Research Progress on Detection Methods of Amphetamines in Human Hair.

Amphetamines are chemical synthetic drugs that are becoming increasingly popular in China. As a common sample in the inspection of poisons, hair has the advantages of easy storage, good stability, and long detection time compared with traditional human body fluid samples （blood, urine）, thus possesses an unique application value in the field of forensic toxicology analysis. By now, methods for detecting amphetamines in human hair have been widely used, and validity of the results has been recognized and adopted by the court. This paper reviews domestic and foreign research progress of the detection of amphetamines in hair samples, including the pretreatment and analytic methods.

Regulatory mechanism of microRNA-377 on CDH13 expression in the cell model of Alzheimer's disease.

OBJECTIVE: To explore whether microRNA-377 could participate in the development of Alzheimer's disease (AD) by regulating CDH13. MATERIALS AND METHODS: In this research, AD model was constructed by the SH-SY5Y cells. The expression levels of microRNA-377 and CDH13 in the AD model were detected by quantitative Real-time polymerase chain reaction (qRT-PCR). The cell viability and apoptosis after knockdown of microRNA-377 and CDH13 were measured by cell counting kit-8 (CCK-8) assay and flow cytometry, respectively. The regulatory mechanism of microRNA-377 on CDH13 was confirmed by dual-luciferase reporter gene assay, qRT-PCR and Western blot. RESULTS: Downregulated microRNA-377 and upregulated CDH13 were observed after successful construction of the AD model. Cell viability in the AD model group was significantly reduced compared with that of the control group. Moreover, downregulated microRNA-377 could further inhibit the cell viability, which was reversed by CDH13 knockdown. Cell apoptosis in the AD model group was enhanced after microRNA-377 knockdown, which was rescued by decreasing the expression level of CDH13. MicroRNA-377 was confirmed to regulate the expression level of CDH13 by dual-luciferase reporter gene assay, qRT-PCR and Western blot. CONCLUSIONS: MicroRNA-377 could regulate the expression level of CDH13 by promoting cell proliferation and inhibiting cell apoptosis, thus participating in the occurrence of the Alzheimer's disease.

Establishing a comprehensive genetic diagnosis strategy for hemophilia B and its application in Chinese population.

INTRODUCTION: To reduce the incidence of hemophilia B (HB) which with no complete cure currently, prenatal diagnosis and preimplantation genetic diagnosis (PGD) are effective and feasible means. However, previous studies about genetic diagnosis in HB mostly just focused on the detection of patients and carriers. Here, we established a comprehensive genetic diagnosis strategy for HB and worked it out in Chinese population. The strategy includes the detection of patients and carriers, prenatal diagnosis, and PGD. METHODS: Seven unrelated HB families from Chinese population involved in this study. Firstly, probands and available members were carried out coagulation laboratory assays, and the clinical information has been recorded. Secondly, we used DNA direct sequencing to screen the whole FIX gene of them. The pathogenicity of novel mutations was verified according to 2015 ACMG-AM guidelines. For prenatal diagnosis, a mix of DNA direct sequencing and STR linkage analysis was employed. To explore a better PGD protocol, Karyomapping was first applied in PGD of HB, comparing with conventional PCR-based methods. RESULTS: Six different pathogenic mutations including 1 novel duplication (c.660_661dup ATCA) were identified. The results of prenatal diagnosis were consistent with birth outcomes. In the PGD case, 4 of 11 embryos were confirmed to be normal and one of them was transferred and led to a healthy birth. CONCLUSIONS: The established genetic diagnosis strategy for HB in our study was comprehensive and well applied in clinic practice. Besides, we recommended that DNA direct sequencing combined with Karyomapping was a better PGD protocol.

Purification and characterization of a novel α-glucosidase from Malbranchea cinnamomea.

OBJECTIVES: To characterize a novel α-glucosidase from the thermophilic fungus Malbranchea cinnamomea. RESULTS: The enzyme was purified to homogeneity with purification fold of 40 and a recovery of 7.2 %. It was a monomer with molecular mass of 65.7 kDa on SDS-PAGE. It was optimally active at pH 6 and 50 °C (measured over 10 min) and exhibited a wide range of substrate specificity with the highest specific activity of 47.4 U mg(-1) for p-nitrophenyl α-D-glucopyranoside (pNPGlu) followed by isomaltose, panose and sucrose, suggesting that the enzyme belongs to the type I α-glucosidases. The K m values of the α-glucosidase for pNPGlu and isomaltose were 1.1 and 19.3 mM, respectively. CONCLUSION: Because of its unique properties, the α-glucosidase may have a potential in several industrial applications.

High-level production of ß-1,3-1,4-glucanase by Rhizomucor miehei under solid-state fermentation and its potential application in the brewing industry.

AIMS: To improve the ß-1,3-1,4-glucanase production by Rhizomucor miehei under solid-state fermentation (SSF) for industrial application. METHODS AND RESULTS: The fermentation conditions for ß-1,3-1,4-glucanase production by R. miehei CAU432 under SSF were optimized using a 'one-factor-at-a-time' method. Under the optimized fermentation conditions, viz. oatmeal (0·45-0·9 mm) as sole carbon source, 5% (w/w) peptone as sole nitrogen source, initial moisture of 80% (w/w), initial culture pH of 5·0, incubation temperature of 50°C and incubation time of 6 days, the highest ß-1,3-1,4-glucanase activity of 20,025 U g(-1) dry substrate was achieved, which represents the highest yield for ß-1,3-1,4-glucanase production ever reported. The crude enzyme was extracted and purified to homogeneity with a purification fold of 4·6 and a recovery yield of 9·0%. The addition of the purified ß-1,3-1,4-glucanase in mash obviously reduced its filtration time (24·6%) and viscosity (2·61%). CONCLUSIONS: The optimal fermentation conditions for maximal ß-1,3-1,4-glucanase production under SSF was obtained, and the enzyme was suitable for application in the malting process. SIGNIFICANCE AND IMPACT OF THE STUDY: The high production yield and excellent capability of the enzyme may enable it great potential in industries, especially in brewing industry.

Astragalus membranaceus lectin (AML) induces caspase-dependent apoptosis in human leukemia cells.

OBJECTIVES: Recently, plant lectins have attracted great interest due to their various biological activities such as anti-cancer, anti-fungal and anti-viral activities. We have reported earlier concerning anti-proliferation of human cancer cell lines by a galactose-binding lectin (AML), from a Chinese herb, ASTRAGALUS MEMBRANACEUS: In the present study, detailed investigations into the mechanism of such anti-proliferation properties have been carried out. MATERIALS AND METHODS: Mechanism of apoptosis initiation in K562 cells by AML was investigated by morphology, flow cytometry and western blot analysis. RESULTS: AML induced apoptosis in a caspase-dependent manner in the chronic myeloid leukemia cell line, K562. Furthermore, we observed that cytotoxicity and apoptosis of K562 cells induced by AML were completely abolished in presence of lactose or galactose. CONCLUSIONS: Our results suggest that AML could act as a potential anti-cancer drug.

A xylose-tolerant beta-xylosidase from Paecilomyces thermophila: characterization and its co-action with the endogenous xylanase.

An extracellular beta-xylosidase from the thermophilic fungus Paecilomyces thermophila J18 was purified 31.9-fold to homogeneity with a recovery yield of 2.27% from the cell-free culture supernatant. It appeared as a single protein band on SDS-PAGE with a molecular mass of approx 53.5 kDa. The molecular mass of beta-xylosidase was 51.8 kDa determined by Superdex 75 gel filtration. The enzyme was a glycoprotein with a carbohydrate content of 61.5%. It exhibited an optimal activity at 55 degrees C and pH 6.5, respectively. The enzyme was stable in the range of pH 6.0-9.0 and at 55 degrees C. The purified enzyme hydrolyzed xylobiose and higher xylooligosaccharides but was inactive against xylan substrates. It released xylose from xylooligosaccharides with a degree of polymerization ranging between 2 and 5. The rate of xylose released from xylooligosaccharides by the purified enzyme increased with increasing chain length. It had a K(m) of 4.3mM for p-nitrophenol-beta-d-xylopyranoside and was competitively inhibited by xylose with a K(i) value of 139 mM. Release of reducing sugars from xylans by a purified xylanase produced by the same organism increased markedly in the presence of beta-xylosidase. During 24-hour hydrolysis, the amounts of reducing sugar released in the presence of added beta-xylosidase were about 1.5-1.73 times that of the reaction employing the xylanase alone. This is the first report on the purification and characterization of a beta-xylosidase from Paecilomyces thermophila.

High-level of xylanase production by the thermophilic Paecilomyces themophila J18 on wheat straw in solid-state fermentation.

The production of extracellular xylanase by a newly isolated thermophilic fungus, Paecilomyces themophila J18, on the lignocellulosic materials was studied in solid-state fermentation (SSF). The strain grew well at 50 degrees C and produced a high-level of xylanase activity using the selected lignocellulosic materials, especially wheat straw. Production of xylanase by P. themophila J18 on wheat straw was enhanced by optimizing the particle size of wheat straw, nitrogen source, initial moisture level, growth temperature and initial pH of the culture medium. Under the optimized conditions, yield as high as 18,580 Ug(-1) of carbon source of xylanase was achieved. No CMCase activity was observed. The xylanase exhibited remarkable stability and retained more than 50% of its original activity at 70 degrees C for 4h at pH 7.0-8.0. Therefore, P. themophila J18 could to be a promising microorganism for thermostable, cellulase-free xylanase production in SSF.

Suppression of two major Fragile X Syndrome mouse model phenotypes by the mGluR5 antagonist MPEP.

Fragile X Syndrome is the most common form of inherited mental retardation worldwide. A Fragile X mouse model, fmr1(tm1Cgr), with a disruption in the X-linked Fmr1 gene, has three substantial deficits observed in several strains: (1) sensitivity to audiogenic seizures (AGS), (2) tendency to spend significantly more time in the center of an open field, and (3) enlarged testes. Alterations in metabotropic glutamate receptor group I signaling were previously identified in the fmr1(tm1Cgr) mouse. In this study, we examined the effect of MPEP, an antagonist of the group I metabotropic glutamate receptor mGluR5, on audiogenic seizures and open field activity of fmr1(tm1Cgr) mice. Genetic analysis revealed synergistic reactions between fmr1(tm1Cgr) and inbred AGS alleles. In addition, AGS sensitivity due to the fmr1(tm1Cgr) allele was restricted during development. Examination of phenotypes combining mGluR5 inhibition and Fmr1 mutation indicated that absence of FMRP may affect mGluR5 signaling through indirect as well as direct pathways. All strains of fmr1(tm1Cgr) mice tested (FVB/NJ, C57BL/6J, and an F1 hybrid of the two) had a more excitable AGS pathway than wild-type, and consequently required more MPEP to achieve seizure suppression. At high doses of mGluR5 antagonists, a Fragile X specific tolerance (loss of drug activity) was observed. The tolerance effect could be overcome by a further increase in drug dose. In open field tests, MPEP reduced fmr1(tm1Cgr) center field behavior to one indistinguishable from wild-type. Therefore, mGluR5 antagonists were able to rescue two of the major phenotypes of the FX mouse. Modulation of mGluR5 signaling may allow amelioration of symptoms of Fragile X Syndrome.

A phenotypic and molecular characterization of the fmr1-tm1Cgr fragile X mouse.

Fragile X Syndrome is the most common form of inherited mental retardation. It is also known for having a substantial behavioral morbidity, including autistic features. In humans, Fragile X Syndrome is almost always caused by inactivation of the X-linked FMR1 gene. A single knockout mouse model, fmr1-tm1Cgr, exists. In this report we further characterize the cognitive and behavioral phenotype of the fmr1-tm1Cgr Fragile X mouse through the use of F1 hybrid mice derived from two inbred strains (FVB/NJ and C57BL/6J). Use of F1 hybrids allows focus on the effects of the fmr1-tm1Cgr allele with reduced influence from recessive alleles present in the parental inbred strains. We find that the cognitive phenotype of fmr1-tm1Cgr mice, including measures of working memory and learning set formation that are known to be seriously impacted in humans with Fragile X Syndrome, are essentially normal. Further testing of inbred strains supports this conclusion. Thus, any fmr1-tm1Cgr cognitive deficit is surprisingly mild or absent. There is, however, clear support presented for a robust audiogenic seizure phenotype in all strains tested, as well as increased entries into the center of an open field. Finally, a molecular examination of the fmr1-tm1Cgr mouse shows that, contrary to common belief, it is not a molecular null. Implications of this finding for interpretation of the phenotype are discussed.

Phosphoryl transfer reaction regulated by amino acid side chains: a model for phosphoproteins.

Through a kinetic study of the reaction of phosphoamino acids as incubated in alcohol, it was found that the inter- and intramolecular phosphoryl transfer reactions were regiospecific and stereoselective. First, the phosphoryl transfer reaction required the regio-specifically neighboring alpha-carboxy group activation of amino acid but not beta-carboxy group. The intramolecular side chain catalytic effects relative to hydrogen for N-phosphoamino acids compared to N-phosphoglycine were a 119-to 4-times enhancement of the phosphoryl transfer reaction, respectively. Secondly, the intramolecular N-->O phosphoryl transfer migration was highly stereoselective, since the reaction rate constant of phosphoallothreonine relative to its diastereomeric threonine was reduced to half. The pentacoordinate transition states modulated by the amino acid side chains were demonstrated by the formation rates of intramolecular pentacoordinate spiro mixed anhydride compounds.

Detection of the organophosphorus nerve agent sarin by a competitive inhibition enzyme immunoassay.

Two artificial antigens, NalphaNepsilon-di(O,O-diisopropyl) phosphoryl L-lysine (DIP)- bovine serum albumin (BSA) conjugate (DIP-BSA) and DIP-KLH (keyhole limpet hemocyanin), were synthesized. Antibodies against sarin (O-isopropyl methylphosphonofluoridate) were obtained after immunization of rabbits with DIP-KLH conjugate. A competitive inhibition enzyme immunoassay (CIEIA) was developed to detect the organophosphorus nerve agent sarin. The antibody solutions could be inhibited by sarin as low as 10(-6) mol/l, and the standard curve was linear over 3 orders of magnitude. The coefficients of intraassay and interassay variation of this method were 5.4-6.2% (n = 11) and 8.0-9.5% (n = 6) at a sarin concentration range of 10(-3)-10(-6) mol/l, respectively. The recovery of sarin in water samples at the concentration of 5 x 10(-5) mol/l was in the range of 96.8-102.5%. The specificity of the antiserum was assessed by comparing the inhibition induced by sarin with soman, Vx, isopropyl alcohol and isopropyl methyl phosphonic acid. The results showed that less than 5 mmol/l soman, 2 mmol/l Vx, 16 mmol/l isopropyl alcohol and 8 mmol/l isopropyl methyl phosphonic acid did not influence the determination of sarin in water samples.