ASU Scholarship Showcase

Linnorm is a novel normalization and transformation method for the analysis of single cell RNA sequencing (scRNA-seq) data. Linnorm is developed to remove technical noises and simultaneously preserve biological variations in scRNA-seq data, such that existing statistical methods can be improved. Using real scRNA-seq data, we compared Linnorm with existing normalization methods, including NODES, SAMstrt, SCnorm, scran, DESeq and TMM. Linnorm shows advantages in speed, technical noise removal and preservation of cell heterogeneity, which can improve existing methods in the discovery of novel subtypes, pseudo-temporal ordering of cells, clustering analysis, etc. Linnorm also performs better than existing DEG analysis methods, including BASiCS, NODES, SAMstrt, Seurat and DESeq2, in false positive rate control and accuracy.

Two distinct monocyte (Mo)/macrophage (Mp) subsets (Ly6C^low and Ly6C^hi) orchestrate cardiac recovery process following myocardial infarction (MI). Prostaglandin (PG) E₂ is involved in the Mo/Mp-mediated inflammatory response, however, the role of its receptors in Mos/Mps in cardiac healing remains to be determined. Here we show that pharmacological inhibition or gene ablation of the Ep3 receptor in mice suppresses accumulation of Ly6C^low Mos/Mps in infarcted hearts. Ep3 deletion in Mos/Mps markedly attenuates healing after MI by reducing neovascularization in peri-infarct zones. Ep3 deficiency diminishes CX3C chemokine receptor 1 (CX3CR1) expression and vascular endothelial growth factor (VEGF) secretion in Mos/Mps by suppressing TGFβ1 signaling and subsequently inhibits Ly6C^low Mos/Mps migration and angiogenesis. Targeted overexpression of Ep3 receptors in Mos/Mps improves wound healing by enhancing angiogenesis. Thus, the PGE₂/Ep3 axis promotes cardiac healing after MI by activating reparative Ly6C^low Mos/Mps, indicating that Ep3 receptor activation may be a promising therapeutic target for acute MI.

Modeling of transcriptional regulatory networks (TRNs) has been increasingly used to dissect the nature of gene regulation. Inference of regulatory relationships among transcription factors (TFs) and genes, especially among multiple TFs, is still challenging. In this study, we introduced an integrative method, LogicTRN, to decode TF–TF interactions that form TF logics in regulating target genes. By combining cis-regulatory logics and transcriptional kinetics into one single model framework, LogicTRN can naturally integrate dynamic gene expression data and TF-DNA-binding signals in order to identify the TF logics and to reconstruct the underlying TRNs. We evaluated the newly developed methodology using simulation, comparison and application studies, and the results not only show their consistence with existing knowledge, but also demonstrate its ability to accurately reconstruct TRNs in biological complex systems.

The low pH of the stomach serves as a barrier to ingested microbes and must be overcome or bypassed when delivering live bacteria for vaccine or probiotic applications. Typically, the impact of stomach acidity on bacterial survival is evaluated in vitro, as there are no small animal models to evaluate these effects in vivo. To better understand the effect of this low pH barrier to live attenuated Salmonella vaccines, which are often very sensitive to low pH, we investigated the value of the histamine mouse model for this application. A low pH gastric compartment was transiently induced in mice by the injection of histamine. This resulted in a gastric compartment of approximately pH 1.5 that was capable of distinguishing between acid-sensitive and acid-resistant microbes. Survival of enteric microbes during gastric transit in this model directly correlated with their in vitro acid resistance. Because many Salmonella enterica serotype Typhi vaccine strains are sensitive to acid, we have been investigating systems to enhance the acid resistance of these bacteria. Using the histamine mouse model, we demonstrate that the in vivo survival of S. Typhi vaccine strains increased approximately 10-fold when they carried a sugar-inducible arginine decarboxylase system. We conclude that this model will be a useful for evaluating live bacterial preparations prior to clinical trials.

Leucine-responsive regulatory protein (Lrp) is known to be an indirect activator of type 1 fimbriae synthesis in Salmonella enterica serovar Typhimurium via direct regulation of FimZ, a direct positive regulator for type 1 fimbriae production. Using RT-PCR, we have shown previously that fimA transcription is dramatically impaired in both lrp-deletion (Δlrp) and constitutive-lrp expression (lrp^C) mutant strains. In this work, we used chromosomal P_fimA-lacZ fusions and yeast agglutination assays to confirm and extend our previous results. Direct binding of Lrp to P_fimA was shown by an electrophoretic mobility shift assay (EMSA) and DNA footprinting assay. Site-directed mutagenesis revealed that the Lrp-binding motifs in P_fimA play a role in both activation and repression of type 1 fimbriae production. Overproduction of Lrp also abrogates fimZ expression. EMSA data showed that Lrp and FimZ proteins independently bind to P_fimA without competitive exclusion. In addition, both Lrp and FimZ binding to P_fimA caused a hyper retardation (supershift) of the DNA-protein complex compared to the shift when each protein was present alone. Nutrition-dependent cellular Lrp levels closely correlated with the amount of type 1 fimbriae production. These observations suggest that Lrp plays important roles in type 1 fimbriation by acting as both a positive and negative regulator and its effect depends, at least in part, on the cellular concentration of Lrp in response to the nutritional environment.

Researchers have iterated that the future of synthetic biology and biotechnology lies in novel consumer applications of crossing biology with engineering. However, if the new biology's future is to be sustainable, early and serious efforts must be made towards social sustainability. Therefore, the crux of new applications of synthetic biology and biotechnology is public understanding and acceptance. The RASVaccine is a novel recombinant design not found in nature that re-engineers a common bacteria ( Salmonella) to produce a strong immune response in humans. Synthesis of the RASVaccine has the potential to improve public health as an inexpensive, non-injectable product. But how can scientists move forward to create a dialogue of creating a 'common sense' of this new technology in order to promote social sustainability? This paper delves into public issues raised around these novel technologies and uses the RASVaccine as an example of meeting the public with a common sense of its possibilities and limitations.

Cancer therapy selects for cancer cells resistant to treatment, a process that is fundamentally evolutionary. To what extent, however, is the evolutionary perspective employed in research on therapeutic resistance and relapse? We analyzed 6,228 papers on therapeutic resistance and/or relapse in cancers and found that the use of evolution terms in abstracts has remained at about 1% since the 1980s. However, detailed coding of 22 recent papers revealed a higher proportion of papers using evolutionary methods or evolutionary theory, although this number is still less than 10%. Despite the fact that relapse and therapeutic resistance is essentially an evolutionary process, it appears that this framework has not permeated research. This represents an unrealized opportunity for advances in research on therapeutic resistance.

Background: Salmonella has been employed to deliver therapeutic molecules against cancer and infectious diseases. As the carrier for target gene(s), the cargo plasmid should be stable in the bacterial vector. Plasmid recombination has been reduced in E. coli by mutating several genes including the recA, recE, recF and recJ. However, to our knowledge, there have been no published studies of the effect of these or any other genes that play a role in plasmid recombination in Salmonella enterica.

Results: The effect of recA, recF and recJ deletions on DNA recombination was examined in three serotypes of Salmonella enterica. We found that (1) intraplasmid recombination between direct duplications was RecF-independent in Typhimurium and Paratyphi A, but could be significantly reduced in Typhi by a ΔrecA or ΔrecF mutation; (2) in all three Salmonella serotypes, both ΔrecA and ΔrecF mutations reduced intraplasmid recombination when a 1041 bp intervening sequence was present between the duplications; (3) ΔrecA and ΔrecF mutations resulted in lower frequencies of interplasmid recombination in Typhimurium and Paratyphi A, but not in Typhi; (4) in some cases, a ΔrecJ mutation could reduce plasmid recombination but was less effective than ΔrecA and ΔrecF mutations. We also examined chromosome-related recombination. The frequencies of intrachromosomal recombination and plasmid integration into the chromosome were 2 and 3 logs lower than plasmid recombination frequencies in Rec[superscript +] strains. A ΔrecA mutation reduced both intrachromosomal recombination and plasmid integration frequencies.

Conclusions: The ΔrecA and ΔrecF mutations can reduce plasmid recombination frequencies in Salmonella enterica, but the effect can vary between serovars. This information will be useful for developing Salmonella delivery vectors able to stably maintain plasmid cargoes for vaccine development and gene therapy.

Background: To be effective, orally administered live Salmonella vaccines must first survive their encounter with the low pH environment of the stomach. To enhance survival, an antacid is often given to neutralize the acidic environment of the stomach just prior to or concomitant with administration of the vaccine. One drawback of this approach, from the perspective of the clinical trial volunteer, is that the taste of a bicarbonate-based acid neutralization system can be unpleasant. Thus, we explored an alternative method that would be at least as effective as bicarbonate and with a potentially more acceptable taste. Because ingestion of protein can rapidly buffer stomach pH, we examined the possibility that the protein-rich Ensure® Nutrition shakes would be effective alternatives to bicarbonate.

Results: We tested one Salmonella enterica serovar Typhimurium and three Salmonella Typhi vaccine strains and found that all strains survived equally well when incubated in either Ensure® or bicarbonate. In a low gastric pH mouse model, Ensure® worked as well or better than bicarbonate to enhance survival through the intestinal tract, although neither agent enhanced the survival of the S. Typhi test strain possessing a rpoS mutation.

Conclusions: Our data show that a protein-rich drink such as Ensure® Nutrition shakes can serve as an alternative to bicarbonate for reducing gastric pH prior to administration of a live Salmonella vaccine.

Salmonella enterica serovar Typhimurium, a gram-negative facultative rod-shaped bacterium causing salmonellosis and foodborne disease, is one of the most common isolated Salmonella serovars in both developed and developing nations. Several S. Typhimurium genomes have been completed and many more genome-sequencing projects are underway. Comparative genome analysis of the multiple strains leads to a better understanding of the evolution of S. Typhimurium and its pathogenesis. S. Typhimurium strain UK-1 (belongs to phage type 1) is highly virulent when orally administered to mice and chickens and efficiently colonizes lymphoid tissues of these species. These characteristics make this strain a good choice for use in vaccine development. In fact, UK-1 has been used as the parent strain for a number of nonrecombinant and recombinant vaccine strains, including several commercial vaccines for poultry. In this study, we conducted a thorough comparative genome analysis of the UK-1 strain with other S. Typhimurium strains and examined the phenotypic impact of several genomic differences. Whole genomic comparison highlights an extremely close relationship between the UK-1 strain and other S. Typhimurium strains; however, many interesting genetic and genomic variations specific to UK-1 were explored. In particular, the deletion of a UK-1-specific gene that is highly similar to the gene encoding the T3SS effector protein NleC exhibited a significant decrease in oral virulence in BALB/c mice. The complete genetic complements in UK-1, especially those elements that contribute to virulence or aid in determining the diversity within bacterial species, provide key information in evaluating the functional characterization of important genetic determinants and for development of vaccines.