Detection of Isoniazid-, Fluoroquinolone-, Amikacin-, and Kanamycin-Resistant Tuberculosis in an Automated, Multiplexed 10-Color Assay Suitable for Point-of-Care Use.

Extensively drug-resistant (XDR) tuberculosis (TB) cannot be easily or quickly diagnosed. We developed a rapid, automated assay for the detection of XDR-TB plus resistance to the drug isoniazid (INH) for point-of-care use. Using a simple filter-based cartridge with an integrated sample processing function, the assay identified a wide selection of wild-type and mutant sequences associated with XDR-TB directly from sputum. Four new large-Stokes-shift fluorophores were developed. When these four Stokes-shift fluorophores were combined with six conventional fluorophores, 10-color probe detection in a single PCR tube was enabled. A new three-phase, double-nested PCR approach allowed robust melting temperature analysis with enhanced limits of detection (LODs). Finally, newly designed sloppy molecular beacons identified many different mutations using a small number of probes. The assay correctly distinguished wild-type sequences from 32 commonly occurring mutant sequences tested in gyrA, gyrB, katG, and rrs genes and the promoters of inhA and eis genes responsible for resistance to INH, the fluoroquinolone (FQ) drugs, amikacin (AMK), and kanamycin (KAN). The LOD was 300 CFU of Mycobacterium tuberculosis in 1 ml sputum. The rate of detection of heteroresistance by the assay was equivalent to that by Sanger sequencing. In a blind study of 24 clinical sputum samples, resistance mutations were detected in all targets with 100% sensitivity, with the specificity being 93.7 to 100%. Compared to the results of phenotypic susceptibility testing, the sensitivity of the assay was 75% for FQs and 100% each for INH, AMK, and KAN and the specificity was 100% for INH and FQ and 94% for AMK and KAN. Our approach could enable testing for XDR-TB in point-of-care settings, potentially identifying highly drug-resistant TB more quickly and simply than currently available methods.

[Chemical constituents contained in ethanol extracts from Acorus tatarinowii and their anti-fatigue activity].

Chemical constituents in ethyl acetate and butanol fractions of ethanol extracts from Acorus tatarinowii were separated by column chromatography. Bufo skeletal muscle fatigue model was established to study the anti-fatigue activity of separated compounds. Five compounds were separated and identified by spectroscopic analysis as acoramone(1),cycloartenone(2),2,4,5-trimethoxyl-2'-butoxy-1,2-phenyl propandiol(3),5-hydroxymethyl furfural(4), and 5-butoxymethyl furfural(5). Compound 3 was a new compound, and compounds 2 and 5 were separated from this plant for the first time. Compound 4 exhibited a notable anti-fatigue activity.

[KLF15/mTOR related proteins involved in effect of aerobic interval training on improving skeletal muscle lesions in rats with type 2 diabetes].

Objective: To investigate the effects of aerobic intermittent exercise on the expressions of KLF15/mTOR related proteins to improve skeletal muscle lesions in type 2 diabetes rats. Methods: The experimental model of type 2 diabetes rats was established by feeding high-fat diet for 4 weeks and intraperitoneal injection of streptozotocin (STZ). After modeling, rats were randomly divided into two groups: diabetes model group (DM), diabetes+exercise group (DE), and normal rats were set as control group (C), 10 rats in each group. Group DE was given 8-week aerobic intermittent treadmill exercise intervention, while group C was not given any intervention. At the end of the experiment, the expressions of KLF15, mTOR, p-mTOR, and cleared caspase-3 in gastrocnemius muscle were detected by Western blot. The histopathologic changes of gastrocnemius were observed under microscope; skeletal muscle cells apoptosis rates and muscle mass were examined respectively using HE staining and TUNEL fluorescence staining. At the same time, changes of blood glucose and serum insulin, and weight were examined in the end of the experiment. Results: ①Compared with group C, the wet weight of gastrocnemius muscle and body weight, ratio of wet gastrocnemius muscle and body weight in group DM were decreased(P＜0.05 or P＜0.01); compared with group DM, the wet weight of gastrocnemius muscle, ratio of wet gastrocnemius muscle and body weight in the group DE were increased significantly (P＜0.05). ②Compared with group C, the fasting blood glucose level of group DM was increased significantly (P＜0.01), while serum insulin level of the group DM was decreased significantly(P＜0.01);compared with group DM, the above indexes were opposite in the group DE with intervention(P＜0.05). ③Compared with group C, the morphology of skeletal muscle cells in group DM was abnormal, the number of muscle nuclei was increased, the transverse lines were blurred and disappeared, the sarcomere was broken, and some muscle fibers were dissolved. Compared with group DM, the abnormal cell morphology, segmental injury of sarcomere and dissolution of muscle fibers in group DE were improved. The sarcolemma was more complete and the arrangement of muscle nuclei was more orderly. ④Compared with group C, the expressions of KLF15 and cleaved caspase-3, cells apoptosis rates in group DM were increased significantly(P＜0.01), while p-mTOR/mTOR level was decreased(P＜0.01) ; compared with group DM, the above indexes were opposite in the group with intervention(P＜0.05 or P＜0.01). Conclusion: Aerobic intermittent exercise is beneficial to improve the skeletal muscle pathological changes in type 2 diabetes rats, which may be due to the effective regulation of KLF15/mTOR related protein expression and the reduction of apoptosis damage.

[The effects of aerobic exercise plus spirulina polysaccharide supplement on learning and memory function of diabetes rats and its mechanism].

Objective: To investigate the effects of aerobic exercise plus spirulina polysaccharide(SP) supplement on the related protein expressions of p75NTR signal in hippocampal and the improvement of learning and memory of type 2 diabetes rats. Methods: The model of type 2 diabetic rats was established by fed high-fat diet for four weeks together with intraperitoneal injecting a low dose of STZ. The model rats were randomly divided into diabetic model group(B),diabetic exercise group(C),diabetic+SP group(D)and diabetic exercise+SP group(E), another normal control group(A)without any intervention was set up,12 rats in each group. The rats in Group C and E were treated with intervention of swimming training for six weeks. The rats in Group D and E were treated with SP intragastrically for 6 weeks. Learning and memory abilities were observed by Morris water maze test. The hippocampus cell apoptosis was observed by Tunnel staining, and BDNF content and the expressions of p75NTR, cleaved caspase-3 of hippocampus were tested by ELISA, Western blot and immunohistochemistry, respectively. At the same time, the changes of blood glucose and levels of serum insulin were examined. Results: ①Compared with Group A at different time points, the body weight of Group B was decreased significantly(P＜0.01). Compared with Group B at different time points, the body weight of Group C,D and E had no difference (P＞0.05). Compared with Group A, levels of the blood glucose and serum insulin Group B were increased significantly(P＜0.01).Compared with Group B, the levels in the intervention groups were decreased significantly (P＜0.01); ②Compared with Group A, the escape latencies of Group B were prolonged significantly(P＜0.01), platform quadrant residence duration and the times of crossing platform were decreased (P＜0.01). Compared with Group B, the escape latencies of the intervention groups were shortened (P＜0.05 or P＜0.01), and the times of crossing platform were increased (P＜0.05 or P＜0.01). ③Compared with Group B, the neural cells apoptosis of the intervention rats was decreased, and the protein expressions of p75NTR and cleaved caspase-3 were decreased (P＜0.05 or P＜0.01), however the expression of BDNF was increased significantly (P＜0.05 or P＜0.01). Conclusion: Aerobic exercise and SP supplement can improve the learning-memory ability of type 2 diabetes rats, and the improvement effect of exercise combined with SP is markedly better than that of exercise and SP alone, the mechanism might be related to better regulating p75NTR signal related protein expressions and then inhibiting apoptosis in hippocampus of rats with type 2 diabetes.

[Effects of acorus tatarinowii Schott and alpha asarone on free radicals and nNOS/NO in hippocampus of rats with fatigue movement].

OBJECTIVE: To study the effects of acorus tatarinowii Schott and its active ingredient-alpha-asarone on learning and memory, free radical metabolism and nNOS/NO signal in hippocampus of rats with fatigue movement. METHODS: Eighty SD male rats were randomly divided into eight groups: control group(A), exercise group(B), exercise + alpha-asarone low, middle and high dose treatment group (C, D, E), exercise + acorus tatarinowii Schott low, middle and high dose treatment group (F, G, H),with ten rats in each group. The rats in group C, D and E were administered with alpha-asarone at the doses of 0.10, 0.50 and 1.00 mg.kg-1.WT-1 by ig. The rats in group F, G and H were administered with the extracts of Acorus tatarinowii Schott of at the doses of 0.12, 1.20 and 4.80 g.kg-1.WT-1 by ig. Learning and memory of rats were tested by the method of water maze experiment, and the activities of SOD and NOS, the contents of MDA were detected by the biochemical methods, and the expression levels of nNOS protein in hippocampus of rats were tested by the method of Western blot in at the end of the experiment. RESULTS: The escape latency and MDA content in hippocampus of rats in groups E and H were lower than those in groups B, C, D, F and G and the numbers of Plateau crossing, SOD and NOS activities and the expression levels of nNOS protein in hippocampus of rats were higher than those in groups B, C, D, F and G(P＜0.01). The activities of SOD in hippocampus of rats in groups A, E and H were A＞E＞H, whereas the contents of MDA were opposite (P＜0.01); the activities of NOS and the expression levels of nNOS protein in hippocampus of group E were lower than those of groups A and H (P＜0.01 or P＜0.05), but there was no significant difference between groups A and H (P＞0.05). There were no significant difference in escape latency and numbers of crossing platform among groups A, E and H (P＞0.05). CONCLUSION: Acorus tatarinowii Schott and alpha-asarone can significantly improve learning and memory of rats with fatigue movement. The mechanism is related to reclaiming the imbalance of free radical metabolism and up-regulating nNOS/NO signal in hippocampus of the rats.

[Effects of Acorus tatarinowii Schott and its active component -5- hydroxymethylfurfural on ERK/CREB signal in hippocampus of exercise-induced fatigue rats].

OBJECTIVE: To investigate the effects of Acorus tatarinowii Schott and its active component 5- hydroxymethyl furfural (HMF) on learning and memory and ERK/CREB signal in hippocampus of rats with exercise-induced fatigue. METHODS: SD rats were randomly divided into normal group (A), exercise group (B), exercise + HMF low, middle and high dose treatment group (C, D, E), exercise + acorus tatarinowii Schott low, middle and high dose treatment group (F, G, H), with ten rats in each group. The rats in group C, D and E were treated with HMF at the doses of 0.10, 1.00 and 3.00 mg. kg-1 by ig. The rats in group F, G and H were treated with the extracts of Acorus tatarinowii Schott at the doses of 0.12, 1.20 and 4.80 g. kg-1 by ig. Learning and memory of rats were tested by the method of water maze experiment, and the expression levels of p-ERK1/2 and p-CREB protein in hippocampus of rats were tested by the method of Western blot in the end of the experiment. RESULTS: The escape latencies of E and H groups were lower than those of groups B, C, D, F and G; and the numbers of plateau crossing were more than those of groups B, C, D, F and G and the expression levels of p-ERK1/2, p-CREB protein were higher than those of groups B, C, D, F and G , respectively(P ＜ 0.01). There was no significant difference in the above indexes among groups A, E and H(P＞0.05) except that the expression levels of p-ERK2 protein in group E were lower than those in group A and H (P＜0.05). CONCLUSION: Acorus tatarinowii and its active component- HMF can improve the learning and memory of rats with exercise-induced fatigue, and the mechanism is related to the up-regulation of ERK / CREB signal in hippocampus of rats with exercise-induced fatigue.

[Protective effects of spirulina on hippocampal injury in exercise-fatigue mice and its mechanism].

OBJECTIVE: To investigate the roles of BDNF/TrkB neurotrophic signaling in hippocampal injury for fatigue rats induced by incremental load exercise and the protective effects and mechanism of spirulina supplement. METHODS: Sixty SD rats were randomly divided into normal control group (NC), normal plus spirulina group(NS), exercise model group (EM), exercise plus spirulina group (ES), and positive control group (PC), 12 rats in each group.Group EM, Group ES and Group PC were applied by treadmill running with high-intensity increasing for three weeks, and Group NC had not any intervention measures.Group ES and Group NS were treated with spirulina at a dose of 300 mg/kg.bw.by intragastric administration.Group PC was gavaged at the same volume of ginseng extract of 1.92 g/kg for three weeks.The expressions of brain-derived neurotrophic factor (BDNF), tyrosine kinase recptor (TrkB), phospho-tyrosine kinase recptor (p-TrkB) were tested by Western blot and immunohistochemical method, and micromorphology changes of hippocampal CA1 were observed by light microscope at the end of the experiment.The general situations of rats such as body weights were recorded during the experiment. RESULTS: Compared with Group NC, Group EM showed significantly decrease in body weight and hippocampal CA1 neurons of the group loosely arrayed and disarrayed and some neurons were shrinked, and even some neurons disappeared.The expressions of BDNF, TrkB and p-TrkB in group EM were increased significantly(P<0.01).Compared with Group EM, body weight of Group ES was increased significantly, and the above mentioned injuries of neurons were improved significantly:the number of neurons and nissl bodies were significantly increased and the neurons arrayed regularly and its morphology was more complete.The expressions of BDNF, TrkB and p-TrkB in the group were increased significantly(P<0.05 or P<0.01).And there was no difference between Group ES and Group PC. CONCLUSIONS: BDNF/TrkB neurotrophic signal pathway could be involved in the repair process of hippocampal nervous damage caused by incremental load exercise for fatigue rats.Spirulina supplement had a protective effect on the damaged nervous through increasing the expressions of BDNF, TrkB and p-TrkB.

[Application of area percent of positive neuron and relative intensity of staining grey level in the image quantitative analysis of rat brain tissues immunohistochemistry].

OBJECTIVE: The acetylcholine expression in hypothalamus arcuate nucleus is detected and then the images are processed and analyzed. The features of the image quantitative analysis of immunohistochemistry (IHC) with the method combining two parameters of area percent of positive neuron (APPN) and relative intensity of staining grey level (RISGL) were investigated. METHODS: Samples were the im-munohistochemical slices of acetylcholine(ACh)expression of hypothalamic arcuate nucleus cholinergic neurons in the process of exercise in-duced immunosuppression, which included twelve groups of "0 w, 2 w, 4 w, 6 w" and three groups of "control, immediately after exercise, 3 hours after exercise" in every week. IHC technology was used to detect the ACh expression. The image quantitative analysis of IHC was con-ducted in accordance with the parameters of ACh total area of positive neuron (TAPN), average intensity of staining grey level (AISGL), APPN, RISGL, APPN/RISGL. Then the differences among APPN, RISGL and traditional parameters in the quantitative analysis were com-pared and the advantages were found. RESULTS: The changes of TAPN and APPN showed almost the same variation. Namely the corresponding significant differences could be found through these two parameters(P < 0.05), but the sensitivity and anti-interference of APPN was higher. The results of AISGL and RISGL were not coincident completely. Furthermore, with the combination of APPN and RISGL, the positive expres-sion could be reflected better than any single parameter. CONCLUSIONS: The parameters of immunohistochemical image analysis, APPN and RIS-GL, can be reliable and accurate in image quantitative analysis of IHC. The combination of APPN and RISGL can not only reflect the expres-sion of positive neurons, but also help analyze its mechanism, which is better than traditional analysis parameters.