Potassium thiocyanate

Determination of total phenolic content (TPC): Amount of TP were assessed using the Folin-Ciocalteu reagent [25 ]. Briefly, the crude extract (50 mg) was mixed with Folin-Ciocalteu reagent (0.5 mL) and deionized water (7.5 mL). The mixture was kept at room temperature for 10 min, and then 20% sodium carbonate (w/v, 1.5 mL) was added. The mixture was heated in a water bath at 40 ^oC for 20 min and then cooled in an ice bath; absorbance was read at 755 nm using a spectrophotometer (U-2001, Hitachi Instruments Inc., Tokyo, Japan). Amounts of TP were calculated using gallic acid calibration curve within range of 10-100 mgL^-1(R² = 0.9986). The results were expressed as gallic acid equivalents (GAE) g/100g of dry plant matter. All samples were analyzed thrice and the results averaged. The results are reported on dry weight basis (DW).
Determination of total flavonoid contents (TFC): The TFC were measured following a previously reported spectrophotometric method [26 (link)]. Briefly, extracts of each plant material (1 mL containing 0.1 mg/mL) were diluted with water (4 mL) in a 10 mL volumetric flask. Initially, 5% NaNO₂ solution (0.3 mL) was added to each volumetric flask; at 5 min, 10% AlCl₃ (0.3 mL) was added; and at 6 min, 1.0 M NaOH (2 mL) was added. Water (2.4 mL) was then added to the reaction flask and mixed well. Absorbance of the reaction mixture was read at 510 nm. TFC were determined as catechin equivalents (g/100g of dry weight). Three readings were taken for each sample and the results averaged.
Determination of reducing power: The reducing power of the extracts was determined according to the procedure described earlier [27 ], with a slight modification. Concentrated extract (2.5-10.0 mg) was mixed with sodium phosphate buffer (5.0 mL, 0.2 M, pH 6.6) and potassium ferricyanide (5.0 mL, 1.0%); the mixture was incubated at 50 ^oC for 20 min. Then 10% trichloroacetic acid (5 mL) was added and the mixture centrifuged at 980 g for 10 min at 5 °C in a refrigerated centrifuge (CHM-17; Kokusan Denki, Tokyo, Japan). The upper layer of the solution (5.0 mL) was decanted and diluted with 5.0 mL of distilled water and ferric chloride (1.0 mL, 0.1%), and absorbance read at 700 nm using a spectrophotometer (U-2001, Hitachi Instruments Inc., Tokyo, Japan). All samples were analyzed thrice and the results averaged.
DPPH^. scavenging assay: 1, 1^’–diphenyl–2-picrylhydrazyl (DPPH) free radical scavenging activity of the extracts was assessed using the procedure reported earlier [28 (link)]. Briefly, to extract (1.0 mL) containing 25 μg/mL of dry matter in methanol, freshly prepared solution of DPPH (0.025 g/L, 5.0 mL) was added. Absorbance at 0, 0.5, 1, 2, 5 and 10 min was measured at 515 nm using a spectrophotometer. The scavenging amounts of DPPH radical (DPPH^.) was calculated from a calibration curve. Absorbance read at the 5th min was used for comparison of radical scavenging activity of the extracts.
Determination of antioxidant activity in linoleic acid system: The antioxidant activity of the tested plant extracts was also determined by measuring the oxidation of linoleic acid [28 (link)]. Five mg of each plant extract were added separately to a solution of linoleic acid (0.13 mL), 99.8% ethanol (10 mL) and 0.2 M sodium phosphate buffer (pH 7, 10 mL). The mixture was made up to 25 mL with distilled water and incubated at 40 ^oC up to 360 h. Extent of oxidation was measured by peroxide value applying thiocyanate method as described by Yen et al. [27 ]. Briefly, ethanol (75% v/v, 10 mL ), aqueous solution of ammonium thiocyanate (30% w/v, 0.2 mL), sample solution (0.2 mL) and ferrous chloride (FeCl₂) solution (20 mM in 3.5% HCl; v/v, 0.2 mL) were added sequentially. After 3 min of stirring, the absorption was measured at 500 nm using a spectrophotometer (U-2001, Hitachi Instruments Inc., Tokyo, Japan). A control contained all reagents with exception of extracts. Synthetic antioxidants butylated hydroxytoluene (BHT) was used as a positive control. Percent inhibition of linoleic acid oxidation was calculated with the following equation: 100 – [(increase in absorbance of sample at 360 h / increase in absorbance of control at 360 h) × 100], to express antioxidant activity.

The SHV-1 and KPC-2 β-lactamases were expressed and crystallized as described previously [18 , 25 ]. As before, we used the C-terminally truncated KPC-2 β-lactamase in which the last 4 residues were removed to facilitate improved crystallization [18 , 26 ]. KPC-2 was crystallized using vapour diffusion using a sitting drop tray with a well solution comprised of 20% PEG 6000, 100 mM potassium thiocyanate, and 100 mM citrate pH 4.0; SHV-1 was crystallized using 25% PEG 6000, 100mM Tris pH 7.5, and 0.56mM Cymal-6. A crystal of SHV-1 was soaked with 50mM avibactam (AstraZeneca, Waltham, Massachusetts, USA) in mother liquor for 40 min prior to transfer to perfluoropolyether for cryo-protection before flash freezing the crystal in liquid nitrogen. KPC-2 crystals were soaked for 8 min with 5mM avibactam in mother liquor (25% PEG 6000, 100mM citrate pH 5.0) and 20% ethylene glycol prior to flash freezing. Data was collected at the Stanford Synchrotron Radiation Lightsource and processed using HKL2000[27 ] resulting in 1.42Å and 1.8Å datasets for SHV-1 and KPC-2, respectively (data collection statistics are shown in Table 1). Starting protein coordinates for SHV-1 and KPC-2 were PDB identifiers 2H5S and 3RXW, respectively; the program MOLREP was used for molecular replacement [28 ]. The structure was refined using REFMAC and COOT[29 , 30 ]. Initial refinement and subsequent density inspection indicated the presence of a covalently bound avibactam in both the SHV-1 and KPC-2 active sites. The program PRODRG[31 ] was used to obtain the topology and refinement parameter files for avibactam for subsequent inclusion of the ligand in refinement. The program PROCHECK was used for structure validation [32 ]. Final refinement statistics are listed in Table 1. The coordinates and structure factors for the KPC-2 and SHV-1 avibactam complexes were deposited with the Protein Data Bank (PDB identifiers 4ZAM and 4ZBE, respectively).

Following reagents were used at different stages of sample processing and DNA isolation: EDTA (0.5 M, pH 8.0), NaCl (5 M), PVPP (Mol wt 40,000), Guanidine thiocyanate (4 M), Sodium-acetate (3 M, pH 5.2), Potassium acetate (5 M, pH 5.2), N-Laurylsarcosine (10%), Glass beads (2.5 mm), Zirconia beads (0.1 mm), Ethanol (96%), Hydrochloric acid (HCl), Sterile deionized water (H₂O). All the chemicals used in this study were purchased from Sigma-Aldrich, USA.

Chloride hexahydrate (NiCl₂·6H₂O), Boric acid (H₃BO₃), ammonium chloride (NH₄Cl), potassium phosphate dibasic anhydrous (K₂HPO₄), Potassium dihydrogen phosphate (KH₂PO₄), Potassium hydroxide (KOH) and polyvinylpyrrolidone (PVP), 25% ammonia (NH₃·H₂O) were bought from Aladdin, and concentrated sulfuric acid (H₂SO₄), copper sulfate pentahydrate (CuSO₄·5H₂O), concentrated nitric acid (HNO₃), ethylenediaminetetraacetic acid, potassium thiocyanate acid (KSCN) and ultrapure water (>18.2 MΩ) were purchased from China National Medicines Corporation Ltd. Ruthenium chloride hydrate (RuCl₃·xH₂O) was obtained from Beijing Innochem Technology Corporation Ltd. All the chemicals were used as received without any other purification.