Efficacy of surgical periodontal treatment with and without photobiomodulation in the treatment of severe periodontitis: An evaluation of periodontal, microbiological, and cytokine levels.

AIM: To compare the efficacy of surgical periodontal treatment (SPT) alone and PDT-assisted surgery in participants with severe periodontitis. MATERIAL AND METHODS: The present clinical trial was completed by 64 participants (n = 32 each). The selection was made according to predefined inclusion and exclusion criteria. Patients in group A were treated with SPT only and participants in group B were treated with SPT adjunct to PDT. Microbiological assessment of P.Gingivalis; T. Forsythia and T.Denticola were evaluated using cultural analysis and periodontal parameters plaque score (PSc), bleeding on probing (BoP) periodontal depth (PD), and clinical attachment loss (CAL) at baseline and post-treatment at 6 months and 12 months were performed. The gingival crevicular fluid (GCF) was collected for the estimation of IL-1ß and tumor necrosis factor-alpha (TNF-α) using an enzyme-linked immunosorbent assay (ELISA). For intra-group comparison and post hoc correction, Student's t-test along with Bonferroni was used. For the difference between follow-ups, an analysis of variance (ANOVA) multiple rank tests were incorporated. RESULTS: The mean age of participants in the SPT group was 55.25±4.6yrs. Whereas, participants treated with PDT adjunct to SPT were 54.88±3.6yrs. Periodontal parameters (BoP, PD, PSc, CAL) showed no significant difference at baseline. At 6 months and 12 months follow-up, a significant difference in all parameters (BoP, PD, PSc, and CAL) was found in participants treated with SPT alone and PDT adjunct to SPT (p<0.05). Inflammatory biomarkers at 6-month and 12-month follow-ups, a statistically significant difference in the level of biomarkers (IL-1ß and TNF-α) were observed in both groups from baseline (p<0.05). However, at baseline, no significant difference was noted in both groups (p> 0.05). The microbiological assessment showed a significant drop in the bacterial count in participants treated with both regimes i.e., SPT alone and PDT adjunct to SPT. CONCLUSION: Photodynamic therapy (PDT) adjunct to surgical periodontal treatment (SPT) in severe periodontitis improves microbiological and periodontal parameters and lowers the level of proinflammatory cytokines.

Evidence of a putative glycosaminoglycan binding site on the glycosylated SARS-CoV-2 spike protein N-terminal domain.

SARS-CoV-2 has rapidly spread throughout the world's population since its initial discovery in 2019. The virus infects cells via a glycosylated spike protein located on its surface. The protein primarily binds to the angiotensin-converting enzyme-2 (ACE2) receptor, using glycosaminoglycans (GAGs) as co-receptors. Here, we performed bioinformatics and molecular dynamics simulations of the spike protein to investigate the existence of additional GAG binding sites on the receptor-binding domain (RBD), separate from previously reported heparin-binding sites. A putative GAG binding site in the N-terminal domain (NTD) of the protein was identified, encompassing residues 245-246. We hypothesized that GAGs of a sufficient length might bridge the gap between this site and the PRRARS furin cleavage site, including the mutation S247R. Docking studies using GlycoTorch Vina and subsequent MD simulations of the spike trimer in the presence of dodecasaccharides of the GAGs heparin and heparan sulfate supported this possibility. The heparan sulfate chain bridged the gap, binding the furin cleavage site and S247R. In contrast, the heparin chain bound the furin cleavage site and surrounding glycosylation structures, but not S247R. These findings identify a site in the spike protein that favors heparan sulfate binding that may be particularly pertinent for a better understanding of the recent UK and South African strains. This will also assist in future targeted therapy programs that could include repurposing clinical heparan sulfate mimetics.

Epitope-Based Peptide Vaccine against Glycoprotein G of Nipah Henipavirus Using Immunoinformatics Approaches.

BACKGROUND: Nipah belongs to the genus Henipavirus and the Paramyxoviridae family. It is an endemic most commonly found at South Asia and has first emerged in Malaysia in 1998. Bats are found to be the main reservoir for this virus, causing disease in both humans and animals. The last outbreak has occurred in May 2018 in Kerala. It is characterized by high pathogenicity and fatality rates which varies from 40% to 70% depending on the severity of the disease and on the availability of adequate healthcare facilities. Currently, there are no antiviral drugs available for NiV disease and the treatment is just supportive. Clinical presentations for this virus range from asymptomatic infection to fatal encephalitis. OBJECTIVE: This study is aimed at predicting an effective epitope-based vaccine against glycoprotein G of Nipah henipavirus, using immunoinformatics approaches. METHODS AND MATERIALS: Glycoprotein G of the Nipah virus sequence was retrieved from NCBI. Different prediction tools were used to analyze the epitopes, namely, BepiPred-2.0: Sequential B Cell Epitope Predictor for B cell and T cell MHC classes II and I. Then, the proposed peptides were docked using Autodock 4.0 software program. Results and Conclusions. The two peptides TVYHCSAVY and FLIDRINWI have showed a very strong binding affinity to MHC class I and MHC class II alleles. Furthermore, considering the conservancy, the affinity, and the population coverage, the peptide FLIDRINWIT is highly suitable to be utilized to formulate a new vaccine against glycoprotein G of Nipah henipavirus. An in vivo study for the proposed peptides is also highly recommended.

Determination of paramagnetic ferrous gel sensitivity in low energy x-ray beam produced by a miniature accelerator.

The INTRABEAM Carl Zeiss Surgical system (Oberkochen, Germany) is a miniature accelerator producing low energy photons (50 keV maximum). The published dosimetric characterization of the INTRABEAM was based on detectors (radiochromic films or ionization chambers) not allowing measuring the absorbed dose in the first millimeters of the irradiated medium, where the dose is actually prescribed. This study aims at determining with Magnetic Resonance Imaging (MRI) the sensitivity of a paramagnetic gel in order to measure the dose deposit produced with the INTRABEAM from 0 to 20 mm. Although spherical applicators are mostly used with the INTRABEAM system for breast applications, this study focuses on surface applicators that are of interest for cutaneous carcinomas. The irradiations at 12 different dose levels (between 2 Gy and 50 Gy at the gel surface) were performed with the INTRABEAM and a 4 cm surface applicator. The gel used in this study is a new « sensitive ¼ material. In order to compare gel sensitivity at low energy with high energy, gels were irradiated by an 18 MV photon beam produced by a Varian Clinac 2100 CD. T2 weighted multi echo MRI sequences were performed with 16 echo times. The T2 signal versus echo times was fitted with a mono-exponential function with 95% confidence interval. The calibration curve determined at low energy is a linear function (r2 = 0.9893) with a sensitivity of 0.0381 s-1.Gy-1, a similar linear function was obtained at high energy (0.0372 s-1.Gy-1 with r2 = 0.9662). The calibration curve at low energy was used to draw isodose maps from the MR images. The PDD (Percent Depth Dose) determined in the gel is within 5%-1mm of the ionization chamber PDD except for one point. The dosimetric sensitivity of this new paramagnetic ferrous gel was determined with MRI measurements. It allowed measuring the dose distribution specifically in the first millimeters for an irradiation with the INTRABEAM miniature accelerator equipped with a surface applicator.

Dendrimer Heparan Sulfate Glycomimetics: Potent Heparanase Inhibitors for Anticancer Therapy.

Heparanase is a mammalian endoglycosidase that cleaves heparan sulfate (HS) polysaccharides and contributes to remodelling of the extracellular matrix and regulation of HS-binding protein bioavailabilities. Heparanase is upregulated in malignant cancers and inflammation, aiding cell migration and the release of signaling molecules. It is established as a highly druggable extracellular target for anticancer therapy, but current compounds have limitations, because of cost, production complexity, or off-target effects. Here, we report the synthesis of a novel, targeted library of single-entity glycomimetic clusters capped with simple sulfated saccharides. Several dendrimer HS glycomimetics display low nM IC50 potency for heparanase inhibition equivalent to comparator compounds in clinical development, and potently inhibit metastasis and growth of human myeloma tumor cells in a mouse xenograft model. Importantly, they lack anticoagulant activity and cytotoxicity, and also inhibit angiogenesis. They provide a new candidate class for anticancer and wider therapeutic applications, which could benefit from targeted heparanase inhibition.

Label-Free Discovery Array Platform for the Characterization of Glycan Binding Proteins and Glycoproteins.

The identification of carbohydrate-protein interactions is central to our understanding of the roles of cell-surface carbohydrates (the glycocalyx), fundamental for cell-recognition events. Therefore, there is a need for fast high-throughput biochemical tools to capture the complexity of these biological interactions. Here, we describe a rapid method for qualitative label-free detection of carbohydrate-protein interactions on arrays of simple synthetic glycans, more complex natural glycosaminoglycans (GAG), and lectins/carbohydrate binding proteins using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The platform can unequivocally identify proteins that are captured from either purified or complex sample mixtures, including biofluids. Identification of proteins bound to the functionalized array is achieved by analyzing either the intact protein mass or, after on-chip proteolytic digestion, the peptide mass fingerprint and/or tandem mass spectrometry of selected peptides, which can yield highly diagnostic sequence information. The platform described here should be a valuable addition to the limited analytical toolbox that is currently available for glycomics.

Panels of chemically-modified heparin polysaccharides and natural heparan sulfate saccharides both exhibit differences in binding to Slit and Robo, as well as variation between protein binding and cellular activity.

Heparin/heparan sulfate (HS) glycosaminoglycans are required for Slit-Robo cellular responses. Evidence exists for interactions between each combination of Slit, Robo and heparin/HS and for formation of a ternary complex. Heparin/HS are complex mixtures displaying extensive structural diversity. The relevance of this diversity has been studied to a limited extent using a few select chemically-modified heparins as models of HS diversity. Here we extend these studies by parallel screening of structurally diverse panels of eight chemically-modified heparin polysaccharides and numerous natural HS oligosaccharide chromatographic fractions for binding to both Drosophila Slit and Robo N-terminal domains and for activation of a chick retina axon response to the Slit fragment. Both the polysaccharides and oligosaccharide fractions displayed variability in binding and cellular activity that could not be attributed solely to increasing sulfation, extending evidence for the importance of structural diversity to natural HS as well as model modified heparins. They also displayed differences in their interactions with Slit compared to Robo, with Robo preferring compounds with higher sulfation. Furthermore, the patterns of cellular activity across compounds were different to those for binding to each protein, suggesting that biological outcomes are selectively determined in a subtle manner that does not simply reflect the sum of the separate interactions of heparin/HS with Slit and Robo.

Characterisation of the interaction of neuropilin-1 with heparin and a heparan sulfate mimetic library of heparin-derived sugars.

Background. Neuropilin-1 (NRP-1) is a multidomain membrane protein with soluble isoforms interacting with a complex network of other membrane receptors, their respective ligands and heparan sulfate (HS). It is involved in the development of vasculature, neural patterning, immunological responses and pathological angiogenesis. Methods. We have characterised the binding of a Fc fusion of rat NRP-1 (Fc rNRP-1) and of a soluble isoform, corresponding to the first four extracellular domains of human NRP-1, shNRP-1, using optical biosensor-based binding assays with a library of heparin derivatives. Selective labelling of lysines protected upon heparin binding allowed their identification by mass spectrometry. Results. Fc rNRP-1 bound to heparin with high affinity (2.5 nM) and fast ka (9.8 × 10(6) M(-1)s(-1)). Unusually, NRP-1 bound both highly sulfated and completely desulfated stretches of heparin and exhibited a complex pattern of preferences for chemically modified heparins possessing one or two sulfate groups, e.g., it bound heparin with just a 6-O sulfate group better than heparin with any two of N-sulfate, 6-O sulfate and 2-O sulfate. Mass-spectrometry based mapping identified that, in addition to the expected the b1 domain, the a1, and c domains and the L2 linker were also involved in the interaction. In contrast, shNRP-1 bound heparin far more weakly. This could only be shown by affinity chromatography and by differential scanning fluorimetry. Discussion. The results suggest that the interaction of NRP-1 with HS is more complex than anticipated and involving a far greater extent of the protein than just the b1-b2 domains. NRP-1's preference for binding long saccharide structures suggests it has the potential to bind large segments of HS chains and so organise their local structure. In contrast, the four domain soluble isoform, shNRP-1 binds heparin weakly and so would be expected to diffuse away rapidly from the source cell.

Isolated subpulmonic fibrous ring, mirror-image dextrocardia and situs solitus in a young lady unreported and a near miss.

Congenital diseases causing obstruction of the right ventricular outflow tract (RVOT) are common, but the isolated subpulmonary membrane/ring is extremely rare and can be difficult to diagnose precisely, especially in adults. We report a case of surgically resected isolated subpulmonic fibrous ring in a lady with mirror-image dextrocardia and abdominal situs solitus that was misdiagnosed by echocardiography as a subaortic membrane.

Diversification of the structural determinants of fibroblast growth factor-heparin interactions: implications for binding specificity.

The functions of a large number (>435) of extracellular regulatory proteins are controlled by their interactions with heparan sulfate (HS). In the case of fibroblast growth factors (FGFs), HS binding determines their transport between cells and is required for the assembly of high affinity signaling complexes with their cognate FGF receptor. However, the specificity of the interaction of FGFs with HS is still debated. Here, we use a panel of FGFs (FGF-1, FGF-2, FGF-7, FGF-9, FGF-18, and FGF-21) spanning five FGF subfamilies to probe their specificities for HS at different levels as follows: binding parameters, identification of heparin-binding sites (HBSs) in the FGFs, changes in their secondary structure caused by heparin binding and structures in the sugar required for binding. For interaction with heparin, the FGFs exhibit K(D) values varying between 38 nM (FGF-18) and 620 nM (FGF-9) and association rate constants spanning over 20-fold (FGF-1, 2,900,000 M(-1) s(-1) and FGF-9, 130,000 M(-1) s(-1)). The canonical HBS in FGF-1, FGF-2, FGF-7, FGF-9, and FGF-18 differs in its size, and these FGFs have a different complement of secondary HBS, ranging from none (FGF-9) to two (FGF-1). Differential scanning fluorimetry identified clear preferences in these FGFs for distinct structural features in the polysaccharide. These data suggest that the differences in heparin-binding sites in both the protein and the sugar are greatest between subfamilies and may be more restricted within a FGF subfamily in accord with the known conservation of function within FGF subfamilies.

Array-based functional screening of heparin glycans.

Array methodologies have become powerful tools for interrogation of glycan-protein interactions but have critically lacked the ability to generate cell response data. Here, we report the development of a slide-based array method exemplified by measurement of activation of fibroblast growth factor signaling by heparin saccharides. Heparan sulfate-deficient Swiss 3T3 cells were overlaid onto an aminosilane-coated slide surface onto which heparin saccharides had been spotted and immobilized. The cells were transiently stimulated with FGF2 and immunofluorescence measured to assess downstream ERK1/2 phosphorylation. Activation of this signaling pathway response was restricted to cells exposed to heparin saccharides competent to activate FGF2 signaling. Differential activation of the overlaid cells by different-sized heparin saccharides was demonstrated by quantitative measurement of fluorescence intensity. This "glycobioarray" platform has significant potential as a generic tool for functional glycomics screening.

Glycomics profiling of heparan sulfate structure and activity.

The heparan sulfate (HS) family of glycosaminoglycans are highly complex and structurally diverse polysaccharides with information encoded within the chains that imparts the ability to bind selectively to a wide range of proteins-the "HS interactome"-and to regulate their biological activities. However, there are two key questions which need to be addressed; first, the extent of structural variation of expressed HS structures-the "heparanome"-in specific biological contexts and second, the degree of functional selectivity exerted by these structures in regulating biological processes. There is a clear need to develop more systematic and high throughput approaches in order to address these questions. Here, we describe a cohort of protocols for profiling different aspects of HS structure and activity, focusing particularly on disaccharide building blocks and larger oligosaccharide domains, the latter representing the functional units of HS chains. A range of other complementary methods in the literature are also discussed. Together these provide a new and more comprehensive toolkit to investigate HS structure and activity in a higher throughput manner in selected biological systems. The implementation of such a glycomics strategy will enable development of a systems biology view of HS structure-function relationships and help to resolve the significant puzzle of the extensive interactome of HS, which remains a key question in the glycobiology field. We anticipate that the next decade will see major advances in our understanding of the complex biology of HS.

Differential scanning fluorimetry measurement of protein stability changes upon binding to glycosaminoglycans: a screening test for binding specificity.

The interaction between glycosaminoglycans (GAGs) and proteins is important for the regulation of protein transport and activity. Here we present a novel method for the measurement of protein-GAG interactions suitable for high-throughput screening, able to discriminate between the interactions of a protein with GAGs of different structures. Binding of proteins to the GAG heparin, a proxy for sulfated regions of extracellular heparan sulfate, was found to enhance the stability of three test proteins, fibroblast growth factors (FGFs)-1, -2, and -18. Chemically modified heparins and heparin oligosaccharides of different lengths stabilized the three FGFs to different extents, depending on the pattern of sugar binding specificity. The method is based on a differential scanning fluorescence approach. It uses a Sypro Orange dye, which binds to exposed core residues of a denatured protein and results in an increased fluorescence signal. It is convenient, requiring low micromolar amounts of protein and ligand compared to other interaction assays, employing only a real-time polymerase chain reaction (PCR) instrument.

Generating heparan sulfate saccharide libraries for glycomics applications.

Natural and semi-synthetic heparan sulfate (HS) saccharide libraries are a valuable resource for investigating HS structure-function relationships, enabling high-throughput glycomics studies. Owing to the difficulty of chemical or in vitro enzymatic synthesis of HS saccharides, the structural diversity displayed in saccharides from tissue or cell sources cannot be readily accessed. In contrast, saccharide libraries can be generated by partial digestion of tissue-derived HS polysaccharide chains and chromatographic fractionation of the resulting saccharide mixtures. Fractionation is initially on the basis of hydrodynamic volume, using size exclusion chromatography. Further fractionation, on the basis of charge using strong anion exchange, can subsequently be applied. Desalting and sample concentration follows each fractionation step. Chromatographic fractions are generated that contain purified, or partially purified, saccharides. Here we describe a comprehensive protocol for generation of structurally diverse natural saccharide libraries from HS variants that is fast (approximately 3 weeks) and reproducible.

Fabrication of carbohydrate surfaces by using nonderivatised oligosaccharides, and their application to measuring the assembly of sugar-protein complexes.

This way up. Dual polarisation interferometry was used to design and characterise a surface on which the orientation and density of immobilised carbohydrates was suitable for studying their interactions with proteins. Lactoferrin was shown to adopt two orientations: "end-on" or "side-on", while for FGF-2 a single monolayer of protein was observed. The new surface can be used to elucidate the binding of proteins to carbohydrates and the geometry of the complexes, a frequently controversial area. Surface-based tools, such as microarrays and optical biosensors, are being increasingly applied to the analysis of carbohydrate-protein interactions. A key to these developments is the presentation of the carbohydrate to the protein target. Dual polarisation interferometry (DPI) is a surface-based technique that permits the real-time measurement of the changes in thickness, refractive index and mass of adsorbates 100 nm thick or less on the surface of a functionalised waveguide. DPI has been used to design and characterise a surface on which the orientation and density of the immobilised carbohydrates is suitable for studying their interactions with proteins and where nonspecific binding is reduced to less than 5 % of total binding. A thiol-functionalised surface was derivatised with a heterobifunctional crosslinker to yield a hydrazide surface. This was treated with oligosaccharides, derived from keratan sulfate (KS) chondroitin sulfate (CS) and heparin, that possess a reducing end. To block the unreacted hydrazide groups, the surface was treated with an aldehyde-functionalised PEG. The heparin DP-10 surfaces were then used to determine the performance of the immobilised DP-10 with respect to binding of two well-characterised proteins, lactoferrin (Lf) and fibroblast growth factor-2. The results show that Lf could adopt two different orientations, at high protein loadings the protein layer thickness corresponded to an "end-on" orientation of Lf, whilst rinsing with buffer saw the Lf molecules adopt a "side-on" configuration. In the case of FGF-2, a single monolayer of protein bound to DP-10 was observed. These results demonstrate that the new surface can be used to resolve key questions relating to the binding of proteins to carbohydrates, including, when used in DPI, the resolution of the geometry of complexes, an area that is frequently controversial.

Towards GAG glycomics: analysis of highly sulfated heparins by MALDI-TOF mass spectrometry.

Glycomics is a developing field that provides structural information on complex populations of glycans isolated from tissues, cells and organs. Strategies employing matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) are central to glycomic analysis. Current MALDI-based glycomic strategies are capable of efficiently analyzing glycoprotein and glycosphingolipid glycomes but little attention has been paid to devising glycomic methodologies suited to the analysis of glycosaminoglycan (GAG) polysaccharides which pose special problems for MALDI analysis because of their high level of sulfation and large size. In this paper, we describe MALDI strategies that have been optimized for the analysis of highly sulfated GAG-derived oligosaccharides. A crystalline matrix norharmane, as well as an ionic liquid 1-methylimidazolium alpha-cyano-4-hydroxycinnamate (ImCHCA), have been used for the analysis of heparin di-, tetra-, hexa- and decasaccharides carrying from 2 to 13 sulfate groups. Information about the maximum number of sulfate groups is obtained using the ionic liquid whereas MALDI-TOF/TOF MS/MS experiments using norharmane allowed the determination of the nature of the glycosidic backbone, and more precise information about the presence and the position in the sequence of N-acetylated residues.

A molecular mechanism for the heparan sulfate dependence of slit-robo signaling.

Slit is a large secreted protein that provides important guidance cues in the developing nervous system and in other organs. Signaling by Slit requires two receptors, Robo transmembrane proteins and heparan sulfate (HS) proteoglycans. How HS controls Slit-Robo signaling is unclear. Here we show that the second leucine-rich repeat domain (D2) of Slit, which mediates binding to Robo receptors, also contains a functionally important binding site for heparin, a highly sulfated variant of HS. Heparin markedly enhances the affinity of the Slit-Robo interaction in a solid-phase binding assay. Analytical gel filtration chromatography demonstrates that Slit D2 associates with a soluble Robo fragment and a heparin-derived oligosaccharide to form a ternary complex. Retinal growth cone collapse triggered by Slit D2 requires cell surface HS or exogenously added heparin. Mutation of conserved basic residues in the C-terminal cap region of Slit D2 reduces heparin binding and abolishes biological activity. We conclude that heparin/HS is an integral component of the minimal Slit-Robo signaling complex and serves to stabilize the relatively weak Slit-Robo interaction.