Risk scores were mainly proved to predict undiagnosed type 2 diabetes mellitus (UT2DM) in a non-invasive manner and to guide earlier clinical treatment. The objective of the present study was to assess the performance of the Finnish Diabetes Risk Score (FINDRISC) for detecting three outcomes: UT2DM, prediabetes, and the metabolic syndrome (MS).This was a prospective, cross-sectional study during which employees aged between 30 and 64, with no known diabetes and working within the faculties of the Lebanese University (LU) were conveniently recruited. Participants completed the FINDRISC questionnaire and their glucose levels were examined using both fasting blood glucose (FBG) and oral glucose tolerance tests (OGTT). Furthermore, they underwent lipid profile tests with anthropometry.P = 0.001, P = 0.003 and P = 0.001) respectively. The AUROC value with 95% Confidence Interval (CI) for detecting UT2DM was 0.795 (0.822 in men and 0.725 in women), 0.621(0.648 in men and 0.59 in women) for prediabetes and 0.710 (0.734 in men and 0.705 in women) for MS. The correspondent optimal cut-off point for UT2DM was 11.5 (sensitivity = 83.3% and specificity = 61.3%), 9.5 for prediabetes (sensitivity = 73.6% and specificity = 43.1%) and 10.5  for MS.Of 713 subjects, 397 subjects  completed the blood tests and thus were considered as the sample population. 7.6% had UT2DM, 22.9% prediabetes and 35.8% had MS, where men had higher prevalence than women for these 3 outcomes (The FINDRISC can be considered a simple, quick, inexpensive, and non-invasive instrument to use in a Lebanese community of working people who are unaware of their health status and who usually report being extremely busy because of their daily hectic work for the screening of UT2DM and MS. However, it poorly screens for prediabetes in this context. Diabetes mellitus (DM) is a chronic metabolic disorder characterized by persistent hyperglycemia . It is pThe prevalence of diabetes, prediabetes, and MS is increasing rapidly worldwide , 6. AccoGiven all these pieces of evidence, early detection and treatment of prediabetes, diabetes, and MS would delay progression to diabetes as well as its relevant health and economic burdens , 7, 13. Till now, several risk assessment tools have been developed for initial screening. However, many of them are not widely accepted and practically used since they may require invasive and expensive blood testing such as the Atherosclerosis Risk in Communities score (ARIC) and the Framingham Offspring score , 15. In This was a prospective, cross-sectional study, conducted between January 2018 and May 2019 over two phases. During the first phase, the staffs working within the faculties of the LU , aged older than 30 to 64 years and who accept to submit the inform consent were recruited by convenience sampling from the campuses of the LU across four regions in Lebanon  and filled the FINDRISC questionnaire. However, individuals who reported having diabetes, women who reported to be pregnant, and people with any physical disability that prevents anthropometric measurements were excluded. In the second phase, faculties were contacted in advance by phone for a blood test analysis appointment.FINDRISC was originally used as a prediction tool to identify patients at risk of developing diabetes over next 10 years . It consBodyweight and waist circumference (WC) were measured for each participant by Nutrition students . Weight Blood Pressure measurement was taken after the participant had been seated and relaxed for 5 min without any distractions, using an automatic monitor  with appropriate cuff’s size. Furthermore, the person’s upper arm was put into the cuff loops, 1 or 2 cm above the elbow, then letting it comfortably rest on the table . Those wParticipants were instructed to fast for at least 12 h and abstain from vigorous exercise in the evening and the morning of the investigation. They were also asked to abstain from caffeine and smoking on the morning of the visit. After ensuring that the fasting period was accomplished completely, the blood sampling procedure was explained by a trained laboratory technician. A total of 3–5 ml of fasting venous blood sample was collected in a serum clot activator tube and centrifuged at 4000 rpm for 10 min on the same day, and then serum was transferred to another tube and stored at -22 °C for biochemical examination. Following this, a load of 75 g of anhydrous glucose in a volume of 200 ml was administered to each individual for the oral glucose tolerance test (OGTT) . After tWe had three outcome variables of interest: UT2DM, Prediabetes, and MS.Individuals who had FBG level ≥ 126 mg/dl (7.0 mmol/l) or 2-h blood glucose (2-h BG) ≥ 200 mg/dl (11.1 mmol/l) were classified as UT2DM.Participants who had impaired fasting glucose (IFG) i.e. blood glucose (BG) ≥ 100 (5.6 mmol/l) and < 126 mg/dl (7.0 mmol/l) or impaired glucose tolerance (IGT) i.e. 2 h blood glucose (2 h BG) ≥ 140 (7.8 mmol/l) and < 200 mg/dl (11.1 mmol/l) were considered as prediabetics.Both UT2DM and Prediabetes were defined according to the latest American Diabetes Association (ADA) criteria .IndividuAbdominal obesity (WC ≥ 102 cm in men and ≥ 88 cm in women)Hypertriglyceridemia (TG ≥ 150 mg/dl or 1.695 mmol/l)Low HDL-C (HDL < 40 mg/dl in men and < 50 mg/dl in women)Elevated blood pressure (Systolic blood pressure SBP > 130 mmHg or Diastolic blood pressure DBP > 85 mmHg) or the use of antihypertensive medication.Elevated blood glucose (FBG ≥ 100 mg/dl).Whereas, MS was defined according to the latest National Cholesterol Education Program Adult treatment panel III (NCEP ATP III) diagnostic criteria (2005revision) . At leasy-axis, and the false-positive rate (1-specificity) was plotted on the x-axis. Then optimal cut-off points were determined by the point with the closest distance to  in the ROC curve which maximizes the sensitivity and specificity of the test (Tradeoff between sensitivity and specificity).Statistical analysis was conducted using SPSS . Descriptive statistics of those who underwent blood tests were expressed as means (± standard deviation) for continuous variables and as proportions for categorical variables. Differences in the socio-demographic (SD) variables between genders were computed using an independent samples t-test for continuous variables and a Chi-square test for categorical variables, while between FINDRISC categories using a one-way ANOVA test for continuous variables and a Chi-square test for categorical variables. To evaluate the FINDRISC accuracy performance we calculated the area under the receiver operating curve (AUROC), sensitivity (the probability that the test is positive for subjects with type 2 diabetes), specificity (the probability that the test is negative for subjects without type 2 diabetes) with 95% CIs . To create the ROC curve, sensitivity was plotted on the P = 0.248). Men had higher BMI , WC , were heavier smokers  and were on BP medication more than women . However, women had a stronger 1st degree family history of T2DM  and were relatively less physically active . The mean values of the blood tests including FBG (P < 0.001), OGTT (P < 0.001), TG (P < 0.001), SBP (P = 0.001) and DBP (P = 0.003) were higher in men than in women, except for HDL-C (P < 0.001), LDL-C (P = 0.006) and TC (P < 0.001)  did not follow the complete blood tests procedures; and therefore, the data of the remaining 397 were used for the validation of the FINDRISC. The sample was composed of 219 women (55.2%) and 178 men (44.8%). The mean age was 48.5 (± 9) and there was no statistical significance between men and women in terms of age (P = 0.001), prediabetes (P = 0.003) and MS (P = 0.001) (Table 30 (7.6%) individuals had UT2DM, 91 (22.9%) had prediabetes and 142 (35.8%) had MS. In addition, Men had statistically higher prevalence of UT2DM (The AUROC curve for detecting UT2DM was 0.795 (95% CI: 0.728–0.862) overall, with 0.822 (0.749–0.895) for men better than, 0.725 (0.589–0.861) for women Fig. . SimilarThe AUROC curve for detecting prediabetes was 0.621 (95% CI: 0.557–0.684) overall, with 0.648 0.563–0.734) for men better than, 0.59 ~ 0.6 (0.492–0.687) for women  overall, with 0.734 (0.661–0.807) for men slightly better than, 0.705 (95% CI: 0.626–0.784) for women , BMI 32 , FBG 110.2 , OGTT 170.6 , TG 227 , HDL-C 35.2 , SBP 138.2  and, DBP 88.2 . Subjects with a FINDRISC category of 15–20 had the highest prevalence of history of elevated blood glucose 22 , UT2DM 11  and, TC 14 . Older individuals 51.4 , current smokers 35  and, those with elevated blood LDL-C levels 126.8  are located within a FINDRISC category of 12–14, with a statistical significant except for smokers. Those with prediabetes 33  and MS 51  had a FINDRISC category of 7–11  of the LU Campuses, but a poor performance regarding prediabetes.The prevalence of UT2DM and prediabetes in our study was 7.6% and 22.9% respectively. However, a recent study carried out in the Bekaa, which is a rural area in Lebanon  found thOriginally, the FINDRISC questionnaire was developed longitudinally as a future predictor of diabetes in the Finnish population  and was P < 0.0001) and 76% of those with UT2DM had MS and that’s why the threshold for MS is localized between the thresholds for prediabetes and UT2DM in our community. To date, only one study assessed the predictive ability of FINDRISC in detecting incident cases of MS (AUC = 0.65) rather than prevalent cases at a cutoff of 12 [In this study, FINDRISC had a good discriminative ability for detecting UT2DM with an AUROC value of 0.795  and a threshold value of 11.5 . Besides, it’s also good at detecting MS in both men and women with an AUROC of 0.7  at a threshold of 10.5 . Whereas, this ability gets weaker in case of prediabetes as evidenced by an AUROC of 0.621  especially in women. Several studies showed similar values and also confirmed that FINDRISC performed better in detecting UT2DM and MS than prediabetes. In a previous cross-sectional study on the general population of the United States of America , the AURff of 12 .It is also worth mentioning that men had always higher AUROC values as well as lower cutoff values than women, specifically for UT2DM and MS in the current study. In other words, men tend to have more risk factors putting them at a higher risk for diabetes, prediabetes, and MS which improves the predictive ability of FINDRISC when compared with women and increases their scoring in FINDRISC and thus limiting their threshold to lower values. In this study, a synergistic interaction for the combined BMI (p < 0.0001), WC (p < 0.0001), smoking (p < 0.0001), could renders men more prone for diabetes with higher prevalence for UT2DM (p = 0.001) and MS (p < 0.0001).The advantage of the FINDRISC relies on its self-report questions so that LU workers that reported to be extremely busy because of their work and daily life stressors can find it easier to fill the FINDRISC quickly and rate their current health status. Being at higher risk based on FINDRIC score would be a sufficient trigger for them to start applying lifestyle changes or to seek health professionals’ help.Some limitations warrant considerations. First, a misclassification bias could be introduced because the diagnosis of diabetes of the respondents was self-reported. Further, the diagnosis of diabetes and prediabetes of the included participants was not confirmed by repeat testing on a separate day as recommended . HoweverThis study has also considerable strengths. To our knowledge, this is the second study that has been carried out in an Arabic country in the Middle East region which has investigated the validity of FINDRISC. A previous study was conducted in Kuwait and showed similar results . AdditioThis cross-sectional study has successfully demonstrated that FINDRISC could be useful as a first-line screening tool that identifies employees with UT2DM, prediabetes, and MS that might benefit from lifestyle modification. FINDRISC model could be also beneficial for community-based interventions and screenings as well as in clinical practice by the health professionals. In future studies, FINDRISC should be validated on a larger and more representative sample of the Lebanese population so Lebanese citizens living in a resource-poor setting like rural areas would benefit the most. Also, FINDRISC should be assessed in a longitudinal study who allows the identification of incident cases of diabetes and MS rather than prevalent cases.


Pramipexole (PPX), a D2-like receptor agonist, is generally used in the treatment of Parkinson's disease and restless leg syndrome. Its neuroprotective effects have been shown against various neurological disorders. Recent research work has demonstrated that PPX exerts neuroprotection through mitochondria. However, the neuromodulator-related effects of PPX against traumatic brain injury (TBI) remain unexplored. The present study, therefore, investigated the mechanism of neuroprotection by PPX against oxidative stress, mitochondrial dysfunction and neuronal damage following TBI in rats. We hypothesized that the neuroprotection by PPX in TBI-subjected rats might involve activation of the Nrf2/HO-1  signaling pathway. PPX was injected intraperitoneally (0.25 mg/kg body weight and 1.0 mg/kg body weight) at different time intervals post-TBI. Several neurobehavioral parameters were assessed at 48 h post-TBI, and the brain was isolated for molecular and biochemical analysis. The results demonstrated that PPX treatment significantly improved the behavioral deficits, decreased the lipid peroxidation rate, increased glutathione levels and decreased 4-hydroxynonenal levels in TBI-subjected rats. PPX also increased the activities of glutathione peroxidase and superoxide dismutase enzymes. In addition, PPX treatment inhibited mitochondrial reactive oxygen species production, restored mitochondrial membrane potential and increased ATP levels after a TBI. Further, PPX treatment reduced the Bax/Bcl2 ratio and translocation of Bax to mitochondria and cytochrome-c to the cytosol. Finally, PPX treatment greatly accelerated the translocation of Nrf2 to the nucleus and upregulated HO-1 protein expression. We conclude that the neuroprotective effects of PPX are mediated by activation of the Nrf2/HO-1 signaling pathway following TBI.This article has an associated First Person interview with the first author of the paper. Editor's choice: We administered pramipexole (PPX) to rats after a traumatic brain injury (TBI) and evaluated various parameters, which indicate that the neuroprotective effects of PPX are mediated by activation of the Nrf2/HO-1 signaling pathway following TBI. A traumatic brain injury (TBI) can have severely detrimental effects on human health. It is associated with difficulties in communication and behavioral impairments, which, in turn, contribute to socioeconomic problems for people of all ages throughout the world . During Experimental evidence indicates that mitochondria are the power plant of a cell. Mitochondria provide energy via oxidative phosphorylation and enable neurons to maintain proper function as well as control various pathological processes . MitochoHO-1; also known as Hmox1), superoxide dismutase family members (SODs) and glutathione reductase (Gsr) in the nucleus  is a transcription factor and important upregulator of the endogenous cell defense mechanism in response to oxidative damage and is involved in restoration of intracellular homeostasis . Nrf2 is nucleus . It has  nucleus  and neur nucleus . Thus, mPramipexole (S)-2-amino-4,5,6,7-tetrahydro-6-propylamino benzothiazole di-hydrochloride monohydrate (PPX) is a dopamine (D3/2) receptor agonist. The US Food and Drug Administration (FDA) has approved it as a drug used for the treatment of Parkinson's disease and restless leg syndrome . It is rTherefore, the present study investigated the neuromodulatory effects of PPX after a TBI. We examined the effects of PPX on TBI-induced oxidative damage and mitochondrial injury in experimental rats. In addition, we explored the neuroprotective effects of PPX on mitochondrial perturbation and its possible ameliorative mechanisms in the brain after a TBI. To the best of our knowledge, this is the first study on TBI that shows that PPX treatment ameliorates oxidative damage and mitochondrial dysfunction, and promotes an antioxidant defense system through activation of the Nrf2/HO-1 signaling pathway. Findings from the study provide new insights into the potential therapeutic value of PPX for TBI.F=11.9, P<0.0001] in the mean grip score compared with the sham rats. However, post-treatment with PPX at 1.0 mg/kg b.wt. significantly increased (P<0.05) the mean grip score in the TBI rats. There was no significant change in mean grip score at a low dose of PPX =11.0, P<0.0001] time on the rotating rod compared with the sham rats. PPX post-treatment significantly increased [0.25 mg/kg b.wt. and 1.0 mg/kg b.wt. (P<0.05 for both doses)] the time spent on the rotarod in the TBI rats =14.7, P<0.0001] to reach the box than did the sham rats. PPX post-treatment [0.25 mg/kg b.wt. (P<0.05) and 1.0 mg/kg b.wt. (P<0.01)] significantly improved the beam balance test performance, and PPX-treated rats were able to cross the beam in a shorter time compared with the TBI rats =6.82, P=0.0014] and an increased LPO rate [measured as µmol/nmol thiobarbituric acid reactive substances (TBARS) formed/h/g tissue or mg protein]  in penumbra tissue as well as isolated mitochondria  of rats in the TBI group compared with those in the sham group. Post-treatment with PPX (1.0 mg/kg b.wt.) significantly restored the GSH levels [tissue (P<0.01) and mitochondria (P<0.05)] and reduced the LPO rate [tissue (P<0.05) and mitochondria (P<0.01)] in brain tissue and isolated mitochondria in the TBI-subjected rats. We also investigated the levels of the carbonyl-containing molecule 4-hydroxynonenal (4-HNE), which is the end product of lipid peroxidation. We observed significantly upregulated (P<0.01) 4-HNE levels in the TBI group compared with the sham group. PPX administration (1.0 mg/kg b.wt.) significantly reduced (P<0.05) the levels of 4-HNE in the TBI rats =12.8, P=0.0001 and F=15.8, P=0.0001, respectively] compared with the sham rats. Post-administration of PPX (1.0 mg/kg b.wt.) significantly increased (P<0.05) the activity of both enzymes in the TBI rats. However, low-dose PPX (0.25 mg/kg b.wt.) failed to exert significant effects.To evaluate the antioxidant effects of PPX against focal TBI, we evaluated biochemical parameters such as lipid peroxidation (LPO), glutathione (GSH), glutathione peroxidase (GPx) and superoxide dismutase 1 (SOD1). As shown in TBI rats B. Next, F=41.6, P=0.0001] in ROS production, as indicated by the increase in DCF fluorescence in the TBI group compared with the sham group. Post-treatment with PPX significantly reduced [0.25 mg/kg b.wt. (P<0.05) and 1.0 mg/kg b.wt. (P<0.001)] ROS production in the TBI rats  fluorescence. TBI induced a significant increase  in the mean fluorescence intensity (MFI) of tetramethylrhodamine ethyl ester (TMRE) dye in the TBI group compared with the sham group. This finding, as mentioned above, is indicative of mitochondrial dysfunction. However, PPX-treated rats showed significantly increased Δψm [0.25 mg/kg b.wt. (P<0.05) and 1.0 mg/kg b.wt. (P<0.01)] compared with the TBI rats, as shown in With regard to the effects of TBI on mitochondrial membrane potential (Δψm), we found a significant decrease  in ATP content in the TBI rats compared with the sham rats. Interestingly, 1.0 mg/kg b.wt. PPX administration increased (P<0.01) ATP content among the TBI rats, but there was no significant change in ATP content with a low dose of PPX  content in the experimental rats' brain tissue. We observed a significant decrease  in TBI rats compared with the sham rats. When we compared the expression of HO-1 in the PPX-treated groups and TBI only group, we observed that PPX post-treatment increased the expression of HO-1 after a TBI.Western blot analysis was applied to detect the expression level of Nrf2 protein in nuclear as well as cytosolic fractions. As shown in P<0.01) compared with the sham rats. Additionally, PPX (1.0 mg/kg b.wt.)-treated rats had a significantly higher number of intact neurons (P<0.05) compared with the TBI only rats. No significant changes were observed at a low dose of PPX , which is reversible. The secondary injury begins after the primary injury, regulates various cascades, and activates neuronal apoptosis and dysfunction . OxidatiDuring normal conditions, antioxidant enzymes such as SOD1 and GPx can inhibit oxygen free radical generation and protect the brain tissue against oxidative damage. In the present study, we found that TBI significantly elevated LPO rate and 4-HNE levels, and reduced GSH levels and the activities of SOD1 and GPx. We demonstrate, for the first time, that PPX significantly reduced LPO rate and 4-HNE, and significantly increased the activities of SOD1 and GPx antioxidant enzymes. Thus, our study results reveal that PPX ameliorates oxidative damage induced by TBI.Loss of mitochondrial function has long been linked with the onset of neurodegenerative pathologies, such as Alzheimer's disease, Parkinson's disease, stroke  and TBI in vivo , Bax and Bak1] play an essential role in the neuronal apoptotic pathway in various neurological diseases. Bax is a pro-apoptotic protein that is involved in TBI-induced apoptosis in the brain, whereas Bcl2 is an anti-apoptotic protein and a strong neuronal death suppressor . Increasin vivo . Based oExcess free radical production can not only modulate oxidation of macromolecules but also initiate apoptosis as single molecules . There ain vitro and in vivo models , obtained from the Central Animal House Facility of Jamia Hamdard, New Delhi, India, were used in the present study. The Institutional Animal Ethics Committee (IAEC) of Jamia Hamdard approved all animal experiments . Experimental rats were kept in an isolated environment in a 12 h light-dark cycle with free access to filtered water and food Table S1 for further details on the numbers of animals in experimental groups. Animals were sacrificed by cervical dislocation after deep anesthetization with an overdose of ketamine and xylazine. The damaged brain tissue area was isolated for analysis of biochemical and molecular assessment at 48 h after TBI.The experimental animals were randomly divided into four groups: sham, TBI, TBI+0.25PPX  and TBI+1PPX . Please refer to TBI was induced by a controlled cortical impact device with minor modifications in our laboratory . In brieThe experimental animals were kept in an isolated chamber at 22±3°C in individual cages . All experimental rats received 0.9% saline (intravenously) to prevent dehydration, and included administration of antibiotic  and analgesic . Standard rodent pellet diet and filtered RO water were kept in the cages throughout the 48-h experimental period so that the experimental animals could easily access it without any physical disturbance due to the craniotomy.PPX , at doses of 0.25 mg/kg b.wt. and 1.0 mg/kg b.wt., was dissolved in 0.9% sterile normal saline and administrated by intraperitoneal injection with repeated doses at 30 min, 8 h, 16 h, 24 h, 32 h 40 h and 48 h following TBI.Motor coordination was evaluated by Omnitech rotarod apparatus  as described previously . ExperimThe grip strength test was performed as described previously . A 50-cmA narrow beam walk was used to analyze the motor impairment of rats as described previously . Rats weg for 20 min at 4°C to obtain post-mitochondrial supernatant (PMS). Mitochondria were isolated by centrifugation using the methods of g for 10 min at 4°C, then the supernatant was collected and again centrifuged at 10,000 g for 10 min at 4°C. Thereafter, the pellet was collected and resuspended in ice-cold buffer B, containing 250 mM sucrose, 10 mM HEPES and 0.1 mM EGTA, pH 7.4, then centrifuged at 12,300 g for 10 min at 4°C. The pellet was resuspended in buffer B and centrifuged at 12,300 g for 15 min. The final obtained pellet was dissolved in buffer C, pH 7.4, containing 250 mM sucrose, 10 mM HEPES and 0.1% fat-free BSA, and centrifuged at 12,300 g for 20 min at 4°C. The final pellet was resuspended in buffer C. Biochemical parameters were performed on the same day after the isolation of mitochondria.Animals were euthanized, and the injured tissue was carefully dissected  from the ipsilateral hemisphere using an adult rat brain slicer matrix  for biochemical and molecular analysis. Brain tissue from the same location was isolated in the sham group rats. Tissue was homogenized in ice-cold 0.1 M phosphate buffer (pH 7.4) to obtain 10% homogenate (w/v) and centrifuged at 21,380 g for 10 min at 4°C, and absorbance was measured at 535 nm. The rate of LPO was measured as µmol/nmol TBARS formed/h/g tissue or mg protein using a molar extinction coefficient (MEC) 1.56×105 M−1 cm−1.LPO was assessed in the injured ipsilateral tissue as well as isolated mitochondria according to the procedure of g at 4°C. A supernatant was used for further analysis to determine the GSH content. For mitochondrial GSH content, mitochondrial samples (300 µg protein) were incubated in 500 µl of 0.1 M/l phosphate buffer (pH 7.5) for 10 min at 4°C, and further steps were performed according to manufacturer guidelines. The results were expressed as GSH activity (ng GSH/mg protein).GSH levels were measured using a reduced GSH colorimetric assay kit according to the manufacturer’s guidelines . Tissue homogenate  was centrifuged at 10,000 2O2. Oxidation of NADPH was measured at 340 nm. The enzyme activity was expressed as nmol NADPH oxidized/min/mg protein using the MEC 6.22×103 M−1 cm−1.GPx activity was measured according to the procedure of −1 cm−1.SOD1 activity was measured using the methods of Mitochondrial ROS production was analyzed by flow cytometry analysis using di-chloro, di-hydro-fluorescein-diacetate (DCFDA) dye and the methods of Δψm was measured by flow cytometry analysis using TMRE dye and the methods of g for 10 min, and then the supernatant was mixed with substrates and transferred into 96-well plates for colorimetric analysis.ATP content was measured using an ATP assay kit  according to the manufacturer’s guidelines. Brain tissue was homogenized in double-distilled water and incubated in a water bath at 90°C for 30 min. After incubation, the homogenate was centrifuged at 10,000 g for 30 min at 4°C. After centrifugation, the supernatant was resolved by 10-15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. Subcellular fraction isolation was performed using fractionation kits  according to the manufacturer’s guidelines. The protein bolts were blocked with 5% donkey serum. After blocking, blots were incubated with primary antibodies, including those against Bcl2 , cyt-c , 4-HNE , β-actin , Bax , Nrf2 , histone H3 , HO-1  and COX-IV  overnight at 4°C. The next day, bolts were washed with PBST  and incubated with appropriate horseradish peroxidase-conjugated secondary antibodies  for 90 min at room temperature. The level of protein expression was visualized by enhanced chemiluminescence. Immunoblot densitometry analysis was performed using ImageJ version 1.50.At 48 h after TBI, injured right ipsilateral brain sample was homogenized in lysis buffer in the presence of protease and phosphatase inhibitor cocktail as reported by 2 in each picture area of the cortex and expressed as the mean of three different brain sections for each group of rats.For Nissl staining, coronal sections were stained with 1% Cresyl Violet  staining solution for 30 min at room temperature. Then, brain sections were washed with regular tap water and a dehydration procedure was performed using ethanol grades  for 5 min at room temperature, before fixing with DPX (Sigma-Aldrich) mounting medium. Brain sections were observed under an inverted Zeiss microscope. The number of intact neurons was counted per mmP-value <0.05.All results from the present study were collected by experimenters blinded to treatment conditions and results were reported as the mean±s.d. for immunoblots. For behavioral and biochemical analyses, values were expressed as the mean±s.e.m. Comparisons among multiple groups were analyzed using one-way ANOVA followed by Tukey's post hoc test, and statistical analysis was performed using Prism software version 6 . Significant differences were defined as


The as-prepared carbon flakes display wrinkled edges and comprise Fe3C nanoparticle and active site of Fe–Nx. These carbon materials exhibit excellent electrocatalytic performance. Towards oxygen reduction reaction (ORR), the optimized sample has Eonset and Ehalf-wave of 0.93 V and 0.83 V in alkaline system, respectively, which are very close to that of Pt/C. This approach may offer a new way to high performance and low-cost electrochemical catalysts.Introduction of both nitrogen and transition metal elements into the carbon materials has demonstrated to be a promising strategy to construct highly active electrode materials for energy shortage. In this work, through the coordination reaction between Fe In recent years, much attention has been focused on the development of facile and applicable methods to fabricate high-activity, low-cost oxygen reduction reaction (ORR) catalysts. This is significant to overcome the challenges in the commercialization and industrialization of hydrogen fuel cells. Nevertheless, Pt/C still acts as a main role in the commercialized ORR catalysts, even though it is relatively expensive ,2,3,4. Tx, M = Cu, Ni, Co, Fe and Mn) and melamine/aniline, can be used to prepare transition metal doped carbon materials with enhanced ORR properties [x and Fe–Cx) [Indeed, transition metal elements have been widely introduced into the carbon materials to achieve high electrochemical performances ,10,11,12operties . More inoperties . Notablyd Fe–Cx) ,17,18. Ud Fe–Cx) ,20. The d Fe–Cx) ,22. So, 3+ coordination strategy using commercial resources as starting materials, can create metallosupramolecular polymer precursors for the fabrication of Fe and N elements co-doped carbon materials with high ORR activity. The organic ligand adopted in this research is 1,3,5–tris(4–aminophenyl)benzene (denoted as TA) possessing three amine groups and conjugated structure. Because of the rigid structure of TA, the as-formed TA–Fe coordinative networks display uniform layer structure. After carbonization, carbon flakes with wrinkled edges (denoted as CNSs) can be easily generated. We have found that Fe3C nanoparticle and active site of Fe–Nx are formed in the carbon matrix, which can promote the ORR activity of the carbon materials. In addition, the feature of this research lies in the simple synthesis of CNSs. It can introduce Fe3C nanoparticle and Fe–Nx into the carbon matrix by one step. This synthetic route has certain universality and representativeness. It is also found that the appropriate molar ratio between amino ligand and Fe3+ was the most important factor that determines the activity of CNSs.In this report, we show that a simple ligand-Fe1,3,5–Tris(4–aminophenyl)benzene, iron(III) chloride hexahydrate and dichloromethane were supplied by Aladdin Company  and directly used. Anhydrous ethanol, anhydrous methanol, KOH, and hydrochloric acid were purchased from Shanghai Chemical Reagent Industry . Nafion (5 wt%) was supplied by Sigma-Aldrich .2 atmosphere. Thus, the corresponding molar ratios between TA and Fe3+ were 1.0:0.6, 1.0:0.8, and 1.0:1.0, respectively. The mixtures became yellow, and suspensions of metallosupramolecular polymers were formed after 12 h of reaction. The yellow powders were collected by centrifugation and washing with a mixed solvent of 30 mL dichloromethane and 30 mL anhydrous methanol three times. After drying in vacuum at 50 °C overnight, TA–Fe precursors were obtained . These precursors were then carbonized at 850 °C for 2 h in argon gas with a heating rate of 5 °C/min to generate the carbon materials. The carbon materials were washed by 6 M HCl for 5 h and 100 mL of ultrapure water three times at room temperature, then dried by freeze-drying overnight. A second carbonization was performed at 850 °C for 2 h in argon gas with a heating rate of 10 °C/min to get the target carbon materials .TA  was firstly dissolved in 100 mL of dichloromethane with vigorous stirring for 3 h. Then, 1.38, 1.84, and 2.30 mL methanol solutions of Iron(III) chloride hexahydrate  were added into the solution drop by drop under vigorous stirring under NThe scanning electron microscopy (SEM) images were observed through an SU-70 microscopy instrument . The FTIR measurements were tested by an AVATAR 360 FTIR  at room temperature. The powder X-ray diffraction (XRD) patterns were measured through a Desktop X-ray Diffractometer  using Cu (600 W) Kα radiation. Raman spectra were tested by a Labram HR800 Evolution . The X-ray photoelectron spectroscopy (XPS) were tested by PHI Quantum-2000 photoelectron spectrometer . The pore volume and Brunauer–Emmett–Teller (BET) were taken through an ASAP 2460 system . Electron paramagnetic resonance (EPR) experiments were conducted on an electron spin resonance spectrometer  at 90 K. Transmission electron microscopic (TEM) measurements were performed using a JEM-2100 microscope . The elemental energy-dispersive X-ray spectroscopy (EDX) were obtained by using a FEI TECNAI F20 microscope .2O and 50 μL of 5 wt% Nafion. The above newly made slurry (4.5 μL) was carefully dropped onto a glassy carbon electrode as working electrode. The ORR performance was tested in newly made KOH aqueous solution (0.1 mol/L) at room temperature. Pt foil and Ag/AgCl (KCl saturation) electrode were separately applied as counter electrode and reference electrode. The potential in this study was relative to the Ag/AgCl electrode.The electrochemical experiments were conducted on an electrochemical workstation , by using the typical three-electrode system. A standard rotating disk electrode with a glassy carbon disk (5 mm in diameter) was applied as working electrode. Before test, CNS–0.6Fe, CNS–0.8Fe, and CNS–1.0Fe (5.0 mg) were dispersed in 1.0 mL of homogeneous solvent with 500 μL of anhydrous ethanol, 450 μL of H3+ can easily induce the formation precipitates. After a pyrolysis treatment of the precursors, carbon materials comprising both Fe3C nanoparticle and Fe–Nx active site can be fabricated easily.The synthetic process of precursor is very simple. As shown in 3+ was verified by FTIR as shown in −1 correspond to the characteristic signals of amino groups of TA, and the peaks at 845, 685, and 600 cm−1 are derived from iron(III) chloride hexahydrate. Comparing the spectra of precursors with TA, the characteristic peak of amino groups shifts and becomes broaden. Also, the characteristic peaks of Fe3+ in the precursors were evidently weakened. These results indicate the coordination between Fe3+ and TA [The typical SEM image of TA–0.8Fe is displayed in + and TA .g’ = 2.00. For TA–0.6Fe, TA–0.8Fe, and TA–1.0Fe, the representative signal of Fe3+ at g’ = 4.25 can be observed, indicating the presence of Fe3+ in the precursor [3/2 and 2p1/2 orbitals of Fe3+, respectively. 3+ [2 than TA–0.6Fe and TA–1.0Fe, which may result in more content of Fe–Nx active site after carbonization.In the EPR spectra a, all sarecursor . The XPS3C nanoparticle  plan of graphitic carbon  derived from disordered graphitic structure and G band (1571 cm−1) derived from ordered carbon structure are calculated to be 0.97, 0.95, and 1.04, respectively. This result indicates that the graphitic degree of CNS–0.8Fe is higher than that of the other samples. The crystalline structures of CNSs were evaluated by the XRD. As displayed in 3C form. All the diffraction peaks are in good agreement with that of the Fe3C (JCPDS Card No.65−2413). These results, in combination with the high-resolution TEM images, prove the presence of Fe3C nanoparticle in the CNSs catalysts, which may promote the ORR activity of carbon materials [aterials .2·g−1, with relevant pore volumes of 0.16, 0.17, and 0.17 cm3·g−1, respectively. Obviously, surface areas of CNSs are resulted from both micro-pore and mesoporous-pore structures. The CNS–0.8Fe has a higher BET surface area than CNS–0.6Fe and CNS–1.0Fe. We consider that the large surface area of CNS–0.8Fe is attributed to the moderate crosslinking degree of the metallosupramolecular polymer networks.The pore character of CNSs was characterized through the physisorption of nitrogen at 77 K. As shown in x, graphitic N, and oxidized N. Notably, Fe–Nx and pyridinic N are recognized to be promising for the improvement of ORR activity [3+ for the 2p1/2 band, and the peak of Fe2+ is detected at 723.2 ± 0.1 eV for the 2p1/2 band. Another two peaks at 714.4 ± 0.1 and 710.5 ± 0.1 eV can be respectively attributed to the binding energies of 2p3/2 orbitals of Fe3+ as well as Fe2+ species. The last signal at 719.6 ± 0.1 eV is the satellite peak. These XPS results, in combination with the XRD results, clearly confirm that the presence of Fe3C nanoparticle, and active sites of Fe–Nx and pyridinic N in the carbon matrix of CNSs. Moreover, as listed in x contents of CNS–0.8Fe are 0.28 and 0.43 at.%, respectively, which are much higher than that of CNS–0.6Fe (0.17 and 0.20 at.%) and CNS–1.0Fe (0.25 and 0.22 at.%). This result indicates that CNS–0.8Fe may have more active sites towards ORR. In summary, the Fe and N elements co-doping effect leads to the generation of both Fe–Nx active site and Fe3C nanoparticle when carbonization, which can greatly improve the catalytic activity towards ORR. That is the main mechanism and contribution of the co-doping effect of Fe and N elements in CNSs.The XPS survey spectra of the CNSs are shown in activity ,29. The 2-saturated 0.1 M KOH solution  of CNS–0.6Fe, CNS–0.8Fe, and CNS–1.0Fe for ORR are 0.89, 0.93, and 0.88 V vs. RHE, separately. The half-wave  of the CNS–0.6Fe, CNS–0.8Fe and CNS–1.0Fe are 0.78, 0.83, and 0.80 V vs. RHE, separately. The above results were confirmed by LSV tests in the newly made O2-saturated solution at the scanned rate of 10 mV/s with the fixed rotation speed of 1600 rpm. As a control experiment, the ORR activity of Pt/C catalyst was also tested with the same experimental condition (onset (0.93 V) and Ehalf-wave (0.83 V) values of CNS–0.8Fe are very close to Pt/C catalyst . The Tafel plots of CNSs were tested  and directly indicating that CNS–0.8Fe owns faster ORR kinetics.The CV curves of CNSs were tested in both Ar or Osolution a. The saondition b. Apparee tested . The TafD/IG value testified that the graphitic degree of CNS–0.8Fe is higher than the others, thus endowing this sample with a better electrical conductivity. Fourth and most importantly, CNS–0.8Fe possesses higher pyridinic N and Fe–Nx contents than CNS–0.6Fe and CNS–1.0Fe, which can provide more active sites towards ORR, thus greatly enhancing the catalytic activity.The evidently improved electrocatalytic performance of CNS–0.8Fe among the three samples can be explained by the following four reasons. First, CNS–0.8Fe has relative higher specific surface area in comparison with CNS–0.6Fe and CNS–1.0Fe, thus resulting in the expose of more active sites. Second, CNS–0.8Fe possesses a better developed lamellar structure than CNS–0.6Fe and CNS–1.0Fe as indicated by the TEM images, which is beneficial for the contact between active sites and oxygen molecules during ORR process. Third, the calculated I2− also with electron transfer numbers (n) values. CNS–0.6Fe, CNS–0.8Fe, CNS–1.0Fe, and the commercial Pt/C displayed higher disk current but minor ring current, as shown in 2− yield ranges of CNS–0.6Fe, CNS–0.8Fe, CNS–1.0Fe, and Pt/C catalyst are 10.57 to 11.90%, 3.99 to 6.22%, 4.66 to 5.93%, and 1.81 to 2.71%, as shown in The LSV curves of CNSs were also collected with different rotation rates. The current density of CNS–0.6Fe, CNS–0.8Fe, and CNS–1.0Fe increase gradually when consecutively changing the rotation speeds from 400 to 1600 rpm, as listed in 2-saturated 0.1 M KOH solution  as reported [Taking CNS–0.8Fe as an example, the durability of CNSs was evaluated at 1600 rpm with consecutive 1000 cycles of CV scan in Osolution . The decreported . This rereported ,35,36.3+ could easily afford metallosupramolecular polymer precursors. After two carbonization processes, carbon flakes with wrinkled edges and active site of Fe–Nx and Fe3C nanoparticle were fabricated. The catalytic activity of the carbon materials towards ORR were detailed investigated. The carbon material of CNS–0.8Fe possessed Eonset = 0.93 V and Ehalf-wave = 0.83 V vs. RHE in alkaline system, which were comparable to Pt/C catalyst. The ligand TA and Fe3+ could generate more content of Fe–NH2 in the precursor at a proper proportion through the coordination reaction and further led to the generation of more content of Fe–Nx active site when carbonization. So, the appropriate molar ratio between amino ligand and Fe3+ was the most important factor that determined the activity of CNSs. We considered that this simple method and conclusion might be of practical interest for the exploration of electrocatalysts with excellent ORR activity.In summary, we prepared a new type of Fe and N co-doped carbon materials through a simple and effective method in one step. Direct coordination between amino ligand and Fe


The stability of the reference gene expression levels was validated using the geNorm, NormFinder, BestKeeper, and RefFinder algorithms. The results indicated that EF-1α was the most stably expressed and suitable reference gene overall and for the heat, cold, and ABA treatments. Additionally, UBQ expression was stable following the salt treatment, whereas TUB was identified as a suitable reference gene for H2O2 and drought treatments. The reliability of the selected reference genes was verified by analyzing the expression of copper/zinc superoxide dismutase (Cu/Zn-SOD) gene in luffa. When the most unstable reference genes were used for data normalizations, the resulting expression patterns had obvious biases when compared with the expression patterns for the most ideal reference genes used alone or combined. These results will be conducive to more accurate quantification of gene expression levels in luffa.Selecting suitable internal reference genes is an important prerequisite for the application of quantitative real-time PCR (qRT-PCR). However, no systematic studies have been conducted on reference genes in luffa. In this study, seven reference genes were selected, and their expression levels in luffa plants exposed to various simulated abiotic stresses [i.e., cold, drought, heat, salt, H The advantages of qRT-PCR include its specificity, sensitivity, and reproducibility as well as the fact it can be completed very quickly and efficiently, making it appropriate for assessing gene expression and rapidly quantifying mRNA transcripts5. However, the accuracy of this quantitative analysis can be influenced by the initial template quantity, the RNA quality, and the enzymatic reaction efficiency7. Therefore, normalizing target gene expression levels against the data for suitable reference genes is essential for minimizing the biases associated with qRT-PCR results.Quantitative real-time PCR (qRT-PCR), which is a molecular technique enabling researchers to quantitatively analyze nucleic acids, was developed based on the common PCR technique and has been widely used in many research fields 18S), β-tubulin (TUB), α-tubulin (TUA), elongation factor 1 alpha (EF-1α), ubiquitin (UBQ), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and actin (ACT) genes9. However, many studies have confirmed there is a lack of universal reference genes for plants. The suitability of specific reference genes depends on the experimental conditions, and the use of unstably expressed reference genes will likely produce biased results and false-positives11. Gutierrez et al.12 revealed that several genes commonly used as reference controls in Arabidopsis thaliana studies are unstably expressed. Additionally, using non-validated reference genes can lead to 100-fold changes in target gene expression levels. Therefore, selecting and validating reference genes under diverse experimental conditions is crucial before a meaningful qRT-PCR analysis can be performed. There are currently several publicly available online statistical tools, including geNorm, NormFinder, BestKeeper, and RefFinder, developed to identify a series of appropriate reference genes as internal controls for normalizing qRT-PCR data.Reference genes, also known as housekeeping genes, refer to genes that are generally stably expressed in different tissues and developmental stages as well as under various experimental conditions. Common reference genes include 18S ribosomal RNA , which belongs to the family Cucurbitaceae, is one of the most important vegetables and widely used medicinal plants in China. Many luffa plant parts and components  have been used in cough remedies and for treating rhinitis and lumbago. Therefore, there may be a considerable market for luffa products, making the commercial production of luffa an economically important industry13. However, the early luffa fruit development stage generally coincides with a period of heavy rainfall and high temperatures, which can damage seedlings , leading to considerable decreases in productivity. Plants are exposed to a variety of abiotic and biotic stresses that can restrict growth and development, with abiotic stresses proven to adversely affect crop yield and quality14. Consequently, the mechanism regulating the adaptation of luffa to abiotic stresses must be elucidated, including the related genes, which necessitates the selection of suitable reference genes for gene expression analyses. To date, the research regarding reference genes appropriate for luffa has been quite limited , and the stability of reference genes has rarely been reported. In the present study, five common reference genes  were cloned from luffa seedlings. These genes along with two previously cloned reference genes, UBQ16 and 18S, were analyzed to identify the most reliable reference genes for normalizing target gene expression via qRT-PCR. Experimental samples were exposed to various stresses, including heat, cold, H2O2, salt, abscisic acid (ABA), and drought. Four algorithms  were applied to evaluate the reference genes and to identify the most stably expressed genes under diverse conditions. Furthermore, to assess the utility of the validated reference genes, the expression levels of copper/zinc superoxide dismutase (Cu/Zn-SOD) gene under the above conditions were examined. The results of these analyses may provide the basis for future qRT-PCR-based investigations of the transcription of important functional genes in luffa.ACT, TUA, TUB, EF-1α, and GAPDH genes were 1134, 1350, 1350, 1344, and 1491 bp long, respectively , Momordica charantia (XP_022143797.1), and Theobroma cacao (XP_017977121.1) sequences. The TUA amino acid sequence was more than 98% identical to Momordica charantia (XP_022153822.1), Cucurbita moschata (XP_022944288.1), and Vitis vinifera (XP_002285721.1) sequences. The TUB amino acid sequence was more than 96% identical to Cucumis sativus (XP_004139067.1), Momordica charantia (XP_022153267.1), and Brassica oleracea (VDD33972.1) sequences. The EF-1α amino acid sequence was more than 96% identical to Cucurbita moschata (XP_022959419.1), Gossypium hirsutum (XP_012468306.1), and Vitis vinifera (XP_002284924.1) sequences. The GAPDH amino acid sequence was more than 96% identical to Cucurbita moschata (XP_022937833.1), Cucumis melo (XP_008456541.1), and Momordica charantia (XP_022143215.1) sequences. These sequence matches confirmed that the obtained sequences corresponded to the ACT, TUA, TUB, EF-1α, and GAPDH genes.The sequencing results indicated that the full-length ORF sequences of the ACT, TUA, TUB, EF-1α, GAPDH, UBQ, and 18S) were selected as candidate reference genes. Information regarding these genes and their qRT-PCR primer pairs are summarized in Table TUB) to 104.2% (18S), and the correlation coefficients varied from 0.988 (ACT) to 0.997 (18S)  were highly expressed, with average Ct values between 20 and 22, whereas two genes (TUB and ACT) were moderately expressed, with Ct values between 24 and 26. Of all analyzed genes, the highest and lowest expression levels were calculated for UBQ (mean Ct of 20.13) and ACT (mean Ct of 26.25), respectively. Additionally, the most and least variable expression were detected for GAPDH and TUA, respectively, suggesting that GAPDH was the most unstably expressed gene, whereas TUA was the most stably expressed gene.The expression levels of the seven reference genes were calculated based on the Ct values . Four algorithms  were used to analyze the expression of relatively stable reference genes.In this study, gene expression was examined in plants exposed to various abiotic stress conditions  were the most stably expressed genes, whereas TUA (M = 0.75) was the least stably expressed gene. Regarding the H2O2 treatment, the most stably expressed genes were UBQ and 18S (M = 0.82) and the least stably expressed gene was GAPDH (M = 1.66). In response to the salt treatment, TUB and TUA (M = 0.71) had the most stable expression levels, whereas 18S (M = 1.75) had the most unstable expression level. For the ABA treatment, EF-1α and TUB (M = 0.56) were detected as the most stably expressed genes and GAPDH (M = 1.26) was identified as the most variably expressed gene. Following the drought treatment, the expression levels were most stable for ACT and 18S (M = 0.52) and least stable for GAPDH (M = 1.37). Overall, EF-1α and TUA (M = 0.73) were the most stably expressed genes in all samples, whereas GAPDH (M = 1.64) was the least stably expressed gene , ACT (CV ± SD = 0.59 ± 0.149) was the most stably expressed. For the cold treatment, all seven reference genes were stably expressed, but ACT (CV ± SD = 1.16 ± 0.298) and EF-1α (CV ± SD = 1.63 ± 0.342) were the most stably expressed. Regarding the salt treatment, the expression of four reference genes was stable, with UBQ (CV ± SD = 2.98 ± 0.56) identified as the most stably expressed gene. For the H2O2 treatment, four reference genes were suitable for normalizing gene expression data, among which TUB (CV ± SD = 2.13 ± 0.494) and EF-1α (CV ± SD = 2.7 ± 0.513) were the most stably expressed. In response to the ABA treatment, five reference genes were stably expressed, with TUA (CV ± SD = 1.9 ± 0.44) and ACT (CV ± SD = 1.87 ± 0.484) detected as the most stably expressed genes. Following the drought treatment, the expression levels of five reference genes were considerably stable, with TUB (CV ± SD = 2.25 ± 0.553) and TUA (CV ± SD = 2.46 ± 0.554) revealed as the most stably expressed genes. An analysis of all treatments indicated that four reference genes were stably expressed, with ACT (CV ± SD = 3.33 ± 0.875) identified as the gene with the most stable expression level  after heat, cold, salt, H2O2, ABA, and drought treatments was analyzed with BestKeeper, geNorm, NormFinder, and RefFinder. The objective of this study was to identify the most stable reference genes for investigating gene expression in luffa plants exposed to abiotic stress factors. The data revealed EF-1α as the most stably expressed gene following the heat, cold, and ABA treatments. This gene, which encodes a polyribosomal protein, is widely expressed in various cells. In many studies on Cucurbitaceae species, EF-1α has been used as an appropriate reference gene. In cucumber, EF-1α, Fbox, CAC, and TIP41 are reportedly stably expressed regardless of the abiotic stress, growth regulator treatment, and nitrogen status31. In another study on cucumber, EF-1α was confirmed as the most stably expressed reference gene32. In an earlier study on zucchini, UFP, EF-1α, RPL36aA, PP2A, and CAC were identified as the ideal reference genes for normalizing expression data33. Ye et al.34 screened and evaluated reference genes for fluorescence-based real-time quantitative PCR analyses of wax gourd and confirmed that EF-1α is the most stably expressed reference gene under cold conditions and in different tissues. In the current study, UBQ was the most stably expressed reference gene following the salt treatment, which is consistent with the results of an earlier study by Jin et al.35. The UBQ gene belongs to the ubiquitin gene family, which affects many important biological processes, including cell cycle regulation, growth and apoptosis, signal transduction, and immune responses. Because of its high sequence homology and the fact it is highly conserved, UBQ has been used as a reference gene for rice36, black fungus37, and cucumber32. Our data also indicated that TUB was the most stably expressed reference gene for the H2O2 and drought treatments. This gene is involved in plant growth and development, with an important role in maintaining cell morphology, promoting intracellular transport, and mediating cell movement and cell division, and has often been used as a reference gene. Kiarash et al.38 determined that TUB is the most appropriate reference gene for wheat plants under drought conditions. Although ACT is the most commonly used reference gene for cucurbit crops41, it was not identified as the best reference gene for any of the conditions analyzed in this study. Furthermore, ACT expression reportedly varies substantially in many other crops42. Kong et al.20 suggested that ACT should be used together with other reference genes for qRT-PCR analyses of melons. In the current study, except for the cold treatment, GAPDH was the most unstably expressed reference gene, implying that GAPDH is not a suitable reference gene for investigations of the effects of abiotic stresses on luffa gene expression. This is consistent with the results of studies by Liu et al.27 and Yu et al.43. Although GAPDH is the most unsuitable reference gene for annual ryegrass and ramie under different stress conditions, it is ideal for studies of Juglans regia L44, L. speciosa45 and Stipa grandis46. Therefore, potential internal reference genes must be evaluated and confirmed as appropriate for different experimental systems and materials before they are used to analyze gene expression.In this study, the expression stability of seven candidate reference genes :8-h dark (18 °C) cycle and 65–75% relative humidity. Seedlings at the third true leaf stage were used for the following treatments. For the heat and cold treatments, the seedlings were incubated at 40 °C and 4 °C, respectively, for 24 h. For the ABA and H2O2 treatments, the seedlings were sprayed with 200 μM ABA and 100 μM H2O2, respectively. Regarding the salt and drought stress treatments, the seedlings were transplanted into full-strength Hoagland’s nutrient solution containing 100 mM NaCl or 15% PEG 6000, respectively, for 24 h. Leaves were then collected at 0, 2, 6, 9, 12, and 24 h after the treatments. Each experiment was completed with three replicates, each comprising five seedlings. All collected leaf samples were immediately frozen in liquid nitrogen and stored at − 80 °C until analyzed.‘Minyan No. 1’, a commercial FTotal RNA was extracted from the frozen leaf samples with the MiniBEST Plant RNA Extration Kit . The NanoDrop ND1000 spectrophotometer  was used to calculate the RNA concentration and assess purity. The RNA samples with a 260/280 nm absorbance ratio of 1.8–2.0 were retained for further analyses. The RNA integrity was evaluated by 1% agarose gel electrophoresis. The PrimeScript II First Strand cDNA Synthesis Kit (TaKaRa) was used to synthesize cDNA.ACT, TUA, TUB, EF-1α, and GAPDH) were identified following a search of an established luffa transcriptome database. Complete open reading frames (ORFs) were obtained for all sequences. Gene-specific primers were designed to clone and verify the ORF sequences of the five genes  for each qRT-PCR primer pair were calculated with a fivefold cDNA dilution series52.The seven genes initially selected as candidate reference genes included the 2O for a final volume of 20 μL. The amplification conditions were as follows: 95 °C for 30 s; 40 cycles of 95 °C for 5 s, 60 °C for 34 s, and 72 °C for 10 s, followed by a melting curve analysis from 65 to 95 °C. A blank control lacking a template was also analyzed. The gene expression levels for each sample were determined based on three replicates.A qRT-PCR assay was completed with SYBR Premix Ex Taq (TaKaRa) and the QuantStudio 7 Flex Real-Time PCR System . The reaction solution consisted of 10 μL SYBR Green PCR Master Mix (TaKaRa), 2 μL cDNA (100 ng), 0.8 μL 10 µM forward primer, 0.8 μL 10 µM reverse primer, 0.4 μL ROX Reference Dye II, and 6 μL ddHn/Vn+1. NormFinder calculates a stability value (SV) for each gene based on the variance analysis, and the gene with the lowest SV is identified as the most stably expressed gene. For both algorithms, the Ct values should first be transformed by 2−ΔCt. BestKeeper mainly determines the stability of reference gene expression based on the standard deviation (SD), Pearson correlation coefficient (r), and the coefficient of variation (CV) of the Ct data for all reference genes. The most stably expressed gene is the one with the lowest SD and CV values. Finally, the reference genes were ranked based on the geometric mean (GM) values calculated with RefFinder (http://www.leonxie.com/referencegene.php).The cycle threshold (Ct) value was recorded for each qRT-PCR analysis of gene expression. Box plots were drawn to visualize the reference gene expression levels and variations. The geNorm, NormFinder, BestKeeper, and RefFinder algorithms were used to assess the expression stability of selected genes. Specifically, geNorm calculates the expression stability value (M) and pairwise variation (V), and the most stably expressed gene is the one with the lowest M value. Moreover, geNorm determines the optimal number of reference genes according to the relative value VCu/Zn-SOD gene were determined and normalized against the data for the two most stable and the least stable reference genes22. The qRT-PCR amplification conditions were as described above and the primer pairs are listed in Table −ΔΔCt method53. Statistical analyses were performed with the SPSS 18 program.To confirm the reliability of the reference genes, the expression profiles of Not applicable.All authors have consented for publication.Supplementary Information


Albizia procera (ApCP) was encapsulated in graphene quantum dots (GQDs). The insecticidal activity of ApCP, with or without GQDs, against two stored grain insect pests, Tribolium castaneum (Herbst) and Rhyzopertha dominica (Fabricius) was explored. Insects were exposed to three concentrations 7.0, 3.5 and 1.7 mg of ApCP per a gram of wheat flour and grains. The insecticidal activity of ApCP encapsulated with GQDs was improved compared to that of ApCP without GQDs for both insect pests. The number of eggs and larvae of T. castaneum was reduced by 49% and 86%, respectively. Larval mortality was increased to 72%, and adult eclosion of T. castaneum was reduced by 98% at a 7.0 mg/g concentration of ApCP with GQDs compared to that of ApCP without GQDs. Exposure to 7.0 mg/g ApCP with GQDs, the number of R. dominica eggs and larvae was reduced by 72% and 92% respectively, larval mortality was increased by 90%, and eclosion was reduced by 97%. The extraction, purification, characterization, quantification and encapsulation of ApCP with GQDs were also studied. Cysteine protease nanocarriers have the potential to control stored grain insect pests.Storing grains remain vulnerable to insect pest attack. The present study developed a biopesticide using biomolecules and their encapsulation in nanoparticles. A 25 kDa cysteine protease extracted from seeds of  The moisture level in the reserve rises due to higher infestation, and grains become heated, causing hot spots. Insects move from hot spots to cooler areas for egg laying, and fungus develops in the grain stock, which causes a decrease in grain quality3.According to an estimate, 25% of food worldwide is damaged as post-harvest losses, out of which 20% of the damage is caused by insect pests through discoloration, change in flavour, weight loss, fungal infestation, reduced nutritional value, and poor germination4. These chemicals minimize production losses caused by insect pests, weeds and microbial diseases5. Pesticides are a low-cost, fast and efficient source of pest control6. However, they are toxic and pollute the environment. Drawbacks also include human intoxication7 and chronic diseases such as cancer, asthma, diabetes, leukaemia, endocrine disorders, and Parkinson’s disease10. Synthetic pesticides can be substituted by botanical pesticides for which research is in progress11. Biopesticides are biodegradable, safe for non-target organisms and high-yield and they may replace conventional pesticides12. Biomolecules such as plant proteins have the insecticidal activity and can be used to develop biopesticides. They are encapsulated in nanocarriers to maximize the molecular absorption to target sites15. Cysteine proteases are proteolytic enzymes involved in chitin degradation of the exoskeleton and peritrophic matrix in the midgut in insects (such as in the cowpea weevil), resulting in mortality16. Catalytic residues of proteolytic activity are Cys-25, His-159, and Asn-17517.Food can be secured by using crop protection chemicals and pesticides21. Nanomaterial synthesis has given rise to quantum dots, which are tiny particles of a few nanometres in size. They are central in nanotechnology and are also called artificial atoms22. Quantum dots are photostable, optically sensitive and can be easily traced. Different sizes of quantum dots are responsive to different frequencies of light23. In agriculture, quantum dots are being applied as nanobiosensors in the detection of plant diseases, quality control of food, and monitoring of food additives, mycotoxins, microbial infestation, allergens, pesticide residues and precision farming26. Graphene-based quantum dots assume novel chemical/physical properties. GQDs (graphene quantum dots) also show low cytotoxicity, excellent solubility, stable photoluminescence, and better surface grafting and pose no environmental hazards as do toxic metal-based quantum dots, instead exhibiting eco-friendly behaviour28. Silica- and metal-based nanoparticles are also used to repel and control insect pests29. In the present study, Albizia procera cysteine protease (ApCP) was encapsulated in graphene quantum dots (GQDs) to increase its insecticidal activity and adsorption to the target site. The formulation was tested against two stored grain insect pests. ApCp encapsulation with GQDs provides biopesticides with directed delivery and environmental safety.Nanotechnology has redefined medicine, engineering, agriculture, the food industry, cosmetics and other fields of scienceA. procera seed coat had a 25 kDa cysteine protease as a major protein constituent. The ApCP sample was separated from the non-protein content by dialysis. SDS-PAGE analyses of reduced and non-reduced samples showed that ApCP had no inter-chain disulfide linkages , followed by Hordeum vulgare EP-B2 (85.7%) and Carica papaya chymopapain (85.7%), as shown in Table ApCP were prepared with closely related plant cysteine proteases  analysis revealed the crystallinity and phase of the material. GQDs exhibited peaks at 19.11° and 32.09° for Graphene oxide and 28.51° for graphite. The peaks matched those listed in the database of JCPDS cards (1906–29). The particle size range was calculated as 17 nm from the X-ray diffraction results by the Scherrer equation  characterization of graphene quantum dots indicated C=C skeletal vibrations of aromatic stretching, carboxyl groups and hydroxyl groups at 1684 and 3400 cm−1 Fig. . There wion Fig. . The parion Fig.  and quanion Fig. .Figure 3ApCP was extracted in phosphate buffer and distilled water to evaluate the best extraction and consequent maximum insecticidal activity. Extracts were tested against T. castaneum and R. dominica, and eggs, larvae, larval mortality and adult eclosion were evaluated. Insecticidal activities of ApCP extracted by phosphate buffer and distilled water were not significant for T. castaneum eggs , larvae , larval mortality  and adults .ApCP extracted by phosphate buffer and distilled water on R. dominica eggs , larvae , larval mortality  and adults  were also similar as control without ApCP  against T. castaneum after 24 hours of treatment showed that the LC50 value with encapsulation was 0.759 mg/L and it was 0.894 mg/L without encapsulation. After 48 hours, the LC50 values were 0.620 mg/L (with encapsulation) and 0.827 mg/L (without). Similar toxicity was observed after 72 hours of treatment, i.e., LC50 values of 0.577 mg/L and 0.634 mg/L with and without encapsulation, respectively. Moreover, GQDs encapsulation did not affect the LC50 of ApCP to T. castaneum  and 0.724 mg/L (without). Similar toxicity was observed after 72 hours of treatment, i.e., LC50 values of 0.449 mg/L and 0.501 mg/L with and without encapsulation, respectively. The toxicities after 24, 48 and 72 hrs were similar (95% FL overlapped with each other). Encapsulation of GQDs was responsible for a slight change in LC50, but the results remained not significant.The contact toxicity data Tables  and 2 ofT. castaneum was subjected to ApCP alone or encapsulated in GQDs. The number of eggs of T. castaneum at 7.0, 3.5 and 1.7 mg/g ApCP, with and without GQD encapsulation, was concentration dependent, i.e., the highest concentration exhibited the lowest number of eggs. ApCP encapsulated in GQDs resulted in a statistically lower number of eggs , in contrast to that of ApCP treatments without GQDs (455.8 ± 31.5) at maximum concentration. Moreover, the control had 667.6 ± 23.1 eggs. The ApCp insecticidal efficacy increased by 49%, 51% and 45.4% for the three tested concentrations with encapsulation compared to that of ApCP without encapsulation  at high concentrations of ApCP , whereas the highest number of larvae was found in the control (284.4 ± 39). ApCP treatments without GQD encapsulation induced a significant decrease in larvae with increasing concentration . However, in treatments without encapsulation treatments, larvae were high in number (275.2 ± 23.4) compared to those in encapsulated treatments at the same concentrations. The difference in insecticidal efficacy (in terms of larvae count) between encapsulated and non-encapsulated ApCP at the three tested concentrations was 85.7%, 80% and 76.8%, respectively . Larval mortality (when comparing the encapsulated and non-encapsulated ApCP) increased by 72.8%, 68% and 67% at the three concentrations, respectively  compared to that of the ApCP treatments . The adult eclosion of the insects decreased by 98.2%, 96.7% and 93.7% for the three concentrations of ApCP with GQD encapsulation compared to that for the three concentrations of ApCP alone  or fed a control diet without ApCP, the results of eggs number were statistically significant . The number of eggs of R. dominica in the highest concentration and in the control was 116.4 ± 9.6 and 362.5 ± 9.2, respectively. The feeding toxicity of the same three concentrations of ApCP without encapsulation against R. dominica also revealed significant differences  from those of treatment with encapsulated ApCP. At the highest concentration, the lowest number of eggs were recorded (418.2 ± 37.9), while 513 ± 32.9 eggs were found at the 3.5 mg/g concentration. The highest number of eggs were documented at the lowest concentration (578 ± 36.1), which was similar to that of the control (677.6 ± 41.6). Insects laid 72.2%, 65.3% and 61.2% fewer eggs at the three concentrations in decreasing order when exposed to ApCP encapsulated with GQDs . Larvae were minimal at the highest concentration (17.4 ± 1.7), and the maximum was observed in the control (314.2 ± 25.6). Treatment with 7.0 mg/mL ApCP without encapsulation resulted in 227 ± 26.3 larvae, and 454 ± 40.5 larvae were observed in the control. The insecticidal efficacy of ApCP after GQD encapsulation was improved by 92.4%, 92% and 92% at the three tested concentrations in order of decreasing concentration (Table ApCP treatment in GQDs (148.4 ± 20.4), and low mortality (33 ± 1.8) was observed in the control . Larval mortality increased by 89.8%, 90% and 88.8% at the three concentrations of ApCP encapsulated with GQDs compared to that for ApCP without GQDs  . Statistically higher adult eclosion of R. dominica was recorded in ApCP without GQD encapsulation treatments . Adults were 78.6 ± 8.2 in number, in contrast to 2.2 ± 0.9 (in encapsulated treatments) at 7.0 mg/g. ApCP encapsulated with GQDs tended to show 97.3%, 94.4% and 94% less adult eclosion at the three tested concentrations in decreasing order  larval count (larvae) and larval mortality of treated insects.The present study reported the insecticidal activity of cysteine protease derived from ApCP is promising against T. castaneum and R. dominica, which are the key pests of warehouse commodities worldwide. The insecticidal activity was tested for the life cycle span of insect pests. Protein-treated insect pests showed a reduction in life cycle parameters including eggs, larvae and ecloded adults. The control experiments showed maximum adult eclosion compared to protein-treated units. Graphene was chosen as a carrier for ApCP molecules to target the basement membrane, chitin, of the exoskeleton and peritrophic membrane of the insect mid gut. Disruption of the peritrophic matrix may affect larval development and other processes such as digestion, digestive enzyme recycling and nutrient absorption35. Small-sized GQDs with encapsulated ApCP can enter into extracellular spaces and/or spaces between the cell membrane, hydrolysing the proteinaceous content of chitin and disrupting normal biochemical processes, leading to insect death . The enzymatic activity of cysteine proteases is due to the catalytic dyad formed by cysteine and histidine residues, which exist as an ion pair (–S–…H+Im–) in the pH range of 3.5–8.0T. castaneum and R. dominica were maintained at the Eco-Toxicology Laboratory in the Department of Entomology, Bahauddin Zakariya University, Multan, Pakistan . Homogenous cultures of R. dominica and T. castaneum were maintained by rearing insects with wheat grains and flour, respectively, at 30 ± 5 °C and 60 ± 5% RH. Insects were separated by sex at the pupal stage by following the standard protocols41.Insects A. procera was separated, ground into a fine powder and suspended in Tris buffer  with 0.15 M NaCl. The mixture was centrifuged at 5300 rpm for 15 min, and the supernatant was collected. Crude extract was dialysed  overnight with the same buffer to remove the salt from the extract. The extract was examined via SDS-PAGE by preparing one-dimensional 12% gels  to see the protein bands. Reduced (β-mercaptoethanol-treated) and non-reduced samples were heated at 95 °C for 5 min in a heating block to denature the samples. The gel was stained with Coomassie brilliant blue R-250 dye (CBBR-250) (Sigma Aldrich) to visualize the protein bands, and the molecular weights were determined by using Protein Ladder .The seed coat of The protein sample was purified through acetone precipitation at a 1:5 (sample: acetone) ratio and centrifuged. The pellet was mixed in 1.0 M ammonium bicarbonate solution. Protein in the extract was determined by reverse phase HPLC using a Tecknochroma C18 column at a 60 min linear gradient of 0.1% TFA in water to 0.1% TFA in ACN at 0.1 mL/min. Protein was quantified by using a Nanodrop spectrophotometer.42. Digested gel pieces were extracted with a 50% acetonitrile/5% formic acid solution and dried in a vacuum concentrator. LC-MS/MS measurements were made by loading samples on a nano-liquid chromatography (n-LC) system (Dionex UltiMate 3000) coupled via electrospray ionization (ESI) to an orbitrap mass spectrometer . The protein solution was loaded on a trapping column  with 2% buffer B, and peptides were eluted (200 mL/min). LC-MS/MS analysis was carried out in data-dependent acquisition (DDA) mode. The raw data were processed with Proteome Discoverer 2.0 . For identification, MS/MS spectra were investigated with Sequest HT against Arabidopsis and the UniProtKB server, while identifications were manually validated.Protein bands stained with Coomassie dye were excised from the gel and reduced with dithiothreitol . In-gel protein digestion was performed overnight with trypsin as per the described protocolApCP were made in Tris buffer solution and poured into beakers. Filter papers were immersed in the three tested concentrations for 30 sec with gentle agitation and then allowed to dry. Treated filter papers were set into petri dishes. Five pairs of newly born T. castaneum and R. dominica adults were placed in the petri dishes in five replicates. The mortalities were recorded after 6, 12, 24 and 72 hours of each concentration for both insects.Petri dishes of 5 cm diameter were washed with distilled water and dried. Filter papers were cut into the same diameter. Three concentrations  of ApCP  were prepared in Tris buffer. These concentrations were mixed with 150 g pre-sanitized wheat flour and grains for T. castaneum and R. dominica, respectively. The control was prepared with wheat flour and grains in Tris buffer. The treated flour, grains and control samples were spread thinly to dry in air. Wheat flour was ground into a fine powder. Treated flour and grains were divided into 5 replicates of 30 g in separate jars in each concentration of ApCP. Each replicate/jar was supplied with 5 pairs of newly born T. castaneum and R. dominica. Treatment jars were placed at 30 ± 5 °C and 60 ± 5% RH. The adults of both insects were removed after one week. Treatment units were checked weekly to determine the changes in life cycle parameters of treated insects. Observations were recorded in terms of the number of eggs and larvae, larval mortality and adult eclosion of treated insects. ApCP was extracted in 100 mL of pH 7 phosphate buffer and distilled water to check the maximum activity of protein. Feeding toxicity assays were conducted for the same life cycle parameters of both insects.Three concentrations of 43 obtained by a refined Hummer’s method44. Two grams graphite powder was mixed with 120 mL sulfuric acid in an ice bath and stirred for 30 min. KMnO4 (11.6 g) was gradually mixed while heating at 30 °C for 2 hrs. Then, 80 mL deionized water was added, and the temperature was raised to 90 °C for 30 min. The temperature was lowered to 60 °C, and 160 mL deionized water was added. Next, 30% H2O2 was added until an orange-yellow solution was obtained. Then, 400 mL 5% HCl was mixed in, and the pH was maintained at 5 by washing through vacuum pump assembly. Blackish grey graphene oxide was obtained. One gram graphene oxide was agitated with 20 mL H2SO4 for 2 hrs in an ice bath. Afterwards, 10 mL 50% KMnO4 was added, the mixture was stirred for 2 hrs, and the temperature was raised to 50 °C for one hour. Then, 80 mL distilled water was added, and the temperature was increased to 90 °C for 30 min. After homogenization of KMnO4, 20 mL H2O2 was added to the ice bath, followed by distilled water until a yellow-brown solution appeared. The solution was ultrasonicated, and the pH was increased to 8 by adding NaOH. HCl was added to lower the pH to 4, giving a dark greenish-yellow mixture. Graphene quantum dots were characterized by Attenuated total reflectance (ATR), X-ray diffraction (XRD), Scherrer equation (www.intanano.com) and atomic force microscopy (AFM). Quantum dots were mixed with distilled water at concentrations of 0.5%, 0.25% and 0.12% and assessed for their contact and feeding toxicity to the stored grain insects using the methods discussed above.Graphene quantum dots were prepared from graphite powderSolutions of 100, 50, 40, 30, 20 and 10 ppm of commercial papaya latex cysteine protease (papain)  were prepared from a 1000 ppm stock solution. A calibration curve of commercial papain was obtained using the standard solution. Solutions were mixed with quantum dots and shaken for 30 min, followed by filtration. Absorbance was recorded by using a UV-Visible spectrophotometer. Encapsulation was optimized by altering parameters such as the weight of GQDs, solution concentrations and temperature (below 50 °C). Different weights of GQDs  were used with a single concentration (100 ppm) at room temperature. Furthermore, a 100 ppm solution was mixed with 0.05 g GQDs at temperatures of 10, 20, 30, 40 and 50 °C. Similarly, 0.05 g GQDs were mixed with 10, 20, 30, 40 and 50 ppm papain solutions at room temperature. Encapsulation efficiency was calculated by the equation below:ApCP was performed with optimized conditions of loading capacity percentage, i.e., weight of GQDs, solution concentration and temperature. ApCP encapsulated with GQDs was subjected to contact and feeding toxicity assays against two stored grain insect pests at the three above-mentioned concentrations by following the previously discussed methods. Papain is the most studied cysteine protease for its insecticidal activity and has a well-explained mode of action in the insect midgut of disrupting the peritrophic matrix. In this study, papain was also used (as a reference protein) to study its insecticidal effects with and without encapsulation of GQDs at a concentration of 3.5 mg/g. The hypothesis of increased insecticidal efficiency with the formulation of proteins with GQDs also proved to be successful for papain encapsulated in GQDs against tested stored grain insect pests.Encapsulation of 45 analysis. For feeding toxicity assays, insects were observed weekly during their life cycle span to evaluate changes in the mean number of eggs and larvae, larval mortality and adult eclosion. Data were subjected to ANOVA, and homogenous groups were further subjected to Tukey’s HSD test through Statistix 8.146.For the contact toxicity bioassay, insects were observed under 6, 12, 24, 48 and 72 hours for mortality and subjected to ProbitSupporting information.


Chroma values of all the tissues reduced (p < 0.05) at 23 °C. At 4 °C, chroma values fluctuated between storage days. The TAC of the peel was the highest (p < 0.05) among the different tissues and continued to increase up to 10 days of storage at 23 °C (3-fold increase). At 4 °C, the highest (p < 0.05) TAC (peel) was observed after 14 days of storage (1.2-fold increase). TPC showed similar results. The highest (p < 0.05) TPC was recorded in the peel after 10 days of storage at 23 °C (2.3-fold increase) and after 14 days of storage at 4 °C (1.3-fold increase), respectively. Total carotenoids in the flesh samples at both storage temperatures were reduced (p < 0.05) after 14 days. Total sugars also decreased during storage. The results of the present study clearly showed that common domestic storage conditions can improve the nutritional quality of plums by increasing the content of bioactive anthocyanins and other phenolic compounds. However, the increase in phytochemicals needs to be counterbalanced with the decrease in total sugars and TA potentially affecting the sensory attributes of the plums.Due to high perishability, plums are harvested at an early stage of maturity to extend postharvest storage life. Storage time and temperature can significantly affect the phytochemical and sugar composition of plums, altering their palatability and nutritional quality. In this study, variations in physiochemical properties , titratable acidity (TA), color (chroma and hue angle)), phytochemical composition , total anthocyanin content (TAC), and carotenoids), and sugars in three different tissues of the Queen Garnet plum (QGP) during storage at two common domestic storage temperatures (4 and 23 °C) were evaluated. There was an increase ( In recent years, the role of dietary phytochemicals in human health has become a major topic of discussion for nutritionists as well as the general public. The consumption of fruits and vegetables rich in phytochemicals, dietary fiber, and vitamins has been associated with a reduced risk in chronic diseases such as type 2 diabetes, hypertension, and cancers ,2,3,4.Prunus, and they are widely consumed around the world due to their attractive appearance, flavor, and aroma. Japanese blood plums  are a good source of dietary phytochemicals such as phenolics  [Plums are a popular stone fruit, belonging to the genus cyanins) ,6, vitamcyanins) , and diecyanins) . Howevercyanins) ,10,11,12Queen Garnet plum (QGP) is a Japanese blood plum cultivar which was developed as a high anthocyanin plum in a Queensland Government breeding program . StudiesMature ripe QGP fruits were obtained from a commercial grower in Queensland, Australia in the 2018/2019 season. Mature fruits were harvested and preliminarily stored at 2 °C for 3 weeks (as a simulated pre-storage). After 3 weeks, approximately 200 plums were transported to the laboratory at Coopers Plains, Queensland, Australia and 90 homogeneous fruits  were subsequently selected. The fruits were randomly divided into 9 groups of 10 fruits. Four groups were stored in a cold room at 4 °C, another 4 groups at room temperature (23 °C), and one group was used as reference for day 0. On 0, 4, 7, 10 and 14 days of storage, a group of fruit was taken randomly from each storage temperature. Each fruit was weighed and cut along a suture into two halves, and the peel was manually separated from the flesh. The flesh was further separated into outer flesh , and the inner flesh (IF) . The weiCommercial standards of cyanidin-3-glucoside (C3G), sorbitol, glucose, fructose, sucrose, lutein, β-carotene, gallic acid, and all other chemicals and solvents (high performance liquid chromatography (HPLC) grade) were purchased from Sigma-Aldrich  unless otherwise stated.The skin and flesh color of the QGP were measured using a Konica Minolta CR-400 Chroma Meter . Variables of color, chroma (C*), and hue angle of the peel were measured randomly at three positions along the equator of the plum, and for IF and OF, the color was measured using the puree. The average of the three values was calculated directly by the instrument and was used as a single variable value of color per plum.The TSS of IF and OF was determined in pureed samples using a digital (0–65%) refractometer  . TA was v/v), 0.1 M HCl. Then, the mixture was shaken on a reciprocating shaker  for 10 min at 200 rpm/min followed by centrifugation  at 3900 rpm for 10 min at 4 °C. The supernatant was collected, and the residue was re-extracted twice using the same procedure as described above. Finally, the supernatants were combined and filtered through a 0.2 μm membrane filter before determining the total phenolic content (TPC) and analyzing anthocyanins. All extractions were done in triplicates.Anthocyanins and non-anthocyanin phenolics were extracted according to Hong et al.  with sliTPC  was determined as reported previously , using aThe analysis was carried out on an ultra-high performance liquid chromatography (UHPLC) system , equipped with a diode array detector (DAD), according to Hong, Netzel, and O’Hare  with sliExtraction of carotenoids from QGP was conducted as described by  with sliv/v)) and mobile phase B (0.1% formic acid in MTBE (v/v)) eluted at a flow rate of 0.6 mL/min for a period of 66 min with a gradient of 80% mobile phase A for 1 min, 70% mobile phase A for 25 min, 30% mobile phase A for 30 min, purging for 5 min, conditioning for 1 min, and re-equilibration for 4 min. Lutein as well as α- and β-carotene were identified by comparing their retention times, molecular masses (from a Shimadzu LC-MS/MS), and specific absorption spectra with those of commercial standards.Carotenoids were analyzed according to Calvo-Brenes, Fanning, and O’Hare  with modSugars in QGP were extracted, identified and quantified according to Hong, et al. .The sugar profiles of the QGP tissues were determined using a Shimadzu Nexera X2 UHPLC system coupled with a Shimadzu MS-8045 triple quadrupole mass spectrometer . Briefly, the multiple reaction monitoring (MRM) mode was applied with optimal collision energy (CE) for individual sugars. The MRM transitions for fructose and glucose were m/z 179.2 → 113.1 (for quantification) and m/z 179.2 → 89.0 (for identification), for sucrose m/z 341.2 → 179.2 (for quantification) and m/z 341.2 → 161.2 → 119.1 (for identification) and sorbitol m/z 181.1 → 119.2 (for quantification) and m/z 181.1 → 113.1 → 101.0 → 89.0 (for identification).p < 0.05) between means were determined using least significant difference (LSD). Data are presented as mean ± standard error (SE).Data were subjected to factorial analysis of variance (ANOVA) for storage time and temperature using IBM SPSS Statistics for Windows, version 25.0 . Significant differences (p > 0.05) up to 10 days of storage, and there was a non-significant (p > 0.05) decrease in TSS on day 14. However, at 4 °C, OF-TSS was significantly (p < 0.05) lower after 14 days of storage compared to day 0 (16.8% vs. 18.5%). The IF-TSS time course plots were similar to that of OF, but TSS after 14 days of storage at both temperatures were significantly (p < 0.05) different to day 0. The TA at 23 °C was significantly (p < 0.05) reduced in both OF and IF, but there was a significant (p < 0.05) increase at 4 °C. Organic acids are needed; they are metabolized for the respiration of plums during storage, and the metabolization is higher at high temperatures, which most likely caused the reduction in total TA [Changes in the physicochemical properties in different tissues of QGP are shown in total TA ,28. Accototal TA , for plup < 0.05) reduced during the storage at 23 °C, but at 4 °C the chroma values fluctuated between storage days. Similar patterns could be observed in IF and OF chroma. Hue angles also varied between the storage days. In both IF and OF, hue angles were higher (significantly and non-significantly) at 4 °C than at 23 °C, with no clear pattern of increase or decrease during the 14 days of storage. However, according to previous studies, both peel and flesh hue angles in black/purple plums reduce significantly during ripening [Fruit color is an important parameter that can be used as an indicator of quality and maturity of most fruits . Consumeripening  and postripening , which iripening . p < 0.05) among the three tissues of QGP  than that at 23 °C, but this was the opposite in the OF-TPC after 14 days of storage. According to Díaz-Mula, Zapata, Guillén, Martínez-Romero, Castillo, Serrano, and Valero [The peel TPC in QGP was much higher than the previously reported value for ‘Black Diamond’, another red fleshed, black peel plum (1205 vs. 270 mg GAE/100 g fresh weight (FW)) . Generald Valero , the fled Valero .p < 0.05) after 10 days of storage at 23 °C (3-fold increase), whilst at 4 °C, the highest anthocyanin content was recorded after 14 days of storage (1.2-fold increase). Of the three tissues analyzed, peel had the highest anthocyanin content , followed by OF and IF . The peel content in the present study is considerably higher than the previously reported highest peel anthocyanin concentration of 538 mg anthocyanins/100g FW [Anthocyanins are the main pigments in Japanese plums having higher anthocyanin concentration in the peel than in the flesh . The ant/100g FW ,10. Thisp < 0.05) difference between the TAC of the peel at 4 and 23 °C at each storage day. C3G was the predominant anthocyanin found in all the tissues. There was an increase (p < 0.05) of C3G towards day 14 in peel (+20%), OF (+16%) and IF (+10%) at 23 °C, and peel (+4%) and IF (+17%) at 4 °C.The highest OF-TAC was observed after 14 days of storage at 4 and 23 °C and highest IF-TAC after 10 days of storage at 4 and 23 °C. There was a significant  between the total carotenoid content in inner and outer flesh, but there was a significant (p < 0.05) difference between the IF and peel and the OF and peel, respectively  in total sugar in OF and IF after 14 days of storage at 4 °C, whereas a significant (p < 0.05) difference could be observed in the peel. At 23 °C, significant (p < 0.05) differences in total sugar were found in OF, IF, and peel after 14 days of storage. Peel had the highest sugar levels, followed by IF and OF.Different tissues have different levels of sugar . Total sp < 0.05) increase in sucrose in the peel (+25%), and a significant (p < 0.05) decrease in OF (−12%) and IF (−17%). These variations suggest that there can be differences in the sugar accumulation and metabolism in plums depending on the cultivar or genotypic [p < 0.05) reduction in sorbitol in all tissues, whereas at 4 °C, sorbitol was only reduced (p < 0.05) in peel and IF but slightly (p > 0.05) increased (+7%) in OF. Sorbitol is a sugar alcohol that can be used as a natural replacement for sucrose and glucose in special diets for diabetic people [Sorbitol, fructose, glucose and sucrose were the main sugars identified in the three different QGP tissues . These senotypic ,42, storc people .The effect of storage temperature (4 and 23 °C) and storage time (up to 14 days) on physicochemical properties, phytochemicals, and sugar composition in different tissues of QGP was demonstrated in the present study. The increase in bioactive anthocyanins in flesh and peel was more prominent at ambient temperature (23 °C) than at refrigerated (4 °C) storage. There was also a decrease in total sugars and TA at ambient storage, which may affect the sensory attributes of QGP. However, the actual impact of this on the overall fruit quality and consumer acceptance needs to be investigated further.


Limited evidence was available for particulate matter with an aerodynamic diameter ≤ 1 μm , diastolic blood pressure (DBP), mean arterial pressure (MAP) and pulse pressure (PP). Moreover, we examined potential effect modifications by demographic, lifestyle and diet factors.This study included 39,259 participants who had completed the baseline survey from Henan Rural Cohort. Participants' exposure to PM1 for all participants during the 3-year before baseline survey was 59.98 μg/m3. Each 1 μg/m3 increase in PM1 concentration was significantly associated with an increase of 4.3%  in odds for hypertension, an increase of 0.401 mm Hg , 0.328 mm Hg , 0.353 mm Hg  and 0.073 mm Hg  in SBP, DBP, MAP and PP, respectively. Further stratified analyses showed that the effect of PM1 on hypertension and blood pressure could be modified by sex, lifestyle and diet.The mean concentration of PM1 increases the risk of hypertension and is associated with elevations in blood pressure in rural Chinese adults, especially in male and those with unhealthy habits.This study suggests that long-term exposure to ambient PM •1 during the study period.Rural cohort participants were exposed to high level of PM•1 was related to increased risk of hypertension and elevated blood pressure.PM•Males and those with unhealthy habits were more susceptible to the adverse effect. Among a2.5) with hypertension, and of particulate matter with an aerodynamic diameter ≤ 10 μm (PM10), PM2.5, and nitrogen dioxide (NO2) with increased diastolic blood pressure (DBP)  . Howeveran PM2.5 . In addian PM2.5 . However1, we investigated the associations between long-term exposure to PM1 and hypertension and four blood pressure component measurements in rural Chinese population. We also examined several potential effect modifications by demographic, lifestyle and diet factors.In this context, to fill in the gap and add to the evidence for adverse effect of long-term exposure to PM22.1We studied participants from The Henan Rural Cohort Study (Registration number: ChiCTR-OOC-15006699), which was established in five rural areas  of Henan Province, China, in July 2015. The detailed descriptions of cohort design and study population were previously published elsewhere . BrieflyThe study complied with the 1975 Declaration of Helsinki and was approved by the ethics committee of Zhengzhou University. Written informed consent was obtained from each participant at their enrollment.2.21 concentrations were predicted at a 0.1° × 0.1° spatial resolution, using satellite remote sensing, meteorology, and land use information. The detailed description of the prediction has been previously published . A 10-fold cross-validation was performed to assess the predictive ability. The results of 10-fold cross-validation showed that R2 and Root Mean Squared Error (RMSE) for daily prediction was 64% and 17 μg/m3. For annual prediction, R2 and RMSE was 82% and 9 μg/m3, respectively. We assigned PM1 concentration estimates for each participant according to the geocoded address of natural village through AutoNavi Map (https://lbs.amap.com/api/webservice/guide/api/georegeo). This is a Chinese web mapping, navigation and location-based services provider and has provided mapping data to Google since 2006. Then we calculated the average during the three-year before the baseline survey for each participant as long-term exposure concentration of PM1.Daily PMublished . In brie2.3Blood pressure was measured using an electronic sphygmomanometer  in the sitting position for three times according to the American Heart Association's standardized protocol . Partici2.4We controlled for potential confounders based on the previous literature on air pollution and blood pressure. Demographic covariates included age and sex. Socioeconomic covariates included education level , marital status (“married/cohabitating” or “widowed/single/divorced/separation”), average monthly income . Health behavior covariates included smoking status (“never smoking” or “ever smoking”), alcohol drinking status (“never drinking” or “ever drinking”), high fat diet (“yes” or “no”), more vegetables and fruits intake (“yes” or “no”), physical activity . Health status covariates included body mass index (BMI), family history of hypertension, and type 2 diabetes. Regarding education level, participants with no schooling or participants who attained up to primary school were considered as low education level, while those who graduated from junior school were considered as medium and those who graduated from senior high school or above were considered as high education level. For smoking status, the responses of “former smoking” and “current smoking” were merged into the variable of “ever smoking” in order to further analysis and make comparison with other studies. This is also the case for drinking status. According to Chinese dietary guidelines , “More v2.5U test for continuous variables and chi-square test for categorical variables. We employed the logistic regression model to examine the association between long-term PM1 exposure and hypertension as a dichotomous outcome. And multivariate linear regression models were employed to investigate associations between PM1 exposure and blood pressure component as a continuous measure. The effect estimates were presented as odds ratios (ORs) for hypertension and changes in mm Hg for blood pressure measures per 1 μg/m3 increment in PM1 concentration, with corresponding 95% confidence intervals (CIs).Descriptive analyses were conducted for all variables. Continuous variables were described as mean ± standard deviations (SD) and categorical variables were expressed as counts and percentage. Differences in the distribution of baseline characteristics between groups were tested using Mann-Whitney 1-hypertension and PM1-BP associations were potentially modified by sex, age, smoking status, drinking status, high-fat diet, more vegetables and fruits intake and physical activity. We did subgroup analyses by each potential modifier, and a cross-product term was added into separate models to assess the significance of interaction terms , Vienna, Austria).3P < 0.001), had a higher level of BMI  than normotensive participants. Also, they were more likely to have family history of hypertension (27.5% versus 15.4% P < 0.001) and diabetes  than normotensive participants. The 3-year average concentration of PM1 for overall cohort was 55.99 μg/m3 (SD:2.06), and the hypertensive participants had similar PM1 exposure levels with normotensive participants .3 increment in PM1 concentration was 1.059  in crude model and 1.043  after fully adjusting for potential confounders (Adjusted model 2). Based on the final adjusted model (Adjusted model 2), each 1 μg/m3 increase in PM1 concentration was associated with an elevation of 0.401  mm Hg in SBP, 0.328  mm Hg in DBP, 0.353  mm Hg in MAP and 0.073  mm Hg in PP . We observed stronger positive associations in males, ever-smokers, ever-drinkers and participants with high level of physical activity. Except for PP, the effect modification by more vegetables and fruits intake and high-fat diet were also observed for SBP, DBP and MAP  in PM2.5 concentration was associated with an OR of 1.11  for hypertension and an increment of 0.60 mm Hg  in SBP  for hypertension, an increment of 1.30 mm Hg  in SBP and an increment of 1.04 mm Hg  in DBP   and therefore PM1 may contain more toxins from anthropogenic emissions, which may even lead to gene damage  in SBP . A study) in DBP . A recen1, 1.09) . However effects . Smallery injury . Additioe damage . Howeverurements . Along wments  development  in individuals younger than 50 years old . Also, this sex-based difference in effect estimates can be related to some behavioral characteristics . Men may accumulate greater PM1 exposures through long periods of physical activity  and the Natural Science Fund of Hubei Province (Granter number: 2018CFB634). Dr. Guo is supported by Career Development Fellowship APP1107107 from the Australian National Health and Medical Research Council (NHMRC). Dr. S. Li is supported by Early Career Fellowship APP1109193 from the Australian NHMRC.The authors acknowledge the cooperation of all the participants and administrators in this study. This research was supported by the Foundation of