Escherichia coli and Staphylococcus aureus), and cells (CD4+ T lymphocytes) from fingerprick volume equivalent of multiple biological specimens such as whole blood, plasma, and peritoneal dialysis effluent with clinically relevant detection and sensitivity.The need for sensitive, robust, portable, and inexpensive biosensing platforms is of significant interest in clinical applications for disease diagnosis and treatment monitoring at the point-of-care (POC) settings. Rapid, accurate POC diagnostic assays play a crucial role in developing countries, where there are limited laboratory infrastructure, trained personnel, and financial support. However, current diagnostic assays commonly require long assay time, sophisticated infrastructure and expensive reagents that are not compatible with resource-constrained settings. Although paper and flexible material-based platform technologies provide alternative approaches to develop POC diagnostic assays for broad applications in medicine, they have technical challenges integrating to different detection modalities. Here, we address the limited capability of current paper and flexible material-based platforms by integrating cellulose paper and flexible polyester films as diagnostic biosensing materials with various detection modalities through the development and validation of new widely applicable electrical and optical sensing mechanisms using antibodies and peptides. By incorporating these different detection modalities, we present selective and accurate capture and detection of multiple biotargets including viruses (Human Immunodeficieny Virus-1), bacteria ( Rapid diagnosis has broad applications in multiple disciplines including clinical medicine, immunoassays, food safety, environmental monitoring, and veterinary medicine1234618+ T lymphocytes, and (iii) detection of bacterial infections such as Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) in various clinical matrices with high sensitivity and specificity. Thus, we demonstrate that paper and flexible material-based platforms can be widely applied to a variety of settings including medical diagnostic and biology laboratories. The combination of these sensing mechanisms on paper and flexible materials offers a promising opportunity to create diagnostic assays that meet the requirements of POC disease detection and treatment monitoring.In this manuscript, we demonstrate different paper and flexible material-based diagnostic platforms with biosensing applications to detect various bioagents in whole blood, serum and peritoneal fluid. These distinct platforms have been integrated with sensing modalities including impedance spectroscopy of biotargets, nanoparticle aggregation using a mobile phone-based detection system, and lensless shadow imaging technology that do not require any signal amplification. As clinical disease models, we present applications of paper and flexible material-based platforms focusing on (i) detection of Human Immunodeficiency Virus-1 (HIV-1) by capturing and detecting multiple HIV-1 subtypes, (ii) CD4A flexible polyester film with two rail silver electrodes in microfluidic channels was employed as an electrical sensing platform to detect viruses (HIV-1) through viral lysate impedance spectroscopy . Here, bTo evaluate the specificity of this platform for HIV-1 detection, we used Epstein-Barr virus (EBV). The impedance magnitude spectra of HIV-1, EBV, and the mixture of HIV-1 and EBV samples were measured and compared with control samples . Control6 copies/mL  and minimum antibody concentration1618i.e., polyester film) to selectively capture and detect viruses (HIV-1) in clinical samples such as whole blood and plasma. However, this platform requires off-chip sample preparation including virus capture using antibodies, washing, and virus lysis. By employing further developments in sample processing steps, this platform technology can be potentially automated for POC applications.Electrical sensing is a powerful modality to develop portable, rapid, and sensitive diagnostic tools231314E. coli and S. aureus as model bacterial pathogens. In this platform, gold nanoparticles were first modified with specific recognition elements to capture pathogens in the solution. We subsequently transferred the modified nanoparticle solution to a disposable paper that allowed distribution of aggregated nanoparticles by utilizing capillary effect on its native structure. thus obtaining uniform distribution for quantitative analysis. To evaluate the chemical modifications on nanoparticle surface, 11-Mercaptoundeconoic acid (MUA), N-Ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) coupling agents, and Lipopolysaccharide Binding Protein (LBP) for recognition of E. coli were used to modify gold nanoparticles. The maximum extinction wavelength peaks of nanoparticle colloids resulted in red-shifts after each surface modification   resulted in red-colored colloids of nanoparticles since there is no recognition site for LBP protein in the solution. Therefore, these experiments showed that blue color formation was not due to non-specific interactions, and it was observed in the presence of target bacteria (E. coli). For quantitative analysis, the solution containing modified nanoparticles and bacteria was also sampled and dried on a paper within 10 minutes, and a mobile phone camera (Sony Ericson i790) was then employed as an image acquisition step for nanoparticle aggregate spots , green (G), and blue (B) pixel values as previously presented2 on the cellulose paper surface. Further, as we demonstrated previously21To this end, the platform was validated by spiking E. coli into three distinct media: DPBS, whole blood serum, and peritoneal dialysis (PD) fluid . These td color) . This cote spots . FurtherE. coli samples were analyzed using statistical methods, and the lowest detectable concentration was observed to be 8 colony forming units (CFUs)/mL for all samples (S. aureus (105 CFUs/mL) into anti-LTA antibody-modified gold nanoparticle solution, (ii) the sampling of E. coli (105 CFUs/mL) into anti-LTA antibody-modified gold nanoparticle solution, and (iii) the sampling of mixed bacteria (S. aureus (105 CFUs/mL) and E. coli (105 CFUs/mL)) into anti-LTA antibody-modified gold nanoparticle solution     . Further < 0.05) . At the  > 0.05) . Since t samples . In addiuisition . WithoutE. coli and S. aureus. In this platform, the nanoparticle aggregation is associated with the repulsive electrostatic forces between nanoparticles, and resulted in a blue color in the presence of the pathogens 2627E. coli is reported as one of the most common species for bacterial infection observed in PD patients29E. coli spiked in PD samples were used as a clinical disease model. In end-stage renal disease (ESRD) patients, kidney replacement or dialysis to preserve any residual renal function is required. It is estimated that more than half a million patients will require dialysis in 2020 with the rising disease prevalence in the USi.e., inflammation of the peritoneum. Peritonitis has to be detected early and treated before it becomes a chronic case. Detecting bacterial infection offers a great promise for the early diagnosis of peritonitis as well as other bacteria-induced diseases such as sepsisFrom bacterial infection perspective, + T lymphocytes in fingerprick whole blood samples for POC HIV diagnosis as a clinical disease model  sensor. These flexible substrates are polyester-based transparency films that are routinely used for printing and lamination. All substrates were first imaged using bright-field (BF) microscopy to visualize any inbuilt debris  using a fluorescent microscope (http://rsb.info.nih.gov/nih-image/). The intensity histograms, corresponding microscope images, and the mean intensity plots were demonstrated in A flexible polyester film with microfluidic channels was employed to selectively and efficiently capture and quantify cells of interest in unprocessed whole blood samples. Here, we evaluated this platform to capture and quantify CD4se model . We havet debris . Some ofcroscope . The subcroscope . To quan+ T cells, this flexible platform was assembled and functionalized to immobilize the capture antibody -based platform can be integrated with CMOS lensless imaging platform for rapid detection of CD4+ T cells (+ T cell counting using fingerprick volume of unprocessed whole blood samples at the POC. These results indicate that the platform has the potential to replace conventional non-disposable glass-based rigid biosensing devices in the future given that the capture efficiency and specificity are further increased beyond 90% by further optimization of the materials and optical properties of the films as a substrate. In the current form, the device processing requires manual pipetting steps for blood injection and channel washing, but the developed platform can potentially be made fully automated by integrating microfluidic devices to disposable cartridges.For the capture of CD4antibody . To optiantibody . The assantibody . The fleantibody . DAPI stlculated . The briectively . The CD4ectively . We obse T cells . Shadows T cells . The CMO+ T cell count is essential for HIV-1 diagnosis and treatment monitoring3334+ T cell count of less than 500 cells/μl+ T cell counting methods require flow cytometer, i.e., a gold standard method, for CD4+ T cell counting in the developed world. This method, however, requires a skilled operator, expensive infrastructure and reagents. The disposable and flexible biosensing platform for efficient counting of CD4+ T cells has potential to address some of these global health challenges at the POC settings.From HIV diagnostic perspective, accurate CD4+ T lymphocytes, E. coli and S. aureus in various matrices including whole blood, plasma, and peritoneal dialysis fluid. We also reported various cellulose paper and flexible substrate-based platforms allowing broadly applicable diagnostic assays through seamlessly integrating optical and electrical sensing modalities. Thus, these various platforms are uniquely able to isolate and detect multiple biotargets selectively, sensitively, and repeatably from diverse biological matrices using antibodies and peptides. Further, these platforms could potentially be adapted and tailored to detect other pathogens and biotargets with well-known biomarkers13343738394041Paper and flexible material-based biosensing platforms are powerful tools to create affordable and disposable POC disease detection and treatment monitoring assays, particularly for the resource-constrained settings. There is a need for their integration with different detection strategies for distinct biotargets from multiple biological and clinical matrices such as whole blood, serum, saline buffer, dialysis effluent or lysates with high sensitivity and specificity. In this manuscript, as clinical models, we demonstrated the isolation and detection of HIV-1 CD4H.S., W.A., F.I., M.Y., and U.D. designed the study and the experiments; H.S., W.A., F.I., M.Y., M.S., M.J., M.H.Z. and N.G.D. performed the experiments; U.A.G. performed the SEM imaging shown in Figures S8 and S9; H.S., W.A. F.I., M.Y., and U.D. wrote the manuscript; D.R.K. supervised the virology aspect of the study and provided HIV samples; H.S., W.A., F.I., M.Y., and U.D. supervised the research.SUPPLEMENTARY INFO


Leishmania (Viannia) braziliensis causing human cutaneous and mucocutaneous leishmaniasis is tested by comparative analysis of infection progression and infectiousness to sandflies in rodent host species previously shown to have high natural infection prevalences in both sylvatic or/and peridomestic habitats in close proximity to humans in northeast Brazil.The possibility that a multi-host wildlife reservoir is responsible for maintaining transmission of Necromys lasiurus, 18 Nectomys squamipes and 18 Rattus rattus) experimentally infected with high (5.5×106/ml) or low (2.8×105/ml) dose L. (V.) braziliensis (MBOL/BR/2000/CPqAM95) inoculum. Clinical signs of infection were monitored daily. Whole animal xenodiagnoses were performed 6 months post inoculation using Lutzomyia longipalpis originating from flies caught in Passira, Pernambuco, after this parasite evaluation was performed at necropsy. Heterogeneities in Leishmania parasite loads were measured by quantitative PCR in ear skin, liver and spleen tissues.The clinical and parasitological outcomes, and infectiousness to sandflies, were observed in 54 colonized animals of three species (18 3 (skin), 2.8×103 (spleen), and 8.9×102 (liver). All three host species, 18/18 N. lasiurus, 10/18 N. squamipes and 6/18 R. rattus, also proved infectious to sandflies in cross-sectional study. R. rattus supported significantly lower tissue parasite loads compared to those in N. lasiurus and N. squamipes, and N. lasiurus appeared to be more infectious, on average, than either N. squamipes or R. rattus.All three rodent species proved to establish infection characterized by short-term self-resolving skin lesions, located on ears and tail but not on footpads (one site of inoculation), and variable parasite loads detected in all three tissues with maximum burdens of 8.1×10A multi-host reservoir of cutaneous leishmaniasis is indicated in this region of Brazil, though with apparent differences in the competence between the rodent species. The results provide preliminary insights into links between sylvatic and peri-domestic transmission cycles associated with overlaps in the rodent species’ ecological niches. Leishmania (V.) braziliensis is the predominant Leishmania species causing cutaneous and mucocutaneous leishmaniasis in humans. Transmitted by Phlebotomine sandflies, questions remain about the epidemiological contributions of the numerous zoonotic and more domestic host species. Domestication of the principal vector and human infection patterns suggest that human infection risk is predominantly peridomestic, whereas control strategies will be more complex if there is a link to a wildlife transmission cycle. Almost no studies have been conducted on the transmission potential of natural hosts of L. (V.) braziliensis. This study evaluates the infectiousness of experimentally infected natural rodent host species, that in different ecological habitats are proposed to act as a single or a multi-host reservoir. Clinical and parasitological development, and the ability to transmit Leishmania to sandflies, was observed under experimental conditions using a single strain of L. (V.) braziliensis isolated from the wild rat, Necromys lasiurus. Xenodiagnoses were performed with laboratory bred sand fly females established from a local population of Lutzomyia longipalpis. All three rodent species developed disseminated subclinical parasitological infections, but clinical signs (lesions) were transient and self-resolving. N. squamipes, N. lasiurus and R. rattus were all infectious when asymptomatic, though their competence in transmission potential appears to differ with R. rattus showing signs of lower susceptibility. These results provide further evidence that a multi-host reservoir is responsible for maintaining transmission with a bridge between infectious sylvatic and peridomestic rodent populations.Across the Americas,  Transmission of zoonotic pathogens may involve one, or typically more than one, reservoir host. Compared to pathogens with single reservoir hosts, those involving multi-host communities usually show reduced transmission rates through a process of zooprophylaxis or “dilution effect” due to heterogeneities in their competence to support pathogen replication and in their infectious duration, resulting in reduced pathogen-host contact, or vector-infectious host contact in the case of vector-borne pathogens , 2, 3. TL. (Viannia.) braziliensis causing, in humans, small simple self-healing cutaneous lesions to disfiguring and destructive lesions known as espundia or mucosal leishmaniasis that can result in irreversible disfigurement of the upper nasal tract. In Brazil the dominant parasite causing cutaneous leishmaniasis is L. (V.) braziliensis and there are approximately 26,000 reported new human cases per year but estimates of annual incidences range from 72,800 to 119,600  as  as Lu. wtrypanum . Howeverg species so anothN. squamipes is the largest rodent of the three species (c. 240gm vs R. rattus 50gm and N. lasiurus 160gm) which may attract relatively more sand flies [Lu. whitmani when fed on their skin lesions [Leishmania, and detection of infection does not necessarily equate to transmission potential .L. (V.) braziliensis involving Atlantic forest small mammals and sand fly vectors, to a more peridomestic cycle involving, not least, the infectious rodents described here, that are associated with overlapping sylvatic and peridomestic habitats (N. lasiurus and N. squamipes) and domestic habitats (R. rattus) respectively [Lu. whitmani demonstrates a degree of domesticity, feeding site and host choice loyalty, potentially limiting vector-host contact to more predominant competent species [N. squamipes [This study provides some preliminary insights into the likely transition from the assumed original transmission cycle of ectively , 15. Theectively . Potenti species , 56. Thiquamipes , with thquamipes . Researc


Coxiella burnetii and localized by positron emission tomography scanning.Few cases of Q fever osteoarticular infection have been reported, with chronic osteomyelitis as the most common manifestation of Q fever osteoarticular infection. Here we present the case of a sternoclavicular joint infection caused by C. burnetii and he was treated with 200 mg doxycycline for 21 days. An immunofluorescence assay of convalescent serum sampled after 2 months revealed very high C. burnetii antibody titers. To localize the site of the infection, we performed positron emission tomography scanning, which revealed intense fluorodeoxyglucose uptake in his right sternoclavicular joint; treatment with 200 mg oral doxycycline daily and 200 mg oral hydroxychloroquine three times daily for 18 months was initiated.A 67-year-old French man from south France was hospitalized for fever and confusion. An examination revealed subclavicular and axillary lymph node enlargement. Computed tomography scanning and transesophageal echocardiogram were normal, and magnetic resonance imaging scanning did not reveal signs of infection. An immunofluorescence assay of an acute serum sample was positive for C. burnetii antibody titers to localize the site of C. burnetii infection.Q fever articular infections may be undiagnosed, and we strongly urge the use of positron emission tomography scanning in patients with high  Coxiella burnetii [C. burnetii has also been implicated in a prosthetic joint infection [Q fever is a potentially life-threatening worldwide zoonosis caused by an obligate intracellular bacterium, burnetii . Infectiburnetii . To dateburnetii , 3. In fburnetii , and 11 burnetii . Osteomyburnetii . C. burnnfection .C. burnetii infections [C. burnetii antibody titers in order to localize the site of C. burnetii infection [C. burnetii, localized by PET scanning.Q fever osteoarticular infection can easily go undiagnosed because of the long evolution of articular involvement, which is accompanied by a low level of laboratory and inflammatory signs . Howeverfections , 7, and nfection , 8. HereC. burnetii; phase I titers for immunoglobulin (Ig) G, IgM, and IgA were 400, 200 and 0, respectively, and phase II titers were 400, 200, and 0, respectively. The diagnosis of acute Q fever infection was made and he was treated with 200 mg oral doxycycline daily for 21 days. IgG anticardiolipin (aCL) antibody levels in the serum sample were very high (216 GPLU), and we suspected valvular heart disease and a possible progression to Q fever endocarditis [C. burnetii by quantitative polymerase chain reaction (qPCR) for the IS1111 and the IS30A spacers [C. burnetii infection was suspected; lymph node biopsies were performed that were negative for C. burnetii by molecular assays. For each sample, we verified the quality of DNA handling and extraction of samples by qPCR for a housekeeping gene encoding beta-actin [C. burnetii by immunohistochemical analysis using a monoclonal antibody against C. burnetii with an immunoperoxidase kit [in situ hybridization (FISH) [C. burnetii was made, and treatment with 200 mg oral doxycycline daily and 200 mg oral hydroxychloroquine three times daily for 18 months was introduced. After 6 months follow-up, the outcome was favorable, with a four-fold decrease in the phase I and phase II IFA titers for IgG.A 67-year-old French man from south France was admitted to our hospital approximately 1 year ago (February 2015) with fever 39 °C) and confusion. He mentioned increased alcohol consumption. An examination revealed subclavicular and axillary lymph node enlargement. Moreover, he presented cerebellar ataxia, with loss of equilibrium and difficulty walking. Laboratory values revealed elevated C-reactive protein (216 mg/L) and liver enzyme levels  and hyponatremia (sodium 123 mEq/L). A liver ultrasound showed hepatomegaly. Computed tomography (CT) scanning was normal and magnetic resonance imaging (MRI) scanning did not reveal signs of infection. Treatment with ceftriaxone and levofloxacin was introduced, but his fever did not resolve. A serum sample was sent to our laboratory in Marseille and an immunofluorescence assay (IFA) was positive for  °C and cC. burnetii vascular infections [C. burnetii were also localized [C. burnetii infection because of the very high IFA titers and IgG-aCL levels [C. burnetii. As Q fever articular infections present a long evolution of articular involvement, accompanied by a low level of inflammatory signs, and can easily remain undiagnosed [C. burnetii in the sternoclavicular joint. Although we did not test the sternoclavicular joint to confirm the diagnosis, a localized infection with C. burnetii is associated with high antibody titers against C. burnetii [We report a case of Q fever sternoclavicular joint infection diagnosed through serology and localized using PET scanning. PET scanning has been previously used for the identification of infectious foci in fections , in the fections , in the fections , and recocalized . In thisL levels . In factL levels . Howeveriagnosed , the PETburnetii . For ourEpidemiological patterns of Q fever osteoarticular infections may consist of sporadic cases that are difficult to diagnose. As with the majority of Q fever osteoarticular infections, our patient was an adult man more than 50-years old . AlthougC. burnetii infections and we strongly urge its use in patients with high C. burnetii antibody titers in order to localize the site of the C. burnetii infection.In conclusion, we confirm the efficacy of PET scanning for the identification of infectious foci in in situ hybridization; IFA, immunofluorescence assay; Ig, immunoglobulin; MRI, magnetic resonance imaging; PET, positron emission tomography; qPCR, quantitative polymerase chain reaction.aCL, anticardiolipin; CT, computed tomography; FISH, fluorescent


Motivation: Interpretation and communication of genomic data require flexible and quantitative tools to analyze and visualize diverse data types, and yet, a comprehensive tool to display all common genomic data types in publication quality figures does not exist to date. To address this shortcoming, we present Sushi.R, an R/Bioconductor package that allows flexible integration of genomic visualizations into highly customizable, publication-ready, multi-panel figures from common genomic data formats including Browser Extensible Data (BED), bedGraph and Browser Extensible Data Paired-End (BEDPE). Sushi.R is open source and made publicly available through GitHub (https://github.com/dphansti/Sushi) and Bioconductor (http://bioconductor.org/packages/release/bioc/html/Sushi.html).Contact:mpsnyder@stanford.edu or dphansti@stanford.edu Genomic science is a rich data-intensive field in which diverse data types are combined to uncover and explore characteristics of sequence elements on a large scale. However, despite a growing set of mature standard visualization techniques and file formats, no comprehensive tools exist to facilitate multi-panel visualization across a broad range of standard genomic data types. To address this deficiency, we developed Sushi.R, a flexible R library that leverages standard visualization techniques and file formats to produce highly customizable publication-quality figures of genomic data within the widespread analysis environment, R.Sushi.R is written exclusively in the R software environment. The Sushi.R package includes 13 example datasets and a vignette detailing the usage of each . DatasetQuantitative and qualitative genomic information can typically be broken down into three data types: features, signals and interactions. Sushi.R provides flexible methods to plot each data type, allowing users to represent virtually any type of genomic data in an aesthetically pleasing, coherent and integrative fashion. A Sushi plot made entirely within R (without any modifications in image-editing software) displaying multiple data types is shown in plotBed, plotGenes and plotManhattan facilitate the visualization of feature data in a host of different formats ranging from heatmaps of feature density to feature pileups  format, feature data can be used to represent sites of transcription factor binding, gene structures, transcript structures, sequence read alignments, Genome-Wide Association Studies (GWAS) hits and data from an array of other sources. The Sushi functions  pileups A, G–K, NplotBedgraph provides flexible methods to plot, overlay and compare signal track data with appropriately represented data from each one of these disparate sources  format or in interaction matrices. Sushi functions e widths B–D.via a number of annotation functions including zoomsregion, zoombox, maptocolor and addlegend, allowing customizable scaling of colors, line types and line widths for flexible quantitative presentation. Zoom inset features facilitate visualization at multiple scales and diverse genomic contexts. Images can be written to all formats supported by R including Encapsulated PostScript (EPS), Portable Document Format (PDF) and Portable Network Graphics (PNG).Sushi plots can easily be combined and augmented The rapid proliferation and complexity of genomics experiments—fueled by high-throughput sequencing—has concomitantly driven demand for analysis and visualization tools that facilitate interpretation and communication of rich and diverse genomic data types. Sushi fills a critical void among currently available visualization tools by providing a means to easily produce sophisticated, customizable, genomic visualizations. Sushi.R will be of great use to the genomic community, as it accelerates our ability to uncover, document and communicate important scientific findings derived from increasingly abundant, and complex, genomic data.Funding: This project is funded by NIH grant U54HG006996 (to M.P.S) and K99HG007356 (to A.P.B). D.H.P. is a Damon Runyon fellow supported by the Damon Runyon Cancer Research Foundation (DRG-2122-12).Conflict of interest: M.P.S. is a cofounder and scientific advisory board (SAB) member of Personalis and also on the SAB of Genapsys.


Diatoms are very efficient in their use of available nutrients. Changes in nutrient availability influence the metabolism and the composition of the cell constituents. Since diatoms are valuable candidates to search for oil producing algae, measurements of diatom-produced compounds can be very useful for biotechnology. In order to explore the diversity of lipophilic compounds produced by diatoms, we describe the results from an analysis of 13 diatom strains. With the help of a lipidomics platform, which combines an UPLC separation with a high resolution/high mass accuracy mass spectrometer, we were able to measure and annotate 142 lipid species. Out of these, 32 were present in all 13 cultures. The annotated lipid features belong to six classes of glycerolipids. The data obtained from the measurements were used to create lipidomic profiles. The metabolomic overview of analysed cultures is amended by the measurement of 96 polar compounds. To further increase the lipid diversity and gain insight into metabolomic adaptation to nitrogen limitation, diatoms were cultured in media with high and low concentrations of nitrate. The growth in nitrogen-deplete or nitrogen-replete conditions affects metabolite accumulation but has no major influence on the species-specific metabolomic profile. Thus, the genetic component is stronger in determining metabolic patterns than nitrogen levels. Therefore, lipid profiling is powerful enough to be used as a molecular fingerprint for diatom cultures. Furthermore, an increase of triacylglycerol (TAG) accumulation was observed in low nitrogen samples, although this trend was not consistent across all 13 diatom strains. Overall, our results expand the current understanding of metabolomics diversity in diatoms and confirm their potential value for producing lipids for either bioenergy or as feed stock. Diatoms are eukaryotic phototrophs and are able to very efficiently sequester water-dissolved carbon dioxide. It is estimated that diatoms are responsible for up to 20% of the global primary production ,2. DiatoDiatoms are very efficient at nutrient utilization and thus successful organisms in various environments  but can Amphitetras antediluviana, Biddulphia biddulphiana, Cerataulina daemon, Chaetoceros simplex, Eunotogramma sp., Hemiaulus sinensis, Leptocylindrus danicus, Rhizosolenia setigera, Thalassionema frauenfeldii, Thalassiosira pseudonana (two strains CCMP1007 and CCMP1335) and Thalassiosira weissflogii (two strains CCMP1587 and CCMP1336) and studied their metabolome under nitrogen-rich and nitrogen-limiting conditions. To fully control the growth medium and nutrient levels diatoms were cultivated in artificial sea water in standard f/2 medium ), phosphatidylcholine PC 34:0 [17:0/17:0]) and 13C-sorbitol as internal standards. To facilitate cell disruption, samples were shaken and incubated in a cooled sonic bath for 10 min. The subsequent addition of a water:methanol (3:1) mixture to the extract resulted in the formation of two liquid phases (polar and non-polar phase). The lipid containing non-polar phase was aspirated, dried in vacuum and kept in –20°C prior to the profiling. The fractionation of lipids was performed by a Waters Acquity UPLC system using a C8 reversed-phase column . For the UPLC gradient the mobile phases consisted of two solvents: solvent A, 1% 1 M NH4Ac and 0.1% acetic acid in water; and solvent B, acetonitrile/isopropanol , with an injection volume of 2 μl. The following gradient profile was applied: 1 min, 55% B; 3 min, linear gradient from 55% B to 75% B; 8 min, linear gradient from 75% B to 89% B; 3 min, linear gradient from 89% B to 100% B. After washing the column for 4.5 min with 100% B, the mixture was set back to 55% B and the column was re-equilibrated for 4.5 min , with a flow rate of the mobile phase of 400 μl/min. The mass spectra were acquired using an Orbitrap mass spectrometer . The spectra were recorded alternating between full-scan and all-ion fragmentation-scan modes, covering a mass range from 100 to 1500 m/z [® 7.5 (GeneData). The software extracted analytical features  from raw files generated by the mass spectrometer. Further processing of the data included the removal of the fragmentation information and chemical noise  as well as the retention time alignment. The output data were normalized to internal standard PC 34:0 due to lower number of co-eluting analytes. Peaks were annotated with help of an in-house database of lipid compounds. Further processing was performed with scripts written in R [Metabolite extraction from diatoms was performed using the method of Giavalisco et al.  and Brom1500 m/z . Chromatten in R , which f13C-sorbitol . The data were normalized to the cell density of every culture. The statistical significance of changes between N-replete and N-limiting conditions was tested with the T-test.The polar fractions of the metabolite extracts were derivatizatized and prepared for the GC-MS-based analysis. Shortly thereafter, the dried extracts were resolved in a mixture of methoxyamine hydrochloride in pyrydine and derivatized with N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA). The samples were analyzed, coupled to an Agilent 6890 gas chromatographer coupled with a time-of-flight mass spectrometer (Agilent Technologies). Details of the method are described by Lisec et al. . ChromatPrior to further analysis and visualization the metabolomic data were log2-transformed. The data are available in Different species and isolates of diatoms were acquired from algal collections and grown under laboratory conditions. Cultures were selected on the basis of their culturing properties  and ability to grow fast at a relatively high temperature of 22°C. To induce changes in lipid metabolism of diatoms, they were cultivated in standard f/2 medium  as well Cerataulina daemon, respectively  on lipid composition of diatoms. As shown in Figs Thalassiosira weissflogii and Thalassiosira pseudonana. Surprisingly there was no specific pattern of changes in content of nitrogen-containing phospholipids, PC and PE. Also other classes of lipids showed no specific changes induced by the low-nitrogen in the growth medium.Whereas specific lipid composition was only slightly influenced by the nitrate availability, a change in lipid concentration was observed. B. biddulphiana, A. antediluviana, and R. setigera. In most samples we detected TAGs of a high mass, which probably represent triacylglycerols with three poly-unsaturated fatty acids. We detected and annotated TAG 66:18, which was observed only in L. danicus, T. weissflogii CCMP1336, and R. setigera, the latter species was especially rich in this compound. There was also a high abundance of TAG 60:15 in lipid extracts.The most abundant group of lipids were TAG with 50 carbon atoms in their acyl chains. The intra-lipid species correlation for these TAG (as well as TAG 48:X and TAG 52:X) formed a high-content block across the heatmap  and reduction of 4−hydroxy−benzoic acid levels in three of four Thalassiosira species (8 to 20 fold less than in controls). These three diatom cultures  also exhibited a distinctive accumulation of many metabolites during the treatment. Even more metabolites showed elevated levels in cells of B. biddulphiana. In contrast, A. antediluviana and H. sinensis showed reduced levels for most of analytes measured. The remaining diatom cultures in this experiment showed mixed phenotypes, in which levels of some analytes were increased and for some these levels dropped in the low nitrogen conditions.The polar phase of each extract was used to measure low molecular size compounds representing mostly primary metabolites. The extracts were chemically derivatized and analysed by CG-MS. Obtained spectra were compared with the Golm Metabolomics Database (gmd.mpimp-golm.mpg.de). This resulted in a list of 96 polar analytes representing primary metabolites . Due to B. biddulphiana, which seemed to accumulate alanine and homoserine. Many other analytes showed elevated levels in cells of nitrogen-limited B. biddulphiana, but the p-value calculated was above the 0.05 threshold.The hierarchical clustering of metabolite changes showed a distinctive cluster of metabolites with reduced levels  were accumulating these compounds during the experiment.Contrasting responses to the N-limitation were observed in the case of xylose, glutamic acid and 2,4−diaminobutanoic acid . These tT. pseudonana CCMP 1335 vs. T. pseudonana CCMP 1007; T. weissflogii CCMP1336 vs. T. weissflogii CCMP 1587) showed high similarity to the profiles of samples from control diatoms grown in nitrogen-replete conditions , which generates tricarboxylic acid cycle intermediates, such as malate, that are used for fatty acid biosynthesis [Nitzschia closterium f. minutissima [Phaeodactylum tricornutum [The regulation of the lipid biosynthesis pathway in diatoms cells under N stress has not yet been studied in detail . Previou enzymes –38. This enzymes ,38, suggynthesis . The N-lynthesis ,41. Receutissima  and PhaeB. biddulphiana and A. antediluviana, which form large cells, displayed the highest amount of lipids per cell. In contrast, T. weisflogii and T. pseudonana, diatoms with relatively small cell size that grow to very high densities, appear to have low lipid content per cell. One possible explanation for this is the previously observed exponential relationship between amount of lipid per cell and cell volume [Diatoms such as l volume .sn–1 and sn–3 in TAG from T. pseudonana [T. pseudonana eicosapentaenoic acid (FA20:5) and docosahexaenoic acid (FA22:6) are the most abundant fatty acids [B. biddulphiana accumulated much less of these fatty acids. The most intense peaks in fatty acid profiles of this diatom correspond to arachidonic acid (FA20:4), while content of the other poly-unsaturated FA (FA20:5 and FA22:6) was significantly smaller [B. biddulphiana is much higher when compared to T. pseudonana. Such differences in fatty acid profiles have been already reported by Dunstan et al. [Thalassiosira and Biddulphia are Mediophyceaen [Thalassiosira and Biddulphia do differ in habitats—Thalassiosira is small-celled planktonic diatom while Biddulphia forms colonies in benthic/epiphytic habitats. These aspects should be taken into account while interpreting the observed molecular phenotype of those diatoms.The most abundant group within TAG are lipids with 48 to 54 C in acyl chains (with different desaturation grades). They are most likely composed of glycerol bound with three of the main cellular fatty acids: palmitic (FA16:0) and stearic (FA18:0) acids and their desaturated derivatives. The abundance of TAG48:0 is in agreement with data obtained previously with approaches focusing on the fatty acids in diatom lipids . These peudonana . Unfortun et al. . In his n et al. . One shophyceaen ,51. Thalsn–1 and sn–2 positions of glycerolipids. [sn–1 position and 16-C fatty acids in the sn–2 position. In the “eukaryotic” pathway, which is localized to the endoplasmic reticulum, FA18:1 (oleic acid) is incorporated into phospholipids in the sn–2 position. These phospholipids are the source of DAG for chloroplast-localized synthesis of galactolipids. Hence galactolipids synthesized according to the “eukaryotic” pathway contain predominantly 18-C fatty acid in both sn–1 and sn–2 positions [sn–2 position [sn–1 position. The plant model of galactosylglycerols synthesis is most likely true for diatoms as well, which is supported the study of Dodson et al. [T. pseudonana, Skeletonema marinoi and P. tricornutum contain primarily 20:5/16-C fatty acids. Similar conclusions about the fatty acid composition can be made for DGDG 36:6. Both galactolipids are the most abundant compounds in their classes in all 13 analysed cultures of diatoms. Radiolabeling experiments showed that in P. tricornutum FA20:5 is synthesized in microsomes [de novo biosynthesis, diacylglycerol acyltransferases catalyze DAG conversion into TAG [In plants galactolipids can be synthesised in two pathways: one “prokaryotic” and the other “eukaryotic”. The main difference between the pathways, other than their subcellular localization, is the enzyme preference for fatty acids at the olipids. . The “prositions . Mono- aposition ,54. Thusn et al.  who showcrosomes . The autcrosomes . Regardlinto TAG . In our T. pseudonana [P. tricornutum [Skeletonema marinoi [Cocconeis scutellum [In recent years there have been several publications presenting metabolic profiles of diatoms, including eudonana , P. triccornutum , Skeleto marinoi  and Cocccutellum . HoweverH. sinensis and A. antediluviana have reduced levels of most of measured compounds, whereas the three Thalassiosira species tend to accumulate various compounds as adaptation mechanisms to low-nitrogen conditions. This contradicts previously published metabolic profiles of T. pseudonana from cultures that were transferred from N-replete to nitrate-free medium [T. pseudonana [Chlamydomonas reinhardtii [T. pseudonana have shown an increase of glycolytic and TCA enzymes in N-limited cells [H. sinensis, B. biddulphiana, and T. pseudonana CCMP1335 but only the profile of Ch. simplex showed a strong increase of citric acid in N-limited cells in comparison to the N-replete control. In our experimental conditions Ch. simpex samples grew very rapidly, which may have resulted in very high demand for N and at the time of harvest strong accumulation of TCA intermediates, including citrate. This could explain the difference between Ch. simpex and other slower growing diatom cultures, which could better adapt to the limited availability of nitrate in the media (e.g. slowed down amino acid synthesis) and consequently the over-accumulation of photosynthetic carbon.The central metabolism of the studied diatoms is also affected by the applied growth conditions with reduced nitrogen level, although the type of response was very different between species . For exae medium . In thiseudonana  and in nnhardtii . Moreoveed cells . IncreasWe have extracted and analysed lipids from 13 different strains and cultures of diatoms in order to explore the diversity of lipid metabolites in these organisms grown under two different conditions, i.e. nitrogen replete and nitrogen deplete conditions. The diatoms analysed represent different species and genera, shape and size, life habitats (benthic and planktonic) and colonization types (free living and chain forming). We identified 142 annotated lipid species mostly from the TAG class, many of them displaying species specificity. The growth in nitrogen-deplete or nitrogen-replete conditions affects lipid accumulation but has no major influence on the lipid profiles. We also observed an increase of TAG accumulation in low nitrogen samples, although this trend was not consistent across all 13 diatom strains. Overall, our results confirm that diatoms display a large diversity of lipid species. This observation is consistent with previous suggestions regarding the value of diatoms for producing lipids for either bioenergy or as feed stock.S1 TableAmphitetras antediluviana; Bidbid, Biddulphia biddulphiana; Cerdea, Cerataulina daemon; Chasim, Chaetoceros simplex; Eunlae, Eunotogramma sp.; Hemsin, Hemiaulus sinensis; Lepdan, Leptocylindrus danicus; Rhiset, Rhizosolenia setigera; Thafra, Thalassionema frauenfeldii; Tp1007, Thalassiosira pseudonana CCMP1007; Tp1335, Thalassiosira pseudonana CCMP 1335; Tw1587, Thalassiosira weissflogii CCMP 1587; Tweiss1336, Thalassiosira weissflogii. The “N” suffix marks samples from low nitrogen growth conditions.Following culture abbreviations were used: Ampant, (XLS)Click here for additional data file.S2 TableAmphitetras antediluviana; Bidbid, Biddulphia biddulphiana; Cerdea, Cerataulina daemon; Chasim, Chaetoceros simplex; Eunlae, Eunotogramma sp.; Hemsin, Hemiaulus sinensis; Lepdan, Leptocylindrus danicus; Rhiset, Rhizosolenia setigera; Thafra, Thalassionema frauenfeldii; Tp1007, Thalassiosira pseudonana CCMP1007; Tp1335, Thalassiosira pseudonana CCMP 1335; Tw1587, Thalassiosira weissflogii CCMP 1587; Tweiss1336, Thalassiosira weissflogii.Following culture abbreviations were used: Ampant, (XLS)Click here for additional data file.


Brassica A and C genomes. These were developed using existing coding DNA sequence (CDS) gene models from the now-published Brassica oleracea TO1000 and Brassica napus Darmor-bzh genome sequence assemblies representing the chromosomes of these species, along with preliminary CDS models from an updated Brassica rapa Chiifu genome sequence assembly. The B. rapa genome sequence scaffolds required splitting and re-ordering to match the expected genome organisation based on a high density SNP linkage map, but the B. oleracea assembly was used unchanged. The resulting B. rapa (A genome) pseudomolecules contained 47,656 ordered CDS models and the B. oleracea (C genome) pseudomolecules contained 54,766 ordered CDS models. Interpolation of B. napus CDS models not already represented by orthologues resulted in 52,790 and 63,308 ordered CDS models in the A and C pan-transcriptomes, an increase of 13,676 overall. Comparison of the organisation of this resource with publicly available genome sequences for B. napus showed excellent consistency for the B. napus Darmor-bzh resource, but more breakdown of collinearity for the B. napus ZS11 resource. CDS datasets comprising the pan-transcriptomes are available with this article (B. rapa) or from public repositories (B. oleracea and B. napus).This data article reports the establishment of the first pan-transcriptome resources for the  Specifications TableValue of the data•B. rapa and B. oleracea representing genome organisation in B. napusProvides an updated pseudomolecule description, with genome sequence scaffolds from •Brassica species containing the A and/or C genomesProvides for the first time pan-transcriptome resources for use in •Brassica A and C genomes as represented in an allopolyploid and its diploid progenitorsProvides insights into the extent of gene content variation between the •Brassica A and C pan-genomes for use in genome evolution studies and Associative Transcriptomics.Provides a hypothetical gene order resource for the 1B. napus for both genome organisation studies B. napus Darmor-bzh indicate that sequence exchanges between the constituent genomes of this allotetraploid species (A genome from an unknown B. rapa and C genome from an unknown B. oleacea) may occur very frequently B. napus, i.e. from B. rapa and B. oleracea. As an improvement on the existing resource based on unigenes assembled across Brassica species Brassica A and C genomes as represented in the progenitor species. As the B. napus genome sequence annotation identified many gene models without orthologues in B. rapa and B. oleracea, we further aimed to interpolate those B. napus-specific CDS models, thus producing pan-transcriptome resources for the Brassica A and C genomes as represented by the union of orthologous genes of B. rapa, B. oleracea and B. napus.Transcriptome-based molecular marker systems have been developed and deployed with great success in the crop species B. rapa Chiifu genome sequence scaffolds represent a major advance on the published version 1 sequences Arabidopsis thaliana gene models were used to perform de novo gene predictions in the new genome assembly of B. rapa, after masking the Class I and Class II transposable elements. The predicted genes with CDS models shorter than 150 bp were filtered out. We further performed homology based gene prediction by aligning A. thaliana, Carica papaya, Populus trichocarpa, Vitis vinifera and Oryza sativa protein sequences to the B. rapa genome. TBLASTN was used to do fast alignment , then Genewise was used to do precise alignment. Additionally, we assembled the Brassica ESTs downloaded from NCBI using PASA and aligned them to B. rapa genome by BLAT. Considering that the fragmented exons in EST data might lead to false results, we filtered out alignments with gaps (introns) that span over 10 kb in length. We then ran GLEAN to merge the gene sets generated from de novo and homology-based predictions, using mRNA-Seq data as the supporting evidence. Finally, the B. rapa gene set was aligned to the TE protein database of Repbase, those hits with e-value>1E−5 and coverage≥50% were filtered out. The remaining gene models were reported as Brassica gene set Version 2.0 . These CDS models were then used in sequence similarity searches using BLAST to identify the highest-scoring significant hit  for each CDS model in both the version 2 B. rapa Chiifu genome sequence scaffolds and the A genome pseudomolecules reported previously B. rapa Chiifu genome sequence B. napus genome via high density transcriptome SNP linkage mapping Brassica A genome. The CDS models from the B. oleracea TO1000 B. oleracea TO1000 assembly was adopted unaltered as representing the Brassica C genome pseudomolecule resource.The version 2 B. rapa Chiifu CDS, along with CDS from the published B. oleracea TO1000 genome sequence e-value 1E−30). This resulted in the mapping and ordering of 47,656 B. rapa CDS models to the A genome and 54,766 B. oleracea CDS models to the C genome. A total of 101,040 CDS models were annotated in the B. napus Darmor-bzh genome B. napus pseudomolecules were mapped onto the respective (B. rapa and B. oleracea-based) genome sequence pseudomolecules by BLAST . B. napus CDS models mapping redundantly with CDS models derived from B. rapa and B. oleracea  were excluded, resulting in the addition of 2165 and 3032 CDS models to the A and C genomes, respectively. Finally, CDS models from the B. napus Darmor-bzh genome sequence that did not have significant  BLAST hits in the (B. rapa and B. oleracea-based) genome sequence pseudomolecules were interpolated based on the positions of flanking gene models that did map. This was done by combining the B. napus Darmor-bzh CDS models׳ sorted location on the B. napus Darmor-bzh chromosome with the mapped location of flanking genes on the B. rapa or B. oleracea-based pseudomolecules using an R script  to perform the following: (1) Sort B. napus CDS models by B. napus Darmor-bzh pseudomolecules, then by their B. rapa or B. oleracea-based pseudomolecules hit locations then (2) CDS models (or runs of adjacent CDS models) that do not have a hit onto the B. rapa or B. oleracea-based pseudomolecules are interpolated onto those pseudomolecules with a three digit suffix starting from the boundary of the point of insertion. When the boundaries are not in the right order, the interpolation starts from the closest boundary number to the mean of the nearest 10 neighbours of the run of CDS models. If there is no mapping in the 10 nearest neighbours, the interpolation starts from the minimum of the boundary numbers. This resulted in the addition of 2969 and 5510 further CDS models to the A and C genomes, respectively. The final AC pan-transcriptome resource therefore comprises a total of 116,098 hypothetically ordered CDS models , 52,790 in the Brassica A genome and 63,308 in the Brassica C genome. This represents an increase of 35,171 over the 80,927 CDS models annotated in the published B. napus Darmor-bzh pseudomolecules, 15,058 over the complement of gene models for B. napus including the 20,113 in sequence scaffolds not incorporated into the B. napus Darmor-bzh pseudomolecules B. rapa and B. oleracea pseudomolecules.The B. napus genome sequence resources. This was conducted by sequence similarity search using BLAST to identify the highest-scoring significant hit  for each CDS model in the pan-transcriptome in each of the B. napus Darmor-bzh and B. napus ZS11 chromosome assemblies. Of the aggregate 116,098 CDS models in the pan-transcriptome, 107,292 (92.4%) returned significant hits  in the B. napus Darmor-bzh assembly and 99,395 (85.6%) returned significant hits in the B. napus ZS11 assembly. The order of these best similarity matches in each resource is illustrated in B. napus Darmor-bzh genome assembly shows excellent collinearity. A small number of local rearrangements can be observed in regions with relative high densities of non-collinear matches, possibly corresponding to paracentromeric regions. In addition, two prominent segments shadowing the main collinearity diagonal can be observed amongst the background of CDS models mapping to non-orthologous positions. Such shadows have been observed in previous studies B. napus ZS11 genome assembly show extensive collinearity, but with more disruption by rearrangements than was observed with B. napus Darmor-bzh resource. Linkage group C6 is also presented in the B. napus ZS11 genome sequence resource in the opposite orientation to the current reference genetic map for the Brassica C genome. These analyses, which together indicate extensive collinearity of the Brassica A and C genomes as represented in the allotetraploid B. napus and representatives of its progenitors, are also consistent with early observations of extensive collinearity, but with some divergence in gene content between orthologous regions of Brassica genomes, including both loss and mobility of coding sequences The order of CDS models in the pan transcriptome was compared with the order of orthologous sequences in two publicly-available


Weissella species play beneficial roles in food fermentation and in probiotic products, others such as Weissella confusa are emerging Gram-positive pathogens in immunocompromised hosts. Weissella species are difficult to identify by conventional biochemical methods and commercial automated systems and are easily misidentified as Lactobacillus and Leuconostoc species. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly being used for bacterial identification. Little, however, is known about the effectiveness of MALDI-TOF MS in identifying clinical isolates of Weissella to the species level. In this study, we evaluated whether the MALDI-TOF MS Bruker Biotyper system could accurately identify a total of 20 W. confusa and 2 W. cibaria blood isolates that had been confirmed by 16s rRNA sequencing analysis. The MALDI-TOF Biotyper system yielded no reliable identification results based on the current reference spectra for the two species . New W. confusa spectra were created by randomly selecting 3 W. confusa isolates and external validation was performed by testing the remaining 17 W. confusa isolates using the new spectra. The new main spectra projection (MSP) yielded reliable score values of >2 for all isolates with the exception of one . Our results showed that the MSPs in the current database are not sufficient for correctly identifying W. confusa or W. cibaria. Further studies including more Weissella isolates are warranted to further validate the performance of MALDI-TOF in identifying Weissella species.Although some  Weissella species are found in various fermented food products and are considered to be potential probiotics  during the period 2000–2014 were obtained from the hospital’s microbiology laboratory. These isolates had initially been identified as either Lactobacillus or Leuconostoc species by traditional or commercial automated identification systems as reported previously , the samples were prepared and analyzed as previously described  as previously described  using Bruker Biotyper MALDI-TOF MS software  as previously described . Using the spectra created by the 3 W. confusa isolates , we further tested the residual 17 W. confusa isolates. We found that all of the W. confusa isolates with the exception of one  were identified correctly with a score >2 using the three newly created MSPs. The highest scores were achieved with MSP created by W. confusa Isolate 1 in the ten W. confusa isolates, MSP created by W. confusa Isolate 2 in the five W. confusa isolates and MSP created by W. confusa Isolate 3 in the W. confusa isolate.The MALDI-TOF Biotyper system with current reference spectra yielded no reliable identification results  among the 20 W. confusa (Isolate 8) and W. cibria (Isolate 22) are illustrated in Figure 1. The characteristic peaks for W. cibria were 3008.1, 3634.1, 4079.8, 4954.5, 5380.2, 6371.5, 7267.7, 7962.6, 9530.7, and 11028.2. The characteristic peaks for W. confusa were 3010.1, 3585.2, 4162.9, 4608.2, 4844.0, 5183.7, 6404.8, 7167.4, 7964.3, 9215.7, and 9686.8.The characteristic spectra of W. confusa and W. cibaria. Using the W. confusa MSPs created by our database, we were able to correctly identify all W. confusa isolates.We found that current reference spectra in the MALDI-TOF database were much less accurate than 16s rRNA sequencing in correctly identifying W. confusa. The 16S rRNA sequencing method is considered the standard identification method, although other methods have shown to be able to accurately identify W. confusa. A novel PCR-based identification method using amplified fragment length polymorphism (AFLP)-derived marker was shown to have 100% specificity in identifying W. confusa (Weissella species are listed in the MALDI-TOF database, namely W. confusa, W. halotolerans, W. kandleri, W. minor, and W. viridescens. W. confusa isolate achieved a score value of 1.7, indicating an identification only at genus level. In our study, all of the W. confusa isolates that had been correctly identified by 16S rRNA sequencing failed to be identified with MALDI-TOF using the current database. The discrepancy between the two identification methods remains to be solved. Further studies comparing the results between 16S rRNA sequencing and MALDI-TOF are warranted.It is notoriously difficult to identify  confusa . MALDI-T confusa . DespiteWeissella species is kimchi, a traditional Korean food. Lactic acid bacteria, belonging to the genera Leuconostoc, Lactobacillus, and Weissella, are considered to be the most likely organisms responsible for kimchi fermentation (W. confusa is involved in the early and middle stages of kimchi fermentation (W. confusa has also been detected in food products from other countries, such as fermented milk products in Ghana, sourdough products in Italy, fermented soy sauce in Taiwan, and in soy sauce brine in Malaysia (The most well-studied food product in association with entation , and stuentation . W. confMalaysia .W. ceti is well known in veterinary medicine for causing weissellosis in rainbow trout (W. confusa has been linked to human invasive infections. Therefore, other Weissella species are probably safer alternatives to W. confusa as probiotics.Although ow trout , to the W. confusa is rare and tends to occur in severely immunocompromised hosts, such as patients who have received organ transplants (W. confusa is underestimated because of the difficulties in making a correct identification with commercial and biochemical methods. The findings also suggest that immunocompromised patients should refrain from consuming fermented foods.Bacteremia due to nsplants . Immunocnsplants . Given tW. confusa (Weissella species were either identified as Lactobacillus or Leuconostoc species by commercial automatic systems or traditional biochemical methods. This finding is in concordance with a phylogenetical study by Leuconostoc species and Lactobacillus species were close to W. confusa in neighbor-joining tree by 16S rRNA sequencing. The applications of 16S rRNA sequencing may lead to a better understanding of the incidence of bacteremia due to Weissella species.In our previous study, the results of 16S rRNA sequencing analysis showed that 10 of 43 blood isolates of catalase-negative, vancomycin-resistant coccobacilli were actually  confusa . In thisWeissella species might be under-estimated. In this study, we did not perform 16S rRNA sequencing on bacteremic isolates of Gram-positive cocci, including viridians group streptococci, Pediococcus, and Gemella species. Some Weissella species were misidentified as these organisms by conventional identification methods (Lactobacillus and Leuconostoc species, which were the most closely relevant species and thus were considered the most possible bacteria W. confusa could be misidentified as (During the study period, the actual number of bacteremia due to  methods . In thisified as .Weissella species. W. confusa is an underestimated cause of bacteremia due to its tendency to be misidentified. More extensive epidemiologic studies with molecular methods are needed to reveal the true prevalence and incidence of invasive W. confusa infections. Also, studies involving more clinical Weissella isolates are needed to explore the effectiveness of the MALDI Biotyper system in identification of Weissella species.The MALDI Biotyper system is less accurate that 16S rRNA sequencing in correctly identifying The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Rho family genes have been reported, including their functions and evolution, a systematic analysis of this family has not been performed in Mollusca or in Bivalvia, one of the most important classes of Mollusca.Rho GTPases are important members of the Ras superfamily, which represents the largest signaling protein family in eukaryotes, and function as key molecular switches in converting and amplifying external signals into cellular responses. Although numerous analyses of Rho, Rac, Mig, Cdc42, Tc10, Rnd, RhoU, RhoBTB and Miro) of thirty Rho GTPase genes in three bivalve species, including nine in the Yesso scallop Patinopecten yessoensis, nine in the Zhikong scallop Chlamys farreri, and twelve in the Pacific oyster Crassostrea gigas. Phylogenetic analysis and interspecies comparison indicated that bivalves might possess the most complete types of Rho genes in invertebrates. A multiple RNA-seq dataset was used to investigate the expression profiles of bivalve Rho genes, revealing that the examined scallops share more similar Rho expression patterns than the oyster, whereas more Rho mRNAs are expressed in C. farreri and C. gigas than in P. yessoensis. Additionally, Rho, Rac and Cdc42 were found to be duplicated in the oyster but not in the scallops. Among the expanded Rho genes of C. gigas, duplication pairs with high synonymous substitution rates (Ks) displayed greater differences in expression.In this study, we systematically identified and characterized a total set (Rho GTPase family genes was performed in scallop and oyster species, and Rho genes in bivalves exhibit greater conservation than those in any other invertebrate. This is the first study focusing on a genome-wide characterization of Rho GTPase genes in bivalves, and the findings will provide a valuable resource for a better understanding of Rho evolution and Rho GTPase function in Bivalvia.A comprehensive analysis of bivalve  Rho GTPases regulates the construction of these elements into a key controller of the mechanical processes of cell motility and phagocytosis [Rho (Ras homolog) GTPases are important small G proteins of the Ras superfamily , the largest signaling protein superfamily found in all eukaryotes , 2. Accoocytosis , 10. In ocytosis , pathogeocytosis  and introcytosis , 15.Similar to other Ras-like proteins, Rho proteins typically consist of a conserved structural backbone of five G-boxes that are involved in GTP-binding and GTPase activity , and RhoRho genes were first isolated from the marine gastropod Aplysia californica in 1985 and were subsequently identified in the human genome [Rho genes have been found in various species [Rho genes originated from an ancestral Rac and were gradually distributed among different subfamilies, of which five  are present in bilaterians, with six (RhoUV) appearing in ecdysozoans [Rho family members (Rnd and RhoDF) are found in chordates. Vertebrate-specific RhoH was the last member identified, completing the entire Rho gene family [Rho GTPase genes arose without further expansion of subfamily members as a result of whole-genome duplication, gene duplication and retrotransposition [RhoDF and Rnd appear to be present only in chordates [n genome . Since t species . These Rysozoans . Two extposition . On the Rho genes were initially identified in invertebrates, the genome-wide understanding of these genes in across taxa is limited. Indeed, only limited studies have been conducted in a few species, including Caenorhabditis elegans, Drosophila melanogaster, Litopenaeus vannamei and Strongylocentrotus purpuratus [Rho genes was determined through both in vivo and in vitro experiments. For instance, transfection of a recombinant plasmid containing the A. californica rho gene into oyster hemocytes was able to reduce the β-adrenoceptor-induced apoptosis [C. elegans, several studies have suggested that Rho GTPases are involved in neuronal migration, axon extension and endocytic recycling [Cdc42 gene was also observed in response to the production of Cd2+-reduced reactive oxygen species (ROS), apoptosis and DNA damage in the shrimp L. vannamei [Drosophila Cdc42 is believed to be necessary for dorsal vessel closure and participates in embryonic heart development [Rho genes; however, a systematic analysis of the complete Rho family has not been undertaken in any invertebrate species.Although rpuratus , in whicpoptosis . In C. eecycling . Up-reguvannamei . Drosophelopment . As a keelopment . All of nd largest phylum in the animal kingdom, comprising approximately 200,000 described extant species [Rho GTPases in the genomes of three bivalve species: the Yesso scallop Patinopecten yessoensis; the Zhikong scallop Chlamys farreri; and the Pacific oyster Crassostrea gigas. Orthologs and paralogs were established through a phylogenetic analysis, and the expression profiles of the genes were analyzed using multiple RNA-seq datasets. To our knowledge, this is the first genome-wide characterization of small GTPases in mollusks, and the results will facilitate a better understanding of Rho evolution and the function of Rho GTPases.Mollusca is the 2 species ; bivalve species . In thisP. yessoensis (SRA027310 and SAMN03654043), C. farreri (unpublished) and C. gigas (GSE31012 and AFTI00000000) were searched using the blastp or tblastn algorithm at the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/blast/) to identify Rho GTPase genes in bivalves using invertebrate and vertebrate Rho protein sequences from NCBI (http://www.ncbi.nlm.nih.gov), GJI (http://genome.jgi.doe.gov/) and Ensembl (http://useast.ensembl.org) as queries  to obtain their genomic structures. ORF (open reading frame) finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) was used to predict amino acid sequences. To further verify the gene identifications, the deduced protein sequences were then analyzed using the blastp algorithm for similarity with known genes. The putative isoelectric (PI) points and molecular weights were computed using the Compute pI/Mw tool (http://web.expasy.org/compute_pi/). The DNA Sequence Polymorphism program DnaSP (http://www.ub.edu/dnasp/) was used to estimate the number of synonymous (Ks) and non-synonymous (Ka) substitutions as well as the Ka/Ks ratio among the C. gigas expanded duplication genes identified in the downstream analysis.The transcriptome and whole-genome sequence databases of  queries . The caphttp://www.ebi.ac.uk/Tools/msa/clustalo/). Conserved domains and motifs were first identified by simple modular architecture research tool (SMART) (http://smart.embl-heidelberg.de/) prediction and further confirmed though sequence alignment with Rho GTPase proteins. Human Rac1, RhoA and Cdc42, with the complete protein architecture resolved, were used as reference sequences. The secondary structure depiction of human Rac1 was used for protein structure annotation. A multiple alignment of the sequences restricted to the core Rho domains was performed using ENDscript (http://endscript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). DNAstar (version 4.05) was used for amino acid sequence identity calculations among bivalve Rho proteins. The summarized identities of bivalve Rho proteins are illustrated using a heat map generated by heatmap.2 in R (http://www.r-project.org/). The Maximum-Likelihood (ML) algorithm in the MEGA 6.0 software (http://www.megasoftware.net/) was used to construct phylogenetic trees of the Rho GTPase genes, and aligned sequences were bootstrapped 1000 times to derive the confidence value for the phylogenic analysis.The identified bivalve Rho GTPase proteins were aligned to previously compiled lists of Rho small GTPases using the Clustal Omega multiple alignment program (Rho genes were constructed using RNA-seq datasets of C. gigas (GSE31012), P. yessoensis (SAMN03654043) and C. farreri (unpublished). After trimming, high-quality reads from multiple RNA-seq datasets were mapped onto the deduced bivalve Rho GTPase genes using a TopHat protocol [C. gigas was performed using t-tests , and p values ≤ 0.05 were considered to be statistically significant. Furthermore, RNA-seq datasets from the digestive gland and gills subjected to different environmental stresses in C. gigas, including digestive gland from oysters challenged with heavy metals , gills challenged with heavy metals , salinity (from 5‰ to 40‰), temperature (from 5°C to 35°C) and exposure to air (up to 11 days), as well as adductor muscles challenged with exposure to air, were analyzed. The differential expression levels of CgRhos were analyzed using edgeR packages (http://bioconductor.org/packages/release/bioc/html/edgeR.html) . The detailed methods and the results of the RT-PCR analysis are provided in The expression profiles of bivalve gigas GSE012, P. yprotocol , 33. Theprotocol . A compaeR.html)  Tables. Rho GTPase genes were identified in the genomes of P. yessoensis, C. farreri and C. gigas, respectively  and C. farreri Rhos (CfRhos) genes were submitted to GenBank under accession numbers KT037718 to KT037753. The ORFs of PyRho genes are 576 to 2121 bp in length, encoding 192 to 707 amino acids, and those of CfRho genes are exactly the same as the corresponding PyRho ORFs; CgRho ORFs are 330 to 2088 bp in length, encoding 110 to 696 amino acids. The predicted molecular weights of these Rho proteins range from 21.37 to 81.51 kDa, with PI from 5.39 to 9.51 (Rho members are summarized in Rho genes are longer than the genes in the oyster. The number of exons could be divided into three categories: most of the Rho genes contain relatively few exons (2–7), though nine exons can be found in RhoBTB genes. In addition, the number of exons in Miro genes are 2–3 times (19–20) comparing those of other Rho genes. The length of the first intron in different bivalve Rho genes varies within a range of 247 to 28,375 bp. Analysis of the genomic structure showed that all of the exon-intron boundaries in the Rho genes are consistent with the GT/AG rule for splicing [C. gigas Rhos  with similarities that are slightly lower (33.5%-48.2%); and a 3rd group containing Rnd, RhoBTB and Miro, with the lowest sequence similarities (8.9%-22.6%) of the investigated groups , Rac1-related subfamily , Cdc42-related subfamily , Rnd subfamily , RhoBTB subfamily (RhoBTB1 and RhoBTB2) and Miro subfamily (Miro1 and Miro2)  .C. farreri, P. yessoensis and C. gigas were analyzed to detect the expression patterns of bivalve Rho GTPase genes  and corresponding real-time PCR validations , followed by CgCdc42 (p = 3.73E-06) and CgRac (p = 0.124) duplications. The integrated expression of C. gigas Cdc42-1 was significantly lower than that of CgCdc42-2, and during developmental, the RPKM of CgCdc42-2 ranged from 96.29 to 474.58, approximately tenfold higher than that of CgCdc42-1. Although differences in expression between these two genes was smaller among adult tissues, no Cdc42-1 transcript was detected in male gonads. CgRho-1 was ubiquitously and highly expressed, not only during developmental stages but also in adult tissues, whereas CgRho-2 mRNA was only barely detected in juvenile and adult tissues. The expression patterns of CgRacs were similar, with slightly more CgRac-2 than CgRac-1 transcripts in the mantle and more CgRac-1 than CgRac-2 transcripts in other tissues.Differential expression patterns were further analyzed among C. gigas . In geneCgRhos in response to environmental stresses, RNA-seq datasets from C. gigas treated with salinity or temperature changes, exposure to air and heavy metals were analyzed. CgCdc42-2, CgRac-2, CgRho-2, CgRhoU, CgRhoBTB and CgRnd were found to be sensitive to at least one of these challenges ; however, the overall differences in expression between the two copies of CgRacs were not statistically significant (p > 0.05). A Ka/Ks rate analysis was performed to further explore the potential selection status of these oyster genes (CgCdc42-1/CgCdc42-2 (Ka/Ks = 0) and CgRho-1/CgRho-2 (Ka/Ks = 0.15). In contrast, a high Ka/Ks ratio (Ka/Ks = 2.45) was observed between the duplication of CgRac-1/CgRac-2, which may indicate that the gene has experienced positive selection [C. gigas, the Ks rate of duplicated genes was 0.026 for CgRacs, 0.084 for CgCdc42s and 1.534 for CgRhos, respectively. Newly duplicated CgRac-2 was found with the most incomplete exon-intron (with three exons missing) and protein (with three G-boxes missing) structures, which indicated that it might be functionally incomplete or even be under a pseudogenization trend in the oyster genome. Nonetheless, based on its selection status  and high expression patterns  and the greatest difference in expression  than its duplicated gene  . Previous studies indicate that expanded genes generated by duplication are less likely to have strongly correlated expression profiles than those that remain in one-to-one relationships among species [CgRho genes.In this study, differences in spatiotemporal expression were detected in the duplicated er genes . Accordielection , 51. In election –54, withelection , 56. In ed genes , 58. The species . In addi species . Such da species , consistRho GTPase genes, encompassing Rho, Rac, Mig, Cdc42, Tc10, Rnd, RhoU, RhoBTB and Miro subfamily members, are herein described in three bivalve species, including nine in P. yessoensis, nine in C. farreri and twelve in C. gigas. Our results showed that bivalve Rho genes might represent the most complete set of Rho genes in invertebrates. The scallops exhibit Rho expression patterns similar to those of C. gigas, whereas more Rho mRNAs were found to be expressed in C. farreri and C. gigas than in P. yessoensis. Gene duplications were found in the C. gigas Rho gene family, and duplication pairs with higher Ks ratios displayed greater differences in expression. This is the first genome-wide investigation of Rho GTPase genes in Mollusca, and our findings will assist in a better understanding of the role of Rho GTPases in Mollusca and in elucidating Rho evolutionary history.In summary, a total of thirty S1 FigThe amino acid sequences of Rho GTPases were aligned using the same procedure as that in (TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Figp ≤ 0.05).The temperature of 20°C and salinity of 30‰ (boxed) were used as the controls. ‘*’ represents significantly different gene expression ((TIF)Click here for additional data file.S4 Figp ≤ 0.05).‘*’ represents significantly different gene expression ((TIF)Click here for additional data file.S5 FigC. gigas which have been challenged with heavy metals  for 12 hours and 9 days were used for CgRho gene expression analysis.The digestive gland (DG) and gills from (TIF)Click here for additional data file.S6 FigDG, digestive gland.(TIF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.


Artemisia tridentata ssp. wyomingensis), and three common native grasses , representing short-, medium-, and deep-rooted growth forms, respectively, across an annual rainfall gradient (220–350 mm) in the Great Basin, USA. We hypothesized that positive shrub-grass relationships would become more frequent at lower rainfall levels, as indicated by greater cover of grasses in shrub canopies than vegetation-free interspaces. We sampled aerial cover, density, height, basal width, grazing status, and reproductive status of perennial grasses in canopies and interspaces of 25–33 sagebrush individuals at 32 sites along a rainfall gradient. We found that aerial cover of the shallow rooted grass, P. secunda, was higher in sagebrush canopy than interspace microsites at lower levels of rainfall. Cover and density of the medium-rooted grass, E. elymoides were higher in sagebrush canopies than interspaces at all but the highest rainfall levels. Neither annual rainfall nor sagebrush canopy microsite significantly affected P. spicata cover. E. elymoides and P. spicata plants were taller, narrower, and less likely to be grazed in shrub canopy microsites than interspaces. Our results suggest that exploring sagebrush canopy microsites for restoration of native perennial grasses might improve plant establishment, growth, or survival (or some combination thereof), particularly in drier areas. We suggest that land managers consider the nurse plant approach as a way to increase perennial grass abundance in the Great Basin. Controlled experimentation will provide further insights into the life stage-specific effectiveness and practicality of a nurse plant approach for ecological restoration in this region.As environmental stress increases positive (facilitative) plant interactions often predominate. Plant-plant associations (or lack thereof) can indicate whether certain plant species favor particular types of microsites  and can provide valuable insights into whether “nurse plants” will contribute to seeding or planting success during ecological restoration. It can be difficult, however, to anticipate how relationships between nurse plants and plants used for restoration may change over large-ranging, regional stress gradients. We investigated associations between the shrub, Wyoming big sagebrush ( Plant spatial distributions reflect biological processes and are studied to gain insight into plant-plant interactions –3. AggreBetter understanding of nurse-beneficiary plant relationships may improve establishment of native plants in ecological restoration settings –17, partIn some cases, native species establishment or growth may require facilitation by other plants . A growiBromus tectorum (cheatgrass) [Artemisia tridentata ssp. wyomingensis (Wyoming big sagebrush), the dominant shrub throughout much of this region at low to mid elevations, has been shown to facilitate common perennial grasses in this region [Because plant-plant interactions often shift from positive at high stress to negative at low stress , 27, 28,atgrass) , 32. Arts region . It creas region , mediatis region , and cons region .A. tridentata would be beneficial as a nurse plant for native perennial grasses throughout the Great Basin region (~32 million hectares) [It is not clear, however, if or to what extent ectares)  that recectares) . Field sectares)  and in tectares) , 37, butectares) , 38. OthA. tridentata and perennial grasses could help restoration practitioners determine where, within the Great Basin, A. tridentata nurse plants have the highest potential to improve growth and establishment of native grasses. We focused on three cool-season perennial grass species that are of interest for use in restoration of Great Basin plant communities. In accordance with the stress gradient hypothesis [Understanding how a Great Basin-wide rainfall gradient affects associations between pothesis , we hypoA. tridentata ssp. wyomingensis-dominated areas across the Great Basin, USA ..A. trideingensis .Bromus tectorum. This species is an invasive, widespread plant of great economic and ecological concern in the Great Basin [Artemisia tridentata spp. wyomingensis (Wyoming big sagebrush), a dominant perennial grass understory of Pseudoroegneria spicata (bluebunch wheatgrass) or Achnatherum thurberianum (Thurber’s needlegrass), and a sub-dominant herbaceous understory of Elymus elymoides (squirreltail) and Poa secunda (Sandberg’s bluegrass). We retained 195 sites that met these criteria.We used a database of 826 candidate sites  to identat Basin , and natat Basin . Other sat Basin  to selecBromus tectorum (cheatgrass). We then visited sites that were selected to represent a rainfall gradient in this region, as well as geographic variability across and within MLRAs. Final selection of our 32 field sites was based on field visits confirming minimal B. tectorum invasion and dominance by native perennial grasses , as well as good accessibility (150–600 m from access roads) and evidence of minimal to moderate recent livestock grazing. All sites were located on public land managed by the Bureau of Land Management (BLM), and evidence of livestock grazing was based on field inspections and BLM grazing records. Sites sampled in 2012 (n = 20), 2013 (n = 9), and 2014 (n = 3) ranged in mean annual rainfall from 220 mm to 378 mm  for each of the 195 candidate sites with a Parameter-Elevation Regressions on Independent Slopes Model (PRISM) one of the most widely used and accurate weather models available  for the Poa secunda, Elymus elymoides, and Pseudoroegneria spicata to represent caespitose bunchgrass growth forms most commonly seen in the Great Basin, namely, shallow-rooted tufted [Pseudoroegneria spicata can reach a rooting depth of 200 cm [E. elymoides and P. secunda are 100 cm [Poa secunda and E. elymoides were common across the entire range of rainfall whereas P. spicata was less common, especially in dry sites (Artemisia tridentata ssp. wyomingensis (Wyoming big sagebrush) is a perennial, evergreen shrub that can live over 100 years [We selected three cool-season, native perennial grass species, d tufted , medium-d tufted , and deed tufted . Pseudorf 200 cm , while te 100 cm  and lesse 100 cm , respectry sites . All thr00 years . These s00 years . The roo00 years .A. tridentata ssp. wyomingensis) and three common native perennial grasses  responded to an annual rainfall gradient. In 2012–2014 field sampling occurred between May 15th and July 30th to capture peak herbaceous biomass. We sampled P. secunda at 29 sites, E. elymoides at 27 sites, and P. spicata at14 sites , we sampled transects in each cardinal direction. On each transect the “canopy” zone extended from shrub base to canopy dripline. When this distance was zero (in the case of asymmetric shrubs), no canopy sampling occurred. A shrub interspace was the region between two large shrubs of any species and in our study could be occupied by herbaceous vegetation and/or very small shrubs. The “interspace” zone extended from the canopy dripline to either a) the mid-point between canopy dripline and the nearest neighboring shrub canopy or b) 200 cm if the nearest neighboring shrub was more than 300 cm away. In cases of overlapping canopies only canopy measurements were taken for that transect. Due to the patchy growth form of P. secunda at our study sites, its aerial cover was visually estimated to the nearest 1% in 20 cm x 20 cm quadrats placed in the canopy (midpoint of canopy zone) and interspace (at both midpoint and end of the interspace) microsites . A third microsite (edge), extended from 10 cm inside to 10 cm outside the canopy dripline (canopy and interspace zones were adjusted accordingly). When no canopy existed , the interspace on that transect extended from shrub base to 200 cm or the midpoint to the nearest neighboring shrub canopy (whichever came first).We also assessed E. elymoides and P. spicata plant recorded in our density counts, we also measured its basal width and height. Basal width provides a measure of plant size that is not affected by current year grazing whereas height reflects both plant size and effects of current year grazing and precipitation. For each plant we also recorded evidence of whether it had been grazed (yes/no). We also recorded flowering (yes/no) as an assay of potential for reproduction. For this measure, we randomly selected E. elymoides and P. spicata plants at 10 sites between May 11 and July 1, 2014   . Due to Bromus tectorum, perennial grass, annual forb and perennial forb cover, as well as bare ground with line-point intercept; basal and canopy gap width of perennial vegetation; and densities of cattle dung, perennial grasses, and A. tridentata shrubs in 2m wide belt transects.We characterized general vegetation conditions at each of the 32 field sites along the three 50-m focal shrub selection line transects described above. Following the methods of Herrick et al. , we asseP. secunda; n = 27 for E. elymoides; n = 10 for P. spicata cover; n = 14 for P. spicata density), pooling shrub-level means. Sample sizes differed among species and response variables because cover and density were too low at some sites to be included in our analyses. We used separate  mixed-effects ANCOVA models with interaction terms to model the responses of perennial grasses to canopy vs. interspace microsites over a continuous rainfall gradient. Our response variables for nine main mixed-effects ANCOVA model analyses were site levels means for cover of P. secunda, E. elymoides, and P. spicata; density of E. elymoides and P. spicata; height of E. elymoides and P. spicata; and basal width of E. elymoides and P. spicata. The categorical predictor variable for all analyses was shrub microsite (canopy or interspace), and the continuous covariate was site-level PRISM rainfall estimate. Our model included a fixed microsite*rainfall interaction term, and the random effects were site and site*rainfall. The analysis for E. elymoides and P. spicata density also included “edge”, a third level of shrub microsite. Since preliminary analyses confirmed there was no year effect on percent cover and density of perennial grasses, we combined field data from 2012, 2013, and 2014. All analyses were conducted with SAS 9.3 [We used sites as replicates in our analyses . We also used t-tests to compare canopy and interspace values for perennial grass cover and density values; when canopy > interspace, we inferred facilitation, and when canopy < interspace we inferred competition. We centered rainfall values around a mean of 0 to avoid obtaining negative extrapolative cover values for lower rainfall. To achieve residual homogeneity of variance and assumptions of normality we log-transformed P. spicata and E. elymoides. For each species our response variable was the number of grasses grazed divided by the total number of grasses in each microsite . The model was run using PROC GLIMMIX [E. elymoides and P. spicata grasses differed among canopy, edge, and interspace microsites. The two separate analyses were conducted using PROC LOGISTIC; we applied the Firth adjustment for small sample sizes [E. elymoides and P. spicata, respectively).We used a mixed logistic regression model to determine if the proportion of grazed plants differed among canopy, edge, and interspace sagebrush microsites for  GLIMMIX , and paile sizes . For eacWe examined variables associated with general site conditions to explore broadscale patterns among rainfall and other site-level covariates. We examined bi-variate (Pearson) correlations between biologically meaningful covariates and cover of focal species in canopy and interspace microsites using PROC CORR . We did Our non-destructive sampling did not require special permissions, and our study did not involve any endangered species.P. secunda aerial cover  to medium  rainfall levels . Differences were no longer significant at the 75th percentile of annual rainfall (310 mm) .We found a significant effect of a microsite*rainfall interaction on mean al cover ; S1 FileE. elymoides cover which was significantly greater in canopy versus interspace microsites over all rainfall levels . Elymus elymoides cover did not change significantly over rainfall values in interspace microsites . In contrast to P. secunda and E. elymoides, there was no significant effect of either microsite or rainfall on P. spicata cover  was greater in canopy than interspace microsites across all rainfall levels  followed the opposite pattern; basal width was greatest in interspace microsites, followed by edge and canopy . Mean basal widths generally differed significantly for canopy-interspace and edge-interspace microsite comparisons up to the 90th percentile of rainfall  was significantly affected only by microsite , as well as interspace vs. edge microsites .The proportion of grazed grasses differed significantly among microsites for both E. elymoides or P. spicata  were correlated with rainfall  shifting to more neutral or competitive relationships in mesic conditions . These results, from across a regional rainfall gradient, provide support for the generality of the stress gradient hypothesis [P. secunda and E. elymoides in moisture-limited areas of the Great Basin. Lack of evidence for positive spatial associations between A. tridentata and the third grass species, P. spicata, however, also indicates the importance of considering species-specific relationships [A. tridentata and native perennial grasses under stressful, moisture-limited conditions [We found that cover of two native perennial grasses, pothesis  and the pothesis , 39, 56.ionships . Our resnditions , 37.A. tridentata on cover of the shallow-rooted grass, P. secunda, shifted to neutral, likely due in part to increased competition with A. tridentata and other herbaceous vegetation for soil moisture. Although sub-canopy cover of the medium-rooted grass, E. elymoides, decreased with increasing rainfall, A. tridentata still had a net facilitative effect on E. elymoides cover over almost the entire range of annual rainfall. Cover of the deep-rooted grass, P. spicata, was unaffected by proximity to A. tridentata nurse shrubs due to either a lack of nurse plant effect or low sample size. Our results are consistent with Davies et al. [E. elymoides cover under A. tridentata canopies at a drier site but no differences in sub-canopy P. spicata cover between dry and mesic sites. Our results for all three grass species contrast with those reported by Reisner et al. [P. secunda and E. elymoides in wetter conditions and positive relationships at drier sites for P. spicata. Our study encompassed a greater geographical extent and more site variability than Reisner et al. [We identified distinctly different facilitative patterns for the different grass species. At high rainfall, the net positive effect of s et al. , who four et al.  who founr et al. , which mElymus elymoides cover in sub-canopy microsites was driven by higher plant density in those microsites, rather than bigger plant sizes. Both E. elymoides and P. spicata sub-canopy microsite plants were taller and had smaller basal widths than those growing in edge or interspace microsites. The narrow, tall stature of sub-canopy plants may be a growth response to the increased ratio of far to near-red light found in sub-canopy conditions [Mechanisms section below). Although reduced plant size could reduce fitness, we found that flowering was not reduced in sub-canopy microsites. Together our size and reproduction results suggest that targeting restoration plantings in sub-canopy microsites might yield smaller plants but not at the expense of establishing a self-sustaining  population.Higher nditions , or it cnditions , 60. AltPoa secunda appears to benefit from growing in A. tridentata canopies at lower rainfall levels, perhaps because it is short-rooted and benefits from hydraulic lift from A. tridentata [A. tridentata at higher rainfall. Additionally, the benefits of sub-canopy microsites might be limited except under the driest conditions because P. secunda enters dormancy sooner than most Great Basin perennial grasses, [E. elymoides generally has a deeper root system than P. secunda but continues to grow later into the summer months. As moisture stress increases over the summer, E. elymoides may increasingly benefit from the improved microsite conditions  in shrub sub-canopies, even at higher rainfall levels. The deep-rooted P. spicata, on the other hand, can survive under a very wide range of moisture conditions [identata . Howevergrasses,  enablinggrasses, , 62. In nditions , which mnditions . To clarElymus elymoides, which only reproduces via seed, may be more likely to establish near or under A. tridentata canopies because shrubs can act as seed traps, simultaneously limiting the dispersal of seeds dropped within shrub canopies and trapping windblown seeds at their periphery [E. elymoides were observed in great numbers under sagebrush canopies (MFH pers. obs). In contrast, because P. spicata produces less seed and propagates mainly by tillering [A. tridentata). Small P. secunda seeds may be trapped by microtopography of the soil surface [Reproductive strategies and seed dispersal patterns also may influence plant spatial patterns. For example, eriphery . The larillering , distrib surface  of eitheArtemisia tridentata sub-canopy microsites may provide physical protection against large herbivore grazing [E. elymoides and P. spicata plants, both of which are palatable to livestock [E. elymoides or P. spicata cover or rainfall. But, because perennial grass densities likely decrease with decreasing rainfall , we cannot rule out the possibility that rainfall and relative grazing stress covary. That is, if livestock activity (as measured by dung density) was indeed not lower at lower rainfall sites, there may be greater relative grazing pressure on fewer plants at those drier sites. Therefore, the shrub-grass associations we observed may have been driven in part by associational defense offered to grasses by growing in shrub canopies, away from grazers. We are conducting further work to explore whether canopy-interspace patterns for these two grasses differ between low and high grazing intensity sites.Herbivory also can mediate plant facilitation and even intensify abiotically-driven facilitation , 21. Art grazing , particu grazing . In our ivestock , 50, werP. secunda and E. elymoides plants were associated with A. tridentata canopy microsites, particularly in drier areas. These patterns suggest that exploiting sub-canopy microsites for restoration seedings or plantings in Great Basin sagebrush communities could improve plant establishment, growth, or survival (or some combination thereof) sensu [E. elymoides distributions may be driven at least partially by trapping of seeds and increased propagule pressure in shrub understories, our results clearly show that plants persist to maturity in understory microsites. Future studies examining responses of plants of different growth forms and at multiple life stages would help clarify the mechanisms behind the patterns we observed.We found that naturally occurring f) sensu , 67. AltOur results are most applicable to areas of the Great Basin that contain shrub overstories but lack a robust perennial understory and are at risk of invasion by undesirable annuals. Typically, however, restoration efforts in the Great Basin target areas where sagebrush canopies are no longer intact such as burned areas. Our results highlight the potential utility of shrubs to serve as nurse plants in restoration settings and provide important ecosystem functions.Based on the results of our study we suggest that land managers in the Great Basin should consider the utility of a nurse plant approach as way to improve the resilience of sagebrush plant communities to disturbance, as well as resistance to invasive species (sensu ). To traS1 TableArtemisia tridentata ssp. wyomingensis.PRISM rainfall refers to annual rainfall values predicted by PRISM data . ESD rai(DOCX)Click here for additional data file.S1 FigP. secunda. 1) Canopy: quadrat placed at approximate midpoint of canopy region; 2) Interspace: quadrat placed at approximate midpoint of transect; 3) Interspace: quadrat placed at end of transect.Numbers correspond to placement of 20 cm x 20 cm quadrats for estimating percent cover of (DOCX)Click here for additional data file.S1 FileMeans were been averaged over shrub-level (subsample) data values.(DOCX)Click here for additional data file.S2 FileP values test the null hypotheses that Pearson’s pairwise correlations between 2 variables = 0 versus the alternative hypothesis that Pearson’s pairwise correlations between 2 variables ≠ 0.(DOCX)Click here for additional data file.