Methodology for Trace and Ultratrace Analysis of Primary Amines

In the study of lipids, or lipidomics, methods for the separation and identification of specific trace compounds are highly sought. Microdroplet techniques have allowed for the ability to handle and detect these trace amounts. The use of low volumes allows for a decrease of the effective mean free path allowing chemical reactions to be carried out efficiently at lower concentrations by performing the reactions in microdroplets as opposed to bulk containers. Microdroplets created on microfluidic devices as segmented flow plugs have the advantage of efficient mixing and minimal dilution or dispersion relative to other nanoliter scale capillary reaction methods. The most sensitive detection techniques are needed for ultratrace analyses. Laser induced fluorescence (LIF) is the most attractive choice for ultratrace analysis. We show two methods for the derivatization and detection of primary amines with 3-(2-furoyl)quinoline-2-carboxaldehyde (FQCA) and with naphthalene-2,3-dicarboxaldehyde (NDA). The method has shown to be successful down to the sub-picomolar level in bulk solutions using an HPLC coupled with a fluorescence detector. The method has been transferred to microfluidic chips to explore the reaction and detection limits of the derivatized amines. Laser induced fluorescence (LIF) by a solid state blue violet laser was used as the detection method for the microfluidic platform. Successful usage of this methodology would allow for ultratrace detection of bioactive amines from small samples

Murine Model of Clostridium difficile Infection with Aged Gnotobiotic C57BL/6 Mice and a BI/NAP1 Strain

The increased incidence and severity of Clostridium difficile infection (CDI) in older adults (age, ⩾65 years) corresponds with the emergence of the BI/NAP1 strain, making elucidation of the host immune response extremely important. We therefore infected germ-free C57BL/6 mice aged 7–14 months with a BI/NAP1 strain and monitored the mice for response. Infected mice were moribund 48–72 h after infection and developed gross and histological cecitis and colitis and elevated concentrations of keratinocyte chemoattractant, interleukin 1β, monocyte chemotactic protein 1, and granulocyte colony-stimulating factor and decreased levels of interferon γ, interleukin 12 p40, interleukin 12 p70, and interleukin 10 compared with controls. We conclude that aged, germ-free C57BL/6 mice are susceptible to fulminant CDI from a BI/NAP1 strain and represent a novel model to further elucidate the host immune response to acute CDI

Effects of adenosine A2A receptor activation and alanyl-glutamine in Clostridium difficile toxin-induced ileitis in rabbits and cecitis in mice

<p>Abstract</p> <p>Background</p> <p>Severe <it>Clostridium difficile </it>toxin-induced enteritis is characterized by exuberant intestinal tissue inflammation, epithelial disruption and diarrhea. Adenosine, through its action on the adenosine A_2Areceptor, prevents neutrophillic adhesion and oxidative burst and inhibits inflammatory cytokine production. Alanyl-glutamine enhances intestinal mucosal repair and decreases apoptosis of enterocytes. This study investigates the protection from enteritis by combination therapy with ATL 370, an adenosine A_2Areceptor agonist, and alanyl-glutamine in a rabbit and murine intestinal loop models of <it>C. difficile </it>toxin A-induced epithelial injury.</p> <p>Methods</p> <p>Toxin A with or without alanyl-glutamine was administered intraluminally to rabbit ileal or murine cecal loops. Animals were also given either PBS or ATL 370 parenterally. Ileal tissues were examined for secretion, histopathology, apoptosis, Cxcl1/KC and IL-10.</p> <p>Results</p> <p>ATL 370 decreased ileal secretion and histopathologic changes in loops treated with Toxin A. These effects were reversed by the A_2Areceptor antagonist, SCH 58261, in a dose-dependent manner. The combination of ATL 370 and alanyl-glutamine significantly further decreased ileal secretion, mucosal injury and apoptosis more than loops treated with either drug alone. ATL 370 and alanyl-glutamine also decreased intestinal tissue KC and IL-10.</p> <p>Conclusions</p> <p>Combination therapy with an adenosine A_2Areceptor agonist and alanyl-glutamine is effective in reversing <it>C. difficile </it>toxin A-induced epithelial injury, inflammation, secretion and apoptosis in animals and has therapeutic potential for the management of <it>C. difficile </it>infection.</p

A global analysis of Y-chromosomal haplotype diversity for 23 STR loci

In a worldwide collaborative effort, 19,630 Y-chromosomes were sampled from 129 different populations in 51 countries. These chromosomes were typed for 23 short-tandem repeat (STR) loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385ab, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, GATAH4, DYS481, DYS533, DYS549, DYS570, DYS576, and DYS643) and using the PowerPlex Y23 System (PPY23, Promega Corporation, Madison, WI). Locus-specific allelic spectra of these markers were determined and a consistently high level of allelic diversity was observed. A considerable number of null, duplicate and off-ladder alleles were revealed. Standard single-locus and haplotype-based parameters were calculated and compared between subsets of Y-STR markers established for forensic casework. The PPY23 marker set provides substantially stronger discriminatory power than other available kits but at the same time reveals the same general patterns of population structure as other marker sets. A strong correlation was observed between the number of Y-STRs included in a marker set and some of the forensic parameters under study. Interestingly a weak but consistent trend toward smaller genetic distances resulting from larger numbers of markers became apparent.Peer reviewe

Exploring the clinical utility of ammonia in critically ill patients with cirrhosis: More to do? Authors' reply

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Identification and Classification of bcl Genes and Proteins of Bacillus cereus Group Organisms and Their Application in Bacillus anthracis Detection and Fingerprinting▿ †

The Bacillus cereus group includes three closely related species, B. anthracis, B. cereus, and B. thuringiensis, which form a highly homogeneous subdivision of the genus Bacillus. One of these species, B. anthracis, has been identified as one of the most probable bacterial biowarfare agents. Here, we evaluate the sequence and length polymorphisms of the Bacillus collagen-like protein bcl genes as a basis for B. anthracis detection and fingerprinting. Five genes, designated bclA to bclE, are present in B. anthracis strains. Examination of bclABCDE sequences identified polymorphisms in bclB alleles of the B. cereus group organisms. These sequence polymorphisms allowed specific detection of B. anthracis strains by PCR using both genomic DNA and purified Bacillus spores in reactions. By exploiting the length variation of the bcl alleles it was demonstrated that the combined bclABCDE PCR products generate markedly different fingerprints for the B. anthracis Ames and Sterne strains. Moreover, we predict that bclABCDE length polymorphism creates unique signatures for B. anthracis strains, which facilitates identification of strains with specificity and confidence. Thus, we present a new diagnostic concept for B. anthracis detection and fingerprinting, which can be used alone or in combination with previously established typing platforms