Comprehensive Analysis of the Dynamics of Aberrant WNT Signaling on Global Gene Expression and Higher Order Nuclear Structure in SW480 Cells

Presenter: Markus Brown                            Status: Graduate Student

Authors: Markus Brown, Janina Lehmann, Darawalee Wangsa, Indika Rajapakse, Yue Hu, Thomas Ried


Abstract: The organization of the genome in the nucleus is complex and dynamic. Features of the nuclear architecture, including the spatial arrangement of genomic sequences, the structure of chromatin, and the accessibility of regulatory DNA elements modulate nuclear processes such as gene transcription DNA replication, DNA repair, RNA processing, and mRNA transport. However, the interplay between nuclear architecture and gene expression is poorly understood. We propose to elucidate the impact of silencing the prominent transcription factor, TCF7L2, the major effector of the WNT signaling pathway, on the transcriptional equilibrium and nuclear architecture of the nucleus in human colorectal cancer cells. Our multidisciplinary approach combines targeted silencing of TCF7L2, 3D-FISH, RNA-seq, and Hi-C chromosome conformation capture, in parallel with mathematical modeling to delineate structure/function relationships in the interphase nucleus. This approach will elucidate the intricacies of a dynamic cellular signaling pathway in three-dimensional space.


Evaluating the bacterial microbiota of reclaimed water using next generation sequencing

Presenter: Prachi Kulkarni                          Status: Graduate Student

Authors: Sapkota AR, Kulkarni P, Claye E, Rosenberg Goldstein RE, Gibbs SG, Pop M, Cummings M, Maddox C, Mongodin EF


Abstract: Over 2.22 billion gallons of wastewater effluent are reclaimed per day in the U.S. However, limited data exist on the pathogens and total bacterial communities that remain in this water, as well as the potential health risks from exposure to this alternative water source. Therefore, the objective of this study was to characterize bacterial communities present in wastewater and reclaimed water using next generation sequencing. Wastewater (from each step of the treatment process), final effluent and reclaimed water samples (n=94) previously collected from four U.S. wastewater treatment plants and their associated reclamation sites were tested. DNA extractions involving both enzymatic and mechanical lyses were performed on all samples, followed by bacterial community profiling using 16S rRNA gene sequencing on the Illumina MiSeq platform.  Raw sequences were processed using PANDAseq to generate high-quality consensus sequences from the MiSeq paired-ends, and taxonomic assignments were performed using a new taxonomic classifier, the MC classifier, able to classify 16S reads to the species level with an accuracy of 98%. Diverse bacterial communities were present in all samples and differing bacterial communities were observed across different sample types. Predominant genera that were detected included the following, to name a few: Shewanella, Pseudomonas, Mycobacterium, Streptococcus, Clostridium, Acinetobacter, Staphylococcus and Legionella. Our approach has generated novel pilot data that can be used in future proposals, the findings of which will assist in the development of risk-based water reuse policies that move us towards a more sustainable water use paradigm and protect human health.


Exosomes of myeloid-derived suppressor cells carry miRNAs

Presenter: Katherine Adams                                   Status: Graduate Student

Authors: Katherine Adams, Ashton Trey Belew, Meghan Burke, Suzanne Ostrand-Rosenberg, Najib El-Sayed, Catherine Fenselau


Abstract: Myeloid-derived suppressor cells (MDSC) accumulate in individuals with cancer where they facilitate tumor progression.  Under inflammatory conditions, which are prevalent in many solid tumors, these cells are immune-suppressive through several mechanisms including polarization of macrophages toward a tumor-promoting phenotype and inhibition of T cell activation.   MDSC release exosomes, 30-100 nm extracellular vesicles that transfer information between cells through their cargo.  These exosomes have been previously shown to contain the calcium- and zinc-binding pro-inflammatory proteins S100-A8 and –A9, nucleic acid binding proteins, and nucleic acids.  Micro-RNAs, or miRNAs, are short, non-coding RNA single stranded sequences that inhibit gene expression by binding to mRNA targets to halt translation.  In the present work, miRNAs from MDSC and their exosomes were isolated and sequenced to begin investigating their role in exosomes.  Four hundred and forty-four miRNAs were identified in murine MDSC and exosomes.  Of those, 54 miRNAs were enriched with a >2 fold change in MDSC versus the exosomes, and 52 miRNAs enriched in the exosomes.  For the 52 miRNAs in exosomes, 2662 target genes were identified using miRBase and TargetScan databases, and these target genes were characterized and highly annotated for ion and metal binding functions using Gene Onotology annotations, DAVID, and KEGG enrichment bioinformatics tools.  Of note is miRNA 203 which was identified as highly enriched in the exosomes and 23 of its known target genes are involved in cancer pathways.  The Let-7 miRNA family was found highly enriched in the MDSC with 50% of the miRNA in the family identified.


Exploring glycoproteins on the exosome surface

Presenter: Sitara Chauhan                          Status: Graduate Student

Authors: Sitara Chauhan,  Dr. Steven Danielson, Dr. Nathan Edwards, Dr. Suzanne Ostrand-Rosenberg, Dr. Catherine Fenselau


Abstract: Myeloid Derived Suppressor Cells (MDSC) accumulate in the tumor microenvironment and contribute to suppression of innate and acquired immune responses against tumor cells. Exosomes shed by MDSC have been shown to carry biologically active proteins and also to suppress immune response (Burke et al J. Prot. Res. 2013). It has been suggested that exosomes communicate via receptor-ligand interactions with target cells in their environment, including parental MDSC. Thus an exploration of their surface proteins is of great interest. We report here the first study ofsurface glycoproteins on exosomes.  Cell surface chemistry provided isolation of accessible glycoproteins. Proteomic analysis of both tryptic peptides and PNGase peptide products identified 81 glycoproteins on the surface of parental MDSC cells. Fifty-one of these are CD (cluster of differentiation) proteins. The strategy was then modified for use on the exosome surface. More than 20 proteins have now been identified from the exosome surface, which carry one or more deamidated versions of the glycosylation N-X-S/T motif for N-glycosylation. Several receptor-ligand binding partners have also been identified between MDSC and their exosomes, which suggest possible modes of communication. Follow-up bioassays are underway to test immunosuppression by exosomal surface glycoproteins.


Fundamental Study of Intact Triubiquitin Chains by Mass Spectrometry

Presenter: Amanda Lee                                Status: Graduate Student

Authors: Amanda E. Lee, Lucia Geis-Asteggiante, Emma K. Dixon, Yan Wang, Tanuja R. Kashyap, David Fushman Catherine Fenselau


Abstract: The presence of different linkages within polyubiquitin chains has been related to different functions within the cell. To interrogate linkages in these chains, most studies to date have used bottom up strategies that leave -GG tags at sites of ubiquitination within a ubiquitin chain after trypsinolysis. However, these tags cannot be used to discern the lengths of polyubiquitins or the location of sequential linkages within the chain. There has been mild success using partial digestions (with trypsin and acid cleavage) to retain linkage information, however, these strategies have their limitations. The most robust proteomic method for analysis of any post-translational modification is top-down. By interrogating intact proteins all modifications can be identified, including ubiquitination. For polyubiquitins this is necessary for retention of all isopeptide linkages and their location within the chain, since they are lost with digestion. Top-down proteomics is limited by the mass accuracy and resolution capabilities of the instrumentation available, however, recent breakthroughs in mass analyzers and fragmentation techniques have allowed for more robust analysis of large proteins (>20,000 Da). In this study, ubiquitin trimers were interrogated using an Orbitrap Fusion Lumos Mass Spectrometer and multiple fragmentation techniques, which, when pulled together, gave definition of isopeptide linkage location and overall chain configuration. The top-down results were comfirmed by middle-down proteomics using time-limited acetic acid hydrolysis to produce large truncation products.


Identification of Grp78/BiP Protein Complexes Using Affinity-based Mass Spectrometry

Presenter: Dapeng Chen                              Status: Graduate Student

Authors: Dapeng Chen, Yan Wang, Eva R. Chin


Abstract: The accumulation of mis-folded proteins activates endoplasmic reticulum (ER) stress and an unfolded protein response to aid in proper protein folding and decrease the cellular protein load.  If persistent, ER stress can induce apoptosis and contribute to cellular pathologies.  We have been investigating the ER stress response in the context of muscle atrophy in amyotrophic lateral sclerosis (ALS).  Grp78/BiP is an ER membrane-located chaperone protein.  Previous studies have shown that protein-protein interactions of Grp78/BiP with target proteins are causally associated with the activation of ER stress. Therefore, identification of Grp78/BiP protein complexes is critical to understanding the molecular mechanisms underlying activation of ER stressand underlying causes of muscle atrophy in ALS.  Mass spectrometry has emerged as a significant tool in protein chemistry research due to its sensitivity, accuracy and efficiency. When combined with antibody-based affinity enrichment methods, mass spectrometry can be used for investigating protein-protein interactions.  The present study focused on developing the workflow to identify Grp78/BiP protein complexes using Grp78/BiP antibody affinity-based mass spectrometry. Moreover, we applied our optimized mass spectrometry strategy to investigate the Grp78/BiP protein complex patterns in skeletal muscle tissues between wild type and amyotrophic lateral sclerosis (ALS) transgenic mice, in which ER stress is severely induced. Our results showed that Grp78/BiP interacted with various non-ER-located proteins such as ATP synthase subunits, ADP/ATP translocase and parvalbumin, suggesting the translocation of Grp78/BiP to other cellular organelles under ER stress conditions.  Based on these observations, we have put forward a hypothesis of how ER stress may induce mitochondrial dysfunction and muscle pathology in ALS mice.  Our mass spectrometry strategy studying protein-protein interaction indicates that Grp78/BiP plays an important role in inducing muscle pathology in ALS and could be a potential therapeutic target to treat muscle atrophy in ALS.


miRNAseq Analysis of Human Vaginal Swabs

Presenter: Steven Smith                              Status: Graduate Student

Authors: Steven Smith


Abstract: Bacterial Vaginosis (BV) is a condition of the human vagina characterized in part by a paucity of Lactobacillus spp. and the presence of a wide array of strict and facultative aerobes such as Gardnerella vaginalis, Atopobium vaginae, and Mobiluncus spp. The etiology of BV remains unknown. Characterization of the vaginal microbiota sampled daily over 10 weeks has shown that some women experience transitions from Lactobacillus-dominated to BV-associated states independently of sexual behavior or other factors. To gain insight into BV etiology and the host regulatory mechanisms that drive these transitions, we are characterizing the types and abundance of a small RNA class known as microRNAs (miRNAs) using miRNAseq. We hypothesize that specific miRNA will be associated with transitions to a BV-associated Community State Type (CST) by affecting specific host functions. To achieve this goal, we first evaluated total RNA extraction and subsequent miRNA detection procedures adequate for vaginal swabs stored in RNAlater. Preliminary results that will be presented show several miRNAs are highly expressed across multiple subjects and sample time points. Furthermore, two miRNAs appear to be differentially expressed as a function of CST. Additional samples representing more longitudinal sampling and metadata are being processed to fully profile the miRNA expression dynamics associated with BV-like states. Further, we are developing novel and improved statistical models for capturing causal relationships between miRNA, host pathways and a BV-associated state.


Annotation of Diverse Gene Families in the Diamondback Terrapin Genome

Presenter: Suraj Jaladanki                           Status: Undergraduate Student

Authors: Dalia Badamo, Peter Blake, Suraj Jaladanki, Amy Voltz


Abstract: The University of Maryland’s Terrapin Genome Project (TGP) works to sequence, assemble, and annotate the diamondback terrapin (Malaclemys terrapin) genome. Others have conducted studies to understand the physiologic responses underlying turtles’ unique characteristics, including resilience to anoxic and freezing conditions, and their potential genomic basis. Our current focus is to annotate the diamondback terrapin for several gene families including: PAX (involved in embryonic development), KCNQ (responsible for ion channel proteins), and FGF (fibroblast growth factors). NCBI’s tblastn tool was used to determine homology of protein sequences against an assembled terrapin genome and genomes of other NCBI annotated species (turtles, chicken, human, and mouse). Phylogenetic analyses through Clustal OMEGA assisted in identifying individual family members in the terrapin. This research can potentially identify genes within the terrapin that function during experimentally adverse conditions such as anoxia. Terrapin genome annotation will also provide an improved understanding of the genomic basis of unique turtle characteristics and the terrapin’s evolutionary relation to other species. This work could translate to the clinical field by leading to improved outcomes in patients undergoing periods of anoxia during strokes and heart attacks. 


Investigating the presence and role of callose in charophycean green algae

Presenter: Kathleen Metz                            Status: Undergraduate Student

Authors: Kathleen Metz


Abstract: Cell wall wounding responses dictate how plants react to biotic and abiotic stresses, and are important to understand due to the implications for food production and the wood processing industry. Callose is a polysaccharide that is deposited at the site of wounding in land plants, protecting against physical and chemical stresses as well as viral infections. Callose is also deposited at the cell plate during cytokinesis, forms plugs in plasmodesmata to regulate homeostasis, and is found in pollen tubes of land plants. Callose has been reported in some green algae, but despite its diverse roles as a cell wall component, little is known about its distribution and function among algae. I have conducted a preliminary search for callose synthase (CALS) genes within eight organisms across the green lineage, using a hidden Markov model created with CALS sequences from seven representative embryophytes. Callose synthase homologs were found in the charophyte green algae Penium margaritaceum, Coleochaete orbicularis, Spirogyra pratensis, and Klebsormidium flaccidum, suggesting conservation of callose synthase throughout the green lineage. Aniline blue staining will be used to confirm the presence of callose in P. margaritaceum under various conditions, such as vegetative growth, wound healing, conjugation and cell division. Transformational approaches may also be applied to investigate functional similarities between callose synthases present in P. margaritaceum and those in A. thaliana.



Mutational profiles and survival of select cancers based on somatic and germline TP53 status.

Presenter: Franklin Ning                              Status: Undergraduate Student

Authors: Franklin Ning, Farzana Walcott, Antonio Tito Fojo


Abstract: Wild-type (WT) TP53, also known as the “guardian of the genome” is a gene whose protein product, p53, has been shown to inhibit proliferation and metastasis of tumorigenic cells. Cancers with mutations in the TP53 gene may consequently have a worse prognosis than those with WT TP53. Here, we analyzed data from The Cancer Genome Atlas (TCGA) database and the International Agency for Research on Cancer’s TP53 database (IARC) to determine if there are differences in the mutational profiles and survival time of cancer patients with somatic or germ line mutations in TP53 vs. WT TP53.  Results indicate the link between TP53 and survivability may not be applicable to all cancers. Adrenocortical cancer (ACC) with mutated somatic TP53 had worse survival compared to ACC with somatic WT TP53. For other cancers such as pancreatic and ovarian, there was no difference in survivability between WT and mutant somatic TP53. C>T single nucleotide variations (SNVs) were also noted to be the most prominent SNV for 15 out of 18 cancer types analyzed, including pancreatic and breast. Renal papillary carcinoma, lung adenocarcinoma, and lung small cell carcinoma though had C>A as the most prominent SNV. The analysis to date thus indicates this relationship of p53 and prognoses is much more complex than expected and may be tumor specific.




Presenter: Travis Wentz                              Status: Undergraduate Student

Authors: Wentz T, Thirunavukkarasu N, Hodge DR, Hammack, T, Brown E, and Sharma SK


Abstract: BoNTs and associated neurotoxin proteins (ANTPs) are assembled to form botulinum neurotoxin complexes. The gene encoding BoNTs are clustered together in close vicinity with either ‘ha’ or orf-x/p-47 genes. The ‘ha’ genes encode for haemagglutinin components (HA33, HA17 & HA70) which are concomitantly synthesized along with BoNTs, often by the alternate sigma factor BotR, a positive regulator. Little is known about the role of the ORF-X/P-47 proteins, the conditions of their expression, or whether they have ability, as is the case for HA, to form and stabilize toxin complexes. We performed alignment and phylogenetic analysis of the ORF-X/P-47 gene cluster proteins to better understand the degree of uniqueness of this cluster to C. botulinum.    METHODS: All available ORF-X2, ORF-X3, and P-47-like proteins identified as containing the Clostridium P-47 domain were gathered through Pfam and PSI-BLAST to create an enriched set for phylogenetic observation; ORF-X1 was also evaluated. Individual gene clusters were obtained for a range of species and all ORF-X/P-47 domain proteins within each cluster were included for alignment and phylogenetic analysis via maximum likelihood phylogeny.  RESULTS P-47, ORF-X2-like, and ORF-X3-like proteins were observed to cluster in to three major clades, each containing a distinct Gram+ taxa consisting of Clostridia and Paenibacilli with ORF-X2-like proteins forming an internal node to ORF-X3-like proteins. In other species P-47 domain proteins were observed to occur in groups of two or more, often flanked by elements indicative of horizontal transfer. PSI-BLAST queries indicated C. botulinum ORF-X1 shares a distant homology with a ~150AA long residue present twice in the N-terminus of ORF-X2 and once in the N-terminus of P-47 & ORF-X3. The twice repeatedN-terminus feature in ORF-X2 was not observed in ORF-X2-like proteins identified in species outside of Clostridia/Paenibacilli. Transmembrane domains were predicted with some consistency in the C-terminus of ORF-X2 & ORF-X3-like proteins. Species specific gene duplications were observed fairly regularly.    CONCLUSIONS: P-47 domain containing proteins exist in a range of organisms beyond Clostridia including fungal and bacterial plant pathogen species, nitrogen fixing bacteria, and several arthropod parasites suggesting broad distribution throughout soil and marine sediment dwelling microorganisms. A fusion event between a P-47 domain containing protein and the N-terminus of ORF-X2 provides the most parsimonious explanation for the difference in length between the protein observed in C.  botulinum (~750AA) and the ORF-X2 grouping proteins observed in other species such as Erwinia tasmaniensis (~550AA). ORF-X1 homology with the N-terminal of P-47 domain containing proteins and fairly conserved synteny of the ORF-X operon within Clostridia offers the possibility of a common origin for all ORF-X proteins and P-47. Recent published findings suggest that ORF-X1 & X2 induced autolysis in E. coli, and may not be physically associated with botulinum toxin complexes. The diversity of species in which similar proteins can be found suggests a horizontal range that exceeds that of BoNT. It is possible that these proteins are unrelated to the toxigenicity of botulinum neurotoxin.