This is a working overview of freeze-thaw, written for readers who want more than a one-paragraph summary but less than a textbook.
This page was last updated on 2025-09-24 and is reviewed periodically as new material appears.
Nicotinamide adenine dinucleotide, commonly abbreviated NAD+, is a dinucleotide coenzyme built from an adenine nucleotide and a nicotinamide nucleotide joined by a pyrophosphate linkage. Its oxidized form carries a positive charge on the nicotinamide ring, while the reduced form, NADH, carries a hydride equivalent. The molecule participates in hundreds of oxidoreductase reactions, where it accepts or donates electrons and protons. Because it can cycle between oxidized and reduced states without net consumption, NAD+ functions as a reusable electron carrier rather than a fuel molecule.
In glycolysis, the tricarboxylic acid cycle, and fatty acid oxidation, NAD+ is reduced to NADH at specific dehydrogenase steps. NADH then delivers electrons to the mitochondrial electron transport chain, mainly at complex I, supporting oxidative phosphorylation and ATP production. The balance between NAD+ and NADH, often expressed as a ratio, influences metabolic flux and redox homeostasis in different cellular compartments. Cytosolic and mitochondrial pools are connected but not identical, and their ratios can differ substantially because of compartment-specific enzymes and transport systems.
In redox reactions, NAD+ accepts a hydride ion, which consists of two electrons and one proton. The hydride adds to the nicotinamide ring at a specific carbon, converting NAD+ into NADH. Dehydrogenase enzymes use this step in glycolysis, the citric acid cycle, and fatty acid oxidation. NADH later donates electrons to the mitochondrial electron transport chain, helping to drive ATP synthesis. The balance between NAD+ and NADH reflects the metabolic state of a cell, and shifts in that balance can alter how pathways operate.
Beyond electron transfer, NAD+ serves as a substrate for enzymes that cleave it and attach its ADP-ribose portion to other molecules. This group includes poly(ADP-ribose) polymerases, CD38, and sirtuins. Such reactions consume NAD+ and can influence its availability for metabolism. Cells replenish NAD+ through a salvage pathway that recycles nicotinamide and through routes starting from tryptophan or vitamin B3 forms. How these synthesis and consumption routes are coordinated across tissues remains an active area of study, and compartment-specific concentrations are difficult to measure directly.
Nicotinamide adenine dinucleotide, abbreviated NAD+, is a dinucleotide built from adenine, nicotinamide, two ribose sugars, and two phosphate groups. The oxidized form carries a positive charge on the nicotinamide ring, while the reduced form, NADH, is neutral. This pair acts as a reversible electron carrier in cells. NAD+ is present in bacteria, plants, animals, and fungi. Its structure allows it to accept and donate electrons without being consumed in the reactions it supports.
| Property | Value | Notes |
|---|---|---|
| Common synonyms | β-NAD+, coenzyme I, DPN | DPN stands for diphosphopyridine nucleotide; older literature uses this term. |
| CAS Registry Number | 53-84-9 | Free acid form of β-nicotinamide adenine dinucleotide. |
| Molecular formula | C21H27N7O14P2 | Anhydrous free acid; molar mass 663.43 g/mol. |
| Appearance | White to off-white powder | Crystalline solid; may absorb moisture from air. |
| Solubility | Freely soluble in water | Insoluble in most nonpolar organic solvents. |
Biosynthesis of NAD+ starts from nicotinamide, nicotinic acid, or nicotinamide riboside through salvage pathways. A rate-limiting enzyme, nicotinamide phosphoribosyltransferase, converts nicotinamide to nicotinamide mononucleotide. Further coupling with ATP yields NAD+. In mammals, the liver and muscle can synthesize NAD+ from dietary precursors, but tissue levels vary widely. Researchers study these pathways to understand age-related changes, metabolic disorders, and neurodegeneration. Direct causal links between NAD+ decline and disease remain an active area of investigation.
NAD+ is a dinucleotide composed of adenine, ribose, and nicotinamide groups joined by phosphate linkages. It serves as a coenzyme in oxidoreductase reactions, cycling between oxidized NAD+ and reduced NADH. The molecule is water-soluble and occurs in all living cells. Its nicotinamide ring accepts hydride ions during catabolic reactions, linking substrate oxidation to electron transport. This redox couple supports ATP production and helps maintain cytosolic and mitochondrial redox balance in many cell types.
Beyond redox catalysis, NAD+ is a substrate for enzymes that transfer ADP-ribose or remove acetyl groups from proteins. Sirtuins and poly(ADP-ribose) polymerases consume NAD+ and release nicotinamide as a byproduct. These reactions connect cellular energy status to gene regulation, DNA repair, and stress responses. Because NAD+ is used rather than merely recycled in such signaling, its concentration reflects both biosynthesis and consumption. The balance between salvage and de novo synthesis pathways determines available pools in different tissues.
Laboratory handling of NAD+ follows standard practices for hygroscopic fine chemicals. Personnel typically avoid inhalation and skin contact, use gloves and eye protection, and work in a ventilated area. Quality control may include ultraviolet absorbance at the nicotinamide maximum, chromatographic purity, water content, and identity confirmation by mass spectrometry. Because commercial preparations can contain counterions, residual solvents, or related nucleotides, a certificate of analysis helps verify the material. Researchers should confirm that the form supplied matches the intended assay.
Measuring NAD+ in biological samples requires care because the molecule is chemically reactive and present at low concentrations in some tissues. Common approaches include enzymatic cycling assays, high-performance liquid chromatography, and liquid chromatography coupled to mass spectrometry. Each method has different sensitivity and specificity, and sample preparation can affect results. Acidic or alkaline extraction steps are used in some protocols, but the choice depends on the analyte and matrix. No single method is universally optimal for every tissue or fluid.
The stability of NAD+ depends on pH, temperature, light exposure, and the presence of degradative enzymes. Aqueous solutions are generally more stable under mildly acidic to neutral conditions and degrade faster under alkaline conditions or prolonged heat. The solid is hygroscopic and should be stored desiccated, often frozen, and protected from repeated freeze-thaw cycles. In laboratory handling, aliquots reduce repeated temperature changes, and chelating agents may limit metal-catalyzed hydrolysis in some buffers. These practices matter because even small amounts of NADH or hydrolysis products can interfere with quantitative assays.
Quality control for NAD+ materials typically combines identity, purity, and water content checks. Identity may be confirmed by ultraviolet spectrum, retention time in chromatography, or mass accuracy, while purity is assessed by HPLC peak area or quantitative nuclear magnetic resonance. Residual water and solvents can affect molar calculations and enzyme assays, so Karl Fischer titration or thermogravimetric analysis may be used. Commercial materials vary in grade and counterion form, and published methods should specify the exact salt or hydrate when reporting concentrations. Regulatory status depends on intended use, with research reagents, dietary ingredients, and clinical products treated under different frameworks.
Quantification of NAD+ in biological samples usually relies on separation techniques coupled to sensitive detection. High-performance liquid chromatography with ultraviolet detection can measure the oxidized form by its absorbance near 260 nm, while mass spectrometry provides greater specificity and can distinguish NAD+ from close analogs. Enzymatic cycling assays use coupled dehydrogenase reactions to amplify signal and estimate NAD+ concentrations in cell or tissue extracts. Because NAD+ and NADH interconvert rapidly, sample preparation must quench metabolism quickly and preserve the redox state before analysis.
Quality control for NAD+ relies on identity, purity, and functional tests. A certificate of analysis may report high-performance liquid chromatography purity, ultraviolet spectrum, water content, and residual solvents. Because NAD+ is hygroscopic, gravimetric values can shift as material absorbs water, so purity should be interpreted alongside storage history. Mass spectrometry confirms molecular identity, while enzymatic assays show whether the material supports dehydrogenase activity. Commercial material is available as the free acid and as salts, and the counterion affects molecular weight, solubility, and how concentrations are calculated.
Laboratory measurement of NAD+ usually begins with rapid sample quenching because the molecule can change form after collection. Enzymatic cycling assays amplify signal through coupled reactions and are suited to small samples. High-performance liquid chromatography with ultraviolet detection separates NAD+ from related nucleotides. Liquid chromatography-mass spectrometry offers higher specificity and can distinguish NAD+ from close analogs. Each method has trade-offs in sensitivity, throughput, and equipment needs, so reported values depend heavily on extraction and detection choices.
Monosynaptic reflex A reflex arc that involves only one synapse between a sensory neuron and a motor neuron, such as the knee-jerk (patellar) reflex. Mossy fiber A type of excitatory input fiber to the cerebellum originating from the spinal cord and brainstem nuclei. They synapse onto granule cells. Motor cortex A region of the frontal lobe involved in planning, controlling, and executing voluntary movements. Motor neuron A type of neuron that transmits signals from the central nervous system to muscles or glands, enabling movement and action. Motor unit A motor neuron and all the muscle fibers it innervates. Varies in size depending on the precision required by the muscle. MRI spectroscopy A specialized MRI technique that measures concentrations of certain brain chemicals or metabolites, aiding in diagnosis of tumors and metabolic disorders. Multisensory integration The process by which the brain combines information from different sensory modalities (e.g., vision and sound) to create coherent perception. Myelin A fatty substance that insulates axons, increasing the speed and efficiency of action potential conduction. Formed by oligodendrocytes in the CNS and Schwann cells in the PNS. Myelination The process of forming a myelin sheath around axons, critical for normal neural function. Occurs progressively during development. Myoclonus Sudden, involuntary muscle jerks or twitches. Can be physiological (e.g., sleep starts) or associated with neurological disorders.
West Berlin, while still under occupation by the Western Allies, viewed itself as part of the Federal Republic and was largely integrated and considered a de facto state. In 1952, following a referendum, Baden, Württemberg-Baden, and Württemberg-Hohenzollern merged into Baden-Württemberg. In 1957, the Saar Protectorate joined the Federal Republic as the state of Saarland. The next major change occurred with German reunification in 1990, in which the territory of the former German Democratic Republic (East Germany) became part of the Federal Republic, by accession of the re-established eastern states of Brandenburg, Mecklenburg-West Pomerania (Mecklenburg-Vorpommern), Saxony (Sachsen), Saxony-Anhalt (Sachsen-Anhalt), and Thuringia (Thüringen), and the reunification of West and East Berlin into a city state. A referendum in 1996 to merge Berlin with surrounding Brandenburg failed to reach the necessary majority vote in Brandenburg, while a majority of Berliners voted in favour.
The release of alarm pheromones near a hive may attract other bees to the location, where they will likewise exhibit defensive behaviors until there is no longer a threat, typically because the victim has either fled or been killed. (Note: A bee swarm, seen as a mass of bees flying or clumped together, is generally not hostile; it has deserted its hive and has no comb or young to defend.) These pheromones do not dissipate or wash off quickly, and if their target enters the water, bees will resume their attack as soon as it leaves the water. The alarm pheromone emitted when a bee stings another animal smells like a banana. Drone bees, the males, are larger and do not have stingers. The female bees (worker bees and queens) are the only ones that can sting, and their stinger is a modified ovipositor. The queen bee has a barbed but smoother stinger and can, if need be, sting skin-bearing creatures multiple times, but the queen does not leave the hive under normal conditions. Her sting is not for the defense of the hive; she only uses it for dispatching rival queens, ideally before they can emerge from their cells. Queen breeders who handle multiple queens and have the queen odor on their hands are sometimes stung by a queen. The stinger consists of three parts: a stylus and two barbed slides (or lancets), one on either side of the stylus. The bee does not push the stinger in but it is drawn in by the barbed slides.
=== Domestic use === When a sanitary potable water supply is plumbed to an unsanitary system, for example lawn sprinklers, a dish washer or a washing machine, a check valve called a backflow preventer is used to prevent contaminated water from re-entering the domestic water supply. Some types of irrigation sprinklers and drip irrigation emitters have small check valves built into them to keep the lines from draining when the system is shut off. Check valves used in domestic heating systems to prevent vertical convection, especially in combination with solar thermal installations, also are called gravity brakes. Rainwater harvesting systems that are plumbed into the main water supply of a utility provider may be required to have one or more check valves fitted to prevent contamination of the primary supply by rainwater. Hydraulic jacks use ball check valves to build pressure on the lifting side of the jack. Check valves are commonly used in inflatables, such as toys, mattresses and boats. This allows the object to be inflated without continuous or uninterrupted air pressure.
Sources: en.wikipedia.org
On 17 May 2022, militias attacked a civilian helicopter that was flying over the Rio area with rifles. No injuries were recorded but the helicopter suffered slight damage. On 20 August of the same year, Delson Lima Neto, brother of one of the leader of the militias, Tandera Danilo Dias Lima, was killed alongside 3 paramilitaries soldiers by the Polícia Militar on Nova Iguaçu, in Baixada Fluminense, after his death the Comando Vermelho conquered the favela of Grão-Pará, in Nova Iguaçu. After the raid, on 23 August, the police found and seized an improvised fighting vehicle (similar to a Caveirão) used by the militias to fight against Comando Vermelho in Nova Iguaçu. According to law student Carlos Gilberto Martins Junior of Brazil in 2023, with emphasis on the State of Rio de Janeiro, there has been an arbitrary use of these powers and attributions, conferred on police institutions, to satisfy the patrimonial aspirations of some of its agents, through territorial domination and violence, to the detriment of the peripheral communities and under the pretext of saving them from the "greater evil" represented by drug trafficking, corroborating the emergence of criminals organizations what conventionally called the "militia". On 6 May 2021, at least 29 people were killed in a shootout between gangsters of Comando Vermelho and police. On May 24 2022, the BOPE raided the Vila Cruzeiro favela in Penha Complex, killing 26 people between civilians and gangsters of Comando Vermelho.
=== Scintigraphy/radionuclide imaging === Scintigraphy can be used to measure the extent and distribution of the amyloid throughout the body, including the liver, kidney, spleen, and heart. A radiolabelled serum amyloid P component can be administered to a patient intravenously and the P component pools to the amyloid deposit proportional to the size of the deposit. The labeling of the P component can then be pictured by a gamma camera. Technetium radionuclide scans can now reliably diagnosis cardiac amyloidosis, with certain scanning methods having greater than 99% sensitivity (but only 91% specific for amyloidosis). In this method of imaging, radiolabeled technetium is injected into the body where it binds to cardiac amyloid deposits. A subsequent scan is taken to determine where the tracer stays, therefore highlighting the amyloid deposition in the heart. This method allows for a noninvasive definitive diagnosis of cardiac amyloidosis (as in the past an endomyocardial biopsy was required)
Early grocery stores were "austere" and tiny by modern standards, with as few as 450 items. Shopping for groceries often involved trips to multiple specialty shops, such as a greengrocer, butcher, bakery, fishmonger and dry goods store, in addition to a general store. Milk and other items of short shelf life were delivered by a milkman. These small retailers were the final links in a "long and tortuous food chain," as most of them were far too small to deal directly with most of the persons who actually harvested, processed, and distributed all that food. During the 1920s, the highly inefficient nature of the American food distribution system meant that the "average urban family spent fully one-third of its budget on food". One of the most important defining features of the modern supermarket is cheap food. The vast abundance of cheap, wholesome food which modern consumers take for granted today was simply unimaginable before the middle of the 20th century, to the point that the first American supermarket customers in the 1930s were overcome with emotion at the sight of so much cheap food. Before the 20th century, food was neither cheap, nor wholesome, nor abundant. For example, in 1812, almost 90 percent of Americans worked in food production, and they struggled to stay alive on food which was often scarce, of poor quality, and riddled with diseases which could and did often kill them.
Afluria (influenza vaccine) -- Argentina, Peru, South Africa, Spain, US Enzira—in various different markets Fluvax—in various different markets Nilgrip—in various different markets Afluria Quadrivalent (influenza vaccine) -- Australia, Canada, New Zealand, US Afluria Quad—in various different markets Afluria Tetra—in various different markets Agrippal (influenza vaccine) -- Argentina, Austria, Brazil, Canada, Chile, Colombia, Germany, Italy, Mexico, Spain, Thailand Agriflu—in various different markets Begripal—in various different markets Chiroflu—in various different markets Fluazur—in various different markets Sandovac—in various different markets Audenz (influenza A (H5N1) vaccine) -- US Fluad (influenza vaccine) -- Argentina, Austria, Australia, Brazil, Canada, Denmark, Germany, Italy, Spain, Switzerland, UK, US Chiromas—Spain Fluad Pediatric (influenza vaccine) -- Canada Flucelvax Quadrivalent (influenza vaccine) -- Germany, Italy, Spain, UK, US Q-VAX (Coxiella burnetii vaccine) -- Australia Rapivab (peramivir) -- Australia, US Antivenoms: (Australia)
Wagyu (Japanese: 和牛, Hepburn: wagyū, lit. 'Japanese cattle') is not the name of a single cattle breed. Today, the term usually refers to four established Japanese breeds of beef cattle: the Japanese Black, Japanese Brown, Japanese Polled, and Japanese Shorthorn. Historically, it was also used for native Japanese cattle; the surviving Mishima and Kuchinoshima cattle populations are not included among the four modern breeds. Beginning in the late nineteenth century, these breeds were developed from regional native cattle crossed with imported stock. The timing of crossbreeding and the imported breeds involved varied by breed and region. The resulting cattle were later established as distinct breeds through selection and closed breeding. The Japanese Black accounts for more than 90% of Wagyu cattle raised in Japan and is the main source of the highly marbled beef commonly associated with Wagyu. Its beef has finely distributed intramuscular fat known as sashi, a high proportion of monounsaturated fatty acids, and a distinctive sweet aroma that develops during cooking. The other three breeds generally produce less heavily marbled beef. Regional names such as Kobe beef, Matsusaka beef, Ōmi beef, and Yonezawa beef refer to brands defined by geographical origin and production or quality standards, not to additional cattle breeds. In Japan, beef carcasses are graded separately for meat yield and meat quality.
Sources: en.wikipedia.org
=== 2008–2009: Beginnings === Riff Raff started rapping in 2005, making homemade CDs of his rapping over other artists' beats, and passing them out at various malls in the Greater Houston Area including Katy Mills Mall. He credited his early influences being Texas veteran rappers Devin the Dude and Paul Wall. Riff Raff took advantage of social media on Myspace, YouTube and WorldStarHipHop in late 2008, releasing songs, freestyle-videos, and sketch clips, which showcased his talent as slapstick and performance art. He did this with the help of his first talent manager, DB da Boss, whose studio provided recording and video services. DB spoke with LA Weekly about the rapper, saying "He was very driven, very ambitious. You couldn't deny his work ethic", going on to say, "Underneath that shell is a very intelligent man." He looked up to Northside Houston rappers from label Swishahouse, who were blowing up around that time, including Paul Wall, Chamillionaire and Slim Thug. He began doing his hair up in braids popular among "Northsiders" like Slim Thug and shopping for bling at TV Jewelry, a store co-owned by Paul Wall and charismatic Vietnamese immigrant "TV" Johnny Dang, from whom Riff Raff also purchased gold grills. Like many other artists from Houston, Riff Raff also spent much of his time selling CDs at colleges and malls, occasionally alongside other local talent such at Fat Tony and Freestyle Bully. In an effort to get on television he went on many auditions, including for a part in a peanut butter commercial intended for a teenager.
=== Aversion therapy === Aversion therapy in alcoholism had its roots in Russia in the early 1930s, with early papers by Pavlov, Galant and Sluchevsky and Friken, and would remain a strain in the Soviet treatment of alcoholism well into the 1980s. In the US a particularly notable devotee was Dr Voegtlin, who attempted aversion therapy using apomorphine in the mid to late 1930s. However, he found apomorphine less able to induce negative feelings in his subjects than the stronger and more unpleasant emetic emetine.
Castor oil is well known as a source of ricinoleic acid, a monounsaturated, 18-carbon fatty acid. Among fatty acids, ricinoleic acid is unusual in that it has a hydroxyl functional group. Because of this functional group, ricinoleic acid is more polar than most fatty acid. The alcohol group underpins the conversion of ricinoleic acid into sebacic acid and 2-octanol. Because of its ricinoleic acid content, castor oil is a valuable chemical in feedstocks, commanding a higher price than other seed oils. As an example, in July 2007, Indian castor oil sold for about USD $0.90 per kilogram ($0.41/lb), whereas U.S. soybean, sunflower, and canola oils sold for about $0.30 per kilogram ($0.14/lb).
Robertson published his findings in the British Medical Journal in 1916 and, with the help of a few like-minded individuals (including the eminent physician Edward William Archibald), was able to persuade the British authorities of the merits of blood transfusion. Robertson went on to establish the first blood-transfusion apparatus at a Casualty Clearing Station on the Western Front in the spring of 1917. Robertson did not test crossmatching so that one died of hemolysis in his 1916 transfusion, and three in 1917. Oswald Hope Robertson, a medical researcher and U.S. Army officer, was attached to the RAMC in 1917, where he became instrumental in establishing the first blood banks in preparation for the anticipated Third Battle of Ypres. He used sodium citrate as the anticoagulant; blood was extracted from punctures in the vein and was stored in bottles at British and American Casualty Clearing Stations along the Front. Robertson also experimented with preserving separated red blood cells in iced bottles. Geoffrey Keynes, a British surgeon, developed a portable machine that could store blood to enable transfusions to be carried out more easily.
complementary DNA (cDNA) Also copy DNA. DNA that is synthesized from a single-stranded RNA template (typically mRNA or miRNA) in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is produced both naturally by retroviruses and artificially in certain laboratory techniques, particularly molecular cloning. In bioinformatics, the term may also be used to refer to the sequence of an mRNA transcript expressed as its DNA coding strand counterpart (i.e. with thymine replacing uracil).
Sources: en.wikipedia.org
NAD+ is an oxidized dinucleotide coenzyme that carries electrons in metabolic reactions. It is also consumed by signaling enzymes, including sirtuins and PARPs. Its reduced form is NADH.
NAD+ is the oxidized form and can accept a hydride equivalent. NADH is the reduced form and donates electrons to the electron transport chain. The two forms cycle between each other during cellular respiration.
In mammals, NAD+ is synthesized mainly through salvage pathways using nicotinamide, nicotinamide riboside, or nicotinic acid. Tryptophan can also contribute through a de novo route. The salvage pathway is often considered the primary source in many tissues.
It indicates the oxidized form, which has a positive charge on the nicotinamide nitrogen. The reduced partner NADH lacks that charge and carries added electrons. The plus sign is part of the standard abbreviation, not a separate ion.