170 results about "Tissue homogenate" patented technology

This document describes an optical sensor unit and a procedure for the specific detection and identification of biomolecules at high sensitivity in real fluids and tissue homogenates. High detection limits are reached by the combination of i) label-free integrated optical detection of molecular interactions, ii) the use of specific bioconstituents for sensitive detection and iii) planar optical transducer surfaces appropriately engineered for suppression of non-specific binding, internal referencing and calibration. Applications include the detection of prion proteins and identification of those biomolecules which non-covalently interact with surface immobilized prion proteins and are intrinsically involved in the cause of prion related disease.

The present invention provides sample collection, shipping, and storage devices and methods of using the same. These devices and methods are useful, for example, for collecting, shipping, and storing biological samples, such as blood, serum, buccal samples, tissue homogenates, or cell lysates in a dry state. The devices and methods facilitate the rapid drying of biological samples collected on the devices, thereby improving the quality of the stored sample, particularly the protein and small molecule components of the stored sample. The present invention further provides methods of recovering biological samples from such devices.

A tissue homogenizer. The tissue homogenizer comprises a first chamber, a pair of blades, a first filter and a second filter. The first chamber has a first opening and a second opening. The blades are disposed in the first chamber. The first filter is disposed in the first chamber between the first opening and the blades. The second filter is disposed in the first chamber between the second opening and the blades. A tissue piece is placed between the first filter and the second filter cut by the blade, and moved by a fluid through the second filter to generate homogenized tissue pieces.

The invention relates to a grape fresh-keeping method using seaweed polysaccharide. The method is as below: cleaning and cutting a raw material of fresh seaweeds, conducting high speed tissue homogenate, leaching with 5-6 times of ethanol with concentration of 65-85% for 8-10 h, conducting pumping filtration, transferring to a rotary evaporator to obtain a seaweed polysaccharide crude extract, extracting the crude extract with 4-6 times of anhydrous ether, and concentrating an aqueous layer by using the rotary evaporator to obtain the seaweed polysaccharide; eliminating pests on fresh grapes and mechanically damaged fruits, soaking the grapes at room temperature in the seaweed polysaccharide solution for 5 min, drying, and packaging with a polyethylene film for storage; and inspecting regularly during storage, and eliminating abnormal fruits timely. The invention has the beneficial that natural seaweed polysaccharide as a protective agent for fresh-keeping of grape is safe, non-toxic and harmless, and can replace chemical fungicides and preservatives for fresh-keeping of grapes, and avoid the problems of environmental pollution and food safety caused by chemical fungicide.

The invention relates to a chemiluminiscence kit for detecting content of hyaluronic acid in human serum, body fluid or tissue homogenate by one-step process, and belongs to the medical field of immunoassay. By solid coating and indirect enzyme labeling technology, the problem that a hyaluronic acid kit is unavailable for one-step detection is solved substantially, operation is more convenient, detection is more accurate, and reagents are more stable. The chemiluminiscence kit for detecting content of the hyaluronic acid in human serum, body fluid or tissue homogenate by one-step process comprises a calibrator, a coated board, an enzyme combination, chemiluminiscence substrate solution A and chemiluminiscence substrate solution B. The sample amount of each of the HA (hyaluronic acid) calibrator and the enzyme combination is 50 microliters in the kit, and reaction time of the one-step process is one hour. Scientific and reasonable range of normal values is determined by strict methodological appraisal, clinical measurement results are compared with a radioimmunoassay kit statistically, and accordingly the chemiluminiscence kit has the technical advantages of high sensitivity, accurate measurement, high repeatability, regent stability and the like and is worthy of vigorous popularization and application to market.

Methods and systems for the use of Surface Enhanced Raman Scattering nanotags (SERS nanotags) to create homogeneous (no-wash), heterogeneous or sequence detection assay platforms. In certain embodiments the SERS nanotags are used in combination with magnetic particles. Multiplexed assay platforms are also disclosed. In certain embodiments, the assay is useful for clinical proteomics. Assay platforms suitable for use within a biological matrix, for example within whole blood or serum are also disclosed. The assay formats described herein may be used to detect any analyte of interest including but not limited to the detection of cells, viruses, bacteria, proteins, DNA, RNA, or small molecules in any type of biological (animal or plant kingdom) or environmental samples including but not limited to whole blood or serum, occult samples, urine, feces, air, drinking water, phage, any organism, multicellular clumps of cells, for example, cancer tissue homogenate.

The invention discloses albumen powder richening in antimicrobial peptide fly maggot, a preparation method for the albumen powder and a feed additive of the albumen powder. The albumen powder richening in the antimicrobial peptide fly maggot is prepared by a method comprising the following steps of: (1) feeding flies, luring the flies to lay eggs and feeding larva by using feed after incubation; (2) conducting ultrasound induction on the larva; (3) continuously feeding, collecting the larva, washing, conducting disinfection treatment, and conducting tissue homogenate smashing; and (4) filtering, treating filtrate at 0-5 DEG C, removing surface lipid compositions, drying to obtain the albumen powder; adding auxiliary materials in the albumen powder, pelletizing to obtain the feed additive. A ultrasound stimulation mode under specified conditions is adopted to induce the larva to enable an antimicrobial peptide active ingredient content in a body of a housefly to obviously improve, and the feed additive has certain growth promotion and disease-resistant strengthening effects on litopenaeus vannamei, crucian and the like.

The invention discloses a cryopreservation method for a tissue block used for cell culture. The method includes: firstly performing homogenate treatment on a tissue into a chyle state, then adding a refrigerating fluid and mixing them uniformly, subpackaging the mixture into a freezing tube containing sucrose, DMSO and glutathione, then placing the freezing tube in a cryopreservation box and letting the box stay overnight in a -80DEG ultra low temperature refrigerator, and finally putting the freezing tube into liquid nitrogen for long-term preservation. During unfreezing, centrifugal washing by an unfreezing solution and a culture solution is performed in order, then the tissue block can be directly subjected to inoculated culture or can be digested into a single cell and is then subjected to culture. The method provided in the invention firstly employs a homogenizer to treat the tissue into a chyle state, thus facilitating permeation of a cryoprotectant into tissue cells and removal of intracellular water. At the same time, the impermeable protective agent sucrose added in the refrigerating fluid is also conducive to removal the water from the tissue cells. The adding of the antioxidant substance glutathione helps alleviate the damage of oxygen free radicals on intracellular protein. After unfreezing, the tissue block is directly inoculated in a culture bottle, and the adherent survival rate is over 96%.

The invention relates to madder extract and a preparation method and an application thereof, wherein the madder extract is prepared from the following steps: pulverizing the rhizome of the madder, and using ethanol for percolation extraction at room temperature; recovering the ethanol and obtaining extract; dissolving the extract into water, and heating and completely dissolving the extract; and then, extracting the extract by using ethyl acetate, and concentrating the extract to obtain the madder ethyl acetate extract. The experiment indicates that the madder ethyl acetate extract and the compound alizarin can effectively reduce the blood glucose value, the GSP value, the TG value and the content of MDA in the tissue homogenate of the mouse with hyperglycemia caused by alloxan, and can obviously increase the SOD activity value of the hepatocyte of the mouse and increase the content of the hepatic glycogen of the tissue. The results prove that the madder ethyl acetate extract and the alizarin have good effect on reducing the content of the blood glucose.

The invention relates to a method for detecting hyaluronic acid in human serum, body fluid or tissue homogenate by a radioimmunoassay method. By a method of using solid phase-coated and europium-marked hyaluronic acid binding protein (HABP-Eu<3+>), reagents such as a first antibody, a second antibody and the like are not needed, a sample is easy to add, reaction is quick, the reagents have long shelf life and are environmental-friendly, europium is stably combined with HABP, storage time can be more than 12 months, and radioactive pollution is avoided.

The application discloses a preparation method of a fish single-cell suspension. The preparation method comprises the following steps: preparing a PBS reagent, a D-Han liquid and an IV type collagenase liquid, preparing a kidney tissue block of a fish, and washing the kidney tissue for more than three times with PBS; putting the kidney tissue block in a flat vessel of an ice bath, adding PBS, shearing the kidney tissue block into small pieces, and putting the tissue blocks in a high throughput tissue centrifuge tube of a burnisher; adding the IV type collagenase liquid into the centrifuge tube and meanwhile, adding PBS to fix the volume to 4ml, putting a zirconium oxide wall to grind, and performing enzymatic digestion on a tissue homogenate; and performing filtration, centrifugalization, washing with PBS, performing centrifugalization to abandon supernate and removing cell debris, filtering the cell suspension and fixing the volume to 3ml with PBS, and storing the cell suspension for later use at 4 DEG C. By adopting an enzymatic hydrolysis grinding method, advantages of a griding method and an enzymatic hydrolysis method are combined. The tissue is more fully treated by enzymatic hydrolysis grinding, so that the problem that the cell yield is low and the cell viability is low for preparing the fish single-cell suspension can be solved.

Relating to the fields of biotechnologies and cosmetic science, the invention provides a technology for using the umbilical cords and placentas of volunteers to prepare a skin care product for skin rejuvenation. Specifically, the umbilical cords and placentas of term delivery volunteers are separated, blood vessels and envelopes therein are removed, a tissue homogenizing machine is employed to make the obtained tissue into homogenate, and then medical grade glycerin, preservative and the like are added to prepare the skin care product. Being rich in trophic factors, the skin care product has the efficacy of promoting the proliferation of epidermal stem cells and hair follicle stem cells, plays an important role in maintaining skin elasticity and health, and has the advantages of no toxic, side effect or adverse reactions, etc. The umbilical placental skin care product provides a good method for delaying aging and restoring skin elasticity.

The present invention relates to methods and processes of inactivating viral contaminants in a biological source material (e.g. a host cell, cell supernatant, cell lysate, blood plasma, tissue homogenate, or other biological materials) with a solution containing one or more alkylamine compounds. In a particular embodiment, the active ingredients are amphipathic, charged amines or amine oxides coupled to saturated hydrocarbon chains of varying lengths.

The invention discloses a homogenate breaking and injecting device. The device comprises an injector and a breaker; the injector comprises an injection cylinder, the front end of the injector is provided with a push rod with a rubber plug; the breaker comprises a motor, a stirring shaft connected with the motor in a driving way and a blade arranged at the end part of the stirring shaft; another end of the push rod is of a cylindrical shape, the motor is arranged in the cylindrical push rod, the blade is arranged adjacent to the inside end plane of the rubber plug and the stirring shaft penetrates through the middle part of the rubber plug and is fixedly connected with the blade. The injector and the breaker are beneficially combined so that parathyroid glands can be completely and uniformly broken in the injector, therefore the technical problem that an existing tissue homogenate breaker is unlikely to be broken can be solved, and the step of sucking the parathyroid gland homogenate broken cannot be broken, so that the parathyroid gland homogenate can be directly injected to a receptor by the homogenate breaking and injecting device.

The invention discloses an auricularia auricula oral liquid and a preparation method thereof. Fresh rhizoma gastrodiae is cleaned and made into homogenate; the homogenate is subjected to deep fermentation with auricularia auricula to obtain an oral liquid containing gastrodin and auricularia polysaccharide. The preparation method comprises the steps of (1) cleaning the fresh rhizome gastrodiae and making the homogenate; (2) culturing slant strains for the auricularia auricular; (3) culturing shake flask liquid seeds by using slant strains; (4) inoculating first order seed into a seed container for culture; (5) pouring the tissue homogenate of the rhizome gastrodiae and culture medium into a fermentation container; inoculating the liquid seeds into the fermentation container after sterilizing and cooling; culturing large-scale agaric mycelium; reaching attenuation limit when the agaric mycelium begins to autolyze, fermentation liquid becomes sticky, and residual sugar decreases to 0.3%; and (6) obtaining homogenous fermentation liquid, canning, sealing, and sterilizing to obtain the auricularia auricular oral liquid. The oral liquid increases the output of auricularia polysaccharide and contains the rhizome gastrodiae, thereby having the effects of enhancing intelligence, nourishing brain, and delaying senility. Therefore, the oral liquid has many healthcare functions and helps improve human health.

The invention provides an earthworm antimicrobial peptide biological pesticide and application and belongs to the field of biochemistry. A technical scheme is as below: extracting earthworm antimicrobial peptide from earthworm body cavity and tissue homogenate by a phosphate buffer; centrifuging a leaching liquor; carrying out ultrafiltration; and concentrating to prepare the earthworm antimicrobial peptide biological pesticide. The biological pesticide of the invention can be effectively applied to control of insect diseases of paddy rice, fruit and vegetable; and the production technology is simple and without generating pollution. The biological pesticide of the invention belongs to a green environment friendly product without pollution, and is especially suitable for production of nuisance-free organic agricultural products.

The invention discloses a solid-phase extracting column, comprising a glass column and a filler. The filler is hydroxypropyl sephadex LH-20. The solid-phase extracting column can be used for pretreatment of biological samples, in particular used for simultaneous concentration and analysis detection of middle and small molecules in serum, urine and tissue homogenate and the molecular weight of middle and small molecules is close. The solid-phase extracting column in the invention can suppress interference effectively, ensure high sensitivity, good specificity and high accuracy of analysis of analytes and has excellent guiding significance to organism metabonomics research and detection of disease. Furthermore the solid-phase extracting column in the invention can be used repeatedly and hardly exist the phenomenon of regeneration. The extracting column is easy in filling, simple in operation. The column has stronger application value in analysis of biological samples.

A method and apparatuses for determining the total content of proteases by means of measuring their enzyme activity after previous deinhibition is disclosed. In biological samples, lysosomal proteases are completely (e.g. in blood serum) or partly (e.g. in tissue homogenates) inhibited. A method proposed for the deinhibition entails immersing a solid support material (e.g. nylon or nitrocellulose) as plastic strip to which is linked, covalently or by adsorption, an inhibitor-binding substance which binds the inhibitor corresponding to the protease more strongly than the protease in the sample for measurement. After the deinhibition has taken place, the plastic strip is removed from the liquid sample for measurement, and the sample for measurement is passed on for measurement of the enzyme activity. Fluorogenic substrates from which the fluorogen 7-amino-4-trifluoromethylcoumarin is eliminated proved to be particularly advantageous for the activity measurement. These substrates make it possible for the sensitivity on measurement in microtitre plates with a fluorescence reader to be at least 10 times higher compared with conventional AMC substrates in blood serum, and thus for the fluorimetric determination of such enzyme activities in blood serum to be efficient with this measuring arrangement which is widely used in clinical laboratories.

Provided herein are methods of recovering single nuclei from a tissue sample comprising chopping or dounce homogenizing the tissue sample in a nuclear extraction buffer at 4° C. to produce a tissue homogenate; centrifuging the tissue homogenate to produce a nuclear pellet; resuspending the nuclear pellet in a nuclear resuspension buffer comprising bovine serum albumin, RNase inhibitor, and salts to produce a resuspension; and filtering the resuspension through a strainer, wherein the single nuclei are present in a supernatant passed through the strainer. The invention also provides a method of single cell sequencing comprising extracting nuclei from a population of cells under conditions that preserve a portion of the outer nuclear envelope and rough endoplasmic reticulum; sorting single nuclei into separate reaction vessels; extracting RNA from the single nuclei; generating a cDNA library, whereby gene expression data from single cells are obtained. The tissue sample may be fresh or frozen.

The invention discloses a preparation method of an iridovirus cell inactivated vaccine for Chinese giant salamanders, and an application method thereof. The vaccine is prepared by the following method: 1, sterilely acquiring the liver of a moribund giant salamander containing frog iridovirus, preparing a tissue homogenate; 2, quickly cracking the tissue homogenate by ultrasonic wave of 20kHz on an ice surface, filtering; 3, centrifugally separating the filtrate to obtain pure virus precipitate, then regulating the virus titer to 105TCID50; 4, culturing the virus cells, collecting a cell culture solution; and 5, and inactivating the cell culture solution by formaldehyde with the final concentration of 0.4% at 37 DEG C for 5-10 hours, thus obtaining the iridovirus cell inactivated vaccine for Chinese giant salamanders. The vaccine is effective when preserved at 4 DEG C within one year. For young giant salamanders and adult giant salamanders below 0.25kg in body weight, dipping bath with the dilute solution of the vaccine is adopted for immunization, and for adult giant salamanders above 0.25kg in body weight, intramuscular injection is adopted for immunization. The vaccine has very strong immunologic specificity, and the immunization rate can reach 80-93%.

The present invention provides sample collection, shipping, and storage devices and methods of using the same. These devices and methods are useful, for example, for collecting, shipping, and storing biological samples, such as blood, serum, buccal samples, tissue homogenates, or cell lysates in a dry state. The devices and methods facilitate the rapid drying of biological samples collected on the devices, thereby improving the quality of the stored sample, particularly the protein and small molecule components of the stored sample. The present invention further provides methods of recovering biological samples from such devices.

The invention discloses solid-phase extraction filler and a preparation method thereof. The filler is filler ODS-CD-HPS bonding beta-cyclodextrin and octadecyl silane mixing functional groups. The invention further discloses a solid-phase extraction column filled with the filler ODS-CD-HPS. The solid-phase extraction column can be applied to pretreatment of biological samples and can be particularly applied to enrichment and analytical detection of medium-polarity and weak-polarity matter in body fluid and tissue homogenate of human bodies. Compared with an existing common solid-phase extraction column based on amorphous silica gel filler, the solid-phase extraction column only needs to be leached with an organic solvent with a small volume, and the extraction recycle rate, reproducibility and extraction efficiency of the medium-polarity and weak-polarity matter are high. Besides, when the solid-phase extraction column is used for pretreatment, leaching and concentrating are completed at a time, and the obtained leached liquid can be enriched and concentrated without extra heating, nitrogen blowing and other steps.

The invention discloses a mussel glycoprotein and a preparation method and an application thereof. The mussel glycoprotein disclosed by the invention is prepared by the following steps: carrying out tissue homogenization of mussel soft tissues; extracting by a buffer solution; carrying out ion exchange column chromatography; carrying out gel column chromatography; purifying by virtue of a fast protein purifying system; concentrating; and freeze-drying. The molecular weight of the mussel glycoprotein is about 16.9kDa and the content of sugar and proteins is respectively 23.54% and 76.46%. The mussel glycoprotein contains O-glucosidic bonds and N-glucosidic bonds. Monosaccharide consists of D-glucose, L-arabinose, D-galactose, D-fructose and D-mannose in a corresponding proportion of 1.00 to 2.01 to 6.93 to 7.27 to 4.05. The protein consists of 15 amino acids. The mussel glycoprotein is remarkable in activity, can be clinically used as an agiogenesis inhibitor and can be used for treating tumor diseases such as prostatic cancer.

The invention relates to a bioactivator preparation method, in particular to a placenta polypeptide preparation method. The placenta polypeptide preparation method comprises the steps of cutting a placenta into pieces, and adding normal saline into the cut placenta for tissue homogenate; mixing the obtained product with protease for enzymatic hydrolysis, and performing centrifugation and collecting centrifugate; performing extraction through reverse micelles to produce placental polypeptides and crude protease extract, subjecting the crude protease extract to ultrafiltration, collecting ultrafiltrate, and drying the ultrafiltrate to produce the placenta polypeptides. The preparation method is simple, production cost is low, equipment investment is little, and large-scale production is facilitated.

The invention provides a method for preparing human placenta extract supernate. The method comprises the following steps: removing excess blood vessels and tissue elements from a full-term eutocous placenta, and preparing placenta tissue homogenate by virtue of mechanical method; cleaning placenta tissue homogenate, and carrying out deep-low-temperature repeated freezing and thawing; carrying outcentrifugation on the repeatedly frozen and thawed placenta tissue homogenate, carrying out filtration, and extracting supernate; and inspecting the supernate, detecting the content of growth factors,and meanwhile, detecting germs, fungi, mycoplasmas and endotoxin, so as to obtain a final supernate product. According to the method, four growth factors are released and highly expressed in the prepared placenta tissue homogenate, and a supernate product with high content of growth factors can be acquired by regulating the volume of the final supernate product; and pollution risks of pathogenicmicroorganisms, the germs, the fungi, the mycoplasmas and the endotoxin are avoided in the preparation process of the supernate, so that the safety and effectiveness are guaranteed and improved.

The invention provides a cowhide processing method for preparing collagen, and belongs to the technical field of biological food. The method is used for performing fine processing on cowhide before collagen preparation, and the cowhide serves as the main raw materials for preparing the collagen. The method comprises the steps that cow dermis tissue is unfrozen, wherein the frozen cow dermis tissue is placed into a beaker and soaked by adding ultrapure water of which the volume is four times of that of the cow dermis tissue until the cow dermis tissue is unfrozen into loosen tissue particles, and the tissue particles are evenly stirred with a glass rod into paste; the cow dermis tissue particles are mashed into even suspension liquid free of obvious cowhide particles through a tissue masher; the pasty tissue particles are transferred to a glass cup of the tissue masher, ten times volume of ultrapure water is added, homogenization is performed for three times according to the operating instruction of the tissue masher until homogenate is completely suspended, and the tissue homogenate is transferred to a plastic drum; glacial acetic acid is added into the tissue suspension plastic drum to regulate the concentration of the solution to be 0.5 milligram / milliliter. According to the method, the pasty liquid obtained by processing the cowhide is used for collagen extraction, and therefore the purification rate and purity of the collagen can be obviously improved.

The invention relates to a method for preparing gram-level high-purity natural thiosulfinate, belonging to the technical field of compound preparation. The method comprises the following steps: preparing an allium tissue homogenate containing active alliinase, keeping the temperature, washing off endogenous thiosulfinate generated in the tissue homogenate with an organic solvent, preparing exogenous ACSO into a solution, adding the solution into the tissue homogenate, keeping the temperature to generate single thiosulfinate or mixed thiosulfinates, and finally, purifying to obtain the product. The method has the advantages of mild reaction conditions, high product purity, high reaction yield, high cleanness and environmental protection, is simple to operate, and conforms to green technological requirements. The purity of the prepared pure thiosulfinate is sufficient for manufacturing drugs and especially diallyl thiosulfinate (garlicin). The research indicates that the garlicin has the maximum antibacterial, antihypertensive, anticancer, antioxidation and other activities in thiosulfinate.

The invention provides a PD-1 antibody detection kit and application thereof. The PD-1 antibody detection kit comprises a solid phase carrier, an antibody trapping agent and a labeled antibody, wherein the antibody trapping agent is a recombinant protein of a PD-1 cytomembrane outer structure, and the amino acid sequence of the antibody trapping agent is SEQ ID NO.1. The PD-1 antibody detection kit can quickly detect a PD-1 antibody in a to-be-detected sample, such as in-vitro serum and tissues homogenate, the specificity of detection is high, the sensitivity of detection is high, and the application range is wide.

The invention provides preparation methods of sheep placenta polypeptide powder and soluble granules. The preparation method of sheep placenta polypeptide powder comprises the following steps: achieving pretreatment to the sheep placenta raw material and realizing tissue homogenate; adding protease and flavourzyme into the homogenate for enzymolysis, deodorization, debittering and enzyme deactivation; conducting centrifugal edulcoration to obtain a sheep placenta polypeptide solution; concentrating and drying to obtain sheep placenta polypeptide dry powder. The preparation method of the soluble granules comprises the step of mixing the sheep placenta polypeptide dry powder and synanthrin or xylose at the proportion of 1:2 to prepare granules. The sheep placenta polypeptide is prepared into soluble granules through adding xylose, synanthrin and the like, so that the water absorbability of the sheep placenta polypeptide powder is reduced, the water solubility of the sheep placenta polypeptide powder is improved and the product flavor is effectively improved. The granules are rich in nutritions, easy to absorb and excellent in flavor and taste, and has the anti-fatigue, immunoregulation, beauty maintaining and young keeping functions. The preparation methods are reasonable in process, simple in manufacture and strong in operability and are suitable for mass production.

The invention discloses an extraction method of sheep embryo active peptide, which sequentially comprises the following steps of: (1) fetching the fresh tissue of 6-16 week sheep embryo, cutting up and adding normal saline; homogenizing by a tissue homogenizing machine; and performing quick freeze thawing at -20 DEG C and 40 DEG C after the homogenization; (2) performing ultrasonic cracking: starting an ultrasonic probe every 30 seconds, letting the ultrasound last for 15 seconds, and repeating for 3-5 times; (3) performing low-temperature high-speed centrifugation for 30 minutes at 4 DEG C, and taking the supernate; and (4) performing centrifugal ultrafiltration of the supernate by use of a 5k ultrafiltration membrane, and clarifying to obtain the ultrafiltrate which is a sheep embryo active peptide solution. Without any acid and alkali treatment, the extraction method disclosed by the invention effectively avoids environmental pollution caused by a chemical method, and also avoids product pollution. Moreover, the method can collect multiple biologically active peptide components to the greatest degree, wherein the molecular weight of the active peptide is effectively controlled below 5,000D, and the bioactivity of the sheep embryo small-molecular peptide is maintained to the greatest degree.