The results show that the KOGM samples in groups III and IV could effectively remove ˙OH, In this paper, konjac oligoglucomannan (KOGM) was obtained with a hydrolysis rate of 56.24% by controlling the hydrolysis conditions. KOGM was passed through a 0.2 kDa dialysis bag, a 3 kDa ultrafiltration tube, and a 5 kDa ultrafiltration tube, creating samples with molecular weights of 0.2–3 kDa (IV), 3–5 kDa (III), and >5 kDa (II), respectively. The  Its main active component is konjac glucomannan (KGM), a water-soluble natural polysaccharide [Konjac is a perennial herbaceous plant belonging to the genus ccharide . KGM is ccharide ,3.The application of KGM is bottlenecked by its large molecular weight, high viscosity, and low solubility. Therefore, much attention has been paid to the degradation of KGM into konjac oligoglucomannan (KOGM), a functional oligosaccharide consisting of 2–10 monosaccharide units. KOGM can be obtained by degradation through enzymatic hydrolysis, chemical method, and physical method . StudiesRecently, it has been learned that KOGMs with different molecular weights differ in structure and biological effect. Ma et al.  demonstrin vivo antioxidant activity [in vitro antioxidant activity of these samples were evaluated in detail.In this paper, konjac gum is hydrolyzed with β-mannanase. By controlling the hydrolysis conditions, KOGM samples were obtained with a hydrolysis rate of 56.24%. Then, the KOGM samples with different molecular weights were separated through dialysis and centrifugal ultrafiltration. The composition, activity ,16,17,1822.1KGM (purity: 95%) was purchased from Wuhan Qingjiang Konjac Products Co., Ltd. β-Mannanase  was purchased from Mianyang Habio Bioengineering Co., Ltd. Mice  were purchased from Shaanxi Medical Laboratory Animal Center. Malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) detection kits were purchased from Shanghai Jianglai Biotechnology Co., Ltd. The dialysis bags were purchased from Shanghai Qiaoxing Trading Co., Ltd. The other reagents were all of analytical grade and produced domestically.2.22.2.1First, 5 g of konjac powder was evenly dispersed into 100 mL of 0.2 mol/L acetate buffer to obtain a 5% (w/v) solution. The solution was mixed thoroughly with β-mannanase (dosage: 30–150 U/g) to initiate the hydrolysis. The mixture reacted for 1–5 h in a water bath at pH of 3.0–7.0 and temperature of 30–70°C. After that, the mixture was taken out and boiled for 10 min to inactivate the enzyme and stop the hydrolysis. Then, the reaction solution was centrifuged at 4,000 rpm to remove the unreacted KGM. The supernatant was taken for a 0.2 h dialysis in a 0.2 kDa dialysis bag to remove monosaccharides and small molecule salts from the sample, creating sample KOGM I. Next, KOGM I was passed through a 5 kDa ultrafiltration tube, producing sample KOGM II with a molecular weight greater than 5 kDa and a dialysate with a molecular weight of less than 5 kDa. The dialysate was further filtered through a 3 kDa ultrafiltration tube, producing sample KOGM III with a molecular weight of 3–5 kDa and sample KOGM IV with a molecular weight of less than 3 kDa. The samples were freeze-dried for further use.2.2.2M1 and M2 are the total contents of reducing sugar before and after hydrolysis, respectively, and M is the content of the KGM.The content of reducing sugar was determined by the dinitrosalicylic acid colorimetric method . The KGM2.2.3To achieve the best degradation effect, it is important to optimize parameters for enzymatic degradation. Assuming all other factors as constants, the commonly used single-factor test examines only one factor, without considering the interaction between parameters. Hence, this test approach cannot judge whether multiple parameters are optimal. As a result, the orthogonal test was selected to determine the optimal parameters.According to the orthogonal test design, the KGM hydrolysis rate was taken as the index to be evaluated. Then, four parameters of enzymatic hydrolysis were optimized, including time (A), temperature (B), pH (C), and enzyme dosage (D). Each factor was tested at three levels . The res2.2.4Mn of KOGM was measured by quantifying the hydrolysate composition through high-performance liquid chromatography. The parameters were configured as follows: chromatographic column, Shodex NH2P-50 4E (4.6 mm × 250 mm); mobile phase, water:acetonitrile = 65:35; flow rate, 1 mL/min; column temperature, 35°C; injection volume, 15 µL; and detector, refractive index detector.The 2.2.52.2.5.1A0, A1, and A2 are the absorbances of the blank control, the sample groups and positive control, and 200 µL of ferrous sulfate solution as the reagent blank.The ˙OH removal capacity of KOGM was tested by the Fenton’s reagent method. First, 150 µL of the KOGM samples with different contents was placed in separate test tubes. Then, 300 µL of 1.8 mmol/L ferrous sulfate solution, 200 µL of 1.8 mmol/L sodium salicylate–ethanol solution, and 25 µL of 0.03% hydrogen peroxide were added in turn into each test tube. The mixture in each tube was shaken well and reacted for 30 min at 37°C. The absorbance of each solution was measured at the wavelength of 510 nm. Vitamin C (Vc) and distilled water were selected as the positive control and the blank control, respectively. The ˙OH removal rate was calculated using the equation , three KOGM experimental groups , and a positive control group (C). In each group, the number of male mice was equal to the number of female mice.The normal control group was fed with 0.25 mL of normal saline every day; the three KOGM groups were fed with 80 mg/kg  KOGM II, KOGM III, and KOGM IV, respectively, every day. The positive control group was fed with 50 mg/kg Vc every day.The mice were free to eat and drink water in the 2-week-long experiment. In the end, the mice were fasted for 12 h. The eyeballs were removed for blood collection, using heparin as an anticoagulant. The mice were killed through cervical dislocation. Then, the livers were taken out and made into 10% liver tissue homogenate. Finally, the SOD activity, GSH-PX activity, and MDA content were measured.Ethical approval: The research related to animal use has been complied with all the relevant national regulations and institutional policies for the care and use of animals.2.2.7n = 3) unless otherwise specified. Except that the orthogonal experiment was expressed by the mean, the others were expressed as mean ± standard deviation (SD). A one-way analysis of variance (ANOVA) was performed using SPSS version 19, and Duncan’s multiple range test was performed to determine the statistical differences among different samples. Differences at P < 0.05 were considered significant, and P < 0.01 was considered extremely significant.All experiments were performed in triplicate . After 2 h, the hydrolysis rate increased slowly (P < 0.05). The trend basically conforms to the kinetic features of the enzymatic reaction. In the beginning, the high concentration of substrate promotes the reaction in the direction of product formation; with the lapse of time, the hydrolysis rate stabilizes due to enzyme inactivation and product inhibition [As shown in hibition .3.1.2The effect of temperature on the hydrolysis rate was tested at time 2 h, pH 5, and an enzyme dosage of 90 U/g.P < 0.05) with temperature before reaching 50°C. This is because the enzymatic hydrolysis of konjac gum requires a certain activation energy. Meanwhile, high temperature makes konjac gum more soluble and KGM less viscous, thereby promoting the mass transfer of the reaction system. The hydrolysis rate peaked at 50°C. However, the enzyme became less stable and active at higher temperatures, causing a reduction in the hydrolysis rate.As shown in 3.1.3The effect of pH on the hydrolysis rate was tested at a time of 2 h, a temperature of 50°C, and an enzyme dosage of 90 U/g.As shown in 3.1.4The effect of enzyme dosage on the hydrolysis rate was tested at a time of 2 h, a temperature of 50°C, and pH 5.P < 0.05), because the enzymatic reaction is insufficient at the low enzyme dosage. The hydrolysis rate reached the maximum when the enzyme dosage reached 90 U/g substrate. The hydrolysis rate did not increase, despite a further increase in enzyme dosage; there was no more substrate left for the extra enzyme molecules to bind with.As shown in 3.29(34) orthogonal test of four factors and three levels. The results of the orthogonal test are listed in K is the sum of the levels under each factor and R = (Kmax − Kmin)/3 is the influence range of each factor.The effect of each parameter on the KGM hydrolysis rate was revealed by the above results of single-factor tests. On this basis, time (A), temperature (B), pH (C), and enzyme dosage (D) were selected for an LR values in 2B2C2D3: a time of 2 h, a temperature of 50°C, pH 5, and an enzyme dosage of 130 U/g.Based on the 3.3Mn was measured  shows that the molecular weight of konjac powder decreased after enzymolysis. The Mn of KOGM IV, which was obtained through 3 kDa centrifugal ultrafiltration, was 773. Hence, KOGM samples with different molecular weights can be acquired efficiently and quickly from KGM through β-mannanase hydrolysis and ultrafiltration.Under the optimal parameter combination, the hydrolysis rate of konjac powder was 56.24%. After enzymolysis, each konjac powder solution was subjected to ultrafiltration and then measured . The res3.43.4.1y = 0.3554x2 + 0.5407x + 27.24 (R² = 0.987). Thus, the IC50 of KOGM IV for the ˙OH was 7.2779 mg/mL.The ˙OH, as the most active radical, causes serious damage to adjacent biomolecules . As show3.4.2y = −1.1857x2 + 23.674x − 18.64 (R² = 0.987). Thus, the IC50 of KOGM IV for the DPPH˙ was 3.5199 mg/mL.DPPH˙ is a stable lipophilic free radical, which has been widely used to evaluate the antioxidant activity of food and pharmaceutical raw materials . As show3.4.3y = −0.0544x2 + 3.5166x + 1.7321 (R² = 0.9852). Thus, the IC50 of KOGM IV for the Despite its inactive nature, the body . As show3.5The SOD and GSH-PX are the most important antioxidant enzymes in the body. They can remove free radicals on time and protect the body from oxidative damage. The MDA is a lipid peroxide produced under the imbalance between the oxidation system and antioxidant system of the body. The growth in the MDA will damage the cells. The MDA has often been used to measure the lipid peroxidation damage .As shown in 4Although there have been many reports about the preparation of KOGM by degradation of KGM, most of them are studies on the degradation process by chemical, physical, and biological methods. There are few studies on the controllable degradation of KGM and the preparation of KOGMs with different molecular weights, and the molecular weight has a great impact on the efficiency of KOGM. In this paper, KGM was hydrolyzed by β-mannanase to prepare KOGM, and the best preparation process was obtained through the study of various factors of enzymatic hydrolysis. At the same time, through the orthogonal experimental analysis of various factors, it was found that the time of enzymatic hydrolysis had a significant impact on the enzymatic hydrolysis, so the controllable degradation of KGM could be realized by controlling the hydrolysis time. At the same time, three kinds of KOGMs with different molecular weights were obtained by centrifugation, dialysis, and ultrafiltration, which is the basis for the follow-up study of the biological function of KOGMs with different molecular weights and further exploration of the preparation of KOGMs with high oxidation resistance.in vitro and in vivo. In vitro experiments showed that KOGM had the ability to scavenge free radicals, and its scavenging ability was positively correlated with the concentration and negatively correlated with the molecular weight of KOGM. This is because the high concentration and low molecular weight of KOGM molecular chain contain more reduced hemiacetal hydroxyl, which can make the highly oxidized hydroxyl free reduction [The metabolism of the human body produces a large number of free radicals. The free radicals of the body are in the dynamic balance of continuous generation and elimination. Once the free radicals in the body are produced too much or the body’s clearance ability is insufficient, the balance will be destroyed, which will cause adverse reactions such as aging and cancer . In thiseduction ; it has eduction .2O2 and O2, GSH-PX can reduce organic peroxide and H2O2 to H2O and nontoxic substances. If SOD and GSH-PX were present, the content of 2O2 would all decrease [in vivo, so that the activity of SOD and GSH-PX can be increased, and then the content of MDA can be reduced.In human body, SOD can reduce decrease . In thisin vitro and in vivo, and the relationship between antioxidant and molecular weight was obtained, which lays a foundation for the preparation of high antioxidant KOGM and edible health products in future.This study showed that KGM can be degraded in a controlled way and KOGMs with different molecular weights can be prepared by the enzymatic method. The antioxidative effect of KOGM was verified by experiments 5in vitro and in vivo tests, KOGM was proved to have certain antioxidant activity, which was negatively correlated with the molecular weight; the antioxidant activity of KOGM was weaker than that of the Vc. Our research shows that the enzymatic method can successfully prepare the KOGMs with different molecular weights and that KOGM with a low molecular weight is a natural antioxidant.In this paper, the KOGM samples were prepared by hydrolyzing konjac powder for 2 h at a temperature of 50°C, pH 5, and a β-mannanase dosage of 130 U/g. The samples were subjected to ultrafiltration, creating three kinds of KOGMs with different molecular weights. Through However, the composition of KOGMs with a low molecular weight is still very complex and requires further study in order to elucidate a specific antioxidant mechanism. At the same time, it is not enough to verify the efficacy only in mice, and the use of the relevant knowledge and equipment in the field of clinical medicine is needed for further study to verify its antioxidant effect.


A case-control study design was used to construct an explanatory set of management factors associated with long-duration bTB herd breakdowns; with a case (n = 191) defined as an investigation into a breakdown of 365 days or longer. Purchase of infected animal(s) had the strongest association as the most likely source of infection for long-duration bTB herd breakdowns followed by badgers and then cattle-to-cattle contiguous herd spread. However, 73.5% (95% CI 61.1–85.9%) of the herd type contributing to the purchase of infection source were defined as beef fattening herds. This result demonstrates two subpopulations of prolonged bTB breakdowns, the first being beef fattening herds with main source continuous purchase of infected animals and a second group of primary production herds  with risk from multiple sources.This study determined farm management factors associated with long-duration bovine tuberculosis (bTB) breakdowns disclosed in the period 23 May 2016 to 21 May 2018; a study area not previously subject to investigation in Northern Ireland. A farm-level epidemiological investigation ( Mycobacterium bovis is a zoonotic disease primarily affecting animals. Although cattle are the main hosts, the disease has been reported in many other farmed and wild animals [Bovine tuberculosis (bTB) caused by  animals . Departm animals . In 2018 animals .In 2016, a bTB eradication strategy for Northern Ireland was published , providiIn a previous publication , data frThe objective of this study was to identify farm-level management factors associated with prolonged bTB herd breakdowns, using data collected during on-farm epidemiological investigations. A case-control study design was used where prolonged duration bTB herd breakdowns (as defined previously ) were coTM . For our study, the 10 Divisional Veterinary Offices (DVOs) were aggregated into three groups according to their geographic location: south east group , west group  and north east group   were disclosed in any Northern Ireland cattle herd. Confirmation of bTB in an LRS was defined as a positive histological and/or bacteriological culture result following laboratory examination. Investigations were carried out by trained Animal Health and Welfare Inspectors (AHWI) who visited each of the bTB breakdown farms. At each farm, an on-site questionnaire was completed (Supplementary Table S2) through face-to-face interview of the farmer, including identification of all herds contiguous to the bTB herd breakdown. Based on the completed questionnaire and local knowledge of the area, the Veterinary Officer (VO) responsible for the bTB herd breakdown, where possible, determined the most likely source of infection for the breakdown. Questionnaire information along with data extracted from APHIS (herd size and location) was collated into Microsoft AccessMallusk) .Fig. 1.Case and control definitions were identical to those used in a previous study . Cases wTM (Microsoft Corporation) and R Version 3.4.0a and Stata/SE 15P ≤ 0.05 level.Microsoft AccessP ≤ 0.25 were then analysed using a multivariable logistic regression model. The resultant model was further refined to produce a reduced multivariable logistic regression model which utilised variables with P ≤ 0.05 from the first multivariable model. Following the fit of the reduced multivariable model, its estimated coefficients were compared to those in the initial multivariable model to determine if there was a magnitude change of >20%. This magnitude change known as Initial analysis was by univariable logistic regression. Any variables containing low numbers <10) of cases at any factor level, which could not have that factor level logically merged with another level were removed after univariable analysis. Remaining variables with  of casesP > 0.25 in the initial univariable analysis were also individually added back in to determine if they contributed to the multivariable model thus producing the preliminary main effects model. The only continuous variable included in the preliminary main effects model was herd size. Fractional polynomial analysis [P < 0.05); however, only those with probable clinical significance were accepted as potential candidates for the model. Interaction terms accepted into the final model had an odds ratio calculated as a linear combination with their associated main effects (β0 + β1 + β2 + β1.β2) and the results placed into Further to this, variables with analysis  was applA total of 2935 bTB herd breakdown investigations were completed during the study period. Supplementary Table S1 provides summary details of the 78 study variables for cases and controls. There were 126 ongoing bTB herd breakdowns at the end of the study period which were removed, leaving 2809 valid bTB herd breakdowns . The results from the final model  demonstrβ0 + β1 + β2 + β1.β2) are shown in The results of the linear combination of the five two-way interaction terms added to the model and their associated main effects . The odds ratio of a case for the baseline group and where there is an increase in herd size transformed by one had was 0.968 (95% CI 0.895–1.047), with cattle to cattle contiguous herd spread of infection source and herd size transformed increase by one was 1.046 (95% CI 0.993–1.173), with purchase of infected animal(s) source and herd size transformed increase by one was 1.119 (95% CI 0.993–1.261), with a carryover of infection source and herd size transformed increase by one was 1.002 (95% CI 0.865–1.161) and with badger infection source and herd size transformed increase by one was 1.071 (95% CI 0.954–1.203).P < 0.001). The odds ratio of a case where a farm purchases store cattle from a cattle market, there was an increase in herd size transformed by one was 1.050 (95% CI 0.950–1.160). vs. non-purchase.One of the key perspectives of this work is to further elucidate chronic long-duration bTB herd breakdowns (>365 days) through the provision of a quantitative characterisation of their infection source thus facilitating the formulation of a more focused disease control policy. Cattle movement has been identified as a consistent herd-level risk factor for bTB . In our Cattle purchase had the strongest association as the most likely infection source  followedet al. generalise the risk from badgers to indicators of badger density/activity [After purchase of infection, badgers formed the next most likely source of a long-duration bTB herd breakdown, which was a risk factor for bTB breakdowns identified in other studies –16. Skucactivity  and, in activity . The finA VO attributing badgers as the most likely infection source also had significant interaction with herd size  which shTwo badger-related variables excluded from the model due to low case numbers were fencing off of badger setts and fencing off of badger latrines; both of which are considered preventive measures. An important observation in this study was that almost one-third (30%) of farms were observed as having badger setts, but only 3% of investigations reported farms where badger setts and/or latrines were fenced off, an observation also reported in other Northern Irish work . WithoutThe third important source of infection was cattle-to-cattle contiguous herd spread. Previously, Denny and Wilesmith reported that in Northern Ireland, the two main associations with bTB breakdowns were the presence of badgers and contiguous neighbours who had confirmed bTB breakdowns  . They alThis study also investigated associations with other common diseases found on Northern Ireland farms. Application of fluke treatment was significantly associated with a reduced odds of developing a prolonged bTB herd breakdown. Co-infection with liver fluke may mask the true bTB infection status of animals making SICCT clearance of the herd difficult . Indeed,et al. stated that the influence of respiratory infections on the susceptibility to infection with M. bovis remains untested but speculated that such infections can facilitate increased aerosol spread [P = 0.054) and given that 73% of these prolonged bTB herd breakdowns were in dairy herds may suggest that other management factors confound this finding.Other disease-related variables investigated were use of IBR (infectious bovine rhinotracheitis) vaccination and leptospirosis vaccination. Skuce l spread . Our stuThe presence of registered pedigree animals on a farm was significantly associated with a reduced odds of developing a prolonged bTB herd breakdown. Association with this variable indicated that herds containing pedigree animals appear to be better at removing th infection. The presence of pedigree animals on a farm is probably indicative of a herd where trade and movement are important to the business, thus providing very strong motivation for a farmer to clear the infection from the herd and employ improved biosecurity measures.M. bovis in contaminated slurry [n = 17) in this category, it would require a guarded interpretation. The results show that herds located in the DVO north east group have the strongest statistical association to the prolonged breakdown case definition. Additionally, there is an interaction between DVO herd group and herd size , but wad slurry ) or witherd size  where reP < 0.05 was used for variable selection and that the final multivariable model contained 16 variables it should be realised that inclusion of at least one spurious association is a possibility. It is also possible with a study design where VOs select a breakdown source of infection there is potential for a degree of subjectivity. However, it is the trained VO with their local knowledge and standardised guidance who are best placed to make these assessments.Given that in this study, a statistical significance cut-off level of One of the central tenets of this work was investigation of disease source and its relationship to long-duration bTB herd breakdowns. The source with the strongest association to long-duration breakdowns was purchase of infection; however, as a source, it applies mainly to beef fattening herds. Beef fattening herds mostly move their stock to an abattoir with very few cattle movements to other herds. However, they do present a risk from the continuous output of infection to local wildlife and to other herds grazed contiguously. In order to reduce the input of infection to these herds, they must have the capability to risk assess their purchases  thus redIn terms of wildlife source, more effort must be placed into breaking the transmission links between cattle and badgers. This could involve an array of methods varying from those directly applied to the badgers through to methodical and efficient fencing of badger setts and latrines. Indeed this work shows that basic segregation methods to separate badgers and cattle using fencing are not being applied. The low levels of fencing off by farmers could be as a result of confused communication, occurring where one section of government responsible for bTB control promotes it as necessary, while another section implementing subsidy payments (Basic Payment Scheme) contradicts it by officially removing these fenced off areas from field maps, potentially affecting payment. It is thus essential that governments do not create contradicting messages with policy implementation and should conceive more effective ways of promoting best practice , such asThe other source shown to be linked to long-duration breakdowns was cattle to cattle contiguous herd spread and without effective biosecure boundaries between herds, this infection route will remain present. This again is an area where it should be possible for the government to intervene, incentivising the good practice of constructing biosecure boundary fences between neighbouring farms and penalising situations where poor fencing risks contiguous disease spread. Effective boundary fencing would form a necessary part of an overall biosecurity package aimed at the structural elements of the ongoing bTB problem.


Capsicum chinense Jacq. were identified as chrysoeriol (1), luteolin-7-O-glucopyranoside (2), and isorhamnetin-7-O-glucopyranoside (3). They had IC50 values of 11.6±2.9, 14.4±1.5, and 42.7±3.5 µg/mL against soluble epoxide hydrolase (sEH), respectively. The three inhibitors (1–3) were found to non-competitively bind into the allosteric site of the enzyme with Ki values of 10.5 ± 3.2, 11.9 ± 2.8 and 38.0 ± 4.1 µg/mL, respectively. The potential inhibitors 1 and 2 were located at the left edge ofa U-tube shape that contained the enzyme active site. Additionally, we observed changes in several factors involved in the binding of these complexes under 300 K and 1 bar. Finally, it was confirmed that each inhibitor, 1 and 2, could be complexed with sEH by the “induced fit” and “lock-and-key” models.Three flavonoids derived from the leaves of  EETs ex vessels . Therefoar range . Due to ar range .Capsicum, and of them Capsicum annuum L. (C. annuum), Capsicum chinense Jacq. (C. chinense), Capsicum frutescens L., Capsicum baccatum L., and Capsicum pubescens Ruiz and Pav. are mainly cultivated [C.chinense or “Bhoot jolokia” and “Bin jolokia” in Assam, “Naga King Chili” in Nagaland, “Umorok” in Manipur, and “Ghost pepper” in the west, which belongs to the family Solanaceace [C. chinense [Capsicum [Tetrastigmahemsleyanum [O-β-D-galactoside and 3,7,3′-tri-O-methylquercetin-4′-O-β-D-apiofuranosyl-(1→2)-O-β-D-glucopyranoside [C. chinense and study of their activity against sEH.There are 30 species in the genus ltivated . C.chinanaceace , is an ianaceace . This franaceace . Capsaicchinense . AdditioCapsicum . The conCapsicum . Recentlsleyanum , and theranoside , have beranoside ,14. Fina2SO4 solution followed by heating to 300℃ in dried air. Nuclear magnetic resonance (NMR) spectra were recorded using the JEOL ECA 500 spectrometer   , Sephadex LH-20 , and C-18  resins. Thin-layer chromatography (TLC) was performed using pre-coated silica gel 60 F254 and RP-18 F254S plates . Spots in the TLC were visualized by spraying with 10% aqueous H, Japan) . AUDA , Korea Atomic Energy Research Institute (KAERI). A sample specimen (RBRC002) was deposited at the Herbarium of RBRC, KAERI, Republic of Korea. The leaves of C. chinense (2 kg) were extracted twice with 95% methanol (36 L) at room temperature for a week. The solution was evaporated under reduced pressure to obtain a methanol extract (~210 g). The brown extract was suspended in distilled water (2.1 L) and successively partitioned with n-hexane, chloroform, ethyl acetate, and BuOH fractions. The chloroform fraction (8.4 g) was chromatographed on a silica gel column and eluted with a gradient system of n-hexane-acetone (0:1→1:1) to give eight fractions (HC1–HC8). The HC6 fraction was re-chromatographed on the C-18 column using an isocratic system of methanol–water (3:1) to afford compound 1 (12 mg). The ethyl acetate fraction (6.4 g) was separated on a silica gel column eluted with a gradient solvent system of chloroform–methanol (0:1→1:2) to achieve ten fractions (HE1–HE10). The HE6 fraction was separated on a Sephadex LH-20 column with 95% methanol to obtain compound 2 (20 mg) and five fractions (HE61–HE65). Compound 3 (7 mg) was purified by C-18 column chromatography with an isocratic solvent system of 50% methanol from the HE64 fraction.The leaves of 0 is the minimum value on the y-axis, a denotes the difference between maximum and minimum values, and b refers to the x value at 50%.sEH  in 25 mM bis-Tris-HCl buffer (pH 7.0) containing 0.1% BSA and 20 µL of compound in MeOH was mixed in a 96-well plate, followed by the addition of 50 µL buffer containing 5 µM PHOME. The reactions were carried out at 37°C for 40 min. The inhibitory ratio was calculated according to the following equations: The docking study of sEH with its inhibitor was performed using Autodock version 4.2. The 3D structure of the inhibitor was developed and minimized by MM2. The single bonds were confirmed as flexible bonds using the AutoDockTools. The 3D structure of the protein was downloaded from the RCSB protein data bank (pdb id: 3ANS). Water, substrate, and the B chain were deleted from the pdb file. Hydrogen atoms and Gasteiger charges were added to the A chain. The grid dimensions were set at 126×126×126 number of points (x×y×z). Docking simulation was performed to dock the inhibitor 25,000,000 times into this grid. These results were visualized using Chimera version 1.14  and AutoDockTools 1.5.6 .A molecular dynamic study was performed to simulate the optimal configuration of sEH with the flavonoids using the Gromacs version 4.6.5. Their Itp and gro files were constructed in the GlycoBioChem PRODRG2 server, and integrated into gro via Gromos96 43a1 force field and topology files of sEH that were generated by utilizing gdb2gmx of Gromacs. The complex was obtained as cubes of water molecules measuring 8.5×8.5×8.5 containing six chloride ions. The energy of the complex was lowered to 10 kJ/mol of maximal force in the steepest descent method. A stable simulated complex was equilibrated at 300 K for 100 ps, and simulated at 1 bar for 100 ps. Finally, the complex was simulated for molecular dynamics for 10 ns.n = 3). Allvalues were analyzed using Sigm aPlot  to determine treatment variations.Data are expressed as the means ± standard deviation  and glycosides (2 and 3) were purified with open column chromatography from chloroform and ethyl acetate fractions, respectively. Their chemical structures were evaluated as chrysoeriol (1) [O-glucopyranoside (2) [O-glucopyranoside (3) [The dried leaves of riol (1) , luteoliside (2) , and isoside (3)  from nuc1–3 were evaluated for binding strength and type of enzyme in vitrofor insight into the sEH inhibition. The inhibitory activity rate on sEH at each concentration of the compound was calculated as the product difference between the presence and absence of the enzyme using Equation (1). 50), these inhibition rate values were fitted by Equation (2). Inhibitors 1–3 were found to have IC50 values of 11.6 ± 2.9, 14.4 ± 1.5, and 42.7 ± 3.5 µg/mL, respectively  at various concentrations of the substrate . The inverse of these concentrations and v0 values were used for the Lineweaver–Burk plot. As indicted in 1–3 exhibited the same non-competitive mechanism of actionin the enzyme with a −1/Km value and different 1/Vmax values , a hydroxyl group and Asp496 (at a distance of 1.7 Å) or Phe497 (at a distance of 2.0 Å). The respective A- and B-rings of the flavonoid kept the π-π interactions with Phe497 at distances of 6.7 and 10.9 Å. The flavonoid glycoside (2) formed four hydrogen bonds between the sugar group and Leu417 and Met419 at distances of 1.9 and 2.0 Å, as well as two hydroxyl groups between the B-ring and His 524 at 1.9 Å and 2.1 Å, respectively. In addition, the B-ring of 2 kept π-π interactions with the furan ring of His420 at a distance of 12.9 Å.In 1894, the “lock-and-key” model was first suggested as the interaction of enzyme (lock) with substrate (key) by Emil Fisher . Moleculectively . They we1 and 2) with sEH.Since the bovine pancreatic trypsin inhibitor in a vacuum was simulated using molecular dynamics (MD) by McCammon in 1977, MD has been one of the state-of-the-art computational techniques for in-depth understanding of the communication between ligand and receptor with structural changes over time . An indu1 and 2 visually confirmed the stable state fitted with sEH during the simulation time with ~9.1×105 kJ/mol potential energy  and luteolin-7-O-glucopyranoside (2) showed inhibitory activities against sEH with IC50 values of 11.6 ± 2.9 and 14.4 ± 1.5 µg/mL, respectively. Through enzyme kinetic study, Lineweaver–Burk and Dixon plots revealed that they were non-competitive inhibitors with Ki values of 10.5 ± 3.2 and 11.9 ± 2.8 µg/mL, respectively. They were visually represented to be bound into the right edge, next to the catalytic site of the enzyme, by the stable Autodock score. In particular, inhibitor 1 was found to form a hydrogen bond with another amino acid (Arg410) in the molecular docking result via dynamic, whereas 2 was confirmed to form hydrogen bonds with common amino acids (Leu417 and Met419) between the molecular docking and dynamics results. These findings confirmed the possibility that the leaves of C. chinense containing flavonoids (1 and 2) were natural sEH inhibitors in vitro and in silico. Finally, the study suggests that the two are suitable for cell-based and in vivo experiments involving cardiovascular disease.Among flavonoids


Together with several chemoinformatic tools that can be used to improve the process of virtual screening, some additional properties of these marine peptides are identified related to their ability to behave as useful drugs. With the further objective of analyzing their bioactivity, some useful parameters for future QSAR studies, their predicted biological targets, and the ADMET  parameters related to the theopapuamides A-D pharmacokinetics are also reported.This work presents the results of a computational study of the chemical reactivity and bioactivity properties of the members of the theopapuamides A-D family of marine peptides by making use of our proposed methodology named Computational Peptidology (CP) that has been successfully considered in previous studies of this kind of molecular system. CP allows for the determination of the global and local descriptors that come from Conceptual Density Functional Theory (CDFT) that can give an idea about the chemical reactivity properties of the marine natural products under study, which are expected to be related to their bioactivity. At the same time, the validity of the procedure based on the adoption of the KID (Koopmans In DFT) technique, as well as the MN12SX/Def2TZVP/H Drug design can be pursued following a methodology driven by advancement and innovation breakthroughs including a combination of experimental and computational strategies. Computational strategies play a pivotal part in advanced therapeutic chemistry, displaying a special potential for changing the early stages in the process of drug discovery, especially in terms of time and cost savings ,2.Bioactive peptides are short amino acid chains that are inactive within the sequence of the parent protein and can be activated through gastrointestinal digestion, food processing, storage, or in vitro hydrolysis by proteolytic enzymes. Since bioactive peptides have the ability to impart a biological effect resulting in a positive impact on body functions or conditions, their application is of relevance for the food industry. Computational chemistry and molecular modeling tools provide methodologies to identify peptide sequences and their 3D molecular structure. Using in silico modeling, these molecular structures can be related to biological activity and targets of interest. These in silico targeted discovery approaches are well known and frequently used in drug discovery in the pharmaceutical industry. Computational chemistry methodologies are based on a broad palette of experience in pharmaceutical drug discovery, and the application of this knowledge towards medicinal chemistry research may lead to new insights through the elucidation of the mechanistic knowledge of potential ingredients such as bioactive peptides.Since the structure of molecules defines the chemical, physical, and biological properties of matter, this information is crucial for understanding, explaining, and predicting chemical reactions and biochemical processes, developing new drugs, and providing crucial insights into the nature of the interactions between drug targets and ligands, which allows predictive models that are suitable for lead discovery and optimization to be constructed. There are numerous reports in the literature showing that from a molecular viewpoint, the bioactivity and the chemical reactivity properties of these peptides are intimately related ,4. For tIt then follows that it is of utmost importance to study the chemical reactivity of natural products , because this is likely to help with the development of some medicines with the aid of tools displayed through computational and theoretical chemistry, as well as molecular modeling. In a very special way, Conceptual Density Functional Theory (DFT) ,6,7 is oAs a part of an ongoing project for the development of new pharmaceutical drugs of marine origin, we are currently investigating new families of peptides obtained from marine sources, hoping that this could be the starting point for the design of potentially helpful therapeutic peptides . Thus, tThe Kohn–Sham (KS) methodology includes the estimation of the molecular energy and density of a given system, as well as the orbital energies, explicitly connected with the frontier orbitals including the Highest Occupied Molecular Orbital (HOMO) and Lowest Unoccupied Molecular Orbital (LUMO) ,25,26,27nctional ,37,38,39y quoted ,21,22,23Following the methodology considered in our previous studies ,21,22,23https://pubchem.ncbi.nlm.nih.gov), which is an open chemistry database that acts as an online free resource of information related to physical, chemical, and biological properties, interactive spectra, and literature references. In order to get a glimpse of the potential therapeutic properties of the considered peptides, Simplified Molecular Input Line Entry Specification (SMILES) notations for the molecular systems under consideration were fed into the online Molinspiration software from Molinspiration Cheminformatics  allowing for the estimation of several molecular properties that are known to be related to drugability and that could be useful in QSAR studies that are commonplace in the process of drug design and development. The results of this determination are presented in The starting molecular structures of the peptides theopapuamides A-D to be studied were obtained from PubChem  model that is accessible within Gaussian 09 . As was A graphical display of the tridimensional optimized molecular structures of the peptides theopapuamides A-D obtained with the aid of the UCSF Chimera Visualization System  are presIt is usually assumed that the goodness of a given density functional can be estimated by comparing the results that it gives with the experimental values that are attempted to be reproduced or with the results that can be obtained through post-Hartree–Fock calculations like MP2, MP4, or CCSD. However, this is not always possible due to the lack of experimental results for the molecular systems that are being studied or the large size of the molecules that prevent some accurate methodologies from being computationally practical. For this reason, we developed a protocol named KID (Koopmans In DFT) ,21,22,23designed ,21,22,23designed ,21,22,23As can be seen from the results in ElectronegativityGlobal hardnessElectrophilicityElectrodonating powerElectroaccepting powerNet electrophilicityTaking into account the KID methodology considered in the previous research being integrated into the finite difference approximation ,21,22,23As a complement of these global reactivity descriptors that arise from Conceptual DFT ,7,53,54,By making use of the mentioned CDFT software tool applied to the results of the calculation of the electronic properties of the peptides theopapuamides A-D, the values of the defined global reactivity descriptors (including the Nucleophilicity (N)) can be obtained, and they are displayed in The global hardness  authors , theopapNucleophilic Fukui functionElectrophilic Fukui functionDual descriptorwhich are the relationships between the electron densities of the neutral, positive, and negative species, as well as between the nucleophilic and electrophilic Fukui functions.The presented global descriptors are a representation of the chemical reactivity of a molecule as a whole. However, local reactivity descriptors have been developed that can give an idea about the differences between the reactivity of each of the atoms that form the molecule. One of the most important groups of such reactivity descriptors are the Fukui functions ,6,7 and The nucleophilic Fukui function, molecule . Thus, aNucleophilic Parr functionElectrophilic Parr functionwhere Another local reactivity descriptor is the Parr functions ,67, whicectively .In order to perform a comparison between the results that can be obtained from the electrophilic and nucleophilic Fukui function analysis for each peptide and the values that are the outcome of the Parr function analysis, the predictions for the specific reactions sites are compiled in a series of tables that are presented as the  program  during tBy looking at the numerical and graphical results presented in The theopapuamides A-D family of cyclodepsipeptides of marine origin are studied by resorting to some techniques of common use in the process of drug discovery and development, showing that these kinds of molecules can be regarded as potential therapeutic drugs. Some chemoinformatic tools are used to obtain information about the potential therapeutic properties of these peptides in the form of bioactivity radar charts, biological targets, and ADME values.With this knowledge in mind, the chemical reactivity of the studied peptides is exhaustively analyzed through the optimization of their structures using an MN12SX/Def2SVP/H2O model chemistry and the determination of their electronic properties by means of a larger model chemistry, namely MN12SX/Def2TZVP/H2O, already used in previous works for the study of peptides, validating their usefulness for these kinds of calculations.The chemical reactivity of the considered molecular systems is subjected to an analysis based on a particular methodology developed by our research group named Conceptual DFT-based Computational Peptidology, the use of the MN12SX density functional being validated once again by resorting to the KID procedure and the in-house software tool CDFT.The analysis of the global and local reactivity descriptors arising from Conceptual DFT together with some proposals like the Nucleophilicity (N) and the Parr functions allows for a complete understanding of the chemical reactivity of the studied peptides, by distinguishing the different chemical reactivities through the analysis of the global descriptors and further identification of the reaction sites or regions within the molecules by resorting to Fukui and Parr functions, as well as the dual descriptor.Finally, a nice agreement is found between the outcome of the numerical and graphical analysis of the Fukui and Parr functions on each peptide, leading to the conclusion that it could be possible to define a new chemical reactivity descriptor analogous to the dual descriptor on the basis of the difference between the Parr functions, which could act as an additional tool for the study of the chemical reactivity of molecular systems through Conceptual DFT-based Computational Peptidology.


However, of the biggest infectious killers, LRI remains the condition with the least funding on a global level.2 In The Lancet Infectious Diseases, the GBD 2017 Lower Respiratory Infections Collaborators3 present modelled trends in LRI incident cases and mortality and estimate the role of 13 modifiable protective and preventive risk factors in these spatiotemporal changes using the Global Burden of Disease (GBD) dataset.As the leading cause of infectious deaths in children,3 report considerable reductions in LRI indicators over the last 30 years, with a 67·2%  reduction in LRI mortality rate and a 32·4% (27·2–37·5) reduction in incidence among children younger than 5 years. Key factors in these reductions have been the roll-out and scale-up of vaccine coverage against Haemophilus influenzae type b (Hib) and pneumococcal infections , improved childhood nutrition and feeding practices, and declines in household air pollution. The impressive scale of the analysis should be commended for filling a vital gap in benchmarking the role of preventive and protective interventions across broad regions.They4 have highlighted the importance of global seasonal variation in the circulation of common viral pneumonia pathogens, with considerable within-country differences. This finding raises important questions about national and regional LRI prevention programming and suggests that more nuanced approaches to vaccination strategies could achieve improved control. With more granular data on the risk factors associated with pneumonia, the same variability would probably be observed in the role and implementation of different prevention and protection interventions for paediatric pneumonia.To translate these estimates into action, intensified commitments are needed to improve the availability of high-quality local epidemiological data on both risks and causes of LRIs—especially how these vary within smaller, subnational regions. Li and colleagues3 improved nutrition-related factors and reductions in household air pollution were the main contributors to impressive reductions in mortality in India. However, absolute differences and change in rates of stunting, wasting, and underweight across India have been heterogenous.6 In Nigeria, low birthweight and short gestation was estimated to have increased LRI mortality in between 1990 and 2017, whereas Hib coverage was the third largest contributor to LRI mortality reduction. A 2016 study reported that the odds of low birthweight was 10·6 times higher in the northwest than in the southwest of Nigeria,7 and the 2018 Demographic and Health Survey, which gathered data from national household surveys, report vaccine coverage to range from 73% for PCV and 74% for Hib in Abuja state to 6% for PCV and 7% for Hib in Sokoto state.8The two countries with the highest estimated numbers of under-5 LRI deaths—India (185 429 deaths [95% UI 167 676–204 328]) and Nigeria (153 069 deaths [115 332–196 193]), which account for 41·8% of the global under-5 LRI mortality (808 920 deaths [747 286–873 591])—serve as important examples. According to the GBD 2017 Lower Respiratory Infections Collaborators,3 present an interesting finding on the role of air pollution. With rapid urbanisation and economic development, reductions in household air pollution occur simultaneously with increasing ambient air pollution outside the home. To date, trials targeted at reducing indoor air pollution in low-income rural settings at a household-level have largely been ineffective,9 although results of the HAPIN trial  in Rwanda, Peru, Guatemala, and India have not been published yet. Evidence would currently suggest that meso-level or macro-level approaches are needed; however, whether these interventions could be done in a manner that results in equitable reductions in LRI morbidity and mortality is unclear.These estimates highlight the need for caution in extrapolating broader conclusions on the roles of different interventions and the public health gains made across heterogeneous countries with little subnational data. To make meaningful, sustainable, and equitable gains, the global health community needs to be mindful of the complex nature of LRI as a disease of poverty and consider the interface between more individualised risks and the wider determinants for health at the community, health-system, and policy levels. For example, the GBD 2017 Lower Respiratory Infections Collaborators3 is an important step in quantifying broader priority areas for the prevention of paediatric pneumonia and in highlighting areas in which successes have been achieved. However, the development of local capacity to collect, analyse, and report on LRI-specific indicators needs to be intensified to ensure high-quality data for stakeholders to act effectively. We hope this work will further galvanise and build on global commitments to accelerate progress towards LRI morbidity and mortality targets and encourage funding to establish sustainable data structures and rigorously evaluate equitable effects of interventions both between and within regions.The GBD 2017 Lower Respiratory Infections Collaborators’ Article


For this purpose, the following were conducted: behavioural studies, the analysis of concentrations of dopamine and its metabolites, and the analyses of mRNA expression and dopamine receptor proteins D1 and D2 in the prefrontal cortex, striatum, hippocampus, and cerebellum of rats. In this study, it was observed for the first time that pre- and postnatal exposure to fluoride ions influenced the phenomenon of morphine dependence in a model expressing withdrawal symptoms. Behavioural, molecular, and neurochemical studies demonstrated that the degenerative changes caused by toxic activity of fluoride ions during the developmental period of the nervous system may impair the functioning of the dopaminergic pathway due to changes in dopamine concentration and in dopamine receptors. Moreover, the dopaminergic disturbances within the striatum and the cerebellum played a predominant role as both alterations of dopamine metabolism and profound alterations in striatal D1 and D2 receptors were discovered in these structures. The present study provides a new insight into a global problem showing direct associations between environmental factors and addictive disorders.Disturbances caused by excess or shortages of certain elements can affect the cerebral reward system and may therefore modulate the processes associated with the development of dependence as was confirmed by behavioural studies on animals addicted to morphine. Earlier publications demonstrated and proved the neurodegenerative properties of both low and high doses of fluoride ions in animal experiments and in epidemiological and clinical studies. The aim of the experiments conducted in the course of the present study was to analyse the effect of pre- and postnatal exposure to 50 ppm F Fluoride is a neurotoxic ion both in vitro and in vivo ,2,3. ChrO-methyl transferase (COMT) to 3,4-dihydroxyphenylacetic acid (DOPAC) or 3-methoxytyramine (3-MT), respectively, and then to the final metabolite—homovanillic acid (HVA). Exposure to abused drugs leads to an increased dopamine release in the nucleus accumbens  which determines the feeling of pleasure.The rewarding effect of various addictive substances is associated with dopaminergic stimulation of mesolimbic structures in brain. Dopamine acts on the D1-like family of dopamine receptors (D1 and D5) and the D2-like family . It is metabolized by the monoaminooxidase-B (MAO-B) enzyme or by catechol-Morphine is one of the strongest drugs of dependence that is used in clinical practice for acute and chronic pain alleviation. It acts primarily on μ opioid receptors. The stimulation of the μ receptor, localized at the neurons producing gamma-aminobutyric acid (GABAergic neurons) in the ventral tegmental area, inhibits GABA release. This leads to disinhibition of dopaminergic neurons, increases the release of dopamine, and causes euphoria, thus promoting the state of dependence . On the Drug dependence, including opioid dependence, is a global, clinical, social, and economic problem. The number of addicted patients is increasing steadily, while the possibilities of effective management are limited. Therefore, further studies are justified to elucidate the novel mechanisms underlying this issue which, in turn, may be helpful in searching for new therapeutic strategies. The main purpose of the present study is to assess whether prenatal and postnatal exposure to fluoride in rats influences morphine dependence. In the first step, behavioural experiments on rats were performed to show the influence of fluoride exposure on the intensity of morphine withdrawal. In the second stage, a series of neurochemical experiments was carried out, in which the levels of dopamine and its three metabolites  in the hippocampus, the striatum, the prefrontal cortex, and the cerebellum were studied using high-performance liquid chromatography with the electrochemical detection (HPLC-ED) method. Next, mRNA and protein expressions of dopamine D1 and D2 receptors were estimated in the same brain structures using real-time quantitative reverse transcription PCR (qRT-PCR), the ELISA test, and immunohistochemical (IHC) reaction, respectively.3,49 = 72.86, p < 0.0001). There were no jumps observed in saline and saline + naloxone  rats. Similarly, no jumps were observed in animals pre- and postnatally exposed to fluoride and acute dose of naloxone. The exposure of rats to increasing doses of morphine and, on the last day of the study, to morphine with naloxone, produced a significant increase in the number of jumps (p < 0.001) in comparison with the saline + naloxone group. The prenatal and postnatal exposure of morphine-dependent rats to fluoride caused a significant increase (p < 0.001) in the number of jumps, both in comparison with fluoride-exposed rats and with morphine-treated rats without fluoride pre-exposure  in the levels of dopamine and two dopamine metabolites–DOPAC and HVA (p < 0.01)—in comparison with saline-treated rats. However, it did not show any significant changes in dopamine and its metabolite concentrations in rats that were pre- and postnatally treated with fluoride. In morphine-withdrawal rats, which were previously treated with fluoride, significant reductions (p < 0.05) in the dopamine concentration and its two metabolites, DOPAC and HVA, were observed in comparison with morphine-or fluoride-treated rats. There were no significant changes in the 3-MT level in the prefrontal cortex of the studied rats.p < 0.05) in dopamine concentration, but not in that of dopamine metabolites, was observed in comparison with saline-treated rats. A significant reduction (p < 0.05) in the dopamine level in morphine-withdrawal rats, which were previously treated with fluoride, was observed in comparison with the rats treated with fluoride or morphine alone. The combination of fluoride pre-treatment with the exposure to morphine induced the increase (p < 0.05) in the level of DOPAC and HVA as compared to fluoride-treated rats. There were no significant changes in the striatal 3-MT level of the studied rats  in dopamine and DOPAC levels. The concentration of 3-MT was significantly increased (p < 0.05) in these animals. There were no significant changes in rats pre- and postnatally treated with fluoride as compared to the saline group. In morphine-withdrawal rats, which were previously treated with fluoride, significant reductions (p < 0.05) in dopamine, DOPAC, and HVA concentrations were demonstrated. In these animals, the level of 3-MT was significantly higher (p < 0.05) than that recorded in fluoride pre-treated rats and was significantly reduced (p < 0.05) in comparison with morphine-treated rats  while the concentrations of dopamine metabolites (3-MT and HVA) were significantly (p < 0.05) reduced in comparison with saline-treated animals. In morphine-withdrawal rats, the level of dopamine was significantly elevated (p < 0.05), yet the concentrations of dopamine metabolites were diversified: DOPAC was significantly increased (p < 0.05), the HVA level was elevated (p < 0.05), and there was no significant alteration in the 3-MT level as compared to the saline-treated group. In rats which were treated with both fluoride and morphine, there was no change in the dopamine level in comparison with the animals treated with fluoride alone or morphine alone, while DOPAC and HVA levels were significantly reduced (p < 0.05)  B.p < 0.05). In the M + F group, a pronounced down-regulation of the receptor was observed in comparison to the fluoride group (decrease of 97%) (p < 0.01). Administration of fluoride resulted in a twofold increase in the expression of D2 vs. C (p < 0.01). In the morphine group, a significant decrease in the expression of said receptor vs. C was observed (72-fold), similar to the M + F group vs. F (175-fold) (p < 0.01)  .p < 0.05). Administration of morphine caused a down-regulation of the studied receptors vs. C (D1: 6× and D2: 1.75×) (p < 0.01). Pre-exposure to fluoride and subsequent morphine dependence resulted in a lowered expression of the D1 receptor vs. F among rats expressing withdrawal symptoms (p < 0.05), but did not affect the expression of the D2 receptor. The M + F group was also significantly different from the M group in terms of the expression of the analysed dopamine receptors (p < 0.01)  ( < 0.01) .p < 0.05), similar to the expression of the D2 receptor (p < 0.05)  .p < 0.01 and p < 0.05, respectively). In the M group, the recorded expression level of the D1 and D2 receptors was, respectively, two times and ten times higher than the level recorded in the control group . The M + F group was characterised by a lower expression of the analysed receptors vs. F  and, in the case of the D1 receptor, the expression was markedly higher than that observed in the M group (p < 0.01)  ( < 0.01) .p < 0.05). The level of protein expression of D1 in the prefrontal cortex was 2 times lower in the M + F group vs. F (p < 0.05) and was 30% lower vs. M (p < 0.05). Exposure to fluoride resulted in an increase in D2 receptor expression by 40% (vs. C) (p < 0.05); the administration of morphine was associated with a decrease in the expression of said receptor vs. C (p < 0.05), similar to what was observed with respect to the MF vs. F groups (p < 0.05)  .p < 0.05). In comparison to all the groups under study, the morphine group was characterised by the lowest D1R expression which was lower than that recorded in the control group, by 33% (p < 0.05).The fluoride group showed a significantly higher D1 expression in the striatum as compared with the other groups (p < 0.05). The M group was characterised by a significantly lower D2 expression in comparison to the C group (p < 0.05), whereas in the M + F group, the level of expression was comparable to that in the F group . The protein expression of the D1 receptor was markedly lower in the M group as compared with the C group (p < 0.05). Exposure to F, when combined with morphine dependence, levelled the expression of D1 to that in the control group, whereas in the case of D2, the expression level was comparable to that of the M group (Exposure to fluoride did not result in significant changes in the protein expression of the D1 receptor (vs. C) in the structure of the hippocampus; however, it caused an increase in the expression of the D2 receptor, by 32% ( M group .p < 0.05), and of D2 by 110% (p < 0.05). Following the administration of naloxone, the morphine-dependent group (M) demonstrated lowered expression of D1R vs. C, by 23%. The M + F group was characterised by a lower level of D1 and D2 expression compared with the F group (p < 0.05)  .The IHC reaction showed that immunoexpression of dopamine receptor 1 in the prefrontal cortex (neocortex) was the highest in brains of fluoride-treated rats .Generally, the striatum portion of the studied rat brains showed a low expression of dopamine receptor 1 B,F,J,N, In the cerebellum of the studied animals D,H,L,P, The IHC reaction showed that immunoexpression of dopamine receptor 2 in the prefrontal cortex (neocortex) seemed to be similar in all studied groups of animals .The striatum of rats treated with fluoride .In the cerebellum cortex of the studied animals D,H,L,P, − levels observed in people living in areas rich in fluoride [In the present study, the effects of pre- and postnatal exposure to fluoride in a model of morphine dependence were studied in rats. A series of behavioural, neurochemical, and molecular experiments and microscopic observations was performed to assess the changes in the intensity of naloxone-induced morphine withdrawal signs in rats and to explore the changes within the dopaminergic system—the most important neurotransmitter in the CNS involved in addiction. To assess the effects of pre- and postnatal exposure to fluoride in rats, the animal model described by Kobayashi et al. (2011) and Morales-González et al. (2010) was chosen ,14. Accofluoride ,16,17. Ifluoride .It was observed in our previous studies, which were performed on a similar experimental model, that long-term fluoride exposure of rats did not produce any changes in the fluoride concentration in the serum, probably because of high urinary removal ; howeverIndeed, it was observed in a behavioural study that pre- and postnatal fluoride exposure significantly increased the intensity of morphine withdrawal signs, manifested as a higher number of jumps in comparison with the animals treated with morphine alone or fluoride alone. This confirms the role of the environmental factors in the development of the state of opioid dependence.The neurochemical analysis showed that pre- and postnatal fluoride exposure produced the effects in the striatum and the cerebellum, but not in the hippocampus and the prefrontal cortex. In the striatum, a significant reduction in the dopamine concentration without the effects on dopamine metabolite levels was observed, while in the cerebellum, a significant increase in the dopamine level and a reduction of dopamine metabolites (3-MT and HVA) were measured. This showed that pre- and postnatal fluoride exposure disturbed the striatal dopamine concentration and inhibited dopamine degradation in the cerebellum. The literature data on the influence of fluoride exposure on dopamine concentration are not homogeneous. Chirumari and Reddy (2007) observed that intraperitoneal administration of fluoride for 14 days (1–10 mg/kg) caused an increase in the level of dopamine and its metabolite (HVA), as well as serotonin and its metabolite in the hippocampus and the neocortex . On the In the case of morphine-dependent rats, during naloxone-induced morphine withdrawal, the inhibition of dopamine concentration in the striatum and the prefrontal cortex was demonstrated. In the hippocampus, the dopamine and DOPAC levels were reduced, while the 3-MT level was elevated. These divergent effects in the hippocampal dopamine metabolites resulted in the lack of changes in the concentration of the final metabolite—HVA. In the cerebellum, a marked increase in dopamine concentration was observed, but the changes in metabolite concentrations were strongly diversified, and some kind of artifact cannot be excluded in that part of the study. Generally, our striatal results confirmed the accepted theory that the appearance of unpleasant effects during morphine withdrawal is a consequence of a reduced dopamine level in the striatum . The dopOne of the most important achievements in neurochemical studies is that pre- and postnatal fluoride exposure in morphine-withdrawal rats significantly disturbs dopamine metabolism, mainly in the striatum and cerebellum. In the striatum, the dopamine concentration was reduced and DOPAC and HVA levels were elevated, confirming the intensification of catabolic processes of dopamine, while in the cerebellum, the opposite effect was observed. The dopamine concentration was higher and its metabolites were reduced, which confirmed the reduction of dopamine degradation in these animals. Additionally, in the hippocampus and the prefrontal cortex of the studied animals, a reduction in dopamine levels, without an effect on dopamine metabolites, was observed in comparison with both fluoride- or morphine-treated rats. Thus, the obtained results demonstrated the existence of strong disturbances in dopamine release  and its degradation (striatum and cerebellum) in naloxone-induced morphine-withdrawal rats which were pre-treated with fluoride.It was also demonstrated in neurochemical experiments that during naloxone-induced morphine withdrawal in fluoride-treated animals, the major enzyme involved in dopamine degradation is MAO, as significant alterations were measured in the levels of DOPAC and HVA. Statistical changes in the level of 3-MT, which is formed by degradation of dopamine by COMT, were observed only in the hippocampus of morphine-withdrawal rats. This effect was similar to our previous study  in whichAs it was easy to predict, the alterations in the dopamine level evoked significant changes in dopamine D1 and/or D2 receptor expression in the studied brain structures. In fluoride-treated rats, despite a poor effect on dopamine concentration and its metabolism in a neurochemical study, a significant increase in mRNA and protein expressions in dopamine D1 and D2 receptors in most studied areas of the brain was observed. The Western blot analyses were consistent with the results obtained from immunohistochemical reactions. These changes might be a result of a long-term adaptation to fluoride exposure. On the other hand, after naloxone-evoked morphine discontinuation, a significant reduction in dopamine D1 and D2 receptors in most studied structures was observed in comparison with saline-treated rats. The pre- and postnatal fluoride exposure in morphine-withdrawal rats produced significant changes in the mRNA and protein expression of dopamine D1 and D2 receptors in the striatum, the structure which is most engaged in morphine withdrawal. Here, the expressions of dopamine D1 and D2 receptors were significantly higher in comparison with morphine-withdrawal rats, but lower in comparison with fluoride-treated rats. In other structures, the expressions of dopamine D1 and D2 receptors were similar to those which were observed in morphine-withdrawal rats.So far, the precise associations between fluoride toxicity and dopaminergic receptors are not recognized; however, the existing evidence on neurotoxic mechanisms of both opioids and fluoride suggests that synergistic effects are possible. For example, it has been documented that a long-term exposure to fluoride alters the neuronal morphology—it induces axon deterioration, myelin sheath degeneration and dark cells with scanty cytoplasm, and vacuolated swollen mitochondria in the neocortex, hippocampus, and cerebellum . It resu2O2). Additionally, dopamine auto-oxidation produces the superoxide anion O2– and H2O2. Both O2– and H2O2 generate highly toxic radicals which disturb cellular processes. This hypothesis was confirmed by Guzmán et al. (2006) and Özmen et al. (2007), who showed that morphine decreased the level of reduced glutathione (GSH) [On the other hand, long-term exposure to morphine influences mainly dopaminergic neurotransmission. As was described in the Introduction, morphine administration via opioid and GABAergic receptors increases the dopamine level while morphine discontinuation produces the opposite effect. It has been demonstrated that higher levels of dopamine (associated with chronic morphine exposure) may act as a neurotoxic agent . Dopaminne (GSH) ,40.Taking into account the above-mentioned data and the results of the present study, it can be concluded that pre- and postnatal exposure to fluoride may disturb the activity of brain and may directly influence the effect of long-term exposure to morphine. It seems that the striatum and the cerebellum were structures damaged the most in the present study.The study was approved by the local Ethics Committee of the Medical University in Lublin  in accordance with the Directive 2010/63/EU on the protection of animals used for scientific purposes.n = 3) received 50 ppm sodium fluoride solution (NaF) in drinking water ad libitum, starting from the first day of gestation. Fluoride dose was adjusted to represent its concentration in rat’s blood similar to that observed in the serum of people environmentally exposed to fluoride compounds [n = 3) received tap water until offspring were weaned. The offspring were fed by the mothers, and those from the experimental group received NaF in drinking water ad libitum. On the 21st post-natal day (PND 21) the offspring were placed in separate cages. From that moment, the young rats still received NaF until PND 60. The control animals received tap water ad libitum in the same period.The study was conducted on Wistar rats. Optimum conditions for the rats were ensured, i.e., a natural day–night cycle and room temperature of 22 ± 1 °C. Mature females were mated with males and then isolated and divided into two groups: the control and experimental. Females from the experimental group  animals. This was obtained by administering increasing doses of morphine  twice a day, for eight consecutive days (PND 60–67). On the next day (PND 68), the next dose of morphine (50.0 mg/kg) was administered, and 1 h later, an opioid receptor antagonist—naloxone —was injected in order to induce morphine withdrawal. Next, the animals were immediately placed in 10-litre glass cylinders. The number of jumps was measured for a period of 30 min. Immediately after the behavioural experiments, the rats were decapitated, and the brains were quickly removed. Four brain structures  were selected for neurochemical and molecular experiments and microscope observation. The obtained tissue was immediately frozen using liquid nitrogen. The samples were stored at −80 °C for neurochemical analysis and Western blot analysis. The samples for the quantitative real-time PCR reaction were placed in Trizol reagent  and stored at −80 °C. The samples of brain tissues for the IHC reaction were fixed in formalin.(1)saline group—rats receiving 0.9% NaCl (n = 6);(2)saline + naloxone group—rats receiving 0.9% NaCl from the prenatal period until adulthood; on the last day of the experiment, they received saline with naloxone (n = 6); these animals were excluded from molecular studies;(3)fluoride group—rats treated with fluoride (NaF) from the prenatal period until adulthood and receiving 0.9% NaCl instead of morphine. They received naloxone on the last day of the experiment (n = 6);(4)morphine group—rats receiving 0.9% NaCl from the prenatal period until adulthood, and then procedure of morphine dependence was performed (n = 6);(5)fluoride + morphine group—rats treated with fluoride (NaF) from the prenatal period until adulthood, F-treated, and then the procedure of morphine dependence was performed (n = 6).In the study, the animals were divided into the following groups:The concentrations of dopamine and its metabolites were measured using high-pressure liquid chromatography with electrochemical detection. The mobile phase was 0.05 M citrate-phosphate buffer of pH 3.5, 0.1 mM of EDTA, 1 mM of sodium octyl sulfonate, and 3.5% methanol. The flow rate was 1 ml/min. Prior to homogenization with 0.1 M perchloric acid containing 0.05 mM of ascorbic acid, all samples were accurately weighted. Then, the samples were centrifuged and filtered using a cellulose membrane (RC 58 0.2-im). The assay was conducted with the use of an HP 1050  equipped with C18 columns. For the purpose of determining the concentration of dopamine and its metabolites, the analysis of standards was conducted in parallel with the analysis of the samples, and then followed by comparison of the obtained peaks.Quantitative mRNA expression of D1R (dopamine receptor D1) and D2R (dopamine receptor D2) genes was performed in a two-step reverse transcription PCR. The GAPDH gene was used as a reference gene. RNA was isolated from the tissue samples kept at −80 °C using the commercial kit RNeasy MiniKit  according to the manufacturer’s instructions. The quality and quantity of RNA were determined with a NanoDrop ND 1000 . The obtained matrix was transcribed into cDNA with the Omniscript RT Kit  according to the manufacturer’s instructions. Quantitative real-time CPR was conducted using a 7500 Fast Real-Time PCR System  and the reagent Power SYBR Green PCR Master Mix , i.e., the buffer solution containing AmpliTaq Gold DNA polymerase, mixture of deoxynucleotides, SYBR Green dye, and ROX reference dye . Monitoring the real-time increase in the CPR reaction product was made possible by fluorescence measurement which is proportional to the concentration of the product in a mixture. For the purpose of further calculations, the mean from the two measurements was used. To calculate the values, the ΔΔCt relative quantification method was used. From the means of the logarithmic expression values, fold change between groups was calculated.The following primer pairs were used: GAPDH forward: ATG ACT CTA CCC ACG GCA AG, reverse: CTG GAA GAT GGT GAT GGG TT; D1R forward: CGC GTA GAC TCT GAG ATT CTG AAT T, reverse: GAG TTA AGG AGC CAC CAC ATC AGT; D2R forward: TGA CAG TCC TGC CAA ACC AGA GAA, reverse: A TGG GCA TGG TCT GGA TCT CAA AGA.Tissue sample homogenates were treated with RIPA lysis buffer containing protease and phosphatase inhibitors . Protein concentration in the filtrate was determined with the BCA method using a commercial Pierce™ BCA Protein Assay Kit . Then, electrophoretic protein fractionation was conducted (SDS-PAGE) using 10% polyacrylamide gel by placing 30 μg protein/well. The fractionated proteins were transferred onto a 0.2 μm PVDF membrane  by a wet transfer. Prior to incubation with antibodies, the membranes were placed in a blocking buffer (5% BSA) for 60 min. The brain protein expression was detected using an antibody against D1R (ab81296) and D2R (ab85367)—rabbit pAb  diluted 1:800 and 1:1000, respectively, and sAb goat anti-rabbit IgG HRP H&L (ab97051) . Expression of β-actin was detected using the β-Actin antibody: sc-47778 . The membranes were developed with an ECL Advance Western Blotting Detection Kit , and subsequently, bands were visualized using the Molecular Imager ChemiDock XRS+ .n = 6 in each studied group) were placed in 4% buffered formalin solution for 24 h. Subsequently, they were washed several times with distilled water and subjected to a dehydration series. The dehydrated samples were placed in paraffin for 3 h and locked in cases. The obtained blocks were cut using a microtome  into sections of 3–5 µm, which were then placed on histological slides . Subsequently, the sections were deparaffinised and hydrated. In order to expose epitopes, the deparaffinised sections were boiled twice in a microwave oven  in 10 nM citrate buffer (pH 6.0). Once cooled and washed with PBS, the preparations were incubated for 60 min at room temperature with a primary antibody against DR1  and DR2  in final solution (1:750). For the purpose of visualisation of the binding site of the primary antibodies, a commercial kit Dako LSAB + System-HRP  was used. The kit is based on the reaction with a biotinylated antibody (against rabbit and mouse proteins), streptavidin marked with peroxidase, and 3 3’ diaminobenzidine (DAB) as a chromogen. In this reaction, brown-coloured precipitate appears in the presence of the antigen. The kit was used according to the manufacturer’s instructions. The preparations were additionally stained with hematoxylin. To visualise the samples and obtain pictures, a light microscope  integrated with a camera was used. For a negative control, specimens were processed in the absence of primary antibodies.The dissected whole brains  using the Graph-Pad Prism Software 5.04. Post-hoc analysis was conducted with a Tukey test. The results are presented as mean values ± S.E.M.p < 0.05.The results of neurochemical and molecular analyses were statistically analysed with Statistica 13 software and are presented as mean values ± SD. The Shapiro-Wilk W test did not show conformity with the normal distribution; therefore, the non-parametric Mann–Whitney U test was used for comparison of the groups. Statistical significance was set at In the present study, it was shown for the first time that pre- and postnatal exposure to fluoride influenced the phenomenon of morphine dependence. Moreover, the dopaminergic disturbances within the striatum and the cerebellum played a predominant role as both the alterations of dopamine metabolism and profound alterations in striatal D1 and D2 receptors were discovered in these structures. The present study provides new insight into the global problem showing direct associations between environmental factors and addictive disorders.


Heterodera medicaginis. This represents the first record of H. medicaginis in Utah and the second report of this nematode in North America.In the spring of 2019, a cyst nematode was discovered from soil samples collected from an alfalfa field in Millard County, Utah. The soil samples were submitted to one of us (SH), who extracted the nematode cysts and sent them to the USDA-ARS, Mycology and Nematology Genetic Diversity and Biology Laboratory (MNGDBL), Beltsville, MD for morphological and molecular identification. Cysts and living nematode juveniles (J2) recovered from the cysts were examined morphologically and molecularly for species identification which indicated that the specimens were  Heterodera medicaginis Kirjanova in the study of H. medicaginis is mostly Europe (Russia and Ukraine) and Asia (Kazakhstan and Uzbekistan)  . This cyekistan) . More reekistan) .The soil sample was collected from Delta, Utah (Millard county) and sent to University of Idaho for initial evaluation, after which it was submitted to USDA-ARS in Beltsville, MD for morphological and molecular identification.Various stages: cysts, second-stage juveniles (J2), and eggs were sent to MNGDBL, Beltsville, MD from the University of Idaho. Juveniles used for morphological observations were separated from soil by sieving and Baermann funnel extraction. Juveniles were fixed in 3% formaldehyde and processed to glycerin by the formalin glycerin method . FemalesLiving nematode juveniles (J2) recovered from cysts were examined morphologically and molecularly for species identification.The ITS 1&2 rDNA region was amplified with primers TW81 and AB28 , producin = 15) included length of body , stylet well developed  with anchor-shaped basal knobs, long basal bulb (75 μm) with three nuclei, tail , and hyaline tail terminus . The lateral field had four distinct lines with few specimens having an aerolated lateral field (n = 5) were dark brown in color, lemon shaped, ambifenestrate with heavy bullae, underbridge and having a zig-zag cyst wall cuticular pattern  and several from Kansas (accession numbers MK093180-MK093186). The mitochondrial Cox1 sequence shared > 99.51% identity to several H. medicaginis sequences from Kansas . Six cloned haplotypes of the Hsp90 short fragment was obtained from the Utah population. These clone sequences varied from 0 to 4 bp among each other and between 3 and 8 bp from those from Kansas (MH798843-MH798846). Based upon the unambiguous similarity of all examined DNA markers with those previously reported for the species, we conclude these populations are from H. medicaginis.DNA sequences from the Utah population were compared with those previously obtained from Kansas and Montana specimens  and RussHeterodera medicaginis. To our knowledge this is the first report of the alfalfa cyst nematode in Utah.Based upon this collective morphological and molecular data, we identify this isolate as