MiR-124 Regulates IQGAP1 and Participates in the Relationship Between Morphine Dependence Susceptibility and Cognition.

Background: Long-term excessive use of morphine leads to addictive diseases and affects cognitive function. Cognitive performance is associated with genetic characteristics.MiR-124 plays a critical regulatory role in neurogenesis, synaptic development, brain plasticity, and the use of addictive substances. As a scaffold protein, IQGAP1 affects learning and memory dose-dependent. However, the role of miR-124 and its target protein as potential addiction biomarkers and the impact on cognitive function have not been fully explored. Method: A total of 40 patients with morphine dependence and 40 cases of healthy people were recruited. We collected basic and clinical information about the two groups. The Generalized Anxiety Disorder Scale (GAD-7), Patient Health Questionnaire-9(PHQ-9), Montreal Cognition Assessment Scale (MoCA), Pittsburgh Sleep Quality Index (PSQI) were used to assess the severity of depression, anxiety, depressive symptoms, cognitive dysfunction, and sleep quality. Results: Compared to the control group, the morphine-dependent group had higher GAD-7, PHQ-9, PSQI scores, and more elevated miR-124 levels but lower MOCA scores and IQGAP1 levels. MiR-124, IQGAP1, the average intake last year were related to OASI scores.MiR-124, IQGAP1, PHQ-9 were associated with MOCA scores. In the multiple regression model, the levels of miR-124 and IQGAP1 were independent factors influencing the severity of morphine dependence. The level of miR-124 was an independent factor influencing the severity of cognitive impairment in patients with morphine dependence. In addition, the luciferase report confirmed that IQGAP1 mRNA is the direct target of miR-124. Conclusion: MiR-124 and its target protein IQGAP1 are involved in the regulation of addiction and cognitive function in patients with morphine dependence.

Simultaneous determination of serum homocysteine, cysteine, and methionine in patients with schizophrenia by liquid chromatography-tandem mass spectrometry.

Schizophrenia is a debilitating psychiatric disorder affecting approximately 1% of the population worldwide. Disturbances in the homocysteine metabolism are an important factor in the pathophysiology of schizophrenia. In this research, a novel validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantification procedure was developed to investigate three significant compounds of homocysteine metabolism: homocysteine, cysteine, and methionine in patients with schizophrenia and healthy controls. Sample preparation involved a reduction with dithiothreitol followed by protein precipitation, and the chromatographic runtime was 2 min. The LC-MS/MS method was validated according to CLSI C62-A and the Chinese Guidance for Liquid Chromatography and Mass Spectrometry Clinical Application. The performance of the method was excellent with a coefficient of variation for precision in the range of 0.5-6.9%, an accuracy of 90.4-101.6%. In addition, the practical applicability of the method was demonstrated by applying it in the routine sample analysis for a schizophrenic patient. Increased homocysteine levels and decreased cysteine levels were observed in the patient with schizophrenia. These results indicate that the activity of the transsulfuration pathway may play a key role in the pathogenesis of schizophrenia.

A simple and rapid LC-MS/MS method for the simultaneous determination of eight antipsychotics in human serum, and its application to therapeutic drug monitoring.

A simple and rapid liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed and used to determine eight antipsychotics (aripiprazole, clozapine, haloperidol, olanzapine, paliperidone, quetiapine, risperidone, and ziprasidone) in human serum for practical clinical usage. Stable isotope-labeled internal standards were used for all drugs to compensate for method variability, including matrix effects, ion extraction and ionization variations. Samples were prepared by simple protein precipitation with methanol. Chromatographic separation was accomplished in less than 3.3 min on a KINTEX C18 column (50 mm × 3.0 mm, 5 µm) using a gradient elution of 2 mM aqueous ammonium formate and methanol at a flow rate of 0.5 mL/min. Quantification was performed by multiple reaction monitoring (MRM) in the positive mode. The method was fully validated according to the latest recommendations of international guidelines. The correlation coefficients of calibration curves were all greater than 0.9945. Internal standard-normalized matrix effects ranged from 96.3% to 115%, and extraction recoveries were between 88.1% and 107%. Coefficients of variation ranged from 1.82 - 13.5% for intra-day precision, 5.69-14.7% for inter-day precision, and the relative error for accuracy did not exceed ±â€¯13.5% for any analyte. The method was successfully applied to routine clinical therapeutic drug monitoring for 2,173 samples.

Improvement in organic solvent resistance and activity of metalloprotease by directed evolution.

Improving enzyme stability in the presence of organic solvent is crucial for non-aqueous catalysis. In this study, directed evolution was applied to improve the tolerance of metalloprotease PT121 towards organic solvent. In presence of acetonitrile and acetone, three mutants (T46Y, H224 F, and H224Y) of PT121 showed excellent solvent stability, which increased their half-lives by 1.2-3.5-fold as compared to the wild-type enzyme. Kinetic constants (KM and kcat values) of the caseinolysis reaction presented H224 F and H224Y mutants have higher affinity than the wild-type, but T46Y mutant were similar to those of the wild-type enzyme. Interestingly, combined mutants T46Y/H224 F and T46Y/H224Y mutants presented awesome stability and excellent caseinolytic activity. Molecular dynamic simulation suggest that improved enzyme stability may be attributed to extensive non-covalent bond network resulting in a more compact structure. Disruption of the disulphide bond formation between Cys-30 and Cys-58 residues in the F56 V mutant is possibly the reason behind its low stability among all the selected mutants. Additionally, T46Y/H224 F and T46Y/H224Y showed a higher peptide synthetic activity in the presence of organic solvents than the wild-type, which renders these mutant enzymes as promising biocatalysts for biotechnological applications.

The g.-762T>C polymorphism of the NPC1L1 gene is common in Chinese and contributes to a higher promoter activity and higher serum cholesterol levels.

Niemann-Pick type C1-like 1 (NPC1L1) protein is responsible for intestinal cholesterol absorption. The aim of the study was to identify genetic polymorphisms of the NPC1L1 gene as well as their functional significance. The method involved screening of promoter and coding regions of the NPC1L1 gene for genetic polymorphisms by direct DNA sequencing of genomic DNA from 50 individuals. Functional studies on promoter polymorphisms were performed using luciferase assay. Association between the polymorphisms and serum cholesterol levels were investigated in 224 individuals. The results showed that in total, 11 single nucleotide polymorphisms were identified. Among them, a promoter polymorphism, g.-762T>C, and a synonymous polymorphism, g.1679C>G, were common (34 and 36%, respectively). These two polymorphisms were highly linked (D' value=0.7459, P-value <0.00001). For the g.-762T>C promoter polymorphism, luciferase assay in HepG2 cell line demonstrated that the -762C allele had a significantly higher promoter activity than the -762T allele (1.30+/-0.22 vs 0.37+/-0.06, 3.5-fold, P<0.05). We also showed that the NPC1L1 promoter activity was downregulated by cholesterol content in both genotypes. When association studies were performed, we found that -762C allele was associated with significantly higher serum total cholesterol and LDL-cholesterol content levels in a recessive model (LDL-cholesterol value=131.2+/-8.1 vs 116.4+/-2.2 mg dl(-1); total cholesterol value=214.7+/-9.0 mg dl(-1) vs 196.9+/-2.6, P-value <0.05, n=224). In conclusion, the C allele at -762 position of the NPC1L1 gene was common in people of Chinese ethnicity. The -762C allele had a higher promoter activity and was associated with a higher serum total cholesterol and LDL-cholesterol level.