Co-expression analysis reveals distinct alliances around two carbon fixation pathways in hydrothermal vent symbionts.

Most autotrophic organisms possess a single carbon fixation pathway. The chemoautotrophic symbionts of the hydrothermal vent tubeworm Riftia pachyptila, however, possess two functional pathways: the Calvin-Benson-Bassham (CBB) and the reductive tricarboxylic acid (rTCA) cycles. How these two pathways are coordinated is unknown. Here we measured net carbon fixation rates, transcriptional/metabolic responses and transcriptional co-expression patterns of Riftia pachyptila endosymbionts by incubating tubeworms collected from the East Pacific Rise at environmental pressures, temperature and geochemistry. Results showed that rTCA and CBB transcriptional patterns varied in response to different geochemical regimes and that each pathway is allied to specific metabolic processes; the rTCA is allied to hydrogenases and dissimilatory nitrate reduction, whereas the CBB is allied to sulfide oxidation and assimilatory nitrate reduction, suggesting distinctive yet complementary roles in metabolic function. Furthermore, our network analysis implicates the rTCA and a group 1e hydrogenase as key players in the physiological response to limitation of sulfide and oxygen. Net carbon fixation rates were also exemplary, and accordingly, we propose that co-activity of CBB and rTCA may be an adaptation for maintaining high carbon fixation rates, conferring a fitness advantage in dynamic vent environments.

TV Networks for College Sports: Implications for Institutional Subsidies.

University athletics because of its reporting structure, regulation by the NCAA, and relationship to the academic enterprise of institutions, provides a unique look into postsecondary institutional behavior. Using a difference-in-difference design, this study tests the introduction of new television networks dedicated to college sports on institutional subsidy levels for athletics. Overall, our findings show that institutions are responsive to the introduction of new college-sports-dedicated television networks. When considering institutions in the Power Five athletic conferences, we find an average decline in subsidy levels of approximately $1.2 million. When considering different types of subsidies, we find significant declines in direct subsidies and student fees, but no significant change for indirect subsidies.

You are Admitted! Early Evidence on Enrollment from Idaho's Direct Admissions System.

In 2015, Idaho adopted the nation's first direct admissions system and proactively admitted all high school graduates to a set of public institutions. This reimagination of the admissions process may reduce barriers to students' enrollment and improve access across geographic and socioeconomic contexts by removing many human capital, informational, and financial barriers in the college search and application process. Despite a lack of evidence on the efficacy of direct admissions systems, the policy has already been proposed or implemented in four other states. Using synthetic control methods, we estimate the first causal impacts of direct admissions on institutional enrollment outcomes. We find early evidence that direct admissions increased first-time undergraduate enrollments by 4-8% (50-100 students per campus on average) and in-state levels by approximately 8-15% (80-140 students) but had minimal-to-no impacts on the enrollment of Pell-eligible students. These enrollment gains were concentrated among 2-year, open-access institutions. We discuss these findings in relation to state contexts and policy design given the emergence of literature highlighting the varied efficacy of similar college access policies.

Cooccurring Activities of Two Autotrophic Pathways in Symbionts of the Hydrothermal Vent Tubeworm Riftia pachyptila.

Genome and proteome data predict the presence of both the reductive citric acid cycle (rCAC; also called the reductive tricarboxylic acid cycle) and the Calvin-Benson-Bassham cycle (CBB) in "Candidatus Endoriftia persephonae," the autotrophic sulfur-oxidizing bacterial endosymbiont from the giant hydrothermal vent tubeworm Riftia pachyptila. We tested whether these cycles were differentially induced by sulfide supply, since the synthesis of biosynthetic intermediates by the rCAC is less energetically expensive than that by the CBB. R. pachyptila was incubated under in situ conditions in high-pressure aquaria under low (28 to 40 µmol · h-1) or high (180 to 276 µmol · h-1) rates of sulfide supply. Symbiont-bearing trophosome samples excised from R. pachyptila maintained under the two conditions were capable of similar rates of CO2 fixation. Activities of the rCAC enzyme ATP-dependent citrate lyase (ACL) and the CBB enzyme 1,3-bisphosphate carboxylase/oxygenase (RubisCO) did not differ between the two conditions, although transcript abundances for ATP-dependent citrate lyase were 4- to 5-fold higher under low-sulfide conditions. δ13C values of internal dissolved inorganic carbon (DIC) pools were varied and did not correlate with sulfide supply rate. In samples taken from freshly collected R. pachyptila, δ13C values of lipids fell between those collected for organisms using either the rCAC or the CBB exclusively. These observations are consistent with cooccurring activities of the rCAC and the CBB in this symbiosis. IMPORTANCE Previous to this study, the activities of the rCAC and CBB in R. pachyptila had largely been inferred from "omics" studies of R. pachyptila without direct assessment of in situ conditions prior to collection. In this study, R. pachyptila was maintained and monitored in high-pressure aquaria prior to measuring its CO2 fixation parameters. Results suggest that ranges in sulfide concentrations similar to those experienced in situ do not exert a strong influence on the relative activities of the rCAC and the CBB. This observation highlights the importance of further study of this symbiosis and other organisms with multiple CO2-fixing pathways, which recent genomics and biochemical studies suggest are likely to be more prevalent than anticipated.

Physiological dynamics of chemosynthetic symbionts in hydrothermal vent snails.

Symbioses between invertebrate animals and chemosynthetic bacteria form the basis of hydrothermal vent ecosystems worldwide. In the Lau Basin, deep-sea vent snails of the genus Alviniconcha associate with either Gammaproteobacteria (A. kojimai, A. strummeri) or Campylobacteria (A. boucheti) that use sulfide and/or hydrogen as energy sources. While the A. boucheti host-symbiont combination (holobiont) dominates at vents with higher concentrations of sulfide and hydrogen, the A. kojimai and A. strummeri holobionts are more abundant at sites with lower concentrations of these reductants. We posit that adaptive differences in symbiont physiology and gene regulation might influence the observed niche partitioning between host taxa. To test this hypothesis, we used high-pressure respirometers to measure symbiont metabolic rates and examine changes in gene expression among holobionts exposed to in situ concentrations of hydrogen (H2: ~25 µM) or hydrogen sulfide (H2S: ~120 µM). The campylobacterial symbiont exhibited the lowest rate of H2S oxidation but the highest rate of H2 oxidation, with fewer transcriptional changes and less carbon fixation relative to the gammaproteobacterial symbionts under each experimental condition. These data reveal potential physiological adaptations among symbiont types, which may account for the observed net differences in metabolic activity and contribute to the observed niche segregation among holobionts.

Hydrogen Does Not Appear To Be a Major Electron Donor for Symbiosis with the Deep-Sea Hydrothermal Vent Tubeworm Riftia pachyptila.

Use of hydrogen gas (H2) as an electron donor is common among free-living chemolithotrophic microorganisms. Given the presence of this dissolved gas at deep-sea hydrothermal vents, it has been suggested that it may also be a major electron donor for the free-living and symbiotic chemolithoautotrophic bacteria that are the primary producers at these sites. Giant Riftia pachyptila siboglinid tubeworms and their symbiotic bacteria ("Candidatus Endoriftia persephone") dominate many vents in the Eastern Pacific, and their use of sulfide as a major electron donor has been documented. Genes encoding hydrogenase are present in the "Ca Endoriftia persephone" genome, and proteome data suggest that these genes are expressed. In this study, high-pressure respirometry of intact R. pachyptila and incubations of trophosome homogenate were used to determine whether this symbiotic association could also use H2 as a major electron donor. Measured rates of H2 uptake by intact R. pachyptila in high-pressure respirometers were similar to rates measured in the absence of tubeworms. Oxygen uptake rates in the presence of H2 were always markedly lower than those measured in the presence of sulfide, as was the incorporation of 13C-labeled dissolved inorganic carbon. Carbon fixation by trophosome homogenate was not stimulated by H2, nor was hydrogenase activity detectable in these samples. Though genes encoding [NiFe] group 1e and [NiFe] group 3b hydrogenases are present in the genome and transcribed, it does not appear that H2 is a major electron donor for this system, and it may instead play a role in intracellular redox homeostasis.IMPORTANCE Despite the presence of hydrogenase genes, transcripts, and proteins in the "Ca Endoriftia persephone" genome, transcriptome, and proteome, it does not appear that R. pachyptila can use H2 as a major electron donor. For many uncultivable microorganisms, omic analyses are the basis for inferences about their activities in situ However, as is apparent from the study reported here, there are dangers in extrapolating from omics data to function, and it is essential, whenever possible, to verify functions predicted from omics data with physiological and biochemical measurements.

Metatranscriptomics profile of the gill microbial community during Bathymodiolus azoricus aquarium acclimatization at atmospheric pressure.

BACKGROUND: The deep-sea mussels Bathymodiolus azoricus (Bivalvia: Mytilidae) are the dominant macrofauna subsisting at the hydrothermal vents site Menez Gwen in the Mid-Atlantic Ridge (MAR). Their adaptive success in such challenging environments is largely due to their gill symbiotic association with chemosynthetic bacteria. We examined the response of vent mussels as they adapt to sea-level environmental conditions, through an assessment of the relative abundance of host-symbiont related RNA transcripts to better understand how the gill microbiome may drive host-symbiont interactions in vent mussels during hypothetical venting inactivity. RESULTS: The metatranscriptome of B. azoricus was sequenced from gill tissues sampled at different time-points during a five-week acclimatization experiment, using Next-Generation-Sequencing. After Illumina sequencing, a total of 181,985,262 paired-end reads of 150 bp were generated with an average of 16,544,115 read per sample. Metatranscriptome analysis confirmed that experimental acclimatization in aquaria accounted for global gill transcript variation. Additionally, the analysis of 16S and 18S rRNA sequences data allowed for a comprehensive characterization of host-symbiont interactions, which included the gradual loss of gill endosymbionts and signaling pathways, associated with stress responses and energy metabolism, under experimental acclimatization. Dominant active transcripts were assigned to the following KEGG categories: "Ribosome", "Oxidative phosphorylation" and "Chaperones and folding catalysts" suggesting specific metabolic responses to physiological adaptations in aquarium environment. CONCLUSIONS: Gill metagenomics analyses highlighted microbial diversity shifts and a clear pattern of varying mRNA transcript abundancies and expression during acclimatization to aquarium conditions which indicate change in bacterial community activity. This approach holds potential for the discovery of new host-symbiont associations, evidencing new functional transcripts and a clearer picture of methane metabolism during loss of endosymbionts. Towards the end of acclimatization, we observed trends in three major functional subsystems, as evidenced by an increment of transcripts related to genetic information processes; the decrease of chaperone and folding catalysts and oxidative phosphorylation transcripts; but no change in transcripts of gluconeogenesis and co-factors-vitamins.

Peculiar citric acid cycle of hydrothermal vent chemolithoautotroph Hydrogenovibrio crunogenus, and insights into carbon metabolism by obligate autotrophs.

The genome sequence of the obligate chemolithoautotroph Hydrogenovibrio crunogenus paradoxically predicts a complete oxidative citric acid cycle (CAC). This prediction was tested by multiple approaches including whole cell carbon assimilation to verify obligate autotrophy, phylogenetic analysis of CAC enzyme sequences and enzyme assays. Hydrogenovibrio crunogenus did not assimilate any of the organic compounds provided (acetate, succinate, glucose, yeast extract, tryptone). Enzyme activities confirmed that its CAC is mostly uncoupled from the NADH pool. 2-Oxoglutarate:ferredoxin oxidoreductase activity is absent, though pyruvate:ferredoxin oxidoreductase is present, indicating that sequence-based predictions of substrate for this oxidoreductase were incorrect, and that H. crunogenus may have an incomplete CAC. Though the H. crunogenus CAC genes encode uncommon enzymes, the taxonomic distribution of their top matches suggests that they were not horizontally acquired. Comparison of H. crunogenus CAC genes to those present in other 'Proteobacteria' reveals that H. crunogenus and other obligate autotrophs lack the functional redundancy for the steps of the CAC typical for facultative autotrophs and heterotrophs, providing another possible mechanism for obligate autotrophy.

Direct Oral Anticoagulants in the Real World: Insights Into Appropriate Prescribing and Medication Use.

Background Direct oral anticoagulants are convenient because of their fixed dosing and without laboratory monitoring. There are instructions on avoidance of moisture, no crushing of capsules, and administration with food for some direct oral anticoagulants. Whether patients adhere to this and are prescribed appropriate doses are unknown. Aims To assess direct oral anticoagulant dosing and medication use. Methods Patients ≥18 years old, receiving a direct oral anticoagulant for any diagnosis, were prospectively included. Nurses at our perioperative anticoagulation clinic helped patients complete a 12-item questionnaire. Results Ninety-three consecutive patients were recruited. Forty-nine were on dabigatran, 18 on apixaban, and 26 were on rivaroxaban. Sixty-two patients (67%) received appropriate direct oral anticoagulant dosing and administered the medication correctly. Eighteen patients (19%) administered the direct oral anticoagulant properly but at an inappropriate dose. Thirteen patients (14%) received an appropriate dose but administered the direct oral anticoagulant inappropriately: 10 (11%) removed dabigatran from its packaging before administration (exposing it to moisture); 2 (2%) did not take rivaroxaban with food; and 1 (1%) crushed the dabigatran capsule. Conclusion Our study demonstrates a large variability in how direct oral anticoagulants are dosed, and how patients take them. Improved medication literacy around direct oral anticoagulants is needed. Our study highlights opportunities that nurses have to improve patients' medication literacy.

Toward establishing model organisms for marine protists: Successful transfection protocols for Parabodo caudatus (Kinetoplastida: Excavata).

We developed protocols for, and demonstrated successful transfection of, the free-living kinetoplastid flagellate Parabodo caudatus with three plasmids carrying a fluorescence reporter gene (pEF-GFP with the EF1 alpha promoter, pUB-GFP with Ubiquitin C promoter, and pEYFP-Mitotrap with CMV promoter). We evaluated three electroporation approaches: (1) a square-wave electroporator designed for eukaryotes, (2) a novel microfluidic transfection system employing hydrodynamically-controlled electric field waveforms, and (3) a traditional exponential decay electroporator. We found the microfluidic device provides a simple and efficient platform to quickly test a wide range of electric field parameters to find the optimal set of conditions for electroporation of target species. It also allows for processing large sample volumes (>10 ml) within minutes, increasing throughput 100 times over cuvettes. Fluorescence signal from the reporter gene was detected a few hours after transfection and persisted for 3 days in cells transfected by pEF-GFP and pUB-GFP plasmids and for at least 5 days post-transfection for cells transfected with pEYFP-Mitotrap. Expression of the reporter genes (GFP and YFP) was also confirmed using reverse transcription-PCR (RT-PCR). This work opens the door for further efforts with this taxon and close relatives toward establishing model systems for genome editing.

Carbon fixation by basalt-hosted microbial communities.

Oceanic crust is a massive potential habitat for microbial life on Earth, yet our understanding of this ecosystem is limited due to difficulty in access. In particular, measurements of rates of microbial activity are sparse. We used stable carbon isotope incubations of crustal samples, coupled with functional gene analyses, to examine the potential for carbon fixation on oceanic crust. Both seafloor-exposed and subseafloor basalts were recovered from different mid-ocean ridge and hot spot environments (i.e., the Juan de Fuca Ridge, the Mid-Atlantic Ridge, and the Loihi Seamount) and incubated with (13)C-labeled bicarbonate. Seafloor-exposed basalts revealed incorporation of (13)C-label into organic matter over time, though the degree of incorporation was heterogeneous. The incorporation of (13)C into biomass was inconclusive in subseafloor basalts. Translating these measurements into potential rates of carbon fixation indicated that 0.1-10 nmol C g(-1) rock d(-1) could be fixed by seafloor-exposed rocks. When scaled to the global production of oceanic crust, this suggests carbon fixation rates of 10(9)-10(12) g C year(-1), which matches earlier predictions based on thermodynamic calculations. Functional gene analyses indicate that the Calvin cycle is likely the dominant biochemical mechanism for carbon fixation in basalt-hosted biofilms, although the reductive acetyl-CoA pathway and reverse TCA cycle likely play some role in net carbon fixation. These results provide empirical evidence for autotrophy in oceanic crust, suggesting that basalt-hosted autotrophy could be a significant contributor of organic matter in this remote and vast environment.

Toxicity and mutagenicity of Gulf of Mexico waters during and after the deepwater horizon oil spill.

The Deepwater Horizon oil spill is unparalleled among environmental hydrocarbon releases, because of the tremendous volume of oil, the additional contamination by dispersant, and the oceanic depth at which this release occurred. Here, we present data on general toxicity and mutagenicity of upper water column waters and, to a lesser degree, sediment porewater of the Northeastern Gulf of Mexico (NEGOM) and west Florida shelf (WFS) at the time of the Deepwater Horizon oil spill in 2010 and thereafter. During a research cruise in August 2010, analysis of water collected in the NEGOM indicated that samples of 3 of 14 (21%) stations were toxic to bacteria based on the Microtox assay, 4 of 13 (34%) were toxic to phytoplankton via the QwikLite assay, and 6 of 14 (43%) showed DNA damaging activity using the λ-Microscreen Prophage induction assay. The Microtox and Microscreen assays indicated that the degree of toxicity was correlated to total petroleum hydrocarbon concentration. Long-term monitoring of stations on the NEGOM and the WFS was undertaken by 8 and 6 cruises to these areas, respectively. Microtox toxicity was nearly totally absent by December 2010 in the Northeastern Gulf of Mexico (3 of 8 cruises with one positive station). In contrast, QwikLite toxicity assay yielded positives at each cruise, often at multiple stations or depths, indicating the greater sensitivity of the QwikLite assay to environmental factors. The Microscreen mutagenicity assays indicated that certain water column samples overlying the WFS were mutagenic at least 1.5 years after capping the Macondo well. Similarly, sediment porewater samples taken from 1000, 1200, and 1400 m from the slope off the WFS in June 2011 were also highly genotoxic. Our observations are consistent with a portion of the dispersed oil from the Macondo well area advecting to the southeast and upwelling onto the WFS, although other explanations exist. Organisms in contact with these waters might experience DNA damage that could lead to mutation and heritable alterations to the community pangenome. Such mutagenic interactions might not become apparent in higher organisms for years.

High frequency of horizontal gene transfer in the oceans.

Oceanic bacteria perform many environmental functions, including biogeochemical cycling of many elements, metabolizing of greenhouse gases, functioning in oceanic food webs (microbial loop), and producing valuable natural products and viruses. We demonstrate that the widespread capability of marine bacteria to participate in horizontal gene transfer (HGT) in coastal and oceanic environments may be the result of gene transfer agents (GTAs), viral-like particles produced by α-Proteobacteria. We documented GTA-mediated gene transfer frequencies a thousand to a hundred million times higher than prior estimates of HGT in the oceans, with as high as 47% of the culturable natural microbial community confirmed as gene recipients. These findings suggest a plausible mechanism by which marine bacteria acquire novel traits, thus ensuring resilience in the face of environmental change.

Using indices of happiness to examine the influence of environmental enhancements for nursing home residents with Alzheimer's disease.

The present study extends the growing behavioral literature on indices of happiness for persons with developmental disabilities to the geriatric population. Data on indices of emotional affect (i.e., happiness) were collected prior to, during, and after each resident was exposed to environmental enhancement activities of various durations. Results showed that every activity improved each resident's level of happiness when compared to pre- and postactivity levels. These outcomes suggest that indexing affect may be as useful for nursing home residents as it has been for individuals with developmental disabilities.