There has been worldwide debate on lymphadenectomy for gastric cancer, with increasing consensus on performing an extended (D2) resection. There is a paucity of data in Australia. Our aim is to compare overall outcomes between a D1 and D2 lymphadenectomy for gastric cancer in a single specialist unit.We performed a retrospective analysis on patients who underwent a curative primary gastric resection for gastric adenocarcinoma between January 1996 and April 2016, primary outcomes included overall survival (OS) and disease-free survival (DFS). Propensity score matching (PSM) analysis was used to balance covariates between D1/D1+ and D2 groups. Kaplan-Meier survival curves of D1/D1+ versus D2 were constructed and evaluated using the log-rank test with subgroup analyses for pathological node (pN) status. Multiple Cox proportional hazards model was used to determine predictors of overall survival.p = 0.008), and 10-year DFS was 35% for D1 and 58.1% for D2 (p = 0.058). Subgroup analysis showed that node-negative (N0) patients had improved 5-year OS for D2 (90.9%), compared to D1/D1+ (76.4%) (p = 0.028). There was no difference in operative mortality between the groups , nor in post-operative complications (p = 0.227). Multiple Cox analysis showed advanced tumor stage (stages III and IV), and lymphadenectomy type (D1) and the presence of postoperative complications were independent predictors of poor overall survival.Two hundred four patients underwent a gastrectomy, 54 had D1/D1+, and 150 had a D2 lymphadenectomy. After PSM, there were 39 patients in each group, the 10-year OS for D1/D1+ was 52.1 and 76.2% for D2 (D2 lymphadenectomy with spleen and pancreas preservation can be performed safely on patients with gastric adenocarcinoma. Significant improvement in overall survival is observed in patients with N0 disease who underwent D2 lymphadenectomy without increasing operative morbidity or mortality. This paper supports the notion of a global consensus for a D2 lymphadenectomy, particularly in the Western context. Gastric cancer is the fifth most common malignancy and the third leading cause of cancer death worldwide . In AustHistorically, there has been controversy regarding the extent of lymph node dissection performed between Eastern and Western countries. East Asian countries  have conIn Australia, there is a relative lack of data on gastric cancer outcomes and more specifically on D1 and D2 lymphadenectomy. Some units have adopted selective criteria for patients undergoing D2 lymphadenectomy, with no significant difference in 5-year survival or mortality .The aim of this study was to evaluate the operative outcomes of patients undergoing D1/D1+ and D2 gastrectomy for gastric adenocarcinoma.n = 150) and D1/D1+ lymphadenectomy (n = 54). Patients with prior gastric resection were not included. A standardized D1/D1+ or D2 lymphadenectomy with spleen/pancreas preservation was performed according to surgeon preference. D1/D1+ or D2 was performed as a standard preference for each surgeon, who are specialists trained in upper gastrointestinal surgery. There was no temporal difference between more D2 resections performed recently. Splenectomy and distal pancreatectomy were performed if there was direct tumor involvement.A retrospective analysis was performed on 204 patients who underwent gastrectomy for gastric adenocarcinoma at our institution between January 1996 and April 2016. Patients were classified into two groups on the basis the type of lymphadenectomy performed: D2 lymphadenectomy (According to the Japanese gastric cancer treatment guidelines 2014 (version 4), the Japanese Gastric Cancer Association (JGCA) defined the extent of lymphadenectomy according to the type of gastrectomy . For totPreoperative demographic data included age, gender, and body mass index (BMI). Pathological data included tumor location, depth of tumor (T), nodal status (N), tumor stage (TNM) stratified according to the American Joint Committee on Cancer (AJCC) 7th edition staging system , and tumPropensity score matching (PSM) analysis was performed. Patients undergoing D1/D1+ lymphadenectomy were matched to patients undergoing D2 lymphadenectomy based on similar estimated propensity scores. This is to balance covariates in the two groups to reduce selection bias . PropensOnly four parameters were used in the matching process as the overall patient numbers in this study are small, with 54 patients in the D1/D1+ group. If the sample size is too small for the propensity model to include all the variables of interest, then it is recommended to choose variables most strongly related to outcome , 20. TheIncreased BMI is associated with higher death rates from all cancers compared to normal weight patients. In addition, there is increased risk with higher BMI and death from gastric cancer in men . CompariThe use of chemotherapy in gastrectomy patients has been shown to affect survival. The United Kingdom Medical Research Council Adjuvant Gastric Infusional Chemotherapy (MAGIC) trial showed a higher likelihood of overall and progression-free survival in those undergoing perioperative chemotherapy . AccordiU test or t test as appropriate. Kaplan-Meier survival curves were constructed and evaluated using the log-rank test. Survival time estimates were expressed as mean (95%CI). Sub-group survival analyses were conducted according to AJCC 7TH Edition pathological node (pN) status. Independent variables that predicted overall survival were assessed with the use of the Cox proportional hazards model that included variables with a univariate p value of less than 0.25. Backward elimination was applied to determine independent variables that significantly impact survival [p < 0.05. All statistical analyses were performed using the Statistical Package for the Social Science (SPSS) version 22.0 software.Preoperative, pathological, treatment, and postoperative data were compared between groups. Results were expressed as the mean (standard deviation) and frequency (%). Categorical variables were compared using the chi-squared or Fisher’s exact test and continuous variables using the non-parametric Mann-Whitney survival . Statist2, p = 0.049; partial gastrectomy, 81 vs 63%, p = 0.011). Lymph node yield was higher in the D2 group .Two hundred four patients with gastric adenocarcinoma underwent primary gastric resection for gastric cancer over a 20-year period from 1996 to 2016 (Table n = 39 each) that had similar preoperative characteristics and tumor pathology (Table p = 0.050).After propensity score matching, 126 patients were excluded, resulting in two patient groups (n = 20 (37%), D2: n = 46 (30.7%). There were 68 patients with recurrence of disease with 17 (25%) occurring in the peritoneum. In the unmatched cohort, before PSM, the mean OS time for all patients was 11.40 years (95%CI 9.82–12.97). Mean OS time for D1/D1+ and D2 groups were 8.48 years (95%CI 5.80–11.16) and 11.97 years (95%CI 10.16–13.78), respectively. The 1-, 3-, 5-, and 10-year OS was 87.7, 75.2, 52.1, and 52.1% for the D1/D1+ group, and 93.0, 77.3, 64.7, and 56.6% in the D2 group respectively . After PSM, mean OS time for all patients was 11.97 years (95%CI 9.52–14.42). Mean OS for D1/D1+ and D2 groups were 7.14 years (95%CI 5.28–9.00) and 14.58 years (95%CI 11.66–17.86) years respectively. The 1-, 3-, 5-, and 10-year OS was 87.9, 74.1, 52.1, and 52.1% and 100, 90.7, 76.2, and 76.2% in the D1/D1+ and D2 group respectively. OS was significantly longer in patients in the D2 group (p = 0.008)  Fig. .Fig. 1Kap = 0.028)  where mean survival time was 9.47 years (95%CI 7.15–11.78) in the D1/D1+ group and 15.90 years (95%CI 12.14–19.66) in the D2 group and 5-year OS was 76.4% (D1/D1+) compared to 90.9% (D2) (p = 0.282), after PSM, the 10-year DFS was 35% for D1/D1+ and 58.1% for D2 (p = 0.058)  (Table p = 0.314). In univariate analysis of factors associated with 30-day mortality, total gastrectomy was a significant variable; however, this association was not present after PSM  nor PSM cohorts (p = 0.227) , proximal/middle tumor location (p = 0.009), and total gastrectomy (p = 0.020) were associated with postoperative complications and mortality. After PSM, advanced age (> 70 yr.) (p = 0.006) and total gastrectomy (p = 0.018) were also associated with post-surgical complications  (ns Table .p = 0.019) stage III /IV tumor (p = 0.000) and the presence of overall complications (Clavien-Dindo ≥ grade II) (p = 0.000) were poor prognostic factors. After adjusting for confounding effects in the multivariable analysis, stage III/IV tumors (HR 3.519 (95%CI 2.067–5.993), p = 0.000) and the presence of overall complications (Clavien-Dindo ≥ grade II) (HR 2.971 (95%CI 1.714–5.149), p = 0.000) were predictors of mortality. After PSM, the univariable analysis showed D1/D1+ lymphadenectomy (p = 0.013), total gastrectomy type, (0.034), stage III/IV tumors (p = 0.000), and overall complications (p = 0.001) were associated with mortality. Multivariable Cox regression analysis identified D1/D1+ (HR 4.353 (95%CI 1.511–12.514), p = 0.006), stage III/IV tumors (HR 4.218 (95%CI 1.560–11.407), p = 0.005), and overall complications (Clavien-Dindo > grade II) (HR 3.849 (95%CI 1.342–11.040), p = 0.012) as independent predictors of mortality. Results from univariable and multivariable analyses are summarized in Table In the unmatched cohort, total gastrectomy . After propensity score matching, OS was 52.1% for D1/D1+ and 76.2% for D2 which was significantly higher (p = 0.008)  [Our results showed improved long-term survival outcomes for D2 lymphadenectomy compared to results reported in Western literature and approaches those reported in Eastern studies. In a meta-analysis of four RCTs, the 5-year OS for D2 resections was 47% . Specifi = 0.53) . Howeverp = 0.028)  compared with the D1/D1+ group 76.4%) (6.4% (p =p = 0.282), after PSM, the 10-year DFS was 35% for D1/D1+ and 58.1% for D2 (p = 0.058)  and 1.2% (after PSM), which is comparable to other units. The 2016 National Oesophago-Gastric Cancer Audit of England and Wales reports 2.2% in-hospital mortality and 30-day mortality rates range between 4.5 and 1.9% . The D2 The overall postoperative complication rate was 61% (unmatched) and 51% (after PSM) Table . The majp = 0.009), this was no longer present after PSM (p = 0.07). Total gastrectomy was associated with postoperative complications in both matched and unmatched cohorts , which has been shown in other studies [p = 0.076), suggesting the influence of confounding factors in the unmatched cohort in which there was an association (p = 0.021)  are listed in Table M p = 0.0. Total g studies . Total gp = 0.008; after PSM, p = 0.006); however, these were mostly Clavien-Dindo grade I and II complications. Older age was not associated with 30-day mortality  vs D2 group (24 kg/m2) (p = 0.049) (Table 2 [Body Mass Index was not associated with post-operative morbidity or mortality. The mean BMI was significantly higher in the unmatched cohort between the D1/D1+ (26 kg/m(Table 2 .Pancreaticosplenectomy was not routinely performed as part of the D2 lymphadenectomy and subsequently not associated with the post-operative complications or 30-day mortality. This is supported by other studies, whereby a D2 lymphadenectomy with spleen preservation avoids operative morbidity without affecting survival . The Jap2, indicate that an extended lymph node dissection with spleen and pancreas preservation can be performed in the Western cohort with no added morbidity.Some surgeons who are proponents of D1/D1+ prefer to do so in Western patients who often have a higher BMI, arguing that a D2 is technically easier in slimmer, Eastern patients. The results for our cohort, with BMI < 30 kg/mThe use of perioperative chemotherapy in the management of gastric cancer has been a major development over the past decade, with increased survival in perioperative chemotherapy versus surgery alone . The numThe results of our morbidity and mortality are similar to those reported in the literature, thus suggesting that identification of high-risk factors may allow more rational patient selection or systemic therapy . In asseOur results indicate that D2 lymphadenectomy with spleen/pancreas preservation is not associated with poor short-term and long-term outcomes whereas a D1/D1+ dissection was one of the factors that predicted poor overall survival in our unit. This finding is comparable to results of a recent meta-analysis  that pooThis study is limited by its retrospective nature with possible selection and information bias. This data represented the experience of a single tertiary academic referral center over two decades and is subject to referral bias. In addition, the number of patients included in this analysis was relatively small resulting in a low powered study; however, this is considered a large series from a single Western center in Australia.Furthermore, it is difficult to adequately account for the surgical techniques employed by the operators including contamination and compliance, which has been an issue in many surgical studies . While tIn comparing patients that underwent a D1/D1+ lymphadenectomy versus the D2 lymphadenectomy in the context of gastrectomy for gastric adenocarcinoma, we used direct comparison of outcomes with multiple regression to adjust for confounding factors. Given there were 150 patients in the D2 group compared to 54 in the D1/D1+ group, there is risk for outcomes to be confounded due to systemic differences between the two groups. In order to address this, we also performed propensity score matching, to estimate the causal effect of D2 lymphadenectomy compared to D1/D1+ and reduce the effect of bias, and furthermore compared the matched groups using a regression model to further adjust for imbalances.The Australian experience within this single unit shows that D2 lymphadenectomy with spleen and pancreas preservation can be performed safely on patients with gastric adenocarcinoma with excellent survival outcomes. Significant improvement in overall survival is observed in patients who underwent D2 lymphadenectomy, without increased surgical morbidity and mortality. This survival benefit was also seen in a subgroup analysis of N0 patients who underwent a D2 lymphadenectomy. This paper supports the notion of global consensus for D2 lymphadenectomy, particularly in the Western context.


In both stimulated and unstimulated samples, in the examined group, IL-1β and IFN-γ had higher concentrations and RANTES had lower concentrations when compared to those in the control group. In the examined group, IL-4 and IL-17A had higher concentrations without stimulation and TNF-α had higher concentrations with stimulation. The GSV samples had higher IL-2, IL-4, IL-12 (p70), and IFN-γ concentrations without stimulation and lower IL-2 and TNF-α concentrations with stimulation when compared to those of the upper limb in the examined group. These observations indicate that the oscillatory flow present in incompetent veins causes changes in the cytokine production by lymphocytes, promoting a proinflammatory profile. However, the relations between immunological cells, cytokines, and the endothelium require more insight.The pathogenesis of chronic venous disease (CVD) remains unclear, but lately inflammation is suggested to have an important role in its development. This study is aimed at assessing cytokines released by lymphocytes in patients with great saphenous vein (GSV) incompetence. In 34 patients exhibiting oscillatory flow (reflux) in GSV, blood was derived from the cubital vein and from the incompetent sapheno-femoral junction. In 12 healthy controls, blood was derived from the cubital vein. Lymphocyte culture with and without stimulation by phytohemagglutinin (PHA) was performed. Interleukins (IL) 1 According to multiple studies, various stages of CVD may affect up to 85% of the population and more advanced clinical changes  classification) occur in about 30% of the population –3. Venou(ICAM-1) . Next, e(ICAM-1) .However, there are very few studies concerning cytokines in this disease . There aThe study has been carried out in accordance with the Declaration of Helsinki and approved by the Bioethical Committee of the Medical University of Silesia (KNW/0022/KB1/31/I/12). All participants gave their written informed consent for the study.The examined group consisted of 34 primary CVD patients with great saphenous vein (GSV) incompetence confirmed by Doppler ultrasound examination. The reflux at the sapheno-femoral junction (reflux time > 0.5 s) was confirmed in all patients in standing position, with blood flow elicited by manual squeezing. The control group included 12 volunteers with healthy GSV confirmed by Doppler ultrasound. The exclusion criteria involved history of venous thrombosis, pregnancy, diabetes, any inflammatory diseases present in the last two weeks, alcohol abuse, smoking, ulceration on the examined limb during the last month, and intake of anti-inflammatory drugs during the last two weeks.Blood samples were obtained from the cubital vein in both groups and collected to vials containing heparin (10 IU/ml of blood). The patients from the examined group then underwent a standard surgical procedure of GSV stripping, with femoral nerve block and additional local anaesthesia. After inguinal incision and visualization of the GSV, the blood sample was obtained from the GSV directly below the incompetent sapheno-femoral junction into a heparinized vial. All samples were immediately transferred to the laboratory, maintaining 37°C. Cultures of lymphocytes were prepared either with lymphocyte-stimulating phytohemagglutinin (PHA) or with medium.g, 30 min), the separated lymphocytes were transferred to another vial and washed twice with phosphate-buffered saline (PBS) . Microscopic morphological assessment of the cell population was performed, and no differences were found between groups. No significant contamination by other cells was found in the samples. A suspension of 2 mln lymphocyte cells/ml of medium  1640, 10% bovine serum, penicillin 100 μg/ml, and 100 μg/ml streptomycin). 0.5 ml of this suspension was added to 0.5 ml of PHA solution (20 μg PHA/ml of medium) and to 0.5 ml of medium for no-stimulation samples. These suspensions were incubated for 24 h at 37°C, 5% CO2 atmosphere, and 99% humidity. After incubation and centrifugation , the supernatant was collected to Eppendorf vials and stored at −80°C.The lymphocytes were separated by the use of Histopaque gradients (1.119 g/ml and 1.077 g/ml). After centrifugation , and 17A , and IL-17A); interleukin 1 receptor α (IL-1ra); tumor necrosis factor-α (TNF-α); interferon-gamma (IFN-γ); and RANTES were assessed. The samples were thawed directly before the Bio-Plex assay. The assay uses magnetic beads with anticytokine immunoglobulins to assess concentrations of many cytokines simultaneously. The samples were processed following the manufacturer's instructions  and read using Bio-Plex Manager™ Software. The statistical analysis was performed with the use of STATISTICA 10.0 software. Because there was no normal distribution of the examined cytokine samples, nonparametric tests were applied . The leukocyte count and lymphocyte percentage had normal distribution; therefore, Student's t-test was applied.Interleukins 13/μl (mean) (3.7–8.8 × 103/μl) in the examined group and 5.9 × 103/μl (4.6–7.5 × 103/μl) in the control group. The lymphocyte percentage was 39% (mean) (22%–47%) in the examined group and 36% (23%–45%) in the control group. There were no statistically significant differences between groups.The examined group consisted of 34 patients, 85% of which were women. The median age was 47 ± 25 (21–68). The patients belonged to clinical CEAP classes C2-C3, with 43% in C2 and 57% in C3. The control group consisted of 12 patients, 92% of which were women. The median age was 36 ± 27 (29–64). The white blood cell count was 5.6 × 10γ were found in the incompetent GSV samples in comparison with the cubital vein samples of the same patients  pg/ml versus 8.7 ± 11.6 (1.3–43.0) pg/ml, p < 0.05; IL-4: 12.6 ± 3.2 (5.2–19.7) pg/ml versus 10.7 ± 2.9 (6.0–18.6) pg/ml, p < 0.01; IL-12 (p70): 26.9 ± 139.3 (11.1–295.8) pg/ml versus 25.7 ± 31.9 (1.9–170.5) pg/ml, p < 0.05; and IFN-γ: 361.3 ± 157.7 (124.7–774.3) pg/ml versus 335.2 ± 165.9 (108.6–543.1) pg/ml, p < 0.05). The above results are presented in Figures 1In the samples cultured without stimulation of the examined group, significantly higher concentrations of IL-2, IL-4, IL-12 (p70), and IFN-p < 0.01)  pg/ml versus 7868 ± 18934 (486.3–55230) pg/ml,  < 0.01) .β, IL-4, IL-17, and IFN-γ concentrations were higher in the examined group (IL-1β: 35.8 ± 62.0 (7.5–446.3) pg/ml versus 10.1 ± 26.9 (5.3–247.0) pg/ml, p < 0.05; IL-4: 10.73 ± 2.97 (6.0–18.61) pg/ml versus 8.45 ± 5.28 (5.19–15.16) pg/ml, p < 0.05; IL-17A: 237.1 ± 107.0 (87–1740) pg/ml versus 140.1 ± 60.6 (79.3–365.2) pg/ml, p < 0.01; and IFN-γ: 335.2 ± 165.9 (108.6–543.1) pg/ml versus 189.3 ± 165.9 (107.4–439.3) pg/ml, p < 0.01) .PHA stimulation resulted in significantly higher concentrations of all examined cytokines in both groups with the exception of IL-12 (p70) and RANTES. IL-12 (p70) had higher concentrations only in the upper limb of the examined group when compared to samples without stimulation, and RANTES concentrations were significantly higher only in the lower limb samples of the examined group. The upper limb samples of the examined group had higher RANTES concentrations with borderline statistical significance (7868 ± 18935 (486.3–55230) pg/ml versus 8394 ± 16724 (595.8–56391) pg/ml, α and IL-2 concentrations in comparison with the lower limb samples (TNF-α: 1899 ± 4204 (319.7–11115) pg/ml versus 1547 ± 1614 (294.4–8314) pg/ml, p < 0.01 and IL-2: 123.3 ± 152.3 (30.6–1805) pg/ml versus 84.3 ± 70.9 (20.0–3139) pg/ml, p < 0.01)  pg/ml versus 122.0 ± 135.5 (32.0–291.0) pg/ml, p < 0.01; TNF-α: 1900 ± 4204 (319.7–11115) pg/ml versus 1170 ± 1879 (159.0–4179) pg/ml, p < 0.05; and IFN-γ: 692.6 ± 523.9 (303.1–4978.26) pg/ml versus 382.3 ± 253.9 (165.0–587.2) pg/ml, p < 0.01)  (RANTES had higher concentrations in controls (43052 ± 18701 (18124–68296) pg/ml versus 8395 ± 16725 (595.8–56392) pg/ml,  < 0.01) .α, interferon-gamma-induced protein-10 (IP-10), IL-15, and granulocyte colony-stimulating factor (G-CSF) concentrations [γ in the samples derived from the incompetent sapheno-femoral junction in comparison to the upper limb veins, exhibiting laminar flow in the same patients. The only other two studies available, comparing varicose veins to general circulation of the same patients, showed that in varicose veins, there are elevated levels of cytokines: endothelial growth factor (EGF), platelet-derived growth factor (PDGF), RANTES, interleukin 6, interleukin 8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1) [One of our hypotheses was that due to the pathological blood flow present in the incompetent sapheno-femoral junction, causing endothelial damage, lymphocytes in contact with the venous wall may exhibit function changes. A study by Zamboni et al. examined 19 cytokines in CVD and showed that the flow correction caused significant amelioration of TNF-trations . This in (MCP-1) , 19. In  (MCP-1) . Our resβ, IL-4, IL-17A, and IFN-γ were found in cubital veins of CVD patients in comparison to the healthy group, further implying proinflammatory condition. However, a leukocyte-recruiting chemokine RANTES had significantly higher concentrations in the healthy group when compared to the varicose patients. In some of the few other available studies on cytokines in CVD, lower cytokine concentrations in varices were also found. Tisato et al. reported that reflux time correlates negatively with the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) [α, and vascular endothelial growth factor (VEGF)) [Higher concentrations of IL-1(GM-CSF) , and Spa (VEGF)) . On the  (VEGF)) . There a (VEGF)) . The ele (VEGF)) ; howeverThe PHA stimulation of lymphocytes was performed in our study in order to establish the potential of the lymphocytes in the incompetent veins to respond to activating factors. PHA is a known lymphocyte T stimulant. The lymphocyte B response to stimulation was therefore not assessed and requires further study, for example, with lipopolysaccharide as a stimulant.α levels in the upper limb of the examined group when compared to the incompetent GSV. Our other observation, in which PHA caused a significant increase in IL-12 (p70) only in the upper limb of the examined group and not in the GSV samples or in the control group, may partially explain these results as IL-12 (p70) on its part stimulates lymphocyte T to produce TNF-α. The higher concentrations of TNF-α in the upper limb of the examined group were also higher in comparison to those of the control group, which requires explanation. In the literature concerning cytokines in CVD, we found that osteoprotegerin (OPG) is released in higher concentrations by endothelial cells in CVD patients and that it correlates positively with reflux time [α, and its production is stimulated by estrogens. Taking into account that females are more prone to CVD and that in our study, the highest TNF-α concentrations were observed in the healthy veins of CVD patients in the stimulated cultures, TNF-α needs further examination as it might play a still unknown role in CVD pathogenesis.Stimulation resulted in significantly higher IL-2 and TNF-lux time , 20. OPGα, in the examined group, higher IL-1β and IFN-γ levels were also found, confirming the earlier observation of elevated production of proinflammatory cytokines in incompetent veins. The production of proinflammatory cytokines in response to stimulation might be of significance, for example, in venous ulcers, where with the progression of venous stasis and vascular permeability, the lymphocytes are exposed to additional stimuli (chemokines and cytokines released by other leukocytes) [Apart from TNF-kocytes) .We also observed that RANTES concentrations were significantly higher after PHA simulation only in the GSV samples. However, even without significant changes in RANTES concentrations in the control group after PHA stimulation, its concentrations remained significantly higher in comparison to those in the CVD patients. This might indicate lower lymphocyte potential to recruit other leukocytes in the varicose veins in response to stimulation.In this study, we did not analyse the relation between cytokine concentrations and dilation of the venous wall. A significant reduction of contractile response to vasoactive agents was shown before, progressing with the severity of the disease, and, interestingly, angiotensin II affinity was significantly lower already in the early stages of the disease . CytokinA small examined group is certainly a limitation of this study. However, the complexity of obtaining lymphocyte cultures involving immediate processing of each acquired sample and strict exclusion criteria prevent creating a large examined group. Similar obstacles were met by other researchers in this field , 18, 20.γ by lymphocytes derived from the incompetent sapheno-femoral junction, suggesting a role of reflux in lymphocyte function. High concentrations of IL-1β, IL-4, IL-17A, and IFN-γ in general circulation of CVD patients also show a proinflammatory tendency in comparison to those of the healthy group. However, RANTES is found in higher concentrations in healthy subjects than in CVD patients, and TNF-α and IL-2 concentrations are higher after stimulation in the healthy veins of the examined group. These observations require further studies of complex interactions between the cytokines, immunological cells, and endothelium subject to pathological blood flow in order to establish their role in CVD pathogenesis. Better understanding of the immunological aspects of this disease may lead to new preventive measures and more effective treatment, for example, with effective anti-inflammatory drugs.Our observations indicate higher production of cytokines IL-2, IL-4, IL-12 (p70), and IFN-


Bacillus strain with a bioprotective potential to alleviate maize fusariosis in South Africa. We selected seven Bacillus isolates  with biosuppressive effects against two maize fungal pathogens (Fusarium graminearum and Fusarium culmorum) based on 16S rDNA gene characterization and lipopeptide gene analysis. The PCR analysis revealed that lipopeptide genes encoding the synthesis of iturin, surfactin, and fengycin might be responsible for their antifungal activities. Few of the isolates also showed possible biosurfactant capability, and their susceptibility to known antibiotics is indicative of their eco‐friendly attributes. In addition, in silico genomic analysis of our best isolate (Bacillus velezensis NWUMFkBS10.5) and characterization of its active metabolite with FTIR, NMR, and ESI‐Micro‐Tof MS confirmed the presence of valuable genes clusters and metabolic pathways. The versatile genomic potential of our Bacillus isolate emphasizes the continued relevance of Bacillus spp. in biological management of plant diseases.The results of this study indicate that the maize rhizosphere remains a reservoir for microbial strains with unique beneficial properties. The study sought to provide an indigenous  HiCrome Bacillus agar is used for rapid identification of Bacillus spp. from a mixed culture by a chromogenic method. Following manufacturer's directions, distinct colonies were randomly selected from the agar plates of each sample. Grams reaction, oxidase activity, and catalase were performed to presumptively ascertain the genera of the selected isolates. Then, 200 isolates were selected and maintained at −80°C in Luria–Bertani (LB) broth with 15% (v/v) glycerol, and a 15 ml LB broth or agar slant of each isolate was kept at 4°C as working culture.Five grams of each soil sample was inoculated in 45 ml of LB (Sigma‐Aldrich L3522) broth and incubated for 16 hr with continuous shaking, at 150 2.32.3.1Fusarium graminearum and Fusarium culmorum were kindly provided by Dr Claire Prigent Combaret (UMR CNRS 5557) Microbial Ecology of Lyon, University Lyon 1, France, and Prof Cristina Cruz, Centre for Ecology, Evolution and Environmental Changes, Faculdade de Ciências da Universidade de Lisboa, Portugal, respectively, and they were maintained on potato dextrose agar (PDA Sigma‐Aldrich P2182) plates. Preliminary detection of the antagonistic activities of the 200 Bacillus isolates against F. graminearum was carried out by multiple confrontation dual culture tests. The protocols of Chen, Chen, Zhang, and Zhu (F. graminearum was placed in the center of freshly prepared PDA plates (90 mm). From the 200 Bacillus isolates initially selected, six fresh colonies from 24 hr LB agar culture were circularly streaked (equidistance 1.5 cm) along each PDA plate at a distance of 1.5 cm from the edge of the plate using a sterile inoculating loop. Control plates consisted of F. graminearum placed on PDA alone. The plates were further incubated at 28°C for 7 days. Thereafter, only 11 isolates  exhibiting strong inhibition were selected for further antifungal confirmatory tests. and Zhu  were sli2.3.2F. graminearum and F. culmorum) in three conditions. (a) Single loop full of bacterial antagonist streaked at the center of PDA 3 days before both fungal agar plugs were inoculated on opposite sides (condition 1); (b) single loop full of bacterial antagonist streaked at the center of plate while simultaneously inoculating both fungal agar plugs on opposite sides (condition 2); and (c) single loop full of bacterial antagonist streaked 3 days after the fungal agar plugs were inoculated on opposite sides (condition 3). The Bacillus isolate with strong inhibition zones (around their streaks) against the two pathogens in the screening plates was selected for further characterization. The antagonistic effect was determined by measuring the zones of inhibition (mm). The percentage of growth inhibition was calculated using the formula.Plates were prepared as described above; however, antagonism was carried out against two fungal pathogens . Plates were incubated from 48 to 72 hr at 37°C  following manufacturer's procedure. DNA quantity and quality were assessed with spectroscopic methods using a NanoDrop 1000 , and the DNA was used as the template for polymerase chain reaction (PCR) analysis.We further chose seven isolates  out of the 11 2.6Bacillus isolates was by 16S rDNA gene sequencing , and 9.5–10.5 μl nuclease‐free water in PCR thermocycler. All the primers utilized in PCR amplification protocols were synthesized by Whitehead Scientific, Integrated DNA Technologies  filters to obtain cell‐free supernatants. About 100 ml of cell‐free supernatant was stored for anti‐pathogen test. Isolate BS10.5 was further grown in 1 L LB broth at 30°C with constant shaking at 200 g for 4 days. After fermentation, the cell filtrate was collected by centrifugation at 6,000 g for 15 min at 4°C, and the supernatant was acid‐precipitated by adjusting to pH 2.0 with 6 M HCl. After an overnight incubation at 4°C, the precipitate was centrifuged at 8,000 g at 4°C for 15 min and the pellet was dissolved in methanol–water (50:50) and then filtered through 0.22 µm PTFE membrane filter to remove larger particles and cell components. The mixture was then concentrated by vacuum evaporator at 45°C and then finally lyophilized.From the seven antagonistic subsets, isolate BS10.5 (NWUMFkBS10.5) was the most effective with the highest number of encoding genes, and it was chosen for further analysis. The production and purification of BS10.5 active metabolites were done according to Gond, Bergen, Torres, and White  with sli2.82.8.1Bacillus cereus ATCC 10876, EF = Enterococcus faecalis ATCC 29212, KP = Klebsiella pneumoniae ATCC 25923, and PA = Pseudomonas aeruginosa ATCC 27853). Control plates included antibiotic disks of norfloxacin (5 μg/disk) and tetracycline (30 μg/disk). The plates were incubated overnight at 25°C and 28°C. Antibacterial activity was observed as inhibition zones around the disk, and the experiment was conducted twice in triplicates.The activity of the cell‐free supernatants was determined by disk diffusion assay. Sterile filter paper disks were impregnated with 60 µl of the cell‐free supernatant of BS10.5, and the disks were placed on a MHA plate previously inoculated with bacterial pathogens (BC = 2.8.2Fusarium pathogens (F. graminearum and F. culmorum) were transferred to the opposite edge of the PDA plates. Nystatin (30 μg/disk) was used as control, and after 7 days of incubation and observation of plates at 25°C, zones of inhibition were recorded.Sterile filter paper disks impregnated with 60 µl of the cell‐free supernatant of BS10.5 were placed at the far edge of a PDA plate, and 5‐mm agar plugs of the 2.9Disk diffusion was employed for the antimicrobial assay following the method described by Chen, Wang, Wang, Hu, and Wang  with mod2.10Using well diffusion, the activity of the BS10.5 extract powder was also compared to the activity of commercial fungicides using PBS as diluent following a modified protocol of Mousa, Shearer, Limay‐Rios, Zhou, and Raizada . 0.2 g/m2.112.11.1−1) with an average of 32 scans. Spectrum was viewed and collated with the OPUS spectroscopy software.Fourier transform infrared (FTIR) spectroscopy identifies the types of chemical bonds and functional groups in compounds, and it is useful in elucidating the components of an unknown sample. Ten microgram of the lyophilized extract of BS10.5 was analyzed with transform alpha (FTIR) KBr‐integrated spectrometer (Bruker). Spectrum reading was at 400–4,000 wavenumbers .Twenty microgram of the lyophilized extract of BS10.5 was dissolved in 0.5 ml of deuterated DMSO (dimethyl sulfoxide), and 2.11.3A high‐resolution mass spectrum was obtained for the lyophilized extract of BS10.5 with an Applied Biosystems 4800 Plus MICRO‐TOF/TOF analyzer  operated in the positive ion mode with an accelerating voltage of 20 kV, 337 nm nitrogen laser for ionization and α‐cyano‐4‐hydroxycinnamic acid for matrix. Bruker compass data analysis was used to process the mass spectrometry data while molecular weights and formulae were characterized by mass spectrum smart formula tools. The FTIR, NMR, and mass spectrometry analyses were carried out at the Chemical Resource Beneficiation/Laboratory Analytical Services of the North‐West University .2.12Bacillus strain NWUMFkBS10.5, (the preferred isolate), was extracted using the Zymo Research ZR Soil Microbe DNA Miniprep Genomic Isolation Kit and sequenced with Illumina MiSeq Reagent Kit v2 microsystem by MR DNA  using their protocols. The Kbase , and close gaps and remove adaptor sequences (Cutadapt v1.0.1). The reads were also assembled into contigs on the Kbase platform using ARAST v0.0.4 (Velvet and Kb_SPAdes v0.0.9). Contigs from the Kbase were uploaded on the RAST Server version (v) 2.0  and PRISM were used to predict the biosynthetic products and genetic clusters present in BS10.5. A Pan‐genome was created on the Kbase platform to run a comparison of the total genes present in BS10.5 and other established biocontrol 2.14A multivariate general linear model was used to analyze treatment means and inhibition rates. Least significant difference test (LSD), Duncan multiple test, and Student–Newman–Keuls (SNK) test were used to compare observed means, pathogen–antagonist relationship, treatment effects, and effect of conditions of inoculation using SPSS statistical software (version 22) at the significance level of 5%.33.1Bacillus agar according to manufacturer's descriptions  and condition 2 compared to condition 3, where the fungal mycelia plug was inoculated on the PDA 3 days before the antagonists were inoculated  are biosurfactant producers Table .3.4SrfC primer. However, only three isolates harbored iturin genes, and no amplification was detected using the fengycin (FenD), Ipdc (indole pyruvate decarboxylase), and Acc deaminase primers. Single predominant amplicon bands were produced from the PCR products from the gel electrophoresis   Figure .3.93.9.1−1, CH stretch at 3,000–2,500 cm−1, NH stretch at 2,500–2,000 cm−1, COO‐ at 1,900–1,500 cm−1, and CC and CN at 1,500–1,000 cm−1 .The proton analysis of the compound showed NH and OH proton at >8.00 ppm, some –CHm Figure , gave a 3.9.3m/z 186.1026, 365.1063, 750.4056, 1,058.6738, 1,477.8184, and 2,095.3363. Mass 1,058.6738 suggests the possible presence of iturin, surfactin, and bacillomycin fragments, while 1,477.8184 represents the mass fragment of fengycin, respectively , a single lantipeptide, a type 3 polyketide synthetase (T3pks), two trans‐AT PKS nonribosomal peptide synthetases, a single bacteriocin nonribosomal peptide synthetase cluster and two unidentified ketide synthetases . The genome has four nonribosomal peptide synthetases (NRPSs), two terpenes, three es Table a–k. In S3.12Bacillus strains with NWUMFkBS10.5 strains confirmed the distinctive status of BS10.5 and its genetic relatedness   showed no sensitivity. This gives an indication of the lowest possible concentration at which the pathogens will remain susceptible. Dose‐dependent effects of lipopeptides have been reported elsewhere  are mostly detected at 1,030.8, 1,044.8, 1,046.8, 1,058.8, 1,060.8, and 1,074.8, that of bacillomycin  at 1,031.7, 1,045.7, 1,053.7, 1,059.7, 1,067.7, 1,069.7, 1,081.7, 1,083.7, 1,097.7, 1,095.7, 1,111.7, 1,527.8, 1,529.9, and 1,543.8, those of fengycins  at 1,449.9, 1,463.9, 1,471.9, 1,477.9, 1,485.9, 1,487.9, 1,491.8, 1,499.9, 1,501.9, 1,505.8, 1,513.9, and 1,515.9  prominently reported in The presence of surfactin, bacillomycin, iturin D, and fengycin D biosynthetic genes, detected in the BS10.5 amplicons during PCR analysis, was confirmed from the WGS data. The BS10.5 genome analysis showed similarity identity above 85% for some biosynthetic genes clusters (BGC). Technically, BGC or biosynthetic genes are considered to be present when the similarity index is 65% and above  . The iturin cluster was predictably found in cluster 6, subcluster BGC0001098     Table  and did 3) Chen, , ericin B. velezensis strains, and this suggests possible evolutionary pressure for the development of competitive antimicrobials among the B. velezensis group. Our goal in this study was to provide a bioprotective Bacillus strain for maize against the onset of fusariosis. B. velezensis NWUMFkBS10.5 was prudentially selected because it harbored functional and metabolically active gene clusters responsible for synthesizing diverse beneficial compounds. To date, no standard effective chemical fungicide exists for the treatment of fusariosis in South Africa. To the best of our knowledge, this is a rare report describing the genomic and biocontrol potential of a native B. velezensis strain peculiar to Africa. Although we did not corroborate our results with in planta experiments in this study, the exhaustive nature of this combinatorial in vitro study affirms the in planta viability of B. velezensis NWUMFkBS10.5. Lastly, the information gathered on NWUMFkBS10.5 will be valuable for its biotechnological manipulation and its probable development into a biofungicide in South Africa.Remarkably, biosynthetic clusters predicted to synthesize compounds such as sessilin, bananamides, cichopeptin, taiwachelin, tolaasin, basiliskamides, kalimantacin, and mersacidin were however identified in strain NWUMFkBS10.5 Table . These cThe authors declare that they have no conflict of interests.AAA designed and performed the in vitro assays/experiments, molecular analysis, in silico genome analysis, analyzed the data, and drafted the manuscript. OSA contributed to the chemical characterization and its data interpretation. OOB participated in the experimental design, supervised the work, and provided funding. All authors contributed to the drafts, final version of the paper, and approved submission.This article does not contain any studies with human participants or animals performed by any of the authors. Click here for additional data file.


Aspergillus niger BCRC31494 has been used in the food fermentation industry because of its thermal and alkaline tolerance. It was cloned and expressed in Pichia pastoris. According to sequence analysis, the gene open reading frame comprises 1,217 bp with five introns (GenBank GQ292753). According to sequence and protein domain analyses, EGLB was assigned to glycosyl hydrolase family 5 of the cellulase superfamily. Several binding sites were found in the promoter region. The purified recombinant enzyme was induced by 0.5% methanol, and it exhibited optimal activity at 70 °C and pH 4. EGLB was stable for 3 h at temperatures below 60 °C, with more than 90% of its activity remaining. The enzyme was specific for substrates with β-1,3 and β-1,4 linkages. In Lineweaver-Burk plot analysis, the mK and maxV values of EGLB for β-D-glucan were 134 mg/mL and 4.68 U/min/mg, respectively. The enzyme activity was increased by 1.86-fold by Co2+ and by 2-fold by Triton X-100 and Tween 80. These favorable properties make EGLB a potential candidate for use in laundry and textile industrial applications.Endoglucanase B (EGLB) derived from  Cellulose, a polymer of glucose with β-1,4 linkages, is the most abundant biomass on Earth. Its abundance and its renewability make it a great candidate energy source and feedstock. After several oil crises, bioethanol has been promoted as an alternative energy source, and the production of bioethanol from cellulosic biomass has been widely explored. Enzymes degrading cellulose are found in microbes, plants, and the digestive tracts of animals .www.cazy.org). Endoglucanase belongs to 17 GH families, namely families 5, 6, 7, 8, 9, 12, 16, 17, 44, 45, 48, 51, 55, 61, 74, 81, and 124. Within GH5, which primarily contains endo-1,4-glucanases, six different subtypes (A1–A6) were proposed  software, and 13 possible eglB glycosylation positions were found. From the result of RPS-BLAST alignment, eglB belongs to glycosidase family 5 of the cellulase superfamily. The proton donor of Glu160 and catalytic nucleophile of Glu266 are consistent with the characteristics of family 5 [eglB was also determined using Signal P 3.0. The result suggested that amino acid residues 1–18 comprise a signal peptide, and the cleavage site was between residues 18 and 19 from the first methionine found in the amino acid sequence. The highly conserved sequence was followed by catalytic nucleophiles, which may be glycosylation sites, and several possible glycosylation sequences were found in this fragment. Another possibility is that these highly conserved sequences were the glycosyl residues of family 5. The glycosidic bond cleavage reaction, including the proton donor, catalytic nucleophile, and five other conserved residues of family 5 are involved belonging to retaining mechanism [The amino acid sequence of the purified EGLB was analyzed using OGPET [Prediction of family 5 . Celluloechanism ,23.P. pastoris and secreted into the medium. After induction with methanol, the carboxymethyl cellulose (CMC) hydrolysis activity of the 70 transformants was analyzed, and 10 transformants with high EGLB activity were selected. Strain 31 had the highest endoglucanase activity, and it was used for subsequent experiments. In our laboratory, Strain 31 was induced with different concentrations  of methanol, and induction with 0.5% methanol yielded the highest enzyme activity  [Syncephalastrum racemosum (70 °C) [Bacillus subtilis BME-15 (50 °C) [The recombinant enzyme EGLB was more stable than the wild-type enzyme. The wild-type enzyme was purified according Vidmar s method . The rel (60 °C) , the the (70 °C) , and the (50 °C) . The recet al. [A. niger displayed thermal stability (up to 50 °C) in acidic conditions. In this study, the thermal stability of EGLB at temperatures as high as 60 °C at pH 5.0 was higher than that reported by Vidmar et al. [endo-β-1,4-glucanase B from A. oryzae KBN616 was stable up to 50 °C and became inactivate rather sharply above 55 °C [A. niger ATCC10574 was stable at temperature between 30 to 50 °C for 30 min [A. niger CBS120.49 was stable at 30 °C for 148 h [A. niger IFO31125 showed thermal stable at 70 °C for 1 h [The thermal stability of EGLB is presented in et al.  reportedr et al. . The theve 55 °C . The Eglr 30 min  and the or 148 h . EGLB in for 1 h  which isendo-β-1,4-glucanase B from A. oryzae KBN616 was stable in pH 3 to 7 [A. niger ATCC10574 was stable between pH 2 to 7 [A. niger CBS120.49 was stable at pH 3.5 to 5 [A. niger in this study was stable at pH 3 to 10, and very stable at alkaline region  and maximum velocity (maxV) of recombinant EGLB for barley β-glucan were 53 mg/mL and 134 U/min/mg, respectively, compared with 32 mg/mL and 4.68 U/min/mg, respectively, for CMC-Na. Kinetic analysis revealed that EGLB has a greater affinity for barley β-glucan than for CMC-Na. The result confirmed that EGLB is a β-1,3-1,4-glucanase. Recently, an endoglucanase from the acidophilic fungus Bispora sp., MEY-1, was also reported as an endo-β-1,3-1,4-glucanase [The substrate specificity and kinetic parameters of EGLB were measured by releasing reducing sugars from several substrates with various linkages . The higlucanase .2+, Triton X-100, and Tween 80 by 186, 201, and 205%, respectively. The enhancement of endoglucanase activity by cobalt ions was previously reported in bacteria [A. niger ATCC10574 (160%) [Trichoderma virde (130%) [et al. [A. niger can be activated by Triton X-100 and Tween 80. However, the enzyme was strongly inhibited by SDS. Partial inhibition (<20%) was observed in the presence of certain metal ions, including Mg2+, Zn2+ and Ca2+, whereas the enzyme activity was not affected by Cu2+ and EDTA.The effects of metal ions and various compounds (10 mM) on EGLB activity are shown in bacteria , and als4 (160%)  and the e (130%) . Surfacte (130%) ,34. Zhen [et al.  divided The purified enzyme was incubated with the added compound for 10 min prior to addition of 0.5% (wt/v) CMC-Na in Na/acetic buffer (pH 5.0) for 30 min at 40 °C. Values represent the relative activity expressed as a percentage of the activity observed in the absence of compound. Values shown are the mean of duplicate experiments, and the variation about the mean was <5%.Aspergillus niger BCRC 31494 and Escherichia coli DH5α were purchased from the Bioresource Collection and Research Center (BCRC), Food Industry Research and Development Institute . Pichia pastoris GS115 was obtained from Invitrogen . PDA slant medium (1.8% Bacto-agar and 2.4% Potato dextrose) was used to culture the fungal spores at 30 °C for 3 days. After inoculating spores (1 × 107) into 250 mL of minimal medium (0.05% (NH4)2SO4·3H2O, 0.02% KH2PO4, 0.02% MgSO4, 0.01% CaCl2·H2O, 0.0001% FeSO4·6H2O, 0.0001% ZnSO4·7H2O, 2 mM citric acid, and 1% bran, pH 3.5) at 37 °C and 150 rpm for 3 days, the mycelia were harvested and used for DNA and RNA isolation. Pichia pastoris GS115 was cultured in YPD medium  at 30 °C and 200 rpm. E. coli DH5α was grown in LB medium  or low salt LB medium  at 37 °C and 200 rpm for DNA manipulation and expression. The plasmid pPICZαC was used as an expression vector and purchased from Invitrogen.A. niger BCRC 31494 was grown in 250 mL of minimal medium at 37 °C and 150 rpm for 3 days. The mycelia were harvested and then ground into a fine powder in liquid nitrogen with a mortar and pestle. The mycelia were then lysed, and total RNA was isolated using TRIzol® and an RNA purification kit (Invitrogen) according to the manufacturer’s protocol. Genomic DNA was isolated from A. niger BCRC 31494 using the Plant Genomic DNA Mini Kit  according to the manufacturer’s protocol. Plasmid DNA isolated from E. coli DH5α was extracted using the Mini M™ Plasmid DNA Extraction System  according to the manufacturer’s protocol.To isolate total RNA, A. niger (NCBI GenBank accession Nos. AJ224452 and XM_001391932). Nested PCR, using genomic DNA as a template, was performed using TaKaLa LA Taq . The PCR reaction product, consisting of partial sequences of the putative eglB, was cloned into yT & A vectors by using a yT & A cloning kit  and sequenced. This sequence was then used to design another set of internal primers against the putative eglB for the next round of nested PCR. Ligation-anchored PCR (LA PCR) was performed using an LA PCR in vitro cloning kit (TaKaRa Shuzo) according to the manufacturer’s protocol. Sequence analysis was performed using DNAMAN software, and sequencing was performed by the TriLigo Company .Genomic DNA was isolated from mycelia cultured for 3 days. Degenerate primers were designed according to two gene sequences of eglB cDNA sequence was amplified using primers eglB-F (5′-ATGAAGTTTCAGAGCACTTT-3′) and eglB-R (5′-TCAGAGATACGTCTCCAGGA-3′) with the Advantage 2 PCR Enzyme System . The amplified DNA fragments were cloned into yT & A vectors (yT & A-eglB) by using the yT & A cloning kit and transformed into E. coli DH5α, and colonies were selected by blue-white selection. Sequence analysis was performed using DNAMAN software, and sequencing was performed by the TriLigo Company.Total RNA was isolated from mycelia cultured for 3 days. The full-length cDNA was obtained using a ReverTra Ace kit  according to the manufacturer’s protocol. Next, the eglB expression vector, two primers, P1 (5′-CCCCGAATTCTGTGCCTCATGGCTCCGGACA-3′), with an added EcoRI site, and P2 (5′-GCTCTAGATTGAGATACGTCTCCAGGA-3′), with an added XbaI site, were designed. The plasmid yT & A-eglB was used as the template, and TaKaLa LA Taq (TaKaRa Shuzo) was employed. The following PCR cycling conditions were used: 94 °C for 1 min; 30 cycles of 94 °C for 30 s, 58 °C for 1 min, and 72 °C for 3 min; and 72 °C for 10 min. DNA fragments amplified by PCR were cut by EcoRI and XbaI and then purified for ligation. The synthesized eglB cDNA was ligated to an EcoRI- and XbaI-cut pPICZαC vector, and the resulting plasmid was designated pPICZαC-eglB. Transformation of E. coli DH5α was performed using aYE607 kit (Yeastern Biotech).To construct the P. pastoris GS115 was performed according to Wu and Letchworth’s method [Transformation of s method . The traPichia colonies, inoculated into YPDZ medium (YPD medium containing 100 μg/mL zeocin), and then grown in a shaking incubator at 200 rpm and 30 °C for 24 h. The culture was inoculated in 3 mL of BMGY medium  and grown in a shaking incubator at 200 rpm and 30 °C for 24 h. The cell pellets were harvested, resuspended in 3 mL of BMMY medium (BMGY medium without 1% glycerol but with 0.5% methanol) to induce expression, and incubated at 30 °C to continue growth. Sterilized pure methanol was added at a final concentration of 1% every 24 h to maintain induction for 4 days. The expressed protein and the endoglucanase of the supernatants and cell pellets were analyzed by SDS-PAGE and stained with Coomassie Blue.A small-scale culture was used for selecting strains with high EGLB activity. A single colony was picked from the purified Pichia colony, No. 31, was inoculated in 25 mL of BMGY medium. The cell pellet was then harvested and resuspended in BMMY medium to induce enzyme expression. Sterilized pure methanol was added at a final concentration of 0.5, 1, 2, or 3% every 24 h to maintain induction for 4 days. The expressed protein and the endoglucanase of the supernatant and cell pellet were analyzed by SDS-PAGE and stained with Coomassie Blue. Mass production of the expressed enzyme was performed by large-scale culturing. The most efficiently expressed single g for 5 min. EGLB was purified by His-bind resin chromatography  according to the manufacturer’s manual. The purified EGLB was pooled, concentrated (Millipore ultrafiltration membrane with a 10-kDa cutoff), and dialyzed with PBS (pH 7.0).After cultivation, the supernatant was collected by centrifugation at 3,000 × Deglycosylation analysis: The purified recombinant EGLB (1 μg) was deglycosylated with 250 U of endoglycosidase H for 1 h at 37 °C according to the manufacturer’s instructions . The deglycosylated and the untreated EGLB were analyzed by SDS-PAGE.The pI of the purified recombinant EGLB was analyzed by PhastSystem PhastGel IEF . The PhastGel IEF gel was silver-stained.The purified enzyme was analyzed by SDS-PAGE according to Laemmli’s method , and theEndoglucanase activity was determined by measuring the increase in the amount of reducing sugar released from CMC-Na using the dinitrosalicylic acid method . Sodium 2HPO4/NaH2PO4 for pH 5.0–8.0, and Tris-HCl for pH 8.0–9.0. EGLB activity was measured as described previously. The optimal temperature and pH of EGLB were determined at different temperatures (from 30 to 80 °C) and in different buffers (pH 3.0–9.0) for 30 min. The buffers used were as follows: 50 mM citrate for pH 3.0–5.0, 50 mM sodium acetate/acetic acid for pH 4.0–6.0, NaThe thermal stability of the purified recombinant enzyme was determined by assessing the residual enzyme activity under standard conditions after incubating the enzyme at different temperatures (30–80 °C) in the absence of substrate for certain periods of time . The pH stability of EGLB was determined by measuring the residual activity under standard conditions  after incubating the enzyme at 37 °C in different buffers  for 2 h in the absence of substrate.Substrate specificity: The substrate specificity of the recombinant EGLB was determined using CMC-Na, avicel, beechwood xylan, laminarin, barley β-glucan, salicin, chitin, locust bean gum, cellobiose, p-nitrophenyl-α-D-glucopyranoside and p-nitrophenyl-β-D-glucopyranoside as substrates at concentrations of 0.5% (w/v). The amount of reducing sugar released was determined by the DNS method described previously.mK and maxV of EGLB were determined via incubation in 50 mM Na-acetate buffer (pH 5.0) at 40 °C with CMC-Na or barley β-glucan at concentrations ranging from 0.001 to 0.1 g/mL. The mK and maxV values were determined from Lineweaver-Burk plots by using standard linear regression techniques.The The effects of metal ions and regents at a concentration 10 mM on EGLB activity were determined by preincubating the enzyme with the individual reagents in 50 mM Na-acetate buffer, pH 5.0 at room temperature for 10 min. The residual activity was then determined. The activity assayed in the absence of metal ions or regents was recorded as 100%.Aspergillus niger (BCRC31494) has been successfully cloned and sequenced. The cloned gene was expressed in Pichia pastoris, the extracellular EGLB was purified, and the properties of the enzyme were characterized. Specifically, the activity of the enzyme was enhanced in the presence of Co2+, Triton X-100, and Tween 80. The recombinant EGLB exhibited high thermotolerance and stability in alkaline conditions, indicating that the enzyme can be effectively used in bioethanol production and in laundry, textile, denim staining, and paper recycling applications in industry.In summary, an EGLB from


Forty-one compounds, accounting for a relative peak area of 87.7% and 94.6% of turmeric and ginger essential oils, respectively, were identified by Gas Chromatography–Mass Spectrometry analysis. Ginger essential oil with α-zingiberene (24.9 ± 0.8%), β-sesquiphelladrene (11.7 ± 0.3%), ar-curcumene (10.7 ± 0.2%), and β-bisabolene (10.5 ± 0.3%) as the main compounds significantly inhibited the seed germination of P. oleracea, L. multiflorum, and C. selloana at the highest dose (1 µL/mL) assayed, as well as the hypocotyl and radicle growth of the weeds. Turmeric essential oil with ar-turmerone (38.7 ± 0.8%), β-turmerone (18.6 ± 0.6%), and α-turmerone (14.2 ± 0.9%) as principal components significantly inhibited the seed germination of C. selloana and hypocotyl and radicle growth of weeds (the latter in particular) at the highest dose, whereas it did not affect either the seed germination or seedling growth of the food crops. Turmeric essential oil can be an effective post-emergent bioherbicide against the tested weeds without phytotoxicity to crops.Ginger and turmeric are two food ingredients that are in high demand due to their flavor and positive effects on health. The biological properties of these spices are closely related to the aromatic compounds they contain. The chemical compositions of their essential oils and their  Human consumption of herbs and spices began in 5000 BC  and has Herbs and spices offer a wide range of flavors that increase sensory variety in food and beverages without additional energy  while prZingiber officinale Rosc.) and turmeric (Curcuma longa L.), two powerful spices, have been widely used for both culinary and medical purposes. Ginger is an underground stem (rhizome) of a perennial herb and is used as a spice for pickles, candies, and as a preserve [Ginger  and butylated hydroxytoluene (BHT), indicating that it may reduce or delay the progression of diseases related with oxidative stress . Ginger arcinoma . These farcinoma , lipid-larcinoma , make giBeneficial health effects of turmeric and especially of curcumin—an orange-yellow-colored, lipophilic polyphenol substance—have been reported . Curcumiin vivo antimutagenic and anticarcinogenic substances. Ginger essential oil is able to significantly increase the levels of phase II carcinogen-metabolizing enzymes uridine 5′-diphospho-glucuronyl transferase and glutathione-S-transferase [Essential oils of these spices also have interesting pharmacological activities, for instance, both essential oils are nsferase , and turnsferase . Furthernsferase .Sitophilus oryzae L. and Rhyzopertha dominica F. [Fusarium verticillioides and F. graminearum, as well as fumonisins (B1 and B2) and zearalenone production [Aspergillus flavus, completely inhibiting conidial germination at 10 µg/mL of ginger essential oil as well as aflatoxin production at 15 µg/mL [Alternaria panax, Botrytis cinerea, Cylindrocarpon destructans, F. oxysporum, Sclerotinia sclerotiorum, and S. nivalis responsible for ginseng root rot disease [Further investigation is necessary in order to know about other potential activities of these essential oils, not only in medicine but also in other remarkable areas like harvest and post-harvest protection of food and crops. Regarding this, turmeric essential oil in edible coatings has been found to improve the shelf-life of cherry tomatoes and raw poultry milk ,21. It hinica F.  and antioduction ,24. Ging15 µg/mL . Finally disease .Parthenium hysterophorus of both hexane and aqueous extracts from ginger has been studied [Curcuma spp., like C. zedoaria essential oil with 1,8-cineole (15.8%) and epi-curzerenone (18.2%) as the main compounds, has also been demonstrated against both lettuce and tomato [C. longa extracts with curcuminoids are able to inhibit the germination and growth of Bidens pilosa [in vitro herbicidal effects against the seed germination and seedling growth of common ragweed (Portulaca oleracea L.), Italian ryegrass (Lolium multiflorum Lam.), barnyardgrass  Beauv.), pampas grass (Cortaderia selloana (Schult. & Schult. f.) Asch. & Graebn.), and tree tobacco (Nicotiana glauca Graham); and finally, to determine whether these essential oils have phytotoxic effects on food crops like tomato (Solanum lycopersicum L.), cucumber (Cucumis sativus L.), and rice (Oryza sativa L.). These studies corroborated the insecticidal and antifungal properties of ginger and turmeric essential oils and their beneficial effects on food crops. However, weeds are also responsible for lost production of food crops. Regarding this, weed management in ginger as well as the herbicidal activity against  studied ,28. The d tomato ; C. longs pilosa . TherefoForty-one compounds in commercial ginger and turmeric essential oils accounting for 94.60% and 87.67% of the total composition, respectively, were identified by Gas Chromatography–Mass Spectrometry analysis. The components were clustered  as homolSesquiterpene compounds represented the main phytochemical group found in both ginger and turmeric essential oils, of which sesquiterpene hydrocarbons (59.6 ± 0.3%) with seven compounds identified were the major set in ginger essential oil, while oxygenated sesquiterpenes (73.9 ± 1.4%) were the principal ones in turmeric essential oil with six components recognized . It is wThe sesquiterpene hydrocarbons α-zingiberene (24.9 ± 0.8%), β-sesquiphelladrene (11.9 ± 0.3%), ar-curcumene (10.7 ± 0.2%) and β-bisabolene (10.5 ± 0.3%), detected in lower percentages in turmeric essential oil , were the main compounds in ginger essential oil. The results obtained were similar to those of recent research  in whichAlthough zingiberene was the major compound in essential oils coming from both fresh and dried ginger rhizomes from Trivandrum (India), fresh ginger essential oil contained more oxygenated sesquiterpenes compared to the dried one which contained large amounts of the sesquiterpene hydrocarbons ar-curcumene (11.0%), β-bisabolene (7.2%), sesquiphellandrene (6.6%), and δ-cadinene (3.5%) .Z. officinale essential oil here analyzed, is a monocyclic sesquiterpene hydrocarbon with natural antioxidant and cytotoxic activities: it is capable of protecting against H2O2-induced cytotoxicity and oxidative DNA damage in neuronal cells [Tetranychus urticae) and whitefly (Bemisia tabaci) [Zingiberene, the chief component of the al cells  as well al cells . Further tabaci) ,38. On t tabaci) .However, different freezing rates and thawing methods can significantly affect the composition of ginger essential oil: gingerol (3.6%) and zingerone (18.3%), the main spicy compounds of fresh ginger, reached maximum percentages when ginger was thawed by an infrared method  or after thawing ginger using an infrared–microwave  method . These rC. longa rhizome and ar-turmerone (21.4%) and the sesquiterpene hydrocarbons α-santalene (7.2%) and ar-curcumene (6.6%) in turmeric essential oil obtained from dry rhizome [C. longa leaves [On the other hand, ar-turmerone (38.7 ± 0.8%), β-turmerone (18.6 ± 0.6%), and α-turmerone (14.20 ± 0.86%), which were not found in ginger oil, were the leading components of turmeric essential oil. The rest of the sesquiterpenes did not reach 1% in either essential oil analyzed . These r rhizome . Higher a leaves , confirma leaves  that ourSitophilus zeamais) and fall armyworm (Spodoptera frugiperda) at low doses [The therapeutic potential of ar-turmerone has been extensively studied due to its numerous beneficial effects such as anti-inflammatory and cytotoxic effects in the treatment of various neurodegenerative disorders ,46. Regaow doses .Monoterpene hydrocarbons were the following main phytochemical group with eight (19.8 ± 0.1%) and nine (5.4 ± 0.7%) compounds identified in ginger and turmeric essential oils, respectively . Camphen1,8-Cineole (1.0 ± 0.0%) was the only oxygenated monoterpene detected in turmeric essential oil. In contrast, this fraction, with ten oxygenated monoterpenes identified, was qualitatively the main phytochemical group found in ginger essential oil. 1,8-Cineole (3.0 ± 0.1%), followed by geranial (3.2 ± 0.0%) and neral (2.1 ± 0.1%), were the main compounds .Rhipicephalus (Boophilus) microplus, and neral and geranial have exhibited anti-inflammatory activity through significant and similar inhibition of the gene NLRP-3 inflammasome-mediated IL-1β secretion, showing use as functional food ingredients [Recent studies  showed tredients .Finally, other compounds such as 6-methyl-5-hepten-2-one (2.1 ± 0.1%), 2-nonanone (0.1 ± 0.0%), and 2-undecanone (0.2 ± 0.0%) were only identified in ginger essential oil .P. oleracea, L. multiflorum, E. crus-galli, C. selloana, and N. glauca.As several studies have indicated that essential oils may be promising herbicides , the effP. oleracea, L. multiflorum, E. crus-galli, and N. glauca at all doses  assayed; however, significant inhibition of the seed germination of C. selloana was achieved in a dose-dependent manner, reaching 81.71% of reduction at the highest dose (1 µL/mL) tested  tested .P. oleracea, L. multiflorum, and E. crus-galli were sensitive to winter savory (Satureja montana L.), which exerted a total inhibitory effect on the seed germination of the three weeds at all doses  tested, and peppermint (Mentha piperita L.), which completely inhibited the seed germination of L. multiflorum and significantly affected the seed germination of P. oleracea and E. crus-galli at the highest dose (1 µL/mL) applied [Previous studies showed that  applied .E. crus-galli and N. glauca, a remarkable decrease in the seed germination of P. oleracea, L. multiflorum, and C. selloana was observed at the highest dose—reductions of 45.35%, 46.67%, and 43.91%, respectively— in relation to the control  in comparison to the control  with respect to the control  assayed (N. glauca was observed at the highest dose (1 µL/mL) applied  as its major compound, affected the seedling growth of Philaris minor Retz. in a concentration-dependent manner, achieving inhibition of both the hypocotyl and radicle development at 1000 ppm and showing less or no effect on the germination of seeds of Triticum aestivum L. [Furthermore, other tivum L. .P. oleracea, significantly inhibited both hypocotyl and radicle growth of the selected weeds at all doses  assayed. The hypocotyl development was reduced without significant differences between doses applied to reach percentages of 56.55% (P. oleracea), 40.45% (L. multiflorum), 39.33% , 97.83% (C. selloana), and 86.23% (N. glauca)  . The radP. oleracea, E. crus-galli, and L. multiflorum because other essential oils such as oregano essential oil with carvacrol (60.4 ± 0.1%), p-cymene (15.5 ± 0.0%), and γ-terpinene (5.2 ± 0.0%) or winter savory essential oil with carvacrol (43.3 ± 0.1%) and thymol (23.2 ± 0.1) as main compounds can completely inhibit the germination of these three weeds at all doses (0.125–1 µL/mL) applied [Thymus mastichina essential oil with 1,8-cineole (49.5 ± 0.4%), linalool (5.7 ± 0.0%), and α-terpineol (5.6 ± 0.0%), which showed significant effects in seedling length depending on the weed and dose [C. selloana. Their use as promising post-emergent alternatives will depend on the phytotoxicity of these essential oils in food crops.Ginger and turmeric essential oils are not suitable as a potent pre-emergent treatment in the control of  applied ,54. ThesSeed germination of tomato, cucumber, and rice was not affected at any dose  applied of ginger essential oil .P. oleracea (45.35%) and L. multiflorum (46.67%)  —weeds co(46.67%) A. These (46.67%) C. Slight(46.67%) .versus other food crops: for instance, chickpea, in which no adverse effect was observed in either seed germination or seedling growth [Curcuma spp. like C. zedoaria have been shown to inhibit the seed germination of lettuce and tomato in a dose-dependent manner  as well as to delay their growth, damaging the root in particular. These results are due to the different chemical compositions of these essential oils, with ar-turmerone (38.7 ± 0.8%), β-turmerone (18.6 ± 0.6%), and α-turmerone (14.2 ± 0.9%) being the main compounds in the turmeric essential oil here analyzed (epi-curzerenone (18.2%) being those in C. zedoaria essential oil [Promising results were obtained with turmeric essential oil against the food crops tested. Neither seed germination nor the hypocotyl growth of tomato, cucumber, and rice were significantly affected by the application of turmeric essential oil at any dose  B,D. The g growth . Howeveranalyzed , and 1,8tial oil .P. oleracea , winter savory (Satureja hortensis L.), and bay (Laurus nobilis L.) in tomato [It is interesting to note that at the highest dose assayed, turmeric essential oil was able to significantly reduce the hypocotyl development of oleracea  as well n tomato .Zingiber officinale Rosc.)  and turmeric (Curcuma longa L.)  essential oils obtained from rhizome and root, respectively, were supplied by Pranarôm S.A. Both were stored at 4 ᵒC until chemical analysis and phytotoxic assays were carried out.Commercial samples of ginger (Portulaca oleracea L.), Italian ryegrass (Lolium multiflorum Lam.), and barnyardgrass  Beauv.) were purchased from Herbiseed (website: www.herbiseed.com), and those of pampas grass (Cortaderia selloana (Schult. & Schult. f.) Asch. & Graebn.) and tree tobacco (Nicotiana glauca Graham) were supplied by the Botanical Garden of Valencia.Mature seeds of the weeds common ragweed (Solanum lycopersicum L.) and cucumber (Cucumis sativus L.) were obtained from Intersemillas S.A. “Albufera-type” rice (Oryza sativa L.) seeds were acquired from Copsemar in Sueca .Mature seeds of the food crops “Muchamiel” tomato  apparatus equipped with an Agilent HP-5MSi (30 m long and 0.25 mm i.d. with 0.25 µm film thickness) capillary column (95% dimethylpolysiloxane/5% diphenyl). The column temperature program was 60 ᵒC for a duration of 5 min, with 3 ᵒC/min increases to 180 ᵒC, then 20 ᵒC/min increases to 280 ᵒC, which was maintained for 10 min. The carrier gas was helium at a flow rate of 1 mL/min. Split mode injection (ratio 1:30) was employed. Mass spectra were taken over the m/z range 30–650 with an ionizing voltage of 70 eV. The resulting individual compounds were identified by MS and their identity was confirmed by comparison of their Kovat’s retention index calculated using co-chromatographed standard hydrocarbons relative to CE. crus-galli, C. selloana, N. glauca, cucumber, and rice) and without  humidity. To evaluate the herbicidal activity of the essential oils, the number of germinated seeds was counted and compared with that of untreated seedlings. Emergence of the radicle (≥ 1 mm) was used as an index of germination and seedling length (hypocotyl and/or radicle) data were recorded after 3, 5, 7, 10, and 14 days in each replicate.Sets of 20 seeds each with five replicates per treatment were homogenously distributed in Petri dishes (9 cm diameter) between two layers of filter paper (Whatman No.1) moistened with 4 mL of distilled water and with 0 (control), 0.125, 0.250, 0.5, and 1 µL/mL of ginger and turmeric essential oils. Petri dishes were sealed with parafilm and incubated in an Equitec EGCS 301 3SHR model germination chamber, according to previous assays , alternap ≤ 0.05.Experiments were performed with five replicates. Data were subjected to one-way analysis of variance (ANOVA) using SPSS statistics 22 software. Tukey’s post hoc test was used when variances remained homogeneous (Levene’s test) and T3 Dunnett’s post hoc test was employed if not, assuming equal variances. Differences were considered to be significant at P. oleracea and L. multiflorum in tomato, cucumber, and rice crops, whereas turmeric essential oil with the oxygenated sesquiterpenes ar-turmerone (38.7 ± 0.8%), β-turmerone (18.6 ± 0.6%), and α-turmerone (14.2 ± 0.9%) can be applied as a post-emergent substance against the weeds tested since no significant phytotoxic effects in tomato, cucumber, or rice were observed. Turmeric essential oil could be a promising alternative in the management of the invasive species C. selloana. More weeds and higher doses of turmeric essential oil must be tested in order to determine any selective herbicide effect.Essential oils from ginger and turmeric, two health-promoting spices, could be used in weed control. Ginger essential oil with high contents of the sesquiterpene hydrocarbons α-zingiberene (24.9 ± 0.8%), β-sesquiphelladrene (11.9 ± 0.3%), ar-curcumene (10.7 ± 0.2%), and β-bisabolene (10.5 ± 0.3%) may be used as a pre-emergent bioherbicide in the control of


Geobacillus sp. strain HTA-462 was cloned and expressed in Escherichia coli BL21 (DE3). Recombinant GSJ was purified and biochemically characterized. The optimum temperature condition of the recombinant enzyme was 65 °C, and the half-life was 84 h at 60 °C, whereas the enzyme was active over the range of pH 6.0–10.0 with maximal activity at pH 7.0. The α-glucosidase activity in shake flasks reached 107.9 U/mL and using 4-Nitrophenyl β-D-glucopyranoside (pNPG) as substrate, the Km and Vmax values were 2.321 mM and 306.3 U/mg, respectively. The divalent ions Mn2+ and Ca2+ could improve GSJ activity by 32.1% and 13.8%. Moreover, the hydrolysis ability of recombinant α-glucosidase was almost the same as that of the commercial α-glucosidase (Bacillus stearothermophilus). In terms of the transglycosylation reaction, with 30% maltose syrup under the condition of 60 °C and pH 7.0, IMOs were synthesized with a conversion rate of 37%. These studies lay the basis for the industrial application of recombinant α-glucosidase.Isomaltose–oligosaccharides (IMOs), as food ingredients with prebiotic functionality, can be prepared via enzymatic synthesis using α-glucosidase. In the present study, the α-glucosidase (GSJ) from  Bifidobacterium and Lactobacillus in the human intestinal tract 2+ or [M + H]+ were assigned.The purified GSJ were dissolved into 0.1% trifluoroacetic acid (TFA). The reagent 2.5-dihydroxy benzoic acid was dissolved in 30/70 (Geobacillus sp. strain HTA-462 was cloned and expressed in E. coli, and the recombinant enzyme was characterized in detail. The α-glucosidase activity in shake flasks was 107.9 U/mL. A detailed biochemical characterization showed that the recombinant α-glucosidase had high activity and stability at high temperatures and in a broad pH range. Additionally, the GSJ showed high α-1, 4-glucosidic hydrolysis and high transglycosylation activity, and converted maltose to IMOs with a yield of 37%. These studies indicated that the recombinant α-glucosidase has a great potential for industrial-scale applications. To the best of our knowledge, this is the first report to express the GSJ gene in E. coli and study IMO synthesis by the recombinant enzyme.In conclusion, α-glucosidase of the


This study investigated whether metabotropic glutamate receptor (mGluR) 5 and 8 are involved in the effect of ultramicronizedpalmitoylethanolamide (um-PEA) on the cognitive behavior and long term potentiation (LTP) at entorhinal cortex (LEC)-dentate gyrus (DG) pathway in mice rendered neuropathic by the spare nerve injury (SNI). SNI reduced discriminative memory and LTP. Um-PEA treatment started after the development of neuropathic pain had no effects in sham mice, whereas it restored cognitive behavior and LTP in SNI mice. 2-Methyl-6-(phenylethynyl) pyridine (MPEP), a selective mGluR5 antagonist, improved cognition in SNI mice and produced a chemical long term depression of the field excitatory postsynaptic potentials (fEPSPs) in sham and SNI mice. After theta burst stimulation (TBS) MPEP restored LTP in SNI mice. In combination with PEA, MPEP antagonized the PEA effect on discriminative memory and decreased LTP in SNI mice. The (RS)-4-(1-amino-1-carboxyethyl)phthalic acid (MDCPG), a selective mGluR8 antagonist, did not affect discriminative memory, but it induced a chemical LTP and prevented the enhancement of fEPSPs after TBS in SNI mice which were treated or not treated with PEA. The effect of PEA on LTP and cognitive behavior was modulated by mGluR5 and mGluR8. In particular in the SNI conditions, the mGluR5 blockade facilitated memory and LTP, but prevented the beneficial effects of PEA on discriminative memory while the mGluR8 blockade, which was ineffective in itself, prevented the favorable action of the PEA on LTP. Thus, although their opposite roles (excitatory/inhibitory of the two receptor subtypes on the glutamatergic system), they appeared to be required for the neuroprotective effect of PEA in conditions of neuropathic pain. Sensory and cognitive disorders are comorbidities of neuropathic pain in both, humans and rodents . The syn= 7.33; p < 0.0001). A single administration of 2-methyl-6-(phenylethynyl)pyridine,, a selective mGluR5 antagonist, was devoid of effect in sham mice (74.5 ± 3.9%) whereas it increased the RI in SNI mice (75.9 ± 6.6%)  SNI mice that received a chronic treatment with vehicle A showed  ± 6.6%) A. MPEP a ± 6.6%) B.2,42 = 221; p < 0.0001) and slope (60–90 min: 257.27 ± 27.15% vs. 15–30 min: 97.87 ± 5.0%)   of the fEPSPs as revealed by one-way ANOVA followed by Dunnet’s post hoc test  and the slope (60–90 min: 192.44 ± 7.55%)  of the fEPSP in vehicle-treated sham mice as indicated by two-way ANOVA followed by the Bonferroni post-hoc test  and the slope (60–90 min: 234.49 ± 3.74%)  of the fEPSPs remained unchanged compared with vehicle-treated sham mice  and slope values (15–30 min: 66.95 ± 7.0% vs. 0–15 min: 95.66 ± 4.03%)  of basal fEPSPs in sham mice. When MPEP (5 nmol) was administered in sham mice chronically treated with PEA, the amplitude (60–90 min: 169.3 ± 7.28% vs. 15–30 min: 93.34 ± 7.38%)  and slope (60–90 min: 185.74 ± 15.53% vs. 15–30 min: 103.31 ± 9.27)  of the fEPSPs post-TBS remained unchanged compared to vehicle-treated sham mice (The theta-burst stimulation (TBS) was applied in the LEC 30 min after the registration of the baseline of field excitatory postsynaptic potentials (fEPSPs) in the DG A. In shahoc test D–G. The hoc test D–G. The ham mice D–G. Howeham mice D–G. Howeham mice D–G.2,42 = 43.2; p > 0.05) and slope (60–90 min: 105.7 ± 5.99% vs. 15–30 min: 97.1 ± 4.1%)  of the fEPSPs did not change after TBS, as assessed by one-way ANOVA followed by Dunnet’s post hoc test  and slope (60–90 min: 193.87 ± 9.85% vs. 15–30 min: 103.64 ± 4.65%)  of EPSPs after TBS as compared to vehicle-treated SNI mice  and the slope (15–30 min: 58.68 ± 8.92% vs. 0–15 min: 72.37 ± 3.68%)  of the fEPSPs before the application of the TBS significantly decreased as assessed by unpaired t-student test  and slope (60–90 min: 114.46 ± 8.57%)  of fEPSPs after TBS in SNI mice. When MPEP (5 nmol) was microinjected into the LEC of SNI mice treated with PEA the amplitude (15–30 min: 94.3 ± 5.5.0% vs. 0–15 min: 95.4 ± 3.78%)  and the slope (15–30 min: 95.77 ± 5.0% vs. 0–15 min: 99.76 ± 3.78%)  of basal fEPSPs did not change  and slope (60–90 min: 154.39 ± 5.21%)  of fEPSPs after TBS  and the slope (60–90 min: 193.61 ± 8% vs. 15–30 min: 102.73 ± 16.65%)  of the fEPSPs remained unchanged in vehicle-treated sham mice, as revealed by unpaired t-student test  and the slope (60–90 min: 229.53 ± 8.26% vs. 15–30 min: 99.99 ± 6.99%)  of the fEPSPs in PEA-treated sham mice as revealed by unpaired t-student test  and the slope (15–30 min: 145.86 ± 6.85% vs. 0–15 min: 107.63% ± 3.76%)  of the basal fEPSPs significantly increased as assessed by unpaired t-student test  and the slope   of the basal fEPSPs in SNI mice chronically treated with PEA  and the slope (60–90 min: 166.6 ± 7.27%)  of basal fEPSPs in vehicle-treated SNI mice which received a single microinjection of MDCPG (100 nmol). Similarly, the application of the TBS did not further potentiated the amplitude (60–90 min: 161.49 ± 0.35%)  and the slope (60–90 min: 155 ± 7.27%)  of fEPSPs in PEA-treated SNI mice  for at least one week before the commencement of experiments. All experimental procedures were approved by the Animal Ethics Committee of University of Campania “L. Vanvitelli”, 1050-2016-PR, 31 October 2016 Organismo Preposto al Benessere Animale, OPBA. Animal care was in compliance with Italian (D.L. 116/92) and European Commission (O.J. of E.C. L358/1 18/12/86) regulations on the protection of laboratory animals. All efforts were made to reduce both animal numbers and suffering.Male C57BL/6J mice  were housed under controlled conditions  started 15 days after the sham or SNI surgery (day 0) and lasted for 15 days. Sham and SNI mice were tested 30 days after surgery. In the novel object recognition test a single administration of MPEP (1 mg/kg i.p.) or MDCPG (50 mg/kg i.p.) has been performed systemically 30 days after the sham or SNI surgery, 15 days after the treatment with vehicle or um-PEA and 60 min before the acquisition trial of the novel object recognition as described below. For the electrophysiological experiments, a single microinjection of MPEP (5 nmol) or MDCPG (100 nmol) was locally microinjected into the LEC at the same time point (30 days after the sham or SNI surgery and 15 days after the treatment with vehicle or um-PEA) and 15 min after the registration of the fEPSP baseline. A schematic illustration showing the timeline of surgery, treatment and experiments in sham and SNI mice is shown in Mice were subjected to 1 h habituation period for exploring the apparatus, consisting of a gray polyvinyl chloride open box  illuminated by a dim light. The day after, each mouse was allowed to explore two identical objects for 5 min (acquisition) and 2 h after the acquisition, one of the two objects was replaced with a new one object. The exploring time was considered as the time the mouse spent with its nose directed, and within 1 cm from the object. The behavior was recorded and analyzed by a video camera . Data were expressed as recognition index (R.I.): the percentage of the time the mouse spent exploring the novel object/ the time the mouse spent exploring the novel object + the time the mouse spent exploring the familiar object.Mice were anesthetized with urethane  and fixed in a stereotaxic device . Body temperature was maintained at 37 °C with a heating pad . Two holes were drilled in the skull over the recording  and stimulation  sites according to the atlas of Franklin and Paxinos  and contUm-PEA (0.8–6.0 μm) was kindly provided by EPITECH Group SpA, Saccolongo (PD). MPEP hydrochloride was purchased from Tocris Bioscience . MDCPG was purchased from Hello Bio . Drugs were dissolved in 0.05% dimethylsulfoxide in artificial cerebrospinal fluid (ACSF) on the day of the experiment. The dose of drugs was chosen according to our and otherin vivoandex vivostudies ,74,75,76p values < 0.05 were considered statistically significant. Statistical analysis was carried out using Prism/Graphpad  software which has calculated the F (degrees of freedom) and t (t-student) related to the number of animals per group (t5).Data were represented as mean ± SEM. Behavioral and electrophysiological data were analyzed by using two-way ANOVA for repeated measures, followed by the Bonferroni post hoc test for comparisons between groups and one-way ANOVA, followed by Dunnett’s multiple comparison post hoc test for multiple comparisons within groups. Moreover, the unpaired t-test was used for single comparison within the group. These preliminary data suggest that a chronic treatment with PEA or a single administration with mGluR5 blockers (or a combination of both) affect discriminative memory and LTP at the LEC-DG pathway in neuropathic pain conditions, in which a decline of LTP and discriminative memory was observed. The mGluR8 blockade, which is ineffective per se, prevents the beneficial effect of PEA on LTP in SNI conditions.The discovery of the molecular mechanisms which make these receptors necessary for the effects of PEA may open the way to the discovery of novel multitarget therapies against cognitive decline associated with chronic pain conditions.


Arabidopsis thaliana. The transcriptional and physiological mechanisms involved, however, remain largely undisclosed. Potassium transporters have been suggested as possible mediators of embryo cell size, controlling turgor pressure during seed maturation. In addition, it has been demonstrated that the expression of K+ transporters is effectively regulated by auxin. Here, we provide evidence for the identification of two Arabidopsis K+ transporters, HAK/KT12 (At1g60160) and KUP4 (At4g23640), that are likely to be implicated in determining seed size during seed maturation and, at the same time, show a differential regulation by indole-3-acetic acid and indole-3-acetamide.The seed is the most important plant reproductive unit responsible for the evolutionary success of flowering plants. Aside from its essential function in the sexual reproduction of plants, the seed also represents the most economically important agricultural product worldwide, providing energy, nutrients, and raw materials for human nutrition, livestock feed, and countless manufactured goods. Hence, improvements in seed quality or size are highly valuable, due to their economic potential in agriculture. Recently, the importance of indolic compounds in regulating these traits has been reported for  Seed size is an essential indicator of plant fitness and a major determinant for agricultural yield. Hence, it is one of the most important traits analyzed for agronomic purposes. Seed formation is divided into two major phases: embryo and endosperm development (or morphogenesis), and seed maturation ,2. MaturDuring the maturation phase, the seed undergoes important transformations ranging from the accumulation of storage compounds in the endosperm (endospermic seeds) or in cotyledons (non-endospermic seeds), to the acquisition of desiccation tolerance and growth arrest ,4,5,6. SSeveral molecular networks controlling seed maturation have been characterized . FirstlyAuxins are essential plant hormones linked to a wide variety of processes associated with plant growth and development ,13,14. CXTH genes is positively regulated by auxin  atoc64-I)  have bee sucrose . The phoeviously . The expv/v)) and a mock solution (0.1% EtOH (v/v)) as a control. Plant material were soaked over 48 h at 4 °C with the corresponding solutions, before the samples were snap-frozen in liquid nitrogen and kept at −80 °C until RNA isolation was executed.Treatment of siliques for subsequent qRT-PCR analysis was performed preparing MS solutions of 100 µM IAA and IAM  and RNase-free dH2O in a total reaction volume of 13.5 µL. Then, 12.5 µL of a mix containing 2.5 mM dNTPs, 40 mM RNAsin, 200 U of Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase and 1 µM of DTT were added. After incubating the solution for 1 h at 37 °C and a subsequent diluted of 1:1 with dH2O, samples were incubated for another 5 min at 80 °C and thereafter stored at −20 °C until they were further processed.Total RNA from siliques was isolated as previously described . The cDN®480 . Specific forward and reverse primers pairs (20–24 bps) were designed using the QuantPrime tool -IAM  as internal standard. The plant material was crushed in a MagNA Lyser Instrument (Roche) at 6,500 rpm for 1 min. After centrifugation , 750 µL of the supernatants were transferred into fresh tubes and the pH adjusted to 2.7 with 1 M hydrochloric acid. The samples were loaded onto OasisTM HLB columns  conditioned with 1 mL methanol and 1 mL water, and equilibrated with 1 mL acidified sodium phosphate buffer . The columns were washed twice with 1.5 mL 5% methanol and subsequently eluted with 2 mL 80% methanol. The eluates were taken to dryness in vacuo. Prior to the mass spectrometric analysis, the evaporated samples were dissolved in 45 µL methanol with 0.1% formic acid (v/v). Ten microliters of the extract were separated using an Ultimate3000 RSLC system . The column used was a 50 mm × 2.1 mm i.d., 1.7 μm, Acquity UPLC BEH C18 column with a 5 mm × 2.1 mm i.d. Acquity UPLC BEH C18 VanGuard pre-column, and a 0.2 μm × 2.1 mm i.d. in-line filter (Waters). The following binary gradient was applied: 0 to 2 min isocratic 98% solvent A (water with 0.1% (v/v) formic acid), 2% B (acetonitrile with 0.1% (v/v) formic acid); 2 to 30 min linear gradient to 5% A, 95% B; at 30 min step gradient to 100% B; isocratic for 1 min. Thereafter, the column was set to 98% A, 2% B and conditioned for 2 min before the next injection. The flow rate was 400 μL min−1. Eluted compounds were analyzed by a microTOF-Q II mass spectrometer  operated in positive electrospray mode. Typical settings were as follows: capillary voltage, −4,500 V; dry gas temperature, 200 °C; dry gas flow, 10 L min-1; funnel, RF 200 Vpp. Ions were detected from m/z 50 to 1000 at a repetition rate of 2 Hz. The instrument was operated in MRM mode. Mass calibration was performed using sodium formate clusters (10 mM solution of NaOH in 50/50% v/v isopropanol/water containing 0.2% formic acid). For data processing the DataAnalysis 4.0 software  was used. The following transitions were recorded to determine analyte contents: IAM, m/z = 175.2 → 130.1 -IAM, m/z = 181.2 → 135.1 . Quantification was achieved by comparing peak areas of the endogenous compounds with those of the internal standards.The LC-MS analysis of endogenous IAM was carried out according to the method established by Novak and co-worker . In briet-test to allow for comparison of two means, or ANOVA followed by Tukey’s B post-hoc test to allow for comparisons among all means. Statistical analysis was conducted using PRISM v5.03 . Significance thresholds are indicated in the figure legends.The obtained data were analyzed using univariate statistics by employing either Student’s


Correction to:British Journal of Cancer (2018) 119, 615-621; 10.1038/s41416-018-0212-9; www.bjcancer.com; published online 21 August 2018The authors have noticed that the final paragraph of the Results section contains errors in the number of patients involved. The correct number of patients is included in the text below. These errors do not affect the Figure referenced.n = 30) in ctDNA was 60.0% (95% CI 40.5–75.0) compared to 80.8 (95% CI 42.4–94.9) in patients without 8q gain (n = 11) in ctDNA .In osteosarcoma, we focused on 8q gain as a specific biological feature of interest. Among the 41 patients with detectable ctDNA in the osteosarcoma cohort, 8q gain was detected in 73.2% (30/41). The 3-year EFS for patients with 8q gain (