CGRP inhibits SARS-CoV-2 infection of bronchial epithelial cells, and its pulmonary levels correlate with viral clearance in critical COVID-19 patients.

Upon infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), patients with critical coronavirus disease 2019 (COVID-19) present with life-threatening respiratory distress, pulmonary damage, and cytokine storm. One unexplored component in COVID-19 is the neuropeptide calcitonin gene-related peptide (CGRP), which is highly abundant in the airways and could converge in multiple aspects of COVID-19-related pulmonary pathophysiology. Whether CGRP affects SARS-CoV-2 infection directly remains elusive. We show that in critical COVID-19 patients, CGRP is increased in both plasma and lungs. Importantly, CGRP pulmonary levels are elevated in early SARS-CoV-2-positive patients and restored to baseline upon subsequent viral clearance in SARS-CoV-2-negative patients. We further show that CGRP and its stable analog SAX directly inhibit infection of bronchial Calu-3 epithelial cells with SARS-CoV-2 Omicron and Alpha variants in a dose-dependent manner. Both pre- and post-infection treatments with CGRP and/or SAX are enough to block SARS-CoV-2 productive infection of Calu-3 cells. CGRP-mediated inhibition occurs via activation of the CGRP receptor and involves down-regulation of both SARS-CoV-2 entry receptors at the surface of Calu-3 cells. Together, we propose that increased pulmonary CGRP mediates beneficial viral clearance in critical COVID-19 patients by directly inhibiting SARS-CoV-2 propagation. Hence, CGRP-based interventions could be harnessed for management of COVID-19.IMPORTANCEThe neuropeptide CGRP is highly abundant in the airways. Due to its immunomodulatory, vasodilatory, and anti-viral functions, CGRP could affect multiple aspects of COVID-19-related pulmonary pathophysiology. Yet, the interplay between CGRP and SARS-CoV-2 during COVID-19 remains elusive. Herein, we show that pulmonary levels of CGRP are increased in critical COVID-19 patients, at an early stage of their disease when patients are SARS-CoV-2-positive. Upon subsequent viral clearance, CGRP levels are restored to baseline in SARS-CoV-2-negative patients. We further show that pre- and post-infection treatments with CGRP directly inhibit infection of Calu-3 bronchial epithelial cells with SARS -CoV-2, via activation of the CGRP receptor leading to decreased expression of both SARS-CoV-2 entry receptors. Together, we propose that increased pulmonary CGRP is beneficial in COVID-19, as CGRP-mediated inhibition of SARS-CoV-2 infection could contribute to viral clearance in critical COVID-19 patients. Accordingly, CGRP-based formulations could be useful for COVID-19 management.

Glycoproteomics of a Single Protein: Revealing Tens of Thousands of Myozyme Glycoforms by Hybrid HPLC-MS Approaches.

Characterization of highly glycosylated biopharma-ceuticals by mass spectrometry is challenging because of the huge chemical space of coexistent glycoforms present. Here, we report the use of an array of HPLC-mass spectrometry-based approaches at different structural levels of released glycan, glycopeptide, and hitherto unexplored intact glycoforms to scrutinize the biopharmaceutical Myozyme, containing the highly complex lysosomal enzyme recombinant acid α-glucosidase. The intrinsic heterogeneity of recombinant acid α-glucosidase glycoforms was unraveled using a novel strong anion exchange HPLC-mass spectrometry approach involving a pH-gradient of volatile buffers to facilitate chromatographic separation of glycoforms based on their degree of sialylation, followed by the acquisition of native mass spectra in an Orbitrap mass spectrometer. Upon considering the structures of 60 different glycans attached to seven glycosylation sites in the intact protein, the large set of interdependent data acquired at different structural levels was integrated using a set of bioinformatic tools and allowed the annotation of intact glycoforms unraveling more than 1,000,000 putative intact glycoforms. Detectable isoforms also included several mannose-6-phosphate variants, which are essential for directing the drug toward its target, the lysosomes. Finally, for the first time, we sought to validate the intact glycoform annotations by integrating experimental data on the enzymatically dissected proteoforms, which reduced the number of glycoforms supported by experimental evidence to 42,104. The latter verification clearly revealed the strengths but also intrinsic limitations of this approach for fully characterizing such highly complex glycoproteins by mass spectrometry.

Tooth acellular extrinsic fibre cementum incremental lines in humans are formed by parallel branched Sharpey's fibres and not by its mineral phase.

In humans, the growth pattern of the acellular extrinsic fibre cementum (AEFC) has been useful to estimate the age-at-death. However, the structural organization behind such a pattern remains poorly understood. In this study tooth cementum from seven individuals from a Mexican modern skeletal series were analyzed with the aim of unveiling the AEFC collagenous and mineral structure using multimodal imaging approaches. The organization of collagen fibres was first determined using: light microscopy, transmission electron microscopy (TEM), electron tomography, and plasma FIB scanning electron microscopy (PFIB-SEM) tomography. The mineral properties were then investigated using: synchrotron small-angle X-ray scattering (SAXS) for T-parameter (correlation length between mineral particles); synchrotron X-ray diffraction (XRD) for L-parameter (mineral crystalline domain size estimation), alignment parameter (crystals preferred orientation) and lattice parameters a and c; as well as synchrotron X-ray fluorescence for spatial distribution of calcium, phosphorus and zinc. Results show that Sharpey's fibres branched out fibres that cover and uncover other collagen bundles forming aligned arched structures that are joined by these same fibres but in a parallel fashion. The parallel fibres are not set as a continuum on the same plane and when they are superimposed project the AEFC incremental lines due to the collagen birefringence. The orientation of the apatite crystallites is subject to the arrangement of the collagen fibres, and the obtained parameter values along with the elemental distribution maps, revealed this mineral tissue as relatively homogeneous. Therefore, no intrinsic characteristics of the mineral phase could be associated with the alternating AEFC incremental pattern.

Determination of highly polar anionic pesticides in beehive products by hydrophilic interaction liquid chromatography coupled to mass spectrometry.

The analysis of highly polar pesticides is challenging due to their unique physicochemical properties, requiring specialized chromatographic techniques for their accurate and sensitive detection. Furthermore, the high level of co-extracted polar matrix components that can co-elute with the analytes can interfere with the analysis. Consequently, there is lack of pesticide monitoring data, as the European Food Safety Authority has pointed out. This article explores the overcoming of such difficulties in the analysis of these compounds. Analytical methodologies for the extraction, clean-up, and direct determination of 11 highly polar anionic pesticides, including glyphosate, glufosinate, ethephon, fosetyl-aluminium, and their related metabolites in complex food matrices such as honey and pollen by hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry were successfully developed and validated. Solid-phase extraction and micro-solid-phase extraction employing strong anion exchange (SAX) cartridges were implemented for clean-up. The automation and miniaturization of SAX clean-up for these compounds were achieved for the first time. For method validation, SANTE/11312/2021 guideline was followed. Recoveries were between 70 and 120%, with RSDs below 20%. Limits of quantitation ranged from 0.005 to 0.020 mg kg-1. Linearity was evaluated from 0.002 to 0.200 mg kg-1. Matrix effects were assessed, showing medium to low signal suppression for most compounds. AMPA and glufosinate presented the highest signal suppression, but it was reduced after SAX clean-up. Analysis of real honey and pollen samples revealed the occurrence of the studied compounds in beehive products and showed the applicability of the validated methodologies for routine control of these complex samples.

Enhancing Proteome Coverage by Using Strong Anion-Exchange in Tandem with Basic-pH Reversed-Phase Chromatography for Sample Multiplexing-Based Proteomics.

Sample multiplexing-based proteomic strategies rely on fractionation to improve proteome coverage. Tandem mass tag (TMT) experiments, for example, can currently accommodate up to 18 samples with proteins spanning several orders of magnitude, thus necessitating fractionation to achieve reasonable proteome coverage. Here, we present a simple yet effective peptide fractionation strategy that partitions a pooled TMT sample with a two-step elution using a strong anion-exchange (SAX) spin column prior to gradient-based basic pH reversed-phase (BPRP) fractionation. We highlight our strategy with a TMTpro18-plex experiment using nine diverse human cell lines in biological duplicate. We collected three data sets, one using only BPRP fractionation and two others of each SAX-partition followed by BPRP. The three data sets quantified a similar number of proteins and peptides, and the data highlight noticeable differences in the distribution of peptide charge and isoelectric point between the SAX partitions. The combined SAX partition data set contributed 10% more proteins and 20% more unique peptides that were not quantified by BPRP fractionation alone. In addition to this improved fractionation strategy, we provide an online resource of relative abundance profiles for over 11,000 proteins across the nine human cell lines, as well as two additional experiments using ovarian and pancreatic cancer cell lines.

Harmonization of L1CAM expression facilitates axon outgrowth and guidance of a motor neuron.

Brain development requires precise regulation of axon outgrowth, guidance and termination by multiple signaling and adhesion molecules. How the expression of these neurodevelopmental regulators is transcriptionally controlled is poorly understood. The Caenorhabditis elegans SMD motor neurons terminate axon outgrowth upon sexual maturity and partially retract their axons during early adulthood. Here we show that C-terminal binding protein 1 (CTBP-1), a transcriptional corepressor, is required for correct SMD axonal development. Loss of CTBP-1 causes multiple defects in SMD axon development: premature outgrowth, defective guidance, delayed termination and absence of retraction. CTBP-1 controls SMD axon guidance by repressing the expression of SAX-7, an L1 cell adhesion molecule (L1CAM). CTBP-1-regulated repression is crucial because deregulated SAX-7/L1CAM causes severely aberrant SMD axons. We found that axonal defects caused by deregulated SAX-7/L1CAM are dependent on a distinct L1CAM, called LAD-2, which itself plays a parallel role in SMD axon guidance. Our results reveal that harmonization of L1CAM expression controls the development and maturation of a single neuron.

Interaction Between the Transferrin Protein and Plutonium (and Thorium), What's New?

Transferrin (Tf) is a glycoprotein that transports iron from the serum to the various organs. Several studies have highlighted that Tf can interact with metals other than Fe(III), including actinides that are chemical and radiological toxics. We propose here to report on the behavior of Th(IV) and Pu(IV) in comparison with Fe(III) upon Tf complexation. We considered UV-Vis and IR data of the M2 Tf complex (M=Fe, Th, Pu) and combined experimental EXAFS data with MD models. EXAFS data of the first M-O coordination sphere are consistent with the MD model considering 1 synergistic carbonate. Further EXAFS data analysis strongly suggests that contamination by Th/Pu colloids seems to occur upon Tf complexation, but it seems limited. SAXS data have also been recorded for all complexes and also after the addition of Deferoxamine-B (DFOB) in the medium. The Rg values are very close for apoTf, ThTf and PuTf, but slightly larger than for holoTf. Data suggest that the structure of the protein is more ellipsoidal than spherical, with a flattened oblate form. From this data, the following order of conformation size might be considered:holoTf

A Pragmatic Guide to Enrichment Strategies for Mass Spectrometry-Based Glycoproteomics.

Glycosylation is a prevalent, yet heterogeneous modification with a broad range of implications in molecular biology. This heterogeneity precludes enrichment strategies that can be universally beneficial for all glycan classes. Thus, choice of enrichment strategy has profound implications on experimental outcomes. Here we review common enrichment strategies used in modern mass spectrometry-based glycoproteomic experiments, including lectins and other affinity chromatographies, hydrophilic interaction chromatography and its derivatives, porous graphitic carbon, reversible and irreversible chemical coupling strategies, and chemical biology tools that often leverage bioorthogonal handles. Interest in glycoproteomics continues to surge as mass spectrometry instrumentation and software improve, so this review aims to help equip researchers with the necessary information to choose appropriate enrichment strategies that best complement these efforts.

WIDENING THE BOTTLENECK OF PHOSPHOPROTEOMICS: EVOLVING STRATEGIES FOR PHOSPHOPEPTIDE ENRICHMENT.

Phosphorylation is a form of protein posttranslational modification (PTM) that regulates many biological processes. Whereas phosphoproteomics is a scientific discipline that identifies and quantifies the phosphorylated proteome using mass spectrometry (MS). This task is extremely challenging as ~30% of the human proteome is phosphorylated; and each phosphoprotein may exist as multiple phospho-isoforms that are present in low abundance and stoichiometry. Hence, phosphopeptide enrichment techniques are indispensable to (phospho)proteomics laboratories. These enrichment methods encompass widely-adopted techniques such as (i) affinity-based chromatography; (ii) ion exchange and mixed-mode chromatography (iii) enrichment with phospho-specific antibodies and protein domains, and (iv) functionalized polymers and other less common but emerging technologies such as hydroxyapatite chromatography and precipitation with inorganic ions. Here, we review these techniques, their history, continuous development and evaluation. Besides, we outline associating challenges of phosphoproteomics that are linked to experimental design, sample preparation, and proteolytic digestion. In addition, we also discuss about the future outlooks in phosphoproteomics, focusing on elucidating the noncanonical phosphoproteome and deciphering the "dark phosphoproteome". © 2020 John Wiley & Sons Ltd.

SAX and Random Projection Algorithms for the Motif Discovery of Orbital Asteroid Resonance Using Big Data Platforms.

The phenomenon of big data has occurred in many fields of knowledge, one of which is astronomy. One example of a large dataset in astronomy is that of numerically integrated time series asteroid orbital elements from a time span of millions to billions of years. For example, the mean motion resonance (MMR) data of an asteroid are used to find out the duration that the asteroid was in a resonance state with a particular planet. For this reason, this research designs a computational model to obtain the mean motion resonance quickly and effectively by modifying and implementing the Symbolic Aggregate Approximation (SAX) algorithm and the motif discovery random projection algorithm on big data platforms (i.e., Apache Hadoop and Apache Spark). There are five following steps on the model: (i) saving data into the Hadoop Distributed File System (HDFS); (ii) importing files to the Resilient Distributed Datasets (RDD); (iii) preprocessing the data; (iv) calculating the motif discovery by executing the User-Defined Function (UDF) program; and (v) gathering the results from the UDF to the HDFS and the .csv file. The results indicated a very significant reduction in computational time between the use of the standalone method and the use of the big data platform. The proposed computational model obtained an average accuracy of 83%, compared with the SwiftVis software.

Automatic Pulse Classification for Artefact Removal Using SAX Strings, a CENTER-TBI Study.

High-resolution, waveform-level data from bedside monitors carry important information about a patient's physiology but is also polluted with artefactual data. Manual mark-up is the standard practice for detecting and eliminating artefacts, but it is time-consuming, prone to errors, biased and not suitable for real-time processing.In this paper we present a novel automatic artefact detection technique based on a Symbolic Aggregate approXimation (SAX) technique which makes it possible to represent individual pulses as 'words'. It does that by coding each pulse with a specified number of letters (here six) from a predefined alphabet of characters (here six). The word is then fed to a support vector machine (SVM) and classified as artefactual or physiological.To define the universe of acceptable pulses, the arterial blood pressure from 50 patients was analysed, and acceptable pulses were manually chosen by looking at the average pulse that each 'word' generated. This was then used to train a SVM classifier. To test this algorithm, a dataset with a balanced ratio of clean and artefactual pulses was built, classified and independently evaluated by two observers achieving a sensitivity of 0.972 and 0.954 and a specificity of 0.837 and 0.837 respectively.

Label-Free Quantitative Phosphoproteomics of the Fission Yeast Schizosaccharomyces pombe Using Strong Anion Exchange- and Porous Graphitic Carbon-Based Fractionation Strategies.

The phosphorylation of proteins modulates various functions of proteins and plays an important role in the regulation of cell signaling. In recent years, label-free quantitative (LFQ) phosphoproteomics has become a powerful tool to analyze the phosphorylation of proteins within complex samples. Despite the great progress, the studies of protein phosphorylation are still limited in throughput, robustness, and reproducibility, hampering analyses that involve multiple perturbations, such as those needed to follow the dynamics of phosphoproteomes. To address these challenges, we introduce here the LFQ phosphoproteomics workflow that is based on Fe-IMAC phosphopeptide enrichment followed by strong anion exchange (SAX) and porous graphitic carbon (PGC) fractionation strategies. We applied this workflow to analyze the whole-cell phosphoproteome of the fission yeast Schizosaccharomyces pombe. Using this strategy, we identified 8353 phosphosites from which 1274 were newly identified. This provides a significant addition to the S. pombe phosphoproteome. The results of our study highlight that combining of PGC and SAX fractionation strategies substantially increases the robustness and specificity of LFQ phosphoproteomics. Overall, the presented LFQ phosphoproteomics workflow opens the door for studies that would get better insight into the complexity of the protein kinase functions of the fission yeast S. pombe.

Drosophila ML-DmD17-c3 cells respond robustly to Dpp and exhibit complex transcriptional feedback on BMP signaling components.

BACKGROUND: BMP signaling is involved in myriad metazoan developmental processes, and study of this pathway in Drosophila has contributed greatly to our understanding of its molecular and genetic mechanisms. These studies have benefited not only from Drosophila's advanced genetic tools, but from complimentary in vitro culture systems. However, the commonly-used S2 cell line is not intrinsically sensitive to the major BMP ligand Dpp and must therefore be augmented with exogenous pathway components for most experiments. RESULTS: Herein we identify and characterize the responses of Drosophila ML-DmD17-c3 cells, which are sensitive to Dpp stimulation and exhibit characteristic regulation of BMP target genes including Dad and brk. Dpp signaling in ML-DmD17-c3 cells is primarily mediated by the receptors Put and Tkv, with additional contributions from Wit and Sax. Furthermore, we report complex regulatory feedback on core pathway genes in this system. CONCLUSIONS: Native ML-DmD17-c3 cells exhibit robust transcriptional responses to BMP pathway induction. We propose that ML-DmD17-c3 cells are well-suited for future BMP pathway analyses.

Dynein and EFF-1 control dendrite morphology by regulating the localization pattern of SAX-7 in epidermal cells.

Our previous work showed that the cell adhesion molecule SAX-7 forms an elaborate pattern in Caenorhabditis elegans epidermal cells, which instructs PVD dendrite branching. However, the molecular mechanism forming the SAX-7 pattern in the epidermis is not fully understood. Here, we report that the dynein light intermediate chain DLI-1 and the fusogen EFF-1 are required in epidermal cells to pattern SAX-7. While previous reports suggest that these two molecules act cell-autonomously in the PVD, our results show that the disorganized PVD dendritic arbors in these mutants are due to the abnormal SAX-7 localization patterns in epidermal cells. Three lines of evidence support this notion. First, the epidermal SAX-7 pattern was severely affected in dli-1 and eff-1 mutants. Second, the abnormal SAX-7 pattern was predictive of the ectopic PVD dendrites. Third, expression of DLI-1 or EFF-1 in the epidermis rescued both the SAX-7 pattern and the disorganized PVD dendrite phenotypes, whereas expression of these molecules in the PVD did not. We also show that DLI-1 functions cell-autonomously in the PVD to promote distal branch formation. These results demonstrate the unexpected roles of DLI-1 and EFF-1 in the epidermis in the control of PVD dendrite morphogenesis.

Evaluating the Long-, Short-, and Oblique-Axis Approaches for Ultrasound-Guided Vascular Access Cannulation.

BACKGROUND: Our goal was to conduct a network meta-analysis of randomized controlled trials to compare the effects of the long-axis (LAX), short-axis (SAX), and oblique-axis (OAX) ultrasound guidance approaches for vascular access cannulation. METHODS: We searched 5 databases, including the Cochrane Central Register of Controlled Trials in the Cochrane Library, Embase, MEDLINE, CINAHL, and Web of Science. Seven randomized clinical trials assessing ultrasound guidance for vascular access cannulation via the LAX, SAX, or OAX approach were included. The primary end point was the first-pass success rate. Secondary end points included the mean time to success and average number of attempts until success. We used random-effects models to calculate weighted mean differences with 95% confidence intervals for continuous outcomes and relative risks with 95% confidence intervals for dichotomous outcomes. RESULTS: There were no significant differences between the LAX, SAX, and OAX techniques with respect to the first-pass success rate, mean time to success, average number of attempts until success, or the incidence of hematoma. CONCLUSION: There was insufficient evidence to definitively recommend the LAX, SAX, or OAX approach for patients undergoing ultrasound-guided vascular access cannulation.

Tip-Based Fractionation of Batch-Enriched Phosphopeptides Facilitates Easy and Robust Phosphoproteome Analysis.

The identification of large numbers of phosphopeptides from complex samples largely relies on sample fractionation to reduce complexity and allow using large amounts of starting material. For such experiments, commonly fractionation of whole cell lysate digests followed by enrichment of phosphopeptides from the single fractions is performed. We evaluated the tip-based fractionation of batch-enriched phosphopeptides as an alternative method. We compared three tip-based fractionation methods employing strong cation exchange (SCX), strong anion exchange (SAX), and C18 material for basic reversed-phase (BRP) fractionation using HeLa whole cell lysate digests. We show that SCX tips are superior to BRP and SAX tips due to a more efficient retention and distribution of phosphopeptides as well as a better resolution. Furthermore, we show that tip-based fractionation results in a similar performance as fractionation followed by phosphopeptide enrichment of the single fractions and outperforms analysis of unfractionated phosphopeptide-enriched samples with long chromatography gradients. Our fractionation approach using SCX tips is straightforward, reproducible, and requires a fraction of time, effort, and instrumentation compared to those of the fractionation of whole cell lysate digests with subsequent enrichment of phosphopeptides from the single fractions.

mir-67 regulates P. aeruginosa avoidance behavior in C. elegans.

Pathogen avoidance behaviors are found throughout the animal kingdom and are important for animal's survival in nature. As a free-living nematode, C. elegans is exposed to a variety of microorganisms, including toxic or pathogenic bacteria, in soil. C. elegans can develop efficient avoidance responses to pathogenic bacteria to minimize the infection risk. However, the role of microRNAs (miRNAs) in pathogen avoidance in C. elegans remains unclear. In this report, we showed that the miRNA mir-67 was involved in a behavioral avoidance response to P. aeruginosa PA14. Exposure to P. aeruginosa PA14 induced the expression of mir-67 in worms. mir-67(n4899) mutants exhibited a reduced ability to avoid P. aeruginosa PA14. By combining quantitative proteomic analysis with miRNA target prediction algorithms, we identified SAX-7/L1CAM, which is transmembrane cell adhesion receptor molecule, as the target of mir-67. Silencing of sax-7 by RNAi on mir-67 mutants rescued avoidance behavioral. Our data demonstrate that the mir-67-SAX-7 pathway modulate the behavioral avoidance response to pathogens, thus providing a new perspective in the role of miRNAs in host-microbe interactions.

Automated platform of µLC-MS/MS using SAX trap column for highly efficient phosphopeptide analysis.

In the current study, a specially designed automated nanoflow liquid chromatography coupled with tandem mass spectrometer (µLC-MS/MS) system for phosphopeptide analysis was reported. The system was established by applying a strong anion exchange (SAX) trap column. At the beginning of the analysis, phosphopeptides were loaded onto SAX trap column at high flow rate. Next, the retained phosphopeptides were eluted onto a C18 analytical capillary column by an acidic buffer with high ionic strength. At last, its performance was evaluated by analyzing the phosphopeptides enriched from the tryptic digest of mouse liver lysate. Compared with conventional automated µLC-MS/MS system using C18 trap column, much more phosphopeptides could be identified by the SAX trap column system. This system was fully automated and had promising application in high throughput phosphoproteomic analysis.

Improving the recognition of grips and movements of the hand using myoelectric signals.

BACKGROUND: People want to live independently, but too often disabilities or advanced age robs them of the ability to do the necessary activities of daily living (ADLs). Finding relationships between electromyograms measured in the arm and movements of the hand and wrist needed to perform ADLs can help address performance deficits and be exploited in designing myoelectrical control systems for prosthetics and computer interfaces. METHODS: This paper reports on several machine learning techniques employed to discover the electromyogram patterns present when performing 24 typical fine motor functional activities of the hand and the rest position used to accomplish ADLs. Accelerometer data is collected from the hand as an aid in identifying the start and end of movements and to help in labeling the signal data. Techniques employed include classification of 100 ms individual signal instances, using a symbolic representation to approximate signal streams, and the use of nearest neighbor in two specific situations: creation of an affinity matrix to model learning instances and classify based on multiple adjacent signal values, and using Dynamic Time Warping (DTW) as a distance measure to classify entire activity segments. RESULTS: Results show the patterns can be learned to an accuracy of 76.64 % for a 25 class problem when classifying 100 ms instances, 83.63 % with the affinity matrix approach with symbolic representation, and 85.22 % with Dynamic Time Warping. Classification errors are, with a few exceptions, concentrated within particular grip action groups. CONCLUSION: The findings reported here support the view that grips and movements of the hand can be distinguished by combining electrical and mechanical properties of the task to an accuracy of 85.22 % for a 25 class problem. Converting the signals to a symbolic representation and classifying based on larger portions of the signal stream improve classification accuracy. This is both clinically useful and opens the way for an approach to help simulate hand functional activities. With improvements it may also prove useful in real time control applications.

Cooperation of binding sites at the hydrophilic domain of cell-surface sulfatase Sulf1 allows for dynamic interaction of the enzyme with its substrate heparan sulfate.

BACKGROUND: Sulf1 is a cell-surface sulfatase removing internal 6-O-sulfate groups from heparan sulfate (HS) chains. Thereby it modulates the activity of HS-dependent growth factors. For HS interaction Sulf1 employs a unique hydrophilic domain (HD). METHODS: Affinity-chromatography, AFM-single-molecule force spectroscopy (SMFS) and immunofluorescence on living cells were used to analyze specificity, kinetics and structural basis of this interaction. RESULTS: Full-length Sulf1 interacts broadly with sulfated glycosaminoglycans (GAGs) showing, however, higher affinity toward HS and heparin than toward chondroitin sulfate or dermatan sulfate. Strong interaction depends on the presence of Sulf1-substrate groups, as Sulf1 bound significantly weaker to HS after enzymatic 6-O-desulfation by Sulf1 pretreatment, hence suggesting autoregulation of Sulf1/substrate association. In contrast, HD alone exhibited outstanding specificity toward HS and did not interact with chondroitin sulfate, dermatan sulfate or 6-O-desulfated HS. Dynamic SMFS revealed an off-rate of 0.04/s, i.e., ~500-fold higher than determined by surface plasmon resonance. SMFS allowed resolving the dynamics of single dissociation events in each force-distance curve. HD subdomain constructs revealed heparin interaction sites in the inner and C-terminal regions of HD. CONCLUSIONS: Specific substrate binding of Sulf1 is mediated by HD and involves at least two separate HS-binding sites. Surface plasmon resonance KD-values reflect a high avidity resulting from multivalent HD/heparin interaction. While this ensures stable cell-surface HS association, the dynamic cooperation of binding sites at HD and also the catalytic domain enables processive action of Sulf1 along or across HS chains. GENERAL SIGNIFICANCE: HD confers a novel and highly dynamic mode of protein interaction with HS.