Methods for detecting contaminants in solutions containing glucose polymers

The invention relates to a method for detecting contaminants of glucose polymers, said contaminants being capable of acting in synergy with one another so as to trigger an inflammatory reaction, characterized in that it comprises an in vitro inflammatory response test using modified cell lines.

Methods for detecting contaminants in solutions containing glucose polymers

The invention relates to a method for detecting contaminants in glucose polymers, said contaminants being capable of triggering, separately or in combination, an inflammatory reaction, characterized in that it includes at least one in vitro inflammatory response test using a cell line enabling at least one inflammatory response factor to be detected, the cell line being either a macrophage or a macrophage-differentiated cell line, or a cell expressing one or more Toll-like receptors (TLRs) or NOD-like receptors such as TLR2, TLR4 or NOD2 and enabling the responses of the receptor(s) to be detected, or a combination thereof.

Biological assay of peptidoglycans

The present invention relates to a biological method for assaying peptidoglycans (PGN) in a sample, particularly a sample of glucose polymers. The PGN assay includes: a) treating the glucose polymer sample by sonication, heating, and/or alkalizing; b) placing the treated sample or a dilution thereof in contact with a recombinant cell expressing an exogenous TLR2 (toll-like receptor 2) and a reporter gene directly dependent on the signaling pathway associated with the TLR2. The reporter gene codes for a colored or fluorescent protein or for a protein the activity of which is measurable with or without a substrate; c) measuring the reporter gene signal; and d) determining the amount of PGN in the sample using a standard curve of the correlation between the amount or PGN and the strength of the reporter gene signal.

METHODS FOR DETECTING CONTAMINANTS IN SOLUTIONS CONTAINING GLUCOSE POLYMERS

The invention relates to a method for detecting contaminants of glucose polymers, said contaminants being capable of acting in synergy with one another so as to trigger an inflammatory reaction, characterized in that it comprises an in vitro inflammatory response test using modified cell lines. 130-. (canceled)31. A method for detecting pro-inflammatory contaminants of glucose polymers comprising contacting a cell line with a composition comprising glucose polymers , the cell line being either a macrophage cell line , a macrophage-differentiated cell line or a cell line expressing one or more Toll-Like Receptors (TLRs) or NOD-like receptors and performing an inflammatory response test to detect an inflammatory response produced by said cell line.32. The method as claimed in claim 31 , wherein the glucose polymers are glucose polymers for peritoneal dialysis claim 31 , for enteral feeding claim 31 , for parenteral feed or the feeding of newborn babies.33. The method as claimed in claim 31 , wherein the cell line is a macrophage cell line or a macrophage-differentiated cell line.34. The method as claimed in claim 33 , characterized in that the inflammatory response test comprises contacting the cells of the cell line with a preparation of glucose polymers that may contain pro-inflammatory contaminants and in measuring production of cytokines of the acute phase of inflammation claim 33 , the production of said cytokines indicating that the preparation contains contaminants capable of triggering an inflammatory reaction.35. The method as claimed in claim 34 , wherein the cytokines of the acute phase of inflammation are selected from the group consisting of TNF-α claim 34 , IL-1β and chemokines.36. The method as claimed in claim 35 , wherein the chemokine is RANTES.37. The method as claimed in claim 34 , wherein a molecule chosen from muramyl dipeptide (MDP) claim 34 , L18-MDP claim 34 , glycolyl-MDP claim 34 , formyl-Met-Leu-Phe or lipopolysaccharide is added to the ...

Methods for detecting contaminants in solutions containing glucose polymers

The invention relates to a method for detecting contaminants of glucose polymers, said contaminants being capable of acting in synergy with one another so as to trigger an inflammatory reaction, characterized in that it comprises an in vitro inflammatory response test using modified cell lines.

BIOLOGICAL ASSAY OF PEPTIDOGLYCANS

The present invention relates to a biological method for assaying peptidoglycans (PGN) in a sample, particularly a sample of glucose polymers. The PGN assay includes: a) treating the glucose polymer sample by sonication, heating, and/or alkalizing; b) placing the treated sample or a dilution thereof in contact with a recombinant cell expressing an exogenous TLR2 (toll-like receptor 2) and a reporter gene directly dependent on the signaling pathway associated with the TLR2. The reporter gene codes for a colored or fluorescent protein or for a protein the activity of which is measurable with or without a substrate; c) measuring the reporter gene signal; and d) determining the amount of PGN in the sample using a standard curve of the correlation between the amount or PGN and the strength of the reporter gene signal. 114-. (canceled)15. A method of assaying peptidoglycans (PGNs) in a sample of glucose polymer , comprising:a) treating the sample of glucose polymer by sonication, heating, and/or alkalization;b) bringing the treated sample or a dilution thereof into contact with a recombinant cell expressing an exogenous TLR2 receptor (Toll-like Receptor 2) and a reporter gene under the direct dependence of the signaling pathway associated with the TLR2 receptor, said reporter gene coding for a colored or fluorescent protein or for a protein whose activity can be measured with or without a substrate;c) measuring the reporter gene signal; andd) determining the amount of PGN in the sample using a calibration curve of the correspondence between the amount of PUN and the intensity of the reporter gene signal.16. The method of claim 15 , wherein the sample is treated by sonication claim 15 , heating claim 15 , and/or alkalization to fragment and disintegrate the PGNs contained in the sample that activate the TLR2 receptor.17. The method of claim 16 , wherein said treatment generates PGNs predominantly having a size of about 120 kDa.18. The method of claim 15 , wherein said ...

Optimized method for decontaminating production of glucose polymers and glucose polymer hydrolyzates

The present invention relates to a method for decontaminating glucose polymers or the hydrolysates of the pro-inflammatory molecules thereof. Said method includes a) providing glucose polymers or the hydrolysates thereof, b) optionally, detecting or assaying the pro-inflammatory molecules in the glucose polymers or the hydrolysates thereof provided in Step a), and c) carrying out the following purifying steps: i. treatment using an enzymatic preparation having detergent properties and clarification properties; ii. treatment using a pharmaceutical-grade activated carbon with very high adsorption properties and “micropore” porosity; iii. optionally, treatment using a second activated carbon with “mesopore” porosity; iv. passing them over a macroporous adsorbent polymer resin having porosity greater than 100 Angstroms; and v. continuous ultrafiltration at 5 kDa.

BIOLOGICAL DOSAGE OF PEPTIDOGLYCANS

The present invention relates to a biological method for the dosage of peptidoglycans in a sample, especially a sample of glucose polymers. 111-. (canceled)12. A method of assaying peptidoglycans (PGNs) in a sample of glucose polymer , comprising:a) enzymatic treatment of the sample of glucose polymer by a lysozyme;b) bringing the treated sample or a dilution thereof into contact with a recombinant cell expressing an exogenous TLR2 receptor (Toll-like Receptor 2) and a reporter gene under the direct dependence of the signaling pathway associated with the TLR2 receptor, the reporter gene coding for a colored or fluorescent protein or for a protein whose activity can be measured;c) measuring the reporter gene signal; andd) determining the amount of PGN in the sample using a calibration curve of the correspondence between the amount of PGN and the intensity of the reporter gene signal.13. The method as claimed in claim 12 , wherein the enzymatic treatment of the sample fragments and disaggregates the PGNs contained in the sample so as to make them capable of activating the TLR2 receptor.14. The method as claimed in claim 12 , wherein the enzymatic treatment of the sample generates PGNs predominantly having a size of approximately 120 kDa.15. The method as claimed in claim 12 , wherein the treatment of the sample comprises incubation of lysozyme at a concentration of approximately 250 to 2500 U/ml in the sample at a glucose polymer concentration of 37.5% (weight/volume) for 30 minutes to 16 h at a temperature of 37° C.16. The method as claimed in claim 12 , wherein the treatment of the sample comprises incubation of lysozyme at a concentration of approximately 250 U/ml in the sample at a glucose polymer concentration of 37.5% (weight/volume) for 2 h at a temperature of 37° C.17. The method as claimed in claim 12 , wherein the reporter gene is a secreted alkaline phosphatase.18. The method as claimed in claim 12 , wherein the cell is a cell of the HEK-Blue™ hTLR2 line.19 ...

Methods for decontaminating circuits for producing glucose polymers and hydrolysates of glucose polymers

The present invention concerns a method for determining the impact of a production step or a purification step on the presence or nature of pro-inflammatory contaminating molecules in glucose polymers or the hydrolysates of same by using an in vitro test of inflammatory response using cell lines. It further concerns an optimised method of producing or purifying glucose polymers or the hydrolysates of same comprising an analysis of the pro-inflammatory contaminating molecules in glucose polymers or the hydrolysates of same and the selection of production or purification steps optimised with respect to the presence and nature of the pro-inflammatory contaminating molecules.

OPTIMISED METHOD FOR DECONTAMINATING THE STARCH USED AS A RAW MATERIAL FOR OBTAINING GLUCOSE POLYMERS INTENDED FOR PERITONEAL DIALYSIS

The present invention concerns a method for decontaminating the starches used as a raw material for the preparation of glucose polymers intended for peritoneal dialysis, the method comprising the following steps: —preparing a Waxy corn starch, —placing the Waxy starch in suspension at a concentration of between 20 and 40% dry matter in a process water at a pH of between approximately 5 and approximately 6, in particular approximately 5.5, —treating the starch suspension with a peracetic acid solution at a concentration equal to or between 100 and 500 ppm, preferably 300 ppm, —dewatering the starch, then dissolving in demineralised water adjusted to a pH of between approximately 5 and approximately 6, in particular approximately 5.5 and at a concentration of between 20 and 40% dry matter, —increasing the temperature to 107° C., then adding an alpha-amylase for 15 minutes, —optionally, treating with an enzymatic preparation having detergent and clarification properties, —filtering the suspension on a bed of diatoms, —treating with an active carbon having a very high adsorption capacity, of pharmaceutical quality, and of “microporous” Porosity, —treating with a second active carbon of “mesoporous” porosity, —optionally, passing over a macroporous adsorbent polymer resin, having a porosity greater than 100 angstroms, —optionally, continuous 5000 Da ultrafiltration, —safety filtration through a sterile filter having a porosity of 0.22 μm. 1. A method for decontaminating starches used as raw material for the preparation of glucose polymers intended for peritoneal dialysis , the method comprising:preparing a waxy starch,suspending the waxy starch at a concentration of between 20 and 40% dry matter in a process water at a pH of between approximately 5 and approximately 6,treating the suspension of starch with a solution of peracetic acid at a concentration of between 100 and 500 ppm,removing excess water from the acid treated starch suspension to form a concentrated ...

BIOLOGICAL ASSAY OF PEPTIDOGLYCANS

The present invention relates to a biological method for assaying peptidoglycans (PGN) in a sample, particularly a sample of glucose polymers. The PGN assay includes: a) treating the glucose polymer sample by sonication, heating, and/or alkalizing; b) placing the treated sample or a dilution thereof in contact with a recombinant cell expressing an exogenous TLR2 (toll-like receptor 2) and a reporter gene directly dependent on the signaling pathway associated with the TLR2. The reporter gene codes for a colored or fluorescent protein or for a protein the activity of which is measurable with or without a substrate; c) measuring the reporter gene signal; and d) determining the amount of PUN in the sample using a standard curve of the correlation between the amount of PGN and the strength of the reporter gene signal. 114-. (canceled)15. A kit for assaying peptidoglycans (PGNs) in a sample of glucose polymers comprising:a recombinant cell expressing an exogenous TLR2 receptor (Toll-like Receptor 2) and a reporter gene under the direct dependence of the signaling pathway associated with the TLR2 receptor, said reporter gene coding for a colored or fluorescent protein or for a protein whose activity can be measured with or without a substrate;either a calibration curve of the correspondence between the amount of PGN and the intensity of the reporter gene signal, or a PGN standard; andoptionally, instructions for use, and a solution for pretreating the sample.16Staphylococcus aureus, Micrococcus luteus, Bacillus subtilisAlicyclobacillus acidocaldarius.. The kit of wherein the PGN standard is derived from a bacterium selected from and17. The kit of claim 15 , further comprising an internal standard that is an agonist of TLR2.17. The kit of claim 16 , wherein the agonist of TLR2 is a lipopeptide.18. The kit of claim 17 , wherein said lipopetide is PAM3Cys-Ser-(Lys)4 trihydrochloride.19. The kit of claim 15 , wherein the recombinant cell is a stably transformed HEK-293 cell ...

OPTIMIZED METHOD FOR DECONTAMINATING PRODUCTION OF GLUCOSE POLYMERS AND GLUCOSE POLYMER HYDROLYZATES

The present invention relates to a method for decontaminating glucose polymers or the hydrolysates of the pro-inflammatory molecules thereof. Said method includes a) providing glucose polymers or the hydrolysates thereof, b) optionally, detecting or assaying the pro-inflammatory molecules in the glucose polymers or the hydrolysates thereof provided in Step a), and c) carrying out the following purifying steps: i. treatment using an enzymatic preparation having detergent properties and clarification properties; ii. treatment using a pharmaceutical-grade activated carbon with very high adsorption properties and “micropore” porosity; iii. optionally, treatment using a second activated carbon with “mesopore” porosity; iv. passing them over a macroporous adsorbent polymer resin having porosity greater than 100 Angstroms; and v. continuous ultrafiltration at 5 kDa. 17-. (canceled)8. A method for decontaminating glucose polymers or the hydrolyzates thereof of the pro-inflammatory molecules thereof , comprising the following steps:a) providing glucose polymers or hydrolyzates thereof;b) optionally, detecting or assaying the pro-inflammatory molecules in the glucose polymers or hydrolyzates thereof provided in step a); and i) treatment by an enzymatic preparation with detergent and clarifying properties,', 'ii) treatment by a pharmaceutical-grade activated carbon with very high adsorption capacity and microporous porosity,', 'iii) treatment by a second activated carbon with mesoporous porosity,', 'iv) passing over a macroporous adsorbent polymer resin having a pore size greater than 100 angstrom, and', 'v) continuous 5 kDa ultrafiltration., 'c) carrying out the following purification steps in the following order9. The method as claimed in claim 8 , wherein the enzymatic preparation with detergent and clarifying properties is an enzymatic preparation with mannanase activity.10. The method as claimed in claim 8 , wherein the pharmaceutical-grade activated carbon with very high ...

METHODS FOR DECONTAMINATING CIRCUITS FOR PRODUCING GLUCOSE POLYMERS AND HYDROLYSATES OF GLUCOSE POLYMERS

The present invention concerns a method for determining the impact of a production step or a purification step on the presence or nature of pro-inflammatory contaminating molecules in glucose polymers or the hydrolysates of same by using an in vitro test of inflammatory response using cell lines. It further concerns an optimised method of producing or purifying glucose polymers or the hydrolysates of same comprising an analysis of the pro-inflammatory contaminating molecules in glucose polymers or the hydrolysates of same and the selection of production or purification steps optimised with respect to the presence and nature of the pro-inflammatory contaminating molecules. 113-. (canceled)14. A method for testing the effectiveness of a purification step or purification steps on the presence of pro-inflammatory molecules in a final preparation of glucose polymers or hydrolysates thereof , the method comprising:a) providing an initial preparation of glucose polymers or hydrolysates thereof, the initial preparation containing pro-inflammatory molecules;b) detecting or assaying the amount of pro-inflammatory molecules in the initial preparation of glucose polymers or hydrolysates thereof provided in step a);c) carrying out the purification step or purification steps on the initial preparation of glucose polymers or hydrolysates thereof provided in step a) to produce the final preparation of glucose polymers or hydrolysates thereof;d) detecting or assaying the amount of pro-inflammatory molecules in the final preparation of glucose polymers or hydrolysates thereof;e) comparing the amount of pro-inflammatory molecules in the initial preparation of glucose polymers or hydrolysates thereof detected or assayed in step b) with the amount of pro-inflammatory molecules in the final preparation of glucose polymers or hydrolysates thereof detected or assayed in step d); and effective if the amount of pro-inflammatory molecules in the final preparation of glucose polymers or ...

Methods for decontaminating circuits for producing glucose polymers and hydrolysates of glucose polymers

La présente invention concerne un procédé permettant de tester l'efficacité d'étape(s) de purification sur la présence ou la nature des molécules pro-inflammatoires dans des polymères de glucose ou leurs hydrolysats en utilisant un test in vitro de réponse inflammatoire à l'aide de lignées cellulaires, la lignée cellulaire étant soit un macrophage ou une lignée cellulaire différenciée en macrophage, soit une cellule exprimant un ou plusieurs récepteurs TLR (Toll Like Receptor) ou NOD (Nucleotide-binding Oligomerization Domain-containing proteins) et permettant de détecter les réponses du ou des récepteurs, soit une combinaison de celles-ci. Elle concerne en outre une méthode de purification optimisée de polymères de glucose ou leurs hydrolysats comprenant une analyse des molécules pro-inflammatoires dans des polymères de glucose ou leurs hydrolysats en utilisant ledit test in vitro de réponse inflammatoire. Elle concerne aussi la sélection d'étapes de purification optimisée au regard de la présence et nature des molécules pro-inflammatoires.

method for detecting proinflammatory contaminants of glucose polymers

Methods for detecting contaminants of solutions containing glucose polymers

Methods for detecting contaminants in solutions containing glucose polymers

L'invention a pour objet un procédé de détection des contaminants des polymères de glucose, contaminants susceptibles d'agir en synergie les uns avec les autres pour déclencher une réaction inflammatoire, caractérisé en ce qu'il comprend un test de réponse inflammatoireà l'aide de lignées cellulaires modifiées.

Methods for detecting contaminants in solutions containing glucose polymers

ABRÉGÉ L'invention vise à prévenir les réactions inflammatoires causés par des contaminants susceptibles d'être amenés par les polymères de glucose lors de la dyalise péritonéale, en proposant un procédé de détection des contaminants pro-inflammatoires de polymères de glucose, lesdits contaminants étant susceptibles de déclencher, séparément ou en combinaison, une réaction inflammatoire, comprenant la mise en présence in vitro d'une préparation de polymère de glucose susceptible de contenir des contaminants pro-inflammatoires avec une ou plusieurs lignées cellulaires exprimant un ou plusieurs récepteurs TLR ou NOD ainsi qu'un gène rapporteur sous la dépendance directe de la voie de signalisation associée au(x) récepteurs(s) TLR et NOD et permettant de détecter les réponses du ou des récepteurs, et la mesure du signal du gène rapporteur, la détection de ce signal indiquant que la préparation contient des contaminants susceptibles d'activer un ou plusieurs récepteurs TLR ou NOD et de déclencher une réaction inflammatoire. CA 2831003 2018-07-17

Detecting proinflammatory contaminants in glucose polymers, comprises testing in vitro inflammatory response using cell line enabling inflammatory response factor to be detected, where cell line is macrophage-differentiated cell line

L'invention a pour objet un procédé de détection des contaminants des polymères de glucose, contaminants susceptibles d'agir en synergie les uns avec les autres pour déclencher une réaction inflammatoire, caractérisé en ce qu'il comprend un test de réponse inflammatoire in vitro à l'aide de lignées cellulaires modifiées.

Optimized method for decontaminating production of glucose polymers and glucose polymer hydrolysates.

La presente invención se refiere a un método para descontaminar polímeros de glucosa o los hidrolisatos de sus moléculas proinflamatorias. Dicho método incluye a) proporcionar polímeros de glucosa o sus hidrolisatos, b) opcionalmente, detectar o someter a ensayo las moléculas proinflamatorias en los polímeros de glucosa o sus hidrolisatos proporcionados en la Etapa a), y c) llevar a cabo las siguientes etapas de purificación: i. tratamiento usando una preparación enzimática que tiene propiedades detergentes y de purificación; ii. tratamiento usando un carbono activado de calidad farmacéutica con propiedades de adsorción muy elevadas y porosidad de "microporo"; iii. opcionalmente, tratamiento usando un segundo carbono activado, con porosidad de "mesoporo"; iv. hacer pasar los mismos sobre una resina polimérica con adsorbente macroporoso que tiene una porosidad mayor de 100 angstrom; y v. ultrafiltración continua a 5 kDa.

Methods for detecting contaminants in solutions containing glucose polymers

The invention relates to a method for detecting contaminants in glucose polymers, said contaminants being capable of triggering, separately or in combination, an inflammatory reaction, characterized in that it includes at least one in vitro inflammatory response test using a cell line enabling at least one inflammatory response factor to be detected, the cell line being either a macrophage or a macrophage-differentiated cell line, or a cell expressing one or more Toll-like receptors (TLRs) or NOD-like receptors such as TLR2, TLR4 or NOD2 and enabling the responses of the receptor(s) to be detected, or a combination thereof.

Peptidoglycan assay

The present invention relates to a biological method for assaying peptidoglycans (PGN) in a sample, particularly a sample of glucose polymers. The PGN assay includes: a) treating the glucose polymer sample by sonication, heating, and/or alkalizing; b) placing the treated sample or a dilution thereof in contact with a recombinant cell expressing an exogenous TLR2 (toll-like receptor 2) and a reporter gene directly dependent on the signaling pathway associated with the TLR2. The reporter gene codes for a colored or fluorescent protein or for a protein the activity of which is measurable with or without a substrate; c) measuring the reporter gene signal; and d) determining the amount of PGN in the sample using a standard curve of the correlation between the amount of PGN and the strength of the reporter gene signal.

Methods for decontaminating circuits for producing glucose polymers and hydrolysates of glucose polymers

The present invention concerns a method for determining the impact of a production step or a purification step on the presence or nature of pro-inflammatory contaminating molecules in glucose polymers or the hydrolysates of same by using an in vitro test of inflammatory response using cell lines. It further concerns an optimised method of producing or purifying glucose polymers or the hydrolysates of same comprising an analysis of the pro-inflammatory contaminating molecules in glucose polymers or the hydrolysates of same and the selection of production or purification steps optimised with respect to the presence and nature of the pro-inflammatory contaminating molecules.

Biological assay of peptidoglycans

The present invention relates to a biological method for assaying peptidoglycans (PGN) in a sample, particularly a sample of glucose polymers. The PGN assay includes: a) treating the glucose polymer sample by sonication, heating, and/or alkalizing; b) placing the treated sample or a dilution thereof in contact with a recombinant cell expressing an exogenous TLR2 (toll-like receptor 2) and a reporter gene directly dependent on the signaling pathway associated with the TLR2. The reporter gene codes for a colored or fluorescent protein or for a protein the activity of which is measurable with or without a substrate; c) measuring the reporter gene signal; and d) determining the amount of PGN in the sample using a standard curve of the correlation between the amount of PGN and the strength of the reporter gene signal.

Optimized method for decontaminating production of glucose polymers and glucose polymer hydrolysates

La présente invention est relative à un procédé de décontamination des polymères de glucose ou leurs hydrolysats de leurs molécules pro-inflammatoires, comprenant : a) fournir des polymères de glucose ou leurs hydrolysats; b) facultativement, détecter ou doser les molécules pro-inflammatoires dans les polymères de glucose ou leurs hydrolysats fournis à l'étape a); c) effectuer les étapes de purification suivantes : i. traitement par une préparation enzymatique à propriétés détergentes et de clarification, ii. traitement par un charbon actif de très haute capacité d'adsorption, de qualité pharmaceutique, de porosité « micropore », iii. facultativement, traitement par un second charbon actif de porosité « mésopore », iv. passage sur une résine adsorbante polymérique macroporeuse, présentant une porosité supérieure à 100 Angström, v. ultrafiltration en continu sur 5 kDa.

Biological dosage of peptidoglycans

The present invention relates to a biological method for the dosage of peptidoglycans in a sample, especially a sample of glucose polymers.

Optimised method for decontaminating the starch used as a raw material for obtaining glucose polymers intended for peritoneal dialysis

The present invention concerns a method for decontaminating the starches used as a raw material for the preparation of glucose polymers intended for peritoneal dialysis, the method comprising the following steps: - preparing a Waxy corn starch, - placing the Waxy starch in suspension at a concentration of between 20 and 40% dry matter in a process water at a pH of between approximately 5 and approximately 6, in particular approximately 5.5, - treating the starch suspension with a peracetic acid solution at a concentration equal to or between 100 and 500 ppm, preferably 300 ppm, - dewatering the starch, then dissolving in demineralised water adjusted to a pH of between approximately 5 and approximately 6, in particular approximately 5.5 and at a concentration of between 20 and 40% dry matter, - increasing the temperature to 107 °C, then adding an alpha-amylase for 15 minutes, - optionally, treating with an enzymatic preparation having detergent and clarification properties, - filtering the suspension on a bed of diatoms, - treating with an active carbon having a very high adsorption capacity, of pharmaceutical quality, and of "microporous" porosity, - treating with a second active carbon of "mesoporous" porosity, - optionally, passing over a macroporous adsorbent polymer resin, having a porosity greater than 100 angstroms, - optionally, continuous 5000 Da ultrafiltration, - safety filtration through a sterile filter having a porosity of 0.22 μm.