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Molar Fraction of Carbon, Nitrogen and Mineral Sources Regulate Antibiotic Biosynthesis Pattern in Pseudomonas Fluorescens CHA0

Authored by Archana G,

Abstract

Fluorescent Pseudomonas species inhibit fungal phytopathogens under laboratory and greenhouse conditions, but in field trials the inconsistent performance is observed, which is thought to be due to variations in abiotic and biotic factors in/around the rhizosphere. Combination of nutrients and mineral components relevant to root zone and its effect on the antibiotic biosynthesis by fluorescent Pseudomonas species important for the understanding of biocontrol activity in the rhizosphere. Using Stat ease 8, and fractional factorial design sixteen combinations of four macronutrient (glucose, citrate, nitrate, phosphate) and three essential elements (Fe, Zn and Mo) obtained, and studied for the main effects and higher order interactions, on the culture growth, antifungal activity toward Rhizoctonia bataticola, and on the biosynthesis of pyrrolnitrin (PRN), 2, 4-diacetyphloroglucinol (DAPG) and pyoluteorin (PLT) in the model biocontrol strain Pseudomonas fluorescens CHA0 (Pf CHA0).

Citrate, glucose, Fe2+, Zn2+ individually and citrate with glucose, found to be supportive while triple combination of citrate, glucose and nitrate is highly inhibitory to Pf CHA0 growth, antifungal activity as well as antibiotic biosynthesis. Principal Component Analysis revealed that 2 day antifungal is positively correlation with PLT maximally followed by OD, PRN and DAPG. Fifth day antifungal activity showed positive correlation maximally with PRN followed by OD, and PLT. Bio-control supporting nutrient combinations showed effective protection of Vigna radiata from R. bataticola. Present study will be helpful for improving the reliability and application potential to the various soil types and different plant rhizosphere types.

Keywords: Nutrients; Antibiotics biosynthesis; Pf CHA0

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Introduction

Many species of root-associated beneficial bacteria have the ability to reduce the severity of diseases caused by soil borne fungal phytopathogens and to increase plant yields under laboratory and greenhouse conditions and are considered important biocontrol inoculants for sustainable agricultural practices [1-3.], Whipps et al. (2001). However, in field trials the inconsistent performance of bacterial biocontrol agents has been observed from site to site and from year to year [4.,5.] which is a major concern for their large-scale application [6]. A thorough understanding the sources of variability in field performance, which could be due to variations in environmental conditions, abiotic and biotic, that the bacterial inoculants confront in the rhizosphere is an important aspect of study. Many factors are found to affect the performance of biocontrol agents, for example, there is a clear link between soil pH and mineral concentrations and the variability in biocontrol activity of both fungal and bacterial inoculants against root diseases [7-9].

Biosynthesis of antimicrobial compounds by bio-control bacteria is modulated by the total concentration and type of carbon (C) source, nitrogen (N) source, amino acids, metal ions such as Fe2+, Mo2+ and Zn2+ and other compounds found abundantly in plant root exudates but lacking in bulk soil [10-13]. C- Sources contribute to the variability of bio-control in different soils and on host crops that differ in root exudates composition Latour et al. (1996). Comparison of root exudates composition of cucumber, tomato, sweet pepper and grass revealed that it contained more organic acids than sugars and citric, malic, and succinic acid were major organic acids and fructose, glucose and to a minor extent, xylose, were major sugars Kuiper et al. (2002). Plant specificity of bio-control bacteria has been demonstrated at both the species and cultivar level which is generally been attributed to differential utilization of the various carbon and nitrogen compounds found in exudates and its effects on growth [14].

Fluorescent pseudomonads are one of the most well- studied group of biocontrol and plant growth promoting rhizobacteria (PGPR) that have the ability to produce diverse antifungal metabolites of different efficacy, mode of action and stability and thereby protect the crop from attack by different fungal pathogens Raaijmakers et al. (2002) [6,15]. Important antifungal metabolites which contribute to biocontrol by fluorescent Pseudomonas include 2,4-diacetylphloroglucinol (DAPG), pyoluteorin (PLT), pyrrolnitrin (PRN), phenazines (PHZ), lipopeptides and hydrogen cyanide Nielsen et al. (2002), Raaijmakers et al. (2002), Haas & Keel (2003) [16,17]. Certain biocontrol strains of Pseudomonas produce multiple antibiotics with overlapping or different degrees of activity against specific pathogens. For instance, the biocontrol agents Pf CHA0 and Pf-5 produce the phenolic compounds DAPG, PLT and PRN Loper et al. (1997), Raaijmakers et al. (2002) [18].

Regulatory pathways responding to these nutritional and environmental factors are generally integrated into to respond to these signals on various additional regulatory elements, global environmental and physiological regulators such as GacS-GacA, RpoS, and CRP [19,20] and there is evidence that rhizospheric microbes such as Pseudomonas sense the availability of diverse nutritional factors as exogenous signal, activate small RNAs (sRNAs) that bind RsmA/ CsrA proteins are typically produced under the positive control of a two-component system termed GacS/GacA for global activation of antibiotic biosynthesis [21], Babitzke & Romeo et al. (2007).

Pf CHA0 respond to exogenous regulatory signal(s) by activating Gac/ Rsm a membrane-bound sensor kinase Laville et al. (1992) [22] the biosynthesis of bacterial auto inducers, rpoD and the cellular levels of mRNAs, stable RNAs (i,e. rRNAs and tRNAs), small RNAs (RsmX, RsmY and RsmZ) when cells reach high population densities [23]; Valverde et al. (2003); Kay et al. (2005). When bacterial cells are growing in batch culture, the GacS-GacA-dependent phenotypes are expressed mostly when the culture is in the transition from exponential to stationary phase Heeb & Haas (2001). An additional level of control may be provided by the relative concentrations of the housekeeping a factor RpoD and the stress and stationary phase a factor RpoS in the bacterial cell. In Pf CHA0, over expression of the housekeeping a factor RpoD enhances DAPG and PLT biosynthesis in vitro and in the rhizosphere, resulting in improved bio control depending on the plant species Maurhofer et al. (1992) [24,25].

Likewise, an rpoS mutant of the related strain Pf-5 shows strongly enhanced PLT and DAPG biosynthesis [26] whereas rpoS over expression in strain Pf CHA0 was found to shut off PLT biosynthesis Haas & Keel, et al. (2003). Two-component regulatory cascade composed of the sensor kinase GacS and the response regulator GacA that positively controls antibiotic biosynthesis upon activation by a yet unidentified quorum sensing-signal Haas & Keel, et al. (2003); Valverde et al. (2003) [22]. Numerous biotic and abiotic signals may also influence the production of these antifungal compounds, including different mineral and carbon sources as well as metabolites released by micro-organisms and plants [27-29]. Quantitative and/ or qualitative differences in the sugar, nitrogen, phosphate and mineral component of root exudates could determine the optimal environment for the effective bio-control mechanism in given crop-pathogen systems [24,30].

Maria Pechy-Tarr et al. [31] reported that rpoN is a key regulator of bio control activity in plant-beneficial pseudomonad and recently, Kumar and Shimizu, 2010 reported that as C/N ratio get increased, and transcript level of rpoN (which encodes σ54) increases in E. coli.So the role of rpoN in bio control activity by fluorescent pseudomonads under varied C/N ratio could not be ignored. The importance of organic acids as carbon sources for growth of Pseudomonas spp in the rhizosphere was shown previously by De Weert et al. [32] who demonstrated that mutants of the good colonizer P. fluorescens WCS365 with mutations in genes encoding malate/quinone oxidoreductase or cisaconitate hydratase, enzymes of the tricarboxylic acid cycle, are poor competitive colonizers of the tomato root compared with the parental strain. Our results add another point to plant specificity i.e. the influence of combination of root exudates components on the biosynthesis of antimicrobial metabolites by fluorescent Pseudomonas and increase its competence and survival efficiency in rhizosphere.

In the present study, we have studied the well-characterized biocontrol agent Pf CHA0 [2,18] to get insight into the pattern of biosynthesis of the antifungal compounds DAPG, PLT and PRN that are major determinants of the biocontrol activity in this strain under the different combinations of nutritional factors. Along with glucose, citrate is also studied as an alternate C source, since citrate is preferred C source Pseudomonas and it is the one of major constituent of root exudates. The results of this study provide

Insight into the possible biosynthetic regulation of antibiotics by complex of organic and inorganic component present in the rhizosphere,

Limited number of factors and their combinations for intensive study in situ,

The factors that can be manipulated to improve bacterial inoculants and

The application prospect of the biocontrol strains based on the rhizosphere and soil type for better bio-control potential.

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Methods

Bacterial maintenance and growth conditions

Pf CHA0 (kindly provided by Dr. Fabio Rezzonico, Agroscope Changins-Wadenswil Zurich, Switzerland, was routinely maintained on King's B medium plates (Hi-media, India) at 28 °C and stored for long term in 0.8% nutrient broth with 0.5% yeast extract (NBY) broth with 40% glycerol at -80 °C. Starter cultures were grown in 10 ml dilute (1/5-strength) NBY broth in 20 ml test tubes for 12 h at 28 °C at 150 rpm, yielding approximately 109 CFU/ml. For further growth, 20 ml of dilute (1/5-strength) NBY broth in 100-ml Erlenmeyer flasks was inoculated with 100 |il of starter bacterial. Autoclaved medium was amended with different combinations of following seven components: filter-sterilized solutions of KNO3 (10mM), KH2PO4 (Pi,10mM), FeSO4.7H2O (Fe2+,0.5mM), Mo7(NH4)6O24.4H2O (Mo2+,0.5mM),ZnS04.7H20 (Zn2+,0.35m) and with autoclaved stock solutions of glucose and citrate to give final concentration of 100mM. Rhizoctonia bataticola causal organism of dry root rot of Vigna radiata, was maintained on potato dextrose agar slants (PDA, Hi-media, India).

Design of experiment for the combinations of nutritional factors

The study of the effect of combinations of nutritional factors for the enhancement of bio-control traits by Pf CHA0 was carried out by using Stat ease 8. For seven factors, a total of 16 combinations were used (Table 1). Each column has equal number of high and low values of given nutrient factor. 50 ml of all the 16 media combination in 250 ml conical flask was inoculated with 100ul of overnight grown bacterial and kept at 26oC in shaking condition at 150 rpm in darkness. After 2 day and 5 days, 20 ml of bacterial was transferred in sterile 50 ml centrifuge tubes for metabolite extraction. Absorbance of the culture was noted at 600nm to monitor the growth of the organism in the medium. pH of all media was in the range of 6.6 to 6.8 at time of inoculation and between 7.5 to 7.8 after 2 day and 5 day of growth.

Extraction, identification and quantification of antifungal metabolites

Antibiotics were extracted from bacterial supernatants and quantified with high-performance liquid chromatography (HPLC, Shimadzu 10) as described by Duffy and Defago, 1997. Metabolites were identified and by comparison with the pure DAPG, PRN and PLT. Metabolite quantity was estimated from standard curves of reference compounds and normalized for the bacterial absorbance at 600nm prior to extraction. Briefly, liquid culture of 20 ml were acidified to pH 2 with 400 to 700 µl of 1 N HCl and extracted with 20 ml of ethyl acetate for 60 min with vigorous shaking at 200 rpm. Phase separation was accelerated by 15 min of centrifugation at 5000 rpm. The organic phase was transferred to a round-bottomed glass flask flash evaporated, and the residue was dissolved in 1 ml of HPLC-grade methanol and quantified by established HPLC procedures Keel et al. (1992]; Maurhofer et al. (1992)[24].

In vitro tests of fungal antagonism

The plate test screening for in vitro antagonism against the plant-pathogenic fungus Rhizoctonia bataticola was performed by placing an agar plug with in the centre of a Potato Dextrose Agar (Hi-media, Mumbai, India] plate and by adding 100^l ethyl acetate extract in the well bored at four places equidistance from centre at plate periphery. The plates were incubated at 30°C for fungal growth and checked for zones of mycelia growth inhibition after approximately 2 and 5 days, when the fungal mycelium had reached the edge of the plate. Percentage of fungal inhibition was calculated as: radial growth of fungus from centre -radial growth of fungus in presence of extract)/radial growths of fungus from centre X 100. All tests were performed 4 times, with new extract used each time.

Principal Component Analysis

PCA was used to establish combinations of variables e.g. OD600 nm, PRN, DAPG and PLT biosynthesis to describe the principal tendencies. PCA study provided the corresponding Eigen values, which were extracted by each factor, and the variance percentages (accounted for and accumulative] corresponding to the principal components by formula Fn=A1 X1+A2 X2+...+An Xn. Given Xobservations on n variables, PCA reduce the dimensionality of the data matrix by finding r new variables, where r is less than n. PCA Analysis revealed that data can be summarizing with just five variables and it contains all 5 principal components and their corresponding Eigen values for 2 day and 5 day.

Seed sterilization and plant inoculation study

Equal sized seeds Vigna radiata were thoroughly washed 3-4 times by sterile distilled water and treated with 1% HgCl2 for 2 minute followed by 2 minute treatment of 70% ethanol with vigorously shaking. Then the seeds were given two or three washes of sterile distilled water and were transferred to sterile petriplates containing wet filter paper. The seeds were incubated in dark. Sterile distil water was again added on the second day to maintain humidity for proper germination. Seeds were germinated up to the time when radical size reached to 1 cm. Overnight grown bacterial culture were used to inoculate germinated sterile seedlings of Vigna radiata.The germinated seedlings were incubated in saline washed bacterial suspension for 30 minutes and then three seedlings were planted in 75 ml Murashige and Skoog medium with in 500 ml glass tube (Hi- Media Ltd, India] and the various supplementations according to different combination types containing 0.7 % agar. The seedlings were allowed to grow for 7 days at 30±2 o C in alternate dark and light period.

Plant protection assay

For the in vitro antifungal assay MS medium was supplemented with biocontrol supportive combinations. The germinated seedlings were incubated in saline washed bacterial suspension for 30 minutes. After bacterial treatment seedling were treated with fungal broth aliquot from same stock broth of grown fungus in potato dextrose broth. Three seedlings were planted in 75ml Murashige and Skoog medium with in 500 ml glass tube (Hi-Media Ltd, India] and the various supplementations according to different combination types containing 0.7 % agar. The seedlings were allowed to grow for 7 days at 30±2 oC in alternate dark and light period.

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Results and Discussion

Effect of nutrient combinations on growth and antifungal activity

Numerous biotic and abiotic signals influence the production of antifungal compounds, including different mineral and carbon sources as well as metabolites released by micro-organisms and plants [27-29]. Quantitative and/or qualitative differences in the sugar, nitrogen, phosphate and mineral component of root exudates could determine the optimal environment for the effective bio-control mechanism in given crop-pathogen systems [24,30]. Our results add another point to plant specificity i.e. the influence of combination of root exudates components and edaphic factors on the growth, biocontrol traits and biosynthesis of antimicrobial metabolites by fluorescent Pseudomonas which will be helpful to increase its competence and survival efficiency in rhizosphere.

Use of 1/5-strength nutrient broth yeast extract broth only and further amendment with different combinations of glucose, citrate, KNO3, KH2PO4, FeSO4, ZnSO4 , and (NH4)6Mo7O24 resulted into the variations in Pf CHA0 response for the growth ,antifungal activity and antibiotics productions. Effect of nutrient combinations on 2 day growth (p=0.0026) and 5 day growth (p=0.011) and antifungal activity by the 2 day (p=0.0082) and 5 day (p=0.0007) ethyl acetate extract for the antibiotics from different nutrient combinations have shown significant variations (Table 1]. The inhibition of R.bataticola by the ethyl acetate extract of different nutrient combinations is shown in (Figure 1]. Combination amended with KNO3, (NH4]6Mo7O24 and ZnSO4 did not supported the Pf CHA0 growth. Combination amended with citrate, FeSO4, glucose and KH2PO4 combination amended with citrate ,FeSO",KNO" and ZnSO" and the combination amended with citrate ,glucose, FeSO4 , KH2PO4, KNO3, (NH4]6Mo7O24 and ZnSO4 have shown highest antifungal activity of 70% on 5 day and high growth( OD 600nm = 3±0.5]. Combination amended with citrate, FeSO4, glucose and KH2PO4 and combination amended with glucose, KH2PO4, KNO3 and ZnSO4 has supported growth on both the days of sampling with OD 600nm value of 4±0.5 but earlier combination supported more to antifungal activity on 5 day

Combination amended only with all inorganic components viz., FeSO4, KH2PO4, (NH4]6Mo7O2, and ZnSO4 showed green fluorescent pigment biosynthesis and antifungal activity up to 40% on 5 day. Combination with Zn, Mo and nitrate only did not support growth because of very low C/N ratio (C/N<1] and the stress effect of heavy metal Zn2+ and Mo2+ and it was not included in the further data analysis and graphs. Combination supplemented with glucose, citrate, Zn2+ and Mo2+ supported well to growth and showed declined growth on 5 day due to low nitrogen content but good antifungal activity on 2 day and 5 day due to very high C/N ratio. The amount of available nitrogen seems to be the crucial factor in deciding the metabolic response especially under N- limitation. Most the pathways used for N assimilation under N- limitation utilize high amount of ATP.

Therefore, it appears critical for the cell to shut down activities of such pathways under certain circumstances to save ATP which causes the cell concentration decreases [33]. Combination without any amendments showed low growth but high antifungal activity on 5 day than on 2 day which suggest that in diluted nutrient broth even growth is low and not much difference in 2 and 5 day growth but antifungal trait get induced on 5 day which indicates that as bacterial ages antifungal traits get intensified and such trend should be followed in every combinations and this combination should be considered as negative control for the amendments. Kumar and Shimizu 2010 reported that, as C/N ratio increases the transcript level of rpoN get increased, which encodes σ54. So the induction of rpoN was thought to be possible reason for the induced antifungal traits.

Effect of nutritional factors and their contribution to growth of Pf CHA0 at 2 day and 5 day is depicted in (Figure 2]. Citrate, glucose, Zn2+, citrate and glucose and triple combination of citrate, glucose and nitrate have shown positive effect on growth. Citrate and Pi, citrate and zinc, Mo2+, citrate and Mo2+, triple combination of citrate, glucose and KNO3 have shown negative effects on growth (Figure 2]. Citrate and Pi individually have shown positive effect and contribution of 9% and 5% respectively on 2 day and 10 % and 1 % respectively on 5 day however the combination of citrate and Pi showed negative effect on growth and contributed to 18% on 2 day and 19 % on 5 day. Contribution order of the factors acting positively on the 2 day growth is, Glucose > Zn2+>citrate +glucose>citrate>Pi>KNO3 while for the 5 day growth the order is Zn2+> citrate+ glucose > citrate > glucose >Pi=KNO3. In our result, citrate and glucose individually and in combination, have shown strong positive effect on Pf CHA0 growth on 2 day and 5 day which is in accordance to previous finding that an organic acid or a tricarboxylic acid cycle intermediate, not glucose, is usually the preferred carbon source in Pseudomonas species [34]. Of the minerals, zinc was found to be supportive much to Pf CHA0 growth on 2 day and 5 days. Sodeberg et al. (1990] explained that the zinc exhibits different bacterial affinity with gram-positive and gram-negative bacteria, it may be ascribed to the difference in the protein constituents of their cell walls zinc and other minerals are essential for growth, they influence cell membrane integrity, and they are key components and/or catalysts of over 300 enzymes and other proteins [35].

However the contribution order of negatively acting factors on 2 day growth is, citrate +Pi = Citrate +glucose + KNO3 > Mo2+ and for 5 day growth order is, citrate +Pi >citrate +glucose + KNO3 >citrate+ Zn2+. Ammonium molybdate has been reported to be a strong inhibitor of acid phosphatase activity [36] and the process of phosphorylation / dephosphorylation plays a crucial role in many metabolic processes [37].

Effect of nutritional factors and their contribution to 2 day and 5 day antifungal activity shown in Figure 2. Citrate has strong positive effect on antifungal activity on 5 day and contribution 29% and 2 day with contribution 14%. However glucose showed more positive effect on 2 day than 5 day with contribution 33% on 2 day and only 3% on 5 day. Citrate with glucose has positive effect and shown 16% contribution on 2 day and 12% on 5 day (Figure 2]. Although carbon sources differentially influence medium acidification during growth [38], which may then indirectly affect antibiotic biosynthesis [39] and bio control activity [9] but in our result we did not observe any significant change medium pH after 2 day and 5 day of growth. On 2 day, Pi, Fe2+, Citrate with Pi, citrate with Fe2+ and citrate with Mo2+ have shown negative effect on antifungal activity. On 5 day only glucose with nitrate and citrate with glucose and nitrate have shown negative effect on antifungal activity. Glucose with nitrate contributed 14% on 2 day and only 1% on 5 day. Citrate with nitrate contributed 6% on 2 day to 15% on 5 day. Contribution of triple combination of citrate, glucose and nitrate was 25% on 5 day but no contribution on 2 day. Citrate with nitrate showed 6% contribution on 2 day and 15% on 5 day.

Contribution order of factors acting positively to antifungal activity on 2 day is, Glucose>citrate>citrate +glucose while on 5 day antifungal activity the order is, citrate> citrate +glucose>Fe2+>glucose>citrate +Pi=citrate+Fe2+. Combination with Fe2+ and citrate have shown increased antifungal activity on 5 day and was thought to be mediated by induction of antifungal traits specifically. It is in support of previous observation that iron stimulates bicontrol activity [13] and cyanide production [2]. However the antifungal activity on 2 day is negatively regulated in following order, Citrate +Pi > citrate +glucose + KNO3>> citrate+Fe2+ and on 5 day in the order citrate + glucose +KNO3> citrate+KNO3> glucose + KNO3>Pi=KNO3. Glucose and citrate individually and in combinations, supported well to growth and antifungal activity. Combination with mineral supplementation of Fe2+, Mo2+, Zn2+ and Pi showed pigmentation after 5 day of growth and 40% antifungal activity and thought to positive effect of Pi on growth and positive effect of Fe2+ on antifungal traits. Combination with glucose, citrate and nitrate and did not have any amended mineral nutrient and have shown more growth (2 day OD600nm > 4.0) and antifungal activity on 2 day and less growth (OD600nm 5 day>3) and antifungal activity on 5 day which could be possible due to high C/N ratio which will induce rpoN and also antifungal traits and have not any interference of heavy metals.

Combination with Fe2+, nitrate and phosphate showed weak growth and antifungal activity which could be possible due to very low C/N ratio but only low growth because of supplementation of Pi and Fe2+. Effect of factors on antifungal Table 2: Biosynthesis of PRN,DAPG and PLT under different nutrient activity showed many variations in effect (+/-] and contribution compare to its effect on growth. Possible reason could be that the growth is result of basic metabolism and cumulative effect of many supportive pathways but antifungal activity is contributed by limited number of pathways so the variation in effect was observed. Citrate and Pi individually have positive effect on the growth but their combination have shown negative effect which show a kind of the shift of bacterial physiology/behaviour to nutrients present individually and in combinations.'

Effect on nutritional factors on antibiotic biosynthesis

PRN biosynthesis under different nutrient combinations is depicted in Table 2. Significant variation in biosynthesis level of PRN was observed across all combinations on 2 day (p=0.0016) and 5 day (p=0.0045). Nutrient combination amended with FeSO4, KH2PO4 and KNO3 , combination amended with,KH2PO4, (NH4)6Mo7O2, FeSO4 and ZnSO4, combination amended with FeSO4, Glucose, KNO3 and (NH4)6Mo7O24, and combination amended with citrate, FeSO4 , glucose, KH2PO4, KNO3, (NH4]6Mo7O24 and ZnSO4 supported PRN biosynthesis on 2 day.Nutrient combination amended with citrate, FeSO4, glucose and KH2PO4, combination amended with citrate,FeSO4, KNO3 and ZnSO4 , combination amended with citrate, FeSO4 ,glucose, KH2PO4, KNO3, (NH4)6Mo7O24 and ZnSO4, combination amended with citrate,glucose,(NH4]6Mo7O24 and ZnSO4 and combination amended with citrate , KH2PO4, KNO3 and (NH4]6Mo7O24 supported PRN biosynthesis on 5 day. It supports the previous observation by Duffy and Defago, 1999 that the production of pyrrolnitrin by Pf CHA0 was stimulated by Mo2+.

It suggests that direct improvement in the bio control effectiveness by NH4-Mo could be indirectly mediated by altered enzymatic activity. Effect of nutritional factors and their contributions to PRN biosynthesis is depicted in Figure 3. Fe2+, Pi, citrate with glucose and triple combination of nitrate, glucose with nitrate showed positive effect on PRN biosynthesis on 2 day. On 5 day, PRN biosynthesis is primarily influenced by citrate. Fe2+ showed strong positive effect on PRN biosynthesis with contribution 29% on 2 day and 13 % on 5 day PRN biosynthesis. It is in support of previous observation that iron stimulates biosynthesis of a variety of antifungal metabolites (e.g., zwittermycin A [11], kanosamine [12], phenazine [13].

Pi showed positive effect with 8% contribution on 2 day but negative effect with 1% contribution on 5 day PRN biosynthesis which is not significant effect and supports the previous finding that Pyrrolnitrin production by Pf CHA0 was not affected by 200 mM phosphate [16]. Contribution order of factors acting positively to PRN biosynthesis on 2 day is, Fe2+ > citrate +glucose >glucose+KNO3 > Pi> KNO3>citrate +Pi while on 5 day PRN biosynthesis the order is, Citrate>Fe2+>citrate+KNO3> citrate+Fe2+ > Zn2+> KNO3=citrate +Pi. The negative effect of factors on PRN biosynthesis on 2 day is in the order, Citrate +glucose + KNO3 >Citrate +Fe2+ > citrate >Zn2+ and on 5 day in the order, glucose + KNO3> citrate+glucose +KNO3>Pi>Citrate +glucose.

The DAP G biosynthesis levels under different nutrient combinations are shown in Table 2. DAPG biosynthesis varies significantly in all 15 combinations on the 2 day (p=0.009] but non significantly on 5 day (p=0.10) Glucose, citrate with Fe2+, citrate with Zn2+ and triple combination of citrate, glucose and nitrate have shown significantly positive effect on DAPG biosynthesis at 2 day (p<0.05].The effect of nutrients on the DAPG biosynthesis on 2 day and 5 day depicted in Figure 3. Positive contribution of factors acting on DAPG biosynthesis on 2 day follows the order, glucose>citrate+ Zn2+.

DAPG biosynthesis on 2 day is stimulated by glucose followed by combination of citrate and Zn2+ confirming the previous observation that increased DAPG biosynthesis by glucose [16]. However the negatively acting factors on DAPG biosynthesis on 2 day follow the order, citrate + Fe2+ > citrate+ glucose +KNO3. On 5 day DAPG is repressed by Pi which supports previous observation that DAPG production by Pf CHA0 was almost abolished by 10 mM phosphate. Growth was increased 5- to 10fold by 100 mM phosphate amendment [16].

The biosynthesis level of PLT under different nutrient combinations shown in Table 2. PLT biosynthesis level varies significantly in all 15 combinations on the 2 day (p=0.020) and 5 day (p=0.0098]. PLT biosynthesis level was high in combination in FeSO4, KH2PO4, (NH4]6Mo7O2, and ZnSO4, combination amended with citrate ,glucose, (NH4]6Mo7O24 and ZnSO4, combination amended with citrate ,glucose, and KNO3 and combination amended with citrate, FeSO4 ,glucose, KH2PO4, KNO3, (NH4]6Mo7O24 and ZnSO4 on 2 day. High PLT biosynthesis on 5 day was observed in the combination amended with citrate, FeSO4, glucose and KH2PO4 , combination amended with ZnSO4, KH2PO4, (NH4]6Mo7O24, FeSO4 and combination amended with citrate, FeSO4 ,glucose, KH2PO4, KNO3, (NH4)6Mo7O24 and ZnSO4 (Table 2). Table 2 shows the significant effect of nutritional factors and their combinations on PLT biosynthesis (p<0.05). Zinc increased PLT biosynthesis confirming the previous finding of Duffy and Defago, 1999 that zinc increased PLT biosynthesis in all fluorescent Pseudomonas strains able to produce this antibiotic, but the level of stimulation varied.

Citrate, glucose and combination of citrate with glucose showed positive effect on 2 day PLT biosynthesis and combination of citrate with glucose and citrate with nitrate and glucose with nitrate showed positive effect on 5 day PLT biosynthesis. Citrate with glucose contributed 37%, citrate with Pi contributed to 18 %, citrate with Fe2+ contributed to 18% and citrate, glucose individually contributed to only 7% on 2 day PLT biosynthesis. For the 5 day, citrate with glucose, citrate with nitrate and glucose with nitrate has shown strong positive effect on PLT biosynthesis. Positive contribution of factors on PLT biosynthesis on 2 day showed following order, citrate+ glucose> glucose=citrate> glucose+ KNO3> citrate+KNO3 while on 5 day PLT biosynthesis the order is, citrate +glucose> citrate+KNO3 > glucose+KNO3.

The negatively acting factors on PLT biosynthesis on 2 day showed the order, citrate +Fe2+ =Citrate +Pi >>citrate+glucose +KNO3 and on 5 day in the order citrate+ glucose +KNO3> KNO3>> glucose>citrate. PLT biosynthesis is maximally repressed by combination of citrate and Fe2+, citrate and phosphate on 2 day confirming the previous observation, that production of PLT by Pf CHA0 was completely inhibited by 100 mM but only slightly reduced by 10 mM phosphate. However on 5 day PLT biosynthesis is maximum repressed by triple combination of citrate, glucose and nitrate followed by nitrate confirming the previous finding that PLT is repressed by glucose [16].

Combination of citrate and glucose has shown positive effect on 2 day and 5 day antifungal activity and well correlated by its positive effect on PRN and PLT production. Glucose showed the positive effect on 2 day antifungal activity and was correlated with its positive effect on DAPG production. Combination of citrate and Fe2+ has shown negative effect on 2 day antifungal activity and it was supported well by negative effect of this combination on 2 day PRN, DAPG and PLT production. Citrate has shown positive effect on 5 day antifungal activity and correlated with its positive effect on 5 day PRN production. Triple combination of citrate, glucose and nitrate has negative effect on antifungal activity and correlated also with its negative effect on PRN and PLT production. The importance of nutrient status to pyoluteorin production is corroborated by the observation that pyrrolquinoline quinone, a cofactor required by glucose and alcohol dehydrogenases, represses pyoluteorin production confirm that pyoluteorin production is linked to the physiological status of the cell [40].

In case of Pf-5 as bacterial ages, the stationary phase and stress response sigma factor RpoS (aS) and Lon protease, are implicated in regulation of antibiotic production. Relative levels of aS and the housekeeping sigma factor RpoD (aD) influence pyoluteorin, 2, 4-diacetylphloroglucinol, and pyrrolnitrin production. Abundant aS is required for the production of pyrrolnitrin but is repressive to pyoluteorin and 2,4-diacetylphloroglucinol production [25,26]. So the role of relative levels of aS and the housekeeping sigma factor RpoD (aD) in the increased antifungal trait on 5 day in low/moderate growth supporting combinations could not be ignored.

Contribution of PRN,DAPG and PLT for antifungal activity

Based on antifungal activity under all combinations and the biosynthesis level of antifungal metabolites, the percentage contribution of PRN,DAPG and PLT to 2 day and 5 day antifungal activity by Pf CHA0 depicted in Figure 4. For the 2 day antifungal activity, PLT has contributed 59%,PRN has contributed 26 % and remaining 15% by DAPG. For the 5 day antifungal activity PRN has contributed 59%,PLT 37% and DAPG only 4%. Regulation of DAPG and PLT production in Pf CHA0 involves a molecular balance in which each antibiotic induces the expression of its own biosynthetic genes while repressing the expression of the biosynthetic genes of the other antibiotic [41,42]. This could be possible reason of not obtaining significant variations in DAPG production under different nutrient combinations on day 5 and day 2. Another possible explanation for these deviations is that other, yet unknown effectors could interfere with the fine-tuned regulation of the DAPG-PLT balance. Similar deviation in DAPG production was obtained by Baehler et al. [41] as they have used GFP-based reporters to study the antibiotic gene expression at the transcriptional level. The possibility of involvement of some post-transcriptional regulatory mechanisms such as those controlled by the GacS/GacA two-component system Haas & Keel (2003) may be involved in the modulation of the observed effects as well.

2 day antifungal found to have maximum positive correlation with PLT followed by PRN however both PLT and PRN itself had shown strong positive correlation with OD600nm. Up to 2 day DAPG is found to be not dependent much on OD600nm and PLT 5 day antifungal found to have maximum positive correlation with PRN followed by PLT however both PLT and PRN itself had shown a positive correlation with OD6OOnm. S day PLT have shown but negative correlation with DAPG production. S day DAPG have shown negative correlation with OD600nm, PRN and PLT which proves the earlier observation by Baehler et al. [41].

Principal Component Aanalysis for 2 day and 5 day antifungal activity

For the 2 day as biplot graph (Figure S) as shown in that 2 day antifungal, OD and PLT biosynthesis showed strong correlation and fall in same zone (-x, +y).While 2 day DAPG and PRN fall in other zone (+x, +y). Based on PCA analysis for the five variables with sixteen different combinations, Pearson correlation between different variables was obtained (Figure S) which shows the degree to which the variables are related with each other. Only the variables with the Pearson value (n) > 0.3S have been considered with significant relatedness between them. 2 day antifungal is positively correlation with PLT maximally (n=0.8l8) followed by OD (n=0.S44), PRN and DAPG.2 day PRN has shown positively correlation with 2 day OD (n=0.44), antifungal activity (n=0.38). DAPG is nearly independent of OD and PLT (n<0.1).2 day PLT has shown high positive correlation with OD6OOnm (n=0.694) and antifungal activity.S day antifungal activity have shown positive correlation with OD (n=0.S22), PRN (n=0.l68) and PLT (n=0.4lS). S day PLT have shown positive correlation with OD (n=0.33) and antifungal activity (n=0.4lS] but negative correlation with DAPG production (n= -0.16). DAPG have shown negative correlation with OD (n= -0.187), PRN (n=- 0. 131) and PLT.

For 2 day the results showed that of the first three components, the first component accounted for about 54.52%, the second component about 22.36% and the third component about 13.88% of the total variance in the data set These three components together accounted for about 90.71% of the total variance and the rest of the components only accounted for about 9.29%.For 5 day biplot graph (Figure 5). shows that PLT and OD shows correlation and falls in same zone (+x, +y] while 5 day antifungal and PRN falls in other zone (+x,-y].For the 5 day PCA analysis first three components, the first component accounted for about 45.72%, the second component about 22.72% and the third component about 19.44% of the total variance in the data set. These three components together accounted for about 87.89 % of the total variance and the rest of the components only accounted for about 12.11% (Figure 5).

Plant protection assay

The plant growth promoting effect of Pf CHA0 on Vigna radiata under biocontrol trait supporting combinations has been shown in Figure 6. Combination with glucose, FeSO4, KNO3 and ZnSO4 supplemented to MS medium and combination with glucose,citrate, nitrate,Pi, ZnSO4, FeSO4 and Mo2+ have shown inhibition of plant growth in compare to non supplemented combination while inhibitory effect was minimized in inoculated with Pf CHA0.Plant inoculation study for the antifungal activity of Pf CHA0 under antifungal traits supportive combinations has been shown in Figure 6. Non supplemented combination supported to plant growth in uninoculated control and growth suppression in R.bataticola treated set. Pf CHA0 have shown highest plant growth promotion out of all combination and MS medium control. Pf CHA0 showed an effective inhibition of R.bataticola and plant protection in combination supplemented with glucose, FeSO4, KNO3 and ZnSO4 supplemented to MS medium and combination with glucose, citrate, nitrate, Pi, ZnSO4, FeSO4 and Mo2+ in compare to un-inoculated control.

In biocontrol supportive combination with Mo and Zn it could be deleterious effect of metals on plant growth which could be the possible reason of less growth in compare to non supplemented combination .Combination 16 have three metals so it has retorted the plant growth effectively in compare to combination 9 which have Fe2+ and Zn2+. Combination 9 retardation effect was minimized by PfCHAO inoculation compare to uninoculated. In bio control supportive combination the effective suppression of R.bataticola by Pf CHA0 was observed. Non Supplemented combination have supported to plant growth in uninoculated control and bio control support against R.bataticola. Pf CHA0 have shown highest plant growth promotion out of all combination and MS medium control. Possible reason could be that this combination does not have any metal which is inhibitory to plant growth and it is also supportive to Pf CHA0 growth in compare to MS medium. Pf CHA0 showed effective bio-control against R.bataticola in bio control supporting combinations.?

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Conclusion

Present work establish the connection between multiple combinations of nutrients and edapic factors relevant to root zone and its effect on the growth, antifungal activity and biosynthetic of pyrrolnitrin, 2, 4-DAPG and pyoluteorin by Pf CHA0. Result showed that citrate, glucose, combination of citrate and glucose, ferrous iron and zinc have positive effect on growth, antifungal activity and antibiotic biosynthesis. Triple combination of citrate, glucose and nitrate inhibited the growth, antifungal activity and antibiotic biosynthesis. Antifungal activity is maximum contributed by pyoluteorin initially but by pyrrolnitrin later stage of growth. Bio-control supporting nutrient combinations showed effective protection of Vigna radiata from R.bataticola infection in plant inoculation assay.

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Acknowledgement

Authors express a gratefully acknowledgement to University Grant Commission-Research Fellowship for Meritorious Student (UGC-RFMS) and Gujarat State Biotech Mission (GSBTM), India, for the financial support.

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juniperpublishers-oajnn-blog · 5 years ago

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In Vitro Radiosensitivity of Adamantinomatous Craniopharyngiomas

Authored by Elfar úlfarsson

Abstract

Background and purpose: Improvements in radiation treatment for craniopharyngiomas are needed. No clear in vivo data exists on radiosensitivity of craniopharyngiomas and in vitro data is lacking. The purpose of this study was to assess the radiosensitivity of adamantinomatous craniopharyngioma in vitro.

Materials and methods: Craniopahryngioma cells from 7 individuals (two different passage number) were seeded in triplicate wells and exposed to 8 photon doses in the range 0-10 Gy in a 137Cs irradiation chamber. The radiosensitivity was measured as clonogenic cell survival. The surviving fraction at 2 Gy (SF2), the mean inactivation dose ( ) and the α/β ratios were calculated from the dose response curve fitted with the Linear Quadratic model.

Results: The SF2, and α/β values for the cell strains ranged from 0.31 to 0.47, 1.65 to 2.44 and 10 to 30 Gy, respectively. The mean, standard deviation and coefficient of variation of the SF2, and α/β values were 0.40 ± 0.07, 17%, 2.04 ± 0.30, 15% and 19 ± 6 Gy, 33%, respectively.

Conclusion: The high α/β value indicates that adamantinomatous craniopharyngioma is among early responding tissues. This supports the clinical praxis of fractionated radiation for those tumors.

Keywords: Craniopharyngioma; Radiosensitivity, Clonogenic cell survival, Alpha/beta ratio, radiation treatment

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Introduction

Craniopharyngioma is a benign neoplasm of epithelial origin located in the sellar and suprasellar region. The adamantinomatous craniopharyngioma represents around 2/3 of all craniopharyngiomas and is the most common pathology in this region in children [1,2]. Other histological subtypes are the squamous papillary and the xanthogranulomatous type [3]. The former occurs almost exclusively in adults. The main treatment options are surgery and conventional radiotherapy (CRT). Surgical cure is hampered by the eloquent surrounding structures and is accomplished in few cases. The acknowledgement of the devastating effect of hypothalamus damage on quality of life has led to more conservative surgery and increased use of adjuvant RT in children [4,5]. CRT has proved its value in controlling craniopharyngiomas; however considerable irradiation to the surrounding tissues is unavoidable by this technique and not without sacrifices.

The decline in intellectual function in children [6], inability to treat the youngest patient group, radiation induced malignant gliomas [7] and 10-years recurrent free survival rates of 32 % - 49 % [8,9] in children stresses the limits in the current CRT.

Gamma Knife radiosurgery (GKRS) has been used as an alternative to CRT to reduce the radiation load on the surrounding tissues. Reports on low morbidity from the anterior visual pathways using this technique, 3.1%(10), underlines the power of photon beam focusing. The main drawbacks are the target volume restrictions and higher risk of geometrical misses compared to CRT. The ultimate limitation of the single session GKRS is that the most effective GKRS-dose, 11.7 - 12.7 Gy, is also the critical toxicity dose for the anterior visual pathway [10,11].

The majority of craniopharyngioma patients receive radiation treatment during their diseased life and around one third of them experience tumor progress [12]. The hazard of repeated irradiation precludes further radiation in most cases and the danger of repeated surgical trauma is well known. The gravity of the situation of late recurrences is reflected in a very high mortality after salvage treatment [13,14]. Thus, improvement in the radiation treatment is clearly needed. No clear data exists on the radiosensitivity of craniopharyngiomas and the optimal dose has not been defined. The lack of detailed dose-response data and standardized way of reporting endpoints disables a thorough interpretation of radiobiological data from the literature. Furthermore in vitro radiosensitivity data, that could be useful in designing new fractionation regimens, is lacking.

The purpose of this study is first to assess the in vitro radiosensitivity of adamantinomatous craniopharyngiomas and second to address the alpha beta ratio (α/β) specifically from a clinical perspective.

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Methods and Materials

Cell cultures

Primary cultures of human craniopharyngioma cells were isolated and prepared from tumour samples in a similar manner as for keratinocytes according to the methods described elsewhere [15,16]. The obtained cultures used for the (irradiation) experiments were plated without feeder cells in passages between 3 and 9 (median 5). All patients harboured histological verified adamantinomtous type of craniopharyngioma. The procedures were in accordance with the ethical standards of the institutional committee on human experimentation and with the Helsinki Declaration of 1975, as revised in 2000.

Clonogenic survival

Appropriate cell numbers were plated for survival using the clonogenic assay technique described previously [17]. The single-cell suspensions were plated into 35 mm plastic petri dishes (Corning, New York) in triplicates to a final medium volume of 3 ml and then left in the incubator for 3-4 h to attach before irradiation.

The cells were irradiated at room temperature with doses of 0-10 Gy at a dose rate of 0.5 Gy/min from a 137Cs-source (Scanditronix, Uppsala, Sweden). The cultures were then incubated for 10-14 days, with a change of medium after 5-7 days. Thereafter colonies were fixed, stained and counted. Radiation survival curves were constructed from two independent experiments. The mean PE for un-irradiated cells was 43, 55, 85, 12, 10, 42 and 36% for case 1 to 7, respectively.

Dose-response models for clonogenic cell survival. The LQ model(18) was used to fit the data with the Maximum Likelihood method, where the probability for clonogenic cell survival S at a dose D is given by [19]:

S = e-(αD+βD2 ) (1)

The surviving fraction at 2 Gy (SF2) was computed from the whole survival curve and was used as a unique measure of cellular radiation sensitivity. The mean activation dose, D , was calculated according to Taylor [20]. The D was chosen since it has been shown to keep the scattering of data smaller than for other parameters such as SF2 and D0 [21].

Assessment of α/β in vivo

From in vivo data, the LQ model can be used to calculate the α/β from isoeffective fractionation regimens. This is based on the assumption that the biological effective dose (BED) is the same if two fractionation regimens result in an equivalent clinical effect [22]. In this study, the same tumour control rates were assumed with 2.0 Gy daily fractions with a total dose of 50 Gy [23] and 11.5 Gy prescription dose at the 50% isodose line using GKRS [10]. For the GKRS, it was assumed as a first reasonable approximation, that the tumor response was related to the mean dose to the tumor which is generally 30 - 50% higher than the dose to the periphery. Thus for a prescribed dose of 11.5 Gy, the mean dose will be in the range from 15 Gy to 17 Gy. Calculation were done for two cases; 1) without- and with correction for repopulation during the time of fractionated treatment, in the second case, two values of Tk (the time after start of the treatment when repopulation starts) were used; for keratinocyte-related malignancies such as head and neck tumors (Tk=21 days) and craniopharyngioma related normal tissues such as mucosa (Tk =7 days) [24]. For each of the two Tk values, the cell doubling time (Tp) was estimated in patient C1 and C2using the formula

Tp=0.693*t/ln(Nt/N0).

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Results

The clonogenic cell survival curves and the radiosensitivity parameters from the seven craniopharyngioma cell strains are presented in Figure 1 and Table 1, respectively. The mean values and the coefficient of variation (CV) for SF2 and D were 0.401 (SE +/- 0.021) and 2.04 (SD +/- 0.08) and 17% and 15%, respectively. The α/β value ranged from 10.3 to 29.9 Gy with CV of 33%. The mean value was 19 Gy (SE +/- 2.4). Acknowledging the fact that other survival models will fit the data in the lower dose region better, we used the entire dose range when fitting the data. This was done since doses in the range of 8-13Gy are being used for these types of tumors.

In Figure 2, α/β values are given as a function of the mean dose to the tumor in GKRS Estimated mean doses to the tumor, for a prescription dose of 11.5 Gy will be in the range from 15 Gy to 17 Gy. Results are shown, without correction for repopulation, as well as corrected with two Tk values, with two sets of Tp and α values.

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Discussion

In this study we present a novel data set on radiosensitivity of adamantinomatous craniopharyngioma cell strains, based on clonogenic survival assays. More than 25 years ago Fertil et al. [25] pointed out a correlation between the radiosensitivity of human cancer cell lines in vitro and the radiocurability of the corresponding tumor types in vivo. Since then histological groups of human cell lines have been characterized by an intrinsic radiosensitivity in vitro [26,27] and this data has been shown to be useful in predicting tumour response to RT [28].

The high variation of α/β within specific tumor group as for adamantinomatous craniopharyngiomas in this study is commonly noted in reports for different human cancer lines in vitro. Taghian et al. [29] reported data on glioblastoma, which is among the most radioresistant tumours. The α/β ranged from 3.7 to 48 Gy. Weichselbaum et al. [30] reported mean α/β for human cancer cell lines with large SD values, reflecting a high variation within each tumour group.

According to SF2 and Dcraniopharyngiomas cell strains are slightly more radiosensitive than the average radiosensitive cancer cell line [27] and the variation is not higher than reported in other cancer cell lines [21,27]. Comparative in vitro data for other benign intracranial tumours is lacking. However, from in vivo data, and with the method used here α/β for meningiomas and vestibularis schwannomas have been calculated to be 3.3 Gy and 2-3 Gy, respectively [31]. In this study, the α/β will be in the range of 4-6 Gy for adamantinomatous craniopharyngiomas, without correction for repopulation and from 6 Gy to more than 20 Gy if reasonable parameters for correction for repopulation are applied (Figure 2). Those values are still much lower than the in vitro values in this study. The reason for this discrepancy could be explained by a lack of correlation between the in vivo and in vitro data, by paucity of reliable in vivo data or both.

Arguments for craniopharyngiomas having high α/β can be found when growth kinetics is considered. It is known that the growth kinetics of the tissue irradiated dominates the radiation response. A higher proliferation indices [32-35] and more dramatic response to irradiation [10,36-39] compared to meningiomas and acustic neurinomas support our findings of a high α/β for craniopharyngiomas. The α/β for cranipharyngioma related tissues, such as mucosa and epithelium is in the range for early responding tissues or 7 - 15 Gy [40-42]. Since there is often a close similarity between the mean lethal dose of tumour and the normal tissues from which they arise, it is likely that α/β for cranipharyngioma is in this high range. If this is the case, one should consider the repopulation time factor in calculating the α/β from clinical data. This results in higher α/β values (Figure 2) [43,44].

The above estimations of α/β values based on in vivo data have to be viewed with caution since the differences in patient and treatment characteristics between clinical studies make it difficult to compare results from different radiation regimens. The main obstacle is that certain parameters that can influence endpoints, such as histological subtypes and tumour volume, are generally not considered in reports on treatment results.

The comparison of iso effective doses for GKRS and CRT could be flawed by differences in tumour volumes treated with those techniques. The general principle is that large tumours (more clonogenic cells) require higher doses than small ones (fewer clonogenic cells) to obtain the same tumour control probability (all clonogenic cells killed, Figure 1). This is also partly explained by the differences in micro-environment between large and small tumors [45,46]. The lack of information on tumour size is more a rule than an exception in studies using CRT for craniopharyngiomas. In viewing the few studies available, it seems that the tumour volume tends to be substantially larger in the CRT studies compared to GKRS studies [10,47-49]. This is a reasonable assumption regarding the radiobiological volume restrictions of GKRS. The implication of this phenomenon in CRT for craniopharyngioma is probably less than for other more solid tumours types due to the large fluid component in craniopharyngiomas.

Taking the arguments above together:

CRT studies tend to include substantially larger tumour volumes and

Larger tumour volumes require higher doses for local control, it is tempting to conclude that a somewhat lower CRT dose than 50 Gy would give the same local control as GKRS for similar tumour volumes. If this line of arguments is correct, the α/β from clinical data, will be larger than the ones presented in Figure 2 and hence even more close to the α/β presented in this study from in vitro data.

It is debated whether benign intracranial tumours benefit from fractionation since the α/β is thought to be similar as for neural tissue or 2 Gy. This is generally supposed to reduce the beneficial effects of fractionation but if the clinical α/β is as high as observed in our study we have strong arguments to apply fractionated radiation regimens to improve the therapeutic ratio. It is however important to notice that improvements in RT are clearly needed.

Even though CRT has been used for craniopharyngiomas for more than 40 years, the toxicity risk for the anterior visual pathway and the hypothalamus has not been reduced with recent improvements in this technique. The major part of those structures is still included in the high dose field prescribed for the tumour and receives doses just below the maximum tolerance level [6,39,48]. Furthermore structures involved in neurocognition are still at risk [6]. Improvements could be made by using fractionation with Gamma Knife® or similar technique. By this approach one could overcome the target volume restrictions of single fraction treatment, avoid critical doses to structures involved in neurocognition and reduce the radiation load on the visual pathway and hypothalamus. Radiobiological data such as presented in this study would then be needed to design new fractionation regimens.

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Conclusion

The high α/β in this in vitro study indicates that the adamantinomatous craniopharyngioma is among early responding tissues and supports the clinical praxis of fractionated radiation. The radiosensitivity parameters, SF2 and , indicate that these cell strains are slightly more radiosensitive than the average radiosensitive cancer cell line. Although some correlation with in vivo data is found, improvements in presenting treatment parameters and results after radiation treatment and further in vitro studies are encouraged to validate this data.

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annieboltonworld · 4 years ago

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Juniper Publishers- Open Access Journal of Environmental Sciences & Natural Resources

Environmental Sciences

Effect of Plant Spacing On Quantitative and Qualitative Characteristics of Fcv Tobacco Hybrids

Authored by Hazrat Usman

Abstract

A field experiment was carried out on “effect of plant spacing on quantitative and qualitative characteristics of FCV tobacco hybrids” at The Tobacco Research Station, Khan Garhi, Mardan during 2014-2015, using randomized complete block design with split plot arrangement, replicated thrice. Treatments included three tobacco hybrids (CSC 4302, CSC 4704 and CSC 447) and three plant spacing (T1=R-R 120 cm and P-P 55 cm, T2= R-R 107 cm and P-P 55cm and T3=R-R 90cm and P-P 60cm). Plant height, leaf area were significantly affected by tobacco hybrids and cured leaf yield (kg ha 1) were significantly affected by spatial. Higher plant height (114.7cm) from CSC hybrid 4302, leaf area (1142 cm2) from CSC hybrid 4704 and higher cured leaf yield (3842 kg ha-1) were recorded from CSC hybrid 447 tobacco hybrids. Higher plant height (107.3 cm) and higher cured leaf yield (4109.7 kg h a-1) were recorded from spatial arrangement T3. It is due to the space for tobacco hybrids are better and the variety CSC-447 performed better. Hence spacing T3 and CSC-447 hybrid is recommended for better growth and higher yield of tobacco crop in Mardan region.

Keywords: Plant Spacing; FCV Hybrids; Tobacco yield; Tobacco Leaf Quality

Introduction

Tobacco is grown for leaf purpose and widely grown as nonfood commercial plant in the world. Tobacco is also one of the major cash crops in Pakistan especially in Khyber Pakhtunkhwa. Tobacco is the only crop in Pakistan whose yield on per unit area is compare with developed countries like USA. However there is struggle for improvement in yield on per unit area Ahmed [1] Tobacco is cultivated in Pakistan on 50,000 hectares with a total leaf production of 108,000 tons. Tobacco has become an important cash crop of Khyber Pakhtunkhwa the agro climatic conditions are extremely suitable for its cultivation. Total area under cultivation in Khyber Pakhtunkhwa is 30,048 ha, which produce 100.78 million kg Pakistan Bureau Statistics [2] Tobacco contributes 4.4% to the GDP of Pakistan. Its export value was 570.2 million rupees which share in all export of Pakistan. Cigarettes and tobacco give 30.6 billion rupee indirect taxes whose share in gross are 5.4% Federal Board of Revenue, 2008. Among growing practices, plant spacing can affect agronomic and chemical traits of Tobacco Alizadeh [3] several researchers recoded high yield on maximum plant densities. However, quality of such leaves was usually lower due to decreased nicotine content Chaplin [4], Campbell [5]

Higher plant density in tobacco produce taller plants with smaller leaves, but also effect on number of leaves per plant. Closer spacing tobacco result in higher yields, increased crop returns Kozunrttx and Lurosrvrcrus., 1975. Plant spacing is required for the optimum yield. Closer spacing of plants resulted in reduction of size, body, thickness and weight per unit area of the leaf, Price of tobacco grown at higher plant densities was also lower, resulting in lower income from such production observed a decrease in total leaf area per plant with increased plant population Bukan [6] Regulate the optimal density is one of the important factors to get the maximum yield due to the climatic conditions of each region and specifications of varieties are cultivated.

The purpose is to determine the optimal density; spacing between plants, so that an appropriate combination of environmental factors provided to achieve maximum performance with possible quality. In considering an appropriate density, mutual ghosting is minimized and light thus photosynthesis is maximized. If the planting spaces are too common, certainly the number of plants per unit area reduced and the yield will be faced with a deficit. Due to the efficiency of water use in the product increase, desirable high density is needed to achieve a high performance Kharazmi [7].

Materials and Methods

Experimental site

Tobacco research station is located in the south west of the district Mardan at 34°12'0N 72°16'0E, altitude of 283 meters (928 feet), and with an elevation of 314 meters above sea level.

Experimental design

The experiment was laid out in RCB design with split plot arrangement, having three tobacco varieties (Main plot) and treatment (sub plot), replicated thrice at the Tobacco Research Station, Khan Garhi, Mardan, during 2014-15. Spatial arrangement was done 120x55cm, 107x55cm and 90x60cm row to row and plant to plant distances respectively.

Nursery management

Nursery was raised on 20th December 2014. Seed rate of 4g ha-1 was used. Bed size will be 10m2. Thinning was done to get optimum and healthy plant stand. Three tobacco hybrids of CSC series i.e. CSC 4302, CSC 447, CSC 4704 were sown.

Field preparation

Before transplantation, land preparation was done using primary and secondary tillage implements. Good and viable seedlings were transplanted in the field and the field was irrigated to establish good root system.

Transplantation

Transplantation was done during the last week of March 2015. Each plot and subplot having five rows, having 10 plants in each row. The row to row distance was 3, 3.5 and 4 feet, while plant to plant distance was 2, 1.8 and 1.8 feet respectively.

Field management

After transplantation irrigation were applied 6-7 times. N:P:K fertilizers were applied with the rate 75:75:75 kg ha-1. After transplantation one dose was applied to the two sides of plants. Topping was done at 24 leaves stage. The parameters studied were plant height, leaf area plant-1, green leaves weight plot-1 (kg), number of green leaves kg-1, cured leaves weight plot-1(kg), number of cured leaves kg-1, cured leaf yield ha-1, nicotine and reducing sugar contents, using the following procedure.

Plant height (cm)

In each plot thirty plants were selected and measured from top to bottom and their mean was calculated.

Leaf area (cm2)

For leaf area, thirty plants randomly were taken then leaf length and breadth was measured. The average leaf size was computed from these plants by multiplying with a common factor of 0.644 derived by Suggs et al. 1960.

Leaf area = Leaf length x leaf breadth x 0.644 2.

Number of green leaves kg-1

The number of green leaves was found by measuring the number of green leaves in each bundle of plot.

Number of green leaves kg-1 = No of leaves in the determined weight x kg

Green weight determined

Number of cured leaves kg-1

The number of cured leaves was found by measuring the number of cured leaves in each bundle plot.

Number of cured leaves kg-1 = No of leaves in the determined weight x kg

Cured weight determined

Cured leaf yield (kg ha-1)

Data concerning leaf yield, the weight of cured leaf in each treatment was taken after each picking. The total cured leaf yield was calculated by the following formula:

Yield (kg ha-1) = Cured leaf weight plot-1 x total no. of plants ha-1

No. of leaves per plot

Nicotine Content (%)

Nicotine was determined by the method of Cundif and Markunas 1964. The nicotine contents were calculated by the following formula:

Nicotine (%) = V1 x N x 32.45 x 100

Weight of sample

Whereas

V1=Volume of titrant for non-acetylated aliquote.

N=Normality of perchloric acid.

4.13. Reducing Sugars (%)

Reducing sugars percentage was estimated as follow:

Reducing sugars = 25 x 100 x 0.05

Titrate x wt. of sample

Data collected was analyzed according to split plot design and means were compared using least significant difference (LSD) test Steel and Torrie 1980

Results and Discussion

Plant height (cm)

Data regarding plant height of tobacco hybrids indicated that different tobacco hybrids had significant effect on plant height of different tobacco hybrids (Table 1). Taller plants (114.7) were reported in plots of CSC-4302 as compared to CSC-4704 and CSC-447. Spatial arrangement of plant had non-significant effect and the interaction as well. The possible reason for this could be due to genetic characteristics of tobacco hybrids and each tobacco hybrid showed different response Bukan [6].

Lsd value(0.05) for varieties=9.4011

Leaf area (cm2)

Lsd value(0.05) for varieties=86.410

Data regarding leaf area of tobacco hybrids indicated that different tobacco hybrids had significant effect on leaf area (Table 2). Maximum leaf area (1142 cm2) was reported in plot of CSC-4704as compared CSC-4302 and CSC-447. Spatial arrangement of plant had non-significant effect and the interaction as well. The possible reason for this could be due to genetic characteristics of tobacco hybrids and each tobacco hybrids showed different response Bukan[6].

Number of green leaves kg-1

Lsd value(0.05) =ns

Number of cured leaves kg-1

Lsd value (0.05) =ns

Data regarding number of green leaves kg-1 of tobacco hybrids indicated that tobacco hybrids, spatial arrangement and interaction had non-significant effect on number of green leaves kg-1 (Table 3). Maximum green leaves kg-1 (17) was reported in plots of CSC-4302 and CSC-4704 as compared to CSC-447. While for spatial arrangement maximum green leaves kg-1 (17 cm) observed in spacing T1 as compared to T2 and T3.

Data regarding number of cured leaves kg-1 of tobacco hybrids indicated that tobacco hybrids, spatial arrangement and interaction had non-significant effect (Table 4). Maximum number of cured leaves kg-1 (115) was reported in plots of CSC- 4704 as compared to CSC-4303 and CSC-447. While for spatial arrangement maximum number of cured leaves kg-1 (115) observed in spacing T1 as compared to T3 and T2.

Cured leaf yield (kg ha'1)

Lsd value (0.05) for spacing=484.48

Data regarding cured leaf yield kg ha-1 of tobacco hybrids indicated that different tobacco hybrids and interaction had non-significant on cured leaf yield kg ha-1 of different tobacco hybrids (Table 5). Spatial arrangement had significant effect on cured leaf yield kgha-1 of different tobacco hybrids. Maximum cured leaf yield kg ha 1 (4109) were reported in plot of spatial T3 arrangement as compared to spatial T2 and T3. The possible reason for this could be due to the genetic adoptability of tobacco hybrids to T3 spatial arrangement Kharazmi [7] and also similar results were reported by Bukan [6-10].

Nicotine contents percentage

Data regarding nicotine contents percentage of tobacco hybrids indicated that tobacco hybrids, spatial arrangement and interaction had non-significant effect on nicotine contents percentage (Table 6). Maximum nicotine contents percentage (2.40) were reported in plots of CSC-4302 as compared to CSC- 4704 and CSC-447. While for spatial arrangement maximum nicotine contents percentage (2.46) observed in spacing T3 as compared to T2 and T1.

Reducing sugar contents percentage

Data regarding reducing sugar contents percentage of tobacco hybrids indicated that tobacco hybrids, spatial arrangement and interaction had non-significant effect on nicotine contents percentage (Table 7). Maximum reducing sugar contents percentage (18.07) were reported in plots of CSC- 4302 as compared to CSC-4704 and CSC-447. While for spatial arrangement maximum reducing sugar contents percentage (19.38) observed in spacing T1 as compared to T2 and T3.

Conclusion

It was concluded from the results of experiment that variety CSC-447 showed better results from the other two varieties. the variety CSC-447 have tremendous potential to improve various traits such as yield and yield related components and therefore variety CSC-447 are recommended to obtain higher yield and the Row to Row 90 cm and plant-plant 60 cm distance are sufficient for tobacco crop and increasing further spacing in tobacco crop were not significant effect on yield and yield related traits. Hence 90x60cm spacing are good to obtain higher FCV leaf yield and of good quality.

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serenavangstuff · 5 years ago

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Journal of Gerontology and Geriatric Medicine-JuniperPublishers

Abstract

When confronting chronic and/or serious illness many individuals wish to be active participants in determining the treatment and care provided to sustain optimal quality of life. As healthcare professionals, our goal is to facilitate a conversation with the individual and/or their family or other advocates that supports that person's life's journey and addresses their physical, social, spiritual, and emotional needs, as well as their values and cultural traditions. Through this discussion, we assist the person in using a systematic approach to identify and coordinate a plan of care to maintain quality of life.

Introduction

The acknowledgement of the complex needs surrounding chronic/serious illness and the importance and benefit of palliative care, led to palliative medicine becoming recognized in 2006 as a board-certified sub-specialty of internal medicine with specialized fellowships for physicians in the United States. Palliative care is a multidisciplinary approach to person-centered care, relying on input from pharmacists, nurses, chaplains, social workers, psychologists, and other allied health professionals in formulating a plan of care to help relieve suffering in all aspects of a seriously ill person's life. This multidisciplinary approach allows the palliative care team to address physical, emotional, spiritual and social concerns that arise with advanced illness.

Palliative care focuses on providing the individual with relief from the symptoms, pain, and physical and emotional stress of a serious or chronic illness- whatever the diagnosis. Palliative care is provided by a team as an extra layer of support by healthcare, ancillary, and spiritual staff. It is appropriate at any age and at any stage in a serious illness and can be provided as the main goal of care or along with curative treatment. Palliative care can be provided across multiple settings in healthcare settings, in the person's home, or as part of community palliative care programs.

Key Elements in a Palliative Care Program

A. Element One: Education

a) Determine who will be the educators.

i. Select a "Champion" educated in the palliative care concept and effective in "having the conversation." This champion will educate other individuals who wish to be a part of the Palliative Care team.

b) Advanced Care Planning Education

i. Leadership Roles

c) Individual (s) who is engaging, enthusiastic and has demonstrated competency in the practice of palliative care

d) Director of Nursing Services/Advanced Nurse Practitioner/Corporate or contracted provider

e) Direct care staff

f) Director of Social Services

g) Rehabilitation therapist

h) Chaplain

i) Training specific to your healthcare environment:

j) Acute care

k) Post-acute care

l) Hospice agency

m) Assisted living

n) Skilled nursing

o) Home care

p) Ensure individuals are sufficiently trained and comfortable in starting the conversation for advance care planning:

q) Suggested times for the conversation may be

i. During care plan/family conference

ii. MDS schedule - 5-day/14-day/30-day/60-day/ quarterly

iii. In assisted living - resident/responsible representative conference/6-month review

iv. Following a significant change in condition

B. Element Two: Create an Advance Directive, along with the individual, that identifies a healthcare agent and stated goals of care

a) Advance care planning means the individual has determined what his/her personal goals, values, religious, cultural beliefs are and what defines their quality of life.

b) The conversation will include a discussion of the treatment(s) the individual may want to receive and what treatment to withhold.

c) This document will provide the health care professional and loved ones with valuable information to coordinate goals of care and discuss chronic disease exacerbation and possible palliative treatment at end of life to alleviate pain and suffering.

a. The POLST (Providers Orders for Life Sustaining Treatment) is an approach to end-of-life planning that states patients' wishes about medical treatments they want to receive and emphasizes:

b. advance care planning conversations between patients, health care professionals and loved ones

c. informed shared decision-making between a patient and his/her health care professional about the treatment the patient would like to receive at the end of life; and

d. Ensuring patient treatment wishes are honored. The decisions from these conversations may be documented as actionable medical orders on a POLST form [1-4].

d) A POLST form is an official Provider's Medical Order that ensures patients' treatment wishes are known and will be followed by health care professionals during a medical crisis when the patient cannot speak for him/herself.

e) The POLST form may vary in appearance from state to state, but an officially signed form by a licensed provider will be honored across state lines by all levels of healthcare.

f) Five Wishes: Five Wishes originated in 1996 as a Florida-only document, combining a living will and health care power of attorney in addition to addressing matters of comfort care and spirituality.

g) A national version of Five Wishes was introduced in 1998.It was originally distributed with support from a grant by the Robert Wood Johnson Foundation.

h) An online version called Five Wishes Online (https:// www.agingwithdignity.org/five-wishes/five-wishes-online) was introduced in April 2011, allowing users to complete the document using an online interface or print out a blank version to complete by hand.

i. Wish 1: The Person I Want to Make Care Decisions for Me When I Can't

ii. Wish 2: The Kind of Medical Treatment I Want or Don't Want

iii. Wish 3: How Comfortable I Want to Be

iv. Wish 4: How I Want People to Treat Me

v. Wish 5: What I Want My Loved Ones to Know

i) Be aware of, and carry out all signature and witness requirements

j) Determine where these documents will be readily assessable

a. Advance Care Planning Document

i. Readily accessible to all staff, and health care proxy

b. Advance Directive

i. At home

a. Post on refrigerator where document can be found

ii. In facility

a. Taped to inside cabinet of individual’s kitchen or bathroom for quick access

b. First page in medical record

c. Emergency binder at general nursing station.

Conclusion

Advance care planning is a process of developing a relevant expression of wishes rather than a single consultation or the signing of a legal document. It is an informed consent, and once completed, is a decision-making document that becomes part of a continuing engagement with the individual, caregiver, and health care provider. The goal of this best practice is to facilitate palliative care advance care planning as part of the individual's care across the continuum of care, especially for those with chronic medical conditions. Included in this continuum of care is acute care, post-acute care, assisted living, skilled nursing, and home care.

An informational brochure accompanies this document and is an example of providing individuals the information they need to begin the conversation of advanced care planning with their medical provider/health care representative. The health care provider trained in the ability to communicate will be able to assist the seriously ill individual and/or their representative to articulate their values and goals of treatment. Inviting the health care proxy to be present at the time of this discussion is encouraged, so that they will also have a clear understanding of their loved one's wishes. "Being Mortal" by Atul Gwande is highly recommended reading as an example of the importance of communicating how we want to be cared for at end of life.

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juniperpublishers-dentistry · 5 years ago

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3D Printing Role in Oral and Maxillofacial Surgery Current and Future Trend- Juniper Publishers

AbstractThe application of three-dimensional (3D) printing enables virtual simulation surgery with tremendous details. Data is usually obtained from detailed computed tomography (CT) scan and the creation of material objects from digital images by depositing layers into 3D structures. That can be used for training, education, surgical planning and prosthetic reconstruction. We report on two patients with complex reconstructive option in maxillofacial surgery where 3D technology was utilized to analyze the tumor size, location, and extension more precisely which drastically aids in better preoperative planning Another case was for fabrication of custom 3D pure titanium TMJ joint for reconstruction with optimal fit and function.Keywords: 3D Printing; Personalized medicine; Personalized surgery; Virtual surgery; Customized prosthesis; Medical models; Custom TMJ; Titanium TMJ implant; Scleroderma

Introduction

Medical applications for 3-D printing are expanding rapidly and are expected to revolutionize health care. 3-D printing is currently $700 million industry with only 11 million (1.6%) being invested in medical applications. In the next 10 years it is expected that 3-D printing will grow to $8.9 billion industry and 1.9 (26%) billion is projected to be spend in medical applications.Medical uses for 3-D printing can be categorized into three segments.

Bioprinting tissue and organ;

Creation of customized prosthetics, implantable devices and medical models

Pharmaceutical drug dosage forms delivery and discovery.

Most reconstructive surgeons are familiar with Charles Hull invention of 3-D printing “stereo lithography” in the early 1980’s. 3-D printing has since evolved and been applied in medicine since the early 2000s. The first applications were used in dental implants and custom prosthetic devices. Since then it’s applications have significantly grown and most recent published reviews describe the use of 3-D printing to produce bone, ears, trachea, blood vessels, tissue organs as well as novel dosage form for pharmaceuticals by personalizing drug printing fabrication at point of care while taking into account patient age, gender, race and clinic response.In this article we will focus on creation of customize prosthesis, implantable devices and anatomical models within the oral maxillofacial surgery practice [1].

Case 1 - Custom 3D pure titanium TMJ prosthesis

64 year old female with history of scleroderma has developed a spontaneous pathologic fracture of her mandibular angle bilaterally over 3 years ago. As a result, she developed significant anterior open bite (Figure 1) with inability to chew food requiring parental feeding for nutrition. She has seen multiple surgeons within the US who have not been able to assist her in her reconstructive needs due to the complexity and surgical limitations. After CT evaluation and virtually ideal occlusion alignment patient seem to have had significant bone resorption bilaterally. Patient also does not have enough proximal condylar head to allow any fixation. Additionally, a stock Total TMJ prosthesis would not be able to reach the distal segment of the mandible bilaterally after it is properly alignment (Figure 2). The only option left is to create custom 3D temporomandibular joint replacement pure titanium, as she exhibited sensitivity to nickel (Figure 3). Currently Biomet custom joint are not FDA approved in the United States however though the compassionate use program we have been able to secure approval from the FDA to custom make the implant to this the patient. Without the 3D printing option for the custom prostheses this patient would continue to suffer and live a life with significant compromised quality of life [2].

Case 2- Custom 3D Titanium Crib

34 year old male with destructive lesion in mandible that was identified as a myxoma after biopsy of his right mandible. Surgical plan was made to undergo partial mandibulectomy with adequate margin. After conversation with the patient we decided to reconstruct with custom 3D printing titanium plate with crib containment. This allows for corticocancellous bone graft from anterior iliac crest with bone marrow aspirate concentrate (BMAC) and platelet rich plasma (PRP). BMAC is a minimally invasive procedure used to collect bone marrow from the patient’s own body (autologous) and concentrates it to the optimal level while keeping all cell types, including adult stem cells, mesenchymal cells and bone morphogenic protein signal. While PRP acts are a stimulator for bone and soft tissue healing via several growth factures enhancing bone maturity and consolidation [3]. This 3D plate allows the patient to obtain optimal cosmetic outcome as we are able mimic the pre-existing contours, width and height of bone making dental implant rehabilitation easier and more predictable (Figure 4-7).

Conclusion

Despite advances 3-D printing there are significant barriers and controversies. Some of which are unrealistic expectation in particularly regarding tissue/organ printed, safety and security issues, and regulatory approvals. Regardless of the challenges 3-D printing is expected to play an important role in the trend towards personalized medicine and revolutionize healthcare. It is through the vision and collaborative support that allows us to service these complex cases.

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juniperpublishers-crdoj · 5 years ago

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GPR119 and GPR131: Functional Difference?

Abstract

Recently, two strategies are generally applied in clinical practice to treat diabetes, namely, glucagon like peptide-1 (GLP-1) analogs and inhibitors of the enzyme dipeptidylpeptidase-IV (DPP-4) that degrades both GLP-1 and glucose dependent insulin tropic polypeptide (GIP). Physiologically, after food ingestion, enteroendocrine cells in the intestinal mucosa may release the incretins, including GLP-1 and GIP, that can stimulate insulin secretion from endocrine pancreas and thereby decrease blood glucose. GLP-1 is produced and released mainly by L-cells located in the distal ileum while GIP is secreted by enteroendocrine K-cells in the proximal gut. However, GIP is not focused in clinics because diabetic patients are mostly GIP resistant. Therefore, development of agent(s) that may enhance GLP-1 pathway received increasing attentions in recent.

Many G protein-coupled receptors (GPCRs) expressed in pancreatic islet and GLP-1 is known to be released in response to activation of two GPCRs, GPCR119 (GPR119) and GPCR 131 (GPR131). Physiologically, GPR119 regulates fatty acid while GPR131 also named as TGR5 is mainly activated by bile acid. Both receptors possess the ability to induce GLP-1 secretion and alleviate diabetes and obesity in animal studies. Interestingly, both receptors coupled Gs protein to activate cAMP signaling pathway. However, many functional variations are observed between GPR119 and GPR131. Therefore, clarification of the difference may help the reduction of adverse effect(s) during development of agent(s).

Herein, we cited the published reports showing the effects of GPR119 or GPR131 activation to conduct the difference between them. Also, we mentioned our opinions to call the attention(s) for avoiding the possible side effects during activation of each receptor.

Keywords: GPR119; GPR131; GLP-1; Diabetes; Obesity

Introduction

Diabetes mellitus (DM) is known as metabolic disorders showing hyperglycemia and hyperlipidemia due to the dysfunction of pancreatic islets [1]. The prevalence of DM in clinics is markedly increased and it will reach approximately 439 million in 2030 [2]. Generally, DM is mentioned to include two main subtypes, Type 1 (Insulin dependent diabetes) and Type 2 (Non-insulin dependent diabetes) subtypes, in addition to others. Clinically, type 2 DM (T2DM) characterized by insulin resistance in addition to hyperglycemia and/or hyperlipidemia is widely considered as metabolic disorder [3]. Many factors, such as reduced insulin secretion from pancreatic dysfunction, inadequate hepatic glucose production and peripheral insulin resistance, are introduced to involve in the development of T2DM [4]. Therefore, therapeutic approaches have been the hot projects to develop critically.

After food ingestion, enteroendocrine cells in the intestinal mucosa may release the hormones that can stimulate insulin secretion from endocrine pancreas and thereby lower blood glucose; known as incretin effect [5]. Two types of incretins were identified in human, including glucose dependent insulinotropic polypeptide (GIP) and glucagon like peptide-1 (GLP-1). Physiologically, GLP-1 is produced and released mainly by L-cells located in the distal ileum while GIP is secreted by enteroendocrine K-cells in the proximal gut [6]. In recent, GLP-1 has become a new target for therapeutics of T2DM due to its insulinotropic activity [5]. However, GIP is not focused in clinics because patients with T2DM are mostly GIP resistant [7]. Two strategies have then been applied in clinical practice to treat T2DM, namely, GLP-1 analogs and inhibitors of the enzyme dipeptidylpeptidase-IV (DPP-4) that degrades both GLP-1 and GIP [5]. However, clinical practice meets some limitations, such as GLP-1 analogs shall be treated by injection only, and the effectiveness of DPP-4 inhibitors is mild [8]. Therefore, development of agent(s) that may enhance GLP-1 pathway received increasing attentions in recent.

Many G protein-coupled receptors (GPCRs) expressed in pancreatic islet and GLP-1 is known to be released in response to activation of two GPCRs, GPCR119 (GPR119) and GPCR 131 (GPR131), in addition to others such as GPR40 and GPR120. All of these GPCRs with a similar genomic sequence were coupled to G-protein while Taq Man Gene Expression Assays showed GPR119, mouse- Mm00731497, rat - Rn01648212 and GPR131, mouse -Mm04212121, rat - Rn00710093 for gene expression [9].

The GPR119 receptor for regulation of fatty acid was described as a class 1 (rhodopsin-type) orphan G-proteincoupled receptor [10]. The oleoylethanolamide (OEA) is identified as a potential endogenous ligand for GPR119 receptor that has been suggested as novel target for treatment of diabetes and obesity [11]. Activation of GPR119 by agonists showed an elevation of cAMP levels to stimulate GLP-1 secretion from cells. Similarly, activation of GPR 131 can result in same changes. GPR131 also named as Takeda G-protein-coupled receptor 5 (TGR5) or G-protein-coupled bile acid receptor 1 (GPBAR1) to bind bile acid in main [12]. Activation of GPR131 (TGR5) receptor may also promote the secretion of GLP-1 [13]. Knockout of GPR131 (TGR5) decreases energy expenditure and elicits obesity in female mice [14]. Similar to GPR119, GPR131 (TGR5) is also suggested as an attractive target for the treatment of diabetes and obesity [15]. However, functional difference between GPR119 and GPR131 remained obscure.

In intestinal L-cells, both GPR119 and GPR131 participated in GLP-1 secretion through cAMP signaling pathway. Gene of GPR119 or GPR131 from L-cells can be identified in pancreatic α-cell line while GLP-1 secretion was stimulated by the activation of GPR131 (TGR5) but not by GPR119 [9]. Selective secretion of GLP-1 by GPR131 (TGR5) agonist for glucose homeostasis has also been demonstrated [16]. Merit of GPR131 (TGR5) activation from basic research to clinical applications has been summarized [17]. However, GPR131 (TGR5) agonist may cause gallbladder filling in mice [18] probably due to the co-released peptide YY (PYY) while GLP-1 and PYY have been shown to act synergistically to slow gastric emptying and inhibit food intake [19]. Interestingly, the recently developed new compound OL3 is a low-absorbed TGR5 agonist that lowers blood glucose without inducing gallbladder filling [20]. But, similar effect from GPR119 agonist is still not reported.

For obesity, GPR119 activation by agonist OEA showed the merits in reduction of feeding behavior [21]. Similar results were observed during activation of GPR131 [15]. The effect on obesity is easily to link with GLP-1 because GLP-1 is known to cause gastric deceleration and increase satiety [22]. However, it has been reported that OEA is able to suppress the food intake to a similar level in both wild-type and GPR119-knockout mice [23]. Additionally, homology clustering analysis showed the closest relatives of GPR119 to be the cannabinoid receptors [24]. Otherwise, GPR131 agonist bile acids induce energy expenditure by promoting intracellular thyroid hormone activation [25]. Therefore, treatment of obesity by GPR119 agonist seems not the same as that induced by GPR131 (TGR5) agonist.

High mRNA levels of GPR131 (TGR5) were detected in human many organs with a gene located on chromosome position 2q35 and the open reading frame of 993 base pairs, encoding 330 amino acids [26]. Recently, functions of GPR131 (TGR5) have been extended to more than the metabolic regulation and included the inflammatory response, cancer and liver regeneration Guo et al. [27]. Therefore, different to GPR119, the agonist(s) of GPR131 (TGR5) will be developed to involve in many functional regulations in the future.

Conclusion

GPR119 and GPR131 (TGR5) have been identified in metabolic regulation for a long time. However, both receptors possess different role in another functional regulation. Development of the ligand(s) both agonist(s) and/or antagonist(s) shall be concerned the difference to avoid the adverse effect(s).

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journalofsportsmedicine · 4 years ago

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The Changes of Homocysteine Serum Level and Body Mass Index of Overweight Young Women after Eight Weeks of Pilates Exercise | Juniper Publishers

Juniper Publishers- Journal of Physical Fitness, Medicine & Treatment in Sports

Introduction

The increasing trend of obesity is one of the most major public health concern and health related issues [1]. According to an epidemiologic study conducted in 199 countries around the world in 2008, 1.46 billion adults were overweight. The scope of the outbreak obesity is varying substantially between nations, largest rise in Oceania and the lowest trend was estimated in India [1]. In the USA, 34 percent of adults aged above 20 suffered from obesity in 2008. in addition, East Mediterranean countries estimated an increased by over 20% [2]. A review study indicated that obesity reached an alarming level in all ages of the East Mediterranean countries. the spread development of obesity has been ranged from 25 to 81.9 percent among adults [3]. In the same vein, a research of systemic review has conducted a study regarding the effect of BMI on cardiovascular diseases which indicated that each 5 unit raised in BMI increases the chances of heart problems by 29 percent [4].

Homocysteine is considered to be associated with microalbuminuria, which is a serious indicator of the risk factor of future cardiovascular disease. Homocysteine is also known to mediate heart attack, whenever its level raises, the risk of arterial artery problems such as atherosclerosis would increase [5,6]. It is a phosphor containing amino acid which is formed during methionine metabolism [7]. Epidemiology studies revealed that high levels of homocysteine in blood plasma could be a risk factor for cardiovascular, heart attack and peripheral vascular diseases and it causes atherosclerosis through three ways of intra-arterial wall damage, interference in the blood-clotting factors, and the oxidation of low density lipoprotein [8]. The prevalence of hyper homo cysteinemia is estimated to be about 5 percent in the general population and 13 to 14 percent among patients with symptoms of atherosclerosis. However, these estimates are based on a cut above the 90th or 95th percentile of total homocysteine distribution in the general population [7,9]. Various surveys have shown that levels of this amino acid are at a high level in obese or overweight as well as those who do not have regular exercise. Yet, there is debate on the effects of physical activity on this factor [8].

The effect of stair climbing with moderate intensity exercise on cardio-respiratory Fitness, blood lipids, and serum homocysteine were examined among sedentary young women. This study showed that these exercises can favorably make some changes in homocysteine cardiovascular risk factors and blood lipids profiles of inactive young women [10]. Vincent also reported that the decrease in homocysteine level caused by resistive exercises among inactive elderly people in aged of 60_80 [11]. Moreover, no change in homocysteine level had been reported among 6 inactive men who participated in a walking activity with low intensity exercise [12]. In addition, another study showed that homocysteine concentration does not lead to significant changes in sub maximal exercise. Hence, the training intensity, sex and age are the factors affecting the above index [13]. Nevertheless, level of participation and different levels of interest in continuing exercise are the most important keys to reduce weight in overweight population although it may be exhausting. Therefore, doing sport activity such as Pilates can have a prominent role in increasing their interest in exercise (Table 1).

A collection of specialized sport activities affecting body and mind by using special equipment is called Pilates. It can increase power or endurance of whole body and even target the deepest muscles. Pilates exercises include effort for one’s mental concentration on body muscles and how they function. Pilates exercise is named after its founder, Joseph Pilates, who developed a series of exercises in the 1920s to encourage physical and mental conditioning [14]. Strength, more body balanced, flexibility and core stability are emphasized in Pilates exercise to control of posture, movement, and breathing [15]. A survey in this domain showed that Pilates exercise among untrained women for 8 weeks leads to decreased serum creatine kinase, LDL, TG and Cholesterol. They also reported that Pilates increases HDL among these people [16]. Moreover, it has been investigated the effect of 24 weeks of Pilates exercise among elderly women and reported that this activity culminates in increased bone strength and decreased body lipid mass [17]. Nonetheless, there is no conclusive result about the effect of Pilates on homocysteine level and it has not been clearly identified whether the intensity employed in Pilates exercises has any effect on the amount of homocysteine of overweight women. Hence, the present research designed to investigate the effect of 8 weeks of Pilates exercise on protein and mRNA level of serum homocysteine and BMI among overweight young women (Table 2).

Participation and Method

Subject recruitment

The study involved 20 overweight women whose BMI was above 25 from “Tandorosti gym” located in Tehran, Iran. The volunteers had the required features for participation in this project, including; no cardiovascular disease, not consuming special medicine, no smoking, not having a regular exercise. Physiologic characteristics of volunteers including height, weight, age, BMI, heart rate, systolic and diastolic blood pressure were recorded. Participations were well informed about the study prior to the experiment and written consent was obtained from them. A double-blind study method was applied, and the participants were randomly assigned into 2 groups of 10; (1) control and (2) Pilates exercise groups. Pilates exercise group performed Pilates exercise 3 times per week for 8 weeks. It contains movements which engaged abdominal muscles, hips, waist, legs and shoulder belt and it was performed on a mat without any specific equipment in three conditions of sitting, standing and lying down. During this period, the control group was also barred from participating in regular physical activity. Prior the exercise protocol and at the end of eighth weeks, BMI was recorded, and blood sample was collected to measure protein or mRNA level of homocysteine. All procedures involving experiments were carried out in strict accordance of the United States Institute of Research guidelines and approved by the Medical Centre Board of Tehran University (Table 3).

Method of body mass index measurement

The body mass index was calculated by measuring height in meters, weight in kilograms and putting them in the following formula:

Height (m)2 / weight (in kilograms) = body mass index

Blood Sample Collection

Blood samples were collected at 24 hours before the pilates exercise and 24 hours after the last exercise session of 8 weeks training in fasting state through the elbow antecubital vein of all subjects for measurement of homocysteine mRNA and protein expressions.

Measurement of Serum Homocysteine protein

The level of serum homocysteine protein was measured by homocysteine measurement Elisa kit (Axis-shield diagonistmade in Germany). ELISA was performed according to the manufacturer’s instructions. The absorbance for homocysteine was determined by using a microplate reader (iMark; Bio - Rad, Hercules, CA, USA) at a wavelength of 450 nm. A set of standard serial dilutions of known concentrations of homocysteine were provided by the manufacturer and were used to construct a standard curve in order to determine the homocysteine levels. Homocysteine which was attached to protein was changed to free homocysteine and then it was changed to S-adenosyl L homocysteine (Figure 1).

RNA purification and mRNA Expression Analysis by Real Time PCR (qPCR)

QIA amp RNA Blood Mini Kit (Qiagen, Germany) was used to isolate total cellular RNA from fresh whole blood. The concentration and purification of isolated RNA were evaluated by 260/280 UV absorption ratios (Gene Quant 1300, UK). Specific amplification fragments of DNA/RNA, Two-step Real time qPCR (quantitative Polymerase Chain Reaction) technique was used to calculate gene expression during the PCR amplification process with application of TaqMan reagent. This method was able to detect small differences between samples compared to other methods [18]. All reagents including probes and primers were obtained from Applied Biosystems, USA. TaqMan probe (known as fluorogenic 5′ nuclease) was chosen to perform qPCR. This probe has a sensitivity of 100% and a specificity of 96.67% [19] and is capable of detecting as few as 50 copies of RNA/ml and as low as 5-10 molecules [20].

Primers were designed by the same company for homocysteine targets

MRT: Hs01090026_m1; Lot no: 4351372, amplifies 61 bp segment from the whole mRNA length of 10558 bp. Beta Actin and GAPDH were used as reference genes. All amplification experiments were done in 3 biological replicates. Amplification program include 15 minutes at 48 °C (reverse transcriptase), 10 minutes at 95 °C activation of ampli Taq gold DNA polymerase, denaturation at 95 °C for 15 second and annealing at 60 °C for 1 minute. Denaturation and annealing steps were performed for 40 cycles. Step-One Plus real time PCR machine, TaqMan Fast Advanced Master Mix and assays were purchased from Applied Biosystems, USA. The fold changes of each target per average of ACTB were calculated and considered as mRNA expression levels of the target gene. Data was analyzed according to Comparative Ct (2−ΔΔCt) method, where amplification of the target and the reference genes were measured in the sample and reference [18].

Statistical method

In this study, the Kolmogorov - Smirnov was used for the normal distribution of data as well as t-test (paired) for comparing pre-test and post-test stages within each group. Independent t-test was used for between-group comparison of indices in pre- and post-test stages. Pearson’s coefficient and linear regression were used to examine the association between indices. All data in significance level of 05.0 ≥P were examined using SPSS version 21 (Chicago, America) (Figure 2).

Results

BMI

Findings of this research show that 8 weeks Pilates exercise leads to a significant decreased of BMI among overweight women (P=0.001).

Homocysteine protein expression quantification

The findings indicate that homocysteine protein expression levels were significantly increased after 8 weeks Pilates exercise among overweight women compared to control group or pretest (P=0.001).

Homocysteine mRNA expression level

The results also indicate that homocysteine mRNA expression levels were significantly increased after 8 weeks Pilates exercise among overweight women compared to control group or pretest (P=0.0001). However, there was no significant change in the amount of these indices in the control group (P≤0.05). Also, there was no significant difference between the Pilates exercise group and the control group in pre-test stage regarding the homocysteine (P=0.004) and BMI (P=0.001) level.

Discussion and Conclusion

This study reports a significant change in homocysteine level among overweight women after eight weeks of Pilates exercise. Although the comparison of two groups in the pre and post-test stages showed that the Pilates group had a lower level of homocysteine in the post-test stage compare to pre-test or control group, there was no significant change in the control group (P=0.004). A study investigated the effect of aerobic exercise on homocysteine level among overweight women which suffered from polycystic ovary syndrome showed that a 6-week aerobic exercise caused a significant decrease in the level of homocysteine [21]. In addition, similar study reported findings in young untrained women [10]. On the other hand, Di Santolo et al, investigated the relationship between recreational sports and homocysteine level among young women and found no significant change in the level of homocysteine [22].

The effect of two types of exercise on homocysteine level have been examined, as well as other cardiovascular diseases risk factors. It was revealed that a 12-week aerobic and resistive exercise does not make any significant change on the level of homocysteine, lipid profile and maximal aerobic power [23]. Furthermore, Bambaeichi et al, investigated the effect of increasing aerobic exercise on homocysteine level in young men and reported that the level of homocysteine did not have a significant change in the experimental group [24]. Various factors such as; intensity, type, or length of activity, gender or age of the participants have been considered to be effective on the divergent findings of the researchers. However, the effect of exercise on homocysteine can be explored from different aspects. During doing physical activity or recovery, amino acids more likely play an important role in anaplerotic functions sustaining the whole metabolic apparatus, as well as synthesis of other proteins, such as carnitine or melatonine. Increasing the energyreleasing reactions in the muscles; more induction in catabolism of amino acids such as methionine would be increasing. Also, regular sport activities increase renovation and repair muscle tissue by metabolic reactions require.

Since methionine is an amino utilized in protein formation which starting material for numerous biochemical molecules. On the other hand, homocysteine is one of the critical biochemical junctures between methionine metabolism and the biosynthesis of the amino acids’ cysteine and taurine, therefore decreasing methionine leads to homocysteine reduction [25]. There is this possibility that Pilates assists in decreasing homocysteine level and changing homocysteine to methionine leads to prevents its accumulation in the blood through increasing absorption of vitamins which are effective in homocysteine cycle especially group B vitamins (which decrease homocysteine during its menatbolosm) [26]. In addition, reduction of oxidative stress indices of regular exercise can also reduce levels of homocysteine [27]. Also, it is likely that insulin has an impact on homocysteine metabolism and its level through affecting activities of CBS, MTHFR enzymes, Systationine Gamma-lyase and betaine homocysteine methyltransferase [28]. Moreover, it seems that sport activities affect insulin role in decreasing homocysteine by reducing CRP and resistance to insulin [29].

The relationship between body composition and homocysteine is also of great importance. Although in the present study no significant relationship was found between them, BMI totally decreased with homocysteine reduction in Pilates group. The relationship between body composition and homocysteine have been investigated among Korean men and women in aged of 30 to 55 which had high homocysteine level. Findings of their study revealed that increase in total body fat content and decrease in LBM have a significant relationship with increased homocysteine. They reported that decrease in LBM is a suitable index for predicting homocysteine increase in different people [30]. Our findings showed that 8 weeks of Pilates exercise causes a significant decrease in BMI level among young overweight women. However, this change was not considered significant in the control group. In this domain, some consistent and contradictory studies can be mentioned. A 4 week of Pilates exercise on body composition have assessed in young girls. They showed that BMI level, waist size and blood pressure significantly decreased after 4 weeks of Pilates exercise [31]. Another study also explored the effect of two sport activities on indices of cardiovascular patients’ body composition. They declared that BMI level and waist-to-hip ratio significantly decreased compared to pre-test [23]. Alternatively, 5-week Pilates exercise on trunk power, endurance and flexibility among low active adult women had no any significant change in BMI level [32].

Furthermore, no significant change also caused after doing 8 weeks Pilates exercise on fitness indices among low-active women [33]. Similarly, Cakmakçi et al found no significant change of BMI as a result of 8 weeks of Pilates exercise among obese women [34]. It seems as if the amount of subcutaneous fat, exercise intensity and the amount of nutrition control of the participants are among the influential factors with contradictory findings. Different factors can be pointed out regarding the effective mechanisms on Pilates exercise. A negative relationship was reported between body composition and the level of VO2max [35]. It appears that sport activity can be effective in decreasing suitable BMI through increasing Lipoprotein Lipaz enzyme, fat tissue, blood flow, active muscles flow, and expanding the activity of hormones effective in metabolism [36-38]. In the similar vein, it has been reported a significant increase in aerobic power as a result of 8 weeks of Pilates exercise among obese women [39]. In conclusion, findings of this study showed that eight weeks of aerobic exercise significantly decreased the amount of homocysteine serum and body mass index in overweight young women. However, despite BMI level changes caused by Pilates, it seems that BMI decrease is not the major and effective factor on homocysteine decrease among overweight women. Probably there are other factors in this domain which have not been measured in this research. It is considered as the limitation of this study and should be taken into consideration in future researches.

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juniperpublisherscasestudies · 3 years ago

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Juniper Publishers- Open Access Journal of Case Studies

Estimation of Superficial Venous Reflux with Duplex Ultrasound and Foot Volumetry

Authored by Helene Zachrisson

Abstract

Objective: To evaluate quantitative duplex ultrasound (DUS) parameters of reflux in patients with isolated great saphenous vein insufficiency.

Methods: 20 limbs were studied. DUS derived reflux time (RT, sec), peak reflux velocity (PRV, cm/s) and reflux volume flow (ml/min) were evaluated and related to expelled volume (EV, ml) and half refilling time (T50, sec) measured by water-based foot volumetry with and without compression of superficial veins.

Results: Reflux volume flow correlated significantly to all hemodynamic parameters assessed by foot volumetry, i.e., EV (p = 0.003), ΔEV (p = 0.006), T50 (p = 0.004) and ΔT50 (p = 0.011). PRV displayed a weaker correlation to foot volumetry parameters EV (p = 0.027) and T50 (p = 0.008). No significant correlation was found between RT and foot volumetry.

Conclusion: These results indicate that reflux volume flow may be a potential parameter in future attempts to quantify reflux using DUS in patients with isolated great saphenous vein insufficiency.

Keywords: Venous insufficiency; Foot volumetry; Duplex ultrasound; Pathophysiology; Anatomical distribution

Introduction

Chronic venous insufficiency is a common condition with clinical signs ranging from minor telangiectasias, varicose veins, edema to more severe stages with skin manifestations as eczema, lipodermatosclerosis and venous ulcers [1-3]. The diagnosis relies on physical examination (C of the CEAP classification, “Clinical Etiology Anatomy Pathophysiology”) [4] as well as noninvasive testing [3,5]. Duplex ultrasound (DUS) is considered to be gold standard and provides diagnostic information about the anatomical distribution of the disease [3,6]. A retrograde flow (reflux time, RT) of more than 0.5 seconds is generally used to define the presence of reflux [6]. However, individual RT does not seem to reflect the magnitude of reflux and the correlation between severity of disease or hemodynamic state and RT is limited [7]. Based on this it has been suggested that RT may be used for detection of reflux but other DUS derived parameters are needed for quantifying venous insufficiency [7,8]. Previous attempts to quantify reflux using DUS has involved peak reflux velocity (m/s), calculated reflux volume flow (ml/min) and reflux volume (ml) [7], however, the optimal method for quantifying reflux by DUS is still unclear [9]. Quantitative information of global venous hemodynamics can be derived from plethysmographic measurements such as strain gauge, photo, air as well as foot volumetry [10-12]. We have shown that it is possible to predict post-interventional outcome in Great Saphenous Vein Incompetence using strain-gauge plethysmography [10].

Foot volumetry may provide accurate information on the magnitude of global venous reflux as well as correlate to C in the CEAP classification [4,12]. The aim of the study was to evaluate DUS derived reflux parameters in patients with isolated great saphenous vein insufficiency (GSV) compared to quantifying plethysmographic measurements using foot volumetry.

Material and Methods

46 consecutive patients referred to Department of Clinical Physiology, Linköping University Hospital for evaluation of venous insufficiency in the lower limb were evaluated according to the study protocol. All patients were investigated with both Duplex ultrasound (DUS) and water-based foot volumetry. Six patients presented with small saphenous vein (SSV) insufficiency. Patients with mixed and/or isolated SSV insufficiency were excluded from the study. Finally, eighteen patients with isolated great saphenous vein (GSV) insufficiency (13 women and 5 men, mean age 59 years, range 40 – 90 years), two with bilateral GSV insufficiency (20 legs) were included in the study. Demographical and clinical data (C in CEAP) [4] is presented in Table 1. The study was approved by the regional ethical review board in Linköping, Sweden, and written informed consent was provided by each participant.

Duplex ultrasound

DUS examinations were performed with ACUSON S2000 system (Siemens Medical Solutions, Malvern, PA, USA) with 9 and 18MHz transducers. The 9MHz transducer was used for assessment of reflux. Patients were examined in the sitting position and superficial (saphenous veins and tributaries), perforator and deep veins (femoral, common femoral, deep femoral, popliteal, and calf veins) were scanned in both longitudinal and transverse planes. A distal manual compression was used to determine the valvular integrity. Normal veins were defined as veins with no reflux, normal reflux time (RT, duration < 0.5sec), or a very short reflux area (between 2 or 3 valves). If a pathologic reflux was detected, i.e., RT > 0.5sec, spectral Doppler measurements was performed along the vein in the longitudinal plane at 60o angle. Measurements were made in the insufficient GSV at the distal thigh level. At least three measurements were conducted at each point (proximal, mid and distal part on the thigh) and the mean value was used in the calculations. The anatomical extent of reflux was carefully noted, and no perforator reflux was noted on the calf or thigh level. Spectral flow velocity and vessel diameter was measured where the vein was straight, and turbulent areas were disregarded. The magnitude of reflux was quantified in several ways. i.e., RT (sec), peak reflux velocity (PRV, m/s) and time average velocity (TAV, m/s) during the first second of reflux. The vessel lumen was considered circular and the cross-sectional area was calculated as the area of a circle (A = Πr2 ) . Thus, reflux volume flow (ml/min) was calculated according to the following:

reflux volume flow (ml / min) = TAV (m / s) × A (cm2 ) × 60.

Hence, three DUS derived parameters were studied: RT (sec), PRV (m/s) and reflux volume flow (ml/min).

Foot volumetry

Global venous hemodynamics were evaluated with water- based foot volumetry [13]. The foot volumeter consisted of an open, water-filled box, which enabled measurements of volume changes during exercise [13]. Patients performed 20 knee bends at the rate of one every two seconds, and after the exercise phase was completed the patients remained completely still during the refilling phase. Expelled volume (EV, ml), and the time taken in seconds for 50% (T50) of the venous volume to be refilled was evaluated both with and without compression of superficial veins either above or below knee. A 10cm wide tourniquet was inflated to 60mmHg to achieve superficial compression.

Statistics

Values are expressed as mean ± SD unless otherwise stated. Spearman correlation coefficient (rho) were calculated for non-parametric linear association between different DUS parameters and foot volumetry measurements. p-values < 0.05 were considered significant. Statistical analyses were carried out using SPSS 24.0 for Windows (Armonk, NY: IBM Corp.).

Results

Isolated great saphenous vein (GSV) insufficiency was detected in 20 legs. DUS measurements displayed a mean vessel diameter of 4.2 ± 2.0mm. RT was 3.3 ± 1.3sec, PRV 45 ± 19cm/s, TAV 13.0 ± 6.9cm/s, and calculated reflux volume flow 128 ± 114ml/ min. EV was 13.6 ± 7.9ml, and T50 was 7.3 ± 6.4sec during foot volumetry without superficial compression. EV increased to 20.1 �� 8.1ml, and T50 increased to 13.0 ± 6.2sec after superficial occlusion. The calculated difference between foot volumetry measurements with and without superficial occlusion, i.e., ΔEV and ΔT50 were 6.5 ± 9.6ml and 5.8 ± 5.0sec respectively.

Table 2 shows the correlation between different DUS derived parameters and foot volumetry measurements. Reflux volume flow correlated negatively with foot volumetry parameters EV (rho = -0.626, p = 0.003) and T50 (rho = -0.613, p = 0.004). Reflux volume flow correlated positively with ΔEV (rho = 0.588, p = 0.006) and ΔT50 (rho = 0.554, p = 0.011). A negative correlation was also found between PRV and EV (rho = -0.493, p = 0.027) as well with T50 (rho = -0.574, p = 0.008). No correlation was detected between RT and foot volumetry parameters. Figure 1 shows reflux volume flow divided into three groups, mild refux (< 30ml/min), moderate reflux (30-100ml/min) and severe reflux (> 100ml/ min). This classification demonstrated a correlation with EV (rho = -0.519, p = 0.019), ΔEV (rho = 0.504, p = 0.023), T50 (rho = -0.589, p = 0.006) and ΔT50 (rho = 0.460, p = 0.041).

Discussion

This study was designed to evaluate different DUS parameters describing reflux in patients with isolated GSV and relate them to alterations in global venous hemodynamics based on water-based foot volumetry. The main findings were that particularly DUS derived reflux volume flow significantly related to global hemodynamic measurements during foot volumetry. Thus, the present study supports previous findings that reflux volume flow may be a potential parameter in future attempts to quantify reflux using DUS in patients with isolated GSV insufficiency.

DUS is a well-established method in the diagnosis of CVI and is able to measure several reflux components [6]. There has been numerous attempts to use one or more of these components as indexes for global reflux severity, although the most useful and reliable parameter for quantification of superficial reflux is still under debate. On the other hand, dynamic measurements of volume changes with plethysmographic methods may provide accurate quantitative information about whole limb venous haemodynamics and abnormalities in the reflux phase [10-12]. Foot volumetry was used in this study and previous findings suggest that it may provide accurate information on the magnitude of global venous reflux as well as correlate both to the clinical severity of the disease and the ambulatory venous pressure [12,14]. A further advantage with foot volumetry is that the method uses a dynamic and quite heavy situation, kneeling, which is supposed to simulate walking. In order to investigate the relation between DUS parameters and global reflux occlusion of the superficial system was accomplished with a tourniquet to evaluate the magnitude of superficial reflux in each patient.

RT was initially viewed as potential quantitative parameter and earlier studies found that patients with ulceration demonstrated a longer total mean RT than those without ulcers [15]. In the present study, no association was found between RT and any parameter of global reflux derived from foot volumetry. This is consistent with more recent studies who have shown RT to be a poor quantifier of individual reflux as well as an inadequate parameter to discriminate between disease severity [7,8,16]. Taken together, RT may primary be viewed as a qualitative parameter. Other parameters, such as, PRV has been found to provide a better quantitative evaluation compared to RT, e.g., PRV > 30cm/s has in previous studies been suggested as a risk factor for venous ulceration and patients with skin changes presented with significant higher PRV [17,18]. In line with this we found that PRV was associated to foot volumetric parameters EV as well as T50.

The parameters derived from DUS that showed best correlation with foot volumetry measurements was volume flow. Reflux volume flow was related to EV and T50 at baseline as well as to changes in global reflux after occlusion of the superficial veins evaluated by ΔEV and ΔT50. Assessment of retrograde flow volume has previously shown that reflux greater than 10ml/sec is related to a high incidence of skin changes [19] and volume flow also seems to correlate with the hemodynamic state measured with air plethysmography [7,8]. Although the association to clinical severity is still uncertain [7] our result suggest that reflux volume flow may provide a reflection of the magnitude of venous incompetence. In an attempt to classify the results of reflux volume flow in mild, moderate and severe reflux a correlation was found with all volumetric parameters, i.e., EV, ΔEV, T50 and ΔT50, when volume flow was divided into < 30ml/min (mild), 30-100ml/min (moderate) and > 100ml/min (severe). This characterization seems to agree with previous studies showing that normal values of EV and T50 appears to center around 16ml and 20sec respectively, although higher age may be associated with lower EV and T50 [14]. Based on the proposed groups, a gradual decrease in both EV and T50 can be seen as reflux volume flow was classified as moderate or severe. Correspondingly, no obvious improvement at group level was found in ΔEV and ΔT50 when volume flow was classified as mild reflux. However, in both moderate and severe reflux, ΔEV and ΔT50 displayed a gradual improvement. This kind of semi classification may be helpful in early diagnostics as well as postoperative follow up.

The purpose of this study was to compare different segmental DUS derived parameters in order to understand which of these parameters best reflected global hemodynamic changes in patients with GSV insufficiency. Based on this, reflux volume flow seemed to provide the most reliable results. Nonetheless, it should be noticed that the information provided by DUS cannot replace the global evaluation derived plethysmografic investigations, especially in patients with a more complex disease, such as, mixed deep and superficial incompetence.

Limitations

The present material is small and further studies are also needed to evaluate the suggested classification in relation to other parameters of global venous reflux as well as the relation between clinical severity and both DUS and plethysmographic data.

We recently developed for selective occlusion of superficial veins validated by ascending phlebology [10]. and this model must be evaluated in larger studies. Thus, larger populations need to be performed to assess DUS derived reflux volume flow with other parameters of global venous reflux and its correlation to clinical severity.

Conclusion

In the present study reflux volume flow appeared to best reflect the magnitude of venous incompetence. Further, a semi quantification derived from DUS investigation in individual vein segments is suggested by using limits for mild reflux < 30ml/min, moderate reflux 30-100ml/min and severe reflux > 100ml/min. Further and larger studies combining DUS and plethysmographic methods are needed to confirm the results as well as to evaluate the possible benefits of quantify reflux with DUS in the clinical practice.

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dargeereads · 2 years ago

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Melissa Schroeder has revealed the cover for Last Love!

Releasing January 26, 2023 Welcome back to Juniper Springs, home of gay ducks, Nerdvana, and the LOLS. Okay, so here’s the thing. I’m Liv, the normal O’Bryan sister. I have two kids I’m raising on my own, and I even have what people think is a boring job. Going to Vegas with my insane sisters is out of character for me, but I take that chance because of my new job. And let’s face it. I lost my husband over five years ago and I just haven’t…well…there’s been no one. But an altercation with some drunken idiots has me almost falling on my butt, until a set of very strong hands save me. Mason Spencer is younger, beyond sexy, and interested in me. So, I give in for one night. Can’t hurt, right? He goes above and beyond my wildest expectations. The next morning, I say thank you and head back to my life. Until I move in next door to him, and my insane dog Houdini keeps showing up on his doorstep so I can’t even avoid him. Worse, Mason tells me he wants more than that one night stand. Worser, he seems to fit right in with my kids and me and I discover that being with him makes me happier than I’ve been in years. But remember, I’m the normal sister, and there’s nothing normal about an older woman with two kids snatching up a young man like Mason. Of course, I’ve realized that there is nothing normal about the town, so maybe I would fit right in. Author Note: Yep, it’s that time again to jump back into Juniper Springs. Home of Little Old Ladies, the Juniper Springs Express, and a seriously younger man with his mind set on his sexy neighbor. There’s a rescue dog with a mohawk who lives up to his name, and a meddling younger sister determined to help Liv find love.

Photographer: Wander Aguiar Model: Clever Huller Preorder today! Amazon: https://amzn.to/3CazyvE Apple Books: https://apple.co/3gagiHJ Nook: https://bit.ly/3UEBfcS Kobo: https://bit.ly/3ExQFd7 Google Play: https://bit.ly/3g8zvtq Goodreads: https://bit.ly/3tATSlX Meet Melissa Schroeder

From an early age, USA Today Bestselling author Melissa loved to read. First, it was the books her mother read to her including her two favorites, Winnie the Pooh and the Beatrix Potter books. She cut her preteen teeth on Trixie Belden and read and reviewed To Kill a Mockingbird in middle school. It wasn't until she was in college that she tried to write her first stories, which were full of angst and pain, and really not that fun to read or write. After trying several different genres, she found romance in a Linda Howard book. Since her first published book, Grace Under Pressure, Mel has had over 60 short stories, novellas, and novels published. She has written in genres ranging from historical to contemporary to futuristic and has worked with 8 publishers although she handles most of her publishing herself. She is best known for her Harmless and Santini series. After years of following her military husband around the country and world, Mel happily lives with her family in horse and wine country in Northern Virginia. Connect with Melissa Website | www.melissaschroeder.net Goodreads | https://bit.ly/3uiGfbx Amazon | https://amzn.to/3iBJJ6A Facebook | https://bit.ly/3FvVGDT Facebook Group | https://bit.ly/3H6Icj4 Instagram | https://bit.ly/3OY8QMN TikTok | https://bit.ly/3gYmVxm Twitter | https://bit.ly/3OXmIaa Bookbub | https://bit.ly/3uiGeUX Pinterest | https://bit.ly/3FlUWAO Newsletter | https://bit.ly/3FkhHW2

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juniperpublishersmicrobiology · 5 years ago

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Blood Serum Affects Polysaccharide Production and Surface Protein Expression in S. Aureus

Authored by Nazrul Islam

Abstract

Background: S. aureus biofilm serves a major role in pathogenesis. Two of the major components of bacterial biofilm are Polysaccharides intercellular adhesions (PIA) and surface proteins. It is not known how PIA and surface proteins expressions are affected in presence of blood serum. Analyses of surface proteins expressions will provide more effective biomarker discovery that might lead to development of antimicrobial therapeutics to meet the challenges of biofilm-related infections.

Method: Secondary cultures of S. aureus Philips, a biofilm-forming bacterium, were generated by inoculating 1 ml of overnight culture into 50 ml of TSB. Bacteria were cultured at several concentrations of blood serum and found that 12.5% supplemented blood serum provide s similar growth curve as normal TSB (100%). One and 2 D SASPAGE were used to separate proteins and the differentially expressed proteins were identified by nano-LC/MS.

Results: Polysaccharide intercellular adhesions production was significantly increased due to the addition of blood serum in the media. We also identified two serum proteins, apolipoprotein and globulin (Fc and Fab), that remained attached with the membrane fraction of bacterial proteins.

Conclusion: These results have strongly demonstrated that blood serum influences the exopolysaccharide expression in S. aureus.

Keywords: Biofilm; Serum; Staphylococcus aureus; Proteome

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Background

A biofilms are micro Colonies of bacteria adhere to each other and to biotic or biotic surfaces, embedded in an extracellular matrix produced by the sessile bacterial cells [1]. Extracellular matrix (ECM) and ECM proteins in bacterial biofilm play crucial roles in contaminating the agricultural produce starting from the field to the packing. In addition, ECM mediates adhesions protect bacteria from external threats and other stressors of adverse environment. Some of the ECM enzymes hydrolyze macro biomolecules into smaller biomolecules which subsequently is taken up by bacteria [2,3].

Both polysaccharide and protein embedded in extracellular matrix of biofilm play critical roles in biofilm stability Martm- Cereceda et al. (2001); Tsuneda et al. (2003). The polysaccharide intercellular adhesns are the major components (90%) of biofilm. Gutberlet et al. (1997); Gross et al. (2001); Weidenmaier & Peschel (2008); Rupp et al. (1995) [4]. Two types of PIA have been reported based on structure,. PIA type I (typically>80%) is a unique linear beta-1, 6 glucosaminoglycan which is predominantly positively charged. PIA type II (typically<20%) is structurally similar to type I, but contains phosphate and ester-linked succinate, and thus carries a mild negative charge Rupp et al. (1995); Mack et al. (1996). The biofilms are stabilized by the linear structure of these PIAs electrostatic interaction between positively and negatively charged residues Mack et al. (1996). In addition, surface proteins appear to play a critical role in contributing to biofilm stability. For example, nearly all S. aureus clinical isolates possess and express the genes necessary for PIA production (ica-operon, described below), yet many do not form biofilms Fitzpatrick et al. (2005, 2006). This implies that surface proteins may act as additional biofilm stabilizers, possibly cooperating with PIA to mediate intercellular adhesion O'Gara (2007).

In antibiotic therapy, biofilm has been found in 65-80% of the bacterial infections, and is considered refractory to host defenses [4]. Staphylococcus s. aureus, a biofilm forming bacteria, is responsible for severe skin infections to such major diseases as bacteremia, endocarditis and osteomyelitis. Under favorable conditions, S. aureus causes serious complications in devices like implants and catheters by producing biofilms on them [5]. Treatment of such infections becomes even more challenging given that several S. aureus strains show resistance to multiple antibiotics (e.g., methicilin and vancomycin). Extracellular matrix (ECM) proteins in bacterial biofilm play crucial roles in biofilm stability. In addition, ECM mediates adhesins to protect bacteria from external threats and also other stressors under adverse environment.

The mechanisms that how bacteria survive in their diverse natural habitats by using ECM and ECM proteins are yet to be fully understood. In a recent study, Floyd et al. [6] studied spatial proteome of surface-associated single-species biofilms formed by uropathogenic Escherichia coli and concluded the presence of at least two regulatory mechanisms controlling type 1 pili expression in response to oxygen availability. Similarly, a recent study on ECM proteome of Bacteroides fragilis, a widely distributed member of the human gut micro biome, identified several lipoproteins, TonB-dependent transporters and auto transporters [7]. Similar to theses investigations, several studies on ECM proteome in E coli were also performed [8-10]. Although these investigations have provided in-depth information about the certain ECM proteins, it is not known how surface proteins are affected in presence of serum. We, therefore, investigated how blood serum affects ECM and polysaccharide production and surface protein expression in S. aureus using proteomic techniques.

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Materials and Methods

Bacterial strain

S. aureus Philips, a biofilm-forming bacterium, was used in this study. In previous studies, we successfully used this strain, which was originally isolated from a patient diagnosed with osteomyelitis Patti et al. (1994); George et al. (2006); George et al. (2007). Secondary cultures was generated by inoculating 1ml of overnight culture into 50ml of TSB and growing at 37 °C with constant rotation in shake flasks for 16 hours. We grew the bacteria at several concentrations of blood serum and found that 12.5% supplemented blood serum similar growth curve as normal TSB. The growth of the bacterial strains was monitored by measuring the absorbance of the broth at 600nm on a spectrophotometer. The cells were then harvested and resuspended in phosphate-buffered saline (D-PBS; 138mm NaCl, 2.7mM KCl, pH 7.4). Cell concentrations was be determined using a Coulter Multisizer.

Measuring PIA

The cell plate was created from one ml of the culture, transferred to a micro tube and centrifuged at 10,000xg for 10 min at 4 °C. One ml of PBS buffer was used to wash the cell plates. Cells were then resuspended in 100|il of 0.5M EDTA, pH 8.0 and boiled in hot water for 10 min at 100 °C. The sample was then centrifuged at 10,000xg for 10 min at 4 °C. The clear supernatant was transferred to a new micro tube. Boiling cells with 0.5M EDTA is the best method known to date for the isolation of crude PIA from staphylococcal cell surface [11]. The crude PIA quantification was performed by a colorimetric method as described elsewhere [12]. Briefly, 50 |il of the crude PIA was transferred to a micro tube and mixed with 25|il of 80% w/v Phenol solution (Sigma-Aldrich) and 1 ml of concentrated sulphuric acid was added. The solution was kept at room temperature for 10 min, and absorbance was read at 490nm. Normalization of the amount of PIA was performed by dividing by the number of cells used for extraction.

Protein extraction

Cells were washed with PBS containing 0.1% sodium azide and then with PBS without azide, followed by a brief wash with digestion buffer containingm10 mm Tris HCl, 1 mm EDTA, 5 mm MgCl2. Approximately 5x109 bacterial cells were resuspended in 1ml of digestion mixture containing 35% raffinose, protease inhibitor cocktail (1 tablet/ml of digestion buffer), lysostaphin (5units/ml) and then incubated at 37 °C for 30 min. Cell debris were removed by centrifugation at 8,000g for 20 minutes and the supernatant was collected. After digestion and centrifugation, the digest was kept at -20 °C overnight and then centrifuged at 8,000g for 20min precipitated raffinose was discarded. After digestion and centrifugation, the protein solution was subjected to ultrafiltration using the Millipore ultrafiltration tube and centrifuged as per manufacturer's instructions. Protein concentration in the solution was determined using 2 D Quant (GE) and the resulting solution will be stored at -80 °C for 2-DE.

Two dimensional gel electrophoresis

In preparation for 2-DE, 150 ng proteins was resolubilized by adding standard sample solubilization buffers containing urea (8M), thiourea (2M), ASB 14 (1%), DTT (1%), and Carrier ampholytes (0.08%).The resulting solution was diluted to the desired volume with destreak rehydration solutions. Rehydration of IPG strips with the sample was carried out in the Immobiline Dry Strip Re-swelling Tray (GE Healthcare) according to the manufacturer's instructions. IPG strips of pH 3-11 (NL 24 cm) were used. The rehydrated strips were subjected to isoelectric focusing (IEF), performed using IPGphor operated at 20°C in gradient mode (97 kVhr). After focusing, the strips were stored at -80°C for later use. Prior to the second dimension SDS-PAGE, IPG strips were equilibrated for 15 minutes in equilibration solution (15 ml) containing 50mm Tris-HCl, pH 8.8, 6 M urea, 30% w/v glycerol, 2% w/v SDS and traces of bromophenol blue with 100 mg/10 ml (w/v) of DTT.

A second equilibration was carried out for 15 minutes by adding iodoacetamide (250mg/10 ml) instead of DTT in equilibration solution. Second dimension vertical SDSPAGE was performed using large format (26.8x20.5 cm) gels (12.5% T/ 2.6% C) according to the manufacturer's instructions. Electrophoresis was carried out with an initial constant voltage of 10 mA/gel applied for 30 minutes followed by 20 mA/gel for overnight until the bromophenol band exits the gel. The gels was stained with Colloidal Coomassie brilliant blue (BioRad). Gels were scanned as 12-bit TIFF images using Biorad GS-800 densitometer and analyzed by Nonlinear Dynamics Same Spots (v.3.2). Spot volumes were normalized by the software to a reference gel. At least three gels (biological replicates) for each treatment was used for analyses.

Protein identification

For mass spectrometric identification, gel spots were excised, destained, and digested with sequencing grade trypsin (Promega). Peptide samples were analyzed by Nano ESI-MS/ MS using LTQ (Finnigan, Thermo, USA). Nano LC was performed at reversed phase conditions using an Ultimate 3000 (Dionex corporation, USA) C18 column with a flow rate of 1-5 microliter/ min in 70-90% acetontrile containing 0.1% formic acid. MS and MS/MS data was collected and interrogated using SEQUEST against the NCBI non-redundant protein database for S. aureus providing peptide tolerance of 1.4 amu. Searched results were filtered using three criteria: distinct peptides, Xcorr vs Charge state (1.50, 2.00, 2.50, 3.00) and peptide probability (0.001). The confirmation of the protein identification was based on the Xcorr value of more than 50 and Sf score for individual peptide of more than 0.8.

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Results and Discussion

Blood serum affects polysaccharides intercellular adhesins

We have developed an experimental protocol for isolation and quantification of polysaccharides intercellular adhesins of S. aureus by boiling cells with 0.5M EDTA, digesting the PIA with concentrated sulphuric acid and phenol, and then measuring absorbance at 490nm. Although isolation of crude PIA by 0.5M EDTA is a routine procedure for PIA purification [13], to our knowledge it has not been reported for crude PIA quantification. We combined the EDTA extraction [13] with determination of sugars and their derivatives by colorimetry [11]. Using this procedure, we were able to reproducibly quantify PIA from S. aureus. As evident from the Figure 1, significantly higher amounts of PIA were observed in presence of blood serum. Similar to these findings, we also observed increased level of PIA in elevated level of NaCl [14].

PIA biosynthesis is mediated by ica operon-encoded enzymes [15,16]. The icaA, D and C gene products are involved in translocation of the growing polysaccharide to the cell surface [17], while IcaB is responsible for deacetylation of the PIA I molecule (providing its positive charge) which is essential for biofilm formation [18]. In contrast, the icaR gene, located upstream of the ica ADBC operon, encodes a transcriptional repressor which plays a central role in the environmental regulation of the ica operon [19]. For example, exposure to NaCl activates the ica operon in an icaR-dependant manner [18-20]. We anticipate that blood serum might have similar effect on the ica operon in an icaR-dependant manner, which is yet to be explored.

Blood serum and fibronectin binding and collagen binding proteins

In the SDSPAGE (Figure 2) (Table 1), several virulence- associated surface proteins were identified such as fibronectin- binding protein (b2), collagen-adhesins precursor (b4, b7), trigger factor (b8). However, serum supplement significantly reduced the abundance of fibronectin binding protein, although the abundance of collagen binding protein was not affected. In a recent report, Shinji H et al. (2011) studied, Fibronectin-binding protein A (FnBPA) and FnBPB, by constructing constructed fnbA and/or fnbB mutant strains and reported that the serum levels of interleukin-6 and nuclear factor kB (NF-kB) activation have no significant reduction in fnbB mutant infection(18)s. It is probable that the NF-kB of serum we used might have reduced the fibronectin-binding protein.

Bacterial strain

Measuring PIA

Protein extraction

Two dimensional gel electrophoresis

Protein identification

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Results and Discussion

Blood serum affects polysaccharides intercellular adhesins

Blood serum and fibronectin binding and collagen binding proteins

Serum proteins in bacterial surface

We identified two serum proteins, apolipoprotein and globulin (Fc and Fab), in the membrane fraction of bacterial proteins. These results were confirmed from both 1D and 2-DE SDS PAGE. The presence of serum proteins in membrane fraction of bacterial protein has raised several questions. If we consider these proteins as a contaminant from the serum, why were we unable to wash out these proteins while we successfully washed out the most abundant serum protein such as albumin? If not a contaminant, what is causing these proteins to remain attached to the bacterial surface? It is known that Fc and Fab motifs of globulin interact with Spa C and Spa D domains of protein A. But the bacterial strain we used was a mutant of proteins A. In addition, by using a deletion mutant of Newman, we confirmed the presence of Fc and Fab with bacterial membrane associated protein (Figure 3). This raise another question of what components of bacteria are causing this Fc and Fab to remain attached with bacterial proteins.

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Conclusion

Polysaccharide intercellular adhesins production was significantly increased due to the addition of blood serum in the media. We identified two serum proteins, apolipoprotein and globulin (Fc and Fab), remained attached with the membrane fraction of bacterial proteins even after several washing procedures, indicating that these proteins might play a critical role in bacterial processes of biofilm formation.

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Author's contributions

Nazrul Islam designed and conducted the experiment, and corresponding author for this manuscript. Julia M. Ross, Khwaja G. Hossain and Mark R. Marten developed the concept.

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Acknowledgement

This research was supported by Grant no. R01AI059369 from the NIH and also an Institutional Development Award (IDeA) under NIH grant no. P20GM103442.

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juniperpublishers-oajnn-blog · 5 years ago

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Use of Sphenopalatine Ganglion Blockade in Chronic Migraine Management

Authored by Michelle Androulakis

Case Report

Chronic migraine (CM) is a debilitating neurological disorder which affects more than 4 million individuals in the United State and 2% of the global population [1] in 2015, the Health Care utilization was estimated at $5.4 billion and the total cost associated with management of comorbidities exceeded over $40 billion in united states [2,3]. Different acute and preventive therapies, which are available for chronic migraineurs, are generally sub-optimally effective and are accompanied by side effects that are difficult to tolerate. Currently, Botulinum toxin therapy (Botox) is the only FDA approved CM preventative therapy, however, it is expensive and up to 9% of patients experience side effects such as neck pain after the injections. Recently, SPG neuromodulation has gained interest among headache specialist in management of CM. A series of SPG blockade using intranasal bupivacaine was efficacious for acute pain reduction in CM. However, further investigation into the long term preventative benefit of SPG block is warranted as this study sample size was too small to reach its statistical significance [4,5].

Sphenopalatine ganglion (SPG) has been a very important target for headache management since the beginning of the 20th century. SPG is the largest extra cranially ganglion of the head and is likely to play an important role in migraine pathogenesis through the activation of trigemino-autonomic reflex [6]. Up to 70% of migraine patients have cranial autonomic symptoms such as eyelid edema, nasal congestion, lacrimation, conjunctival injection, rhinorrhea, and facial swelling [7]. SPG modulation via electrical stimulation, microvascular decompression, surgical or radiofrequency ablation, and radiosurgical lesion have been performed for head pain in operating room settings, however, adverse effects of these interventions can be extensive. The SPG is located just posterior/superior to the tail of the middle turbinate on the lateral nasal wall and superior to the pterygopalatine fossa.

Manipulation of this region was often very challenging, as there is no direct access to the SPG and it is covered by a thin layer of nasal mucosa (1-1.5mm). New methods to modulate the SPG with a topical, intranasal approach have proven to be among the safest, least invasive, and least costly of all SPG interventions in headache management.

Recently, several new devices have been developed which facilitate a more accurate and effective delivery of the local anesthetics into the SPG. The risks of this procedure are typically minimal and may include minor discomfort during and after the procedure, a numbing or burning sensation, bitter taste from the anesthesia, bleeding from the nose (rarely), and lightheadedness. These side effects typically resolve within minutes to a few hours. There is also a very small risk of allergic reactions.

Use of SPG block has been recommended by American Headache Society (AHS) as part of comprehensive headache management plan. Indeed, repetitive SPG blockade twice a week for 6 weeks provides an alternative migraine prophylaxis for those with chronic migraine but could not tolerate (i.e. needle phobia) or unresponsive to Botox therapy. SPG block generally provides a better outcome for treatment of CM with head pain in frontal and/orbital regions, and may also help CM patients with coexisting medication overuse headaches to wean off excessive use of pain medications.

Nausea, which has been suggested as one of the main contributing factors for migraine chronification, is also another possible symptom that can be relieved with a series of SPG block. The area postrema area, located at the infer posterior part of IV ventricle, is responsible for nausea and vomiting through its connection to the nucleus of the solitary tract. The superior salivatory nucleus (SSN) provides preganglionic parasympathetic innervation to SPG, but also receives inputs from multiple areas, such as nucleus of solitary tract, limbic, and cortical regions. Repetitive intranasal SPG blockade with bupivacaine may reduce nausea and vomiting via inhibition of superior salivatory nucleus given its direct connection with the nucleus of solitary tract.

The exact mechanism of SPG neuro modulation remains to be elucidated. It has been postulated that inhibition of the parasympathetic outflow from the SPG would inhibit pain and autonomic symptoms that accompanying recurrent migraine attacks. This inhibition of parasympathetic outflow would decrease activation of perivascular nociceptors in the cranial and meningeal vasculature, especially in the frontal regions of the brain [8-10]. Additionally, modulation of the SPG may in turn modulate brain networks activity involved in pain processing. In a recent resting state functional MRI connectivity study, our group demonstrated that a series of SPG block treatment in chronic migraine significantly improved two intrinsic resting state functional connectivity networks (manuscript in preparation). This increase in functional connectivity coherence may represent that after effective treatment, reorganization of resting state brain networks to normalized states may occur.

Additionally, reduced parasympathetic outflow due to repetitive SPG inhibition may help to restore baseline homeostasis of brain networks involved in pain processing, via improved mesocorticolimbic modulation [11-13]. A large double blinded, randomized, placebo controlled clinical trial is warranted to evaluate the efficacy of repetitive SPG block in CM (Figure 1 & 2).

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juniperpublishersajop · 4 years ago

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The Explanation of Magnetic Metal Carbon Mesocomposites Synthesis Peculiarities by Means of Mesoscopics Notions

Abstract

Mechanism of mesoparticles modification reactions are considered with the application of such notions as charges quantization, phase coherence, interference and annihilation. On the base of theoretical Mesoscopics ideas the formation of covalent bonds because of the interference of negative charges quants in modification reactions is discussed. The hypothesis about possibility of annihilation at the interaction of positive and negative charges quants in redox processes is presented. The magnetic metal carbon mesoscopic composites synthesis (for example, initial metal carbon Mesocomposites) is realized by mechanochemical method at the grinding of metal oxides microscopic particles with polyvinyl alcohol macromolecules. Then in the result the Copper or Nickel Carbon mesocomposites which have the following atomic magnetic moments: for Copper – 1,3 μB, for Nickel – 1,8 μB are obtained. The investigations are carried out on the analysis examples of processes of Copper and Nickel Carbon mesoparticles modification by the compounds containing p, d elements. In the middle of its such substances as polyethylene polyamine, ammonium iodide, ammonium polyphosphate (APP), silica (SiO2), aluminum oxide, iron oxide, nickel oxide and copper oxide are used. It’s noted that the red ox processes are accompanied by the metal atomic magnetic moments growth, that is explained by the electron shift on high energetic levels because of the annihilation phenomenon. The hypothesis concerning to the passing of two phenomena (annihilation and interference) at redox processes is proposed.

Keywords: Macro Molecules; Nanoparticles; Suspension; Emulsions; Polymers; Toxicity

Abbreviations: NCPs: Cationic Polymers; DEX: Cationic Dextran; PLL: Poly-L-lysine; WHO: World Health Organization; PEG: Polyethylene Glycol; PDMAEMA: Poly(2-N,N-dimethylaminoethylmethacrylate)

Introduction

The process occurs at the charge’s quantization with the certain phase coherence and then with the chemical bond’s formation because of interference as well as in the red ox processes possible annihilation takes place [1,2]. If chemical reactions are realized without the changes of atoms oxidation states, then the negative charges quants quantization and the interference are carried out. However, the most reactions flow with the changes of elements oxidation states and then according to known schemes of reduction-oxidation processes it’s necessary to take into consideration of positive charge quants.At the interaction of positive charge quants with the negative charge quants the annihilation phenomenon with the electromagnetic radiation or/and the direct electromagnetic field is possible. Also, the interaction of positive charges with the formation of “dark hole” must not excluded (Figure 1). In this case the explosion with diffusion of many most quantity of energy into surroundings is possible. The phenomena of charge quantization, interference and annihilation are considered on the examples of metal carbon mesoparticles interactions with reagents containing p, d elements. At these investigations the basic method of researches is x-ray photoelectron spectroscopy.

Results and Discussion

The production of Metal Carbon mesoscopic composites is carried out with the using of mechanochemical interaction between microscopic particles of metal oxides and macromolecules of polymers at the active medium presence [3]. At the mesoscopic composites production, the sign variable loadings are applied. These loadings are appeared at the grinding with pressing. At the common grinding of Copper oxide particles with Polyvinyl alcohol (PVA) particles (or concentrated water solution) the metallic phase clusters falls between macromolecules of polyvinyl alcohol or, in other words, into reservoir (according to mesoscopic notions), in which banks are PVA macromolecules. The metal (example – Copper) within cluster has the positive charge. Therefore, the negative charge quants are directed to positive charged atom. In our case the negative charged quants from polyvinyl alcohol acetate and hydroxyl groups are transferred to copper positive charge quants. As a result, the annihilation with the electromagnetic direct field formation takes place. In this process the acetic acid and water are formed, and also the banks structures are changed: the poly acetylene and carbine fragments are appeared. There are unpaired electrons on joints of these fragments. The process of pair electron division and the shift of electrons on the high atomic levels for metal are explained by the annihilation origin. In this case the metal atomic magnetic moment growth is observed in the dependence on the electrons number which participates in red ox process.

The hypothesis about possibility of annihilation at the interaction of positive and negative charges quants in red ox processes is confirmed by the examples of processes of Copper and Nickel Carbon mesocomposites modification with application such substances as polyethylene polyamine, ammonium iodide, ammonium polyphosphate (APP), silica (SiO2), aluminum oxide, iron oxide, nickel oxide and copper oxide [3-5]. In the case, when polyethylene polyamine and ammonium iodide are applied, the connection reactions take place. At the interactions of polyethylene polyamine with mesoparticles the C=N bond formation is explained by the interference of negative charges quants. When the mesoparticles modification reactions with the using APP, SiO2, metal oxides are carried out, the redox processes are realized. In these cases, the modifiers reduction reactions take place. The structures of metal carbon mesoscopic composites with active carbon shells are defined by means of the complex of methods including x-ray photoelectron spectroscopy, transition electron microscopy with high permission, electron microdiffraction and EPR spectroscopy. In correspondent reactions the element reduction for reagents and Nickel or Copper atomic magnetic moments growth in mesoparticles take place. Below in (Table 1) the examples of metal atomic magnetic moments changes for mesoparticles modified by APPh or silica after the mechanochemical modification processes are given. The presence of unpaired electrons on mesoparticles carbon shells in above systems is determined by means of electron paramagnetic resonance (EPR) (Table 2).

Cu C NC – APPh (or SiO2) and Ni C NC – APPh (or SiO2).

The metal atomic magnetic moment growth proceeds owing to the redox processes with above chemical compounds. In papers [4,5] it’s shown that the reduction reactions of Phosphorus and Silicon from correspondent substances at the interaction on the interphase boundary with mesoparticles are realized.

The relations of mesoparticles to above oxides are changed from 1:1 to 1:0,2 depending on the qualitive spectra obtaining, for example, the relations of 1:1 and 1:0,5 for system “Ni/C NC – Al2O3” leads to full mask of mesoparticles. Therefore, the quantity of aluminum oxide is decreased to the relation 1:0,2. In accordance with Al3s spectra Aluminum is completely reduced during the modification process, and Nickel atomic magnetic moment is increased to 4,8μB (Table 3). In this case the reduction process is related to not only Aluminum oxide but also to Nickel oxide from metal cluster of mesoparticles (Ni C MC). Therefore, the reduction processes are stipulated by the electron transport from carbon shell of mesoparticles in direction to Al+3 and Ni+2 of atoms in correspondent oxides. Exceptional properties of modified Metal Carbon nanostructures with magnetic characteristics lead to the property’s improvement of nanostructured polymeric coatings [6-8]. For example, the introduction of 0,008% Cu C MC into the melamine-formaldehyde resins stimulates the polarization growth in two times (on the AFM data). Similar results can be received at the combination of phase coherency and interference of charges quants during the preparation process of modified polymeric materials. The decreasing of nanostructures activity is possible when the modification is carried out with the ultrasound processing or the violation of phase coherency takes place at the nanostructures quantities changes [9,10].

Conclusion

The present investigation has fundamental character. It’s based on the ideas concerning to the change of Metal Carbon mesoscopic composites reactivity. The investigations are dedicated the mechanochemical red ox processes in which the electron transport from mesoscopic composite cluster to carbon shell takes place. In this case the electron delocalization is found. For the first time the metal carbon mesoscopic composite modification by mechanochemical process with the using of active substances including also bioactive systems is possible. The activity of metal carbon mesoscopic composites is caused by the structure and composition of correspondent composites, which contain the delocalized electrons and double bonds on the surface of carbon shell. Thus, at the mechanic chemical reduction/oxidation synthesis the changes of element oxidation states as well as the increasing of metal atomic moment for cluster can be appeared. At the same time, the modifiers elements and functional groups are discovered in carbon shell of mesoscopic composites modified. The creation of reactive mesoscopic materials with regulated magnetic characteristics which can find the application as modifiers of materials properties, catalysts for different processes, effective inhibitors of corrosion, sorbents, stimulators of plant growth, is very topical. These facts open new era for further investigations and development of metal carbon mesoscopic composites application fields.

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annieboltonworld · 6 years ago

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Juniper Publishers

Juniper Publishers Inc. is established with the aim of spreading quality scientific information to the research community throughout the universe. Juniper Publishers Inc, as Open Access publishers, strive to offer the best in class online science publications. Juniper Publishers Inc as an open Access process eliminates the barriers associated with the older publication models, thus matching up with the rapidity of the twenty-first century. Juniper Publishers Inc’s main areas of interest lie in the fields of science, engineering and other related areas. Juniper Publishers is a platform for professors and researchers who aspire to give out quality information based on their research and expertise, in an attempt to aid scholars/researchers in their field of interest with the latest information.

Juniper Publishers mainly focuses on the requirements of scientists, students and research scholars, Juniper Publishers aspire to be the leading provider in Open Access Publications, with an array of prestigious academic international journals, dedicated to serving the engineering, scientific and medical communities. Juniper Publishers publish articles of various formats (i.e., HTML, PDF, Digital, Audio conversion and Video development) and they can be accessed freely by scientists, students and the research community immediately after publication.

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laceyspencer · 4 years ago

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Analysis of Satisfaction of Small Cucumber Contract Famers in Predominantly Agriculture areas in Sri Lanka-Juniper Publishers

Introduction

With the ever-increasing competition of the agricultural business world, most of the private sector contracts farming providers are also in the phase of increasing their profits. The techniques of contract farming which are used for the profit enhancement is occasionally giving positive outcomes as well as negative outcomes. Efficiencies and productive farmer community vital assets for achieving a higher level of performance in an organization [1]. Satisfied farmer community leads to enhance effort to cultivation performance, so that maintaining of satisfied contract farmer in an organization is crucial factor. To maintain the well-being of an organization management tries to create a highly satisfied farmer community. Therefore, organizational management places a noticeable reliance on their individual farmer performance to gain higher productive cultivation [2,3].

These conditions are leading the contract farmers in the organization to going through a condition of farmer’s job satisfaction which directly affects day today cultivation practices. Together with these facts, there is a new trend in the agricultural academic societies in search of factors that are affecting the level of job satisfaction condition as well as searching the relationships among the job satisfaction conditions and the contract farmer’s performances [4].

The aim of this research study is to identify the factors effect on job satisfaction level of the contract farmers on small cucumber cultivation in predominant agriculture areas such a s Ampara, Thabutthegama & Pollonnaruwa in Sri Lanka. When a contract farmer satisfied about the farming contract they are tending to motivate to do higher effort to the cultivation performance. Ultimately it increases organizational overall performance by increasing continues proper supply to export market [5]. On the other hand, a satisfied farming community & their effort & commitment to cultivation practices are vital for successfulness of organizational goals & objectives. Therefore, the address research problem of this study is that; Satisfaction factors which effect on contract farming [6].

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Problem Statement

Small cucumber export market mainly depend on continues product supply to the market. The continues repetition of contract farming in small cucumber ensure continues small cucumber supply to the market. When there is an insufficient supply, company must import Small cucumber from India and USA and re-export by spending huge cost. Farmers are tending to renew their contract agreement when farmer are satisfied with the existing contract farming activities. Analysis of farmer satisfaction on small cucumber contract farming is vital to increase the profitability of the company [7-9].

During the past few decades, in this small cucumber export private company contract farmer’s repetition of cultivating small cucumber level has decreased significantly. According to the Supply chain department’s sources in 2016 since the organization has implemented lots of strategies changes to the contract farming of the organization, most of the farmers has faced various organizational challenges and those challenges has decreased the satisfaction level of the contract farmers of the organization. According to Bommanahalli and Rangappa, (2016) contract farmers found to be using improved farming practices in maize production. According to Aziri [10] research emphasize that contract farming enhances farmers knowledge, attitude & agronomic practices about small cucumber farming.

As indicated by the Asian Development Bank (2008) a “farmer– endeavor” association for industrialization of agribusiness can be deciphered as the model of agreement cultivating that is generally drilled in numerous nations. It is by and large acknowledged that economical, efficient, and impartially executed contract cultivating can bring advantages to the two ranchers and the endeavors [11- 15].

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Justification of the Problem

It is identified that satisfaction of contract farmers to work to achieve higher amount of cultivation & repetition, as well as dissatisfaction of contract farmers will decrease the cultivation area & increase the non-repetition. Because of dissatisfied contract farmers continuous small cucumber export is breaking that problem of the finally decrease the organizational profit & reputation [16]. According to the Organization 2016 financial report it was then found a significant reduction in the repetition of small cucumber contract farming of the organization by 45% compared to the previous year (2015).

It was also found that there is a significantly higher percentage of small cucumber production deduction compared to the previous years and the previous record prevailing with the records of the organization. All in all, these all factors and figures are providing enough evidences for the occurrence of lower satisfaction level with the contract farmers of the organization [17-20]. As per the Asian Development Bank Report in 2015 underlined that farmer– endeavor organizations ought to be built up to pull in more speculations into horticulture and provincial regions, and to assemble a market-situated rural innovation augmentation framework. The administration has constantly considered contract cultivating as an essential intends to reinforce farmer– endeavor associations [21-23]. As indicated by the Lieping [24] the way that ranchers are unwilling to go into contract cultivating additionally captures the improvement of farmer– endeavor organizations. Numerous examinations additionally detailed that the satisfaction rate of agreement cultivating in the China was low [25].

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Objectives of the Study

The researcher is trying to achieve the below mentioned objectives by conducting this research study [26].

General Objective

a) To study the existing small cucumber contract farming cultivation under leading export pvt. group of company and analysis the farmer satisfaction towards the of contract farming to reduce farmer dropout rate [27].

Specific Objectives

a) To ascertain the existing small cucumber contract farming under leading export company

b) To analysis of farmer satisfaction toward small cucumber contract farming.

c) To give recommendation and suggestion to enhance contact farming of small cucumber [28].

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Research Design and Research Methodology

The key motive of this descriptive research method and the descriptive research design is mainly to investigate the relationship of the contract farmer’s job satisfaction level and the impact of those job satisfaction levels towards the contract farming in small cucumber cultivation [29]. The specific organization selected for the simplification of the overall work was the main small cucumber export private company of Sri Lanka.

This research base on to seek the “Impact of contract farmer’s satisfaction level on the small cucumber cultivation: a study of small cucumber export organization of Sri Lanka. Primary data was collected from pre-tested structured questioner, which specially design and given selected group. Secondary data was collected from previous researches, journals, and government publications etc which are specifically focused on the farmer satisfaction and the contract farming [30-33]. As mentioned in the conceptual model and as elaborated in the hypothesis also, the independent variables were the contract farmer ‘s satisfaction’. As per the dependent variable, the most significant variable of the research which was the ‘small cucumber cultivation related performance’ was used. It does mean whether repetition or not.

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Population, Sample and Sampling

In this study to seek the influence of farmer satisfaction towards the small cucumber cultivation. Mainly focus group was the currently engaging farmers of the organization. Though it is obvious that the compilation of data from the all districts and making necessary data bases for the purpose of the accuracy of the data and the accuracy of the data interpretation when it comes to the generalization of the final results and discussions; the chosen study areas were restricted to the Ampara, Pollonnaruwa & Thambutthegama districts due to the time limitation in collecting the data and the budget constraints of the study. After all the final sample of the study would be a compilation of the given below [34-38].

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Data Collection Techniques

In a quantitative research there are several data collection methods, there two source of data such as Primary data and secondary data. Primary data gathered from actual research events. Secondary data gathered from previous articles, journals, government publications. According to this research secondary data was analyzed by a quantitative manner. Pre-tested questioner did help in achieving this task. It also could be two ways such as personally administrated and interview. Since the limitations pertaining to the research as well as to the researcher (time constraint and the budget constraint) the method which was used for the collection of data was that the use of pre tested questionnaire by interviewed of 20 contract farmers in one district for the purpose of obtaining the data has first been granted the required permission from the upper management of the organization [35].

The sample was selected through simple random sampling and the areas from the Ampara, Pollonnaruwa & Thambutthegama districts were chosen through purposive sampling. The key motive of using the simple random sampling for the sample selection was that to maintain the accuracy of the data which has been generated and to increase the probability for the research to be published in peer reviewed journals or any relevant standard publication materials as a final step of the carrying out of a successful research [36].

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Results and Discussion

Frequency Distribution of Age

According to the Pearson correlation test for age r= -0.0048 (95% confidence interval). When age is increasing job satisfaction will decrease. Have a negative correlation. There were 34.4% contract farmers in 31-40 years’ age range. In the range of 41-50 age categories, there were 33.3% of the respondents, and in 51- 60 years’ category 24.5% of the contract farmers. It was observed that only 7.8% of the respondents were in the age range of above 60 years (Figure 1).

Frequency Distribution of Job status

According to the analyzed data most of the contract’s farmers (76%) only engage with agriculture.24% contract farmers doing agriculture simultaneously with other status such as government, private employment. According to the Pearson correlation value job status and job satisfaction has positive relationship. (r=0.0638, p=0.0000 at 5% significant level) (Figure 2).

Frequency Distribution of Civil Status

94% of contract farmers were married and only 6% of contract farmers were single (Figure 3).

Frequency Distribution of Agricultural Experience

According to the sample 38.9% of farmers have 13-16 years in agricultural experience. In the range of 9-12 experience categories, there were 33.3% of the respondents, and in 5-8 years category 16.7% of the contract farmers. It was observed that 8.9% of the respondents were in the 17-20 range and only 2.2% of contract farmers above 21 years experiences (Figure 4).

Cultivation

More than half (61.1%) of contract farmers have 1-5 years’ work experience in small cucumber cultivation.6-10 years it was 26.7% and 11-15 category there was 11.1% and only 1.1% contract farmers have 16-20 years small cucumber cultivation experiences. According to the correlation value r=0.1299 (at 95% confidence interval) experiences positively correlated with job satisfaction (Figure 5).

Frequency Distribution of Different Cultivation Pattern

There was only 1.1% very small amount of contract farmers cultivate only in small cucumber. 85.6% very high amount of contract farmers cultivate small cucumber with paddy cultivation. Other contract farmers are cultivating small cucumber with paddy and pumpkin (Figure 6).

Variation of Small Cucumber Cultivation Land with Different Seasons

According to the secondary data (organizational annual reports 2014-2017) it clearly shows land areas diminishing with the time [37]. Land amount in Yala season greater than comparing with yala-off and maha season. Lowest amount of small cucumber land can be seen in maha season (Figure 7). Contract farmers’ satisfaction about small cucumber cultivation practices special reference to land preparation. In small cucumber cultivation very, simple land preparation can identify. Contract farmers were highly satisfied with land preparation practices.it was 57%. 33% were satisfied with land preparation (Figure 8).

Contract farmers’ satisfaction about small cucumber cultivation practices special reference to harvesting practices (Figure 9). Daily harvesting is vital in small cucumber cultivation. It is labor extensive practice and somewhat difficult practice comparing to other crops. 37% of contract farmers under the dissatisfied category and 33% under the highly dissatisfied level. There were only 7% contract farmers satisfied about harvesting practices [38,39].

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Contract Farmers’ Satisfaction About Income Receiving

More than half (57%) of contract farmers highly dissatisfied with their income coming from small cucumber cultivation.33% of contract farmers under the dissatisfied category there were no any contract farmers who satisfied with income receiving from small cucumber cultivation (Figure 10).

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Contract Farmers’ Satisfaction About Farm Gate Price

Majority (77%) of contract farmers dissatisfied with their farm gate price.17% of contract farmers are highly dissatisfied with farm gate price [40-42]. There was no any contract farmer who satisfied with farm gate price (Figure 11).

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Contract Farmers’ Satisfaction About Pest Control Practices

In small cucumber cultivation pest control is crucial practice. 50% of contract farmers were dissatisfied about pest control practices.13% were highly dissatisfied with pest control practice. No any contract farmer satisfied with pest control practice (Figure 12).

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Contract Farmers’ Satisfaction About Timely Receiving Money

Majority (57%) of contract farmer dissatisfied about timely money receiving. 23% contract farmers highly dissatisfied about timely money receiving factor (Figure 13). Wilcoxon sign rank test for Training, Yield, Income, Farm gate price, Pest control, Money receiving. According to the Wilcoxon sign rank test p value is less than 0.05 (<0.05=p) in analyzed factors. Training, yield, income, farm gate price, control practices and timely money receiving factors were significantly affect on contract farmer job satisfaction (Table 1).

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Conclusion and Recommendations

Contract farmers are the key persons and main responsible person’s development of cultivation. This become true when the contract farmer satisfied with their cultivation, it will directly have affected to the effective and efficiency of the contract farmers at the field. To achieve organizational goals and objectives it is vital to identify the individual contract farmer satisfaction level toward the cultivation. In this research, basically its main objective was to study the existing Small cucumber contract farming cultivation under pvt group of company and analysis the farmer satisfaction towards the of contract farming to reduce farmer dropout rate job satisfaction level.

Here in this study consider about 90 contract farmers in Ampara, Pollonnaruwa & Thambutthegama areas. There is only limited number of researches are available to identify the satisfaction level of contract farmer with special reference to the Sri Lankan agricultural private organization. The aid of survey method for data gathering and it was utilized Pearson correlation analysis; Wilcoxon sign rank test and descriptive statistics for data analysis. One of the major conclusions that can be drawn from the study result is that difficulties occurred in cultivation practices especially in harvesting and pest controlling. Contract farmers satisfied about their training received and yield from small cucumber cultivation. Poor organizational practices like lower farm gate price, money not receiving timely to contract farmer reduce continues small cucumber contract farming.

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Recommendations

The following recommendations are forwarded to organization to enhance the satisfaction performance of the contract farmers to the highest level.

a) According to the analyzed result concluded that the young age was significant in their satisfaction. Hence, in the farmer selection process give priority to that young farmer with agricultural background.

b) Enhance small cucumber farm gate price.

c) Develop standard system for effective money transferring to contract farmer.

Ex- “On the Spot Money Transferring Method”

a) Develop innovative methods for harvesting and pest controlling.

Enhance the awareness about intergraded pest management instead to chemical application among contract farmers.

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juniperpublisherswb · 4 years ago

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Juniper publishers-The Sustainability of the Cultivation of Quinua in Peru-Approximations After the International Year of the Quinoa (AIQ)

Introduction

Thanks to the enormous publicity and diffusion of the AIQ, carried out at global level led by the governments of Peru, Bolivia, Ecuador, Argentina and France (Group of countries of the International Committee of the AIQ), the world has known quinoa, as a food of high nutritional value and with nutritional and medicinal properties that could contribute to improve food security in developing countries, promoting its cultivation that is highly adaptable to most of the climatic conditions of the orb. This generated a high demand for the product and, consequently, increased the prices of quinoa, generating temporary profits for the producers.

The question is, what is the situation of producers of family farming systems? After a stage of economic prosperity, after the AIQ, they have returned to the reality of a policy that protects neither prices nor genetic resources. Currently more than 70 countries are in the experimental phase and others have already started large-scale production. The losers will be the small producers who produce in conditions of high climatic, financial adversity and state lack of protection. The countries of South America and specifically Peru, must now face the global competition of highly technician and industrialized countries that easily based on improvements and even genetic manipulation of quinoa can exceed the average productivity of the original countries.

The consequences can be irreversible due to the uncontrolled exit of genetic material. In Peru, the lack of national policies for the protection of genetic resources is evident; For example, Puno, which is the largest quinoa producing area in the country to date, could not achieve the denomination of origin, or Plant Breeders’ Certificates (COV). On the other hand, the high genetic instability of quinoa can be a negative factor; commercial varieties registered in Peru, when they leave to another country for multiplication purposes, can be adapted by very easily changing their phenotypic characteristics and could be registered and patented as a new variety.

Despite having some natural advantages and comparative advantages, in general production is stationary, with low quality and lack of standardization of the product, individualized and small sale, limited access to markets and lack of community rural industries. However, one of the causes of the limited development of the local industry is the reduced local market, both for the grain (low levels of consumption per capita) and for the products derived and little demanding with quality and innovation. There is weak pressure from local consumers towards the supply of more and better products from the food industry, which use quinoa as an input. That is one of the causes of the limited development of that industry. Therefore, it will be essential to generate competitive advantages through forms of association, production and marketing that enable the development of differentiation factors based on research, innovation and the development of products in which quinoa is paramount for its multiple benefits. Meanwhile, the role of the State is relevant in terms of support and generation of a regulatory framework and with the promotion of public policies for the productive sector of quinoa.

The objective of this article is to analyze the impacts generated in family farming systems of highland areas of Peru in terms of technological innovations, management and conservation of resources: soil, water, genetic resources and an approach to the sustainability of production of quinoa, after AIQ 2013.

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National Production and Regional Performance of Quinoa Production

According to official MINAGRI figures (2014), the quinoa production of 2014 was 114,000 tons, a figure that reflects a growth of 119% compared to 2013 that reached 52,000 tons. This increase was mainly in the regions of Arequipa (522%), Puno (23%) and Junín (173%), based on the largest sowings executed and, consequently, the highest yields obtained

At the regional level, on the coast in the departments of Lambayeque, La Libertad, Ica, Tacna and Lima, the growth rate of quinoa production was 24% per year, while in the departments of the southern highlands (Arequipa, Apurímac, Ayacucho , Cusco, Moquegua and Tacna) was 18.7% annual average. In the case of Puno, it grew at a slower pace, and its participation in national production decreases each year [1].

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Case of the Expansion of Quinoa Cultivation in Ayacucho

Agriculture practiced in the high-Andean tropical zones (> 2500 masl) is based on the management of biodiversity and different farming systems in a wide range of socio-economic and environmental scenarios that allow the self-sustainability of small and medium farmers in rural communities ( Fonte and Vanek, 2010). The production area of quinoa in Ayacucho according to Gómez and Aguilar (2014) and Tapia et al. (2014) corresponds to the inter-Andean valley agro-ecological zone. The production of quinoa is practiced from 2500 to 3800 masl [2]. At the level of the Ayacucho region in the 1992/93 season the cultivated area was 123 ha, in 2003/04 2140 ha, increasing to 5768 ha in the 2012/2013 season. At the provincial level, the growth of the cultivated area in Huamanga stood out, from 244 to 2536 hectares, which represents an increase of 939% in the last 10 agricultural seasons [3]. According to the [4] in the 2014/15 campaign in Ayacucho the total area planted reached 11 115 hectares, of which 6429 have been planted in the province of Huamanga. Currently, quinoa is planted as monoculture, with a predominance of conventional production systems that involve the intensive use of soils, intensification of agricultural mechanization, indiscriminate use of synthetic fertilizers and pesticides, use of improved varieties with predominance of white quarries displacing the color quinoa and the local ecotypes, and the reduction of areas of other traditional food crops [5].

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Main Markets and Customers

The commercialization of the quinoa grain has three types of market: internal regional market (district and provincial fairs and in the departmental capital); external regional market (production goes outside the departmental scope to supply the demand of the national market); and finally export market to the different countries that demand quinoa [1]. Peruvian quinoa is exported to the international market as conventional and organic, being for the last five years (2010-2014) 75.4% of conventional type, with an annual growth rate of 67%. The annual growth rate of organic quinoa was 82%. In 2014, it was possible to export 27,200 tons of conventional quinoa and 8,900 tons of organic quinoa (The United States is the largest importer of Peruvian quinoa, with a tariff of 0% established for the entry of quinoa via the TLC).

However, despite the preferences of the international market for healthy products, the preference for organic quinoa has not been as decisive, since in practice more conventional quinoa was exported. This is another valid argument to analyze the possibility of massive production of quinoa in other countries with higher technology support (protection measures and subsidies and financing) which could easily displace domestic production. Perhaps it should be considered as a more favorable possibility (less possibilities of direct competition in the production of quinoa due to its unfavorable agro-ecological characteristics for the production of quinoa, but, comparative advantages due to the technological level reached in the food industry) trade with countries like Brazil that also through the application of the Agreement of Economic Complementation, exports have a 0% tariff like Uruguay and Paraguay. Likewise, South Africa with which Peru is in the process of negotiating an TLC; also, Asian and Oceanian markets who demand and prefer natural products.

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Annual Per Capita Consumption of Quinoa

Despite the immense campaign of diffusion made in favor of the consumption of the quinoa this has not had significant increases, according to general data provided by the MINAGRI in 2012 the annual per capita consumption was 1.20 kg / person / year. According to IICA [1] in a study carried out by ADEX (2001), it is estimated that per capita consumption at the national level is 0.52 kg / year, with the urban population registering a relatively low consumption (according to the results of IV CENAGRO, the main destination of the national production of quinoa per planting But, according to the FAO-ALADI [6], the consumption of quinoa estimated for 2012 in kg / person / year in Peru was 1.15 kg. area would be for self-consumption 68% of the total, 31% is destined for sale, and 1% for seed).

According to studies carried out by IICA [1] in the regional area of Puno and Junín, annual per capita consumption of 3 kg / person / year; while Junín would reach 3.6 Kg at an urban level and 15 Kg / person / year at rural level, which would merit reviewing the aforementioned figures since they are quite far from the national average provided by MINAGRI, with the aforementioned averages at the Puno level and Junín would be consuming an average of 9 kg / person / year. But if confirmed these figures can be an excellent indicator of the increase of family consumption of quinoa at rural and urban level with a clear tendency to improve food and nutrition security [7].

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Loss of Biodiversity and Global Competition for Quinoa Production

After the IYQ, more than 70 countries have quinoa genetic material from Peru and Bolivia, which are the two main countries of quinoa. In countries like USA, France, Holland, England with economic and technological support of their governments are in the research phase and several countries in the production phase [8].

Under these conditions, will farmers mainly have family farming systems, possibilities to compete with high technology and large industry in developed countries? Despite the natural and comparative advantages of having a huge variability of quinoa genes and as demonstrated, they can be adapted to almost all life zones existing in Peru; however, under current conditions, the producers of the altiplano and the inter-Andean valleys will have little chance of competing with the quinoa product and its derivatives if the developed countries begin the production of quinoa on a large scale. Production at the coastal level and marine Yunga would have better possibilities; However, the limiting factor that has not yet been solved is the phytosanitary issue (high incidence of pests obliges the indiscriminate use of pesticides). The average yield reached in the country is 1.2 t / ha, considering that some varieties have a productive potential of 9 t / ha, highly technician countries with protectionist agricultural policies can reach these roofs via genetic improvement and export quinoa with high added value and on a large scale. Possibly it will not be news that in a few years the original countries are forced to import quinoa [9,10].

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#sustainability Climate Change crops Ecosystem food quality Ecology Biodiversity

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