Protein Kinase C Controls the Excitability of Cortical Pyramidal Neurons by Regulating Kv2.2 Channel Activity.

The family of voltage-gated potassium Kv2 channels consists of the Kv2.1 and Kv2.2 subtypes. Kv2.1 is constitutively highly phosphorylated in neurons and its function relies on its phosphorylation state. Whether the function of Kv2.2 is also dependent on its phosphorylation state remains unknown. Here, we investigated whether Kv2.2 channels can be phosphorylated by protein kinase C (PKC) and examined the effects of PKC-induced phosphorylation on their activity and function. Activation of PKC inhibited Kv2.2 currents and altered their steady-state activation in HEK293 cells. Point mutations and specific antibodies against phosphorylated S481 or S488 demonstrated the importance of these residues for the PKC-dependent modulation of Kv2.2. In layer II pyramidal neurons in cortical slices, activation of PKC similarly regulated native Kv2.2 channels and simultaneously reduced the frequency of action potentials. In conclusion, this study provides the first evidence to our knowledge that PKC-induced phosphorylation of the Kv2.2 channel controls the excitability of cortical pyramidal neurons.

Neuritin promotes neurite and spine growth in rat cerebellar granule cells via L-type calcium channel-mediated calcium influx.

Neuritin is a neurotrophic factor that is activated by neural activity and neurotrophins. Its major function is to promote neurite growth and branching; however, the underlying mechanisms are not fully understood. To address this issue, this study investigated the effects of neuritin on neurite and spine growth and intracellular Ca2+ concentration in rat cerebellar granule neurons (CGNs). Incubation of CGNs for 24 h with neuritin increased neurite length and spine density; this effect was mimicked by insulin and abolished by inhibiting insulin receptor (IR) or mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (ERK) activity. Calcium imaging and western blot analysis revealed that neuritin enhanced the increase in intracellular Ca2+ level induced by high K+ , and stimulated the cell surface expression of CaV 1.2 and CaV 1.3 α subunits of the L-type calcium channel, which was suppressed by inhibition of IR or mitogen-activated protein kinase kinase/ERK. Treatment with inhibitors of L-type calcium channels, calmodulin, and calcineurin (CaN) abrogated the effects of neuritin on neurite length and spine density. A similar result was obtained by silencing nuclear factor of activated T cells c4, which is known to be activated by neuritin in CGNs. These results indicate that IR and ERK signaling as well as the Ca2+ /CaN/nuclear factor of activated T cells c4 axis mediate the effects of neuritin on neurite and spine growth in CGNs. OPEN PRACTICES: Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/ Cover Image for this issue: doi: 10.1111/jnc.14195.

Functions and the related signaling pathways of the neurotrophic factor neuritin.

Neuritin is a member of the neurotrophic factor family, which is activated by neural activity and neurotrophins, and promotes neurite growth and branching. It has shown to play an important role in neuronal plasticity and regeneration. It is also involved in other biological processes such as angiogenesis, tumorigenesis and immunomodulation. Thus far, however, the primary mechanisms of neuritin, including whether or not it acts through a receptor or which downstream signals might be activated following binding, are not fully understood. Recent evidence suggests that neuritin may be a potential therapeutic target in several neurodegenerative diseases. This review focuses on the recent advances in studies regarding the newly identified functions of neuritin and the signaling pathways related to these functions. We also discuss current hot topics and difficulties in neuritin research.

Neuritin Enhances Synaptic Transmission in Medial Prefrontal Cortex in Mice by Increasing CaV3.3 Surface Expression.

Neuritin is a neurotrophic factor involved in neural development and synaptic plasticity. However, its role in modulating synaptic transmission remains unclear. Here, we investigated the effects of neuritin on miniature excitatory postsynaptic currents (mEPSCs) and glutamate release in the medial prefrontal cortex (mPFC) in mice. Incubation of mPFC slices with neuritin for 45 min significantly increased mEPSC frequency and glutamate release as measured by high-performance liquid chromatography, which was mimicked by insulin and abrogated by an insulin receptor (IR) inhibitor. Neuritin-induced upregulation of synaptic transmission was correlated with activation of ERK, and inhibition of mitogen-activated protein kinases/extracellular signal-regulated kinases (MEK/ERK) activity attenuated the neuritin-induced increase in mEPSC frequency and glutamate release. T-type calcium channel inhibitors but not the L-type inhibitor abolished the inward calcium current and the effects of neuritin on mEPSC frequency and glutamate release. Western blotting of membrane proteins showed that neuritin promoted surface expression of CaV3.3 α-subunit, which was also eliminated by inhibition of IR or MEK/ERK activity. The effects of neuritin on mEPSC frequency, glutamate release, and CaV3.3 α-subunit expression were inhibited by an intracellular protein-transport inhibitor. These results confirm involvement of the IR and ERK signaling pathway, and provide novel insights into the mechanisms of neuritin function in synaptic transmission.

Effect of 1.8 GHz radiofrequency electromagnetic radiation on novel object associative recognition memory in mice.

Mounting evidence suggests that exposure to radiofrequency electromagnetic radiation (RF-EMR) can influence learning and memory in rodents. In this study, we examined the effects of single exposure to 1.8 GHz RF-EMR for 30 min on subsequent recognition memory in mice, using the novel object recognition task (NORT). RF-EMR exposure at an intensity of >2.2 W/kg specific absorption rate (SAR) power density induced a significant density-dependent increase in NORT index with no corresponding changes in spontaneous locomotor activity. RF-EMR exposure increased dendritic-spine density and length in hippocampal and prefrontal cortical neurons, as shown by Golgi staining. Whole-cell recordings in acute hippocampal and medial prefrontal cortical slices showed that RF-EMR exposure significantly altered the resting membrane potential and action potential frequency, and reduced the action potential half-width, threshold, and onset delay in pyramidal neurons. These results demonstrate that exposure to 1.8 GHz RF-EMR for 30 min can significantly increase recognition memory in mice, and can change dendritic-spine morphology and neuronal excitability in the hippocampus and prefrontal cortex. The SAR in this study (3.3 W/kg) was outside the range encountered in normal daily life, and its relevance as a potential therapeutic approach for disorders associated with recognition memory deficits remains to be clarified.

Growth differentiation factor-15 promotes glutamate release in medial prefrontal cortex of mice through upregulation of T-type calcium channels.

Growth differentiation factor-15 (GDF-15) has been implicated in ischemic brain injury and synapse development, but its involvement in modulating neuronal excitability and synaptic transmission remain poorly understood. In this study, we investigated the effects of GDF-15 on non-evoked miniature excitatory post-synaptic currents (mEPSCs) and neurotransmitter release in the medial prefrontal cortex (mPFC) in mice. Incubation of mPFC slices with GDF-15 for 60 min significantly increased the frequency of mEPSCs without effect on their amplitude. GDF-15 also significantly elevated presynaptic glutamate release, as shown by HPLC. These effects were blocked by dual TGF-ß type I receptor (TßRI) and TGF-ß type II receptor (TßRII) antagonists, but not by a TßRI antagonist alone. Meanwhile, GDF-15 enhanced pERK level, and inhibition of MAPK/ERK activity attenuated the GDF-15-induced increases in mEPSC and glutamate release. Blocking T-type calcium channels reduced the GDF-15 induced up-regulation of synaptic transmission. Membrane-protein extraction and use of an intracellular protein-transport inhibitor showed that GDF-15 promoted CaV3.1 and CaV3.3 α-subunit expression by trafficking to the membrane. These results confirm previous findings in cerebellar granule neurons, in which GDF-15 induces its neurobiological effects via TßRII and activation of the ERK pathway, providing novel insights into the mechanism of GDF-15 function in cortical neurons.

GDF-15 enhances intracellular Ca2+ by increasing Cav1.3 expression in rat cerebellar granule neurons.

GDF-15 (growth/differentiation factor 15) is a novel member of the TGF (transforming growth factor)-ß superfamily that has critical roles in the central and peripheral nervous systems. We reported previously that GDF-15 increased delayed rectifier outward K(+) currents and Kv2.1 α subunit expression through TßRII (TGF-ß receptor II) to activate Src kinase and Akt/mTOR (mammalian target of rapamycin) signalling in rat CGNs (cerebellar granule neurons). In the present study, we found that treatment of CGNs with GDF-15 for 24 h increased the intracellular Ca(2+) concentration ([Ca(2+)]i) in response to membrane depolarization, as determined by Ca(2+) imaging. Whole-cell current recordings indicated that GDF-15 increased the inward Ca(2+) current (ICa) without altering steady-state activation of Ca(2+) channels. Treatment with nifedipine, an inhibitor of L-type Ca(2+) channels, abrogated GDF-15-induced increases in [Ca(2+)]i and ICa The GDF-15-induced increase in ICa was mediated via up-regulation of the Cav1.3 α subunit, which was attenuated by inhibiting Akt/mTOR and ERK (extracellular-signal-regulated kinase) pathways and by pharmacological inhibition of Src-mediated TßRII phosphorylation. Given that Cav1.3 is not only a channel for Ca(2+) influx, but also a transcriptional regulator, our data confirm that GDF-15 induces protein expression via TßRII and activation of a non-Smad pathway, and provide novel insight into the mechanism of GDF-15 function in neurons.

Neuritin reverses deficits in murine novel object associative recognition memory caused by exposure to extremely low-frequency (50 Hz) electromagnetic fields.

Animal studies have shown that electromagnetic field exposure may interfere with the activity of brain cells, thereby generating behavioral and cognitive disturbances. However, the underlying mechanisms and possible preventions are still unknown. In this study, we used a mouse model to examine the effects of exposure to extremely low-frequency (50 Hz) electromagnetic fields (ELF MFs) on a recognition memory task and morphological changes of hippocampal neurons. The data showed that ELF MFs exposure (1 mT, 12 h/day) induced a time-dependent deficit in novel object associative recognition memory and also decreased hippocampal dendritic spine density. This effect was observed without corresponding changes in spontaneous locomotor activity and was transient, which has only been seen after exposing mice to ELF MFs for 7-10 days. The over-expression of hippocampal neuritin, an activity-dependent neurotrophic factor, using an adeno-associated virus (AAV) vector significantly increased the neuritin level and dendritic spine density. This increase was paralleled with ELF MFs exposure-induced deficits in recognition memory and reductions of dendritic spine density. Collectively, our study provides evidence for the association between ELF MFs exposure, impairment of recognition memory, and resulting changes in hippocampal dendritic spine density. Neuritin prevented this ELF MFs-exposure-induced effect by increasing the hippocampal spine density.

[Methodology research and preliminary assessment of Mycobacterium tuberculosis detection by immunomagnetic beads combined with functionalized fluorescent quantum dots].

OBJECTIVE: To establish a detection method for Mycobacterium tuberculosis (MTB) by immunomagnetic beads combined with functionalized fluorescent quantum dots technology, and to investigate the optimal test condition and the diagnostic value of this method. METHODS: MTB standard strain H37Rv was used as detection object. Nanobeads and quantum dots were prepared by using wet chemical method, and conjugated separately with MTB binding peptide H8 to obtain immunomagnetic beads and functionalized fluorescent quantum dots, which could react with H37Rv simultaneously and form a ternary complex structure. Based on measurement of the fluorescence value and observation under fluorescence microscopy to determine if MTB existed in the sample, a new detection method of MTB using nanotechnology was established. The optimal detection concentration and reaction time of immunomagnetic beads and quantum dots were investigated, and the detection limit and specificity of this detection method were evaluated by using bacterial suspension and simulation sputum samples. RESULTS: By fluorescence microscopy examination, it was found that conjugated immunomagnetic beads and functionalized fluorescent quantum dots both bound with H37Rv and formed the ternary complex structure. The fluorescent value ratio of the experimental group and the control group could be 4:1. The best detection concentration of immunomagnetic beads and functionalized fluorescent quantum dots was 100 mg/L and the optimal incubation time was 2 h. The detection limit of H37Rv bacterial suspension and simulation sputum sample were both 10(3) CFU/ml. The detection results for 3 non-mycobacteria were all negative, while for the 12 types of NTM, only Mycobacterium parafortuitum, Mycobacterium aurum, Mycobacterium smegmatis and Mycobacterium fortuitum were positive, and others were all negative. CONCLUSION: The detection method of immunomagnetic beads combined with fluorescence quantum dots can be a new detection method for MTB, but the clinical value needs to be evaluated further.

[Cross-resistance between rifampin and rifabutin in multidrug resistant Mycobacterium tuberculosis complex strains].

OBJECTIVE: To study the cross-resistance between rifampin and rifabutin in multidrug resistant Mycobacterium tuberculosis complex strains, and therefore to provide laboratory data for using rifabutin in the treatment of multidrug resistant tuberculosis. METHODS: The MIC(90) of rifabutin and rifampin against 99 multidrug resistant Mycobacterium tuberculosis clinical strains were determined by microplate assays. Statistical analysis was performed by using the χ(2) test and the t test. RESULTS: The cross-resistance rate between rifampicin and rifabutin was 85.9% (85/99), but the MIC(90) of rifabutin (≤ 16 mg/L, median 2 mg/L) was significantly lower than that of rifampicin (≥ 2 mg/L, median > 32 mg/L). The cross-resistance rate increased with the resistance level of rifampicin. The cross-resistance strains in the lower and the medium groups were 0/9 and 5/9 respectively, while the strains of the high rifampicin-resistant group were almost all cross-resistant (98.8%, 80/81). CONCLUSION: Rifabutin had activities against rifampin resistant Mycobacterium tuberculosis complex strains in vitro, and therefore may be used as an alternative for the treatment of multidrug resistant tuberculosis.

[Identification and evaluation of a new nucleic acid amplification test target for specific detection of Mycobacterium tuberculosis complex].

OBJECTIVE: To identify and evaluate a new nucleic acid amplification (NAA) test target for specific detection of Mycobacterium tuberculosis (MTB) complex (MTC). METHODS: MTC-specific fragment was obtained by ISSR genotyping technology. Primer pairs were designed based on the sequences of MTC-specific fragment and tested in 211 mycobacterial strains including 107 MTC strains and 104 nontuberculous mycobacteria (NTM) strains. IS6110 element (specific identification of MTC strains) and 16s rRNA gene (specific identification of Mycobacterium) amplification were used as a control to evaluate the efficacy of the NAA test target in the detection of MTC strains. RESULTS: One MTC-specific fragment with the length of 588 bp, located in 315947 - 316534 of the genome from MTB reference strain H(37) Rv, were obtained, cloned and sequenced. MTC-specific primer pairs MTCF/R were designed based on these sequences. All 211 mycobacterial strains accurately produced the genus-specific 16s rRNA amplicon. All MTC strains were positive in the MTCF/R PCR amplification while 99% MTC strains (106/107) were positive in the amplification of IS6110 sequences. All NTM strains were negative in both IS6110 and MTCF/R PCR amplification. CONCLUSIONS: The MTC-specific fragment developed in this study can be used as a new NAA test target to correctly distinguish MTC from NTM.

[In vitro activities of clofazimine against different drug-resistant types of Mycobacterium tuberculosis].

OBJECTIVE: To study the in vitro antituberculous activities of clofazimine (CLF) to different drug-resistant types of Mycobacterium tuberculosis. METHODS: The minimal inhibitory concentration (MIC) of CLF and isoniazid (INH), rifampicin (RFP), ofloxacin (OFLX), amikacin (AK), and capreomycin (CPM) against sensitive, single-drug resistant (SDR), poly-drug resistant (PDR), multi-drug resistant (MDR), and extensive-drug resistant (XDR) Mycobacterium tuberculosis strains isolated clinically were determined by microplate assays. RESULTS: The MICs of CLF for sensitive, SDR, PDR, MDR and XDR strains of clinically isolated Mycobacterium tuberculosis were 0.06 - 4.00 mg/L, 0.03 - 4.00 mg/L, 0.06 - 8.00 mg/L, 0.06 - 8.00 mg/L, 0.03 - 8.00 mg/L. For the sensitive group, the MIC of CLF (0.06 - 4.00 mg/L) was higher than that of INH (0.06 - 0.25 mg/L) and RFP (0.06 - 0.25 mg/L), while there was no significant difference among OFLX (0.06 - 2.00 mg/L), AK (0.06 - 4.00 mg/L), and CPM (0.50 - 4.00 mg/L). For the single-drug resistant group, there was no significant difference among CLF (0.03 - 4.00 mg/L), INH (0.06 - 0.25 mg/L), and RFP (0.06 - 0.25 mg/L), but the MIC of CLF was lower than that of OFLX (0.25 - 8.00 mg/L), AK (0.06 - 4.00 mg/L), and CPM (0.50 - 8.00 mg/L). For the MDR group, there was no significant difference between CLF (0.06 - 8.00 mg/L) and AK (0.25 - 8.00 mg/L), but the MIC of CLF was lower than that of OFLX (0.125 - 8.00 mg/L) and CPM (0.50 - 8.00 mg/L). For the XDR group, the MIC of CLF (0.03 - 8.00 mg/L) was lower than that of others. CONCLUSION: CLF showed good in vitro activity against Mycobacterium tuberculosis, especially MDR and XDR strains.

[Screening of the binding peptides of Mycobacterium tuberculosis H(37)Rv].

OBJECTIVE: To screen the Mycobacterium tuberculosis H(37)Rv binding peptide using phage-displayed random peptide libraries, and to analyze the binding capacity of the peptide with Mycobacterium tuberculosis. METHODS: Inactive Mycobacterium tuberculosis H(37)Rv was used for screening of the binding peptide from the Ph.D.-7 peptide library, and Mycobacterium smegmatis was used for reverse screening during the 2(nd) to 4(th) rounds of screening. After 4 rounds of screening, single phages were randomly selected for DNA sequencing. The selected clones were tested by indirect enzyme linked immunosorbent assay (ELISA). The peptide of positive clone, which showed the highest affinity, was synthesized in vitro with fluorescent markers. The specific combination of the peptide with 16 mycobacterium standard strains and 3 other microbes (Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans) were observed by fluorescence microscopy. RESULTS: After 4 rounds of biopanning, remarkable enrichment of the phages that specifically bind with target molecules were observed. Single phages were randomly selected for sequencing analysis and 5 sequences were obtained. Five phages with different sequences were detected using indirect ELISA and all of them were found to be positive clones. Phage 8 showed the highest affinity with target molecule. The peptide of phage H8 was synthesized in vitro with fluorescent markers, and it was confirmed that the peptide could bind with H(37)Rv and other 15 mycobacterium including Mycobacterium smegmatis, but not with 3 other microbes. CONCLUSIONS: By using phage-displayed random peptide libraries, we obtained the binding peptide of H(37)Rv. It was shown that the peptide could bind with Mycobacterium tuberculosis specifically, which provided a new way for the detection of Mycobacterium tuberculosis in vitro.

[Fast identification of mycobacteria in microtiter liquid culture].

OBJECTIVE: This research was to establish a method for fast identification of mycobacteria in microtiter liquid culture and to evaluate its clinical value. METHODS: 2-thiophenecarboxylic acid hydrazide (TCH) and paranitrobenzoic acid (PNB) at different concentrations were added into liquid culture in 96-well plate. Different mycobacterium standard strains were incubated in liquid culture with PNB and TCH for 7 to 10 days. According to the growth assay for 15 mycobacterium strains and 30 mycobacterium tuberculosis strains, the best PNB and TCH concentration were determined. A total of 424 clinical mycobacterium isolates were identified by microtiter liquid culture at the best PNB and TCH concentration. The results of microtiter liquid culture were compared with those of PCR and DNA sequencing. RESULTS: The best concentration of PNB was 200 µg/ml in microtiter liquid culture. Compared with the results of PCR, the sensitivity and specificity for identification of mycobacterium tuberculosis complex in microtiter liquid culture were 97.8% (306/313) and 100.0% (107/107) respectively and those for non-tuberculosis mycobacteria in microtiter liquid culture were 100.0% (107/107) and 96.5% (306/317) respectively. The best concentration of TCH was 0.5 µg/ml. Compared with the results of PCR, the sensitivity of mycobacterium tuberculosis in microtiter liquid culture was 100.0% (305/305). The specificity remained under and more studies were needed. CONCLUSION: In microtiter liquid culture with PNB and TCH, mycobacteria can be identified in 7 to 10 days. The results were accurate and the process was simple without expensive equipments. This method meets clinical needs and can be used in all level hospitals in China.

[Evaluation of microscopic observation drug susceptibility for drug susceptibility testing of mycobacterium tuberculosis in smear-positive sputum].

OBJECTIVE: To evaluate microscopic observation drug susceptibility (MODS) for mycobacterium tuberculosis drug susceptibility in smear-positive sputum. METHODS: Drug susceptibility of mycobacterium tuberculosis in 275 smear-positive sputum samples collected from TB patients were detected directly by MODS. The susceptibility of seven antimicrobials including streptomycin, isoniazid, rifampicin, ethambutol, levofloxacin, amikacin and capromycin were detected MODS. At the same time the sputum sample were cultured in MGIT 960 tube and the positive isolates were tested for drug susceptibility by MGIT 960 system. The results of MODS were analyzed and compared with that of MGIT 960. RESULTS: Of 275 smear-positive sputum, MODS detected 235 (85.45%). Results of MODS were obtained in a median time of 18 days (5 - 39 d). For the 235 MODS-positive samples, the compliance rates of MODS to MGIT of 7 drugs were 90.21% (212/235), 88.09% (207/235), 93.62% (220/235), 87.23% (205/235), 92.34% (217/235), 88.51% (208/235) and 86.81% (204/235) respectively. The sensitivity of MODS method were 83.33% (90/108), 85.11% (120/141), 90.74% (98/108), 85.71% (78/91), 86.73% (85/98), 76.92% (40/52) and 77.08% (37/48). The specificities of MODS method were 96.06% (122/127), 92.55% (87/94), 96.06% (122/127), 88.19% (127/144), 96.35% (132/137), 91.80% (168/183) and 89.30% (167/187) respectively. CONCLUSION: MODS is an optimal alternative method for direct and rapid drug susceptibility of sputum with high accuracy in a timely and affordable way in resource-limited settings.

[The effects of gene mutation related to drug resistance and drug efflux pump in extensively drug-resistant tuberculosis clinical isolates].

OBJECTIVE: To explore the effects of 2 major drug-resistant mechanisms in clinically isolated strains of extensively drug-resistant tuberculosis (XDR-MTB). METHODS: Genomic DNA of 10 XDR-MTB strains isolated from Shanghai Pulmonary Hospital were extracted. The main gene mutations related to drug resistance and 15 SNPs unique to XDR-MTB clinical isolate KZN605 reported by the Broad Institute in USA were detected by sequencing. The changes of minimal inhibition concentration (MIC) of XDR-MTB isolates were detected before and after the addition of efflux pump inhibitors verapamil, CCCP and reserpine in liquid cultures. RESULTS: The mutation of rpoB, katG and rpsL occurred in all XDR-MTB strains. The mutation of gyrA, gyrB and rrs occurred in 9 strains, 2 strains and 6 strains respectively. There was no mutation of tlyA in all the strains. Most of the SNPs in KZN 605 strains were not detected in the clinical strains. The clinical strains showed no significant changes of MICs, except 1 strain for which the MIC of ofloxacin decreased by 16 times after addition of the efflux pump inhibitors. CONCLUSIONS: The gene mutations related to drug resistance are the key mechanism for the clinical XDR-MTB strains, while the efflux pumps partly play a role in the drug resistance to fluoroquinolones. The detailed mechanism of efflux pump mediated drug resistance to other anti-TB drugs needs further study.

[Selecting and affinity analysis of DNA aptamers to MPT64 protein from Mycobacterium tuberculosis].

OBJECTIVE: To obtain DNA oligonucleotide aptamers which can specifically bind to MPT64 protein from Mycobacterium tuberculosis by SELEX technology. METHODS: An in vitro synthesized 78 per random DNA library was subjected to 12 rounds of selection by SELEX (Systematic evolution of ligands by exponential enrichment) method against MPT64 protein. Binding of the aptamers to the protein was examined by biotin-streptavidin-horseradish peroxidase system. DNAMAN package was employed to analyze the sequences and the second structures of the aptamers. Moreover, target protein was bound to one aptamer and another aptamer modified with biotin together forming a sandwich-like complex, which was captured in microwell, to be tested in negative group including BCG and reference strains from nontuberculous mycobacteria, and positive group including H37Rv, Mycobacterium bovis reference strain, and clinical strains from Mycobacterium tuberculosis. RESULTS: After 12 rounds of selection, high-affinity aptamers to MPT64 was obtained. The OD value at 450 nm of affinity of aptamers to MPT64 protein was from 0. 492 to 1.243, in which 73.3% was over 1.0. Pocket and stem-loops was the basis of aptamers binding to MPT64 protein by the analysis of structures,with several GC pairs among bridges between pocket and stem-loops. The analysis of the sandwich-like complex system based on two aptamers and protein showed that the positive percentage was 87. 9% in the positive group while the negative percentage was 85.7% in the negative group, with positive H37Rv and Mycobacterium bovis, and negative BCG, when the cut-off value for a positive response was 0.61 OD. CONCLUSION: A set of aptamers with considerable binding affinity to MPT64 protein were successfully selected from the initial random DNA library.

[Evaluation of pyrosequencing for the detection of rpoB gene mutation in Mycobacterium tuberculosis].

OBJECTIVE: To detect the mutations of rpoB gene in Mycobacterium tuberculosis by pyrosequencing and to evaluate the values on detection of rifampin resistance in clinical isolates. METHODS: Using the new technology of pyrosequencing, the mutations in the rifampin resistance determining region (RRDR) of rpoB gene were analyzed. The results were compared with those obtained from methods of the absolute concentration and the minimum inhibitory concentration (MIC). RESULTS: Among the 150 Mycobacterium tuberculosis clinical isolates, 84 were susceptible and 66 resistant to RIF. 54 of the 66 resistant isolates were multidrug-resistant (MDR) strains. Ser531Leu and His526Asp or Tyr, including twelve different genotypes and six codons, were the most common mutations. In the drug susceptibility testing, the accordance rates of the pyrosequencing and the absolute concentration method as well as MIC were 92.7% and 97.8% respectively. CONCLUSION: Not only is the pyrosequencing technology a fast, sensitive and high throughput method in detecting rifampin resistance in Mycobacterium tuberculosis, but also a useful tool in the research of rifampin resistance mechanism.