Anaerobic Ammonium-Oxidizing (Anammox) Bacteria and Associated Activity in Fixed-Film Biofilters of a Marine Recirculating Aquaculture System

Microbial communities in the biological filter and waste sludge compartments of a marine recirculating aquaculture system were examined to determine the presence and activity of anaerobic ammonium-oxidizing (anammox) bacteria. Community DNA was extracted from aerobic and anaerobic fixed-film biofilters and the anaerobic sludge waste collection tank and was analyzed by amplifying 16S rRNA genes by PCR using anammox-selective and universal GC-clamped primers. Separation of amplified PCR products by denaturing gradient gel electrophoresis and sequencing of the different phylotypes revealed a diverse biofilter microbial community. While Planctomycetales were found in all three communities, the anaerobic denitrifying biofilters contained one clone that exhibited high levels of sequence similarity to known anammox bacteria. Fluorescence in situ hybridization studies using an anammox-specific probe confirmed the presence of anammox Planctomycetales in the microbial biofilm from the denitrifying biofilters, and anammox activity was observed in these biofilters, as detected by the ability to simultaneously consume ammonia and nitrite. To our knowledge, this is the first identification of anammox-related sequences in a marine recirculating aquaculture filtration system, and our findings provide a foundation for incorporating this important pathway for complete nitrogen removal in such systems.     

The P1812A and P25T BRCA1 and the 5164del4 BRCA2 mutations: occurrence in high-risk non-Ashkenazi Jews

Founder mutations in the BRCA1 and BRCA2 genes have been discovered in the Ashkenazic Jewish population, but a founder mutation(s) has not been discovered among non-Ashkenazi Jews (NAJ). Two BRCA1 mutations (P1812A, P25T), and a BRCA2 mutation (5164del4) have been detected in NAJ high-risk families. We studied the prevalence of these three mutations in 270 high-risk NAJ families, including 85 from Iraq/Iran, 67 from North Africa, 27 from Yemen, 50 from the Balkan region, and 41 with mixed ancestry. The three mutations were detected only in individuals related to the original families. We conclude that the P1812A and P25T BRCA1 and 5164del4 BRCA2 mutations are not likely to be founder mutations in NAJ high-risk families. We also assessed the pathogenicity of the BRCA1 P1812A mutation in vitro using reporter gene assays in yeast and mammalian cells. We found that the BRCA1 P1812A variant activity assays yielded a slightly reduced reporter gene activity. Thus, there is some uncertainty as to the pathogenicity of BRCA1 P1812A.     

Distance-Decay Relationships Partially Determine Diversity Patterns of Phyllosphere Bacteria on Tamrix Trees across the Sonoran Desert

Dispersal limitation in phyllosphere communities was measured on the leaf surfaces of salt-excreting Tamarix trees, which offer unique, discrete habitats for microbial assemblages. We employed 16S rRNA gene pyrosequencing to measure bacterial community dissimilarity on leaves of spatially dispersed Tamarix specimens in sites with uniform climatic conditions across the Sonoran Desert in the Southwestern United States. Our analyses revealed diverse bacterial communities with four dominant phyla that exhibited differential effects of environmental and geographic variables. Geographical distance was the most important parameter that affected community composition, particularly that of betaproteobacteria, which displayed a statistically significant, distance-decay relationship.     

Dynamic features of allosteric Ca2+ sensor in tissue-specific NCX variants

The Na(+)-Ca(2+) exchanger (NCX) mediated Ca(2+) fluxes are essential for handling Ca(2+) homeostasis in many cell-types. Eukaryotic NCX variants contain regulatory CBD1 and CBD2 domains, whereas in distinct variants the Ca(2+) binding to Ca3-Ca4 sites of CBD1 results either in sustained activation, inhibition or no effect. CBD2 contains an alternatively spliced segment, which is expressed in a tissue-specific manner although its impact on allosteric regulation remains unclear. Recent studies revealed that the Ca(2+) binding to Ca3-Ca4 sites results in interdomain tethering of CBDs, which rigidifies CBDs movements with accompanied slow dissociation of "occluded" Ca(2+). Here we investigate the effects of CBD2 variants on Ca(2+) occlusion in the two-domain construct (CBD12). Mutational studies revealed that both sites (Ca3 and Ca4) contribute to Ca(2+) occlusion, whereas after dissociation of the first Ca(2+) ion the second Ca(2+) ion becomes occluded. This mechanism is common for the brain, kidney and cardiac splice variants of CBD12, although the occluded Ca(2+) exhibits 20-50-fold difference in off-rates among the tested variants. Therefore, the spliced exons on CBD2 affect the rate-limiting step of the occluded Ca(2+) dissociation at the primary regulatory sensor to shape dynamic features of allosteric regulation in NCX variants.     

A Dominant Negative Heterozygous G87R Mutation in the Zinc Transporter, ZnT-2 (SLC30A2), Results in Transient Neonatal Zinc Deficiency

Zinc is an essential mineral, and infants are particularly vulnerable to zinc deficiency as they require large amounts of zinc for their normal growth and development. We have recently described the first loss-of-function mutation (H54R) in the zinc transporter ZnT-2 (SLC30A2) in mothers with infants harboring transient neonatal zinc deficiency (TNZD). Here we identified and characterized a novel heterozygous G87R ZnT-2 mutation in two unrelated Ashkenazi Jewish mothers with infants displaying TNZD. Transient transfection of G87R ZnT-2 resulted in endoplasmic reticulum-Golgi retention, whereas the WT transporter properly localized to intracellular secretory vesicles in HC11 and MCF-7 cells. Consequently, G87R ZnT-2 showed decreased stability compared with WT ZnT-2 as revealed by Western blot analysis. Three-dimensional homology modeling based on the crystal structure of YiiP, a close zinc transporter homologue from Escherichia coli, revealed that the basic arginine residue of the mutant G87R points toward the membrane lipid core, suggesting misfolding and possible loss-of-function. Indeed, functional assays including vesicular zinc accumulation, zinc secretion, and cytoplasmic zinc pool assessment revealed markedly impaired zinc transport in G87R ZnT-2 transfectants. Moreover, co-transfection experiments with both mutant and WT transporters revealed a dominant negative effect of G87R ZnT-2 over the WT ZnT-2; this was associated with mislocalization, decreased stability, and loss of zinc transport activity of the WT ZnT-2 due to homodimerization observed upon immunoprecipitation experiments. These findings establish that inactivating ZnT-2 mutations are an underlying basis of TNZD and provide the first evidence for the dominant inheritance of heterozygous ZnT-2 mutations via negative dominance due to homodimer formation.     

High mortality and female‐biased operational sex ratio result in low encounter rates and moderate polyandry in a spider

The taxonomy of the Old World bat genus Otomops (Chiroptera: Molossidae) has been the subject of considerable debate. The failure of classical morphological studies to provide consistent patterns regarding interspecific relationships within Otomops has limited any understanding of the evolutionary history of the genus. We used traditional and geometric morphometric approaches to establish the species limits of taxa from sub‐Saharan Africa, the Arabian Peninsula, and Madagascar. Morphometric data supported the recent recognition of three distinct Afrotropical taxa: Otomops madagascariensis from Madagascar; Otomops martiensseni s.s. from southern, eastern, central, and western Africa; and an undescribed taxon from north‐east Africa and the Arabian Peninsula. Analyses of craniodental measurements and landmark‐based data showed significant cranial size and shape divergence between the three taxa. Cranial size and shape variation within Afro‐Arabian Otomops were strongly influenced by altitude, seasonality of precipitation, and precipitation in the driest month. Based on morphometric patterns and molecular divergence estimates, we suggest that morphological evolution within Afro‐Arabian Otomops occurred in response to the fluctuating climate during the Pleistocene on the one hand, and the increasing aridity and seasonality over north‐eastern Africa on the other. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, •• , ••–••.     

Molecular identification, phylogenetic status, and geographic distribution of Culicoides oxystoma (Diptera: Ceratopogonidae) in Israel

Culicoides oxystoma (Diptera: Ceratopogonidae) is an important vector species, reported mainly from Asia, with high potential to transmit viral diseases affecting livestock. In Japan, many arboviruses have been isolated from C. oxystoma, suggesting it as a key player in the epidemiology of several Culicoides-borne diseases. Over the years, C. oxystoma has also been reported in the Middle East region, including Israel. In this region, however, C. oxystoma cannot be easily distinguished morphologically from its sibling species included in the Culicoides schultzei complex. We therefore used genomic data for species identification and phylogeny resolution. Phylogenetic analyses based on internal transcribed spacer 1 (ITS-1) of ribosomal DNA and the mitochondrial gene encoding cytochrome oxidase subunit I (COI) showed that C. oxystoma from Israel is closely related to C. oxystoma from Japan. Using differential probing PCR, we showed that C. oxystoma is distributed all over the country, especially in Mediterranean climate regions. Culicoides oxystoma is less common or even absent in arid regions, while the other genetic cluster of C. schultzei complex was found only in the east of the country (mostly arid and semiarid regions). The molecular finding of C. oxystoma in wide geographical regions, together with its high proportion in the general Culicoides population and its vectoring potential, imply that it may be an important vector species in the Middle East.     

A common Ca2+-driven interdomain module governs eukaryotic NCX regulation

Na(+)/Ca(2+) exchanger (NCX) proteins mediate Ca(2+)-fluxes across the cell membrane to maintain Ca(2+) homeostasis in many cell types. Eukaryotic NCX contains Ca(2+)-binding regulatory domains, CBD1 and CBD2. Ca(2+) binding to a primary sensor (Ca3-Ca4 sites) on CBD1 activates mammalian NCXs, whereas CALX, a Drosophila NCX ortholog, displays an inhibitory response to regulatory Ca(2+). To further elucidate the underlying regulatory mechanisms, we determined the 2.7 ? crystal structure of mammalian CBD12-E454K, a two-domain construct that retains wild-type properties. In conjunction with stopped-flow kinetics and SAXS (small-angle X-ray scattering) analyses of CBD12 mutants, we show that Ca(2+) binding to Ca3-Ca4 sites tethers the domains via a network of interdomain salt-bridges. This Ca(2+)-driven interdomain switch controls slow dissociation of "occluded" Ca(2+) from the primary sensor and thus dictates Ca(2+) sensing dynamics. In the Ca(2+)-bound conformation, the interdomain angle of CBD12 is very similar in NCX and CALX, meaning that the interdomain distances cannot account for regulatory diversity in NCX and CALX. Since the two-domain interface is nearly identical among eukaryotic NCXs, including CALX, we suggest that the Ca(2+)-driven interdomain switch described here represents a general mechanism for initial conduction of regulatory signals in NCX variants.