Relative normalized expression of AKR17A1 in <i>Anabaena</i> 7120 exposed to butachlor, cadmium, UV-B, heat, salt, drought and arsenic.

<p>Transcript levels were determined by qRT-PCR. The 16S rRNA gene was used as an internal control for normalizing the variations in cDNA amounts. Biological triplicates were averaged. Bars indicate SE.</p

Steady-state kinetic parameters of recombinant His-tagged AKR17A1.

<p>Kinetic constants are mean values of three independent experiments. Values in parentheses indicate standard error of the mean (SEM). The activity of AKR17A1 at various concentrations of substrates tested is given as Fig C in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137744#pone.0137744.s003" target="_blank">S3 File</a>.</p

Sequence alignment of <i>Anabaena</i> 7120 aldo- keto reductase All2316 and members of AKR13 family using PROMALS3D multiple sequence and structure alignment server (prodata.swmed.edu/promals3d/).

<p>Representative sequences are colored according to predicted secondary structures (red: alpha-helix, blue: beta-strand). Consensus predicted secondary structure symbols: alpha-helix: h; beta-strand: e. Consensus amino acid symbols are: conserved amino acids are in bold and uppercase letters; aliphatic (I, V, L): l; aromatic (Y, H, W, F): @; hydrophobic (W, F, Y, M, L, I, V, A, C, T, H): h; alcohol (S, T): o; polar residues (D, E, H, K, N, Q, R, S, T): p; tiny (A, G, C, S): t; small (A, G, C, S, V, N, D, T, P): s; bulky residues (E, F, I, K, L, M, Q, R, W, Y): b; positively charged (K, R, H): +; negatively charged (D, E):-; charged (D, E, K, R, H): c.</p

Growth behavior of <i>E</i>. <i>coli</i> BL21 (DE3) cells transformed with pET21a and pET21a-AKR in response to (a) cadmium (0.3 mM) (b) mannitol (600 mM), (c) heat (50°C), (d) NaCl (600 mM), (e) UV-B (10 min) and (f) arsenic (6 mM) stress using liquid culture assay.

<p>The mean of three independent replicates is plotted with error bars indicating standard deviations. (a-f) represents the growth curves of <i>E</i>. <i>coli</i> strains in LB medium supplemented with 100 μg ml⁻¹ ampicillin and 0.2 mM IPTG.</p

Absorption spectrograms showing degradation of butachlor by NADPH dependent AKR17A1.

<p>The analysis was performed in triplicates. The absorption spectra for each samples was recorded in 1 mL sample volume containing 50 mM potassium phosphate buffer (pH 7.4), 10 mM β-mercaptoethanol, 0.1 mM butachlor and either 0.12 mM NADPH, 5μg AKR or both.</p

Percent composition of butachlor and metabolites as determined by GC-MS analysis.

<p>The GC-MS measurements were carried out with the metabolites extracted from the reaction mix containing 50mM potassium phosphate buffer pH 7.4, 0.4 mM butachlor and 10mM 2-mercaptoethanol, (1) neither NADPH nor AKR, (2) NADPH (0.12mM), (3) AKR (5 μg), or (4) both NADPH (0.12mM) and AKR (5 μg).</p

Spot assay of <i>E</i>. <i>coli</i> strain BL21 (DE3) transformed with vector alone (BL/pET21a) and recombinant plasmid (BL/ pET21a-AKR) under different abiotic stresses.

<p>3 μL of sequential dilutions (only growth at dilution level 10⁻² is shown here) were spotted on the Luria Bertani (LB) plates supplemented with varying concentration of (a) cadmium, (b) mannitol, (c) arsenic and (d) NaCl. For (e) UV-B and (f) heat (50°C),cells were pretreated for different time intervals and then spotted on the LB plates. All spot tests were performed in triplicate.</p

GC charts of the reaction products of AKR17A1 with butachlor used as substrate.

<p>(a) Control assay devoid of enzyme AKR17A1 and cofactor (NADPH); (b) reaction with NADPH alone; (c) enzymatic reaction with AKR17A1 alone; and (d) enzymatic reaction with both AKR17A1 and cofactor NADPH.</p

A Novel Aldo-Keto Reductase (AKR17A1) of <i>Anabaena</i> sp. PCC 7120 Degrades the Rice Field Herbicide Butachlor and Confers Tolerance to Abiotic Stresses in <i>E</i>. <i>coli</i>

<div><p>Present study deals with the identification of a novel aldo/keto reductase, AKR17A1 from <i>Anabaena</i> sp. PCC7120 and adds on as 17^th family of AKR superfamily drawn from a wide variety of organisms. AKR17A1 shares many characteristics of a typical AKR such as— (i) conferring tolerance to multiple stresses like heat, UV-B, and cadmium, (ii) excellent activity towards known AKR substrates (isatin and 2-nitrobenzaldehyde), and (iii) obligate dependence on NADPH as a cofactor for enzyme activity. The most novel attribute of AKR17A1, first reported in this study, is its capability to metabolize butachlor, a persistent rice field herbicide that adversely affects agro-ecosystem and non-target organisms. The AKR17A1 catalyzed- degradation of butachlor resulted into formation of 1,2-benzene dicarboxylic acid and 2,6 bis (1,1, dimethylethyl) 4,-methyl phenol as the major products confirmed by GC-MS analysis.</p></div

Tetanus toxoid-loaded cationic non-aggregated nanostructured lipid particles triggered strong humoral and cellular immune responses

<p>In the present investigation, non-aggregated cationic and unmodified nanoparticles (TT-C-NLPs4 and TT-NLPs1) were prepared of about 49.2 ± 6.8-nm and 40.8 ± 8.3-nm, respectively. In addition, spherical shape, crystalline architecture and cationic charge were also noticed. Furthermore, integrity and conformational stability of TT were maintained in both TT-C-NLPs4 and TT-NLPs1, as evidenced by symmetrical position of bands and superimposed spectra, respectively in SDS-PAGE and circular dichroism. Cellular uptake in RAW264.7 cells indicating the concentration-dependent internalisation of nanoparticles. Qualitatively, CLSM exhibited enhanced cellular uptake of non-aggregated TT-C-NLPs4 owing to interaction with negatively charged plasma membrane and clevaloe mediated/independent endocytosis. In last, <i>in vivo</i> immunisation with non-aggregated TT-C-NLPs4 elicited strong humoral (anti-TT IgG) and cellular (IFN-γ) immune responses at day 42, as compared to non-aggregated TT-NLPs1 and TT-Alum following booster immunisation at day 14 and 28. Thus, non-aggregated cationic lipid nanoparticles may be a potent immune-adjuvant for parenteral delivery of weak antigens.</p