ASU Scholarship Showcase

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.

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.

Background: Healthy individuals on the lower end of the insulin sensitivity spectrum also have a reduced gene expression response to exercise for specific genes. The goal of this study was to determine the relationship between insulin sensitivity and exercise-induced gene expression in an unbiased, global manner.

Methods and Findings: Euglycemic clamps were used to measure insulin sensitivity and muscle biopsies were done at rest and 30 minutes after a single acute exercise bout in 14 healthy participants. Changes in mRNA expression were assessed using microarrays, and miRNA analysis was performed in a subset of 6 of the participants using sequencing techniques. Following exercise, 215 mRNAs were changed at the probe level (Bonferroni-corrected P<0.00000115). Pathway and Gene Ontology analysis showed enrichment in MAP kinase signaling, transcriptional regulation and DNA binding. Changes in several transcription factor mRNAs were correlated with insulin sensitivity, including MYC, r=0.71; SNF1LK, r=0.69; and ATF3, r= 0.61 (5 corrected for false discovery rate). Enrichment in the 5’-UTRs of exercise-responsive genes suggested regulation by common transcription factors, especially EGR1. miRNA species of interest that changed after exercise included miR-378, which is located in an intron of the PPARGC1B gene.

Conclusions: These results indicate that transcription factor gene expression responses to exercise depend highly on insulin sensitivity in healthy people. The overall pattern suggests a coordinated cycle by which exercise and insulin sensitivity regulate gene expression in muscle.

Although insulin resistance in skeletal muscle is well-characterized, the role of circulating whole blood in the metabolic syndrome phenotype is not well understood. We set out to test the hypothesis that genes involved in inflammation, insulin signaling and mitochondrial function would be altered in expression in the whole blood of individuals with metabolic syndrome. We further wanted to examine whether similar relationships that we have found previously in skeletal muscle exist in peripheral whole blood cells. All subjects (n=184) were Latino descent from the Arizona Insulin Resistance registry. Subjects were classified based on the metabolic syndrome phenotype according to the National Cholesterol Education Program’s Adult Treatment Panel III. Of the 184 Latino subjects in the study, 74 were classified with the metabolic syndrome and 110 were without. Whole blood gene expression profiling was performed using the Agilent 4x44K Whole Human Genome Microarray. Whole blood microarray analysis identified 1,432 probes that were altered in expression ≥1.2 fold and P<0.05 after Benjamini-Hochberg in the metabolic syndrome subjects. KEGG pathway analysis revealed significant enrichment for pathways including ribosome, oxidative phosphorylation and MAPK signaling (all Benjamini-Hochberg P<0.05). Whole blood mRNA expression changes observed in the microarray data were confirmed by quantitative RT-PCR. Transcription factor binding motif enrichment analysis revealed E2F1, ELK1, NF-kappaB, STAT1 and STAT3 significantly enriched after Bonferroni correction (all P<0.05). The results of the present study demonstrate that whole blood is a useful tissue for studying the metabolic syndrome and its underlying insulin resistance although the relationship between blood and skeletal muscle differs.

Background: Although the effect of the fat mass and obesity-associated (FTO) gene on adiposity is well established, there is a lack of evidence whether physical activity (PA) modifies the effect of FTO variants on obesity in Latino populations. Therefore, the purpose of this study was to examine PA influences and interactive effects between FTO variants and PA on measures of adiposity in Latinos.

Results: After controlling for age and sex, participants who did not engage in regular PA exhibited higher BMI, fat mass, HC, and WC with statistical significance (P < 0.001). Although significant associations between the three FTO genotypes and adiposity measures were found, none of the FTO genotype by PA interaction assessments revealed nominally significant associations. However, several of such interactive influences exhibited considerable trend towards association.

Conclusions: These data suggest that adiposity measures are associated with PA and FTO variants in Latinos, but the impact of their interactive influences on these obesity measures appear to be minimal. Future studies with large sample sizes may help to determine whether individuals with specific FTO variants exhibit differential responses to PA interventions.

Background: Obesity is a metabolic disease caused by environmental and genetic factors. However, the epigenetic mechanisms of obesity are incompletely understood. The aim of our study was to investigate the role of skeletal muscle DNA methylation in combination with transcriptomic changes in obesity.

Results: Muscle biopsies were obtained basally from lean (n = 12; BMI = 23.4 ± 0.7 kg/m[superscript 2]) and obese (n = 10; BMI = 32.9 ± 0.7 kg/m[superscript 2]) participants in combination with euglycemic-hyperinsulinemic clamps to assess insulin sensitivity. We performed reduced representation bisulfite sequencing (RRBS) next-generation methylation and microarray analyses on DNA and RNA isolated from vastus lateralis muscle biopsies. There were 13,130 differentially methylated cytosines (DMC; uncorrected P < 0.05) that were altered in the promoter and untranslated (5' and 3'UTR) regions in the obese versus lean analysis. Microarray analysis revealed 99 probes that were significantly (corrected P < 0.05) altered. Of these, 12 genes (encompassing 22 methylation sites) demonstrated a negative relationship between gene expression and DNA methylation. Specifically, sorbin and SH3 domain containing 3 (SORBS3) which codes for the adapter protein vinexin was significantly decreased in gene expression (fold change −1.9) and had nine DMCs that were significantly increased in methylation in obesity (methylation differences ranged from 5.0 to 24.4 %). Moreover, differentially methylated region (DMR) analysis identified a region in the 5'UTR (Chr.8:22,423,530–22,423,569) of SORBS3 that was increased in methylation by 11.2 % in the obese group. The negative relationship observed between DNA methylation and gene expression for SORBS3 was validated by a site-specific sequencing approach, pyrosequencing, and qRT-PCR. Additionally, we performed transcription factor binding analysis and identified a number of transcription factors whose binding to the differentially methylated sites or region may contribute to obesity.

Conclusions: These results demonstrate that obesity alters the epigenome through DNA methylation and highlights novel transcriptomic changes in SORBS3 in skeletal muscle.

Background: Mutual policing is an important mechanism for reducing conflict in cooperative groups. In societies of ants, bees, and wasps, mutual policing of worker reproduction can evolve when workers are more closely related to the queen's sons than to the sons of workers or when the costs of worker reproduction lower the inclusive fitness of workers. During colony growth, relatedness within the colony remains the same, but the costs of worker reproduction may change. The costs of worker reproduction are predicted to be greatest in incipient colonies. If the costs associated with worker reproduction outweigh the individual direct benefits to workers, policing mechanisms as found in larger colonies may be absent in incipient colonies.

Results: We investigated policing behavior across colony growth in the ant 'Camponotus floridanus.' In large colonies of this species, worker reproduction is policed by the destruction of worker-laid eggs. We found workers from incipient colonies do not exhibit policing behavior, and instead tolerate all conspecific eggs. The change in policing behavior is consistent with changes in egg surface hydrocarbons, which provide the informational basis for policing; eggs laid by queens from incipient colonies lack the characteristic hydrocarbons on the surface of eggs laid by queens from large colonies, making them chemically indistinguishable from worker-laid eggs. We also tested the response to fertility information in the context of queen tolerance. Workers from incipient colonies attacked foreign queens from large colonies; whereas workers from large colonies tolerated such queens. Workers from both incipient and large colonies attacked foreign queens from incipient colonies.

Conclusions: Our results provide novel insights into the regulation of worker reproduction in social insects at both the proximate and ultimate levels. At the proximate level, our results show that mechanisms of social regulation, such as the response to fertility signals, change dramatically over a colony's life cycle. At the ultimate level, our results emphasize the importance of factors besides relatedness in predicting the level of conflict within a colony. Our results also suggest policing may not be an important regulatory force at every stage of colony development. Changes relating to the life cycle of the colony are sufficient to account for major differences in social regulation in an insect colony. Mechanisms of conflict mediation observed in one phase of a social group's development cannot be generalized to all stages.

Introduction: Decreased insulin sensitivity blunts the normal increase in gene expression from skeletal muscle after exercise. In addition, chronic inflammation decreases insulin sensitivity. Chronic kidney disease (CKD) is an inflammatory state. How CKD and, subsequently, kidney transplantation affects skeletal muscle gene expression after exercise are unknown.

Methods: Study cohort: non-diabetic male/female 4/1, age 52±2 years, with end-stage CKD who underwent successful kidney transplantation. The following were measured both pre-transplant and post-transplant and compared to normals: Inflammatory markers, euglycemic insulin clamp studies determine insulin sensitivity, and skeletal muscle biopsies performed before and within 30 minutes after an acute exercise protocol. Microarray analyses were performed on the skeletal muscle using the 4x44K Whole Human Genome Microarrays. Since nuclear factor of activated T cells (NFAT) plays an important role in T cell activation and calcineurin inhibitors are mainstay immunosuppression, calcineurin/NFAT pathway gene expression was compared at rest and after exercise. Log transformation was performed to prevent skewing of data and regression analyses comparing measures pre- and post-transplant performed.

Result: Markers of inflammation significantly improved post-transplantation. Insulin infusion raised glucose disposal slightly lower post-transplant compared to pre-transplant, but not significantly, thus concluding differences in insulin sensitivity were similar. The overall pattern of gene expression in response to exercise was reduced both pre-and post-transplant compared to healthy volunteers. Although significant changes were observed among NFAT/Calcineurin gene at rest and after exercise in normal cohort, there were no significant differences comparing NFAT/calcineurin pathway gene expression pre- and post-transplant.

Conclusions: Despite an improvement in serum inflammatory markers, no significant differences in glucose disposal were observed post-transplant. The reduced skeletal muscle gene expression, including NFAT/calcineurin gene expression, in response to a single bout of exercise was not improved post-transplant. This study suggests that the improvements in inflammatory mediators post-transplant are unrelated to changes of NFAT/calcineurin gene expression.

Background: The extracellular sunscreen scytonemin is the most common and widespread indole-alkaloid among cyanobacteria. Previous research using the cyanobacterium Nostoc punctiforme ATCC 29133 revealed a unique 18-gene cluster (NpR1276 to NpR1259 in the N. punctiforme genome) involved in the biosynthesis of scytonemin. We provide further genomic characterization of these genes in N. punctiforme and extend it to homologous regions in other cyanobacteria.

Results: Six putative genes in the scytonemin gene cluster (NpR1276 to NpR1271 in the N. punctiforme genome), with no previously known protein function and annotated in this study as scyA to scyF, are likely involved in the assembly of scytonemin from central metabolites, based on genetic, biochemical, and sequence similarity evidence. Also in this cluster are redundant copies of genes encoding for aromatic amino acid biosynthetic enzymes. These can theoretically lead to tryptophan and the tyrosine precursor, p-hydroxyphenylpyruvate, (expected biosynthetic precursors of scytonemin) from end products of the shikimic acid pathway. Redundant copies of the genes coding for the key regulatory and rate-limiting enzymes of the shikimic acid pathway are found there as well. We identified four other cyanobacterial strains containing orthologues of all of these genes, three of them by database searches (Lyngbya PCC 8106, Anabaena PCC 7120, and Nodularia CCY 9414) and one by targeted sequencing (Chlorogloeopsis sp. strain Cgs-089; CCMEE 5094). Genomic comparisons revealed that most scytonemin-related genes were highly conserved among strains and that two additional conserved clusters, NpF5232 to NpF5236 and a putative two-component regulatory system (NpF1278 and NpF1277), are likely involved in scytonemin biosynthesis and regulation, respectively, on the basis of conservation and location. Since many of the protein product sequences for the newly described genes, including ScyD, ScyE, and ScyF, have export signal domains, while others have putative transmembrane domains, it can be inferred that scytonemin biosynthesis is compartmentalized within the cell. Basic structural monomer synthesis and initial condensation are most likely cytoplasmic, while later reactions are predicted to be periplasmic.

Conclusion: We show that scytonemin biosynthetic genes are highly conserved among evolutionarily diverse strains, likely include more genes than previously determined, and are predicted to involve compartmentalization of the biosynthetic pathway in the cell, an unusual trait for prokaryotes.

Investigation into the causes underlying the rapid, global amphibian decline provides critical insight into the effects of changing ecosystems. Hypothesized and confirmed links between amphibian declines, disease, and environmental changes are increasingly represented in published literature. However, there are few long-term amphibian studies that include data on population size, abnormality/injury rates, disease, and habitat variables to adequately assess changes through time. We cultured and identified microorganisms isolated from abnormal/injured and repressed tissue regeneration sites of the endangered Ozark Hellbender, Cryptobranchus alleganiensis bishopi, to discover potential causative agents responsible for their significant decline in health and population. This organism and our study site were chosen because the population and habitat of C. a. bishopi have been intensively studied from 1969–2009, and the abnormality/injury rate and apparent lack of regeneration were established.

Although many bacterial and fungal isolates recovered were common environmental organisms, several opportunistic pathogens were identified in association with only the injured tissues of C.a. bishopi. Bacterial isolates included Aeromonas hydrophila, a known amphibian pathogen, Granulicetella adiacens, Gordonai terrae, Stenotrophomonas maltophilia, Aerococcus viridans, Streptococcus pneumoniae and a variety of Pseudomonads, including Pseudomonas aeruginosa, P. stutzeri, and P. alcaligenes. Fungal isolates included species in the genera Penicillium, Acremonium, Cladosporium, Curvularia, Fusarium, Streptomycetes, and the Class Hyphomycetes. Many of the opportunistic pathogens identified are known to form biofilms. Lack of isolation of the same organism from all wounds suggests that the etiological agent responsible for the damage to C. a. bishopi may not be a single organism. To our knowledge, this is the first study to profile the external microbial consortia cultured from a Cryptobranchid salamander. The incidence of abnormalities/injury and retarded regeneration in C. a. bishopi may have many contributing factors including disease and habitat degradation. Results from this study may provide insight into other amphibian population declines.