ASU Scholarship Showcase

Recent studies suggest a role for the microbiota in autism spectrum disorders (ASD), potentially arising from their role in modulating the immune system and gastrointestinal (GI) function or from gut–brain interactions dependent or independent from the immune system. GI problems such as chronic constipation and/or diarrhea are common in children with ASD, and significantly worsen their behavior and their quality of life. Here we first summarize previously published data supporting that GI dysfunction is common in individuals with ASD and the role of the microbiota in ASD. Second, by comparing with other publically available microbiome datasets, we provide some evidence that the shifted microbiota can be a result of westernization and that this shift could also be framing an altered immune system. Third, we explore the possibility that gut–brain interactions could also be a direct result of microbially produced metabolites.

There is a growing body of scientific evidence that the health of the microbiome (the trillions of microbes that inhabit the human host) plays an important role in maintaining the health of the host and that disruptions in the microbiome may play a role in certain disease processes. An increasing number of research studies have provided evidence that the composition of the gut (enteric) microbiome (GM) in at least a subset of individuals with autism spectrum disorder (ASD) deviates from what is usually observed in typically developing individuals. There are several lines of research that suggest that specific changes in the GM could be causative or highly associated with driving core and associated ASD symptoms, pathology, and comorbidities which include gastrointestinal symptoms, although it is also a possibility that these changes, in whole or in part, could be a consequence of underlying pathophysiological features associated with ASD. However, if the GM truly plays a causative role in ASD, then the manipulation of the GM could potentially be leveraged as a therapeutic approach to improve ASD symptoms and/or comorbidities, including gastrointestinal symptoms.

One approach to investigating this possibility in greater detail includes a highly controlled clinical trial in which the GM is systematically manipulated to determine its significance in individuals with ASD. To outline the important issues that would be required to design such a study, a group of clinicians, research scientists, and parents of children with ASD participated in an interdisciplinary daylong workshop as an extension of the 1st International Symposium on the Microbiome in Health and Disease with a Special Focus on Autism (www.microbiome-autism.com). The group considered several aspects of designing clinical studies, including clinical trial design, treatments that could potentially be used in a clinical trial, appropriate ASD participants for the clinical trial, behavioral and cognitive assessments, important biomarkers, safety concerns, and ethical considerations. Overall, the group not only felt that this was a promising area of research for the ASD population and a promising avenue for potential treatment but also felt that further basic and translational research was needed to clarify the clinical utility of such treatments and to elucidate possible mechanisms responsible for a clinical response, so that new treatments and approaches may be discovered and/or fostered in the future.

Widespread contamination of groundwater by chlorinated ethenes and their biological dechlorination products necessitates the reliable monitoring of liquid matrices; current methods approved by the U.S. Environmental Protection Agency (EPA) require a minimum of 5 mL of sample volume and cannot simultaneously detect all transformative products. This paper reports on the simultaneous detection of six chlorinated ethenes and ethene itself, using a liquid sample volume of 1 mL by concentrating the compounds onto an 85-µm carboxen-polydimenthylsiloxane solid-phase microextraction fiber in 5 min and subsequent chromatographic analysis in 9.15 min. Linear increases in signal response were obtained over three orders of magnitude (∼0.05 to ∼50 µM) for simultaneous analysis with coefficient of determination (R²) values of ≥ 0.99. The detection limits of the method (1.3–6 µg/L) were at or below the maximum contaminant levels specified by the EPA. Matrix spike studies with groundwater and mineral medium showed recovery rates between 79–108%. The utility of the method was demonstrated in lab-scale sediment flow-through columns assessing the bioremediation potential of chlorinated ethene-contaminated groundwater. Owing to its low sample volume requirements, good sensitivity and broad target analyte range, the method is suitable for routine compliance monitoring and is particularly attractive for interpreting the bench-scale feasibility studies that are commonly performed during the remedial design stage of groundwater cleanup projects.

Dehalococcoides mccartyi strains are of particular importance for bioremediation due to their unique capability of transforming perchloroethene (PCE) and trichloroethene (TCE) to non-toxic ethene, through the intermediates cis-dichloroethene (cis-DCE) and vinyl chloride (VC). Despite the widespread environmental distribution of Dehalococcoides, biostimulation sometimes fails to promote dechlorination beyond cis-DCE. In our study, microcosms established with garden soil and mangrove sediment also stalled at cis-DCE, albeit Dehalococcoides mccartyi containing the reductive dehalogenase genes tceA, vcrA and bvcA were detected in the soil/sediment inocula. Reductive dechlorination was not promoted beyond cis-DCE, even after multiple biostimulation events with fermentable substrates and a lengthy incubation.

However, transfers from microcosms stalled at cis-DCE yielded dechlorination to ethene with subsequent enrichment cultures containing up to 10⁹ Dehalococcoides mccartyi cells mL^-1. Proteobacterial classes which dominated the soil/sediment communities became undetectable in the enrichments, and methanogenic activity drastically decreased after the transfers. We hypothesized that biostimulation of Dehalococcoides in the cis-DCE-stalled microcosms was impeded by other microbes present at higher abundances than Dehalococcoides and utilizing terminal electron acceptors from the soil/sediment, hence, outcompeting Dehalococcoides for H₂. In support of this hypothesis, we show that garden soil and mangrove sediment microcosms bioaugmented with their respective cultures containing Dehalococcoides in high abundance were able to compete for H² for reductive dechlorination from one biostimulation event and produced ethene with no obvious stall. Overall, our results provide an alternate explanation to consolidate conflicting observations on the ubiquity of Dehalococcoides mccartyi and occasional stalling of dechlorination at cis-DCE; thus, bringing a new perspective to better assess biological potential of different environments and to understand microbial interactions governing bioremediation.

Although emerging evidence indicates that deep-sea water contains an untapped reservoir of high metabolic and genetic diversity, this realm has not been studied well compared with surface sea water. The study provided the first integrated meta-genomic and -transcriptomic analysis of the microbial communities in deep-sea water of North Pacific Ocean. DNA/RNA amplifications and simultaneous metagenomic and metatranscriptomic analyses were employed to discover information concerning deep-sea microbial communities from four different deep-sea sites ranging from the mesopelagic to pelagic ocean. Within the prokaryotic community, bacteria is absolutely dominant (~90%) over archaea in both metagenomic and metatranscriptomic data pools. The emergence of archaeal phyla Crenarchaeota, Euryarchaeota, Thaumarchaeota, bacterial phyla Actinobacteria, Firmicutes, sub-phyla Betaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria, and the decrease of bacterial phyla Bacteroidetes and Alphaproteobacteria are the main composition changes of prokaryotic communities in the deep-sea water, when compared with the reference Global Ocean Sampling Expedition (GOS) surface water. Photosynthetic Cyanobacteria exist in all four metagenomic libraries and two metatranscriptomic libraries. In Eukaryota community, decreased abundance of fungi and algae in deep sea was observed. RNA/DNA ratio was employed as an index to show metabolic activity strength of microbes in deep sea. Functional analysis indicated that deep-sea microbes are leading a defensive lifestyle.

Background: Buffering to achieve pH control is crucial for successful trichloroethene (TCE) anaerobic bioremediation. Bicarbonate (HCO3−) is the natural buffer in groundwater and the buffer of choice in the laboratory and at contaminated sites undergoing biological treatment with organohalide respiring microorganisms. However, HCO3− also serves as the electron acceptor for hydrogenotrophic methanogens and hydrogenotrophic homoacetogens, two microbial groups competing with organohalide respirers for hydrogen (H2). We studied the effect of HCO3− as a buffering agent and the effect of HCO3−-consuming reactions in a range of concentrations (2.5-30 mM) with an initial pH of 7.5 in H2-fed TCE reductively dechlorinating communities containing Dehalococcoides, hydrogenotrophic methanogens, and hydrogenotrophic homoacetogens.

Results: Rate differences in TCE dechlorination were observed as a result of added varying HCO3− concentrations due to H2-fed electrons channeled towards methanogenesis and homoacetogenesis and pH increases (up to 8.7) from biological HCO3− consumption. Significantly faster dechlorination rates were noted at all HCO3− concentrations tested when the pH buffering was improved by providing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) as an additional buffer. Electron balances and quantitative PCR revealed that methanogenesis was the main electron sink when the initial HCO3− concentrations were 2.5 and 5 mM, while homoacetogenesis was the dominant process and sink when 10 and 30 mM HCO3− were provided initially.

Conclusions: Our study reveals that HCO3− is an important variable for bioremediation of chloroethenes as it has a prominent role as an electron acceptor for methanogenesis and homoacetogenesis. It also illustrates the changes in rates and extent of reductive dechlorination resulting from the combined effect of electron donor competition stimulated by HCO3− and the changes in pH exerted by methanogens and homoacetogens.

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: The use of culture-independent nucleic acid techniques, such as ribosomal RNA gene cloning library analysis, has unveiled the tremendous microbial diversity that exists in natural environments. In sharp contrast to this great achievement is the current difficulty in cultivating the majority of bacterial species or phylotypes revealed by molecular approaches. Although recent new technologies such as metagenomics and metatranscriptomics can provide more functionality information about the microbial communities, it is still important to develop the capacity to isolate and cultivate individual microbial species or strains in order to gain a better understanding of microbial physiology and to apply isolates for various biotechnological applications.

Results: We have developed a new system to cultivate bacteria in an array of droplets. The key component of the system is the microbe observation and cultivation array (MOCA), which consists of a Petri dish that contains an array of droplets as cultivation chambers. MOCA exploits the dominance of surface tension in small amounts of liquid to spontaneously trap cells in well-defined droplets on hydrophilic patterns. During cultivation, the growth of the bacterial cells across the droplet array can be monitored using an automated microscope, which can produce a real-time record of the growth. When bacterial cells grow to a visible microcolony level in the system, they can be transferred using a micropipette for further cultivation or analysis.

Conclusions: MOCA is a flexible system that is easy to set up, and provides the sensitivity to monitor growth of single bacterial cells. It is a cost-efficient technical platform for bioassay screening and for cultivation and isolation of bacteria from natural environments.

We have previously hypothesized a biological pathway of activity-dependent synaptic plasticity proteins that addresses the dual genetic and environmental contributions to schizophrenia. Accordingly, variations in the immediate early gene EGR3, and its target ARC, should influence schizophrenia susceptibility. We used a pooled Next-Generation Sequencing approach to identify variants across these genes in U.S. populations of European (EU) and African (AA) descent. Three EGR3 and one ARC SNP were selected and genotyped for validation, and three SNPs were tested for association in a replication cohort. In the EU group of 386 schizophrenia cases and 150 controls EGR3 SNP rs1877670 and ARC SNP rs35900184 showed significant associations (p = 0.0078 and p = 0.0275, respectively). In the AA group of 185 cases and 50 controls, only the ARC SNP revealed significant association (p = 0.0448). The ARC SNP did not show association in the Han Chinese (CH) population. However, combining the EU, AA, and CH groups revealed a highly significant association of ARC SNP rs35900184 (p = 2.353 x 10^-7; OR [95% CI] = 1.54 [1.310–1.820]). These findings support previously reported associations between EGR3 and schizophrenia. Moreover, this is the first report associating an ARC SNP with schizophrenia and supports recent large-scale GWAS findings implicating the ARC complex in schizophrenia risk. These results support the need for further investigation of the proposed pathway of environmentally responsive, synaptic plasticity-related, schizophrenia genes.

Background: Immunosignaturing is a new peptide microarray based technology for profiling of humoral immune responses. Despite new challenges, immunosignaturing gives us the opportunity to explore new and fundamentally different research questions. In addition to classifying samples based on disease status, the complex patterns and latent factors underlying immunosignatures, which we attempt to model, may have a diverse range of applications.

Methods: We investigate the utility of a number of statistical methods to determine model performance and address challenges inherent in analyzing immunosignatures. Some of these methods include exploratory and confirmatory factor analyses, classical significance testing, structural equation and mixture modeling.

Results: We demonstrate an ability to classify samples based on disease status and show that immunosignaturing is a very promising technology for screening and presymptomatic screening of disease. In addition, we are able to model complex patterns and latent factors underlying immunosignatures. These latent factors may serve as biomarkers for disease and may play a key role in a bioinformatic method for antibody discovery.

Conclusion: Based on this research, we lay out an analytic framework illustrating how immunosignatures may be useful as a general method for screening and presymptomatic screening of disease as well as antibody discovery.