To assess the association between plasma retinol-binding protein 4 (RBP4) levels both in the first trimester and second trimester and risk of gestational diabetes mellitus (GDM).Plasma RBP4 levels and insulin were measured among 135 GDM cases and 135 controls nested within the Peking University Birth Cohort in Tongzhou. Multivariable linear regression analysis was conducted to assess the influence of RBP4 levels on insulin resistance. Conditional logistic regression models were used to compute the odds ratio (OR) and 95% confidence interval (CI) between RBP4 levels and risk of GDM.P < 0.05). Plasma RBP4 concentrations in the first and second trimester were associated with fasting insulin, homeostasis model assessment for insulin resistance (HOMA-IR), and the quantitative insulin sensitivity check index (QUICKI) in the second trimester . With adjustment for diet, physical activity, and other risk factors for GDM, the risk of GDM increased with every 1-log μg/L increment of RBP4 levels, and the OR (95% CI) was 3.12 (1.08–9.04) for RBP4 in the first trimester and 3.38 (1.03–11.08) for RBP4 in the second trimester.The GDM cases had significantly higher levels of RBP4 in the first trimester than controls (medians: 18.0 μg/L vs 14.4 μg/L; Plasma RBP4 levels both in the first trimester and second trimester were dose-dependently associated with increased risk of GDM. RBP4 gene could enhance insulin sensitivity  [The primary outcomes of interest were insulin resistance and GDM. Homeostasis model assessment (HOMA) was used to estimate insulin resistance [HOMA-IR = insulin/(22.5elucose)] . The qualucose)] . GDM waslucose)] . Women w− 1) [Covariates were collected at the first prenatal visit, such as maternal characteristics regarding demographic information, pre-pregnancy weight, pregnancy history, smoking status, alcohol intake, family history of diabetes. Dietary intake was recorded for two non-consecutive days and average daily intake of calories was calculated. Physical activity was evaluated using the last 7-day, short form of the International Physical Activity Questionnaire and quantified using metabolic equivalents of task (MET-min week− 1) . Each su2 test. Multivariable linear regression analysis was conducted to estimate the associations of RBP4 levels in the first and second trimester with fasting insulin, HOMA-IR and QUICKI in the second trimester. Then, study subjects were divided into four groups  according to quartiles of RBP4 levels among control group, and the lowest quartile (Q1) was used as the reference group. Conditional logistic regression models were used to compute the odds ratio (OR) and 95% confidence interval (CI) between RBP4 levels and risk of GDM. Three models were established to assess the robustness of the results. In model 1, we adjusted for maternal age, education, occupation, gestational weeks of RBP4 measurements in the first trimester, pre-pregnancy BMI, systolic blood pressure (SBP), and diastolic blood pressure (DBP) at the first prenatal visit, and GWG before OGTT. We additionally adjusted for biochemical indicators involving total cholesterol, triglyceride, HDL, LDL, GFR, ALT, and AST in model 2. Further adjustment for daily intake of calories and weekly physical activity time was conducted in model 3. We also performed tests for linear trend by entering the median value of each RBP4 level as a continuous variable in the models. Restricted cubic spline regressions were used with three knots to examine possible nonlinear relationships between RBP4 levels and risk of GDM. In the sensitivity analysis, we converted all continuous covariates into categorical covariates  to test the robustness of our results. Moreover, we conducted a subgroup analysis to test if maternal age  could modify the association between RBP4 and GDM by adding an interaction term to the regression models. Unconditional logistic regression models were applied to compute the results. All analyses were performed with SAS 9.4 and two-sided P values < 0.05 was considered to be statistically significant.Continuous variables were presented as medians  and analyzed for between-group differences using Wilcoxon rank sum test. Categorical variables were analyzed for between-group differences using the χP > 0.05).The characteristics of the study subjects are presented in Table P < 0.05; Table P < 0.001), after adjustment for potential confounders, including biochemical indicators, daily intake of calories, and physical activity  and second trimester  after adjusting for maternal age, education, occupation, gestational weeks of RBP4 measurements in the first trimester, pre-pregnancy BMI, GWG before OGTT, SBP, and DBP. After additional adjustment for biochemical indicators, subjects in the highest quartile of RBP4 had 3.72-fold  and 3.25-fold  higher risk of developing GDM, respectively. In addition, further adjusting for daily intake of calories and physical activity didn’t attenuate this association, and the adjusted OR (95% CI) was 3.71 (1.27~10.84) for RBP4 in the first trimester and 3.30 (1.03~10.52) for RBP4 in the second trimester. We also estimated the risk of GDM associated with every 1-log μg/L increment in RBP4 levels, and the OR (95% CI) in model 3 was 3.12 (1.08~9.04) for RBP4 in the first trimester and 3.38 (1.03~11.08) for RBP4 in the second trimester. Findings of the sensitivity analysis showed that our results were robust after converting all continuous covariates into categorical covariates in the models . In the subgroup analysis, we found that maternal age did not modify the association between RBP4 levels and GDM . Another study also found RBP4 levels were significantly correlated with HOMA-IR in the first trimester in healthy pregnant women  [Mechanism studies found that RBP4 was involved in the pathogenesis of diabetes by inducing insulin resistance through several etiologic pathways. Increasing serum RBP4 could induce the expression of the phosphoenolpyruvate kinase from the liver and impair insulin signaling in muscle . Further< 0.001) . However< 0.001) . The con< 0.001) . FurtherThe relationship between RBP4 levels in early pregnancy and the risk of GDM have been assessed by limited studies and inconclusive results were yielded. A prospective study found that compared with the first quartile of RBP4 in the first trimester, adjusted ORs of GDM for the second, third and fourth quartile were 1.54, 3.05 and 6.36 respectively . AnotherSeveral limitations of the current study should be considered. First, although we adjusted for several potential confounders such as diet and physical activity, we cannot exclude the possibility of unmeasured confounders. Second, RBP4 is generally present in free and transthyretin-bound forms for the transport of retinol. Previous study reported that the RBP4:retinol ratio and the RBP4:transthyretin ratio might be more informative than RBP4 levels alone when assessing insulin resistance during pregnancy . We did In summary, we reported for the first time a dose-dependent association between RBP4 levels both in the first trimester and second trimester and GDM risk. In addition, plasma RBP4 in the first and second trimester is associated with insulin resistance in the second trimester. Further research is warranted to determine the underlying mechanism and examine if a reduction in RBP4 concentrations could lower the risk of GDM.Additional file 1: Figure S1. The dose-response analysis between GDM and RBP4 levels in the first trimester (A) and the second trimester (B) with restricted cubic spline. Conditional logistic regression models were adjusted for maternal age, education, occupation, gestational weeks of RBP4 measurements in the first trimester, pre-pregnancy BMI, GWG before OGTT, SBP, DBP, total cholesterol, triglyceride, HDL, LDL, GFR, ALT, AST, daily intake of calories, and weekly physical activity time. Bold lines are ORs and dashed lines are 95% CIs. The horizontal line is the reference line. Abbreviation: GDM, gestational diabetes mellitus; RBP4, retinol-binding protein 4.Additional file 2: Table S1. Comparison of the baseline characteristics between GDM cases included in the analysis and all GDM cases. Abbreviation: GDM, gestational diabetes mellitus; Gweek, gestational week; BMI, body mass index; GWG, gestational weight gain; OGTT, oral glucose tolerance test; PA, physical activity; SBP, systolic blood pressure; DBP, diastolic blood pressure; HDL, high-density lipoprotein; LDL, low-density lipoprotein; GFR, glomerular filtration rate; ALT, alanine transaminase; AST, aspartate aminotransferase.Additional file 3: Table S2. Comparison of the baseline characteristics between controls included in the analysis and all controls. Abbreviation: GDM, gestational diabetes mellitus; Gweek, gestational week; BMI, body mass index; GWG, gestational weight gain; OGTT, oral glucose tolerance test; PA, physical activity; SBP, systolic blood pressure; DBP, diastolic blood pressure; HDL, high-density lipoprotein; LDL, low-density lipoprotein; GFR, glomerular filtration rate; ALT, alanine transaminase; AST, aspartate aminotransferase.Additional file 4: Table S3. Odds ratio  of GDM associated with different levels of RBP4 in the first trimester and second trimester after converting all continuous covariates into categorical covariates in the models. Models were adjusted for maternal age , education , occupation , gestational weeks of RBP4 measurements in the first trimester , pre-pregnancy BMI , GWG before OGTT , SBP , DBP , total cholesterol , triglyceride , HDL , LDL , GFR , ALT , AST , daily intake of calories  and weekly physical activity time . Abbreviation: GDM, gestational diabetes mellitus; RBP4, retinol-binding protein 4.Additional file 5: Table S4. Odds ratio  of GDM associated with different levels of RBP4 in the first trimester and second trimester stratified by maternal age. Unconditional logistic regression models were adjusted for maternal age, education, occupation, gestational weeks of RBP4 measurements in the first trimester, pre-pregnancy BMI, GWG before OGTT, SBP, DBP, total cholesterol, triglyceride, HDL, LDL, GFR, ALT, AST, daily intake of calories, and weekly physical activity time. Abbreviation: GDM, gestational diabetes mellitus; RBP4, retinol-binding protein 4.


A fundamental understanding of the phonon transport mechanism is important for optimizing the efficiency of thermoelectric devices. In this study, we investigate the thermal transport properties of the oxidized form of phosphorene called phosphorene oxide (PO) by solving phonon Boltzmann transport equation based on first-principles density functional theory. We reveal that PO exhibits a much lower thermal conductivity (2.42–7.08 W/mK at 300 K) than its pristine counterpart as well as other two-dimensional materials. To comprehend the physical origin of such low thermal conductivity, we scrutinize the contribution of each phonon branch to the thermal conductivity by evaluating various mode-dependent quantities including Grüneisen parameters, anharmonic three-phonon scattering rate, and phase space of three-phonon scattering processes. Our results show that its flexible puckered structure of PO leads to smaller sound velocities; its broken-mirror symmetry allows more ZA phonon scattering; and the relatively-free vibration of dangling oxygen atoms in PO gives rise to additional scattering resulting in further reduction in the phonon lifetime. These results can be verified by the fact that PO has larger phase space for three-phonon processes than phosphorene. Furthermore we show that the thermal conductivity of PO can be optimized by controlling its size or its phonon mean free path, indicating that PO can be a promising candidate for low-dimensional thermoelectric devices. The efficiency of a thermoelectric device can be expressed using a dimensionless quantity, ZT defined asS, σ, T, λ, and κ represent the Seebeck coefficient, electrical conductivity, absolute temperature, electron and lattice thermal conductivities, respectively. In order to increase ZT value, we need to increase the power factor S2σ, and at the same time to decrease thermal conductivities. Seebeck coefficient S is usually much higher in semiconductor than in metal, since S is strongly related to differential total electron density of state (DOS), which is maximized near band gaps, where DOS changes abruptly. On the other hand, it is, unfortunately, impossible to adjust σ high, and λ low simultaneously to enhance the thermoelectricity, because they are coupled to each other and governed by the Wiedemann-Franz law. In semiconductors, the phononic contribution of the thermal conductivity (κ) is much larger than its electronic contribution (λ). Moreover, κ is partially decoupled from σ, and thus its reduction is a useful strategy for high-performance thermoelectric materials. Recently, a few studies proposed that introduction of particular defect structures increases σ/(λ + κ) since the thermal conductivity decreases much more than its electrical counterpart, although the defects usually reduce both4. Another interesting research showed that making low-dimensional structures enhances thermoelectric power. In the low-dimensional materials, S can be further increased due to its unique properties, such as sudden changes in the DOS5, and quantum confinement effects7. Moreover, such structural redesign may affect thermal conductivity. κ can be lowered more while keeping σ by forming superlattice or nanocrystal structures9, because the mean free path of phonon is longer than that of electron. To realize state-of-the-art thermoelectric devices, therefore, one should design a quasi low-dimensional or superlattice structure with a semiconductor, which has a low lattice thermal conductivity. It is, for example, known that the best thermoelectric materials are Bi2 Sb3/Sb2 Te3 superlattice10 and layered SnSe structure11, which exhibit the highest ZT values.The thermoelectric effect is one of the promising next-generation energy harvesting technologies and has been studied extensively in recent decades13, would fit in a category satisfying conditions mentioned above. Due to its puckered structure representing strong anisotropy, its mechanical, electrical and thermal properties shows remarkably different directional behavior, especially along the armchair and zigzag directions19. There have been several reports showing that phosphorene has a high ZT value along the armchair direction21.Phosphorene, which has recently attracted attentions in various fields related to materials research25. Although there were a few proposals to prevent phosphorene from being oxidized26, they are not easily applicable, and thus it is still difficult to use phosphorene as a thermoelectric device. Another way to overcome the poor air stability of phosphorene is to use the oxidized structure itself as a device. It was reported that a particular structure of phosphorene oxide (PO) is a semiconductor with a direct band gap of 0.6~0.88 eV25, which is slightly smaller than that of its pristine counterpart, phosphorene. It was also reported that PO has a relatively small electron effective mass of 0.18 me24, which is similar to that of phosphorene along the armchair direction and much smaller than along the zigzag direction. In addition, phosphorene oxide exhibits some interesting physical properties such as non-symmorphic phase protected band structure25.However, the poor stability of phosphorene under ambient air conditions has been a crucial problem for its various applications including thermoelectric devices. Earlier studies reported that phosphorene can be easily oxidized in air because of the lone-pair electrons in each phosphorus atomMoreover, PO satisfies the conditions mentioned above for good thermoelectric materials. In this paper, we report our investigation on the thermal transport properties of the oxidized structure of phosphorene by solving the Boltzmann transport equation based on first-principles density functional theory. PO shows significantly lower lattice thermal conductivity than phosphorene. Furthermore, we present our analysis of why such low lattice thermal conductivity was observed in PO. Our analysis includes the evaluation of the phonon relaxation time, Grüneisen parameters, and phase space of the three-phonon processes, all of which enable us to understand the fundamental origin of the low thermal conductivity of PO.25, we found the equilibrium structure of PO, as shown in Fig. 24. We expanded this equilibrium structure to 4 × 4 × 1 super cell to investigate the thermal properties.In an earlier studyω ≤ 200 from (a), the acoustic modes exhibit anisotropic dispersion along the Γ-Y (zigzag) and Γ-X (armchair) directions. To clearly describe such an anisotropic behaviors in the dispersion, we computed the group velocity vg of the three acoustic branches along these two directions. As shown in Fig. 27, MoS2 (4.2–6.8 km/s)28, graphene (3.7–6.0 km/s)29, and phosphorene (4.0–7.8 km/s)16. We found that vg of the LA phonon mode along the zigzag direction is larger than that along the armchair direction, as we easily understand since the LA mode depends strongly on the elastic modulus of the material. Due to the puckered structure shown in Fig. Figure In a carrier transport phenomenon in a crystal solid, not only the group velocity but the carrier lifetime or scattering rate also play a crucial role in determining its transport properties. The evaluation of the carrier lifetime requires anharmonic phonon-phonon scattering or Umklapp process evolving three phonons, since the momentum-conserved phonon scattering process described by only two phonons is not relevant to the phonon lifetime.We noticed that the Grüneisen parameter (GP), which is usually considered for the thermal expansion behaviors, gives us a useful information on the anharmonic phonon scattering rate. The mode-dependent GP is defined asγZA becomes very large in the long wavelength region near the Γ point as in other 2D materials30 as described by membrane effect31. It is also clear that the GPs are largely anisotropic and discontinuous at the Γ point, reflecting its structural features similar to the sound velocity. It was derived that the mode-dependent anharmonic phonon lifetime is inversely proportional to the mode-dependent GP squared or 35. Thus LA mode of PO, which has smaller GP values than the TA or ZA, may exhibit longer phonon lifetime than the other acoustic modes. Therefore, the LA mode plays an important role in PO’s thermal transport behavior as we will later describe in detail.Figure κPO(T) of PO, we solved phonon Boltzmann equation using the iterative approach mentioned in the Method section. We also applied the same method to evaluate the lattice thermal conductivity κP(T) of phosphorene for comparison. Figure a/T with a single parameter a, which is expected from the three-phonon anharmonic process36. Our predicted room-temperature thermal conductivity values of phosphorene, which are 146 W/mK and 65 W/mK along the zigzag and armchair directions, respectively, agree reasonably well with previously reported values ranging from 110 to 153 W/mK and from 33 to 64 W/mK along the respective directions37. Both phosphorene and PO exhibit that their lattice thermal conductivities along their armchair directions are approximately 2.5 times smaller than those along the zigzag directions, owing to the structural anisotropy mentioned above. Although PO is structurally similar to phosphorene, we observed that the thermal conductivity values of PO are much lower than those of phosphorene over the whole temperature range regardless of the transport directions. For example, at room temperature (T = 300 K), we found 38, MoS2 (23.2~34.5 W/mK)41, stanene (11.6 W/mK)42, graphene (3000~6000 W/mK)44, and hexagonal boron nitride (hBN) (350~600 W/mK)45. Low thermal conductivity is one of sufficient conditions for high-performance thermoelectric materials. In this sense, PO can be a new candidate for 2D thermoelectric materials.To investigate the temperature dependence of the lattice thermal conductivity κPO and summarized in Table 46. For example, in graphene and hBN, whose room-temperature thermal conductivities have been reported to be a few thousands46 and a few hundreds45 in W/mK, their ZA modes contribute to their thermal conductivities by about 75%46. We found, on the other hand, that the contribution of the ZA mode in PO becomes less significant to be only 15~17%, whereas the contribution from the other modes including the LA and TA modes becomes dominant. This is because PO is not a perfect 2D structure, but a puckered one, allowing more phonon-phonon scattering of the ZA mode. Thus, puckered structures would be advantageous for a low-dimensional thermoelectric application.To explore what causes such a low lattice thermal conductivity in PO, we first estimated the contributions of various phonon modes to the τ with the Umklapp scattering rate 1/τU. Figure To further analyze the low lattice thermal conductivity of PO, we evaluated the relaxation times of phosphorene and PO as a function of frequency. In general, there are various sources giving rise to phonon scattering, for example, anharmonic phonon-phonon or Umklapp (U) scattering, phonon-electron scattering, impurity effect, boundary effect, isotope effect, and so on. Among these scattering sources, we took only the phonon-phonon scattering into account, since the other sources were much smaller in PO. Therefore, we simply replaced the total scattering rate 1/P3 for the anharmonic three-phonon scattering process introduced by Lindsay and Broido47. The three-phonon scattering is allowed only when the energy and momentum conservation conditions are satisfied. Thus, the phonon scattering rate of each phonon state is proportional to the number of available final states. In other words, the phonon lifetime of each mode ωj(q) is inversely correlated with the mode-dependent phase space P3(ωj(q)) defined by48BZ is the volume of BZ; 49 for absorption (+) and emission (−) processes. Difference of a factor two between two coefficients was introduced to avoid double counting. To discuss the physical phenomena of phonon scattering in more detail, we analyzed the allowed phase space q″ with q ± q′ − G, where G vectors are the reciprocal lattice vectors to describe Umklapp processes. Figure P3 values as a function of frequency ωj evaluated over the Brillouin zone. It is clear that PO has a much larger P3 value than phosphorene, indicating that more scattering processes occur in PO than in phosphorene. This explains why PO has a much shorter relaxation time than phosphorene.Here the momentum conservation was already imposed to replace 20. Therefore, optimization of grain size in nanocrystalline structures or nanowire approach can be an effective way to enhance the thermoelectric figure of merit. To explore the size dependence of thermal conductivity of PO, we evaluated the cumulative thermal conductivity κ(l) by summing up all the contributions from phonon modes with MFP smaller than l. Figure κ(l) as a function of phonon MFP along both zigzag and armchair directions at various temperatures. As shown in the figure, κ(l) curves resemble a logistic function when MFP is given in a logarithmic scale, and thus we fit our calculated data to a single parametric function given as50l0 is the fitting parameter determining the characteristic value of the MFP, yielding κ(l = l0) = κ/2. The l0 values are fitted to be 40.26 (39.98), 15.30 (16.46), 9.44 (10.48), and 6.71 (7.64) nm at 200, 400, 600, and 800 K, respectively, for the zigzag (armchair) direction.To design high-performance thermoelectric devices, most important strategy is to reduce the thermal conductivity while remaining its electronic counterpart. Usually the lattice thermal conductivity of a system can be decreased by reducing the size of the system, since the size reduction results in shortening of its phonon MFP. On the other hand, electron MFP is much smaller than that of phonon especially in semiconductors. For example, the electron MFP of phosphorene is known to be around only a few nm at 300 KWe presented our investigation on the thermal transport properties of phosphorene oxide (PO) as well as phosphorene using the first-principles calculations combining with the semiclassical Boltzmann transport theory. We found that the thermal conductivity of PO is much lower than those of other two-dimensional materials including phosphorene, revealing that oxidation is responsible for the reduction in the thermal conductivity. The thermal conductivity of PO depends strongly on its transport directions, and were calculated to be 2.42 and 7.08 W/mK along the armchair and the zigzag directions at 300 K. Similar to phosphorene, PO is structurally characterized by the flexible puckered structure, which leads to lower-frequency acoustic phonon modes and smaller sound velocities than genuine 2D flat materials. In addition, PO possesses no mirror symmetry allowing more ZA phonon scattering. Furthermore, we identified that nearly-free vibration of dangling oxygen atoms gives rise to additional scattering resulting in further reduction in the phonon lifetime. Spontaneous oxidation of phosphorene greatly reduces its thermal conductivity, which can be additionally optimized by controlling the size, and thus PO can be a promising candidate for use in low-dimensional thermoelectric devices.51 as implemented in the Vienna ab initio simulation package (VASP)53. We employed the projector augmented wave potentials55 to describe the valence electrons, and treated exchange-correlation functional within the generalized gradient approximation of Perdew-Burke-Ernzerhof 56. The plane-wave kinetic energy cutoff was selected to be 500 eV, and c = 20 Å was chosen for the lattice constant of the direction perpendicular to the plane to minimize the interlayer interaction. The Brillouin zone (BZ) of each structure was sampled using a 11 × 11 × 1 k-point grid for the primitive unit cells of phosphorene and PO.To investigate the thermal property of PO, we performed first-principles calculations based on density functional theoryκPO(T) of PO, we solved the phonon Boltzmann transport equation using an iterative approach proposed by Omini et al.57, which implemented in the ShengBTE code50. The thermal conductivity tensor καβ, where α and β denote x, y or z, can be obtained fromkB is the Boltzmann constant, Ω the unit cell volume, and N the Γ-centered q-point grids. In the summand, f0 is the Bose-Einstein distribution function; and ωj,q and vj,q are respectively the phonon frequency and group velocity of the phonon mode with the branch index j and wavevector q. Lj,q was introduced to compensate the phonon distribution deviated from f0 in the presence of the temperature gradient. This quantity with a dimension of length can be expressed asuj,q is the correction to the SMRT approach due to the deviation of the phonon population at the specific mode with j and q from the Bose-Einstein statistics. Since it is nonlinearly coupled with Lj,q, we solved Eq. (uj,q and thus Lj,q. For more complete expressions of Lj,q, and uj,q, and their evaluation approach, see the reference [ShengBTE_2014]. The phonon dispersion relations of phosphorene and PO were calculated by applying the finite displacement method58 to their respective 6 × 6 × 1 supercells. We also computed their third-order interatomic force constants and phonon relaxation times to evaluate their correpsonding lattice thermal conductivities59 using 4 × 4 × 1 supercell. We took up to the fourth nearest neighboring interaction and the corresponding BZ was sampled by a 2 × 2 × 1 k-point grid. Note that the cross-section area should be determined in order to evaluate the thermal conductivity. As usually done in two dimensional cases, the thicknesses of phosphorene and PO were set to 5.5 and 8.0 Å,respectively, approximately corresponding to interlayer distances in their bulk configurations.To precisely evaluate the temperature dependence of the lattice thermal conductivity Low Lattice Thermal Conductivity of a Two-Dimensional Phosphorene Oxide


To explore the effects and underlying biological mechanisms of tetrahedral DNA nanostructures (TDNs) on the proliferation and osteogenic differentiation of periodontal ligament stem cells (PDLSCs).Real‐time cell analysis (RTCA) and CCK8 were used to screen the best concentration of TDN for PDLSCs. Cell proliferation and osteogenic differentiation were assessed after PDLSCs were treated with TDN. Data were analysed using one‐way ANOVA.Tetrahedral DNA nanostructures could play a crucial role in accelerating the proliferation of PDLSCs and had the strongest promotive effect on PDLSCs at a concentration of 250 nmol/L. Simultaneously, the osteogenic differentiation of PDLSCs could be promoted significantly by TDNs and the finding displayed that the Wnt/β‐catenin signalling pathway might be the underlying biological mechanisms of TDNs on promoting the osteogenic differentiation of PDLSCs.Tetrahedral DNA nanostructure treatment facilitated the proliferation of PDLSCs, significantly promoted osteogenic differentiation by regulating the Wnt/β‐catenin signalling pathway. Therefore, TDNs could be a novel nanomaterial with great potential for application to PDLSC‐based bone tissue engineering. With the improvement in materials and technology, more and more therapies are being developed to rebuild bone defects, but the results remain unsatisfactory.Since the structure DNA nanotechnology was put forward by Seeman in 1982, DNA nanomaterials have both aroused wide public concern and become a research hotspot in related fields because of their programmability, controllability and good mechanical performance.Considering all of this evidence, a hypothesis was proposed that TDNs may facilitate proliferation and promote osteogenic differentiation of PDLSCs by regulating the Wnt/β‐catenin signalling pathway. Therefore, PDLSCs were exposed to TDNs, with the purpose of systematically exploring the role of TDNs in the proliferation and osteogenic differentiation of PDLSCs. The results indicated that TDNs had a tremendous potential for application in PDLSC‐based bone regeneration.22.1As shown in Table 2.22, pH 8.0), and the mixture was heated at 95°C for 10 minutes and then incubated at 4°C for 20 minutes. The successful synthesis of TDNs needed to be corroborated using polyacrylamide gel electrophoresis . In addition, the configuration of TDNs was displayed by using atomic force microscopy (AFM) and transmission electron microscope (TEM).Tetrahedral DNA nanostructures were prepared as reported previously with four pre‐designed DNA single strands.2.3The periodontal ligament tissue samples came from the extracted third molars, healthy and with no inflammation, of adults between 18 and 25 years old. We received the informed consent of the donors, and we followed the guidelines of the ethics committee of the University of Sichuan. The isolation and culture of cells have been described in previous studies.2.4Cell counting kit‐8 (CCK‐8) and real‐time cell analysis (RTCA) were used to monitor the PDLSCs growth and choose the optimum concentration of TDNs. The PDLSCs were placed into a 16‐well plate with 5000 cells per well. After cultured with regular growth medium for 24 hours, these cells were exposed to the TDNs solutions with concentrations of 0, 125, 250 and 375 nmol/L. Then we used the CCK‐8 and RTCA to monitor cell proliferation for 24‐48 hours and choose the optimum effect concentration on the PDLSCs.2.5g for 5 minutes. We dislodged the supernatant lightly and added the ice‐cold 70% ethyl alcohol to fix the PDLSCs overnight. The samples washed with PBS were mixed with RNase A and dyed with PI. A flow cytometer  was applied to detect the DNA contents. Finally, two computer software packages, WinMDI2.9 and WinCycle, were employed to analyse the results.We used RTCA and CCK8 to analyse the effect of TDNs on PDLSC proliferation. Besides that, we also applied a cell cycle detection kit  to explore the effect on the cell cycle of PDLSCs. After TDN exposure for 24 hours, the PDLSCs were trypsinized by 0.25% trypsin and centrifuged at 10 300 2.64 cells/cm into a six‐well plate. Under the traditional cell culture conditions, PDLSCs were cultivated in regular α‐MEM growth medium for one day. After that, the medium was changed to the induction medium, which included l‐ascorbic acid 2‐phosphate (0.05 mmol/L), 10% FBS, β‐glycerophosphate (10 mmol/L), dexamethasone (100 nmol/L) and α‐MEM medium. Then the PDLSCs were exposed to the 250 nmol/L TDNs solution to promote the osteogenic differentiation, while the same volume of TM buffer was put into the control groups. The induction time would last for 3 and 7 days, and the medium should be replaced every 3‐4 days.When PDLSCs were fused to 85%, the cells were seeded with the density of 3 × 102.7After the PDLSCs were treated with TDNs for 7 days, a BCIP/NBT ALP colour development kit  was applied to observe the roles played by TDNs in the osteogenic differentiation of PDLSCs. The cells were rinsed with PBS thrice to clean up residual medium. Then 4% cold paraformaldehyde was employed to fix the PDLSCs samples for 20 minutes. After washed thrice again with PBS, the cells were stained with ALP colour buffer solution for about 2 hours at 37°C until the colour of the samples became dark blue. Rinsed by PBS, the PDLSCs samples were observed with a fluorescent microscope.2.8The PDLSCs exposed to 250 nmol/L TDNs for 2 weeks as previously described, the calcium deposition that was considered as a late marker of the PDLSCs osteogenic differentiation was detected with Alizarin Red staining. Washed with PBS, the PDLSCs were fixed by 4% cold paraformaldehyde for 20 minutes. After that, the Alizarin Red S dye liquor  was applied to dye the samples for a half hour at room temperature and a fluorescent microscope was used to survey the calcium deposition.2.9RUNX 2), ALP, osteopontin (OPN), quantitative RT‐PCR, was employed. (Table GAPDH) was characterized as the housekeeping gene.For the sake of evaluating, the expression of inflammatory and osteogenic genes, such as runt‐related transcription factor 2  was used to harvest the total proteins. In order to make these collected proteins denature and depolymerize, the 1/4 (v/v) ratio of loading buffer was added and the mix was boiled for 4‐5 minutes. After that, the 10% SDS‐PAGE was performed to separate total albumens into different molecular weight albumens. Subsequently, the target albumens that included the osteogenic differentiation proteins, such as RUNX 2, OPN, and the Wnt signalling pathway‐related proteins, such as β‐catenin, LEF 1 and GSK‐3β, were transferred to a polyvinylidene fluoride membrane. Then the membranes containing these target proteins were immersed in the mixture solutions that included 2.5% bovine serum albumin and 2.5% skimmed milk powder for about 50 minutes to block albumens and incubated overnight with primary antibodies (Abcam) at 4°C. Next day, after rewarming for 60 minutes at 37°C, these membranes were washed thrice with TBST and incubated with secondary antibodies  for 1 hour. After being washed with TBST, the bands of albumens were visualized by an ECL chemiluminescence detection system . The housekeeping control protein was GAPDH.2.11+; Nikon, Tokyo, Japan) was used to capture the images.Periodontal ligament stem cells were seeded on the sterile 20 mm cell slides  and treated with 250 nmol/L TDNs solution for 1 and 3 days. The PDLSCs samples were rinsed with PBS and fixed with 4% (w/v) cold paraformaldehyde solution. Then we used the 0.5% TritonX‐100 to permeate the cytomembrane and 5% goat serum to block the cells. The PDLSCs samples were incubated with primary antibodies  overnight at 4°C. Next day, after rewarming the samples for 30 minutes, the DyLight 488 secondary antibody (1:300 dilution) was added to incubate PDLSCs for 1 hour and DAPI was employed to stain the cell nucleus. Finally, a confocal laser microscope  was applied to make statistical analysis for all the experiment data, when P values <0.05, they were statistically significant. All experiments were repeated at least three times.The result comparisons between experimental groups and control groups were obtained using the student's 33.1In Figure 3.2For the sake of exploring the influence of TDNs and choosing the optimum effective concentration for PDLSCs, a RTCA was performed. As shown in Figure 3.3P < 0.001) between the TDN‐treated group and the control group in the number of S phase and G2 phase cells. The analysis of Figures We used flow cytometry to explore the role of TDNs on the PDLSCs by detecting cell cycle changes. After PDLSCs were exposed to 250 nmol/L TDNs for one day, there was a significant difference (3.4As typical osteogenic‐related markers, OPN, RUNX2 and ALP were chosen to demonstrate that TDNs could facilitate the osteogenic differentiation of PDLSCs at the gene and protein levels.After the gene evaluation, WB and immunofluorescence were performed to detect the albumen contents of OPN and RUNX2 regarded as osteogenic‐related markers. Following TDN treatment for 3 and 7 days, it can be seen in Figure 3.5As an early marker of osteogenic differentiation, the ALP activity of PDLSCs treated with TDNs for 7 days was performed in order to evaluate the part played by TDNs on the osteogenic differentiation of PDLSCs.In addition, calcium nodules, as late indicators of osteogenic differentiation, were detected by Alizarin Red staining to assess the role played by TDNs on PDLSCs.3.6The mechanism through which the osteogenic differentiation of PDLSCs was increased by TDNs is still not clear. But some previous studies have demonstrated that the Wnt/β‐catenin signalling pathway plays a significant role in the osteogenic differentiation of PDLSCs.4As a common clinical problem, targeted repair and reconstruction of bone defects, especially alveolar bone defects, remain a challenge. Although a variety of surgical bone augmentation techniques, such as distraction osteogenesis and autogenous bone graft, have been skilfully and routinely applied to the reconstruction of bone defects, surgeons increasingly prefer bone tissue engineering due to the large trauma and high technical sensitivity required for surgical techniques. Compared with BMSCs, PDLSCs are considered as the ideal seed cells in the oral bone regeneration, owing to the advantages of small trauma and low immunogenicity. In addition, various novel biological materials are applied in PDLSC‐based tissue engineering to provide bone space for seed cells, simulate the growth microenvironment and provide nutrients or related stimulators to promote PDLSCs biological behaviours, such as proliferation and differentiation. In our experiments, TDNs, as novel DNA nanomaterials, were employed to stimulate PDLSCs to regulate its biological behaviours. The results demonstrated that TDNs could facilitate the proliferation of PDLSCs and the optimum work concentration was 250 nmol/L. In addition, the findings also demonstrated that TDNs could greatly enhance the osteogenic differentiation of PDLSCs by regulating the Wnt/β‐catenin pathway Figure .As a new three‐dimensional structure of DNA nano‐biomaterials, TDNs have garnered attention in the field of biomedicine because its structure could be functionalized and designed to respond to the stimulation of the surrounding environment. It could not only be used as a stimulator to adjust biological behaviours such as differentiation, anti‐inflammatory response, proliferation and migration of cells, but could also be applied as a biosensor in molecular diagnosis and as a special carrier to transport ligands and drugs such as siRNA, AS1411 aptamers and DOX for molecular and targeted drug delivery.Tetrahedral DNA nanostructure, as a new DNA nanomaterial, was applied in the PDLSC‐based nanoengineering system for the first time. The results of this investigation demonstrated that TDNs could play a crucial role in accelerating the proliferation of PDLSCs and had the strongest promotive effect on PDLSCs at a concentration of 250 nmol/L. Simultaneously, the most obvious finding of this study was that the osteogenic differentiation of PDLSCs could be promoted significantly with TDNs by regulating the Wnt/β‐catenin pathway. Therefore, TDNs could be a novel nanomaterial that could regulate some cell biological behaviours with a tremendous potential for application in PDLSC‐based bone tissue engineering, such as promoting osteogenic differentiation to restore bone defect.There is no conflict of interest.


Tospovirus (family Peribunyaviridae), which is composed by viruses with tripartite RNA genome that infect plants and are transmitted by thrips (order Thysanoptera). Previous studies have suggested several reassortment events between these two viruses, and some speculated that they may share one of their genomic segments. To better understand the intimate evolutionary history of these two viruses, we sequenced the genomes of the first TCSV and GRSV isolates ever reported. Our analyses show that TCSV and GRSV isolates indeed share one of their genomic segments, suggesting that one of those viruses may have emerged upon a reassortment event. Based on a series of phylogenetic and nucleotide diversity analyses, we conclude that the parental genotype of the M segment of TCSV was either eliminated due to a reassortment with GRSV or it still remains to be identified.Tomato chlorotic spot virus (TCSV) and groundnut ringspot virus (GRSV) share several genetic and biological traits. Both of them belong to the genus  Tospovirus , a genus that solely encompasses the “bunyaviruses” that have plants as hosts and are propagatively transmitted by thrips (order Thysanoptera) [The tospoviruses have a great impact on agriculture since they can cause from mild to severe symptoms in their plant hosts . These vnoptera) ,3. Sincenoptera) ,5. WhileThe main criterion for determination of new tospovirus species resides in the amino acid (aa) sequence of the nucleocapsid (N) protein that is encoded in the S RNA . New isoN genes of isolates BR-03 (TCSV) and SA-05 (GRSV). Both isolates were initially identified from infected tomato in Brazil and groundnut in South Africa, respectively [Gn and Gc genes of BR-03 and SA-05 were sequenced and revealed 92% aa identity. However, when looking in further detail, the phylogenetic distance between BR-03 and SA-05 based on the glycoprotein sequences was similar to that observed between tomato spotted wilt virus (TSWV) isolates, which was unexpected and questioned the N protein-based species demarcation [Tomato chlorotic spot virus (TCSV) and groundnut ringspot virus (GRSV), although serologically related, were first suggested as members of different tospovirus species in the early 1990s after sequencing the ectively . Sequencectively . Such obectively , the Gn arcation . The reaarcation . This isarcation . From all these results abovementioned, we started wondering whether all GRSV and TCSV isolates sequenced so far have been sharing a highly conserved M segment and, more importantly, whether the BR-03 and SA-05 isolates could be considered as parental genotypes of TCSV and GRSV, respectively. To increase the reliability of our analyses, the complete genome of the original BR-03 and SA-05 isolates were high-throughput sequenced in this work. After several analyses, our results suggested that TCSV may have incorporated the M segment of GRSV and that the parental genotype of TCSV was eliminated or remains to be identified.Nicotiana benthamiana leaf material infected with isolates BR-03 (TCSV) or SA-05 (GRSV) were kept frozen (−80 °C) as a virus inoculum stock since the early 1990s at the Wageningen University, Netherlands. These frozen leaves were ground and used for mechanical inoculation of wild type N. benthamiana plants as previously described [escribed . InoculaReads generated by the Illumina platform were trimmed and de novo assembled using the software CLC Genome Workbench 6.5.2 . Contigs corresponding to virus sequences were identified using Blastx  against NSm genes from TCSV, GRSV, and TSWV isolates obtained from the NCBI portal using 1000 bootstrap replicates. TSWV sequences were included in this analysis for comparison. Maximum likelihood (ML) trees were constructed using the amino acid sequences coded by the S, M, and L segments of TCSV, GRSV, and TSWV available at the NCBI portal. For the S and M segments that code for more than one protein, the amino acid sequences were concatenated. Only tospovirus isolates with all open reading frames (ORFs) sequenced were included in this analysis. As done previously, TSWV isolates were included for comparison since they display an intraspecific genetic distance between each other. Multiple alignments were done using the program MUSCLE [A preliminary neighbor-joining tree was constructed using fragments of m MUSCLE  and the m MUSCLE   between TCSV and GRSV was estimated by Markov chain Monte Carlo (MCMC) Bayesian analysis for each segment using BEAST v2.5.0 . Due to ee prior ,24. The ee prior  was usedNSm genes. Thus, these sequences were used to evaluate whether TCSV and GRSV isolates indeed share a highly identical M segment. Regarding genetic distance, NSm genes of TSWV isolates (most abundant on public databases) were included for comparison. Interestingly, the trees generated on the NSm genes showed that the interspecific diversity between TCSV and GRSV isolates is comparable to the intraspecific diversity between TSWV isolates  and SA-05 (GRSV) available on public databases. To circumvent this problem and further substantiate our findings, we have sequenced the complete genome of these isolates from infected With the availability of BR-03 and SA-05 genomes, phylogenetic trees were built with protein sequences derived from the three RNA segments. In the trees based on protein sequences from the S and L segments, TCSV and GRSV isolates clustered separately, forming two groups a. In conL (L segment) and of NSs and N (S segment) was within the expected value with an observed/expected ratio ~ 1 or > 1. Differently, the Gn, Gc, and NSm genes (M segment) showed an observed/expected ratio < 1 with high significance as seen in the p-value graphic , and L segments, respectively. Additionally, they presented narrow 95% high posterior density (95% HPD) intervals: 7.8493E-5 to 4.0956E-4, 1.5095E-4 to 4.4141E-4, and 1.1819E-4 to 3.2171E-4 for the S, M (NSm), and L segments, respectively. The mean TMRCA estimated between TCSV and GRSV was 548.52 years  for the S segment and 571.68 years  for the L segment. Interestingly, but somewhat expected, the mean TMRCA of the M segment was much more recent, being 101.95 years . The fact that the 95% HPD interval of the TMRCA between GRSV and TCSV for the M segment do not overlap with those from the S and L segments states that the TMRCA of the M segment is more recent. Focusing on the cluster containing intercalated GRSV and TCSV isolates (M segment), the first reported TCSV reassortment event may have happened about 38.01 years ago  as seen in To test whether the M segment from TCSV and GRSV are indeed more closely related than the S and L segments, the TMRCA was estimated by Bayesian phylogenetic analysis. The mean substitution rates were similar, with 2.4392E-4, 2.9163E-4, and 2.1783E-4 substitutions/site/year for the S, M  of these viruses as they were in the 1990s. The focus was to understand why they share a highly conserved M segment, while their S and L genomic segments are more genetically distant. Previous works have observed this trait between these two tospoviruses ,26. WhilOur analyses revealed that the TCSV and GRSV M segments exhibit the lowest diversity accumulation in comparison with the S and L segments. There are two possibilities that could explain this variation. Either the coding sequences of the M segment are highly functional at the RNA level, constraining the introduction of mutations, or a reassortment event took place. The first possibility seems very unlikely due to the fact that the substitution rate estimated is similar for the three RNA segments. Based on the nucleotide diversity analysis, the M segment of TCSV isolates presented less diversity accumulation than GRSV isolates, suggesting that the TCSV isolates are reassorted tospoviruses. In previous works, GRSV isolates found in the U.S were reported as reassortants, containing the M segment of TCSV ,27. To cNSm gene/M segment trees. The reason why this reassorted M segment was fixed in all known TCSV isolates remains to be investigated. In any case, it may have increased the virus adaptation to both plant and invertebrate hosts since the parental genotypes of TCSV have not been reported so far. It is worthy to notice that in recent years, TCSV have significantly increased its spread to other regions of the Americas [Reassortment events appear to be frequent between TCSV and GRSV, given that TCSV sequences are scattered throughout the TCSV and GRSV cluster for Americas ,30,31,32


We developed a mathematic empirical model for describing the small field penumbra in order to analyze the potential dose perturbation caused by overlapping field to avoid the dose calculation errors in linear accelerator-based radiosurgery. n and t for the best fitting of the LSRE model. Geometric and mathematical methods were used to derive the model parameters.  A ball phantom was fabricated for measuring penumbra at 4 different gantry angles in the coplanar plane. A least square root estimation (LSRE) Model was created to fit the measured penumbra dose profile and to predict the penumbra dose profile at any gantry angles. The Sum of Squared Errors (SSE) was used for finding the parameters D(š) denotes the dose profile D(š) at the penumbra region, T is the penumbra height , n is the parameter for curvature, š = x − Wd/2 , and t is the radiation transmission of the collimator. Geometric analysis establishes the correlation between the penetration depth of the exposure and its effect on the penumbra region in ball phantom. The penumbra caused by an exposure at any arbitrary angles can be geometrically derived by using a one-variable quadratic equation.  The results showed that the larger the gantry angle of the field, the more the expansion of the penumbra dose profile. The least square root estimation model for describing small field penumbra is as follows:  The dose distribution in penumbra region of small field can be created by the LSRE model and the potential overdosage or underdosage owing to overlapping field perturbation can be estimated. With the adoption of advanced technologies in modern radiotherapy such as stereotactic radiosurgery, stereotactic body radiation therapy, and intensity-modulated radiation therapy, there is an increased interest in the small-field dosimetry of photon beams. A beamlet used in linear accelerator-based radiosurgery is the smallest field formed by one single leaf of the MLC facing the opposite one and is only a portion of the target of dose delivery . All theThe analysis of small field penumbra is important in linear accelerator-based radiosurgery. It includes geometric, transmission, and photon scattered components. Geometric penumbra originates from the radiation source when it is not a single point. Transmission penumbra occurs when the beam passes through the edge of the jaw or MLC before it reaches the full attenuation point of the jaw and the MLC. Scattered radiation from the former two components is added to form the total penumbra, namely, the physical penumbra. The physical penumbra width is defined as the lateral distance from the central axis between 20% and 80% of the central axis dose at a reference depth. The height of a penumbra is defined as the intersection point of the central axis with the dose profile and is usually normalized to be 100%.We design a mathematical model to fit the penumbra in small field used in linear accelerator-based radiosurgery. The penumbra perturbation was also investigated by our model when one field is overlapped by another segment created by MLC. The dose perturbation at the field edges could lead to a potential monitor unit calculation error; therefore, this penumbra perturbation has to be accounted for in order to obtain the correct dose modification especially in the situation of split field .Establishment of a calibration curve for future dose quantification of irradiated filmsSingle exposures of the standard reference field and with double exposure at four different gantry anglesEstablishment of a mathematical model, the least square root estimation (LSRE) model, and using the LSRE model to fit the penumbra dose profiles obtained in the previous stepUsing the Sum of Square Errors (SSE) to find the values of the LSRE model parametersGeometric derivation of the LSRE penumbra parameters at any gantry angle by using a transformation equationEstimating the dose curve expansion owing to overlapping field perturbation in split field by using LSREThe experiment was conducted in the following steps:The details of each step are described in the following sections.Zeff tissue equivalence of 6.98 with a fast polymerization in the image-forming process. Its absorption peaks are independent of radiation energy. Its higher sensitivity in the dose range of 2–800 cGy and an inhomogeneity smaller than 2% after radiation exposure make it suitable for dosimetry analysis. The film processing and dose profile measurements followed the international protocols [2 field size at an SAD of 100 cm. Graded doses of 5, 10, 15, 40, 60, 80, 100, 150, and 200 cGy were given to obtain the Hurter-Driffield calibration curve (H-D curve).We used GAF Chromic EBT2 films  for the experiment. The film has a rotocols . A preexrotocols . This waAll exposed films were then scanned with an Epson Expression 10000XL scanner in the 48-bit RGB mode (16 bits per color), and the data were saved as tagged image file format (TIFF) and analyzed by the VariSoft imaging procession software. The films were scanned in the landscape orientation to reduce the optical density (OD) variation to ≤2% . The expB0 is the background density, namely, the scanner signal of the unexposed film, and B is the scanner signal of the exposed film.The OD of the film was derived by the following equation:A red filter was placed on top of the GAF films before scanning to increase the slope of the H-D curve, thereby raising the resolution of the dose-OD curves .2 on dimension 10 × 10 cm2 GAF film was conducted in a regular 30 × 30 cm2 polystyrene solid phantom at the depth of 5 cm with 100 cGy as the standard reference of penumbra profile. The other four films with dimension 10 × 10 cm2 were exposed each with a radiation field 4 × 2 cm2 on GAF film in a regular 30 × 30 cm2 polystyrene solid phantom at the depth of 5 cm with 100 cGy as the first exposure and then follows the 6 × 4 cm2 field at four different gantry angles in ball phantom separately. We chose a 4 × 2 cm2 field for two reasons: firstly, to simulate the commonly used calculation grid size in the treatment planning systems; secondly, to mark dose profile measurement direction for a smaller filed size used in linear accelerator-based radiosurgery. The penumbra dose profile produced by this single exposure was used as the original standard reference data.The single exposure of a radiation field 4 × 2 cm3 dent on the equator plane to hold the film =1/x . For details, please refer to A mathematical model, named the least square root estimation model (LSRE model), was developed for modeling the penumbra dose profile perturbation. The goal was to build a mathematical model which could predict the penumbra profiles at any gantry angles in coplanar irradiation. The LSRE model originated from the proportion function D(š) denotes the dose profile D(š) at the penumbra region; T is the penumbra height representing the intersection of the beam central axis with the penumbra curve and is between 100 and 99.8%; n is the parameter which gives the curve the curvature, and the greater the n is, the flatter the penumbra would be; š = x − Wd/2, where š and  x are numbers on the x axis in the unit of cm, Wd/2 is the half-field width or the width from the central axis to the 50% dose point of the penumbra region; and t is the radiation transmission of the collimator.The least square root estimation model is expressed as follows:2 dose profile on the 4 cm side after 6 × 4 cm2 field double exposures at 4 different gantry angles in a coplanar irradiation. It fits the penumbra (PenumbraD(š)) dose profile from the central axis to the end of the curve.The LSRE model was used to fit the penumbra of the single exposure standard reference curve and the 4 × 2 cmWe need a tool to determine whether a best fit to the penumbra profile had been achieved or not by the LSRE. The Sum of Square Errors (SSE) provides an ideal measurement of the differences between the measured and calculated data by the LSRE model . The lowxi} is the observation and The SSE is defined as follows:The total SSE isn that leads to the lowest SSE would result in the best fit of the penumbra curvature; the t with the lowest SSE is derived separately and it affects the height of the penumbra tail.The n and t only for the dose profiles at gantry angles of 30°, 45°, 60°, and 90°. Eventually, we aim to obtain ns and ts at any gantry angles. For this, we need to find the lengths of n and t were dependent only on the penetration depth θ. It is the distance where the radiation traverses inside the phantom before intersecting with the penumbra edge of the previous field. It is the only factor controlling the dose contribution from the larger superimposing field onto the smaller field along 2 field. x and −x at an arbitrary gantry angle of  θ. For the detailed geometric derivation of So far, we have the best fits of parameters geometry . ed¯ is When the film was scanned with a red color filter, the calibration curve was steeper and thus with a higher OD-dose resolution. Radiation dose was then derived by the OD method  was 19.8 mm after single irradiation at a gantry angle of 0 in the flat polystyrene solid phantom. The FWHM became 20.3 mm, 21.1 mm, 21.3 mm, and 21.6 mm after second exposures at gantry angles of 30°, 45°, 60°, and 90° in the ball phantom, respectively , at gantry angles of 30°, 45°, 60°, and 90°, are , , , and , respectively.Parameters equation ). The ri t = 0.2 . n and tn, T, and t . After the second exposures, the parameters  were , , , and  at gantry angles of 30°, 45°, 60°, and 90°, respectively.The measured curves of the left-side dose region can also be estimated by adjusting the T, and t . Parametn and t with the smallest SSE which gives the best LSRE model fitting of the measured data at the gantry angle of 45°. The best fit at other gantry angles can be done in the same manner.Figures eμϖ− with the x is a straight line  (for (fored¯ quations .(5)p2−2sn and t were shown in Figures n and t can be found in Figures The regression curves for slopes versus x is the distance from the beam edge, namely, center of penumbra, and A + B = 0.5.We developed the LSRE model for studying the penumbra perturbation incurred by field overlapping in IMRT. This model has several advantages for penumbra profile fitting. Several functions, such as cosine  and expoβ are constants. The weak point of this type of penumbra function is the discrete nature in describing the penumbra curve. On the contrary, the LSRE is based on an inverse square root function [x axis (A, B, α, and function , 13 and  [x axis .n and t can be derived easily for the measured and predicted penumbra at any gantry angles once given the gantry angle and then the slope and eventually n and t. For example, if we replace x with the slope from y = 283467x − 3.1361, then we get the y value as n (x with the slope of a known gantry angle from y = 63.098x2 + 8.9481x + 0.2101, then we get the y value as t (n and t in the LSRE model can then be used to predict the dose profile with precision for any gantry angles at coplanar irradiation. This method enables radiation physicists and treatment planning system programmers to obtain penumbra dose perturbation data by using a mathematical process.Using our model, lue as n ; if we rlue as t . The parPenumbra region dose data are important in radiotherapy planning systems for monitor unit calculation especially in linear accelerator-based radiosurgery. The precise position of the MLC and the penumbra shape are two important factors for treatment monitor unit calculation. The MLC-related dose penumbra in the treatment planning system must be calibrated to avoid underdosage or overdosage.2 split field calculated in For example, the penumbra expansion perturbed by overlapping fields in 4 × 2 cmMany lung cancer patients who undergo radiation therapy are treated with higher-energy photons such as 10 MV or higher to obtain a deeper penetration and better dose uniformity. However, lower energy such as 6 MV photon beams should be preferred over higher energies photons because of the significant loss of lateral dose equilibrium for high-energy beams in the low-density medium. Any gains in radial dose uniformity across steep density gradients for higher-energy beams must be weighed carefully against the lateral beam degradation due to penumbra widening. The LSRE model can be applied to predict the penumbra perturbation of both high- and low-energy photon beams in lesions surrounded by low-density organ such as lung.Our study shows how to perform an accurate calculation of the penumbra perturbation caused by an overlapping field at different gantry angles by using a mathematical model in linear accelerator-based radiosurgery. The LSRE model is the first continuous equation which describes the dose profile across the entire penumbra. The clinical significance of this study is that the treatment monitor units can be overestimated or underestimated during linear accelerator-based radiosurgery if the penumbra dose profile is not correctly calculated in the treatment planning system. The LSRE model offers a mathematical approach to correct the penumbra and makes it unnecessary to do tedious physics experiments.


Bacterial ventriculitis is a common complication in children with temporary external ventricular drains (EVD) and the diagnosis is challenging. The present study compared the diagnostic accuracy of novel cerebrospinal fluid (CSF) marker - CD64 expression on neutrophils measured as neutrophil CD64 index (CD64in) to routine laboratory CSF and blood markers for bacterial ventriculitis in children with EVD.We conducted a prospective, observational study, enrolling children with EVD. CD64in in CSF together with CSF markers  and blood markers , and procalcitonin (PCT)) were studied at the time of suspected bacterial ventriculitis. CD64in was measured by flow cytometry. Diagnostic accuracy determined by the area under the receiver–operating characteristic (ROC) curves (AUC) was defined for each marker.Thirty-three episodes of clinically suspected ventriculitis in twenty-one children were observed during a 26-month period. Episodes were classified into those with microbiologically proven ventriculitis (13 episodes) and into those with microbiologically negative CSF (20 episodes). CD64in and leukocyte count were the only CSF markers that could differentiate between groups with diagnostic accuracy of 0.875 and 0.694, respectively. Among blood markers only CRP and band neutrophils differentiated between groups with diagnostic accuracy of 0.792 and 0.721, respectively.CD64in in CSF is a promising diagnostic marker of bacterial ventriculitis in children with EVD as it has higher diagnostic accuracy than routine blood and CSF markers for diagnosing bacterial ventriculitis at the time of clinical suspicion. External ventricular drains (EVD), which divert cerebrospinal fluid (CSF) externally, are often used in children as temporary emergency tools to control raised intracranial pressure secondary to acute hydrocephalus caused by intracranial haemorrhage, neoplasm obstruction of the CSF circulation, or trauma . BacteriDiagnosing bacterial ventriculitis in children with EVD is challenging due to frequent reoperations, blood contamination of CSF, presence of chemical ventriculitis and elevation of blood laboratory markers by concomitant systemic infection , 12. DiaClinical signs can be confounded by the primary neurological insult, critical care treatment , seizures, electrolyte disturbances and non-surgical infection . ChangesSome novel markers of bacterial infection were already studied in CSF with promising results –21. RoutIn an adult population with EVD early high serum PCT is the most reliable indicator of bacterial ventriculitis , 22, 23.An additional CSF marker with high diagnostic accuracy for bacterial ventriculitis would be of great value. CD64 is a high-affinity and restricted isotype-specificity FcγRI receptor on neutrophils. Its expression is substantially up-regulated during infections, induced by inflammatory cytokines interferon-gamma and granulocyte colony-stimulating factor . Until rThe present study compared the diagnostic accuracy of CD64 expression on neutrophils measured as neutrophil CD64 index (CD64in) to that of routine laboratory CSF  and blood markers  for bacterial ventriculitis in children with EVD.This prospective observational study was conducted at Department of Paediatric Surgery and Intensive Care of University Medical Centre Ljubljana, Slovenia during a 26-month period (December 2011–January 2014). Twenty-one consecutive patients with external ventricular drainage with thirty-three episodes of clinically suspected ventriculitis were eligible for enrolment. Clinical suspicion of ventriculitis was based on Centers for Disease Control and Prevention’s National Healthcare Safety Network (CDC/HSN) 2017 definition of meningitis or ventriculitis . InclusiCSF samples for flow cytometry (0.5 ml) were obtained at the time of suspected bacterial ventriculitis, together with samples for routine laboratory tests . CSF EDTA-anticoagulated samples were immediately transported to the flow cytometry laboratory or stored refrigerated (4 °C) during the nights and weekends up to 36 h, according to manufacturer’s reference and our previous study . Expressp < 0.05.Statistical analysis was performed using MedCalc for Windows, version 17.0.4 . Data were presented as the median with interquartile range (IQR). T-test for normally distributed data and Mann-Whitney test for not normally distributed data were used for comparing parametric variables between groups. Chi-square test was used for comparing nonparametric variables between groups. Receiver operating characteristic (ROC) curves for each studied marker were constructed to determine the optimal sensitivity, specificity, positive and negative predictive value, cut-off value and diagnostic accuracy from the area under the ROC curve (AUC). The cut-off values with the greatest sum of sensitivity and specificity were determined by the statistical program. Differences were considered to be statistically significant at a level of p = 0.52, p = 0.37, and p = 0.75; respectively). The patients’ characteristics are summarized in Table Staphylococcus epidermidis 3, Staphylococcus haemolyticus 2, Staphylococcus capitis 1, Streptococcus mitis 1, Streptococcus cristatus 1, and Enterococcus gallinarum 1), and 4 Gram-negative pathogens .There were 13 episodes of microbiologically proven ventriculitis (ventriculitis group) and 20 episodes with microbiologically negative CSF (no-ventriculitis group). There were no statistically significant differences between groups in age, gender, and number of drainage days  in children with internal ventricular shunts . In adulSeveral novel CSF markers of bacterial ventriculitis in children and adults have already been studied. A cytokine interleukin-6 (IL-6) was found to be a reliable marker for prediction of bacterial infection in adult neurosurgical patients with EVD after subarachnoid haemorrhage, when performed on daily basis , 20. In Gram-positive bacteria, mainly coagulase-negative staphylococci, were predominant causative pathogens in our children with ventriculitis. Coagulase-negative staphylococci can be considered as contaminants in adults but not in neonates and infants, in whom they can cause serious infection , 32. TheSeveral limitations of this study merit consideration. Firstly, diagnosis of bacterial ventriculitis was based on results of microbiological cultures of CSF. Although only children with clinical signs of infection were included, it is still possible that some cases of colonisation were recognized as infection. Gram stain is usually done at the time of suspicion of bacterial ventriculitis at our institution, since the result is quickly obtained, but negative result does not exclude bacterial infection . Gram stOur study showed that CD64in has the highest diagnostic accuracy among all routine blood and CSF markers for diagnosing bacterial ventriculitis in children with EVD. Since clinical diagnosis is very difficult in this specific population, a novel CSF marker with high negative predictive value, such as CD64, would be useful when added to routine markers for differentiating children who require antibiotic treatment and taking microbiological culture, from those who do not. Furthermore, since many novel CSF markers have recently been studied with promising results, a combination of these markers, including CD64, would be of great value in this population. With simplification and extension of availability of flow cytometry, CD64in measurement might be routinely done in these children, but because of very challenging diagnosis of ventriculitis in this population, it should be viewed with caution.


In contrast, CSF concentrations of t-tau and S100B were not affected by EM. There was a correlation between total neutrophil count in CSF and Aβ42 . Absolute number of mononuclear cells in the CSF correlated with CSF t-tau . Both correlations remained significant after adjustment for age, blood leukocytes, serum CRP, CSF leukocytes, and CSF protein concentration.Biomarkers classically studied in Alzheimer’s disease have been analyzed in numerous central nervous system infections in adults, but there are scarce data on these biomarkers in children. Enteroviruses appear to be the most common cause of aseptic meningitis throughout the world. The aim of the study was to investigate neuroinflammatory properties of non-polio enteroviruses by measuring CSF concentrations of biomarkers that are involved in neuropathological pathways of neurodegenerative disorders. We measured Aβ42, t-tau, and S100B concentrations in 42 children with enteroviral meningitis (EM) compared to control group without central nervous system infection. We found enteroviral meningitis (EM) to reduce CSF concentration of Aβ42 , 737.6–1559.5 vs. median, 459.4 pg/mL; IQR, 312.0–662.0,  Enteroviruses are ubiquitous, and most individuals encounter at least one of these viruses at some time in their lives. The overwhelming majority of viral meningitis cases of any ages are caused by non-polio enteroviruses. Given non-polio enteroviruses are so common and they are known to exhibit strong neurotropism, it is well worth analyzing brain injury following enteroviral central nervous system (CNS) infection.Biomarkers classically studied in neurodegenerative disorders such as Alzheimer’s disease (AD) are being investigated in CNS infections of bacterial and viral origin , 2. TradIn the current study, we aimed at investigating neuroinflammatory properties of non-polio enteroviruses by measuring CSF concentrations of S100B and classic AD biomarkers: Aβ42, t-tau in children with enteroviral meningitis (EM).The study was conducted on children with meningitis caused by enteroviruses, who were hospitalized in the tertiary teaching hospital in Bialystok, Poland, during a single seasonal outbreak. The control group included children suspected of having a CNS infection, but evaluation of CSF ruled out the diagnosis.CSF samples were centrifuged and stored at − 80 °C for analysis. Biomarker concentrations were measured with ELISA kits. Aβ42 was analyzed using INNOTEST β-AMYLOID(1-42) ELISA kit . Total tau levels were measured with INNOTEST h-tau ELISA kit . Protein S100B was analyzed with Human S100B ELISA kit . Values below the detection limit were estimated as the detection limit divided by two.U test. Correlations were calculated using Spearman’s rank correlation coefficient and adjusted using a general multiple regression model. A p value of less than 0.05 was considered statistically significant.Statistical analysis was conducted using Statistica version 12 (TIBCO Software Inc.). Data are presented as median and interquartile range (IQR). Differences between the groups were analyzed using the Mann–Whitney n = 40) or stool samples (n = 2). Viral culture followed by sequencing revealed echovirus 30 to be a single cause of the outbreak. The baseline characteristics of the study groups and main results are presented in Table Forty-two children with EM were included in this study. The diagnosis of EM was confirmed by the presence of increased cell count in the CSF (> 5 cells/μL) and the detection of enterovirus RNA in CSF (42 increased in EM (Table Concentration of Aβ42 in CSF was significantly reduced in EM patients. Interestingly, CSF concentrations of t-tau and S100B were not affected by the infection (Fig. We did not find any significant correlations between the biomarkers and serum CRP, blood leukocytes, CSF leukocytes, or CSF protein concentration. However, CSF t-tau moderately correlated with Aβ42 in the EM group and controls (Fig. The absolute number of neutrophils in CSF strongly and negatively correlated with CSF Aβ42 in EM Fig. a. There In this study, CSF Aβ42 concentration was decreased in EM. Similar observations were made in bacterial meningitis and HSV-1 encephalitis –11. The Tau concentration in the CSF is a marker of brain parenchymal damage. Increased CSF tau levels are observed in brain hemorrhage, traumatic brain injury, severe bacterial meningitis, and HSV-1 encephalitis , 14, 15.We hypothesize that the correlations between CSF Aβ42, t-tau, neutrophils, and mononuclear cells may reflect the natural course of inflammatory response in EM. Neutrophils can predominate early in the course of aseptic meningitis . As the Interestingly, the t-tau/Aβ42 ratio was higher in the EM group. The ratio over 0.52 was suggested to be a robust marker of an AD profile in the CSF . Here, wSeveral limitations of this study must be acknowledged. This is a cross-sectional study, so correlations we found do not necessary mean causation. This is, however, the first study to analyze CSF Aβ42, t-tau, and S100B concentrations in children with aseptic meningitis. The infection was caused by a single etiological agent making the study group homogenous, aiding in data interpretation.Our data suggest that there may be an important relation between cerebrospinal fluid leukocytes, Aβ42, and t-tau. Previous studies have already acknowledged that CNS infections have an impact on biomarkers of neurodegeneration in CSF, but failed to give insights on underlying processes. Here, we report that neutrophils may play a role in amyloid metabolism, and that mononuclear cells are linked to t-tau concentrations in children with enteroviral meningitis.


Troponin is a key regulatory protein in muscle contraction, consisting of three subunits troponin C (TnC), troponin I (TnI), and troponin T (TnT). Calcium association to TnC initiates contraction by causing a series of dynamic and conformational changes that allow the switch peptide of TnI to bind and subsequently cross bridges to form between the thin and thick filament of the sarcomere. Owing to its pivotal role in contraction regulation, troponin has been the focus of numerous computational studies over the last decade. These studies elegantly supplemented a large volume of experimental work and focused on the structure, dynamics and function of the whole troponin complex, individual subunits, and even on segments of the thin filament. Molecular dynamics, Brownian dynamics, and free energy simulations have been used to elucidate the conformational dynamics and underlying free energy landscape of troponin, calcium, and switch peptide binding, as well as the effect of disease mutations, small molecules and post-translational modifications such as phosphorylation. Frequently, simulations have been used to confirm or explain experimental observations. Computer-aided drug discovery tools have been employed to identify novel potential calcium sensitizing agents binding to the TnC-TnI interface. Finally, Markov modeling has contributed to simulating contraction within the sarcomere on the mesoscale. Here we are reviewing and classifying the existing computational work on troponin and its subunits, outline current gaps in simulations elucidating troponin's role in contraction and suggest potential future developments in the field. Troponin (Tn) is a three-subunit protein complex that resides on the thin actin filament in muscle cells. Its three subunits, troponin C (TnC), troponin I (TnI), and troponin T (TnT) have separate roles in facilitating muscle contraction  simulations and helped elucidate the functional importance of these motions. Molecular dynamics numerically integrates Newton's equations of motion and allows for the simulation of trajectories of biomolecular atoms and molecules  and fast-skeletal troponin C (sTnC) have been simulated using molecular dynamics. The Li group simulated a full troponin complex and demonstrated that the inter-linker region of sTnC was flexible in simulations, in contrast to what the static model suggested  and dilated (DCM) cardiomyopathy. A key use of MD is to study the dynamics of the cTn complex and its subunits in the presence of these mutations. HCM- and DCM-associated mutations that exist on the regulatory domain of cTnC have been in the focus of various conventional MD simulations. Early short simulations were run for 5 ns on the DCM-associated mutation D75Y in calcium-free cNTnC and showed that the D75Y mutation would lead to a reduction in contraction through stabilization of the closed state  on clusters derived from MD simulations were performed on cNTnC. This technique was able to identify a calcium-sensitizing compound, NSC147866, from the NCI II diversity set , challenging simulations to correctly account for those differences. In general, design of additional computational experiments that are verifiable All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Subclinical hypothyroidism (SCH) plays a crucial role in the development and progression of coronary heart disease (CAD). Retinol-binding protein 4 (RBP4) is an adipokine correlated with cardiovascular diseases. Recent studies found that RBP4 levels are increased in patients with SCH. However, the relationship of RBP4 with CAD in SCH patients remains unclear. A total of 199 SCH patients (148 with CAD and 51 without CAD) and 102 healthy controls were enrolled in this study. Serum RBP4 was increased in SCH patients than controls. Moreover, serum RBP4 was higher in SCH patients with CAD. Although there was no significant difference of metabolic parameters between SCH patients with and without CAD, serum RBP4 was positively correlated with body mass index, total cholesterol, and low-density lipoprotein cholesterol, as well as thyroid stimulating hormone. Multivariable logistic regression analyses revealed elevated RBP4 was correlated with increased risk for CAD in SCH patients. Serum RBP4 levels were also increased as the number of stenosed vessels increased. Furthermore, increased RBP4 was positively correlated with the severity of CAD quantified by the Gensini score. Our findings demonstrate that serum RBP4 is associated with the presence and severity of CAD in patients with SCH. Despiteetc. , severaletc. . Epidemietc. . Subclinetc. . SCH is etc. . The preetc. . The Rotetc. , indicatRetinol-binding protein 4 (RBP4) is an approximately 21-kDa secreted protein that transports retinol (vitamin A) in circulation . RBP4 haP < 0.01), but lower FT4 (P = 0.005).The anthropometric and biochemical characteristics are presented in vs. 132.0 nmol/L, P = 0.037) and TSH , but lower T3 levels . Moreover, the RBP4 concentrations were also increased in patients with CAD . No statistically significant differences were found between these two groups regarding age, sex, BMI, smoking, hypertension, diabetes, blood pressure, and the levels of TC, TG, HDL-C, LDL-C, FBG, ALT, AST, Cr, UA, FT3, FT4, and T4.SCH patients were divided into 148 patients with CAD (mean age 65.0 ± 11.0 years) and 51 patients without CAD (mean age 62.7 ± 10.7 years). When compared to those without CAD, patietns with CAD had higher ApoA , LDL-C , and BMI . Serum RBP4 were also positively related to BMI  in patients without CAD. We then analyzed the association of serum RBP4 with thyroid function. As shown in r = 0.257, P < 0.001), but negatively correlated with T3 . The correlation of RBP4 with TSH  and T3  remained significant even after adjustments for potential confounders including age, sex, smoking, hypertension, diabetes, BMI, and the levels of Cr, FBG, TC, TG, HDL-C, and LDL-C.We next evaluated the association of RBP4 with clinical parameters in SCH patients . In CAD P < 0.0001). Univariate and multivariate logistic regression demonstrated that elevated RBP4 concentration was associated with the presence of CAD even after adjustment for the above mentioned potential confounders (P < 0.001) (P = 0.001).As shown in < 0.001) . SimilarP = 0.015). Linear regression analysis revealed that RBP4 concentrations were also increased as the stenosed vessels increased   even after adjustment for the aforementioned confounders. Moreover, serum RBP4 levels were positively correlated with the Gensini score , which is also an indicator for the severity of CAD  . The trey of CAD .vs. 54.35 μg/mL, P = 0.621). After exclusion of patients with diabetes, patients with elevated RBP4 concentrations were correlated with higher risk of CAD even after adjustment for the aforementioned confounders . Similar results were obtained by using serum RBP4 as a continuous variable . In addition, there was also no significant difference of serum RBP4 between men and women . Elevated serum RBP4 levels were correlated with higher risks of CAD both in male and female patients.Stratified analyses were further conducted according to diabetes and sex status . We firsOur present study demonstrated that serum RBP4, a well-known adipokine with adverse effects on cardiovascular system, was not only correlated with thyroid dysfunction, but also strongly associated with CAD in patients with SCH. Among 199 patients with SCH in our study, subjects with elevated serum RBP4 levels were correlated with nearly 1.7-fold increase in the risk of CAD. Furthermore, we also found that RBP4 concentration was positively correlated with the severity of CAD.et al. [in vivo and in vitro studies are needed to delineate the effects and mechanisms of RBP4 in thyroid dysfunction.RBP4 has been shown to correlate with insulin resistance, and its circulating level elevates in diabetes, obesity, and metabolic disorders . Thyroidet al.  reportedet al. . Therefoet al. . In factet al. . FurtherOur results demonstrated that RBP4 levels were positively correlated with TC, LDL-C, and BMI, which are all risk factors for CAD. Increasing numbers of clinical studies have shown that increased circulating RBP4 are not only correlated with established cardiovascular risk factors such as obesity and dyslipidemia, but also correlated with the prevalence of atherosclerotic diseases and CAD . We demoMoreover, RBP4 could also promote the abnormal proliferation and migration of vascular smooth muscle cells, which is important for the formation of coronary atherosclerosis . A recenThe strength of the study is that the CAD patients and non-CAD patients were identified by coronary angiography. Several limitations of the present study should also be considered. Firstly, selection bias might underpower our results due to the cross-sectional design. Secondly, as our study was performed in Chinese Han population, our findings need to be confirmed in other regions and ethnicities. Thirdly, the relatively small sample size may cause a potential low power of our results.The study population was composed of patients with SCH and healthy control subjects. A total of 199 patients with SCH were recruited from inpatients admitted to the First Affiliated Hospital of Nanjing Medical University. The patients were received coronary angiography because of angina pectoris or other symptoms or signs of cardiovascular disease. The control subjects were selected during the same period in the same hospital from the health examination centre. All subjects included in this study had no history of significant concomitant diseases, including hyperthyroidism, thyroidectomy, previous anti-thyroid therapy, severe hepatic or renal diseases, bleeding disorders, previous thoracic irradiation therapy, autoimmune disease, and malignant diseases. SCH was defined as elevated TSH levels with normal total or free T4 levels. Hypertension was defined as resting systolic blood pressure (SBP) above 140 mmHg and/or diastolic blood pressure (DBP) above 90 mmHg or in the presence of active treatment with antihypertensive agents. Diabetes mellitus was defined as fasting blood glucose (FBG) >7.0 mmol/L or a diagnosis with diet adjustment or anti-diabetic drug therapy. Dyslipidemia was defined according to guideline of the National Cholesterol Education Program (Adult Treatment Panel III). Written informed consent was obtained from each participant and this study was approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University.Two cardiologists who were unaware of the patients included in this study assessed the angiograms. CAD was defined as luminal diameter narrowing estimated visually at least 50% in any epicardial coronary artery, including the left main coronary artery, left anterior descending, left circumflex, or right coronary artery. CAD patients were divided into single-, double-, and triple-vessel disease subgroups according to the number of significantly stenosed vessels. The severity of CAD was assessed with the Gensini score system based on the degree of luminal narrowing and its geographic importance . The GenVenous blood sample was collected and separated into serum and cellular fractions within 2 h by centrifugation at 3000 g for 10 min. The supernatant (serum) was collected and further centrifuged at 10000 g for 15 min to completely remove the cell debris. The obtained serum was stored at -80 °C before further analysis. Total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and Apolipoprotein A (ApoA) levels were measured enzymatically on a chemistry analyzer . Glucose levels were measured by a glucose oxidase method . TSH, free triiodothyronine (FT3), Triiodothyronine (T3), free thyroxine(FT4), T4 were measured by an electrochemiluminescence method .Serum RBP4 levels were assayed in duplicate by using an a sandwich enzyme-linked immunosorbent assay (ELISA) kit  according to the manufacturer's protocol. The intra- and inter-assay coefficients of variance were 2.32% and 2.95%, respectively. The analytic sensitivity of the assays was 0.021 ng/mL.t test. Skewed data, including body mass index (BMI), SBP, DBP, TC, TG, ApoA, FBG, alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine (Cr), TSH, FT4 and Gensini score were expressed as median and quartile ranges, and comparisons were analyzed by the Mann-Whitney U test. Pearson 2χ test was used to compare qualitative variables represented as frequencies. The correlations between serum RBP4 level and other variables were calculated using Spearman correlation coefficient and partial correlation coefficient adjusted for age, sex, smoking, alcohol intake, BMI, Cr, FBG, TC, TG, and LDL-C, as appropriate. Univariate analysis and multivariate logistic regression analysis were taken to determine the variables that independently contributed to the presence of CAD. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. Linear regression analysis was used to test for trend of the changes of serum RBP4 concentrations across the severity of coronary angiography . Receiver operating characteristic (ROC) curve analysis was used to determine the optimum cut-off level of RBP4 best predicting CAD. All tests were two-sided and P < 0.05 was considered statistically significant. Statistical analyses were performed using PASW 18.0 .Normality of distribution was assessed using the Kolmogorov-Smirnov test. Data for age, LDL-C, HDL-C, uric acid (UA), FT3, T3, T4 and RBP4 were normally distributed parameters and presented as the mean ± standard deviation, and comparisons were analyzed by Student’s In summary, our findings support the point that serum RBP4 is associated with the presence and severity of CAD in SCH patients. Further prospective and experimental studies are needed to delineate whether increased RBP4 may participate in the development of coronary atherosclerosis in hypothyroidism.Written informed consent was obtained from each participant and this study was approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University.