O-Glycome Beam Search Arrays for Carbohydrate Ligand Discovery.

O-glycosylation is a post-translational modification of proteins crucial to molecular mechanisms in health and disease. O-glycans are typically highly heterogeneous. The involvement of specific O-glycan sequences in many bio-recognition systems is yet to be determined because of a lack of efficient methodologies. We describe here a targeted microarray approach: O-glycome beam search that is both robust and efficient for O-glycan ligand-discovery. Substantial simplification of the complex O-glycome profile and facile chromatographic resolution is achieved by arraying O-glycans as branches, monitoring by mass spectrometry, focusing on promising fractions, and on-array immuno-sequencing. This is orders of magnitude more sensitive than traditional methods. We have applied beam search approach to porcine stomach mucin and identified extremely minor components previously undetected within the O-glycome of this mucin that are ligands for the adhesive proteins of two rotaviruses. The approach is applicable to O-glycome recognition studies in a wide range of biological settings to give insights into glycan recognition structures in natural microenvironments.

Syringe Decriminalization Advocacy in Red States: Lessons from the North Carolina Harm Reduction Coalition.

PURPOSE OF REVIEW: Syringe access programs (SAPs) are cornerstone harm reduction interventions for combatting the national opioid epidemic. The goal of this paper is to describe effective advocacy strategies for enacting syringe decriminalization legislation to foster the expansion of SAPs in high-need areas amidst political opposition. RECENT FINDINGS: Decades or research shows that SAPs prevent the transmission of HIV among people who inject drugs (PWID) and are a cost-effective tool for linking PWID to medical care, health education, and social services. In the USA, state laws criminalizing distribution and possession of syringes impede the expansion of SAPs into areas where they are sorely needed. In 2016, North Carolina became the first state to legalize SAPs with a Republican super majority. This paper distills strategies for community organizations seeking to advance syringe decriminalization legislation in politically conservative states with histories of prioritizing punitive sanctions over public health responses to drug use.

Access to Syringe Services Programs - Kentucky, North Carolina, and West Virginia, 2013-2017.

The Appalachian region of the United States is experiencing a large increase in hepatitis C virus (HCV) infections related to injection drug use (IDU) (1). Syringe services programs (SSPs) providing sufficient access to safe injection equipment can reduce hepatitis C transmission by 56%; combined SSPs and medication-assisted treatment can reduce transmission by 74% (2). However, access to SSPs has been limited in the United States, especially in rural areas and southern and midwestern states (3). This report describes the expansion of SSPs in Kentucky, North Carolina, and West Virginia during 2013-August 1, 2017. State-level data on the number of SSPs, client visits, and services offered were collected by each state through surveys of SSPs and aggregated in a standard format for this report. In 2013, one SSP operated in a free clinic in West Virginia, and SSPs were illegal in Kentucky and North Carolina; by August 2017, SSPs had been legalized in Kentucky and North Carolina, and 53 SSPs operated in the three states. In many cases, SSPs provide integrated services to address hepatitis and human immunodeficiency virus (HIV) infection, overdose, addiction, unintended pregnancy, neonatal abstinence syndrome, and other complications of IDU. Prioritizing development of SSPs with sufficient capacity, particularly in states with counties vulnerable to epidemics of hepatitis and HIV infection related to IDU, can expand access to care for populations at risk.

Unravelling glucan recognition systems by glycome microarrays using the designer approach and mass spectrometry.

Glucans are polymers of d-glucose with differing linkages in linear or branched sequences. They are constituents of microbial and plant cell-walls and involved in important bio-recognition processes, including immunomodulation, anticancer activities, pathogen virulence, and plant cell-wall biodegradation. Translational possibilities for these activities in medicine and biotechnology are considerable. High-throughput micro-methods are needed to screen proteins for recognition of specific glucan sequences as a lead to structure-function studies and their exploitation. We describe construction of a "glucome" microarray, the first sequence-defined glycome-scale microarray, using a "designer" approach from targeted ligand-bearing glucans in conjunction with a novel high-sensitivity mass spectrometric sequencing method, as a screening tool to assign glucan recognition motifs. The glucome microarray comprises 153 oligosaccharide probes with high purity, representing major sequences in glucans. Negative-ion electrospray tandem mass spectrometry with collision-induced dissociation was used for complete linkage analysis of gluco-oligosaccharides in linear "homo" and "hetero" and branched sequences. The system is validated using antibodies and carbohydrate-binding modules known to target α- or ß-glucans in different biological contexts, extending knowledge on their specificities, and applied to reveal new information on glucan recognition by two signaling molecules of the immune system against pathogens: Dectin-1 and DC-SIGN. The sequencing of the glucan oligosaccharides by the MS method and their interrogation on the microarrays provides detailed information on linkage, sequence and chain length requirements of glucan-recognizing proteins, and are a sensitive means of revealing unsuspected sequences in the polysaccharides.

Generation and characterization of ß1,2-gluco-oligosaccharide probes from Brucella abortus cyclic ß-glucan and their recognition by C-type lectins of the immune system.

The ß1,2-glucans produced by bacteria are important in invasion, survival and immunomodulation in infected hosts be they mammals or plants. However, there has been a lack of information on proteins which recognize these molecules. This is partly due to the extremely limited availability of the sequence-defined oligosaccharides and derived probes for use in the study of their interactions. Here we have used the cyclic ß1,2-glucan (CßG) of the bacterial pathogen Brucella abortus, after removal of succinyl side chains, to prepare linearized oligosaccharides which were used to generate microarrays. We describe optimized conditions for partial depolymerization of the cyclic glucan by acid hydrolysis and conversion of the ß1,2-gluco-oligosaccharides, with degrees of polymerization 2-13, to neoglycolipids for the purpose of generating microarrays. By microarray analyses, we show that the C-type lectin receptor DC-SIGNR, like the closely related DC-SIGN we investigated earlier, binds to the ß1,2-gluco-oligosaccharides, as does the soluble immune effector serum mannose-binding protein. Exploratory studies with DC-SIGN are suggestive of the recognition also of the intact CßG by this receptor. These findings open the way to unravelling mechanisms of immunomodulation mediated by ß1,2-glucans in mammalian systems.

Carbohydrate sequence of the prostate cancer-associated antigen F77 assigned by a mucin O-glycome designer array.

Monoclonal antibody F77 was previously raised against human prostate cancer cells and has been shown to recognize a carbohydrate antigen, but the carbohydrate sequence of the antigen was elusive. Here, we make multifaceted approaches to characterize F77 antigen, including binding analyses with the glycolipid extract of the prostate cancer cell line PC3, microarrays with sequence-defined glycan probes, and designer arrays from the O-glycome of an antigen-positive mucin, in conjunction with mass spectrometry. Our results reveal F77 antigen to be expressed on blood group H on a 6-linked branch of a poly-N-acetyllactosamine backbone. We show that mAb F77 can also bind to blood group A and B analogs but with lower intensities. We propose that the close association of F77 antigen with prostate cancers is a consequence of increased blood group H expression together with up-regulated branching enzymes. This is in contrast to other epithelial cancers that have up-regulated branching enzymes but diminished expression of H antigen. With knowledge of the structure and prevalence of F77 antigen in prostate cancer, the way is open to explore rationally its application as a biomarker to detect F77-positive circulating prostate cancer-derived glycoproteins and tumor cells.

Galactose recognition by the apicomplexan parasite Toxoplasma gondii.

Toxosplasma gondii is the model parasite of the phylum Apicomplexa, which contains numerous obligate intracellular parasites of medical and veterinary importance, including Eimeria, Sarcocystis, Cryptosporidium, Cyclospora, and Plasmodium species. Members of this phylum actively enter host cells by a multistep process with the help of microneme protein (MIC) complexes that play important roles in motility, host cell attachment, moving junction formation, and invasion. T. gondii (Tg)MIC1-4-6 complex is the most extensively investigated microneme complex, which contributes to host cell recognition and attachment via the action of TgMIC1, a sialic acid-binding adhesin. Here, we report the structure of TgMIC4 and reveal its carbohydrate-binding specificity to a variety of galactose-containing carbohydrate ligands. The lectin is composed of six apple domains in which the fifth domain displays a potent galactose-binding activity, and which is cleaved from the complex during parasite invasion. We propose that galactose recognition by TgMIC4 may compromise host protection from galectin-mediated activation of the host immune system.

The human epithelial carcinoma antigen recognized by monoclonal antibody AE3 is expressed on a sulfoglycolipid in addition to neoplastic mucins.

The term human epithelial carcinoma antigen (HCA) has been applied collectively to mucin-type high molecular weight (>1000kDa) glycoproteins that are over-expressed in epithelial cancers. Since the 1990s, over 40 monoclonal antibodies have been raised that recognize HCA. There has been evidence that the antigenic determinants are mostly carbohydrates, but details have been elusive. Here we have carried out carbohydrate microarray analyses of one of the monoclonal antibodies, AE3, that has been regarded the 'most carcinoma specific' in respect to its ability to detect HCA in sera of patients with epithelial cancers. The microarrays encompassed a series of 492 sequence-defined glycan probes in the form of glycolipids and neoglycolipids. We have thus established that the antigen recognized by antibody AE3 is a carbohydrate sequence distinct from the A, B, H, Lewis(a/b), Lewis(x/y) and T antigens, but that it is strongly expressed on the monosulfated tetra-glycosyl ceramide, SM1a, Galß1-3GalNAcß1-4(3-O-sulfate)Galß1-4GlcCer. This is the first report of an anti-HCA to be characterized with respect to its recognition sequence and of the occurrence of the antigen on a glycolipid as well as on glycoproteins. Knowledge of a discrete glycan sequence as target antigen now opens the way to its exploration as a serologic cancer biomarker, namely to determine if the antigen elicits an autoantibody response in early non-metastatic cancer, or if it is shed and immunochemically detectable in more advanced disease.

Altered receptor specificity and cell tropism of D222G hemagglutinin mutants isolated from fatal cases of pandemic A(H1N1) 2009 influenza virus.

Mutations in the receptor-binding site of the hemagglutinin of pandemic influenza A(H1N1) 2009 viruses have been detected sporadically. An Asp222Gly (D222G) substitution has been associated with severe or fatal disease. Here we show that 222G variants infected a higher proportion of ciliated cells in cultures of human airway epithelium than did viruses with 222D or 222E, which targeted mainly nonciliated cells. Carbohydrate microarray analyses showed that 222G variants bind a broader range of α2-3-linked sialyl receptor sequences of a type expressed on ciliated bronchial epithelial cells and on epithelia within the lung. These features of 222G mutants may contribute to exacerbation of disease.

Implant rehabilitation of edentulous maxilla in digital dentistry: A case report utilizing CAD/CAM technologies.

The replacement of missing teeth utilizing dental implants and digital dental technologies has gained significant popularity in daily clinical practice over the last decade. Partially dentate patients present more anatomical references to guide the implant position and prosthetic reconstruction as compared to completely edentulous arches. Therefore, the management of edentulous maxilla using implant digital dentistry represents a challenging clinical situation where a thorough treatment plan is paramount to achieve a final prosthetic result that meets both functional and esthetic requirements. This case report discusses the oral rehabilitation of an edentulous maxilla and partially dentate mandible using a digital workflow for both the surgical and prosthetic phases of the implant therapy. Protocols for clinical assessment, treatment planning, and restorative management are described to provide a predictable and prosthetic-driven treatment for implant-supported prostheses.

Impact of a community-based naloxone distribution program on opioid overdose death rates.

BACKGROUND: In August 2013, a naloxone distribution program was implemented in North Carolina (NC). This study evaluated that program by quantifying the association between the program and county-level opioid overdose death (OOD) rates and conducting a cost-benefit analysis. METHODS: One-group pre-post design. Data included annual county-level counts of naloxone kits distributed from 2013 to 2016 and mortality data from 2000-2016. We used generalized estimating equations to estimate the association between cumulative rates of naloxone kits distributed and annual OOD rates. Costs included naloxone kit purchases and distribution costs; benefits were quantified as OODs avoided and monetized using a conservative value of a life. RESULTS: The rate of OOD in counties with 1-100 cumulative naloxone kits distributed per 100,000 population was 0.90 times (95% CI: 0.78, 1.04) that of counties that had not received kits. In counties that received >100 cumulative kits per 100,000 population, the OOD rate was 0.88 times (95% CI: 0.76, 1.02) that of counties that had not received kits. By December 2016, an estimated 352 NC deaths were avoided by naloxone distribution (95% CI: 189, 580). On average, for every dollar spent on the program, there was $2742 of benefit due to OODs avoided (95% CI: $1,237, $4882). CONCLUSIONS: Our estimates suggest that community-based naloxone distribution is associated with lower OOD rates. The program generated substantial societal benefits due to averted OODs. States and communities should continue to support efforts to increase naloxone access, which may include reducing legal, financial, and normative barriers.

N-glycolyl GM1 ganglioside as a receptor for simian virus 40.

Carbohydrate microarrays have emerged as powerful tools in analyses of microbe-host interactions. Using a microarray with 190 sequence-defined oligosaccharides in the form of natural glycolipids and neoglycolipids representative of diverse mammalian glycans, we examined interactions of simian virus 40 (SV40) with potential carbohydrate receptors. While the results confirmed the high specificity of SV40 for the ganglioside GM1, they also revealed that N-glycolyl GM1 ganglioside [GM1(Gc)], which is characteristic of simian species and many other nonhuman mammals, is a better ligand than the N-acetyl analog [GM1(Ac)] found in mammals, including humans. After supplementing glycolipid-deficient GM95 cells with GM1(Ac) and GM1(Gc) gangliosides and the corresponding neoglycolipids with phosphatidylethanolamine lipid groups, it was found that GM1(Gc) analogs conferred better virus binding and infectivity. Moreover, we visualized the interaction of NeuGc with VP1 protein of SV40 by molecular modeling and identified a conformation for GM1(Gc) ganglioside in complex with the virus VP1 pentamer that is compatible with its presentation as a membrane receptor. Our results open the way not only to detailed studies of SV40 infection in relation to receptor expression in host cells but also to the monitoring of changes that may occur with time in receptor usage by the virus.

Neoglycolipid probes prepared via oxime ligation for microarray analysis of oligosaccharide-protein interactions.

Neoglycolipid technology is the basis of a microarray platform for assigning oligosaccharide ligands for carbohydrate-binding proteins. The strategy for generating the neoglycolipid probes by reductive amination results in ring opening of the core monosaccharides. This often limits applicability to short-chain saccharides, although the majority of recognition motifs are satisfactorily presented with neoglycolipids of longer oligosaccharides. Here, we describe neoglycolipids prepared by oxime ligation. We provide evidence from NMR studies that a significant proportion of the oxime-linked core monosaccharide is in the ring-closed form, and this form selectively interacts with a carbohydrate-binding protein. By microarray analyses we demonstrate the effective presentation with oxime-linked neoglycolipids of (1) Lewis(x) trisaccharide to antibodies to Lewis(x), (2) sialyllactose analogs to the sialic acid-binding receptors, siglecs, and (3) N-glycans to a plant lectin that requires an intact N-acetylglucosamine core.

Low kV versus dual-energy virtual monoenergetic CT imaging for proven liver lesions: what are the advantages and trade-offs in conspicuity and image quality? A pilot study.

PURPOSE: Single-energy low tube potential (SE-LTP) and dual-energy virtual monoenergetic (DE-VM) CT images both increase the conspicuity of hepatic lesions by increasing iodine signal. Our purpose was to compare the conspicuity of proven liver lesions, artifacts, and radiologist preferences in dose-matched SE-LTP and DE-VM images. METHODS: Thirty-one patients with 72 proven liver lesions (21 benign, 51 malignant) underwent full-dose contrast-enhanced dual-energy CT (DECT). Half-dose images were obtained using single tube reconstruction of the dual-source SE-LTP projection data (80 or 100 kV), and by inserting noise into dual-energy projection data, with DE-VM images reconstructed from 40 to 70 keV. Three blinded gastrointestinal radiologists evaluated half-dose SE-LTP and DE-VM images, ranking and grading liver lesion conspicuity and diagnostic confidence (4-point scale) on a per-lesion basis. Image quality (noise, artifacts, sharpness) was evaluated, and overall image preference was ranked on per-patient basis. Lesion-to-liver contrast-to-noise ratio (CNR) was compared between techniques. RESULTS: Mean lesion size was 1.5 ± 1.2 cm. Across the readers, the mean conspicuity ratings for 40, 45, and 50 keV half-dose DE-VM images were superior compared to other half-dose image sets (p < 0.0001). Per-lesion diagnostic confidence was similar between half-dose SE-LTP compared to half-dose DE-VM images (p ≥ 0.05; 1.19 vs. 1.24-1.32). However, SE-LTP images had less noise and artifacts and were sharper compared to DE-VM images less than 70 keV (p < 0.05). On a per-patient basis, radiologists preferred SE-LTP images the most and preferred 40-50 keV the least (p < 0.0001). Lesion CNR was also higher in SE-LTP images than DE-VM images (p < 0.01). CONCLUSION: For the same applied dose level, liver lesions were more conspicuous using DE-VM compared to SE-LTP; however, SE-LTP images were preferred more than any single DE-VM energy level, likely due to lower noise and artifacts.

Correlation of hepatic fractional extracellular space using gadolinium enhanced MRI with liver stiffness using magnetic resonance elastography.

PURPOSE: To compare MR hepatic fractional extracellular space (fECS) to liver stiffness (LS) with magnetic resonance elastography (MRE) for evaluation of liver fibrosis. METHODS AND MATERIALS: 71 consecutive patients with suspected chronic liver disease underwent standard liver MRI with MR elastography and additional delayed Gd-DTPA-enhanced sequences at 5 and 10 min in order to calculate hepatic fECS (%) and LS (kilopascals, kPa). Two radiologists blinded to clinical history examined MR images and calculated fECS and LS in identical locations for every patient. Interobserver agreement was calculated using the intraclass correlation coefficient. Pearson's correlation was calculated for LS and fECS measures, as was the area under the receiver operatic curve (AUROC), sensitivity and specificity of fECS to predict liver stiffness ≥2.93 and ≥5 kPa. The sensitivity of fECS for detecting fibrosis was separately analyzed in the subgroup of patients without anatomic findings of cirrhosis. RESULTS: Substantial to excellent interobserver agreement for both LS and fECS measurements was seen with intraclass correlation of 0.88 (95% CI 0.81-0.92) for LS, 0.77 (95% CI 0.66-0.85) for fECS5 and 0.76 (95% CI 0.64-0.84) for fECS10. A significant correlation was found between MRE and fECS5 (r = 0.47, p < 0.0001) and fECS10 (r = 0.44, p < 0.0001). The performance of fECS improved for detection of advanced fibrosis (≥5 kPa) with AUROC, sensitivity and specificity of 0.72, 38%, and 94% for fECS5 and 0.72, 67%, and 66% for fECS10. CONCLUSION: fECS correlates modestly with MRE-determined LS. fECS at MRI is a simple calculation to perform and may represent a practical way to suggest the presence of fibrosis during routine liver evaluation.

Preparation of neoglycolipids with ring-closed cores via chemoselective oxime-ligation for microarray analysis of carbohydrate-protein interactions.

Affinities of most oligosaccharide-protein interactions are so low that multivalent forms of ligand and protein are required for detecting interactions. The neoglycolipid (NGL) technology was designed to address the need for microscale presentation of oligosaccharides in a multivalent form for studying carbohydrate-protein interactions, and this is now the basis of a state-of-the-art carbohydrate microarray system. NGL technology involves conjugating oligosaccharides by reductive amination to the aminolipid 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (DHPE). Other than ring-opening of the monosaccharide residues at reducing ends, oligosaccharides remain intact, and the NGLs derived from trisaccharides or larger oligosaccharides have performed well for the majority of carbohydrate-recognition systems that have the peripheral or backbone regions of oligosaccharides as recognition motifs. However, ring-opening of reducing end monosaccharides limits applicability to very short oligosaccharides (di- and trisaccharides) and, potentially, to N-glycans recognized by proteins such as Pisum sativum agglutinin (pea lectin) that require both intact core and backbone regions for strong binding. This chapter describes a method for preparing NGLs (designated AO-NGLs) from reducing oligosaccharides by chemoselective oxime-ligation to a new lipid reagent, N-aminooxyacetyl-DHPE. Microarray analyses of the AO-NGL derived from Lewis x (Le(x)) trisaccharide probed with anti-Le(x) antibodies indicate that a significant proportion of the core monosaccharide linked to lipid is in ring-closed form. Thus, AO-NGLs have broadened the applicability of NGLs as probes in studies of carbohydrate-protein interactions.

The neoglycolipid (NGL)-based oligosaccharide microarray system poised to decipher the meta-glycome.

The neoglycolipid (NGL) technology is the basis of a state-of-the-art oligosaccharide microarray system. The NGL-based microarray system in the Glycosciences Laboratory Imperial College London (http://www3.imperial.ac.uk/glycosciences) is one of the two leading platforms for glycan microarrays, being offered for screening analyses to the broad biomedical community. Highlighted in this review are the sensitivity of the analysis system and, coupled with mass spectrometry, the provision for generating 'designer' microarrays from glycomes to identify novel ligands of biological relevance. Among recent applications are assignments of ligands for apicomplexan parasites, pandemic 2009 influenza virus, polyoma and reoviruses, an innate immune receptor against fungal pathogens, Dectin-1, and a novel protein of the endoplasmic reticulum, malectin; also the characterization of an elusive cancer-associated antigen. Some other contemporary advances in glycolipid-containing arrays and microarrays are also discussed.

Attitudes of North Carolina law enforcement officers toward syringe decriminalization.

BACKGROUND: North Carolina, like much of the U.S. South, is disproportionately affected by HIV and hepatitis. This persistently high disease burden may be driven in part by laws that criminalize the possession and distribution of syringes for illicit drug use. Legal change to decriminalize syringes may reduce infection rates in the state, but is unlikely absent support from law enforcement actors. METHODS: We analyzed the responses of 350 North Carolina law enforcement officers to a confidential, anonymous survey. The survey instrument collected data regarding self-reported needle-stick injury (NSI), blood borne disease risk perception and attitudes toward syringe decriminalization. RESULTS: 82% of respondents reported that contracting HIV was a "big concern" for them. 3.8% of respondents reported ever receiving a job-related NSI, a rate of 36 NSI per 10,000 officer-years. Majorities of respondents reported positive views regarding syringe decriminalization, with approximately 63% agreeing that it would be "good for the community" and 60% agreeing that it would be "good for law enforcement." Black and female officers were significantly less likely to agree that on-the-job NSI was a "big concern" and significantly more likely to agree that it would be good for law enforcement. CONCLUSIONS: These findings suggest that many North Carolina LEOs understand the public health benefits of syringe access programs and may be inclined to support syringe decriminalization legislation. Further research is indicated to determine the causes of observed differences in perceptions of bloodborne disease risk and attitudes toward syringe decriminalization by race and sex.

Changes in the hemagglutinin of H5N1 viruses during human infection--influence on receptor binding.

As avian influenza A(H5N1) viruses continue to circulate in Asia and Africa, global concerns of an imminent pandemic persist. Recent experimental studies suggest that efficient transmission between humans of current H5N1 viruses only requires a few genetic changes. An essential step is alteration of the virus hemagglutinin from preferential binding to avian receptors for the recognition of human receptors present in the upper airway. We have identified receptor-binding changes which emerged during H5N1 infection of humans, due to single amino acid substitutions, Ala134Val and Ile151Phe, in the hemagglutinin. Detailed biological, receptor-binding, and structural analyses revealed reduced binding of the mutated viruses to avian-like receptors, but without commensurate increased binding to the human-like receptors investigated, possibly reflecting a receptor-binding phenotype intermediate in adaptation to more human-like characteristics. These observations emphasize that evolution in nature of avian H5N1 viruses to efficient binding of human receptors is a complex multistep process.