Precise characterization method of antibody‐conjugated magnetic nanoparticles for pathogen detection using stuffer‐free multiplex ligation‐dependent probe amplification

Antibody‐conjugated magnetic nanoparticles (Ab‐MNPs) have potential in pathogen detection because they allow target cells to be easily separated from complex sample matrices. However, the sensitivity and specificity of pathogen capture by Ab‐MNPs generally vary according to the types of MNPs, antibodies, and sample matrices, as well as preparation methods, including immobilization. Therefore, achieving a reproducible analysis utilizing Ab‐MNPs as a pathogen detection method requires accurate characterization of Ab‐MNP capture ability and standardization of all handling processes. In this study, we used high‐resolution CE‐single strand conformational polymorphism coupled with a stuffer‐free multiplex ligation‐dependent probe amplification system to characterize Ab‐MNPs. The capture ability of Ab‐MNPs targeting Salmonella enteritidis and nine pathogens, including S. enteritidis, was analyzed in phosphate buffer and milk. The effect of storage conditions on the stability of Ab‐MNPs was also assessed. The results showed that the stuffer‐free multiplex ligation‐dependent probe amplification system has the potential to serve as a standard characterization method for Ab‐MNPs. Moreover, the precise characterization of Ab‐MNPs facilitated robust pathogen detection in various applications.     

Rapid and sensitive detection of lower respiratory tract infections by stuffer‐free multiplex ligation‐dependent probe amplification

Lower respiratory tract infection is one of the most common infectious diseases. However, conventional methods for detecting infectious pathogens are time‐consuming, and generally have a limited impact on early therapeutic decisions. We previously reported a rapid and sensitive method for detecting such pathogens using stuffer‐free multiplex ligation‐dependent probe amplification coupled with high‐resolution CE‐SSCP. In this study, we report an application of this method to the detection of respiratory pathogens. As originally configured, this method was capable of simultaneously detecting seven bacterial species responsible for lower respiratory tract infections, but its detection limit and assay time were insufficient to provide useful information for early therapeutic decisions. To improve sensitivity and shorten assay time, we added a target‐specific preamplification step, improving the detection limit from 50 pg of genomic DNA to 500 fg. We further decreased time requirements by optimizing the hybridization step, enabling the entire assay to be completed within 7 h while maintaining the same detection limit. Taken together, these improvements enable the rapid detection of infectious doses of pathogens (i.e. a few dozen cells), establishing the strong potential of the refined method, particularly for aiding early treatment decisions.     

Editorial Board: Electrophoresis 10'14

Electrophoresis 2014, 35, 1504–1508. DOI: 10.1002/elps.201400001 Combination of DNA biobarcode assay with micro‐capillary electrophoretic analysis on a chip allows us to perform breast and colorectal cancer cell detection with high sensitivity, multiplexity, and accuracy.

     

Synthesis of novel 2,4,6,8,10-pentaaza[3.3.3]propellane derivatives

Propellanes and azapropellanes are attractive compounds for their unique structure and valuable applications but there are few synthetic studies for highly N-substituted azapropellanes. Several novel derivatives of 2,4,6,8,10-pentaaza[3.3.3]propellane, such as 3, 4, and 5 were successfully synthesized from readily available diethyl tartrate. Most of the synthetic steps were efficient. Their propellane structures were established by spectroscopic data and confirmed with single crystal X-ray analyses.     

Synthesis of tris(β,β,γ-oximinoalkyl)amines from aliphatic nitro compounds and methyl vinyl ketone

A general strategy for the assembly of previously unknown tris(β,β,γ-oximinoalkyl)amines from aliphatic nitro compounds and methyl vinyl ketone is described. The strategy involves N,N-bis(siloxy)enamines as key intermediates. The latter are accessible by double silylation of alkylnitro compounds. Nickel(II) and copper(II) complexes of tris(β,β,γ-oximinoalkyl)amines are prepared and structurally characterized.     

Rapid colorimetric detection of Salmonella typhimuriumusing a selective filtration technique combined with antibody–magnetic nanoparticle nanocomposites

Detection of pathogenic bacteria that pose a great risk to human health requires a rapid, convenient, reliable, and sensitive detection method. In this study, we developed a selective filtration method using monoclonal antibody (MAb)–magnetic nanoparticle (MNP) nanocomposites for the rapid and sensitive colorimetric detection of Salmonella typhimurium. The method contains two key steps: the immunomagnetic separation of the bacteria using MAb–MNP nanocomposites and the filtration of the nanocomposite-bound bacteria. Color signals from the nanocomposites remaining on the membrane were measured, which reflected the amount of bacteria in test samples. Immunomagnetic capture efficiencies of 8 to 90 % for various concentrations of the pathogen (2?×?10⁴–2?×?10¹ cells) were obtained. After optimization of the method, 2?×?10¹ cells of S. typhimurium in pure culture solution was detectable as well as in artificially inoculated vegetables (100 cells/g). The method was confirmed to be highly specific to S. typhimurium without cross-reaction to other pathogenic bacteria and could be concluded within 45 min, yielding results in a shorter or similar time period as compared with recently reported antibody immobilized on magnetic-particle-based methods. This study also demonstrated direct application of MAb–MNP nanocomposites without a dissociation step of bacteria from magnetic beads in colorimetric assays in practice.     

Phosphorescent Cyclometalated Iridium(III) Complexes That Contain Substituted 2‐Acetylbenzo[b]thiophen‐3‐olate Ligand for Red Organic Light‐Emitting Devices

We report the synthesis of a new class of thermally stable and strongly luminescent cyclometalated iridium(III) complexes 1 – 6 , which contain the 2‐acetylbenzo[b]thiophene‐3‐olate (bt) ligand, and their application in organic light‐emitting diodes (OLEDs). These heteroleptic iridium(III) complexes with bt as the ancillary ligand have a decomposition temperature that is 10–20 % higher and lower emission self‐quenching constants than those of their corresponding complexes with acetylacetonate (acac). The luminescent color of these iridium(III) complexes could be fine‐tuned from orange (e.g., 2‐phenyl‐6‐(trifluoromethyl)benzo[d]thiazole (cf₃bta) for 4 ) to pure red (e.g., lpt (Hlpt=4‐methyl‐2‐(thiophen‐2‐yl)quinolone) for 6 ) by varying the cyclometalating ligands (C‐deprotonated C^N). In particular, highly efficient OLEDs based on 6 as dopant (emitter) and 1,3‐bis(carbazol‐9‐yl)benzene (mCP) as host that exhibit stable red emission over a wide range of brightness with CIE chromaticity coordinates of (0.67, 0.33) well matched to the National Television System Committee (NTSC) standard have been fabricated along with an external quantum efficiency (EQE) and current efficiency of 9 % and 10 cd A^?1, respectively. A further 50 % increase in EQE (>13 %) by replacing mCP with bis[4‐(6H‐indolo[2,3‐b]quinoxalin‐6‐yl)phenyl]diphenylsilane (BIQS) as host for 6 in the red OLED is demonstrated. The performance of OLEDs fabricated with 6 (i.e., [(lpt)₂Ir(bt)]) was comparable to that of the analogous iridium(III) complex that bore acac (i.e., [(lpt)₂Ir(acac)]; 6 a in this work) [Adv. Mater.­ 2011 , 23, 2981] fabricated under similar conditions. By using ntt (Hnnt=3‐hydroxynaphtho[2,3‐b]thiophen‐2‐yl)(thiophen‐2‐yl)methanone) ligand, a substituted derivative of bt, the [(cf₃bta)₂Ir(ntt)] was prepared and found to display deep red emission at around 700 nm with a quantum yield of 12 % in mCP thin film.     

The frequency of CFVD speed report for highway traffic

The control signals of cellular networks have been used to infer the traffic conditions of the road network. In particular, consecutive handover events are being used to estimate the traffic speed. During traffic congestion, consecutive handover events may be rare because vehicles move slowly, and thus very few or no speed reports would be generated from the congested area.However, the traffic speed report rate during traffic congestion has not been investigated in the literature. In this paper, we present an analytic model to estimate the speed report rate from cellular network signaling in steady traffic conditions, that is, the traffic speed and flow are assumed constant. Real field trial data were used to validate our analytic model. In addition, computer simulations were conducted to study how speed reports are generated in dynamic traffic conditions when traffic speed and flow change rapidly. Our study indicates that in a typical cell of length 1.5 km with a typical expected call holding time of 1 min, no speed report was generated from a congested three‐lane highway. Our study demonstrates that the lack of speed reports from consecutive handover events during rush hours indicates severe traffic congestion, and new methods that can estimate traffic speed from cellular network data during severe traffic congestion need to be developed. Copyright © 2013 John Wiley & Sons, Ltd.     

Phytofabrication of silver nanoparticles from Limonia acidissima leaf extract and their antimicrobial,antioxidant and its anticancer prophecy

Green synthesis of nanoparticles by eco-friendly methods is a recent technique which draws the attention of researchers because of the reward over many conventional chemical methods. The present work focuses on aqueous Limonia acidissima leaf extract in synthesizing silver nanoparticles and its applications in a simple way. The silver nanoparticles formed were characterized by Infrared, Ultra violet-visible, X-ray diffraction, transmission electron microscopic, and atomic force microscopic techniques. The powder X-ray diffraction studies and transmission electron microscopic images reveal that the silver nanoparticles synthesized were approximately 10–40 nm and have a spherical structure. The nanoparticles were assayed for their antibacterial, antifungal and antioxidant activity. The antimicrobial studies for the silver nanoparticles show a maximum zone of inhibition of 8.8 mm for Bacillus subtilis bacteria and 8.5 mm for Candida albicans fungi at 3 and 1 μg/mL respectively. In-silico ADMET studies reveal that the toxicity, bioactivity, pharmacokinetics and drug-likeness properties of Limonia acidissima leaf extract is good. The molecular docking studies show that the microbial activity is high for Bacillus subtilis and Candida albicans showing the coincidence of the in silico and in vitro studies as expected. The free radical scavenging activity of nanoparticles is 80 for 100 μg/mL. The 50% of inhibition of silver nanoparticles against human breast cancer cell lines is 18 μg/mL. It is evident that silver nanoparticles would be helpful in treating cancer cell lines and have great perspectives in the biomedical sector.     

Investigation of Antifungal Mechanisms of Thymol in the Human Fungal Pathogen,Cryptococcus neoformans

Due to lifespan extension and changes in global climate, the increase in mycoses caused by primary and opportunistic fungal pathogens is now a global concern. Despite increasing attention, limited options are available for the treatment of systematic and invasive mycoses, owing to the evolutionary similarity between humans and fungi. Although plants produce a diversity of chemicals to protect themselves from pathogens, the molecular targets and modes of action of these plant-derived chemicals have not been well characterized. Using a reverse genetics approach, the present study revealed that thymol, a monoterpene alcohol from Thymus vulgaris L., (Lamiaceae), exhibits antifungal activity against Cryptococcus neoformans by regulating multiple signaling pathways including calcineurin, unfolded protein response, and HOG (high-osmolarity glycerol) MAPK (mitogen-activated protein kinase) pathways. Thymol treatment reduced the intracellular concentration of Ca²⁺ by controlling the expression levels of calcium transporter genes in a calcineurin-dependent manner. We demonstrated that thymol decreased N-glycosylation by regulating the expression levels of genes involved in glycan-mediated post-translational modifications. Furthermore, thymol treatment reduced endogenous ergosterol content by decreasing the expression of ergosterol biosynthesis genes in a HOG MAPK pathway-dependent manner. Collectively, this study sheds light on the antifungal mechanisms of thymol against C. neoformans.