Evaluation of propidium monoazide (PMA) treatment directly on membrane filter for the enumeration of viable but non cultivable Legionella by qPCR

A PMA (propidium monoazide) pretreatment protocol, in which PMA is applied directly to membrane filters, was developed for the PCR-based quantification (PMA-qPCR) of viable Legionella pneumophila. Using this method, the amplification of DNA from membrane-damaged L. pneumophila was strongly inhibited for samples containing a small number of dead bacteria.     

<Emphasis Type="Italic">parkin</Emphasis>mutation dosage and the phenomenon of anticipation: a molecular genetic study of familial parkinsonism

Background  

parkin mutations are a common cause of parkinsonism. Possessing two parkin mutations leads to early-onset parkinsonism, while having one mutation may predispose to late-onset disease. This dosage pattern suggests that some parkin families should exhibit intergenerational variation in age at onset resembling anticipation. A subset of familial PD exhibits anticipation, the cause of which is unknown. The aim of this study was to determine if anticipation was due to parkin mutation dosage.     

枯草芽孢杆菌HS-A38产抗菌脂肽Bacillomycin D的组分分析及鉴定

对一株枯草芽孢杆菌HS-A38产生的脂肽类物质进行分离鉴定及抑菌活性研究。通过酸沉淀分离和有机溶剂抽提的方法,从枯草芽孢杆菌HS-A38发酵液中得到脂肽粗提物LP,产率为1.956 g/L。利用薄层色谱和茚三酮染色法确定该脂肽粗提物中存在四个组分,分别为LP1、LP2、LP3和LP4;抑菌活性检测显示,组分LP3对两株海洋致病菌副溶血性弧菌和铜绿假单胞杆菌的活性较高。组分LP3经硅胶柱层析纯化分离后,应用反相高效液相色谱(RP-HPLC)和基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)对该组分做进一步纯化和鉴定。分析表明,LP3样品在保留时间20 min~28 min产生单峰团LP3-1,其纯度为85.24%;经MALDI-TOF-MS分析和数据比对,组分LP3-1中的主要成分为杆菌霉素Bacillomycin D。     

Clinical and environmental isolates of Legionella pneumophila serogroup 1 cannot be distinguished by sequence analysis of two surface protein genes and three housekeeping genes

We used gene sequencing to determine whether clinical (sporadic, epidemic, and endemic) and environmental isolates of Legionella pneumophila serogroup (sg) 1 belong to specific lineages. A total of 178 clinical and environmental L. pneumophila sg 1 isolates, defined by pulsed-field gel electrophoresis and epidemiological data as sporadic, epidemic, or endemic, were analyzed for polymorphisms in five gene fragments. The fragments belonged to three housekeeping genes (coding for aconitase [acn], aspartate-beta-semialdehyde dehydrogenase [asd], and RNA polymerase beta subunit [rpoB]) and two surface protein genes (coding for the macrophage infectivity potentiator [mip] and the major outer membrane protein [mompS]). The phylogenetic tree inferred from sequence polymorphisms of the five genes identified two large clusters, one consisting of 133 poorly differentiated strains and containing two smaller clusters (10 and 2 strains) unrelated to each other and the other consisting of 42 strains. Clinical and environmental isolates could not be distinguished on this basis, and no link between genetic background and epidemiological type was found, suggesting that other factors are responsible for differences in pathogenicity.     

Analysis of proteomic changes in colored mutants of Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

The yeast Xanthophyllomyces dendrorhous synthesizes astaxanthin as its most prevalent xanthophyll derivative. Comparisons between the protein profiles of mutant lines of this yeast can provide insight into the carotenogenic pathway. Differently colored mutants (red, orange, pink, yellow, and white) were obtained from this yeast species, and their protein profiles were determined using two-dimensional polyacrylamide gel electrophoresis (2DE). Individual proteins differentially expressed were identified using mass spectrometry. The red mutants hyperproduced total carotenoids (mainly astaxanthin), while in white and orange mutants, mutagenesis affected the phytoene dehydrogenase activity as indicated by the accumulation of phytoene. Inactivation of astaxanthin synthase after the mutagenic treatment was evident in β-carotene accumulating mutants. Differences in the proteomic profiles of wild-type X. dendrorhous and its colored mutants were demonstrated using 2DE. Of the total number of spots detected in each gel (297–417), 128 proteins were present in all strains. The red mutant showed the greatest number of matches with respect to the wild type (305 spots), while the white and yellow mutants, which had reduced concentrations of total carotenoids, presented the highest correlation coefficient (0.6) between each other. A number of differentially expressed proteins were sequenced, indicating that tricarboxylic acid cycle and stress response proteins are closely related to the carotenogenic process.     

Inhibition of proton pumping by zinc ions during specific reaction steps in cytochrome c oxidase

Cytochrome c oxidase (CytcO) is a redox-driven proton pump in the respiratory chain of mitochondria and many aerobic bacteria. The results from several studies have shown that zinc ions interfere with both the uptake and release of protons, presumably by binding near the orifice of the proton entrance and exit pathways. To elucidate the effect of Zn2+ binding on individual electron and proton-transfer reactions, in this study, we have investigated the reaction of the fully reduced R. sphaeroides CytcO with O2, both with enzyme in detergent solution and reconstituted in phospholipid vesicles, and, with and without, Zn2+. The results show that addition of Zn2+ at concentrations of < or = 250 microM to the outside of the vesicles did not alter the transition rates between intermediates PR (P3)-->F3-->O4. However, proton pumping was impaired specifically during the P3-->F3, but not during the F3-->O4 transition at Zn2+ concentrations of < or = 25 microM. Furthermore, proton pumping during the P3-->F3 transition was typically impaired with the "as isolated" CytcO, which was found to contain Zn2+ ions at microM concentration. As has already been shown, Zn2+ was also found to obstruct proton uptake during the P3-->F3 transition, presumably by binding to a site near the orifice of the D-pathway. In this work we found a KI of approximately 1 microM for this binding site. In conclusion, the results show that Zn2+ ions bind on both sides of CytcO and that binding of Zn2+ at the proton output side selectively impairs proton release during the P3-->F3 transition.     

Bioassay-guided discovery of antibacterial agents: in vitro screening of Peperomia vulcanica, Peperomia fernandopoioana and Scleria striatinux

Background

The global burden of bacterial infections is high and has been further aggravated by increasing resistance to antibiotics. In the search for novel antibacterials, three medicinal plants: Peperomia vulcanica, Peperomia fernandopoioana (Piperaceae) and Scleria striatinux (Cyperaceae), were investigated for antibacterial activity and toxicity.

Methods

Crude extracts of these plants were tested by the disc diffusion method against six bacterial test organisms followed by bio-assay guided fractionation, isolation and testing of pure compounds. The minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations were measured by the microdilution method. The acute toxicity of the active extracts and cytotoxicity of the active compound were performed in mice and mammalian cells, respectively.

Results

The diameter of the zones of inhibition (DZI) of the extracts ranged from 7?C13?mm on Escherichia coli and Staphylococcus aureus of which the methylene chloride:methanol [1:1] extract of Scleria striatinux recorded the highest activity (DZI?=?13?mm). Twenty-nine pure compounds were screened and one, Okundoperoxide, isolated from S. striatinux, recorded a DZI ranging from 10?C19?mm on S. aureus. The MICs and MBCs indicated that the Peperomias had broad-spectrum bacteriostatic activity. Toxicity tests showed that Okundoperoxide may have a low risk of toxicity with an LC₅₀ of 46.88???g/mL.

Conclusions

The antibacterial activity of these plants supports their use in traditional medicine. The pure compound, Okundoperoxide, may yield new antibacterial lead compounds following medicinal chemistry exploration.     

Acid-degradable cationic dextran particles for the delivery of siRNA therapeutics

We report a new acid-sensitive, biocompatible, and biodegradable microparticulate delivery system, spermine modified acetalated-dextran (Spermine-Ac-DEX), which can be used to efficiently encapsulate siRNA. These particles demonstrated efficient gene knockdown in HeLa-luc cells with minimal toxicity. This knockdown was comparable to that obtained using Lipofectamine, a commercially available transfection reagent generally limited to in vitro use due to its high toxicity.     

Uropathogenic Escherichia coli P and Type 1 fimbriae act in synergy in a living host to facilitate renal colonization leading to nephron obstruction

The progression of a natural bacterial infection is a dynamic process influenced by the physiological characteristics of the target organ. Recent developments in live animal imaging allow for the study of the dynamic microbe-host interplay in real-time as the infection progresses within an organ of a live host. Here we used multiphoton microscopy-based live animal imaging, combined with advanced surgical procedures, to investigate the role of uropathogenic Escherichia coli (UPEC) attachment organelles P and Type 1 fimbriae in renal bacterial infection. A GFP+ expressing variant of UPEC strain CFT073 and genetically well-defined isogenic mutants were microinfused into rat glomerulus or proximal tubules. Within 2 h bacteria colonized along the flat squamous epithelium of the Bowman's capsule despite being exposed to the primary filtrate. When facing the challenge of the filtrate flow in the proximal tubule, the P and Type 1 fimbriae appeared to act in synergy to promote colonization. P fimbriae enhanced early colonization of the tubular epithelium, while Type 1 fimbriae mediated colonization of the center of the tubule via a mechanism believed to involve inter-bacterial binding and biofilm formation. The heterogeneous bacterial community within the tubule subsequently affected renal filtration leading to total obstruction of the nephron within 8 h. Our results reveal the importance of physiological factors such as filtration in determining bacterial colonization patterns, and demonstrate that the spatial resolution of an infectious niche can be as small as the center, or periphery, of a tubule lumen. Furthermore, our data show how secondary physiological injuries such as obstruction contribute to the full pathophysiology of pyelonephritis.