Fat oxidation (‘FATMAX’ and steady state exercise protocols), body composition, cardio-metabolic and blood measures  were assessed at baseline, week 4 and 8. Following 8 weeks of dGTE+, maximal fat oxidation (MFO) significantly improved from 154.4 ± 20.6 to 224.6 ± 23.2 mg·min−1 (p = 0.009), along with a 22.5% increase in the exercise intensity at which fat oxidation was deemed negligible . Steady state exercise substrate utilisation also improved for dGTE+ only, with respiratory exchange ratio reducing from 0.94 ± 0.01 at week 4, to 0.89 ± 0.01 at week 8 (p = 0.004). This corresponded with a significant increase in the contribution of fat to energy expenditure for dGTE+ from 21.0 ± 4.1% at week 4, to 34.6 ± 4.7% at week 8 (p = 0.006). LDL-c was also lower  for dGTE+ by week 8 (p = 0.038). No other significant effects were found in any group. Eight weeks of dGTE+ improved MFO and substrate utilisation during exercise, and lowered LDL-c. However, body composition and cardio-metabolic markers in healthy, overweight individuals who maintained regular physical activity were largely unaffected by dGTE.This study investigated the effect of decaffeinated green tea extract (dGTE), with or without antioxidant nutrients, on fat oxidation, body composition and cardio-metabolic health measures in overweight individuals engaged in regular exercise. Twenty-seven participants (20 females, 7 males; body mass: 77.5 ± 10.5 kg; body mass index: 27.4 ± 3.0 kg·m Green tea (GT) polyphenols have been widely investigated for their potential therapeutic health benefits from an antioxidant , anti-in−1 EGCG, have reported significant improvements in whole-body fat oxidation rates in untrained and overweight individuals both at rest [−1 EGCG [In terms of fat oxidation, studies with administration of GT extract (GTE) for <7 days, providing between 270 and 375 mg·d at rest ,18, duri at rest , as well at rest . Elevate at rest ,19 suppo at rest ), inferr[−1 EGCG ,22, sugg−1 EGCG) for 3 weeks did not improve fat oxidation during exercise in trained individuals [−1 EGCG) significantly improved total fat oxidation rates during exercise, and reduced body fat percentage in one study [−1 EGCG [In the longer term (>3 weeks), the effects of GTE supplementation on fat oxidation are equivocal. Low-dose GTE supplementation , with b−1 GTE) for 8 weeks was not effective in improving body fat percentage, BMI, blood pressure, triglyceride and LDL-c levels over that obtained through introduction of aerobic training in overweight/obese, previously sedentary females [−1 GTE; 595.8 mg·d−1 EGCG) for 12 weeks did not improve resting energy expenditure, body mass, BMI, waist to hip ratio (WHR) or fat mass over that explained through sustained inclusion of a low-energy diet in overweight woman [−1 GTE; ~225 mg·d−1 EGCG) significantly improved body mass, BMI, body fat percentage, visceral fat area (VFA) and adiponectin levels above that obtained through introduction of aerobic training in previously sedentary overweight women [With regards to cardio-metabolic risk factors, studies lasting 8-12 weeks also provide equivocal findings. Low-dose GTE supplementation , improving the bioavailability and effectiveness of GTE through standardised consumption away from food [In addition to the range of EGCG doses used, confounding factors such as GTE type and bioavailability may, in part, explain the varying findings observed. Only a handful of studies have employed a dGTE formula ,18,25,26rom food  and/or irom food ,32,33,34rom food ,36. Elserom food . HoweverTherefore, this study aimed to assess the longer-term impact of moderate dGTE supplementation (with or without antioxidant nutrients) on fat oxidation, body composition and cardio-metabolic risk factors in overweight, but healthy individuals engaged in regular exercise. It was hypothesised that fat oxidation would be improved with dGTE , resulting in improved body composition and cardio-metabolic risk factors compared with placebo.ClinicalTrials.gov (Identifier: NCT04628624). Ethical approval was obtained from the Faculty of Science and Technology Research Ethics Panel, Anglia Ruskin University (FST/FREP/17/703). Following a priori power calculation assessment , and registered with  Germany ; using α Germany ,25), a m2; initial relative peak oxygen uptake 2peak): 30.2 ± 5.8 mL·kg−1·min−1) satisfactorily completed the intervention, with study power deemed sufficient based on a priori analysis. Participant characteristics for each intervention group are displayed in Thirty-one participants were therefore invited to take part in the intervention study. However, 2 participants withdrew at week 4 for personal reasons, and data for 2 participants were excluded based on not meeting supplement adherence or testing criteria. Therefore, a total of 27 participants , age: 43 ± 8 years; body mass: 77.5 ± 10.0 kg; height: 1.68 ± 0.06 m; BMI: 27.4 ± 3.0 kg·m2 and/or a body fat percentage in the overweight category for age/gender based on guidelines set out by the American College of Sports Medicine (ACSM) [2peak between 20 and 45 mL·kg−1·min−1 during pre-screening testing. Participants with a known history of cardio-metabolic disorders, blood- and liver-related disorders, and recent viral infections were not eligible for study inclusion. Participants were also required to not be consuming other nutritional or thermogenic supplements  or medication for at least 4 weeks prior to beginning the study intervention.This study employed a randomised, repeated-measures, double-blinded, placebo-controlled design over an 8 week period. Following a full study briefing and provision of written, informed consent, volunteers took part in an initial pre-screening assessment with full familiarisation to the main study testing procedures outlined below. Study eligibility required volunteers to: satisfactorily complete a general health screen questionnaire; be over 18 years of age and ‘recreationally active’  with a BMI between 25.0 and 29.9 kg·me (ACSM) . As a me−1, of which 50% carbohydrate, 20% protein, 30% fat), and arrive in a euhydrated, fasted state (~10 hours) having refrained from consuming caffeinated products prior to testing. All testing procedures were carried out in the Cambridge Centre for Sport and Exercise Sciences, Human Physiology Laboratory, Anglia Ruskin University under controlled environmental conditions . In addition to initial pre-screening, participants visited the laboratory at baseline (week 0), and at the end of weeks 4 and 8 of the intervention period. All testing was undertaken in the morning, with participants being tested at the same time of day across the intervention. In the week leading into each laboratory visit, participants were required to complete dietary intake and activity diaries see . In addiUpon arrival, participants rested for ~10 min in a supine position prior to duplicate assessment of resting heart rate and blood pressure . Following this, a venous whole blood sample was collected from participants by a qualified phlebotomist into triplicate 4 mL serum clot activator Vacuette™ tubes . Samples were centrifuged for 10 min at 2000 rcf according to manufacturer recommendations, with aliquotted serum pipetted into sterile, nonpyrogenic, polypropylene cryovials  and frozen at −80 °C for later assessment of blood analytes see .Participants then underwent body composition assessment, with height measured using a stadiometer . Body mass and body fat percentage were assessed through the use of bioelectrical impedance analysis scales . Body fat percentage was additionally confirmed using an 8-site skinfold calliper assessment ). Specific girth measures were undertaken for abdomen, thigh and calf locations, as well as waist to hip ratio (WHR) conforming to ISAK/ACSM guidelines. Sagittal abdominal diameter (SAD) was also measured using a Holtain-Kahn abdominal caliper , with visceral fat area (VFA) estimated from validated equations . AnthropMAX’) test using a Lode Excalibur sport cycle-ergometer . The FATMAX protocol was based on original research [Participants were fitted with a Polar FS2 telemetric heart rate (HR) monitor  and a suitable face-mask for expired air assessment  prior to an initial resting period. Resting expired air was then measured using a Metalyser 3B automated gas-analyser , after which participants underwent a maximal fat oxidation  of <2 mL·kg−1·min−1 with increasing workload; (ii) attainment of 95% of age-predicted HRmax (220-age); (iii) a respiratory exchange ratio (RER) > 1.05; and (iv) an RPE >9  along with evidence of volitional fatigue symptoms. 2peak was estimated from the highest 30 sec moving average oxygen consumption.Briefly, participants completed a graded exercise test to volitional exhaustion, starting at 30 W (acting as a warm-up stage) with 20 W increments every 3 min. Cadence was maintained between 75 and 85 rpm throughout. Breath-to-breath expired air analysis was undertaken throughout the test, along with telemetric HR and self-assessed rating of perceived exertion (RPE)  in the lOX; mg·min−1) were derived from 2 and carbon dioxide (2) expired air assessment (L·min−1) using stoichiometric equations [Fat oxidation rates , a 2nd-order polynomial curve was fitted from which maximal fat oxidation rate , maximal fat oxidation exercise intensity  and minimum fat oxidation exercise intensity  were derived, based on previous research [Using individual FATresearch .2 and HR comparable to pre-exercise conditions), participants underwent a fixed-intensity exercise test , using the same cycle-ergometer, with cadence maintained between 75 and 85 rpm for 60 min. Perceived exertion and HR were measured at 5 min and each 15 min thereafter. Breath-to-breath expired air was continuously measured throughout the submaximal exercise period, with 60 sec average data recorded upon test completion. Fat oxidation rates were derived from the previous stoichiometric Equation (1). Carbohydrate oxidation rates  and energy expenditure  were derived from the following equations [Following a standardised 30 min rest period (with resting quations :(2)CHOOXOX and CHOOX rates, respectively. Contributions of both carbohydrate and fat to EE were calculated on the basis of caloric conversion of estimated total CHOOX and FATOX (g·h−1) expressed as a percentage of EE .Additionally, both fat and carbohydrate oxidation stability (expressed as a %) were assessed on the basis of the duration maintained within ±10% of mean FATwww.randomizer.org accessed on 26 February 2021). Participants were randomly assigned to an 8 week intervention  of either: (i) placebo ; (ii) decaffeinated green tea extract ); or (iii) a novel decaffeinated green tea extract formula , with quercetin (50 mg·d−1) and α-lipoic acid , also matched weighted with additional bulking agent). Following baseline assessment, participants were allocated, in a double-blinded manner, to one of three intervention groups using a random number generator , and verified through Nutraceuticals Group Europe  with dGTE sourced from Changsha Active Ingredients Group Inc. , based on our previous research . All supwww.MyFitnessPal.com accessed on 26 February 2021), validated in previous research [www.phenol-explorer.eu accessed on 26 February 2021) as a means to estimate dietary quercetin intake.Following the pre-screening session, participants included in the main intervention completed a 7 day food diary using a smart phone application , at ~60–65% 2peak (~75–80%HRmax) in accordance with guidelines set out by the American College of Sports Medicine [In conjunction with this, participants were also provided with a standard activity log and HR monitor, and requested to track all exercise activity prior to the start of the intervention , and each week across the 8 week study period. Participants recorded exercise activity, mean session HR, duration and overall session rating of perceived exertion (sRPE) from which training load, monotony and strain were determined as previously described ,49. To sMedicine ,50.−1); triglycerides ; total cholesterol ; and HDL-c , with LDL-c  derived from the Friedewald equation [All blood samples were analysed independently in conjunction with the NIHR Cambridge Biomedical Research Centre, Core Biochemical Analysis Laboratory (CBAL), Addenbrookes Hospital, Cambridge. Serum samples were assessed at week 0, 4 and 8 using a Siemens Dimension EXL analyser  for the following analytes (between-batch imprecision (BBI) included): glucose (BBI: 3.6% at 3.5 mmol·Lequation  as follo−1 and 7.0% at 135.0 pmol·L−1. An updated Homeostasis Model Assessment (HOMA) was employed to estimate steady state beta cell function (%B), insulin sensitivity (%S) and insulin resistance (IR) using a HOMA2 calculator  based on previous research [−1, 4.5% at 466 µmol·L−1). Serum leptin and adiponectin were assessed using an AutoDELFIA analyser (Perkin Elmer LAS (UK) Ltd., Beaconsfield, Buckinghamshire, UK), employing in-house two-site microtiter plate-based DELFIA assay , with antibodies and standards supplied by R&D systems . Additionally, serum insulin was assessed using a sandwich chemiluminescence immunoassay via a DiaSorin Liaison XL analyser , Italy), with BBI noted as 11.0% at 34.0 pmol·Lresearch . Serum g−1, 6.4% at 282 U·L−1); alanine aminotransferase ; aspartate aminotransferase ; and bilirubin . As a means to detect any acute elevations in liver enzymes, participants also had an additional blood sample collected at week 2 of the intervention as a precautionary measure. An important feature of the current study, and with recent concerns around potential for high-dose catechin-related hepatic toxicity, a liver enzyme panel was also carried out using a Siemens Dimension EXL analyser  for the following analytes: alkaline phosphatase  also reported . Data are reported as the mean ± SE.Statistical analyses were performed using SPSS . Normality of data was verified by the Shapiro–Wilk test. Outliers were identified by inspection of box plots > 1.5 interquartile range in SPSS. Baseline measures were assessed using a between-groups analysis of variance (ANOVA), or where applicable  an independent samples t-test was employed. A mixed-design repeated-measures ANOVA  were performed for main analyses, with Bonferroni post-hoc comparisons where applicable. Where sphericity was violated a Greenhouse–Geisser correction was applied. An alpha level of ≤0.05 was employed for statistical significance, with effect size . In addition, no differences were reported between mean energy intake compared with expected maintenance caloric intake (p > 0.05) overall or within groups at week 4 or 8, demonstrating dietary consistency.Dietary analysis comparisons across the intervention are shown in −1  compared with 1882 ± 96 kcal·d−1  for dGTE, and 1975 ± 96 kcal·d−1  for PL. Macronutrient intake was also comparable, with an average fat intake of 1.0 ± 0.1 g·kg−1·day−1 for dGTE+ compared 0.9 ± 0.1 g·kg−1·day−1 for dGTE and 0.9 ± 0.1 g·kg−1·day−1 for PL. Likewise, average carbohydrate intake was 2.7 ± 0.2 g·kg−1·day−1 for dGTE+ compared with 3.0 ± 0.2 g·kg−1·day−1 for dGTE and 2.7 ± 0.2 g·kg−1·day−1 for PL. Similarly, average protein intake was comparable between groups at 1.1 ± 0.1 g·kg−1·day−1 for dGTE+, 1.2 ± 0.1 g·kg−1·day−1 for dGTE, and 0.9 ± 0.0 g·kg−1·day−1 for PL. Across the 8 week intervention, average daily energy intake for dGTE+ was 1918 ± 66 kcal·d−1, with no differences reported between groups . Mean adherence to the supplement intervention was 95.4 ± 1.0%. Within groups, adherence rates were 93.0 ± 1.1%, 96.3 ± 0.8% and 96.9 ± 1.3% for PL, dGTE and dGTE+, respectively, with a significant difference reported between PL and dGTE+ (p = 0.04) only.Estimation of dietary quercetin highlighted a mean overall intake of 20.6 ± 8.5 mg·dp > 0.05) demonstrating relative consistency. Mean weekly duration was reported at 189.7 ± 22.4 min, 204.0 ± 20.9 min and 196.4 ± 29.1 min for PL, dGTE and dGTE+, respectively (p > 0.05), meeting expected recommendations. No significant differences were observed for mean HR compared to training recommendations (75–80%HRmax) for PL (82 ± 2%HRmax), dGTE (79 ± 2%HRmax) and dGTE+ (79 ± 1%HRmax), respectively (p > 0.05).Exercise monitoring training load comparisons are shown in p > 0.05). A significant interaction effect was found for maximal fat oxidation , with post-hoc analysis highlighting a 45.4% increase in MFO for dGTE+ at week 8 (p = 0.009) compared to baseline only. This was supported with a noted trend at week 4 for dGTE+ (p = 0.063) also compared to baseline. Similarly, dGTE+ resulted in 49.4% increase in relative MFO  by week 8 compared to baseline (p = 0.024), with a trend noted compared to week 4 (p = 0.059). dGTE had a positive, but non-significant impact on MFO (+12.7%) and MFO·FFM−1 (+13.9%), whereas PL resulted in a non-significant reduction in MFO (−11.8%) and MFO·FFM−1 . Mean fat oxidation measures are shown in p = 0.037, ηp2 = 0.24), but not by week 4 (p = 0.113). Normalised MFO was significantly greater with dGTE+ only (+0.65 ± 0.25 fold change) compared with PL  across the intervention. Similarly, normalised relative MFO was significantly different between groups by week 8  but not by week 4 (p = 0.073), again with a highlighted increase for dGTE+ (+0.76 ± 0.24 fold change) compared with PL . In terms of normalised fold change , a signiMAX or HR at FATMAX within or between groups (p > 0.05). However, a positive interaction effect was observed for FATMIN , with dGTE+ resulting in a 22.5% increase in FATMIN by week 8 compared with week 4 (p = 0.003) and a trend noted compared to baseline (p = 0.056). When expressed as normalised fold change, a significant effect was found for FATMIN by week 8 , with dGTE+ demonstrating a +0.18 ± 0.08 fold change compared with PL . Throughout the intervention, as expected, 2peak was maintained for PL: 2.5 ± 0.2 L·min−1, 2.4 ± 0.1 L·min−1 and 2.4 ± 0.1 L·min−1; dGTE: 2.4 ± 0.2 L·min−1, 2.5 ± 0.2 L·min−1, and 2.4 ± 0.2 L·min−1; and dGTE+: 2.1 ± 0.2 L·min−1, 2.3 ± 0.2 L·min−1, and 2.1 ± 0.2 L·min−1, with no differences reported within or between groups at week 0, 4 and 8, respectively (p > 0.05).No significant effects were found for FAT2peak, 2: 1.4 ± 0.1 L·min−1; week 4: 59.8 ± 1.6% 2peak, 2: 1.4 ± 0.1 L·min−1; week 8: 58.7 ± 1.5% 2peak,2: 1.4 ± 0.1 L·min−1; p > 0.05), with no differences reported within or between groups, or compared to set exercise intensity (p > 0.05). No significant differences were reported at baseline for all variables (p > 0.05). A significant interaction effect was found for mean RER , with post-hoc analyses revealing a reduction in RER by week 8 for dGTE+ compared with week 4 (p = 0.004). When normalised data were considered, a significant overall between-group effect was found for RER , highlighting a difference between dGTE and dGTE+ by week 8 (p = 0.034).Mean steady state exercise measures are shown in OX during steady state exercise , no significant post-hoc analyses were found, despite a +28.4% increase in steady state FATOX by week 8 with dGTE+ (p > 0.05). However, a significant interaction was found with relative FATOX , with a +54.2% increase in FATOX·FFM−1 by week 8 compared to week 4 for dGTE+ only (p = 0.036). Normalised data also highlighted a significant between-group effect for FATOX by week 8  with a +0.84 ± 0.48 fold change for dGTE+ compared with both PL (−0.22 ± 0.14) and dGTE (−0.22 ± 0.15). However, post-hoc analyses only revealed a trend between PL and dGTE (p = 0.064) and dGTE and dGTE+ (p = 0.063). A similar pattern was found for normalised FATOX·FFM−1 , with no significant post-hoc analyses .Whilst a significant interaction effect was also found for mean FATp > 0.05). Similarly, mean fat oxidation stability did not significantly improve with either dGTE formula, despite an observed trend  by week 8 for dGTE+. No significant differences were observed for normalised data for carbohydrate or fat oxidation stability at week 4 or 8 (p > 0.05). No significant differences were reported for EE within or between groups (p > 0.05). However, a significant interaction effect was found for both the contribution of carbohydrate  and the contribution of fat  to EE, highlighting a −17.8% reduction in carbohydrate contribution (p = 0.008) and a +64.8% increase in fat contribution (p = 0.006) to EE for dGTE at week 8 compared to week 4, respectively. When normalised data were considered, a significant between-group effect was observed for both carbohydrate  and fat  contribution to EE by week 8. However, post-hoc analyses only indicated a difference between dGTE and dGTE+ for carbohydrate contribution to EE (p = 0.043), but not fat contribution (p = 0.083).No significant effects were found for mean carbohydrate oxidation stability, despite a noted -11.9% reduction by week 8 for dGTE+ (p > 0.05). No significant differences were observed for body mass, BMI, body fat, fat-free mass, fat mass, WHR, SAD, VFA or blood pressure within or between groups (p > 0.05). A main effect was reported for waist circumference , with a −0.8 ± 0.2 cm relative decrease at week 4 compared to baseline for PL only (p = 0.019). A main effect was also reported for abdominal circumference , with a −1.6 ± 0.4 cm relative decrease by week 4 for dGTE compared to baseline (p = 0.004). Resting HR was significantly reduced by week 4 for dGTE+ compared with dGTE only .Mean body composition, resting HR and blood pressure measures are shown in p > 0.05), except adiponectin. Mean adiponectin levels for dGTE+ were significantly greater compared to PL at baseline (p = 0.036), week 4 (p = 0.021) and week 8 (p = 0.049). No other main effects were found for all variables, except for LDL-c, where a main interaction effect was observed . However, post-hoc analyses did not reveal any significant differences for LDL-c within or between groups despite a relative −0.38 ± 0.22 mmol·L−1 reduction for dGTE+, and a relative +0.30 ± 0.12 mmol·L−1 increase for dGTE over the intervention. When data were normalised, a significant interaction effect was found for LDL-c over the intervention  highlighting the difference between dGTE and dGTE+ only (p = 0.038).Mean serum cardio-metabolic markers, including normalised fold change, are shown in −1, −8.22 ± 7.43 U·L−1 and −0.89 ± 1.78 µmol·L−1, respectively, by week 8 for PL (p > 0.05). However, ALP significantly reduced by −6.67 ± 3.03 U·L−1 over time  for PL only (p ≤ 0.025 for both baseline and week 4 compared with week 8). When data were normalised, a significant between-group effect was reported overall for ALT only , with the highlighted difference between PL and dGTE+ (p = 0.015) likely explained by the relative decrease for ALT in PL over time. All liver enzymes, and bilirubin, were unaffected by both dGTE and dGTE+ supplementation.Mean serum liver function markers, including normalised fold change, are shown in MIN (+22.5%) was observed with dGTE+ over the last 4 weeks of this study. Metabolic changes were also observed by week 8 for dGTE+, with a large effect size reported for improvements in steady state substrate utilisation, evidenced by lower mean RER (−5.3%), improved relative FATOX (+55.2%) and increased the contribution of fat to total EE (+64.8%). Collectively, these findings suggest that a novel dGTE+ formula positively influenced fat oxidation in overweight individuals, particularly in the final 4 weeks of this study.This study aimed to assess the longer-term impact of moderate dGTE supplementation (with or without antioxidant nutrients) on fat oxidation, body composition and cardio-metabolic risk factors in overweight, but healthy individuals engaged in regular exercise. The findings from the current study demonstrated that a moderate dose of dGTE consumed over 8 weeks did not result in a statistically significant change on fat oxidation rates, but did produce a non-significant increase in both MFO (+12.7%) and relative MFO (+13.9%). In contrast, the use of a novel dGTE+ formula, containing antioxidants in addition to dGTE, had a significant impact (moderate–large effect size) on MFO (+45.5%) and relative MFO (+49.4%) by week 8 of supplementation in recreationally active, overweight individuals. Additionally, a large effect size for FATOX measures, concerns have been noted in the literature pertinent to reliability and variability [OX by +28.4%, which was comparable to previous work undertaken [OX rates in the current study were comparable to previous research with untrained [−1). As such, normalised data were also assessed to confirm or support findings. When data were normalised, taking into consideration individual responses to the intervention, MFO, relative MFO and FATMIN were all significantly enhanced with dGTE+ overall compared with PL, but not dGTE. However, for steady state exercise, normalised data only confirmed improved substrate utilisation for dGTE+ based on a reduced RER and the contribution of carbohydrate to total EE compared with dGTE. Whilst this further supports the contention that dGTE+ improved fat oxidation dynamics and substrate utilisation efficiency, data should be interpreted with caution based on individual ‘responders’ to the protocol. Overall FATMAX was not improved with dGTE+, despite a +24% increase in the latter 4 weeks of this study. This may be explained with the wide range of FATMAX values observed across this study (range: 24.1 to 52.4% 2peak).With regards to the methodological approaches to FATiability ,54,55. Tdertaken , despitentrained , active ntrained ,58,59 orntrained ,61,62, a−1 EGCG) [−1 EGCG) [−1), although these findings were based on pooled data from only two randomised controlled trials and should be interpreted with caution. Interestingly, beneficial effects of GTE have been mainly observed with healthy, active, untrained or sedentary individuals using acute (<2 days) moderate EGCG dose [Previous studies have reported mixed findings on the effect of GTE on fat oxidation, with several observing improvements in whole-body fat oxidation rates (+17–35% improvement employing a dose range of 270–400 mg·d−1 EGCG) ,19,25 an−1 EGCG) ,24,26,63−1 EGCG)  highlighGCG dose ,17,18,19GCG dose ,66. It iGCG dose ,22,23,24OX during exercise [Collectively, these studies indicate that EGCG (both caffeinated and decaffeinated) may be effective in recreationally active or less trained individuals, potentially via thermogenic mechanisms pertinent to COMT inhibition and increased lipolytic activity leading to enhanced FATexercise ,14, althexercise  and/or sexercise ,68. Howeexercise . Future In the current study, the specific use of dGTE did not significantly enhance fat oxidation measures in agreement with previous research ,22,26,63OX, it is feasible the observed metabolic benefits associated with dGTE+ may be specific to LA. Whilst numerous mechanisms have been proposed, LA supports mitochondrial efficiency as a cofactor for α-ketoacid dehydrogenases [OX and subsequent weight loss. However, whilst a recent meta-analysis supports the contention that LA may elicit small, meaningful weight reduction benefits, studies ranging from 8–52 weeks have employed higher LA doses between 300 and 1800 mg·d−1 [The additional inclusion of LA in the dGTE+ formula may have also enhanced EGCG stability through local antioxidant mechanisms, leading to increased EGCG availability. However, as EGCG bioavailability was not ascertained in the current study, this cannot be confirmed. As the use of dGTE only did not significantly influence FATogenases . Reducedogenases ,72,73. Aogenases ,72. Ther0 mg·d−1 , which mOX by week 8, any changes in body composition attributed to EGCG mechanisms may require longer interventions based on overall relative patterns in body composition indices with dGTE+. Indeed, a previous meta-analysis [Whilst the use of dGTE+ impacted on fat oxidation, by the end of the intervention, neither dGTE supplementation influenced overall body composition measures. This was despite relative, albeit small reductions in overall mean body mass (−0.50 kg), body fat percentage (−0.50%), fat mass (−0.37 kg), abdominal circumference (−0.77 cm) or SAD (−0.61 cm), particularly for dGTE+ compared with PL. Furthermore, the relative reduction in estimated VFA with dGTE+ was ~3-fold greater than for both dGTE and PL, albeit non-significant. Maintenance of habitual exercise only marginally impacted on body composition indices as evidenced by a significant reduction in waist and abdomen circumference for PL and dGTE at week 4 within group only. Collectively, the results indicate that when habitual exercise and dietary intake are controlled for, dGTE supplementation did not favour improved body composition indices. As dGTE+ only enhanced FATanalysis  highligh−1) [−1) [MAX  ,67,75, a−1)  also int−1) [−1) ,13,29, p−1) [−1) ,29. Inte V.O2max ,76,77) i studies ,78) were−1) was comparable to previous studies [−1 [−1) when capsulated or decaffeinated green tea products are used [−1) and triglycerides (−0.23 mmol·L−1) in longer-term clinical trials (>12 weeks) [−1 with LA alone, which is comparable to that observed in the current study. Cardio-metabolic variables were largely unaffected by either dGTE supplementation, with the exception of a medium effect size reported for LDL-c which was ~11% lower with dGTE+ by week 8, and further supported via normalised values. The relative reduction in total LDL-c across the intervention with dGTE+  . In the 2 weeks)  have repBoth leptin and adiponectin levels were unaffected across the intervention with dGTE or dGTE+. This agrees with a recent meta-analysis  demonstrIt is also noteworthy, when considering normalised values, that mean insulin, HOMA-IR and leptin changes were elevated with PL across the intervention , with negligible changes observed for dGTE or dGTE+. It is therefore unclear whether the small changes observed for PL were related to dietary composition differences, or whether dGTE acted in a ‘protective’ manner. However, collectively, these results support previous findings that GTE was ineffective in improving cardio-metabolic function ,27,28, bWhilst dGTE+ also resulted in a significant reduction in resting HR by week 4 compared to dGTE, but not by week 8, this did not impact on blood pressure measurements. This is somewhat surprising as a recent meta-analysis on green tea  highlighted studies (≤12 weeks) resulting in a reduction of −1.32 mmHg for SBP (non-significant) and -1.78 mmHg for DBP (deemed significant), with lower observed effects for capsulated green tea, and green tea  consumed over longer periods (> 12 weeks) . In the An important aspect of the current study was the inclusion of liver enzyme and bilirubin measures. This was undertaken based on current concerns with the potential for GTE-induced hepatotoxicity . HoweverIt is important to note several limitations of the current study. As previously mentioned, EGCG bioavailability was not undertaken, and as no LA-specific intervention was included, findings should be interpreted with caution. Further studies investigating the specific effects of LA with or without dGTE are therefore warranted. In the current study a standardised dGTE (70% EGCG) product was sourced based on our previous research . Whilst −1) was comparable between groups, and similar to previously reported research [In a similar context, consideration to dietary consistency and total polyphenol intake is also warranted. In the present study, relative dietary intake consistency was established in the 7 day period leading into each testing session. Furthermore, estimated quercetin intake  was effective in improving MFO and FAT


Absence of cortical alignment in wedge-shaped and multifragmentary fractures (Fx) results in decreased fixation stability. The aim of this study was to compare the outcome using 2.0- vs. 3.5-mm screws for open reduction and internal fixation (ORIF) in dislocated, wedge-shaped or fragmentary midshaft clavicle fractures.Patients suffering from AO/OTA 15 2.A-C midshaft clavicle fractures were operatively treated between 2008 and 2018. 2.0- or 3.5-mm cortical screws were used to restore anatomic alignment in dislocated, wedge-shaped and fragmentary clavicle fractures. Data of radiologic outcome were collected until fracture consolidation was identified.80 consecutive patients with a mean age of 44.5 ± 16.3 years, who were operatively treated for dislocated midshaft clavicle fractures were enrolled. 40 patients were treated using 2.0-mm and 40 patients using routine 3.5-mm cortical screws, respectively. Time to fracture consolidation was 12.8 ± 7.8 months. No mal- or non-unions occurred during routine follow-up until 18 months postoperatively.Restoring anatomic alignment in wedge or fragmentary clavicle fractures can ultimately be addressed using cortical screw augmentation. Both groups showed comparable results with respect to fracture reduction, fixation and stability as well as time to consolidation of the fracture, while the 2.0-mm screw diameter was associated with easier handling of small Fx fragments. In daily clinical routine, the majority of clavicle fractures can be treated conservatively. However, the degree of dislocation and shortening of the clavicle needs to be considered regarding decision-making. Open Fx, infection or non-union of the Fx are regarded as evident indications for surgical treatment. However, there has been a considerable increase in operative treatment of clavicle Fx even exceeding the rising incidence of clavicle Fx . From a In this retrospective cohort study, patients suffering from midshaft clavicle Fx , who were operatively treated in our level I trauma center from September 1st 2008 until January 1st 2018 were identified. The AO/OTA classification was used to determine indication for surgical treatment. Preoperative X-ray imaging in two planes was performed in all patients. Whole-body computed tomography (CT) was performed in patients who were transferred to our emergency room to rule out additional injury . Operatip = 0.969). 42 Fx occurred on the left and 38 on the right hand side. 40 consecutive patients were assigned to the 2.0-mm cortical screw group  and another 40 consecutive patients were treated by 3.5 mm  cortical lag screw augmentation. Overall, 80 consecutive patients were enrolled for statistical follow-up. 25 of 40 (62.5%) group I patients and 32 of 40 (80%) group II patients completed all follow-up visits and were eventually considered for statistical evaluation  with a mean age of 44.5 ± 16.3 (range 25.1–81.01) years were operatively treated for dislocated midshaft clavicle Fx (AO/OTA 15.2A-C) between September 1st 2008 and January 1st 2018. The patients showed a comparable mean age of 43.6 ± 16.0  compared to 45.8 ± 15.4   in group I (83.1 ± 19.2) as compared to group II (93.4 ± 35.8). 40 patients received 2.0-mm cortical screw augmentation and 40 patients were treated with 3.5-mm cortical lag screw augmentation. 65 patients were treated using a superior locking compression plate  and 15 patients using a superior clavicle fracture plate . Overall, the mean Fx healing time was 12.8 ± 7.4 months. The time for Fx healing in group I (2.0 mm) accounted for 10.5 ± 5.5 months, whereas a fracture healing time of 14.6 ± 8.5 months was recorded for group II (3.5 mm). Thus, there was no statistically significant difference for fracture healing in group I (2.0 mm) as compared to group II (3.5 mm) (p = 0.10). The type of the used plate osteosynthesis did not influence the time until bony union was achieved . Implant removal was performed upon the patients’ explicit request after a mean of 18.9 ± 7.1 months in 33/80 patients . No mal- or non-unions, as well as no re-Fx of affected clavicles were detected throughout the follow-up appointments, respectively  of 722 nonsmokers/33.3% (n = 73) of 219 smokers which goes along with results from Clement et al. [n = 33). High irritation rates after osteosynthesis of the clavicle are reported in the literature, yet from our experience patients profit from implant removal [p = 0.013) can be explained by the easier handling of 2.0-mm screws in small fragmentary clavicle fractures, which represents the two senior surgeons’ opinion . Obviously, the faster surgical duration is in turn owed to the surgeons’ experience as well as complexity of the fracture itself.However, the majority of clavicle Fx can still be treated conservatively, although there has been a clear trend towards operative treatment in the last years . One majt et al. . With tht et al. . With ret et al. , 12, 13.t et al. , 10. Symt et al. . Operatit et al. –19. Knowt et al. , 8, 20.  removal . Howeverp = 0.10), yet without a statistical significance. Also, the type of the locking compression plate used did not interfere with the time until Fx healing was achieved, yet this study suggests using preformed locking compression plates instead of reconstruction plates, since they showed an inferior outcome from a biomechanical point of view [Our study results suggest that cortical lag screw augmentation results in additional fixation stability in wedge-shaped and fragmentary clavicle Fx. In direct comparison, the use of 2.0- and 3.5-mm cortical screws did not show a significant advantage and the diameter of the cortical screw was chosen upon the surgeon’s preference. However, time until Fx healing was shorter in the 2.0-mm group  until fracture healing or after implant removal, which is why complementing an additional cortical lag screw augmentation using 2.0-mm screws can be advised to achieve adequate stability in wedge-shaped and fragmentary midshaft clavicle Fx.


Whiteflies are significant pests of cotton and vegetables in southeastern USA. In previous studies, we isolated and identified a new strain of entomopathogenic fungus that caused epizootics among whiteflies in cotton fields of Southern Georgia, USA. The objective of this study was to test the level of tolerance of this new strain to environmental conditions as compared to commercial fungal strains. We exposed the new strain and three commercially available strains of biopesticides  to different temperatures and strong ultraviolet (UV) radiation before examining post-treatment viability and virulence against a common model organism for insect pathology, the greater wax moth larvae. We found that the new strain had similar levels of activity to commercial strains at moderate temperatures, but higher tolerance than PFR-97 to extremely low and high temperatures and strong UV intensity. These findings suggest that the new fungal strain has potential for commercial development as an alternative to PFR-97 for managing certain types of insect pests.Cordyceps javanica (wf GA17) was observed causing widespread epizootics among whiteflies in Southern Georgia in 2017. The tolerance of conidia to environmental factors including variable temperature and ultraviolet (UV) light was compared between this strain and three commercial strains of entomopathogenic fungi . Under 10–30 °C, C. javanica wf GA17 responded similarly to other fungi, with the highest virulence against Galleria mellonella at 25 °C, followed by 20, 30, and 15 °C; lowest virulence was observed at 10 °C. At 35 °C and 40 °C, C. javanica wf GA17 had lower tolerance than M. brunneum F52 and B. bassiana GHA, but was superior to C. fumosorosea Apopka97 in conidia viability and post-treatment virulence. After exposure to −20 °C for 56 d, C. javanica wf GA17 exhibited lower germination than M. brunneum F52 and lower virulence than M. brunneum F52 and B. bassiana GHA, but higher germination and virulence than C. fumosorosea Apopka97. Following exposure to strong UV light, viability and virulence of all fungi were reduced with increasing exposure periods. Increased environmental tolerance of C. javanica wf GA17 over C. fumosorosea Apopka97 suggests that the new strain could have applicability for commercial pest management.A new strain of  Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), comprises a complex of biotypes, and infests a wide range of vegetable, ornamental, and field crops and may cause significant crop losses at high population densities [B. tabaci MEAM1  occurred in vegetable and cotton fields in southern Georgia in USA in 2017, during which a widespread naturally occurring epizootic of fungal pathogens was observed among whitefly populations [Cordyceps javanica  .The whitefly, ensities ,2. The pensities ,4. An ouulations . The funavanica, ), which The entomopathogenic fungi (EPF) can provide safe and effective suppression of arthropod pests. They have a broad host range and therefore some of them have been commercially produced as biocontrol agents . InfectiBeauveria bassiana  Vuill. strain GHA decreased more slowly on the leaf undersides (9–11% per day) compared with the upper leaf surface without shade (47% per day). Thus, these environmental factors are important considerations when evaluating the commercial potential of EPF species and strains.The efficacy of EPF in the field may be affected by various ecological factors, including solar radiation (primarily shortwave ultraviolet (UV)), rain, temperature, humidity, surface chemistry, and phylloplane microbiota ,8,9,11. C. javanica wf GA17 to environmental factors has not been reported, but it is crucial for considerations of potential commercialization and field applications. Therefore, the objective of this project was to compare the environmental tolerance of C. javanica wf GA17 to three commercialized EPF strains (Cordyceps fumosorosea (formerly Isaria fumosorosea) (Wize) Apopka strain 97, B. bassiana strain GHA, and Metarhizium brunneum Petch (formerly Metarhizium anisopliae (Metchnikoff) Sorokin) strain F52. Specifically, we assessed the effect of environmentally relevant temperatures on the virulence of C. javanica wf GA17, as well as its tolerance to temperature extremes (high and low) and UV light.The tolerance of M. brunneum strain F52 (Mb F52), C. fumosorosea strain Apopka97 (Cf Apopka97), B. bassiana strain GHA (Bb GHA), and the new strain of C. javanica wf GA17 (Cj wf GA17), were used in this study. Strains Cf Apopka97, Bb GHA, and Mb F52 were cultured from the commercial products PFR-97 , BotaniGard 22WP , and Met52 EC , respectively. In contrast, Cj wf GA17 was isolated during the natural epizootic affecting whiteflies (B. tabaci MEAM1) in southern Georgia, USA, in September 2017  locus; accession no. MN461230 for translation elongation factor (tef) gene sequences for identification of Cj wf GA17); it was deposited to the USDA–ARS Collection of Entomopathogenic Fungal Cultures (ARSEF) (accession no. ARSEF 14327).Four entomopathogenic fungal strains, 6 viable conidia mL−1 with 0.05% Silwet L-77 as the solvent. Conidia viability was tested the day before experiments by spreading 0.1 mL suspension of 106 conidia mL−1 in PDA with 1% yeast extract (60 mm), incubating at 25 °C for 16 h, and observing the germination rate of the first 200 conidia encountered at 400× magnification [All fungi were cultured in parallel on potato dextrose agar (PDA) for 7–10 days at 25 °C with a 14:10 (L: D) (14 h light: 10 h dark) photoperiod followed by storage at 4 °C for up to 2 weeks. Conidia were harvested by washing with sterile 0.05% Silwet L-77  after gently scraping the surface with a sterilized blade. The mixtures were transferred to centrifuge tubes (50 mL) provisioned with six glass beads (6 mm) that facilitated mixing and fragmenting conidia clusters. Conidia suspensions were vigorously vortexed for 5 min until conidia clusters were broken apart and thoroughly mixed. Using a hemocytometer at 400× magnification, an average count from at least four samples was used to estimate the conidia concentration of each fungus. The fungal suspensions were serially diluted from the original concentration to 10fication . Conidiafication . G. mellonella under six environmentally relevant temperatures , modified based on the study of Miętkiewski et al. [6 viable conidia mL−1 and the untreated control received 1 mL of 0.05% Silwet L-77. All treatments were applied to 30–mL clear plastic Solo cups filled with 10 g of autoclaved soil in a loamy sand texture , and 0.4 mL of distilled water was added after treatment, resulting in final soil moisture of 14% (volume by weight). Immediately after application, a single last instar G. mellonella was added to each cup. Each cup was then covered with a tight fitting lid and placed on cafeteria trays, which were bagged with wet paper towels to maintain moisture before being placed in growth chambers maintained at 14:10 (L:D) photoperiod and randomly assigned to a given temperature. The percentage of insect morality and signs of mycosis  [The four fungal strains and an untreated control were tested against i et al. . Each tr6 viable conidia mL−1 suspension in 0.05% Silwet L-77) in a 2 mL sterile Nalgene cryogenic vial  that was subsequently exposed to specified temperature treatments for different lengths of time. After treatment, the vial was vortexed for 1 min before taking samples to check conidia viability and virulence. A 0.1 mL aliquot was spread over PDA with 1% yeast extract (60 mm Petri dish) and incubated at 25 °C for 16 and 40 h to score the germination rate in separate dishes; two germination dishes were prepared for each observation. The virulence of conidia after temperature treatment was tested in Petri dishes , and an untreated control was used in parallel for each time point. An aliquot of 0.1 mL conidia suspension was applied to a 35–mm Petri dish lined with a single layer of sterile filter paper , and one last instar G. mellonella larva was added after application. The untreated control received 0.1 mL sterile 0.05% Silwet L-77 and the insect only. There were three replicates of 10 dishes per treatment. After treatment, the dishes were arranged in trays and placed in a growth chamber maintained at 25 °C . Insect mortality and mycosis were evaluated at 7 and 14 dpi. The entire experiment was repeated twice.Conidia viability and virulence of the four EPF strains were tested at low (−20 °C) and high (35 and 40 °C) temperatures. The −20 °C treatments were performed in a laboratory freezer, while the 35 °C and 40 °C replicates were tested in water baths. The EPF were exposed to different conditions for different lengths of time based on preliminary tests. At −20 °C, both conidia viability and virulence were observed at 0 and 56 days post treatment. At 35 °C, conidia viability was evaluated at 0, 2, 4, 8, 12, 24, 48, and 72 h of treatment; fungal virulence was checked at 0, 12, 24, and 48 h. At 40 °C, conidia viability was observed at 0, 0.5, 1, 2, 3, 4, 6, and 8 h and virulence was examined at 0, 1, 2, and 4 h of treatment. Tests were conducted by placing 1.5 mL of conidia suspension  and incubated at 25 °C for 16 and 40 h to score the germination rate in separate dishes; two germination dishes were prepared for each observation. The virulence of conidia after UV treatment was tested in Petri dishes as described previously for the temperature experiments. A complete set of fungal dishes that were not exposed to UV was also assessed in parallel to evaluate conidial germination and virulence. Insect mortality and mycosis were evaluated at 7 and 14 dpi. The entire experiment was repeated twice.Conidia suspensions of the four EPF were exposed to UV light to compare the UV tolerance of the new strain and the commercial strains. The experiment was carried out under a Labconco Purifier Class II biosafety cabinet  equipped with a 254 nm UV lamp . A 1.5 m50) at 16 h incubation for 35 and 40 °C treatment was calculated with probit analysis ; lack of overlap in the 95% fiducial limits (F.L.) of LT50 was used to indicate significant difference between fungi.Experimental design in all experiments was completely randomized, and responses  were analyzed using a two-way (temperature and fungus) factorial design under a linear mixed model analysis with multiple mean comparisons following the Tukey’s test (α = 0.05)  . Respons4,158 = 50.75, p < 0.0001) and fungal strain , and there was no significant interaction between temperature and fungus  , while fungal strains did not affect mortality ; there was no significant interaction between the two factors  , and no mycosis was observed in any fungal treatment. Therefore, only data observed at 10–30 °C are presented below. At 7 dpi, insect mortality was significantly affected by both temperature (F 0.1761) A. At bot 0.9906) B. All fu4,158 = 53.30, p < 0.0001), whereas fungal strain only had an effect at 20 °C; Cj wf GA17 and Cf Apopka97 exhibited lower mycosis than Mb F52 and Bb GHA  . Mycosis development followed a similar trend as mortality. At 7 dpi, all fungi had the highest mycosis at 25 °C; mycosis was lower at 20 and 30 °C, but the effects were similar. Very low mycosis occurred at 10 °C and 15 °C. At 14 dpi, mycosis levels were significantly affected by temperature , but not by fungus ; there was a significant interaction between these factors   C. There = 0.024) D. Cj wf 6,305 = 106.32, p < 0.0001) and fungal strains ; there were significant interactions between time and strain  (50) was 26.28 h (95% F.L.: 25.74–26.83 h) for Mb F52, 4.34 h (95% F.L.: 4.22–4.46 h) for Cj wf GA17, 2.72 h (95% F.L.: 2.66–2.78 h) for Cf Apopka97, and 19.49 h (95% F.L.: 19.14–19.84 h) for Bb GHA. These data suggested that Mb F52 had the highest heat tolerance, followed by Bb GHA, then Cj wf GA17, and Cf Apopka97. Delayed germination after 40 h incubation followed the same pattern as 16 h germination, and it appeared in Mb F52, Cj wf GA17, Cf Apopka97, and Bb GHA by 72 h (10.6 ± 6.3%), 24 h (1.2 ± 0.5%), 12 h (0.7 ± 0.4%), and 48 h (3.5 ± 1.1%) of heat treatment, respectively.At 35 °C, conidia viability before treatment was ≥95% for all strains. Conidia germination varied significantly due to both time of heat exposure (F 0.0001) A. A sign6,139 = 185.42, p < 0.0001) and fungi  affected 16 h germination, and had significant interactions  (50) was 2.24 h (95% F.L.: 2.20–2.27 h) for Mb F52, 0.91 h (95% F.L.: 0.89–0.93 h) for Cj wf GA17, 0.70 h (95% F.L.: 0.69–0.72 h) for Cf Apopka97, and 2.52 h (95% F.L.: 2.48–2.56 h) for Bb GHA. These data suggested that, at 40 °C, Bb GHA had the highest germination rate, followed by Mb F52, then Cj wf GA17, and Cf Apopka97 still had the lowest tolerance. However, delayed germination at 40 h was observed in Mb F52, Cj wf GA17, Cf Apopka97, and Bb GHA by 8 h (12.3 ± 2.3%), 4 h (0.3 ± 0.2%), 2 h (0.3 ± 0.3%), 8 h (0.9 ± 0.5%) of heat treatment, respectively, indicating similar thermotolerance of Bb GHA and Mb F52 at 40 °C.At 40 °C, conidia viability before treatment was >98% for all fungi. Similar to 35 °C, both time of heat treatment (F 0.0001) B. A sign3,79 ≥ 15.26, p < 0.0001) and fungal strains , in causing both insect mortality and mycosis at both 7 and 14 dpi . At 7 dpi, decline in mortality started after 48, 12, 12, and 24 h of heat treatment for Mb F52, Cj wf GA17, Cf Apopka97, and Bb GHA, respectively  and fungal strain , with a significant interaction between the factors   A–D. At 7 0.0252) A,C. At 7 0.0252) B,D. At 11,39 = 161.47, p < 0.0001), and the level of reduction varied with fungal strain  , ranging from no impact on Mb F52, Cj wf GA17, and Bb GHA, to significant suppression on Cf Apopka97  , the 56-day treatment had reduced germination of all fungi (F 0.0206) A. After  0.0336) B,C.p > 0.05). Although Cf Apopka97 had lower mycosis (76.7 ± 6.1%) than other fungi  for the 56-day exposure , there was no difference between days 0 and 56 in any fungus .At the 14 dpi observations, insect mortality was ≥ 85.1% and mycosis was ≥ 76.7% in all treatments; compared with 0 day, there was no treatment effect in reducing either mortality or mycosis ; except for Bb GHA being marginally more tolerant than other fungi at 5 min UV , there was no differences among fungi in UV susceptibility otherwise (data not shown). After UV exposure and subsequent 40 h incubation, germination was significantly inhibited by UV exposure , regardless of fungal strains (p > 0.05). Both 5 min and 10 min UV treatment decreased conidia germination of all fungi; the inhibition was more pronounced at 10 min, with only 0.2% germination of Mb F52 and 1.0% germination of Bb GHA and no germination of Cj wf GA17 and Cf Apopka97 , regardless of fungal strain (p > 0.05). At 14 dpi, insect mortality was affected by both time length of UV treatment  and fungal strain ; mycosis was affected by UV treatment , but not by fungal strain (p > 0.05) , but not in mycosis (p > 0.05). Compared to observations before UV treatment, a significant decline in mortality and mycosis appeared in all treatments except after 5 min exposure of Mb F52 and Cj wf GA17 (reduced mycosis). Compared to 5 min UV, the 10 min treatment resulted in lower mortality and mycosis in all fungi, except there was no difference in mortality caused by Bb GHA. For 5 min UV, Cf Apopka97 and Bb GHA had lower mortality than Mb F52 and Cj wf GA17; for 10 min, Cf Apopka97 had lower mortality than Mb F52 and Bb GHA, but was not different from Cj wf GA17  and were significantly affected by UV treatments at both 5 min and 10 min lengths (F > 0.05) B,C. Ther wf GA17 B. Howeve wf GA17 C.B. bassiana and four strains of M. anisopliae on the legume flower thrips, Megalurothrips sjostedti, ranged between 25 and 30 °C; activities of all tested fungal strains were low at 15 and 35 °C. In addition, Miętkiewski et al. [B. bassiana, M. anisopliae, and Paecilomyces fumosoroseus (C. fumosorosea) at 35 °C under which G. mellonella was exposed to pine litter as a “bait”; the highest activity of M. anisopliae occurred at 30 °C, B. bassiana at 25 °C, and activity of P. fumosoroseus appeared at 15–25 °C only, with optimum growth at 27.5, 25, and 25 °C, respectively. This pattern is similar to our findings, except that in our test, M. brunneum was less tolerant to high temperatures and had lower virulence at 30 °C than at 25 °C, which was probably due to strain difference. Cabanillas and Jones [Isaria sp. (Cordyceps sp.), which was later identified as Isaria poprawskii sp. nov. [C. javanica, did not grow at constant 35 °C for 7 days, but it had optimum growth at 30 °C. This is different from the optimum virulence at 25 °C and reduced virulence at 30 °C in our test, which suggests that the two species differ in temperature tolerance.Under environmentally relevant temperatures (10–30 °C), virulence of Cj wf GA17 followed a similar pattern as other fungi, with the highest virulence at 25 °C, followed by 20 and 30 °C, delayed virulence at 15 °C, and low virulence at 10 °C. However, in early observations (7 dpi), Cj wf GA17 and Cf Apopka97 tended to have lower virulence than Mb F52 and Bb GHA. At 30 °C, in late observations 14 dpi), all fungi had reduced mycosis than at 25 °C; particularly, Mb F52 was lower than other fungi; at 35 °C, no fungi showed mycosis development. This was probably due to inhibition of fungal viability and virulence, and competition from saprophytic fungi and bacteria, which outgrew the entomopathogenic fungi under the high temperature. Ekesi et al.  found th dpi, allsp. nov. , a speciI. fumosorosea (C. fumosorosea) strain ARSEF 3889 had lower tolerance than M. brunneum ARSEF 1187 and B. bassiana ARSEF 252, while M. brunneum ARSEF 1187 had lower LT50 of conidial germination than B. bassiana ARSEF 252 (1.83 versus 3.54 h) at 45 °C for 48 h germination. In our study, Mb F52 had higher LT50 than Bb GHA at 35 °C (26.3 versus 19.5 h), but slightly lower LT50 at 40 °C (2.2 versus 2.5 h). Such a difference was probably due to differences in strains and temperatures.The strain Cj wf GA17 showed lower tolerance than Mb F52 and Bb GHA to high temperatures of 35 and 40 °C in both viability and virulence. Similarly, Souza et al.  found thB. bassiana and M. anisopliae in response to heat stress [Isaria sp. (Cordyceps sp.) did not grow when held at a constant 35 °C for 7 days, but was able to recover and grow when transferred to 25 °C; no recovery or growth occurred at either constant 40 °C or transfer from 40 to 25 °C. This indicated that, in short-time heat stress, germination and growth of the fungus may be suppressed and retarded; for longer time or higher temperature of exposure, however, the damage may become irreversible and the fungus may lose viability permanently.Superior heat tolerance in Cj wf GA17 over Cf Apopka97 was observed at the high temperatures of 35 °C and 40 °C in both conidia viability and virulence. However, these advantages may be compromised or diminished at even longer exposure to higher temperatures, given that neither of the fungi survived long-term exposure (lost viability by 24 h at 35 °C and 4 h at 40 °C or earlier). Previously, there have been several reports in delayed germination of t stress ,24,25,26t stress , Isaria B. bassiana strain ARSEF 252 was more cold-active than Metarhizium spp. isolates at 5 °C. The trend is different from our findings, yet this may be explained in that we used different strains and investigated lower temperatures.Similar to tolerance of high temperatures, Cj wf GA17 had higher viability and virulence than Cf Apopka97 and lower virulence than Bb GHA and Mb F52 at −20 °C. However, Cj wf GA17 only had lower germination than Mb F52; while Bb GHA had the lowest germination, its virulence was not affected, probably because the amount of survived conidia was adequate to infect the host. Fernandes et al.  found thB. bassiana GHA had marginally higher viability than other fungi. Fargues et al. [B. bassiana isolates had stronger tolerance to ultraviolet B (UV-B) (≥295 nm) than M. anisopliae and I. fumosorosea (C. fumosorosea) isolates; the latter isolates were most susceptible. Our results followed a similar pattern, but the intensity of UV light used (254 nm) in our treatments was too strong to more clearly separate the fungi in their UV susceptibility. The impact of solar radiation on fungal activity should be further investigated under longer wavelengths of UV.The effect of UV on Cj wf GA17 virulence was similar to other fungi. Although Cj wf GA17 had higher insect mortality than Cf Apopka97 and Bb GHA after 5 min exposure, it caused similar levels of mycosis to other fungi. In contrast, Cf Apopka97 caused lower mortality than Mb F52 and Cj wf GA17 after 5 min treatment and lower than Mb F52 and Bb GHA after 10 min treatment, although the mycosis levels were not different. It is worthwhile to mention that s et al.  found thDelayed germination after 40 h incubation appeared in all fungi exposed to UV light for 5 min. Delayed germination of EPF in response to UV light has been reported in other studies ,29,30,31B. tabaci (MEAM1) and a similar effect against Aphis gossypii in the laboratory , which indicate its good potential for managing foliar-feeding pests such as whiteflies. However, weather conditions and the time of spray should be taken into account in field applications to avoid direct exposure of fungal propagules to heat and UV radiation. This study provides a valuable reference for forthcoming field studies on post-application persistence and efficacy of Cj wf GA17 as compared to Cf Apopka97 for managing whiteflies.Overall, under environmentally important temperatures, Cj wf GA17 responded similarly to commercial strains, with optimum virulence at 25 °C. Fungal virulence and viability were inhibited by high temperatures. The tolerance of Cj wf GA17 to extremely low or high temperatures was inferior to Mb F52 and Bb GHA, but it was superior to Cf Apopka97. In tolerance of UV light, it had similar response to other fungi, except marginally lower viability than Bb GHA. The advantage of Cj wf GA17 over Cf Apopka97 in environmental tolerance makes it a good alternative to, or even a replacement of, Cf Apopka97 in pest management. Previously, this new strain showed higher virulence than Cf Apopka97 against The United States Department of Agriculture (USDA) has passed on the patent potential of the research findings reported in this manuscript.


Their structures were assigned based on the interpretation of their NMR and HRMS data. The compounds exhibited quorum sensing inhibition (QSi) and antifouling activities against several strains of bacteria and microalgae. To our knowledge, the QSi activity of this type of bromotyrosine metabolite is described here for the first time.Four new brominated tyrosine metabolites, aplyzanzines C–F ( Verongiida a well-studied group. In addition to their bioactivities, the brominated tyrosine metabolites found in Verongiida are considered as chemotaxonomic markers + ion cluster at m/z 726, 728, 730, 732, and 734 in a 1:3:5:3:1 ratio, indicating the presence of four bromine atoms in the molecule. Analysis of the [M + H]+ peak at m/z 729.9170 suggested a C24H31N3O3Br4 formula for 1, indicating nine degrees of unsaturation. The 1H-NMR spectrum . The 13C NMR spectrum revealed the presence of only one carboxyl at δC 168.3, and eight signals between δC 119.1 and 155.7, similar to those reported for aplyzanzine A + ion as a cluster at m/z 669, 671, 673, 675, and 677 in a 1:4:6:4:1 ratio, indicating the presence of four bromine atoms in the molecule. Analysis of the [M + H]+ peak at m/z 672.8610 suggested the molecular formula C21H24N2O3Br4 for compound 2, indicating nine degrees of unsaturation. When compared to aplyzanzine C (1), the molecular formula lacked 57 mu corresponding to a C3H7N fragment. Examination of the 1D and 2D NMR spectra . The structure 2 was thus assigned to aplyzanzine D.NMR spectra of aplyzanzine C  and D (2) exhibited similar optical rotation values of respectively +26° and +27.4°. Comparison with similar compounds containing an l-tyrosine residue previously reported in the literature, such as suberedamines A  , anomoia +47.5°) , indicat3) is a minor colorless compound obtained from both the precipitate and the butanolic phases. The HRESIMS data showed the [M + H]+ ion as a cluster at m/z 681, 683, 685, 687, and 689 mu, indicating the presence of four bromine atoms in the molecule again. Analysis of the [M + H]+ peak at m/z 684.8616 suggested the molecular formula C22H24N2O3Br4 for this compound, indicating ten degrees of unsaturation. The difference of molecular formulae with aplyzanzine C (1) suggested the loss of the dimethylamine fragment. The same dibromophenolic moieties found in aplyzanzine C (1) were observed on the 1D and 2D NMR spectra (3)2 at δH 2.91 (δC 42.7) on the 1H NMR spectrum of compound 3 but present on the spectrum of compound 1. Regarding the UV spectra of compounds 1–3, aplyzanzines C (1) and D (2) exhibited the same UV spectra with a maximum of absorption at 207 nm (in MeOH), but the spectrum of aplyzanzine E (3) displayed two maxima at 207 and 284 nm, suggesting the presence of additional π system confirmed by NMR signals of two olefinic protons at δH 6.96 (δC 138.4) and δH 6.60 (δC 120.7). Their coupling constant of 15 Hz is characteristic of a trans configuration of the double bond between CH-2 and CH-3. HMBC correlations of these two protons with the aromatic C-4 at δC 133.3 and the amidic carbonyl C-1 at δC 168.9 allowed to assign the position of this double bond between these two fragments. These data are consistent with those reported for psammaplysenes + ion cluster at m/z 622, 624, 626, 628, and 630 mu, indicating the presence of four bromine atoms in the molecule again. Analysis of the [M + H]+ signal at m/z 627.8020 suggested the molecular formula of 4 as C19H17NO3Br4, indicating ten degrees of unsaturation. Examination of the 1D and 2D NMR spectra, similar to those of aplyzanzine E (3) (3) but lacking the propylamine fragment. This is consistent with the loss of 57 mu of the [M + H]+ ion, corresponding to a fragment C3H7N. The UV absorption spectra are also similar to the same maxima at 208 and 285 nm. The 1H NMR spectrum also displayed a coupling constant of 16Hz between the two olefinic CH at δH 6.97 (δC 138.8) and δH 6.64 (δC 120.7) and thus confirmed the trans configuration of this double bond.Aplyzanzine F (ne E (3) , showed 5) + Aplyzanzine D (2): colourless varnish (16.7 mg); c 1.0, MeOH); UV (MeOH) λmax (log ε) 206 (4.77) nm; 1H and 13C NMR data, m/z = 672.8610 [M+H]+ .Aplyzanzine E (3): colourless compound (1.4 mg): UV (MeOH) λmax (log ε) 207 (4.48), 284 (3.79) nm; 1H and 13C NMR data, m/z = 684.8616 [M + H]+ .Aplyzanzine F (4): colourless varnish (1.0 mg): UV (MeOH) λmax (log ε) 208 (4.83), 285 (4.15) nm; 1H and 13C NMR data, m/z = 627.8020 [M + H]+ .Purealidin A (5): colourless compound (1.5 mg): 1H NMR data, m/z = 519.0208 [M + H]+ .6: colourless compound (4.0 mg): 1H NMR data, m/z = 444.9745 [M + H]+ .V. harveyi strain CIP 104774. The 96-well microplates in liquid broth media was used for the bioguided isolation using several Vibrio harveyi strains [V. harveyi BB120 wild-type and its derived double mutants  obtained from Bassler’s laboratory [The extracts and compounds were screened on agar plate using the wild  strains : V. harvboratory ,61, tripVibrio proteolyticus (ATCC 15338), Vibrio aesturianus (ATCC 35048), and Polaribacter irgensii (ATCC 700398) [All compounds were tested against three marine bacterial strains commonly found in biofilms,  700398) . Bacteri 700398) . All assPrior to experiments, bacteria were pre-grown in sterile Marine Bacterial Medium (MBM) with 0.5% peptone  in sterile filtered  natural seawater, at 20 °C.8 colony forming units/mL) were aseptically added to the microplate wells containing compound (0.01–10 µg/mL), and the plates were incubated for 48 h at 21 °C prior to assessment of bioactivity. Media only  was used as a blank, and a commercial antifoulant (Seanine 211) was used as a positive control. Bacterial suspensions  for bacterial growth, which is defined as the lowest concentration which results in a decrease in OD, compared to the blank. The bacterial solutions were emptied from the microplates, and the bacterial adhesion assay was performed using aqueous crystal violet staining method on the microplates. The MIC for bacterial adhesion was defined as the lowest concentration of compound that, after 48-h incubation, produced a decrease of the OD at 595 nm compared to the blank.Cylindrotheca closterium (AC 170), Halamphora coffeaeformis (AC 713), and Porphyridium purpureum (AC 122) [All compounds were tested for their capability to inhibit the adhesion and/or growth of 3 microalgal species known to be involved in surface colonization: (AC 122) . All the(AC 122) . All assIn order to evaluate the algal growth and adhesion inhibition, compounds were diluted in methanol and transferred to black 96-multiwell plates. After evaporation of the carrier solvent, F/2 medium was added to reach the same final concentrations tested for the antibacterial activity. The same controls than for the antibacterial test were carried out. Microalgal stock solutions were prepared using the chlorophyll analysis methodology described by Chambers et al. (2011) . The preMicroalgal adhesion was determined in an analogous manner where the non-attached algal cells were removed prior to methanol addition (100 μL), releasing chlorophyll a from the remaining algal biofilms. Results were expressed as MIC values.1–4), along with the known 2-aminoimidazolic compounds, purealidin A (5) and 6, were isolated and identified from the French Polynesian sponge Pseudoceratina sp. 2081. Biological evaluations were carried out to measure the capacity to inhibit V. harveyi’s QS and antifouling activities against 3 strains of microalgae and 3 strains of marine bacteria. Aplyzanzine E (3), purealidin A (5) and (6) displayed QSi activity against V. harveyi, and also against fouling bacteria. To the best of our knowledge, QSi activity of this type of bromotyrosine derivatives is described here for the first time. Aplyzanzine E (3) and purealidin A (5) reduced the adhesion of bacteria with a MIC smaller than 0.2 µM. Aplyzanzine E (3), purealidin A (6) inhibited the growth of V. aesturianus, with MIC, respectively, of 0.01 and 0.22 µM. Aplyzanzine C (1) was found to be the most active of these compounds against microalgae: it inhibits adhesion and growth of the three microalgae strains tested with a MIC of 0.14 µM. Aplyzanzine F (4) and purealidin A (5) are specific to H. coffeaeformis, inhibiting adhesion and growth, respectively, with a MIC of 0.02 µM. Four new bromotyrosine metabolites, the aplyzanzines C–F (1 and 3 have very complementary activities that allow the sponge Pseudoceratina sp. 2081 to fight microfouling in a holistic way. Defence synergies combining physical, mechanical or chemical parade are part of the strategies of living systems to survive in nature. This also explains why some marine organisms synthesize an arsenal of chemicals that will have in the same time targeted- and broad-spectrum activities. The biosynthesis of some of these compounds is constitutive, while for others, the synthesis will be induced by the season or by the presence of colonizing organisms. In this study, we found that compounds


Epoxy resin plays a key role in composite matrices and DGEBA is the major precursor used. With the aim of favouring the use of bio resources, epoxy resins can be prepared from lignin. In particular, diglycidyl ether of isoeugenol derivatives are good candidates for the replacement of DGEBA. This article presents an effective and eco-friendly way to prepare epoxy resin derived from isoeugenol (BioIgenox), making its upscale possible. BioIgenox has been totally characterized by NMR, FTIR, MS and elemental analyses. Curing of BioIgenox and camphoric anhydride with varying epoxide function/anhydride molar ratios has allowed determining an optimum ratio near 1/0.9 based on DMA and DSC analyses and swelling behaviours. This thermoset exhibits a Tg measured by DMA of 165 °C, a tensile storage modulus at 40 °C of 2.2 GPa and mean 3-point bending stiffness, strength and strain at failure of 3.2 GPa, 120 MPa and 6.6%, respectively. Transposed to BioIgenox/hexahydrophtalic anhydride, this optimized formulation gives a thermoset with a Tg determined by DMA of 140 °C and a storage modulus at 40 °C of 2.6 GPa. The thermal and mechanical properties of these two thermosets are consistent with their use as matrices for structural or semi-structural composites. Epoxy thermosets result from a polymerization reaction between an epoxy resin and a hardener, mainly polyamine or acid anhydride. Although epoxy resins are more expansive than the unsaturated polyester resins, they play a key role in the market of paints, glue, varnishes or in the sectors of transport and building due to their high adhesive and mechanical properties .Nowadays, diglycidyl ether of bisphenol A (DGEBA) represents 90% of the commercial epoxy resins. Its synthesis results from the reaction between two molecules of epichlorohydrin and a molecule of bisphenol A (BPA). However, the use of DGEBA has two major drawbacks: BPA used in its synthesis is petro-sourced and, secondly, BPA, an endocrine disruptor ,3, is reWith the aim of avoiding BPA reactant and of favouring the use of bio resources, many studies have been carried out. For instance, because of their abundance and low toxicity, plant oils are one of the bio-based feedstocks frequently used. Recently oxidation of unsaturated triglycerides derived from soybean  or linseEpoxy resins derived from lignin are prepared according to two ways. Raw lignin can be epoxidized ,8,9 and Within this context, our research team has recently described both the synthesis of an epoxy monomer derived from isoeugenol, named as GlycidylEther Epoxy isoeugenol (GEEpiE) , and its1H NMR, 13C {1H} NMR, IR, mass spectrometry and elemental analysis, has allowed to suppress the chromatography purification step. Indeed, it has been shown that both the by-products obtained contain allylic groups easily converted into epoxide functions (ep) and leading thus also to diepoxy monomers. The resulting monomers mixture named as BioIgenox (BI) has subsequently been cured with bio-based camphoric anhydride and hexahydrophtalic anhydride. The resulting materials have been characterized by dynamic mechanical analysis (DMA) and differential scanning calorimetry (DSC) analyses and their swelling behaviour were measured. In order to optimize their thermal and mechanical properties, different formulations with epoxide function/anhydride molar ratio varying from 1/0.5 to 1/1, have been investigated. These results have then been discussed with the aim of developing a bio-based epoxy matrice for semi-structural or structural composites. Bending properties are also determined for the most promising ratio.Thus, this work proposes a preparation improvement of the bio-based epoxy resin in order to make its scale-up possible. In particular, an extensive characterization of the reaction by-products by 3, ≥95% purity, Sigma-Aldrich, St. Louis, MO, USA), tetrabutylammonium hydrogen sulfate , epichlorohydrine , and Oxone® (Sigma-Aldrich) were purchased and used as received. Natural isoeugenol , diglycidyl ether of bisphenol A , camphoric anhydride , hexahydrophtalic anhydride  and 1,2-dimethylimidazole  were purchased and used without any further purification. These reagents are depicted in Dichloromethane , acetone , sodium hydrogen carbonate .FTIR spectra were recorded using a Bruker  Alpha spectrophotometer fitted with an ATR module ALPHA–P equipped with a mono-reflection diamond crystal in the 4000–375 cm1H and 13C {1H} nuclear magnetic resonance (NMR) spectra were obtained using a Bruker Avance III 500 MHz spectrometer  working at 500 and 125 MHz, respectively, at room temperature and using CDCl3 as solvent. External references were trimethylsilane (TMS) for 1H and 13C {1H} NMR.Mass Spectrometry (MS) analysis was carried out using an HPLC Ultimate 3000 RSLC instrument from DIONEX  coupled through an electrospray ionisation (ESI) interface to an ion-trap AmaZon SL mass spectrometer from Bruker for MS detection. Methanol was used as the solvent.Elemental analyses were performed on a CHNS/O Thermo Electron Flash EA 1112 Series apparatus.−1) with a sample mass of 10 ± 3 mg. To study the curing reaction, samples were heated from 20 °C to 200 °C at a heating rate of 5 °C min−1. The determination of glass transition temperatures (Tg) is carried out on the samples cured in the oven. After a first heating from 20 °C to 180 °C, the Tg is determined on the second heating from 20 °C to 190 °C at a heating rate of 10 °C min−1 and using the TA Universal Analysis 2000 software.Differential scanning calorimetry (DSC) analyses were performed on a TA Instruments  Discovery DSC under nitrogen flow (50 mL minE′), loss modulus (E″) and loss factor (tan(δ)) were measured using a METRAVIB DMA50 apparatus at 5 °C intervals, under isothermal conditions. A stabilization of 3 min was maintained before each measurement to ensure a homogeneous temperature inside the specimens. The temperature was varied between 40 °C and 210 °C with a heating rate of 10 °C min−1 between each plateau temperature. The frequency of the excitation varied from 0.5 Hz to 5 Hz. A sinusoidal tensile displacement was applied on the sample with a peak-to-peak amplitude of 8 μm and a mean amplitude of 40 μm. For each batch, two specimens were systematically tested. A very good reproducibility of the measurements was observed. For reason of readability, in the present paper, results are presented in the Figure 4 for one specimen only.Samples for dynamic mechanical analysis (DMA) tests were cut to the dimensions of 60 × 10 × 1.7 mm. Storage modulus (E′ is the storage modulus at Tg + 30 °C (MPa), Φ is the front factor (approximated to 1 in the Flory theory), R is the gas constant (8314 cm3 MPa mol−1 K−1 and T is the absolute temperature at Tg + 30 °C (K). ρ is expressed in mol cm−3.The rubber elasticity theory defines the crosslinking density as :ρ=E′ϕRT0) was immersed in THF at room temperature for 48 h. The weight of swollen sample (w1) was measured after wiping the surface with a filter paper. The swelling ratio (S) was calculated as follows:The swelling behaviour of the cured sample was studied by a solvent immersion method. The dry sample (weight wL is the distance between supports (span), b is the width of the sample, h is the thickness of the sample and f is the deflection.Some of the cut specimens were also characterized under three-point bending tests following the ASTM D790 standard. Tests were performed using a universal testing machine MTS criterion 45 equipped with a 1 kN full-range load sensor. Three samples were tested at a constant crosshead displacement rate of 10 µm/s. The deflection was measured at mid-span, on the bottom face of the specimen, using a micrometre laser sensor. The support span was 40 mm. The ultimate stress (E) was determined using the slope of the stress-strain curve in the strain range from 0.1% to 0.3%. The mean values and standard deviation were then calculated.The three-point bending modulus  and a solution of NaOH  in ethanol (200 mL) are introduced in a round-bottom flask of 1000 mL equipped with a refrigerant and a dropping funnel and heated at 80 °C under argon atmosphere. Epichlorohydrin  is added dropwise for 1 h and the mixture is stirred for 3 h. After cooling at room temperature, the mixture is filtered. The solid is rinsed with toluene (3 × 20 mL). The resulting organic solution is washed with water, saturated NaCl solution, and dried over anhydrous MgSOenol see , with a −1): 3486 (O−H), 3019 (aromatic C−H), 1653 (C = C), 1601, 1583, 1509, 1464 (aromatic C = C), 910 (epoxy ring GEiE).FTIR : δ ppm 1.88 , 2.74 and 2.89 , 3.08 , 3.29 , 3.39 , 3.57 , 3.88 , 4.02–4.28 , 4.39 , 5,75 , 6.13 , 6.34 , 6.82–6.95  (See H9″) See .13C {1H} NMR : δ 150.0 , 147.5 , 132.6 , 130.9 , 124.6 , 119.0 , 114.6 , 109.5 , 71.8 (C12″), 71.5 (C11′), 70.7 (C12′), 70.3 (C11), 70.7 (C11″) 69.0 (C13″), 56.2 , 50.6 (C12), 45.4 (C13), 18.7  (See C1″) See .The mixture of 1,3-di (isoeugenyloxy)propan-2-ol and 2,3-di (isoeugenyloxy)propan-1-ol (DiEP1 and DiEP2 depicted in −1): 3474 (O−H), 3019 (aromatic C−H), 1653 (C = C), 1602, 1584, 1510, 1463 (aromatic C = C).FTIR : δ ppm 1.90 , 3.42 , 3.85 , 4.13–4.26 , 4.41 , 5,74 , 6.13 , 6.34 , 6.82–6.95  (See H9″) See .13C{1H} NMR  δ 149.8 , 147.3 , 132.3 , 130.6 , 124.3 , 118.8 , 114.8 , 109.1 , 71.5 (C12″), 71.3 (C11′), 71.0 (C12′), 69.1 (C11″), 69.9 (C13″), 55.8 , 18.4 (C1′ C1″) (See C1″) See .Elemental analysis: C calculated 66.33%, found 64.46%. H calculated 6.78%, found 6.86%.m/z, ES+, [M + Na+]) calculated 407.183, found: 407.181.MS  is gradually added during 1 h and the stirring is maintained for 4 h. Organic phase is poured in a separating funnel, washed with water, saturated NaCl solution, and dried over anhydrous MgSO4 leading, after evaporation under vacuum to produce a brown oil that solidifies to afford BioIgenox, as a brown waxy past (83%).BioIgenol : 3496 (O−H), 3027 (aromatic C−H), 1608, 1592, 1514, 1455 (aromatic C = C), 912 (epoxy ring).FTIR : δ ppm 1.41 , 2.71 and 2.86 , 2.94 , 3.35 , 3.46 , 3.51 , 3.81 , 3.95–4.28 , 4.36 , 6.69–6.93  (See H9″) See .13C {1H} NMR : δ 150.1 , 148.1 , 131.5 , 118.5 , 114.2 , 108.7 , 71.0 (C12″), 70.4 (C11′), 69.1 (C12′), 68.6 (C11), 67.0 (C11″), 59.5 , 58.9 , 55.9 , 50.2 (C12), 44.9 (C13) 17.9  (See C1″) See .−1. The remaining mixture was cured in an oven at 150 °C for 2 h, before being cooled down by leaving the mold at room temperature for one hour.Polyepoxy polymers were obtained via thermal curing according to the following protocol: Diepoxy monomers containing different molar ratio of anhydride hardener were ground in a mortar at room temperature and heated (120 °C for CA and 45 °C for HHPA) until a homogeneous mixture was obtained. DMID catalyst was then added with a molar ratio of 0.025 with respect to epoxide function (ep). Polymerization reaction was studied using DSC. A few milligrams of this mixture were heated from room temperature to 180 °C at a heating rate of 5 °C minThe reaction of isoeugenol with epichlorohydrin leads, as expected, to glycidyl ether isoeugenol . Two by-N2 mechanism occurring in the basic conditions [−1. The C=C vinyl stretching band is present at 1655 cm−1, and the oxirane ring from GEiE is measured at 910 cm−1.The phenolate anion formed in the basic conditions of the reaction acts as nucleophilic reactant toward the oxirane function of GEiE. Depending on the oxirane carbon which is attacked, DiEP1 and DiEP2 can be obtained. According to a Snditions  the less1H NMR, 13C {1H} NMR, mass spectrometry, and elemental analysis have confirmed the formation of the dimers DiEP1 and DiEP2. In particular, 13C {1H} NMR shows five different signals attributed to the glycerol chain carbons . As expected, the mass spectrum is in accordance with DiEP1 and DiEP2 structures . It is noteworthy that DiEP1 and DiEP2 were not separated by a chromatography column.DiEP1 and DiEP2 were isolated from the BioIgenol mixture by chromatography column. 1H NMR, 13C {1H} NMR, FTIR have confirmed the formation of the three diepoxy GEEpiE, DiEpiE1 and DiEpiE2. In particular 1H NMR spectra show the disappearance of the allylic protons  and the appearance of the epoxy protons . In the 13C NMR spectra, the signals of allylic carbons  are replaced by those of epoxy carbons .DiEP1 and DiEP2 contain both allylic groups easily converted into epoxide functions and leading thus also to diepoxy monomers. Thus, BioIgenol mixture has been directly oxidized into BioIgenox as depicted in To avoid the purification of crude GEEpiE, the GEEpiE/(DiEpiE1and DiEpiE2) fractions have been estimated. Indeed the purification by chromatography of 10 g of crude GEEpiE involves an entire working day, requires more than 2 L of elution solvents and finally leads to only 6 g of pure GEEpiE.1H NMR signals intensities allows the estimation of GEEpiE/(DiEpiE1and DiEpiE2) fractions. By measuring the intensity of the signal H12 (IH12) and total intensity of aromatic protons  (IAr), the respective fractions of GEEpiE (x1) and of DiEpiE (x2 = 1 − x1) are determined by resolving the following equation:The x1 varying from 0.85 to 0.55 and on the basis of five experiments.GEEpiE was always found to be the major product of the mixture with −1. DSC thermograms are depicted in BioIgenox has been cured with different ratio of bio-based camphoric or hexahydrophtalic anhydride  in orderWhen curing with CA, the DSC curves exhibit the maximum of the heat release associated to the reticulation reaction at 140 °C whatever the ratio. It is noteworthy that the endothermic peak at about 100 °C which is attributed to the mixture melting, decreases with the epoxide function/CA molar ratio. This is in accordance with the physical properties of the CA which is crystallized contrary to the waxy paste of BioIgenox.HHPA reacts with BioIgenox at a lower temperature than CA see , as it wg significantly increases when epoxide function/CA varies from 1/0.5 to 1/0.8 while it remains quite constant for the 1/0.9 and 1/1 ratios. For the most crosslinked systems, Tg cannot be accurately determined using DSC because the change of the Cp value associated to Tg is not well pronounced. It is well known that DMA is more sensitive to the changes occurring at the Tg when compared to DSC [g values  curve. These values, synthetized in The DSC curves for the BI/CA thermoset series obtained after 2 h of curing at 150 °C in oven, are shown in d to DSC ,27. Thusg values . Three dE″ curve) when epoxide function/CA varies from 1/0.5 to 1/0.9. The change for the higher ratio is then very slight.As usually observed, the Tg values obtained from these methods differ up to 30 °C from one another on the same run. Whatever the method used, the same tendency as for the DSC results is observed with a significant increase from 75 °C to 145 °C  at the peak) is lower. This may be explained by a higher disparity in chains length. Moreover, HHPA-cured thermoset has a higher modulus at 40 °C and a higher damping ability. This effect could be explained by the higher crosslinking density with a formulation involving HHPA.These results show that both BioIgenox/CA and BioIgenox/HHPA bio-based formulations studied are promising candidates to replace DGEBA based matrices used for high-performance composites.In the course of our research in the development of high-performance epoxy materials, the synthesis of a bio-based epoxy resin has been improved with the aim of making its scale-up possible. The removal of chromatography purification step has led to the obtaining of a monomers mixture named BioIgenox (BI), which has been totally characterized by spectrometric and elemental analyses.BioIgenox and camphoric anhydride have been cured with varying epoxide function/anhydride molar ratios (1/0.5 to 1/1). Based on DMA and DSC analyses and swelling behaviours, the optimum ratio has been found to be near 1/0.9. This optimized formulation has been transposed to BioIgenox/hexahydrophtalic anhydride. Both thermosets exhibit thermal and mechanical behaviours consistent with their use as matrices for structural or semi-structural composites. Namely, BioIgenox/CA and BioIgenox/HHPA thermosets have Tg determined by DMA of 165 °C and 140 °C, respectively, and storage modulus at 40 °C of 2.2 and 2.6 GPa, respectively. The quasi-static bending properties are comparable, even higher, to the one obtained with the very classical and widespread DGEBA-based epoxy systems.Finally, this study demonstrates an effective and ecofriendly way to prepare bio-based epoxy matrices able to replace petro-sourced DGEBA with comparable performances.


Relationships between FFA species and various parameters were evaluated. Plasma concentrations of myristate (14:0), palmitoleate (16:1), palmitate (16:0), linoleate (18:2), oleate (18:1c9), cis-vaccenate (18:1c11), eicosapentaenoate (20:5), arachidonate (20:4) and docosahexanoate (22:6) were reduced in the T1DM group ; α-linolenate (18:3) and dihomo-γ-linolenate (20:3) concentrations were unchanged. The saturated/unsaturated FFA ratio, n-3/n-6 ratio, de novo lipogenesis index /linoleate (only found in diet)) and elongase index  were increased in the T1DM group . The stearoyl-CoA desaturase 1 (SCD1) index 1  and index 2 (oleate/stearate) were reduced in T1DM (p < 0.0001 for both). The delta-(5)-desaturase (D5D) index (arachidonate/dihomo-γ-linolenate) was unchanged. Age and sex had no effect on plasma FFA concentrations in T1DM, while SCD1 index 1 was positively correlated (p = 0.098) and elongase index negatively correlated with age (p = 0.0363). HbA1c was negatively correlated with all plasma FFA concentrations measured except α-linolenate and dihomo-γ-linolenate. Correlations were observed between plasma FFA concentrations and cholesterol and HDL concentrations, but not LDL concentration or diabetes duration. Collectively, these results aid our understanding of T1DM and its effects on lipid metabolism.Type-1 diabetes mellitus (T1DM) is associated with metabolic changes leading to alterations in glucose and lipid handling. While T1DM-associated effects on many major plasma lipids have been characterised, such effects on plasma free fatty acids (FFA) have not been fully examined. Using gas chromatography–mass spectrometry, we measured the plasma concentrations of FFA species in individuals with T1DM (  •Plasma concentrations of major FFA species are lower in T1DM compared to controls.•Plasma FFA concentrations negatively correlates with HbA1c in T1DM.•The SCD1 index is reduced in T1DM.•Lipogenesis, elongase, n3/n6, saturated/unsaturated indices are increased in T1DM.•Collectively, the data highlight specific changes in lipid metabolism in T1DM Dysregude novo lipogenesis activity. Thus, to gain insight into how T1DM influences the plasma FFA profile, we measured and compared plasma concentrations of major FFA species in individuals with T1DM and an age/sex-matched control group without diabetes. In addition, we explored how plasma FFA concentrations and lipid indices compare with sex, age and the concentrations of glycated haemoglobin A1c (HbA1c) and diverse lipids.FFAs are important signalling molecules and as well as being an important source of energy, impact on numerous physiological processes, including the regulation of inflammation, the management of oxidative stress, the composition of cellular membranes and signal transduction, as recently reviewed by us . Thus, c22.1g for 20 min at 4 °C. The plasma was snap frozen in liquid nitrogen and stored at −40 °C until analysis. Plasma concentration of human serum albumin (HSA), HbA1c, fasting glucose, triglycerides, cholesterol, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) were measured with routine methods.A total of 44 individuals with T1DM and 44 controls were recruited from Leeds Teaching Hospital Trust following approval by the Leeds West Research Ethics Committee (REC: 09/H1307/12). We excluded individuals with a history of acute coronary syndrome or those that had experienced a stroke within 3 months of enrolment, individuals who had received prior treatment with aspirin, clopidogrel, warfarin or non-steroidal anti-inflammatory drugs or who had a current treatment with any drug other than insulin, individuals with a history of deep venous thrombosis or pulmonary embolism, previous or current history of upper gastrointestinal pathology, malignancy or coagulation disorders, individuals with abnormal liver function tests  or abnormal thyroid function tests and individuals with proteinuria, advanced nephropathy or clinical signs of neuropathy or retinopathy (except for those with background changes). Written informed consent was obtained. All blood samples were collected after a light breakfast  and within 2 h the blood was centrifuged at 2400 ×2.2In order to characterise the FFAs present in each sample, they were converted to fatty acid methyl esters (FAMEs) and analysed by gas chromatography–mass spectrometry (GC–MS) analysis. This was performed by thawing the citrated plasma samples and spiking them with an internal standard  to allow for normalisation. Dole's protocol was used to extract the FFAs .2.3t-tests or, for continuous parameters, using Pearson's correlation test. The significance threshold was set at p ≤ 0.05. Statistical analyses were performed and graphs were generated with Prism 8.2.1 . Data are represented as mean ± standard deviation (SD).Differences between groups were analysed using multiple Student's 33.1p = 0.0359). There was no difference in the concentrations of triglycerides, cholesterol, LDL, HDL and cholesterol ratio between the groups. In the T1DM group, HbA1c was higher (p < 0.0001) and HSA was lower (p = 0.0413) compared to controls.In addition to collected demographic information, plasma concentration of triglycerides, cholesterol, LDL, HDL, HbA1c and HSA were measured and the cholesterol ratio was calculated subjects with T1DM and the controls. Summaries of this data are presented in 3.2p < 0.0001), palmitoleate , palmitate , linoleate , oleate , cis-vaccenate , stearate , eicosapentaenoate , arachidonate  and docosahexaenoate .A FAME analysis was carried out on plasma samples from both T1DM and control groups to identify and quantify the major FFA species (p = 0.0166 and p = 0.0089 respectively). The de novo lipogenesis index was calculated as the ratio of palmitate  with linoleate . The elongase index, which indicates elongase enzyme activity was calculated as the oleate/palmitoleate (18:1c9/16:1) ratio. The activity of stearoyl-CoA desaturase 1 (SCD1) was calculated using SCD1 indices 1 and 2, which reflect the palmitoleate/palmitate (16:1/16:0) ratio and the oleate/stearate (18:1c9/18:0) ratio, respectively. The delta-(5)-desaturase (D5D) index was calculated with the arachidonate/dihomo-γ-linolenate (20:4/20:3) ratio. The de novo lipogenesis index and the elongase index were higher (p < 0.0001 and p = 0.0008 respectively) and the SCD1 index 1 and 2 were lower (p < 0.0001 for both) in the T1DM group compared to the controls. The D5D index was unchanged between the groups.Different plasma FFA indices were then calculated and compared between the T1DM and control groups (3.3p < 0.0001 for myristate (14:0), palmitate (16:0), linoleate (18:2), oleate (18:1c9), cis-vaccenate (18:1c11) and stearate (18:0), p = 0.0145 for eicosapentaenoate (20:5), p = 0.0281 for arachidonate (20:4) and p = 0.0017 for docosahexanoate (22:6)). We then explored the relationships between HbA1c and the different FFA indices . The saturated/unsaturated FFA ratio did not show any correlation with HbA1c but the n-3/n-6 ratio positively associated with HbA1c (p = 0.0023). The SCD1 index negatively associated with HbA1c whether measured using either palmitoleate/palmitate (16:1/16:0) or oleate/stearate ratios . The D5D index was not associated with HbA1c but the elongase and de novo lipogenesis indices each positively correlated with HbA1c (p = 0.0013 and < 0.0001 respectively). We then assessed the relationship between HbA1c concentration and plasma concentrations of cholesterol, HDL, LDL and between HBA1c concentration and the cholesterol/HDL ratio and diabetes duration (Table S6). No associations between these respective parameters were found.We examined the relationship between HbA1c concentration and plasma concentrations of the major FFA species ( indices . The pla3.4p = 0.0098), while the elongase index negatively correlated with age . In controls, plasma concentrations of palmitate (16:0), linoleate (18:2), oleate (18:1c9), cis-vaccenate (18:1c11) and stearate (18:0) were negatively correlated with age . In addition, total plasma FFA concentration, total plasma concentration of saturated, unsaturated and n-6 FFAs (but not n-3) were negatively correlated with age , while the D5D index was positively correlated with age . We examined the association between different lipids and the plasma concentrations of FFA species in T1DM , and negatively associated with the de novo lipogenesis index (p = 0.0003). HDL positively associated with the plasma concentrations of myristate (14:0), palmitate (16:0), linoleate (18:2), cis-vaccenate (18:1c11), stearate (18:0), total FFAs and total saturated FFAs  and negatively associated with the de novo lipogenesis index (p = 0.0258). The cholesterol ratio positively associated with the SCD1 index 1 (p = 0.0424).The presence of sex or age-specific differences in the plasma concentrations of major FFA species was assessed. No differences in the plasma concentration of specific FFA species or in the indices monitored were found between males and females with T1DM . There were no associations between age and the plasma concentration of major FFA species in T1DM, but the SCD1 index 1 positively correlated with age (4trans), oleate (18:1 cis), stearate (18:0) and arachidonate (20:4) and they found elevated levels of oleate, elaidate and stearate in individuals with T1DM and good glycaemic control compared to healthy controls, while unchanged plasma FFA levels were found in individuals with T1DM and bad glycaemic control compared to healthy control. Here, FFAs were extracted from plasma taken from 45 individuals with T1DM and 45 controls and their concentrations were determined using GC–MS. Our cohort was age and sex-matched, but BMI was slightly higher in the T1DM group. HSA concentration was also lower in the T1DM group compared to the controls, which is in agreement with a study that found that insulin withdrawal in women with T1DM leads to reduced HSA synthesis .This research was funded by the Amélie I.S. Sobczak: Conceptualisation, Methodology, Formal analysis, Investigation, Data curation, Writing - original draft, Writing - review & editing, Visualisation. Samantha J. Pitt: Writing - review & editing, Funding acquisition. Terry K. Smith: Conceptualisation, Methodology, Validation, Resources, Writing - review & editing, Supervision. Ramzi A. Ajjan: Conceptualisation, Resources, Writing - review & editing, Supervision, Project administration, Funding acquisition. Alan J. Stewart: Conceptualisation, Methodology, Resources, Writing - original draft, Writing - review & editing, Supervision, Project administration, Funding acquisition.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.