Transcriptomics Reveals the Putative Mycoparasitic Strategy of the Mushroom Entoloma abortivum on Species of the Mushroom Genus Armillaria

During mycoparasitism, a fungus—the host—is parasitized by another fungus—the mycoparasite. The genetic underpinnings of these relationships have been best characterized in ascomycete fungi. However, within basidiomycete fungi, there are rare instances of mushroom-forming species parasitizing the reproductive structures, or sporocarps, of other mushroom-forming species, which have been rarely investigated on a genetic level. One of the most enigmatic of these occurs between Entoloma abortivum and species of Armillaria, where hyphae of E. abortivum are hypothesized to disrupt the development of Armillaria sporocarps, resulting in the formation of carpophoroids. However, it remains unknown whether carpophoroids are the direct result of a mycoparasitic relationship. To address the nature of this unique interaction, we analyzed gene expression of field-collected Armillaria and E. abortivum sporocarps and carpophoroids. Transcripts in the carpophoroids are primarily from E. abortivum, supporting the hypothesis that this species is parasitizing Armillaria. Most notably, we identified differentially upregulated E. abortivum b-trefoil-type lectins in the carpophoroid, which we hypothesize bind to Armillaria cell wall galactomannoproteins, thereby mediating recognition between the mycoparasite and the host. The most differentially upregulated E. abortivum transcripts in the carpophoroid code for oxalate decarboxylases—enzymes that degrade oxalic acid. Oxalic acid is a virulence factor in many plant pathogens, including Armillaria species; however, E. abortivum has evolved a sophisticated strategy to overcome this defense mechanism. The number of gene models and genes that code for carbohydrate-active enzymes in the E. abortivum transcriptome was reduced compared to other closely related species, perhaps as a result of the specialized nature of this interaction

The English Language Learner Program at Burns Latino Studies Academy: Why Does it Fall Short in Servicing Students?

Burns Latino Studies Academy has been labeled an “at risk” institution frequently reported on by the media. Inadequate funding for English Language Learners (ELL) is just one of the shortcomings cited, and has resulted in the school receiving extra support in the wake of a 2007 lawsuit filed by the Center for Children’s Advocacy. Since then, Burns has secured both additional ELL funding and a new ELL coach. However, a number of challenges remain. The current study asks the question: Why does Burns Latino Studies Academy’s bilingual program fall short of fully serving the needs of its ELL students? Employing a qualitative research approach, it provides a more comprehensive narrative of the many institutional barriers present at Burns. In-depth interviews with Principal Brase, three early childhood education teachers, and the recently hired ELL coach, along with classroom observations, point to two key factors, the state of Burns’ bilingual classrooms and the quality of interactions with students identified as English Language Learners. Preliminary findings suggest both the need to provide greater individual attention to “new arrival” students and the need to enhance ELL instructional technology in order to improve bilingual literacy. Based on these findings, the current study recommends that Burns Academy pair with family care centers in the community. It has also identified free online resources to assist in communication with ELL students and a potential private donor of new computers for the targeted student group at Burns Academy. Lastly, a donation from the company, Techroot, has been secured to replace the school’s recently destroyed ELL instructional equipment, and the research team is currently working with Trinity College’s La Voz Latina student group to act as an outreach and mentoring resource for ELL students at Burns Academy

Design and Characterization of a Microfluidic Flow Cytometer

The development and characterization of a microfluidic flow cytometer is described in this dissertation for rapid, inexpensive fluorescence and scatter based cellular enumeration for biomedical diagnostic and monitoring applications. Microfluidic cytometry enables a portable, economical platform for performing point-of-care cellular assays that are impractical with large conventional flow cytometry systems. Microchips were fabricated from glass using standard photolithography and wet chemical etching techniques. These chips were used in conjunction with static optical and electronic components to construct a robust instrument capable of accepting disposable microchips. Flow cytometry measurements on-chip have demonstrated cellular analysis at rates greater than 2 kHz using vacuum driven flow, and positive pressure driven flow cytometry has been performed to measure cells at 12 kHz. Measurement precision has been investigated using commercially available calibration beads, and results demonstrate that the precision of chip based cytometry measurements is close to that seen using conventional instrumentation. Cell discrimination based on immunophenotyping has been demonstrated in mixtures of cultured cells, peripheral blood mononuclear cells, and whole blood to demonstrate the applicability of this instrument for clinical cellular diagnostic and monitoring applications. Comparison studies have been performed using conventional cytometry instrumentation to assess the accuracy of our microfluidic system for immunophenotyping applications. Efforts have also been made to incorporate erythrocyte lysis on-chip in order to rapidly analyze whole blood in an automated fashion. Chemical erythrocyte lysis followed by leukocyte cytometry has been integrated on a single monolithic device to demonstrate the ability to accurately measure cell subtype ratios from a whole blood sample while reducing off-chip sample processing steps. Further, applications in blood dosimetry are described to reveal potential advantages of performing rapid blood cell counts for therapeutic monitoring and bioterrorism applications using only a fingerstick of whole blood. The technology described in this dissertation may be applied to numerous applications requiring the measurement of relative cell populations from biological fluids. Further development and optimization of this instrument has the potential to increase the accessibility of clinical cellular analyses in resource poor settings by providing rapid, low-volume, inexpensive clinical testing at the point-of-care.Doctor of Philosoph

Synchronous Chip-to-Chip Communication with a Multi-Chip Resonator Clock Distribution Network

Superconducting digital circuits are a promising approach to build packaged-level integrated systems with high energy-efficiency and computational density. In such systems, performance of the data link between chips mounted on a multi-chip module (MCM) is a critical driver of performance. In this work we report a synchronous data link using Reciprocal Quantum Logic (RQL) enabled by resonant clock distribution on the chip and on the MCM carrier. The simple physical link has only four Josephson junctions and 3 fJ/bit dissipation, including a 300 W/W cooling overhead. The driver produces a signal with 35\,GHz analog bandwidth and connects to a single-ended receiver via 20 $\Omega$ Nb Passive Transmission Line (PTL). To validate this link, we have designed, fabricated and tested two 32$\times$32 mm$^2$ MCMs with eight 5$\times$5 mm$^2$ chips connected serially and powered with a meander clock, and with four 10$\times$10 mm$^2$ chips powered with a 2 GHz resonant clock. The meander clock MCM validates performance of the data link components, and achieved 5.4 dB AC bias margin with no degradation relative to individual chip test. The resonator MCM validates synchronization between chips, with a measured AC bias margin up to 4.8 dB between two chips. The resonator MCM is capable of powering circuits of 4 million Josephson junctions across the four chips with a projected 10 Gbps serial data rate.Comment: 8 pages, 8 figure

Improving Taxonomic Delimitation of Fungal Species in the Age of Genomics and Phenomics

Species concepts have long provided a source of debate among biologists. These lively debates have been important for reaching consensus on how to communicate across scientific disciplines and for advancing innovative strategies to study evolution, population biology, ecology, natural history, and disease epidemiology. Species concepts are also important for evaluating variability and diversity among communities, understanding biogeographical distributions, and identifying causal agents of disease across animal and plant hosts. While there have been many attempts to address the concept of species in the fungi, there are several concepts that have made taxonomic delimitation especially challenging. In this review we discuss these major challenges and describe methodological approaches that show promise for resolving ambiguity in fungal taxonomy by improving discrimination of genetic and functional traits. We highlight the relevance of eco-evolutionary theory used in conjunction with integrative taxonomy approaches to improve the understanding of interactions between environment, ecology, and evolution that give rise to distinct species boundaries. Beyond recent advances in genomic and phenomic methods, bioinformatics tools and modeling approaches enable researchers to test hypothesis and expand our knowledge of fungal biodiversity. Looking to the future, the pairing of integrative taxonomy approaches with multi-locus genomic sequencing and phenomic techniques, such as transcriptomics and proteomics, holds great potential to resolve many unknowns in fungal taxonomic classification

Improving Taxonomic Delimitation of Fungal Species in the Age of Genomics and Phenomics

Species concepts have long provided a source of debate among biologists. These lively debates have been important for reaching consensus on how to communicate across scientific disciplines and for advancing innovative strategies to study evolution, population biology, ecology, natural history, and disease epidemiology. Species concepts are also important for evaluating variability and diversity among communities, understanding biogeographical distributions, and identifying causal agents of disease across animal and plant hosts. While there have been many attempts to address the concept of species in the fungi, there are several concepts that have made taxonomic delimitation especially challenging. In this review we discuss these major challenges and describe methodological approaches that show promise for resolving ambiguity in fungal taxonomy by improving discrimination of genetic and functional traits. We highlight the relevance of eco-evolutionary theory used in conjunction with integrative taxonomy approaches to improve the understanding of interactions between environment, ecology, and evolution that give rise to distinct species boundaries. Beyond recent advances in genomic and phenomic methods, bioinformatics tools and modeling approaches enable researchers to test hypothesis and expand our knowledge of fungal biodiversity. Looking to the future, the pairing of integrative taxonomy approaches with multi-locus genomic sequencing and phenomic techniques, such as transcriptomics and proteomics, holds great potential to resolve many unknowns in fungal taxonomic classification

The application of the skin virome for human identification

The use of skin virome offers a unique approach for human identification purposes in instances where a viable and statistically relevant human DNA profile is unavailable. The skin virome may act as an alternative DNA profile and/or an additional form of probative genetic material. To date, no study has attempted to investigate the human virome over a time series across various physical locations of the body to identify its diagnostic potential as a tool for human identification. For this study, we set out to evaluate the stability, diversity, and individualization of the human skin virome. An additional goal was to identify putative viral signatures that can be used in conjunction with traditional forensic STR loci. In order to accomplish this, human viral metagenomes were collected and sequenced from 42 individuals at three anatomical locations (left hand, right hand, and scalp) across multiple collection periods over a 6-month window of time. Assembly dependent and independent bioinformatic approaches, along with a database centered assessment of viral identification, resulted in three sets of stable putative viral markers. In total, with the three sets combined, we identified 59 viral biomarker regions, consisting of viral species and uncharacterized viral genome assemblies, that were stable over the sampling period. Additionally, we found the abundance profiles of these 59 viral biomarkers, based on presence or absence, to be significantly different across subjects (P \u3c 0.001). Here we demonstrate that not only is the human virome applicable to be used for human identification, but we have identified many viral signatures that can putatively be used for forensic applications, thus providing a foundation to the novel field of forensic virology

Poplar Allene Oxide Synthase 1 Gene Promoter Drives Rapid and Localized Expression by Wounding

Promoters play critical roles in controlling the transcription of genes and are important as tools to drive heterologous expression for biotechnological applications. In addition to core transcription factor-binding motifs that assist in the binding of RNA polymerases, there are specific nucleotide sequences in a promoter region to allow regulation of gene expression. The allene oxide synthase (AOS) gene family are cytochrome P450s that are responsive to a variety of environmental stress, making them good candidates for the discovery of inducible promoters. Populus AOS homologs separate phylogenetically into two clades. Based on the 19 promoter motifs with significant abundance differences between the two clades, Clade I AOS genes are likely more responsive to hormones, salt, and pathogen, whereas clade II homologs are likely inducible by water stress. In this study, an upstream promoter from a Clade I poplar AOS encoding gene (AOS1) was cloned and used to drive the expression of a ß-glucuronidase (GUS) gene in Arabidopsis. AOS is an essential enzyme in the lipoxygenase pathway that is responsible for the production of many nonvolatile oxylipins in plants, including the jasmonates, which are regulatory phytohormones coordinating a variety of biological and stress response functions. Consistent with AOS transcript expression patterns, we found that the poplar AOS1 promoter drives rapid and localized expression by wounding. The study provides insight on the responsive elements in the poplar AOS promoters, but more importantly identifies a strong wound-inducible and localized promoter for future applications. Key Message: Populus AOSs separate phylogenetically into two clades, which show significant abundance differences in 19 promoter motifs. * AOS1 is predominantly expressed in growing vascular tissue in Populus * Populus AOS1 promoter drives rapid and localized expression by wounding in Arabidopsis

Root and crown rot pathogens found on dry beans grown in Mozambique

Dry edible beans are a vital food source in Mozambique, East Africa—one that alleviates hunger and malnutrition and adds value to the economy. In recent years, root/crown rot (RCR) pathogens have emerged as limiting constraints in dry bean production. Not much has been characterized concerning the causal agents of RCR in Mozambique. The purpose of this study was to identify the primary pathogen(s) associated with RCR dry bean samples collected at breeder nursery sites and farmer fields in Mozambique using molecular sequencing and culture-based methods. Sequencing revealed, not surprisingly, an increased diversity of fungal/oomycete operational taxonomic units when compared to culture-based methods of diversity. Species of Fusarium, mainly F. oxysporum, were the dominant taxa detected in RCR dry beans through sequencing the ITS rDNA region and partial EF-1α gene. Collectively, 333 fungi and/or Oomycetes were isolated in culture during the 2014–2015 growing seasons and tested for pathogenicity on healthy bean seedlings. Fusarium species were identified by both morphological and molecular characters. At least 60% of the isolates inoculated on common bean were recognized as potentially pathogenic. From both isolation frequency and pathogenicity testing, F. oxysporum and related species play an important role in the bean RCR complex. We found similar results from dry beans grown in the two main bean-growing regions of Mozambique. These findings will allow breeders to screen for resistance to F. oxysporum in greenhouse grown bean plants as well as within field grown bean cultivars