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Pseudomonas aeruginosa is one of the three most pathogenic bacteria that frequently cause life-threatening opportunistic human infections, pneumonia, and lower respiratory tract infections in immunocompromised hosts, particularly in the burns ward. The present study aimed to establish a loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of P. aeruginosa-specific gene hypothetical protein (GenBank ID: 882161). The gene was obtained through local and online BLAST, and specific primers were designed for this gene. Reaction conditions were optimized at 65°C for 30 min and 80°C for 2 min, whereas the reaction system contained 5.2 mM Mg2+, 8 U Bst 2.0 DNA polymerase, 1.4 mM deoxyribonucleotide and 0.2 and 1.6 µM of the outer and inner primers, respectively. The LAMP method was evaluated using 150 P. aeruginosa and 170 non-P. aeruginosa strains. Positive reactions were observed on 150 P. aeruginosa strains, whereas all non-P. aeruginosa strains exhibited negative results. Plasmids with the specific gene and mouse blood with P. aeruginosa were used for sensitivity assay. The detection limit of LAMP was 1 bacterium/reaction. Results indicated that the LAMP method targeted to hypothetical protein is a fast, specific, sensitive, inexpensive and suitable method for detection of P. aeruginosa.

A phytochemical investigation on the flowers of Sophora davidii resulted in the isolation of three unusual matrine–adenine hybrids, sophovicines A–C, together with biogenetically related analogue sophocarpine. Their structures and absolute configurations were determined by NMR analysis, single-crystal X-ray diffraction, and electronic circular dichroism (ECD) data. Since sophovicines represent the first example of matrine-adenine hybrids, a putative biosynthetic pathway toward sophovicines A–C was proposed. In addition, computational molecular modeling suggested that compounds sophovicines B and C may have potent activities against human cytomegalovirus (HCMV). So, the inhibit effects of isolates on HCMV were evaluated. The results show that sophovicines B and C can inhibit HCMV replication effectively with IC50 values of 7.12 and 7.32 μM, respectively.

Algicidal bacteria as part of the microbial populations in natural waters, is possible toinhibit the growth of algae or kill algae, dissolve algal cells in a direct or indirect way. In this study,four algicidal bacteria were isolated from cyanobacteria bloom water of Lake Dianchi, designated asDCJ-1, DCJ-2, DCJ-3, DCJ-4 respectively. The algicidal range and algicidal activity assay of the fouralgicidal bacteria showed that they have different algicidal range on the 10 tested algal species, whichstrain DCJ-2 has the best algicidal activity. The results indicated that bacterial strain DCJ-2 has highalgicidal activity against the Plectonema boryanum and exhibited algicidal activity through directattack. The lytic effect of strain DCJ-2 against Plectonema boryanum was time-dependent. It could be a potential bio-agent to control the blooms of cyanobacteria.

Quantitative analysis of H129-ΔTK-tdT monosynaptic tracing. Helper AAVs and H129-ΔTK-tdT were sequentially injected at VPM (a), nRT (b) or VTA (c) of wild type C57BL/6, PV-Cre or DAT-Cre mice respectively, as described in Fig.4-6. The brains’ corona slices were observed with an interval of 120 μM (every 4 slices), and the amounts of the labeled cell at the indicated brain regions were counted. The starter cells at the injection site were detected at Day32 as tdTomato and GFP co-expressing neurons (opened circle, each circle represents one mouse), and the tdTomato positive neurons at other brain regions were observed at Day 32 (filled triangle, each circle represents one mouse). (PDF 109 kb)

Anterograde transmission of H129-G4 from retina to CNS (a) Schema of the simplified mouse visual pathway. LGN, lateral geniculate nucleus; LP, lateral posterior thalamic nucleus; SC, superior colliculus; V1 and V2, primary and secondary visual cortex. (b-d) H129-G4 tracing from retina. H129-G4 was injected into the right retina of wild-type C57BL/6 mice, and images were obtained at 6 dpi. Representative images at LGN, LP (b) and visual cortex (c) are shown. Selected regions are magnified correspondingly, and representative single neurons are presented (d). (PDF 1100Â kb)

H129-ΔTK-tdT alone in various brain regions. (a) Parameters for H129-ΔTK-tdT injection. (b-g) Representative labeling results. H129-ΔTK-tdT was injected into the indicated brain regions of wild type C57BL/6 as listed in the table (a), and the brains were perfused at 10 dpi. The coronal brain slice throughout the brain were observed after staining for NeuN (blue), GFAP (green) and tdTomato (red). Representative images of upstream and downstream region of the injection sites are shown. The white arrows indicated the injection sites. The neurons in the upstream brain region labeled by virus terminal invasion are indicated by the white arrowheads (e3). OB, olfactory bulb; Pir, piriform cortex; MOE, main olfactory epithelium; M1, primary motor cortex; DG, dentate gyrus; LEC, lateral entorhinal cortex; LGN, lateral geniculate nucleus; V1, primary visual cortex; VTA, ventral tegmental area; LDTg, laterodorsal tegmental nucleus. (PDF 2370 kb)

Absence of labeled cells upstream of nRT-PV neurons. H129-Î TK-tdT and AAV-DIO-TK-GFP were injected into the nRT of PV-Cre mice as shown in Fig.5. The animals were perfused Day 32, and the coronal brain slices throughout the entire brains were observed. The representative images of S1, the upstream region of nRT, are shown. (PDF 929Â kb)

In vitro replication of the H129-ΔTK-tdT. To determine the growth property of H129-ΔTK-tdT which lacks TK, fetal mouse hippocampal and cortical neurons (Neuron) or Vero cells were infected with H129-ΔTK-tdT or the TK competent strain H129-tdT at an MOI of 0.02. At the indicated time point, virus titers in the cell culture were determined by standard plaque forming assay. Shown is the representative data from 3 independent experiments, and presented as the Mean ± SD from triplicates. (PDF 183 kb)

The microfluidic plate (a) The schematic structure diagram of the microfluidic system. (b) Axons through the microchannels. Freshly isolated fetal mouse hippocampal and cortical neurons were seeded into one chamber of the microfluidic plate, and cultured for 7 days with positive hydrostatic pressure in the soma chamber. Then the plate was disassembled and stained with antibodies against Tau and Map2. Shown is the representative image from 3 plates. (c) Pre- and post-synaptic markers in the afferent chamber. Neurons were sequentially plated into both chambers at Day 1 and Day 5 respectively, and cultured for additional 7 days with positive hydrostatic pressure in the efferent chamber. The plate was disassembled on Day 12 and stained for pre-synaptic marker synaptophysin (SYP) and post-synaptic marker PSD-95. The nuclei were counterstain with Hoechst dye. Shown is the representative image from 3 plates. (d) No inter-compartment leakage between the chambers. Vero cells were cultured in one chamber with positive hydrostatic pressure, and H129-G4 was added into the opposite chamber to a final concentration of 2.5 × 109 pfu/ml. The GFP signal in the Vero cell culture chamber was monitored, and show is the representative image at 72 hpi. (PDF 676 kb)

Invasion and transmission of H129-G4 in the VPM-S1 circuit. (a) Simplified schema of the VPM-S1 circuit. VPM, ventral posteromedial thalamic nucleus; nRT, nucleus of reticular thalamus; S1, primary somatosensory cortex; IV, V and VI, layer 4, 5 and 6 of the cortex. (b) Representative tracing results of H129-G4 in VPM-S1 circuit. H129-G4 (1 × 106 pfu in 200 nl) was injected into the VPM of wild-type C57BL/6 mice together with Alexa Fluor 594-conjugated CTB (CTB, red). The animals were perfused at the indicated time points, and representative images of the coronal brain slice at the VPM-S1 regions are shown. The boxed areas are magnified and presented in the right panel. The layers of the S1 cortex were determined according to NeuN staining and indicated by the dotted lines. (PDF 503 kb)

Terminal invasion of H129-G4 in vitro (a) Dose related incidence of H129-G4 terminal invasion. Fetal mouse hippocampal and cortical neurons were cultured as described above, H129-G4 was added to the terminal chamber at the indicated final concentrations, and GFP signal was monitored daily. Representative images at 48 hpi from 3 plates at each concentration group are shown. The labeled neurons are indicated with the dotted boxes and magnified in the lower panels. Scale bar = 100 μm. (b) Census of H129-G4 terminal invasion. Hippocampal and cortical neurons, trigeminal ganglion (TG) and dorsal root ganglion neurons (DRG) were cultured in microfluidic plates at the indicated cell amount. H129-G4 was added to the axonal terminal chamber at different final concentrations, and the GFP positive neurons in each plate were counted. Data were from 3 plates under each condition, and results are presented as mean ± SD. (c) Terminal invasion of VSV. Similarly, VSV-GFP was added into the terminal chamber of hippocampal and cortical neurons to a final concentration of 1 × 107 pfu/ml. The representative image from 3 plates at 48 hpi is shown. Scale bar = 100 μm. (PDF 429 kb)

Invasion and transmission of H129-G4 in the visual pathway. Different amount of H129-G4 was injected into the left LGN (a) or V1 (b) of wild-type C57BL/6 mice together with CTB, respectively. The animals were perfused at the indicated time points, and the coronal brain slices throughout the entire brains were observed. Representative images at V1, LGN and retinas are presented. (PDF 1400Â kb)

A new flavonoid glycoside, eriodictyol 7-O-(6″-caffeoyl)-β-D-glucopyranoside (1), along with 14 known compounds, were isolated from the whole plants of Elsholtzia bodinieri. All of the structures were determined by spectroscopic methods and chemical transformation. Compound 1 and luteolin (9) exhibited potent anti-HCV activities with a selective index of 135.85 and 20.84, respectively.

Hepatitis C virus (HCV) is a prevalent and globally distributed human pathogen that currently infects an estimated 170 million people. Chronic HCV infection significantly increases the risk of chronic hepatitis. The virus exhibits a very high degree of genetic diversity that is classified six genotypes and sub-classified more than 80 subtypes by phylogenetic analysis. The various genotypes and subtypes of HCV have been associated with different epidemiological and geographical spread patterns. Genotypes 1 and 2 are globally distributed; genotype 3 is predominant in Asia, North America and parts of Europe; similar regional patterns of endemic diversity have been found for genotype 4 in Europe, Middle East and Central Africa, for genotype 5 in parts of Africa and Europe, and for genotype 6 in Southeast Asia and North America. Up to date, four HCV genotypes, including genotype 1, 2, 3 and 6, were identified in China. Genotypes 1b and 2a were mainly found in Northern China. The fast spreading of genotypes 3 and 6 in Southern and South-Western China was reported by recent studies. It's deduced that Yunnan may become an important source of HCV epidemic and spread, which will cause great changes in the distribution of HCV genotypes and subtypes, and a variety of transmission. With the development of evolution theory and related analysis methods, such as coalescent theory and evolutionary molecular, it is possible to understand the characters of virus evolution and migration. For HCV, the further understanding on the prevalence and evolution characters of various genotypes is very important for the deep investigation of HCV epidemic and the development of prevention strategy.

The landfill leachate is a kind of complicated waste water which is hard to be treated at present. In this paper seven dominant stains that can effectively degrade COD in leachate were isolated by increasing the concentration of leachate. The influence of treated time, temperature, pH on treatment efficiency of leachate with these dominant stains were studied. The results showed that five bacterial strains, one Actinomyces strain and one mould strain possess fairly high rate to eliminate COD in leachate. The optimum time of degradation of COD in leachate was 3 days, and the pH was suitable for strains which the best was 7. In a broad temperature range from 25°C to 35°C, the COD in leachate could be effectively degraded by all of seven strains.

The incidence of algae blooms is increasing in China and worldwide, causing serious environmental problem. A protozoan P1 was isolated from the water samples of Lake Dianchi, which had potent grazing effect on some kind of cyanobacteria. It should be belonged to Ciliophore, Oligohymenophorea, Philasterida based on morphological characters observed by the optical microscope. The main influencing factors of the protozoan grazing properties including: algae species, protozoan density, culture temperature, time and pH value were investigated in batch experiments. The results showed that the protozoan P1 had strong ability to graze the cells of Microcystis wesenbergii. The greater the initial protozoan density, the faster the degradation of chlorophyll-a. The removal rate of chlorophyll-a after 4 days was 78% at the optimum temperature 25°C, pH 7 when using a lower inoculation quantity of 2%. The protozoan P1 was easy to cultivate and reproduce rapidly. It could be applied in the ecological environment to control the Microcystis water bloom.

Two new ursane-type triterpenoid saponins, bodiniosides M (1) and N (2), along with three known saponins, oblonganosides I (3), pseudobuxussaponin B (4) and bodinioside A (5), were isolated from the aerial parts of Elsholtzia bodinieri. The structures of compounds 1 and 2 were characterized by spectroscopic data as well as acid hydrolysis and GC analysis as 3-O-β-D-xylopyranosyl-19α-hydroxy-23-acetoxy-urs-12(13)-en-28-oic acid 28-O-α-L-rhamnopyranosyl-(1-2)-β-D-glucopyranoside and 3-O-β-D-glucopyranosyl-2α,19α-dihydroxy-urs-12(13)-en-28,20β-lactone. Compounds 1 and 5 exhibited potent anti-HCV activities in vitro with a selective index of 6.53 and 4.41, respectively.

Cyanobacteria are widespread in eutrophic freshwater lakes and can produce potent toxins which pose serious risk for human and animal health, aquatic ecosystem sustainability and economic vitality. There are 9 major lakes, which eutrophication and related ecological problem had occurred in most of these lakes in Yunnan, China. In this study, water samples were collected at different sampling time from five freshwater plateau lakes, which located in the middle area of Yunnan province, to assess the cyanobacteria diversity vary with the seasons. The morphological character of cyanobacteria were observed by light microscope (LM), the genetic diversity of cyanobacteria had been demonstrated by constructed phylogenetic trees based on DNA sequence of cyanobacteria 16S-23S rRNA internal space (ITS).The results showed that a variety of cyanobacterial species were distributed in five plateau freshwater lakes. The cyanobacterial population consisted mainly of several Microcystis species as dominants in Dianchi Lake and the dominant species of cyanobacteria found in June to October of 2009 was Microcystis aeruginosa. The diversity of cyanobacteria in Caohai was higher than that of Waihai in Dianchi Lake.

Single-strand conformation polymorphism (SSCP) method for detecting of HVR1 quasi-species of HCV is necessary for hepatitis C diagnosis. In this study, we described a rapid and sensitive SSCP method to analyze HCV quasi-species using common equipment. After RT-PCR reaction, denaturing was conducted with heating and Chilling. By varying the electrophoresis temperature, voltage and total acrylamide concentration，the extent of cross-linking in the gels (1-2.5%) and the concentration in glycerol ( 0-10％), SSCP method was established. With the optimized procedure, variants representing 2-3% of the whole quasi-species could be detected. By isolating visible bands from electrophoresis gel followed by sequencing, the genetic variability of HCV can be concluded. This method has high accuracy and reproducibility, simplicity and low cost, which could be a useful tool in laboratory practice.

Inorganic antibacterial materials consist of the antibacterial ions, the additives and the carrier. In this study, we synthesized a new inorganic antibacterial material, of which Ag+ was selected to be the antibacterial ion, lanthanum nitrate served as the additives, and the white carbon black was chosen as the carrier, which was prepared by a sol-gel method. The as-synthesized antibacterial material was characterized by inductively coupled plasma, particle size measurement instrument, and enumeration tests. The result showed that this material has loose and dispersive structure, good thermal and light stability. The possible antibacterial mechanism was also proposed through all the experimental data in this study.

Inherited cardiomyopathy is the most common hereditary cardiac disease. It also causes a significant proportion of sudden cardiac deaths in young adults and athletes. So far, approximately one hundred genes have been reported to be involved in cardiomyopathies through different mechanisms. Therefore, the identification of the genetic basis and disease mechanisms of cardiomyopathies are important for establishing a clinical diagnosis and genetic testing. Next-generation semiconductor sequencing (NGSS) technology platform is a high-throughput sequencer capable of analyzing clinically derived genomes with high productivity, sensitivity and specificity. It was launched in 2010 by Life Technologies of USA, and it is based on a high density semiconductor chip, which was covered with tens of thousands of wells. NGSS has been successfully used in candidate gene mutation screening to identify hereditary disease. In this review, we summarize these genetic variations, challenge and application of NGSS in inherited cardiomyopathy, and its value in disease diagnosis, prevention and treatment.

Human/simian immunodeficiency virus (HIV/SIV) infection can cause severe depletion of CD4(+) T cells in both plasma and mucosa; it also results in damage to the gut mucosa barrier, which makes the condition more conducive to microbial translocation. In this study, we used SIV-infected Chinese rhesus macaques to quantify the extent of microbial translocation and the function of immune cells in the entire gastrointestinal tract and to compare their differences between rapid and slow progressors. The results showed that in the slow progressors, microbial products translocated considerably and deeply into the lamina propria of the gut; the tissue macrophages had no significant differences compared with the rapid progressors, but there was a slightly higher percentage of mucosal CD8(+) T cells and a large amount of extracellular microbial products in the lamina propria of the intestinal mucosa of the slow progressors. The data suggested that although microbial translocation increased markedly, the mucosal macrophages and CD8(+) T cells were insufficient to clear the infiltrated microbes in the slow progressors. Also, therapies aimed at suppressing the translocation of microbial products in the mucosa could help to delay the progression of SIV disease.

Hepatitis C virus (HCV) is the etiological factor for Hepatitis C, which is one of the most important pathogenic factors of chronic liver diseases, cirrhosis and even hepatocellular carcinoma. HCV infection brings great threat to human health. Host genetic background could impact HCV infection, viral clearance, and treatment. Recently, some genome-wide association studies (GWAS) of HCV patients were performed. The results showed that single nucleotide polymorphisms (SNPs) of the IL28B gene, which encodes protein IFN-lambda3, are associated with viral clearance and treatment effectiveness of HCV patients who were cured by PEG-IFNalpha combined with ribavirin (RBV). IFN-lambda3 interacts with its acceptor, a heterodimer (IFN-lambdaR1 x IL-10R2), and upregulates the IFN-stimulated gene factors (ISGF). IFN-lambda3 plays roles in antiviral, antitumor, and immunoloregulation, and thus it might become a potential drug for Hepatitis C treatment. However, the mechanism of the IL28B gene in HCV infection and treatment is unclear, and further studies are needed to reveal the veils and provide theoretical basis for developing a new antiviral drug in clinic.

Hepatitis C virus is a prevalent and globally distributed human pathogen that seriously harmful to public health. However, the development of therapy and vaccine was impeded by the lack of suit small animal models. Herein, we introduce the characters of HCV replication. Taken the HCV cellular receptors as the viewpoint, the potentiality of tupaia as hepatitis C animal model is discussed at the molecular level by comparing of present animal model.

The use of tree shrews (Tupaia belangeri) in human disease studies demands essential research tools, in particular cellular markers and their monoclonal antibodies for immunological studies. Here we cloned the full-length cDNAs encoding CD3E from total RNA of the spleen, liver and peripheral blood of tree shrews and analyzed their structural characteristics in comparison with other mammals by Discovery Studio software. The results showed that the open reading frame sequence of tree shrew CD3E was 582 bp, encoding 194 amino acids. The overall structure of tree shrew CD3E protein was similar to its counterparts of other mammals, intracellular and transmembrane domain highly conserved. However, detailed analysis revealed two potential glycosylation sites and different surface charges in the extracellular domain. Availability of the entire open-reading-frame and related sequence information would therefore facilitate the preparation of monoclonal antibodies against tree shrew CD3 and further studies for its function.