50 results about "Endotoxin removal" patented technology

The invention discloses humanized III-type collagen as well as a preparation method and application thereof, and belongs to the technical field of bioengineering. According to the method, firstly, on the basis of an original humanized III-type collagen gene, cell adhesion activity is taken as an evaluation index, a better cell adhesion sequence is screened out and spliced and integrated with the original collagen gene, and brand-new humanized III-type collagen is obtained. And constructing a recombinant expression vector of the humanized III type collagen containing the adhesion sequence by taking the probiotic EcN as a host cell, and introducing the recombinant expression vector into the EcN to obtain a strain of functional recombinant probiotic. Compared with a traditional escherichia coli engineering bacterium BL21 (DE3), the recombinant probiotics have good cell adhesion activity while efficiently expressing the humanized type III collagen, the bacterial endotoxin content of the recombinant humanized type III collagen obtained through purification meets the industrial standard, and the recombinant probiotics have good application prospects. The purification cost caused by industrial removal of endotoxin can be avoided, so that the production cost is reduced, and the method has a good application prospect.

The invention discloses a high-stability endotoxin-free PHA nano-emulsion and a preparation method thereof, and belongs to the technical field of biodegradable high polymer material compositions and processing thereof. The nano-emulsion is characterized in that the endotoxin content is not higher than 0.10 EU / g, the median particle size d50 is 100-200 nm, and the PDI is not higher than 0.15, and the nano-emulsion is stable in long-term storage. The preparation method is a continuous solvent-free process and comprises the steps of melt extrusion, high-pressure thermal emulsification and deep endotoxin removal. By adopting the specific pH-insensitive cationic polymer emulsifier and the crystallization inhibitor, the chemical contradiction that an efficient alkaline purification process is incompatible with a nano-emulsion stable system is solved, physical aging is inhibited, and an unexpected technical effect is obtained. The invention also discloses an O / W type cream containing the nano emulsion.

The invention relates to the technical field of plasmid extraction, in particular to an automatic extraction method of endotoxin-free large-volume plasmid DNA and a kit. According to the kit provided by the invention, a magnetic bead adsorption technology and a buffer system with a unique separation effect are adopted, and specially modified protein is combined with magnetic beads, so that endotoxin and protein impurities can be adsorbed; an endotoxin removing solution is adopted, so that endotoxin can be efficiently removed; the method comprises the following steps: firstly, preparing plasmid DNA, then, uniquely embedded nucleic acid adsorption magnetic beads have very strong affinity to the plasmid DNA under a certain condition and are not easy to adsorb endotoxin, and when the condition is changed, the nucleic acid adsorption magnetic beads release the adsorbed plasmid DNA, so that the purpose of rapidly separating and purifying the plasmid DNA is achieved. The plasmid DNA extracted by utilizing the kit and the extraction method disclosed by the invention is high in yield, good in purity and extremely low in endotoxin content, and can be suitable for transfecting various cells and various conventional operations including enzyme digestion, PCR (Polymerase Chain Reaction), sequencing, connection and other various downstream experiment requirements.

The invention relates to the technical field of biomacromolecule separation and purification engineering, and discloses a chromatographic separation method for removing endotoxin in a vaccine preparation, which comprises the following steps: preparing an equilibrium buffer solution containing citrate with specific concentration and L-arginine hydrochloride; providing a mixed mode chromatography medium with hydrophobic and ion exchange complex ligands and balancing with a buffer; according to the method, through the synergistic effect of 8.0-12.0 mmol / L of citrate and 150-250 mmol / L of L-arginine hydrochloride, the endotoxin micelle structure is destroyed and is induced to be dissociated into monomers, and meanwhile, L-arginine is used for inhibiting monomer aggregation, so that the monomers are diffused into inner holes of the medium in a small size to be adsorbed, and the endotoxin micelle structure is separated into the medium. Therefore, the problem of separation caused by size overlapping of endotoxin micelles and antigens is solved, and double breakthrough of deep removal of endotoxin and high recovery rate of antigens is realized.

The invention discloses a method for purifying recombinant X VII type collagen expressed by pichia pastoris fermentation, and relates to the technical field of protein purification methods. The purification method comprises the following steps: centrifuging a recombinant X VII type collagen fermentation liquid fermented and expressed by pichia pastoris engineering bacteria, filtering the obtained centrifugate, then carrying out ultrafiltration concentration, cooling the obtained ultrafiltration concentrated liquid to 2-10 DEG C, then adding Triton X-114, maintaining the material liquid at 2-10 DEG C, carrying out chromatography, leaching and extraction, carrying out ultrafiltration concentration on the extraction liquid, and then carrying out freeze drying to obtain the recombinant X VII type collagen. The recombinant X VII type collagen freeze-dried powder is obtained. The purification method provided by the invention is simple and convenient in whole operation process, can effectively remove endotoxin, can prepare the high-purity recombinant X VII type collagen freeze-dried powder, and is convenient for industrial large-scale application.

The invention discloses a magnetic bead method endotoxin-free plasmid extraction kit, relates to the technical field of plasmid extraction and endotoxin removal, including: solution I, solution II, solution III, magnetic bead liquid I, magnetic bead liquid II, cleaning solution and eluent; solution I includes: sucrose, EDTA, Tris-HCl, NaCl; solution II includes: KO H, SDS, EDTA; solution III includes: potassium acetate, sodium acetate, trehalose, pH is 5-5.6; magnetic bead liquid I includes: NaCl, sodium citrate, magnetic beads I, pH is 5.0-5.5; magnetic bead liquid II includes: guanidine hydrochloride, urea, anhydrous ethanol, isopropanol, magnetic beads II, pH is 5.5-6.5. The present invention prepares an endotoxin-free plasmid extraction kit based on a double magnetic bead method, which has the advantages of convenience, low cost and good safety, can remove endotoxins in the plasmid extraction process, and can also be used to directly remove endotoxins in plasmids, and the obtained plasmids can meet the requirements of molecular biology experiments and cell experiments such as enzyme cutting, sequencing, transfection and virus packaging. At the same time, full automation of endotoxin-free plasmid extraction can be achieved.

The invention relates to the technical field of biology, in particular to a purification method of a bacteriophage preparation and application of the bacteriophage preparation in industrial scale production. The purification method of the bacteriophage preparation comprises the following steps: removing impurities such as bacteria and bacterial fragments in the bacteriophage preparation through grading membrane microfiltration; a hollow fiber ultrafiltration system is adopted to remove impurities including free lipopolysaccharide (LPS) and improve the titer of the bacteriophage at the same time; free LPS is further rapidly and accurately removed through a novel compound chromatography medium of molecular exclusion and charge adsorption, and a high-purity bacteriophage preparation can be obtained. According to the method, the endotoxin level in a phage preparation can be lower than 20 EU / 10 < 9 > PFU (the removal rate is greater than 99%), and the phage recovery rate is greater than 63.5%. The purification method provided by the invention has the advantages that the use of an organic solvent is reduced or avoided to the greatest extent, the technological process is green and environment-friendly, the endotoxin removal rate is high, and time and cost are saved.

The invention discloses a method for purifying recombinant type I mussel mucin and removing endotoxin, and relates to the technical field of preparation of recombinant type I mussel mucin. According to the method, recombinant I-type mussel mucin fermentation liquor which is subjected to fermentation induced expression by escherichia coli engineering bacteria is adopted as a raw material, and the purification efficiency can be greatly improved, the content of endotoxin can be reduced, and the quality of the mussel mucin can be improved by combining Triton X-100 and disodium ethylene diamine tetraacetate, controlling the cooling temperature of feed liquid and optimizing the endotoxin removal process and the addition amount of Triton X-114. Therefore, the invention provides the method for purifying the recombinant I-type mussel mucin of which the purity is greater than 95% and the endotoxin content is less than 5EU / mg.

The invention belongs to the technical field of biology, and provides a pseudomonas aeruginosa endotoxin specific binding polypeptide and application thereof. The amino acid sequence of the polypeptide is shown as SEQ ID No. 1, SEQ ID No. 3 or SEQ ID No. 4 in a sequence table. Experiments prove that the polypeptide has excellent affinity with pseudomonas aeruginosa endotoxin, and compared with an existing anti-endotoxin clinical drug polymyxin B, the polypeptide has obviously better pseudomonas aeruginosa endotoxin detoxification capability. On the basis, the invention further provides application of the polypeptide in removal or separation or analysis of the endotoxin of the pseudomonas aeruginosa and application of the polypeptide in preparation of a detoxification medicine for the endotoxin of the pseudomonas aeruginosa. The polypeptide can be widely applied to the biomedical fields of biological separation, biological detection, disease diagnosis and treatment and the like.

The invention relates to the technical field of hemodialysis, in particular to a preparation method and system for a low-endotoxin hemodialysis concentrated solution, and the method comprises the steps: obtaining a preparation data set, extracting a temperature sequence, a pH value sequence and an endotoxin content index value, and calculating a membrane flux distribution sequence and an adsorbent activity distribution sequence. Performing primary dialysis path optimization to generate a first completely optimized network; identifying a target path node pair according to the target endotoxin content value, and performing secondary path extension to generate a second completely optimized network; and finally identifying a temperature and pH value regulation and control sequence corresponding to the target path node to complete preparation. By optimizing the network, the test workload is remarkably reduced, the endotoxin removal efficiency and the process parameter control precision are improved, and the problems that in the prior art, the endotoxin removal efficiency is low, and the product batch stability is poor are solved.

The present application relates to a method for removing endotoxin from alginate oligosaccharide and alginate oligosaccharide with endotoxin removed, aiming to provide a method for efficiently removing endotoxin from alginate oligosaccharide with simple operation and easy industrialization. The technical points are: insoluble particles are removed by centrifugation, mixed Lewis acid is added for synergistic treatment, the treated solution is acidified to form a precipitate, the precipitate is washed with deionized water, an alkaline solution is added to dissolve the precipitate and filter, the filtrate is adsorbed by an adsorption medium, then alginate oligosaccharide is eluted with an organic solvent, and the alginate oligosaccharide with endotoxin removed is obtained after drying. The present application removes endotoxin from alginate oligosaccharide by using mixed Lewis acid synergistic treatment and adsorption by an adsorbent, no impurities are introduced during the preparation process, the endotoxin content in the obtained alginate oligosaccharide is low, the operation is simple, and the preparation process is suitable for industrial production.

This invention discloses a highly stable, endotoxin-free PHA nanoemulsion and its preparation method, belonging to the field of biodegradable polymer material compositions and their processing technology. This nanoemulsion has an endotoxin content of no more than 0.10 EU / g and a median particle size of d... 50 Characterized by a nanoparticle size of 100-200 nm and a PDI not exceeding 0.15, it exhibits long-term storage stability. Its preparation method is a continuous solvent-free process, including melt extrusion, high-pressure thermal emulsification, and deep endotoxin removal steps. This invention resolves the chemical incompatibility between efficient alkaline purification processes and the stable nanoemulsion system by employing specific pH-insensitive cationic polymer emulsifiers and crystallization inhibitors, and also inhibits physical aging, achieving unexpected technical effects. This invention also discloses an O / W type cream containing this nanoemulsion.

The invention discloses a salmonella bacteriophage Y1 with a wide lysis spectrum and a preparation method of a low-endotoxin preparation of the salmonella bacteriophage Y1. The preservation number of the salmonella bacteriophage Y1 is CCTCC M 20242911. The method comprises the following steps: adding a chitosan solution into a bacteriophage suspension, uniformly mixing, and precipitating overnight at room temperature. The salmonella phage Y1 disclosed by the invention is high in cracking capacity and can be applied to preparation of drugs for preventing and treating salmonella. The method disclosed by the invention is low in cost, simple in used equipment, simple and convenient in operation procedure, stable in effect, easy for large-scale industrial production, low in endotoxin preparation 3, good in endotoxin removal effect and small in influence on the activity of the bacteriophage.

The invention discloses a method for removing endotoxin in chicken infectious bursal disease virus VP2 protein. The method comprises the following steps: firstly, crushing a VP2 protein expression strain; adding calcium chloride into the treated VP2 protein, and standing; returning the temperature, adding sodium carbonate into the VP2 protein subjected to temperature returning, and standing; and returning the temperature of the VP2 protein added with the sodium carbonate, taking supernate, adding calcium chloride into the supernate, and standing. By setting the adding concentration of calcium chloride, the adding concentration of sodium carbonate and the treatment sequence and times of calcium chloride and sodium carbonate, the endotoxin in the VP2 protein is efficiently and rapidly removed by using calcium chloride and sodium carbonate. By adopting the method disclosed by the invention, the endotoxin removal rate reaches 99.91%, the agar diffusion titer is improved compared with that before treatment, and the animal vaccine preparation standard is met. The method disclosed by the invention is short in treatment period, large in treatment capacity, low in removal cost and suitable for industrial mass production.

The utility model discloses an endotoxin removing device. The endotoxin removing device comprises a tank body and an endotoxin removing assembly, the endotoxin removal assembly is mounted in the tank body and is used for removing endotoxin in pure steam formed by heating and evaporating raw water; the endotoxin removing assembly comprises a first shell, a second shell and a filter screen; the corresponding end parts of the first shell and the second shell are connected to form a frame structure; the filter screens are installed on the two sides of the frame structure, and the filter screens and the frame structure form a hollow frame structure. The frame structure is filled with a silk screen. According to the device, endotoxin in pure steam can be removed, and the purity of the pure steam is improved.

The invention discloses an endotoxin removing device based on collagen microsphere production and a using method thereof, and relates to the field of collagen microsphere production, the technical scheme is that the endotoxin removing device comprises a base, a filtering tank and a treatment tank, the filtering tank and the treatment tank are located on the base, and a reciprocating pipeline assembly is installed between the filtering tank and the treatment tank; a collagen microsphere solution can be transported between the two tank bodies in a reciprocating manner, so that the collagen microsphere solution entering and exiting from the treatment tank can be filtered by the filtering tank; a liquid inlet pipe is mounted at the upper part of the outer side wall of the filtering tank, a liquid discharge pipe is mounted at the lower part of the outer side wall of the filtering tank, and a filtering plate is mounted in the middle of the filtering tank; and an endotoxin adsorption column is mounted in the filtering tank, so that two times of filtering can be completed by only one filtering tank in the whole process, the cost is saved, the size of the device is reduced, and the device is very beneficial to being used in small factories.

The invention relates to the technical field of biology, in particular to a method for removing bacteriophage endotoxin, which comprises the following steps: step 1, selecting ion exchange resin as an adsorption material; step 2, removal pretreatment: putting ion exchange resin into a chromatographic column, and washing and soaking the ion exchange resin in the chromatographic column with a 0.5-1M sodium hydroxide standard solution; and step three, removing endotoxin from the bacteriophage: adjusting the pH value of the ion exchange resin to 7.2-7.4, adding the bacteriophage of which the endotoxin is to be removed into the chromatographic column, and standing for 10-15 minutes. According to the method, the ion exchange resin is used as an adsorption material and is loaded into the chromatographic column to remove the phage endotoxin, other equipment is not needed, and the operation is simple; the ion exchange resin is high in durability and stability and can be repeatedly used and stably stored, so that the cost is reduced; meanwhile, the removal process is efficient and rapid.

The present invention discloses a nanomagnetic bead for removing endotoxins from protein samples, as well as a preparation method and application thereof. The nanomagnetic bead comprises an inner core, an intermediate layer, and an outer layer; the inner core is a magnetic core, the intermediate layer is a metal oxide layer, and the outer layer is a boronic acid ligand layer. The metal oxide layer coordinates with the phosphate group in the endotoxin, and the boronic acid ligand layer combines with the cis-diphenol structure in the endotoxin. The nanomagnetic beads of the present invention achieve an endotoxin-clearing effect through a dual mechanism. The nanomagnetic beads for removing endotoxins from protein samples of the present invention can reduce the endotoxin content in the protein to below 0.5 EU / mL, meet the raw material requirements in the pharmaceutical field, and the protein will not be denatured. It has the advantages of simple and rapid operation, low cost, high endotoxin removal rate, and high protein recovery rate.

The invention relates to a method for early-stage removal of endotoxin in plasmid DNA preparation. The method comprises the following steps: (1) carrying out microfiltration on a lysis neutralization solution to obtain a filtrate; (2) concentrating the filtrate to obtain a concentrated solution, exchanging the obtained concentrated solution with purified water or a 10 * TE buffer solution, and adjusting the pH value to 4.4-5.6 with acetic acid to obtain an acidic solution; (3) adding zinc sulfate with the final concentration of 0.5-1M into the acidic solution, and incubating at 10-20 DEG C for 20-60 minutes to obtain an incubation solution; and (4) carrying out microfiltration on the incubation liquid, and carrying out ultrafiltration replacement on the obtained filtrate to remove the precipitate. The early clearing method for endotoxin in plasmid DNA preparation is efficient, economical and safe, endotoxin pollution can be controlled from the source, the proportion and yield of superhelix plasmids are considered, and the quality of plasmid DNA is improved. The early clearing method for endotoxin in plasmid DNA preparation can simplify the downstream purification process, and is especially suitable for large-scale industrial production of high-quality plasmid DNA.

The present invention discloses a PES membrane modification method, a PES membrane, and its application in endotoxin removal. The modification method, based on membrane surface treatment and chemical modification, significantly increases the number of amino groups on the PES membrane surface through a Schiff base / Michael addition reaction, thereby increasing the positive surface charge of the PES membrane. The method offers advantages such as high efficiency and energy saving, ease of operation, easy process control, and ease of scale-up and integration. The modified PES membrane positively modifies the original PES membrane surface, significantly increasing its positive surface charge, enabling electrostatic adsorption to remove endotoxins present in intravenous medications. Furthermore, the modified membrane introduces a large number of amino or hydroxyl groups, which readily bind to lipopolysaccharide molecules, onto the surface. These groups can be used to immobilize endotoxins on the membrane surface through molecular forces for removal, thereby ensuring that the modified membrane can remove endotoxins over a wider pH range.

The invention discloses a method for removing endotoxin by hydrophobic chromatography, which comprises the following specific steps: adding TritonX-114 into a protein sample to be subjected to endotoxin removal, and stirring at 0-4 DEG C to completely dissolve the TritonX-114 to prepare a sample to be detected; sequentially cleaning the filler in the hydrophobic chromatographic column with isopropanol, water and sodium hydroxide, and balancing the hydrophobic chromatographic column with a buffer solution; loading the sample to be detected into the balanced hydrophobic chromatographic column, and collecting a flow-through sample containing target protein; and cleaning the filler in the hydrophobic chromatographic column by using the buffer solution again. According to the method, the endotoxin is removed by using the hydrophobicity of the endotoxin by using a hydrophobic chromatography method, the endotoxin residue in the protein raw material can be reduced by 90% or more, the protein loss is less, the residual amount of TritonX-114 in the harvested protein sample is less, the influence of the TritonX-114 residue on downstream use is reduced, and the method has the characteristics of simple process and easiness in operation, and is suitable for industrial production. And the method is suitable for large-scale production.

A method for depletion or removal of endotoxins from a known or suspected endotoxin-containing source by virtue of a solid phase extraction material in an essentially aqueous system comprising the steps of—providing a known or suspected endotoxin-containing source, —contacting the known or suspected endotoxin-containing source with a positively charged solid phase material having a surface on which ferric iron is immobilised, wherein the solid phase extraction material has immobilised the ferric iron by (2-aminoethyl)amine (TREN) ligand—incubating the known or suspected endotoxin-containing source for a period of time sufficient to bind endotoxin to the porous solid phase material, —separating the solid phase material from the essentially aqueous system, —optionally isolating the essentially aqueous system freed or depleted from endotoxin.

The invention discloses a method for removing endotoxin in biosynthetic recombinant small RNA (Ribonucleic Acid), which comprises the following steps of: separating and purifying total RNA expressed by escherichia coli in FPLC (Fluorescence Programmable Logic Controller) by using an anion exchange column, desalting and concentrating by using an ultrafiltration membrane, and removing the endotoxin in a recombinant small RNA solution by using an ultrafiltration method. Meanwhile, the effects of different endotoxin removal methods are also compared, and finally, the effects of different methods are comprehensively evaluated from the three aspects of sample flux, the recovery rate of the recombinant small RNA and the endotoxin removal rate. Other impurities are not introduced in the process of removing the endotoxin by the ultrafiltration method, the flux is high, the durability is good, the stability is excellent, and the content of the endotoxin is 1.2-5.8 EU / ml, which is lower than the endotoxin limit requirement of biosynthetic protein preparations similar to the production process in Pharmacopoeia. The invention provides an economical, efficient and rapid method for removing the endotoxin of the recombinant small RNA, and powerful support is provided for wide application of the recombinant small RNA.

The present invention relates to the field of protein purification. In particular, the invention concerns methods for increasing the filtration capacity of virus filters, by combined use of endotoxin removal and cation-exchange media in the prefiltration process.

The invention provides a method for efficiently purifying recombinant collagen and removing endotoxin, and belongs to the technical field of collagen purification. Affinity chromatography, anion exchange chromatography, ultrafiltration concentration and dialysis filtration are combined, the affinity chromatography technology is used for primarily purifying recombinant collagen and removing endotoxin, and the anion exchange chromatography technology is used for further purifying recombinant collagen and removing endotoxin. And finally, carrying out ultrafiltration concentration and dialysis filtration to remove small molecular impurities and residual salts so as to achieve the purposes of efficiently purifying the recombinant collagen and removing endotoxin. The method improves the purification efficiency, has flexibility and expandability, can improve the quality and consistency of the product, and has good practicability.

The application discloses a method for removing endotoxin in pullulan and application thereof, and mainly comprises the following steps: crude pullulan extract is precipitated and washed by an organic solvent to obtain pullulan; after being dissolved, a surfactant is added into the solution to make endotoxin aggregates depolymerize into monomolecular form; then, the solution is filtered through an ultrafiltration membrane to remove endotoxin and residual small molecular impurities and inorganic salts; finally, the polysaccharide solution is precipitated by ethanol, dried and crushed to obtain refined pullulan; the endotoxin removal method adopted by the prepared pullulan has little influence on the molecular weight of the pullulan, the endotoxin content of the obtained pullulan is not higher than 0.05 EU / mg, the prepared pullulan meets the medical standard, has high biological safety, the operation steps are simple, the pullulan is easy to realize large-scale production, and the pullulan has the advantages of rapidness, simplicity, low cost and industrialization; and the technical scheme of the application is favorable to promoting the application of the pullulan in the field of tissue engineering.

The present invention relates to the field of protein purification. In particular, the invention concerns methods for increasing the filtration capacity of virus filters, by combined use of endotoxin removal and cation-exchange media in the prefiltration process.

The invention relates to a fusion protein for improving the antiviral titer of prokaryotically expressed porcine interferon-alpha, and belongs to the technical field of animal medicine. The fusion protein is an sIL-2-Thy alpha 1 fusion protein, and is a high-purity sIL-2-Thy alpha 1 protein obtained by connection and recombinant expression of an sIL-2 gene and a Thy alpha 1 gene, chromatographic purification by using Ni < 2 + > affinity resin, removal of a label by enzyme digestion and removal of endotoxin. The sIL-2-Thy alpha 1 fusion protein and the sIFN-alpha protein are combined into an antiviral composition, so that the inhibition effect of the sIFN-alpha on the swine vesicular stomatitis virus (VSV) can be remarkably enhanced, and a remarkable synergistic effect is achieved. The composition has unique advantages in the aspect of preventing and treating VSV infection. In addition, through innovative fusion protein design, the antiviral efficacy of interferon is improved, and the interferon has wide application prospects.

It is necessary to establish a purification method effective for the removal of endotoxin, in order to obtain thermolysin containing a reduced amount of contaminant endotoxin. On the premise of the establishment, accurate measurement of the amount of endotoxin that contaminates thermolysin is required. The present invention addresses the problem of providing a novel measurement method which enables accurate measurement of the amount of endotoxin that contaminates thermolysin. The present invention also addresses the problem of providing thermolysin containing a reduced amount of contaminant thermolysin, and the use thereof. There is provided thermolysin containing contaminant endotoxin in an amount of 1 EU / mg or less. There is also provided a method for measuring endotoxin that contaminates thermolysin, the method including pretreatment of deactivating the thermolysin. An enzyme preparation including the thermolysin according to the present invention as an active ingredient has a high utility value in the field of regenerative medicine.

To provide a method for depletion or removal of endotoxins from a known or suspected endotoxin-containing source.SOLUTION: A method for depletion or removal of endotoxins from a known or suspected endotoxin-containing source comprises the steps of: in an essentially aqueous system, bringing the source into contact with a positively charged solid phase material having a surface on which ferric iron is immobilized by a (2-aminoethyl)amine (TREN) ligand; incubating the source for a period of time sufficient to bind the endotoxins to the porous solid phase material; separating the solid phase material from the essentially aqueous system; and optionally isolating the aqueous system freed or depleted from the endotoxins.SELECTED DRAWING: Figure 1