After incorporating 2 wt % ZB in the PP/APP-LDH system, the flame retardant property was further improved. Polypropylene composites with 20 wt % APP-LDH and 2 wt % ZB showed a 58% PHRR reduction. In addition, thermogravimetric analyzer (TGA) results indicated that the addition of APP-LDH and ZB improved the temperature at 50% weight loss (T50%) and the char formation of the materials significantly.We found in our previous study that layered double hydroxides (LDHs) which undergo aqueous miscible organic solvent treatment (AMOST) can tune the hydrophobicity surface of LDHs to be hydrophobic, and then the solvent mixing method can be used to prepare polymer/LDH nanocomposites. However, flame retardant property is not very high if LDHs are only used. In this present work, ammonium polyphosphate (APP) intercalated LDHs and zinc borate (ZB) was incorporated into a polypropylene (PP) matrix using the solvent mixing method. The structures, morphologies, and performance of the composites were characterized carefully. The peak heat release rate (PHRR) reduction of PP containing 10 and 20 wt % APP-LDH reached 27% and 55%, respectively, which increased up to 63% compared with PP/CO Polypropylenes (PPs) are involved in a wide range of applications, such as the automotive, home appliance, and construction industries, etc., due to their ease of processing, excellent mechanical, electrical, chemical resistance, and non-toxicity . However2 adsorbents [2 [Among the variety of flame retardants, layered double hydroxides (LDHs) have been regarded as effective flame retardants as well as electrocatalysts , CO2 adssorbents , and supsorbents , due to sorbents ,7,8,9. Bsorbents ,11, whicsorbents , phosphosorbents ,14, carbsorbents , borate-sorbents , Ni(OH)2bents [2 , or intubents [2 , and so bents [2 . Some rebents [2 ,21,22,233B10O18·14H2O morphology—a combination of rods and particles by solution method—and turn its surface hydrophobic so that it can be uniformly dispersed in the polymer matrix. In addition, it is reported that zinc borates (ZBs) combined with LDH can function as a synergistic additive promoting formation of a char residue, as well as acting as a protective layer on the surface of polymers ,25. ZincIn this work, we take advantage of the layered structure of LDHs and the superior flame retardant performance of APP, synthesizing APP intercalated LDHs and investigate both the fire resistance and thermal stability of APP-LDH. On the basis of this, the synergistic effect of LDHs and zinc borate is considered as well.3)2·6H2O , Al(NO3)3·9H2O , APP , Na2B4O7·10H2O , ZnSO4·7H2O , H3BO3 , acetone , sodium hydroxide , xylene , and hexane .Materials were procured as follows: polypropylene , Mg2·6H2O and 4.70 g Al(NO3)3·9H2O in 50 mL deionized water and NaOH (4 M) was added into an APP solution (25 g APP in 50 mL deionized water), the pH of the system was ~10. The obtained mixture slurry was sealed in an autoclave and reacted at 100 °C for 24 h. After the reaction, the slurry was centrifuged and washed with water until its pH was neutral condition. Then it was treated with acetone to tune the LDH surface from hydrophilic to hydrophobic. For the PP/APP-LDH nanocomposites, the resultant AMO-LDH was directly used without drying. Mg3Al-CO3 LDH was synthesized similarly. Mg2B4O7·10H2O, ZnSO4·7H2O and H3BO3 as raw materials. In detail, the mixture of 19.07 g of Na2B4O7·10H2O, 6.18 g of H3BO3, and 50 mL of deionized water was stirred at 70 °C. Then 14.38 g of ZnSO4·7H2O was added into the mixture solution and further stirred for 8 h at 90 °C. The obtained white precipitates were similarly treated with LDH.Zinc Borate with uniform morphology was prepared at 90 °C using NaThe solvent mixing method was used to synthesize PP/APP-LDH nanocomposites. In detail, a certain amount of PP and APP-LDH  was added into 100 mL xylene. Refluxing at ~140 °C for at least 2 h to obtain dissolved PP solution with highly dispersion of LDH particles. Then the hot solution was poured into 100 mL hexane. After being filtered and dried, the PP/APP-LDH nanocomposites were obtained. Polypropylene/APP-LDH/ZB nanocomposites were prepared in the same way.−1, Kyoto, Japan). Fourier Transform Infrared Spectroscopy (FT-IR) spectra were recorded on a Bruker VERTEX 70 FT-IR spectrophotometer . Scanning electron microscopy-energy dispersive spectrometer (SEM-EDS) analyses were recorded on a Hitachi SU–8010 machine . 27Al NMR analysis was performed on a Bruker Advance 500 spectrometer  at a resonance frequency of 130.44 MHz with a 4 mm triple-resonance magic angle spinning (MAS) probe; the sample spinning rate was 10 kHz. Differential scanning calorimetry (DSC) analyses were performed using a Shimadzu TA-60WS instrument , heating rate was 10 °C·min−1, nitrogen flow rate was ca. 50 mL·min−1.X-ray diffraction (XRD) results were performed on a Shimadzu XRD–7000S instrument , 2θ degree from 2° to 70°, scanning rate 5° min−1, and the air flow rate was 20 mL min−1. Experiments were undertaken from 20 to 600 °C. The flammability performance was investigated using a microscale combustion calorimeter , with sample mass of 5 mg and heating rate of 1 °C s−1, and nitrogen flowing of 80 cm3 min−1. Experiments were undertaken from room temperature to 700 °C.The thermal stabilities were evaluated using a TGA Q50 machine , the heating rate was 10 °C·min3Al-CO3 LDH, and Mg3Al-APP LDH. Mg3Al-CO3 LDH exhibited the typical reflections of LDHs; the sharp basal reflection (00l) harmonics at relatively low 2θ angles indicated a well-crystallized structure of LDH. The characteristic Bragg reflection of (003) was observed at 11.36°, corresponding to a 0.78 nm interlayer spacing. For APP-LDH, the (003) diffraction peak moved to a lower angle (2θ = 9.34°), with an interlayer distance of 0.93 nm, which can be attributed to the intercalation of the relatively larger molecule of APP in the LDH interlayers. Nevertheless, Bragg reflections with low intensity were observed in Mg3Al-APP LDH. This is perhaps because the APP-LDH become more amorphous, and thus there is insufficient coherence along the platelet stacking axis (c-axis) to observe any high intensity (00l) Bragg reflection [flection .27Al solid state NMR analysis was performed, as shown in In order to have a better understanding of the LDH structure, 3Al-CO3 LDH, and Mg3Al-APP LDH are displayed in −1 was due to the OH stretching of water molecules, and the bending vibration around 1643 cm−1 was also due to the interlayer of H2O. The adsorption peak at 562 cm−1 belongs to MO, OMO, and MOM lattice vibrations (M represents Mg and Al). The band at 1365 cm−1 is attributed to the interlayer of CO32−. Besides, the bands at 1141, 907, and 1440 cm−1 are attributed to the vibrations of P–O and N–H group, respectively, demonstrating the presence of phosphate in the obtained APP-LDH nanoparticles [Fourier transform infrared spectroscopy spectra of APP, Mgarticles . The spe3Al-CO3 LDH and Mg3Al-APP LDH. Both LDHs have formed plate-like nanoparticles with plate-like shapes with a diameter around 100~300 nm, according to the SEM images, and normal distribution of particles, which is consistent with previous studies [3Al-APP LDH, whereas almost no P elements were found in the Mg3Al-CO3 LDH, further confirming that APP molecule existed in the LDH interlayers. The EDS results are in agreement with both XRD and FT-IR results. studies ,31. Howe3B10O18·14H2O (File No. 32-1461), indicating that the ZB was synthesized successfully. The FT-IR spectra of ZB exhibited some bands and can be assigned as follows –O (means three coordinate boron). The peak at 1213 cm−1 represent the bending of B–O–H. The peaks at 1113 cm−1, 833 cm−1, and 759 cm−1 are the stretching of B(4)–O (means four coordinate boron). The band at 639 cm−1, 519 cm−1, and 578 cm−1 are derived from B(3)–O. The peak at 455 cm−1 is due to the B(4)–O bending. Fourier transform infrared spectroscopy results further confirmed the successful synthesis of ZB. As seen in  follows c,d [32,3Scanning electron micrographs of the samples are shown in T10%), the temperature at 50% weight loss (T50%), and the char residual percentage at 600 °C, as listed in T10% of PP/10% APP-LDH nanocomposites increased by 30 °C; however, the PP nanocomposites with 20% APP-LDH exhibited a reduction in T10%, which was probably attributed to the decomposition of the LDH particles, as shown in T10% decreased. The char layer catalyzed by APP-LDH slowed down the heat release rate upon the ignition of PP, and thus protected the inside polymer matrix from being degraded by the flame. As to the T50%, both PP nanocomposites increased no matter with 10% or 20% LDH loading. However, the T50% of PP/10% APP-LDH increased by 52 °C, while it only increased 7 °C for PP/20% APP-LDH  and heat release capacity (HRC) values decreased significantly , but the value of T50% increased by at least 30 °C no matter with 2, 4, or 6 wt % ZB together. When the LDH loading increased to 20 wt % LDH, the T0.5 of PP/LDH/ZB composites increased 10, 27, and 29 °C, respectively. Differential scanning calorimetry (DSC) analysis of pure PP and PP/LDH/ZB composites was studied as well. As shown in In order to further increase the flame retardant properties of PP polymers, at the same time, to not affect the thermal stability of the composites caused by the high additives loading, ZB was added to PP/LDH systems and the thermal stability and flame retardant performance was studied systematically. Thermogravimetric analyzer (TGA) analyses for PP and PP/LDH/ZB composites are shown in −1, after incorporation of 10 wt % and 20 wt % APP-LDH, and the PHRR value decreased by 27% and 55%, respectively  and elongation at break (EB) was studied, and the results are shown in T50% increased by 52 °C and 7 °C, respectively. We also found that addition of a very small amount of ZB can enhance the flame retardancy and thermal stability further. This is because ZB can form a viscous substance and make the carbon layer more sealed. Therefore, it is a significant attempt to improve the flame retardant and thermal stability properties of PP by using halogen free additives.In this contribution, the PP/APP-LDH and PP/APP-LDH/ZB nanocomposites were synthesized by solvent mixing method successfully. Both APP-LDH and ZB dispersed well within the PP matrix after aqueous miscible organic solvent treatment (AMOST). Microscale combustion calorimeter (MCC) and TGA results displayed that the APP-LDH had an outstanding flame retardant and thermal stability property for PP. With 10 and 20 wt % addition, the PHRR reduction reached to 27% and 55%, and the


To investigate the risk factors for post-pancreatectomy hemorrhage (PPH).The incidence, outcome, and risk factors for PPH were evaluated in 1169 patients who underwent pancreatectomy.2 (OR 3.70), absence of diabetes mellitus , and pancreatoduodenectomy (OR 3.06) were risk factors for PPH after all pancreatectomies. The PPH incidence was 0%, 1%, 2%, 6%, and 20% in patients with risk scores of 0 (n = 65), 1 (n = 325), 2 (n = 455), 3 (n = 299), and 4 (n = 25), respectively. The differences between risk-score groups 0–2 (2%) and 3–4 (7%) were significant . In patients who had undergone pancreatoduodenectomy, postoperative pancreatic fistula  and absence of DM (OR 3.45) were risk factors for PPH. There was no significant association between POPF and PPH after distal pancreatectomy (P = 0.28). The incidence of POPF post-pancreatoduodenectomy was 20%. BMI ≥ 25 kg/m2 (OR 3.17), serum albumin < 3.5 g/dl (OR 1.77), absence of DM (OR 1.75), distal extrahepatic bile duct carcinoma (OR 4.05), and carcinoma of the papilla of Vater (OR 5.19) were risk factors for POPF post-pancreatoduodenectomy.The incidence and mortality rates of PPH were 3% and 11% in all pancreatectomies, 4% and 11% in pancreatoduodenectomy, 1% and 20% in distal pancreatectomy, and 3% and 0% in total pancreatectomy, respectively. Male sex [odds ratio (OR) 2.32], body mass index (BMI) ≥ 25 kg/mOur study clarified the preoperative risk factors for PPH and recommends using a risk scoring system that includes “absence of DM” for predicting PPH.The online version of this article (10.1007/s00423-019-01830-w) contains supplementary material, which is available to authorized users. Recently, the surgical technique for pancreatectomy has improved; however, pancreatectomy is associated with many complications postsurgery –4. AlthoBetween 2005 and 2018, 1169 patients underwent pancreatectomy at the Department of Surgery, Institute of Gastroenterology, Tokyo Woman’s Medical University. Parameters that were evaluated included the incidence, outcomes, time of onset, location, severity, grade, and risk factors of PPH. Clinical data that were collected and examined included age; sex; preoperative BMI; white blood cell (WBC) count; serum levels of Alb, C-reactive protein (CRP), and hemoglobin A1c (HbA1c); preoperative drainage for obstructive jaundice; preoperative treatment (chemotherapy or chemoradiotherapy); operative procedure, duration of surgery; amount of intraoperative blood loss; vascular resection; pancreatojejunostomy (PJ) anastomosis method; falciform ligament wrapping of the gastroduodenal artery (GDA) stump; clipping of the GDA stump; PJ stent; pathological tissue type; pancreatic fistula; delayed gastric emptying (DGE); and postoperative bile leakage. The significant factors identified by multivariate analysis to cause PPH were defined as risk factors for PPH. The risk score was calculated, with 1 point assigned to each risk factor, and the incidence of PPH was examined for each risk score number.2 (range 14.3–36.8 kg/m2); 421 patients (36%) had diabetes mellitus (DM); 675 patients (57%) underwent pancreatoduodenectomy (PD); 211 patients (18%) had POPF grade B  or C ; 279 (24%) patients underwent HDL wrapping of the GDA stump; none of the patients with omental grafts underwent GDA stump wrapping, and all patients underwent vascularized graft placement of the GDA stump using the falciform ligament; 26 (2%) patients underwent clipping of the GDA stump.The patients’ characteristics for all pancreatectomies are shown in Supplemental Table The PPH, POPF, and DGE grades were defined using ISGPS guidelines , 15, 16 HbA1c level ≥ 6.3% was defined as DM in this study because the normal value of HbA1c in Japan is determined to be ≤ 6.2%.Pancreatectomy with typical lymph node dissection for carcinoma involves regional lymph node dissection, and partial lymph node dissection was performed for inflammation and low malignant tumors. Regional lymph node dissection involves excision of the plexus in the right half of the SMA and dissection of the hepatoduodenal ligament from the left and right hepatic artery bifurcation to the upper margin of the pancreas. The reconstruction method was the Child’s procedure. All patients underwent PJ anastomosis for gastroenterological reconstruction, and no patient underwent pancreatogastrostomy. Usually, the GDA stump was double ligated, and beginning in 2018, additional clipping  was performed on the GDA stump. Moreover, beginning in 2010, the GDA stump was wrapped with the falciform ligament. We performed duct-to-mucosa anastomosis for PJ until 2016 and modified Blumgart anastomosis after 2017. The surgeon chose PJ stenting if the diameter of the PJ anastomosis was narrow, reconstruction was difficult, and the remaining pancreas was soft.The rate of inpatient death related to PPH during hospitalization was used as the mortality rate in this study.P value < 0.05 was considered statistically significant. All the analyses were performed using JMP version 12.1.9 for Windows .Univariate and multivariate logistic regression analyses were performed to determine independent predictors of PPH in patients who had undergone pancreatectomy. Only factors that were significant on univariate analysis were subjected to multivariate analysis. A The incidence of PPH grades B or C was 3% in all 1169 patients undergoing pancreatectomy, 4% in the 675 patients undergoing PD, 1% in the 406 patients undergoing distal pancreatectomy (DP), and 3% in the 88 patients undergoing total pancreatectomy (TP). The incidence of late onset time, extraluminal location, high severity, bleeding from pseudoaneurysm, and interventional radiology treatment were 96%, 74%, 81%, 66%, and 63%, respectively. The median onset time of all PPH and bleeding from pseudoaneurysms from the initial surgery was 22 and 21 days, respectively. Mortality among patients with PPH was 11%. These patients died from multiple organ dysfunction caused by bleeding from pseudoaneurysms. The overall mortality rate within 30 and 90 days was 0.3% (3/1169) and 0.6% (7/1169), respectively. Mortality from PPH within 30 and 90 days was 0.2% (2/1169) and 0.3% (3/1169), respectively.2 (OR 3.70), absence of DM (OR 3.62), and PD (OR 3.06) were significant independent risk factors for PPH in all patients undergoing pancreatectomy (P < 0.05). When assessing using these risk factors, the incidence of PPH after all pancreatectomies in patients with a risk score of 0 (n = 65), 1 (n = 325), 2 (n = 455), 3 (n = 299), and 4 (n = 25) was 0%, 1%, 2%, 6%, and 20% (P < 0.0001), respectively .In multivariate analysis, male sex (odds ratio [OR] 2.32), BMI ≥ 25 kg/mP < 0.05). There was no significant difference in the incidence of PPH between patients with and without falciform ligament wrapping of the GDA stump . The incidence of POPF after PD was 20%.Because PD was risk factor of PPH after all pancreatectomies, we evaluated risk factors for PPH after PD, excluding DP and TP. In multivariate analysis, POPF (OR 31.7) and absence of DM (OR 3.45) were independent risk factors for PPH after PD (2 (OR 3.17), Alb < 3.5 g/dl (OR 1.77), absence of DM (OR 1.75), distal extrahepatic bile duct carcinoma (OR 4.05), and carcinoma of the papilla of Vater (PVC) (OR 5.19) were independent risk factors for POPF after PD.Because POPF was risk factor for PPH after PD, we assessed risk factors for POPF after PD. In multivariate analysis, BMI ≥ 25 kg/mP = 0.84]. Unlike with PD where the OR of 31.7, there was also no significant association between POPF and PPH after DP .The incidence of POPF after DP was 19% (78/406). There was no significant difference in the incidence of POPF between DP and PD [20% (133/675), This study indicates that absence of DM (HbA1c level ≤ 6.2%) is an independent risk factor for PPH after all pancreatectomies and PD and for POPF after PD; a risk scoring system including the absence of DM may be useful for predicting PPH before surgery. By determining the value of HbA1c level (≤ 6.2%), we can predict PPH more easily. This is a new finding that has not been reported before.In recent years, PPH has a low frequency, but it has a poor outcome. POPF, DGE, wound infection, and abdominal abscess are major complications of PD , 4, 17. Some previous reports about intraoperative and postoperative factors showed that vascular resection, pancreaticogastrostomy, postoperative bile leakage, postoperative abdominal infection, and especially POPF were independent risk factors for PPH –12. PPH Insulin acts to promote secretion of pancreatic exocrine cells . It has Previous literatures reported that omental flaps or grafts around various anastomoses after PD could reduce the incidence of POPF and PPH , 31. In Patients from different periods over the 14-year span of the study underwent different diagnostic and treatment modalities owing to the advances in techniques that occurred over time; these variations may have skewed the outcomes of patients treated during the different periods of the study. Moreover, our investigation had a retrospective design and was performed at a single institution; the biases inherent in such settings cannot be completely excluded.2, absence of DM (HbA1c level ≤ 6.2%), and PD are independent risk factors for PPH after all pancreatectomies. A risk scoring system including the new preoperative risk factor “absence of DM” would be useful for predicting PPH.Male sex, BMI ≥ 25 kg/mESM 1(DOCX 31 kb)


Moreover, the cytokine pattern was modified by the three treatments, with a down regulation of interleukin (IL)-13 secretion. These results showed the contribution of these growth factors in the lymphocytes MLNs immune maturation during the neonatal period.Breast milk, due to its large number of nutrients and bioactive factors, contributes to optimal development and immune maturation in early life. In this study, we aimed to assess the influence of some growth factors present in breast milk, such as transforming growth factor-β2 (TGF-β2), epidermal growth factor (EGF), and fibroblast growth factor 21 (FGF21), on the immune response development. Newborn Wistar rats were supplemented daily with TGF-β2, EGF, or FGF21, throughout the suckling period. At day 14 and 21 of life, lymphocytes from mesenteric lymph nodes (MLNs) were isolated, immunophenotyped, and cultured to evaluate their ability to proliferate and release cytokines. The main results demonstrated that supplementation with TGF-β2, EGF, or FGF21 modified the lymphocyte composition in MLNs. At day 14, all supplementations were able to induce a lower percentage of natural killer (NK) cells with the immature phenotype (CD8 At time of birth, the intestine is immature, not only anatomically, but also metabolically and immunologically ,2,3. IntBreast milk is the gold standard to feed the newborn because it includes a rich number of components, which are essential for optimal growth and development . It alsoOn the one hand, the transforming growth factor-β (TGF-β) family members have multifunctional actions involved in maintaining intestinal homeostasis, regulating inflammation, and allergy development ,11,12. TOn the other hand, one of the most abundant GFs in breast milk is the epidermal growth factor (EGF), the concentration of which is 500 times more than other GFs present in breast milk . Its conEGF and TGF-β together with the immunosuppressive interleukin (IL)-10, also present in breast milk, are involved in the functional development of the gastrointestinal mucosa, tolerance acquisition, and inflammation downregulation in damaged intestinal cells ,7.In recent years, new components present in breast milk have been discovered. This is the case of the fibroblast growth factor 21 (FGF21), which belongs to the hormone-like subgroup within the FGF superfamily, and has been found in rodent and human milk . The FGFOverall, because TGF-β2, EGF, and FGF21 are biologically active factors found in breast milk and are suggested to be involved in neonatal intestine maturation; our hypothesis is that they could also have an important role in the immune development process in early life. Therefore, the main objective of the current study was to ascertain whether the effect of a daily supplementation with these compounds could promote immune response development, during the suckling period in rats. The effects of the supplementations on mesenteric lymph nodes (MLNs), an inductor site of the gut associated lymphoid tissue (GALT), were studied specifically during and at the end of the suckling period, in terms of lymphocyte phenotypic composition and on the immune functionality, such as their lymphoproliferative and cytokine production abilities.ad libitum. The pregnant rats were monitored daily and allowed to deliver naturally. The day after birth was reported as day 1. The studies were performed in accordance with institutional guidelines, for the care and use of laboratory animals, and were approved by the Ethical Committee for Animal Experimentation (CEEA) at the University of Barcelona (UB) and Catalonia Government .Pregnant Wistar rats (G15) were obtained from Janvier Labs , and were individually housed in cages under controlled conditions of temperature and humidity in a 12:12 h light:dark cycle with access to food and water n = 27 pups/group). This sample size was required for each group, as previous studies have demonstrated the remarkable role of variability among litters [The suckling pups were unified in litters of nine pups per mother and were randomized into four groups, formed by three litters each ( litters . The AppBesides the reference group (REF group), three supplemented groups based on nutritional intervention were created: the transforming growth factor-β2 (TGF-β2), epidermal growth factor (EGF), and the fibroblast growth factor 21 (FGF21). All animals were identified daily, weighed, and supplemented by oral gavage with a volume of 10 mL/kg/day during the suckling period, from day 1 to day 21 of age. The suckling pups were separated from their mothers 30 min before oral administration, to allow gastric emptying. All daily handling was done at the same period of the day, to avoid modifications in biological rhythms. All actions were performed as described in previous studies in the group, References ,20.®, Rocky Hill, NJ, USA). The products were reconstituted according to the manufacturer’s recommendations.TGF-β2, EGF, and FGF21 groups were supplemented with recombinant human TGF-β2, recombinant rat EGF, and recombinant human FGF21 , and the milk intake by pups within 4−14 days of age . Finally®, Merial, Barcelona, Spain) and xylazine hydrochloride ; and body length  was measured. These data allowed the calculation of morphologic variables, such as the body mass index  and Lee index, for assessing obesity in rats ((g1/3/cm) × 1000).Body weight was registered daily throughout the study. Two end points were established, at day 14 and at the end of the suckling period at day 21. At these times, prior to sacrifice, the pups were anesthetized with intramuscular ketamine  were obtained through a ventral laparotomy. The SI was weighed, measured, and divided into three equal length portions. Gut washes (GWs) were obtained from the distal SI. Briefly, the intestine was flushed with cold PBS and cut into 5 mm pieces. The tissue was incubated with PBS , centrifuged , and later, supernatant was collected and stored at −20 °C until Igs quantification.The IgA and IgM content were quantified in GWs from day 21 of study, by ELISA Quantitation Set , as previously described in Reference . GWs werl-glutamine , 1% penicillin streptomicin , and 0.05 mM 2-β-mercaptoethanol . MLNs lymphocytes were obtained in sterile conditions by passing the tissue through a cell strainer . The cell suspensions were centrifuged , and the pellet was resuspended with complete RPMI medium. The cell counts and viabilities were determined using an automated cell counter, after staining the cells with trypan blue , following usual laboratory procedures, as described in Reference [The MLNs were placed in complete culture medium, containing Roswell Park Memorial Institute  10% fetal bovine serum , 1% eference . The lym®, Trasadingen, Switzerland), which were pre-incubated with 200 μL/well of monoclonal antibodies (mAbs) anti-CD3  and anti-CD28  . Then, MLNs lymphocyte suspensions (5 × 104 cells/mL) were added to each well. A total of eight wells were used for each sample: four pre-incubated with the mitogenic mAbs  and four without pre-incubation . The plates were incubated for 48 h, at 37 °C and 5% CO2. Four hours before the end time of the incubation, 5-bromo-2’-deoxyuridine  was added to measure its incorporation as an indicator of DNA synthesis in a colorimetric immunoassay.MLNs cell activation was performed in sterile conditions, using 96-well tissue culture plates /Abs-NSC) reference group.The plates were centrifuged  and supernatants were collected and stored at −80 °C, until cytokine analysis. After fixation, the anti-BrdU mAbs and peroxidase conjugated anti-BrdU mAbs and 3,3’,5,5’-tetramethylbenzidine (TMB) were added, following the manufacturer’s recommendations of the BrdU Cell Proliferation Assay Kit . The absorbance (Abs) was measured at 450 nm . Data were expressed as a proliferation rate as follows: Proliferation rate = (A/B), where, A = ((Abs-SC − Abs-NSC)/Abs-NSC) ® Multiplex Immunoassay, according to the manufacturer’s instructions  and as previously described in Reference [® 4.2 software . The limits of detection were as follows: 2.10–8600 pg/mL for IL-2; 0.85–3500 pg/mL for IL-4; 14–55,700 pg/mL for IL-10; 3.17–13,000 pg/mL for IL-13; 2.61–2675 pg/mL for IL-17A; 4.35−17,800 pg/mL for IFN-γ; and 3.08−12,600 pg/mL for TNF-α.Supernatants collected after the cell stimulation process described above, were used for the quantification of IL-2, IL-4, IL-10, IL-13, IL-17A, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α concentration using a ProcartaPlexeference . Each an4 cells) were immunophenotyped by multiple immunofluorescence staining technique. Mouse anti-rat mAbs conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin-chlorophyll-a protein (PerCP), allophycocyanin (APC) or APC-cyanine(Cy)7 were used, as in previous studies [+ TCRαβ−), natural killer T (NKT) cells (NKR-P1A+ TCRαβ+), and TCRαβ+ cells (TCRαβ+ NKR-P1A−) could be differentiated, which in combination with the TCRγδ+ cells (obtained from Panel 2) constituted the total of T cells. B cells (CD45RA+) were identified with Panel 4. The mAbs used in this study are detailed in the Lymphocytes from MLNs . Results were expressed as percentages of positive cells in the lymphocyte population, selected according to their forward- and side-scatter characteristics (FSC/SSC) using previous studies protocols [Briefly, cells were incubated with a mixture of 10 μL of saturating concentrations of each mouse anti-rat mAbs in PBS pH 7.2, containing 2% FBS and 0.1% sodium azide , at 4 °C in darkness for 20 min. For T reg evaluation, an intracellular staining was performed. For that, cells previously labeled with anti-CD4-PE and anti-CD25-FITC mAbs, were fixed/permeabilized using a specific buffer kit . Then, intracellular staining with anti-Foxp3-APC mAb was carried out, under the same conditions as extracellular staining. After washing, all stained cells were fixed with 0.5% paraformaldehyde  and stored at 4 °C in darkness, until analysis by flow cytometry. For each sample, a positive control staining using each isotype matched mAbs and a negative control without staining were included. Analyses were performed in a Gallios™ Cytometer  in the CCiT-UB. Data were assessed by FlowJorotocols , or in ap < 0.05. Data in the text, tables, and figures are expressed as the mean ± standard error of the mean (S.E.M).Statistical analyses were performed using the IBM Social Sciences Software Program . Levene’s and Shapiro−Wilk tests were applied to assess variance equality and normal distribution, respectively. When the results demonstrated equality of variance and normal distribution, a one-way analysis of variance (ANOVA) test was performed. Nonparametric tests were carried out when normal distribution and equality of variance did not exist. Specifically, Kruskal–Wallis and Mann–Whitney U tests were used to assess significance for independent samples. Significant differences were established at p < 0.05) and the supplementations did not affect this growth pattern , and compared with those receiving the vehicle (REF). Body weight increased during suckling period in the REF group . Regarding the Lee index, only the animals from the REF group and those supplemented with TGF-β2, showed significant differences associated with age (p < 0.05), but the supplementation with EGF and FGF21 did not display such a significant age difference.All groups increased BMI, SI relative weight, and SI relative length with age  is the only source of food. Nevertheless, none of the interventions  showed significant differences regarding IgA or IgM, compared to the REF group .+ cells and only ~3% TCRγδ+ cells; B cells counted for about 15–18%, NK and NKT cells represented subpopulations with less than 3% of total lymphocytes, and Treg cells were ~4% (The proportion of the main lymphocyte subsets in MLN was established during (day 14) and at the end of (day 21) suckling period. On both days, most of the MLN cells (~75%) were T cells, the majority of them being TCRαβwere ~4% .+ cells, compared to the REF group (p < 0.05). Moreover, the REF and groups supplemented with TGF-β2 and EGF decreased the NK cell proportion, in comparison to the same group at 14 days (p < 0.05). The animals from the REF and FGF21 groups increased the percentage of NKT cells with age, and only those from the EGF group were able to decrease the proportion of TCRγδ+ and B cell percentages at day 21 (p < 0.05 vs. day 14).Although at 14 days there were no differences among the supplemented groups, some differences could be seen at 21 days . At the + cells and the CD8αα/CD8αβ ratio from MLNs lymphocytes, were determined as indicators of immune maturation  or to any of the supplementations, compared to the REF group , when values from day 21 were compared to day 14 (p < 0.05). The TCRγδ+ CD8− cell percentage . Moreover, the TCRγδ+ CD8+ cell proportion in the EGF group (p < 0.05). In relation to the NKT population (CD8- and CD8+ subsets), only the NKT CD8− subset showed changes associated with age for all groups , and that only supplementation with TGF-β2 induced a significant decrease compared with the REF group at 21 days (p < 0.05 vs. REF). The phenotype of the intestinal NK cells based on CD8 expression, could be considered as immature (NK CD8+) or more mature (NK CD8−). The three supplementations were able to decrease the proportion of NK cells expressing CD8 at 14 days , none of the supplemented groups induced significant differences, when they were compared to the REF group at the same age. The αE+ cells were present in a proportion lower than 5% at both studied times . The REF group increased (~15%) αE+ B cells subset proportion, with respect to the same group at 14 days, whereas all supplemented groups decreased (~20%) this subset with age (p < 0.05). In addition, the supplementation with EGF and FGF21, decreased the percentage of αE expression in CD4+ cells with age. Although there was a tendency to increase with age, the proportion of CD62L+ in B cells in all groups, it was only significant in the TGF-β2 group (p < 0.05).The percentages of integrin αE studied . None of− CD25+ CD4+ cells) and Treg cells (Foxp3+ CD25+ CD4+ cells) were also studied, and no changes were found due to diets (day 21), with all groups together having a mean percentage of 1.22 ± 0.12 and 3.81 ± 0.11, respectively.At the end of the suckling period, activated CD4 cells . IL-4 levels were ~50% lower in the EGF group at day 21, than those at day 14 (p < 0.05). The groups that received EGF and FGF21 for 21 days, decreased the IL-10 content ~1.5 times with respect to their values at day 14 (p < 0.05). IL-13 secretion was lower at day 21 with respect to day 14, in the EGF and FGF21 groups (p < 0.05), and the three GFs groups showed lower values than those present in the REF group (p < 0.05). The lower levels of IFN-γ production at day 21 compared to day 14 were only significant in the REF group, which accounted for a 50% reduction (p < 0.05). Moreover, TNF-α release decreased (~30%) with age in the REF, TGF-β2, and FGF21 groups, whereas supplementation with EGF increased TNF-α production (p < 0.05 vs. REF group).The pattern of cytokines released from MLNs cells at day 14, showed a high secretion of IFN-γ, followed by IL-10. In the conditions tested, the secretion of IL-2, IL-4, IL-13, IL-17A, and TNF-α was lower than 90 pg/mL. IL-10/TNF-α (anti‑inflammatory/pro-inflammatory cytokines) and IFN-γ/IL-4 (Th1/Th2) ratios were also calculated. No changes in the cytokine pattern at day 14 were detected by GFs supplementation. However, at day 21, some changes appeared due to supplementations and due to age. IL-2 production was 5–10 times higher due to age in the REF, TGF-β2, and EGF groups (p < 0.05).The IL‑10/TNF-α ratio decreased according to age in the EGF and FGF21 groups, and in the EGF group, values were significantly lower than the REF group  has a wide range of biological activities and among them, it has an important role in cell proliferation and differentiation. TGF-β acts as a cytokine having predominantly suppressive effects on the growth of T and B lymphocytes. In this study, the supplementation of suckling rats with TGF-β2 did not have a significant influence on unspecific MLNs proliferative cell response. This result contrasted with a report showing that suckling rats receiving a whey-enriched TGF-β formula, down-regulated the MLNs lymphocyte proliferative response to specific antigen after being sensitized . On the +) on day 14 at levels observed later (day 21); which indicated a positive action on the intestinal immune maturation. This developmental pattern, decreasing CD8 expression in the NK cell surface with age, has previously described in rat neonatal intraepithelial lymphocytes (IEL) and MLN cells [+ cells were observed due to TGF-β2 supplementation, a lower CD8αα/CD8αβ ratio at day 21, compared to day 14 appeared. This ratio has been described to be reduced according to age in healthy MLNs cells from suckling rats [+ T cell proportions when their lactating mothers were treated with mAbs, against TGF-β twice a week from delivery until weaning [+ CD25+ Foxp3+) cell proportion. However, some authors have suggested that oral tolerance induced by breastfeeding could depend on TGF-β signaling from breast milk, which would be able to up-regulate Foxp3+ cells [In the current study, the TGF-β2 supplementation was already able to modulate MLN lymphocyte composition in suckling rats. It decreases the proportion of the immature phenotype of the intestinal NK cells  is a peptide that modulates a variety of biological responses, such as cell proliferation and differentiation . It is kFinally, focusing on fibroblast growth factor 21 (FGF21), recent studies showed that its function is not limited to the regulation of metabolism, but that it is also involved in the protection of multiple physiological processes, such as oxidation, inflammation, atherosclerosis, and aging processes . A recen+ cells and the decrease in the CD8αα/CD8αβ cell ratio in MLNs, as well as TGF-β2 and EGF. This is the first time that the immunomodulatory effect of FGF21 has been demonstrated in early life.It is known that FGF21 is present in breast milk and does not contribute to systemic levels in mouse neonates, but appears to act locally on the mouse neonatal intestine ; and it −) and reducing IL-13 production, which could be useful in avoiding allergic processes. Further studies should be carried out to establish the effect of the supplementation of TGF-β2, EGF, or FGF21 on the response of suckling pups in other parts of the intestinal immune system, such as in the intestinal epithelium or even at the systemic level.In conclusion, our study demonstrated that supplementation with TGF-β2, EGF, or FGF21 during the suckling period had an immunoregulatory effect. Although some specific effects appeared, the three growth factors were able to modulate similar aspects of MLN cells, such as promoting lymphocyte maturation, as observed by increasing NK cells with a more mature phenotype (CD8


Thyme EO showed the highest level of total phenolics, the highest antioxidant capacity, and exhibited the stronger antibacterial activity, results that were emphasized by both chemometric methods used. Among tested essential oils, the one of parsley was distinguished by a low total TPC, weak antioxidant activity, and weak antibacterial activity against S. enteritidis (ATCC 13076); lovage EO by low TPC, weak antioxidant activity, but moderate antibacterial activity; and basil EO by low TPC, moderate antioxidant activity, and weak antibacterial activity against L. monocytogenes (ATCC 19114).This study is focused on the comparison and classification of parsley, lovage, basil, and thyme essential oils (EOs) based on their chemical composition, total phenolic content, antioxidant and antibacterial activities by using appropriate chemometric methods: Principal component analysis (PCA) and hierarchical cluster analysis (HCA). The results showed that parsley, lovage, and thyme EOs are rich in monoterpene hydrocarbons, but basil EO is rich in oxygenated monoterpenes and phenylpropanoids, and that both PCA and HCA separated essential oils into two main groups of which one contains two sub-groups. β-Phellandrene was the major component identified in parsley and lovage EOs, estragole was the major component in basil EO, and  Essential oils are aromatic and volatile liquids distilled from different plant materials . NumerouDue to negative perceptions of synthetic preservatives among consumers, the interest in essential oils focusing on their application in food preservation has grown considerably in the last few years . EssentiOur previous study  has showv/w), was recorded in leaves of thyme , followed by basil , lovage , and parsley . The volatile compounds detected by ITEX-GC/MS analysis in the four essential oils with their percentage composition are summarized in The highest essential oil concentration, as calculated on the basis of dry plant weight (p-menthatriene (13.9%), 1,8-cineole (12.5%), β-myrcene (12.7%), α-pinene (6.5%), and 2,6-dimethylstyrene (6.4%), all of them belonging to the monoterpene hydrocarbons class. 1,3,8-p-Menthatriene is a characteristic volatile constituent of parsley leaves that contribute to the parsley aroma [p-menthatriene, β-myrcene, and α,p-dimethylstyrene being the major components. In a study on essential oil from dried leaves of Estonian parsley, Vokk et al., 2011 [p-menthatriene, and β-myrcene.Parsley essential oil: Twenty-three compounds, representing 99.8% of the total detected constituents, were identified in the essential oil of parsley and grouped on the basis of their chemical structure into six classes . The most abundant constituents were monoterpene hydrocarbons (97.2%), followed by oxygenated monoterpenes (0.9%), phenylpropanoids (0.8%), others (0.5%), sesquiterpene hydrocarbons (0.4%), and unidentified compounds (0.2%). The major components identified in parsley EO were β-phellandrene (32.4%), 1,3,8-ey aroma . A similey aroma  in essenl., 2011  have notLovage essential oil: Eighteen compounds were identified in the essential oil of lovage representing 100% of the total detected constituents. The most abundant constituents were monoterpene hydrocarbons (83.6%), followed by oxygenated monoterpenes (15.6%), others (0.5%), and phenylpropanoids (0.3%). The major components identified in lovage EO were β-phellandrene (53.9%), β-terpinyl acetate (12.7%), β-myrcene (11.4%), and trans-β-ocimene (7.0%); the β-terpinyl acetate belongs to the oxygenated monoterpenes class and the rest of them to monoterpene hydrocarbons. The study of Bylaiteù et al., 1998  has evidBasil essential oil: Twenty-four compounds were identified in the essential oil of basil representing 99.9% of the total detected constituents. The most abundant constituents were oxygenated monoterpenes (46.4%), followed by phenylpropanoids (41.2%), monoterpene hydrocarbons (10.7%), sesquiterpene hydrocarbons (1.6%), and unidentified compounds (0.1%). The major components identified in basil EO were estragole (40.9%), 1,8-cineole (24.3%), and linalool acetate (19.8%), first of them belonging to phenylpropanoids and the others to oxygenated monoterpenes. In a study on essential oil extracted from dried leaves of Polish basil, Calín-Sánchez et al., 2012  have foup-cymene (49.8%), γ-terpinene (18.6%), thymol (8.7%), and β-myrcene (5.4%); the thymol belongs to the oxygenated monoterpenes class and the rest of them to monoterpene hydrocarbons.Thyme essential oil: Nineteen compounds were identified in the essential oil of thyme representing 96.5% of the total detected constituents. The most abundant constituents were monoterpene hydrocarbons (83.5%), followed by oxygenated monoterpenes (9.7%), unidentified compounds (3.5%), others (3.0%), sesquiterpene hydrocarbons (0.2%), and phenylpropanoids (0.2%). The major components identified in thyme EO were p-cymene, and α-pinene. Borugă et al., 2014 [p-cymene as the major components of essential oil from thyme cultivated in Romania. In another study, Calín-Sánchez et al., 2013 [p-cymene, caryophyllene, and α-terpinene as the major constituents of Polish essential oil. Agili, 2014 [p-cymene, and carvacrol as the major constituents of essential oil extracted from thyme collected from Saudi Arabian markets. The main constituents found by Soto-Mendívil et al., 2006 [A number of studies have focused on the chemical composition of essential oil extracted from dried leaves of thyme. Contrary to our study, Jamali et al., 2013  have foul., 2013  have idel., 2006  in essen−1 EO), followed by essential oil of lovage (3.0 ± 0.15 mg GAE 100 µL−1 EO), basil (2.9 ± 0.13 mg GAE 100 µL−1 EO), and parsley (2.2 ± 0.05 mg GAE 100 µL−1 EO). Phenolics are a class of compounds that act as free radical scavengers and are responsible for the antioxidant activity of essential oils [−1 EO), followed by the one of basil (105.0 ± 1.09 µM TE mL−1 EO), parsley (39.4 ± 0.02 µM TE mL−1 EO), and lovage (35.9 ± 0.27 µM TE mL−1 EO).p < 0.05) between TEAC and phenylpropanoids-C4. These findings show that phenylpropanoids (41.2%) are in fact mainly responsible for the antioxidant activity of basil EO.It is well known that phenolic compounds act as antioxidants ,26, but p < 0.0001) between values obtained using the two tests since the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were similar, except those of parsley EO against L. monocytogenes. The lower the MIC/MBC value, the higher the antibacterial activity of an essential oil. Of the four essential oils tested, basil EO has proved to be more effective against S. enteritidis than L. monocytogenes and the others conversely. The results show that thyme EO was the most bacteriostatic/bactericidal against S. enteritidis, followed equally by lovage and basil EOs, and then by parsley EO. Thyme EO was also the most bacteriostatic/bactericidal against L. monocytogenes, followed instead by essential oils of lovage, parsley, and basil. The essential oil of parsley showed similar MIC and MBC values against S. enteritidis (22.68 µL EO mL−1); a MIC value of 5.14 µL EO mL−1 and MBC value of 10.80 µL EO mL−1 against L. monocytogenes. Essential oils of lovage, basil, and thyme revealed similar bacteriostatic and bactericidal activities against S. enteritidis  and L. monocytogenes . Jamali et al., 2013 [−1 for MIC and 540 µg EO mL−1 for MBC in essential oil extracted from dried leaves of thyme tested against L. monocytogenes; an antibacterial activity much lower than the one found in the current study.17 in 10). The first principal component (PC1) explained 53% of the total variance while the second one (PC2) showed a further 41%. Basil and thyme EOs instead were positioned very distinctly based on their characteristics constituents: Estragole (No. 33), 1,8-cineole (No. 16), and linalool acetate (No. 25) for basil EO (bottom-left plot), respectively p-cymene (No. 14), γ-terpinene (No. 20), and thymol (No. 38) for thyme EO (upper-left plot). Similar to PCA, the hierarchical cluster analysis (HCA) based on the Euclidean distance between groups  in order to better visualize and identify their grouping trends. The PCA biplot a indicatn groups b indicatChemical PCA and HCA analyses of compound class: In this case, PCA and HCA c,d reveaPCA and HCA analysis of total phenolic content and antioxidant activity: It was interesting to note that performing PCA and HCA based only on total phenolic contents and antioxidant capacities a,b, a clS. enteridis while basil EO .The current study was conducted to determine the chemical composition of four essential oils extracted from herbs belonging to the families Apiaceae (parsley and lovage) and Lamiaceae (basil and thyme), their total phenolic content (TPC), antioxidant activity (TEAC), as well as minimum inhibitory and bactericidal concentrations (MIC/MBC) against two of the most common bacteria responsible for food poisoning (http://www.solina-group.ro). Essential oils were obtained by hydrodistillation, dried leaves (50 g) of each plant material being extracted for 3 h with 750 mL distilled water in a Clevenger-type apparatus . The essential oils were dried over anhydrous sodium sulphate and stored at 4 °C until analysis. The extraction yield was calculated as the volume of essential oil (mL) per dried leaves weight (g) and multiplied by 100 [Commercially available leaves of parsley, lovage, basil, and thyme, in the dried state, were purchased from a company that markets food ingredients , as previously reported by our group . Sample m/z was scanned.A Zebron ZB-5 ms capillary column  was used for the chromatographic separation of volatile compounds. The column oven temperature program was set as follows: From 40 °C (kept at this temperature for 2 min) to 160 °C at 4 °C/min, then raised to 240 °C at 15 °C/min (kept at this temperature for 5 min). The ion source, injector, and interface temperatures were set at 250 °C. Helium was used as carrier gas, the constant flow rate being 1 mL/min. The split ratios were 1:200 for parsley EO, 1:250 for lovage and basil EOs, and 1:300 for thyme EO. The MS mode used was Electron Impact (EI) ionization, while a mass range from 40 to 650 www.pherobase.com [www.flavornet.org [The volatile compounds were tentatively identified by comparing their mass spectra with those in the NIST27 and NIST147 libraries (considering a minimum similarity of 85%) and by retention indices obtained from rnet.org  (for colv/v); lovage EO:MeOH, 1:50 (v/v); basil EO:MeOH, 1:50 (v/v); and thyme EO:MeOH, 1:250 (v/v)) were transferred into a 16 mL glass bottle with a rubber stopper. Then, 6 mL of distilled water and 0.5 mL of 2 N Folin-Ciocalteu phenol reagent were added and immediately vortexed . After 4 min, 1.5 mL of 0.71 M sodium carbonate aqueous solution and 1.9 mL distilled water were added to the mixture. The incubation of the test sample was carried out in the dark at room temperature for 2 h. The absorbance value was read at 725 nm against blank sample using a double beam UV-VIS spectrophotometer . The blank sample was prepared with methanol and treated identically to the test sample. Three readings per sample were taken. The result was expressed in mg gallic acid equivalents (GAE) 100 µL−1 EO.The total phenolic content was measured using the method described by Socaci et al., 2014 . One hunv/v); lovage EO:MeOH, 1:100 (v/v); basil EO:MeOH, 1:300 (v/v); and thyme EO:MeOH, 1:500 (v/v)) were transferred into a 16 mL glass bottle with a rubber stopper, then 2850 μL of ABTS working solution were added and vortexed. The incubation of the test sample was carried out in the dark, at room temperature for 2 h. The absorbance value was read at 734 nm against methanol using a double beam UV-VIS spectrophotometer . The blank sample was prepared with methanol and treated identically to the test sample. Three readings per sample were taken. The absorbance value of the test sample is extracted from the absorbance value of the blank sample; the final result was expressed in µM Trolox equivalent (TE) mL−1 EO.The Trolox equivalent antioxidant capacity assay was used to measure the antioxidant activity of essential oils; it was performed according to the method described by Thaipong et al., 2006 . The stoSalmonella enteritidis  and Listeria monocytogenes . Each strain was grown into a test tube containing 10 mL sterile nutrient broth  at 37 °C for 24 h in the case of S. enteritidis and at 37 °C for 48 h in the case of L. monocytogenes. The purity of the inoculum was confirmed by plating on appropriate selective media and microscopic examination of the Gram-stained smear . A loopful of inoculum was transferred by streaking onto a selective medium: XLD agar  for S. enteritidis and Palcam agar base  with added Palcam selective supplement for L. monocytogenes. Plates were incubated at 37 °C for 24 h in the case of S. enteritidis and at 37 °C for 48 h in the case of L. monocytogenes. Bacterial morphology was confirmed by optical microscopy. Several colonies were collected with a sterile inoculating loop, transferred into sterile saline solution (8.5 g L−1), and adjusted to match the turbidity of a McFarland 0.5 standard (1.5 × 108 CFU mL−1) [serial 10-fold dilutions  were prepared using the sterile saline solution as diluent.The following microorganisms were tested: FU mL−1) . Then, t5 CFU mL−1) was added to each well. Concentrations ranging from 0.01 to 47.62 µL EO mL−1 were thus reached. For the negative control, eight parts 50% ethanol and one part Tween 80 were mixed with one part of the saline solution, while gentamicin  was considered the positive control. Microplates were incubated at 37 °C for 21 h in the case of S. enteritidis and at 37 °C for 45 h in the case of L. monocytogenes. A volume of 20 μL resazurin aqueous solution (0.2 mg mL−1) was added to each well. Microplates were subsequently incubated at 37 °C for 2 h in the case of S. enteritidis and at 37 °C for 23 h in the case of L. monocytogenes. The lowest concentration that retained the blue color was considered the concentration that completely inhibited bacterial growth (MIC). Three replicates were run for each essential oil. The result was expressed in µL EO mL−1, respectively µg gentamicin (GE) mL−1.The test was performed according to the method described by Semeniuc et al., 2017 . Eight pminimum bactericidal concentration, 10 µL of the dilution representing the MIC and the more concentrated two dilutions were plated on the appropriate selective media  using a Drigalski spatula and incubated for 48 h at 37 °C. A colony counter  was used to determine the relative number of bacterial colonies. The lowest concentration of the antimicrobial agent causing negative growth (fewer than three colonies) was considered to be the concentration that completely killed bacteria [−1.To determine the bacteria . The resHierarchical clustering (HCA) and correlation analysis were carried out using IBM SPSS Statistics software, version 19.0 . Cluster analysis was run using the complete linkage method. Correlation analysis between was performed via Pearson’s correlation. Principal component analysis (PCA) was carried out using Unscrambler software, version 9.7 .L. monocytogenes (ATCC 19114) but basil EO was more effective against S. enteritidis (ATCC 13076). The highest amount of total phenolics and antioxidant capacity were found in thyme EO; this essential oil also exhibited the stronger antibacterial activity. Parsley EO instead had a low total phenolic content (TPC), weak antioxidant activity, and weak antibacterial activity against S. enteritidis (ATCC 13076); lovage EO by low TPC, weak antioxidant activity, and moderate antibacterial activity; basil EO by low TPC, moderate antioxidant activity, and weak antibacterial activity against L. monocytogenes (ATCC 19114).Monoterpenes hydrocarbons were the most abundant chemical compounds in parsley, lovage, and thyme EOs, while in basil, oxygenated monoterpenes and phenylpropanoids prevailed. Regarding the antibacterial activity, all tested essential oils showed inhibitory and bactericidal effects on both bacteria; most of them were more effective against


The role of interleukin-12 (IL-12), interleukin-23 (IL-23), and interleukin-17 (IL-17) has been recognized in psoriasis pathogenesis, and new drugs targeting this axis have already been developed which may provide a new therapeutic approach for patients with moderate to severe psoriasis.  To compare the direct and indirect evidences of the efficacy and safety of brodalumab, secukinumab, ixekizumab, ustekinumab, guselkumab, tildrakizumab, and risankizumab in the short-term treatment of moderate to severe plaque psoriasis using network meta-analysis (NMA).  A comprehensive literature search was performed in PubMed, EMBASE, and Cochrane Central Register of Controlled Trials for the available relevant studies. NMA was conducted by Stata 15.0 software using relative risks (RR) with 95% confidence interval to assess the clinical effectiveness and safety. Ranked the efficacy and safety for each drug accordance with the surface under the cumulative ranking curve (SUCRA).  This meta-analysis included 28 studies. All the interventions performed better than placebo in short-term achievement. Based on the result of SUCRA, ixekizumab 80 mg every 2 weeks ranked the highest in short-term achievement of PASI 75 (SUCRA = 93.0%). Brodalumab 210 mg ranked the highest in short-term achievement of PASI 100 (SUCRA = 85.0%). Secukinumab 300 mg ranked the highest in short-term achievement of sPGA 0/1 or IGA 0/1 or PGA 0/1 (SUCRA = 98.1%). In terms of having a risk of adverse events, the rates were higher in brodalumab, secukinumab, ixekizumab, and ustekinumab 45 mg compared with placebo. Ixekizumab 80 mg every 4 weeks ranked the highest in the risk of adverse events during short-term treatment (SUCRA = 4.5%). Guselkumab 50 mg ranked the highest in the risk of serious adverse events during short-term treatment (SUCRA = 25.9%). Ixekizumab 80 mg every 4 weeks ranked the highest in the risk of discontinuations due to adverse events during short-ter treatment (SUCRA = 10.7%).  IL-17, IL-12/23, and IL-23 inhibitors had high efficacy in the achievement of PASI 75, PASI 100, and sPGA 0/1 or IGA 0/1 or PGA 0/1 in moderate to severe plaque psoriasis after 12 or 16 weeks of treatment. IL-17 inhibitors showed superior efficacy. However, its clinical safety was poor. Risankizumab appeared to have relatively high efficacy and low risk. The clinical tolerance of other biological agents needs to be further observed. The complex interactions of these cytokines ultimately lead to epidermal hyperplasia, recruitment of neutrophils, and angiogenesis [Psoriasis is a common chronic inflammatory skin disease whose main pathological manifestations were inflammation, hyperproliferation of the epidermis, altered maturation of the epidermis, and vascular alterations . The preogenesis . Recentlogenesis . TherefoFortunately, IL12/23, IL-17, and newer IL-23p19 antagonists have been produced and applied in clinics which shows a translational revolution in the treatment and management of psoriasis. Several studies have summarized the efficacy and safety data of IL-17 and IL-23 agents, but the results are inadequate , 9. In aOur NMA was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) statement .A computer-based literature search was performed to identify the available relevant studies published before August 1, 2018, in PubMed, EMBASE, and Cochrane Central Register of Controlled Trials (CENTRAL), and clinical trials registered at ClinicalTrials.gov were searched for details of any relevant clinical trials in progress. We used the terms “ustekinumab or stelara or CNTO 1275 or guselkumab or tildrakizumab or SCH 900222 or MK-3222 or risankizumab or BI 655066 or secukinumab or cosentyx or AIN 457 or brodalumab or siliq or AMG-827 or lumicef or ixekizumab or taltz or LY2439821” and “psoriasis.” Vocabulary and syntax were adapted to be appropriate for each database. Standardized filters were applied for study designs, including the Cochrane highly sensitive search strategy for randomized controlled trials. Language was restricted to English. Comments, editorials, and letters were removed. The search strategy was shown in Supplementary We determined the inclusion and exclusion criteria before the search. The included studies should fulfill the following criteria: study design was limited to randomized, double-blind, placebo-controlled trials; adult patients (age > 18 years) are of either sex with a diagnosis of moderate to severe plaque psoriasis; the study should provide at least one efficacy outcome for short-term treatment: (1) 75% or greater reduction from baseline in the psoriasis area and severity index (PASI 75), (2) 100% reduction from baseline in the psoriasis area and severity index (PASI 100), (3) static physician's global assessment score of 0 or 1 (sPGA 0/1), (4) modified investigator's global assessment score of 0 or 1 (IGA 0/1), and (5) physician's global assessment score of 0 or 1 (PGA 0/1); the study should provide at least one safety outcome for short-term treatment: (1) one or more adverse events (AEs), (2) one or more serious adverse events (sAEs), and (3) discontinuations due to AEs. The exclusion criteria were as follows: the patients with psoriasis were under 18 years of age.Two independent investigators abstracted the data using a standard data extraction form, and any disagreement will be resolved by a third author. The following information will be extracted from each included article: author, year of publication, journal, drug, time to evaluate, primary endpoint, details of the interventions, sample size, male proportion, age, duration of psoriasis, involved body surface area (%), and baseline psoriasis area and severity index (PASI) score.Two authors independently assessed the quality of each included study in accordance with the Cochrane handbook of systematic reviews of interventions 5.1.0 (updated March 2011), which covers the following: (1) random sequence generation (selection bias), (2) allocation concealment (selection bias), (3) blinding of participants and treatment providers (performance bias), (4) blinding of outcome assessors (detection bias), (5) incomplete outcome data (attrition bias), (6) selective reporting (reporting bias), and (7) other biases. Disagreements between the review authors over the risk of bias in particular studies were resolved by a third review author.Clinical data were synthesized through narrative review with tabulation of the results of the included studies. Where sufficient clinically and statistically homogenous data were available, data were pooled using appropriate meta-analytic techniques.Stata V.15.0   in a freIn total, 6801 publications matching the search criteria were identified. After removing the duplicate publications, titles and abstracts of the remaining 4945 publications were screened. 4899 publications were irrelevant records, and 46 publications were ultimately determined with eligibility. Based on the inclusion and exclusion criteria, 23 articles including 28 randomized, double-blind, placebo-controlled clinical trials were assessed in this meta-analysis –36: 5 trThe characteristics of the included studies are shown in We summarized the risk of bias in Among the 28 studies, the patients in 22 studies , 31–36 w11 studies , 33–36 uOne study  did not 21 studies , 29–36 sFour studies , 16, 23 One study  did not The evidence to judge the other biases was not enough, so we regarded the other biases of all the studies as an unclear risk of bias.We built 6 networks involving 6 major outcomes. And each network plot involved 13 different dosages of 7 different biologics. The summarized network plots of the comparisons are provided in (1) PASI 75. With regard to PASI 75, all the interventions performed better than placebo, and the effect size was the strongest for ixekizumab 80 mg every 2 weeks  and secukinumab 300 mg . In the mixed comparisons, ixekizumab 80 mg every 2 weeks was superior compared with ixekizumab 80 mg every 4 weeks , ustekinumab 90 mg , ustekinumab 45 mg , guselkumab 100 mg , guselkumab 50 mg , and brodalumab 140 mg . Secukinumab 300 mg was more effective than secukinumab 150 mg , ustekinumab 45 mg , guselkumab 100 mg , and brodalumab 140 mg . Risankizumab 150 mg performed better than ustekinumab 45 mg  and brodalumab 140 mg . There was no significant difference in the efficacy of ixekizumab 80 mg every 2 weeks and secukinumab 300 mg . Network meta-analysis results for PASI 75 are presented in (2) PASI 100. With regard to PASI 100, all the interventions were significantly superior than placebo. The effect size was the strongest for ixekizumab 80 mg every 2 weeks  and brodalumab 210 mg . In the mixed comparisons, brodalumab 210 mg was more effective than brodalumab 140 mg , ustekinumab 90 mg , ustekinumab 45 mg , tildrakizumab 200 mg , and tildrakizumab 100 mg . Brodalumab 140 mg performed better than ustekinumab 90 mg  and ustekinumab 45 mg . Ixekizumab 80 mg every 2 weeks was found to be more efficacious than tildrakizumab 200 mg  and tildrakizumab 100 mg . Network meta-analysis results for PASI 100 are presented in (3) sPGA 0/1 or IGA 0/1 or PGA 0/1. With regard to sPGA 0/1 or IGA 0/1 or PGA 0/1, the biologics involved were statistically significantly superior to placebo. The effect size was the strongest for secukinumab 300 mg  and secukinumab 150 mg . In the mixed comparisons, secukinumab 300 mg was more effective than secukinumab 150 mg , ustekinumab 90 mg , tildrakizumab 200 mg , risankizumab 150 mg , guselkumab 100 mg , and brodalumab 210 mg . Brodalumab 210 mg performed better than ustekinumab 90 mg  and ustekinumab 45 mg . Network meta-analysis results for sPGA 0/1 or IGA 0/1 or PGA 0/1 responses are presented in (4) AEs. In terms of having a risk of AEs, the rate was higher in secukinumab 300 mg, secukinumab 150 mg, ustekinumab 45 mg, brodalumab 210 mg and brodalumab 140 mg, ixekizumab 80 mg every 4 weeks, and ixekizumab 80 mg every 2 weeks compared to placebo. The effect size was the strongest for ixekizumab 80 mg every 4 weeks  and ixekizumab 80 mg every 2 weeks . In the mixed comparisons, secukinumab 150 mg was more likely to result in AEs than ustekinumab 90 mg , tildrakizumab 200 mg , and tildrakizumab 100 mg . There was no significant difference between ixekizumab 80 mg every 2 weeks and ixekizumab 80 mg every 4 weeks . Network meta-analysis results for AEs are presented in (5) sAEs. In terms of having a risk of sAEs, no significant difference was observed between these biologics and placebo. Besides, in the mixed comparisons, the rate was higher in ixekizumab 80 mg every 4 weeks compared to risankizumab 150 mg . Network meta-analysis results for sAEs are presented in (6) Discontinuations due to AEs. In terms of having a risk of discontinuations due to AEs, ustekinumab 45 mg and risankizumab 150 mg present a relatively lower risk than placebo; RR were 0.47  and 0.22 , respectively. In the mixed comparisons, ixekizumab 80 mg every 4 weeks was more likely to result in discontinuations due to AEs than ustekinumab 45 mg , risankizumab 150 mg , and guselkumab 50 mg . Network meta-analysis results for discontinuations are presented in The summarized efficacy and safety ranking of the 13 interventions according to their surface under the cumulative ranking curves (SUCRA) are shown in According to the SUCRA, the ranking for the short-term risk of adverse events from high to low was as follows: ixekizumab 80 mg every 4 weeks (SUCRA: 4.5%), ixekizumab 80 mg every 2 weeks (SUCRA: 7.5%), secukinumab 150 mg (SUCRA: 22.7%), brodalumab 210 mg (SUCRA: 23.7%), secukinumab 300 mg (SUCRA: 33.9%), brodalumab 140 mg (SUCRA: 38.1%), ustekinumab 45 mg (SUCRA: 41.0%), guselkumab 100 mg (SUCRA: 63.4%), risankizumab 150 mg (SUCRA: 67.6%), ustekinumab 90 mg (SUCRA: 75.2%), guselkumab 50 mg (SUCRA: 76.2%), tildrakizumab 100 mg (SUCRA: 88.8%), and tildrakizumab 200 mg (SUCRA: 90.2%). The ranking for the short-term risk of serious adverse events from high to low was as follows: guselkumab 50 mg (SUCRA: 25.9%), ixekizumab 80 mg every 4 weeks (SUCRA: 27.5%), secukinumab 150 mg (SUCRA: 30.7%), secukinumab 300 mg (SUCRA: 35.6%), brodalumab 140 mg (SUCRA: 38.4%), tildrakizumab 200 mg (SUCRA: 41.1%), guselkumab 100 mg (SUCRA: 49.4%), ixekizumab 80 mg every 2 weeks (SUCRA: 50.8%), ustekinumab 90 mg (SUCRA: 52.5%), ustekinumab 45 mg (SUCRA: 61.1%), brodalumab 210 mg (SUCRA: 63.9%), tildrakizumab 100 mg (SUCRA: 70.8%), and risankizumab 150 mg (SUCRA: 92.8%). The ranking for the short-term risk of discontinuations due to adverse events from high to low was as follows: ixekizumab 80 mg every 4 weeks (SUCRA: 10.7%), ixekizumab 80 mg every 2 weeks (SUCRA: 14.8%), guselkumab 100 mg (SUCRA: 32.1%), tildrakizumab 200 mg (SUCRA: 35.4%), secukinumab 300 mg (SUCRA: 42.2%), secukinumab 150 mg(SUCRA: 43.5%), ustekinumab 90 mg (SUCRA: 49.7%), brodalumab 140 mg (SUCRA: 54.2%), tildrakizumab 100 mg (SUCRA: 58.6%), brodalumab 210 mg (SUCRA: 63.0%), ustekinumab 45 mg (SUCRA: 79.0%), guselkumab 50 mg (SUCRA: 84.6%), and risankizumab 150 mg (SUCRA: 92.6%).Inconsistency refers to the difference between direct and indirect evidences, which will affect the authenticity of network meta-analysis. We used the relative odds ratio (ROR) with 95% CIs to calculate the absolute difference between direct and indirect evidences. If the ROR is close to 1, or the 95% CIs include 0, the effect estimations for direct and indirect evidences are consistent. The results of PASI 100, sPGA 0/1 or IGA 0/1 or PGA 0/1, AEs, sAEs, and discontinuations due to AEs showed no significant inconsistencies in all closed loops which revealed the consistency model's conclusions were robust. For PASI 75, there was statistical loop inconsistency in the loop containing placebo, ustekinumab 45 mg, and ustekinumab 90 mg in the combined results of direct and indirect evidences . Inconsistency plots in closed loops for all the outcomes are shown in Considered PASI 100 and sPGA 0/1 or IGA 0/1 or PGA 0/1 responses are more relevant to the psoriasis patient, we performed sensitivity analyses with these outcomes by excluding the trials at a high risk of bias to evaluate the robustness of our findings. Results were consistent with the main analysis for the efficacy outcomes. And the forest plots are detailed in Supplementary Comparison-adjusted funnel plots of all the outcomes in network meta-analysis are shown in In recent years, biological agents have been widely used in dermatology, especially in patients with chronic psoriasis. Many clinical trials have shown that biological agents can quickly control the illness and improve life quality. Meanwhile, the safety of biological agents has also attracted the attention of dermatologists. Therefore, we summarized and evaluated the short-term therapeutic efficacy and safety of IL-17, IL-12/23, and IL-23 biological agents for the treatment of moderate to severe plaque psoriasis. We extracted direct and indirect evidences from 28 trials with 19840 patients into this meta-analysis. Among these studies, except for 7 phase II trials , 30, 32,In order to make the efficacy and safety data of these medications comparable, we performed a NMA to compare the indirect evidences in this quantitative meta-analysis. We found that all biologics involved were more efficacious than placebo in achieving PASI 75, PASI 100, and sPGA 0/1 or IGA 0/1 or PGA 0/1 responses. IL-17 inhibitors achieved outstanding performance in the treatment effect at 12 weeks compared with IL-12/23 and IL-23 inhibitors. For PASI 75, ixekizumab 80 mg every 2 weeks ranked first, followed by secukinumab 300 mg, ixekizumab 80 mg every 4 weeks, secukinumab 150 mg and brodalumab 210 mg. For PASI 100, brodalumab 210 mg ranked first, followed by ixekizumab 80 mg every 2 weeks and ixekizumab 80 mg every 4 weeks. Studies showed that about 42% of patients treated with brodalumab 210 mg reached PASI 100 at 12 weeks, while only 0.4% of patients treated with placebo reached PASI 100 at 12 weeks . For sPGOur findings indicated efficacy and safety differences among the biologic agents used in moderate to severe plaque psoriasis which could provide valuable references to clinicians. The strengths in, firstly, our research included the latest biological agents for psoriasis treatment. Tildrakizumab had been licensed for treating psoriasis patients by the FDA in March 2018, and risankizumab had been approved by the FDA in Apirl 2019. Secondly, our study included a large number of patients and high quality of RCTs availabled at present. We compared different drugs at the dose level which not only increased the credibility of our research but also provided more information to the clinicians. Thirdly, previous studies took more attention on PASI 75 and PASI 90. However, PASI 100 is more relevant to the patients. In order to better reflect the efficacy of these drugs, we included PASI 100 in the evaluation of the primary outcomes.Some potential limitations could affect the interpretation of our findings. Firstly, a placebo-controlled study was conducted during the induction period; however, the study design of the maintenance stage varied and usually the lack of a placebo-control group which increased the difficulty to extract and analyze all the data. So we evaluated the primary endpoints at the end of the induction period (12-16 weeks), whether the biologic agents can ultimately improve the quality of life of psoriasis patients is still unclear and requires more researches to continue. Secondly, some studies lack details of randomization sequence generation, allocation concealment, and blinding, which could reduce the reliability of our results. Therefore, to ensure the authenticity of the results, we made a sensitivity analysis with these outcomes by excluding the trials at a high risk of bias to evaluate the robustness of our findings. Results were consistent with the main analysis for the efficacy outcomes which could strengthen our analysis. Thirdly, few head to head trials available in our NMA, and most of the analyses were based on the indirect comparisons which may limit the consistency evaluation. Fourthly, there were more Americans and Canadians involved in the trials than Asians, so the results of this analysis may not be generalized to all the people in the world. Finally, the analysis has not considered the different medical histories of the patients, which may affect the findings and results.IL-17, IL-12/23, and IL-23 inhibitors had high efficacy in the achievement of PASI 75, PASI 100, and sPGA 0/1 or IGA 0/1 or PGA 0/1 in moderate to severe plaque psoriasis after 12 or 16 weeks of treatment. IL-17 inhibitors brodalumab, secukinumab, and ixekizumab showed superior efficacy in current clinical trials. However, the clinical safety of IL-17 inhibitors was weaker than that of IL-12/23 and IL-23 inhibitors. Ustekinumab was the only IL-12/23 inhibitor included in this study which performed mediocre in both efficacy and tolerance. The IL-23 inhibitor risankizumab is an excellent performer with high efficacy and low risk. The clinical tolerance of other biological agents needs to be further observed. These results may provide a new choice for a clinical treatment of plaque psoriasis. However, further clinical head to head trials are needed to confirm the long-term efficacy and safety of action of the interventions. And treatment decisions should also be based on the associated cost-effectiveness.


Hemileia vastatrix uredospores and collected them at 0, 12, 24 and 48 hours post-inoculation (HPI) to evaluate the relative expressions of genes encoding cytosolic glutamine synthetase (1CaGS), plastid glutamine synthetase (2CaGS), nitrate reductase (CaNR), and asparagine synthetase (CaAS). The genes exhibited distinct patterns of transcriptional modulation for the different genotypes and N nutritional regimes. The resistant genotype (I59) presented high levels of transcription in response to pathogen inoculation for CaNR and 1CaGS genes, evaluated under N sufficiency in the initial moments of infection (12 HPI). The gene 1CaGS also showed a peak at 48 HPI. The susceptible genotype (CV99) showed increased transcript rates of CaNR at 12 and 24 HPI in response to rust inoculation. The transcriptional patterns observed for CV99, under N suppression, were high levels for CaAS and 2CaGS at all post-inoculation times in response to coffee leaf rust disease. In addition, 1CaGS was up-regulated at 48 HPI for CV99. Cultivar I59 showed high transcript levels at 12 HPI for CaAS and peaks at 24 and 48 HPI for 2CaGS in inoculated samples. Consequently, total chlorophyl concentration was influenced by N suppression and by rust infection. Regarding enzyme activities in vitro for glutamine synthetase and CaNR, there was an increase in infected coffee leaves (I59) and under N sufficiency. Moreover, CV99 was modulated in both N nutritional regimes for GS activity in response to rust. Our results indicate that N transport genes trigger a differential modulation between genotypes through the action of rust disease.This study evaluated the transcriptional profile of genes related to nitrogen (N) assimilation in coffee plants susceptible and resistant to rust fungi under N sufficiency and N suppression. For this purpose, we inoculated young coffee leaves with  Coffea arabica L. , caused by the basidiomycete Hemileia vastatrix Berk & Br, is one of the most aggressive ones. This fungus mainly attacks coffee tree leaves, causing losses in the coffee production of around 35–50%  can also affect the plant responses to diseases, depending on the species and the type of pathogen (Nitrogen (N) is an essential macronutrient for plants, playing an important role in defense mechanisms against diseases . Generalpathogen .Nitrogen use includes uptake, assimilation, translocation, and remobilization processes . GlutamiIn some pathosystems, it has been demonstrated that genes related to N metabolism have their transcriptional activity modulated, as in the case of anthracnose and common bean interaction , corn smasparagine synthetase (AS)  , supportbidopsis . It has bidopsis .H. vastatrix, using susceptible and resistant coffee cultivars. Genes encoding glutamine synthetase (CaGS1 and CaGS2), nitrate reductase (CaNR), and CaAS had their transcriptional profile evaluated by real-time quantitative PCR (RT-qPCR) analysis. In addition, we quantified glutamine synthetase and CaNR in vitro activities and chlorophyl concentration in infected coffee leaves. This is the first study to provide valuable information on molecular responses involved in the N assimilation of coffee plants in response to CLR, the main disease that affects coffee production.Within this context, the aim of this study was to evaluate the transcriptional activity of genes related to N assimilation during N-sufficiency and -suppression treatments in leaves infected with C. arabica L. cv. IAPAR59 (I59) and Catuaí Vermelho IAC 99 (CV99), considered resistant and susceptible to rust fungi, respectively . Leaves were collected, immediately frozen by immersion in liquid nitrogen, and stored at −80 °C for further analysis. The experimental design was completely randomized, with biological triplicates represented by pools of leaves from seven plants.CaGS1), plastid glutamine synthetase (CaGS2), CaNR and CaAS , using ESTs of Coffea spp. dbEST (NCBI) and CafEST (C. canephora genome (Expressed sequence tags (ESTs) coding for cytosolic glutamine synthetase  and the absence of genomic DNA contamination was verified by PCR using GAPDH primers  following the manufacturer’s instructions in a final volume of 13 µL, using 2.5 µg of total RNA.Total RNA was isolated as described by  primers . The RNASpecific primers were designed using the Primer Express software v. 3.0 , aimed to obtain amplicons of 100–150 bp with an annealing temperature of 60 ± 2 °C . To confThe transcriptional abundance of N metabolism genes was analyzed by RT-qPCR, performed in a 7,500 Fast Real-Time PCR System  using the kit 2x SYBR Green/ROX qPCR Master Mix  according to the manufacturer’s instructions. Each reaction was performed in a volume of 25 μL, with 12.5 μL SYBR Green/ROX qPCR Master Mix, 0.5 μL primer (10 μm), 10.5 μL nuclease-free water, and one μL cDNA (five ng/μL). The amplification conditions were 10 min for 95 °C, followed by 40 cycles of amplification 95 °C for 30 s and 60 °C for 60 s. The amplification specificity of each reaction was verified and determined by dissociation curve analysis.−ΔΔCt method t method , where ΔThe GS activity was determined according to the hydroximate biosynthetic methodology described by −1, MgCl2 20 mmol·L−1, dithiothreitol 5 mmol·L−1, ascorbic acid 10 mmol·L−1, EDTA 5 mmol·L−1, 2% polyvinylpolypyrrolidone, FAD 10 µm, and leupeptine 10 µm. The reaction was incubated for 5 min in ice, and subsequently, the samples were centrifuged for 15 min at 4 °C. The supernatant was kept on ice and in the dark. After this, 400 µL extract were added to two mL of reaction buffer solution . Samples without the addition of NADH were used as reaction control. The samples were kept in a water bath at 30 °C for 30 min in the dark, and the reaction was stopped with the addition of 150 µL zinc sulfate 0.4 M. Subsequently, we added 750 µL of 1% sulfanilamide solution in 3 M HCl and 750 µL of 0.02% of N-(1-naphthyl) ethylenediamine dihydrochloride . After 2 min, the samples were centrifuged at 3,000g for 2 min, and the absorbance was evaluated in a spectrophotometer at 540 nm. Absorbance readings were performed in triplicate using one mL of reaction solution, applying a standard curve of NaNO2. The enzymatic activity was calculated in µM NO2/g based linear regression, using NaNO2 concentrations between 0 and 10 µmol·L−1, and presented a correlation coefficient (r2) over 0.99.The NR activity was measured and adapted according to several protocols . Plant ta, and chlorophyl b were determined in leaf extracts following the protocol described by Chlorophyll concentration, chlorophyl p < 0.05. Means were separated according to Tukey’s test (p < 0.05). All statistical analyses were performed using the ExpDes package  to determine differences between treatments at  package  in the R package .−1 for I59 and CV99, respectively. Plants under N suppression had their N content reduced and presented 24.95 g·kg−1 (I59) and 26.74 g·kg−1 (CV99). These results confirm the different plants nutritional status.The H. vastatrix, used as control to confirm rust infection, with symptoms after 45 days post-inoculation. The coffee plant I59, considered a resistant cultivar, showed typical responses of an incompatible reaction. The pictures in the C. arabica assimilation genes were differentially regulated according to the resistance to CLR and to N nutritional regimes. As a consequence of CLR inoculation, we observed different expression patterns for all genes between genotypes. The CaNR gene had the highest transcriptional level observed in both N conditions  than in the inoculated leaves (black bars)   and 2B.CaAS transcriptional profiles were similar for leaves under mock and fungus inoculation treatments for both cultivars under N sufficiency , that is, 55, 30 and 25 µg/mL, as shown in The total chlorophyl concentration in coffee leaves under N sufficiency differed between the different post-inoculation times  and 5C. Therefore, according to our results under N suppression, the total chlorophyl concentrations decreased significantly in comparison to the treatment with N sufficiency  and 5D. As reviewed by CaNR and CaGS1 in the initial moment of pathogen inoculation (12 HPI)   and 1E, H. vastatrix did not influence the expression patterns for CaNR and CaGS1 for both genotypes under N suppression, except for CV99 at 48 HPI for CaGS1  in response to Pseudomonas syringae pv. syringae in tobacco (Colletotrichum lindemuthianum) recognition by inducing GS1α, which resulted in glutamine accumulation in infected leaf tissues.Regarding GS genes, under N sufficiency, genotype . Here, Gpression  and 2H.  tobacco . TaverniA. thaliana, low N availability severely compromises the plant’s ability to express pathogen-induced resistance  of GS in vitro activity for each hour post-inoculation (HPI) and genotype. Averages followed by the same capital letter, compare differences between mock and rust inoculated coffee leaves at the same HPI, did not differ significantly by the Tukey test (p < 0.05). Averages followed by the same lowercase letter, compare differences between the treatments (mock and inoc) at different HPI, did not differ significantly by the Tukey test (p < 0.05).The vertical bars indicate the values presented by the mean ± standard error  of NR in vitro activity for each hour post-inoculation (HPI) and genotype. Averages followed by the same capital letter, compare differences between mock and rust inoculated coffee leaves at the same HPI, did not differ significantly by the Tukey test (p < 0.05). Averages followed by the same lowercase letter, compare differences between the treatments (mock and inoc) at different HPI, did not differ significantly by the Tukey test (p < 0.05).The vertical bars indicate the values presented by the mean ± standard error  of total chlorophyl concentration, a and b for each post-inoculation time and genotype. *Averages followed by the same lowercase letter in the column did not differ significantly by the Tukey test (p < 0.05).Data are expressed as the mean ± standard error (Click here for additional data file.10.7717/peerj.8320/supp-7Supplemental Information 7Click here for additional data file.


The obtained mPEG45−PLAla copolymers showed distinct transition temperatures and degradation abilities. It was found that slight changes in the length of hydrophobic side groups had a decisive effect on the gelation behavior of the polypeptide hydrogel. Longer hydrophobic ends led to a lower gelation temperature of gel at the same concentration, which implied better gelation capability. The hydrogels showed rapid gelling, enhanced biocompatibility, and better degradability. Therefore, this thermosensitive hydrogel is a promising material for biomedical application.Thermosensitive gels are commonly used as drug carriers in medical fields, mainly due to their convenient processing and easy functionalization. However, their overall performance has been severely affected by their unsatisfying biocompatibility and biodegradability. To this end, we synthesized poly( The application of hydrogels in the biomedical field has rapidly increased over the past decade, including use in three-dimensional 3D) cell culture ,2,3, druD cell cuco-glycolic acid) (PLGA) [Thermosensitive hydrogels can be generated from block copolymers consisting of hydrophilic poly(ethylene glycol) (PEG) and hydrophobic moieties such as poly(lactic acid) (PLA) ,14, poly) (PLGA) ,16,17, p) (PLGA) ,19,20, p) (PLGA) , and so ) (PLGA) ,23,24, f) (PLGA) , and cel) (PLGA) ,27. At b) (PLGA) . For exa) (PLGA) ,30,31. T) (PLGA) , aiming ) (PLGA) .In 2012, Qian’s team prepared a bionic hydrogel composed of three components , nano-hydroxyapatite (n-HA), and collagen), which showed satisfactory efficacy in skull repair . In 2015co-leucine)−poloxamer−poly (PAL−PLX−PAL) hydrogels, which exhibited a sustained drug release pattern without producing evident inflammatory effects [block-poly mPEG-b-PLAF hydrogel was synthesized by the ring-opening polymerization (ROP) reaction of l-alanine N-carboxyanhydrides (l-Ala NCA) and l-phenylalanine NCA, with amino-terminated mPEG (mPEG-NH2) as a macroinitiator. Encapsulated with combretastatin A4 (CA4) and doxorubicin (DOX), mPEG−b−PLAF hydrogel had an excellent inhibitory effect on the proliferation of B16F10 melanoma cells [Towards this aim, peptide systems have been developed to avoid evoking the formation of an acidic microenvironment during degradation, and thus reduce the damages to surrounding tissues and minimize the adverse effects toward the bioactivity of the loaded protein or cells. Apart from this, the gelation performance of the polypeptide copolymer can be tuned by copolymerization with various hydrophobic or hydrophilic amino acid monomers, which widens the clinical applications. In 2011, Jeong’s team reported the synthesis of poly (mPEG−PLAla) polymers with different degrees of polymerization (DPs) were synthesized and named mPEG45−PLAla30, mPEG45−PLAla22, and mPEG45−PLAla14. The influence of different DPs on the gelation properties of hydrogels was studied. It was found that hydrogels with higher DP showed better gelation ability than those with lower DP. In addition, the gelation behavior, tissue safety, and cytotoxicity of these polymers in vitro and in vivo were also tested and evaluated. The study revealed that these hydrogels had notable potential for employment in the biomedical field.In this work, mPEG−poly solvent, as shown in l-Ala NCA initiated by mPEG45-NH2. The mPEG45−PLAla copolymers with different DPs were synthesized by altering the feed amount. The DPs of PLAla were determined by contrasting the integral of the methyl peaks of side chains (−CH3) with the methylene peak of PEG (−CH2CH2O−). The DPs of mPEG45−PLAla30, mPEG45−PLAla22, and mPEG45−PLAla14 were 30, 22, and 14, respectively.Triphosgene and 1H-NMR) spectrum of block copolymer is shown in The typical proton nuclear magnetic resonance  of mPEG45−PLAla30 and mPEG45−PLAla22 were 5 °C and 15 °C, respectively, at the concentration of 5.0 wt. %. The lower CGT of mPEG45−PLAla30 could be explained by longer hydrophobic end than that of mPEG45−PLAla22 [The tube inversion method was used to determine the transition temperatures. Polypeptides were solubilized in a phosphate-buffered saline (PBS) solution, and went through a solution–gel transition with the change in temperature. The samples were defined as gel if they stayed still when the vial was inverted for as long as 30 s. The phase diagrams of mPEG−PLAla22 .45−PLAla30 and mPEG45−PLAla22 solutions in PBS (5.0 wt. %) at 4 °C and 37 °C. Although all copolymers were dispersed in PBS at 4 °C, the mPEG45−PLAla14 formed a clear solution while mPEG45−PLAla30 and mPEG45−PLAla22 showed a turbid state. When temperature rose to 37 °C, mPEG45−PLAla30 and mPEG45−PLAla22 were observed to be stably gelled within 5 min, while mPEG45−PLala14 remained in a viscous flow state. This was because mPEG45−PLala14 had better water solubility. Further, the morphology of the mPEG45−PLAla30 hydrogel (5.0 wt. %) was observed by scanning electron microscope (SEM). As can be seen in 45−PLAla30 gel, which was due to the 3D network structure of the gel. These pores showed a size of about 100 μm and were evenly distributed within the hydrogel, making them suitable as a depot for therapeutic agents. These findings prove that mPEG45−PLAla30 and mPEG45−PLAla22 hydrogels feature good stability at body temperature and are suitable for biomedical applications.Solution phase behaviors of the three copolymers at different temperatures were investigated. The images in G′) and loss modulus (G″) changes of the three copolymers were obtained by dynamic mechanical analysis. G′ represents the systematic gel-like behavior of the elastic component of the complex modulus, and G″ is an index of the viscous component of the complex modulus and a measure of the sol-like behavior. The intersection of G′ and G″ reflects the sol–gel transition. As shown in G′ of mPEG45−PLAla30 and mPEG45−PLAla22 obviously increased along with rising temperatures. However, only a minor increment in G′ was seen in mPEG45−PLAla14, and no significant change in G′ was detected, even when the temperature rose to about 50 °C, implying that there was no sol–gel modification of mPEG45−PLAla14 within the experimental temperature range. The reason was that hydrophilic mPEG was longer than the hydrophobic end made up of polypeptides. Therefore, with a longer hydrophilic segment, mPEG45−PLAla14 showed better water solubility than mPEG45−PLAla30 and mPEG45−PLAla22, thus featuring less obvious sol–gel change. Notably, the length of the hydrophobic side chain had a remarkable effect on the gelation function of the mPEG−peptide block copolymer. Thermally induced storage modulus , which reflected the viscous state of hydrogels. As the temperature increased above CGTs, G′ was sharply elevated and surpassed G″, confirming the gel formation. This result was in accordance with the findings in In 13C-NMR), dynamic light scattering (DLS), and circular dichroism (CD). As shown in In order to study the mechanism of sol–gel transition, the nuclear magnetic peak changes, diameter changes, and conformation evolution of polypeptides in response to temperature were tested by carbon nuclear magnetic resonance  of mPEG45−PLAla30 was about 31.4 nm. When the temperature rose to 20 °C and 50 °C, the number swiftly increased to 95.9 nm and 356.2 nm, respectively. The dramatic changes in particle size could be explained by the interaction between the shell of PEG and core of polypeptides caused by the dehydration processof PEG [As seen in ssof PEG .45−PLAla30 during the sol–gel transition process. The two typical bands corresponding to the β-sheet conformation, a positive Cotton band at 195 nm and a negative Cotton band at 226 nm, were clearly shown. The above results revealed that the synthesized thermosensitive hydrogel had good physicochemical properties and a clear solution−gel transformation mechanism.In 45−PLAla30 hydrogel (5.0 wt. %) was evaluated in PBS and PBS with elastase K or α-chymotrypsin. As shown in Since hydrogels are often used in the biomedical field, biodegradability is a decisive factor for clinical application. If the degradation rate of the hydrogel is too high, it will lead to a rapid release of the payload. In contrast, if the hydrogel requires a long time to degrade, then it will probably remain at the injection site even after all drugs are released, which is inconvenient for further treatment. The in vitro degradation of mPEG45−PLAla30 solution (5.0 wt. %) was subcutaneously injected into Sprague−Dawley (SD) rats through 21-gauge syringe needles. In The degradation process of hydrogels in vivo was monitored for 28 days, and images were taken at 10 min, 7, 14, 21, and 28 days after hydrogel treatment. At 4 °C, 500.0 μL of mPEG45−PLAla30 in vitro was evaluated by methyl thiazolyl tetrazolium (MTT) assay and hemolysis test. In 45−PLAla30 (100.0 ìg mL−1) for 24 h retained almost 100% viability, confirming its excellent biocompatibility. Moreover, the effect of different concentrations of gel on hemolysis was also investigated. In this part, hemolysis was measured spectroscopically according to previously reported methods [45−PLAla30, which further confirmed the outstanding biocompatibility. Overall, the findings above revealed that the hydrogel showed little toxicity, both in vitro and in vivo. Therefore, it is a promising material for usage in clinical practice.The cytotoxicity of hydrogel mPEG methods . As showMn) = 2000 g mol−1, was purchased from Sigma-Aldrich . The mPEG45−NH2 was synthesized conforming to the previously reported protocol in our work [N,N-Dimethylformamide (DMF) was stored over calcium hydride (CaH2) and purified by vacuum distillation. All the other reagents and solvents were bought from Sinopharm Chemical Reagent Co. Ltd., Beijing, China, and used as obtained.mPEG−OH, number-average molecular weight (our work . THF andThe sol–gel transition behavior of the copolymers in PBS (pH 7.4) was determined by inverting test method with a temperature increment of 2 °C per step. Samples with concentrations ranging from 3.0−7.0 wt. % were dissolved in PBS and stirred at 0 °C for 12 h. The copolymer solution (0.2 mL) was introduced into the test tube with an inner diameter of 10.0 mm. The sol–gel transition temperature was recorded if no flow was observed within 30 s after inverting the test tube. Each data point was the average of three measurements.−1. The heating rate was 1 °C min−1.Rheological experiments were performed on a US 302 Rheometer . The copolymer solution was placed between parallel plates of 25.0 mm in diameter with a gap of 0.5 mm. To prevent the evaporation of water, the outer edge of the sandwiched sample was sealed by a thin layer of silicon oil. The data were collected under a controlled strain γ of 1% and a frequency of 1 rad s45−PLAla30 hydrogel was incubated in PBS (5.0 wt. %) in vials (diameter = 16.0 mm) at 37 °C for 10 min. PBS solutions (pH 7.4) containing 0.2 mg mL−1 elastase K or 0.2 mg mL−1 α-chymotrypsin were used as degradation media, and hydrogels incubated in PBS were only used as a control. Different solutions (2.0 mL) were added to the top of the gels at 37 °C and the entire medium was changed daily. The weight of the remaining gel was measured daily.For this, 0.5 mL of mPEG45−PLAla30 PBS solutions (5.0 wt. %) were injected into the dorsal subcutaneous area of the rats using a 21-gauge needle. The rats were sacrificed on day 7, day 14, day 21, and day 28, respectively, to monitor the degradation behavior of the gel. The experiments on animals were carried out according to the guide for the care and use of laboratory animals, provided by Jilin University, Changchun, China, and the procedure was approved by the local Animal Ethics Committee.SD rats  were used for the detection of gel degradation in vivo. Rats were anesthetized by inhalation of ether before 0.5 mL of mPEGv/v) FBS, penicillin (50.0 IU mL−1), and streptomycin (50.0 IU mL−1) at 37 °C in a 5.0% (v/v) carbon dioxide atmosphere. L929 cells with a density of 6000 cells per well were planted in 96-well plates in 180.0 μL of DMEM. After incubation for 24 h, 20.0 μL of copolymer solutions at different concentrations (31.3–1000.0 μg mL−1) were added. L929 cells were incubated with copolymers for another 24 h before 20.0 ìL of PBS solution containing MTT (0.05 mg mL−1) was added and incubated for a further 4 h. Then, the media was replaced with 160.0 μL of dimethyl sulfoxide (DMSO). The absorbance of the solution was measured on a Bio-Rad 680 microplate reader  at 490 nm. Cell viability (%) was calculated according to the following Equation (1).The relative cytotoxicity was assessed by MTT viability assay against L929 mouse fibroblasts cells. L929 cells were cultured in complete Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10.0%  were synthesized by ROP of l-Ala NCA monomer initiated by mPEG45−NH2. The effect of different DP on the solution−gel transition was investigated. It is worth noting that mPEG45−PLAla30 had a lower sol–gel transition temperature than mPEG45−PLAla22, attributed to the longer hydrophobic segment. In addition, mPEG−PLAla hydrogel had good stability and high mechanical strength after gelation. Moreover, mPEG−PLAla hydrogel did not cause any tissue damage, inflammatory reaction, or hemolysis reaction during degradation, confirming its good biocompatibility. In summary, the mPEG−PLAla hydrogel designed in our study had improved biocompatibility, appropriate degradation, and gelation ability. Therefore, the polypeptide hydrogels showed promise for application in tissue repair and regeneration, cell 3D culture, and treatment of cancer. Moreover, by loading functional drugs, the hydrogels can also be used for postoperative recovery and anti-infection. Based on the above findings, polypeptide thermosensitive hydrogels have broad prospects in the biomedical field.In this work, thermosensitive hydrogels mPEG