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Publications in peer reviewed journals
Astrobiology as a framework for investigating antibiotic susceptibility: a study of Halomonas hydrothermalis2017 - J R Soc Interface, 126: online only
Physical and chemical boundaries for microbial multiplication on Earth are strongly influenced by interactions between environmental extremes. However, little is known about how interactions between multiple stress parameters affect the sensitivity of microorganisms to antibiotics. Here, we assessed how 12 distinct permutations of salinity, availability of an essential nutrient (iron) and atmospheric composition (aerobic or microaerobic) affect the susceptibility of a polyextremotolerant bacterium, Halomonas hydrothermalis, to ampicillin, kanamycin and ofloxacin. While salinity had a significant impact on sensitivity to all three antibiotics (as shown by turbidimetric analyses), the nature of this impact was modified by iron availability and the ambient gas composition, with differing effects observed for each compound. These two parameters were found to be of particular importance when considered in combination and, in the case of ampicillin, had a stronger combined influence on antibiotic tolerance than salinity. Our data show how investigating microbial responses to multiple extremes, which are more representative of natural habitats than single extremes, can improve our understanding of the effects of antimicrobial compounds and suggest how studies of habitability, motivated by the desire to map the limits of life, can be used to systematically assess the effectiveness of antibiotics.
Adaptability as the key to success for the ubiquitous marine nitrite oxidizer Nitrococcus2017 - Sci Adv, 3: e1700807
Nitrite-oxidizing bacteria (NOB) have conventionally been regarded as a highly specialized functional group responsible for the production of nitrate in the environment. However, recent culture-based studies suggest that they have the capacity to lead alternative lifestyles, but direct environmental evidence for the contribution of marine nitrite oxidizers to other processes has been lacking to date. We report on the alternative biogeochemical functions, worldwide distribution, and sometimes high abundance of the marine NOB Nitrococcus. These largely overlooked bacteria are capable of not only oxidizing nitrite but also reducing nitrate and producing nitrous oxide, an ozone-depleting agent and greenhouse gas. Furthermore, Nitrococcus can aerobically oxidize sulfide, thereby also engaging in the sulfur cycle. In the currently fast-changing global oceans, these findings highlight the potential functional switches these ubiquitous bacteria can perform in various biogeochemical cycles, each with distinct or even contrasting consequences.
Vitamin and Amino Acid Auxotrophy in Anaerobic Consortia Operating under Methanogenic Conditions.2017 - mSystems, 5: e00038-17
Syntrophy among Archaea and Bacteria facilitates the anaerobic degradation of organic compounds to CH4 and CO2. Particularly during aliphatic and aromatic hydrocarbon mineralization, as in the case of crude oil reservoirs and petroleum-contaminated sediments, metabolic interactions between obligate mutualistic microbial partners are of central importance. Using micromanipulation combined with shotgun metagenomic approaches, we describe the genomes of complex consortia within short-chain alkane-degrading cultures operating under methanogenic conditions. Metabolic reconstruction revealed that only a small fraction of genes in the metagenome-assembled genomes encode the capacity for fermentation of alkanes facilitated by energy conservation linked to H2 metabolism. Instead, the presence of inferred lifestyles based on scavenging anabolic products and intermediate fermentation products derived from detrital biomass was a common feature. Additionally, inferred auxotrophy for vitamins and amino acids suggests that the hydrocarbon-degrading microbial assemblages are structured and maintained by multiple interactions beyond the canonical H2-producing and syntrophic alkane degrader-methanogen partnership. Compared to previous work, our report points to a higher order of complexity in microbial consortia engaged in anaerobic hydrocarbon transformation. IMPORTANCE Microbial interactions between Archaea and Bacteria mediate many important chemical transformations in the biosphere from degrading abundant polymers to synthesis of toxic compounds. Two of the most pressing issues in microbial interactions are how consortia are established and how we can modulate these microbial communities to express desirable functions. Here, we propose that public goods (i.e., metabolites of high energy demand in biosynthesis) facilitate energy conservation for life under energy-limited conditions and determine the assembly and function of the consortia. Our report suggests that an understanding of public good dynamics could result in new ways to improve microbial pollutant degradation in anaerobic systems.
Ammonia-oxidising archaea living at low pH: Insights from comparative genomics.2017 - Environ. Microbiol., 12: 4939-4952
Obligate acidophilic members of the thaumarchaeotal genus Candidatus Nitrosotalea play an important role in nitrification in acidic soils, but their evolutionary and physiological adaptations to acidic environments are still poorly understood, with only a single member of this genus (Ca. N. devanaterra) having its genome sequenced. In this study, we sequenced the genomes of two additional cultured Ca. Nitrosotalea strains, extracted an almost complete Ca. Nitrosotalea metagenome-assembled genome from an acidic fen, and performed comparative genomics of the four Ca. Nitrosotalea genomes with 19 other archaeal ammonia oxidiser genomes. Average nucleotide and amino acid identities revealed that the four Ca. Nitrosotalea strains represent separate species within the genus. The four Ca. Nitrosotalea genomes contained a core set of 103 orthologous gene families absent from all other ammonia-oxidizing archaea and, for most of these gene families, expression could be demonstrated in laboratory culture or the environment via proteomic or metatranscriptomic analyses respectively. Phylogenetic analyses indicated that four of these core gene families were acquired by the Ca. Nitrosotalea common ancestor via horizontal gene transfer from acidophilic representatives of Euryarchaeota. We hypothesize that gene exchange with these acidophiles contributed to the competitive success of the Ca. Nitrosotalea lineage in acidic environments.
Abiotic Conversion of Extracellular NH2OH Contributes to N2O Emission during Ammonia Oxidation.2017 - Environ. Sci. Technol., 22: 13122-13132
Abiotic processes involving the reactive ammonia-oxidation intermediates nitric oxide (NO) or hydroxylamine (NH2OH) for N2O production have been indicated recently. The latter process would require the availability of substantial amounts of free NH2OH for chemical reactions during ammonia (NH3) oxidation, but little is known about extracellular NH2OH formation by the different clades of ammonia-oxidizing microbes. Here we determined extracellular NH2OH concentrations in culture media of several ammonia-oxidizing bacteria (AOB) and archaea (AOA), as well as one complete ammonia oxidizer (comammox) enrichment (Ca. Nitrospira inopinata) during incubation under standard cultivation conditions. NH2OH was measurable in the incubation media of Nitrosomonas europaea, Nitrosospira multiformis, Nitrososphaera gargensis, and Ca. Nitrosotenuis uzonensis, but not in media of the other tested AOB and AOA. NH2OH was also formed by the comammox enrichment during NH3 oxidation. This enrichment exhibited the largest NH2OH:final product ratio (1.92%), followed by N. multiformis (0.56%) and N. gargensis (0.46%). The maximum proportions of NH4+ converted to N2O via extracellular NH2OH during incubation, estimated on the basis of NH2OH abiotic conversion rates, were 0.12%, 0.08%, and 0.14% for AOB, AOA, and Ca. Nitrospira inopinata, respectively, and were consistent with published NH4+:N2O conversion ratios for AOB and AOA.
Depth distribution and assembly of sulfate-reducing microbial communities in marine sediments of Aarhus Bay2017 - Appl Environ Microbiol, 83: e01547-17
Most sulfate-reducing microorganisms (SRM) present in subsurface marine sediments belong to uncultured groups only distantly related to known SRM and it remains unclear how changing geochemical zones and sediment depth influence their community structure. We mapped the community composition and abundance of SRM by amplicon-sequencing and quantifying dsrB, which encodes dissimilatory sulfite reductase subunit beta, in sediment samples covering different vertical geochemical zones ranging from the surface sediment to the deep sulfate-depleted subsurface at four locations in Aarhus Bay, Denmark. SRM were present in all geochemical zones including sulfate-depleted methanogenic sediment. The biggest shift in SRM community composition and abundance occurring across the transition from bioturbated surface sediments into non-bioturbated sediments below, where redox fluctuations and input of fresh organic matter due to macrofaunal activity are absent. SRM abundance correlated with sulfate reduction rates determined for the same sediments. Sulfate availability showed weaker correlation with SRM abundances and no significant correlation with the composition of the SRM community. The overall SRM species diversity decreased with depth, yet we identified a subset of highly abundant community members that persists across all vertical geochemical zones of all stations. We conclude that subsurface SRM communities assemble by persistence of members of the surface community and that the transition from the bioturbated surface sediment to the unmixed sediment below is a main site of assembly of the subsurface SRM community.
Bottled aqua incognita: Microbiota assembly and dissolved organic matter diversity in natural mineral waters2017 - Microbiome, 5: 126
Background: Non-carbonated natural mineral waters contain microorganisms that regularly grow after bottling despite low concentrations of dissolved organic matter (DOM). Yet, the compositions of bottled water microbiota and organic substrates that fuel microbial activity, and how both change after bottling, are still largely unknown.
Results: We performed a multifaceted analysis of microbiota and DOM diversity in twelve natural mineral waters from six European countries. 16S rRNA gene-based analyses showed that less than ten species-level operational taxonomic units (OTUs) dominated the bacterial communities in the water phase and associated with the bottle wall after a short phase of post-bottling growth. Members of the betaproteobacterial genera Curvibacter, Aquabacterium, and Polaromonas (Comamonadaceae) grew in most waters and represent ubiquitous, mesophilic, heterotrophic aerobes in bottled waters. Ultrahigh-resolution mass spectrometry of DOM in bottled waters and their corresponding source waters identified thousands of molecular formulae characteristic of mostly refractory, soil-derived DOM.
Conclusions. The bottle environment, including source water physicochemistry, selected for growth of a similar low-diversity microbiota across various bottled waters. Relative abundance changes of hundreds of multi-carbon molecules were related to growth of less than ten abundant OTUs. We thus speculate that individual bacteria cope with oligotrophic conditions by simultaneously consuming diverse DOM molecules.
Identification of secondary metabolite gene clusters in the Pseudovibrio genus reveals encouraging biosynthetic potential toward the production of novel bioactive compounds2017 - Front Microbiol, 8: 1494
Increased incidences of antimicrobial resistance and the emergence of pan-resistant 'superbugs' have provoked an extreme sense of urgency amongst researchers focusing on the discovery of potentially novel antimicrobial compounds. A strategic shift in focus from the terrestrial to the marine environment has resulted in the discovery of a wide variety of structurally and functionally diverse bioactive compounds from numerous marine sources, including sponges. Bacteria found in close association with sponges and other marine invertebrates have recently gained much attention as potential sources of many of these novel bioactive compounds. Members of the genus Pseudovibrio are one such group of organisms. In this study, we interrogate the genomes of 21 Pseudovibrio strains isolated from a variety of marine sources, for the presence, diversity and distribution of biosynthetic gene clusters (BGCs). We expand on results obtained from antiSMASH analysis to demonstrate the similarity between the Pseudovibrio-related BGCs and those characterized in other bacteria and corroborate our findings with phylogenetic analysis. We assess how domain organization of the most abundant type of BGCs present among the isolates (Non-ribosomal peptide synthetases and Polyketide synthases) may influence the diversity of compounds produced by these organisms and highlight for the first time the potential for novel compound production from this genus of bacteria, using a genome guided approach.
Kinetic analysis of a complete nitrifier reveals an oligotrophic lifestyle.2017 - Nature, 549: 269-272
Nitrification, the oxidation of ammonia (NH3) via nitrite (NO2(-)) to nitrate (NO3(-)), is a key process of the biogeochemical nitrogen cycle. For decades, ammonia and nitrite oxidation were thought to be separately catalysed by ammonia-oxidizing bacteria (AOB) and archaea (AOA), and by nitrite-oxidizing bacteria (NOB). The recent discovery of complete ammonia oxidizers (comammox) in the NOB genus Nitrospira, which alone convert ammonia to nitrate, raised questions about the ecological niches in which comammox Nitrospira successfully compete with canonical nitrifiers. Here we isolate a pure culture of a comammox bacterium, Nitrospira inopinata, and show that it is adapted to slow growth in oligotrophic and dynamic habitats on the basis of a high affinity for ammonia, low maximum rate of ammonia oxidation, high growth yield compared to canonical nitrifiers, and genomic potential for alternative metabolisms. The nitrification kinetics of four AOA from soil and hot springs were determined for comparison. Their surprisingly poor substrate affinities and lower growth yields reveal that, in contrast to earlier assumptions, AOA are not necessarily the most competitive ammonia oxidizers present in strongly oligotrophic environments and that N. inopinata has the highest substrate affinity of all analysed ammonia oxidizer isolates except the marine AOA Nitrosopumilus maritimus SCM1 (ref. 3). These results suggest a role for comammox organisms in nitrification under oligotrophic and dynamic conditions.
AmoA-targeted polymerase chain reaction primers for the specific detection and quantification of comammox Nitrospira in the environment2017 - Front Microbiol, 8:1508
Nitrification, the oxidation of ammonia via nitrite to nitrate, has always been considered to be catalyzed by the concerted activity of ammonia- and nitrite-oxidizing microorganisms. Only recently, complete ammonia oxidizers ('comammox'), which oxidize ammonia to nitrate on their own, were identified in the bacterial genus Nitrospira, previously assumed to contain only canonical nitrite oxidizers. Nitrospira are widespread in nature, but for assessments of the distribution and functional importance of comammox Nitrospira in ecosystems, cultivation-independent tools to distinguish comammox from strictly nitrite oxidizing Nitrospira are required. Here we developed new PCR primer sets that specifically target the amoA genes coding for subunit A of the distinct ammonia monooxygenase of comammox Nitrospira. While existing primers capture only a fraction of the known comammox amoA diversity, the new primer sets cover as much as 95% of the comammox amoA clade A and 92% of the clade B sequences in a reference database containing 326 comammox amoA genes with sequence information at the primer binding sites. Application of the primers to 13 samples from engineered systems (a groundwater well, drinking water treatment and wastewater treatment plants) and other habitats (rice paddy and forest soils, rice rhizosphere, brackish lake sediment and freshwater biofilm) detected comammox Nitrospira in all samples and revealed a considerable diversity of comammox in most habitats. Excellent primer specificity for comammox amoA was achieved by avoiding the use of highly degenerate primer preparations and by using equimolar mixtures of oligonucleotides that match existing comammox amoA genes. Quantitative PCR with these equimolar primer mixtures was highly sensitive and specific, and enabled the efficient quantification of clade A and clade B comammox amoA gene copy numbers in environmental samples. The measured relative abundances of comammox Nitrospira, compared to canonical ammonia oxidizers, were highly variable across environments. The new comammox amoA-targeted primers enable more encompassing future studies of nitrifying microorganisms in diverse habitats. For example, they may be used to monitor the population dynamics of uncultured comammox organisms under changing environmental conditions and in response to altered treatments in engineered and agricultural ecosystems.
In situ architecture, function, and evolution of a contractile injection system.2017 - Science, 6352: 713-717
Contractile injection systems mediate bacterial cell-cell interactions by a bacteriophage tail-like structure. In contrast to extracellular systems, the type 6 secretion system (T6SS) is defined by intracellular localization and attachment to the cytoplasmic membrane. Here we used cryo-focused ion beam milling, electron cryotomography, and functional assays to study a T6SS in Amoebophilus asiaticus The in situ architecture revealed three modules, including a contractile sheath-tube, a baseplate, and an anchor. All modules showed conformational changes upon firing. Lateral baseplate interactions coordinated T6SSs in hexagonal arrays. The system mediated interactions with host membranes and may participate in phagosome escape. Evolutionary sequence analyses predicted that T6SSs are more widespread than previously thought. Our insights form the basis for understanding T6SS key concepts and exploring T6SS diversity.
Allspice and Clove As Source of Triterpene Acids Activating the G Protein-Coupled Bile Acid Receptor TGR5.2017 - Front Pharmacol, 8: 468
Worldwide, metabolic diseases such as obesity and type 2 diabetes have reached epidemic proportions. A major regulator of metabolic processes that gained interest in recent years is the bile acid receptor TGR5 (Takeda G protein-coupled receptor 5). This G protein-coupled membrane receptor can be found predominantly in the intestine, where it is mainly responsible for the secretion of the incretins glucagon-like peptide 1 (GLP-1) and peptide YY (PYY). The aim of this study was (i) to identify plant extracts with TGR5-activating potential, (ii) to narrow down their activity to the responsible constituents, and (iii) to assess whether the intestinal microbiota produces transformed metabolites with a different activity profile. Chenodeoxycholic acid (CDCA) served as positive control for both, the applied cell-based luciferase reporter gene assay for TGR5 activity and the biotransformation assay using mouse fecal slurry. The suitability of the workflow was demonstrated by the biotransformation of CDCA to lithocholic acid resulting in a distinct increase in TGR5 activity. Based on a traditional Tibetan formula, 19 plant extracts were selected and investigated for TGR5 activation. Extracts from the commonly used spices Syzygium aromaticum (SaroE, clove), Pimenta dioica (PdioE, allspice), and Kaempferia galanga (KgalE, aromatic ginger) significantly increased TGR5 activity. After biotransformation, only KgalE showed significant differences in its metabolite profile, which, however, did not alter its TGR5 activity compared to non-transformed KgalE. UHPLC-HRMS (high-resolution mass spectrometry) analysis revealed triterpene acids (TTAs) as the main constituents of the extracts SaroE and PdioE. Identification and quantification of TTAs in these two extracts as well as comparison of their TGR5 activity with reconstituted TTA mixtures allowed the attribution of the TGR5 activity to TTAs. EC50s were determined for the main TTAs, i.e., oleanolic acid (2.2 ± 1.6 μM), ursolic acid (1.1 ± 0.2 μM), as well as for the hitherto unknown TGR5 activators corosolic acid (0.5 ± 1.0 μM) and maslinic acid (3.7 ± 0.7 μM). In conclusion, extracts of clove, allspice, and aromatic ginger activate TGR5, which might play a pivotal role in their therapeutic use for the treatment of metabolic diseases. Moreover, the TGR5 activation of SaroE and PdioE could be pinpointed solely to TTAs.
Unexpected genomic features in widespread intracellular bacteria: evidence for motility of marine chlamydiae.2017 - ISME J, 10: 2334-2344
Chlamydiae are obligate intracellular bacteria comprising important human pathogens and symbionts of protists. Molecular evidence indicates a tremendous diversity of chlamydiae particularly in marine environments, yet our current knowledge is based mainly on terrestrial representatives. Here we provide first insights into the biology of marine chlamydiae representing three divergent clades. Our analysis of single-cell amplified genomes revealed hallmarks of the chlamydial lifestyle, supporting the ancient origin of their characteristic developmental cycle and major virulence mechanisms. Surprisingly, these chlamydial genomes encode a complete flagellar apparatus, a previously unreported feature. We show that flagella are an ancient trait that was subject to differential gene loss among extant chlamydiae. Together with a chemotaxis system, these marine chlamydiae are likely motile, with flagella potentially playing a role during host cell infection. This study broadens our view on chlamydial biology and indicates a largely underestimated potential to adapt to different hosts and environments.
'Candidatus Cochliophilus cryoturris' (Coxiellaceae), a symbiont of the testate amoeba Cochliopodium minus.2017 - Sci Rep, 1: 3394
Free-living amoebae are well known for their role in controlling microbial community composition through grazing, but some groups, namely Acanthamoeba species, also frequently serve as hosts for bacterial symbionts. Here we report the first identification of a bacterial symbiont in the testate amoeba Cochliopodium. The amoeba was isolated from a cooling tower water sample and identified as C. minus. Fluorescence in situ hybridization and transmission electron microscopy revealed intracellular symbionts located in vacuoles. 16S rRNA-based phylogenetic analysis identified the endosymbiont as member of a monophyletic group within the family Coxiellaceae (Gammaprotebacteria; Legionellales), only moderately related to known amoeba symbionts. We propose to tentatively classify these bacteria as 'Candidatus Cochliophilus cryoturris'. Our findings add both, a novel group of amoeba and a novel group of symbionts, to the growing list of bacteria-amoeba relationships.
Biphasic Metabolism and Host Interaction of a Chlamydial Symbiont.2017 - mSystems, 2: e00202-16
Chlamydiae are obligate intracellular bacteria comprising well-known human pathogens and ubiquitous symbionts of protists, which are characterized by a unique developmental cycle. Here we comprehensively analyzed gene expression dynamics of Protochlamydia amoebophila during infection of its Acanthamoeba host by RNA sequencing. This revealed a highly dynamic transcriptional landscape, where major transcriptional shifts are conserved among chlamydial symbionts and pathogens. Our data served to propose a time-resolved model for type III protein secretion during the developmental cycle, and we provide evidence for a biphasic metabolism of P. amoebophila during infection, which involves energy parasitism and amino acids as the carbon source during initial stages and a postreplicative switch to endogenous glucose-based ATP production. This fits well with major transcriptional changes in the amoeba host, where upregulation of complex sugar breakdown precedes the P. amoebophila metabolic switch. The biphasic chlamydial metabolism represents a unique adaptation to exploit eukaryotic host cells, which likely contributed to the evolutionary success of this group of microbes. IMPORTANCE Chlamydiae are known as major bacterial pathogens of humans, causing the ancient disease trachoma, but they are also frequently found in the environment where they infect ubiquitous protists such as amoebae. All known chlamydiae require a eukaryotic host cell to thrive. Using the environmental chlamydia Protochlamydia amoebophila within its natural host, Acanthamoeba castellanii, we investigated gene expression dynamics in vivo and throughout the complete chlamydial developmental cycle for the first time. This allowed us to infer how a major virulence mechanism, the type III secretion system, is regulated and employed, and we show that the physiology of chlamydiae undergoes a complete shift regarding carbon metabolism and energy generation. This study provides comprehensive insights into the infection strategy of chlamydiae and reveals a unique adaptation to life within a eukaryotic host cell.
Crenothrix are major methane consumers in stratified lakes.2017 - ISME J, 9: 2124-2140
Methane-oxidizing bacteria represent a major biological sink for methane and are thus Earth's natural protection against this potent greenhouse gas. Here we show that in two stratified freshwater lakes a substantial part of upward-diffusing methane was oxidized by filamentous gamma-proteobacteria related to Crenothrix polyspora. These filamentous bacteria have been known as contaminants of drinking water supplies since 1870, but their role in the environmental methane removal has remained unclear. While oxidizing methane, these organisms were assigned an 'unusual' methane monooxygenase (MMO), which was only distantly related to 'classical' MMO of gamma-proteobacterial methanotrophs. We now correct this assignment and show that Crenothrix encode a typical gamma-proteobacterial PmoA. Stable isotope labeling in combination swith single-cell imaging mass spectrometry revealed methane-dependent growth of the lacustrine Crenothrix with oxygen as well as under oxygen-deficient conditions. Crenothrix genomes encoded pathways for the respiration of oxygen as well as for the reduction of nitrate to N2O. The observed abundance and planktonic growth of Crenothrix suggest that these methanotrophs can act as a relevant biological sink for methane in stratified lakes and should be considered in the context of environmental removal of methane.
Capturing the genetic makeup of the active microbiome in situ.2017 - ISME J, 9: 1949-1963
More than any other technology, nucleic acid sequencing has enabled microbial ecology studies to be complemented with the data volumes necessary to capture the extent of microbial diversity and dynamics in a wide range of environments. In order to truly understand and predict environmental processes, however, the distinction between active, inactive and dead microbial cells is critical. Also, experimental designs need to be sensitive toward varying population complexity and activity, and temporal as well as spatial scales of process rates. There are a number of approaches, including single-cell techniques, which were designed to study in situ microbial activity and that have been successively coupled to nucleic acid sequencing. The exciting new discoveries regarding in situ microbial activity provide evidence that future microbial ecology studies will indispensably rely on techniques that specifically capture members of the microbiome active in the environment. Herein, we review those currently used activity-based approaches that can be directly linked to shotgun nucleic acid sequencing, evaluate their relevance to ecology studies, and discuss future directions.
Evaluating the Detection of Hydrocarbon-Degrading Bacteria in 16S rRNA Gene Sequencing Surveys.2017 - Front Microbiol, 8: 2460
Hydrocarbonoclastic bacteria (HCB) play a key role in the biodegradation of oil hydrocarbons in marine and other environments. A small number of taxa have been identified as obligate HCB, notably the Gammaproteobacterial genera Alcanivorax, Cycloclasticus, Marinobacter, Neptumonas, Oleiphilus, Oleispira, and Thalassolituus, as well as the Alphaproteobacterial genus Thalassospira. Detection of HCB in amplicon-based sequencing surveys relies on high coverage by PCR primers and accurate taxonomic classification. In this study, we performed a phylogenetic analysis to identify 16S rRNA gene sequence regions that represent the breadth of sequence diversity within these taxa. Using validated sequences, we evaluated 449 universal 16S rRNA gene-targeted bacterial PCR primer pairs for their coverage of these taxa. The results of this analysis provide a practical framework for selection of suitable primer sets for optimal detection of HCB in sequencing surveys.
Vibrational Spectroscopy for Imaging Single Microbial Cells in Complex Biological Samples.2017 - Front Microbiol, 8: 675
Vibrational spectroscopy is increasingly used for the rapid and non-destructive imaging of environmental and medical samples. Both Raman and Fourier-transform infrared (FT-IR) imaging have been applied to obtain detailed information on the chemical composition of biological materials, ranging from single microbial cells to tissues. Due to its compatibility with methods such as stable isotope labeling for the monitoring of cellular activities, vibrational spectroscopy also holds considerable power as a tool in microbial ecology. Chemical imaging of undisturbed biological systems (such as live cells in their native habitats) presents unique challenges due to the physical and chemical complexity of the samples, potential for spectral interference, and frequent need for real-time measurements. This Mini Review provides a critical synthesis of recent applications of Raman and FT-IR spectroscopy for characterizing complex biological samples, with a focus on developments in single-cell imaging. We also discuss how new spectroscopic methods could be used to overcome current limitations of single-cell analyses. Given the inherent complementarity of Raman and FT-IR spectroscopic methods, we discuss how combining these approaches could enable us to obtain new insights into biological activities either in situ or under conditions that simulate selected properties of the natural environment.
HuR small-molecule inhibitor elicits differential effects in adenomatosis polyposis and colorectal carcinogenesis2017 - Cancer Res., 77: 2424-2438
HuR is an RNA-binding protein implicated in immune homeostasis and various cancers, including colorectal cancer. HuR binding to AU-rich elements within the 3' untranslated region of mRNAs encoding oncogenes, growth factors, and various cytokines leads message stability and translation. In this study, we evaluated HuR as a small-molecule target for preventing colorectal cancer in high-risk groups such as those with familial adenomatosis polyposis (FAP) or inflammatory bowel disease (IBD). In human specimens, levels of cytoplasmic HuR were increased in colonic epithelial cells from patients with IBD, IBD-cancer, FAP-adenoma, and colorectal cancer, but not in patients with IBD-dysplasia. Intraperitoneal injection of the HuR small-molecule inhibitor MS-444 in AOM/DSS mice, a model of IBD and inflammatory colon cancer, augmented DSS-induced weight loss and increased tumor multiplicity, size, and invasiveness. MS-444 treatment also abrogated tumor cell apoptosis and depleted tumor-associated eosinophils, accompanied by a decrease in IL18 and eotaxin-1. In contrast, HuR inhibition in APCMin mice, a model of FAP and colon cancer, diminished the number of small intestinal tumors generated. In this setting, fecal microbiota, evaluated by 16S rRNA gene amplicon sequencing, shifted to a state of reduced bacterial diversity, with an increased representation of Prevotella, Akkermansia, and Lachnospiraceae Taken together, our results indicate that HuR activation is an early event in FAP-adenoma but is not present in IBD-dysplasia. Furthermore, our results offer a preclinical proof of concept for HuR inhibition as an effective means of FAP chemoprevention, with caution advised in the setting of IBD.
The life sulfuric: Microbial ecology of sulfur cycling in marine sediments.2017 - Environ Microbiol Rep, 9: 323-344
Almost the entire seafloor is covered with sediments that can be more than 10,000 m thick and represent a vast microbial ecosystem that is a major component of Earth's element and energy cycles. Notably, a significant proportion of microbial life in marine sediments can exploit energy conserved during transformations of sulfur compounds among different redox states. Sulfur cycling, which is primarily driven by sulfate reduction, is tightly interwoven with other important element cycles (carbon, nitrogen, iron, manganese) and therefore has profound implications for both cellular- and ecosystem-level processes. Sulfur-transforming microorganisms have evolved diverse genetic, metabolic, and in some cases, peculiar phenotypic features to fill an array of ecological niches in marine sediments. Here, we review recent and selected findings on the microbial guilds that are involved in the transformation of different sulfur compounds in marine sediments and emphasize how these are interlinked and have a major influence on ecology and biogeochemistry in the seafloor. Extraordinary discoveries have increased our knowledge on microbial sulfur cycling, mainly in sulfate-rich surface sediments, yet many questions remain regarding how sulfur redox processes may sustain the deep-subsurface biosphere and the impact of organic sulfur compounds on the marine sulfur cycle. This article is protected by copyright. All rights reserved.
Giant viruses with an expanded complement of translation system components.2017 - Science, 6333: 82-85
The discovery of giant viruses blurred the sharp division between viruses and cellular life. Giant virus genomes encode proteins considered as signatures of cellular organisms, particularly translation system components, prompting hypotheses that these viruses derived from a fourth domain of cellular life. Here we report the discovery of a group of giant viruses (Klosneuviruses) in metagenomic data. Compared with other giant viruses, the Klosneuviruses encode an expanded translation machinery, including aminoacyl transfer RNA synthetases with specificities for all 20 amino acids. Notwithstanding the prevalence of translation system components, comprehensive phylogenomic analysis of these genes indicates that Klosneuviruses did not evolve from a cellular ancestor but rather are derived from a much smaller virus through extensive gain of host genes.
Members of the Oral Microbiota Are Associated with IL-8 Release by Gingival Epithelial Cells in Healthy Individuals.2017 - Front Microbiol, 8: 416
The triggers for the onset of oral diseases are still poorly understood. The aim of this study was to characterize the oral bacterial community in healthy humans and its association with nutrition, oral hygiene habits, and the release of the inflammatory marker IL-8 from gingival epithelial cells (GECs) with and without stimulation by bacterial endotoxins to identify possible indicator operational taxonomic units (OTUs) associated with inflammatory marker status. GECs from 21 healthy participants (13 females, 8 males) were incubated with or without addition of bacterial lipopolysaccharides (LPSs), and the oral microbiota was profiled using 16S rRNA gene-targeted sequencing. The basal IL-8 release after 6 h was between 9.9 and 98.2 pg/ml, and bacterial communities were characteristic for healthy oral microbiota. The composition of the oral microbiota was associated with basal IL-8 levels, the intake of meat, tea, white wine, sweets and the use of chewing gum, as well as flossing habits, allergies, gender and body mass index. Additionally, eight OTUs were associated with high basal levels of IL-8 and GEC response to LPS, with high basal levels of IL-8, and 1 with low basal levels of IL8. The identification of indicator bacteria in healthy subjects with high levels of IL-8 release is of importance as they may be promising early warning indicators for the possible onset of oral diseases.
Variant profiling of evolving prokaryotic populations.2017 - PeerJ, e2997
Genomic heterogeneity of bacterial species is observed and studied in experimental evolution experiments and clinical diagnostics, and occurs as micro-diversity of natural habitats. The challenge for genome research is to accurately capture this heterogeneity with the currently used short sequencing reads. Recent advances in NGS technologies improved the speed and coverage and thus allowed for deep sequencing of bacterial populations. This facilitates the quantitative assessment of genomic heterogeneity, including low frequency alleles or haplotypes. However, false positive variant predictions due to sequencing errors and mapping artifacts of short reads need to be prevented. We therefore created VarCap, a workflow for the reliable prediction of different types of variants even at low frequencies. In order to predict SNPs, InDels and structural variations, we evaluated the sensitivity and accuracy of different software tools using synthetic read data. The results suggested that the best sensitivity could be reached by a union of different tools, however at the price of increased false positives. We identified possible reasons for false predictions and used this knowledge to improve the accuracy by post-filtering the predicted variants according to properties such as frequency, coverage, genomic environment/localization and co-localization with other variants. We observed that best precision was achieved by using an intersection of at least two tools per variant. This resulted in the reliable prediction of variants above a minimum relative abundance of 2%. VarCap is designed for being routinely used within experimental evolution experiments or for clinical diagnostics. The detected variants are reported as frequencies within a VCF file and as a graphical overview of the distribution of the different variant/allele/haplotype frequencies. The source code of VarCap is available at https://github.com/ma2o/VarCap. In order to provide this workflow to a broad community, we implemeted VarCap on a Galaxy webserver, which is accessible at http://galaxy.csb.univie.ac.at.
Cultivation and characterization of Candidatus Nitrosocosmicus exaquare, an ammonia-oxidizing archaeon from a municipal wastewater treatment system.2017 - ISME J, 5: 1142-1157
Thaumarchaeota have been detected in several industrial and municipal wastewater treatment plants (WWTPs), despite the fact that ammonia-oxidizing archaea (AOA) are thought to be adapted to low ammonia environments. However, the activity, physiology and metabolism of WWTP-associated AOA remain poorly understood. We report the cultivation and complete genome sequence of Candidatus Nitrosocosmicus exaquare, a novel AOA representative from a municipal WWTP in Guelph, Ontario (Canada). In enrichment culture, Ca. N. exaquare oxidizes ammonia to nitrite stoichiometrically, is mesophilic, and tolerates at least 15 mm of ammonium chloride or sodium nitrite. Microautoradiography (MAR) for enrichment cultures demonstrates that Ca. N. exaquare assimilates bicarbonate in association with ammonia oxidation. However, despite using inorganic carbon, the ammonia-oxidizing activity of Ca. N. exaquare is greatly stimulated in enrichment culture by the addition of organic compounds, especially malate and succinate. Ca. N. exaquare cells are coccoid with a diameter of ~1-2 μm. Phylogenetically, Ca. N. exaquare belongs to the Nitrososphaera sister cluster within the Group I.1b Thaumarchaeota, a lineage which includes most other reported AOA sequences from municipal and industrial WWTPs. The 2.99 Mbp genome of Ca. N. exaquare encodes pathways for ammonia oxidation, bicarbonate fixation, and urea transport and breakdown. In addition, this genome encodes several key genes for dealing with oxidative stress, including peroxidase and catalase. Incubations of WWTP biofilm demonstrate partial inhibition of ammonia-oxidizing activity by 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), suggesting that Ca. N. exaquare-like AOA may contribute to nitrification in situ. However, CARD-FISH-MAR showed no incorporation of bicarbonate by detected Thaumarchaeaota, suggesting that detected AOA may incorporate non-bicarbonate carbon sources or rely on an alternative and yet unknown metabolism.
Lifestyle and horizontal gene transfer-mediated evolution of Mucispirillum schaedleri, a core member of the murine gut microbiota2017 - mSystems, 2: e00171-16
Mucispirillum schaedleri is an abundant inhabitant of the intestinal mucus layer of rodents and other animals and has been suggested to be a pathobiont, a commensal that plays a role in disease. In order to gain insights into its lifestyle, we analyzed the genome and transcriptome of M. schaedleri ASF 457 and performed physiological experiments to test traits predicted by its genome. Although described as a mucus inhabitant, M. schaedleri has limited capacity for degrading host-derived mucosal glycans and other complex polysaccharides. Additionally, M. schaedleri reduces nitrate and expresses systems for scavenging oxygen and reactive oxygen species in vivo, which may account for its localization close to the mucosal tissue and expansion during inflammation. Also of note, M. schaedleri harbors a type VI secretion system and putative effector proteins and can modify gene expression in mucosal tissue, suggesting intimate interactions with its host and a possible role in inflammation. The M. schaedleri genome has been shaped by extensive horizontal gene transfer, primarily from intestinal Epsilon- and Deltaproteobacteria, indicating that horizontal gene transfer has played a key role in defining its niche in the gut ecosystem.
Genomic repertoire of the Woeseiaceae/JTB255, cosmopolitan and abundant core members of microbial communities in marine sediments.2017 - ISME J, 5: 1276-1281
To date, very little is known about the bacterial core community of marine sediments. Here we study the environmental distribution, abundance and ecogenomics of the gammaproteobacterial Woeseiaceae/JTB255 marine benthic group. A meta-analysis of published work shows that the Woeseiaceae/JTB255 are ubiquitous and consistently rank among the most abundant 16S rRNA gene sequences in diverse marine sediments. They account for up to 22% of bacterial amplicons and 6% of total cell counts in European and Australian coastal sediments. The analysis of a single-cell genome, metagenomic bins and the genome of the next cultured relative Woeseia oceani indicated a broad physiological range, including heterotrophy and facultative autotrophy. All tested (meta)genomes encode a truncated denitrification pathway to nitrous oxide. The broad range of energy-yielding metabolisms possibly explains the ubiquity and high abundance of Woeseiaceae/JTB255 in marine sediments, where they carry out diverse, but yet unknown ecological functions.
Microbial nutrient niches in the gut.2017 - Environ. Microbiol., 4: 1366-1378
The composition and function of the mammalian gut microbiota has been the subject of much research in recent years, but the principles underlying the assembly and structure of this complex community remain incompletely understood. Processes that shape the gut microbiota are thought to be mostly niche-driven, with environmental factors such as the composition of available nutrients largely determining whether or not an organism can establish. The concept that the nutrient landscape dictates which organisms can successfully colonize and persist in the gut was first proposed in Rolf Freter's nutrient niche theory. In a situation where nutrients are perfectly mixed and there is balanced microbial growth, Freter postulated that an organism can only survive if it is able to utilize one or a few limiting nutrients more efficiently than its competitors. Recent experimental work indicates, however, that nutrients in the gut vary in space and time. We propose that in such a scenario, Freter's nutrient niche theory must be expanded to account for the co-existence of microorganisms utilizing the same nutrients but in distinct sites or at different times, and that metabolic flexibility and mixed-substrate utilization are common strategies for survival in the face of ever-present nutrient fluctuations.
A 12-week intervention with nonivamide, a TRPV1 agonist, prevents a dietary-induced body fat gain and increases peripheral serotonin in moderately overweight subjects.2017 - Mol Nutr Food Res, 5: 1600731
A bolus administration of 0.15 mg nonivamide has previously been demonstrated to reduce energy intake in moderately overweight men. This 12-week intervention investigated whether a daily consumption of nonivamide in a protein-based product formulation promotes a reduction in body weight in healthy overweight subjects and affects outcome measures associated with mechanisms regulating food intake, e.g. plasma concentrations of (an)orexigenic hormones, energy substrates as well as changes in fecal microbiota.
Nineteen overweight subjects were randomly assigned to either a control (C) or a nonivamide (NV) group. Changes in the body composition and plasma concentrations of satiating hormones were determined at fasting and 15, 30, 60, 90, and 120 min after a glucose load. Participants were instructed to consume 0.15 mg nonivamide per day in 450 mL of a milk shake additionally to their habitual diet. After treatment, a group difference in body fat mass change (-0.61 ± 0.36% in NV and +1.36 ± 0.38% in C) and an increase in postprandial plasma serotonin were demonstrated. Plasma metabolome and fecal microbiome read outs were not affected.
A daily intake of 0.15 mg nonivamide helps to support to maintain a healthy body composition.
Convergent patterns in the evolution of mealybug symbioses involving different intrabacterial symbionts.2017 - ISME J, 3: 715-726
Mealybugs (Insecta: Hemiptera: Pseudococcidae) maintain obligatory relationships with bacterial symbionts, which provide essential nutrients to their insect hosts. Most pseudococcinae mealybugs harbor a unique symbiosis setup with enlarged betaproteobacterial symbionts ('Candidatus Tremblaya princeps'), which themselves contain gammaproteobacterial symbionts. Here we investigated the symbiosis of the manna mealybug, Trabutina mannipara, using a metagenomic approach. Phylogenetic analyses revealed that the intrabacterial symbiont of T. mannipara represents a novel lineage within the Gammaproteobacteria, for which we propose the tentative name 'Candidatus Trabutinella endobia'. Combining our results with previous data available for the nested symbiosis of the citrus mealybug Planococcus citri, we show that synthesis of essential amino acids and vitamins and translation-related functions partition between the symbiotic partners in a highly similar manner in the two systems, despite the distinct evolutionary origin of the intrabacterial symbionts. Bacterial genes found in both mealybug genomes and complementing missing functions in both symbioses were likely integrated in ancestral mealybugs before T. mannipara and P. citri diversified. The high level of correspondence between the two mealybug systems and their highly intertwined metabolic pathways are unprecedented. Our work contributes to a better understanding of the only known intracellular symbiosis between two bacteria and suggests that the evolution of this unique symbiosis included the replacement of intrabacterial symbionts in ancestral mealybugs.
Happens in the best of subfamilies: establishment and repeated replacements of co-obligate secondary endosymbionts within Lachninae aphids.2017 - Environ. Microbiol., 1: 393-408
Virtually all aphids maintain an obligate mutualistic symbiosis with bacteria from the Buchnera genus, which produce essential nutrients for their aphid hosts. Most aphids from the Lachninae subfamily have been consistently found to house additional endosymbionts, mainly Serratia symbiotica. This apparent dependence on secondary endosymbionts was proposed to have been triggered by the loss of the riboflavin biosynthetic capability by Buchnera in the Lachninae last common ancestor. However, an integral large-scale analysis of secondary endosymbionts in the Lachninae is still missing, hampering the interpretation of the evolutionary and genomic analyses of these endosymbionts. Here, we analysed the endosymbionts of selected representatives from seven different Lachninae genera and nineteen species, spanning four tribes, both by FISH (exploring the symbionts' morphology and tissue tropism) and 16S rRNA gene sequencing. We demonstrate that all analysed aphids possess dual symbiotic systems, and while most harbour S. symbiotica, some have undergone symbiont replacement by other phylogenetically-distinct bacterial taxa. We found that these secondary associates display contrasting cell shapes and tissue tropism, and some appear to be lineage-specific. We propose a scenario for symbiont establishment in the Lachninae, followed by changes in the symbiont's tissue tropism and symbiont replacement events, thereby highlighting the extraordinary versatility of host-symbiont interactions.
Metabolic and physiological interdependencies in the Bathymodiolus azoricus symbiosis.2017 - ISME J, 11: 463–477
The hydrothermal vent mussel Bathymodiolus azoricus lives in an intimate symbiosis with two types of chemosynthetic Gammaproteobacteria in its gills: a sulfur oxidizer and a methane oxidizer. Despite numerous investigations over the last decades, the degree of interdependence between the three symbiotic partners, their individual metabolic contributions, as well as the mechanism of carbon transfer from the symbionts to the host are poorly understood. We used a combination of proteomics and genomics to investigate the physiology and metabolism of the individual symbiotic partners. Our study revealed that key metabolic functions are most likely accomplished jointly by B. azoricus and its symbionts: (1) CO2 is pre-concentrated by the host for carbon fixation by the sulfur-oxidizing symbiont, and (2) the host replenishes essential biosynthetic TCA cycle intermediates for the sulfur-oxidizing symbiont. In return (3), the sulfur oxidizer may compensate for the host's putative deficiency in amino acid and cofactor biosynthesis. We also identified numerous 'symbiosis-specific' host proteins by comparing symbiont-containing and symbiont-free host tissues and symbiont fractions. These proteins included a large complement of host digestive enzymes in the gill that are likely involved in symbiont digestion and carbon transfer from the symbionts to the host.The ISME Journal advance online publication, 1 November 2016; doi:10.1038/ismej.2016.124.
Stable isotope techniques for the assessment of host and microbiota response during gastrointestinal dysfunction2017 - J Pediatr Gastroenterol Nutr, 64: 8-14
The International Atomic Energy Agency convened a technical meeting on environmental enteric dysfunction (EED) in Vienna (28th – 30th October 2015; https://nucleus.iaea.org/HHW/Nutrition/EED_Technical_Meeting/index.html) to bring together international experts in the fields of EED, nutrition and stable isotope technologies. Advances in stable isotope labelling techniques open up new possibilities to improve our understanding of gastrointestinal dysfunction and the role of the microbiota in host health. In the context of EED, little is known about the role gut dysfunction may play in macro- and micronutrient bioavailability and requirements and what the consequences may be for nutritional status and linear growth. Stable isotope labelling techniques have been used to assess intestinal mucosal injury and barrier function, carbohydrate digestion and fermentation, protein derived amino acid bioavailability and requirements, micronutrient bioavailability and to track microbe-microbe and microbe-host interactions at the single cell level. The non-invasive nature of stable isotope technologies potentially allows for low-hazard, field deployable tests of gut dysfunction that are applicable across all age-groups. The purpose of this review is to assess the state-of-the-art in the use of stable isotope technologies and to provide a perspective on where these technologies can be exploited to further our understanding of gut dysfunction in EED.
Pediatric obesity is associated with an altered gut microbiota and discordant shifts in Firmicutes populations2017 - Environ. Microbiol., 1: 95-105
An altered gut microbiota has been linked to obesity in adulthood, although little is known about childhood obesity. The aim of this study was to characterize the composition of the gut microbiota in obese (n = 42) and normal-weight (n = 36) children aged 6 to 16. Using 16S rRNA gene-targeted sequencing, we evaluated taxa with differential abundance according to age- and sex-normalized body mass index (BMI z-score). Obesity was associated with an altered gut microbiota characterized by elevated levels of Firmicutes and depleted levels of Bacteroidetes. Correlation network analysis revealed that the gut microbiota of obese children also had increased correlation density and clustering of operational taxonomic units (OTUs). Members of the Bacteroidetes were generally better predictors of BMI z-score and obesity than Firmicutes, which was likely due to discordant responses of Firmicutes OTUs. In accordance with these observations, the main metabolites produced by gut bacteria, short chain fatty acids (SCFAs), were higher in obese children, suggesting elevated substrate utilisation. Multiple taxa were correlated with SCFA levels, reinforcing the tight link between the microbiota, SCFAs and obesity. Our results suggest that gut microbiota dysbiosis and elevated fermentation activity may be involved in the etiology of childhood obesity.


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subset: commoncrawl


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After a brief hiatus due to a hectic work schedule, we are back here on The Weekly SLAM! just in time to talk politics.
With the U.S. presidential election yesterday, I started thinking about which two wrestlers I'd most vote for if there were to somehow be a professional wrestling election. And I'm not talking about how they personally view politics. So the well-publicized political leanings of the likes of Kane and Val Venis don't really mean a whole lot in this one.
I'd like to create a wrestling system where, much like the real thing, there are a couple of parties who would try to get elected to dictate their platform on how professional wrestling should be run.
Going across the well-divided lines of this business, we'd have the Sport Party and the Entertainment Party.
The Sport Party would represent the pure sport-nature of pro wrestling. Going back to the old day where there wasn't 18 "comedy" skits on each show and men wrestled for 60 minutes at a time, night-in, night-out.
The Entertainment Party would represent the current and modern adaptation of the business. They support the entertainment aspect of things, the pomp and circumstance that make pro wrestling a big arena attraction these days. They'd cater to the younger demographic but still have the ties necessary to know what makes this so great.
Now, we have to ask: Would we want current wrestlers in the role? Past wrestlers? Executives? It's a lot to chew on.
After going through a list of candidates in my head, I think the Sport Party would be best represented in our inaugural election by the ticket of Ric Flair and Ricky Steamboat. What pair better represents the true sport of professional wrestling? I can't think of any. These two have forgotten more about the sport in a night than most of us will ever know.
Here's a big question, though: Who represents the Entertainment Party? I've been going back-and-forth on this one and it's tough to nail it down. Who should the Entertainment Party represent?
After much debate, the Entertainment Party would be represented by Daniel Bryan and Dolph Ziggler. Now, here's the reasoning on this one. I know it's tough not to include John Cena, but he's more of the working man. He takes his orders and goes. With the ticket of Bryan and Ziggler, we get both the best elements of entertainment, but also great wrestling, to create a broader appeal. Plus, Ziggler as VP, as a rising star, would be a great presidential candidate in the future.
You always have to look toward the future.
So who do you like to represent professional wrestling and move this sport forward into the future? Do you cast your vote for the Sport Party of Flair/Steamboat, or do you like the Entertainment Party ticket of Bryan/Ziggler?
And no, Linda McMahon wouldn't win this election, either.
Polling Station
From our last edition of The Weekly SLAM!, here were our poll results:
Question: Should Vince McMahon still be wrestling at age 67?
So I guess the conclusion I draw from this is that our readers don't like to see old men get beat up. So that's good. Chalk one up for society at large on that one.
Star of the Week: Raw fans in Birmingham, England
That's right, this week's Star of the Week isn't a wrestler, or even a personality. I want to give a big gold star to the fans in attendance for this week's edition of Monday Night Raw in Birmingham, England, who greatly added to the show with their enthusiasm, a level we haven't seen on a Raw show in months.
The reaction for Bryan was nothing short of electric, as was the participation of the crowd during the Vickie Guerrero/Cena segment. A dead crowd can really hurt a show and a live one can certainly enhance it, as was the case here. Kudos to those in Birmingham!
What To Watch For
If you forgot TNA Turning Point is this weekend, well, you're not alone, folks. The show features a ladder match for the TNA World Heavyweight Championship between Jeff Hardy and Austin Aries. While the match should be fantastic, I feel bad for them because they are going to kill themselves for the 1,100 people in the Impact Zone and about 8,000 people on pay-per-view.
That match should be great, but it's not a one-trick pony: The co-main event is a triple threat No. 1 contender bout between James Storm, AJ Styles and Bobby Roode. If that match isn't great, I'll eat my hat. The rest of the card is looking pretty good right now, too. If you're a fan of good professional wrestling, Turning Point should deliver.
Tweet of the Week
This week's featured Tweet comes to us from Smackdown play-by-play voice Josh Mathews, who happened to notice a certain similarity from his broadcast colleagues during Raw on Monday:
Josh Mathews: I spy... Matching suits, ties & shirts. All @MichaelCole needs is a black cowboy hat. pic.twitter.com/jWHt51Kz
YouTube of the Week
Conviniently, this week's featured video comes to us direct from WWE, where the superstars themselves weigh-in on which wrestler they think could be the actual president.
Matt Bishop can be reached at email@example.com. Please include your name and hometown. Click here to follow Matt on Twitter. Should be a good one!.


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I decided I need to post at least one here in case you aren't the link following type! That way I will lure you in (my diabolical plan!).
Before you read, though, you will need an adjustment to your mental image of the Hayes. I want you to think of this version of the Hayes:
Source: WW Norton
Dearest Ruddy, [Remember what I told you - President Hayes in his official portraits doesn't make a good "Ruddy," but the first one does!]
I cannot tell you how happy your first letter made me- I felt certain - satisfied that you loved me as dearly and truly as in all the past years- How rapidly time passes away we are now almost old folks and yet it is but a few months in feeling- I was down street to day and when I came home found your dear letter and read it and reread it till I almost knew it by heart- I can sympathize with your feelings as the men were sworn in- what a solemn sight it is- and oh how my heart fills with joy and feelings too great for utterance crowd upon me as I think of our brave men- This morning as I was going down street a young man dressed in Guthrie Grey- spoke to me How do you do Mrs Hayes- I looked closely at him and recognised [sic] the boy or man that used to be in the meat store- He grasped my hand warmly and I felt that he was not a poor boy but one of our defenders-
My greatest happiness now would be to feel that I was doing some thing for the comfort and happiness of our men I feel that in giving you up- (for dearest it is hard to feel we may be parted) I have tried to do cheerfully and without a murmur what was my duty- If I could only follow you whereever [sic] you are called to go no privation or trial would cause the slightest discontent- you would find Ruddy that your foolish little trial of a wife was fit to be a soldiers wife.
Ruddy last night I dreamed that Dr Muscroft was Surgeon of the Regiment- I awoke in an agony- if I could only feel that brother- Joe would be with you- I could see you go cheerfully- you do not know how intense my feeling is upon the subject dearest do all you can I know you have confidence in him- and oh how it would brighten my heart-
Mother says she never felt before that you were really away from us- she is pretty well and sends much love to you-
Birchie says tell Papa- That I have prayed for him and asked God to take care of him- and he must pray for himself and say I love him so much Webb says- Ask him if he wants me and Birch up there- I wish he would say, yes- that he prays for him every night and if you want any nuts he will bring them to you Webb says to tell you Ruddy says his prayers and says Now I lay me down to sleep- Ruddy says- sends the letter to him- and give him some nuts-
It is well dearest that you have expressed the wish to have me with you- for I had settled in my own mind- that as long as I can be with you I will but it was such a happiness to know that you in the midst of all the hurry and bustle of camp life thought of it. We have had two letters from Uncle which I will send- also the notice of your election to the Presidency of the Y- M G. Association.
We are all well- hope to see you soon- I will have everything ready for you as far as I know-
Good bye my dearest- May it not be long till we shall be together.
Your loving Wife


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A little more than 20 years ago, Congress did something that, today, is hard to imagine. Lawmakers from both parties and across the political spectrum found common ground and passed, by a near-unanimous vote, the Religious Freedom Restoration Act, which firmly commits the federal government to protecting and promoting our "inalienable right" to freely exercise religion. As President Clinton remarked when he signed the legislation into law, "the power of God is such that even in the legislative process, miracles can happen."
Last week, the Supreme Court agreed to hear two cases that are testing the strength of this commitment. The arguments and decisions in the Hobby Lobby and Conestoga Wood lawsuits will say a lot about the health, and the future, of what Clinton called our "first freedom."
These cases involve religious liberty challenges by two family businesses to the rule in the
The Green family has not confined its Christian beliefs to Sunday worship, and has instead expressed them through the operation of its Hobby Lobby stores for nearly 40 years. Similarly, the Hahn family's Mennonite faith has animated and sustained its woodworking business for generations.
Like millions of religious believers and groups, these challengers reject the idea that religious faith and religious freedom are simply about what we believe and how we pray, and not also about how we live, act and work. At the heart of these two cases is the straightforward argument that federal law does not require us to "check our faith at the door" when we pursue vocations in business and commerce.
There should be no question about the sincerity of the religious beliefs at issue. These are not cases where the profit-focused managers of publicly traded mega-companies are cynically trying to save a few bucks or to gain a competitive edge.
As many would-be Sunday shoppers know, the Green family "walks the walk." Signs on Hobby Lobby stores' doors say that they close on Sundays "to allow employees time for worship and to spend time with their families." Their stores do not carry shot glasses, lewd greeting cards or vulgar posters, and the background music is Christian. Hobby Lobby contributes generously to charities and starts full-time employees at nearly double the minimum wage. When the Greens and Hobby Lobby do this, and many other things, they are living out their faith and exercising their religion.
Hobby Lobby also provides excellent health insurance, which includes coverage for most — but not all — contraceptives. However, because of the Greens' firm belief in the dignity of human life and about when and how it begins, Hobby Lobby cannot provide coverage for some of the required drugs because they could cause an abortion.
The government and others argue that the Greens' religious beliefs are irrelevant because they've freely chosen to enter the rough-and-tumble world of commerce and that, in any event, the exercise of religion is for individuals, not corporations. But Hobby Lobby's lawyers at the Becket Fund for Religious Liberty will be on solid ground when they explain to the court that both of these arguments are misguided.
The issue is not whether groups, associations and corporations have religious freedom rights under federal law. Of course they do. After all, religious hospitals, schools, social service agencies and churches are not "individuals," but it would be bizarre to say that they don't exercise religion.
And the question should not be whether legal protections for religious liberty stop at the sanctuary door or evaporate when a person is trying to make a living or a business is aiming to make a profit. At a time when we talk a lot about corporate responsibility and worry about the feeble influence of ethics and values on Wall Street decision-making, it would be strange if the law were to welcome sermonizing from
The Religious Freedom Restoration Act reaffirmed an idea that is deeply rooted in America's history and traditions — namely, that politics and policy should respect and, whenever possible, make room for religious commitments and conscientious objections. True, religious liberty is not absolute, and, in a pluralistic society like ours, not all requests for exemptions and accommodations can, or should, be granted. Some religious liberty lawsuits will, and should, fail, but not simply because they involve what happens at work on Monday and not what happens in services on the Sabbath.
What Clinton said when he signed the act into law is worth remembering today: "Let us never believe that the freedom of religion imposes on any of us some responsibility to run from our convictions." He challenged us instead to "bring our values back to the table of American discourse to heal our troubled land." The Greens and Hobby Lobby, by refusing to check their faith at the marketplace door, are doing just that.
Richard W. Garnett is a professor of law at the University of Notre Dame.


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For the sewing section:
Work surfaces for machines and sewing
Cutting island (under the counter top I will hang my scissors and rotary cutters)
Shelves for fabric tubs
Ruler rack
Storage for buttons, threads,
Book shelf for magazines and quilt books.
Storage for batts and backing.
And I am going to use an armoire for storing WIPS and quilts waiting for new homes.
Have I missed anything?
This room is starting from scratch. It is large and has two closets. I am also using the room for painting and scrapbooking and I am putting in a desk and filing cabinet dedicated to DH's part-time business. Any suggestions for these other hobby areas would be appreciated too!
Thanks folks!


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25% off for a friend. $10 for you. Refer someone today.
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GER 323 - The Teaching of German II (0)
This course continues the transition from student of German to teacher of German. Topics focus on the teaching of German in the middle school. Students will engage in pedagogical practices intended to create environments that encourage active engagement in learning foreign languages. Students will select and utilize authentic foreign language instruction materials including the literature of the target language appropriate for all levels of learners. Students will plan, implement, teach, and assess foreign language curriculum in the target language to demonstrate an understanding of the relationships among central concepts of learning and teaching foreign languages, including the ability to communicate high expectations and create meaningful learning experiences for all students.
Generally taken during the junior year.
Prerequisite: GER 322 or permission.
Offered fall term.


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Princess Grace dies
Grace Kelly, an American actress who married Rainier III, Prince of Monaco, and became European royalty, died on Sept. 14,1982, at age 52 after suffering a stroke and crashing her car. Kelly appeared in many films in the 1950s, including Alfred Hitchcock's "Dial M for Murder" and "Rear Window." She won an Academy Award for her role in "The Country Girl" in 1954. She quit acting at 26 when she married Prince Rainier.
Associated Press, Photofest
Copyright © 2019, Los Angeles Times


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Shop For Mary Kay Perfume in Watertown, SD
Mary Kay beauty products go beyond just makeup and skin care – our fragrances enhance beauty and confidence for both men and women with a selection of pleasing, attractive scents. From clean or woody to floral or fruity, we have a perfect scent for anyone in Watertown. Eau de Parfums by Mary Kay are long-lasting, magnetic perfumes for women to make a subtle, beautiful impression, while Eau de Toilette fragrances are more casual, inexpensive products for simple everyday wear. Masculine fragrances from MKMen® create an irresistible scent for men. If you need perfume in Watertown or a gift idea, get in touch with a Mary Kay Independent Beauty Consultant today.


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The U.S. Environmental Protection Agency doesn't necessarily have farm interests lined up in its crosshairs, but it's not exactly ignoring agriculture as a target of opportunity either.
"We've had it pretty good in agriculture for a long time," said Shannon Ferrell, assistant professor at Oklahoma State University, during the recent Rural Economics Outlook Conference on the Stillwater campus. "EPA went after the low-hanging fruit first, the big polluters," Ferrell said. "Now, they're going after the rest."
Are they picking on agriculture? Ferrell says not exactly, although some programs, such as concentrated animal feeding operations (CAFO), may be priorities. "Everyone is subject to pressure," he said. "A lot of small businesses are getting squeezed by environmental regulations" that have already hit big business.
He said when the industry asks if "they are coming after us," we have to determine: "Who are they and who is us?" "They" could be the EPA, the federal government or interest groups, he said. And "we" may be hard to identify. "We talk about ag as one unified front, but from the outside we look different." He said outsiders see divisions within the ag industry — livestock versus grain, for instance.
To minimize damaging effects of over-regulation, Ferrell said agriculture should try to develop a more unified voice and learn more about how the process works. "We're good at discussing our needs with the House and Senate but we are not as cognizant of administrative law (as we should be). An incredibly large amount of regulations are enacted or proposed every year."
He said agricultural interests should be aware of those proposals and understand the effects they could have on the industry. "We need to do a better job of monitoring the administrative process," he said. "We need to improve advocacy, establish a dialogue and educate officials. And farmers need to develop some semblance of unity."
He said the Clean Air Act is an issue farm interests need to follow, especially as it relates to particulate matter. EPA recently considered a more strict particulate matter standard (PM10). Ferrell said the proposed standard could have created a significant hardship on the livestock industry, for instance. According to one estimate by the National Cattlemen's Beef Association (NCBA), the proposed standard would have required implementation of particulate matter controls across most of the western U.S.
Ferrell said regulating particulate matter is a lot easier with point sources. "It's harder with non-point sources."
Solutions to reduce particulate matter in agricultural production include a "buffet of practices," including reduced tillage, transgenic seed, wind breaks and other practices that reduce trips across a field, as well as dampening feedlots and slowing down equipment at livestock operations.
The standard is revised every five years, Ferrell said, and the change with the higher assessment has "been taken off the table," for now. A sticking point for this and other regulations is that economic impact does not come into play, "only health."
He said EPA is "still gathering data on air and particulates with several evaluation sites in Oklahoma. After the study, we will know more about pollution and animal production. As we see CAFO facilities grow, we will see more potential for particulate matter."
Water issues also will be critical, Ferrell said.
"Ammonia may be the next great regulatory frontier. Currently, 80 percent of ammonium emissions come from agriculture. Those emissions can form particulates that may be deposited on water." That could put ammonium under regulations of the Clean Water Act.
Ferrell said Oklahoma faces "a lot of tough decisions on how to allocate water resources," during a five-year process to develop a water plan. Infrastructure upgrades will be part of the discussion, he said.
"We need more robust state funding for infrastructure. We also need to look at best water use. Is it better to use it or leave it in place?" Ferrell said current water use policy that reduces allocation if the allocation is not all used encourages use.
He said better monitoring will be necessary to improve both water quantity and quality.
Landowner liability for hazardous waste clean-up also deserves attention, Ferrell said. He cautioned anyone buying property to be aware of any hazardous material problems before buying.
"Before buying property do an environmental audit," he suggested. "Conducting an environmental audit can provide a landowner with very important defenses if pollution is later discovered on the property, but the audit must be done before the purchase. You can't do that after you buy it."
Ferrell, both an agricultural economist and an attorney by training, said farmers may wonder if dealing with the EPA will bring "production restraints or business as usual. Will we have constraints on what we can do on the land?" he asked.
Acquiring as much information as possible about the regulatory process is an important step, as is monitoring regulations, he said.
"Also, unplug emotion from the process. Step back and see where you stand."


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Lil Boosie "The Bad Ass" Goes Shoppin + Borrows Stack Of Money From His Daughter! "She Gonna Be A Millionaire Kid"
Off The Real Talk Vol. 9 DVD
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8 Nights - 9 Days
From- $1,937 Inquiry
Flights & Transport
8 Breakfast
1 Lunch
1 Dinner
Visa & Taxes
5 % Govt. Tax would be extra on above cost.(INR + USD)
*All prices mentioned above are Per person
*CWB=Child With Bed
*CNB=Child No Bed
*Infant will not be given a seat in the flight or in the Vehicle, so do not count the infant in number of passengers
*Booking will be honored only after receiving the payment in the name of FLAMINGO TRANSWORLD PVT LTD as per the payment schedule.
Room 1
Above rates are subject to change with our prior notice.
ROE will be considered on the day when you make final payment.
The airfare & taxes based on the fare structure existing as on January'17.
Any further increase in airfare due to increase in the fuel price, change in government regulations, taxes, etc., charged by the airline will have to be borne by the passengers. FLAMINGO will not be held responsible for them.
Above rates are subject to availability & confirmation by the airline & hotel
Price is valid for minimum 20 Passengers traveling together. Flamingo reserves right to cancel a change date of departure in case of inadequate members in the Group.
International Hotel Standard Check in time 1400 hrs Check out Time 1200 hrs
There is No Refund for unutilized Service
Room Rates based on Standard Rooms. Supplement cost will be applicable for Sea facing/Lake Facing / Deluxe / Suite Rooms.
Meals Doesn't Include any Water Bottles , Beverages Hard/Soft drinks, Fruit Juices.
Optional sightseeing has to be booked in advance on your own by paying an additional cost.
Overseas Medical insurance is must for Schengen Visas and Also must for Travel. The cost for the same upto the age of 60 years is included in the cost. Additional cost must be paid for travelers of and above the age of 60 years.
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All services of the tour including changes in Schedule of mode of travel may have an impact on tour Itineraries and the company reserves a right to change / modify / vary/ alter in the tour Itinerary and/or tour tariff accordingly.
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The Changing Views of TV and Video
- Jul 29, 2013 4:22 PM EST
- Comments
This year's Fortune Brainstorm Tech put much focus on television and how it will evolve in the future as it faces new competition from video services such as YouTube, mobile video, and over-the-top services. YouTube is gaining traction among younger viewers in particular, as are new services like Aereo, but representatives from the traditional networks and cable companies were optimistic about both existing and new business models. James Murdoch, deputy chief operating officer of 21st Century Fox, declared, "it couldn't be a more exciting time to be in television." Read More »
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