Adults with disabilities often have worse health outcomes than do their peers without disabilities. While education is a key determinant of health, there is little research available on the health disparities across education levels among adults with disabilities in developing countries. We therefore examined the association between health outcomes and education among adults with disabilities in Shanghai, China.We used the health examination records of 42,715 adults with disabilities in Shanghai in 2014. Five health outcomes, including two diseases (fatty liver and hemorrhoids) and three risk factors (overweight [body mass index ≥ 24]), high blood glucose, and high blood lipid), were evaluated. Descriptive statistics and Pearson’s chi-square test were used to assess differences in participants’ demographic and disability characteristics. Pearson’s chi-square test and Fisher’s exact test were conducted to compare the prevalence of each health outcome among the different education levels. Finally, logistic regression analyses were conducted to explore the association between education and health outcomes after adjusting for sociodemographic characteristics.p < 0.001); however, there were no significant disparities in hemorrhoids across the education levels among adults with intellectual disabilities.People with an elementary school or lower degree had the highest prevalence of overweight (52.1%) and high blood glucose (20.8%), but the lowest prevalence of hemorrhoids (18.6%) and fatty liver (38.9%). We observed significant differences in the association between education and health outcomes across disability types. For example, in physically disabled adults, higher education was related to higher odds of hemorrhoids (Compared with people without disabilities, adults with disabilities in Shanghai have relatively poor health. The association between education and health outcomes differed according to the health condition and disability type. To reduce the prevalence rate of overweight and high blood glucose among people with disabilities, tailored health promotion initiatives must be developed for people with lower education levels. In contrast, specific attention should be paid to the prevention of hemorrhoids and fatty liver among more-educated people with disabilities. Our study provides important evidence for targeting educational groups with specific disability types for health promotion and intervention. The World Report on Disability has revealed that more than one billion people worldwide—about 15% of the total global population—live with some kind of disability . The numIndividuals with disabilities tend to have poorer health, achieve lower education levels, have fewer economic opportunities, and have a higher likelihood of poverty than do individuals without disabilities. For example, individuals with mental disabilities have a risk of preventable death four to six times that of the general population . MoreoveEducation is a significant determinant of health . A positChina has more than 1.3 billion people—about one-fifth of the world’s population. According to the Sixth China National Census and the Second China National Sample Survey on Disability, around 85.02 million Chinese people live with some form of disability . The impShanghai is the most developed city in China; it has a population of around 24.15 million and approximately 423,520 disabled people . The ShaThis study aims to examine health disparities in China by comparing health outcomes of adults with disabilities versus those without disabilities and outcomes among disabled adults with different education levels. Our primary hypothesis was that more highly educated adults with disabilities would have better health outcomes. These findings can help assess the significance of these disparities and target interventions toward disabled adults across specific educational levels.n = 2, 896). The disability types were classified as: visual, hearing, speech, physical, intellectual, mental, and multiple disabilities. However, in this study we combined the hearing and speech disability populations, as in the studies by In this study, we selected all health data from SHDPRCIP on individuals aged 25 or older who received health examinations in 2014. If people underwent two or more examinations, we only used the most recent data. We limited the analyses to participants 25 years of age or older because education level is more stable thereafter . All recThe annual health checkup included physical examination, diagnostic imaging, and laboratory tests. The physical examination consisted of basic measurements  and organ checkups . The diagnostic imaging involved chest X-ray, electrocardiogram (ECG), and abdominal ultrasound scan . Finally, the laboratory tests involved routine blood tests, blood biochemical tests, routine urine tests, and immunological tests. During the checkup, a medical practitioner in each department initially gathered preliminary results. Subsequently, all data obtained from the checkup, including the laboratory test results, were integrated and the principal doctor provided the final report to the person with disability.The institutional review board (IRB) of the Fudan University School of Public Health (IRB #2015-08-0563) approved this study, and prior approval was obtained from the SHDPF about the use of the data in SHDPRCIP.Two diseases (fatty liver and hemorrhoids) and three risk factors  were selected as dependent variables to reflect the health outcomes of adults with disabilities.The two diseases were classified using the Tenth Revision of the International Classification of Diseases (ICD-10), with the code K76.0 for fatty liver and I84 for hemorrhoids . Hemorrh2  of ≥24 kg/m2 . High bl2 . Using t2 , we defiThe authors defined education level as elementary school education or below, or graduated from middle school, high school, college, or higher. The classification was based on China’s educational system and in accordance with the International Standard Classification of Education .Demographic characteristics were regarded as a covariate in this study, including gender , age , residence permit , and marital status . Disability severity was also included as a covariate in this study, using the four-level classification system in China: level 1 indicates severe disability, level 2 moderately severe disability, level 3 moderate disability, and level 4 mild disability .SPSS Statistics 22.0 was used to analyze all data. The percentage of each education level was calculated for all demographic, disability type, and disability severity groups. Descriptive statistics and Pearson’s chi-squared test were conducted to determine differences in demographic and disability characteristics according to education level. We also compared the distributions of education level between all people without disability in Shanghai, people with disability in Shanghai, and the sample of people with disability in this study to ensure the representativeness of the sample. The data for the first two groups were from the China Statistical Yearbook 2015  and the p-value <0.05 was regarded as statistically significant.Logistic regression analysis was used to explore differences in health outcomes according to education level after adjusting for covariates. The correlation coefficient of the independent variable and covariates was <0.5, indicating no significant multicollinearity. Individuals with an elementary school education or below were considered as the reference group; odds ratios (ORs) and 95% confidence intervals (CIs) were estimated for the other three education groups. n = 2,896), we were left with an analytical sample of 42,715 individuals with disabilities.In 2014, 46,108 disabled individuals received health examinations. Of these, we selected the 45,611 individuals who were 25 years of age or older. After excluding those with missing data (p <0.001). Most participants (72.7%) were aged 50–69 years old, and their average age was 56.7 ±10.5 years. The majority of participants had an urban residence permit (79.1%). However, among participants with an elementary school education or below, only 60.9% had an urban residence permit. Almost all (98.4%) of the disabled adults with a college or higher degree had an urban residence permit. About half of the participants (50.2%) were physically disabled, followed by people with visual disabilities (23.8%). In terms of the severity of the disability, most participants were evaluated as level 4 or level 3, which accounted for 50.4% and 28.8% of the study sample, respectively. As for marital status, the majority of participants (81.6%) were married; only 11.8% of participants were unmarried; and, 6.6% were divorced or widowed.As for disability type, people with visual disability had the highest prevalence of hemorrhoids (33.1%), fatty liver (42.2%), and high blood lipids (61.6%). Participants with mental disability had the highest prevalence of overweight (59.5%) and high blood glucose (22.2%), but the lowest prevalence of fatty liver (39.3%). Regarding disability severity, participants with severe disability (level 1) had the highest prevalence of fatty liver (41.7%) and high blood lipids (59.9%). People with moderately severe disability (level 2) had the highest prevalence of overweight (50.3%) and high blood glucose (21.1%), but the lowest prevalence of fatty liver (39.5%) and high blood lipids (57.9%). People with mild disability (level 4) had the highest prevalence of hemorrhoids (28.5%).p < .01; visual aOR = 0.71, p < .001; physical aOR = 0.90, p < .01). Among people with visual and physical disabilities, those with a college education or higher had significantly lower odds of having high blood glucose  than did those with an elementary school education or below.p < .001; high school aOR = 1.22, p < .001; college or higher aOR = 1.27, p < .05). Interestingly, education level was not significantly related to any health outcomes in adults with intellectual or multiple disabilities.In contrast, compared with the other educational levels, people with an elementary school education or below had significantly lower odds of hemorrhoids for hearing/speaking, visual, physical, and mental disabilities. Among people with visual disabilities, the odds of having fatty liver rose with education level  and information . Despite these limitations, this is one of the first studies in China to examine education as a determinant of health among individuals with disabilities. While acknowledging the complex relation between education and health, future research should leverage our study to design effective interventions and prevention methods to improve the health outcomes of people with disability in China and other developing countries.There may be a complex relationship between education and health, especially among adults with disabilities in China. The association between education and health outcomes differed according to the health condition and disability type. Compared with people without disabilities, adults with disabilities in Shanghai have relatively poor health. To reduce the prevalence rate of overweight and high blood glucose among people with disabilities, tailored health promotion initiatives must be developed for people with lower education levels. In contrast, specific attention should be paid to the prevention of hemorrhoids and fatty liver among more-educated people with disabilities. Recognition of the complexity of this association should be embedded in future prospective research and intervention programs to address health and education disparities in people with disabilities in China.10.7717/peerj.6382/supp-1Data S1Click here for additional data file.10.7717/peerj.6382/supp-2Supplemental Information 1Click here for additional data file.10.7717/peerj.6382/supp-3Table S1χ2 test was conducted to compare the demographic and disability characteristics of participants by different education levels. a, p = 1.14896317009354E–75 b, p = 0.000 c, p = 0.000 d, p = 0.000 e, p = 5.40799275230419E–22 f, p = 1.09432772270004E–90.Note: Level 1 indicates the most severe disabilities. Levels 2 and 3 indicate moderately severe to moderate disabilities. Level 4 represents mild disabilities. ∗Click here for additional data file.10.7717/peerj.6382/supp-4Table S2χ2 test and Fisher’s exact test were conducted to compare the prevalence of each health outcome in different education levels. a, p = 8.08620222642061E–11 b, p = 4.12037312310817E–92 c, p = 0.000107210522360043 d, p = 0.000144998989757823 e, p = 1.62261094666859E–08 f, p = 3.12302572411109E–06 g, p = 2.94192672999227E–10 h, p = 5.51669620111951E–11 i, p = 0.000111660991424272 j, p = 2.04694838804187E–42 k, p = 0.0000127694131019277 l, p = 3.46362411394606E–11 m, p = 0.000185619627816675 n, p = 7.79213264752363E–26 o, p = 2.40217947128789E–06 p, p = 2.26450131270884E–57 q, p = 1.02180388285092E–06.∗Click here for additional data file.10.7717/peerj.6382/supp-5Table S31Adjusted for gender, age, hukou, marital status, disability type, and disability severity. 2Adjusted for gender, age, hukou, marital status and disability severity. OR, odds ratio; CI, confidence interval a, p = 1.82610338670946E–09 b, p = 3.59421532803415E–65 c, p = 5.21541397814597E–12 d, p = 5.34560735401499E–73 e, p = 0.000485652226983674 f, p = 0.0000228939475230585 g, p = 1.46498546264525E–38 h, p = 3.36865066099832E–07 i, p = 5.65373183403489E–07 j, p = 2.05001733219406E–08 k, p = 0.0000069875277559385 l, p = 2.0130374120598E–08 m, p = 5.73649683295901E–16 n, p = 9.50661598792444E–08 o, p = 9.00383872834067E–14 p, p = 0.000111084046409749 q, p = 3.22739499284614E–08 r, p = 1.23882486591996E–44 s, p = 0.0000848185284451649 t, p = 2.57456171857619E–56 u, p = 2.12628072004128E–33 v, p = 0.0000379636759741926 w, p = 9.13903589573368E–07.Click here for additional data file.


L* and b*‐color co‐ordinates) than tarhana or kishk. Compared with SCpCSB, FMBW had numerically higher levels of protein, lactose, and lactic acid, lower levels of Ca, Fe, Zn, and Mg, and lower pH. Tarhana had highest mean levels of starch, and on reconstitution (133 g/kg) had highest water holding capacity, viscosity during pasting and cooling, yield stress (σ0), consistency coefficient (K), and viscosity on shearing from 20 to 120 s−1 at 60°C. Reconstituted FMBW, kishk, and SCpCSB had similar pasting and flow behavior properties. Overall, the composition , pasting and flow behavior characteristics of FMBW were closer to those SCpCSB and kishk than to tarhana. The results suggest that the FMBW powder, on appropriate supplementation with Ca, Fe, Zn and Mg, could be used for the development of customized fortified blended foods for specific groups.Dehydrated blends of milk and cereal are reconstituted and consumed as a nutritious soup or porridge in many regions; the composition and reconstitution behavior of the blends are likely to impact on nutritional quality and consumer acceptability of the soup/porridge. Experimental samples of dried fermented milk‐bulgur wheat blend (FMBW) and commercial samples of dried dairy‐cereal blends, namely kishk, tarhana, and super cereal plus corn–soy blend (SCpCSB) were compared for composition, color, water sorption, and reconstitution characteristics. FMBW blends had higher contents of protein, Ca, lactose and lactic acid, lower levels of salt (NaCl) and Fe, and a lighter, more‐yellow color (higher  Tarhana is produced using a similar approach to kishk but substitutes wheat bulgur with wheat flour, and additionally includes condiments such as seasonings, vegetables, and/or lentils  from a blend of fermented milk and wheat and with a gross composition and reconstitution behavior similar to SCpCSB, using the formulation and processing technology that has been traditionally applied in the manufacture of kishk and tarhana. To approach this objective, available commercial samples of SCpCSB, kishk and tarhana were analyzed for composition and consistency and the resultant data were used as a guide in formulation of the FMBW.22.1Five commercial brands of tarhana powder and three of kishk powder were procured from Syria, Greece, Turkey, and Lebanon. Super cereal plus (corn–soy blend) was kindly supplied by Concern Worldwide.® 207 (Streptococcus thermophilus) and YC380 (Lactobacillus delbrueckii subsp. bulgaricus) were obtained from Chr. Hansen Ireland Ltd.Bulgur wheat  was obtained from a local retail store (Tesco Sores Ltd.). Buttermilk powder  was obtained from Glanbia Ingredients Plc. Low heat skim milk powder  was manufactured using a pilot‐scale NIRO Tall‐Form Dryer in Moorepark Technology Limited (Teagasc), as described previously , inoculated with direct‐vat starter cultures, incubated at 42°C until the pH dropped to 4.6, cooled to 15°C, and stored at 4°C overnight. Fermented milk (FM) and bulgur wheat (1 mm particle size) were blended at a weight ratio of 80:20 for 5 min , incubated at 35°C for 24 hr  to form a dough which was manually rolled into thin layers (0.5–1.0 cm) and dried for 48 hr at 46°C  to a moisture content of 60–70 g/kg. The resultant dried cake was manually broken into pieces and milled  to a powder with a particle size of 1 mm, vacuum‐packed in polythene liners (Henkelman Polar 80 Floor Standing Vacuum Packer) and stored at 15°C.Fermented milk‐bulgur wheat blends were prepared, as described in detail by Shevade et al. . BrieflyThe FMBW was produced on three separate occasions, using a separate batch of freshly prepared FM on each occasion.2.32.3.1aw) was measured at room temperature .Powders were analyzed in triplicate, as described by O'Callaghan et al.  for prot2.3.2L*, a*, and b* values, were measured on powdered samples using a CR‐400 Chroma Meter (Konica Minolta), which had been calibrated using the Minolta calibration plate. The L* value is indicative of lightness, varying from 0 (black) to 100 (white), whereas a*‐ and b*‐ values represent the variation in color from green  to red , and of blue  to yellow , respectively. A sample of powder (~20 g) was uniformly distributed in a Petri dish, and color measurements were made at three different locations along the sample surface.The color space co‐ordinates, namely the 2.3.3Water sorption and desorption behavior, as a function of relative humidity (RH) in the range 5%–85%, were measured gravimetrically using an SPS11 automatic multi‐sample moisture sorption analyzer , as described previously  were reconstituted in distilled water, cooked , cooled to 21°C while stirring at 120 rpm, and centrifuged  at 12,500 2.4.2® RT 10 Magnetic Stirrer, IKA‐Werke GmbH) at room temperature, loaded in the calorimeter, and scanned on heating from 20 to 95°C at 5°C/min. An empty pan was used as a reference. The temperatures at gelatinization onset (To), peak (Tp), and end (Te) were obtained for each sample endotherm from the system software .Gelatinization characteristics were analyzed in triplicate using differential scanning calorimetry  as described by Shevade et al. . Triplic2.4.3−1, heated to 95°C over 10 min, held at 95°C for 25 min, and cooled to 30°C in 10 min while constantly shearing (160 s−1). The viscosity was measured dynamically and the following parameters were calculated from the resultant viscosity/time curves: peak viscosity (Vp); viscosity after heating to 95°C (V95), holding at 95°C (Vh) and cooling to 30°C (Vc); breakdown viscosity (BRV = Vp − Vh) which denotes the viscosity decrease during holding; and setback viscosity (SBV = Vc − Vh) corresponding to the viscosity increase during cooling.Reconstituted powder (133 g/kg) was pasted in a controlled stress rheometer , fitted with a starch pasting cell, comprising a measuring cup and stirrer, as described previously  was cooked, as described for WHC, and cooled to 46°C in 5 min while constantly stirring (120 rpm). A sample of the cooled soup (120 g) was loaded into the chamber of a Bostwick Consistometer (CR Instruments Limited), which was preheated to 46°C. The extent flow (mm) in 30 s on release of the sample from the chamber was recorded  sweep, from 18 to 120 s−1 over 20 min. The changes in shear stress  and viscosity  were recorded. The resultant σ versus γ˙ data were fitted to the Herschel–Bulkley model using TA data analysis software :σ0, K, and n represent yield stress (Pa), consistency coefficient (Pa.s), and flow behavior index (n), respectively , cooked, and cooled to 60°C while stirring at 120 rpm. Rheology was measured in a subsample (11 g) of the cooled product as described previously  were included as an observation.The data were analyzed using a randomized complete block design which incorporated the three different powder types and replicate samples of each powder type . Analysis of variance (ANOVA) was carried out using the general linear model (GLM) procedure of SAS 9.3 to determine the effect of powder type on each response variable SAS, . Tukey's33.13.1.1The levels of fat, protein, and starch in the kishk and tarhana powders were within the range reported previously for experimental tarhana  had significantly higher mean levels of protein, lactose, lactic acid and Ca, and lower mean contents of salt and Fe, than tarhana or kishk powders. Compared with tarhana, kishk had higher mean levels of protein, fat, lactic acid, salt, ash, Ca, Fe, and Zn, and a lower mean content of starch. Overall, tarhana had lowest mean levels of protein, fat, lactic acid, Ca, Mg, Zn, ash, highest mean starch content, and pH. The data suggest a higher ratio of fermented milk‐to‐wheat in FMBW than in kishk or tarhana  and minerals WFP, .3.1.2L*, a*, b*) of the powders are shown in Table L* (63.5–90.5), a* (−0.7 to 5.4) and b* (15.8–37.2) for all powders were comparable to those previously reported for kishk and tarhana, i.e., 48–89, −0.14 to 28.1, and 1.43–52.88, respectively , and characteristics of starch granules , porosity, and processing conditions , hydration of starch molecules, granule swelling, and an increase in viscosity  during stirring and heating, and hence is likely to impact on the viscosity of the resultant soup/porridge , and its relatively low concentration of lactic acid which correlated negatively with WHC. The results are consistent with those of other studies which reported an increase in the storage modulus, G′, of wheat, maize, and potato starch pastes as starch content was increased in the range 75–125 g/kg  and cooling to 30°C is shown Figure g Figure b. Factorg Figure . In contg Figure a. The dig Figure . This isg Figure a,b. Parbg Figure  and subsg Figure . Increasg Figure . The preeal WFP, , suggesteal WFP, .V95, Vh, Vc, BRV, and SBV than FMBW, kishk, or SCpCSB . All products displayed a yield stress (σ0) and shear thinned , K and η120s-1 than FMBW or kishk, which were similar  when reconstituted (133 g/kg) and cooked as a soup. Tarhana powders had highest mean values of starch and pH, lowest contents of lactic acid and Ca, and highest values of water holding capacity (WHC) and viscosity on reconstitution and cooking. The kishk samples were closer to FMBW and SCpCSB than to tarhana for most of the measured parameters. The results highlight the importance of formulation on the composition of dehydrated fermented milk‐cereal blends and their consistency when reconstituted. Consistency is likely to impact on consumer acceptability and satiety aspects of the reconstituted powders when cooked and, hence, their effectiveness as a nutrient vector.An experimental fortified blended food base product (FMBW) was prepared by blending fermented milk  with parboiled dehulled wheat  at a weight ratio of 4:1 and drying the blend . The composition and reconstitution behavior of the FMBW were compared with those of commercial dehydrated dairy‐cereal blends, namely super cereal plus corn–soy blend (SCpCSB), tarhana and kishk. Compared with SCpCSB, FMBW had numerically higher contents of protein, lactose, lactic acid, and salt, lower levels of fat, Ca, Mg, Fe, and Zn, and similar pasting viscosity profile, and flow behavior properties (yield stress, The authors have no conflict of interest.This study did not involve any human or animal testing. Click here for additional data file. Click here for additional data file.


Twenty-nine adolescent patients with symptomatic lumbar isthmic spondylolisthesis were included. For each patient, two age-matched, gender-matched and BMI-matched controls were enrolled. Comparison analyses detected higher values in the case group for the following parameters: CL , T1 Slope , PT , PI , SS , LL , SVA , L5 Slope , and L5I . Conversely, TS-CL , cSVA , and TLK  were smaller in the case group. Slippage percentage was most correlated with PI , followed by PT  and L5I . Results of binary logistic regression showed that the main risk factor of isthmic spondylolisthesis was PI . Further subgroup analysis also showed that PI was the main risk factor of isthmic spondylolisthesis in the female adolescents  and in the male adolescents . PI was the main risk factor for adolescent symptomatic isthmic spondylolisthesis in the Chinese Han adolescents. The greater PI indicated the higher the progressive risk of spondylolisthesis. In these isthmic spondylolisthesis adolescents, the body always inclined forward and lumbar and cervical lordosis increased.This case-control study aimed to investigate differences in the sagittal spinal parameters between the  Isthmic spondylolysis is defined as a unilateral or bilateral defect in the pars interarticularis, which frequently occurs at L4 or L5 vertebrae2. Based on different populations, the incidence of spondylolysis varied from 4.4% to 39.7%4. Adolescent athletes were more susceptible to isthmic spondylolysis5. Several factors were associated with the susceptibility to spondylolysis, including ethnic heterogeneity6, genetic background7, occupation5, and sagittal balance8. Spondylolisthesis is defined as the forward slippage of vertebrae with respect to the underlying vertebrae, which frequently occurs in patients with bilateral pars defects. This abnormality usually occurred after walking age, and the adaptation both in the sagittal balance of the spine and lumbar-pelvic-femoral complex was associated with spondylolisthesis. Furthermore, it was assumed that the acquisition of bipedalism coupled with vertical stance was the prerequisite to spondylolisthesis8.Adolescents suffering from lumbar isthmic spondylolisthesis or spondylolysis always complain of low back pain9. Sagittal spine alignment was associated with Health-related quality of life (HRQoL)10, especially in elderly individuals12. Based on 492 consecutive Caucasian patients, aged on average 51.9 ± 16.8 years, Schwab et al.12 observed that threshold values for severe disability  were comprised of , , and . It is well-known that sagittal spinal alignment changes with age, and pelvic-spine parameters vary in different populations. For example, a relatively smaller pelvic incidence (PI) was observed in the Chinese Han population than in Caucasian populations14.In adults, several studies have investigated the sagittal alignment in different populations16. Insufficient information was reported regarding the normal sagittal alignment range in adolescents, especially concerning the association of abnormal sagittal alignment with lumbar diseases such as lumbar isthmic spondylolisthesis. Therefore, this study aimed to detect the difference of sagittal alignment between adolescent symptomatic lumbar isthmic spondylolisthesis patients and general adolescents in the Chinese Han population.In adolescents where no aforementioned clinical evidence was observed, previous radiographic studies proposed that the average values of PT, PI, sacral slope(SS), and other sagittal parameters should be slightly smaller when compared with normal adults2 and 22.34 ± 1.45 kg/m2, respectively. Table There were 13 male and 16 female adolescents in the case group. The control group comprised of 26 males and 32 females. The averaged slip percentage was 36.97 ± 8.95%. No difference was observed in terms of age (P = 0.780), height (P = 0.332), weight (P = 0.256), BMI (P = 0.798), and gender distribution (P = 0.589). The average age in the case and control groups was 14.03 ± 1.50 years and 14.14 ± 1.68 years, respectively. The average height was 155.10 ± 11.95 cm and 157.57 ± 10.69 cm, the average weight was 53.38 ± 7.55 kg and 55.33 ± 7.36 kg, and the average BMI was 22.15 ± 1.52 kg/mComparison analyses detected higher values in the case group for the following parameters cervical lordosis , T1 Slope , PT , PI , SS , LL , SVA , L5 Slope , and L5 Incidence . Conversely, T1 Slope minus Cervical Lordosis , C2-C7 plumbline , and thoracolumbar kyphosis  were smaller in the case group. There was no difference in C1-C2 cervical lordosis , or thoracic kyphosis . Table For the male group, comparison analyses detected higher values in the case group for the following parameters including CL , PT , PI , SS , L5 Slope , and L5I . Conversely, TS-CL  and TLK  were smaller in the case group. While there was no difference in C1-C2 (P = 0.890), T1 Slope (P = 0.397), cSVA (P = 0.216), LL (P = 0.749), and SVA (P = 0.215) , T1 Slope , PT , PI , LL , TK , SVA , L5 Slope , and L5I . Conversely, cSVA  was smaller in the case group. While there was no difference in C1-C2 (P = 0.240), and TLK (P = 0.754) , followed by PT  and L5I . In the male group, slippage percentage was correlated with SVA . However, no significant correlation was observed in the female group  with the following parameters excluded   with the following parameters excluded   with the following parameters excluded  Table .Table 6BTo the best of our knowledge, no report has been focused on the sagittal spinal alignment in isthmic adolescent lumbar spondylolisthesis patients in the Chinese Han population. Comparison analyses detected higher values in our case group for the following parameters: CL (P < 0.001); T1 Slope (P = 0.001); PT (P < 0.001); PI (P < 0.001); SS (P < 0.001); LL (P = 0.042); SVA (P = 0.009); L5 Slope (P < 0.001); and L5I (P < 0.001). Correlation analysis detected that slippage percentage was most correlated with PI , followed by PT  and L5I . Binary logistic regression showed that the main risk factor of isthmic spondylolisthesis was PI .13. In adults, Yin et al.17 reported that elevated PI and small sacral table angle (STA) played vital roles in lumbar spondylolysis in the Chinese Han population. In our study, the increased PI was also observed in adolescents with lumbar isthmic spondylolisthesis . Previous studies reported that the PI in the Chinese Han population ranged from 40 to 50 degrees18, which was smaller than that found in Caucasian15 as well as in Korean populations19. Our study also detected a similar PI (43.60 ± 9.669°) in normal adolescents. In addition, this study also observed higher PT  and SS  in the case group, which was in accordance with the previous findings in adult spondylolisthesis patients17. In both the female and male group, the greater values of PI, PT, and SS also presented in lumbar isthmic spondylolisthesis patients.It is commonly accepted that the sagittal spine misalignment plays a vital role in the mechanisms of several spine disorders. For example, it has been reported that young lumbar disc herniation patients demonstrated smaller LL, TK, and SS in the Chinese Han population17. There was a significant correlation between PI and other sagittal parameters, including SS, PT and LL. PI increase was accompanied by an increase in both SS and PT, with a greater increase in SS than in PT. For those individuals of higher PI, larger LL always occurred to guarantee C7 plumb line behind the femoral head and to maintain the posture balance20. However, the larger LL and SS could exert relatively higher forward shear force on an isthmic of L5, which, coupled with the defect of pars interarticularis, would lead to lumbar spondylolisthesis. Moreover, the increased shear force also led to a larger L5 slope in the control group. Further subgroup analysis also detected that PI was the main risk factor of isthmic spondylolisthesis, both in female adolescents  and in male adolescents .Moreover, logistic regression analysis indicated that the main risk factor of isthmic spondylolisthesis was PI , which was similar to findings in previous reports21. LL is essential to maintain an upright posture in human being. Reports have shown that the lumbar lordosis angle increased until 14 or 16 years of age, and that the increased disc wedging angle resulted in the increase of LL22. Therefore, the relatively young average age might explain the smaller LL than previously reported LL values18. Similarly, the small TK and TLK values were also observed when compared with the corresponding values in adults.As for the LL, increased LL was observed in the case groups . Further subgroup analysis also detected higher LL in the female isthmic spondylolisthesis . Similarly, a greater degree of LL was also observed in isthmic spondylolisthesis patients (−55° ± 6°) than in degenerative spondylolisthesis patients (−43° ± 13°), as well as in the controls (−48° ± 12°) in Koreans (P = 0.004)et al.23 introduced the L5I. It was inferred that L5I was significantly associated with isthmic spondylolisthesis. Based on 138 healthy volunteers, Zhu et al.24 reported mean values of L5I in adolescents (17.63 ± 8.65°) and adults (16.43 ± 7.64°). They also reported that there was a positive correlation between L5I and PI (r = 0.818), and established a linear formula to evaluate an ideal L5I based on PI (L5I = 0.725PI-12.757). Thus, the larger the PI, the larger also the L5I. Therefore, the greater L5I  in the case group might result from the greater PI . Additionally, the further subgroup analysis also demonstrated higher L5I values both in the male and female isthmic spondylolisthesis patients.Initially, Roussouly versus 13.99 ± 6.537°, P = 0.001), and a greater CL . However, there was no difference in C1-C2 (P = 0.301). Smaller cSVA  and smaller TS-CL  were demonstrated in the case group. Recently, an increased number of studies was focused on cervical sagittal alignment. Hiyama et al.25 proposed that cervical sagittal alignment could be affected by thoracic deformity. Similarly, Hwang et al.26 demonstrated that there was a significant association between TK and cervical sagittal alignment. Moreover, another study suggested that the cervical sagittal alignment was correlated with the global sagittal spine alignment rather than regional thoracic kyphosis27. Given the larger SVA  and T1 slope  in the case group, we also inferred that the increased lordosis in cervical sagittal plane might compensate for the forward inclined trunk. So, the horizontal gaze can be guaranteed. In addition, the further subgroup analysis also demonstrated greater cervical lordosis in both the male and the female isthmic spondylolisthesis patients.This study also compared the cervical sagittal alignment between the two groups. In the case group, we observed a greater T1 Slope  between slip percentage and PI. Another study also reported that slip percentage was correlated with PI 29. Therefore, we inferred that progression of isthmic lumbar spondylolisthesis in adolescents was associated with a greater PI. Positive correlations were also detected in PT , L5I  and cSVA , which presumably results from the compensation for the forward inclined body to maintain sagittal balance. However, slippage percentage was only correlated with SVA  in the male population. In the female population, no significant correlation was observed. It should be noticed that the small sample size might have failed to reflect the correlation when subgroup analysis was performed based on gender difference.As for slippage percentage, correlation analysis detected that it was most correlated with PI , followed by PT  and L5I  , CL , cSVA, TS-CL (T1 Slope minus CL is the difference between T1 Slope and CL) 31, TLK (thoracolumbar kyphosis).Thoracic kyphosis and Lumbar lordosis parameters: TK (thoracic kyphosis)23) , and L5I , and T1 Slope .32. Figure Slip percentage was assessed in those lumbar isthmic spondylolisthesis patientsDemographic details on age, height, weight, BMI, and gender were collected. The radiographic films were measured independently by two researchers. The parameters measured were as follows:t test was employed to assess the difference between groups. Count data distribution was assessed by Chi-square test. Correlation analysis was performed to assess the associations between slip percentage and other parameters in the case group.Statistical analyses were performed using SPSS 19.0 statistics software . Descriptive statistics were listed in the form of mean ± SD (standard deviation). An independent-sample P < 0.05 was considered as the significant level.To identify the main risk factors of adolescent symptomatic isthmic spondylolisthesis, multiple logistic regression models were constructed using sagittal lumbosacral parameter variables and global sagittal alignment parameters that were of significance in univariate analysis. In addition, there are gender differences in several aspects, such as growing speed, and skeletal structure. Subgroup analysis was also performed based on gender differences.The data sets generated during the current study are available from the first author (Jian Zhao) on request.


To investigate the optimal dose of pretreated-dexmedetomidine in fentanyl-induced cough (FIC) suppression.2, ASA I or II, were equally randomized into four groups (n = 45) to receive intravenous pretreatment of dexmedetomidine with 0 (group 1), 0.3 (group 2), 0.6 (group 3) and 0.9 (group 4) mcg/kg over 10 mins, respectively. After the pretreatment, all patients were given a 5-s intravenous injection of fentanyl 4 mcg/kg. The symptoms of irritating cough including the severity and onset time were recorded for 1 min after fentanyl injection. General anesthesia induction was completed with midazolam, propofol and cisatracurium, then endotracheal tube or laryngeal mask was inserted and connected to an anesthesia machine. MAP, HR and SpO2 at the beginning of pretreatment (T0), 3 min (T1), 6 min (T2), 9 min (T3) and 12 min (T4) after the beginning of pretreatment were recorded. Side effects of dexmedetomidine, such as bradycardia, hypertension, hypotension, and respiratory depression were also recorded during the course.Patients of 180 undergoing elective surgery with general anesthesia, aged 18–65 years, BMI 18.5–30 kg/mP > 0.05) and between groups 3 and 4 (P > 0.05). The incidence and severity of cough in groups 3 and 4 were significantly lower than those in groups 1 and 2 (P < 0.05). Compared to T0, HR at T2 (P < 0.05), T3 (P < 0.01), and T4 (P < 0.01) decreased significantly and MAP at T4 decreased significantly (P < 0.05) in group 4. Bradycardia occurred in 1 case and respiratory depression occurred in 1 case in group 4. Compared to group 1, the onset time of cough in the other 3 groups were delayed significantly (P < 0.05).Totally 168 patients completed the study. The incidences of cough were 52.4, 42.9, 11.9, and 14.3% in groups 1, 2, 3, and 4, respectively, with no significant differences between groups 1 and 2 , April 13, 2017.This study was registered in  Fentanyl is used widely for general anesthesia induction due to its rapid onset, intensive analgesia and cardiovascular stability; however, an irritating cough may be caused after its intravenous (IV) administration . The inc2-adrenoceptor agonist effect [2-adrenoceptor agonist, is widely used for its particular virtues, such as favorable sedative and analgesic effects. It can also reduce central sympathetic outflow and stress response [The mechanism of FIC has not been elucidated definitely, although various studies have been conducted to suppress or alleviate this side effect , 9. A prt effect . Dexmederesponse . A previresponse . While aresponse . HoweverClinicalTrials.gov (NCT03126422).This prospective, randomized, double-blind, controlled clinical trial was approved by the Institutional Research Ethics Committee of the Affiliated Hospital of Yangzhou University, Yangzhou, China. All the participants provided written informed consent following principles of the Helsinki Declaration. Also, this study was registered in 2, and scheduled for elective surgeries under general anesthesia between Oct 2017 and May 2018, were enrolled in the study. Exclusion criteria were patients with bradycardia , hypoteThis study was randomly assigned to four groups with 45 patients each depending on the 10-min pretreated dose of dexmedetomidine, using computer-generated random numbers: group 1 (0 mcg/kg), group 2 (0.3 mcg/kg), group 3 (0.6 mcg/kg) and group 4 (0.9 mcg/kg).− 1•h− 1 was transfused. The vertical distance from the drip bottle to the venous access was 80 cm in all the cases in this study. The IV cannula was connected to T-connectors for drugs infusion and injection in the operating theater. All patients were monitored with electrocardiogram, noninvasive blood pressure, and SpO2 during the whole study.No premedication was used in all patients. Venous access was established on the wrist cephalic vein of the nondominant hand with a 20-G intravenous cannula after patients came into the pre-operation room and Ringers’ solution of 8 ml•kg2 fell below 95% or decreased by 5% from initial value throughout the study. At 10 min after the beginning of pretreatment infusion, the pumping rate of dexmedetomidine was continued at 0.5 mcg•kg− 1•h− 1 in all the groups. Meanwhile, fentanyl  4 mcg/kg with the injection time of 5 s was given to all the patients. A stopwatch was used to control the time.Anesthesia induction was standardized and the procedure consisted of the following. Dexmedetomidine  was diluted with normal saline to a concentration of 4 mcg/ml. Patients were given dexmedetomidine by pumping at an intravenous dose of 0, 0.3, 0.6, and 0.9 mcg/kg over 10 mins in groups 1, 2, 3, and 4, respectively. In group 1, normal saline was used and the infusion rate was set at 50 ml/h. All the pretreatments were prepared and implemented by an experienced anesthesiologist who was not involved in data collection. Also, all the priming drugs and the infusion pumps were covered with a piece of sheet. Oxygen supply through facemask was given to all the patients. Assisted ventilation was supplied if SpOAfter fentanyl injection, the symptoms of irritating cough including the severity and onset time (the time from the end of fentanyl injection to the beginning of coughing) of cough were recorded for 1 min. Any occurrence of cough was identified as coughing. According to the number of coughs within 1 min after fentanyl injection , the sev2 were recorded at T0, 3 min (T1), 6 min (T2), 9 min (T3) and 12 min (T4) after the beginning of pretreatment. Side effects of dexmedetomidine, such as bradycardia, hypertension, hypotension, and respiratory depression were recorded during the course. Ephedrine was used if MAP < 60 mmHg or the decrease of MAP > 30% of the basal data. Atropine was used if HR < 50 beats/min or the decrease of HR > 30% of the basal data. The relevant measures taken to deal with the side effects were also recorded. The above recordings were done by another anesthesiologist who was unaware of the grouping criteria.General anesthesia induction was continued following cough cessation or 1 min after fentanyl injection with midazolam 0.05 mg/kg, propofol 1.5–2.5 mg/kg and cisatracurium 0.2 mg/kg to facilitate endotracheal intubation or laryngeal mask insertion. Mechanical ventilation was controlled with tidal volume of 8 ml/kg, at a respiratory rate of 12 breaths/min. The beginning of pretreated-dexmedetomidine use was recorded at 0 min (T0). MAP, HR, and SpOIn our preliminary study, the incidence of FIC was 48%. A power analysis was performed using the incidence of FIC as the primary variable. We hypothesized that certain dose of 10-min dexmedetomidine priming infusion could reduce the incidence of FIC to 15%. To detect this deference with 90% power at a 5% significance level, 40 patients would be necessary in each group. Therefore, we recruited 45 patients for each group to allow missing data.P value of < 0.05 was considered statistically significant.Statistical analysis was performed using Statistical Product for Social Sciences (SPSS) software 19.0 for windows. Data were expressed as mean ± SD, number, proportion, or percentage. Quantitative variables were analyzed using one-way ANOVA with repeated measures between groups. One-way ANOVA and post Hoc Bonferroni multiple comparison test were used to compare differences of vital signs between groups after dexmedetomidine infusion and fentanyl injection. Ordinal data were compared with the Kruskal-Wallis test followed, when indicated, with Dunn’s multiple comparison tests. n = 42) and completed the study  . Compared to groups 1 and 2, the incidence of cough in groups 3 and 4 decreased significantly (P < 0.05). (Table There were 22 (52.4%), 18 (42.9%), 5 (11.9%), and 6 (14.3%) patients had coughs in groups 1, 2, 3, and 4, respectively. No significant differences between groups 1 and 2 and between groups 3 and 4 were found (P > 0.05). The severity of cough decreased significantly in groups 3 and 4 compared to groups 1 and 2 (P < 0.05).The severity of cough in the four groups was shown in Table P < 0.05). However, there were no significant differences about it between groups 2, 3, and 4.The onset time of cough was 11.8 ± 4.5 s, 17.5 ± 6.5 s, 17.4 ± 5.7 s, and 17.2 ± 5.8 s in groups 1, 2, 3, and 4, respectively (Table P > 0.05). In group 4, HR at T2 (P < 0.05), T3 (P < 0.05) and T4 (P < 0.01) decreased significantly compared to T0   The trigger stimulus and bronchial hyper-irritability theory might be a reason. Opioid receptors have been identified in the trachea, bronchi, and alveolar walls. The opioid receptors can be activated by fentanyl and airway smooth muscles can be triggered to constrict. Histamine and neuropeptides may be released by action on the prejunctional μ-opioid receptors after fentanyl injection. Irritating-cough then is produced , 19; (2)lubility .Medications or mechanical measures have been used to relieve FIC , 21–23. 2-adrenergic receptors of dexmedetomidine and the according decreased release of catecholamine. After pretreatment in group 4, HR decreased to below 50 beats/min in 1 patient and was treated with atropine effectively. SpO2 was seen decreasing to below 95% in 1 case and increased to 100% after pressurized auxiliary ventilation during dexmedetomidine infusion also in group 4. The onset time of FIC in group 3 was about 17.4 s later after fentanyl injection in this study. The peak plasma concentration of fentanyl in lung parenchyma could decrease over time because of its high lipid solubility and the absorption of other tissues. A low plasma concentration of fentanyl might not induce FIC. Prolonging the injection time of fentanyl over the time to reach the threshold of its plasma concentration could reduce the incidence and severity of FIC further.In this study, we applied different doses of pretreated-dexmedetomidine intravenously to explore the optimal dose of dexmedetomidine in suppressing FIC. This study demonstrated that intravenous pretreatment of dexmedetomidine 0.6 mcg/kg bolus given over 10 mins could effectively decrease the incidence and severity of FIC. As was seen in group 4 in our study, compared to T0, MAP at T4 decreased significantly and HR at T2, T3, and T4 decreased significantly in group 4. This may be due to the exciting effects of αThere are still some limitations in this study. First, the judgment index of FIC incidence and degree is subjective, for no objective indicators have been found till now. Second, this study focused on clinical manifestation of the inhibitory effect of dexmedetomidine on FIC, while the specific physiological mechanism needs further study.In conclusion, the pretreated intravenous infusion of dexmedetomidine 0.6 mcg/kg bolus given over 10 mins before a 5-s injection of fentanyl 4 mcg/kg can suppress FIC effectively without side effects.


Ambiguous decision-making involves different processes. However, few studies have focused on the evaluation process. In this study, event-related potentials (ERPs) and event-related spectrum perturbation (ERSP) techniques were used to explore the neural dynamics underlying the evaluation process of ambiguous options through an ambiguous choice task. Some important results emerged. We found a preference for lotteries with low ambiguity regardless of reward amount, suggesting that subjects were averse to ambiguity in our paradigm. Our electroencephalography (EEG) results clarified the neural dynamics underlying the evaluation process. In the time domain, lotteries with both a larger reward and lower ambiguity elicited a larger P3. In the time-frequency domain, larger amplitudes of delta activity at 200–400 ms and 500–600 ms post-stimulus were elicited by lotteries with low ambiguity. Moreover, lotteries with a larger reward elicited larger amplitudes of delta activity at 400–600 ms post-stimulus. Our ERPs and ERSP results suggested that individuals in our paradigm evaluated ambiguity and reward separately, and then integrated their evaluation to form subjective values of different lotteries. Decision-making under uncertainty permeates our daily life. According to the precise likelihood of outcome, economists divide two types of uncertain events: risk and ambiguity. For risk, the outcome probability corresponds to a point estimation. For ambiguity, the outcome probability is either unknown  or an inThe focus of previous neuroimaging studies has been on contrasting the neural mechanism related to decision-making under risk and ambiguity. Decision-making under ambiguity elicits greater activity in the amygdala, orbitofrontal cortex, lateral prefrontal cortex, anterior insular cortex, posterior inferior frontal gyrus, and posterior parietal cortex, and less activity in the striatum . One funEvent-related potentials (ERPs) and event-related spectral perturbations (ERSPs) have millisecond-level temporal resolution, which is useful in exploring the evaluation process of ambiguous options. Existing ERP studies have been mainly focused on neural correlates underlying the choice and feedback stage of decision-making under uncertainty  components in the time domain and time-frequency domain can be used to explore the process of ambiguous option evaluation. In the time domain, the relevant component during the evaluation stage is P3 . The P3 A total of 25 healthy volunteers  from Nankai University participated in this study. Sample size was determined by power analysis. All participants were right-handed, native Chinese speakers. The participants had normal or corrected-to-normal vision and no history of psychiatric or neurological disorders. Each participant signed written informed consent forms and received a base payment of 30 Chinese yuan  for participation, plus a bonus of 0–60 CNY based on his/her decision. The study protocol was approved by the Ethics Committee of Nankai University. The procedures were performed in accordance with approved guidelines and the Declaration of Helsinki. Materials and data related to this study will be made available upon request.The participants performed an ambiguous choice task. In each trial, participants were presented with two lotteries sequentially with varying ambiguity levels and varying reward amounts. Participants had to indicate which lottery they preferred. Each lottery appeared on the screen in the form of a “pie” painted partly red and partly green . All pieAt the beginning of the experiment, participants were told that each lottery corresponded with a unique bag. Therefore, we provided four sealed bags associated with four types of lotteries. Before the task, participants were asked to sign their names on the sealed bags. This method was used to ensure the relative numbers of red and green balls could not have been adjusted by experimenters during the task. At the end of the experiment, three trials were randomly selected by computers. Based on the lotteries they chose in these trials, participants then drew a ball from each corresponding bag . In addition to their participation fee, they were paid according to the lotteries and number of balls of the target color.The EEG recording was performed in a small, sound-attenuated, electrically shielded chamber. After the EEG electrodes were attached, the participants sat in a comfortable chair that was approximately 100 cm in front of a 23-inch (58.42 cm) computer monitor. Before the tasks began, all participants read the instructions carefully and were asked to take eight practice trials. The entire experiment comprised 80 test and eight practice trials. Only the test trials were used for EEG analysis. The trials occurred within four blocks of 20 trials. Each block was separated by a break, the duration of which was determined by the participants. All 80 trials were performed within 15–25 min, during which the trials were randomly presented. The E-Prime software was used to control the display of stimuli and acquisition of behavioral data .The EEG data were recorded continuously with a 40-channel NuAmps DC amplifier . According to the International 10-20 System, 32 active Ag/AgCl electrodes were used. The EEG was sampled at 1,000 Hz using a 22-bit A/D converter. The reference and ground electrodes were positioned at AFz, and the impedances of all electrodes were kept below 10 kΩ.Preprocessing of EEG data was performed with the EEGLAB 14.1.1 tool , implemeClean EEG data were analyzed in the time domain. The 1,000-ms epochs were extracted, starting at 200 ms before the presentation of the stimulus. A 200-ms pre-stimulus period was used as baseline, and the accepted epochs were baseline-corrected. The P3 was scored as the mean voltage from 500 to 600 ms post-stimulus at Pz, corresponding to the 100-ms time window surrounding the peak.Time-frequency analysis was performed using the Fieldtrip toolbox  built-inp were corrected using the Greenhouse-Geisser correction when the sphericity assumption was violated. A value of p < 0.05 was considered significant. Significant interaction was analyzed using the simple effect model. Statistics were analyzed using the SPSS 19.0 software.For all analyses, the values of F = 452.859, p < 0.001, partial η2 = 0.950], as a higher subjective value was noted for 20 CNY (mean ± SEM = 30.860 ± 1.392) versus 10 CNY (mean ± SEM = 9.140 ± 1.119). A significant main effect was also found for ambiguity , as a higher subjective value was noted for low ambiguity (mean ± SEM = 27.840 ± 1.808) versus high ambiguity (mean ± SEM = 12.160 ± 1.492). A significant interaction effect was revealed . For high ambiguity, a significant main effect was observed with reward amount , as a higher subjective value was noted for 20 CNY (mean ± SEM = 21.760 ± 1.007) than 10 CNY (mean ± SEM = 2.560 ± 0.663). For low ambiguity, a significant main effect was also observed with reward amount , as a higher subjective value was observed for 20 CNY (mean ± SEM = 39.960 ± 0.040) than 10 CNY (mean ± SEM = 15.720 ± 1.053). For 20 CNY, a significant main effect was observed with ambiguity level , as a higher subjective value was noted for low ambiguity (mean ± SEM = 39.960 ± 0.040) than high ambiguity (mean ± SEM = 21.760 ± 1.007). For 10 CNY, a significant main effect was also observed with ambiguity level , as a higher subjective value was noted for low ambiguity (mean ± SEM = 15.720 ± 1.053) than high ambiguity (mean ± SEM = 2.560 ± 0.663).The subjective value of a lottery was defined as the frequency with which it was selected by the participants. The subjective value was analyzed using two-way repeated measures ANOVA (rmANOVA) with ambiguity levels (high vs. low) and reward amounts (10 vs. 20 CNY) as within-subject factors . A signiF = 1.818, p = 0.184, partial η2 = 0.070] for the various conditions . We found no significant main effects  for reward amount, with a larger amplitude of P3 for 20 CNY (mean ± SEM = 2.730 ± 0.376 μV) than 10 CNY (mean ± SEM = 0.403 ± 0.417 μV). Moreover, a significant main effect was also found  for ambiguity level, with a larger amplitude of P3 for low ambiguity (mean ± SEM = 2.283 ± 0.430 μV) than high ambiguity (mean ± SEM = 0.824 ± 0.373 μV). However, no significant interaction effects were found.A two-way rmANOVA was performed with ambiguity (high vs. low) and reward (10 vs. 20 CNY) as factors . A signiF = 11.822, p = 0.002, partial η2 = 0.330] for ambiguity level, with a larger amplitude for low ambiguity (mean ± SEM = 2.379 ± 0.259 dB) than high ambiguity (mean ± SEM = 1.733 ± 0.218 dB). However, we found no significant main effects for reward or significant interaction effects.A two-way rmANOVA was performed on the delta power from 200 to 300 ms with ambiguity (high vs. low) and reward (10 vs. 20 CNY) as factors . Only onF = 7.646, p = 0.011, partial η2 = 0.242] for ambiguity, with a larger amplitude for low ambiguity (mean ± SEM = 2.444 ± 0.255 dB) than high ambiguity (mean ± SEM = 1.864 ± 0.219 dB).A two-way rmANOVA was performed on the delta power from 300 to 400 ms with ambiguity (high vs. low) and reward (10 vs. 20 CNY) as factors . Only onF = 9.846, p = 0.004, partial η2 = 0.291] for reward amount, with a larger amplitude for 20 CNY (mean ± SEM = 2.252 ± 0.200 dB) than 10 CNY (mean ± SEM = 1.502 ± 0.194 dB).A two-way rmANOVA was performed on the delta power from 400 to 500 ms with ambiguity (high vs. low) and reward (10 vs. 20 CNY) as factors . Only onF = 6.772, p = 0.016, partial η2 = 0.220] for ambiguity level, with a larger amplitude for low ambiguity (mean ± SEM = 2.485 ± 0.212 dB) than high ambiguity (mean ± SEM = 1.854 ± 0.160 dB). Moreover, a significant main effect was found  for reward, with a larger amplitude for 20 CNY (mean ± SEM = 2.658 ± 0.191 dB) than 10 CNY (mean ± SEM = 1.681 ± 0.199 dB). However, no significant interaction effects were found.A two-way rmANOVA was performed on the delta power from 500 to 600 ms with ambiguity (high vs. low) and reward (10 vs. 20 CNY) as factors . A signiAmbiguous decision-making involves different processes, from evaluation to feedback. Previous behavioral studies have suggested that ambiguity aversion occurs because of a lower subjective value with high variance of the mean outcome during the evaluation process. However, this speculation lacks supportive evidence from neural dynamics analyses. In this study, ERP and ERSP techniques were used to explore the neural dynamics underlying the evaluation process of ambiguous options through an ambiguous choice task. Some important results have emerged. We found a preference for lotteries with low ambiguity regardless of reward amount, suggesting that subjects were averse to ambiguity in our paradigm. Our EEG results clarified the neural dynamics underlying the evaluation process. In the time domain, both lotteries with larger rewards and those with low ambiguity elicited a larger P3. In the time-frequency domain, larger amplitudes of delta activity at 200–400 and 500–600 ms post-stimulus were elicited by lotteries with low ambiguity. Moreover, lotteries with larger rewards elicited a larger amplitude of delta activity at 400–600 ms post-stimulus.Our behavioral data showed that most participants disliked lotteries with high ambiguity and small rewards. This finding is consistent with previous studies regarding decision-making under ambiguity . In our In the time domain, we observed an obvious P3 component peaking at approximately 500–600 ms following the presentation of the lotteries on the screen. This component reflects the stimulus categorization process (for reviews, see In the time-frequency domain, delta activity is sensitive to reward evaluation during reward anticipation processing (for review, see In summary, to our knowledge, this study is the first to investigate the neural dynamics underlying the evaluation process of lotteries under ambiguity. Our ERPs and ERSP results suggest that individuals in our paradigm evaluated ambiguity and reward separately. The ambiguity level was evaluated at 200–400 ms and the reward was evaluated at 400–500 ms after the lotteries were presented on the screen. At 500–600 ms after the stimulus, individuals integrated the evaluation of ambiguity and reward to form a subjective value of the different lotteries. These findings shed light on our understanding of the temporal course of processing ambiguous options. Furthermore, our findings provide neural dynamic evidence of the emergence of ambiguity avoidance during the evaluation process. One limitation of this study should be mentioned. Although the evaluation of ambiguity was earlier than that of reward in our task, whether individuals in other tasks evaluate ambiguity first is unclear. Future studies should explore the impacting factor of evaluation sequence during the process of ambiguous decision-making.The datasets generated for this study are available on request to the corresponding author.This study was carried out in accordance with the recommendations of the Ethics Committee of Business of Nankai University committee with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Ethics Committee of Business of Nankai University committee.CZ, JP, PW, and JL designed the experiment. CZ and JP carried out the experiment, analyzed the data, wrote the paper, and contributed equally to this work. CZ, JP, JL, YW, and PW revised the paper.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.


Type 2 diabetes (T2D) is known to be associated with high BMI and waist circumference (WC). These measures do not discriminate well between skeletal muscle (SM) and body fat (BF), which may have opposite influences.We conducted a secondary analysis of population-based data from 58,128 aged 18–85 yrs from Scottish Health Surveys  and Health Surveys for England , excluding pregnant women and insulin-treated diabetes. Logistic regression was used to assess associations of known T2D, and of screened HbA1c > 48 mmol/mol (> 6.5%), with sex-specific quintiles of BMI, WC, and BF% and SM% estimated by validated anthropometric equations, adjusted for age, sex, smoking, ethnicity, country, and survey year.As expected, ORs for having known T2D rose with quintiles of BMI  and WC . Compared to the lowest BF% quintile, OR for having T2D in highest BF% quintile was 11.1 (95% CI = 8.4–14.6). Compared to the highest SM% quintile, OR for having T2D in lowest SM% quintile was 2.0 (1.7–2.4). Of 72 adults with T2D/HbA1c > 6.5% in the lowest quintile of BF%, 27 (37.5%) were in quintile 1 of SM%. Similar patterns of OR were observed for having HbA1c > 6.5% in those without known T2D.Estimated BF% associates strongly with T2D. Low SM% also has a significant association, suggesting a neglected aspect of aetiology within T2D. These two simple measures with biological relevance, available from data collected in most health surveys, may be more useful than the purely statistical terms BMI. It is gsleading . WC is asleading . In mostsleading , 8, but sleading , 10, WC sleading . A largesleading .In principle, increased body fat is likely to promote T2D by impairing insulin sensitivity, and possibly insulin secretion, particularly when there is ectopic fat in the liver, muscle, and pancreas . HoweverAccurate measurement of body composition requires complex methods that are not practically applicable in large surveys. Some indirect field methods such as bioelectrical impedance have been used in large studies, but they gain little or no advantage over anthropometry, when compared against reference methods such as magnetic resonance imaging (MRI) . Most epThe present study explores the associations of T2D and glycated haemoglobin (HbA1c) with BF and SM calculated from equations validated against MRI, in a large database of national health surveys.n = 92,216) and Health Survey for England (HSE) collected in 2003–2006 and 2008–2013 . The surveys followed identical methods. Subjects younger than 18 years or over 85 years were excluded, because they were outside the age range of populations used to derive the equations for estimating BF and SM. Pregnant women and patients with insulin-treated diabetes were also excluded, leaving 58,128  with complete data for anthropometric, BF, SM, and T2D, and 38,349 with HbA1c screened in individuals without a previous diagnosis of T2D  collected in 2003 and 2008–2011 . The trained nurses also measured weight, height, and waist and hip circumferences by calibrated instruments. Participants were asked to wear light clothing and stand straight in a relaxed position, feet 25–30 cm apart. WC was measured midway between the iliac crest and lowest rib, and HC at the largest circumference around the buttocks.Percentage body fat was calculated using validated published equations for men: BF% = 0.567 × WC (cm) + 0.101 × age (years)−31.8 and for women: BF% = 0.439 × WC (cm) + 0.221 × age (years)−9.4 . SM was HbA1c was measured using non-fasting blood samples. Diabetes mellitus, assumed to be T2D as insulin-treated subjects were excluded, was identified first from participants reporting that the diagnosis had been confirmed by a doctor or a nurse, or secondly as newly diagnosed on the basis of having HbA1c > 48 mmol/mol (> 6.5%) without previously diagnosed T2D.P < 0.05.Group differences were assessed by independent t test for continuous variables and by chi-squared test for categorical variables. The associations of T2D or HbA1c > 6.5% (dependent variables) with BMI, WC, BF, and SM (independent variables) were assessed by logistic regression analysis to estimate odds ratios (ORs) and 95% confidence intervals (CIs). Independent variables were categorised into quintiles. Data were adjusted for age, sex, smoking status (categorised into non-smokers and current or ex-smokers), ethnicity (categorised into white Caucasians or others), survey year, and country. Analyses were conducted using SPSS (version 23.0). The null hypothesis was rejected when 2 ± 4.5 v.s. 27.3 kg/m2, ± 5.6), and HC (103.9 cm ± 10.4 v.s. 103.8 cm ± 10.1). Men were taller  and heavier , and had larger WC , SM , and SM% , while women had higher BF than men  and BF% .Men and women had similar mean (± SD) age , BMI . Of 72 adults with T2D/HbA1c > 6.5% in the lowest quintile of BF%, 27 (37.5%) were in quintile 1 of SM%.Tables Very similar BF% and SM% quintile patterns were observed for prevalences and OR of having HbA1c > 6.5% without known T2D Tables .Further analysis was conducted by combining the individuals with known T2D and those with HbA1c > 6.5% without known diabetes, as the dependent variable. The combined prevalence of T2D again rose with higher quintiles of BF%  fat accumulation . The invThere was also evidence of significant associations of T2D and HbA1C > 6.5% with both low SM% and high BF%, but this combination does not always co-exist within individuals. This suggests that these two measures detect different individuals with T2D, which could point towards T2D being promoted by different body-composition phenotypes, potentially demanding different approaches to treatment and prevention.High BF%, particularly intra-abdominal adipose tissue, may have a key intermediary role in the development of insulin resistance and subsequent T2D. It is now recognised that intra-abdominal fat is an active endocrine organ producing a number of adipocytokines such as adiponectin, leptin, resistin, and interleukins, which play a crucial role in appetite and energy regulation . An imbaWe have previously shown that the prevalence of metabolic syndrome was increased in those with either low BMI and high WC, or high BMI and low WC, compared to individuals who had low BMI and low WC . HoweverThe significant association of HbA1c and T2D with low SM% demonstrated here has not been well documented in the literature. In a cross-sectional study of Koreans, SM was 2–4-fold lower in individuals with T2D than those without  and an NThere are certain limitations to the present study, including the cross-sectional design, but that should not diminish the validity of the new analysis for BF% and SM%, particularly as the analyses for BMI and WC are in line with published data. The previous studies have shown slightly variable associations, e.g., stronger associations with WC and incidence of T2D in long term follow-up studies , than inThe use of existing survey data is economical, but has drawbacks, because the number of collected variables is fixed and may limit analyses. For example, we did not have information on the duration of diabetes, but we did attempt to reduce associated errors by excluding insulin-treated patients (likely to have longer duration of diabetes). Measurement errors are always possible. Accuracy is important at individual level to avoid misclassification of subjects in categories of interest such as BMI, but the sizes of errors in measuring height, weight, or waist circumference are not great enough to cause frequent misclassification, which is more severely affected in self-reported than in measured data . When anIn conclusion, estimated BF% associates with T2D. Low SM% is also a significant factor. These two simple measures, available from data collected in most health surveys, may be more useful than using BMI or WC for assessing T2D. These results suggest two distinct body-composition phenotypes driving type-2 diabetes, with implications for prevention and treatment strategies. Further exploration would be valuable, especially confirmation in longitudinal follow-up studies.


To provide a scientific basis for the prevention and treatment of cervical cancer (CC), we investigated the distribution characteristics and genotypes of human papillomavirus (HPV) and the prevalence of multiple HPV infections in women of different ages seeking management for abnormal cytology in Foshan City.Screening for the 21 genotypes of HPV was carried out in 9945 females seeking management of abnormal cervical cytology results using rapid flow-through hybridization of nucleic acid molecules. The overall prevalence, genotype distribution and age-specific prevalence were examined.Our results indicate that the prevalence of overall, high-risk, intermediate-risk, and low-risk HPV infections was 13.5%, 12.1%, 1.3%, and 1.9%, respectively. Of the 1346 women who tested positive, 89.5% were positive for a single HPV genotype, and 10.5% were positive for ≥2 genotypes. The most frequently detected HPV genotype was HPV-16 (2.9%), followed by HPV-52 (2.9%), HPV-58 (1.5%), and HPV-CP8304 (1.0%). The highest infection prevalence was found in patients 21–30 years old .The prevalence of HPV infection in women seeking management for abnormal cytology in Foshan City is highest in the younger population (21–30 years old). Similar to most previous surveys, HPV-58 and HPV-52 infections are as common as HPV-16 infection. Cervical cancer (CC) is the third most common cancer among females around the word , with moHere, we investigate cervical HPV infections among women seeking management of abnormal screening results to determine the age-specific and genotype-specific prevalence of HPV in the Nanhai area of Foshan.From January 2013 to July 2017, 9945 women who visited gynecologists or oncologists at affiliated Nanhai Hospital of Southern Medical University to seek management of abnormal cervical cytology results were enrolled; all patients provided informed consent. Inclusion criteria were as follows: (1) mentally and physically healthy; (2) age over 20 years; (3) permanent residency in the Nanhai area; (4) had participated in sexual intercourse at least once; (5) not pregnant or menstruating at the time of enrolment; (6) no history of total hysterectomy; (7) no history of cervical surgery; and (8) willingness to be tested for HPV genotypes.Physicians used a swab to swipe the region of the uterine cervix to ensure that cervico-vaginal cells were obtained. The collected samples were placed separately into vials containing 3 ml of preservation solution  and stored at 4 °C until they were transported to the laboratory of Nan Hai Hospital affiliated with Southern Medical University, China, within 2 weeks for HPV DNA detection and typing. DNA extraction was conducted according to the MagPure Fast Blood DNA KF Kit instruction manual .HPV detection and genotype analysis were performed using the HPV GenoArray Test Kit for 21 Types ; this chip technology can identify 21 different HPV genotypes, including 13 high-risk subtypes , 2 intermediate-risk subtypes (66 and 53), and 6 low-risk subtypes . In brief, 1 μl of sample DNA was mixed with 23.25 μl of PCR Mix and 0.75 μl of DNA Taq polymerase (5 U/μl), as provided in the kit. Both positive and negative controls supplied in the kit were included in every PCR test. Amplification was performed using an ABI VeritiDx PCR system . The PCR conditions were as follows: initial denaturation at 95 °C for 9 min, followed by 40 cycles of denaturation at 95 °C for 20 s, annealing at 55 °C for 30 s and extension at 72 °C for 30 s, with a final extension at 72 °C for 5 min. Amplicons were stored at 4 °C until denaturation, which was carried out in the thermal cycler at 95 °C for 5 min. The denatured product was applied onto the nylon membrane that contained immobilized HPV probes (HybriMem). The hybridization process was performed according to the manufacturer’s protocol. The assay utilized Hybribio’s proprietary Flow-through Hybridization Technology , which actively directs targeting molecules toward the immobilized probes within the membrane fibers, with the complementary molecules being retained by the formation of duplexes. After a stringent wash, hybridization was detected by the addition of the streptavidin horseradish peroxidase conjugate, which binds to biotinylated PCR products and cleaves a substrate (nitro-blue tetrazolium-5-bromo-4-chloro- 3-indolylphosphate) to generate a blue-purple precipitate at the probe dot. Multiple-type infection was defined as one that was positive for at least 2 subtypes.The detection limit of the assay is 500 copies per reaction.P values < 0.05 were considered significant.All statistical analyses were performed using Excel and SPSS 19.0. Prevalence and 95% confidence intervals (CIs) were computed for any HPV infection, any high-risk type, any low-risk type, single- and multiple-type HPV infection, and individual genotypes. Data were stratified by age . The Pearson Chi-squared test was used to assess the statistical significance of any differences in prevalence across age groups. A total of 9945 individuals  were included in the study. Genotype screening of the study participants indicated that the prevalence of overall, high-risk, intermediate-risk, and low-risk HPV infection was 13.5% %, 10.6 , 1.2%  and 1.7% , respectively. As shown in Fig. Of the 1346 women positive for HPV, 89.5% (1205/1346) were positive for a single HPV type, and 10.5% (141/1346) were positive for ≥2 types. Of those carrying more than one type, 87.9% (124/141) were infected with two genotypes, 9.9% (14/141) with three, and 2.8% (4/141) with four or more. Of the women infected by multiple HPV genotypes, 1 was infected by multiple low-risk (low-low) HPV genotypes, accounting for 0.7% of the patients with multiple HPV genotype infections, 0.1% of the HPV-positive cases, and 0.0% of all cases. In addition, 7 were infected by a combination of multiple high- and intermediate-risk subtypes (high-intermediate) HPV genotypes, accounting for 5.0% of the patients with multiple HPV genotype infections, 0.5% of the HPV-positive cases, and 0.1% of all cases; 122 were infected by multiple high-risk (high-high) HPV genotypes, accounting for 86.5% of the multiple HPV genotype infections, 9.1% of the HPV-positive cases, and 1.2% of all cases.2 = 22.4, p = 0.001 for any HPV; χ2 = 16.2, p = 0.003 for single-type infection; χ2 = 14.3, p = 0.006 for multiple-type infection). The prevalence of HPV 16 and HPV 52 also differed among the age groups. As shown in Fig. The women were divided into five age groups: 21–30 years old, 31–40 years old, 41–50 years old, 51–60 years old, and > 60 years old. Overall, HPV prevalence in women 21–30 years old, 31–40 years old, 41–50 years old, 51–60 years old, and > 60 years old was 16.2% , 12.8%, 12.1% , 15.5%  and 14.8% , respectively. As shown in Fig. Most HPV infections are transient and resolve spontaneously. However, CC generally develops with persistent high-risk HPV infection , 5. ThusFor example, the most frequently detected HPV genotype among women in Zhejiang province  was HPV-Moreover, the distribution of HPV genotypes and their prevalence exhibited age-related differences. The highest overall prevalence of HPV was found in women 21–30 years old , and the lowest HPV prevalence was found in women 41–50 years old . The main reason for these findings may be the natural history of HPV infection and that the probability of acquisition cervical HPV infection is highest in young sexually active individuals and declines with age . The higMoreover, according to similar studies of women seeking management for abnormal cervical cytology results in Shanghai  and ChaoThere are several limitations of the data in the present study. First, the prevalence data from the age groups over 60 are unreliable due to the fewer number of samples than the total number considered from the other age groups. Second, our study specifically recruited women seeking management for abnormal cervical cytology results and was not population-based. Therefore, our results only reflect the prevalence of HPV of women in Foshan City seeking management for abnormal cytology. In addition, we did not collect lifestyle information from every patient and did not investigate a detailed disease report for the participants. In the future, we will study the connection between lifestyle and HPV infection.In summary, our results suggest that the prevalence of HPV infection varies in women seeking management for abnormal cytology of different age groups. The prevalence of HPV infection in women seeking management for abnormal cytology is highest in the younger age group (21–30 years old). Moreover, HPV-16, − 52, − 58 and -CP8304 are the most common HPV subtypes among women seeking management of abnormal screening results in this region.


Bacteriocins produced by lactic acid bacteria (LAB) can be considered as viable alternatives for food safety and quality, once these peptides present antimicrobial activity against foodborne pathogens and spoilage bacteria. Fermented foods, such as artisanal sausages and cured meats, are relevant sources of LAB strains capable of producing novel bacteriocins, with particular interest by the food industry.Lactobacillus curvatus 12, L. curvatus 36 and Weissella viridescens 23) were obtained from calabresa by presenting promising bacteriocinogenic activity, distinct genetic profiles  and wide inhibitory spectrum. Among these strains, L. curvatus 12 presented higher bacteriocin production, reaching 25,000 AU/mL after incubation at 25, 30 and 37 °C and 6, 9 and 12 h. Partially purified bacteriocins from L. curvatus 12 kept their inhibitory activity after elution with isopropanol at 60% (v/v). Bacteriocins produced by this strain were purified by HPLC and sequenced, resulting in four peptides with 3102.79, 2631.40, 1967.06 and 2588.31 Da, without homology to known bacteriocins.Three LAB strains  contains supplementary material, which is available to authorized users. Lactic acid bacteria (LAB) can be considered as a major component of the microbiota of fermented meats, playing an important role to develop specific flavors and textures . DespiteNowadays, several studies have been reporting on isolation of bacteriocinogenic LAB from different sources, in order to investigate their potential as natural biopreservatives in different food products. Thus, the present study aimed to isolate bacteriocinogenic LAB strains from artisanal meats produced in Minas Gerais state, Brazil, and to characterize their produced bacteriocins towards their antimicrobial features and structure.6 colony forming units per gram (CFU/g) for choriço, 6.4 × 106 CFU/g for bacon and calabresa and 5.6 × 107 CFU/g for lombo defumado. A total of 94 LAB isolates was obtained from the tested samples and selected due to the presence of inhibition halo against Listeria monocytogenes 72. After the confirmatory assays, 17 of them presented bacteriocinogenic activity. These isolates were obtained from calabresa, being confirmed as LAB .LAB counts in the examined samples were 5.3 × 10Lactococcus garvieae , Lactobacillus curvatus  and Weissella viridescens .Repetitive element palindromic-Polymerase Chain Reaction (rep-PCR) and Random Amplified Polymorphic DNA (RAPD) allowed the determination of five genetic profiles, leading to the selection of five representative LAB strains. Based on Basic Local Alignment Search Tool (BLAST) analysis, the sequencing of 16S rRNA allowed the identification of . L. curvatus strains presented similar genetic profiles regarding bacteriocin related genes: both presented amplified products for sakTA and sakTB, and L. curvatus 36 also presented positive results for plaW and plaS. W. viridescens strains also presented similar genetic profiles, with positive results for ped, plaW, and sakTA and sakTB; plaS was also recorded in W. viridescens31. L. garvieae 32 presented positive results only for entP and sakTA.Results for amplification of bacteriocin related genes in the tested LAB strains are presented in Table 1L. curvatus 12, L. curvatus 36 and W. viridescens 23) presented a high potential in inhibiting the Gram positive targets usually associated with human infections and usual focus of bacteriocin studies, like L. monocytogenes (from different serotypes), Enterococcus spp. and Staphylococcus; low frequencies of inhibition were observed for LAB (except Enterococcus spp.), and no inhibitory activity was recorded against the tested Gram negative bacteria. L. garvieae 32 and W. viridescens 31 presented inhibitory activity against only one L. monocytogenes strain   and Tween 80, while the CFS of the other strains kept their inhibitory activity at different levels (Table Treatment of the cell free supernatant (CFS) with different enzymes resulted in complete inactivation of the inhibitory activity Table . None of2) Table . pH, tem2) Table . TemperaGrowth and bacteriocin production by the bacteriocinogenic strains cultured in de Man, Rogosa and Sharpe (MRS) broth at 25, 30 and 37 °C are presented in Fig. L. curvatus 12 was the strains that presented less influence of the tested variation on its inhibitory activity, being decreased only at some pH variations , some carbohydrates sources  and absence of manganese sulphate (MnSO4) and Tween 80. The inhibitory activity of W. viridescens 23 was totally inhibited when the strain was cultured at pH 10 and 12, and in absence of MnSO4, and L. curvatus 36 was highly affected by the MRS composition, once only the variations on MgSO4 did not result in a loss or decrease of its inhibitory activity.Table L. curvatus 12 when compared to the other tested LAB strains, L. curvatus 36 and W. viridescens 23.These obtained data Fig. , Table 4L. monocytogenes 72 were assessed in two steps: first, in target cells at stationary phases, and second, in the beginning of their log growth phase (after 3 h). Considering the first approach, CFS of L. curvatus 12 determined complete inhibition of L. monocytogenes 72. CFS from L. curvatus 36 and W. viridescens 23 were able to reduce L. monocytogenes 72 populations to approximately 102 CFU/mL. Based on the second approach, Fig. L. monocytogenes 72.The inhibitory effects of the produced bacteriocins on L. monocytogenes 72 .L. curvatus 12 presented inhibitory activity against L. monocytogenes 72. The extract submitted to a high-performance liquid chromatography (HPLC) using a C4 column also presented inhibitory activity against L. monocytogenes 72 and resulted in one partially isolated peak  and seasoning, stuffed in intestinal casings and subjected to smoking and curing; lombo defumado is usually produced with the pork tenderloin, added with cure salts, and subjected to smoking and curing; and bacon is usually produced with pork belly, added with cure salts and subjected to smoking and mild curing  and from Sigma-Aldrich, instead of glucose), organic nitrogen source , and other chemicals . The MRS variations were assessed individually. Cultures were incubated at 37 °C for 24 h, when the bacteriocin activity was determined against L. monocytogenes 72 by a quantitative assay (as described above).Also, the selected strains were transferred to 10 mL of MRS broth (BD), incubated at 37 °C for 24 h, and their cells were obtained by centrifugation  for 18 h at 37 °C, when the cells were centrifuged , washed two times with sterile 0.85% NaCl (w/v) and resuspended in 10 mL of NaCl 0.85% (w/v). Equal volumes of L. monocytogenes 72 suspensions and CFS (obtained as described above) of the bacteriocinogenic strains were mixed and incubated at 37 °C for 1 h. Then, L. monocytogenes 72 was ten-fold diluted  and pour plated in BHI agar (BD), followed by incubation at 37 °C for 24, when colonies were enumerated and results were expressed as CFU/mL. Target cell suspensions mixed with NaCl 0.85% (w/v) were considered as controls.6 CFU/mL. Then, aliquots of 100 mL of the inoculated BHI were distributed in four flasks and incubated at 37 °C for 12 h. After the initial 3 h of incubation, 20 mL of the CFS of the bacteriocinogenic strains were individually inoculated to the target cultures. A target culture without CFS adding was considered as growth control. In each hour, aliquots of the cultures were obtained and subjected to spectrophotometry at 600 nm  [Also, the target strain was inoculated in BHI (BD) at approximately 101, Bell) .g, 1 h, 4 °C), and the obtained pellet was re-suspended in 30 mL of phosphate buffer . Partial separation of proteins was performed by SepPakC18 hydrophobic column , with a gradient of different concentrations of isopropanol  in PB . All obtained fractions were tested for bacteriocin activity by the spot-on-the-lawn method against L. monocytogenes 72, as described above. Fractions that presented bacteriocinogenic activity were selected and freeze dried. Before use, the dried material was resuspended in ultrapure water .Bacteriocinogenic strains were cultured in 1000 mL of MRS (BD), at 37 °C for 24 h. CFS was obtained as described above. Proteins were precipitated by adding ammonium sulphate to 300 mL of the CFS, in order to obtain 40, 60 and 80% of saturation. The mixtures were stirred for 4 h in orbital shaker at 4 °C, centrifuged , incubated at 37 °C for 24 h and the CFS was obtained as described above. CFS was precipitated by adding ammonium sulphate 40%, incubated at 4 °C for 2 h, centrifuged  and the obtained pellet was re-suspended in MiliQ water (Merck Millipore). The obtained extract was tested for its inhibitory activity against L. monocytogenes 72 using the spot-on-the-lawn assay, as described above. The obtained extract was subjected to a HPLC using a C4 column  and mobile phase A (0.1% Formic acid in water) and B (0.1% Formic acid in acetonitrile). The system was equilibrated with 5% B and the absorbed substances were eluted with a linear gradient from 5 to 80% B. The fractions were collected and dried in SpeedVac (Thermo Fisher) for 1 h, and the pellet was re-suspended in MiliQ water (Merck Millipore) and tested for its inhibitory activity against L. monocytogenes 72 by the spot-on-the-lawn methodology, as described above.Based on previous results, L. monocytogenes 72 were dried. Then, the fractions were solubilized in 400 μL of 0.1% formic acid, and analyzed in a nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS) using the nanoAcquity UPLC system . Briefly, aliquots of 1 μL of sample were injected and the gradient was linearly varied 2–30% B (v/v) in 59 min, 30–85% in 5 min at a flow rate of 0.3 μL/min in nanoAcquity UPLC BEH C18 column . The eluted peptides were automatically injected into a mass spectrometer model MAXIS 3G , acting in online mode with a CaptiveSpray ionization source. Peptide analysis was performed using an appropriate method (IE_GCF_01-02-2017), with the drying gas flow rate of 3 L/min, temperature of ionization source of 150 °C and the transmission voltage of 2 kV.HPLC fractions that presented inhibitory activity against Lactobacillus protein bank, deposited in the Uniprot Consortium using the PEAKS application version 7.0  [http://bioserv.rpbs.univ-paris-diderot.fr).The raw data were converted to a mass list and compared against the  Canada) . The parAdditional file 1:Table S1. PCR primers used for detection of bacteriocin-related genes in lactic acid bacteria isolated from calabresa, a fermented sausage. (DOCX 19 kb)


Psammosilene tunicoides (Caryophyllaceae) is a narrowly distributed and endemic plant species in southwestern China. The overexploitation of natural P. tunicoides has led to the destruction of many populations. Population and genetic studies will provide crucial data for the protection and management of P. tunicoides. In this study, we develop simple sequence repeat markers of P. tunicoides to analyze population diversity.P. tunicoides were isolated with FIASCO. Eleven polymorphic and 10 monomorphic primers were developed. The 11 polymorphic primers were tested in three P. tunicoides populations, yielding two to nine alleles per locus. Levels of observed heterozygosity varied from 0.000 to 1.000, and levels of expected heterozygosity ranged from 0.000 to 0.615. In addition, three of these loci were successfully amplified, and showed polymorphism, in three Silene species.Microsatellite loci of P. tunicoides.These microsatellite markers can be valuable tools to investigate the genetic diversity and population structure of  Psammosilene tunicoides W. C. Wu & C. Y. Wu (Caryophyllaceae), a perennial and monotypic herb endemic to southwestern China, was described more than 500 years ago and is highly valued in traditional Chinese medicine for pain relief, coagulative effects, and promoting blood circulation   database contains no SSR sequences for P. tunicoides based on Sanger sequencing data. In this study, we report the development and characterization of 11 novel polymorphic genomic SSR markers for P. tunicoides. Additionally, we cross‐amplified these loci in three species of the genus Silene L.: S. gracilicaulis C. L. Tang, S. huguettiae Bocquet, and S. gonosperma (Rupr.) Bocquet.As molecular markers, microsatellites  are DNA motifs composed of one to six nucleotides, which have gained considerable importance in plant genetics analysis and breeding due to their many desirable attributes, including codominant inheritance, stability, extensive genome coverage, and amenability to automation. SSRs have been found ubiquitously in genetic diversity research, genome evolution, species conservation, and marker‐assisted selection breeding . Then, using the universal adapter pair  combined with its fragment, the digested product was placed at 37°C. This mixture was amplified by PCR using a reaction program containing an initial denaturation of 94°C for 3 min; followed by 20 cycles of 94°C for 30 s, 55°C for 60 s, and 72°C for 60 s; with a final extension of 72°C for 8 min.Total genomic DNA was extracted from silica gel–dried leaf tissue from seven samples of Appendix  using thAppendix . Approxi15/(AG)15 probes, and hybridization products were enriched using magnetic beads. The collected enriched products were used as a template to conduct a PCR reaction according to the above program. After the amplified product was purified, the purified product was ligated into a pGM‐T vector (Tiangen Biotech) and transformed using Trans1‐T1 Phage Resistant Chemically Competent Cells  to carry out amplification and expression. Positive strains were selected using Luria–Bertani medium containing ampicillin. A total of 359 positive clones were selected and sequenced using the ABI PRISM 3730XL DNA sequencer , including 217 sequences containing an SSR. Out of these 217 sequences, 46 successfully yielded clear bands; the others showed multi‐banding patterns or no amplification.PCR product hybridization was performed with 5′‐biotinylated (AC)P. tunicoides that were collected from three locations in China: Yunnan, Sichuan, and Guizhou , 2.5 μL of 10× PCR buffer, 2.0 μL of MgCl2 (25 mM), 0.5 μL of dNTP mixture (10 mM each), and 17.8 μL ddH2O. The amplification reaction was conducted using the following protocol: initial denaturation at 95°C for 3 min; followed by 30 cycles of 95°C for 30 s, 45–56°C for 1 min , and expected heterozygosity (He) were calculated by GenAlEx 6.5  show amplified bands, with lower Ho and He in these loci ; GenBank accession numbers are provided in Table