Conversion of boreal forests to agricultural systems: soil microbial responses along a land-conversion chronosequence.

BACKGROUND: Boreal regions are warming at more than double the global average, creating opportunities for the northward expansion of agriculture. Expanding agricultural production in these regions will involve the conversion of boreal forests to agricultural fields, with cumulative impacts on soil microbial communities and associated biogeochemical cycling processes. Understanding the magnitude or rate of change that will occur with these biological processes will provide information that will enable these regions to be developed in a more sustainable manner, including managing carbon and nitrogen losses. This study, based in the southern boreal region of Canada where agricultural expansion has been occurring for decades, used a paired forest-adjacent agricultural field approach to quantify how soil microbial communities and functions were altered at three different stages post-conversion (< 10, > 10 and < 50, and > 50 years). Soil microbial functional capacity was assessed by quantitative PCR of genes associated with carbon (C), nitrogen, and phosphorous (P) cycling; microbial taxonomic diversity and community structure was assessed by amplicon sequencing. RESULTS: Fungal alpha diversity did not change, but communities shifted from Basidiomycota to Ascomycota dominant within the first decade. Bacterial alpha diversity increased, with Gemmatimonadota groups generally increasing and Actinomycetota groups generally decreasing in agricultural soils. These altered communities led to altered functional capacity. Functional genes associated with nitrification and low molecular weight C cycling potential increased after conversion, while those associated with organic P mineralization potential decreased. Stable increases in most N cycling functions occurred within the first decade, but C cycling functions were still changing 50 years post conversion. CONCLUSIONS: Microbial communities underwent a rapid shift in the first decade, followed by several decades of slower transition until stabilizing 50 years post conversion. Understanding how the microbial communities respond at different stages post-conversion improves our ability to predict C and N losses from emerging boreal agricultural systems, and provides insight into how best to manage these soils in a way that is sustainable at the local level and within a global context.

Using metatranscriptomics to better understand the role of microbial nitrogen cycling in coastal sediment benthic flux denitrification efficiency.

Spatial and temporal variability in benthic flux denitrification efficiency occurs across Port Phillip Bay, Australia. Here, we assess the capacity for untargeted metatranscriptomics to resolve spatiotemporal differences in the microbial contribution to benthic nitrogen cycling. The most abundant sediment transcripts assembled were associated with the archaeal nitrifier Nitrosopumilus. In sediments close to external inputs of organic nitrogen, the dominant transcripts were associated with Nitrosopumilus nitric oxide nitrite reduction (nirK). The environmental conditions close to organic nitrogen inputs that select for increased transcription in Nitrosopumilus (amoCAB, nirK, nirS, nmo, hcp) additionally selected for increased transcription of bacterial nitrite reduction (nxrB) and transcripts associated with anammox (hzo) but not denitrification (bacterial nirS/nirk). In sediments that are more isolated from external inputs of organic nitrogen dominant transcripts were associated with nitrous oxide reduction (nosZ) and changes in nosZ transcript abundance were uncoupled from transcriptional profiles associated with archaeal nitrification. Coordinated transcription of coupled community-level nitrification-denitrification was not well supported by metatranscriptomics. In comparison, the abundance of archaeal nirK transcripts were site- and season-specific. This study indicates that the transcription of archaeal nirK in response to changing environmental conditions may be an important and overlooked feature of coastal sediment nitrogen cycling.

Temporal profiling resolves the drivers of microbial nitrogen cycling variability in coastal sediments.

Here we describe the potential for sediment microbial nitrogen-cycling gene (DNA) and activity (RNA) abundances to spatially resolve coastal areas impacted by seasonal variability in external nutrient inputs. Three sites were chosen within a nitrogen-limited embayment, Port Phillip Bay (PPB), Australia that reflect variability in both proximity to external nutrient inputs and the dominant form of available nitrogen. At three sediment depths (0-1; 1-5; 5-10 cm) across a 2 year study key genes involved in nitrification (archaeal amoA and bacterial ß-amoA), nitrite reduction (clade I nirS and cluster I nirK, archaeal nirK-a), anaerobic oxidation of ammonium (anammox 16S rRNA phylogenetic marker) and nitrogen fixation (nifH) were quantified. Sediments impacted by a dominance of organic nitrogen inputs were characterised at all time-points and to sediment depths of 10 cm by the highest transcript abundances of archaeal amoA and archaeal nirk-a. Proximity to a dominance of external nitrate inputs was associated with the highest transcript abundances of nirS which temporally co-varied with seasonal changes in sediment nitrate. Sediments isolated from external inputs displayed the greatest depth-specific decrease in quantifiable transcript abundances. In these isolated sediments bacterial ß-amoA transcripts were temporally associated with increased sediment ammonium levels. Across this nitrogen limited system variability in the abundance of bacterial ß-amoA, archaeal amoA, archaeal nirk-a or nirS transcripts from the sediment surface (0-1 and 5 cm) demonstrated a capacity to improve our ability to monitor coastal zones impacted by anthropogenic nitrogen inputs. Specifically, the spatial detection sensitivity of bacterial ß-amoA transcripts could be developed as a metric to determine spatiotemporal impacts of large external loading events. This temporal study demonstrates a capacity for microbial activity metrics to facilitate coastal management strategies through greater spatial resolution of areas impacted by external nutrient inputs.

The founding charter of the Omic Biodiversity Observation Network (Omic BON).

Omic BON is a thematic Biodiversity Observation Network under the Group on Earth Observations Biodiversity Observation Network (GEO BON), focused on coordinating the observation of biomolecules in organisms and the environment. Our founding partners include representatives from national, regional, and global observing systems; standards organizations; and data and sample management infrastructures. By coordinating observing strategies, methods, and data flows, Omic BON will facilitate the co-creation of a global omics meta-observatory to generate actionable knowledge. Here, we present key elements of Omic BON's founding charter and first activities.

Oral health in primary care: Integration of enhanced oral health education in a nurse practitioner residency program.

ABSTRACT: Access to oral health care is challenging, especially for vulnerable populations and those in rural and underserved areas. The purpose of this brief report is to discuss the implementation strategies of enhanced content in oral health educational materials and share results of acquisition of knowledge and confidence in oral health content by residents in a nurse practitioner residency program. The method included the use of 3 surveys of 34 questions administered at different time points measuring confidence levels and sustainability. Results showed the average level of confidence obtained in Posttest1 and in Posttest 2 after 6 months is greater than the pretest. The paired-sample t-test provides significant evidence of improving mean responses for Questions 10, 11, 16, and 32 in Posttest 1 (p-values: .04 each) and Questions 10, 17, 25, and 31 in Posttest2 (p-values: .04 each) compared with the pretest. In conclusion, the addition of enhanced oral health educational materials in our program resulted in improved knowledge and confidence in the residents to incorporate oral health care in their practices.

The Ecobiomics project: Advancing metagenomics assessment of soil health and freshwater quality in Canada.

Transformative advances in metagenomics are providing an unprecedented ability to characterize the enormous diversity of microorganisms and invertebrates sustaining soil health and water quality. These advances are enabling a better recognition of the ecological linkages between soil and water, and the biodiversity exchanges between these two reservoirs. They are also providing new perspectives for understanding microorganisms and invertebrates as part of interacting communities (i.e. microbiomes and zoobiomes), and considering plants, animals, and humans as holobionts comprised of their own cells as well as diverse microorganisms and invertebrates often acquired from soil and water. The Government of Canada's Genomics Research and Development Initiative (GRDI) launched the Ecobiomics Project to coordinate metagenomics capacity building across federal departments, and to apply metagenomics to better characterize microbial and invertebrate biodiversity for advancing environmental assessment, monitoring, and remediation activities. The Project has adopted standard methods for soil, water, and invertebrate sampling, collection and provenance of metadata, and nucleic acid extraction. High-throughput sequencing is located at a centralized sequencing facility. A centralized Bioinformatics Platform was established to enable a novel government-wide approach to harmonize metagenomics data collection, storage and bioinformatics analyses. Sixteen research projects were initiated under Soil Microbiome, Aquatic Microbiome, and Invertebrate Zoobiome Themes. Genomic observatories were established at long-term environmental monitoring sites for providing more comprehensive biodiversity reference points to assess environmental change.

Web services for data warehouses: OMOP and PCORnet on i2b2.

Objective: Healthcare organizations use research data models supported by projects and tools that interest them, which often means organizations must support the same data in multiple models. The healthcare research ecosystem would benefit if tools and projects could be adopted independently from the underlying data model. Here, we introduce the concept of a reusable application programming interface (API) for healthcare and show that the i2b2 API can be adapted to support diverse patient-centric data models. Materials and Methods: We develop methodology for extending i2b2's pre-existing API to query additional data models, using i2b2's recent "multi-fact-table querying" feature. Our method involves developing data-model-specific i2b2 ontologies and mapping these to query non-standard table structure. Results: We implement this methodology to query OMOP and PCORnet models, which we validate with the i2b2 query tool. We implement the entire PCORnet data model and a five-domain subset of the OMOP model. We also demonstrate that additional, ancillary data model columns can be modeled and queried as i2b2 "modifiers." Discussion: i2b2's REST API can be used to query multiple healthcare data models, enabling shared tooling to have a choice of backend data stores. This enables separation between data model and software tooling for some of the more popular open analytic data models in healthcare. Conclusion: This methodology immediately allows querying OMOP and PCORnet using the i2b2 API. It is released as an open-source set of Docker images, and also on the i2b2 community wiki.

Primer selection influences abundance estimates of ammonia oxidizing archaea in coastal marine sediments.

Quantification of the α-subunit of ammonia monooxygenase (amoA) through PCR is an established technique for estimating the abundance of ammonia oxidizing archaea (AOA) in environmental samples. This study quantified AOA with two established primer sets in 1 cm increments from the sediment surface (0-1 cm) to a depth of 10 cm at two locations within Port Phillip Bay (PPB), Australia. Primer choice had a significant effect on within sample estimates of AOA with copy numbers ranging from 102 to 104 copies per ng DNA. Variation in AOA abundance patterns with increasing sediment depth were site and primer specific. Sequence mismatches between the primer binding region of the isolated amoA sequences from PPB and Nitrosopumilus maritimus SCM1 were identified and may explain the high variation identified between primer estimates. Our results highlight the need for testing multiple primer pairs that target different regions of the AOA amoA sequence prior to large-scale marine sediment environmental studies.

A transcriptomics approach uncovers novel roles for poly(ADP-ribosyl)ation in the basal defense response in Arabidopsis thaliana.

Pharmacological inhibition of poly(ADP-ribose) polymerase (PARP) or loss of Arabidopsis thaliana PARG1 (poly(ADP-ribose) glycohydrolase) disrupt a subset of plant defenses. In the present study we examined the impact of altered poly(ADP-ribosyl)ation on early gene expression induced by the microbe-associate molecular patterns (MAMPs) flagellin (flg22) and EF-Tu (elf18). Stringent statistical analyses and filtering identified 178 genes having MAMP-induced mRNA abundance patterns that were altered by either PARP inhibitor 3-aminobenzamide (3AB) or PARG1 knockout. From the identified set of 178 genes, over fifty Arabidopsis T-DNA insertion lines were chosen and screened for altered basal defense responses. Subtle alterations in callose deposition and/or seedling growth in response to those MAMPs were observed in knockouts of At3g55630 (FPGS3, a cytosolic folylpolyglutamate synthetase), At5g15660 (containing an F-box domain), At1g47370 (a TIR-X (Toll-Interleukin Receptor domain)), and At5g64060 (a predicted pectin methylesterase inhibitor). Over-represented GO terms for the gene expression study included "innate immune response" for elf18/parg1, highlighting a subset of elf18-activated defense-associated genes whose expression is altered in parg1 plants. The study also allowed a tightly controlled comparison of early mRNA abundance responses to flg22 and elf18 in wild-type Arabidopsis, which revealed many differences. The PARP inhibitor 3-methoxybenzamide (3MB) was also used in the gene expression profiling, but pleiotropic impacts of this inhibitor were observed. This transcriptomics study revealed targets for further dissection of MAMP-induced plant immune responses, impacts of PARP inhibitors, and the molecular mechanisms by which poly(ADP-ribosyl)ation regulates plant responses to MAMPs.

Combining clinical and genomics queries using i2b2 - Three methods.

We are fortunate to be living in an era of twin biomedical data surges: a burgeoning representation of human phenotypes in the medical records of our healthcare systems, and high-throughput sequencing making rapid technological advances. The difficulty representing genomic data and its annotations has almost by itself led to the recognition of a biomedical "Big Data" challenge, and the complexity of healthcare data only compounds the problem to the point that coherent representation of both systems on the same platform seems insuperably difficult. We investigated the capability for complex, integrative genomic and clinical queries to be supported in the Informatics for Integrating Biology and the Bedside (i2b2) translational software package. Three different data integration approaches were developed: The first is based on Sequence Ontology, the second is based on the tranSMART engine, and the third on CouchDB. These novel methods for representing and querying complex genomic and clinical data on the i2b2 platform are available today for advancing precision medicine.

Introducing BASE: the Biomes of Australian Soil Environments soil microbial diversity database.

BACKGROUND: Microbial inhabitants of soils are important to ecosystem and planetary functions, yet there are large gaps in our knowledge of their diversity and ecology. The 'Biomes of Australian Soil Environments' (BASE) project has generated a database of microbial diversity with associated metadata across extensive environmental gradients at continental scale. As the characterisation of microbes rapidly expands, the BASE database provides an evolving platform for interrogating and integrating microbial diversity and function. FINDINGS: BASE currently provides amplicon sequences and associated contextual data for over 900 sites encompassing all Australian states and territories, a wide variety of bioregions, vegetation and land-use types. Amplicons target bacteria, archaea and general and fungal-specific eukaryotes. The growing database will soon include metagenomics data. Data are provided in both raw sequence (FASTQ) and analysed OTU table formats and are accessed via the project's data portal, which provides a user-friendly search tool to quickly identify samples of interest. Processed data can be visually interrogated and intersected with other Australian diversity and environmental data using tools developed by the 'Atlas of Living Australia'. CONCLUSIONS: Developed within an open data framework, the BASE project is the first Australian soil microbial diversity database. The database will grow and link to other global efforts to explore microbial, plant, animal, and marine biodiversity. Its design and open access nature ensures that BASE will evolve as a valuable tool for documenting an often overlooked component of biodiversity and the many microbe-driven processes that are essential to sustain soil function and ecosystem services.

A numerical similarity approach for using retired Current Procedural Terminology (CPT) codes for electronic phenotyping in the Scalable Collaborative Infrastructure for a Learning Health System (SCILHS).

BACKGROUND: Interoperable phenotyping algorithms, needed to identify patient cohorts meeting eligibility criteria for observational studies or clinical trials, require medical data in a consistent structured, coded format. Data heterogeneity limits such algorithms' applicability. Existing approaches are often: not widely interoperable; or, have low sensitivity due to reliance on the lowest common denominator (ICD-9 diagnoses). In the Scalable Collaborative Infrastructure for a Learning Healthcare System (SCILHS) we endeavor to use the widely-available Current Procedural Terminology (CPT) procedure codes with ICD-9. Unfortunately, CPT changes drastically year-to-year - codes are retired/replaced. Longitudinal analysis requires grouping retired and current codes. BioPortal provides a navigable CPT hierarchy, which we imported into the Informatics for Integrating Biology and the Bedside (i2b2) data warehouse and analytics platform. However, this hierarchy does not include retired codes. METHODS: We compared BioPortal's 2014AA CPT hierarchy with Partners Healthcare's SCILHS datamart, comprising three-million patients' data over 15 years. 573 CPT codes were not present in 2014AA (6.5 million occurrences). No existing terminology provided hierarchical linkages for these missing codes, so we developed a method that automatically places missing codes in the most specific "grouper" category, using the numerical similarity of CPT codes. Two informaticians reviewed the results. We incorporated the final table into our i2b2 SCILHS/PCORnet ontology, deployed it at seven sites, and performed a gap analysis and an evaluation against several phenotyping algorithms. RESULTS: The reviewers found the method placed the code correctly with 97 % precision when considering only miscategorizations ("correctness precision") and 52 % precision using a gold-standard of optimal placement ("optimality precision"). High correctness precision meant that codes were placed in a reasonable hierarchal position that a reviewer can quickly validate. Lower optimality precision meant that codes were not often placed in the optimal hierarchical subfolder. The seven sites encountered few occurrences of codes outside our ontology, 93 % of which comprised just four codes. Our hierarchical approach correctly grouped retired and non-retired codes in most cases and extended the temporal reach of several important phenotyping algorithms. CONCLUSIONS: We developed a simple, easily-validated, automated method to place retired CPT codes into the BioPortal CPT hierarchy. This complements existing hierarchical terminologies, which do not include retired codes. The approach's utility is confirmed by the high correctness precision and successful grouping of retired with non-retired codes.

Taking advantage of continuity of care documents to populate a research repository.

OBJECTIVE: Clinical data warehouses have accelerated clinical research, but even with available open source tools, there is a high barrier to entry due to the complexity of normalizing and importing data. The Office of the National Coordinator for Health Information Technology's Meaningful Use Incentive Program now requires that electronic health record systems produce standardized consolidated clinical document architecture (C-CDA) documents. Here, we leverage this data source to create a low volume standards based import pipeline for the Informatics for Integrating Biology and the Bedside (i2b2) clinical research platform. We validate this approach by creating a small repository at Partners Healthcare automatically from C-CDA documents. MATERIALS AND METHODS: We designed an i2b2 extension to import C-CDAs into i2b2. It is extensible to other sites with variances in C-CDA format without requiring custom code. We also designed new ontology structures for querying the imported data. RESULTS: We implemented our methodology at Partners Healthcare, where we developed an adapter to retrieve C-CDAs from Enterprise Services. Our current implementation supports demographics, encounters, problems, and medications. We imported approximately 17 000 clinical observations on 145 patients into i2b2 in about 24 min. We were able to perform i2b2 cohort finding queries and view patient information through SMART apps on the imported data. DISCUSSION: This low volume import approach can serve small practices with local access to C-CDAs and will allow patient registries to import patient supplied C-CDAs. These components will soon be available open source on the i2b2 wiki. CONCLUSIONS: Our approach will lower barriers to entry in implementing i2b2 where informatics expertise or data access are limited.

Whole genome sequencing reveals potential targets for therapy in patients with refractory KRAS mutated metastatic colorectal cancer.

BACKGROUND: The outcome of patients with metastatic colorectal carcinoma (mCRC) following first line therapy is poor, with median survival of less than one year. The purpose of this study was to identify candidate therapeutically targetable somatic events in mCRC patient samples by whole genome sequencing (WGS), so as to obtain targeted treatment strategies for individual patients. METHODS: Four patients were recruited, all of whom had received > 2 prior therapy regimens. Percutaneous needle biopsies of metastases were performed with whole blood collection for the extraction of constitutional DNA. One tumor was not included in this study as the quality of tumor tissue was not sufficient for further analysis. WGS was performed using Illumina paired end chemistry on HiSeq2000 sequencing systems, which yielded coverage of greater than 30X for all samples. NGS data were processed and analyzed to detect somatic genomic alterations including point mutations, indels, copy number alterations, translocations and rearrangements. RESULTS: All 3 tumor samples had KRAS mutations, while 2 tumors contained mutations in the APC gene and the PIK3CA gene. Although we did not identify a TCF7L2-VTI1A translocation, we did detect a TCF7L2 mutation in one tumor. Among the other interesting mutated genes was INPPL1, an important gene involved in PI3 kinase signaling. Functional studies demonstrated that inhibition of INPPL1 reduced growth of CRC cells, suggesting that INPPL1 may promote growth in CRC. CONCLUSIONS: Our study further supports potential molecularly defined therapeutic contexts that might provide insights into treatment strategies for refractory mCRC. New insights into the role of INPPL1 in colon tumor cell growth have also been identified. Continued development of appropriate targeted agents towards specific events may be warranted to help improve outcomes in CRC.

Integrated genomic characterization reveals novel, therapeutically relevant drug targets in FGFR and EGFR pathways in sporadic intrahepatic cholangiocarcinoma.

Advanced cholangiocarcinoma continues to harbor a difficult prognosis and therapeutic options have been limited. During the course of a clinical trial of whole genomic sequencing seeking druggable targets, we examined six patients with advanced cholangiocarcinoma. Integrated genome-wide and whole transcriptome sequence analyses were performed on tumors from six patients with advanced, sporadic intrahepatic cholangiocarcinoma (SIC) to identify potential therapeutically actionable events. Among the somatic events captured in our analysis, we uncovered two novel therapeutically relevant genomic contexts that when acted upon, resulted in preliminary evidence of anti-tumor activity. Genome-wide structural analysis of sequence data revealed recurrent translocation events involving the FGFR2 locus in three of six assessed patients. These observations and supporting evidence triggered the use of FGFR inhibitors in these patients. In one example, preliminary anti-tumor activity of pazopanib (in vitro FGFR2 IC50≈350 nM) was noted in a patient with an FGFR2-TACC3 fusion. After progression on pazopanib, the same patient also had stable disease on ponatinib, a pan-FGFR inhibitor (in vitro, FGFR2 IC50≈8 nM). In an independent non-FGFR2 translocation patient, exome and transcriptome analysis revealed an allele specific somatic nonsense mutation (E384X) in ERRFI1, a direct negative regulator of EGFR activation. Rapid and robust disease regression was noted in this ERRFI1 inactivated tumor when treated with erlotinib, an EGFR kinase inhibitor. FGFR2 fusions and ERRFI mutations may represent novel targets in sporadic intrahepatic cholangiocarcinoma and trials should be characterized in larger cohorts of patients with these aberrations.

Simultaneous characterization of somatic events and HPV-18 integration in a metastatic cervical carcinoma patient using DNA and RNA sequencing.

OBJECTIVE: Integration of carcinogenic human papillomaviruses (HPVs) into the host genome is a significant tumorigenic factor in specific cancers including cervical carcinoma. Although major strides have been made with respect to HPV diagnosis and prevention, identification and development of efficacious treatments for cervical cancer patients remains a goal and thus requires additional detailed characterization of both somatic events and HPV integration. Given this need, the goal of this study was to use the next generation sequencing to simultaneously evaluate somatic alterations and expression changes in a patient's cervical squamous carcinoma lesion metastatic to the lung and to detect and analyze HPV infection in the same sample. MATERIALS AND METHODS: We performed tumor and normal exome, tumor and normal shallow whole-genome sequencing, and RNA sequencing of the patient's lung metastasis. RESULTS: We generated over 1.2 billion mapped reads and identified 130 somatic point mutations and indels, 21 genic translocations, 16 coding regions demonstrating copy number changes, and over 36 genes demonstrating altered expression in the tumor (corrected P < 0.05). Sequencing also revealed the HPV type 18 (HPV-18) integration in the metastasis. Using both DNA and RNA reads, we pinpointed 3 major events indicating HPV-18 integration into an intronic region of chromosome 6p25.1 in the patient's tumor and validated these events with Sanger sequencing. This integration site has not been reported for HPV-18. CONCLUSIONS: We demonstrate that DNA and RNA sequencing can be used to concurrently characterize somatic alterations and expression changes in a biopsy and delineate HPV integration at base resolution in cervical cancer. Further sequencing will allow us to better understand the molecular basis of cervical cancer pathogenesis.

Ectomycorrhizal fungi contribute to soil organic matter cycling in sub-boreal forests.

Soils of northern temperate and boreal forests represent a large terrestrial carbon (C) sink. The fate of this C under elevated atmospheric CO2 and climate change is still uncertain. A fundamental knowledge gap is the extent to which ectomycorrhizal fungi (EMF) and saprotrophic fungi contribute to C cycling in the systems by soil organic matter (SOM) decomposition. In this study, we used a novel approach to generate and compare enzymatically active EMF hyphae-dominated and saprotrophic hyphae-enriched communities under field conditions. Fermentation-humus (FH)-filled mesh bags, surrounded by a sand barrier, effectively trapped EMF hyphae with a community structure comparable to that found in the surrounding FH layer, at both trophic and taxonomic levels. In contrast, over half the sequences from mesh bags with no sand barrier were identified as belonging to saprotrophic fungi. The EMF hyphae-dominated systems exhibited levels of hydrolytic and oxidative enzyme activities that were comparable to or higher than saprotroph-enriched systems. The enzymes assayed included those associated with both labile and recalcitrant SOM degradation. Our study shows that EMF hyphae are likely important contributors to current SOM turnover in sub-boreal systems. Our results also suggest that any increased EMF biomass that might result from higher below-ground C allocation by trees would not suppress C fluxes from sub-boreal soils.