Round robin investigation of methods for recovering human enteric viruses from sludge

To select a tentative standard method for detection of viruses in sludge the American Society for Testing and Materials D19:24:04:04 Subcommittee Task Group initiated round robin comparative testing of two procedures that, after initial screening of several methodologies, were found to meet the basic criteria considered essential by the task group. Eight task group member laboratories agreed to perform round robin testing of the two candidate methods, namely, The Environmental Protection Agency or low pH-AlCl3 method and the Glass or sonication-extraction method. Five different types of sludge were tested. For each particular type of sludge, a single laboratory was designated to collect the sludge in a single sampling, make samples, and ship it to the participating laboratories. In most cases, participating laboratories completed all the tests within 48 h of sample arrival. To establish the reproducibility of the methods, each laboratory tested each sludge sample in triplicate for the two candidate virus methods. Each processed sludge sample was quantitatively assayed for viruses by the procedures of each individual round robin laboratory. To attain a more uniform standard of comparison, a sample of each processed sample from all laboratories was reassayed with one cell line and passage number by a single laboratory (Environmental Protection Agency Environmental Monitoring and Support Laboratory, Cincinnati, Ohio). When the data were statistically analyzed, the Environmental Protection Agency method was found to yield slightly higher virus recoveries for all sludge types, except the dewatered sludge. The precisions of both methods were not significantly different.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron microscopy of the skin of the teleost,Hippoglossoides elassodon

Summary The normal skin of the pleuronectid fish, Hippoglossoides elassodon, is described by light and electron microscopy. The epidermis consists of 5 to 9 layers of cells, the majority of which are squamous cells and the minority mucous cells. The squamous cells are characterized by numerous desmosomes and associated cytoplasmic filaments. The mucous cells accumulate mucous droplets in vacuoles of Golgi origin and are observed apparently in the process of releasing their content at the free surface. The dermis consists of alternating lamellae composed of typical collagen fibers. Pigment cells are of three types: melanophores, iridophores (guanophores), and lipophores.This work was supported by Public Health Service Research Grant CA-08158 from the National Cancer Institute.     

Isolation and identification of pathogenic Naegleria from Florida lakes.

Five cases of primary amoebic meningoencephalitis associated with swimming in freshwater lakes have been recorded in Florida over the past 14 years. The present study demonstrated that pathogenic Naegleria, the causative agent, is relatively widespread. Twelve of 26 lakes sampled only once yielded the amoeba. Populations in three of five lakes sampled routinely reached levels of one amoeba per 25 ml of water tested during the hot summer months. Overwintering in freshwater lake bottom sediments was demonstrated, showing that thermal-discharge pollution of waters plays a miniscule, if any, role in the maintenance of pathogenic Naegleria in nature in this semitropical area.     

Immunohistochemistry in the evaluation of neovascularization in tumor xenografts

Angiogenesis, or neovascularization, is known to play an important role in the neoplastic progression leading to metastasis. CD31 or Factor VIII-related antigen (F VIII RAg) immunohistochemistry is widely used in experimental studies for quantifying tumor neovascularization in immunocompromised animal models implanted with transformed human cell lines. Quantification, however, can be affected by variations in the methodology used to measure vascularization including antibody selection, antigen retrieval (AR) pretreatment, and evaluation techniques. To examine this further, we investigated the microvessel density (MVD) and the intensity of microvascular staining among five different human tumor xenografts and a mouse syngeneic tumor using anti-CD31 and F VIII RAg immunohistochemical staining. Different AR methods also were evaluated. Maximal retrieval of CD31 was achieved using 0.5 M Tris (pH 10) buffer, while maximum retrieval of F VIII RAg was achieved using 0.05% pepsin treatment of tissue sections. For each optimized retrieval condition, anti-CD31 highlighted small vessels better than F VIII RAg. Furthermore, the MVD of CD31 was significantly greater than that of F VIII RAg decorated vessels (p<0.001). The choice of antibody and AR method has a significant affect on immunohistochemical findings when studying angiogenesis. One also must use caution when comparing studies in the literature that use different techniques and reagents.     

<Emphasis Type="Italic">Yr32</Emphasis> for resistance to stripe (yellow) rust present in the wheat cultivar Carstens V

Stripe or yellow rust of wheat, caused by Puccinia striiformis f. sp. tritici, is an important disease in many wheat-growing regions of the world. A number of major genes providing resistance to stripe rust have been used in breeding, including one gene that is present in the differential tester Carstens V. The objective of this study was to locate and map a stripe rust resistance gene transferred from Carstens V to Avocet S and to use molecular tools to locate a number of genes segregating in the cross Savannah/Senat. One of the genes present in Senat was predicted to be a gene that is present in Carstens V. For this latter purpose, stripe rust response data from both seedling and field tests on a doubled haploid population consisting of 77 lines were compared to an available molecular map for the same lines using a non-parametric quantitative trait loci (QTL) analysis. Results obtained in Denmark suggested that a strong component of resistance with the specificity of Carstens V was located in chromosome arm 2AL, and this was consistent with chromosome location work undertaken in Australia. Since this gene segregated independently of Yr1, the only other stripe rust resistance gene known to be located in this chromosome arm, it was designated Yr32. Further QTLs originating from Senat were located in chromosomes 1BL, 4D, and 7DS and from Savannah on 5B, but it was not possible to characterize them as unique resistance genes in any definitive way. Yr32 was detected in several wheats, including the North American differential tester Tres.     

Dietary input of microbes and host genetic variation shape among-population differences in stickleback gut microbiota

To explain differences in gut microbial communities we must determine how processes regulating microbial community assembly (colonization, persistence) differ among hosts and affect microbiota composition. We surveyed the gut microbiota of threespine stickleback (Gasterosteus aculeatus) from 10 geographically clustered populations and sequenced environmental samples to track potential colonizing microbes and quantify the effects of host environment and genotype. Gut microbiota composition and diversity varied among populations. These among-population differences were associated with multiple covarying ecological variables: habitat type (lake, stream, estuary), lake geomorphology and food- (but not water-) associated microbiota. Fish genotype also covaried with gut microbiota composition; more genetically divergent populations exhibited more divergent gut microbiota. Our results suggest that population level differences in stickleback gut microbiota may depend more on internal sorting processes (host genotype) than on colonization processes (transient environmental effects).     

Caspase-mediated cleavage of the exosome subunit PM/Scl-75 during apoptosis

Recent studies have implicated the dying cell as a potential reservoir of modified autoantigens that might initiate and drive systemic autoimmunity in susceptible hosts. A number of subunits of the exosome, a complex of 3'→5' exoribonucleases that functions in a variety of cellular processes, are recognized by the so-called anti-PM/Scl autoantibodies, found predominantly in patients suffering from an overlap syndrome of myositis and scleroderma. Here we show that one of these subunits, PM/Scl-75, is cleaved during apoptosis. PM/Scl-75 cleavage is inhibited by several different caspase inhibitors. The analysis of PM/Scl-75 cleavage by recombinant caspase proteins shows that PM/Scl-75 is efficiently cleaved by caspase-1, to a smaller extent by caspase-8, and relatively inefficiently by caspase-3 and caspase-7. Cleavage of the PM/Scl-75 protein occurs in the C-terminal part of the protein at Asp369 (IILD³⁶⁹↓G), and at least a fraction of the resulting N-terminal fragments of PM/Scl-75 remains associated with the exosome. Finally, the implications of PM/Scl-75 cleavage for exosome function and the generation of anti-PM/Scl-75 autoantibodies are discussed.     

Identification of DNA sequence variation in Campylobacter jejuni strains associated with the Guillain-Barré syndrome by high-throughput AFLP analysis

Background  

Campylobacter jejuni is the predominant cause of antecedent infection in post-infectious neuropathies such as the Guillain-Barré (GBS) and Miller Fisher syndromes (MFS). GBS and MFS are probably induced by molecular mimicry between human gangliosides and bacterial lipo-oligosaccharides (LOS). This study describes a new C. jejuni-specific high-throughput AFLP (htAFLP) approach for detection and identification of DNA polymorphism, in general, and of putative GBS/MFS-markers, in particular.     

Novel Concepts for the Design and Manufacture of Solid Supports for Synthesis of Peptides

Two novel concepts for the design and manufacture of polymer supports for solid phase synthesis of peptides are described. The first concept involves the encapsulation of polymers within the hole of short pieces of capillary tubing often referred to as seed beads. This provides a rigid exo-skeleton for the support of soft polymer gel and other mechanically fragile polymer based matrices. The rigidity of the support provides a polymeric media that is particularly suited to continuous flow based peptide synthesis. The second concept complements this by providing an inexpensive approach to the preparation of spherical polymer particles by coating commercially available impervious hollow glass microspheres with polymer. The added advantage of this approach lies in the buoyancy of the resultant polymer particles, which facilitates handling on a large scale.