Multi-omics study reveals associations among neurotransmitter, extracellular vesicle-derived microRNA and psychiatric comorbidities during heroin and methamphetamine withdrawal.

Despite decades of research in the field of substance withdrawal, molecular biomarkers and related mechanistic study have generally been lacking. In addition to known neurotransmitters, circulating miRNAs are found in small vesicles known as exosomes within blood that have diagnostic potential and are known to contribute to psychiatric disorders. The aim of this work was to characterize the changes in neurotransmitter and exosomal miRNA profiles during heroin and methamphetamine withdrawal using a cross-sectional study design, and to determine their associations to psychiatric comorbidities in a large group of patients with substance use disorders (SUDs). Using weighted gene co-expression network analysis, a series of known, conserved, and novel exosomal miRNAs were identified as being associated with the severity of anxiety and depression, as well as the concentrations of neurotransmitters GABA, choline, and serotonin. Bioinformatics analyses established that the differences in the miRNA profile target signaling pathways are significantly associated with developmental and intellectual abnormalities. Notably, a set of dysregulated miRNA signatures including hsa-mia-451a and hsa-mir-21a resulted in an AUC of 0.966 and 0.861, respectively, for predicting the patients with SUDs. Furthermore, hsa-miR-744a-5p was positively correlated with serotonin, and its important role in maintaining neuronal development and function was revealed using an in vitro human induced pluripotent stem cells derived neuronal model. Our results suggest that the miRNA content of circulating exosomes represent a biomolecular "fingerprint" of the progression of substance withdrawal and may uncover the putative mechanism of how these exosomal miRNAs contribute to psychiatric symptoms.

Metformin modulates microbiota-derived inosine and ameliorates methamphetamine-induced anxiety and depression-like withdrawal symptoms in mice.

BACKGROUND: Metformin exhibits therapeutic potential in behavioural deficits induced by methamphetamine (METH) in rats. Emerging studies suggest gut microbiota may impact psychiatric symptoms, but there is no direct evidence supporting metformin's participation in the pathophysiology of withdrawal symptoms via modulation of gut microbiota. METHODS: In order to define the functional impacts of gut microbiota and metformin to the behavioural deficits during METH withdrawal, we utilized a combination of fecal microbiota transplantation (FMT), high-throughput sequencing, and untargeted metabolomics technologies. RESULTS: First, METH addicts exhibited higher α diversity and distinct microbial structures compared to healthy controls. In particular, the relative abundance of Rikenellaceae was positively correlated with the severity of anxiety and depression. Second, both human-to-mouse and mouse-to-mouse FMTs confirmed that METH-altered-microbiota transplantation is sufficient to promote anxiety and depression-like behaviours in recipient germ-free mice, and these behavioural disturbances could be ameliorated by metformin. In-depth analysis revealed that METH significantly altered the bacterial composition and structure as well as relative abundance of several bacterial taxa and metabolites, including Rikenellaceae and inosine, respectively, whereas add-on metformin could remodel these alterations. Finally, the inosine complementation successfully restored METH-induced anxiety and depression-like behaviours in mice. CONCLUSION: This study demonstrates that METH withdrawal-induced anxiety and depression-like behaviours are reversible and transmissible via gut microbiota in a mouse model. The therapeutic effects of metformin on psychiatric manifestations are associated with microbiota-derived metabolites, highlighting the role of the gut microbiota in substance use disorders and the pathophysiology of withdrawal symptoms.

The Association of Altered Gut Microbiota and Intestinal Mucosal Barrier Integrity in Mice With Heroin Dependence.

The gut microbiota is believed to play a significant role in psychological and gastrointestinal symptoms in heroin addicts. However, the underlying mechanism remains largely unknown. We show here that heroin addicts had a decrease in body mass index (BMI) and abnormal serum D-lactic acid (DLA), endotoxin (ET) and diamine oxidase (DAO) levels during their withdrawal stage, suggesting a potential intestinal injury. The gut microbial profiles in the mouse model with heroin dependence showed slightly decreased alpha diversity, as well as higher levels of Bifidobacterium and Sutterella and a decrease in Akkermansia at genus level compared to the control group. Fecal microbiota transplantation (FMT) further confirmed that the microbiota altered by heroin dependence was sufficient to impair body weight and intestinal mucosal barrier integrity in recipient mice. Moreover, short-chain fatty acids (SCFAs) profiling revealed that microbiota-derived propionic acid significantly decreased in heroin dependent mice compared to controls. Overall, our study shows that heroin dependence significantly altered gut microbiota and impaired intestinal mucosal barrier integrity in mice, highlighting the role of the gut microbiota in substance use disorders and the pathophysiology of withdrawal symptoms.

Integration of Molecular Inflammatory Interactome Analyses Reveals Dynamics of Circulating Cytokines and Extracellular Vesicle Long Non-Coding RNAs and mRNAs in Heroin Addicts During Acute and Protracted Withdrawal.

Heroin addiction and withdrawal influence multiple physiological functions, including immune responses, but the mechanism remains largely elusive. The objective of this study was to investigate the molecular inflammatory interactome, particularly the cytokines and transcriptome regulatory network in heroin addicts undergoing withdrawal, compared to healthy controls (HCs). Twenty-seven cytokines were simultaneously assessed in 41 heroin addicts, including 20 at the acute withdrawal (AW) stage and 21 at the protracted withdrawal (PW) stage, and 38 age- and gender-matched HCs. Disturbed T-helper(Th)1/Th2, Th1/Th17, and Th2/Th17 balances, characterized by reduced interleukin (IL)-2, elevated IL-4, IL-10, and IL-17A, but normal TNF-α, were present in the AW subjects. These imbalances were mostly restored to the baseline at the PW stage. However, the cytokines TNF-α, IL-2, IL-7, IL-10, and IL-17A remained dysregulated. This study also profiled exosomal long non-coding RNA (lncRNA) and mRNA in the plasma of heroin addicts, constructed co-expression gene regulation networks, and identified lncRNA-mRNA-pathway pairs specifically associated with alterations in cytokine profiles and Th1/Th2/Th17 imbalances. Altogether, a large amount of cytokine and exosomal lncRNA/mRNA expression profiling data relating to heroin withdrawal was obtained, providing a useful experimental and theoretical basis for further understanding of the pathogenic mechanisms of withdrawal symptoms in heroin addicts.

Plasma metabolites changes in male heroin addicts during acute and protracted withdrawal.

BACKGROUND: Heroin addiction and withdrawal have been associated with an increased risk for infectious diseases and psychological complications. However, the changes of metabolites in heroin addicts during withdrawal remain largely unknown. METHODS: A total of 50 participants including 20 heroin addicts with acute abstinence stage, 15 with protracted abstinence stage and 15 healthy controls, were recruited. We performed metabolic profiling of plasma samples based on ultraperformance liquid chromatography coupled to tandem mass spectrometry to explore the potential biomarkers and mechanisms of heroin withdrawal. RESULTS: Among the metabolites analyzed, omega-6 polyunsaturated fatty acids (linoleic acid, dihomo-gamma-linolenic acid, arachidonic acid, n-6 docosapentaenoic acid), omega-3 polyunsaturated fatty acids (docosahexaenoic acid, docosapentaenoic acid), aromatic amino acids (phenylalanine, tyrosine, tryptophan), and intermediates of the tricarboxylic acid cycle (oxoglutaric acid, isocitric acid) were significantly reduced during acute heroin withdrawal. Although majority of the metabolite changes could recover after months of withdrawal, the levels of alpha-aminobutyric acid, alloisoleucine, ketoleucine, and oxalic acid do not recover. CONCLUSIONS: In conclusion, the plasma metabolites undergo tremendous changes during heroin withdrawal. Through metabolomic analysis, we have identified links between a framework of metabolic perturbations and withdrawal stages in heroin addicts.

Psychiatric Comorbidities and Liver Injury Are Associated With Unbalanced Plasma Bile Acid Profile During Methamphetamine Withdrawal.

Background: The pathogenesis of methamphetamine usedisorders (MUDs) remains largely unknown; however, bile acids may play arole as potential mediators of liver injury and psychiatric comorbidities.The aim of this study was to characterize bile acid (BA) profiles in plasmaof patients with MUDs undergoing withdrawal. Methods: Liver functions and psychiatric symptoms wereevaluated in a retrospective cohort (30 MUDs versus 30 control subjects) andan exploratory cohort (30 MUDs including 10 subjects each at the 7-day,3-month, and 12-month withdrawal stages versus 10 control subjects). BAcompositions in plasma samples from MUD patients in the exploratory cohortwere determined by gas-liquid chromatography. Results: Both psychiatric comorbidities andmethamphetamine-induced liver injury were observed in patients in both MUDcohorts. The plasma concentrations of the total BA, cholic acid (CA), andchenodeoxycholic acid (CDCA) were lower in MUD patients relative tocontrols. The maximum decline was observed at the 3-month stage, withgradual recovery at the 12-month stage. Notably, the ratios of deoxycholicacid (DCA)/CA and lithocholic acid (LCA)/CDCA were statistically significantat the 3-month stage comparing with controls. Significant correlations werefound between the LCA/CDCA and taurolithocholic acid (TLCA)/CDCA ratios andthe levels of alanine transaminase and aspartate aminotransferase, andbetween the LCA/CDCA ratio and the HAM-A score. Conclusion: BA profile during METH withdrawal weremarkedly altered, with these unbalanced BAs being associated with liverinjury. The associations between BA profiles and psychiatric symptomssuggest an association between specific BAs and disease progression,possibly through the liver-brain axis.

[Hematological and genotypic features of 100 patients with hemoglobin E disorders from Yunnan Province].

OBJECTIVE: To explore hematological and genotypic characteristics of patients with hemoglobin E (Hb E) disorders from Yunnan Province. METHODS: One hundred individuals with Hb E disorders indicated by high performance liquid chromatography (HPLC) were subjected to genetic testing through multiple gap-PCR and reverse dot-blotting analysis. RESULTS: All patients were found to harbor a mutation to the 26th codon of the ß -globin chain (HBB: c.79G>A). Ninety patients were heterozygotes, and 10 co-inherited c.79G>A and an α -thalassemia mutation (7 α α /-α3.7, 2 α α /--SEA and 1 -α 3.7/-α3.7). Hematological characteristics of the heterozygotes were: Hb A2 (26.02±3.64)%, Hb F(1.35±1.25)%, MCV(78.83±4.68) fl, MCH(26±1.54) pg, MCHC (329.65±10.73) g/L, HGB (141.08±16.53) g/L, while that of the co-inherited cases was decided by the type of α -thalassemia mutation. CONCLUSION: Hb E can be effectively detected by HPLC. The type of α -thalassemia mutations will determine hematological features of co-inherited cases. Hb E disorders may be missed by relying only on routine blood test upon prenatal screening.

Capillary electrophoresis-integrated immobilized enzyme microreactor utilizing single-step in-situ penicillinase-mediated alginate hydrogelation: Application for enzyme assays of penicillinase.

Rapid, low-cost and efficient assays for penicillinase activity and inhibition are of vital importance for therapeutics and diagnostics of bacterial resistance to antibiotics. Herein we report a novel approach for on-line enzyme assays for penicillinase utilizing capillary electrophoresis-integrated immobilized enzyme reactors (CE-IMERs). The CE-IMERs are fabricated based on penicillinase-mediated alginate hydrogelation, allowing single-step in-situ encapsulation of enzymes without any additional manipulation process. We show that the fabricated CE-IMERs have high enzyme loading capacity with approximately 61.8% of the original penicillinase in the sol mixture being encapsulated in the "egg-box" hydrogel matrix. Excellent intraday and interday stability and batch-to-batch reproducibility are proved, indicating the reliability of our method for accurate on-line enzyme assays for penicillinase. Enzymatic activities and inhibition of immobilized penicillinase are analyzed, the results of which are in good agreement with those using free enzymes. The proposed method is successfully used for determination of penicillin in pork samples, indicating the potential applications for analysis of complicated real samples.

Single-Step In Situ Acetylcholinesterase-Mediated Alginate Hydrogelation for Enzyme Encapsulation in CE.

A novel capillary electrophoresis-integrated immobilized enzyme reactor (CE-integrated IMER) is developed using single-step in situ acetylcholinesterase (AChE)-mediated alginate hydrogelation and enzyme encapsulation. Alginate hydrogelation with "egg-box" structure is triggered inside a capillary with releasing of Ca2+ by changing the pH of the sol solution, which is accomplished in situ by AChE-catalyzed hydrolysis reaction of acetylthiocholine to produce acetic acid. AChE and any other enzyme initially contained in the sol solution [e.g., xanthine oxidase (XO)] are efficiently encapsulated as the hydrogel network grows, forming CE-integrated IMERs without any additional manipulation process. The proposed method facilitates the analysis of different kinds of enzymes using the same IMER depending on the substrate injected for CE analysis. Approximately 68% of the original enzyme in the sol mixture can be encapsulated, indicating high loading capacity for the CE-integrated IMERs. The IMERs exhibit excellent intraday and interday stability and batch-to-batch reproducibility, and these characteristics imply the reliability of the proposed IMERs for accurate online enzyme assays. Enzymatic activities and inhibition of immobilized AChE and XO are analyzed, and the results are compared with those using free enzymes. The feasibility of the proposed method for potential application in real sample analysis is demonstrated by the successful application of the IMERs in detecting organophosphorus pesticides in apple juice samples using AChE-catalyzed reactions. The proposed method is a simple, efficient, and universal approach for online CE assays with immobilized enzymes, which can be widely applied in bioanalysis.

Screening Platform Based on Robolid Microplate for Immobilized Enzyme-Based Assays.

A facile, cost-effective, and high-throughput screening method was developed for enzyme-based assays based on Robolid/Microplate (RLMP) platform. The RLMP platform is constructed by immobilizing enzyme on commercial robolids and combining it with a standard 96-well microplate to achieve high-throughput analysis. The initiation and quenching of enzymatic reaction can be performed by simply sandwiching or unsealing the enzyme-immobilized robolids and the sample-containing microplate. This platform enables measurements of multiple target analytes simultaneously based on immobilized enzymatic reactions, with analysis time independent of the number of wells in the microplate. Using urea as the model analyte, we have shown that the RLMP platform exhibits large linear detection range of up to 10 mM, fast analysis time of 30 min/96 samples, as well as good reproducibility and stability. Measurements of urea in human urine and serum samples were performed using the RLMP platform and were compared with the commercial urea test kit. A good correlation was found between the two methods. This study shows that the present RLMP platform has promising prospects for detection of clinical markers and application in disease diagnosis and biochemical analysis.

Enzyme and inhibition assay of urease by continuous monitoring of the ammonium formation based on capillary electrophoresis.

We present here an easy-to-operate and efficient method for enzyme and inhibition assays of urease, which is a widely distributed and important enzyme that catalyzes the hydrolysis of urea to ammonia and CO2 . The assay was achieved by integrating CE technique and rapid on-line derivatization method, allowing us to continuously drive the sample to the capillary, thus to measure the amount of the product ammonia from the beginning to the end of the reaction. The method exhibits excellent repeatability with RSD as low as 2.5% for the initial reaction rate (n = 5), with the LOD of ammonia of 20 µM (S/N = 5). The enzyme activity as well as the inhibition of urease by Cu2+ were investigated using the present method. The results show that Cu2+ is a noncompetitive inhibitor on urease, in accordance with the result published in the literature. The enzyme activity and inhibition kinetic constants were obtained and were found to be consistent with the results of traditional off-line enzyme assays. Our study indicates that the present approach is a reliable and convenient method for analysis of the urease activity and inhibition kinetics by continuous on-line monitoring of the ammonium formation based on CE.

Determination of the inhibitory effect of green tea extract on glucose-6-phosphate dehydrogenase based on multilayer capillary enzyme microreactor.

Natural herbal medicines are an important source of enzyme inhibitors for the discovery of new drugs. A number of natural extracts such as green tea have been used in prevention and treatment of diseases due to their low-cost, low toxicity and good performance. The present study reports an online assay of the activity and inhibition of the green tea extract of the Glucose 6-phosphate dehydrogenase (G6PDH) enzyme using multilayer capillary electrophoresis based immobilized enzyme microreactors (CE-IMERs). The multilayer CE-IMERs were produced with layer-by-layer electrostatic assembly, which can easily enhance the enzyme loading capacity of the microreactor. The activity of the G6PDH enzyme was determined and the enzyme inhibition by the inhibitors from green tea extract was investigated using online assay of the multilayer CE-IMERs. The Michaelis constant (Km ) of the enzyme, the IC50 and Ki values of the inhibitors were achieved and found to agree with those obtained using offline assays. The results show a competitive inhibition of green tea extract on the G6PDH enzyme. The present study provides an efficient and easy-to-operate approach for determining G6PDH enzyme reaction and the inhibition of green tea extract, which may be beneficial in research and the development of natural herbal medicines. Copyright © 2016 John Wiley & Sons, Ltd.

Genetic heterogeneity of the ß-globin gene in various geographic populations of Yunnan in southwestern China.

OBJECTIVES: The aim of this study was to investigate the geographic distribution of ß-globin gene mutations in different ethnic groups in Yunnan province. METHODS: From 2004 to 2014, 1,441 subjects with hemoglobin disorders, identified by PCR-reverse dot blot and DNA sequencing, were studied according to ethnicity and geographic origin. Haplotypes were examined among 41 unrelated thalassemia chromosomes. RESULTS: Eighteen ß-thalassemia mutations and seven hemoglobin variants were identified for 1,616 alleles in 22 different ethnic groups from all 16 prefecture-level divisions of Yunnan. The prevalence of ß-thalassemia was heterogeneous and regionally specific. CD 41-42 (-TCTT) was the most prevalent mutation in the populations of northeastern Yunnan. CD 17 (A>T) was the most common mutation in the populations of southeastern Yunnan, especially for the Zhuang minority, whereas Hb E (CD 26, G>A) was the most prevalent mutation in populations of southwestern Yunnan, especially for the Dai minority. Among the seven types of haplotypes identified, CD 17 (A>T) was mainly linked to haplotype VII (+ - - - - - +) and IVS-II-654 (C>T) was only linked to haplotype I (+ - - - - + +). CONCLUSION: Our data underline the heterogeneity of ß-globin gene mutations in Yunnan. This distribution of ß-globin mutations in the geographic regions and ethnic populations provided a detailed ethnic basis and evolutionary view of humans in southern China, which will be beneficial for genetic counseling and prevention strategies.

[Analysis of the EEG information of rats epileptic model using unstable periodic orbits].

In order to further research into the EEG information of rats epileptic model, we applied different nonlinear dynamic methods. After having analyzed the EEG signal of rat falling sickness by means of approximate entropy and correlation dimension, we adopted a the new method, unstable periodic orbits, which was used to analyze complex activity of neurons system to look for the change regularity of change in the EEG signal in the whole course of rat's falling sickness. We found period 1 orbits and period 2 orbits to be statistically significant in the data of ictal time of epilepsy. At the same time, we found period 1 orbits to be statistically significant in the data of preictal time of epilepsy.

What is the maximum duration to perform the hemorheological measurement for the human and mammals.

The hemorheological performance of mammals and human will change with the time after the blood is withdraw from the subject. The whole blood apparent viscosity, erythrocyte deformability and aggregation of some mammals and human were measured. Our results showed that the hemorheological characteristics of mice, rats, guinea pigs, dogs and human began to change respectively after the threshold time of 0.5, 1, 3, 7, 12 h when the blood were stored in vitro. Moreover there exist a relationship: t=3.18W(0.32) between the threshold time (t) and the animal weight (W) and the related coefficient is 0.992.

[Nonlinear analysis on the EEG information of rat epileptic model].

The aim of this study was to develop a new method of epileptic prediction using nonlinear dynamic theory. When rat was falling sickness, its EEG was researched by using approximate entropy and correlation dimension. The results showed the approximate entropy and correlation dimension during epileptic seizure are obviously lower than those before seizure and after seizure. The span of time before seizure is a special phase. Before the seizure symptom appeared, the complexity of EEG had begun declining. Thus, the outbreak of epilepsy could be predicted in short time using nonlinear dynamic methods.

[Design of MC-III low frequency pulsed strong magnetic fields generator].

In this paper, We designed and accomplished a low frequency pulsed strong magnetic fields generator, which provides a pulsed magnetic field with the intensity range from 0.1-2.5 T and the adjusted time interval of pulse. This device is easy to operate and performs reliably. It can work steady for a long time and has been successful used in the experiments of biological effects of electromagnetics.