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Vibrio cholerae

Vibrio cholerae is a species of Gram-negative, facultative anaerobe and comma-shaped bacteria. The bacteria naturally live in brackish or saltwater where they attach themselves easily to the chitin-containing shells of crabs, shrimp, and other shellfish. Some strains of V. cholerae are pathogenic to humans and cause a deadly disease called cholera, which can be derived from the consumption of undercooked or raw marine life species or drinking contaminated water.

V. cholerae was first described by Félix-Archimède Pouchet in 1849 as some kind of protozoa. Filippo Pacini correctly identified it as a bacterium and from him, the scientific name is adopted. The bacterium as the cause of cholera was discovered by Robert Koch in 1884. Sambhu Nath De isolated the cholera toxin and demonstrated the toxin as the cause of cholera in 1959.

The bacterium has a flagellum (a tail like structure)at one pole and several pili throughout its cell surface. It undergoes respiratory and fermentative metabolism. Two serogroups called O1 and O139 are responsible for cholera outbreaks. Infection is mainly through drinking contaminated water or ingestion of food contaminated with faecal matter from an infected person, therefore is linked to sanitation and hygiene. When ingested, it invades the intestinal mucosa which can cause diarrhea and vomiting in a host within several hours to 2–3 days of ingestion. Ringers lactate and Oral rehydration solution combined with antibiotics such as fluoroquinolones and tetracyclines are the common treatment methods in severe cases.

V. cholerae has two circular DNA. One DNA produces the cholera toxin (CT), a protein that causes profuse, watery diarrhea (known as "rice-water stool"). But the DNA does not directly code for the toxin as the genes for cholera toxin are carried by CTXphi (CTXφ), a temperate bacteriophage (virus). The virus only produces the toxin when inserted into the bacterial DNA. Quorum sensing in V. cholerae is well studied and it activates host immune signaling and prolongs host survival, by limiting the bacterial intake of nutrients, such as tryptophan, which further is converted to serotonin. As such, quorum sensing allows a commensal interaction between host and pathogenic bacteria.

Initial observations
During the third global pandemic of cholera (1846–1860), there was extensive scientific research to understand the etiology of the disease. The miasma theory, which posited that infections spread through contaminated air, was no longer a satisfactory explanation. The English physician John Snow was the first to give convincing evidence in London in 1854 that cholera was spread from drinking water – a contagion, not miasma. Yet he could not identify the pathogens, which made most people still believe in the miasma origin.

V. cholerae was first observed and recognized under microscope by the French zoologist Félix-Archimède Pouchet. In 1849, Pouchet examined the stool samples of four people having cholera. His presentation before the French Academy of Sciences on 23 April was recorded as: "[Pouchet] could verify that there existed in these [cholera patients] dejecta an immense quantity of microscopic infusoria." As summarised in the Gazette medicale de Paris (1849, p 327), in a letter read at the 23 April 1849 meeting of the Paris Academy of Sciences, Pouchet announced that the organisms were infusoria, a name then used for microscopic protists, naming them as the 'Vibrio rugula of Mueller and Shrank', a species of protozoa described by Danish naturalist Otto Friedrich Müller in 1786.

Identification of the bacterium
An Italian physician, Filippo Pacini, while investigating cholera outbreak in Florence in the late 1854, identified the causative pathogen as a new type of bacterium. He performed autopsies of corpses and made meticulous microscopic examinations of the tissues and body fluids. From feces and intestinal mucosa, he identified many comma-shaped bacilli. Reporting his discovery before the Società Medico-Fisica Fiorentina (Medico-Physician Society of Florence) on 10 December, and published in the 12 December issue of the Gazzetta Medica Italiana (Medical Gazette of Italy), Pacini stated:"Le poche materie del vomito che ho potuto esaminare nel secondo e terzo caso di cholera ... e di più trovai degli ammassi granulosi appianati, simili a quelli che si formano alla superficie delle acque corrotte, quando sono per svilupparsi dei vibrioni; dei quali di fatto ne trovai alcuni del genere Bacterium, mentre la massima parte, per la loro estrema piccolezza, erano stati eliminati con la decantazione del fluido. [From the few samples of vomit that I was able to examine in the second and third cases of cholera ... and in addition I found smoothed granular masses, similar to those which form on the surface of dirty waters, when they are about to develop vibrios; of which in fact I found some of the genus Bacterium, while the greatest part, because of their extreme smallness, had been eliminated with the removal of the liquid. ]"Pacini thus introduced the name vibrioni (Latin vībro means "to move rapidly to and fro, to shake, to agitate"). A Catalan physician Joaquim Balcells i Pascual also reported such bacterium around the same time. The discovery of the new bacterium was not regarded as medically important as the bacterium was not directly attributed to cholera. Pacini also stated that there was no reason to say that the bacterium caused the disease since he failed to create a pure culture and perform experiments, which was necessary 'to attribute the quality of contagion to cholera'. The miasma theory was still not ruled out.

Rediscovery
The medical importance and relationship between the bacterium and the cholera disease was discovered by German physician Robert Koch. In August 1883, Koch, with a team of German physicians, went to Alexandria, Egypt, to investigate the cholera epidemic there. Koch found that the intestinal mucosa of people who died of cholera always had the bacterium, yet he could not confirm if it was the causative agent. He moved to Calcutta (now Kolkata), India, where the epidemic was more severe. It was from here that he isolated the bacterium in a pure culture on 7 January 1884. He subsequently confirmed that the bacterium was a new species, and described it as "a little bent, like a comma." He reported his discovery to the German Secretary of State for the Interior on 2 February, and it was published in the Deutsche Medizinische Wochenschrift (German Medical Weekly).

Although Koch was convinced that the bacterium was the cholera pathogen, he could not entirely procure critical evidence that the bacterium produced the symptoms in healthy subjects (an important element in what was later known as Koch's postulates). His experiment on animals using his pure bacteria culture did not lead to the appearance of the disease in any of the subjects, and he correctly deduced that animals are immune to the human pathogen. The bacterium was by then known as "the comma bacillus." It was only in 1959, in Calcutta, that Indian physician Sambhu Nath De isolated the cholera toxin and showed that it caused cholera in healthy subjects, hence fully proving the bacterium-cholera relationship.

Taxonomy
Pacini had used the name "vibrio cholera", without proper binomial rendering, for the name of the bacterium. Following Koch's description, a scientific name Bacillus comma was popularised. But an Italian bacteriologist Vittore Trevisan published in 1884 that Koch's bacterium was the same as that of Pacini's and introduced the name ''Bacillus cholerae.  A German physician Richard Friedrich Johannes Pfeiffer renamed it as Vibrio cholerae'' in 1896. The named was adopted by the Committee of the Society of American Bacteriologists on Characterization and Classification of Bacterial Types in 1920. In 1964, Rudolph Hugh of the George Washington University School of Medicine proposed to use the genus Vibrio with the type species V. cholerae (Pacini 1854) as a permanent name of the bacterium, regardless of the same name for protozoa. It was accepted by the Judicial Commission of the International Committee on Bacteriological Nomenclature in 1965, and the International Association of Microbiological Societies in 1966.

Characteristics
V. cholerae is a highly motile, comma shaped, gram-negative rod. The active movement of V. cholerae inspired the genus name because "vibrio" in Latin means "to quiver". Except for V. cholerae and V. mimicus, all other vibrio species are halophilic. Initial isolates are slightly curved, whereas they can appear as straight rods upon laboratory culturing. The bacterium has a flagellum at one cell pole as well as pili. It tolerates alkaline media that kill most intestinal commensals, but they are sensitive to acid. It is an aerobe while all other Vibrios are facultative anaerobes, and can undergo respiratory and fermentative metabolism. It measures 0.3 μm in diameter and 1.3 μm in length with average swimming velocity of around 75.4 μm/sec.

Pathogenicity
V. cholerae pathogenicity genes code for proteins directly or indirectly involved in the virulence of the bacteria. To adapt the host intestinal environment and to avoid being attacked by bile acids and antimicrobial peptides, V. cholera uses its outer membrane vesicles (OMVs). Upon entry, the bacteria sheds its OMVs, containing all the membrane modifications that make it vulnerable for the host attack.

During infection, V. cholerae secretes cholera toxin (CT), a protein that causes profuse, watery diarrhea (known as "rice-water stool"). This cholera toxin contains 5 B subunits that plays a role in attaching to the intestinal epithelial cells and 1 A subunit that plays a role in toxin activity. Colonization of the small intestine also requires the toxin coregulated pilus (TCP), a thin, flexible, filamentous appendage on the surface of bacterial cells. Expression of both CT and TCP is mediated by two component systems (TCS), which typically consist of a membrane-bound histidine kinase and an intracellular response element. TCS enable bacteria to respond to changing environments. In V. cholerae several TCS have been identified to be important in colonization, biofilm production and virulence. Quorum regulatory small RNAs (Qrr RNA) have been identified as targets of V. cholerae TCS. Here, the small RNA (sRNA) molecules bind to mRNA to block translation or induce degradation of inhibitors of expression of virulence or colonization genes. In V. cholerae the TCS EnvZ/OmpR alters gene expression via the sRNA coaR in response to changes in osmolarity and pH. An important target of coaR is tcpI, which negatively regulates expression of the major subunit of the TCP encoding gene (tcpA). When tcpI is bound by coaR it is no longer able to repress expression tcpA, leading to an increased colonization ability. Expression of coaR is upregulated by EnvZ/OmpR at a pH of 6,5, which is the normal pH of the intestinal lumen, but is low at higher pH values. V. cholerae in the intestinal lumen utilizes the TCP to attach to the intestinal mucosa, not invading the mucosa. After doing so it secretes cholerae toxin causing its symptoms. This then increases cyclic AMP or cAMP by binding (cholerae toxin) to adenylyl cyclase activating the GS pathway which leads to efflux of water and sodium into the intestinal lumen causing watery stools or rice watery stools.

V. cholerae can cause syndromes ranging from asymptomatic to cholera gravis. In endemic areas, 75% of cases are asymptomatic, 20% are mild to moderate, and 2-5% are severe forms such as cholera gravis. Symptoms include abrupt onset of watery diarrhea (a grey and cloudy liquid), occasional vomiting, and abdominal cramps. Dehydration ensues, with symptoms and signs such as thirst, dry mucous membranes, decreased skin turgor, sunken eyes, hypotension, weak or absent radial pulse, tachycardia, tachypnea, hoarse voice, oliguria, cramps, kidney failure, seizures, somnolence, coma, and death. Death due to dehydration can occur in a few hours to days in untreated children. The disease is also particularly dangerous for pregnant women and their fetuses during late pregnancy, as it may cause premature labor and fetal death. A study done by the Centers for Disease Control (CDC) in Haiti found that in pregnant women who contracted the disease, 16% of 900 women had fetal death. Risk factors for these deaths include: third trimester, younger maternal age, severe dehydration, and vomiting Dehydration poses the biggest health risk to pregnant women in countries that there are high rates of cholera. In cases of cholera gravis involving severe dehydration, up to 60% of patients can die; however, less than 1% of cases treated with rehydration therapy are fatal. The disease typically lasts 4–6 days. Worldwide, diarrhoeal disease, caused by cholera and many other pathogens, is the second-leading cause of death for children under the age of 5 and at least 120,000 deaths are estimated to be caused by cholera each year. In 2002, the WHO deemed that the case fatality ratio for cholera was about 3.95%.

Cholera illness and symptoms
V. cholerae infects the intestine and causes diarrhea, the hallmark symptom of cholera. Infection can be spread by eating contaminated food or drinking contaminated water. It also can spread through skin contact with contaminated human feces. Not all infection indicate symptoms, only about 1 in 10 people develop diarrhea. The major symptoms include: watery diarrhea, vomiting, rapid heart rate, loss of skin elasticity, low blood pressure, thirst, and muscle cramps. This illness can get serious as it can progress to kidney failure and possible coma. If diagnosed, it can be treated using medications.

Disease occurrence
V. cholerae has an endemic or epidemic occurrence. In countries where the disease has been for the past three years and the cases confirmed are local (within the confines of the country) transmission is considered to be "endemic." Alternatively, an outbreak is declared when the occurrence of disease exceeds the normal occurrence for any given time or location. Epidemics can last several days or over a span of years. Additionally, countries that have an occurrence of an epidemic can also be endemic. The longest standing V. cholerae epidemic was recorded in Yemen. Yemen had two outbreaks, the first occurred between September 2016 and April 2017, and the second began later in April 2017 and recently was considered to be resolved in 2019. The epidemic in Yemen took over 2,500 lives and impacted over 1 million people of Yemen. More outbreaks have occurred in Africa, the Americas, and Haiti.

Preventive measures
When visiting areas with epidemic cholera, the following precautions should be observed: drink and use bottled water; frequently wash hands with soap and safe water; use chemical toilets or bury feces if no restroom is available; do not defecate in any body of water and cook food thoroughly. Supplying proper, safe water is important. A precaution to take is to properly sanitize. Hand hygiene is an essential in areas where soap and water is not available. When there is no sanitation available for hand washing, scrub hands with ash or sand and rinse with clean water. A single dose vaccine is available for those traveling to an area where cholera is common.

There is a V. cholerae vaccine available to prevent disease spread. The vaccine is known as the, "oral cholera vaccine" (OCV). There are three types of OCV available for prevention: Dukoral®, Shanchol™, and Euvichol-Plus®. All three OCVs require two doses to be fully effective. Countries who are endemic or have an epidemic status are eligible to receive the vaccine based on several criteria: Risk of cholera, Severity of cholera, WASH conditions and capacity to improve, Healthcare conditions and capacity to improve, Capacity to implement OCV campaigns, Capacity to conduct M&E activities, Commitment at national and local level Since May the start of the OCV program to May 2018 over 25 million vaccines have been distributed to countries who meet the above criteria.

Treatment
The basic, overall treatment for Cholera is re-hydration, to replace the fluids that have been lost. Those with mild dehydration can be treated orally with an oral rehydration solution (ORS). When patients are severely dehydrated and unable to take in the proper amount of ORS, IV fluid treatment is generally pursued. Antibiotics are used in some cases, typically fluoroquinolones and tetracyclines.

Genome
V. cholerae (and Vibrionaceae in general) has two circular chromosomes, together totalling 4 million base pairs of DNA sequence and 3,885 predicted genes. The genes for cholera toxin are carried by CTXphi (CTXφ), a temperate bacteriophage inserted into the V. cholerae genome. CTXφ can transmit cholera toxin genes from one V. cholerae strain to another, one form of horizontal gene transfer. The genes for toxin coregulated pilus are coded by the Vibrio pathogenicity island (VPI), which is separate from the prophage.

The larger first chromosome is 3 Mbp long with 2,770 open reading frames (ORFs). It contains the crucial genes for toxicity, regulation of toxicity, and important cellular functions, such as transcription and translation.

The second chromosome is 1 Mbp long with 1115 ORFs. It is determined to be different from a plasmid or megaplasmid due to the inclusion of housekeeping and other essential genes in the genome, including essential genes for metabolism, heat-shock proteins, and 16S rRNA genes, which are ribosomal subunit genes used to track evolutionary relationships between bacteria. Also relevant in determining if the replicon is a chromosome is whether it represents a significant percentage of the genome, and chromosome 2 is 40% by size of the entire genome. And, unlike plasmids, chromosomes are not self-transmissible. However, the second chromosome may have once been a megaplasmid because it contains some genes usually found on plasmids, including a P1 plastid-like origin of replication.

Bacteriophage CTXφ
CTXφ (also called CTXphi) is a filamentous phage that contains the genes for cholera toxin. Infectious CTXφ particles are produced when V. cholerae infects humans. Phage particles are secreted from bacterial cells without lysis. When CTXφ infects V. cholerae cells, it integrates into specific sites on either chromosome. These sites often contain tandem arrays of integrated CTXφ prophage. In addition to the ctxA and ctxB genes encoding cholera toxin, CTXφ contains eight genes involved in phage reproduction, packaging, secretion, integration, and regulation. The CTXφ genome is 6.9 kb long.

Ecology and epidemiology
The main reservoirs of V. cholerae are aquatic sources such as rivers, brackish waters, and estuaries, often in association with copepods or other zooplankton, shellfish, and aquatic plants.

Cholera infections are most commonly acquired from drinking water in which V. cholerae is found naturally or into which it has been introduced from the feces of an infected person. Cholera is most likely to be found and spread in places with inadequate water treatment, poor sanitation, and inadequate hygiene. Other common vehicles include raw or undercooked fish and shellfish. Transmission from person to person is very unlikely, and casual contact with an infected person is not a risk for becoming ill. V. cholerae thrives in an aquatic environment, particularly in surface water. The primary connection between humans and pathogenic strains is through water, particularly in economically reduced areas that do not have good water purification systems.

Nonpathogenic strains are also present in water ecologies. The wide variety of pathogenic and nonpathogenic strains that co-exist in aquatic environments are thought to allow for so many genetic varieties. Gene transfer is fairly common amongst bacteria, and recombination of different V. cholerae genes can lead to new virulent strains.

A symbiotic relationship between V. cholerae and Ruminococcus obeum has been determined. R. obeum autoinducer represses the expression of several V. cholerae virulence factors. This inhibitory mechanism is likely to be present in other gut microbiota species which opens the way to mine the gut microbiota of members in specific communities which may utilize autoinducers or other mechanisms in order to restrict colonization by V. cholerae or other enteropathogens.

Outbreaks of Cholera cause an estimated 120,000 deaths annually worldwide. There has been roughly seven pandemics since 1817, the first. These pandemics first arose in the Indian subcontinent and spread.

Diversity and evolution
Two serogroups of V. cholerae, O1 and O139, cause outbreaks of cholera. O1 causes the majority of outbreaks, while O139 – first identified in Bangladesh in 1992 – is confined to Southeast Asia. Many other serogroups of V. cholerae, with or without the cholera toxin gene (including the nontoxigenic strains of the O1 and O139 serogroups), can cause a cholera-like illness. Only toxigenic strains of serogroups O1 and O139 have caused widespread epidemics.

V. cholerae O1 has two biotypes, classical and El Tor, and each biotype has two distinct serotypes, Inaba and Ogawa. The symptoms of infection are indistinguishable, although more people infected with the El Tor biotype remain asymptomatic or have only a mild illness. In recent years, infections with the classical biotype of V. cholerae O1 have become rare and are limited to parts of Bangladesh and India. Recently, new variant strains have been detected in several parts of Asia and Africa. Observations suggest these strains cause more severe cholera with higher case fatality rates.

Natural genetic transformation
V. cholerae can be induced to become competent for natural genetic transformation when grown on chitin, a biopolymer that is abundant in aquatic habitats (e.g. from crustacean exoskeletons). Natural genetic transformation is a sexual process involving DNA transfer from one bacterial cell to another through the intervening medium, and the integration of the donor sequence into the recipient genome by homologous recombination. Transformation competence in V. cholerae is stimulated by increasing cell density accompanied by nutrient limitation, a decline in growth rate, or stress. The V. cholerae uptake machinery involves a competence-induced pilus, and a conserved DNA binding protein that acts as a ratchet to reel DNA into the cytoplasm. There are two models of genetic transformation, sex hypothesis and competent bacteria.

title: Vibrio cholerae


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Antihydrogen

Antihydrogen () is the antimatter counterpart of hydrogen. Whereas the common hydrogen atom is composed of an electron and proton, the antihydrogen atom is made up of a positron and antiproton. Scientists hope that studying antihydrogen may shed light on the question of why there is more matter than antimatter in the observable universe, known as the baryon asymmetry problem. Antihydrogen is produced artificially in particle accelerators.

Experimental history
Accelerators first detected hot antihydrogen in the 1990s. ATHENA studied cold in 2002. It was first trapped by the Antihydrogen Laser Physics Apparatus (ALPHA) team at CERN in 2010, who then measured the structure and other important properties. ALPHA, AEgIS, and GBAR plan to further cool and study atoms.

1s–2s transition measurement
In 2016, the ALPHA experiment measured the atomic electron transition between the two lowest energy levels of antihydrogen, 1s–2s. The results, which are identical to that of hydrogen within the experimental resolution, support the idea of matter–antimatter symmetry and CPT symmetry.

In the presence of a magnetic field the 1s–2s transition splits into two hyperfine transitions with slightly different frequencies. The team calculated the transition frequencies for normal hydrogen under the magnetic field in the confinement volume as:

 * fdd = $2,466,061,103,064 kHz$
 * fcc = $2,466,061,707,104 kHz$

A single-photon transition between s states is prohibited by quantum selection rules, so to elevate ground state positrons to the 2s level, the confinement space was illuminated by a laser tuned to half the calculated transition frequencies, stimulating allowed two photon absorption.

Antihydrogen atoms excited to the 2s state can then evolve in one of several ways:

 * They can emit two photons and return directly to the ground state as they were
 * They can absorb another photon, which ionizes the atom
 * They can emit a single photon and return to the ground state via the 2p state—in this case the positron spin can flip or remain the same.

Both the ionization and spin-flip outcomes cause the atom to escape confinement. The team calculated that, assuming antihydrogen behaves like normal hydrogen, roughly half the antihydrogen atoms would be lost during the resonant frequency exposure, as compared to the no-laser case. With the laser source tuned 200 kHz below half the transition frequencies, the calculated loss was essentially the same as for the no-laser case.

The ALPHA team made batches of antihydrogen, held them for 600 seconds and then tapered down the confinement field over 1.5 seconds while counting how many antihydrogen atoms were annihilated. They did this under three different experimental conditions:

 * Resonance: exposing the confined antihydrogen atoms to a laser source tuned to exactly half the transition frequency for 300 seconds for each of the two transitions,
 * Off-resonance: exposing the confined antihydrogen atoms to a laser source tuned 200 kilohertz below the two resonance frequencies for 300 seconds each,
 * No-laser: confining the antihydrogen atoms without any laser illumination.

The two controls, off-resonance and no-laser, were needed to ensure that the laser illumination itself was not causing annihilations, perhaps by liberating normal atoms from the confinement vessel surface that could then combine with the antihydrogen.

The team conducted 11 runs of the three cases and found no significant difference between the off-resonance and no laser runs, but a 58% drop in the number of events detected after the resonance runs. They were also able to count annihilation events during the runs and found a higher level during the resonance runs, again with no significant difference between the off-resonance and no laser runs. The results were in good agreement with predictions based on normal hydrogen and can be "interpreted as a test of CPT symmetry at a precision of 200 ppt."

Characteristics
The CPT theorem of particle physics predicts antihydrogen atoms have many of the characteristics regular hydrogen has; i.e. the same mass, magnetic moment, and atomic state transition frequencies (see atomic spectroscopy). For example, excited antihydrogen atoms are expected to glow the same color as regular hydrogen. Antihydrogen atoms should be attracted to other matter or antimatter gravitationally with a force of the same magnitude that ordinary hydrogen atoms experience. This would not be true if antimatter has negative gravitational mass, which is considered highly unlikely, though not yet empirically disproven (see gravitational interaction of antimatter). Recent theoretical framework for negative mass and repulsive gravity (antigravity) between matter and antimatter has been developed, and the theory is compatible with CPT theorem.

When antihydrogen comes into contact with ordinary matter, its constituents quickly annihilate. The positron annihilates with an electron to produce gamma rays. The antiproton, on the other hand, is made up of antiquarks that combine with quarks in either neutrons or protons, resulting in high-energy pions, that quickly decay into muons, neutrinos, positrons, and electrons. If antihydrogen atoms were suspended in a perfect vacuum, they should survive indefinitely.

As an anti-element, it is expected to have exactly the same properties as hydrogen. For example, antihydrogen would be a gas under standard conditions and combine with antioxygen to form antiwater, 2.

Production
The first antihydrogen was produced in 1995 by a team led by Walter Oelert at CERN using a method first proposed by Charles Munger Jr, Stanley Brodsky and Ivan Schmidt Andrade.

In the LEAR, antiprotons from an accelerator were shot at xenon clusters, producing electron-positron pairs. Antiprotons can capture positrons with probability about $$, so this method is not suited for substantial production, as calculated. Fermilab measured a somewhat different cross section, in agreement with predictions of quantum electrodynamics. Both resulted in highly energetic, or hot, anti-atoms, unsuitable for detailed study.

Subsequently, CERN built the Antiproton Decelerator (AD) to support efforts towards low-energy antihydrogen, for tests of fundamental symmetries. The AD supplies several CERN groups. CERN expects their facilities will be capable of producing 10 million antiprotons per minute.

Low-energy antihydrogen
Experiments by the ATRAP and ATHENA collaborations at CERN, brought together positrons and antiprotons in Penning traps, resulting in synthesis at a typical rate of 100 antihydrogen atoms per second. Antihydrogen was first produced by ATHENA in 2002, and then by ATRAP and by 2004, millions of antihydrogen atoms were made. The atoms synthesized had a relatively high temperature (a few thousand kelvins), and would hit the walls of the experimental apparatus as a consequence and annihilate. Most precision tests require long observation times.

ALPHA, a successor of the ATHENA collaboration, was formed to stably trap antihydrogen. While electrically neutral, its spin magnetic moments interact with an inhomogeneous magnetic field; some atoms will be attracted to a magnetic minimum, created by a combination of mirror and multipole fields.

In November 2010, the ALPHA collaboration announced that they had trapped 38 antihydrogen atoms for a sixth of a second, the first confinement of neutral antimatter. In June 2011, they trapped 309 antihydrogen atoms, up to 3 simultaneously, for up to 1,000 seconds. They then studied its hyperfine structure, gravity effects, and charge. ALPHA will continue measurements along with experiments ATRAP, AEgIS and GBAR.

In 2018, AEgIS has produced a novel pulsed source of antihydrogen atoms with a production time spread of merely 250 nanoseconds. The pulsed source is generated by the charge exchange reaction between Rydberg positronium atoms -- produced via the injection of a pulsed positron beam into a nanochanneled Si target, and excited by laser pulses -- and antiprotons, trapped, cooled and manipulated in electromagnetic traps. The pulsed production enables the control of the antihydrogen temperature, the formation of an antihydrogen beam, and in the next phase a precision measurement on the gravitational behaviour using an atomic interferometer, the so-called Moiré deflectormeter.

Larger antimatter atoms
Larger antimatter atoms such as antideuterium, antitritium , and antihelium are much more difficult to produce. Antideuterium, antihelium-3 (3helium)  and antihelium-4 (4helium) nuclei have been produced with such high velocities that synthesis of their corresponding atoms poses several technical hurdles.

license: Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/

title: Antihydrogen


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Cable transport

Cable transport is a broad class of transport modes that have cables. They transport passengers and goods, often in vehicles called cable cars. The cable may be driven or passive, and items may be moved by pulling, sliding, sailing, or by drives within the object being moved on cableways. The use of pulleys and balancing of loads moving up and down are common elements of cable transport. They are often used in mountainous areas where cable haulage can overcome large differences in elevation.

Aerial transport
Forms of cable transport in which one or more cables are strung between supports of various forms and cars are suspended from these cables.

 * Aerial tramway
 * Chairlift
 * Funitel
 * Gondola lift
 * Ski lift
 * Zip line

Cable railways
Forms of cable transport where cars on rails are hauled by cables. The rails are usually steeply inclined and usually at ground level.
 * Cable car
 * Funicular

Other
Other forms of cable-hauled transport.
 * Cable ferry
 * Surface lift
 * Elevator

History
Rope-drawn transport dates back to 250 BC as evidenced by illustrations of aerial ropeway transportation systems in South China.

Early aerial tramways

The first recorded mechanical ropeway was by Venetian Fausto Veranzio who designed a bi-cable passenger ropeway in 1616. The industry generally considers Dutchman Adam Wybe to have built the first operational system in 1644. The technology, which was further developed by the people living in the Alpine regions of Europe, progressed and expanded with the advent of wire rope and electric drive.

The first use of wire rope for aerial tramways is disputed. American inventor Peter Cooper is one early claimant, constructing an aerial tramway using wire rope in Baltimore 1832, to move landfill materials. Though there is only partial evidence for the claimed 1832 tramway, Cooper was involved in many of such tramways built in the 1850s, and in 1853 he built a two-mile-long tramway to transport iron ore to his blast furnaces at Ringwood, New Jersey.

World War I motivated extensive use of military tramways for warfare between Italy and Austria.

During the industrial revolution, new forms of cable-hauled transportation systems were created including the use of steel cable to allow for greater load support and larger systems. Aerial tramways were first used for commercial passenger haulage in the 1900s.

The first cable railways
The earliest form of cable railway was the gravity incline, which in its simplest form consists of two parallel tracks laid on a steep gradient, with a single rope wound around a winding drum and connecting the trains of wagons on the tracks. Loaded wagons at the top of the incline are lowered down, their weight hauling empty wagons from the bottom. The winding drum has a brake to control the rate of travel of the wagons. The first use of a gravity incline isn't recorded, but the Llandegai Tramway at Bangor in North Wales was opened in 1798, and is one of the earliest examples using iron rails.

The first cable-hauled street railway was the London and Blackwall Railway, built in 1840, which used fibre to grip the haulage rope. This caused a series of technical and safety issues, which led to the adoption of steam locomotives by 1848.

The first Funicular railway was opened in Lyon in 1862.

The Westside and Yonkers Patent Railway Company developed a cable-hauled elevated railway. This 3½ mile long line was proposed in 1866 and opened in 1868. It operated as a cable railway until 1871 when it was converted to use steam locomotives.

The next development of the cable car came in California. Andrew Hallidie, a Scottish emigre, gave San Francisco the first effective and commercially successful route, using steel cables, opening the Clay Street Hill Railroad on August 2, 1873. Hallidie was a manufacturer of steel cables. The system featured a human-operated grip, which was able to start and stop the car safely. The rope that was used allowed the multiple, independent cars to run on one line, and soon Hallidie's concept was extended to multiple lines in San Francisco.

The first cable railway outside the United Kingdom and the United States was the Roslyn Tramway, which opened in 1881, in Dunedin, New Zealand. America remained the country that made the greatest use of cable railways; by 1890 more than 500 miles of cable-hauled track had been laid, carrying over 1,000,000 passengers per year. However, in 1890, electric tramways exceeded the cable hauled tramways in mileage, efficiency and speed.

Early ski lifts
The first surface lift was built in 1908 by German Robert Winterhalder in Schollach/Eisenbach, Hochschwarzwald and started operations February 14, 1908.

A steam-powered toboggan tow, 950 ft in length, was built in Truckee, California, in 1910. The first skier-specific tow in North America was apparently installed in 1933 by Alec Foster at Shawbridge in the Laurentians outside Montreal, Quebec.

The modern J-bar and T-bar mechanism was invented in 1934 by the Swiss engineer Ernst Constam, with the first lift installed in Davos, Switzerland.

The first chairlift was developed by James Curran in 1936. The co-owner of the Union Pacific Railroad, William Averell Harriman owned America's first ski resort, Sun Valley, Idaho. He asked his design office to tackle the problem of lifting skiers to the top of the resort. Curran, a Union Pacific bridge designer, adapted a cable hoist he had designed for loading bananas in Honduras to create the first ski lift.

More recent developments
More recent developments are being classified under the type of track that their design is based upon. After the success of this operation, several other projects were initiated in New Zealand and Chicago. The social climate around pollution is allowing for a shift from cars back to the utilization of cable transport due to their advantages. However, for many years they were a niche form of transportation used primarily in difficult-to-operate conditions for cars (such as on ski slopes as lifts). Now that cable transport projects are on the increase, the social effects are beginning to become more significant. In 2018 the highest 3S cablecar has been inaugurated in Zermatt, Switzerland after more than two years of construction. This cablecar is also called the "Matterhorn Glacier ride" and it allows passengers to reach the top of the Klein Matterhorn mountain (3883m)

Comparison with other transport types
When compared to trains and cars, the volume of people to transport over time and the start-up cost of the project must be a consideration. In areas with extensive road networks, personal vehicles offer greater flexibility and range. Remote places like mountainous regions and ski slopes may be difficult to link with roads, making cable transport project a much easier approach. A cable transport project system may also need fewer invasive changes to the local environment.

The use of Cable Transport is not limited to such rural locations as skiing resorts; it can be used in urban development areas. Their uses in urban areas include funicular railways, gondola lifts, and aerial tramways.

Accidents

 * A cable car accident in Cavalese, Italy, on 9 March 1976 is considered the worst aerial lift accident in history. The car crashed off the rails and fell 200 meters down a mountainside, also crashing through a grassy meadow before coming to a halt. The tragedy caused the death of 43 people, and four lift officials were jailed for charges regarding the accident.
 * On April 15, 1978, a cable car at Squaw Valley Ski Resort in California came off from one of its cables, dropping 75 feet (23 m) and violently bouncing up. It collided with a cable which sheared through the car. Four people were killed and 31 injured.
 * The Singapore cable car crash of 29 January 1983 occurred when a drilling rig passed beneath the cable car system linking the Singapore mainland with Sentosa island. The derrick of the drilling rig aboard the ship MV Eniwetok struck the cables, causing two of the gondolas to fall into the sea below. There were 7 fatalities.
 * On February 3, 1998, twenty people died in Cavalese, Italy, when a United States Marine Corps EA-6B Prowler aircraft, while flying too low, against regulations, cut a cable supporting a gondola of an aerial tramway. Those killed, 19 passengers and one operator, were eight Germans, five Belgians, three Italians, two Poles, one Austrian, and one Dutch. The United States refused to have the four Marines tried under Italian law and later court-martialed two of them with minimal charges in their country.
 * The Kaprun disaster was a fire that occurred in an ascending train in the tunnel of the Gletscherbahn Kaprun 2 funicular in Kaprun, Austria, on 11 November 2000. The disaster claimed the lives of 155 people, leaving 12 survivors (10 Germans and two Austrians) from the burning train. It is one of the worst cable car accidents in history.
 * A cable car derailed and crashed to the ground in the Nevis Range, near Fort William, Scotland, on 13 July 2006, seriously injuring all five passengers. Another car on the same rail also slid back down the rails when the crash happened. Following the incident, 50 people were left stranded at the station whilst the staff and aid helped the passengers of the crashed car.
 * On Wednesday 25 July 2012, passengers of the London cable car were stuck 90 meters in the air when a power failure caused the gondola to stop over the River Thames. The fault happened at 11:45am and lasted for about 30 minutes. No passengers were injured, but this was the first problem to ever hit the London's new cable car link.

title: Cable transport


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Talk:Vibrio cholerae

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History: Pacini and Koch
Looking up on cholera in my encyclopedia set, I found a little bit on the bacteria Vibrio comma. I believe this was possibly an older name for Vibrio cholerae? Nonetheless, the text reads that Vibrio comma was first dicovered in 1883 by the German physician and Bacteriologist Robert Koch. The encyclopedia in question is the Funk and Wagnalls New Encyclopedia, Volume 6, pages 79-80. The copyright year is 1979. CanbekEsen 05:46, 10 April 2007 (UTC )

The discovery of Vibrio Cholerae was made by Pacini in 1854, who found curved bacteria in intestinal contents of cholera victims. However, Robert Koch, who studied cholera in Egypt during 1883, demonstrated that the cholerae disease was caused by a comma-shaped organism, which he named Kommabazillen. This work by Koch initially gained alot of attention and overshadowed the work of Pacini. For several decades, the name Vibrio Comma was also used. Finally, the pioneering work of Pacini was recognized and the name was changed back to Vibrio Cholerae.

Read more in: J.B. KAPER, J.G. MORRIS, JR., AND M.M. LEVINE, Cholera, CLINICAL MICROBIOLOGY REVIEWS, Jan. 1995, p. 48–86

85.224.21.147 (talk) 22:48, 23 November 2007 (UTC)Dabalk

Oxygen Usage?
Anyone know? aerobe, anaerobe, facultative, etc? This guy ( http://trishul.sci.gu.edu.au/courses/ss12bmi/micro_groups/fac_anaerobes.html ) seems to say it is a facultative anaerobe. —Preceding unsigned comment added by 134.84.140.127 (talk) 21:01, 22 January 2009 (UTC)
 * Added "facultative" to the description. --Synaptophysin (talk) 16:49, 19 September 2011 (UTC)

Domain, not kingdom
The Taxon Bacteria was reassigned the rank of domain a number of years ago now, so I have corrected this. The Mysterious El Willstro (talk) 03:33, 22 April 2011 (UTC)

title: Talk:Vibrio cholerae


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Protein folding

Protein folding is the physical process by which a protein, after synthesis by a ribosome as a linear chain of amino acids, changes from an unstable random coil into a more ordered three-dimensional structure. This structure permits the protein to become biologically functional.

The folding of many proteins begins even during the translation of the polypeptide chain. The amino acids interact with each other to produce a well-defined three-dimensional structure, known as the protein's native state. This structure is determined by the amino-acid sequence or primary structure.

The correct three-dimensional structure is essential to function, although some parts of functional proteins may remain unfolded, indicating that protein dynamics are important. Failure to fold into a native structure generally produces inactive proteins, but in some instances, misfolded proteins have modified or toxic functionality. Several neurodegenerative and other diseases are believed to result from the accumulation of amyloid fibrils formed by misfolded proteins, the infectious varieties of which are known as prions. Many allergies are caused by the incorrect folding of some proteins because the immune system does not produce the antibodies for certain protein structures.

Denaturation of proteins is a process of transition from a folded to an unfolded state. It happens in cooking, burns, proteinopathies, and other contexts. Residual structure present, if any, in the supposedly unfolded state may form a folding initiation site and guide the subsequent folding reactions.

The duration of the folding process varies dramatically depending on the protein of interest. When studied outside the cell, the slowest folding proteins require many minutes or hours to fold, primarily due to proline isomerization, and must pass through a number of intermediate states, like checkpoints, before the process is complete. On the other hand, very small single-domain proteins with lengths of up to a hundred amino acids typically fold in a single step. Time scales of milliseconds are the norm, and the fastest known protein folding reactions are complete within a few microseconds. The folding time scale of a protein depends on its size, contact order, and circuit topology.

Understanding and simulating the protein folding process has been an important challenge for computational biology since the late 1960s.

Primary structure
The primary structure of a protein, its linear amino-acid sequence, determines its native conformation. The specific amino acid residues and their position in the polypeptide chain are the determining factors for which portions of the protein fold closely together and form its three-dimensional conformation. The amino acid composition is not as important as the sequence. The essential fact of folding, however, remains that the amino acid sequence of each protein contains the information that specifies both the native structure and the pathway to attain that state. This is not to say that nearly identical amino acid sequences always fold similarly. Conformations differ based on environmental factors as well; similar proteins fold differently based on where they are found.

Secondary structure

Formation of a Protein secondary structure|secondary structure is the first step in the folding process that a protein takes to assume its native structure. Characteristic of secondary structure are the structures known as alpha helices and beta sheets that fold rapidly because they are stabilized by intramolecular hydrogen bonds, as was first characterized by Linus Pauling. Formation of intramolecular hydrogen bonds provides another important contribution to protein stability. α-helices are formed by hydrogen bonding of the backbone to form a spiral shape (refer to figure on the right). The β pleated sheet is a structure that forms with the backbone bending over itself to form the hydrogen bonds (as displayed in the figure to the left). The hydrogen bonds are between the amide hydrogen and carbonyl oxygen of the peptide bond. There exists anti-parallel β pleated sheets and parallel β pleated sheets where the stability of the hydrogen bonds is stronger in the anti-parallel β sheet as it hydrogen bonds with the ideal 180 degree angle compared to the slanted hydrogen bonds formed by parallel sheets.

Tertiary structure
The α-Helices and β-Sheets are commonly amphipathic, meaning they have a hydrophilic and a hydrophobic portion. This ability helps in forming tertiary structure of a protein in which folding occurs so that the hydrophilic sides are facing the aqueous environment surrounding the protein and the hydrophobic sides are facing the hydrophobic core of the protein. Secondary structure hierarchically gives way to tertiary structure formation. Once the protein's tertiary structure is formed and stabilized by the hydrophobic interactions, there may also be covalent bonding in the form of disulfide bridges formed between two cysteine residues. These non-covalent and covalent contacts take a specific topological arrangement in a native structure of a protein. Tertiary structure of a protein involves a single polypeptide chain; however, additional interactions of folded polypeptide chains give rise to quaternary structure formation.

Quaternary structure
Tertiary structure may give way to the formation of quaternary structure in some proteins, which usually involves the "assembly" or "coassembly" of subunits that have already folded; in other words, multiple polypeptide chains could interact to form a fully functional quaternary protein.

Driving forces of protein folding

Folding is a spontaneous process that is mainly guided by hydrophobic interactions, formation of intramolecular hydrogen bonds, van der Waals forces, and it is opposed by conformational entropy. The process of folding often begins co-translationally, so that the N-terminus of the protein begins to fold while the C-terminal portion of the protein is still being synthesized by the ribosome; however, a protein molecule may fold spontaneously during or after biosynthesis. While these macromolecules may be regarded as "folding themselves", the process also depends on the solvent (water or lipid bilayer), the concentration of salts, the pH, the temperature, the possible presence of cofactors and of molecular chaperones.

Proteins will have limitations on their folding abilities by the restricted bending angles or conformations that are possible. These allowable angles of protein folding are described with a two-dimensional plot known as the Ramachandran plot, depicted with psi and phi angles of allowable rotation.

Hydrophobic effect

Protein folding must be thermodynamically favorable within a cell in order for it to be a spontaneous reaction. Since it is known that protein folding is a spontaneous reaction, then it must assume a negative Gibbs free energy value. Gibbs free energy in protein folding is directly related to enthalpy and entropy. For a negative delta G to arise and for protein folding to become thermodynamically favorable, then either enthalpy, entropy, or both terms must be favorable.

Minimizing the number of hydrophobic side-chains exposed to water is an important driving force behind the folding process. The hydrophobic effect is the phenomenon in which the hydrophobic chains of a protein collapse into the core of the protein (away from the hydrophilic environment). In an aqueous environment, the water molecules tend to aggregate around the hydrophobic regions or side chains of the protein, creating water shells of ordered water molecules. An ordering of water molecules around a hydrophobic region increases order in a system and therefore contributes a negative change in entropy (less entropy in the system). The water molecules are fixed in these water cages which drives the hydrophobic collapse, or the inward folding of the hydrophobic groups. The hydrophobic collapse introduces entropy back to the system via the breaking of the water cages which frees the ordered water molecules. The multitude of hydrophobic groups interacting within the core of the globular folded protein contributes a significant amount to protein stability after folding, because of the vastly accumulated van der Waals forces (specifically London Dispersion forces). The hydrophobic effect exists as a driving force in thermodynamics only if there is the presence of an aqueous medium with an amphiphilic molecule containing a large hydrophobic region. The strength of hydrogen bonds depends on their environment; thus, H-bonds enveloped in a hydrophobic core contribute more than H-bonds exposed to the aqueous environment to the stability of the native state.

In proteins with globular folds, hydrophobic amino acids tend to be interspersed along the primary sequence, rather than randomly distributed or clustered together. However, proteins that have recently been born de novo, which tend to be intrinsically disordered, show the opposite pattern of hydrophobic amino acid clustering along the primary sequence.

Chaperones

Molecular chaperones are a class of proteins that aid in the correct folding of other proteins in vivo. Chaperones exist in all cellular compartments and interact with the polypeptide chain in order to allow the native three-dimensional conformation of the protein to form; however, chaperones themselves are not included in the final structure of the protein they are assisting in. Chaperones may assist in folding even when the nascent polypeptide is being synthesized by the ribosome. Molecular chaperones operate by binding to stabilize an otherwise unstable structure of a protein in its folding pathway, but chaperones do not contain the necessary information to know the correct native structure of the protein they are aiding; rather, chaperones work by preventing incorrect folding conformations. In this way, chaperones do not actually increase the rate of individual steps involved in the folding pathway toward the native structure; instead, they work by reducing possible unwanted aggregations of the polypeptide chain that might otherwise slow down the search for the proper intermediate and they provide a more efficient pathway for the polypeptide chain to assume the correct conformations. Chaperones are not to be confused with folding catalyst proteins, which catalyze chemical reactions responsible for slow steps in folding pathways. Examples of folding catalysts are protein disulfide isomerases and peptidyl-prolyl isomerases that may be involved in formation of disulfide bonds or interconversion between cis and trans stereoisomers of peptide group. Chaperones are shown to be critical in the process of protein folding in vivo because they provide the protein with the aid needed to assume its proper alignments and conformations efficiently enough to become "biologically relevant". This means that the polypeptide chain could theoretically fold into its native structure without the aid of chaperones, as demonstrated by protein folding experiments conducted in vitro; however, this process proves to be too inefficient or too slow to exist in biological systems; therefore, chaperones are necessary for protein folding in vivo. Along with its role in aiding native structure formation, chaperones are shown to be involved in various roles such as protein transport, degradation, and even allow denatured proteins exposed to certain external denaturant factors an opportunity to refold into their correct native structures.

A fully denatured protein lacks both tertiary and secondary structure, and exists as a so-called random coil. Under certain conditions some proteins can refold; however, in many cases, denaturation is irreversible. Cells sometimes protect their proteins against the denaturing influence of heat with enzymes known as heat shock proteins (a type of chaperone), which assist other proteins both in folding and in remaining folded. Heat shock proteins have been found in all species examined, from bacteria to humans, suggesting that they evolved very early and have an important function. Some proteins never fold in cells at all except with the assistance of chaperones which either isolate individual proteins so that their folding is not interrupted by interactions with other proteins or help to unfold misfolded proteins, allowing them to refold into the correct native structure. This function is crucial to prevent the risk of precipitation into insoluble amorphous aggregates. The external factors involved in protein denaturation or disruption of the native state include temperature, external fields (electric, magnetic), molecular crowding, and even the limitation of space (i.e. confinement), which can have a big influence on the folding of proteins. High concentrations of solutes, extremes of pH, mechanical forces, and the presence of chemical denaturants can contribute to protein denaturation, as well. These individual factors are categorized together as stresses. Chaperones are shown to exist in increasing concentrations during times of cellular stress and help the proper folding of emerging proteins as well as denatured or misfolded ones.

Under some conditions proteins will not fold into their biochemically functional forms. Temperatures above or below the range that cells tend to live in will cause thermally unstable proteins to unfold or denature (this is why boiling makes an egg white turn opaque). Protein thermal stability is far from constant, however; for example, hyperthermophilic bacteria have been found that grow at temperatures as high as 122 °C, which of course requires that their full complement of vital proteins and protein assemblies be stable at that temperature or above.

The bacterium E. coli is the host for bacteriophage T4, and the phage encoded gp31 protein appears to be structurally and functionally homologous to E. coli chaperone protein GroES and able to substitute for it in the assembly of bacteriophage T4 virus particles during infection. Like GroES, gp31 forms a stable complex with GroEL chaperonin that is absolutely necessary for the folding and assembly in vivo of the bacteriophage T4 major capsid protein gp23.

Fold switching
Some proteins have multiple native structures, and change their fold based on some external factors. For example, the KaiB protein switches fold throughout the day, acting as a clock for cyanobacteria. It has been estimated that around 0.5–4% of PDB (Protein Data Bank) proteins switch folds.

Protein misfolding and neurodegenerative disease
A protein is considered to be misfolded if it cannot achieve its normal native state. This can be due to mutations in the amino acid sequence or a disruption of the normal folding process by external factors. The misfolded protein typically contains β-sheets that are organized in a supramolecular arrangement known as a cross-β structure. These β-sheet-rich assemblies are very stable, very insoluble, and generally resistant to proteolysis. The structural stability of these fibrillar assemblies is caused by extensive interactions between the protein monomers, formed by backbone hydrogen bonds between their β-strands. The misfolding of proteins can trigger the further misfolding and accumulation of other proteins into aggregates or oligomers. The increased levels of aggregated proteins in the cell leads to formation of amyloid-like structures which can cause degenerative disorders and cell death. The amyloids are fibrillary structures that contain intermolecular hydrogen bonds which are highly insoluble and made from converted protein aggregates. Therefore, the proteasome pathway may not be efficient enough to degrade the misfolded proteins prior to aggregation. Misfolded proteins can interact with one another and form structured aggregates and gain toxicity through intermolecular interactions.

Aggregated proteins are associated with prion-related illnesses such as Creutzfeldt–Jakob disease, bovine spongiform encephalopathy (mad cow disease), amyloid-related illnesses such as Alzheimer's disease and familial amyloid cardiomyopathy or polyneuropathy, as well as intracellular aggregation diseases such as Huntington's and Parkinson's disease. These age onset degenerative diseases are associated with the aggregation of misfolded proteins into insoluble, extracellular aggregates and/or intracellular inclusions including cross-β amyloid fibrils. It is not completely clear whether the aggregates are the cause or merely a reflection of the loss of protein homeostasis, the balance between synthesis, folding, aggregation and protein turnover. Recently the European Medicines Agency approved the use of Tafamidis or Vyndaqel (a kinetic stabilizer of tetrameric transthyretin) for the treatment of transthyretin amyloid diseases. This suggests that the process of amyloid fibril formation (and not the fibrils themselves) causes the degeneration of post-mitotic tissue in human amyloid diseases. Misfolding and excessive degradation instead of folding and function leads to a number of proteopathy diseases such as antitrypsin-associated emphysema, cystic fibrosis and the lysosomal storage diseases, where loss of function is the origin of the disorder. While protein replacement therapy has historically been used to correct the latter disorders, an emerging approach is to use pharmaceutical chaperones to fold mutated proteins to render them functional.

Experimental techniques for studying protein folding
While inferences about protein folding can be made through mutation studies, typically, experimental techniques for studying protein folding rely on the gradual unfolding or folding of proteins and observing conformational changes using standard non-crystallographic techniques.

X-ray crystallography
X-ray crystallography is one of the more efficient and important methods for attempting to decipher the three dimensional configuration of a folded protein. To be able to conduct X-ray crystallography, the protein under investigation must be located inside a crystal lattice. To place a protein inside a crystal lattice, one must have a suitable solvent for crystallization, obtain a pure protein at supersaturated levels in solution, and precipitate the crystals in solution. Once a protein is crystallized, X-ray beams can be concentrated through the crystal lattice which would diffract the beams or shoot them outwards in various directions. These exiting beams are correlated to the specific three-dimensional configuration of the protein enclosed within. The X-rays specifically interact with the electron clouds surrounding the individual atoms within the protein crystal lattice and produce a discernible diffraction pattern. Only by relating the electron density clouds with the amplitude of the X-rays can this pattern be read and lead to assumptions of the phases or phase angles involved that complicate this method. Without the relation established through a mathematical basis known as Fourier transform, the "phase problem" would render predicting the diffraction patterns very difficult. Emerging methods like multiple isomorphous replacement use the presence of a heavy metal ion to diffract the X-rays into a more predictable manner, reducing the number of variables involved and resolving the phase problem.

Fluorescence spectroscopy
Fluorescence spectroscopy is a highly sensitive method for studying the folding state of proteins. Three amino acids, phenylalanine (Phe), tyrosine (Tyr) and tryptophan (Trp), have intrinsic fluorescence properties, but only Tyr and Trp are used experimentally because their quantum yields are high enough to give good fluorescence signals. Both Trp and Tyr are excited by a wavelength of 280 nm, whereas only Trp is excited by a wavelength of 295 nm. Because of their aromatic character, Trp and Tyr residues are often found fully or partially buried in the hydrophobic core of proteins, at the interface between two protein domains, or at the interface between subunits of oligomeric proteins. In this apolar environment, they have high quantum yields and therefore high fluorescence intensities. Upon disruption of the protein's tertiary or quaternary structure, these side chains become more exposed to the hydrophilic environment of the solvent, and their quantum yields decrease, leading to low fluorescence intensities. For Trp residues, the wavelength of their maximal fluorescence emission also depend on their environment.

Fluorescence spectroscopy can be used to characterize the equilibrium unfolding of proteins by measuring the variation in the intensity of fluorescence emission or in the wavelength of maximal emission as functions of a denaturant value. The denaturant can be a chemical molecule (urea, guanidinium hydrochloride), temperature, pH, pressure, etc. The equilibrium between the different but discrete protein states, i.e. native state, intermediate states, unfolded state, depends on the denaturant value; therefore, the global fluorescence signal of their equilibrium mixture also depends on this value. One thus obtains a profile relating the global protein signal to the denaturant value. The profile of equilibrium unfolding may enable one to detect and identify intermediates of unfolding. General equations have been developed by Hugues Bedouelle to obtain the thermodynamic parameters that characterize the unfolding equilibria for homomeric or heteromeric proteins, up to trimers and potentially tetramers, from such profiles. Fluorescence spectroscopy can be combined with fast-mixing devices such as stopped flow, to measure protein folding kinetics, generate a chevron plot and derive a Phi value analysis.

Circular dichroism
Circular dichroism is one of the most general and basic tools to study protein folding. Circular dichroism spectroscopy measures the absorption of circularly polarized light. In proteins, structures such as alpha helices and beta sheets are chiral, and thus absorb such light. The absorption of this light acts as a marker of the degree of foldedness of the protein ensemble. This technique has been used to measure equilibrium unfolding of the protein by measuring the change in this absorption as a function of denaturant concentration or temperature. A denaturant melt measures the free energy of unfolding as well as the protein's m value, or denaturant dependence. A temperature melt measures the denaturation temperature (Tm) of the protein. As for fluorescence spectroscopy, circular-dichroism spectroscopy can be combined with fast-mixing devices such as stopped flow to measure protein folding kinetics and to generate chevron plots.

Vibrational circular dichroism of proteins
The more recent developments of vibrational circular dichroism (VCD) techniques for proteins, currently involving Fourier transform (FT) instruments, provide powerful means for determining protein conformations in solution even for very large protein molecules. Such VCD studies of proteins can be combined with X-ray diffraction data for protein crystals, FT-IR data for protein solutions in heavy water (D2O), or quantum computations.

Protein nuclear magnetic resonance spectroscopy
Protein nuclear magnetic resonance (NMR) is able to collect protein structural data by inducing a magnet field through samples of concentrated protein. In NMR, depending on the chemical environment, certain nuclei will absorb specific radio-frequencies. Because protein structural changes operate on a time scale from ns to ms, NMR is especially equipped to study intermediate structures in timescales of ps to s. Some of the main techniques for studying proteins structure and non-folding protein structural changes include COSY, TOCSY, HSQC, time relaxation (T1 & T2), and NOE. NOE is especially useful because magnetization transfers can be observed between spatially proximal hydrogens are observed. Different NMR experiments have varying degrees of timescale sensitivity that are appropriate for different protein structural changes. NOE can pick up bond vibrations or side chain rotations, however, NOE is too sensitive to pick up protein folding because it occurs at larger timescale.

Because protein folding takes place in about 50 to 3000 s−1 CPMG Relaxation dispersion and chemical exchange saturation transfer have become some of the primary techniques for NMR analysis of folding. In addition, both techniques are used to uncover excited intermediate states in the protein folding landscape. To do this, CPMG Relaxation dispersion takes advantage of the spin echo phenomenon. This technique exposes the target nuclei to a 90 pulse followed by one or more 180 pulses. As the nuclei refocus, a broad distribution indicates the target nuclei is involved in an intermediate excited state. By looking at Relaxation dispersion plots the data collect information on the thermodynamics and kinetics between the excited and ground. Saturation Transfer measures changes in signal from the ground state as excited states become perturbed. It uses weak radio frequency irradiation to saturate the excited state of a particular nuclei which transfers its saturation to the ground state. This signal is amplified by decreasing the magnetization (and the signal) of the ground state.

The main limitations in NMR is that its resolution decreases with proteins that are larger than 25 kDa and is not as detailed as X-ray crystallography. Additionally, protein NMR analysis is quite difficult and can propose multiple solutions from the same NMR spectrum.

In a study focused on the folding of an amyotrophic lateral sclerosis involved protein SOD1, excited intermediates were studied with relaxation dispersion and Saturation transfer. SOD1 had been previously tied to many disease causing mutants which were assumed to be involved in protein aggregation, however the mechanism was still unknown. By using Relaxation Dispersion and Saturation Transfer experiments many excited intermediate states were uncovered misfolding in the SOD1 mutants.

Dual-polarization interferometry
Dual polarisation interferometry is a surface-based technique for measuring the optical properties of molecular layers. When used to characterize protein folding, it measures the conformation by determining the overall size of a monolayer of the protein and its density in real time at sub-Angstrom resolution, although real-time measurement of the kinetics of protein folding are limited to processes that occur slower than ~10 Hz. Similar to circular dichroism, the stimulus for folding can be a denaturant or temperature.

Studies of folding with high time resolution
The study of protein folding has been greatly advanced in recent years by the development of fast, time-resolved techniques. Experimenters rapidly trigger the folding of a sample of unfolded protein and observe the resulting dynamics. Fast techniques in use include neutron scattering, ultrafast mixing of solutions, photochemical methods, and laser temperature jump spectroscopy. Among the many scientists who have contributed to the development of these techniques are Jeremy Cook, Heinrich Roder, Terry Oas, Harry Gray, Martin Gruebele, Brian Dyer, William Eaton, Sheena Radford, Chris Dobson, Alan Fersht, Bengt Nölting and Lars Konermann.

Proteolysis
Proteolysis is routinely used to probe the fraction unfolded under a wide range of solution conditions (e.g. fast parallel proteolysis (FASTpp).

Single-molecule force spectroscopy
Single molecule techniques such as optical tweezers and AFM have been used to understand protein folding mechanisms of isolated proteins as well as proteins with chaperones. Optical tweezers have been used to stretch single protein molecules from their C- and N-termini and unfold them to allow study of the subsequent refolding. The technique allows one to measure folding rates at single-molecule level; for example, optical tweezers have been recently applied to study folding and unfolding of proteins involved in blood coagulation. von Willebrand factor (vWF) is a protein with an essential role in blood clot formation process. It discovered – using single molecule optical tweezers measurement – that calcium-bound vWF acts as a shear force sensor in the blood. Shear force leads to unfolding of the A2 domain of vWF, whose refolding rate is dramatically enhanced in the presence of calcium. Recently, it was also shown that the simple src SH3 domain accesses multiple unfolding pathways under force.

Biotin painting
Biotin painting enables condition-specific cellular snapshots of (un)folded proteins. Biotin 'painting' shows a bias towards predicted Intrinsically disordered proteins.

Computational studies of protein folding
Computational studies of protein folding includes three main aspects related to the prediction of protein stability, kinetics, and structure. A 2013 review summarizes the available computational methods for protein folding.

Levinthal's paradox
In 1969, Cyrus Levinthal noted that, because of the very large number of degrees of freedom in an unfolded polypeptide chain, the molecule has an astronomical number of possible conformations. An estimate of 3300 or 10143 was made in one of his papers. Levinthal's paradox is a thought experiment based on the observation that if a protein were folded by sequential sampling of all possible conformations, it would take an astronomical amount of time to do so, even if the conformations were sampled at a rapid rate (on the nanosecond or picosecond scale). Based upon the observation that proteins fold much faster than this, Levinthal then proposed that a random conformational search does not occur, and the protein must, therefore, fold through a series of meta-stable intermediate states.

Energy landscape of protein folding
The configuration space of a protein during folding can be visualized as an energy landscape. According to Joseph Bryngelson and Peter Wolynes, proteins follow the principle of minimal frustration, meaning that naturally evolved proteins have optimized their folding energy landscapes, and that nature has chosen amino acid sequences so that the folded state of the protein is sufficiently stable. In addition, the acquisition of the folded state had to become a sufficiently fast process. Even though nature has reduced the level of frustration in proteins, some degree of it remains up to now as can be observed in the presence of local minima in the energy landscape of proteins.

A consequence of these evolutionarily selected sequences is that proteins are generally thought to have globally "funneled energy landscapes" (a term coined by José Onuchic) that are largely directed toward the native state. This "folding funnel" landscape allows the protein to fold to the native state through any of a large number of pathways and intermediates, rather than being restricted to a single mechanism. The theory is supported by both computational simulations of model proteins and experimental studies, and it has been used to improve methods for protein structure prediction and design. The description of protein folding by the leveling free-energy landscape is also consistent with the 2nd law of thermodynamics. Physically, thinking of landscapes in terms of visualizable potential or total energy surfaces simply with maxima, saddle points, minima, and funnels, rather like geographic landscapes, is perhaps a little misleading. The relevant description is really a high-dimensional phase space in which manifolds might take a variety of more complicated topological forms.

The unfolded polypeptide chain begins at the top of the funnel where it may assume the largest number of unfolded variations and is in its highest energy state. Energy landscapes such as these indicate that there are a large number of initial possibilities, but only a single native state is possible; however, it does not reveal the numerous folding pathways that are possible. A different molecule of the same exact protein may be able to follow marginally different folding pathways, seeking different lower energy intermediates, as long as the same native structure is reached. Different pathways may have different frequencies of utilization depending on the thermodynamic favorability of each pathway. This means that if one pathway is found to be more thermodynamically favorable than another, it is likely to be used more frequently in the pursuit of the native structure. As the protein begins to fold and assume its various conformations, it always seeks a more thermodynamically favorable structure than before and thus continues through the energy funnel. Formation of secondary structures is a strong indication of increased stability within the protein, and only one combination of secondary structures assumed by the polypeptide backbone will have the lowest energy and therefore be present in the native state of the protein. Among the first structures to form once the polypeptide begins to fold are alpha helices and beta turns, where alpha helices can form in as little as 100 nanoseconds and beta turns in 1 microsecond.

There exists a saddle point in the energy funnel landscape where the transition state for a particular protein is found. The transition state in the energy funnel diagram is the conformation that must be assumed by every molecule of that protein if the protein wishes to finally assume the native structure. No protein may assume the native structure without first passing through the transition state. The transition state can be referred to as a variant or premature form of the native state rather than just another intermediary step. The folding of the transition state is shown to be rate-determining, and even though it exists in a higher energy state than the native fold, it greatly resembles the native structure. Within the transition state, there exists a nucleus around which the protein is able to fold, formed by a process referred to as "nucleation condensation" where the structure begins to collapse onto the nucleus.

Modeling of protein folding

De novo or ab initio techniques for computational protein structure prediction can be used for simulating various aspects of protein folding. Molecular dynamics (MD) was used in simulations of protein folding and dynamics in silico. First equilibrium folding simulations were done using implicit solvent model and umbrella sampling. Because of computational cost, ab initio MD folding simulations with explicit water are limited to peptides and small proteins. MD simulations of larger proteins remain restricted to dynamics of the experimental structure or its high-temperature unfolding. Long-time folding processes (beyond about 1 millisecond), like folding of larger proteins (>150 residues) can be accessed using coarse-grained models.

Several large-scale computational projects, such as Rosetta@home, Folding@home and Foldit, target protein folding.

Long continuous-trajectory simulations have been performed on Anton, a massively parallel supercomputer designed and built around custom ASICs and interconnects by D. E. Shaw Research. The longest published result of a simulation performed using Anton as of 2011 was a 2.936 millisecond simulation of NTL9 at 355 K. Such simulations are currently able to unfold and refold small proteins (<150 amino acids residues) in equilibrium and predict how mutations affect folding kinetics and stability.

In 2020 a team of researchers that used AlphaFold, an artificial intelligence (AI) protein structure prediction program developed by DeepMind placed first in CASP, a long-standing structureprediction contest The team achieved a level of accuracy much higher than any other group. It scored above 90% for around two-thirds of the proteins in CASP's global distance test (GDT), a test that measures the degree of similarity between the structure, predicted by a computational program, and the empirical structure, determined experimentally in a lab. A score of 100 is considered a complete match, within the distance cutoff used for calculating GDT.

AlphaFold's protein structure prediction results at CASP were described as "transformational" and "astounding". Some researchers noted that the accuracy is not high enough for a third of its predictions, and that it does not reveal the physical mechanism of protein folding for the protein folding problem to be considered solved. Nevertheless, it is considered a significant achievement in computational biology and great progress towards a decades-old grand challenge of biology, predicting the structure of proteins.

title: Protein folding


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Word play

Word play or wordplay (also: play-on-words) is a literary technique and a form of wit in which words used become the main subject of the work, primarily for the purpose of intended effect or amusement. Examples of word play include puns, phonetic mix-ups such as spoonerisms, obscure words and meanings, clever rhetorical excursions, oddly formed sentences, double entendres, and telling character names (such as in the play The Importance of Being Earnest, Ernest being a given name that sounds exactly like the adjective earnest).

Word play is quite common in oral cultures as a method of reinforcing meaning. Examples of text-based (orthographic) word play are found in languages with or without alphabet-based scripts, such as homophonic puns in Mandarin Chinese.

Techniques

 * Tom Swifties: A form of humorous writing where adverbs are chosen to reflect the nature of the situation in a punning way. "Hurry up and get to the back of the ship," Tom said sternly.
 * Wellerisms: Using linguistic fossils and set phrases. Example: "We'll have to rehearse that," said the undertaker as the coffin fell out of the car.
 * Unpaired words: Deliberate use of unusual or obsolete antonyms, such as "I was well-coiffed and sheveled," (back-formation from "disheveled").

 * Spoonerism: An accidental and often humorous transposition of initial letters or sounds, such as "a flock of bats" instead of "a block of flats" or "a bunny phone" instead of "a funny bone".
 * Malapropism: Replacing a word with a different word that sounds similar, either unintentionally or for comedic effect. For example, saying "He is the very pineapple of politeness." instead of pinnacle
 * Anthimeria: Altering a word's regular part of speech. This can occur naturally with the evolution of a language, but can also be done for emphasis or comedic effect. For example, saying "The thunder would not peace at my bidding." using the noun peace as a verb, or "The little old lady turtled across the street."
 * Double entendre: Words or phrases with multiple meanings are used ambiguously with a humorous or sexual (or both) result. For example, May West's "Marriage is a fine institution, but I"m not ready for an institution." and the Groucho Marx line "If I said you had a beautiful body, would you hold it against me?"
 * Portmanteau: Combining two words to create a new word, such as smoke and fog to make smog.

Examples
Most writers engage in word play to some extent, but certain writers are particularly committed to, or adept at, word play as a major feature of their work. Shakespeare's "quibbles" have made him a noted punster. Similarly, P.G. Wodehouse was hailed by The Times as a "comic genius recognized in his lifetime as a classic and an old master of farce" for his own acclaimed wordplay. James Joyce, author of Ulysses, is another noted word-player. For example, in his Finnegans Wake Joyce's phrase "they were yung and easily freudened" clearly implies the more conventional "they were young and easily frightened"; however, the former also makes an apt pun on the names of two famous psychoanalysts, Jung and Freud.

An epitaph, probably unassigned to any grave, demonstrates use in rhyme.
 * Here lie the bones of one 'Bun'
 * He was killed with a gun.
 * His name was not 'Bun' but 'Wood'
 * But 'Wood' would not rhyme with gun
 * But 'Bun' would.

Crossword puzzles often employ wordplay to challenge solvers. Cryptic crosswords especially are based on elaborate systems of wordplay.

An example of modern word play can be found on line 103 of Childish Gambino's "III. Life: The Biggest Troll"."H2O plus my D, that's my hood, I'm living in it"

Rapper Milo uses a play on words in his verse on "True Nen"

 * Keep any heat by the fine China dinner set
 * Your man's caught the chill and it ain't even winter yet

A farmer says, "I got soaked for nothing, stood out there in the rain bang in the middle of my land, a complete waste of time. I'll like to kill the swine who said you can win the Nobel Prize for being out standing in your field!".

Eminem is known for the extensive wordplay in the lyrics of his music.

The Mario Party series is known for its mini-game titles that usually are puns and various plays on words; for example: "Shock, Drop, and Roll", "Gimme a Brake", and "Right Oar Left". These mini-game titles are also different depending on regional differences and take into account that specific region's culture.

Related phenomena
Word play can enter common usage as neologisms.

Word play is closely related to word games; that is, games in which the point is manipulating words. See also language game for a linguist's variation.

Word play can cause problems for translators: e.g., in the book Winnie-the-Pooh a character mistakes the word "issue" for the noise of a sneeze, a resemblance which disappears when the word "issue" is translated into another language.

title: Word play


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Osvaldo Ardiles

Osvaldo César Ardiles (born 3 August 1952), often referred to in Britain as Ossie Ardiles, is an Argentine football manager, pundit and former midfielder who won the 1978 FIFA World Cup as part of the Argentina national team.

A competitive and skilled midfielder, Ardiles became a cult hero in England, along with Glenn Hoddle and compatriot Ricardo Villa, as a player for Tottenham Hotspur. He left England for a period on loan as a result of the outbreak of the Falklands War in 1982, thus missing most of the 1982–83 English season.

After retirement, Ardiles began his management career in England, coaching Swindon Town, Newcastle United and West Bromwich Albion, before returning to Tottenham to become the first Premier League manager from Argentina. As manager of Spurs in the mid-1990s, he played several matches utilizing a formation that had five forwards, a formation that had not been used in English football since the 1960s. During his career, Ardiles has also coached in Mexico, Croatia, Japan, Syria, Saudi Arabia, Malaysia, Israel, Paraguay and his native Argentina. In Ireland, he is a pundit for RTÉ Sport.

Club career
Ardiles was born in Córdoba, and played for Instituto de Córdoba from a young age. As a youngster, Ardiles played football in the streets and was given the nickname Pitón (python) by his brother because of his snake-like dribbling skills. He was named as El Gráfico's best player of the interior in 1974, and abandoned his law degree studies in order to play professional football.

He also played for Club Atlético Belgrano and Huracán. After the 1978 World Cup he moved to England to play for Tottenham Hotspur where he spent ten seasons.

He helped Tottenham win the FA Cup in his third season there (1980–81), and collaborated with pop duo Chas & Dave as well as the rest of the Tottenham players for a song, "Ossie's Dream". He played a big part in another FA Cup triumph the following year, but did not play in the final because it had already been arranged with the Spurs management that he would leave early to join up with Argentina's 1982 World Cup squad.

In the wake of the Falklands War between Britain and Argentina it became difficult for him to return to White Hart Lane and he went on loan to Paris Saint-Germain in France. After one season in Paris, he returned to Tottenham, helping the club to win the UEFA Cup in 1984 (coming on as a substitute in the second leg of the final). In the autumn of 1987, he was caretaker coach under caretaker manager Doug Livermore of Tottenham between the resignation of David Pleat and the appointment of Terry Venables. Ardiles left Spurs in 1988. He then played for Blackburn Rovers, Queens Park Rangers and Swindon Town, before being appointed as manager of Swindon Town in July 1989. He played part of the 1989 American Soccer League season with the Fort Lauderdale Strikers.

On 7 February 2008, Ardiles, along with his fellow countryman Ricardo Villa, was inducted into the Tottenham Hotspur Hall of Fame.

International career
Ardiles was called up to the Argentina senior team by manager César Luis Menotti in 1975. He was a member of the World Cup winning squad in 1978. At the 1982 World Cup he wore the number 1 shirt, as Argentina's policy at the time was to number their players alphabetically by surname, with an exception made so Diego Maradona could wear his preferred number 10.

Management career
In July 1989, Ardiles moved into football management with second division Swindon Town when Lou Macari resigned to join West Ham in July 1989. He wowed fans by replacing the long ball style which had been so successful with a new "Samba style", which saw the Town playing attacking football. Part of this change was the new "diamond formation" which Ardiles implemented: a 4–4–2 style with left-sided, right-sided, attacking and defensive midfielders.

Ten months after he had joined, Ardiles led Swindon to their highest ever league position, finishing fourth in the second division. After beating Blackburn in the first leg of the play-off semi-final, the fans paid tribute with a tickertape reception in the second leg. Swindon went on to win promotion to the top flight for the first time in their history—beating Sunderland in the Play-Off Final—only to have the promotion taken from them ten days later, when the Football League demoted them for irregular payments to players.

The following season, Ardiles was told to sell players to keep the club alive and Wembley hero Alan McLoughlin was the first big-money departure. With Swindon rocked by their pre-season troubles, their form deserted them. By the end of February, relegation threatened, and when Newcastle offered Ardiles the chance to become their new boss, he accepted, becoming the club's first foreign manager. But his time on Tyneside was not a success and he lasted 12 months in the job before being sacked, with the Magpies bottom of the second division, though they achieved safety under his successor Kevin Keegan.

In June 1992 Ardiles replaced Bobby Gould as manager of West Bromwich Albion, who had just missed out on the third division playoffs in 1991–92. At the end of the 1992–93 season, Ardiles guided Albion to victory over Port Vale in the Division Two playoff final. Shortly afterwards he walked out of the Hawthorns to return his former club Tottenham as manager, but his management spell was nowhere near as successful as his spell as a player. Tottenham finished 15th in the Premiership and despite the expensive acquisition of Jürgen Klinsmann and Ilie Dumitrescu in the 1994 close season, Ardiles was sacked in October 1994 with Tottenham languishing in the bottom half of the Premier League. They had just been punished for financial irregularities committed during the late 1980s: with a 1-year FA Cup ban, £600,000 fine and 12 league points deducted. The punishment was later amended to a £1.5million fine and six points deducted but the FA Cup ban and points deduction were later quashed.

Ardiles became coach of J. League Division 1 side Yokohama F. Marinos in January 2000, but was sacked in June 2001 following a poor start to the season. From 2003 to 2005 he coached Tokyo Verdy, with whom he won the 2004 Emperor's Cup, In July 2005 Ardiles was fired after a nine-game winless streak. In mid-2006 he moved to Israel to coach Beitar Jerusalem, from which he quit after only a few months in charge on 18 October 2006 due to severe differences of opinion with the club's board of directors. After a small break he was appointed Club Atlético Huracán manager in his native Argentina in September 2007, he steered the club to 7th in the table before resigning at the end of the Apertura 2007.

He joined Paraguayan club Cerro Porteño in May 2008 but was sacked in August of the same year after a string of poor results and was replaced by Pedro Troglio.

Media career
Ardiles was enlisted by RTÉ Sport for their squad of pundits ahead of the 2010 FIFA World Cup in South Africa. He returned to RTÉ's team for the 2014 FIFA World Cup in Brazil.

Ardiles played Carlos Rey in the 1981 World War II film Escape to Victory.

Personal life
He married fellow Argentine Silvia Navarro in December 1973. Ardiles' cousin, José, was killed during the first Argentinian bombing raid of the Falklands War on 1 May 1982.

In January 2014, Ardiles and Ricardo Villa were involved in a car crash in the Falkland Islands during the filming of Camilo Antolini's 30 for 30 documentary White, Blue and White. Ardiles sustained minor injuries in the accident, and required more than 20 stitches in his head.

Player
Huracán
 * Primera División runner-up: 1976

Tottenham Hotspur
 * FA Cup: 1980–81, 1981–82
 * FA Charity Shield: 1981 (shared)
 * UEFA Cup: 1983–84
 * Football League Cup runner-up: 1981-82

Argentina
 * FIFA World Cup: 1978

Individual
 * World Soccer World 11: 1978, 1982, 1983
 * Football League 100 Legends list (as the only Argentinian)
 * Football League First Division PFA Team of the Year: 1979
 * Tottenham Hotspur F.C. Hall of fame
 * English Football Hall of Fame: 2009
 * Golden Foot: 2013
 * IFFHS Argentina All Times Dream Team: 2021

Manager
Swindon Town
 * Promotion to Division One: 1990

West Bromwich Albion
 * Promotion to Division One: 1993

Shimizu S-Pulse
 * Nabisco Cup: 1996

Yokohama F. Marinos
 * J-League first stage: 2000

Tokyo Verdy
 * Emperor's Cup: 2004–05

Individual
 * J. League Manager of the Year: 1998

title: Osvaldo Ardiles


text:
TDM

TDM may refer to:
 * TDM, a 2023 Indian Marathi comedy film
 * TDM (Macau) (Teledifusão de Macau), a Macanese radio and television network
 * The Yamaha TDM, a motorcycle model
 * Target Disk Mode, a boot mode on certain Macintosh computers
 * Team deathmatch, a mode of gameplay in video games
 * Technical death metal, a subgenre of heavy metal music
 * Telecomunicações de Moçambique, a Mozambican telecommunications and Internet service provider
 * Teresian Daughters of Mary, a Filipino Roman Catholic institute
 * Text data mining, a process of deriving high-quality information from text
 * The Difference Machine, an album by English rock band Big Big Train
 * Therapeutic drug monitoring, a branch of clinical chemistry
 * Time-division multiplexing, a method of transmitting and receiving independent signals over a common signal path
 * Toad Data Modeler, a database design tool
 * Transportation demand management, also traffic demand management or travel demand management
 * DanTDM, British YouTube personality

title: TDM


text:
XNS

XNS or xns may refer to:

Computing

 * Xerox Network Systems, an early computer networking protocol suite
 * Extensible Name Service, an XML-based digital identity architecture

Other uses

 * Kanashi language (ISO 639:xns), a Sino-Tibetan language
 * Navtech System Support (IATA code: XNS), in the List of airline codes
 * Christians (XNS), in the List of acronyms: X

title: XNS