Recent advances in the synthesis of extensive libraries of heparan sulfate oligosaccharides for structure-activity relationship studies.

Heparan sulfate (HS) is a linear, sulfated and highly negatively-charged polysaccharide that plays important roles in many biological events. As a member of the glycosaminoglycan (GAG) family, HS is commonly found on mammalian cell surfaces and within the extracellular matrix. The structural complexities of natural HS polysaccharides have hampered the comprehension of their biological functions and structure-activity relationships (SARs). Although the sulfation patterns and backbone structures of HS can be major determinants of their biological activities, obtaining significant amounts of pure HS from natural sources for comprehensive SAR studies is challenging. Chemical and enzyme-based synthesis can aid in the production of structurally well-defined HS oligosaccharides. In this review, we discuss recent innovations enabling the syntheses of large libraries of HS and how these libraries can provide insights into the structural preferences of various HS binding proteins.

Functional glycoproteomics by integrated network assembly and partitioning.

The post-translational modification (PTM) of proteins by O-linked ß-N-acetyl-D-glucosamine (O-GlcNAcylation) is widespread across the proteome during the lifespan of all multicellular organisms. However, nearly all functional studies have focused on individual protein modifications, overlooking the multitude of simultaneous O-GlcNAcylation events that work together to coordinate cellular activities. Here, we describe Networking of Interactors and SubstratEs (NISE), a novel, systems-level approach to rapidly and comprehensively monitor O-GlcNAcylation across the proteome. Our method integrates affinity purification-mass spectrometry (AP-MS) and site-specific chemoproteomic technologies with network generation and unsupervised partitioning to connect potential upstream regulators with downstream targets of O-GlcNAcylation. The resulting network provides a data-rich framework that reveals both conserved activities of O-GlcNAcylation such as epigenetic regulation as well as tissue-specific functions like synaptic morphology. Beyond O-GlcNAc, this holistic and unbiased systems-level approach provides a broadly applicable framework to study PTMs and discover their diverse roles in specific cell types and biological states.

Chondroitin 4-O-sulfation regulates hippocampal perineuronal nets and social memory.

Glycan alterations are associated with aging, neuropsychiatric, and neurodegenerative diseases, although the contributions of specific glycan structures to emotion and cognitive functions remain largely unknown. Here, we used a combination of chemistry and neurobiology to show that 4-O-sulfated chondroitin sulfate (CS) polysaccharides are critical regulators of perineuronal nets (PNNs) and synapse development in the mouse hippocampus, thereby affecting anxiety and cognitive abilities such as social memory. Brain-specific deletion of CS 4-O-sulfation in mice increased PNN densities in the area CA2 (cornu ammonis 2), leading to imbalanced excitatory-to-inhibitory synaptic ratios, reduced CREB activation, elevated anxiety, and social memory dysfunction. The impairments in PNN densities, CREB activity, and social memory were recapitulated by selective ablation of CS 4-O-sulfation in the CA2 region during adulthood. Notably, enzymatic pruning of the excess PNNs reduced anxiety levels and restored social memory, while chemical manipulation of CS 4-O-sulfation levels reversibly modulated PNN densities surrounding hippocampal neurons and the balance of excitatory and inhibitory synapses. These findings reveal key roles for CS 4-O-sulfation in adult brain plasticity, social memory, and anxiety regulation, and they suggest that targeting CS 4-O-sulfation may represent a strategy to address neuropsychiatric and neurodegenerative diseases associated with social cognitive dysfunction.

Efficient platform for synthesizing comprehensive heparan sulfate oligosaccharide libraries for decoding glycosaminoglycan-protein interactions.

Glycosaminoglycans (GAGs) are abundant, ubiquitous carbohydrates in biology, yet their structural complexity has limited an understanding of their biological roles and structure-function relationships. Synthetic access to large collections of well defined, structurally diverse GAG oligosaccharides would provide critical insights into this important class of biomolecules and represent a major advance in glycoscience. Here we report a new platform for synthesizing large heparan sulfate (HS) oligosaccharide libraries displaying comprehensive arrays of sulfation patterns. Library synthesis is made possible by improving the overall synthetic efficiency through universal building blocks derived from natural heparin and a traceless fluorous tagging method for rapid purification with minimal manual manipulation. Using this approach, we generated a complete library of 64 HS oligosaccharides displaying all possible 2-O-, 6-O- and N-sulfation sequences in the tetrasaccharide GlcN-IdoA-GlcN-IdoA. These diverse structures provide an unprecedented view into the sulfation code of GAGs and identify sequences for modulating the activities of important growth factors and chemokines.

Synthesis of a Systematic 64-Membered Heparan Sulfate Tetrasaccharide Library.

Heparan sulfate (HS) has multifaceted biological activities. To date, no libraries of HS oligosaccharides bearing systematically varied sulfation structures are available owing to the challenges in synthesizing a large number of HS oligosaccharides. To overcome the obstacles and expedite the synthesis, a divergent approach was designed, where 64 HS tetrasaccharides covering all possible structures of 2-O-, 6-O- and N-sulfation with the glucosamine-glucuronic acid-glucosamine-iduronic acid backbone were successfully produced from a single strategically protected tetrasaccharide intermediate. This extensive library helped identify the structural requirements for HS sequences to have strong fibroblast growth factor-2 binding but a weak affinity for platelet factor-4. Such a strategy to separate out these two interactions could lead to new HS-based potential therapeutics without the dangerous adverse effect of heparin-induced thrombocytopenia.

Expedient Synthesis of a Library of Heparan Sulfate-Like "Head-to-Tail" Linked Multimers for Structure and Activity Relationship Studies.

Heparan sulfate (HS) plays important roles in many biological processes. The inherent complexity of naturally existing HS has severely hindered the thorough understanding of their structure-activity relationship. To facilitate biological studies, a new strategy has been developed to synthesize a HS-like pseudo-hexasaccharide library, where HS disaccharides were linked in a "head-to-tail" fashion from the reducing end of a disaccharide module to the non-reducing end of a neighboring module. Combinatorial syntheses of 27 HS-like pseudo-hexasaccharides were achieved. This new class of compounds bound with fibroblast growth factor 2 (FGF-2) with similar structure-activity trends as HS oligosaccharides bearing native glycosyl linkages. The ease of synthesis and the ability to mirror natural HS activity trends suggest that the new head-to-tail linked pseudo-oligosaccharides could be an exciting tool to facilitate the understanding of HS biology.

Automated Solid Phase Assisted Synthesis of a Heparan Sulfate Disaccharide Library.

Heparan sulfate (HS) regulates a wide range of biological events, including blood coagulation, cancer development, cell differentiation, and viral infections. It is generally recognized that structures of HS can critically impact its biological functions. However, with complex structures of naturally existing HS, systematic investigations into the structure-activity relationship (SAR) of HS and efforts to unlock their "sulfation code" have been largely limited due to the challenges in preparing diverse HS oligosaccharide sequences. Herein, we report an automated machine-aided solid-phase strategy that significantly expedited the assembly of HS disaccharides. The key strategically protected advanced disaccharide intermediates were immobilized onto Synphase lanterns. Divergent deprotections and sulfations of the disaccharides were achieved on the lanterns in high yields. In addition, the full synthetic process was automated, enabling the reproducible production of HS disaccharides. A library of 16 HS disaccharides with diverse sulfation patterns was prepared via this method. Compared to the traditional HS synthesis, this new strategy led to a reduction of 50% of the number of synthetic steps and over 80% of the number of column purification steps needed from the disaccharide intermediates, significantly improving the overall synthetic efficiency. The potential utility of the method was highlighted in a microarray study using the synthetic HS disaccharide library with fibroblast growth factor-2 (FGF-2), which yielded insights into the SAR of HS/FGF-2 interactions.

Building diversity in the speech-language pathology workforce through a blended online Master of Speech Pathology.

Purpose: A lack of diversity in the speech-language pathology profession is widely recognised internationally. The role of speech-language pathology education in reproduction of this homogeneity and as a barrier to diversification is little understood. The potential of blended online education to increase access to education and diversify the speech-language pathology profession is yet to be explored. The purpose of the study was to investigate the profile of students engaging in the first blended online speech-language pathology course in Australia, including motivations for studying, experiences of studying via a blended online mode, and intentions for future practice.Method: Data were collected from 44 students in the first Australian blended online Master of Speech Pathology via an online survey. Quantitative data were analysed using descriptive statistics in SPSS and a content analysis of the qualitative data was conducted using NVivo.Result: With over 92% of participants choosing the course because it was offered online, flexible blended online study provided opportunity for those who would not have otherwise accessed speech-language pathology education, including rural students and those balancing work and home commitments. Participants found face to face elements of the course essential to their learning, but also the most challenging to manage and to fit around other commitments. Participants' ideal work scenarios revealed that 82.9% wished to practice within 50kms of their current location, indicating that provision of tertiary education to people in rural areas is contributing to establishing and retaining a rurally focussed speech-language pathology workforce.Conclusion: Blended online study provides an opportunity to diversify the speech-language profession by providing access to students such as rural students and those balancing work and home commitments with tertiary education. By reducing barriers to studying speech-language pathology, flexible learning is helping address issues of access and equity in service delivery across Australia.

Tools for mammalian glycoscience research.

Cellular carbohydrates or glycans are critical mediators of biological function. Their remarkably diverse structures and varied activities present exciting opportunities for understanding many areas of biology. In this primer, we discuss key methods and recent breakthrough technologies for identifying, monitoring, and manipulating glycans in mammalian systems.

Conversational speech of school-age children after syllable-timed speech treatment for stuttering.

Purpose: The purpose of this laboratory study was to investigate whether rhythmic speech was primarily responsible for stuttering reductions in four school-aged children after the instatement stage of the Westmead Program of syllable-timed speech (STS) intervention. The study was designed to inform further development of the program. Reduction in variability of vowel duration is a marker of STS, and it was predicted that this would be present in the children's conversational speech after Stage 1 of the program if they were using STS. To strengthen such a finding, it was also predicted that there would be no reduction in articulation rate, sentence complexity, and utterance length after treatment, as there is evidence that reductions in these can reduce stuttering. Perceptual judgments of speech quality after treatment were also made by independent listeners.Method: Participants were four children, ages 8-11 years, who completed Stage 1 of an STS program and whose stuttering had reduced significantly. Pre-treatment (PRE) and post-treatment (POST) within-clinic audio-visual recordings of conversational speech were analysed for percentage of syllables stuttered, variability of vowel duration, articulation rate, and length and complexity of utterance. Four blinded listeners made perceptual judgments of speech quality in the POST recordings.Result: Recordings of all children showed that variability of vowel duration clearly reduced from the PRE to POST speech samples. Importantly, articulation rate and language use were not compromised. Some possible indicators of rhythmicity were identified in one child in the perceptual study.Conclusion: The findings suggest that STS was primarily responsible for the clinically significant reductions in stuttering after Stage 1 of the program. There is an urgent need for more evidence-based interventions for stuttering in this age group and further development of STS interventions is warranted.

Psychological characteristics of early stuttering.

PURPOSE: The purpose of this study was to use psychological measures of pre-schoolers who stutter and their parents to inform causal theory development and influence clinical practices. This was done using data from a substantive clinical cohort of children who received early stuttering treatment. METHOD: The cohort (N = 427) comprised parents and their children who were treated with the Lidcombe Program, the Westmead Program, and the Oakville Program. The study incorporated demographic information, stuttering severity, and child and parent psychological measures prior to treatment. RESULT: The cohort revealed nothing unusual about behavioural and emotional functioning, or the temperaments, of pre-school children that would influence treatment, be targeted during treatment, or influence causal theory development. However, a third of parents were experiencing moderate to high life stressors at the time of seeking treatment, and half the parents failed first-stage screening for Anankastic Personality Disorder. CONCLUSION: The present results are consistent with a number of previous reports that showed that the population of pre-schoolers who stutter have no unusual psychological profiles. Hence, these results suggest that the association between mental health and stuttering later in life is a consequence of the disorder rather than being a part of its cause. The finding of the life stress of parents who seek stuttering treatment for pre-school children has potential clinical importance and warrants further investigation. Further psychological research is required about parents of pre-school children who stutter, because half the parents in the cohort failed the screener for Anankastic Personality Disorder. This is of interest because a previous study associated screening failure for another personality disorder (Impulsive Personality Disorder) with treatment dropout for early childhood stuttering.

Predictors of Lidcombe Program treatment dropout and outcome for early stuttering.

BACKGROUND: Information is available about what predicts Lidcombe Program treatment time, but nothing is known about what predicts treatment prognosis. AIMS: To investigate the predictors of treatment dropout and treatment outcome for children who were treated for early stuttering with the Lidcombe Program (N = 277). METHODS & PROCEDURES: A total of 32 variables were used as predictors in regression analyses of short- and medium-term Lidcombe Program outcome, and of treatment dropout. OUTCOMES & RESULTS: Regression analyses associated children who have better language skills and easy temperament with better treatment outcome, although only a small portion of the variance of treatment outcome was accounted for by these variables. There was an association between treatment dropout and parental scores on a personality screening tool relating to their impulsivity. CONCLUSIONS & IMPLICATIONS: Variables identified as predictors of Lidcombe Program treatment outcome were statistically significant, but not clinically significant. They did not account for a clinically substantive portion of treatment outcomes. Findings about parental impulsivity and their relationship with intervention drop-out require replication with prospective methods and comprehensive assessment of parent psychological status. This is particularly important because parents are involved in conducting all early interventions. What this paper adds What is already known on the subject Information is available about what predicts Lidcombe Program treatment time, but nothing is known about what predicts Lidcombe Program treatment outcome. What this paper adds to existing knowledge There are predictors of Lidcombe Program treatment outcome that are statistically significant, but none are clinically significant. What are the potential or actual clinical implications of this work? Clinicians can tell parents that nothing has been found that can assist with making prognostic indications about treatment outcome for their children.

Sulfated glycans engage the Ang-Tie pathway to regulate vascular development.

The angiopoietin (Ang)-Tie pathway is essential for the proper maturation and remodeling of the vasculature. Despite its importance in disease, the mechanisms that control signal transduction through this pathway are poorly understood. Here, we demonstrate that heparan sulfate glycosaminoglycans (HS GAGs) regulate Ang-Tie signaling through direct interactions with both Ang ligands and Tie1 receptors. HS GAGs formed ternary complexes with Ang1 or Ang4 and Tie2 receptors, resulting in potentiation of endothelial survival signaling. In addition, HS GAGs served as ligands for the orphan receptor Tie1. The HS-Tie1 interaction promoted Tie1-Tie2 heterodimerization and enhanced Tie1 stability within the mature vasculature. Loss of HS-Tie1 binding using CRISPR-Cas9-mediated mutagenesis in vivo led to decreased Tie protein levels, pathway suppression and aberrant retinal vascularization. Together, these results reveal that sulfated glycans use dual mechanisms to regulate Ang-Tie signaling and are important for the development and maintenance of the vasculature.

Photoaffinity Probes for the Identification of Sequence-Specific Glycosaminoglycan-Binding Proteins.

Glycosaminoglycan (GAG)-protein interactions mediate critical physiological and pathological processes, such as neuronal plasticity, development, and viral invasion. However, mapping GAG-protein interaction networks is challenging as these interactions often require specific GAG sulfation patterns and involve transmembrane receptors or extracellular matrix-associated proteins. Here, we report the first GAG polysaccharide-based photoaffinity probes for the system-wide identification of GAG-binding proteins in living cells. A general platform for the modular, efficient assembly of various chondroitin sulfate (CS)-based photoaffinity probes was developed. Systematic evaluations led to benzophenone-containing probes that efficiently and selectively captured known CS-E-binding proteins in vitro and in cells. Importantly, the probes also enabled the identification of >50 new proteins from living neurons that interact with the neuroplasticity-relevant CS-E sulfation motif. Several candidates were independently validated and included membrane receptors important for axon guidance, innate immunity, synapse development, and synaptic plasticity. Overall, our studies provide a powerful approach for mapping GAG-protein interaction networks, revealing new potential functions for these polysaccharides and linking them to diseases such as Alzheimer's and autism.

Expedient Synthesis of Core Disaccharide Building Blocks from Natural Polysaccharides for Heparan Sulfate Oligosaccharide Assembly.

The complex sulfation motifs of heparan sulfate glycosaminoglycans (HS GAGs) play critical roles in many important biological processes. However, an understanding of their specific functions has been hampered by an inability to synthesize large numbers of diverse, yet defined, HS structures. Herein, we describe a new approach to access the four core disaccharides required for HS/heparin oligosaccharide assembly from natural polysaccharides. The use of disaccharides rather than monosaccharides as minimal precursors greatly accelerates the synthesis of HS GAGs, providing key disaccharide and tetrasaccharide intermediates in about half the number of steps compared to traditional strategies. Rapid access to such versatile intermediates will enable the generation of comprehensive libraries of sulfated oligosaccharides for unlocking the "sulfation code" and understanding the roles of specific GAG structures in physiology and disease.

Delphi Study: ASPAN Adult Patient Pain and Comfort Practices.

PURPOSE: The American Society of PeriAnesthesia Nurses (ASPAN) is responsible for establishing evidence-based standards to guide perianesthesia nursing practice. The ASPAN model for evidence-based practice acknowledges the potential for the Delphi technique to identify priorities for perianesthesia research. The purpose of this Delphi study was to generate a consensus on pain and comfort among a panel of experts. DESIGN: ASPAN convened a panel of experts to provide recommendations based on seven categories, this led to the development of a questionnaire to build consensus. METHODS: Survey conducted among panel of experts to obtain consensus. Two survey rounds were completed. FINDINGS: A consensus was obtained reaching a 70% benchmark for an acceptance. CONCLUSIONS: The results found a consensus on topics required for education and competency among perianesthesia nurses including transfer and discharge criteria related to pain and comfort, resources for perianesthesia nurses, policy guidelines, and the management of the special needs of perianesthesia patients.

Three Lidcombe program clinic visit options: a phase II trial.

PURPOSE: Weekly clinic visits are recommended in the Lidcombe Program Treatment Guide (Packman et al., 2015). That specification is based on traditional speech-language pathology practices rather than empirical research, and two studies have suggested that such a format does not always occur in clinical communities. This research was conducted to determine the relative efficacy of different Lidcombe Program models of clinic visits. METHOD: Thirty-one children were randomized to three different service delivery models: twice-weekly, weekly and fortnightly (once every two weeks) clinic visits. All children were treated with the Lidcombe Program following manualised procedures. Measures of percentage syllables stuttered were obtained from beyond clinic audio recordings pre- and post-randomization. RESULTS: Results showed that the twice-weekly and fortnightly treatment formats were not suitable for all families. However, the fortnightly outcomes at 9 months post-randomization were comparable with those attained during weekly clinic visits. CONCLUSIONS: These results justify further, large-scale clinical trialling to compare weekly Lidcombe Program clinic visits with schedules involving less frequent clinic visits.

Long noncoding RNA HOTAIR promotes invasion of breast cancer cells through chondroitin sulfotransferase CHST15.

The long noncoding RNA HOTAIR plays significant roles in promoting cancer metastasis. However, how it conveys an invasive advantage in cancer cells is not clear. Here we identify the chondroitin sulfotransferase CHST15 (GalNAc4S-6ST) as a novel HOX transcript antisense intergenic RNA (HOTAIR) target gene using RNA profiling and show that CHST15 is required for HOTAIR-mediated invasiveness in breast cancer cells. CHST15 catalyzes sulfation of the C6 hydroxyl group of the N-acetyl galactosamine 4-sulfate moiety in chondroitin sulfate to form the 4,6-disulfated chondroitin sulfate variant known as the CS-E isoform. We show that HOTAIR is necessary and sufficient for CHST15 transcript expression. Inhibition of CHST15 by RNA interference abolished cell invasion promoted by HOTAIR but not on HOTAIR-mediated migratory activity. Conversely, reconstitution of CHST15 expression rescued the invasive activity of HOTAIR-depleted cells. In corroboration with this mechanism, blocking cell surface chondroitin sulfate using a pan-CS antibody or an antibody specifically recognizes the CS-E isoform significantly suppressed HOTAIR-induced invasion. Inhibition of CHST15 compromised tumorigenesis and metastasis in orthotopic breast cancer xenograft models. Furthermore, the expression of HOTAIR closely correlated with the level of CHST15 protein in primary as well as metastatic tumor lesions. Our results demonstrate a novel mechanism underlying the function of HOTAIR in tumor progression through programming the context of cell surface glycosaminoglycans. Our results further establish that the invasive and migratory activities downstream of HOTAIR are distinctly regulated, whereby CHST15 preferentially controls the arm of invasiveness. Thus, the HOTAIR-CHST15 axis may provide a new avenue toward novel therapeutic strategies and prognosis biomarkers for advanced breast cancer.

O-GlcNAcylation of core components of the translation initiation machinery regulates protein synthesis.

Protein synthesis is essential for cell growth, proliferation, and survival. Protein synthesis is a tightly regulated process that involves multiple mechanisms. Deregulation of protein synthesis is considered as a key factor in the development and progression of a number of diseases, such as cancer. Here we show that the dynamic modification of proteins by O-linked ß-N-acetyl-glucosamine (O-GlcNAcylation) regulates translation initiation by modifying core initiation factors eIF4A and eIF4G, respectively. Mechanistically, site-specific O-GlcNAcylation of eIF4A on Ser322/323 disrupts the formation of the translation initiation complex by perturbing its interaction with eIF4G. In addition, O-GlcNAcylation inhibits the duplex unwinding activity of eIF4A, leading to impaired protein synthesis, and decreased cell proliferation. In contrast, site-specific O-GlcNAcylation of eIF4G on Ser61 promotes its interaction with poly(A)-binding protein (PABP) and poly(A) mRNA. Depletion of eIF4G O-GlcNAcylation results in inhibition of protein synthesis, cell proliferation, and soft agar colony formation. The differential glycosylation of eIF4A and eIF4G appears to be regulated in the initiation complex to fine-tune protein synthesis. Our study thus expands the current understanding of protein synthesis, and adds another dimension of complexity to translational control of cellular proteins.