This study was aimed at establishing a method of ultra-fast quantitative PCR for Brucella detection.We used an exogenous recombinant plasmid as the internal reference and targeted the T4SS secretion system,an important Brucella viru-lence factor,to design specific primers and probes.The sensitivity,specificity,and repeatability of this method were evaluated,and a standard curve was constructed.The coincidence rate of detection findings with this method versus quantitative PCR was determined.This method markedly decreased the detection time to only 10 minutes.The standard curve demonstrated a good linear relationship(Y=-3.410 7x+38.357,R2=0.998 5)with a low minimum detection limit of 10 copies/μL.The method exhibited good specificity and did not specifically amplify several common clinical bacteria other than Brucella.The de-tection of three concentrations of positive plasmids yielded coefficients of variation(CVs)of 0.20％to 0.91％,thus demonstra-ting the method's excellent repeatability.Furthermore,140 clinical samples were analyzed concurrently with the fluorescence PCR method,which yielded a 100％compliance rate and consistent results.Our findings indicated that the Brucella ultra-fast quantitative PCR was ultrafast;had high sensitivity,high specificity,and good specificity;and can be used for the clinical de-tection of Brucella and emergency investigation of epidemics.Therefore,this method is valuable for the early diagnosis of Bru-cella.

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This study was aimed at establishing a method of ultra-fast quantitative PCR for Brucella detection.We used an exogenous recombinant plasmid as the internal reference and targeted the T4SS secretion system,an important Brucella viru-lence factor,to design specific primers and probes.The sensitivity,specificity,and repeatability of this method were evaluated,and a standard curve was constructed.The coincidence rate of detection findings with this method versus quantitative PCR was determined.This method markedly decreased the detection time to only 10 minutes.The standard curve demonstrated a good linear relationship(Y=-3.410 7x+38.357,R2=0.998 5)with a low minimum detection limit of 10 copies/μL.The method exhibited good specificity and did not specifically amplify several common clinical bacteria other than Brucella.The de-tection of three concentrations of positive plasmids yielded coefficients of variation(CVs)of 0.20％to 0.91％,thus demonstra-ting the method's excellent repeatability.Furthermore,140 clinical samples were analyzed concurrently with the fluorescence PCR method,which yielded a 100％compliance rate and consistent results.Our findings indicated that the Brucella ultra-fast quantitative PCR was ultrafast;had high sensitivity,high specificity,and good specificity;and can be used for the clinical de-tection of Brucella and emergency investigation of epidemics.Therefore,this method is valuable for the early diagnosis of Bru-cella.

OBJECTIVE: To investigate the potential mechanisms of electroacupuncture (EA) to prevent ischemic stroke. METHODS: The method of middle cerebral artery occlusion (MCAO) was employed to establish a rat model of ischemic stroke. Seventy-eight Sprague-Dawley rats were divided into the sham group, MCAO + EA control (EC) group, and MCAO + EA (EA) group according to a random number table (n=26 per group). EA was applied to the acupoints of Baihui (DU 20) and Shenting (DU 24) 5 min and 6 h, respectively after the onset of MCAO. Rats in the sham and EC groups received only light isoflurane anesthesia for 30 min after MCAO. The neuroprotective effects of EA were evaluated by rota-rod test, neurological deficit scores and infarct volumes. Additionally, Nissl staining and immunostaining were performed to examine brain damage, rod formation, cellular apoptosis, and neuronal loss induced by ischemia. The activities of caspase-3, and expression levels of cofilin and p-cofilin in mitochondria and cytoplasm after ischemic injury were determined by Western blot. RESULTS: Compared with the EC group, EA significantly improved neuromotor function and cognitive ability after ischemic stroke (P<0.05 or P<0.01). Therapeutic use of EA also resulted in a significant decrease of cofilin rod formation and microtubule-associated protein-2 (MAP2) degradation in the cortical penumbra area compared with the EC rats (P<0.01). Furthermore, Western blot analysis showed that EA stimulation significantly inhibited mitochondrial translocation of cofilin and caspase-3 cleavage (P<0.05 or P<0.01). Additionally, brain damage (infarct volume and neuropathy), cellular apoptosis and neuronal loss induced by ischemia were remarkably suppressed by EA in the cortical penumbra of rats (P<0.05 or P<0.01). CONCLUSION EA treatment after ischemic stroke may attenuate ischemic brain injury and cellular apoptosis through the regulation of mitochondrial translocation of cofilin, a novel mechanism of EA therapy.

A capacitively coupled contactless conductivity detector for high performance liquid chromatography was developed. The detector was consisted of a signal generator, a signal amplifier, and a pair of tubular electrodes. A plastic connecting tube following the separation column was threaded through the two tubular electrodes. When the separated components passed the region between two tubular electrodes, they would be sensed. The instrumental parameters of this detector were investigated, including the internal diameter of connecting tube,the frequency and voltage of excitation signal, electrodes length, and the gap between two electrodes. The 0.5 mm of internal diameter of connecting tube, 70 kHz of excitation frequency, 60 V of excitation voltage,10 mm of electrodes length and 1.5 mm of gap between two electrodes were finally found to be the best working conditions for the detector. This HPLC-C4D(capacitively coupled contactless conductivity detector) was applied to the analysis of oleic and linoleic acids in Brucea javanica oil. The chromatographic peak area was linear with concentration for oleic and linoleic acids in 5-1000 μg/mL, and the limit of detection reached 2.5 μg/mL and 1.0 μg/mL,respectively. The results demonstrate that the present method is sensitive and accurate, proving this homemade contactless conductivity detector has a great application potential for pharmaceutical analysis in future development.

OBJECTIVE: To investigate the effect of rosuvastatin on homocysteine (Hcy) induced mousevascular smooth muscle cells(VSMCs) dedifferentiation and endoplasmic reticulum stress(ERS). METHODS: VSMCs were co-cultured with Hcy and different concentration of rosuvastatin (0.1, 1.0 and 10 μmol/L). Cytoskeleton remodeling, VSMCs phenotype markers (smooth muscle actin-α, calponin and osteopontin) and ERS marker mRNAs (Herpud1, XBP1s and GRP78) were detected at predicted time. Tunicamycin was used to induce, respectively 4-phenylbutyrate(4-PBA) inhibition, ERS in VSMCs and cellular migration, proliferation and expression of phenotype proteins were analyzed. Mammalian target of rapamycin(mTOR)-P70S6 kinase (P70S6K) signaling agonist phosphatidic acid and inhibitor rapamycin were used in Rsv treated VSMCs. And then mTOR signaling and ERS associated mRNAs were detected. RESULTS: Compared with Hcy group, Hcy+ Rsv group (1.0 and 10 μmol/L) showed enhanced α-SMA and calponin expression (<0.01), suppressed ERS mRNA levels (<0.01) and promoted polarity of cytoskeleton. Compared with Hcy group, Hcy+Rsv group and Hcy+4-PBA group showed suppressed proliferation, migration and enhanced contractile protein expression (<0.01); while tunicamycin could reverse the effect of Rsv on Hcy treated cells. Furthermore, alleviated mTOR-P70S6K phosphorylation and ERS (<0.01)were observed in Hcy+Rsv group and Hcy+rapamycin group, compared with Hcy group; while phosphatidic acid inhibited the effect of Rsv on mTOR signaling activation and ERS mRNA levels (<0.01). CONCLUSIONS Rosuvastatin could inhibit Hcy induced VSMCs dedifferentiation suppressing ERS, which might be regulated by mTOR-P70S6K signaling.
Actins ; metabolism ; Animals ; Calcium-Binding Proteins ; metabolism ; Cell Dedifferentiation ; drug effects ; Cells, Cultured ; Endoplasmic Reticulum Stress ; drug effects ; Heat-Shock Proteins ; metabolism ; Homocysteine ; Membrane Proteins ; metabolism ; Mice ; Microfilament Proteins ; metabolism ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; drug effects ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Rosuvastatin Calcium ; pharmacology ; TOR Serine-Threonine Kinases ; metabolism ; X-Box Binding Protein 1 ; metabolism

OBJECTIVETo investigate the change of early serum TNF-alpha and IL-6 levels in acute cerebral infarction and its significances.

METHODSSerum TNF-alpha and IL-6 levels in 30 health subjects and 35 patients with acute cerebral infarction (ACI) within 6 hours of onset were measured by enzyme linked immunosorbent assay (ELISA); neurological deficits scores (NDS) in all cases were determined, and Spearman test was used for correlation.

RESULTSThe serum levels of TNF-alpha and IL-6 in ACI group were markedly higher than those in health subjects and there was a positive correlation of TNF-alpha and IL-6 levels with 6 h NDS (rs=0.89 and 0.93, P<0.001) and with NDS progression (rs=0.90 and 0.91, P<0.001). Early serum TNF-alpha and IL-6 levels in progressive cerebral infarction (PCI) group were evidently higher than those in stable cerebral infarction (SCI)[(49.56+/-12.12) pg/L compared with (24.30+/-7.4) ng/L and (39.76+/-7.88) ng/L compared with (20.78+/-6.28) ng/L, respectively, P<0.01)].

CONCLUSIONThe early serum levels of TNF-alpha and IL-6 in ACI markedly increase and are closely correlated with disease severity; which may be of value in PCI risk evaluation.

Acute Disease ; Adult ; Aged ; Biomarkers ; blood ; Cerebral Infarction ; blood ; Early Diagnosis ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Interleukin-6 ; blood ; Male ; Middle Aged ; Time Factors ; Tumor Necrosis Factor-alpha ; blood