High uric acid (UA) level and high salt intake are reportedly associated with cardiovascular disease. This study investigated the association between UA and urinary sodium excretion, as well as its interaction on the risk of prehypertension. A total of 1869 participants without hypertension were recruited from a previously established cohort in Shaanxi Province, China. The participants were classified as normotensive or prehypertensive on the basis of their blood pressure. Increasing quartiles of sodium excretion were associated with high urinary UA/creatinine levels in prehypertensive participants. Estimated sodium excretion positively correlated with urinary UA/creatinine excretions in the prehypertensive group. In addition, the multivariate-adjusted odds ratios for prehypertension compared with normotension were 1.68 (1.27–2.22) for sodium excretion and 1.71 (1.21–2.42) for serum UA. Increasing sodium excretion and serum UA were associated with higher risk of prehypertension. Compared with the lowest quartiles, the highest sodium excretion and serum UA quartiles entailed 3.48 times greater risk of prehypertension. Sodium excretion is associated with urinary UA excretion in prehypertensive participants. The present study shows that high levels of salt intake and serum UA simultaneously are associated with a higher risk of prehypertension. According to the Seventh Report of the Joint National Committee (JNC-7) guideline, prehypertension is characterized by a systolic blood pressure (SBP) ranging from 120 mm Hg to 139 mm Hg and/or a diastolic blood pressure (DBP) ranging from 80 mm Hg to 90 mm Hg1. The prevalence of prehypertension is rapidly increasing worldwide. In the InterASIA study, the prevalence of prehypertension is 21.9% among Chinese adults 2, and prehypertension is emerging as an independent risk factor for cardiometabolic disorders, including metabolic syndrome, diabetes, chronic kidney disease, stroke, and cardiovascular diseases4.Hypertension contributes to the burden of heart disease, stroke, and kidney failure, and it is one of the leading causes of morbidity and mortality. Prehypertension, a state between normotension and hypertension, is a strong predictor of hypertension5. Dietary salt intake by itself, even without causing hypertension or volume overload, might be deleterious, resulting in cardiac remodeling, renal fibrosis, and left ventricular hypertrophy8. Several mechanisms, including endothelial dysfunction, oxidative stress, inflammation, insulin resistance, and neurogenically mediated increase in peripheral resistance, contribute to the harmful effects of dietary salt10. Recent studies have shown that increased salt intake may be associated with the pathogenesis of prehypertension13. However, data on the association between dietary salt intake and prehypertension are lacking.Excessive dietary salt intake plays an important role in the onset and maintenance of hypertension, whereas restricted salt intake lowers blood pressure (BP)14. Epidemiological studies have identified serum UA is an important risk factor for cardiovascular disease and hypertension17. For example, Puddu et al.17 found that serum UA could predict not only short-term but also long-term incidence of cardiovascular events as well as cardiovascular death and all-cause mortality. Recent studies have shown that increased sodium intake significantly lowers serum UA19. However, no research has focused on the relationship between dietary salt intake and UA levels, especially urinary UA excretion, in prehypertensive participants. Furthermore, the relationship between UA and prehypertension and the synergistic effects of UA and dietary salt intake on the risk of prehypertension remain unclear to date.Uric acid (UA) is the metabolic end product of purine degradation in humans; xanthine oxidase is the enzyme responsible for UA production and free radical damageIn the present study, we used our previously established cohort that has been followed up for 30 years to examine the possible associations between urinary sodium excretion, which was used as surrogate for salt intake, and serum and urinary UA levels in prehypertensive participants. We particularly sought to investigate the interactions between urinary sodium excretion and serum UA on the risk of prehypertension in Chinese young adults.21. To explore the BP trajectory and its risk factors from children to adults, we made the long-term follow-ups of this cohort in 1989, 1992, 1995, 2005, 2013, and 2017  in Hanzhong, Shaanxi, ChinaIn this study, we followed up this cohort from April to July 2017, and a total of 2780 were followed up this time. The total rate of this follow-up was 60.3%, which was very rare for such a long-term follow-up. The participant selection process is described in Fig. http://www.clinicaltrials.gov). Date of registration: 12/04/2016.The present study complied with the Declaration of Helsinki, and the research protocol was approved by the Ethics Committee of the First Affiliated Hospital of Xi’an Jiaotong University (code: 2015–128). Upon admission, all subjects provided their informed consent. Trial Registration Number: NCT02734472  and normotension (SBP <120 mm Hg and DBP <80 mm Hg).BP was measured by six trained staff members using a standard mercury sphygmomanometer as previously describedg for 10 min, and then stored at −80 °C until analysis. Total cholesterol, triglyceride, high-density lipoprotein (HDL), low-density lipoprotein (LDL), serum creatinine, and blood glucose were measured using an automatic biochemical analyzer . Serum UA level was measured with a Hitachi clinical chemistry analyzer with the uricase HMMPS method. Five samples were used to evaluate the intra-assay and inter-assay coefficients of variation (CV), which ranged from 2.3% to 4.5% and from 3.2% to 6.4%, respectively. Estimated glomerular filtration ratio (eGFR) was calculated using the Modification of Diet in Renal Disease formula24. Hyperuricemia was defined as serum UA level of ≥420 μmol/L for men and ≥360 μmol/L for women.Blood samples were obtained by peripheral venous puncture, immediately centrifuged at 3000 × 26. A brief description of the validation of the Kawasaki formula is provided in the article by Mente et al.27.A morning fasting midstream urine sample was collected from each participant and frozen at −20 °C to −40 °C. All urine samples were shipped in ambient packaging with the use of ice boxes to the clinical laboratory at the First Affiliated Hospital of Xi’an Jiaotong University in Xi’an, China. Urinary sodium, creatinine, UA, and albumin were measured by an automatic biochemical analyzer  at a certified clinical lab. The intra- and inter-assay CVs were 0.42% and 1.69% for urinary sodium, 1.25% and 2.17% for urinary creatinine, 0.64% and 2.13% for urinary albumin, and 3.1% and 5.2% for urinary UA, respectively. The Kawasaki formula was used to estimate 24-hour urinary sodium excretion, and the estimate was used as surrogate for salt intakeP < 0.05 was considered statistically significant.Data are expressed as means ± standard deviation (SD) for normally distributed values, as median (25th and 75th percentile) for non-normally distributed values, and as percentages. The Kolmogorov–Smirnov test was used to determine whether the continuous variables were normally distributed. Statistical significance of difference among the groups was calculated using χ2-test for categorical variables, Student’s t-test for continuous variables in normally distributed data, and Mann–Whitney test for non-normally distributed data. One-way ANOVA was employed to compare the mean values across the sodium excretion quartile groups. Multiple linear regression and Pearson correlation analyses were performed to determine the strength of association between serum and urinary UA levels and other continuous parameters. The adjusted odds ratio (OR) and 95% confidence interval (CI) were calculated to estimate the risk for prehypertension of single/combined status of sodium excretion and serum UA, and quartiles of sodium excretion and serum UA by logistic regression analyses. All statistical analyses were performed using SPSS for Windows version 16.0 . A two-sided All raw experimental data used in this study is available from the corresponding authors on request.A total of 1869 individuals who met the inclusion criteria were finally followed up and included in the current analyses. Of these 1869 individuals, 842 (45.1%) were identified as prehypertensive while 1027 (54.9%) as normotensive.P for trend <0.001). No significant difference was observed in serum UA across each quartile of urinary sodium excretion in prehypertensive participants (P for trend = 0.951).Firstly, a total of 842 prehypertensive subjects were analyzed to examine the association between sodium excretion and serum and urinary UA levels. Table r = 0.496, P < 0.001) but not with serum UA  in the prehypertensive group  and gender .Estimated sodium excretion, the substitute for salt intake, positively correlated with urinary UA/creatinine excretions  did not remain in the final model  and prehypertensive (n = 842) subjects in the current analysis. Table 7 and preel Table .Table 2CP for trend <0.001). In the multivariate model, further adjusting for BMI, fasting glucose, total cholesterol, triglycerides, LDL, HDL, serum creatinine, alcohol consumption, smoking status, diabetes and physical activity, the ORs (95% CI) were 1.00, 1.15 (0.87–1.52), 1.46 (1.11–1.91) and 1.68 (1.27–2.22) (P for trend = 0.001). The effect of serum UA on the risk of prehypertension was also estimated by the quartiles of serum UA. The quartiles of serum UA were defined as follows: quartile 1: < 219.2 μmol/L; quartile 2: 219.2–271.1 μmol/L; quartile 3: 271.1–322.2 μmol/L; and quartile 4: > 322.2 μmol/L. After adjustment for multiple confounders, compared with the lowest serum UA quartile, the ORs of prehypertension were 1.22 , 1.46  and 1.71 ; P = 0.019 for trend.We further assessed the individual effects of salt intake and serum UA on prehypertension risk Table . In an aP = 0.046). The ORs of prehypertension were about 3.48 times as much as in the highest sodium excretion and serum UA quartiles (sodium excretion >5.88 g/day and serum UA >322.2 µmol/L) than in the lowest sodium excretion and serum UA quartiles (sodium excretion <4.02 g/day and serum UA <219.2 µmol/L).Finally, the synergistic effects of salt intake and serum UA on the risk of prehypertension were estimated after controlling for confounders Fig. . A formaIn this cohort of Chinese individuals with prehypertension, urinary sodium excretion showed no association with serum UA but significantly correlated with urinary UA. To the best of our knowledge, this study is the first to examine the relationship of salt intake to serum and urine UA levels in prehypertensive subjects.Homo sapiens, allowing them to maintain BP when access to sodium is scarce28. This theory was demonstrated in uricase-deficient rat models showing an increase in BP from hyperuricemia in the context of a low-salt diet29. Additional rat models have supported this hypothesis, showing that UA upregulates and activates epithelial sodium channels in nephrons30. Hou et al.31 have recently reported in a cross-sectional study of 1668 Chinese participants that high salt intake enhances associations of blood UA with hypertension and related cardiovascular risk. However, limited studies focused on salt intake and UA. One large longitudinal cohort study conducted in 4062 participants from Netherlands found that higher sodium intake predicted lager longitudinal increase of serum UA. It was reported that each 1 gram higher sodium intake was associated with a 1.4 μmol/L increase in serum UA32. Another trial performed in 27 men showed that increasing sodium intake from 20 mEq/day to 200 mEq/day decreased UA levels by 1 mg/dL33. In addition, a randomized crossover trial of 103 adults with prehypertension or stage I hypertension showed that 30 days of low versus high sodium intake (60 versus 180 mmol/day) significantly decreased serum UA19. In this study, we showed that the serum UA levels were similar between each quartile of estimated sodium excretion in prehypertensive subjects. Furthermore, sodium excretion was not correlated with serum UA and hyperuricemia in the unadjusted and adjusted analyses. The discrepant results of these studies may be attributed to their different study populations, designs, sample sizes, and racial differences.Although the relationship between salt intake and hypertension is well established, its relationship with UA remains controversial. One proposed hypothesis states that higher UA levels represent an evolutionary advantage in 32. Approximately 10% of filtered UA is excreted34. To the best of our knowledge, this present study is the first to demonstrate that urinary excretions of UA were significantly associated with sodium excretion in prehypertensive subjects. The mechanism by which sodium intake increases urinary excretion of UA remains unclear. It is possible that the relationship between sodium intake and urinary urate excretion results from effects of sodium intake on glomerular filtration rate and excretion or absorption of urate. Previous physiology studies have shown that reabsorption of sodium and urate accompanies one another at different sites in the nephron36. Thus, it is possible that decreased renal reabsorption of sodium from excess sodium intake contributes to a decrease in urate reabsorption. This hypothesis has been evidenced by our interventional study showing that urinary UA excretions were markedly increased during high-salt intake, which was further reinforced by the observation that urinary UA positively correlated with urinary sodium excretion37. Furthermore, a Spanish study also found a directly correlation between the clearance of UA and fractional excretion of sodium, indicating the potential interaction of sodium and UA excretion38. Finally, this relationship may reflect action of the renin-angiotensin system, as uric acid is inversely related to vascular resistance39 and renal blood flow40. Similarly, angiotensin II has been shown to decrease urate excretion after an acute infusion42. Determining the molecular mechanism and signaling molecules responsible for the effects of salt intake on urinary UA can be of great interest.UA is a product of the metabolic breakdown of purine nucleotides. Approximately 70% of UA is excreted into the urine but is easily filtered into the renal tubule, and about 90% of filtered UA is reabsorbed by the S1 segment of the proximal convoluted tubule43. Jiang et al.44 described that the OR for prehypertension is 1.36 in subjects with UA ≥365 μmol/L compared with those with UA <215.9 μmol/L after adjusting for many confounders. The other two cross-sectional studies demonstrated that serum UA was independently related to the prevalence of prehypertension in Chinese adults46. In contrast, Vucak et al.47 determined that no association existed between elevated serum UA level and prehypertension; this might be because of higher background rate of prehypertension with increasing age that would contribute to a reduction in the odd ratios for serum UA. Recently, a prospective cohort study demonstrates that serum UA is an independent predictor for developing prehypertension48. In addition, Soletsky et al.49 reported that UA reduction rectifies prehypertension in obese adolescents. In the present study, we consistently showed that higher serum UA category was significantly associated with an increased OR for the presence of prehypertension, compared with the reference group. And the observed positive association between serum UA and prehypertension consistently occurred when serum UA was considered as a continuous variable.A limited number of studies have examined the relationship serum UA and prehypertension, and findings are conflicting. One US study found a positive association between serum UA and prehypertension with an OR of 1.96 for the top category of serum UA levels compared with the lowest5. However, clinical trials scarcely examined the relationship between salt intake and prehypertension. Moinuddin et al.11 showed that daily salt intake of prehypertensives (21.2 ± 1.2 g/day) was significantly greater than normotensive subjects (9.0 ± 0.5 g/day). This result is similar to our study, which found that compared with participants without prehypertension, those with prehypertension tended to have higher urinary sodium excretions . We further observed that the risk of prehypertension was significantly increased with the increasing quartiles of sodium excretion. In addition, Forman et al.32 in a large, prospective, population-based cohort, found that a higher sodium intake is associated with an increased risk of developing hypertension, particularly in those individuals who have higher levels of serum UA. Our results also showed that salt intake significantly interacted with serum UA. Taken both serum UA and sodium excretion into consideration to assess prehypertension, we found that the risk of prehypertension in the highest quartiles of serum UA and sodium excretion was 3.48  times greater than in the lowest quartile. In other words, participants with higher serum UA levels and salt intake simultaneously were more likely to have a higher risk for prehypertension.Previously, ample evidence suggests that excess salt intake is positively associated with elevated blood pressure and it can be lowered with reductions in dietary saltet al.27. Finally, the study was a single-center, cross-sectional study. A multi-center, prospective trial will be conducted to further understand and confirm the conclusions.This study has limitations that deserve mention. Firstly, since the study population was included from our previously established cohort, all participants in the present work were middle aged and youth between the ages of 35 and 48 years during the follow-up at 2017. In addition, 24-h urinary sodium excretion was estimated using spot urine samples. Estimated sodium excretions may change according to the urine sampling time because urinary sodium excretion has a circadian rhythm and may also be influenced by the time at which food is consumed. Thus, a single measurement may be insufficient to assess the sodium excretion of individuals. However, spot urine samples are practical at general medical facilities, and the reliability of the findings obtained may be improved using a calculation formula incorporating the estimated 24-hour urinary creatinine excretion based on age, height, and body weight. The validation of the Kawasaki formula was conducted by Mente In conclusion, the present study showed that urinary sodium excretion was significantly associated with urinary UA excretions in prehypertensive individuals. However, we failed to find a significant relationship between sodium excretion and serum UA in this Chinese population. In addition, elevated serum UA and sodium excretions appeared to be associated with the development of prehypertension. Individuals with higher serum UA levels and sodium excretions simultaneously had a higher risk for prehypertension. Further clinical trials that include participants with hypertension to investigate evaluate the joint effects of salt intake and serum UA on hypertension and other cardiovascular diseases can be of significant interest. Our findings supported the need for the development of a salt reduction programme.Supplementary Information


We sought to identify the MB signaling pathway of formoterol and the differences in signaling between these two ligands that result in the differential induction of MB. While formoterol and clenbuterol increased cAMP, only formoterol increased the phosphorylation of Akt and its downstream target eNOS. The increase in Akt phosphorylation was Gβγ- and PI3K-dependent, and the increase in eNOS phosphorylation was Gβγ- and Akt-dependent. Only formoterol increased cGMP. Formoterol induced MB as measured by increases in uncoupled cellular respiration and PGC-1α and NDUFS1 mRNA expression and was blocked by inhibitors of Gβγ, Akt, NOS, and soluble guanylate cyclase. To identify distinct receptor-ligand interactions leading to these differences in signaling, we docked formoterol and clenbuterol to six structures of the β2AR. Compared to clenbuterol, the methoxyphenyl group of formoterol interacted more frequently with V114 and F193, while its formamide group interacted more frequently with C191. These data indicate that the unique structural features of formoterol allow it to interact with the β2AR to activate the Gβγ-Akt-eNOS-sGC pathway to induce MB.Mitochondrial dysfunction is associated with numerous acute and chronic degenerative diseases. The beta-2 adrenergic receptor (β However, in disease states, dysfunctional mitochondria lead to metabolic defects and subsequent derangements in survival2, proliferation3, and differentiation7. One therapeutic strategy to treat mitochondrial dysfunction is the induction of mitochondrial biogenesis (MB)9. By generating new mitochondria, MB increases cellular respiration and ATP, reduces pathologic oxidative stress, and promotes cell repair and regeneration11.Mitochondria play numerous roles in cellular homeostasis, including energy metabolism, synthesis of key biomolecules, regulation of reactive oxygen species, and apoptosis9. In particular, GPCRs are attractive targets for the identification of therapeutics that induce MB because GPCR ligands represent numerous clinically approved receptor agonists12.A number of signaling molecules have been shown to induce MB, including transcription factors, kinases, cyclic nucleotides, and G protein-coupled receptors (GPCRs)2AR) agonist, as a potent and efficacious inducer of MB in vitro and in vivo13. Furthermore, in a mouse model of bilateral ischemic reperfusion-induced acute kidney injury formoterol stimulated MB with increased mitochondrial proteins and accelerated recovery of renal function14. Based on the success of formoterol, other β2AR agonists were screened for induction of MB. Although several agonists were able to induce MB similar to formoterol, several other agonists, including clenbuterol, were unable to induce MB at any concentration15. These data suggest that a subset of biogenic β2AR agonists modulates distinct signaling pathways from non-biogenic β2AR agonists to induce MB.Previous work in our laboratory identified formoterol, a beta-2 adrenergic receptor  and the signaling pathway responsible for formoterol-induced MB. Furthermore, we explored their chemical differences to identify key functional groups and structural differences that result in their differing abilities to induce MB. We found that formoterol, but not clenbuterol, activates the Gβγ-Akt-eNOS-sGC signaling pathway and that this pathway is necessary for the transcriptional and functional changes associated with formoterol-induced MB. Molecular modeling showed that formoterol stretches further across the binding pocket than clenbuterol, allowing for more frequent interactions with V114, F193, and C191 at specific chemical features. Additionally, the methoxyphenyl and formamide groups displayed distinct interaction fingerprints with the β2AR that may lead to the activation of Gβγ-dependent signaling.Because both formoterol and clenbuterol, a non MB inducer, are selective β2AR couples to the stimulatory G protein Gαs and the inhibitory G-protein Gαi, both of which affect the activity of adenylate cyclase and therefore the accumulation of cAMP. To assess the effects of formoterol and clenbuterol on cAMP accumulation, RPTC were co-treated for 1 h with 30 nM formoterol or 30 nM clenbuterol in the presence of 100 μM IBMX, a phosphodiesterase inhibitor to prevent cyclic nucleotide degradation. These concentrations ensure selective activation of the β2AR while also exerting the previously observed effects on MB. Maximal cAMP accumulation occurs at 1 h (data not shown)15. Both formoterol and clenbuterol increased cAMP relative to vehicle controls . Formoterol increased Akt phosphorylation while clenbuterol did not ion Fig. .Figure 320. Both gallein and MK2206 attenuated formoterol-induced eNOS phosphorylation . To examine any differences in cGMP accumulation, RPTC were treated with formoterol or clenbuterol in the presence of 100 μM IBMX for 1 h, and cGMP was determined by ELISA. Formoterol, but not clenbuterol, increased levels of cGMP relative to vehicle control Fig. .21. RPTC were then treated with formoterol or clenbuterol for 24 h, and FCCP-OCR consumption was measured. This time point was chosen based on previous studies identifying that formoterol induces MB at 24 h15. Formoterol alone increased FCCP-OCR, in agreement with previous studies  and NADH-ubiquinone oxidoreductase core subunit S1 (NDUFS1) was assessed using RT-qPCR. Formoterol alone caused a small but significant increase in PGC-1α and NDUFS1 Fig. , which w2AR agonists with the receptor. In addition to formoterol and clenbuterol, other ligands with known efficacy for MB were considered, including fenoterol, ritodrine, and terbutaline15. Ligands were docked to three inactive structures of the β2AR  and three active structures of the β2AR . Due to its longer methoxyphenyl group, formoterol was able to extend across the binding pocket to be in proximity to TM2, TM3, ECL2, and TM5 compared to clenbuterol . The resulting values were used to generate a heatmap for the interactions between the receptor and specific structural features of the ligand. Interaction pairs with more positive values (blue) indicated a greater importance for β2AR-mediated MB  enantiomer of fenoterol is a Gαs-biased ligand; however, the  enantiomer activates both Gαs and Gαi, potentially enabling it to activate Gβγ-dependent signaling pathways like formoterol22. Comparing -fenoterol to -fenoterol showed that the former was more likely to interact with V114 and F193 at F11 and engage in hydrogen bonding interactions with S203, S207, and the backbone of C191  enantiomer of fenoterol with a greater capacity to activate Gβγ-dependent signaling is more capable of inducing MB than the Gαs-biased  enantiomer. Comparing -fenoterol to clenbuterol showed a similar interaction profile to formoterol vs. clenbuterol -fenoterol, although interactions with V114 had a greater tendency to be aromatic than seen with formoterol. -fenoterol also had more hydrogen bonding interactions at F1, particularly with S207, S203, and the backbone of C191.To confirm the importance of the interactions identified above, we repeated the ligand interaction analysis for other ligands previously tested for MB. Fenoterol is a β1 Figure . Becausel Figure . InteracS,R’)-fenoterol to ritodrine -fenoterol interacted more frequently with F193 than ritodrine. Because these ligands share a common feature F11 but differ in biogenic status, the interaction of F193 with F11 is important for β2AR-mediated MB.To confirm the importance of interactions at F11, we compared . At feature F1, terbutaline interacted more frequently with S203, S207, and N293. When considered with the interaction profiles of formoterol and -fenoterol, these data suggest that interactions with S203 and S207 at feature F1 are important for β2AR-mediated MB. Interestingly, terbutaline also interacted more frequently with F193 at F8, F19, F10, and F11 than clenbuterol, suggesting that the 2,5-hydroxyl substituted ring of terbutaline and fenoterol facilitates interactions with F193 at features F8-F11.To confirm the importance of interactions at F1, we compared terbutaline to clenbuterol Figure . Like ri9. Here, we show that in RPTC, formoterol, but not clenbuterol, activates the Gβγ-Akt-eNOS-sGC signaling pathway and that this pathway is necessary for formoterol-induced MB -epicatechin and erythropoietin31. For such compounds, the role of Akt seems to be limited to the phosphorylation of eNOS, leading to NO generation. Nitric oxide is a potent inducer of MB in vitro and in vivo through the activation of sGC and subsequent cGMP accumulation32. However, because NO is a free radical, sustained NOS activation can increase oxidative and proteotoxic stress and can inhibit complex I of the electron transport chain34. Thus, both the signaling cascade activated by a compound and the duration of that signaling contribute to the therapeutic potential of inducers of MB.Prolonged activation of Akt enhances cellular survival but can lead to a decrease in mitochondrial function35. In contrast, gallein also prevents inflammatory cell infiltration17, leading to enhanced recovery following ischemic acute kidney injury36. Although neither study assessed mitochondrial activity, both regeneration following injury and inflammatory cell chemotaxis are enhanced by increases in mitochondrial activity38.Interestingly, there are conflicting data regarding the role of Gβγ in acute organ injury, which is frequently characterized by mitochondrial dysfunction. Inhibition of Gβγ by gallein inhibits RPTC proliferation and thereby exacerbates ischemic acute kidney injury2AR is a prototypical class A GPCR and has been extensively studied for its role in cellular signaling and the structural features that enable such signaling. This study is the first to examine the receptor-ligand interactions that distinguish mitochondrial biogenic β2AR agonists from non-biogenic β2AR agonists. As expected for two agonists of the β2AR, formoterol and clenbuterol have a common pharmacophore, and many of the interactions showed little preference for formoterol or clenbuterol. Nonetheless, the structural dissimilarities of the two compounds led to several distinct receptor-ligand interactions. In particular, interactions with V114 and F193 tended to occur more frequently at the methoxyphenyl group on formoterol, while C191 and its peptide backbone interacted more frequently with the formamide group of formoterol. To enable these interactions to occur, formoterol binds to the β2AR in a conformation that places it near the deeper pocket residues of TM3 and TM5 as well as near the shallower residues of TM2, ECL2, and ECL4. Our observation of the proximity to ECL2 and ECL4 with formoterol is in agreement with NMR studies showing that formoterol weakens the ionic interaction between D192 and K30539. Furthermore, ECL2 flexibility is important to ligand activity at the β2AR and for other GPCRs, such as the D2 dopamine receptor40.The β2AR agonists with the receptor further supported the role of a subset of receptor ligand interactions in stabilizing conformations of the β2AR that lead to MB. When compared to the non-biogenic agonist clenbuterol or the partial agonist of MB ritodrine, the hydroxyphenyl group (F11) of -fenoterol was more likely to engage in contact interactions with F193 and hydrogen bond donor interactions with C191 and its backbone. Additionally, its 3,5-hydroxyl groups (F1) were more likely to act as hydrogen bond donors for S203 and S207. These results are in agreement with previous docking studies of fenoterol with the β2AR41. Similarly, when compared to clenbuterol, the partial agonist of MB terbutaline had more contact interactions between its tert-butyl group (F8-F11) and F193 and hydrogen bond donor interactions between its 3,5-hydroxyl groups (F1) and S203, S207, and N293. Previous docking studies with terbutaline identified interactions between terbutaline and F193 but not between S203, S207, or N29342. However, those studies generated homology models of the rat β2AR from 2RH1, which represents an inactive conformation of the receptor43, while our study employed both active and inactive structures of the β2AR.Modeling interactions of other β2AR44, particularly regarding the conformational shift of TM6. Indeed, numerous studies using NMR and mass spectrometry have shown that functionally similar agonists can effect distinct active conformations of the receptor47. Additionally, the conformational flexibility of the β2AR allows for multiple “active” conformations that may lead to differences in effector coupling. Among its active conformations, formoterol may stabilize a set of biogenic β2AR conformations that are thermodynamically unfavorable for clenbuterol.Previous NMR studies have shown that formoterol and clenbuterol engage different conformations of the β2AR agonist formoterol but not by the non-biogenic β2AR agonist clenbuterol in metabolically competent primary cells. This Gβγ-Akt-eNOS-sGC pathway is necessary for the transcriptional and functional changes associated with MB. We also identified distinct structural features and ligand interactions that may allow formoterol to activate this pathway. Together, these data can facilitate the development of novel β2AR agonists that selectively stimulate the Gβγ-Akt-eNOS-sGC pathway to induce MB and recovery from acute and chronic degenerative diseases.In conclusion, this study identified a distinct signaling pathway activated by the mitochondrial biogenic βAnti-GAPDH antibody was purchased from Fitzgerald Antibodies  and was used at a dilution of 1:1,000. Anti-phospho-Akt (Ser473), anti-Akt, and anti-phospho-eNOS (Ser1177) antibodies were purchased from Cell Signaling  and were all used at a dilution of 1:1,000. Anti-eNOS antibody (1:500 dilution), anti-mouse IgG , and anti-rabbit IgG  antibodies were purchased from Abcam . MK2206 and GDC0068 were purchased from SelleckChem . Gallein, LY294002, L-NAME, and ODQ were purchased from Tocris . All other chemicals were purchased from Sigma .49. Three days after initial playing, dedifferentiated RPTCs were trypsinized and replated on XF-96 polystyrene culture microplates  at a density of 18,000 cells/well and were maintained at 37 °C for 3 days before pharmacological manipulation. For other RPTC experiments, isolated renal proximal tubules were plated in 35 mm dishes and used at confluence 6 days after initial plating. All experiments were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All procedures were approved by the Institutional Animal Care and Use Committees of the Medical University of South Carolina and the University of Arizona, and appropriate efforts were made to reduce animal suffering.Female New Zealand white rabbits (1.5–2.0 kg) were purchased from Charles River Laboratories . RPTCs were isolated via the iron oxide perfusion method, and RPTCs were cultured under improved conditions as described previously50. RPTCs in 96-well assay plates were treated with vehicle control (dimethylsulfoxide (DMSO), <0.5%) or with experimental compounds. Basal OCR was measured, followed by injection of 10 μM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) to allow for the measurement of uncoupled OCR (FCCP-OCR), a marker of MB.The oxygen consumption rate (OCR) of RPTCs was measured using the Seahorse Bioscience XF-96 Extracellular Flux Analyzer as previously describedFreshly isolated RPTCs were suspended in protein lysis buffer  containing protease inhibitors and phosphatase inhibitors . Following sonication, protein was quantified using a bicinchoninic acid assay, subjected to SDS-PAGE, transferred onto nitrocellulose membranes, and incubated with primary and secondary antibodies. Membranes were detected using chemiluminescence and processed using ImageJ  software.To measure RNA expression, RPTC were scraped in TRIzol , and RNA was isolated using a phenol-based centrifugation method. cDNA was reversed transcribed from 5 μg RNA using the iScript Advanced cDNA Synthesis Kit , diluted 1:10, and 5 μL added to a real-time SYBR green quantitative polymerase chain reaction master mix (BioRad). Changes in gene expression were calculated based on the Δ-Δ threshold cycle method. The following primers were used: PGC1α forward (AGGAAATCCGAGCCGAGCTGA), PGC1α reverse (GCAAGACGGAGACACATCAAA), NDUFS1 forward (AGATGATTTGGGAACAACAG), NDUFS1 reverse (TAGGGCTTAGAGGTTAGAGC), tubulin forward (CTCTCTGTCGATTACGGCAAG), and tubulin reverse (TGGTGAGGATGGAGTTGTAGG).To measure mtDNA copy number, RPTC were scraped in phosphate buffered saline, and DNA was extracted using the DNeasy Blood and Tissue kit . PCR products were amplified from 50 ng of cellular DNA using a real-time SYBR green quantitative polymerase chain reaction master mix (BioRad). For estimation of mtDNA, the NADH dehydrogenase subunit 6 (ND6) gene was used and normalized to tubulin. The following primers were used: ND6 forward (ACTGCGATGGCAACTGAGGAGTAT), ND6 reverse (ACCATAACTATACAACGCCGCCAC), tubulin forward (CTCTCTGTCGATTACGGCAAG), and tubulin reverse (TGGTGAGGATGGAGTTGTAGG).RPTCs in 35-mm dishes were treated with vehicle control or the compound of interest for 1 h. RPTCs were then harvested according to the manufacturer’s protocol . Levels of cAMP and cGMP were measured using a commercially available enzyme-linked immunosorbent assay kit. Values were normalized to protein as quantified by a bicinchoninic acid assay followed by normalization to vehicle control for each biological replicate.Modeling, simulations, and visualizations were performed using MOE (Molecular Operating Environment) version 2015.1001 . The structural files used as input for analysis and docking simulations were PDB codes 3NYA, 3NY8, 4LDE, 4LDL, 4LDO, and 5D5B. Before analysis and simulations, all atoms and molecules other than the receptor and the ligand were removed. The receptor and all ligands were protonated at pH 7.4 in MOE. Initial placement calculated 30 poses per molecule using triangle matching placement with London dG scoring. All 30 poses were then refined using induced fit with London dG scoring. Ligand interactions were assessed using the protein-ligand interaction fingerprint (PLIF) function in MOE. To score interaction frequencies, interactions with individual atoms were tabulated. Interactions with the active ligand formoterol were assigned a value of 1, while interactions with the inactive ligand clenbuterol were assigned a value of -1, and interactions for all poses across all crystal structures were added to generate overall ligand interaction frequencies at structural features.Data are expressed as means ± S.E.M. (N ≥ 3) for all experiments. Each N represents a biological replicate. Multiple comparisons of normally distributed data were analyzed by one-way analysis of variance, as appropriate. Single comparisons were analyzed with the Wilcoxon signed rank test where appropriate. The criterion for statistical differences was p < 0.05 for all comparisons.Supplementary Information


Giardia duodenalis is an important zoonotic pathogen in both public and veterinary health, and has been genotyped into at least eight assemblages (A–H), each with a distinct host range. In recent years, this intestinal protozoan parasite has been identified widely in humans and various other animals, and has even been recorded in environmental contaminants. Along with whole genome sequencing of G. duodenalis, multilocus sequence typing is increasingly being used to characterize G. duodenalis isolates. Here, we review the epidemiology, genotyping, and subtyping of G. duodenalis from humans and a wide range of other animals, as well as from wastewater, in China. Giardia is one of the most common intestinal parasites of both humans and a diverse range of other animals  includes humans and domestic, farmed, and wild animals  and cysts that are resistant to environmental degradation, which are excreted with feces and transmitted onwards via the fecal-oral route , each of which have a distinct host range  and variable  genes was originally used to assess the G. duodenalis assemblages  have been studied genomically  analysis using both conserved , ruminants, companion animals, domestic animals, wildlife, and even in the environment in China  and B isolates have been found in China, with subtype AII and assemblage B being the dominant genotypes  and B (subtype BIV) subtypes have been identified in NHPs, with subtype BIV identified as the dominant subtype , and have mainly been documented in Henan and Heilongjiang Provinces  tools are increasingly being used for characterizing G. duodenalis infections in humans and animals. Access to these methods is especially important for gaining a better understanding of some host-adapted assemblages  that are pathogenic in humans, and for assessing the zoonotic assemblages (A and B) with infective potential. Therefore, a comprehensive and systematic study based on MLST analysis should be carried out to allow a full assessment of the burden of giardiasis of animal origin in humans.Most molecular investigations of LZ conceived the idea for the review and revised the manuscript. JL and HW wrote the manuscript, and JL, HW, and RW reviewed and abstracted the data from each selected article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In dry savannas, the increased water-use efficiency in plants relaxes precipitation-driven constraints and increases woody growth. In wet savannas, the increase of carbon allocation to tree roots results in faster recovery rates after disturbance and a greater likelihood of reaching sexual maturity. Our proposed framework can be tested using a mixture of experimental and earth observational techniques. At a local level, changes in precipitation, burning regimes or herbivory could be driving woody encroachment, but are unlikely to be the explanation of this continent-wide phenomenon.Savanna ecosystems are an integral part of the African landscape and sustain the livelihoods of millions of people. Woody encroachment in savannas is a widespread phenomenon but its causes are widely debated. We review the extensive literature on woody encroachment to help improve understanding of the possible causes and to highlight where and how future scientific efforts to fully understand these causes should be focused. Rainfall is the most important determinant of maximum woody cover across Africa, but fire and herbivory interact to reduce woody cover below the maximum at many locations. We postulate that woody encroachment is most likely driven by COThe online version of this article (doi:10.1007/s00442-017-3807-6) contains supplementary material, which is available to authorized users. Over the past 60 years, growing evidence suggests that savannas throughout the world are being altered by a phenomenon known as ‘woody encroachment’  and unstable (wet) states has been questioned. Staver et al. , b showe2 would increase tree growth and encourage a closed forest system. This in turn would lower grass fuel loads and increase humidity, which lowers the flammability of the landscape, creating a positive feedback system in maintaining a forest. In contrast, in areas where fire occurs more frequently, tree maturity rates decrease due to top-killing, which prevents canopy closure and allows C4 grasses to thrive  experiments shows an increase under elevated CO2, it is lower than expected due to the effects of rising temperatures  and wet (unstable) savannas, particularly with regards to the role that water plays in altering the tree–grass competition  and dry (stable) savannas are caused by two separate mechanisms Below is the link to the electronic supplementary material.


Gallus gallus domesticus) to learn to avoid distasteful food that was associated with one of three color signals  that varied in both hue and in brightness in order to assess which of these traits most influenced their ability to learn avoidance. Our results show that while chicks learned to avoid all three colors, avoidance was based on the hue, not brightness of the different signals. We found that yellow was the most effective for avoidance learning, followed by red, and finally white. Our results suggest that while these three colors are commonly used in aposematic signaling, predators’ ability to learn avoidance differs among them. These results may explain why yellow is among the most common signals across aposematic taxa.A few colors, such as red and yellow, are commonly found in aposematic (warning) signaling across taxa, independent of evolutionary relationships. These colors have unique traits  that aid in their differentiation, and perhaps, their effectiveness in promoting avoidance learning. This repeated use calls into question the influence of selection on specific warning colors adopted by aposematic prey-predator systems. To disentangle the influence of color characteristics on this process, we trained week-old chickens ( Convergent phenotypic evolution, where traits have evolved independently, is widespread in the natural world, and as such, provides an intriguing opportunity to examine the evolutionary pressures driving such patterns. Convergence can be found in a wide variety of taxa from plants  and animAposematic (warning) signaling is widespread throughout Animalia , and desAs research into sensory ecology has progressed, our understanding of the constituent components of signaling has also improved. Aposematic signaling has increasingly been understood to not be comprised solely of conspicuous colors, but rather a complex combination of features that aid in predator recognition, learning, and avoidance –13. EverWhile predators of aposematic species are undoubtedly quite varied and possess a variety of visual systems, birds are commonly used in aposematism studies –17. The This research was approved by the University of Mississippi IACUC and conducted under University of Mississippi IACUC 14–026.Dendrobates tinctorius (with the exception of red). Bodies were black and legs were blue. White and yellow colors were extracted from photographs of D. tinctorius from Grand Matoury and Kaw in French Guiana, respectively, while the red color was extracted from photographs of Oophaga pumilio from Bastimentos, Panama. These represent colors common in aposematic signaling, but they also represent low (red), medium (yellow), and high (white) brightness  where a value of 1 JND would mean differences could be observed under the modeled vision system when objects are stationary under bright conditions [We analyzed spectra using the  package  to examinditions . Howevernditions .We used the average avian vision system for visual contrasts and chicken double cone sensitivity to calculate achromatic contrasts under the D65 (standard daylight) illuminant . We condGallus gallus domesticus; male cornish rock crosses from the commercial supplier, Ideal Poultry). These were housed in wire brooders (approximately 1m x 1m x 30cm) together while not being tested and fed a commercial corn meal mash. To examine how naïve predators learn color signals, chicks were equally divided  into three different treatments: white signal, yellow signal, and red signal. Trials were done in 30cm x 60cm wooden compartments under full spectrum lighting. Chicks were placed individually into a compartment and allowed to habituate for 2 hours. Chicks were food-deprived during this acclimation period to ensure motivation to feed during the trials.In order to assess how predators learn avoidance, we used one-week-old chickens (Tenebrio molitor) from a petri dish with a printed brown frog on a tan background below the transparent dish. The training phase was completed once the chick had eaten three consecutive times, after which we allowed the birds to rest for a period of 5 minutes.Once in the compartment and after the 2-hour acclimation period, training consisted of teaching the chicks to eat a dried mealworm (D. tinctorius (described above) with either a white, yellow, or red  for each comparison of chromatic contrasts was found to be quite distinct . Pairwise comparisons yielded significant differences when comparing results from the yellow treatment and white treatment . While none of the other pairwise comparisons yielded significant differences, the average hesitation time between the red and white treatments did trend towards significant (p = 0.092). When comparing whether there was a difference between signals in how often chicks learned to avoid signals, we found that red was not significantly different from white (p = 0.498) or yellow (p = 0.157). However, when comparing yellow and white, we did find a significant difference (p = 0.016) with chicks more likely to learn to avoid yellow than white  being the most important factor in determining avian predator learning ,24. TherWhile this research was conducted with domestic chickens in a laboratory setting, it is important to give this research context to wild populations and patterns, particularly because prior studies have demonstrated that chicks can be neophobic to novel signals . InteresThis research provides interesting insight into the evolution and proliferation of aposematic signals in the wild. Aposematic signaling is prevalent throughout the animal kingdom, but a number of colors continually evolve independently as signal components . Our resOophaga pumilio) color may be an important component in mate choice [Of course, our research does raise the question as to why suboptimal signals such as red or white may persist in aposematic species. While we examined the influence of color on aposematic signaling, these colors likely have a wide variety of functions outside of warning signaling. Research suggests that, for example, in the Strawberry Poison Frog Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


Engineered molecules based on human cytokines have potential uses in research and medicine. Related research article Moraga I, Spangler JB, Mendoza JL, Gakovic M, Wehrman TS, Krutzik P, Garcia KC. 2017. Synthekines are surrogate cytokine and growth factor agonists that compel signaling through non-natural receptor dimers. eLife6:e22882. doi: 10.7554/eLife.22882As with John Lennon and Paul McCartney, pairings can sometimes exceed the sum of their parts. Many biological processes also rely on proteins forming pairs: for example, a wide variety of cells (particularly those involved in the immune system) release small proteins called cytokines to communicate with other cells, and typically it takes two cytokine receptors to detect one of these proteins. Now, in eLife, Christopher Garcia and colleagues at Stanford University School of Medicine – including Ignacio Moraga as first author – report that they have developed a new class of molecule called synthekines that bind to different pairs of cytokine receptors than the cytokines they are based on .Each cytokine interacts with a unique cytokine receptor pair and activates signaling pathways that lead to changes in gene expression . In mammCytokines bind to a region of cytokine receptors known as the ligand-binding domain, which sticks out from the cell surface. This causes the signaling domains of two receptors, which are inside the cell, to interact with one another and activate Jak-STAT signaling. Moraga et al. first tested whether ‘unnatural’ cytokine receptor pairings can trigger Jak-STAT signaling. To that end, they devised an experimental system in which the signaling domains of 10 different cytokine receptors were each fused to one of two ligand-binding domains. These ligand-binding domains were compatible with a single cytokine that, in turn, could induce any one of the 10x10=100 possible pairings of signaling domains, depending on which two were fused to the ligand-binding domains.Moraga et al. discovered that most combinations of ligand-binding and signaling domains were able to activate Jak-STAT signaling. This confirms that the pairing of cytokine receptors is sufficient to trigger signaling inside cells and that the specificity of the interaction between cytokines and ligand-binding domains is what determines which receptors can form pairs. The researchers also showed that a cytokine receptor can even trigger signaling when forced to form a pair with a completely different class of receptor molecule, such as a receptor tyrosine kinase.Moving beyond proof-of-principle, Moraga et al. developed a way to produce ‘unnatural’ receptor pairings in native human cells. Based on the biophysics of the interactions between cytokines and their receptors, they designed mutant versions of three cytokines that were capable of binding to only one component of their ‘natural’ receptor pairs. Two of these mutant cytokines were then linked together to create synthetic cytokines (named synthekines) that can cause one receptor for each mutant cytokine to form a pair that would not naturally occur .The experiments show that Jak-STAT signaling activity downstream of two synthekines called SY1 and SY2 were distinct from those of their parent cytokines. The responses driven by synthekines generally had lower overall levels of Jak-STAT activity compared to the responses triggered by their parent cytokines. Furthermore, the patterns of STAT protein activity differed. For example, STAT1 is the most active STAT protein in the response to SY2, but least active in the response to its parent cytokines. Furthermore, a technique called high-dimensional mass cytometry revealed that other signaling pathways, particularly downstream of a protein called phosphoinositide 3-kinase, were altered. Thus, synthekines are not just a watered down combination of their parent cytokines but, instead, are new biological entities with unique signaling properties. This makes them useful research tools and promising drug candidates.There are clear precedents for the current work  have only begun to be explained, and their bioactivity in animals, particularly regarding therapeutic uses, has yet to be addressed. The latter will require the generation of synthekines that are compatible with rodents and other animal models because the current versions are made from human constituents. However, there appear to be no major obstacles to doing this.All told, this work stands as a triumph of scientific imagination, a lucid illustration that we need not be constrained by the rules of engagement prescribed within the genome. After all, given the ever-growing practicality of synthetic biology, we are living in a time where, in the words of Lennon and McCartney, there's “nothing you can make that can't be made” .


In the semi-classical view of a Kerr nonlinear medium, the amplitude of two-mode hybrid correlations of this kind is subject to a limit determined by the hybrid maximally entangled state. Whereas the degree of correlation and squeezing is determined by the dressing effects of the input laser fields participating in the SP-FWM process. We also find that the variations in magnitude of three-mode hybrid intensity-noise correlation and intensity-difference squeezing are consistent with nonlinear cross-Kerr processes. Such a three-mode hybrid signal may have potential applications in long-distance communication, dense coding, all-optical communication and quantum storage on photonic chips.We report the generation of three-mode hybrid intensity-noise correlation and intensity-difference squeezing of spontaneous parametric four-wave mixing (SP-FWM) and fourth-order fluorescence (FL) signals in the heteronuclear-like (three-level Λ-type) molecular structure of a Pr In addition, classical3 and non-classical4 photon correlations have been achieved under various experimental conditions. Recently, hybrid entangled states5, which involve entanglement between classical and non-classical photons, have been identified as useful resources for long-distance communications6 and optical quantum information processing8, as such a hybrid state acts as a new type of qubit9. This approach has received considerable attention because of its incorporation of the advantages of both classical and non-classical photon states, especially for loophole-free Bell inequality tests using inefficient detectors10, near-deterministic quantum teleportation using linear optics and hybrid qubits8 and information transfer between different types of qubits11. Thus, the generation of hybrid entangled states is crucial. The most widely used technique for the generation of entangled photon pairs is spontaneous parametric downconversion (SPDC) (χ(2) nonlinearity) using a nonlinear crystal12. However, the photons generated via SPDC have a very wide bandwidth (~THz), making it difficult to realize effective coupling between photons and atoms. Such coupling is essential for long-distance communications13. The filter-driven four-wave mixing (FWM) effect (χ(3) nonlinearity) can induce a high repetition rate of an ultrafast fibre laser14, and FWM can induce the energy transfer of different waves with a self-stabilizing function, as first reported by Liu et al.15, and can be used as a basis for the development of multi-wavelength fibre lasers18. Moreover, spontaneous parametric FWM (SP-FWM) using a rare-earth-ion-doped Pr3+:Y2SiO5 (Pr3+:YSO) crystal preserves “atom-like” properties, with a long coherence time (0.1–1 s) and a narrow spectral width (~MHz)19. In such a nonlinear crystal, the dopant cations are localized at different vacancy sites arising from dipole-dipole interactions, giving rise to different energy-level configurations20. Therefore, when multiple Zeeman energy levels are involved in the atomic systems of the nonlinear crystal, the effects of dressing fields can be used to control the process22. Moreover, an SP-FWM process generates pairs of Stokes and anti-Stokes photons accompanied by a simultaneously generated fourth-order fluorescence (FL) signal. Recently, hybrid-cascaded sources from the SP-FWM and spontaneous Raman scattering processes in atomic ensembles using nonlinear processes of different orders have been studied23. Intensity-noise correlated and intensity-difference squeezed beams have important applications in quantum metrology and gravitational-wave detection. In addition, Jietai Jing’s group has experimentally demonstrated that increasing the number of modes of entanglement enhances the degree of correlation and squeezing19, and this phenomenon also shows potential for application to increase the information rate in dense coding24.The generation of entangled photons is of particular interest in fundamental studies of quantum mechanics and has potential applications in information processing and long-distance communications. In recent decades, scientists have created various photon states, such as coherent states25. In this paper, we focus on a hybrid state with three-mode intensity-noise correlation and intensity-difference squeezing of SP-FWM and hybrid FL signals in a Pr:YSO crystal using the nonlinear cross-Kerr effect under polarized dressing fields as Kerr non linearity is directly proportional to the square of the electric field. We investigated the differences between the three-mode hybrid correlation and squeezing effects induced by the cross-Kerr effect under linearly and circularly polarized dressing fields. We found that the degree of three-mode hybrid correlation and squeezing induced by the cross-Kerr effect decreases when the dressing field is changed from a linear polarization to a circular polarization. We also found that the FL signal interacts with the Stokes/anti-Stokes photon pairs and that the degree of hybrid intensity-noise correlation and intensity-difference squeezing can be controlled by controlling the emission of the hybrid FL signal. Our results obtained in this atom-like medium indicate several obvious advantages, such as an improved integration ability compared to atomic vapours and a long coherence time compared to other traditional solid materials. These achievements also have potential applications in all-optical communication and quantum storage on photonic chips.Motivated by these achievements, two-mode intensity-noise correlation and intensity-difference squeezing of SP-FWM and fourth-order FL signals have been investigated3+) 3-mm YSO (Y2SiO5) crystal was used in the experiment. Coupling can occur between Pr3+ ions localized at different cation vacancies as a result of induced dipole-dipole interactions; consequently, one can treat two such ions as a heteronuclear-like molecule. Therefore, we can construct a Λ-type three-level system  pumped by an injection-locked single-mode Nd:YAG laser and a Continuum Powerlite DLS 9010 with a 10 Hz repetition rate and a 5 ns pulse width were used to generate the pumping field E1 and the dressing field E2 with frequency detunings of m〉 and |n〉 and ωi  is the laser frequency. The pumping field E1  and the dressing field E2 were coupled into the crystal in opposite directions. A quarter-wave plate (QWP) was used to control the polarization state of the dressing field E2, and the power P2 and detuning Δ2 of the dressing field E2 could be modified by changing the parameters of the dye lasers. The Stokes and anti-Stokes photons were produced under the phase-matching condition k1 + k2 = kS + kaS and the energy conservation condition ω1 + ω2 = ωS + ωaS, where k and ω denote the wave vectors and transition frequencies, respectively, of the pumping field, the dressing field and the generated photon pairs. Meanwhile, the hybrid photons were obtained via a Kerr nonlinear interaction under the influence of the higher photonic flux of the pump beam E1, which passed through the nonlinear crystal as shown in Fig. E1 (pumping field) and E2 (dressing field). The weak Stokes field (ES), anti-Stokes field (EaS) and fluorescence field (FL) constitute the hybrid signal, whose propagation can be described as evolution under the Heisenberg picture27; see the Methods section for further details.A rare-earth-ion-doped (0.05% PrE2 increases from 2 mW (shown in (c1)) to 6 mW (in (c2)) when the polarization state of E2 remains fixed at 0° (linear polarization), the detuning of E2 is set to Δ2 = 0 GHz, and the power of E1 (P1) is held fixed. Figure 28 with increasing power, as shown in Fig. D1 and D2, between the SP-FWM signal (left peak) and the FL signal (right peak) in Fig. 31. We can make use of these properties of the FL signal to selectively monitor the input dressing laser’s polarization, power and frequency detuning in a Λ-type system to control the degree of hybrid three-mode intensity-noise correlation and intensity-difference squeezing.Figure E2 fixed at 0° and the detuning of E2 set to Δ2 = 0 GHz. Under the cross-Kerr effect with the dressing field E2 in the linearly polarized state, Eqs   E2 fixeω  between the Stokes and anti-Stokes signals. Finally, the intensity differences ω between the Stokes and FL signals  is approximately 1.5 at τ = 0, which corresponds to Figure By contrast, Fig. In other words, because the Stokes and anti-Stokes photons are produced under conditions of phase matching and energy conservation, the Stokes and anti-Stokes signals are determined by each other, so the degree of correlation in the SP-FWM process is large. By contrast, there is no phase matching or energy conservation between the pumping field and the FL signal, so although the hybrid Stokes/SP-FWM+FL and anti-Stokes/SP-FWM+FL signals affect each other, their degrees of correlation via the cross-Kerr processes are smaller than that of the SP-FWM process.ζ represents the bandwidth of the source laser and is constant. By contrast, the correlation shapes obtained from the coherent and Kerr processes together are sharp and described by Figure The SP-FWM process dominates in Fig. In the following paragraphs, we will discuss the three-mode correlation and squeezing results of the three signals. In the Λ-type system shown in Fig. θ = 0°) or circularly (θ = 45°) polarized state. Figure 2 of E2 are fixed to 6 mW and 0 GHz, respectively. Figure Here, we first study the differences in hybrid three-mode intensity-noise correlation and intensity-difference squeezing induced by the cross-Kerr effect with the dressing field in a linearly E2, whic 0° Fig.  to 15°  in Eqs  and (12), in Eq. . Thus, t dB Fig.  to −1.28 dB Fig.  and foll dB Fig. , reduce E2 changes from 0° to 45°, the anti-Stokes signal becomes small, as the Stokes and anti-Stokes signals are produced and determined by each other and the correlation between them is still strong. However, the amount of FL in the hybrid signal that is created by the Stokes and anti-Stokes signals will be reduced; hence, the degree of correlation between the Stokes/anti-Stokes and hybrid FL signals will decrease as the polarization of E2 changes from 0° to 45°. This effect will induce three-mode correlation changes, as shown in Fig. As the polarization of E2 remain fixed at 0° and 0 GHz, respectively. Figure E1 in Eqs   E2 rema, in Eqs  and (8).2 changes from −200 GHz to +200 GHz while the power and polarization of E2 remain fixed at 6 mW and 0°, respectively. The three-mode signals from PMT1, PMT2 and PMT3 , the detuning terms  in Eqs  and 8)  2 changeg in Eq. , \docume3+:Y2SiO5 crystal under the polarized cross-Kerr effect. The degree of three-mode hybrid correlation and squeezing degree induced by the cross-Kerr effect was found to decrease as the dressing field was changed from being linearly polarized to being circularly polarized. It was found that the degree of hybrid intensity-noise correlation and intensity-difference squeezing can be controlled by controlling the amount of FL emission from two Kerr processes. In particular, the two-mode correlation obtained from the interaction of the SP-FWM and Kerr processes is lower than that of the maximally entangled Stokes/anti-Stokes state. The variations in magnitude of the two- and three-mode intensity-noise correlation and intensity-difference squeezing are consistent with each other. Such a three-mode hybrid signal may have potential applications in long-distance communication, dense coding, all-optical communication and quantum storage on photonic chips.In conclusion, the two- and three-mode hybrid intensity-noise correlation and intensity-difference squeezing of SP-FWM and FL signals was studied by selectively controlling the polarization, power and frequency detuning of the input dressing laser in a Λ-type three-level atomic system in a PrIn this experiment, the correlation was calculated from the intensities of the signals obtained from three processes: coherent signals were obtained from the SP-FWM process, and non-coherent (hybrid) signals were obtained from two cross-Kerr nonlinear processes. The SP-FWM process produces Stokes and anti-Stokes signals. The two cross-Kerr nonlinear processes produce two hybrid FL+Stokes and FL+anti-Stokes signals via the interaction of the FL with the Stokes and anti-Stokes photons, respectively.To generate the hybrid signal, we used a scheme similar to the one explained in ref. n0 + n2|E|2, where n0 is the weak-field linear refractive index term and n2 = 3χ(3)/2n0 is a nonlinear refractive index term that is proportional to the field strength |E|2. Finally, the nonlinear refractive index can be characterized as n0 + 3χ(3)/2n0|E|2.Cross-Kerr nonlinearity is essential for the generation of an entangled state. The Kerr nonlinearity is characterized by a refractive index of i represents S-FL, FL-S, aS-FL or FL-aS). We can write the density matrixθ is the rotation angle of the QWP’s axis from the x axis, the cx,y are the anisotropy factors in different directions in the crystal, Gi|2 is the Rabi frequency of the field Ei.Because of the coherence of the nonlinear medium, the interaction between the SP-FWM and FL outputs results in a hybrid signal (SP-FWM+FL), whose propagation can be described as follows:33.The field intensities of these hybrid signals are given by mentclass2pt{minimquations ), and coquations ). The sequations ); similaquations ). Such Kpure perturbation theory to obtain the Stokes and anti-Stokes signals under the weak-field approximation. Then, we incorporate dressing perturbation theory (DPT), in which the dressing terms of a strong field are included. In DPT, the density matrix elements of the Stokes and anti-Stokes signals and the associated FL signals are derived using the Liouville equation v is the group velocity; and g is the nonlinear gain. For a Λ-type three-level system with two strong pumping fields E1 and E2, when the self-dressing effect of E1 and the external-dressing effect of E2 are considered, the third-order nonlinear density matrix elements of ES and EaS obtained via the perturbation chains E2 into its components along the x and y axes. Thus, considering the different Clebsch-Gordan coefficients for various transitions, the Rabi frequency |G2|2 in Eqs ,,10\documE1 and E2 fields open, the fourth-order FL signal is generated as depicted in Fig. In the Λ-type three-level system shown in Fig. E2 are considered, the corresponding equation obtained by modifying Eq.  and EaS, under the dressing conditions of the driving fields E1 and E2 and the light-matter interaction induced by the finite pulses shown in Fig. For two weak multimode fields These three nonlinear processes, the SP-FWM process and the two cross-Kerr nonlinear processes, create the hybrid FL state; hence, the degree of hybrid intensity-noise correlation and intensity-difference squeezing can be controlled by controlling the amount of FL in the hybrid signal.Q = cosh2(κL) is the gain coefficient. The intensity of the signal  , was recorded using digital oscilloscopes and PMTs. Here, the 〈Ii〉 are the average intensities of the anti-Stokes, Stokes and FL beams, and the δIi(τ) are the corresponding intensity fluctuations. Then, the intensity fluctuations were averaged by a fast gated integrator over 10 pulses. The temporal waveform correlations between the Stokes, anti-Stokes and FL signals were investigated based on Eqs  an an3+:YSOd on Eqs  under a d on Eqs , 20) an an3+:YSOd on Eqs .g1, g2 and g3 are the coupling coefficients and X1, X2, and X3 and Y1, Y2, and Y3 are the intensities and phases, respectively, of the three corresponding signals.Furthermore, the three-mode entanglement condition is given by


This study aims to investigate the role of thymic stromal lymphopoietin (TSLP) in the pathogenesis of lumbar disc degeneration (LDD).Nucleus pulposus tissues were collected from 77 LDD patients (the case group), in addition, normal tissues were extracted from 21 patients suffering from lumbar fractures (the control group). Immunohistochemistry was applied in order to detect TSLP positive expression. In accordance with varying transfection, the cells were divided into TSLP-siRNA, TSLP-siRNA + TSLPR-siRNA, control, blank, anti-TSLPR, and IgG groups. Western blotting was used in order to detect TSLP expression in tissues, and TSLP and type II collagen (COL2AL) in cell culture media were detected using enzyme linked immunosorbent assay (ELISA). Cell viability was measured using a MTT assay. Aggrecan levels were detected using antonopulos, and cell apoptosis was determined using flow cytometry.P < .05)TSLP-positive expression was found to be significantly higher in the case group compared with the control group. LDD patients’ Pfirrmann grades and preoperative visual analogue scale (VAS) scores were associated with TSLP-positive rate. Cells transfected with TSLP-siRNA and TSLPR-siRNA plasmids exhibited lower TSLP and thymic stromal lymphopoietin receptor (TSLPR) protein expression compared with the control and blank groups. Compared with the control and blank groups, there was significantly higher cell viability, lower cell apoptosis, and higher COL2AL and Aggrecan levels in the TSLP-siRNA, anti-TSLPR, and TSLP-siRNA+TSLPR-siRNA groups; there were significant differences between the TSLP-siRNA, anti-TSLPR, and TSLP-siRNA+TSLPR-siRNA groups and IgG group (all Our study provides evidence for the hypothesis that TSLP could reflect the histological severity of LDD, and TSLP-siRNA and, TSLPR-siRNA could inhibit apoptosis of nucleus pulposus cells. The evident information obtained from the investigation could lead the way for new therapeutic approaches regarding LDD treatment. The current treatments to alleviate the LDD pain include nonsurgical therapy and surgical therapy. Bone marrow mesenchymal stem cells (BMSCs) transplantation and axial dispersion treatment applied have been reported to have an apparent reversal effect on LDD in animal experiments. At present, a significant correlation between immune responses and the development and progression of LDD has been discovered. This has raised considerable concerns like sciatica, paraesthesia, numbness, or weakness in LDD treatment. Thymic stromal lymphopoietin (TSLP), utilization as a cell growth factor, was initially discovered in mouse thymic stromal cell line (MTSC). Lumbar disc degeneration (LDD), the main cause of lumbago and backache, has a serious impact on general public health.,8 It has been widely reported that TSLP can aid in activating the antigen recognition molecules such as dendritic cells (DC cells) that aid in transforming T cells into T Helper Type 2 cells (Th2-type). There have been numerous studies over the recent years, which primarily focused on exploring the association between TSLP and allergic inflammatory diseases such as asthma, allergic skin, and mucous membrane disease.,9 However, TSLP activation in DC cells and the differentiation of Treg cells had a significant impact on the development and progression of autoimmune diseases. As LDD is associated with inflammatory diseases, this study intends to explore the specific role and mechanism of TSLP in LDD so as to discover the effective targets in LDD treatment, which could further improve the chance of successful prevention and diagnosis of LDD.TSLP, a 4-helix bundle structural protein, is similar in structure with interleukin-7 (IL-7). There are many similarities between TSLP and IL-7 in terms of features, but TSLP can stimulate lymphocyte proliferation without relying on IL-7.22.1The study was performed with consent from all participating patients. This study was conducted in accordance with the relative ethical standards and approvals from the Ethics Committee of Peking University First Hospital, following the standard of The National Research Council.2.2 According to the Pfirrmann grade system, patients were divided into type I (n = 14), type II (n = 20), type III (n = 21), and type IV (n = 22). In adherence with a 100 mm visual analogue scale (VAS) for testing sciatic nerve pain intensity, the average preoperative VAS score was 6.30 ± 1.88. This could be further divided into mild (≤3.4), moderate (3.4–7.5), and severe (≥7.5). The inclusion criteria were as follows: patients without long-term manual labour history; patients without any history of spinal injury; patients who didn’t experience constant backache; patients without lumbago and spine malformation. Exclusion criteria: patients with tumor and autoimmune diseases, chronic infectious diseases, and patients with leg pain but without osphyalgia.Between June 2013 and January 2015, nucleus pulposus tissues of 77 LDD patients  who were hospitalized in Peking University First Hospital were elected for the case group. Among them, Spengler typing was used for categorizing the patients into 25 cases of protrusion, 29 cases of rupture type, and 23 cases of free type.P > .05).Additionally, nucleus pulposus tissues of 21 lumbar vertebral bone fracture patients  were extracted as controls. These patients did not experience any back and leg pain. Patients in the control group had no clinical manifestations and symptoms of LDD as well as no significant degenerative changes in nucleus pulposus tissues post magnetic resonance imaging (MRI) examination. There was no statistical difference observed in terms of age and sex, between the case group and the control group  was added and fully mixed with 10 μL of biotin goat anti-rabbit secondary antibody . Two individuals independently estimated the immunohistochemistry results using the double blind method.Following this, the mixture was used to process the sections over a period of 30 minutes, and then underwent 4 °C overnight inoculation. For developing color diaminobenzidine (DAB) chromogenic  was used. In regards to mouse IgG as blank control, sections were observed under an optical microscope. The cytoplasm was stained using TSLP. Yellow-like particles were regarded as TSLP-positive. From each section, 5 high-power fields (×400) were chosen, and 100 cells were counted in each field. The percentage of TSLP-positive cells/total cells >10% was regarded as positive, while <10% was negative.2.4The tissue protein from both the case group and the control group were extracted by using the conventional splitting method, and the concentration was testified using a bicinchoninicacid (BCA) kit. 1× Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer was added to tissue and the protein were boiled to obtain protein degeneration. SDS-PAGE gel electrophoresis was performed in order to obtain equivalent protein samples. Next, the protein was transferred onto a polyvinylidenefluoride (PVDF) membrane and sealed with bovine serum albumin (50 g/L) for 1 hour. Subsequently, TSLP antibody  and thymic stromal lymphopoietin receptor (TSLPR) antibody  were added to culture the protein at 4 °C overnight. The proteins were washed with TBST buffer solution and cultured with HRP-labeled secondary antibody  for 1 hour. Next, the proteins were washed with TBST another time. Finally, the protein was developed with electro-chemic luminescence (ECL) and photographed under a microscope. Consequently, the above-mentioned method was applied to verify the effectiveness of silencing TSLPR and TSLP.2.5−ΔCt. After which, the percentages of expressions related to TSLPR and TSLP genes in β-actin were analyzed in order to identify how much TSLPR and TSLP gene were silenced by siRNA. Each experiment was repeated 3 times.TSLPR siRNA and TSLP siRNA were coconstructed by the laboratory and Shanghai Genechem Genetic Biological Technology Co., Ltd . TSLPR-specific target sequence was used to design 3 different siRNAs in order to avoid an off-target effect. Similarly, 3 different types of siRNAs were designed for TSLPR as well as for negative control. In order to enhance siRNA stability, a dTdT-suspended end was also designed. For RNA interference the above mentioned sequences were connected with lentivirus vectors pGLVU6/GFP. The bacteria transformed and bacterial plasmids were extracted in large quantities. Two hundred and fifty microliter of serum-free Opti-MEM was used to dilute equivalent amount of corresponding siRNA or negative control . The siRNA and negative controls were then introduced in cells to increase the plasmid concentration to 50 nM, which was cultured at room temperature for 5 minutes. This was followed by dilution with 250 μL of serum-free Opti-MEM and mixing with, a cultured 5 μL amount of Lipofectamine 2000 at room temperature for 5 minutes Opti-MEM and Lipofectamine 2000 then fully mixed. The mixture was further cultured for another 20 minutes and introduced into culture wells containing cells. After culturing the cells for 6 to 8 hours, the culture medium was replaced with a completely new medium to continue the cell culturing process. After a day, the total RNA underwent reverse transcription according to the specification of miScript II RT Kit . The qRT-PCR system was also in accordance with specification of a kit  as well as the primer sequence was shown in Table 2.62 for culturing at 37 °C. After the cells had been isolated and cultured for a week, the cells were passaged and cultured if the cell fusion reached up to 80%. The nucleus pulposus cells were accordingly divided into the TSLPR-siRNA + TSLP-siRNA groups (cells transfected with TSLPR siRNA1 and TSLP-siRNA2), the TSLP-siRNA group (cells transfected with TSLP-siRNA2), the control group (cells transfected with negative control siRNA), and the blank group (cells received no treatment). Two hundred fifty microliter of serum-free Opti-MEM (Gibco Company) diluted equivalent corresponding with siRNA plasmid or negative control siRNA, was then introduced to raise the plasmid concentration up to 50 nM. After culturing at room temperature for 5 minutes the process was followed by dilution with 250 μL of serum-free Opti-MEM and mixing with 5 μL of Lipofectamine 2000, culturing at room temperature for 5 minutes. At the point when Opti-MEM and Lipofectamine 2000 were fully mixed, the mixture was then further cultured for 20 minutes and added to culture wells containing cells in them. After culturing the cells for 6 to 8 hours, the culture medium was replaced with completely new medium to continue cell culturing. At the same time, the nucleus pulposus cells that were not transfected by any plasmids and were still in good condition were inoculated into 6-well plates for culturing for 24 hours. Followed by the addition of the cells to anti-TSLPR (5 μg/m) or homologous and unrelated IgG antibody , for diving them into the anti-TSLPR group and the IgG group.The nucleus pulposus tissues of the LDD patients were extracted and cut into small pieces (0.5 mm in length) in aseptic conditions. The tissue pieces were rinsed with Hams F12 + 10% FBS  3 times, and were accordingly inoculated in a culture flask with a basal area of 20 cm2.7Once the nucleus pulposus were cultured with TSLP for 48 hours, the cells were then cultured on a sterile cover slip. Immunofluorescence staining was employed in order to detect the expression of TSLP after transfection. Once the cells multiplied and fused, the cover slip was fixed by immersing in 4% paraformaldehyde at room temperature for 10 minutes, and followed by sealing with block buffer for 45 minutes. Subsequently, TSLP-monoclonal antibody was added to cells  to be diluted and cultured at 4 °C overnight. Fluorescein-labeled CY-3 goat anti-rabbit secondary antibody  was added in order to dilute the cells and culture the solution at room temperature in the dark. After observation under a fluorescence microscope, cells exhibiting red fluorescence were deemed as TSLP-positive and the TSLP-positive rate was calculated.2.8The culture medium was collected 48 hours after nucleus pulposus cells were transfected with TSLPR siRNA. The concentrations of TSLP and COL2AL in serum samples were testified according to the specification of ELISA kit . The operation was conducted as follows: the antibody was diluted to the concentration of 1 μg/mL using the ELISA coating buffer, and subsequently added into 96-well plates at 4 °C overnight (concentration of 100 μL/well). The coating buffer was removed and the plates were washed 3 times. A total of 150 μL of blocking solution was added in each well to culture the proteins for 1 hour. Next, the plates were washed, and 100 μL of diluted standard or serum samples was added to culture the protein for 2 hours, with blank control set up using the aforementioned method. After the plates were washed, TSLP antibody or COL2AL antibody was added to each well to culture the proteins for 1 hour. At this point in the operation, the plates were washed again, and 100 μL of the substrate solution was added in each well to react with the proteins for 15 minutes at room temperature. The reaction was terminated with the addition of 50 μL sulfuric acid (2 M). The optical density (OD) value at 450 nm of each well were read and recorded using a microplate reader, and TSLP AND COL2AL concentration were calculated based on standard curves.2.95/mL. The cells were inoculated in 12-well plates (0.2 mL/well), and cultured continuously in a humidified incubator . The cells were placed over 96-well plates and then counted based on the fact that each 200 μL contained 200 cells. For the cells in each group a total of 4 96-well plates and 8 duplicate wells were prepared; there were also 4 time intervals points, namely 0, 24, 48, and 72 hours. Once the nucleus pulposus cells in each well adhered to the cell wall, the culture medium was suctioned out and cells were added with 20 μL of MTT (5 g/L). After culturing the cells for another 4 minutes, dimethylsulfoxide (DMSO) (150 μL/well) was added and fully mixed with the cells. Followed by testing the OD value at 570 nm using an enzyme-linked immunosorbent assay 10 minutes later. The result was recorded with time as the abscissa and OD value as ordinate, on the basis of which a precise cell growth curve was drawn.A MTT assay was applied to detect the cell activity and after the cells had proliferated and fused a Hams F12 was used for diluting the concentration of nucleus pulposus cells to be 1 × 102.10One milliter of nucleus pulposus cells supernatant from the digestive tract was collected after cell proliferation and fusion, and was mixed with 0.5 mL of hydrochloric acid (4 mol/L) and 0.5 mL of protection solution. The cells were underwent a warm bath and hydrolysis at 110 °C for 6 hours, which was followed by the addition of 10 mL of distilled water (pH 6.0) for filtration. The 0.2 mL of filtered solution was collected and 0.4 mL of distilled water, 0.4 mL of sodium carbonate (4 mol/L), and 0.5 mL of 2% acetylacetonate were added to the solution. After mixing the solution, it was boiled for 20 minutes. Once the mixture cooled down 0.5 mL of Her reagent was introduced. The OD value was testified at 530 nm using a spectrophotometer (Type: 721).2.114 nucleus pulposus cells were resuspended in binding buffer. The cells were then labeled using Annexin V-PE and 7-AAD , they were detected using a FACS Calibur flow cytometer. Cells in the Annexin V+/7-AAD− quadrant were considered as early apoptotic cells, while the cells in the Annexin V+/7-AAD+ quadrant were considered as late apoptotic cells. Both the early and late apoptotic cells were counted together.After inducing apoptosis in cells of each group 10 ng/mL IL-1β for 24 hours, the cells were collected and washed by PBS 3 times. 1 × 102.12t test. The rank-sum test was used for ranked data analysis. The counting data were represented as percentage or rate and compared using a χ2 test. P < .05 was considered to be statistically significant.Clinical data obtained from all patients using a follow-up were encoded onto Microsoft excel (version: 2010) to establish a database. SPSS 20.0 software  was applied for statistical analysis. Measurement data were represented in terms of standard and deviation and highlighted using a 33.1χ2 = 5.119, P < .001).The immunohistochemistry results showed that TSLP was positively expressed in the case group Fig. A while i3.2χ2 = 8.971, P = .030). The TSLP-positive expression rate of patients with lumbar disc herniation, lumbar spondylolisthesis, and lumbar spinal stenosis were 56%, 62.07%, and 65.22%, respectively. However, no correlation was established between the TSLP-positive rate and the degeneration type. In terms of VAS scores, patients were divided into mild (n = 8), moderate (n = 23), and severe (n = 46), and the TSLP-positive expression rates were 12.50%, 30.43%, and 84.78%, respectively. The TSLP-positive expression rate elevated with an increase in the VAS score  .As shown in Fig. 3.4−ΔΔCt methods were used to calculate and analyze the relative expressions of TSLP and TSLPR gene, and the inhibitory effect of 3 different TSLP siRNAs and 3 different TSLPR siRNAs. Compared with the control siRNA group, the TSLP mRNA-inhibition rate of TSLP siRNA-1 was 63%, the TSLP mRNA-inhibition rate of TSLP siRNA-2 was 90%, and the TSLP mRNA-inhibition rate of TSLP siRNA-3 was 25% . Compared to the TSLP-siRNA group, the TSLP-positive rate decreased in the TSLP-siRNA + TSLPR-siRNA group . However, there was no difference between the TSLPR-siRNA group and the anti-TSLPR group . Compared with the TSLP-siRNA group, there was a notable decrease in TSLP concentration level in the TSLP-siRNA + TSLPR-siRNA group (P < .05). Besides, the TSLP-siRNA+TSLPR-siRNA group showed the lowest TSLP level among the 6 groups . The cell viability increased significantly at 48 and 72 hours  compared with 24 hours. After cell transfection, the cell viability was higher in the TSLP-siRNA group, the TSLP-siRNA + TSLPR-siRNA group, and the anti-TSLPR group than the control group and the blank group at 48 and 72 hours . There were no statistical differences observed among the control group, the bank group, and the IgG group . Both the TSLP-siRNA group and the anti-TSLPR group showed poorer cell viability than the TSLP-siRNA + TSLPR-siRNA group (P < .05)A growth curve was drawn using a MTT assay to show the cell viability after cell transfection. The measured OD values of transfected nucleus pulposus cells are shown in Table 3.8P < .05), while no difference was observed between the blank group and the control group. The transfection of TSLP-siRNA and TSLPR-siRNA plasmids could promote nucleus COL2AL expression and aggrecan in pulposus cells . The TSLP-siRNA + TSLPR-siRNA group had lower apoptosis rates compared with the TSLP-siRNA group and the anti-TSLPR group (both P < .05). The results indicate that TSLP siRNA, TSLPR-siRNA, and anti-TSLPR could inhibit apoptosis of nucleus pulposus cells , which is the antigen-presenting cells (APC) maintain the immunoregulatory functioning in the induction of Th2 cell polarization and excitation hypersensitivity inflammation. A possible trajectory of the mechanism could be that TSLP induces the expression of INF super-family protein OX40L in DC cells; DC cells promote the generation of Th2 cells in the absence of IL-12 through the interaction between DC and T cell surface receptors OX40L.,24 Furthermore, previous study has revealed that TSLP-DC could promote the polarization of Th2 central memory cells at the local inflammation spot and the expression of prostaglandin D2 receptors and other active substances, and therefore could accelerate the process of allergy inflammatory participation. A previous study found that increased level of TSLP could be induced by human epithelial cells through costimulation of inflammatory cytokines, such as TNF-α, IL-1β, and Th2 cytokines. TSLPR is a novel receptor subunit related to sequence to IL-2 receptor common gamma chain. TSLP is capable of binding to a heterodimer receptor complex composed of the IL-7 receptor alpha chain. Therefore, TSLP expression is positively proportional to TSLPR.LDD leads to high health care costs and has serious implications on general human health. The results of the present study were confirmed by Rodrigues et al, who considered discs with a Pfirrmann grade lower than or equal to II to be normal, and those with a Pfirrmann grade higher than II to be degenerated. The VAS score is frequently used in clinical medicine in order to estimate the severity of subjective symptoms. TSLP could result in acceleration of allergy inflammatory; therefore, it is reasonable to assume that a TSLP-positive rate is associated with a higher VAS score.The present study found that TSLP-positive rate is associated with a higher Pfirrmann grade and higher VAS score. Pfirrmann disc degeneration grade is a morphologic disc degeneration grading system. Alteration of matrix components is related to inflammatory cells infiltration and hemorrhage. As a result, lesser inflammation caused by TSLP siRNA can result in an increase of the matrix component, causing increased secretion of Aggrecan and COL2AL. In addition, TSLP-siRNA could inhibit the apoptosis and induction of nucleus pulposus cells in order to reduce the apoptosis rate. The main components of the extracellular matrix disc include collagen with strong toughness (whereas type II collagen covers a larger proportion), proteoglycans, elastin, and water. The occurrence of LDD results in unclear boundaries between annulus and nucleus, reductions in the synthesis of nucleus proteins and carbohydrates, changes in the collagen arrangement and type, and an obvious decrease of the corresponding biological role. It has been demonstrated that IL-1β and TNF-α could induce the apoptosis of nucleus pulposus cells, as well as promote the expression of TSLP.,36 RNA interference (RNAi) is a widely used technique that specifically inhibits the expression of the corresponding gene sequences. Thus, apoptosis of nucleus induced by IL-1β was decreased after the inhibition of TSLP activity. Similarly, we observed that anti-TSLPR could promote cell viability, increase COL2AL and Aggrecan levels, and prohibit cell apoptosis. Studies have shown that upregulation of eosinophil surface expression of TSLPR significantly enhanced sensitivity of eosinophils in order to TSLP-mediated degranulation. TSLP signaling was blocked by intratracheal administration of anti-TSLPR antibody before sensitization. Hence, anti-TSLPR coupled with blocked TSLP signaling, had the similar function as TSLP-siRNA.By the means of this study, it has been highlighted that TSLP-siRNA transfected human nucleus pulposus cells can promote the viability of nucleus pulposus cells and synthesis of COL2AL and Aggrecan. The matrix components are responsible for COL2AL, Aggrecan, and cartilage mechanical properties. Aggrecan, a protective factor in preventing degradation of collagen fibrils and the use of an aggrecanase inhibitor could affect overall cartilage protection.5In summary, the TSLP positive expression rates of LDD patients in the case group were significantly higher compared with the control group. The TSLP expression indirectly reflects the degree of degeneration for histological level in intervertebral disc. The transfection of TSLP-siRNA or anti-TSLPR significantly inhibits the apoptosis and induction of nucleus pulposus cells, could potentially serve as a new target for LDD treatment. TSLP is a growth factor secreted outside the cell; however, the study fails to detect TSLP levels in blood samples. Consequently, the TSLP level determined in the study could not be completely accurate in comparison. Hence, further analysis and investigation are required to confirm the results our study.