Assessment of Four Solvents for Extraction and Analysis of the Chemical Composition of Sansevieria Extrafoliar Nectar Drops by Gas Chromatography-mass Spectrometry

In the latter part of the 20th century, much effort was devoted to elucidating the chemical constituents of floral and extrafloral nectar secretions, with the primary aim of understanding their ecological roles, especially in regards to attracting pollinators. But, nearly all these studies focused on determining sugar and amino acid constituents. Only a few studies have reported more comprehensive assessments of the organic chemical constituents of plants, with none of those reporting such efforts for Sansevieria taxa (common houseplants known to purify air by bioaccumulating pollutants). To address this knowledge gap, we evaluated the efficacy of four organic solvents with distinct polarities (dichloromethane (DCM), ethyl acetate, toluene and hexane) to extract the most diverse suite of organic compounds from extrafoliar nectar drops (n = 12) secreted by motherin-law’s-tongue plant (Sansevieria spp.). Each solvent exhibited unique extraction efficiencies, with DCM extracting the greatest number of unique compounds (141), followed by hexane (113), ethyl acetate (58) and toluene (43). Compound class distributions varied with solvent type, with aliphatic hydrocarbons dominating in all but the ethyl acetate extracts. We detected 105 unique aliphatic compounds in the DCM extracts, followed by 69, 28 and 9 in the hexane, toluene and ethyl acetate extracts, respectively. Alcohols predominated in the ethyl acetate extracts. We are aware of no published studies reporting such analytical determinations of the organic compound inventories of Sansevieria extrafoliar nectars. Validation of this methodology provides the impetus to study various other plant secretions of known and unknown utility

The hetZ Gene Regulates Heterocyst Formation in Anabaena sp. strain PCC 7120

To form a complex multicellular organism, stem cells must differentiate into each cell/tissue type along proper spatiotemporal scales. The study of differentiation and organismal development has historically been conducted in prokaryotes due to their genetic and morphological simplicity. Anabaena sp. strain PCC 7120 is a multicellular filamentous cyanobacterium that differentiates a morphologically distinct secondary cell type, the heterocyst, in response to a lack of combined environmental nitrogen. Heterocysts are regularly spaced along filaments and fix atmospheric dinitrogen to maintain organismal viability in its absence. Previous work suggested that the hetZ gene is involved in heterocyst differentiation, but the insertional mutants created produced inconsistent phenotypes, so a specific role was not assigned. In this work, a clean hetZ mutant incapable of heterocyst differentiation was generated and the mutation was complemented with the reintroduction of hetZ alone. Overexpression of hetZ bypassed a mutation of hetR, the master regulator of heterocyst differentiation that controls biological pattern formation, but not a mutation of hetP, a regulator of commitment to a differentiated cell fate, which places hetZ roughly between these processes. A protein-protein interaction study showed that HetZ interacts with both HetR and itself. Assessment of transcriptional fusions between the hetZ, hetR, hetP, and patS (an inhibitor of HetR) promoter regions and GFP, and overexpression of HetR in a hetZ mutant resulted in the differentiation of heterocyst-like cells, together indicated that HetZ may act in concert with HetR as an early regulator of development. Taken together, these data describe a non-linear pathway of regulation leading to heterocyst development governed by both HetR and HetZ

Exploring Molecular Simulations of a Plausible Prebiotic Reduced Phospholipid Using Hyperchem Software

How the first cells emerged from the primordial milieu is one of the great questions in science. Biomolecular emergence scenarios abound in the literature, but the lack of bioaccessible phosphate and molecular oxygen on the primordial Earth has posed formidable challenges for deducing emergence pathways. One idea gaining wide acceptance invokes delivery of the phosphide mineral schreibersite ((Fe,Ni)3P) to Earth via meteorite impacts ca. 4.2 billion years ago, whereupon they were corroded to reduced phosphorous oxyacids and phosphonates in primordial aquatic environments. We previously proposed that these reduced phosphorus forms could readily combine with putative geochemical species in shallow mineral-rich alkaline hydrothermal systems to form reduced phospholipid analogs of contemporary phosphate-based phospholipids (Fitch, N.W., K.L. Even, L.J. Leinen and M.O. Gaylor. 2016. Plausible prebiotic assembly of a primitive reduced phospholipid from meteoritic phosphorus on the primordial earth. Proceedings of the South Dakota Academy of. Science 95:176.). Lacking resources to empirically validate this idea, we explored “water box” simulations of the proposed phospholipid structure using the HyperChem software package. Simulation results showed the hydrophobic tails migrating away from water molecules, while hydrophilic heads migrated towards them, resulting in quasistacking behaviors consistent with those of known amphiphiles in water. Inspired by these results, we are now investigating more complex primordial simulation scenarios

Identification and Characterization of Fungal Isolates from Land-applied Sewage Sludge

Approximately eight million dry tons of sewage sludge is generated in the U.S. each year, with more than half of that now land-applied as the primary method of disposal. Despite the proliferation of this practice, little is known about the microbial constituents of these noxious materials. To address this knowledge gap, we isolated and characterized fungi present in archived samples of land-disposed sewage sludge collected from the Snoqualmie National Forest (Washington State). Sludge samples were resuspended in sterile water and 15 fungal isolates were selected and purified on sabouraud dextrose agar plates supplemented with 50 mg/L of chloramphenicol. Fungal morphology was assessed and photodocumented following growth on sabouraud dextrose agar, potato dextrose agar, yeast maltose agar, and malt extract agar. Additionally, pH and temperature tolerance was assessed by growth in liquid cultures of sabouraud dextrose broth from 0 ºC to 50 ºC and determined from pH 2 to 11 at 28 ºC. Carbon source utilization was assessed using Biolog Filamentous Fungi plates. Amplification and sequencing of the ITS region, commonly used in fungal phylogenetic analysis, is in progress to identify each isolate. Phenotypic assessments of growth, carbon utilization, and lactophenol cotton blue staining revealed that sludge-associated fungi are quite varied in size and appearance, but commonly grow in a mesophilic range (10-40 ºC and pH 4-10). Interspecies variation is further evident in the percentage of 31 carbon sources utilized (63-100%). To our knowledge, this work represents the first reported assessment of the fungal community in sewage sludge wastes disposed in the Snoqualmie National Forest

Characterizing the Volatilome of Land-disposed Sewage Sludge Under Seasonal Temperature Regimes

About eight million dry tons of sewage sludge waste is generated in the US annually, with more than half of that now land-disposed on agricultural and forested lands. Though containing essential plant nutrients, sludge also harbors complex mixtures of volatile organic compounds (VOCs) that result in toxic emissions therefrom. While ecotoxic impacts to sludged ecosystems are a primary concern, the stifling emissions are most obvious to and disconcerting for the public, which has led to increasing concerns for the safety of this practice. The large-scale disposal of sludge in the temperate rainforests of the Puget Sound Watershed has resulted in acute toxicity to macrobiota, and despite escalating concerns for detrimental impacts, little is known about the total VOC inventories, or “volatilomes,” of these complex wastes. To address this knowledge gap, we characterized VOC emissions from forest-disposed sewage sludge over a range of seasonal temperature regimes. We also incubated sludge samples at the more extreme 100 o C to assess the “complete volatilome.” After 1-hr incubations in gastight vials, VOCs accumulated in the headspace were sampled with a gastight syringe and analyzed with gas chromatography-mass spectrometry to generate distinct chemical fingerprints of sludge sample volatilomes over the range of temperatures. Total integrated chromatographic peak areas increased with temperature, indicating increased VOC production. Sludge volatilomes were dominated by a multitude of aliphatics and aromatics, with comparatively lesser emissions of alcohols, esters, aldehydes, terpenes, and nitrogen-, sulfur-, and halogen-containing compounds

LIN28B Underlies the Pathogenesis of a Subclass of Ewing Sarcoma

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EWS-FLI1 Utilizes Divergent Chromatin Remodeling Mechanisms to Directly Activate or Repress Enhancer Elements in Ewing Sarcoma

SummaryThe aberrant transcription factor EWS-FLI1 drives Ewing sarcoma, but its molecular function is not completely understood. We find that EWS-FLI1 reprograms gene regulatory circuits in Ewing sarcoma by directly inducing or repressing enhancers. At GGAA repeat elements, which lack evolutionary conservation and regulatory potential in other cell types, EWS-FLI1 multimers induce chromatin opening and create de novo enhancers that physically interact with target promoters. Conversely, EWS-FLI1 inactivates conserved enhancers containing canonical ETS motifs by displacing wild-type ETS transcription factors. These divergent chromatin-remodeling patterns repress tumor suppressors and mesenchymal lineage regulators while activating oncogenes and potential therapeutic targets, such as the kinase VRK1. Our findings demonstrate how EWS-FLI1 establishes an oncogenic regulatory program governing both tumor survival and differentiation