Molecular Differentiation of Astragalus Species and Varieties from the Western United States: The Chloroplast DNA Bridge Between Evolution and Molecular Systematics

Locoweeds are the most widespread poisonous plant problem in the world and have been reported in the Western United States since the 1800s, causing tremendous losses in livestock. Consumption of locoweeds by grazing animals stimulates the neurological disease, locoism, characterized by weight loss, ataxia, and lack of muscular coordination. The name locoweed is used for Astragalus and Oxytropis species known to contain swainsonine, the toxic principle produced by the plant endophytic fungus Undifilum. Astragalus includes 2,500-3,000 species and many varieties that have almost identical morphological characteristics that overlap among species, leading to improper identification. Therefore, the aim of this study was to develop effective markers for differentiation of Astragalus at low taxonomic level. Genomic DNA extraction, followed by PCR amplification were carried out with species and varieties of Astragalus. Primers targeted regions from the chloroplast genome (DE, trnH-psbA, rpl32-trnL) and the nuclear genome from the plants, (ITS1-ITS4) and phylogenetic trees were generated. Sequences from the chloroplast genome of Astragalus species and populations within a species had sufficient variation to support taxonomic differentiation at intraspecific and interspecific levels. The noncoding rpl32-trnL region of the chloroplast genome represent more specific differentiation of populations within a species than other chloroplast noncoding regions tested in this study, due to high numbers of potentially informative characteristics. All primer sets used in this study successfully amplified across species and varieties of Astragalus, and their combined sequences constructed well supported concatenated phylogenetic tree. This molecular systematics study aimed to provide a reliable plant identification system using high-resolution molecular markers at low taxonomic levels, to investigate the evolutionary diversification of locoweed, to improve understanding of the plant-fungal relationship, and ultimately, benefit management of locoweed

Field Evaluation of Two External Attachment Location of Radio Transmitters on Non-Venomous Rat Snakes (elaphe obsolete)

The objective of this study was to determine the effectiveness of external attachment of radio transmitters at one of two locations on mature rat snakes (Elaphe obsolete). Transmitters were attached to mature snakes (n = 10; 136.7cm ± 6.4) on either the ventral surface (n = 5) or dorsal-lateral surface of the rib cage (n = 5), approximately 25cm cranially to the cloaca. Transmitters (18mm × 8mm × 2mm) were attached by one drop of acrylamide gel glue to the adhering side of camouflage duct tape (20cm × 30mm), a single drop of glue on the exposed side of the transmitter, and secured by wrapping the tape to the snake’s body with the transmitter in the appropriate location. A second piece of duct tape (20cm × 40mm) overlapped and secured the first piece of tape with the transmitter. Snakes were placed in a 0.1ha plastic fence enclosure, (128.5cm ± 0.5 height, and 17.1° ± 0.5 inward slope) in an unimproved pasture with numerous hides, water and food. Snakes (n = 4) shed their skin and the transmitter, within 6- 17d post-attachment (11.7d ± 2.4). Snakes (n = 4) escaping the enclosure and not located, ranged from 1- 21d post-attachment, (12.3d ± 4.7). There was no difference (p \u3e 0.05) in functional days snakes were located by radio telemetry due to attachment site or sex. Transmitter reception distance was typically \u3c 50m and often problematic. Results of this study suggest that the location of external attachment of transmitters had no influence on duration of effectiveness. However, shedding and limited telemetry range under these conditions should be considered to determine if the methodology is appropriate for the desired objectives

Genetic Relationships in the Toxin-Producing Fungal Endophyte, \u3ci\u3eAlternaria oxytropis\u3c/i\u3e Using Polyketide Synthase and Non-Ribosomal Peptide Synthase Genes

The legume Oxytropis sericea hosts a fungal endophyte, Alternaria oxytropis, which produces secondary metabolites (SM), including the toxin swainsonine. Polyketide synthase (PKS) and non-ribosomal peptide synthase (NRPS) enzymes are associated with biosynthesis of fungal SM. To better understand the origins of the SM, an unannotated genome of A. oxytropis was assessed for protein sequences similar to known PKS and NRPS enzymes of fungi. Contigs exhibiting identity with known genes were analyzed at nucleotide and protein levels using available databases. Software were used to identify PKS and NRPS domains and predict identity and function. Confirmation of sequence for selected gene sequences was accomplished using PCR. Thirteen PKS, 5 NRPS, and 4 PKS-NRPS hybrids were identified and characterized with functions including swainsonine and melanin biosynthesis. Phylogenetic relationships among closest amino acid matches with Alternaria spp. were identified for seven highly conserved PKS and NRPS, including melanin synthesis. Three PKS and NRPS were most closely related to other fungi within the Pleosporaceae family, while five PKS and PKS-NRPS were closely related to fungi in the Pleosporales order. However, seven PKS and PKS-NRPS showed no identity with fungi in the Pleosporales or the class Dothideomycetes, suggesting a different evolutionary origin for those genes

Role of Sequence and Structure of the Hendra Fusion Protein Fusion Peptide in Membrane Fusion

Viral fusion proteins are intriguing molecular machines that undergo drastic conformational changes to facilitate virus-cell membrane fusion. During fusion a hydrophobic region of the protein, termed the fusion peptide (FP), is inserted into the target host cell membrane, with subsequent conformational changes culminating in membrane merger. Class I fusion proteins contain FPs between 20 and 30 amino acids in length that are highly conserved within viral families but not between. To examine the sequence dependence of the Hendra virus (HeV) fusion (F) protein FP, the first eight amino acids were mutated first as double, then single, alanine mutants. Mutation of highly conserved glycine residues resulted in inefficient F protein expression and processing, whereas substitution of valine residues resulted in hypofusogenic F proteins despite wild-type surface expression levels. Synthetic peptides corresponding to a portion of the HeV F FP were shown to adopt an α-helical secondary structure in dodecylphosphocholine micelles and small unilamellar vesicles using circular dichroism spectroscopy. Interestingly, peptides containing point mutations that promote lower levels of cell-cell fusion within the context of the whole F protein were less α-helical and induced less membrane disorder in model membranes. These data represent the first extensive structure-function relationship of any paramyxovirus FP and demonstrate that the HeV F FP and potentially other paramyxovirus FPs likely require an α-helical structure for efficient membrane disordering and fusion

Proteomic analysis of the endophytic fungus Undifilum oxytropis

The filamentous Ascomycete fungus Undifilum oxytropis is an endophyte of locoweed plants of the genera Oxytropis that produces a toxic alkaloid swainsonine. Swainsonine, an alpha-mannosidase inhibitor causes a general toxicosis and neurological problems (locoism) when consumed by grazing animals. Swainsonine is also being assessed for its anti-cancer properties. While the ecology of U. oxytropis has been studied, little is known about the genetics and proteomics of any swainsonine-producing fungus. To help understand the proteins in U. oxytropis, the proteome of U. oxytropis was analyzed using 2-dimensional electrophoresis (2-DE). Proteins from U. oxytropis mycelia were extracted and separated by in-gel isoelectric focusing (IEF). The entire immobilized pH gradient (IPG) strip was cut into a set of gel sections and each gel section was digested with trypsin and then identified using liquid chromatography tandem mass spectrometry (LC-MS/MS). 2-DE maps were also developed for U. oxytropis to define its proteome. In the isoelectric point (pI) range of 3-11 and 10-250 kDa ranges, more than 450 spots were detected in 2-DE silver-stained gels, and 52 proteins were identified by LC-MS/MS. Most of the identified proteins were involved in energy production, oxidoreductase activity, carbohydrate metabolic process, amino acid and cellular ketone metabolic process. A large group of identified proteins were related to stress proteins and heat shock proteins. This work presents the first two-dimensional reference map of this alkaloid-producing fungus. Details of the proteome serve as a baseline for further study of this swainsonine-producing fungus and are essential for a reverse genetic analysis of the fungus.Keywords: Undifilum oxytropis fungus, two-dimensional gel electrophoresis, proteome reference map, liquid chromatography tandem mass spectrometry, swainsonin

Scanning the Data Environment at the University of Massachusetts Medical School

Objective Environmental scanning constitutes a primary mode of organizational learning” (Choo 1999). In a step toward active development of research data support services for its community, the Lamar Soutter Library at the University of Massachusetts Medical School has undertaken extensive environmental scanning to better understand the strengths, weaknesses, opportunities and challenges of an academic biomedical institution with respect to research data. Given the variety of potential data services that an academic library may deploy, the information gathered from these activities will identify and prioritize new library activities. Method Environmental scanning activities include a survey of student’s experiences and attitudes with research data management; faculty and administrator interviews (via the DuraSpace 2014 eScience Institute program); and the identification of existing local services and policy documents related to research data. Results from these activities are analyzed by the Library Data Services Advisory Group and the eScience Institute working group to plot a formal roadmap for library-based data services. Results Students, faculty, administrators, and existing documentation together reveal a variety of attitudes, assumptions, and avenues for the handling of research data on campus. They identify potential activities where the library might play a role, some expected and some unexpected. Conclusion Information gathered during environmental scanning activities at the University of Massachusetts Medical School informs the development and prioritization of library-based research data support services

Timing Termination of a Biofumigant Cover Crop for Weed Suppression in Chile Pepper

Overwinter mustard cover crops incorporated into soil may suppress early-season weeds in chile pepper (Capsicum annuum). However, the potential for mustard cover crops to harbor beet leafhoppers (Circulifer tenellus) is a concern because beet leafhoppers transmit beet curly top virus to chile pepper. The objectives of this study were to determine the amounts of a biopesticidal compound (sinigrin) added to soil from ‘Caliente Rojo’ brown mustard (Brassica juncea) cover crops ended on three different days before beet leafhopper flights during spring and to determine the effects of the cover crop termination date on weed densities and hand-hoeing times for chile pepper. To address these objectives, a field study was conducted in southern New Mexico. In 2019–20, the cover crop was ended and incorporated into soil 45, 31, and 17 days before beet leafhopper flights. In 2020–21, cover crop termination occurred 36, 22, and 8 days before beet leafhopper flights. Treatments also included a no cover crop control. Cover crop biomass and sinigrin concentrations were determined at each termination. Chile pepper was seeded 28 days after the third termination date. Weed densities and hand-hoeing times were determined 28 and 56 days after chile pepper seeding. In 2019–20, the third termination (17 days before beet leafhopper flights) yielded the maximum cover crop biomass (820 g⋅m−2) and greatest sinigrin addition to soil (274 mmol⋅m−2). However, only the second termination (31 days before beet leafhopper flights) suppressed weeds in chile pepper. In 2020–21, the third termination (8 days before beet leafhopper flights) yielded the maximum cover crop biomass (591 g⋅m−2) and greatest sinigrin addition to soil (213 mmol⋅m−2), and it was the only treatment that suppressed weeds. No cover crop treatment reduced hand-hoeing times. These results indicate that overwinter mustard cover crops can be ended to evade beet leafhopper flights and suppress weeds in chile pepper

The Influence of Overbuilt Versus Game-Permitted American Football Facemasks on Peripheral Visuomotor Ability in NCAA Division I Football Athletes

Topics in Exercise Science and Kinesiology Volume 2: Issue 1, Article 9, 2021. Heavier facemask reinforcement has been shown to impair reaction time. While overbuilt facemasks are illegal for gameplay at all levels of competition, empirical rationale for this has not be realized and it is unknown if overbuilt facemasks are worse for peripheral vision than permitted ones. Thus, the purpose of this study was to investigate the effects of wearing an overbuilt facemask versus game permitted facemasks on peripheral visuomotor ability in collegiate football players. Male NCAA Division I football players (n=18) completed a 60-second peripheral visuomotor test on a Dynavision D2 visuomotor board under the following conditions: 1) Baseline (No helmet), 2) Helmet + Light reinforced facemask, 3) Helmet + Medium reinforced facemask, 4) Helmet + Heavy reinforced facemask, 5) Helmet + Overbuilt facemask. Overall peripheral reaction time (PRT) and PRT separated by rings and quadrants were analyzed. Points of application: 1) Regardless of facemask reinforcement, wearing protective headgear impairs peripheral reaction time (PRT) compared to baseline with no helmet. Addition of an overbuilt facemask significantly impairs PRT compared to NCAA permitted facemasks. 2) An overbuilt facemask exhibits the most visuomotor decrement in far peripheral visual fields. 3) Decreases in visuomotor ability while wearing an overbuilt facemask are most pronounced in lower regions of visual field

Swainsonine Biosynthesis Genes in Diverse Symbiotic and Pathogenic Fungi

Swainsonine—a cytotoxic fungal alkaloid and a potential cancer therapy drug—is produced by the insect pathogen and plant symbiont Metarhizium robertsii, the clover pathogen Slafractonia leguminicola, locoweed symbionts belonging to Alternaria sect. Undifilum, and a recently discovered morning glory symbiont belonging to order Chaetothyriales. Genome sequence analyses revealed that these fungi share orthologous gene clusters, designated “SWN,” which included a multifunctional swnKgene comprising predicted adenylylation and acyltransferase domains with their associated thiolation domains, a β-ketoacyl synthase domain, and two reductase domains. The role of swnK was demonstrated by inactivating it in M. robertsii through homologous gene replacement to give a ∆swnK mutant that produced no detectable swainsonine, then complementing the mutant with the wild-type gene to restore swainsonine biosynthesis. Other SWN cluster genes were predicted to encode two putative hydroxylases and two reductases, as expected to complete biosynthesis of swainsonine from the predicted SwnK product. SWN gene clusters were identified in six out of seven sequenced genomes of Metarhzium species, and in all 15 sequenced genomes of Arthrodermataceae, a family of fungi that cause athlete’s foot and ringworm diseases in humans and other mammals. Representative isolates of all of these species were cultured, and all Metarhizium spp. with SWN clusters, as well as all but one of the Arthrodermataceae, produced swainsonine. These results suggest a new biosynthetic hypothesis for this alkaloid, extending the known taxonomic breadth of swainsonine producers to at least four orders of Ascomycota, and suggest that swainsonine has roles in mutualistic symbioses and diseases of plants and animals