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20 | JULY 2016 | GENengnews.com | Genetic Engineering & Biotechnology News which are different types of mesoderm." Recently, Dr. Kyba and colleagues pro- posed to interrogate whether individual MESP1-producing cells have both poten- tials. "We wanted to know whether the early cells that produce cardiac and blood cells are intrinsically different, so that a cell could be- come either one of the two—and retrospec- tively, whether they still had those develop- mental opportunities open," says Dr. Kyba. RNA-seq opened the possibility of examin- ing the intrinsic differences between cells that superfcially appear similar but might have dif- ferent developmental potentials. In cells intrin- sically programmed to become one lineage or another, single-cell expression patterns would reveal different clusters for the genes involved in cardiovascular development than for those associated with blood development. An alternative possibility involves cells that do not intrinsically have these different fates but are still in a plastic state. "Such cells would have the same expression pattern of the detectable genes," proposes Dr. Kyba. "One would not be able to cluster them." In single-cell RNA-seq whole-genome transcriptomic analyses, Dr. Kyba and col- leagues found that the genes did not clus- ter into two or more different groups. "We were surprised because the cells looked as if they were homogeneous," recalls Dr. Kyba. However, particularly during early develop- ment, when gene expression profles are set to change dramatically, capturing key tran- scription factors is known to be more impor- tant than surveying the totality of the genes from the genome. "When we selected the key master regula- tors of the blood and the cardiac lineages and looked at those specifc genes, we found that cells segregated," reports Dr. Kyba. "It was possible to discern cells destined to become cardiac from the ones destined to become blood lineages." This analysis revealed that in cells appearing superfcially homogeneous, whole-transcriptome analysis might obscure very small subsets of genes that are the key drivers of later developmental decisions. Microbiome Communities "We initially used RNA-seq to study gene function and genome organization in indi- vidual organisms," says Karsten Zengler, Ph.D., associate professor of pediatrics at the University of California, San Diego. "We recently started using it more in the commu- nity context." Researchers in Dr. Zengler's lab have per- formed several microbiome studies to under- stand the dynamics of individual members of the microbiome over time. One of the major challenges in microbiology has been the fact that the vast majority of the micro- bial species cannot be cultured using routine laboratory methods, but studies on micro- bial populations promise to circumvent the need to culture individual microorganisms. "The idea is not so much to isolate mi- croorganisms, but to elucidate what they do in their natural environment," explains Dr. Zengler. Exploring microbial populations is ideally positioned to collect information that would otherwise be diffcult or impos- sible to collect from the individual constitu- ents of the population. "The most challeng- ing aspect of using RNA-seq is that we have very little material to work with," notes Dr. Zengler, who also works on the human skin microbiome. In a recent study, Dr. Zengler and col- leagues interrogated complex microbial communities. The scientists were able to ex- tract the genomes of individual community members and transcriptomic information to unveil details about metabolic interactions between species. This work relied on a com- bined systems biology and molecular biology approach that helped the scientists charac- terize the metabolic fux at the community level. The scientists were also able to predict the effects of metabolic perturbations. This proof-of-concept study promises experimental strategies in which individual community members can be depleted or en- riched to achieve a desired effect, such as ther- apeutic benefts. "Using this concept, which we developed with our collaborators, we can now identify microorganisms that control the microbial community on skin," asserts Dr. Zengler. "By placing selected microbes into an ointment, we might be able to treat skin diseases such as atopic dermatitis." Deliberately adding specifc microorgan- isms back into microbial communities promis- es therapeutic avenues for a variety of medical conditions. "Ultimately, the goal is to explore how the microbiome is originally assembled," concludes Dr. Zengler. "If we understand why an organism is present or absent, this would allow us to intervene in a targeted manner to modulate health and disease outcome." Edge toward Co-Expression Networks "Co-expression is the ideal approach to look at how gene sets interact and to deter- mine functional output," says Jesse Gillis, Ph.D., assistant professor at the Cold Spring Harbor Laboratory. Major efforts in Dr. Gil- lis' lab are focusing on understanding gene variants that are relevant for disease and on using single-cell gene expression to capture the function of genes and their interactions in complex networks. In the frst major single-cell co-expression analysis, Dr. Gillis and colleagues examined single-cell RNA-seq data from 31 studies, in- cluding 163 different cell types, to character- ize co-expression replicability. "Surprisingly, we found that one of the ways genes interact in cells is not very cell-specifc," informs Dr. Gillis. "While some cell specifcity is involved, the interaction tends to be fairly binary." In this analysis, single-cell network con- nectivity emerged as a signifcant predictor of function. To further explore the factors shap- ing this connectivity, Dr. Gillis and colleagues performed RNA-seq analyses on 126 cortical interneurons. This experimental design cap- tured co-expression patterns and provided an ideal setting to identify factors that interfere with measurements and to implement ap- proaches to control for them. "A major challenge that is being recog- nized, but has not been fully solved, is that RNA-Seq Continued from page 18 OMICS The image, provided by Michael Kyba, Ph.D., and Sunny Chan, Ph.D., of the University of Minnesota, illustrates a single Mesp1-induced cell (green) captured by the Fluidigm C1 microfuidic system. The cell is colored for clarity purposes. There are 96 of these fuid microcells in the device. After cell capture, the RNA is extracted and collected from each cell. Ultimately, an RNA-seq library is created. IMPROVING THE PHARMACOKINETIC PROFILE OF DRUGS CHEMICAL GLYCOSYLATION OF PEPTIDES ADVANTAGES OF CHEMICAL GLYCOSYLATION • Homogeneous products: chemical synthesis yields well-defi ned glycopeptides • Most chemically synthesized peptide drugs can be easily adapted to glycosylation • Competitive production costs WWW.BACHEM.COM Bachem and GlyTech, Inc. Two pioneers in their respective fi elds collaborating to advance innovation in drug development. Interferon β-1a is a glycosylated 166 amino acid protein and an approved drug substance to treat multiple sclerosis.