The Development of a Novel qPCR Assay-Set for Identifying Fecal Contamination Originating from Domestic Fowls and Waterfowl in Israel.

The emerging microbial source tracking (MST) methodologies aim to identify fecal contamination originating from domestic and wild animals, and from humans. Avian MST is especially challenging, primarily because the Aves class includes both domesticated and wild species with highly diverse habitats and dietary characteristics. The quest for specific fecal bacterial MST markers can be difficult with respect to attaining sufficient assay sensitivity and specificity. The present study utilizes high throughput sequencing (HTS) to screen bacterial 16S rRNA genes from fecal samples collected from both domestic and wild avian species. Operational taxonomic unit (OTU) analysis was then performed, from which sequences were retained for downstream quantitative polymerase chain reaction (qPCR) marker development. Identification of unique avian host DNA sequences, absent in non-avian hosts, was then carried out using a dedicated database of bacterial 16S rRNA gene taken from the Ribosomal Database Project. Six qPCR assays were developed targeting the 16S rRNA gene of Lactobacillus, Gallibacterium, Firmicutes, Fusobacteriaceae, and other bacteria. Two assays (Av4143 and Av163) identified most of the avian fecal samples and demonstrated sensitivity values of 91 and 70%, respectively. The Av43 assay only identified droppings from battery hens and poultry, whereas each of the other three assays (Av24, Av13, and Av216) identified waterfowl species with lower sensitivities values. The development of an MST assay-panel, which includes both domestic and wild avian species, expands the currently known MST analysis capabilities for decoding fecal contamination.

Microbial Source Tracking in Adjacent Karst Springs.

Modern man-made environments, including urban, agricultural, and industrial environments, have complex ecological interactions among themselves and with the natural surroundings. Microbial source tracking (MST) offers advanced tools to resolve the host source of fecal contamination beyond indicator monitoring. This study was intended to assess karst spring susceptibilities to different fecal sources using MST quantitative PCR (qPCR) assays targeting human, bovine, and swine markers. It involved a dual-time monitoring frame: (i) monthly throughout the calendar year and (ii) daily during a rainfall event. Data integration was taken from both monthly and daily MST profile monitoring and improved identification of spring susceptibility to host fecal contamination; three springs located in close geographic proximity revealed different MST profiles. The Giach spring showed moderate fluctuations of MST marker quantities amid wet and dry samplings, while the Zuf spring had the highest rise of the GenBac3 marker during the wet event, which was mirrored in other markers as well. The revelation of human fecal contamination during the dry season not connected to incidents of raining leachates suggests a continuous and direct exposure to septic systems. Pigpens were identified in the watersheds of Zuf, Shefa, and Giach springs and on the border of the Gaaton spring watershed. Their impact was correlated with partial detection of the Pig-2-Bac marker in Gaaton spring, which was lower than detection levels in all three of the other springs. Ruminant and swine markers were detected intermittently, and their contamination potential during the wet samplings was exposed. These results emphasized the importance of sampling design to utilize the MST approach to delineate subtleties of fecal contamination in the environment.

Up-regulation of secretory leukocyte protease inhibitor (SLPI) in the brain after ischemic stroke: adenoviral expression of SLPI protects brain from ischemic injury.

Secretory leukocyte protease inhibitor (SLPI) is a 12-kDa secreted protein initially identified from epithelial cells as an inhibitor of leukocyte serine proteases. In the present study, we described the identification of SLPI expression in ischemic cortex by suppression subtractive hybridization strategy. Our full-length rat SLPI cDNA shares 81% and 63% amino acid sequence identity with its mouse and human homologs, respectively, and with several polymorphisms to previous reported rat sequences. Northern blot analysis confirmed that SLPI mRNA was significantly induced in the ischemic brain tissue at 12 h (5.1-fold increase over sham controls, n = 4, p < 0.05), peaked at 2 days (26.1-fold increase, p < 0.001), and sustained up to 5 days (5.1-fold increase, p < 0.05). SLPI was localized in neurons and astrocytes in the peri-infarct zone from 24 to 72 h after middle cerebral artery occlusion by means of immunohistochemical and confocal microscopy analysis. Administration of a recombinant adenovirus overexpressing SLPI (Adv/SLPI) into the cortical tissue resulted in up to 58.4% reduction in ischemic lesion over controls at the site of Adv/SLPI expression (p < 0.01, n = 8) and significantly improved functional outcome (p < 0.01). These data suggest that the ischemia-induced expression of SLPI might play a neuroprotective role in focal stroke, possibly because of rapid inhibition of activated proteases and its suppression in inflammatory response.

c-FLIP(L) is a dual function regulator for caspase-8 activation and CD95-mediated apoptosis.

Activation of the caspase cascade is a pivotal step in apoptosis and can occur via death adaptor-mediated homo-oligomerization of initiator procaspases. Here we show that c-FLIP(L), a protease-deficient caspase homolog widely regarded as an apoptosis inhibitor, is enriched in the CD95 death-inducing signaling complex (DISC) and potently promotes procaspase-8 activation through hetero-dimerization. c-FLIP(L) exerts its effect through its protease-like domain, which associates efficiently with the procaspase-8 protease domain and induces the enzymatic activity of the zymogen. Ectopic expression of c-FLIP(L) at physiologically relevant levels enhances procaspase-8 processing in the CD95 DISC and promotes apoptosis, while a decrease of c-FLIP(L) expression results in inhibition of apoptosis. c-FLIP(L) acts as an apoptosis inhibitor only at high ectopic expression levels. Thus, c-FLIP(L) defines a novel type of caspase regulator, distinct from the death adaptors, that can either promote or inhibit apoptosis.