Naegleria fowleri Detected in Grand Teton National Park Hot Springs.

The free-living thermophilic amoeba Naegleria fowleri (N. fowleri) causes the highly fatal disease primary amoebic meningoencephalitis. The environmental conditions that are favorable to the growth and proliferation of N. fowleri are not well-defined, especially in northern regions of the United States. In this study, we used culture-based methods and multiple molecular approaches to detect and analyzeN. fowleri and other Naegleria spp. in water, sediment, and biofilm samples from five hot spring sites in Grand Teton National Park, Wyoming, U.S.A. These results provide the first detections of N. fowleri in Grand Teton National Park and provide new insights into the distribution of pathogenic N. fowleri and other nonpathogenic Naegleria spp. in natural thermal water systems in northern latitudes.

Development and performance of a comprehensive targeted sequencing assay for pan-ethnic screening of carrier status.

Identifying individuals as carriers of severe disease traits enables informed decision making about reproductive options. Although carrier screening has traditionally been based on ethnicity, the increasing ethnic admixture in the general population argues for the need for pan-ethnic carrier screening assays. Highly multiplexed mutation panels allow for rapid and efficient testing of hundreds of mutations concurrently. We report the development of the Pan-Ethnic Carrier Screening assay, a targeted sequencing assay for routine screening that simultaneously detects 461 common mutations in 91 different genes underlying severe, early-onset monogenic disorders. Mutation selection was aided by the use of an extensive mutation database from a clinical laboratory with expertise in newborn screening and lysosomal storage disease testing. The assay is based on the Affymetrix GeneChip microarray platform but generates genomic DNA sequence as the output. Analytical sensitivity and specificity, using genomic DNA from archived control cultures and from clinical specimens, was found to be >99% for all mutation types. This targeted sequencing assay has advantages over multiplex PCR and next-generation sequencing assays, including accuracy of mutation detection over a range of mutation types and ease of analysis and reporting of results.

Conserved regional 3' grouping of rare codons in the coding sequence of ocular prosecretory mitogen lacritin.

PURPOSE: Lacritin is a prosecretory mitogen in tears and, although a tear protein, it promotes basal tearing and lacrimal gland secretion. Since scale up is relevant to its potential use in the treatment of dry eye, we explored various mutagenic strategies to alter the stability, solubility, and translational efficiency of nascent lacritin, and discovered 3' clustering of rare human codons. METHODS: Site-directed mutagenesis of lacritin coding cDNA "pLAC" generated 24 different nonsynonymous and 13 synonymous mutations. Nonsynonymous mutations altered amino acids with nonpolar, basic or acidic side chains to serine. Synonymous mutation progressively optimized human codons that are rare or uncommon in Escherichia coli without changing the amino acid specified. These changes were validated by sequencing and protein production, and analyzed via the "rare codon calculator" (RCC). Nonhuman primate and nonprimate lacritin coding sequences were extracted from Ensembl, and analyzed via RCC using codon usage appropriate for each species. RESULTS: Superior yields were obtained by modification of individual hydrophobic residues or a predicted salt bridge, suggesting that production was limited by lacritin stability. Accordingly, elimination of rare codons increased yields less effectively. Importantly, RCC analysis of human, nonhuman primate (mouse lemur) and nonprimate (cat, tree shrew) lacritin coding sequences revealed remarkable 3' clustering of rare codons, unlike human lipocalin-1 and 21 other widely expressed human tear genes. CONCLUSIONS: Lacritin protein yields were improved primarily by hydrophobic or salt bridge mutagenesis and less so by elimination of rare codons. The 3' clustering of rare codons is conserved in all lacritin orthologs examined.

Thermocrinis minervae sp. nov., a hydrogen- and sulfur-oxidizing, thermophilic member of the Aquificales from a Costa Rican terrestrial hot spring.

A thermophilic bacterium, designated strain CR11(T), was isolated from a filamentous sample collected from a terrestrial hot spring on the south-western foothills of the Rincón volcano in Costa Rica. The Gram-negative cells are approximately 2.4-3.9 microm long and 0.5-0.6 microm wide and are motile rods with polar flagella. Strain CR11(T) grows between 65 and 85 degrees C (optimum 75 degrees C, doubling time 4.5 h) and between pH 4.8 and 7.8 (optimum pH 5.9-6.5). The isolate grows chemolithotrophically with S(0), S(2)O(2)(3)(-) or H(2) as the electron donor and with O(2) (up to 16 %, v/v) as the sole electron acceptor. The isolate can grow on mannose, glucose, maltose, succinate, peptone, Casamino acids, starch, citrate and yeast extract in the presence of oxygen (4 %) and S(0). Growth occurs only at NaCl concentrations below 0.4 % (w/v). The G+C content of strain CR11(T) is 40.3 mol%. Phylogenetic analysis of the 16S rRNA gene sequence places the strain as a close relative of Thermocrinis ruber OC 1/4(T) (95.7 % sequence similarity). Based on phylogenetic and physiological characteristics, we propose the name Thermocrinis minervae sp. nov., with CR11(T) (=DSM 19557(T) =ATCC BAA-1533(T)) as the type strain.

Anaerobic oxidation of methane: mechanisms, bioenergetics, and the ecology of associated microorganisms.

Microbially mediated anaerobic oxidation of methane (AOM) moderates the input of methane, an important greenhouse gas, to the atmosphere by consuming methane produced in various marine, terrestrial, and subsurface environments. AOM coupled to sulfate reduction has been most extensively studied because of the abundance of sulfate in marine systems, but electron acceptors otherthan sulfate are more energetically favorable. Phylogenetic trees based on 16S rRNA gene clone libraries derived from microbial communities where AOM occurs show evidence of diverse, methanotrophic archaea (ANME) closely associated with sulfate-reducing bacteria, but these organisms have not yet been isolated as pure cultures. Several biochemical pathways for AOM have been proposed, including reverse methanogenesis, acetogenesis, and methylogenesis, and both culture-dependent and independent techniques have provided some clues to howthese communities function. Still, questions remain regarding the diversity, physiology, and metabolic restrictions of AOM-related organisms.

Monoclonal antibody to a cancer-specific and drug-responsive hydroquinone (NADH) oxidase from the sera of cancer patients.

Monoclonal antibodies were generated in mice to a 34-kDa circulating form of a drug-responsive hydroquinone (NADH) oxidase with a protein disulfide-thiol interchange activity specific to the surface of cancer cells and the sera of cancer patients. Screening used Western blots with purified 34-kDa tNOX from HeLa cells and the sera of cancer patients. Epitopes were sought that inhibited the drug-responsive oxidation of NADH with the sera of cancer patients, but which had no effect on NADH oxidation with the sera of healthy volunteers. Two such antisera were generated. One, designated monoclonal antibody (mAb) 12.1, was characterized extensively. The NADH oxidase activity inhibited by mAb 12.1 also was inhibited by the quinone site inhibitor capsaicin (8-methyl- N-vanillyl-6-noneamide). The inhibition was competitive for the drug-responsive protein disulfide-thiol interchange activity assayed either by restoration of activity to scrambled RNase or by cleavage of a dithiodipyridine substrate, and was uncompetitive for NADH oxidation. Both the mAb 12.1 and the postimmune antisera immunoprecipitated drug-responsive NOX activity and identified the same 34-kDa tNOX protein in the sera of cancer patients that was absent from sera of healthy volunteers, and was utilized as immunogen. Preimmune sera from the same mouse as the postimmune antisera was without effect. Both mouse ascites containing mAb 12.1 and postimmune sera (but not preimmune sera) slowed the growth of human cancer cell lines in culture, but did not affect the growth of non-cancerous cell lines. Immunocytochemical and histochemical findings showed that mAb 12.1 reacted with the surface membranes of human carcinoma cells and tissues.