62 results about "Adenosine Phosphates" patented technology

The invention discloses a novel biosynthesis path for producing 3 '-phosphoadenosine-5'-phosphosulfuric acid and application, and belongs to the technical field of bioengineering. According to the invention, through recombinant expression and purification of ribose kinase, ribose phosphate pyrophosphate kinase, ribose phosphate transferase, adenine thiotransferase, adenosine phosphate sulfate kinase and polyphosphate kinase 2, a six-enzyme cascade reaction taking ribose and adenosine as substrates in vitro is constructed, the molar conversion rate reaches 22.85%, and the escherichia coli and bacillus subtilis PAPS cell factory can be used for preparing the recombinant ribose phosphate pyrophosphate kinase, the ribose phosphate transferase, the adenine thiotransferase, the adenosine phosphate sulfate kinase and the polyphosphate kinase 2. The yield of PAPS in a 5-L fermentation tank reaches 7.60 g / L and 5.03 g / L respectively. The PAPS synthesis route provided by the invention provides an extensible, economical and efficient solution for the production of sulfated compounds.

The invention provides a yeast rich in polyphosphate and application of the yeast in synthesis of adenosine triphosphate, and the content of polyphosphate is 15-35 wt% based on the dry matter mass of the yeast rich in polyphosphate; according to the method disclosed by the invention, the yeast is subjected to phosphorus starvation culture and then is subjected to phosphorus supplementary culture, so that the yeast is enriched with polyphosphates, and the yeast rich in polyphosphates is obtained; then, the yeast rich in polyphosphate is used as a biocatalyst to synthesize ATP, that is, self-related enzyme systems, mainly including adenosine kinase, endo-polyphosphate kinase and excision-polyphosphate kinase, in the yeast rich in polyphosphate are used for replacing exogenous polyphosphate kinase and adenosine kinase, and in addition, ATP is synthesized by using the yeast rich in polyphosphate as a biocatalyst. According to the method for synthesizing ATP by using the yeast rich in polyphosphate as the biocatalyst, exogenous adenosine kinase and exogenous polyphosphate kinase do not need to be additionally added, the cost is low, the efficiency of converting adenosine into ATP is high, the batch-to-batch stability of the yeast is realized, and the enzyme activity of the self-related enzyme system is high.

The invention discloses a method for catalytically synthesizing adenosine triphosphate by using microcrystalline cellulose carrier purification immobilized enzyme, which comprises the following steps: preparing bacterial suspension by using escherichia coli wet thalli and Tris-HCL buffer solution, crushing, centrifuging, and taking supernate to obtain crude enzyme; adding a microcrystalline cellulose carrier and the wet thalli into the crude enzyme liquid, and washing the immobilized enzyme after immobilization to obtain the immobilized enzyme; the method comprises the following steps: adding immobilized enzyme adenosine kinase and immobilized polyphosphate kinase into a substrate system with the volume of 1L according to a weight ratio, and starting a catalytic reaction under the conditions that the pH is 5.5-7.5 and the temperature is 20-40 DEG C; after catalysis is finished, the immobilized enzyme adenosine kinase and the immobilized polyphosphate kinase are subjected to solid-liquid separation with reaction liquid through a Buchner funnel, the immobilized enzyme continues to be subjected to catalytic reaction, and the method for preparing the immobilized enzyme is simple in mode, low in cost, easy to industrially produce, high in stability and capable of being repeatedly used for many times, and products are easy to separate.

The invention relates to an adenosine triphosphate optical probe, in particular to an adenosine triphosphate (ATP) optical probe as well as a preparation method and application thereof. Specifically, the invention provides an adenosine triphosphate optical probe, which comprises an adenosine triphosphate sensitive polypeptide and an optically active polypeptide, and the optically active polypeptide is located in the sequence of the adenosine triphosphate sensitive polypeptide.

The present invention relates to the field of enzyme engineering, and more particularly to enzyme mutants, enzyme compositions or their immobilized enzymes and their applications and methods for preparing L-carnitine. The present invention provides a simple L-carnitine holoenzyme preparation process, the overall process of which utilizes bulk betaine (N,N,N-trimethylglycine) and acetic acid as raw materials. L-carnitine is directly obtained by two-step continuous conversion by carboxyl reductase (CAR) and aldolase (DERA). This route can be completed in one step using a crude enzyme solution, or by utilizing a recyclable immobilized enzyme, and the reduced coenzyme II (NADPH) and adenosine triphosphate (ATP) used in the first step of the reaction can also be recycled using corresponding enzymes to further reduce production costs. Therefore, the patented L-carnitine preparation process has many advantages, such as being fully green, simple in process, inexpensive to produce, and easy to scale up.

The invention relates to the technical field of biosynthesis, in particular to a biosynthesis process of adenosine triphosphate and equipment thereof. Comprising the following steps: optimizing an adenosine triphosphate synthetase gene sequence, constructing an expression vector, and introducing the expression vector into a yeast host cell; sub-packaging the engineering yeast group in a pore plate for parallel culture, detecting the acid yield and substrate conversion rate data of each micropore, and screening out elite strains; fermenting elite strains, and dynamically feeding materials by an online monitoring and feedback control system; the yeast strain is subjected to precise transformation through codon optimization and gene overexpression driven by a strong promoter; elite strains are quickly identified by adopting high-throughput screening in cooperation with miniaturized parallel fermentation and real-time optical detection; in the fermentation process, a self-control feedback fed-batch technology is introduced, and a culture environment is stabilized in an optimal physiological state through real-time monitoring and dynamic regulation, so that the production potential of engineering bacteria is fully exerted, and the yield of adenosine triphosphate, the substrate conversion rate and the product purity are improved.

The application belongs to the technical field of molecular biology, and particularly relates to metabolic markers for distinguishing hypertrophic cardiomyopathy and left ventricular hypertrophy and application. The application analyzes the metabolome of hypertrophic cardiomyopathy (HCM) patients, left ventricular hypertrophy (LVH) patients caused by non-hypertrophic cardiomyopathy and healthy people, finds that carnitine C16:2, carnitine C14:0, carnitine C16:0, carnitine C18:0 and carnitine C18:1 are differentially expressed in HCM patients and LVH patients, pyridoxine phosphate, ito acid, adenosine monophosphate, adenosine diphosphate and adenosine triphosphate are differentially expressed in HCM patients and healthy people, lysine base, methyl glutamic acid, acetyl proline, malate semialdehyde and citramalate are differentially expressed in LVH patients and healthy people, and ROC curve analysis shows excellent prediction effect, strong specificity and high sensitivity.

The invention provides a beta-galactosidase compound and a method. The beta-galactosidase compound comprises beta-galactosidase and cyclic adenosine monophosphate combined with amino acid residues of the beta-galactosidase. According to the invention, small molecular cyclic adenosine monophosphate acts on lactase molecules, and the catalytic activity of lactase is significantly enhanced through unique intermolecular interaction. Compared with a traditional lifting means, the method has the advantages that the lifting efficiency is higher, an ideal lactase activity level can be achieved in a shorter time, and a powerful guarantee is provided for lactose digestion and utilization.

This disclosure relates to the use of glycogen phosphatase B agonists in the treatment of neurodegenerative diseases. Specifically, this disclosure provides the use of glycogen phosphatase B agonists (preferably adenosine monophosphate) in the preparation of medicaments for the prevention or treatment of neurodegenerative diseases. This disclosure demonstrates that glycogen phosphatase B agonists (AMPs) hold promise as a safe and effective treatment option for patients with neurodegenerative diseases.

The invention belongs to the field of insecticidal and acaricidal agents, and relates to an insecticidal and acaricidal composition. The composition contains an active component A and an active component B, and the weight part ratio of the active component A to the active component B is (1: 99)-(99: 1); the active component A is selected from a compound I; the active component B is selected from inhibitors influencing the growth of chitin synthetase 1 (CHS1) mites, mitochondrial adenosine triphosphate (ATP) synthetase inhibitors, mitochondrial electron transfer complex (III) inhibitors, mitochondrial electron transfer complex (I) inhibitors, acetyl-CoA carboxylase inhibitors and mitochondrial electron transfer complex (II) inhibitors; a trifluoroethyl sulfide type acaricide or other types of insecticidal and acaricide; the composition disclosed by the invention has the advantages of obvious synergism, resistance delay and the like, and can be used for preventing and treating various pests.

The invention relates to non-naturally occurring mutated arylsulfotransferases comprising (i) an amino acid substitution in at least one amino acid position selected among positions 6, 7, 8, 9, 11, 17, 20, 33, 62, 97, 138, 195, 236, 239, 244, 263, and combinations thereof, wherein the position is relative to the amino acids sequence of rat arylsulfotransferase IV SEQ ID NO: 1 and (ii) an amino acid sequence having at least 60% sequence identity with amino acids sequence SEQ ID NO: 1. The mutated arylsulfotransferase may have a sulfotransferase activity for converting adenosine 3′,5′-bisphosphate (PAP) into 3′-phosphoadenosine-5′-phosphosulfate (PAPS) enhanced compared to the wild-type enzyme.

The invention relates to a traditional Chinese medicine composition for treating ischemic heart failure and a preparation method thereof, and belongs to the field of traditional Chinese medicine pharmacy, and the traditional Chinese medicine composition is prepared from the following raw material medicines in parts by weight: 5-15 parts of cassia twig, 5-15 parts of bighead atractylodes rhizome, 5-15 parts of poria cocos, 6-12 parts of rhizoma zingiberis, 5-15 parts of acanthopanax, 10-30 parts of fructus polygoni orientalis, 3-9 parts of verbena, 6-12 parts of dogwood and 3-9 parts of honey-fried licorice root. The composition can remarkably reduce the level of N-terminal proB-type natriuretic peptide, remarkably improve pathological changes of heart tissues of model rats, regulate and control the heart functions of the model rats, improve the systolic function of the heart, relieve expression of inflammatory factors such as interleukin-1beta, interleukin-6 and tumor necrosis factor-alpha of the rats and improve the anti-inflammatory effect of the rats. The action mechanism of the compound relates to a plurality of pathways such as extracellular matrix receptor interaction, a chemotactic factor signal pathway, a relaxin signal pathway, a cyclic adenosine monophosphate signal pathway, a nuclear factor-kB signal pathway, fatty acid metabolism and a Toll-like receptor signal pathway, and the compound is an effective medicament for treating ischemic heart failure.

The present invention belongs to the technical field of preparing functional materials for molecular recognition, adsorption and separation, and discloses a method for preparing a hydrophilic dual-receptor MOFs nanosheet surface imprinted adsorbent and its application in the selective separation of adenosine monophosphate. The present invention targets the structural characteristics of a typical nucleoside compound adenosine monophosphate (AMP) molecule, designs and synthesizes Zr-MOFs nanosheets coordinated with its specific metal ion as a matrix material, and uses 5-(2-methoxyvinyl)-2′-deoxyuridine (AcrU) as a functional monomer that undergoes specific base complementary pairing. The arrangement and orientation of the template molecules are precisely fixed through an ordered assembly strategy. Finally, the large number of double bonds modified on the surface of the Zr-MOFs nanosheets and a hydrophilic cross-linking agent are utilized to photoinitiate the construction of a stable hydrophilic dual-receptor MOFs nanosheet surface imprinted adsorbent (D-MIPs), thereby enhancing the recognition and separation effect of nucleoside compounds and achieving the selective separation and enrichment of nucleoside compounds such as AMP in biological samples such as urine and blood.

The present invention relates to the field of biosynthesis technology, and more particularly to a β-nicotinamide mononucleotide production system and a method for preparing β-nicotinamide mononucleotide in a dual-enzyme collaborative manner. The β-nicotinamide mononucleotide production system comprises an NRK-HS01 whole-cell catalyst, an ADK whole-cell catalyst, nicotinamide riboside, adenosine triphosphate, and MgCl2. The present invention couples the NRK-HS01 whole-cell catalyst with an ATP regeneration system (ADK whole-cell catalyst) to catalyze the conversion of nicotinamide riboside and adenosine triphosphate to produce NMN in a single step. This method achieves an NMN molar yield exceeding 99%, reduces ATP costs by 50%, and reduces feedback inhibition of the synthesis reaction caused by the accumulation of the byproduct ADP. This method also enables the recycling of ATP, thereby improving synthesis efficiency and saving production costs. This method is of great significance for the industrial development of biosynthesis of NMN.

The invention discloses a recombinant plant lactobacillus for overexpressing an adenosine triphosphate binding cassette transporter protein gene and application of the recombinant plant lactobacillus. Belongs to the technical field of bioengineering. The invention aims to improve the cold-resistant growth capability of the plant lactobacillus, increase the capability of producing lactic acid and degrading nitrite, and improve the integrity and permeability of the plant lactobacillus cells. The invention provides a recombinant lactobacillus plantarum, which is characterized in that the lactobacillus plantarum is used as an original strain, and a nucleic acid molecule as shown in SEQ ID NO.1 is overexpressed. The method is used for fermenting products in food industry.

The application discloses a salt-tolerant adenylate kinase mutant and application thereof. Through modification on an amino acid sequence of a wild-type adenylate kinase (adenylate kinase, EC 2.7.4.3) from Bacillus subtilis (strain 168), three salt-tolerant adenylate kinase mutants are obtained; the mutant adenylate kinase has higher salt tolerance compared with the wild-type adenylate kinase, and can tolerate a salt concentration of 300-2000 mmol / L in an adenosine diphosphate reaction system.

The invention belongs to the technical field of biological detection and analysis, and particularly relates to an adenosine phosphate photoaffinity probe as well as a preparation method and application thereof. The probe comprises an AMP-N (adenosine monophosphate-N) photoaffinity probe, an ADP-N (adenosine monophosphate-N) photoaffinity probe and an ATP-N (adenosine monophosphate-N) photoaffinity probe, the phosphoric acid part of adenosine monophosphate is modified with a photoaffinity group diaziridine with a small volume and alkynyl capable of carrying out a biological orthogonal reaction, and a connecting bond capable of cracking under an acidic condition is designed between adenosine monophosphate and a photoreactive group. The probe is high in stability and good in marking efficiency, can specifically mark the adenosine phosphate binding protein and identify the binding site of the adenosine phosphate binding protein, is suitable for screening, functional research and affinity analysis of the adenosine phosphate binding protein and discovery and verification of related drug targets, and has a wide application prospect.

The application provides an adenosine triphosphate and nitric oxide dual-response near-infrared fluorescent probe and a preparation method and application thereof. The near-infrared fluorescent probe has the structure shown in the following formula I, is responsive to both adenosine triphosphate and nitric oxide, and has high sensitivity and selectivity.

The application discloses a cyclic GMP-AMP synthetase mutant and a method for preparing 2',3'-cyclic guanosine monophosphate-adenosine monophosphate by using the cyclic GMP-AMP synthetase mutant, wherein the amino acid sequence of the cyclic GMP-AMP synthetase mutant has any one of the following mutations: P361A, L377M, K432R, S221Y / F357L / I496V, S221L / Y248F / K432H / S434T, F357L / P361A / L377M / S434T, F357L / P361A / S434T / H437T or H437T / S434T, compared with a wild type as shown in SEQ ID NO. 1. The activity of the cGAS mutant of the application can reach 4 times of the wild type, raw materials of the preparation method are economical and easy to obtain, the operation is simple, green, environment-friendly, pollution-free, the synthesis efficiency is high, and industrial development is easy.

The invention belongs to the technical field of biosensing and underwater detection, and particularly relates to an underwater adenosine triphosphate detection system and method. According to the system, a nanogold modified screen-printed electrode is used as a sensing platform, based on an electrochemical aptamer sensing principle, an ATP specific nucleic acid aptamer is fixed in an Au-S bond self-assembly mode to form a molecular recognition interface, and when ATP is combined with the aptamer, characteristics such as interface electron transfer resistance are changed, so that the ATP specific nucleic acid aptamer is identified. The high-sensitivity and high-selectivity detection of ATP is realized by detecting the peak current change of the electroactive reporter molecule; meanwhile, an epoxy resin-silicone rubber-silane coupling agent composite packaging system is adopted, a compact protection layer is formed in a non-working area of the electrode through a stepped heating curing process, and the three parts cooperate to ensure the sealing performance and long-term stability in an underwater environment, so that underwater in-situ detection in a real sense is realized; the target object degradation in the sample collection and storage process is avoided. The method has important value in the fields of marine environment monitoring, underwater biological research and the like.

The invention belongs to the field of insecticides and acaricides, and relates to a composition containing alanine-isoxazoline compounds and acaricides. The composition contains an active component A and an active component B, wherein the weight part ratio of the active component A to the active component B is (1: 100)-(100: 1); the active component A is selected from a compound I, a compound II or a stereoisomer thereof; the active component B is selected from inhibitors influencing the growth of chitin synthetase 1 (CHS1) mites, mitochondrial adenosine triphosphate (ATP) synthetase inhibitors, mitochondrial electron transfer complex (III) inhibitors, mitochondrial electron transfer complex (I) inhibitors, acetyl-CoA carboxylase inhibitors and mitochondrial electron transfer complex (II) inhibitors; a trifluoroethyl sulfide type acaricide or other types of insecticidal and acaricide; the composition disclosed by the invention has the advantages of obvious synergism, resistance delaying and the like, and can be used for preventing and treating various pests.

The invention discloses a method for detecting antibacterial and antiviral effects, which is suitable for detecting the antibacterial and antiviral effects of the surface of a material to be detected. The bacteriostatic and antiviral effect detection method comprises the following steps: providing a plurality of samples to be detected, the samples to be detected are made of materials to be detected, and each sample to be detected is provided with a surface to be detected; respectively coating probiotics on the to-be-detected surfaces, and detecting the to-be-detected surfaces by using an adenosine triphosphate (ATP) measuring device to generate a plurality of original values; placing the to-be-detected samples in a constant temperature environment for a first time, and detecting the to-be-detected surface by using the adenosine triphosphate measuring device to generate a plurality of first detection values; calculating a plurality of first suppression values by using the first detection values and the original values; and confirming the antibacterial and antiviral effects of the surface of the to-be-detected material according to the first detection values.

The present invention addresses the problem of providing: a composition that increases the amount of intracellular ATP and the amount of intracellular NAD; and a composition that improves motor performance. More specifically, the present invention provides a composition for increasing the amounts of intracellular adenosine triphosphate (ATP) and intracellular nicotinamide adenine dinucleotide (NAD), the composition being obtained by combining (a) and (b): (a) an NAD precursor or a pharmaceutically acceptable salt thereof; and (b) one or more substances selected from the group consisting of uridine and uridine precursors, or one or more pharmaceutically acceptable salts thereof, and / or one or more substances selected from the group consisting of inosine and inosine precursors, or one or more pharmaceutically acceptable salts thereof.　Furthermore, the present invention provides a composition for enhancing muscle strength, the composition being characterized by inclluding a combination of (c) and (d): (c) one or more substances selected from the group consisting of β-alanine and β-alanine precursors, or one or more pharmaceutically acceptable salts thereof; and (d) one or more substances selected from the group consisting of uridine and uridine precursors, or one or more pharmaceutically acceptable salts thereof, and / or one or more substances selected from the group consisting of inosine and inosine precursors, or one or more pharmaceutically acceptable salts thereof.

The application discloses a method for preparing 2'-methoxy-uridine triphosphate, which comprises the following steps: taking 2'-methoxy-uridine acid as a substrate, taking adenosine triphosphate as an initial phosphate donor, constructing a phosphate circulation system, adding an enzyme catalyst, and adopting one-pot enzyme method to perform enzyme catalysis reaction, so as to obtain the end product 2'-methoxy-uridine triphosphate. The phosphate circulation system is composed of adenosine triphosphate, adenosine diphosphate and phosphate compounds. The enzyme catalyst catalyzes the dephosphorylation of the phosphate compounds to provide inorganic phosphate to the adenosine diphosphate, and the adenosine triphosphate is regenerated to realize the circulation energy supply. The method adopts high-activity enzyme combination of specific sources, has high conversion rate of the substrate 2'-methoxy-uridine acid, and has high conversion rate of the final 2'-methoxy-uridine triphosphate up to 92.6%, and has the advantages of high synthesis efficiency, simple steps, green environmental protection and controllable cost.

The invention discloses a method for visually detecting various phosphates, which comprises the following steps: firstly, synthesizing three MOF (Metal Organic Framework) nano-enzymes, namely PCN-222 (Fe), PCN-223 (Fe) and PCN-224 (Fe) with peroxidase-like activity, catalyzing a colorless substrate 3, 3 ', 5, 5'-tetramethyl benzidine (TMB) into oxTMB in a blue oxidation state by the three MOF nano-enzymes in the presence of H2O2, and generating remarkable colorimetric response; then, the five phosphates, namely ATP (adenosine triphosphate), ADP (adenosine diphosphate), AMP (adenosine monophosphate), PPi (pyrophosphoric acid) and Pi (inorganic phosphate), are simultaneously distinguished and detected by utilizing the principle that different phosphates have different regulation degrees on the peroxidase-like activity of the MOF nano-enzyme. In combination with a smartphone application program 'ColorMax', real-time visual analysis of phosphate is realized, and an efficient and convenient method is provided for distinguishing and detecting different phosphates.

The invention relates to a property-changed glucose-1-adenosine monophosphate transferase mutant and application thereof, and belongs to the technical field of enzyme engineering. The glucose-1-phosphoadenylyltransferase mutant is subjected to mutation and deletion of different sites, the property of the obtained glucose-1-phosphoadenylyltransferase mutant is changed, particularly, the thermal stability of the glucose-1-phosphoadenylyltransferase mutant is improved, the residual enzyme activity of the glucose-1-phosphoadenylyltransferase mutant at 30 DEG C is remarkably improved, and the residual enzyme activity of the glucose-1-phosphoadenylyltransferase mutant at 65 DEG C is also greatly improved. Therefore, in the process of catalyzing glucose-1-phosphate to generate ADP-glucose, the glucose-1-phosphate adenylyl transferase mutant disclosed by the invention can tolerate a high-heat environment generated by thallus fermentation, and has wide application value in industrial application.

The present disclosure relates to uses and methods implementing a cell free extract (CFE) having an activity for converting adenosine 2',3'-cyclic phosphate 5'-phosphosulfate (cyclo-PAPS) into 3'-phosphoadenosine-5'-phosphosulfate (PAPS) useful for sulfating a substrate.

The present invention discloses a recombinant Lactobacillus plantarum that overexpresses an ATP-binding cassette transporter protein gene and its application. This relates to the field of bioengineering technology. The present invention aims to improve the cold-resistant growth ability of Lactobacillus plantarum, increase its ability to produce lactic acid and degrade nitrite, and improve the integrity and permeability of Lactobacillus plantarum cells. Lactiplantibacillus plantarum ), using Lactobacillus plantarum as the starting strain, overexpressing the nucleic acid molecule shown in SEQ ID NO. 1. It is used for fermentation products in the food industry.

The invention relates to a 3 '-phosphoadenosine-5'-phosphoryl sulfuric acid biosynthesis system based on ATP (adenosine triphosphate) regeneration and enzyme immobilization and application. The invention firstly provides an enzyme compound which comprises ATP (adenosine triphosphate) regeneration enzyme and PAPS (Phosphatidylcholine) synthetase; the ATP regeneration enzyme is immobilized PPK labeled by streptavidin-biotin; the PAPS synthetase is composed of immobilized ATPS labeled by streptavidin-biotin, APSK and PPA, and the immobilized ATPS labeled by streptavidin-biotin, the APSK and The mass ratio of the ATP regeneration enzyme to the PAPS synthetase is (1-1.5): 2. The invention provides a method for synthesizing 3 '-adenosine-5'-phosphoryl sulfuric acid by using an enzyme compound, so that high-efficiency and low-cost synthesis of PAPS is realized, and the problems of high cost and poor conversion efficiency of a traditional PAPS synthesis method are effectively solved. The invention further provides a 3 '-adenosine-5'-phosphoryl sulfuric acid biosynthesis system based on ATP regeneration and enzyme immobilization on the basis of the enzyme compound, and the system utilizes yeast cells as an ATP supply source to realize efficient conversion from adenosine to ATP.

This invention discloses a liver-protective composition containing burdock root aqueous extract, its preparation method, and its application. The composition comprises six traditional Chinese medicinal herbs: burdock root aqueous extract, jujube seed extract, lily extract, wolfberry extract, white chrysanthemum extract, and poria cocos extract; seven nutrients: vitamin D3, vitamin E, inositol, choline, pyrrolizidine quinoline quinone, magnesium glycine, and zinc citrate; and three flavoring agents: semaside, mogroside, and adenosine monophosphate (AMP). AMP has both umami enhancement and AMPK activation effects. The composition is prepared into powder or tablets using low-temperature freeze-drying or low-temperature spray drying. The composition of this invention has a significant protective effect against chemically induced liver damage and sleep deprivation-induced liver damage, and is suitable for liver protection and health maintenance in people who stay up late, overuse their eyes, experience high mental stress, and are middle-aged and elderly.