Emilie Gillon

Annabelle Varrot

Anne Imberty
email: anneimberty@cermav.cnrs.fr

LecB, a High Affinity Soluble Fucose-Binding Lectin from Pseudomonas aeruginosa

Keywords: Pseudomonas aeruginosa, soluble lectin, fucose, blood-group oligosaccharide, microcalorimetry

LecB/PA-IIL (Pfam PF07472) from bacterium Pseudomonas aeruginosa is a fucose-binding lectin with unusual high affinity for glycans. The occurrence of LecB and related proteins is limited to few opportunistic bacterial species, some of them being responsible for severe infections in immune compromised patients. This lectin is therefore of interest as a target for the design of anti-infectious compounds, but can also be used for research and biotechnology. LecB is a small protein that can be produced in good quantity in recombinant system and purified by affinity chromatography.

Introduction

Pseudomonas aeruginosa is one of the main human opportunistic pathogens and is responsible of infections such as folliculitis, keratitis, otitis but also pneumopathies in cystic fibrosis patients resulting in morbidity and mortality [START_REF] Govan | Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia[END_REF][START_REF] Mesaros | Pseudomonas aeruginosa: resistance and therapeutic options at the turn of the new millennium[END_REF]. Among several virulence factors, P. aeruginosa produces two soluble lectins named LecA (or PA-IL) and LecB (or PA-IIL) that are specific for galactose and fucose, respectively [START_REF] Gilboa-Garber | Pseudomonas aeruginosa lectins[END_REF]. LecB consists of four subunits of 11.73 kDa, each with a high affinity binding site for L-fucose and fucose-containing glycoconjuguates [START_REF] Gilboa-Garber | Identification and characterization of Pseudomonas aeruginosa PA-IIL lectin gene and protein compared to PA-IL[END_REF]. The lectin also binds to D-mannose but with lower affinity. LecB is produced in the cytoplasm of P. aeruginosa but has been shown to be located in the outer membrane and involved in biofilm formation [START_REF] Tielker | Pseudomonas aeruginosa lectin LecB is located in the outer membrane and is involved in biofilm formation[END_REF]. The crystal structure of LecB in complex with fucose [START_REF] Mitchell | Structural basis for oligosaccharidemediated adhesion of Pseudomonas aeruginosa in the lungs of cystic fibrosis patients[END_REF] and with mannose [START_REF] Loris | Structural basis of carbohydrate recognition by the lectin LecB from Pseudomonas aeruginosa[END_REF] uncovered the molecular basis of its ligand recognition. LecB forms homotetramer, each monomer presenting a fucose binding site with two calcium ions that are directly involved in carbohydrate coordination. LecB displays exceptionally high affinities for fucose, with Kd values in the micromolar range [START_REF] Mitchell | High affinity fucose binding of Pseudomonas aeruginosa lectin PA-IIL: 1.0 Å resolution crystal structure of the complex combined with thermodynamics and computational chemistry approaches[END_REF]. It binds to a large range of fucosylated antigens (ABH, Lewis, P and I systems) present on human tissues and in human milk [START_REF] Perret | Structural basis for the interaction between human milk oligosaccharides and the bacterial lectin PA-IIL of Pseudomonas aeruginosa[END_REF][START_REF] Wu | Interactions of the fucose-specific Pseudomonas aeruginosa lectin, PA-IIL, with mammalian glycoconjugates bearing polyvalent Lewis(a) and ABH blood group glycotopes[END_REF] with highest reported affinity in the nM range for Lewis a epitope [START_REF] Perret | Structural basis for the interaction between human milk oligosaccharides and the bacterial lectin PA-IIL of Pseudomonas aeruginosa[END_REF], and for biantennary N-glycans presenting two H-type epitopes at the extremities of branches [START_REF] Sommer | The virulence factor LecB varies in clinical isolates: consequences for ligand binding and drug discovery[END_REF]. LecB is a target of interest for anti-adhesion and anti-biofilm compounds and the search for glyco-derived drugs with potential therapeutical application is very active [START_REF] Boukerb | Anti-adhesive properties of glycoclusters against Pseudomonas aeruginosa lung infection[END_REF][START_REF] Wagner | Novel strategies for the treatment of Pseudomonas aeruginosa infections[END_REF]. As other lectins with specificity towards human glycans, LecB is also of biotechnical interest for the purification of glycoproteins or the labelling of glycoconjugates on tissues. LecB is now included in the few lectin-arrays proposed in different laboratories for the fine characterization of glycans. Due to its propensity to easily form crystals, LecB has been utilized as a template for structural determination of smaller biomolecules that are difficult to crystallize by themselves : after attachment of a fucose residue, modified DNA duplexes or short anti-bacterial peptides could be co-crystallized with LecB and structurally characterized [START_REF] Baeriswyl | X-ray crystal structures of short antimicrobial peptides as Pseudomonas aeruginosa Lectin B complexes[END_REF]15]. Most studies have been performed using LecB from P. aeruginosa PAO1 strain. However, recent works demonstrated sequence variations in LecB when analyzing the many isolates from clinical or environmental origin. Three different clusters of sequences were identified, represented by LecB from PAO1, PA7 and PA14 virulent strains [START_REF] Sommer | The virulence factor LecB varies in clinical isolates: consequences for ligand binding and drug discovery[END_REF][START_REF] Boukerb | Genomic rearrangements and functional diversification of lecA and lecB lectincoding regions impacting the efficacy of glycomimetics directed against Pseudomonas aeruginosa[END_REF]. Some of the amino acid variations are located near the fucose binding site but affect only slightly the specificity and affinity of LecB. LecB-like lectins are also present in a limited number of bacteria from the Ralstonia, Chromobacterium and Burkholderia species. CV-IIL from Chromobacterium violaceum is very similar to LecB [START_REF] Pokorná | Unusual entropy driven affinity of Chromobacterium violaceum lectin CV-IIL towards fucose and mannose[END_REF], while RS2L from Ralstonia solanacearum is specific for mannoside [START_REF] Sudakevitz | A new Ralstonia solanacearum high affinity mannose-binding lectin RS-IIL structurally resembling the Pseudomonas aeruginosa fucose-specific lectin PA-IIL[END_REF]. Such variation in specificity has been demonstrated to be due to one amino acid variation at position 22 [START_REF] Adam | Engineering of PA-IIL lectin from Pseudomonas aeruginosa -Unravelling the role of the specificity loop for sugar preference[END_REF]. Burkholderia cenocepacia hosts four different LecBlike proteins with dimeric association of lectin and extension by several other proteins [START_REF] Lameignere | Structural basis for mannose recognition by a lectin from opportunistic bacteria Burkholderia cenocepacia[END_REF][START_REF] Šulák | Burkholderia cenocepacia BC2L-C is a super lectin with dual specificity and proinflammatory activity[END_REF]. The details of production and characterization listed here can be therefore applied, with small adjustments to the whole family of LecB-like lectins.

Materials

Prepare all solutions using analytical grade reagents and ultrapure water and then filter on 0.22 µM membrane. Follow waste disposal regulations.

LecB Production

Methods

LecB Production

Transformation and Preculture

1. The plamid pET25b-LecB has been obtained from template DNA from Pseudomonas aeruginosa ATCC 33347 as previously described [START_REF] Mitchell | High affinity fucose binding of Pseudomonas aeruginosa lectin PA-IIL: 1.0 Å resolution crystal structure of the complex combined with thermodynamics and computational chemistry approaches[END_REF] and is available upon request. 2. Add 1 µL of pET25b-LecB plasmid to 100 µL of competent BL21(DE3) cells before 10 mn incubation on ice. 3. Heat shock at 42 °C for 45 s before a 3 min stay on ice. 4. Add 900 µL of liquid LB and incubate horizontally for 1 hr at 37 °C with shaking at 180 rpm. 5. Spread 100 µL on a petri dish with LB-agar and 100 µg/mL ampicilline and incubate at 37 °C overnight. 6. Transfer 1 colony to 10 mL liquid LB containing 100 µg/mL ampicilline and incubate overnight with shaking at 180 rpm.

Culture

1. Inoculate 1 L of liquid LB supplemented with 100 µg/mL ampicilline in 3 liters Erlenmeyer with the overnigth preculture. 2. Incubate at 37 °C with shaking at 180 rpm until OD600nm reached 0.6. 3. Switch the temperature to 30 °C, induce expression by adding 0.25 mM IPTG and culture for 3 hours at 180 rpm.

4. Centrifuge at 6,000 ×g, 4 °C for 15 min. 5. Resuspend the pellet in 35 mL liquid LB, transfer in 50 mL sterile conical tube and centriguge 6,000 ×g for 10 min. 6. Remove supernatant and store the pellet at -20 °C untill use.

LecB Purification

1. Resuspend the pellet in 30 mL of equilibration buffer. 2. Break the cells using a one shot table top cell disruptor at a pressure of 1.9 kbar. 3. Centrifuge 24,000 ×g, 30 min at 4 °C to clarify the sample. 4. Filter the supernatant on 0.45 µm with syringe filter. 5. Equilibrate a 10 mL D-Mannose agarose resin packed in C10/10 column connected to automated chromatography system such as NGC system with 5 column volumes (CV) of equilibration buffer at a flowrate of 1 mL/min. 6. Load the filtered supernatant at a flow rate of 1 mL/min and collect the flowthrough. 7. Rinse the column with the equilibration buffer to wash out contaminants until the baseline of OD280nm on the chromatogram stabilized to a low value (3 mL/min flowrate). 8. Elute the protein with the elution buffer using a flowrate of 3 mL/min and collect fraction of 5 mL. 9. Rinse the column with 3 CVs of ultrapure water at a flowrate of 3 mL/min and 2 CVs of 20 % ethanol at flowrate of 1mL/min prior storage at 4 °C. 10. Collect the fractions of the eluted peak and dialyzed against ultrapure water for 1 week, changing the bath each morning and evening. 11. Lyophilize protein and store at -20 °C (see Note 2).

Quality Control by Evaluation of Affinity

Isothermal titration calorimetry (ITC) is an analytical technique, which is considered as the gold standard for analyzing intermolecular interactions in solution. It is widely used for quality control in biotechnology since it gives access to quantitative analysis of the interaction of a protein with its ligand. A single experiment allows to determine stoichiometry (n), binding enthalpy (∆H), and affinity (i.e. dissociation constant, Kd).

Since the method is based on the heat released or absorbed in the binding process, it is very well suited for protein carbohydrate interaction, where the large number of hydrogen bonds result in strong exothermic signal [START_REF] Dam | Thermodynamic studies of lectin-carbohydrate interactions by isothermal titration calorimetry[END_REF].

Sample Preparation :

1. Prepare 400 µL of LecB at 50 µM in ITC buffer from the lyophilized protein.

Weight 1.5× excess amount of the necessary protein, dissolve in the corresponding volume of buffer, centrifuge 7,000 ×g for 5 min. 2. Recover the supernatant and determine the precise concentration by spectroscopy at 280 nm using a molecular weight of 11,732 daltons per monomer and a theoretical molar extinction coefficient of 6,990 corresponding to Absorbance 0.1 % (= 1 g /L) of 0.596. 3. Use the Origin software of the microcalorimeter for data analysis (see Note 4). The default parameters with a "one-site" model is fully compatible with the present experiments. Data will be obtained as indicated in Fig. 2 when clicking on the "ITC data" button (see Note 5). 4. Quality control is performed by comparing the obtained thermodynamic of binding with literature [START_REF] Sabin | Binding of different monosaccharides by lectin PA-IIL from Pseudomonas aeruginosa: Thermodynamics data correlated with X-ray structures[END_REF]. LecB was reported to have a submicromolar affinity for aMeFuc (Kd = 430 nM) with strong enthalpy (∆H = -41.3 kJ/mol) and a stoichiometry corresponding to approximately one binding site per monomer (n = 0.77). 1. LecB is a calcium-dependent lectin and it is advised to maintain a CaCl2 concentration of 100 µM through the purification process. If needed, this concentration can be decreased to 10 µM. 2. LecB is a very robust protein that can be lyophilized and stored at -20 °C for months or years. It is only recommended to aliquot in small quantities to avoid repeating cycles of defreezing and refreezing. 3. For a perfect ITC measurement, the main recommendation is to avoid buffer mismatch. Use exactly the same buffer for the protein solution and the sugar solution. 4. Alternatives to Origin are available in the public domain. The suite of integrated software packages NITPIC, SEDPHAT and GUSSI offer powerful treatment of data with automated shape analysis of the injection peaks [START_REF] Brautigam | Integration and global analysis of isothermal titration calorimetry data for studying macromolecular interactions[END_REF]. It also allows for integrating results from different calorimetric titration experiments into a global analysis 5. In the Origin software, the default units are calories. This could be change to international units Watt and Joules using the Tab ITC and then Display Watt/Joules.

Figure 1 :

 1 Figure 1: Alignment of amino acid sequences for LecB sequences and crystal structure of LecBPA14 in complex with a-methyl-fucoside (5A6X). Protein is represented by ribbon, monosaccharides by sticks and calcium ions by spheres.

3 .

 3 Ajust the concentration to 50 µM with the ITC buffer. 4. Prepare 70 µL of 0.5 mM of α-methyl fucoside (αMeFuc) in the same buffer as the lectin (see Note 3). 5. Degas all samples. 3.3.2 ITC Experiment 1. Fill the measuring cell with 50 µM LecB solution and the syringe with 0.5 mM αMeFuc solution. Cell volume is 200 µL and syringe volume is 40 µL. 2. Setup the parameters to DP 8, injection number 20, injection volume of 1 µL for the first one and 2 µL for all following ones with an injection delay of 120 s.

Figure 2 :

 2 Figure 2: Typical ITC experiment of titration of LecB PAO1 by α-methyl-L-fucoside with thermogram (top) and integrated data (bottom). The observed stoichiometry of 0.835 indicates here that 16.5% of the protein is inactive. This does not affect the determination of thermodynamics constant, since they are expressed as a function of the concentration of ligand. The enthalpy of binding is -11.05 Kcal/mol (-46kJ/mole) with a weak unfavorable contribution of entropy ∆S of -8.3 cal/mol/deg (ie T∆S = -10.3 kJ/mol) which is in full agreement with previous data [23].

Quality Control by Evaluation of Affinity

	5. Plasmid: pET25b-LecB
	6. Lennox L Broth Base.
	7. 100 mg/mL ampicilline.
	8. 1 M Isopropyl-β-D-thiogalactopyranoside (IPTG).
	9. 1 L polycarbonate centrifuge bottle.
	10. 50 mL sterile conical centrifuge tubes.
	11. Centrifuge with adapted rotors.
	2.2 LecB Purification
	1. Endonuclease such as Benzonase or Denarase at 250 units/µL .
	2. Rotating wheel.
	3. One shot table top cell disruptor (Constant systems Ltd).
	4. 35 mL centrifuge tube and centrifuge wth rotor going to 24,000 ×g
	5. 0.45 µm PES syringe filter.
	6. C10/10 column with flow adapter (GE Healthcare Life Sciences).
	7. Equilibration buffer : 20 mM Tris pH 7.5 100 mM NaCl and 100 µM CaCl2 (see Note
	1).
	8. Elution buffer : 20 mM Tris pH 7.5 100 mM NaCl and 100 mM D-Mannose.
	9. Low or Medium-Pressure Chromatography Systems such as Akta Prime (GE
	Healthcare Life Sci) or NGC (Bio-Rad).
	10. 5mL collection tubes.
	11. Snakeskin dialysis tubing 3500 MWCO (Thermo Fisher Scientific).
	12. 20 % ethanol
	2.3
	1. Dry block heater at 42 °C.
	2. Incubation shaker with cooling option.
	3. 250 mL and 3 L baffled Erlenmeyer flasks.
	4. Competent bacteria : Escherichia coli BL21(DE3)

1. ITC200 Microcalorimeter (Malvern Panalytical). 2. Nanodrop 2000 Spectrophotometer (Thermo Scientific). 3. ITC buffer : 20 mM Tris pH 7.5 100 mM NaCl and 100 µM CaCl2. 4. α-methyl fucopyranoside (SIGMA 32198).

Acknowledgements

The authors acknowledge support by the ANR PIA Glyco@Alps (ANR-15-IDEX-02), Labex ARCANE and CBH-EUR-GS (ANR-17-EURE-0003), and the French Cystic fibrosis association Vaincre la Mucoviscidose.


Sakonwan Kuhaudomlarp

Emilie Gillon

Annabelle Varrot

Anne Imberty
email: anneimberty@cermav.cnrs.fr

LecA (PA-IL), a Galactose-Binding Lectin from Pseudomonas aeruginosa

Keywords: Lectins, Protein-Carbohydrate Interactions, X-ray crystallography, Surface Plasmon Resonance, Pseudomonas aeruginosa

LecA/PA-IL (Pfam PF07828) is a soluble galactose-binding lectin from bacterium Pseudomonas aeruginosa. The lectin is specific for α-galactose present on glycosphingolipids of the globoside family and has therefore been proposed to play a role in cell adhesion and in internalization of bacteria in epithelial cells. The lectin has also direct toxic activity. Search for high affinity inhibitors can be performed on the recombinant lectin, with use of surface plasmon resonance assays and structural studies.

Introduction

Pseudomonas aeruginosa is a Gram-negative bacterium and a leading pathogen responsible for acute as well as chronic infections of immune-compromised patients and patients suffering from cystic fibrosis [START_REF] Lyczak | Lung infection associated cystic fibrosis[END_REF]. Among other virulent factors, P. aeruginosa produces LecA, a calcium-dependent galactose-specific soluble lectin, also referred to as PA-IL [START_REF] Gilboa-Garber | Pseudomonas aeruginosa Lectins[END_REF], the production of which is regulated by quorum sensing [START_REF] Winzer | The Pseudomonas aeruginosa Lectins PA-IL and PA-IIL Are Controlled by Quorum Sensing and by RpoS[END_REF]. LecA was initially identified and characterized in the cytoplasm of P. aeruginosa but large quantities are present on the outer membrane of the bacterium, suggesting it may play a role in adhesion [START_REF] Glick | The intracellularl localization of Pseudomonas aeruginosa lectins[END_REF]. LecA is involved in biofilm development [START_REF] Diggle | The galactophilic lectin, LecA, contributes to biofilm development in Pseudomonas aeruginosa[END_REF]. It also has strong cytotoxic effect and has been demonstrated to induce permeability effect in epithelial cells, resulting in injury in lungs [START_REF] Chemani | Role of LecA and LecB lectins in Pseudomonas aeruginosa-induced lung injury and effect of carbohydrate ligands[END_REF] and intestines [START_REF] Laughlin | The key role of Pseudomonas aeruginosa PA-I lectin on experimental gut-derived sepsis[END_REF]. The lectin is specific for globosides, i.e. glycosphingolipids terminated by an α-galactose (α-Gal) residue. The interaction with cell membrane glycolipids result in signalling through protein phosphorylation [START_REF] Zheng | The Pseudomonas aeruginosa lectin LecA triggers host cell signalling by glycosphingolipid-dependent phosphorylation of the adaptor protein CrkII[END_REF] and bacteria intracellular invasion through membrane invagination [START_REF] Eierhoff | A lipid zipper triggers bacterial invasion[END_REF]. The lectin LecA (51 kDa) is composed of four subunits of 121 amino acids. Its binds to α-Gal monosaccharide and terminal α-Gal on oligosaccharides with moderate µM affinity [START_REF] Blanchard | Structural basis of the preferential binding for globo-series glycosphingolipids displayed by Pseudomonas aeruginosa lectin I[END_REF]. The presence of aromatic group on the sugar anomeric position, either in α or β configurations, yield to higher affinity, with KD of 5 µM for β-galactosides through CH-π interaction between His50 and the aromatic ring of the galactoside aglycone [START_REF] Kadam | CH-π "T-Shape" Interaction with Histidine Explains Binding of Aromatic Galactosides to Pseudomonas aeruginosa Lectin LecA[END_REF]. The first X-ray structure of LecA in complex with α-Gal and calcium was reported in 2003 (Fig. 1) [START_REF] Cioci | Structural basis of calcium and galactose recognition by the lectin PA-IL of Pseudomonas aeruginosa[END_REF], which has since provided a solid framework for the successive development of inhibitors against LecA and subsequently P. aeruginosa infection, in particular the development of aromatic glycoside inhibitors [START_REF] Kadam | CH-π "T-Shape" Interaction with Histidine Explains Binding of Aromatic Galactosides to Pseudomonas aeruginosa Lectin LecA[END_REF][START_REF] Rodrigue | Aromatic thioglycoside inhibitors against the virulence factor LecA from Pseudomonas aeruginosa[END_REF], multivalent galactoside inhibitors [START_REF] Kadam | A glycopeptide dendrimer inhibitor of the galactose-specific lectin LecA and of Pseudomonas aeruginosa biofilms[END_REF][START_REF] Visini | Structural insight into multivalent falactoside binding to Pseudomonas aeruginosa lectin LecA[END_REF][START_REF] Cecioni | Achieving High Affinity towards a Bacterial Lectin through Multivalent Topological Isomers of Calix[4]arene Glycoconjugates[END_REF][START_REF] Cecioni | Rational Design and Synthesis of Optimized Glycoclusters for Multivalent Lectin-Carbohydrate Interactions: Influence of the Linker Arm[END_REF][START_REF] Novoa | A LecA ligand identified from a galactoside-conjugate array inhibits host cell invasion by Pseudomonas aeruginosa[END_REF][START_REF] Bergmann | Multivalency effects on Pseudomonas aeruginosa biofilm inhibition and dispersal by glycopeptide dendrimers targeting lectin LecA[END_REF] and a covalent inhibitor targeting the carbohydrate binding site in LecA [START_REF] Wagner | Covalent Lectin Inhibition and Application in Bacterial Biofilm Imaging[END_REF]. Moreover, a structure of LecA in complex with melibiose (αGal1-6Glc) unravelled a secondary binding site that could be used as an additional target for inhibitor design [START_REF] Blanchard | Secondary sugar binding site identified for LecA lectin from Pseudomonas aeruginosa[END_REF].

Materials

2.1

Gene Sequence GenBank ID:NP_251260.1|PA-I galactophilic lectin [Pseudomonas aeruginosa PAO1].

2.2

Protein production in Escherichia coli 1. BL21 (DE3) competent cells. 2. Lysogeny broth (LB) (Invitrogen) and LB agar containing 100 µg/mL ampicillin 3. Isopropyl β-D-1-thiogalactopyranoside (IPTG).

2.3

Protein Purification 1. Pierce™ D-galactose agarose column (ThermoFisher). 2. NGC ™ Chromatography System (BIO-RAD) with ChromLab software. 3. One Shot Cell disruptor (Constant Systems Ltd, UK). 4. Buffer A (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 100 µM CaCl2). (see Note 1) 5. Buffer B (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 100 mM EDTA).

2.4

Surface Plasmon Resonance: 1. BIACORE™ X100 SPR system (GE Healthcare). 2. BIACORE sensor chip CM5 (GE Healthcare). 3. 1× PBS-P+ (10 mM phosphate buffer, 2.7 mM KCl, 0.137 M NaCl, 0.05% Tween-20, 0.1 mM CaCl2). 4. 10 mM acetate buffer pH 4.5. 5. 1× PBS buffer containing 100 µM CaCl2. 6. 4-nitrophenyl β-D-galactopyranoside (β-pNP-Gal, MW 301.25, Sigma-Aldrich). 7. Amine Coupling Kit (GE Healthcare) containing 1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride (EDC) (750 mg), N-hydroxysuccinimide (NHS) (115 mg), and ethanolamine-HCl pH 8.5 (1 M, 10.5 ml) in separate vials.

2.5

Protein Crystallisation 1. 24 or 48 wells VDX Plate with sealant 24 well (Hampton Research) or SuperClear™, pregreased 24 well plates (Jena Bioscience). 2. Siliconized glass circle cover slides (22 mm, Hampton research). 3. Crystallisation solution for LecA in complex with Gal (1.5 M ammonium sulfate, 100 mM sodium acetate pH 4.7, 5% 2-methyl-2,4-pentanediol (MPD) and 2% glycerol). (see Note 2) 4. D-galactose (Gal, MW 180.16, Sigma-Aldrich).

Methods

3.1

LecA Production

Transformation and Preculture

1. The gene encoding LecA was obtained from template DNA from Pseudomonas aeruginosa ATCC 15692. The gene was successfully ligated into an expression vector pET25b to create a recombinant plasmid called pET25b-PAIL, which is available upon request. 2. 1µL of the pET25b-PAIL plasmid was mixed with 100 µL of competent BL21(DE3) cells, followed by incubation on ice for 10 min.

3. The plasmid was transformed into the competent cells by heat shock at 42 ° for 45 s, then rest on ice for 3 min. 4. The transformed cells were inoculated in 900 µL liquid LB and incubated for 1 h at 37 °C with agitation at 180 rpm. 5. 100 µL of the liquid culture was spread on LB agar containing 100 µg/ml ampicillin and incubated at 37 °C overnight to select for cells containing the recombinant plasmid. 6. In order to prepare precultures, cell colonies containing the recombinant plasmid were inoculated in 10 mL liquid LB containing 100 µg/mL ampicillin and incubated at 37 °C overnight with agitation at 180 rpm.

Culture

1. 2.5 mL of the preculture was transferred into 1 litre of liquid LB containing 100 µg/mL ampicillin. 2. The 1-litre culture was incubated at 37°C with agitation at 180 rpm until OD600 of 0.6 was reached. 3. The expression of LecA was induced by adding 0.5 mM IPTG to the culture, then further incubated at 30 °C for 3 hours with agitation at 180 rpm. 4. The cells were harvested by centrifugation (6000 ×g, 4 °C, 10 min), then re-suspended in liquid LB and centrifuged again in 50 mL falcons at 6000 x g for 10 min. 5. The pellets were stored at -20 °C until required.

LecA Purification by Affinity Chromatography

1. The cell pellet from 1L of culture was re-suspended in 30 mL of Buffer A supplemented with 1 μl of benzonase and incubated at room temperature for 10 minutes. 2. The cells were lysed by a cell disruptor at 1.9 kbar. 3. The lysate was centrifuged (24,000 x g, 30 min, 4 °C). The supernatant was filtered with 0.45 µm syringe filter. 4. D-Gal agarose 5-mL column was pre-equilibrated with Buffer A (3 column volumes, 3 mL/min). 5. The filtered supernatant containing LecA was injected onto the pre-equilibrated column at low flow to promote interaction of LecA with Gal on the column. 6. The column was washed with Buffer A to remove unbound proteins (approximately 3 CVs or until Abs280 reached 0). 7. Bound LecA protein was eluted with 100% Buffer B and collected as 5-mL fractions. 8. The column was rinsed with milliQ water + 0.02% sodium azide and stored in 20% ethanol. 9. The collected protein fractions were combined and dialyzed in 5 L of milliQ water for 3 days at 4 °C while changing the water twice per day. The dialysed protein was lyophilized and stored at -20 °C until required. (see Note 3)

Surface Plasmon Resonance (SPR)

SPR is a label-free analytical technique for detecting intermolecular interactions between an analyte in solution and an immobilised ligand on the SPR sensor surface. The method is based on the change in the refractive index of the biosensor surface, which occurs when the analyte interacts with the immobilised ligand. SPR has a broad range of sensitivity detection (nM-mM) and requires relatively small quantity of samples per analysis. SPR has been used for analysis of various protein-carbohydrate interactions [START_REF] Duverger | Carbohydrate-lectin interactions assessed by surface plasmon resonance[END_REF]. SPR experiments enable thermodynamics (i.e. dissociation constant, KD) and/or kinetics parameters (kon and koff) to be determined. We have previously demonstrated the use of SPR for characterisation of LecA and their prospective glycomimetics compounds [START_REF] Cecioni | Achieving High Affinity towards a Bacterial Lectin through Multivalent Topological Isomers of Calix[4]arene Glycoconjugates[END_REF][START_REF] Cecioni | Rational Design and Synthesis of Optimized Glycoclusters for Multivalent Lectin-Carbohydrate Interactions: Influence of the Linker Arm[END_REF][START_REF] Cecioni | Selectivity among two lectins: Probing the effect of topology, multivalency and flexibility of "clicked" multivalent glycoclusters[END_REF]. Herein, we provided a methodology for preparation of immobilised LecA biosensor surface and an example of SPR analysis of an interaction between immobilised LecA and a glycomimetic compound. Set up a manual run on BIACORE X100 Control software and the temperature of the system at 25 °C. 1x PBS-P+ is used as a running buffer for chip activation, LecA immobilization and subsequent analyses. 2. Mix thoroughly an equal volume of EDC and NHS in an SPR tube (a minimum volume required for one injection is 130 μL). Ensure that there is no air bubble formed in the solution. (see Note 6) 3. Inject the EDC/NHS mixture at 10 µL/min with a contact time of 540 s to activate the docked CM5 chip (both on flow cell 1 and 2). Follow the change in the response on sensorgram during the injection. Repeat this step until the response relative to the preinjection baseline is 100-400 resonance units (RU). 4. Fix LecA on the activated surface on flow cell 2 only by injecting 100 µg/mL LecA in 10 mM acetate buffer pH 4.5 at 10 µL/min with a contact time of 540 s. Repeat this step until the response relative to the baseline reaches ~10,000 RU. 5. Inject 1 M ethanolamine-HCl pH 8.5 at 10 µL/min with a contact time of 540 s on flow cell 1 and 2 to deactivate remaining active groups and remove non-covalently bound LecA. Flow cell 1 is referred to as a reference flow cell (no LecA) and flow cell 2 as an active flow cell (containing LecA).

Sample Preparation

6. β-pNP-Gal was used as a positive control to monitor the activity of immobilized LecA on the CM5 chip prior to analysis of analytes of interest. KD of β-pNP-Gal was previously reported as 26.1 µM [START_REF] Rodrigue | Aromatic thioglycoside inhibitors against the virulence factor LecA from Pseudomonas aeruginosa[END_REF], which is sufficiently good for its use as a positive control. Figure 2 shows a typical sensorgram response after an injection of 0.1 mM β-pNP-Gal at 30 μL/min flow rate with a contact time of 30 s and dissociation time of 60 s. 7. For determination of kinetic or affinity parameters, multiple injections of a range of concentrations of an analyte of interest are required. Fig. 3A represents an example of multiple cycle kinetic experiment of compound 1 (60 s contact time and 60 s dissociation time, flow rate = 30 µL/min). The sensorgram for compound 1 has a square-like shape, indicating very fast association and dissociation of the compound from immobilised LecA, which hindered the determination of the kinetic parameters. However, the binding response and KD could be determined by plotting the binding responses at equilibrium against the corresponding analyte concentrations (Fig. 3B). (see Note 7)

Figure 3. A multicycle kinetic experiment for compound 1 for determination of KD. (A) Sensorgrams of multicycle injections of a range of concentrations of compound 1. (B)

The binding responses that were determined from the sensorgrams in (A) were plotted against the compound concentrations. KD is determined as the concentration at which half of the maximum response is reached

3.3

LecA Crystallisation

We demonstrated below a detailed methodology for co-crystallisation of LecA with Gal following our previously published protocol from Cioci et al. [START_REF] Cioci | Structural basis of calcium and galactose recognition by the lectin PA-IL of Pseudomonas aeruginosa[END_REF], which reported the first structure of LecA in complex with Gal (PDB code 1OKO) (Fig. 1). Gal drop. Ensure that the drop is hanging inside the well but not in contact with the crystallisation solution. Press gently on the cover slip to ensure good contact between the cover slip and the grease on top of the well. 5. Close the plate lid and store the plate at 19 °C. Monitor the formation of protein crystals every 2-3 days until the crystals are formed.

Sample preparation

Notes

1. LecA is a calcium-dependent lectin and it is advised to maintain a CaCl2 concentration of 100 µM throughout the purification and the SPR experiment. If needed, this concentration can be decreased to 10 µM.

2. LecA can also be crystallised using PEG as a precipitant. For example, the crystallisation conditions for LecA in complex with melibiose (αGal1-6Glc) is 15% PEG 5000 MME, 100 mM KSCN, 100 mM sodium acetate pH 4.6 [START_REF] Blanchard | Secondary sugar binding site identified for LecA lectin from Pseudomonas aeruginosa[END_REF]. The favourite crystallization pH is 4.6 but some crystals can also be obtained at basic pH with 100 mM Tris 8.5.

[18] 3. LecA is a very robust protein that can be lyophilised and stored at -20 °C for months or years. It is only recommended to aliquot in small quantities to avoid defreezing and refreezing. 4. Preparation of LecA in acetate buffer pH 4.5 is required to ensure that the protein is positively charged (theoretical pI of LecA is 5.04), allowing electrostatic interaction with the negatively charge on the chip surface, which provides efficient means for concentrating positively charged protein on the surface. However, LecA has limited stability in the acetate buffer and therefore the protein solution should be prepared as shortly as possible before injection. 5. For a perfect SPR measurement, the main recommendation is to avoid buffer mismatch by using the running buffer to prepare the analyte solution. It is strongly advised not to use organic buffering agent such as HEPES during chip activation and LecA fixation because the organic agent could react with the activated surface. 6. NHS/EDC mixture should be mixed shortly before injection. 7. For the evaluation of the binding response, the BIACORE system automatically performs reference subtraction, where the absolute response of the active flow cell (flow cell containing immobilised LecA) is subtracted by that recorded on the reference flow cell (the flow cell without LecA). During data analysis such as kinetic and affinity determination, double referencing is also performed, where reference subtraction, as well as the subtraction of blank injections (zero analyte concentrations) are performed. 8. LecA can also be prepared in 20 mM Tris-HCl or HEPES-NaOH pH 7.5 supplemented with 0.1 mM CaCl2 for ligands that to be in basic solution. 9. LecA ligand can also be added to the crystallization solutions instead of to the protein solution but it requires much more ligand and is not recommended for precious ligand.

Figure 1 .

 1 Figure 1. Crystal structure of LecA in complex with Gal. (A) The overall structure of LecA tetramer in complex with Gal.The protein backbone of a tetrameric LecA is shown in cartoon representation (yellow), Gal is shown as grey cylinders and calcium ion as green spheres. (B)The Gal binding site in a LecA monomeric subunit. Hydrogen bonds are shown in black dash lines and contacts with calcium ion in red dash lines. Calcium is represented as a light green sphere and Gal in grey cylinder.

.

Figure 2 .

 2 Figure 2. A typical sensorgram for β-pNP-Gal. The analyte was injected at the flow rate of 30 μl/min with a contact time of 30s and dissociation time of 60s. The report point for the binding response was automatically detected by BIACORE X100 control software when the equilibrium is reached (in this case, at 88s as indicated by a cross). The spikes indicate the buffer mismatches caused by the analyte.

  1. Prepare LecA solution from the lyophilized protein powder (approx. 2 mg) from section 3.1.3 in 500 μL of 1x PBS buffer containing 100 µM CaCl2. 2. Centrifuge the solution at 7000 ×g (5 min) to remove precipitated protein. 3. Determine the protein concentration in the supernatant in mg/mL using absorbance at 280 nm (absorbance coefficient of LecA is 2.191). The concentration should be at least 1 mg/mL. From this stock solution, prepare 500 µL of 100 µg/mL LecA in 10 mM acetate buffer pH 4.5. (see Note 4) 4. Prepare 100 µL of 0.1 mM solution of β-pNP-Gal in 1× PBS-P+. (see Note 5) 5. Add 10 mL filtered milliQ water into EDC and NHS vials from GE Healthcare to dissolve the compounds. Store them separately at -20 °C in Eppendorf tubes as 100 µL aliquots until required.

3.2.2 LecA Immobilisation on SPR Chip Using Amine Coupling Method

1. Pre-equilibrate BIACORE X100 SPR system with 1× PBS-P+ and dock a new CM5 chip.

  Add 1 µL of 50 mM galactose to 24 µL of LecA at 10 mg/mL and incubate for 1 hour at room temperature (see Note 9). 2. Aliquots 1 mL of the crystallisation solution into a 24 wells crystallisation plate. 3. Mix 1 µL of protein-ligand solution from 3.3.1 with 1 μL of the crystallization solution on a 22-mm cover slip to create a crystallisation drop. 4. Close the well containing crystallisation solution with the cover slip containing the LecA-

	1. Prepare LecA solution from the lyophilised protein powder (approx. 2 mg) from subsection
	3.1.3 in milliQ water supplemented with 0.1 mM CaCl2 (see Note 8).
	2. Centrifuge the solution at 7,000 x g (5 min) to remove precipitated protein.
	3. Determine the protein concentration in the supernatant in mg/mL using absorbance at 280
	nm (absorbance coefficient of LecA is 2.191). The final concentration should be around 10
	mg/mL.
	4. Prepare a 50 mM solution of Galactose in milliQ water.
	5. Prepare crystallization solution containing 1.5 M ammonium sulphate, 100 mM sodium
	acetate pH 4.7, 5% 2-methyl-2,4-pentanediol (MPD) and 2% glycerol (1 mL per well).
	3.3.2. Hanging Drop Vapour Diffusion
	1.

Acknowledgements

The authors acknowledge support by the ANR PIA Glyco@Alps (ANR-15-IDEX-02), Labex ARCANE and CBH-EUR-GS (ANR-17-EURE-0003), and the french cystic fibrosis association Vaincre la Mucoviscidose.


Rochelle Ackerley

Yrsa B Sverrisdttir

Frank Birklein

Mikael Elam

Håkan D Olausson

Heidrun H Krämer
email: heidrun.kraemer@neuro.med.uni-giessen.de

Yrsa B Sverrisdόttir

Cutaneous warmth, but not touch, increases muscle sympathetic nerve activity during a

Keywords: homeostasis, MSNA, touch, warmth, vibration, C-fiber

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

As we go about our daily activities, humans encounter external somatosensory stimuli that impact our body. In parallel, the efferent sympathetic system regulates various bodily processes to maintain homeostasis. It is hypothesised that C-fibre somatosensory afferents provide input to the insula about the state of the body and the sympathetic drive acts on this as required [START_REF] Craig | How do you feel? Interoception: the sense of the physiological condition of the body[END_REF][START_REF] Craig | How do you feel--now? The anterior insula and human awareness[END_REF]. In general, muscle sympathetic nerve activity (MSNA) is associated with the control of blood flow and skin sympathetic nervous activity (SSNA) controls body temperature via sweat gland activity [START_REF] Macefield | Sympathetic microneurography[END_REF]. It is well-known that MSNA to skeletal muscle increases during muscle contraction and is related to the onset and duration of muscle fatigue [START_REF] Seals | Regulation of muscle sympathetic nerve activity during exercise in humans[END_REF][START_REF] Seals | Influence of force on muscle and skin sympathetic nerve activity during sustained isometric contractions in humans[END_REF]. This has been studied under controlled conditions, where isometric muscle contraction (e.g. a hand-grip task) produces a clear increase in MSNA that can be recorded at a remote site (e.g. peroneal nerve of the leg), using multi-unit microneurography [START_REF] Mark | Microneurographic studies of the mechanisms of sympathetic nerve responses to static exercise in humans[END_REF][START_REF] Saito | Responses in muscle sympathetic nerve activity to sustained hand-grips of different tensions in humans[END_REF][START_REF] Seals | Influence of force on muscle and skin sympathetic nerve activity during sustained isometric contractions in humans[END_REF].

MSNA is associated with the regulation of homeostatic mechanisms via vasoconstriction, especially in the cardiovascular system. During isometric exercise, MSNA is primarily increased via the muscle metaboreflex; however, MSNA may be altered by other factors. For example, MSNA can be modulated by respiration [START_REF] Hagbarth | Pulse and Respiratory Grouping of Sympathetic Impulses in Human Muscle Nerves[END_REF][START_REF] Eckberg | Respiratory modulation of muscle sympathetic and vagal cardiac outflow in man[END_REF], changes in body position [START_REF] Delius | Manoeuvres Affecting Sympathetic Outflow in Human Skin Nerves[END_REF], and by mental stress [START_REF] Wallin | Comparison of sympathetic nerve activity in normotensive and hypertensive subjects[END_REF][START_REF] Anderson | Dissociation of sympathetic nerve activity in arm and leg muscle during mental stress[END_REF]. MSNA can also be altered by different stimuli applied to the body. The infusion of hypertonic saline causes long lasting muscle pain and induces consistent increases or decreases in MSNA, depending on the individual [START_REF] Kobuch | Inter-Individual Responses to Experimental Muscle Pain: Baseline Physiological Parameters Do Not Determine Whether Muscle Sympathetic Nerve Activity Increases or Decreases During Pain[END_REF], which may be due to a combination of incoming peripheral nociceptive signals and the way the individual reacts to this. MSNA has been shown to increase during painful heat and cold [START_REF] Kregel | Sympathetic nervous system activity during skin cooling in humans: relationship to stimulus intensity and pain sensation[END_REF][START_REF] Lautenschläger | The impact of baroreflex function on endogenous pain control: a microneurography study[END_REF]. The onset of skin cooling can decrease the general level of MSNA [START_REF] Kregel | Sympathetic nervous system activity during skin cooling in humans: relationship to stimulus intensity and pain sensation[END_REF]) and whole body warming has been shown to increase it [START_REF] Niimi | Effect of heat stress on muscle sympathetic nerve activity in humans[END_REF]. Conversely, recording MSNA at sites local to the skin stimulation have found the opposite effect, where innocuous cold can increase MSNA, [START_REF] Ishida | Suppression of activation of muscle sympathetic nerve during non-noxious local cooling after the end of local cooling in normal adults[END_REF], whereas skin warming can decrease it [START_REF] Takahashi | Local heat application to the leg reduces muscle sympathetic nerve activity in human[END_REF], which was thought to be part of a local regulatory reflex. The activation of Aβ lowthreshold mechanoreceptors via vibration can decrease MSNA [START_REF] Strzalkowski | Cutaneous Mechanoreceptor Feedback from the Hand and Foot Can Modulate Muscle Sympathetic Nerve Activity[END_REF], as can transcutaneous electrical skin stimulation [START_REF] Goswami | Forebrain organization representing baroreceptor gating of somatosensory afferents within the cortical autonomic network[END_REF].

The above studies have measured the MSNA response induced from applying somatosensory stimuli directly to the skin, whereas less is known about how MSNA is regulated under ongoing stimulation. It has been demonstrated that in the presence of isometric exercise (hand-grip), local skin heating produces augmented MSNA [START_REF] Ray | Augmentation of exercise-induced muscle sympathetic nerve activity during muscle heating[END_REF][START_REF] Kuipers | Interactive effect of aging and local muscle heating on renal vasoconstriction during isometric handgrip[END_REF]. In contrast, the response to somatosensory stimuli different from local heating has been little investigated. As C-fibres are the afferent part of a homeostatic reflex arc while the autonomic nervous system is the efferent part [START_REF] Craig | How do you feel? Interoception: the sense of the physiological condition of the body[END_REF][START_REF] Craig | How do you feel--now? The anterior insula and human awareness[END_REF], we predicted that activating the C-fibre system to different degrees would modify MSNA. The present study focused on the body's ability to react to and perceive applied somatosensory stimuli during ischemic muscle fatigue.

The present work utilizes the technique of microneurography to record MSNA, while a hand-grip task was used to generate muscle exhaustion and concurrent somatosensory stimuli were applied to the arm. We aimed to preferentially stimulate C-warm fibres (and to some extent C-mechano-heat responsive nociceptors; Ackerley and Watkins 2018) through the application of a warm heat pack, C-tactile (CT) fibres through gentle stroking [START_REF] Löken | Coding of pleasant touch by unmyelinated afferents in humans[END_REF][START_REF] Ackerley | Human C-tactile afferents are tuned to the temperature of a skin-stroking caress[END_REF], and mechanoreceptive Aβ fibres using a vibratory stimulus [START_REF] Strzalkowski | Cutaneous Mechanoreceptor Feedback from the Hand and Foot Can Modulate Muscle Sympathetic Nerve Activity[END_REF]. We hypothesised that the different somatosensory stimuli would modulate MSNA, physiological and behavioural measures differentially, where conditions with increased thin fibre afference would have stronger effects on the measures, due to their homeostatic relevance

Methods

The investigation was performed on healthy participants, in accordance with the World Medical Association Declaration of Helsinki, and following approval of the local ethics committee of the University of Gothenburg. Prior to the experiment participants were given standard information and written, informed consent was obtained. We used the in vivo technique of microneurography [START_REF] Vallbo | Microneurography: how the technique developed and its role in the investigation of the sympathetic nervous system[END_REF]) to gain axonal recordings of vasoconstrictor outflow from humans during somatosensory stimulation with concurrent muscular fatigue, and additional physiological and behavioural measures were gained.

Multi-unit, MNSA microneurographical recordings were made from the left leg in 35 participants (age range 20-40 years; 20 male). MSNA has local muscle and body-wide influence, but in general, MSNA is similar across the body [START_REF] Hansen | Muscle metaboreflex triggers parallel sympathetic activation in exercising and resting human skeletal muscle[END_REF][START_REF] Ray | Sympathetic nerve activity to nonactive muscle of the exercising and nonexercising limb[END_REF][START_REF] Boulton | A comparison of muscle sympathetic nerve activity to non-contracting muscle during isometric exercise in the upper and lower limbs[END_REF]. It is therefore possible to measure MSNA on a well-accessible site (e.g. the peroneal nerve of the leg), while the stimulation is carried out at another part of the body (e.g. the arm), as in the present study. Such a setup has been used in previous work [START_REF] Saito | Responses in muscle sympathetic nerve activity to sustained hand-grips of different tensions in humans[END_REF][START_REF] Seals | Regulation of muscle sympathetic nerve activity during exercise in humans[END_REF][START_REF] Seals | Influence of force on muscle and skin sympathetic nerve activity during sustained isometric contractions in humans[END_REF][START_REF] Ray | Sympathetic nerve activity to nonactive muscle of the exercising and nonexercising limb[END_REF]. A muscle fatigue hand-grip task was chosen as it is known to produce clear muscle exhaustion, but without exercise effects (e.g. increasing body temperature, sweating) and is effective in provoking MSNA. The hand-grip dynamometer consisted of two wooden blocks joined together (size: 10 x 5 cm) with a force monitor in the middle, which was capable of recording the time and pressure of the grip. Each participant first did a hand-grip muscle fatigue calibration. The calibration provided a standard level of muscle fatigue per participant, before commencing the experiment. Here, participants were instructed to apply their maximum hand grip for 60 s, during which a custom-written MATLAB (The Mathworks, Natick, MA) program determined the 30% maximum voluntary contraction. This 30% maximum voluntary contraction represented the participantspecific grip level that the participant was required to maintain during the muscle fatigue experimental task until muscular exhaustion. Throughout the muscle fatigue task, hand-grip force was presented on a monitor to the participant, as well as the experimenter, so that it could be corrected continuously.

The hand-grip task was used as a muscle fatigue stimulus during four experimental conditions. Each participant completed two out of the four conditions, in one experimental visit. Thus, they completed one condition using the muscle fatigue task on one arm, then after a break, they completed a second condition using the muscle fatigue task on the other arm. We chose not to conduct all four conditions in single participants, as microneurography combined with the fatigue task was already challenging, as the processes was mentally and physically tiring. Further, we did not want to perform the hand-grip muscle fatigue task on recently-fatigued muscle and it is not recommended to perform microneurography in the same nerve without a gap of ~3 months.

The four conditions during a muscle fatigue task were: (1) 'control', where the muscle fatigue task was completed alone with no concurrent intervention; (2) 'warmth', where a warming heat pack (size: 10 x 7 cm, average temperature 40 o C measured with a contact thermometer; Fluke; Germany) was placed on the arm doing the hand-grip; (3) 'brush', where slow brushing with a soft brush (backand-forth brushing traversing 10 cm skin in ~1 s, brush width: 7 cm) was carried out on the arm doing the hand-grip; (4) 'vibration', where 50 Hz tactile vibration (vibrometer area: 4 x 1 cm) was carried out on the arm doing the hand-grip. Each one of these concurrent stimuli was carried out in the middle of the dorsal forearm and the concurrent somatosensory stimuli were applied on the same arm as the hand-grip muscle fatigue task. Participants were assigned to two out of the four conditions in a pseudo-random order. The first hand used for the muscle fatigue hand-grip task was randomised for order between (right or left) participants. The different somatosensory stimuli started at the beginning of the hand-grip and were applied during the entire duration of the hand-grip, muscle fatigue task. Three types of responses were recorded in these experiments: (1) microneurography, multi-unit MSNA responses, (2) physiological haemodynamic responses, and (3) behavioural responses, as detailed below.

(1) The multi-unit recordings were made with low-impedance, insulated tungsten electrodes (200 µm shaft and 5 µm tip diameters; FHC, Bowdoin, ME) inserted into the peroneal nerve, posterior to the fibular head. An uninsulated reference electrode was inserted subcutaneously nearby. For further details of the microneurography and MSNA criteria, see [START_REF] Vallbo | Somatosensory, proprioceptive, and sympathetic activity in human peripheral nerves[END_REF][START_REF] Low | Clinical Autonomic Disorders. In: Microneurography and autonomic dysfunction[END_REF][START_REF] Vallbo | Microneurography: how the technique developed and its role in the investigation of the sympathetic nervous system[END_REF]). Once the electrode had entered a muscle nerve fascicle (as indicted by responses to manual muscle stimulation and no response to skin touch), small adjustments were made in the electrode location to maximize recordings of spontaneous MSNA. During the experiment, MSNA data were amplified (x 50,000), filtered (band pass 700-2000 Hz) and fed through a discriminator for audio monitoring. A mean voltage (integrated), smoothed signal was obtained by passing the nerve signal through a resistance-capacitance circuit (time constant 0.1 s). Burst identification was performed automatically and checked visually by the experimenter. Three MSNA measures were taken from this signal: the burst frequency (number of bursts per minute), the burst incidence (number of bursts occurring per hundred heartbeats) and the burst area (total neural activity as a product of the area under the burst over a minute, in arbitrary units). Burst incidence illustrates central control (the relative amount of cardiac cycles filled with MSNA bursts) whereas burst frequency reflects the degree of activity reaching the effector organs [START_REF] Sundlöf | The variability of muscle nerve sympathetic activity in resting recumbent man[END_REF].

(2) Heart rate (measured as beats per minute) was taken before and during the task, using electrocardiography via chest electrodes. Mean arterial blood pressure was also measured (middle finger cuff; Finapres 2300, Louisville, KY), however, this measure was only reliable in around half of the participants over the time-course of the muscle fatigue task and was therefore only used to ensure normal haemodynamic function from readings taken at the beginning of the task, and not analysed further.

(3) Two behavioural measures were recorded: the duration of the hand-grip (in seconds) of the muscle fatigue task, and a pain rating when the task ended. Participants rated their level of pain at the point of muscle exhaustion to the hand-grip on a visual analogue scale, with anchors from 'no pain' (equating to '0' in our data) on the left to 'most intense pain imaginable' (equating to '100') on the right.

MSNA and heart rate were recorded throughout the experiment and average baseline resting values for these were taken over a 5 minute period before the first muscle fatigue task for each participant. After the first task was completed, the participant rested for at least 10 minutes and second average baseline values were gained over a one minute period before starting the second muscle fatigue task on the other arm. The responses gained in a participant's specific task were compared to the baseline period occurring directly before that task. In the analysis, the average MSNA for burst frequency, incidence, and area, as well as the heart rate, from both the first and last minute of the muscle fatigue task were compared to the specific task average baseline readings per participant. Due to the typical variation seen in these physiological responses, these data were normalised to the averaged baseline values (which were set at 100%). The MSNA and heart rate data presented in the current study show the average responses from the included participants as an increase or decrease from the 100% baseline measures.

Data Analysis

To compare differences and correlations in the data, statistical tests were carried out using SPSS (version 23; IBM, Armonk, NY). The first analysis step was to check that the two pre-test baselines did not differ using paired t-tests. As a stable baseline was required over time, the criteria for exclusion was if a participant had two or more measures that varied by >30% in MSNA (out of the burst frequency, burst incidence, and burst area) or that the heart rate varied by >10%, between both baselines. The majority of participants did not differ significantly; however, seven participants were excluded as they displayed inconsistent baseline physiological (MSNA and/or heart rate) measurements. Since ischemic muscle pain was seen as a prerequisite to move from homeostasis to provoke an autonomic response, participants with insufficient pain rating (<5 on our numeric rating scale) were excluded as well. Two participants had insufficient pain ratings, but they were in the group of seven excluded participants, as they also showed inconsistent baseline physiological measurements. For the included participants, each of the MSNA, heart rate, and behavioural measures, per condition, were tested for normality using a Kolmogorov-Smirnov test and were found to be normally distributed. Statistical effects are presented at the p < 0.05 level and significance stated to three decimal places.

The second analysis step was to investigate whether MSNA and heart rate showed changes due to the muscle fatigue task. To assess changes over time, the baseline normalised MSNA and heart rate were compared to the same measure from (i) the first-minute of the task and (ii) the last-minute of the task, using one-sample t-tests. Then, the measures were entered into separate general linear model analyses of variance (ANOVAs) as dependent variables, with the condition type as the independent variable (with four levels corresponding to the different conditions: control, warm, brush, vibration). A full-factorial model was used to assess the main effect of condition on each of the measures and the sex of the participant was included as a covariate. We also provide the effect sizes as partial Eta squared and the observed power (computed using alpha = 0.05). When a significant main effect was found, pairwise comparisons between the different conditions were tested using the Tukey method, which corrects the p value for the multiple comparisons. A correlation analysis was also conducted on the endpoint responses from all the measures to determine which measures were positively or negatively correlated, using Pearson's test.

Results

The study was a cross-over design and 28 of the participants could be included in the full analysis. The numbers of participants in each condition were: control (no intervention): n = 16, warm: n = 11, brush: n = 17 and vibration: n = 12. Examples of MSNA, electrocardiography, and heart rate from different participants are displayed in Figure 1.

Overall, there was a significant increase in all the MSNA measures, compared to the resting baseline, for the hand grip task and concurrent warmth, brushing, vibration and during no concurrent stimulation (all p < 0.001; Figure 2). The MSNA recordings showed significant main effects for condition type in mean burst frequency (Figure 2A, Table 1), mean burst incidence (Figure 2B, Table 1), and The table shows the statistical details per test and the only significant effects found where for condition on all three MNSA variables and on pain intensity ratings (highlighted in bold). mean burst area (Figure 2C, Table 1). There were no effects of sex on any of the MSNA variables (Table 1). The main effect of condition relied mainly on the warm condition because this induced higher MSNA responses than the control and vibration condition for burst frequency (control: p = 0.013; vibration p = 0.003; Figure 2A) and burst incidence (control: p = 0.023; vibration p = 0.007; Figure 2B). Additionally, the warm condition induced a larger burst area than the brush condition (control: p = 0.003; vibration p = 0.001, warmth p = 0.004; Figure 2C). No differences were found between the vibration, brush and control conditions.

Heart rate increased during the last minute in all the conditions, as compared to the resting baseline (all p < 0.001; Fig. 3a). However, there were no between-condition effects for heart rate, or for sex (Table 1). The duration of the task did not differ between the conditions (Fig. 3c). Conversely, there was a significant main effect of the condition type on the end pain ratings (Fig. 3b, Table 1). In the warm condition, pain intensity was significantly higher than in the control and vibration conditions; additionally, the concurrent brushing condition showed increased pain ratings over the vibration condition (all p < 0.05; Fig. 3b). Again, there was no significant effect of sex on either the duration of hand grip or on the pain intensity rating.

A correlation analysis was conducted between all end-point measures consisting of MSNA, heart rate, and behavioural parameters. Table 2 shows the significant correlations between these measures in bold text. Many significant correlations were found: as expected, all of the MSNA measures were significantly positively correlated with each other, and the burst frequency and burst area correlated better with the other measures than the burst incidence. Heart rate correlated positively with all of the measures, except for the burst incidence. The pain ratings correlated significantly with all of the measures except for the task duration, and the task duration measure only correlated significantly with heart rate (see Table 2).

Table 2: Correlations between the end-point physiological and behavioural measures.

The table shows Pearson's R and significance values for each measure comparison. Significant correlations are highlighted in bold.

Discussion

Concurrent somatosensory stimulation during a muscle fatigue handgrip paradigm induced between-condition differences in MSNA, heart rate, and pain, with warmth inducing higher MSNA burst frequency, burst incidence, and burst area compared to the control and vibration condition, and higher burst area compared to the brush condition. MSNA parameters correlated with the experimentallyinduced pain intensity from muscle exhaustion, linking the central processing of the task with the MSNA regulatory output.

The concurrent sensory stimuli were chosen to preferentially activate different types of skin afferents, as we hypothesised that increased thin-fibre afferents would have a larger homeostatic effect. The application of warmth to the skin is an effective C-fibre stimulus and this increased MSNA the most. However, we cannot say whether this mechanism was due to (1) the direct influence of increasing muscle temperature, (2) a more general reflex arc from somatosensory input provoking increased sympathetic output, or (3) a combination of these two and/or other mechanisms. Exercise increases local muscle temperature, which is transmitted by thin muscle afferents [START_REF] Hertel | Responses of group IV and group III muscle afferents to thermal stimuli[END_REF][START_REF] Kumazawa | Thin-fibre receptors responding to mechanical, chemical, and thermal stimulation in the skeletal muscle of the dog[END_REF] and this increases MSNA, but additional warmth augments it even further [START_REF] Ray | Augmentation of exercise-induced muscle sympathetic nerve activity during muscle heating[END_REF][START_REF] Kuipers | Interactive effect of aging and local muscle heating on renal vasoconstriction during isometric handgrip[END_REF]). Nearby skin heating may sensitise the muscle mechanoreflex induced from the hand-grip and [START_REF] Kuipers | Interactive effect of aging and local muscle heating on renal vasoconstriction during isometric handgrip[END_REF] proposed that this mechanism caused increased MSNA during concurrent arm-warming. [START_REF] Ray | Augmentation of exercise-induced muscle sympathetic nerve activity during muscle heating[END_REF] postulated that the mechanism was through increased activity of mechanoreceptive muscle afferents that were also sensitive to temperature. However, these studies and our present work all find increased MSNA during warming with hand-grip and it cannot be discounted that skin afferents, especially C-warm and C-mechano-heat sensitive nociceptors (that can be activated through innocuous heat; Ackerley and Watkins 2018) will send innocuous afferent information about skin conditions. Therefore, if the participant feels warmth, the corresponding somatosensory signal will be present.

Although we cannot be sure about the underlying mechanism behind warmth eliciting additional MSNA during hand-grip, we provide new insights into the regulation of MSNA during muscle fatigue with other types of somatosensory stimuli. It is known that different somatosensory stimuli have specific afferent pathways, where touch and nociception are largely separate [START_REF] Mcglone | Discriminative and affective touch: sensing and feeling[END_REF][START_REF] Marshall | Spinal signalling of C-fiber mediated pleasant touch in humans[END_REF], although it is thought that the insula is a target for Cfibre afferents providing input about physiological body conditions [START_REF] Craig | How do you feel? Interoception: the sense of the physiological condition of the body[END_REF][START_REF] Craig | Interoception: the sense of the physiological condition of the body[END_REF]. Presently, we found no additional increases or decreases of MSNA with tactile stimulation during hand-grip. A previous study showed decreased MSNA with vibration alone [START_REF] Strzalkowski | Cutaneous Mechanoreceptor Feedback from the Hand and Foot Can Modulate Muscle Sympathetic Nerve Activity[END_REF]), but it seems that in our experiment, the overriding effect was an increase in MSNA due to the hand-grip. However, MSNA was generally lower during vibration and was significantly lower than MSNA with concurrent warmth. Vibration, which effectively activates Aβ mechanoreceptive afferents, reduced pain in the hand-grip task, as compared to warmth or brushing, where decreased pain may lead to reduced MSNA. Transcutaneous electrical nerve stimulation activates large diameter cutaneous nerve fibres pain and can decrease pain, by modulating the central transmission of pain [START_REF] Melzack | Pain mechanisms: a new theory[END_REF], especially for inputs converging on the same spinal level [START_REF] Hollman | Effect of transcutaneous electrical nerve stimulation on the presser response to static handgrip exercise[END_REF]. Such electrical skin stimulation can also decrease MSNA [START_REF] Goswami | Forebrain organization representing baroreceptor gating of somatosensory afferents within the cortical autonomic network[END_REF]) and thus may be more effective than vibration, but more further work would need to be carried out to evaluate this.

We believed that brushing touch, which stimulates both C-fibre (CT afferents) and Aβ mechanoreceptive afferents (although is an effective CT stimulus, [START_REF] Löken | Coding of pleasant touch by unmyelinated afferents in humans[END_REF][START_REF] Ackerley | Human C-tactile afferents are tuned to the temperature of a skin-stroking caress[END_REF], could have either increased MSNA (due to C-fibre activation and its involvement in homeostasis) or decreased it (due to such stroking being pleasant and relaxing). However, we found no significant effect during hand-grip and that may have been due to competing effects.

In contrast to a previous investigation on heat pain [START_REF] Liljencrantz | Slow brushing reduces heat pain in humans[END_REF], the addition of CT-optimal stimulation by brushing was not sufficient to reduce pain from muscle fatigue in our hand-grip task. However, we found that MSNA burst area was significantly lower during brushing than warmth. Recent studies indicate that pathophysiological mechanisms mediating the occurrence and strength (burst area) of sympathetic bursts are different [START_REF] Kienbaum | Two sites for modulation of human sympathetic activity by arterial baroreceptors?[END_REF]). It has also been shown that whole body heating leads to an increase of MSNA activity, but leaves the burst area unchanged [START_REF] Keller | Heat stress enhances arterial baroreflex control of muscle sympathetic nerve activity via increased sensitivity of burst gating, not burst area, in humans[END_REF]. Therefore, the decrease in MSNA burst area during brushing may be part of a wider homeostatic mechanism.

We conclude that warmth is an additional relevant homeostatic input because thermal signals are important during exercise, and need to be counteracted. The tactile stimuli did not decrease MSNA, as compared to the control, possibly because it did not provide a strong enough or relevant homeostatic drive that needed counteracting. Further studies could investigate whether SSNA changes under such conditions, as well as testing other somatosensory stimuli (e.g. cooling) and measuring local and non-local MSNA with and without isometric exercise, to see whether sympathetic efference can be modulated, which could help in situations such as physiotherapy and rehabilitation.

Figure 1 :

 1 Figure 1: Examples of integrated MSNA, ECG, and heart rate

Figure 2 :

 2 Figure 2: End-point MSNA responses to evoked muscle pain over the conditions Microneurographic recordings of muscle sympathetic activity showing the normalised mean sympathetic (A) burst frequency, (B) burst incidence and (C) burst area. The responses were taken in the last minute of the hand-grip task for each participant and normalised to each participant's pre-task resting baselines (100%). There was a significant increase in all of the MSNA measures over the conditions, as compared to the resting baseline (all p < 0.001). The significances shown indicate the between-condition effects from the separate measures (* p < 0.05, ** p < 0.01). Error bars are +SEM.

Figure 3 :

 3 Figure3: Heart rate, duration of hand-grip and end-point pain ratings over the conditions (A) Normalised mean heart rate was taken in the last minute of the hand-grip task for each participant and normalised to each participant's pre-task resting baselines (100%). There was a significant increase in heart rate over all of the conditions, as compared to the resting baseline (all p < 0.001), but no between-condition effects. (B) Mean hand-grip duration showed no significant effect of condition on the task duration (participants were instructed to continue to grip until exhaustion). (C) Mean pain ratings on a visual analogue scale ranging from 0 to 100 obtained at the end of each condition. There was a significant effect of condition on the pain ratings and betweencondition significances are shown (* p < 0.05, ** p < 0.01). Error bars are +SEM.

Table 1 : Statistical effects for condition and sex on each measure.

 1

Acknowledgements:

We thank Tomas Karlsson and Karin Saar for their excellent technical assistance.