Providencia sp. strain X1 showing the highest xylanase activity among six bacterial isolates was isolated from saw-dust decomposing site. Strain X1 produced cellulase-free extracellular xylanase, which was higher in wheat bran medium than in xylan medium, when cultivated at pH 8.0 and 35°C. Zymogram analysis of crude preparation of enzymes obtained while growing on wheat bran and birchwood xylan revealed the presence of seven and two distinct xylanases with estimated molecular weight of 33; 35; 40; 48; 60; 75; and 95 kDa and 33 and 44 kDa, respectively. The crude xylanases were produced on wheat bran medium and showed optimum activity at pH 9.0 and 60°C. The thermotolerance studies showed activity retention of 100% and 85% at 40°C and 60°C after 30 min preincubation at pH 9.0. It was tolerant to lignin, ferulic acid, syringic acid, and guaiacol and retained 90% activity after ethanol treatment. The enzyme preparation was also tolerant to methanol and acetone and showed good activity retention in the presence of metal ions such as Fe2+, Mg2+, Zn2+, and Ca2+. The crude enzyme preparation was classified as endoxylanase based on the product pattern of xylan hydrolysis. Pretreatment of kraft pulp with crude xylanases for 3 h at 60°C led to a decrease in kappa number by 28.5%. The properties of present xylanases make them potentially useful for industrial applications. The xylan is the most abundant renewable polysaccharide after cellulose and its hydrolysis by xylanases into simple compounds, mainly xylose, is important for several biotechnological applications. The global demand of xylanase has increased over the years due to its potential application in various industrial processes such as in animal feed, food processing, textiles, pharmacy, and pulp and paper industry [Bacillus spp. due to their ability to produce cellulase-free, thermostable and alkaline xylanase [Xylan is the major component of the hemicellulosic complex found in the plant cell wall. Complete degradation of xylan requires a coordinated action of several hydrolytic enzymes, among which xylanases E.C. 3.2.1.8) are sufficient to hydrolyze .8 are suxylanase .Bacillus spp. [Cellulase-free xylanases functioning at high temperature and pH are required for application in pulp and paper industry, where they reduce toxic wastes, making the bleaching process environmentally friendly. However, relatively high cost of enzyme production has hindered the industrial application of the enzymatic process. In microbial-based enzyme production processes, approximately 30–40% of production of many industries is attributed to the cost of the growth substrates . Xylanaslus spp. , 7, 8.  Providencia sp. strain X1 from saw-dust decomposing site. It produces multiple xylanases when grown on wheat bran medium. The crude enzyme preparation was alkali- and thermotolerant in nature. Although, a highly purified enzyme is a prerequisite for characterization and structural studies; however, from application perspective, using purified enzyme is not economical for applications like biobleaching . Also, iChemicals like birchwood xylan, 3,5-dinitrosalicylic acid (DNSA), carboxymethyl cellulose (CMC), Congo red, and D-xylose were purchased from Sigma . Other reagents used in this work were of analytical grade.3 3.0, K2HPO4 0.5, MgSO4·7H2O 0.2, MnSO4·H2O 0.02, FeSO4·H2O 0.02, and CaCl2·2H2O 0.02, agar powder 16.0 and yeast extract 5.0 [2CO3, and 1.0% birchwood xylan (w/v) as a source of carbon. After incubation at 35°C for 48 h, six bacterial colonies of distinct morphology (named as X1-X6) were isolated, and their purity was verified by microscopic observation after Gram-staining. Serial dilutions of 10% (w/v) soil sample collected from saw-dust decomposing site, located around a saw mill in Lucknow, Uttar Pradesh, India, were used for screening of the bacteria. Agar plates were made by using modified Berg's mineral salts (BMSs) medium that contained (g/L): NaNOract 5.0 , adjusteAll isolated xylanolytic bacteria were further tested for their xylanase activity by growing them on agar plate and in broth medium. All six isolates were grown on BMS agar plate which contained birchwood xylan . After incubation for 48 h at 35°C, xylan hydrolysis zone was measured by Congo red staining method . These bμL culture broths were added to 9.0 mL distilled water and the dilution tubes were heated at 80, 90 and 100°C in water bath for 30 min. Samples (50 μL) were spread onto nutrient agar plates and incubated at 35°C for 48 h. Similarly halotolerance was assayed by adding 8.0% NaCl (w/v) to the media. Molecular characterisation of bacterium was done by 16S rRNA gene sequencing. Total DNA was isolated from bacterium using kit . The 16S rRNA gene of DNA sample was PCR amplified using 16S rRNA universal primers: 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGTTACCTTGTTACGACTT-3′) at annealing temperature of 56°C (35 cycles). The PCR product was purified by gel extraction  and was sequenced in an ABI 3130 genetic analyzer using Big Dye Terminator version 3.1 cycle sequencing kit. The task of sequencing was outsourced to M/s. Eurofins Genomic, a professional company in Bangalore, India. The 16S rRNA gene sequences were compared with available sequences using NCBI-BLAST. A phylogenetic tree was constructed using Bio Informatics Bacterial Identification (BIBI) software (http://umr5558-sud-str1.univ-lyon1.fr/lebibi/lebibi.cgi).The selected bacterium was characterized by conventional and molecular approaches. Morphological and biochemical tests were performed according to standard method . Physiol2CO3. Flasks were inoculated with 1.0 mL of overnight grown pre-culture with an inoculum size of 1 × 106 CFU/mL on nutrient agar plate and incubated at 35°C, 120 rpm for 120 h . The culture broths were withdrawn every 24 h and measured for change of bacterial growth and enzyme activity.Xylanase was produced by selected bacterium in liquid culture condition using BMS broth containing wheat bran and birchwood xylan as production substrate. Erlenmeyer conical flasks (250 mL) containing 100 mL of BMS broth (pH 8.0) supplemented with 1.0% (w/v) of wheat bran or xylan were autoclaved for 20 min at 121°C. The pH of the medium was adjusted using 2.0% NaCulture broth (2 mL) was withdrawn to measure bacterial growth by taking absorbance at 620 nm in a glass cuvette with 1.0 cm path length . μM of reducing sugars equivalent to D-xylose per minute under the assay conditions. Cellulase activity was assayed by the same method except that low viscosity carboxymethyl cellulose (CMC) was used as substrate. One unit of cellulase activity was defined as the quantity of enzyme releasing 1 μM reducing sugar equivalent to glucose per minute under the assay conditions. The culture broth was centrifuged at 10,000 ×g for 30 min and xylanase activity in culture supernatant was determined by measuring the released reducing sugars formed by enzymatic hydrolysis of birchwood xylan. Briefly, 0.5 mL of supernatant and 1.5 mL of 1.0% (w/v) birchwood xylan prepared in 100 mM phosphate buffer, pH 8.0, was incubated at 50°C for 15 min. The reaction was terminated by adding 2.0 mL of 3,5-dinitrosalicylic acid (DNS) reagent. The color development due to formation of reducing sugars was monitored at 540 nm , and quaμg protein) were subjected to SDS-PAGE [β-mercaptoethanol, or any other reducing agents and without boiling to ensure that all active isoforms present there may be detected. After electrophoresis, the gel was cut in two parts. One part was stained with Coomassie brilliant blue R-250 staining and the other portion was used for zymogram analysis. The gel for zymogram analysis was washed two times for 30 min at 4°C in 100 mM sodium phosphate buffer (pH 7.0) containing 25% isopropanol to remove SDS and renature the protein. The gel was then incubated in the same buffer containing 1.0% birchwood xylan solution at 37°C for 30 min to allow the enzyme to digest xylan. Zymogram was developed by staining the gels in Congo red solution  for 15 min. The gel was then destained with NaCl solution (1 M) until zones of clearance and then immersed in 0.5% (w/v) acetic acid to stop the reaction. Presence of halos around the protein bands was considered as enzyme activity. The molecular weight of proteins was determined by comparing them with standard protein marker . Total protein concentration in crude xylanase preparations was measured by the method of Bradford [Crude enzyme preparations obtained after ammonium sulfate precipitation (80% saturation) and ultrafiltration  were used for zymogram analysis . SamplesSDS-PAGE  using 10Bradford  using thThe optimum pH for xylanase activity was determined by incubating the crude enzyme preparation with 1.0% (w/v) birchwood xylan prepared in 100 mM of citrate buffer (pH 4.0–6.0), phosphate buffer (pH 6.0–8.0), Tris-HCl buffer (pH 8.0–9.0), or glycine-NaOH buffer (pH 9.0–11.0) at 50°C for 15 min. To determine the stability of enzyme at different pH, the crude xylanase preparation was preincubated in the above mentioned buffers of pH 4.0–11.0 at 37°C for 30 min without substrate, and the remaining activity was measured in phosphate buffer (pH 8.0) at 50°C. The optimum temperature for xylanase activity was determined by incubating the crude enzyme preparation with 1.0% (w/v) birchwood xylan prepared in Tris-HCl buffer (pH 9.0) for 15 min at 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, and 100°C. Thermostability of the xylanase was determined by preincubating the crude enzyme preparation at the above mentioned temperatures in Tris-HCl buffer (pH 9.0) without substrate for 30 min, and then residual activity was measured at 60°C. 4, MnSO4, MgCl2, ZnSO4, CuSO4, and CaCl2) at 2 mM final concentration in a 2.0 mL reaction mixture. Similarly, the effect of organic solvents on enzyme activity was determined using ethanol, acetone, and methanol at 25% (v/v) concentration in a 2.0 mL reaction mixture. The activity of enzyme without any metal and solvent was considered as control and was taken as 100%.The effect of metal ions on crude xylanase activity was determined by addition of metal ions  and methanolic extract of crude xylanase preparation, control, and treated xylan were applied on TLC plate and developed in a solvent mix consisting of n-butanol-acetic acid-water (6 : 4 : 3 v/v/v). The TLC plate was sprayed with 99.5% ethanol-concentrated sulfuric acid mix (1 : 1 v/v) and visualized after heating at 100°C.The exo/endoxylanolytic activity of the crude enzyme preparation of strain X1 was examined by carrying out thin layer chromatography (TLC) on silica gel plates. The crude xylanase preparation, control (standard xylan), and treated xylan (1 mL of 1% xylan incubated with 0.5 mL of crude xylanase for 30 min at 50°C) were extracted with methanol . AliquotOne gram unbleached hardwood Kraft pulp , was washed extensively and treated with 25 mL of crude xylanase preparation (10 IU/mL) at 60°C (pH 9.0) for 3 h at 100 rpm. At the end of reaction, control and enzyme treated pulp samples were filtered, washed with tap water, and dried in an oven at 50°C to a constant weight. Reducing sugars released from untreated and enzyme-treated pulp were measured according to Miller . Kappa nProvidencia sp.; however, maximum homology (99%) was observed with Providencia rettgeri  which are morphologically distinct and formed clear zones on xylan-agar plate were isolated from saw-dust decomposing soil. Strain X1 which showed the highest clearance was selected for further characterization. The property was corroborated by measurement of xylanase activity, which again was the highest (17.4 ± 0.4 IU/mL) in strain X1 . The extrettgeri . The 16S620 2.4) in this medium, compared to (OD620 1.3) in xylan medium at 48 h  after 48 h of incubation . The enz Figures . High prns MK001 , 23. Thens MK001 . Providencia sp. strain X1 as has also been reported earlier from Myceliophthora sp. [Zymogram technique was employed to observe the presence of multiple forms of xylanase expressed in presence of wheat bran and birchwood xylan. The zymogram analysis of crude xylanase preparation of organism grown on wheat bran contained multiple xylanases as indicated by the presence of halos around seven distinct proteins bands corresponded to estimated molecular weight of 33, 35, 40, 48, 60, 75, and 95 kDa . In conthora sp. . Multiplhora sp. , 27. To hora sp. . Bacillus spp. exhibited the highest activity within pH range of 6.0–8.0 [Bacillus sp. strain 41 M-1 and Bacillus sp. AR-009 [Bacillus sp. AR-009 retained only 54% of its activity after 1 h [The crude xylanase preparation was active in the pH range of pH 4.0–11.0 (>60% activity) with the highest activity at pH 9.0 . Also, t 6.0–8.0 –31. Alth. AR-009 , 33 showBacillus spp. showed the highest activity in the temperature range of 50–55°C [The activity of crude xylanase preparation was the highest at 60°C. The enzyme retained more than 80% and 40% of the maximum activity when assayed at 70°C and 80°C, respectively . The sta 50–55°C , 30, 34. 50–55°C , 36. Dif2+, Mg2+, Zn2+, and Ca2+ ions at final concentration of 2 mM showed a partial inhibition on activity of the crude xylanase (retaining 93–98% of activity), whereas addition of 2 mM of Mn2+ strongly inhibited the xylanase activity by 65%  [Providencia sp. strain X1 in presence of metal ions  and hydrophilic solvents enhances its suitability for paper pulping applications. The addition of Fey by 65% . Mn2+ haurans S7 . In conteptively . The stabutanol) . Impuritβ-glucosidase by lignin preparation. Cellulase displayed varying levels of inhibition while xylanases displayed consistent inhibition and β-glucosidase was the least affected. The effect of soluble lignin on xylanase activity was investigated at lignin concentration ranging from 0 to 1.5 mg/mL. The result showed that inhibition in xylanase activity was observed at low lignin concentration with a 10% reduction in activity at 0.25 mg/mL of lignin. However, no increase in inhibition was observed at higher concentrations of lignin . The inhρ-coumaric acid and gallic acid on xylanase activity and found 85% and 84% inhibition at low concentration (0.1%) of ρ-coumaric acid and gallic acid. Kaya et al. [The kraft pulp contains trace amount of low molecular weight phenolic compounds, which may potentially affect the activity of enzyme. In the present study, four phenolic compounds, namely, ferulic acid, syringic acid, guaiacol, and phenol, were tested to see their effect on xylanase activity at concentrations ranging from 0 to 10 mg/mL. These compounds are either lignin degradation products or naturally present in plant biomass. Addition of ferulic acid, syringic acid, and guaiacol increased the activity of xylanase by 91%, 21%, and 26%, respectively, as compared to control at concentration of 10 mg/mL . Conversa et al.  found tha et al. . Providencia sp. strain X1 xylanase released a range of xylooligosaccharides from xylan, suggesting it to be an endoxylanase. Similarly, xylanase produced by Bacillus halodurans S7 was an endoxylanase [The thin layer chromatography (TLC)  study rexylanase .Providencia sp. strain X1 xylanase was studied by treatment of pulp with crude enzyme (10 IU/mL) after incubation of 0 to 3 h. The results reveal gradual increase of reducing sugars with time. The amount of reducing sugars present in filtrate of untreated pulp was 59 μg/mL and it increased to 185, 455, and 460 μg/mL in filtrate of treated pulp after 1, 2, and 3 h, respectively (Cellulosimicrobium sp. MTCC 10645 was significantly greater with increasing time. Kappa number is the measurement of the amount of lignin present in the pulp. Kappa number of untreated pulp was 13.7. After treating it with enzyme, it decreased to 9.8, which indicates that the kappa number of pulp decreased by 28.5% after 3 h incubation. Biobleaching of hardwood kraft pulp with crude xylanase dose of 20 IU g−1 pulp from Bacillus megaterium and B. pumilus ASH has resulted in a decrease in kappa number by 11 to 13.6% after 3 h treatment at 50°C and 60°C, respectively [Providencia sp. strain X1 led to 28.5% reduction in kappa number. The biobleaching efficiency of ectively . Kamble Providencia sp. strain X1 to be a potential source of xylanase for pulp biobleaching. The pulp and paper industry requires cellulase-free and thermoalkalophilic xylanase that can be produced at low cost. In the present study, the isolated bacterial strain X1 produces xylanases using wheat bran as a substrate. The xylanases produced by present isolate are cellulase-free and are alkali and thermotolerant. They are also tolerant to higher lignin concentrations as well as its degradation products and to metal ions and solvents. The significant reduction in kappa number of pulp by crude xylanase preparation suggested


Streptococcus pneumoniae can colonize the human nasopharynx and cause conjunctivitis and otitis media. Different deletions in the capsular polysaccharide biosynthesis locus and different multilocus sequence types have been described for nonencapsulated strains. Draft genome sequences were generated to provide insight into the genomic diversity of these strains.Nonencapsulated  Streptococcus pneumoniae is a prerequisite for pneumococcal pneumonia, meningitis, and otitis media , which are caused by deletions of cps. In one of these variations, the cps genes are replaced by the gene for pneumococcal surface protein K, which is involved in murine nasal colonization ; S. pneumoniae MNZ14 and MNZ85 ; and S. pneumoniae MNZ41 . Genomic DNA was isolated with a DNeasy kit (Qiagen), and double-stranded DNA (dsDNA) was quantified with a Qubit fluorometric assay (Invitrogen). Multiplexed paired-end libraries (2 × 150 bp) were prepared using a Nextera XT DNA sample preparation kit (Illumina). Sequencing was done with an Illumina MiSeq. CLC Genomics Workbench v6.0 software was used for quality trimming of the reads and de novo assembly.Five nonencapsulated strains of of MNZ11 , and S. 50, and total sequence length, respectively, for each strain were as follows: 54, 73.44 kb, and 2.02 Mb for MNZ11b; 72, 61.10 kb, and 2.13 Mb for MNZ14; 66, 50.17 kb, and 1.95 Mb for MNZ37; 97, 38.75 kb, and 2.13 Mb for MNZ41; and 97, 41.84 kb, and 2.15 Mb for MNZ85. Each scaffold from each strain had an average coverage of >146×. Open reading frames were identified and annotated with the NCBI Prokaryotic Genomes Automatic Annotation Pipeline (An average of 7.86 million paired-end reads per strain was collected. The number of scaffolds, scaffold Ncps (These genome sequences represent a diverse sample of nonencapsulated pneumococci. Previously reported deletions of cps  are confASJF00000000, ASJO00000000, ASJP00000000, ASJQ00000000, and ASJW00000000 for strains MNZ85, MNZ14, MNZ37, MNZ41, and MNZ11b, respectively.The GenBank accession no. for version 1 of these sequences are


Houttuynia cordata Thunb (HCT) is commonly used in Taiwan and other Asian countries as an anti-inflammatory, antibacterial and antiviral herbal medicine. In this study, we investigated the anti-human lung cancer activity and growth inhibition mechanisms of HCT in human lung cancer A549 cells.0/G1 and Sub-G1 cell (apoptosis) populations and HCT increased DNA fragmentation and DNA condensation as revealed by DAPI staining and the Comet assay. HCT induced activation of caspase-8 and caspase-3. Fas/CD95 protein levels were increased in HCT-treated A549 cells. The G0/G1 phase and apoptotic related protein levels of cyclin D1, cyclin A, CDK 4 and CDK 2 were decreased, and p27, caspase-8 and caspase-3 were increased in A549 cells after HCT treatment.In order to investigate effects of HCT on A549 cells, MTT assay was used to evaluate cell viability. Flow cytometry was employed for cell cycle analysis, DAPI staining, and the Comet assay was used for DNA fragmentation and DNA condensation. Western blot analysis was used to analyze cell cycle and apoptotic related protein levels. HCT induced morphological changes including cell shrinkage and rounding. HCT increased the G0/G1 phase arrest and Fas/CD95-dependent apoptotic cell death in A549 cellsThe results demonstrated that HCT-induced G In Taiwan, 26 individuals per 100,000 died from lung cancer each year, based on reports from the “People Health Bureau of Taiwan”. Surgery, radiotherapy, and chemo-therapy are used for treating lung cancer patients . HoweverMorphological changes in apoptotic cells include cell membrane blebbing, DNA or chromatin condensation, and caspase activation -10. PrevHottuynia cordata Thunb (HCT), also called E-Sung-Cho is a Chinese herb used to treat several different diseases  [za etc.) -24. In aza etc.) ,26, viraza etc.) -29. Manyza etc.) ,31 and aza etc.) ,33. HCT za etc.) . In thisHouttuynia cordata Thunb (yield: 6.73% of dry wt.) was obtained by 48 h incubation at room temperature. The ethanol extract was filtered through a 0.45 μm filter , lyophilized and kept at 4°C. The dried extract was re-solubilized in PBS before use as previously described [Ethanol extract of escribed ,33.RPMI-1640 cell culture medium , DAPI , low-melting agarose, MTT -2,5-diphenyltetrazolium bromide), and DMSO (dimethyl sulfoxide) were purchased from Sigma . FBS , penicillin/streptomycin, PI (propidium iodide) and trypsin-EDTA were obtained from Life Technologies . Proteinase K was purchased from Roche Diagnostics Gmbh . Ac-DEVE-pNA and Ac-IETD-pNA were purchased from R&D Systems Inc., . All other chemicals used were of analytical grade.Human lung cancer A549 cells were obtained from the Bioresource Collection and Research Center , originally from the American Type Culture Collection . Cells were maintained in RPMI-1640 containing 100 mL/L FBS with 100,000 U/L penicillin and 100 mg/L streptomycin.A549 cells were plated onto 96-well plates and incubated with HCT  for 24 and 48 h. MTT was added to each well then incubated for an additional 4 h at 37°C. The blue formazan product was dissolved in 100 μL of DMSO. The plates were read at O.D.570 nm using a spectrophotometric plate reader . The experiments were performed in triplicate (n = 3). Cell viability was calculated as O.D. of drug-treated sample/O.D. of none treated sample × 100% as previously described ,35.For cell cycle and apoptosis determination, A549 cells were plated onto 24-well plates and incubated with HCT  for 24 h. Cells were fixed gently in 70% ethanol at 4°C and then re-suspended in phosphate-buffered saline (PBS) containing 40 μg/ml PI, 0.1 mg/ml RNase and 0.1% Triton X-100 for 30 min at 37°C. Cell cycle transition and apoptosis were then analyzed by flow cytometry  as previously described .A549 cells were plated onto 24-well plates and treated with HCT (0 and 500 μg/ml) for 24 h. After HCT treatment, cells were fixed in 4% paraformaldehyde and then incubated with 1 μg/ml of DAPI staining solution in darkness. The apoptotic cells were observed by fluorescence microscopy  as previously described ,35.4 cells were mixed with 150 μL 0.75% low-melting agarose held at 37°C and layered onto a pre-treated slide with 1.5% regular agarose. After agarose were solidified on a chilled plate, the slides were transferred to the same lysis buffer, held at room temperature for 4 h and stained with propidium iodide as previously described [A549 cells were plated onto 24-well plates and incubated with HCT (0 and 500 μg/ml) for 24 h, 1 × 10escribed .A549 cells were plated onto T-75 flasks and treated with HCT  for 24 h. Total cell lysates were prepared as previously described. Thirty μg of total protein applied to SDS-PAGE and transferred onto a polyvinylidene fluoride membrane . After blocking, the blots were incubated with the appropriate dilution of specific monoclonal antibodies for cyclin D1, cyclin A, CDK 4, CDK 2, p27, caspase-8 and caspase-3 . Blots were washed and then incubated with horseradish peroxidase-conjugated secondary antibody . Protein expressions were detected using enhanced chemiluminescence kits  as previously described .A549 cells were plated onto T-75 flasks and incubated with HCT  for 24 h. A549 cells were collected in a protein lysis buffer . Cell lysates were centrifuged at 15,000 × g at 4°C and then incubated with caspase-3 and caspase-8 specific substrates (Ac-DEVE-pNA and Ac-IETD-pNA) with reaction buffer in a 96-well plate at 37°C for 1 h. Caspase activity was determined by measuring O.D. 405 nm of the released pN using a spectrophotometric plate reader  as previously described. The experiments were performed in triplicate [Fas/CD95 cell surface antigen expression was measured by flow cytometry. A549 cells were plated onto 24 well and treated with HCT  for 12 h. Cells were collected and rinsed in PBS. Fas/CD95 was analyzed by direct immune-fluorescence staining. FITC-conjugated anti-Fas/CD95 and its FITC- conjugated isotype mAb  were analyzed using a flow cytometer as previously described .t-test was used for single variable comparison. For multiple variable comparisons, data were analyzed by one-way ANOVA followed by Dunnett’s test. P < 0.05 was considered significant.The experiments were performed in triplicate (n = 3) and all data were expressed as the mean ± standard error. Student’s p < 0.001, compared to HCT 0 μg/ml group). The inhibition of HCT in A549 viability was 15.73%, 27.13%, and 58.57% for 125, 250, and 500 μg/ml at 24 h; 29.32%, 50.81%, and 69.60% for 125, 250, and 500 μg/ml at 48 h, respectively. HCT also induced morphological changes seen as cell shrinkage and rounding  treated A549 cells was determined by MTT assay for 24 and 48 h. As shown in Figure 0/G1 phase, and the proportion of cells in the G0/G1 phase increased markedly in a concentration-dependent manner. As shown in Figure 0/G1 by HCT  treatment was 43.48%, 45.06%, 58.59%, and 63.25%, respectively. In addition, A549 cells treated with HCT  showed a significant increase in the sub-G1 apoptotic cell population, and the apoptotic cell population was 4.53%, 13.07%, 18.77% and 31.89%, respectively . Figure 0/G1 phase. Figure 0/G1 phase, decrease of CDK4 was the most significant one. HCT decreased protein levels of CDK4 by 0%, 20%, 70%, and 90%, respectively.We investigated the protein levels of the GEffects of HCT on nuclear morphological change were examined by DAPI staining and DNA damage by the Comet assay. As shown in Figure We determined if HCT would stimulate the caspase-9, caspase-8 and caspase-3 activity in A549 cells. Figure et. al. indicated that quercitrin and isoquercitrin isolated from methanolic extracts of Houttuynia cordata showed excellent DPPH radical scavenging activities with IC50 values of 31 μM and 63 μm, respectively [The death receptor apoptotic pathway has previously been proposed as an anti-cancer drug target in human lung cancer ,40. Tradectively . Also, Hectively  which haet. al. suggested that ethanolic extract of HCT induces human leukemic HL-60 and Molt-4 cell apoptosis through a mitochondrial apoptotic pathway [Previous studies have shown that HCT induces human lymphoblastic leukemic Molt-4 cell death through an endoplasmic reticulum stress pathway . Banjerd pathway .1-3) [kip1, belongs to the Cip/Kip (kinase inhibitor protein) family, inhibits most CDKs [kip1[kip1) or down-regulation of cyclins required for CDK activation (e.g. cyclin D1) are mechanisms of inhibition of cell cycle progression [0/G1 arrest. HCT induced apoptosis through caspase8/caspase-3 activation . CDKs  is paired with the cyclins  . Activat1-3)  and leadost CDKs . Dysreguost CDKs . p27kip1DKs [kip1, cyclin DKs [kip1, has beegression . Our resn Figure A,B and uIn conclusion, this study demonstrated that HCT has anti-lung cancer activity by modulating G0/G1 arrest and stimulating the Fas/CD95 protein level, which leads to caspase-8 and caspase-3 activation resulting in the induction of apoptosis in human lung cancer A549 cells -2,5-diphenyltetrazolium bromide; FBS: Fetal Bovine Serum; PBS: Phosphate-Buffered Saline; DMSO: Dimethyl Sulfoxide; PI: Propidium Iodide; PVDF: Polyvinylidene Fluoride Membrane.HCT: The authors declare that they have no competing interests related to this work.0/G1 relative protein levels assay and caspase activities assay. SCT helped to draft the manuscript. SFP carried out the DAPI staining and Comet assay. YRZ performed immunostaining assay for Fas/CD95 protein levels. All authors read and approved the final manuscript.YFC conceived for the study, and participated in its design, performed the statistical analysis and figure drawing, coordination, draft and revise the manuscript. JSY helped to draw the figures and drafted the manuscript. WSC carried out the cell viability, cell cycle transition, G


A complete musculoskeletal examination should include the specific tests to detect hypermobile individuals, especially when physiotherapy affecting joint mobility is planned. Joint hypermobility (JH) is defined as an excessive range of motion of joints, taking into consideration the subject’s gender, age, and ethnic background.The goal of this study was to assess the prevalence of JH in girls with idiopathic scoliosis (IS), taking into account the age, curve pattern and curve size. The study group included 147 Caucasian girls with IS, aged 9-18 years (mean 13.6 ±2.2), Cobb angle range 11°-65°, mean 27.9 ±11.9, comprising 42 single thoracic, 31 single lumbar and 74 double curve scoliosis. According to the Cobb angle, there were 70 mild (10-24º), 54 moderate (25-40º) and 23 severe (> 45º) curves. The control group included 147 girls aged 9-18 years (mean 13.1 ±.3), selected at random from the group of 300 girls free of IS (angle of trunk rotation <5°). The presence of JH was assessed with the 9-point Beighton scale [JH was diagnosed in 24.5% of IS girls, whilst in the control group, it was diagnosed in 15.1% (p=0.04). The prevalence of JH was significantly (p=0.03) lower in IS girls aged 16-18 years in comparison to younger individuals (9-15). There was no difference regarding JH occurrence among girls with mild, moderate and severe scoliosis (p=0.8). No significant differences in JH prevalence was observed among girls with single thoracic, single lumbar and double curve scoliosis (p=0.68). The number of vertebrae within curvature did not influence the prevalence of JH (p=0.13). JH appeared more often in IS girls than in healthy controls. Its prevalence decreased with age. No relation between JH prevalence and curve pattern, curve size or number of vertebrae within curvature was found.


Plasmodium falciparum and Plasmodium vivax.Glucose-6-phosphate-dehydrogenase deficiency (G6PDd) rates are unknown in malaria-infected Cambodian patients. These data are key to a rational drug policy for malaria elimination of From September 2010–2012, a two-year survey of G6PDd and haemoglobinopathies assessed by quantitative enzyme activity assay and haemoglobin electrophoresis, respectively, was conducted in malaria-infected patients presenting to 19 health centres throughout Cambodia.Plasmodium falciparum was present in 1,443 (59.9%) and P. vivax in 965 (40.1%) patients. Mean G6PD activity was 11.6 (CI 95%: 11.4-11.8) U/g Hb, G6PDd was present in 13.9% of all patients  and severe G6PDd (including WHO Class I and II variants) was more common in western  versus eastern  Cambodia (P = 0.01). Of 997/2,408 (41.4%) had a haemoglobinopathy. Mean haemoglobin concentrations were inversely related to age: 8.1 g/dL < five years, 8.7 g/dL five to 14 years, and 10.4 g/dL >15 years (P <0.001).A total of 2,408 confirmed malaria patients of mean age 26.7 (range 2–81) years were recruited from mostly western Cambodia ; males outnumbered females by 3.9:1. G6PDd prevalence, anaemia and haemoglobinopathies were common in malaria-infected patients. The deployment of primaquine in Cambodia should be preceded by primaquine safety studies paralleled with evaluations of easy to use tests to detect G6PDd. Plasmodium falciparum, because it kills mature gametocytes, and as anti-relapse treatment against Plasmodium vivax by killing liver hypnozoites [P. falciparum without prior G6PD testing believing that AHA would be clinically mild [Controlling malaria remains a significant global health challenge , especianozoites ,4. Primanozoites , a scenanozoites . Neverthnozoites . Indeed,lly mild .Cambodia has low transmission of essentially falciparum and vivax malaria in an overall ratio of about 1:1 but there is seasonal and geographical variation. Approximately half of the population, ~3,000,000, is estimated to be at risk of malaria  and publG6PDd is a common, X-linked hereditary enzyme deficiency affecting approximately 400 million people worldwide , mainly The G6PDd prevalence rates in Cambodia vary between 13.4 and 26.1% in males and 3.1 and 4.3% in females, depending on the sampled population  to prioritize its anti-malarial drug policy and to conduct future research on the safety of primaquine. Results of a G6PDd survey in malaria patients are reported herein.P. falciparum and non-P. falciparum parasites . Malaria-positive patients or their legal guardians were asked if they would be interested to join the study. If signed informed consent was obtained, patients were allocated a study number and had blood taken. The study protocol was reviewed and approved by the Ethics Committee of the Cambodian Ministry of Health .The study took place from 2010 to 2012 at 19 public health facilities from across Cambodia  complete blood count (CBC), quantitative determination of G6PD activity and haemoglobin electrophoresis, and, (ii) in vitro anti-malarial drug sensitivity testing and detection of molecular markers related to anti-malarial drug resistance.Five ml of venous blood were collected into ACD-coated tubes, stored in a fridge before transport to IPC within 24 to 48 hours at +4°C in cool boxes. At IPC, repeat malaria blood films were made, stained with 3% Giemsa solution for 30 to 45 minutes, and checked for The CBC was determined using a CellDyn 3200 analyzer™  after daily standardization with three different controls of all the standard parameters.Determination of the G6PD enzyme activity was performed on the fresh blood within a maximum of 48 hours after sample collection, using the Trinity Biotech quantitative G6PD assay™  adapted on the Integra 400 analyzer™ , according to the manufacturer’s instructions and as described previously . The relG6PD deficiency was classified according to the WHO classification expressing the G6PD enzyme activity as a percentage of the population defined mean, 11.8 U/g Hb for Cambodia : (i) Cla2 control (ref PN4778), (ii) normal HbA and F and abnormal HbS and C , and (iii) increased haemoglobin A2 fraction (ref PN4779).The capillary electrophoresis was performed with the MINICAP system™  according to the manufacturer’s instructions and using controls of human blood for every run: (i) normal Hb A2 =1.8-3.4%, HbF <1%, (ii) homozygous HbE: HbE > 85%, HbA = 0%, HbA2 = 3.4-5.3%, HbF <15%, (iii) heterozygous HbE: HbE = 25-30%, HbA = 68-73%, HbA2 = 2.5-4.5%, HbF < 1%, (iv) mixed heterozygous HbE and α-thalassaemia: HbA = 73-85%, HbA2 = 3.7-4.5%, HbF < 1%,, (v) heterozygous HbE and β-thalassaemia: HbA = 0-10%, HbA2 = 2.2-4.4%, HbF = 30-65%, HbE = 0-15%, and (vi) β-thalassaemia: HbA > 90%, HbA2 = 2.6-6.5, HbF = 3.0-7.4%, HbE = 0%.Briefly, red cell haemolysate was automatically prepared on the instrument and charged molecules were separated according to their electrophoretic mobility in an alkaline buffer (pH 9.4). Relative quantification and presumptive identification of the haemoglobin fractions were performed automatically by the software. The HbA fraction was centred in the middle of the review window and resulting electrophoregrams evaluated visually for pattern abnormalities (identified in zones Z1 to Z15) to classify haemoglobinopathies as: (i) normal profile: HbA >97%, HbAP-values are two-sided and were considered statistically significant if <0.05.Data were entered and verified using Microsoft Excel 2010 software and analysed using MedCalc software  and XLSTAT for Windows XP . Continuous variables were compared using an independent-sample analysis of variance (ANOVA) or Mann–Whitney test (skewed data). For categorical variables, Chi-squared or Fisher’s exact tests were used to assess significant differences in proportions. All reported For the logistic regression, some variables were grouped in different categories: (1) G6PD deficiency enzyme activity into two groups: severe deficient  and mild deficient/normal ; (2) sampling locations in three areas: western Cambodia , eastern Cambodia  and central Cambodia ; and, (3) ages in three groups: <five years, five to 14 years and >15 years.P-values <0.25 from the bivariate analyses were initially introduced into the model and removed following a backwards-stepwise selection procedure to leave only those variables those with a P-values <0.05 in the final model. Odds ratios (OR) and their 95% confidence intervals (CI) are reported for these significant explanatory variables.Variables with From September 2010 to September 2012, 2,408 malaria-positive patients were recruited from western , eastern , and central  Cambodia. The mean age was 26.7 years (SD: ± 12.0) with a range of two to 81 years, distributed as follows: (i) 0.5% < five years, (ii) 9.9% five to 14 years, and (iii) 89.6% >15 years. The male: female ratio was 3.9:1.Plasmodium falciparum was present in 1,443 (59.9%) and P. vivax in 965 (40.1%) patients; species distribution is shown in Figure P. falciparum and P. vivax infections for age (P <0.0001), sex (P <0.0001), G6PDd class (P <0.0001) and the detection haemoglobinopathy  more frequent in female patients , but (ii) less frequent in severe G6PDd patients  and patients with a haemoglobinopathy .P <0.001), (ii) females 9.1 g/dL vs males 10.5 g/dL (P <0.001), (iii) severe (Class I and II) G6PDd: 10.0 g/dL vs 10.7 g/dL for mild/normal G6PD, P = 0.004), (iv) patients with homozygous E: 8.8 g/dL vs 10.2 g/dL  and vs 10.1 g/dL heterozygous HbE/heterozygous HbE and α-or β-thalassaemia (P <0.001). The mean Hb concentrations by G6PDd classes were: 11.1 g/dL for Class I (n = 18), 10.6 g/dL for Class II (n = 179), 9.5 g/dL for Class III (n = 140), 10.2 g/dL for Class IV  and 8.0 g/dL for Class V (n = 122).For all patients, the mean Hb concentration was 10.1 g/dL (95% CI: 9.9-10.2) and was significantly lower in: (i) younger age groups: 8.1 g/dL < five years, 8.7 g/dL five to 14 years, and 10.4 g/dL >15 years (vs 8.7% (P <0.0001). G6PDd was found more commonly in western compared to eastern Cambodia (Table The mean G6PD enzyme activity was 11.6 (CI 95%: 11.4-11.8) U/g Hb Figure ; the proP = 0.0007), and (ii) vivax-infected patients , but (iii) more likely in haemoglobinopathy patients: .Logistic regression showed that Class I and II deficiency was less likely in (i) females . Heterozygous Hb E accounted for 862/997 (86.4%) of all the haemoglobinopathies. The prevalence of homozygous HbE (5.6%) was highest in MondulKiri province (13.2%), following by Preah Vihear province (9.8%), Battambang province (7.0%) and Sihanouk province (6.8%). The prevalence of heterozygous HbE  (35.8%) ranges from 30.0% in Kampot province to 49.3% in Ratanakiri province.P. vivax and artemisinin-resistant P. falciparum, primaquine is an essential drug [For countries moving towards elimination of ial drug ,29,30. Pial drug  and the ial drug  is not yTo date, most of G6PD studies in Cambodia have been conducted in a single site or in particular healthy populations  using different methods to measure G6PD enzyme activity and different enzyme cut-offs to classify individuals as G6PDd Table  20,23,2,26,28, wHealth centres distributed throughout Cambodia were selected for geographical completeness in this study; although not a randomized sample, the species ratio was broadly similar to the recent national data. Most patients were young male adults (50% were aged 18–30 years), with anaemia (mean Hb about 10 g/dL and 74% had Hb <12 g/dL). Data observed in the present study show that anaemia was more frequent in children under five years of age, in females, in patients with G6PDd or with HbE-related haemoglobinopathies.The high proportion of malaria-infected, anaemic patients reinforces the importance of not only detecting G6PDd but also to measure the haemoglobin concentration before starting primaquine because 0.75 mg/kg of primaquine, the recommended weekly dose in G6PDd vivax patients, has produced falls of 20-30% in haemoglobin concentration ,31.Before deploying primaquine in Cambodia, carefully conducted safety studies are needed for both low and weekly dose primaquine in falciparum and vivax patients, respectively, supported by studies to validate G6PD RDTs that have extremely low rates of missing G6PDd. Daily primaquine dosing in a RDT-diagnosed G6PD normal patient who is actually G6PDd would probably cause life threatening AHA in severe Southeast Asian G6PDd variants and may lead to a fall of confidence in primaquine by the population .The G6PDd prevalence 13.9%) reported here is very similar to previous studies in healthy individuals ,24,26-283.9% repoP. vivax is still substantial, and where primaquine is urgently needed.As expected, severe G6PDd was more frequent (~three-fold) in males whilst mild G6PDd was more frequent in females (~two-fold). G6PDd differs geographically with severe deficiency more common in western Cambodia. These data are of major concern for the safe use of primaquine as G6PDd is most prevalent in these areas, especially Pailin province, where artemisinin-resistant falciparum parasites are present and emerging, where Haemoglobinopathies were also very common, affecting just under half of malaria patients and was associated with moderate anaemia. Heterozygous HbE was the most frequent haemoglobinopathy  and was more evenly distributed across Cambodia compared to G6PDd. Interestingly, like severe G6PDd, it protected patients against vivax malaria, consistent with data from others .P. vivax. Many malaria patients were anaemic, raising questions whether primaquine-induced AHA would be well tolerated. Combined primaquine safety and G6PD point of care evaluation studies are needed most urgently in Cambodia.This study has shown that G6PDd prevalence rates and enzyme activity in malaria patients are consistent with those in healthy individuals and that severe G6PDd is prevalent mostly in western areas of Cambodia where artemisinin resistance is present. Haemoglobinopathies were common and contributed to anaemia but also to protection against The authors declare that they have no competing interests.DM contributed to the design and coordination of the study, assisted with data entry, analysis and interpretation. SKi supervised the field study. NK, CB, PC, LC, BW, AK and PT were involved in laboratory work. NK, CB, WRJT and DM wrote the first draft of the paper. SKh, SS, RL, SL, SM, NC, CMC and SD gave constructive advice. All authors read and approved the final manuscript.Walter RJ Taylor and Didier Ménard share senior authorship.


In this paper, we concluded that transient acidosis reperfusion conferred cardioprotection against myocardial ischemia reperfusion injury in isolated rat hearts through activating PI3K-Akt-eNOS pathway. During myocardial ischemia, tissue pH significantly declines and returns to normal after reperfusion . RecentlIschemic postconditioning, a novel strategy of cardioprotection consisting of the application of brief cycles of ischemia-reflow at the onset of reperfusion, represents a promising approach to protect the myocardium against ischemia and reperfusion injury , 5. And The experimental procedures conformed to the Guide for the Care and Use of Laboratory Animals published by National Institute of Health of the People's Republic of China and approved by the Institutional Animal Ethics Committee.4, 1.2 KH2PO4, 1.25 CaCl2, 25.0 NaHCO3, and 11.0 Glucose, equilibrated with 95% O2 and 5% CO2 to create perfusate pH 7.4, and maintained at 37°C as previously described [Male Sprague-Dawley rats (450–550 g) were heparinised and then anaesthetized with urethan (700 mg/kg). The hearts were rapidly excised and mounted onto a Langendorff apparatus and perfused with modified Krebs-Henseleit bicarbonate buffer (KHB) that contained (in mM) 115.0 NaCl, 5.0 KCl, 1.2 MgSOescribed . Flow ran = 12 per group). In control group, hearts were stabilized for 30 min and then subjected to no-flow global ischemia by inflation of the coronary balloon for 30 min followed by 120 min of reperfusion with normal KHB (C group). Postconditioning was achieved by 6 cycles of 15 s reperfusion and 15 s occlusion after no-flow global ischemia (IPO group). In the acidotic reperfusion group, hearts were perfused with KHB adjusted at pH 6.9 for the first 3 min of reperfusion (H+ group). The acidotic perfusion buffer was adjusted by equilibrating with 80% O2/20% CO2 (pH 6.9). To test whether alkali abolished the protection of ischemia postconditioning, hearts were postconditioned with alkalotic buffer which was equilibrated with 100% O2 (pH 7.8) (IPO + OH−). In OH− group, hearts were only reperfused with alkalotic buffer without ischemia postconditioning. Finally, PI3k specific inhibitor wortmannin (100 nmol/L) was added to the acidotic perfused and infused into heart for the first 3 min of reperfusion after ischemia (H+ + wort group). In wort group, hearts were reperfused with normal KHB cotreatment of wort (100 nmol/L). After the first 3 min of reperfusion with respective treatment, all groups were switched to buffer equilibrated with 5% CO2. In all hearts, reperfusion lasted for 120 min.Rats were randomly divided into 7 groups , 0.5% deoxycholic acid, 10 mmol/L NaF, 1 mmol/L PMSF, 25 mg/mL leupeptin, and 2 mg/mL aprotinin) for 30 min then sonicated and centrifuged at 12000 g for 20 min at 4°C. Protein concentrations were determined using the Bradford assay . Samples containing equal amounts were separated on a 10% SDS-polyacrylamide gel, and then proteins were transferred to PVDF membrane overnight at 4°C. Membranes were blocked with 5% nonfat milk in Tris-buffered saline (TBS)-Tween for 1 hr and were incubated with anti-Akt or anti-eNOS antibodies and GAPDH  at 1 : 1000 dilution overnight at 4°C. After washing with phosphate buffered saline-Tween (PBST) three times for 30 min, membranes were then incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG at 1 : 2000 dilution for 1 h. Protein bands were developed with enzymatic chemiluminescence, and images were measured by a densitometer with analysis software.Free 15-F2t-isoprostane (15-F2t-IsoP), a specific marker of oxidative stress, was measured by using an enzyme-linked immunoassay kit  as described . PerfusaP ≤ 0.05.Data are presented as means ± standard error of the mean (S.E.M.). Data were analysed by the ANOVA within the same group and between groups. Multiple comparisons of group means were analyzed by Tukey's test. The analysis was performed using statistical software package . Significant difference was defined as max and −dp/dtmax were lower compared with its baseline data before ischemia. IPO treatment significantly decreased LVEDP and increased +dp/dtmax and −dp/dtmax at the time of reperfusion (30 min) as compared with the I/R group . When postconditioning was performed with alkalotic perfusate, it was no longer protective. Acidic reperfusion (H+ group) also improved cardiac function, similar to the effect of postconditioning. However, this protection was reverted by PI3k inhibitor wort (P < 0.05) (P > 0.05).As shown in  < 0.05)  althoughP < 0.01) (P < 0.05) (P < 0.05) (+ group) mimicked the protection of postconditioning, which was reverted by PI3k inhibitor wortmannin (P < 0.05) (P < 0.05).Postconditioning decreased infarct size from 34.6% ± 2.3% of risk zone in control hearts to 10.8% ± 1.4% ( < 0.01) . However < 0.05) . However < 0.05) . Acidic  < 0.05)  FurthermP < 0.05) (P < 0.05), this indicated that the dosage of wortmannin used in this study was enough to inhibit PI3k activation.Postconditioning significantly increased nitric oxide release at the time point of reperfusion (30 minutes) compared with that of control group ( < 0.05) , which wAs shown in Figures P < 0.01 or P < 0.05 versus C, P < 0.05, IPO + OH− versus PO; P > 0.05, IPO + OH− versus control, 15-F2t-isoprostane, which was reverted by wortmannin. Wortmannin alone did not affect level of both perfusate and tissue free 15-F2t-isoprostane compared with control group.Compared with the control group, the levels of free 15-F2t-IsoP were significantly decreased in both the perfusate and heart tissues in IPO group  which will induce the release of NO –17. NO cAdditionally, restoration of oxygenation during brief reperfusion causes mitochondria to produce ROS which could induce oxidative stress and subsequently lead to the myocardial damage during reperfusion, while acidosis during reperfusion has been reported to attenuate ROS generation . AdminisIn summary, acidic reperfusion exactly mimics the protection of postconditioning. Acidosis confers cadioprotection by inhibiting ROS production and increasing NO release through activating PI3k-Akt-eNOS pathway.


To investigate the potential of nevirapine 200 mg once-daily regimen and evaluate the influence of patient characteristics on nevirapine concentrations.This was a prospective, multicentre cohort study with 532 HIV-infected patients receiving nevirapine as a part of their initial antiretroviral therapy. Plasma samples were collected at trough or peak time at the end of week 2 (lead-in period) and week 4, 12, 24, 36, and 48 (steady-state period), and nevirapine concentrations were determined using a validated HPLC method. Potential influencing factors associated with nevirapine concentrations were evaluated using univariate and multivariate logistic regression.v.s. 4.86 µg/mL, for ALT <1.5×ULN and ≥1.5×ULN, respectively, P = 0.045). No significant difference was observed in median nevirapine trough concentration between lead-in and steady-state periods in patients with baseline ALT and AST level ≥1.5×ULN , which was different from the patients with ALT/AST level <1.5ULN. The median trough concentrations were significantly higher in HIV/HCV co-infected patients than those without HCV at week 48 .A total of 2348 nevirapine plasma concentrations were collected, including 1510 trough and 838 peak values. The median nevirapine trough and peak concentration during the lead-in period were 4.26 µg/mL (IQR 3.05–5.61) and 5.07 µg/mL (IQR 3.92–6.44) respectively, which both exceeded the recommended thresholds of nevirapine plasma concentrations. Baseline hepatic function had a moderate effect on median nevirapine trough concentrations at week 2 (4.25 µg/mL The 200 mg once-daily regimen of nevirapine might be comparable to twice-daily in plasma pharmacokinetics in Chinese population. Hepatic function prior to nevirapine treatment and HIV/HCV coinfection were significantly associated with nevirapine concentrations.NCT00872417Clinicaltrial.gov ID:  Nevirapine is a human immunodeficiency virus type 1 (HIV-1) specific non-nucleoside reverse transcriptase inhibitor that binds directly to the viral reverse transcriptase of HIV-1 to block polymerase activity by causing disruption of the enzymes catalytic site Ctrough) and higher peak concentrations (Cmax). Because high nevirapine concentrations are associated with increased adverse events The substantial benefits conferred by cART, however, require strict patient adherence to the prescribed medication because poor adherence will lead to virologic failure. Adherence might be improved with more convenient dosing regimen. A possible measure for simplifying antiretroviral therapy is the use of once-daily dosing regimen. The long plasma elimination half-life of nevirapine (∼25 to 30 h) after multiple dosing may justify once daily dosing throughout the therapy with equivalent efficacy to that of twice daily regimen i.e. 200 mg twice daily, had excessively high plasma concentrations, which may contribute to the higher prevalence of hepatotoxicity in these patients. Another prospective study Ctrough and liver toxicity among Chinese HIV-infected patients, especially in males. To date, Food and Drug Administration approves one-pill, once-daily nevirapine extended-release tablets for use in combination with other antiretroviral agents for treatment of HIV-1 infection in adults. In the context of attempts to simplify treatment regimen while securing efficacy, reducing toxicity and enhancing adherence, there is great interest in nevirapine 200 mg once daily dosing for the treatment of Chinese HIV-infected patients in a long run. The present study aims to investigate the potential of nevirapine 200 mg once daily regimen in Chinese HIV-infected patients by comparing nevirapine plasma concentrations during the lead-in period with the steady-state concentrations. The influence of patient characteristics was evaluated on nevirapine concentrations.Our group has found that the pharmacokinetic profiles of nevirapine in Chinese patients are different with those in Caucasians and blacks Five hundred and thirty-two (n = 532) eligible HIV-infected patients were included and completed this study . DemograCtrough were available during the 48-week period, of which 290 at week 2, 270 at week 4, 247 at week 12, 244 at week 24, 233 at week 36 and 226 at week 48. Ctrough was 4.26 µg/mL (IQR 3.05–5.61) at the end of week 2 when the patients were administered with nevirapine 200 mg once daily, significantly lower than those in the later weeks when patients were receiving twice-daily dosing  (P<0.0001). The steady-state nevirapine Ctrough at week 4, 12, 24, 36 and 48 were 5.90 µg/mL (IQR 4.54–8.14), 6.21 µg/mL (IQR 4.68–8.15), 6.23 µg/mL (IQR 4.56–8.47), 6.15 µg/mL (IQR 4.51–8.07) and 6.37 µg/mL (IQR 4.72–8.64), respectively  shows that the mean nevirapine Ctrough (±SD) at the end of week 2 (4.59±1.81 µg/mL) was significantly lower than the levels at week 4 (6.40±2.59 µg/mL), week 12 (6.59±2.16 µg/mL), week 24 (6.85±2.83 µg/mL), week 36 (6.58±2.35 µg/mL), and week 48 (6.76±2.68 µg/mL) (One hundred and five HIV-infected adults (n = 105) had 6 consecutive 8 µg/mL) .Cmax of nevirapine were obtained, of which 167 at week 2, 159 at week 4, 137 at week 12, 129 at week 24, 126 at week 36 and 120 at week 48. It was demonstrated in Cmax of 5.07 µg/mL (IQR 3.92–6.44) during the lead-in period was significantly lower than the steady-state levels  (P<0.0001). The observed Cmax of nevirapine at week 4, 12, 24, 36 and 48 were 6.89 µg/mL (IQR 5.23–8.84), 6.34 µg/mL (IQR 4.72–8.03), 6.32 µg/mL (IQR 5.40–7.85), 6.48 µg/mL (IQR 5.19–8.35) and 6.91 µg/mL (IQR 5.49–8.56), respectively   . There wCtrough of nevirapine between lead-in and steady-state periods in patients with baseline ALT level ≥1.5×ULN  and baseline AST level ≥1.5×ULN , whereas the median Ctrough during lead-in period was significantly lower than the steady-state levels in patients with ALT/AST <1.5 ULN prior to initial treatment . The median Ctrough of nevirapine at week 48 in patients with HCV coinfection was significant higher than those without HCV coinfection . The difference was not observed in the patients with HBV coinfection  and higher (6.69 versus 5.74 µg/mL) compared with the 200 mg twice daily. The 2NN sub-study confirmed this findings and showed that Cmin (3.26 versus 4.44 µg/mL) was lower and Cmax (7.88 versus 6.55 µg/mL) was higher in the 400 mg once-daily dosing Cmax might result in a higher incidence of toxicity in patients with nevirapine once-daily dosing than those assigned twice-daily administration The efficacy and safety of nevirapine 400 mg once daily in treatment of HIV-infected patients had been assessed in several studies Gonzalez de Requena et al.Wang J et al.Ctrough and liver toxicity among Chinese HIV-infected patients, especially in males (P = 0.015).The liver toxicity is one of the most common hypersensitivity reactions to nevirapine and may be associated with its plasma concentrations. 1/2 (30.94 h), AUC0–12 h (92.82 µg·h/mL), Cl/F (0.71 L/h), Cmax (10.09 µg/mL) and Ctrough (7.88 µg/mL), respectively Ctrough in Chinese males and females (8.95 µg/mL and 6.59 µg/mL) were significantly higher than the levels in Caucasians and blacks The steady-state pharmacokinetic study in 15 Chinese patients with HIV infection Ctrough of nevirapine 200 mg once daily at the end of week 2 was significantly lower than the twice-daily dosing in later weeks . Similarly, the median Cmax of nevirapine during the lead-in period was also lower than the steady-state levels . The decreased nevirapine concentrations with 200 mg once daily dosing may suggest a lower incidence of liver toxicity than twice daily. To test this assumption, an small trial in seven treatment-naïve Chinese HIV-infected patients was carried out by our team. The seven HIV+ adults patients were administered with nevirapine 200 mg once daily as a part of initiating antiretroviral therapy. Till now, they maintained a high level of efficacy and comparable tolerability during 2-month follow up compared with the twice-daily dosing regimen.The present study showed that the median Vries-Sluijs et al.Ctrough of 3.0 µg/mL Ctrough at 3.9 µg/mL for Chinese patients with HIV infection, higher than the commonly recommended 3.0 µg/mL. The current study demonstrated that median Ctrough  and Cmax  of nevirapine in Chinese HIV-infected patients receiving 200 mg once daily were above the recommended thresholds of nevirapine concentrations, suggesting that nevirapine 200 mg once daily regimen may produce adequate viral inhibition in Chinese HIV-infected patients. The ongoing pilot trial in Chinese patients confirmed this assumption. Certainly, we cannot rule out the possibility that some patients will have nevirapine levels lower than the target thresholds due to inter-individual variability and unpredictable features, which may result in virologic treatment failure and even drug resistance. So routine therapeutic drug monitoring should be carried out.On the other hand, there are some concerns about virologic failure which was related to low nevirapine plasma levels in clinical practice. The relationship between nevirapine concentration and virologic response has been explored in previous studies. The INCAS trial v.s. 4.25 µg/mL, for ALT level≥v.s.<1.5×ULN, P = 0.045) and might exert an influence on the metabolism and clearance of nevirapine at steady state, because no significant difference was observed in median Ctrough of nevirapine between lead-in and steady-state periods in patients with baseline ALT and AST level ≥1.5×ULN , which was different from the patients with ALT/AST level <1.5×ULN. Gender, weight, HBV or HCV coinfection, baseline CD4 cell counts and viral load appeared to have no significant influence on nevirapine plasma concentrations. Consistently, the univariate and multivariate logistic regression models showed that none of the examined factors was found to predict nevirapine trough concentration at week 2 lower than 3.0 µg/mL or 3.9 µg/mL.Previous studies have found ethnicity, gender, weight and underlying hepatic disease to be predictive of nevirapine plasma concentrations v.s. 6.15 µg/mL, P = 0.004). We assumed that a high incidence of liver toxicity in Chinese patients and particularly a great risk of severe hepatotoxicity in HIV/HCV coinfected patients were both significantly associated with plasma nevirapine exposure. This finding was consistent with our previous study The nevirapine trough concentration increased gradually in HIV/HCV coinfected patients during the follow-up periods and finally was significantly higher than those patients without HCV coinfection at week 48  and serum creatinine level >1.5×ULN. Another important exclusion criterion was non-adherence to the study treatment regimen which was defined as less than 95% adherence.Patients were recruited from January 2009 to December 2010. Male and female antiretroviral-naïve patients with documented HIV-1 infection were eligible for inclusion if they were between the age of 18 and 65 years with CD4+T cell count <350 cells/mmThe cohort study was approved by institutional review boards and carried out in accordance with the Declaration of Helsinki and the principles of Good Clinical Practice. Written informed consent was obtained from each patient and the study protocol was approved by the ethics committee of Peking Union Medical College Hospital. The protocol for this trial and supporting CONSORT checklist are available as supporting information; see All the patients received a standard ART regimen based on nevirapine together with two nucleoside reverse transcriptase inhibitors including stavudine plus lamivudine or zidovudine plus lamivudine. Nevirapine  was administered at 200 mg once daily for 2 weeks and 200 mg twice daily thereafter. The bioequivalence of nevirapine in reference to Viramune® from Boehringer Ingelheim was demonstrated in the previous study During the treatment period, patients were monitored at baseline, week 2, 4, 12, 24, 36, and 48 for clinical features (particularly severe adverse events), plasma nevirapine concentrations and laboratory values including blood routine examination, hepatic and renal function, and hepatitis B (HBV) or hepatitis C (HCV) serological state.Ctrough and/or 2 h post-ingestion for Cmax. All samples were collected in spray dry powdered EDTA tubes and centrifuged in the real time to obtain plasma, which were stored at −80°C and thawed at the day of analysis. The exact time of nevirapine dose and blood sampling was recorded.Blood samples were drawn prior to the next drug administration for The nevirapine concentration in plasma was determined by a validated HPLC assay modified from a previous study Plasma HIV-1 RNA viral load was measured by either bDNA Analyzer System 340  or the COBAS Ampliprep/TaqMan 48  according to the manufacturer's instructions. The measurement ranges were 50–500,000 copies/mL and 40–1,000,000 copies/mL, respectively.PBMCs were stained with combinations of immunofluorescent monoclonal antibodies FITC-CD3 and PEcy5-CD4  followed by flow cytometer analysis  to determine the number of CD4+T cells (CD4+CD3+).Statistical analyses were performed with Statistical Product and Service Solutions for Windows . Mean , median  and frequencies (%) were used to describe characteristics of patients as appropriate. Normal distribution of values was examined by Kolmogorov-Smirnov methods. Categorical variables were tested with Chi-Square or Fisher's exact test, and continuous variables were tested with Kruskal-Wallis or student's t test or one-way ANOVA.trough at the 6 follow-up visits in the 105 patients. An independent t-test or Mann-Whitney U test was used when two groups or two variables were compared.The differences between lead-in and steady-state periods for nevirapine plasma concentrations were assessed by the Kruskal-Wallis H test. ANOVA was used to determine whether there were significant differences in nevirapine CFactors affecting nevirapine plasma exposure were estimated using univariate stratification analysis. Risk factors predicting nevirapine trough concentration at week 2 lower than the recommended cut-off thresholds were evaluated using univariate and multivariate logistic regression. Odd ratios (OR) and 95% confidence intervals (95% CI) were also obtained.For all tests, P<0.05 was considered to be statistically significant.Checklist S1CONSORT Checklist.(DOC)Click here for additional data file.Protocol S1Trial Protocol.(DOC)Click here for additional data file.


The proposed method has been applied successfully to the determination of methyl sulfonyl methane (MSM) (1), tinidazole (2), rofecoxib (3), and nimesulide (4) in pure forms, laboratory-prepared mixtures, pharmaceutical dosage forms, spiked human plasma samples, and in volunteer's blood. The method showed linearity over concentration ranging from 1 to 150 μg/mL, 10 to 1000 ng/mL, 1 to 1800 ng/mL, and 30 to 2100 ng/mL for standard solutions of 1, 2, 3, and 4, respectively, and over concentration ranging from 5 to 150 μg/mL, 10 to 1000 ng/mL, 10 to 1700 ng/mL, and 30 to 2350 ng/mL in spiked human plasma samples of 1, 2, 3, and 4, respectively. The method showed good accuracy, specificity, and precision in both laboratory-prepared mixtures and in spiked human plasma samples. The proposed method is simple, does not need sophisticated instruments, and is suitable for quality control application, bioavailability, and bioequivalency studies. Besides, its detection limits are comparable to other sophisticated chromatographic methods. A simple spectrofluorometric method has been developed, adapted, and validated for the quantitative estimation of drugs containing  Further HPLC-MS . 3) is a nonsteroidal anti-inflammatory drug (NSAID) introduced in 1998 with lots of fanfare claiming it a selective COX-2 inhibitor with minimal side effects, and 3 was withdrawn in September, 2004 because of safety concerns regarding its untoward cardiovascular side effects leading to several deaths [Rofecoxib  is a sulfonanilide analogue clinically used anti-inflammatory agent; it is not related to conventional NSAIDs, which usually present a carboxyl or hydroxyl functional group [4 in the presence of its compendia-related substances [Nimesulide  to the analysis of various compounds containing α-methylene carbonyl groups [α-adduct fluorophore [α-methylene groups adjacent to other functional groups.Nakamura and Tamura described the application of the reaction of l groups . Nakamurorophore . This reα to carbonyl functional groups. This paper formulates our continuous effort to extend the utility of NMNCl to determine different classes of drugs containing active methylene α to various groups such as cyclic ketone,  sulfoxide (PPIs), and sulfone/sulfonamide (1–4) and even to groups that would produce active methylene upon hydration, for example, methyne group, such as levonorgestrel and ethinyl estradiol (unpublished results). All previous publications based on the fluorophore produced by reaction with NMNCl described the utility of this reagent to react with drugs containing active methylene α-methylene sulfone groups.This paper describes the application and validation of the reaction of NMNCl with some drugs containing 1, 2, 3, and 4   and founμg/mL, 10 ng/mL, 1 ng/mL, and 30 ng/mL, for standard solutions and 5 μg/mL, 10 ng/mL, 10 ng/mL, and 30 ng/mL, for plasma samples of 1, 2, 3, and 4, respectively.Quantitation limits were practically determined according to the ICH topic Q2B (R1)  and founThe accuracy of the proposed method was studied according to the ICH topic Q2B (R1) , by prepThe precision of the method was judged by performing intraday and interday triplicate analyses of different concentrations covering the linearity range of each drug in spiked human plasma samples. The results are reported as S.D. and coefficient of variation (C.V.) in 1, 2, and 3 and two synthetic mixtures of 4 were prepared to contain the possible interfering substances used during pharmaceutical formulations. These mixtures were analyzed using the proposed method and the results, were expressed as % recovery ± S.D., and were as follows: 99.8% ± 3.0 for 1, 100.3% ± 2.8 for 2, 99.6% ± 3.5 for 3, and 100.7% ± 1.7 for 4.To study the specificity of the proposed method, three synthetic mixtures of All the pharmaceutical preparations available in the local market for each drug were analyzed using the proposed method. The results, expressed as % recovery ± S.D., are illustrated in 2 and 4, in spiked human plasma samples with good accuracy and precision, encouraged the investigator to study its application for monitoring the drug level in the blood of a volunteer receiving 2 or 4 therapy. The level of 2 and 4 was monitored in the blood of volunteers, and their concentrations were found to be 48 μg/mL and 35 μg/mL, respectively, that lie in the therapeutic levels of 2 (47.7 ± 7.5  μg/mL) and 4 (38 ± 10.6 μg/mL).The success in the application of the highly sensitive proposed procedure for the determination of The proposed method makes use of the high sensitivity and specificity of the fluorometric analysis to reach low limits of detection and quantitation for all the studied drugs in standard solutions, synthetic mixtures, pharmaceutical preparations, spiked human plasma samples, and patient's or volunteer's blood. The method is simple; it gives results comparable to those obtained by other techniques that require elaborate instrumentation and time-consuming sample preparation procedure.The method showed good accuracy and precision suitable for quality assurance and could be recommended for bioequivalency and bioavailability studies as well as for validation of cleaning methodology prior to line clearance during manufacture of said dosage forms.The proposed method application could be extended to cover all available pharmaceutical preparations for each of the chosen drugs.Shimadzu RF 5301 PC spectrofluorometer.1, 2, 3, and 4 working standards were supplied by Eva Pharma for Pharmaceutical Industries and Medical Appliances, Egypt, Medical Union Pharmaceuticals (MUP), Egypt, October Pharma, Egypt, and Sigma Pharmaceutical Industries, Egypt, respectively.Plasma samples were purchased from the Central Blood Bank of Tanta University Hospital.1N-Methylnicotinamide chloride was obtained from Sigma Chemicals Co. Formic acid, sodium hydroxide, methanol, and all other chemicals were of analytical grade. Water used was doubly distilled.MSM (1): MSM 1 g tablets (Eva Pharma).Tinidazole (2): Fasigyn 500 mg tablets (Pfizer) and Protozol 500 mg tablets (MUP).Rofecoxib (3): Romacox 25 mg tablets (October Pharma). Nimesulide (4): Sulide 50 mg and 100 mg tablets (Alkan Pharma), Nimalox 100 mg tablets (Sigma) and sulidan 100 mg tablets ).1, methanol for 3, and ethanol for 2 and 4 to contain 10 mg/mL, 10 mg/mL, 0.2 mg/mL, and 0.25 mg/mL for 1, 2, 3, and 4, respectively.Stock standard solutions were prepared in distilled water for 15 mg/mL, 0.1 to 10 μg/mL, 0.01 to 20 μg/mL and 0.1 to 250 μg/mL for 1, 2, 3, and 4, respectively.Aliquots of the stock solution were diluted quantitatively with the same solvent to obtain serial standard solutions in concentration ranging from 0.1 to 1 along various excipients, additives, and other nonactive ingredients commonly used in pharmaceutical formulations were prepared. The first mixture contained 1000 mg 1, 135 mg starch, 60 mg gelatin, and 8.0 mg magnesium stearate. The second mixture contained 1000 mg 1, 75 mg lactose, 30 mg starch, 60 mg gelatin, 8.0 mg magnesium stearate, and 42 mg talc. The third mixture contained 100 mg avicel instead of lactose and gelatin.Three synthetic mixtures containing 2 were prepared. The first mixture contained 500 mg 2, 200 mg cellose, 72 mg starch, 8.0 mg magnesium stearate, 8.0 mg polyethylene glycol and 1.0 mg titanium dioxide. The second mixture contained 500 mg 2 and, 100 mg lactose, 60 mg starch, 60 mg gelatin, 8.0 mg magnesium stearate and 72 mg talc. The third mixture contained 160 mg avicel instead of lactose and gelatin.Three synthetic mixtures containing 3 were prepared. The first mixture contained 25 mg 3, 100 mg croscarmellose, 83.5 mg lactose, and 8.0 mg stearate. The second mixture contained 25 mg 3, 60 mg citric acid, 80 mg sodium citrate, and 10% sorbitol solution. The third mixture contained 25 mg 3, 235 mg lactose, 60 mg gelatin, 8.0 mg magnesium stearate, and 72 mg talc.Three synthetic mixtures containing 4 along with various excipients and additives were prepared. The first mixture contained 100 mg 4, 200 mg lactose, 60 mg starch, 60 mg gelatin, 8.0 mg magnesium stearate, and 72 mg talc. The second mixture contained 260 mg avicel instead of lactose and gelatin.Two synthetic mixtures containing 1, 2, 3, or 4 was extracted with 100 mL of distilled water for 1, methanol for 2, or ethanol for 3 and 4, filtered, and the first 10 mL of the filtrate was rejected. Aliquots of the filtrate were diluted with the same solvents to obtain serial dilutions in concentrations ranging from 0.1 to 25 mg/mL, 0.1 to 10 μg/mL, 0.01 to 20 μg/mL and 0.1 to 25 μg/mL, for 1, 2, 3, and 4, respectively.Each synthetic mixture containing 1–4, was transferred with the aid of several portions of distilled water for 1, methanol for 3 or ethanol for 2, 4 to a 100 mL volumetric flask and the volume was completed with the same solvent. The resulting solution was filtered and the first 10 mL of the filtrate was rejected. Aliquots of the filtrate were diluted with the same solvents to obtain 100 μg/mL, 6 μg/mL, 5 μg/mL and 6 μg/mL solutions, for 1, 2, 3 and 4, respectively.Twenty tablets were finely powdered, a quantity of the powder, equivalent to one tablet of 1), in methanol (for 2), and in ethanol (for 3 and 4) in concentrations ranging from 1 to 150 mg/mL, 0.01 to 1 mg/mL, 0.001 to 2.0 mg/mL and 0.01 to 2.5 mg/mL of 1, 2, 3, and 4, respectively.Serial standard solutions were prepared in distilled water  were mixed with 1800 μL methanol and centrifuged for 15 minutes to separate the precipitated protein. The clear supernatant was filtered through Millipore filter (0.45 μm) to obtain solutions in concentration range of 0.01–1.5 mg/mL, 0.1–10 μg/mL, 0.01–20 μg/mL, and 0.1–25 μg/mL for 1, 2, 3, and 4, respectively.Two hundred 2 and 4 in plasma samples (cf. preparation of assay solutions of drugs in plasma samples).Blood sample was withdrawn in a test tube to which heparin was previously added and dried. The sample was centrifuged to separate plasma and then treated as previously mentioned under preparation of assay solutions of −4 N HCl. Aliquots of this solution were diluted with distilled water to obtain 1.0 mM, 2.0 × 10−1 mM, 4.0 × 10−1 mM, and 5.0 × 10−1 mM solutions.Ten mM solution NMNCl reagent was prepared by dissolving 17.262 g NMNCl in one liter of 104, the pH of the reaction product was adjusted to 10.0 before completing to volume with distilled water. The intensity of the resulting fluorescence was measured at the optimal wavelengths indicated in One milliliter of each drug standard solutions, assay solutions of synthetic mixtures, assay solutions of pharmaceutical preparations, assay solutions of plasma samples, or the assay solution of the volunteer's plasma was transferred to 10.0 mL screw-capped test tube. Solutions of sodium hydroxide and NMNCl were added. The mixture was cooled (in ice) for the indicated time, then the pH was adjusted using formic acid and heated for the indicated time and then was cooled in ice for 5 minutes  and cooling and heating times are indicated in


Corynebacterium glutamicum.Purine nucleotides exhibit various functions in cellular metabolism. Besides serving as building blocks for nucleic acid synthesis, they participate in signaling pathways and energy metabolism. Further, IMP and GMP represent industrially relevant biotechnological products used as flavor enhancing additives in food industry. Therefore, this work aimed towards the accumulation of IMP applying targeted genetic engineering of CDW-1. Deletion of the pgi gene encoding glucose 6-phosphate isomerase in combination with the deactivated AMP and GMP generating reactions, however, resulted in significantly decreased IMP pools (13 μmol gCDW-1). Targeted metabolite profiling of the purine biosynthetic pathway further revealed a metabolite shift towards the formation of the corresponding nucleobase hypoxanthine (102 μmol gCDW-1) derived from IMP degradation.Blocking of the degrading reactions towards AMP and GMP lead to a 45-fold increased intracellular IMP pool of 22 μmol gThe purine biosynthetic pathway is strongly interconnected with various parts of the central metabolism and therefore tightly controlled. However, deleting degrading reactions from IMP to AMP and GMP significantly increased intracellular IMP levels. Due to the complexity of this pathway further degradation from IMP to the corresponding nucleobase drastically increased suggesting additional targets for future strain optimization. IMP and GMP  and , were obtained from Cambridge Isotope Laboratories . All other chemicals and reagents of analytical grade were purchased from Sigma-Aldrich, Merck , Fluka  or Roth .Yeats extract, beef extract, polypeptone, casamino acids, and brain heart infusion (BHI) were purchased from Difco Laboratories . 99% enriched glucose, -1 agar. Second precultures and main cultures were grown on minimal medium (pH 7.2) containing per liter: 15.0 g glucose, 4.0 g KH2PO4, 16.0 g Na2HPO4, 500 mg MgCl2 · 6H2O, 300 mg 3,4-dihydroxibenzoic acid, 100 mg CaCl2 · 2H2O, 100 μg cyanocobalamin, 750 μg thiamine, 4 μg pyridoxal phosphate, 100 μg biotin, 400 μg calcium pantothenate, 2 μg folic acid, 400 μg nicotinic acid, 200 μg 4-aminobenzoic acid, 400 μg pyridoxine · HCl, 2 mg inositol, 10 mg FeCl2 · 4H2O, 1 mg ZnCl2, 100 μg CuCl2, 20 μg NiCl2 · 6H2O, 20 μg Na2MoO4 · 2H2O, 500 μg boric acid, 100 μg KI, 100 μg CoCl2 · 6H2O and 10 mg MnCl2 · 4H2O. For tracer studies, naturally labeled glucose was replaced by 99% 13C enriched [1-13C] or  glucose. Mutants of C. glutamicum exhibiting deletions of adenylosuccinate synthetase (purA) or IMP dehydrogenase (guaB2) were grown on minimal media additionally supplemented with 200 mg L-1 adenine sulfate or 80 mg L-1 guanine hydrochloride, due to the introduced auxotrophies.The first precultures were grown on complex media (pH 6.8) containing per liter: 10.0 g glucose, 2.5 g NaCl, 2.0 g urea, 5.0 g yeast extract, 5.0 g beef extract, 5.0 g polypeptone and 20.0 g casamino acids. For agar plates, the complex medium was supplemented with 20.0 g LC. glutamicum contained per liter: 37 g BHI, 26.4 g (NH4)2SO4 and 40 g glucose for BHI++ and 37 g BHI and 46 g sorbitol for BHIS medium.Media used for transformation experiments of 13C] glucose or  glucose for analysis of glucose 6-phosphate isomerase (pgi) deletion mutants and the volume was adjusted to 5 mL in 50 mL baffled shake flasks.Cultivations were carried out in baffled shake flasks on an orbital shaker  at 230 rpm and 30°C. First precultures were grown on complex medium (25 mL medium in 250 mL baffled flask) for 8 h and used for inoculation of the second precultures (25 mL medium in 250 mL baffled flask). Second precultures and main cultures were performed in minimal medium. Main cultivations were performed in duplicates in minimal medium using 500 mL baffled shake flasks with 50 mL medium. For tracer experiments, which were conducted under the same conditions as main cultivations, naturally labeled glucose was replaced by [1-660nm) using a photometer  which was gravimetrically correlated with the cell dry weight The correlation factor was determined as 0.414 gCDW OD660nm-1. Extracellular substrates and secreted products were analyzed after 3 min centrifugation at 16,000 g. Quantification of glucose, α-ketoglutarate and trehalose was performed in diluted supernatants by HPLC . Separation was performed using an Aminex HPX-87H Bio-Rad column , at 55°C with a constant flow rate of 0.8 mL min-1 (7 mM H2SO4 as mobile phase). Refraction index (RI) detection was used for quantification of sugars, and UV detection at 218 nm for organic acids and at 254 nm for quantification of supplemented adenine and guanine. Determination of amino acids was performed as described previously [-1. Gradient elution was performed using 5 mM ammonium formate solution at pH 5.8 (eluent A) and acetonitrile:methanol:water mixture  applying the following gradient: an initial isocratic step with 0% B (3.5 min) was followed by an increase to 30% within 10 min and to 100% within 1 min. The flushing step at 100% B was performed for 5 min. The column was reconditioned at 0% B for another 5 min.Biomass concentration was determined by measuring the optical density of the culture at 660 nm (ODeviously . Extracei.e. nucleotides, nucleosides and nucleobases, by LC-ESI-MS/MS was performed as described previously [Extraction and quantification of intracellular purine intermediates, eviously .The mass isotopomer distributions of amino acids derived from cellular protein and of trehalose secreted during cultivation were determined by mass spectrometry–gas chromatography. Sample preparation and instrument settings were performed as described previously ,51,52. Iin vivo metabolic fluxes of the C. glutamicum strains was carried out by fitting simulated mass isotopomer distributions to experimentally determined labeling patterns of amino acids derived from cellular protein and trehalose secreted during cultivation. The calculations were based on the approach of Yang et al. [C. glutamicum ΔpurA ΔguaB2 purFK348QΔpgi and C. glutamicum ΔpurA ΔguaB2, were considered likewise.Calculation of the g et al.  implemeng et al.  and implMeasured GC-MS data were corrected for naturally occurring isotopes in the corresponding amino acid fragments and derivatization reagent respectively ,56. A Mo13C-labeled cell extract as internal standard as described previously [Mean concentration values for purine intermediates were calculated from four biological replicates for each strain for two biomass concentrations and corrected using eviously . The metThe authors declare that they have no competing interests.SP, KS, EH were involved in conception and design of experiments. SP, TB, SZ performed the targeted strain engineering and cultivation experiments. SP carried out analytical quantitation supported by DAV and KS. SP, KS, DAV and EH were involved in manuscript preparation. KS and SP were involved in data analysis and interpretation. KS drafted and revised the manuscript. All authors read and approved the final manuscript.


Learning and memory systems are intimately involved in drug addiction. Previous studies suggest that galanin, a neuropeptide that binds G-protein coupled receptors, plays essential roles in the encoding of memory. In the present study, we tested the function of galnon, a galanin receptor 1 and 2 agonist, in reward-associated memory, using conditioned place preference (CPP), a widely used paradigm in drug-associated memory. Either before or following CPP-inducing morphine administration, galnon was injected at four different time points to test the effects of galanin activation on different reward-associated memory processes: 15 min before CPP training (acquisition), immediately after CPP training (consolidation), 15 min before the post-conditioning test , and multiple injection after post-tests (reconsolidation and extinction). Galnon enhanced consolidation and extinction processes of morphine-induced CPP memory, but the compound had no effect on acquisition, retrieval, or reconsolidation processes. Our findings demonstrate that a galanin receptor 1 and 2 agonist, galnon, may be used as a viable compound to treat drug addiction by facilitating memory extinction process. Drug addiction, characterized by persistent drug-seeking behaviors, is frequently conceptualized as a disorder of maladaptive memory Galnon, a galanin receptor 1 and 2 agonist, can easily penetrate the blood-brain-barrier and resist enzymatic degradation because of its low molecular weight and lipophilic properties To integrate these data, and test for a potent therapeutic for drug-addictive behavior, we more specifically studied galnon's role in the different phases of encoding reward-associated memory. Memory processes are generally divided into five phases: acquisition, consolidation, retrieval, reconsolidation, and extinction ad libitum. All mice were handled individually and sham injected intraperitoneally (i.p.) once daily for a week. After one week of habituation, 9 week old mice were used for the further experiments. The experimental protocols (Permit Number: 200910011) were approved by the Xi'an Jiaotong University Laboratory Animal Administration Committee and performed according to the Xi'an Jiaotong University Guidelines for Animal Experimentation and also conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.C57BL/6J male mice (8 weeks old) weighing 20–25 g were obtained from Beijing Vital River Laboratories. They were housed in groups of four under constant temperature (23±2°C) and maintained on a 12 hour light/dark cycle (lights on at 7 a.m.). Food and water was available Morphine hydrochloride (Mor) was purchased from Qinghai Pharmaceutical Group and dissolved in sterile 0.9% saline. Galnon  was synthesized by Shanghai Hanhong Chemical Limited Company. Galnon was initially dissolved in 100% DMSO, then diluted in 0.9% saline and briefly sonicated to obtain a uniform solution. In the final solution, the concentration of DMSO was not more than 1%, which has been previously demonstrated to not affect animal behavior The CPP chambers consisted of two identical wooden compartments  with different visual and tactile cues. White walls with a stainless-steel mesh floor and black walls with a stainless-steel bar floor  were separated by a sliding wood panel with a 5 cm×7 cm door in the center of the base. The door remained open during the test phase, but was shut during the training phase to prevent movement of mice between two compartments. Night vision equipped cameras were used to digitally record the behavior of mice; time spent in the two compartments and distance traveled was measured by Shanghai Jiliang software.pre-conditioning test (pre-test), mice are allowed to move freely between the two compartments for 15 min to determine any baseline preferences for the W or B compartment prior to morphine administration. During the training period, mice were treated once daily for six consecutive days with three cycles of alternating i.p. injections of morphine in compartment W and then saline in compartment B. Immediately following each injection, mice were immediately confined for 40 min to either compartment W or B compartment for morphine or saline injections, respectively. In the control group, saline was given every training day, irrespective of compartment. The post-conditioning test (post-test) was given 24 h following all three training cycles. Like the pre-test, the door separating the B and W compartments was left open and mice freely moved 15 min in the post-test. All compartments were cleaned and wiped dry between animal runs. The counterbalanced design for day of treatment was used: half of the mice were injected morphine before they were placed in white walls with a stainless-steel mesh floor compartment, and half received saline prior to placement in the black walls with a stainless-steel bar floor compartment in the same day. The time spent in the white compartment  during the post-conditioning test minus the time in the CS+ during the pre-conditioning test, assigned as “Post CS+ minus Pre CS+”, was used as an index of preference The CPP paradigm is divided into three phases. In the Locomotor activity was measured as the total distance traveled in a separate chamber (43×43×43 cm) for 1 h using the automated Smart system .Before testing the effect of galnon on the acquisition of CPP memories, two possibilities must be excluded: 1.) high doses galnon affect locomotor activity of mice; 2.) galnon itself might induce CPP, independent of morphine administration. To test the former, two doses of galnon  were injected 15 min before mice were placed into the locomotor chambers for 1 hr. Automated systems measured the total distance traveled. The dose that did not affect total distance traveled was used to test the effects of galanin activation on CPP and CPA formation. Much like the morphine experiments, this control study was performed by confining animals to a chamber for 40 min following galnon injection during training days.To more directly test if galanin activation affects the acquisition of morphine-induced CPP, galnon or saline was administered 15 min before morphine or saline was injected on each of the six training days  . FollowiTo test if galnon affects the consolidation of morphine-induced CPP, different groups of mice received saline or different doses of galnon  immediately after each training day . A post-The studies in experiment 1 tested if 10 mg/kg galnon acutely affected locomotion 15 min after the compound was injected. Here, the distances traveled were recorded twice in morphine-galnon5, morphine-galnon10 and morphine-saline group (control group) to assess how prior galnon treatment can affect activity as measured 24 h later; two tests were performed immediately after injection of saline for 40 min in compartment B  .To test if galanin activation affected retrieval of morphine-induced CPP, galnon (5 mg/kg) or saline was given 15 min before the post-test that followed the third completed training cycle .Two doses of galnon (5 and 10 mg/kg) were used in these studies, which tested if galanin activation influenced reconsolidation and extinction of morphine-induced CPP memories. When all the training cycles were completed, daily post-conditioning tests, which are similar to the pre-test , were performed with galnon or saline injected immediately following each post-test . These pOne-way analysis of variance (ANOVA) was performed in retrieval experiment and the experiments to determine the effect of galnon on locomotor activity and whether galnon itself had CPP or CPA effects . Two-way = 5.395, p = 0.011; saline (n = 10) vs 5 mg-galnon (n = 9), p = 0.696; saline vs 10 mg-galnon (n = 10), p = 0.005). As such, we chose 5 mg/kg galnon as a suitable dose to test if galanin activation itself influences the proper expression of CPP. In the absence of morphine, expression of CPP in animals injected with 5 mg/kg galnon or saline did not differ  = 5.395, p<0.001; 5 mg-galnon (n = 8) vs saline (n = 8), p = 0.908), which also suggested that galnon itself did not have CPP or CPA effects. However, the degree of CPP induced by morphine was significantly different than that in animals injected with either galnon or saline , p<0.001; saline vs 5 mg-morphine, p = 0.001; As shown in  = 6.450, p = 0.006; test day: F = 2.888, p = 0.066; treatment × test day: F = 5.710, p = 0.001. Post hoc analysis: Post-test 2: F = 9.468, p = 0.001; saline-saline (n = 8) vs all others p<0.01; and Post-test 3: F = 5.111, p = 0.015; saline-saline vs others all p<0.05). Moreover, 5 mg/kg galnon did not significantly affect morphine-induced CPP effect  vs morphine-saline (n = 8), p = 0.197; Post-test 3: morphine-saline vs morphine-galnon5, p = 0.470). Collectively, these results demonstrate that galnon has no effect on the acquisition of morphine-induced CPP.To test if galanin activation affects acquisition of morphine-induced CPP memory, galnon was administered 15 min before each training day's morphine or saline injection . Fig. 1D = 4.118, p = 0.007; test day: F = 5.582, p = 0.007; treatment × test day: F = 2.214, p = 0.035. Post hoc analysis: Post-test 1: F = 3.034, p = 0.029; saline-saline (n = 8) vs morphine-saline (n = 9), morphine-galnon0.5, morphine-galnon5, morphine-galnon10, p = 0.870, 0.083, 0.023, 0.039; Galnon was administered at three doses  immediately following training episodes to test if galanin activation influences the consolidation phase of morphine-induced CPP. All mice underwent three cycles of conditioning to specific chambers with morphine and saline . Two dif = 3.056, p = 0.028; saline-saline vs morphine-saline, p = 0.021. Post-test 3: F = 4.260, p = 0.006; saline-saline vs morphine-saline, p = 0.044;  = 0.031, p = 0.970; treatment × test day: F = 2.392, p = 0.110; Similar to the results in experiment 1 see , the CPPAs such, galnon promotes the consolidation process of morphine-induced CPP without interfering with underlying locomotor function. = 7.784, p = 0.002; morphine-galnon5 (n = 12) vs morphine-saline (n = 10), p = 0.260), and all morphine treatment groups expressed the CPP effect  vs morphine-galnon5, morphine-saline, p = 0.001, 0.017).To test if galanin activation affects the retrieval of CPP memory, animals were injected with 5 mg/kg galnon 15 min before the post-test that followed the third completed training cycle . As illu = 6.658, p = 0.005; test day: F = 3.571, p<0.001; treatment × test day: F = 1.542, p = 0.046. Post hoc analysis: Post-test 10: F = 3.726, p = 0.038; saline-saline(n = 10) vs morphine-galnon5 (n = 9), morphine-saline (n = 10), p = 0.602, 0.015; and Post-test 11: F = 2.976, p = 0.069). This data suggests that galanin also promotes the extinction of morphine-induced CPP.As shown in  = 5.481, p = 0.010; saline-saline vs morphine-galnon5, morphine-saline, p = 0.003, 0.045). This rescue was not transient, as it persisted through Post-test 14 and Post-test 15  = 3.771, p = 0.036; saline-saline vs morphine-galnon5, morphine-saline, p = 0.014, 0.048. Post-test 15: F = 3.464, p = 0.046; saline-saline vs morphine-galnon5, morphine-saline, p = 0.035, 0.036). Additionally, this rescue appeared to require morphine as it was not present on the day prior, Post-test 12, when saline was injected immediately prior to the post-test  = 3.442, p = 0.048; saline-saline vs morphine-galnon5, morphine-saline, p = 0.102, 0.016).Immediately prior to Post-test 13, 3 mg/kg of morphine was injected to test if extinction of CPP could be reinstated; morphine induced a robust and immediate rescue of CPP  = 6.526, p = 0.005; test day: F = 4.209, p<0.001; treatment × test day: F = 2.247, p = 0.001. Post hoc analysis: Post-test 8: F = 1.251, p = 0.302; and Post-test-9: F = 3.709, p = 0.038; saline-saline vs morphine-galnon10 (n = 10), morphine-saline, p = 0.235, 0.011). CPP was also reinstated by 3 mg/kg morphine; however, this CPP rescue with morphine did not persist in the days following repriming  = 2.706, p = 0.145; saline-saline vs morphine-galnon10, morphine-saline, p = 0.306, 0.044. post-test-11: F = 6.862, p = 0.004; saline-saline vs morphine-galnon10, morphine-saline, p = 0.001, 0.047. Post-test 12: F = 3.848, p = 0.034; saline-saline vs morphine-galnon10, morphine-saline, p = 0.363, 0.011. Post-test 13: F = 4.874, p = 0.016; saline-saline vs morphine-galnon10, morphine-saline, p = 0.131, 0.004).With higher doses of galnon (10 mg/kg), CPP was extinguished two days earlier than the 5 mg/kg group  = 3.233, p = 0.089; treatment × test day: F = 0.317, p = 0.581).As shown in The present study examined the effect of galnon, a galanin receptor 1 and 2 agonist, on morphine-induced reward memory using the CPP paradigm. Our results directly show that galnon enhances the consolidation process of morphine-induced CPP without affecting either the acquisition or retrieval processes.Experiment 1: Acquisition”, and got a similar conclusion–galnon can not influence the acquisition process of CPP. One possibility is that galnon may partially counteract the effects of the addictive drug in a protocol that did not inject galnon before saline conditioning; if galnon was also given before saline conditioning, as it is the case in the present work, then galnon similarly affected both training processes. In addition, the galanin receptor 1 exhibits mostly opposite behavioral effects as compared to the galanin receptor 2 receptor Many studies have demonstrated that the galanin system mediates learning and memory processes. For example, galanin is over-expressed in Alzheimer's disease, administration of galanin interferes with proper consolidation of several learning and memory tasks, and galanin transgenic mice exhibit marked deficits in the Morris water maze  However, for the reconsolidation process, there is a possibility that post-test trial employed in experiment 4 is not strong enough to reactivate the original memory and thus reconsolidation fails. For examples, some investigators demonstrated that an established morphine-induced CPP could be interrupted by anisomycin or cycloheximide (protein synthesis inhibitors) when animals experience the conditioned context and the drug, rather than only after contextual recall Based on the above analysis, we conclude that galnon has no effect on the reconsolidation process but does facilitate the extinction process of morphine-induced CPP. Because only one extinction training paradigm was used in our experiment, more research is needed to confirm the current positive findings using other training methods of extinction, such as longer training procedure (similar to acquisition training, but mice only received saline In summary, our work illustrates that galnon, a galanin receptor 1 and 2 agonist, enhances consolidation and extinction of morphine-induced CPP memory. Curiously, however, galnon had no effect on acquisition, retrieval, or reconsolidation processes. The facilitated effect of galnon on extinction may be of potential value in the treatment of drug addiction.