Opioid use is a widespread epidemic, and traumatic stress exposure is a critical risk factor in opioid use and relapse. There is a significant gap in our understanding of how stress contributes to heroin use, and there are limited studies investigating individual differences underlying stress reactivity and subsequent stress-induced heroin self-administration. We hypothesized that greater individual vulnerability to stress would predict higher demand for heroin self-administration in a within-subjects rodent model of stress and heroin use comorbidity. Male rats were exposed to inescapable intermittent swim stress (ISS) and individual biological (corticosterone) or behavioral  measures were assessed before and after the stress episode. Individual demand for self-administered heroin  was assessed using a behavioral economics approach followed by extinction and reinstatement tests triggered by stress re-exposure, non-contingent cue presentations, and yohimbine . We found that behavioral, biological, and a combination of behavioral and biological markers sampled prior to and after the stress episode that occurred weeks before the access to heroin self-administration predicted the magnitude of individual demand for heroin. Non-contingent presentation of cues, that were previously associated with heroin, reinstated heroin seeking in extinction. For the first time, we show that individual biological response to an ecologically relevant stressor in combination with associated behavioral markers can be used to predict subsequent economic demand for heroin. People subjected to traumatic experiences are especially susceptible to the abuse of depressants, including opioids, but there is evidence of individual vulnerability or resilience to such stresses. Traumatic psychological stress, often accompanied by physical trauma, can include harassment, sexual abuse, bullying, domestic violence, life-threatening events, and other individually subjective negative experiences. It is known that stress is an important factor in the development of addiction disorders, and it is generally understood what critical neurobiological substrates underlie this mechanism  to induce long-lasting effects of stress. The ISS protocol is derived from both the learned helplessness and forced swim test (FST) models  and were reviewed and approved by the University of New Hampshire Institutional Animal Care and Use Committee.A total of 24 male adult (PD 70–90) and 12 juvenile (PD 28–32) Sprague Dawley rats  were used in the study. Juvenile Sprague Dawley males served as social exploration stimuli in the social exploration tests. Upon arrival at the vivarium, adults were single housed and acclimated to a colony for at least 1 week prior to experimentation. The vivarium was maintained on a 12 h light/dark cycle with lights on at 07:00. For all rats, food and water were available Social exploration pretest and posttest were conducted in identical test chambers, which consisted of a plastic tub cage , wire lid, and 3 cm of wood shaving bedding free of food and water. The room was lit by cool fluorescent bulbs and light penetration into the test chamber was 200–300 lx. Juvenile stimuli were used for a maximum of four tests and adults were never exposed to the same juvenile more than once. A camera that was mounted above the apparatus recorded behavior during each test session  painted with flat black enamel. Test sessions were video recorded from a camera mounted above the apparatus and processed using Ethovision XT software .ISS was conducted in two acrylic cylinders  with a 6.35 mm galvanized wire mesh at the bottom of each cylinder. Cylinders were suspended over a tank  filled with water maintained at 15 ± 1°C. The apparatus was controlled by a Med-PC interface and software . Space heaters, above and in front of each cylinder, circulated warm air (~36°C) in and around the cylinders to limit the effects of hypothermia during the inter-trial intervals.The FST was conducted in acrylic cylinders . The water was filled to 30 cm height and was kept at 24°C. Test sessions were video recorded and quantified as described above.Self-administration chambers  measuring 30 cm × 20 cm × 20 cm (l × w × h), were enclosed in a sound- and light-attenuating cubicle equipped with an exhaust fan. Each chamber was equipped with two retractable levers, two stimulus lights, and a house light. The infusion pump, that was located outside of the sound-attenuating cubicle, was equipped with a syringe that was connected to a swivel inside the chamber and that was further extended through a spring leash suspended over the ceiling of the chamber on a balanced metal arm. Lighting in the test room was maintained at 400–500 lx and exposure to light was minimized during the transfer from the colony.Diamorphine hydrochloride (generously provided by NIDA Drug Supply Program) and yohimbine hydrochloride  were mixed in a 0.9% sterile saline solution.Experimental progression is shown in r = 0.93). The following behaviors of the adult directed toward the juvenile were quantified: sniffing , pinning (minimum of two fore-paws against juvenile), allogrooming, and chasing. A total sum of these measures comprised the reported social exploration score.Social exploration consisted of a 1 h acclimation to the test chamber after which a juvenile was placed into the cage for 3 min. Social exploration tests were analyzed in real time by two experimenters , and time spent in the center vs. perimeter (thigmotaxis) were measured using Ethovision XT 8.5. Ethovision software was calibrated to identify and track the center point of the white subject against the black arena background using the static subtraction method  at the default five samples/s for the Open Field Template within the program. The following parameters were defined in Ethovision for the locomotor variables. The open-field apparatus was divided into two portions: the center consisted of a central 60 cm × 60 cm square , while the remaining surrounding area of the apparatus consisted of the perimeter. The path length was calculated as total distance moved (in cm) of the center point of the subject throughout the entire arena and movement was defined when the center point changed position above a threshold of 1 cm/s. All dependent measures were divided into the first 5 min  and last 5 min  of the test. Behaviors during the second 5 min bin were used for data analyses.ISS was administered 24 h after pretests between 07:00 and 12:30. Each swim trial consisted of a 5-s forced swim in which the cylinder was submerged to a depth of 25 cm. One-hundred trials were presented at a variable 60 s (10–110 s) inter-trial-interval. Immediately following ISS, rats were towel-dried and returned to the vivarium to heated cages (heating pad placed underneath cages) to promote vasodilation for blood collection that occurred 30 min post-ISS and was performed in a separate room.via a widely used and reliable serial time-sampling procedure in which behavior is scored as either immobile, swim or climb in 5 s intervals and reported as mean counts for each behavior .The FST was administered immediately following the post-ISS social exploration and open field tests. During the FST, rats were forced to swim for 5 min in 24 ± 1°C. Water depth was kept at 30 cm. This depth level forced rats to swim because it avoids the possibility of body support by the tail touching the bottom of the cylinder . FST videos were analyzed via lateral tail vein incision with a #11 scalpel into a capillary tube. The first incision was made in the distal 2 cm of the tail with subsequent incisions made at least 1 cm rostral of the previous. All samples were collected within 3 min and rats were returned to a home cage within 5 min  and processed in duplicates. Corticosterone concentrations were read at 405 nm on a BioTek microplate reader using Gen5 software . The intra-assay and inter-assay coefficients were 6% and 8%, respectively.Twenty-four hours before initiation of behavioral tests, a baseline blood sample was collected from the lateral tail vein; all samples were collected between 09:00 and 13:00. Second blood collection occurred 30 min after ISS test. Additional blood collections were performed 30 min post-FST when circulating corticosterone concentration peaks . Immediately following the surgery, catheters were flushed with 0.2 mL mixture of 30 U/ml heparin and 50 mg/mL of cefazolin (antibiotic) diluted in sterile saline (0.9% NaCl). Atipamezole hydrochloride  diluted in saline was used to terminate anesthesia. To manage post-surgical pain, butorphanol tartrate  was administered immediately after the surgery and daily for the next two recovery days. Starting from the day after surgery, catheters were flushed daily with 0.2 mL heparinized saline (30 U/ml) and cefazolin (50 mg/mL) mixture. Catheter patency was assessed when patency loss was suspected or upon completion of the self-administration phase with an infusion of 0.05 ml xylazine . This xylazine concentration produces clear motor ataxia within 5–10 s  and xylazine (20 mg/ml) mixture . A polyurethane catheter  with a rounded tip and double suture beads  was implanted into the right external jugular vein. The other end of the catheter was subcutaneously placed around the shoulder and exited below the scapula via a raised dipper, lever retraction, and commencement of a timeout . Following the timeout, a randomly selected lever was inserted with the condition that the same lever could not be presented more than twice in a row. This protocol was repeated for 60 sucrose deliveries. Sessions lasted 65–80 min depending on individual performance. Training continued until a lever press was made on at least 80% of the lever insertions for two consecutive days . After rats met this criterion, they were surgically implanted with an intravenous catheter as described earlier. Following 7 days of recovery, lever press training continued as described above, but the response contingency was changed to a variable ratio (VR3) schedule of reinforcement where on average every third response was followed by a sucrose delivery (range = 1–6 presses). At least 80% of the 60 available sucrose deliveries had to be earned to move to the self-administration phase; this occurred after 3–5 sessions. This protocol ensures high rates of responding, yet both levers have similar reinforcement history to avoid any potential bias of differential lever press training in later phases.Rats were trained to lever press over three daily sessions. At the start of each session, the house-light was turned on and a randomly selected lever (right or left) was inserted. A lever press or lapse of 15 s resulted in sucrose delivery  After recovery from surgery, all rats were retrained to lever press on both levers using a variable schedule of reinforcement (VR3) and after meeting a criterion transitioned to daily 12 h heroin self-administration . Each session began with a termination of the house light, insertion of both levers, and a 0.9 s infusion to flush approximately 90% of catheter volume. Completion of the required response resulted in a ~1 s infusion of heroin, retraction of both levers, and illumination of both cue lights above each lever for a 20 s timeout. Additional 5 min timeout with levers retracted and house light illuminated was instituted every 55 min to mitigate possible overdose-related deaths. All rats self-administered the exact dose of heroin using a variation in infusion duration that was automatically controlled by the program based on their pre-session weight. To closely simulate drug taking conditions observed in humans, all self-administration sessions were conducted during the night cycle which corresponds to rodents’ active phase (19:00–07:00). Access to heroin self-administration commenced 18–22 days after the initial ISS-induced stress episode. After 8 days of heroin self-administration on VR3 as described above, heroin was earned on fixed ratio (FR) schedule of reinforcement that was escalated daily using the following sequence: 1, 3, 5, 8, 12, 18, 26, 38, 58, 86, 130, 195, 292, 438, and 657. Subsequently, rats were allowed to self-administer heroin on VR3 as described above for an additional 5 days.Extinction training was identical to self-administration sessions except that active lever responding had no programmed consequences; no heroin, cues, or lever retractions. Reinstatement tests commenced on the day after last (10th) extinction session at the usual time of self-administration or extinction sessions (19:00). There were a total of five 1-h reinstatement tests with two intervening daily extinction sessions between these tests. Reinstatement triggers included abbreviated ISS re-exposure , non-contingent cue presentations, and yohimbine . Abbreviated ISS and yohimbine were administered 30 min before the beginning of each test. Non-contingent cue presentations were identical to the cues that were associated with heroin infusions and consisted of both cue lights turned on, and both levers retracted for 20 s. These cue triggers were presented at the start of the session and every 5 min from the beginning of the session thereafter. The order of reinstatement tests for each rat was assigned using a Latin square design.N = 16). Some individual corticosterone datum was not available because of failed sampling attempts or not enough serum for the analysis . Statistical analyses involving corticosterone data were restricted to subjects with available data. Individual demand for heroin was derived from the amount of heroin consumed (mg/kg) over each FR schedule of reinforcement . The main advantage of using essential value is that it is a unifying measure based on several critical parameters forming exponential-demand equation. For example, essential value takes into consideration consumption when the price of a reinforcer is low , when the price of the reinforcer is high , and the slope of the demand curve—also referred to as elasticity. Using this approach that we previously demonstrated in nicotine self-administration study, it is possible to plot individual demand curves for each subject, and most importantly, derive a single value of individual demand for heroin that is based on performance over a range of schedules of reinforcement . Responding on active and inactive levers was analyzed using ANOVAs (performed using GraphPad Prism). Individual effects were analyzed using linear mixed-effects modeling with maximum likelihood fit . Individual effects were analyzed by building a model with a maximum likelihood fit from a baseline that does not include any predictors other than an intercept. The model was then built by adding one predictor or a combination of predictors and comparing it to a baseline. The model fit was declared significant when its addition improved the model by accounting for significantly more variance . The proportion of variance explained by the factors is reported as the marginal R2. Additional model fitting criteria like AIC and BIC are presented in t-tests by comparing the responding on the active lever during reinstatement to the average of active lever presses on the last two extinction sessions. Reinstatement tests involved yohimbine were assessed using linear mixed-effects modeling followed by ANOVA.The difference between pre- and post-stress behaviors were assessed using t(15) = 0.42, p = 0.68; t(15) = 4.48, p < 0.001), significantly increased time spent in the perimeter of open field (t(15) = 4.85, p < 0.001), and significantly decreased total distance traveled during the open field  = 6.42, p < 0.0001; ISS did not affect social exploration behavior  = 10.13, p < 0.0001), Lever  = 212.6, p < 0.0001), and there was no interaction  = 1.205, p = 0.30). Active lever responding was consistently higher over the first eight sessions of heroin self-administration  = 409.7, p < 0.0001; Bonferroni comparisons; F = 0.5653, p = 0.68) and there was not Session by Lever interaction  = 0.8616, p = 0.4886). During extinction active lever responding decreased from 315.19 (SD = 209) on session 1–44.44 (SD = 35.72) on last session 10 (During the first eight sessions of heroin self-administration responding on levers varied by Session (ssion 10 .p < 0.01). The difference in the open field center activity explained approximately 43% of the variance in demand for heroin with the effect size (f2) equal to 0.75  and Immobility (p < 0.01) during the 5 min FST, administered a day after the ISS exposure, also significantly predicted an increased demand for heroin. Because climbing and immobility measures had an almost perfect negative relation with each other (R2 = 0.97), we are only showing and discussing climbing activity to avoid redundancy. Climbing activity during the FST explained approximately 50% of the variance in demand for heroin . The fact that a combination of these measures improves the predictive ability of the linear model indicates that these measures explain a different portion of the variance in heroin demand and complement each other in their predictive qualities. Predicted values from the linear model that included open-field center difference score and climbing measure from FST as predictors for heroin demand are visualized in The detailed statistical output from the tests below is provided in F = 3.89, p < 0.05; Dunnett’s tests; p < 0.01; R2 = 0.37; f2 = 0.58; p = 0.032; R2 = 0.29; f2 = 0.41; The detailed statistical output from the model fitting tests below is provided in p < 0.01; R2 = 0.56; f2 = 1.27; 2 change = +0.13 when compared to a corresponding behavioral marker alone. Climbing during FST and corticosterone after the FST together explain 67% of the variance in demand for heroin  = 1.52, p = 0.14; t(15) = −3.68, p < 0.01; t(5) = −3.73, p < 0.01; data not shown). Importantly, this is the first demonstration of heroin-associated cues presented non-contingently during extinction reinstating heroin seeking in rats with a history of stress exposure.Abbreviated intermittent stress swim re-exposure , administered before the 60 min extinction test, did not reinstate active lever responding when compared to the average of responding on the last two extinction sessions  = 2.625; p = 0.089; There was trending but not significant effect of yohimbine dose on active lever responding in extinction  corticosterone response after ISS and FST was higher than at baseline; (b) the magnitude of the corticosterone response after ISS and FST combined predicted the magnitude of the demand for heroin; (c) behavioral markers strategically sampled around the time of stress exposure predicted the demand for heroin; and (d) a combination of biological and behavioral markers greatly improved the ability to predict individual demand for heroin, when compared to biological or behavioral markers alone. Furthermore, for the first time, we show that heroin-associated cues, which were non-contingently presented during extinction, reinstated heroin seeking in extinction in rats with a history of stress exposure. Importantly, higher climb counts from FST, that was administered the day after ISS, predicted a higher magnitude of cue-triggered reinstatement. Overall, these findings show that the individual biological response to stress and individual behaviors sampled around the time of stress exposure can be used as predictors for the magnitude of stress-induced heroin self-administration and cue-triggered reinstatement of drug seeking in extinction. a priori by the experimenter so that each subject receives the identical amount of stress and therefore allows direct comparison across groups or individual subjects  preclinical model of drug self-administration in conjunction with a well-validated economic demand approach to study individual differences in stress-induced heroin drug taking. Early preclinical models of drug self-administration used unlimited access, often resulting in overdose-related deaths . Importantly, the variance observed at the highest doses indicates that some rats had a much greater response to yohimbine that others  in subsequent demand for heroin provides a framework for a variety of future studies that can further investigate behavioral, biological, or mediating factors underlying this effect.All procedures were in accordance with the Guide for the Care and Use of Laboratory Animals, Eighth Edition  and were reviewed and approved by the University of New Hampshire Institutional Animal Care and Use Committee.NS, TK, RD and SC contributed to the conception and design of the study. NS, TK, CD and EH conducted the study. NS, RD and SC contributed to writing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Our results suggest that maternal consumption of total sulfur amino acids exceeding the NRC 2012 recommendations by 25% during late gestation and lactation benefits sow productivity and piglet neonatal performance. Moreover, increased consumption of sulfur amino acids by both sows and post-weaned piglets improved their ability to counteract the adverse effects by lipopolysaccharide (LPS) exposure. In addition, OH-Met showed a better response than DL-Met in both neonatal and weaned piglets. Taken together, our findings indicate that it might be necessary to update the recommendations for sulfur amino acids for gestating and lactating sows. Attention should also be given to sulfur amino acids supply during an inflammatory challenge as often encountered by piglets early in life.n = 10/treatment) for a 3 × 2 factorial design [diets  and challenge ]. OH-Met and/or DL-Met supplementation increased (p ≤ 0.05) piglets’ body weight gain during day 0–7 and day 7–14. Sow’s milk quality was improved in the supplemented treatments compared to the CON. The LPS challenge decreased (p ≤ 0.05) piglets’ performance from 35 to 63 d and increased (p ≤ 0.05) the levels of aspartate aminotransferase, total bilirubin, IL-1β, IL-6, TNF-a, and malondialdehyde. Plasma albumin, total protein, total antioxidant capacity and glutathione peroxidase decreased post-challenge. The results were better with OH-Met than DL-Met. The increase of Met consumption, particularly as OH-Met increased piglets’ growth performance during the lactation phase and the challenging period.This study determined the effects of increased consumption of sulfur amino acids (SAA), as either DL-Met or Hydroxy-Met (OH-Met), by sows and piglets on their performance and the ability of the progeny to resist a lipopolysaccharide (LPS) challenge. Thirty primiparous sows were fed a diet adequate in SAA (CON) or CON + 25% SAA, either as DL-Met or OH-Met from gestation day 85 to postnatal day 21. At 35 d old, 20 male piglets from each treatment were selected and divided into 2 groups ( Livestock husbandry practices have prompted an expanded use of synthetic amino acids in animal diets to enhance the performance and carcass quality of livestock, as well as to minimize the environmental impact caused by nitrogen excretion ,2,3,4. TConventional sources of supplemental methionine used in animal feeds are either DL-methionine (DL-Met) or 2-hydroxy-4-methylthiobutanoic acid (OH-Met) . AlthougEscherichia coli 0149 [E. coli infection, the bacterial endotoxin lipopolysaccharide (LPS) activates cells of the innate immune system leading to inflammation. Prolonged and/or excessive inflammation caused by stimulants such as LPS can adversely affect animal productivity by inducing muscle catabolism, anorexia and oxidative stress that is damaging to various organs [In addition, weaning, when performed at very young age, is a period of physiological stress for piglets that predisposes them to enteric disease caused by pathogens such as oli 0149 . During s organs . Accordis organs ,20. Meths organs ,22. Alsos organs ,16, we hThis study first aimed to compare the effects of an increase in dietary methionine as OH-Met or DL-Met on the performance of sows and their progeny during gestation and lactation. The second objective of this study was to assess the effects of LPS-induced inflammation and oxidative stress on piglets’ responses when they were fed increased levels of total SAAs, through either DL-Met or OH-Met diets, for a prolonged period of time during the post-weaning period.n = 10 pens/treatment, 1 sow/pen) on gestation d 85 based on their body weight and backfat thickness ). In total, 30 primiparous sows (Landrace × Yorkshire) were randomly allocated to three treatment groups  for a 3 × 2 factorial design trial that included the dietary treatments  and immunological challenge ) by intraperitoneal injection. The dose of LPS was chosen in accordance with previous studies [n = 5 pens/groups, 2 piglets/pen) were allowed free access to the same mash diets as before the immunological challenge for 4 weeks. The body weight and feed intake were measured biweekly from day 35 to day 63.On the morning of day 35, 60 male piglets from the three dietary treatments (20 piglets/treatment) were selected according to their body weight . The ini studies . Blood wThe concentrations of fat, lactose, protein, and nonfat solid in milk were analyzed using an ultrasonic milk analyzer  as described previously . The freThe concentrations of interleukin-1 beta (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a) in plasma were measured using an ELISA kit with catalog numbers PLB00B, P6000B, and PTA00 from R&D Systems (USA). The concentrations of total protein (TP), albumin (ALB) and total bilirubin (TBIL) and activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using a colorimetric method with specific assay kits  from the Nanjing Jiancheng Bioengineering Institute of China ,26. The Statistical analysis was performed using XLSTAT (Version 2015.3.01.19199). Growth performance data, backfat thickness and litter size generated from the sows and progeny during the lactation period were analyzed by one-way ANOVA using the Equation (1).An ANCOVA model was applied to body weight data generated during the LPS challenge, taking the treatments  and the challenge  as qualitative variables and the body weight at d 35 as a quantitative variable, according to Equation (2): i, challenge j; The growth performance data and biochemistry data generated during the challenge were submitted to a two-way ANOVA using the following model described in Equation (3):p ≤ 0.05 was considered significant, and p ≤ 0.10 was considered to have a tendency toward difference. If there was a significant effect, a Tukey test was used for post hoc comparisons of means. Results were presented as means with SEM.p > 0.05) were found in body weight and backfat thickness of the sows among the three groups at lactation d 0 and d 21. However, sow body weight loss between lactation d 0 and d 21 tended to be reduced with OH-Met (p = 0.08) compared to CON, whereas a significant effect of OH-Met was observed on the loss in back fat thickness (p ≤ 0.05). The average daily feed intake was not affected by dietary treatments from gestation d 85 to gestation d 114 and from d 0 to d 21 of lactation. Litter size at birth was not different among the treatments.p = 0.10) by 62% in comparison to the CON. During lactation day 7–14, in comparison to the CON, DL-Met resulted in a 55% increase in piglet body weight gain, whereas OH-Met treatment resulted in a 63% increase. No significant effect of the treatments was observed on piglet mortality during the lactation period. At 35 d old, after two weeks of post-weaning feed supply, the piglets’ body weight, feed intake and feed conversion ratio were not significantly different between treatments. However, OH-Met tended (p = 0.09) to increase body weight in comparison to the CON (The piglets’ body weights at birth was not significantly different between the three treatments . However the CON . p = 0.07) and lactose (p = 0.09) concentrations on lactation d 0. Nonfat solid content was significantly increased (p ≤ 0.05) with both DL-Met and OH-Met, in comparison to the CON. On lactation day 14, milk fat content was similar between treatments. Milk protein concentration was significantly increase with OH-Met supplementation (5.64 ± 0.34%) in comparison to the CON (4.48 ± 0.23%), while DL-Met led to intermediate results (5.22 ± 0.52%). Lactose and nonfat solids were significantly increased with the increasing HO-methionine level but not with DL-Met in comparison to control.Milk composition at lactation d 0 and d 14 is presented in p ≤ 0.05) the milk free amino acid concentrations of cystine, glutamic acid, isoleucine, leucine, lysine, methionine, threonine, tyrosine, α-amino-n-butyric acid, β-amino-isobutyric acid, and taurine and decreased (p ≤ 0.05) ornithine and 3-methyl histidine concentrations at lactation d 0 and/or 14 (p ≤ 0.05) milk free amino acid concentrations of cystine, isoleucine, leucine, lysine, methionine, tyrosine, and α-amino-n-butyric acid but lower (p ≤ 0.05) ornithine and β-amino-isobutyric acid concentrations at lactation d 0 and/or 14  and feed intake (p ≤ 0.05) and tended to decrease the gain-to-feed ratio (p ≤ 0.10) of piglets from day 35 to 49 and from day 49 to 63, respectively. Notably, the changes in the growth performance variables with the LPS challenge were attenuated (p ≤ 0.05) in piglets fed DL-Met and OH-Met compared to pigs fed the CON. Piglets fed increased levels of SAAs had higher body weight, body weight gain and gain-to-feed ratios than piglets fed the CON (p < 0.05) under the LPS challenge. The highest performance was obtained with OH-Met.The growth performance of the piglets was significantly affected by the LPS challenge, diet, or their interaction . The 6 gp ≤ 0.05) activity of AST and concentration of TBIL and decreased (p ≤ 0.05) concentrations of ALB and TP in plasma at 4, 12, and/or 24 h post-LPS challenge. Meanwhile, the LPS challenge led to increased plasma levels of pro-inflammatory cytokines IL-1β, IL-6 and TNF-a at 4 h post-LPS challenge compared with the saline-treated group. Changes to most of these plasma biochemical variables due to the LPS challenge were alleviated in piglets fed DL-Met or OH-Met compared to pigs fed the CON. Piglets fed OH-Met exhibited similar values as the CON for some plasma biochemistry variables in comparison to piglets fed DL-Met at various time points after the LPS challenge. The plasma biochemical variables of the piglets were significantly affected by the LPS challenge, diet, or their interaction . Comparep ≤ 0.05) activities of T-AOC (27.0–36.0%) and GPX (~15.7%), and an increased (p ≤ 0.05) MDA (36.8–57.9%) concentration in plasma at 12 and/or 24 h post-LPS challenge. Notably, changes to plasma T-AOC and GPX activities due to the LPS challenge were prevented (p < 0.05), but the MDA concentration was not alleviated in piglets fed DL-Met and OH-Met. The plasma antioxidant variables of the piglets were significantly affected by the LPS challenge or diet . CompareThe present study shows that maternal and neonatal methionine supplementation, particularly in the form of OH-Met, during late gestation, lactation, and postweaning, can improve the performance of sows and their progeny. Dietary supplementation of both DL-Met and OH-Met did not affect body weight, backfat thickness, feed intake and litter size at birth for the sows, but OH-Met supplementation reduced the loss of body weight and backfat thickness of sows during lactation. A previous study also showed that dietary supplementation of DL-Met and OH-Met during lactation did not affect the body weight and backfat thickness of sows, but the loss of body weight and backfat during lactation was not calculated . BecauseThirteen free amino acids present in milk were affected by maternal dietary methionine supplementation. Among them, five essential amino acids  and a nonessential amino acid (tyrosine) are used by cells to synthesize proteins and play important roles in the activation of protein biosynthesis . Thus, iMaternal dietary DL-Met and/or OH-Met supplementation also increased free amino acids in milk that were associated with redox control (cystine and taurine), which might enhance the ability of piglets to cope with stress . AdditioAnother novel finding in the current study was that methionine, particularly in the form of OH-Met, alleviated LPS-induced adverse effects in piglets. The LPS challenge reduced body weight gain, feed intake and feed utilization efficiency throughout the study in piglets, in accordance with earlier studies . StrikinMaternal and neonatal methionine supplementation during late gestation, lactation and postweaning improved the performance of sows and their progeny. This was mainly associated with improved milk quality, along with increased essential  and reduction-oxidative control (cystine and taurine) amino acids and decreased energy (glutamic acid) and muscular proteolysis markers (3-methyl histidine) amino acids in milk. Moreover, prolonged supplementation of methionine in the postweaning diet of the progeny improved their ability to counteract LPS-induced negative effects, which may be due to the enhancement of anti-inflammation and antioxidant capacities of piglets. Finally, better results were obtained with dietary supplementation of OH-Met in comparison to DL-Met for most of these variables, which indicates that OH-Met might be a better methionine source than DL-Met for pig production. This novel finding indicated that the current methionine requirement from NRC (2012) may be underestimated and could have meaningful impacts on swine nutrition and health.


IgG4-related disease (IgG4-RD) is a systemic immune-mediated fibro-inflammatory condition, which could involve multiple structures, including the pancreas, salivary glands, and lymph nodes. However, cases of IgG4-RD involving the bilateral orbits, salivary glands, submandibular glands, lymph nodes, and prostate are rare.A 51-year-old man complaining of bilateral exophthalmos, reduced vision, and weight loss of 15 kg over 2 years presented to our department for evaluation.18F-fluoro-2-deoxy-D-glucose (18F-FDG) positron emission tomography/computed tomography (PET/CT), which revealed diffuse increased FDG uptake in many structures, he was diagnosed with IgG4-related disease involving the bilateral orbits, salivary glands, submandibular glands, lymph nodes, and prostate.Based on the elevated serum IgG4 level, postoperative pathology, and the features of Because of the significant bilateral proptosis and exposure keratoconjunctivitis in the right eye, bilateral soft-tissue masses located in the orbits were resected, and the patient was started on oral methylprednisolone with gradual tapering.The patient's symptoms gradually relieved after the operation and glucocorticoid therapy. Four months later, cranial axial CT revealed remarkable narrowing of soft-tissue masses in the bilateral orbits, and his serum IgG4 level reduced sharply.IgG4-RD should be considered in cases of diffuse FDG uptake in the bilateral orbits, salivary glands, submandibular glands, lymph nodes, and prostate on PET/CT. Although the presentation of IgG4-RD on 18F-fluoro-2-deoxy-D-glucose positron emission tomography/computed tomography (18F-FDG PET/CT) has been reported, cases of IgG4-RD involving the bilateral orbits, salivary glands, submandibular glands, lymph nodes, and prostate with intense diffuse FDG uptake are rare.–9IgG4-related disease (IgG4-RD) is a systemic immune-mediated fibro-inflammatory condition, characterized by tumor-like swelling, with variable degrees of “storiform” fibrosis, and lymphoplasmacytic infiltration enriched with IgG4-positive plasma cells.2A 51-year-old man presented with bilateral exophthalmos, reduced vision, and weight loss of 15 kg over 2 years. He had a history of type 2 diabetes, which was well-controlled with metformin. Physical examination revealed decreased visual acuity of 0.5 in the right eye and 0.2 in the left eye, elevated intraocular pressure of 22 mmHg in the right eye and 23 mmHg in the left eye, bilateral exophthalmos, ocular motility disturbance, and exposure keratoconjunctivitis in the right eye. Contrast-enhanced cranial axial CT  and imaged for 2.5 minutes per bed position, approximately 1 hour after the injection, with a Gemini 16 PET/CT scanner . Whole-body 18F-FDG PET/CT showed increased FDG uptake in the soft-tissue masses located in the bilateral orbits , the possibility of IgG4-RD increased.Subsequently, the patient was administered Because of the severe bilateral proptosis and exposure keratoconjunctivitis in the right eye, soft-tissue masses located in the bilateral orbits were resected. The postoperative pathology showed fibrous hyperplasia with infiltrations of many lymphocytes and plasma cells. Immunohistochemical staining revealed that the lymphocytes and plasma cells were positive for IgG4 (more than 50 cells/high-power field), IgG, CD20, CD3ε, CD23, CD138, with a Ki-67 proliferation index of about 5% to 10%, but negative for CD10 and CyclinD1. No IgH/Igκ gene rearrangement peaks were detected. Combined with the aforementioned morphological findings and immunohistochemical staining and gene rearrangement results, the diagnosis of IgG4-RD was confirmed.The patient was started on 40 mg of oral methylprednisolone daily, which was gradually tapered. His symptoms gradually relieved after the surgery and glucocorticoid therapy. Four months later, cranial axial CT Fig. C revealeThe patient provided informed consent for publication of this case.3–4,10 In addition, elevated serum concentrations of IgG4 (> 135 mg/dL) are found in 60% to 70% of the patients with IgG4-RD.,12 Although IgG4-RD occurs more commonly in middle-aged and older men, the disease extent and severity appear to be similar between men and women. In a series of 125 patients with biopsy-proven IgG4-RD, the number of involved organs, degree of serum IgG4 elevation, or damage from IgG4-RD did not differ between men and women. The pathogenesis of IgG4-RD is unclear; findings consistent with both autoimmune and allergic disorders are present.,15–21 It has been postulated that IgG4 plays a role in tolerance to allergens and responses to certain infectious agents. However, its physiologic role is poorly understood. There is an emerging consensus that IgG4 antibodies in this disease are not pathogenic but represent a down-regulatory response to other primary processes instead.IgG4-RD is a systemic immune-mediated fibro-inflammatory condition, characterized by tumor-like swelling of the involved structures, lymphoplasmacytic infiltration enriched with IgG4-positive plasma cells, and variable degrees of fibrosis, with a characteristic “storiform” pattern.–8,23,24 The pancreas, lymph nodes, and salivary glands are the most commonly involved structures in IgG4-RD. In addition, the orbit is also commonly involved (17%–23%) in IgG4-RD, called IgG4-related ophthalmic disease, including IgG4-related dacryoadenitis and IgG4-related orbital myositis.,3,9,25 The typical ocular manifestation is proptosis or swelling of the ocular region, most commonly caused by dacryoadenitis . Orbital myositis (involvement of the extraocular muscles), orbital pseudotumors , or a combination of these disorders can also result in exophthalmos. The less common ocular manifestations of IgG4-RD are scleritis, obstruction of the nasolacrimal duct, and compression of the infraorbital and trigeminal nerves.–29 In our patient, the primary ocular manifestations were bilateral proptosis and reduced vision. In addition, the salivary glands, submandibular glands, lymph nodes, and prostate were involved.IgG4-RD can involve one or multiple structures (60%–90%), including the pancreas (autoimmune pancreatitis [AIP]), biliary tract (sclerosing cholangitis), lacrimal and salivary glands , lymph nodes (lymphadenopathy), retroperitoneum , kidney , thyroid (Riedel thyroiditis), and mediastinum (fibrosing mediastinitis).,30 Although elevated serum IgG4 levels are not diagnostic, the serum IgG4 level should be measured because isolated elevated levels are a significant diagnostic aid.,32The diagnosis of IgG4-RD is based on the characteristic histopathologic findings on biopsy and immunohistochemical staining results, including lymphoplasmacytic tissue infiltration of mainly IgG4-positive plasma cells and lymphocytes, accompanied by fibrosis, with storiform features, and often accompanied by obliterative phlebitis and modest tissue eosinophilia. Generally, CT of the chest, abdomen, and pelvis are performed for patients diagnosed with IgG4-RD because of the high frequency of subclinical disease. If available, 18F-FDG PET/CT can also be highly effective in determining the disease extent and should be considered at baseline.,30,33 A prospective cohort study evaluating the value of 18F-FDG PET/CT in characterizing IgG4-RD revealed that 18F-FDG PET/CT can reveal more organ involvement compared to conventional evaluations, including physical examination, ultrasonography, and CT. Moreover, comprehensive understanding of all involvement can aid the biopsy-site selection. In addition, 18F-FDG PET/CT can monitor the therapeutic response after 2–4 weeks of steroid-based therapy. The specific characteristics and pattern of IgG4-RD on 18F-FDG PET/CT include diffusely elevated FDG uptake in multiple structures, including the pancreas, salivary glands, and retroperitoneal region.,30 Most patients with IgG4-RD respond to glucocorticoids within several weeks, typically with symptomatic improvement, reduction in the size of masses or organ enlargement, improvement in organ function, and often, reduction in serum IgG4 levels.,34 The natural history of IgG4-RD has not been well-defined. Some cases improve temporarily without treatment, but most cases progress at variable rates.,34 In 2010, Vege et al reported that 47% of the patients with type 1 AIP (the first disease recognized to be associated with IgG4-RD) experienced a relapse and that diffuse pancreatic swelling and proximal biliary involvement can predict recurrence.Although the diagnosis of IgG4-RD requires the presence of characteristic findings in the biopsy specimen of the affected tissues, additional organ involvement can be identified through a careful history, physical examination, routine laboratory testing, and selected imaging.18F-FDG PET/CT images with intense diffuse FDG uptake in the bilateral orbits, salivary glands, submandibular glands, lymph nodes, and prostate. This report suggests that IgG4-RD should be considered in cases of diffuse FDG uptake in the bilateral orbits, salivary glands, submandibular glands, lymph nodes, and prostate on PET/CT. In addition, 18F-FDG PET/CT is a useful diagnostic modality for patients with IgG4-RD, which can evaluate organ involvement, aid in biopsy-site selection, and monitor the therapeutic response.In conclusion, we presented rare Data curation: Ping Dong.Resources: Ping Dong, Li Wang.Supervision: Lin Li.Writing–original draft: Ping Dong, Li Wang.Writing–review & editing: Li Wang, Lin Li.


Escherichia coli (E. coli) strains obtained after cleaning and disinfection. On those field isolates antibiotic susceptibility was monitored and susceptibility to commonly used active components of farm disinfectants  was tested.Farm disinfectants are widely used in primary production, but questions have been raised if their use can select for antimicrobial resistance. The present study examined the use of disinfectants in poultry and pig husbandry and its contribution to the antibiotic and disinfectant susceptibility of E. coli strains were susceptible to in-use concentrations of formaldehyde, benzalkoniumchloride and a formulation of peracetic acid and hydrogen peroxide, indicating that the practical use of disinfectants did not select for disinfectant resistance. Moreover, the results showed no indications for the selection of antibiotic resistant bacteria through the use of disinfectants in agricultural environments.This study showed a high resistance prevalence (> 50%) for ampicillin, sulfamethoxazole, trimethoprim and tetracycline for both production animal categories, while for ciprofloxacin only a high resistance prevalence was found in broiler houses. Disinfectant susceptibility results were homogenously distributed within a very small concentration range. Furthermore, all E. coli disinfectant susceptibility.Our study suggests that the proper use of disinfectants in agricultural environments does not promote antibiotic resistance nor reduce The online version of this article (10.1186/s12917-019-2044-0) contains supplementary material, which is available to authorized users. Biocidal products are frequently used chemicals with the aim to inactivate microorganisms  harmful Despite the increasing use of disinfectants, bacteria seem to remain susceptible to these disinfection products when used correctly. Their in-use concentrations are normally far above the minimum inhibitory concentration (MIC) of wildtype isolates , as oppo. Laboratory-based adaptation experiments have shown that step-wise exposure of initially susceptible bacteria to subinhibitory concentrations of benzalkoniumchloride, chlorhexidine, triclosan and some commercial disinfectants may lead to decreased susceptibility to either antibiotics or disinfectants , ac8, acE. cg for 10 min and the supernatant was discarded. The remaining pellet was resuspended in 10 mL Ringers solution . Next, inocula were diluted with Ringer solution to an optical density at 600 nm (OD600) corresponding with a viable count of 1–5 × 108 CFU/mL. To control the inoculum concentration, enumerations on PCA were carried out by using a spiral plater .The selected isolates were cultured on PCA at 37 °C for 24 h. Per agar plate, one colony was picked and used to inoculate 10 mL of Tryptone Soya Broth  and grown at 37 °C for 16 h to obtain fresh liquid cultures. Subsequently, liquid cultures were centrifuged at 5000 To check the reproducibility and repeatability of the assay, eight isolates were tested in triplicate, on two different occasions. From then on, each isolate was tested only once.The MICs of each active component  or given formulation (D50) for the selected isolates were determined with a broth microdilution method based on the method described by Knapp et al. (2015) .8 CFU bacterial /mL) were added to the TSB in the microtiter plate, resulting in a total volume of 100 μL. Final concentration ranges were as follows: 0.213–0.007 g/L BKC, 1.480–0.046 mL/L F, 20–0.625 mL/L GA and 20–0.125 mL/L D50. As a positive control, 50 μL of each bacterial suspension was added to 50 μL TSB without disinfectant. To check for possible contamination, wells without bacterial suspension and disinfectant served as blank. After inoculation, plates were incubated for 24 h in a shaking incubator (100 rpm) at 37 °C. After incubation, the MICs were read. The MIC was defined as the lowest concentration of active components or formulation where no growth was visually observed. In every experiment the E. coli reference strains for antibiotic susceptibility (ATCC 25922) and disinfectant susceptibility (ATCC 10536) were used as controls.A 96-well microtiter plate with U-shaped wells  was filled with 50 μL TSB containing twofold dilutions of the active component or formulation. Fifty microliters of the field isolates .2O2’ and ‘use of other active components’) were tested univariable for all antibiotics (n = 13 by combining sulfamethoxazole and trimethoprim resistance). Those variables with univariable P-values of < 0.20 were retained for further analysis in a multivariable model. Subsequently, with the retained variables, a multivariable logistic regression model was constructed using the stepwise backward elimination procedure starting with the global model and gradually excluding all non-significant factors. Multivariate binary logistic regression models were used for each antibiotic. As multiple models were tested to evaluate the effect of the different active components on the different types of antibiotic resistance a bonferroni correction for multiple testing was performed. P-values ≤0.0038 (after Bonferroni correction) were considered as significant. All statistics were performed using SPSS Statistics 25.0 .For both animal categories the antibiotic resistance prevalence and the accompanying 95% confidence interval was calculated for each antibiotic based on the standard error of the binomial distribution in Microsoft Excel . The association between active components used  during disinfection and antibiotic resistance at each farm was tested by means of binary logistic regression analysis taking the corresponding antibiotic use (TI100) into account as co-variable. First, the independent variables Additional file 2:Questionnaire 1: Measuring both cleaning and disinfection practices and antibiotic usage at broiler farms. It describes the set of questions asked to all participating poultry farmers related to the applied cleaning and disinfection protocol and antibiotic use. (DOCX 68 kb)Additional file 3:Questionnaire 2: Measuring both cleaning and disinfection practices and antibiotic usage at pig farms. It describes the set of questions asked to all participating pig farmers related to the applied cleaning and disinfection protocol and antibiotic use. (DOCX 68 kb)


Pseudomonas exotoxin A (PE) are in use clinically for the treatment of hairy cell leukemia, and under trial for the treatment of other cancers. In an effort to improve the efficacy of PE-based RITs, we evaluated the potential of combination therapy with several common antibiotics  on human cell lines HEK293, OVCAR8, and CA46. Antibiotics were selected based on their potential to inhibit mitochondrial protein synthesis and disrupt energy metabolism in cancer cells.Recombinant immunotoxins (RITs) are antibody-toxin fusion proteins that can selectively eliminate populations of cells expressing specific surface receptors. They are in evaluation as therapeutic agents for cancer. RITs based on Tetracycline, chloramphenicol, linezolid, and fusidic acid alone killed cultured human cells at high concentrations. At high but nontoxic concentrations of each antibiotic, only chloramphenicol treatment of the Burkitt’s lymphoma cell line CA46 showed enhanced cytotoxicity when paired with an anti-transferrin receptor/PE RIT. This result, however, could not be replicated in additional Burkitt’s lymphoma cell lines Ramos and Raji. Although the six antibiotics we tested are not promising candidates for RIT combination therapy, we suggest that fusidic acid could be considered independently as a potential cancer therapeutic.The online version of this article (10.1186/s13104-019-4337-6) contains supplementary material, which is available to authorized users. Pseudomonas exotoxin A (PE) [Recombinant immunotoxins (RITs) are genetically engineered, chimeric proteins comprised of an antibody joined to a cytotoxic protein –3. They n A (PE) . Severaln A (PE) , and onen A (PE) . Althougn A (PE) .Recent improvements to the design and construction of PE RITs have reduced their immunogenicity and off-target effects while substantially retaining activity . The cytDysregulated bioenergetics have been identified as a hallmark of cancer . One potIn this study, we attempted to determine if a combination therapy of PE-based RITs and antibiotics targeting bacterial protein synthesis might exhibit enhanced cytotoxicity. We tested FDA-approved antibiotics tetracycline, chloramphenicol, streptomycin, linezolid, fusidic acid, and kanamycin. Alone, tetracycline, chloramphenicol, linezolid, and fusidic acid killed cultured human cells at high concentrations. High, nontoxic concentrations of most antibiotics did not enhance the activity of the HB21-LR RIT, an anti-transferrin receptor scFv combined with PE24 , 18. Onl2 and 37 °C in DMEM with 4.5 g/l glucose, 1 mM sodium pyruvate, 10% FBS, and 2 mM l-glutamine. All cells were thawed and grown from liquid nitrogen stocks prepared from an early passage of the original cell lines obtained as a kind gift from the laboratory of Dr. Ira Pastan . All cell lines evaluated are sensitive to HB21-LR.Human-derived cell lines HEK293 (embryonic kidney), OVCAR8 (ovarian serous adenocarcinoma), and the Burkitt’s lymphoma cell lines CA46, Raji, and Ramos were grown in culture at 5% COSix antibiotics were selected in this study: chloramphenicol, tetracycline, fusidic acid, kanamycin, linezolid, and streptomycin. Chloramphenicol  stocks were prepared in 200 proof ethanol to a concentration of 154.4 mM. Fusidic acid  stocks were prepared in 200 proof ethanol to a concentration of 96.8 mM. Linezolid  stocks were prepared in DMSO to a concentration of 59.3 mM. Kanamycin  stocks were prepared in ultrapure water to a concentration of 85.8 mM. Streptomycin  stocks were prepared in ultrapure water to a concentration 34.3 mM. Tetracycline  stocks were prepared in ultrapure water to a concentration of 104 mM. Prepared stocks were sterile filtered through a 0.22 µm syringe filter.The anti-transferrin receptor/PE24 RIT HB21-LR was prepared as described , diluted50). A two-tailed paired T-test was utilized to evaluate significant differences in EC50 values by comparing experimental conditions to a control plate not treated with antibiotic. P values less than 0.01 were considered significant.Cell viability was evaluated using the WST-8 reagent . Assays were performed essentially as described . The 0% Common antibiotics that inhibit bacterial translation are known to affect mitochondrial protein synthesis , and mig50 values for cell lines sensitive to antibiotics are shown in Additional file Human cell lines HEK293, OVCAR8, and CA46 were evaluated for cytotoxic responses to the six antibiotics at least twice to determine a high but nontoxic dose of the antibiotic. Example viability responses are shown for HEK293 . Chloramphenicol was further evaluated on the Burkitt’s lymphoma cell lines Ramos and Raji  that was more than ten times greater than the therapeutic blood plasma concentration of the antibiotic 15–46 µM). The 500 µM concentration of chloramphenicol was also well above the toxic blood plasma concentration of the drug, 77 µM . Raji an00 µM tha5–46 µM. Fusidic acid was the only antibiotic consistently evaluated at concentrations below the therapeutic blood plasma range of 58–387 µM. Although fusidic acid did not enhance the cytotoxicity of HB21-LR at the concentrations we tested, the low concentration required to diminish cell viability suggests that higher doses might be worth evaluating. Recent studies have suggested that fusidic acid, and derivatives thereof, may have therapeutic utility for the treatment of cancers , 28. We Only the anti-transferrin receptor/PE24 RIT HB21-LR was evaluated. It is not known if RITs with other targets, other forms of PE, or other toxins may behave differently.Only six antibiotics were evaluated. Other antibiotics may display different effects.Only a single dose of antibiotic was evaluated in combination with HB21-LR. A more extensive evaluation of different combinations could reveal additional effects.Only the human cell lines HEK293, OVCAR8, CA46, Raji, and Ramos were evaluated. Other human cell lines may exhibit different responses to treatment.Additional file 1. HEK293 survival in response to antibiotic treatment. Survival of HEK293 cells in response to six antibiotics was evaluated. Each antibiotic was evaluated at least twice. Representative graphs are shown here. Error bars indicate standard error of six replicates. Data were fit to a straight line  or a four-parameter sigmoid function . The vertical dotted line indicates the concentration of antibiotic selected for evaluation in combination with HB21-LR  or a four-parameter sigmoid function . The vertical dotted line indicates the concentration of antibiotic selected for evaluation in combination with HB21-LR  or a four-parameter sigmoid function . The vertical dotted line indicates the concentration of antibiotic selected for evaluation in combination with HB21-LR . The survival of HEK293, OVCAR8, and CA46 cells in response to six antibiotics was evaluated. The survival of Raji and Ramos cells were evaluated in response to chloramphenicol. Each antibiotic tested was evaluated on each cell line at least twice. Where cytotoxicity was observed, data were fit to a four-parameter sigmoid function. The EC50 was extracted from the curve fit and is presented in tabular format here. Estimates were taken for those antibiotics where complete cell killing was not achieved. If no toxicity was observed, that is indicated.Additional file 6. Maximum antibiotic concentrations evaluated. The survival of cells was evaluated in response to the antibiotics chloramphenicol, tetracycline, fusidic acid, kanamycin, linezolid, and streptomycin. The maximum concentration of antibiotic tested on cells is shown here.Additional file 7. HEK293 survival in response to antibiotic/RIT combination treatment. Survival of HEK293 cells was evaluated in response to combination treatment with antibiotic and HB21-LR. Antibiotic concentrations are shown in Table Additional file 8. OVCAR8 survival in response to antibiotic/RIT combination treatment. Survival of OVCAR8 cells was evaluated in response to combination treatment with antibiotic and HB21-LR. Antibiotic concentrations are shown in Table Additional file 9. CA46 survival in response to antibiotic/RIT combination treatment. Survival of CA46 cells was evaluated in response to combination treatment with antibiotic and HB21-LR. Antibiotic concentrations are shown in Table Additional file 10. Raji and Ramos survival in response to chloramphenicol/RIT combination treatment. Survival of cells Raji and Ramos cells was evaluated in response to combination treatment with chloramphenicol and HB21-LR. Antibiotic concentrations are shown in Table


Bacillus subtilis SL-44 played a significant role in the reduction of wilt disease severity on pepper plants. To evaluate biological control mechanism of B. subtilis SL-44 on wilt disease caused by Rhizoctonia solani, the activities of the related enzymes were detected in the pepper seedling with different treatment in this study. Fluorescence microscopy combined with different dyes showed that B. subtilis SL-44 induced a large amount of active oxygen and callose accumulation in pepper leaves. The defense-related enzyme activities in pepper were improved significantly when treated with B. subtilis SL-44, including peroxidase, catalase, superoxide dismutase, polyphenol oxidase, and phenylalanine ammonia lyase. The activity of chitinase and β-1,3-glucanase in B. subtilis SL-44-treated pepper was also enhanced. Furthermore, the expression level of pepper-resistance gene CaPIN II was significantly increased in B. subtilis SL-44 treatment. Besides, B. subtilis SL-44 filtrate led to the death of the pathogenic fungus by fracturing the mycelia and leaking of the cell contents. Surfactin, iturin, and fengycin were found in B. subtilis SL-44 crude extracts, which could be effective antifungal compounds against R. solani. The results suggest that B. subtilis SL-44 could not only activate induced systemic resistance of pepper seedling against wilt disease caused by R. solani by jasmonic acid-dependent signaling pathway but also produce antifungal compounds to inhibit or even damage the mycelium growth of R. solani. The findings of this study provide novel guidance in plant protection development.Pepper seedling wilt disease is the main cause of crop yield reduction. Biocontrol agents are widely used to control plant diseases caused by pathogenic fungi and activate plant defense systems. Our preliminary work showed that  Rhizoctonia solani is responsible for pepper yield losses. Usually, these pathogens are controlled by chemical pesticides, which cause environmental pollution and potential drug resistance from long-time usage. In organic agriculture, there are restrictions on the use of more pesticides. Therefore, the application of biological control in the prevention of phytopathogen infection has gradually gained attention because it is environment-friendly. In our previous works, Bacillus subtilis SL-44 (SL-44) has been studied with strong antifungal activity against the mycelial growth of pathogenic fungus Rhizoctonia solani. Furthermore, SL-44 also has the significant capability of phosphate dissolving, IAA producing, and root colonizing and could significantly increase pepper’s biomass and promote the growth of the pepper , superoxide dismutase (SOD), polyphenol oxidase (PPO), and phenylalanine ammonia lyase (PAL) and expression of defense-related genes enhanced by Bacillus sp. in soybean, tomato, and Arabidopsis thaliana (Capsicum annuum pathogenesis-related protein (CaPR) genes indicate that plant systemic immunity is elicited through SA or JA signaling pathway by different treatments  medium for growth at 37°C, 180 rpm for 24 h in a shaker until it reached 108 cfu/ml  were purchased from a local market in Shihezi, Xinjiang of China.The pathogenic fungus 8 cfu/ml . Pepper Pepper seed surface was sterilized with 6% sodium hypochlorite, then washed four times with sterile distilled water, and then kept at 25°C for 3 days, until germinated on sterilized filter paper. A 7-day-old pepper seedling was transferred into a flowerpot  containing a sterile vermiculite and perlite mixture  and placed in a growth chamber . Eight plants were found in each tray and eight trays per treatment. Water and 30 ml Hoagland’s solution were added regularly for normal growth.R. solani), SL&R.s (SL-44 inoculation and R. solani infection), SA , and MeJA (pepper foliage treated with 0.1 mM methyl jasmonate). Method of microorganism inoculation is as follows: 5 ml per flowerpot of the overnight cultured SL-44 broth was pipetted into the 2 cm depth soil around root of the pepper seedlings with a sterile syringe; the mature R. solani hyphae cultured in the potato dextrose liquid medium for 5 days was taken out, and the hyphae ball with a diameter of about 1 cm was selected and buried at a depth of 2 cm around the root of the pepper seedling; when pepper seedling was inoculated with both biocontrol bacteria and plant pathogens, the fungus was inoculated at first; and staged spray 5 ml of salicylic acid at a concentration of 0.5 mM onto the leaves of the pepper on each flowerpot. After salicylic acid was completely absorbed by leaves, it can be used as samples of SA treatment. The method of MeJA treatment was same with SA treatment but concentration of methyl jasmonate was 0.1 mM.The 35-day-old pepper seedlings were treated in six treatments: CK (control group treated with water), SL-44 (inoculated with SL-44), R.s . Six mature leaves are collected and tested by 3, 3′-diaminobenzidine (DAB) staining to detect HThe leaves of pretreated were stored in a 50% (v/v) ethanol solution for microscopic examination. The coloration was observed under a 40 × magnification using a ZEISS optical microscope and photographed.λ = 385 nm) and photographed.According to the method of g for 20 min at 4°C. The crude enzyme extracts (supernatants) were collected and stored in a refrigerator at −20°C for detection.Samples were collected 1, 3, 5, 7 and 9 days after inoculation (dai). About 0.5 g of pepper leaves of fresh weight was ground in a pre-cooled mortar. The grounded powder was added into 3 ml different PBS buffers with the corresponding pH required for different enzyme activity assays. The homogenate was centrifuged at 10,000 × 470 per minute is a POD enzyme activity unit. The activities of catalase (CAT), PAL, and PPO were tested by UV absorption, the amount of enzyme that reduces the A240 by 0.1 per minute is CAT enzyme activity unit. The amount of enzyme that changes A290 by 0.01 per minute is PAL enzyme activity unit. The increase of A398 by 0.01 per minute was defined as a PPO enzyme activity unit. All of these enzyme activity detection methods were according to The method of activity detection of SOD was photoreduction of nitroblue tetrazolium (NBT). One SOD enzyme activity unit refers to the amount of enzyme needed to inhibit 50% NBT photoreduction reaction. The method of guaiacol was used for POD detection, 0.01 increase in Ag for 30 min at 4°C; and the crude enzyme extract obtained was stored in the refrigerator at −20°C until use.About 0.5 g of treated pepper leaves (3 dai) was placed in a pre-cooled mortar and quickly ground to powder with liquid nitrogen protection; 5 ml of 0.05 M acetic acid-sodium acetate buffer (pH 5.0) was added to the extract and then centrifuged at 8,000 × 540 was determined by UV-VIS Spectrophotometer  and repeated three times for each treatment.C. annum pathogenesis-related 1 (CaPR1), C. annum basic β-1,3-glucanase (CaPR2), C. annuum pathogenesis-related protein 4 (CaPR4), and C. annuum proteinase inhibitor II (CaPIN II)  to verify the differential expression of selected genes. Expression of aPIN II)  have beeaPIN II) . CaACT1 Total RNA was isolated using RNAprep Pure Plant Kit . The first strand cDNA was reverse transcribed using PrimeScript™ RT reagent Kit with gDNA Eraser . The Oligo(dT) primer was synthesized by GENEWIZ .Premix Ex Taq™ II according to the instruction. The extracted sample to be tested in condition of pre-denatured at 95°C for 30 s, and then the PCR cycle is started, and followed by 40 cycles. Dissociation curve analysis at 95°C for 0 s, 65°C for 15 s, and 95°C for 0 s. All genes evaluated were identical for the qRT-PCR parameters. The qRT-PCR experiments were conducted twice with three replicates for each treatment.Quantitative RT-PCR reactions were performed on the Light Cycler 96 system  using SYBR®R. solani, SL-44 was inoculated into 50 ml NB medium and placed in a shaking incubator with 170 rpm at 30°C. After culturing for 36 h, the bacterial suspension was collected and centrifuged at 10,000 × g for 10 min at 4°C. The supernatant was filtered using a 0.22 μm filter. R. solani was inoculated on PDA, grown, and covered with agar medium surface, and then we used a 10-mm diameter punch holes in the fungus-coated medium to obtain a 10-mm diameter mycelial disk of R. solani. About 10-mm diameter mycelium of R. solani was mixed with SL-44 filtrate and incubated at 28°C for 24 h. The same size of mycelium was added into sterile water as a control. The experiment was repeated three times for each treatment. The sample was observed under an optical microscope  to observe changes in the mycelium.To detect the disruption of the culture filtrate of SL-44 against the pathogenic fungus g for 20 min) and removed the cells, the supernatant was filtered using a 0.22 μm sterile filter, and then the filtered supernatant was mixed with the equal volume of n-butanol. The antibacterial lipopeptide in the supernatant was extracted into the n-butanol phase for three times, then concentrated with rotary evaporation to dry at 50°C, and residual was dissolved in a small amount of sterile water prior to use  with an ultraviolet detector. A reversed phase column Agilent ZORBAX Eclipse Plus C18  was used and the detection wavelength was 230 nm. Mobile phase A was HPLC grade methanol and B was 0.1% acetic acid solution, A:B = 40:60 at a flow rate of 0.8 ml/min at 40°C.To further determine lipopeptides in antimicrobial crude extracts, the molecular weight of the crude extracts was determined by LC-MS/MS . The column was a liquid column Thermo HYPERSIL GOLD C18 . Mobile phase A was a 0.1% formic acid solution and mobile phase B was 100% acetonitrile at a flow rate of 0.3 ml/min. Mass spectrometry detection conditions are as follows: cone voltage 3 kV, electrospray temperature 300°C, capillary temperature 350°C, and scan range 800–1,600 m/z. Data were acquired using a positive ion mode.p < 0.05) to compare the difference in enzyme activity of control and treatments and defense-related gene expression of different treatments in the same time intervals .All data were presented using mean ± standard deviation (SD). Significant differences between the means were determined using one-way analysis of variance (ANOVA) and Duncan’s multiple range tests  in pepper leaves treated with SL-44 was lower than those treated with water in 24 hpi   ; 1034.92 A (C13) ,C, respe A (C13) , fengyci A (C13) , fengyci A (C13) , and fen A (C13)  and are  A (C13) . Based oR. solani in the world has become an urgent problem to be solved. The use of chemical fungicide to control this disease has been limited because of drug resistance, environmental pollution, and restricted for use in organic agriculture. In previous study, the use of SL-44 as a biological control agent and its effect on R. solani were examined and the result obtained was promising  in early defense against bacterial soft rot in tomato  by activating two signaling pathways, the JA/ET-dependent (major) signaling pathway and the SA-dependent (minor) signaling pathway. When the SA-dependent signaling pathway is activated, the contents of pathogenesis-related (PR) proteins, such as chitinase and β-1,3-glucanase, will increase in the plant. It was reported that  disease . In the CaPR1, CaPIN II, CaPR2, and CaPR4 were discovered from pepper (Capsicum annuum L.) by researchers  and/or whitefly, which indicated that SA and JA signaling pathways in AG and below ground (BG) were induced by above-ground (AG) whitefly infestation  were not only activated both in SAR and ISR systems that are incompatible in pepper but also showed significant difference on expression between the whitefly-treated and control group except CaPR4 gene . B. subtilis SPB1 and found that it had a strong inhibitory effect on the growth of R. solani and R. bataticola. B. amyloliquefaciens XZ-173 on R. solanacearum QL-Rs1115. B. amyloliquefaciens FZB42 has an antagonistic effect on corn and wheat on F. graminearum. Antifungal compounds can also resist pathogenic bacteria by inducing plant resistance to prevent pathogen infection (B. subtilis UMAF6639 secretes lipopeptides to protect plants against powdery mildew by activating JA- and SA-dependent defense responses (R. solani and induce pepper defense resistance such as to trigger cellular defense response, induce defense-related enzyme enhancement, and stimulate the JA-dependent signaling pathway.Several diseases . They hadiseases . Antimicthe host . In this. solani . This re. solani , iturin . solani ,C, and f. solani ,B and B . solani  accordin. solani . Liu et nfection . B. subtesponses . In thisRhizoctonia solani in pepper. SL-44 is a beneficial bacterium that has successfully performed the outstanding act of biological control in pepper seedling via two main strategies. On the one hand, SL-44 induced pepper plant systemic resistance by activating JA-mediated signaling pathway and enhanced plant immunity to defense pathogen aggression in pepper. On the other hand, it also produced lipopeptides such as fengycin and iturin that inhibit mycelium growth of R. solani and can also damage this pathogenic fungus. This work will provide novel guidance in plant protection development.In conclusion, this study gives a preliminary explanation to prevention mechanism of SL-44 against wilt disease caused by All datasets generated for this study are included in the article/supplementary material.ZW was conceived and supervised the study as corresponding author. YH designed and performed the experiments in a major part and wrote the manuscript. YL and JD assisted for performing experiments and analyzing data. XL revised the important academic content of the article. CL assisted on supervising the study. All authors approved the final article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The design, while strengthening the concrete structure, also aims to monitor common SHM parameters such as strain and cracks. This was achieved by weaving the carbon fiber together with optical fiber, based on a specialized technique that uses an embroidery setup where both the carbon and optical fiber are woven on a water dissolvable polymer substrate. The performance of the sensing scheme was characterized in-situ utilizing the OFDR based technique and the results presented. The sensors embedded on a custom made concrete block were subjected to varying strain via a three point bending test to destruction and the results discussed. The intended dual-achievement of the scheme thus proposed in SHM and strengthening the C3 is demonstrated. The suitability of the OFDR scheme for C3 is combined with a fibre Bragg grating (FBG)-based approach, and discussed in detail.An Optical Frequency Domain Reflectometry (OFDR) based fiber optic sensor scheme “embedded” in concrete for the purpose of structural health monitoring (SHM) of carbon concrete composites (C This arises from the fact that CRC possesses a considerable amount of advantages over SRC, such as light weight, longer lifespan, flexibility (easier to mould), efficiency and immunity to risk of corrosion [The project Carbon Concrete Composite (Corrosion . In addiThe use of fiber optic sensors (FOSs) for Structural Health Monitoring (SHM) of reinforced concrete structures is state of the art and within this domain, FOSs are utilized mainly to monitor corrosion of the reinforcing steel and to measure the deformation of the reinforced concrete. For example, fiber optic strain sensors , moistur3 building cycle, both a new embroidery integration process for fabrication and the property of the bond between suitable OGF and carbon fiber reinforcement structures need to be investigated.The rapid uptake of carbon fiber reinforcement by Civil engineering creates a demand, but more of an opportunity, for smart sensors to be embedded in the structure itself for real-time measurement of the structural integrity. The direct integration of FOSs into carbon fiber structure for monitoring the condition of carbon fiber concrete, therefore, represents a new field of research. Compared to CFRP applications, when integrating FOSs in carbon fiber reinforced concrete structures, the highly alkaline environment in concrete and the concrete mechanical stress places special demands on the optical fiber as well as on the bond between optical fiber and carbon fiber reinforcement. Also, the optical glass fiber (OGF) can be destroyed and/or cracking can be experienced by concrete chemical stress induced micro defects on the surface of glass fiber resulting from manufacturing defects or stress corrosion. Thus, the mechanical properties of the OGF must be carefully evaluated in this context. Furthermore, the sensor region can be detached from the carbon reinforcement as a result of mechanical stress, or the composite can be slowly decomposed as a result of the highly alkaline environment in concrete. Thus, in order to ensure functionality across the CPrevious work by the authors  has focuThe FCS, as illustrated schematically in 2 have been fabricated using the embroidery technique reported above. Furthermore, the measurement of the spatial strain profile along the OGF was achieved based on an OFDR technique using Corning SMF28E XB fiber with acrylate coating and two Fibercore SM1500 fibres (with FBG sensors and acrylate coating) that had been integrated in the FCS, which is illustrated in In order to investigate the performance of the FOSs, FCSs with 1600 tex and dimension of 500 × 110 mm2) were placed in a mould with dimensions of 500 × 400 × 40 mm3, which was half filled with liquid concrete. After the FCS samples were fixed at the correct position, the mould was then completely filled with concrete. Following this, the liquid concrete blocks were shaken for five minutes to obtain an even distribution and the concrete was left to set for at least 28 days. After the concrete was complety set, the concrete blocks were seperated into two blocks, where each block incorporated a FCS sample. Furthermore, to protect the optical fiber in the FCS from breackage when integrating the FCS sample into the concrete, the optical fiber was protected at the FCS/concrete interface using plastic tubes and FC/PC pigtails were spliced to the endfaces of the fibers of the FCSs. The FCS sample before (a) and during embedding (b) is illustrated in Upon completion of the fabrication, the FCSs were embedded in concrete blocks in order to evaluate their performance. To do so, first, two FCS samples . A summary of the thus determined values of three exemplary samples is presented in The performance of the integrated OFSs was evaluated using a 3-point bending test rig, as illustrated in In order to test the concrete specimens under practical conditions, three bending cycles (between 500 and 1500 N) were first carried out with the help of the 3-point bending test rig. The three bending cycles at the beginning of the test procedure were performed in order to specify the hysteresis of the FOSs embedded in the FCS. Following the three bending cycles, the concrete specimens were tested until failure, i.e., breakage. An example of the strain and force applied to a concrete specimen as a function of time using the 3-point bending test is presented in The results of FBG sensor measurements from the FCS are shown in The result of the OFDR measurement is shown in In this communication, the sensor performance of FCS with integrated FOSs under practical conditions has been evaluated. To do so, the FCSs have been embedded in concrete blocks and the sensor performance was evaluated using a 3-point bending test. During embedding in concrete the FCS was closely monitored and only negligible attenuation variation of the light propagating in the optical fiber was detected, which can be attributed to connector tolerances. Moreover, for the evaluation of the FCS, an FBG technique was also applied in addition to the proposed OFDR-based technique. By using the FBG sensors, it could be verified that (i) the response of the FOSs embedded in the FCS has a linear response to applied strain of 0.44 nm/% with a relatively low hysteresis (0.011%) and that (ii) FCSs with two OGFs placed perpendicularly could in principle be used, for example, to discriminate between transverse and longitudinal forces and/or to compensate for temperature of the FOSs, depending on the positon of each OGF sensor relative to the load. Moreover, the OFDR technique verifies that the spatial strain profile of the optical fiber embedded in FCS can be successfully monitored within concrete elements. When performing the 3-point bending test the optical fibers utilized for OFDR measurements were loaded four times and the four load points were illustrated as four strain peaks. Furthermore, the OFDR readings verify that for large loads, the strain propagates from the load points along the FCS and thus causes a broadening of the measured strain peaks. Thus, for large loads the spatial strain resolution of the FCS is limited. Based on the obtained results, currently an appropriate theoretical model is developed in order to model the sensor performance of the FCS embedded in concrete.


The effect of silica fume (SF) in concrete on mechanical properties and dynamic behaviors was experimentally studied by split Hopkinson pressure bar (SHPB) device with pulse shaping technique. Three series of concrete with 0, 12%, and 16% SF as a cement replacement by weight were produced firstly. Then the experimental procedure for dynamic tests of concrete specimens with SF under a high loading rate was presented. Considering the mechanical performance and behaviors of the concrete mixtures, those tests were conducted under five different impact velocities. The experimental results clearly show concrete with different levels of SF is a strain-rate sensitive material. The tensile strength under impact loading of the tested specimens was generally improved with the increasing content of SF levels in concrete. Additionally, the tensile strength under impact loading of the concrete enhances with the increase of the strain rates. Finally, failure modes, dynamic tensile strength, dynamic increase factor (DIF), and critical strain are discussed and analyzed. These investigations are useful to improve the understanding of the effect of SF in concrete and guide the design of concrete structures. Concrete is currently the most commonly used construction and building material. Dynamic resistance is a fundamental basis for the evaluation of structural safety. The responses of concrete to transient dynamic loading (including compressive and tensile loading) are of interest in both academic and engineering fields, such as bridge construction, hydraulic engineering, constructional engineering, and so on ,2,3. To Due to the different mixing methods and amounts, the influence of SF on the mechanical properties and behaviors of concrete is quite different. Recent investigations have tried to improve the mechanical properties and behaviors of concrete by using SF for cement replacement. Pedro et al.  investig2 to 104 s−1. Many factors have obvious influences on the strain rate sensitivity of concrete. The strain rate sensitivities are mainly measured by strength or the strains at the maximum stress [Split Hopkinson pressure bar (SHPB) device is an effective technique to analyze and characterize the mechanical properties and dynamic behaviors of brittle materials at high strain rate. In recent years, researchers studied the dynamic mechanical properties of brittle materials, such as rock or rock-like materials ,21,22,23m stress ,35,36,37This study uses Brazilian disk (BD) specimens containing different levels of SF, using an SHPB test device, and proposes to enhance the understanding of SF in concrete on the mechanical properties under impact loading. At the same time, it intends to promote and evaluate the use of SF to replace cement in concrete. For those reasons, three series of concrete mixtures with different SF levels were produced and tested. The materials’ strains and derived testing strain rates were recorded by resistance strain gauges which were placed on the surface of elastic bars. The influences of the strain rates on the mechanical properties and dynamic behaviors of the tested specimens with different mixture proportions of SF were studied. This present study is organized as follows: 3. The supplementary cementitious materials for concrete are OPC and SF. The particle size of fine aggregates ranges from 0.5 mm to 2.5 mm with continuous gradation, of which the density is 2.64 g/cm3. The water is potable water. Coarse aggregates are natural crushed stones having rough surfaces and angular shapes. The maximum particle size of coarse aggregates is 10 mm, of which the density is 2.65 g/cm3. In order to study the influences of SF on the mechanical properties of concrete, the concrete was distributed into I, II, and III series. Details of the mix proportions of the three series of concrete are listed in During this experimental study, five kinds of materials were consumed: cement, fine aggregates, coarse aggregates, water, and SF. In this study, the cement is ordinary Portland cement (OPC), supplied by Anhui Conch Cement Company, Wuhu City, Anhui Province, China. The specific density of the OPC is 3.15 g/cmThe concrete was produced and cured in accordance with BS EN 12390-2  and EN 1Φ 75 mm × 37 mm for dynamic loading tests, and Φ 75 mm × 150 mm for quasi-static loading tests. Firstly, the cuboid-shaped concrete was cored into a cylindrical shape with a diameter of 75 mm and then cut into a Brazilian disk (BD) shape with a thickness of 37 mm. The BD specimens’ ends were ground to achieve the parallelism of the specimen surface. To determine the effect of SF in concrete, significant effort was made to distinguish the BD specimens with different SF content. Three specimens for each test were prepared to guarantee the reproducibility and reduce the discreteness of those experimental results. A total of 45 specimens  were prepared for high strain rates impact loading tests. Another 18 specimens  were prepared for quasi-static loading tests. Images of the concrete BD specimens is shown in The geometries of the concrete specimens are The operating principle of the SHPB equipment is based on one-dimensional wave transmission theory, which is satisfied with two hypotheses: the stress and strain propagate uniformly along the axis, and the inertia and friction effect of the specimen can be ignored. It is necessary to reduce the diameter of specimens to obey the rationality of the above assumptions. In the SHPB test, the size of the specimens is usually 75 mm with little fluctuation. Moreover, the concrete specimens usually contain aggregate particles. To ensure the reasonableness and accuracy of the mechanical properties, the minimum diameter of the specimens must not be less than three times the maximum aggregate size. In addition, from the perspective of reducing the inertia effect of the specimens, the length of the specimen should be as small as possible.Quasi-static loading tests were conducted by an MTS testing machine . Φ 74 mm-diameter straight taper variable cross-section SHPB device at Hohai University, Nanjing, China. The SHPB test device is comprised of the following parts: three elastic bars , power systems (including an air compressor and pressure vessel) which is propelled by a gas gun, buffer (energy-absorbing device), and data processing systems . The bullet’s velocities  can be captured by light-electric tachometers. The technical parameters and accuracy of the SHPB testing system is listed in Φ 74 mm-diameter SHPB system are presented in 2), Young’s modulus (GPa), and elastic wave velocities (km/s); 2); Dynamic loading tests under high strain rates were carried out by the SHPB test system. The experimental procedure was conducted by a quations :(1)σs(t)Before stress is uniformly reached, tested specimens can be fractured in SHPB tests. Therefore, modification of the incident pulse technique is required to match the elastic response. In this work, the pulse-shaping technique  was applied in the SHPB tests. The pulse-shaping copper disks can improve the stress wave shapes through attenuating high-frequency oscillations of the incident stress waves ,19. The 2), Young’s modulus (GPa), and wave velocity (km/s). In this experimental study, the specimens’ strain rates were controlled by changing the gas pressure of the SHPB device power system. Specimens were subjected to impact loadings under the gas pressure determined by 0.15 MPa, 0.2 MPa, 0.25 MPa, 0.3 MPa, and 0.35 MPa to obtain the dynamic tensile strength of the concrete containing different SF levels under a wide range of strain rates. Those gas pressures correspond to impact velocities of 5.88 m/s, 7.38 m/s, 9.26 m/s, 10.46 m/s, and 11.37 m/s.The experiments were conducted by an SHPB device with a pulse-shaping technique under five different impact velocities. Mechanical test results of the prepared BD concrete specimens are reported below and the experimental results are exhibited using the tables and figures in this section.During the dynamic loading tests, it is essential to distinguish which test is valid. Three necessary conditions were summarized by Chen et al. details can be seen in .Based on the impact velocities, tests were classified into five groups: e.g., Group 1, the launcher pressure is 0.15 MPa, corresponding to velocity = 5.88 m/s. Under the action of dynamic loading, failure will occur at a larger stress value compared with quasi-static loading. Stress–strain behavior is one of the important characteristics for concrete-like materials, which can reflect their strength and deformation properties during loading processes . In ordeThe mechanical properties and dynamic behaviors of brittle materials relate to the strain rate . TherefoThe dynamic increase factor (DIF) can be defined as the dynamic strength divided by the quasi-static strength. Usually, the DIF can be employed to describe brittle materials’ sensitivity to the strain rate similarly to dynamic compressive tests :(6)DIF=fain rate : (7)DIF=The relationship between the tensile DIF of three series of concrete and the strain rate is presented in The strain at the peak of stress is defined as the critical strain. Previous studies have shown that there are many disputes about whether the critical strain is related to the strain rate ,46,47. WAccording to studies from Bischoff and Perry ,49, withIn this study, the effect of SF in concrete on the mechanical properties and dynamic behaviors of concrete under impact loading were investigated. With SF replacing cement, a series of changes have taken place in the physical structure and chemical composition of concrete. SF is particularly recommended as an alternative to moderate amounts of cement to obtain high-performance concrete with better mechanical properties. The SF in concrete gives better results and performance on mechanical properties under dynamic tensile loading. The dynamic tensile strength of specimens increases with the increase of the strain rate due to the excellent physical and mechanical properties of SF, and the stress-strain behaviors of concrete have a significant sensitivity to the strain rate. From the above results, it is observed that strain rate sensitivity is one of the important factors affecting the performance of concrete under impact loading. The failure mode of specimens will change with the increase of the strain rate. However, the strain rate sensitivity of the critical strain has little relationship with concrete series. In addition, the impact stress with respect to the cracking of concrete is a major issue under dynamic loading, but difficult to attain. Using more advanced instruments to obtain the impact stress, and proposing the most plausible explanation, are the important targets for the next research step.


Aconite Lateralis Radix Praeparata (Fuzi) and Pinelliae Rhizoma (Banxia) are a combination often used to treat cardiovascular diseases in ancient and modern clinical practice. However, eighteen antagonisms based on traditional Chinese medicine (TCM) theory often abided against such combination therapy. Therefore, exploring whether coadministration of the two herbs can be used in adriamycin- (ADR-) induced cardiomyopathy and clarifying the potential mechanism could help to guide its clinical application. Echocardiography experiments revealed that either Fuzi, Banxia, or their combination had effect on ADR-induced heart dysfunction, while high dose Fuzi exerted positive inotropic effect associated with restored PKA levels. Moreover, low dose Fuzi significantly reduced QT/QTc prolongation, inhibited cardiac apoptosis, and upregulated protein expression of PKA. However, combination of Fuzi and Banxia greatly aggravated QT/QTc prolongation and cardiomyocyte apoptosis in ADR rats compared with each drug alone, which was accompanied by a marked decrease in PKA, pSer346 levels. Similarly, Banxia alone treatment promoted cardiac apoptosis and downregulated protein levels of PKA and pSer346. Additionally, high dose Fuzi treatment also produced proapoptotic effect. Taken together, our study has provided the first direct evidence that combination of Fuzi, a positive inotropic agent, with Banxia promoted cardiac apoptosis in an ADR induced rat model of cardiomyopathy, which may be associated with suppression of PKA/β2AR-Gs signaling. This study also provides scientific language for better understanding of the risks and limitations of combination of Fuzi and Banxia in clinical applications. Duality of toxic and efficacious performance is a nature of drugs, and there could be some undesirable effects in the process of curing disease; traditional Chinese medicine (TCM) is no exception . ClinicaAconite Lateralis Radix Praeparata (Fuzi) and Pinelliae Rhizoma (Banxia) are representative of incompatible pairs recorded in “the eighteen antagonisms”, a special rule for TCM incompatibility in formulas, which suggests strictly compatibility prohibition in clinical prescriptions because mutual antagonism results in an undesirable consequence . The rats were housed in a room with a 12 h light and dark cycle and controlled ambient temperature (22±2°C) and humidity (50-60%). They were allowed for ad libitum access to food and water. All the animal experiments were performed in accordance with approved guidelines specified by the animal ethics committee of Tianjin University of Traditional Chinese Medicine .−1) over a period of two weeks, with total cumulative dose of 15mg·kg−1 body weight. According to the account of Chinese pharmacopoeia document, the adult dose of Fuzi was 3-15g crude drug and the given dosage in rats was 5.4g/kg which was equivalent to 4-fold maximum clinical dose. In treatment group, rats were given low dose Fuzi , Banxia , Fuzi and Banxia combination , and high dose Fuzi  once a day concurrently with ADR during the 2-week period via intragastric administration (n = 15/group). Control and ADR animals received the vehicle  only.ADR-induced HF was generated as our previous work . ADR was3 – LVEDd3] – 13.6; stroke volume (SV) = LVEDV – LVESV; cardiac output  was determined by multiplying HR by SV. For each M-mode tracing, at least three consecutive cardiac cycles were sampled.Transthoracic echocardiographic determinations were conducted in the lateral decubitus position through the visualSonics Vevo 2100 with a 21 MHz transducer (Canada). Echocardiography was performed in anesthetized animals using isoflurane . Time-motion mode measurements were performed in the parasternal long-axis view. Some of the measured parameters included diastolic/systolic septal wall thickness (IVSd/IVSs), diastolic/systolic posterior wall thickness of the left ventricle (LVPWd/LVPWs), end-diastolic and end-systolic diameter of the LV (LVEDd/LVEDs), and end-diastolic and end-systolic volume of the LV (LVEDV/LVESV). The following parameters were determined: ejection fraction (EF) = (LVEDd – LVEDs) / LVEDd × 100%; LV mass = 1.04 × , rabbit anti-PKA polyclonal antibody , and rabbit anti-tubulin primary antibody . Blots were washed and incubated in peroxidase-conjugated goat anti-rabbit IgG . Blots were developed with the enhanced chemiluminescence method (ECL) following the manufacture's instruments  and then visualized by Vilber Fusion FX7 RT-ECL scanner . The levels of pSer346 and pSer from densitometry were normalized to β2-AR levels whereas others proteins were normalized to the tubulin levels.Equal amounts of protein  among groups. In ADR-injected rats, PKA and GRK2 were significantly decreased in rat as compared to vehicle-injected control rats (Figures As shown in Figures  Figures . Adminis Figures . By cont Figures . In addi Figures .β2AR-Gs signaling.In the present study, we reported for the first time that the incompatible pairs of Fuzi and Banxia treatment aggravated ADR-induced cardiomyopathy in a rat model. The main findings of this study included that combination of Fuzi and Banxia was detrimental against the improving effect of Fuzi or Banxia alone on ADR-induced prolongation of QT and QTc intervals. Fuzi and Banxia combined treatment greatly increased myocardial apoptosis and the underlying mechanism may be associated with the suppression of PKA/The utility of ADR antineoplastic agent in the clinic is compromised by the risk of cardiotoxicity . CurrentNowadays, Fuzi is widely used in the treatment for HF due to lots of efficacy such as improving the myocardial contraction, dilation of blood vessels, and increasing the blood flow . ConsistMultiple mechanisms are involved in ADR-induced HF. ADR-induced cardiomyopathy is strongly linked to an increase in cardiac oxidative stress, which subsequently activates apoptotic signaling leading to cardiomyocyte apoptosis , whereasβ2AR by increasing cAMP levels in HEK 293T cells [β2AR signaling, which indicated that cardiac inhibition of FB was more likely related to Banxia against the inotropic response of Fuzi, suggesting further the incompatibility of Fuzi and Banxia in ADR-induced cardiomyopathy. On the other hand, GRK2 has been implicated as a causal factor of HF in various experimental models [β1AR is by far the predominant AR subtype in the heart, where it promotes cardiac contractility, it comes as no surprise that most studies have focused on associations of the β1AR with HF [β1AR is associated with ADR-induced cardiomyopathy [It is now generally accepted that, as the major effector of cAMP, PKA is the master regulator of cardiac muscle contraction . We prev3T cells . Howeverl models , but itsl models , it is t with HF . Indeed,myopathy . HoweverImportantly, a recent study demonstrated that blocking directly the PKA pathway almost completely prevented cancer cell growth, especially of detached anoikis-resistant cells, indicating that PKA is a potential chemotherapeutic target to prevent cancer cell metastasis . In factβ2AR signaling. In conclusion, the present study provided scientific rational for incompatibility of Fuzi and Banxia, which helps to understand the risks and limitations when using incompatible pairs in clinical applications.Combination of Fuzi and Banxia antagonized the cardioprotection of Fuzi on ADR-treated rats, which might be related to the prolongation of QT and QTc intervals, the promotion of cardiac cell apoptosis, and inhibition of Gs-biased