Euphorbia bicolor (Euphorbiaceae) latex extract induced peripheral analgesia, partly via TRPV1, in hindpaw-inflamed male and female rats. The present study reports that E. bicolor latex extract also can evoke analgesia via reduction of oxidative stress biomarkers and proinflammatory cytokines/chemokines in a rat model of orofacial pain. Male and female rats were injected with complete Freund's adjuvant (CFA) into the left vibrissal pad to induce orofacial inflammation, and mechanical allodynia was measured by the von Frey method. Twenty-four hours later, rats received one injection of E. bicolor latex extract or vehicle into the inflamed vibrissal pad. Mechanical sensitivity was reassessed at 1, 6, 24, and/or 72 hours. Trigeminal ganglia and trunk blood were collected at each time point. In the trigeminal ganglia, ROS were quantified using 2′,7′-dichlorodihydrofluorescein diacetate dye, Nox4 protein was quantified by Western blots, and cytokines/chemokines were quantified using a cytokine array. AOPPs were quantified in trunk blood using a spectrophotometric assay. E. bicolor latex extract significantly reduced orofacial mechanical allodynia in male and female rats at 24 and 72 hours, respectively. ROS, Nox4, and proinflammatory cytokines/chemokines were significantly reduced in the trigeminal ganglia, and plasma AOPP was significantly reduced in the trunk blood of extract-treated compared to vehicle-treated rats. In vitro assays indicate that E. bicolor latex extract possessed antioxidant activities by scavenging free radicals. Together our data indicate that the phytochemicals in E. bicolor latex may serve as novel therapeutics for treating oxidative stress-induced pain conditions.Recent studies have reported that the transient receptor potential V1 ion channel (TRPV1), a pain generator on sensory neurons, is activated and potentiated by NADPH oxidase-generated reactive oxygen species (ROS). ROS are increased by advanced oxidation protein products (AOPPs), which activate NADPH oxidase by upregulating Nox4 expression. Our previous studies reported that  Pain is a major submodality of the somatosensory system that serves as a warning to alert the organism to actual injury or the threat of injury. However, pain also can develop in the absence of injury or continue following the resolution of injury leading to a transition from acute to chronic pain. Acute and chronic pain manifest as the development and maintenance of hyperalgesia and/or allodynia. The International Association for the Study of Pain defines hyperalgesia as an increased sensitivity to noxious stimuli, while allodynia is defined as an increased sensitivity to nonnoxious stimuli. The transition mechanism from acute to chronic pain is not entirely understood, and once chronic pain has developed, it is hard to treat without the long-term use of addictive opioid-based narcotics. The identification of nonopioid pharmaceutical targets is needed to improve chronic pain management.A potential target for chronic pain management may be managing the noxious effects of oxidative stress on peripheral sensory neurons. Patients with spinal cord injury and diabetic neuropathy , 2 suffein vivo ROS is attributed to advanced oxidation protein products (AOPPs), dityrosine-containing cross-linking protein products formed primarily because of oxidative stress [via NADPH oxidase 4-dependent ROS production leading to the development of hyperalgesia [Recent studies have reported that ROS may cause pain through activation of the transient receptor potential V1 ion channel (TRPV1), a pain generator in peripheral sensory neurons. It was reported that ROS, such as nitric oxide, can activate TRPV1 , 11 to ie stress . AOPPs ae stress , 15. Noxe stress . Indeed,ralgesia .α), interleukin 6 (IL-6), and interleukin 1 beta (IL-1β) [via TRPV1 as TNFα enhances capsaicin responses in sensory neurons through neuronal production of prostaglandins [α, IL-1β, and IL-6 increase neuronal excitability via TRPV1 [α receptors are coexpressed with TRPV1 in sensory neurons and IL-1β and IL-6 increase TRPV1 expression in sensory neurons via the extracellular signal-regulated kinase pathway [The development of hyperalgesia and allodynia also involves sensitization of sensory neurons by inflammatory cytokines and chemokines. Tissue injury induces the release of bradykinin, which leads to the release of tumor necrosis factor alpha (TNF (IL-1β) . Cytokin (IL-1β) –21. Thisglandins . Also, Tia TRPV1 , 23. Fur pathway , 25.Euphorbia species are known to possess phytochemicals with antioxidant and anti-inflammatory properties [Euphorbia bicolor (Euphorbiaceae) latex extract evokes long-lasting peripheral analgesia that does not involve opioid receptors but occurs in part through TRPV1, in a rat inflammatory pain model [Euphorbia species display antioxidant and anti-inflammatory properties and E. bicolor is able to induce robust analgesia, we hypothesized that E. bicolor latex extract evokes analgesia via downregulation of oxidative stress biomarkers and proinflammatory cytokines in a rat model of orofacial pain.Many operties –33. We pin model . Given tE. bicolor (Euphorbiaceae) plants were collected from prairies in Denton County, Texas, USA, and a voucher specimen was placed in the Texas Woman's University Herbarium. Fresh latex was collected from the stem, leaf, and inflorescence bracts and extracted in 80% methanol (1 : 40 w/v) at room temperature for two days. The extract was centrifuged at 3500 rpm for 20 minutes, and the supernatant was filtered through Whatman #54 filter paper and stored at -20°C for future use.μL of latex extract was mixed with 1.6 mL of 7.5% sodium carbonate and 2 mL of Folin–Ciocalteu reagent (diluted 10 times in deionized water). The reaction mixtures were incubated at room temperature for 1 hour. The absorbances were measured at 765 nm. A gallic acid standard curve was used to estimate the total phenolic content in the latex extract, and the results were expressed as mg gallic acid equivalents (GAE)/g of latex fresh weight (FW).Total phenolic content of the latex extract was determined by the Folin–Ciocalteu method . 400 μL et al. [μL of latex extract was mixed with 500 μL of 2% aluminium chloride prepared in ethanol and incubated at room temperature for 1 hour. The absorbances were measured at 430 nm. A quercetin standard curve was used to estimate the total flavonoid content in the latex extract, and the results were expressed as mg quercetin equivalents (QE)/g of latex FW.Total flavonoid content was determined by the method of Ordonez et al. . 500 μL μL of latex extract, 3 mL of 4% (v/v) vanillin-methanol, and 1.5 mL of hydrochloric acid were mixed and vortexed thoroughly and allowed to stand at room temperature for 15 minutes. The absorbances were measured at 500 nm. The total proanthocyanidin content of the latex extract was estimated using a gallic acid standard curve and expressed as mg GAE/g of latex FW.Total proanthocyanidin content was determined by the method of Aiyegoro and Okoh . 500 μL et al. [μL of supernatant was mixed with 1.5 mL of chloroform. The standard was prepared by adding 200 μL of 2000 μg/mL of linalool in methanol to 1.5 mL of chloroform, and serial dilutions were prepared in the range of 100 mg/200 μL-1 mg/200 μL. The sample and standard mixtures were vortexed thoroughly and allowed to stand for three minutes, after which 100 μL of sulfuric acid (H2SO4) was added. The sample mixture was incubated at room temperature for 1.5-2 hours in the dark, and the standard was incubated for 5 minutes at room temperature in the dark. A reddish-brown precipitate formed at the end of the incubation. The supernatants were decanted from both sample and standard mixtures, and the precipitates were dissolved in 1.5 mL of 95% (v/v) methanol and vortexed thoroughly until dissolved completely. The absorbances were measured at 538 nm using 95% (v/v) methanol as a blank. The total terpenoid concentration of the latex extract was estimated as linalool equivalents using a linalool standard curve.Total terpenoid content was estimated by the modified method of Ghorai et al. . The latin vitro assays were employed to determine the in vitro antioxidant and radical scavenging activities of E. bicolor latex extract. All assays were performed according to the original methods specified for each assay and modified by Basu and Maier [A series of nd Maier .μg/mL–100 μg/mL) of 1 mL latex extract were mixed with 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of 1% w/v K3Fe(CN)6. The reaction mixtures were incubated at 50°C for 20 min, after which 2.5 mL of 10% w/v trichloroacetic acid (TCA) was added and the mixtures were centrifuged at 3000 rpm for 10 min. 2.5 mL of supernatants was mixed with 2.5 mL deionized water and 0.5 mL of 0.1% w/v FeCl3. The results were presented as absorbances measured at 700 nm. Butylated hydroxylated toluene (BHT) was used as a positive control.Ferric reducing power of the latex extract was performed by the method of Oyaizu . Increaset al. [μg/mL-100 μg/mL) of 1 mL latex extract were mixed with 1 mL of ABTS solution. The reaction mixtures were incubated at room temperature for 7 minutes, and absorbances were measured at 734 nm. The results were presented as percent inhibition of ABTS radical calculated according to equation  (ABTS) scavenging activity was determined by the method of Re et al. . ABTS woet al. [μg/mL–100 μg/mL) of 0.05 mL latex extract were mixed with 2.95 mL of 0.1 mM DPPH in methanol, thoroughly vortexed, and incubated in the dark for 30 minutes. The absorbances were recorded at 517 nm, and results were presented as percent inhibition of DPPH radical calculated according to equation  radical scavenging activity was determined using a modified method of Gülçin et al. . Increaset al. [μg/mL-100 μg/mL) of 4 mL latex extract were mixed with 600 μL of 4 mM H2O2 solution prepared in 0.1 M phosphate buffer (pH 7.4). The reaction mixtures were incubated for 4 minutes, and the absorbances were recorded at 230 nm against blank solution containing latex extract without H2O2. The results were presented as percent inhibition of H2O2 radical calculated according to equation  of 1 mL latex extract were mixed with 2 mL of sodium nitroprusside prepared in phosphate-buffered saline (PBS). The reaction mixtures were incubated at 25°C for 150 min, after which 0.5 mL of Griess reagent was added to the incubating mixture. The absorbances were measured at 540 nm, and results were presented as percent inhibition of NO radical calculated according to equation (Nitric oxide (NO) scavenging activity was determined by the modified method of Balakrishnan et al. . Increas50 values, i.e., the concentration required to scavenge 50% of free radicals, were calculated by linear regression analysis. Pearson's correlations were performed between the IC50 values of radical scavenging activities and total phenolic, flavonoid, proanthocyanidin, and terpenoid contents of the latex extract.ICad libitum access to food and water. Rats were maintained in the animal facility for a minimum of five days before testing. All studies were conducted under the approval of the Texas Woman's University Institutional Animal Care & Use Committee and under the strict guidelines of Committee for Research and Ethical Issues of the International Association for the Study of Pain and Animal Welfare Act, implementing Animal Welfare Regulations, and the principles of the Guide for the Care and Use of Laboratory Animals. The experimenters were blind to the treatment groups during behaviour testing, and all rats were acclimated to the testing room and apparatus twenty-four hours prior to testing.A total of 70 male and 70 female (250-350 g) adult Sprague-Dawley rats  were separated by sex and housed in cages in a 12 : 12 hour light : dark cycle with Sensitivity to a nonnoxious mechanical (touch) stimulus was measured as the force to withdraw from contact with a blunt von Frey filament , as previously characterized . For thiμL complete Freund's adjuvant  into the left vibrissal pad to induce inflammation. Mechanical allodynia was then confirmed 24 hours following CFA injections. Rats received one injection of either E. bicolor latex extract  or vehicle  into the inflamed vibrissal pad. Mechanical allodynia was then reassessed at 1, 6, and 24 hours in males and 1, 6, and 72 hours in females. The latex extract concentration and time points were selected based on our previous report that the onset of extract-induced analgesia in male and female rats with hindpaw inflammation occurs at 6 and 72 hours, respectively [For the experiment, baseline mechanical sensitivity was recorded followed by injections of 50 ectively .n = 10 per time point, per sex, and per treatment) were injected with CFA into the left vibrissal pad and saline into the right vibrissal pad. At 1, 6, and 24 hours in males and 1, 6, and 72 hours in females, rats were rapidly decapitated under brief gas anesthesia . A separate control group of rats  received only one saline injection into the left vibrissal pad. 24 hours post saline injection, male and female rats were rapidly decapitated under brief gas anesthesia . Trunk blood was collected in BD Vacutainer® spray-coated K2EDTA collection tubes on ice . Immediately after collection, the cells were removed from plasma by centrifugation at 1000–2000 x g for 10 minutes. The separated plasma was then transferred into clean polypropylene tubes and stored at −80°C. The trigeminal ganglia (TGs) were bilaterally extracted from the same rats and either immediately used or stored at −80°C.A total of 60 male and 60 female rats (E. bicolor latex extract or vehicle (n = 10 per sex and per treatment) by the modified method of Witko-Sarsat et al. [μL of 1.16 M potassium iodide (KI) was added, followed by 20 μL of acetic acid after 2 min. The absorbance of the reaction mixture was measured at 340 nm against a blank containing 2 mL of PBS, 10 μL of KI, and 20 μL of acetic acid. The AOPP level was determined as μmol·L−1 chloramine-T equivalents by using a chloramine-T linear curve ranging from 0 to 100 μM.The level of AOPP was quantified in the plasma of male and female rats treated with either t et al. . For thiE. bicolor latex extract or vehicle (n = 4 per sex and per treatment) by the method of Chung et al. [2DCFDA), a reduced form of fluorescein used as ROS indicator. The nonfluorescent H2DCFDA is converted into the highly fluorescent 2′,7′-dichlorofluorescein when the acetate groups of H2DCFDA are cleaved by intracellular oxidation. H2DCFDA detects levels of hydrogen peroxide, peroxyl radicals, and peroxynitrite in dissociated cells and intact tissues [μL PBS for 30 minutes at 37°C. After 30 minutes, the background fluorescence was detected (Biotek's Synergy HT fluorimeter) at 485 nm excitation and 535 nm emission. After recording the background reading, H2DCFDA was added into each well at a final concentration of 10 μM. The plates were incubated for another 30 minutes at 37°C, after which the fluorescence was remeasured. The level of ROS was detected as the intensity of fluorescence after subtracting the background fluorescence. TGs from the saline-treated vibrissal pad were used as internal controls and data normalization.The level of ROS was detected in the TGs isolated from male and female rats treated with either g et al. , using a tissues . FreshlyE. bicolor latex extract or vehicle (n = 4 per sex and per treatment) were homogenized in radioimmunoprecipitation (RIPA) lysis and extraction buffer containing Halt protease inhibitor cocktail  to prevent proteolysis. Tissue homogenization was performed at 6.5 m/s, 3 times for 10 seconds each, in a FastPrep®-24 homogenizer . Homogenates were centrifuged at 13000 rpm for 15 minutes at 4°C. The protein concentration was determined by using Pierce BCA™ protein assay kit. Protein lysate (30 μg) was resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer onto a polyvinylidene difluoride (PVDF) membrane  using Bio-Rad's Western blot unit. Membranes were blocked with 5% bovine serum albumin (BSA) in TBS-Tween 20 and probed with monoclonal anti-Nox4  overnight, followed by incubation with secondary antibody  at room temperature for one hour. To confirm the selectivity of the antibody, blots were preincubated with blocking peptide  for Nox4. Immunoreactive bands were detected with a Licor Odyssey imaging system. Beta actin was used as an internal control to normalize protein expression, and the percentage of β-actin was calculated based on at least four independent experiments.TGs isolated from male and female rats treated with either μg of protein extracted from vehicle- or extract-treated TGs from male or female rats  was mixed with a cocktail of biotinylated detection antibodies. The nitrocellulose membranes were blocked according to the manufacturer's protocol. The protein and antibody mixtures were incubated with the membrane containing immobilized antibodies for 29 rat cytokines. Bound protein was detected with streptavidin conjugated to horseradish peroxidase (HRP). Membranes were washed and developed with chemiluminescent detection reagents. The cytokine/chemokine spots were detected with a Bio-Rad ChemiDoc™ MP imaging system.A Proteome Profiler Rat Cytokine Array Kit, column A , containing 4 membranes coated with 29 cytokines/chemokines  was probIn vitro antioxidant and radical scavenging activities were analyzed by ordinary two-way ANOVA followed by Bonferroni post hoc analysis with statistical significance tested at p ≤ 0.05. AOPP and ROS levels in saline- and CFA-treated animals were analyzed by the Student t-test with a statistical significance tested at p ≤ 0.05. Western blot bands were quantified by densitometry using the Licor Odyssey imaging system Image Studio 2.0, and cytokine/chemokine levels were quantified by densitometry using ImageJ version 1.8.0 . Densitometry data were analyzed by two-way ANOVA followed by Bonferroni post hoc analysis with statistical significance tested at p ≤ 0.05.All data were analyzed using GraphPad Prism software version 7 . Behavioural data were presented as means ± SEM grams and analyzed by repeated measures two-way analysis of variance (ANOVA) followed by Bonferroni post hoc analysis. E. bicolor latex extract significantly contained more total phenolics (TPC), proanthocyanidins (TPrC), and terpenoids (TTC) than total flavonoids (TFC)  = 24.34; p ≤ 0.05)  = 902.7; p ≤ 0.05)  = 6729; p ≤ 0.05)  = 100787; p > 0.05)  = 0.34; p > 0.05)  = 2983; p > 0.05). ≤ 0.05) . The in  ≤ 0.05) . The ext ≤ 0.05) . Additio)  , while t > 0.05) . The lat > 0.05)  and was 50 values of the radical scavenging activities of the E. bicolor latex extract  = 10.71; p ≤ 0.05) quenched 50% of ABTS, DPPH, H2O2, and NO radicals , ABTS (18.2 ± 13 μg/mL), H2O2(50 ± 6.6 μg/mL) < DPPH (68.4 ± 3.8 μg/mL). ABTS radical scavenging activity showed high correlations with flavonoid (R2 = 0.866) and proanthocyanidin (R2 = 0.861) contents of the latex extract. The DPPH (R2 = 0.929) and NO (R2 = 0.975) radical scavenging activities of the extract showed high correlations with phenolics (R2 = 0.929) and terpenoids (R2 = 0.979). H2O2 radical scavenging activity showed low to negative correlations with polyphenolics, indicating that other phytochemicals are involved. NO radical scavenging activity showed positive and high correlations with phenolics (R2 = 0.975) and terpenoids (R2 = 0.999)  .E. bicolor latex extract significantly reduced orofacial mechanical sensitivity in male rats  = 38.6; p ≤ 0.05) at 24 hours  = 15.71; p ≤ 0.05) at 72 hours (p > 0.05).24 hours  and in f72 hours  as compat = 14.66, df = 18; p ≤ 0.05)   = 24.18; p ≤ 0.05). E. bicolor extract treatment significantly decreased the AOPP levels in males at 6 and 24 hours  = 212.8; p ≤ 0.05)  = 395.5; p ≤ 0.05)    = 0.02; p > 0.05). Extract treatment significantly decreased ROS levels in males at 6 and 24 hours  = 63.43; p ≤ 0.05)  = 12.36; p ≤ 0.05)    = 1.96; p > 0.05). Extract treatment significantly decreased Nox4 expression at 24 hours posttreatment in male rats  = 36.24; p ≤ 0.05)  = 23.03; p ≤ 0.05)   and fema ≤ 0.05)  rats as  ≤ 0.05)  and at 1 ≤ 0.05) . CFA inf df = 6)  and fema df = 6)  rats com ≤ 0.05)  and at 7 ≤ 0.05) . Further df = 6)  and fema df = 6)  rats com ≤ 0.05)  and at 7 ≤ 0.05) .E. bicolor latex extract significantly downregulated several proinflammatory cytokines , chemotactic cytokines (MIP-1α and MIP-3α), and proinflammatory chemokines  and upregulated an anti-inflammatory chemokine (TIMP) in the trigeminal ganglia 1 hour post E. bicolor latex extract treatment compared to vehicle controls  = 125.2; p ≤ 0.05) (α) and proinflammatory chemokines  were significantly downregulated compared to vehicle controls  = 256.7; p ≤ 0.05)  = 12.64; p ≤ 0.05)  . At 6 ho ≤ 0.05) . At 24 h ≤ 0.05) .E. bicolor latex extract significantly downregulated several proinflammatory cytokines  and proinflammatory chemokines (MIG) in the trigeminal ganglia 1 hour post E. bicolor latex extract treatment compared to vehicle controls  = 189.5; p ≤ 0.05)  = 0.85; p > 0.05)  . No sign > 0.05)  or 72 hoEuphorbia possess antioxidant and anti-inflammatory activities [E. bicolor as a potential phytomedicine. Here, we report for the first time that E. bicolor latex extract (1) possesses in vitro antioxidant and free radical scavenging activities; (2) reduces orofacial mechanical allodynia in both male and female rats; (3) downregulates the oxidative stress biomarkers AOPP, ROS, and Nox4; and (4) alters the expression levels of pro- and anti-inflammatory cytokines/chemokines.Pain and inflammatory conditions are treated mainly with opioids and nonsteroidal anti-inflammatory drugs (NSAIDs), which are responsible for adverse reactions, such as gastrointestinal disturbances, renal damage, respiratory depression, and possible dependence –50. Thestivities –57, we hE. bicolor latex extract displayed in vitro free radical scavenging activities by inhibiting ABTS, DPPH, H2O2, and NO. The ferric reducing power of the latex extract was significantly more potent than that of the BHT standard. The DPPH radical scavenging activity of the latex extract was found to be significantly lower than ABTS scavenging activity. This could be explained by the fact that the latex extract contains more hydrophobic than hydrophilic phytochemicals with antioxidant properties, since the ABTS assay is more suitable for hydrophilic antioxidants while the DPPH assay is more applicable for hydrophobic antioxidants [2O2 radical scavenging activities. Therefore, the differences in the correlations between IC50 values and polyphenolic contents explain why DPPH, H2O2, and NO radical scavenging activities of the extract are not as potent as their corresponding standards.oxidants . In addiE. bicolor latex extract significantly reduced orofacial mechanical sensitivity in both male and female rats at 24 hours and 72 hours, respectively, which correspond to the same onset time of analgesia observed in our previous studies on hindpaw-inflamed male and female rats [Sida cordifolia [Hyptis pectinata [Hyptis fruticosa [Ocimum basilicum [Acmella oleracea [Syzygium cumini [The orofacial region is innervated by the trigeminal nerves and is one of the most densely innervated regions of the body making trigeminal system-associated pain, such as migraine, headache, temporomandibular joint disorder, and trigeminal neuralgia, difficult to manage. In the present study, ale rats . In suppale rats . For exardifolia , Hyptis ectinata , 62, Hypruticosa , Ocimum asilicum , Acmellaoleracea , and Syzm cumini  reduce ovia activation of the MAP kinase signalling pathway, which further confirms the antioxidant properties of estrogens [Elevated AOPP levels are linked to many diseases, such as cancer , chronicstrogens . TherefoE. bicolor latex extract reduced plasma AOPP levels in male rats at 6 and 24 hours and in female rats at 72 hours. Together, these data indicate that the antioxidant activity of E. bicolor latex extract correlates to the onset of analgesia but varies by tissue type and sex. Composite methanol extract of Aegle marmelos, Azadirachta indica, Murraya koenigii, Ocimum sanctum leaves, and Syzygium cumini fruits [Anthyllis henoniana flowers [E. bicolor latex extract on the AOPP levels in both male and female rats. To the best of our knowledge, this is the first study that reports the sex differences in AOPP level.Treatment with i fruits  and ethy flowers  also redet al. [E. bicolor latex extract significantly reduced CFA-evoked ROS levels in the trigeminal ganglia of both male and female rats and along the same time course as that for the AOPP level reduction. Other Euphorbia species also display antioxidant activity by reducing ROS levels. For example, Ricinus communis reduces ROS generation in macrophage cells [E. supina reduces ROS generation in fibrosarcoma cells [E. bicolor latex extract treatment decreased Nox4 expression in both male and female rats at 24 hours and 72 hours, respectively. The time point for decreased Nox4 protein expression corresponded to that of analgesia onset in both male and female rats, indicating that the extract-induced analgesia may be working at least in part by downregulating Nox4 protein. Since it has been reported that ROS can activate TRPV1 [E. bicolor evokes analgesia in part via TRPV1 [E. bicolor latex extract treatment reduces AOPP, Nox4, and ROS levels leading to reduced TRPV1 activity contributing to analgesia. Thus, E. bicolor latex phytochemicals can reduce oxidative stress effects on TRPV1, thus reducing pain.Generated ROS as a result of nerve injury or inflammation in peripheral tissues are known to induce pain behaviours –81. In oet al.  also repge cells  and E. sma cells . The proma cells . In rodema cells . Nox4 hama cells . In the te TRPV1 , 11 and ia TRPV1 , it is pE. bicolor latex extract on AOPP-mediated activation of TRPV1 via Nox4-dependent production of ROS. Other studies [et al. reported that stimulation of proalgesic transient receptor potential ankyrin 1 (TRPA1) channel led to the activation of NOX1/2 in the soma of trigeminal ganglion neurons [et al. found that capsaicin regulates Ecto-NOX1  expression through TRPV1 and ROS in human platelets [E. bicolor latex extract in different pain conditions.The present study focused on the effects of  studies , 87, 88  neurons . The TRPlatelets . Therefoα, TNF-α, CINC-2α/β, CNTF, IL-17, IL-1β, IL-13, TIMP-1, LIX, L-selectin, IFN-γ, and RANTES. Durham et al. showed that several cytokines/chemokines, including LIX, L-selectin, TIMP-1, and VEGF, were significantly upregulated in male rats at 2 hours post-TNFα injection [α, modulate the expression of other proinflammatory cytokines within the trigeminal ganglion, thus promoting sensitization of neurons [β and IL-17, which are known to directly activate nociceptors [α, at 6 and 24 hours postinjection correlated to a reduction in pain behaviour in male rats.ROS are also known to modulate proinflammatory cytokines –93. In tnjection . Similar neurons . In the iceptors , 97. Whiγ (MIG), macrophage inflammatory protein 1α (MIP-1α), and MIP-3α, play an important role in rheumatoid arthritis pathology [E. bicolor latex extract significantly increased tissue inhibitors of metalloproteinase (TIMP) level in the trigeminal ganglia of female rats at 72 hours. Others have reported that intrathecal injection of TIMPs significantly reduces neuropathic pain [E. bicolor latex phytochemicals may be effective against neuropathic pain and osteoarthritis.Chemokines, such as monokine induced by interferon-athology –102. In athology –105. No hic pain  and inhihic pain . TogetheE. bicolor latex treatment. In female rats, fewer cytokines/chemokines, such as CNTF, lL-1β, IL-1ra, IL-2, IL-3, IL-10, L-selectin, and MIG, were significantly altered at 1 hour posttreatment. While lL-1β was no longer detectable in males after 1 hour, it was present and lowered by E. bicolor treatment in females at 72 hours. During acute inflammatory responses, resident leukocytes present in the resting tissues modulate the levels of circulating cytokines and recruit blood leukocytes. Scotland et al. reported that the numbers of leukocytes present in the naïve peritoneal and pleural cavities were higher in female than in male rodents, indicating that increased leukocyte population in female rats may contribute to the recognition and elimination of infectious stimuli without recruiting neutrophils or producing excessive cytokines [The present study also reports a sexually dimorphic modulation of cytokines/chemokines by ytokines . The resE. bicolor latex extract (E. bicolor latex phytochemicals at the site of an injury significantly reduces oxidative stress by reducing AOPP and ROS levels and Nox4 protein expression in parallel with reduced orofacial mechanical allodynia in both male and female rats. Our previous study reported that E. bicolor latex extract induced long-lasting peripheral analgesia in both male and female rat model of inflammatory pain without involving the opioid receptors [E. bicolor latex phytochemicals may be effective at alleviating acute or chronic pain that is driven by nociceptor activation, oxidative stress, and/or inflammation.ROS are key players in the development of peripheral and central sensitization in various pain etiologies, including neuropathic, inflammatory, opioid-induced, and orofacial pain. We provide evidence for multiple nonopioid mechanisms that contribute to peripheral analgesia induced by  extract . We propeceptors . Togethe


Leishmania (L.) major, depends on an immune response with well-regulated cytokine signaling. Here we describe a pathogen-mediated mechanism of evading CXCL10, a chemokine with diverse antimicrobial functions, including T cell recruitment. Infection with L. major in a human monocyte cell line induced robust CXCL10 transcription without increasing extracellular CXCL10 protein concentrations. We found that this transcriptionally independent suppression of CXCL10 is mediated by the virulence factor and protease, glycoprotein-63 (gp63). Specifically, GP63 cleaves CXCL10 after amino acid A81 at the base of a C-terminal alpha-helix. Cytokine cleavage by GP63 demonstrated specificity, as GP63 cleaved CXCL10 and its homologs, which all bind the CXCR3 receptor, but not distantly related chemokines, such as CXCL8 and CCL22. Further characterization demonstrated that CXCL10 cleavage activity by GP63 was produced by both extracellular promastigotes and intracellular amastigotes. Crucially, CXCL10 cleavage impaired T cell chemotaxis in vitro, indicating that cleaved CXCL10 cannot signal through CXCR3. Ultimately, we propose CXCL10 suppression is a convergent mechanism of immune evasion, as Salmonella enterica and Chlamydia trachomatis also suppress CXCL10. This commonality suggests that counteracting CXCL10 suppression may provide a generalizable therapeutic strategy against intracellular pathogens.Clearance of intracellular pathogens, such as Leishmania (L.) spp. employ to suppress host chemokines: a Leishmania-encoded protease cleaves chemokines known to recruit T cells that fight off infection. We observe that other common human intracellular pathogens, including Chlamydia trachomatis and Salmonella enterica, reduce levels of the same chemokines, suggesting a strong selective pressure to avoid this component of the immune response. Our study provides new insights into how intracellular pathogens interact with the host immune response to enhance pathogen survival.Leishmaniasis, an infectious disease that annually affects over one million people, is caused by intracellular parasites that have evolved to evade the host's attempts to eliminate the parasite. Cutaneous leishmaniasis results in disfiguring skin lesions if the host immune system does not appropriately respond to infection. A family of molecules called chemokines coordinate recruitment of the immune cells required to eliminate infection. Here, we demonstrate a novel mechanism that  Leishmania have served as a paradigm  and 0.4 million cases of visceral leishmaniasis annually (VL)  cells and CD8+ cytotoxic T lymphocytes . CXCL10 is part of a family of highly homologous chemokines, including CXCL9 and CXCL11, which bind to and activate the CXCR3 chemokine receptor  to invasive Salmonellosis  gondii . GP63 cleavage of CXCL10 occurs throughout in vitro infection and abrogates CXCR3-dependent T cell migration. Furthermore, we observed CXCL10 suppression with other intracellular pathogens, including S. enterica and C. trachomatis, demonstrating that diverse intracellular pathogens have developed convergent mechanisms to suppress CXCL10.Here, we report that L. major manipulation of host immunity, we measured secreted levels of 41 cytokines following infection of lymphoblastoid cell lines (LCLs) with L. major. LCLs constitutively produce CXCL10, and incubation with L. major reduced CXCL10 levels by >90%  restored CXCL10 suppression  and CXCL11 , genetic relatedness (leishmania vs. viannia subgenera), and clinical manifestation  , L. donovani , L. venezuelenesis , and L. braziliensis . Therefore, CXCL10 suppression is found in Leishmania spp. encompassing diverse geographic origins, genetic backgrounds, and clinical manifestations.Finally, we tested whether this mechanism of immune evasion is conserved across t CXCL10  includinToxoplasma (T.) gondii, Plasmodium (P.) bergei, Salmonella (S.) enterica serovar Typhimurium, Chlamydia (C.) trachomatis, Mycobacterium (M.) marinum, Mycobacterium (M.) smegmatis, Staphylococcus (S.) aureus, and Cryptococcus (C.) neoformans. CXCL10 suppression of at least 80% was observed with two additional intracellular pathogens: S. Typhimurium and C. trachomatis. In contrast, other pathogens, including the extracellular pathogens S. aureus and C. neoformans, exhibited modest to no suppression of CXCL10  cells, CD8+ T cells, dendritic cells, and CD4+ Th1 cells. With the early upregulation of CXCL10 transcript  on chromosomes 28 and 31, in addition to the tandem array of gp63 genes on chromosome 10  cells after secondary infection, and antibody blockade of CXCR3 prevents recruitment of circulating CD4+ T cells to the site of infection , selecting for transfected cells with 1000 μg/mL Geneticin, and collecting highly expressing CXCR3 cells by FACS. Cells were maintained in RPMI 1640 media (Sigma) supplemented with 10% FBS, 1% Penicillin/Streptomycin, 0.23% Glucose, 10 mM HEPES, 1 mM Sodium Pyruvate, and 250 μg/mL Geneticin. The A2EN cell line was provided by Raphael Valdivia and maintained in Keratinocyte serum free media  supplemented with 10% heat-inactivated FBS, Epidermal Growth Factor 1–53, and Bovine Pituitary Extract.LCLs from the International HapMap Project , NR-48819], L. major Δgp63 , L. major Δgp63+1 , L. major Friedlin V1 [(MHOM/IL/80/FN) NR-48815], L. tropica [(MHOM/AF/87/RUP) NR-48820], L. donovani [(MHOM/SD/62/1S) NR-48821], L. venezuelensis [(MHOM/VE/80/H-16) NR-29184], L. braziliensis [(MHOM/BR/75/M2903) ATCC-50135]. Parasites were maintained at 27°C in M199 media , supplemented with 100 μ/ml penicillin/streptomycin, and 0.05% Hemin . Cultures were split 1:20 every 5 days into 10 mL of fresh culture media. To prepare parasites for infection, 8 mL of a 5-day-old culture was spun at 1,200 g for 10 min and washed with 5 mL of HBSS prior to counting promastigotes with a hemocytometer and resuspending at the indicated concentration.The following Leishmania major infections of LCLs and THP-1 monocytes, 1 × 105 cells were placed in 100 μl of RPMI 1640 assay media as described above, with no penicillin/streptomycin added. In the case of THP-1 monocytes, cells were then stimulated with 1 μg/mL of LPS derived from Salmonella enterica serovar Typhimurium S-form‘ . Finally, 1 × 106L. major promastigotes were added in 50 μL of RPMI 1640 assay media for a multiplicity of infection (MOI) of 10. Culture supernatants and cell pellets or mRNA were collected after 24 h of infection. For phorbol 12-myristate 13-acetate (PMA) differentiation of THP-1 monocytes, 1.2 × 106 cells were placed in 2 mL of complete RPMI 1640 media supplemented with 100 ng/mL of PMA for 8 h after which the RPMI media was replaced and cells allowed to rest for 36 h. Parasites were then washed and counted as described above and added at an MOI of 20. At 24 h post-infection, the culture supernatant was removed, spun at 1,200 g for 10 min to separate extracellular parasites, and stored at −20°C for downstream cytokine analysis. Cells were then washed 3 times with 1 mL of PBS followed by one additional wash with 2 mL of RPMI media to remove the remaining extracellular promastigotes. At 48 h post-infection the culture supernatant was collected and stored for downstream analysis. To assess intracellular CXCL10 in differentiated THP-1 monocytes, at 24 h post-infection, cells were removed from the plate by pipetting with cold PBS. The concentration of living cells was determined using 7AAD staining and counting cells with a Guava EasyCyte flow cytometer. Cells were then lysed with RIPA buffer supplemented with protease inhibitor tablet  and 10 μM 1,10-phenanthroline. For downstream mRNA extraction , cells were stored in 1 mL of RNAprotect  at −20°C.For Salmonella enterica serovar Typhimurium 14028s, Chlamydia trachomatis serovar L2, and Toxoplasma gondii (RH and Prugniaud A7) were performed as described previously  prior to infection with 400,000 bacteria per well. Cells were spun at 100 x g for 5 min prior to incubation at 33°C for 3 h, after which 50 μl of streptomycin in RPMI media was added for a final concentration of 200 μg/ml streptomycin with 10% FBS, and incubation was continued at 33°C for 24 h. Cell death was monitored by 7AAD staining and quantified using a Guava easyCyte HT flow cytometer. To harvest supernatants, LCLs were centrifuged at 200 x g for 5 min prior to removing supernatant and storing at −80°C prior to quantifying chemokines by ELISA.Screening GM18524 CXCL10 after infection with S. Typhimurium and C. trachomatis in LCL HG02647 was performed in 24 well-plate format. For S. Typhimurium infection, 5 × 105 cells were washed with antibiotic free RPMI assay media and plated in 500 μl of RPMI assay media prior to infection at MOI 30. At 1 h post-infection, gentamycin was added at 50 μg/mL to kill the remaining extracellular bacteria. At 2 h post-infection, gentamycin was diluted to 18 μg/mL to prevent killing of intracellular bacteria. For C. trachomatis infection, 2 × 105 cells were washed and plated in 500 μl of RPMI assay media prior to infection at MOI 5 followed by centrifugation at 1,500 g for 30 min. For S. Typhimurium infection of THP-1 monocytes, cells were washed once with antibiotic free RPMI assay media and resuspended at a concentration of 1 × 105 in 100 μl of RPMI assay media on a 96-well-plate. Cells were then treated with 1 μg/mL of LPS diluted in RPMI assay media or the equivalent volume of media and S. Typhimurium added at an MOI of 10. At 1 h post-infection, gentamycin was added at 50 μg/mL. At 2 h post-infection, gentamycin was diluted to 25 μg/mL. For C. trachomatis infection of A2EN cells, 1 × 105 cells were plated in a 96 well-plate the day prior to infection. C. trachomatis was added at an MOI of 5 and centrifuged for 30 min at 1,500 g. For all S. Typhimurium infections, culture supernatants were harvested at 24 h post-infection. For C. trachomatis infection, culture supernatants were collected cells at 72 h post-infection to assess cytokine production.Confirmation of suppression by L. major. The following cytokines and chemokines were included: eotaxin, MCP-1, MCP-3, MIP-1α, MIP-1β, RANTES, Fractalkine, IL8, IL10, IL12p40, IL12p17, IL13, IL17A, IL1RA, IL1α, IL4, IL6, 7, EGF, FGF2, FLT-3L, G-CSF, GM-CSF, PDGF-AA, PDGF-BB, VEGF, IFNα2, IFNγ, TNFα, TNFβ, sCD40L, IL15, IL1β, IL2, IL3, IL5, IL8, TGFα, and MDC (Millipore). Cytokines below or above the limit of detection were excluded from analyses.Cytokines were detected by ELISA, Luminex, western blot, or total protein as indicated. Luminex platform based assay for detection of multiple cytokines from Millipore was used to screen To interrogate the effect of GP63 on individual substrates, the following recombinant chemokines were used: human CXCL10 , mouse CXCL10 , human CXCL11 , human CXCL9 , human CXCL8 , and human CCL22 . To separate chemokines based on size, all chemokines except for CXCL9 (which antibody required non-reducing conditions) were incubated at 97°C for 10 min in SDS-loading buffer in the presence of βME prior to loading in a 4–20% bis-tris polyacrylamide gels and running at 120 V for 1 h. For total protein, the Colloidal Blue Staining kit  was used per manufacturer's protocol. For western blotting, protein was transferred to a PVDF membrane using a Hoefer TE77X semi-dry transfer system. LiCor Odyssey (TBS) Blocking Buffer  was used to block for 1 h at room temperature. The following primary antibodies were used to detect chemokines: human CXCL9 , human CXCL11 (MAB672-SP), human CXCL8 , human CCL22 . Primary antibodies were detected with IRDye secondary antibodies (Li-Cor) and developed using a LiCor Odyssey Infrared Imaging System . Relative protein quantification based on band intensity was performed using the FIJI gel analysis function  were designed using the L. major Friedlin reference gene 10.0480 which has 100% sequence similarity with the gp63-1 gene in L. major Seidman WT and Δgp63+1  kit. Incubation with TURBO DNase was extended to 1 h and repeated twice. RNA cleanup was performed with the RNeasy MinElute Cleanup Kit . Reverse transcriptase was carried out using 1 μg RNA per sample with the iScript Reverse Transcriptase Kit  in 20 μl. cDNA was diluted 1:5 prior to adding to PCR reaction with ITaq universal SYBR Green supermix , 50 nM of each primer, and 4 μl cDNA for gene targets or 1 μl cDNA for housekeeping gene in a final volume of 10 μl. To determine the relative expression of L. major WT, Δgp63, or Δgp63+1. After 12 h of pre-incubation, 600 μl of CXCL10/conditioned media mix was added to a 24 well-plate. Five hundred thousand Jurkat T cells stably transfected with CXCR3 were seeded onto the apical membrane of the 5 μm transwell insert , and allowed to incubate at 37°C for 4 h. The transwell insert was removed and the concentration of cells in the basal chamber determined using a Guava easyCyte HT flow cytometer (Millipore).RPMI 1640 media (Sigma) was supplemented with 2% FBS and CXCL10 at a starting concentration of 100 nM. This was pre-incubated at a ratio of 1:1 with conditioned media from either 5) were treated with 100 ng/mL PMA in 200 mL on a poly-D lysine treated 8-well-chamber slide for 8 h and subsequently allowed to rest for 1 day prior to infection. Cells were then infected at MOI 20 with the indicated L. major strain. At 24 h post-infection, media was removed and cells were fixed with 4% paraformaldehyde for 20 min. After washing 3 times with PBS, cells were incubated in blocking and permeabilization buffer  for 30 min at room temperature after which the blocking buffer was aspirated and replaced with 5 μM DAPI in PBS for 30 min. Cells were then washed three times with PBS prior to mounting. Images were acquired using a fluorescence microscope (Leica DM4B) with attached CCD microscope camera (Leica DFC3000g) at 400x total magnification. One field of view was recorded for each well and image quantification was performed by two independent experimenters using the cell counter plug-in in FIJI . One hour prior to transfection, media was replaced with fresh FreeStyle media. Transfection was performed with 2.5 total μg of endotoxin free plasmid DNA using the Lipofectamine 3000 Transfection Reagent Kit per manufacturer's instructions. Transfected HEK293Ts were incubated at 37°C for 48 h prior to harvesting culture supernatant and storing in polypropylene, low-binding tubes  at −80°C until use.L. major Fd sequence (Q4QHH2-1) was inserted with the Leishmania specific secretion signal and GPI anchor motif removed (V100-N577) , and contains C-terminal Myc and Flag epitope tags. For GP63, a codon optimized plasmid was obtained from GenScript on the pcDNA3.1/Hygro plasmid backbone. Following a kozak sequence and secrecon to enhance secretion Kozak, , GP63-1 CXCL10 exposed to GP63 for 5 h, along with a negative (untreated) control was delivered in PAGE loading buffer at an approximate concentration of 30 ng/uL. Mass spectrometry was carried out by the Duke Proteomics and Metabolomics Shared Resource. Molecular weight analysis of intact and cleaved CXCL10 from gel loading buffer was performed using a ZipChip CE system  coupled to a Q Exactive HF Orbitrap mass spectrometer (Thermo Scientific). Ammonium acetate was added to the sample to a final concentration 0.1 M, and 5 μL of the loading buffer was pipetted manually into a HR ZipChip. Capillary electrophoresis (CE) separation was performed at 500 V/cm with a 30 s injection in Metabolite BGE . Mass spectrometry used positive electrospray with 120,000 Rs scan, 500–2,000 m/z, 3e6 AGC target and 100 ms max ion injection time. Mass deconvolution was performed in Proteome Discoverer 2.2.Homo sapiens taxonomy selected, semitryptic specificity, along with fixed modification carbamidomethyl (C) and variable modifications oxidated (M), and deamidated (NQ). The results of the database searches were compiled into Scaffold v4 for curation. Using the search results as a spectral library, Skyline v4.1 was used to extract peak intensities for peptides which looked to be a part of the cleavage region (residues 74–91) or non-cleaved region (residues 48–68), in order to more definitively localize the specific cleavage location (Panoramaweb.org (https://goo.gl/4xsLsF).Tandem mass spectrometric sequencing of the cleaved and uncleaved fragments of CXCL10 after GP63 treatment, as well as an untreated control sample, were performed after gel separation on a 4–12% NuPAGE gel (Invitrogen). Gel bands were isolated after colloidal Coomassie staining, destained in acetonitrile/water, reduced with 10 mM DTT, alkylated with 20 mM iodoacetamide, and digested overnight at 37°C with 300 ng sequencing grade trypsin (Promega) in 50 mM ammonium bicarbonate at pH 8. Peptides were extracted in 1% formic acid and dried on a speedvac, then resuspended in a total of 10 μL 97/2/1 v/v/v water/acetonitrile/TFA. Four microliter of each sample was injected for analysis by LC-MS/MS using a 90 min, 5–30% MeCN LC gradient and a top 12 DDA MS/MS method with MS1 at 120 k and MS2 at 15 k resolution. The data files were searched on Mascot v 2.5 with the UniProt database (downloaded November 2017) and location . Intensit-test, one-way ANOVA, and two-way ANOVA with Tukey's post-hoc test were used as appropriate where indicated. The number of biological replicates (N) are indicated in the figure legend for each experiment and defined as follows. For in vitro cell culture and protein assessment each well of cells or chemokine prior to experimental manipulation (such as infection with parasite or addition of chemokine and/or inhibitor) was treated as a unique biological replicate. When technical replicates, repeated use of the same biological sample in a readout assay, were used they are indicated in the figure legend text and averaged values were combined into the single biological replicate prior to calculating statistics.All statistical analysis was performed using GraphPad Prism. Unpaired Student's All datasets generated for this study are included in the manuscript and/or All authors critically reviewed the manuscript and contributed input to the final submission. AA, DK, KG, and RR wrote the manuscript. AA, DK, RR, JS, SR, and JT contributed to strategy and project planning. AA, KG, ET, KP, AM, BS, JS, JT, RR, and DK carried out experiments and analysis.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.


Botrytis cinerea in lettuce. Spraying mature lettuce leaves, with inulin-type fructans derived from burdock or chicory was as effective in reducing grey mold disease symptoms caused by Botrytis cinerea as spraying with oligogalacturonides (OGs). OGs are well-known defense elicitors in several plant species. Spraying with inulin and OGs induced accumulation of hydrogen peroxide and levels further increased upon pathogen infection. Inulin and OGs were no longer able to limit Botrytis infection when plants were treated with the ethylene signaling inhibitor 1-methylcyclopropene (1-MCP), indicating that a functional ethylene signaling pathway is needed for the enhanced defense response. Soluble sugars accumulated in leaves primed with OGs, while 1-MCP treatment had an overall negative effect on the sucrose pool. Accumulation of γ-aminobutyric acid (GABA), a stress-associated non-proteinogenic amino acid and possible signaling compound, was observed in inulin-treated samples after infection and negatively affected by the 1-MCP treatment. We have demonstrated for the first time that commercially available inulin-type fructans and OGs can improve the defensive capacity of lettuce, an economically important species. We discuss our results in the context of a possible recognition of fructans as Damage or Microbe Associated Molecular Patterns. The concept of “Sweet Immunity” postulates that sugar metabolism and signaling influence plant immune networks. In this study, we tested the potential of commercially available inulin-type fructans to limit disease symptoms caused by  In a rapidly changing world, there is a need for high yielding, stress tolerant crops. Pest attacks account for an estimated loss of $20–40 billion worldwide . BesidesIn primed plants, plant immune signaling pathways are activated after pathogen infection, involving all the major defense phytohormones: jasmonic acid (JA), salicylic acid (SA), abscisic acid (ABA), and ethylene in a pathosystem-specific manner ,12,13. ISoluble sugar levels are tightly controlled by transporters and enzymes. Among them, invertases are able to irreversibly split Suc into glucose (Glc) and fructose (Fru) and play a central role in source-sink partitioning and plant development ,40. Thesin vivo was linked to pathogen resistance in plant species such as agave and wheat [Arctium lappa) roots (burdock fructooligosaccharides (BFOs)) proved to be effective in boosting plant resistance towards biotic attacks in pathosystems such as Nicotiana tabacum-tobacco mosaic virus and Cucumis sativus-Colletotrichium orbiculare [Lactuca sativa) against the model necrotrophic fungus Botrytis cinerea, able to infect more than 200 plant species [Here, we focus on fructans, which accumulate in approximately 15% of land plants. These oligo- and polysaccharides are synthesized by elongating the Fru part of Suc with Fru moieties via β(2-1) and/or β(2-6) linkages, resulting in three major subclasses: inulins (β(2-1) bonds), levans (β(2-6) bonds), and graminans (mixed bonds) . Fructannd wheat ,56 and ebiculare ,58. Thus species , and to Lactuca sativa var. Gisela) and Botrytis cinerea strain B05.10 [−1) was used to spray 45 day-old lettuce plants and inoculate them 3 days after priming with Botrytis. To confirm that the observed effect is not associated with impurities  3–6 inulin type fructans), present in our fructan formulation, BFOs priming was compared with the subsequent controls: fully hydrolyzed BFOs (H BFOs), BFOs dialyzed with a 1 kDa-cutoff membrane  (D BFOs), hydrolyzed and dialyzed BFOs (HD BFOs). As a positive control, oligogalacturonides (OGs) was selected, which is a well-characterized DAMPs (Damage-Associated Molecular Patterns) [−1. Conductivity tests estimated the molarity of a 5 g·L−1 BFOs solution between 50 and 55 mM. Thus, to take possible osmotic effects into account, sorbitol 50 mM was used as osmotic control. After priming, fully-grown sprayed leaves were detached and inoculated. Botrytis, but no positive results were found   at a conre found . Cichorium intybus) at similar concentrations. −1 still led to significantly decreased disease symptoms  derived from chicory  assay [Botrytis inoculation (uninfected) was sampled 1 DPI. Results are shown in 2O2 accumulation was detected and this trend was maintained at 1 DPP, indicating a steady production of H2O2 over the first 24 h. At 3 DPP, H2O2 levels in inulin samples decreased compared to the mock treatment, whereas H2O2 levels of OGs samples remained steady  assay . Leaves d steady A. After  control A. Botrytis infection [Botrytis in lettuce. To test this, we treated mock, OGs, and inulin-primed plants with 10 ppm 1-MCP (1-methylcyclopropene), a competitive inhibitor of ethylene signaling [p = 0.904, Mann–Whitney u-test), suggesting that ethylene signaling is not required for basal resistance in mature lettuce leaves against Botrytis. To test whether 1-MCP application may have a direct effect on Botrytis growth and sporulation, we fumigated PDA plates inoculated with Botrytis with 10 ppm 1-MCP and looked for differences in mycelial growth compared to untreated plates after 7 and 12 days. No differences were detected at either time-point tested (Botrytis growth on the plate. We tried the same experiment using 10 µM AVG (2-aminoethoxyvinyl glycine), an ethylene biosynthesis inhibitor which blocks synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC) [Botrytis. Since in lettuce, ethylene signaling was shown to be activated in response to nfection , we hypoignaling , for 24 t tested C, indicaid (ACC) . We obtaFru, Glc, and Suc content of leaf samples collected immediately before inoculation (0 DPI or 3 DPP) and 1 day post inoculation (1 DPI) were analyzed. To properly take into account the effect of priming, samples taken immediately before the priming event  were also collected. To distinguish between detachment and pathogen effects, uninfected control leaves were sampled at 1 DPI. Results are summarized in Botrytis. Results collected for CWI activity show that priming treatments had no effect on activity levels of invertases, but their activity was enhanced by the 1-MCP treatment in all samples, although with a smaller magnitude in the case of CWI after inulin priming  and insoluble  acidic invertases was measured in our samples in order to assess their involvement in priming and response to  priming A. At 1 D priming B. Import priming B. This s priming C. At 1 D priming D. A sign priming D. Our daBotrytis infection triggered Pro accumulation (γ-Aminobutyric acid (GABA) levels in leaf samples from the previous 1-MCP experiment were quantified. Results obtained at 0 DPI did not show differences between priming treatments for both the 1-MCP treated and untreated leaves, although a decreasing trend following 1-MCP treatment can be recognized in this respect A. Data cmulation D. 1-MCP mulation D. These Botrytis in lettuce  ,74, and reatment . Our datreatment ,76. Altoe-points . In gene cinerea . Thus, iwith OGs  is not aand bean ,80. ThisBotrytis resistance in Arabidopsis [Botrytis  [Botrytis resistance in pepper leaves in the absence of priming [Botrytis resistance was demonstrated in species such as Arabidopsis and tomato [Ethylene and JA are the most important phytohormones in modulating resistance against necrotrophs . Ethylenbidopsis , and our tomato) ,81,82. H priming . Taken t priming . JA invod tomato ,85. AlthThe changes in soluble sugars content after priming suggest that the OGs treatment triggers accumulation of Suc, Glc, and Fru in lettuce leaves A,C,E. ToThe interaction between ethylene and sugar metabolism represents an interesting topic in present day plant physiology, although the regulatory mechanisms may greatly depend on the source and sink status of the organs, the developmental stage, and on the species studied ,87. On tInvertases can modulate Suc/hexose balances. As such, they can greatly influence sugar metabolism and sugar signaling events. Our data on acidic invertase activities revealed no significant changes following priming treatments A,C, althChenopodium rubrum [Arabidopsis ein4 mutants [Botrytis [Ethylene negatively affects CWI activity in cell suspensions of m rubrum  and incr mutants , suggestBotrytis . AccordiBotrytis . Such a Botrytis , but aftBotrytis pathosystem, the GABA shunt was shown to be critical in replenishing the TCA (Tricarboxylic Acid) cycle and eventually maintaining cell viability during Botrytis infection in ABA-deficient sitiens mutants of tomato [2O2 may fulfil complementary roles, maintaining cell viability on the one hand and preventing rapid spreading of the pathogen on the other hand [2O2 accumulation, it will be interesting to dedicate future research to the link between ABA and fructan priming/perception. Interestingly, 1-MCP impairs GABA accumulation in OGs and inulin treated leaves after infection  were grown in a Conviron® chamber with a temperature of 21/16 °C (day/night), 12 h/12 h day/night light regime, light intensity of 170 µmol·m−2·s−1, and 80% RH (Relative Humidity). For experiments, fully mature leaves from 45 days old plants were used. Botrytis cinerea B05.10 strain was used in our trials [5 spores mL−1 with the help of a stereomicroscope Olympus 3000 .Lettuce plants  as described [For priming treatments, four mature leaves from each plant were selected, and sprayed with different solutions. From 4 to 6 plants per treatment were used. All the solutions were prepared in Milli Q water supplemented with 0.0001% Tween 20 as surfactant. Three days after priming, two 5 µL drops of the above mentioned d leaves . Inoculaescribed . Lesions2O2 was performed according to Daudi et al. [Detection of Hi et al. , with mii et al. . Anti-ethylene treatments were done with an ethylene perception inhibitor  and an ethylene biosynthesis inhibitor  24 h before inoculation. Lettuce plants were placed in airtight sealed Plexiglas boxes and administered with 10 ppm 1-MCP  for 24 h. Dry 1-MCP powder was wetted with water after sealing the boxes by gently shaking. For the AVG treatments, plants were sprayed 4 h before inoculation with 10 µM AVG  supplemented with 0.0001% Tween 20 as surfactant. Leaves were detached 24 h after the 1-MCP or AVG treatment and prior to the inoculation treatment.® anion and cation exchange resins. Rhamnose 20 µM was added as internal standard. The sample was then analyzed by HPAEC-IPAD Dionex 3000  according to Shiomi et al., [To extract and quantify Glc, Fru and Suc, a circular shape (3.5 cm diameter) of leaf material with an approximate weight of 200 mg was taken from primed and infected leaves. In the case of infected leaves, a circular leaf disc (3.5 cm diameter) surrounding the lesion was sampled. This material was ground in liquid nitrogen and then heated at 90 °C in 0.5 mL water, and subsequently passed through Dowex et al., . Arctium lappa) roots according to the protocol described by Hao et al. [BFOs were extracted from freeze-dried burdock  in 60 mL NaAc buffer pH 5 for 60 min at 40 °C. The solution was then boiled for 8 min at 90 °C and centrifuged . The resulting supernatant was diluted in 40 mL acetone and centrifuged . This step was repeated twice. Finally, the obtained pellet was washed two times in 80% acetone and dried with the use of a liofilizator .OGs were extracted from polygalacturonic acid sodium salt (Sigma). 800 mg of powder were incubated with 10 mg of pectinase extracted from 3, 0.01% Polyclar and 2 mM β-mercaptoethanol (β-ME). 1 µL of 200 mM phenylmethylsulfonyl fluoride (PMSF) was added to each sample. The homogenized solution was centrifuged at 13,000× g for 15 min, and pellet (CWI-enriched) and supernatant (VI-enriched) were separated. De-salting and invertase activity measurements were carried out as described [For VI and CWI activity assays, 50 mg of grounded lettuce leaf material was dissolved in 150 µL of extraction buffer 50 mM NaAc buffer at pH 5, with 10 mM NaHSOescribed .g for 5 min. 45 µL of supernatant was diluted with 30 µL of a 25 mM NorValine solution . GABA was then injected on a reverse HPLC system  and derivatized before column injection as O-Phtalaldehyde and separated on a YMC Triart C18 column with the use of buffer “A”  and buffer “B” (Acetonitrile:MeOH:water 45:40:15) with the following gradient: from 0 to 6 min = 96% A 4% B; from 6 to 18 min = 92% A 8% B; from 18 to 32 min = 85% A 15% B; from 32 to 50 min = 67% A 33% B; from 50 to 53 min = 100% B. GABA was detected with a fluorescence detector at λex = 230 nm and λem = 450 nm.GABA was extracted from 50 mg of ground lettuce leaf in 100 µL water and subsequently sonicated for 10 min. Samples were then centrifuged at 13,000× g for 5 min. The supernatant was diluted 10 times in water containing 20 µM serine . The sample was then vortexed and centrifuged at 13,000× g for 5 min, transferred to a vial with a conical glass insert and analyzed by HPAEC-IPAD  in order to determine the Pro content after separation on a CarboPac® PA100 anion exchange column. The flow rate was 0.25 mL·min−1. Pro was eluted with the following gradient: 9 mM NaOH from 0 min to 15 min; 0.5 M NaAc from 15 to 17 min; 90 mM NaOH from 17 to 23 min. Quantification was performed on the peak areas with the external standard, consisting of 20 µM Pro. Pro was extracted from 50 mg of ground lettuce leaf with 100 µL water and subsequently sonicated for 10 min. Samples were then centrifuged at 13,000× u-test for categorized data. Other data were analyzed by using one-way ANOVA followed by Tukey’s post-hoc test, or two-tailed Student’s t-test when appropriate. Disease scoring data were analyzed by using two-tailed Mann–Whitney 2O2 accumulation, sugar metabolism, and GABA dynamics, as well as the clear connections with ethylene signaling, opens new research avenues to address the role of fructans in the sweet immunity context.In this study, we show that inulin-type fructans are promising priming agents in the context of disease control, an observation that should stimulate research on fructans and on other carbohydrates as potential substitutes for pesticides use in field and postharvest applications ,28. In t


To underpin these data, microbial communities were sequenced and characterised across the four reactors. Dominant operational taxonomic units (OTUs) in the 100% RS unit were Bacteroidetes/Firmicutes, whereas the 30:70 RS:DM unit was dominated by Proteobacteria/Spirochaetes, suggesting major microbial community shifts occur with DM additions. However, community richness was lowest with 100% RS (despite higher specific yields), suggesting particular OTUs may be more important to yields than microbial diversity. Further, ambient VFA and VS levels were significantly higher when no DM was added, suggesting DM-amended reactors may cope better with higher organic loading rates (OLR). Results show that RS AD without DM addition is feasible, although co-digestion with DM will probably allow higher OLRs, resulting in great RS throughput in farm AD units.Anaerobic digestion (AD) uses a range of substrates to generate biogas, including energy crops such as globally abundant rice straw (RS). Unfortunately, RS is high in lignocellulosic material and has high to C:N ratios (~80:1), which makes it  a comparatively poor substrate for AD. Co-digestion with dairy manure (DM) has been promoted as a method for balancing C:N ratios to improve RS AD whilst also treating another farm waste and co-producing a potentially useful fertiliser. However, past co-digestion studies have not directly compared RS AD microbial communities with and without DM additions, which has made it hard to assess all impacts of DM addition to RS AD processes. Here, four RS:DM ratios were contrasted in identical semi-continuous-fed AD bioreactors, and 100% RS was found to produce the highest specific methane yields (112 mL CHThe online version of this article (10.1007/s00253-018-9243-7) contains supplementary material, which is available to authorized users. Anaerobic digestion (AD) uses a range of substrates to generate biogas, including food waste, energy crops, wastewater sludge and animal slurry. Lignocellulosic material, such as rice straw (RS), is not an ideal substrate as it is comparatively recalcitrant to AD and tends to generate lower biogas yields compared with many other crops  co-digestion with RS in bench-scale bioreactors fed at different RS:DM ratios. Reactor performance was monitored, and microbial communities were characterised using 16S rDNA amplicon sequencing to assess how eubacteria and archaea composition and diversity vary as a function of DM additions and RS co-digestion conditions.Four 2.5 L reactors with control towers were used as the AD units, each with working volumes of 2.0 L. The glass airtight-sealed reactors consisted of a heating jacket set to 37 °C, a biogas sampling bag and a paddle stirrer. Overall, the reactors were operated for 150 days of which the first 75 days were used to acclimate the reactors to different RS:DM mixes. The same reactors had been operated for over 250 days prior to this experiment at different RS organic loading rates (OLRs); therefore, microbial communities were already acclimated to RS before DM amendments were commenced.During acclimation with DM, the sludge inoculum was operated in draw-fill mode  with a 25-day hydraulic retention time (HRT) and an OLR of 1.0 g VS/L/day  fed oncTotal solids (TS), volatile solids , moisture content (MC), ash content (AC) and total C and N were analysed using American Public Health Association Standard Methods APHA . VFA ana4), gas samples were directly injected into a Carlo Erba HRGC 5160 GC-FID fitted with a HP-PLOT Q column maintained at 35 °C with hydrogen as the carrier gas and Atlas software. A seven-point calibration was performed before and after each analysis session by injecting neat standards spanning expected methane concentrations . All injections were made in triplicate and the standard calibration required a minimum R2 of 0.99. The volume of biogas was collected over time in 1.0 L Supel-Inert Multi-Layer Foil bags (Sigma Aldrich) before daily extraction using a 1 L gas tight syringe (SGE). Biogas and methane analysis was normalised to specific production (mL/g VS/day), corrected for moisture, standard temperature and pressure (STP) and headspace volume, according to the VDI Standard: 4630 . Biogas samples were collected at the same time of day and analysed immediately. To quantify methane content (% CHrd: 4630 .Samples for DNA extraction (2 mL each) were collected in triplicate from the reactors after 0, 37 and 75 days of operations after acclimation. Raw DM samples also were collected in triplicate to compliment the reactor samples. Samples were always stored at − 20 °C before further processing. For each sample, DNA was extracted following the instructions of the FastDNA SPIN Kit for Soil . Extracted DNA was assessed for purity and quantified using a Qubit 2.0 Fluorometer (Invitrogen) to ensure adequate quantity and quality prior to sequencing.Sequencing of extracted DNA (concentration of 1 and 10 ng/μL and a volume of 10–20 μL) was undertaken by LGC Genomics GmbH in Berlin, Germany. Analysis consisted of PCR amplification using universal forward primer (U341F) CCTAYGGGRBGCASCAG and universal reverse primer (U806R) GGACTACNNGGGTATCTAAT, targeting the V3-V4 16S DNA region . All statistical analyses and figure plots were conducted or developed using Microsoft Excel and Minitab 17 . PRIMER 7  was used to perform principal coordinates analysis (PCoA) and distance-based linear modelling (DistLM) of log-transformed, normalised, RS composition data.Analysis of variance (ANOVA) with Tukey comparison was used to compare mean CHAlpha diversity and beta diversity, based on weighted UniFrac distances, were calculated in QIIME 1.9.1. PRIMER 7  was used for principal component analysis (PCA), metric-multidimensional scaling (MDS), heat map development to visualise beta diversity, permutational analysis of variance (PERMANOVA) and for analysis of similarities (ANOSIM). To compare microbial diversity with performance data, RELATE, BEST and DistLM (distance-based linear model) were used for weighted UniFrac distances, employing Bray-Curtis distance (after square root transformation) . Group significant differences were compared in STAMP v2.1.3 using the 4 content, VS, pH and total VFA data were tallied and summarised  in biogas yields were apparent when comparing sequential HRTs of operating time. Biogas production ‘stability’ was achieved after the third HRT, after which specific gas yields, CH4 yields ranged from ~48 to 112 mL CH4/g VS/day. For both parameters, decreasing the RS:DM ratio resulted in significantly lower specific yields (p < 0.001). Further, percent CH4 content within biogas from RS100 and RS90 were ~51 and 48% CH4, respectively, which are significantly higher than in RS30 and RS70 .Mean specific biogas yields ranged from ~115 to 222 mL/g VS/day across reactors, whereas specific CHp = 0.003). RS30 also had the lowest mean VS residuals (4.7 g VS/L), significantly lower than the other three reactors . Differences in pH among reactors also were significant. RS30 had the highest mean pH (6.3), although all four reactors had pHs > 6.0. Differences in total VFA levels among reactors also were apparent; i.e. RS30 VFA levels were significantly lower than the other three reactors (mean of 97 ppm), which ranged from 401 to 506 ppm (p < 0.003). Although various VFAs were measured throughout the experiment, individual acids were not always present; therefore, only formic, acetic, isobutyric and propionic acid had adequate data to be statistically compared. Of these, propionic acid in RS30 was significantly lower (37 ppm) than the other three acids, which were between 246 and 319 ppm (p < 0.003). Time course data , significantly higher than the other three reactors, which ranged between 23 to 26% , HRT effects are apparent, albeit were not significant (7.3% at p = 0.276). The influence on beta-diversity of physiochemical operating conditions was shown by a significant 0.67 correlation  with pH (0.725), followed by %CH4, total VFAs, formic and isobutyric acid combined (0.724), providing the BEST correlations. DistLM analysis showed a range of variables were significantly related to beta-diversity, including biogas yield (p = 0.012), %CH4 (p = 0.039) and pH (p = 0.002).To determine whether the groupings and correlations seen in Fig. p < 0.001) followed by RS30, RS70 and RS90, although only RS100 had significantly lower OTUs than RS30 (p = 0.041). Chao1 estimations also showed that raw DM had significantly higher OTUs than any reactor (p < 0.001), which means it has a higher number of OTUs only sequenced once. Both these estimates show that as DM addition decreases, reactor community richness decreases . Shannon’s score considers both richness and evenness, and it was higher for RS30 and DM (5.6 and 5.5). All diversity scores observed here, even in the reactor without DM addition, were quite high , especially compared with Zhao et al.  through RS70 (35 OTUs) to RS30 (38 OTUs). The DM samples also had a higher number of rare OTUs as their predominant OTU total was the lowest at 26.RS100, RS90 and RS70 were generally similar in phyla and dominated by Methanobacterium and Methanosarcina always were the main methanogen guilds, including OTUs identified as 005 and 044/002, with abundances ranging from 2.2 to 3.5 and 1.1 to 2.4%, respectively. Interestingly, these two Archaea taxa were below detection limits in DM samples. Methanobrevibacter (OTU 052) was dominant in the DM samples (3.4%), but was lower than 0.2% in all the reactors.Methanogens were present in all reactors, ranging from 4.5 to 6.2% in terms of relative abundance. Highest abundances were seen in RS90 and the lowest in RS100. Proteiniphilum (OTU 012) was significantly more abundant in RS100 and RS90  than in RS30 (0.1% at p < 0.001 and 0.008). Anaerolineaceae abundance in RS100 and RS90 was 6.6 and 6.9%, lower in RS70 (4.4%), but significantly lower in RS30 (2.0% at p = 0.031 versus RS100 and 0.019 versus RS90). Christensenellaceae was significantly higher in RS90 (3.4% for OTU 033) than in RS30 (p = 0.007). Ruminococcus (OTU 083) generally had low abundance (~1%), but was significantly higher (p = 0.022) in RS100 (0.9% than RS30 (< 0.1%)). Rhodobacteraceae was highest in RS90 (1.6%) and lowest in RS30  with 0.8 and 1.0% in RS100 and RS70. Christensenellaceae (OTU 023) was 5.0 and 5.9% in RS100 and RS90, but was highest in RS70 (7.8%) and lowest in RS30 (0.1%).Spirochaetaceae (OTU 026) was highest in RS30 (11.3%), which was significantly higher than in the other reactors, versus RS100 (> 0.1%), RS90 (1.0%) and RS70 (1.2%) at ≤ 0.006. Clostridium (OTU 027) and Clostridiales (OTU 074) in RS30 (0.7 and 1.2%) were significantly higher than in any other reactor , implying that higher DM addition might result in slightly higher putative pathogen levels than at lower DM additions. Peptostreptococcaceae (OTU 015 & 016) were both < 1.0% in RS100, RS90 and RS70, but were significantly higher in RS30 at 2.8 and 2.4% .Relative abundance of 4 yields were significantly greater at higher RS:DM ratios, although actual %CH4 content in the biogas was fairly similar across DM additions. In contrast, increasing DM additions resulted in slightly higher pH, greater VS% removals and lower levels of VS and VFA accumulation. Lower specific CH4 yields were unexpected as co-digestion with manure is touted as a method for enhancing RS AD processes , which are six times higher than those used here. Therefore, at higher OLRs, the benefits of C:N balancing and extra nutrients may overweigh lower degradability of DM used for co-digestion. Therefore, we suspect the relative value of adding DM to RS AD systems depends on many factors, and specific CH4 yields with and without DM may differ dramatically depending on the specific RS and DM. To underpin results herein, 16S rDNA amplicon sequencing was performed to compare raw DM microbial communities with RS AD communities as a function of RS:DM in the reactor feed.However, DM addition did have positive benefits here that are reflected in non-methane AD performance data, which also was observed by Babaee et al. . Manure 4 production, which is consistent with higher RS in an AD unit generating higher gas yields and VFAs whilst lower RS leads to higher pH. In fact, ANOSIM and PERMANOVA show that RS:DM ratio was the dominant factor in determining reactor performance and also beta-diversity of the microbial community.As RS content in the reactor feed was decreased from RS100 to RS30, reactor performance moved from higher biogas and VFA production to more stable pH and increased VS% removal in the systems Fig. b. Furthe. We see this here in richness and evenness data on alpha-diversity as indicated by Chao1, OTU numbers and Simpson and Shannon Indices. As DM level in the feed was increased, more diverse communities were apparent , which do not use cellulose  showed a similar pattern, being more abundant with a higher RS feed. Xia et al.  was higher in RS90 than RS100 and was significantly higher than in any other condition, although there are few cultivated species of this facultative anaerobe (Paster Christensenellaceae that can degrade cellulose (Fontes and Gilbert Ruminococcus (OTU 083) was in relatively low abundance, but was significantly higher in RS100 than RS30, which may be explained by its association with cellulose hydrolysis and its potential use of cellobiose  and Clostridiales (OTU 074) also were more evident in RS30, which is interesting given they are both higher in raw DM and are prospective pathogens. The presence of Clostridium and Clostridiales in RS30 may simply be legacy of higher DM additions, which may suggest that if DM is too high, strains in DM of potential health concern may prevail in AD digestates. Peptostreptococcaceae (family) (OTU 015 and 016) also were high in RS30 at 2.8 and 2.4%. Li et al.  was highest in RS70 (7.8%). There is currently only one described species of Christensenellaceae, which was found to favour gut environments . Overall, this implies co-digestion AD systems with higher DM might cope better at higher OLRs due to greater fermentation versatility, although Zealand et al.


Fish gelatin hydrolysates have been shown to possess various biological activities due to their unique Gly-Pro-Y and Gly-X-Hyp sequences. In the current study, fish gelatin was extracted from non-extruded milkfish scale (FSG1) or extrusion-pretreated milkfish scale (FSG2); extracted gelatins were hydrolyzed with different combinations of Flavourzyme and Alcalase to give four different hydrolysates, namely: FSGH1 (FSG1 hydrolyzed with Flavourzyme), FSGH2 , FSGH3 (FSG2 hydrolyzed with Flavourzyme), and FSGH4 . The extrusion-pretreatment process enhanced the extraction yield of gelatin from fish scale. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared (FTIR) analyses showed the extracts FSG1 and FSG2 possessed characteristics of gelatin. Moreover, the physicochemical characteristics of FSGH1–FSGH4 were examined by analyses of their degree of hydrolysis, amino acid composition, UV spectrum, FTIR spectrum, molecular weight, and RP-HPLC profile. Additional biological functional analyses showed that all of the studied gelatin hydrolysates FSGH1–FSGH4 possessed antioxidant activity dose-dependently as revealed by DPPH scavenging, ABTS scavenging, and reducing power analyses. In addition, FSGH2 and FSGH4 showed higher angiotensin-I-converting enzyme (ACE)-inhibitory activity as compared to FSGH1 and FSGH3. Taken together, FSGH2 and FSGH4 showed high antioxidant activity and potent anti-ACE activity. Due to the potential antioxidant and antihypertensive properties of FSGH2 and FSGH4, further research is needed to explore their possible use as natural supplementary raw materials in food and nutraceutical products. ROS interact with one another in biological systems. The uncontrolled generation of ROS often correlates directly with molecular markers of many disease conditions including atherosclerosis, diabetes, inflammation, neurodegenerative diseases, and cancer [Reactive oxygen species (ROS) in the forms of superoxide anion  is an important aquacultured fish in the Indo-Pacific region, particularly the Philippines, Indonesia, and Taiwan [Large quantities of fish are harvested annually worldwide, and approximately 50% of protein-rich fish processing byproducts are discarded or used as fishmeal and animal feed without any attempt to recover the essential nutrients . The fisd Taiwan . Accordid Taiwan . The extSaurida spp.) scales ranged from 9.1–10.9% (dry basis), which are similar to that of FSG2. However, these investigations used a high temperature (70–90 °C) and a long duration (1–5 h) for the extraction of gelatin [The proximate compositions of raw milkfish scale on a wet weight basis are presented in  gelatin . Process−1 (amide I), 1541–1558 cm−1 (amide II), 1237–1239 cm−1 (amide III), 3304–3315 cm−1 (amide A), and 2922–2940 cm−1 (amide B) [−1 (amide I), 1558/1556 cm−1 (amide II), 1242/1240 cm−1 (amide III), 3350/3402 cm−1 (amide A), and 2935/2935 cm−1 (amide B) were present, indicating that gelatins were extracted by this process. The extracted FSG1 and FSG2 were further utilized for the preparation of gelatin hydrolysates.The physicochemical properties of the gelatin extracts FSG1 and FSG2 were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared (FTIR) analyses. The SDS-PAGE graphs of FSG1 and FSG2 presented in amide B) ,24,25,26amide B)  or diffeamide B) . In the 2 in polypeptide chains of collagen [Enzymatic hydrolysis is widely used in the food industry, and this technique normally does not involve the use of any toxic chemicals or solvents. Thus, in the present study, the extracted gelatins FSG1 and FSG2 were hydrolyzed using industrial food-grade proteinases, namely Alcalase and Flavourzyme, to obtain gelatin hydrolysates. Alcalase is an endoprotease that has been used in a number of studies involving gelatin/collagen hydrolysis owing to its broad specificity as well as the high degree of hydrolysis that can be achieved in a relatively short time under moderate conditions . Flavourcollagen . The maxcollagen . Here, acollagen . As showcollagen . The ordcollagen . The RP-collagen . Previoucollagen . Here, w50 values (concentration of sample capable of scavenging 50% of DPPH) of FSGH1, FSGH2, FSGH3, FSGH4, and vitamin C for DPPH radical scavenging activity were 800, 1280, 1790, 2100, and 10.9 μg/mL, respectively  were FSGH1 (70.4 ± 1.6 μM) > FSGH2 (66.8 ± 1.2 μM) > FSGH3 (56.8 ± 0.8 μM) > FSGH4 (53.2 ± 0.6 μM). A similar pattern was also found for the DPPH scavenging properties of these four gelatin hydrolysates. The reducing power of a compound is generally related to the presence of reductones (antioxidants), which exert an antioxidant potential by donating a hydrogen atom, thereby breaking the free radical chain [3+/ferricyanide complex to the Fe2+ form, and Fe2+ can be monitored by measuring the formation of Prussian blue at 700 nm. B. subtilis A26, Raja clavata crude alkaline protease extract, Alcalase, and Neutrase ranged from 0.25–0.85 at a concentration of 4000 μg/mL [In the present study, four gelatin hydrolysates FSGH1–FSGH4 were subjected to analyses with 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), and reducing power analyses to determine their antioxidant activities. The scavenging effects of FSGH1–FSGH4 and vitamin C (as a positive control) on DPPH free radicals are shown in ectively A. In gen80 μg/mL . Another20 μg/mL . These r complex ,41. The al chain . In the 00 μg/mL . Our res00 μg/mL .50 value of 0.002 ± 0.000 μg/mL, followed by FSGH2 with an IC50 value of 472 ± 12 μg/mL, FSGH4 with 547 ± 1 μg/mL, FSGH3 with 592 ± 0 μg/mL, and then FSGH1 with 762 ± 8 μg/mL. The observed results suggest that FSGH2 and FSGH4 possibly contained more potent ACE-inhibitory peptides than the other gelatin hydrolysates. Previous studies suggested that the difference in ACE inhibitory activity might be attributed to the differences in chain length and amino acid sequences of peptides as well as to their hydrophobicity [The ACE inhibitory activities of FSGH1–FSGH4 were investigated, and the results are shown in hobicity . Severalhobicity . In the hobicity . On the hobicity ,46. TherFish scale from milkfish was obtained from a factory of a local fishery in Tainan, Taiwan. Fresh fish scale was kept on ice and transported to the laboratory immediately. Fish scale was mixed with 0.1 N NaOH to remove non-collagenous proteins , and the2O until the final moisture content reached 27% as a preconditioning step. Extrusion cooking was performed using a model single-screw extruder  equipped with a screw diameter of 74 mm, a screw length-to-diameter (L/D) ratio of 3.07:1, and a rounded die opening (5 mm) at the end of the extruder. Heating of the barrel was controlled by an electric heating element jacketing the barrel and thermal probe, and the barrel temperature was set at 135 °C. The screw speed was kept constant at 360 rpm. The feed rate was set at 11.4 kg/h to ensure stable operation of the extruder. The extrudate was collected at the die end and kept at 50 °C in a hot air oven for 30 min to remove extra moisture. After the extrusion-cooking procedure, the extrudate was ground into fine particles (<20 mesh), sealed in aluminum foil bags, and stored at 4 °C for further extraction experiments.The collagen  in fish scale is tightly linked to hydroxyapatite , and botThe Lowry assay was performed according to a method described previously . Varying2O with a sample ratio of 1:10 (w/v) and shaken in a water bath at 50 °C for 1 h. The mixture was centrifuged at 10,200× g for 10 min, the supernatant was collected, determined its protein concentration by the Lowry method, lyophilized, and then two gelatins, namely FSG1 (non-extruded) and FSG2 (extrusion-pretreated), were obtained. The yield of gelatin was expressed as weight of protein extracted by hot water/weight of fish scale, dry basis, and calculated by Equation (1) below.The non-extruded fish scale powder or fish scale extrudate powder was soaked in ddHA detailed description of the extrusion-pretreatment of fish scale and extraction of gelatin from fish scale extrudate is depicted in The determinations of the moisture, fat, ash, and crude protein were performed using the following AOAC (1984)  procedurSDS-PAGE was performed according to the method described by Laemmli (1970) . A 4% st2. The FTIR spectra were obtained using a FT-730 spectrometer . The signals were automatically collected using 32 scans over the range of 4000 to 400 cm−1 at a resolution of 2 cm−1 and were compared to a background spectrum collected from the KBr alone at room temperature.Two milligrams of protein powder were ground evenly with approximately 100 mg KBr until particles measured < 2.5 μm in size. The transparent KBr pieces were made at 500 kg/cmBacillus licheniformis—is a liquid preparation of serine endopeptidase that has a broad specificity and can hydrolyze most peptide bonds with higher specificity for aromatic , acidic (Glu), sulfur-containing (Met), aliphatic (Leu and Ala), hydroxyl (Ser), and basic (Lys) amino acid residues [w/w)) or with Alcalase + Flavourzyme  and the ratio of protein to Flavourzyme was also 100:6 (w/w)) for 2 h at 50 °C in a batch reactor, heated at 98 °C for 10 min to inactivate the protease, then cooled by cold flowing water, and finally centrifuged at 1200× g for 15 min. The supernatant was lyophilized and the resulting hydrolysates were utilized for further experiments. For digestion of fish scale gelatin, two proteases, namely, Alcalase and Flavourzyme, were used. Alcalase—an alkaline bacterial protease produced from residues . Flavourresidues . GelatinThe DH of samples was determined by the OPA method  with sli2-To = Number of free -NH2 groups at 0 min of hydrolysis;NH2-Tx = Number of free -NH2 groups in the supernatant after x minutes of protease-catalysed hydrolysis for each experimental point;NH2-Total = Number of -NH2 groups resulting due to acid hydrolysis (complete hydrolysis of protein is assumed).NHA 0.01 g sample of protein was hydrolyzed in 0.6 mL of 6 M HCl in an evacuated and sealed tube at 110 °C for 24 h. The hydrolysate was dried at 65 °C under vacuum. Dry hydrolysate was dissolved in 2 mL of 0.25 M sodium citrate buffer (pH 2.2) and then filtered through 0.45-μm PVDF filters. A Shimadzu LC-10A high-performance liquid chromatography system  equipped with a dual-pump LC-10AT binary system , a fluorescence detector RF-10AXL , and a Shim-pack AMINO-Na column (100 mm × 6.0 mm) was used to conduct the analysis. Amino acid analysis was done using pre-column fluorescence derivatization with OPA. The individual amino acid content was based on the area of the corresponding peak on the elution curves of the samples and standards, as determined by software , and the sample’s amino acid composition was expressed on the basis of residues per 1000 total residues. The contents of methionine and cystine and/or cysteine were determined by HPLC analysis after converting them into methionine sulfone and cysteic acid, respectively  . DetermiThe UV absorption spectra of samples were studied following a method reported previously  with sliThe molecular weight analysis of the gelatin hydrolysates was conducted using a Superdex Peptide HR 10/30 column  with a Shimadzu HPLC system . The chromatography conditions were: eluent 0.02 M sodium phosphate and 0.25 M sodium chloride, at pH 7.20; flow rate 0.5 mL/min, sample concentration 1%; injection volume 45 μL; temperature 25 °C; and wavelength 214 nm. Standard proteins used for calibration of MW are aprotinin (6511 Da) and gly-gly-gly (189 Da).v/v) and solvent B contained TFA  in acetonitrile (ACN):double-distilled water . Both mobile phases A and B were filtered through a 0.45 μm nylon membrane filter and degassed prior to any analytical run. Peptides were first eluted with 100% solvent A for 2 min, followed by a linear gradient from 0 to 25% of solvent B for 40 min at a flow rate of 0.5 mL/min. The separation was monitored at a wavelength of 214 nm.The hydrophobicity of peptides from gelatin hydrolysates was studied using reversed-phase HPLC according to a previous method . A Shima® 6600 System  equipped with a nanoelectrospray ion source was used . The hydrolysates (dissolved in ddH2O) were desalted on a C18 trap column  for 4.5 min at a flow rate of 10 μL/min. The samples were then dried and redissolved in ddH2O containing 0.1% FA. Next, aliquots (10 μL) were injected using an autosampler. The sample was subsequently separated by a C18 resolving column  at a flow rate of 300 nL/min. The mobile phases consisted of water with 0.1% formic acid (A) and 100% acetonitrile with 0.1% formic acid (B), respectively. Separation of the peptides was accomplished using a linear gradient of 5% to 30% B over 90 min, followed by 30% to 60% for 6 min, and 60% to 90% for another 6 min. The mass spectrometer was operated in the information-dependent acquisition (IDA) mode in which the initial MS scan recorded the mass to charge (m/z) ratios of ions over the mass range from 350 to 1500 Da, and then the 20 most abundant ions were automatically selected for subsequent collision-activated dissociation. All MS/MS data were searched against the UniProtKB/Swiss-Prot database using the Mascot program . The following search parameters were used: “none” enzyme, oxidation (M), oxidation (HW), and deamidated (NQ); peptide mass tolerance ±0.05 Da; fragment mass tolerance ±0.03Da.An UltiMate 3000 RSLCnano LC Systems  connected to a TripleTOFcontrol represents absorbance of the methanol solution of DPPH without the sample and Asample is absorbance of the methanol solution of DPPH with tested samples.The DPPH radical scavenging activity was determined by a previously described method . In briecontrol represents absorbance of ABTS without the sample and Asample is absorbance of ABTS with tested samples. Trolox was prepared as a standard. A calibration curve of scavenging percentage against different concentrations of Trolox standard was prepared. The ABTS radical scavenging activity of samples was expressed as Trolox equivalent antioxidant capacity (TEAC), which represents the concentration (μM) of Trolox.The ABTS radical scavenging activity was determined according to a previous method . The ABTg for 10 min). Finally, 0.5 mL of the supernatant solution was mixed with 0.5 mL of ddH2O and 0.1 mL of FeCl3 (0.1%), and was then stood for 10 min. The absorbance was measured at 700 nm using an ELISA reader .Reducing power was measured according to a previous method . BrieflyThe ACE-inhibiting activities of samples were assayed by measuring the concentration of hippuric acid liberated from HHL according to a previous method , with sot-test or one-way analysis of variance (ANOVA), followed by Duncan’s Multiple Range tests. The differences were considered to be statistically significant if p < 0.05.Experiments were performed at least three times. All data are presented as mean ± standard deviation (SD). Statistical evaluation of data was performed by Student’s In this investigation, we found that an extrusion-pretreatment process increased the extraction yield of gelatin from milkfish scale samples. Moreover, FSG2  was more susceptible to enzyme digestion as compared to FSG1 , indicating that extrusion pretreatment enhanced the hydrolysis efficiency of gelatin hydrolysates. Application of these findings could be economically beneficial in the food and fishery industries. Four gelatin hydrolysates FSGH1–FSGH4 showed unique characteristics as revealed by degree of hydrolysis, amino acid composition, UV spectrum, FTIR spectrum, MW, RP-HPLC profile, and LC-ESI/MS/MS analyses. Biological functional analyses revealed that all of the studied gelatin hydrolysates FSGH1–FSGH4 showed antioxidant and anti-ACE activities. The gelatin hydrolysates obtained from combined enzyme digestion  had lower antioxidant activities than those obtained from digestion with Flavourzyme alone, indicating that the use of Flavourzyme alone to obtain gelatin hydrolysate with high antioxidant activity is feasible. Among FSGH1–FSGH4, FSGH2 and FSGH4 showed high anti-ACE activity, which might be attributed to their higher DH, lower MW, and higher hydrophobic amino acid contents. As such, these gelatin hydrolysates may have potential as supplementary raw materials in the food and nutraceutical industries. In addition, our findings provide a method of reusing fish byproduct waste, which might be of value in the fishery industry. Further in vivo experiments on FSGH2 and FSGH4 regarding their antioxidant and antihypertensive capacities are warranted.