Mutant) constructed for protein crystallization, limiting the study of the dynamic structural features of authentic CETP involved in lipid transport under physiological conditions. In this study, we conducted comprehensive molecular dynamics (MD) simulations of both authentic CETP (CETPAuthentic) and CETPMutant. Considering the structural differences between the N- and C-terminal domains of CETPAuthentic and CETPMutant, and their crucial roles in lipid transfer, we identified the two domains as binding pockets of the ligands for virtual screening to discover potential lead compounds targeting CETP. Our results revealed that CETPAuthentic displays greater flexibility and pronounced curvature compared to CETPMutant. Employing virtual screening and MD simulation strategies, we found that ZINC000006242926 has a higher binding affinity for the N- and C-termini, leading to reduced N- and C-opening sizes, disruption of the continuous tunnel, and increased curvature of CETP. In conclusion, CETPAuthentic facilitates the formation of a continuous tunnel in the “neck” region, while CETPMutant does not exhibit such characteristics. The ligand ZINC000006242926 screened for binding to the N- and C-termini induces structural changes in the CETP unfavorable to lipid transport. This study sheds new light on the relationship between the structural and functional mechanisms of CETP. Furthermore, it provides novel ideas for the precise regulation of CETP functions.Current structural and functional investigations of cholesteryl ester transfer protein (CETP) inhibitor design are nearly entirely based on a fully active mutation (CETP Cholesteryl ester transfer protein (CETP) encoded by the CETP gene, is a 476-residue-long plasma glycoprotein that plays a crucial role in facilitating the hetero exchanges of cholesteryl esters (CE) and triglycerides (TG) . The devIn light of the current intense clinical interest in the functional regulation of CETP, there is a pressing need to gain a deeper understanding of its structure and function, particularly regarding the molecular basis for the lipid transfer mechanism ,9,10. X-Experimental studies have demonstrated that inhibition of CETP reduces cholesterol uptake by cells within atherosclerotic plaques and enhances cholesterol effluvia . CETP-dehttps://www.ncbi.nlm.nih.gov/clinvar/, accessed on 25 July 2023) [Authentic and CETPMutant, with a primary focus on stability, flexibility, hydrophobicity, and residue reorientation, all of which are closely related to the lipid transfer function. In addition, virtual screening and MD simulations were performed based on the authentic CETP structure against the ZINC library [As noted above, recent X-ray, EM, and MD studies have revealed several important physical attributes of CETP and a substantial molecular basis for neutral lipid transport mediated by CETP ,12,14. Hly 2023) . Moreovely 2023) ,28,29. Aly 2023) ,30. The ly 2023) ,24,31. I library . The mosMutant and CETPAuthentic reach a plateau after approximately 40 ns, oscillating with minor variations  are generally stable in an aqueous solution, which may serve as indirect evidence supporting the tunnel hypothesis [Authentic further suggests the possibility of tunnel transfer.The distinct configuration adjustments were further explored by the superimposition of typical conformations from MD simulations. The initiating conformations of CETPla1–Gly4 . With re by 4.5° . In contectively . Regardipothesis . In thisMutant can penetrate the HDL particle surface through the major anchoring role of residues Trp105, 106, and 162, associated with the large-scale structural alterations in the N-terminal end (flaps Ω4–6) that give rise to an opening  for the hetero exchanges of neutral lipids [Mutant and CETPAuthentic (0 ns), the maximum distances between Trp106 and Trp162 (dTrp106-Trp162) are 16.30 Å and 16.35 Å, respectively. After 30 ns, they reduce slightly, while subsequently reaching plateaus (15.57 Å and 15.92 Å) at 80 ns ; however, the opposite occurs for the authentic one (~8% ↑) (Authentic. The maximum distance between residues Phe301 and Met412 (dPhe301-Met412) has been used to further estimate the structural differences. As Phe301-Met412 of CETPAuthentic is a constant ~10 Å during 100 ns MD simulations, with residue Phe301 slightly deviating from residue Met412. By contrast, the mutant one reaches a plateau at ~25 ns with a decrement of ~2 Å. In the beginning, residue Phe301 in CETPMutant orients towards the backbone of residue Met412. Then, it gradually gets close to residue Met412 in the first ~25 ns, with dPhe301-Met412 leveling off at 7.9 Å. This is in accord with the aspirations of hydrophobic SASA in that the “C-opening” region of CETPAuthentic retains hydrophobicity and is more conducive to the development of an opening. The spatial size of CE’s rigid steroid ring  and the van der Waals radius of the hydrogen atom (~2.4 Å) [Mutant, leading to an obstruction of CETP-mediated lipid exchange. However, this situation does not occur in the authentic case, because of its good hydrophobicity and larger value of dPhe301-Met412. Furthermore, residues Phe301 and Met412 are located in flexible flapΩ1 and stable α-helix, respectively. During the simulations, the hydrophobic area of flap Ω1 increases by 4% in CETPAuthentic, while it decreases by 13% in CETPMutant  [Differing from the N-terminal end, the C-terminal distal region (flaps Ω1–3) of CETP pore P1 . In P1 t pore P1 . The mot pore P1 ,37,38. Call RMSF  and a re results , the hyd (~8% ↑) . The dat(~2.4 Å)  suggest TPMutant . This suly 2023) ,26.Authentic is more flexible and exposed to solvent, with a larger increase in hydrophobic SASA (~9%)  allows an estimation of the size of the “neck”. As Phe265-Met433 is constant around 16.5 Å in CETPAuthentic, while it decreases to ~10.5 Å in CETPMutant. This corresponds to the obvious enlargement of the internal cavity volume of CETPAuthentic, which might provide a broad space for transfer. The significant structural changes in the “neck” may result from the residue replacement (C131A N88D N240D N341D) required for crystallization due to their close proximity. In MD simulations, residue Phe265 in CETPAuthentic orients towards residue Met433 at 50 ns, with dPhe265-Met433 and angle being 17.4 Å and 77.7°. Note that the angle is defined with the extreme edge carbon of a side-chain of residue Phe265 and the Cα atoms of residues Phe265 and Met260. After ~70 ns, dPhe265-Met433 slightly decreases to reach a plateau (17.0 Å), with an angle of 71.6°. At 80 ns, residue Phe265 orients towards the C-terminal end, and residues Phe265 and Met433 are pulled apart, giving rise to an open wider duct. During that time, dPhe265-Met433 and angle changed to 17.6 Å and 45.7°. These structural alterations in CETPAuthentic might be beneficial for lipid transfer. In CETPMutant, dPhe265-Met433 has a smaller contraction, but the orientation of residue Phe265 remains unchanged over the 100 ns MD simulations. These results demonstrate that CETPAuthentic, rather than CETPMutant, facilitates the required conformational changes (continuous tunnel), corroborating the tunnel mechanism.The amphipathic Helix X of CETP is located about 50~60 Å from the N-terminal domain end, and its closed state permits a more sustained CE transfer ,15,39. CSA (~9%) . HoweverSA (~9%) . This prtransfer . And thetransfer ,14,38. Atransfer . For exatransfer . Upon a Authentic to discover the potential CETP inhibitors from the bioactive compounds in the ZINC database via the cDocker algorithm [Eint) of both N- and C-termini, the top eight ligands were selected for further analysis, including ZINC000002010603, ZINC000006248133, ZINC000005871812, ZINC000002261174, ZINC000003526223, ZINC000005871644, ZINC000007067674, and ZINC000006242926. Subsequently, each docked complex was further refined by 100 ns MD simulations. Previous CETP inhibitors were found to bind near the narrowing neck of the hydrophobic central tunnel, potentially hindering the connection between the N- and C-terminal pockets . Howeverlgorithm . Based obindG) of CETP N-terminus and ZINC000002010603, ZINC000006248133, ZINC000005871812, ZINC000002261174, ZINC000003526223, ZINC000005871644, ZINC000007067674, and ZINC000006242926, which encompass the electrostatic energy (ΔEele), van der Waals interactions (ΔEvdw), polar solvation energy (ΔGGB), and non-polar solvation energy (ΔGsur), are summed to −23.6 ± 0.2, −25.4 ± 2.1, −25.4 ± 2.1, −35.0 ± 1.7, −0.2 ± 0.2, −26.3 ± 2.3, −19.7 ± 2.8, and −32.6 ± 1.5 kcal·mol−1, respectively (bindG) of CETP C-terminus and ZINC000002010603, ZINC000006248133, ZINC000005871812, ZINC000002261174, ZINC000003526223, ZINC000005871644, ZINC000007067674, and ZINC000006242926 are summed to −32.9 ± 2.3, −38.8 ± 1.6, −45.3 ± 1.3, −33.5 ± 1.8, −46.5 ± 1.3, −39.4 ± 1.4, −43.4 ± 1.0 and −42.2 ± 1.4 kcal·mol−1, respectively . In the DCCM plot, blue indicates a significant positive correlation, red indicates obvious anti-correlation, and white regions indicate less correlation. The binding of ZINC000006242926 to the CETP N-terminus significantly weakens the negative correlation between residues Lys180–Pro246 located near the “neck” region and the Ω4, Ω5, and Ω6 regions , probablPrevious research has shown that the hydrophobic interactions between the CETP inhibitors torcetrapib and anacetrapib and the CETP tunnel center may alter the flexibility of CETP, the structure of the distal β-barrel domain, and the structure–function relationship between phospholipids and Helix X, resulting in the inhibitors blocking the connection between the N-terminal and C-terminal pockets and disrupting the interaction of CETP with lipoproteins to reduce the transport of CE ,23,30,34Mutant” throughout this work. To obtain the authentic structure  was used as a starting structure . For con, −1 Å−1 . −1 Å−1.Correct ionization and low-energy conformers of the ‘Drugs-Now’ subset of the ZINC database  were obt+ counter-anions were placed to neutralize the system [−1 Å−2 position restraints on the backbone. Finally, the systems were equilibrated in an isothermal–isobaric ensemble (NPT) at 310 K and 1 Bar. All MD simulations were performed using periodic boundary conditions, and the cutoff radius for coulomb and van der Waals interactions was set to 8.0 Å. The simulation time step of 2.0 fs was used, and coordinates were collected every 10.0 ps. In order to improve sampling and collect more data, each system was repeated three times, with different random number generator seeds each time.Each system was sufficiently equilibrated by 100.0 ns MD simulations, using AMBER18 software  ,52 and Ae system . The steThe cpptraj module of Amber Tools 18 was used to perform the dynamic analysis . FpocketbindG) were calculated using the molecular mechanics–generalized Born surface area method (MM/GBSA) which can be used for various drug–ligand systems without additional regression [All values of binding free energies  consists of electrostatic energy (ΔEele), van der Waals (ΔEvdw), polar solvation energy (ΔGGB), non-polar solvation energy (ΔGsur), and entropy contribution (−TΔS) [The binding free energy (Δn (−TΔS) . The valMutant, with four mutated residues and four missed residues), constructed for protein crystallization. In this work, comprehensive MD simulations of CETPAuthentic and CETPMutant were performed to evaluate their structural differences influencing CETP-mediated lipid exchange, including stability, flexibility, hydrophobicity, and residue reorientation.Numerous X-ray, EM, and MD studies conducted thus far have revealed some important physical attributes of CETP and the lipid-exchange mechanisms behind these behavioral effects. However, their conclusions are based on artificial CETP  is similar in CETPAuthentic and CETPMutant, supporting the existence of an N-opening. The configurations of the C-terminal distal region (flaps Ω1–3) in CETPAuthentic and CETPMutant are different in aqueous solutions, with considerable differences in hydrophobic SASA. The authentic one retains even more hydrophobicity and is more likely to form an opening, with a larger maximum distance between residues Phe301 and Met412 (dPhe301-Met412), which might be the key residues for the formation of a “C-opening”. In all MD systems, Helix X in either CETPAuthentic or CETPMutant adopts the closed state, and this motion should be a necessary condition for lipid exchange. Regarding the “neck” region, CETPAuthentic rather than CETPMutant facilitates the formation of a continuous tunnel, with a constant distance between residues Phe265 and Met433 (dPhe265-Met433). In CETPAuthentic, residue Phe265 orients away from residue Met433 and provides a more conducive environment for lipid exchange to overcome the potential barrier formed by residues Phe265 and Met433. In short, there are obvious spatial distinctions between authentic CETP and the mutant construct. CETPAuthentic, rather than CETPMutant, supports the tunnel mechanism, and further studies on the mechanisms of CETP-mediated lipid exchange should fully consider this point of view.Our explicit solvent MD simulations show that CETPCryo-EM and MD results indicated that the N-terminal and C-terminal structures are flexible and have important roles in the lipid-transfer function. Eight small molecules were selected as potential ligands, which bind to the N- and C-terminal domains of CETP. Based on the complicated MD simulations and MM/GBSA calculations, the ligand ZINC000006242926 was chosen for further investigation to assess its influence on the structural dynamics of CETP. The mutant structures of CETP in complex with the inhibitor torcetrapib, which has shown a significant effect on plasma lipoprotein levels in clinical trials, as well as being an analog of structurally different inhibitor families, have revealed that the inhibitors bind to the protein by forming hydrophobic interactions, thereby obstructing the connection between the N- and C-terminal pockets . Further


Hydrocarbon-based products have many household and commercial uses and exposure to these substances is common. Severe clinical effects can occur if these products are ingested. This study investigated the characteristics and trends of hydrocarbon ingestions reported to United States Poison Centers.Data from the National Poison Data System were analyzed for cases of hydrocarbon ingestion among individuals < 20 years old reported to United States Poison Centers from January 1, 2000 through December 31, 2021.p < 0.0001) by 66.5% from 2000 to 2021. The greatest rate decrease was observed among lamp oils , followed by gasolines .There were 284,085 hydrocarbon ingestions reported during the 22-year study period in which a hydrocarbon was the first-ranked substance. Most of these cases occurred among children < 6 years old (83.2%), males (64.6%), at a residence (96.5%), were single-substance exposures (98.3%), and were managed on-site rather than in a health care facility (74.9%). However, 4.5% of cases were associated with a serious medical outcome, including 34 deaths. Thirty-two deaths were among children < 6 years old and most were associated with aspiration. Gasolines accounted for 24.6% of total cases, followed by lubricating oils and/or motor oils (19.9%), other types of hydrocarbons (14.9%), lamp oils (11.3%), and lighter fluids and/or naphtha (10.3%). The rate of hydrocarbon ingestions among United States youth < 20 years old decreased significantly  of cases associated with a serious medical outcome, including death. Most deaths were among children < 6 years old. This underscores the need to increase primary prevention efforts, especially for young children. Hydrocarbon-based products contain petroleum distillates and have many household and commercial uses and exposure to these substances is common. Severe clinical effects can occur if these products are ingested , which is maintained by America’s Poison Centers (formerly known as the American Association of Poison Control Centers). This national database collects standardized information from telephone calls to PCs across the US and its territories  were obtained from the NPDS. Hydrocarbons in the following NPDS generic substance categories were included: diesel fuels, gasolines, kerosenes, lamp oils, lighter fluids and/or naphtha, lubricating oils, mineral seal oil, mineral spirits, other types of halogenated hydrocarbons, other types of hydrocarbons, turpentine, and unknown types of hydrocarbon. Among the cases provided by the NPDS, 297,640 met study inclusion criteria. Cases (referred to as ingestion cases in this article) were included if at least one route of exposure was (1) ingestion or (2) aspiration (with ingestion), which resulted in the exclusion of 120,492 cases associated with other routes of exposure. The following cases were also excluded from the study: (1) exposure occurred outside the 50 US states or District of Columbia (n = 1451), or (2) medical outcome was coded as “confirmed non-exposure” (n = 355) or “unrelated effect” (n = 5044), reason for exposure coded as “adverse reaction—food” (n = 207) or “adverse reaction—drug” (n = 92) or “unintentional—bite/sting” (n = 11) or “unintentional—therapeutic error” (n = 484) or “unintentional—food poisoning” (n = 172). The NPDS defines “confirmed non-exposure” as an exposure in which “there is reliable and objective evidence that the exposure never occurred and that any symptoms exhibited by the patient were not related to the reported exposure,” and “unrelated effect” as a report for which “based upon all the information available, the exposure was probably not responsible for the effect(s)”  < 6 years, (2) 6–12 years, and (3) 13–19 years. The reason for exposure was grouped as (1) unintentional-general (includes exploratory behaviors), (2) unintentional-other , (3) unintentional-unknown, (4) intentional-suspected suicide, (5) intentional-misuse, (6) intentional-abuse, (7) intentional-unknown, (8) other, and (9) unknown. The NPDS defines intentional-misuse as “an exposure resulting from the intentional improper or incorrect use of a substance for reasons other than the pursuit of a psychotropic effect,” and intentional-abuse as “an exposure resulting from the intentional improper or incorrect use of a substance where the patient was likely attempting to gain a high, euphoric effect or some other psychotropic effect, including recreational use of a substance for any effect.”  Exposure sites were grouped into (1) residence (includes other or own residence), (2) school, (3) other , and (4) unknown.The level of health care received was based on NPDS-defined categories and was grouped into (1) no health care facility (HCF) treatment received, (2) treated/evaluated at a HCF and released, (3) admitted , (4) refused referral/did not arrive at a HCF, and (5) unknown   no effect (no symptoms developed as a result of the exposure), (2) minor effect , (3) moderate effect , (4) major effect (symptoms are life-threatening or result in significant disability or disfigurement), (5) death, (6) not followed , and (7) unknown   and SAS 9.4  statistical software. Population-based rates were calculated using US Census Bureau July 1 intercensal and postcensal population estimates from 2000 through 2021  and occurred most frequently among males (64.6%), at a residence (96.5%), and were managed on-site rather than in a HCF (74.9%)  of these. A hydrocarbon was the first-ranked substance in 284,085 of the cases, averaging 12,913 cases per year (Table Most (96.9%) exposures were unintentional, and the most common reason for exposure was unintentional-general (89.8%) Table . The reaApproximately three-fourths 74.1%) of hydrocarbon ingestions did not receive care at a HCF; however, 4.0% were admitted to a HCF (including 3.8% to a CCU or non-CCU and 0.2% directly to a psychiatric facility)   than ingestion without aspiration. Children who experienced aspiration (with ingestion) were more likely to have a serious medical outcome , including death , or be admitted to a CCU or non-CCU  than children who ingested a hydrocarbon without aspiration.n = 15, 44.1% of deaths) and two  years. Thirty-two deaths involved a single substance, and 25 deaths were associated with aspiration (with ingestion). Twenty-three of the 25 aspiration-related deaths were among children < 6 years old. Lamp oils was the leading hydrocarbon category associated with ingestion-related fatality, accounting for 12 deaths (35.3%), followed by gasolines , mineral spirits , kerosenes , lighter fluids , other types of hydrocarbons , and 1 fatality (2.9%) each for: diesel fuels, turpentine, and unknown types of hydrocarbons. Males  and children < 6 years old  were more likely to experience a serious medical outcome than females and youth 6–19 years old, respectively.Most hydrocarbon ingestions were associated with no effect (34.2%) or minor effect (20.9%) Table . The proAmong youth < 20 years old, the gasolines category was the most common hydrocarbon category associated with ingestion cases (24.6%), followed by lubricating oils and/or motor oils (19.9%), other types of hydrocarbons (14.9%), lamp oils (11.3%), and lighter fluids and/or naphtha (10.3%) , followed by lighter fluids and/or naphtha (17.5%) and gasolines (15.0%). Similarly, the proportion of HCF admissions was highest for ingestions involving lamp oils (32.0%), followed by lighter fluids and/or naphtha (17.9%) and gasolines (12.5%). Compared with other substances, lamp oils were more likely to be associated with a serious medical outcome , death , or admission to a CCU or non-CCU . In addition, lamp oils were more likely than other hydrocarbons to be associated with aspiration (with ingestion)  than ingestion without aspiration.n = 297,640, including first-ranked and non-first-ranked cases) reported to US PCs from 2000 to 2021. The rate of hydrocarbon ingestions among youth < 20 years old per 100,000 US population decreased significantly by 66.5% (p < 0.0001) from 25.8 in 2000 to 8.6 in 2021  from 32.4 in 2000 to 10.5 in 2021 and children < 6 years old  from 75.5 in 2000 to 24.8 in 2021 (trend by sex not shown in figure). The rate of ingestion decreased among all hydrocarbon categories, with the greatest decrease observed among lamp oils  from 3.8 in 2000 to 0.8 in 2021, followed by gasolines  and lubricating oils and/or motor oils   from 1.2 in 2000 to 0.4 in 2021 and 64.3% from 5.5 in 2000 to 2.0 in 2021, respectively. Among the age groups, children < 6 years old demonstrated the greatest decrease in the rate of a serious medical outcome  from 3.6 in 2000 to 1.2 in 2021 and rate of admission to a CCU or non-CCU (− 65.7%) from 16.1 in 2000 to 5.5 in 2021  cases involved children < 6 years old, and consistent with this age group, most (89.8%) ingestions were related to exploratory behavior , involved a single substance (98.3%), and occurred in the home (96.5%). This information is consistent with previous findings and is helpful for targeting prevention efforts  promulgated a rule under the authority of the Poison Prevention Packaging Act (PPPA) Law  that manConsistent with previous publications demonstrating the severe clinical outcomes of hydrocarbon aspiration  of cases associated with a serious medical outcome, including 34 deaths during the study period. Thirty-two of these deaths were among children < 6 years old and most were associated with aspiration (with ingestion). This underscores the need to increase primary prevention efforts, especially for young children.


We established the I/R model in mice and used BT2, a highly specific BCAA dehydrogenase (BCKD) kinase inhibitor to promote BCAA metabolism. It was further verified by lentivirus knocking down PPM1K in neurons. We found that BCAA levels were elevated after I/R injury due to dysfunctional oxidative degradation caused by phosphorylated BCKD E1α subunit (BCKDHA). Additionally, the level of phosphorylated BCKDHA was determined by decreased PPM1K in neurons. We next demonstrated that BCAA could induce oxidative stress, lipid peroxidation, and ferroptosis in primary cultured cortical neurons in vitro. Our results further showed that BT2 could reduce neuronal ferroptosis by enhancing BCAA oxidation through inhibition of BCKDHA phosphorylation. We further found that defective BCAA catabolism could induce neuronal ferroptosis by PPM1K knockdown. Furthermore, BT2 was found to alleviate neurological behavior disorders after I/R injury in mice, and the effect was similar to ferroptosis inhibitor ferrostatin-1. Our findings reveal a novel role of BCAA in neuronal ferroptosis after cerebral ischemia and provide a new potential target for the treatment of ischemic stroke.Ischemic stroke is a neurological disorder caused by vascular stenosis or occlusion, accounting for approximately 87% of strokes. Clinically, the most effective therapy for ischemic stroke is vascular recanalization, which aims to rescue neurons undergoing ischemic insults. Although reperfusion therapy is the most effective treatment for ischemic stroke, it still has limited benefits for many patients, and ischemia-reperfusion (I/R) injury is a widely recognized cause of poor prognosis. Here, we aim to investigate the mechanism of protein phosphatase Mg Stroke is a significant cause of death and disability in the world, with approximately 795,000 people suffering from new or recurrent stroke every year, approximately 87% of which are ischemic strokes . ReperfuNeuronal death is significant in I/R injury and the main cause of neurological deficits. Thus, uncovering the mechanism of neuronal injury and loss is urgently needed. Ferroptosis, a novel form of regulated cell death, is characterized by the accumulation of iron-mediated lipid peroxide of polyunsaturated fatty acids (PUFA) . The bioMetabolic dysfunction of amino acids is closely related to the occurrence of ferroptosis  and tighAdult male C57BL/6 mice  and Sprague-Dawley rats  were housed in a pathogen-free facility with a 12-h light/dark cycle and allowed free access to food and water. Rats were randomly assigned to the middle cerebral artery occlusion (MCAO) group or sham group and underwent metabolomics analysis after surgery.We used 3,6-dichlorobenzo[b]thiophene-2-carboxylic acid (BT2) or Fer-1 to assess the influence of BCAA and ferroptosis on cerebral I/R damage in mice. BT2  and Fer-1  were dissolved in a vehicle buffer (VEH) containing 5% dimethyl sulfoxide , 10% cremophor EL , and 85% sodium bicarbonate . BT2 was injected intraperitoneally (i.p.) at a dosage of 40 mg/kg/day, as previously described . Fer-1 wMCAO and cerebral I/R models were established as previously described , 18. RatAfter surgery, animals were initially examined for neurological deficits when fully awake according to Longa’s five-tiered grading system, as follows: 0, no deficit; 1, failure to extend contralateral forepaw; 2, spin longitudinally; 3, falling to the right side; 4, unable to walk spontaneously. A score of 1–3 points was the criterion for successful modeling .After anesthetizing mice at the indicated time points with 1% pentobarbital sodium (100 mg/kg. i.p.), samples were quickly collected. For tissue sampling, the ischemic border tissue was rapidly separated. The ischemic border was defined as the approximately 500-μm-wide region containing generally morphologically normal cells that surrounds the core . Tissues−1, and the gas flow rate through the column was 1 mL min−1. The initial temperature was kept at 50 °C for 1 min, then raised to 310 °C at a rate of 10 °C min−1, then kept for 8 min at 310 °C. The injection, transfer line, and ion source temperatures were280, 280 and 250 °C, respectively. The energy was −70 eV in electron impact mode. The mass spectrometry data were acquired in full-scan mode with the m/z range of 50–500 at a rate of 12.5 spectra per second after a solvent delay of 6.30 min [Rat brain samples were harvested 24 h after operation in each of the MCAO and sham groups (six rats per group). Liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) were performed as we previously described . The ult6.30 min .After reperfusion, brain tissues and serum were immediately collected for detection of BCAA or stored at −80 °C. Tissues were homogenized according to the manufacturer’s instructions, and the supernatant was analyzed with a commercial kit  to determine relative levels of BCAA. The absorbance of each sample was measured at 450 nm with a multimode microplate reader  and calculated based on the standard curve.−∆∆Ct method. Primer sequences are listed in Supplementary Table Total RNA was extracted and purified from cultured cells or tissues using TRizol . A reverse transcriptase kit  was used to reverse total RNA into cDNA. qRT-PCR was performed on a Bio-Rad CFX system  using SYBR Green Master Mix . All gene expression values were normalized to β-actin mRNA levels in each sample using the 2+/Mn2+ dependent 1 K , Acyl-CoA synthetase long chain family member 4 , prostaglandin-endoperoxide synthase 2 , xCT , ferritin heavy chain , glutathione peroxidase 4 , Ferroportin , Transferrin receptor , and β-actin  at 4 °C. After incubation with appropriate horseradish peroxidase-conjugated secondary antibodies  for 2 h, bands were developed and visualized using VisionWorks . The results were analyzed with ImageJ software .Total protein was extracted and purified from brain tissues or cultured cells with radioimmunoprecipitation assay lysis buffer according to the manufacturer’s instructions. Proteins were separated in sodium dodecyl sulfate polyacrylamide gels and transferred onto a polyvinylidene difluoride membrane, which was blocked with 5% nonfat milk. The following primary antibodies were used overnight for blots: BCAA dehydrogenase , BCKD kinase , BCKD E1α subunit (BCKDHA) phosphorylated S293 , BCKDHA , protein phosphatase Mg2After washing with PBS, sections were blocked with 10% donkey serum, 0.5% bovine serum albumin (BSA), and 0.25% Triton X-100 in PBS for 1 h. Next, sections were incubated with the following primary antibodies overnight at 4 °C: PPM1K (1:200), p-BCKDHA S293 (1:200), 4-hydroxynonena , ionized calcium binding adapter molecule 1 , glial fibrillary acidic protein , and neuronal nuclei . Subsequently, slides were incubated with corresponding Alexa Fluor 594-, Alexa Fluor 488-, or Alexa Fluor 647-labeled secondary antibodies. After counterstaining with 4’,6-diamidino-2-phenylindole  for 10 min, slides were washed three times with PBS and covered using 50% glycerol in PBS. Images were subsequently taken using a confocal microscope  and quantitatively analyzed by ImageJ software. Sections from four animals in each group were immunohistochemically stained and used for double- or triple-labeling experiments.4 g for 10 min, the supernatant of samples was used to detect contents of MDA , GSH, oxidized GSH , and iron levels .To quantify levels of malondialdehyde (MDA), GSH, and iron, tissues, and cells were collected and assayed with kits according to the manufacturer’s instructions. Samples were homogenized after reperfusion or culture. After centrifuging at 4 °C and 1.2 × 10Primary cortical neurons were isolated from embryonic day 15 (E15)–E16 mouse embryos as previously described . In brieNeurons were cultured for 24 h with BCAA  at 2.5, 5, 10, 20, 50, and 100 mmol/L (mM) in the medium. RSL3  and erastin  stock solutions were dissolved in DMSO at a storage concentration. These reagents were added into the medium for 2 h at 5 μM (RSL3) or 10 μM (erastin) to induce ferroptosis in vitro. Cells were pretreated with BT2  or Fer-1  for 4 h and then BCAA was added for an additional 24 h of culture. The control group was cultured in medium containing 0.1% DMSO for 24 h.The shPPM1K-mCherry lentivirus targeting the mouse PPM1K was prepared by OBiO Technology Corp.,Ltd . Lentivirus expressing scramble shRNA (vector) was used as a negative control. For lentivirus transduction, primary cortical neurons were seeded on the poly-L-lysine coated plates or dishes as above. Three days postseeding, the medium was removed and added the fresh medium containing shPPM1K or vector lentivirus at a certain multiplicity of infection (MOI). Twenty-four hours post-transduction, the medium containing lentivirus was replaced with a fresh culture medium. Transduced neurons were incubated for 3 days and then harvested to detect the transduction efficiency. The efficiency of Lenti-shPPM1K was validated by qRT-PCR and Western blotting. Finally, based on the pre-experimental results, an MOI of 20 was selected to continue transduction and used for subsequent observation and analysis. The sequences of shRNA were provided in Table 4 cells per well), and their viability was assessed after treatment using a Cell Counting Kit-8 (CCK-8) assay . Briefly, 10 μL of reagent was incubated with cells in a 96-well plate at 37 °C for 1 h and then the absorbance was measured at 450 nm by a multimode microplate reader (Tecan Infinite M200).Cells were seeded onto 96-well plates . The absorbance of each well at 450 nm was measured with a multimode microplate reader (Tecan Infinite M200).Cells were seeded onto six-well plates at a density of 5 × 10Cells were grown on cell glass slides, rinsed with PBS, fixed with 4% PFA, and washed with PBS again. Next, the slides were blocked with 3% BSA with 0.3% Triton X-100 for 1 h, and incubated with the following primary antibodies overnight at 4 °C: ACSL4, 4-HNE, NeuN , and beta III tubulin . After washing slides with PBS three times, they were incubated with corresponding secondary antibodies and DAPI in the dark. Finally, slides were mounted with glycerol and imaged using a confocal microscope (Olympus FV 3000).Following all interventions, cells grown in confocal dishes were stained with 2 μM C11-BODIPY 581/591  for 30 min at 37 °C. Lipid ROS levels in primary neuronal cells were analyzed using a confocal microscope (Olympus FV 3000). Nonoxidized C11 was observed under the 594-nm excitation channel, while oxidized C11 was observed under the 488-nm excitation channel. Lipid ROS levels were assessed as the mean fluorescence intensity (MFI) of the 488-nm excitation channel.Cerebral blood flow (CBF) of each group was monitored 24 h after cerebral I/R, using a laser speckle Doppler flowmeter . The images and data analysis were processed with the RFLSI analysis software (RWD Life Science). The change in CBF was quantitated and expressed as a relative blood flow that was calculated as the ratio of the region of interest (ROI) areas between the ipsilateral and contralateral hemispheres .At 1, 3, 5, 7 and 14 days after reperfusion, the neurological functions of mice in each group were evaluated according to Longa scores and the Garcia scale , which aTo assess functional impairment and recovery, mice underwent a CatWalk XT automated quantitative gait analysis  at 1, 3, 5, 7, and 14 days after I/R, and were trained 3 days before the experiment as previously described , 27. BriAfter reperfusion for 24 h, mice were anesthetized and their fresh brains were removed. Subsequently, whole-brain tissue was evenly cut into 1-mm coronal sections with a metallic brain matrix, and the resulting sections were incubated in 1% TTC  for 30 min at 37 °C in the dark. Subsequently, sections were fixed with 4% PFA and stored until imaging. The percentage of infarct volume was calculated according to the formula: cerebral infarction volume/total cerebral volume × 100%.t-test and Wilcoxon tests were used to analyze the two groups. For multiple comparisons, one-way ANOVA was performed, followed by Tukey’s post hoc test. Assessment of neurological scores and gait test results were analyzed by two-way repeated measures ANOVA. Cumulative survival rates were compared among the four groups using the Kaplan–Meier method and log-rank tests. All experiments were performed at least three independent times. A value of P < 0.05 was used to indicate statistical significance.All statistical analyses were performed using GraphPad Prism 9 software . Data are presented as the mean ± standard deviation (SD). Independent sample two-tailed Student’s The other detailed materials and methods and full and uncropped western blots are presented in the P < 0.05 and values of variable importance in the project > 1 calculated by orthogonal partial least-squared discriminant analysis were identified as differential metabolites. The resulting differential metabolites were visualized in a volcano plot and heatmap of hierarchical clustering  resulted in obvious clustering of the samples Fig. , which ring Fig. . A totalTo further explore its potential mechanisms in cerebral ischemia, we performed metabolic pathway analysis of differential metabolites using the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic library. The results indicate significant differences in metabolic pathways related to amino acids and lipid fatty acids Fig. . DiffereAcsl4, Lpcat3, Ptgs2, Tfrc, and Gls2 were significantly increased after cerebral I/R, while those of Gpx4 and xCT were decreased  and β-oxidation (Acaa2) were increased 24 h after cerebral I/R, while there were no differences in Acsl1, Cpt1b, or Acadm levels  analysis in mice. IF staining of NeuN showed smaller and irregular NeuN-positive fluorescence signals in the ischemic area after I/R. We observed a band between ischemic and normal tissue Fig.  that wasAcadm, Acaa2, and Acsl1 were significantly increased  and Bcat1 was slightly decreased, while Bcat2 mRNA expression was significantly decreased , lentivirus vectors (Lenti-vector), or vectors carrying PPM1K shRNA (Lenti-shPPM1K). The effect and efficiency of knocking down of Lenti-shPPM1K was verified by qRT-PCR and western blotting Fig. . Compareons Fig. . Furthersed Fig. , while msed Fig. . There woa4 Fig. . Proteinoa4 Fig. . In addioa4 Fig. . Taken tNext, we tested whether BT2 or Fer-1 could rescue the neuronal injury induced by BCAA. Double staining of 4-HNE and NeuN displayed high numbers of 4-HNE- and NeuN-positive cells in the ischemic border and ischemic core areas of VEH group mice. BT2 and Fer-1 obviously decreased 4-HNE expression Fig. . WesternWe further monitored the CBF of each group 24 h after cerebral I/R, and the results showed that BT2 and Fer-1 obviously alleviated the CBF reduction compared with the VEH group Fig. . FurtherTo further investigate the effect of BT2 and Fer-1 on functional recovery after cerebral I/R, several scorings were evaluated. We first observed the change of mortality within 14 days after cerebral I/R Fig. . The resAlthough previous studies demonstrated the significance of ferroptosis in ischemic disease, the underlying mechanisms remain unknown , 28. OurA growing body of studies have implicated abnormal BCAA metabolism in I/R injury, multiple metabolic diseases, and tumors , 36–39. In the heart, abnormal BCAA catabolism resulted in elevated circulating and tissue BCAA levels, which directly inhibited PDH activity and glucose metabolism, causing a shift from glucose oxidative metabolism to FAO . In vitrOur findings reveal that PPM1K expression was reduced in neuronal cells following cerebral I/R injury. Because PPM1K is reportedly highly expressed in the brain, mainly in neurons , its decPpm1k-KO mice display deficient BCAA catabolism that promotes heart failure and is associated with induced oxidative stress and metabolic disturbances; however, BT2 significantly mitigated cardiac dysfunction in these mice [To further verify that elevated BCAA mediated ferroptosis after cerebral I/R, we enhanced BCAA metabolism pharmacologically to observe neurological recovery and changes in ferroptosis. Our results show that BT2 or Fer-1 intervention after cerebral I/R markedly reduced cerebral infarct volumes and neuronal death, and improved CBF and neurological recovery after cerebral I/R. BT2 inhibition of BCKDK activity can effectively activate BCKDH and significantly reduce p-BCKDHA levels . Consistese mice . Gabapenese mice , reducedese mice . In the In conclusion, our findings show that BCAA mediated neuronal ferroptosis in cerebral I/R injury by regulating BCKDHA Ser293 phosphorylation, providing new understanding of the biochemistry driving ferroptosis in ischemic stroke. Moreover, pharmacological intervention of BCAA metabolism to inhibit ferroptosis may be a novel strategy for treatment of ischemic stroke.Supplementary MaterialFigure S1Figure S2Figure S3Figure S4Original Data FileA reproducibility checklist


Pseudomonas aeruginosa persister cells to withstand host innate immunity. We found that persister cells resist MAC-mediated killing by the complement system despite being bound by complement protein C3b at levels similar to regular vegetative cells, in part due to reduced bound C5b, and are engulfed at a lower rate (10- to 100-fold), even following opsonization. Once engulfed, persister cells resist killing and, contrary to regular vegetative cells which induce a M1 favored  macrophage polarization, they initially induce a M2 favored macrophage polarization , which is skewed toward M1 favored polarization  by 24 h of infection, once persister cells awaken. Overall, our findings further establish the ability of persister cells to evade the innate host response and to contribute chronic infections.Bacterial persister cells—a metabolically dormant subpopulation tolerant to antimicrobials—contribute to chronic infections and are thought to evade host immunity. In this work, we studied the ability of  Recurrent and persistent bacterial infections, chronic infections, are of major importance across the world as they remain in the host for extended periods of time, and as such, are detrimental to patients and a burden on the health care system. The recalcitrance of chronic infections is aided by the characteristics of persister cells. Persister cells are present in most bacterial species, and depending on the bacterial growth phase, they can make up to 1% of the overall bacterial population . PersistDespite the possible contribution to chronic bacterial infections, the interactions between bacterial persister cells and the host innate immune system are still poorly understood. Typically, during an infection, macrophages are the first immune cells to recognize pathogens via their pattern recognition receptors . MacrophS. aureus is exposed to host oxidative stress  patients  in the median forward-scattering (cell size) or side-scattering (cell granulation) between regular vegetative cells and persisters, the latter had a narrower peak, indicating a less heterogeneous population, regarding cell size  and cells exposed to ciprofloxacin (20 mg/L) in saline were named persister cells (A2). Subsequently, isolated/selected cells were stained with SYTO9 and propidium iodide and the % of dead cells was calculated (B). Download FIG S1, PDF file, 0.4 MB.Cells remaining following selection. Regular vegetative cells and persister cells of Copyright © 2023 Hastings et al.2023Hastings et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. aeruginosa persister cells with human complement both for MAC-mediated killing  and/or C5b (initiating protein of the MAC formation) to bind persister cells, we used an anti-C3 antibody to detect and quantify the C3b binding to the cells (P > 0.05) of C3b binding between persister and regular vegetative cell populations albeit persister cells having a clear bi-modal pattern of binding (P > 0.05)  .P. aeruginosa persister cells with and without prior-opsonization. THP-1 macrophages were exposed to the same inoculum of bacteria, whether regular or persister, for 30, 60, 90, and 180 min and engulfment was evaluated based on intracellular bacterial viability (P. aeruginosa were engulfed significantly less (P < 0.001) by THP-1 macrophages compared to regular vegetative cells, with an overall 100-fold decrease (P > 0.05) nor to the level of regular vegetative cells  by a total of 1.4 Logs (P. aeruginosa cells were active in the macrophages upon engulfment and found that it was decreased at time zero by 5.6-fold ± 1.7 and no change at time 24 (1.1 ± 0.4) in persisters compared to regular vegetative cells. These findings provide evidence that the only surviving cells within the macrophages 24 h postinfection are persister cells and are supported by previous findings for Salmonellatyphimurium where once engulfed the bacteria adopt a nongrowing antibiotic tolerant state and can reside for extended periods of time within the macrophages ::GFPmut were isolated and resuspended in RPMI medium. Constitutive fluorescence (A) and absorbance were monitored for 24 h. Results were analyzed according to T-test  and are presented as mean ± SD. Download FIG S2, PDF file, 0.5 MB.Bacterial growth and metabolism of regular vegetative and persister cells. Bacterial populations of Copyright © 2023 Hastings et al.2023Hastings et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.00056-23.3FIG S3P. aeruginosa regular vegetative and persister cells. The relative expression level for P. aeruginosa persister and regular vegetative cells was compared to uninfected controls. Results shown consist of at least 4 experiments. The CT value of the housekeeping gene gadph remained constant throughout the different treatments (P > 0.5 by ANOVA and no difference between treatments by Tukey’s multiple-comparison test). A significant change was considered to occur when a 2-fold change in the relative expression level occurred. Download FIG S3, PDF file, 0.7 MB.Macrophage cytokine gene expression by macrophages. The relative gene expression of 4 cytokines IL-6 (A), TNF (B), CXCL-8 (C), and IL-10 (D) was quantified for: 0.5, 1.5, and 24 h of infection (MOI of 10) with Copyright © 2023 Hastings et al.2023Hastings et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vivo, where it is known that the mRNA coding for cytokines becomes unstable resulting in a reduction of translation  resist both MAC-mediated complement killing and macrophage killing albeit being opsonized by C3b, and (ii) elicit the polarization of macrophages toward a M2 response, which switches to a M1 response upon persister awakening. To our knowledge, this is the first comprehensive study of the overall innate immune response to P. aeruginosa persister cells.In this study, we describe a mechanism of the effect of persister cells on the immune response, while describing their resilience to other aspects of innate immunity namely, MAC-mediated complement killing and macrophage killing. Our study provides evidence that P. aeruginosa and E. coli persister cells compared to regular—metabolically active—bacterial cells  state  and the chaperone protein SurA were present in higher abundance in P. aeruginosa cells reverting from a persister state  state  which hass state . The reser state . In the er state  while Oper state , and itser state . A similhibition  and the hibition . It has eins Tuf  and LpD eins Tuf . Persistin blood .S. aureus persister cells  proteins to attack host phagocytes , while persister cells initially induce a polarization favoring M2, more specifically M2b , which is then skewed toward M1 polarization, by 24 h of infection, once the internalized persister cells revert into an awakened metabolically active state.From our findings, we propose that the mechanism of infection and immune system modulation between regular vegetative cells and persister cells is distinct . RegularIn conclusion, we found that in addition to being tolerant to antibiotics, persister cells are also resilient to an immune system attack/response where they are not eliminated by MAC-mediated killing, as demonstrated by the decrease of bound C5b, despite being bound by C3b, and elicit an intermediate anti-inflammatory response by triggering macrophage M2b favored polarization. This study sheds further light as to how persister cells modulate the immune response and survive in the host during an infection. By escaping/resisting the immune response, persister cells can later become active dividing cells and reinfect the host, further confirming that these cells are involved in chronic and recurrent infections. Despite these advances, there remain many unknowns relating to persister cell behavior when infecting a host, and how the immune response to persister cells occurs in other bacterial species.Staphylococcus aureus ATCC 6538, P. aeruginosa PA14, P. aeruginosa MPAO1 attTn7::P(A1/04/03)::GFPmut, and E. coli BW25113 were used. Overnight cultures were grown on Lennox media  in Erlenmeyer flasks at 37°C with aeration.In this study, S. aureus, P. aeruginosa, and E. coli were prepared on 100% LB agar and incubated at 37°C for 24 h. Planktonic overnight cultures were prepared by removing an isolated colony from the streak plate and inoculating it into 100% LB broth and grown at 37°C with agitation (220 rpm) for a period of 24 h. Cells were then collected , washed twice with saline , and subsequently resuspended in either saline (0.85% NaCl) or ciprofloxacin (20× MIC) in saline to a final OD600 of 1.6. Ciprofloxacin was used as means to induce oxidative DNA damage which results in an accumulation of persister cells (g for 5 min at 4°C). The cells were then resuspended in saline and washed two times by centrifugation . These 2 washes were performed to remove lysed dead cells, as ciprofloxacin was previously demonstrated to lyse cells of P. aeruginosa, E. coli, and Enterobacter cloacae , where after a 15-min incubation, the cells were washed (to remove excess of stain) with PBS and resuspended. Bacterial fluorescence (from the stains) was measured using a SpectraMax I3x Multi-Mode plate reader, Molecular Devices. We also used dead cells—cells exposed to 70% ethanol for 30 min—as a control.Persister cells were isolated as described previously –6. Brief–er cells . Culture cloacae –61. Oncecloacae – and onlyruginosa , 33, 34.P. aeruginosa regular and persister cells were isolated as above, collected by centrifugation, and washed in 0.85% saline three times by centrifugation . Each population was then resuspended in RPMI at a final OD600 of 1.5. Each population was added to a 96-well plate and changes in absorbance (595 nm) was monitored every hour, for 24 h in a microtiter plate reader (Beckman DTX880) at 37°C.P. aeruginosa MPAO1 attTn7::P(A1/04/03)::GFPmut (g for 5 min at 4°C). Each population was then resuspended in RPMI to a final OD600 of 1.5. Each population was added to a 96-well plate and changes in fluorescence  were monitored for 24 h, at 30 min intervals .Regular and persister cells of ::GFPmut , constitP. aeruginosa, S. aureus, and E. coli were collected by centrifugation, washed in 0.85% saline three times by centrifugation . Samples were then resuspended in 0.85% saline at a final concentration of 107 cells/mL, and subsequently in either 90% complete human serum, PBS, or heat-inactivated serum. Cells were then incubated at 37°C, and cell viability was determined by adding 10 μL of 10 mM EDTA to stop the reaction at 0, 0.75, 1.5, 3, and 24 h of incubation followed by serial dilutions and plating of bacteria on 1:2 PCA with MgCl2·7H2O for 48 h at 37°C. Controls consisted of cells exposed to heat-inactivated human serum , unbound proteins from the serum were decanted, and cells were resuspended in PBS. Serial dilutions of each population were performed, and each dilution was stained. Bacterial cells were stained with BacLight Red , and complement protein C3b was labeled with fluorescent Anti-C3 antibody (35 μg MP Bio). Samples were imaged using an epifluorescence microscope (Olympus BX46) at ×100 magnification. Experiments were performed in quadruplicate with five images being taken per sample. Images were analyzed using Intensity Luminance V1 software  then resuspended in 0.85% saline at a final concentration of 107 cells/mL, and subsequently incubated in either 90% complete human serum or PBS for a period of 1.5, 3, or 24 h. The complement reaction was then stopped with 10 mM EDTA, and the cells were washed in 0.85% saline three times by centrifugation . The cell pellets were resuspended in PBS and then ELISAs were executed per manufacturer’s instructions to measure membrane bound C5b .software . To asse5 cells/mL. THP-1 monocytes were seeded at a concentration of 5 × 105 cells/mL onto 24-well plates and differentiated into M0 Macrophages via the introduction of 100 nM phorbol 12-myristate 13-acetate (PMA) for 3 days at 37°C 5% CO2, after which they were ready for the experimental procedures  for 1 h to remove any remaining extracellular bacteria , followed by resuspension in PBS, and subsequently used to infect THP-1 macrophages as described above. Experiments were performed in quadruplicate.f 30 min . The comTo assess the elimination of intracellular bacteria, infections were performed for a period of 90 min as described above. Once infections were stopped, and following the gentamicin exposure, infection media was replenished, and the cultures were incubated for further 24 h. After incubation, macrophages were lysed with 10% Triton X-100 for 45 min and intracellular bacteria viability was quantified as described above . ExperimTo assess the size of persister cells relative to regular vegetative cells, persister cells were isolated as above, stained with BacLight Red  and then fixed with paraformaldehyde. Flow cytometry was performed using the BD Accuri C6 Plus system and the data were analyzed with the flow cytometry software FlowJo. Similarly, to assess the elimination of persister cells, THP-1 macrophages were infected as above with prestained bacteria for 90 min or 24 h, followed by fixation with paraformaldehyde and flow cytometry analysis as above. To determine macrophage polarization, THP-1 macrophages were infected as above with either regular or persister cells for 90 min followed by immunostaining for M1/M2 cell-surface marker proteins CD80 , CD86 , and CD206 , and DAPI (Thermo Fisher Scientific) DNA staining for 30 min. The cells were then fixed with paraformaldehyde and flow cytometry analysis was performed with the Bio-Rad ZE5 Cell Analyzer and FlowJo. Experiments were performed in quadruplicate.P. aeruginosa 16s rRNA were quantified. To quantify the relative expression, infections were performed as described above. At the end of the incubation with gentamicin, TRIzol reagent was added to macrophages, the contents of each well were collected, and RNA was isolated using the Zymo RNA purification kit (Zymo Research). A total of 0.5 μg of RNA was used for cDNA synthesis, and cDNA was generated using QScript cDNA Synthesis kit. Quantitative reverse transcriptase PCR (qRT-PCR) was performed with an Eppendorf Mastercycler Ep Realplex instrument  and the Kapa SYBR Fast qPCR kit  with the oligonucleotides for THP-1 cells (obtained from Qiagen) and P. aeruginosa 16s rRNA  for P. aeruginosa : IL-10 (BMS2152), TNF-α (BMS223HS), IL-6 (BMS213HS), and CXCL-8 (KHC0081). At the end of each assay, the absorbance of each sample was determined at 450 nm . Cytokine concentrations were determined using standard curves generated in each assay, then accounting for the dilution factor. Experiments were performed at least in quadruplicate.Macrophages were infected with All data were analyzed using GraphPad Prism 9.3.1. One-way ANOVA was performed for multivariant analysis followed by Tukey’s or Dunnett’s multiple-comparison tests.


Buspirone, as a partial agonist for a 5-hydroxytryptamine (serotonin) receptor 1A (5-HT1A), has been prescribed as an anxiolytic drug for patients. In addition, the lowering effect of serotonin on blood pressure was reported in hypertensive animal model. We investigated the therapeutic mechanism of buspirone against lipid metabolism disturbed by hypertension of early stage via hypertensive and obese animal model.  The levels of various biomarkers related to lipid metabolism and hypertension were estimated through the measurement of body weight and fat weight, blood analysis, western blotting, immunohistochemistry, and staining methods. μM compared with untreated differentiated control. Body weight and abdominal fat weight were lowered in spontaneously hypertensive rats (SHRs) administered with buspirone of 10 mg/kg/day for 4 weeks than 8-week untreated group. Triglyceride (TG) level was decreased in SHRs administered with buspirone of 5 and 10 mg/kg/day compared to 8-week untreated group. High-density lipoprotein (HDL)-cholesterol concentration was elevated by buspirone 10 mg/kg/day treatment compared to 8-week untreated group. Blood pressures in SHRs were lowered by buspirone treatments of 5 and 10 mg/kg/day compared with 8-week untreated group. Protein levels for peroxisome proliferator-activated receptor δ (PPARδ), 5′ adenosine monophosphate-activated protein kinase (AMPK), and PPARγ coactivator-1 alpha (PGC-1α) were increased both in C2C12 cells treated by buspirone of 100 μM and in SHRs administered by buspirone of 1, 5, and 10 mg/kg/day compared to untreated control cells and 8-week untreated group. Fat cell numbers decreased in 8-week untreated group were increased in SHRs administered by buspirone treats of 1, 5, and 10 mg/kg/day. Protein expression levels for angiotensin II type 1 receptor (AT1R) and vascular cell adhesion molecule 1 (VCAM1) were increased in 8-week untreated group compared to 4-week group, however, they were decreased by buspirone treatments of 1, 5, and 10 mg/kg/day.  The lipid accumulation was lowered in differentiated 3T3-L1 cells by buspirone treatments of 50 and 100 δ dependent catabolic metabolism producing energy, and eventually, the ameliorated lipid metabolism could normalize high blood pressure. Buspirone may induce the losses of body weight and abdominal fat weight through the activation of PPAR Moreove2C12 mouse myoblasts were purchased from Korean Cell Line Bank . Reagents for cell culture such as media, fetal bovine serum (FBS), and antibiotic–antimycotic solution (AA) were supplied from WELGENE, Inc. . PRO-PREP™ protein extraction solution and pre-stained protein marker were bought from Intron Biotechnology . Hematoxylin and eosin (H&E), Oil Red O, sodium succinate, nitroblue tetrazolium (NBT), potassium phosphate monobasic, sodium phosphate dibasic, and protease inhibitor cocktail were supplied from Sigma-Aldrich . A Clarity™ Western ECL Substrate Kit was obtained from Bio-Rad . X-ray film was bought from Agfa , and the developer and fixer reagents were purchased from Carestream Health, Inc. . Buspirone was purchased from Boryung Pharmaceutical Co., Ltd. . SHRs were purchased from Doo-Yeol Biotech , while the animal diets were purchased from Central Lab. Animal Inc. . Primary antibodies against 5′ adenosine monophosphate kinase (AMPK), phosphorylated AMPK (p-AMPK), and endothelial nitric oxide synthase (eNOS) were bought from Cell Signaling Technology, Inc. . Primary antibodies for peroxisome proliferator-activated receptor delta (PPARδ) and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) were supplied from Abcam . Primary antibodies for vascular cell adhesion molecule 1 (VCAM1) and secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. . Primary antibody for angiotensin II type 1 receptor (AT1R) was supplied from Abcam . VECTASTAIN® Elite® ABC-HRP Kit and DAB peroxidase substrate kit were bought from Vector Laboratories, Inc. . Reagents to quantify the concentrations of total cholesterol and high-density lipoprotein cholesterol (HDL cholesterol) were bought from Kyowa Medex Co., Ltd. . Reagent to estimate the level of triglyceride (TG) was supplied from Abcam .3T3-L1 mouse embryonic fibroblasts and C2C12 myoblasts were cultured in Dulbecco's Modified Eagle's Medium (DMEM) including 10% FBS and 1% AA solution in 37°C, 5% CO2 incubator. The medium was replaced with fresh one every 48–72 hours. When the C2C12 myoblasts reached to 80% confluence, they were placed in differentiation medium containing 1% FBS and 1% AA and differentiated to myotubes for 72 hours. Buspirone of 100 μM, as a final concentration, was treated to the cells at differentiation stage for 48 hours and incubated for 24 hours. 3T3-L1 preadipocytes were cultured in DMEM containing 10% calf serum and 1% AA solution at 37°C in a 5% CO2 incubator. The medium was replaced every 48–72 hours. 3T3-L1 cells between 8 and 17 passages were plated at a density of 5 × 104 cells per well in 24-well culture dishes in DMEM containing 10% calf serum and 1% AA solution. When 3T3-L1 cells reached confluence, the existed medium was replaced with differentiation medium. The differentiation medium contained 0.0125 μmol/mL dexamethasone, 12.5 μmol/mL 3-isobutyl-1-methylxanthine, 10 μg/mL insulin, and 10% FBS. Buspirone of 50 μM and 100 μM, as a final concentration, was added to the differentiation medium. After differentiation for 2 days, the medium was replaced with insulin medium, which included 10 μg/mL insulin and 10% FBS. After incubation in insulin media for 2–4 days, the medium was replaced with maintenance medium, which contained only 10% FBS. Buspirone is added to the differentiation medium step.CThree-week-old male SHRs were bred in standardized conditions  with a regular day/night 10/14 hours) cycle and free access to water and a laboratory diet. After 1 week of acclimation, the assigned rats were divided into five groups: 4w , 8w , buspirone 1 mg/kg/day, buspirone 5 mg/kg/day, and buspirone 10 mg/kg/day. The occurrence of hypertension in SHRs has been known to begin at the 6 weeks age , 10. How/14 hoursBuspirone was administered to SHRs for 4 weeks starting at 4 weeks of age. Buspirone was administered to SHRs via drinking water in a 500 mL bottle, and the method to make drinking water containing buspirone is as follows: we measured mean body weight per each experimental group once a week during whole experimental period, and the weight of buspirone to be dissolved was calculated based on mean body weight per each group and average daily water intake amount per rat. An average consumption amount of drinking water per rat body weight 100 g has been known 10 mL/day . Thus, tThis animal experiment started at 4 weeks of age to elucidate the effects and the action mechanism of buspirone on the hypertension of early stage. Blood pressure was measured with tail cuff plethysmography every week using a BP-2000 Blood Pressure Analysis System™ in all SHRs .Animals in group 4w were euthanized at 4 weeks of age, whereas rats in the other groups were euthanized after 4 weeks of treatment at 8 weeks of age. Blood samples were acquired from the inferior vena cava, and skeletal muscle and visceral fat tissues were dissected properly and weighed. Harvested organs were stored in a −80°C refrigerator or in 10% (w/v) formalin for immersion fixation. All animal experiments were in accordance with the Animal Experiment Ethics Guide of Guro Hospital, Korea University. All animal experiments were accomplished according to the Korea University Animal Science Rules and Regulations, and the protocols were approved by the Korea University Institutional Animal Care and Use Committee .μg were loaded onto 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels. Protein blotting to nitrocellulose membranes was done for 90 minutes at 100 volts, and the membranes were blocked overnight in 5% skimmed milk solution and washed three times for 10 minutes with Tris-buffered saline containing 0.05% tween 20 (TBS-T). The membranes were incubated with primary antibodies at room temperature for 2 hours. Dilution conditions for primary antibodies were as follows: PPARδ, AMPK, p-AMPK (at Thr172), and PGC-1α were 1 : 1000. After the additional washing three times for 10 minutes, with TBS-T, the membranes were incubated with horseradish peroxidase conjugated secondary antibodies at room temperature for 1 hour. Dilution conditions for secondary antibodies were as follows: anti-rabbit IgG antibodies for PPARδ, AMPK, p-AMPK, and PGC-1α were 1 : 10,000. Afterward, the membranes were washed three times for 10 minutes with TBS-T and once with TBS for 10 minutes, and the membranes were treated with chemiluminescent substrate and enhancer solutions. Images were acquired manually using developer and fixer reagents, and the results were analyzed by ImageJ program.Cells and tissues were homogenized with protein extracting solution, and protein amounts of cell and tissue extracts were measured by the Bradford method. The extracted proteins of 10 μm thick) using microtome, and the slices were stained with H&E. They were further visualized using an optical microscope  and photographed.Visceral fat tissues, isolated from all SHRs, were harvested and fixed in 4% paraformaldehyde. The samples were embedded in paraffin and cut into slices  for 1 hour and washed with phosphate buffered saline (PBS). Secondary antibody was added to slides and reacted for 30 minutes. After washing them with PBS, the slides were reacted with premixed VECTASTAIN ABC reagent solution for 30 minutes. After PBS washing, the slides were reacted with DAB substrate solution until color appeared. The slides were washed by tab water, dried, and mounted. Observation and photograph acquisition were done by optical microscope .Aorta tissue slides were manufactured from frozen sections, and then the tissues on the slides were fixed with 4% paraformaldehyde solution for 20 minutes. The slides were treated with 0.3% HμL), 0.2 M sodium succinate (125 μL), 10 mg/mL NBT (25 μL), and distilled water (235 μL) per reaction, and samples were incubated for 20 minutes at a 37°C. Enzyme solution (90 μL) was added to the prewarmed incubation solution (410 μL), and the reaction mixture was incubated for 30 minutes at a 37°C. After the reaction is over, the absorbance of the reaction mixture was measured at 550 nm. Enzyme activity was calculated using the following formula:Succinate dehydrogenase (SDH) activity assay is a modified method of previously reported protocol . Skeletat-test, and to compare means of three or more groups was used one-way ANOVA test and followed by Bonferroni's multiple comparisons test. The p value of <0.05 was considered significant.Experimental results are expressed as mean ± SEM (standard error of mean). Statistically significant differences between two groups were calculated by the unpaired μM compared to the untreated differentiated cells  compared to the 8w untreated group Figures . In abdoThe systolic and diastolic blood pressure levels and the TG concentration were decreased by the buspirone treat of 5 and 10 mg (0.05 and 0.1 mg/mL) compared to the untreated 8w Figures . The diaμm2) were measured in abdominal fat tissue slides stained with H&E reagents, the adipocytes numbers in the 8w untreated group decreased to less than 50% of that in the 4w group. However, the adipocytes numbers increased in SHRs treated with buspirone 1, 5, and 10 mg/kg/day  than in the 8w untreated SHRs  compared to the 8w untreated group; however, there were no differences among buspirone 1, 5, and 10 mg/kg/day  treated groups  compared to the 8w untreated group; however, there were no differences among buspirone 1, 5, and 10 mg/kg/day  treated groups ; however, there were no differences among buspirone 1, 5, and 10 mg/kg/day  treated groups  . In partin vivo studies reported that blood pressure level is proportional to obesity or weight gain grade [Many clinical and in grade –15, morein grade –19.In addition, increased visceral fat mass induces an increase in levels of cholesterol and TGs , 20, andAlthough buspirone has been clinically prescribed to cure anxiety disorders, it lowered the blood pressure of healthy volunteers in clinical trial , also siδ, AMPK, and PGC-1α are involved in catabolic metabolism producing ATP.Generally, the elevation of energy consumption rate is an important factor in obesity and hypertension, and PPARδ is an essential modulator for lipid biosynthesis and oxidative phosphorylation; PPARδ gene transduction lowered fat accumulation in adipose and liver tissues and body weight of obese animals, and its activation by an agonist also decreased lipid contents in fat and liver and increased fatty acid oxidation in adipose tissue [δ [δ activation [δ is involved in the restoration of endothelial dysfunction, and vasodilation in animals fed a high-fat diet [δ functions as an upper regulator for AMPK in various cell types [PPARe tissue . In specissue [δ , also fitivation . Furtherfat diet . In addill types , 31.AMPK senses the energy state (ATP:ADP ratio) in cells and then maintains an energy balance via the modulation of ATP production and consumption—namely, AMPK functions as a key regulator for energy homeostasis . AMPK acα via phosphorylation [α is a coactivator that participates in various physiological processes, such as mitochondrial biogenesis, thermogenesis, and fatty acid metabolism, and is highly expressed in brown adipose tissue, heart, and skeletal muscle, wherein mitochondrial numbers and energy-producing catabolic metabolism are elevated [α affects lipid metabolism and metabolic diseases as follows. PGC-1α overexpression increases fatty acid oxidation in liver tissue and skeletal muscle cells; however, PGC-1α decreases the triacylglycerol content in hepatic tissue [α does an important role in an obesity-induced disease; thus, obesity downregulates PGC-1α expression, and the diminished PGC-1α level decreases the expression of mitochondrial genes and, finally, insulin resistance is induced [α overexpression ameliorates angiotensin II-induced hypertension via the regulation of eNOS [α and is an essential component in both the citric acid cycle and the electron transport system in mitochondria, and the elevations of the mitochondrial fraction and oxidative phosphorylation in skeletal muscle are intimately correlated with weight loss [δ, AMPK, and PGC-1α in skeletal muscle and abdominal fat tissues from SHRs and elevated the SDH activity in the skeletal muscle. Thus, we can suppose that buspirone activated an energy-producing catabolic procedure in abdominal fat and skeletal muscle tissues, and then the stimulated pathway induced the decrease of fat mass and body weight, and it might contribute to the amelioration of high blood pressure. Moreover, buspirone directly induced the normalization of hypertension through reducing the expressions for AT1R and VCAM1 related to atherosclerosis. The correlation among hypertension, AT1R, and VCAM1 has been reported as follows. Angiotensin II induces vasoconstriction after binding to AT1R, and vasoconstriction-induced hypertension provokes atherosclerosis, which causes cardiovascular diseases [In addition, the administration of metformin, an AMPK activator, reduced pulmonary artery pressure in a pulmonary hypertension animal model , and AMPrylation . PGC-1α elevated . So, PGCc tissue , 39. In  induced . Moreove of eNOS . SDH is ght loss , 43. In diseases , 45. VCAdiseases . Moreovediseases . So, theδ–AMPK–PGC-1α in hypertensive and obese animals (In conclusion, buspirone may ameliorate the early stage of dyslipidemia, obesity, and hypertension via the pathway of PPAR animals .N number in the C2C12 cell experiments shall be supplemented in a later study.In this study, because of feeding normal diet to SHRs, enough obesity did not induce. Thus, in a future study, SHRs should be fed high-fat diet to make more a proper hypertensive and obese rat model, and additional markers such as adiponectin and blood glucose concentration should be estimated. In addition, the more evident lowering effect of buspirone on hypertension shall be proved through a later study using old aged SHRs. Also, the small


Root system architecture (RSA) plays a pivotal role in efficient uptake of essential nutrients, such as phosphorous (P), nitrogen (N), and water. In soils with heterogeneous nutrient distribution, root plasticity can optimize acquisition and plant growth. Here, we present evidence that a constitutive RSA can confer benefits for sorghum grown under both sufficient and limiting growth conditions. Our studies, using P efficient SC103 and inefficient BTx635 sorghum cultivars, identified significant differences in root traits, with SC103 developing a larger root system with more and longer lateral roots, and enhanced shoot biomass, under both nutrient sufficient and deficient conditions. In addition to this constitutive attribute, under P deficiency, both cultivars exhibited an initial increase in lateral root development; however, SC103 still maintained the larger root biomass. Although N deficiency and drought stress inhibited both root and shoot growth, for both sorghum cultivars, SC103 again maintained the better performance. These findings reveal that SC103, a P efficient sorghum cultivar, also exhibited enhanced growth performance under N deficiency and drought. Our results provide evidence that this constitutive nature of RSA can provide an avenue for breeding nutrient- and drought-resilient crops.The online version contains supplementary material available at 10.1007/s42994-023-00112-w. It has been estimated that, by 2050, the global human population will reach 10 billion  genes were discovered. Equivalent PSTOL1 genes were earlier identified in rice, as P efficiency genes  cultivars, SC103 and BTx635, were used in this study. These cultivars represent widely employed sorghum breeding lines in the United States and Brazil  sodium hypochlorite for 20 min, rinsed with 18 Mega-ohm water (MΩ), and germinated, in darkness, for 4 days at 27 °C on moist germination paper . Uniform seedlings were transferred, depending on the specific experiment, to hydroponic solution, silica sand or Sunshine #1 potting mix  as the appropriate growth system for RSA studies. Plants were grown in a walk-in growth chamber with controlled climate conditions of 16 h/8 h (light/dark), 27 °C/22 °C (day/night) temperature, a light intensity of approx. 350 µmol m3)2, 1.3 NH4NO3, 0.58 K2SO4, 0.58 KCl, 0.56 KNO3, 0.85 MgSO4; and micronutrients (at μM levels): 2.5 H3BO3, 9.1 MnCl2, 0.6 CuSO4, 2.4 ZnSO4, 0.8 Na2 MoO4, 100 Fe-HEDTA. Nutrient solution phosphate (Pi) concentrations were 0, 2.5, 10 and 200 µM. Experiments were first conducted to establish the appropriate Pi concentrations for severe plant P deficiency (0 and 2.5 μM), moderate Pi deficiency (10 μM) and sufficient plant Pi status (200 μM): the quantitative indicators of P deficiency are a strong inhibition of shoot growth and a moderate stimulation of root growth, resulting in an increase in root:shoot ratio  and 400 µM as low N (LN) conditions. As N was added as Ca(NO3)2, to maintain the same Ca2+ concentration in the LN media, as used in the SN media, 900 µM CaCl2 was supplied in LN media. The nutrient solution was aerated continuously and renewed every 3 days. The solution pH value was maintained at 5.7.After germination, uniform sorghum seedlings were transplanted to 100 L polypropylene containers and grown hydroponically in a specially designed pouch system, as described by  containing silica sand and nutrient solutions of different [Pi] were added to the top of the pots. The plants were grown for 28 days, using the above-described nutrient solutions, except that the Pi concentration used for control (CK) and low Pi (LP) conditions were adjusted to 500 and 75 µM, respectively, and the N concentration used for LN media was reduced to 600 µM. For experiments conducted with plants grown in silica sand, the appropriate concentrations of N and P for sufficiency and deficiency were determined empirically, by measuring and comparing shoot biomass for plants grown on LN and SN nutrient solution. For plants grown in sand watered with LP and SP solutions, shoot growth and root traits were evaluated as described for P deficiency in hydroponically grown plants in 2D pouches. Given that nutrient availability in silica sand is restricted to the pores within the sand, the nutrient concentrations in the sand experiments were adjusted to ensure sufficient or deficient levels. For LP, a concentration of 75 μM was determined to be moderately P deficient, whereas moderate P deficiency symptoms developed in plants grown in hydroponic media with 10 μM Pi. Regarding LN, 600 μM N generated moderately N deficient sand-grown plants, whereas 400 μM N in hydroponic media resulted in moderate N deficiency symptoms.To evaluate drought performance of the sorghum cultivars, plants were grown in plastic pots, as above, containing 1.2 kg Sunshine #1 potting mix. Plants were fertilized biweekly using water-soluble fertilizer , with a standard N–P–K (20–20–20) treatment. Pot water content was measured using an HydroSense II Handheld Soil Moisture Sensor , based on the volumetric water content (VWC) for porous media. Pots were initially filled with 18 MΩ water and then allowed to drain to establish soil ‘field capacity’. Seedlings were then transplanted, with one seedling per pot, and 200 mL of 18 mΩ water (corresponding to near-full field capacity) was added, daily, for the first 14 days.After the initial two-week growth period, plants were divided into two treatment groups: well-watered (WW) and water stressed (WS). The WW plants continued to receive 200 mL of 18 MΩ water, daily, per pot, to maintain the water content near the full field capacity. For the WS plants water was withheld, and fourteen days later leaf wilting was observed, coincident with a measured potting mix water content of 7.5% VWC. Plants were subsequently removed from the pots and their root systems were soaked in water to remove potting mix, and then thoroughly cleaned by washing using low water pressure. Finally, RSA measurements were determined , were assessed. Root morphology traits, derived from the hydroponic pouch system included average root diameter (cm), total root system surface area (cm2), total root system length (cm), and total root system volume (cm3). Root morphology traits, derived from experiments in which plants were grown in silica sand or potting mix, included total root system length (cm), root system surface area (cm2), and total root system volume (cm3), were assessed.Phenotyping experiments, using a hydroponic pouch system, utilized a Nikon D7200 DSLR camera with a 50 mm lens and a 2D root imaging platform. Raw images were collected and stored in a Plant Root Imaging and Data Acquisition (PRIDA) program, and then extracted as TIFF files for further image processing and root trait computation , root dry weight (RDW), and the root:shoot ratio (R/S). After weight measurements, the root and shoot samples were ground and digested, using a solution of concentrated sulfuric acid and selenium at 330 °C. Total P and N concentrations were then determined, using a Skalar SAN-plus segmented flow analyzer , following the manufacturer’s protocols.https://imagej.nih.gov/ij/). Proportion of root cross sections occupied by aerenchyma was calculated using AA divided by SCA. Sample data were normalized, based on a previously described method  or low Pi  conditions. Growth protocols were as described above. Primary and crown roots were excised at 5 cm from the root-stem junction and at 5 cm from the root tip, respectively. Lateral roots were excised at 5 cm from the primary-lateral root junction and 5 cm from the lateral root tip. All root samples collected were ~ 5 cm in length. Root samples were placed into 3D-printed polylactic acid molds for embedding (Atkinson and Wells t test or Tukey’s honest significant difference (HSD) tests, where appropriate. ANOVA was performed using the “Anova” function, as implemented in the “car” package . This nutrient delivery system was used to provide Pi distribution to the root systems of these sorghum cultivars. Both cultivars exhibited significantly larger root systems in response to Pi deficiency Fig. A. An incTraits associated with RSA, including primary root length, total root system length, total root system surface area, root system width Fig. , and aveAs demonstrated by the data presented in Fig. P < 0.001) and P content (P < 0.001) in SC103 seeds, compared with BTx635 . This observed increase in plant P content, in SC103, is a reflection of its larger root system. Based on these findings, the lack of a difference in shoot and root P concentration between both cultivars, and the much larger P content in SC103, is consistent with P acquisition being a major contributor to enhanced P efficiency in SC103.Under sufficient Pi conditions (200 µM), SC103 exhibited a significant enhancement in P uptake (72% more shoot Pi content) than BTx635. As expected, Pi stress reduced uptake in both cultivars; however, P uptake was still significantly higher in SC103. Here, SC103 accumulated 50%, 64% and 43% more P in their shoots, compared with BTx635, under the three imposed Pi stress conditions, respectively  and P acquisition efficiency (PAE), PAE accounts for the majority of the genetic contribution to P efficiency. Specifically, we determined in our study that PAE contributes 82% to the overall PAE, whereas PUE accounts for 18% , total root cortical aerenchyma area (AA) and the proportion of root cross sectional area occupied by aerenchyma (AA/CSA), were evaluated Figs. A, E. HowA similar pattern was also observed for lateral root anatomy, near both the stem-root junction and at least 5 cm back from the lateral root tip. Again, SC103 had the larger AA and AA/CSA ratio, compared with BTx635, with these differences being enhanced under LP conditions  and low N (LN) conditions , and the test group was subjected to water stress (WS), by withholding water Fig. A. As migOsMYB4P, an R2R3-type MYB transcriptional activator, in rice seedlings increased seminal and lateral root length and density, which enhanced phosphate acquisition  versus induced genetic component of the SC103 RSA traits could facilitate trait engineering into target crops due to a reduced impact by genome × environment interactions. In this manner, crop improvement be would available across a wider range of soil types and agroecologies. Lastly, as SC103 is more efficient in terms of P and N acquisition and drought resilience, this could allow for the efficient breeding for all three traits from one genetic source.The grain yield differential, between SC103 and BTx635, established under low Pi field conditions, was clearly reflected in the measured RSA and growth characteristics, determined under LP, LN, and WS conditions Figs. , 7, 8, 9Supplementary file1Supplementary Fig. 1 Analysis of seed P and N in SC103 and BTx635. (A) Seed P concentration, (B) seed N concentration, (C) seed P content, and (D) seed N content. Supplementary Fig. 2 Crown root anatomy of sorghum cultivars SC103 and BTx635 grown for 10 days in hydroponics under sufficient Pi  and low Pi  conditions. Representative transverse images of crown root cross sections, collected 5 cm below the stem-root junction (A), and ~5 cm from the crown root tip (E) . Histograms represent root cross-sectional area, total root cortical aerenchyma area (AA), and proportion of root cross section occupied by aerenchyma, near the stem-root junction  and ~5 cm from the root tip  of the SC103 (green) and BTx635 (orange) crown root, respectively. Data shown are means ± SE (n=6). Asterisks indicate significant differences between the cultivars, under the same condition, as determined by Student’s t-test: For these assays, significance differences are indicated as follows: * P < 0.05; ** P < 0.01; *** P < 0.001. Different lowercase letters indicate significant differences (P < 0.05) between the two Pi concentrations, in the same genotype, as determined by Tukey’s HSD tests. CSA, cross section area (mm2); AA, total root cortical aerenchyma area (mm2). Scale bar applies to all images. Supplementary Fig. 3 SC103 and BTx635 RSA and growth data used to develop the radar plots in Fig. 8 B-D. Histograms represent shoot dry weight (A), root dry weight (B), root:shoot ratio (C), total root system length (D), total root system surface area (E), root system volume (F), shoot N content (G), shoot P content (H), root N content (I), root P content (J). Data were collected from plants grown in pots with silica sand as substrate under control (CK), low Pi , or low N . Data shown are means ± SE (n=6). Asterisks indicate significant differences between the cultivars, under the same condition, as determined by Student’s t-test: For these assays, significance differences are indicated as follows: * P < 0.05; ** P < 0.01; *** P < 0.001. Different lowercase letters indicate significant differences (P < 0.05) between the two Pi concentrations, in the same genotype, as determined by Tukey’s HSD tests (PPTX 17516 KB)Supplementary file2 (DOCX 25 KB)Below is the link to the electronic supplementary material.


Temperature affects seed germination and seedling growth, which is a critical and complex stage in plant life cycle. However, comprehensive metabolic basis on temperature implicating seed germination and seedling growth remains less known. Here, we applied the high-throughput untargeted metabolomic and advanced shotgun lipidomic approaches to profile the Arabidopsis 182 metabolites and 149 lipids under moderate  and extreme high  temperatures. Our results showed that a typical feature of the metabolism related to organic acids/derivates and amines was obviously enriched at the moderate temperature, which was implicated in many cellular responses towards tricarboxylic acid cycle (TCA), carbohydrates and amino acids metabolism, peptide biosynthesis, phenylpropanoid biosynthesis and indole 3-acetate (IAA) biosynthetic pathway. Whereas, under extreme high temperatures, there was no seed germination, but 148 out of total 182 metabolites were highly enriched, involving in the galactose metabolism, fatty acid degradation, tryptophan/phenylalanine metabolism, and shikimic acid-mediated pathways especially including alkaloids metabolism and glucosinolate/flavone/flavonol biosynthesis. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) also exhibited the gradually increased tendency from moderate temperatures to extreme high temperatures; whereas phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylglycerol (PG), monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG) and sulfoquinovosyldiacylglycerol (SQDG) were contrary to decrease. Another typical feature of the distinguished metabolites between 22°C and 28°C, the TCA, disaccharides, nucleotides, polypeptides, SQDG and the biosynthesis of fatty acids and glucobrassicin-mediated IAA were obviously decreased at 28°C, while amino acids, trisaccharides, PE, PC, PA, PS, MGDG, DGDG and diacylglycerol (DAG) preferred to enrich at 28°C, which characterized the alteration of metabolites and lipids during fast seedling growth. Taking together, our results provided the comprehensive metabolites phenotyping, revealed the characteristics of metabolites necessary for seed germination and/or seedling growth under different temperatures, and provided insights into the different metabolic regulation of metabolites and lipid homeostasis for seed germination and seedling growth. As the Lipids constitute membrane structure and also mediate multiple cellular signaling transduction. In seed germination and seedling growth, PA showed the lowest levels in imbibition and followed an increasing trend during seedling growth, while PE molecules showed the highest levels in imbibition and a general decreasing trend at later stages . Heat stMetabolomic and lipidomic analysis have become a powerful technology to comprehensively clarify plant dynamic development , plant rIn this study, we investigated the comprehensive metabolic and lipidomic profiles during seed germination and seedling growth through the systematic biology approaches of both high-throughput untargeted metabolomics and advanced ESI-based lipidomics. We demonstrated the characteristics of metabolites and lipids in response to moderate (22°C and 28°C) and extreme high (34°C and 40°C) temperatures during the both stages of seedling establishment, and the distinguished characteristics between 22°C and 28°C during seedling growth. Our findings provided a molecular characteristic phenotype for seedling establishment under heat stress, revealed the metabolic pathways of TCA, carbohydrates metabolism, peptide biosynthesis, and lipid homeostasis were crucially implicated in seed germination and seedling growth under heat stress, providing cues to further study functional mechanism of these cellular metabolic pathways.22.1Arabidopsis thaliana ecotype Columbia (Col) were surface-sterilized and kept in the dark at 4°C for 3 days. The seeds were then sown onto filter papers with pre-soaked sterile water, and placed at 22 °C under light for 3 hours before transferring to the dark chamber for another 21 hours at 22°C, 28°C, 34°C and 40°C, respectively.Seed germination was carried out according to the previous study . Briefly2.2Total metabolites and lipids from 50 mg samples with whole tissue were extracted with methyl tert-butyl ether (MTBE) and methanol (MeOH). Briefly, 50 mg plant materials were mixed with 225 μL of ice-cold MeOH and homogenized for three times. The homogenate was then sonicated in an ice bath for 10 min. Added 750 μL pre-cooled MTBE and vortexed the mixture for 30 s and then kept on ice for 1 hour. The samples were vortexed for 30 s after adding 188 μL deionized water, and centrifuged at 14000 g for 10 min at 4°C. All experimental steps were performed on ice. The upper organic phase (800 μL) was carefully transferred to new tubes. The lower phase (200 μL) was transferred to additional new tubes. Repeat the above extraction steps twice. Finally, the three extractions were combined and dried with nitrogen gas.The upper phase dried extract was dissolved in chloroform: methanol: 300 mmol/L ammonium acetate  for lipids analysis. The lower phase dried extract was dissolved in methanol: water (1:1) for metabolites analysis.2.34AC) and 0.02% formic acid, and phase B was acetonitrile: water  with 5 mM NH4AC and 0.02% formic acid. The flow rate was 0.3 mL/min. The ion source parameters were set as follows: scan range, 50-1200 Da; source temperature, 300°C; curtain gas, 35; collision gas, medium; two ion source gases, 60; ion spray voltage, 5500/-4500V; DP, 60; CE, 35 ± 15.The Shimadzu Nexera UHPLC system equipped with the 6600 QTOF mass spectrometer (AB Sciex) was applied for untargeted metabolomics analysis. 1 uL aliquots of sample was injected into a Waters Acquity UPLC T3 column  maintained at 45°C by gradient elution. Mobile phase A was deionized water with 5 mM ammonium acetate , identified according to the database, and some of them were confirmed by ZenoTOF 7600 (AB Sciex). The relative amount was comparatively quantified according to the peak intensity. The data of shotgun lipidomics was processed by LipidView Software (AB Sciex) for identification and quantification.The raw data of metabolomics was preprocessed by MetDNA2 , three subclasses of galactosyldiacylglycerides  and 2 ceramides  were identified  was applied to analyze the metabolites which drove the group separation. PC1 and PC2 captured 73.6% and 10.9% of the total variance in the metabolites of FW-normalized dataset, respectively  and ethylene. All the precursor and intermediates for auxin biosynthesis were detected, which showed that the precursor (tryptophan) and intermediates of the two bypasses including tryptamine, indole-3-acetonitrile, indole-3-pyruvate and indole-3-acetyl-beta-1-D-glucoside to form IAA were enriched at 34°C and 40°C . In our 3.5Phospholipids function not only as membrane structure but also as messengers to play vital roles for plant development. The volcano plot was used to analyze the significant differences between 22~28°C and 34~40°C, and 104 lipid species were obviously altered among 149 lipid species identified  and PGs species , and all the species of PSs  and PAs  tended to have high levels at moderate temperatures); whereas, other species of PEs and most PCs species tended to have high levels at extreme high temperature 4A, 5AThe degree of lipid unsaturation was closely related to membrane lipid fluidity, and might affect the physiological efficiency of membrane. The acyl fatty acid (FA) chain length and unsaturation of phospholipids were analyzed in response to moderate and extreme high temperatures, respectively. All the PC species with long FA chain and more unsaturated degree were accumulated at high temperatures; whereas, the high saturated PC species with short FA chain length (PC 32:0 and PC 32:1) tended to be abundant at extreme high temperature 5B. Thes3.6DAG plays important roles in lipid metabolism, which could generate TAG and were also as substrate to biosynthesize both the phospholipids intermediates PA and galactolipid MDGD. DAG was enriched at 28°C, compared with other temperatures, but DAG did not show significant difference between moderate and extreme high temperatures . In thisPG is essential component of the chloroplast membranes and also as substrate to biosynthesize galactolipids, which showed the similar altered tendency to galactolipids .3.7Based on the distinguished profiles of metabolome and lipidome during seed germination and seedling growth at moderate and extreme high temperatures, we further analyzed the metabolomic and lipidomic differences between the moderate temperatures of 22°C and 28°C. A total of 59 metabolites and 50 lipids were significantly altered between 22°C and 28°C, which included 23 amino acids, 10 sugars/derives, 9 organic acids, 5 CoA, 2 purine/derives, 2 nicotine/derives, 1 indole, 1 phenol, 13 PEs, 10 PCs, 7 PAs, 5 MGDGs, 4 DGDGs, 4 DAGs, 3 PGs, 2 PSs, 1 SQDG and 1 ceramide , TCA , phosphoenolpyruvate pool , FAs metabolism , nucleotide metabolism , bioactive cyclin dipeptides kyotorphin (l-tyrosyl-l-arginine) and tripeptides  , was reported to completely improve seed germination . EthylenOur study demonstrated the metabolic alterations of seed germination under heat stress, including sugars, amino acids, plant hormones and lipids Figure 7Bangia fuscopurpurea) sublethal exposure to high-temperature stress altered membrane fluidity via changes in fatty acid composition, which resulted in the acquisition of heat stress memory and better survival  metabolites and specific lipids were highly enriched at extreme high temperatures , which involved in the galactose metabolism, fatty acid degradation, tryptophan/phenylalanine metabolism, and shikimic acid-mediated pathways  and phospholipids metabolism . Whereas, TCA, carbohydrates and amino acids metabolism, peptide biosynthesis, phenylpropanoid biosynthesis, IAA biosynthetic pathway and lipids  were featured with high levels at 22°C and 28°C, especially the metabolites of amino acids, trisaccharides, PE, PC, PA, PS, MGDG, DGDG and DAG preferred to enrich at 28°C. In addition, the TCA molecules, disaccharides, nucleotides, polypeptides, biosynthesis of fatty acids, glucobrassicin-mediated IAA and SQDG were obviously high at 22°C. Our results characterized the distinguished metabolites and related regulatory metabolic pathways and the tendency of cellular metabolism pathways in response to different temperature conditions from 22°C to 34°C during seed germination and seedling growth. Our comprehensive metabolome and lipidome especially indicated that 1) phytohormones, such as the bypass of auxin biosynthesis might be activated under different temperatures, and SA might be implicated in the heat stress response; 2) most lipids tended to accumulate under moderate temperatures (22°C and 28°C), whereas the severe heat (34°C and 40°C) contrarily resulted in the decrease of most lipids; 3) most metabolites were induced to be highly enriched for resistance against severe heat stress (34°C and 40°C); 4) organic acids/derivates and amines implicating in cellular TCA, carbohydrates and amino acids metabolism, peptide biosynthesis, phenylpropanoid biosynthesis and IAA biosynthetic pathway were obviously enriched at the moderate temperature (22°C and 28°C). Heat stress is an increasing risk for agricultural crops due to global warming. Our results are crucial to study the comprehensive metabolites and lipids phenotyping, providing the cues to functional innovation with heat tolerance.The original contributions presented in the study are included in the article/WQ and HY carried out the assays and data analysis of metabolites and lipids. YZ and HY performed the preparation of plants materials. QC and TL extracted peaks and confirmed the structural data. SW and LC preformed the standard rectification of UPLC-MS/MS. HT guided for the data analysis. HY, WQ, YZ and HT wrote the manuscript. All the authors reviewed the manuscript. HY and HT were responsible for contact and communication. All authors contributed to the article and approved the submitted version.