Phenazine virulence factor binding to extracellular DNA is important for Pseudomonas aeruginosa biofilm formation

Bacterial resistance to conventional antibiotics necessitates the identification of novel leads for infection control. Interference with extracellular phenomena, such as quorum sensing, extracellular DNA integrity and redox active metabolite release, represents a new frontier to control human pathogens such as Pseudomonas aeruginosa and hence reduce mortality. Here we reveal that the extracellular redox active virulence factor pyocyanin produced by P. aeruginosa binds directly to the deoxyribose-phosphate backbone of DNA and intercalates with DNA nitrogenous base pair regions. Binding results in local perturbations of the DNA double helix structure and enhanced electron transfer along the nucleic acid polymer. Pyocyanin binding to DNA also increases DNA solution viscosity. In contrast, antioxidants interacting with DNA and pyocyanin decrease DNA solution viscosity. Biofilms deficient in pyocyanin production and biofilms lacking extracellular DNA show similar architecture indicating the interaction is important in P. aeruginosa biofilm formation

Characterization of Botulinum Neurotoxin Type A Neutralizing Monoclonal Antibodies and Influence of Their Half-Lives on Therapeutic Activity

Botulinum toxins, i.e. BoNT/A to/G, include the most toxic substances known. Since botulism is a potentially fatal neuroparalytic disease with possible use as a biowarfare weapon (Centers for Disease Control and Prevention category A bioterrorism agent), intensive efforts are being made to develop vaccines or neutralizing antibodies. The use of active fragments from non-human immunoglobulins (F(ab')2, Fab', scFv), chemically modified or not, may avoid side effects, but also largely modify the in vivo half-life and effectiveness of these reagents. We evaluated the neutralizing activity of several monoclonal anti-BoNT/A antibodies (mAbs). F(ab')2 fragments, native or treated with polyethyleneglycol (PEG), were prepared from selected mAbs to determine their half-life and neutralizing activity as compared with the initial mAbs. We compared the protective efficiency of the different biochemical forms of anti-toxin mAbs providing the same neutralizing activity. Among fourteen tested mAbs, twelve exhibited neutralizing activity. Fragments from two of the best mAbs (TA12 and TA17), recognizing different epitopes, were produced. These two mAbs neutralized the A1 subtype of the toxin more efficiently than the A2 or A3 subtypes. Since mAb TA12 and its fragments both exhibited the greatest neutralizing activity, they were further evaluated in the therapeutic experiments. These showed that, in a mouse model, a 2- to 4-h interval between toxin and antitoxin injection allows the treatment to remain effective, but also suggested an absence of correlation between the half-life of the antitoxins and the length of time before treatment after botulinum toxin A contamination. These experiments demonstrate that PEG treatment has a strong impact on the half-life of the fragments, without affecting the effectiveness of neutralization, which was maintained after preparation of the fragments. These reagents may be useful for rapid treatment after botulinum toxin A contamination

Preferential Entry of Botulinum Neurotoxin A Hc Domain through Intestinal Crypt Cells and Targeting to Cholinergic Neurons of the Mouse Intestine

Botulism, characterized by flaccid paralysis, commonly results from botulinum neurotoxin (BoNT) absorption across the epithelial barrier from the digestive tract and then dissemination through the blood circulation to target autonomic and motor nerve terminals. The trafficking pathway of BoNT/A passage through the intestinal barrier is not yet fully understood. We report that intralumenal administration of purified BoNT/A into mouse ileum segment impaired spontaneous muscle contractions and abolished the smooth muscle contractions evoked by electric field stimulation. Entry of BoNT/A into the mouse upper small intestine was monitored with fluorescent HcA (half C-terminal domain of heavy chain) which interacts with cell surface receptor(s). We show that HcA preferentially recognizes a subset of neuroendocrine intestinal crypt cells, which probably represent the entry site of the toxin through the intestinal barrier, then targets specific neurons in the submucosa and later (90–120 min) in the musculosa. HcA mainly binds to certain cholinergic neurons of both submucosal and myenteric plexuses, but also recognizes, although to a lower extent, other neuronal cells including glutamatergic and serotoninergic neurons in the submucosa. Intestinal cholinergic neuron targeting by HcA could account for the inhibition of intestinal peristaltism and secretion observed in botulism, but the consequences of the targeting to non-cholinergic neurons remains to be determined

Toward biosensors for the detection of circulating microRNA as a cancer biomarker: An overview of the challenges and successes

Considerable attention has been dedicated to developing feasible point-of-care tests for cancer diagnosis and prognosis. An ideal biomarker for clinical use should be easily assayed with minimally invasive medical procedures but possess high sensitivity and specificity. The role of microRNAs (miRNAs) in the regulation of different cellular processes, the unique altered patterns in cancer patients and presence in body fluids in the stable form, points to their clinical utility as blood-based biomarkers for diagnosis, prognosis, and treatment of cancer. Although a variety of selective and sensitive laboratory-based methods are already exist for the detection of circulating miRNA, having a simple, low-cost and rapid assay, which could be routinely used in clinical practice, is still required. Among different approaches that have developed for circulating miRNA detection, biosensors, due to the high sensitivity, ease of use, short assay time, non-toxic experimental steps, and adaptability to point-of-care testing, exhibit very attractive properties for developing portable devices. With this view, we present an overview of some of the challenges that still need to be met to be able to use circulating miRNAs in clinical practice, including their clinical significance, sample preparation, and detection. In particular, we highlight the recent advances in the rapidly developing area of biosensors for circulating miRNA detection, along with future prospects and challenges

Analysis of Ground Water Fluoride Content and its Association with Prevalence of Fluorosis in Zarand/Kerman: (Using GIS)

Abstract: Statement of Problem: The concentration of fluoride in water is usually higher in areas around the coal mines. Zarand region in the south-east of Iran is known for its coal mines. Some studies have shown the high prevalence of fluorosis and some studies reported high levels of fluoride in the region. Objectives: This study aimed to use Geographic Information System (GIS) to assess the relationship between water fluoride content and the prevalence of fluorosis and its spatial distribution in Zarand region. Materials and Methods: This cross-sectional study aimed to recruit 550 people aged 7-40 years in Zarand. Dental examination for fluorosis was conducted based on the Dean’s Index. The level of fluoride in the water was determined in samples of water taken from 35 areas. Information on fluorosis and fluoride content was mapped on GIS. Results: Most participants lived in rural areas (87.25%) and had an educational status of high school level (66%). About 23% of the examined people had normal teeth, 10% had severe and 67% had mild to moderate fluorosis. Distribution of severe fluorosis was higher in areas with higher levels of fluoride in the water according to GIS map. Conclusions: GIS map clearly showed a positive relationship between the prevalence and severity of fluorosis with the level of fluoride in water in Zarand. The GIS analysis may be useful in the analysis of other oral conditions

A Comparison of Differently Synthesized Gold-coated Magnetic Nanoparticles as 'Dispersible Electrodes'

Gold-coated magnetic nanoparticles (Au@MNPs) have attracted significant interest in electrochemistry in recent years. This is especially the case with their application as dispersible electrodes where modified Au@MNPs are dispersed into a solution, selectively bind to the analyte of interest and are then brought to an electrode via application of a magnetic field for measurement. This paper characterizes four types of Au@MNPs with different sizes, shapes, and method of synthesis as dispersible electrodes. The Au@MNPs were characterized by transmission electron microscopy and X-ray photoelectron spectroscopy and scanning electron microscopy. In addition, the electrochemical behaviour of Au@MNPs was investigated using cyclic voltammetry. The four sorts of Au@MNPs were evaluated with regards to the three main features required in the dispersible electrodes approach, well-defined morphology, well-defined electrochemistry and fast response to a magnetic field. The Cubic-Au@MNPs, which presents the simplest synthetic route, showed the best electrochemical stability and performance, responding quickly to a magnet and had a well defined shape

Switching "on and off" faradaic electrochemistry at an otherwise passivated electrode using gold-coated magnetic nanoparticles

Herein we combined the use of self-assembled monolayer (SAM) modified electrodes and gold coated magnetic nanoparticles (Au@MNPs) to modulate faradaic electrochemistry when nanoparticles are absorbed on and removed from, a passivated electrode using an external magnetic field. Substantial faradaic electrochemistry of potassium ferricyanide in solution is first prevented by modifying gold electrodes with 11-mercaptoundecanoic acid to form a passivating SAM. Restoration of the faradaic electrochemistry is then achieved by introducing Au@MNPs which are brought to the surface using an external magnetic field. The faradaic electrochemistry can again be suppressed by removing Au@MNPs from the SAM using another external magnetic field

Analysis of Ground Water Fluoride Content and its Association with Prevalence of Fluorosis in Zarand/Kerman: (Using GIS)

STATEMENT OF PROBLEM: The concentration of fluoride in water is usually higher in areas around the coal mines. Zarand region in the south-east of Iran is known for its coal mines. Some studies have shown the high prevalence of fluorosis and some studies reported high levels of fluoride in the region. OBJECTIVES: This study aimed to use Geographic Information System (GIS) to assess the relationship between water fluoride content and the prevalence of fluorosis and its spatial distribution in Zarand region. MATERIALS AND METHODS: This cross-sectional study aimed to recruit 550 people aged 7-40 years in Zarand. Dental examination for fluorosis was conducted based on the Dean's Index. The level of fluoride in the water was determined in samples of water taken from 35 areas. Information on fluorosis and fluoride content was mapped on GIS. RESULTS: Most participants lived in rural areas (87.25%) and had an educational status of high school level (66%). About 23% of the examined people had normal teeth, 10% had severe and 67% had mild to moderate fluorosis. Distribution of severe fluorosis was higher in areas with higher levels of fluoride in the water according to GIS map. CONCLUSIONS: GIS map clearly showed a positive relationship between the prevalence and severity of fluorosis with the level of fluoride in water in Zarand. The GIS analysis may be useful in the analysis of other oral conditions

Quantitative determination of target gene with electrical sensor

Integrating loop-mediated isothermal amplification (LAMP) with capacitively coupled contactless conductivity detection (C4D), we have developed an electrical sensor for the simultaneous amplification and detection of specific sequence DNA. Using the O26-wzy gene as a model, the amount of initial target gene could be determined via the threshold time obtained by monitoring the progression of the LAMP reaction in real time. Using the optimal conditions, a detection limit of 12.5 copy/μL can be obtained within 30 min. Monitoring the LAMP reaction by C4D has not only all the advantages that existing electrochemical methods have, but also additional attractive features including being completely free of carryover contamination risk, high simplicity and extremely low cost. These benefits all arise from the fact that the electrodes are separated from the reaction solution, that is C4D is a contactless method. Hence in proof of principle, the new strategy promises a robust, simple, cost-effective and sensitive method for quantitative determination of a target gene, that is applicable either to specialized labs or at point-of-care