Evidences of lateral gene transfer between archaea and pathogenic bacteria

Acquisition of new genetic material through horizontal gene transfer has been shown to be an important feature in the evolution of many pathogenic bacteria. Changes in the genetic repertoire, occurring through gene acquisition and deletion, are the major events underlying the emergence and evolution of bacterial pathogens. However, horizontal gene transfer across the domains i.e. archaea and bacteria is not so common. In this context, we explore events of horizontal gene transfer between archaea and bacteria. In order to determine whether the acquisition of archaeal genes by lateral gene transfer is an important feature in the evolutionary history of the pathogenic bacteria, we have developed a scheme of stepwise eliminations that identifies archaeal-like genes in various bacterial genomes. We report the presence of 9 genes of archaeal origin in the genomes of various bacteria, a subset of which is also unique to the pathogenic members and are not found in respective non-pathogenic counterparts. We believe that these genes, having been retained in the respective genomes through selective advantage, have key functions in the organism’s biology and may play a role in pathogenesis.     

Electromembrane extraction of trace amounts of naltrexone and nalmefene from untreated biological fluids

Nalmefene and naltrexone are used to block the effects of narcotics and alcohol. In the present work, for the first time a microextraction technique was presented to reduce matrix interferences and improve detection limits of the drugs in urine and plasma samples. Electromembrane extraction (EME) followed by high performance liquid chromatography (HPLC) coupled with ultraviolet (UV) detection was optimized and validated for quantification of nalmefene and naltrexone from biological fluids. The membrane consists 85% of 2-nitrophenyl octyl ether (NPOE) and 15% di-(2-ethylhexyl) phosphate (DEHP) immobilized in the pores of a hollow fiber. A 100 V electrical field was applied to make the analytes migrate from sample solution with pH 2.0, through the supported liquid membrane (SLM) into an acidic acceptor solution with pH 1.0 which was located inside the lumen of hollow fiber. Extraction recoveries in the range of 54% and 75% were obtained in different biological matrices which resulted in preconcentration factors in the range of 109-149 and satisfactory repeatability (2.0相似文献   

A computational study of atomic oxygen-doped silicon carbide nanotubes

We investigated the properties of atomic oxygen-doped (O-doped) models of representative (6,0) and (4,4) silicon carbide nanotubes (SiCNTs) by density functional theory (DFT) calculations of isotropic and anisotropic chemical shielding (CS) parameters of Si-29, O-17 and C-13 atoms for the optimized structures. The calculated parameters indicated the effects of O-doping on the electronic environments of the first neighboring atoms of the doped sites. Comparing the results of the zigzag and armchair models also indicated that the latter model detects more effects of the O-doping than the former one.     

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum‐free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.Download video file.^{(69M, mov)}     

Proteomic analysis of the Mexican lime tree response to "Candidatus Phytoplasma aurantifolia" infection

"Candidatus Phytoplasma aurantifolia" is the causative agent of witches' broom disease in the Mexican lime tree (Citrus aurantifolia L.), and is responsible for major tree losses in Southern Iran and Oman. The pathogen is strictly biotrophic, and, therefore, completely dependent on living host cells for its survival. The molecular basis of compatibility and disease development in this system is poorly understood. We applied a proteomics approach to analyse gene expression in Mexican limes infected with "Ca. Phytoplasma aurantifolia". Leaf samples were collected from healthy and infected plants and were analysed using 2-DE coupled with MS. Among 800 leaf proteins that were detected reproducibly in eight biological replicates of healthy and eight biological replicates of infected plants, 55 showed a significant response to the disease. MS resulted in identification of 39 regulated proteins, which included proteins that were involved in oxidative stress defence, photosynthesis, metabolism, and the stress response. Our results provide the first proteomic view of the molecular basis of the infection process and identify genes that could help inhibit the effects of the pathogen.     

Purification of immature neuronal cells from neural stem cell progeny

Large-scale proliferation and multi-lineage differentiation capabilities make neural stem cells (NSCs) a promising renewable source of cells for therapeutic applications. However, the practical application for neuronal cell replacement is limited by heterogeneity of NSC progeny, relatively low yield of neurons, predominance of astrocytes, poor survival of donor cells following transplantation and the potential for uncontrolled proliferation of precursor cells. To address these impediments, we have developed a method for the generation of highly enriched immature neurons from murine NSC progeny. Adaptation of the standard differentiation procedure in concert with flow cytometry selection, using scattered light and positive fluorescent light selection based on cell surface antibody binding, provided a near pure (97%) immature neuron population. Using the purified neurons, we screened a panel of growth factors and found that bone morphogenetic protein-4 (BMP-4) demonstrated a strong survival effect on the cells in vitro, and enhanced their functional maturity. This effect was maintained following transplantation into the adult mouse striatum where we observed a 2-fold increase in the survival of the implanted cells and a 3-fold increase in NeuN expression. Additionally, based on the neural-colony forming cell assay (N-CFCA), we noted a 64 fold reduction of the bona fide NSC frequency in neuronal cell population and that implanted donor cells showed no signs of excessive or uncontrolled proliferation. The ability to provide defined neural cell populations from renewable sources such as NSC may find application for cell replacement therapies in the central nervous system.     

Evaluation of the effects of weak and moderate static magnetic fields on the characteristics of human low density lipoprotein in vitro

It has been suggested that exposure to electromagnetic fields may be a risk factor for cardiovascular disease in humans. Low density lipoprotein (LDL) modifications such as peroxidation and aggregation have been implicated in the pathogenesis of atherosclerosis. The present study investigated the effects of weak (0.125–0.5 mT) and moderate (1–4 mT) static magnetic fields (SMFs) on LDL oxidation, aggregation and zeta potential in vitro. Our results demonstrated that magnetic flux densities of 0.25 and 0.5 mT decreased, and magnetic flux densities of 3 and 4 mT increased the zeta potential and LDL oxidation in comparison with the control samples. All doses of SMFs increased the LDL aggregation in a time‐ and dose‐dependent manner. It is concluded that SMFs can alter the susceptibility of LDL to oxidation and this alteration is dependent on the applied magnetic flux density. The SMF, in addition to its role in the production and stabilization of free radicals and promotion of lipid peroxidation, may influence the metabolism of lipoproteins and their interaction with other molecules such as apolipoproteins, enzymes and receptors through the alteration of the LDL zeta potential and its particles tendency to aggregation. Bioelectromagnetics 34:397–404, 2013. © 2012 Wiley Periodicals, Inc.     

Association of Mycobacterium infections in patients with Mendelian susceptibility to mycobacterial disease with venous thromboembolism

An association between a hypercoagulable state and Mendelian susceptibility to mycobacterial disease (MSMD) has been established in a few studies; resultant thrombosis is considered rare. In a case‐control study, the prevalence of factor V Leiden, prothrombin G20210A and methylenetetrahydrofolate reductase (MTHFR) C677T, A1298C mutations were investigated in mycobacterium‐infected patients. The study comprised 30 patients with mycobacterial infections (invasive, disseminated and/or recurrent infections with Bacille Calmette–Guerin or non‐tuberculosis mycobacteria and Mycobacterium Tuberculosis with positive results for acid‐fast bacilli and tuberculin skin tests) and 30 normal healthy controls. Forty female (66.7%) and 20 male subjects (33.3%) aged from 3 to 70 years were recruited into this study. Genotyping of targeted genes was performed by RT‐PCR and cytokine TNF‐α concentrations were quantified using a commercially available ELISA kit. Significant associations between mycobacterial infection and TNF‐α production after stimulating peripheral blood mononuclear cells with LPS alone and with IFN‐γ plus LPS were identified. Moreover, genotyping analysis in the studied population revealed a significant association between MTHFR c.677C>T (OR, 3.28; 95% CI, 1.35–7.92; P < 0.05), MTHFR c.1298A>C (OR, 2.33; 95% CI, 1.10–4.93; P < 0.05) and mycobacterial infection in affected patients, indicating susceptibility to venous thromboembolism according to previous studies. Additionally, mycobacterium‐infected patients had a significantly greater prevalence of MTHFR C677T and A1298C mutations than controls.     

Biochemical and spectroscopic studies of human melanotransferrin (MTf): electron-paramagnetic resonance evidence for a difference between the iron-binding site of MTf and other transferrins

Melanotransferrin (MTf) is a member of the transferrin (Tf) family of iron (Fe)-binding proteins that was first identified as a cell-surface marker of melanoma. Although MTf has a high-affinity Fe-binding site that is practically identical to that of serum Tf, the protein does not play an essential role in Fe homeostasis and its precise molecular function remains unclear. A Zn(II)-binding motif, distinct from the Fe-binding site, has been proposed in human MTf based on computer modelling studies. However, little is known concerning the interaction of its proposed binding site(s) with metals and the consequences in terms of MTf conformation. For the first time, biochemical and spectroscopic techniques have been used in this study to characterise metal ion-binding to recombinant MTf. Initially, the binding of Fe to MTf was examined using 6M urea gel electrophoresis. Although four different iron-loaded forms were observed with serum Tf, only two forms were found with MTf, the apo-form and the N-monoferric holo-protein, suggesting a single high-affinity site. The presence of a single Fe(III)-binding site was also supported by EPR results which indicated that the Fe(III)-binding characteristics of MTf were unique, but somewhat comparable to the N-lobes of human serum Tf and chicken ovo-Tf. Circular dichroism (CD) analysis indicated that, as for Tf, no changes in secondary structure could be observed upon Fe(III)-binding. The ability of MTf to bind Zn(II) was also investigated using CD which demonstrated that the single high-affinity Fe-binding site was distinct from a potential Zn(II)-binding site.