Showing papers by "Kettering University published in 2013"

Microtubule attachment and spindle assembly checkpoint signalling at the kinetochore.

Abstract: In eukaryotes, chromosome segregation during cell division is facilitated by the kinetochore, a multiprotein structure that is assembled on centromeric DNA. The kinetochore attaches chromosomes to spindle microtubules, modulates the stability of these attachments and relays the microtubule-binding status to the spindle assembly checkpoint (SAC), a cell cycle surveillance pathway that delays chromosome segregation in response to unattached kinetochores. Recent studies are shaping current thinking on how each of these kinetochore-centred processes is achieved, and how their integration ensures faithful chromosome segregation, focusing on the essential roles of kinase-phosphatase signalling and the microtubule-binding KMN protein network.

Systematic evaluation of spliced alignment programs for RNA-seq data

Abstract: High-throughput RNA sequencing is an increasingly accessible method for studying gene structure and activity on a genome-wide scale. A critical step in RNA-seq data analysis is the alignment of partial transcript reads to a reference genome sequence. To assess the performance of current mapping software, we invited developers of RNA-seq aligners to process four large human and mouse RNA-seq data sets. In total, we compared 26 mapping protocols based on 11 programs and pipelines and found major performance differences between methods on numerous benchmarks, including alignment yield, basewise accuracy, mismatch and gap placement, exon junction discovery and suitability of alignments for transcript reconstruction. We observed concordant results on real and simulated RNA-seq data, confirming the relevance of the metrics employed. Future developments in RNA-seq alignment methods would benefit from improved placement of multimapped reads, balanced utilization of existing gene annotation and a reduced false discovery rate for splice junctions.

Intestinal Microbiota Containing Barnesiella Species Cures Vancomycin-Resistant Enterococcus faecium Colonization

Abstract: Bacteria causing infections in hospitalized patients are increasingly antibiotic resistant. Classical infection control practices are only partially effective at preventing spread of antibiotic-resistant bacteria within hospitals. Because the density of intestinal colonization by the highly antibiotic-resistant bacterium vancomycin-resistant Enterococcus (VRE) can exceed 10(9) organisms per gram of feces, even optimally implemented hygiene protocols often fail. Decreasing the density of intestinal colonization, therefore, represents an important approach to limit VRE transmission. We demonstrate that reintroduction of a diverse intestinal microbiota to densely VRE-colonized mice eliminates VRE from the intestinal tract. While oxygen-tolerant members of the microbiota are ineffective at eliminating VRE, administration of obligate anaerobic commensal bacteria to mice results in a billionfold reduction in the density of intestinal VRE colonization. 16S rRNA gene sequence analysis of intestinal bacterial populations isolated from mice that cleared VRE following microbiota reconstitution revealed that recolonization with a microbiota that contains Barnesiella correlates with VRE elimination. Characterization of the fecal microbiota of patients undergoing allogeneic hematopoietic stem cell transplantation demonstrated that intestinal colonization with Barnesiella confers resistance to intestinal domination and bloodstream infection with VRE. Our studies indicate that obligate anaerobic bacteria belonging to the Barnesiella genus enable clearance of intestinal VRE colonization and may provide novel approaches to prevent the spread of highly antibiotic-resistant bacteria.

Adult-specific functions of animal microRNAs

Abstract: MicroRNAs (miRNAs) are ~22 nt RNAs that coordinate vast regulatory networks in animals and thereby influence myriad processes. This Review examines evidence that miRNAs have continuous roles in adults in ways that are separable from developmental control. Adult-specific activities for miRNAs have been described in various stem cell populations, in the context of neural function and cardiovascular biology, in metabolism and ageing, and during cancer. In addition to reviewing recent results, we also discuss methods for studying miRNA activities specifically in adults and evaluate their relative strengths and weaknesses. A fuller understanding of continuous functions of miRNAs in adults has bearing on efforts and opportunities to manipulate miRNAs for therapeutic purposes.

Widespread and extensive lengthening of 3′ UTRs in the mammalian brain

Abstract: Remarkable advances in techniques for gene expression profiling have radically changed our knowledge of the transcriptome. Recently, the mammalian brain was reported to express many long intergenic noncoding (lincRNAs) from loci downstream from protein-coding genes. Our experimental tests failed to validate specific accumulation of lincRNA transcripts, and instead revealed strongly distal 3' UTRs generated by alternative cleavage and polyadenylation (APA). With this perspective in mind, we analyzed deep mammalian RNA-seq data using conservative criteria, and identified 2035 mouse and 1847 human genes that utilize substantially distal novel 3' UTRs. Each of these extends at least 500 bases past the most distal 3' termini available in Ensembl v65, and collectively they add 6.6 Mb and 5.1 Mb to the mRNA space of mouse and human, respectively. Extensive Northern analyses validated stable accumulation of distal APA isoforms, including transcripts bearing exceptionally long 3' UTRs (many >10 kb and some >18 kb in length). The Northern data further illustrate that the extensions we annotated were not due to unprocessed transcriptional run-off events. Global tissue comparisons revealed that APA events yielding these extensions were most prevalent in the mouse and human brain. Finally, these extensions collectively contain thousands of conserved miRNA binding sites, and these are strongly enriched for many well-studied neural miRNAs. Altogether, these new 3' UTR annotations greatly expand the scope of post-transcriptional regulatory networks in mammals, and have particular impact on the central nervous system.

Systematic improvement of a classical molecular model of water.

Abstract: We report the iAMOEBA (“inexpensive AMOEBA”) classical polarizable water model. The iAMOEBA model uses a direct approximation to describe electronic polarizability, in which the induced dipoles are determined directly from the permanent multipole electric fields and do not interact with one another. The direct approximation reduces the computational cost relative to a fully self-consistent polarizable model such as AMOEBA. The model is parameterized using ForceBalance, a systematic optimization method that simultaneously utilizes training data from experimental measurements and high-level ab initio calculations. We show that iAMOEBA is a highly accurate model for water in the solid, liquid, and gas phases, with the ability to fully capture the effects of electronic polarization and predict a comprehensive set of water properties beyond the training data set including the phase diagram. The increased accuracy of iAMOEBA over the fully polarizable AMOEBA model demonstrates ForceBalance as a method that allo...

Spatially isotropic four-dimensional imaging with dual-view plane illumination microscopy

Abstract: Optimal four-dimensional imaging requires high spatial resolution in all dimensions, high speed and minimal photobleaching and damage. We developed a dual-view, plane illumination microscope with improved spatiotemporal resolution by switching illumination and detection between two perpendicular objectives in an alternating duty cycle. Computationally fusing the resulting volumetric views provides an isotropic resolution of 330 nm. As the sample is stationary and only two views are required, we achieve an imaging speed of 200 images/s (i.e., 0.5 s for a 50-plane volume). Unlike spinning-disk confocal or Bessel beam methods, which illuminate the sample outside the focal plane, we maintain high spatiotemporal resolution over hundreds of volumes with negligible photobleaching. To illustrate the ability of our method to study biological systems that require high-speed volumetric visualization and/or low photobleaching, we describe microtubule tracking in live cells, nuclear imaging over 14 h during nematode embryogenesis and imaging of neural wiring during Caenorhabditis elegans brain development over 5 h.

FGF4 is required for lineage restriction and salt-and-pepper distribution of primitive endoderm factors but not their initial expression in the mouse

Abstract: The emergence of pluripotent epiblast (EPI) and primitive endoderm (PrE) lineages within the inner cell mass (ICM) of the mouse blastocyst involves initial co-expression of lineage-associated markers followed by mutual exclusion and salt-and-pepper distribution of lineage-biased cells. Precisely how EPI and PrE cell fate commitment occurs is not entirely clear; however, previous studies in mice have implicated FGF/ERK signaling in this process. Here, we investigated the phenotype resulting from zygotic and maternal/zygotic inactivation of Fgf4. Fgf4 heterozygous blastocysts exhibited increased numbers of NANOG-positive EPI cells and reduced numbers of GATA6-positive PrE cells, suggesting that FGF signaling is tightly regulated to ensure specification of the appropriate numbers of cells for each lineage. Although the size of the ICM was unaffected in Fgf4 null mutant embryos, it entirely lacked a PrE layer and exclusively comprised NANOG-expressing cells at the time of implantation. An initial period of widespread EPI and PrE marker co-expression was however established even in the absence of FGF4. Thus, Fgf4 mutant embryos initiated the PrE program but exhibited defects in its restriction phase, when lineage bias is acquired. Consistent with this, XEN cells could be derived from Fgf4 mutant embryos in which PrE had been restored and these cells appeared indistinguishable from wild-type cells. Sustained exogenous FGF failed to rescue the mutant phenotype. Instead, depending on concentration, we noted no effect or conversion of all ICM cells to GATA6-positive PrE. We propose that heterogeneities in the availability of FGF produce the salt-and-pepper distribution of lineage-biased cells.

Nuclear positioning in muscle development and disease.

Abstract: Muscle disease as a group is characterized by muscle weakness, muscle loss, and impaired muscle function. Although the phenotype is the same, the underlying cellular pathologies, and the molecular causes of these pathologies, are diverse. One common feature of many muscle disorders is the mispositioning of myonuclei. In unaffected individuals, myonuclei are spaced throughout the periphery of the muscle fiber such that the distance between nuclei is maximized. However, in diseased muscles, the nuclei are often clustered within the center of the muscle cell. Although this phenotype has been acknowledged for several decades, it is often ignored as a contributor to muscle weakness. Rather, these nuclei are taken only as a sign of muscle repair. Here we review the evidence that mispositioned myonuclei are not merely a symptom of muscle disease but also a cause. Additionally, we review the working models for how myonuclei move from two different perspectives: from that of the nuclei and from that of the cytoskeleton. We further compare and contrast these mechanisms with the mechanisms of nuclear movement in other cell types both to draw general themes for nuclear movement and to identify muscle-specific considerations. Finally, we focus on factors that can be linked to muscle disease and find that genes that regulate myonuclear movement and positioning have been linked to muscular dystrophy. Although the cause-effect relationship is largely speculative, recent data indicate that the position of nuclei should no longer be considered only a means to diagnose muscle disease.

Multidimensional gas chromatography: fundamental advances and new applications

Abstract: ■ CONTENTS Main Types of MDGC 558 Heart-Cutting Two-Dimensional Gas Chromatography 558 Comprehensive Two-Dimensional Gas Chromatography 558 Fundamental Advances 558 New Modes of Implementation 558 Hybrid MDGC 558 Smart MDGC Interfaces 559 High-Speed GC × GC 559 High Temperature GC × GC 559 Coupling SFC to GC × GC 559 GC × GC Hardware Development and Method Optimization 559 New GC × GC Modulators 559 Comparison of Thermal and Flow Modulation 560 Studying the Precision of Low Duty Cycle Modulation GC × GC 560 Influence of Experimental Conditions on GC × GC Separations 560 Improved Mass Spectrometric Detection for GC × GC 561 Ionic Liquid Stationary Phases and MDGC 562 Unique Combinations of GC × GC Stationary Phases 562 GC × GC Retention Models 562 Predicting GC × GC Retention Times 562 Retention Indices and GC × GC 562 GC × GC Data Analysis 563 Improved 2-D Peak Integration 563 Multivariate Analysis of GC × GC Chromatograms 563 Applications of MDGC 564 Fuels and Industrial Chemicals 564 Examining Low Volatility/High Complexity Mixtures 564 Petroleum Biomarker Determination 565 Composition of Coal-Derived Materials 565 Alternative Fuel Characterization 566 GC × GC Separations of Miscellaneous Fuels and Industrial Mixtures 567 Environmental Analysis 567 Atmospheric Analysis 567 Water Analysis 567 Soil and Sediments 568 Biological Matrices 568 Commercial Products 569 Foods, Flavors, and Fragrances 569 Essential Oils 569 Personal Care Products 570 Agricultural Products 570 Wine Analysis 571 Biological Studies 572 Drug Analysis 572 VOC Emissions Resulting From Decaying Flesh 572 VOC Emissions From Specific Organisms 572 Analysis of Cells and Biofluids 573 Metabolomics 573 Summary of Instrument Configurations used for Applications 574 Conclusions 575 Author Information 575 Corresponding Author 575 Notes 575 Biographies 575 References 575

Nonsense-Mediated Decay of Alternative Precursor mRNA Splicing Variants Is a Major Determinant of the Arabidopsis Steady State Transcriptome

Abstract: The nonsense-mediated decay (NMD) surveillance pathway can recognize erroneous transcripts and physiological mRNAs, such as precursor mRNA alternative splicing (AS) variants. Currently, information on the global extent of coupled AS and NMD remains scarce and even absent for any plant species. To address this, we conducted transcriptome-wide splicing studies using Arabidopsis thaliana mutants in the NMD factor homologs UP FRAMESHIFT1 (UPF1) and UPF3 as well as wild-type samples treated with the translation inhibitor cycloheximide. Our analyses revealed that at least 17.4% of all multi-exon, protein-coding genes produce splicing variants that are targeted by NMD. Moreover, we provide evidence that UPF1 and UPF3 act in a translation-independent mRNA decay pathway. Importantly, 92.3% of the NMD-responsive mRNAs exhibit classical NMD-eliciting features, supporting their authenticity as direct targets. Genes generating NMD-sensitive AS variants function in diverse biological processes, including signaling and protein modification, for which NaCl stress–modulated AS-NMD was found. Besides mRNAs, numerous noncoding RNAs and transcripts derived from intergenic regions were shown to be NMD responsive. In summary, we provide evidence for a major function of AS-coupled NMD in shaping the Arabidopsis transcriptome, having fundamental implications in gene regulation and quality control of transcript processing.

Erythropoietin derived by chemical synthesis.

Abstract: Erythropoietin is a signaling glycoprotein that controls the fundamental process of erythropoiesis, orchestrating the production and maintenance of red blood cells. As administrated clinically, erythropoietin has a polypeptide backbone with complex dishomogeneity in its carbohydrate domains. Here we describe the total synthesis of homogeneous erythropoietin with consensus carbohydrate domains incorporated at all of the native glycosylation sites. The oligosaccharide sectors were built by total synthesis and attached stereospecifically to peptidyl fragments of the wild-type primary sequence, themselves obtained by solid-phase peptide synthesis. The glycopeptidyl constructs were joined by chemical ligation, followed by metal-free dethiylation, and subsequently folded. This homogeneous erythropoietin glycosylated at the three wild-type aspartates with N-linked high-mannose sialic acid–containing oligosaccharides and O-linked glycophorin exhibits Procrit-level in vivo activity in mice.

LRP-6 is a coreceptor for multiple fibrogenic signaling pathways in pericytes and myofibroblasts that are inhibited by DKK-1

Abstract: Fibrosis of vital organs is a major public health problem with limited therapeutic options. Mesenchymal cells including microvascular mural cells (pericytes) are major progenitors of scar-forming myofibroblasts in kidney and other organs. Here we show pericytes in healthy kidneys have active WNT/β-catenin signaling responses that are markedly up-regulated following kidney injury. Dickkopf-related protein 1 (DKK-1), a ligand for the WNT coreceptors low-density lipoprotein receptor-related proteins 5 and 6 (LRP-5 and LRP-6) and an inhibitor of WNT/β-catenin signaling, effectively inhibits pericyte activation, detachment, and transition to myofibroblasts in vivo in response to kidney injury, resulting in attenuated fibrogenesis, capillary rarefaction, and inflammation. DKK-1 blocks activation and proliferation of established myofibroblasts in vitro and blocks pericyte proliferation to PDGF, pericyte migration, gene activation, and cytoskeletal reorganization to TGF-β or connective tissue growth factor. These effects are largely independent of inhibition of downstream β-catenin signaling. DKK-1 acts predominantly by inhibiting PDGF-, TGF-β–, and connective tissue growth factor-activated MAPK and JNK signaling cascades, acting via LRP-6 with associated WNT ligand. Biochemically, LRP-6 interacts closely with PDGF receptor β and TGF-β receptor 1 at the cell membrane, suggesting that it may have roles in pathways other than WNT/β-catenin. In summary, DKK-1 blocks many of the changes in pericytes required for myofibroblast transition and attenuates established myofibroblast proliferation/activation by mechanisms dependent on LRP-6 and WNT ligands but not the downstream β-catenin pathway.

Clinical activity of ipilimumab for metastatic uveal melanoma: a retrospective review of the Dana-Farber Cancer Institute, Massachusetts General Hospital, Memorial Sloan-Kettering Cancer Center, and University Hospital of Lausanne experience.

Abstract: BACKGROUND Uveal melanoma exhibits a high incidence of metastases; and, to date, there is no systemic therapy that clearly improves outcomes. The anticytotoxic T-lymphocyte–associated protein 4 (anti-CTLA-4) antibody ipilimumab is a standard of care for metastatic melanoma; however, the clinical activity of CTLA-4 inhibition in patients with metastatic uveal melanoma is poorly defined. METHODS To assess ipilimumab in this setting, the authors performed a multicenter, retrospective analysis of 4 hospitals in the United States and Europe. Clinical characteristics, toxicities, and radiographic disease burden, as determined by central, blinded radiology review, were evaluated. RESULTS Thirty-nine patients with uveal melanoma were identified, including 34 patients who received 3 mg/kg ipilimumab and 5 who received 10 mg/kg ipilimumab. Immune-related response criteria and modified World Health Organization criteria were used to assess the response rate (RR) and the combined response plus stable disease (SD) rate after 12 weeks, after 23 weeks, and overall (median follow-up, 50.4 weeks [12.6 months]). At week 12, the RR was 2.6%, and the response plus SD rate was 46.%; at week 23, the RR was 2.6%, and the response plus SD rate was 28.2%. There was 1 complete response and 1 late partial response (at 100 weeks after initial SD) for an immune-related RR of 5.1%. Immune-related adverse events were observed in 28 patients (71.8%) and included 7 (17.9%) grade 3 and 4 events. Immune-related adverse events were more frequent in patients who received 10 mg/kg ipilimumab than in those who received 3 mg/kg ipilimumab. The median overall survival from the first dose of ipilimumab was 9.6 months (95% confidence interval, 6.3-13.4 months; range, 1.6-41.6 months). Performance status, lactate dehydrogenase level, and an absolute lymphocyte count ≥1000 cells/μL at week 7 were associated significantly with survival. CONCLUSIONS In this multicenter, retrospective analysis of 4 hospitals in the United States and Europe of patients with uveal melanoma, durable responses to ipilimumab and manageable toxicity were observed. Cancer 2013;119:3687–3695. © 2013 American Cancer Society.

Th2 differentiation is necessary for soft tissue fibrosis and lymphatic dysfunction resulting from lymphedema

Abstract: Lymphedema is a dreaded complication of cancer treatment. However, despite the fact that >5 million Americans are affected by this disorder, the development of effective treatments is limited by the fact that the pathology of lymphedema remains unknown. The purpose of these studies was to determine the role of inflammatory responses in lymphedema pathology. Using mouse models of lymphedema, as well as clinical lymphedema specimens, we show that lymphatic stasis results in a CD4 T-cell inflammation and T-helper 2 (Th2) differentiation. Using mice deficient in T cells or CD4 cells, we show that this inflammatory response is necessary for the pathological changes of lymphedema, including fibrosis, adipose deposition, and lymphatic dysfunction. Further, we show that inhibition of Th2 differentiation using interleukin-4 (IL-4) or IL-13 blockade prevents initiation and progression of lymphedema by decreasing tissue fibrosis and significantly improving lymphatic function, independent of lymphangiogenic growth factors. We show that CD4 inflammation is a critical regulator of tissue fibrosis and lymphatic dysfunction in lymphedema and that inhibition of Th2 differentiation markedly improves lymphatic function independent of lymphangiogenic cytokine expression. Notably, preventing and/or reversing the development of pathological tissue changes that occur in lymphedema may be a viable treatment strategy for this disorder.

Paralog-selective Hsp90 inhibitors define tumor-specific regulation of HER2

Abstract: Specific inhibitors against Hsp90 paralogs show that Hsp90 and Grp94 regulate HER2 in a cell-specific and proteome alteration-driven manner.

Human pluripotent stem cells: an emerging model in developmental biology

Abstract: Developmental biology has long benefited from studies of classic model organisms. Recently, human pluripotent stem cells (hPSCs), including human embryonic stem cells and human induced pluripotent stem cells, have emerged as a new model system that offers unique advantages for developmental studies. Here, we discuss how studies of hPSCs can complement classic approaches using model organisms, and how hPSCs can be used to recapitulate aspects of human embryonic development ‘in a dish’. We also summarize some of the recently developed genetic tools that greatly facilitate the interrogation of gene function during hPSC differentiation. With the development of high-throughput screening technologies, hPSCs have the potential to revolutionize gene discovery in mammalian development.

Clinically-translated silica nanoparticles as dual-modality cancer-targeted probes for image-guided surgery and interventions

Abstract: Early diagnosis and treatment of melanoma are essential to minimizing morbidity and mortality. The presence of lymph node metastases is a vital prognostic predictor, and accurate identification by imaging has important implications for disease staging, prognosis, and clinical outcome. Sentinel lymph node (SLN) mapping procedures are limited by a lack of intraoperative visualization tools that can aid accurate determination of disease spread and delineate nodes from adjacent critical neural and vascular structures. Newer methods for circumventing these issues can exploit a variety of imaging tools, including biocompatible particle-based platforms coupled with portable device technologies for use with image-guided surgical and interventional procedures. We describe herein a clinically-translated, integrin-targeting platform for use with both PET and optical imaging that meets a number of key design criteria for improving SLN tissue localization and retention, target-to-background ratios, and clearance from the site of injection and the body. The use of such agents for selectively probing critical cancer targets may elucidate important insights into cellular and molecular processes that govern metastatic disease spread. Coupled with portable, real-time optical camera systems, we show that pre-operative PET imaging findings for mapping metastatic disease in clinically-relevant larger-animal models can be readily translated into the intraoperative setting for direct visualization of the draining tumor lymphatics and fluorescent SLN/s with histologic correlation. The specificity of this platform, relative to the standard-of-care radiotracer, 18F-FDG, for potentially discriminating metastatic disease from inflammatory processes is also discussed in the setting of surgically-based or interventionally-driven therapies.

Homeostatic control of Argonaute stability by microRNA availability.

Abstract: Homeostatic mechanisms regulate the abundance of several components in small-RNA pathways. We used Drosophila and mammalian systems to demonstrate a conserved homeostatic system in which the status of miRNA biogenesis controls Argonaute protein stability. Clonal analyses of multiple mutants of core Drosophila miRNA factors revealed that stability of the miRNA effector AGO1 is dependent on miRNA biogenesis. Reciprocally, ectopic transcription of miRNAs within in vivo clones induced accumulation of AGO1, as did genetic interference with the ubiquitin-proteasome system. In mouse cells, we found that the stability of Ago2 declined in Dicer-knockout cells and was rescued by proteasome blockade or introduction of either Dicer plasmid or Dicer-independent miRNA constructs. Notably, Dicer-dependent miRNA constructs generated pre-miRNAs that bound Ago2 but did not rescue Ago2 stability. We conclude that Argonaute levels are finely tuned by cellular availability of mature miRNAs and the ubiquitin-proteasome system.

MicroRNA-205 controls neonatal expansion of skin stem cells by modulating the PI(3)K pathway

Abstract: Skin stem cells are amplified in early development. Yi and colleagues found that miR-205 promotes neonatal skin stem cell expansion by directly targeting negative regulators of PI(3)K–Akt signalling to maintain proliferation.

A membrane-associated β-catenin/Oct4 complex correlates with ground-state pluripotency in mouse embryonic stem cells

Abstract: The maintenance of pluripotency in mouse embryonic stem cells (mESCs) relies on the activity of a transcriptional network that is fuelled by the activity of three transcription factors (Nanog, Oct4 and Sox2) and balanced by the repressive activity of Tcf3. Extracellular signals modulate the activity of the network and regulate the differentiation capacity of the cells. Wnt/β-catenin signaling has emerged as a significant potentiator of pluripotency: increases in the levels of β-catenin regulate the activity of Oct4 and Nanog, and enhance pluripotency. A recent report shows that β-catenin achieves some of these effects by modulating the activity of Tcf3, and that this effect does not require its transcriptional activation domain. Here, we show that during self-renewal there is negligible transcriptional activity of β-catenin and that this is due to its tight association with membranes, where we find it in a complex with Oct4 and E-cadherin. Differentiation triggers a burst of Wnt/β-catenin transcriptional activity that coincides with the disassembly of the complex. Our results establish that β-catenin, but not its transcriptional activity, is central to pluripotency acting through a β-catenin/Oct4 complex.

A Vision for Vaccines Built from Fully Synthetic Tumor-Associated Antigens: From the Laboratory to the Clinic

Abstract: Cancer cells may be distinguished from normal cells by cell surface displays of aberrant levels and types of carbohydrate domains. Accordingly, these tumor-associated carbohydrate antigens (TACAs) represent promising target structures for the design of anticancer vaccines. Over the past 20 years, our laboratory has sought to use the tools of chemical synthesis to develop TACA-based anticancer vaccine candidates. We provide herein a personal accounting of our laboratory’s progress toward the long-standing goal of developing clinically viable fully synthetic carbohydrate-based anticancer vaccines.