Serum samples of 16 individuals were used in this study. The concentration of folate and vitamin B12 has been determined at t=0 and at 1, 8, and 13 years after storage at −80°C. The folate concentrations in serum samples remained stable at −80°C. The concentration of vitamin B12 was decreasing during the time of the study to about 50%. The correlation of the folate and also of the vitamin B12 concentrations in the stored samples compared with the starting values was still good. Therefore, although the concentration of vitamin B12 decreased upon storage, reliable comparative analyses can still be performed.In epidemiological and nutrition research, it is very important to evaluate the stability of biomarkers as function of both storage time and temperature. In this study, the stability of folate and vitamin B Folate 12 are important biomarkers in clinical medicine and in epidemiologic studies. Both vitamins can be measured by a variety of methods, but the use of autoanalyzers have become common practice [The measurements of folate and to a lower extend vitamin B12 have been measured with a commercially dedicated autoimmunoanalyzer. The stability of folate and vitamin B12 in serum samples was determined after a long-term storage of up to 13 years at −80°C.In the present study, folate and vitamin BFor the stability study, serum samples of 16 human volunteers (blood donors) were used. Samples were obtained from the Central Blood Laboratory of the Red Cross  with written permission of the volunteers. After blood withdrawal, serum samples were prepared within two hours, divided in aliquots, and stored at −20°C and −70°C. In this study, only samples that have been stored at −70°C were used.12 at t=0 have been determined within 4 hours after centrifugation. For long-term stability, samples have been stored for 1, 8, and 13 years, at −70°C for one year and at −80°C for the remaining period. Samples stored at −70°C were kept in a freezer equipped with temperature recorder and sound alarm. From 1 year onwards, the samples were stored at −80°C in a freezer equipped with an automatic temperature registration system with an alarm function. In The initial concentrations of folate and vitamin Bt=0. The measurements were performed as a single measurement, except on day 0 (duplicate measurements). At time points 0 and 1 year, folate and vitamin B12 have been determined with an autoanalyzer . A reference serum (AHSL-1) was included in each assay. During the first year of storage, the inter assay variation for folate was 1.9% (N=8). The inter assay variation for vitamin B12 was 3.4% (N=8).The serum samples were kept on ice for less than 1 hr before the first measurements at 12 have been determined in serum with an immunoanalyzer . A pretreatment converts the folate polyglutamic acid forms to the monoglutamic acid form predominant in serum. The measurements were performed as a single measurement, with dedicated kits from Beckman-Coulter. At time point 8 years, the intra-assay variation was determined with a reference serum (Biorad number: 40192). For folate, the intra-assay variation was 1.1% (N=2). The intra-assay variation for vitamin B12 was 4.3% (N=2). At time point 13 years, the intra-assay variation was determined with three reference sera . For folate, the mean intra-assay variation was 1.6% (N=4). The mean intra-assay variation for vitamin B12 was 2.7% (N=4).At time points 8 and 13 years, folate and vitamin Bt=0 were determined with a t-test for two-samples assuming equal variances.The statistical differences  of the values from the initial value at t=0), the initial concentrations of folate and vitamin B12 of the 16 volunteers were determined. The mean concentration for folate at t=0 was 6.1 ±1.7 ng/mL, and the median value was 5.8 ng/mL, with a range of 3.9–10.5 ng/mL. The mean concentration for vitamin B12 at t=0 was 471 ± 75 pg/mL, and the median value was 464 pg/mL, with a range of 381–709 pg/mL.At the start of the study (12 in human serum was followed during 13 years. In t=0 (before freezing of the samples).The stability of both folate and vitamin BThe levels for folate are rather constant during 13 years of storage at −80°C. Somewhat lower values, but statistically significant, were observed at time points 1 and 13 years of 94.4% and 92.0%, respectively.12, an almost linear decrease was observed during the time period of storage. The mean level of vitamin B12 decreased from 100% to 85.6%, 62.2%, and 50.5% at time points 1, 8, and 13 years, respectively.For vitamin By = 0.870x + 1.02, y = 0.703x + 3.53, and y = 0.552x + 5.11 for 1, 8, and 13 years, respectively. The correlation coefficients (R2) were 0.961, 0.867, and 0.889 for 1, 8, and 13 years, respectively. The statistics between 2 sets of data are P=0.0019 for 0 versus 1 year, P=0.155 for 0 versus 8 years, and P=0.031 for 0 versus 13 years as determined with a t-test for equal variances.In 12 concentrations of the 16 volunteers before the storage of the sera and after storage at 1, 8, and 13 years are shown. Although the concentration of vitamin B12 decreased substantially with the duration of storage, as shown in y = 0.829x + 2.80, y = 0.600x + 33.7, and y = 0.401x + 48.5 for 1, 8, and 13 years, respectively. The correlation coefficients (R2) were 0.894, 0.890, and 0.865 for 1, 8, and 13 years, respectively. The statistics between the 2 sets of data are P=1.7∗E − 8 for 0 versus 1 year, P=1.2∗E − 11 for 0 versus 8 years, and P=3.0∗E − 12 for 0 versus 13 years as determined with a t-test for equal variances.In 12 were performed in the past [12, only one long-term stability study was found [Several short-term stability studies on folate and vitamin Bthe past –17. But the past . After 6as found  and show12 were measured after 1, 8, and 13 years of storage at −80°C that that the measurements of folate still can be done without any major change in the concentration. For vitamin B12, there is a consistent decrease to about 50% after 13 years of storage at −80°C. It must be noted that from 8 years on, the autoanalyzer and reagents were changed so that a possible level change could be expected, although a comparative study was performed that showed the same results for both methods. In a previous report after 1 year of storage [12 was already visible, but then it was not feasible to make a strong conclusion based on only one time point. Now after 13 years, it is clear that this decrease was consistent.The present study shows in which folate and vitamin B storage , the dec12 has only little effect on the rank order and correlations with the starting concentrations. Rather good correlation coefficients (R2) were found between 0.87 and 0.89 after storage. For folate, the concentrations of the stored samples remain close to the starting concentrations with good correlations with the starting concentrations; for example, the correlation coefficient (R2) at 13 years was 0.89.For epidemiological studies, however, this decrease in concentration of vitamin BThere has been one study on the long-term stability of B vitamins  in which12 status did not show any decline, as measured with a microbiological assay [In the same study, vitamin Bal assay . This is12, can still be used for data analyses, although the concentration of vitamin B12 decreases steadily with longer storage time.In conclusion, this study shows that long-term storage of serum samples at −80°C, intended for future measurements of folate and vitamin B


CYP2D6 and CYP2C19 genes that result in decreased enzymatic activity have been documented. Here we describe a patient case of VEN associated palpitations and asthenia. The patient takes VEN extended release 150 mg twice daily. Genotyping confirmed the patient is a poor metabolizer for CYP2D6 and an intermediate metabolizer for CYP2C19. We propose that the palpitations and asthenia are related to sustained VEN exposure due to reduced metabolism.Cardiotoxicity has been extensively reported in venlafaxine (VEN) overdoses. Asthenia is also among the common side effects described for this antidepressant. VEN is metabolized mainly by CYP2D6 and to a minor extent by CYP2C19 to the major active metabolite O-desmethylvenlafaxine (ODV). Altered pharmacokinetic parameters in patients with polymorphisms in the CYP2D6  and CYP2C19 (∗2/∗2) which corresponded to poor metabolizer phenotype for both enzymes. Serum level of VEN was extremely elevated and the concentrations of its metabolites were altered as a result of the reduced activity of CYP2D6 and CYP2C19. It seems that the deceased had elevated VEN blood levels for a prolonged period of time before cardiac arrest happened. The authors concluded that the unintended death of the subject could be attributed to the impaired metabolism of VEN [VEN is a serotonin-norepinephrine reuptake inhibitor (SNRI) prescribed to treat depression . VEN is m of VEN . In the The patient is a 58-year-old Caucasian male with a history of depression and complex posttraumatic stress disorder (PTSD) that presented for a medical therapy management (MTM) consult. The patient expressed feeling intermittent palpitations associated with exertional dyspnea and added that he had been experiencing asthenia and lack of endurance since early childhood; however his fatigue symptoms worsened when he started VEN around twelve years ago. The patient was treated for bipolar disorder, which he believes was misdiagnosed. Additionally, he disclosed that many of the factors contributing to his PTSD in the past are no longer present and for these reasons he has been weaned off from some of the medications prescribed for bipolar disorder and PTSD. Patient's current medications are as follows: venlafaxine XL 150 mg twice daily, adalimumab 40 mg every 15 days, and lamotrigine 125 mg daily. He described his weakness as being unable to do any type of physical activity for a prolong period of time and he experiences pain in his calves by just standing. Contributing to his fatigue, the patient has a history of ankylosing spondylitis. The patient was recently started on coenzyme Q10 400 mg daily and L-carnitine 300 mg daily by the referring physician to aid with his fatigue prior to the MTM consult. He has no history of diabetes, dyslipidemia, or hypertension.On clinical examination, the palpitations were not associated with QTc prolongation (QTc = 443 msec). Electrocardiogram (holter) examination revealed symptoms of skipped beats and heart racing which were associated with sinus rhythm and occasional premature atrial beats. He was in sinus tachycardia at 101 beats per minute with normal intervals and normal axis. There were no other abnormalities. He has no associated syncope or presyncope. He also underwent a dobutamine stress test. His ejection fraction response was from 60% at rest to 80% at peak stress. Peak heart rate was 150 bpm, which was 93% of the maximum predicted heart rate. His hemoglobin A1C was borderline elevated at 5.5%; blood glucose was 111 mg/dL . Blood urea nitrogen, creatinine, and urinary profile were all within normal limits. Antinuclear antibodies and other immunology blood testing were unremarkable. Patient's alpha-tocopherol was elevated at 24.9 mg/L. Also serum calcium level was slightly elevated at 10.3 mg/dL. Vitamin D levels were not available; however a pelvic X-ray suggested normal bone mineral density. CYP1A2, CYP2B6, CYP2C19, CYP3A4, and OPRM1 was compiled using a custom xTAG assay (Luminex Molecular Diagnostics). Analysis of CYP2D6 was completed using xTAG kits. The following genetic variants can be observed using this assay: CYP1A2: −3860G>A, −2467>delT, −739T>G, −729C>T, −163C>A, 125C>G, 558C>A, 2116G>A, 2473G>A, 2499A>T, 3497G>A, 3533G>A, 5090C>T, 5166G>A, and 5347C>T; CYPB6: ∗1, ∗4, ∗6, and ∗9; CYP2C19: ∗1, ∗2, ∗3, ∗4, ∗5, ∗6, ∗7, ∗8, and ∗17; CYP2C9: ∗1, ∗2, ∗3, ∗4, ∗5, and ∗6; CYP2D6: ∗1, ∗2, ∗2A, ∗3, ∗4, ∗5, ∗6, ∗7, ∗8, ∗9, ∗10, ∗11, ∗12, ∗14, ∗15, ∗17, and ∗41, gene duplication; CYP3A4: ∗1, ∗13, ∗15A, and ∗22; OPRM1 118A>G. The patient carried CYP2D6 ∗4/∗9 and CY2C19 ∗1/∗2 consistent with poor CYP2D6 metabolizer and intermediate CYP2C19 metabolizer phenotypes.Genomic testing was performed from a buccal swab sample using a commercial laboratory. Briefly, genomic DNA was isolated from a buccal swab and the relevant genomic regions were amplified by polymerase chain reaction. Analysis ofPatient's genetic test results established a phenotype of CYP2D6 poor metabolizer and CYP2C19 intermediate metabolizer. Due to reports of cardiotoxicity in VEN overdose and in poor metabolizer patients , 13, ourμg/L [μg/L) [P < 0.0001) [It has been suggested that cardiotoxicity can be expected in patients on VEN and with poor metabolism in the CYP2D6 enzyme , 13. VENμg/L ; howeverL [μg/L) . ODV undL [μg/L) . NDV, a L [μg/L) . VEN eliL [μg/L) . EliminaL [μg/L) . NDV is L [μg/L) , 23. CYPL [μg/L) , 12, 25.L [μg/L) –10. The L [μg/L) . In a st 0.0001) . Our pat 0.0001) . A simil 0.0001) . However 0.0001)  and adal 0.0001) . TherefoThe patient also is experiencing severe asthenia. Even though our patient has a significant past medical history of weakness and fatigue, the patient reported worsening of his symptoms after VEN initiation. Several reports of asthenia in patients on VEN have been published , 32–35. In summary, we present a case of severe asthenia and palpitations. VEN appears to be associated with these side effects in a CYP2D6 poor and CYP2C19 intermediate metabolizer. Several studies mentioned in this report have suggested cardiotoxicity associated with VEN overdose and in CYP2D6 poor metabolizers. This report supports the clinical utility of determining a patient's genotype in order to optimize patient therapy by choosing optimal agents and preventing side effects or toxicity. Finally, the involvement of a pharmacist with expertise in pharmacogenomics can be helpful in guiding adjustments based on these increasingly recognized “gene-drug” interactions.


Ribes nigrum L.) and bilberry (Vaccinium myrtillus L.) juices against bacterial strains Asaia lannensis and Asaia bogorensis isolated as spoilage of commercial soft drinks. The composition of fruit juices was evaluated using chromatographic techniques HPLC and LC-MS. The adhesion to glass, polystyrene, and polyethylene terephthalate in two different culture media was evaluated by luminometry and the plate count method. The major anthocyanins in the V. myrtillus were petunidin-3-glucoside, malvidin-3-glucoside, cyanidin-3-glucoside, and delphinidin-3-glucoside, while in R. nigrum delphinidin-3-rutinoside and cyanidin-3-rutinoside were detected. The LC-MS analysis showed presence of anthocyanins , phenolic acids (chlorogenic and neochlorogenic acids), flavonols , and flavanols (procyanidin B2 and procyanidin type A2). Additionally, in the bilberry juice A type procyanidin trimer was detected. The adhesion of Asaia spp. cells depended on the type of medium, carbon sources, and the type of abiotic surfaces. We noted that the adhesion was significantly stronger in minimal medium containing sucrose. The addition of bilberry and black currant juices notably reduced bacterial growth as well as cell adhesion to polyethylene terephthalate surfaces.The aim of the study was to evaluate the activity of high-polyphenolic black currant ( Escherichia coli, Proteus vulgaris), Helicobacter pylori, and bacterial etiological factors of oral diseases  [Nowadays, consumers are increasingly interested in their health and expect the foods, besides possessing the sensory attractiveness, to have health-promoting effects. Numerous studies indicate that a diet rich in berries and their preserves positively affects human health. Regular consumption of fruits may delay ageing processes and reduce the risk of various illnesses, such as cancer, cardiovascular and lung diseases, rheumatoid arthritis, Alzheimer's dementia, or Parkinsonism –3. Fruitum spp.) –12. Asaia [ Asaia spp. cells are able to grow in soft drinks supplemented with different preservatives  [ Asaia species on solid surfaces of a production line can be a source of secondary contamination of final products [Lately, numerous reports detailed various spoilage microorganisms in soft drinks, for example, acetic acid bacteria belonging to the genus Asaia –15. The products . Asaia spp. cells.The initial, key step leading to biofilm formation is bacterial adhesion to the surface. This is the complex process, influenced by various physical and chemical properties of microbial cells, media, and abiotic surfaces. Among these factors, modification of media could be changed in order to prevent biofouling in soft drinks technology. New antimicrobial strategy is the use of berry juices to inhibit or reduce bacterial adhesion. The application of native and low-priced fruits with additional potential as health-promoting agents is especially interesting. Therefore, the aim of our study was to investigate antibacterial and antiadhesion activities of juices from bilberries and black currants againstR. nigrum L.) and bilberry (V. myrtillus L.) fruits were freshly harvested from the local orchard and forests around Lodz . The fruits were washed with sterile water, lightly air-dried, and frozen at −20°C for one month. The fresh juice was squeezed out from defrosted fruits using extractor MES3000 . Cloudy juice was clarified using Whatman qualitative paper-filter and then by 0.45 μm filtration (Filter-Bio). Immediately after preparation, the clear juice was added to the culture media to the final concentration of 10% (v/v).The black currant ( Asaia bogorensis ISD1 (GenBank KP234014), A. bogorensis ISD2 (GenBank KP234015), A. bogorensis FFMW (GenBank KC756841), A. lannensis IFMW (GenBank KP234011), A. lannensis IFCW (GenBank KP234012), and A. lannensis FMW1 (GenBank HQ917850) isolated from spoiled flavored mineral water and isotonic drinks. These strains were identified using morphological, physiological, and genetic methods described by Kregiel and coworkers [The study used the six bacterial strains:oworkers , 17. The1) (0.3% (w/v) peptone, 0.3% (w/v) yeast extract) and the minimal medium (M2) (0.3% (NH4)2PO4 (w/v), 0.3% KH2PO4 (w/v), 0.3% MgSO4  × 7H2O (w/v), and 0.05% (w/v) yeast extract). In both media, carbohydrates, glucose, fructose, and sucrose (2% w/v), were used as a carbon source. The sterile media (20 cm3) were poured aseptically into 25 cm3 Erlenmeyer flasks covered with a textile cloth in order to ensure aerobic conditions. Sterile carriers were placed vertically in a liquid culture medium in such a way that half of the carrier was immersed in the medium, and the other part was above the liquid.The adhesion was investigated in liquid culture media: the rich GC medium   and polyethylene terephthalate (PET)  slides measuring 76 × 26 mm. These materials are certified by Polish National Institute of Public Health and approved for contact with food. The white glass slides (G)  were used as the reference material. Carriers were sterilized in two-step process. First, the carriers were kept in the 70% ethanol solution for 3 hours. Subsequently, they were placed in a laminar chamber and subjected to UV irradiation for 2 hours. Asaia spp. attachment and biofilm formation were carried out in two stages. The first stage involved the selection of a culture medium, a carbon source, and an abiotic material where bacteria demonstrated the strongest adhesion abilities. In the second stage, we checked the effect of fruit juices on the growth and adhesion abilities of Asaia spp. For this purpose, the culture medium containing selected carbon source, with proper carrier, was supplemented with 10% (v/v) of black currant or bilberry juice.Studies on the5 ÷ 106 CFU/cm3. The adhesion ability of the bacterial strains was evaluated according to the method described by Kregiel (2013) [3 (w/v), and after incubation at 25°C for 92 h the colonies were counted. The number of colony forming units (CFU) per cm3 (of liquid media) or per cm2 (of carriers) was calculated. On the basis of the results, the relative adhesion coefficient A (%) was calculated using formula A (%) = (Na/Np) × 100%, where Na is the number of attached cells to a carrier and Np is the number of planktonic cells in the culture medium.At the beginning of the experiments culture media were inoculated with standardized bacterial suspensions, to obtain cell concentration 10l (2013) . For lumThe organic acids and carbohydrates profiles of the tested fruit juices were determined using high performance liquid chromatography (HPLC), according to the method described by Gutarowska and Czyżowska (2009) . In addip < 0.05).Means were calculated from the data obtained from three independent experiments, and the standard deviations (SD) were calculated. The mean values of the adhesion results were compared using one-way repeated measures analysis of variance with Tukey test . Statistical significance was set at the conventional level of 5%  for medium M1 and medium M2, are presented in Figures  Asaia strains significantly increased in culture media supplemented with sucrose (p < 0.05) in comparison to media containing glucose or fructose. It was noted that the minimal M2 medium was a more favorable environment for the Asaia spp. adhesion and biofilm formation compared to the rich M1 medium. The results for adhesion in M2 medium, with reference to those obtained for the adhesion in M1 medium, were significantly higher (p < 0.05). The average value of A (%) for cells adhesion in M2 medium with sucrose was 1.72 ± 0.26%, while for the same medium but with fructose and glucose the results were slight lower and equaled 1.10 ± 0.23% (p = 0.00001) and 0.80 ± 0.19% (p = 0.00004), respectively. The highest value of A (%) was noted for A. lannensis IFCW strain on PET surface, which was 4.54 ± 0.37%. Figures 2) obtained for bacterial adhesion in M1 and M2 media, respectively. The obtained results confirmed that the more favorable environment for biofilm formation is the minimal medium M2 with sucrose. Average value of the RLU for rich M1 medium with sucrose (1784 ± 257 RLU/cm2) was statistically lower (p = 0.001) in comparison to minimal medium M2 with the same carbohydrate (3923 ± 447 RLU/cm2).To determine the level of bacterial adhesion, two main analytical methods, namely, plate count and luminometry, were used. The evaluation ofA (%) (2 ( A. lannensis strains showed slightly stronger adhesion in culture media containing sucrose. The mean A (%) values for A. lannensis strains adhesion to PET surface in culture media with sucrose were 1.23 ± 0.61% (M1) and 3.24 ± 1.05% (M2) while for the A. bogorensis strains 1.12 ± 0.36% (p = 0.05) and 2.36 ± 0.74% (p = 0.02) were noted, respectively.Additionally, to assess the differences between the adhesion abilities of all bacterial strains to all tested carriers in all culture media containing different carbon sources, the mean values and standard deviations calculated from obtained results of A (%)  and the A (%) , 1.90 ± 0.23% (PS), and 2.80 ± 0.21% (PET), while for rich M1 medium 1.18 ± 0.71%, 1.82 ± 1.01%, and 1.17 ± 0.23% were noted, respectively. Performed ANOVA test showed that the results are statistically different. Obtained p values, in comparison to glass, for the M1 medium were 0.02 (PS) and 0.01 (PET), while the results for M2 medium were less than 0.01 for both PS and PET. The results of RLU measurement also showed that slightly better surface for biofilm formation in M2 medium with sucrose is PET. Asaia spp. adhesion were obtained by Kregiel (2013) and Kregiel et al. (2014), where, after incubation, the adhesion to plastic materials was several times higher in comparison to the glass surface [The similar results for surface , 17.A (%) and RLU/cm2, the values obtained by these two methods showed differences.Of course, there are different techniques that can be used in the analysis of the microbial adhesion to abiotic surfaces, but neither method is perfect. The plate count technique in particular allows determining culturable microorganisms, while luminometric methods enable estimating total biological material on the abiotic surfaces. This approach is based on bacterial ATP quantification and can be used to evaluate not only the total number of adhering cells, but all biomass: bacteria that are able and unable to grow, extracellular polymeric substances, or adhered organic material from culture media. Thus, comparing the results of the relative coefficient  Listeria monocytogenes adhesion and biofilm formation [ Asaia spp., we choose rich M1 medium with glucose and minimal M2 medium with sucrose, respectively.The type of material, its roughness, and hydrophobicity significantly affect bacterial attachment and biofilm development. The plastic materials used in our study were characterized by low surface energy  in comparison to hydrophilic glass surface (70 mN/m at 20°C) , 20. Whaormation . TherefoThe carbohydrate profiles of the fruit extracts indicated that the main sugars were glucose and fructose. In the bilberry juice, fructose concentration was 1.94 g/100 mL, while glucose equaled 0.76 g/100 mL. Respectively, for the black currant juice, the values were 0.60 g/100 mL and 0.54 g/100 mL. According to the literature, in the majority of native fruit juices, the content of saccharides is limited only to glucose, fructose, and sucrose. The variability of determined saccharide contents in fruit juices from berries stemmed from differences in variety, stage of ripeness, and climatic conditions .μg of cyanidin-3-glucoside per one mL. The highest concentration of these compounds in the Vaccinium myrtillus juice was noted for petunidin-3-glucoside (2.48 μg/mL) and malvidin-3-glucoside (2.41 μg/mL), cyanidin-3-glucoside (1.83 μg/mL), and delphinidin-3-glucoside (1.78 μg/mL). The major anthocyanins in the Ribes nigrum juice were delphinidin-3-rutinoside (2.04 μg/mL) and cyanidin-3-rutinoside (1.99 μg/mL). The presence of anthocyanins was also confirmed by LC-MS ( Ribes nigrum [ Vaccinium myrtillus fruits [ R. nigrum the average content of anthocyanin amounts to 3500 mg/L [Vaccinium macrocarpon) juice, in particular, helps in maintaining the health of the urinary tract [ Escherichia coli [ Vaccinium angustifolium exhibit strong antiproliferation activity against human prostate and mouse liver cancer cell lines [The polyphenolic profiles in fruit juices were determined using HPLC method and the results are presented in Figures by LC-MS . Twenty-s nigrum –26 and Vs fruits –30. The 500 mg/L . In rela500 mg/L . Despite500 mg/L , 33 and 500 mg/L , 35, ant500 mg/L , 37, neu500 mg/L , and ant500 mg/L  activiti500 mg/L . Researccoside 2. μg/mL, cry tract . PACs arry tract . The anthia coli  and othenoside 1.9 μg/mL. ll lines . Charactll lines . Generalll lines –52. Morell lines .A (%) results and quite high RLU values, for the next stage of this study, based on the effect of fruit juices on the growth and adhesion of Asaia spp., we chose M1 medium with glucose for growth analysis and M2 medium with sucrose with PET carriers for adhesion investigation.Due to the higher 1 medium without fruit juices varied depending on the strain with mean value of 7.02 ± 2.41 × 109 CFU/cm3 ( A. bogorensis ISD2 (1.08 ± 0.23 × 1010 CFU/cm3) and A. bogorensis ISD1 (9.97 ± 1.45 × 109 CFU/cm3) while the lowest number of the bacteria was detected for A. lannensis FMW1 (2.67 ± 1.70 × 109 CFU/cm3). The addition of R. nigrum and V. myrtillus juices caused a slight reduction in the number of viable bacterial cells. The average count in M1 medium with 10% (v/v) bilberry juice and black currant juice was 2.60 ± 1.35 × 109 CFU/cm3 and 4.37 ± 2.85 × 109 CFU/cm3, respectively. The obtained results suggested that A. bogorensis showed higher sensitivity to fruit juices than A. lannensis strains.The growth in M CFU/cm3 . After 1 P. aeruginosa, Staph. aureus, E. coli, L. monocytogenes, and Salmonella spp. Polyphenols are able to suppress a number of microbial virulence factors, such as reduction of host ligands adhesion, inhibition of biofilm formation, and neutralization of bacterial toxins, and show synergism with antibiotics [ Salmonella spp. membranes, cell penetration, and reaction with cellular proteins [ E. coli and Salmonella spp. Phenolic extracts containing tannins and their derivatives showed strong antibacterial effect against Staph. aureus, H. pylori, C. perfringens, B. cereus, Klebsiella spp., and Proteus spp. [According to the literature, polyphenols from various fruit demonstrate antibacterial activities, especially against pathogenic strains:ibiotics . The actibiotics . In addiibiotics . The peribiotics . It was proteins . Accordiproteins . In Puupproteins . It was eus spp. , 58. HowIt is well known that luminometric measurements in an environment of fruit juices that are rich in polyphenols may carry a margin of error. Luminometry is based on the reaction of enzymatic oxidation of luciferin to oxyluciferin and the presence of antioxidants can influence the final results. It has been documented that polyphenols present in green tea can inhibit the enzymatic activities . Therefo Asaia spp. was performed during cultivation in M2 medium with PET carriers. Results, expressed as adhesion relative coefficient A (%) and RLU/cm2, were presented in Figures A (%) calculated for the sixth day of incubation with 10% (v/v) juice showed significant decrease in the adhesion and biofilm formation ( V. myrtillus juice. Luminometric results (RLU/cm2) also confirmed significant reduction of adhesion (p < 0.05). Additionally, the differences were noted for antiadhesive activities of tested juices. The values ranged from 1460 ± 102 RLU/cm2 to 9800 ± 520 RLU/cm2 (Av = 4252 ± 2748 RLU/cm2) for the control sample and for adhesion in the presence of V. myrtillus and R. nigrum from 14 ± 5 RLU/cm2 to 160 ± 34 RLU/cm2 (Av = 70 ± 58 RLU/cm2) and from 600 ± 54 RLU/cm2 to 1900 ± 187 RLU/cm2, respectively (Av = 1218 ± 474 RLU/cm2). Thus, bilberry juice inhibited biofouling of all tested Asaia spp. bacteria, while in the presence of black currant juice we noted the antiadhesive effect for A. bogorensis strains in particular.The effect of the bilberry and black currant juices on the adhesion properties oformation . This paadhesion . There wVaccinium macrocarpon) [ Staph. mutans was also documented [ Asaia spp. It was documented that, in the presence of cranberry juice, the attachment of A. bogorensis cells to plastic surfaces was significantly lower [ V. macrocarpon confirmed the presence of type A proanthocyanidin [ Asaia spp. cells.The use of fruit juice not only brings antiadhesive effects, but also has other health benefits. The prohealth action of berry juices has been known in folk medicine. However, antiadhesive properties of fruit juices were documented scientifically mainly for cranberry (ocarpon) , 41, 54.cumented . The recly lower . Howeverly lower . The chrcyanidin , 62. Thu Asaia. Particularly V. myrtillus juice characterized by a higher content of polyphenols including A type proanthocyanidin showed strong antiadhesive and bacteriostatic properties. The high content of bioactive compounds with proven health-promoting properties makes them a valuable supplement of soft drinks, as well as interesting alternative to artificial additives to keep the microbial stability of final products.The results presented in this study suggest that bilberry and black currant juices show high antiadhesive and antibacterial activity against food-spoiled bacteria belonging to the genus


The geo-accumulation studies indicated a moderate to high levels of buildup of some elements in the sewage sludge. Principal components analysis (PCA) indicated strong spatial and weak temporal variations in the concentrations of the elements. Therefore, the sludge disposal operations, based on the element concentrations, geoaccumulation and economic potential are suggested for each WWTP. Sludge from W1 and W2 were found suitable for agricultural usage, while that from W3 showed a higher economic potential for the recovery of precious metals. This study concludes that a comprehensive analysis of the elements in the sewage sludge could provide critical information for the disposal and management of the sludge.The limited information about the sludge quality has made its management a top environmental challenge. In the present study, occurrence and the spatiotemporal variations of 52 inorganic elements were investigated in the sludge samples from three wastewater treatment plants (WWTPs) in Xiamen city, China. The results showed, the occurrence of 49 elements with the concentrations in the range of >125–53500 mg kg According to NBSC5 and Yang et al.6, 6.25 million tons dry sludge (DS) was produced in 2013 in China, of which, >84% was disposed improperly. The safe disposal of the municipal sewage sludge has become a major environmental challenge in China and in many countries of the world9.China is a populous country with a population of over 1.3 billion. With the rapid industrialization, urbanization, and economic growth, China is facing growing environmental issues11. However, the high concentration of metals in sludge is a great concern14, that may pose adverse impact on human and other living organisms when their bioavailability exceeds the threshold limit 16. These metals mainly originate from the aqueous phase of the wastewater, and then concentrate in the sludge during the treatment processes like precipitation, coagulation, adsorption etc. For the sludge with higher levels of metals, the resource recovery, including the major industrial elements, precious metals, and rare earth elements, has been suggested as an alternative, not only to reduce the pollutant loadings but also to exploit the economic potential of the sludge before its disposal18. The sewage sludge, which is the end-of-life product, would now be considered the “urban mines”. Therefore, it is necessary to investigate the loads of the metallic elements to achieve the proper disposal of sewage sludge9.Sewage sludge is a rich source of phosphorous and nitrogen, and could be value-added as fertilizeret al.19 collected sludge samples from 107 municipal wastewater treatment plants (WWTPs) located in 48 cities in China in 2006. The results showed that the average concentrations of As, Cd, Cr, Cu, Hg, Ni, Pb, and Zn were 20.2, 1.97, 93.1, 218.8, 2.13, 48.7, 72.3, and 1058 mg·kg−1, respectively. Dong et al.20 reported the heavy metal occurrence in sewage sludge in China, with concentrations up to 293.7, 181.7, 114.8, 40.3, 1453.9 mg kg−1 DS for Cr, Cu, Ni, Pb and Zn, respectively. In another study, carried out on industrial sewage sludge, Wu et al.21 reported the concentrations up to 172300, 237, 2225, and 1700 mg kg−1 DS for Cd, Cu Ni and Zn, respectively. In addition, Suanon et al.23 reported concentration of 64, 73.1, 604.1, 1102.1, 483.9, and 2060.3 mg kg−1 DS for Cd, Co, Cu, Cr, Ni and Zn, respectively, in the urban sewage sludge. As it can be noticed that most of the studies investigated a limited number of metals, which are generally with greater environmental concerns, and they are frequently listed in the environmental monitoring methods, standards, or regulations25. In order to better assess the environmental and ecotoxicological risks and to appreciate the economic opportinuity for their recovery, it is necessary to investigate a broad range of elements in the sludge.Several studies have reported the occurrence of metallic elements, mainly heavy metals in the sewage sludge in China. Yang Therefore, the present work aimed to (1) investigate the occurrence of 52 elements, including industrial metals, precious metals, rare earth elements and phosphorous in sewage sludge, (2) understand the temporal and spatial variations, (3) appreciate the quality of sludge and evaluate the geoaccumulation index, and (4) to indicate the proper sludge disposal methods. To our knowledge, this is the first investigation on such a broad range of inorganic elements in the sewage sludge in China.26. The average daily excess sludge was 1691.7, 214.5, and 243.7 tones, with the dry weight of 53.3, 4.5 and 8.1 tones, respectively for W1, W2 and W326. The treatment process varied from one plant to another. Briefly, in W1, the treatment included screening, grit chamber, biological aerated filter, and UV disinfection. In W2, screening, grit chamber, orbal oxidation ditch, secondary settling tank, and UV disinfection were involved; while in W3, screening, grit chamber hydrolyzing pond, anaerobic/anoxic/oxic (A2O) reactor, secondary settling tank, and chemical disinfection were the main treatment processes26.Three municipal WWTPs (W1-3) located in Xiamen, China were investigated in this study. The wastewater in W1 was nearly 100% domestic wastewater, while in W2 and W3, the industrial wastewater contributed to 20% and 50%, respectively along with domestic wastewater. W1, W2, and W3 served around 1.0, 0.35, and 0.30 million inhabitants, respectivelyth (F), May 8th (M), August 11th (A), and November 12th (N) 201426. Sludge samples were concentrated by centrifugation (5000 g) at 4 °C for 15 min, and dried via lyophilization. The dried sludge was then grounded in clean mortar and sieved by passing through a mesh size <0.15 mm. The obtained powders were kept in polyethylene bags for physicochemical characterization and determination of elements.Activated sludge samples in W1 and the return sludge samples in W2 and W3 were seasonally collected from each WWTP on February 2027 using a multi-parameter meter . The total carbon (TC), total nitrogen (TN), and total sulfur (TS) were determined by a macro elemental CNHS/O Analyzer . The pre-detection of the elements in the sludge samples was performed using scanning electron microscopy  with energy dispersive X-ray (EDX) spectroscopy (Genesis XM2).Physicochemical parameters, including pH, electrical conductivity (EC), and C, N, and S, were analysis. The values of pH and EC were determined in the filtrate of dissolved sludge sample in the milliQ water at ratio 1/5 (v/v)3 and HCl at the ratio of 3:1) at 90 °C for a minimum of 2 h according to the European Norm 3051A28. The cleanup was then completed following the recommendations by Westerhoff et al.17. All volumetric wares, including glass calibrate cylinders, were carefully acid washed and rinsed following the same recommendations. The nitric acid and hydrochloride acid were analytical grade .All digestion vessels (polytetrafluoroethylene) were cleaned by leaching with hot 10 mL aqua regia (mixture of HNO28. The digestion program is described in the Table Sludge sample (0.100 g) and freshly prepared aqua regia (12.0 mL) were placed in the digestion tube. The reaction mixture was kept for 30 min at room temperature, and then the digestion was completed under microwave at 180 °C according to the European Norm 3051AInductively coupled plasma mass spectroscopy  and inductively coupled plasma optical emission spectrometry  were used for the elemental quantification. Stock solutions of element standards were purchased from National Center of Analysis and Testing for Nonferrous Metals and Electronic Materials (NCATN). The target elements were analyzed in four groups: G1 , G2 , G3 , and G4 . The detection limits and the recoveries of target elements are shown in Table Principal components analysis, conducted by R software (version 3.2.2), was used to identify spatial and temporal variability of the 52 elements in the sewage sludge. Data were first normalized using the vegan package to alleviate negative effect caused by some elements which distributed extremely non-uniform. The correlation among multiple elements was carried out using Pearson’s correlation coefficient assay in SPSS 21.0. Results obtained were then used to construct a heatmap using R software with heatmap package.p > 0.05). However, the pH values in W3 were higher than W1 (p = 0.014). The relatively high values of EC (0.73–1.81 mS cm−1) indicated the high concentration of charged particles such as Cl−, Na+, K+, NH4+, NO3−, SO42−, HCO3− etc. in the sludge samples29. Sludge samples were C, N and S-rich, with the average concentrations of 260.4, 36.5, and 22.3 g kg−1 DS, respectively. The EDX image  to 53500 mg kg−1 DS (Fe)  to 2230 mg kg−1 DS (for Zn in W3). Regarding the elements with great environmental concerns such as As, Cd, Co, Cr, Cu, Ni, Pb, and Zn, the concentrations ranged from 3.9–5.2 mg kg−1 DS, 9.1–61.4 mg kg−1 DS, 5.46–48.7 mg kg−1 DS, 36.2–1600.0 mg kg−1 DS, 64.8–1390.0 mg kg−1 DS, 14.9–610.0 mg kg−1 DS, 42.8–63.4 mg kg−1 DS, and 231–2230 mg kg−1 DS, respectively. The concentrations of the major industrial elements were within the similar ranges described in the studies of the domestic sewage sludge in the US17 and China22.Out of 52 investigated elements, 49 were detected in the sludge. The most widely used industrial elements like Al, Fe, P, Ca, K, Mg, Na, Mn and W etc. were detected at very high concentrations in the sludge samples with an average annual concentration ranging from 125 mg kg−1 DS, 2.18–14.1 mg kg−1 DS, and 1.22–5.59 mg kg−1 DS for Pd, Ag and Au, respectively. However, the detected concentrations have economic attraction, considering the critical and precious nature of these metals. The detected Au and Ag concentrations were within the similar concentration range with the samples from US17 and Switzerland18.Precious metals i.e. Pd, Ag, Au, Ru, and Pt were detected in the sludge samples Table , while t−1 DS (Ho) to 439 mg kg−1 DS (Ce). The application of rare earth elements in the manufacturing industry was probably the major source to the sewage sludge via wastewater treatment process. As shown in the Yearbook of Xiamen Special Economic Zone30, the mechanical and electronic are the most important industries in Xiamen, where the rare earth elements were widely used besides other wide range of technological applications31. In addition, it is known that the Chinese rare earth elements reserve represents 50% of the world reserves32, which makes China the leader of the world in the rare earth element production33. As a consequence, the residues of these metals could accumulate in the sewage sludge.A total of 16 rare earth elements, including Ce, Nd, La, Y, Pr, Sc, Sm, Gd, Dy, Er, Yb, Eu, Ho, Tb, Tm, and Lu, were also analyzed in the present study. Fifteen were detected, while Lu was below the detection limit Table . As showThe relative distribution of element concentrations amongst the four seasons, which highlight the temporal variation of the elements, is displayed in Fig. p < 0.01 for the majority of the elements with the exception of Pr, Dy, Er, Yb, Eu, Tb, and Tm with 0.01 < p < 0.05; and Al, Fe, V, Ir, and Pt with p > 0.01. The significant spatial variation of the elements was likely due to the difference of the wastewater sources and the difference in the treatment processes in WWTPs. Indeed, the land usage type in W1 is mainly residential or commercial and service, while there are some manufacturing and industrial areas in W2 and W326. The wastewater was nearly 100% domestic wastewater in W1, while W2 and W3 contained 20% and >50% industrial wastewater besides domestic wastewater, respectively. Consequently, the sludge from W3 showed higher concentration of most of the metals, as they are mainly used in the manufacturing and industrial processes34. In addition, as previously stated, the treatment processes in the WWTPs also vary from one WWTP to another. Biological aerated filters + UV disinfection (BAF + UVdis) were used in W1, Orbal oxidation ditches + UV disinfection (O-OD + UVdis) in W2 while anaerobic/anoxic/oxic + disinfection (A2O + dis) were applied in W3 WWTP.The relative distribution of each element in W1-3 based on the average concentration from four sampling seasons is shown in Fig. 25. The regulated heavy metal concentrations for Cd, Cr, Cu Ni and Zn in the sludge adaptable for agricultural purposes are 5, 600, 800, 100 and 2000 mg kg−1 for acidic soils (pH ≤ 6.5) and 20, 1000, 1500, 200 and 3000 mg kg−1 for alkaline soil (pH ≥ 6.5), respectively. Therefore, the sludge from W1 and W2 could be used for the land application after properly stabilization. However, the heavy metals accumulation in the soil resulting from the repeated land application, together with the elemental bioavailability and bio-toxicity, needs to be furthered evaluated. In addition, the issue of emerging contaminants and persistent organic pollutants should also be taken into account and investigated during sludge stabilization before any application to the soil. Due to higher heavy metal concentrations, the sludge from W3 cannot be directly applied to the land. Due to the higher concentrations of precious elements in W3, the valuable elements are worthy to be recovered. Therefore, finding cost effective technology for the recovery of precious elements in W3 would be a good alternative.Generally, the concentrations of the elements of greater environmental concern such as Cd, As, Ni, Cr, Cu and Zn in W1 and W2 met with Chinese standard 4 mg kg−1), although, the concentrations were standardized. Therefore, PCA was then performed based on the major industrial elements, precious metals, and rare earth elements.PCA was performed to show the variation and correlation of the occurrence of elements in different samples. Figure As shown in Fig. p value > 0.05. Generally, the elements were clustered into two apparent groups. Five rare earth elements  showed strong and positive correlation (r2 > 0.5) with eight industrial heavy metals . These elements also showed a strong and positive correlation with pH. This could be explained by the fact that the pH plays an important role in the concentration and accumulation of elements in the soil or sludge35. Other physicochemical parameters didn’t show any significant correlation with the elements concentrations in the sludge. In addition, rare earth elements, including Dy, Er, Ho, Yb and Tm, also showed strong correlation with each other. The strong correlations among the elements might be due to the similarity in their usage and origin.The correlation among the elemental concentrations and environmental parameters was carried out by SPSS. The heatmap Fig.  was prodIgeo was used to evaluate the level of soil, sludge and sediment contaminations by comparing with the background concentration of a given element in the studied sample to its concentration in the upper continental crust (UCC) concentration37. Igeo was calculated via the equation (1)39.40 were applied to evaluate the contamination of individual element in each sludge sample.es Table 40 were aIgeo ≤ 1) (Table Igeo ≤ 2), while W and Cd indicated moderate to strong accumulation (1 ≤ Igeo ≤ 3) in all the WWTPs. In the samples from W3, there was moderate accumulation of Cr, Cu, and Zn, (0 ≤ Igeo ≤ 2). This is noteworthy that some precious metals, including Pd, Au, Ag and Ru, showed strong accumulation (Igeo ≤ 4) in the sludge from all WWTPs, indicating their enrichment in the sludge.Generally, the target elements were not accumulated in most of the studied sludge samples (1) Table . HoweverIn this study, the occurrence of 52 elements was investigated in the sludge samples from three WWTPs in Xiamen city, China over one year. There were 49 elements detected, including major industrial elements, precious metals, and rare earth elements. The concentrations of most elements showed strong spatial variations but little seasonal variations. The spatial variations were mainly due to the difference of the wastewater source as well as treatment processes in WWTPs. Sludge from W1 and W2, which mainly received domestic wastewater, had relatively low elemental concentrations and could be suitable for the agricultural land application after a thorough stabilization and sequestration of potential emerging contaminants and persistent organic pollutants. However, sewage sludge from W3, which received more than 50% of the industrial wastewater, was found with relatively higher elemental concentrations and would pose an environmental risk for the agricultural usage. The high geoaccumulation index in W3 suggested that the elemental recovery might be a better option for the disposal of sewage sludge from W3. The investigation on a broad range of elements in this study provided useful information to assess the environmental risks and to evaluate the potential value of their recoveries, which can be very important for the sludge disposal and management.Supplementary Information


This paper details the development and assessment of a new empirical creep model that belongs to the limited ranks of models reproducing full creep curves. The important features of the model are that it is fully standardised and is universally applicable. By standardising, the user no longer chooses functions but rather fits one set of constants only. Testing it on 7 contrasting materials, reproducing 181 creep curves we demonstrate its universality. New model and Theta Projection curves are compared to one another using an assessment tool developed within this paper. Over the last 50 years, creep modelling has become an area of significant expertise as components are being designed with more precision for increasingly extreme environments. As such, engineers and scientists have endeavored to understand, describe and predict the phenomenon of creep focusing on the rupture time and minimum strain rate of a material . Simply These creep curve models at some point in their implementation require a user to define functions from data trends . This isIn developing a model the goal is it for it to be universally applicable. This means the model should be able to reproduce creep curves for an array of materials over a range of conditions. It thus raises the question of “how many materials and conditions must it reproduce accurately before the model can be accepted?” To address this, the new model has been applied to seven materials including a pure element and a range of alloys with different compositions, crystal structures and microstructures. The new model was used to reproduce 181 creep curves sourced from Swansea University’s historical database covering tests conducted from the early 1990s through to the current day.Developing a new model requires the ability to assess how accurate it is. There is no standardised approach to judging how accurate a creep model is compared to real data. As such, there are a range of curve shapes and vast differences in magnitudes, meaning a combined quantitative-qualitative approach needs to be used. One has been developed here with pass/fail criteria and applied to the new model. A case study using Theta Projection as a comparison is also provided.2.5S where S is the standard deviation of the residual log time [2 value reflecting the qualitative fit [y is the measured data and x the predicted value and correspondingly Numerically defining the fit of a function is a complex problem as there are many ways of doing so requiring a balance between quantitative and qualitative errors ,9,10. Thlog time . This metive fit ,9,10, anLooking at It is from the more classical representation of data seen in c*Q [cQ obtained at constant σ, the normalised activation energy is obtained at constant σ/σN, where σN can be the temperature dependant Ultimate Tensile Strength (UTS), Proof Stress, or Yield Strength. This approach to activation energy was used as it provides a more complex link between stress and temperature, and has been found to provide values more consistent with proposed creep mechanisms than the traditional cQ [This model utilises the normalised activation energy, Qc* . Unlike ional Qc ,5,12,13.The underlying premise of this model is the observation from numerous creep datasets that for a given strain, the time to strain is related to the normalised stress by a power relationship:t(ε) vs. normalised stress (1 − σ/σN) collapses into a master curve for all stress-temperature combinations. This master curve can be described by a power equation of the form P(ε)y = M(ε)·x  as seen in Equation (4). Examination of the values for M and P over progressive strains showed that these values can be reproduced for each strain using a Frechet and Lognormal distribution with μ = 0, such that:1-A7A are constants obtained from fitting the defined functions M(ε) and P(ε). If the constants are known, then for a given stress-temperature combination the whole strain curve can be predicted.For a given strain, the time to strain f) vs. 1/Tln vs. (1t(ε)exp(−Q − σ/σN) was graphed and a power based trend line of the form Py = Mx was fitted. The values for M(ε) and P(ε) at the specific strains were then fitted to the previously mentioned functions and all constants obtained.Taking the raw creep dataset for a material, the normalised activation energy is obtained from the gradient of c*/RT) vs. (1t(ε)exp(−Q − σ/σN) was graphed and a power based trendline of the form Py = Mx was fitted. The values for M(ε) and P(ε) at these strains were then fitted to Equations (5) and (6). 1 = 1.68 × 10−5, A2 = 0.0046, A3 = 2.09, and A4 = 4, A5 = 1, A6 = 110, A7 = 11).Inconel 100 is used in this article to provide a detailed case study of the new model. It is a material with a composition of 15% wt Co, 10% wt Cr, 5.5% wt Al, 4.5% wt Ti, 3% wt Mo, 1% wt V, 0.18% wt C, 0.06% wt Zr, 0.014% wt B, balance Ni. A total of 22 creep tests ranging over stresses of 150–400 MPa and temperatures of 1073–1223 K were available from Swansea University’s historical database. Taking the raw data from tests, the time to 0.1, 0.2, 0.3, 0.5, 0.7, 1, 2, 3, 4% strain was extracted. For each strain, et al. [2* = Q4* = Qc*, A1 = 0.0114, A2 = 4.66 × 1010, A3 = 0.0981, A4 = 4.69 × 109, n1 = 0.92, n2 = 5.29, n3 = 3.29, and n4 = 4.91. The results of both creep prediction methods are shown in Having obtained all the constants, the model can now be used to reproduce creep curves with the user input being normalised stress and temperature values. Each predicted curve was then evaluated by the MoF. As a further means of comparison the Theta Projection method was used on the same data and the MoF evaluated. The form of the Theta Projection function was taken from Harrison et al.  with Q2*To further illustrate the differences between methods we consider the lowest common pass MoF of the methods. For the New Model this is MoF = 0.607, and so for Theta Projection MoF = 0.603. These are shown in From c* and constants A1–A7 were determined . The MoF values for pure copper are shown in Pure copper is known to be a difficult material to creep life due to changes in mechanism and, in some cases, the appearance of sigmoidal primary creep. Applying the New Model to pure copper, the same process was followed where QThe focus of this section is not on the accuracy of the New Model, but rather on a particular observation. Looking at The performance of the New Model has been evaluated thoroughly and been compared to Theta Projection evaluated by MoF. The model reproducing non-logarithmic primary creep without modification for the case of pure copper has also been discussed. Having a good understanding of the model and its implementation we consider the seven materials and 181 creep curves as a full set of data.Al2124. This material has a composition of 3.76% wt Cu, 1.33% wt Mg, 0.49% wt Mn, 0.08% wt Fe, <0.01% wt Zn, 0.02% wt Si, <0.02% wt Cr, and Al for the remaining balance.Al2419. This material has a composition of 6.06% wt Cu, 0.24% wt Mn, 0.13% wt Fe, 0.06% wt Si, 0.16% wt Zr, 0.01% wt Zn, 0.09% wt V, 0.09% wt Ti, 0.01% Mg, and Al for the remaining balance.Al7010. This material has a composition of 6.14% wt Zn, 2.43% wt Mg, 1.62% wt Cu, 0.13% wt Zr, 0.07% wt Fe, 0.01% wt Mn, 0.05% wt Si, 0.04% wt Ti, 0.01% Cr, and Al for the remaining balance.Al8090. This material has a composition of 2.34% wt Li, 1.14% wt Cu, 0.64% wt Mg, 0.1% wt Zr, 0.03 wt Fe, 0.02% wt Si, 0.02% wt Ti, <0.02% wt Zn, <0.01% Mn, <0.01% wt Ni, <0.01% wt Cr, and Al for the remaining balance.Pure Copper.IN100. The composition of this super alloy has already been stated above.NIM105. Nimonic 105 is a material with a composition of 20% wt Co, 14.85% wt Cr, 4.98% wt Mo, 4.79% wt Al, 1.23% wt Ti, <0.15% wt Si, 0.04% wt Mn, 0.01% wt Cu, 0.095% wt Zr, 0.125% wt C, 0.004% wt S, 0.006% wt B, <0.001% wt Pb, <0.001% wt Ag, <0.001% wt Bi, and the remaining balance Ni.The results seen in Traditionally, creep curves are displayed on a logarithmic scale where primary creep is not easily recognisable. Using this representation often skews the visual perception of the accuracy of a model as logarithmic scale skews the data and suppresses much of the finer detail. With the desire to design to more precise conditions it is important to reproduce the creep curve and its features on the true scale.In developing a model, the issues of “burden of proof” and how to measure accuracy have been addressed. Although some readers may disagree with the way it has been done here, it is important to note there is no established standard and very little advice onto how to proceed under these circumstances. Using IN100 the New Model was described and compared to the more familiar Theta Projection. Furthermore the incidental reproduction of sigmoidal primary creep of Pure Copper raised the idea that the model may be able to deal with non-traditional primary creep unlike other existing models.This model hinges upon the power relationship between a time to strain and the normalised stress. How effective the model will be in predicting curves can be initially assessed from the data spread and variation from this initial power relationship.i.e., A1–A3 is obtained from a single line fit, and A4–A7 from another. Unlike many other creep models these constants once defined remain constant unless region splitting is observed.The second most obvious concern is the number of constants. Although there are a total of seven constants these are obtained simultaneously from only two fits P(ε) appears to be a complex function, it is a common function which in MS Excel simplifies to “A4*LOGNORM.DIST + A7”. On rare occasion multiple fits can be obtained for this function but incorrect values produced unphysical curves which were easily identified.Although The simplicity of the model is that the user only defines the normalised stress and temperature conditions to produce full strain-time creep curves.f), nor is easily differentiated to produce έmin. This model seeks to reproduce the whole curve rather than predicting two points within the curve.This model is based on a continuous function that does not terminate at fracture (tIn looking at this New Model, we can observe:It is acknowledged that this model is not a perfect physical description of creep, but is important as it adds another tool for design engineers to predict creep and advance our understanding of this phenomenon.Examining the range of temperatures and normalised stresses over which the power relationship holds. This will define the range over which this method can be used to predict whole creep curves. If this master curve is widely applicable then shorter tests may be sufficient to predict long term creep curves.The ability of this model to predict the transition in copper from logarithmic primary creep to sigmoidal needs further investigation. This needs to be examined across other materials and conditions to establish if this is reliable or specific to the case examined.Future research and application of this model may include:


Harsh demanding has been exposed on the concentration of aflatoxin M1 (AFM1) and chloramphenicol (CAP) in milk. In this study, we developed a new method based on background fluorescence quenching immunochromatographic assay (bFQICA) to detect AFM1 and CAP in milk. The detection limit for AFM1 was 0.0009 ng/mL, while that for the CAP was 0.0008 ng/mL. The assay variability was determined with 3 AFM1 standards , and the actual detection value was 0.2497, 0.5329, and 1.0941, respectively. For the assay variability of 3 CAP standards , the actual detection value was 0.0996, 0.3096, and 0.4905, respectively. The recovery rate of AFM1 was 99.7%–101.7%, while that for CAP was 95.3%–97.6%. For the test stability, AFM1 and CAP showed satisfactory test stability even at month 5. Compared with the sensitivity of liquid chromatography-mass spectrometry (LC-MS) method, no statistical difference was noticed in results of the bFQICA. Our method is convenient for the detection of AFM1 and CAP in milk with a test duration of about 8 minutes. Additionally, an internal WiFi facility is provided in the system allowing for quick connection and storage in the intelligent cell phone.  For example, aflatoxin M1 (AFM1), the hydroxylated metabolite of aflatoxin B1, is a carcinogenic substance detected in milk and dairy products . Long-techemical .Currently, several methods have been developed for the detection of AFM1 and CAP, such as enzyme linked immunosorbent assay (ELISA), high performance liquid chromatography (HPLC), and gold immune chromatographic assay (GICA) –9. TheseRecently, a quantitative assay named background fluorescence quenching immune chromatographic assay has been developed based on the fluorescence quenching and nitrocellulose membrane background signals. The method has been used for the alpha fetoprotein in clinical practice. Unlike the conventional GICA assay, in the background fluorescence quenching immunochromatographic assay (bFQICA), the fluorescence donors are fluorescein that are precoated on the entire nitrocellulose membrane and quenching occurs between the gold particles and nitrocellulose membrane . In thisThe bFQICA analyzer was provided by the Simp Bio-Science Co., Ltd. . The reference substance and the monoclonal antibody of AFM1 were purchased from Rohi Biotech Co., Ltd. . The AFM1-bovine serum albumin (BSA) conjugate, reference substance and the monoclonal antibody of CAP, CAP-BSA conjugate, and the goat anti-mouse IgG were purchased from Rohi Biotech Co., Ltd. .2O) as previously described [GNPs were made through trisodium citrate reduction of hydrogen tetrachloroaurate (iii) hydrate (HAuCl4·3Hescribed . After pF0), test line, and control line (F1/F0) [The test strip consisted of sample pad, background fluorescence  .μg/mL, 0.60 μg/mL, 0.90 μg/mL, and 1.20 μg/mL, respectively. For the preparation of GNP-labeled CAP antibody, CAP was labeled by GNP solution (pH 7.5) with a concentration of 0.30 μg/mL, 0.60 μg/mL, 0.90 μg/mL, and 1.20 μg/mL. Ten minutes later, the mixture (1 mL) was add to an Eppendorf tube, followed by adding 100 μL PBS and incubating for 2 hrs. Afterwards, the mixture was centrifuged at 12,000 r/min for 10 min. The pellet was suspended in 3% bovine serum albumin (BSA) (1 mL) solution, and the supernatant was washed using PBS after centrifugation. Subsequently, the mixture was mixed with 0.5 μg/mL gold based goat anti-mouse antibody (20 μL). Finally, 10 μL product was transferred into a test tube and dehydrated for 12 hrs under vacuum.For the preparation of GNP-labeled AFM1 antibody, AFM1 antibody was labeled by GNP solutions (pH 7.0) with a concentration of 0.30 For the preparation of AFM1 or CAP coupled antibody, the AFM1-BSA was diluted using PBS buffer into a concentration of 0.30 mg/mL, 0.50 mg/mL, and 0.70 mg/mL, respectively, while that of CAP-BSA was 0.20 mg/mL, 0.40 mg/mL, and 0.60 mg/mL, respectively. Subsequently, the solution was coated onto the test line of the nitrocellulose membrane and kept at room temperature for 8 hrs. On this basis, three test strips coated with AFM1-BSA  and CAP-BSA  on the test line were obtained.μL each mixture was add to each test tube with gold probe based AFM1. Part of the mixture (60 μL) was added onto the sample port on the test line to obtain the reading. The optimal density of GNP-labeled AFM1 antibody and concentration of AFM1 coupled antibody were obtained in presence of significant difference between F1/F0 at four concentrations.The reference substance of AFM1  or CAP  diluted by PBS was add to each test tube. After that, 100 μL test samples were add to the small cup containing GNPs-labeled antibody. Then 60 μL mixture was dripped onto the hole foe  was added onto the sample port on the test line to obtain the F1/F0. After reading, the curves were established using concentration of AFM1 or CAP standard as the x-axis and F1/F0 at each concentration as y-axis.Using serial dilutions of the standard solution, we established a standard curve for the analysis of known samples with AFM1 or CAP in the range of 0–2.0 ng/mL. In brief, 100 μL milk was directly add to the small cup containing the GNP-labeled antibody, and then 60 μL of mixture was added onto the test line.Upon preparation of the test strips, AFM1 or CAP standards at various concentrations were used to assess the performance of bFQICA, including assay limit of blank and variability, test repeatability, and concentration recovery, as well as test stability. For the detection of AFM1 and CAP, 100 μm). The flow speed was 0.25 mL/min.LC-MS detection was performed according to the conventional description. The samples were extracted using the methyl cyanide-Mcilvaine buffer and then subjected to the Eclipse XDB-C18 column . For CAP, the formula was Y = −0.2519χ2 + 0.5353χ + 0.6558 (r = 0.9923), where χ is concentration of AFM1 or CAP standard solution and Y is the corresponding F1/F0.The formula of AFM1 was as follows: To test the limit of blank, a blank sample of AFM1 or CAP (0 ng/mL) was analyzed with the bFQICA system. The test was repeated 20 times, and a detection limit of 0.0009 ng/mL was obtained for AFM1, while that for the CAP was 0.0008 ng/mL. The assay variability was determined with 3 AFM1 standards at 0.25 ng/mL, 0.5 ng/mL, and 1.0 ng/mL, and the actual detection value was 0.2497 (RSD = 0.21%), 0.5329 (RSD = 0.13%), and 1.0941 (RSD = 0.15%), respectively. For the assay variability of 3 CAP standards at 0.10 ng/mL, 0.30 ng/mL, and 0.50 ng/mL, the actual detection value was 0.0996 (RSD = 1.76%), 0.3096 (RSD = 1.03%), and 0.4905 (RSD = 0.26%), respectively.To assess the test repeatability of the system, the concentration of AFM1 and CAP in the milk obtained in a local supermarket was determined. The concentration of AFM1 was 0.008 ng/mL (RSD = 0.54%), while that for the CAP was 0.0011 ng/mL (RSD = 0.45%).μL, 30 μL, and 10 μL, respectively. Afterwards, the mixture was dripped on the sample port of the test strip. The recovery rate of AFM1 was 100.1% (RS = 0.20%), 99.7% (RS = 0.35%), and 101.7% (RS = 0.87%), respectively (The milk samples (1 mL) were add to an Eppendorf tube, followed by addition of 10 ng/mL AFM1 or CAP of a volume of 50 ectively . The recectively .In this section, we determined the test stability by AFM1 or CAP standard and the standard of the same batch at months 1, 2, 3, 4, and 5, respectively. Both AFM1 and CAP showed satisfactory test stability even at month 5 .P > 0.05, The test milk containing AFM1 or CAP was subject to bFQICA detection and LC-MS detection. The detected concentration of AFM1 and CAP was listed in Food safety has been a great concern worldwide. Harsh demanding has been exposed on the concentration of AFM1 and CAP in milk. To date, several methods have been developed for the detection of these drugs such as ELISA and LC-MS. In this study, we developed a new method for the detection of AFM1 and CAP in milk which was more convenient with high specificity and sensitivity.The presence of AFM1 and CAP in milk has been a concern in some countries, which promotes the emergence of determination of these chemicals using various methods, such as ELISA, HPLC, and immunochromatographic assay. For example, in a previous study , Behfar μg/mL, while that for the CAP was 0.4 mg/mL and 0.6 μg/mL, respectively. Meanwhile, method validation was also confirmed in our study. The test efficiency showed no difference compared with the standard method proposed by SFDA. The detection method was stored in the two-dimensional codes, and quantitative test strips were established for CAP and AFM1, respectively. For the sensitivity, compared with the LC-MS method that had been considered as the golden standard with high accuracy, no statistical difference was noticed in the results obtained from bFQICA. This validated the accuracy of our method.Using this method, we confirmed that the AFM1 antigen coating concentration was 0.5 mg/mL in the test line, and the corresponding GNP-labeled AFM1 antibody concentration in the test tube was 1.2 As is known to all, false positive samples were noticed which may be related to the application of such technique and enzyme labeling , 17. TheIn this study, we developed a new method for the detection of AFM1 and CAP in milk which was more convenient with high specificity and sensitivity. The detection limit for AFM1 was 0.0009 ng/mL, while that for the CAP was 0.0008 ng/mL. Besides, the method showed satisfactory stability and test efficiency.


N = 21), MSA (N = 11), and age-matched controls  were tested on the prosaccade and antisaccade tasks using an infrared eye tracker. Twenty of these subjects were retested ~7 months later. Saccade latencies, error rates, and longitudinal changes in saccade latencies were measured. Both PD and MSA patients had greater antisaccade error rates than C subjects, but MSA patients exhibited longer prosaccade latencies than both PD and C patients. With repeated testing, antisaccade latencies improved over time, with benefits in C and PD but not MSA patients. In the prosaccade task, the normal latencies of the PD group show that basic sensorimotor oculomotor function remain intact in mid-stage PD, whereas the impaired latencies of the MSA group suggest additional degeneration earlier in the disease course. Changes in antisaccade latency appeared most sensitive to differences between MSA and PD across short time intervals. Therefore, in these mid-stage patients, increased antisaccade errors combined with slowed prosaccade latencies might serve as a useful marker for early differentiation between PD and MSA, and, antisaccade performance, a measure of MSA progression. Together, our findings suggest that eye movements are promising biomarkers for early differentiation and progression of parkinsonian disorders.Current clinical diagnostic tools are limited in their ability to accurately differentiate idiopathic Parkinson’s disease (PD) from multiple system atrophy (MSA) and other parkinsonian disorders early in the disease course, but eye movements may stand as objective and sensitive markers of disease differentiation and progression. To assess the use of eye movement performance for uniquely characterizing PD and MSA, subjects diagnosed with PD ( Parkinsonian disorders refer to a group of diseases linked to basal ganglia dopamine insufficiency. The most common form is idiopathic Parkinson’s disease (PD) and the remaining syndromes are grouped as atypical Parkinsonism (AP) . AP encoSpecifically, due to early overlapping symptoms, PD and MSA pose challenges in differential diagnosis, critical for early disease prognosis. Currently, no biomarkers can differentiate between the etiologies and clinical measures lack sensitivity and objectivity to uniquely characterize early stages of PD and MSA , 5. TherNote that MSA is characterized by glial cytoplasmatic inclusions in different regions including the putamen, caudate nucleus, substantia nigra, locus ceruleus, pontine nuclei, inferior olivary nucleus, Purkinje cell layer of the cerebellum, and intermediolateral cell columns , and theEye movements are a highly sensitive and objective measures used to test various levels of nervous system function, including cognitive status, making them a suitable measure for modeling PD and MSA disease course . Particul-DOPA , MSA , and controls (N = 20) were recruited from the UTHealth Movement Disorders Clinic  = 1.84, p = 0.17 for age; Fisher’s exact test, p = 0.66 for gender and p = 0.20 for race/ethnicity]. However, clinical characteristics were significantly different among groups  = 0.09, p = 0.77 for age; Fisher’s exact test, p = 0.39 for gender, and p = 0.36 for race/ethnicity; F = 0.46, p = 0.50 for H&Y; F = 0.07, p = 0.79 for UPDRS Total; and F = 0.14, p = 0.71 for UPDRS Motor.All 52 participants were tested at time point 1 (T1) on the eye movement tasks, and 20 returned at a second time point (T2) on average 7.4 months later (range 4.6–13.8). Demographics were similar among the three groups . MSA participants made more errors than controls at T1 . MSA participants did not make more errors than PD participants , and there was no significant difference between PD and control groups in prosaccade error rate .At T1, the direction error rate (±SE) for prosaccade was 0.6% (±0.4), 1.5% (±0.5), and 2.17% (±1.0), for control, PD, and MSA groups, respectively (Table p = 0.05), there was a significant difference among groups in error rate . Both PD and MSA participants made more errors than controls at T1 . There was no significant difference between PD and MSA groups in antisaccade error rate .The direction error rate (±SE) for antisaccade task test at T1 was 18.2% (±3.0%), 43.2% (±4.9%), and 42.6% (±8.0%), for control, PD, and MSA groups, respectively (Table 2(1) = 0.23, p = 0.63] nor interaction effect between group and time point . In addition, there were no differences among groups  or significant changes across time point collapsed across groups .Of the 20 subjects who were tested again at T2, the average prosaccade error rate changed from 0.4% (±0.4%) to 1.7% (±0.8%) for control group, 1.4% (±0.8%) to 1.5% (±0.8%) for PD group, and 1.5% (±0.7%) to 2.7% (±2.7%) for MSA group Table . There w2(2) = 5.53, p = 0.063], the paired comparison showed that PD and MSA participants had a 2.5- and 2.7-fold higher antisaccade error rates than controls, respectively . Additionally, time point was not significant . The change in antisaccade error rate (time point) was not significantly affected by age  nor by group .The average antisaccade error rate changed from 16.2% (±2.0) to 12.4% (±2.5) for control group, 37.6% (±7.8) to 34.8% (±8.8) for PD group, and 37.8% (±10.8) to 45.7% (±12.8) for MSA group (Table p < 0.001), the mean latencies were different among groups . MSA patients were significantly slower than PD  and controls , while there was no difference in mean latency between PD and controls .At T1, the average prosaccade latency (±SE) for correct trials was 260 ms (±10) for control, 267 ms (±8) for PD, and 318 ms (±20) for MSA group (Table p < 0.001), the antisaccade latencies were not significantly different among groups .In the antisaccade task, the average latency was 431 ms (±19) for controls, 446 ms (±18) for PD group, and 456 ms (±30) for MSA group (Table p = 0.37), the change in latency between time points for each group, i.e., interaction between group and time point, was not significant . The main effect of time point was not significant . Consistent with T1, the group effect was significant , with the MSA group having the slowest prosaccade latencies.For participants who were retested, average prosaccade latency (±SE) was 241 ms (±12) at T1 and 235 ms (±6) at T2 for controls, 247 ms (±10) at T1 and 254 ms (±9) at T2 for PD group, and 332 ms (±34) at T1 and 308 ms (±24) at T2 for MSA group  = 49.42, p < 0.001], the changes in latency between time points among groups, interaction between group and time point, were similar . Overall, participants had a faster response time at T2 compared to T1 ; however, the MSA patients were not faster at T2 compared to T1 . Unlike the T1 analysis, the group effect was significant , with the MSA group having the slowest latencies at T2 .Average antisaccade latency (±SE) changed from 409 ms ±37) at T1 to 378 ms (±38) at T2 for controls, 430 ms (±31) to 407 ms (±27) for PD group, and 445 ms (±38) to 440 ms (±33) for MSA group (Table 7 at T1 tGiven prior work showing that age  and dise2(1) = 1.35, p = 0.25]. For antisaccades, as reported above, there was no significant difference between the two groups, even when controlling for a significant effect of age. After controlling for age and H&Y score together, there again was no significant difference between the two groups .For prosaccades, as reported above, there was no significant difference between the two groups. After controlling for age and H&Y score together, there remained no significant differences between the two groups . Thus, when controlling for both age and H&Y score, MSA participants were on average 29 ms slower than PD participants. For antisaccades, as reported above, there was no significant difference between the groups, even when controlling for a significant effect of age. After controlling for age and H&Y score together, there again was no significant difference between the two groups .For prosaccades, as reported above, there was a significant difference between the two groups (MSA slower than PD). After controlling for age and H&Y score together, there remained a significant difference between the two groups [At a single time point, the study revealed two important findings: (1) error rates of MSA and PD patients in the antisaccade task were higher than controls, suggesting a behavioral biomarker for risk of a Parkinsonian disorder and (2) MSA patients show slower prosaccade latency than both controls and PD patients, suggesting a useful early behavioral marker for distinguishing MSA from PD. In addition, across a relatively short time frame (~7 months), repeated testing suggested antisaccade performance might also distinguish MSA from PD and may be a sensitive measure of the progression of cognitive deficit in MSA.High antisaccade error rates of PD and MSA reflect the common frontal brain deficits characteristic of both groups. These deficits  result in impaired inhibition of impulsive or reflexive responses and impaired generation of voluntary movements. With difficulties suppressing reflexive responses as well as with initiating correct voluntary saccades, the observed high error rates in PD and MSA compared to controls may be a useful behavioral biomarker to distinguish normal aging from Parkinsonian syndromes.Much previous work suggests voluntary saccade deficits in PD , 22, 35,The significantly slower reflexive latencies of MSA patients likely reflect the underlying differences in MSA versus PD pathology. Specifically, impairments to visual cortex and brainstem regions in MSA could result in more impaired reflexive oculomotor function. Previous work demonstrated slower reflexive latencies of both MSA-P and PD patients and suggested equivalent slowing . AlthougFrom the longitudinal data, there was no significant group by time point interactions for any of the dependent measures, suggesting that no significant changes occur over a short interval (~7 months) that could be used to distinguish between MSA and PD. Although the group–time point interactions were non-significant, there was an additional group effect that emerged in antisaccade latency. Overall, participants had shorter latencies in the repeated testing sessions; however, while C and PD showed greater benefits (shorter latencies) during retesting, the MSA group did not Figures C,D. ThesGiven the faster progression of disease in MSA patients, in our mid-stage patients, there was a significant difference in clinical severity, as measured by the H&Y. Thus, it is possible that differences in clinical severity were the cause of slower prosaccade latencies in our MSA group. Terao and colleagues have shown that there are changes in PD eye movement measures with disease progression . HoweverThese biomarkers may only be useful for early-to-moderate PD patients. Prospective investigations at an earlier time point in the disease course are critical to determine whether the observed changes in dependent measures can differentiate PD and MSA groups before definitive clinical diagnosis. Additionally, it is important to note that our MSA group is composed almost equally of MSA-P and MSA-C patients, which differs from the phenotype that is predominant in countries like Japan (MSA-C). Future investigations using homogeneous subtype populations, more time points, or a longer time interval would be helpful to discern group differences in the progression of disease.As new neuroprotective agents are developed, early and accurate diagnosis of Parkinsonian disorders becomes paramount. Our study indicates that increased antisaccade errors combined with slowed prosaccade latencies might serve as a behavioral biomarker for early differentiation between PD and MSA. Additionally, changes in antisaccade performance may prove useful for tracking MSA disease progression and evaluating potential interventions.Subjects provided written informed consent in accordance with the Declaration of Helsinki and were enrolled into a study approved by the Committee for the Protection of Human Subjects, the Institutional Review Board at the University of Texas Health Science Center at Houston.SB: data collection, analysis, and manuscript preparation. EK: data collection, analysis, and manuscript preparation and review. SR: data collection and initial analysis. NM: data collection and manuscript review. SP: data analysis, manuscript preparation, and review. AC: statistical analysis, interpretation, manuscript preparation, and review. JS: clinical testing, clinical data collection, manuscript preparation, and review. MS: subject recruitment, clinical testing, evaluation and diagnoses, and manuscript review. AS: study formulation, design, data collection and analysis, and manuscript preparation. All authors: contributed by drafting parts of the work, have approved the final version, and agreed to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.


In vitro, coculture of infected macrophages with MSC increased parasite load on macrophages in comparison with controls (macrophages without MSCs). In vivo, BALB/c mice were infected with 2 × 106Leishmania amazonensis (Josefa strain) promastigotes in the footpad. 7 and 37 days after infection, animals were treated with 1 × 105 MSCs, either intralesional (i.l.), i.e., in the same site of infection, or intravenously (i.v.), through the external jugular vein. Control animals received the same volume (50 µL) of phosphate-buffered saline by i.l. or i.v. routes. The lesion progression was assessed by its thickness measured by pachymetry. Forty-two days after infection, animals were euthanized and parasite burden in the footpad and in the draining lymph nodes was quantified by the limiting dilution assay (LDA), and spleen cells were phenotyped by flow cytometry. No significant difference was observed in lesion progression, regardless of the MSC route of administration. However, animals treated with i.v. MSCs presented a significant increase in parasite load in comparison with controls. On the other hand, no harmful effect due to MSCs i.l. administered was observed. The spleen cellular profile analysis showed an increase of IL-10 producing T CD4+ and TCD8+ cells in the spleen only in mice treated with i.v. MSC. The excessive production of IL-10 could be associated with the disease-aggravating effects of MSC therapy when intravenously administered. As a conclusion, in the current murine model of L. amazonensis-induced cutaneous disease, MSCs did not control the damage of cutaneous disease and, depending on the administration route, it could result in deleterious effects.Cutaneous leishmaniasis remains both a public health and a therapeutic challenge. To date, no ideal therapy for cutaneous leishmaniasis has been identified, and no universally accepted therapeutic regimen and approved vaccines are available. Due to the mesenchymal stromal cell (MSC) immunomodulatory capacity, they have been applied in a wide variety of disorders, including infectious, inflammatory, and allergic diseases. We evaluated the potential effects of bone marrow MSC therapy in a murine model of cutaneous leishmaniasis.  Leishmania (Leishmania) amazonensis is one of the etiological agents of the tegumentary leishmaniasis in Latin America  can stimulate endogenous repair of injured tissues, modulate immune responses, and are recruited to inflammation sites through chemotactic mechanisms not completely understood . The MSCrotozoan –12. Therrotozoan –12, we hL. amazonensis is an immunopathology with direct participation of CD4+ T cells in lesion development  were maintained in our own facilities with water and food L. amazonensis (MHOM/BR/00/Josefa strain) obtained by puncture of amastigotes from the lesion of infected BALB/c mice. The promastigotes were maintained at 26°C in M199 medium  containing 10% fetal bovine serum  until the fifth passage in culture to perform infection experiments. Infections were carried out with stationary phase culture, between the fourth and fifth days of culture.The parasites used in the experiments presented in this paper were 2). The dissected pieces were washed with PBS, cut into smaller fragments, and subsequently digested with type I collagenase (1 mg/mL in DMEM/10 mM HEPES) for 30–40 min at 37°C. Any gross remnants that persisted after collagenase digestion were poured off between 1 and 3 min, and the supernatant was transferred to a new tube containing fresh medium and centrifuged at 400 × g for 10 min at 25°C. The pellets were resuspended in 3.5 mL Dulbecco’s Modified Eagle’s Medium  containing 1% penicillin and streptomycin, with concentration of 5,000 IU/mL and 5,000 µg/mL, respectively , 20% of inactivated FBS  and 15 mM HEPES , seeded in T25 flasks (4 mL per flask) and incubated at 37°C in a humidified atmosphere containing 5% CO2. On day 3 of culture, the medium was changed, and non-adherent cells were removed. Adherent cells exhibited similar proliferation rates and, upon reaching 80% confluence, they were passaged with a 0.25% trypsin-EDTA solution  and maintained in DMEM with 10% FBS (complete medium). Approximately 1 × 106 cells were characterized as MSCs at the third passage according to the consensus of the International Society of Cell Therapy , which were anesthetized with intravenous ketamine (25 mg/kg) and xylazine (2 mg/kg). Tissues were collected, rinsed with phosphate-buffered saline (PBS), transferred to a Petri dish, and cut into small pieces , 5 µg/mL ascorbic acid 2-phosphate , and 10 mM/L β-glycerolphosphate  , 2.5 µg/mL insulin, and 50 µg/mL indomethacin   . AdipocyMO, USA)  for deteg, 4°C. Afterward, 10 µL of anti-CD16/32 antibody were added in cells (Fc receptor blocker), remaining for 15 min at 4°C. At the end of incubation, the cells were washed with PBS (100 µL/well), the plate was centrifuged at 300 × g for 4 min at 4°C and the supernatant was discarded. Subsequently, the cells were incubated with 10 µL of solution containing the anti-CD11b/FITC, anti-Sca-1/APC, anti-CD44/Pecy5, anti-CD45/PE, anti-CD49/PE, and anti-CD34/EFluor660 for 30 min at 4°C under light protection. Thereafter, the cells were washed with 100 µL of PBS and again centrifuged at 300 × g for 4 min at 4°C. After discarding the supernatant, the cells were resuspended in 200 µL of 1% PFA and transferred to FACS tubes containing 100 µL of PBS. The cells were analyzed using the FACSCalibur BD flow cytometry apparatus (BectonDickenson). Data were analyzed using FlowJo software 8.7  following standard procedures. Cells were plated in 96-well plates and then centrifuged for 4 min at 300 × 5 cells per well. The cells were incubated for 1 h at 37°C and 5% of CO2 for adhesion of macrophages to the plate. Thereafter, the plate was washed with warm PBS three consecutive times to remove the non-adherent cells, and then, 400 µL of RPMI with 10% of FBS supplemented with glutamine, pyruvate, and non-essential amino acids were added. After 24 h, the wells were washed again with PBS to remove B1 lymphocytes and 300 µL of RPMI with 10% of supplemented FBS were added.Syringes containing 5 mL of RPMI  medium were injected in the peritoneal cavity of BALB/c mice to obtain the macrophages. The medium containing the macrophages was withdrawn from the cavity and cells were counted with Tripan blue and transferred to a 24-well plate at the concentration of 5 × 105 cells per well. The infection was performed with 2.5 × 106 of L. amazonensis per well (ratio of 5:1) during 4 h with stationary promastigotes of L. amazonensis. After the infection time, the wells were washed with PBS and 5 × 104 MSC were added to some wells and left for 48 h at 37°C and 5% of CO2. The plate was washed again with warm PBS three times. Finally, the plate was fixed and stained with a fast panoptic kit . The infection was analyzed by optical microscopy .The peritoneal cells were plated in a concentration of 5 × 106 promastigotes of L. amazonensis on the right rear footpad. 7 and 37 days after infection, animals were treated with 1 × 105 MSCs, either intralesional (i.l.) (into the plantar cushion of the right rear footpad) or intravenously (i.v.), through the external jugular vein. Control animals received the same volume (50 µL) of PBS by i.l. or i.v. routes. MSCs (1 × 105 cells) were administered in 50 µL by intravenous (i.v.) injection or 20 µL by intralesional (i.l.) injection. The animals were sedated with sevoflurane  for i.v. or i.l. administration.One week after being received from the UFF, the animals were infected by subcutaneous route with 20 µL of PBS containing 2 × 10The lesion growth was weekly monitored with a pachymeter in millimeter scale . The measurement of the uninfected footpad was then deducted. Forty-two days after infection, mice were euthanized and the infected footpad of each animal was removed, placed in a 70% alcohol solution for 1 min, and individually weighed. Additionally, the right popliteal lymph node was removed and placed in a vial with 1 mL of M199 medium. Footpads homogenates were obtained by manual maceration of the lesions with addition of 1 mL of M199 medium and, after sedimentation of the heavier particles, 10 µL of the supernatants were recovered and resuspended in 990 µL of medium, thereby obtaining a pre-dilution of 1:100. The number of parasites in the footpads, spleens, lymph nodes, and livers was determined by the limiting dilution assay (LDA). The macerates were diluted into 96-well culture plates  and incubated at 26°C for 15 days. Promastigotes cultures were observed with an optical microscope  and the last well containing promastigotes was observed and noted (limiting dilution assay).6 cells per well in 24-well plates) to characterize T cells , regulatory T cells , and production of cytokines . The plates were centrifuged at 300 × g for 4 min. 100 µL of FACS buffer (PBS in 10% FBS) were added to the pellet and the cells were centrifuged again.The cells of the macerate of the popliteal lymph node were quantified by counting in a Neubauer chamber  incubation was done for 5 min, followed by surface labeling with anti-CD4/Pecy7 (1:200) and anti-CD25/APC (1:200) for additional 30 min. The volume was made up to 200 µL in FACS buffer; the cells were centrifuged, followed by fixation in a kit fixation buffer (eBioscience) during 1 h. Then, the cells were washed and 100 µL of permeabilization buffer (eBioscience) kit were added to the pellet and incubated for 1 h, followed by centrifugation. Then, the cells were incubated with anti-FCR for 20 min and intracellular labeling was performed with anti-FoxP3/AlexaFluor 488 (1:50) incubating for 30 min. The cells were washed in permeabilization buffer and resuspended in FACS buffer for flow cytometry analysis .In the cytokine characterization plate, the cells were stimulated during 4 h with PMA (phorbol 12-myristate 13-acetate) (20 ng/mL), ionomycin (1000 ng/mL), and monensin (5 µL/mL) to inhibit cytokines secretion. Following the stimulation, the cells were washed in FACS buffer. Blockage of non-specific labeling with anti-FCR (1:100) incubation was done for 5 min, followed by surface labeling with anti-CD4/PeCy7 (1:200), anti-CD8/FITC for 30 min. The volume was filled to 200 µL in FACS buffer; the cells were centrifuged and further washed in FACS buffer. 100 µL of 1% paraformaldehyde was added to the pellet for fixation, which was left for 1 h incubation, followed by centrifugation. 100 µL of permeabilization buffer were added to the pellet (PBS and 0.1% saponin) and left for 1 h incubation, followed by centrifugation. The cells were incubated with anti-FCR for 5 min and then intracellular labeling was performed with anti-IL-10-PE (1:60) and anti-IFN-γ/APC (1:60) incubating for 30 min. The cells were washed in permeabilization buffer and resuspended in FACS buffer for flow cytometry . The results were analyzed using software SUMMIT V4.3 Build 2445.Concentrations of the cytokines present in the footpad homogenate supernatants were determined by the ELISA using commercial kits (BD OptEIA) according to the datasheet instructions. Recombinant murine IL-10, IL-4, and TGF-β were used to generate the standard curves. The detection limits for these tests are 31.3 pg/mL for TGF-beta and IL-10 and 15.72 pg/mL for IL-4.in vitro data, Student’s t-test was used. Two-way ANOVA following Bonferroni’s post hoc test was used to compare all other parameters. P-values of 0.05 or less were considered meaningful. For in vivo experiments, we compared both groups, i.l. and i.v. This comparison between i.l. and i.v. was carried out besides of the differences between the two treatment protocols, according to the reviewer suggestion. All tests were analyzed using the statistical software package GraphPad Prism v6.0 .No formal sample size calculation was performed to determine the numbers of animals per group; the sample size was based on the experience of our laboratory in previous studies using this model of cutaneous leishmaniasis . To compin vitro conditions (data not shown). Additionally, MSCs expressed cell surface markers, such as: CD44, CD49 and Sca-1, bud did not express CD11b, CD45 and CD34  and infected macrophages in coculture with MSC (MΦ + Leish + MSC)  Figure C. The sas Figure D. This rin vivo, BALB/c mice were infected and treated as described in the methodology. In relation to the lesion growth, treatment with MSCs by intravenous or intralesional route did not induce protection when compared with the controls  and Treg cells (CD4+ CD25+ FoxP3+) by quantification of CD25+ and FoxP3+ markers in CD4+ T cells. In both groups, the percentages of effector T cells were similar  (in vitro and fail in skin hypersensitivity tests using Leishmania antigens (negative Montenegro reaction), although some patients continue to respond to other antigens, such as tuberculin and lepromin  . Patientlepromin . Accordi regions , thus raT. cruzi  in resistant mice  anti-IL-4 monoclonal antibodies were used. However, this treatment did not promote resolution of the infection, which did not evidence a relation of IL-4 with the pathogenesis of the disease in this model. Regarding the levels of IL-4 and TGF-β in our study, we did not obtain significant differences among treated groups, which suggest the lack of pathogenesis modulation by IL-4 and TGF-β. The high concentration of IL-10 ; however, without the increase of IL-10 level, the parasite load did not rise. The concomitant increase of IL-10 and decrease of IFN-γ may be the main factor of failure of our therapy.Previous observations have shown that parasite-specific Th1 cells promoted protective immunity to nfection . In addiic phase . Studiesarasites . The decL. amazonensis infection, Treg cells are important in the control of lesion and parasite load in resistant mice model using C57BL6  in the spleen , but it did not protect against the lesion as expected based on C57BL6 mice model. The increase of Treg did not affect growth lesion, suggesting that lesion and Treg on BALB/c mice may not be related. Furthermore, Treg cells were not contra-protective either in our model, since the increase of Treg in intralesional groups (PBS i.l. and MSC i.l.) did not result in the increase of parasite load in the intralesional groups. Interesting, the groups that presented an increase of Treg  showed a decrease of TCD8+ producing IFN-g, which suggests a possible function for Treg cells in BALB/c mice.Corroborating with the literature , 40, we L. amazonensis infection. Therefore, more studies must be conducted to find therapies that are effective against leishmaniasis due to the toxicity of the current pharmacological treatments used in the clinical setting. Besides, as it has been reported, there are significant concerns in that the application of mesenchymal stem cell may suppress antimicrobial immunity with increased risk of infection  when compared with controls (PBS i.l. and i.v.) did not show protective effect against nfection .L. amazonensis in BALB/c mice. Therefore, these results cannot be extrapolated to other leishmaniasis models, mouse strain or directly to the clinical scenario. Second, two doses of (105) have been used and the effects of MSCs were evaluated 5 days after the second administration. Based on pilot studies, higher number of MSCs may increase the risk of embolism. Therefore, we decided to administer this dose twice. Nonetheless, in future studies with different doses, different ranges between MSC administration and analysis at earlier and later time points would be interesting. Third, MSCs from different sources exhibited distinct effects (Further studies are necessary to a complete understanding of the treatment using MSC against leishmaniasis. First, experimental cutaneous leishmaniasis was induced by  effects , 44; howL. amazonensis, bone marrow MSC therapy, in comparison with PBS, did not result in any significant difference in lesion progression, regardless of the administration route. Intravenous, but not intralesional administration of MSCs, increased the parasite load. BM-MSC therapy is not effective as a treatment against murine leishmaniasis.In the current murine model of cutaneous disease induced by Conceived and designed the experiments: HG and PR. Performed the experiments: JCP, TR, JS, JK, JESP, AF-M, MM, and LC. Analyzed data: JCP, TR, and JS. Scientific discussion: TR, JS, JCP, SC, DG, BD, PR, and HG. Contributed reagents/materials/analysis tools: HG, PR, and BD. Wrote the paper: TR, JS, JCP, SC, DG, BD, PR, and HG.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.