flank on days 0, 21, and 42. Blood was collected on days 0 (pre-bleed), 34 (13 days post-boost 1), and 56 (14 days post-boost 2). Mice were monitored and weighed regularly after vaccination. Portions of sera from each group were pooled and stored along with individual samples at −80 °C.600 of 0.5 in sterile PBS. Microtiter plates (Nunc Maxisorp) were coated with the bacterial suspension (100 µL per well) and the plates were tightly sealed to prevent drying. After 18–24 h at 37 °C, the wells were washed three times with PBS and blocked with 200 µL/well of 5% Difco skim milk in wash buffer (PBS + 0.05% Tween20) for 2 h at 23 °C with agitation. After 3 washes, serum that had been serially diluted in PBS + 0.05% Tween20 + 1% skim milk (antibody buffer) was added (100 µL/well) and incubated for 2 h at 23 °C with agitation. The plates were washed 3 times and incubated with HRP-conjugated goat anti-mouse IgM+IgG+IgA (Millipore AP501P) or HRP-conjugated goat anti-mouse IgG  (100 µL/well), diluted 1:5000 in antibody buffer. After 1 h, the wells were washed and 100 µL of TMB substrate  was added. The plates were incubated in the dark for 20 min and the reaction was stopped by adding 100 µL of 2 M H2SO4. Absorption at 450 nm was measured using a BioTek EPOCH2NS spectrophotometer . The serum antibody titer, defined as the dilution that yielded an OD450 value 4-fold greater than the assay background (antigen-coated wells with no serum/antibody added), was calculated using a linear regression analysis of the OD readings from the 3 to 4 Log10-transformed dilutions using the GraphPad Prism 9.0 software. The dilution series for the post-vaccination sera was started at 1:500, which generated no background signal in the uncoated (no antigen) control wells. Mouse anti-LPS  or rabbit anti-E.coli  paired with HRP-conjugated donkey-anti-rabbit IgG  served as positive controls. To screen for ETEC protein-specific antibodies, ELISA plates were coated overnight at 4 °C with 100 µL/well of recombinant proteins, MipA, Skp, ETEC_2479, or EtpA (4 μg/mL in pH 9.6 carbonate buffer). The rest of the assay was as per the whole-cell ELISA.ELISA. An ELISA assay using whole ETEC as the coating antigen was adapted from a published protocol . ETEC cuLTB ELISA. The detection of LTB-specific antibodies was performed according to a published protocol . Plates t-test using the GraphPad Prism 9.0 software. p ≤ 0.05 was considered to be significant.Statistics. Statistical significance was calculated by a two-tailed, unpaired Student’s 600 1.5) in 1.0 mL CFA broth cultures. AMT was added for 1 h, after which, the bacteria were removed from the culture and exposed to 1 J/cm2 of UVA light. The number of CFU remaining was measured by plating dilutions on LB agar. At 1 J/cm2 of UVA, the reduction in CFU/mL was AMT dose-dependent, but no AMT dose killed all of the bacteria in the culture, with residual CFU remaining in some experiments even at the highest AMT dose (2 (600 relative to the starting culture over 72 h. Similar CFU/mL were detected in the live (untreated) and AMT only cultures , indicating that AMT at the applied doses was not toxic in the absence of UVA light. Similarly, as shown in 2 did not kill ETEC (open symbols), UVA at 2 and 4 J/cm2 killed the cultures completely in combination with 50 µg/mL of AMT (closed symbols). Therefore, an AMT dose of ≥ 10 µg/mL combined with at least 2 J/cm2 of UVA  and formalin-killed. To test their capacity to repair psoralen adducts, samples of bacteria from five independent PUVA-inactivated cultures were diluted into fresh media. Approximately 1 × 108 inactivated bacteria yielded no growth  over 72 h at 37 °C. These results strongly suggest that ETEC were not capable of reverting to replication competency after treatment with 50 µg/mL of AMT + 2 J/cm2 of UVA according to the described protocol and at a small scale.For vaccine purposes, the inactivation method should reproducibly sterilize a culture. As shown in 2 of UVA were KBMA, as determined by an MTS assay. Although the level of dehydrogenase enzyme activity was decreased relative to the live bacteria and varied among the independent PUVA-inactivated cultures, MTS activity was consistently detected in the PUVA-inactivated cultures, but not in the heat- or formalin-inactivated cultures. Frozen and thawed PUVA–ETEC remained KBMA, indicating that the property was stable to the short-term frozen storage of vaccine stocks in glycerol-containing media  state, as reported for other psoralen-inactivated bacteria . As showng media b. A timeng media c. Identi2 of UVA light) agglutinated the RBC in a manner that was qualitatively indistinguishable from live bacteria. In contrast, a reduced capacity of formalin–ETEC to agglutinate RBC under the same experimental conditions was apparent. H10407-P failed to agglutinate RBC due to the absence of the gene encoding CFA/I. Similar results were obtained using the slide assay, with two fields captured using a 4× lens shown for each sample  in the presence of mannose  . The ELISA assay was tested using broadly reactive rabbit anti-E. coli and mouse-anti-LPS antibodies. As shown in We next evaluated the vaccines’ immunogenicity in mice. In order to measure the humoral response generated against a broad repertoire of H10407 ETEC antigens after vaccination, an ELISA was developed using whole, untreated bacteria as the coating antigen, according to a published method . To eval8 CFU equivalents ± 0.25 µg dmLT. The dmLT dose was based on previous studies in mice that demonstrated an effective range of 0.10–1.0 µg of dmLT per vaccine dose [As an initial test of its immunogenicity in mice, the PUVA–ETEC vaccine was administered with or without a dmLT adjuvant in an IM prime-boost regimen using a vaccine dose of 1 × 10ine dose . BALB/c 8 CFU equivalents to 1 × 107 CFU equivalents per mouse. The mice in both groups initially lost weight after receiving the 1 × 108 dose  PUVA–ETEC, (2) PUVA–ETEC + dmLT, (3) formalin–ETEC, and (4) formalin–ETEC + dmLT. In contrast to the high-dose vaccines, there was no adverse effect in terms of body weight change after vaccination with 1 × 10ivalents b. Sera fivalents . Both vap < 0.01 for all ± dmLT comparisons). Therefore, both vaccines induced robust serum IgG antibodies that recognized a heterologous strain with a different colonization factor profile relative to the vaccine strain, presumably reflecting an abundance of shared protein and polysaccharide antigens. The PUVA–ETEC and formalin–ETEC vaccines were prepared from strain H10407 (078-H11). Serum pools from each vaccination group were next screened against ETEC B7a (O148:H28) [p = 0.002 and p = 0.005 for the 1:100 and 1:300 serum dilutions, respectively). Mouse anti-EtpA antibody is shown in Having determined that the polyclonal anti-ETEC serum antibody response (representing a multitude of antigens) was similar for the two vaccines, we tested the reactivity of the immune sera against individual ETEC proteins. ETEC proteins that are conserved across clinical isolates were chosen for this analysis. The pooled sera were first screened against Mip, Skp, and ETEC_2479, which were identified in a screen for immunogenic outer membrane proteins that mediated protection against several ETEC strains in mice ,48. As sGiven the KBMA status of the psoralen-inactivated ETEC, we hypothesized that the PUVA–ETEC vaccine might induce LT-specific antibodies by virtue of the secretion or presentation of surface-associated LT  to the iIn the current study, we explored the potential of psoralen inactivation as an alternative to formalin to produce an inactivated vaccine for ETEC, for which whole-cell vaccine platforms provide a safe and relatively inexpensive means for targeting a large repertoire of antigens, including proteins conserved across clinical strains ,53. We dWe found that several properties of PUVA–ETEC were not shared by formalin–ETEC. First, PUVA–ETEC were killed but metabolically active (KBMA), as previously reported for other psoralen-inactivated Gram-negative and Gram-positive bacteria ,22,23,54uvrABC locus, which renders bacteria highly susceptible to UV-induced DNA damage by disabling the nucleotide excision repair system (NER) [2 of UVA light. To our knowledge, we are the first to use AMT to photochemically inactivate bacteria. Because our goal was the complete and irreversible inactivation of bacterial replication, we used AMT doses that were 10–100 fold higher on a molar basis than those reported for other psoralen drugs used to inactivate bacteria for the purpose of vaccine production [E. coli were inefficient at repairing inter-strand crosslinks induced by a different psoralen drug, 8-methoxypsoralen [uvr mutant background remains an option for minimizing the potential for reversion to replication competency, which would present a significant safety concern.As reported previously for other bacteria ,22,23,54em (NER) ,55. In ooduction ,23,54. Soduction , the higpsoralen . NeverthShigella [A key outcome of our study is evidence to support the hypothesis that PUVA preserves proteins in native-like form, which we determined using protein adhesive function as a proxy for protein structural and antigenic integrity. An MRHA assay demonstrated that PUVA–ETEC retained its CFA/I-mediated adhesive function, which was indistinguishable from that of live ETEC, while the activity of formalin–ETEC was severely reduced, strongly suggesting that PUVA preserved while formalin compromised the CFA/I protein integrity. While it is clear that anti-CFA/I antibody responses are generated in animals and humans in the context of formalin-inactivated ETEC vaccines ,41, and Shigella ,14,16. Shigella [To compare the immunogenicity in mice, we chose an IM prime/boost regimen. Although there was no difference between the anti-ETEC serum antibody response measured by screening immune sera against whole bacteria, we considered that any reduction in antibody responses against individual proteins would be masked in the context of screening against the large repertoire of antigens displayed by the whole bacteria. When the sera were screened against the individual proteins that were chosen for conservation among the ETEC clinical isolates, IgG specific for Mip, Skp, ETEC_2479, and EtpA was detected after PUVA–ETEC vaccination, but the IgG levels were minimal after vaccination with formalin–ETEC. Previous studies with those recombinant proteins delivered to mice have demonstrated the induction of functional (adhesion blocking) antibodies and protection against a challenge with ETEC, including against heterologous strains ,48,58, iThe immune sera from the PUVA– and formalin–ETEC-vaccinated mice had high titers of IgG that recognized the vaccine strain (H10407) as well as B7a, a strain with a different serotype and distinct set of adhesins and colonization factors . It is wBacillus anthracis, which presented protective antigen (PA), a secreted protein, to the immune system in animal models [Listeria monocytogenes produced listeriolysin O, promoting the cytosolic delivery of a heterologous antigen in a mouse model [Although we demonstrated that the KBMA state is a property of psoralen-inactivated ETEC, we did not formally address the implications of sustained metabolic activity for an ETEC vaccine. There are obvious implications, including the potential for toxin production to cause adverse effects, which could be addressed using strains with mutations that detoxify but leave the intrinsic antigenicity and adjuvant activity of LT intact ,31. Intel models . Similarse model . The prese model  and meas7 CFU, but not 1 × 108 CFU, equivalents of PUVA–ETEC via the intramuscular-route-augmented ETEC-specific serum IgG titers. Similar studies with Campylobacter and Shigella vaccines demonstrated the antigen dose-dependence of dmLT adjuvant activity [8 bacteria, with and without dmLT, caused weight loss, which was not observed at the 1 × 107 dose (Our results with dmLT recapitulate the results of studies demonstrating an adjuvant effect that was antigen dose-dependent . DmLT (0activity ,61. We c107 dose . AlthougIn summary, the current study fills a gap in the knowledge by directly comparing the properties and immunogenicity of psoralen-inactivated bacteria, first described more than a decade ago for their potential to improve whole-cell bacterial vaccines ,19, to f


Medulloblastoma is a cancerous brain tumor that affects mostly children. Among the four groups defined by molecular characteristics, Group 3, the least well characterized, is also the least favorable, with a survival rate of 50%. Current treatments, based on surgery, radiotherapy, and chemotherapy, are not adequate and the lack of understanding of the different molecular features of Group 3 tumor cells makes the development of effective therapies challenging. In this study, the problem of medulloblastoma is approached from a metabolic standpoint in a low oxygen microenvironment. We establish that Group 3 cells use both the mitochondrial glycerol-3 phosphate (G3PS) and malate-aspartate shuttles (MAS) to produce NADH. Small molecules that target G3PS and MAS show a greater ability to decrease cell proliferation and induce apoptosis specifically of Group 3 cells. In addition, as Group 3 cells show improved respiration in hypoxia, the use of Phenformin, a mitochondrial complex 1 inhibitor, alone or in combination, induced significant cell death. Furthermore, inhibition of the cytosolic NAD+ recycling enzyme lactate dehydrogenase A (LDHA), enhanced the effects of the NADH shuttle inhibitors. In a 3D model using Group 3 human cerebellar organoids, tumor cells also underwent apoptosis upon treatment with NADH shuttle inhibitors. Our study demonstrates metabolic heterogeneity depending on oxygen concentrations and provides potential therapeutic solutions for patients in Group 3 whose tumors are the most aggressive. Pediatric cancers are a leading cause of death by disease in children and young people . Medullo+/NADH) and nicotinamide adenine dinucleotide phosphate (NADP+/NADPH), which are essential for maintaining cellular redox homeostasis and modulating several biological processes, including cellular metabolism [Hypoxia plays an important role in a broad range of solid tumors , 7. Canctabolism –15.+ [+ than LDHA/B because they enable glycolysis-derived pyruvate to be oxidized in mitochondria. The reducing equivalents transferred from cytosolic NADH to mitochondrial NAD+ by the MAS and G3PS are ultimately used to reduce oxygen at complex IV of the electron transport chain. Thus, when oxygen is limited, the only mechanism for regenerating cytosolic NAD+ is through the LDH reaction that produces lactate.Cells employ three different mechanisms to oxidize NADH and regenerate cytosolic NAD+ . The firTo study the role of MAS and G3PS in Group3 cells in terms of cancer cell fitness, we tested the effect of mitochondrial GPDH inhibitor (iGP-1) targeting G3PS as well as aminooxyacetate acid (AOAA) targeting GOT1/2 Fig. . We testThe HDMB-03 cell line was purchased from the DSMZ (ACC740). The D-458 cell line was provided by Dr. C. Pouponnot (Institut Curie - France). Both cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco-BRL) supplemented with 20% fetal bovine serum with penicillin G (50U/ml) and streptomycin sulfate (50 µg/ml).2 200 anaerobic workstation  set at 6% and 1% oxygen, 94% nitrogen and 5% carbon dioxide were used for hypoxic conditions.An INVIVOPhenformin, rotenone, antimycin A, oligomycin, and 2, 4-Dinitrophenol (DNP) were from Sigma , iGP-1 from Merck  and AOAA from Selleckchem  and GNE-140 from MedChemExpress .Affimetrix Human Gene 1.1 ST Array profiling of 763 primary medulloblastoma samples (GSE85217) was used for identification of Medullobastoma subtypes .2 for colony formation. After 7 days, colonies were fixed with 10% (v/v) methanol for 15 min and stained with 5% Giemsa  for 30 min for colony visualization.Cells  were plated on 60-mm dishes and incubated at 37 °C, 5% COThe Glycerol 3-Phosphate colorimetric Assay Kit (MAK207), NAD/NADH quantitation kit (MAK037), and Malate assay kit (MAK067) were used according to the manufacturer’s instructions .Cells were plated at 100,000 cells/well and treated the following day. At specific times, cells were detached using trypsin-EDTA, suspended in their conditioned medium and evaluated for viability and proliferation using an automatic cell counter .Human iPS cells  were maintained in self-renewal on a layer of geltrex , in E8 Basal Medium  supplemented with E8 supplement (50 X). All cells were mycoplasma-free. iPSCs were dissociated with EDTA (Invitrogen) 0.5 mM, pH 8.0, for 3 min incubation, to maintain cell clusters. Cerebellar organoids were cultured as described by Muguruma et al.  and IshiOrganoids were treated from days 71 or 79 of differentiation, with three doses of 100 μM iGP1, 1 mM AOAA, 5 µM GNE-140, 100 µM Phenf, and two combos iGP1/GNE-140/AOAO, iGP1/GNE-140/Phenf or DMSO as a control. After the drug treatment, organoids were fixed in PFA 4%, permeabilized with 0.5% Triton X-100 for 30 min and then blocked for 1 h at room temperature. Primary antibodies against cleaved Caspase 3  were incubated overnight and a secondary antibody was coupled to AlexaFluor 594 for 1 h . Annexin V  was incubated 24 h before imaging. Tumoroids were imaged with a Nikon confocal spinning disk microscope. Images were processed with Imaris for 3D reconstruction.The cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were obtained using a Seahorse XF96 extracellular flux analyzer from Seahorse Bioscience  following the manufacturer’s instructions. OCR and ECAR were measured in real time in Nx, Phx, or Hx. Cells were deprived of glucose for 1 h, then glucose (G–10 mM), oligomycin (O–1 µM), 2,4-Dinitrophenol (DNP–10 µM), and Rotenone + Antimycin A (R/A–1 µM) were injected at the indicated times. Protein standardization was performed after each experiment, with no noticeable differences in protein concentration and cell phenotype.® Phenotype Microarray™ system  evaluating the cell’s ability to metabolize 367 substrates. Cells were cultured for 48 h in normoxia or hypoxia, then transferred at seeding densities of 20,000 cells/well to the PM-M1 to 4 plates in a phenol red-free RPMI-1640-based medium depleted of carbon energy sources , supplemented with 0.3 mM glutamine, 5% FCS, 100 U/mL penicillin, and 100 µg/mL streptomycin. Cells were then incubated for 24 h at 37 °C under 5% CO2 in hypoxia or normoxia before adding Biolog Redox Dye Mix MA, sealing the plate with tape to prevent gas transfer, and incubating at 37 °C in the Omnilog® automated incubator-reader  for 24 h to kinetically measure tetrazolium reduction. Data was collected using PMM Kinetics software with subtraction of the average values of three negative control wells (background), then, analysis was conducted with the opm package in R (Version 3.4.4).Metabolic profiling was studied by using the Omnilogg for 5 min, followed by two subsequent extractions of the insoluble pellet with 0.5 mL 80% methanol, with centrifugation at 16,000 g for 5 min at 4 °C. The 5-mL metabolite extract from the pooled supernatants was dried down under nitrogen gas using an N-EVAP . Dried pellets were resuspended using 20 μL HPLC-grade water for mass spectrometry. A 7-μL sample was injected and analyzed using a 5500 QTRAP triple quadrupole mass spectrometer (AB/SCIEX) coupled to a Prominence UFLC HPLC System (Shimadzu) via selected reaction monitoring of a total of 300 endogenous water-soluble metabolites for steady-state analyses of samples [http://www.metaboanalyst.ca). Univariate statistical differences in the metabolites between two groups were analyzed using a two-tailed Student t-test.To determine the relative levels of intracellular metabolites, extracts were prepared and analyzed by LC/MS-MS. Triplicate 10-cm plates (∼80% confluent) were incubated in Hx for 72 h. Metabolites were extracted on dry ice with 4 mL 80% methanol ( −80 °C), as described previously . Insolub samples . The norCells were lysed in 1.5x SDS buffer and the protein concentration determined using the BCA assay. 40 µg of protein from whole cell extracts was resolved by SDS-PAGE and transferred onto a PVDF membrane (Millipore). Membranes were blocked in 5% non-fat milk in TN buffer  and incubated in the presence of the primary and then secondary antibodies in 5% non-fat milk in TN buffer. Rabbit monoclonal anti-GPD2 antibody (ab188585) was from Abcam . Rabbit monoclonal anti-MDH2 antibody (D8Q5S) was from Cell Signaling technology. Mouse anti-b-tubulin, HSP90 and b -actin were from Sigma. ECL signals were normalized to either b-tubulin or HSP90. After washing in TN buffer containing 1% Triton-X100 and then in TN buffer, immunoreactive bands were visualized with the ECL system (Amersham Biosciences).For live imaging, human MB tumor organoids were observed with an Evos optical microscope with GFP fluorescence (Venus). Immunofluorescence staining was performed in 96-well flat-bottom microplates (Greiner Bio-One: 655090). Blocking and antibodies solutions consisted of PBS supplemented with 2% Fetal Bovine Serum. Human MB tumor organoids were harvested and washed once with ice cold culture medium. Human MB tumor organoids were post-fixed for 1 h in 4% PFA, permeabilized with 0.5% Triton X-100 (SIGMA: T8787) for 30 min and then blocked for 1 h at room temperature (RT). Primary antibody against Caspase 3 active (cleaved) form  was incubated overnight. Secondary antibody coupled to AlexaFluor 594  was incubated for 1 h at RT. Human MB tumor organoids were mounted and imaged with a Nikon confocal microscope. Images were processed with Imaris for 3D reconstruction and measurement.p values are indicated. All categorical data used numbers and percentages. Quantitative data were presented using the median and range or mean. All statistical tests were two-sided, and p values < 0.05 indicated statistical significance while p-values between 0.05 and 0.10 indicated a statistical tendency.All values are the means ± SEM. Statistical analyses were performed using the ordinary oneway ANOVA and 2-way ANOVA tests in Prism. The GPD2 gene expression, encoding mGPDH, and MDH2, encoding mitochondrial malate dehydrogenase (MDH2), were different between the four groups  and malate-aspartate shuttle (MAS), respectively Fig. . We consups Fig. . GPD2 exoup Fig. . Across i et al. , alpha, mma Fig. . Such he2), Phx (6%) and Hx (1%) microenvironments. We observed no significant increase in mGPDH and MDH2 mRNA expressions after 72 h in Group 3 MB cells . The profiles obtained are clearly different. HDMB03 cells produced more pyruvate and asparagine, suggesting that they may promote glycolysis and the TCA cycle. iGP1 clearly impacted HDMB03 metabolism via a reduction in the production of D-sedoheptulose-7-phosphate, D-ribose-1/5-phosphate, and S-adenosyl-methionine, suggesting a direct impact on the lipopolysaccharide, pentose phosphate and s-adenosylmethionine metabolic (SAM) pathways  and aminooxyacetate (AOAA) to block mGPDH and mMDH pathways, respectively. Similar G3P accumulation and NAD/NADH ratio were observed in the presence of both siRNA targeting mGPDH and iGP1 in HDMB03 Group 3 MB cells  in low oxygen concentrations Fig. , in combWe then hypothesized that the reason for these increases in proliferation and viability observed in the presence of iGP-1 was due to metabolic changes. Thus, we quantified both mitochondrial respiration and glycolysis with the Seahorse XF by measuring the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR). Interestingly, the overall respiration of Group 3 MB cells (HDMB03 and D-458) in Phx and Hx appeared greater than in Nx Fig. . In paraIn the presence of iGP1, the respiratory capacity was almost always increased in both cells, especially under Phx Fig. . GlycolyRecently, Di Magno et al. proposed a therapeutic potential for phenformin (Phenf) in medulloblastoma in the SHH group . The autTo better identify the dual function of Phenf  on Group 3 cells, we compared its action in Hx on cell metabolism, proliferation, and viability. We also used Rotenone, a potent and specific inhibitor of mitochondrial complex 1, as a control. Cells were challenged with Phenformin (Phenf – 3 µM and 100 µM) and Rotenone (2.5 µM). In HDMB03 cells, Rotenone was less potent to block the respiration compared to Phenf 3 µM and 100 µM (Supplementary We then hypothesized that reactive oxygen species (ROS), generated by mGPDH , may be Taken together, these results showed that mGPDH inhibition by iGP1 allows Group 3 MB cells to switch to glycolysis, thereby increasing their cell growth. Although cells from Group 3 oxidize G3P via mGDPH under Phx or Hx, this activity tends to stunt the cells in their development, probably due to ROS production. The results demonstrate that blocking mGPDH with iGP1 as the only inhibitor cannot be used to specifically kill Group 3 MB cells but should be combined with another inhibitor of a similar nature, a specific respiratory inhibitor such as Phenf.We used the MAS inhibitor, AOAA, to block mMDH activity in both cell groups. We also combined the two inhibitors iGP1 and AOAA in Hx to totally block both the MAS and mGPDH shuttles. All cells showed sensitivity to 1 mM AOAA in both proliferation and viability Fig.  suggestiRespiration analysis showed that the combination AOAA+iGP-1 was required to block mitochondrial respiration in Group 3 MB cells Fig.  and alsoThese results indicate that the functioning of Group 3 cells relies on both shuttles since both inhibitors were needed to observe an inhibition effect, suggesting a high dependency on NAD+/NADH metabolism for Group 3 MB cells.We hypothesized that the other NAD+ production pathway generated by lactate dehydrogenase could be used when both shuttles are blocked and therefore addressed blocking it as well.LDHA and LDHB gene expressions were different within the 4 groups  , LDHA anups Fig. . Gene exmma Fig. . These rWe therefore used GNE-140, which is a potent inhibitor of LDHA, to block the transformation of pyruvate to lactate. It also blocks LDHB, which allows the reverse transformation. The results obtained with Omnilog clearly showed that all cells, regardless of group, were avid for glucose but also for glucose-6 phosphate and Fructose-6 phosphate Fig. . This abFinally, we tested GNE-140 with the different combinations previously used, to block any potential for NAD+ production and any switch to lactate production. The addition of GNE-140 clearly potentiated the effects of the AOAA+iGP-1 combo Fig. , regardlThese results strongly suggest that i) Group 3 MB cells are definitely very sensitive when one or more metabolic pathways are blocked in hypoxia, ii) the addition of GNE-140 strongly sensitizes all cell lines, even the most refractory ones such as D-458 Group 3 MB cells, and finally iii) we have characterized new therapeutic leads to potentially target the most treatment-refractory cells (Group 3 MB).Finally, we sought to validate the drug responses identified in 2D on an organoid-derived model of human Group 3 MB in which cancer cells are GFP/Venus positive . Human GThese findings suggest that the drug responses observed in 2D can be recapitulated when assayed with the human Group 3 MB tumor organoids. Interestingly, we found that AOAA, to a less extend, and GNE140, two specific inhibitors of NAD+ production, rapidly reduce the number of tumor cells. The drug combo, certainly more drastic for the cell, is also clearly effective.2, an estimated physiological setting of 6% O2, and a tumor pathophysiological setting of 1% O2. While Bernauer et al.’s review [Our study studies Group 3 (HDMB03 and D-458) MB cells in a laboratory setting of 21% Os review  clearly s review , 29 and s review  cancers s review , 32.2 and between 6% and 1% O2, for example the impact of iGP-1 on respiration (OCR) and glycolysis (ECAR), respectively . We have thus clearly highlighted the Achilles heel of the Group 3 MB cells: the hypoxic metabolism. If blocking mGPDH alone did not appear to be the best therapeutic approach, blocking MAS with AOAA or concomitantly with mGPDH seems to be a very promising approach confirmed by the use on human Group 3 MB tumor organoids. We note that AOAA has already been administered in trials relating to Huntington’s disease  and tinnSimilarly, GNE-140, the LDHA/LDHB inhibitor, was also a very good candidate for Group 3 MB cells. Significant evidence exists to support the development of LDH inhibitors as a therapeutic option for cancer treatment –38, but Finally, our results support previous findings that a 3D model presents heterogeneous conditions of normoxia, physioxia and hypoxia, with kinetics closer to pathological conditions than a 2D model. This coculture method better reflects the tumor microenvironment and provides a good platform for screening and testing treatment plans. It would be worthwhile testing our different drugs in conjunction with current radio- and chemo-therapies  , 40, butOrganoids thus appear to be a promising tool, paving the way for valuable advances in the development of new treatments for Group 3 patients with high levels of MYC and Otx2.Suppl. Figures Legendsaj-checklistUncropped Western blotsSuppl. Figure 1Suppl. Figure 2Suppl. Figure 3Suppl. Figure 4Suppl. Figure 5Suppl. Figure 6