Tobacco rattle virus (TRV) infection. In the Pacific Northwest, this virus is transmitted by the stubby root nematode (SRN) within the genus Paratrichodorus. Remediating CRS affected fields is a major challenge that can be mitigated by growing plant varieties that are resistant to TRV infection. Growing alfalfa has been shown to reduce TRV levels in CRS infested fields over time but the development of a potato cultivar with these same capabilities would be of great economic benefit to potato growers. Castle Russet is a new potato clone that does not develop symptoms of CRS disease. To assess its ability to reduce soil virus load, Castle Russet, tobacco var. “Samsun NN”, alfalfa var. “Vernema”, and Russet Burbank potato were grown for a period of 1 to 3 months in soils containing viruliferous SRN populations at two different inoculation pressures (60 nematodes/pot and 1060 nematodes/pot) in greenhouse pot experiments. SRN population size and the presence of TRV were assessed over several months post inoculation. Results indicate that plant host and length of exposure significantly influence SRN population dynamics, whereas the TRV infection status of bait plants was significantly affected by both of these factors as well as inoculation pressure. These results suggest that both alfalfa var. “Vernema” and Castle Russet are resistant to TRV infection and may potentially be used to eliminate the virus from fields affected by CRS.Corky ringspot (CRS) is a widespread potato tuber necrotic disease caused by  Tobacco rattle virus (TRV) infection. This viral pathogen is vectored by stubby root nematodes (SRNs) (Paratrichodorus and Trichodorus spp.) in the Trichodoridae family that reside in the soil as obligate ectoparasites (Corky ringspot disease (CRS), also commonly referred to as “spraing” in Europe, is an important potato necrotic tuber syndrome caused by arasites . Symptomarasites . The lac3 of soil) can cause substantial CRS damage to a potato crop if left untreated  and systemic nematicides  can effectively reduce the population of the SRN vector in the soil, complete eradication is nearly impossible. SRN are mobile organisms that can survive deep in the soil profile re-emerging after pesticide application has subsided . FurtherStrategies to remediate land affected by CRS generally involve moving infected fields out of potato production and cropping varieties of alfalfa var. “Vernema” or Scotch spearmint var. “770”  as rotatUsing greenhouse pot experiments, we have examined the potential of the TRV resistant potato cultivar, Castle Russet, to cleanse soils containing viruliferous SRNs of TRV infection relative to a known host (tobacco), a non-host , and an unrelated popular potato variety (Russet Burbank). The objectives of this study were to assess how host plant genotype, cultivation time, and nematode inoculation pressure influenced nematode fecundity and the presence of TRV through multiple generations of SRN.Two independent experiments were carried out in a greenhouse at the USDA-ARS Temperate Tree Fruit and Vegetable Research Unit in Prosser, WA. The average temperature during the trials was 25°C with a 12-hour photoperiod. The plants were grown in 10 L plastic pots filled with a reconstituted sandy silt loam soil composed of 84% sand, 10% silt and 6% clay . The soi2 plastic pot filled with Sunshine #1 potting mix  and plants were allowed to develop in a growth chamber . Alfalfa var. “Vernema” was direct seeded into Sunshine potting mix #1 in a 72-cell tray. In Experiment 1, plants of Russet Burbank and Castle Russet (aka POR06V12-3) were propagated first in tissue culture. The tissue culture media was prepared by dissolving in 1 L of double distilled water 4.41 g of MS Modified BC Potato Basal Medium, 8 g of Plant TC agar , and 25 g of sucrose. The mixture was autoclaved at 120°C for 20 min. Tissue culture plantlets were kept for 1.5 months in the tissue culture media to develop a massive root system, and were then transferred to a 72-cell tray in Sunshine potting mix # 1. The foliage was pruned to induce vigorous growth. In Experiment 2, disease-free nuclear tubers were produced from tissue cultured plants and stored at 5.5°C until the dormancy of the tubers was broken.For both experiments, tobacco var. “Samsun NN” seed was bulk sown into a 10 cmParatrichodorus allius was maintained on TRV infected Samsun NN in a sandy silt loam soil (described above) in the greenhouse . The identity of the nematode was confirmed using light microscopy after nematode extraction . Experiment 2 was conducted beginning in September 2016. In total, 15 tubers of Russet Burbank and Castle Russet were planted in early August 2016. Approximately four weeks post planting all plants had emerged; 5 tobacco and 15 alfalfa were transplanted from potting mix into the sandy silt loam described above. Two weeks post-transplant, 1,060 P. allius in 5 mL of water were pipetted into three holes in each pot (~350 SRN/hole). Subjects within each experiment were assigned treatment categories using a randomized block design.An overview of the experimental procedure can be observed in One month post inoculation a number of replicates were destructively harvested and processed for evaluation of TRV infection. The number of replicates harvested was as follows: 10 tobacco, 10 alfalfa, 6 Russet Burbank and 6 Castle Russet for Experiment 1; 5 tobacco, 5 alfalfa, 5 Russet Burbank and 5 Castle Russet for Experiment 2. After plants were removed from the pots, a 250 cm³ sample of soil was collected and nematodes were isolated from the soil by sieving and sugar-centrifugal flotation . NematodTaq High Fidelity DNA Polymerase kit, Life Technologies, Carlsbad, CA), 2.5 μl of RediLoad (Invitrogen), 0.5 μl of 20 μM solutions of each forward and reverse primers TRV 2/1, respectively (Taq HiFi Mix (Life Technologies), and 2 μl of nucleic acid extracts. Thermal cycling  consisted of 15 min at 50°C, 5 min at 94°C, then 30 cycles of 15 sec at 94°C, 1 min at 58°C, 30 sec at 72°C, and a final incubation for 5 min at 72°C. Ten microliters of reaction products were resolved by electrophoresis in 1.5% agarose  gels in 10 mM Tris-acetate, 1 mM EDTA buffer (pH 8) for approximately 40 min at 100 V, stained with ethidium bromide, and observed under UV light.The harvested plants were washed and split into foliar, root and stolon/tuber (if present) sub-samples. For each sub-sample, approximately 0.5 to 1 g of tissue was grounded in grinding bags . A total nucleic acid extraction was performed using a modified Dellaporta procedure . Nucleicectively , 6.5 μl Two months post inoculation, the above procedure was repeated for the following number of replicates: 10 alfalfa, 6 Russet Burbank and 6 Castle Russet for Experiment 1; 5 alfalfa, 5 Russet Burbank and 5 Castle Russet for Experiment 2. Here, plants were destructively harvested, the nematode Rf was calculated from soil samples, and new tobacco bait plants were added. In addition, the aforementioned bait plants were destructively harvested and evaluated for TRV infection by RT-PCR. Three months post inoculation, the above procedure was performed on the remaining replicates  for Experiment 1; and  for Experiment 2. Again, plants were destructively harvested, the Rf was calculated from soil samples, and new tobacco bait plants were added. Additionally, the bait plants from the two-month evaluation were also tested. Four months post inoculation the bait plants from the three-month evaluation were destructively harvested and tested for TRV infection.All statistical analyses were performed within the R statistical computing environment . GeneralYijk is the individual observation of the dependent variable, μ is the overall mean, plant hosti is the effect of the ith plant host, monthj is the effect of the jth month, experimentk is the effect of the inoculation pressured applied in either of the kth experiment and ε is the random error term. The significance of each factor was tested using type III analysis of variance based upon likelihood ratio tests for the negative binomial and binomial variables (nematode count and RT-PCR detection of TRV respectively) and sum of squares in the case of log(Rf) using the car library in R (In which ary in R .α < 0.05) as implemented using the agricolae package in R  groupings of genotype at the final sampling time point within each experiment were determined using Tukey’s honest significant distance  = 50.14, phost< 7.45e−11) and exposure period (χ2month (1) = 26.09, pmonth< 3.27−07) both had a significant influence on SRN population size, whereas all three variables  appeared to significantly affect the nematode Rf  = 21.25, phost < 8.70−12; Fmonth  = 35.55, pmonth < 1.34E−08; Finoculation  = 6.76, pinnoculation < 0.002).The capacity of tobacco var. Samsun NN, alfalfa var. “Vernema”, Russet Burbank potato and Castle Russet potato to sustain populations of viruliferous SRN was investigated in two independent greenhouse trials that differed by SRN inoculation pressure . Overallχ2 tests (SRN counts) and sum of squares estimation  indicate plant host was the most influential factor, followed by the length of exposure, whereas inoculation pressure appeared to exhibit the smallest effect on both nematode count and Rf. Each plant species appeared to support increases of SRN populations throughout both experiments , carrot , watermelon , common groundsel (Senicio vulgaris), pigweed , Coast fiddleneck (Amsinckia intermedia), Longsping sandbur (Cenchrus longspinus), Redstem filaree (Erodium cicutarium) and Nicotiania clevelandi (Statistics from both likelihood ratio eriments  with Toberiments . In the eriments . Both cuevelandi .χ2root (3) = 130.22, proot = 4.85e−28; χ2foliar (3) = 121.87, pfoliar = 3.05E−26), length of time cultured on the plant host (χ2root (1) = 25.92, proot = 3.55e−07; χ2foliar (1) = 14.48, pfoliar = 0.00014), and the SRN inoculum pressure (χ2root (1) = 20.83, proot = 5.03e−06; χ2foliar (1) = 40.32, pfoliar = 2.16e−10). Plant host was the most influential variable associated with infection status in both plant root and shoot tissue (χ2root (3) = 130.22, proot = 4.85e−28; χ2foliar (3) = 121.87, pfoliar = 3.05E−26), whereas the length of exposure (χ2root (1) = 25.92, proot = 3.55e−07) and inoculation pressure (χ2foliar (1) = 40.32, pfoliar = 2.16e−10) was the next most influential variable associated with TRV detection in the root and shoot tissue, respectively. As observed in previous studies (The virulence of SRNs was evaluated over a three-month period by using RT-PCR to detect TRV from total nucleic acids extracted from foliage and root tissue of tobacco bait plants. The presence of TRV was significantly influenced by plant host ( studies , tobacco studies . In the  studies . However studies . Russet  studies . These r studies . The mec studies . Based u


Glutamicibacter nicotianae MSSRFPD35 was isolated and identified from Canna indica rhizosphere grown in distillery effluent contaminated sites. It showed high phenol degrading efficiency up to 1117 mg L–1 within 60 h by the secretion of catechol 1,2-dioxygenase via ortho intradial pathway. The strain MSSRFPD35 possess both the catechol 1,2 dioxygenase and catechol 2,3 dioxygenase coding genes that drive the ortho and meta pathways, but the enzymatic assay revealed that the strain cleaves catechol via ortho pathway. Haldane’s kinetic method was well fit to exponential growth data and the following kinetic parameter was obtained: μ∗ = 0.574 h–1, Ki = 268.1, Ks = 20.29 mg L–1. The true μmax and Sm were calculated as 0.37 h–1 and 73.76 mg L–1, respectively. The Haldane’s constant values were similar to earlier studies and healthy fitness depicted in correlation coefficient value R2 of 0.98. Phenol degrading kinetic’s was predicted using Haldane’s model as qmax 0.983, Ki′ 517.5 and Ks′ 9.152. Further, MSSRFPD35 was capable of utilizing different monocyclic and polycyclic aromatic hydrocarbons and to degrade phenol in the presence of different heavy metals. This study for the first time reports high phenol degrading efficiency of G. nicotianae MSSRFPD35 in the presence of toxic heavy metals. Thus, the strain G. nicotianae MSSRFPD35 can be exploited for the bioremediation of phenol and its derivatives polluted environments, co-contaminated with heavy metals.Biodegradation of phenol using bacteria is recognized as an efficient, environmentally friendly and cost-effective approach for reducing phenol pollutants compared to the current conventional physicochemical processes adopted. A potential phenol degrading bacterial strain  Phenol and its derivatives namely nitrophenol, halogenated phenol, alkylphenol, etc. are widely used in several industrial plants like petrochemical, phenol resin, pharmaceuticals, paint, textile, leather, pulp mills, coal conversion, and leather processing units . Gallons–1 (–1 (–1 (IS: 2490-1974) and 5 mg L–1 in public sewers (IS: 3306-1974)   and concfe forms .In addition to phenol, the effluent released from industries contain heavy metals like copper, lead, cadmium, chromium, etc., as co-contaminants in different composition and concentration which are highly toxic, persistent, and non-degradable in nature . The conAlthough numerous physio-chemical methods such as water chlorination, flocculation, photocatalysis, Fenton’s reaction, ozonisation, chemical oxidation, activated carbon adsorption, reverse osmosis, ion exchange with resin, etc., are widely adopted for the removal of phenol and heavy metal contaminants from industrial waste-waters  these aportho cleavage pathway which converts catechol into intermediate cis, cis muconic acid or the meta cleavage pathway that converts catechol to 2-hydroxymuconic semialdehyde (2-HMSA)   during pradation . Pseudompathways . Pseudompathways . Bacterionas sp. , Burkholeria sp. , Kocuriauria sp. , Acinetocter sp. , Arthrobcter sp. , Bacillullus sp. , Halomononas sp. , ArthrobW1, etc. .–1, and limited studies have reported the biological degradation efficiency of phenol under the influence of different heavy metals such as chromium, etc., till date  to isolate and characterize potential phenol degrading bacterial isolates, (ii) to evaluate kinetic models to determine the degradation efficiency of potential isolates, (iii) to identify molecular and metabolic pathways involved in the degradation of phenol, and (iv) to determine the impact of heavy metals pollutants in phenol biodegradation.Degradation kinetics of microbial bioremediation in bioreactor offers evidence for optimum design and efficient bioremediation of phenol contaminated effluents . Generalill date . Moreove2.2H2O, ZnSO4.H2O, (CH3COO)2Pb.3H2O, HgCl2, CdCl2, CuSO4.5H2O, NiCl2.6H2O, MnCl2.4H2O, and K2Cr2O7 were purchased from Merck Millipore, Taq DNA polymerase Master Mix from Ampliqon and PCR purification Kit from Favorgen Biotech Corp. were used.Davis minimal medium (DMM), 4-aminoantipyrine, potassium ferrocyanide, catechol, sodium hydroxide pellets used in this study were purchased from HiMedia Laboratories Pvt. Ltd. Mumbai, phenol, 2-mercaptoethanol, ammonium hydroxide, glycerol, disodium EDTA, primers, acids and solvents were procured from Sigma, Aldrich, Tris Base, Agarose from Bio Basic Inc. All the following heavy metals CoClCanna indica grown in sites contaminated by effluents from Distillery plant located in Vuyyuru, Krishna District, Andhra Pradesh, India (16°21′52.7″N 80°51′52.2″E). Three individual C. indica plants were uprooted and the rhizosphere soils from each sample were collected aseptically in sterile polythene bags and pooled. About 10 g of rhizosphere soil was used for the isolation of phenol degrading bacterial isolates by aseptically dispensing it into 100 ml of DMM containing dipotassium phosphate (7 g L–1), monopotassium phosphate (2 g L–1), ammonium sulfate (1 g L–1), sodium citrate (0.5 g L–1) and magnesium sulfate (0.1 g L–1) with final pH 7.0 supplemented with 200 mg L–1 of phenol and enriched by incubating in the shaker at 160 rpm at 30°C for 24 h. Then 1 ml of the enriched soil suspension sample was serially diluted and spread on DMM plate amended with 200 mg L–1 of phenol as the sole source of carbon with 1.8% agar and incubated for 48 h at 30°C and the colony-forming units (CFU) were counted. Diverse individual colonies were selected and streaked in 200 mg L–1 phenol amended DMM medium and stored in 25% glycerol (v/v) at −80°C for further analysis.Phenol degrading bacterial strains were isolated from rhizosphere soils of –1 of phenol were screened to determine the phenol degrading efficiency by inoculating in DMM agar plates amended with different phenol concentration of 600, 700, 800, 900, 1000, and 1200 mg L–1. The inoculated plates were incubated at 30°C for 72 h and the isolates with efficiency to grow in phenol concentration of 1200 mg L–1 were selected and maintained as pure culture  . The fingerprinting profiles were analyzed using GelJv.2 DNA tool by normalization, recognition, and assignment of bands on the gel by the Dice coefficient  . Each 20fficient . The cluThe 16S rRNA gene was amplified from the representative isolates of the BOX-PCR cluster groups using universal primers 27F (5′-AGA GTT TGA TCM TGG CTC AG-3′) and 1492R (5′-TAC GGH TAC CTT GTT ACG ACT T-3′) . Each 20–1 phenol. The culture inoculated broths were incubated in 30°C at 160 rpm for 96 h, the sample were withdrawn at every 24 h and analyzed for phenol concentration. A 1 ml of culture was centrifuged at 8000 rpm for 10 min to remove the cell debris and 50 μl of supernatant was made up to 1 ml and used to determine the phenol residual content through modified 4-amino antipyrine method at absorbance 510 nm by adding 25 μl of 0.5 N NH4OH to sample, followed by 15 μl of PBS (pH-6.9), 11 μl of 4-aminoantipyrine, and 11 μl of potassium ferrocyanide and incubated at 30°C room temperature for 15 min. The appearance of dark red color indicated positive reaction and intensity was measured using MultiskanTM GO Microplate Spectrophotometer at 500 nm  grown in DMM broth with phenol (200 mg L–1) was inoculated in DMM medium amended with phenol concentrations of 0 to 1117 mg L–1, flask without inoculation served as controls and the assay was performed in triplicates. The inoculated flasks were incubated on a rotary shaker at 160 rpm for 120 h at 30°C. At every 12 h time interval samples were withdrawn from each conical flask and the growth rate was measured as absorbance at 600 nm using MultiskanTM GO Microplate Spectrophotometer , the cell growth was follows first-order kinetics.The growth absorbance of MSSRFPD35 was converted into dry biomass using linear coefficient derived from growth absorbance vs. dry biomass  to t (a time when maximum biomass reached), it becomeswhen integrating Eq. (2) with limit X0 – initial biomass, X – biomass at time t, and Eq. (3) rewritten aswhere X/X0) vs. time.The experimental specific growth rate (μ) was calculated from the slope of a semi-logarithmic plot of dry biomass lnS0) was used to predict various kinetic parameters of Haldane’s kinetic model using non-linear regression analysis.Experimental μ was calculated for each initial phenol concentration and this μ value against different initial phenol concentration  in Eq. (5), .max is true maximum growth rate, calculated from Eq. (7).Where μThe phenol degradation rate from each sample was estimated by using 4-aminoantipyrine method as described above . Reductit0 to t (when complete depletion of phenol)Integrate Eq. (9) with time S0 – initial phenol concentration, S – phenol concentration at time t.where q) initial phenol concentration (S0) was acquired from the slope of the linear curve by plotting between −ln(S/S0) and time. The phenol degradation kinetics was acquired by experimental degradation rate of individual phenol concentration . Primers were designed for catechol 1,2-dioxygenase gene between 321 bp and 730 bp AC12O-F (5′-ATC GAA GGC CCT TAC TAC-3′); AC12O-R (5′-AAG TAC AGC TGG GCG GTG A-3′) and for catechol 2,3-dioxygenase between 3 bp to 819 bp AC23O-F (5′-GAG CAA AGA GAT CGC AAA CC-3′); AC23O-R (5′-GTA GAT CTC GAT GCG GTG GT-3′) using Primer3Plus program  and catechol 2,3-dioxygenase (PJJ43550). The nucleotide sequences of these enzymes from  program . PCR was program .–1 of phenol and harvested at late exponential phase (48 h) by centrifugation at 8000 rpm at 4°C for 10 min. The bacterial cells were washed and resuspended in Tris-HCl buffer (pH 7.6) and kept in ice to avoid heat generation during sonication for 4 min (1 or 2 min off) and to achieve complete lysis. The intracellular crude enzyme present in supernatant were separated from cell debris by centrifugation at 8000 rpm 4°C for 10 min and the cell-free extracts were stored at −20°C. Catechol 1,2-dioxygenase (Ortho enzymatic cleavage assay) and catechol 2,3-dioxygenase (Meta enzymatic cleavage assay) enzyme activities were spectrophotometrically determined using 3.5 ml of quartz cuvette as described by The strain MSSRFPD35 was inoculated in DMM medium containing 1000 mg LOrtho cleavage pathway was performed using 0.7 ml of distilled water, 2 ml of 50 mM Tris-HCL, 0.1 ml of 100 mM β-mercaptoethanol, 0.1 ml of 1 mM catechol and 0.1 ml of cell-free extract. The formation of aromatic ring cleavage product cis, cis muconic acid was measured spectrophotometrically at an absorbance of 260 nm. Similarly, the enzymatic assay for meta cleavage pathway was carried out using 0.6 ml of distilled water, 2 ml of 50 mM Tris-HCl, 0.2 ml of 100 mM catechol and 0.2 ml of cell-free extract mixed well and the increase in absorbance was measured at 375 nm for 5 min. The formation of 2-HMSA from catechol indicates the presence of catechol 2,3-dioxygenase activity at an absorbance of 375 nm. Enzyme activity and specific activities were calculated by equation described by The enzymatic assay for 2.4H2O – 800 mg L–1; iron (II) FeSO4 – 400 mg L–1; Zinc (II) ZnSO4.H2O – 600 mg L–1; lead (II) (CH3COO)2 Pb.3H2O – 200 mg L–1; cadmium (II) CdCl2 – 50 mg L–1; chromium (VI) K2Cr2O7 – 50 mg L–1; nickel (II) NiCl2.6H2O – 20 mg L–1; copper (II) CuSO4.5H2O – 20 mg L–1; cobalt (II) CoCl2.2H2O – 10 mg L–1; and mercury (II) HgCl2 – 10 mg L–1 in DMM medium with 1000 mg L–1 of phenol. The flasks were inoculated with 5% inoculum of G. nicotianae MSSRFPD35 (8 log CFU ml–1) and incubated on a rotary shaker at 160 rpm for 72 h at 30°C. DMM medium without heavy metals and inoculation were maintained as control and all the treatments were performed in triplicates. The samples from flasks were collected at every 24 h intervals and phenol concentration was measured.Heavy metal tolerance and phenol degradation potential of MSSRFPD35 were determined using manganese (II) MnCl–1; cinnamic acid 100 mg L–1; 1-chloro-2,4-dinitro benzene 50 mg L–1; 4-nitrophenol 50 mg L–1; catechol 100 mg L–1; 1-naphthol 50 mg L–1; naphthylamine 50 mg L–1; and gallic acid 200 mg L–1 was screened by inoculating 5% inoculum (8 log CFU/ml) of MSSRFPD35 in DMM broth containing phenol derivatives at the above-mentioned concentration and incubated at 30°C on a rotary shaker at 160 rpm. Bacterial growth was measured at 600 nm absorbance  after 72 h of incubation. Individual derivatives in DMM without inoculum served as negative control and were performed in triplicates.Growth of MSSRFPD35 in different phenol derivatives like tannic acid 50 mg L–1, 110 mg L–1 and 240 mg L–1) of phenol in sterile distilled water and 2.5 ml inoculum with 8 log10 CFU ml–1 of G. nicotianae MSSRFPD35 were added into the respective flask under aseptic condition and mixed thoroughly to get a uniform suspension of soil, phenol, and the inoculum. The treatments without microbial inoculum served as control and were performed in triplicates. The flasks were kept in 30°C for 10 days with gentle mixing at every 24 h. About 500 mg of soil samples were collected at the time of inoculation and on the 10th day of incubation from each treatment and suspended in 500 μl of distilled water, vigorously mixed and phenol was extracted by collecting the aqueous phase of the mixture and the phenol concentration was determined from the extracted samples as described above  and accession are provided, Canna indica rhizosphere soil suspension spread plated on DMM agar medium amended with 200 mg L–1 of phenol yielded 7.3 log10 CFU ml–1 after 48 h of incubation at 30°C. Around 128 different morphotype bacterial colonies were isolated and the phenol tolerance assay revealed that 11 isolates which were the most dominant colonies could grow in 1000 mg L–1 of phenol concentration and were coded as MSSRFPD27, MSSRFPD28, MSSRFPD29, MSSRFPD30, MSSRFPD35, MSSRFPD36, MSSRFPH100, MSSRFPH124, MSSRFPH134, MSSRFPH139, and MSSRFPH145. The BOX-PCR fingerprinting analysis showed the existence of polymorphism by amplifying three distinct patterns among the isolates. Dendrogram generated based on BOX-PCR profile with Dice coefficient and the UPGMA clustering method with 90% tolerance level revealed the existence of three diverse cluster groups  at 85% similarity coefficient , II (MSSRFPD28 and MSSRFPD36), and III (MSSRFPD29 and MSSRFPD35), showed 99.9% identity to ficiency .–1. Among the three strains, MSSRFPD35 rapidly degrade the 1000 mg L–1 of phenol completely within 48 h of incubation, followed by MSSRFPD30 and MSSRFPD36 in 60 h and 96 h, respectively  to calculate growth rate  and substrate (Sm) concentration at which it occurred were calculated by Eqs. (7) and (6) as 0.37 h–1 and 73.76 mg L–1, respectively. Here graphically predicted growth rate μ∗ (0.574) and true growth rate μmax (0.37) which were overestimated by 55% are shown in Si was calculated from the slope of the plot between negative logarithmic substrate concentration, −ln(S/Si) and time, t. The qs values clearly showed an inhibition effect which was reduced when Si was increased. The kinetic parameters were q∗ 1.244, Ks′ 9.152, Ki′ 517.5, Sm′ 68.820 and true maximum degradation rate qmax 0.983 obtained , Zn (600 mg L–1), Pb (200 mg L–1), Ni (20 mg L–1). The phenol degradation was 99% in the presence of heavy metals Pb and Ni, 83% and 93% in the presence of Mn and Zn, respectively, with the time frame of 72 h (–1) and Co (10 mg L–1) amended medium had an inhibitory effect on phenol degradation, were in only 24% and 43% of phenol were degraded, respectively, in 72 h of incubation. While Cd (50 mg L–1), Cr (50 mg L–1), and Hg (200 mg L–1) completely inhibited the growth of MSSRFPD35 and no phenol degradation was observed. The strain MSSRFPD35 grew in the presence of aromatic compounds like 4-nitrophenol (50 mg L–1), gallic acid (200 mg L–1), cinnamic acid (100 mg L–1), naphthol (50 mg L–1), tannic acid (50 mg L–1), naphthylamine (50 mg L–1), and catechol (100 mg L–1) by utilizing all of these as sole carbon source, but was not able to utilize 1-chloro-2,4-dinitrobenzene and failed to grow in the amended medium.The phenol degrading efficiency of MSSRFPD35 was not affected in the presence of heavy metal ions such as Mn  species. The potential of phenol degradation is a specific trait of the individual strain. Several studies reported that degree of phenol degradation and other functional traits varies among the same group, due to its habitat, presence of respective gene and its level of expression , 4-nitrophenol and 4-bromophenol phenol derivatives  and true (μmax) maximum growth rate were demonstrated in earlier studies conducted using substrate phenol (∗ (0.574) and true growth rate μmax (0.37) were overestimated. However, compared to other bacteria the μmax of MSSRFPD35 was second higher after Alcaligenes sp. TW1  in the presence of K2Cr2O7 at the concentration of 100 to 200 μg ml–1. Similarly, P. putida at the concentration of 150 mg L–1 and reported heavy metal ions such as iron, aluminum and zinc had no effect on the phenol biodegradation rate. Bacillus at maximum concentrations of 1000 mg L–1 and reduced Cr (VI) to Cr (III). This study revealed the potential of MSSRFPD35 in degrading phenol in the presence of different heavy metals. B. subtilis KCMRG5 and P. rhodesiae KCMR5 were able to degrade phenol in the presence of heavy metals. P. fluorescens (BBN1) and P. corrugata (BBB2) were reported to degrade PAH which were able to tolerate 993.6 mg L–1-Pb (NO3)2, 717.83 mg L–1-ZnSO4 and 499.36 mg L–1-CuSO4, respectively  can be found in the article/in vitro experiments, molecular analysis and manuscript correction. PR supervised and supported the study, revised the manuscript and approved for publishing. All authors contributed to the article and approved the submitted version.PD collected the samples, designed and performed the experiments, data analysis and drafted the manuscript. JS and AA supported sample collection, contributed to 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.


In vivo, they undergo metabolic degradation including gut microbial metabolism yielding urolithins. Only limited data is available about compounds being present in blood after intake of an extract from French oak wood, Robuvit®. In the course of a randomized, double-blind, controlled clinical investigation, 66 patients undergoing hysterectomy received placebo or 300 mg Robuvit® per day before and over 8 weeks after surgery. Serum and blood cell samples were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The number of urolithin producers and the urolithin levels increased after intake of Robuvit®. In serum samples, the median concentration of urolithin A was 14.0 ng/ml [interquartile range (IQR) 57.4] after 8 weeks. Urolithin B was determined at 22.3 ng/ml (IQR 12.6), urolithin C at 2.66 ng/ml (IQR 2.08). In blood cells, lower concentrations and only urolithins A and B were detected. A statistically significant association of lower post-surgical pain scores with metabotype A was detected (p < 0.05). To conclude, supplementation with French oak wood extract raised urolithin generation in patients and suggested health advantages for urolithin-producers.Ellagitannins are signature constituents of oak wood and their consumption has been associated with various health benefits.  Numerous studies investigating the impact of polyphenol-rich diets attributed these nutritional components health-promoting effects such as in cardiovascular and metabolic disease prevention as well as anti-inflammatory, anti-tumor, and neuroprotective activities  and hydrolysable tannins (ellagitannins) . EllagitEllagitannins are structurally variable and complex . Recentl®, a registered proprietary extract from French oak (Quercus robur L.) wood. Robuvit® contains e.g., gallic acid, ellagic acid, vescalagin, and castalagin. The extract is standardized according to its HPLC profile and its polyphenol content (> 40%). The specific phenolic composition and quantity have been previously analyzed and described in detail . Urolithin C and urolithin D were obtained from Biozol . Methanol (BAKER ANALYZED LC-MS Reagent), water (HiPerSolv CHROMANORM for HPLC MS grade), acetonitrile (Ultra Gradient HPLC Grade), and L(+) ascorbic acid were purchased from VWR . Formic acid, ammonium formate, ß glucuronidase from Helix pomatia , ethyl acetate, disodium ethylenediaminetetraacetic acid (EDTA), magnesium sulfate (anhydrous), sodium acetate, and Dulbecco's phosphate buffered saline were all obtained from Sigma Aldrich.The analytical standards caffeic acid, (+) catechin, ellagic acid, ferulic acid, gallic acid, trans para-coumaric acid, 3,4-dihydroxybenzoic acid (protocatechuic acid), urolithin A, urolithin B [3-hydroxyl-6H-benzo(c)chromen-6-one], 4-hydroxyl-3-methoxycinnamaldehyde , trans-3,5-dimethoxy-4-hydroxycinnamaldehyde , scopoletin, syringic aldehyde, syringic acid, vanillic acid, vanillin, and the internal standard 1,2,3-13C3 ferulic acid were prepared in methanol, aliquoted and kept at −80°C. They were diluted as needed with methanol to yield a working solution containing all analytes. The working solution containing the IS was prepared separately by diluting the stock solution of the IS to yield a concentration of 2 µg/ml in a mixture of methanol and water . The IS working solution was aliquoted and stored at −20°C.Stock solutions (1 mg/ml) of each analytical standard and the internal standard (IS) 1,2,3-Human serum and blood cells for the calibration curves and quality control samples were obtained from a blood bank . Serum originated from six different donors, was pooled, and stored in aliquots at −20°C. Blood cells were obtained by centrifugation  of whole blood and discarding of the upper layer. Blood cells of four different donors were pooled, aliquoted, and kept at −20°C.®, batch number Q086X, provided by Horphag Research Ltd., Geneva, Switzerland) three times daily (daily dose 300 mg) or placebo. Patient blood samples were obtained before surgery (t = 0) and four (t = 4) and eight (t = 8) weeks after surgery. At t = 4 and t = 8, samples were drawn approximately 12 h after the last intake of Robuvit® or placebo. Patients had given written informed consent, the study had been approved by the Ethics Committee of the University Hospital and the Faculty of Medicine, Comenius University Bratislava (10.01.2011) and has been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. The study has been registered with the study ID number ISRCTN11457040.Human serum and blood cells from 66 hysterectomy patients were provided by Prof. Z. Ďuračková . Samples were collected in the course of a randomized, double-blind, placebo-controlled clinical study detailed earlier . BrieflySamples were analyzed by HPLC MS/MS using two different chromatographic methods. The LC System (Agilent 1200 series) comprised a pump model BinPump SL G1312B, a degasser G1379B and an autosampler HiP ALS SL+ G1312B. A Pursuit PFP column  (method 1) and a Zorbax SB C18 column  (method 2) were used for the chromatographic separation . The mobile phase consisted of an ammonium formate buffer (5 mM) containing 0.065% formic acid (pH 3.2) (A) and methanol with 0.2% formic acid (B).For the quantification of urolithin A, urolithin B, (+) catechin, syringic acid, syringaldehyde, ferulic acid, caffeic acid, gallic acid, vanillic acid, para-coumaric acid, protocatechuic acid, sinapic aldehyde, scopoletin, coniferyl aldehyde, and vanillin, the solvent flow was 0.5 ml/min and the injection volume 5 µl. The gradient elution started at 20% B (0–1 min) and gradually increased to 60% B (2 min), was held constant until 6 min, increased further to 90% B (8 min), and 95% B (9 min) was kept until 13 min.Urolithin C, urolithin D, and ellagic acid were analyzed using a solvent flow of 0.3 ml/min and an injection volume of 2.5 µl. The gradient elution started with the same initial solvent composition as method 1 (20% B), was increased to 80% B (1.2 min), and held constant until 1.8 min. The ratio of solvent B was raised to 95% until 2 min and maintained for 5 min.For analyte detection a Triple Quadrupole LC/MS G6460A, equipped with an electrospray ion (ESI) source with JetStream , was used. The mode of detection was a dynamic multiple reaction monitoring (dMRM) with a cycle time of 750 ms in a positive and negative ionization mode.+ and [M−H]− in positive and negative ionization mode, respectively, for all analytes. Fragmentor voltage, collision energy, and cell accelerator voltage were optimized individually for each compound or transition by manually injecting standard substances in methanol (5 µg/ml)  and 500 V (negative). For the fragmentation of precursor ions in the collision cell, nitrogen was used as collision gas. The most abundant precursor ions were [M+H]Helix pomatia for 30 min at 37°C on a horizontal shaker (120 rpm). The incubation was stopped with ice. Sixteen microliters of formic acid and 50 µl of the IS working solution (2 µg/ml) were added and samples were vortexed. After addition of 1 ml ethylacetate samples were shaken for 5 min on a rotating mixer. Following phase separation by centrifugation for 5 min at 3,000 x g , 600 µl of the upper organic layer were evaporated under nitrogen until dryness and reconstituted with 200 µl of a mixture of methanol and water . Before LC MS/MS analysis, the extracted samples were centrifuged at 18,000 x g for 15 min (4°C).Serum samples were prepared by liquid liquid extraction after enzymatic hydrolysis of metabolic conjugates with β glucuronidase. Briefly, 500 µl of human serum samples were acidified with 4 µl formic acid (pH 5) and incubated with 8 µl aqueous β glucuronidase solution  from Helix pomatia, the samples were incubated for 30 min at 37°C under gently shaking. Subsequently, 50 µl of the IS working solution (2 µg/ml) was added, as well as 2.5 ml acetonitrile containing 1% acetic acid. After vortexing, QuEChERS salts (2 g magnesium sulfate and 500 mg sodium acetate) were added to each sample. The samples were thoroughly mixed on a Multivortex  and centrifuged to facilitate phase separation ; 850 µl of the upper layer were transferred to a vial containing 10 µl of ascorbic acid solution (10 mg/ml) and evaporated until dryness using a vacuum centrifuge . The residues were reconstituted with 100 µl of a mixture of methanol and water . Prior to LC MS/MS analysis, the extracted samples were centrifuged at 18,000 x g for 15 min (4°C).Human blood cells were extracted based on a adapted quick, easy, cheap, effective, rugged and safe (QuEChERS) approach as previously described  with somCalibration curves were prepared from spiked human pooled serum or blood cells and plotted using weighted linear regression (1/conc²). Study samples were extracted and quantified accordingly. If analytes were already present in the blank matrix, the calibration curves of the respective compounds were shifted along the y-axis depending on the response of the zero-samples (containing IS) as described previously .A validation of the method for the quantification of urolithin A, urolithin B, urolithin C, urolithin D, syringic acid, syringaldehyde, ferulic acid, caffeic acid, gallic acid, vanillic acid, para-coumaric acid, protocatechuic acid, sinapic aldehyde, scopoletin, coniferyl aldehyde, and vanillin in serum was performed according to For descriptive statistics of not normally distributed data, the median and interquartile range (IQR) was calculated instead of mean and standard deviation (SD). Statistical analyses were performed using R version 3.6.0 for Windows. Student's t-test was used for comparison of group differences in case of normally distributed data according to Shapiro-Wilk test. Not normally distributed data was analyzed using Mann-Whitney-U tests. A p-value < 0.05 was considered to be statistically significant.® (n = 33) or placebo (n = 33) after a medically indicated hysterectomy surgery procedure. The detailed flow diagram of the study has been published earlier  therefore only samples of 30 (t = 0) and 20 (t = 4) patients were available for analysis.Of the 70 patients recruited for the study (age 52.9 ± 9.4 years) 66 were randomized to receive three times daily either 100 mg of the oak wood extract Robuvit earlier . Blood sSerum and blood cells were analyzed separately as previously described  using a For serum, the lower limits of quantification (LLoQ), corresponding to the lowest data point of the calibration range, was lowest for syringaldehyde (0.3 ng/ml (1.5 nM); For blood cell samples, the LLoQ were generally higher than for serum ; interquartile range (IQR) 20.1 (130.2)], vanillic acid , scopoletin , and urolithins A , and B . In the serum of all patients  ferulic acid, protocatechuic acid, scopoletin and vanillic acid were found. In contrast, only two patients had measurable concentrations of p-coumaric acid and syringaldehyde  suggesting that the degree of urolithin conjugation was close to 100%.®, ferulic acid was detected at a median concentration of 7.81 ng/g (IQR 2.28), scopoletin at 16.64 ng/g (IQR 3.28), and vanillic acid at 54.58 ng/g (IQR 10.58). In contrast, compared to serum, lower concentrations of urolithins A and B were present in blood cells  and urolithin C was not found at all in blood cells.In blood cell samples, only five different polyphenols were determined Table 3.® and placebo group , 1.11 (IQR 0.27) and 1.58 (IQR 0.46), respectively, at t = 8.® the compound was not enriched in blood cells. A similar picture was seen for urolithin B with no data for t = 0 and ratios of 0.111 (IQR 0.029) and 0.153 (IQR 0.096) after 4 and 8 weeks, respectively  questionnaires  were avabotype A Figure 5The mean reduction of nociceptive points was minimal  in patients with metabotype 0 (n = 6). In contrast, patients with metabotype A reported a mean decline of −1.14  which differed by 0.98 score points (95% CI 0.05–1.90) from metabotype 0 and was statistically significantly lower (p = 0.039). The mean improvement of patients with metabotype B was even more pronounced , but not statistically significantly different from metabotype 0 due to high interindividual data variability.When metabotypes were classified based on the presence of urolithins in blood cells, similar results were observed. The mean reduction of nociceptive points was low  in patients with metabotype 0. Patients with metabotype A reported a mean decline of −1.40 (SD 0.84) which differed by 1.1 score points (95% CI 0.32–1.88) from metabotype 0 and was statistically significantly lower (p = 0.009). The mean improvement of patients with metabotype B was −1.33 (SD 1.86), which was not statistically significantly different from metabotype 0.In the present study, oak wood constituents and metabolites were determined in serum and blood cell samples of patients before and during the recovery phase after hysterectomy. For the first time, the concentrations and distribution of urolithins between serum and blood cells was analyzed and a statistically significant association between the urolithin metabotype and post-surgical pain scores was discovered.® focused on the detection of ellagic acid and urolithins in plasma samples. Compounds were quantified as ellagic acid equivalents due to the lack of reference standards for urolithin glucuronides  compared the earlier investigation (1 h). This suggests a rapid systemic elimination of ellagic acid, which has indeed been observed in human studies  were determined, followed by urolithin A (14.0 ng/ml) and urolithin C (2.66 ng/ml). This corresponded to mean serum levels of 451 nM (urolithin A), 153 nM (urolithin B), and 26 nM (urolithin C). Compared to other ellagitannin sources such as pomegranate, strawberries, or raspberries, the urolithin concentrations observed in the present study were comparatively high in relation to the administered dose  and urolithin B (2.89 ng/g) levels were quantified in blood cells. These concentrations are comparable to values of 2 ng/g reported for prostate tissue samples  expression  so that the sample number available for analysis was too low and data variability too high to confirm statistically significant differences in urolithin concentrations between the study groups. Isourolithin A was not quantified in this study. The characterisation of the study participants' metabotypes could have been refined with this additional information, since isourolithin A is another biomarker of metabotype B . The patients/participants provided their written informed consent to participate in this study.ZD and PH contributed conception and design of the study. LV and OS-C developed the analytical method, performed the blood sample and data analysis. VF, MJ, and ZD handled the patients, collected and analyzed clinical patient data. PH wrote the first draft of the manuscript. LV, ZD, and OS-C wrote sections of the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.This research project was funded by an educational grant of Horphag Research, Geneva, Switzerland. This publication was funded by the German Research Foundation (DFG) and the University of Würzburg in the funding program Open Access Publishing.ZD and PH received an educational grant of Horphag Research, Geneva, Switzerland. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. The remaining 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.


Anthracycline anticancer drugs, such as doxorubicin (DOX), can induce cardiotoxicity supposed to be related to mitochondrial damage. We have recently demonstrated that a branched-chain amino acid (BCAA)-enriched mixture (BCAAem), supplemented with drinking water to middle-aged mice, was able to promote mitochondrial biogenesis in cardiac and skeletal muscle. To maximally favor and increase oxidative metabolism and mitochondrial function, here we tested a new original formula, composed of essential amino acids, tricarboxylic acid cycle precursors and co-factors (named α5), in HL-1 cardiomyocytes and mice treated with DOX. We measured mitochondrial biogenesis, oxidative stress, and BCAA catabolic pathway. Moreover, the molecular relevance of endothelial nitric oxide synthase (eNOS) and mechanistic/mammalian target of rapamycin complex 1 (mTORC1) was studied in both cardiac tissue and HL-1 cardiomyocytes. Finally, the role of Krüppel-like factor 15 (KLF15), a critical transcriptional regulator of BCAA oxidation and eNOS-mTORC1 signal, was investigated. Our results demonstrate that the α5 mixture prevents the DOX-dependent mitochondrial damage and oxidative stress better than the previous BCAAem, implying a KLF15/eNOS/mTORC1 signaling axis. These results could be relevant for the prevention of cardiotoxicity in the DOX-treated patients. Ppargc1a and Ppargc1b, which is likely responsible for blocking transcription of mitochondrial genes ), 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin and 10% fetal bovine serum   stock solution dissolved in 30 mM Aldrich) ,16. MCF-Klf15, eNOS, and Raptor knockdown experiments, HL-1 cells were transfected with 50–100 nM Klf15, eNOS, and Raptor siRNA SMARTpool  or siGENOME nontargeting siRNA using Dharmafect 1 transfection reagent. After 24 h transfection, cells were treated with 1% α5 for 24 h and 1 μM DOX for 16 h. Transfection efficacy was determined with siGLO-RISC-free non-targeting siRNA and siRNA uptake by fluorescence detection (absorbance/emission 557/570). Proteins were then extracted for western blotting analysis.For the study of phospho-proteins, HL-1 cells were treated with 1% α5 for 2 h and 1 μM DOX for the last 60 min. For n = 10 mice) fed with standard diet  and receiving a single i.p. saline injection (vehicle); (2) the α5 group (n = 10 mice) fed with standard diet and α5 supplementation (1.5 mg/g body weight/day in drinking water) receiving a single i.p. saline injection (vehicle). α5 mixture was dissolved in tap water, after calculating the average daily drinking volume 2 weeks before the start of treatment and stored at 4 °C before daily administration; (3) the DOX group (n = 10 mice) fed with standard diet and receiving i.p. DOX (Doxo-HCl from Sigma-Aldrich) injection at 20 mg/kg, a dose that had been shown cardiotoxic . Th. Th14]. ted mice ,23.The activity was measured spectrophotometrically at 412 nm at 30 °C in left ventricle extracts ,25. TissOxygen consumption was measured as described ,27. Mito2O2 release was measured in the presence of horseradish peroxidase (HRP), using the Amplex Red Hydrogen Peroxide/Peroxidase Assay kit . Fluorometric measures were made using a Fusion Universal Microplate Analyzer  with excitation filter at 550 nm and emission filter at 590 nm. H2O2 production, calculated from a standard curve, was expressed as nmol/ min/mg protein as described  reagent (Sigma). HL-1 were seeded into 96-well culture plates at density 20,000 cells/well (100 μL). The purple formazan crystals were dissolved in 5% SDS/0.1 M HCl (100 μL/well), and the absorbance was recorded on a microplate reader  at a wavelength of 570 nm. Each test was repeated at least four times in quadruplicates.To quantify MCF7 cell growth, acid phosphatase assay was used as described . Brieflyt-test. Data are presented as the means ± standard deviations (SD), unless otherwise specified. A statistically significant difference was accepted at p < 0.05.Statistical analysis was performed with a one-way ANOVA followed by Student-Newman-Keuls’ test or Student’s PGC-1α, nuclear respiratory factor 1 (NRF1), mitochondrial transcription factor A (Tfam), cytochrome c (cyt c), and cytochrome c oxidase complex IV (COX IV) over the basal value, only PGC-1α expression was statistically increased by BCAAem  precursors, and cofactors on differentiating HL-1 cells . For thiy BCAAem A. Convery BCAAem A. Notably BCAAem A. Excepty BCAAem A. Hence,y BCAAem B.2O2 release  was markedly increased by DOX treatment; α5 supplementation prevented this effect  and ability to eliminate superoxides through SOD activity demonstrated that α5 supplementation prevented the oxidative stress induced by DOX, with beneficial effects also when added alone (glutathione peroxidase 1 (GPX1) and superoxide dismutase 1 (SOD1) genes was increased in DOX-treated compared to untreated HL-1 cells , a marker of oxidative DNA damage, in cells exposed to DOX E. The α5X1 genes D and by X1 genes E. FigureX1 genes . No effeX1 genes .in vitro results, an acute in vivo DOX treatment was performed as described . Be. Be7]. Bin vivo [Because we and others found that specific amino-acid combinations improve mitochondrial homeostasis in pathological conditions through eNOS and mTORC1 activity ,25,32, win vivo . Our datEmphasizing this hypothesis, Sestrin2 has been recently proposed as a leucine sensor for the mTORC1 pathway, since high Sestrin2 levels inhibit mTORC1 activity when intracellular leucine concentrations are low . ConversKlf15 mRNA levels were significantly reduced by DOX treatment, and this reduction was fully prevented by the α5 supplementation  has recently emerged as a critical transcriptional regulator of amino acid metabolism, particularly BCAA catabolism , especially in the heart , as wellentation F. No sigentation F. Given  failure ,42, we i failure ,44. NotaTo investigate more this hypothesis and to verify our findings specifically in cardiomyocytes, we sought to assess the impact of KLF15, eNOS, and mTORC1 on mitochondrial homeostasis in HL-1 cells exposed to DOX and α5. First, although eNOS gene expression was only slightly decreased by 1 µM DOX exposure, (Ser1177)-eNOS phosphorylation was markedly reduced  A,B. α5 Klf15 siRNA or nontargeting siRNA. The silencing efficacy was measured both at mRNA and protein levels -S6 phosphorylation -BCKDH phosphorylation both in DOX-treated and untreated cells, while DOX was unable to change phosphorylation when added alone —with specific siRNAs reduced PGC1-α and COX IV levels per se and abolished the ability of α5 supplementation to rescue the reduced protein levels when administered with DOX  is a highly effective and frequently used antineoplastic drug since its introduction in the 1960s, it causes a dose-related cardiotoxicity that can lead to severe heart failure. If the heart has been damaged by DOX, treatment options are few. Typically, DOX-induced cardiomyopathy and heart failure are refractory to conventional therapy . Increasin vivo, we found that the dietary supplementation of α5 activates a KLF15/Akt/eNOS/mTORC1 signaling axis -Akt phosphorylation. An extensive scientific literature demonstrates that the Akt-eNOS activation with drugs and food supplements mitigates the DOX-induced cardiac damage both in animals and humans ,60,61,62in vivo measurements of heart physiology in mice treated with DOX and α5. Our principal purpose, however, was to first assess the ability of the novel α5 formula to prevent the cardiac mitochondrial damage induced by DOX and to characterize the molecular mechanisms involved. Our results offer to study cardiac activity with more adequate coming in vivo studies. A second limit of this study is the absence of exploration on the neoplastic potential of α5, although this seems to be cast doubt by increasing evidence that supplementation of amino acids—especially the BCAAs—is beneficial mainly in liver cancer, yet their role in the nutritional support of cancer patients remains to be clearly defined [in vivo models whether the α5 supplementation can affect the antineoplastic efficacy of DOX. Nevertheless, the exposure of MCF7 breast cancer cell line to α5 mixture did not promote MCF7 cell proliferation, as assessed with two different assays  confirm that acute DOX treatment induces mitochondrial dysfunction in both cardiac tissue and cardiomyocytes, (2) demonstrate that a designer amino acid formula, that we named α5—after five abundances, among TCA intermediates and co-factors—markedly prevents this damage, (3) suggest that eNOS and mTORC1 signaling pathways are crucially involved in the action of this protector, and (4) show that KLF15, a specific transcription factor particularly important in cardiac development and circadian regulation, plays a relevant role in controlling these signaling axis. These findings highlight that dietary α5 supplementation might be a strategy in DOX-treated patients to prevent cardiomyopathy, acting on a complex signaling network.


Cudrania tricuspidata (C. tricuspidata) is widespread throughout East Asia and in China and Korea, and it is widely used as a traditional remedy against eczema, mumps, and tuberculosis. With regard to the aforementioned medical efficacy, various studies are continuously being conducted, and it has been reported that C. tricuspidata extract has various actions against inflammation, diabetes, obesity, and tumors. Therefore, we evaluated antiplatelet effects using derrone in C. tricuspidata. We examined the effect of derrone on the phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and inositol 1, 4, 5-triphosphate receptor I (IP3RI), and on the dephosphorylation of cytosolic phospholipase A2 (cPLA2), mitogen-activated protein kinases p38 (p38MAPK), and Akt, which affects platelet function and thrombus formation. Various agonists-induced human platelets were inhibited by derrone without cytotoxicity, and it also decreased the intracellular calcium level through the signaling molecule phosphorylations. In addition, derrone inhibited glycoprotein IIb/IIIa (αIIb/β3) affinity. Thus, in the present study, derrone suppressed human platelet aggregation and thrombin-induced clot formation. In normal circulation of blood, platelets are necessary for hemostasis and thrombosis. . The carαIIb/β3 activation, but its phosphorylation suppressed its activity [3RI [2+]i mobilization i concentration i) was elevated from 100.5 ± 0.4 nM to 755.5 ± 10.2 nM by collagen (2.5 μg/mL). However, derrone (15 to 60 μM) reduced the collagen-increased [Ca2+]i levels (μM) increased inositol 1, 4, 5-triphosphate receptor type I (IP3RI) phosphorylation. This shows that derrone decreased [Ca2+]i levels through phosphorylation of IP3RI. In addition, we explored whether derrone is involved in the inhibition of dense granule secretion. Serotonin is not synthesized in megakaryocytes or platelets, but is taken up from plasma and stored in dense granules in platelets. Upon stimulation by agonists, high [Ca2+]i levels facilitate the myosin light chain and pleckstrin phosphorylation to trigger δ-granule and α-granule release, and the serotonin released by exocytosis acts as a platelet agonist on the 5-hydroxytryptamine 2 (5-HT2) receptor on platelets [δ-granule release, and, as shown in μM) dose-dependently inhibited collagen-stimulated serotonin secretion. It is known that ERK phosphorylation is involved in influx from extracellular Ca2+ [μM) inhibited collagen-induced phosphorylation of ERK2 (42 kDa).As shown in i levels . Next, wlatelets . We evallar Ca2+ ; therefoμg/mL) increased the TXA2 generation to 55.8 ± 4.8 ng/108 platelets from 1.5 ± 0.2 ng/108 platelets. However, derrone inhibited TXA2 production , were investigated. The cPLA2 has been reported to hydrolyze arachidonic acid from membrane of platelets, and p38MAPK activates cPLA2 to act as an enzyme. As shown in Figures 2 and p38MAPK phosphorylation in a dose-dependent manner.As shown in oduction . To idenαIIb/β3 affinity, the binding of fibrinogen to αIIb/β3 is increased  regulates actin, but its phosphorylation by kinases inhibits affinity , 11. As  Figures , we inve Figures . Next, wαIIb/β3 in the previous experiment, we examined whether derrone affects fibrin clot retraction using PRP. As shown in μM) effectively delayed the thrombin-stimulated fibrin clot, with inhibitory degrees of 79.8%, 59.4%, 44.5%, 42.8%, and 34.7%, respectively . These data indicate that regulation of signaling molecules such as IP3RI (Ser1756) and VASP  influences the clot retraction delay.On the platelet surface,  Figures  by upreg Figures  and elev Figures . Moreovetraction , throughC. tricuspidata, administration of extract of C. tricuspidate (50 and 100 mg/kg) reduced hyperaggregated platelet aggregation without any hepatotoxicity in high-fat diet- (HFD-) fed rats. Moreover, a decrease in thromboxane A2 production was observed in vivo [L, euchrestaflavanone A, and cudraxanthone B [L and euchrestaflavanone A increased only the cAMP levels.According to the results of studies on the antiplatelet effects of  in vivo . In addinthone B –28. Theiin vitro and did not confirm the antiplatelet effect in vivo. Although the experiment was conducted using a low concentration (15–45 μM) of derrone in vitro, it is difficult to reach this concentration in blood by ingestion. Moreover, since our study is not an in vivo study using the human body, it is difficult to prove its effect in the human body; thus, we cannot prove that these effects would be the same in people with high and low platelet counts or cardiovascular disease. However, based on the in vitro effect, we suggest that derrone has the potential to inhibit thrombosis-mediated CVDs.Our study had some limitations, in that it was conducted αIIb/β3 affinity, and clot retraction, which were achieved by the regulation of IP3RI (Ser1756), VASP (Ser157 and Ser239), cPLA2 (Ser505), and p38MAPK. In addition, derrone increases cAMP and cGMP levels in human platelets. These two cyclic nucleotides are key mediators of the antiplatelet effects. Thus, we confirmed that derrone could be a potential phytochemical for the prevention of platelet-mediated illnesses.In conclusion, we confirmed that derrone suppressed collagen-stimulated platelet aggregation through downregulation of intracellular calcium concentration,


Heortia vitessoides Moore, which is an oligophagous pest of Aquilaria sinensis (Lour.), was characterized (HvATG8). Multiple sequence alignment showed that HvATG8 possesses highly conserved domain structures. Stage- and tissue-specific expressions indicated that HvATG8 is highly expressed in prepupal, pupal, and adult stages and in the midgut of larvae and abdomen of adults. Lack of function of HvATG8 by RNA interference resulted in a significant decrease in survival rate and an increase in abnormal or nonviable phenotypes in H. vitessoides. Transition rate from larval to pupal stages was 33.0% and from pupal to adult stages was 15.0% after injection. Reduction of ATG8 expression reduced survival of H. vitessoides. Therefore, HvATG8 possibly plays a key role in normal growth stage of H. vitessoides. HvATG8 suppression downregulates HvATG3 expression, suggesting that the two genes are interconnected. Further, HvATG8 expression increased by 20-hydroxyecdysone treatment, starvation, and extreme temperature exposure. Starvation also altered expression of other ATGs in H. vitessoide. This study may be used to guide research on molecular mechanisms of autophagy in insects.Autophagy is a highly conserved and regulated process in eukaryotic cells and remodels cytoplasm, recovers essential nutrients, and disposes of unwanted cytoplasmic components. Autophagy-related gene (ATG) 8, identified in  Aquilaria sinensis (Lour.) Gilg  is not only a traditional and precious natural spice, but it is also the only medicinal plant that can produce aloes [A. sinensis is the principal source of Chinese agarwood, a resinous heartwood formed in response to fungal infection, a moth whose leaf-eating larvae use A. sinensis as a sole food source [A. sinensis, causing serious financial losses [H. vitessoides [ce aloes ,2. The tce aloes . A. sined source . The motl losses ,5,6. Conl losses . Howeverl losses , but fewessoides .Autophagy is a self-digesting system of cells, which plays a pivotal role in maintaining the stability of the intracellular environment . Three dDrosophila melanogaster [Haemaphysalis longicornis [Galleria mellonella [D. melanogaster, mammals, and Caenorhabditis elegans [Autophagy is induced during intracellular structural remodeling to respond to environmental stresses, to encapsulate and digest nonfunctional cell components into lysosomes, and to recycle nutrients. These activities are critical for the maintenance of normal cell metabolism and function ,13. Undenogaster ,17,18, wnogaster , e.g., igicornis  and the llonella . New resllonella . Further elegans . Recent  elegans . G. mellonella [Bombyx mori [Aedes aegypti [Spodoptera litura [H. vitessoides (HvATG8). Autophagy-related protein 8 is one of two ubiquitin-like proteins involved in autophagy that communicates protein lipidation with phosphatidylethanolamine (PE) by the ubiquitin-activating enzyme E1-like enzyme ATG7 and ubiquitin-conjugating enzyme E2-like protein ATG3 ,26. The llonella , Bombyx byx mori , Aedes a aegypti , and Spoa litura . The chaHvATG8 homolog was identified, cloned, and characterized by silencing HvATG8 with RNAi as well as immunofluorescence targeting. Then, the transcription pattern was defined for responses to treatment with 20E, starvation, and extreme temperatures. Through the analysis of expression patterns and functions, the ATG8 status of H. vitessoides under unfavorable environments and the morphological changes and survival rates caused by silenced ATG8 were clearly demonstrated, further confirming the importance of studying ATG8; this provides theoretical support for further exploration of its biological functions and application of ATG8 insecticides.In this study, a H. vitessoides individuals were fed in a laboratory at a temperature of 26 °C and relative humidity of 70% ± 2%. Insects were sustained under a 14:10 h diurnal cycle. The larvae were reared with A. sinensisata leaves to promote into adults. We transferred mature larvae into plastic containers with sand at a relative humidity of 12% for later research. The larval stage of this insect has 5 instar stages; the entire larval stage takes 16–18 days for completion: approximately 10 days of the pupal stage and 2–4 days of the adult stage. There are slight differences depending on the environment. For stage-specific expression profiling of the target gene, 90 first-instar larvae , 6 of the third- to fifth-instar larvae , 45 instar larvae , 6 pupae, and 6 adults  were selected. Selecting a representative stage, it was observed whether the target gene has an important role in metamorphosis and whether it is expressed throughout the stage. All of the abovementioned steps were repeated thrice. One-day-old fifth-instar (L5D1) larvae were dissected into the head, epidermis, fat body, foregut, midgut, and hindgut; 1-day-old adults were dissected into the head, antenna of male and female, thorax, abdomen, leg, and wing. All samples were quickly frozen in liquid nitrogen and stored at −80 °C for total RNA extraction and cDNA synthesis.Concerning starvation treatment, 50 stochastically selected L5D1 larvae for each experimental group were deprived of food for 96 h and sampled at 12 h intervals for analysis.H. vitessoides transcriptome . Corresponding pairs of HvATG8-specific primers were designed by Primer Premier 5.0  to verify the sequences. PCR amplification conditions were as follows: 95 °C for 5 min, 34 cycles of 30 s at 95 °C, 58 °C for 30 s, 72 °C for 2 min, and 72 °C for 10 min. The PCR product was gel purified and then roped into a pClone007 simple vector , translated into Escherichia coli DH5α competent cells , and sequenced to confirm the target gene. The ATG8 amino acid sequences of other insect species were retrieved from the National Center for Biotechnology Information database. Sequence editing and multiple sequence alignment were performed with Clustal Omega  and DNAMAN 6.0. Physicochemical properties of the ATG8 amino acid sequence were predicted using the ExPASy online server (http://web.expasy.org/compute_pi/). A neighbor-joining tree was generated using MEGA-X [Through the 3035102; ), the ATH. vitessoides larvae and adults by using the E.Z.N.A. Total RNA Kit II  according to the manufacturer’s instructions. RNA concentrations were detected using a NanoDrop™ 2000 spectrophotometer . First-strand cDNA was synthesized with 2 µg total RNA from each sample using the Prime Script RT reagent kit with a gDNA eraser  and then directly stored at −20 °C for later use.Total RNA was isolated from 2O. Amplification conditions were as follows: initial denaturation at 95 °C for 5 min, 40 cycles at 95 °C for 10 s, 60 °C for 20 s, and cooling at 40 °C for 30 s. Negative controls were nontemplate reactions (cDNA replaced with diethyl-pyrocarbonate water). For RT-qPCR, the LightCycler® Real-Time PCR System was used. Three biological replicates and three technical replicates were used in RT-qPCR. The 2−ΔΔCt method was used in calculating the quantities of ATG8 mRNA [Primers were designed by Primer Premier 5.0  and synthesized by TSINGKE Biotech Co., Ltd. All primer sequences are shown as TG8 mRNA .HvATG8 and green fluorescent protein were used with T7 RNA polymerase to form dsATG8 (314 bp) and dsGFP  fragments. The DNA template was removed, followed by dsRNA annealing and single-stranded RNA (ssRNA) removal through nuclease digestion. Then, dsRNA was purified on the basis of the manufacturer’s purification protocol (Promega). After purification, dsRNA was dissolved in nuclease-free water, quantified using a NanoDrop 2000 spectrophotometer , and, in order to ensure the purity and integrity, 1.5% agarose gel electrophoresis was used. The reagents for RNAi experiments in dsRNA synthesis were acquired from the T7 RiboMAX™ Express RNAi System . To obtain the DNA template, primers with the T7 polymerase promoter sequence were designed for routine PCR. DNA templates for HvATG8 solution was diluted to a concentration of 3 μg/μL for injection, and each larva was injected with 1 μL of this solution. L5D1 insects were injected at the lateral internodal membrane by FemtoJet . The same concentration and dosage of dsGFP and DEPC were injected into controls [HvATG8 transcript expression levels were recorded every 12 h until the 96th h by RT-qPCR.The dscontrols . At leasThe treated insects should be checked discreetly for phenotypic changes. Individuals that did not react to brush touching within 1 min were considered dead. Surviving individuals were retained and continued to be monitored. We purchased 20E from Shanghai Yuanye Biotechnology Co., Ltd. , then diluted to 10 mg/ml with dimethyl sulfoxide and stored at −20 °C. The stock solution was diluted to a concentration of 1 μg/μL with 1× phosphate-buffered saline (PBS), and the injection volume was 1 μL. There were 30 larvae in each treatment that were injected between the membrane of the seventh and eighth segments. Each trial included three biological replicates and three technical replicates. Insects were collected in 1× PBS at 12, 24, 36, 48, and 60 h after injection and then reserved at −80 °C.L5D1 larvae were divided into three groups (n = 50) and deprived of food for 96 h. Larvae were collected at 12 h intervals and stored at −80 °C until further use.L5D1 larvae were selected in each group (n = 30). Larvae were exposed for 2 h to temperatures of 0 °C, 3 °C, 5 °C, 10 °C, 26 °C, 30 °C, 35 °C, 37 °C, and 40 °C. p < 0.05. One-way analysis of variance and Tukey’s test or two-way analysis of variance and Students’ t-test with SPSS 18. Excel (Microsoft) was used for primary statistical analysis. Experimental data are rendered as mean ± standard error through three independent replicates. We used GraphPad Prism 8 to create charts. Differences were considered statistically significant with H. vitessoides transcriptome data, ATG8 sequences were identified through keyword search and online comparison and designated as HvATG8 (GenBank accession number: MN788362). The sequence has an open reading frame of 357 bp length and encodes 118 amino acids, and the theoretical molecular mass is 29.54 kDa with a predicted isoelectric point of 5.27 , S. litura SlATG8 , Spodoptera. frugiperda SfATG8 , Trichoplusia ni TnATG8 , Helicoverpa armigera HaATG8 , and Ostrinia furnacalis OfATG8 . Amino acid sequences of ATG8 among insects appear highly conserved , Coleoptera (Group II), Diptera (Group III), Blattaria, Hemiptera, and Homoptera (Group IV). HvATG8 was most closely related to ATG8 in S. frugiperda with strong bootstrap support  when larvae were exposed to high or low temperatures. Among all temperatures, the highest HvATG8 expression level was detected at 40 °C, which was approximately 70 times that detected at 26 °C  from the transcriptome of adult H. vitessoides was identified in this study A. aegypti mosquitoes, autophagy plays a pivotal role in maintaining the egg maturation cycle [HvATG8 indicates that HvATG8 is expressed at all stages of development and that its expression is higher at prepupal–adult stages but lower at larval stages  undergo autophagic degradation [G. mellonella, the expressions of GmATG8 are detected in midgut, ovary, malpighian tubules, fat body, and silk gland, with the highest expression level in the midgut of larvae [HvATG8 expression was the highest in the midgut of larvae -injected larvae experienced difficulties during ecdysis, including a higher death rate [HvATG8 declined to a minimum at 72 h before increasing  injection caused HvATG8 silencing with peak inhibition at 72 h. Decrease in HvATG8 expression decreased HvATG3 expression, and the determination of the underlying mechanism for this observation warrants further research. RT-qPCR showed that HvATG8 expression could be caused by 20E. Its expression level is upregulated after starvation treatment, and ATGs in H. vitessoides are sensitive to starvation. Expression level also increased in response to extreme temperatures. Thus, extreme temperatures can trigger autophagy. In this study, RNAi-mediated targets were identified for pest control. The ATG8 sequence was successfully identified using the


Macadamia has increasing commercial importance in the food, cosmetics, and pharmaceutical industries. However, the toxic compound hydrogen cyanide (HCN) released from the hydrolysis of cyanogenic compounds in Macadamia causes a safety risk. In this study, optimum conditions for the maximum release of HCN from Macadamia were evaluated. Direct headspace analysis of HCN above Macadamia plant parts  was carried out using selected ion flow tube–mass spectrometry (SIFT-MS). The cyanogenic glycoside dhurrin and total cyanide in the extracts were analyzed using HPLC-MS and UV–vis spectrophotometer, respectively. HCN released in the headspace was at a maximum when Macadamia samples were treated with pH 7 buffer solution and heated at 50 °C for 60 min. Correspondingly, treatment of Macadamia samples under these conditions resulted in 93–100% removal of dhurrin and 81–91% removal of total cyanide in the sample extracts. Hydrolysis of cyanogenic glucosides followed a first-order reaction with respect to HCN production where cyanogenesis is principally induced by pH changes initiating enzymatic hydrolysis rather than thermally induced reactions. The effective processing of different Macadamia plant parts is important and beneficial for the safe production and utilization of Macadamia-based products. Macadamia-based commercial products have rapidly increased in recent years. In addition to Macadamia nuts, Macadamia flowers, husks, leaves, and shells are now widely used as a source of functional foods, beverages, and raw materials in cosmetics, feed, and other applications. Abundant antioxidant substances, such as polyphenols, can be extracted from Macadamia skin and husks for utilization in the food and pharmaceutical industries , mol L−1 versus heating time (min) plots for pH 2, 3, 4, 5, 6, and 7. At pH 8 and 9, the 90 min data point had to be excluded. The calculated empirical rates of hydrogen cyanide production (d[HCN]/dt) at different pH , followed by the leaves (513 ± 0.6 vppb), husks ; 256 ± 0.4 vppb, (Oc); 476 ± 0.7 vppb, (A16), and nuts (5.8 ± 0.1 vppb) at p < 0.05. Interestingly, the hydrogen cyanide concentration in Macadamia flowers was approximately 2–3 times higher than the highest concentration of cyanide detected in M. ternifolia cotyledon (~4800–6500 vppb) [Macadamia.00 ppbv) . Previou00 ppbv) . The flo00 ppbv) ,46,47. FMacadamia leaves (513 ± 0.6 vppb) is within the concentration range of cyanide (364–1403 vppb) detected in the leaf tissue of M. ternifolia, M. integrifolia, and M. tetraphylla species during their early to mid-developmental stages (3rd–4th week) [The concentration of hydrogen cyanide in  513 ± 0. ppbv is  513 ± 0. ppbv is th week) ,49,50.Macadamia husks are the fleshy green fibrous pericarp covering the conical or spherical hard brown shell enclosure of Macadamia nuts [Macadamia flowers, there are no available published data reported for the hydrogen cyanide concentration in Macadamia husks for comparison. Moreover, the hydrogen cyanide concentration of husks analyzed from three different Macadamia varieties were significantly different: 21 ± 0.1 vppb for variety 695; 256 ± 0.4 vppb for variety Oc; and 476 ± 0.7 vppb for variety A16  was lower than the reported cyanide concentrations in commercially used seeds (~74 vppb) of M. integrifolia or M. tetraphylla and significantly lower than the concentration detected in M. ternifolia (~4800 vppb), which is considered to be inedible.Seeds of  variety ,43. In tMacadamia plant part extracts with different treatments. Fresh, untreated Macadamia flower (424.4 ± 0.6 mg/L) had the highest amount of dhurrin compared to the fresh, untreated leaves (97.1 ± 0.6 mg/L) (.6 mg/L) A, fresh .6 mg/L) C.Macadamia flower (417.7± 0.8 mg/L), leaves (167 ± 2 mg/L), nuts (67.1 ± 0.6 mg/L), and husks . After full sample treatment using the optimized conditions , significant amounts of dhurrin and cyanide were removed in the analyzed extracts as shown in The cyanide concentration in the extracts have the same trend as the dhurrin concentration. Macadamia flower and leaf samples. On the other hand, treating the Macadamia flower and leaf samples with buffered solution at pH 7 without heating (buffered-only), resulted in significant removal of dhurrin , treatment produced about similar removal effectivity (It is interesting to note that heating samples at 50 °C for 60 min without pH adjustment (heated-only samples) had little to no effect on the removal of dhurrin A or cyan dhurrin  showed t cyanide . Howeverectivity  of dhurrMacadamia samples were 50 °C, 60 min at pH 7. Under these treatment conditions, trace amounts of hydrogen cyanide could still be detected in the headspace directly above the different Macadamia plant part samples using SIFT-MS. The measured hydrogen cyanide in the headspace of the treated samples were 12,535 ± 11 vppb (flower), 513 ± 0.6 vppb (leaves), 6 ± 0.1 vppb (nuts), 476 ± 0.7 vppb (husk A16), 256 ± 0.4 vppb (husk Oc), and 21 ± 0.1 vppb (husk 695). Treatment of Macadamia samples under these optimum conditions produced 93–100% removal of dhurrin and 81–91% removal of total cyanide in the sample extracts. Treatment by pH 7 adjustment (buffered-only) without heating also resulted in an effective removal of dhurrin (86–100%) and total cyanide (88–89%) in Macadamia extract similar to the full, optimized treatment conditions. Heating the samples alone at 50 °C for 60 min without pH adjustment was not effective in the hydrolysis and removal of cyanogenic glycoside dhurrin and total cyanide in Macadamia samples. The varying concentration of generated hydrogen cyanide could be correspondingly attributed to the concentration of cyanogenic glycosides (such as dhurrin) from the different parts of the Macadamia plant and their subsequent hydrolysis to hydrogen cyanide. Cyanogenic glycosides were greatest in Macadamia flowers, followed by the leaves and husks (depending on variety), and lowest in nuts. The results indicate that the hydrolysis of cyanogenic glycosides in Macadamia is predominantly induced by pH changes rather than by heat. This further suggests that the enzymatic hydrolysis involved in cyanogenesis is chiefly pH-directed rather than thermally induced. In addition, the hydrolysis reaction of cyanogenic glycosides could be described as a first-order reaction with respect to the in vitro production of hydrogen cyanide.The optimum conditions for the maximum release of hydrogen cyanide in Macadamia. Moreover, the evaluated optimum conditions for the hydrolysis of dhurrin and removal and release of hydrogen cyanide could be helpful for the effective processing of different parts of Macadamia. Such information provides some guidelines toward the safe production, utilization, and consumption of Macadamia-based products.These results provide further insights into the cyanogenic systems in


Taraxacum officinale L.) roots, leaves, and flowers have a long history of use in traditional medicine. Compared to the above organs, dandelion fruits are the least known and used. Hence, the present paper was aimed at the phytochemical analysis of T. officinale fruit extract and estimating its antiradical, antiplatelet, and antioxidant properties related to hemostasis. Methanolic extract of fruits (E1), enriched with polyphenols /g), was successfully separated into cinnamic acids  and flavonoids  extracts. Flavonoid extract was further divided into four fractions characterized by individual content: A , B , C , and D . High DPPH radical scavenging activity was evaluated for fractions A and B (A > B > Trolox), medium for extracts (Trolox > E3 > E2 > E1), and low for fractions C and D. No simple correlation between polyphenol content and antiradical activity was observed, indicating a significant influence of qualitative factor, including higher anti-oxidative effect of flavonoids with B-ring catechol system compared to hydroxycinnamic acids. No cytotoxic effect on platelets was observed for any dandelion preparation tested. In experiments on plasma and platelets, using several different parameters , the highest antioxidant and antiplatelet potential was demonstrated by three fruit preparations–hydroxycinnamic acids extract (E2), flavonoid extract (E3), and luteolin fraction (A). The results of this paper provide new information on dandelion metabolites, as well as their biological potential and possible use concerning cardiovascular diseases.Dandelion ( The occurrence of some civilization diseases, such as atherosclerosis, diabetes, heart attack, and some cancers, has been associated with elevated levels of oxidative stress and dysfunctional hemostasis. Hemostasis is defined as a sequence of reactions that maintain the fluidity of circulating blood, while also preventing its outflow in the event of a break in the vessel ,2. HowevT. officinale, as a source of bioactive compounds bestowing anti-inflammatory, antioxidative, choleretic, diuretic, hepatoprotective, and immunostimulatory effects , where Ablank and Asample are the absorbance values of the blank and test samples at t = 30 min, respectively.Inhibition (%) = 100 × [ of samples on DPPH, the slope of the sample linear curve, i.e., the absorbance inhibition (%) vs. concentration (µg/mL), was divided by the slope of the standard linear curve.50 value of extracts and fractions, defined as the concentration of sample necessary to cause 50% inhibition was determined from the sample linear curves as absorbance inhibition (%) vs. concentration (µg/mL), with Trolox used as a positive control.The ICHuman blood was obtained from a Medical Center in Lodz (Poland), and the biological material came from regular, non-smoking/non-drinking alcohol and medication-free donors. The blood was collected into tubes with citrate/phosphate/dextrose/adenine (CPDA) anticoagulant. All experiments were approved by the University of Lodz Committee for Research on Human Subjects and carried out under permission number 2/KBBN-UŁ/II/2016.8/mL. The protein concentration was calculated by measuring the absorbance of tested samples at 280 nm according to Whitaker and Granum [Plasma and blood platelets were isolated from fresh human blood by differential centrifugation as described previously . The plad Granum . Each saT. officinale L. fruit extracts and fractions were prepared with 50% DMSO (v/v). The final concentration of DMSO in test samples was lower than 0.05%, and its effects were determined in all experiments. The extracts and fractions were then taken for use in different models to evaluate the antithrombotic properties. Briefly:Stock solutions of the 2O2/3.8 mM Fe2SO4/2.5 mM EDTA .The plasma was pre-incubated for five minutes at 37 °C with dandelion extracts (E1–E3) and fractions (A–D) at two concentrations, 10 and 50 µg/mL, and then treated with 4.7 mM HThe blood platelets were incubated for 30 min at 37 °C with dandelion extracts (E1–E3) and fractions (A–D) at final concentrations of 10 and 50 µg/mL.2O2/3.8 mM Fe2SO4/2.5 mM EDTA (25 min at 37 °C).The blood platelets were pre-incubated for five minutes at 37 °C with dandelion extracts (E1–E3) and fractions (A–D) at two concentrations, 10 and 50 µg/mL, and then treated with 4.7 mM HThe blood platelets were pre-incubated (25 min at 37 °C) with dandelion extracts (E1–E3) and fractions (A–D) at two concentrations, 10 and 50 µg/mL, and then treated with thrombin at a final concentration of 5 U/mL (5 min at 37 °C).Whole blood was incubated (30 min at 37 °C) with dandelion extracts (E2 and E3) and fraction A at final concentrations of 10 and 50 µg/mL.p-nitrophenol produced was measured at 405 nm, using SPECTROstar Nano Microplate Reader . A control group was platelets without the addition of plant preparations; and its absorbance was expressed as 100%.Adhesion of blood platelets to fibrinogen was determined colorimetrically by measuring acid phosphatase activity . Blood p2+ and stained with anti-CD61/PerCP, anti-CD62/PE, or PAC-1/FITC antibodies . Appropriate isotype controls were then prepared: the resting blood samples were stained with anti-CD61/PE and isotype control antibodies marked with FITC or PE. Finally, all samples were fixed with 1% CellFix .Changes in the activation and reactivity of resting and stimulated blood platelets were studied in the whole blood model . Fresh bThe platelets were counted using an LSR II Flow Cytometer  based on the fluorescence of 10,000 platelets (CD61/PerCP positive objects). The platelets were distinguished from other blood cells by a forward light scatter (FCS) vs. side light scatter (SSC) plot on a log/log scale (first gate) and by positive staining with monoclonal anti-CD61/PerCP antibodies (second gate). The percentages of CD62P-positive and PAC-1-positive platelets were calculated relative to the total number of platelets (CD61-positive cells) presented in each sample. Non-specific antibody binding was determined using the isotype control antibodies IgG1/PE and IgM/FITC. All results were analyzed using BD FACSDiva software .Plasma (50 µL) was incubated in a measuring cuvette , then a 100 µL of thrombin at the final concentration of 5 U/mL was added. The TT was measured using an Optic Coagulation Analyser . The procedure was carried out as described by Malinowska et al . Time unPlasma (50 µL) was incubated in a measuring cuvette , then a 100 µL of Dia-PT liquid  was added. The PT was measured using an Optic Coagulation Analyser . The procedure was carried out as described by Malinowska et al . Time un2 was added. The APPT was measured using an Optic Coagulation Analyser . The procedure was carried out as described by Malinowska et al [Fifty microliters of Dia-PTT liquid  were added to 50 µL of human plasma, followed by incubation at 37 °C (3 min). Afterward, a 50 µL of 25 mM CaClka et al . Time un® was used to analyze the thrombus formation process under flow conditions. Platelet thrombus formation was measured using the PL-chip microchip coated with collagen. Whole blood (400 µL) anticoagulated with BAPA (benzylsulfonyl-D-arginyl-prolyl-4-amidinobenzylamide) was incubated with the tested fractions . Subsequently, samples (340 µL) were transferred to the PL-chip. The results were taken as AUC10 i.e., Area Under the Curve [T-TAShe Curve .The cytotoxicity of dandelion fruit preparations against blood platelets was examined by measuring extracellular LDH activity. Firstly, the 270 µL of 0.1 M phosphate buffer, 10 µL of supernatant, and 10 µL of nicotinamide-adenine-dinucleotide (NADH) were added to the microtiter plate and then incubated . Thereafter, 10 µL of pyruvate was added and the absorbance was immediately measured at 340 nm using a SPECTROstar Nano Microplate Reader . The measurement was carried out for 10 min, and it was repeated every minute .v/v) trichloroacetic acid in 0.25 M HCl and 0.37% (v/v) thiobarbituric acid in 0.25 M HCl were transferred to the mixture. Then, the samples were incubated in the boiling water bath for 10 min, followed by cooling in an ice bath and centrifugation  [−1 cm−1). The results were expressed as nmol/mL of plasma, or nmol/108 blood platelets.Lipid peroxidation was determined by measuring the concentration of thiobarbituric acid reactive substances (TBARS). The procedure was carried out according to Bartosz . After i, 18 °C) ,50. Abso−1 cm−1). The results were expressed as nmol/mg of plasma protein, or nmol/mg of platelet protein.The level of protein carbonyl groups in plasma was measured by the addition of 2,4-dinitrophenylhydrazine (DNPH), which in the dark binds with this functional group. The resulting colored compound was assayed spectrophotometrically at 375 nm using a SPECTROstar Nano Microplate Reader . The procedure was carried out according to Levine et al . and Bar−1 cm−1). The results were expressed as nmol/mg of plasma protein, or nmol/mg of platelet protein.Thiol group level was measured using Ellman’s reagent, i.e., 5,5′-dithiobis-(2-nitrobenzoic) acid (DTNB). The procedure was carried out according to Ando and Steiner ,53. The p < 0.05. To eliminate uncertain data, the Q-Dixon test was performed.Data distribution was checked by normal probability plots, and the homogeneity of variance by Levene’s test. Differences within and between groups were assessed by the Kruskal-Wallis test; for the sake of clarity, only the differences between the tested preparations and the control/control positive were marked. Additionally, in


Plasmodium falciparum in the four provinces with the highest malaria burden in Pakistan, after introducing artemether–lumefantrine as first-line treatment in 2017.The spread of artemisinin resistance in the Greater Mekong Subregion of Southeast Asia poses a significant threat for current anti-malarial treatment guidelines globally. The aim of this study was to assess the current prevalence of molecular markers of drug resistance in Plasmodium falciparum infections were confirmed by rapid diagnostic test or microscopy. Plasmodium falciparum positive isolates (n = 179) were screened by Sanger sequencing for single nucleotide polymorphisms (SNPs) in the P. falciparum kelch 13 (pfk13) propeller domain and in P. falciparum coronin (pfcoronin). SNPs in P. falciparum multidrug resistance 1 (pfmdr1) N86Y, Y184F, D1246Y and P. falciparum chloroquine resistance transporter (pfcrt) K76T were genotyped by PCR-restriction fragment length polymorphism.Samples were collected during routine malaria surveillance in Punjab, Sindh, Baluchistan, and Khyber Pakhtunkhwa provinces of Pakistan between January 2018 and February 2019. pfk13 propeller domain or in pfcoronin. The pfmdr1 N86, 184F, D1246 and pfcrt K76 alleles associated with reduced lumefantrine sensitivity were present in 83.8% (150/179), 16.9% (29/172), 100.0% (173/173), and 8.4% (15/179) of all infections, respectively. The chloroquine resistance associated pfcrt 76T allele was present in 98.3% (176/179) of infections.No artemisinin resistance associated SNPs were identified in the P. falciparum sensitivity to artemether and/or lumefantrine in Pakistan, including a first baseline assessment of polymorphisms in pfcoronin. No mutations associated with artemisinin resistance were observed in pfk13 or pfcoronin. However, the prevalence of the pfmdr1 N86 and D1246 alleles, that have been associated with decreased susceptibility to lumefantrine, remain high. Although clinical and molecular data suggest that the current malaria treatment guidelines for P. falciparum are presently effective in Pakistan, close monitoring for artemisinin and lumefantrine resistance will be critical to ensure early detection and enhanced containment of emerging ACT resistance spreading across from Southeast Asia.This study provides an update on the current prevalence of molecular markers associated with reduced  Plasmodium vivax, 15% by Plasmodium falciparum, and 1% were mixed P. falciparum and P. vivax infections.Despite ongoing global efforts to control and eradicate malaria, the disease remains a significant contributor to morbidity and mortality worldwide. According to the World Health Organization (WHO), there were over 228 million cases of malaria in 2018, resulting in approximately 405,000 deaths [P. falciparum resistance to artemisinin and its partner drugs spreading across Southeast Asia [P. falciparum kelch 13 (pfk13) gene [pfk13 C580Y mutation has been reported in Guyana [pfk13 resistance mutations (P574L and A675V), which are common in southeast Asia and have been associated with delayed parasite clearance, have been identified in Rwanda [pfk13 has been reported in Eastern India after treatment with artesunate-sulfadoxine-pyrimethamine (AS + SP) [Malaria control relies on a handful of interventions including prompt and effective treatment with artemisinin-based combination therapy (ACT); a strategy currently threatened by ast Asia , 4. Arteast Asia , 6, and ast Asia , 8. It i13) gene , 10. Posn Guyana  and Papun Guyana , and cann Rwanda –16. ReceAS + SP) , 18.pfk13, SNPs in the P. falciparum actin-binding protein coronin (pfcoronin) gene  have recently been identified to confer reduced in vitro sensitivity to artemisinin [pfcoronin mutants could emerge as a non-pfk13 type of artemisinin resistance in natural settings [P. falciparum multidrug resistance 1 (pfmdr1) and P. falciparum chloroquine resistance transporter (pfcrt) genes have been associated with decreased susceptibility to chloroquine, and important ACT partner drugs including lumefantrine, mefloquine, and amodiaquine [The availability of molecular markers for both artemisinin- and partner drug resistance has greatly aided close surveillance of the emergence and spread of ACT resistance. In addition to emisinin . Whilst emisinin , it has settings . Moreovediaquine –27.P. falciparum infections in 2007. However, after rapid increases in AS + SP treatment failure rates in Somalia and Sudan (ranging from 12% to 22%) [P. falciparum malaria [After establishment of chloroquine resistance in Pakistan, the WHO recommended AS + SP for treatment of uncomplicated  to 22%) , and the to 22%) , 18, thepfcoronin gene and prevalence of SNPs in the pfk13 propeller region, that have been associated with reduced sensitivity to artemisinin, as well as the proportions of infections harbouring the pfmdr1 N86, 184F, D1246 and pfcrt K76 alleles that have been associated with reduced sensitivity to lumefantrine [This study aims to provide an update on the current prevalence of molecular markers associated with drug resistance after introduction of AL as first-line treatment, in the four provinces with the highest malaria burden in Pakistan. This includes a first baseline assessment of polymorphisms in the fantrine , 28–36.Seventeen government and private hospitals in the four provinces with the highest malaria burden in Pakistan (Khyber Pakhtunkhwa (KPK), Sindh, Balochistan, and Punjab) were invited to provide blood samples during routine malaria surveillance. Ten facilities responded and were included in this study: Bannu, Kohat, Karak, Dera Ismail Khan, Frontier Region (FR) Khyber, FR Dera Ismaeel (DI) Khan, and Hangu in KPK province; Karachi in Sindh Province; Quetta in Balochistan Province; and Dera Ghazi Khan in Punjab Province Fig. . SymptomApproximately 5 ml of whole blood was collected in EDTA tubes from 209 patients with malaria infections confirmed by rapid diagnostic test and/or microscopy. Samples were shipped to Karolinska Institutet, Stockholm, Sweden for subsequent molecular analysis. DNA was extracted from 200 μl of whole blood using the QIAamp DNA mini kit , following the manufacturer’s guidelines. Extracted DNA was stored at − 20 °C.pfk13-propeller domain was amplified with Q5 high-fidelity polymerase  using a previously described nested polymerase chain reaction (PCR) protocol [P. falciparum 3D7 positive control and a no template control was included in each PCR. Amplified PCR products were separated by electrophoresis in 1.5% agarose gels stained with GelRed® . The 849-base pair fragment was extracted from the gel and purified using GeneJET Gel Extraction Kit . The purified PCR products from clinical isolates and 3D7 positive control were sent for bidirectional Sanger sequencing at Macrogen, Netherlands. The sequencing data was analysed using Sequencher version 5.1 software . Generated sequences were aligned with the reference sequence of 3D7 (PlasmoDB gene ID: PF3D7_1343700). Nested PCR and sequencing were repeated in case of poor-quality sequencing output.The 849 base pair fragment covering amino acid positions 407–689 in the pfcoronin where all three SNPs associated with artemisinin resistance were reported [2, 1 × GoTaq reaction buffer, and 0.75U GoTaq® DNA polymerase , using the following cycling program: 30 s at 90 °C; followed by 30 cycles of 20 s at 90 °C, 30 s at 45 °C, 1.30 min at 68 °C. For the nested PCR, 1 µL of primary PCR products were amplified under the same conditions using the following cycling program: 30 s at 90 °C; followed by 45 cycles of 15 s at 94 °C, 30 s at 50 °C, 1 min at 68 °C. A 3D7 positive control and a no template control was included in each PCR. The nested PCR products were sent for direct bidirectional Sanger sequencing at Macrogen and analysed as described above with 3D7 as a reference (PlasmoDB gene ID: PF3D7_1251200).A 368 base pair fragment covering the region in the N-terminal WD40 domain of reported , was amppfmdr1 N86Y, Y184F, and D1246Y and pfcrt K76T were genotyped following nested PCR-restriction fragment length polymorphism (RFLP) protocols as summarized in [®  and documented with a GelDoc™ system.SNPs at positions rized in . Laboratpfmdr1 haplotype analysis; frequencies for haplotypes were otherwise calculated as for the SNPs.The prevalence of each SNP was defined as the proportion of isolates containing the allele of interest, including mixed infections. Isolates containing mixed SNP results at more than one position were excluded from the P. falciparum cases from 10 different sites across the country were enrolled in this study between January 2018 and February 2019  were male and 59 (33%) were female, with ages ranging from two to 65 years, and a median age of 25 years.In total, 209 uncomplicated pfk13 propeller domain and pfcoronin were 97.7% (175/179) and 100% (179/179), respectively, of which 121 (69.1%) and 149 (83.2%) generated good quality sequences. No artemisinin resistance associated SNPs were identified in either of the analysed fragments, hence all successfully sequenced samples showed wild-type genotypes in both pfk13 and pfcoronin.PCR success rates for the nested PCR amplifying the pfmdr1 N86Y and pfcrt K76T were 100% (179/179); PCR success rates for pfmdr1 Y184F and D1246Y were 96.1% (172/179) and 96.6% (173/179), respectively , 16.9% (29/172), 100.0% (173/173), and 8.4% (15/179) of all infections, respectively, with similar distribution across the four provinces  of the samples, followed by pfmdr1 YYD in 22.9% (38/166), and pfmdr1 NFD in 15.1% (25/166) of the samples. The pfcrt 76T allele that is associated with chloroquine resistance was found in 98.3% (176/179) of the samples.PCR success rates for the nested PCRs for determining SNPs in pfcoronin gene which have been associated with decreased in vitro sensitivity to artemisinin.Pakistan has set a target to reduce the malaria burden by 75% in high endemic districts and to eliminate malaria in low endemic districts by 2020 . Early spfk13 propeller domain, nor in pfcoronin. One other study has to date, assessed the prevalence of polymorphisms in pfk13 in Pakistan [No SNPs associated with artemisinin resistance were observed in the Pakistan . This stPakistan . HoweverPakistan . EspeciaPakistan , 40.P. falciparum in the whole of the WHO Eastern Mediterranean Region, covering Afghanistan, Djibouti, Pakistan, Somalia and Sudan [pfk13 and pfcoronin, the pfmdr1 N86 and D1246 alleles which have been associated with reduced sensitivity to lumefantrine [pfmdr1 N86 allele in particular, as well as increased pfmdr1 copy number, have shown to be significant independent risk factors for recrudescence in patients treated with AL [pfmdr1 N86 has increased since the introduction of ACT in Pakistan in 2007, with a prevalence ranging from 55 to 67% in 2005–2007, and 96% in 2011,  of the chloroquine resistance associated essments , 50, 51,P. falciparum sensitivity to artemether and/or lumefantrine in Pakistan. No mutations associated with artemisinin resistance were observed in pfk13 or pfcoronin. However, the prevalence of the pfmdr1 N86 and D1246 alleles, that have been associated with decreased susceptibility to lumefantrine, remain high. Although clinical and molecular data suggest that the current malaria treatment guidelines for P. falciparum are presently effective, close monitoring for artemisinin and lumefantrine resistance will be critical to ensure early detection and enhanced containment of emerging ACT resistance spreading across from Southeast Asia.This study provides an update on the current prevalence of molecular markers associated with reduced