A Method for Production of Human Adenosine Deaminase 1 (ADA1) in Pichia pastoris Provides Needed Quality Control Material for Clinical Laboratories.

OBJECTIVE: Adenosine deaminase (ADA) plays a major role in maintaining metabolic homeostasis via catalysis of hydrolytic deamination of adenosine to inosine. The ADA1 isoenzyme of ADA is an analyte tested in clinical laboratories; however, lack of quality control (QC) material in terms of enzyme homogeneity, stability, and coverage of the clinically relevant analytical measurement range (AMR), poses a challenge for adequate monitoring of this analyte. The aim of this study was to address the need for manufacture of QC material through recombinant expression of catalytically active ADA1 in eukaryotic cells (Pichia pastoris GS115). METHODS: The coding region of ADA1 gene was amplified by PCR and ligated into plasmid pPICZαA, followed by transfer into P. pastoris using electroporation. Recombinant ADA1 produced by P. pastoris was purified using a Ni-NTA resin column, yielding 5 mL of purified ADA1 with an activity of 4200.6 U/L. Purified ADA1 protein was added to human donor serum as the appropriate matrix for QC materials preparation. RESULTS: One hundred vials of lyophilized ADA1 were prepared at clinically significant concentrations at 41.6 U/L and 115.5 U/L (50 vials each). Both concentrations were homogenous and stable at room temperature (RT, 22-24°C) for at least 7 d, at 4°C for 3 months, and at -20°C for 12 months. Reconstituted aliquots of QC material were found to be stable at -20°C for up to 60 d and should be used within 8 h or 48 h when stored at RT or 4°C, respectively. CONCLUSION: Success of this ADA1 expression system presents a potential solution to increase production options available to clinical laboratories.

Inflammation Pharmacological Reaction and YMDD Mutational Patterns in Lamivudine Therapeutics Hepatitis B Virus.

Background/Aims: Emergence of tyrosine-methionine-aspartate-aspartate (YMDD) motif in reverse transcriptase is a serious problem in chronic hepatitis B(CHB) patients after Lamivudine (LAM) therapy. However, the relationship between inflammation pharmacological reaction and YMDD mutational patterns of CHB has not been well-characterized. The aim of this study was to investigate the inflammation pharmacological reaction and different YMDD mutants patterns of CHB patients. Methods: We investigated the inflammation pharmacological reaction and YMDD mutational patterns through biochemical, serological and virological detection among 83 CHB patients, including 25 YMDD mutants, 25 under detection, and 33 control patients without YMDD mutants. Results: Prevalence of YMDD mutation patterns is different. Among 25 YMDD mutants patients, YIDD was the dominant mutation (72%), followed YVDD (16%) and the hybrid YIDD + YVDD (12%). The time course during the YMDD mutations was also different. 52.4% patients developed the mutation less than 12 months after the LAM therapy. Serum hepatitis B virus (HBV) DNA level in patients with YMDD mutants were significantly higher than that in control and negative groups. Serum HbsAg and HbeAg in patients with YMDD mutants were also higher than those in control and negative groups, despite no significant difference was found forserum HbeAb. ALT and AST levels were also significantly higher in mutants group. Conclusions: Illuminating inflammation pharmacological reaction and YMDD mutational patterns of CHB during pathological process may have implications for future therapy in YMDD mutation patients. This may have impact on the choice of treatment strategies for lamivudine-resistant HBV.

Performances Evaluation of Four Systems for Homocysteine Determination by LC-MS/MS Reference Method.

BACKGROUND: The aim of this study is to verify the analytical performance of four homocysteine detection systems made in China and to explore the comparability of homocysteine detection systems by isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) reference method. METHODS: The intra-batch precision, inter-batch precision, accuracy, and linear range of four homocysteine detection systems were evaluated. The ID-LC-MS/MS reference method was used to evaluate the comparability and accuracy of fresh frozen serum samples in four different detection systems of homocysteine. The ID-LC-MS/MS reference method is used to assign samples as calibrators to calibrate each system. The variation and deviation of fresh serum samples between different systems before and after calibration were compared. RESULTS: The intra-batch imprecision of the four detection systems was less than 5%, and the coefficient of variation of inter-batch imprecision was less than 6.7%. The precision met the clinical requirements. Before calibration, the results measured by detection system 2 are consistent with the ID-LC-MS/MS reference method, which meets the requirements of accuracy verification. The regression equation of R² ≥ 0.975 in the regression equation of linear analysis of the four systems, the linearity of the four detection systems is good in the range of evaluation concentration, and all of them can meet the declared linear range. The absolute average bias of fresh serum measured by the four detection systems after calibration decreased from 3.76 µmol/L, 0.96 µmol/L, 1.30 µmol/L, -1.56 µmol/L to 0.31 µmol/L, 0.28 µmol/L, 0.4 µmol/L, 0.40 µmol/L, respectively. The relative average bias decreased from 22.6%, 7.50%, 11.0% and -8.50% to 1.98%, 1.78%, 2.59%, 2.34%, respectively. After calibration, the slope and intercept of the regression curve of the fresh serum measured by the four detection systems and the reference method are closer to 1 and 0 than before calibration. CONCLUSIONS: The precision, reference interval, and linear evaluation of the four detection systems are good. The ID-LC-MS/MS reference method assigning fresh frozen serum samples as calibrators can improve the accuracy and comparability of the results of different detection systems.

Expression, purification and identification of isotope-labeled recombinant cystatin C protein in Escheichia coli intended for absolute quantification using isotope dilution mass spectrometry.

Isotope-labeled proteins are expected to be used as internal standard proteins in the field of protein quantification by isotope dilution mass spectrometry (ID/MS). To achieve the absolute quantification of Cystatin C (Cys C) based on ID/MS, we aims to obtain 15N isotope-labeled recombinant Cys C (15N-Cys C) protein. Firstly, the Cys C gene was optimized based on the preferred codons of Escherichia coli, and inserted into the pET-28a(+) expression plasmid. Then, the plasmid was transformed into TOP10 and BL21 strains, and 15N-Cys C was expressed in M9 medium using 15N as the only nitrogen source. 15N-Cys C was detected by SDS-PAGE, protein immunoblotting and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). The characteristic peptides obtained from 15N-Cys C were analyzed by a Q Exactive Plus MS system. Results showed that 53.06% of the codons were optimized. The codon adaptation index of the Cys C genes increased from 0.31 to 0.95, and the GC content was adjusted from 64.85% to 54.88%. The purity of 15N-Cys C was higher than 95%. MALDI-TOF MS analysis showed that the m/z of 15N-Cys C had changed from 13 449 to 14 850. The characteristic peptides showed that 619.79 m/z (M+2H)2+ was the parent ion of 15N-Cys C and that the secondary ions of 15N-labeled peptides from y+5 to y+9 were 616.27 m/z, 716.33 m/z, 788.39 m/z, 936.43 m/z, and 1052.46 m/z, respectively. In conclusion, we successfully expressed, purified and identified of 15N-Cys C protein in Escheichia coli intended for absolute quantification using ID/MS.

Determination of serum 25-hydroxyvitamin D status among population in southern China by a high accuracy LC-MS/MS method traced to reference measurement procedure.

OBJECTIVE: We aimed to describe the 25-hydroxyvitamin D (25(OH)D) status of southern Chinese individuals by a high-accuracy liquid chromatography tandem mass spectrometry (LC-MS/MS) method which can trace to reference measurement procedure. MATERIALS AND METHODS: From January 2018 to June 2019, a total of 4775 southern Chinese individuals were evaluated in our study. The serum levels of parathyroid hormone (PTH) were detected simultaneously in 162 cases. 25(OH)D was determined by LC-MS/MS, and PTH was detected using routine automated analysers. The distribution of the concentration, prevalence and seasonal variability of 25(OH)D in males and females of different age groups were studied. RESULTS: The mean 25(OH)D concentration in our study was 32.57 ng/mL (4.20-101.40 ng/mL). The global 25(OH)D concentration in males was higher than that in females of different age group. The prevalence of vitamin D deficiency (< 20 ng/mL) in females (16.65%) was higher than that in males (6.83%). The prevalence of vitamin D deficiency (< 20 ng/mL) was most common in winter (22.98% of all women and 15.49% of all men). 25(OH)D concentrations were higher in those from whom blood samples were collected in summer and autumn than in winter and spring. 25(OH)D2 was detected in 672 serum samples (14.07%). In addition, there was a negative correlation between the concentrations of 25(OH)D and serum PTH (r = - 0.149, P < 0.05). CONCLUSION: Our study demonstrated that the average serum 25(OH)D concentration in southern Chinese individuals was higher than that in other Chinese cohorts by a high-accuracy LC-MS/MS method. The global 25(OH)D concentration in males was higher than that in females of different ages, and the prevalence of vitamin D deficiency in females was higher than that in males. Seasonal change was an important aspect of 25(OH)D concentration in young and middle-aged people but became less relevant for that in older subjects. 25(OH)D2 detection was of minor practical significance in our study. In addition, we also found that there was a negative correlation between the serum levels of 25(OH)D and PTH in southern Chinese individuals.

Evaluation of a bracketing calibration-based isotope dilution liquid chromatography-tandem mass spectrometry candidate reference measurement procedure for 17α-hydroxyprogesterone in human plasma.

A candidate reference measurement procedure (RMP) based on isotope dilution coupled with liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) was developed and validated for the quantification of 17α-hydroxyprogesterone (17-OHP) in human plasma. 17-OHP spiked with a deuterium-labeled internal standard was extracted from plasma by liquid-liquid extraction with 1 mL n-hexane/ethyl acetate (3:2, v/v). Reversed-phase chromatography and positive electrospray ionization were used in the ID-LC-MS/MS. Gradient elution coupled with use of a C18-packed ultrahigh-performance liquid chromatography column allowed complete baseline resolution of 17-OHP from its structural analogue desoxycorticosterone in 6 min. To determine the 17-OHP level in human plasma, a bracketing calibration method was used to give higher accuracy and precision. The limit of detection and the lower limit of the measuring interval for the candidate RMP were 2.1 pg/mL (6.4 pmol/L) and 4.6 pg/mL (13.9 pmol/L), respectively. Extraction recovery was determined to be (96.08 ± 3.03)% (n = 3). Imprecision (intra-assay and interassay) was 4.03% or less at 0.83, 15.19, 64.22, and 313.46 ng/mL (2.51, 45.97, 194.34, and 948.56 nmol/L, respectively). Recoveries ranged from 98.05% to 102.24%. When comparing our RMP results with those obtained with an established RMP via International Federation of Clinical Chemistry and Laboratory Medicine external quality assessment scheme for reference laboratories in laboratory medicine (RELA) samples, we found that the biases ranged from -1.99% to 3.08% against the targets. No interference was observed, and the linear response ranged from 0.47 to 958.63 ng/mL (1.42 to 2900.90 nmol/L). Moreover, the candidate RMP was used to measure the concentration of 17-OHP in human plasma and was compared with an immunoassay using 40 plasma samples. The performance of the method meets the needs of an RMP (total coefficient of variation of 5% or less and bias of 3.08% or less). This method can be used for reference material value assignment of 17-OHP in human plasma matrix. It could also serve as an accurate reference baseline for routine methods to increase the accuracy and precision of certain clinical laboratory measurements. Graphical abstract Selected ion chromatograms obtained by liquid chromatography-tandem mass spectrometry with a C18 column for 17α-hydroxyprogesterone (17-OHP) from a plasma sample.

Tauroursodeoxycholic acid inhibits endoplasmic reticulum stress, blocks mitochondrial permeability transition pore opening, and suppresses reperfusion injury through GSK-3ß in cardiac H9c2 cells.

This study investigates whether inhibition of endoplasmic reticulum (ER) stress prevents opening of the mitochondrial permeability transition pore (mPTP) and evaluates the corresponding signaling pathways involved in this process. Exposure of cardiac H9c2 cells to 800 µM H2O2 for 20 min opened mPTP in response to oxidative stress, as demonstrated by quenching of tetramethylrhodamine ethyl ester (TMRE) fluorescence. Oxidative stress-induced mPTP opening was rescued by the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) in a dose-dependent manner at low concentrations. The PI3K and PKG inhibitors LY294002 and KT5823 inhibited the effect of TUDCA on mPTP opening, suggesting the involvement of PI3K/Akt and PKG signaling pathways. TUDCA significantly increased glycogen synthase kinase 3 (GSK-3ß) phosphorylation at Ser-9, with peak effect at 30 µM TUDCA. The level of GRP78 (ER chaperone) expression was significantly upregulated by 30 µM TUDCA. TUDCA-induced increases in Akt and GSK-3ß phosphorylation were inhibited by LY294002, whereas KT5823 suppressed TUDCA-induced increases in VASP and GSK-3ß phosphorylation. Oxidative stress severely affected cell morphology and ultrastructure. TUDCA prevented H2O2-induced ER swelling and mitochondrial damage. TUDCA boosted the viability of cells disrupted by ischemia/reperfusion (I/R), indicating that TUDCA eased reperfusion injury. However, TUDCA did not improve the viability of cells expressing the constitutively active GSK-3ß mutant (GSK-3ß-S9A-HA) that were subjected to I/R, suggesting an essential role of GSK-3ß inactivation in TUDCA-mediated cardioprotection against reperfusion damage. These data indicate that ER stress inhibition prevents mPTP opening and attenuates reperfusion injury through GSK-3ß inactivation. The PI3K/Akt and PKG pathways may mediate GSK-3ß inactivation.

[Characteristic of Ultrafine Particles Transferring Through Building Envelopes].

Penetration and transmission characteristics of outdoor particulate matter through building envelope structure into indoor and its influencing factors were studied by experimental and numerical simulation methods. With the aid of fast mobility particle spectrometer (fast mobility particle sizer, FMPS), particle number concentrations were measured and particle penetration rates were obtained. The effects of slit size and flow pressure on the infiltration process were studied. Compared with numerical simulation and experimental results, the trend was consistent. Experiment and simulation results showed that when the slit was 1 mm high, the penetration rate of particulates with small particle size was small. Its leading influence factor was Brownian diffusion movement, with the increase of particle size, the penetration rate increased. Particle penetration rate was enhanced with the increase of inlet pressure and particle size, but decreased with the increase of slit length. Simulation results showed that the particle penetration rate was enhanced with the increase of slit height. Among all the factors, slit height was the dominant one. When the particle size was more than 30 nm, the penetration rate was close to 1. When the slit height was reduced to 0.25 mm, the penetration rate of particles with size of near 300 nm reached the maximum of 0.93. With the increase of the particle size, particle penetration rate showed a trend of decrease, and gravity settling began to dominate. The experiment result showed that when the slit height changed, the dominant factors of particles subsidence to the wall were changed. At low concentration in a certain range, the particle number concentration had little effect on the penetration rate. The range of particle number concentration of inside and outside I/O ratio was 0.69- 0.73. The correlation coefficient R2 was 0.99. The linear correlation was obvious. The particle penetration rate in slit straight way was significantly greater than that at the corner of the channel.

[Ultrafine particle number concentration and size distribution of vehicle exhaust ultrafine particles].

Ultrafine particle (UFP) number concentrations obtained from three different vehicles were measured using fast mobility particle sizer (FMPS) and automobile exhaust gas analyzer. UFP number concentration and size distribution were studied at different idle driving speeds. The results showed that at a low idle speed of 800 rmin-1 , the emission particle number concentration was the lowest and showed a increasing trend with the increase of idle speed. The majority of exhaust particles were in Nuclear mode and Aitken mode. The peak sizes were dominated by 10 nm and 50 nm. Particle number concentration showed a significantly sharp increase during the vehicle acceleration process, and was then kept stable when the speed was stable. In the range of 0. 4 m axial distance from the end of the exhaust pipe, the particle number concentration decayed rapidly after dilution, but it was not obvious in the range of 0. 4-1 m. The number concentration was larger than the background concentration. Concentration of exhaust emissions such as CO, HC and NO showed a reducing trend with the increase of idle speed,which was in contrast to the emission trend of particle number concentration.

[Experimental study on the size spectra and emission factor of ultrafine particle from coal combustion].

The emission characteristics of ultrafine particles released from pulverized coal combustion were studied, the size spectra of ultrafine particles (5.6-560 nm) were measured with FMPS (fast mobility particle sizer) on a self-built aerosol experiment platform. Meanwhile, a particle dynamic evolution model was established to obtain the particle deposition rate and the emission rate through the optimized algorithm. Finally, the emission factor was calculated. The results showed that at the beginning of particle generation, the size spectra were polydisperse and complex, the initial size spectra was mainly composed of three modes including 10 nm, 30-40 nm and 100-200 nm. Among them, the number concentration of mode around 10 nm was higher than those of other modes, the size spectrum of around 100-200 nm was lognormal distributed, with a CMD (count median diameter) of around 16 nm. Then, as time went on, the total number concentration was decayed by exponential law, the CMD first increased and then tended to be stable gradually. The calculation results showed that the emission factor of particles from coal combustion under laboratory condition was (5.54 x 10(12) ± 2.18 x 10(12)) unit x g(-1).