ASU Scholarship Showcase

Biological Soil Crusts (BSCs) are organosedimentary assemblages comprised of microbes and minerals in topsoil of terrestrial environments. BSCs strongly impact soil quality in dryland ecosystems (e.g., soil structure and nutrient yields) due to pioneer species such as Microcoleus vaginatus; phototrophs that produce filaments that bind the soil together, and support an array of heterotrophic microorganisms. These microorganisms in turn contribute to soil stability and biogeochemistry of BSCs. Non-cyanobacterial populations of BSCs are less well known than cyanobacterial populations. Therefore, we attempted to isolate a broad range of numerically significant and phylogenetically representative BSC aerobic heterotrophs. Combining simple pre-treatments (hydration of BSCs under dark and light) and isolation strategies (media with varying nutrient availability and protection from oxidative stress) we recovered 402 bacterial and one fungal isolate in axenic culture, which comprised 116 phylotypes (at 97% 16S rRNA gene sequence homology), 115 bacterial and one fungal. Each medium enriched a mostly distinct subset of phylotypes, and cultivated phylotypes varied due to the BSC pre-treatment. The fraction of the total phylotype diversity isolated, weighted by relative abundance in the community, was determined by the overlap between isolate sequences and OTUs reconstructed from metagenome or metatranscriptome reads. Together, more than 8% of relative abundance of OTUs in the metagenome was represented by our isolates, a cultivation efficiency much larger than typically expected from most soils. We conclude that simple cultivation procedures combined with specific pre-treatment of samples afford a significant reduction in the culturability gap, enabling physiological and metabolic assays that rely on ecologically relevant axenic cultures.

The heterocyclic indole-alkaloid scytonemin is a sunscreen found exclusively among cyanobacteria. An 18-gene cluster is responsible for scytonemin production in Nostoc punctiforme ATCC 29133. The upstream genes scyABCDEF in the cluster are proposed to be responsible for scytonemin biosynthesis from aromatic amino acid substrates. In vitro studies of ScyA, ScyB, and ScyC proved that these enzymes indeed catalyze initial pathway reactions. Here we characterize the role of ScyD, ScyE, and ScyF, which were logically predicted to be responsible for late biosynthetic steps, in the biological context of N. punctiforme. In-frame deletion mutants of each were constructed (ΔscyD, ΔscyE, and ΔscyF) and their phenotypes studied. Expectedly, ΔscyE presents a scytoneminless phenotype, but no accumulation of the predicted intermediaries. Surprisingly, ΔscyD retains scytonemin production, implying that it is not required for biosynthesis. Indeed, scyD presents an interesting evolutionary paradox: it likely originated in a duplication event from scyE, and unlike other genes in the operon, it has not been subjected to purifying selection. This would suggest that it is a pseudogene, and yet scyD is highly conserved in the scytonemin operon of cyanobacteria. ΔscyF also retains scytonemin production, albeit exhibiting a reduction of the production yield compared with the wild-type. This indicates that ScyF is not essential but may play an adjuvant role for scytonemin synthesis. Altogether, our findings suggest that these downstream genes are not responsible, as expected, for the late steps of scytonemin synthesis and we must look for those functions elsewhere. These findings are particularly important for biotechnological production of this sunscreen through heterologous expression of its genes in more tractable organisms.

Cyanobacteria are considered good models for biohydrogen production because they are relatively simple organisms with a demonstrable ability to generate H₂ under certain physiological conditions. However, most produce only little H₂, revert readily to H₂ consumption, and suffer from hydrogenase sensitivity to O₂. Strains of the cyanobacteria Lyngbya aestuarii and Microcoleus chthonoplastes obtained from marine intertidal cyanobacterial mats were recently found to display much better H₂ production potential. Because of their ecological origin in environments that become quickly anoxic in the dark, we hypothesized that this differential ability may have evolved to serve a role in the fermentation of the photosynthate. Here we show that, when forced to ferment internal substrate, these cyanobacteria display desirable characteristics of physiological H₂ production. Among them, the strain L. aestuarii BL J had the fastest specific rates and attained the highest H₂ concentrations during fermentation of photosynthate, which proceeded via a mixed acid fermentation pathway to yield acetate, ethanol, lactate, H₂, CO₂, and pyruvate. Contrary to expectations, the H₂ yield per mole of glucose was only average compared to that of other cyanobacteria. Thermodynamic analyses point to the use of electron donors more electronegative than NAD(P)H in Lyngbya hydrogenases as the basis for its strong H₂ production ability. In any event, the high specific rates and H₂ concentrations coupled with the lack of reversibility of the enzyme, at the expense of internal, photosynthetically generated reductants, makes L. aestuarii BL J and/or its enzymes, a potentially feasible platform for large-scale H₂ production.

Quiescin sulfhydryl oxidase 1 (QSOX1) is a highly conserved disulfide bond-generating enzyme that is overexpressed in diverse tumor types. Its enzymatic activity promotes the growth and invasion of tumor cells and alters extracellular matrix composition. In a nude mouse-human tumor xenograft model, tumors containing shRNA for QSOX1 grew significantly more slowly than controls, suggesting that QSOX1 supports a proliferative phenotype in vivo. High throughput screening experiments identified ebselen as an in vitro inhibitor of QSOX1 enzymatic activity. Ebselen treatment of pancreatic and renal cancer cell lines stalled tumor growth and inhibited invasion through Matrigel in vitro. Daily oral treatment with ebselen resulted in a 58% reduction in tumor growth in mice bearing human pancreatic tumor xenografts compared to controls. Mass spectrometric analysis of ebselen-treated QSOX1 mechanistically revealed that C165 and C237 of QSOX1 covalently bound to ebselen. This report details the anti-neoplastic properties of ebselen in pancreatic and renal cancer cell lines. The results here offer a “proof-of-principle” that enzymatic inhibition of QSOX1 may have clinical relevancy.

Soil surface temperature, an important driver of terrestrial biogeochemical processes, depends strongly on soil albedo, which can be significantly modified by factors such as plant cover. In sparsely vegetated lands, the soil surface can be colonized by photosynthetic microbes that build biocrust communities. Here we use concurrent physical, biochemical and microbiological analyses to show that mature biocrusts can increase surface soil temperature by as much as 10 °C through the accumulation of large quantities of a secondary metabolite, the microbial sunscreen scytonemin, produced by a group of late-successional cyanobacteria. Scytonemin accumulation decreases soil albedo significantly. Such localized warming has apparent and immediate consequences for the soil microbiome, inducing the replacement of thermosensitive bacterial species with more thermotolerant forms. These results reveal that not only vegetation but also microorganisms are a factor in modifying terrestrial albedo, potentially impacting biosphere feedbacks on past and future climate, and call for a direct assessment of such effects at larger scales.

Soils are arguably the most microbially diverse ecosystems. Physicochemical properties have been associated with the maintenance of this diversity. Yet, the role of microbial substrate specialization is largely unexplored since substrate utilization studies have focused on simple substrates, not the complex mixtures representative of the soil environment. Here we examine the exometabolite composition of desert biological soil crusts (biocrusts) and the substrate preferences of seven biocrust isolates. The biocrust's main primary producer releases a diverse array of metabolites, and isolates of physically associated taxa use unique subsets of the complex metabolite pool. Individual isolates use only 13−26% of available metabolites, with only 2 out of 470 used by all and 40% not used by any. An extension of this approach to a mesophilic soil environment also reveals high levels of microbial substrate specialization. These results suggest that exometabolite niche partitioning may be an important factor in the maintenance of microbial diversity.

S-cysteinylated albumin and methionine-oxidized apolipoprotein A-I (apoA-I) have been posed as candidate markers of diseases associated with oxidative stress. Here, a dilute-and-shoot form of LC–electrospray ionization–MS requiring half a microliter of blood plasma was employed to simultaneously quantify the relative abundance of these oxidized proteoforms in samples stored at −80 °C, −20 °C, and room temperature and exposed to multiple freeze-thaw cycles and other adverse conditions in order to assess the possibility that protein oxidation may occur as a result of poor sample storage or handling. Samples from a healthy donor and a participant with poorly controlled type 2 diabetes started at the same low level of protein oxidation and behaved similarly; significant increases in albumin oxidation via S-cysteinylation were found to occur within hours at room temperature and days at −20 °C. Methionine oxidation of apoA-I took place on a longer time scale, setting in after albumin oxidation reached a plateau. Freeze–thaw cycles had a minimal effect on protein oxidation. In matched collections, protein oxidation in serum was the same as that in plasma. Albumin and apoA-I oxidation were not affected by sample headspace or the degree to which vials were sealed. ApoA-I, however, was unexpectedly found to oxidize faster in samples with lower surface-area-to-volume ratios. An initial survey of samples from patients with inflammatory conditions normally associated with elevated oxidative stress-including acute myocardial infarction and prostate cancer—demonstrated a lack of detectable apoA-I oxidation. Albumin S-cysteinylation in these samples was consistent with known but relatively brief exposures to temperatures above −30 °C (the freezing point of blood plasma). Given their properties and ease of analysis, these oxidized proteoforms, once fully validated, may represent the first markers of blood plasma specimen integrity based on direct measurement of oxidative molecular damage that can occur under suboptimal storage conditions.

Mastigocoleus testarum strain BC008 is a model organism used to study marine photoautotrophic carbonate dissolution. It is a multicellular, filamentous, diazotrophic, euendolithic cyanobacterium ubiquitously found in marine benthic environments. We present an accurate draft genome assembly of 172 contigs spanning 12,700,239 bp with 9,131 annotated genes with an average G+C% of 37.3.

To address the need to study frozen clinical specimens using next-generation RNA, DNA, chromatin immunoprecipitation (ChIP) sequencing and protein analyses, we developed a biobank work flow to prospectively collect biospecimens from patients with renal cell carcinoma (RCC). We describe our standard operating procedures and work flow to annotate pathologic results and clinical outcomes. We report quality control outcomes and nucleic acid yields of our RCC submissions (N=16) to The Cancer Genome Atlas (TCGA) project, as well as newer discovery platforms, by describing mass spectrometry analysis of albumin oxidation in plasma and 6 ChIP sequencing libraries generated from nephrectomy specimens after histone H3 lysine 36 trimethylation (H3K36me3) immunoprecipitation. From June 1, 2010, through January 1, 2013, we enrolled 328 patients with RCC. Our mean (SD) TCGA RNA integrity numbers (RINs) were 8.1 (0.8) for papillary RCC, with a 12.5% overall rate of sample disqualification for RIN <7. Banked plasma had significantly less albumin oxidation (by mass spectrometry analysis) than plasma kept at 25°C (P<.001). For ChIP sequencing, the FastQC score for average read quality was at least 30 for 91% to 95% of paired-end reads. In parallel, we analyzed frozen tissue by RNA sequencing; after genome alignment, only 0.2% to 0.4% of total reads failed the default quality check steps of Bowtie2, which was comparable to the disqualification ratio (0.1%) of the 786-O RCC cell line that was prepared under optimal RNA isolation conditions. The overall correlation coefficients for gene expression between Mayo Clinic vs TCGA tissues ranged from 0.75 to 0.82. These data support the generation of high-quality nucleic acids for genomic analyses from banked RCC. Importantly, the protocol does not interfere with routine clinical care. Collections over defined time points during disease treatment further enhance collaborative efforts to integrate genomic information with outcomes.

Insulin-like growth factor 1 (IGF1) is an important biomarker for the management of growth hormone disorders. Recently there has been rising interest in deploying mass spectrometric (MS) methods of detection for measuring IGF1. However, widespread clinical adoption of any MS-based IGF1 assay will require increased throughput and speed to justify the costs of analyses, and robust industrial platforms that are reproducible across laboratories. Presented here is an MS-based quantitative IGF1 assay with performance rating of >1,000 samples/day, and a capability of quantifying IGF1 point mutations and posttranslational modifications. The throughput of the IGF1 mass spectrometric immunoassay (MSIA) benefited from a simplified sample preparation step, IGF1 immunocapture in a tip format, and high-throughput MALDI-TOF MS analysis. The Limit of Detection and Limit of Quantification of the resulting assay were 1.5 μg/L and 5 μg/L, respectively, with intra- and inter-assay precision CVs of less than 10%, and good linearity and recovery characteristics. The IGF1 MSIA was benchmarked against commercially available IGF1 ELISA via Bland-Altman method comparison test, resulting in a slight positive bias of 16%. The IGF1 MSIA was employed in an optimized parallel workflow utilizing two pipetting robots and MALDI-TOF-MS instruments synced into one-hour phases of sample preparation, extraction and MSIA pipette tip elution, MS data collection, and data processing. Using this workflow, high-throughput IGF1 quantification of 1,054 human samples was achieved in approximately 9 hours. This rate of assaying is a significant improvement over existing MS-based IGF1 assays, and is on par with that of the enzyme-based immunoassays. Furthermore, a mutation was detected in ∼1% of the samples (SNP: rs17884626, creating an A→T substitution at position 67 of the IGF1), demonstrating the capability of IGF1 MSIA to detect point mutations and posttranslational modifications.