We found that physiological cerebrospinal concentrations of HCy (0.5 µM) induce an increase in synaptic proteins, whereas higher doses of HCy (30–100 µM) decrease their levels, thereby increasing oxidative stress and causing excitatory transmission hyperactivity, which are all considered to be neurotoxic effects. We also observed that normal cerebrospinal concentrations of HCy slow the aggregation kinetic of Aβ40, whereas high concentrations accelerate its aggregation. Finally, we studied the effects of HCy and HCy + Aβ42 over long-term potentiation. Altogether, by studying an ample range of effects under different HCy concentrations, we report, for the first time, that HCy can exert beneficial or toxic effects over neurons, evidencing a hormetic-like effect. Therefore, we further encourage the use of HCy as a biomarker and modifiable risk factor with therapeutic use against AD and other types of dementia.Alzheimer’s Disease (AD) is the primary cause of dementia among the elderly population. Elevated plasma levels of homocysteine (HCy), an amino acid derived from methionine metabolism, are considered a risk factor and biomarker of AD and other types of dementia. An increase in HCy is mostly a consequence of high methionine and/or low vitamin B intake in the diet. Here, we studied the effects of physiological and pathophysiological HCy concentrations on oxidative stress, synaptic protein levels, and synaptic activity in mice hippocampal slices. We also studied the in vitro effects of HCy on the aggregation kinetics of Aβ Homocysteine (HCy) is a sulfur-containing amino acid and a byproduct of methyl-transfer reactions due to methionine metabolism . Levels HHCy is a risk factor for vascular and neurodegenerative diseases ,7,8,9 anIn 2015, we published a novel study demonstrating, for the first time, that an L-methionine rich diet, a known protocol to increase HCy levels . Several1-40 (Aβ40) aggregation kinetics. Our results show that: (1) High HCy concentrations cause an increase in oxidative stress, (2) HCy induces differential changes in the levels of pre- and post-synaptic proteins, (3) high HCy concentrations induce a toxic overactivation of excitatory transmission in the CA1 hippocampal region, and (4) low HCy concentrations cause slower Aβ40 aggregation, whereas elevated HCy increases the aggregation kinetics. Overall, these results indicate that HCy shows an adaptative response characterized by a biphasic dose response, which is consistent with a hormetic-like effect [We performed studies to determine the effects of different HCy concentrations on oxidative stress, synaptic protein levels, and synaptic activity in hippocampal slices of two-month-old wild-type mice, as well as in vitro Aβe effect . Here, we effect . Our finIt has been widely reported that HCy increases protein oxidation in the cerebral cortex and hippocampus  in both in vitro and in vivo studies ,6,20. WeOxidative stress can affect proteins and their cellular structures. For this reason, as a first approach to study the effects of homocysteine on the hippocampal synapses, we evaluated synaptic protein levels. Hippocampal slices from two-month-old wild-type mice were treated with increasing concentrations of HCy, and the protein levels were detected by immunoblot. As pre-synaptic markers, the levels of synaptotagmin 1 and synaptotagmin 2 (SYT1 and SYT2), Synaptophysin (SYP), and phospho-Synapsin (pS553-SYN) were evaluated A,B. All To analyze the post-synaptic compartment, the levels of the AMPA receptor subunit, GluA2, as well as the NMDA receptor subunits, GluN2A and GluN2B, and the postsynaptic density protein, PSD-95, were measured C,D. It hTo determine if changes in the synaptic structure result in functional alterations, we evaluated the electrophysiological response to different HCy concentrations. To do so, we recorded field excitatory post-synaptic potentials (fEPSP) over synaptic transmission in the CA3–CA1 circuitry in acute hippocampal slices. After 20 min of basal stable recording with aCSF, the perfusion was changed by different concentrations of HCy for 20 min (continuous line), after which the perfusion was changed back to aCSF (dotted line) to washout HCy A. The la40 peptide at room temperature under constant stirring. To correlate the kinetic curve with specific types of aggregates, we used the Aβ40 peptide, which has been shown to present a more defined lag-phase, allowing us to recognize, through the kinetics, which Aβ species are present at the determined time points [40 aggregation alone or co-incubated with HCy for 0, 3, 4, and 6 h. At the initial point of the aggregation course (0 h), Aβ40 alone was not aggregated and remained as monomers, which gradually formed oligomers (3 h), individual fibrils (4 h), and large aggregates (6 h), as expected [40. For Aβ40 + 100 µM HCy, at 3 h, we observed the fast formation of aggregates, such as protofibrils and small fibrils, unlike Aβ40 alone. At 4 h of incubation, there was a dramatic increment in the presence of fibrils in comparison to those observed at the same time point for 0.5 µM HCy or Aβ40 alone. At 6 h, the Aβ40 + 100 µM HCy formed huge and dense aggregates. Most importantly, for the Aβ40 + 30 µM HCy condition, at the same time point, we observed small fibers and also oligomers, suggesting a disaggregation process. This could be very interesting and deserves to be further studied. The lowest HCy concentration clearly showed slower aggregation kinetics compared to every other condition, even when compared with Aβ40 aggregation alone. Moreover, the sizes of the Aβ40 fibrils formed in the presence of 0.5 µM HCy at 6 h were clearly smaller than those observed with Aβ40 alone, suggesting that this concentration of HCy delays Aβ40 aggregation. In 40 alone continuously aggregates. By comparison, the aggregation kinetics are faster under the Aβ40 + 100 µM HCy condition and slower under the Aβ40 + 0.5 µM HCy condition, as shown in 40 + 30 µM HCy kinetics present an intermediate rate of aggregation between the previous two conditions, similar to the aggregation kinetics observed for Aβ40 alone. This indicates that higher concentrations of HCy accelerate the aggregation process of Aβ40, forming larger aggregates. On the contrary, low concentrations of HCy seem to slow the aggregation kinetics, favoring the presence of smaller aggregates. This is in accordance with the conclusions of 40 peptide aggregation, depending on the HCy concentration used. In this way, an increase in HCy levels, like the one seen in HHCy, would favor the formation of high molecular weight aggregates , whereas low concentrations favor the formation of low molecular weight aggregates . The present research is the first in vitro study on the direct effect of different HCy concentrations over Aβ40 aggregation. These groundbreaking results will help us understand the toxic mechanisms of HCy in amyloidogenic diseases.It has been reported that HHCy can cause an increase in Aβ load and cognitive deficits in vivo ,44,45,46e points ,48. The e points . The aggexpected . Interes42 oligomers completely abolish LTP [42 oligomers on LTP. We observed that Aβ42 generates an 8.3% ± 0.01% decrease of fEPSP compared to the baseline. Remarkably, when 1 μM Aβ42 oligomers plus 100 μM HCy are used, LTP is generated, with an intermediate effect of 168.7% ± 0.01% potentiation over the baseline—a similar value to the one observed under the control condition (181.9% ± 0.02%)  of hippocampal slices subjected to theta burst stimulation (TBS). Similar to the effects under paired pulse stimulation A,B, 100 lish LTP ,52. Ther± 0.02%) A,B. Ther40 aggregation. On the other hand, high HCy concentrations increase oxidative stress, decrease synaptic protein levels, and promote faster Aβ40 aggregation, among others. This non-linear dose-response behavior is called a hormetic effect. Hormetic-like effects of HCy were first described by Lipton et al. in 1997 [In this study, we showed that HCy can exert protective or toxic effects depending on its concentration. We report that HCy at lower concentrations increases ATP production and synaptic protein levels while slowing Aβ in 1997 . Since t in 1997 ,55,56,57 in 1997 , a diffe in 1997 . Differe in 1997 . This reRecently, high HCy levels were classified as a modifiable risk factor and a biomarker of AD and other types of dementia ,59,60,61We detected increased n-Tyr levels with 30 and 100 µM HCy, as well as increased ROS content in hippocampal slices . IntriguAn increase in Nrf2 levels, an antioxidant response protein, corroborates that 1 h exposure to high HCy concentrations (30–100 µM) is enough to induce an increase in oxidative stress. Since we are examining the response in whole hippocampal slices with diverse cell types, we cannot pinpoint which cell type is responsible for the elevated Nrf2 levels. Nonetheless, it has been described that retinal Müller glial cells, and not retinal ganglion cells, are the cells responsive to oxidative stress, with an increase in Nrf2 levels . TherefoWe found that exposure to 0.5 μM HCy generates an increase in the levels of PSD95 and its subunits GluN2A and GluA2. Higher concentrations of HCy seem to cause a decrease in the relative levels of GluN2A and the pre-synaptic proteins SYT1 and SYP, and similar effects were noted by Chai et al. . HCy hasIn hippocampal slices of wild-type mice, we observed that high levels of HCy increase the fEPSP. A concentration-dependent effect is induced by HCy, in which lower concentrations  have a gradient effect on fEPSP. There is an important body of evidence that demonstrates HCy to cause an increase in NMDA currents, which could lead to excitotoxicity ,24,65,6642 is the most neurotoxic species of Aβ peptide found in patients with AD [o, it is often preferable to use the Aβ40 peptide [42 has much faster aggregation kinetics than Aβ40 [40 also has a longer lag phase that allows for the clear identification of monomeric, oligomeric, and fibrillar forms, which would not be clearly identifiable when using Aβ42 [40 rather than Aβ42 , co-incubated with the same amount of Aβ40 oligomers, clearly produced larger aggregates , which avoids the HCy-induced excitotoxicity caused by high HCy concentrations. This would explain why we do not observe the depression caused by Aβ42 alone nor the glutamatergic overactivation  caused by HCy alone.On the another hand, it has been established that Aβ species ,78. Therl levels , unlike for Aβ40 A. Functi40 aggregation, whereas high concentrations of HCy (1) cause an increase in oxidative stress, (2) reduce the levels of pre-synaptic and post-synaptic proteins, (3) increase fEPSP over the basal condition and LTP (likely related to NMDAR overactivity), and (4) favor Aβ40 aggregation, a condition known to be toxic. Altogether, our results show the hormetic-like effects of HCy over neuronal structure and function, which are perceived to have beneficial properties at low concentrations and toxic effects at high concentrations. From this data, we cannot determine the exact threshold concentration at which HCy stops being beneficial and begins to be toxic. Thus, a more detailed analysis should be performed.Altogether, we have established that low concentrations of HCy (1) increase ATP production, (2) increase synaptic protein levels, and (3) slow AβMonitoring HCy levels and treating HHCy should become the gold standard procedures for the elderly population. This would not only make it easier to detect at-risk populations but would also allow medical professionals to create personalized therapies according to the needs of each patient. This treatment, an increase in the intake of B family vitamins, would also provide an easy and affordable way to tackle dementia .The primary antibodies used were anti-3-nitrotyrosine , goat anti-4HNE , mouse anti-Nrf2 (A-10) , mouse anti-PGC1α , mouse anti-Syt1/2 (H-9) , mouse anti-Syp , goat anti-pSyn Ia/b , mouse monoclonal anti-PSD95 , mouse anti-GluN2A , mouse anti-GluN2B , mouse anti-GluA2 , goat anti-synaptophysin , and rabbit polyclonal anti-Arc . Anti-Actin  or GAPDH  were used as the loading control.1-40 and Aβ1-42 peptides, corresponding to human sequences, were obtained from Genemed Synthesis, Inc. . Aβ peptide stock solutions were prepared by dissolving freeze-dried aliquots of Aβ in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at 1 mM. Immediately before use, the peptide film was dissolved in dimethyl sulfoxide (DMSO) at 5 mM and was then diluted in PBS to a final concentration of 100 μM [Synthetic Aβf 100 μM . Thiofla40, 0.5% sodium deoxycholate, and 1% SDS) supplemented with a protease inhibitor cocktail (Sigma-Aldrich P8340) and phosphatase inhibitors  using a Potter homogenizer and then passed sequentially through different caliber syringes. Protein samples were centrifuged at 14,000 rpm at 4 °C twice for 15 min. Protein concentrations were determined using a BCA Protein Assay Kit . Twenty micrograms of samples were resolved by 10% SDS-PAGE and transferred to a PVDF membrane. These reactions were followed by incubation with anti-mouse, anti-goat, or anti-rabbit IgG peroxidase-conjugated antibodies  developed using an ECL kit .Brain slices treated with HCy or control were dissected on ice and immediately frozen at −150 °C or processed, as detailed previously. Briefly, the slices were homogenized in a RIPA buffer  Triton X-100, pH = 7.4) using a luciferin/luciferase bioluminescence assay kit  ,82,83. TROS content was measured using the fluorescent dye CM-H2DCFDA. Briefly, hippocampal samples diluted in Triton Buffer were added to a black 96-well plate in duplicate followed by the addition of 25 μM DCF. Then, the plate was incubated for 5 min and examined in a BioTek Synergy HT.1-40 peptide at the indicated concentrations were diluted in PBS pH 7.4 and incubated at different times under constant stirring (1200 rpm) at room temperature. Aβ1-40 was used to measure the kinetics since it has a slower aggregation curve, which allows for the identification of different aggregation states. For co-incubation experiments, homocysteine (HCy) was used at 0.5, 30, and 100 μM. To quantify the amyloid formation, the Th-T fluorescence method was used [1-40 alone or in the presence of HCy, the samples were taken every hour and diluted in 50 mM sodium phosphate buffer pH 6.0 and 0.1 mM Th-T. The fluorescence was monitored at an excitation of 450 nm and an emission of 485 nm using a JASCO spectrofluorometer. The binding of Th-T to amyloids produces a shift in its emission spectrum and an increase in the fluorescence signal, which is proportional to the number of amyloid fibrils formed.Aliquots of Aβwas used . Briefly42 peptides corresponding to wild-type human Aβ were obtained from Genemed Synthesis, Inc. . An Aβ peptide stock solution was prepared by dissolving freeze-dried aliquots of Aβ in 1,1,1,3,3,3-hexafluoro-2-propanol  at 1 mM. For oligomer preparation, the peptide film was dissolved in dimethyl sulfoxide  at 5 mM and was then diluted in PBS to a final concentration of 100 μM. This preparation was incubated overnight to allow the formation of Aβ oligomers and then centrifuged  to eliminate any formed fibril [Synthetic Aβd fibril .3, 10 D-glucose, 2.69 KCl, 1.25 KH2PO4 2.5 CaCl2, 1.3 MgSO4, y 2.60 NaHPO4) using a vibratome  and incubated in ACSF for 1 h at room temperature. From dissection onwards, the slices were constantly maintained under 5% CO2/95% O2 conditions. In all experiments, picrotoxin  was added to the ACSF perfusion media to suppress inhibitory GABAA transmission. Slices were then transferred to an experimental chamber (2 mL), superfused  with gassed ACSF, and visualized by trans-illumination with a binocular stereomicroscope . The experiments were carried out at room temperature (21–22 °C), measured at the recording chamber. To evoke field excitatory postsynaptic potentials (fEPSPs), SC fibers were activated by bipolar cathodic stimulation generated by a stimulator electrode  and connected to an isolation unit  [Hippocampal slices were prepared according to standard procedures, from 2-month-old male mice. Transverse slices (350 µm) from the dorsal hippocampus were cut under freshly prepared cold artificial cerebrospinal fluid  . Bipolar Israel) . To geneElectron microscopy was performed as previously described . Fresh aData analysis was performed using the Prism 8 software (GraphPad Software Inc.) All results are expressed as the mean ± standard error. Data were tested for normality using Shapiro–Wilkinson and Kolmogorov–Smirnov tests. For statistical analysis, normally distributed data were analyzed by a one-way ANOVA with post hoc tests performed using the Bonferroni test. Non-normally distributed data were analyzed by a Kruskal–Wallis test with post hoc tests performed using Dunn’s test.


This study investigated a new category of transparent encapsulant materials for light-emitting diodes (LEDs). It comprised a phenyl group that contained siloxane-modified epoxy (SEP-Ph) hybridized with a cyclic tetrafunctional siloxane-modified epoxy (SEP-D4) with methylhexahydrophthalic anhydride (MHHPA) as a curing agent. The SEP-Ph/SEP-D4 = 0.5/0.5 (sample 3) and SEP-D4 (sample 4) could provide notably high optical transmittance (over 90% in the visible region), high-temperature discoloration resistance, low stress, and more crucially, noteworthy sulfurization resistance. The lumen flux retention of the SEP encapsulated surface mounted device LEDs remained between approximately 97% and 99% after a sulfurization test for 240 h. The obtained comprehensive optical, mechanical, and sulfurization resistance proved the validity and uniqueness of the present design concept with complementary physical and chemical characteristics. Light-emitting diodes (LEDs) have become an essential light source because of their compact size, high efficiency, high brightness, low power consumption, and long operating lifetime. The performance of LEDs is greatly affected by the properties of their encapsulation materials, such as high optical transmittance, thermal- and photostability, low stress, and gas permeation resistance ,3,4,5,6.Transparent epoxy resins cured with anhydrides possess good optical clarity, strong adhesion, and high mechanical strength; however, their poor thermal- and photostability, as well as high thermal stress result in discoloration of and catastrophic damage to LED devices, which restricts their application. By contrast, silicone resins have excellent thermal- and photostability, and are thus usually applied in mid- to high-power LED packaging; however, their weak adhesion and high gas permeability, mainly originating from their highly flexible siloxane backbone, which allows for gas diffusion ,11, are 3) and diphenylsilanediol (Ph2Si(OH)2), enhanced ultraviolet stability was achieved compared with an anhydride-cured cycloaliphatic epoxy -cyclotetrasiloxane, 1H NMR: 0.04 ppm , 0.48 ppm , 0.17–2.18 ppm , 3.12 ppm , and EEW = 184.The synthesis of 1,3,5,7-tetramethyl-1,3,5,7-tetra[ethyl]-cyclotetrasiloxane (SEP-D4) was conducted according to the hydrosilylation reaction ,25. Sche1H-NMR analysis was performed on a NMR spectrometer  with CDCl3 as the solvent and TMS as a reference. Differential scanning calorimetry  analysis was conducted under a nitrogen atmosphere. Approximately 10 mg of the testing sample was sealed in an aluminum pan. The samples were heated from 40 to 300 °C at a rate of 10 °C/min. The onset temperature, peak temperature, and enthalpy of the exothermic peak of the curing compositions were recorded. Modulated DSC  analysis was conducted under a nitrogen atmosphere. Approximately 10 mg of the testing sample was sealed in an aluminum pan. The sample was equilibrated at −60 °C, modulated at ±1.00 °C every 60 s as well as isothermally for 5 min, and then heated to 250 °C at a heating rate of 5 °C/min. The glass transition temperature (Tg) was determined by the inflection point of the reverse heat flow curve. Thermal degradation properties were measured using a TGA  from 30 °C to 800 °C at a heating rate of 10 °C/min. Dynamic mechanical analysis  was performed at a heating rate of 5 °C/min from −100 to 280 °C at a fixed frequency of 1 Hz in single cantilever mode. The dimensions of the test specimen were 17.5 mm (L) × 12.8 mm (W) × 3.2 mm (T). Optical transmittance from 350 to 800 nm was measured using UV/Vis spectrometer  and the sample thickness was 2 mm. Thermal mechanical analysis  was conducted at a heating rate of 10 °C/min from −50 °C to 250 °C. Optical properties of the LED were measured using an integrating sphere  with a driving current of 150 mA.In this study, EMC3030  SMD LEDs bonded with a 350 mA blue LED chip were used for the optical-device testing vehicle. Before encapsulation, the lead frame was baked at 140 °C for 2 h to remove moisture, followed by the compositions being dispensed into the cavity of the EMC3030 SMD LED. The samples in this study were cured in a high-temperature oven with curing profiles of 130 °C for 1 h and 150 °C for 2 h. The commercial silicone was cured with curing profiles of 100 °C for 1 h and 150 °C for 5 h. After encapsulation, the optical property of luminous flux was measured using the integrating sphere.This study performed a sulfurization test on the SMD LEDs encapsulated by the transparent siloxane-modified epoxy compositions and commercial silicone to evaluate their sulfurization resistance. The encapsulated LEDs were attached to the wall side of a 100-mL glass sample flask; 0.2 g of sulfur powder was placed in the flask and closed, and then the flask was placed in a high-temperature oven at 70 °C. Changes in color of the SMD LEDs’ silver electrodes were observed during the sulfurization test period. Moreover, the light-output performance of the LED was recorded using the integrating sphere.Tg) was determined using the inflection point of the reverse heat flow curve of mDSC, as displayed in Tg was evidence of the blending of SEP-Ph and SEP-D4 being miscible with no obvious phase separation. By contrast, the reverse heat flow curve of SEP-D4 did not exhibit any significant inflection, which was probably because of the highly rigid network restricting the molecular mobility.The DSC analysis see a revealeHigh optical transmittance and thermal discoloration resistance are basic requirements for transparent materials in optical-device applications. The thermal decomposition of samples 1, 2, 3, and 4 were studied using thermogravimetric analysis (TGA). Tg (α1) of samples 1–4 were 101, 94, 89, and 64 ppm/°C, respectively. Sample 1 exhibited the highest α1 because of the flexible siloxane backbone of SEP-Ph, and it was reduced through incorporating a rigid structure of SEP-D4. A reason for this could be that introducing this rigid structure helped to restrict the segment motion of the transparent siloxane-modified epoxy, thereby lowing α1. This study noticed that the α1 values of samples 1–4 were lower than that of the epoxy siloxane polymer that was prepared and investigated by Morita et al. [In addition, the thermal dimensional stability and mechanical properties of transparent siloxane-modified epoxy compositions are critical for their application ,28. Thera et al. . AccordiDynamic mechanical properties were evaluated using DMA analysis, which was conducted from −100 to 280 °C. The storage modulus is the elastic component of a material and is related to its stiffness. ρ is the crosslinking density, E′ is the storage modulus at temperature T (T = Tg + 50 K and represents absolute temperature), and R is the gas constant (8.314 J·K−1·mol−1).The crosslinking densities of samples 1–4 were studied according to the storage modulus in a rubbery state. In general, a polymer material with a high crosslinking density usually exhibits a high storage modulus in the rubbery region. The crosslinking densities of samples 1–4 were calculated using the following equation ,31 and tThe results indicated that as the quantity of SEP-D4 increased, the crosslinking density also increased, because of the increasing crosslinking point. Sample 4 had the highest crosslinking density among the prepared transparent siloxane-modified epoxy materials. A high crosslinking density is believed to be beneficial for reducing the free volume and network flexibility of siloxane-modified epoxy materials, and thus improving their sulfurization resistance ,33,34.Tg values of samples 1–4 were 17, 25, 61, and 200 °C, and their peak tanδ values were 0.352, 0.348, 0.253, and 0.183, respectively. According to these results, this study concluded that increasing the amount of SEP-D4 results in a higher glass transition temperature, lower tanδ, and lower network flexibility, thereby, decreasing material’s energy dissipation ability.Tanδ, which is defined as the ratio of loss modulus to storage modulus, is usually used to estimate the damping property of a material. Damping is the dissipation of energy in a material under cyclic loading. The glass transition temperature can also be determined using the peak value of tanδ see d. In thiThe sulfurization resistance of the four transparent siloxane-modified epoxy compositions was evaluated with a sulfurization test of the encapsulated EMC3030 SMD LEDs at 70 °C. The results were compared with those of a commercial silicone encapsulant and are displayed in In addition, the sulfurization resistance behaviors of the EMC3030 SMD LEDs encapsulated by samples 1–4 were found to be correlated with crosslinking density, and are illustrated in Adding appropriate amounts of SEP-D4 into the SEP-Ph composition led to increased crosslinking density and reduced network flexibility. Therefore, acceptable sulfurization resistance could be obtained. Notably, after the LEDs were exposed to sulfur gas for 24 h, those encapsulated with samples 3 and 4 exhibited higher flux retention than that encapsulated with only trifluoropropyl phenyl silicone rubber or even with an additional protective polysilazane top layer . The mucIn summary, this study investigated a siloxane-modified epoxy material with high transparency, good thermal discoloration resistance, low thermomechanical stress, and promising sulfurization resistance through incorporating SEP-D4 into SEP-Ph and an MHHPA curing agent composition. SEP-D4 exhibited a high capacity with SEP-Ph and MHHPA in various equivalent ratios. The results indicated that both samples 3 and 4 had high transmittance (approx. 90%) in the visible light region, high thermal discoloration resistance . Furthermore, the lumen flux retention of the LED encapsulated by these compositions approached 97%–99% after a sulfurization test for 240 h. The comprehensive properties of the transparent siloxane-modified epoxies of samples 3 and 4 demonstrated promising properties for LED package applications.


The incidence of cervical cancer is increasing annually worldwide. Low-dose naltrexone (LDN) has been reported to delay tumor progression, but the mechanism remains unclear. Here, we found that low-dose naltrexone could upregulate the expression of OGFr. Additionally, LDN could suppress the abilities of colony formation, migration and invasion in cervical cancer cells. LDN could also inhibit cervical cancer progression in mice model. Moreover, LDN indirectly reduced the expressions of PI3K, pAKT and mTOR in vitro and in vivo. Therefore, LDN may be considered a potential treatment option for cervical cancer. Approximately 570 000 cases of cervical cancer and 311 000 deaths from the disease occurred in 2018 Many viruses are associated with the development of this tumor and that there isone reason for chronic infection, inflammation and the role of cytokines ininflammation and carcinogenesis The opioid growth factor (OGF) and its receptor, OGFr, play a regulatory role in cell proliferation, and maintain homeostasis through a tonically active negative feedback mechanism. OGF is an inhibitory growth factor that is autocrine and paracrine produced and targets normal and abnormal replicating tissues In the present study, we found that OGFr was low expressed in tumors of patients with cervical cancer. Low-dose naltrexone could upregulate the expression of OGFr. Additionally, LDN could suppress the abilities of colony formation, migration and invasion. LDN could also inhibit cervical cancer progression in mice model. Moreover, LDN indirectly reduced the expressions of PI3K, pAKT and mTOR in vitro and in vivo. Therefore, LDN may be considered a potential treatment option for cervical cancer.2. The siRNA- OGFr sequence: 5’-CCCTGGACTACTTCATGTT-3.The human cervical cancer cell lines Hela and Siha  were cultured in DMEM and MEM  with 10% fetal bovine serum, 2% penicillin and streptomycin at 37 °C under 5% COSDS loading buffer. Samples were boiled for 15 min at 95 ° Western blot analysis was performed as described previously Hela and Siha cells at a density of 200 per group were seeded into 35 mm Petri dishes and cultured for 24 h. For treatment with LDN, medium containing 1.24 (Hela) or 1.83 (Siha) mg/mL LDN  was added. The medium were changed every 3 days for all groups. Visible colonies were observed after approximately 14 days. Then, the colonies formed were fixed with 4% formaldehyde for 20 min, followed by visualization treatment with 0.3% crystal violet staining for 5–8 min. After washing twice with PBS, the clones were scanned, photographed, and counted. Colony formation rate = (number of colonies/numbers of seeded cells) × 100%.Scratch assays were performed as described previously 4) were seeded on Matrigel in the upper chamber, and the bottom chamber was filled with 30% FBS culture medium as chemoattractant. Cells that invaded through the Matrigel-coated membrane after 24 h were fixed with paraformaldehyde and stained with 0.4% crystal violet. The fold change in invasion was calculated by dividing the number of cells in LDN treated cells by the number of cells in the control cells.Invasion assays were performed as described previously Total RNA from tissue samples was isolated with TRIzol . qPCR was performed in the CFX96 Real-Time PCR Detection System  using TransStart Top Green qPCR SuperMix  according to the manufacturer's instructions. The primers used for PCR amplification were as follows: PIK3CA forward, 5′- CCACGACCATCATCAGGTGAA -3′ and reverse, 5′-CCTCACGGAGGCATTCTAAAGT-3′; PDK1 forward, 5′- CTGTGATACGGA TCAGAAACCG -3′ and reverse, 5′-T TCCACCAAACAATAAAGAGTGCT -3′; AKT1 forward, 5′- TCCTCCTCAAGAATGATGGCA′ and reverse, 5′- GTGCGTTCGATGACAGTGGT -3′; mTOR forward, 5′- TCCGAGAGATGAGTCA AGAGG -3′ and reverse, 5′- CACCTTCCACTCCTATGAGGC -3′; and VEGFR2 forward, 5′- GTGATCGGAAATGACACTGGAG -3′ and reverse, 5′- CATGTTGGTCACTAACAGAAGCA -3′and β-actin forward, 5′- CATGTACG TTGCTATCCAGGC-3′and reverse, 5′- CTCCTTAATGTCACGCACGAT-3′. qPCR data were analyzed using the comparative Ct method, and the expression of target genes was normalized to that of β-actin.6 Hela cells (in 0.1 mL solution) were injected into nude mice at the right flank via subcutaneous injections (five mice/group). The mice were randomly divided into four groups, including the control, the concentrations of LDN 0.5 mg/kg, 5 mg/kg, and 10 mg/kg. Intraperitoneal injection of naltrexone dissolved in physiological saline with corresponding concentration began the day after tumor cell implantation. Tumor growth and body weights were measured every 3 days, and the tumor volume was calculated with the formula: (length × width2)/2 (mm3). Thirty-six days after the tumor was implanted, retro-orbital blood collection was performed before euthanizing the mice to collect and weigh the tumors.BALB/C nude mice (4–6 weeks old) were purchased from Charles River . The Ethics Committee of the China Medical University approved the experiments. A total of 4 × 10t-tests. A p-value of ≤0.05 was considered statistically significant. p ≤0.05, ≤0.01, ≤0.001 and≤0.0001 are represented by single, double, triple and four asterisks respectively.All statistical analyses were performed using GraphPad Prism Software. The results are expressed as mean values ± SD. Each assay was performed in at least three independent replicates. Significant differences between two groups were assessed using paired two-tailed Student's To check the expression of OGFr in patients’ tumor with cervical cancer, western blot was performed in 12 patients. We found that OGFr was low expressed in cervical cancer tissues A. FurtheNaltrexone has a strong blocking effect on OGFr, and we detected the expression of OGFr when cervical cancer cells treated with LDN. We found that treatment with LDN increased the OGFr expression in Hela and Siha A&B. WhenFurthermore, we extended our findings to a xenograft model in vivo, in which cervical cancer cells are subcutaneously injected into mice. We noticed that LDN could inhibit tumor growth A&B, as cPI3K/AKT/mTOR signaling pathway was involved in oncoprotein-mediated upregulation of EMT-related transcription factors in NSCLC cells. To explore whether the LDN effect was mediated the PI3K/AKT/mTOR signaling pathway, we detected the expression of VEGFR, PI3K, PDK1, AKT and mTOR by qPCR and western. As shown in Naltrexone, is a kind of non-specific opioid receptor antagonist, including the classical opioid receptors can adversely affect opioid receptors  and the non-classical ζ-receptor  Low dose naltrexone (LDN) gradually play a role of treatment in the wider fields, such as cancer, immune related diseases, complications related to diabetes The PI3K/AKT/mTOR signaling pathway regulates the basic functions of cells, such as proliferation, migration, survival and angiogenesis, and also plays an important role in the response to hypoxia and energy consumption. Activation of this signaling pathway is associated with the development of cancer The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


They were cultivated under constant (con) or parabolic (dyn) blue  and/or red  light-emitting diode (LED) photosynthetic photon flux densities (PPFDs) at 12, 16, and 20 h photoperiods, maintaining consistent daily light integrals (DLIs) for each light component in all treatments, at 2.3 and 9.2 mol m–2 per day for blue and red light, respectively. The obtained results and principal component analysis (PCA) confirmed a significant impact of the light spectrum, photoperiod, and parabolic profiles of PPFD on the physiological response of lettuce. The 16 h photoperiod resulted in significantly higher content of xanthophylls  in lettuce leaves under both constant and parabolic blue light treatments . Lower PPFD levels under a 20 h photoperiod (BdynRdyn 20 h) as well as higher PPFD levels under a 12 h photoperiod (BdynRdyn 12 h) had a pronounced impact on leaf gas exchange indices , xanthophylls, soluble sugar contents, and antioxidant properties of lettuce leaves. The parabolic PPFD lighting profile over a 16 h photoperiod (BdynRdyn 16 h) led to a significant decrease in Ci/Ca, which resulted in decreased Pr and Tr, compared with constant blue or red light treatments with the same photoperiod (BconRdyn and BdynRcon 16 h). Additionally, constant blue lighting produced higher α + β-carotene and anthocyanin (ARI) content and increased carotenoid to chlorophyll ratio (CRI) but decreased biomass accumulation and antioxidant activity.This study aimed to evaluate the effect of dynamic red and blue light parameters on the physiological responses and key metabolites in lettuce and also the subsequent impact of varying light spectra on nutritive value. We explored the metabolic changes in carotenes, xanthophylls, soluble sugars, organic acids, and antioxidants; the response of photosynthetic indices [photosynthetic (Pr) and transpiration (Tr) rates]; and the intracellular to ambient CO The effects of light on plants are complex, as it controls photosynthesis, plant growth, and developmental processes. Plants generally absorb radiation in the visible electromagnetic spectrum; however, not the entire spectrum of light is beneficial . Under c+ to form NADPH, whereas the dark-dependent reactions involve the fixation of CO2 into carbohydrates via the Calvin–Benson cycle, regenerating ADP and NADP+ , located in the thylakoid membrane, absorbing red and blue light, induce the process of photosynthesis. Moderate stress caused by different environmental factors, including light, influences the metabolism of carotenoids  was grown in a growth chamber in peat substrate  . The average concentrations of nutrients (mg L–1) in the substrate were as follows: N, 110; P2O5, 50; and K2O, 160. The microelements Fe, Mn, Cu, B, Mo, and Zn were also present. Electrical conductivity (EC) varied between 1.0 and 2.5 mS cm–1 (±0.03 m). One seed was seeded into a 120 ml vessel, and 28 plants for each treatment were analyzed. Plants were watered as needed. Experiments were performed in a walk-in controlled environment growth chamber (4 × 6 m). Day/night temperatures of +21/17 ± 2°C were established, and the relative humidity was maintained at 50–60%.Lettuce (–2 per day for B and R) was maintained for each light component in all treatments  of blue  and red , were used for lettuce lighting. Plants grew under constant (con) or parabolic (dyn) blue (B) and/or red (R) LED photosynthetic photon flux density (PPFD) at 12, 16, and 20 h photoperiods, but the same daily light integral  were extracted using 80% acetone (1 g of sample ground with liquid N2 and 10 ml of solvent), centrifuged , and filtered through a 0.45-μm nylon membrane syringe filter . The contents of carotenes, xanthophylls, and chlorophylls were evaluated using a Shimadzu HPLC (Japan) instrument equipped with a diode array detector (SPD-M 10A VP) on a YMC Carotenoid column  . The mobile phase consisted of A  and B (100% ethyl acetate). The gradient was as follows: 0 min; 20% B, 2.5 min; 22.5% B, 20–22.5 min; 50% B, 24–26 min; 80% B, 31–34 min; 100% B, 42–47 min; and 20% B, flow rate 1 ml min–1 , carotenes (carotenes α and β), and chlorophylls . Sugars were analyzed according to –1.About 0.5 g of fresh plant tissue was ground and diluted with deionized Hproteins  was perf2O (1:10) (w/v) and heated in a water bath for 30 min at +50°C. The extract was centrifuged at 10,000 rpm for 15 min and filtered through a 0.22 μm PTPE syringe filter . Organic acids were analyzed according to –1. The peak was detected at 210 nm and identified using an external calibration method.Approximately 0.5 g of fresh plant tissue was homogenized and diluted with deionized H2+ reducing antioxidant power (FRAP) assay , ABTS , and an FeP) assay . Extract–1 of fresh plant weight.The DPPH free radical scavenging activity was determined by mixing a diluted extract with 0.06 M methanolic DPPH solution and monitoring radical quenching every minute for 16 min to measure the absorbance at 515 nm . The results are presented as DPPH free radical scavenging activity, μmol g2S2O8, allowing the mixture to stand in the dark at room temperature for 16 h before use. The working solution was diluted to obtain an initial absorbance of AU 0.700 at 734 nm ; 100 μl of the samples were mixed with 2 ml ABTS solution and absorbance was monitored for 11 min. The results are presented as ABTS free radical scavenging activity, μmol g–1 of fresh plant weight.The ABTS radical solution was prepared by mixing 50 ml of 2 mM ABTS with 200 μl 70 mM K3 × 6H2O at 10:1:1 (v/v/v). Twenty microliters of the sample was mixed with 3 ml of working solution and incubated in the dark for 30 min. Then, the absorbance at 593 nm was read. The antioxidant power was expressed as Trolox equivalent antioxidant capacity  and Fe2+ antioxidant capacity (Fe2+ μmol g–1 of fresh plant weight).For the FRAP assay, the working reagent was prepared by mixing acetate buffer , a solution of 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) in 40 mM HCl, and 20 mM FeCl2 m–2 s–1), transpiration rate , and the intercellular to ambient CO2 concentration ratio (Ci/Ca) were measured on the third developed leaf, using a portable photosynthesis system  under the conditions at +21°C, with a CO2 concentration of 400 μmol mol–1 and 60% relative humidity. Artificial irradiation was supplied to the leaf using different LEDs (665 and 470 nm), but their respective intensities were the same for PPFD 1,000 μmol m–2 s–1. Photosynthesis was measured from 9 to 12 a.m.Photosynthetic rate  from 9 to 12 a.m. Reflection spectra obtained from the leaves were used to calculate the carotenoid reflectance index (CRI) and the anthocyanin reflectance index (ARI).CRI, which shows carotenoid to chlorophyll ratio, was evaluated using the following formula:ARI evaluates changes in anthocyanin amount:where ρ510, ρ550, and ρ570 represent the leaf reflectance integrated over a 10 nm wavelength band centered on 510, 550, and 570 nm, respectively.At the end of the experiment, plant fresh mass (FW) was determined by harvesting leaves from five different plants per light treatment. Leaf area (LA) was determined using a leaf area meter . Dry weight (DW) was weighed after tissue dehydration at +70°C for 48 h .p = 0.05. Multivariate principal component analysis (PCA) was performed. The results are presented in a PCA scatterplot that indicates distinct levels of metabolites, antioxidant activity, and photosynthetic indices in lettuce subjected to constant or dynamic red and blue light at different photoperiods and a correlation circle (based on Pearson’s correlation matrix) that summarizes the metabolic relations between investigated metabolites, antioxidants, and photosynthetic systems.Data were processed using the XLSTAT software , using one-way analysis of variance, ANOVA, and Tukey’s (HSD) test at confidence level Ci/Ca, Pr, and Tr , sucrose, or maltose (–1 DW) than sucrose, compared with other treatments (1.1–2.6 mg g–1 DW)  .–1 DW) were found in lettuce, with folic and fumaric acids present as a second major group (0.19–0.05 mg g–1 DW), while oxaloacetic, succinic, and ascorbic acids were present in the smallest amounts (0.034–0.003 mg g–1 DW)  . The acc+, and FRAP . Significantly, the highest antioxidant capacity was found under a 20 h photoperiod using the FRAP assay (12.0 mmol TE g–1); however, a significant decrease (18.8%) was conditioned only under constant blue (BconRdyn 16 h) treatments. The ABTS+ assay showed an antioxidant activity of 5.5 mmol TE g–1, whereas the DPPH∙ assay showed no significant differences in lettuce extracts compared with the 12- or 16 h parabolic blue and red light profiles (BdynRdyn).The trends of antioxidant activity in response to lighting treatments, based on different assays, were similar . ConstanThe PCA score scatterplot  shows thCi/Ca, Pr, Tr, Chl a, DPPH, oxalic, and fumaric acid values and low or medium malic acid, sucrose, and α- and β-carotene content. All analyzed metabolites were in cluster 1 or cluster 2, except for Chl a (C3) and oxalic acid (C6). The highest similarity among metabolites was found in cluster 1. Fumaric acid, anthocyanin amount, violaxanthin, and neoxanthin were grouped into cluster C1-1; succinic acid, oxaloacetic acid, carotenes α + β, lutein + zeaxanthin, and ascorbic acid were grouped into cluster C1–2; and maltose, carotenoid/chlorophyll ratio, and folic acid were grouped into cluster C1–3.The agglomerative hierarchical cluster (AHC) analysis was used to divide the responses to the light profile and photoperiod data into groups of increasing dissimilarity . These dCi/Ca) and the 12 h photoperiod was found  or constant red (BdynRcon) light was observed. A negative correlation between antioxidant properties , growth parameters , and constant blue light (BconRdyn) was found, while a parabolic light profile (BdynRdyn) led to a positive correlation.A negative correlation between xanthophylls  and the 12 h photoperiod treatment was found, with the opposite pattern for the 16 h photoperiod treatment. A positive correlation between photosynthetic response  and photoperiod (Ci/Ca increased under the constant blue (BconRdyn) or constant red (BdynRcon) light treatments compared with the other light treatments. The same was true for antioxidant properties  and growth parameters  under the parabolic light profile BdynRdyn, with the opposite responses observed for treatments with the constant blue (BconRdyn) light profile.A positive correlation was found between xanthophylls  and photoperiod, with the content of these compounds increasing as the length of the light cycle increased (12–16 h). The opposite pattern was observed between photosynthetic response . The trend of the response of carotenes and chlorophylls to all lighting conditions in this study was the same  led to a significant increase in fructose and glucose  treatment, which showed a significantly higher fresh weight and new leaf area formation , xanthophylls, soluble sugar contents, and antioxidant properties of lettuce leaves. Parabolic lighting (BdynRdyn 16 h) led to a significant decrease in Ci/Ca, resulting in decreased photosynthetic and transpiration rates, compared with constant blue or red light PPFD over the same photoperiod . Moreover, under constant blue lighting, higher carotenes α + β, anthocyanin (ARI) content, and CRI were obtained, but these were accompanied by decreased biomass accumulation and antioxidant activity.The results obtained and the PCA analysis confirmed the significant impact of both photoperiod and the parabolic profiles of PPFD distribution on lettuce physiological response. A 16 h photoperiod resulted in significantly higher xanthophyll content  in lettuce leaves under both constant and parabolic blue light treatments . Lower and higher PPFD levels under a 20 and 12 h photoperiod , respectively, and maintaining the same DLI had a pronounced reducing impact on photosynthetic indices (Pr, Tr, The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.GS: data analysis, writing of the manuscript. AV: joint coordination of the experiment, modeling of light parameters, and data summarizing JM: spectrophotometric analysis, biometric measurements, and data summarizing. PH: chromatographic analysis. KL: photosynthesis and optical indices measurements, data summarizing. AB and PD: the realization of lighting schedules in vegetative experiments, data analysis All authors read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


More than 15% of piglets and about 10% of newborn humans suffer from intrauterine growth retardation (IUGR), which refers to growth lag, developmental restriction and impaired organs in the fetus. IUGR exhibits programming consequences and exerts permanent negative effects on postnatal growth and health. Dietary supplemented curcumin, as the main natural polyphenol isolated from the natural antioxidant (turmeric), might show possible effects on antioxidant capacity, and the meat quality of IUGR pigs. Therefore, in our present study, 12 normal birth weight (NBW) and 24 IUGR neonatal female piglets were selected and fed control diets supplemented 0 (NBW), 0 (IUGR) and 200 (IUGR + Cur) mg/kg curcumin from 26 to 115 days of age (n = 12). The growth performance, meat quality, redox status and its related Nrf2 pathway were determined to test the hypothesis that curcumin may play beneficial roles against IUGR-induced oxidative stress. This study suggested that curcumin could serve as a potential natural antioxidant in nutrition interventions of IUGR offspring to enhance the redox status and improve the meat quality of leg muscles. These results attained from IUGR pig models can also provide some useful theoretical references for IUGR offspring in humans.p < 0.01) but had no difference on 115 d among NBW, IUGR and IUGR + Cur groups (p > 0.05). Compared with NBW and IUGR groups, a significant decrease in drip loss (24 h and 48 h) was observed in the IUGR + Cur group (p < 0.01). IUGR pigs had higher concentrations of malondialdehyde (MDA) (p < 0.01) and protein carbonyl (PC) (p = 0.03) and lower activities of glutathione peroxidase (p = 0.02), catalase (p < 0.01) and peroxidase (p = 0.02) in leg muscles than NBW pigs. Dietary-added 200 mg/kg curcumin decreased concentrations of MDA and PC and improved the activities of catalase, superoxide dismutase (SOD) and peroxidase as compared to the IUGR group (p < 0.05). Additionally, dietary curcumin enhanced protein (NQO1) and mRNA expression of genes , heme oxygenase-1 (HO-1), glutathione S-transferase (GST) and catalase (CAT)) as compared to the IUGR group (p < 0.05). These results suggest that dietary curcumin could serve as a potential additive to enhance redox status and improve meat quality of IUGR growing pigs via the Nrf2 signal pathway. Intrauterine growth retardation (IUGR) exhibits programming consequences and may induce oxidative stress in growing animals and humans. This study was conducted to investigate the hypothesis that dietary curcumin may protect growing pigs from IUGR-induced oxidative stress via the Nrf2 pathway. Twelve normal birth weight (NBW) and 24 IUGR female piglets were selected and fed control diets supplemented 0 (NBW), 0 (IUGR) and 200 (IUGR + Cur) mg/kg curcumin from 26 to 115 days of age (n = 12). Growth performance, meat quality, redox status and its related Nrf2 pathway were determined. Results showed that IUGR pigs exhibited decreased body weight on 0 d, 26 d and 56 d ( Intrauterine growth retardation (IUGR) refers to growth lag, developmental restriction or impaired organs in fetuses. More than 15% of piglets and about 10% of newborn humans suffer from IUGR ,3. RecenCurcuma longa), has received much interest in both culinary and medical fields [Among natural antioxidants, turmeric, a rhizomatous herbaceous perennial plant .At the time of parturition (114 days gestation), 12 NBW (1.49 ± 0.02 kg) and 24 IUGR female (0.76 ± 0.02 kg) piglets (Duroc× Landrace× Large White) were selected from 12 sows with similar litter sizes. One NBW and two IUGR female piglets were chosen from each litter according to the previous studies ,18,19. Ag for 10 min at 4 °C and stored at −80 °C until analysis. The muscle (biceps femoris) of the left hind leg samples were frozen in liquid nitrogen and stored at −80 °C for further determination. For the meat quality, the muscle samples were stored at 4 °C. During this feeding trial, pigs were housed on the concrete floors with water and feed ad libitum. After weaning, each group was housed in one pen, while 2 pigs were housed in one pen during 56–115 d. At the end of the feeding trial, 6 pigs (close to the average body weight) from each group were chosen. After fasting for 12 h, pigs were euthanized by electrical stunning and exsanguinated. The blood was centrifuged at 3000 The body weights were weighed at 0, 26, 56 and 115 d to calculate the average daily gain (ADG) during 0–26 d, 26–56 d and 56–115 d. The feed intake was measured during 56–115 d to calculate the average daily feed intake (ADFI) and feed:gain ratio (F:G) on a wet basis. 45min) and 24 h (pH24h) post-slaughter using a pH meter , each sample with three replicates. The color of fresh meat was measured using a Minolta chromameter  with 8 mm measuring diameter and 8° illumination angle (CIE standard illuminant D65). The CIE L* (light index), a* (red index) and b* (yellow index) were collected from three different orientations on the cut surface of each leg muscle chop. Calibration was carried out prior to each color determination using a white standard plate. The drip loss was determined as described in our previous publication with some modifications [Muscle pH value was determined at 45 min , superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), glutathione peroxidase (GPX) and the level of glutathione (GSH), thyrotropin (TSH), malondialdehyde (MDA) and protein carbonyls (PC) in the supernatant were determined with corresponding commercial kits purchased from the Nanjing Jiancheng Bioengineering Institute . The activities of T-AOC, SOD, CAT, POD and GPX were expressed as units (U) per milligram of protein. The concentrations of MDA and PC were expressed as nanomoles per gram of protein. The levels of TSH and GSH were expressed as millimoles and milligram per gram of protein, respectively.The samples of biceps femoris were homogenized in ice-cold 0.90% sodium chloride buffer (w:v) as described by Zhang et al. . The sup3:HClO4 = 4:1, v:v) was added to the muscle sample (about 1.0 g), stewing for 12 h at room temperature, and then was treated with high temperature until digested completely. The reaction condition of high temperature was as follows: 90 °C for 30 min; 120 °C for 30 min; 160 °C for 120 min and 210 °C for 210 min. The digest was diluted with 5% HNO3 solution to the optimal level for further determination. After the external matrix-matched standard curves were determined via corresponding standards, which were diluted with 5% HNO3 solution, the blanks and prepared solutions of samples were analyzed with inductively coupled plasma optical emission spectrometry (ICP-OES).The mineral concentrations  in the muscles were determined as the methods of Demirbaş et al.  and WangTM, Grand Island, NY, USA) according to the manufacturer’s guidelines. The SYBR Green PCR reaction system was 10 μL in total, which was composed of 5 μL ChamQ SYBR qPCR Master Mix (2×), 0.2 μL forward and reverse primers, 0.2 μL ROX Reference Dye 1 (50×), 1 μL cDNA and 3.6 μL ddH2O. The relative mRNA expressions of target genes were calculated using the 2−ΔΔCt method as previously reported by Livak et al. [Total RNA from the leg muscle samples was extracted using Trizol Reagents . The integrity of RNA was examined by 1% agarose gel. The RNA concentration was determined, and its quality was verified (ratios of absorption including 260/280 nm and 263/230 nm between 1.90 and 2.05) via a NanoDrop ND-2000 UV spectrophotometer . The RNA (each sample 2 μg) was reverse-transcribed into cDNA using PrimeScript™ RT Reagent Kit  according to the manufacturer’s protocol. The cDNA was stored at −20 °C for further determination. The specific primers for the target genes related to the antioxidant and Nrf2 signal pathway, including CAT, SOD, glutathione S-transferase (GST), HO-1, kelch-like ECH-associated protein 1 (Keap1), nuclear factor erythroid 2 related factor 2 (Nrf2), NAD (P)H dehydrogenase, quinone 1 (NQO1), glutamate-cysteine ligase catalytic subunit (GCLC), glutamate-cysteine ligase modifier subunit (GCLM) and gamma-glutamyltransferase 1 (GGT1), were synthesized by Invitrogen Biotech Co. Ltd.  and are listed in The Western blotting analysis was conducted according to our previous method with some modifications . Brieflyp < 0.05. All statistic procedures were done in the SPSS (20.0) package.To estimate the effects of the treatments on the response variables evaluated, a one-way ANOVA was used. To evaluate the difference among groups, multiple comparisons were conducted using the Duncan test. Pens were used as experimental units for the analysis of ADFI and F:G, and for other parameters, each pig was used as the experimental unit. Statistical significance was considered at p < 0.01), but no significant difference on 115 d was found among the NBW, IUGR and IUGR + Cur groups (p > 0.05). Similarly, IUGR pigs had lower ADG (p < 0.05) during 0–26 d (p < 0.01) and 26-56 d (p = 0.02) than NBW pigs, while they showed no significant difference during 56-115 d (p > 0.05). Dietary-added 200 mg/kg curcumin did not affect the body weights, ADG and F:G of IUGR pigs (p > 0.05); however, there was significantly decreased ADFI during 56–115 d than both the NBW and IUGR groups (p < 0.01). As indicated in p > 0.05). However, compared with the NBW and IUGR groups, a significant decrease was observed in drip loss at 24 h and 48 h in the IUGR + Cur group (p < 0.01). In addition, the a* value of pigs in the IUGR + Cur group was significantly increased as compared to the NBW group (p = 0.02).Effects of dietary curcumin on meat quality in leg muscles are presented in p < 0.01) and PC (p = 0.03) levels in leg muscles and had significantly lower GPX (p = 0.02), CAT (p < 0.01) and POD (p = 0.02) activities than NBW pigs but did not show significant difference in GSH and TSH contents (p > 0.05). Compared with the IUGR group, dietary-added 200 mg/kg curcumin significantly decreased MDA (p < 0.01) and PC (p = 0.03) contents in the muscles and increased the CAT (p < 0.01), SOD (p = 0.04) and POD (p = 0.02) activities. There were no significant differences in antioxidant enzyme activities  and contents of MDA, PC, GSH and TSH between the NBW and IUGR + Cur groups (p > 0.05).Effects of dietary curcumin on antioxidant capacity of leg muscles in IUGR growing pigs are shown in p > 0.05).Effects of curcumin on mineral concentrations of leg muscles in IUGR growing pigs are summarized in p < 0.05). Compared with the IUGR group, dietary-supplemented 200 mg/kg curcumin significantly increased mRNA expressions of GST, HO-1 and CAT of leg muscles (p < 0.05), while it did not show significant effects on SOD mRNA expression (p > 0.05). There were no significant differences in mRNA expressions of SOD, GST, HO-1 and CAT in leg muscles of pigs between the IUGR + Cur and NBW groups (p > 0.05).As shown in p > 0.05). However, pigs in the IUGR + Cur group had significantly higher Nrf2 protein expression than those of the NBW group (p < 0.05) and significantly higher NQO1 protein expression than those of the IUGR group (p < 0.05).Effects of curcumin on protein expressions of the Keap1/Nrf2 signal pathway in leg muscles are shown in p > 0.05). However, pigs in the IUGR + Cur group had significantly higher mRNA expressions of Nrf2, NQO1 and GGT1 than those of the IUGR group (p < 0.05) and significantly increased GCLM and GGT1 mRNA expressions than those of the NBW group (p < 0.05). There were no significant differences in the Keap1 and GCLC mRNA expressions among the three groups (p > 0.05).Effects of curcumin on gene expressions of the Keap1/Nrf2 signal pathway in leg muscles, including Keap1, Nrf2, NQO1, GCLC, GCLM and GGT1, are shown in IUGR can result in low birth weights and show postnatal catch-up growths after birth, which are considered to be important risk factors for later developments of chronic metabolic diseases ,26. Thes45min and pH24h); drip loss (24 and 48 h) and meat color , were determined. Results indicated that IUGR showed no significant effects on these parameters of meat quality; however, pigs in the IUGR+Cur group had higher a* values as compared to that in the NBW group, which was in agreement with the results of Zhang et al., who reported that dietary-supplemented 0–200 mg/kg curcumin showed a significant linear and quadratic increase in a* values of breast muscles in broilers [IUGR may also affect meat quality as well as growth performance via causing changes in the physiology and metabolism ,35. In obroilers . Mancinibroilers . In our broilers . 2− to H2O2 and further reduce to H2O, while GSH and TSH concentrations and T-AOC reflect the nonenzymatic antioxidant defense system [The decreased drip loss might be related to enhanced antioxidant capability and reduced oxidative stress ,14. Oxide system . TherefoMineral concentrations in serum and tissues, including Zn, Fe, Cu, Mn and Se, are closely involved in the enzymatic antioxidant defense system, since they are important components of many antioxidant enzymes or act as enzyme activators . For exaTo further explore effects of IUGR-induced oxidative stress and the beneficial effects of dietary curcumin, the mRNA expressions of SOD, GST, HO-1 and CAT in leg muscles were analyzed. Results showed that the IUGR pigs had lower mRNA expressions of HO-1 and CAT, while dietary-supplemented 200mg/kg curcumin significantly increased GST, HO-1 and CAT mRNA expressions. In consistent with our present study, Ferencz et al. reported that the mRNA expressions of HO-1, HO-2 and CAT in low birth weight neonates were considerably lower than that in high birth weight neonates . Our preAdditionally, mRNA expressions of some antioxidant genes such as HO-1 are closely related to the Keap1/Nrf2 pathway ,45. Nrf2Our results indicated that IUGR pigs exhibited growth inhibitions before 56 d and showed postnatal catch-up growth during 56–115 d. In addition, IUGR could induce oxidative stress in leg muscles, and dietary-supplemented 200 mg/kg curcumin could improve meat color, increase water-holding capacity and attenuate the oxidative stress of pigs via the Nrf2 signal pathway. This study suggested that curcumin could serve as a potential natural antioxidant in nutrition interventions of IUGR offspring to improve the redox status of leg muscles and benefit the meat quality.


Agrobacterium tumefaciens-mediated gene transfer is the most commonly used method for plant genetic engineering. However, during the period of A. tumefaciens culture, the effects of Agrobacterium culture before inoculation on genetic transformation are poorly understood. In the present study, we investigated the factors that affect the genetic transformation efficiency during Agrobacterium culture using Tamarix hispida as transgenic plant material. Agrobacterium treatment with spermidine (Spe), azacitidine (5-AzaC), dithiothreitol (DTT), or acetosyringone (AS) alone all significantly improved the efficiency of T-DNA transfer. Treatment with 5-AzaC reduced DNA methylation in Agrobacterium to induce the expression of virulence (vir) family genes, including virA, virB1, virC1, virD2, virD4 virE2, and virG. Spe treatment significantly induced the expression of all the studied genes, including virA, virB1, virC1, virD1, virD2, virD4, virE2, and virG. DTT treatment decreased reactive oxygen species accumulation. AS treatment activated the expression of the genes virA, virB1, virC1, virD1, virD2, virD4 and virG. All these effects resulted in increased T-DNA transfer. Additionally, combined Spe, 5-AzaC, DTT, and AS treatment improve Agrobacterium infection to a greater extent compared with their use alone, increasing T-DNA transfer by more than 8-fold relative to no treatment. Therefore, to improve genetic transformation, pretreatment of Agrobacterium during the culture period is important for improving genetic transformation efficiency. Agrobacterium tumefaciens- or Agrobacterium rhizogenes-mediated plant transformation  were generated by using Agrobacterium-mediated transformation , into the co-cultivation medium could highly improve transgenic efficiency , and supplementation with of ROS scavenging factors in the co-cultivation medium, such as dithiothreitol (DTT), sodium thiosulfate (STS), and L-cysteine (L-Cys), can reduce ROS accumulation and increase the efficiency of transgene delivery. For instance, Genetic transformation is a method that transfers DNA of interest into the cell of an organism resulting in genetic alteration, and has been used in many areas of biotechnology, such as studies of gene function, genetic improvement, and molecular breeding. To deliver foreign genes into host plants, three methods are mainly used: ormation , protoplormation , and parormation . Among tormation . This geormation . Howeverficiency . During Vitis vinifera somatic embryo growth, which increased transgenic resistant somatic embryos induction rate, suggesting that Spe plays a positive role in T-DNA transfer. The above studies significantly improved the efficiency of T-DNA transfer and indicated that the importance of activating the T-DNA transfer capability of Agrobacterium.Agrobacterium. Our results also indicated that treatment of Agrobacterium in culture before inoculation is relatively important for T-DNA transfer efficiency and should be considered in Agrobacterium-mediated transformation studies.However, most previous studies on improvement of T-DNA transfer assessed the period of the transformation and co-cultivation. The treatments were mainly conducted during co-cultivation time or were added to the co-cultivation medium. In the present study, we identified and studied factors that might affect the infection capability of T. hispida were collected from the Turpan Botanical Garden of the Chinese Academy of Sciences. The seeds of T. hispida were surface-sterilized in 33% (v/v) sodium hypochlorite and grown on Murashige-Skoog (MS) solid medium containing 2% sucrose for germination. Two-month-old seedlings were used for transient transformation investigation.The seeds of Agrobacterium tumefaciens (EHA105) harboring pCAMBIA1301 was picked and cultured in LB liquid culture supplied with 100 μg/ml rifampicin and 50 μg/ml Kanamycin at 28°C until the OD600 reached 0.8 (about 24 h). The culture was diluted 100-fold to a volume of 50 ml and used for the following treatments. To determine the effects of Spe, the diluted cultures were supplied with 0, 1, 3, 5, and 7 mM Spe, and then cultured at 28°C until the OD600 reached 0.6–0.7 (about 7–8 h). For 5-AzaC treatment, the diluted cultures were added with 0, 10, 20, and 30 μM5-AzaC, and cultured at 28°C until the OD600 reached 0.6–0.7 (about 7–8 h). To test the effect of AS, the diluted cultures containing 0, 100, 120, 150, and 200 μM AS were cultured at 28°C until the OD600 reached 0.6–0.7 (about 7–8 h). For DTT supplementation, the diluted cultures were supplied with 0, 50, 100, and 150 μM DTT, and cultured at 28°C until the OD600 reached 0.6–0.7 (about 7–8 h). After cultivation, the cultures were centrifuged at 2,800 × g to pellet the Agrobacterium cells, and then used for transient transformation.A single colony of Agrobacterium was performed according to the method described by A. tumefaciens EHA105 cells were centrifuged at 2,800 × g for 10 min, and then adjusted to an OD600 of 0.8 using a solution comprising 1/2 MS +10 mM CaCl2+.5% (w/v) sucrose + 100 μM acetosyringone + Tween20 , pH 5.8 (transformation solution). The plants were soaked in the transformation solution with shaking at 90 rpm for 2 h at 25°C. The plants were then washed quickly with distilled water for 10 s to remove the excessive A. tumefaciens cells. After washing, the plants were planted vertically on 1/2 MS solid medium . After culture for 72 h, the plants were washed with distilled water and wiped with sterile filter paper to remove excessive water. These plants were harvested for GUS staining and GUS activity assays. Each sample contains at least 10 seedlings, and three biological repeats were performed.Transient transformation mediated by 2PO4-Na2HPO4, pH 7.4, 10 mM β-mercaptoethanol, 10 mM EDTA, 0.1% Triton X-100, 0.1% sodium lauryl sarcosine). Then, 1 mM 4-methylumbelliferyl-β-d-glucuronide (MUG) was added into the extraction buffer, the enzyme reaction was performed at 37°C, and was stopped by adding 500 μl of 0.2 M Na2CO3. After stopping the enzyme reaction, the fluorescence of 4-methylumbelliferone was checked using a DyNA Quant fluorometer , and a protein standard curve was generated using the Bradford assay to calculate the GUS activity.GUS activity was measured following the method described by The ROS content was determined using a Hydrogen Peroxide Content measuring kit , according to the manufacturer’s protocol. The DNA methylation content was determined using Methylated DNA Quantification Kit (Fluorometric) according to the manufacturer’s protocol . Three independent biological replications were performed.T. hispida or Agrobacterium cells was isolated using the Trizol reagent (Promega) and treated with DNaseI (RNase free) to remove DNA contamination. The integrality of RNA was analyzed using agarose gel electrophoresis. For the synthesis of T. hispida cDNA, 1 μg of total RNA from each sample was reverse-transcribed using a random primer (9 mer) as synthesis primers with a PrimeScript™ RT reagent Kit  in volume of 10 μl according to its instruction. The products were subsequently diluted to 100 μl and used as the template for qRT-PCR. For T. hispada, two internal references were used, including β-tubulin (GenBank number: FJ618519) and α-tubulin (GenBank number: FJ618518). For Agrobacterium cells, 16S rRNA was used as internal reference. All the primers used are shown as -ΔΔCt method . data were compared using one-way analysis of variance (ANOVA) and differences were considered statistically significant at P < 0.05.Agrobacterium cells treated with 5-AzaC before transformation significantly elevated β-glucuronidase (GUS) expression and activity in transient transformed T. hispida plants , supplementation with 5 and 7 mM Spe significantly increased GUS expression and activity  medium for vir genes were studied. The results showed that except for virD1, all the studied vir genes were significantly induced by treatment with 5 mM Spe  region. VirA and virG families serve as transcriptional activators for vir gene transfer and transformation of higher plants . Heilongjiang Touyan Innovation Team Program (Tree Genetics and Breeding Innovation Team) and the Overseas Expertise Introduction Project for Discipline Innovation (B16010).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Although melatonin has been reported to play an important role in regulating metabolic events under adverse stresses, its underlying mechanisms on germination in aged seeds remain unclear. This study was conducted to investigate the effect of melatonin priming (MP) on embryos of aged oat seeds in relation to germination, ultrastructural changes, antioxidant responses, and protein profiles. Proteomic analysis revealed, in total, 402 differentially expressed proteins (DEPs) in normal, aged, and aged + MP embryos. The downregulated DEPs in aged embryos were enriched in sucrose metabolism, glycolysis, β-oxidation of lipid, and protein synthesis. MP (200 μM) turned four downregulated DEPs into upregulated DEPs, among which, especially 3-ketoacyl-CoA thiolase-like protein (KATLP) involved in the β-oxidation pathway played a key role in maintaining TCA cycle stability and providing more energy for protein translation. Furthermore, it was found that MP enhanced antioxidant capacity in the ascorbate-glutathione (AsA-GSH) system, declined reactive oxygen species (ROS), and improved cell ultrastructure. These results indicated that the impaired germination and seedling growth of aged seeds could be rescued to a certain level by melatonin, predominantly depending on β-oxidation, protein translation, and antioxidant protection of AsA-GSH. This work reveals new insights into melatonin-mediated mechanisms from protein profiles that occur in embryos of oat seeds processed by both aging and priming. Triticum aestivum L.) [Oryza sativa L.) [Seeds are the genetic materials for cultivation of almost all crop species in agriculture. Seed germination represents the most crucial initiation for crop development and growth, greatly relying on seed physiological quality, and influences the subsequent seedling performances in various environments . Howeverivum L.)  and ricetiva L.)  seeds shtiva L.) ,7,8. Howtiva L.) . TherefoCucumis sativa L.) [Zea maize L.) [Seed priming, a technique applied by imbibing seeds with water or various chemical reagents  to trigger the germination-related events before radicle protrusion, is widely used to reinforce vigor and enhance germination and emergence of aged seeds . The imptiva L.)  and maizaize L.) . HoweverProteomic approaches have successfully created new avenues to detect changes in various cellular processes, understand biological functions of individual proteins, and elucidate metabolic mechanisms during seed aging and priming . EmbryonMelatonin (N-acetyl-5-methoxytryptamine) is a pleiotropic molecule ubiquitously distributed in diverse kingdoms, covering bacteria, insects, animals, and plants . MelatonAvena sativa L.) is an important cereal crop possessing high levels of valuable nutrients such as protein, unsaturated fatty acids, soluble dietary fiber and minerals [Oat  of oat seeds showed a reverse “S-shaped” curve with prolonged aging duration . Aging oThe 48-days aging duration significantly decreased the GP from 100% (C1) to 70% (C2) in oat seeds, with similar changes shown in seed vigor index (VI) and germination index (GI) A,B. PrimThe phenotype of seedling development on the 10th day was repressed after 48-days aging duration at 45 °C, with more abnormal seedlings and dead seeds C. MeanwhThe TEM images of embryonic cells’ ultrastructure in oat seeds during aging and melatonin priming were acquired . The embSeed aging resulted in damage to embryonic cells, with incomplete cytoplasmic membrane and plasmolysis, and mainly altered the ultrastructure of the cell nucleus and mitochondria. The structural response of the nucleus to aging was that the boundary between the nucleus and cytoplasm became blurred and indistinct with only the location of the nuclear region being vaguely distinguished, nucleolus disappeared, and the double-layer nuclear membrane was destroyed. Mitochondria swelled and burst, with their inner and outer membranes damaged, crista disappeared, and the matrix become thin similar to “vacuole” structure D–F.Furthermore, the ultrastructure of embryonic cells in melatonin-primed seeds was observed to evaluate whether melatonin could prevent these alterations caused by aging. The results indicated that 200 μM melatonin could restore the integrity of the cellular structure. The boundary between the nucleus and cytoplasm was obvious, and explicit nuclear region, clear inner and outer layers of the nuclear membrane, and visible nucleolus were found. The structure of mitochondria returned to typical, with internal crista and complete double-layer membrane G–I. HoweIn summary, the significant phenotypes in germinability, seedling growth, and the altered ultra-microstructure suggested that various physiological or molecular changes could take place after the processes of seed aging and subsequent melatonin priming.2O2), superoxide anion (O2−·), and hydroxyl radical (OH·)) production and the malondialdehyde (MDA) accumulation were significantly induced by seed aging, increasing by 26.6%, 148.8%, 45.5%, and 110.1%, respectively  significantly reduced the content of H2O2 (by 53.1%), OH· (by 44.5%), and MDA (by 46.6%), which were also significantly lower than those in M200 , and APX in C2 were significantly lower than those in C1, and no significant differences were generated in the activities of CAT, monodehydroascorbate reductase (MDHAR), and glutathione reductase (GR) between C1 and C2 . M200 siTo uncover the differential proteins involved in aging and melatonin priming, a global analysis of the iTRAQ-based quantitative proteome was performed, using embryos from non-aged, aged and aged + MP oat seeds. A total of 2643 proteins were identified , which wp-value ≤ 0.05 were considered upregulated, whereas those with a FC < 0.67 and p-value < 0.05 were downregulated. Taking into account these criteria, among the total 2643 identified proteins, a total of 402 DEPs were identified, among which 145 DEPs that included 55 upregulated and 90 downregulated DEPS were found in aged seeds as compared with non-aged oat seeds  and protein alterations during the melatonin priming processes . Proteins with a fold change (FC) ≥ 1.5 and at seeds . Howeverat seeds .In total 70 DEPs were shared by the aging process and at least one of the melatonin priming processes . In summIn addition, a total of 225 DEPs were upregulated, of which 47 (20.9%), 54 (24.0%), 33 (14.7%), and 47 (20.9%) DEPs were upregulated only in “C2 vs. C1”, “M200 vs. C2”, “M1000 vs. C2”, and “M1000 vs. M200”, respectively. Meanwhile, one, three, and 17 upregulated DEPs were, respectively, shared by “C2 vs. C1” and “M200 vs. C2”, “C2 vs. C1” and “M1000 vs. C2”, “M200 vs. C2” and “M1000 vs. C2” and one upregulated DEP was shared by “C2 vs. C1”, “M200 vs. C2”, and “M1000 vs. C2” B. Among Avena sativa, more than half of the DEPs were uncharacterized or predicted proteins, therefore, functional analysis was focused on the 195 DEPs with annotated function  pathway analysis was also performed. According to the Top 10 KEGG pathways , the resTo validate the proteomic data, avoiding the possible error of a single test method, qRT-PCR was performed to further verify the correspondence between protein abundance and their expression at mRNA transcriptional level . A totalVigna radiata roots under chilled and rewarmed conditions [Seed aging during storage is irreversible and results into the decrease or even loss of seed vigor, which in turn leads to serious agricultural problems . Delayednditions . SimilarSeed embryo is an important tissue where seed physiological aging and germination occur in oat. Here, the iTRAQ-based quantitative proteomics was adopted in order to study the embryonal protein changes during the process of seed aging and melatonin priming. On the basis of the proteomic data, a total of 402 DEPs were identified in the two processes and these DEPs were found to be related to multiple pathways, mainly including carbohydrate and energy metabolism , protein synthesis (ribosome), and amino acid metabolism.Seed aging is a naturally occurring event during storage, and priming treatment could promote germination and improve seedling consistency. When subjected to priming, seed embryonal cells transform from a static state into a highly active metabolic state, in which various physiological and cellular processes are initiated, including energy supply, protein synthesis, amino acid metabolism, and stress-related activities . AccordiCoix lacryma-jobi L.) seed during storage [Many DEPs were significantly enriched in sucrose metabolism, glycolysis, TCA cycle, and fatty acid metabolism. Sucrose synthase (SuSy) and sucrose-phosphate synthase (SPS) are two key enzymes in plant sucrose metabolism , respect storage . In agreLactuca sativa L.) seeds, there was a strong correlation between glycolysis related byproducts and aging. Therefore, our findings suggested that the disordered energy metabolism caused by PGK and PEPCase2 could result in seed aging in oat. Whereas, the data also showed that 200 μM melatonin priming significantly upregulated the expressions of PEPCase2, enolase 2 , and pyruvate kinase  by approximately 5.32-, 7.78- and 15.62-folds, respectively as compared with aged oat seeds , including three key enzymes of pyruvate dehydrogenase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase (DLD) . Acetyl-Arabidopsis mutants with β-oxidation destruction was impaired, when they germinated on medium without sucrose [Lipid is metabolized via β-oxidation to form acetyl-CoA, which enters into the TCA cycle and is eventually converted to hexose for seedling growth after germination . It was  sucrose . 3-Ketoa sucrose . In thisP5CS1 improved drought tolerance in rice [Proline is deemed to clean ROS and reduce the damage caused by abiotic stress in plants. Under adverse conditions, cells can control proline content by increasing proline synthase expression or inhibiting proline degradation. Chen et al.  found th in rice . The datAspartate aminotransferase (AspAT) catalyzes the reversible transamination between aspartate and 2-oxalate to produce glutamate and oxaloacetate, which plays a key role in the distribution of carbon and nitrogen in plants . GlutamaSHMT genes were found in Arabidopsis [SHM1 was reported to play a key role in regulating cell damage caused by abiotic stress. Additionally, the level of methionine synthetase 1 enzyme , involved in methionine metabolism, was increased by 2.27-fold after aging  and serine hydroxymethyltransferase  were upregulated by 2.31- and 2.09-folds, respectively, which played key roles in serine biosynthesis . Seven Sbidopsis , and SHMer aging , and thier aging , suggestThe deleterious effects of aging on seed germination are related to the protein synthesis system. As the organelles catalyzing protein synthesis, ribosomes consist of a large subunit and a small subunit . In thisVigna mungo L.) seeds was upregulated during artificial aging, indicating that aging promoted the production of damaged proteins and the substantial destruction of storage proteins, therefore, higher active 26S proteasome was required to perform the cleanup task. During aging of oat seeds, proteasome subunit beta type  was identified as a downregulated protein, and melatonin altered the expression of the other proteasome subunit alpha type   and significantly decreasing heat shock protein 83 , involved in endoplasmic reticulum protein processing, suggested that aging affected the protein processing, and then further could lead to protein damage. Nevertheless, melatonin increased the expression of 70 kDa heat shock cognate protein 1 (HSC-I), another molecular chaperone that helps protect cells against multiple stresses by repairing damaged proteins, indicating that the protein folding, denaturation, and degradation were improved in melatonin primed seeds. In addition, several identified DEPs related to proteolysis were also detected in this study. The ubiquitin-proteasome pathway is in charge of the elimination of proteins induced by misfolding, destruction, potential toxicity, and various cellular stresses in all phases of normal plant developmental events . As an i1J3K4N2) . The PSA2+ and has a role in the signal transduction pathway. CDPK is responsible for protein phosphorylation by transferring ATP phosphate to protein substrates, therefore, protein phosphorylation plays an essential role in many signaling pathways, such as cold, heat shock, salt, and ABA stress [Calcium signaling is an important regulator in response to stress in many plants . CalciumA stress . In thisVarious antioxidant mechanisms have evolved in plants to adapt to stress, such as ROS scavenging enzymes and proteins. In this study, several DEPs were identified, including thioredoxin (TRX), glutaredoxin (GRX), peroxiredoxin (PRX), and glutathione S-transferase (GST), which participated in the GPX pathway and the PRX/TRX pathway. TRX, a small conservative protein involved in plant oxidative stress response, operated as the antioxidant to restrict stress through scavenging hydrogen peroxide and certain radicals directly or served as a reductant for the regulation of several ROS-related enzymes, such as CAT, GR, GPX, and PRX . SimilarOat (cultivar “Cayuse”) seeds were available from Rytway Ecotechnology Company , with the original GP and the moisture content (MC), respectively, being 100% and 8.9% (fresh weight basis). Upon reception, seeds with uniform sizes and weights were selected through a 2.5 mm sieve, dehulled  and adjusted to 10% MC according to the saturated salt solution equilibrium static weighing method. Then, seeds were immediately sealed in hermetical aluminum foil bags  and stored at −20 °C in the dark prior to further experimentation.Oat seeds of 10% MC in bags (approx. 25 g each) were incubated in an electric thermostatic cistern  at 45 °C for 48 d to prepare the aged seeds of 70% GP  was 1:3, and melatonin solutions were changed at 12-hour intervals. After the scheduled priming treatments, seeds were washed in distilled water three times, surface-dried with filter paper, and air-dried back to 10% MC at 20 °C and 33% relative humid atmosphere. Three groups were prepared: (a) normal control, non-aged and non-primed seeds (C1); (b) aging control, aged but non-primed seeds (C2); and (c) melatonin treatments, aged seeds primed with various concentrations of melatonin . After melatonin priming treatments, the dried seeds were used to determine germinability, including GP, VI, and GI.A single layer of aged seeds was placed into a petri dish (110 × 110 mm) with a single layer of filter paper , to which the embryos were tightly attached. After that, aged seeds were primed with 1, 10, 100, 200, 500, and 1000 μM of melatonin solutions  at 20 °C for 24 h in darkness. The ratio of seed weight to melatonin solution volume  trichloroacetic acid, and then centrifuged at 15,000× g for 20 min at 4 °C. Reaction mixture contained 2.5 mL of 0.5% thiobarbituric acid in 5% (w/v) trichloroacetic acid and 2.5 mL of supernatant. The mixture was incubated at 100 °C for 15 min, immediately cooled to 25 °C and centrifuged at 4000× g for 20 min. The MDA content was determined by measuring absorbance at 532 nm and 600 nm.MDA content was determined to evaluate the LPO according to methods from Bailly et al. . Embryo 2O2, O2−·, and OH· were determined, respectively, using H2O2, O2−·, and OH· Assay Kit, according to the manufacturer’s instructions.The ROS including Hg for 20 min at 4 °C. As for APX activity assay, 0.5 mM AsA was added into the phosphate buffer. The resulting supernatant was used for assays of antioxidant enzyme activities.For extraction of antioxidant enzymes, embryo samples (0.2 g) were ground into powder with liquid nitrogen, homogenized in 4 mL of phosphate buffer , and then centrifuged at 15,000× 2O2 extinction [2O2. MDHAR was determined by measuring the decrease in absorbance at 340 nm as a result of NADH oxidation [2O2, 0.5 mM AsA, and enzyme extract. GR activity was assayed by measuring the decrease in absorbance at 340 nm due to NADPH oxidation [2, 0.5 mM GSSG, 1.5 mM NADPH, and enzyme extract. The protein content was quantified referring to the protein quantitative kit. All assays were repeated four times.SOD activity was assayed on the basis of its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) . One unitinction . Supernaxidation . Reactioxidation . Reactioxidation . Reactioxidation . ReactioAlready prepared embryo samples (stored at −80 °C) were used for protein extraction, with two biological replicates . The embryos (~0.2 g) were ground into powder in liquid nitrogen, extracted with 200 μL of cold lysis buffer , suspended using an ultrasonic processor  for 15 min, and then centrifuged at 13,000× rpm for 20 min at 4 °C. The supernatant was mixed well with 800 μL of cold acetone (containing 10 mM DTT), and incubated for ~2 h before being centrifuged at 13,000× rpm for 20 min at 4 °C. The collected precipitate was resuspended with 800 μL of cold acetone (containing 10 mM DTT), and then centrifuged at 13,000× rpm for 20 min at 4 °C. The pellet was air-dried and resuspended with 100 μL of lysis buffer. Protein concentration was quantified, and then stored at −80 °C for the next use.Proteins (100 µg) were mixed with 10 mM DTT and incubated at 37 °C for one hour, and then 55 mM iodoacetamide was added to alkylate at room temperature for one hour in the dark. After reduction and alkylation, 2 μg of trypsin  was added at a protein/trypsin ratio of 50:1, and then digested at 37 °C for 12 h, followed by centrifugation at 12,000× rpm for 15 min. After trypsin digestion, acidulation was conducted by adding an equal volume of 0.1% formic acid, then purified by a Strata-X C18 column  for three times, washed twice with 0.1% formic acid + 5% acetonitrile, and eluted with 0.1% formic acid + 80% acetonitrile. Peptides were evaporated to dryness by vacuum centrifugation, and then the dried peptides were reconstituted in 0.5 M TEAB solution (pH 8.5).2O. Then, the differentially labeled peptide mixtures were pooled and dried by vacuum centrifugation.According to the manufacturer’s protocol, proteins were labeled with an 8-plex iTRAQ Reagent Multiplex Kit . For labeling, one unit of iTRAQ reagent was thawed and reconstituted in 24 μL of isopropanol, and proteins were labeled with isobaric tags as follows: 113- and 114-tag for C1, 115- and 116-tag for C2, 117- and 118-tag for M200, and 119- and 121-tag for M1000. The labeled samples were incubated at 25 °C for 1 h, and the reaction was stopped with 100 μL of ddH4FA, pH 10.0, adjusted with NH3·H2O), loaded onto a Durashell C18 column , and fractionated using a high performance liquid chromatography (HPLC) system (Thermo Dionex Ultimate 3000 BioRS). Buffer A and buffer B  were used to develop a gradient elution. Peptides were eluted at a flow rate of 1 mL/min with a gradient of 5% B in 7 min, 25% B in 16 min, and 5% B in 25 min. Elution was monitored by measuring the absorbance at 214 nm. A total of 12 fractions were collected at 1-min interval, desalted with a C18 column  and dried in a vacuum concentrator for subsequent analysis.Pooled iTRAQ-labeled peptide mixtures were reconstituted with 100 μL of buffer A , and separated by a nano LC-MS/MS system connected to a Q-exactive HF-X mass spectrometer . Peptides were loaded onto the eksigent Chromxp Trap Column  by an autosampler, with a flow rate of 10 μL/min for 5 min, and then eluted onto an analytical C18 column . The samples were eluted with a gradient of buffer B  at a flow rate of 300 nL/min using the following elution program: 5% to 30% buffer B for 0 to 65 min, 30% to 50% buffer B for 65 to 70 min, 50% to 80% buffer B for 70 to 85 min, 80% to 5% buffer B for 85 to 90 min.The mass spectrometer was operated in the data-dependent mode to switch automatically between MS and MS/MS acquisition, and data was acquired using an ion source gas 1 of 5 psi, curtain gas of 35 psi, ion spray voltage floating of 2.5 kV, and interface heater temperature of 150 °C. The MS1 scan spectra were collected in the range 350–1500 m/z for 250 milliseconds, and mass tolerance was 50 mDa; the MS2 spectra were collected in the range 100–1500 m/z for 100 milliseconds; and dynamic exclusion was set to 12 s.Avena sativa, raw files were searched against a Uniprot protein database containing all plants . For protein identification, the following parameters were set: precursor ion mass tolerance ± 10 ppm; fragment ion mass tolerance ± 0.02 Da, maximum missed cleavages for trypsin digestion were set to two, carbamidomethyl (C) and iTRAQ8plex  were set as fixed modifications, and oxidation (M) and acetyl  were set as dynamic modifications. Peptides with a false discovery rate (FDR) of 0.01 and at least one unique peptide included for iTRAQ labeling quantification were selected for further analysis. The DEPs were identified on the basis of FC and p-value calculated through T-test of each protein, performed using Proteome Discoverer 2.1. In this study, identified proteins with FC ≥ 1.5 (or ≤0.67) and p-value ≤ 0.05 were considered as DEPs.Raw data from MS/MS were analyzed by Proteome Discoverer 2.1  using the Paragon algorithm and ProGroup algorithm to perform search engine. Due to the unavailable database for proteins of http://www.uniprot.org/). KEGG (Kyoto Encyclopedia of Genes and Genomes) database (http://www.genome.jp/kegg/pathway.html) was used to predict the main metabolic pathways.The DEPs were used for COG (cluster of orthologous groups of proteins) category and annotation. GO (gene ontology) annotation of DEPs, including the biological process, cellular component, and molecular function, was performed using Uniprot  and 1.0% denatured agarose gel electrophoresis. cDNA was synthesized using the PrimeScript™ RT reagent Kit with gDNA Eraser . The qRT-PCR was performed using the SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) , following the manufacturer’s instructions, with the ABI7900 Real-Time PCR thermal cycler (Applied Biosystems). Procedure of cycle conditions was as follows: 95 °C for 30 s, then 40 cycles at 95 °C for 5 s and 60 °C for 40 s, the final cycle of 95 °C for 10 s, 60 °C for 60 s, and 95 °C for 15 s. Gene ACTIN2 was selected as the reference gene to normalize the expression level of target genes, which were calculated using the 2Ct−∆∆ method. qRT-PCR determination was performed in three biological replicates and three technical replicates. The primer sequences used in this study are listed in Total RNA from embryos was extracted using a TRNzol Kit , according to the manufacturer’s instructions. The quantity of total RNA was confirmed by NanoDropp < 0.05 level. All analyses were performed by SPSS Statistics software (version 17.0).Results were presented as the means ± SE. Statistical analyses of the data were performed using one-way ANOVA test and the Duncan’s test was conducted to compare the means of different treatments at the On the basis of the conjoint analysis of ultrastructure, physiology, and proteomics, we confirmed a putative schematic pathway that could perform during melatonin-mediated germination in aged oat seeds . To summ