Intubation using direct laryngoscopy is a risky and painful procedure that is associated with undesirable hemodynamic changes such as tachycardia, hypertension, and arrhythmia. Recently, intravenous oxycodone was introduced and used for the control of acute postoperative pain and to attenuate intubation-related hemodynamic responses (IRHRs), but there is insufficient information regarding its proper dosage. We investigated the attenuating effects of different doses of oxycodone and fentanyl on IRHRs.95), which attenuated all IRHR changes to less than 20% over baseline values in 95% of male patients at 1 minute after intubation, oxycodone 0.1 mg/kg was injected for the first patient 1 hour before intubation, and the next dose for each subsequent patient was determined by the response of the previous patient using Dixon up-and-down method with an interval of 0.01 mg/kg. After obtaining the predictive oxycodone ED95, 148 patients were randomly allocated to groups receiving normal saline (group C), oxycodone ED95 (group O1), oxycodone 2 × ED95 (group O2), or fentanyl 2 μg/kg (group F). We recorded the incidence of “success” as a less than 20% change from baseline values in all IRHRs 1 minute after intubation.For calculating oxycodone effective dose (ED95 was 0.091 (0.081–0.149) mg/kg. The incidence of “success” was highest in group O2 (75.7%), followed by group O1 (62.2%) and group F (45.9%) with significant differences between the groups (P < .001). The systolic, diastolic, mean arterial pressure, and heart rate were not significantly different among groups after administration of either oxycodone or fentanyl. The percentage hemodynamic changes of the group O2 were significantly lower than those of groups F and O1, but the absolute percentage hemodynamic changes were not significantly different among groups F, O1, and O2. The recalculated oxycodone ED95 with probit analysis (0.269 mg/kg) was needed to prevent any arterial pressure and heart rate changes.The predictive oxycodone ED95 (0.269 mg/kg) is also effective and hemodynamically safe for preventing all IRHRs.Oxycodone 0.182 mg/kg is more effective in attenuating all IRHRs than fentanyl 2 μg/kg with safe hemodynamic changes. Further research is required to determine if the recalculated oxycodone ED Therefore, various pharmaceutical interventions such as opioids, beta-adrenergic blockers, and antihypertensive drugs have been used and studied to attenuate intubation-related hemodynamic responses (IRHRs).Endotracheal intubation using direct laryngoscopy is a risky and painful procedure that causes adverse stimulation and is associated with undesirable hemodynamic changes such as tachycardia, hypertension, and arrhythmia. Oral oxycodone has been used in patients with cancer pain and chronic pain. Recently, intravenous oxycodone was introduced and used for the control of acute postoperative pain, and because intravenous oxycodone provides better postoperative analgesic effect than fentanyl, it can help prevent or attenuate IRHRs.Oxycodone  is a semisynthetic opioid with an agonistic activity on the mu, kappa, and delta receptors.–9 and the pharmacokinetics of oxycodone have a somewhat different effect depending on age and sex.,11However, there is insufficient information about the appropriate dosage of oxycodone for this purpose,50 and ED95, respectively). We then investigated the actual attenuating effects of intravenous oxycodone ED95, 2 × ED95, and fentanyl on these IRHRs.Therefore, in this study, we calculated the effective doses (EDs) of intravenous oxycodone that could attenuate all IRHR changes to less than 20% over baseline values in 50% and 95% of male adult patients  on January 4, 2016. Written informed consent was obtained from all participants, a legal surrogate, or the parents or legal guardians of participants who were minors. This study was conducted in accordance with the tenets of the Declaration of Helsinki.This prospective, randomized, controlled and double-blinded study was approved by the Institutional Review Board of Chosun University Hospital, and registered with the Clinical Research Information Service  physical status I or II, and who were scheduled to undergo elective surgery under general anesthesia. We excluded patients with cardiopulmonary disease, neurovascular disease, brain disease, mental disorders, renal or hepatic function abnormalities, recent medication of monoamine oxidase inhibitor, or a body mass index (BMI) above 25%.2 6 L/min via a facial mask.After premedication with intramuscular midazolam (0.05 mg/kg), patients were transported to the operating room. Before the induction of anesthesia, standard patient monitoring devices to obtain electrocardiograms, non-invasive blood pressure, end-tidal partial pressure of carbon dioxide, and peripheral pulse oximetry were applied. A catheter was then installed in the radial artery to continuously monitor blood pressure. Patients were premedicated with ramosetron 0.3 mg for prevention of postoperative nausea and vomiting under preoxygenation with O2.1,13 We performed the Dixon up-and-down method until we obtained 8 crossover points, which were manifested as crossover from “failure” to “success.”Intravenous oxycodone 0.1 mg/kg was administered slowly over 2 minutes for the first patient 20 minutes before intubation, and anesthesia was induced with propofol 2 mg/kg and rocuronium 0.9 mg/kg 2 minutes before intubation. One minute after intubation, “success” or “failure” was determined based on whether the IRHRs were significantly different from their baseline values. We defined “success” as all changes of systolic, diastolic, or mean arterial pressures  and heart rate (HR) being less than 20%, and “failure” as any change in these parameters being above 20%. The subsequent patient received the next oxycodone dose, which was decreased or increased with an interval of 0.01 mg/kg by the Dixon up-and-down method, depending on the “success” or “failure” of the previous patient, respectively.2.250 and ED95 using a probit regression model in “Part 1,” 148 patients were randomly allocated to one of 4 groups receiving normal saline (group C), oxycodone ED95 (group O1), oxycodone 2 × ED95 (group O2), or fentanyl 2 μg/kg (group F), by using a random numbers table obtained via a computer program , 5, and 2 minutes before intubation, just before intubation, and 1, 3, 5 minutes after intubation. We also recorded the percentage changes and absolute percentage changes of SAP, DAP, MAP, and HR between the values at baseline and those at 1 minute after intubation. The age, ASA physical status, height, weight, and BMI of each patient were noted.Patients and investigators were blinded to the study medications. A noninvestigating nurse loaded them into indistinguishable numbered syringes with the same volume (5 mL) and randomized the medications using a random number table.The primary endpoint was the incidence of “success,” 1 minute after intubation. The secondary endpoints were the changes in SAP, DAP, MAP, and HR values, 1 minute after intubation.2.3α = 0.05 and β = 0.2 and using an expected effect size of 0.28. The effect size was calculated with the expected proportion of “success” for groups C (0.15), F (0.25), O1 (0.25), and O2 (0.35), which was based on a previous pilot study, owing to lack of other evidence for calculating the effect size. The study needed a total of 140 patients, so we enrolled 148 patients, allowing for an assumed 5% dropout rate.For the analysis of Part 2, the necessary sample size for chi-square tests using G∗Power software  was calculated by taking the level of statistical significance as SPSS  was used for statistical analysis. All measured values are presented as mean ± standard deviation, mean  or number (percentage) of patients [n (%)].50 of oxycodone was calculated with the mean values of the midpoint dose of all independent pairs of patients who manifested as crossover from “failure” to “success” after 8 crossover points. A probit regression model was used to calculate the predictive oxycodone ED50 and ED95, and the 95% CIs. In Part 2, the chi-square test was used for analysis of the incidence of “success” and the ASA physical status classification. A 1-way ANOVA followed by Scheffe post-hoc test was used for analysis of hemodynamic responses, percentage changes and absolute percentage changes, ages, height, weight, and BMI. A probit regression model was used to recalculate the predictive oxycodone ED95 with data from groups C, O1, and O2. P values < .05 were considered statistically significant.In Part 1, ED33.150, which was calculated using the Dixon up-and-down method, was 0.074 ± 0.008 mg/kg in male patients  mg/kg and 0.091  mg/kg in male patients, respectively . It was highest in group O2 (75.7%), followed by group O1 (62.2%) and group F (45.9%), while it was lowest in group C, at 16.7%.The incidence of “success” was significantly different among the groups (Table P < .05) after propofol injection, while the SAP and MAP were not significantly different.SAP, DAP, and MAP did not decrease after administration of either oxycodone or fentanyl, but they decreased after propofol injection in all groups , and these hemodynamic trends continued until 5 minutes after intubation. They were lowest in group O2, followed by groups O1 and F, but there were no significant differences among these groups, except for SAP. SAP of group O2 was significantly lower than that of group F at 1 minute after intubation .SAP, DAP, and MAP increased immediately after intubation, and subsequently declined progressively , while those of groups O1, O2, and F were not significantly different. Three minutes after intubation, the mean HR of group O2 was significantly lower than that of group C (P = .03). HR was not significantly different among the groups at the other time points.HRs of all groups did not decrease after administration of oxycodone, fentanyl, or propofol, but they increased immediately after intubation and subsequently declined progressively Fig. D. One miPercentage changes and absolute percentage changes of SAP, DAP, MAP, and HR at 1 minute after intubation were significantly lower in groups F, O1, and O2, compared with those of group C Fig. A and B. 95), which was derived from the oxycodone data in this randomized controlled study. Recalculated oxycodone ED95 values were 0.152, 0.232, 0.188, and 0.217 mg/kg for attenuating changes of SAP, DAP, MAP, and HR, respectively.Probit analysis was performed to derive the optimal dose in the oxycodone and normal saline groups Fig. . We foun495, which was defined as a dose for attenuating all IRHR changes to less than 20% over baseline values in 95% of the patients, was 0.091 mg/kg in male patients. Oxycodone 2 × ED95 (0.182 mg/kg) was more effective in preventing significant IRHRs than fentanyl 2 μg/kg, with less changes in arterial pressures and HR.We demonstrated that the predictive intravenous oxycodone ED95 (0.091 mg/kg) and 2 × ED95 (0.182 mg/kg) in this study, to determine the effective or optimal dose of oxycodone to minimize IRHRs.–9 IRHR changes in these previous studies showed striking similarities to this study, increasing immediately after intubation and subsequently declining progressively. Intravenous oxycodone above 0.1 mg/kg was more effective than oxycodone below 0.1 mg/kg to maintain arterial pressures at 1 minute after intubation with non-significant differences.,8 Oxycodone 0.14 mg/kg and 0.2 mg/kg showed an attenuating effect on arterial pressures comparable with fentanyl 2 μg/kg, which is known to be preferable to minimize the IRHRs in patients without hypertension.,9,14 However, the HR after intubation remained significantly higher than the baseline values at all measured points, even though patients received either oxycodone or fentanyl.,8 Lee at al reported that the maximal change of HR from the baseline value was not significantly different between oxycodone (0.2 mg/kg) and fentanyl (2 μg/kg) groups 5 minutes after following intubation, but the maximal changes above 20% was higher in the group receiving oxycodone (53%) than the group receiving fentanyl (47%). Bostan and Eroglu suggested that esmolol 1 mg/kg might provide reliable protection against increases in both HR and SAP compared with fentanyl 1 μg/kg. In this case, the addition of a β adrenergic blocker would be helpful to prevent intubation-related tachycardia if oxycodone of less than 0.2 mg/kg was used. Korpinen et al showed that esmolol (a β adrenergic blocker) 2 mg/kg before induction was effective in mitigating HR increase 1 minute after intubation in patients with a premedication of intravenous oxycodone 0.1 mg/kg 1 hour before anesthesia.There are a few studies that used oxycodone doses of 0.1 or 0.2 mg/kg, which are similar to the doses of oxycodone ED95 using a less than 20% change as a cut-off level of all IRHRs. Recently, Park et al reported oxycodone ED95 with less than 15% changes as a cut-off level for individual IRHRs. They reported that oxycodone ED95 for attenuating the changes of SAP and MAP were 0.159  mg/kg and 0.219  mg/kg, respectively. However, oxycodone ED95 for preventing HR change could not be calculated, and they suggested that it was far higher than their experimental dose when it was calculated using a less than 20% change cut-off level.There has been no report on intravenous oxycodone ED95 was 0.091  mg/kg in male patients using a less than 20% change cut-off level for all IRHRs. However, the oxycodone ED95 and 2 × ED95 showed actual preventing effects at rates of 62.2% and 75.7%, respectively. Even though these actual attenuating effects did not reach 95%, those of oxycodone 0.182 mg/kg (2 × ED95) increased to 94.6% and 89.2% for attenuating changes of SAP and MAP to less than 20%, respectively. The recalculated oxycodone ED95, which was derived oxycodone and normal saline group data, was 0.152 and 0.188 mg/kg for attenuating changes of SAP and MAP, respectively, which were within 95% CI of the results of Park study. In addition, the recalculated oxycodone ED95 was 0.269 mg/kg for attenuating all IRHRs, and 0.217 mg/kg for attenuating changes of HR.This study showed that the predictive oxycodone ED,8 Even though the hemodynamic complications of oxycodone up to 0.2 mg/kg have been reported to be comparable to those of fentanyl (2 μg/kg), the incidence of both hypotension (systolic blood pressure below 90 mm Hg) and bradycardia (HR below 60 beats/min) was about 33%.,9 Our results for IRHRs were also similar to those of previous reports.–9 In this study, we did not observe any significant hypotension and bradycardia after administration of oxycodone (0.091 mg/kg and 0.182 mg/kg) because it was administered slowly over 2 minutes. The administration of oxycodone can induce a significantly higher incidence of desaturation compared with that of fentanyl in patients without pre-oxygenation. However, we did not observe significant desaturation because all patients were pre-oxygenated.Oxycodone as high as 0.269 mg/kg might be used in order to completely eliminate a significant increase in all IRHRs considered in this study, but complications such as hypotension, bradycardia, and respiratory depression would have to be considered at this higher dose of oxycodone. The incremental dosage of oxycodone up to 0.2 mg/kg showed significantly less changes of IRHRs with more suppressed trends compared to those seen with lower doses of oxycodone or fentanyl. Kautto reported that 2 μg/kg of fentanyl 3.5 minutes before endotracheal intubation only decreased HR but larger doses of fentanyl decreased both HR and blood pressure. In our hospital, we commonly use 2 μg/kg of fentanyl for preventing IRHRs. Oxycodone has been reported to be effective for controlling preoperative acute pain such as bone fracture, and preoperative administration was associated with the beneficial effect of reducing postoperative opioid consumption without an increase in side effects. Therefore, we assumed that intravenous oxycodone also could be used effectively to attenuate IRHRs, and we chose fentanyl 2 μg/kg and intravenous oxycodone for this study.Various doses of fentanyl  were used to study the effect on preventing morbidity associated with IRHRs.,20 While intravenous oxycodone has an onset similar to that of fentanyl, it has a longer action duration (4 hours 52 minutes) than that of fentanyl.,20 The peak effect of fentanyl and oxycodone is achieved within 5 minutes and 15 to 30 minutes after intravenous injection, respectively.,22 Thus, we administrated fentanyl and oxycodone at 5 and 20 minutes before anesthesia induction, respectively, with consideration of the patient's safety and peak effect time.The onset and action duration of fentanyl is 2 to 3 minutes and 3 hours 39 minutes, respectively. reported that age is a key factor affecting the pharmacokinetics of oxycodone in older patients aged >70 years. Sex also is a contributor to differences in oxycodone serum concentration, which is lower in women than in men. Second, we did not exclude patients using routine opioids preoperatively, which can alter IRHRs. However, none of these patients were enrolled in this study. Third, all patients were premedicated with intramuscular midazolam. Intramuscular midazolam can influence IRHRs with stabilized hemodynamics and an analgesic effect during the induction of anesthesia. However, we ignored this effect in our study because it is a positive effect on IRHRs and we use intramuscular midazolam routinely as an anxiolytic, except where contraindicated. Fourth, we did not record the Mallampati grades of all patients. If patients had high Mallampati grades, intubation within 15 seconds would have failed, and we excluded these patients in the final statistical analysis. Fifth, a relatively small sample size was used for calculating the predictive oxycodone ED95 in part 1. The sample size was enough for the up-and-down methods, but the small sample size might have led to wide range of confidence intervals. Therefore, the predictive oxycodone ED95 showed a 75.7% “success” rate in part 2, and the recalculated oxycodone ED95 with probit analysis (0.269 mg/kg) was needed to prevent any arterial pressure and HR changes. Finally, we did not evaluate intraoperative hemodynamics and postoperative effects regarding emergent or postoperative pain. Oxycodone may affect intraoperative hemodynamics as well as the intraoperative and postoperative requirement for opioids, because the onset time of oxycodone and duration of action of intravenous oxycodone are about 3 minutes and 4 hours, respectively.,19,24 Therefore, further research is required to obtain the predictive ED50 and ED95 of oxycodone-based on age and sex, and to investigate the intraoperative and postoperative effects on hemodynamics and the analgesic requirements using these doses of oxycodone. In addition, further research is required to determine whether the recalculated oxycodone ED95 (0.269 mg/kg) for preventing all IRHRs is also effective and has hemodynamic safety with larger sample size.There are some limitations associated with the present study. First, the enrolled patients were all male adults aged 20 to 65 years. The individual titration of oxycodone dosage is important, particularly in older adults. Liukas et al95, for attenuating all changes of hemodynamic responses to less than 20%, was 0.091 mg/kg in male patients. The administration of oxycodone 0.182 mg/kg (2 × ED95) 20 minutes before intubation was more effective in attenuating IRHRs than fentanyl 2 μg/kg 5 minutes before intubation, and resulted in smaller hemodynamic changes. However, oxycodone 0.182 mg/kg (2 × ED95) was not sufficient to blunt increases in HR after intubation; oxycodone above 0.2 mg/kg, or a combination with a β adrenergic blocker would be required. In addition, we recommend that slow injection and pre-oxygenation should be performed after the administration of oxycodone to minimize the risk of complications before intubation. Moreover, our results provide guidelines for preoperative intravenous oxycodone use and its reference dosage in patients requiring control acute pain before surgery. Oxycodone may provide effective preoperative pain control as well as attenuation of IRHRs without the addition of other analgesics for these purposes in patients who received intravenous oxycodone preoperatively.In conclusion, the predictive intravenous oxycodone EDConceptualization: Ki Tae Jung, Keum Young So, Sang Hun Kim.Data curation: Ki Tae Jung, Jong Sik Seo, Sang Hun Kim.Formal analysis: Gi-Ho Koh, Ki Tae Jung, Keum Young So, Jong Sik Seo, Sang Hun Kim.Investigation: Ki Tae Jung, Keum Young So, Jong Sik Seo, Sang Hun Kim.Methodology: Sang Hun Kim.Project administration: Ki Tae Jung, Keum Young So, Sang Hun Kim.Resources: Sang Hun Kim.Software: Gi-Ho Koh, Sang Hun Kim.Supervision: Ki Tae Jung, Keum Young So, Sang Hun Kim.Validation: Gi-Ho Koh, Sang Hun Kim.Visualization: Ki Tae Jung, Sang Hun Kim.Writing – original draft: Gi-Ho Koh.Writing – review and editing: Ki Tae Jung, Keum Young So, Jong Sik Seo, Sang Hun Kim.Sang Hun Kim orcid: 0000-0003-3869-9470.


We allowed separate populations of the common bed bug, Cimex lectularius L., to feed once on a rabbit before and after it was injected subcutaneously with 0.3 mg/kg of ivermectin, and bed bug morbidity and mortality were recorded. Ivermectin levels in the rabbit were measured using high-performance liquid chromatography and mass spectroscopy. Ivermectin blood levels of ∼2 ng/mL caused reductions in bed bug fecundity, and levels of >8 ng/mL caused bed bug death and long-term morbidity including reductions in refeeding, mobility, reproduction, and molting. Gut bacterial cultures from the fed bed bugs showed that ivermectin altered the bed bug gut microbiome.Human bed bug infestations have undergone a recent global resurgence. The human antiparasitic drug ivermectin has been proposed as a strategy to help control bed bug infestations, but  Cimex lectularius Linnaeus (1758) is a cosmopolitan anthropophilic hematophagous temporary human ectoparasite [Cimex lectularius has made a recent global resurgence, and current bed bug control is significantly hampered by an increasing resistance of the insect to pesticides used in their control [The common bed bug, Linnaeus 758 is a parasite . Cimex l control –6. Bed bPediculus humanus capitis (head lice), Pediculus humanus humanus (body lice), Pthirus pubis (pubic lice), and Sarcoptes scabiei (scabies). Ivermectin is not currently used in the control of C. lectularius, but ivermectin has been shown to kill bed bugs at plasma levels that have been reported in humans taking the drug [The pharmaceutical drug ivermectin is one of the most common antiparasitic drugs given to human, and is used to treat many human ectoparasites including the drug –11. Iverthe drug .in vitro feedings where blood samples are spiked with ivermectin and fed to bed bugs in an artificial feeding system [in vitro conditions likely do not replicate in vivo conditions principally for two reasons: pharmacokinetic studies of ivermectin in humans report ivermectin levels as plasma concentration, but bed bugs consume whole blood meals, and the secondary metabolites of ivermectin likely have antiparasitic effects that may persist beyond the parent compound. Ivermectin added to defibrinated in vitro blood samples will not undergo appreciable metabolism, so any secondary metabolites with toxic effects against bed bugs cannot be studied. Any ivermectin that accumulates intracellularly in hematogenous cells under in vivo conditions would not be measured in the plasma concentration. The in vivo concentration of ivermectin in the blood that is required to cause bed bug toxicity is unknown.The existing research involving ivermectin and bed bugs has focused on g system , 10. TheThe objective of our study was to record bed bug mortality and the insects interest and ability to refeed, fecundity, activity level, and molting after the insects fed on a rabbit injected with ivermectin. Our secondary objectives were to correlate blood ivermectin levels in the rabbit with bed bug morbidity and mortality and to determine if ivermectin affects the bed bug gut microbiome.We received Institutional Animal Care and Use Committee's (IACUC) approval. We used a two-year-old New Zealand white neutered male rabbit weighing 4.54 kg. Ivermectin (1% sterile solution)  at 0.3 mg/kg was injected subcutaneously into the left shoulder of the rabbit by a veterinarian. The rabbit was trained to sit in the lap of a handler while the insects fed on a shaved left hip. The same rabbit was used for both experiments which were separated by more than a month.C. lectularius L. was used in the experiments. These insects were first collected in a New Haven, Connecticut, apartment in 2009 and have been maintained under laboratory conditions by feeding on a human volunteer. Populations were kept in small glass canning jars with open mouths covered by sheer fabric secured by a metal band. Inside each jar were vertical cardboard wafers where insects established natural refuges. Insects were exposed to natural daylight cycles at a temperature of ∼24°C (75°F) and 40–50% RH conditions.Ridge-strain Featherweight surface forceps were used to move insects. Twelve populations of five males, five females, and 10 mixed age instar nymphs were placed into 55 × 25 mm (4 dram) glass vials which contained a single piece of 10 × 40 mm card for perching. To keep stress low, vials were populated with 20 fully sated insects seven days before each test and left undisturbed. A 60 × 60 mm square of sheer fabric was secured over the vial mouth with an elastic band. When inverted, the card slid to the vial mouth, allowing insects to feed through the fabric.Populations of 5 females, 5 males, and 10 nymphs fed once on the rabbit at specific postinjection time intervals 0 (control—just before the rabbit was given ivermectin), 1, 6, 12, 24, 36, 48, 72, 96, 120, 144, and 168 hours were taken. All bed bugs fed to repletion. The experiment was repeated twice (Test 1 and Test 2).Test 1 recorded the health of the bed bugs up to day 45 after feeding. Test 2 recorded the health of the bed bug up to day 64 after feeding but also included a breath test on day 14—performed by gently exhaling onto the insects and observing levels of stimulation and opportunities to feed on an investigator at days 16 and 31. Bed bugs were categorized as healthy ; reactive (cognizant to stimuli without response); immobile ; paralyzed ; and dead (no movement).g for 20 minutes, and 500 microliters of supernatant was transferred to a clean tube and speed vacuumed to dryness. It was then reconstituted with 50 microliters of reconstitute solvent  and centrifuged at 16,000 g for 20 minutes. Five microliters were used for high-performance liquid chromatography (HPLC) and mass spectrometry (MS). The chromatography was performed with a reversed-phase C18 column . Ivermectin was separated from blood endogenous components using 10% 0.5 mM ammonium formate containing 0.1% formic acid in the isocratic mode at 0.2 ml/min. The column was set at 35°C. Ivermectin was detected by a Thermo Scientific TSQ Quantum Ultra with HESI-II probe using ESI positive ionization mode; spray voltage of 3000 V; capillary temperature of 200°C; vaporizer temperature of 300°C; sheath gas pressure of 40; aux gas pressure 10; skimmer offset 10 V; SRM setup: Q1: 0.7 FWHM, Q3: 0.7 FWHM, and Q2: 1.5 mTorr (Ar); scan width: 0.002 m/z; scan time at 0.02 s.For Test 2, 0.5 mL of blood was drawn from the marginal ear vein at 0, 6, 24, 36, 48, and 72 hours post-ivermectin injection. This blood was centrifuged at low speed, and the whole blood sample was frozen to −52°C and shipped on dry ice to Case Western Reserve University Proteomics Core facility, Cleveland, Ohio, for testing. The ivermectin concentration was analyzed using a modified method reported previously . In brie0, 101, and 102, and 0.1 ml was spread onto blood agar culture plates (Sigma-Aldrich Inc.) with 2 plates per dilution. Plates were incubated at 25°C for 3 days and then photographed.Bed bugs from Test 2 had their gut contents cultured after the morbidity and mortality was recorded on day 64 after ivermectin injection. The healthiest appearing adults were used first, and if no adults were alive, then the healthiest appearing nymphs were used. Bed bugs were surface disinfected to minimize external contaminants by twice vortexing the insects for one minute in a sterile saline solution and pipetting off the fluid. Insects were then exposed to benzalkonium chloride for 1 minute and rinsed again in sterile saline. Rinsates (0.1 mL) from the last washing were spread onto blood agar culture plates to assess the disinfection protocol and found to reproduce no colonies after 72 hours. Bed bugs were thoroughly crushed with a sterile glass rod in 1 ml sterile saline. Serial dilutions were prepared at 10N − 1” chi-squared test was used to assess for differences between bed bug morbidity and mortality for those harmed for females, males, and nymphs for each feeding time after injection compared to the control group . Comparison of proportions was used to determine differences in nymphal molting and the number of eggs laid for each feeding time after injection compared to the control group. We used ANOVA to compare group differences in bed bug mortality and incapacitation rates between females, males, and nymphs for feeding times after injection.Comparison of proportions using the “The morbidity and mortality results are summarized in Tables p < 0.05).Both morbidity and mortality were noted in the 6-hour post-ivermectin injection feeding group in Test 1. After 45 days, the mortality rate was 0/30 (0%), 2/30 (7%), 8/30 (27%), 7/30 (23%), 8/30 (27%), 12/30 (40%), 10/30 (30%), 8/30 (27%), 5/30 (17%), 2/30 (7%), 1/30 (3%), and 3/30 (10%) for the feedings done at 0, 1, 6, 12, 24, 36, 48, 72, 96, 120, 144, and 168 hours, respectively. Peak bed bug mortality occurred at 36 hours after ivermectin injection. The mortality rates for bed bugs fed 6–96 hour after ivermectin injection were significantly different from controls (p < 0.05). There was unexpectedly high mortality in all the adult females after feeding making it more difficult to ascertain differences between the experimental groups with the controls.Test 2 ivermectin levels in the blood were 0, 2.1, 8.3, 10.4, 12.4, and 18.3 ng/mL for 0, 6, 24, 36, 48, and 72 hours after injection, respectively. The Test 2 day 64 mortality rate was 6/20 (30%), 5/20 (25%), 9/21 (43%), 9/20 (45%), 4/20 (20%), 12/20 (60%), 8/20 (40%), 8/20 (40%), 10/31 (48%), 8/20 (40%), 10/20 (50%), and 8/20 (40%) for the feedings done at 0, 1, 6, 12, 24, 36, 48, 72, 96, 120, 144, and 168 hours, respectively, demonstrating a dose-dependent effect of ivermectin. Only the 36-hour post-ivermectin injection feeding group mortality was significantly different from that of the control group . In Test 2 after 64 days, the overall incapacitation rate was 30%, 25%, 43%, 45%, 20%, 60%, 80%, 90%, 90%, 40%, 60%, and 40% for the feedings done at 0, 1, 6, 12, 24, 36, 48, 72, 96, 120, 144, and 168 hours, respectively. Peak bed bug harm occurred in the groups fed 36–96 hours after ivermectin injection where there was significantly more harm than controls (p < 0.05). The incapacitation rate, or the number of insects that were harmed, is the number of bed bugs that were categorized as reactive, immobile, paralyzed, and dead over the number of healthy insects. The Test 1 day 45 incapacitation rate was 0%, 0%, 50%, 55%, 55%, 95%, 100%, 100%, 55%, 45%, 25%, and 20% for the feedings done at 0, 1, 6, 12, 24, 36, 48, 72, 96, 120, 144, and 168 hours, respectively. Peak bed bug harm occurred in the groups fed 36–72 hours after ivermectin injection. All bed bugs that fed after 6 hours after ivermectin injection had significantly more harm than controls  practices.C. lectularius when fed on a rabbit injected with ivermectin. Ivermectin blood levels of ∼2 ng/mL cause changes in bed bug fecundity, and levels of >8 ng/mL are required for significant bed bug mortality. Ivermectin causes alterations in the bed bug gut microbiome.Ivermectin causes dose-dependent toxicity in


Allium cepa L. and Allium × cornutum (Clementi ex Visiani 1842), members of the family Amaryllidaceae. The identification of flavonoids and anthocyanins, and their individual quantities, was determined by high-performance liquid chromatography (HPLC). The potency of both extracts to scavenge free radicals was determined by the DPPH  radical-scavenging activity and oxygen radical absorbance capacity (ORAC) methods. The DNA protective role was further tested by the single-cell gel electrophoresis (COMET) assay and by Fenton’s reagent causing double-strand breaks on the closed circular high copy pUC19 plasmid isolated from Escherichia coli. In the presence of both extracts, a significant decrease in DNA damage was observed, which indicates a protective role of Allium cepa and Allium × cornutum on DNA strand breaks. Additionally, cytotoxicity was tested on glioblastoma and breast cancer cell lines. The results showed that both extracts had antiproliferative effects, but the most prominent decrease in cellular growth was observed in glioblastoma cells.Here, we report a comparative study of the phytochemical profile and the biological activity of two onion extracts, namely  Allium covers more than 750 species distributed all over the northern hemisphere [Allium species, Allium cepa L. (the common onion) is one of the oldest plants cultivated worldwide [A. × cornutum (Clementi ex Visiani 1842) is a triploid hybrid onion (2n = 3x = 24) originating from three parental species , traditionally cultivated in coastal Croatia under the name ‘Ljutika’  [A. × cornutum have not been studied so far. This motivated us to identify and quantify major phenolic compounds present in the bulbs of A. × cornutum and A. cepa and to assess the biological activity  of these widely used plants.Oxidative stress is the disturbance between the production of reactive oxygen species (ROS) and antioxidant defenses. Overproduction of ROS can cause damage to important biomolecules, such as DNA, proteins, lipids, and carbohydrates, resulting in a variety of diseases . For thimisphere . The memmisphere . Among oorldwide ,9,10. Thorldwide . The mosorldwide ,13. In aorldwide . A. × coshallot) . Due to A. × cornutum and A. cepa methanolic extracts (1) (retention time (tR) for A. cepa 33.26 min; tR for A. × cornutum 33.30 min) and quercetin 4′-monoglucoside (2) . The three minor peaks were identified as myricetin (3) , quercetin aglycone (4) , and isorhamnetin (5) . Two main quercetin conjugates, (1) and (2), together account for about 80% of the total flavonol content in both onions (Two major and three minor fine peaks were successfully resolved by high-performance liquid chromatography (HPLC) UV-VIS analysis of flavonols in extracts . The resh onions .1) and (2) were observed in A. × cornutum, which agrees with the higher total phenolic content (TPC) (A. × cornutum TPC 6.63 ± 0.31 mg caffeic acid (CA)/g dry weight (DW); A. cepa TPC 6.24 ± 0.23 CA/g DW) accounted for in this onion species.Similar concentrations of the two quercetin conjugates were previously observed in other studies ,17,18,19Our obtained TPC values show a good correlation to those reported by Santas et al. . In theiA. cepa and four in A. × cornutum were identified. Three anthocyanins, equal in both onions, were further identified as peonidin glucoside (6), petunidin glucoside (7), and malvidin glucoside (8). Interestingly, the fourth anthocyanin, delphidin glucoside (9), was found only in the A. × cornutum extract  and the oxygen radical absorbance capacity (ORAC) assay. Both onions showed mutually similar DPPH and ORAC values at a concentration of 100 μg/mL . Howeverompounds ,27,28,29Taking into account that both onions have a relatively high amount of phenolic compounds, we wanted to further investigate their potential in protecting DNA from ROS. In that sense, the pUC19 plasmid that was exposed to Fenton’s reagent served as a biological probe.A. cepa and A. × cornutum, where it can be seen that lower extract concentrations correlate to poor DNA protection and an increase in open circular and linear forms of DNA. On the contrary, higher concentrations show significant free radical scavenging activity and the ability to maintain a supercoiled form of DNA. Our results clearly indicate that the preservation of an intact circular plasmid is concentration-dependent (5–100 µg/mL).Hydroxyl radicals generated by the Fenton reaction cause the oxidatively induced DNA strand breaks to yield their open circular or relaxed form . Figure Since both extracts have a relatively high amount of phenolic compounds (quercetin and its derivatives) which act as antioxidants, we presume that they are responsible for promoting good scavenging activity. Several other previous studies have, in turn, also reported that high phenolic content prevents DNA damage caused by hydroxyl radicals ,30.The antioxidant activity of phenolic compounds is associated with the total number and position of the hydroxyl functional groups which are able to reduce highly oxidizing free radicals such as superoxides, peroxyls, aloxyls, and hydroxyls . Other t2O2, 200 μM) resulted in a significant increase of DNA damage compared to the untreated control (*** p < 0.001) . Incubation of leukocytes with the DNA damage-inducing reagent (H< 0.001) .A. × cornutum or A. cepa extracts showed only a moderate level of DNA damage, indicating that the presence of phytochemicals in onions can also provoke oxidative DNA damage. This is considered to be a structural property of phenolic compounds which can act as both antioxidants and prooxidants at the same time. In the presence of oxygen, some transition ionic metals, such as Cu2+ and Fe2+ or Fe3+, can cause the oxidation of flavonoids, leading to the formation of ROS, which can, in turn, damage DNA [2O2, a significant decrease in DNA damage was observed (p < 0.0001 for A. × cornutum and A. cepa).Cells treated with 100 μg/mL mage DNA . When le●) that cause DNA strand breaks and fragmentations. Thanks to the high levels of phenolic compounds, especially quercetin and its derivatives, A. × cornutum and A. cepa are able to catch and inactivate those radicals before they can induce any DNA damage  have a cytotoxic effect on breast and glioblastoma cancer cell lines, cells were treated with onion extracts for 4, 24, 48, and 72 h, and the amount of metabolically active cells was measured by MTT -2,5-diphenyl tetrazolium bromide) assay.To evaluate whether p < 0.001) the number of viable cells, albeit A. × cornutum showed a slightly stronger inhibitory effect on all cancer cell lines. The most striking observation was the low viability of glioblastoma which, among other tested cancer cells, showed the highest susceptibility in the presence of both onion extracts. Our results indicate that both plants, A. × cornutum and A. cepa, are effective inhibitors of tumor cell proliferation, which correlates with the study of Boivin et al. [Allium vegetables on different cancer cell lines.As shown in n et al. , who havAllium extracts used in their study also displayed strong inhibitory effects against glioblastoma cancer cells. Most importantly, the authors showed that onion extract had no impact on normal fibroblast growth, suggesting that their selective antiproliferative activity was exclusively against tumor cells. Similar results were obtained by Yang et al. [Allium species. These findings are in agreement with all known anticancer properties of Allium species noticed in many other epidemiological studies [It is worth noting that the g et al.  as they g et al.  tested t studies .2O2), ethyl acetate, ethanol, methanol, ferric chloride (FeCl3), caffeic acid, phosphate-buffered saline (pH 7.4), dimethyl slulfoxide (DMSO), ascorbic acid, hydrochloric acid , sodium chloride (NaCl), sodium hydroxide (NaOH), sodium lauryl sarcosine, disodium salt ethylene-diamin-tetra-acetic acid (Na2EDTA), Trizma base, Triton X-100, Folin-Ciocalteu reagent, 2,2-diphenyl-picrylhydrazyl (DPPH), 6-hydrohy-2,5,7,8-tetramethyl-2-carboxylic acid (Trolox), fluoroshield with 4′,6-diamino-2-phenylindole (DAPI), quercetin 4′-monoglucoside, quercetin, isorhamnetin and myricetin were obtained from Sigma-Aldrich . pUC19 plasmid DNA was purchased from Invitrogen Life Technologies . Quercetin 3,4′-diglucoside was obtained from Polyphenols AS , and malvidin-3-O-glucoside chloride was obtained from Extrasynthese . All chemicals and reagents were AR or HPLC grade.Low melting point (LMP), normal melting point (NMP) agarose, hydrogen peroxide . After 30 min of extraction with magnetic stirring at room temperature (ca. 20 °C), the extract was centrifuged at 3000 rpm for 15 min. All three supernatants were pooled and dried under vacuum using a rotary evaporator (ca. 50 °C). After evaporating solvent at rotavap, samples were dried in a vacuum oven until they reached constant weight. The dry residues were dissolved with 10% DMSO. For HPLC analysis, the solution was filtered through a 0.45 µm nylon filter disc prior to analysis.The phenolic compounds were extracted from homogenized dry plant material (10 g) using 70% methanol water (3PO4) and solvent B  was applied at a flow rate of 0.8 mL/min as follows: 0–0.5 min 96% A and 4% B; 0.5–40 min 50% A and 50% B; 40–45 min 40% A and 60% B; 45–60 min 0% A and 100% B; 60–68 min 0% A and 100% B; 68–70 min 96% A and 4% B; 70–80 min 96% A and 4% B. Detection of the elution peaks was at λ = 360 nm. Flavonoid compounds were identified on the basis of their retention times and quantified using external standard calibration curves. Standards for identification purposes were: quercetin, quercetin 4′-monoglucoside, and quercetin 3,4′-diglucoside prepared in methanol. The resultant concentrations are expressed as mg/100 g of dry weight.High-performance liquid chromatography (HPLC) measurement of flavonols were carried out using the Perkin Elmer HPLC system  consisting of a binary pump Series 200, an autosampler, Peltier column oven Series 200, UV-VIS detector Series 200, and UltraAqueous C18 column . TotalChrom Workstation software , was used to process the chromatographic data. After filtration through a 0.45 µm syringe filter, the extract was injected directly through a 20 µL fixed loop into a guard of the C18 column. Each sample was injected three times in order to check its reproducibility. A gradient consisting of solvent A , consisting of a Star 9010 pump, a Rheodyne 7125 syringe loading sample injector, a 500-LC module for a column oven, a ProStar 330 photodiode array detector, and a Star Chromatography workstation, version 5. The separation was carried out using a Kinetex C18 core-shell column (150 × 4.6 mm), filled with 5 µm particles, and furnished with the SecurityGuard ULTRA Cartridge UHPLC C18 for 4.6 mm ID column , both termostated at 35 °C. Two eluents were used: A was 0.3% HClO6 mL/min . SamplesA. × cornutum and A. cepa methanolic extracts was determined using Folin-Ciocalteu method described by Singleton and Rossi [v/v) and 50 µL of onion extract. After 3 min of incubation, 1 mL of saturated sodium carbonate was added after which the reaction mixture was incubated for another 60 min in the dark and the absorbance was measured at 725 nm using the Perkin Elmer UV-VIS Lambda Bio 40 spectrophotometer . Caffeic acid served as the standard and results were expressed as mg of CA equivalents per g of dry weight. All measurements were carried out in triplicate and the results are expressed as mean values ± SD.The total phenolic content (TPC) of nd Rossi . BrieflyC(0) is the absorbance of the control at t = 0 min, and AA(t) is the absorbance of the antioxidant at t = 1 h. All measurements were performed in triplicate.Antioxidant capacity of extracts was evaluated using the DPPH method previously described by Kulišić, Dragović-Uzelac, and Miloš . This meThe assay was performed in Perkin-Elmer LS55 spectrofluorimeter, using 96-well white polystyrene microtiter plates . Each reaction contained 190 µL of fluorescein (160 µM), 60 µL 2,2'-Azobis(2-methyl-propionamidine) dihydrochloride (AAPH) (150 mM), and 30 µL of plant extracts or reference standard Trolox (6.25–50 µM). All experimental solutions and samples were prepared in a phosphate buffer . The measurements were performed in triplicate at 37 °C using the excitation wavelength of 485 nm, and the fluorescence decay was monitored at 530 nm during the period of 60 min. The obtained fluorescence decay curves were analyzed by FL WinLab software  and the resultant area under the curve (AUC) of each standard or sample was acquired after the blank AUC subtraction . The stag for 30 min and washed twice with phosphate-buffered saline (PBS). Cell viability was determined with trypan blue exclusion assay. Leukocytes (2 × 106 cells/mL) were then incubated in RPMI-1640 medium  and supplemented with A. × cornutum and A. cepa methanolic extract (100 μg/mL) for 30 min at 37 °C in a humidified atmosphere with 5% CO2. Cells growing in a medium supplemented with 1% DMSO were used as the negative control and those incubated with 200 μM of H2O2  served as the positive control. After incubation, cells were centrifuged at 300× g for 5 min at 4 °C and washed twice with PBS. Supernatant was discarded and the pellet was placed on ice and resuspended in LMP agarose. All treatments were performed in duplicate.Blood samples were obtained from a healthy female donor . Venous blood was collected into heparinized vacutainer tubes  under sterile conditions. Leukocytes were isolated by gradient centrifugation with a Histopaque-1077 (Sigma) at 400× g, 5 min, 4 °C) and the supernatant was removed. The pellet was resuspended in the RPMI medium and the cells were counted. Approximately 1 × 106 cells/mL were mixed with 100 µL 0.5% LMP agarose and placed on a precoated slide. After solidification of the agarose, the slides were immersed in fresh ice-cold lysis solution  overnight at 4 °C. Alkaline denaturation was performed in an electrophoresis buffer solution  for 20 min. Electrophoresis was carried out in a chilled electrophoresis buffer for 20 min . The slides were then washed three times for 5 min in 0.4 M Tris-Cl, pH 7, treated with ethanol for another 5 min, and dried. For comet analysis, the slides were stained with DAPI (5 µg/mL). A total of 100 comets (50 cells from each of the two replicated slides) were scored visually in five classes according to tail size and intensity  as depicted in n1–n4 are the number of comets with damage levels 1, 2, 3 and 4. DI values can indicate various situations, from all undamaged cells  to highly damaged cells .The alkaline comet assay was carried out according to the procedure of Singh et al. . Convent2O2, 50 µM ascorbic acid, and 80 µM of FeCl3). The reaction mixture was then incubated for 30 min at 37 °C and the DNA was analyzed on a 1% agarose gel.DNA nicking assay was performed using supercoiled pUC19 plasmid DNA by the method of Prakash et al. A reaction mixture containing different concentrations of plant extracts (10–100 µg/mL) and pUC19 plasmid DNA (0.5 µg) was incubated for 10 min at room temperature, followed by the addition of Fenton’s reagent  containing 4.5 g/L glucose, 10% fetal bovine serum (FBS), and 1% antibiotics .Cells were purchased from ATCC . Cancer cell lines (breast cancer cell line MDA-MB-231 and human glioblastoma cell line-A1235) were cultured in a humidified atmosphere with 5% CO4/mL. The cells were then plated in 96-well plates at a density of 11,000 cells/well and incubated overnight. The cells were treated with A. × cornutum and A. cepa methanolic extract at a concentration of 100 µg/mL in a complete medium (in triplicate) for 4, 24, 48 and 72 h. Then, the MTT assay was performed in such a manner that after the treatment with onion extracts, the cells were incubated with 0.5 g MTT/L at 37 °C for 2 h. After that, the medium was removed and dimethylsulphoxide (10% DMSO) was added and incubated for another 10 min at 37 °C while shaking. The degree of formazan formation, an indicator of living and metabolically active cells, was measured photometrically at 570 nm. The data was calculated in relation to the untreated control (100%) from three independent measurements.Cells were resuspended in a diluted solution of trypan blue and counted by a binocular inverted microscope, MOTIC AE30 , using Neubauer chambers. The cell number was calculated according to the formula: number of counted cells × 10t-test was used to analyze data and to discriminate statistically significant results.Results are expressed as mean values with depicted standard deviation. Microsoft Excel Student A. × cornutum. The HPLC analysis revealed two major quercetin conjugates  as the most abundant flavonols in A. × cornutum and A. cepa extracts. Additionally, we successfully identified three anthocyanins in both onions, while the fourth anthocyanin, delphinidin glucoside, was observed only in A. × cornutum. Overall, A. × cornutum showed slightly higher concentrations of all identified phenolic compounds, which may be the cause of the higher bioactivity profile accounted for in this onion.This was the first known thoroughly comprehensive study of the phytochemical composition and biological activity of A. × cornutum and A. cepa methanolic extracts have a comparable composition and concentrations of phenolic compounds, as well as antioxidant, antigenotoxic and antiproliferative effects. This research demonstrated that A. × cornutum and common onions should be considered as important sources of natural antioxidants that have a beneficial protective effect on human health.Our results clearly indicate that both onions have strong protective effects on the DNA molecule, as was proven with several different in vitro experiments. Both onions have also shown strong antiproliferative activity on human cancer cell lines; however, the susceptibility of glioblastoma cells against both onion extracts was higher than that of a breast cancer cell line. The antiproliferative activity of the tested methanol extracts could be mediated by the induction of apoptosis, alterations of the cell cycle, or some other mechanism, while further research is needed to clarify the exact mechanism(s) of their antiproliferative activity in vitro. Therefore, it can be concluded that


The subsequent L5-S1 disc degeneration associated with long fusion arthrodesis terminating at L5 in patients with adult scoliosis has been a common concern. However, few studies paid attention to its preoperative predictors, especially in spinopelvic parameters. The purpose of the present study was to clarify the preoperative predictors of subsequent L5-S1 disc degeneration after long fusion arthrodesis terminating at L5 in patients with adult scoliosis on spinopelvic parameters.In this retrospective study, we enrolled 67 patients with adult scoliosis, and the patients were divided into disc degeneration group (DD) and no disc degeneration group (NDD), based on the presence or absence of subsequent L5-S1 disc degeneration. The status of L5-S1 disc was evaluated by a modified version of radiographic classification. Characteristics and spinopelvic parameters of preoperative patients were collected as potential predictors for subsequent lumbosacral disc degeneration after long fusion arthrodesis terminating at L5 in patients with adult scoliosis. Multivariate logistic regression analysis and the receiver operating characteristic curve were used to identify the preoperative predictors, with an adjusted odds ratio (OR) and 95% confidence intervals (CI).P <  0.001), and the other 31 patients were divided into group NDD (preoperative and last follow-up score 0.87 ± 0.49). There was no statistical difference in preoperative score (P = 0.583) of lumbosacral disc between two groups; however, significant statistical difference showed in last follow-up score (P <  0.001). Multivariate logistic regression identified three preoperative predictors: pelvic incidence (PI) (P = 0.018), sagittal vertical axis (SVA) (P = 0.024), and sacrum-femoral distance (SFD) (P = 0.023). PI < 48.5° , SVA > 4.43 cm , and SFD > 5.65 cm  showed satisfied accuracy for predicting subsequent L5-S1 disc degeneration.Thirty-six patients (53.73%) with subsequent L5-S1 disc degeneration were divided into group DD . PI < 48.5°, SVA > 4.43 cm, and SFD > 5.65 cm were preoperative predictors for the subsequent L5-S1 disc degeneration. More attention should be paid to prevent the L5-S1 disc from degeneration when these preoperative predictors exist, especially with two or more. Adult scoliosis, a spinal deformity with Cobb angle greater than 10° after skeletal maturation, occurs in two types: idiopathic adolescent scoliosis in adulthood and de novo scoliosis –4.The distal fusion segment extending to the sacrum is undisputed for patients with adult scoliosis with presented severe lumbosacral disc degeneration, instability of lumbosacral segment, L5 spondylolysis, and nerve compression at L5-S1 needing decompression . Long fuTherefore, the purpose of our study was to clarify the preoperative predictors of subsequent degeneration in L5-S1 disc after long fusion arthrodesis terminating at L5 in patients with adult scoliosis and to provide evidence for surgical strategy. Besides, spinopelvic parameters have been addressed in the current study because of their important role in subsequent lumbosacral disc degeneration.This retrospective study was approved by the Institutional Ethics Board of the Third Hospital of Hebei Medical University. The retrospective study included a consecutive series of 67 patients with adult scoliosis who underwent surgical treatment at our institution from May 2004 and March 2016. The inclusion criteria were as follows: (1) presence of adult scoliosis with Cobb angle greater than 10°, (2) posterior-only surgical instrumented procedure terminating at L5, (3) no history of any spinal surgery, (4) fixed segments greater than or equal to four, and (5) follow-up period more than 2 years. The exclusion criteria were as follows: (1) lack of completed clinical data, (2) disability of lower limb, and (3) tumor or inflammation involving the spine. Finally, 49 women and 18 men with mean age at surgery of 59.24 years  were reviewed in the study. Forty-one patients (61.19%) had de novo scoliosis in adulthood and 26 patients (38.81%) had a history of idiopathic adolescent scoliosis without treatment. The mean follow-up period was 4.85 years . Clinical data, including age, gender, body mass index (BMI), number of instrumented vertebrae, and spinopelvic parameters, were collected as potential predictors for subsequent lumbosacral disc degeneration.Spinopelvic parameters were measured on anteroposterior and lateral radiographs of the entire spine Fig. . The sagt test; if the data was not in accord with normal distribution or homoscedasticity, Mann-Whitney U test would be used. Categorical data including gender and number of instrumented vertebrae was analyzed by chi-square test. The statistical significance was set as P value less than 0.05. The potential predictors which showed P <  0.05 in univariate regression analysis were analyzed by multivariate logistic regression analysis with adjusted odds ratio (OR) and 95% confidence intervals (CI). PI, SVA, and SFD were entered into the logistic regression again to identify the correlation between subsequent disc degeneration and the predictors after adjusting for potential confounding factors. The sensitivity and specificity in PI, SVA, and SFD were evaluated by the receiver operating characteristic curve, and, at the same time, cutoff value in the three parameters was obtained to achieve good predicting.The data was analyzed by statistical software, the SPSS 21.0 software . Results are presented as the mean ± standard deviation (SD). Quantitative data including age, BMI, degenerative degree, and spinopelvic parameters was analyzed by independent P <  0.001). However, no significant statistical difference was found in preoperative average score between the two groups  (P = 0.583). The number of instrumented vertebrae was four in three patients, five in four patients, six in 11 patients, seven in eight patients, eight in 28 patients, nine in six patients, ten in four patients, and 11 in three patients. There was no significant statistical difference in instrumented vertebrae number between group DD (7.56 ± 1.64) and group NDD (7.52 ± 1.54) (P = 0.927). Significant statistical difference was not found in average follow-up period (P = 0.377), BMI (P = 0.207), and gender (P = 0.355) , SVA  and SFD  showed significant difference. In multivariate logistic regression, PI , SVA , and SFD  were identified to be predictors for subsequent L5-S1 disc degeneration (Table P = 0.008), SVA , and SFD  were shown in receiver operating characteristic curve analysis  . It may be caused by the reason that the number of patients with instrumented vertebrae > 10 (three patients with fusion from T7 to L5) in our study was limited. Unexpectedly, all the three patients developed into subsequent L5-S1 disc degeneration. The relationship between number of instrumented vertebrae and subsequent L5-S1 disc degeneration should be further studied.The preoperative SVA > 4.43 cm was identified as a preoperative predictor for the subsequent L5-S1 disc degeneration. Many previous studies proved that the subsequent L5-S1 disc degeneration was highly correlated with sagittal imbalance , 22–24. PI, an anatomical parameter, being not affected by lumbar degeneration diseases after the end of bone growth, has been mentioned in previous studies and played a significant role in disc degeneration , 22, 25.Several limitations in the current study needed to be pointed out. First, the study was limited by its retrospective nature. Second, follow-up period in the study was medium-term. However, medium-term follow-up was not enough to evaluate the status of disc degeneration because the process was gradual. Third, the radiographic classification of the disc is divided into only four grades, so a more detailed classification method would be proposed and applied in this study. What is more, the number of patients with adult scoliosis after long fusion arthrodesis terminating at L5 in this study was small. Thus, further multicenter studies with a large sample would be preformed to clarify the correlation between the preoperative predictors and the subsequent L5-S1 disc degeneration.The prevalence of the subsequent L5-S1 disc degeneration in this study was 57.3% (36 of 67 patients). The PI < 48.5°, SVA > 4.43 cm, and SFD > 5.65 cm were identified as the preoperative predictors for the subsequent L5-S1 disc degeneration. Spine surgeons should pay more attention in choosing the surgical strategy when the preoperative predictors exist in patients, especially with two or more.


However, the full commercialization of Li-S batteries is still hindered by dramatic capacity fading resulting from the notorious “shuttle effect” of polysulfides. Herein, we first describe the development of a facile, inexpensive, and high-producing strategy for the fabrication of N-, O-, and S-tri-doped porous carbon (NOSPC) via pyrolysis of natural wheat straw, followed by KOH activation. The as-obtained NOSPC shows characteristic features of a highly porous carbon frame, ultrahigh specific surface area (3101.8 m2·g−1), large pore volume (1.92 cm3·g−1), good electrical conductivity, and in situ nitrogen (1.36 at %), oxygen (7.43 at %), and sulfur (0.7 at %) tri-doping. The NOSPC is afterwards selected to fabricate the NOSPC-sulfur (NOSPC/S) composite for the Li-S batteries cathode material. The as-prepared NOSPC/S cathode delivers a large initial discharge capacity (1049.2 mAh·g−1 at 0.2 C), good cycling stability (retains a reversible capacity of 454.7 mAh·g−1 over 500 cycles at 1 C with a low capacity decay of 0.088% per cycle), and superior rate performance (619.2 mAh·g−1 at 2 C). The excellent electrochemical performance is mainly attributed to the synergistic effects of structural restriction and multidimensional chemical adsorptions for cooperatively repressing the polysulfides shuttle.Recently, lithium-sulfur (Li-S) batteries have been greeted by a huge ovation owing to their very high theoretical specific capacity (1675 mAh·g Wheat straw, a natural subsidiary product of wheat, is readily available, widespread, and generally cheap, which is a fascinating starting material for fabricating high value-added nano-materials, such as hierarchically porous N-rich carbon for lithium ion batteries, nano-silica, activated carbon for supercapacitors, and wheat straw carbon-matrix-wrapped sulfur composites ,32,33,342 atmosphere with a heating-up velocity of 5 °C·min−1. Then, the activation was carried out by calcinating a mixture of the above obtained product and KOH with a weight ratio of 1:5 at 800 °C for 120 min in a sealed box-type furnace under N2 flow at a heating-up velocity of 5 °C min−1. After cooling to the ambient temperature spontaneously, the activated carbon was collected, neutralized with 0.5 M HCl solution, washed by DI water until a pH equaling to 7.0, and afterwards dried at 105 °C in an oven for 24 h, and the consequent samples were named as NOSPC. As a comparison, the unactivated wheat straw carbon (marked as WSC) was also produced via pyrolysis of wheat straw at 400 °C for 90 min, and followed by at 800 °C for 120 min under N2 atmosphere in a sealed box-type furnace except with KOH activation (the other steps are the same as that of NOSPC).The N-, O-, and S-tri-doped porous carbon (NOSPC) was produced via directly pyrolysis of wheat straw followed by KOH activation. In detail, the wheat straw was first cut into small segments with shears (about 3 cm), washed by deionized water and afterwards dried at 60 °C for 12 h. Twenty g of wheat straw was put in the plumbago crucible and pyrolyzed in a sealed box-type furnace at 400 °C for 90 min under NThe NOSPC-sulfur (NOSPC/S) composite was fabricated via the representative melt-diffusion strategy. In detail, the above NOSPC and sulfur powder with a weight ratio of 35:65 were ground in a mortar for 30 min to ensure uniform mixing. This mixture was placed in a small glass bottle, and then transferred into 80 mL Teflon-lined stainless steel autoclave filled with Ar protection in a glove box. The stainless steel autoclave was fetched out from the glove box and put in a drying oven, finally heated at 155 °C for 12 h, and subsequently for an extra 2 h at 220 °C. As a comparison, the WSC/S composite containing the same sulfur mass ratio was also produced by the above technique.2 atmosphere from indoor temperature to 800 °C with a heating-up velocity of 10 °C·min−1. The specific surface area, pore volume, and pore size distribution of the samples were determined by N2 gas adsorption at 77 K, with a self-propelled adsorption apparatus . To study surface chemical ingredients and function groups of the samples, X-ray photoelectron spectroscopy (XPS) tests were carried out on a K-Alpha 1063 Ultra spectrometer . Elemental analysis was done using a EuroEA3000  analyzer.The structures and morphologies of the as-prepared materials were characterized by scanning electron microscope (SEM)  provided with an X-ray energy dispersive spectrometer (EDS), X-ray diffractometer , and Raman spectra . The sulfur contents in NOSPC/S and WSC/S composites were gauged by a thermogravimetric analyzer  in a N−2. CR2025 coin cells were assembled in an Ar-filled glove box , with H2O and O2 levels below 0.1 ppm. The electrolyte was 1 mol·L−1 lithium bis-(trifluoromethanesulfonyl)imide (LiTFSI) in a mixture of equivalent volumes of 1,3-dioxolane (DOL) and dimethoxyethane (DME) with 0.1 mol·L−1 LiNO3 additives, and the related amount of electrolyte was 25 μL for a coil cell. The separator was a Celgard 2400 membrane and the anode was lithium foil. The galvanostatic discharge/charge tests were implemented within the voltage scope of 1.7–2.8 V versus Li+/Li at indoor temperature by employing a cell testing system . The specific capacity of the cell was based on active sulfur (1 C = 1675 mA·g−1). The cyclic voltammogram (CV) test was carried out at the sweep speed of 0.2 mV·S−1 within the voltage scope of 1.6–3.0 V by a PARSTAT 4000 electrochemistry workstation . The electrochemical impedance spectroscopy (EIS) measurement was performed via PARSTAT 4000 electrochemistry workstation between the frequency ranges from 100 kHz to10 mHz with a 5 mV response excursion.The cathode electrodes were prepared by mixing the carbon/sulfur composites, acetylene black, and PVDF binder with a weight ratio of 8:1:1, and the mixture was dispersed into NMP solvent to form the electrode slurry. The slurry was uniformly casted on the Al foil with a scraper blade. Then, the Al foil was dried at 50 °C overnight under the vacuum. The Al foil was chopped into round pieces with a diameter of 1 cm for use as the working electrode. The sulfur areal mass loading of the cathode electrodes was about 1.0–1.2 mg·cmWheat straw, a subsidiary product of mature wheat, has a natural multilayer structure composed of three different kinds of polymers, namely, cellulose, hemicellulose, and lignin. These polymers cross-link mutually and constitute the stem of wheat straw ,38. The The structures and morphologies of as-prepared WSC, NOSPC, WSC/S, and NOSPC/S were first characterized by SEM. As illustrated in After the sulfur encapsulation process, the SEM images of WSC/S and NOSPC/S composites are displayed in 2 atmosphere from indoor temperature to 800 °C. As displayed in In order to quantify the sulfur amount in the WSC/S and NOSPC/S composites, TGA measurements were performed in an N−1 and 1590 cm−1 (D and G bands), respectively, which implies their good electrical conductivity. The D band usually represents disordered carbon structure associated with the edge sites, defects, and holes, while the G band is a typical character of crystalline graphite structure corresponding to sp2 bonding. Moreover, the relative intensity of D band (ID) and G band (IG) can indicate the disorder degree of carbon materials [D/IG (1.08) than that of WSC (1.01), on account of the heteroatomic functional groups on the surface of NOSPC, as well as more defects and highly porous structure caused by KOH activation [−1 which can be assigned to the S-S bond [The Raman spectroscopy was also used to examine the structures of the WSC, NOSPC, WSC/S, and NOSPC/S, as shown in aterials ,41. The S-S bond , while t2 adsorption/desorption isotherms derived from BET tests were obtained to quantitatively confirm the porous structure of WSC, NOSPC, and NOSPC/S. As illustrated in 2 adsorption/desorption isotherms for original WSC and NOSPC exhibit a high adsorption uptake in the relative pressure of P/P0 < 0.1, and a hysteresis loop within the scope of 0.5–0.99 P/P0, indicating that both the WSC and NOSPC are typically hybrid micro/mesoporous carbon. It is worth noting that the micropores of NOSPC are more luxuriant than those of WSC  and larger pore volume (1.92 cm3·g−1), which was caused by the KOH activation. In addition, the micropore volume of WSC (0.221 cm3·g−1) was also much less than that of NOSPC (1.457 cm3·g−1), which is consistent with the results of 2·g−1 and 0.026 cm3·g−1, respectively). Meanwhile, from The Ne of WSC . Moreove NOSPC 1.7 cm3·g−18 [The XPS was utilized to analyze the chemical constituents and functional groups of NOSPC and NOSPC/S, and the corresponding outcomes were displayed in 8 . In the 8 ,24. The 8 ,20,21, b8 ,27,36.−1 within the voltage scope of 1.6–3.0 V. During the cathodic scan, there are two noteworthy reduction peaks roughly at 2.3 and 2.0 V, relating to the conversion of elemental sulfur to dissoluble lithium polysulfides  and further from polysulfides to indissoluble Li2S2/Li2S, respectively [2S2/Li2S to low-order lithium polysulfides and afterwards to high-order lithium polysulfides, respectively [The electrochemical properties of CR2025 coin cells assembled with lithium foils as the anodes and NOSPC/S and WSC/S composites as the cathodes were then studied. ectively ,18. In tectively ,46. Rema−1 (with a coulombic efficiency of 98.1%), contrasting to the 435.6 mAh·g−1 of the WSC/S cathode (with a coulombic efficiency of 92.5%). The higher active material utilization of the NOSPC/S cathode mainly results from the highly homogeneous decentralization of elemental sulfur in the pores of NOSPC, thus leading to a preferable electric contact between carbon and sulfur. At the same time, as illustrated in −1 after 100 cycles at 0.2 C, while a pretty inferior discharge capacity of 326.3 mAh·g−1 is delivered for the WSC/S cathode. All in all, in comparison with the WSC/S cathode, the NOSPC/S cathode exhibits improved reversible capacity and cycling performance, which could be ascribed to the highly porous structure and in situ N-, O-, and S-tri-doping of NOSPC.The initial galvanostatic charge/discharge profiles and cycling behaviors of the WSC/S and NOSPC/S composites at 0.2 C are presented in −1 at 0.5, 1.0, and 2 C, respectively. Importantly, a reversible capacity of 799.5 mAh·g−1 for the NOSPC/S cathode is recovered when the current is switched back to 0.2 C, indicating a favorable high-rate performance. Besides, As shown in −1 was obtained at 1 C after the activation in the first two charged/discharged cycles at 0.05 C. Notably, the NOSPC/S cathode exhibited a distinguished cyclability by maintaining the high discharge capacities of about 585.9 mAh·g−1 after 200 cycles and 454.7 mAh·g−1 after 500 cycles at 1 C, respectively. Compared with the initial discharge capacity, the fading rate is only 0.088% over 500 cycles at 1 C, and the corresponding average coulombic efficiency is about 95.6%. Moreover, after 500 cycles, the decrease in coulombic efficiency is probably due to the consumption of a small amount of LiNO3 additive after prolonged cycling [The long-range cycling performance of the NOSPC/S cathode was also tested at a high rate of 1 C, as shown in  cycling . Besides cycling ,49,50,51 cycling ; 2) the the−1 wa cycling ,45; and  cycling ,27,36.o, which includes the interface impedance between active materials and current collector, the active materials’ intrinsic resistance, as well as the electrolytes’ ionic resistance [ct, which corresponds to the kinetic resistance of electrochemical reaction at the electrode-electrolyte interface [o (1.88 Ω) value of the NOSPC/S electrode is much lower than that of the WSC/S electrode (9.82 Ω). It is because sulfur in the WSC/S electrode mainly exists on the surface of the WSC, while most of the sulfur permeates the pores of the NOSPC. Moreover, compared with the WSC/S electrode, the Rct of the NOSPC/S electrode markedly decreases from 46.36 to 18.63 Ω. This is due to the conductive NOSPC can lower the resistance for electrons transport throughout the electrode, and the highly porous structure can facilitate ion transfer, thus leading to the decrease of Rct.In order to further comprehend the interfacial charge transfer and ion diffusion process of the WSC/S and NOSPC/S electrodes, the EIS tests were implemented with button cells before discharge, as displayed in sistance ,4. The snterface . Dependi−1 at 0.2 C), good cycling stability (retains a reversible capacity of 454.7 mAh·g−1 over 500 cycles at 1 C with 0.088% capacity decay per cycle), and superior rate performance (619.2 mAh·g−1 at 2 C). This work will shed light on the reasonable design of carbon/sulfur composite materials with unique physical properties and surface chemistries, offering priceless guidance for the progress of advanced Li-S batteries for the practical applications.In summary, a novel N-, O-, and S-tri-doped porous carbon (NOSPC) has been triumphantly synthesized by pyrolyzing of natural wheat straw followed by KOH activation for the first time. Various test analyses demonstrate that the resulting NOSPC possesses a highly porous carbon framework, ultrahigh specific surface area, large pore volume, good electrical conductivity, and in situ N-, O-, and S-tri-doping. Due to the synergistic effects of physical confinement and multidimensional chemical adsorptions for repressing the polysulfides shuttle, the as-obtained NOSPC/S composite for the Li-S batteries delivers dramatically enhanced electrochemical properties including large initial discharge capacity (1049.2 mAh·g