Patterns and determinants of the global herbivorous mycobiome.

Despite their role in host nutrition, the anaerobic gut fungal (AGF) component of the herbivorous gut microbiome remains poorly characterized. Here, to examine global patterns and determinants of AGF diversity, we generate and analyze an amplicon dataset from 661 fecal samples from 34 mammalian species, 9 families, and 6 continents. We identify 56 novel genera, greatly expanding AGF diversity beyond current estimates (31 genera and candidate genera). Community structure analysis indicates that host phylogenetic affiliation, not domestication status and biogeography, shapes the community rather than. Fungal-host associations are stronger and more specific in hindgut fermenters than in foregut fermenters. Transcriptomics-enabled phylogenomic and molecular clock analyses of 52 strains from 14 genera indicate that most genera with preferences for hindgut hosts evolved earlier (44-58 Mya) than those with preferences for foregut hosts (22-32 Mya). Our results greatly expand the documented scope of AGF diversity and provide an ecologically and evolutionary-grounded model to explain the observed patterns of AGF diversity in extant animal hosts.

No time to die: Comparative study on preservation protocols for anaerobic fungi.

Anaerobic fungi (AF, phylum Neocallimastigomycota) are best known for their ability to anaerobically degrade recalcitrant lignocellulosic biomass through mechanic and enzymatic means. While their biotechnological potential is well-recognized, applied research on AF is still hampered by the time-consuming and cost-intensive laboratory routines required to isolate, maintain, and preserve AF cultures. Reliable long-term preservation of specific AF strains would aid basic as well as applied research, but commonly used laboratory protocols for AF preservation can show erratic survival rates and usually exhibit only moderate resuscitation success for up to one or two years after preservation. To address both, the variability, and the preservation issues, we have set up a cross-laboratory, year-long study. We tested five different protocols for the preservation of AF. The experiments were performed at three different laboratories (Austria, Germany, Switzerland) with the same three morphologically distinct AF isolates (Anaeromyces mucronatus, Caeocmyces sp., and Neocallimastix cameroonii) living in stable co-culture with their naturally occurring, syntrophic methanogens. We could show that handling greatly contributes to the variability of results, especially in Anaeromyces mucronatus. Cryopreservation of (mature) biomass in liquid nitrogen had the highest overall survival rates (85-100%, depending on the strain and laboratory). Additionally, preservation on agar at 39°C had surprisingly high survival rates for up to 9 months, if pieces of agar containing mature AF thalli were resuscitated. This low-cost, low-effort method could replace consecutive batch cultivation for periods of up to 6 months, while long-term preservation is best done by cryopreservation in liquid nitrogen. Regardless of the method, however, preserving several replicates (>three) of the same strain is highly advisable.

Effect of Growth Media on the Diversity of Neocallimastigomycetes from Non-Rumen Habitats.

Anaerobic fungi (AF), belonging to the phylum Neocallimastigomycota, are a pivotal component of the digestive tract microbiome of various herbivorous animals. In the last decade, the diversity of AF has rapidly expanded due to the exploration of numerous (novel) habitats. Studies aiming at understanding the role of AF require robust and reliable isolation and cultivation techniques, many of which remained unchanged for decades. Using amplicon sequencing, we compared three different media: medium with rumen fluid (RF), depleted rumen fluid (DRF), and no rumen fluid (NRF) to enrich the AF from the feces of yak, as a rumen control; and Przewalski's horse, llama, guanaco, and elephant, as a non-rumen habitats. The results revealed the selective enrichment of Piromyces and Neocallimastix from the feces of elephant and llama, respectively, in the RF medium. Similarly, the enrichment culture in DRF medium explicitly manifested Piromyces-related sequences from elephant feces. Five new clades (MM1-5) were defined from llama, guanaco, yak, and elephant feces that could as well be enriched from llama and elephant samples using non-conventional DRF and NRF media. This study presents evidence for the selective enrichment of certain genera in medium with RF and DRF from rumen as well as from non-rumen samples. NRF medium is suggested for the isolation of AF from non-rumen environments.

Simultaneous Metabarcoding and Quantification of Neocallimastigomycetes from Environmental Samples: Insights into Community Composition and Novel Lineages.

Anaerobic fungi from the herbivore digestive tract (Neocallimastigomycetes) are primary lignocellulose modifiers and hold promise for biotechnological applications. Their molecular detection is currently difficult due to the non-specificity of published primer pairs, which impairs evolutionary and ecological research with environmental samples. We developed and validated a Neocallimastigomycetes-specific PCR primer pair targeting the D2 region of the ribosomal large subunit suitable for screening, quantifying, and sequencing. We evaluated this primer pair in silico on sequences from all known genera, in vitro with pure cultures covering 16 of the 20 known genera, and on environmental samples with highly diverse microbiomes. The amplified region allowed phylogenetic differentiation of all known genera and most species. The amplicon is about 350 bp long, suitable for short-read high-throughput sequencing as well as qPCR assays. Sequencing of herbivore fecal samples verified the specificity of the primer pair and recovered highly diverse and so far unknown anaerobic gut fungal taxa. As the chosen barcoding region can be easily aligned and is taxonomically informative, the sequences can be used for classification and phylogenetic inferences. Several new Neocallimastigomycetes clades were obtained, some of which represent putative novel lineages such as a clade from feces of the rodent Dolichotis patagonum (mara).

Characterization and rank assignment criteria for the anaerobic fungi (Neocallimastigomycota).

Establishing a solid taxonomic framework is crucial for enabling discovery and documentation efforts. This ensures effective communication between scientists as well as reproducibility of results between laboratories, and facilitates the exchange and preservation of biological material. Such framework can only be achieved by establishing clear criteria for taxa characterization and rank assignment. Within the anaerobic fungi (phylum Neocallimastigomycota), the need for such criteria is especially vital. Difficulties associated with their isolation, maintenance and long-term storage often result in limited availability and loss of previously described taxa. To this end, we provide here a list of morphological, microscopic, phylogenetic and phenotypic criteria for assessment and documentation when characterizing newly obtained Neocallimastigomycota isolates. We also recommend a polyphasic rank-assignment scheme for novel genus-, species- and strain-level designations for newly obtained Neocallimastigomycota isolates.

Aestipascuomyces dupliciliberans gen. nov, sp. nov., the First Cultured Representative of the Uncultured SK4 Clade from Aoudad Sheep and Alpaca.

We report on the isolation of the previously-uncultured Neocallimastigomycota SK4 lineage, by two independent research groups, from a wild aoudad sheep rumen sample (Texas, USA) and an alpaca fecal sample (Baden-Württemberg, Germany). Isolates from both locations showed near-identical morphological and microscopic features, forming medium-sized (2-5 mm) white filamentous colonies with a white center of sporangia, on agar roll tubes and a heavy biofilm in liquid media. Microscopic analysis revealed monocentric thalli, and spherical polyflagellated zoospores with 7-20 flagella. Zoospore release occurred through an apical pore as well as by sporangial wall rupturing, a duality that is unique amongst described anaerobic gut fungal strains. Isolates were capable of growing on a wide range of mono-, oligo-, and polysaccharide substrates as the sole carbon source. Phylogenetic assessment based on the D1-D2 28S large rRNA gene subunit (D1-D2 LSU) and internal transcribed spacer-1 (ITS-1) regions demonstrated high sequence identity (minimum identity of 99.07% and 96.96%, respectively) between all isolates; but low sequence identity (92.4% and 86.7%, respectively) to their closest cultured relatives. D1-D2 LSU phylogenetic trees grouped the isolates as a new monophyletic clade within the Orpinomyces-Neocallimastix-Pecoramyces-Feramyces-Ghazallamyces supragenus group. D1-D2 LSU and ITS-1 sequences recovered from the obtained isolates were either identical or displayed extremely high sequence similarity to sequences recovered from the same aoudad sheep sample on which isolation was conducted, as well as several sequences recovered from domestic sheep and few other herbivores. Interestingly, members of the SK4 clade seem to be encountered preferably in animals grazing on summer pasture. We hence propose accommodating these novel isolates in a new genus, Aestipascuomyces (derived from the Latin word for "summer pasture"), and a new species, A. dupliciliberans. The type strain is Aestipascuomycesdupliciliberans strain R4.

Isolation, identification and characterization of lignocellulolytic aerobic and anaerobic fungi in one- and two-phase biogas plants.

Aerobic and anaerobic fungi are among the most effective plant biomass degraders known and have high potential to increase the efficiency of lignocellulosic biomass utilization, such as for biogas generation. However, limited information is available on their contribution to such industrial processes. Therefore, the presence of fungi along the biogas production chain of one-phase and two-phase biogas plants in Germany was analyzed. Seventeen aerobic species of Zygomycota, Ascomycota and Basidiomycota were identified, including efficient producers of lignocellulases, such as Trichoderma capillare isolated from a hydrolysis tank and Coprinopsis cinerea from fibers separated from pressed digestate. Five anaerobic fungal species of the phylum Neocallimastigomycota (comprising two novel clades) were present in an slightly acidic fermenter of a biogas plant fed with cow manure displaying endoglucanase transcriptional activity. The broad fungal presence demonstrated in this study can serve developing bioaugmentation systems with relevant lignocellulolytic fungi to improve biogas production from recalcitrant fiber material.

Exploring Hospitals' Use of Facebook: Thematic Analysis.

BACKGROUND: Although health care organizations such as hospitals and clinics have widely embraced social media as a means to educate the community on health topics and increase patient loyalty and satisfaction, little is known about the content these organizations actually share when using social media channels. OBJECTIVE: This study aimed to explore the types of content US hospitals post on their Facebook pages and how hospitals' Facebook activities differ with regard to content types. METHODS: We collected and thematically analyzed more than 1700 Facebook posts made over a 3-month period by 17 US hospitals. During the first phase, the 2 researchers coded a set of 159 posts and created an initial thematic web of content. During the second phase, the researchers coded the remaining posts and then revised, refined, and validated the initial web of content accordingly. Coding consensus was achieved on 1184 of the 1548 analyzable sampled posts (76.49%). RESULTS: We identified a list of 13 unique health social media post themes and classified those themes into 3 thematic groups that included announcing, sharing, and recognizing activities. The most frequently used theme was sharing health information, which appeared in 35.81% (424/1184) of the posts analyzed. Such posts sought to provide health tips and advice to community members. Recognizing special days and recognizing employees were the second and third most frequently used themes, respectively, with 14.95% (177/1184) and 11.82% (140/1184) of the posts containing those themes. The frequency of these themes was surprising as the content was geared more toward stakeholders internal to the organization, although most previous literature has focused on social media as a tool to connect with external stakeholders. In addition, we found many of the posts involved more than one theme, and selected sets of themes co-occurred frequently. For example, 25.4% (45/177) of the posts recognizing special days also included content to share health information, and approximately 38% (32/85) of the posts announcing research activities also included content to share health information. Finally, we found similarities and differences between the sampled hospitals in terms of the types of content they posted more frequently on their Facebook pages. CONCLUSIONS: Hospitals use Facebook as an inexpensive way to educate people on health and wellness topics and to communicate different types of information and news to the public audience. Hospitals and clinics that are expanding their social media activities or are starting to embark on social media strategies can use the results of this study to better formulate their activities on Facebook.

B2A Polypeptide in Foot and Ankle Fusion.

Foot and ankle fusion procedures often incorporate autogenous bone graft to help achieve bony union. Pain and morbidity associated with graft harvest have resulted in decreased autograft use as alternative bone graft substitutes have become available. Recently B2A peptide-coated ceramic granules have been developed and investigated. B2A, a bioactive synthetic multi-domain peptide acting on bone morphogenetic protein receptors of osteoblast precursor cells, amplifies the cell response to bone morphogenetic proteins. Use of B2A-granule has the additional benefit of eliminating autogenous bone graft donor site morbidity. The surgical technique of subtalar arthrodesis incorporating B2A-granule is described.

Continuous Infragluteal Sciatic Nerve Block for Postoperative Pain Control After Total Ankle Arthroplasty.

Continuous peripheral nerve blocks (CPNB) for postoperative pain control are being used more frequently for total ankle arthroplasty. The purpose of this study was to compare the amount of opioid analgesia used in patients with an infragluteal CPNB to that of patients with no CPNB for postoperative pain management after total ankle replacement. A retrospective cohort study was performed on 78 consecutive patients who had a total ankle arthroplasty from October 2006 to June 2013. The primary outcome measure was opioid analgesia use in the first 48 hours postoperatively. The mean postoperative narcotic use in oral morphine equivalents was 64.6 mg in the CPNB group compared to 129.6 mg in the no CPNB group (P < .001). Using an infragluteal CPNB as a means of postoperative pain control in patients undergoing a total ankle replacement is associated with significantly decreased opioid use compared to patients receiving no CPNB. LEVELS OF EVIDENCE: Therapeutic, Level IV, case series.

Design and control of a bio-inspired soft wearable robotic device for ankle-foot rehabilitation.

We describe the design and control of a wearable robotic device powered by pneumatic artificial muscle actuators for use in ankle-foot rehabilitation. The design is inspired by the biological musculoskeletal system of the human foot and lower leg, mimicking the morphology and the functionality of the biological muscle-tendon-ligament structure. A key feature of the device is its soft structure that provides active assistance without restricting natural degrees of freedom at the ankle joint. Four pneumatic artificial muscles assist dorsiflexion and plantarflexion as well as inversion and eversion. The prototype is also equipped with various embedded sensors for gait pattern analysis. For the subject tested, the prototype is capable of generating an ankle range of motion of 27° (14° dorsiflexion and 13° plantarflexion). The controllability of the system is experimentally demonstrated using a linear time-invariant (LTI) controller. The controller is found using an identified LTI model of the system, resulting from the interaction of the soft orthotic device with a human leg, and model-based classical control design techniques. The suitability of the proposed control strategy is demonstrated with several angle-reference following experiments.

The use of sediment toxicity identification evaluation methods to evaluate clean up targets in an urban estuary.

The Ballona Creek Estuary (BCE) in Los Angeles, California, is in a highly urbanized watershed, is contaminated by a variety of chemicals, and has prevalent sediment toxicity. Sediment cleanup targets for BCE have been established for Cu, Cd, Pb, Zn, chlordane, DDTs, PCBs, and PAHs, based on sediment quality guidelines. A sediment toxicity identification evaluation (TIE) was conducted to examine how these targets corresponded to toxicity observed with the estuarine amphipod Eohaustorius estuarius. Whole sediment and porewater TIEs were used to identify the cause of toxicity. Passive samplers were deployed to determine the bioavailable fraction of contaminants. Spiked sediment tests were conducted to determine the thresholds of toxicity for selected constituents. Toxicity was found to be widespread but temporally and spatially variable. Whole sediment and porewater TIEs both indicated pyrethroid pesticides were the most likely contaminant group contributing to the toxicity. Concentrations of the chemicals listed for cleanup were found to often exceed target values but were not observed at concentrations likely to cause toxicity. Bioavailable fractions of the target chemicals quantified using passive samplers did not exceed toxicity thresholds. Spiked sediment tests established 10 day LC50s for 4,4' DDE, 4, 4' DDT, α-chlordane, and cyfluthrin at >3050 µg/g, 266 µg/g, >2120 µg/g, and 0.33 µg/g organic carbon (OC), respectively. The cyfluthrin LC50 was within the range of concentrations observed in the estuary sediments, but LC50s for the other 3 chemicals were orders of magnitude greater than observed levels. The combination of TIE, sediment chemistry and the results from spiked sediment exposures indicate pyrethroid pesticides are more likely the cause of the observed toxicity than any of the contaminants targeted for cleanup. The results of this study indicate the importance of using a TIE approach to determine chemicals of concern and dose-response information to set cleanup targets, rather than using sediment quality guidelines. Integr Environ Assess Manag 2014;10:260-268. © 2013 SETAC.

Safety, efficacy and efficiency of laser-assisted IVF in subfertile mutant mouse strains.

In the present report we studied the safety, efficacy and efficiency of using an infrared laser to facilitate IVF by assessing fertilization, development and birth rates after laser-zona drilling (LZD) in 30 subfertile genetically modified (GM) mouse lines. We determined that LZD increased the fertilization rate four to ten times that of regular IVF, thus facilitating the derivation of 26 of 30 (86.7%) GM mouse lines. Cryopreserved two-cell stage embryos derived by LZD-assisted IVF were recovered and developed to blastocysts in vitro at the same rate as frozen-thawed embryos derived by regular IVF. Surprisingly after surgical transfer to pseudopregnant recipients the birth rate of embryos derived by LZD-assisted IVF was significantly lower than that of embryos derived by regular IVF. However this result could be completely mitigated by the addition of 0.25 M sucrose to the culture medium during LZD which caused the oocyte to shrink in volume relative to the perivitelline space. By increasing the distance from the laser target site on the zona pellucida, we hypothesize that the hyperosmotic effect of sucrose reduced the potential for laser-induced cytotoxic thermal damage to the underlying oocytes. With appropriate preparation and cautious application, our results indicate that LZD-assisted IVF is a safe, efficacious and efficient assisted reproductive technology for deriving mutant mouse lines with male factor infertility and subfertility caused by sperm-zona penetration defects.

Regional assessment of marine and estuarine sediment toxicity in Southern California, USA.

Sediment toxicity was investigated at 222 stations in the Southern California Bight (SCB) during 2008. This represented the first time that assessment methods established by California's new Sediment Quality Objectives program were employed in a survey of this scale. The goal was to determine the extent and magnitude of sediment toxicity in the SCB, how toxicity compared among specific environments, and whether toxicity has changed over the last decade. Two toxicity tests were used: the 10-day amphipod whole sediment survival test with Eohaustorius estuarius and a 48-h embryo development test with the mussel Mytilus galloprovincialis exposed at the sediment-water interface. Less than 1 % of the area of the SCB was found to be toxic to the amphipod test. No toxicity was found in offshore stations, but 14 % of embayment areas were toxic to the amphipods. The mussel test identified 13 % of the embayment areas to be toxic. Estuary and marina locations had the greatest areal extent of toxicity for both tests. The two toxicity methods agreed that sediments were not toxic at over half of the stations tested. The mussel test showed a greater magnitude of response than the amphipod. Sediment toxicity was shown to have declined in both extent and magnitude from levels measured in 1998 and 2003.

Decision making under time pressure, modeled in a prospect theory framework.

The current research examines the effects of time pressure on decision behavior based on a prospect theory framework. In Experiments 1 and 2, participants estimated certainty equivalents for binary gains-only bets in the presence or absence of time pressure. In Experiment 3, participants assessed comparable bets that were framed as losses. Data were modeled to establish psychological mechanisms underlying decision behavior. In Experiments 1 and 2, time pressure led to increased risk attractiveness, but no significant differences emerged in either probability discriminability or outcome utility. In Experiment 3, time pressure reduced probability discriminability, which was coupled with severe risk-seeking behavior for both conditions in the domain of losses. No significant effects of control over outcomes were observed. Results provide qualified support for theories that suggest increased risk-seeking for gains under time pressure.

Using gene expression to assess the status of fish from anthropogenically influenced estuarine wetlands.

The diverse mixture of contaminants frequently present in estuaries complicates their assessment by routine chemical or biological analyses. We investigated the use of gene expression to assess contaminant exposure and the condition of southern California estuarine fish. Liver gene expression, plasma estradiol concentrations, and gonad histopathology were used to study biological condition in longjaw mudsuckers (Gillichthys mirabilis). Metals, legacy organochlorine pesticides, PCBs, and contaminants of emerging concern were detected in sediments and whole fish. Overall gene expression patterns were characteristic to each of four sites investigated in this study. Differentially expressed genes belonged to several functional categories including xenobiotic metabolism, detoxification, disease, and stress responses. In general, plasma estradiol concentrations were similar among fish from all areas. Some fish gonads had pathologic changes (e.g., infection, inflammation) that could indicate weakened immune systems and chronic stress. The differential expression of some genes involved in stress responses correlated with the prevalence of histologic gonad lesions. This study indicates that gene expression is a promising tool for assessing the biological condition of fish exposed to environmental contaminants.

Modeling the Impact of Control on the Attractiveness of Risk in a Prospect Theory Framework.

Many decisions involve a degree of personal control over event outcomes, which is exerted through one's knowledge or skill. In three experiments we investigated differences in decision making between prospects based on a) the outcome of random events and b) the outcome of events characterized by control. In Experiment 1, participants estimated certainty equivalents (CEs) for bets based on either random events or the correctness of their answers to U.S. state population questions across the probability spectrum. In Experiment 2, participants estimated CEs for bets based on random events, answers to U.S. state population questions, or answers to questions about 2007 NCAA football game results. Experiment 3 extended the same procedure as Experiment 1 using a within-subjects design. We modeled data from all experiments in a prospect theory framework to establish psychological mechanisms underlying decision behavior. Participants weighted the probabilities associated with bets characterized by control so as to reflect greater risk attractiveness relative to bets based on random events, as evidenced by more elevated weighting functions under conditions of control. This research elucidates possible cognitive mechanisms behind increased risk taking for decisions characterized by control, and implications for various literatures are discussed.

Expanding the criteria for BRCA mutation testing in breast cancer survivors.

PURPOSE: Every year approximately 25% of women diagnosed with breast cancer are younger than 50 years of age, and almost 10% of them have a BRCA mutation. Not all potential carriers are identified by existing criteria for BRCA testing. We estimated the costs and benefits of different BRCA testing criteria for women with breast cancer younger than 50 years. METHODS: We developed a Markov Monte Carlo simulation to compare six criteria for BRCA mutation testing: (1) no testing (reference); (2) medullary breast cancer in patients younger than 50 years; (3) any breast cancer in patients younger than 40 years; (4) triple negative (TN) breast cancer in patients younger than 40 years; (5) TN breast cancer in patients younger than 50 years; (6) any breast cancer in patients younger than 50 years. Net health benefits were life expectancy and quality-adjusted life expectancy, and primary outcome was the incremental cost-effectiveness ratio (ICER). The model estimated the number of new breast and ovarian cancer cases. RESULTS: BRCA mutation testing for all women with breast cancer who were younger than 50 years could prevent the highest number of breast and ovarian cancer cases, but with unfavorable ICERs. Testing women with TN breast cancers who were younger than 50 years was cost-effective with an ICER of $8,027 per year of life gained ($9,084 per quality-adjusted life-year), and could reduce subsequent breast and ovarian cancer risks by 23% and 41%, respectively, compared with the reference strategy. CONCLUSION: Testing women with TN breast cancers who were younger than 50 years for BRCA mutations is a cost-effective strategy and should be adopted into current guidelines for genetic testing.

Examining the relations among narcissism, impulsivity, and self-defeating behaviors.

A recent meta-analysis (S. Vazire & D. C. Funder, 2006) suggested that narcissism and impulsivity are related and that impulsivity partially accounts for the relation between narcissism and self-defeating behaviors (SDB). This research examines these hypotheses in two studies and tests a competing hypothesis that Extraversion and Agreeableness account for this relation. In Study 1, we examined the relations among narcissism, impulsivity, and aggression. Both narcissism and impulsivity predicted aggression, but impulsivity did not mediate the narcissism-aggression relation. In Study 2, narcissism was related to a measure of SDB and manifested divergent relations with a range of impulsivity traits from three measures. None of the impulsivity models accounted for the narcissism-SDB relation, although there were unique mediating paths for traits related to sensation and fun seeking. The domains of Extraversion and low Agreeableness successfully mediated the entire narcissism-SDB relation. We address the discrepancy between the current and meta-analytic findings.

Comparison of methods for evaluating acute and chronic toxicity in marine sediments.

Sublethal test methods are being used with increasing frequency to measure sediment toxicity, but little is known about the relative sensitivity of these tests compared to the more commonly used acute tests. The present study was conducted to compare the sensitivity of several acute and sublethal methods and to investigate their correlations with sediment chemistry and benthic community condition. Six sublethal methods (amphipod: Leptocheirus plumulosus survival, growth, and reproduction; polychaete: Neanthes arenaceodentata survival and growth; benthic copepod: Amphiascus tenuiremis life cycle; seed clam: Mercenaria mercenaria growth; oyster: Crassostrea virginica lysosome destabilization; and sediment-water interface testing with mussel embryos, Mytilus galloprovincialis) and two acute methods (amphipod survival with Eohaustorius estuarius and L. plumulosus) were used to test split sediment samples from stations in California. The test with Amphiascus proved to be the most sensitive sublethal test and the most sensitive overall, identifying 90% of the stations as toxic. The Leptocheirus 10-d test was the most sensitive of the acute tests, identifying 60% of the stations as toxic. In general, the sublethal tests were not more sensitive to sediments than the acute tests, with the sublethal tests finding an average of 35% of the stations to be toxic while the acute found 44%. Of the sublethal tests, only the Amphiascus endpoints and Neanthes growth significantly (p