ASU Scholarship Showcase

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: 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.

Background: Heterogeneity within cell populations is relevant to the onset and progression of disease, as well as development and maintenance of homeostasis. Analysis and understanding of the roles of heterogeneity in biological systems require methods and technologies that are capable of single cell resolution. Single cell gene expression analysis by RT-qPCR is an established technique for identifying transcriptomic heterogeneity in cellular populations, but it generally requires specialized equipment or tedious manipulations for cell isolation.

Results: We describe the optimization of a simple, inexpensive and rapid pipeline which includes isolation and culture of live single cells as well as fluorescence microscopy and gene expression analysis of the same single cells by RT-qPCR. We characterize the efficiency of single cell isolation and demonstrate our method by identifying single GFP-expressing cells from a mixed population of GFP-positive and negative cells by correlating fluorescence microscopy and RT-qPCR.

Conclusions: Single cell gene expression analysis by RT-qPCR is a convenient means for investigating cellular heterogeneity, but is most useful when correlating observations with additional measurements. We demonstrate a convenient and simple pipeline for multiplexing single cell RT-qPCR with fluorescence microscopy which is adaptable to other molecular analyses.

Core-shell microgels containing sensors/dyes in a matrix were fabricated by two-stage free radical precipitation polymerization method for ratiometric sensing/imaging. The microgels composing of poly(N-isopropylacrylamide) (PNIPAm) shell exhibits a low critical solution temperature (LCST), underwent an entropically driven transition from a swollen state to a deswollen state, which exhibit a hydrodynamic radius of ∼450 nm at 25°C (in vitro) and ∼190 nm at 37°C (in vivo). The microgel’s ability of escaping from lysosome into cytosol makes the microgel be a potential candidate for cytosolic delivery of sensors/probes. Non-invasive imaging/sensing in Antigen-presenting cells (APCs) was feasible by monitoring the changes of fluorescence intensity ratios. Thus, these biocompatible microgels-based imaging/sensing agents may be expected to expand current molecular imaging/sensing techniques into methods applicable to studies in vivo, which could further drive APC-based treatments.

Cellular heterogeneity plays a pivotal role in a variety of functional processes in vivo including carcinogenesis. However, our knowledge about cell-to-cell diversity and how differences in individual cells manifest in alterations at the population level remains very limited mainly due to the lack of appropriate tools enabling studies at the single-cell level. We present a study on changes in cellular heterogeneity in the context of pre-malignant progression in response to hypoxic stress. Utilizing pre-malignant progression of Barrett’s esophagus (BE) as a disease model system we studied molecular mechanisms underlying the progression from metaplastic to dysplastic (pre-cancerous) stage. We used newly developed methods enabling measurements of cell-to-cell differences in copy numbers of mitochondrial DNA, expression levels of a set of mitochondrial and nuclear genes involved in hypoxia response pathways, and mitochondrial membrane potential. In contrast to bulk cell studies reported earlier, our study shows significant differences between metaplastic and dysplastic BE cells in both average values and single-cell parameter distributions of mtDNA copy numbers, mitochondrial function, and mRNA expression levels of studied genes. Based on single-cell data analysis, we propose that mitochondria may be one of the key factors in pre-malignant progression in BE.

Background: In Africa and Asia, sugarcane is the host of at least seven different virus species in the genus Mastrevirus of the family Geminiviridae. However, with the exception of Sugarcane white streak virus in Barbados, no other sugarcane-infecting mastrevirus has been reported in the New World. Conservation and exchange of sugarcane germplasm using stalk cuttings facilitates the spread of sugarcane-infecting viruses.

Methods: A virion-associated nucleic acids (VANA)-based metagenomics approach was used to detect mastrevirus sequences in 717 sugarcane samples from Florida (USA), Guadeloupe (French West Indies), and Réunion (Mascarene Islands). Contig assembly was performed using CAP3 and sequence searches using BLASTn and BLASTx. Mastrevirus full genomes were enriched from total DNA by rolling circle amplification, cloned and sequenced. Nucleotide and amino acid sequence identities were determined using SDT v1.2. Phylogenetic analyses were conducted using MEGA6 and PHYML3.

Results: We identified a new sugarcane-infecting mastrevirus in six plants sampled from germplasm collections in Florida and Guadeloupe. Full genome sequences were determined and analyzed for three virus isolates from Florida, and three from Guadeloupe. These six genomes share >88% genome-wide pairwise identity with one another and between 89 and 97% identity with a recently identified mastrevirus (KR150789) from a sugarcane plant sampled in China. Sequences similar to these were also identified in sugarcane plants in Réunion.

Conclusions: As these virus isolates share <64% genome-wide identity with all other known mastreviruses, we propose classifying them within a new mastrevirus species named Sugarcane striate virus. This is the first report of sugarcane striate virus (SCStV) in the Western Hemisphere, a virus that most likely originated in Asia. The distribution, vector, and impact of SCStV on sugarcane production remains to be determined.

Four genomovirus genomes were recovered from thrips (Echinothrips americanus) collected in Florida, USA. These represent four new species which are members of the Gemycircularvirus (n = 2), Gemyduguivirus (n = 1), and Gemykibivirus (n = 1) genera. This is the first record, to our knowledge, of genomoviruses associated with a phytophagous insect.

Intercellular interactions play a central role at the tissue and whole organism level modulating key cellular functions in normal and disease states. Studies of cell-cell communications are challenging due to ensemble averaging effects brought about by intrinsic heterogeneity in cellular function which requires such studies to be conducted with small populations of cells. Most of the current methods for producing and studying such small cell populations are complex to implement and require skilled personnel limiting their widespread utility in biomedical research labs. We present a simple and rapid method to produce small populations with varying size of epithelial cells (10–50 cells/population) with high-throughput (~1 population/second) on flat surfaces via patterning of extracellular matrix (ECM) proteins and random seeding of cells. We demonstrate that despite inherent limitations of non-contact, drop-on-demand piezoelectric inkjet printing for protein patterning, varying mixtures of ECM proteins can be deposited with high reproducibility and level of control on glass substrates using a set of dynamically adjustable optimized deposition parameters. We demonstrate high consistency for the number of cells per population (~1 cell standard error of mean), the population’s size (~0.2 coefficient of variation) and shape, as well as accurate spatial placement of and distance between colonies of a panel of metaplastic and dysplastic esophageal epithelial cells with differing adhesion and motility characteristics. The number of cells per colony, colony size and shape can be varied by dynamically varying the amount of ECM proteins deposited per spatial location and the number of spatial locations on the substrate. The method is applicable to a broad range of biological and biomedical studies including cell-cell communications, cellular microenvironment, migration, and stimulus response.

With the advent of metagenomics approaches, a large diversity of known and unknown viruses has been identified in various types of environmental, plant, and animal samples. One such widespread virus group is the recently established family Genomoviridae which includes viruses with small (∼2–2.4 kb), circular ssDNA genomes encoding rolling-circle replication initiation proteins (Rep) and unique capsid proteins. Here, we propose a sequence-based taxonomic framework for classification of 121 new virus genomes within this family. Genomoviruses display ∼47% sequence diversity, which is very similar to that within the well-established and extensively studied family Geminiviridae (46% diversity). Based on our analysis, we establish a 78% genome-wide pairwise identity as a species demarcation threshold. Furthermore, using a Rep sequence phylogeny-based analysis coupled with the current knowledge on the classification of geminiviruses, we establish nine genera within the Genomoviridae family. These are Gemycircularvirus (n = 73), Gemyduguivirus (n = 1), Gemygorvirus (n = 9), Gemykibivirus (n = 29), Gemykolovirus (n = 3), Gemykrogvirus (n = 3), Gemykroznavirus (n = 1), Gemytondvirus (n = 1), Gemyvongvirus (n = 1). The presented taxonomic framework offers rational classification of genomoviruses based on the sequence information alone and sets an example for future classification of other groups of uncultured viruses discovered using metagenomics approaches.