Cary 300 uv vis spectrophotometer

Enzyme assays were performed in a 1 mL reaction volume consisting of 50 mM Tris-HCl (pH 8.5), 2 mM MnCl₂ or MgCl₂, 1 mM trisodium DL-isocitrate, and 0.5 mM NAD(P)⁺ at a temperature of 25 °C. The increase in NAD(P)H absorbance was monitored at 340 nm using a Cary 300 UV-Vis spectrophotometer (Varian, Polo Alto, CA, USA) with an extinction coefficient (ε340) of 6.22 mM⁻¹·cm⁻¹. One unit (U) of enzyme activity was defined as the generation of 1 μmol of NADPH per minute. Protein concentrations were determined using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). The enzyme’s kinetic parameters were determined by measuring its activity at various concentrations of NADP⁺ or NAD⁺. The apparent kinetic parameters were calculated using nonlinear regression analysis with Prism 5.0 software (GraphPad Software, San Diego, CA, USA). All experiments were conducted at least three times to ensure reproducibility.

The activities of wild-type and mutant enzymes were assayed by a modification of the method by Cvitkovitch et al.58 (link). Activity assays were carried out in 25°C 1-ml cuvettes (1-cm light path) containing 35 mM Tris-HCl buffer (pH 7.5), 2 mM MgCl₂ or MnCl₂, 1.5 mM DL-isocitrate, and 1.0 mM NAD⁺. The increase in NADH was monitored at 340 nm with a thermostated Cary 300 UV-Vis spectrophotometer (Varian, USA), using a molar extinction coefficient of 6.22 mM⁻¹cm⁻¹. One unit of enzyme activity represented the reduction of 1 µM of NAD⁺ per minute. Protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad, USA) with bovine serum albumin as the standard.

Oxygen consumption rate was measured at 30 °C using a Clark-type oxygen electrode (Oxygraph System Hansatech Instruments), with 1 mL of air-saturated respiration buffer (0.1 M phthalate KOH, pH 5.0), 0.5% glucose. Yeast W303-1B cells were cultured at 28 °C in YP medium supplemented with glucose at the non-repressing concentration of 0.6%, until glucose exhaustion, in the presence of isopropylbenzaldehyde thiosemicarbazone (mHtcum) at different concentrations (5, 10, 25, and 50 µM). The control was treated with the same volume of the solvent DMSO. Oxygen consumption was normalized to the dry weight of the cells. Cytochrome profiles were determined spectrophotometrically at room temperature (Varian Cary300 UV-VIS Spectrophotometer), by recording reduced oxidized cytochrome spectra of yeast W303-1B cells cultured for oxygen consumption rate determination.

To determine the mRNA levels of the target genes, total RNA was extracted from the clinical serum, cells or tissues using the Trizol reagent (Invitrogen). Reverse transcription reactions were performed using 5 μg of total RNA following the standard protocol supplied with the SYBR Premix Ex Taq II (TaKaRa, Daliang, China). The resulting cDNA was used for PCR, and GAPDH was used as a loading control. All the reactions had a hot start of 5 min at 9 °C and a final elongation step at 72 °C for 10 min. The primers used were as follows: GAPDH, 5′-ATGGGGAAGGTGAAGGTCG-3′ (sense) and 5′-GGGGTCATTGATGGCAACAATA-3′ (antisense); miR-34, 5′-GGCAGTGTCTTAGCTGGTTGT-3′ (sense) and 5′-TGGTGTCGTGGAGTCG-3′ (antisense); miR-449, 5′-ACACTCCAGCTG GGTGGCAGTGTATTGTTA-3′ (forward) and 5′-TGGTGTCGTGGAGTCG-3′ (reverse); U6, 5′-CTCGCTTCGGCACA-3′ (sense) and 5′-AACGCTTCACGAATTTGCGT-3′ (antisense). Amplified products were separated on agarose gels, visualized and quantified using a thermostated Cary 300 UV-Vis spectrophotometer (Varian, Sunnyvale, CA, USA).

The phase structure of samples was identified using Bruker D8 Advance X-ray diffract meter (XRD, BRUKER Co., Karlsruhe, Germany) with Cu Kα1 radiation (k = 0.15418 nm). The morphology of the as-prepared samples was studied with a Tecnai-G2-F20 transmission electron microscopy (TEM, FEI Co., Hillsboro, OR, USA) and Hitachi New Generation SU8220 field emission scanning electron microscopy (FESEM, HITACHI Co., Kyoto, Japan). The ultraviolet-visible diffuse reflection spectroscopy (UV-vis DRS) was measured by the Cary 300 UV-vis spectrophotometer (Varian Co., Palo Alto, CA, USA). X-ray photoelectron spectroscopy (XPS) data were collected from the K-Alpha photoelectron spectroscope (Thermo Fisher Scientific, Waltham, MA, USA) with monochromatic Al Ka radiation (200 W). Edinburgh FS5 fluorescence lifetime spectrophotometer was used to obtain the stable state photoluminescence (PL) spectra and time-resolved photoluminescence (TRPL) spectra (Edinburgh Instruments Co., Edinburgh, UK). The electron paramagnetic resonance (EPR) spectra were obtained using an EPR spectro-meter (E300, BRUKER Co., Karlsruhe, Germany) with a modulation amplitude of 1 G and microwave power of 3.99 mW.