Studies of the human CCR3 chemokine receptor: development of a cell line stably expressing CCR3, receptor purification and characterization, and phosphopeptide enrichment methods to study the CCR3 GPCR signaling pathway

Studies of the human CCR3 chemokine receptor: development of a cell line stably expressing CCR3, receptor purification and characterization by mass spectrometry, and phosphoproteomic methods to study the CCR3 GPCR signaling pathway. Chemokine Receptors are a class of G Protein coupled receptors (GPCRs) or transmembrane (TM) serpentine receptors present on cell membranes which act as gate keepers and signal transducers for the cells. Cellular homeostasis is maintained by GPCRs by controlling the movement of various signals and molecules from exterior to interior of the cell. Chemokine receptor subtype 3 has a critical role in homeostasis in organ systems in human body, a novel target in age-related macular degeneration disease progression, and act as co-receptor for HIV entry into cells. Their roles range from the mediation of early stage allergic and inflammatory responses, to host cell defenses and related physiological roles. Little is known about their structure - function properties at the receptor level and the downstream signaling events after the receptor is stimulated. My dissertation focuses on Chemokine receptor subtype 3 (CCR3). CCR3 expressing cell lines available to date are mortal cell line, meant for single use assay purpose with limited/transient CCR3 receptor expression. These cell lines are not a viable option for CCR3 receptor expression-purification and stabilization for biophysical and related structural studies. My work focused on developing a human endothelial kidney (HEK 293S) cell line stably expressing human CCR3 using a tetracycline inducible mammalian protein expression vector. This cell line is immortal and can be propagated for cell culture scale-up for semi-preparative scale purification of CCR3. The HEK 293S CCR3 cell line was used specifically for two purposes, as elaborated in this dissertation. The first is to overexpress the human CCR3 receptor for purification and characterization, by establishing a standard membrane protein purification method for human CCR3 membrane protein. Cellular membrane protein expression in human cells is one of the tough challenges in protein biochemistry. Membrane proteins lose their structural and functional integrity once removed from their lipid bilayer environment in cell membranes; a membrane protein is stabilized in its native biophysical environment. As a fundamental pre-requisite for maintaining the near-native conditions around the membrane protein molecule during its extraction and purification, one has to maintain its biophysical integrity and to preserve the structural and functional features of the protein during the overall extraction and purification processes. The purified human CCR3 has several uses, such as 1) in protein sequencing to identify any possible sequence variants and post translational modifications in the protein, 2) to obtain data useful in receptor modeling and structure-based drug discovery efforts of CCR3, 3) biophysical characterization of the receptor at a single molecule level and its dimer and oligomeric states, and 4) immobilizing GPCRs on surfaces for ligand/drug screening with SPRI-based methods. All the above said uses require human CCR3 receptor purified in significant quantities; micrograms to milligrams. Here we successfully characterized a limited amino acid sequence of the purified CCR3 GPCR by mass spectrometry based methods. In the second objective, we were able to successfully adapt the laboratory developed CCR3 expressing HEK 293S cell line to stable isotope amino acid enriched DMEM supplemented with 10 % dialyzed FBS cell culture media. The C13 and N15 labeled Arginine (+10 Dalton) and Lysine (+8 Dalton) isotopic enrichment of the cell line was greater than ninety-five percent. This cell line was intended for tht study of CCR3 receptor downstream signaling events by phosphoproteomics studies. Temporal phosphorylation of the signaling protein in the cell is the rationale behind global cellular phosphorylation analysis. Protein phosphorylation is the second most common post-translation modification (PTM) after acetylation. Reversible phosphorylation is critical in the functional aspects of cellular proteome and the signaling events involving biochemical pathways. The overall phosphopeptide enrichment for complex SILAC HEK 293S whole cell lysate protein digested peptide samples is developed with titanium dioxide nanoparticles (TiO2) as a metal ion affinity resin. The developed SILAC workflow can be applied to study temporal phosphorylation dynamics for different cellular physiological states, following CCR3 receptor stimulation. In summary, the results emphasize overall stable cell line development of CCR3 receptor expression in HEK 293S, analytical methods in CCR3 characterization, and methods in phosphopeptide enrichment from complex protein samples such as HEK 293S cell lines

Molecular Modeling Studies of Curcumin Analogs as Anti-Angiogenic Agents

Angiogenesis plays a pivotal role in the metastasis of cancer: curcumin showed excellent anti-angiogenesis activity on metastatic tumors. Several curcumin analogues have been synthesized and studied, and their biological activity was reported in the literature. One class of potent analogues are aromatic enones. In Dr Bowen's laboratory sixty three compounds were synthesized and in the laboratory of Dr Jack Arbizer (Emory University, Atlanta, GA) they were tested for their anti-angiogenic activity with an SVR endothelial cell growth assay developed by Dr Arbizer. The precise mechanism or the specific biological target on which these analogs exert their inhibition potential as anti-angiogenic agents is unknown. Therefore, structure-based molecular modeling is not a possibility. However, ligand based molecular modeling methods are available for studying and predicting which compounds among the sixty three can be further optimized for selectivity and desired property. Computational studies were carried out to identify which structural features within the series of analogues are significantly important for activity. Initially, pharmacophore modeling was carried out in Molecular Operating Environment (MOE) software to identify the Interaction Pharmacophore Elements (IPE) and their relative geometry in three-dimensional space. Two different three dimensional quantitative structural Activity Relationship (3D-QSAR) studies, Comparative Molecular Field Analysis (CoMFA), and Comparative Molecular Similarity Indices Analysis (CoMSIA) were carried out with this dataset. SYBYL (versions 7.2 and 7.3) were used for the development of the models. Forty six compounds were used as the calibration or the training set. The model yielded a cross validated q2 of 0.289 for CoMFA and 0.146 for CoMSIA analyses. Eleven compounds were used as the test set (or the prediction) set to externally validate the QSAR models and their robustness. The predictions of the model are acceptable with a few outliers

Capacity Gaps in Post Disaster Waste Management: Case Study in Sri Lanka

Disaster waste is one of the major consequences aftermath of any disaster, impacts on public and environment, rescue and emergency services, provision of lifeline support and socio-economic recovery of affected areas. Thus, management of wastes created by disasters has become an increasingly important issue to be addressed in responding to a disaster. This chapter intends to present the prevailing gaps in disaster waste management and approaches to minimize the impacts on disaster management at developing countries with special emphasis to Sri Lankan context. Findings revealed that, unavailability of single point responsibility and provisions for disaster waste in existing policies and capacity constraints of the prevailing peace time solid waste management practices as major capacity gaps. Establishment of a regulatory body and enforceable rules and regulations with necessary levels of capacities were identified with seven areas for capacity building for post disaster waste management. The research enabled to attain sustainable post disaster waste management for future resilience

Paying it Forward: A Strategy for Developing a Culture of Lifelong Engagement and Inclusion for First-Generation Students

This project aims to propose a strategy for VCU to increase the engagement and connection among First-Generation VCU students to create lifelong commitment as alumni through VCU’s Office of Development and Alumni Relations (DAR). The team proposes to expand DAR’s outreach through collaboration with key VCU stakeholders (e.g., YOU First at VCU). By developing a positive undergraduate experience, students’ loyalty to VCU will result in greater engagement as alumni, paying it forward

Does Transit Service Reliability Influence Ridership?

69A3551747127This research focused on analyzing the association between transit service reliability indicators and ridership. Further, the effect of road network, demographic, socioeconomic, and land use characteristics on transit service reliability was analyzed. The analysis was conducted at a bus stop level. Bus arrival/departure and ridership data from the Charlotte Area Transit System (CATS) was obtained. The road network, demographic, socioeconomic, and land use characteristics were captured within 0.25-mile and 0.50-mile buffers. Pearson correlation analysis was conducted to understand the association between road network, demographic, socioeconomic, and land use characteristics and bus transit service reliability measures. The results show that bus transit service reliability has a substantial impact on ridership and is influenced by road network, demographic, socioeconomic, and land use characteristics within the bus stop vicinity. The findings help public transportation agencies to effectively utilize available resources, plan, and provide equitable services to all riders

Adaptive Evolution of Escherichia coli to an α-Peptide/β-Peptoid Peptidomimetic Induces Stable Resistance.

Antimicrobial peptides (AMPs) and synthetic analogues thereof target conserved structures of bacterial cell envelopes and hence, development of resistance has been considered an unlikely event. However, recently bacterial resistance to AMPs has been observed, and the aim of the present study was to determine whether bacterial resistance may also evolve against synthetic AMP analogues, e.g. α-peptide/β-peptoid peptidomimetics. E. coli ATCC 25922 was exposed to increasing concentrations of a peptidomimetic (10 lineages), polymyxin B (10 lineages), or MilliQ water (4 lineages) in a re-inoculation culturing setup covering approx. 500 generations. All 10 lineages exposed to the peptidomimetic adapted to 32 × MIC while this occurred for 8 out of 10 of the polymyxin B-exposed lineages. All lineages exposed to 32 × MIC of either the peptidomimetic or polymyxin B had a significantly increased MIC (16-32 ×) to the selection agent. Five transfers (≈ 35 generations) in unsupplemented media did not abolish resistance indicating that resistance was heritable. Single isolates from peptidomimetic-exposed lineage populations displayed MICs against the peptidomimetic from wild-type MIC to 32 × MIC revealing heterogeneous populations. Resistant isolates showed no cross-resistance against a panel of membrane-active AMPs. These isolates were highly susceptible to blood plasma antibacterial activity and were killed when plasma concentrations exceeded ≈ 30%. Notably, MIC of the peptidomimetic against resistant isolates returned to wild-type level upon addition of 25% plasma. Whole-genome sequencing of twenty isolates from four resistant lineages revealed mutations, in murein transglycosylase D (mltD) and outer-membrane proteins, which were conserved within and between lineages. However, no common resistance-conferring mutation was identified. We hypothesise that alterations in cell envelope structure result in peptidomimetic resistance, and that this may occur via several distinct mechanisms. Interestingly, this type of resistance result in a concomitant high susceptibility towards plasma, and therefore the present study does not infer additional concern for peptidomimetics as future therapeutics

Bcl11b sets pro-T cell fate by site-specific cofactor recruitment and by repressing Id2 and Zbtb16

Multipotent progenitor cells confirm their T cell–lineage identity in the CD4^–CD8^– double-negative (DN) pro-T cell DN2 stages, when expression of the essential transcription factor Bcl11b begins. In vivo and in vitro stage-specific deletions globally identified Bcl11b-controlled target genes in pro-T cells. Proteomics analysis revealed that Bcl11b associated with multiple cofactors and that its direct action was needed to recruit those cofactors to selective target sites. Regions near functionally regulated target genes showed enrichment for those sites of Bcl11b-dependent recruitment of cofactors, and deletion of individual cofactors relieved the repression of many genes normally repressed by Bcl11b. Runx1 collaborated with Bcl11b most frequently for both activation and repression. In parallel, Bcl11b indirectly regulated a subset of target genes by a gene network circuit via the transcription inhibitor Id2 (encoded by Id2) and transcription factor PLZF (encoded by Zbtb16); Id2 and Zbtb16 were directly repressed by Bcl11b, and Id2 and PLZF controlled distinct alternative programs. Thus, our study defines the molecular basis of direct and indirect Bcl11b actions that promote T cell identity and block alternative potentials

Single-Cell Expression Profiling Reveals a Dynamic State of Cardiac Precursor Cells in the Early Mouse Embryo

In the early vertebrate embryo, cardiac progenitor/precursor cells (CPs) give rise to cardiac structures. Better understanding their biological character is critical to understand the heart development and to apply CPs for the clinical arena. However, our knowledge remains incomplete. With the use of single-cell expression profiling, we have now revealed rapid and dynamic changes in gene expression profiles of the embryonic CPs during the early phase after their segregation from the cardiac mesoderm. Progressively, the nascent mesodermal gene Mesp1 terminated, and Nkx2-5+/Tbx5+ population rapidly replaced the Tbx5low+ population as the expression of the cardiac genes Tbx5 and Nkx2-5 increased. At the Early Headfold stage, Tbx5-expressing CPs gradually showed a unique molecular signature with signs of cardiomyocyte differentiation. Lineage-tracing revealed a developmentally distinct characteristic of this population. They underwent progressive differentiation only towards the cardiomyocyte lineage corresponding to the first heart field rather than being maintained as a progenitor pool. More importantly, Tbx5 likely plays an important role in a transcriptional network to regulate the distinct character of the FHF via a positive feedback loop to activate the robust expression of Tbx5 in CPs. These data expands our knowledge on the behavior of CPs during the early phase of cardiac development, subsequently providing a platform for further study

Phylogenetically and spatially close marine sponges harbour divergent bacterial communities

Recent studies have unravelled the diversity of sponge-associated bacteria that may play essential roles in sponge health and metabolism. Nevertheless, our understanding of this microbiota remains limited to a few host species found in restricted geographical localities, and the extent to which the sponge host determines the composition of its own microbiome remains a matter of debate. We address bacterial abundance and diversity of two temperate marine sponges belonging to the Irciniidae family - Sarcotragus spinosulus and Ircinia variabilis – in the Northeast Atlantic. Epifluorescence microscopy revealed that S. spinosulus hosted significantly more prokaryotic cells than I. variabilis and that prokaryotic abundance in both species was about 4 orders of magnitude higher than in seawater. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) profiles of S. spinosulus and I. variabilis differed markedly from each other – with higher number of ribotypes observed in S. spinosulus – and from those of seawater. Four PCR-DGGE bands, two specific to S. spinosulus, one specific to I. variabilis, and one present in both sponge species, affiliated with an uncultured sponge-specific phylogenetic cluster in the order Acidimicrobiales (Actinobacteria). Two PCR-DGGE bands present exclusively in S. spinosulus fingerprints affiliated with one sponge-specific phylogenetic cluster in the phylum Chloroflexi and with sponge-derived sequences in the order Chromatiales (Gammaproteobacteria), respectively. One Alphaproteobacteria band specific to S. spinosulus was placed in an uncultured sponge-specific phylogenetic cluster with a close relationship to the genus Rhodovulum. Our results confirm the hypothesized host-specific composition of bacterial communities between phylogenetically and spatially close sponge species in the Irciniidae family, with S. spinosulus displaying higher bacterial community diversity and distinctiveness than I. variabilis. These findings suggest a pivotal host-driven effect on the shape of the marine sponge microbiome, bearing implications to our current understanding of the distribution of microbial genetic resources in the marine realm.This work was financed by the Portuguese Foundation for Science and Technology (FCT - http://www.fct.pt) through the research project PTDC/MAR/101431/2008. CCPH has a PhD fellowship granted by FCT (Grant No. SFRH/BD/60873/2009). JRX’s research is funded by a FCT postdoctoral fellowship (grant no. SFRH/BPD/62946/2009). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript