Simultaneous detection of 9 respiratory pathogens using a newly developed multiplex real-time PCR panel based on an automatic molecular detection and analysis system.

Timely identification of respiratory pathogens guides specific treatment, reduces hospital costs and minimizes the excessive use of antibiotics. A new multiplex real-time PCR panel was developed based on an automatic molecular detection and analysis system (AutoMolec system), consisting of three separate internally controlled assays. Mycoplasma pneumoniae, Chlamydia pneumoniae, adenovirus, human metapneumovirus, influenza B virus, respiratory syncytial virus and human parainfluenza virus 1-3 may be directly detected in original samples. The system's clinical performance was evaluated by comparison with an approved commercial kit, using 517 clinical samples. The limit of detection of the AutoMolec mRT-PCR panel ranged from 4 × 10-4 ∼3.3 TCID50/mL and no cross-reaction with common respiratory pathogens was observed. The AutoMolec mRT-PCR panel had 99.09% sensitivity and 100.0% specificity and overall detection consistency was 99.61%, making it comparable to that of the commercial kit. Therefore, the AutoMolec mRT-PCR panel has great potential for routine screening of respiratory infection in China.

[Ultra-performance liquid chromatography for determination of the contents of biogenic amines in the seminal plasma].

OBJECTIVE: To establish an ultra-performance liquid chromatography (UPLC) method for determination of the contents of the three biogenic amines putacine, spermidine and spermine in human seminal plasma. METHODS: Seminal plasma samples were extracted with 5% trichloroacetic acid and processed by pre-column derivatization with dansyl chloride. Chromatographic separation was performed with a C18 (2.1×50 mm,1.7 µm) chromatographic column using water and acetonitrile for mobile-phase gradient elution at a flow rate of 0.3 ml/min, a detection wavelength of 245 nm, a column temperature of 35℃ and an injection volume was 3.0 µl. The contents of putacine, spermidine and spermine in the seminal plasma of 52 healthy sperm donors (the normal group) and 23 azoospermia patients (the AS group) were measured, and their correlation with routine semen parameters were analyzed. RESULTS: The three biogenic amines showed a good linearity (r ≥ 0.999), with a lower detection limit of 0.03－0.08 µg/ml. The relative standard deviation (RSD) of precision was ≤ 0.72% and the average recovery rate was 79.74%－108.87%. The normal group, compared with the AS patients, showed significantly higher contents of putrescine (ï¼»8.19 ± 7.85ï¼½ vs ï¼»2.43 ± 1.38ï¼½ mg/ml, P < 0.05), spermidine (ï¼»77.30 ± 32.58ï¼½ vs ï¼»31.99 ± 16.21ï¼½ mg/ml, P < 0.05) and spermine (ï¼»246.44 ± 83.99ï¼½ vs ï¼»166.15 ± 79.28ï¼½ mg/ml, P < 0.05). However, the contents of the three biogenic amines in the seminal plasma exhibited no significant correlation with the routine semen parameters in the normal group. CONCLUSIONS: The ultra-performance liquid chromatography method we established, with the advantages of high sensitivity and reproducibility and short peak-time, can quickly and accurately determine the contents of biogenic amines in the seminal plasma.

[Correlation of seminal plasma biochemical indexes with routine semen parameters in sperm donors].

OBJECTIVE: To investigate the relationship of seminal plasma biochemical indexes with routine semen parameters and that between seminal plasma biochemical indexes. METHODS: Using the automatic biochemical analyzer, we measured the contents of neutral α-glucosidase (NAG), fructose hexokinase (Fru) , citric acid (CA), acid phosphatase (ACP), (zinc) Zn, uric acid (UA), lactase dehydrogenase (LDH) and α-hydroxybutyrate dehydrogenase (α-HBDH) in the seminal plasma of 84 sperm donors in the Human Sperm Bank of Hebei Province. We analyzed the correlation between these indexes and that between routine semen parameters and these indexes. RESULTS: Sperm concentration and total sperm count were correlated positively with the contents of seminal plasma NAG, ACP, Zn, CA, LDH and α-HBDH (P<0.05) but negatively with Fru (P<0.05), the percentage of progressively motile sperm positively with seminal plasma Zn (P<0.05), and CA positively with NAG, Zn, LDH, α-HBDH and ACP (P<0.01) but negatively with Fru (P<0.01), NAG positively with Zn, LDH, α-HBDH and ACP (P<0.05) but Fru negatively with ACP (P<0.01), Zn positively with LDH, α-HBDH and ACP (P<0.01), and LDH positively with α-HBDH and ACP (P<0.01) but UA negatively with ACP (P<0.05). CONCLUSIONS: Biochemical indexes in the seminal plasma of healthy men are not only closely related to each other, but also to some routine semen parameters.

Displacement of the retina after idiopathic macular hole surgery with different internal limiting membrane peeling patterns.

AIM: To explore retinal displacement after surgical treatment for idiopathic macular hole (IMH) with different internal limiting membrane (ILM) peeling patterns. METHODS: Totally 22 eyes from 20 patients with IMH were randomly allocated into two groups, N-T group (11 eyes) and T-N group (11 eyes). For patients in N-T group, ILM was peeled off from nasal to temporal retina. For patients in T-N group, ILM was peeled off from temporal to nasal retina. Preoperative, postoperative 1, 3, and 6mo, autofluorescence fundus images were collected for manual measurement of distances of fixed nasal (N), temporal (T), superior (S), and inferior (I) retinal points (bifurcation or crossing of retinal vessels) around the macula to the optic disc (OD). These were respectively defined as N-OD, T-OD, S-OD, and I-OD. The retinal displacement, macular hole closure rate, and best corrected visual acuity (BCVA) were compared between the two groups after surgery. RESULTS: At postoperative 1, 3, and 6mo, the macula slipped toward the OD, manifested by the decreased T-OD, N-OD, S-OD, and I-OD (P<0.05). No significant difference was found in the T-OD, N-OD, S-OD, and I-OD between N-T group and T-N group. IMH closure rate was 100% both in N-T group and T-N group. There was no significant difference in BCVA between two groups (P<0.05). CONCLUSION: The macula slips toward the OD after successful macular hole surgery. The two different ILM peeling pattern show similar visual outcome and retinal displacement, which means ILM peeling directions are not the influencing factor of postoperative retinal displacement.

CD51 distinguishes a subpopulation of bone marrow mesenchymal stem cells with distinct migratory potential: a novel cell-based strategy to treat acute myocardial infarction in mice.

BACKGROUND: Experimental and clinical trials have demonstrated the efficiency of bone marrow-derived mesenchymal stromal/stem cells (bMSCs) in the treatment of myocardial infarction. However, after intravenous injection, the ineffective migration of engrafted bMSCs to the hearts remains an obstacle, which has an undesirable impact on the efficiency of cell-based therapy. Therefore, we attempted to identify a marker that could distinguish a subpopulation of bMSCs with a promising migratory capacity. METHODS: Here, CD51-negative and CD51-positive cells were isolated by flow cytometry from Ter119-CD45-CD31-bMSCs and cultured in specifically modified medium. The proliferation ability of the cells was evaluated by 5-ethynyl-2'-deoxyuridine (EdU) staining or continuously monitored during culture, and the differentiation potential was assessed by culturing the cells in the appropriate conditioned media. Wound healing assays, transwell assays and quantitative polymerase chain reaction (qPCR) were used to measure the migratory ability. The mice were subjected to a sham operation or myocardial infarction (MI) by permanently occluding the coronary artery, and green fluorescent protein (GFP)-labelled cells were transplanted into the mice via intravenous infusion immediately after MI. Heart function was measured by echocardiography; infarct myocardium tissues were detected by triphenyl tetrazolium chloride (TTC) staining. Additionally, immunofluorescence staining was used to verify the characteristics of CD51+bMSCs and inflammatory responses in vivo. Statistical comparisons were performed using a two-tailed Student's t test. RESULTS: In this study, the isolated CD51-bMSCs and CD51+bMSCs, especially the CD51+ cells, presented a favourable proliferative capacity and could differentiate into adipocytes, osteocytes and chondrocytes in vitro. After the cells were transplanted into the MI mice by intravenous injection, the therapeutic efficiency of CD51+bMSCs in improving left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) was better than that of CD51-bMSCs. Compared with CD51-bMSCs, CD51+bMSCs preferentially migrated to and were retained in the infarcted hearts at 48 h and 8 days after intravenous injection. Accordingly, the migratory capacity of CD51+bMSCs exceeded that of CD51-bMSCs in vitro, and the former cells expressed higher levels of chemokine receptors or ligands. Interestingly, the retained CD51+bMSCs retained in the myocardium possessed proliferative potential but only differentiated into endothelial cells, smooth muscle cells, fibroblasts or cardiomyocytes. Transplantation of CD51+bMSCs partially attenuated the inflammatory response in the hearts after MI, while the potential for inflammatory suppression was low in CD51-bMSC-treated mice. CONCLUSIONS: These findings indicated that the CD51-distinguished subpopulation of bMSCs facilitated proliferation and migration both in vitro and in vivo, which provided a novel cell-based strategy to treat acute MI in mice by intravenous injection.

Activity-Dependent Sulfhydration Signal Controls N-Methyl-D-Aspartate Subtype Glutamate Receptor-Dependent Synaptic Plasticity via Increasing d-Serine Availability.

AIMS: Reactive sulfur species, including hydrogen sulfide (H2S) and its oxydates, have been raised as novel redox signaling molecules. The present study aimed at examining whether endogenous sulfhydration signal is required for long-term potentiation (LTP), a cellular model for memory. RESULTS: In this study, we found that increased synaptic activity triggered sulfide generation and protein sulfhydration. Activity-triggered sulfide production was essential for N-methyl-D-aspartate subtype glutamate receptor (NMDAR)-dependent LTP via maintaining the availability of d-serine, a primary coagonist for synaptic NMDARs. Genetic knockdown of cystathionine ß-synthase, not cystathionine γ-lyase, impaired LTP. H2S increased NMDAR-dependent LTP via sulfhydration and disinhibition of serine racemase (SR), a main synthetase of d-serine. We found that polysulfides also increased NMDAR-dependent LTP and NMDAR activity. In aged rats, the level of H2S and SR sulfhydration decreased significantly. Exogenous supplement of H2S restored the sulfhydration of SR, followed by the improvement of age-related deficits in LTP. Furthermore, boost of H2S signal in vivo improves hippocampus-dependent memory. Innovation and Conclusion: Our results provide a direct evidence for the biological significance of endogenous sulfhydration signal in synaptic plasticity. Exogenous supplement of H2S could be considered as the new therapeutic approach for the treatment of neurocognitive dysfunction after aging. Antioxid. Redox Signal. 27, 398-414.

Hydrogen Sulfide Promotes Surface Insertion of Hippocampal AMPA Receptor GluR1 Subunit via Phosphorylating at Serine-831/Serine-845 Sites Through a Sulfhydration-Dependent Mechanism.

AIMS: Hydrogen sulfide (H2 S) has been widely accepted as a gas neuromodulator to regulate synaptic function. Herein, we set out to determine the effect of H2 S on α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) and its mechanism. METHODS: BS(3) protein cross-linking, Western blot, patch clamp, and biotin-switch assay. RESULTS: Bath application of H2 S donor NaHS (50 and 100 µM) rapidly promoted surface insertion of hippocampal AMPAR GluR1 subunit. This effect can be abolished by dithiothreitol (DTT) and mimicked by Na2 S4 , indicating that a sulfhydration-dependent mechanism may be involved. NaHS increased APMAR-mediated EPSC and led to an elevation of GluR2-lacking AMPAR content. Notably, NaHS did not increase the sulfhydration of AMPAR subunits, but it significantly increased the phosphorylation of GluR1 at serine-831 and serine-845 sites. Postsynaptic signal pathways that control GluR1 phosphorylation, such as protein kinase A (PKA), protein kinase C, and calcium/calmodulin-dependent protein kinases II (CaMKII), were sulfhydrated, activated by NaHS, and these effects can be occluded by DTT. H2 S increased S-sulfhydration of protein phosphatase type 2A (PP2A), which may be partially involved in the activation of signal pathways. CONCLUSION: Our data suggest that H2 S promotes surface insertion of AMPARs via phosphorylation of GluR1, which depends on a sulfhydration-mediated mechanism.

Methylprednisolone exerts neuroprotective effects by regulating autophagy and apoptosis.

Methylprednisolone markedly reduces autophagy and apoptosis after secondary spinal cord injury. Here, we investigated whether pretreatment of cells with methylprednisolone would protect neuron-like cells from subsequent oxidative damage via suppression of autophagy and apoptosis. Cultured N2a cells were pretreated with 10 µM methylprednisolone for 30 minutes, then exposed to 100 µM H2O2 for 24 hours. Inverted phase contrast microscope images, MTT assay, flow cytometry and western blot results showed that, compared to cells exposed to 100 µM H2O2 alone, cells pretreated with methylprednisolone had a significantly lower percentage of apoptotic cells, maintained a healthy morphology, and showed downregulation of autophagic protein light chain 3B and Beclin-1 protein expression. These findings indicate that methylprednisolone exerted neuroprotective effects against oxidative damage by suppressing autophagy and apoptosis.

Fast Micro-Differential Evolution for Topological Active Net Optimization.

This paper studies the optimization problem of topological active net (TAN), which is often seen in image segmentation and shape modeling. A TAN is a topological structure containing many nodes, whose positions must be optimized while a predefined topology needs to be maintained. TAN optimization is often time-consuming and even constructing a single solution is hard to do. Such a problem is usually approached by a "best improvement local search" (BILS) algorithm based on deterministic search (DS), which is inefficient because it spends too much efforts in nonpromising probing. In this paper, we propose the use of micro-differential evolution (DE) to replace DS in BILS for improved directional guidance. The resultant algorithm is termed deBILS. Its micro-population efficiently utilizes historical information for potentially promising search directions and hence improves efficiency in probing. Results show that deBILS can probe promising neighborhoods for each node of a TAN. Experimental tests verify that deBILS offers substantially higher search speed and solution quality not only than ordinary BILS, but also the genetic algorithm and scatter search algorithm.

Differential Evolution with an Evolution Path: A DEEP Evolutionary Algorithm.

Utilizing cumulative correlation information already existing in an evolutionary process, this paper proposes a predictive approach to the reproduction mechanism of new individuals for differential evolution (DE) algorithms. DE uses a distributed model (DM) to generate new individuals, which is relatively explorative, whilst evolution strategy (ES) uses a centralized model (CM) to generate offspring, which through adaptation retains a convergence momentum. This paper adopts a key feature in the CM of a covariance matrix adaptation ES, the cumulatively learned evolution path (EP), to formulate a new evolutionary algorithm (EA) framework, termed DEEP, standing for DE with an EP. Without mechanistically combining two CM and DM based algorithms together, the DEEP framework offers advantages of both a DM and a CM and hence substantially enhances performance. Under this architecture, a self-adaptation mechanism can be built inherently in a DEEP algorithm, easing the task of predetermining algorithm control parameters. Two DEEP variants are developed and illustrated in the paper. Experiments on the CEC'13 test suites and two practical problems demonstrate that the DEEP algorithms offer promising results, compared with the original DEs and other relevant state-of-the-art EAs.

Interactions between N-ethylmaleimide-sensitive factor and GluR2 in the nucleus accumbens contribute to the expression of locomotor sensitization to cocaine.

Many studies have reported a withdrawal-dependent increase in synaptic AMPA receptor (AMPAR) levels in the nucleus accumbens (NAc) of cocaine-sensitized rats; however, the exact relationship between the expression of sensitization and altered AMPAR surface expression in the NAc has not yet been investigated. We demonstrated that the expression of behavioral sensitization was negatively controlled by N-ethylmaleimide-sensitive factor (NSF)-GluR2 interactions in the NAc. The upregulation of NSF-GluR2 interactions, which may be resulted by the increase in NSF S-nitrosylation after withdrawal from cocaine, was associated with the changes in the expression of behavioral sensitization. Disruption of NSF-GluR2 interactions in the NAc with a specific peptide, TAT-pep-R845A, increased the locomotor response of rats to cocaine by decreasing GluR2 surface insertion. In contrast, prevention of GluR2-containing AMPARs removal from synapses with Pep2-EVKI attenuated the expression of behavioral sensitization. Similarly, treatment with the nitric oxide donor, S-Nitroso-N-acetyl-DL-penicillamine (SNAP), attenuated the expression of locomotor sensitization by promoting GluR2 surface expression. This effect was mediated by the binding of S-nitrosylated NSF to GluR2, which promoted the surface expression of AMPARs. Noticeably, exogenous injection of SNAP into NAc also attenuated the expression of cocaine-induced conditioned place preference. Thus, these results indicate that increased NSF-GluR2 interactions in the NAc after withdrawal from cocaine attenuated the expression of behavioral sensitization and serves as a negative regulatory mechanism in drug-exposed individuals.

A specific and rapid colorimetric method to monitor the activity of methionine sulfoxide reductase A.

Considerable evidence indicates that methionine sulfoxide (MetO) reductase A (MsrA) plays an important role in cytoprotection against oxidative stress and serves as a potential drug target. To screen for MsrA regulators, a rapid and specific assay to monitor MsrA activity is required. Most of current assays for MsrA activity are based on the reduction of radioactive substrates such as [3H]-N-acetyl-MetO or fluorescent derivatives such as dimethylaminoazo-benzenesulfonyl-MetO. However, these assays require extraction procedures and special instruments. Here, we developed a specific colorimetric microplate assay for testing MsrA activity quickly, which was based on the fact that MsrA can catalyze the reduction of methyl sulfoxides and simultaneously oxidize dithiothreitol (DTT), whose color can be produced by reacting with Ellman's reagent (dithio-bis-nitrobenzoic acid, DTNB). The corresponding absorbance change at 412nm was recorded with a microplate reader as the reaction proceeded. This method to monitor MsrA activity is easy to handle. Our findings may serve as a rapid method for the characterization of recombinant enzyme and for the screening of enzyme inhibitors, pharmacological activators, gene expression regulators and novel substrates.

Resveratrol preconditioning increases methionine sulfoxide reductases A expression and enhances resistance of human neuroblastoma cells to neurotoxins.

Methionine sulfoxide reductases A (MsrA) has been postulated to act as a catalytic antioxidant system involved in the protection of oxidative stress-induced cell injury. Recently, attention has turned to MsrA in coupling with the pathology of Parkinson's disease, which is closely related to neurotoxins that cause dopaminergic neuron degeneration. Here, we firstly provided evidence that pretreatment with a natural polyphenol resveratrol (RSV) up-regulated the expression of MsrA in human neuroblastoma SH-SY5Y cells. It was also observed that the expression and nuclear translocation of forkhead box group O 3a (FOXO3a), a transcription factor that activates the human MsrA promoter, increased after RSV pretreatment. Nicotinamide , an inhibitor of silent information regulator 1 (SIRT1), prevented RSV-induced elevation of FOXO3a and MsrA expression, indicating that the effect of RSV was mediated by a SIRT1-dependent pathway. RSV preconditioning increased methionine sulfoxide(MetO)-reducing activity in SH-SY5Y cells and enhanced their resistance to neurotoxins, including chloramine-T and 1-methyl-4-phenyl-pyridinium. In addition, the enhancement of cell resistance to neurotoxins caused by RSV preconditioning can be largely prevented by MsrA inhibitor dimethyl sulfoxide. Our findings suggest that treatment with polyphenols such as RSV can be used as a potential regulatory strategy for MsrA expression and function.