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Divine Joy
A selection of articles related to divine joy.
Original articles from our library related to the Divine Joy. See Table of Contents for further available material (downloadable resources) on Divine Joy.
- Dionysos – A Gift of Abundance and Divine Inspiration
- At the end of the summer the four elements have worked their alchemy; a good balance between fertile earth, air of the right temperature, the fire of the sun and gentle rain showers will now have resulted in an abundant harvest. Flowers proudly open up to the...
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- When you hear the word Tantra, what images come to mind? Turbans and Sitar music? Mind-blowing sex? Most people in the Western world have heard the word Tantra, but few know anything in detail about what it means. The situation in India isn't much different....
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- Candlelight flickers over the Beltaine revels. Food is laid out in the circle for the feast. Only one rule – no one can feed themselves. Each is dependent on friends and loved ones for sustenance, joy and delight. After an hour of laughter and revels and way...
Paganism & Wicca >> Daily Life
- The Aeonic Perspective of the Enochian Temples
- The initiatory system of the Enochian Temples divides the path of initiation into four major stages, distinguished primarily by the types and sources of the influences they are sensitive to. Briefly, these stages are: 1. Lunar-planetary, focused in Yesod and...
Magick >> Enochian
- The Doctrine of Signatures as fingerprint of the Divine
- Researching substances in laboratories is a modern way of trying to understand the universe. The various components of a subject are taken apart, isolated and carefully studied. This is contrary to the old days, where people would observe a complete plant,...
Earth Mysteries >> Herbalism
- The Religious Experience: A Wiccan Viewpoint
- What is religion? Religion is a set of beliefs which allow us to understand and categorize our world and our place in it. A set of beliefs which define our culture, our expectations, our views of people and behaviors we expect. I have found several different...
Religion & Philosophy >> Religions
Divine Joy is described in multiple online sources, as addition to our editors' articles, see section below for printable documents, Divine Joy books and related discussion.
Suggested Pdf Resources
- Mar 20, 2011 servants beloved of God, may divine joy and peace be with you,.
- ec.europa.eu
- Developing One-pointedness
- Concentration endows one with divine joy, wisdom beyond measure, inner vision , insight into the deeper truths,. Chapter XIII. Developing One-pointedness.
- www.sssbpt.info
- Artist Bio
- www.knowlesstudio.com
- Dev dev Shivananda (God of gods Shivananda) Translated by P.R.
- Take care of me one who lives in the heart of devotees and is the lord of God,. Take care of me one who has divine form . has divine joy and is Shivananda.
- stotraratna.awardspace.com
- Preface to the Third Issue
- O Lord, Divine Builder of this marvel, my heart overflows with joy and gratitude when I think of it, and my hope has no bounds.
- anti-matters.org
Suggested News Resources
- Hindus celebrate holy birthday with prayers
- The actual birthday is on Sunday, August 21, during which prayers conclude and devotees make sweets and food to share.
Suggested Web Resources
- Home Page Intro | DivineJoy
- divinejoy.org
- Divine Joy - Spiritual Guidance
- Divine Joy Channeling by Zorriah through Abby Isadora Haydon August 17, 2005 . Greeting and salutations, you lovely beings. You are beings of light and joy.
- www.spiritualguidance.com
- Divine Joy / On First Principles « My Side of the Street
- Divine Joy / On First Principles. Divine Joy / On First Principles This is where David Bentley Hart opens his discussion about Divine Joy.
- jeremiahaandrews.com
- Being Filled With Divine Joy: By St. Justin Popovich « Milk & Honey
- May 9, 2010 And when man looks into himself- behold, he is completely filled with divine joy, and there is not a trace of grief or fear of death in him.
- theinnerkingdom.wordpress.com
- Divine Joy singing "Good Time" - YouTube
- Mar 27, 2011 Quartet groups, divine Joy atlanta, Divine joy gospel, Gospel, Divine Joy Singing.
- www.youtube.com
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I stop always 1 night on a road trip. Well maintained hotel (not always the case with this brand), hard to find this standards for the asked price. My only complaint would be for very loud housekeeping (not so early but if you advertise the ability to keep the room until noon...) Very easy to access. Not much to do... More
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Massively parallel tag sequencing reveals the complexity of anaerobic marine protistan communities
© Stoeck et al. 2009
Received: 14 May 2009
Accepted: 03 November 2009
Published: 03 November 2009
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© Stoeck et al. 2009
Received: 14 May 2009
Accepted: 03 November 2009
Published: 03 November 2009
Recent advances in sequencing strategies make possible unprecedented depth and scale of sampling for molecular detection of microbial diversity. Two major paradigm-shifting discoveries include the detection of bacterial diversity that is one to two orders of magnitude greater than previous estimates, and the discovery of an exciting 'rare biosphere' of molecular signatures ('species') of poorly understood ecological significance. We applied a high-throughput parallel tag sequencing (454 sequencing) protocol adopted for eukaryotes to investigate protistan community complexity in two contrasting anoxic marine ecosystems (Framvaren Fjord, Norway; Cariaco deep-sea basin, Venezuela). Both sampling sites have previously been scrutinized for protistan diversity by traditional clone library construction and Sanger sequencing. By comparing these clone library data with 454 amplicon library data, we assess the efficiency of high-throughput tag sequencing strategies. We here present a novel, highly conservative bioinformatic analysis pipeline for the processing of large tag sequence data sets.
The analyses of ca. 250,000 sequence reads revealed that the number of detected Operational Taxonomic Units (OTUs) far exceeded previous richness estimates from the same sites based on clone libraries and Sanger sequencing. More than 90% of this diversity was represented by OTUs with less than 10 sequence tags. We detected a substantial number of taxonomic groups like Apusozoa, Chrysomerophytes, Centroheliozoa, Eustigmatophytes, hyphochytriomycetes, Ichthyosporea, Oikomonads, Phaeothamniophytes, and rhodophytes which remained undetected by previous clone library-based diversity surveys of the sampling sites. The most important innovations in our newly developed bioinformatics pipeline employ (i) BLASTN with query parameters adjusted for highly variable domains and a complete database of public ribosomal RNA (rRNA) gene sequences for taxonomic assignments of tags; (ii) a clustering of tags at k differences (Levenshtein distance) with a newly developed algorithm enabling very fast OTU clustering for large tag sequence data sets; and (iii) a novel parsing procedure to combine the data from individual analyses.
Our data highlight the magnitude of the under-sampled 'protistan gap' in the eukaryotic tree of life. This study illustrates that our current understanding of the ecological complexity of protist communities, and of the global species richness and genome diversity of protists, is severely limited. Even though 454 pyrosequencing is not a panacea, it allows for more comprehensive insights into the diversity of protistan communities, and combined with appropriate statistical tools, enables improved ecological interpretations of the data and projections of global diversity.
Molecular surveys of protistan diversity research, traditionally based on amplification of small subunit (SSU) rRNA (SSU rRNA) gene fragments from environmental samples, clone library construction and Sanger sequencing have discovered protistan novelty at all levels of taxonomic hierarchy [1]. At the same time, such surveys indicated that we have described only a very small fraction of the species richness of protistan communities [2]. There are few SSU rRNA gene surveys of any community that are reasonably complete [3, 4]; the majority appear to be no more than small samples from apparently endless lists of species present at any locale studied. (e.g. [1, 2, 5–9]). This is not only detrimental to the exploration of the true richness and complexity of protistan communities, but also hampers comparative analyses of protistan communities in an ecological and biogeographical context [10–12]. Massively parallel tag sequencing (454 sequencing, pyrosequencing) is a promising remedy and offers a means to more extensively sample molecular diversity in microbial communities [13]. For example Sogin et al. [14] analyzed up to 23,000 tags per sample of the V6 hypervariable region of the bacterial SSU rRNA genes from deepwater masses of the North Atlantic and hydrothermal vents in the NE Pacific. The study revealed that bacterial communities are one to two orders of magnitude more complex than previously reported, with thousands of low abundant populations accounting for most of the phylogenetic diversity detected in this study (the so called rare biosphere). This was confirmed by Huber et al. [15] who analyzed nearly 700,000 bacterial and ca. 200,000 archaeal V6 tag sequences obtained from two biogeochemically distinct hydrothermal vents. These data sets demonstrated that these distinct population structures reflect the different local biogeochemical regimes, corroborating previous indications that environmental factors and geographic separation lead to non-random distributions of microbes (see [16] for review, but see also [17]). Pyrosequencing has subsequently unveiled the richness and complexity of soil bacterial communities [18], human [19] and Macaque [20] gut microbiota. In the project described in this paper we applied the 454 sequencing technique to eukaryotes to analyze the complexity of microbial eukaryotic communities in two environmentally contrasting anoxic basins (Cariaco and Framvaren).
Summary of recovery of pyrosequencing tags for Framvaren (FV) and Cariaco (CAR) samples, along with accompanying metadata.
Sample number-sample name
N 454-reads total
Total eukaryotic tags > 100 bp
Unique eukaryotic tags
Total protistan tags (incl. Fungi)
Unique protistan tags (incl. Fungi)
Total and (unique) unassignable tags at 85%
Total and (Unique) Archaeal tags
0 (0)
2 (2)
2 (2)
0 (0)
4 (3)
6 (5)
2 (2)
Total and (Unique) Bacterial tags
0 (0)
0 (0)
1 (1)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
Temperature °C
Depth (m)
Salinity [80]
Nitrate (μmol/l)
Silicate (μmol/l)
Ammonium (μmol/l)
O2 (μmol/l)
H2S (μmol/l)
Bacteria (× 106 cells/ml)
Bact Production (H3-Leu, mg/m2/d)
Chlorophyll a (μg/l)
DNA conc (ng/μl)
Water volume sampled (/)
Sampling date
The number of high-quality eukaryotic reads we obtained from each sample ranged from 16,256 (FV3) to 38,280 (FV1). After dereplication (consolidating all sequences that are identical in primary structure into one OTU), the numbers of unique eukaryotic tags ranged from 3,765 (FV3) to 5,983 (CAR1). After exclusion of metazoan tags, we were left with numbers of unique tags ranging from 2,070 (CAR4) to 5,597 (CAR1), most of which could be assigned to protists and fungi (Table 1) for further analyses. The number of tags from non-eukaryotic domains was only marginal (0-0.02% of total tag reads, see Table 1) indicating the high domain-specificity of the primers used.
Interestingly, in nearly all dominant phyla occurring at both locales, the taxon composition in the non-sulfidic anoxic water depth was distinctly different from the sulfidic waters below the redoxcline (Figures 4a-f). For example, in the Cariaco Basin, unique tags assigned to Polycystinea accounted for 31% of all protistan tags in the interface (CAR1), while below the interface this number ranged between 3% (CAR3) and 7% (CAR4). However, generally, the genotype diversity in the sulfidic waters was decisively higher in a variety of taxon groups compared to the anoxic, non-sulfidic waters. Ciliophora tag proportion varied more than five-fold between CAR1 and CAR4, Cercozoa 18-fold between CAR1 and CAR2, Bacillariophyta seven-fold, Ascomycota nearly 10-fold, Heterokontophyta and Chlorophyta ca. seven-fold between these samples (Figure 4). This, even though to a lesser extent, was also true for the Framvaren Fjord. Just to mention a few examples, the proportion of Dinozoa-assigned tags decreased from 42% in the interface (FV1) to 14% below the interface (FV2), ascomycota increased nearly three-fold, Cercozoa increased ca. four-fold and Haptophyta, Chlorophyta and Heterokontophyta nearly five-fold (Figure 4). Figures 5, 6, 7, 8, 9 show the lower-rank taxonomic groups of dominant phyla displayed in Figure 4 that predominantly account for the differences in genotype richness between the non-sulfidic and the sulfidic water samples. While for example coscinodiscophycidae Bacillariophyta were missing from the non-sulfidic waters at both locales, they accounted for up to ca. 4% of all Bacillariophyta in the sulfidic waters (CAR3, Figure 7). This was also true for other taxonomic groups like Xanthophyceae and Phaeophyceae (Figure 9), Chlorodendrales (Figure 8); oligohymenophorean ciliates increased noticeably in the sulfidic waters at both locales (Figure 6), just to mention a few examples.
When comparing higher-taxonomic groups (phylum-level, Figure 4) found in Framvaren vs. Cariaco we noted that Radioloaria (all of which were exclusively Polycystinea), which are typically pelagic marine protists primarily found in the open ocean and very scarce or absent altogether in coastal waters [33], were the only higher taxon group that was detected in all Cariaco samples but in none of the Framvaren samples. Conversely, the proportion of Cercozoa in Framvaren samples was noticeably higher than in Cariaco. Differences between Framvaren samples and Cariaco samples become more pronounced when looking at lower taxon levels (Figures 5, 6, 7, 8, 9). For examples Blastodiniales and Noctilucales (Dinozoa, Figure 5) were exclusively found in all Cariaco samples. Rhizosoleniophycid Bacillariophyta (Figure 7) and Pseudoscourfieldiales (Chlorophyta, Figure 8) were noticeably higher in all Cariaco samples and Choricystis (Chlorophyta, Figure 8) genotypes were much more diverse in Framvaren.
The application of the 454 sequencing technique to the investigation of protistan communities in two anoxic marine basins revealed three significant findings. First, even a sampling effort that was one to two orders of magnitude larger than that achieved by environmental clone library construction and Sanger sequencing, was not successful in retrieving all unique SSU rRNA gene sequences present in a single sample (Figure 1). Up to 5,600 unique tags could be identified in a 7-L water sample from the Cariaco basin without reaching saturation (sample CAR1). However, this is unlikely to reflect the true species richness, because (i) not all SSU rRNA gene copies within a species are necessarily identical [34, 35], (ii) some of the observed tag variability may be due to extreme variability of the V9 region in specific taxonomic groups, and [36] even when minimizing the effect of sequencing and PCR errors using a systematic trimming procedure (see Methods section and [14]) the accuracy of the 454 pyrosequencing strategy (GS-technology) is 99.75% - 99.5% for small subunit rRNA genes [37]. Indeed, in sample CAR1 the number of OTUs drops from 5,600 to ca. 2,600 when phylotypes are clustered based on one nucleotide difference (accounting for ca. 0.8% sequence similarity). Thus, about half of the unique protistan tags retrieved from this sample are potentially afflicted with an error and/or represent the same taxon. The detected number of unique tags would likely represent an overestimation of taxon richness. On the other hand, clustering OTUs at ten nucleotide differences (OTUs10 nt, reflecting ca. 8% sequence similarity) resulted most likely in an underestimation because different taxa may be lumped together into the same OTU. Consequently, it is reasonable to assume that the true taxon richness is reflected in the range between OTUs1 nt (ca. 1,700 in sample CAR1) and OTUs5 nt (ca. 1,200 in CAR1).
Interestingly, even the number of detected OTUs10 nt exceeded previous parametric and non-parametric richness estimates from the same sites, based on clone-library derived OTUs called at 99% or 98% sequence similarity, respectively [10, 38, 39]. Explanations for this may be several fold: (i) even though the sample sizes obtained from previous Cariaco and Framvaren clone libraries were relatively large, the sample size may still have been too small to obtain adequate resolution of the complex communities. If so, this makes previous clone library-based richness estimates severe underestimations; (ii) the statistical error of previous richness estimates may be too large, which cannot be assessed due to a lack of good confidence intervals; [36] abundance-based richness estimates may not reflect the true community richness or relative species abundance in a sample but rather the PCR-amplicon richness. The reasoning for the latter is that in contrast to bacteria, the copy number of SSU rRNA genes varies widely among protists [8, 40, 41]. Thus, the relative amplicon copy number after PCR does not necessarily reflect the relative abundance of a specific taxon in a sample, rendering abundance-based species richness estimates highly erroneous. It is likely that these factors and probably other factors that we cannot account for at present resulted in severe richness underestimations. We hypothesize that the protistan richness in marine anoxic waters by far exceeds previous estimates, and that anaerobic protistan communities are substantially more complex than previously reported. It will be interesting to further investigate how sequence divergence of a hypervariable SSU rRNA gene region translates into taxonomic entities. This will help interpreting the vast diversity of tags generated by massively parallel tag sequencing.
Most of the observed complexity was found in the low-abundance populations. Even when calling OTUs at five nucleotide differences, the proportion of rare OTUs (represented by less than 10 tags) ranges between 71% and 81% in FV samples and between 78% and 83% in CAR samples (data not shown), indicating that the high number of rare taxa is not an artifact based on high intra-species heterogeneity in the V9 region. This corroborates, to a somewhat lesser extent, the previous findings in the bacterial world [14, 15, 18]. The origin and meaning of this complexity is still unclear [42, 43]. Actually, to date there is no evidence that this high frequency of low-abundant genotypes describes a true diversity. It could result from the amplification of detrital or extracellular DNA. On the other hand, it is reasonable to assume that a liter of water is only inhabited by a few individuals of a protist species that never meet in this volume and are therefore subjected to allopatric speciation. The result would be tremendous microheterogeneity that is reflected in these rare genotypes. One hypothesis suggests that these rare genotypes (if real) may represent a large genomic pool, which helps the protistan community to react to any biotic or abiotic changes [43]. In this seed-bank scenario, the species that are best adapted to prevailing environmental conditions would always be abundant in a community.
The second significant finding is the phylum-richness of protistan communities that is missed by the clone library/Sanger sequencing approach. Previous environmental protistan diversity surveys in the same sites of the Framvaren Fjord ([10] and Behnke et al. unpublished, accession numbers [DQ310187 to DQ310369 and EF526713 to EF527205]) did not retrieve any sequences assigned to Apusozoa, Chrysomerophytes, Centroheliozoa, Eustigmatophytes, hyphochytriomycetes, Ichthyosporea, Oikomonads, Phaeothamniophytes, and rhodophytes, all of which have been recovered with the massively parallel tag sequencing approach. Similarly, a vast array of higher taxon ranks detected in this tag-sequencing project could not be detected with an extensive clone library sampling in Cariaco ([26, 30] Edgcomb et al. in preparation). Interestingly, the tags that could be assigned to taxonomic groups not detected via clone libraries all account for <1% of the unique protistan tags, explaining why they have been missed with the clone library approach [26, 30]. Regarding taxonomic groups that were represented by large relative abundances of tags (e.g. alveolates and stramenopiles), the 454 data sets corroborate well with clone library-obtained data. Evidence of and tentative explanations for the dominance of these taxonomic groups in anoxic marine systems have already been intensively discussed elsewhere (e.g. [30, 44, 45]).
The broad taxonomic representation of 454 tags nicely demonstrates the efficiency of the primers used to target the hypervariable V9 region of eukaryote SSU rRNA genes. However, up to 50% of unique 454 tag sequences in our data sets were metazoa. This is a general problem also observed in SSU clone libraries (even though probably to a lesser extent) and not specific to 454 technology [46–48]. The consequence is that this large proportion of potential non-target tags has to be taken into account when designing protistan diversity studies using 454 technology. Either sequencing effort needs to be increased 1.5-fold to get the desired number of protistan tags, or group-specific 454 primers need to be applied subsequently to focus on selected protistan groups.
Our findings also reveal that higher sampling efforts can be obtained in a cost- and time-efficient way by the application of pyrosequencing, which therefore paints a substantially more comprehensive picture of protistan communities. The degree of undersampling inherent in most published clone library-based studies may be so high that it is possible that they cannot be compared in a meaningful manner to other equivalent surveys of diversity. Getting a comprehensive picture of a microbial community is critical to addressing fundamental questions in protistan ecology on the basis of molecular diversity surveys. Such questions include for example, determining the true richness and evenness of microbial communities, which is important in defining microbial ecosystem dynamics [15], and determining the biogeographic distribution of specific taxonomic groups, the stability of protistan communities over time, as well as local patchiness of protists. All of these community attributes are cornerstones for understanding microbial diversity, ecology, and evolution [16, 49, 50].
Some of these subjects frame the third important finding of this study. The eight sites sampled differed markedly in community composition. Based on community membership, it appears that protistan communities from the supersulfidic Framvaren Fjord with an interface located in the photic zone are distinct from the ones of a less sulfidic anoxic deep-sea site. Similarly, anaerobic protistan communities exposed to hydrogen sulfide are distinct from those that thrive in sulfide-free oxygen-depleted habitats. Even though we cannot unequivocally identify H2S as the single most important driving force shaping these protistan communities using this dataset, this observation is not unexpected: H2S-detoxification requires specific adaptation that is not necessarily present in all facultative or strictly anaerobic protists [51, 52]. For example, Atkins et al. [53] found a significant difference in the hydrogen sulfide tolerance of different hydrothermal vent species they isolated, including the closely related sister taxa Cafeteria and Caecitellus. Cafeteria strains isolated by these authors could tolerate up to 30 mM sulfide under anoxic conditions over the 24 hr course of their experiment, Rhynchomonas nasuta could tolerate up to 5 mM sulfide, and Caecitellus could only tolerate up to 2 mM sulfide. Symbioses between protists and sulfide-oxidizing bacteria are another adaptive strategy observed in micro-oxic environments with high hydrogen sulfide concentrations. For example, the peritrich ciliate Zoothamnium niveum found in mangrove channels of the Caribbean Sea depends on its sulfur oxidizing ectobionts for detoxification of its immediate environment [54]. Scanning electron microscopy has revealed a visible diversity of ectobiotic prokaryotic associations with ciliates in the anoxic water column of Cariaco, and these associations are likely to be dependent on the distinct chemical nature of the basin's water column (see Additional file 1). The environmental selection pressure that acts on the phylogenetic composition of protistan communities can be of interest for the design of environment-specific phylo-chips (for example of application see Sunagawa et al. [55] that may help to monitor the global distribution of specific protistan communities.
The temporal and spatial resolution of our sampling strategy is insufficient to deduce temporal and spatial patterns in protistan communities under study. Yet, possible explanations for the observation that in the Cariaco deep-sea basin, samples collected from the same depth at two different points in time are distinctively less similar to each other (samples CAR2 and CAR4 in Figure 3, UPGMA), compared to the shallow Framvaren Fjord (samples FV2 and FV4) are obvious: Surface waters of the Cariaco Basin are subject to strong seasonal upwelling, driving as much as 13-fold excursions in net primary production (NPP) between upwelling and non-upwelling seasons [22]. This causes significant seasonal variations in vertical carbon fluxes, which seems to be not only very important for the dynamics of viral [27] and bacterial communities [56] in such systems, but also for protistan communities, even though the exact mechanisms for how vertical carbon flux variations may act on protistan communities are largely unknown. One possibility could be that due to selective interactions of protist with specific bacteria [57–59], changes in vertical carbon flux that have a direct influence on bacteria can act indirectly on protistan communities.
Only a few taxa accounted for most of these metazoan tags, which belonged predominantly to copepods, cnidaria, ctenophores, molluscs and polychaetes (Additional file 2). Copepods can survive anoxia and high hydrogen sulfide concentrations for long periods of time [60]. Also several molluscs [61], cnidarians, ctenophores [62] and polychaetes are tolerant of anoxia [63]. Even Bryozoa that were detected in three of the samples (Additional file 2) are capable of thriving under anoxic conditions [64]. Thus, the detection of metazoan sequences in anoxic environments retrieved by domain (Eukarya)-specific PCR primers is not surprising. Yet, with the exception of copepods, which we can observe frequently at least in the oxic-anoxic interfaces of our sampling sites we did not confirm the presence of these metazoan taxa in the water samples under study by visual inspection. This is mainly due to the fact that we only screened 20-μl aliquots microscopically (for protistan target taxa). Because of this, small forms (life stages) of larger metazoans or small metazoans like bryozoa represented in our amplicon libraries may have been easily overlooked. It is reasonable to assume that the metazoan amplicons may represent a mixture of allochtonous material (see the detection of an hymenoptera phylotype in FV4 that is represented by nearly 5,000 amplicons) and autochtonous organisms. However, taking into account the low proportion of unique metazoan taxa when clustered at 5 nt differences and the high likelihood of the indigenous nature of most of the metazoans represented by the non-protistan tags it is reasonable to consider contamination in general as an insignificant issue.
This study shows that when 454 pyrosequencing of the V9 region is paired with rigorous downstream data processing, this method is more time- and cost-efficient, and produces a much more comprehensive picture of the protist community than Sanger sequencing of clone libraries, allowing for better estimates of community complexity. While direct comparison of the Framvaren and Cariaco communities is complicated by multiple physico-chemical differences between these two sampling locations, it is possible to distinguish protistan communities on the basis of community composition in the supersulfidic Framvaren Fjord with an interface located in the photic zone from those in the deep-sea anoxic and less sulfidic environment. Furthermore, protistan populations in the sulfide-free oxic/anoxic interface in both Framvaren and Cariaco are distinct from those that are exposed to hydrogen sulfide. However, the specific environmental factors structuring protistan communities remain unknown.
This study combined high-throughput parallel tag sequencing with a highly conservative bioinformatic analysis pipeline to investigate protist community complexity in two contrasting anoxic marine ecosystems (Framvaren Fjord, Norway and Cariaco Basin, Venezuela). Our data suggest that both ecosystems under study are highly variable regarding the dynamics of protistan communities on a spatial and temporal scale. However, high-resolution sampling will be necessary to reliably assess the true extent of this variability. Furthermore, this study illustrates that our current understanding of the ecological complexity of protist communities, and of the global species richness and genome diversity of protists, is severely limited. A deep sequencing of ca. 250,000 V9 SSU rRNA gene tags in total allowed us to recover eukaryotic taxonomic groups that were not detected by previous clone library-based diversity surveys of both sampling sites. Furthermore, the number of detected OTUs far exceeded previous richness estimates from the same sites based on clone libraries and Sanger sequencing, even when tag sequences were clustered at up to ten nucleotide differences (reflecting ca. 8% sequence similarity). Our data highlight the magnitude of the under-sampled protistan gap in the eukaryotic tree of life, and support the notion that not only in bacteria but also in protists over 90% of the observed diversity was represented by rare OTUs that had less than 10 sequence tags assigned to them. Even though 454 pyrosequencing is not a panacea, it allows for more comprehensive insights into the diversity of protistan communities, and combined with appropriate statistical tools, enables improved ecological interpretations of the data and projections of global diversity.
Samples were collected from two locales, the Cariaco Basin, located on the northern continental shelf of Venezuela, and the Framvaren Fjord located in Southwest Norway. Sampling protocols for both sites were as described elsewhere, as well as the protocols for measurement of physico-chemical and biological parameters [10, 26]. Depths of samples, volumes of water collected, and physicochemical characteristics at the time of sampling are presented in Table 1. The sampling design accounted for three features: (i) temporal effects (same site sampled at different seasons), (ii) local patchiness (same depth sampled at two distant sites at each locale), [36] environmental factors (vertical water column gradient at each site and distinct locale-characteristics). Cariaco samples were collected at the Cariaco Time Series Station A (10.30°N, 64.40°W) and at Station C (10.40°N, 65.35°W). These two stations are located in the eastern and western sub-basins of the Cariaco system, respectively. Cariaco samples were collected from Station A at the depth corresponding to the oxic/anoxic interface (250 m, oxygen and sulfide not detectable) in January 2005 (CAR1) and from below the interface (300 m) in January 2005 (CAR2) and May 2005 (CAR4). Sample CAR3 was collected at Station C from below the interface (320 m) ca 100 km distant from Station A, in January 2005. Framvaren Fjord samples were collected in the central basin (58.09°N, 06.45°E) from the oxic/anoxic interface at 20 m sampled in September of 2005 (FV1, oxygen and sulfide not detectable), and from below the interface (36 m) in September 2005 (FV2) and May of 2004 (FV4). The sample FV3 was taken in the upper basin (58.11°N, 06.45°E, ca. 3 km distant from the sampling location in the central basin) from below the interface at 36 m in September 2005.
DNA was isolated from environmental samples and quality-checked as described previously [26]. In short, samples were taken with Niskin bottles and drawn onto 0.45 μm Durapore membranes (Millipore, Billerica MA, USA) under anoxic conditions with no prefiltration step. Samples were frozen immediately in liquid nitrogen until further processing in the laboratory. The nucleic acid extraction protocol employed a high-salt extraction buffer (100 mM Tris HCl (pH 8), 100 mM sodium phosphate buffer (pH 8), 1.5 M NaCl, 100 mM EDTA (pH 8.)) with 1% cetyl trimethylammonium bromide. Approximately 3 ml of this buffer was added to one filter and the total genomic DNA was extracted using chloroform-phenol extraction and isopropanol precipitation. In order to minimize bias caused by sampling the extracts from three filters per sample site were combined prior to polymerase chain reaction-amplification. Our strategy targeted the V9 hypervariable region of the SSU rRNA genes [65]. This region was chosen because it is (i) among the most variable of eukaryotic SSU rRNA hypervariable regions [66], represents a good marker for the taxonomic complexity of protistan communities, (ii) allowed for the use of conserved PCR-primers that target most described major eukaryote lineages, [36] has only marginal length variability among different taxonomic groups (127-150 bp) and (iv) could be fully sequenced using the Roche GS FLX system (up to 250 bp-reads) developed by 454 Life Sciences ([65], Stoeck T., Richards T, and Bass D., unpublished). PCR amplification and pyrosequencing followed the protocol of Amaral-Zettler et al. [65]. The PCR primers we used flanked the V9 region of eukaryote SSU rRNA genes. These primers were 1,380F (forward 1), 1,389F (forward 2), and 1,510R (reverse). Separate 1380F/1510R and 1389F/1510R reactions were run for each sample to recover the broadest eukaryotic diversity possible. The 454 Life Science's A or B sequencing adapters were fused to the 5' end of the primers. For each individual environmental DNA extract we ran three independent 30-μl PCR reactions with reaction mix consisting of 5 U of Pfu Turbo polymerase (Stratagene, La Jolla, CA, USA), 1× Pfu reaction buffer, 200 μm dNTPs (Pierce Nucelic Acid Technologies, Milwaukee, WI, USA), a 0.2 μM concentration of each primer in a volume of 100 μl, and 3-10 ng genomic DNA as template. The PCR protocol employed an initial denaturation at 94°C for 3 min; 30 cycles of 94°C 30 s, 57°C for 45 s, and 72°C for 1 min; and a final 2 min extension at 72°C. PCR products from the same DNA sample were pooled and cleaned by using the MinElute PCR purification kit (Qiagen, Valencia, CA, USA). The quality of the products was assessed on a Bioanalyzer 2100 (Agilent, Palo Alto, CA, USA) using a DNA1000 LabChip (Agilent). Only sharp, distinct amplification products with a total yield of >200 ng were used for 454 sequencing. The fragments in the amplicon libraries were bound to beads under conditions that favor one fragment per bead. The emulsion PCR (emPCR, [67]) was performed by emulsifying the beads in a PCR mixture in oil, with PCR amplification occurring in each droplet, generating >10 million copies of a unique DNA template. After breaking the emulsion, the DNA strands were denatured, and beads carrying single-stranded DNA clones were deposited into wells on a PicoTiter-Plate (454 Life Sciences) for pyrosequencing on a Genome Sequencer FLX system (Roche, Basel, Switzerland) at the Marine Biological Laboratory (Woods Hole, MA, USA). In total, we recovered 251,648 sequence reads for the eight samples that were subjected to quality control. Removal of low quality sequences [14] left us with 222,593 high-quality reads for further consideration. Tag sequences have been deposited in the National Center for Biotechnology Information (NCBI) Short Read Archive (SRA) under the accession number SRP001212.
We developed three automated analysis pipelines to analyze quality-checked 454 reads: The first pipeline for taxonomic assignment of V9 tags, the second pipeline for clustering V9 tags at different sequence similarity levels and dereplication, and the third pipeline to construct a global tag-matrix for sample comparison.
The first pipeline was aimed at assigning taxonomy to our 454 tags and included four steps. First, 454 reads were preprocessed to remove reads with more than 1 ambiguity (N) and short sequences (having fewer than 100 nucleotides after the proximal primer), as well as all sequences having mismatches with the PCR primers. Second, each remaining sequence was compared through similarity searches, using the program BLASTN (version 2.2.21, [68]), against a reference database including every SSU rRNA sequence longer than 800 nt (561,000 sequences) extracted from 1,300,000 SSU rRNA genes present in the EMBL/GenBank database, with three longest sequences selected to represent each family (as described in their respective EMBL entries). This served to remove tags that matched with at least 70% similarity to sequences from Archaea, Bacteria or Metazoa. Third, the remaining sequences were blasted against all publicly available SSU rRNA gene sequences of protists, fungi and viridiplantae (170,000 sequences), requesting up to 150 best hits, using the BLAST parameters: -m 7 -r 3 -q -2 -G 6 -E 6. Parameter -m 7 allowed for an XML output, which was easier to analyze. The other parameters were selected after running 1,500 test BLAST runs using tags extracted from longer, well known sequences in order to finely tune the blast search to the characteristics of the domain analyzed. Fourth, the blast output was parsed to extract Best and Highest hits at a series of thresholds for sequence similarity. Sequence similarity was calculated as the sum of identities for non-overlapping (if any) HSP (High Scoring Pairs, see the BLAST documentation) divided by the length of the query sequence; this is a much more efficient method than simply taking the first HSP into account as is usually done. Best hit was the most similar target sequence that had a good taxonomy associated with the sequence (i.e. the Organism Classification (OC) field in the EMBL entries). The Highest Hit was the sequence with the highest similarity overall. Also, every sequence above the designated threshold was used to build a list of taxa (i.e. the contents of the OC field in the EMBL entries), which allowed for verification of whether the taxonomic assignment of the best hit was in global agreement with the next most similar sequences. Results at thresholds of 70, 75, 80, 85, 90, 95, 98 and 99% similarity were stored as tabulated files for further analyses. A manual examination of the relationships between threshold and qualities of taxonomic assignment led us to choose the 80% similarity threshold for assigning a given tag to a taxon (see the results). The reasoning for this similarity threshold is based on GenBank sequence data analyses. Therefore, we extracted the V9 regions from a random selection of 100 full-length eukaryote SSU rRNA gene sequences with a described taxonomy. A BLASTN analysis of the V9 fragments against the GenBank nr database revealed that the short V9 fragments could reliably be assigned to order-level when the closest BLAST hit (the original respective full-length sequence excluded) was at least 80% (see also [33, 34]). Taxonomy of protists is according to Adl et al. [69] and for fungi according to Hibbett et al. [70]. We note that because Synurophyceae and Chrysophyceae are hardly distinguishable even when full-length 18S rRNA gene sequences are available, we united tags that were putatively assigned to Synurophyceae with Chrysophyceae to Heterokontophyta.
The second pipeline was dedicated to the clustering of tags at a given level of similarity. This is usually done by first using a multiple sequence alignment (MSA) program (usually MAFFT [71] or MUSCLE [72] to align the tags, followed by the calculation of a distance matrix (using QuickDist [14] for example) and finally statistical analyses. Our experience with the V9 domain indicated that none of the MSA programs was able to output alignments of high enough quality. We therefore implemented a completely new approach (Shahbazkia & Christen, in preparation). Our key hypothesis was that the greater frequency at which a given sequence occurs, the more likely it represents a real sequence. Conversely, there is a probability that a sequence found only once is the result of a PCR or sequencing error, or due to the presence of variations in some operons within a single genome [73]. First a python program allowed for a strict dereplication, i.e. clustering strictly identical sequences. This led to a 5 to 10 fold reduction in the number of sequences. Strict dereplication allowed for the second step, but also allowed for the construction of rank abundance curves. The resulting file (of strictly dereplicated tags) was sorted by decreasing abundances of tags in each cluster. Then, instead of computing a percentage of similarity between sequences (which is difficult because we don't know how to implement a good substitution matrix for hypervariable regions of rRNA sequences) we implemented a Levenshtein distance calculation for clustering sequences. Levenshtein distance [74] is a measure of the similarity between two strings, which we will refer to as the source string (s) and the target string (t). The distance is the number of deletions, insertions, or substitutions required to transform s into t. Taking successively each dereplicated tag, the following tags were clustered with this representative if they had a Levenshtein of k or less (k ranging from 1 to 10). A number of checks were performed to analyze such clusters. A comparison of these clusters to the taxonomic assignments performed by the first pipeline showed an almost perfect agreement when taxonomic assignments had been possible by BLAST (k = 1,2,3). Above these k values many non-assigned tags could be assigned to clusters containing assigned tags.
Comparisons of operational taxonomic units (OTUs) based on V9 domains and (almost) complete SSU rRNA sequences are almost impossible on large data sets of sequences because none of the multiple sequence alignment software is able to properly align SSU rRNA sequences within their divergent domains, and this problem is exacerbated for short divergent tag sequences (Guillou & Christen unpublished). For this reason, published 454 studies have relied heavily on BLAST alignment to public sequences to cluster tags. We used a completely new algorithm (Shahbazkia & Christen, unpublished) that directly clusters tag sequences having less than k differences (k = 0, 1....10) and does not rely on a multiple sequence alignment. We validated this approach in a separate analysis (Guillou and Christen unpublished) by demonstrating that our tag clustering method based on word counting instead of percent sequence similarity identified correctly the almost full-length sequences of a separate large, well-curated SSU rRNA alignment from which tags were extracted, and that using the clustering approach here, the same cluster ID was attributed to sequences that were phylogenetically close to the original tag sequence (Guillou and Christen, unpublished).
A final pipeline was designed for the global statistical analysis of all eight samples. The entire data set consisting of all eight samples was this time considered and globally treated as described above in pipelines 2 and 3. This led to the construction of an abundance matrix at various clustering values as explained above where each column was a given sample and each line a cluster, values being either the number of occurrences of the tag in the sample, or simply 1 or 0 to indicate presence or absence of sequences belonging to that cluster. However, we here refrained from further analysis of the abundance-matrix, because due to different genome sizes and rRNA gene copy numbers among protists [75] and PCR primer selectivity [30] the abundance of PCR-amplicons from a sample does not necessarily reflect the relative abundance of the respective organisms in this sample.
The script for data analyses (Linux, Windows and Macintosh operating systems) is provided online http://bioinfo.unice.fr/biodiv/protist_data/.
We calculated the Jaccard index, based on incidence (Jincidence) of unique OTUs as obtained from the third data processing pipeline described above, as a measure of community similarity between the eight samples under study using the program package SPADE [76]. Analyses were performed as recommended by the authors. Similarity values were transformed into a distance matrix and used for an Unweighted Pair Group Method with Arithmetic Mean analysis (UPGMA) of the eight unique libraries [77].
Data from the authors cited as unpublished are available from the authors upon request.
operational taxonomic unit
polymerase chain reaction
multiple sequence alignment
deoxyribonucleic acid
ribonucleic acid
Unweighted Pair Group Method with Arithmetic Mean
net primary production
Cariaco Basin
Framvaren Fjord
Basic Local Alignment Search Tool
small subunit ribosomal RNA.
The International Census of Marine Microbes and the W.M. Keck Foundation award to the Marine Biological Laboratory at Woods Hole (MA) supported the pyrosequencing part of this study. We would like to thank L Graham Murphy and E Andreischeva for assistance with pyrosequencing. Further financial support came from a grant from the Deutsche Forschungsgemeinschaft to TS (STO414/3-1). Support for the unpublished work on Cariaco Basin protists came from NSF MCB-0348407 to VE (collaborative project with S Epstein at Northeastern University, Boston, MA, USA). Financial support to AC was provided by NSF MCB-0348045. Financial support to RC was provided by the ANR-Biodiversité project Aquaparadox. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We would like to thank H R Shahbazkia (DEEI-FCT Universidade do Algarve Campus de Gambelas, Faro, Portugal) for programming parts of the clustering algorithm, the captain and crew of the B/O Hermano Gines and the staff of the Fundacion La Salle de Ciencias Naturales for their assistance during our fieldwork in Venezuela and HW Breiner for technical assistance. We are grateful to M Scranton and G Taylor for some of our Cariaco metadata. We would like to thank four anonymous reviewers who substantially helped to improve the manuscript.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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BETHLEHEM For more than 30 years, the Bethlehem Police Department has been using land provided by Callanan Industries to train their officers in shooting their weapons.
Last week, Callanan Industries donated the land off Old Quarry Road to the Town of Bethlehem with the intent it be used as a shooting range for Bethlehem police. A small ceremony took place on Friday, Dec. 13, after the donation was accepted at the previous town board meeting.
"We've become very fond of that facility because of its proximity to Town Hall," said Bethlehem Police Chief Louis Corsi. "It saves us a lot of money in logistics by moving people back and forth from the facility, so in the long run it's going to be really good for the Town of Bethlehem."
Since the location has been used for so many years, much of the department's equipment is already on site. Come spring, Corsi said he would like to see the space cleaned up and possibly purchase some new equipment.
Since the town now owns the land, the Bethlehem Police Department can apply for state and federal grants to help upgrade the space.
Corsi said Bethlehem is now one of only two municipalities within Albany County to have their own shooting range. Other agencies such as officers from the Albany County Sheriff's Office, the State Police, the Albany division of the Federal Bureau of Investigation and Secret Service sometimes use the facility for shooting practice.
Andy Stokes, a retired employee of Callanan Industries, said getting the land donated had been a pet-project of his for years. Stokes was invited to the donation event and took the lead on behalf of Callanan Industries.
"Since the company's founder, Peter Callanan, first began the stone quarry on his South Bethlehem farm and was awarded the state's first road-building contract, we have been members of the Town of Bethlehem and members of the Bethlehem community," Stokes said. "We are proud to keep Bethlehem strong and growing, and a safe place to live and work.
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Part of my "We Dont Disco Like We Used To" series and a commision for David Czyz. Original image of David by David Czyz. I tried to keep it as close to the original as possible, but then I got carried away and got this. His original image was a self portrait with pencil. Yes the lines take as long as you think they would take with a mouse.
Full detail here [link]
Full detail here [link]
Awesome man, i love the colors and the details
nice! how long did this take?
I really like this work. Love the style on his hair.
hair is niice!
As long as it seems it took, it's damn impressive
I faved just for the hair
Hah, looks nice bro!
nice lines, must have been a Chinese sweatshop of a time doing his hair hah
well done
well done
Thats some real tight line work right there. Very nice chief!
Nice work ...


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Elliott House
From $200 / night
Description from the owner
Description from the owner
Bedrooms: 3/
Bathrooms: 1/
Sleeps: 7/
Type: House
This is a 3 bedroom and 1 bath home. It will sleep 7 guest. Main floor bedroom has a double bed. Upstairs bedrooms one has a double and the other has two twins. There is also a rollaway bed in the closet for extra sleeping.There is a large deck with patio furniture an a charcoal grill or Gas gill. Firepit in the backyard. Inside you have a large living room that can be closed off for more sleeping. Dishwasher, coffee maker, dishes,cable tv, linens. There is a toy room for the small children,games and books for the older kids Newer carpeting was put in all the bedrooms and living room. This home is walking distance to town.TripAdvisor property ID 3639237
About the owner
About the owner
caroline merz
caroline merz
- Response rate : 70%
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- Calendar updated: 8 months ago
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RENTING - Fri to Fri
Nearby Attractions: - Golf,boating, walking the boardwalk, putt-putt,-farmers market, dining, shopping, charter boat fishing with Whitney Charter call 616-846-6325 for rates and times, state park beach, water front stadium, ice cream shop! Take a short ride on the Trolley to the beach and Lake Michigan. Visit the Historic Train, or in the evening watch the Muscial Fountain. There is a paddle boat cruise close by where you can cruise up river into Spring Lake and out to Lake Michigan.
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Hi all,
I am extremely confused--I have been playing Ghosts for 360 since launch, got up to 8th prestige, now when I try to play it is as if I have never played before--advertises DLC and camos that I already had, puts me at rank 1, doesn't have ANY of my progress. Does anyone know what to do about this issue???
You probably got reset, because you were either boosting, cheating, exploiting, or playing on the opposite team of your clan.
Nope, not in a clan, never boosted, cheated, or exploited. Anyone from activision here? I thought this is where we could post questions for responses-should I phone instead?


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Even as huge losses and eroding customer confidence have led to a situation that would cause anyone to grimace, McDonald's Japan says it has come up with a plan to turn that frown upside down.
Starting next Monday, the bargain-priced "¥0 for smiles" offer will return to the menus of every McDonald's Holdings Co. (Japan) outlet across the country. Read More »


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I am rather disappointed that there is no documentation for singing up to iTunes U as a museum or art gallery, etc.
I was stage two in the sign up process when it started asking me if I was working for a Higher Education or K12; and really we are neither and I was scared to continue as all the questions are directed very much towards schools.
It seems as if iTunes U is created foremost for Schools, Collages and University and the 'beyond campus' as Apple calls it is put in is an undocumented bolt on?
I have Google searched and spent all morning on these forums trying to find anything to help me. But....nothing. Can anyone help me out?
All we would like to do is having something like the Smithsonia American Art Museums page.
A home page with a "what's new" leading you to various channels and a list of top downloads.
Retrieving data ...


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Anyone can start a business. But to start a business that succeeds and continues to flourish isn't so easy. Did you know at least half the new companies started go out of business before their third anniversary, and one in four new enterprises don't survive longer than one year.
If you're going to start a business that has staying power, you need to be informed and take advantage of resources, tools and workshops.
The Sierra Economic Development Corp. (SEDCorp) is offering affordable classes for entrepreneurs and small business owners in Auburn, Grass Valley and Placerville in February.
New for February are state and federal agencies bringing a series of government workshops for small business owners to Auburn. The workshops will run from noon-2 p.m. each Thursday in February.
Feb 17 — You Want To Start A Business? Department of Treasury, IRS. Learn what you need to know about federal income and payroll taxes as a small business owner; running your business from your home; new tax credits and incentives.
Feb 24 — How to Do Business with the California State Government and Unraveling the Mysteries of eProcurement, state Dept. of General Services. You will learn: How to register as an interested vendor within eProcurement; get certified with the state and learn where contracting opportunities are available.
Cost is $10 per workshop. SEDCorp, a regional nonprofit, who is in the business of supporting the growth of small businesses through training workshops and small business loans, is providing the Government workshops in SEDCorp's Training Room 560 Wall Street, Suite F, Auburn. SEDCorp also offers other business development classes in Auburn, Grass Valley and Placerville. Classes fill up fast and there is limited seating. Call 530-823-4703 or visit sedcorp.biz for class details.


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David Yates Commits to WB's 'Tarzan'; Henry Cavill, Charlie Hunnam, Alexander Skarsgård, and Tom Hardy Eyed for Lead
Posted on Wednesday, November 7th, 2012 by Angie Han
When you're coming off four massively lucrative installments of one of the most successful cinematic series of all time, where do you go next? If you're Harry Potter helmer David Yates, apparently, to the African jungle.
The filmmaker has just committed to Warner Bros.' live-action Tarzan, one of several films in the works based on Edgar Rice Burroughs' character. And he's wasting no time getting the pieces together, meeting with up-and-coming talents like Henry Cavill, Charlie Hunnam, Alexander Skarsgård, and Tom Hardy for the lead role. More details after the jump.


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Dreamworks (DWA) +4.4% AH after announcing a new distribution deal with 20th Century Fox, following earlier speculation that it might ink a deal with Sony (SNE). DWA says the five-year agreement with Fox "presents more favorable economics... taking advantage of lower costs associated with the emerging digital distribution landscape and managing domestic television distribution in-house."
Dreamworks (DWA) +4.4% AH after announcing a new distribution deal with 20th Century Fox,...
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Comments (1)
I think Sony being at 945.37B means that this company has major issues. They were once2.3T. After removing Linux from Tue PS3 They have angered many people which has lead to attacks mnd consumers leaving some call it a double whammy I also understand that major disasters hit the area. But, they could have over come those had they stayed honest .Maybe Sony aught t re-think there priorities .
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Swordtail facts?
Hi I am thinking of putting swordtails in my tank instead of platys. How many is the minimum amount of swordtails necessary for them to be happy? (my tank description is in my signature) I don't want any babies, cuz then I'll have to take care of them, so preferably I'd like to have all males. If this is not possible in my tank size, how many females minimum should I have to a male, and do I have to take care of the babies, or can I just let the other fish eat them (cruel I kno but I just don't have the space :-()? Please help!
For 29 gal. One male and three or four females would be bout right.IMHO
In larger tank's, you could place more males but male to female ratio is same.
Male swordtail's are aggressive with other males ,especially when wanting to breed and one male usually comes out the winner, while subdominant males are often chased,nipped at, and possibly injured.
Fish prefer cool water with some movement and are largely herbivores with occasional meaty foods.
thanks..but I still have a few questions. Can I fit the 3 female and 1 male swordtails in my 29 gal tank with all the other fishes, or will it be too crowded? Also, what do I do with the fry in my tank if they breed? I don't want more fish, but I find it rather cruel to just let them die. :-(
anyone?? :-(
I think you could safely put 3 swords in your tank, 2 females and 1 male, but try to get juveniles and allow them to grow and become used to the other inhabitants.
As for any fry that may come along, don't worry about it, you will only ever see the survivors and therefore will never know if there were any others. As for being "cruel", it's just a word and has absolutely no relevance in the animal world.
Just had a bit of a laugh when I read your profile............boomerang throwing, good for you, perhaps you could come and teach me!
sure :-D
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40 year old woman | Libertyville, IL, United States | Seeking men 36-45 within 50 miles
Must love the outdoors!
Just because I'm a scientist for a pharmaceutical company doesn't mean I'm a fan of Breaking Bad. Who's got time for TV when I can be outdoors running or riding my horse? Yes – I admit it – I'm a (gasp) nature lover. In fact, if you're going to try to get me to work out in a gym, just walk (or type) away – because I cannot even stand how downright restrictive the building is!Growing up in a suburb of Chicago, I was raised by more-traditional parents who had a good relationship. And, now that I've accomplished many of my professional goals, I'm looking for a wonderful long-term relationship with a man who can – in Jerry McGuire lingo – "complete me." (Basically, if this were a classified ad, it'd read "SWF seeks S?M for loving marriage, unfailing faithfulness, and a lifelong commitment. Jerks and meanies need not apply.)Pretty much I love any outdoor activities – I love surrounding myself with nature – like gardening, going horseback riding, camping, hiking, bird watching, and sometimes all in the same weekend. In addition to having a great appreciation for homecooked meals others may make – it's reminiscent of my upbringing –I also love cooking. Then there's the fact that I like to have a great beer or glass of wine with that good food!
Camping , Coffee and conversation , Cooking , Dining out , Gardening/Landscaping , Hobbies and crafts , Movies/Videos , Music and concerts , Exploring new areas , Travel/Sightseeing , Watching sports , Wine tasting
Other types of exercise, Running, Walking / Hiking
I exercise 3-4 times per week.
I have Horses
Firkin, Indiana Dunes, Kettle Moraine State Forest
I enjoy observing different kinds of birds, campfires, movies, animals, flowers, eating sweets (and those homecooked meals), and running – so I can eat more sweets (and larger portions of those homecooked meals!) and still keep fit.
I love horseback riding, running, camping, hiking, bird watching, movies, cooking, and gardening.
Team of Rivals
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|What she is looking for
|5'7" (170cms)
|5'8" (172cms) to 6'5" (195cms)
|Body type:
|Athletic and toned
|About average, Athletic and toned
|No preference
|Light brown
|No preference
|No Way
|No Way
|Social Drinker
|Social Drinker
|Other profession
|No preference
I am a scientist for a pharmaceutical company.
|I'll tell you later
|No preference
|Never Married
|No preference
|Have kids:
|Wants kids:
|White / Caucasian
|White / Caucasian
I am attracted to the more rough hewn man – the sight of nice, strong arms can make me (momentarily) weak in the knees. One who's back-to-nature and isn't just fit – he also has to have at least as much muscle as I do.
|Christian / Catholic
|Spiritual but not religious
|Graduate degree
|Bachelors degree, Graduate degree


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Presidents and the Constitution
Walter Dellinger moderated a panel of legal scholars who talked about the issue of presidential obligation to follow the law. Among… read more
Walter Dellinger moderated a panel of legal scholars who talked about the issue of presidential obligation to follow the law. Among the topics of discussion were the role of the Justice Department, congressional restraints on presidential power, and the George W. Bush administration's actions on torture and wire taps. Following their remarks, panelists answered audience members' questions. close
People in this video
- Consuelo Maria Callahan Judge U.S. Court of Appeals->Ninth Circuit
- David D. Cole Professor Georgetown University Law Center
- Walter E. Dellinger III Attorney
- Kenneth W. Starr President Baylor University
- Kathleen M. Sullivan Professor Stanford University->Law School
- John C. Yoo Professor University of California, Berkeley->School of Law
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Re: Paying for insurance only when you "need" it?[ Edited ]
12-27-2012 12:52 AM - edited 12-27-2012 12:55 AM
Be careful, many times you cannot get medical insurance if you have cancelled a self-insured plan in the last 6-12 months. They share info through systems (MIB.com for instance) just like the credit bureaus do and especially if it is within the same state you may have a real hard time finding someone even willing to take you.
Also beware starting Jan 2014 you will be required to have medical insurance or face government fines/penalties.
(759 Views)


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Mercedes-Benz SL65 AMG Repair and Maintenance
A guide to problems, costs, maintenance and repair for your Mercedes-Benz SL65 AMG
Verified by mechanics, reported by drivers like you
Ask a mechanic in our community
Never overpay. Check prices with our estimator
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Related Content
Mercedes-Benz SL65 AMG Problems
Engine misfires (rough running) and lack of power are commonly caused by ignition coil pack or ignition module faults. Proper diagnoses should isolate the cause and prevent unnecessary replacement of good parts.
Leaks in the pneumatic system will cause problems with the dynamic seats and the trunk and/or door closing assist.
The early versions of the central gateway module (which allows different systems to communicate with each other) have software problems.
Mercedes-Benz SL65 AMG Questions
transmission seams to perform good until this coming out of gear ocurs
transmission seams to perform fine ,problem is corrected if car slows down to like 10 miles per hr it automatically goes back in gear,happens all the time,any info on this flaw?
can not move the gear lever stuck ip, batteries are ok, do not start, esp lights and it says that it is defective?
Mercedes-Benz SL65 AMG Recalls
In a crash that deploys vehicle air bags to protect occupants, the passenger seat air bag may not deploy, increasing risk of injury. Dealers will replace the passenger seat air bag free of charge to resolve the problem.
The occupant detection system may unintentionally shut off the passenger front air bag due to a sensitivity issue in the sensor mounted within the passenger seat cushion. Dealers will replace the passenger side seat cushion which includes the sensor free of charge to resolve the concern.
There is a faulty software calibration that affects several areas including shutting off the fuel supply in a collision. This could cause a fire hazard during an accident. Dealers will reprogram this software. The Mercedes-Benz recall number is 2008 070001. This recall began during August, 2008.
Mercedes-Benz SL65 AMG Reviews
when car is sound it gets five stars,however that is wish-full thinking. frustrated because it seams only the dealerships can fix this car and it costs huge amounts to fix just about any problem,haven't been able to find one person or shop that can work on this car,sure would like to? anyone in the Suffolk county NY area that knows how to fix this car,please contact Jim at email@example.com
Excellent speed and drive, this car has been 15% of it's life in the dealer with problems. Motor, tranny, suspension, ect, But due to it's speed and looks I will no change this car. I got the extended 7/100K MB CPO Warranty and intent to use all. I will get rid of this car after the warranty and POOR SUCKER who buys this car with out warranty.


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Re: IQ only. Stylus 1 or XZ-1?
Started Dec 22, 2013 | Discussions thread
Shop cameras & lenses ▾
Contributing Member • Posts: 939 • Gear list
in technology
- NEW beats the OLD.
Time / Age adds humans more experience, accuracy, individuality... simply every good things. BUT its the opposite on the gears side.
You might say "xz-1 is more pocketable!" yes... however it does not reach as far as Stylus does.... you can say "a microphone can be attached to xz-1, but can´t Stylus 1" and I would say, dont buy a mic. for XZ-1 buy even a second hand camcorder by that mic.´s price
IQ says stylus on my mind.
sure its personal
Ben O Connor's gear list:Ben O Connor's gear list
Olympus XZ-1 Panasonic Lumix DMC-GM1 Olympus M.Zuiko Digital ED 40-150mm 1:4-5.6 R Olympus M.Zuiko Digital 45mm F1.8 Panasonic Leica DG Summilux 15mm F1.7 ASPH +3 more
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We offer discount to nonprofits, government agencies, and federal contractors
In December 2015, DHS conducted nationwide raids to deport Central American families. AILA has condemned these inhumane tactics.
Stay Up to Date
AILA Doc No. 12020866 | Dated February 8, 2012 | File Size: 62 KDownload the Document
DOS Visa Bulletin for March 2012. Employment-based priority dates continued to move forward for the month of March. Section C addresses advance notification of diversity (DV) immigrant category rank cut-offs which will apply in March.
[[To print the PDF on this page please use the print function in the PDF reader.]]
Cite as AILA Doc. No. 12020866.Open the Document
American Immigration Lawyers Association
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Washington, DC 20005
Copyright © 1993-2016
American Immigration Lawyers Association.


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Mike Miller
Mike Miller, owner and versatile racer, was proud to announce the formation of his new SCCA Trans Am Pro-Racing team, Miller Racing in 2012.
Mike Miller is no stranger to the racing world or the SCCA. He has raced various cars over the past 35 years. In 1982, after making his way through several different racing categories, he debuted in SCCA Super-Vee open-wheel racing with the guidance of legendary racer, Dan Gurney. From '82-'86, Miller honed his driving and component-development skills with several teams. He finished third in Super-Vee behind Michael Andretti and Ed Pimm. Miller's resume includes driving in Formula Atlantic, Cam Am, Trans Am, and even NASCAR Winston Cup. He currently serves as CEO of Orchid Orthopedic Solutions and owns Trick Titanium, a manufacturer of Titanium engine valves and other Ti components along with Titanium Bellhousings used in Top Fuel dragsters all over the world.
Most recently, Miller drove for Stealth Racing in the Grand Am Series with co-driver Pete Halsmer. In hopes of harnessing that same winning spirit, the two enjoyed in Trans Am in the late 1980's driving for legendary owner Jack Roush, Miller and Halsmer will drive in the new Trans Am TA2 category backed by a list of impressive corporate partners. In one season, Halsmer finished second in the drivers' championship while Miller was driving to rookie-of-the-year honors. The pair gave Roush another manufacturer's championship. The duo hopes to bring their years of experience and Miller Racing's professional equipment to another championship season. "I am excited to jump in and race once again with Pete as a teammate and test driver. I know Pete will push us to peak performance" said Mike Miller. Halsmer mirrors Miller's enthusiasm and knowledge of the sport. 2012 brought exceptional results for Miller Racing consisting of five, first place wins and 15 podium finishes. By the conclusion of 2012, consistently solid performance put Miller Racing in a position to win the series. However, prior to Mid-Ohio, seasoned driver Pete Halsmer sustained a serious back injury requiring Miller Racing to pull a car from the race. As a result, a 2012 series overall 1st place overall was not possible.
Cameron Lawrence
Cameron Lawrence's racing career started at the age of 7 when he began racing Quarter Midgets in the central Florida area. He worked his way up through 3 different classes while learning car control and fundamental driving skills. During those years Cameron won numerous races while simultaneously competing in 2 classes. At the age of 12, he won the state championship in the most competitive class in which we competed, Senior Honda.
His local track was closed the following year and Cameron decided to focus on baseball. It wasn't until after he graduated from high school that Cameron took up racing again.
Cameron Lawrence recently just completed his second full season in GTA. After finishing 2nd in points while earning V8StockCar Rookie of the Year he worked on improving for 2012. In only his second season he became V8StockCar GTA Points Champion. Also during the 2012 season Cameron competed in 5 SCCA Trans-Am Series races. In those 5 race weekends he finished 2nd a total of three times and finished off the year with a win at Road Atlanta.
In 2013, Cameron Lawrence won eight of ten races and finishing second in the other two. Cameron's point total of 210 was 106 point ahead of the second place finisher in the series. Cameron won eight pole position starts and led the most laps in six of the ten races. As a result, Cameron became the youngest Champion in the history of Trans Am.
For more information about Cameron Visit his Website: www.CameronLawrenceRacing.com
Joe Stevens
Joe Stevens, lifelong automobile enthusiast, is starting his racing career with Miller Racing's 2015 TA2 program. A graduate from UTI as well as highly acclaimed racing schools such as the Bondurant School of High Performance Driving and the Gary Curtis Racing School. Joe built and drove an Audi A4 track day car which has provided a further foundation for him to step into the 2015 Trans Am TA2 Series. The Miller Racing Team is excited to put Joes training into use out on the track this season.
For mor information you can follow Joe on twitter @StevensSpring and or visit his website http://www.racejs.com/.


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21 Mar 2014 at 10:31
Used car dealers are feeling more confident about the state of the market, according to new figures.
CAP Automotive, a company which provides car cost information, found that in a survey of about 100 independent used car retailers, more than half said sales are increasing.
The dealers have found that the balance of supply and demand has also got better, according to the survey.
In recent months, dealers have encountered a shortage of high quality stock, meaning they were having to compete harder and pay a higher price for the best cars.
However, the new CAP figures show that only a minority of dealers are struggling to find suitable cars now, with the lowest figure recorded since summer 2013.
August last year saw the highest vote of confidence in market conditions since CAP began measuring feeling among dealers, but that level of optimism has now been reached again.
Copyright Press Association 2014
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Where Baitfish Don't Belong
- By: Ted Williams
Wild trout water more beautiful than northern Maine's Big Reed Pond doesn't exist. It is embraced by one of the few remaining old-growth forests in the East. It is one of about 307 lakes in the nation (305 in Maine) that still sustain native brook trout undefiled by hatchery genes and one of only 14
Robbed by RAT's
- By: Ted Williams
You're losing more than money when you have to pay to fish public water
Under the Influence of Ethanol
- By: Ted Williams
America's corn-based ethanol program carries high costs in fish, wildlife and tax dollars
Reforming the Corps of Engineers
- By: Ted Williams
The Corps has tried to control nature. Now it's time to control the Corps.
Pits in the Crown Jewels
- By: Ted Williams


text:
Translation of rider in Spanish:
- 1 1.1 (on horseback)Example sentences1.2 (on bicycle) 1.3 (of subway, bus) (American English)
pasajero, (-ra) (masculine, feminine)
- The shop provided saddles, boots, and other riding equipment for riders at south Florida racetracks.
- As he intended, he is once again the best young rider in the race.
- Reece now wants to develop his riding and hopes to become a top dressage rider.
usuario, (-ria) (masculine, feminine)
- 2 2.1 (appended statement)cláusula (feminine) adicional(condition) condición (feminine)Example sentences
Example sentences2.2 (Law)
- A few years ago the US introduced a pernicious rider to their new telecommunications legislation.
- Just as a rider to that, policing operations, in their widest context, normally can go on for years.
- With an environmental rider to the 1999 budget, they succeeded: the date was bumped four years to 2005.
recomendación (feminine) (del jurado)
- At his inquest the jury added a rider to its verdict urging further research into Roaccutane and its side-effects.
- No evidence was called on their behalf, and when convicting the jury added this rider.
- The committee was forced to add a number of riders to this verdict, however.
Definition of rider in:
- British & World English dictionary
- US English dictionary
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Presenting the Single Best Review of Gwyneth Paltrow's Cookbook, Courtesy of Ruth Bourdain
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The Tiki's Mug by PhostexWatch
©2007-2016 Phostex
Everyone should do at least one bit of tiki art in their lives. I've done mine, now go do yours.
Hi Phostex, great artwork! I want to ask you for permission to use it on the cover of my first formal album of surf music. What procedure should I follow? greetings. This is my page. jimmystrings.bandcamp.com/
You can e-mail me at email@example.com. There, we could discuss your budget, and I'd be willing to go reasonably low, given that this is an existing piece.
Love this piece. Oddly, my favorite part is the paint splatter eruption.
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I have been in the hospitality business for over ten years, by now it is second nature to me to be a host and make people feel right at home in an instant. Love the ocean, the beach, music, a night around town, working out, bike rides. South beach is an amazing place almost magical. I would recommend it to anyone who is looking for adventure and unlimited fun to come check us out and find out for yourself why we call it the "Magic City"


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Grab your copy today distributed via VPal, iTunes, Amazon, Google Play & all outlets!
Check out this soundcloud uploaded by VP records.
Tenna Star singer and songwriter, an artist with powerful vocals that always thrills the audience. Tenna Star born in Kingston Jamaica to a family of nine children grew up in the Red Hills Road area where he spent most of his life.
His singing career and everything he has done in the music business he owes it all to his mother who is the back bone for everything. His early stages of development as a singer came from his mother who he heard singing all the time.
The work of God was taught and sang daily in his house, his first band was a gospel group which was formed by his mother, called the Tenn's brothers which consisted of Tenna Star, his older brother and his younger brother Mark Tenn who is also a prominent force in the music business. After leaving high school Tenna began doing odd jobs here and there mainly graphic designing, which he is good at, but singing was always in his blood and every opportunity he got he would sing.
Check out this soundcloud uploaded by VP Records.
Harmonie Deja is a singer, songwriter from North-West London, and one of the freshest female artists to come onto the UK Reggae music scene. Currently under the Starrvybz Entertainment Label, the singing songstress has already made a name for herself with songs such as, 'Really Together' a modern cover version of the Reggae hit by Marcia Griffiths and Bob Andy which also features Tony Douglas, and one of her more current releases 'Hunger'
is taking the radio airways by storm and also more recently for 2015 'Life' a combination featuring Harmonie Deja alongside Frankie Paul.
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Miss Melody is a singer/ songwriter born in the South American country of Guyana but also representing London and Hackney where she grew up and now lives.
She started writing songs at a young age and rapidly developed her talent through working with a wide catalogue of underground and unsigned artists where she learned that expression of her talent was necessary.
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STARmeterSEE RANK
Up 147,331 this week
Robert Rhodin
Robert Rhodin was born on November 25, 1972 in Stalowa Wola, Podkarpackie, Poland as Robert Kolodziej. He is known for his work on Commander Hamilton (1998), Hamilton (2001) and Lilla Jönssonligan på kollo (2004). See full bio »
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Dark ADL.! The history of the ADL and the Mossad!!
- Uploaded by Deadspace76 on Sep 8, 2009
- Views: 209
A short film about The Dark origins of the ADL, and it's ties to the Mossad. Watch an ex-Jewish Mossad Agent spill the beans..!!! To sign up at:http://theforbiddentruth.net/index.php?referrerid=1060, and get ready to be shocked.!!!!


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Ingredients (serves 4)
Ingredients (serves 4)
4 Portions of Salmon skin on (approximately 150 to 200g each)
2 tbs Soy Sauce
1 tbs Sesame Seeds
½ Red Chilli finely chopped
1/2" of Ginger finely grated
1 Garlic clove crushed
1 tsp runny Honey
¼ Head of Chinese leaf
½ Red Onion peeled
1 Carrot peeled
½ Cucumber
4 Spring Onions
1 handful Bean sprouts
1 Sprig of Fresh Coriander and Mint
2 tsp Thai fish sauce
1 tsp Sugar
½" of Ginger grated
1 Lime zest and juice
½ Red Chilli finely chopped
Place the soy, chilli, honey, ginger and garlic into a bowl and whisk tog`ether.
Dip each salmon portion into the soy mixture.
Place the salmon skin side up onto a grill rack and place under a hot grill for 4 minutes. Carefully turn the fish and sprinkle with the sesame seeds and cook for a further 3 minutes or until fully cooked.
Shred the Chinese leaf, finely slice the onion and cut the carrot and cucumber into thin strips.
Mix all this in a bowl with the bean sprouts.
Roughly chop the herbs and mix.
Make the dressing by whisking all the ingredients together.
Toss with the raw vegetables last minute and serve with the salmon and steamed rice.
By NIALL MURRAY from City of Glasgow College
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Respond to Review of Evolution Fast Food
by cheddargurl
Review: My sister suggested going to Evolution as a family, I ordered a cheeseburger and sweet potato fries. The place has a unique and artsy feel. The cashier was very friendly, and the food was Delicious. The ketchup was really good, it didn't taste all sugary and artificial.I've definitively gone back many more times. My whole family loved it. And I even rode the bus with sister just to go.
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Bubble Shooter with a fun twist: the bubbles are on a rotating platform. See if you can knock them all out. Very addicting.
Jewel Search - 5Players onlinePlayers: 530 daily
You travel a lot to find precious stones. You have to find them in the mountains in all around the world and save them. Make your drema real and find as many jewelry as you can!
Bubble Cannon 2 - 13Players onlinePlayers: 546 daily
Can you bounce, reflect and pop your way through 10 levels to achieve a state of Zen? This game is intentionally hard - make every shot count!
Monkey GO HappyPlayers: 17 daily
Have you ever wished you had a MONKEY??? Well they're hard work you know! You have to spend all your time entertaining them.
Bejeweled BlitzPlayers: 17 daily
Swap adjacent gems to create sets of 3 gems of the same color lined up horizontally or vertically.
Flower ReactionPlayers: 29 daily
Blossom the maximum amount of flowers with just one click. Try to make a chain reaction of exploding flowers and record the highscore!
Sarah QuestPlayers: 28 daily
Join Sarah Quest on her first adventure as she works her way through the tombs of Egypt to discover treasure and save her family's legacy!
Crystal Balls - 3Players onlinePlayers: 418 daily
Stack as many balls as possible with matching colors & gain points. Stack 3 or more matching colors balls horizontally, vertically or diagonally to clear them.
Tropical GemsPlayers: 14 daily
Come to tropical island! Rotate gems to make matches of three or more and record the best highscore!
Egypt Puzzle - 2Players onlinePlayers: 90 daily
Truly original, innovative and extremely addictive gameplay will keep you moving these bricks for hours on end. Crack the intriguing puzzles of the past and unlock the secrets of the pharaohs!
Cover Orange 2:Player PackPlayers: 6 daily
This cute logical game has finally got its first Player Pack! Can you make it through the all 80 levels?
Shanghai Dynasty - 1Players onlinePlayers: 255 daily
This is a great puzzle game that will have you pulling your hair out for hours!
Lock 'n' RollPlayers: 38 daily
Lock 'n' Roll is an intriguing and addictive puzzle game requiring a combination of luck and skill.


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Showing 1-3 of 3 Dude Ranches
Wilstem Guest Ranch
Wilstem, "A Southern Indiana Property of Distinction," is a secluded 1,100-acre working ranch in southern Indiana's rolling hills. Choose from cabins, cottages, houses or the extraordinary Lodge. All units include a fireplace and a full kitchen and some feature hot tubs and whirlpools. Enjoy horseback riding on 30 miles of trails along with bird... more
Crazyhorse Ranch & Lodge
Crazyhorse Ranch & Lodge offers guided horseback riding excursions. Don't worry, the horses aren't wild or crazy. The ranch's herd of gaited walking horses are great for beginning riders or experts. Trips are usually one to two hours, but longer trips can be arranged. ... more
Elk Ridge Ranch
Discover a ranch experience unlike any other in Southern Indiana at the Elk Ridge Ranch. This 120-acre working ranch offers a range of charming, rustic accommodations and activities to make your stay unforgettable. Enjoy fun hay rides and chuck wagon dinners, and a close proximity to nearby golf courses and much more!... more
Showing 1-3 of 3 Dude Ranches