Aspergillus foetidus in soybean residues was optimized using 2 × 23 factorial designs. The experimental data was fitted into a polynomial model for xylanase activity. Statistical analyses of the results showed that variables pH and the interaction of pH and temperature had influenced the production of xylanase, with the best xylanase production level (13.98 U/mL) occurring at fermentation for 168 hours, pH 7.0, 28°C, and 120 rpm.Enzymatic hydrolysis is an important but expensive step in the process to obtain enzyme derived products. Thus, the production of efficient enzymes is of great interest for this biotechnological application. The production of xylanase by They randomly catalyze the endohydrolysis of 1,4-.8 are foβ-xylanases, endo-1,3-β-xylanases, and cellobiohydrolases. The major enzymes of this family are endo-1,4-β-xylanases; however, substrate specificity studies revealed that these may not be entirely specific for xylan and may not be active on low molecular mass cellulose substrates. In effect, it has been found that the replacement of one or two xylose residues by glucose is normally tolerated by the xylanases of this family, with this generally resulting in a lowered catalytic efficiency [Family 10 consists of endo-1,4-ficiency .β-sheet structure [In contrast to other families, family 11 is monospecific, it consists solely of xylanases. Moreover, these xylanases are “true xylanases” as they are exclusively active on D-xylose containing substrates. They have a lower catalytic versatility than family 10 xylanases and indeed the products of their action can be further hydrolyzed by the family 10 enzymes. These xylanases are characterized by a high pI, low molecular weight, and a tructure .α-helix (~3–6%) and high percentage of β-sheets (~43–48%). This suggests xylanase from Aspergillus foetidus is classified as a member of family 11. These results are in accordance with literature, in which xylanases present approximately 3–5% of α-helix structures and a higher percentage of β-sheets [Supplementary studies (data not shown) revealed a low percentage of α-helix ~–6% and h-sheets ~3–48%. ThAmong the many microbial sources, filamentous fungi are especially interesting as they secrete these enzymes into the medium and their xylanase levels are very much higher than those found in yeasts and bacteria . SeveralXylanases are extensively used in the paper and pulp industry, as well as in baking, animal feed, biofuels production, fruit and vegetable processing, manufacture of bread, food, and drinks, textiles, xylitol production, saccharification of agricultural, and industrial and municipal wastes among other utilities , 8. The Brazil is the second biggest producer of soybean worldwide, harvesting 96.5 kton in 2016, just behind United States, with 106.9 kton in 2016 . TherefoThus, the experimental design statistical approach for enzyme production using a response surface methodology (RSM) is an alternative strategy to reduce the production cost. Recently, RSM has been utilized successfully to improve product yield and to reduce development time and cost of biotechnological processes . Aspergillus foetidus strain isolated from Brazilian Savannah, grown in submerged fermentation, using soybean residue as substrate.In this work, we employed Central Composite Design (CCD) for the planned statistical optimization of xylanase activity of anSoybean residue was autoclaved at 121°C for 2 hours and thoroughly washed with tap water. After autoclaving, it was dried at 65°C for 48 hours and then grounded to form a relatively homogeneous blend. A fine powder was obtained and used as a substrate for xylanase production . Aspergillus foetidus was obtained from the microorganisms' collection of Laboratory of Enzymology from University of Brasília. The Aspergillus foetidus was kindly provided by Professor Dr. Edivaldo Ximenes, Depositary Microorganisms Center: Collection of microorganisms for phytopathogens and weeds control from Embrapa Genetic Resources, accredited by Genetic Heritage Management Council by CGEN deliberation n° 67 published in the DOU in 13.09.2004, Section 1, page 53, linked to the project “Biotechnological processes in application of hollocelulases from filamentous fungi”, Process n° 010237/2015-1, University of Brasília. It is maintained in potato dextrose agar (PDA) medium at −80°C.The fungi7 spores/mL.The spore concentration was determined by counting under a microscope with a Newbauer chamber and adjusted with sterile saline solution (0.9%) to a final concentration of 1 × 107 spores/mL) was inoculated into 250 mL Erlenmeyer flasks containing 50 mL of liquid medium  at pH 7.0 with 2% (w/v) of soybean residue. The cultures were incubated at 28°C with constant agitation at 120 rpm for 7 days.For xylanase production, an aliquot (1 mL) of spore suspension  and stored at −20°C. The resulting filtrate, here called crude extract, was used as a source of xylanase.μL of enzyme solution with 100 μL of birchwood xylan (10 mg/mL) in 50 mM sodium acetate buffer, pH 5.4 at 50°C for 30 min [μmol of reducing sugar released per min per milliliter (IU/mL). Xylose was used as standard.The xylanase activity was determined by mixing 50 r 30 min . The relr 30 min . The absProtein concentration was measured by the method of Bradford, using bovine serum albumin as standard .X1), temperature (X2), and pH (X3) at enzymatic production of xylanase in medium with soybean residue, two factorial designs were employed (FD1 and FD2). For both, a 23 factorial design with three center points and axial points was employed (Y).To analyze the effects of the agitation (employed . The facCenter points were defined based on previous methodology used in our laboratory (data not shown); axial, −1, and +1 points were determined in order to evaluate significant differences. FD2 was planned aiming to increase the difference observed.p < 0.05) were considered.The Design-Expert® software, version 9.0.6.2, was used for regression and graphical analysis of the data. Only the factors with significance higher than or equal to 5%  were performed and are shown in results section. Aspergillus foetidus xylanase specific activity was assessed every day, for 20 days  [The highest xylanase activity was after 15 days of culture 11.84 U/mL). On the 7th day of culture, the xylanase activity was 9.72 U/mL, and specific activity had its maximum value, which was 810.41 U/mg during the period of analysis. A specific activity of purified U/mL. OnThe specific activity is an important parameter to assess the enzymatic activity in relation to the amount of proteins in the sample. In general, high specific activity represents the highest level of target enzyme. For this reason, the 7th day was chosen for this study. Aspergillus foetidus cultivated with soybean residue is consistent with previous reports, which xylanases from Aspergillus sp. cultivated in different residues also present high activity levels, as described below.The xylanase specific activity from Aspergillus fumigatus, 1055.6 U/g and 558.3 U/g in wheat bran and soybean, respectively, after 5 days of solid state fermentation. In the same work, he found for Aspergillus niger, a specific xylanolytic activity of 1285.0 U/g, 484.2 U/g, and 1050.0 U/g using as residue wheat bran, soybean, and wheat bran with sugarcane bagasse, respectively [ Aspergillus fumigatus submerged liquid culture with sugarcane bagasse a xylanolytic activity of 53.1 U/mg [ Aspergillus niger solid state fermentation found for xylanase activity 13.24 U/mL [Delabona et al. (2013) found a specific xylanolytic activity forectively . Supplem3.1 U/mg ; and Far.24 U/mL .The amount of protein oscillated during the culture period, suggesting this result may include other enzymes besides xylanase, which are concomitantly produced and also participate in the substrate degradation process. The induction profile followed the pH variation, with maximum value of 5.92 on the 1st day of culture and minimum value of 2.94 on the 4th day. Seventh day pH was 3.20. This result indicates a xylanase production in acidic medium. Aspergillus foetidus grown on soybean residue under submerged liquid culture was assessed. Variation on agitation, temperature, and pH effects on xylanase activity were evaluated using the statistical design of experiments and RSM analysis. The activity of the xylanase present in the crude extract produced by filamentous fungus2, and pH ∗ temperature  had a significant effect on the xylanase production (p < 0.05). Interactions between agitation and temperature (AB) and interactions between agitation and pH (AC) had no significance (p > 0.05). Under the levels tested in the factorial design, the variables agitation and temperature did not interact with each other. Subsequently, the xylanase activity is not significantly modified.In the first study, the independent variables pH, pHR2) was calculated to be 0.88, indicating that 88% of the variability in the response could be explained by the model. In addition, the F test  was satisfactory for the prediction of the model used to describe response surface plot of the enzyme activity as a function of pH and temperature  found a maximum activity at 60°C and optimum pH of 4.0 for xylanase produced by A. fumigatus under solid state fermentation (SSF) [Other studies of xylanase report optimum pH, temperature, and agitation at specific values. De Souza Moreira et al. 2013) found an optimum pH and temperature of pH 6.0, 50°C at 120 rpm and pH 5.0, 45°C at 120 rpm for xylanases produced by13 found on (SSF) .p value, indicating that the model is sufficiently accurate to predict the factors responses within the ranges studied. The “lack-of-fit F-value” of 1.38 implies that the lack of fit is not significant relative to the pure error. There is a 47% (p = 0.47) chance that a “lack of-fit F-value” this large could occur due to noise.The lack-of-fit test did not result in a significant R-Squared” of 0.242 is not as close to the “Adj R-Squared” of 0.732 as one might normally expect (difference is more than 0.2). This may indicate a large block effect or a possible problem with the model. “Adeq Precision” measures the signal-to-noise ratio. A ratio of 7.91 indicates an adequate signal (a ratio greater than 4 is desirable). This model can be used to navigate the design space.The “Pred Y1 = −81.111 + 0.431∗A + 1.784∗B + 12.937∗C + 0.006∗A∗B + 0.009∗A∗C − 0.153∗B∗C − 0.003∗A2 − 0.027∗B2 − 0.748∗C2, where Y1 is the predicted xylanase activity in U/mL; A, B, and C are the coded variables of agitation, temperature, and pH, respectively. The equation in terms of actual factors can be used to make predictions about the response for given levels of each factor.The response equation obtained is the following: ∗ pH and pH2 showed a negative effect, within the range evaluated. Furthermore, the significance of the interaction effect between pH and temperature revealed synergistic effect of these variables; that is, the variables pH and temperature well-adjusted could modify the xylanase activity. The pH effect was higher than the temperature effect, as can be verified for the coefficient values listed in  Aspergillus niger and cellulase from Aspergillus heteromorphus, respectively. The authors found that the change in temperature was less importante than changes in pH, within the range evaluated. As the pH varies, the charge of the substrate and ionic components of the substrate changes, affecting the activity of the enzymes [The negative effect of the factors means that an increase in one of them will reduce the enzymatic activity. For xylanase activity, pH showed a positive effect while temperature  enzymes , 21.In the second study of xylanase, the range of agitation, pH, and temperature were expanded in relation to the range of the previous study, in order to evaluate a possible positive effect in the xylanase activity.F-value of 4.39 implies the model is significant at a high confidence level , indicating the significance of the model. The coefficient of variation (R2 = 0.84) indicates a high correlation between the experimentally observed and predicted values and indicates the degree of precision with which the treatments are compared.The computed ce level . The proF-value” of 3.27 implies the lack of fit is not significantly relative to the pure error. There is a 25.04% (p = 0.2504) chance that a “lack-of-fit F-value” this large could occur due to noise. Additionally, “Adeq Precision” of 5.88 indicates an adequate signal. This model can be used to represent the design space.The “lack-of-fit 2, and temperature2  had a significant effect on the xylanase production (p > 0.05).The independent variable pH, pHoduction . The othY2 = −54.322 + 0.113∗A + 1.997∗B + 10.506∗C + 0.000∗A∗B − 0.001∗A∗C − 0.056∗B∗C − 0.000∗A2 − 0.028∗B2 − 0.713∗C2, where Y2 is the predicted xylanase activity and A, B, and C are the coded variables of agitation, temperature, and pH, respectively. The equation in terms of actual factors can be used to make predictions about the response for given levels of each factor.The response equation obtained for the second model was as follows: ∗ temperature on xylanase production, the three-dimensional contour plot was used to assess the effects of pH and temperature on xlylanase production . There were very small differences in the predicted response and the activity observed during the experiments, confirming that the results are in accordance of the RSM study. The xylanase from Aspergillus niger and Aspergillus flavus of 10.50 and 11.92 U/mL, respectively, using as residue wheat bran 0.5% and corncob 0.5%. Supplementing it, Naseeb et al. (2015) found for A. fumigatus pea peel substrate under Solid State Fermetation (SSF) 13.97 U/mL [Guimaraes et al. (2013) found a xylanolitic activity for.97 U/mL , 23. Aspergillus foetidus to produce high level of xylanase enzyme in a liquid medium. This study highlighted a newly isolated strain A. foetidus which could produce xylanase in soybean residue medium, which is cheap and abundant. The best conditions of xylanase production were pH 7.0, 120 rpm, and 28°C (168 hours). In future studies, statistical optimization of medium, physical factors, and scaling up studies in bioreactor should be used as an alternative to contribute toward the economics of biotechnological processes.In this study, we investigated the feasibility of filamentous fungus


Calcium (Ca)- magnesium (Mg) imbalance is implicated in prostate cancer. Ca/Mg ratio increases or decreases with proliferation or apoptosis, respectively. The study examined whether this Ca/Mg imbalance exists in BPH patients and the effect of a phytotherapeutic drug on the Ca/Mg ratio.Croton membranaceus (60 mg/day) for 3 months were examined for serum Ca, Mg, phosphate, parathyroid hormone (PTH), vitamin D, prostate specific antigen (PSA) levels and renal function tests (RFT) before (BT) and after treatment (AT) alongside thirty (30) controls. Twenty (20) trace element including Mg and Ca were determined in the drug by neutron activation analysis (NAA).Thirty (30) BPH patients who used the ethanolic root extract of p = 0.002). Mg, Ca/Mg ratio BT, AT and control were significantly different . After treatment, Mg and Ca/Mg ratio were not different from controls. The prevalence of Ca/Mg imbalance was 80% (BT), 13.3% (AT) and 3.3% (control group).RFT, PTH and vitamin D for BT, AT and controls (C) were normal. Mean PSA was 1.0 ± 0.64 (C), 27.9 ± 19.0 (BT) and 16.2 ± 11.8 ng/mL (AT) (Ca/Mg ratio imbalance is associated with BPH. This has previously not been demonstrated. The imbalance was significantly corrected after treatment with the phytotherapeutic drug. Epidemiological studies of a cohort in Taiwan associated drinking water Ca and Mg levels, to prostate cancer (PCa) deaths. The authors concluded that Mg from the drinking water or other dietary sources was protective against prostate cancer . Major t2+ signaling is therefore important in regulating other physiological functions such as cell proliferation and differentiation in conjunction with Mg  were also examined by neutron activation analysis.2SO4. The final chromogen was read at 450 nm on a BioTek plate reader . TPSA was calculated from the calibration curve.Total PSA (TPSA) was performed using AccuBind total PSA ELISA kits purchased from MonoBind Inc.  according to the manufacturer’s instructions. The ELISA plate was coated with highly specific monoclonal anti-PSA antibodies. In brief, 25 μl of samples together with standards were aliquoted into designated wells and incubated with 100 μl antibody-HRP enzyme conjugate at room temperature for 30 min. The plate was then washed with a wash buffer after which 100 μl of 3,3′,5,5′- tetramethylbenzidine (TMB)/hydrogen peroxide (substrate) was then added to react with the HRP. After 15 min incubation, the reaction was terminated by the addition of concentrated HPTH and vitamin D levels were estimated by ELISA techniques using Sunlong Biotech Co. Ltd. . In brief, microtiter plates coated with PTH and vitamin D antibodies were used. PTH and vitamin D from the blood samples reacted with the antibodies immobilized on the plate and the immunogen was further developed using a second antibody-horseradish peroxidase conjugate. The final chromogen was read at 450 nm.Serum concentration of Ca, Mg, phosphorous (P), urea, creatinine, sodium, potassium and albumin were determined by standard analytic methods on the Mindray BS 300 Chemistry analyzer  using commercial kits from Elitech (France). All assays were performed with standards and controls.Croton membranaceus) alongside international standards and controls were irradiated in the Ghana Research Reactor – 1 (GHARR-1) operating at 15 KW and a thermal flux of 5 × 1011 n.cm -2. S-1. Samples were irradiated for 5 min followed by 10 min of counting. The radioactivity measurement of induced radionuclide was performed by a PC interfaced with the γ-ray spectrometric equipment. The following trace elements were analyzed: As, Cl, Co, Cr, Cu, Cd, Hg, Pb, Ni, Fe, Mn, I, V, Al, Mg, Se, K, Na, Ca, and ZnIt has been suggested that Mg and other trace elements (TE) supplementation may play a role in shrinking an enlarged prostate. To this end, TE analysis was performed to ascertain whether the phytotherapeutic drug could possible play the role of a TE supplement. In brief, TE were determined by neutron activation analysis (NAA) at the Ghana Atomic Energy Commission. Samples of de-capsulated extract  and Microsoft Excel 2013 were used for the statistical analysis. Data was presented as mean ± standard deviation. Statistical analysis between groups was determined by student’s t-test. Categorical variables were expressed as proportion. Correlation analysis was performed with Pearson correlation test. The level of significance was 0.050.P = 0.002)  and after treatment cases (AT) were 1.0 ± 0.64, 27.9 ± 19.0 and 16.2 ± 11.8 ng/mL, respectively. There was a significant difference before and after treatment (p = 0.0001). Similarly, Mg BT and AT were different (p = 0.0031). Mg concentration differences were no more significantly different after treatment compared to the control or BT , but higher for the control group (0.8 ± 0.1 mmol/L). Control group and BT group Mg differences were significant  Table . Similarp = 0.0001) and BT and AT (0.0001). However, the ratio after treatment and that of the control group no longer showed significant differences . However, this correlation did not exist after treatment with levels similar to the control group. Similarly, there was a positive correlation between Mg and P before treatment . Correlation no longer existed after treatment with levels similar to the control group (Table There was a positive correlation between Ca and Mg before treatment (up Table .Table 4Pr = -0.884). This correlation existed moderately in the test groups . This correlation also existed in the disease groups to a moderate extent . However, the correlation was moderate in the disease group  were randomly selected compared to 2.5% in the control group made up of 341 hospital staff . Other dCroton membranaceus) was analyzed for its trace element content. It was noted that the extract was high in Ca, followed by potassium and Mg. Indeed the Ca level was 18 times that of Mg; nevertheless Ca absorption is always tightly regulated. Although the serum Mg level was significantly low while the Ca/Mg ratio was significant high before treatment, the Mg level significantly increased to that of the controls and the Ca/Mg ratio significantly reduced to the level of the controls after treatment.The phytotherapeutic drug that was administered , is debatable. The former is very unlikely considering the RDA for Ca and Mg and the overall 60 mg intake of the plant extract drug which is far below the RDA. Nevertheless, the imbalance and its association with the Ca channels remain plausible.Ca channels and the Ca/Mg ratio is a developing area of focus. The Ca/Mg ratio in this study was significantly raised in the BPH group compared to the control group. Studies have shown that inadequate Mg levels, relative to Ca levels (i.e. high Ca/Mg ratio) are associated with greater risk of prostate disorders. Among high-grade prostate cancer cases compared to controls, serum Mg levels were significantly lower, while the Ca/Mg ratio was significantly higher. An elevated Ca/Mg ratio was therefore associated with an increased risk of high-grade prostate cancer and elevated Mg was significantly associated with a lower risk . HoweverCroton membraneus on BPH-1 cells have recently been demonstrated [It has been recently demonstrated that a high Ca/Mg ratio is pro-inflammatory and Mg increase reverses this imbalance through gate-keeping activities of TRPM6/7 gene in an apoptotic drive. The apoptotic mechanism of this phytotherapeutic drug made of nstrated . Therefonstrated .The PSA levels were also reduced significantly after treatment and symptoms improved significantly by the International Prostate Symptoms Score (IPSS) assessment as published elsewhere . The corCa/Mg ratio imbalance is associated with BPH similar to observations in PCa. This has previously not been demonstrated. The imbalance was corrected after treatment. Although apoptosis is suspected to be one of the mechanisms for the action of this pytotherapeutic drug, the actual mechanism is yet to be elucidated.


AbstractIshigakicoccusgen. n.shimadaisp. n. is described based on the adult female morphology. This new species was found in the nest of a rare Japanese ant, Acropygayaeyamensis Terayama & Hashimoto, 1996, in Ishigaki Is., Japan. It resembles Capitisetellamigrans  and Pseudorhizoecusproximus Green, 1933. However, the new species differs from those two species in having small multilocular pores, large 3-5 locular pores on the medial area of ventral abdomen, and two different-sized body setae. This new species is also the first record of a potential trophobiont of Acropygayaeyamensis. A key to Japanese species of Rhizoecidae is also provided. Rhizoecidae is a family of Coccomorpha (Hemiptera: Sternorryncha) which members mostly live underground, feeding on plant rootlets , and 11 species distributed in five genera have been recorded from Japan   . Trophobfounding , the meafounding , and thefounding . This obomically . NeverthAcropygayaeyamensis which showed a close association with the ant .Specimens of the root mealybug species used in this study were originally collected by a collaborator of this study, Mr. Taku Shimada, on 16 February 2015, from a nest of PageBreakTaxon classificationAnimaliaHemipteraRhizoecidaeTribeWilliams, 1969Rhizoecus Künckel d’Herculais, 1878Xenococcini  where the type species was collected first. The suffix coccus is commonly used in naming Coccomorpha genera. Gender: masculine.Named after the island . Paratypes, same data as Holotype, 3♀ (NSMT-I-Ho 00074–76).Holotype, adult female. Japan, Okinawa-pref., PageBreakst : 18.4–29.5 µm long, 2nd : 13.7–18.4 µm long, 3rd : 15.8–26.3 µm long, 4th : 17.1–22.1 µm long and 5th : 36.8–41.3 µm long. The fifth segment with three fleshy setae. Anterior spiracles each 20.8–28.7 µm wide across atrium; posterior spiracles each 16.8–29.7 µm wide across atrium. Legs well developed, length of posterior pair: coxa 45.8–55.2 µm long, trochanter + femur 123–133 µm long, tibia + tarsus 112–122 µm long, claw 33.4–39.4 µm long. Ratio of lengths of tibia + tarsus to trochanter + femur 1.05–1.11. Claw digitules setose, each 3.2–6.1 µm long.Body elongate oval, slightly pyriform, 721–895 µm long, 379–632 µm wide. Eye spot absent. Antennae 5 segmented, 1Dorsum: Two types of body setae present. Type I: relatively well developed setae, 19.7–58.9 µm long, some setae with a bent or rarely with a knobbed apex. Type II: relatively slender setose setae, 11.0–32.8 µm long, each with a pointed apex. Both types of setae distributed in transverse segmental rows. Multilocular pores each about 2.9–3.7 µm in diameter, each with 5–6 loculi, present on head and thorax, but rarely present on abdomen  and Pseudorhizoecusproximus Green, 1933, in lacking ostioles and tritubular and/or bitubular pores, and having a pyriform to round body shape. However, the former is clearly distinguishable from those two species by having small multilocular pores on both surfaces of the body, 3–5 large locular pores on the medial area of the ventral abdomen, and two different-sized body setae. These morphological differences are here considered as good for the establishment of a new genus. Based on some morphological similarity, the genus and species is tentatively placed in the subtribe Ripersiellina Kozár 2007 of the tribe Rhizoecini Williams, 1969, in which Capitisetellamigrans and Pseudorhizoecusproximus are also placed (The genus and species described in this paper slightly resembles two other species of the monotypic genera of o placed .PageBreakXenococcini have been frequently found in Acropyga ants’ nests (Acropyga ants. However, the new species described in this paper is clearly different from all of them in having small multilocular pores, 3–5 locular pores, and 5 segmented antennae. Although the exact phylogenetic position of this new species is still unclear, but morphological features of the specimens indicate that this species apparently does not belong to tribe Xenococcini.In east and southeast Asia, many species in tribe s’ nests , and theAcropygayaeyamensis workers grabbing and carrying the root mealybug individuals by their mandibles when he collected the species (Figure Acropygayaeyamensis and the morphological features of the species, i.e. the lack of wax on the body surface (Figure Acropygayaeyamensis, even though it does not appear to be a xenoccocine species. Similar associations between Acropyga ants and scale insects that are not members of the tribe Xenococcini have been sometimes, but not commonly, observed in the neotropics, the United States, and Australia (Acropygortheziawilliamsi LaPolla & Miller, 2008, which belongs to the family Ortheziidae, not Rhizoecidae, showed a close trophobiotic association with Acropygamyops (Acropyga ants, including non-xenococcine trophobiont and/or potential trophobiont species of Acropyga ants, related co-evolution and co-speciation analyses and behavioral ecological studies may provide new insights into the evolution and maintenance of this unique obligate mutualism.The collector of the species, Mr. Taku Shimada, observed some s Figure . This bee Figure , which ie Figure  indicateustralia . One of gamyops . Intensi


Escherichia coli O157:H7, were examined for their physiological and morphological characteristics. Degree of bacterial lysis and host range of isolated bacteriophages was determined against 55 isolates of E. coli O157:H7. Morphology of phages was examined under transmission electron microscope. Phage growth parameters, particularly rate of adsorption, rise period, latent period, and burst size were also determined. The stability of isolated phages was tested at acidic and alkaline pH, at high temperatures, and in cold storage. A total of 7 phages were isolated which showed lytic activity against 50 out of 55 isolates of E. coli O157:H7. Based on the morphology, phages were classified into Myoviridae or Siphoviridae family. Phages had a rise period between 19 and 40 min, a short latent period between 12 and 30 min, and a large burst size (89–631 virions per infected cell), indicating high lytic activity. Phages remained stable for 24 h at a wide pH (1–11) and temperature range (40–60°C) and for 90 d in cold storage. Characterization of bacteriophages, with a diverse host range of E. coli O157:H7, could aid in the development of effective biocontrol strategies for this pathogen in the food industry.Bacteriophages, recovered from beef cattle environment and specifically targeting Escherichia coli O157:H7 is an important foodborne pathogen that lives commensally in the rumen of cattle and other food animals such as sheep and goat [ E. coli O157:H7 could mediate its transfer to water and food products, which could result in human infections upon consumption. In a typical year in the United States (US), E. coli O157:H7 causes an estimated 63,000 foodborne illnesses, 2,100 hospitalizations, and 20 deaths, imposing an economic burden of $271 million [and goat , 6–8. Di million , 10. Adv million –13. Some million . E. coli O157:H7 at the preharvest and processing level could play a significant role in preventing the introduction of this pathogen into the food chain. Various approaches, such as diet and probiotic therapy, vaccination, and antibiotics, against E. coli O157:H7 have been evaluated to control its prevalence in food animals [ E. coli O157:H7, limits the effectiveness of these strategies [ E. coli O157:H7 persistence in foods [ E. coli O157:H7 in the food and animal industry.Reduction of animals , 10, 29.rategies , 30. Simin foods –34. Howe E. coli O157:H7 could reduce this problem. Bacteriophages are ubiquitous, naturally abundant viruses, which invade and kill specific bacteria. They are recognized as gastrointestinal (GI) commensal microorganisms and have been isolated from various sources [ in vitro conditions, virulent bacteriophages have shown the potential for selective elimination of E. coli O157:H7 [ E. coli O157:H7 can be prevented by oral administration of O157:H7-infecting phages [ E. coli O157:H7 has also been documented in various foods such as cantaloupe, spinach, lettuce, tomato, broccoli, and ground beef [ E. coli O157:H7-specific bacteriophages completely reduced pathogen counts on the surface of beef steaks. Additionally, studies have shown that phages can effectively eliminate E. coli O157:H7 in biofilms on various food-processing surfaces such as stainless steel and high-density polyethylene [ E. coli O157:H7, very little is known about the morphology, physiology, and characteristics of specific phages. At the same time, selection of effective bacteriophages for food and animal industry applications also requires an understanding of their survival under various stress conditions. The present study was aimed at isolating E. coli O157:H7-specific bacteriophages from beef cattle operations and determining their physiomorphological characteristics, including survival under various pH, high temperatures, and cold storage conditions.Targeted use of bacteriophages against sources , 36. Alm sources  demonstr O157:H7 , 39. Addund beef –42. In aund beef , a cocktethylene . Even th E. coli O157:H7 ATCC 43895  and incubated statically at 37°C for 18 h.Isolation of bacteriophages was carried out using the host strain,CC 43895 . Host raisolates . Two of n = 67) and bovine fecal (n = 60) samples were collected from beef cattle operations in Oklahoma, during the summer months, and used for isolation of bacteriophages specific to E. coli O157:H7 [E. coli O157:H7 ATCC 43895). After incubation, 2 mL suspension was centrifuged at 12,000 rpm for 10 min to remove cellular debris and fecal material. The supernatant was filtered using 0.45 µ syringe filter  and the filtrate plated on NZCYM agar  via double-layer agar method, as described by Sambrook et al. [µL) of E. coli O157:H7 was suspended in molten (0.75%) NZCYM agar and poured onto an NZCYM agar plate which was allowed to solidify for 2–5 min. The plate was then streaked with the phage, as horizontal and vertical lines, using a sterile platinum loop and incubated at 37°C for 18–20 h. After incubation, 5 mL SM buffer  was poured onto the agar plates to elute any plaques along the lines. The agar containing the plaques was then scraped with a sterile platinum loop to release the phages and the elution was centrifuged at 12,000 rpm for 15 min. The resulting supernatant was filtered using a 0.22 µ syringe filter, to which 0.1% chloroform  was added to make a phage working-stock solution which was stored at 4°C until further use. Plaque size (mm) of individual phages was determined by performing plaque assay [Over a period of two years, water  and by performing plaque assay [Prior to an experiment, phage titers were determined , as plaque assay . E. coli O157:H7 isolates (µL) of each bacterial isolate was added to 5 mL of molten (0.75%) NZCYM agar and was overlaid onto an NZCYM agar plate. Ten microliters of the respective phage solution (~108 PFU mL−1) was spotted onto each bacterial agar plate. The plates were incubated at 37°C for 18 h and examined for clear zones on the bacterial lawn, to determine the host range of the respective phages. Bacterial sensitivity to a bacteriophage was established by bacterial lysis at the spot where the phage was deposited. Based on the degree of clarity on the bacterial lawn, the spots were differentiated into three categories: clear (+++), turbid (++), or no reaction (—)  .µL) of E. coli O157:H7 in 5 mL molten (0.75%) NZCYM agar and was poured onto a fresh NZCYM agar plate. Phage stock (prepared above) was serially diluted in SM buffer supplemented with 1 M calcium . 10 µL of each dilution was spotted onto the bacterial agar plate (prepared above) and incubated at 37°C for 18 h. Following incubation, phages were eluted in 10 µL SM-Ca by carefully pipetting up and down (7–10 times) onto the webbed spot. Each eluted phage sample was negatively stained with 2% phosphotungstic acid  on carbon-coated grids  and examined under the transmission electron microscope . Morphology (shape), capsid diameter (nm), and tail length (nm) of the phages were determined by electron micrographs at a magnification of 50,000x .Phage morphology was determined using transmission electron microscopy. Sample was prepared by suspending an overnight culture (100 8 PFU mL−1 (Stability of isolated phages was tested against a wide pH (1–11) and temperature (−80 to 90°C) range to determine their survival under acidic, alkaline, high temperature, and cold storage conditions. All phage stability studies were conducted with a phage population of about 10PFU mL−1 .Stability studies for acidic and alkaline conditions were conducted according to the methods described by Niu et al. . BrieflyµL) was suspended in 900 µL PBS and the suspension was incubated at 40, 60, 70, and 90°C for 60 min in a dry bath . Phage population at each temperature was determined every 10 min during the 60-minute incubation.For thermal stability , workingFor cold storage stability , 1 mL st E. coli O157:H7 (109 CFU mL−1) was centrifuged at 12,000 rpm for 2 min. After discarding the supernatant, the pellet was resuspended in 900 µL PBS and 100 µL of the phage-stock solution (108 PFU mL−1) added to the suspension to achieve a 0.1 multiplicity of infection (MOI). This bacteria-phage suspension was used for the adsorption and one-step growth kinetics study.Phage adsorption assay  was perfµL aliquot was sampled every 20 min by adding to 900 µL PBS and centrifuging for 2 min at 12,000 rpm. The supernatant containing unadsorbed phages was filtered through 0.2 µ filter and plated using double-layer agar technique. Percent of unadsorbed phages, at each 20-minute interval, was calculated as the ratio of PFU mL−1 in the supernatant to the initial PFU mL−1 at 0 min. The percent of adsorbed phages was determined by subtracting the percent of unadsorbed phages from 100.For phage adsorption study, the bacteria-phage suspension was incubated at 37°C for 80 min. After taking an initial sample at 0 min, a 100 μL) at 0 min, incubated at 37°C for 60 min. To obtain the one-step growth curve, a 100 μL sample was collected every 5 min, serially diluted, and plated on NZCYM agar using double-layer agar method. The rise period and latent period, as well as the burst size of each phage, were calculated by fitting the one-step growth curve into a 4-parameter sigmoidal model [Pt = Po + α/(1 + et − tr)/b)− at time t (min), Po was the initial log phage-density at time 0 (min), tr was the rise period (min), α was the log density (PFU mL−1) of virions per infected cell, and b was the maximum rate of exponential phage growth (min−1). The burst size (PFU cell−1) and latent period (min) were calculated as 10α and tr − 1/b, respectively. The latent period was defined as the time-interval between the end of adsorption and the beginning of the first burst , indicated by minimal increase in phage titer [For one-step growth experiment, the bacteria-phage suspension was incubated at 37°C for 10 min to allow the phages to adsorb to the bacterial cell . After ial model  as folloge titer , 55. Thege titer . The burge titer , 56.10 PFU mL−1. Statistical analysis was performed to determine the effect of pH and high temperature on phage survival. Data were analyzed to determine the analysis of variance (ANOVA) using Duncan's multiple range test . Significant differences between results were estimated at P < 0.05.All the experiments were repeated three times and the mean values of the three replicates obtained. Surviving phage populations, obtained in each study, were converted to log E. coli O157:H7, were isolated and designated as P-1 to P-7. The isolated phages formed medium-sized (ca. 0.3–0.5 mm in diameter) clear plaques on the lawn of its host bacteria . All the isolated phages formed clear (+++) spots on the lawn of 49 (89%) E. coli O157:H7 isolates, indicating strong lytic activity and a wide host range  and a short contractile tail (ca. 115 nm) with a base plate and several tail fibers . The ove−1) after 2 h of incubation. Of the seven phages tested, three  survived at pH 1 over a period of 24 h, with a population of 4.4–5.9 logs PFU mL−1 (−1) after 1 h of incubation (P < 0.05) loss in viability (0.9 logs PFU mL−1) after 24 h of incubation. All the phages survived well at pH 7 and 9 with no significant (P < 0.05) loss in viability (0.1–1.9 logs PFU mL−1) after 24 h of incubation (−1) in titers after 24 h of incubation (−1) during the 24 h period.All the isolated phages were tested against a wide pH range (1–11), over a period of 24 h, to determine their stability under acidic and alkaline conditions Figures . In the PFU mL−1 , while Pcubation . Among acubation . Phages cubation . Among aP < 0.05) affected at higher temperatures of 70 and 90°C  was observed over a period of 90 d. Phages P-6 and P-7 were the most stable, with minor (0.6 logs PFU mL−1) loss in titers during the 90-day storage (−1) in population (−1) but maintained their viability over a period of 90 d (−1) after 90-day storage (−1) in population after 90-day storage.Phages were also tested for cold storage stability and found to be more stable at refrigeration temperature (4°C) than at frozen temperatures (−20 and −80°C) over a period of 90 d Figures . At 4°C, storage . At −20°pulation . Phages  of 90 d . Phages  storage . ComparePhage growth properties were determined by one-step growth kinetics and phage adsorption rate. Results from the adsorption study revealed that, for all the phages, it took 20 min for approximately 41% of the initial population, and 80 min for approximately 55% of the initial population, to be adsorbed to the host cell surface . Of the One-step growth kinetics of phages, observed experimentally over a period of 60 min, is shown in  E. coli O157:H7.Several studies have demonstrated the efficacy of bacteriophages against foodborne pathogens; however, limited information is available on their general characteristics and physiology. At the same time, the stability of bacteriophages under various stress conditions, such as acidic and alkaline pH, high temperatures, and cold storage, has not been very well studied. Phage application in the food industry or food-animal industry requires them to be stable in acidic, alkaline, and high or low temperature conditions. For example, food-animal production practices, such as high temperature processing of animal feed, acidic or alkaline environment of the animal GI tract, or antimicrobials used at the farm level, could affect the viability of phages during application. Similar challenges exist at the food-processing level such as high temperature processing, fermentation, and high pressure processing. Additionally, use of multiple-hurdle control strategies is gaining popularity, where phages with high lytic activity may be used in combination with commercial sanitizers, which would require them to be stable and active in concert with the sanitizers. It is therefore important to determine the stability of bacteriophages in adverse conditions to select highly effective phages for application in the food industry and food-animal industry. In the present study, physiomorphological characteristics of bacteriophages, isolated from beef cattle environment, and their stability at a wide pH and temperature range along with their growth and lytic properties were determined to identify and select effective phages against E. coli O157:H7, including the WT isolates  compared to the phages in other studies , 60. Niut low pH –63, whict low pH  showed at low pH . The abit low pH , 66. It t low pH , 68.−1) after 60 min of incubation at 60°C. However, in the current study, no significant decrease in phage activity was observed at 60°C for 60 min. Additionally, five of the seven phages  also survived at 70°C for 10 min. Different phage strains behave differently to heat treatments [ Morganella morganii, which is sensitive to high temperature [Studies have shown that high temperature inactivates phages due to nucleic acid and protein denaturation , 69. Yameatments  used theperature  and may perature , explainBacteriophage viability at refrigerated and frozen temperatures was determined to ensure their survival during storage or shipment. All the phage isolates maintained their viability over a storage period of 90 d at 4, −20, and −80°C. Previous studies have revealed that tailed phages are resistant to refrigeration temperatures and can retain viability for more than 10–12 years at 4°C , 74. In −1 for many Myoviridae and Siphoviridae phages [ E. coli O157:H7 in the food chain. However, one concern with this approach is the emergence of phage-resistant bacteria, particularly with application of phages in animals or on the farm. Use of phage cocktails that are regularly updated with new or different phages could potentially address the issue, by maintaining selective pressure on bacterial host [The efficacy of phages to infect bacterial cells was measured using phage growth kinetics and adsorption rate. Isolated phages had a long rise period 19–40 min) and a short latent period (12–30 min) with a large burst size (89–631 virions per infecting cell), suggesting that they had a high progeny production rate. The average burst size of tested phages was about 329 phages per infected cell, which is more than the typical range of 50–100 PFU celle phages , 78. Fure phages , 79. The min and e phages . Bacteriial host , 82. E. coli O157, and their survivability under various stress conditions. This information will afford the ability to determine the application of phage-based interventions. Bacteriophages isolated in this study showed a wide range of host specificity towards E. coli O157:H7 isolates, along with high lytic activity, and pH and thermal stability and could therefore be used as biocontrol agents in the food industry.This study provides a better understanding of the infection kinetics of seven phages, targeting


Asthma is a global problem. Prevalence varies among different countries and cities. We aimed to obtain the prevalence, describe the characteristics, and discover factors that may relate to asthma in Hangzhou.This cross-sectional study was conducted in Hangzhou. The subjects were children aged 14 years and younger. A control group of non-asthma children that matched in age and sex with each asthmatic patient was also randomly selected and interviewed. International Study of Asthma and Allergies in Childhood and National Epidemiology study of Asthma and Allergies in China questionnaires were used in this survey.We have questionnaired 13,877 children, and 665 (4.8 %) children were diagnosed asthma. The guardians regarded the cost of asthma management affordable in 49.4 %, tolerable in 46.9 %, and intolerable in 3.7 %. Both guardians and children have been absent from work or school due to children’s asthma.p values were <0.05).Respiratory tract infection was the most common trigger of asthma attacks (85.1 %). Other common causes included cold air, house dust, exercise, fish and shrimp, pollen, and et al. Interestingly, we also found in children 6 years and older, some triggers happened more than that in children 5 years and younger. Those factors included exercise, emotional changes, house dust, pollen, renovation works in the home, mosquito–repellent incense and pets . Exclusive breastfeeding within first 6 months and keeping animals had higher percentage in non-asthma children than in asthma .We compared some factors may relate to asthma development. Higher percentage of family history of asthma, personal history of allergy , comorbidities , caesarean birth and complications ever happened during pregnancy were discovered in asthma children than in non-asthma children  and LTRA.Inhaled corticosteroid (ICS)/ICS + long-acting beta2 agonists (LABA) was applied to 46.2 % of patients. Traditional Chinese medicine (TCM) was used in 44.2 % of asthma children, while leukotriene receptor antagonist (LTRA) was used in 36.4 % of them. The adherence scored higher in TCM than in ICS/ICS + LABA (In conclusion, we conducted an epidemiology study in Hangzhou. The prevalence of childhood asthma was 4.8 %. Asthma was an economic and social burden to both children and guardians. Risk factors of asthma development may include caesarean birth, personal history of allergy and concomitant allergic diseases. Exclusive breastfeeding within first 6 months and keeping animals might be protecting factors. TCM was really popular in China besides ICS/ICS + LABA and LTRA. Asthma is characterized by variable symptoms of wheeze, shortness of breath, chest tightness and/or cough, and by variable expiratory airflow limitation, causing poor quality of life, emotional disorders, missing of schooling days and parents’ working days, and economic burdens. It is a common, chronic respiratory disease affecting 1–18 % of the population in different countries . HoweverThis cross-sectional study was conducted in Hangzhou, the capital of Zhejiang Province in the southeast coast of China. The predetermined sample size was 12,000 subjects, calculating from prevalence of 1.54 % in 2000 [International Study of Asthma and Allergies in Childhood (ISAAC) and National Epidemiology study of Asthma and Allergies in China (NEAAC) questionnaires were used in this survey. In the first stage, the ISAAC questionnaires were filled in by all the children or their parents from selected communities, kindergartens, primary schools, and grade one in junior middle schools. If any of the answers to questions of “wheeze, ever”, “wheeze, last 12 months”, “‘he, he’ sound, last 12 months”, or “diagnosis of asthma, ever” were positive, the children were included as possible asthma patients. They were identified immediately by doctors on spot or called back by phone to doctors in the hospitals in charge of this investigation to make sure the diagnosis of asthma according to Global Initiative for Asthma (GINA) 2010 .In the second stage, asthma patients were interviewed again. In-depth questionnaires (NEAAC questionnaire) were filled in by investigators. In addition, a control group of non-asthma children that matched in age and sex with each asthmatic patient individually was also randomly selected and interviewed. If the selected control child was not interviewed because of guardian’s refusal or other reasons, a second control would be selected and interviewed. NEAAC questionnaire were completed by investigators. The Survey started in September 2010 and finished in April 2011.The study was approved by the Ethics Committee of Children’s Hospital, Zhejiang University School of Medicine. Each questionnaire was approved by the child’s guardian. Investigators were doctors or medical students, who were trained before the program. The questionnaires were reviewed in completeness and logicality, and were demanded to be revised properly by interviewing again.P values of less than 0.05 by using two-sided tests. Data were analyzed by SPSS 19.0 .Data were double entered using epi-info system. Data skewed distributed were expressed as medians (25th–75th interquartile ranges). Data normal distributed were expressed as mean ± standard deviation. McNemar test was applied for data comparison between asthma and control groups. Mann-Whitney U test was applied for quantitative data comparison between subgroups in asthma children. Chi-square test was applied for qualitative data comparison between subgroups in asthma children. Variables were considered statistically significant at n = 210), 6–14 years (n = 455).We have actually questionnaired 13,877 children. A total of 2438 possible asthma patients were selected and 665 (4.8 %) children were diagnosed asthma. Among asthma children, 34 (5.1 %) children were diagnosed cough variant asthma. The male to female ratio was 1:0.58 (420:245). The mean age was 6.9 ± 2.9 year. The prevalence of asthma children was higher in boys than that in girls in some ages , 2000–5000 yuan 32.9 %), 5000–10,000 yuan (18.9 %), 10,000–30,000 yuan (5.4 %) and more than 30,000 yuan (0.9 %) in the year of spending most on asthma-related expenditures. The family yearly incomes were less than 60,000 yuan (14.2 %), 60,000–120,000 yuan (44.5 %), 120,000–240,000 yuan (25.15 %) and more than 240,000 yuan (16.2 %). The guardians regarded the cost affordable in 49.4 %, tolerable in 46.9 %, and intolerable in 3.7 %. Both guardians and children have been absent from work or school due to children’s asthma , followed by cough (85.4 %), shortness of breath (34.3 %), nocturnal waking (16.8 %), and chest tightness (16.1 %).p = 0.012), emotional changes , house dust , pollen , renovation works in the home , mosquito–repellent incense  , and pets . Five kinds of triggers were inhaled causes.In 85.1 % asthmatic children, respiratory tract infection was a trigger of asthma attacks. Other causes included cold air (53.1 %), house dust (20.3 %), exercise (14.6 %), fish and shrimp (14.0 %), pollen (11.1 %), et al. .We compared some factors may relate to asthma development Table .Higher pP values were <0.05).Additionally, higher percentages of caesarean birth and complications ever happened during pregnancy were found in asthma than in non-asthma .Exclusive breastfeeding within first 6 months and keeping animals, including cats, dogs, birds, pigs, horses, cows, et al. had higher percentage in non-asthma children than in asthma . Although patients ever used desensitization therapy seemed to have more proportion of more than 30,000 yuan (12.5 %) of the most expensive yearly cost of asthma .The therapies ever used as controller treatment were inquired. On the top of the list, inhaled corticosteroid (ICS) or ICS + long-acting beta2 agonists (LABA) was applied to 46.2 % of patients. As Chinese people, traditional Chinese medicine (TCM) using in asthma patients was the second most frequently used (44.2 %). And the third one was leukotriene receptor antagonist (LTRA) (36.4 %). Some have tried desensitization therapy (7.2 %). The ages of patients who ever used LTRA, ICS/ICS + LABA, TCM and desensitization therapy were 6.7 ± 2.9, 7.0 ± 2.8, 7.4 ± 3.0, and 8.3 ± 2.6, separately. Significant statistical differences were found in ages between every two controller groups  and LTRA (P = 0.006). No statistical significance was found between other groups.Adherence was assessed by parents using a scale from 0 to 10 points, which indicating not following doctor’s advice at all to completely following doctor’s advice. For ICS/ICS + LABA, TCM, LTRA, and desensitization therapy, the adherence scored 10 (8–10), 10 (10–10), 10 (8–10) and 10 (9–10), respectively. Data were expressed as medians (25th–75th interquartile ranges). We also found the adherence scored higher in TCM than in ICS/ICS + LABA (P = 0.021). Maternal education did not affect adherences of other therapies. Paternal education and monthly income was not related to adherence of asthma medicine .We explored some factors which we think may affect adherences of different therapies. Those factors included maternal or paternal education and income. We found only maternal education was related to adherence of ICS. Mothers holding college or above degrees had poorer adherence of ICS than those holding middle school or below degrees  was the reason on the top of the list. However, only 2.7 % of patients actually showed adverse effects, such as uncomfortable in throat or mycotic stomatitis. No major adverse effects or mortality occurred in our study. The second reason was parents believing it was the time to cease medicine for well-controlled asthma (24.8 %) Table .Table 3RIn this study, we found the prevalence of childhood asthma was 4.8 % in Hangzhou. This data was higher than the nationwide prevalence of 1.54 % about 10 years ago . The preIt should be noticed that, as a developing country, about a half of the family yearly income were 60,000–120,000 yuan in our study. However, in the most costive year, asthma-related expenditure would be as high as more than 30,000 yuan in 0.9 % of families. That was why 3.7 % of guardians regarded the cost intolerable. In a systematic review, authors showed the mean annual cost/person, including direct and indirect cost on asthma management, was 151 to 4158 US dollars [Increased prevalence and heavy burden make early diagnosis and early treatment of asthma really important. In this study, we explored some factors that may be important for asthma development. We found the percentages of caesarean birth in asthma were higher than that in control group. In our country, the caesarean section rate rose dramatically from 3.4 % in 1988 to 39.3 % in 2008 , 11. In For children already diagnosed asthma, how to prevent the onset of acute asthma is still an interesting topic. In this study, we found respiratory tract infection was still a very important trigger of asthma attacks. Cold air, house dust, exercise, sea food and pollen were also important triggers. A significant negative correlation between asthma hospitalizations and daily mean temperature was found in an 8-year study . Here weWe found concomitant allergic diseases, including atopic dermatitis, food allergy, allergic rhinitis, and urticaria had higher percentages in asthma children than in non-asthma children. Similar results were found in other literatures –19. AsthWe also found exclusive breastfeeding within first 6 months and keeping animals had higher percentage in control group than in asthma patients. Most of results about breastfeeding and asthma encouraged breastfeeding. There were evidences that breastfeeding is protective for asthma (5–18 years) . BreastfThe treatment of ICS/ICS + LABA as a controller treatment was only used in less than a half in this study. The proportion of ICS/ICS + LABA using was lower than most studies in other areas . This coThere were some limitations to our study. In investigating of side effects, we could not include the effect of height or body weight growth as this was a cross-sectional survey. Additionally, in answering some questions about things happened many years ago, recall bias occurred. This happened more easily in big children when answering about pregnancy, breastfeeding, and so on. In this study, we did not include the severity assessment which could have helped us to analyze some important aspects like the choose of therapies.In conclusion, we conducted an epidemiology study in Hangzhou. The prevalence of childhood asthma was 4.8 %. Asthma was an economic and social burden to both children and guardians. Respiratory tract infection was the most common trigger of asthma attacks (85.1 %). Other common causes included cold air, house dust, exercise, fish and shrimp, pollen, and et al. In children 6 years and older, some triggers happened more than that in children 5 years and younger. The triggers were exercise, emotional changes and five inhaled causes, including house dust, pollen, renovation works in the home, mosquito–repellent incense, and pets. Risk factors of asthma development may include caesarean birth, personal history of allergy , and concomitant allergic diseases . Exclusive breastfeeding within first 6 months and keeping animals might be protecting factor of asthma development. The used of ICS/ICS + LABA, TCM, LTRA, and desensitization therapy was 46.2 %, 44.2 %, 36.4 % and 7.2 %. The adherence scored higher in TCM than in ICS/ICS + LABA and LTRA.


E. coli lipopolysaccharide (LPS) affected two dissociable constructs of motivational behavior, ie, effort and reward sensitivity. After familiarization with 5 effort levels, participants made a series of accept/reject decisions on whether the stake offered  was ‘worth the effort’ . Effort and reward levels were parametrically modulated to dissociate their influence on choice. Overall, 29 healthy young males were administered LPS  or placebo . The effort-stake task, and self-reported depression and fatigue were assessed prior to LPS/placebo injection, 2 and 5 h post injection. Cytokines and sickness symptoms were assessed hourly till 8 h after LPS injection. LPS transiently increased interleukin-6 and tumor necrosis factor-α, sickness symptoms, body temperature and self-reported fatigue, and depression post injection relative to baseline and placebo. These changes were accompanied by LPS-induced decreases in acceptance rates of high-effort options, without significantly affecting reward sensitivity 2 h post injection, which were partially recovered 5 h post injection. We suggest that LPS-induced changes in motivation may be due to alterations to mesolimbic dopamine. Our behavioral paradigm could be used to further investigate effects of inflammation on motivational behavior in psychiatric and chronic illnesses.Sickness behavior in humans is characterized by low mood and fatigue, which have been suggested to reflect changes in motivation involving reorganization of priorities. However, it is unclear which specific processes underlying motivation are altered. We tested whether bacterial endotoxin  Studiesα) or acute inflammation challenges. These studies demonstrated altered reward learning  paradigm ; and cancer-related fatigue  at a dose of 2 ng/kg or saline (0.9% NaCl) intravenously. They were randomly assigned to the LPS or placebo condition on the morning of testing by an unaffiliated research nurse and deblinding of conditions took place after all data had been collected. To control for individual differences in baseline performance, behavioral testing took place at three time points: session 1: 45 min before injection; session 2: 2 h post injection and; session 3: 5 h post injection. ‘Timing was based on previous experiences from our group showing that sickness symptoms are limited 2 h after LPS administration, whereas cytokine levels are still high . The human endotoxemia sessions took place 1 week before the administration of Fluenz and were therefore not confounded by this second part of the clinical trial. Participants received either N=14, placebo group: N=15).Thirty healthy, non-smoking Caucasian males aged 18–35 years old  without any medical/psychiatric history or current use of (prescription) drugs were recruited by the Radboud University Medical Centre Intensive Care Research Unit (see After estimation of maximum voluntary contraction (MVC) see . BeginniParticipants were presented with a series of offers, in the form of the apple trees, and they had to select YES or NO, by lightly squeezing the right or left dynamometer, depending on if they evaluated the stake offered to be ‘worth the effort’. Twenty-five possible offers ) were each sampled four times in a pseudo-random order, totaling 100 trials in each session .To make each judgment behaviorally relevant, participants were told that 26 decisions would be randomly selected for them to perform immediately following the decision phase. These 26 trials were then presented during an execution phase. If the offer was accepted, participants had 5 s to reach the required effort level and hold it for 2 s. If they were successful, they received feedback stating how many apples they had won. If the offer was rejected, the tree appeared on screen with the message ‘offer rejected’, meaning they were not able to attempt to win the apples offered . ParticiPhysical sickness symptoms were measured before LPS administration (T=0) and at 30-min intervals until 8 h after LPS administration (17 measures). Participants were asked to rate from 0 (absent) to 5 (very severe) the severity of six common symptoms: nausea, headache, muscle aches, back pain, shivers, and vomiting. Self-reported mood was assessed using the depression and fatigue subscales of the profile of moods state questionnaire , and stored at −80 °C until analysis. Plasma concentrations of cytokines of interest (TNFα and IL-6) were measured using a simultaneous (entered together in one batch) Luminex assay . Statistical analyses to calculate plasma concentrations were performed using Graphpad Prism version 5.0 . Lower detection limits in plasma and intra-assay coefficients of variation (C.V.) were; 0.22 pg/ml (C.V. 1.54%) for TNFα and <0.86 pg/ml (C.V. 0.92%) for IL-6. We initially also aimed at assessing IL1-Ra and IL1-b because of the suggested association between IL1 and fatigue .EDTA-anticoagulated blood was collected at: 1, 1.5, 2, 3, 4, 6, and 8 h after LPS administration, centrifuged (2000 The percentage of accepted offers for each of the 25 conditions (5 effort and 5 stake levels) was the key variable for each participant. We first tested whether LPS induced a change in choice behavior between session 1 and session 2. Acceptance rates were entered into a 5 (effort level) × 5 (stake level) × 2 (time) × 2 (groups) AVOVA. On the basis of our hypothesis, we were specifically interested in whether LPS affected effort and/or stake sensitivity. Accordingly, our tests of interests were the effort × time × stake × group interaction and, if significant, the effort × time × group and stake × time × group interactions. If LPS induced a significant change in choice behavior, we subsequently ran an ANOVA comparing session 1 and session 3 to test whether the LPS-induced changes in choice behavior recovered to baseline.These analyses were repeated with generalized estimating equation (GEE) using a binary logistic model and exchangeable working correlation structure, which is better suited for binomially distributed categorical outcomes. Effort level, stake level, and trial (1–100) were mean centered and entered as continuous variables, session and group (LPS/placebo) were entered as factors, and the model contained all main effects and interactions.α and IL-6) were entered into a repeated-measures ANOVA with the factors time (8 levels) and group.A total score for sickness symptoms was calculated for each subject at each time point. Febrile response and sickness symptoms were entered into a repeated-measures ANOVA with the factors time (17 levels) and group. Plasma concentrations of cytokines  and group was performed for fatigue and depression scores separately. Similar to our behavioral analysis, we first assessed LPS-induced changes between session 1 and 2. If significant, we assessed whether changes recovered to baseline by comparing session 1 and 3. β’s were standardized by dividing by the standard error to minimize the impact of inflated β’s  as dependent variable for each subject. The ated β’s . Change To assess whether LPS effects were explained by alterations in perceived physical demand, we assessed LPS effects on total scores of the NASA task-load index questionnaire (sum of each section) using a repeated-measures ANOVA with the factors effort, time, and group. We additionally assessed LPS effects on the NASA task-load index subscales ‘physical demand’  and ‘effort’ (how hard did you have to work to accomplish the task?) using repeated-measures ANOVA with the factors effort, time and group, and on the sickness symptom ‘muscle aches’ using repeated-measures ANOVA with the factors time and group. When no groups’ differences were found, we tested whether LPS effects on behavior remained significant when the NASA subscales or the sickness symptom ‘muscle aches’ were added as covariate into the ANOVA on acceptance rates.β’s from the binomial logistic regression and other variables that may confound the behavioral results  within the LPS group using Pearson’s correlation analysis. Total sickness symptoms and febrile response could not be added as covariate in the ANOVA because they were significantly affected by LPS (see ‘Results’) and therefore violate the assumption of homogeneity of regression slopes =131.2, p<0.001, effort: F=117.2, p<0.001).Results are presented in =1.81, p=0.028), with significant group effects on effort-related acceptance rates =3.2, p=0.016), but not stake-related acceptance rates =0.4, p>0.7). Breakdown of the interaction by group revealed that effort-related acceptance rates were reduced in the LPS group =3.12, p<0.001; effort × time: F=4.93, p=0.002; stake × time: F=1.77, p=0.15), but not in the placebo group =1.17, p=0.29; effort × time: F=1.13, p=0.34; stake × time: F=2.28, p=0.11). Further breakdown of the effort × time × group interaction by session revealed that there was no between group difference at session 1 on any of the effort levels , and that the LPS group accepted less offers than the placebo group for the highest effort level during session 2 (80%: T(27)=−2.695, p=0.012; 62.5%: T(27)=−1.843, p=0.076). This between group difference on effort was not present during session 3, compatible with partial recovery =1.295, p=0.196; effort × time × group =0.366, p=0.833; stake × time × group =0.999, p=0.411).LPS effects on stake differed significantly from LPS effects on effort . Breakdown of the interaction by group revealed that effort but not stake-related acceptance rates were reduced in the LPS group , whereas the trend observed for the stake × time × group interaction was driven by a trend in the placebo group . Further breakdown of the effort × time × group interaction by session revealed that there was a between group difference at session 2  but not at session 1  or session 3  (These findings were confirmed with GEE (stake × effort × time × group:  p=0.14) .p>0.05)  . All sub=18.9, p<0.001), which peaked at 1.5 h post injection. Importantly, sickness symptoms were significantly higher in the LPS group relative to the placebo group at session 2, whereas no group differences were observed at session 1 prior to injection, nor at session 3 at 5 h post injection (session 1 T(27)=1.37, p>0.1; session 2: T(27)=2.15, p<0.05; session 3: T(27)=0.30, p>0.7, LPS, but not placebo, induced an increase in sickness symptoms (group × time: F°C (mean±SD) increase in temperature =13.4, p<0.001) and marked increases in all cytokines of interest at session 2 =32.48, p<0.001; TNFα: F=88.83, p<0.001) (LPS resulted in a 1±0.6 p<0.001) . Tempera=10.997, p=0.003; fatigue time × group: F=23.6, p<0.001). Specifically, the LPS group reported feeling significantly more depressed and fatigued than the placebo group during session 2 (depression: T(27)=3.609, p<0.001; fatigue: T(27)=4.806, p<0.001), but not session 1 (depression: T(27)=1.517, p=0.141; fatigue: T(27)=0.915, p=0.368). The time × group interaction effect was no longer significant for fatigue or depression when looking at session 3 vs session 1 (depression: T(27)=1.579, p=220; fatigue: T(27)=2.477, p<0.127). However, direct comparisons revealed that the LPS group remained significantly more depressed than the placebo group during session 3, but only marginally more fatigued (depression: T(27)=2.221, p=0.035; fatigue: T(27)=1.942, p=0.063) suggesting only partial recovery for fatigue =1.11, p=0.30).The LPS group did not differ from the placebo group on the total NASA score or any of the NASA subscales =1.802, p=0.029), and no relationships were observed between ‘physical demand’ and effort sensitivity (r(14)=0.34, p=0.02). However, adding the subscale ‘effort’ as covariate to the main analyses resulted in a trend for the effort × stake × time interaction =1.65, p=0.054). Change in NASA effort was also marginally correlated with change in effort sensitivity   .=1.70, p=0.044, p>0.05, Adding muscle aches a covariate in the analysis of decisions did not affect our main result , it may have captured behavior that is not necessarily reflected by the subjective reports of fatigue and depression  induced robust increases with little variation in multiple cytokines that strongly interact with one another, making it difficult to disentangle which cytokine is responsible for the observed change in behavior. Future studies that induce more variation, eg, by using different dosages or selectively stimulate or inhibit cytokines could provide more insight on the role of specific cytokines on change in behavior. Although many studies have suggested relationships between motivational behavior and mood, actual reports of these relationships have been limited (vs low-reward/low-effort) and showed that after LPS the high-effort option was still favored, more so when the probability of gaining the reward was also high. There was no difference between LPS and placebo conditions in the number of high-effort choices. Although our results may at first appear to contradict these findings, both studies demonstrated that reward sensitivity was not affected by LPS. In addition, the two studies differed in important ways.To the best of our knowledge, there has only been one previous human study that investigated effort and reward processing during inflammation , and they chose to perform the high-effort actions to gain a higher reward at the same rate as after placebo. In our design, participants only had the option to perform actions they considered ‘worth the effort’ for the reward offered, ie, they could choose to do nothing, which led to a reduction in choice of high-effort options during inflammation. Furthermore, our paradigm parametrically modulated effort and reward, rather than offering two-option choices. In this way, our task allowed participants to gain the high rewards at a lower effort level, meaning gaining the largest reward was not contingent on putting in the highest effort. Second, the previous human study , but also targets at the central level, affecting dopamine function, for patients groups in which immune-alterations are less prominent . This inherently also induce sickness symptoms that could affect the blindness of the conditions and potentially confound task performance. Although we show that our effects are specific to effort and not reward-based decisions, and that sickness symptoms were not directly correlated with effort-based choice within the LPS group, it is unfortunately statistically impossible to dissociate sickness symptoms from behavioral alterations, as these factors are not independent . Simply In summary, experimental endotoxemia reduced, otherwise, healthy participant’s willingness to engage in high-effort options, while reward sensitivity was not significantly altered. This change in motivation was not due to the task being perceived as more effortful. Endotoxemia concomitantly induced an increase in subjective reports of depression and fatigue. The behavioral paradigm used in this study provides a human model to further investigate brain mechanisms underlying inflammation effects on motivational behavior. A better understanding of these mechanisms in humans will be important for further development and testing of pharmaceutical targets to treat motivational deficits in psychiatric disorders.This research was funded by a Wellcome Trust Principal Fellowship awarded to Prof Masud Husain and supported by the Oxford NIHR BRC and CRF. This work was also supported by an EFRO Grant (2011-013287). The authors declare no conflict of interest.


Cymbopogon validus and Hyparrhenia hirta species with the aim of determining the trace metals concentrations in selected traditional plants consumed in Eastern Cape, South Africa. The trace metal concentration (mg/kg) in the samples of dry Cymbopogon validus leaves (DCVL) showed Cu of 12.40 ± 1.000; Zn of 2.42 ± 0.401; Fe of 2.50 ± 0.410; Mn of 1.31 ± 0.210; Pb of 3.36 ± 0.401 mg/kg, while the samples of fresh Hyparrhenia hirta flowers (FHHF) gave Cu of 9.77 ± 0.610; Zn of 0.70 ± 0.200; Fe of 2.11 ± 0.200; Mn of 1.15 ± 0.080; Pb of 3.15 ± 0.100 mg/kg. Abundance of metal concentrations follows the order: Cu > Fe > Pb > Mn > Zn in the flower samples of Cymbopogon validus and Hyparrhenia hirta species. The concentrations of trace metals in both plant parts were below the permissible limits (PL) set by WHO. It is suggested that pharmacovigilance be carried out periodically to improve the quality, safety, and efficiency of various herbal products.FAAS was used for the analysis of trace metals in fresh and dry plant parts of Medicinal plants have played an indisputable role in the development of human race and culture, for example, religions and different ceremonies. Majority of cultures  around the world use plants directly as medicines for disease treating purposes. Medicinal plants are plants that contain substances that could be used by man because of their ability to exert a modifying benefit to the physiology of sick mammals  or plant Erythroxylum coca, which has provided the chemical structure for the synthesis of Procaine and other related anaesthetics [Medicinal plants can be used as blueprints for the production of synthetic drugs of a similar structure, for instance, the plant alkaloid Cocaine extracted fromsthetics . MP's thsthetics . Complemsthetics , 7. Cymbopogon validus belongs to the family Poaceae. Cymbopogon species occur in large quantities in tropics and subtropics with unlimited scattering ranging from mountains and grasslands to dry lands [ Cymbopogon species are in large demand worldwide due to their wide industrial uses which include raw materials for confectionery, perfumery, and pharmaceuticals [ Hyparrhenia belongs to the family Poaceae; it is a genus of 55 grass species such as .The speciesry lands . Its speeuticals  and theseuticals , 9. In Eeuticals . The gen Hyparrhenia hirta using high performance liquid chromatography (HPLC) were the rare diastereoisomeric flavonolignans tricin 4′-O-(erythro-beta-guaiacylglyceryl)ether and tricin 4′-O-(threo-beta-guaiacylglyceryl)ether together with their 7-O-glucosides [ In vitro studies of flavonolignans have been established to have a wide variety of biological and pharmacological actions comprising antiallergic, antimicrobial , anti-inflammatory, antioxidant, anticancer, and antidiarrhoea activities, alongside the capability to induce DNA mutation in the mixed lineage leukaemia gene [Many of these grass species are commonly acknowledged as thatching grasses . They arucosides . In vitrmia gene , 9, 11.Medicinal plants that are grown on polluted fields, near roadways or metal mining sites, possess greater chances of amassing heavy metals from the soil, water, and air. In general, the topography, the geochemical soil features, impurities in the soil, water, and air, and other development, transport, and storage conditions can significantly affect the properties and the quality of herbal plants and their formulations . High leCymbopogon validus and Hyparrhenia hirta) collected from environmentally diverse region of Komga road near King William's Town, Eastern Cape of South Africa, using flame atomic absorption spectrometry (FAAS). The medicinal plants are used for variety of biological and pharmacological activities amongst the populace.In view of this, the study seeks to evaluate the levels of trace metals  present in different parts of the medicinal plants  were collected from Komga road (roadside) near King William's Town. Taxonomic identification of Cymbopogon validus and Hyparrhenia hirta was done by Dr. T. Dold and the specimens voucher was deposited in Selmar Schonland Herbarium Grahamstown (GRA) at Rhodes University. The voucher number was PR/Pl 03. The plant sample parts are leaves and flowers for both fresh and dry plants. The plants were then air-dried, ground to be in a powder form, and stored in fastened air tight bottles for further investigation. Trace metal analysis was carried out according to AOAC [Fresh and dry to AOAC  using fl to AOAC .3) solution. The standardization curve for each element was linear by plotting the measured absorbance and concentration (mg/kg) and a correlation coefficient was achieved. Accurately, 1.00 g of plant samples (pulverized) was weighed and placed into a digestion tube; 30 mL of 55% nitric acid (HNO3) and 10 mL of 32% hydrochloric acid (HCl) in 3 : 1 mole ratio were added [All glassware and digestion vessels were soaked overnight in 20% nitric acid and washed with deionised water. Standard solutions of lead (Pb), manganese (Mn), iron (Fe), zinc (Zn), and copper (Cu) were obtained by dilution of 1000 mg/L stock solutions  with 5% nitric acid  Model Ice 3500 with Shimadzu, Wizard software. Optimized operating conditions for FAAS (Flame Atomic Absorption Spectrophotometer) of various heavy metals are recorded in p < 0.05).All experiments were completed in triplicate for precision and accuracy of the results. Concentration of each metal was obtained from the calibration functions of each replicate and articulated in mg/kg on a dry/fresh-weight basis of the plant sample and results were reported as mean values ± standard deviation (mean ± SD). ANOVA was used for statistical analysis in the study  were found to be as follows: Cu: 6.01 ± 0.530; Zn: 0.79 ± 0.010; Fe: 3.39 ± 0.280; Mn: 1.17 ± 0.071; Pb: 1.83 ± 0.028 mg/kg; Cymbopogon validus leaves (CVL) showed the following: Cu: 8.85 ± 0.281; Zn: 1.60 ± 0.141; Fe: 2.65 ± 0.010; Mn: 1.31 ± 0.085; Pb: 3.14 ± 0.135 mg/kg, while in samples of Hyparrhenia hirta flower (HHF) the following was observed: Cu: 7.79 ± 0.275; Zn: 0.71 ± 0.020; Fe: 3.13 ± 0.148; Mn: 1.13 ± 0.021; Pb: 2.33 ± 0.014 mg/kg; Hyparrhenia hirta leaves (HHL) gave the following: Cu: 6.62 ± 0.106; Zn: 0.67 ± 0.177; Fe: 2.76 ± 0.071; Mn: 1.15 ± 0.053; Pb: 2.12 ± 0.192 mg/kg. Abundance of metal concentrations follows the following order: Cu > Fe > Pb > Mn > Zn in both flower samples of Cymbopogon validus and Hyparrhenia hirta while Cu > Pb > Fe was observed for leave samples of both medicinal plant species. The results indicate the presence of copper, lead, and iron in moderate concentrations; Zn and Mn recorded the lowest concentration in samples taken from Komga road.The mean trace metal concentration (mg/kg) in the samples of Cymbopogon validus leaves (8.85 ± 0.281 mg Cu/kg) followed by Hyparrhenia hirta flower (7.79 ± 0.275 mg Cu/kg) and the lowest concentration was found in Hyparrhenia hirta leaves for Zn (0.67 ± 0.177 mg Zn/kg) followed by Hyparrhenia hirta flower (0.71 ± 0.020 mg Zn/kg). The accumulation of metals by roots, leaves, and flowers increases with increasing available metal concentration in the biota [Among the medicinal plant species, the average concentration of Cu was the highest inhe biota . Also, the biota . Contamihe biota . Even th Cymbopogon validus 12.40 ± 1.000 mg Cu/kg; fresh flowers of Cymbopogon validus contain 11.60 ± 0.810 mg Cu/kg, followed by fresh Hyparrhenia hirta flowers 9.77 ± 0.610 mg Cu/kg and fresh Hyparrhenia hirta leaves 8.94 ± 1.410 mg Cu/kg while very low concentration was observed in dry Cymbopogon validus flowers 0.42 ± 0.060 mg Cu/kg as displayed in High levels of copper concentrations were found in dry leaves ofCopper is one of the crucial elements for plants and animal and essential mirconutrients for neutrological syetems and nervous system myelin sheaths . VariousNevertheless, disproportionate ingestion of Cu could lead to the rupturing of erythrocytes and nephrotoxic effects; also, its constant consumption through food crops or herbal medicines may lead to the copper damage in human beings  leading  Cymbopogon validus (2.42 mg Zn/kg) and in low concentrations as it ranges from 0.63, 0.77, and 0.95 mg Zn/kg for dry Cymbopogon validus flowers, dry Hyparrhenia hirta leaves, and fresh Cymbopogon validus flowers, respectively, as seen in Tables Zinc is an important component of the human body but its excessiveness may be a suggestion of metal poisoning and growth impedance . The higIn general, the obtained results revealed that the plant part species studied had concentrations lower than 50 mg/kg, the FAO/WHO permissible limit stipulated for zinc in herbal medicines , 30, 31. Hyparrhenia hirta 4.12 ± 0.410 mg Fe/kg and the lowest concentration of Fe was observed in fresh Hyparrhenia hirta flowers 2.11 ± 0.200 mg Fe/kg, including fresh flower of Cymbopogon validus 3.53 ± 0.310 mg Fe/kg, dry flowers of Cymbopogon validus 3.22 ± 0.710 mg Fe/kg, fresh leaves of Cymbopogon validus containing 2.80 ± 0.410 mg Fe/kg, and dry leaves of Hyparrhenia hirta containing 3.05 ± 0.201 mg Fe/kg  acts as a catalytic agent . It also Cymbopogon validus flowers, Cymbopogon validus leaves, Hyparrhenia hirta flowers, and Hyparrhenia hirta leaves with average levels of 1.83 ± 0.028, 3.14 ± 0.135, 2.33 ± 0.014, and 2.12 ± 0.192 mg Pb/kg, respectively, as displayed in It is worth noting that excessive iron in the body can lead to prolonged effects that may be lethal for homeostasis . ToxicitThe Pb levels can be found in the following order: DCVL > FHHF > FCVL > FHHL > FCVF > DHHL > DHHF > DCVF. The medicinal plant part species (flower and leaf) contained tremendously lower concentrations of Pb when compared to the permissible limit of 10 mg/kg established by WHO . Lead is Gunnera perpensa, a medicinal plant from Mabandla Village, KwaZulu-Natal Province, South Africa, has been reported to have values above the permissible limit set by FAO/WHO [Concentration of lead above permissible limits in medicinal plants and herbs has been reported in previous studies. Example of such is medicinal herbs in Jordan which were found to be 13.9, 13.1, and 16.9 mg Pb/kg on a dry weight basis, respectively . Pb cont FAO/WHO  in edibl FAO/WHO . Likewis FAO/WHO , 43. Hyparrhenia hirta and Cymbopogon validus is in an array of 0.93 ± 0.210 mg Mn/kg to 1.40 ± 0.110 mg Mn/kg for flowers of Cymbopogon validus and 1.11 ± 0.005 mg Mn/kg to 1.19 ± 0.080 mg Mn/kg for leaves of Hyparrhenia hirta as displayed in Tables Manganese is among the principal minerals associated with carbohydrate and fat metabolism . The con Cymbopogon validus and Hyparrhenia hirta plant part species contain lower levels of Mn. Mtunzi and coworkers [ Berkheya setifera collected from Mabandla Village, KwaZulu-Natal Province of South Africa, to accumulate Mn above the permissible limit on dry mass basis. A study by Sheded et al. [ Justicia adhatoda and 105.56 mg/kg in Achyranthes aspera on dry mass basis.For medicinal plants, the WHO  limit isoworkers  reportedd et al.  indicated et al.  reportedMn is vital in regular reproductive activities and normal functioning of the central nervous system and also acts as cofactor for many enzymes. Elevated concentrations of manganese cause adverse effects mostly on the lungs and on the brain . Its defThe results of FAAS analysis indicated that trace metals were present in varied concentrations in the eight samples of the traditional medicinal plant species commonly used in Eastern Cape, South Africa. The levels of trace metals in the plant species were of lower concentrations than internationally accepted permissible levels. Toxic metals accumulation varies significantly among plant species; toxic element uptake by plants is predominantly dependent on plant species and soil quality and its intrinsic controls . Highly  Cymbopogon validus and Hyparrhenia hirta leaves and flowers contain trace metals: iron, copper, zinc, manganese, and lead in different degrees. The results indicate the presence of copper, lead, and iron in moderate concentrations; Zn and Mn recorded the lowest concentrations. The mineral contents in Cymbopogon validus parts for Cu and Zn are in the following order: DCVL > FCVF > FCVL > DCVF, whereas in Hyparrhenia hirta for Cu and Pb they are in the order: FHHF > FHHL > DHHF > DHHL. Comparison of the results with the defined permissible concentration limits by WHO showed that the levels of elements content in the locally available medicinal plant parts species under investigation fall below the permissible range and, hence, contribute no metal toxicity to the surrounding populace. Human health of the immediate users may not be affected by these medicinal plant part species when consumed orally or used for diet preparation. It is suggested that farming of herbal medicinal plants within the diameter closer to the vicinity of industries and high human activities be discouraged, as there might be probability of heavy metals uptake by medicinal plant parts, which will in turn end up in the food chain and finally to humans via herbs consumption. Furthermore, there should be a constant investigation into the effectiveness and safety measures of medicinal plants collected around anthropogenic activities while using the medicinal herbs for healthcare purposes.Medicinal plant (MP) continues to play a crucial part, to a large extent, in the healthcare scheme of the world's populace. Fresh and dry


Lumbar spinal stenosis (LSS) refers to narrowing of the lumbar central spinal canal, lateral recess, and/or neuro-foramina. Radiographic LSS plays an important role in clinical LSS but is not solely accountable for the presence of symptoms. We sought to characterise clinical LSS and to determine factors associated with presence of symptoms of LSS in patients with radiographic LSS in a sub Saharan Africa setting.After prior ethical clearance, a case control study was done in a tertiary hospital in Douala-Cameroon, including 105 patients with radiographic LSS: 57 with symptoms of LSS (cases) and 58 with no symptoms (controls). Spinal stenosis was assessed using computed tomography (CT) scans. Data were analysed using SPSS version 23.p < 0.001) and a high waist circumference (p = 0.002) were significantly associated with presence of LSS symptoms in persons with radiographic LSS. After adjusting for body mass index, a positive family history of low back pain (p = 0.004), vertebra lesion at L2 (p = 0.034), L3 (p = 0.002), L4 (p = 0.025) and multiple (p = 0.008) levels, degenerative disc protrusion (p = 0.044), disc lesion at L3-L4 (p = 0.001), L4-L5 (p = 0.011) and multiple (p = 0.046) levels were significantly associated with presence of symptoms of LSS in persons with radiographic LSS.The mean age of our study participants was 53.4 ± 13.1 years. The mean age of onset of symptoms of LSS was 50.3 ± 11.6 years and the most common symptoms were Low back pain (100.0%), radicular symptoms (98.2%) and neurogenic claudication (98.2%). Obesity (Characteristics of clinical LSS have been described in this sub-Saharan Africa population. Obesity, a high waist circumference and a positive family history of low back pain are significantly associated with presence of symptoms of LSS in persons with radiographic LSS. Lumbar spinal stenosis (LSS) refers to narrowing of the lumbar central spinal canal, lateral recess, and/or neuro-foramina , 2. The Clinical LSS (symptomatic LSS) is diagnosed by the presence of neurogenic claudication, radicular symptoms, or both, with or without low back pain, in the presence of radiographic LSS , 15. BioBased on the pathophysiology of LSS, there is an expected correlation between radiographic features and symptoms of LSS. There has been conflicting results on the association between radiographic findings of stenosis and symptoms of LSS , 21–23. The aims of this study were to describe the characteristics of clinical LSS and to determine factors associated with presence of symptoms of LSS in patients with radiographic LSS in a sub-Saharan Africa setting.We carried out a case control study in the Radiology Unit of the Douala General Hospital, Cameroon, from December 2014 to April 2015. After ethical clearance from an institutional review committee, we targeted patients aged 21 years and above, undergoing a lumbar spine or abdomino-pelvic CT scan during the study period. We excluded persons with a history of spine surgery and persons with an ankle-brachial index ≤0.90.Cases included patients with confirmed clinical LSS . They were consecutively enrolled into the study after consent was obtained. Controls were recruited from patients referred to our radiology unit for abdomino-pelvic CT scans for other conditions, and included patients with no clinical evidence of LSS who had radiographic LSS on supplementary spine CT scan analysis. They were consecutively matched to cases in a ratio of 1:1 based on gender, and after consent was obtained, they were enrolled into the study Fig. All study participants were assessed by the principal investigator and findings were filled into a pretested data collection sheets. Cases were assessed within 2 h before lumbar CT scan was done and data collected included demographic characteristics, history of symptoms and physical findings. Controls were assessed after supplementary analysis of their abdomino-pelvic CT scans showed radiographic LSS. Assessment was done when these patients presented for their CT scan results within 3 days; data collected included demographic characteristics and anthropometric measurements.2, overweight as 25.0 ≤ BMI < 30 kg/m2, normal weight as a BMI 18.5 ≤ BMI < 25 kg/m2 and underweight as BMI < 18.5 kg/m2 [Occupation was classified using the international standard classification of occupations (ISCO-08) . Pain wa.5 kg/m2 . Waist c.5 kg/m2 . Sitting.5 kg/m2 . The par.5 kg/m2 .i.)2 [Central canal stenosis: presence of any of the following; antero-posterior diameter of central canal <10 mm , 40, tra2 .ii.)0 [Lateral stenosis: presence of any of the following; depth of lateral recess ≤3 mm , 43, hei0 , 45.iii.)Foraminal stenosis; antero-posterior diameter of the foramen ≤3 mm , 46.CT scan  imaging was used to assess LSS. In order to minimise investigator bias, CT scan images of participants recruited into the study were analysed by a radiologist blinded to the group of the participant. The items assessed included: type(s)  and level(s) of vertebral lesion, type(s) and level(s) of disc disease, and ligamentum flavum hypertrophy. Biometric measurements of the lumbar vertebrae were automated generated CT measurements. The criteria used to define stenosis were as follows:t-test. Bivariate analysis was done using logistic regression to estimate the odds ratio of having symptoms of LSS. BMI class and waist circumference class were significantly different between cases and controls . Therefore, on multivariate analysis using logistic regression, in order to deal with multicollinearity, we adjusted the odds ratio of having symptoms of LSS for BMI class only. Analysis was done using Statistical Package for Social Sciences (SPSS), version 23 Inc., Chicago, Illinois, USA. Statistical significance was set at α ≤ 0.05.Statistical significant difference between proportions was assessed using the Pearson’s chi-squared test, and between means using independent samples student’s ls Table , and werDuring the study period, we received a total of 67 cases of confirmed clinical LSS. Of these, we excluded 10 cases: 6 did not meet the inclusion criteria and 4 declined to participate. A total of 57 cases  were recruited into the study, with a participation rate of 93.4% for cases. We had 77 cases of radiographic LSS, out of the 256 abdomino-pelvic CT scans of patients without clinical LSS evaluated. Seventy-four were eligible for matching, 64 were consecutively matched to cases and 6 declined to participate. We recruited of total 58 controls  into the study, with a participation rate of 90.6% for controls Fig. p = 0.128). Sixty- five (56.5%) participants had central stenosis (56.1% cases and 56.9% controls), 48 (41.7%) had foraminal stenosis (40.4% cases and 43.1% controls) and 2 (1.7%) had lateral stenosis (3.5% cases and no controls) Table The mean age of our study participants was 53.4 ± 13.1 years; 55.2 ± 12.9 years for cases and 51.5 ± 13.1 years for controls ; 20.0% degenerative disc and protrusion; 13.9% disc herniation. Most common lumbar spine disc level affected included L5-S1 disc level (59.1%) and L4-L5 disc level (48.7%); more than one disc levels were affected in 45.2% of participants Fig. p = 0.488). Low back pain (100.0%), radicular symptoms (98.2%) and neurogenic claudication (98.2%) were the most common presenting symptoms. Low back pain was described as cramping by 77.2% of cases and 57.9% of cases said the timing of pain was intermittent; with 8.8% of cases reporting nocturnal symptoms. Cases who admitted having reduced walking distances constituted 98.2%, while cases who admitted having impaired routine daily activities constituted 96.5% Table The mean age of onset of symptoms of LSS was 50.3 ± 11.6 years; 49.3 ± 12.4 years for females and 51.4 ± 10.7 years for males (p < 0.001), grand multi-parity , obesity , and high waist circumference  were significantly associated with presence of LSS symptoms in persons with radiographic LSS. Radiographic features of vertebra lesion at L3 level , degenerative disc protrusion , degenerative disc herniation , L3-L4 disc lesion , L4-L5 disc lesion  and multi-level disc lesion  were also significantly associated with presence of LSS symptoms in persons with radiographic LSS. However, age , sitting height , relative sitting height , type of radiographic stenosis (p = 0.991), type of vertebra lesion (p = 0.261), type of disc lesion (p = 0.197) and ligamentum flavum hypertrophy  were not significantly associated with presence of symptoms of LSS in these persons.On bivariate analysis, clinical features of a positive family history of chronic low back pain , vertebra lesion at L2 , L3 , L4  and multiple  levels, degenerative disc protrusion , disc lesion at L3-L4 , L4-L5  and multiple  levels were significantly associated with presence of symptoms of LSS in persons with radiographic LSS Table On multivariate analysis adjusted for BMI class, a positive family history of low back pain , attesting that degenerative changes observed in LSS are part of the normal aging process [The mean age of onset of symptoms of LSS was found in our study was similar to reports published on a Caucasian population , thus no process , 51, andIn our study, symptoms of LSS were worsened by standing erect in 98.2% of cases, and relieved by leaning forward/sitting/stooping in 96.5% of cases, consistent with reports of an important dynamic component in LSS . HirasawObesity and central obesity has been associated to the occurrence of symptoms in LSS as found in our study. Knutsson et al.  suggestePersons who had vertebra lesions at multiple levels and disc lesions at multiple levels had significant higher odds of having symptoms of LSS compared to persons who had at one level as found in other studies , 59, butWe found no clear association between radiographic and clinical LSS. The type of radiographic stenosis, type of vertebra lesion and type of disc disease were not significantly associated with presence of LSS symptoms in persons with radiographic LSS, confirming findings in other studies –30, thouThe observed ambiguous correlation between radiographic and clinical LSS can be accounted by a number of reasons. There is no consensus on the diagnostic criteria for radiographic LSS . DiffereOur study had strengths and limitations. To the best of our knowledge, this is the first study in sub-Saharan Africa to determine factors associated with presence of symptoms of LSS in radiographic LSS. We explored a wide range of clinical and radiographic variables. However, we did not assess psychological factors such as depression and anxiety which may impact presence of symptoms in radiographic LSS , 63. DesCharacteristics of clinical LSS have been described in this sub-Saharan Africa population. Anatomic stenosis plays an important role in clinical LSS but is not solely accountable. Obesity, high waist circumference, a positive family history of low back pain, vertebra lesion at L2, L3, L4 and multiple levels, degenerative disc protrusion, disc lesion at L2-L3, L3-L4, L4-L5 and multiple levels are significantly associated with presence of symptoms of LSS in persons with radiographic LSS.