Primer Bias in Finding Fungal Diversity in an Artificial Biome

Studying soil microbial diversity in tropical ecosystems is of high importance to understanding global nutrient cycling. Soil microbes, as decomposers, are primary contributors to terrestrial nutrient cycling. In tropical systems, warm, moist conditions create ideal environments for microbial growth year-round. However, the ability to study these topics in the field is limited by the remoteness and ruggedness of many tropical forest locations, combined with the highly changeable and difficult-to-control conditions present in the field. To that end, proxy study systems have been created that allow researchers to approximate natural conditions, but with better accessibility in terms of location and controllability. / Biosphere 2 is a facility owned by the University of Arizona dedicated to recreating several ecosystems. Among the ecosystems present in this environment are examples of several tropical rainforest biomes, including both lowland, deep-soil forest, as well as highland-type, focusing primarily on Neotropical species. Thus, it is an ideal place to carry out studies on topics like soil nutrient cycling that would be challenging in a natural rainforest biome./ Young et al. 2019 recently did a study where they examined N2O production and microbial community composition and dynamics of the rainforest ecosystem in Biosphere 2 during the dry season, profiling the metabolic and genetic backgrounds of the bacterial and archaeal species present in the soil. However, a similar treatment was not done for the fungi of the soil, leaving their contribution unexamined. The soil fungal biota of the Biosphere 2 itself is not well-understood, and generally consists of whatever fungal symbionts and phoresy that was brought in with the construction of the soil and floral communities. / While fungal sequences from these samples exist, sequencing was completed using two different sets of primers: one is the standard for ITS sequences, and another set was designed by the Joint Genome Institute (JGI), but may be biased against particular taxonomic groups. / In order to better understand the soil fungal communities in the Biosphere 2 rainforest biome, and determine if there are significant sources of bias in the primer sets used, I will analyze the data using several R statistical analysis packages. The sequences will be denoised and cleaned using the DADA2 package. I will use a sequencing dataset built from an artificial mock community of known species (U’ren et al .2019) to test for bias in the primers of both the standard and JGI varieties. Next, I will obtain the standard ITS data from the author, as well as the JGI-primer ITS data from the JGI open-access data portal. Then, the Phyloseq data package will be used to create illustrative graphs to visualize the data from these sources to illustrate the phylogenetic data relevant to the study from both genomic datasets, with the artificial community serving as a control. To examine the diversity present in the data, genetically distinct OUT clusters will be created using the UPARSE-OTU algorithm. After this, the putative OTUs will be filtered through the ITSx software to remove any sequences not bearing the ITS2 region. Afterwards, the remaining sequences will be identified using BLAST against existing sequence databases. From these identified sequences, trees that graphically illustrate the diversity can be created using the tree-based alignment selector toolkit (T-BAS). From these data, both the selection bias of the JGI ITS2 primers as well as the overall diversity of the Biosphere 2 fungal community can be illustrated. / Overall, this study will help increase our understanding of both the ecology of soil fungi in the rainforest biome of Biosphere 2, as well as how the JGI primer set compares in genetic bias to standard primer sets

Johtamiskulttuurin kehittäminen valmennusprosessina: Case tutkimus Balentor Oy

Tässä tutkimuksessa tarkastellaan johtamiskulttuuria ja miten sitä voidaan kehittää ulkoisen valmennusprosessin avulla. Tutkimuksessa kuvataan, kuinka johtamiskulttuuri koostuu johtamisen rakenteista ja käytänteistä sekä johtamiseen liittyvistä uskomuksista ja asenteista. Johtamiskulttuuri vaikuttaa laajasti organisaation toimintoihin sekä menestymiseen, joten sen kehittäminen on tärkeää organisaation kilpailukyvyn parantamiseksi. Tutkimuksen taustalla toimii ajatus, että ulkopuolisena tarkkailijana yritysvalmentaja on erityisasemassa ja voi tarkastella kohdeorganisaatiota objektiivisesti sekä räätälöidä valmennusta asiakkaan tarpeiden mukaan ja näin ollen olla hyvä kumppani johtamiskulttuurin kehittämisessä. Tutkimuksessa tarkastelun alla ovat johtamiskulttuurin valmennusprosessin eri vaiheet sekä niihin liittyvät keskeiset asiat. Tutkimuksen tarkoituksena oli luoda johtamiskulttuurin valmentamisen malli, mikä avaa valmennusprosessin vaiheita ja niihin liittyviä asioita prosessimaiseen kaavioon. Tavoitteena oli analysoida johtamiskulttuurin valmennusprosessia ja selvittää tarkemmin, mitkä asiat ovat keskeisiä valmennusprosessin eri vaiheiden kannalta sekä miten näitä asioita voidaan hoitaa. Tämä tutkimus toteutettiin laadullisena case-tutkimuksena, missä kohdeorganisaatio oli Balentor Oy. Balentor Oy toimii organisaatioiden kehityskumppanina ja muutoksen vauhdittajana. Empiirinen aineisto kerättiin haastattelemalla kolmea Balentor Oy:n työntekijää. Kaksi haastateltavaa työskentelevät yritysvalmentajina ja yksi työskentelee tutkimusjohtajana. Haastattelut toteutettiin teemahaastatteluina ja niitä varten muodostettiin haastattelurunko, minkä mukaan käytiin johtamiskulttuurin valmennusprosessin vaiheita järjestelmällisesti. Kerätty aineisto analysoitiin teoriaohjaavalla sisällönanalyysillä ja se esitetään taulukossa, johon kerättiin alaluokat, yläluokat, pääluokat ja kokoava käsite. Tutkimuksen tulokseksi laadittiin johtamiskulttuurin valmennusprosessin malli. Tulokset osoittivat, että johtamiskulttuuria voidaan kehittää selkeästi rakennetun valmennusprosessin avulla. Valmennusprosessissa korostuu johtamiskulttuurin tavoitetilan määrittäminen, johtamiskulttuurin nykytilan kartoittaminen suhteessa tavoitetilaan ja muutoksen toteuttamisen toimenpiteet. Empiirinen aineisto toi kirjallisuudesta eroavaa tietoa esiin. Näin ollen valmennusprosessin mallia varten yhdisteltiin kirjallisuutta ja empiiristä aineistoa. Empiirisen aineiston pohjalta yritysvalmennuksessa korostuu sen prosessinomaisuus, jotta osaaminen ja vastuu saadaan siirrettyä organisaatioon järjestelmällisesti. Tutkimus avaa johtamiskulttuurin valmennusprosessia ja esittää syventävää tietoa prosessin eri vaiheista. Kaaviota voidaan hyödyntää valmennusprosessin suunnittelussa ja läpiviennissä sekä sillä voidaan esittää asiakkaalle, että mitä valmennuskonseptilla tarkoitetaan ja mitä sillä voidaan tehdä

Thermoelectric magnetohydrodynamic control of melt pool dynamics and microstructure evolution in additive manufacturing

Large thermal gradients in the melt pool from rapid heating followed by rapid cooling in metal additive manufacturing generate large thermoelectric currents. Applying an external magnetic field to the process introduces fluid flow through thermoelectric magnetohydrodynamics. Convective transport of heat and mass can then modify the melt pool dynamics and alter microstructural evolution. As a novel technique, this shows great promise in controlling the process to improve quality and mitigate defect formation. However, there is very little knowledge within the scientific community on the fundamental principles of this physical phenomenon to support practical implementation. To address this multiphysics problem that couples the key phenomena of melting/solidification, electromagnetism, hydrodynamics, heat and mass transport, the lattice Boltzmann method for fluid dynamics was combined with a purpose-built code addressing solidification modelling and electromagnetics. The theoretical study presented here investigates the hydrodynamic mechanisms introduced by the magnetic field. The resulting steady-state solutions of modified melt pool shapes and thermal fields are then used to predict the microstructure evolution using a cellular automata based grain growth model. The results clearly demonstrate that the hydrodynamic mechanisms and, therefore, microstructure characteristics are strongly dependent on magnetic field orientation

Study of thermoelectric magnetohydrodynamic convection on solute redistribution during laser additive manufacturing

Melt pools formed in laser additive manufacturing (AM) are subject to large thermal gradients, resulting in the formation of thermoelectric currents due to the Seebeck effect. When in the presence of an external magnetic field, a Lorentz force is formed which drives fluid flow in the melt pool. This Thermoelectric Magnetohydrodynamics (TEMHD) phenomenon, can have a significant impact on the melt pool morphology and can alter the microstructural evolution of the solidification process. By coupling steady-state mesoscopic melt pool calculations to a microscopic solidification model, predictions of the resulting microstructure for multiple deposited layers have been obtained. The results indicate that the magnetic field can have a transformative effect on the microstructure and solute redistribution. This study highlights the theoretical potential for using magnetic fields as an additional control system to tailor AM microstructures

Mass spectrometry assays of plasma biomarkers to predict radiographic progression of knee osteoarthritis

Introduction: Biomarkers to identify osteoarthritis (OA) patients at risk for disease progression are needed. As part of a proteomic analysis of knee synovial fluid from normal and OA patients, differentially expressed proteins were identified that could represent potential biomarkers for OA. This study aimed to use mass spectrometry assays to identify representative peptides from several proteins in synovial fluid and peripheral blood, and assess their levels as biomarkers of OA progression. Methods: Multiplexed high throughput selected reaction monitoring (SRM) assays were developed to measure tryptic peptides representative of 23 proteins in matched serum and synovial fluid samples from late OA subjects at the time of joint replacement. Subsequently plasma samples from the baseline visit of 173 subjects in an observational OA cohort were tested by SRM for peptides from nine of these proteins: afamin, clusterin, cartilage oligomeric matrix protein, hepatocyte growth factor, kallistatin, insulin-like growth factor binding protein, acid labile subunit, lubricin, lumican, and pigment epithelium-derived factor. Linear regression was used to determine the association between the peptide biomarker level at baseline and change in joint space width (ΔJSW) from baseline to 30 months, adjusting for age and sex. Results: In the matched cohort, 17 proteins could be identified in synovial fluid and 16 proteins were detected in serum. For the progression cohort, the average age was 62 and average ΔJSW over 30 months was 0.68 mm. A high correlation between different peptides from individual proteins was observed, indicating our assays correctly measured their target proteins. Peptides representative of clusterin, lumican and lubricin showed statistically significant associations with joint space narrowing after adjustment for age and sex. Partial R2 values showed clusterin FMETVAEK and lubricin LVEVNPK peptide biomarkers explains about 2 to 3% of the variability of ΔJSW, similar to that explained by age. A biomarker score combining normalized data for both lubricin and clusterin peptides increased the model R2 to 0.079. Conclusions: Our results suggest that when combined, levels of peptides representative of clusterin and lubricin in plasma are as predictive of OA progression as age. Replication of these findings in other prospective OA cohorts is planned. Electronic supplementary material The online version of this article (doi:10.1186/s13075-014-0456-6) contains supplementary material, which is available to authorized users

Proteins other than the locus of enterocyte effacement-encoded proteins contribute to Escherichia coli O157:H7 adherence to bovine rectoanal junction stratified squamous epithelial cells

Background: In this study, we present evidence that proteins encoded by the Locus of Enterocyte Effacement (LEE), considered critical for Escherichia coli O157 (O157) adherence to follicle-associated epithelial (FAE) cells at the bovine recto-anal junction (RAJ), do not appear to contribute to O157 adherence to squamous epithelial (RSE) cells also constituting this primary site of O157 colonization in cattle. Results: Antisera targeting intimin-γ, the primary O157 adhesin, and other essential LEE proteins failed to block O157 adherence to RSE cells, when this pathogen was grown in DMEM, a culture medium that enhances expression of LEE proteins. In addition, RSE adherence of a DMEM-grown-O157 mutant lacking the intimin protein was comparable to that seen with its wild-type parent O157 strain grown in the same media. These adherence patterns were in complete contrast to that observed with HEp-2 cells (the adherence to which is mediated by intimin-γ), assayed under same conditions. This suggested that proteins other than intimin-γ that contribute to adherence to RSE cells are expressed by this pathogen during growth in DMEM. To identify such proteins, we defined the proteome of DMEM-grown-O157 (DMEM-proteome). GeLC-MS/MS revealed that the O157 DMEM-proteome comprised 684 proteins including several components of the cattle and human O157 immunome, orthologs of adhesins, hypothetical secreted and outer membrane proteins, in addition to the known virulence and LEE proteins. Bioinformatics-based analysis of the components of the O157 DMEM proteome revealed several new O157-specific proteins with adhesin potential. Conclusion: Proteins other than LEE and intimin-γ proteins are involved in O157 adherence to RSE cells at the bovine RAJ. Such proteins, with adhesin potential, are expressed by this human pathogen during growth in DMEM. Ongoing experiments to evaluate their role in RSE adherence should provide both valuable insights into the O157-RSE interactions and new targets for more efficacious anti-adhesion O157 vaccines

Modulating Meltpool Dynamics and Microstructure using Thermoelectric Magnetohydrodynamics in Additive Manufacturing

Meltpool modulation in Selective Laser Remelting Additive Manufacturing via an oscillating magnetic field generates Thermoelectric Magnetohydrodynamics (TEMHD) flow. Numerical predictions show that the resulting microstructure can be significantly altered. A multi-scale numerical model captures the meso-scale melt pool dynamics coupled to microscale solidification showing the microstructure evolution and solute redistribution. The results highlight the complex interaction of the various physical phenomena and also show the method's potential to disrupt the epitaxial growth defect. The model predictions are supported by preliminary experimental results that demonstrate the dependency of the melt pool depth on magnetic field orientation. The results highlight how a time-dependent field has the potential to provide an independent control mechanism to tailor microstructures

Moment-based boundary conditions for straight on-grid boundaries in three dimensional lattice Boltzmann simulations

In this article, moment‐based boundary conditions for the lattice Boltzmann method are extended to three dimensions. Boundary conditions for velocity and pressure are explicitly derived for straight on‐grid boundaries for the D3Q19 lattice. The method is compared against the bounce‐back scheme using both single and two relaxation time collision schemes. The method is verified using classical benchmark test cases. The results show very good agreement with the data found in the literature. It is confirmed from the results that the derived moment‐based boundary scheme is of second‐order accuracy in grid spacing and does not produce numerical slip, and therefore offers a transparent way of accurately prescribing velocity and pressure boundaries that are aligned with grid points in three‐dimensional

High abundance synovial fluid proteome: distinct profiles in health and osteoarthritis

The development of increasingly high-throughput and sensitive mass spectroscopy-based proteomic techniques provides new opportunities to examine the physiology and pathophysiology of many biologic fluids and tissues. The purpose of this study was to determine protein expression profiles of high-abundance synovial fluid (SF) proteins in health and in the prevalent joint disease osteoarthritis (OA). A cross-sectional study of 62 patients with early OA (n = 21), patients with late OA (n = 21), and control individuals (n = 20) was conducted. SF proteins were separated by using one-dimensional PAGE, and the in-gel digested proteins were analyzed by electrospray ionization tandem mass spectrometry. A total of 362 spots were examined and 135 high-abundance SF proteins were identified as being expressed across all three study cohorts. A total of 135 SF proteins were identified. Eighteen proteins were found to be significantly differentially expressed between control individuals and OA patients. Two subsets of OA that are not dependent on disease duration were identified using unsupervised analysis of the data. Several novel SF proteins were also identified. Our analyses demonstrate no disease duration-dependent differences in abundant protein composition of SF in OA, and we clearly identified two previously unappreciated yet distinct subsets of protein profiles in this disease cohort. Additionally, our findings reveal novel abundant protein species in healthy SF whose functional contribution to SF physiology was not previously recognized. Finally, our studies identify candidate biomarkers for OA with potential for use as highly sensitive and specific tests for diagnostic purposes or for evaluating therapeutic response