Establishment and Application of a 42-plex Microhaplotype Assay in Forensic Genetics.

OBJECTIVES: To establish and forensically verify a 42 microhaplotypes (mircohaps, MHs) multiplex assay system based on next-generation sequencing (NGS), and to explore the application value of this system in the practice of forensic genetics. METHODS: A total of 42 highly polymorphic MHs were selected from previous studies, and sequenced by the MiSeq FGxTM platform to verify the repeata-bility, sensitivity, specificity, stability, and mixture analysis ability of the detection system. Through population genetic investigation of 102 unrelated Chinese Han individuals in Liyang City, Jiangsu Province, China, the application value of this system in forensic genetics was evaluated. RESULTS: The sequencing repeatability of the 42-plex MHs assay was 100% and the sensitivity was as low as 0.062 5 ng. The system had the ability to withstand the interference of indigo (≤2 500 ng/µL), humic acid (≤9 ng/µL), hemoglobin(≤20 µmol), and urea (≤200 ng/µL) and to detect mixtures of 2 people (1∶19), 3 people (1∶1∶9) and 4 people (1∶1∶1∶9). Based on 102 individual data, the combined power of discrimination and the combined power of exclusion were 1-3.45×10-30 and 1-3.77×10-11, respectively, and the average effect value of alleles was 2.899. CONCLUSIONS: The 42-plex MHs assay was successfully established in this study and this system has high repeatability and sensitivity, good anti-jamming ability and mixture analysis ability. The 42 MHs are highly polymorphism and have good application value in individual identification and paternity testing.

Efficiency Analysis of Uncle-Nephew Relationship Identification by Increasing STR Markers and Adding Reference Samples.

OBJECTIVES: To estimate the system efficiency of uncle-nephew relationship identification by increasing STR markers and adding reference samples based on the test results of simulated data and real samples, so as to provide references for selecting the appropriate number of STRs and reference samples for uncle-nephew relationship identification. METHODS: Five common models of uncle-nephew relationship identification were constructed by adding different reference samples. In each model, the likelihood ratio (LR) for 10 000 pairs of uncle-nephew relationships and 10 000 pairs of unrelated individuals were simulated by detecting 19, 39 or 55 STRs, and the system efficiency at different thresholds was simulated. The samples of the Han population in Zhejiang were collected, and 55 autosomal STRs were obtained by using SiFaSTRTM 23plex kit, Goldeneye® DNA ID 22NC kit and AGCU 21+1 PCR amplification kit. When 19, 39 and 55 STRs were detected, the LR of each model and system efficiency under different thresholds were calculated and compared with the simulation results. RESULTS: Under the same detection system, the calculated results of simulated data and corresponding true samples were basically consistent. In the same model, there was a positive correlation between the system efficiency of uncle-nephew relationship identification and the number of STRs detected. Moreover, the system efficiency of introducing relatives was higher than identifying only two individuals. The order of preference for the introduction of relatives was the full sibling (or mother) of the uncle and the full sibling (or mother) of the nephew. CONCLUSIONS: The system efficiency of uncle-nephew relationship identification could be improved by increasing the number of STRs and introducing known relatives, which would provide the basis for selecting the most appropriate detection system and reference individuals in actual cases.

Genetic Polymorphism of 16 X-STR Loci in Xinjiang Uygur Population.

OBJECTIVES: To study the genetic polymorphism and population genetic parameters of 16 X-STR loci in Xinjiang Uygur population. METHODS: The Goldeneye® DNA identification system 17X was used to amplify 16 X-STR loci in 502 unrelated individuals (251 females and 251 males). The amplified products were detected by 3130xl genetic analyzer. Allele frequencies and population genetic parameters were analyzed statistically. The genetic distances between Uygur and other 8 populations were calculated. Multidimensional scaling and phylogenetic tree were constructed based on genetic distance. RESULTS: In the 16 X-STR loci, a total of 67 alleles were detected in 502 Xinjiang Uygur unrelated individuals. The allele frequencies ranged from 0.001 3 to 0.572 4. PIC ranged from 0.568 8 to 0.855 3. The cumulative discrimination power in females and males were 0.999 999 999 999 999 and 0.999 999 999 743 071, respectively. The cumulative mean paternity exclusion chance in trios and in duos were 0.999 999 997 791 859 and 0.999 998 989 000 730, respectively. The genetic distance between Uygur population and Kazakh population was closer, and the genetic distance between Uygur and Han population was farther. CONCLUSIONS: The 16 X-STR loci are highly polymorphic and suitable for identification in Uygur population, which can provide a powerful supplement for the study of individual identification, paternity identification and population genetics.

Application of SifaInDel 45plex System in the Han and Mongolian Populations.

OBJECTIVES: To investigate the genetic polymorphism of InDel loci in SifalnDel 45plex system in the Han population in Jiangsu Province and the Mongolian population in Inner Mongolia, and to evaluate the effectiveness of the system in forensic medicine. METHODS: SifaInDel 45plex system was used for genotyping in blood samples of 398 unrelated individuals from the above two populations, and allele frequencies and population genetic parameters of the two populations were calculated respectively. Eight intercontinental populations in the gnomAD database were used as reference populations. The genetic distances between the two studied populations and eight reference populations were calculated based on the allele frequencies of 27 autosomal-InDels (A-InDels). The phylogenetic trees and multidimensional scaling (MDS) analysis diagrams were constructed accordingly. RESULTS: Among two studied populations, the 27 A-InDels and 16 X-InDels showed no linkage disequilibrium between each other and the allele frequency distributions were in Hardy-Weinberg equilibrium. The CDP of the 27 A-InDels in two studied populations were all higher than 0.999 999 999 9, and the CPEtrio were all less than 0.999 9. The CDP of the 16 X-InDels in Han in Jiangsu and Mongolian in Inner Mongolia female and male samples were 0.999 997 962, 0.999 998 389, and 0.999 818 940, 0.999 856 063, respectively. The CMECtrio were all less than 0.999 9. The results of population genetics showed that the Jiangsu Han nationality, Inner Mongolia Mongolian nationality and East Asian population clustered into one branch, showing closer genetic relationship. The other 7 intercontinental populations clustered into another group. And the above 3 populations displayed distant genetic relationships with the other 7 intercontinental populations. CONCLUSIONS: The InDels in the SifaInDel 45plex system have good genetic polymorphism in the two studied populations, which can be used for forensic individual identification or as an effective complement for paternity identification, and to distinguish different intercontinental populations.

Astragaloside IV ameliorates intermittent hypoxia-induced inflammatory dysfunction by suppressing MAPK/NF-κB signalling pathways in Beas-2B cells.

PURPOSE: Intermittent hypoxia is a characteristic pathological change in obstructive sleep apnoea (OSA) that can initiate oxidative stress reaction and pro-inflammatory cytokine release. The purpose of this study was to assess the effect and protective mechanism of Astragaloside IV (AS-IV) in intermittent hypoxia-induced human lung epithelial Beas-2B cells. METHODS: Human lung epithelial Beas-2B cells were exposed to intermittent hypoxia or normoxia in the absence or presence of AS-IV. MTT assay was performed to determine the cell viability. The levels of reactive oxygen species (ROS), lactate dehydrogenase (LDH), malonaldehyde (MDA), and superoxide dismutase (SOD) were measured to evaluate oxidative stress. The levels of cytokines interleukin (IL)-8, IL-1ß, and IL-6 were evaluated by enzyme-linked immunosorbent assay and real-time PCR. The expression of Toll-like receptor 4 (TLR4), mitogen-activated protein kinase (MAPK), and nuclear transcription factor-kappa B (NF-κB) signalling pathways was analysed by western blot. RESULTS: The results showed that AS-IV significantly reduced the levels of ROS, LDH, MDA, IL-8, IL-1ß, and IL-6, and increased the level of SOD in intermittent hypoxia-induced Beas-2B cells. It also suppressed the phosphorylation of MAPKs, including P38, c-Jun N-terminal kinase and extracellular signal-regulated kinase, and inhibited the activation of the NF-κB signalling pathway by reducing the phosphorylation of IκBα and p65. CONCLUSIONS: AS-IV attenuates inflammation and oxidative stress by inhibiting TLR4-mediated MAPK/NF-κB signalling pathways in intermittent hypoxia-induced Beas-2B cells.

Selective extraction of catecholamines by packed fiber solid-phase using composite nanofibers composing of polymeric crown ether with polystyrene.

For the first time, electrospun composite nanofibers comprising polymeric crown ether with polystyrene (PCE-PS) have been used for the selective extraction of catecholamines - dopamine (DA), norepinephrine (NE) and epinephrine (E) - prior to their analysis by high-performance liquid chromatography-electrochemical detection. Using a minicartridge packed with PCE-PS composite nanofibers, the target compounds were extracted effectively from urine samples to which diphenylborinic acid 2-aminoethyl ester was added as a complexing reagent. The extracted catecholamines could be liberated from the fiber by the addition of acetic acid. A good linearity was observed for catecholamines in the range of 2.0-200 ng mL(-1) (NE, E and DA). The detection limits of catecholamines (signal-to-noise ratio = 3) were 0.5 ng mL(-1) (NE), 0.2 ng mL(-1) (E) and 0.2 ng mL(-1) (DA), respectively. Under the optimized conditions, the absolute recoveries of the above three catecholamines were 90.6% (NE), 88.5% (E) and 94.5% (DA). The repeatability of extraction performance was from 5.4 to 9.2% (expressed as relative standard deviation). Our results indicate that the proposed method could be used for the determination of NE, E and DA in urine.

[Distribution of Inflammatory Cells and Expression of PSGL-1 in Infant Brainstem Tissue Related Fatal Brainstem Encephalitis].

OBJECTIVE: To explore the distribution of inflammatory cells and positive expression of P-se- lectin glycoprotein ligand-1 (PSGL-1) in infant brainstem tissue from hand-foot-mouth disease related fatal brainstem encephalitis. METHODS: Twenty brainstem samples from infants suffered from brainstem en- cephalitis were collected as the experimental group. Ten brainstem samples from infants died of non- brain diseases and injuries were collected as the control group. The distribution of inflammatory cells and the expression of PSGL-1 in the two groups were examined by immunohistochemical method. The characteristics of the positive cells were observed. RESULTS: In brainstem tissue of the experimental group, there were sleeve infiltrations of inflammatory cells around the vessels and in the glial nodule. Microglia was the most and following was neutrophils around the vessels and in the glial nodule. There was a significant statistical difference among microglias, neutrophils and lymphocytes (P < 0.05). There was no sleeve infiltration in the control group. PSGL-1 protein was expressed widely in inflammatory cells in the experimental group, especially in the inflammatory cells around the vessels and in the glial nodule. But PSGL-1 positive staining could be observed significantly less in the control group comparing with the experimental group (P < 0.05). CONCLUSION: Microglia is the main type of inflammatory cells involved in the progress of the fatal disease. Moreover, PSGL-1 could participate in the pathogenesis of hand-foot-mouth disease related fatal brainstem encephalitis.

Unidirectional charge transfer in di-cobalt valence tautomeric compound finely tuned by ancillary ligand.

A dinuclear valence tautomeric compound containing a cationic structure with crystallographically distinguishable hs-Co(II) and ls-Co(III) centers undergoes unidirectional charge transfer.

Thermally induced and photoinduced valence tautomerism in a two-dimensional coordination polymer.

Herein reported is the first two-dimensional coordination polymer capable of undergoing thermally induced and photoinduced valence tautomeric transitions.

The study of adsorption characteristics of electrospun polymer nanofibers for benzenes in water.

The adsorption properties of benzene, p-dichlorobenzene and nitrobenzene on polymer nanofibers were studied. Compared with polyacrylonitrile nanofiber, polystyrene (PS) nanofiber presented better adsorption performance. Langmuir and Freundlich adsorption models were used for the mathematical description of adsorption equilibria, and Freundlich isotherms fitted better. Kinetic studies showed that the adsorption of PS nanofiber followed pseudo first-order model. Various thermodynamic parameters such as standard free energy (delta G), enthalpy (delta H) and entropy (delta S) were calculated for predicting the adsorption nature of PS nanofiber for three benzenes, which indicated that the adsorption was spontaneous and a physical process. The regeneration efficiency maintains over 80% after five cycles of adsorption/desorption tests. It showed that PS nanofibers are promising candidates for adsorption and removal of aromatic hydrocarbons from water.

Application of nanofiber-packed SPE for determination of salivary-free cortisol using fluorescence precolumn derivatization and HPLC detection.

Salivary cortisol has emerged as an easy-to-collect biologic marker of stress in many researches. In this study, we present a method for the determination of salivary-free cortisol using HPLC method with fluorescence precolumn derivatization, which is based on a novel extraction from the strongly acidic medium (fluorescent derivatives of cortisol in sulfuric acid medium) by electrospun polystyrene nanofibers packed SPE. For high-throughput sample extraction, an array pretreatment device based on nanofibers packed SPE micro-column was designed. The LOD of cortisol was 0.01 microg/L (S/N=3). The RSDs (n=6) for all analytes were below 8.0%, and the recoveries were 110, 102.4, and 99.4% (n=3) for saliva spiked with 0.1, 10, and 20 microg/L of cortisol, respectively. The proposed method was then successfully applied in the determination of free cortisol in human saliva. The salivary cortisol concentrations in the real samples ranged from 0.22 to 7.45 microg/L. The nanofiber-packed SPE overcame the low extraction recovery and bad clean-up effect of the conventional methods, and increased the sensitivity and selectivity of the method.

Design of packed-fiber solid-phase extraction device for analysis of the drug and its metabolite in plasma.

A mini-column packed with 1 mg electrospun polystyrene nanofibers (about 200 approximately 400 nm in diameter) was designed for simple, fast extraction of drugs, diazepam and its major metabolite, N-desmethyldiazepam for the analysis of them in human and dog plasma. Ttrezodone was selected as internal standard. The drugs adsorbed on the solid phase could be desorpted with 50 microl of the methanol and then monitored by liquid chromatography coupled to an ultraviolet detector. Parameters influencing the extraction efficiency such as fiber packing amount, eluted solvent, and pH of the sample were decided. The time for the pretreatment of 0.5 ml plasma sample was less than 10 min. The detection limits of diazepam and N-desmethyldiazepam in plasma could be as low as 1 microg/L. The intra- and inter-day precision, calculated from quality control (QC) samples, was less than 9.1%. The method was evaluated by its application in determination of dog plasma samples from three beagles after a single dose oral of diazepam. The technique was validated by comparison with conventional plasma analysis. It was observed that the mini-column offers improved limits of detection and reduced sample preparation time as compared to conventional method. For its simplicity and sensitivity, the method may be used in therapeutic drug monitoring and pharmacology study.

Performance of electrospun nanofibers for SPE of drugs from aqueous solutions.

A novel extraction technique was reported. The solid phase material, nanofiber, was prepared by electrospinning using polystyrene. Twenty different drugs (10 microg/L in water) were extracted using 1 mg of nanofibers within 5 min. The analytes can be desorpted from the fibers with 50 microL of the methanol and then monitored by LC coupled to a UV detector. Packed-fiber SPE (PFSPE) provide high recoveries (>50%) for some relatively non-polar drugs (log P >1.5) (n-octanol-to-water partition ratio), and relatively low recoveries (9.9-39.8%) for the drugs within the log P window below 1. Experimental optimization of the technique has been carried out using seven representative drugs, edaravone, cinchonine, quinine, voriconazole, chlordiazepoxide, verapamil, and rutonding. Except for edaravone, the maximum yields of seven drugs (0.2 microg/L) from water samples were approximately 100%, and were 33.7-88.2% from human plasma. The advantageous aspect of the technique encompasses high throughput, high sensitivity, simplicity, low cost, and green chemistry.

Evaluation of tumor markers biochip C12 system in the diagnosis of gastric cancer and the strategies for improvement: analysis of 100 cases.

BACKGROUND/AIMS: A C12 biochip system using 12 tumor markers has been developed in China for serum diagnosis of common cancers. This work is to evaluate this C12 system in the diagnosis of gastric cancer. METHODOLOGY: Sera from 100 gastric carcinoma patients were screened for 12 tumor markers including carcinoembryonic antigen, alpha-fetoprotein, carbohydrate antigen 19-9, carbohydrate antigen 242, cancer antigen 15-3, cancer antigen 125, prostate specific antigen, free-PSA, neuron-specific enolase, human chorionic gonagotropin-beta, human growth hormone, and ferritin, using the C12 biochip system. The most relevant tumor marker and the contribution of the tumor markers to the improvement of diagnosis were determined. RESULTS: The overall diagnostic rate of C12 biochip system was 37%, and 7.8%, 29.4%, 35.5% and 50%, respectively, for stages I, II, III and IV patients. The differences in diagnostic rates between stage I (7.8%) and stage IV (50%) reached statistical significance (chi-square test, Chi2=7.20, p<0.01). Among all the 12 markers, carbohydrate antigen 19-9 had the highest positive rate up to 23%, against which any form of combinations of 5 most relevant tumor markers (2, 3, 4 or 5 markers combined) could not significantly improve the diagnostic rate. CONCLUSIONS: The C12 biochip system has some value in the diagnosis of advanced stage gastric cancer, but less sensitive in early gastric cancer.

[Effect of intravenous immunoglobulin and vitamin C on progression of experimental autoimmune myocarditis in mice].

OBJECTIVE: To evaluate the effect of intravenous immunoglobulin (IVIG) and vitamin C on the progression of experimental autoimmune myocarditis(EAM). METHODS: Fifty-two Balb/c mice were randomized into six groups: The blank group received no treatment, the remaining 5 groups were immunized with 100mug emulsified porcine myosin at d 1 and d 7. Different agents were injected from d 1, SVitC group:150 mg/kg*d(-1)vitamin C; LVitC group: 300 mg/kg*d(-1)vitamin C; IVIG group: 1 g/kg*d(-1)IVIG; IVIG+VitC group: 1 g/kg*d(-1)IVIG and 150 mg/kg*d(-1)vitamin C; The control group same volume of normal saline. All mice were sacrificed at d 21, and serum TNF-alpha levels were detected with enzyme linked immunosorbent assay (ELISA). The ratio of heart to body weight(C/W), spleen to body weight(S/W) and kidney to body weigh(R/W) were calculated. The spleens and heart were examined pathologically and/or immunohistochemically. RESULT: Compared with those of control group, inflammatory cells infiltration in the myocardium and calcification in the pericardiume in SVitC and LVitC groups were extenuated. There were inflammatory cells infiltrating in the myocardium sparely and no calcification in the pericardium in IVIG and IVIG+VitC groups. The size of spleens enlarged especially in IVIG and IVIG+VitC groups. White and red pulps of spleens were hyperplastic microscopically. The C/W of treatment groups decreased significantly compared with that of control group. The S/W of therapy groups and control group was significantly higher than that of blank group; and the S/W of IVIG and IVIG + VitC groups was significantly higher than that of SVitC and LVitC groups. The R/W in each groups had no significant difference. The TNF-alpha level in SVitC and LVitC groups was a little lower than that in control group; TNF-alpha level in IVIG and IVIG+VitC groups was significantly lower than that of control group. Wide fluorescence stripe was found along extracellular matrix surrounding the damaged cardiomyocytes of control group. Both density and intensity of fluorescence in SVitC and LVitC groups were lower than those of control group. There were much wider fluorescence stripe and strengthened intensity in IVIG and IVIG + VitC groups. The myofilaments were in wild disorder and sarcomere had severe breakage in control group. Moreover, chondriosome hypertrophy and vacuolar degeneration were found. The damage lessened in SVitC and LVitC groups. Both myofilaments and sarcomeres in IVIG and IVIG + VitC groups were almost normal, and the chondriosome was normal. CONCLUSION: IVIG and vitamin C have some protective and therapeutic effect on the progression of EAM by decreasing pathological damage of myocardium and depressing TNF-alpha production, and IVIG combined with vitamin C is more effective.

[Effect of maternal autoimmune thyroid disease on intellectual development of infants].

OBJECTIVE: To study the effect of maternal Hashimoto's disease (an autoimmune thyroid disease) on intellectual development of infants. METHODS: From July 2001 to June 2003, 21 infants born by mothers suffered from Hashimoto's disease were followed up with provincial neonatal disease screening network system. Their thyroid function was assessed and their mental development was evaluated with Gesell development schedules. RESULT: (1) Among the 21 infants, 8 showed normal thyroid function, 11 showed hyperthyrotropinemia, 2 cases had congenital hypothyroidism, which showed significant differences from those born by healthy mothers. (2) The mental and psychomotor development of infants whose mothers suffered from Hashimoto's disease lagged behind those with the healthy mothers (P <0.05). CONCLUSION: Maternal Hashimoto's disease may affects infants' thyroid function and mental development.

[Viraemia and extraintestinal involvement after rotavirus infection].

OBJECTIVE: To study the incidence of viraemia and extraintestinal organ damage in children with acute rotavirus (RV) gastroenteritis. METHODS: Eighty-three children with acute rotavirus gastroenteritis were hospitalized from October 2002 to March 2003, whose blood and fecal samples were obtained on admission. Rotavirus RNA (encoding the VP7 outer capsid protein) were detected in blood and fecal samples by nest reverse transcription-polymerase chain reaction (RT-PCR). According to the result of blood RV-RNA, the patients were divided into RV-RNA positive group and RV-RNA negative group. The differences between these two groups in the severity of gastroenteritis and extraintestinal organ damage were analyzed. RESULTS: Eighty-two of 83 stool samples from the children with rotavirus infection were positive for rotavirus RNA. Sixteen of 83 blood samples were positive for rotavirus RNA with a positive rate of 19.3%. The nucleotide sequence of cloned cDNAs, resembling part of the VP7 gene, was identical from paired blood and fecal samples. There were no significant differences between blood RV-RNA positive group and blood RV-RNA negative group in the rate and degree of fever, diarrhea, dehydration, metabolic acidosis, hypokalemia and myocardial damage (P>0.05); while the incidences of liver damage, rash, lower respiratory tract infection and the central nervous system involvement in the blood RV-RNA positive group were significantly higher than those in the blood RV-RNA negative group (P<0.05). CONCLUSION: Viraemia is present in the children with acute rotavirus gastroenteritis. Viraemia might be an important mechanism by which rotavirus spread to the extraintestinal sites resulting in organs damage.

Induction of Bcl-2 and Bax was related to hyperphosphorylation of tau and neuronal death induced by okadaic acid in rat brain.

Abnormal hyperphosphorylation of the cytoskeletal protein tau is a characteristic feature of neurodegeneration in Alzheimer's disease (AD) brain. Okadaic acid (OA), a protein phosphatase inhibitor, induces neuronal death and hyperphosphorylation of tau. In the present study using a model of microinjection of OA into rat frontal cortex, we aimed to investigate if OA-induced hyperphosphorylation of tau and neuronal death are related to the expression of Bcl-2, an apoptosis inhibitor, or Bax, an apoptosis inducer. Immunohistochemistry and Western blot analysis showed that OA injection dose- and time-dependently induced the expression of Bcl-2 and Bax protein in the surrounding of OA injection areas, which were similar with that of AT8 immunostaining, a marker of hyperphosphorylated tau. However, the ratios of Bcl-2 over Bax had a negative relationship to the expression of AT8. Furthermore, double fluorescent staining showed that AT8-positive neurons mainly costained with terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick-end labeling, a marker of DNA damage, indicating that tau hyperphosphorylation may be associated with DNA damage in the neurons of rat brain. In the areas more adjacent to the OA injection site, most neurons with AT8-positive staining showed vulnerability to OA toxicity and could be triple-stained with Bcl-2 and Bax or double-stained with Bcl-2. However, in the areas further from the OA injection site, neurons with few AT8-positive staining showed resistance to OA toxicity and only stained with Bcl-2, but not Bax. The results suggest that the ratios of Bcl-2 over Bax expression may have an effect on tau hyperphosphorylation and neuronal death following OA injection.

[Relationship between serum dehydroepiandrosterone levels and female precocious puberty].

OBJECTIVE: To investigate the relationship of serum dehydroepiandrosterone (DHEA) levels and female precocious puberty. METHODS: The serum levels of DHEA and dehydroepiandrosterone sulfate (DHEAS) were measured by ELISA in 60 idiopathic central precocious puberty (ICPP) girls, 62 premature thelarche (PT) girls and 31 age-matched health prepuberty girls. Bone age,volume of uterus and ovary, DHEA and DHEAS were re-measured in 3, 12 months after treatment with Diphereline in ICPP girls. RESULT: (1) The Log(DHEA) and Log(DHEAS) were (0.81 +/-0.36)microg/L and (2.31 +/-0.31)microg/L in ICPP group, (0.72 +/-0.30)microg/L and (2.31 +/-0.28)mg/L in PT group, and (0.32 +/-0.26)microg/L and (2.16+/-0.27)microg/L in controls (P <0.05). However, no significant differences were found between ICPP and PT group (P >0.05). Moreover, the serum levels of DHEA and DHEAS in precocious puberty girls with Tanner III stage were significant higher than those with Tanner II stage (P <0.05). (2) With bivariate correlation analysis, Log(DHEA) was positively correlated with height, bone age, volume of uterus and ovary (r=0.429, 0.339, 0.217, 0.282; all P<0.05), while no significant correlation with Log(LH peak), Log(FSH peak) and BMI (r=0.135, -0.165, 0.059). Log(DHEAS) was positively correlated with height,bone age and volume of ovary (r=0.319, 0.210, 0.181; P <0.05), while no correlated with Log(LH peak), Log(FSH peak), volume of uterus and BMI (r=0.012, -0.173, 0.146 and 0.081 respectively). (3) Serum Log (DHEA) and Log(DHEAS) of 32 ICPP were decreased from (0.83 +/-0.35) microg/L and (2.27 +/-0.30)microg/L to (0.68 +/-0.44)microg/L and (2.11 +/-0.43)microg/L (P<0.05) 3 months after treatment. The serum Log(DHEA) and Log(DHEAS) in 12 months after treatment were (0.78 +/-0.30)microg/L and (2.40+/-0.34)microg/L, which was not significantly different with that before treatment (P>0.05). However, the volume of uterus and ovary, bone age/age in 12 months after treatment were significantly different with those before treatment (2.82 +/-1.52 compared with 1.09 +/-0.50 ml, 3.15 +/-1.13 compared with 1.18 +/-0.42 ml, 1.43 +/-0.23 compared with 1.25 +/-0.12, all P<0.05). CONCLUSION: (1) The serum levels of DHEA and DHEAS are increased in precocious puberty girls with the development of Tanner stage. (2) Serum levels of DHEA and DHEAS are declined transiently when the hypothalamic-pituitary-gonadal axis is inhibited. (3) Serum DHEA is associated with the acceleration of growth and bone age in precocious puberty girls.

Determination of protoberberine alkaloids in Rhizoma Coptidis by ERETIC ¹H NMR method.

An alternative quantification approach called ERETIC (Electronic REference To access In vivo Concentrations) utilizing electronic reference-based proton nuclear magnetic resonance (¹H NMR) spectroscopy techniques has been successfully introduced in our present study to simultaneously determine the contents of five major active protoberberine alkaloids (berberine, coptisine, jatrorrhizine, palmatine and epiberberine) in Rhizoma Coptidis, one of the most commonly used traditional Chinese medicines (TCM). The NMR experimental conditions including deuterated solvent, relaxation delay time, and ERETIC transmitter power level were optimized, and the developed method was validated in terms of precision, reproducibility, stability, accuracy, recovery, and limit of quantification (LOQ). The recoveries of the five tested alkaloids ranged between 89.94 and 97.72% for berberine, 90.87 and 100.05% for coptisine, 98.35 and 107.57% for jatrorrhizine, 95.37 and 101.26% for palmatine, and 93.18 and 98.00% for epiberberine, respectively, and LOQ was 0.1 mM for berberine. The high universality, accuracy, reproducibility, and efficiency of the ERETIC method demonstrated in this work suggest that this method could serve as a highly potential quantification alternative for quality assessment of TCM.