A Useful Guide to Lectin Binding: Machine-Learning Directed Annotation of 57 Unique Lectin Specificities.

Glycans are critical to every facet of biology and medicine, from viral infections to embryogenesis. Tools to study glycans are rapidly evolving; however, the majority of our knowledge is deeply dependent on binding by glycan binding proteins (e.g., lectins). The specificities of lectins, which are often naturally isolated proteins, have not been well-defined, making it difficult to leverage their full potential for glycan analysis. Herein, we use a combination of machine learning algorithms and expert annotation to define lectin specificity for this important probe set. Our analysis uses comprehensive glycan microarray analysis of commercially available lectins we obtained using version 5.0 of the Consortium for Functional Glycomics glycan microarray (CFGv5). This data set was made public in 2011. We report the creation of this data set and its use in large-scale evaluation of lectin-glycan binding behaviors. Our motif analysis was performed by integrating 68 manually defined glycan features with systematic probing of computational rules for significant binding motifs using mono- and disaccharides and linkages. Combining machine learning with manual annotation, we create a detailed interpretation of glycan-binding specificity for 57 unique lectins, categorized by their major binding motifs: mannose, complex-type N-glycan, O-glycan, fucose, sialic acid and sulfate, GlcNAc and chitin, Gal and LacNAc, and GalNAc. Our work provides fresh insights into the complex binding features of commercially available lectins in current use, providing a critical guide to these important reagents.

Inhibition of Clostridium difficile and Antibiotic-Resistant Subtypes by Compound RNW03.

Clostridium difficile infection is mainly the result of antibiotic usage which disturbs the normal microbial flora. Antibiotic-resistant strains of C. difficile have emerged which have limited the number of therapeutic options. We sought to determine if compound RNW03, a broad spectrum anti-microbial formula, would be effective in inhibiting C. difficile and its antibiotic-resistant subtypes. Compound RNW03 was titrated and added to standard concentrations of C. difficile. The wells were incubated in an anaerobic chamber for 48 hours followed by a cell count. Compound RNW03 proved to be effective in preventing the growth of seven different C. difficile strains, including those that were antibiotic-resistant strains in dilutions of 32- to 64-fold.

Hematopoietic Stem and Progenitor Cells Exhibit Stage-Specific Translational Programs via mTOR- and CDK1-Dependent Mechanisms.

Hematopoietic stem cells (HSCs) require highly regulated rates of protein synthesis, but it is unclear if they or lineage-committed progenitors preferentially recruit transcripts to translating ribosomes. We utilized polysome profiling, RNA sequencing, and whole-proteomic approaches to examine the translatome in LSK (Lin-Sca-1+c-Kit+) and myeloid progenitor (MP; Lin-Sca-1-c-Kit+) cells. Our studies show that LSKs exhibit low global translation but high translational efficiencies (TEs) of mRNAs required for HSC maintenance. In contrast, MPs activate translation in an mTOR-independent manner due, at least in part, to proteasomal degradation of mTOR by the E3 ubiquitin ligase c-Cbl. In the near absence of mTOR, CDK1 activates eIF4E-dependent translation in MPs through phosphorylation of 4E-BP1. Aberrant activation of mTOR expression and signaling in c-Cbl-deficient MPs results in increased mature myeloid lineage output. Overall, our data demonstrate that hematopoietic stem and progenitor cells (HSPCs) undergo translational reprogramming mediated by previously uncharacterized mechanisms of translational regulation.

Familial Angiofibrohistiocytic Hamartoma Syndrome.

A 27-year-old Hispanic man presented with multiple papules and nodules measuring up to 10 cm in diameter. These lesions were widespread (Figure 1), but not on mucosal epithelium. At birth, the patient had had multiple hypopigmented macules that had progressed to papules and nodules over time. Dermatoscopic examination of these papules and nodules showed prominent capillaries. New lesions were constantly developing but were slow-growing. Other family members had similar lesions, including the patient's three brothers, father, paternal aunt, and a 3-year-old niece (daughter of the 2nd eldest brother), although not as extensively as in this patient. The paternal grandparents were not affected (Figure 2). As the patient had been raised in rural Mexico with limited financial resources, access to health care was limited, so the condition had been left undiagnosed and untreated for most of the patient's life.

Tau Protein in Oral Mucosa and Cognitive State: A Cross-sectional Study.

Neurodegenerative diseases are characterized by the presence of abnormal aggregates of proteins in brain tissue. Among them, the presence of aggregates of phosphorylated Tau protein (p-Tau) is the hallmark of Alzheimer's disease (AD) and other major neurodegenerative disorders such as corticobasal degeneration and frontotemporal dementia among others. Although Tau protein has previously been assumed to be exclusive to the central nervous system, it is also found in peripheral tissues. The purpose of this study was to determine whether there is a differential Tau expression in oral mucosa cells according to cognitive impairment. Eighty-one subjects were enrolled in the study and classified per Mini-Mental State Examination test score into control, mild cognitive impairment (MCI), and severe cognitive impairment (SCI) groups. Immunocytochemistry and immunofluorescence revealed the presence of Tau and four p-Tau forms in the cytoplasm and nucleus of oral mucosa cells. More positivity was present in subjects with cognitive impairment than in control subjects, both in the nucleus and cytoplasm, in a speckle pattern. The mRNA expression of Tau by quantitative real-time polymerase chain reaction was higher in SCI as compared with the control group (P < 0.01). A significantly higher percentage of immunopositive cells in the SCI group was found via flow cytometry in comparison to controls and the MCI group (P < 0.01). These findings demonstrate the higher presence of p-Tau and Tau transcript in the oral mucosa of cognitively impaired subjects when compared with healthy subjects. The feasibility of p-Tau quantification by flow cytometry supports the prospective analysis of oral mucosa as a support tool for screening of proteinopathies in cognitively impaired patients.

CD99 is a therapeutic target on disease stem cells in myeloid malignancies.

Acute myeloid leukemia (AML) and the myelodysplastic syndromes (MDS) are initiated and sustained by self-renewing malignant stem cells; thus, eradication of AML and MDS stem cells is required for cure. We identified CD99 as a cell surface protein frequently overexpressed on AML and MDS stem cells. Expression of CD99 allows for prospective separation of leukemic stem cells (LSCs) from functionally normal hematopoietic stem cells in AML, and high CD99 expression on AML blasts enriches for functional LSCs as demonstrated by limiting dilution xenotransplant studies. Monoclonal antibodies (mAbs) targeting CD99 induce the death of AML and MDS cells in a SARC family kinase-dependent manner in the absence of immune effector cells or complement, and anti-CD99 mAbs exhibit antileukemic activity in AML xenografts. These data establish CD99 as a marker of AML and MDS stem cells, as well as a promising therapeutic target in these disorders.

The Presence of Alpha-Synuclein in Skin from Melanoma and Patients with Parkinson's Disease.

BACKGROUND: The misfolding and prion-like propagation of the protein α-synuclein (α-syn) is the leading molecular signature in Parkinson's disease (PD). There is a significant coincidence of PD and melanoma that may suggest a shared pathophysiology. This study compared the presence of α-syn in neural crest-derived tissues, such as nevi, melanoma, skin tags, and skin biopsies from patients with PD and healthy controls. METHODS: Biopsies from participants with PD were obtained from patients from a tertiary referral center for dermatology and neurology in Mexico and a private dermatopathology center in Florida between January 2015 and March 2016. Biopsies from 7 patients with melanoma, 15 with nevi, 9 with skin tags, 8 with PD, and 9 skin biopsies from healthy volunteers were analyzed for immunohistochemical determination of α-syn and tyrosinase. All analyses were performed by pathologists who were blinded with respect to the clinical diagnosis. RESULTS: In healthy controls, positive α-syn status was restricted to scattered cells in the basal layer of the epidermis and accounted for 1 ± 0.8% of the analyzed area. In patients with PD, there was increased staining for α-syn PD (3.3 ± 2.3%), with a higher percentage of positive cells in nevi (7.7 ± 5.5%) and melanoma (13.6 ± 3.5%). There was no increased staining in skin tags compared with healthy controls. CONCLUSION: Patients with PD and melanoma have increased staining for α-syn in their skin. The authors propose that neurons and melanocytes, both derived from neuroectodermal cells, may share protein synthesis and regulation pathways that become dysfunctional in PD and melanoma.

Complex N-linked glycans serve as a determinant for exosome/microvesicle cargo recruitment.

Exosomes, also known as microvesicles (EMVs), are nano-sized membranous particles secreted from nearly all mammalian cell types. These nanoparticles play critical roles in many physiological processes including cell-cell signaling, immune activation, and suppression and are associated with disease states such as tumor progression. The biological functions of EMVs are highly dependent on their protein composition, which can dictate pathogenicity. Although some mechanisms have been proposed for the regulation of EMV protein trafficking, little attention has been paid to N-linked glycosylation as a potential sorting signal. Previous work from our laboratory found a conserved glycan signature for EMVs, which differed from that of the parent cell membranes, suggesting a potential role for glycosylation in EMV biogenesis. In this study, we further explore the role of glycosylation in EMV protein trafficking. We identify EMV glycoproteins and demonstrate alteration of their recruitment as a function of their glycosylation status upon pharmacological manipulation. Furthermore, we show that genetic manipulation of the glycosylation levels of a specific EMV glycoprotein, EWI-2, directly impacts its recruitment as a function of N-linked glycan sites. Taken together, our data provide strong evidence that N-linked glycosylation directs glycoprotein sorting into EMVs.

Mapping posttranscriptional regulation of the human glycome uncovers microRNA defining the glycocode.

Cell surface glycans form a critical interface with the biological milieu, informing diverse processes from the inflammatory cascade to cellular migration. Assembly of discrete carbohydrate structures requires the coordinated activity of a repertoire of proteins, including glycosyltransferases and glycosidases. Little is known about the regulatory networks controlling this complex biosynthetic process. Recent work points to a role for microRNA (miRNA) in the regulation of specific glycan biosynthetic enzymes. Herein we take a unique systems-based approach to identify connections between miRNA and the glycome. By using our glycomic analysis platform, lectin microarrays, we identify glycosylation signatures in the NCI-60 cell panel that point to the glycome as a direct output of genomic information flow. Integrating our glycomic dataset with miRNA data, we map miRNA regulators onto genes in glycan biosynthetic pathways (glycogenes) that generate the observed glycan structures. We validate three of these predicted miRNA/glycogene regulatory networks: high mannose, fucose, and terminal ß-GalNAc, identifying miRNA regulation that would not have been observed by traditional bioinformatic methods. Overall, our work reveals critical nodes in the global glycosylation network accessible to miRNA regulation, providing a bridge between miRNA-mediated control of cell phenotype and the glycome.

Cross-platform comparison of glycan microarray formats.

Carbohydrates participate in almost every aspect of biology from protein sorting to modulating cell differentiation and cell-cell interactions. To date, the majority of data gathered on glycan expression has been obtained via analysis with either anti-glycan antibodies or lectins. A detailed understanding of the specificities of these reagents is critical to the analysis of carbohydrates in biological systems. Glycan microarrays are increasingly used to determine the binding specificity of glycan-binding proteins (GBPs). In this study, six different glycan microarray platforms with different modes of glycan presentation were compared using five well-known lectins; concanavalin A, Helix pomatia agglutinin, Maackia amurensis lectin I, Sambucus nigra agglutinin and wheat germ agglutinin. A new method (universal threshold) was developed to facilitate systematic comparisons across distinct array platforms. The strongest binders of each lectin were identified using the universal threshold across all platforms while identification of weaker binders was influenced by platform-specific factors including presentation of determinants, array composition and self-reported thresholding methods. This work compiles a rich dataset for comparative analysis of glycan array platforms and has important implications for the implementation of microarrays in the characterization of GBPs.

Molecular imaging using fluorescent lectins permits rapid endoscopic identification of dysplasia in Barrett's esophagus.

Barrett's esophagus is an example of a pre-invasive state, for which current endoscopic surveillance methods to detect dysplasia are time consuming and inadequate. The prognosis of cancer arising in Barrett's esophagus is improved by early detection at the stage of mucosal carcinoma or high-grade dysplasia. Molecular imaging methods could revolutionize the detection of dysplasia, provided they permit a wide field of view and highlight abnormalities in real time. We show here that cell-surface glycans are altered in the progression from Barrett's esophagus to adenocarcinoma and lead to specific changes in lectin binding patterns. We chose wheat germ agglutinin as a candidate lectin with clinical potential. The binding of wheat germ agglutinin to human tissue was determined to be specific, and we validated this specific binding by successful endoscopic visualization of high-grade dysplastic lesions, which were not detectable by conventional endoscopy, with a high signal-to-background ratio of over 5.

Identification of a conserved glycan signature for microvesicles.

Microvesicles (exosomes) are important mediators of intercellular communication, playing a role in immune regulation, cancer progression, and the spread of infectious agents. The biological functions of these small vesicles are dependent on their composition, which is regulated by mechanisms that are not well understood. Although numerous proteomic studies of these particles exist, little is known about their glycosylation. Carbohydrates are involved in protein trafficking and cellular recognition. Glycomic analysis may thus provide valuable insights into microvesicle biology. In this study, we analyzed glycosylation patterns of microvesicles derived from a variety of biological sources using lectin microarray technology. Comparison of the microvesicle glycomes with their parent cell membranes revealed both enrichment and depletion of specific glycan epitopes in these particles. These include enrichment in high mannose, polylactosamine, α-2,6 sialic acid, and complex N-linked glycans and exclusion of terminal blood group A and B antigens. The polylactosamine signature derives from distinct glycoprotein cohorts in microvesicles of different origins. Taken together, our data point to the emergence of microvesicles from a specific membrane microdomain, implying a role for glycosylation in microvesicle protein sorting.

Development of an oregano-based ointment with anti-microbial activity including activity against methicillin-resistant Staphlococcus aureus.

Increasing antibiotic resistance has prompted a search for new compounds with anti-microbial activity. In the authors' previous study, oregano extract was identified as one of the most potent anti-microbial compounds. The disk diffusion method was employed to assess the degree of inhibition against various microorganisms, and the bacteriostatic or bactericidal mechanism of action. Disk diffusion studies showed that oregano was found to be bacteriostatic for Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus, (MRSA) but bacteriocidal for seven other microorganisms. Pseudomonas aeruginosa could not be inhibited by oregano. An ointment consisting of 1-10% oregano could inhibit most organisms except for Proteus mirabilis and Proteus vulgaris, which required 20% and Pseudomonas which could not be inhibited even at the highest concentration of 80%. Oregano extracts can be formulated into an ointment that shows broad antimicrobial activity. Additional testing to assess tissue toxicity and other adverse reactions would be needed prior to human testing.

Alternative zippering as an on-off switch for SNARE-mediated fusion.

Membrane fusion between vesicles and target membranes involves the zippering of a four-helix bundle generated by constituent helices derived from target- and vesicle-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). In neurons, the protein complexin clamps otherwise spontaneous fusion by SNARE proteins, allowing neurotransmitters and other mediators to be secreted when and where they are needed as this clamp is released. The membrane-proximal accessory helix of complexin is necessary for clamping, but its mechanism of action is unknown. Here, we present experiments using a reconstituted fusion system that suggest a simple model in which the complexin accessory helix forms an alternative four-helix bundle with the target-SNARE near the membrane, preventing the vesicle-SNARE from completing its zippering.

Distinct domains of complexins bind SNARE complexes and clamp fusion in vitro.

In regulated exocytosis, the core membrane fusion machinery proteins, the SNARE proteins, are assisted by a group of regulatory factors in order to couple membrane fusion to an increase of intracellular calcium ion (Ca(2+)) concentration. Complexin-I and synaptotagmin-I have been shown to be key elements for this tightly regulated process. Many studies suggest that complexin-I can arrest the fusion reaction and that synaptotagmin-I can release the complexin-I blockage in a calcium-dependent manner. Although the actual molecular mechanism by which they exert their function is still unknown, recent in vivo experiments postulate that domains of complexin-I produce different effects on neurotransmitter release. Herein, by using an in vitro flipped SNARE cell fusion assay, we have identified and characterized the minimal functional domains of complexin-I necessary to couple calcium and synaptotagmin-I to membrane fusion. Moreover, we provide evidence that other isoforms of complexin, complexin-II, -III, and -IV, can also be functionally coupled to synaptotagmin-I and calcium. These correspond closely to results from in vivo experiments, providing further validation of the physiological relevance of the flipped SNARE system.

Energetics and dynamics of SNAREpin folding across lipid bilayers.

Membrane fusion occurs when SNAREpins fold up between lipid bilayers. How much energy is generated during SNAREpin folding and how this energy is coupled to the fusion of apposing membranes is unknown. We have used a surface forces apparatus to determine the energetics and dynamics of SNAREpin formation and characterize the different intermediate structures sampled by cognate SNAREs in the course of their assembly. The interaction energy-versus-distance profiles of assembling SNAREpins reveal that SNARE motifs begin to interact when the membranes are 8 nm apart. Even after very close approach of the bilayers (approximately 2-4 nm), the SNAREpins remain partly unstructured in their membrane-proximal region. The energy stabilizing a single SNAREpin in this configuration (35 k(B)T) corresponds closely with the energy needed to fuse outer but not inner leaflets (hemifusion) of pure lipid bilayers (40-50 k(B)T).

Hidradenocarcinoma: a histological and immunohistochemical study.

The diagnosis of hidradenocarcinoma is difficult due to a combination of factors including inconsistent nomenclature/ classification, rarity of the neoplasm, and variable morphology of cells composing the neoplasm. Immunohistochemistry has not been previously performed on a series of hidradenocarcinomas. We evaluated six cases of hidradenocarcinoma histologically and immunohistochemically using antibodies to gross cystic disease fluid protein-15 (GCDFP-15), carcino-embryonic antigen (CEA), epithelial membrane antigen (EMA), S-100 protein, keratin AE1/3, cytokeratin 5/6, p53, bcl-1, bcl-2, and Ki67. Histology suggested concurrent eccrine and apocrine differentiation of the cases. Ki67 and p53 staining was strongly positive in five of six tumors. The neoplasms stained with antibodies to CEA, S-100 protein, GCDFP-15, EMA, bcl-1, and bcl-2 in no consistent pattern. All tumors studied stained positively for keratin AE1/3 and cytokeratin 5/6. In making the diagnosis of hidradenocarcinoma, it may be unnecessary to separate hidradenocarcinoma into eccrine and apocrine categories, and although Ki67 and p53 may be helpful, histological parameters remain paramount.

A clamping mechanism involved in SNARE-dependent exocytosis.

During neurotransmitter release at the synapse, influx of calcium ions stimulates the release of neurotransmitter. However, the mechanism by which synaptic vesicle fusion is coupled to calcium has been unclear, despite the identification of both the core fusion machinery [soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)] and the principal calcium sensor (synaptotagmin). Here, we describe what may represent a basic principle of the coupling mechanism: a reversible clamping protein (complexin) that can freeze the SNAREpin, an assembled fusion-competent intermediate en route to fusion. When calcium binds to the calcium sensor synaptotagmin, the clamp would then be released. SNARE proteins, and key regulators like synaptotagmin and complexin, can be ectopically expressed on the cell surface. Cells expressing such "flipped" synaptic SNAREs fuse constitutively, but when we coexpressed complexin, fusion was blocked. Adding back calcium triggered fusion from this intermediate in the presence of synaptotagmin.

Cutaneous angiosarcoma: a case series with prognostic correlation.

BACKGROUND: Cutaneous angiosarcoma (CA) is a rare and aggressive endothelial-derived sarcoma. Few large studies have examined the clinicopathologic and prognostic attributes of CA. OBJECTIVES: We sought to discern the potential prognostic significance of a variety of demographic features (i.e., age, sex, location), histologic attributes (i.e., depth of invasion, tumor necrosis, tumor cell morphology, margin status, mitoses), and follow-up data (i.e., tumor recurrence, metastases) in CA. METHODS: The statistical influence of age, sex, anatomic location, tumor depth of invasion, tumor cell morphology, presence or absence of necrosis, number of mitoses, and margin status on time to tumor recurrence and metastases were examined in a series of 47 patients with CA. Angiosarcoma arising within the breast, in a previously irradiated anatomic site, and a pre-existing vascular malformation or one associated with a lymphedematous extremity were excluded from study. RESULTS: Most of the patients were men (76%), with an average age of 75.1 years (range: 59-92 years). The most common location was the head and neck region (96%). The most common presentation was of a rapidly expanding erythematous patch, and the most common clinical impression was angiosarcoma. The average external diameter of the tumor was 5.3 cm (range: 1.1-8.9 cm). The most common histologic pattern was characterized by anastomosing dissecting sinusoids lined by atypical endothelial cells (64%) with 15% of cases showing a diffuse epithelioid or spindle cell proliferation and 21% showing a mixture of the 2 histologic patterns. The average depth of tumor invasion was 2.86 mm (range: 1.8->6.0 mm). Of the tumors, 78% had a mitotic rate that exceeded 3/mm(2). Follow-up was available in 37 of the patients and ranged from 6 to 65 months. The 5-year local recurrence rate was 84% and the overall 5-year survival was 34%. Most patients died as a result of their disease with widespread pulmonary, cardiac, and/or brain metastases. CONCLUSIONS: Of the gross and histologic features, external diameter (>5 cm), depth of invasion (>3 mm), mitotic rate (>3 HPF), positive surgical margins, tumor recurrence, and metastases correlated with adverse outcome by univariate analysis and, with the exception of mitotic rate, by multivariate analysis. Of the foregoing, tumor diameter, depth of invasion, positive margins, metastases, and tumor recurrence were the most robust predictors of outcome. None of the demographic factors was associated with outcome. This study confirms the poor prognosis of patients with CA. Among all demographic and histologic patterns examined for prognostic significance, tumor diameter, tumor depth of invasion, margin status, tumor recurrence, and metastases emerged as the most important determinants of outcome.

Muir-Torre syndrome: role of the dermatopathologist in diagnosis.

Muir-Torre syndrome (MTS) is an autosomal dominantly inherited disorder characterized by sebaceous lesions and visceral malignancies. The defect is thought to be the result of a mutation in mismatch repair genes and associated with microsatellite instability. Two cases whose diagnoses were suggested first by the dermatopathologist are discussed. The first is a 47-year-old white man who over the past 6 years developed multiple sebaceous lesions. Due to the number of sebaceous lesions and their morphology, the possible diagnosis of MTS was suggested by the dermatopathologist. Subsequently, a lesion in the right colon was found during colonoscopy that proved to be a poorly differentiated cecal adenocarcinoma. A pedigree analysis revealed other family members afflicted with multiple malignancies. Genetic testing of the colonic adenocarcinoma showed microsatellite instability. The second patient is a 50-year-old white man who underwent biopsy of a skin lesion that showed features of both a sebaceous hyperplasia and sebaceous adenoma. Because of the mixed, unusual features of the lesion, the dermatopathologist suggested the diagnosis of MTS. It was later confirmed that the patient had a history of malignancies of the colon and kidney as well as a family history significant for multiple malignant neoplasms. These cases demonstrate the important role of the dermatopathologist in alerting the clinician to the possibility of Muir-Torre syndrome when the diagnosis of a sebaceous neoplasm is made, especially when unusual histologic features are observed.