Journal articles on the topic 'Approximately 436 B.C.-358 B.C'

In vitro antagonistic potential of eleven isolates of Bacillus spp. against two phytopathogenic bacteria and one fungus was tested in order to identify potential biocontrol agents in vegetable crops. The Bacillus spp. isolates demonstrated different levels of antagonistic effect against the tested pathogenic microorganisms. Data in the study proved Xanthomonas vesicatoria to be more sensitive to Bacillus spp. strains than Clavibacter michiganensis subsp. michiganensis. Ten B. subtilis strains induced growth inhibition of X. vesicatoria, while a strain of B. pumilus did not affect the growth of that bacterium. The largest inhibition zones against X. vesicatoria were induced by strains B-319, B-325 and B-358. The pathogenic strain C. michiganensis subsp. michiganensis was most inhibited by two B. subtilis strains (B-338 and B-348) with mean inhibition zone diameters of up to 20 mm. B. subtilis strain B-319 which was the best in inhibiting X. vesicatoria, showed the lowest inhibitory effect on C. michiganensis subsp. michiganensis. The largest growth inhibition percentage of Verticillium sp. (PGI approximately 70%) was induced by B. subtilis strains B-310 and B-322. The other B. subtilis strains showed PGI values ranging from 45% to 68%, while B. pumilus strain B-335 had the least antagonistic potential (PGI =34.43%) against the pathogen. This study identified at least one suitable biocontrol candidate, B. subtilis strain B-358, as effective in vitro against all three vegetable pathogens.

A dilution/quench technique was used to monitor the time course of chemical modification on the heparin-cofactor (a) and progressive thrombin-inhibitory (b) activities of human antithrombin III. Treatment of antithrombin III (AT III) with 2,4,6-trinitrobenzenesulphonate at pH 8.3 and 25 degrees C leads to the loss of (a) at 60-fold more rapid rate than the loss of (b). This is consistent with previous reports [Rosenberg & Damus (1973) J. Biol. Chem. 248, 6490-6505; Pecon & Blackburn (1984) J. Biol. Chem. 259, 935-938] that lysine residues are involved in the binding of heparin to AT III, but not in thrombin binding. Treatment of AT III with phenylglyoxal at pH 8.3 and 25 degrees C again leads to a more rapid loss of (a) than of (b), with the loss of the former proceeding at a 4-fold faster rate. The presence of heparin during modification with phenylglyoxal significantly decreases the rate of loss of (a). Full loss of (a) correlates with the modification of seven arginine residues per inhibitor molecule, whereas loss of (b) does not commence until approximately four arginine residues are modified and is complete upon the modification of approximately eleven arginine residues per inhibitor molecule. This suggests that (the) arginine residue(s) in AT III are involved in the binding of heparin in addition to the known role of Arg-393 at the thrombin-recognition site [Rosenberg & Damus (1973) J. Biol. Chem. 248, 6490-6505; Jörnvall, Fish & Björk (1979) FEBS Lett. 106, 358-362].

Two hundred thirty-five oriented cores were taken in a stratigraphic sequence of 575 m of red sediments from the Namurian Maringouin and Shepody formations. These samples were taken from the south-dipping limb of the Maringouin Anticline on the Maringouin Peninsula in eastern New Brunswick. An additional 59 samples of the Maringouin and Shepody formations were taken from the north-dipping limb for separate fold tests. Progressive thermal demagnetization to temperatures as high as 700 °C permitted the resolution of an intermediate-temperature, syn-folding B component and a high-temperature, pre-folding C component. The C-component mean for the Maringouin Formation yields a north paleopole at 32.4°N, 121.3°E. The Shepody Formation mean yields a paleopole at 35.7°N, 124.3°E. The circles of confidence (α95) for the Maringouin and Shepody formations are 3.8 °and 4.6°, respectively. Plotting sample polarity versus stratigraphie position yields the first coherent magnetic stratigraphy from the late Paleozoic of North America, with five discrete reversed-polarity and five discrete normal-polarity zones. The magnetic field in Namurian times had approximately 50% normal and 50% reversed polarity.

Embryo bisection has been used to produce identical twins, to increase the pregnancy rate per embryo, and for preimplantation diagnosis. However, the invasive nature of splitting might affect development decreasing embryo survival. In general, after bisection or biopsy, embryos are transferred to surrogate mothers and their competence evaluated in terms of implantation and pregnancy maintenance. However, this makes it difficult to evaluate the immediate response of each embryo to bisection. Our aim was to evaluate embryo growth during the 5 days following bisection by using an extended in vitro culture system. We postulated that bisected blastocysts are able to counteract the injury and expand in size until Day 13 of in vitro culture. Two experiments were performed. First, two different culture systems were evaluated to determine the best to support embryo development from Day 9 to 13. One system consisted of conventional culture in plastic (CCP), while the other one included co-culture with endometrial cells derived from a cycling cow (CC). Both used SOFaa supplemented with 3 mg mL–1 of fatty acid-free BSA and 2% FBS in 4-well dishes. Twenty-six nonbisected in vitro-derived blastocysts were cultured. Embryo size and survival were recorded daily. All living embryos were measured with Micrometrics™ SE Premium software and statistical analyses were performed using the Kruskal–Wallis test. From Day 9 to 11, blastocysts cultured in the CCP system had smaller diameters than those cultured in CC [Day 9: CC 358 µm, CCP 277 µm (P = 0.04); Day 10: CC 456 µm, CCP 340 µm (P = 0.005); and Day 11: CC 535 µm, CCP 408 µm (P = 0.02)]. However, on Day 12 and 13, no difference was observed in embryo diameters [(Day 12: CC 560 µm, CCP 411 µm (P = 0.1) and Day 13: CC 470 µm, CCP 474 µm (P = 0.9)]. Additionally, embryos with diameters less than 200 µm on Day 9 did not develop further independent of the culture system (P < 0.001). Thus, in the second experiment, to determine embryo size after bisection, only well-expanded grade 1 blastocysts >200 µm were used in the conventional CCP system. Twenty four Day 8 bisected (B) and nonbisected (C) blastocysts were cultured from Day 9 until 13. In the bisected group, one-half was kept for further gene expression analysis. Significant differences were observed in embryo diameter between both groups on Day 9 and 10 of culture [Day 9: B 321 µm, C 277 µm (P = 0.05); D10: B 436 µm, C 340 µm (P = 0.01)]. However, on Days 11, 12, and 13, no differences in diameter were observed (Day 11: B 411 µm, C 408 µm (P = 0.8); Day 12: B 394 µm, C 411 µm (P = 0.5); Day 13: B 316 µm, C 474 µm (P = 0.3)]. In conclusion, we show that bovine embryos are capable of developing in vitro until Day 13, and embryo diameter on Day 9 impacts on the subsequent in vitro survival of nonsplit embryos, regardless of culture system. Finally, on Day 11 of culture, the split embryos were able to overcome the injury caused by the bisection procedure and expanded in size similar to controls, until at least Day 13 of culture.

trans-syn Photodimer of polyoxyethylene bisthymine (1) [1,1′-(ethylenedioxydiethylene)dithymine], trans-syn-1,8-(ethylenedioxyethylene)hexahydro-4a,4b-dimethylcyclobuta[1,2-d:4,3-d′]dipyrimidine-2,4,5,7(3H,6H)-tetrone (2), C16H22N4O6, Mr = 366.4, triclinic P{\bar 1}, a = 7.900 (4), b = 10.061 (8), c = 12.048 (4) Å, α = 71.37 (5), β = 78.13 (13), γ = 72.17 (5)°, V = 857.7 (9) Å3, Z = 2, Dx = 1.418 g cm−3, μ = 0.103 mm−1, F(000) = 388, R = 0.0953 for 1482 observed reflections; 1,1′-(1,2-phenylenedioxydiethylene)dithymine (3), C20H22N4O6, Mr = 414.4, triclinic, P{\bar 1}, a = 8.558 (2), b = 11.077 (2), c = 11.437 (3) Å, α = 108.57 (2), β = 94.37 (2), γ = 99.77 (2)°, V = 1003.1 (4) Å3, Z = 2, Dx = 1.377 g cm−3, μ = 0.096 mm−1, F(000) = 436, R = 0.0443 for all reflections; trans-syn photodimer of (3), 1,8-(1,2-phenylenedioxydiethylene)hexahydro-4a,4b-dimethylcyclobuta[1,2-d:4,3-d′]dipyrimidine-2,4,5,7(3H,6H)-tetrone (4), C20H22N4O6, Mr = 414.4, monoclinic, P21/n, a = 10.357 (4), b = 14.578 (5), c = 12.676 (2) Å, β = 97.99 (3)°, V = 1895 (1) Å3, Z = 4, Dx = 1.453 g cm−3, μ = 0.102 mm−1, F(000) = 872, R = 0.0628 for 1077 observed reflections. The bisthymines (1) and (3) on photodimerization produce cyclobutane-linked trans-syn thymine dimers as the exclusive products. The planar thymines stack one above the other in (1), while they are approximately perpendicular in (3). Planarities of the thymines are altered on photodimerization. The stereochemistry around the cyclobutane ring in photoproducts (2) and (4) is highly strained. The distortions in the bond lengths and angles due to this strain are concentrated mainly on the C5 and C6 atoms, which form the interpyramidal bonds. This strain is comparatively less in the rigid catechol-linked dimer (4). The N3 atom of thymines forms hydrogen bonds in all structures. Photoproducts show closer packing compared with the parent compounds.

1529 Background: Approximately 30 million people worldwide are estimated to have liver cirrhosis (LC) associated with hepatitis C virus (HCV). In addition, patients with HCV-associated liver cirrhosis (HCV-LC) have a high risk of developing hepatocellular carcinoma (HCC). Namely, 60–80% of the patients may develop HCC in 10 years. So, preventing these patients from developing HCC is an urgent problem to be solved. To find a way to prevent the development of hepatocellular carcinoma (HCC) from hepatitis C-virus associated liver cirrhosis (HCV-LC), an analysis of the HCV-LC patients who had reduction therapy of ALT levels was performed. Patients and Methods: Seventy-four consecutive HCV-LC patients of Child Stage A were followed for > 10 years for the development of HCC. They were divided into two groups: In group A, the reduction therapy for ALT levels is aggressively performed and in group B, the reduction therapy was not performed aggressively. Results: Initial ALT was higher in group A than in group B, but there was no significance. After reduction therapy, ALT level was significantly high in group A in the first year. However, there were no significant differences between ALT levels in groups A and B of after the second year. In total, 39 patients out of 74 (52.7%) developed HCC within 13 years from the beginning of the study and 35 patients did not developed HCC in the same observation period. The incidence of HCC development in group B [65.7% (23/35)] was significantly higher than in group A [41.0% (16/39)] (p=0.039). The median HCC developing time in group A (12.8 years) was significantly longer than in group B (3.8 years) (p=0.0013). Multivariate analysis demonstrated that reduction therapy and ALT levels were the significant factors affecting HCC development. The incidence of HCC development in group B was 5.8 times higher than in group A. The high and unclassified ALT groups were 4.6 times and 2.2 times higher than in the low ALT group. Conclusion: The chances of surviving for more than 10 years without developing HCC in the HCV-LC patients of Child Stage A were far more favorable in group A than in group B. These results suggest that aggressive reduction therapy for ALT levels in HCV-LC patients could significantly prevent HCC development. No significant financial relationships to disclose.

Data on the trends in the prevalence of chronic liver disease (CLD) in Korea are scarce. This study aimed to evaluate whether the CLD prevalence changed between 1998–2001 and 2016–2017. Data were extracted from the Korea National Health and Nutrition Examination Survey (1998–2001 to 2016–2017; n=25,893). Non-alcoholic fatty liver disease (NAFLD) was defined as a hepatic steatosis index >36 in the absence of any other evidence of CLD. The definition of alcoholrelated liver disease (ALD) was excessive alcohol consumption (≥210 g/week for men and ≥140 g/week for women) and an ALD/NAFLD index >0. The prevalence of NAFLD increased from 18.6% (95% confidence interval [CI], 17.8–19.5%) in 1998–2001 to 21.5% (95% CI, 20.6–22.6%) in 2016–2017. During the same time period, increases were observed in the prevalence of obesity (27.0 vs. 35.1%), central obesity (29.4 vs. 36.0%), diabetes (7.5 vs. 10.6%), and excessive drinking (7.3 vs. 10.5%). ALD prevalence also increased from 3.8% (95% CI, 3.4–4.2%) to 7.0% (95% CI, 6.4–7.6%). In contrast, chronic hepatitis B decreased from 5.1% (95% CI, 4.6–5.5%) to 3.4% (95% CI, 3.0–3.8%). The prevalence of chronic hepatitis C was approximately 0.3% in 2016–2017. The prevalence of NAFLD and ALD increase among Korean adults. Our results suggest potential targets for interventions to reduce the future burden of CLD.

ObjectiveThis investigation aimed at examining whether circulating inflammatory biomarkers C-reactive protein (CRP), interleukin-6 (IL6), and alpha 1-antichymotrypsin (ACT) were related to cerebrovascular disease (CVD) assessed by MRI.MethodsThe study included nondemented elderly participants of a community-based, multiethnic cohort, who received baseline MRI scans and had CRP (n = 508), ACT (435), and IL6 (N = 357) measured by ELISA. Silent brain infarcts and white matter hyperintensities (WMH) were derived from all available MRI scans at baseline, approximately 4.4 years after blood sample collection for inflammatory biomarkers. Repeated assessments of infarcts and WMH, as well as microbleeds assessment, were performed at follow-up MRI visits around 4.5 years later. Cross-sectional and longitudinal relationship between inflammatory biomarkers and CVD were analyzed using appropriate logistic regression models, generalized linear models, or COX models.ResultsAfter adjusting for age, sex, ethnicity, education, APOE genotype, and intracranial volume, 1 SD increase in log10IL6 was associated with infarcts on MRI {odds ratio [OR] (95% confidence interval [CI]) = 1.28 [1.02–1.60], p = 0.033}, and 1 SD increase in log10CRP and log10ACT was associated with microbleeds (OR [95% CI] = 1.46 [1.02–2.09], p = 0.041; and 1.65 [1.11–2.46], p = 0.013; respectively). One SD increase in log10ACT was also associated with larger WMH at the follow-up MRI (b = 0.103, p = 0.012) and increased accumulation of WMH volume (b = 0.062, p = 0.041) during follow-up. The associations remained significant after additional adjustment of vascular risk factors and excluding participants with clinical stroke.ConclusionsAmong older adults, increased circulating inflammatory biomarkers were associated with the presence of infarcts and microbleeds, WMH burden, and progression of WMH.

Eucalyptus spp. are grown on approximately 2 million ha in China and is the most important genus in forest plantations worldwide. An outbreak of cankers and shoot blight was observed for the first time on Eucalyptus grandis in China during May of 2008. Symptoms included dieback of shoots and branches, lesions and canker formation on the stems, and clear or yellow-to-red exudates on stems and branches. Isolations were made from affected trees in Shilin County from May to July of 2008. Diseased samples were plated on potato dextrose agar (PDA) and incubated at 26°C. Fungal isolates developed copious, white, aerial mycelium that became dark gray after 5 to 6 days and formed black pycnidia after 14 days. Conidia were hyaline, aseptate, thin walled, fusiform, and measured 19 to 28 × 4 to 6 μm. Ascospores were hyaline, aseptate, and widest from the middle to upper third (17 to 28 × 6 to 13 μm). Identity was confirmed by analysis of the rDNA internal transcribed spacer region (ITSI-5.8S-ITS2) with primers ITS1 and ITS4. BLAST searches showed 99 to 100% identity with Botryosphaeria dothidea isolates from GenBank (Accession Nos. FJ358703 and EU080916). Representative sequences of B. dothidea from eucalyptus from China were deposited into GenBank (Accession Nos. FJ517657 and FJ517658). On the basis of morphological and molecular results, the fungus isolated from diseased eucalyptus wood was confirmed to be B. dothidea. Pathogenicity tests were conducted by stem inoculation of 10-month-old E. grandis seedlings. Two experiments were conducted using two inoculation techniques. In the first experiment, 2-mm-diameter actively growing mycelium plugs of B. dothidea were applied to 2-mm-long bark wounds on the middle point of the stems, and control seedlings were inoculated with sterile PDA plugs in a similar fashion as above. Inoculated and control seedlings were inoculated in a greenhouse and watered as needed. In the second experiment, segments of branches (averaging 18 mm in diameter and 30 cm long) were inoculated with 5-mm-diameter plugs of actively growing mycelium. Control segments of branches were inoculated as previously described. The branches were incubated at 26°C in moist chambers. There were five replicate seedlings per inoculation technique. After 20 days, all E. grandis seedlings showed leaf wilting, Dark, vascular stem tissue was observed. Symptoms were more abundant on the segment of branches. After 6 days, vascular necroses that developed on the inoculated plants were 5.2 ± 1.2 cm. B. dothidea was reisolated from all inoculated symptomatic tissue; no symptoms were visible in the control seedlings and no fungus was isolated from them. These results confirm previous reports of B. dothidea causing canker and dieback symptoms of Eucalyptus species in Australia (2), the United States (1), and South Africa (3). To our knowledge, this is the first report of B. dothidea causing canker disease on eucalyptus in China. References: (1) E. L. Barnard et al. Plant Dis. 71:358, 1987. (2) E. M. Davison and C. S. Tay. Plant Dis. 67:1285, 1983. (3) H. Smith et al. Plant Pathol. 43:1031, 1994.

Curly dock (Rumex crispus L.) is a perennial deciduous plant in the family Polygonaceae. It is widely distributed in grasslands and orchards and is an important weed that is traditionally used as a medicinal herb. During the summers of 2000 and 2001, a severe anthracnose disease was observed on leaves of mature curly dock in the foothills near the western coastal area of Muchangpo, Ungchon, the district of Chungnam in Korea. Initial symptoms usually appeared in June as a small number of slightly soaked spots on leaves. Typical symptoms, generally observed in late July and August following a long, rainy, hot period, consisted of a number of brown leaf spots that expanded and often twisted, resulting in discoloration of whole parts to blackish brown or slightly reddish brown and defoliation. A fungus, which was isolated from the leaf lesion, was identified as Colletotrichum destructivum O'Gara based on previous descriptions (1,3). The fungus was characterized by conidia, which were long, relatively narrow, and straight to slightly curved with abruptly tapered and obtuse ends, complex appressoria, and cultures with apricotto salmon-colored sectors that lacked sclerotia. Conidial size ranged from 4.0 to 6.4 μm (average 4.8) × 10 to 23 μm (average 16.5). Setae were slender and straight but frequently flexuous, subulate, brown, and variable in length. C. destructivum has a teleomorph, Glomerella glycines (Hori) Lehman & Wolf, but the species is not well known, and the connection has not been studied in detail. The isolate has been deposited in the IMI Culture Collection as isolate IMI387103. The dimensions of conidia from the isolate matched those of C. destructivum N150 (GenBank Accession No. AF325064) isolated from Nicotiana tabacum (3). C. destructivum is distinguishable from C. gloeosporioides, whose spores are short and cylindrical with obtuse apices tapering slightly to a truncate base. Pathogenicity of the isolate was determined on 5-week-old leaves of curly dock. Leaves were inoculated with a conidial suspension of the fungus (approximately 1 × 106 conidia per ml), placed in a moist chamber for 3 days, and subsequently transferred to a growth chamber maintained at 25°C. Within 7 days after inoculation, symptoms appeared that were similar to those originally observed on leaflets. Uninoculated control (sprayed only with distilled water) leaves exposed to the same environmental conditions remained healthy. C. destructivum was consistently reisolated from infected leaves. C. destructivum has been reported as a pathogen on approximately 15 genera, including Medicago sativa, Trifolium spp., Cuscuta spp., and N. tabacum (1,3), and two fungal species, C. erumpens and C. rumicis-crispi, have been reported to cause anthracnose on R. crispus. To our knowledge, R. crispus represents a previously unreported host for C. destructivum causing anthracnose, although C. gloeosporioides has been reported as a pathogen of R. crispus in Korea (2). References: (1) A. P. Baxter et al. S. Afr. Tydskr. Plantk. 2:259, 1983. (2) B. S. Kim et al. Korean J. Plant Pathol. 14:358, 1998. (3) S. Shen et al. Mycol. Res. 105:1340, 2001.

Abstract Tissue factor–induced blood coagulation was studied in 20 individuals, for varying periods of time during 54 months, in contact pathway–inhibited whole blood at 37°C and evaluated in terms of the activation of various substrates. After quenching over time with inhibitors, the soluble phases were analyzed for thrombin–antithrombin III (TAT) complex formation, prothrombin fragments, platelet activation (osteonectin release), factor Va generation, fibrinopeptide (FP) A and FPB release, and factor XIII activation. TAT complex formation, for 35 experiments, showed an initiation phase (up to 4.6 ± 0.6 minutes) in which thrombin was generated at an average rate of 0.93 ± 0.3 nM/min catalyzed by about 1.3 pM prothrombinase yielding approximately 26 nM thrombin. During a subsequent propagation phase, thrombin was generated at a rate of 83.9 ± 3.8 nM/min by about 120 pM prothrombinase, reaching ultimate levels of 851 ± 53 nM. Clot time, determined subjectively, occurred at 4.7 ± 0.2 minutes and correlated with the inception of the propagation phase. The thrombin concentrations associated with the transitions to rapid product formation are 510 ± 180 pM for platelet activation (1.9 ± 0.2 minutes), 840 ± 280 pM for factor XIII activation and factor Va generation (2.2 ± 0.6 minutes), 1.3 ± 0.4 nM for FPA release (2.5 ± 0.7 minutes), 1.7 ± 0.5 nM for FPB release and prethrombin 2 (2.8 ± 0.8 minutes), 7.0 ± 2.2 nM for thrombin B chain (3.6 ± 0.2 minutes), and 26 ± 6.2 nM for the propagation phase of TAT formation (4.6 ± 0.6 minutes). These results illustrate that the initial activation of thrombin substrates occurs during the initiation phase at less than 2 nM thrombin (0.2%). Most thrombin (96%) is formed well after clotting occurs.

Abstract Abstract 3581 Thioredoxin-interacting protein (TXNIP) binds and inactivates thioredoxin (TRX) leading to increased reactive oxygen species (ROS) production. It has been previously shown that patients with CLL have high levels of ROS compared to normal individuals and that TRX has a protective function in CLL. In an initial pilot study of 17 patients (Koshy et al., AACR Annual Meeting, 2010) we found that TXNIP levels (avg. 0.82 ± 0.18, range 0.45–1.15) and ROS (avg. 7967 ± 2515, range 2340–12340) were positively correlated (ρ = 0.92). Although not statistically significant, TXNIP expression was higher in patients under observation than those receiving treatment (0.89 ± 0.18 vs. 0.76 ± 0.15). For further insights into the role of TXNIP in CLL, we chose the TCL-1 transgenic mouse model of B-CLL. These animals develop a CD5+ B-cell leukemia and express therapeutic targets and display sensitivity to therapeutic agents relevant in the treatment of CLL. Mice develop a clonal leukemia characterized by splenomegaly and increased lymphocyte counts in peripheral blood at a median age of 10 months. To first determine if TXNIP was altered in the progression of CLL in the TCL-1 mice, we isolated peripheral blood (PB) and bone marrow (BM) from mice of varying ages; 3 months (disease free), 7 months (pre-leukemic phase) and 11 months (end-stage disease). As shown previously, end-stage animals developed severe splenomegaly (1.32±0.82g vs. 0.16±0.08; p<0.05) compared to disease-free. These animals also demonstrated a 1.4 fold increase in TXNIP RNA in BM (0.62±0.08 vs. 0.87±0.12, p<0.05) and 1.5 fold in PB cells (0.81±0.06 vs. 1.18±0.20; p<0.05) associated with a 3.2 fold increase in ROS in BM (15212±1522 vs. 46812±11563, p=0.07)and 3.9 fold increase in PB (11150±560 vs. 39230±8671; p=0.7) compared to animals with no evidence of disease. Although ROS levels are regulated by several cellular redox mechanisms, we have shown in a breast cancer model, that TXNIP upregulation play a major role in ROS control through its interaction with TRX. We chose to alter the ROS levels by antioxidant therapy on animals with end-stage disease. The reason for this choice was to assess a faster way to interfere with the final product of TXNIP upregulation, rather than the protein itself. We wanted to prove that by reducing ROS we would have changed the course of the disease. For this purpose, we administered the antioxidant, N-acetylcysteine (NAC), in the drinking water and measured the changes in both ROS and TXNIP. Mice of approximately 10 months of age were given fresh NAC water every other day. After a treatment period of 4 weeks animals were sacrificed. Peripheral blood and splenocytes were stained for ROS and B220 expression. Bone marrow cells were stained for ROS, B220 and bone marrow lineage markers and evaluated by flow cytometry. Upon gross examination of animals, spleen weight was found to be reduced 2-fold in NAC treated animals compared to that of untreated (0.69±0.40 g vs. 1.3±0.77, p<0.05) although still higher than that expected of a healthy animal of the same age (approximately 0.25g). We found that ROS was reduced in the spleen 1.5 fold (59±29 vs. 81±41; p<0.05). B220 expression was increased 1.8 fold (1155±611 vs. 836±416; p<0.05) in spleen cells. In peripheral blood, we found that NAC reduced ROS levels 1.7 -fold (51±19 vs. 29±14; p<0.05) and B220 was decreased by 1.5-fold (857±213 vs. 575±105). Total bone marrow ROS levels were decreased 2- fold by NAC treatment (79±31 vs. 38±13; p<0.01). In the bone marrow, low levels of ROS expression have been associated with primitive hematopoietic stem cells. Indeed, in our studies we found the bone marrow could be separated into a ROShi and ROS lo population and that NAC treatment increased the percentage of ROSlo cells. The ratio of ROShi/ROSlo was decreased by 1.5 fold (1.7±0.4 vs. 1.1±0.2; p<0.05) This ROSlo population encompassed the Lin−, c-kit+, Sca-1+ cells. However while NAC treatment induced changes in ROS levels, as expected, TXNIP RNA levels were not found to differ between NAC-treated and untreated animals in any of the cell types tested. In conclusion, both TXNIP and ROS levels were increased with progression of the disease. Antioxidant treatment of animals with end-stage disease resulted in decreased ROS in all cell types tested, associated with phenotype changes of the disease. This finding sets the ground for further work in identifying therapeutic tools that interfere with TXNIP-ROS axis. Disclosures: Turturro: Celgene: Speakers Bureau; Genentech: Speakers Bureau; Spectrum Pharmaceuticals: Speakers Bureau.

A ocupação extensiva e, por vezes, indevida do solo pode resultar em uma série de problemas ambientais que aceleram a degradação de uma bacia hidrográfica, com consequências diretas na qualidade dos seus recursos hídricos. Esse trabalho avalia o impacto do uso e cobertura da terra em parâmetros quantitativos e de qualidade das águas superficiais da bacia hidrográfica do rio Pirapozinho. A produção cartográfica consistiu de mapa de uso e cobertura da terra, declividade, rede de drenagem, mapa de área de preservação permanente e mapa de Índice de Qualidade da Água (IQA). Tanto a coleta quanto as análises das águas superficiais da bacia foram desenvolvidas seguindo os procedimentos do Standard Methods. Os resultados das análises foram comparados com os valores de referência CONAMA, n° 357/05, alterada pela Resolução 410/2009 e pela 430/2011, sendo os padrões estabelecidos para rios de classe 2, mesma classe que se enquadra os corpos hídricos inseridos na bacia em estudo. Conclui-se que há um desequilíbrio ambiental na bacia do rio Pirapozinho. O mapa de uso e cobertura aponta que aproximadamente 90% da área da bacia encontra-se ocupada por pastagem e culturas. Outro aspecto é que mais de 84% da área de preservação permanente encontra-se desprovida de vegetação arbórea. Embora o mapa do IQA revele que a água da bacia possa ser classificada como boa (IQA entre 52 e 79) predominantemente, as análises laboratoriais apontam que há pontos na área com contaminação. Isto pode ser atribuído a atividades industriais e agropastoris desenvolvidas na bacia em estudo. Recomenda-se ações de manejo sustentável na bacia do rio Pirapozinho para a recuperação dos seus recursos naturais. A B S T R A C TExtensive and unduly occupation of soil may result in several environmental problems those contribute to accelerate the degradation of a hydrographic basin bringing diretly consequences to quality of its water resources. This paper evaluates the impact of land use and occupation on quantitative parameters and of superficial waters quality of the hydrographic basin of the Pirapozinho river. The cartographic products designed consisted of land use and occupation map, declivitity map, drainage network map, permanent preservation area map and map of Water Quality Index (WQI) of hydrographic basin. Both collection and analysis of the superficial waters samples were developed following the Standard Method procedure. The results of the laboratorial analyses were compared with the reference values at CONAMA 357/05 and 430/11 resolutions. We conclude that there is an environmental disequilibrium at the hydrographic basin of the Pirapozinho river. The land use and occupation map shows that approximately 90% of the area of the basin is occupied by pasture and crop fields. Other finding is that there is no arboreal vegetation in 84% of the permanent preservation area of the hydrographic basin. Although the WQI map revels that water of the basin can be classified as good (WQI among 52 and 79) predominately, the laboratorial analyses point out taht there is some points in area with contamination of water. This may be related to the industrial and agricultural activities performed in the area. We recommend sustainable management actions in the Pirapozinho river basin to recovery of its natural resources.Keywords: Cartographic production; water quality; environmental analysis; hydrographic basin.

Arsenic and Protein Expression: It might help to know the mechanism of As toxicity is described Introduction One of the largest public health problems at present is the drinking of water containing levels of Inorg-As that are known to be carcinogenic. The chronic ingestion of Inorg-As can results in skin cancer, urinary bladder cancer, lungs cancer, kidneys cancer, liver cancer, and cancer of other human organs 1-6. The molecular mechanisms of the carcinogenicity and toxicity of inorganic arsenic are not well understood 7–9. Many mechanisms of arsenic toxicity and carcinogenicity have been suggested 1, 7, 10 including chromosome abnormalities 11, oxidative stress 12, 13, altered growth factors 14, cell proliferation 15, altered DNA repair 16, altered DNA methylation patterns 17, inhibition of several key enzymes 18, gene amplification 19 etc. Some of these mechanisms result in alterations in protein expression. Proteomics is a powerful tool developed to enhance the study of complex biological system 20. This technique has been extensively employed to investigate the proteome response of cells to drugs and other diseases 21, 22. A proteome analysis of the Na-As (III) response in cultured lung cells found in vitro oxidative stress-induced apoptosis 23. In one of the study, hamsters were exposed to sodium arsenite (173 mg As/L) in drinking water for 6 days and several protein spots were over expressed and several were under expressed in the livers and urinary bladders of hamsters (Fig.) 24, 25. Hamsters were exposed to sodium arsenite (173 mg As/L) in drinking water for 6 days. The control hamsters were given tap water. The spot pairs of (A) equally expressed, (B) overexpressed, and (C) under expressed proteins in the liver tissues were shown. The amount of the protein is proportional to the volume of the protein peak. Transgelin was down-regulated, and GST-pi was up-regulated in the urinary bladder tissues of hamsters. In the liver tissues ornithine aminotransferase (OAT) was up-regulated, and senescence marker protein 30 (SMP 30), and fatty acid binding protein (FABP) were down-regulated. Down-regulation of transgelin has been noted in the urinary bladders of rats having bladder outlet obstruction 26. Ras-dependent and Ras-independent mechanisms can cause the down regulation of transgelin in human breast and colon carcinoma cell lines and patient-derived tumor samples 27. The loss of transgelin expression has been found in prostate cancer cells 28 and in human colonic neoplasms 29. It has been suggested that the loss of transgelin expression may be an important early event in tumor progression and a diagnostic marker for cancer development 26-29. Figure. Three-dimentional simulation of over-and under expressed protein spots in the livers of hamsters using Decyder software. Over-expression of GST-pi has been found in colon cancer tissues 30. Strong expression of GST-pi also has been found in gastric cancer 31, malignant melanoma 32, lung cancer 33, breast cancer 34 and a range of other human tumors 35. GST-pi has been up-regulated in transitional cell carcinoma of human urinary bladder 36. OAT has a role in regulating mitotic cell division and it is required for proper spindle assembly in human cancer cell 37. Ornithine amino transferase knockdown in human cervical carcinoma and osteosarcoma cells by RNA interference blocks cell division and causes cell death 37. It has been suggested that ornithine amino transferase has a role in regulating mitotic cell division and it is required for proper spindle assembly in human cancer cells 37. SMP 30 expressed mostly in the liver. By stimulating membrane calcium-pump activity it protects cells against various injuries 38. High levels of saturated, branched chain fatty acids are deleterious to cells and resulting in lipid accumulation and cytotoxicity. FABP expression has protected the cells against branched chain saturated fatty acid 39. Proteomics would be a powerful tool to know the unknown cellular mechanisms of arsenic toxicity in humans. References. NRC (National Research Council). (2001). Arsenic in Drinking Water. Update to the 1999 Arsenic in Drinking Water Report. National Academy Press, Washington, DC. Chen, C. J., Chen, C. , Wu, M. M., Kuo, T. L. (1992). Cancer potential in liver, lung, bladder, and kidney due to ingested inorganic arsenic in drinking water. Br. J. Cancer 66, 888-892. Hopenhayn-Rich, C., M.L. Biggs, A. Fuchs, et al. 1996. Bladder cancer mortality with arsenic in drinking water in Argentina. Epidemiology 7: 117–124. International Agency for Research on Cancer. (1987). In IARC Monograph on the Evaluation of Carcinogenicity Risk to Humans. Overall Evaluation of Carcinogenicity:An Update of IARC Monographs 1–42 (Suppl. 7). Lyon, France: International Agency for Research on Can-cer, pp. 100–106. Rossman, T.G., Uddin, A.N., and Burns, F.J. (2004). Evidence that arsenite acts as a cocarcinogen in skin cancer. Toxicol. Appl. Pharmacol. 198: 394–404. Smith, A.H., Hopenhayn-Rich, C., Bates, M.N., et al. (1992). Cancer risks from arsenic in drinking water. Environ. Health Perspect. 97: 259–267. Aposhian, H.V. & Aposhian, M.M. (2006). Arsenic toxicology: five questions. Chem. Res. Toxicol. 19: 1–15. Goering, P.L., Aposhian, H.V., Mass, M.J., et al. (1999). The enigma of arsenic carcinogenesis: role of metabolism. Toxicol. Sci. 49: 5–14. Waalkes, M.P., Liu, J., Ward, J.M., et al. (2004). Mechanisms underlying arsenic carcinogenesis: hypersensitivity of mice exposed to inorganic arsenic during gestation. Toxicology 198: 31–38. Kitchin, K. T., Recent advances in arsenic carcinogenesis: modes of action, animal model systems, and methylated arsenic metabolites. Appl. Pharmacol. 2001, 172, 249-261. Beckman, G., Beckman, L., Nordenson, I., Chromosome aberrations in workers exposed to arsenic. Environ. Health Perspect. 1977, 19, 145-146. Yamanaka, K., Hoshino, M., Okanoto, M., Sawamura, R., et al., Induction of DNA damage by dimethylarsine, a metabolite of inorganic arsenics, is for the major part likely due to its peroxyl radical. Biophys. Res. Commun. 1990, 168, 58-64. Yamanaka, K., Okada, S., Induction of lung-specific DNA damage by metabolically methylated arsenics via the production of free radicals. Health Perspect. 1994, 102, 37-40. Simeonova, P., Luster, M. I., Mechanisms of arsenic carcinogenicity:Genetic or epigenetic mechanisms? J. Environ. Pathol. Toxicol. Oncol. 2000, 19, 281-286. Popovicova, J., Moser, G. J., Goldsworthy, T. , Tice, R. R., Carcinogenicity and co-carcinogenicity of sodium arsenite in p53+/- male mice. Toxicologist 2000, 54, 134. Li, J. H., Rossman, T. G., Mechanism of co-mutagenesis of sodium arsenite with N-methyl-N-nitrosourea. Bi Trace Elem. 1989, 21, 373-381. Zhao, C. Q., Young, M. R., Diwan, B. A., Coogan, T. P., et , Association of arsenic-induced malignant transformation with DNA hypomethylation and aberrant gene expression. Proc. Natl. Acad. Sci. USA 1997, 94, 10907-10912. Abernathy, C. O., Lui, Y. P., Longfellow, D., Aposhian, H. , et al., Arsenic: Health effects, mechanisms of actions and research issues. Environ. Health Perspect. 1999, 107, 593-597. Lee, T. C., Tanaka, N., Lamb, P. W., Gilmer, T. M., et al., Induction of gene amplification by arsenic. Science 1988, 241, 79-81. Lau, A. T., He, Q. Y., Chiu, J. F. (2003). Proteomic technology and its biomedical applications. Acta Biochim. Bioph Sin. 35, 965-975. Jungblut, P. R., Zimny-Arndt, U., Zeindl-Eberhart, E., Stulik, J., Koupilova, K., Pleissner, K. P., Otto, A., Muller, E. C., Sokolowska-Kohler, W., Grabher, G., Stoffler, G. (1999). Proteomics in human disease: cancer, heart and infectious diseases. Electrophoresis 20, 2100-2110. Hanash, S. M., Madoz-Gurpide, J., Misek, D. E. (2002). Identification of novel targets for cancer therapy using expression proteomics. Leukemia 16, 478-485. Lau, A. T., He, Q. Y., Chiu, J. F. (2004). A proteome analysis of the arsenite response in cultured lung cells: evidence for in vitro oxidative stress-induced apoptosis. J. 382, 641-650. Chowdhury, U. K., Aposhian, H. V. (2008). Protein expression in the livers and urinary bladders of hamsters exposed to sodium arsenite. A N. Y. Acad. Sci. 1140, 325-334. Chowdhury, U.K. Expression of proteins in the tissues of hamsters exposed to sodium arsenite. Int. J. of Toxicol., 2021, 1, 1-8. Kim, H-J., Sohng, I., Kim, D-H., Lee, D-C., et al., 2005. Investigation of early protein changes in the urinary bladder following partial bladder outlet obstruction by proteomic approach. J. Korean Med. Sci. 20, 1000-1005. Shields, J.M., Rogers-Graham, K., Der, C.J., 2002. Loss of transgelin in breast and colon tumors and in RIE-1 cells by Ras deregulation of gene expression through Raf-independent pathways. J. Biol. Chem. 277, 9790-9799. Yang, Z., Chang, Y- J., Miyamoto, H., Ni, J., et al., Transgelin functions as a suppressor via inhibition of ARA54-enhanced androgen receptor transactivation and prostate cancer cell grown. Mol. Endocrinol. 2007, 21, 343-358. Yeo, , Kim, D- K., Park, H. J., Oh, T. Y., et al., Loss of transgelin in repeated bouts of ulcerative colitis-induced colon carcinogenesis. Proteomics 2006, 6, 1158-1165. Tsuchida, S., Sekine, Y., Shineha, R., Nishihira, T., et al., Elevation of the placental glutathione S-transferase form (GST-PI) in tumor tissues and the levels in sera of patients with cancer. Cancer Re 1989, 43, 5225-5229. Tsutsumi, M., Sugisaki, T., Makino, T., Miyagi, N., et al., Oncofetal expression of glutathione S-transferase placental form in human stomach carcinomas. 1987, 78, 631-633. Mannervik, B., Castro, V. M., Danielson, U. H., Tahir, M. K., et , Expression of class Pi glutathione transferase in human malignant melanoma cells. Carcinogenesis (Lond.) 1987, 8, 1929-1932. Di llio, C., Del Boccio, G., Aceto, A., Casaccia, R., et al,. Elevation of glutathione transferase activity in human lung tumor. Carcinogenesis (Lond.) 1988, 9, 335-340. Sreenath, A. S., Ravi, K. K., Reddy, G. V., Sreedevi, B., et al., Evidence for the association of synaptotagmin with glutathione S- transferase: implications for a novel function in human breast cancer. Clinical Biochem. 2005, 38, 436-443. Shea, T. C., Kelley S. L, Henner, W. D., Identification of an anionic form of glutathione transferase present in many human tumors and human tumor cell lines. Cancer Res. 1988, 48, 527-533. 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Since the summer of 2006, bacterial boll rot of cotton has been observed on fruits of ‘Xinluzao 31’ (Xinluzao 6 × Acala) in Xinjiang Province. It resulted in as much as 20% yield loss in several fields. Symptoms do not appear on the outer carpel. In the infected cotton bolls, fibers do not mature completely and seed tissue exhibits brown necrotic coloration. Lint and seeds from 24 surface-disinfested cotton bolls were triturated and plated onto King's medium B (KB). Plates were incubated at 28°C for 48 h. Forty eight strains with yellow pigmentation on KB were characterized. All were nonfluorescent on KB, gram negative, facultatively anaerobic, unable to produce indole from tryptophan, able to reduce nitrate to nitrite, and produce acid from glucose, cellobiose, lactose, melibiose, and melonate. In addition, 16S rDNA in seven strains was amplified with universal primers (1). The PCR products were cloned into pGEM-T easy vector and sequenced. A BLAST search of the seven sequences against the GenBank nucleotide library indicated 100% identity with the 16S rDNA sequence of Enterobacter agglomerans strain A80. Then an additional primer pair, pagF and pagR (3), was used for more specific amplification of Pantoea agglomerans 16S rDNA, which resulted in single highly specific fragments of approximately 1 kb. On the basis of morphological, physiological, biochemical characteristics, and 16S rRNA gene sequence analysis, we identified the bacterium to be P. agglomerans. To confirm pathogenicity, cell suspensions (1 × 108 CFU/ml) of eight representative strains were used to inoculate cotton at peak bolling stage in the field. Cell suspensions, or water as the control, were applied to stigma scars, wall sutures, and scratch wounds on bracts, calyxes, and bolls. Alternatively, a needle was used to puncture through a drop of suspension placed on the boll wall suture and bracts. At least 20 bolls or flowers were inoculated with each bacterial strain per inoculation method. Infection occurred only when bacterial injections breached the endocarp of the boll either through the carpel wall or a suture between carpel sections. Disease symptoms developed 1 week postinoculation. The inoculated organism was reisolated from the diseased tissues. P. agglomerans is generally regarded to be a soil saprophyte or leaf epiphyte, but strains can opportunistically infect plants triggering gall formations or human wounds causing septic arthritis. The disease symptoms and pathogen characteristics observed in this study are identical to those reported in the United States (2). To our knowledge, this is the first report of P. agglomerans causing boll rot of cotton in China. References: (1) S. Manulisi and I. Barash. Mol. Plant Pathol. 4:307, 2003. (2) E. G. Medrano et al. J. Appl. Microbiol. 103:436, 2007. (3) S. Vorwerk et al. Agric. For. Entomol. 9:57, 2007.

AbstractCarcass quality was evaluated in 35 male and 41 female lambs, 38 expressing the callipyge phenotype (Cg) and 38 controls expressing the normal phenotype (N). Lambs were from Romanov and Suffolk × Romanov ewes mated to two rams heterozygous for the callipyge locus and slaughtered at approximately 44 kg live weight. The Cg lambs reached slaughter weight at 163 days of age, 13 days younger, and had a dressing proportion 0·524 , 0·029 points higher. Also they had more leg (341 v. 308 g/kg), less loin (315 v. 328 g/kg) and less shoulder (340 v. 360 g/kg) than the N lambs. The leg, loin and shoulder wholesale cuts and half the carcass of Cg lambs had 81, 113, 77, and 92 g/kg more lean and 52, 98, 59, and 72 g/kg less fat than N lambs, respectively. The Cg lambs also had larger loin-eye area (18·5 v.13·1 cm2), less backfat at the C (4·1 v. 6·1 mm) and at the GR locations (11·0 v. 15·2 mm) and less internal fat (202 v. 339 g) than the N lambs. Dissection of the 12th rib indicated that Cg lambs had more muscle (574 v. 462 g/kg), less fat (276 v. 358 g/kg) (both P < 0·001) and similar (V > 0·05) bone (155 v. 265 g/kg) to N lambs. Colour of the longissimus muscle was significantly paler in Cg lambs. Intramuscular fat and dry matter of longissimus muscle were significantly lower (49 v. 120 and 247 v. 253 g/kg) in Cg than in N lambs. Cg lambs were significantly different (P < 0·05) from N lambs for all traits studied except age at slaughter, tissues at the GR location, proportion of bone in the 12th rib and the L* colour of the longissimus muscle. Breed type effect was significant only for age at slaughter, whereas the effect of sex was significant for all traits except for proportion of lean in the leg, b* colour and intramuscular fat content of the longissimus muscle. No interactions were significant except phenotype × breed type on fat thickness over the longissimus muscle. Post-mortem ageing of the longissimus muscle for 9 or 15 days caused a significant reduction in muscle toughness in Cg lambs compared with those aged for 3 days (113 v. 98 Newtons) but the muscle was still tougher than that from N lambs (with a shear force of 64 and 51 Newtons at 3 and 9 or 15 days post mortem). The principle component analysis including carcass conformation and composition traits as well as meat quality traits was indicative of a distinctly different carcass ofCg compared with N lambs. The first two principal components explained 0·70 of the total variation.

In March 2013, symptoms of mild leaf curling, mosaic, and interveinal yellowing were observed in tobacco (Nicotiana tabacum) plants grown in a row surrounding the exterior of a greenhouse containing a tomato crop in Guía de Isora, Tenerife (Canary Islands, Spain). The tobacco plants were found lightly infested by the whitefly (Hemiptera: Aleyrodidae) Bemisia tabaci. The greenhouses in this area are devoted to the commercial production of tomato. The farmers grow some tobacco plants inside and outside of them as a reservoir of parasitoids and depredators of B. tabaci. This insect is the natural vector of the main viruses severely affecting tomato in the Canary Islands, the begomovirus Tomato yellow leaf curl virus and the crinivirus Tomato chlorosis virus (ToCV). ToCV was detected in Spain in 1997 (2) and has become established in most of the coastal provinces of eastern and southern continental Spain and in the Canary Islands. Approximately 50% of the tomato plants grown inside the greenhouse close to the tobacco plants showed typical ToCV symptoms, and infection by this virus was confirmed in the seven plants tested by reverse transcription (RT)-PCR using specific coat protein gene (CP) primers (see below). Total RNA was extracted with TRIzol Reagent (Invitrogen) from leaves of five tobacco plants showing the symptoms mentioned above and analyzed by dot-blot hybridization using digoxigenin-labeled RNA probes to the CP gene of ToCV. Positive signal was obtained for all five plants. RT-PCR reactions were performed with specific primers for the detection of ToCV, MA380(+) (5′-GTGAGACCCCGATGACAGAT-3′) and MA381(-) (5′-TACAGTTCCTTGCCCTCGTT-3′), specific to the CP gene (ToCV RNA 2) (3), and MA396(+) (5′-TGGTCGAACAGTTTGAGAGC-3′) and MA397(-) (5′-TGAACTCGAATTGGGACAGA-3′), specific to the RNA-dependent RNA polymerase (RdRp) gene (ToCV RNA 1) (1). DNA fragments of the expected size (436 and 763 bp, respectively) were obtained, thus supporting the presence of ToCV in the symptomatic samples. The amplified product of the RdRp gene fragment from one sample was directly sequenced (Macrogen Inc., South Korea) and resulted closely related to ToCV isolates from Sudan (GenBank Accession No. JN411686, 99.6% nt identity) and Spain (DQ983480, 99.4% nt identity), thereby confirming the infection by this virus. Partial sequence of the ToCV isolate from tobacco was deposited in GenBank under accession no. KJ175084. In addition, all five plants resulted positive when analyzed by ELISA for Tomato spotted wilt virus and Potato virus Y and by PCR for Tomato yellow leaf curl virus (data not shown), all three viruses reported to infect naturally tobacco. Although tobacco has been reported as an experimental host of ToCV (4), to our knowledge, this is the first report of this species as a natural host of this virus. The finding of ToCV infecting tobacco raises the question of whether this virus could emerge as a pathogen of this crop and questions the use that farmers make of tobacco as reservoirs of natural enemies for whitefly control in tomato. References: (1) G. Lozano et al. J. Virol. 83:12973, 2009. (2) J. Navas-Castillo et al. Plant Dis. 84:835, 2000. (3) H. P. Trenado et al. Eur. J. Plant Pathol. 118:193, 2007 (4) W. M. Wintermantel and G. C. Wisler. Plant Dis. 90:814, 2006.

Abstract Abstract 3298 Introduction: Thrombotic thrombocytopenic purpura (TTP) is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and microvascular thrombosis. TTP is usually due to acquired, autoimmune deficiency of ADAMTS13, a metalloprotease that cleaves von Willebrand factor (VWF) and inhibits the growth of platelet thrombi. Most patients respond to treatment with plasma exchange, but inhibitory autoantibodies and persistent ADAMTS13 deficiency are associated with an increased risk of refractory or relapsing disease. Therefore, ADAMTS13 activity and inhibitor assays can be useful for diagnosis, prognosis, and monitoring the response to therapy. ADAMTS13 assays currently use the fluorogenic substrate FRETS-VWF73, which absorbs/emits at 340 nm/430 nm. These spectral properties make FRETS-VWF73 subject to interference from plasma proteins, hemoglobin and bilirubin. To avoid this problem, plasma is diluted at least 1:20, which reduces assay sensitivity to 5% of normal ADAMTS13 levels and prevents the detection of some clinically relevant inhibitors. We have addressed these limitations by developing FRETS-rVWF71, a recombinant fluorogenic substrate with chromophores that absorb/emit in the near infrared. Methods: The substrate polypeptide corresponds to VWF residues Gln1599-Arg1668, with mutation N1610C to introduce a reactive thiol and K1617R to remove an amino group that competes with the N-terminus for chemical modification. This peptide was expressed in E. coli as a thioredoxin-(His6)-fusion protein, purified by Ni2+-NTA chromatography, and digested with TEV protease to remove the thioredoxin. After modification at Cys1610 with DyLight 633-maleimide (abs 638 nm, em 658 nm) and at the N-terminus with IRDye QC-1 N-hydroxysuccinimidyl ester (abs 500–800 nm), the substrate FRETS-rVWF71 was purified by C18-HPLC. Assays were performed with 1 μM FRETS-rVWF71 under physiological buffer conditions (50 mM HEPES, pH 7.4, 150 mM NaCl, 10 mM CaCl2) to facilitate the assay of samples containing up to 95% plasma (Figure, panel A). Inhibitor assays were performed by preincubating equal volumes of pooled normal plasma and serially diluted patient plasma, followed by addition of an equal volume of buffer containing FRETS-rVWF71. Product generation was monitored in a fluorescence microplate reader with 635 nm excitation and 660 nm emission filters. Results: Serum and matched samples of plasma anticoagulated with citrate or heparin had equivalent ADAMTS13 activity that was stable indefinitely at −20°C. Bilirubin (>20 mg/dL) did not inhibit ADAMTS13 activity. As reported, hemoglobin was a weak inhibitor (EC50 approximately 1g/dL). Neither bilirubin nor hemoglobin interfered with product detection. Healthy donors (Li+-heparin plasma, n = 96) had a mean ADAMTS13 activity of 107.1 ± 18% (SD). Intra-assay and inter-assay coefficients of variation (CV) were <2%. No significant differences were observed by gender (male 104.9 ± 16%, n = 51; female 109.6 ± 16%, n = 45) or ethnicity (African American 102.7 ± 24%, n = 22; Caucasian 108.2 ± 17%, n = 48; Hispanic 108.4 ± 15%, n = 26). Results with FRETS-rVWF71 and FRETS-VWF73 correlated well with an inter-assay CV of 3.8%. For patients with idiopathic TTP, assays with FRETS-rVWF71 allowed accurate measurement of ADAMTS13 activity levels with a limit of detection of <0.5%. Inhibitor assays with FRETS-rVWF71 in minimally diluted plasma gave inhibitor titers approximately 3-fold higher than assays with FRETS-VWF73 at the 1:20 dilution required for that substrate. For example, a patient with an inhibitor titer of 4.8 U/ml in the FRETS-rVWF71 assay (Figure, Panel B) had an inhibitor titer of 1.8 U/ml in a FRETS-VWF73-based assay. Conclusions: The use of chromophores that absorb/emit in the near infrared avoids interference from blood proteins, bilirubin or hemoglobin. The combination of brighter chromophores and compatibility with undiluted plasma makes ADAMTS13 activity assays with FRETS-rVWF71 substantially more sensitive than with FRETS-VWF73. Higher sensitivity allows discrimination between very low levels of activity that may influence the risk of relapse in congenital or acquired TTP. Optimized detection of ADAMTS13 inhibitors will facilitate the monitoring of antibody responses to therapy and should help to determine why some patients with acquired TTP relapse and others do not. Disclosures: No relevant conflicts of interest to declare.

Biosurfactants are natural substances produced by several bacterial and fungal organisms that are amphiphilic and are extracellular (a part of the cell membrane). Biosurfactants can reduce the stress between solids and liquids on the surface and at the end. Biosurfactants have several properties, i.e. they are stable, less harmful, as well as readily degradable, and extremely eco-friendly. Biosurfactants also have a wide range of industrial uses because they are a versatile category of chemical substances. The principal justification for conducting such research was the isolation of possible biosurfactants containing bacteria. Sampling was performed for the isolation of bacteria producing biosurfactants from different oil-polluted sites That is to say, experiment for emulsification, test for oil spreading, test for drop collapse, and measure for hemolysis. The capability to produce biosurfactants was seen in 22 different isolates from polluted sites B1, B2, and B3. Through different biochemical tests and Gram staining, it was identified that isolated bacterial strains are Pseudomonas spp and that is Pseudomonas aeruginosa. The procedure used as characterizing biosurfactants was the TLC plate’s procedure, by using TLC plates process yellow dots emerged after spraying on silica gel plates with an throne and ninhydrin reagents. These yellow spots confirmed the presence and production of rhamnolipid in the biosurfactant. Hence, it was concluded that identified strains in the study can be helpful in the heavy metals, pesticides, and hydrocarbons bio-degradation and bioremediation. These may also be used as biological control agents to protect plants from various pathogens, resulting in improved crop yields. Introduction Biosurfactants are natural substances produced by several bacterial and fungal organisms that are amphiphilic and are extracellular (a part of the cell membrane) (Chen et al., 2007; Ghayyomiet al., 2012). Main purpose of the bio-surfactantsgeneration or production is a consequence of financial availability (Van Dyke et al., 1993 It is reported that almost 50 percent of the world's surfactants are used because of the need for cleaning agents as well as the rate of growth grows every day (Deleu and Paquot, 2004). Appropriate use of bio-surfactants will control environmental emissions what these are the most dangerous, constantly rising gradually and disrupting the routine maintenance of life every day. Awareness campaign initiatives have been introduced and also increase for environmental laws, various innovative approaches need to be implemented and even the issue of pollution focused entirely. Developing appropriate advanced technologies to help clear up chemicals and toxins from the ecosystem, like hydrocarbons (both inorganic and organic). Studies on biosurfactants are being launched by scholars and researchers with significant health issues like adverse environmental effects, air contamination, environmental change, and waste management (Makkar and Cameotra, 2002 Biosurfactants contribute to expanded demand for such microbial products as alternatives to chemical surfactants (Benatet al., 2000). Microbes seem to have the capability to degrade contaminants, but their biodegradation is limited leading to hydrophobicity, low solubility in water, and inadequate bioavailability, of such pollutants (Patil, et al., 2012). GhayyomiJazeh, Mishraet. al (2001) those bacteria that produce biosurfactants were isolated from the site of petroleum spills and afterward, 160 strains and as well as 59 strains were able to produce biosurfactants have shown better performance in a test for hemolysis of blood, and 45 strains with positive findings within oil spread experiment were applied in the laboratory to isolate and segregate the media cultured Banat process (Rahman et al., 2002) These were observed and researched that biosurfactants of Pseudomonas aeruginosa spp are most likely to disrupt the bonding of hydrocarbons like nonadecane, octa, Hexa, and hepta, in marine Water contaminated with oil spills up To approximately 47%, 53%, 73% and 60%(Abrar et al., 2020). Current study concluded that the isolated strain having the ability to degrade hydrocarbon as well as the ability to degrade the heavy metal. The strain also can protect the plant from various diseases. The present research found that the isolated strain is capable of degrading hydrocarbon while also being capable of degrading the heavy metal. As well as the strain does have the capability to defend plants from different diseases. Material And Methods Area of Study The investigation was conducted at HazaraUniversity(HU) Microbiology Laboratory, MansehraPakistan. Assemblage of Samples Thehomestay area of the city Mansehra Pakistan which is named as a township, where oil spills arose, oil spills soil samples were obtained as well as sampling from various Mansehra automobile workshops were also done. Sterilized bags of polythene were being used to collect samples of the soil, after thatthe sample was taken towards the Hazara University (HU) Mansehra Microbiology Laboratory to examine and extract bacterial strains that could develop biosurfactants. The soil temperature at the time of sample selection was around 30 ° C. The pH was also verified by Galvano science companies at the time of selection by pH meter, and the pH being reported was 7. Preparation of Media 15 x 100 mm Petri dishes were being used to prepare the media. Agar plates were thoroughly cleaned with water from the tap and then carefully covered in aluminum foil following cleaning then placed within autoclave at 121°C for about 15 min at 15 psi for sterilization. The nutrient agar which contains 0.5% NaCl, 0.3% beef extract, 0.5% peptone, and 1.5% agar, in 500 ml of distilled water, 14 g of the nutrient agar media (Merck) were dissolved. The nutrient level used mainly for the production of non-fastidious species. Nutrient agar is widely known as it's capable of growing a variety of bacteria types and provides nutrients required for the growth of bacteria. Upon sufficient dissolution of such nutrient agar in distilled water, these were then sterilized by autoclaving for 15 min at 15 psi in the autoclave and held at 121 °C Upon autoclaving, pouring of the media was done in laminar flow hood, and then packed and placed for yet more use in a fridge at 4°C. 2.4 Preparation of serial dilution The bacteria are isolated using the serial dilution process. During this process, 10 test tubes were taken and distilled water (9ml) was added in each tube. After that tubes were put for 15 minutes in the autoclave machine at 121°C. After that 1gm of a crude oil sample from the soil was added in a test tube containing distilled water. Further, 1 ml of the solution was taken from the first test tube and poured to the adjoining tubes for the preparation dilution as under . Afterward, 10μl of the solution was pipetted from both the dilution of and shifted for spread culture techniques, then incubated the plates at 37°C for 48hrs. Biosurfactants extraction Firstly, in nutrient broth solution theculture of bacteria was added and inoculated with oil, the bacterial colony was then incubated at the temperature of 25°C in a shaking incubator just for 7 days. Incubation after seven days of trembling. Thebacterial Crop was then taken and centrifuged at 5000rpm at temperature 4°C for 20minutes. Following centrifugation, the supernatant was collected and then mixed in the equivalent amount in Methanol: Chloroform. White sediment was then retained and collected for further use . Bacterial Colonies Isolation 1 g of the soil polluted with oil was diluted serially up to 106 dilutions.10 μl of 104 and 106 dilutions for spread culture were transferred to the MSM agar plates and nutrient agar. The plates were then incubated at 37°C for 48hrs. Twenty-two morphologically separate colonies were separated for further specific examination just after the incubation and processed by using the technique of streak plate. Screening of Isolates’ Biosurfactants Behavior To check the activity of biosurfactants produced by the bacterial species the following methods of screening were done. Hemolytic Activity of Biosurfactants for Erythrocytes Blood agar containing 5% of blood was prepared as after the fresh isolates were added and inoculated on blood agar plates, then the plates were taken and placed in the incubator at temperature 37°C for 48hrs (Rashediet al., 2005). Thereafter the observation of clear zone in the colonies indicated the existence of bacterial species that produce biosurfactants. This experiment was undertaken to control the ability of isolated bacteria to induce blood agar hemolysis. Three forms of hemolysis usually involve; alpha, beta, and hemolysis of the gamma. The agar underneath the species is dark greenish, then it is Alpha, the yellowish color produced in beta hemolysis and gamma hemolysis does not affect the bacterial sppwhichadded on the plates (Anandaraj and Thivakaran, 2010). Bio-surfactant identification with process of CTAB MSM (Mineral salt agar medium) with (2%) of glucose serving both as carbon source, (0.5 mg / ml) acetyl-tri-methyl-ammonium-bromide (CTAB), and methylene blue (MB: 0.2 mg/ml) are used to detect anionic bio-surfactants (Satpute et al., 2008). For this method, thirty microliters (30μl) of cell-free supernatant were added to each of the wells of the methylene blue agar plate that comprises of borer (4 mm in diameter). after that, the incubation of the plates was done for 48-72 hrs at 37°C. Just after incubation in each of the wells, a dark blue halo zone was being used to show the successful anionic bio-surfactant production. Table 1: Composition of MSM Media S. No Ingredients Amount (gm/L) I Potassium dihydrogen phosphate (KH2PO4) II Magnesium Sulfate (MgSO4) III Iron Sulfate (FeSO4) IV Sodium Nitrate (NaNO3) V Calcium Chloride (CaCl2) VI Ammonium Sulfate (NH4)2SO4 Technique for Spreading of Oil A sufficient number of isolated bacteria were inoculated into a solution of 100ml nutrient broth. Over 3 days, the culture was incubated at 37 ° C in a rotating shaker incubator (150 rpm). After that biosurfactants synthesis was checked in culture suspensions (Priya and Usharani, 2009; Anandaraj and Thivakaran, 2010). For this process, thirty milliliters (30ml) of distilled water was added in a Petri dish. In the middle of the distilled water, 1 milliliter (1ml) of diesel oil was added, and then a centrifuged twenty microliter (20μl) culture was introduced to the middle of a plate, which was isolated from oil spilled soil or local oily groundwater. The species producing the bio-surfactant displace the hydrocarbons and disperse it even in the water. Then it was calculated and analyzed within 1 mint (Ali et al., 2013). Technique for Drop collapse In this process, 96-wellsformed in each of the plates of nutrient agar. Afterwards, all the 96-wells of microliter plates was then filled withmineral oil of about 2ml. Then stabilized the plate at 37oC for 1 hour, after which the oil surface was filled with 5μl of supernatant culture. Therefore, the drop shape was taken to be observed on the oil surface after 1min. The drop which was collapsed, generated by the supernatant culture which is used to signify positive(+ive) outcome and the drops which stayed the same and displayed no changeindicates negative(-ive) outcome. And was taking distilled water as a control(Plaza et al., 2006). Emulsification index The emulsification index was calculated, as stated by the process followed by Cooper and Goldenberg (1981) In this process, 2 ml of kerosene oil was taken and inserted in each of the test tubes to the same amount of cell-free supernatant, and then homogenized for 2 min in a vortex at high speed and allowed for 24 hours to stand. The emulsification steadiness was then determined after the 24 hours, and the emulsification value was estimated by measuring the emulsified layer height by the total liquid layer height, then multiplied by 100. Quantification for the Dry weight of Biosurfactants The bacterial colony was inserted and inoculated in the nutrient broth medium, followed by oil and centrifuged at 5000rpm and after that, the supernatant was clutched and treated with chloroform and methanol and mixed. The white colored deposits were taken and used for the furtherprocess of dry weight. Afterwards, took the clean Petri plate and determined the empty plate weight. Next, the sediment was poured onto Petri plates. Now, for the drying process the hot air oven was used and set the 100ºC of temperature for 30minutes and the plates were put in the oven. After the drying process, the plates were weighted again. The dry weight was calculated for the biosurfactants using the formula which described below: Selected strains Identification and their characterization Instead, various basic biochemical methods were used to identify the isolated bacterial strains. Various biochemical tests, such as Gram staining, Oxidase test, Urease test.Catalase test, Methyl red test, Motility test, Indole test, Starch hydrolysis, Citrate test, Spore staining, Gelatin hydrolysis. Then afterwards, for the preliminary characterization of the biosurfactant, the thin layer chromatography process was used. Physical characterization of the strains selected Gram staining First, on the slide, using the wire loop the bacterial pure culture was taken, and smear was prepared on the slide, and then a drop of purified water was applied. Then, the sterile loop or needle was correctly mixed the bacterial colony and purified water, then mixed up until it is somewhat turbid. Then, spirit lamp was used to fixed the bacterial smear on slide and cooled to room temperature. With this glass slide was loaded with solution of crystal violet and stood for 1minute anddistilled water was applied on slide. Meanwhile the slide was submerged for 1 minute with the iodine solution, and then flushed and rinsed with water. Therefore, decolorizer of about 1 to 2 drops(5 percent acetone and 95 percent alcohol) were added to the slide’s smear and stand for 30seconds, and then treated with water. After then slide was rinsed with safranin for 60seconds, and then treated with water anddry in air. Microscopic analysis was done with 100x objective lenses using emersion oil on smear. Cell morphology The isolates of the bacterial cell were gram stained on slides and then the slides were observed under the light microscope, showing the shape and color of the cells. Biochemical characterization of the selected strains Catalase test Aim of this study is to identify, evaluate and examine that, whether or not the microbes are capable of producing catalase enzymes, while catalase is a protective enzyme, i.e. catalase has the potential to protect against the lethal chemicals known as (H2O2). In this study a bacterial culture that was clarified overnight was used. This culture has been smeared on a glass slide, and 3 percent hydrogen peroxide (H2O2) has been applied and observed on smear. Effects have been observed for bubble formation. Citrate test This study was performed to check the amount or ingest the citrate as the carbon and energy supply for growth and metabolism. Medium containing bromothymol blue and sodium citrate as pH indicator, bacterial was introduced. Ammonium chloride is also present in this medium used as a nitrogen source. Results were noted with variations of color from green to blue. Urease test The capability of urease enzyme for degrading urea was calculated in this bacterial capacity test. Bacterial culture was taken and inoculated for 48 hours at 37 ° C in urease broth, and then color was observed. Methyl red test Through using the process known as mixed acid fermentation which is used to evaluate the bacteria's acid production. The bacterial culture was taken and introduced in the broth of MR-VP and then incubated for 3days at a temperature of 37°C. Two (2) to three (3) drops of Methyl red were added in the broth medium after the incubation period. The change in broth color was observed for final results after a few seconds. Indole test Through using the process to assess the bacteria 's capability to crash indole from tryptophane molecules. After the 24 hours of incubated, taken the fresh inoculum of bacteria and then inserted into the tryptone medium, 24 hours of incubation of about 30oC, 2ml of the tryptone broth medium was added into a sterile test tube. Kovac's reagent was taken to be added (few drops) in sterile test tube and stimulated for a few minutes, and variations of color were detected. Gelatin test It is the approach assess to figure out the use of enzymes known as gelatins from bacterial organisms that precipitate the gelatin. Fresh inoculum of bacteria was taken after 24 hours, and inserted into the media of gelatin agar. This was incubated for around 24 hours, so the temperature did not exceed 30 ° C. Media was observed after incubation time. Starch hydrolysis Several of the micro-organisms that use the starch as a carbon energysource. Therefore, this method has been used to assess whether or not bacteria may use starch as a source of carbon. The bacterial fresh inoculum was spread on the petri starch agar plates, and after that the plate was incubated for 24 hours andmaintained the temperature at 30 to 35 ° C, then gradually applying the supplements of iodine to the plates to flow the change, and then examining the plates. Preliminary characterization of the strains selected Experimental characterization of the bio-surfactant was performed by using the process of TLC (Anandaraj et al., 2010). On a silica gel plate, crude portion of the rudimentary bio-surfactant was separated using Methanol: Chloroform: water (CH3OH: CHCl3: H2O) in the ratio of as an eluent with a different color producing reagents. Ninhydrin reagent (0.5 g ninhydrin in 100ml anhydrous acetone) was used to find bio-surfactant lipopeptide as red spots and anthrone reagent (1 g anthrone in 5ml sulfuric acid combined with 95ml ethanol) as yellow spots to identify rhamnolipid bio-surfactant (Yin et al., 2008). Results and Discussion Isolation of bacteria At first, twenty-two (22) strains from a polluted soil sample were isolated from nutrient agar media.Mixed culture provided by these colonies, so they were taken and smeared on the plates of nutrient agar and then fresh inoculum was collected and stored at temperature of 4oC for the further analysis. Bio-surfactants (surface-active compounds)are formed by a variety of amphiphilic bacterial and fungal organisms that are extracellular (a part of the cellular membrane) (Chen et al., 2007). Screening of Isolated strains for biosurfactant producing colonies Different experiments were carried out to identify, isolate and screen bacteria that are capable of generating bio-surfactants and that is Oil spreading technique(OST), blood hemolysis test(BHT), CTAB test, Emulsification operation. There were twenty-two distinct isolates observed in the current research. And the B1, B2 and B3culture were taken and selected from the twenty-two (22) strains isolated from the polluted spot, which were found to produce biosurfactant. And the oil spreading technique showed promising results for these strains. And strain B2 showed a greater displacement of oil and this is 4 mm. Oil spreading method is quick and often easy to handle, and this technique requires no particular equipment, only a very small amount of sample is used. This approach can be applied when the production and quantity of biosurfactant is small (Plaza et al., 2006) and (Youssef et al., 2004) Only bacterial cultures have been allocated and screened for bacterial species that can generate or use biosurfactants. Just three (3) strainsamong them presented the best results.Those 3 strain,s (B1, B2 and B3) were selected as an additional analysis. Blood hemolysis test On the petri plates of blood agar, the . Isolated bacteriaof B1, B2 and B3 were taken andstreak at the temperature about 37°C for 48 hours. Strain B1 demonstrated β (Beta) hemolysis after the incubation cycle and B2 and B3strains demonstrated γ (Gamma) hemolysis. The B1 strain had an emulsification index of about 74 percent and that was very high as compared to 70 percent for B2 and about 53 percent for B3 respectively. Around the same time, B1 strain showed β (Beta) hemolysis and γ (Gamma) hemolysis was shown bystrains B2 and B3 on the platesof bloodagar. The β hemolysisshowed by the strain B1 in the blood agar test, and the strain B2 and B3 showed γ (Gamma) hemolysis. It is determined that 20 percent strains that are the bestproducer of rhamnolipid have not fully lysed the blood, because the ability of the producer strains capacity not be responsible for the hemolytic activity. According to many researchers, who have shown that this is not such an effective tool for biosurfactant detection due to many bioproducts that may also induce red blood cell lysis, that is not so sufficient to be the surface-active molecule (Youssef et al., 2004). (Rashedi and others, 2005). Table2 Blood Hemolysis Test CTAB agar plate test This test confirms the anionic biosurfactants development. After plate incubation at a temperature of 37 ° C for 72 hours, dark blue hollow zone was existedaround each of the B1 strains wells, which clearly indicated the positive (+ive) development of anionic Biofactant. In addition, the B1 and B2 strains showed positive (+ I ve) results and, in the CTAB analysis, the B3 strain was found to be negative (-ive). The growing microorganisms when secreted the anionic biosurfactants on the plates of CTAB (cetyl-tri-methyl-ammonium-bromide) and methylene blue, then as a result the dark blue-purple insoluble ion pairs formed on the plates. The halo zone around each of the colonies was developed that can recognize rhamnolipid production and that was dark blue in colour, and could correlate with production of rhamnolipid (Siegmund et al., 1991). As indicated in (Fig1) Fig1: B1 positive on CTAB agar plate Oil Spreading Technique The oil was displaced by B1, B2and B3 strains in this test strain and showed a zone that was so clear. The bacterial strains capable of developing biosurfactant were tested and separated from the sample of soil which was oil spilled and brought from the District of Mansehra, Pakistan and from automobile workshops of Mansehra. As shown in (Fig.2). Fig.2: Results of Oil Spreading by B1, B2 and B3Table 3;.Test for oil spreads Bacterial culture Formation of zone (mm) Readings B,1 B,2 B,3 Drop-collapse technique During this process the drop shape was observed at the oil surface. As seen in Fig 3, the collapsed drop was provided by the supernatant culture B1 , B2 and B3.. Emulsification index Emulsification stability was measured with the use of kerosene oilin this test, and then observed the results. Since this emulsification index was calculated by dividing the height of the emulsion layer by the total height of the liquid layer and then multiplying by 100, as shown in the formulation below. Emulsification index Emulsification stability was measured with the use of kerosene oilin this test, and then observed the results. Since this emulsification index was calculated by dividing the height of the emulsion layer by the total height of the liquid layer and then multiplying by 100, as shown in the formulation below. Fig 3: Result of Drop-collapse test Table 4: The activity of Biosurfactant emulsification Dry weight of bio-surfactants In this examination, white-colored sediment was collected. Then measured the weight of the sterile Petri plate which was empty in the first step. Then, the sediment was poured into plates. The plates were taken and weighted after 30 minutes of drying on a hot air oven, following the process of drying. The weight of biosurfactants (dry weight) was measured using the following formulations: Fig 4: Dry weight of biosurfactants Table: 5: Dry weight of the biosurfactants Bacterial Culture Weight of the plate (g) biosurfactant in The plate after drying (g) Dry weight of Biosurfactant (g) B,1 B,2 B,3 Identification of selected strains and their characterization Gram staining For structural applications, and stroke analysis gram staining method was used.(Fig.5) shows findings from the process of gram staining. Fig 5: Microscopic view of Gram staining Biochemical identification of bacterial strains and their characterization Specific biochemical studies were performed to identify the species for further recognition and characterization. The bio-surfactant producing microorganism was found to be Pseudomonas aeruginosa after conducting various characterizations and the biochemical tests(Eric Deziel et al., 1996), Which can be used to further analyze and study the industrial development of the biosurfactant. Rhamnolipid is also isolated and produced from the Pseudomonas aeruginosa species on the silica gel plate (Rashedi et al., 2005), a form of biosurfactants highly recommended for processes of bioremediation. All the findings collected from biochemical testing were labeled as Berge 's Manual and it revealed that the protected microorganism was (Pseudomonas aeruginosa). Results of biochemical test were tabulated in (Table.5) Table 6: Bacterial strain identification Tests B1 B2 B3 Gram staining Negative Negative Negative Oxidases Positive e Positive Positive Catalase Positive Positive Positive Indole Positive Negative Negative Citrate Positive Negative Negative Urease Negative Positive Negative Nitrate Positive Positive Positive Motility Positive Positive Positive Gelatin hydrolysis Positive Negative Negative Lactose Negative Positive Positive Methyl red Negative Positive Positive Voges Proskauer Negative Negative Negative Fig 6: Results of biochemical tests(A) Methyl red and Voges Proskauer tests (b) catalase tests (c) oxidase tests (d) indole tests (e) citrate tests (g) lactose tests (h) urease tests Preliminary bacterial strain’s characterization The plates showed yellow dots, when sprayed with anthrone reagent. It indicated the existence of biosurfactants of rhamnolipid in the organism on the plate of TLC as seen in theFig.7 Fig 7: Biosurfactant characterization by TLC Conclusion Biosurfactant development is exciting and perceptible across industries to clean up oil waste and pollutants, particularly in the ecosystem.Compared with chemical surfactants, the biosurfactants are less harmful. It plays an important role in defining the advantages and the importance of industrial applications. Therefore, it is not possible to disregard the growing role and importance of biosurfactants in environmental sustainability.Biosurfactant formulations which can be used for bacterial, fungal, and viral organisms as growth inhibitors. Such biosurfactant inhibition properties can make them components that are applicable to Numerous illnesses that are used as medicinal agents. Therefore it was decided that the described strain could be used as a potential source for heavy metal bioremediation pesticide and hydrocarbon polluted sites. And also used as shielding the plant from different pathogens, contributing to improved crop yields. There is no doubt that the biosurfactants are a multifunctional, advanced, versatile, long-lasting and updated type not only for the twenty-first century but beyond. Conflict of interest The authors declared that they have no conflict of interest and the paper presents their own work which does not been infringe any third-party rights, especially authorship of any part of the article is an original contribution, not published before and not being under consideration for publication elsewhere. References Ali, S.R.; Chowdhury, B.R.; Mondal, P. and Rajak, S. “Screening and characterization of biosurfactants producing microorganism from natural environment (Whey spilled soil)”. Nat. Sci. Res. 2013, 3(13), 34–64. 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Hamsters were exposed to sodium arsenite (173 mg As/L) in drinking water for 6 days. Equal amounts of proteins from urinary bladder or liver extracts of control and arsenic-treated hamsters were labeled with Cy3 and Cy5 dyes, respectively. After differential in gel electrophoresis and analysis by the DeCyder software, several protein spots were found to be down-regulated and several were up regulated. Our experiments indicated that in the bladder tissues of hamsters exposed to arsenite, transgelin was down-regulated and GST-pi was up-regulated. The loss of transgelin expression has been reported to be an important early event in tumor progression and a diagnostic marker for cancer development [29-32]. Down-regulation of transgelin expression may be associated with the carcinogenicity of inorganic arsenic in the urinary bladder. In the liver of arsenite-treated hamsters, ornithine aminotransferase was up-regulated, and senescence marker protein 30 and fatty acid binding protein were down-regulated. The volume ratio changes of these proteins in the bladder and liver of hamsters exposed to arsenite were significantly different than that of control hamsters. Introduction Chronic exposure to inorganic arsenic can cause cancer of the skin, lungs, urinary bladder, kidneys, and liver [1-6]. The molecular mechanisms of the carcinogenicity and toxicity of inorganic arsenic are not well understood [7-9). Humans chronically exposed to inorganic arsenic excrete MMA(V), DMA(V) and the more toxic +3 oxidation state arsenic biotransformants MMA(III) and DMA (III) in their urine [10, 11], which are carcinogen [12]· After injection of mice with sodium arsenate, the highest concentrations of the very toxic MMA(III) and DMA(III) were in the kidneys and urinary bladder tissue, respectively, as shown by experiments of Chowdhury et al [13]. Many mechanisms of arsenic toxicity and carcinogenicity have been suggested [1, 7, 14] including chromosome abnormalities [15], oxidative stress [16, 17], altered growth factors [18], cell proliferation [19], altered DNA repair [20], altered DNA methylation patterns [21], inhibition of several key enzymes [22], gene amplification [23] etc. Some of these mechanisms result in alterations in protein expression. Methods for analyzing multiple proteins have advanced greatly in the last several years. In particularly, mass spectrometry (MS) and tandem MS (MS/MS) are used to analyze peptides following protein isolation using two-dimensional (2-D) gel electrophoresis and proteolytic digestion [24]. In the present study, Differential In Gel Electrophoresis (DIGE) coupled with Mass Spectrometry (MS) has been used to study some of the proteomic changes in the urinary bladder and liver of hamsters exposed to sodium arsenite in their drinking water. Our results indicated that transgelin was down-regulated and GST-pi was up-regulated in the bladder tissues. In the liver tissues ornithine aminotransferase was up-regulated, and senescence marker protein 30, and fatty acid binding protein were down-regulated. Materials and Methods Chemicals Tris, Urea, IPG strips, IPG buffer, CHAPS, Dry Strip Cover Fluid, Bind Silane, lodoacetamide, Cy3 and Cy5 were from GE Healthcare (formally known as Amersham Biosciences, Uppsala, Sweden). Thiourea, glycerol, SDS, DTT, and APS were from Sigma-Aldrich (St. Louis, MO, USA). Glycine was from USB (Cleveland, OH, USA). Acrylamide Bis 40% was from Bio-Rad (Hercules, CA, USA). All other chemicals and biochemicals used were of analytical grade. All solutions were made with Milli-Q water. Animals Male hamsters (Golden Syrian), 4 weeks of age, were purchased from Harlan Sprague Dawley, USA. Upon arrival, hamsters were acclimated in the University of Arizona animal care facility for at least 1 week and maintained in an environmentally controlled animal facility operating on a 12-h dark/12-h light cycle and at 22-24°C. They were provided with Teklad (Indianapolis, IN) 4% Mouse/Rat Diet # 7001 and water, ad libitum, throughout the acclimation and experimentation periods. Sample preparation and labelling Hamsters were exposed to sodium arsenite (173 mg) in drinking water for 6 days and the control hamsters were given tap water. On the 6th day hamsters were decapitated rapidly by guillotine. Urinary bladder tissues and liver were removed, blotted on tissue papers (Kimtech Science, Precision Wipes), and weighed. Hamster urinary bladder or liver tissues were homogenized in lysis buffer (30mMTris, 2M thiourea, 7M urea, and 4% w/w CHAPS adjusted to pH 8.5 with dilute HCI), at 4°C using a glass homogenizer and a Teflon coated steel pestle; transferred to a 5 ml acid-washed polypropylene tube, placed on ice and sonicated 3 times for 15 seconds. The sonicate was centrifuged at 12,000 rpm for 10 minutes at 4°C. Small aliquots of the supernatants were stored at -80°C until use (generally within one week). Protein concentration was determined by the method of Bradford [25] using bovine serum albumin as a standard. Fifty micrograms of lysate protein was labeled with 400 pmol of Cy3 Dye (for control homogenate sample) and Cy5 Dye (for arsenic-treated urinary bladder or liver homogenate sample). The samples containing proteins and dyes were incubated for 30 min on ice in the dark. To stop the labeling reaction, 1uL of 10 mM lysine was added followed by incubation for 10 min on ice in the dark. To each of the appropriate dye-labeled protein samples, an additional 200 ug of urinary bladderor liver unlabeled protein from control hamster sample or arsenic-treated hamster sample was added to the appropriate sample. Differentially labeled samples were combined into a single Microfuge tube (total protein 500 ug); protein was mixed with an equal volume of 2x sample buffer [2M thiourea, 7M urea, pH 3-10 pharmalyte for isoelectric focusing 2% (v/v), DTT 2% (w/v), CHAPS 4% (w/v)]; and was incubated on ice in the dark for 10 min. The combined samples containing 500 ug of total protein were mixed with rehydration buffer [CHAPS 4% (w/v), 8M urea, 13mM DTT, IPG buffer (3-10) 1% (v/v) and trace amount of bromophenol blue]. The 450 ul sample containing rehydration buffer was slowly pipetted into the slot of the ImmobilinedryStripReswelling Tray and any large bubbles were removed. The IPG strip (linear pH 3-10, 24 cm) was placed (gel side down) into the slot, covered with drystrip cover fluid (Fig. 1), and the lid of the Reswelling Tray was closed. The ImmobillineDryStrip was allowed to rehydrate at room temperature for 24 hours. First dimension Isoelectric focusing (IEF) The labeled sample was loaded using the cup loading method on universal strip holder. IEF was then carried out on EttanIPGphor II using multistep protocol (6 hr @ 500 V, 6 hr @ 1000 V, 8 hr @ 8000 V). The focused IPG strip was equilibrated in two steps (reduction and alkylation) by equilibrating the strip for 10 min first in 10 ml of 50mM Tris (pH 8.8), 6M urea, 30% (v/v) glycerol, 2% (w/v) SDS, and 0.5% (w/v) DTT, followed by another 10 min in 10 ml of 50mM Tris (pH 8.8), 6M urea, 30% (v/v) glycerol, 2% (w/v) SDS, and 4.5% (w/v) iodoacetamide to prepare it for the second dimension electrophoresis. Second dimension SDS-PAGE The equilibrated IPG strip was used for protein separation by 2D-gel electrophoresis (DIGE). The strip was sealed at the top of the acrylamide gel for the second dimension (vertical) (12.5% polyacrylamide gel, 20x25 cm x 1.5 mm) with 0.5% (w/v) agarose in SDS running buffer [25 mMTris, 192 mM Glycine, and 0.1% (w/v) SDS]. Electrophoresis was performed in an Ettan DALT six electrophoresis unit (Amersham Biosciences) at 1.5 watts per gel, until the tracking dye reached the anodic end of the gel. Image analysis and post-staining The gel then was imaged directly between glass plates on the Typhoon 9410 variable mode imager (Sunnyvale, CA, USA) using optimal excitation/emission wavelength for each DIGE fluor: Cy3 (532/580 nm) and Cy5 (633/670 nm). The DIGE images were previewed and checked with Image Quant software (GE Healthcare) where all the two separate gel images could be viewed as a single gel image. DeCyde v.5.02 was used to analyze the DIGE images as described in the Ettan DIGE User Manual (GE Healthcare). The appropriate up-/down regulated spots were filtered based on an average volume ratio of ± over 1.2 fold. After image acquisition, the gel was fixed overnight in a solution containing 40% ethanol and 10% acetic acid. The fixed gel was stained with SyproRuby (BioRad) according to the manufacturer protocol (Bio-Rad Labs., 2000 Alfred Nobel Drive, Hercules, CA 94547). Identification of proteins by MS Protein spot picking and digestion Sypro Ruby stained gels were imaged using an Investigator ProPic and HT Analyzer software, both from Genomic Solutions (Ann Arbor, MI). Protein spots of interest that matched those imaged using the DIGE Cy3/Cy5 labels were picked robotically, digested using trypsin as described previously [24] and saved for mass spectrometry identification. Liquid chromatography (LC)- MS/MS analysis LC-MS/MS analyses were carried out using a 3D quadrupole ion trap massspectrometer (ThermoFinnigan LCQ DECA XP PLUS; ThermoFinnigan, San Jose, CA) equipped with a Michrom Paradigm MS4 HPLC (MichromBiosources, Auburn, CA) and a nanospray source, or with a linear quadrupole ion trap mass spectrometer (ThermoFinnigan LTQ), also equipped with a Michrom MS4 HPLC and a nanospray source. Peptides were eluted from a 15 cm pulled tip capillary column (100 um I.D. x 360 um O.D.; 3-5 um tip opening) packed with 7 cm Vydac C18 (Vydac, Hesperia, CA) material (5 µm, 300 Å pore size), using a gradient of 0-65% solvent B (98% methanol/2% water/0.5% formic acid/0.01% triflouroacetic acid) over a 60 min period at a flow rate of 350 nL/min. The ESI positive mode spray voltage was set at 1.6 kV, and the capillary temperature was set at 200°C. Dependent data scanning was performed by the Xcalibur v 1.3 software on the LCQ DECA XP+ or v 1.4 on the LTQ [27], with a default charge of 2, an isolation width of 1.5 amu, an activation amplitude of 35%, activation time of 50 msec, and a minimal signal of 10,000 ion counts (100 ion counts on the LTQ). Global dependent data settings were as follows: reject mass width of 1.5 amu, dynamic exclusion enabled, exclusion mass width of 1.5 amu, repeat count of 1, repeat duration of a min, and exclusion duration of 5 min. Scan event series were included one full scan with mass range of 350-2000 Da, followed by 3 dependent MS/MS scans of the most intense ion. Database searching Tandem MS spectra of peptides were analyzed with Turbo SEQUEST, version 3.1 (ThermoFinnigan), a program that allows the correlation of experimental tandem MS data with theoretical spectra generated from known protein sequences. All spectra were searched against the latest version of the non redundant protein database from the National Center for Biotechnology Information (NCBI 2006; at that time, the database contained 3,783,042 entries). Statistical analysis The means and standard error were calculated. The Student's t-test was used to analyze the significance of the difference between the control and arsenite exposed hamsters. P values less than 0.05 were considered significant. The reproducibility was confirmed in separate experiments. Results Analysis of proteins expression After DIGE (Fig. 1), the gel was scanned by a Typhoon Scanner and the relative amount of protein from sample 1 (treated hamster) as compared to sample 2 (control hamster) was determined (Figs. 2, 3). A green spot indicates that the amount of protein from sodium arsenite-treated hamster sample was less than that of the control sample. A red spot indicates that the amount of protein from the sodium arsenite-treated hamster sample was greater than that of the control sample. A yellow spot indicates sodium arsenite-treated hamster and control hamster each had the same amount of that protein. Several protein spots were up-regulated (red) or down-regulated (green) in the urinary bladder samples of hamsters exposed to sodium arsenite (173 mg As/L) for 6 days as compared with the urinary bladder of controls (Fig. 2). In the case of liver, several protein spots were also over-expressed (red) or under-expressed (green) for hamsters exposed to sodium arsenite (173 mg As/L) in drinking water for 6 days (Fig. 3). The urinary bladder samples were collected from the first and second experiments in which hamsters were exposed to sodium arsenite (173 mg As/L) in drinking water for 6 days and the controls were given tap water. The urinary bladder samples from the 1st and 2nd experiments were run 5 times in DIGE gels on different days. The protein expression is shown in Figure 2 and Table 1. The liver samples from the 1st and 2nd experiments were also run 3 times in DIGE gels on different days. The proteins expression were shown in Figure 3 and Table 2. The volume ratio changed of the protein spots in the urinary bladder and liver of hamsters exposed to arsenite were significantly differences than that of the control hamsters (Table 1 and 2). Protein spots identified by LC-MS/MS Bladder The spots of interest were removed from the gel, digested, and their identities were determined by LC-MS/MS (Fig. 2 and Table 1). The spots 1, 2, & 3 from the gel were analyzed and were repeated for the confirmation of the results (experiments; 173 mg As/L). The proteins for the spots 1, 2, and 3 were identified as transgelin, transgelin, and glutathione S-transferase Pi, respectively (Fig. 2). Liver We also identified some of the proteins in the liver samples of hamsters exposed to sodium arsenite (173 mg As/L) in drinking water for 6 days (Fig. 3). The spots 4, 5, & 6 from the gels were analyzed and were repeated for the confirmation of the results. The proteins for the spots 4, 5, and 6 were identified as ornithine aminotransferase, senescence marker protein 30, and fatty acid binding protein, respectively (Fig. 3) Discussion The identification and functional assignment of proteins is helpful for understanding the molecular events involved in disease. Weexposed hamsters to sodium arsenite in drinking water. Controls were given tap water. DIGE coupled with LC-MS/MS was then used to study the proteomic change in arsenite-exposed hamsters. After electrophoresis DeCyder software indicated that several protein spots were down-regulated (green) and several were up-regulated (red). Our overall results as to changes and functions of the proteins we have studied are summarized in Table 3. Bladder In the case of the urinary bladder tissue of hamsters exposed to sodium arsenite (173 mg As/L) in drinking water for 6 days, transgelin was down-regulated and GST-pi was up-regulated. This is the first evidence that transgelin is down-regulated in the bladders of animals exposed to sodium arsenite. Transgelin, which is identical to SM22 or WS3-10, is an actin cross linking/gelling protein found in fibroblasts and smooth muscle [28, 29]. It has been suggested that the loss of transgelin expression may be an important early event in tumor progression and a diagnostic marker for cancer development [30-33]. It may function as a tumor suppressor via inhibition of ARA54 (co-regulator of androgen receptor)-enhanced AR (androgen receptor) function. Loss of transgelin and its suppressor function in prostate cancer might contribute to the progression of prostate cancer [30]. Down-regulation of transgelin occurs in the urinary bladders of rats having bladder outlet obstruction [32]. Ras-dependent and Ras-independent mechanisms can cause the down regulation of transgelin in human breast and colon carcinoma cell lines and patient-derived tumorsamples [33]. Transgelin plays a role in contractility, possibly by affecting the actin content of filaments [34]. In our experiments loss of transgelin expression may be associated or preliminary to bladder cancer due to arsenic exposure. Arsenite is a carcinogen [1]. In our experiments, LC-MS/MS analysis showed that two spots (1 and 2) represent transgelin (Fig. 2 and Table 1). In human colonic neoplasms there is a loss of transgelin expression and the appearance of transgelin isoforms (31). GST-pi protein was up-regulated in the bladders of the hamsters exposed to sodium arsenite. GSTs are a large family of multifunctional enzymes involved in the phase II detoxification of foreign compounds [35]. The most abundant GSTS are the classes alpha, mu, and pi classes [36]. They participate in protection against oxidative stress [37]. GST-omega has arsenic reductase activity [38]. Over-expression of GST-pi has been found in colon cancer tissues [39]. Strong expression of GST-pi also has been found in gastric cancer [40], malignant melanoma [41], lung cancer [42], breast cancer [43] and a range of other human tumors [44]. GST-pi has been up-regulated in transitional cell carcinoma of human urinary bladder [45]. Up-regulation of glutathione – related genes and enzyme activities has been found in cultured human cells by sub lethal concentration of inorganic arsenic [46]. There is evidence that arsenic induces DNA damage via the production of ROS (reactive oxygen species) [47]. GST-pi may be over-expressed in the urinary bladder to protect cells against arsenic-induced oxidative stress. Liver In the livers of hamsters exposed to sodium arsenite, ornithine amino transferase was over-expressed, senescence marker protein 30 was under-expressed, and fatty acid binding protein was under-expressed. Ornithine amino transferase has been found in the mitochondria of many different mammalian tissues, especially liver, kidney, and small intestine [48]. Ornithine amino transferase knockdown inhuman cervical carcinoma and osteosarcoma cells by RNA interference blocks cell division and causes cell death [49]. It has been suggested that ornithine amino transferase has a role in regulating mitotic cell division and it is required for proper spindle assembly in human cancer cells [49]. Senescence marker protein-30 (SMP30) is a unique enzyme that hydrolyzes diisopropylphosphorofluoridate. SMP30, which is expressed mostly in the liver, protects cells against various injuries by stimulating membrane calcium-pump activity [50]. SMP30 acts to protect cells from apoptosis [51]. In addition it protects the liver from toxic agents [52]. The livers of SMP30 knockout mice accumulate phosphatidylethanolamine, cardiolipin, phosphatidyl-choline, phosphatidylserine, and sphingomyelin [53]. Liver fatty acid binding protein (L-FABP) also was down- regulated. Decreased liver fatty acid-binding capacity and altered liver lipid distribution hasbeen reported in mice lacking the L-FABP gene [54]. High levels of saturated, branched-chain fatty acids are deleterious to cells and animals, resulting in lipid accumulation and cytotoxicity. The expression of fatty acid binding proteins (including L-FABP) protected cells against branched-chain saturated fatty acid toxicity [55]. Limitations: we preferred to study the pronounced spots seen in DIGE gels. Other spots were visible but not as pronounced. Because of limited funds, we did not identify these others protein spots. In conclusion, urinary bladders of hamsters exposed to sodium arsenite had a decrease in the expression of transgelin and an increase in the expression of GST-pi protein. Under-expression of transgelin has been found in various cancer systems and may be associated with arsenic carcinogenicity [30-33). Inorganic arsenic exposure has resulted in bladder cancer as has been reported in the past [1]. Over-expression of GST-pi may protect cells against oxidative stress caused by arsenite. In the liver OAT was up regulated and SMP-30 and FABP were down regulated. These proteomic results may be of help to investigators studying arsenic carcinogenicity. The Superfund Basic Research Program NIEHS Grant Number ES 04940 from the National Institute of Environmental Health Sciences supported this work. Additional support for the mass spectrometry analyses was provided by grants from NIWHS ES06694, NCI CA023074 and the BIOS Institute of the University of Arizona. Acknowledgement The Author wants to dedicate this paper to the memory of his former supervisor Dr. H. VaskenAposhian who passed away in September 6, 2019. He was an emeritus professor of the Department of Molecular and Cellular Biology at the University of Arizona. 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Ключові слова: пам’ять, ідентичність, національна ідентичність, комеморація, кмеморативні практики. Анотація Національна ідентичність, з одного боку, є однією з фундаментальних ідентичностей, тобто, вона може служити орієнтиром для людини навіть тоді, коли інші (професійні, родинні, класові) швидко змінюються внаслідок трансформації суспільства. З іншого боку, зміни суспільства, його соціальних інститутів (як агентів впливу) змінюють зміст і форми національної ідентичності членів суспільства. Відтак усталення тієї чи іншої моделі ідентичності здійснюється в певному соціальному контексті, який задає не тільки спектр альтернатив, але й набір різних комеморативних практик. У статті обґрунтовується взаємообумовленість смислоконституювання пам’яттєвого та ідентичнісного дискурсів, із урахуванням того, що пам’яттєвий дискурс, з одного боку, віддзеркалює характерні риси національної ідентичності, а з іншого – формує своєрідну смислову основу останньої, моделює її, впливаючи на зміни з різним ступенем інтенсивності. Доведено, що національна ідентичність формується за допомогою колективної пам’яті та комеморативних практик. Посилання Anderson, 2001 – Anderson B. Voobrazhaemыe soobshchestva. Razmыshlenyia ob ystokakh y rasprostranenyy natsyonalyzma [Imaginary communities. Reflections on the origins and spread of nationalism]. M. : Kanon-press, 2001. 288 s. [in Russian] Assman, 2012 – Assman A. Prostory spohadu. Formy ta transformatsii kulturnoi pam’iati [Spaces of memory . Forms and transformations of cultural memory] / per. z nim. K. Dmytrenko, L. Doronicheva, O. Yudin. K.: Nika-Tsentr, 2012. 440 s. (Seriia «Zmina paradyhmy». Vyp. 15). [in Ukrainian] Assman, 2014 – Assman A. Dlynnaia ten proshloho. Memoryalnaia kultura y ystorycheskaia polytyka [Long shadow of the past. Memorial culture and historical politics] / per. s nem. B. Khlebnykova. M.: Novoe lyteraturnoe obozrenye, 2014. 323 s. [in Russian] Brubeiker, 2012 – Brubeiker R. Эtnychnost bez hrupp [Ethnicity without groups]. M.: Yzdatelskyi dom Vsshei shkolы эkonomyky, 2012. 408 s. [in Russian] Briubeiker, 2006 – Briubeiker R. Pereobramlenyi natsionalizm. Status natsii ta natsionalne pytannia v novii Yevropi [Perebramlennyi natsionalizm [Text]: status of the nation and the national question in the new Europe] / Per. z anhl. Lviv : Kalvariia, 2006. 280 s. [in Ukrainian] Vahner-Patsyfy, Shvarts, 2011 – Vahner-Patsyfy R., Shvarts B. Memoryal veteranov Vetnama: pamiaty trudnoho pryshloho [Vietnam Veterans Memorial: Commemorating a Difficult Newcomer] // Polytycheskaia kontseptolohyia. 2011. № 2. S. 155–192. [in Russian] Vasylev, 2009 – Vasylev A.H. 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Kommemoratsyia ystorycheskykh sobytyi kak ynstrument symvolycheskoi polytyky: Vozmozhnosty sravnytelnoho analyza [Commemorating Historical Events as a Tool of Symbolic Politics: Possibilities for Comparative Analysis] // Polytyia. 2017. № 4 (87). S. 6-11. [in Russian] Malynova, 2018 – Malynova O.Yu. Kommemoratsyia stoletyia revoliutsyy(i) 1917 hoda v RF: analyz stratehyi kliuchevykh mnemonycheskykh aktorov [Commemoration of the centenary of the revolution (s) of 1917 in the Russian Federation: analysis of the strategies of key mnemonic actors] // Polys. Polytycheskye yssledovanyia. 2018. № 1. S. 9–25. [in Russian] Mehyll, 2007 – Mehyll A. Ystorycheskaia epystemolohyia [Historical epistemology] / Perevod Kukartsevoi M., Kashchaeva V., Tymonyna V. M.: «Kanon+»; ROOY «Reabylytatsyia», 2007. 480 s. [in Russian] Nahorna, 2005 – Nahorna L. Identychnist natsionalna [National identity] // Entsyklopediia istorii Ukrainy: T. 3: E-I / Redkol.: V.A. Smolii (holova) ta in. NAN Ukrainy. 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Nitrogen is a paramount important essential element for all living organisms. It has been found to bea crucial structural component of proteins, nucleic acids, enzymes and other cellular constituents which are inevitable for all forms of life. In the atmosphere, the percentage of nitrogen is very high (N2, 78%) compared to other inorganic gases. However, most organisms have practically no direct access to this nitrogen. While plants can not directly uptake nitrogen from atmosphere, they are capable of assimilating other forms of nitrogen, for example ammonium (NH4+) and nitrate (NO3-). For agricultural crop production, artificial fixation of nitrogen is heavily utilized and it is an expensive process that requires high temperatures (at least 400 °C) and pressures (around 200 atm). It has been conspicuously demonstrated that indiscriminate use of fertilizer hampers soil physical, chemical and micro biological properties and also a potential risk to environment e.g. water quality. Besides, chemically manufactured fertilizers are depleted from soils in various ways, for instance; denitrifying bacteria, volatilization, and leaching. Consequently, it results relatively poor availability of nitrogen to get into plants. On the flipside, only 1-2% of the nitrogen fixation in the world occurs through the natural process of lightening. Notably, microbial fixation is well characterized in diazotrophs for example; Rhizobia and Frankia, and blue-green algae. Against the backdrop, we are accentuated on an environmentally friendlyand themost sustainable approach to increase productivity for legume and non-legume crops. Till today, the term biological nitrogen fixation (BNF) has received much attention as a sustainable alternative; this process facilitates atmospheric nitrogen to convert into ammonia by rhizobia in specialized plan organs termed “root nodules”. This review article seeks to better understand plant mechanisms involved in the development of root nodules in soybean. Soybean (Glycine max) is one of the most important oil crops and a source of animal feed protein in the world. It has a salient feature to fix atmospheric nitrogen through symbioses with compatible rhizobia that yields to determinate type nodule (Oldroyd, Murray et al. 2011). Biological nitrogen fixation in soybean nodules reduces the use of chemical nitrogen fertilizers resulting in cost-savings to producers and minimizes environmental damage due to nitrogen run-off. A better understanding of how nodules form and function is important for selection or generation of soybean genotypes with better nitrogen fixation capacity. Soybean nodules originate from root cortex via de novo cell differentiation (Oldroyd 2013). Consequently, two major nodule development zones are formed for instance; the nodule primordium (Npr) in the middle and it is encircled by nodule parenchyma (Npa). At later time point, the Npr gives rise to N-fixation zone and the Npa holds vascular bundles. It is not clear what early signaling pathways driving the conspicuous development of the nodule zones. My research is aimed at filling this knowledge gap by illustrating the molecular signatures that paves the way to cellular differentiation in root nodule development in soybean. Based on initial evidence obtained by the Subramanian lab, we hypothesize that microRNAs (miRNAs) play important regulatory roles in spatio-temporal expression of their target genes during nodule developmental in soybean. For instance, the regulation of auxin sensitivity by miR160 has been found to be crucial for formation of nodule primordia and vasculature in the parenchyma (Marie Turner 2013). Against this backdrop, this review article focused on nuclear and cytoplasmic transcriptome as well as miRNA profiles of parenchyma and primordial tissues and determine the relative abundance and differentially expressed mRNAs and regulatory role of miRNAs in cell differentiation and nodule development. Root nodule a sustainable alternative to fix atmospheric nitrogen Atmospheric nitrogen percentage is very high (N2, 78%) compared to other inorganic gases (Mary Elvira 1932). However, most of the organisms have practically no direct access to this nitrogen. Nevertheless, plants can not directly uptake nitrogen from atmosphere but they are capable of assimilating only very specific forms of nitrogen, for example ammonium (NH4+) and nitrate (NO3-) (Bytnerowicz and Fenn 1996, Peter M. Vitousek 1997) (Sponseller, Gundale et al. 2016). Virtually, nitrogen has been found to be a crucial structural component of proteins, nucleic acids, enzymes, and other cellular constituents which are inevitable for all forms of life (O'Brien, Vega et al. 2016). For agricultural crop production, artificial fixation of nitrogen is heavily utilized. It is an expensive process that requires high temperatures (approx. 400 °C) and pressures (approx. 200 atm) (Witschi 2000). It has been conspicuously demonstrated that indiscriminate use of N fertilizer hampers the diversity of the bacterial community and decreases soil C and N concentrations (Verzeaux, Alahmad et al. 2016). Notably, it has been demonstrated as a potential risk to environment e.g. water quality (Zhao, Sha et al. 2016) (Sponseller, Gundale et al. 2016). Besides, chemically manufactured fertilizers are depleted from soils in various ways, for instance; denitrifying bacteria, volatilization, and leaching (Johnson 1996, Peter M. Vitousek 1997). Consequently, it results relatively poor availability of nitrogen to get into plants. On the flipside, over 90 % of the nitrogen fixation in the world occurs through the natural process of lightening and microorganisms. Furthermore, microbial fixation is well characterized in diazotrophs for example; Rhizobia and Frankia, and blue-green algae (Cheng 2008). It has been demonstrated that Bradyrhizobium strains substantially escalated soybean grain yield, and protein content up to 57% and 26%, respectively (Zimmer, Messmer et al. 2016). Against the backdrop, we are accentuated on an environmentally friendly and a sustainable approach to increase the productivity for legume and non-legume crops. Literature mining depicted that biological nitrogen fixation in soybean nodules reduces the use of chemical nitrogen fertilizers resulting in cost-savings to producers and minimizes environmental damage due to nitrogen run-off. Rhizobia infection leads to the root nodule development In the natural environment, plants are continuously confronted with pathogenic and symbiotic microbes. Symbioses involves mutual exchange of diffusible signal molecules, first endophytic bacteria (rhizobia) are attracted by the plant root exudates flavonoids which are perceived and triggered the bacterial nodulation (nod) genes. Consequently, the bacteria synthesize specific lipochito-oligosaccharides, called nodulation (Nod) factors. This signal is perceived by the LysM receptor like kinase of host plant, it induces the root hair curling, and bacteria get access into the host epidermis through infection threads (ITs) and initiate cell division within the root cortex, leading to the progression of the root nodule meristem. In later stages of the interaction, bacteria are released from the infection threads into the plant cells, surrounded by membrane of plant origin. These bacteria multiply within the host cells and differentiate into the nitrogen fixing bacteroids (Udvardi and Day 1997) (Oldroyd 2013). Till now, integration of genetic and genomic approaches has revealed twenty-six genes to be involved in nodule development of Medicago truncatualaand Lotus japonicum (Kouchi, Imaizumi-Anraku et al. 2010). In addition, deep sequencing of the Medicago truncatularoot transcriptome has uncovered thousands of genes to be induced during Nod factor signaling and its resulting ethylene (ET) biosynthesis throughout the multiple development stages of indeterminate nodule (Larrainzar, Riely et al. 2015). Albeit the molecular mechanism of such regulation is not well understood. There has been a large-scale transcriptome analysis of B. japonicum-inoculated and mock-inoculated soybean root hairs. It has showed that a total of 1,973 soybean genes differentially expressed during root hair infection, particularly NFR5 and NIN genes (Libault, Farmer et al. 2010). Nevertheless, the signaling mechanisms directing the cellular differentiation of nodule are not known. Soybean root nodule organogenesis Soybean (Glycine max) has a genome size of 1.1 to1.5 Gb, it is partially diploidized tetraploid. It is one of the most important oil crops and a source of animal feed protein in the world (soybase.org/sb_about.php). It has a salient feature to fix atmospheric nitrogen through symbioses with compatible rhizobia that yields to determinate type nodule (Udvardi and Day 1997) (Oldroyd, Murray et al. 2011). Notwithstanding of the economic and environmental importance, there has been very few studies about quantitative trait loci (QTL) that controlling BNF traits, for instance nodule number, ration of nodule dry weight with nodule number, and shoot dry weight (SDW). It has been reported via composite interval mapping that approximately six QTLs bears very small effect on BNF traits (Santos, Geraldi et al. 2013). Besides, it has been demonstrated in earlier studies that nodules originate from root cortex via de novo cell differentiation into two different cell types, parenchymal and primordium (Celine Charon 1997) (Oldroyd&Downie 2008; Oldroyd 2013). In addition, early nodulin genes in legume for instance; Enod 40 gene reported to be expressed in root pericycle during the rhizobia infection and later it occupied in the dividing cortical cells (H. Kouchi and S. Hata 1993). Among the two major nodule development zones, the nodule primordium (Npr) in the middle which is encircled by nodule parenchyma (Npa). At later time point, the Npr gives rise to N-fixation zone and the Npa holds vascular bundles. Lately, a β- expansin gene, GmEXPB2 fused with GUS reporter gene which was observed to be preferentially expressed in nodule vascular trace and nodule vascular bundles. It indicated that GmEXPB2 might be crucial for nodule organogenesis. Over expression of GmEXPB2 contrast to suppressed GmEXPB2 transgenic lines found to be escalated nodule number, nodule mass and nitrogenase activity. It further suggested that GmEXPB2 might have influenced over root architecture, nodule formation and development, and profoundly yielding to biological N2 fixation (Li, Zhao et al. 2015). Even though, it is not clear what early signaling pathways driving the conspicuous development of the nodule zones. Against the back drop, to understand the regulation of auxin sensitivity by miR160 which is believed to be crucial for the formation of nodule primordia (Marie Turner 2013). Figure 1 a. Illustrating the progression of root nodule development through Rhizobial bacterial infection in the plant root leading to the determinate nodule (Oldroyd 2013). b. Nodule development zones A. Nodule primordial zone (Enod 40 gene) in the middle B. surrounding parenchyma (Enod 2 gene), differentiated from cortex (collected from Sen Subramanian lab). Regulatory small RNAs biogenesis and its molecular functions Regulatory small RNAs are ranged between 20 to 24 nucleotides which are ubiquitous elements of endogenous plant transcriptomics, a common response to exogenous viral infections and introduced double-stranded RNA (Axtell 2013). Three core enzymes families, for instance; RNAdependent RNA polymerase (RDR), Dicer like (DCL), and Argonaute (AGO) proteins paves the way of small RNA biogenesis and function in plants. Firstly, ribonuclease type III or DICERLIKE1 involves in the yield of a fold-back precursor RNA or primary miRNA (primiRNA) transcripts using an RNA templates in the nuclei. Later, the resulting miRNA-miRNA duplex which is originated in nucleus then translocated into cytoplasm. The guided miRNAmolecule is incorporated into ARGONAUTE (AGO) to form an active RISC complex to specific target RNAs that are complementary to the miRNA, and this process eventually follows up mRNA cleavage, represses the translation of the mRNAs or Chromatin modification. This phenomenon accentuated as an inhibition or silencing of the gene expression, which play a crucial role in the developmental process in plant and animal (Chapman and Carrington 2007) (Axtell 2013). Fig. 2 Regulation of gene expression events via RISC complex (modified from https://www.google.com/?gws_rd=ssl#q=mirna+picture+in+plants accessed on 7th February, 2016) Fig. 3 Gene expression events occurring in typical plant cell (modified https://www.google.com/search?q=transcription+and+translation accessed on 7th February, 2016) It has been found in several studies that most plant miRNAs are non-coding RNA, and small 21-24 nucleotide long (Cuperus, Fahlgren et al. 2011). It requires DCL1-clade DCL for their biogenesis and AGO1-clade AGO for their function (Wu, Zhou et al. 2010, Manavella, Koenig et al. 2012). In rice (Oryza sativa), DCL3 has been reported in the biogenesis of 24nt long miRNA that incorporated in AGO4 to regulate the target gene expression primarily through mRNA cleavage (Wu, Zhou et al. 2010). Argonaute proteins (AGO) form RNA inducing silencing complexes (RISC) with small RNAswhich is known as post-transcriptional gene silencing. It has typically four domains, for instance:N-terminal, PAZ, MID and PIWI domains. The MID-PIWI lobes are belongs to the C-terminus. It has been studied that MID-domains contains the specificity loop to recognize and bind to the 5’-phosphate of smRNAs. The PIWI domains contained the catalytic active site D-E-D-H/D. PAZ domain anchored the 2-nt overhang at the 3’ end of miRNAs. The N-terminal domain involved in the separation of miRNA-miRNA duplex and the slicer activity of the mRNA (Song, Smith et al.2004). There has been an expansion and duplications of AGO family members during plantevolution (Singh, Gase et al. 2015). The functional diversification of AGOs is indicating sRNAdirected regulatory pathways. The binding preference of AGO and sRNA is mainly assigned by the sequence of sRNA. In Arabidopsis, 10 AGO have been extensively studied (Liu et al. 2014). It has been demonstrated that AtAGO10 like AtAGO1, it recognized distinct structural features in miR165/miR166 duplex than involved by AtGO1. AtAGO10 found to regulate shoot apical meristem by decoying miR165/miR166 and subsequent repression of homeodomain-leucinezipper (HD-ZIP) gene expression (Zhu, Hu et al. 2011). Notably, 22 AGO proteins have been reported in Soybean (Glycine max). It has been found that genome duplication in Soybean resulted such a proliferation of AGOs. For example: its genome encodes two copies of AGO1, AGO2, AGO5, AGO4/9, AGO6 and AGO7 (Xiang Liu 2014). However, the molecular function of the plant AGO genes yet not very clear. There are several miRNA families that are conserved across the vast evolutionary distances from flowering plants to mosses (Cuperus, Fahlgren et al. 2011). It has been observed in another study that miRNA, and its target pairing found to be stable for a prolonged periods of plant evolution. On the flip side, another group demonstrated that conserved plant miRNAs and their targets are to somehow flexible. For instance; miR159 is a highly conserved miRNA that targets not only a subset of MYB mRNAs but also observed to target a non MYB mRNA, SGN-U567133 (Buxdorf, Hendelman et al. 2010). A mutant tomato transgenic line (miR159-resistant line) showed higher level of the SGN-U567133 transcript and exhibited defects in leaf and flower development. This result suggests that miR159 involves in a post-transcriptional regulation. Additionally, it is found to be crucial for the normal tomato development. Recently, the identification of miRNAs in the regulation of photoperiodic pathways in soybean have been reported through high throughput sequencing and qRT-PCR. Six libraries were constructed using Illumina Solexa, for instance; 0, 8, and 16 h under short day treatment, similar time points considered for the long the long day treatment. A total of 163 miRNAs families were reported which covered 318 plant miRNAs, and unclassified 81 novel predicted miRNAs. As expected, significant differences in abundance between short day and long day treatment was observed (Wenbin Li 2015). These findings provided evidence of miRNA in the regulation of flowering time that ultimately affects the seed yield and quality of soybean. The complex regulatory network of miRNA-mRNA interactions during viral infection has been revealed via small RNA seq (sRNA), degradome seq, and genome-wide transcriptome analysis. There has been a total of 253 soybean miRNAs found to be two-folds abundance compared with mock-inoculated control demonstrated through sRNA seq analysis. Among them 105 miRNAs were identified as potential targets of 125 transcripts that has been validated by degradome seq analyses. In addition, 2679 genes were detected via genome wide transcriptomic analysis. These genes have been differentially expressed during infection of soybean mosaic virus and among them 71 genes projected to induce in defense response (Hui Chen 2016). These findings suggested the regulatory role miRNA that governed the target gene expression during viral infection. Furthermore, the regulatory role of microRNAs (miRNAs) during Soybean- Bradyrhizobium japonicum mutualistic association was studied first by Subramanian et al. 2008. They sequenced approximately 350000 small RNAs of soybean root sample which were inoculated with B. japonicum. It helps to detect 20 conserved miRNAs loci based on the similarity to miRNAs in another plant species. In addition, 35 novel miRNAs were identified based on potential hairpin forming precursors in Soybean EST as well as shotgun genomic sequences (Subramanian, Fu et al. 2008). These findings advocated the potential role of miRNAs in the regulation of legumerhizobiumsymbiosis. In another study, 120 hairpin-forming precursor genes have been identified in soybean by Turner et al. In addition, they reported three novel miRNAs for instance; miR160, miR164 and miR393 found to be involved in auxin signaling (Turner, Yu et al. 2012). Moreover, the plant hormone auxin is thought to have a pivotal role in nodule organogenesis in determinate and indeterminate type of nodule. It indicates a redundancy and diversity of miRNAs family members that governs the formation of root nodule. It has been illustrated that auxin receptor gene family hushed by over expressed microRNA393. These plant roots found to be hypersensitive to auxin and yielded normal nodule. This observation advocated that only minimal/reduced auxin signaling is required for determinate nodule development. Likewise, overexpressed microRNA160 hushed a set of repressor auxin response transcription factor. These plant roots were hypersensitive to auxin and observed not to be reluctant in epidermal responses to rhizobia. Notably, it yielded to lower sized nodule primordium (Marie Turner 2013). This observation indicated that auxin hypersensitivity inhibits nodule organogenesis Organ specific expression of profile of miRNA and the potential targets were also studied. Two genes (Glyma10g10240 and Glyma17g05920) which were the target of miR169 but detected to be highly expressed in soybean nodule. Likewise, three potential targets of gma-new-miR13587 demonstrated to be highly expressed in the nodules than in the roots. As expected, gma-newmiR13587 found to be poorly expressed in the nodules than in the roots (Turner, Yu et al. 2012). There was an inverse expression pattern observed in between roots and nodules. Li et al., studied the transgene expression of three novel miRNAs namely, miR482, miR1512, and miR1515 in Soybean. They noticed a significant increase of nodule numbers while root length and later root density were normal in all tested miRNA lines. As expected, there were differential expression of these miRNAs in supernodulating and nonnodulating soybean mutants. They reported that 6 novel miRNAs decoyed 22 predicted target genes. And it was estimated via real time polymerase chain reaction and qRT-PCR (Li, Deng et al. 2010). It advocates that miRNAs have the signatory roles in soybean nodule development. Sequencing of small RNAs and Parallel analysis of RNA ends (PARE) libraries revealed to identify 284 nodule miRNAs, more than 500 target genes, and including 178 novel soybean miRNAs. It has been reported that ENOD93 only found to be expressed in nodule tissue not in other plant parts of Soybean. Ectopic expression of miR393j-3p and RNAi silencing approach to ENOD93 expression showed a significant reduction in nodule formation (Zhe Yan 2015). Therefore, this study showed a list of miRNAs and their potential target of nodulation genes. In the model legume (Medicago truncatula), 25 conserved miRNA families and 100 novel miRNA reads were detected by high-throughput sequencing. The expression of MtHAP2-1 (encodes a CCAAT binding transcription factor) to meristematic zones was restricted by miR169a which is found to be critical for the development of indeterminate type of nodule (Combier, Frugier et al. 2006). In another study, HDZIPIII transcripts were inhibited by overexpression of miR166, it dropped the number of symbiotic nodule and lateral root (Boualem, Laporte et al. 2008). To get insights into key genes of nodule zones, transcript profiles of specific cells/tissues were investigated at different time points from indeterminate nodules of M. truncatulausing laser capture micro dissection. It has been demonstrated from the comprehensive gene expression map that selected genes enriched in different cell/tissue types (Limpens, Moling et al. 2013). These findings indicated that organ specific gene expression could be controlled by the presence or absence of miRNAs. Recently, Agrobacterium rhizogenesmediated hairy root transformation has been applied as tool for exploring cell type specific gene expression in tomato. Cell type or tissue specific promoter introduced into INTACT and TRAP constructs via gateway cloning technology to develop binary vectors. INTACT method used to capture biotin tagged nuceli from specific cell types and TRAP method used for profiling of mRNAs or foot printing of individual ribosomes (Ron 2014). TRAP methodology is not required tissue fixation or single cell suspension. It has been successfully used to date in organisms ranging from D. melanogaster to mice and human cultured cells. Multiple ribosomes or Polyribosomes (polysomes) are engaged in translation on a single mRNA. To evaluate the translation state of an mRNA, ribosomal subunits, ribosomes, and polysomes can be isolated from detergent-treated cell extracts (Heiman, Kulicke et al. 2014). In this study, we would perform polysome isolation deploying gene cassettes ENOD40p:HF-GFP-RPL18 for primordial tissues, and ENOD2p:HF-GFP-RPL18 for parenchymal tissues in Glycine max root nodules that express an epitope tagged version of ribosomal protein L18. Over the last one decade, there has been several microarrays-based studies which characterized transcriptional variations deployed in nodule formation. It has been embedded with couple of shortcomings, for instance; relative late time points study, incomplete representation of plant genes,discrimination of close paralogs, and reduced sensitivity. Lately, next generation sequencingtechnology have widened the horizon of transcription analyses in different legume species to detectsymbiosis induced changes in late nodule developmental stages. Against this backdrop, we areaccentuated to reveal early transcriptional changes induced in determinate type of soybean noduleby Bradyrhizobium japonicum. In determinate type of nodule, two major nodule development zones are formed for instance, the nodule primordium (Npr) in the middle and it is encircled by nodule parenchyma (Npa). At later time point, the Npr converted to N-fixation zone and the Npa contained vascular bundles. Of these facts, it is not clear what early signaling pathways driving the conspicuous development of thenodule zones. In this context, mechanisms regulate the distinct gene expression profiles in Npr andNpa cell types has not understood clearly. The proposed research study is aimed at filling this knowledge gap byillustrating the molecular signatures that paves the way to cellular differentiation in root noduledevelopment in soybean considering four different time points (5 dai, 7 dai, 10 dai& 14 dai). The hypothesisis microRNAs(miRNAs) play important regulatory roles in spatio-temporal expression of their target genesduring nodule developmental in soybean. For example, a gradient of microRNA localizationbetween nodule primordium and parenchyma cells could result in distinct differentiation of thesecell types. To test this hypothesis, one has to obtain both cell type-specific miRNA andtranscriptome (miRNA target) profiles. Since, the majority of miRNA regulation occurs in thecytoplasm, we reasoned that comparison of nuclear and ribosomal transcriptome profiles wouldreveal genes whose expression is potentially regulated by post transcriptional mechanisms such asmiRNA cleavage. Combining this information with cell type-specific miRNA profiles, andto test the above hypothesis and identify key miRNA-target pairs important for nodule celldifferentiation. The use of translating ribosome affinity purification (TRAP) of nodule zonecells, namely from parenchyma and primordial tissues, to obtain cytoplasmic transcriptomes data. Techniques to determine cell type specific expression profiles: TRAP methods TRAP is termed translating ribosome affinity purification, combines cell-type-specific transgene expression with affinity purification of translating ribosomes. It supersedes the need for tissue fixation, and facilitates to study the cell type-specific mRNA profiles of any genetically defined cell type. It has been successfully used to date in organisms ranging from D. melanogaster to mice, and human cultured cells. Multiple ribosomes or Polyribosomes (polysomes) are engaged in translation on a single mRNA. To evaluate the translation state of an mRNA, ribosomal subunits, ribosomes, and polysomes can be isolated from detergent-treated cell extracts. In this study, the polysome isolation using gene cassettes ENOD40p:HF-GFP-RPL18 for primordial tissues, and ENOD2p:HF-GFP-RPL18 for parenchymal tissues in Glycine max root nodules that express an epitope tagged version of ribosomal protein L18 RPL18(Heiman, Kulicke et al. 2014, Ron 2014). Relative abundance and differentially expressed mRNAs profile in two different tissue specific zones would help to understand the effect of regulatory role of miRNAs in cell differentiation and nodule development. References: Axtell, M. J. (2013). "Classification and comparison of small RNAs from plants." Annu Rev PlantBiol 64: 137-159. 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Plant Cell 23(2): 431-442. Hiroshi Kouchi1, K.-i. T., Rollando B. So2, Jagdish K. Ladha2 and Pallavolu M. Reddy2 (1999). "Rice ENOD40: isolation and expression analysis in rice and transgenic soybean root nodules." The Plant Journal 18(2): 121-129. Johnson, D. S. O. a. G. V. (1996). "Fertilizer Nutrient Leaching and Nutrient Mobility: A Simple Laboratory Exercise." Nat. Resour. L. ife Sci. Educ 25(2): 128-131. Kouchi, H. and Hata, S. (1993) Isolation and characterization of novel nodulin cDNAs representing genes expressed at early stages of soybean nodule development. Gen. Genet. 238, 106–119. Li, H., et al. (2010). "Misexpression of miR482, miR1512, and miR1515 increases soybean nodulation." Plant Physiol 153(4): 1759-1770. Manavella, P. A., et al. (2012). "Plant secondary siRNA production determined by microRNAduplexstructure." Proc Natl Acad Sci U S A 109(7): 2461-2466. Marie Turner, e. a. (2013). 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IntroductionDespite claims that the importance of distance has been reduced due to technological and communications improvements (Cairncross; Friedman; O’Brien), the ‘power of place’ still resonates, often intensifying the role of geography (Christopherson et al.; Morgan; Pratt; Scott and Storper). Within the film industry, there has been a decentralisation of production from Hollywood, but there remains a spatial logic which has preferenced particular centres, such as Toronto, Vancouver, Sydney and Prague often led by a combination of incentives (Christopherson and Storper; Goldsmith and O’Regan; Goldsmith et al.; Miller et al.; Mould). The emergence of high end television, television programming for which the production budget is more than £1 million per television hour, has presented new opportunities for screen hubs sharing a very similar value chain to the film industry (OlsbergSPI with Nordicity).In recent years, interventions have proliferated with the aim of capitalising on the decentralisation of certain activities in order to attract international screen industries production and embed it within local hubs. Tools for building capacity and expertise have proliferated, including support for studio complex facilities, infrastructural investments, tax breaks and other economic incentives (Cucco; Goldsmith and O’Regan; Jensen; Goldsmith et al.; McDonald; Miller et al.; Mould). Yet experience tells us that these will not succeed everywhere. There is a need for a better understanding of both the capacity for places to build a distinctive and competitive advantage within a highly globalised landscape and the relative merits of alternative interventions designed to generate a sustainable production base.This article first sets out the rationale for the appetite identified in the screen industries for co-location, or clustering and concentration in a tightly drawn physical area, in global hubs of production. It goes on to explore the latest trends of decentralisation and examines the upturn in interventions aimed at attracting mobile screen industries capital and labour. Finally it introduces the Scottish screen industries and explores some of the ways in which Scotland has sought to position itself as a recipient of screen industries activity. The paper identifies some key gaps in infrastructure, most notably a studio, and calls for closer examination of the essential ingredients of, and possible interventions needed for, a vibrant and sustainable industry.A Compulsion for ProximityIt has been argued that particular spatial and place-based factors are central to the development and organisation of the screen industries. The film and television sector, the particular focus of this article, exhibit an extraordinarily high degree of spatial agglomeration, especially favouring centres with global status. It is worth noting that the computer games sector, not explored in this article, slightly diverges from this trend displaying more spatial patterns of decentralisation (Vallance), although key physical hubs of activity have been identified (Champion). Creative products often possess a cachet that is directly associated with their point of origin, for example fashion from Paris, films from Hollywood and country music from Nashville – although it can also be acknowledged that these are often strategic commercial constructions (Pecknold). The place of production represents a unique component of the final product as well as an authentication of substantive and symbolic quality (Scott, “Creative cities”). Place can act as part of a brand or image for creative industries, often reinforcing the advantage of being based in particular centres of production.Very localised historical, cultural, social and physical factors may also influence the success of creative production in particular places. Place-based factors relating to the built environment, including cheap space, public-sector support framework, connectivity, local identity, institutional environment and availability of amenities, are seen as possible influences in the locational choices of creative industry firms (see, for example, Drake; Helbrecht; Hutton; Leadbeater and Oakley; Markusen).Employment trends are notoriously difficult to measure in the screen industries (Christopherson, “Hollywood in decline?”), but the sector does contain large numbers of very small firms and freelancers. This allows them to be flexible but poses certain problems that can be somewhat offset by co-location. The findings of Antcliff et al.’s study of workers in the audiovisual industry in the UK suggested that individuals sought to reconstruct stable employment relations through their involvement in and use of networks. The trust and reciprocity engendered by stable networks, built up over time, were used to offset the risk associated with the erosion of stable employment. These findings are echoed by a study of TV content production in two media regions in Germany by Sydow and Staber who found that, although firms come together to work on particular projects, typically their business relations extend for a much longer period than this. Commonly, firms and individuals who have worked together previously will reassemble for further project work aided by their past experiences and expectations.Co-location allows the development of shared structures: language, technical attitudes, interpretative schemes and ‘communities of practice’ (Bathelt, et al.). Grabher describes this process as ‘hanging out’. Deep local pools of creative and skilled labour are advantageous both to firms and employees (Reimer et al.) by allowing flexibility, developing networks and offsetting risk (Banks et al.; Scott, “Global City Regions”). For example in Cook and Pandit’s study comparing the broadcasting industry in three city-regions, London was found to be hugely advantaged by its unrivalled talent pool, high financial rewards and prestigious projects. As Barnes and Hutton assert in relation to the wider creative industries, “if place matters, it matters most to them” (1251). This is certainly true for the screen industries and their spatial logic points towards a compulsion for proximity in large global hubs.Decentralisation and ‘Sticky’ PlacesDespite the attraction of global production hubs, there has been a decentralisation of screen industries from key centres, starting with the film industry and the vertical disintegration of Hollywood studios (Christopherson and Storper). There are instances of ‘runaway production’ from the 1920s onwards with around 40 per cent of all features being accounted for by offshore production in 1960 (Miller et al., 133). This trend has been increasing significantly in the last 20 years, leading to the genesis of new hubs of screen activity such as Toronto, Vancouver, Sydney and Prague (Christopherson, “Project work in context”; Goldsmith et al.; Mould; Miller et al.; Szczepanik). This development has been prompted by a multiplicity of reasons including favourable currency value differentials and economic incentives. Subsidies and tax breaks have been offered to secure international productions with most countries demanding that, in order to qualify for tax relief, productions have to spend a certain amount of their budget within the local economy, employ local crew and use domestic creative talent (Hill). Extensive infrastructure has been developed including studio complexes to attempt to lure productions with the advantage of a full service offering (Goldsmith and O’Regan).Internationally, Canada has been the greatest beneficiary of ‘runaway production’ with a state-led enactment of generous film incentives since the late 1990s (McDonald). Vancouver and Toronto are the busiest locations for North American Screen production after Los Angeles and New York, due to exchange rates and tax rebates on labour costs (Miller et al., 141). 80% of Vancouver’s production is attributable to runaway production (Jensen, 27) and the city is considered by some to have crossed a threshold as:It now possesses sufficient depth and breadth of talent to undertake the full array of pre-production, production and post-production services for the delivery of major motion pictures and TV programmes. (Barnes and Coe, 19)Similarly, Toronto is considered to have established a “comprehensive set of horizontal and vertical media capabilities” to ensure its status as a “full function media centre” (Davis, 98). These cities have successfully engaged in entrepreneurial activity to attract production (Christopherson, “Project Work in Context”) and in Vancouver the proactive role of provincial government and labour unions are, in part, credited with its success (Barnes and Coe). Studio-complex infrastructure has also been used to lure global productions, with Toronto, Melbourne and Sydney all being seen as key examples of where such developments have been used as a strategic priority to take local production capacity to the next level (Goldsmith and O’Regan).Studies which provide a historiography of the development of screen-industry hubs emphasise a complex interplay of social, cultural and physical conditions. In the complex and global flows of the screen industries, ‘sticky’ hubs have emerged with the ability to attract and retain capital and skilled labour. Despite being principally organised to attract international production, most studio complexes, especially those outside of global centres need to have a strong relationship to local or national film and television production to ensure the sustainability and depth of the labour pool (Goldsmith and O’Regan, 2003). Many have a broadcaster on site as well as a range of companies with a media orientation and training facilities (Goldsmith and O’Regan, 2003; Picard, 2008). The emergence of film studio complexes in the Australian Gold Coast and Vancouver was accompanied by an increasing role for television production and this multi-purpose nature was important for the continuity of production.Fostering a strong community of below the line workers, such as set designers, locations managers, make-up artists and props manufacturers, can also be a clear advantage in attracting international productions. For example at Cinecitta in Italy, the expertise of set designers and experienced crews in the Barrandov Studios of Prague are regarded as major selling points of the studio complexes there (Goldsmith and O’Regan; Miller et al.; Szczepanik). Natural and built environments are also considered very important for film and television firms and it is a useful advantage for capturing international production when cities can double for other locations as in the cases of Toronto, Vancouver, Prague for example (Evans; Goldsmith and O’Regan; Szczepanik). Toronto, for instance, has doubled for New York in over 100 films and with regard to television Due South’s (1994-1998) use of Toronto as Chicago was estimated to have saved 40 per cent in costs (Miller et al., 141).The Scottish Screen Industries Within mobile flows of capital and labour, Scotland has sought to position itself as a recipient of screen industries activity through multiple interventions, including investment in institutional frameworks, direct and indirect economic subsidies and the development of physical infrastructure. Traditionally creative industry activity in the UK has been concentrated in London and the South East which together account for 43% of the creative economy workforce (Bakhshi et al.). In order, in part to redress this imbalance and more generally to encourage the attraction and retention of international production a range of policies have been introduced focused on the screen industries. A revised Film Tax Relief was introduced in 2007 to encourage inward investment and prevent offshoring of indigenous production, and this has since been extended to high-end television, animation and children’s programming. Broadcasting has also experienced a push for decentralisation led by public funding with a responsibility to be regionally representative. The BBC (“BBC Annual Report and Accounts 2014/15”) is currently exceeding its target of 50% network spend outside London by 2016, with 17% spent in Scotland, Wales and Northern Ireland. Channel 4 has similarly committed to commission at least 9% of its original spend from the nations by 2020. Studios have been also developed across the UK including at Roath Lock (Cardiff), Titanic Studios (Belfast), MedicaCity (Salford) and The Sharp Project (Manchester).The creative industries have been identified as one of seven growth sectors for Scotland by the government (Scottish Government). In 2010, the film and video sector employed 3,500 people and contributed £120 million GVA and £120 million adjusted GVA to the economy and the radio and TV sector employed 3,500 people and contributed £50 million GVA and £400 million adjusted GVA (The Scottish Parliament). Beyond the direct economic benefits of sectors, the on-screen representation of Scotland has been claimed to boost visitor numbers to the country (EKOS) and high profile international film productions have been attracted including Skyfall (2012) and WWZ (2013).Scotland has historically attracted international film and TV productions due to its natural locations (VisitScotland) and on average, between 2009-2014, six big budget films a year used Scottish locations both urban and rural (BOP Consulting, 2014). In all, a total of £20 million was generated by film-making in Glasgow during 2011 (Balkind) with WWZ (2013) and Cloud Atlas (2013), representing Philadelphia and San Francisco respectively, as well as doubling for Edinburgh for the recent acclaimed Scottish films Filth (2013) and Sunshine on Leith (2013). Sanson (80) asserts that the use of the city as a site for international productions not only brings in direct revenue from production money but also promotes the city as a “fashionable place to live, work and visit. Creativity makes the city both profitable and ‘cool’”.Nonetheless, issues persist and it has been suggested that Scotland lacks a stable and sustainable film industry, with low indigenous production levels and variable success from year to year in attracting inward investment (BOP Consulting). With regard to crew, problems with an insufficient production base have been identified as an issue in maintaining a pipeline of skills (BOP Consulting). Developing ‘talent’ is a central aspect of the Scottish Government’s Strategy for the Creative Industries, yet there remains the core challenge of retaining skills and encouraging new talent into the industry (BOP Consulting).With regard to film, a lack of substantial funding incentives and the absence of a studio have been identified as a key concern for the sector. For example, within the film industry the majority of inward investment filming in Scotland is location work as it lacks the studio facilities that would enable it to sustain a big-budget production in its entirety (BOP Consulting). The absence of such infrastructure has been seen as contributing to a drain of Scottish talent from these industries to other areas and countries where there is a more vibrant sector (BOP Consulting). The loss of Scottish talent to Northern Ireland was attributed to the longevity of the work being provided by Games of Thrones (2011-) now having completed its six series at the Titanic Studios in Belfast (EKOS) although this may have been stemmed somewhat recently with the attraction of US high-end TV series Outlander (2014-) which has been based at Wardpark in Cumbernauld since 2013.Television, both high-end production and local broadcasting, appears crucial to the sustainability of screen production in Scotland. Outlander has been estimated to contribute to Scotland’s production spend figures reaching a historic high of £45.8 million in 2014 (Creative Scotland ”Creative Scotland Screen Strategy Update”). The arrival of the program has almost doubled production spend in Scotland, offering the chance for increased stability for screen industries workers. Qualifying for UK High-End Television Tax Relief, Outlander has engaged a crew of approximately 300 across props, filming and set build, and cast over 2,000 supporting artist roles from within Scotland and the UK.Long running drama, in particular, offers key opportunities for both those cutting their teeth in the screen industries and also by providing more consistent and longer-term employment to existing workers. BBC television soap River City (2002-) has been identified as a key example of such an opportunity and the programme has been credited with providing a springboard for developing the skills of local actors, writers and production crew (Hibberd). This kind of pipeline of production is critical given the work patterns of the sector. According to Creative Skillset, of the 4,000 people in Scotland are employed in the film and television industries, 40% of television workers are freelance and 90% of film production work in freelance (EKOS).In an attempt to address skills gaps, the Outlander Trainee Placement Scheme has been devised in collaboration with Creative Scotland and Creative Skillset. During filming of Season One, thirty-eight trainees were supported across a range of production and craft roles, followed by a further twenty-five in Season Two. Encouragingly Outlander, and the books it is based on, is set in Scotland so the authenticity of place has played a strong component in the decision to locate production there. Producer David Brown began his career on Bill Forsyth films Gregory’s Girl (1981), Local Hero (1983) and Comfort and Joy (1984) and has a strong existing relationship to Scotland. He has been very vocal in his support for the trainee program, contending that “training is the future of our industry and we at Outlander see the growth of talent and opportunities as part of our mission here in Scotland” (“Outlander fast tracks next generation of skilled screen talent”).ConclusionsThis article has aimed to explore the relationship between place and the screen industries and, taking Scotland as its focus, has outlined a need to more closely examine the ways in which the sector can be supported. Despite the possible gains in terms of building a sustainable industry, the state-led funding of the global screen industries is contested. The use of tax breaks and incentives has been problematised and critiques range from use of public funding to attract footloose media industries to the increasingly zero sum game of competition between competing places (Morawetz; McDonald). In relation to broadcasting, there have been critiques of a ‘lift and shift’ approach to policy in the UK, with TV production companies moving to the nations and regions temporarily to meet the quota and leaving once a production has finished (House of Commons). Further to this, issues have been raised regarding how far such interventions can seed and develop a rich production ecology that offers opportunities for indigenous talent (Christopherson and Rightor).Nonetheless recent success for the screen industries in Scotland can, at least in part, be attributed to interventions including increased decentralisation of broadcasting and the high-end television tax incentives. This article has identified gaps in infrastructure which continue to stymie growth and have led to production drain to other centres. 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