It can be difficult to recognise new instances of an unfamiliar face. Recognition errors in this particular situation appear to be viewpoint dependent with error rates increasing with the angular distance between the face views. Studies using front views for comparison have shown that recognising faces rotated in yaw can be difficult and that recognition of faces rotated in pitch is more challenging still. Here we investigate the extent to which viewpoint dependent face recognition depends on the comparison view. Participants were assigned to one of four different comparison view groups: front, ¾ yaw (right), ¾ pitch-up (above) or ¾ pitch-down (below). On each trial, participants matched their particular comparison view to a range of yaw or pitch rotated test views. Results showed that groups with a front or ¾ yaw comparison view had superior overall performance and more successful generalisation to a broader range of both pitch and yaw test views compared to groups with pitch-up or pitch-down comparison views, both of which had a very restricted generalisation range. Regression analyses revealed the importance of image similarity between views for generalisation, with a lesser role for 3D face depth. These findings are consistent with a view interpolation solution to view generalisation of face recognition, with front and ¾ yaw views being most informative. An ability to successfully recognise new instances and views of an unfamiliar face is critical for most of our interactions involving faces, including common tasks such as face matching, face classification and face identification. While theories of familiar face recognition suggest that accurate identification should occur regardless of viewpoint, for example, see , this abMuch of the past research into face recognition has focused on the front or “full-face” view with good reason  axis, which show performance costs in terms of both accuracy/sensitivity and response latency. While these viewpoint dependent costs are known to increase with the angular distance between the probed viewpoints , 4], , , 11], , , they al, , 11], The results of Favelle et al.  and FaveTypically, in the study of viewpoint effects, view is changed between study and test along a single axis . Few studies have examined the problem of face recognition when such rotations can occur about orthogonal axes. Wallraven, Schwaninger, Schumacher and Bülthoff  examinedA canonical view of an object is a view that is most informative or representative of the visual aspects of that object and thus results in superior object naming and recognition compared to other views . In faceIn studies where the learning and test views are identical, a “same-view advantage” has been found for ¾ yaw (compared to front and profile) views  a front face view, (ii) a ¾ view from the right (yaw-right), (iii) a ¾ view from above (pitch-up), and (iv) a ¾ view from below (pitch-down). This will allow us to distinguish between the competing accounts of the three-quarter view advantage. The angular distance between a yaw ¾ view and a front or pitch rotated test view will be the same as that between a pitch ¾ view and a front or yaw rotated test view. Thus, if angular distance is the primary determinant of this three-quarter view advantage  then theIn addition to the angular distance between the different face views, their image similarity , 24], a, a24], aImage similarity may be estimated in a variety of ways–such as by an observer simply rating the similarity of the two images (a high-level subjective measure) or by comparing the number of pixels in the two images that correspond to the face  a) a14]) aSpecifically, to address these aims we used a sequential matching task to measure generalisation of face recognition from a particular comparison view to multiple test views. Comparison (or first) view will be examined as a between-subjects group factor. The comparison view presented on each trial will differ for each group . The test faces will be the same for all groups and consist of different views rotated in yaw from 75° left of 0° through to 75° right of 0° (in 15° increments) and of views rotated in pitch from 75° below 0° through to 75° above 0° .M = 22.4 years, SD = 5.4). Note that one participant did not indicate their age, so N = 79 for the descriptive statistics for age. All had normal or corrected-to-normal vision and none were familiar with the face stimuli used in the experiment.A total of 80 undergraduate psychology students from the University of Wollongong served as participants for this experiment and received course credit for participation . Their ages ranged from 18 to 51 years (d’) and reaction time (RT) measured in milliseconds.The experiment had a mixed design with one factor (comparison view) manipulated between subjects and two within subjects factors (test view axis and test view angle). The between subjects factor of comparison view had four levels . The two within subjects factors were test view axis with four levels  and test view angle with six levels . These two factors were combined factorially. Since 0° was common to all axes, this resulted in 21 different test view combinations. The dependent variables were recognition sensitivity (2 = .2 or larger in the context of a 4 (test axis) x 5 (test angle), within-subjects ANOVA. See We have previously observed large effect sizes for the main effects and interactions of a similar study of test view axis and angle using front comparison views . A powerStimuli were images of 9 Caucasian female faces taken from a database of high-quality digital face images . ). 14]). All images were viewed in the centre of the computer screen against a white background. The visual area subtended by 0° face images was 14.7° x 19.2°. For the yaw rotated viewpoints the height of the face image remained constant, however face width increased as the viewpoint was rotated further away from 0°. Face width remained constant for pitch camera rotations, however face height decreased as the viewpoint was rotated further away from 0° (for both pitch-up and pitch-down conditions). The smallest image for the pitch-up camera condition was at a viewpoint of 75°, which produced a visual angle of 14.7° x 9.2°. The rectangular patterned mask used in the experiment subtended a visual area of 18° x 22° and was composed of various elements taken from the stimuli used in the task.www.tarrlab.org) guided the trial sequence.Full colour images were presented on a 48 cm flat-screen monitor with a resolution of 1024 x 768 pixels. Trials were run on a Macintosh G4 computer and RSVP experimental software . The experiment began with a set of written instructions and a practice session of 14 trials using different faces shown under the same conditions. After the practice trials, participants had the opportunity to ask any further questions before continuing on to the experiment. The experiment consisted of 378 trials . In half of the trials the two faces presented were the same, regardless of viewpoint . In the remainder the two faces were different; the different face was randomly selected from the images of the remaining 8 face models . Trial type was presented in random order. Participants were given 6 self-timed rest periods spaced equally throughout the experiment. The experiment lasted approximately 30 minutes.Each trial began with a fixation cross displayed for 500 ms followed by the presentation of face 1 for 250 ms. Face 1 was always shown at the same view . Following the presentation of Face 1, a mask was presented immediately after for 500 ms to control stimulus exposure. Face 2 (one of 21 views rotated in either pitch or yaw) was then presented for 250 ms, followed by a second presentation of the mask for 500 ms. Following the second mask the screen remained blank for 2 s or until a response was made by the participant (see d’) and RT for correct trials. Participants’ “same/different” responses were converted into hits and false alarm rates, where a hit was a correct “same” response to a test face, and a false alarm was an incorrect “same” response to a “different” test face. Hits and false alarm rates were converted into z-scores and used to calculate d’ using the formula d’ = z(Hits)–z  (see ). The daNext we investigated the possible relationships between matching sensitivity and three potential predictors  via a multiple regression analysis.F = 10.40, p < .001, ηp2 = .29. Post hoc pairwise comparisons showed that overall sensitivity was higher when the comparison stimuli were front or ¾ yaw faces compared to ¾ pitch-up or ¾ pitch-down faces . On average, sensitivity was not significantly different for front face compared to the ¾ yaw comparison stimuli nor was it significantly different for the ¾ pitch-up and ¾ pitch-down comparison stimuli (both p = 1.0).F = 60.92, p < .001, ηp2 = .45in which overall sensitivity was greatest for the yaw rotated test stimuli, with no significant difference in performance for left and right yaw rotated test stimuli . Overall sensitivity for yaw rotated test stimuli was significantly higher than that for either pitch-up or pitch-down rotated test stimuli (both p < .001), and overall sensitivity for the pitch-up test stimuli  was lower than that for the pitch-down test stimuli .The omnibus mixed design ANOVA also revealed performance differences across the test views in line with the pattern of results found in our previous research , 14]. T. T14]. TF = 59.22, p < .001, ηp2 = .44. A linear contrast revealed a significant linear decrease in sensitivity as the test view angle increased from 15° to 75°, F = 149.2, p < .001, ηp2 = .66.Overall performance was viewpoint dependent, shown by a significant main effect of test view angle, F = 20.18, p < .001, ηp2 = .44, between group and test view angle, F = 10.40, p < .001, ηp2 = .29 and between test view axis and test view angle, F = 10.76, p < .001, ηp2 = .12. These interactions were, however, all qualified by a significant three-way interaction, F = 3.88, p < .001, ηp2 = .13. This three-way interaction was examined with a series of four separate fully-within subject ANOVAs.There were significant two-way interactions between group and test view axis, We conducted separate 4  x 5  ANOVAs on the d’ data for each of the comparison view groups  = 12.43, p < .001, ηp2 = .40. Post hoc comparisons show greater overall sensitivity for the yaw, compared to the pitch, rotated test views . On average, sensitivities to the left and right yaw rotated test views were not significantly different . Sensitivities to the pitch-up and pitch-down rotated test views were also not significantly different on average . There was also a significant main effect of test view angle, F = 26.98, p < .001, ηp2 = .59. A significant linear contrast revealed a linear decrease in sensitivity as test view angle increased from 15° to 75°, F = 100.77, p < .001, ηp2 = .84.For this front view comparison group, the 4 x 5 repeated measures ANOVA showed a significant main effect of test view axis on matching sensitivity, F = 3.59, p < .001, ηp2 = .16, which replicated previous research . A. AF = 84.62, p < .001, ηp2 = .82 which showed the same pattern as the omnibus ANOVA . However sensitivity for yaw test views was significantly higher than that for either pitch-up or pitch-down test views (both p < .001). Matching sensitivity for pitch-up test views  was lower than for pitch-down test views . The main effect of test view angle was significant, F = 20.24, p < .001, ηp2 = .52 with a significant linear contrast showing an overall linear decrease in sensitivity as the test view angle increased from 15° to 75°, F = 41.35, p < .001, ηp2 = .69.For this ¾ yaw comparison group there was also a significant main effect of test view axis on matching sensitivity, F = 5.14, p < .001, ηp2 = .21. For this ¾ yaw comparison group, performance was best for both the 45° yaw-left and 45° yaw-right test views . Matching sensitivity for the yaw test views appeared to decline as the angular distance increased from either of these two optimal test views (see p = .65); 2) significant differences between the pitch and yaw test views  and 3) significant differences between the pitch-up and pitch-down test views (p < .001).These main effects were qualified by a significant test view axis by test view angle interaction, F = 36.79, p < .001, ηp2 = .66, this appeared to be markedly different to the effects found for the front and ¾ yaw comparison groups. Overall sensitivity for matching pitch-down , yaw-left  and yaw-right  test views was not significantly different . Overall sensitivity to pitch-up test views  was, however, significantly lower to that for the other axis test views . The main effect of test view angle, F = 11.63, p < .001, ηp2 = .38, with a significant linear contrast showing an overall linear decrease in sensitivity as test view angle increased from 15° to 75°, F = 19.96, p < .001, ηp2 = .51.While there was also a significant main effect of test view axis for the ¾ pitch-down comparison group  = 4.78, p < .001, ηp2 = .20. For this ¾ pitch-down comparison group, the test view angle appeared to have little effect on matching sensitivity for the yaw test views–both the yaw-left and yaw-right functions appeared to be relatively flat. There was clearer evidence of viewpoint dependence in the matching performance for the pitch test views. Specifically, matching performance was best for pitch-down test views from 30° to 60° (mean d’s of 2.1–2.5 were found near the 45° pitch-down comparison view), while performance for pitch-up test views decreased steadily with increasing test view angles—reaching chance  for the largest (75°) pitch-up view angle. The linear contrasts for the four different test view axis conditions were not significantly different to each other .There was also a significant test view axis by test view angle interaction, F = 11.20, p < .001, ηp2 = .37 showed a pattern analogous to the pitch-down group , yaw-left  and yaw-right  tests views . Overall sensitivity for pitch-down test views  was poorer than for the views along the other three axes . There was also a main effect of test view angle, F = 10.72, p < .001, ηp2 = .36, with a significant linear contrast showing an overall linear decrease in sensitivity as angle of test view increased from 15° to 75°, F = 14.24, p = .001, ηp2 = .43.The main effect of test view axis for the ¾ pitch-up comparison group, F = 9.84, p < .001, ηp2 = .34. For this ¾ pitch-up view comparison group, the best matching sensitivity was found for the 45° pitch-up test view (the mean d’ = 3.0 found for this test view here appears to represent a “same-view advantage”), with a steep declines seen in performance as the pitch test view angle either increased or decreased. Again test view angle appeared to have little effect on matching sensitivity along the yaw test view axis–both the yaw-left and yaw-right functions appear relatively flat. Linear contrast analyses showed no significant difference in the linear functions for rotation for yaw-left, yaw-right and pitch down test views . The linear functions for test views along the pitch-up axis were steeper than those for both yaw and pitch-down test views . However, the performance function for test view rotation was not linear and appeared quadratic, with a peak at the ¾ pitch-up view.There was also a significant test view axis by test view angle interaction, F = .53, p = .67, ηp2 = .02, no interaction between group and test view angle, F = .87, p = .58, ηp2 = .03, and no 3-way interaction between group, test view angle and test view axis, F = .99, p = .49, ηp2 = .04.F = .6.22, p < .001, ηp2 = .20 and F = 24.07, p < .001, ηp2 = .24 respectively. Post hoc comparisons showed that in the ¾ yaw and the front view comparison groups, RT was faster for the yaw (compared to the pitch) test views. However, in the ¾ pitch-up comparison group, RT was similar for the pitch-up and yaw test views and these RTs were all faster than those for the pitch-down test views. The complementary pattern was seen in the ¾ pitch-down comparison group, where RT was similar for the ¾ pitch-down and yaw test views and these RTs were all faster than for the pitch-up test views.However, both the main effect of test view axis and the interaction between group and test view axis were found to reach significance, F = 26.51, p < .001, ηp2 = .26 and F = 1.94, p = .04, ηp2 = .03 respectively. In general viewpoint dependent costs to reaction time were greater for the pitch (compared to the yaw) test views  = 66.43, p < .001, ηp2 = .47. Post hoc comparisons showed that RTs for yaw axis test views  were faster than those for the pitch-up and pitch-down test views  .The main effect of test view angle and the interaction between test view angle and test view axis were also found to reach significance, iews see . OverallWe next conducted a multiple regression analysis to investigate the hypothesis that matching performance (d’) would be a function of the following four variables: (i) the angular distance between the test and comparison views, (ii) the difference in image pixels, (iii) their rated image similarity; and (iv) the difference in the perceived 3-D shape of these face views.Angular distance was calculated from the comparison view to the test view through the intersection of the axes  was also obtained from a sample of independent participants. The mean perceived depth differences between the 4 comparison views and 21 test views were then calculated separately for each participant see .Prior to interpreting the results, the data were evaluated for appropriate use in a multiple regression analysis. Inspection of residuals plots indicated that the assumptions of normality, linearity and homoscedasticity of residual were met. Mahalanobis distance and tolerance statistics indicated that multivariate outliers and multicollinearity of predictors were not of concern.R2 = .782, adjusted R2 = .77, F = 70.84, p < .001. A combined effect of this magnitude, f2 = .61 can be considered large . E. E16]. EA second aim of this study was to determine whether any of the comparison views tested had canonical status for generalisation of face recognition across views. Previous studies have shown that front and ¾ yaw views generalise well to other yaw rotations , 39], h, h39], hAssessing the value of intrinsic properties available in various views requires using same-view matching tasks rather than the generalisation  task used here. And while this is an avenue for future research, we can venture some suggestions as to why ¾ yaw and front views allowed for good generalisation of face recognition across a wide range of test views. Ratings of perceived face depth  were larThere are clear implications of this research for security systems and photo identity (ID) documents used in those systems. Successful view generalisation of faces is a task central to security and law enforcement work in which novel views of unfamiliar faces  have to be matched to mug shots or other photo ID information. More often than not the photo ID image is a front view and increasingly it is the case that matching has to occur to pitch views . Unfamiliar face matching is difficult enough under ideal image conditions of well lit, close distance, front views . However, not all viewpoint dependent effects show the same pattern and generalisation of face recognition performance is very much reliant on the comparison face view. The current results show that we can generalise well from ¾ yaw and front views to almost all yaw and more test views from an orthogonal (pitch) axis than either of the ¾ pitch comparison views examined here (which do not provide much more than a same view advantage). Previous findings of poorer face recognition with views rotated up or down in the pitch axis are not specific to front view generalisation or to static image comparisons ; this poS1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.S3 Appendix(DOCX)Click here for additional data file.S1 DatasetData is provided for each participant across each test view. Key to the labels for the test views: “zero” refers to the front view, “pd”, “pu”, “ym” and “y” refer to pitch down, pitch up yaw left and yaw right respectively. The number in each test view label refers to the rotation in degrees away from zero.(XLSX)Click here for additional data file.


In addition, 84.96% and 72.18% of the patients achieved weight loss of ≥5% and ≥10%, respectively. Subgroup analyses showed that weight loss was significantly greater in patients with ≥28 kg m−2 of baseline body mass index and 0.60 mg of beinaglutide doses . HbA1c reductions were significantly greater in patients with ≥9.0% baseline HbA1c and in those administered 0.40–0.48 mg of beinaglutide doses . Weight loss at 3 months was positively correlated with baseline BMI and the dose of beinaglutide. Positive determinants for HbA1c reduction after 3 months were baseline HbA1c and the dose of beinaglutide.A total of 314 patients were included in the study. After 3 months of treatment with beinaglutide, there were significant reductions in body weight , HbAThese observational results confirmed the benefits of beinaglutide in weight loss and glycaemic control and support the use of beinaglutide as an effective treatment for T2DM. China is the country with the largest number of diabetic patients in the world 1c), fasting plasma glucose (FPG) and postprandial plasma glucose (PPG) in patients with T2DM Beinaglutide (formerly known as benaglutide), a GLP‐1RA, is recombinant human glucagon‐like peptide‐1 and shares 100% homology with human GLP‐17‐36)  6. The e1c, body weight, blood pressure, and lipid profiles in a real‐world setting in China. The authors hypothesized that the efficacy of beinaglutide in RCTs will also be observed in the real‐world study.Real‐world studies are very important to further evaluate the performance of new drugs once they reach the market. Such studies can complement RCTs and assess their results in a more general patient population. The real‐world effects of beinaglutide have not been reported to date. Thus, the purpose of this study was to examine the effectiveness of beinaglutide on HbA1c, 2‐h PPG, FPG, body weight, BMI, waist circumference (WC), C‐peptide, heart rate, systolic blood pressure (SBP), diastolic blood pressure (DBP), triglycerides, low‐density lipoprotein cholesterol (LDL‐C), high‐density lipoprotein cholesterol (HDL‐C), anti‐hypertensive and lipid‐lowering therapies and history of previous anti‐diabetic medications. Moreover, the dosage of beinaglutide and concomitant anti‐diabetic therapies was recorded. The values of HbA1c, FPG, 2‐h PPG, C‐peptide, triglycerides, LDL‐C and HDL‐C were extracted from standardized laboratory test results. Body weight, WC, heart rate, SBP and DBP were measured by proficient nurses using standard equipment.This was a multicentre, observational, retrospective, open‐label study conducted in two hospitals in Hebei Province (northern China). The GLP‐1RA beinaglutide was launched in February 2017 in China. Since then, data from T2DM patients treated with beinaglutide under routine clinical practice conditions were consecutively collected until March 2018, i.e. over a time frame of 14 months. Data were extracted from the electronic medical record system and gathered in an Excel datasheet by the researchers. Adult T2DM patients (≥18 years) were eligible for the study. The exclusion criteria were patients with type 1 diabetes mellitus and those who refused to provide informed consent. The following data were collected at baseline and/or three subsequent visits : age, sex, diabetes duration, HbAThe primary study objective was to assess the effectiveness of beinaglutide in controlling glycaemia after 3 months of treatment. Secondary objectives included assessing changes in body weight, the proportions of patients with weight loss ≥ 5% and ≥10% and other clinical parameters related to diabetes after 3 months of treatment. Adverse events, hypoglycaemic events and discontinuation of beinaglutide were also tracked.This study was approved by the local ethical committees and performed in accordance with the Declaration of Helsinki (revised in 2013).1c or weight. Efficacy analysis included all patients who received at least one dose of beinaglutide and had at least one postbaseline measurement for HbA1c or body weight. Missing values were imputed using the last observation carried forward (LOCF) method. For paired samples, the paired t‐test or Wilcoxon matched‐pairs signed‐rank test was used for two time points, and repeated‐measures analysis of variance or the Friedman test followed by Dunn's multiple‐comparisons test was used for multiple time points. The mean (SD), mean difference, standard error (SE) and 95% confidence interval (CI) were calculated for each time point. Subgroup analyses for paired samples were performed with the same statistical methods. The changes in HbA1c and weight were assessed by analysis of covariance, adjusting for baseline measures and the dose of beinaglutide as covariates. A multivariate linear regression model was applied to identify determinants of HbA1c reduction and weight loss. Independent variables included age, sex, diabetes duration, baseline HbA1c, BMI, SBP, triglycerides, LDL‐C, HDL‐C and the dose of beinaglutide. p < 0.05 was considered statistically significant (two tailed). Data were analysed using spss 23 software .The Kolmogorov–Smirnov test was used to determine if values were normally distributed. Data for continuous variables were expressed as the mean (standard deviation [SD]). Data for categorical variables were expressed as percentages (%). Baseline characteristics are reported on the basis of the full analysis set (FAS), which included all patients who met the eligibility criteria and had baseline measures for HbABased on the data without imputation for missing values (i.e. observed data only), a sensitivity analysis was conducted to assess whether the LOCF method for handling missing data might have influenced any critical conclusions.1c and body weight (unknown reasons) and were excluded from the analysis. The remaining 314 patients were included in the FAS %, 2‐h PPG was 14.23 (3.23) mmol L−1 and FPG was 9.25 (1.77) mmol L−1. Baseline characteristics of the FAS population are summarized in Table From January 2017 to March 2018, data from 323 patients treated with beinaglutide were extracted from the electronic medical record system. Of those, nine patients lacked baseline data for HbAAt baseline, 215 patients (68.5%) used beinaglutide monotherapy, 96 (30.6%) used beinaglutide in combination with insulin glargine, two (0.6%) in combination with metformin and one (0.3%) in combination with insulin glargine and metformin. At baseline, 37.6% of the patients were prescribed a daily dose of 0.2 mg, 52.3% received 0.3 mg, 5.5% received 0.4 mg, 0.5% received 0.45 mg and 4.1% received 0.6 mg of beinaglutide. Following a period of dose escalation of 1–2 weeks, prescriptions of 0.2, 0.3, 0.4, 0.42, 0.48 and 0.6 mg of beinaglutide were given to 31.2%, 5.7%, 2.1%, 11.3%, 22.7% and 27.1% of the patients, respectively. In addition, 96.0% of patients carried out supplemental lifestyle interventions.1c, 2‐h PPG, FPG, BMI, WC, heart rate, SBP, DBP, triglycerides and LDL‐C and a significant elevation in HDL‐C . In subgroups, patients receiving beinaglutide monotherapy or beinaglutide in combination with insulin glargine showed similar results (p < 0.001). C‐peptide showed a significant reduction from baseline to 3 months in the total population of patients (p = 0.0016) and the patients who received monotherapy (p < 0.0001), but not in those receiving beinaglutide in combination with insulin glargine (p = 0.4464).Clinical parameters at baseline and the 3‐month follow‐up are presented in Table p < 0.0001). After 3 months, 84.96% and 72.18% of patients attained weight loss of ≥5% and ≥10%, respectively. Subgroup analyses showed similar results. After 3 months, body weight was decreased by −9.98 kg (−8.97 to −10.99) and −12.81% (−11.64 to −13.97) in beinaglutide monotherapy patients and by −10.30 kg (−9.43 to −11.16) and 13.33% (−12.28 to −14.38) when administered in combination with insulin glargine . Furthermore, at 3 months, the proportions of patients with weight loss of ≥5% and ≥10% were 80.75% and 68.45% for monotherapy and 97.37% and 85.53% for the combination therapy with insulin glargine, respectively.Temporal trends in body weight change are shown in Figure 1c, 2‐h PPG and FPG are shown in Figure 1c after 1 month was −0.26% (95% CI −0.15 to −0.38), with a further gradual reduction over time reaching −2.87% (−2.62 to −3.11) after 3 months of treatment (p < 0.0001). The average changes in 2‐h PPG and FPG after 3 months reached −5.46 mmol L−1 (−4.96 to −5.95) and −3.04 mmol L−1 (−2.78 to −3.31) , respectively. The statistical results of the subgroups showed a similar trend. For patients receiving beinaglutide monotherapy, the average change in HbA1c, 2‐h PPG and FPG gradually decreased over 3 months and reached −2.83% (−2.53 to −3.12), −5.44 mmol L−1 (−4.88 to −5.99) and −2.97 mmol L−1 (−2.67 to −3.27)  after 3 months of treatment, respectively. For patients receiving combination therapy with beinaglutide and insulin glargine, the average change in HbA1c, 2‐h PPG and FPG after 3 months reached −2.94% (−2.49 to −3.39), −5.24 mmol L−1 (−4.27 to −6.21) and −3.10 mmol L−1 (−2.55 to −3.64) , respectively.Temporal trends in HbA1c, 2‐h PPG, FPG and body weight are reported in Tables Detailed results  of the temporal trends in HbAp = 0.097), age (p = 0.157), diabetes duration (p = 0.157) or baseline HbA1c categories (p = 0.949). The changes in HbA1c also did not vary significantly by sex (p = 0.471), age (p = 0.249), diabetes duration (p = 0.169) or baseline BMI categories (p = 0.964). However, the changes in body weight were significantly greater in patients with ≥28 kg m−2 of baseline BMI and in patients receiving beinaglutide 0.60 mg ; and the changes in HbA1c were significantly greater in patients with ≥9.0% baseline HbA1c and in patients receiving beinaglutide of 0.40–0.48 mg   and vomiting (18.2%). Other reported adverse events were dizziness (17.2%) and headache (8.3%). These adverse events mainly occurred in the first month after initiation of treatment, and most were mild to moderate. A total of 14 symptomatic hypoglycaemic events were recorded within the 3 months prior to beinaglutide treatment, but no such events were reported after beinaglutide treatment. A total of 26 patients (8.3%) discontinued beinaglutide therapy within 3 months owing to gastrointestinal side effects (5.7%), treatment failure (2.2%) or unaffordable cost (0.3%).Sensitivity analysis performed on the completer's data without LOCF imputation was generally consistent with the conclusions of the aforementioned study results (data not shown).1c after 3 months of treatment. These observations confirmed the effectiveness of beinaglutide in a real‐world setting.The present study is the first multicentre, observational, retrospective, open‐label analysis of beinaglutide effectiveness in T2DM patients, mostly with overweight and obesity, in China. These data demonstrated a mean weight loss of 10.05 kg and a mean reduction of 2.87% in HbAThe patients included in the present study represented a general T2DM population in a real‐world setting in China. Baseline data indicated that beinaglutide therapy was commonly prescribed for patients with overweight and obesity who had a relatively short disease duration. Notably, 27.7% of the patients were newly diagnosed with T2DM, indicating that beinaglutide was generally accepted as a second‐line treatment.−2 is defined as overweight, and a BMI ≥ 28.0 kg m−2 is categorized as obese For the Chinese people, a BMI between 24.0 and 27.9 kg m1c in this study was higher than mean reductions reported in the previous RCTs of beinaglutide, in which mean reductions from baseline between 0.7% and 1.2% have been reported 1c reductions in real‐world groups have usually been smaller than those in RCTs according to Edelman and Polonsky's study 1c levels might predict an early response to GLP‐1RAs in terms of HbA1c reductions 1c reduction was positively correlated with baseline HbA1c levels; that is, i.e. patients with higher baseline HbA1c levels had a greater HbA1c reduction after 3 months of treatment. Compared with previous RCTs of beinaglutide (mean baseline HbA1c between 7.97% and 8.05%) 1c levels (9.02%) in this study may be an important reason for the increased efficacy observed with beinaglutide treatment.The mean reduction in HbA1c reductions were more significant in the relatively high‐dose groups (0.40–0.48 and 0.60 mg), and this finding was consistent with that of previous studies of GLP‐1RAs This discrepancy might also be explained by a change in the formulation of beinaglutide. In previous RCTs, beinaglutide lyophilized powder was used for injection; that is, i.e. the powder had to be dissolved and then subcutaneously injected with a disposable sterile syringe Decreased fasting C‐peptide levels after beinaglutide monotherapy suggested potential efficacy for improving hyperinsulinaemia and β‐cell rest. As a short‐acting meal‐time GLP‐1RA, beinaglutide has a short half‐life (15 min) and duration of action (2 h) The present study also provides new information about the benefits of beinaglutide on other health indicators in a real‐world setting. After 3 months of treatment, significant reductions in heart rate, SBP, DBP, total cholesterol and LDL‐C were observed, whereas HDL‐C increased significantly. These improvements in cardiovascular and lipid profiles were related to beinaglutide treatment and only partially contributed by concomitant anti‐hypertensive or lipid‐lowering treatments. These benefits together with weight loss implied a positive impact of beinaglutide on overall cardiovascular outcomes. However, this comprehensive beneficial efficacy was not unexpected, as indicated by previous studies of GLP‐1RAs The safety characteristics of beinaglutide were also consistent with previous findings of GLP‐1RAs The present study has significant strengths, such as extensive clinical information and minimal exclusion criteria, resulting in more fruitful data gained from a wide range of T2DM patients. As an observational and retrospective study, the lack of randomization and a control group are the main limitations of this study. In addition, the follow‐up period was limited to the first 3 months from the start of beinaglutide. Additional RCTs and real‐world studies are needed to evaluate the effectiveness and safety of long‐term beinaglutide treatment.1c, blood pressure and lipid profiles after 3 months of treatment with beinaglutide. A trend of improvement in hyperinsulinaemia and β‐cell rest also emerged. Such benefits were observed despite a wide range of patient baseline characteristics. These observational results suggest that beinaglutide may be an effective treatment for T2DM, especially for patients with overweight and obesity, in clinical practice.In summary, this study confirmed the effectiveness of beinaglutide on Chinese T2DM patients in a real‐world setting. Significant improvements were observed in body weight, HbAStudy design was performed by Y. Z. and Y. J. Data collection and analysis were conducted by C. Z., X. L., M. Y., L. T. and X. Z. Manuscript writing was carried out by Y. Z. and Y. J. All authors revised and approved the final version of the manuscript.The authors acknowledge Yale Duan, Ning Du and Guiyu Zhao  for statistical and editorial assistance.This study was supported by the Natural Science Foundation of Hebei Province (grant no. H2013209053). The English‐language editing fee and publication charge were funded by Shanghai Benemae Pharmaceutical Corporation.The authors have declared that they have no conflicts of interest associated with this study.Figure S1 Changes in HbA1c and body weight after 3 months of beinaglutide treatment. (A) Changes in HbA1c according to baseline HbA1c category . (B) Changes in HbA1c according to the dose of beinaglutide . (C) Changes in body weight according to baseline BMI category . (D) Changes in body weight according to the dose of beinaglutide . Data are least squares means. The bars show lower limits of 95% CIs.Table S1 Temporal trends of clinical parameters in total patients.Table S2 Temporal trends of clinical parameters in patients receiving beinaglutide monotherapy.Table S3 Temporal trends of clinical parameters in patients receiving beinaglutide combination therapy with insulin glargine.Table S4 The determinants of HbA1c reduction after 3‐month beinaglutide treatment.Table S5 The determinants of weight loss after 3‐month beinaglutide treatment.Click here for additional data file.


Epidemiological evidence suggests sex difference in serum uric acid (SUA) and alanine aminotransferase (ALT) might be a potential explanation for the gender difference in prevalence of non-alcoholic fatty liver disease (NAFLD). However, few epidemiology data in China have tested this hypothesis.We conducted a cross-sectional study to assess the joint associations between SUA and serum ALT with NAFLD among elderly Chinese men and women.P < 0.01). In addition, the ORs of NAFLD among participants with high SUA levels and high serum ALT was 10.75 (95% CI 3.56–32.46) for men and 7.96 (95% CI 2.83–22.39) for women, compared with those with low SUA levels and low serum ALT.Among 7569 participants with a mean age of 59.8 years (± 13.4 years), 56.6% of women and 43.4% of men were diagnosed as NAFLD, respectively. A positive association between SUA and NAFLD prevalence was observed in both men and women. NAFLD prevalence was 2.74 times (95% CI 2.00–3.76) higher for men and 4.60 times (95% CI 3.39–6.24) higher for women with the highest quintiles of SUA levels compared to those with the lowest levels. SUA levels were significantly associated with prevalence of mild- and severe-steatosis (SUA levels were positively associated with NAFLD prevalence, and the association was slightly stronger in women than in men. A significant joint association of SUA and serum ALT with NAFLD prevalence was observed in all participants, which was slightly stronger in men than in women. Non-alcoholic fatty liver disease (NAFLD), a multi-factorial disease, was formed by the interactions of genetics, diet and lifestyle . IncreasAlanine aminotransferase (ALT), a hepatic enzyme, is mostly presented in the liver and commonly used for indicating the presence of liver disease. ALT is a specific marker of liver inflammation and hepatocellular injury . Recent However, only few studies specifically have evaluated the joint associations of serum SUA levels and ALT with risk of NAFLD in China. Thus, we conducted a population-based cross-sectional study, including 7569 elderly men and women, to evaluate the joint associations of SUA levels and serum ALT on NAFLD prevalence.The study was compounded by patients who underwent physical examination at the Third hospital of Xiangcheng during March to December 2015. Participants were excluded if they met the following criteria: (1) taking anti-hypertensive agents, or anti-diabetic, lipid-lowering or hypouricemic agents within 24-h before physical examination; (2) with alcohol consumption greater than 140 g/week for men and 70 g/week for women; (3) with chronic liver disease, such as cirrhosis, liver cancer, viral hepatitis, autoimmune hepatitis, and taking hepatotoxic medicines. A total of 7569 participants, 3126 men and 4443 women, with a mean age of 60 years were finally included in the analyses.Clinical examinations included physical examination, measurement of blood pressure (BP), and anthropometry assays. General medical history was collected by face-to-face interview by trained physicians.Venous blood samples were drawn in the morning after an overnight fasting, and serum samples were separated for the analysis of biochemical assays without frozen. Biochemical variables, including serum ALT, aspartate aminotransferase (AST), triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), SUA, and fasting plasma glucose (FPG), were analyzed in the Lab of the Third Hospital of Xiangcheng using an Olympus AU640 auto analyzer . Standard laboratory assay methods and quality control methods were conducted.NAFLD was diagnosed by abdominal ultrasound according to the conventional criterion . Hepatict tests were performed to compare the differences of continuous variables between men and women, and the categorical variables were compared by using Chi square tests.We calculated means ± standard deviations (SDs) for continuous variables and frequency (percentage) for categorical variables. The independent-samples We stratified the total population by the quintiles of SUA levels: 230 μmol/L, 271 μmol/L, 312 μmol/L, 366 μmol/L, and 366 μmol/L. The quintiles of SUA levels for men was 278 μmol/L, 317 μmol/L, 355 μmol/L, 402 μmol/L and 403 μmol/L, and for women they were 213 μmol/L, 244 μmol/L, 276 μmol/L, 320 μmol/L and 321 μmol/L. The quartiles of ALT levels were 14 U/L, 18 U/L, 24 U/L and 25 U/L; and for men they were 16 U/L, 19 U/L, 27 U/L and 28 U/L, and for women: 14 U/L, 16 U/L, 23 U/L and 24 U/L. Hyperuricemia was defined as SUA level higher than 420 μmol/L in men and higher than 360 μmol/L in women [P value < 0.05 was considered as statistically significant.All statistical analyses were conducted using the SPSS software package version 21.0 . Two-tailed 2 (SD: 2.73) were included, the average SUA levels were significantly higher in men than in women, 343 μmol/L (± 77.6) for men and 270 μmol/L (± 70.9) for women   aged 60 (± 13) years with a mean BMI of 23.93 kg/m.3% aged P-values < 0.01), and especially significantly higher SUA levels . The prevalence of NAFLD was 30.93% among men and 28.40% for women . 14.7% of men had hyperuricemia and 8.2% were also with hyper-ALT levels (serum ALT levels > 40 U/L) . Also, the prevalence of mild- and severe-steatosis was increased from 7.1/9.1% to 15.1/33.1% as the SUA levels elevated from Q1 towards Q5 .Similar characteristics were observed in women. Participants in the highest quintile of SUA (SUA ≥ 321 μmol/L) were older, with high BMI, SBP, and DBP. Serum biochemical parameters, such as TG, TC, LDL-C, AST, ALT, and FPG were positively associated with SUA levels, and HDL-C levels were negatively related to SUA levels . Also, the prevalence of mild- and severe-steatosis elevated from 3.6 to 12.0% and 5.8 to 37.6%, respectively, along with the SUA levels elevated from Q1 to Q5 . The ORs for the Q2 and Q5 compared to Q1 were 2.41 (95% CI 1.97–2.95) and 10.9 (95% CI 8.88–13.4), respectively. While the risk of NAFLD significantly increased by 5 times  for men and 10 times  for women as the SUA levels increased from Q1 to Q5. Further adjustment for BMI, history of cardiovascular related diseases, DBP, serum AST, TG, TC, HDL and FPG, the positive associations and linear trends between uric acid and prevalence of NAFLD were persisted  to 3.48 (95% CI 2.50–4.86) as SUA levels increased from Q2 to Q5 for all participants, with the ORs of mild steatosis significantly increasing to 2.54 (95% CI 1.68–3.84) for men and 4.17 (95% CI 2.67–6.51) for women. Similar associations of SUA levels with severe-steatosis were observed for all participants, along with the increase of SUA levels to Q5, OR increased to 2.95 (95% CI 1.99–4.37) in men, and 4.93 (95% CI 3.41–7.12) in women, but the association was significant only for the Q3–Q5 in men, and the trend was persisted  and 7.99 for women (95% CI 6.49–9.84). All these trends and associations persisted after further adjustment for other confounders (Table Subjects with both high ALT (> 40 U/L) and high SUA  levels had a 27-time higher prevalence of NAFLD than subjects with both low ALT and low SUA levels after adjustment for age and sex among all participants, further adjustment of other confounders did not change the association  suggested the stronger association of SUA with elevated ALT in women than in men, and the similar associations was found among NAFLD patients as the SUA level was higher than 5.6 mg/dL . In the Although there is evidence suggesting the involvement of uric acid in NAFLD’s pathogenesis, the question that remains is whether hyperuricemia is a causal factor in NAFLD or merely a marker for its presence. Evidence also supported uric acid as a powerful antioxidant , which cThis is the first study to investigate the sex-specific joint associations of SUA and ALT with NAFLD prevalence in elderly Chinese. However, the present study has some limitations. First, the diagnosis of NAFLD was conducted by ultrasonographic examination instead of liver biopsy, which might omit mild steatosis. However, ultrasonographic examination is widely used to screen NAFLD in epidemiological research as it is non-invasive, safe, widely available and portable, and the sensitivity and specificity for detecting hepatic steatosis is acceptable; Second, the assessment of NAFLD degree was conducted by ultrasound, which might induce subjectivity and inconsistency. Although ultrasound might not be the golden standard evaluator for NAFLD degree, liver ultrasonography is a noninvasive method which is most commonly used to evaluate the degree of steatosis for NAFLD in the practice . To reduIn our study, we found that there was a significant positive association between SUA and NAFLD in both men and women, and the association was slightly stronger in women. Furthermore, significant joint associations of SUA and ALT with NAFLD prevalence were observed in all participants. Our findings suggested the combination of serum SUA and ALT might have potential clinical values for the earlier prevention and screening of NAFLD. Further well-designed and large-scaled prospective studies are warranted to confirm these findings.


ATP7B gene, encoding a P-type copper transporting ATPase.Wilson disease (OMIM # 277900) is a autosomal recessive disorder characterized by accumulation of copper in liver and brain. The accumulation of copper resulting in oxidative stress and eventually cell death. The disease has an onset in a childhood and result in a significant neurological impairment or require lifelong treatment. Another serious consequence of the disease is the development of liver damage and acute liver failure leading to liver transplant. The disorder is caused by mutations in the ATP7B gene were amplified and sequenced by Sanger method. Sequencing data were analyzed and compared with the ATP7B gene sequence published in Ensembl (ENSG00000123191) by using BioEdit software to detect mutations.We performed genetic analysis of three unrelated patients from three different Vietnamese families. These patients had clinical features such as numbness of hands and feet, vomiting, insomnia, palsy, liver failure and Kayser–Fleischer (K–F) rings and were diagnosed with Wilson disease in the Human Genetics Department, Vietnam National Children’s Hospital. The entire coding region and adjacent splice sites of ATP7B gene were found. Among of these, three mutations were novel: c.750_751insG (p.His251Alafs*19) in exon 2, c.2604delC (p.Pro868Profs*5) in exon 11, and c.3077 T > A (p.Phe1026Tyr) in exon 14. Our results of the mutations associated with Wilson disease might facilitate the development of effective treatment plans.In this study, five mutations in the The online version of this article (10.1186/s12881-018-0619-4) contains supplementary material, which is available to authorized users. Wilson disease OMIM #277900) is characterized by an accumulation of copper in many organs and tissues . Patient77900 is ATP7B gene were identified as the cause of Wilson disease in 1993 and this gene is still the only one associated with the disorder [ATP7B maps on 13q14.3-q21, contains 21 exons and encodes a 1465-amino acids membrane protein [ATP7B gene. Over 776 mutations have been detected in the ATP7B gene until now (The Human Gene Mutation Database. http://www.hgmd.cf.ac.uk/ac/index.php). Wilson disease invariably results in severe disability and death, especially for patients with no apparent clinical manifestations who are untreated. The identification of mutations in the ATP7B gene is the one of the useful diagnostic tools for diagnosis and treatment orientation for patients with Wilson disease.Mutations in the disorder –12. ATP7 protein –13. ATP7 protein . Wilson ATP7B gene of three patients’ families with Wilson disease by sequencing the entire coding region and adjacent splice sites of the ATP7B gene. Information about mutations in the ATP7B gene will be helpful for efficiently diagnosing Wilson disease and providing early therapeutic intervention for patients.In this study, we identified mutations in the He was an 8-year-old boy who was hospitalized with clinical features such as numbness of hands and feet, vomiting, insomnia, palsy and Kayser–Fleischer (K–F) rings. Biochemical indices of the blood serum revealed 0.0032 mg/dL serum ceruloplasmin , 49 μg/dL serum free copper , 20.1 IU/L ALT  , 26.82 IU/L AST (aspartate aminotransferase) , and PT (prothrombin time) of 49% . In addition, biochemical indices of the urine revealed 580 μg/24 h urinary copper . The patient had liver failure and severe neurological symptoms. He was diagnosed with Wilson disease in the Human Genetics Department, Vietnam National Children’s Hospital. His parents and his half brother had a normal phenotype. However, we did not collect a sample from his father for genetic analysis.She was an 8-year-old girl who was hospitalized with clinical features such as oedema, elevated serum transaminase, liver failure, and Kayser–Fleischer rings. Biochemical indices of the blood serum revealed 0.019 mg/dL serum ceruloplasmin , 116.9 IU/L ALT  , 139.1 IU/L AST (aspartate aminotransferase) , and PT (prothrombin time) of 22% . In addition, biochemical indices of the urine revealed 150 μg/24 h urinary copper . She was diagnosed with Wilson disease in the Human Genetics Department, Vietnam National Children’s Hospital. Her parents and her younger sister had a normal phenotype.He was a 10-year-old boy who was hospitalized with clinical features such as oedema, jaundice, acute liver failure, and Kayser–Fleischer rings. Biochemical indices of the blood serum revealed 0.092 mg/dL serum ceruloplasmin , 11.6 μg/dL serum free copper , 55.7 IU/L ALT  , 129.0 IU/L AST (aspartate aminotransferase) , and PT (prothrombin time) of 17% . In addition, biochemical indices of the urine revealed 3639 μg/24 h urinary copper . He was diagnosed with Wilson disease in the Human Genetics Department, Vietnam National Children’s Hospital. His parents and his younger sister had a normal phenotype.Genomic DNA was isolated from peripheral blood samples (including sample from patients and their families) using a Qiagen DNA blood mini kit  following the manufacturer’s guidelines. The DNA concentration was determined using a Thermo Scientific NanoDrop spectrophotometer .ATP7B gene were amplified and analysed by direct sequencing. Oligonucleotide primers were synthesized and purchased from IDT (USA) . DNA was denatured at 95 °C for 12 min followed by 35 cycles of denaturation for 1 min at 95 °C, annealing for 1 min at 60–65 °C, and extension for 1 min at 72 °C, and a final extension for 7 min at 72 °C. PCR amplification was carried out on an ABI 9700 GeneAmp PCR system (USA).All 21 exons and exon-intron boundaries of the ATP7B gene sequence published in Ensembl (ENSG00000123191) by using BioEdit software to detect mutations.DNA sequencing was performed in both directions, initiated from forward and reverse primers, which had been used in an initial PCR reaction. PCR products were purified with Qiagen Purification kit  and sequenced on ABI PRISM 3130 Genetic Analyser machine (USA). Sequencing data were analysed by using SeqScape 2.5 software, Chromas or equivalent software and compared with the The consequence of any novel nonsynonomous nucleotide variations that were identified within exons were evaluated with the in silico analysis tools sorting intolerant from tolerant (SIFT) prediction , polypheATP7B gene were identified in patient 1. Genetic analysis in his mother showed that she had a heterozygous mutation in exon 14 (p.Phe1026Tyr) but did not have the mutation in intron 12. This result was found the in genotype of the patient’s half brother , and Wilson Disease Mutation Database  were used to determine that the p.Phe1026Tyr change was novel. In addition, c.2866-2A > G was a known mutation with code number CS136099 in The Human Gene Mutation Database (http://www.hgmd.cf.ac.uk/ac/index.php) and its gnomAD frequency was 0.012190% (Table A heterozygous mutation in intron 12 (c.2866-2A > G) and a heterozygous mutation in exon 14 (p.Phe1026Tyr) of the Genetic analysis showed that patient 1 had a p.Phe1026Tyr mutation, which was located in exon 14 and the phosphorylation domain (P-domain) of the ATP7B protein. In addition, the patient also carried a mutation in intron 12 (c.2866-2A > G). Patient 1 had the very typically symptoms of the disease such as numbness of hands and feet, vomiting, insomnia, palsy and Kayser–Fleischer rings. Blood and urine biochemical tests manifested that the index of serum ceruloplasmin was very low at 0.0032 mg/dL . This result could be explained by the fact that the patient carried two mutations that were severely affecting the function of protein ATP7B. The mutation p.Phe1026Tyr was located in exon 14 and the phosphorylation domain (P-domain) of the ATP7B protein, which was supposed to have a significant effect on normal protein function. Forbes et al.  has giveATP7B gene were identified in patient 2. In these mutations, c.314C > A (p.Ser105*) (with gnomAD frequency of 0.008128%) was a known mutation and c.750_751insG (p.His251Alafs*19) (with a gnomAD frequency of 0.004730%) was a novel mutation. Genetic analysis of her family exposed that her father had the heterozygous mutation p.Ser105* and her mother had the heterozygous mutation p.His251Alafs*19  of the ATP7B gene in many populations, such as Chinese Indian populations, and mutations in exons 2–5 associated with severe phenotypes have been found in the Indian population [ATP7B mutants and that truncating mutations in the ATP7B gene were associated with an early onset of Wilson disease. Gromadzka et al. [Patient 2 carried two heterozygous mutations, p.Ser105* and p.His251Alafs*19, in exon 2, leading to complete loss of the protein function and early-onset disease. The study by Huong et al.  on Vietnpulation –26. The pulation  revealeda et al.  noticed a et al. , who repATP7B gene. Patient 3 also had a heterozygous mutation p.Ser105*. Genetic analysis has shown that the heterozygous mutation p.Ser105* was inherited from his father and that the heterozygous mutation p.Pro868Profs*5 was inherited from his mother  was detected in exon 11 of the Patient 3 carried two severe truncating mutations, which is similar to patient 2. In addition, both patients (patients 2 and 3) had liver damage with early onset. Kalach et al.  and WilsIn both patients 2 and 3, who had the combination of a p.Ser105* mutation and a p.His251Alafs*19 or p.Pro868Profs*5 mutation, the liver was severely damaged with high expression levels of liver enzymes, such as aminotransferases


The following article highlights the importance of an indispensable process in cellulose fibers (UFC100) modification which may change the biopolymer properties—drying. The reader is provided with a broad range of information considering the drying process consequences on the chemical treatment of the cellulose. This research underlines the importance of UFC100 moisture content reduction considering polymer composites application with the employment of a technique different than thermal treating. Therefore, a new hybrid chemical modification approach is introduced. It consists of two steps: solvent exchange (with ethanol either hexane) and chemical treatment . With the use of Fourier-transform infrared spectroscopy (FT-IR), it has been proven that the employment of different solvents may contribute to the higher yield of the modification process as they cause rearrangements in hydrogen bonds structure, swell the biopolymer and, therefore, affect its molecular packing. Furthermore, according to the thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC), the improvement in fibers thermal resistance was noticed, e.g., shift in the value of 5% temperature mass loss from 240 °C (regular modification) to 306 °C (while solvent employed). Moreover, the research was broadened with cellulose moisture content influence on the modification process—tested fibers were either dried (D) or not dried (ND) before the hybrid chemical treatment. According to the gathered data, D cellulose exhibits elevated thermal resistance and ND fibers are more prone to the MA modification. What should be emphasized, in the case of all carried out UFC100 treatments, is that a decrease in moisture contend was evidenced—from approximately 4% in case of thermal drying to 1.7% for hybrid chemical modification. This is incredibly promising considering the possibility of the treated fibers application in polymer matrix. Nowadays, cellulose fibers are the subject of various research studies ,10,11,12Therefore, cellulose powder requires drying before incorporation into the polymer matrix in order to obtain the best possible mechanical and thermal properties. According to the literature, water evolution upon the conventional thermal treatment might be divided into the three following stages : (i) phy2O molecules are released from the cellulose surface [During the physical loss of water stage, physical desorption of moisture occurs and rather no chemical elimination of water is present before achieving approximately 220 °C . What is surface . It is a surface .By increasing the temperature, a chemical loss of water starts. At this moment, in dehydration reactions the anhydrocellulose is being formed ,21. ThesAround 600 °C and in oxygen-free conditions, the third stage is entered. Then the liquid fraction that consists mainly of water along with small amounts of acetylaldehyde, proprionaldehyde, furan, acetone, butanedione, and methanol is created . At thisMoreover, according to available information, it might be claimed that water content importantly promotes cellulose degradation. The hydrolysis reaction of the glucosidic bonds contributes to the incorporation of unstable acetalic chain-ends and the molecular weight decrease. Furthermore, taking into consideration the fact that water is a major product of the cellulose thermal decomposition, especially at the initial thermal treatment, the regarded process may be attributed to autocatalytic reactions ,28,29,30From the material science point of view, the processes described above are mostly important considering the first and second water loss stages. The first of them concerns the problem of cellulose drying before powder incorporation into the polymer matrix. Then, the chemical loss of water (220–550 °C) step may help to understand the thermal behavior of prepared polymer composites regarding the initial moisture content in cellulose fibers.This research is a follow-up of previously performed projects concerning the employment of cellulose fibers in polymer composites ,32. This® UFC100 Ultrafine Cellulose for Paper and Board Coating from J. Rettenmaier and Soehne  was the type of cellulose used in this research. It is in a powder form  and its density is about 1.3 g/cm3. It is insoluble in water and fats. Nevertheless, this material exhibits a high water binding capacity (even at high temperatures and shearing forces). Its average fiber length is about 8 μm. the pH value varies between 5 and 7.5.The Arbocel® . It is a white solid substance with a molecular mass of 98.06 g/mol and is commonly used in cellulose chemical modification. When dissolved in water it turns into maleic acid. Its melting point is somewhere in the region of 51–56 °C, the initial boiling point −200 °C and density is 1.48 g/cm3 at 20 °C. Vapor pressure of a compound is 0.2 hPa at 22 °C. The reagent was a commercial product of the highest purity available.The provided cellulose fibers were modified with maleic anhydride (MA) provided by Sigma-Aldrich® . All of them are colorless substances and their properties are listed in Solvents employed in the experiments, such as acetone (A), ethanol (99.9%) solution (E) and hexane (H), were bought from ChempurIn this research a new hybrid chemical treatment approach is proposed. The regular surface modification of cellulose fibers was broadened with the solvent exchange in the filler, from water, to either ethanol or hexane. This approach was adapted from another study carried out by Vuoti et al.  which coSolvent exchange was performed before and after the surface modification of cellulose fibers with MA in order to observe its effect on, subsequently, chemical modification of cellulose fibers and properties of polymer composite samples. Moreover, solvents of different polarity employment influence were examined (ethanol and hexane chosen on the basis of sorption experiments—data available in the Solvent exchange: cellulose fibers were put into the flask and the solvent was poured (cellulose to solvent ratio—1:10). Then, such prepared dispersion was mixed with the dipol (400 rpm for ethanol and 1000 rpm for hexane) at room temperature. After 8 h, the mixing was switched off and the dispersion was left for the next 16 h in an ambient condition. When the time was up, the solvent was distilled in a vacuum rotary evaporator at 40 °C .Modification with maleic anhydride: cellulose was put into the acetone and MA solution  for 2 h  in a rotary evaporator. When the process of stirring was finished, acetone was removed with the vacuum distillation process . Then, the sample was subjected to the heat in a vacuum oven at 100 °C, 440 mbar for 4 h.® oven  at 70 °C  and then after finishing the whole modification process at 40 °C .Between the steps of solvent exchange and modification with MA, cellulose fibers were stored in a Binder® oven; crystallizer 70 mm × 40 mm) before being analyzed. Fourier transform infrared spectroscopy (FT-IR) absorbance spectra were investigated within the 4000–400 cm−1 range . The experiment was performed with the use of a Thermo Scientific Nicolet 6700 FT-IR spectrometer  equipped with diamond Smart Orbit ATR sampling accessory.Cellulose fibers were dried for 24 h at 100 °C . Cellulose fibers were dried for 24 h at 100 °C  before being analyzed.In this method, the near-infrared region of the electromagnetic spectrum was used. The measurements were carried out in the range of 10,000–4000 cm3/min).Thermogravimetric analysis (TGA) was used in order to get acquainted with the thermal degradation process of cellulose fibers, detecting the mass loss as a function of raising temperature in the range of 25–600 °C  and the second step was isothermal (100 °C) in an Ar atmosphere (60 cm3/min) for 120 min. The mass decrease was detected.What is more, isothermal experiment was also carried out in order to observe the water evaporation process for neat cellulose fibers. The first step of the measurement was the heating of an oven from 25 to 100 °C  before being investigated.A Mettler Toledo TGA/DSC 1 STARe System  equipped with Gas Controller GC10  was employed in this investigation. Activation energy values were calculated with the use of Broido’s method . Cellulo® oven; crystallizer 70 mm × 40 mm) before being analyzed. Differential scanning calorimetry (DSC) investigation was performed in a temperature range from −20 to 200 °C  prior to analysis of the water evaporation process establishing its enthalpy (ΔH) and temperature of the peak (Tpeak). Additionally, the Mettler Toledo TGA/DSC 1 STARe System equipped with a Gas Controller GC10 was employed.Cellulose fibers were dried for 24 h at 100 °C . For each experiment, approximately 1.5 g of cellulose sample and 50 mL of Hydranal Solvent E were taken. The water content in the natural filler was established. The measurement was taken using the TitroLine Alpha  device. Cellulose fibers were dried for 24 h at 100 °C  and, then, left in ambient conditions for 45 min before being investigated (it was impossible to carry out an accurate measurement immediately after taking the sample out of a dryer—too high water absorption rate).Fischer titration was carried out with the use of Hydranal Solvent E and Hydranal Titrant 5E supplied by Fluka−1 [−1 [−1 [−1 [−1 occurs. It is assigned to –C=O bonds present in the structure of maleic acid [Analyzing the FT-IR spectra of cellulose , one sho−1  and 1030−1 [−1 , C–H str [−1 [−1 , and –CO [−1 [−1 . Moreoveeic acid . Therefo−1 confirms the introduction of acid, and the signal at 1636 cm−1 indicates that the sample also contains vinyl groups [−1 is less visible than for not dried natural filler. It indicates some information about the yield of the carried-out treatment—UFC100 not dried before the MA grafting are modified in a more efficient way. This phenomenon is also confirmed for all of the performed chemical hybrid modifications , UFC100/ND/MA/2/H (I1720 = 0.024), and UFC100/ND/MA/2/E (I1720 = 0.022). Moreover, when having a closer look at tabularized values, one may observe the huge difference between the dried and not dried fibers, considering the intensity of the absorption band at 1720 cm−1, e.g., UFC100/ND/MA/0 with I1720 = 0.035 and UFC100/D/MA/0 with I1720 = 0.007. It may be also claimed that the different effect of the solvent employment for dried and not dried fiber modification may be observed. In the case of not dried fibers, solvents cause a decrease in the amount of grafted maleic anhydride. On the other hand, considering the dried fibers, employment of ethanol and hexane contributes to the improvement in the yield of the modification process.According to −1 [−1 [−1 [−1 [−1 [Furthermore, considering the differences caused only be the hexane and ethanol treatment, according to −1 , C–H str−1 [−1 , C–C/C–O [−1 [−1 , C–H/C=O [−1 [−1 , and C–O [−1 [−1 . This mi [−1 [−1 . Therefo−1 corresponds with H2O molecules absorbed by the natural filler, as well as –OH groups present in the cellulose structure [−1 is decreased in comparison with the neat, dried cellulose. Therefore, it may be expected that modified fibers would have lower capability of water bonding as the fibers have been hydrophobized via a performed treatment [According to tructure . It may reatment .−1 were found to be significantly impacted by the water uptake. Although, there is no clear chemical explanation for the observed changes, several hypotheses could bring elements of understanding. It could be inherent to the material which is known to be subjected to chemical intrinsic variability or be assigned to non-specific experimental variation [Surprisingly, also the C–H stretching bands situated at 2935 and 2900 cmariation .−1 (C–H stretching of the second overtone) [−1 (C–H stretching and C–H deformation vibrations) [−1 (O–H stretching of the first overtone) [−1 (O–H stretching vibrations) [Considering the Near Infrared Spectroscopy (NIR) spectra of cellulose fibers, some signals characteristic of this substance can be recognized, e.g., 8165 cmvertone) , 7290 cmrations) , 6472 cmvertone) , and 475rations) . Peaks a−1 may be observed. At the same time, for fibers dried prior to chemical grafting, a decrease at this absorption band is noted. The mentioned signal is assigned to O–H stretching vibrations, but it also indicates the development of intramolecular hydrogen bonding [−1  [−1 (C=C bonds) [According to  bonding . This med water)  and 4274C bonds) .−1 which may describe some changes in the interactions between the fibers via hydrogen bonds and the recombination of these bonds. Therefore, it is suspected that solvents are not affecting the chemical structure of cellulose but they are altering interactions between the natural fibers. This may be a consequence of solvent molecules adsorption on the fibers surface.Nevertheless, considering the data gathered in −1 (C–H) [−1  [−1 (–OH water) [On the other hand, shifts visible in case of NIR spectra are stronger than the ones noticed with the use of FT-IR, e.g., 8165 cm−1 (C–H) , 6706 cm, water) , and 519H water) . This su−1 is assigned to the >C=O bond [−1 is higher in the case of all treatments performed for UFC100 not dried before the MA treatment. Therefore, it may be said once more that, while fibers are not subjected to high temperatures prior to the chemical modification process, the reaction yield is improved.What is more, the absorption band at approximately 4754 cmC=O bond  which isAccording to the thermogravimetric analysis, some differences in thermal degradation process between modified cellulose samples are visible. First of all, MA treated natural fibers exhibit a lower mass decrease after the second decomposition step in comparison with UFC100/D/1440 (reference sample). Furthermore, it may be suspected that not dried and then modified cellulose fibers start to decompose at lower temperatures (approximately 200 °C) which is earlier than in the case of UFC100/D/MA/0 and according to the data available in different studies ,68.What should be undoubtedly emphasized is the similar shape of UFC100/D/1440 and UFC100/D/MA/0 curves. This may be caused by lesser grafting of MA on the surface of dried cellulose fibers in comparison with not dried ones, which has been described in the previous sections, as well as a consequence of the partial hornification process . It is r05% is decreased for all samples modified with MA, e.g., UFC100/ND/MA/0—T05% = 240 °C, UFC100/D/MA/0—T05% = 284 °C, UFC100/ND/1/H—T05% = 304 °C, UFC100/D/1/H—T05% = 306 °C, in comparison with specimens treated only with solvents, e.g., UFC100/ND/1/E—T05% = 304 °C, UFC100/D/1/E—T05% = 300 °C. T05% is considered as a temperature at which 5% mass loss, which is more than water content in the cellulose samples, is detected. According to TGA data, water evaporation step mass loss varies between 1.2% and3.6% for MA treated UFC100/D and between 1.4% and 2.2% in case of MA modified UFC100/ND.05% in the case of MA treated specimens, e.g., for not dried cellulose fiber—from 240 °C (UFC100/ND/MA/0) up to 280 °C (UFC100/ND/MA/1/H), for dried natural filler—from 284 °C (UFC100/D/MA/0) up to 305 °C (UFC100/D/MA/1/H). In both cases, the highest improvement is observed in case of sample modified with hexane before the MA treatment. What is also interesting, in the case of the dried cellulose fibers T05% varies between 284 and 306 °C (for MA modified samples T05% = 284 and 305 °C) and for not dried ones—T05% is between 240 and 304 °C (in case of MA modified samples T05% = 240 and 280 °C). This proves the increased thermal stability of dried fibers, probably, mostly thanks to the hornification process [What is more, solvent treatment also caused an increase in T process .50% and T80% values). Nevertheless, it may be observed that most of the solvent treated fibers exhibit elevated T90%, e.g., UFC100/ND/MA/2/E (T90% = 562 °C), UFC100/D/MA/1/H (T90% = 560 °C), in comparison with the reference samples: UFC100/ND/MA/0 (T90% = 496 °C) and UFC100/D/MA/0 (T90% = 478 °C). Moreover, the residual weight in each TGA graph is assigned to the pyrolysis reaction products, which were not oxidized, as the experiment was performed in an argon atmosphere. A complexity of cellulose thermal degradation was widely described in literature [According to the data gathered in terature ,72.Furthermore, in order to assess the differences between the modified samples in a more precise way, the activation energies of decomposition steps were calculated . Presente fibers . Therefo05% values in case of these samples.What is more, it may be observed that in case of fibers dried before the MA treatment, the values are elevated in comparison with not dried cellulose, e.g., UFC100/ND/MA/0—reatment . This coreatment . MoreoveFurther results of a thermal analysis indicate some more information concerning the water content in analyzed cellulose fibers. Water evolution from cellulose occurs both physically through desorption and chemically by elimination reactions. Three distinct temperature regimes may be distinguished: (i) loss of absorbed water at low temperatures (<220 °C), (ii) loss of chemical water at moderate-to-high temperatures (220–550 °C), and (iii) loss of chemical water at high temperatures (>550 °C) . NeverthOn the basis of data gathered in peak towards the higher values in case of only solvent treatment—Tpeak of UFC100/ND/1/H, UFC100/ND/1/E, UFC100/D/1/H, and UFC100/D/1/E are in the region of 94 °C. This value is achieved by merely few samples, e.g., UFC100/D/MA/0, UFC100/D/MA/1/H, but most of the obtained results are at temperatures below 90 °C. Moreover, specimens treated with only hexane or ethanol exhibit also an improvement in ΔH in comparison with the other analyzed samples.What could be also observed is the shift of TA higher enthalpy change value means more energy required to desorb the water from the cellulose fibers as dehydration heat increases considerably when the samples exhibit a raised moisture level . This leFischer titration experiment ,80 helpeIn the literature, it is described that various chemical treatments ,82,83 anMoreover, in the case of not dried UFC100, employment of different solvents causes a decrease in moisture content even to approximately 1.5% which is the lowest of detected values. A slightly different effect may be observed in the case of UFC100 dried before the MA treatment. Here, while ethanol or hexane are used, a more varied impact on absorbed water amount may be noticed.On the other hand, when one compares an impact of hexane and ethanol employment in the modification process, it might be observed that the lowest moisture content values are noted in the case of the following specimens: UFC100/ND/MA/1/E (1.65%), UFC100/ND/1/E (1.79%), and UFC100/D/1/E (1.77%). In each of mentioned modification processes, ethanol was used. It is believed that due to the strong interactions between cellulose and ethanol, in comparison with hexane-fiber interaction forces, the biopolymer is swelled well and the fiber structure becomes more available to the solvent. As a consequence, a higher amount of water is desorbed and the fiber reactivity is improved—the effect of cellulose structure opening and reorganization of hydrogen bonds ,84,85,86−1 was attributed to the bonded maleic anhydride, e.g., UFC100/ND/MA/0 with I1720 = 0.035 and UFC100/D/MA/0 with I1720 = 0.007 is a perfect confirmation for the UFC100 reactivity changes influenced by the drying process. Nevertheless, the effect of drying may be overlapped with the type of solvent employment, e.g., in the case of UFC100/ND/MA/1/E and UFC100/D/MA/1/E specimens there is almost no difference in the intensities at 1720 cm−1. Considering thermal properties, it may be said that MA treatment lowers the thermal stability of cellulose fibers, as T05% is decreased for all samples modified with MA, e.g., UFC100/ND/MA/0—T05% = 240 °C, UFC100/D/MA/0—T05% = 284 °C, UFC100/ND/1/H—T05% = 304 °C, UFC100/D/1/H—T05% = 306 °C, in comparison with specimens treated with only solvents, e.g., UFC100/ND/1/E—T05% = 304 °C, UFC100/D/1/E—T05% = 300 °C. What is more, solvent treatment also caused an increase in T05% in the case of MA treated specimens, e.g., for not dried cellulose fibers—from 240 °C (UFC100/ND/MA/0) up to 280 °C (UFC100/ND/MA/1/H), for dried natural filler—from 284 °C (UFC100/D/MA/0) up to 305 °C (UFC100/D/MA/1/H). Obtained results are incredibly promising considering the possibility of the treated fibers application in polymer matrixes, as sufficient hydrophobization level and decrease in water content have been only partially achieved in previous research studies [In this study, via hybrid chemical modification, cellulose fibers of decreased moisture content were produced. On the basis of the gathered results, it may be claimed that ethanol employment highly contributes to the lowering of water absorption ability of cellulose, e.g., UFC100/ND/MA/1/E (1.65%), UFC100/ND/1/E (1.79%), and UFC100/D/1/E (1.77%). Moreover, it was shown that cellulose fibers not dried before the hybrid chemical modification are more prone to MA grafting. The intensity of the absorption band at 1720 cm studies ,32.


Escherichia coli (E. coli) BL21 (DE3). The maximum fusion protein (92% purity) was obtained with the yield of 89.14 mg/L fermentation culture after purification with Ni-NTA Sepharose column. The hybrid C–L was cleaved from the fusion protein by SUMO-protease, and 17.54 mg/L pure active C–L was obtained. Furthermore, the purified C–L showed identical antibacterial and hemolytic activity to synthesized C–L. Stability analysis results exhibited that the activity of C–L changed little below 80 °C for 20 min, but when the temperature exceeded 80 °C, a significant decrease was observed. Varying the pH from 5.0 to 10.0 did not appear to influence the activity of C–L, however, pH below 4.0 decreased the antibacterial activity of C–L rapidly. Under the challenge of several proteases , the functional activity of C–L was maintained over 50%. In summary, this study not only supplied an effective approach for high-level production of hybrid peptide C–L, but paved the way for its further exploration in controlling infectious diseases of farm animals or even humans.The hybrid peptide cecropin A (1–8)–LL37 (17–30) (C–L), derived from the sequence of cecropin A (C) and LL-37 (L), showed significantly increased antibacterial activity and minimized hemolytic activity than C and L alone. To obtain high-level production of C–L, the deoxyribonucleic acid sequence encoding C–L with preferred codons was cloned into pET-SUMO to construct a fusion expression vector, and overexpressed in  In recent years, more and more efforts have been paid on developing new safe therapeutic compounds to cope with the increasing microbial resistance to conventional antibiotics ,2. AntimHyalophora cecropia, is a small linear helical peptide with broad bioactivities. It shows antibacterial, anti-inflammation and anticancer activities, and does not lyse eukaryotic cells [The human cathelicidin peptide, LL37 (L), plays a critical role in the process of antimicrobial infection and wound healing, but also exhibits undesirable hemolytic activity against host cells ,10,11. Tic cells ,12,13. Hic cells ,15. In tEscherichia coli (E. coli) expression system is effective to produce proteins and peptides, due to its high production, relative simplicity of the DNA manipulation, and low cost [E. coli, recombinant C–L was purified, and its antibacterial and hemolytic activity and stability were also assayed.low cost ,17,18,19low cost ,21,22, alow cost . In the The schematic of construction of the expression vector was depicted in E. coli BL21 (DE3) cells for expression. The gene of hybrid peptide C–L was expressed as a fusion protein in recombinant E. coli BL21 (DE3) cells with 1.5 mM IPTG induction. After cell disruption and centrifuge, the supernatant was analyzed by 16% tricine-SDS-PAGE. As shown in The recombinant plasmid pET-SUMO–C–L was transformed into The soluble fusion protein SUMO–C–L with His-tag enabled the affinity purification through Ni-NTA Sepharose affinity column. Tricine-SDS-PAGE staining with Coomassie blue , lane 3 Staphylococcus aureus) S. aureus ATCC25923 was found around the cylinder to which had been added recombinant C–L, while no antibacterial activities of SUMO–C–L and sodium phosphate buffer (PBS) were observed (The recombinant C–L was purified from the cleavage solution with Ni-NTA Sepharose affinity column. Approximately 17.84 mg/L recombinant C–L was collected. As shown in observed .p < 0.01). Only very low hemolytic activities of C–L to sheep erythrocyte cells were observed in this test  while the HC50 of C and L was 169 and 32 µg/mL, respectively (p < 0.01). Additionally, recombinant C–L exhibited similar antibacterial activity to synthesized C–L against E. coli CVCC245, S. aureus ATCC25923, and Listeria monocytogenes CVCC1599 (p > 0.05). The HC50 of recombinant C–L to sheep erythrocyte cells was agreed with that of synthesized C–L (p > 0.05). It is important that antibacterial display little or no hemolytic activity for their safe application in internal medicine.The antibacterial and hemolytic activity (HC50) of C, L, and C–L was determined through microbroth dilution method . When coS. sureus ATCC25923 were determined by inhibition zone assays. The results of heat stability assay confirmed that synthesized C–L was heat stable, as the antimicrobial activity of C–L was fully retained even after exposing it to 80 °C for 20 min; however, temperature above 80 °C reduced the activity of synthesized C–L significantly  cells with the yield of 89.14 mg SUMO–C–L per liter fermentation liquid, which was extremely high, indicating the effectiveness of SUMO as a fusion partner.To date, various expression systems have been established for the production of recombinant AMPs ,20,23. Aroteases . The exproteases . Several[E. coli ,26. Thiooduction ,28,29. Toduction  in ferme50 >128 μg/mL was determined as no cytotoxicity) was successfully expressed in E. coli BL21 (DE3) and purified with Ni-NTA Sepharose column.It is worth noting that the soluble SUMO–C–L was not active, thus, it was necessary to release the recombinant C–L by enzymatic cleavage. SUMO protease had the ability to cut off the SUMO tag specifically, consequently allowing the recombinant C–L to retain the native N-terminus which was a critical feature for antimicrobial activity . In the The need to apply C–L in human or livestock prompted us to test for interference factors against its activity. The high thermal stability over a temperature range from 37 to 80 °C revealed the possibility of applying C–L in human or farm animals, where the body temperature in which the product must function is close to 40 °C for both poultry  and pigsKnowledge about the peptide function in the pH range of the gastrointestinal tracts is of crucial importance in the efficient application of an oral agent for animals. The pH in the stomach, small intestine, and large intestine of piglets 1 week postweaning was measured, and the values ranged from 1.6 to 4.4, 3.2 to 5.8, and 5.9 to 6.5, respectively . In the Streptococcus bovis HC5) when it was processed by heat and proteinases treatments [It is also important to assess the interference effects of gastric enzymes and pancreatic enzymes on C–L activity, such as pepsin and trypsin, respectively . Additioeatments , which iE. coli CVCC245, S. aureus ATCC25923, and Listeria monocytogenes (L. mono.) CVCC1599. The plasmid pET-SUMO  was used as cloning vector. E. coli DH 5α and BL21 (DE3)  were cultivated and selected in Luria-Bertani (LB) broth containing 50 μg/mL kanamycin at 37 °C, as the host for gene manipulation and expression of fusion protein, respectively. DNA restriction enzymes, T4 DNA ligase, Taq DNA polymerase, and SUMO protease were purchased from Invitrogen .Three pathogenic microorganisms were obtained from China Veterinary Culture Collection . They were Hybrid antibacterial peptide C–L derived from selected parental peptides (C and L) was designed using APD2 by fully considering its structure–activity relationship [E. coli (http://www.kazusa.or.jp/codon/) was synthesized  and cloned into pMD18-T . Plasmid DNA was isolated using a TIAN prep Midi Plasmid Kit . The gene of C–L was amplified using Taq DNA polymerase (TaKaRa), primer C-L-F (5′-GCCGATGAAATGGAAACTGT-3′) and C-L-R (5′-GCGTACTCACTATTAGTTCCTCAG-3′). The PCR condition was 94 °C for 5 min for hot start, followed by denaturing at 94 °C for 30 s, annealing at 54 °C for 30 s, extension at 72 °C for 45 s, and finally, incubation at 72 °C for 10 min. The PCR products were separated by 2% gel electrophoresis, purified with DNA gel extraction kit (Tiangen), and inserted into the linearized pET-SUMO plasmid (Invitrogen) by TA cloning using T4 DNA Ligase (TaKaRa). The ligation mixture was transformed into E. coli Mach1TM-T1R cells, and the recombinant plasmid was verified by PCR amplification and sequencing.The C–L gene with appropriate codons for E. coli BL21 (DE3). The recombinant expression strain was cultivated in LB broth containing 50 μg/mL kanamycin at 37 °C with shaking (200 rpm) to an optical density (OD600) of 0.6~0.8. Isopropyl β-d-1-thiogalactopyranoside (IPTG) (1.5 mM) was then added to induce the expression of the recombinant protein at 37 °C for 5 h. The cells were harvested by centrifuging at 13,000× g for 5 min, and resuspended in lysis buffer , and disrupted by sonication at 200 W for 80 cycles . After centrifuge at 13,000× g for 5 min at 4 °C, the supernatant was collected for tricine-SDS-PAGE analysis.The pET-SUMO–C–L plasmid was transformed into competent 3PO4, 500 mM NaCl, pH 7.4) and the bound protein was eluted by a linear gradient of imidazole from 10 to 400 mM in binding buffer at 2 mL/min. The eluted fractions were analyzed by tricine-SDS-PAGE, and dialyzed overnight at 4 °C against 100 mM NaCl. And the fusion protein content was quantified by bicinchoninic acid (BCA) method with three replicates [The supernatant was purified with a 10 mL Ni-NTA Sepharose column . The column was pre-equilibrated with binding buffer  at 30 °C for 3 h. The recombinant C–L protein was purified with Ni-NTA Sepharose column. The enzymatic solution and dialyzed fractions were analyzed by tricine-SDS-PAGE.S. aureus ATCC25923 as indicator strain. A dilution of the strain was spread on MH plates, cylinders were placed on the agar surface, and 100 μL of purified recombinant SUMO–C–L (100 μg/mL) and C–L (20 μg/mL) was added to each cylinder. The same volume of PBS was used as negative control. The inhibition zone was measured after incubation overnight at 37 °C. Additionally, the minimal inhibitory concentrations (MICs) of C, L, and C–L were also determined according to the Clinical and Laboratory Standards Institute (CLSI) guidelines using S. aureus ATCC 25923, E. coli CVCC245, and L. mono. CVCC1599 as indicator strains. The indicator strains were cultured at 37 °C to logarithmic stage and diluted to the concentration of 1 × 106 cfu/mL with Mueller–Hinton (MH) broth medium, and 180 µL culture was dispensed into per well of 96-well microtiter plate. Peptides were then serially diluted, and dilutions were added into the wells . Each assay was performed in triplicate. After incubation at 37 °C for 12 h, the plate was assessed by measuring the OD600. The MIC was defined as the lowest concentration  required to prevent the growth of bacteria [Antibacterial activity of recombinant C–L was assayed by the agar diffusion method with v/v) Triton X-100  added were used as negative/positive control. Assays were performed in triplicate.Peptide concentrations that caused 50% hemolysis of sheep erythrocytes at 540 nm were measured to evaluate the hemolytic activities . Sheep eS. aureus ATCC25923 was used as the indicator strain and the inhibition zones were recorded.The stability of the purified recombinant C–L and synthesized C–L at different pH values (2.0–11.0) and different temperatures  was evaluated. The susceptibility of C–L to pepsin, trypsin, and proteinase K (10 μg/mL in PBS) was measured by adding it to a solution of the peptide in PBS (1 μM) . StabiliE. coli. SUMO fusion technology was successfully used to eliminate the toxicity and proteolytic degradation of C–L peptide. High recovery yield of recombinant C–L with potential antimicrobial activity was obtained after efficient cleavage of SUMO protease and simple purification. The purified C–L displayed identical antibacterial and hemolytic activities to synthesized C–L, which is significantly superior to the parental peptides. Antimicrobial activity was displayed at the temperature and pH ranges commonly found in the farm animal body. In addition, C–L was, to some extent, resistant to proteolytic digestion of several common enzymes. These findings indicate that the E. coli expression system and SUMO fusion strategy have great potential for high-level expression of various kinds of valuable peptides and proteins. Furthermore, as a novel hybrid peptide with excellent properties, C–L is expected to serve as a useful antimicrobial or even therapeutic agent for animals or possibly humans.In summary, an effective approach was created for overproducing a novel hybrid peptide C–L in