Bacteriophages evolve enhanced persistence to a mucosal surface.

The majority of viruses within the gut are obligate bacterial viruses known as bacteriophages (phages). Their bacteriotropism underscores the study of phage ecology in the gut, where they modulate and coevolve with gut bacterial communities. Traditionally, these ecological and evolutionary questions were investigated empirically via in vitro experimental evolution and, more recently, in vivo models were adopted to account for physiologically relevant conditions of the gut. Here, we probed beyond conventional phage-bacteria coevolution to investigate potential tripartite evolutionary interactions between phages, their bacterial hosts, and the mammalian gut mucosa. To capture the role of the mammalian gut, we recapitulated a life-like gut mucosal layer using in vitro lab-on-a-chip devices (to wit, the gut-on-a-chip) and showed that the mucosal environment supports stable phage-bacteria coexistence. Next, we experimentally coevolved lytic phage populations within the gut-on-a-chip devices alongside their bacterial hosts. We found that while phages adapt to the mucosal environment via de novo mutations, genetic recombination was the key evolutionary force in driving mutational fitness. A single mutation in the phage capsid protein Hoc-known to facilitate phage adherence to mucus-caused altered phage binding to fucosylated mucin glycans. We demonstrated that the altered glycan-binding phenotype provided the evolved mutant phage a competitive fitness advantage over its ancestral wild-type phage in the gut-on-a-chip mucosal environment. Collectively, our findings revealed that phages-in addition to their evolutionary relationship with bacteria-are able to evolve in response to a mammalian-derived mucosal environment.

Functional Microarray Platform with Self-Assembled Monolayers on 3C-Silicon Carbide.

Currently available bioplatforms such as microarrays and surface plasmon resonators are unable to combine high-throughput multiplexing with label-free detection. As such, emerging microelectromechanical systems (MEMS) and microplasmonics platforms offer the potential for high-resolution, high-throughput label-free sensing of biological and chemical analytes. Therefore, the search for materials capable of combining multiplexing and label-free quantitation is of great significance. Recently, interest in silicon carbide (SiC) as a suitable material in numerous biomedical applications has increased due to its well-explored chemical inertness, mechanical strength, bio- and hemocompatibility, and the presence of carbon that enables the transfer-free growth of graphene. SiC is also multifunctional as both a wide-band-gap semiconductor and an efficient low-loss plasmonics material and thus is ideal for augmenting current biotransducers in biosensors. Additionally, the cubic variant, 3C-SiC, is an extremely promising material for MEMS, being a suitable platform for the easy micromachining of microcantilevers, and as such capable of realizing the potential of real time miniaturized multiplexed assays. The generation of an appropriately functionalized and versatile organic monolayer suitable for the immobilization of biomolecules is therefore critical to explore label-free, multiplexed quantitation of biological interactions on SiC. Herein, we address the use of various silane self-assembled monolayers (SAMs) for the covalent functionalization of monocrystalline 3C-SiC films as a novel platform for the generation of functionalized microarray surfaces using high-throughput glycan arrays as the model system. We also demonstrate the ability to robotically print high throughput arrays on free-standing SiC microstructures. The implementation of a SiC-based label-free glycan array will provide a proof of principle that could be extended to the immobilization of other biomolecules in a similar SiC-based array format, thus making potentially significant advances to the way biological interactions are studied.

Lectin Activity of the TcdA and TcdB Toxins of Clostridium difficile.

Clostridium difficile is a major cause of hospital-acquired antibiotic-associated diarrhea. C. difficile produces two cytotoxins, TcdA and TcdB; both toxins are multidomain proteins that lead to cytotoxicity through the modification and inactivation of small GTPases of the Rho/Rac family. Previous studies have indicated that host glycans are targets for TcdA and TcdB, with interactions thought to be with both α- and ß-linked galactose. In the current study, screening of glycan arrays with different domains of TcdA and TcdB revealed that the binding regions of both toxins interact with a wider range of host glycoconjugates than just terminal α- and ß-linked galactose, including blood groups, Lewis antigens, N-acetylglucosamine, mannose, and glycosaminoglycans. The interactions of TcdA and TcdB with ABO blood group and Lewis antigens were assessed by surface plasmon resonance (SPR). The blood group A antigen was the highest-affinity ligand for both toxins. Free glycans alone or in combination were unable to abolish Vero cell cytotoxicity by TcdB. SPR competition assays indicate that there is more than one glycan binding site on TcdB. Host glycoconjugates are common targets of bacterial toxins, but typically this binding is to a specific structure or related structures. The binding of TcdA and TcdB is to a wide range of host glycans providing a wide range of target cells and tissues in vivo.

Lectin activity of Pseudomonas aeruginosa vaccine candidates PSE17-1, PSE41-5 and PSE54.

Pseudomonas aeruginosa is an opportunistic pathogen that causes nosocomial infections most commonly in immunocompromised, cystic fibrosis (CF) and burns patients. The pilin and Pseudomonas lectins 1 (PA-IL) and 2 (PA-IIL) are known glycan-binding proteins of P. aeruginosa that are involved in adherence to host cells, particularly CF host airways. Recently, new P. aeruginosa surface proteins were identified by reverse vaccinology and tested in vivo as potential vaccine antigens. Three of these, namely PSE17-1, PSE41-5 and PSE54, were screened for glycan binding using glycan arrays displaying glycan structures representative of those found on human cells. Surface plasmon resonance was used to confirm the lectin activity of these proteins, and determined affinities with several host glycans to be in the nanomolar range. PSE17-1 binds hyaluronic acid and sialyl Lewis A and X. PSE41-5 binds terminal ß-linked galactose structures, Lewis and ABO blood group antigens. PSE54 binds to ABO blood group antigens and some terminal ß-linked galactose. All three proteins are novel lectins of P. aeruginosa with potential roles in infection of host cells.

Glycan:glycan interactions: High affinity biomolecular interactions that can mediate binding of pathogenic bacteria to host cells.

Cells from all domains of life express glycan structures attached to lipids and proteins on their surface, called glycoconjugates. Cell-to-cell contact mediated by glycan:glycan interactions have been considered to be low-affinity interactions that precede high-affinity protein-glycan or protein-protein interactions. In several pathogenic bacteria, truncation of surface glycans, lipooligosaccharide (LOS), or lipopolysaccharide (LPS) have been reported to significantly reduce bacterial adherence to host cells. Here, we show that the saccharide component of LOS/LPS have direct, high-affinity interactions with host glycans. Glycan microarrays reveal that LOS/LPS of four distinct bacterial pathogens bind to numerous host glycan structures. Surface plasmon resonance was used to determine the affinity of these interactions and revealed 66 high-affinity host-glycan:bacterial-glycan pairs with equilibrium dissociation constants (K(D)) ranging between 100 nM and 50 µM. These glycan:glycan affinity values are similar to those reported for lectins or antibodies with glycans. Cell assays demonstrated that glycan:glycan interaction-mediated bacterial adherence could be competitively inhibited by either host cell or bacterial glycans. This is the first report to our knowledge of high affinity glycan:glycan interactions between bacterial pathogens and the host. The discovery of large numbers of glycan:glycan interactions between a diverse range of structures suggests that these interactions may be important in all biological systems.

Biophysical characterization and structural determination of the potent cytotoxic Psathyrella asperospora lectin.

A lectin with strong cytotoxic effect on human colon cancer HT29 and monkey kidney VERO cells was recently identified from the Australian indigenous mushroom Psathyrella asperospora and named PAL. We herein present its biochemical and structural analysis using a multidisciplinary approach. Glycan arrays revealed binding preference towards N-acetylglucosamine (GlcNAc) and, to a lesser extent, towards sialic acid (Neu5Ac). Submicromolar and millimolar affinity was measured by surface plasmon resonance for GlcNAc and NeuAc, respectively. The structure of PAL was resolved by X-ray crystallography, elucidating both the protein's amino acid sequence as well as the molecular basis rationalizing its binding specificity. Proteins 2017; 85:969-975. © 2016 Wiley Periodicals, Inc.

Functional and structural characterization of a heparanase.

We report the structural and functional characterization of a novel heparanase (BpHep) from the invasive pathogenic bacterium Burkholderia pseudomallei (Bp), showing ∼24% sequence identity with human heparanase (hHep). Site-directed mutagenesis studies confirmed the active site resi-dues essential for activity, and we found that BpHep has specificity for heparan sulfate. Finally, we describe the first heparanase X-ray crystal structure, which provides new insight into both substrate recognition and inhibitor design.

Cancer stem cell marker glycosylation: Nature, function and significance.

Glycans are essential for the maintenance of normal biological function, with alterations in glycan expression being a hallmark of cancer. Cancer stem cells (CSCs) are a subset of cells within a tumour capable of self-renewal, cellular differentiation and resistances to conventional therapies. As is the case with stem cells, marker proteins present on the cell surface are frequently used to identify and enrich CSCs, with the expression of these markers statistical correlating with the likelihood of cancer recurrence and overall patient survival. As such CSC markers are of high clinical relevance. The majority of markers currently used to identify CSC populations are glycoproteins, and although the diverse biological roles for many of these markers are known, the nature and function of the glycan moiety on these glycoproteins remains to be fully elucidated. This mini-review summarises our current knowledge regarding the types and extent of CSC marker glycosylation, and the various roles that these glycans play in CSC biology, including in mediating cell adhesion, metastasis, evading apoptosis, tear shear resistance, tumour growth, maintaining pluripotency, self-renewal, trafficking, maintaining stability, maintaining enzymatic activity and aiding epithelial mesenchymal transitioning. Given that CSCs markers have multiple diverse biological functions, and are potentially of significant diagnostic and therapeutic benefit the search for new markers that are uniquely expressed on CSCs is vital to selectively target/identify this subset of cancer cells. As such we have also outlined how high-throughput lectin microarrays can be used to successfully profile the glycosylation status of CSC and to identify glyco-markers unique to CSCs.

The three-dimensional structure of the extracellular adhesion domain of the sialic acid-binding adhesin SabA from Helicobacter pylori.

The gastric pathogen Helicobacter pylori is a major cause of acute chronic gastritis and the development of stomach and duodenal ulcers. Chronic infection furthermore predisposes to the development of gastric cancer. Crucial to H. pylori survival within the hostile environment of the digestive system are the adhesins SabA and BabA; these molecules belong to the same protein family and permit the bacteria to bind tightly to sugar moieties Lewis(B) and sialyl-Lewis(X), respectively, on the surface of epithelial cells lining the stomach and duodenum. To date, no representative SabA/BabA structure has been determined, hampering the development of strategies to eliminate persistent H. pylori infections that fail to respond to conventional therapy. Here, using x-ray crystallography, we show that the soluble extracellular adhesin domain of SabA shares distant similarity to the tetratricopeptide repeat fold family. The molecule broadly resembles a golf putter in shape, with the head region featuring a large cavity surrounded by loops that vary in sequence between different H. pylori strains. The N-terminal and C-terminal helices protrude at right angles from the head domain and together form a shaft that connects to a predicted outer membrane protein-like ß-barrel trans-membrane domain. Using surface plasmon resonance, we were able to detect binding of the SabA adhesin domain to sialyl-Lewis(X) and Lewis(X) but not to Lewis(A), Lewis(B), or Lewis(Y). Substitution of the highly conserved glutamine residue 159 in the predicted ligand-binding pocket abrogates the binding of the SabA adhesin domain to sialyl-Lewis(X) and Lewis(X). Taken together, these data suggest that the adhesin domain of SabA is sufficient in isolation for specific ligand binding.

A New Cytotoxic Steroidal Glycoalkaloid from the Methanol Extract of Blumea lacera Leaves.

PURPOSE: Blumea lacera (B. lacera) (Asteraceae) is a well-known Bangladeshi medicinal plant. This study aimed to identify and characterize constituents associated with the significant cytotoxic activity of this plant that we reported previously. Here, we describe the isolation and characterization of a new steroidal glycoalkaloid (SGA) 1, the evaluation of its cytotoxic activity, apoptotic potential, and effect on cell cycle in comparison to analogous steroidal glycoalkaloids (SGAs). METHODS: SGA 1 was isolated using C18 SPE and HPLC, and subsequently structurally characterized using 1D and 2D NMR, MS and other spectroscopic methods, along with a comparative inspection of the literature. Cytotoxic activity of 1 and seven SGA analogues and steroidal alkaloids (SAs), (ß-solamarine, α-solanine, ß-solamargine, α-solasonine, khasianine, solasodine, tomatidine HCl) were evaluated for their cytotoxicity against two healthy (NIH3T3 and VERO) and four human cancer (AGS, HT-29, MCF-7 and MDA-MB-231) cell lines using the MTT assay. Cytotoxic SGAs were further evaluated for apoptosis-inducing potential and cell cycle arresting ability against breast cancer cells (MCF-7) using the FITC Annexin V and propidium iodide (PI) assay. RESULTS: Bioactivity guided fractionation of the methanol extract of B. lacera led to isolation of compound 1: (25R)-3ß-{O-ß-D-glucopyranosyl-(1 → 4)-O-α-L-rhamnopyranosyl-(1 → 4)-[O-α-L-rhamnopyranosyl-(1 → 2)]-α-L-rhamnopyranosyl}-22αN-spirosol-5-ene. SGA 1 was the most cytotoxic compound against a number of human cancer cell lines with an IC50 of 2.62 µM against MCF-7 cells. It displayed the highest apoptotic potential (32% AV+/PI-) on MCF-7 cells compared to other cytotoxic SGA analogues and a slight, but significant cell cycle arresting effect. CONCLUSIONS: A new SGA 1 was isolated from B. lacera and its cytotoxic activity, as well as that of other SAGs, was evaluated. SAR investigations on SGA 1, in relation to SGA analogues, show that the number and nature of sugar moieties along with the linkages of the sugar to the aglycone are crucial for cytotoxic and apoptotic activity. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Mushroom lectins: specificity, structure and bioactivity relevant to human disease.

Lectins are non-immunoglobulin proteins that bind diverse sugar structures with a high degree of selectivity. Lectins play crucial role in various biological processes such as cellular signaling, scavenging of glycoproteins from the circulatory system, cell-cell interactions in the immune system, differentiation and protein targeting to cellular compartments, as well as in host defence mechanisms, inflammation, and cancer. Among all the sources of lectins, plants have been most extensively studied. However, more recently fungal lectins have attracted considerable attention due to their antitumor, antiproliferative and immunomodulatory activities. Given that only 10% of mushroom species are known and have been taxonomically classified, mushrooms represent an enormous unexplored source of potentially useful and novel lectins. In this review we provide an up-to-date summary on the biochemical, molecular and structural properties of mushroom lectins, as well as their versatile applications specifically focusing on mushroom lectin bioactivity.

G2/M cell cycle arrest by an N-acetyl-D-glucosamine specific lectin from Psathyrella asperospora.

A new N-acetyl-D-glucosamine (GlcNAc) specific lectin was identified and purified from the fruiting body of the Australian indigenous mushroom Psathyrella asperospora. The functional lectin, named PAL, showed hemagglutination activity against neuraminidase treated rabbit and human blood types A, B and O, and exhibited high binding specificity towards GlcNAc, as well as mucin and fetuin, but not against asialofetuin. PAL purified to homogeneity by a combination of ammonium sulfate precipitation, chitin affinity chromatography and size exclusion chromatography, was monomeric with a molecular mass of 41.8 kDa, was stable at temperatures up to 55 °C and between pH 6-10, and did not require divalent cations for optimal activity. De novo sequencing of PAL using LC-MS/MS, identified 10 tryptic peptides that revealed substantial sequence similarity to the GlcNAc recognizing lectins from Psathyrella velutina (PVL) and Agrocybe aegerita (AAL-II) in both the carbohydrate binding and calcium binding sites. Significantly, PAL was also found to exert a potent anti-proliferative effect on HT29 cells (IC50 0.48 µM) that was approximately 3-fold greater than that observed on VERO cells; a difference found to be due to the differential expression of cell surface GlcNAc on HT29 and VERO cells. Further characterization of this activity using propidium iodine staining revealed that PAL induced cell cycle arrest at G2/M phase in a manner dependent on its ability to bind GlcNAc.

Expression, solubilisation, and purification of a functional CMP-sialic acid transporter in Pichia pastoris.

Membrane proteins, including solute transporters play crucial roles in cellular function and have been implicated in a variety of important diseases, and as such are considered important targets for drug development. Currently the drug discovery process is heavily reliant on the structural and functional information discerned from high-resolution crystal structures. However, membrane protein structure determination is notoriously difficult, due in part to challenges faced in their expression, solubilisation and purification. The CMP-sialic acid transporter (CST) is considered to be an attractive target for drug discovery. CST inhibition reduces cancer cell sialylation and decreases the metastatic potential of cancer cells and to date, no crystal structure of the CST, or any other nucleotide sugar transporter exists. Here we describe the optimised conditions for expression in Pichia pastoris, solubilisation using n-nonyl ß-d-maltopyranoside (NM) and single step purification of a functional CST. Importantly we show that despite being able to solubilise and purify the CST using a number of different detergents, only NM was able to maintain CST functionality.

Identification and biochemical characterization of two functional CMP-sialic acid synthetases in Danio rerio.

Sialic acids (Sia) form the nonreducing end of the bulk of cell surface-expressed glycoconjugates. They are, therefore, major elements in intercellular communication processes. The addition of Sia to glycoconjugates requires metabolic activation to CMP-Sia, catalyzed by CMP-Sia synthetase (CMAS). This highly conserved enzyme is located in the cell nucleus in all vertebrates investigated to date, but its nuclear function remains elusive. Here, we describe the identification and characterization of two Cmas enzymes in Danio rerio (dreCmas), one of which is exclusively localized in the cytosol. We show that the two cmas genes most likely originated from the third whole genome duplication, which occurred at the base of teleost radiation. cmas paralogues were maintained in fishes of the Otocephala clade, whereas one copy got subsequently lost in Euteleostei (e.g. rainbow trout). In zebrafish, the two genes exhibited a distinct spatial expression pattern. The products of these genes (dreCmas1 and dreCmas2) diverged not only with respect to subcellular localization but also in substrate specificity. Nuclear dreCmas1 favored N-acetylneuraminic acid, whereas the cytosolic dreCmas2 showed highest affinity for 5-deamino-neuraminic acid. The subcellular localization was confirmed for the endogenous enzymes in fractionated zebrafish lysates. Nuclear entry of dreCmas1 was mediated by a bipartite nuclear localization signal, which seemed irrelevant for other enzymatic functions. With the current demonstration that in zebrafish two subfunctionalized cmas paralogues co-exist, we introduce a novel and unique model to detail the roles that CMAS has in the nucleus and in the sialylation pathways of animal cells.

Characterisation of CMP-sialic acid transporter substrate recognition.

CMP-sialic acid transporter: We report an in-depth, multidisciplinary, structural study that has identified the amino acid residues intimately involved in CMP-sialic acid transporter (CST) substrate specificity. Our data provide a significant contribution towards a better understanding the structure-function relationship of this important family of transporters and the rational design of CST inhibitors.

Assessment of glycan interactions of clinical and avian isolates of Campylobacter jejuni.

BACKGROUND: Campylobacter jejuni strain 11168 was demonstrated to have a broad specificity for eukaryotic surface glycosylation using glycan array analysis. The initial screen indicated that sialic acid and mannose are important binding partners after environmental stress, while galactose and fucose structures are likely to be involved in persistent infection. RESULTS: In this broader study, five additional human/clinical isolates and six chicken isolates were fully assessed to determine their glycan binding capacity using an extended glycan array. C. jejuni 11168 was rescreened here due to the presence of glycoaminoglycan (GAG) and other structures that were not available on our previous glycan array. The current array analysis of additional C. jejuni strains confirmed the growth condition dependent differences in glycan binding that was previously observed for C. jejuni 11168. We noted strain to strain variations, particularly for the human isolates C. jejuni 520 and 81116 and the chicken isolate C. jejuni 331, with the majority of differences observed in galactose, mannose and GAG binding. Chicken isolates were found to bind to a broader range of glycans compared to the human isolates, recognising branched mannose and carageenan (red seaweed) glycans. Glycan array data was confirmed using cell-based lectin inhibition assays with the fucose (UEA-I) and mannose (ConA) binding lectins. CONCLUSIONS: This study confirms that all C. jejuni strains tested bind to a broad range of glycans, with the majority of strains (all except 81116) altering recognition of sialic acid and mannose after environmental stress. Galactose and fucose structures were bound best by all strains when C. jejuni was grown under host like conditions confirming the likelihood of these structures being involved in persistent infection.

Carbohydrate binding and gene expression by in vitro and in vivo propagated Campylobacter jejuni after immunomagnetic separation.

Campylobacter jejuni is an important human food-borne intestinal pathogen, however relatively little is known about its mechanisms of pathogenesis or pathogen-host interactions. To monitor changes in gene expression and glycan binding of C. jejuni within a common avian host, an immunomagnetic separation technique (IMS) was utilised to directly isolate infecting C. jejuni 81116 from a chicken host. An average of 10(5) cells/g was re-isolated from chicken caecal samples by IMS technique. The in vivo passaged strains were used successfully in evaluation of carbohydrate binding through the use of a glycan array and were further suitable for transcriptome analysis. The glycan microarray analysis demonstrated differences in binding to negatively charged glycans of laboratory grown strains of C. jejuni compared with strains isolated after in vivo passage. The in vivo passaged strains showed marked up-regulation of chemotaxis receptors and toxin genes. The optimised Campylobacter IMS technique described in this study allowed isolation directly from an animal host. Changes in gene expression and glycan binding at an in vivo level can also be identified by using this method.

Mushroom Natural Products in Neurodegenerative Disease Drug Discovery.

The variety of drugs available to treat neurodegenerative diseases is limited. Most of these drug's efficacy is restricted by individual genetics and disease stages and usually do not prevent neurodegeneration acting long after irreversible damage has already occurred. Thus, drugs targeting the molecular mechanisms underlying subsequent neurodegeneration have the potential to negate symptom manifestation and subsequent neurodegeneration. Neuroinflammation is a common feature of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and multiple sclerosis, and is associated with the activation of the NLRP3 inflammasome, which in turn leads to neurodegeneration. Inflammasome activation and oligomerisation is suggested to be a major driver of disease progression occurring in microglia. With several natural products and natural product derivatives currently in clinical trials, mushrooms have been highlighted as a rich and largely untapped source of biologically active compounds in both in vitro and in vivo neurodegenerative disease models, partially supported by successful clinical trial evaluations. Additionally, novel high-throughput methods for the screening of natural product compound libraries are being developed to help accelerate the neurodegenerative disease drug discovery process, targeting neuroinflammation. However, the breadth of research relating to mushroom natural product high-throughput screening is limited, providing an exciting opportunity for further detailed investigations.

Picomolar detection of carbohydrate-lectin interactions on piezoelectrically printed microcantilever array.

Recent advances in micro-electromechanical systems (MEMS) has allowed unprecedent perspectives for label-free detection (LFD) of biological and chemical analytes. Additionally, these LFD technologies offer the potential to design high resolution and high throughput sensing platforms, with the promise of further miniaturization. However, the immobilization of biomolecules onto inorganic surfaces without impacting their sensing abilities is crucial for designing these LFD technologies. Currently, covalent functionalization of self-assembled monolayers (SAMs) present promising pathways for improving assay sensitivity, reproducibility, surface stability and proximity of binding sites to the sensor surface. Herein, we investigate the use of chemical vapor deposition of 3-(glycidyloxypropyl)-trimethoxysilane (GOPTS) as a versatile SAM for the covalent functionalization of a SiO2 microcantilever array (MCA) for carbohydrate-lectin interactions with picogram sensitivity. Additionally, we demonstrate glycan immobilization to MCA is feasible using traditional piezoelectric microarray printer technology. Given the complexity of the glycome, the ability to spot samples in a high-throughput manner establishes our MCA as robust, label-free, and scalable means to analyze carbohydrate-protein interactions These findings demonstrate that GOPTS SAMs provide a suitable biofunctionalization route for MEMS and provides the proof of principle that can be extended to various LFD technologies toward a truly high-throughput and high-resolution platform.

Sugar-decorated carbon dots: a novel tool for targeting immunomodulatory receptors.

Interactions between sialic acid (Sia) and sialic acid-binding immunoglobulin-like lectins (siglecs) regulate the immune system, with aberrations contributing to pathologies such as autoimmunity, infectious disease and cancer. Over the last decade, several multivalent Sia ligands have been synthesized to modulate the Sia-binding affinity of proteins/lectins. Here, we report a novel class of multivalent siglec probes through the decoration of α(2,6)-sialyllactose ligands on inherently fluorescent carbon dots (CD). We show that the preference of α(2,3)-linked Sia for siglec-1 can be altered by increasing the multivalence of Sia ligands present on the CD, and that a locally high glycan concentration can have a direct effect on linkage specificity. Additionally, micromolar (IC50 ∼ 70 µM) interaction of α(2,6)-sialyllactose-CD (6-CD) with siglec-2 (CD22) revealed it was capable of generating a significant cytotoxic effect on Burkitt's Lymphoma (BL) Daudi B cells. This phenonomen was attributed to 6-CD's ability to form trans interactions with CD22 on masked BL Daudi cells as a direct result of clustering of the Sia moiety on the CD surface. Overall, our glycoengineered carbon dots represent a novel high affinity molecular probe with multiple applications in sialoglycoscience and medicine.