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Vitamin D Deficiency: Know The Signs And Symptoms And Why The Sunshine Vitamin Is Important For You – NDTV

Vitamin D deficiency can cause weak bones and muscle pain

Vitamin D deficiency is quite common. As many as one billion people across the world have low levels of the sunshine vitamin in their blood. Vitamin D is an important nutrient for the body. It required for absorbing calcium and helps in building bones. The vitamin is synthesised in the body in the presence of sunlight.

Building a strong immunity is an important role played by Vitamin D. A strong immunity enables you to fight off illness causing bacteria and viruses. Vitamin D directly interacts which cells that fight infection and thus if you fall sick too often, it could be a sign of Vitamin D deficiency.

Also read:Can Vitamin D Help Fight Acne? Let's Find Out And Know The Best Sources Of Vitamin D

Vitamin D helps in improving absorption of calcium. If you experience bone or back pain regularly, then it could be a sign of Vitamin D deficiency. Studies have also found a link between deficiency of the sunshine vitamin and chronic back pain.

Studies have found that Vitamin D increases production of compounds which are crucial for forming new skin that is a part of the wound-healing process. Slow healing of wounds after an injury or surgery could be indicative of low levels of Vitamin D.

Calcium absorption and bone metabolism are two of the most important functions of Vitamin D. According to healthline.com, people who are diagnosed with bone loss in old age may be deficient in Vitamin D, along with loss of calcium and other minerals.

Also read:These Are The 3 Most Important Minerals For Strong Bones

One of the many causes of Vitamin D deficiency could be muscle pain. The receptor of Vitamin D is present in nerve cells known as nociceptors. These nerve cells sense pain.

Excessive tiredness and fatigue, despite living a healthy lifestyle and sleeping well, could be a sign of Vitamin D deficiency.

Vitamin D deficiency can cause tiredness and fatiguePhoto Credit: iStock

Deficiency of Vitamin D could be linked to depression, especially in older adults. Some studies have found that taking supplements can improve mood and reduce feelings of depression.

Severe hair loss is surely associated with a nutrition deficiency, including low levels of Vitamin D. Female pattern hair loss female-pattern hair loss or alopecia areata could be linked to deficiency of Vitamin D.

Also read:7 Foods That Can Boost Your Hair Growth Naturally

What can you do about Vitamin D deficiency?

In prescribed amounts, taking Vitamin D supplements can help in meeting deficiency of Vitamin D. Spending five to 10 minutes under the sun every day can also be beneficial, as the body synthesises Vitamin D in the presence of sunlight.

Disclaimer: This content including advice provides generic information only. It is in no way a substitute for qualified medical opinion. Always consult a specialist or your own doctor for more information. NDTV does not claim responsibility for this information.

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Vitamin D Deficiency: Know The Signs And Symptoms And Why The Sunshine Vitamin Is Important For You - NDTV

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Chat and a cuppa offer for those isolated due to coronavirus – Keighley News

A FRIENDLY chat on the phone beckons Keighley people at the Conversation Caf.

Keighley Healthy Living is hosting the caf as part of its health and wellbeing support services for local people during the coronavirus pandemic.

Melanie Hey, chief officer at KHL, said people could book a regular weekly slot with one of the volunteers, who would call them up, and could also give support and signpost information in many areas.

Melanie said: The KHL team are missing seeing our regular groups and our local community each week. We appreciate the importance of having regular contact and meeting people.

"Although we have moved many of our groups online we know one of the benefits of attending a group is the opportunity to have a good chat and catch up with people.

"We hope that our conversation cafes will help to support people to feel connected and we look forward to hearing from you.

Anyone who would like to book a weekly chat or a one-off chat, is asked to make themselves a cuppa, pick up the phone and call 01535 677177.

KHL also runs several online groups, a YouTube channel, a Facebook page and a regular newsletter.

Visit khl.org.uk or the Facebook page for further information.

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Chat and a cuppa offer for those isolated due to coronavirus - Keighley News

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Chinese alchemical elixir poisoning – Wikipedia

In Chinese alchemy, elixir poisoning refers to the toxic effects from elixirs of immortality that contained metals and minerals such as mercury and arsenic. The official Twenty-Four Histories record numerous Chinese emperors, nobles, and officials who died from taking elixirs in order to prolong their lifespans. The first emperor to die from elixir poisoning was likely Qin Shi Huang (d. 210 BCE) and the last was Yongzheng (d. 1735). Despite common knowledge that immortality potions could be deadly, fangshi and Daoist alchemists continued the elixir-making practice for two millennia.

The etymology of English elixir derives from Medieval Latin elixir, from Arabic (al-iksr), probably from Ancient Greek (xrion "a desiccative powder for wounds"). Elixir originated in medieval European alchemy meaning "A preparation by the use of which it was sought to change metals into gold" (elixir stone or philosopher's stone) or "A supposed drug or essence with the property of indefinitely prolonging life" (elixir of life). The word was figuratively extended to mean "A sovereign remedy for disease. Hence adopted as a name for quack medicines" (e.g., Daffy's Elixir) and "The quintessence or soul of a thing; its kernel or secret principle". In modern usage, elixir is a pharmaceutical term for "A sweetened aromatic solution of alcohol and water, serving as a vehicle for medicine" (Oxford English Dictionary, 2nd ed., 2009). Outside of Chinese cultural contexts, English elixir poisoning usually refers to accidental contamination, such as the 1937 elixir sulfanilamide mass poisoning in the United States.

Dn "cinnabar; vermillion; elixir; alchemy" is the keyword for Chinese immortality elixirs. The red mineral cinnabar (dnsh lit. "cinnabar sand") was anciently used to produce the pigment vermilion (zhhng ) and the element mercury (shuyn "watery silver" or gng ).

According to the ABC Etymological Dictionary of Old Chinese, the etymology of Modern Standard Chinese dn from Old Chinese *tn (< *tlan?) "red; vermillion; cinnabar", gn in dngn from *tn-kn (< *tlan-klan?) "cinnabar; vermillion ore", and zhn from *tan "a red flag" derive from Proto-Kam-Sui *h-lan "red" or Proto-Sino-Tibetan *tja-n or *tya-n "red". The *t- initial and *t- or *k- doublets indicate that Old Chinese borrowed this item. (Schuessler 2007: 204).

Although the word dan "cinnabar; red" frequently occurs in oracle script from the late Shang Dynasty (c. 16001046 BCE) and bronzeware script and seal script from the Zhou Dynasty (1045256 BCE), paleographers disagree about the graphic origins of the logograph and its ancient variants and . Early scripts combine a dot or stroke (depicting a piece of cinnabar) in the middle of a surrounding frame, which is said to represent:

Many Chinese elixir names are compounds of dan, such as jndn (with "gold") meaning "golden elixir; elixir of immortality; potable gold" and xindn (with "Daoist immortal") "elixir of immortality; panacea", and shndn (with "spirit; god") "divine elixir". Bs zh yo "drug of deathlessness" was another early name for the elixir of immortality. Chinese alchemists would lindn (with "smelt; refine") "concoct pills of immortality" using a dndng (with "tripod cooking vessel; cauldron") "furnace for concocting pills of immortality". In addition, the ancient Chinese believed that other substances provided longevity and immortality, notably the lngzh "Ganoderma mushroom".

The transformation from chemistry-based waidan "external elixir/alchemy" to physiology-based neidan "internal elixir/alchemy" gave new analogous meanings to old terms. The human body metaphorically becomes a ding "cauldron" in which the adept forges the Three Treasures (essence, life-force, and spirit) within the jindan Golden Elixir within the dntin (with "field") "lower part of the abdomen".

In early China, alchemists and pharmacists were one and the same. Traditional Chinese medicine also used less concentrated cinnabar and mercury preparations, and dan means "pill; medicine" in general, for example, dnfng semantically changed from "prescription for elixir of immortality" to "medical prescription". Dan was lexicalized into medical terms such as dnj "pill preparation" and dnyo "pill medicine".

The Chinese names for immortality elixirs have parallels in other cultures and languages, for example, Indo-Iranian soma or haoma, Sanskrit amrita, and Greek ambrosia.

In Chinese history, the alchemical practice of concocting elixirs of immortality from metallic and mineral substances began circa the 4th century BCE in the late Warring states period, reached a peak in the 9th century CE Tang dynasty when five emperors died, and, despite common knowledge of the dangers, elixir poisoning continued until the 18th century Qing dynasty.

The earliest mention of alchemy in China occurs in connection with fangshi ("masters of the methods") specialists in cosmological and esoteric arts employed by rulers from the 4th century BCE (De Woskin 1981: 19).

The 3rd-century BCE Zhanguo Ce and Han Feizi both record a story about King Qingxiang of Chu (r. 298263 BCE) being presented a busi zhi yao "immortality medicine". As the chamberlain was taking the elixir into the palace, a guard asked if it was edible and when he answered yes, the guard grabbed and ate it. The king was angered and condemned the guard to death. A friend of the guard tried to persuade the king, saying, "After all the guard did ask the chamberlain whether it could be eaten before he ate it. Hence the blame attaches to the chamberlain and not to him. Besides what the guest presented was an elixir of life, but if you now execute your servant after eating it, it will be an elixir of death (and the guest will be a liar). Now rather than killing an innocent officer in order to demonstrate a guest's false claim, it would be better to release the guard." This logic convinced the king to let the guard live (Needham and Ho 1970: 316).

Qin Shi Huang, the founder of the Qin dynasty (221206 BCE), feared death and spent the last part of his life seeking the elixir of life. He reportedly died from elixir poisoning (Wright 2001: 49). The first emperor also sent Xu Fu to sail an expeditionary fleet into the Pacific seeking the legendary Mount Penglai where the busi zhi shu "tree of deathlessness" grew, but they never returned.

Interest in elixirs of immortality increased during the Han dynasty (206 BC220 AD). Emperor Wu (15687 BCE) employed many fangshi alchemists who claimed they could produce the legendary substance. The Book of Han says that around 133 BCE the fangshi Li Shaojun said to Emperor Wu, "Sacrifice to the stove [zao ] and you will be able to summon ' things ' [i.e. spirits]. Summon spirits and you will be able to change cinnabar powder into yellow gold. With this yellow gold you may make vessels to eat and drink out of. You will then increase your span of life. Having increased your span of life, you will be able to see the [xian ] of [Penglai] that is in the midst of the sea. Then you may perform the sacrifices feng [] and shan [], and escape death." (tr. Waley 1930: 2).

Wei Boyang's c. 142 Cantong qi, which is regarded as the oldest complete alchemical book extant in any culture, influenced developments in elixir alchemy. It listed mercury and lead as the prime ingredients for elixirs, which limited later potential experiments and resulted in numerous cases of poisoning. It is quite possible that "many of the most brilliant and creative alchemists fell victim to their own experiments by taking dangerous elixirs" (Needham et al. 1976: 74). There is a famous story about animal testing of elixirs by Wei Boyang. Wei entered the mountains to prepare the elixir of immortality, accompanied by three disciples, two of whom were skeptical. When the alchemy was completed he said, "Although the gold elixir is now accomplished we ought first to test it by feeding it to a white dog. If the dog can fly after taking it then it is edible for man; if the dog dies then it is not." The dog fell over and died, but Wei and his disciple Yu took the medicine and immediately died, after which the two cautious disciples fled. Wei and Yu later revived, rejoiced in their faith, took more of the elixir and became immortals (Needham and Ho 1970: 322).

Elixir ingestion is first mentioned in the c. 81 BCE Discourses on Salt and Iron (Pregadio 2000: 166).

During the turbulent Six dynasties period (220589), self-experimentation with drugs became commonplace, and many people tried taking poisonous elixirs of immortality as well as the psychoactive drug Cold-Food Powder. At this time, Daoist alchemists began to record the often fatal side effects of elixirs. In an unusual case of involuntary elixir poisoning, Empress Jia Nanfeng (257300) was forced to commit suicide by drinking "jinxiaojiu" "wine with gold fragments" (Needham and Ho 1970: 326).

The Daoist scholar Ge Hong's c. 320 Baopuzi lists 56 chemical preparations and elixirs, 8 of which were poisonous, with visions from mercury poisoning the most commonly reported symptom (Needham et al. 1976: 8996).

The individuals who experimented with Six Dynasties alchemy often had different understandings and intentions. A single alchemical formula could be interpreted as being "suicidal, therapeutic, or symbolic and contemplative", and its implementation might be "a unique, decisive event or a repeated, ritual phantasmagoria" (Strickmann 1979: 192).

Emperor Ai of Jin (r. 361365) died at the age of twenty-five, as the result of his desire to avoid growing old. The Book of Jin says the emperor practiced bigu "grain avoidance" and consumed alchemical elixirs, but was poisoned from an overdose and "no longer knew what was going on around him" (Needham and Ho 1970: 317). In a sardonic sense, the emperor fulfilled his desire since the elixir "did actually prevent him from growing any older" (Ho 2000: 184).

Emperor Daowu (r. 371409), founder of the Northern Wei dynasty, was cautiously interested in alchemy and used condemned criminals for clinical trials of immortality elixirs (like Mithridates VI of Pontus r. 12063 BCE) . The Book of Wei records that in 400, he instituted the office of the Royal Alchemist, built an imperial laboratory for the preparation of drugs and elixirs, and reserved the Western mountains for the supply of firewood (used in the alchemical furnaces). "Furthermore, he ordered criminals who had been sentenced to death to test (the products) against their will. Many of them died and (the experiments gave) no decisive result." (tr. Needham and Ho 1970: 321).

Many texts from the Six dynasties period specifically warned about the toxicity of elixirs. For instance, the Shangqing School Daoist pharmacologist Tao Hongjing's 499 Zhen'gao (, Declarations of the Perfected) describes taking a White Powder elixir.

When you have taken a spatulaful of it, you will feel an intense pain in your heart, as if you had been stabbed there with a knife. After three days you will want to drink, and when you have drunk a full hu [about 50 liters] your breath will be cut off. When that happens, it will mean that you are dead. When your body has been laid out, it will suddenly disappear, and only your clothing will remain. Thus you will be an immortal released in broad daylight by means of his waistband. If one knows the name of the drug [or, perhaps, the secret names of its ingredients] he will not feel the pain in his heart, but after he has drunk a full hu he will still die. When he is dead, he will become aware that he has left his corpse below him on the ground. At the proper time, jade youths and maidens will come with an azure carriage to take it away. If one wishes to linger on in the world, he should strictly regulate his drinking during the three days when he feels the pain in his heart. This formula may be used by the whole family. (tr. Strickmann 1979: 137138)

Within this context, Strickmann says a prospective Daoist alchemist must have been strongly motivated by faith and a firm confidence in his posthumous destiny, in effect, "he would be committing suicide by consecrated means." Tao Hongjing's disciple Zhou Ziliang (497516) had repeated visions of Maoshan divinities who said his destiny was to become an immortal, and instructed him to commit ritual suicide with a poisonous elixir composed of mushrooms and cinnabar (Strickmann 1994: 40). In 517, Tao edited the Zhoushi mingtong ji (Records of Mr. Zhou's Communications with the Unseen) detailing his disciples visions.

The Liang dynasty founder Emperor Wu (r. 502549) was cautious about taking elixirs of immortality. He and Tao Hongjing were old friends, and the History of the Southern Dynasties says the emperor requested him to study elixir alchemy. After Tao had learned the secret art of making elixirs, he was worried about the shortage of materials. "So the emperor supplied him with gold, cinnabar, copper sulphate, realgar, and so forth. When the process was accomplished the elixirs had the appearance of frost and snow and really did make the body feel lighter. The emperor took an elixir and found it effective." (tr. Needham et al. 1976: 120). Tao spent his last years working on different elixirs and presented three to the emperor, who had refused immortality elixirs from Deng Yu (who claimed to have lived 30 years without food, only consuming pieces of mica in stream water).

Emperor Wenxuan (r. 550559) of the Northern Qi dynasty was an early skeptic about immortality elixirs. He ordered alchemists to make the jiuhuan jindan (Ninefold Cyclically Transformed Elixir), which he kept in a jade box, and explained, "I am still too fond of the pleasures of the world to take flight to the heavens immediatelyI intend to consume the elixir only when I am about to die." (tr. Needham and Ho 1970: 320).

At least five Tang dynasty (618907) emperors were incapacitated and killed by immortality elixirs. In the 9th century Tang imperial order of succession, two father-son emperor pairs died from elixirs: first Xianzong (r. 805820) and Muzong (r. 820824), then Wuzong (r. 840846) and Xuanzong (r. 847859). In historic recurrences, the newly enthroned emperor understandably executed the Daoist alchemists whose elixirs had killed his father, and then subsequently came to believe in other charlatans enough to consume their poisonous elixirs (Ho 2000: 184).

Emperor Xianzong (r. 805820) indirectly lost his life due to elixir poisoning. The Xu Tongzhi (Supplement to the Historical Collections) says, "Deluded by the sayings of the alchemists, [Xianzong] ingested gold elixirs and his behaviour became very abnormal. He was easily offended by those officials whom he daily met, and thus the prisons were left with little vacant space." (tr. Needham and Ho 1970: 317). In response, an official wrote an 819 memorial to the throne that said:

Of late years, however, (the capital) has been overrun by a host of pharmacists and alchemists ... recommending one another right and left with ever wilder and more extravagant claims. Now if there really were immortals, and scholars possessing the Tao, would they not conceal their names and hide themselves in mountain recesses far from the ken of man? ... The medicines of the sages of old were meant to cure bodily illnesses, and were not meant to be taken constantly like food. How much less so these metallic and mineral substances which are full of burning poison! ... Of old, as the Li Chi says, when the prince took physic, his minister tasted it first, and when a parent was sick, his son did likewise. Ministers and sons are in the same position. I humbly pray that all those persons who have elixirs made from transformed metals and minerals, and also those who recommend them, may be compelled to consume (their own elixirs) first for the space of one year. Such an investigation will distinguish truth from falsehood, and automatically clarify the matter by experiment. (abridged, tr. Needham and Ho 1970: 318)

After the emperor rejected this appeal, the palace eunuchs Wang Shoucheng and Chen Hongzhi assassinated him in 820.

When Xianzong's son and successor Emperor Muzong (r. 820824) came to the throne, he executed the alchemists who had poisoned his father, but later began to take immortality elixirs himself. An official wrote Muzong an 823 memorial that warned:

Medicines are for use against illnesses, and should not be taken as food. ... Even when one is ill medicines must be used with great circumspection; how much more so when one is not ill. If this is true for the common people how much more so will it be for the emperor! Your imperial predecessor believed the nonsense of the alchemists and thus became ill; this your majesty already knows only too well. How could your majesty still repeat the same mistake? (tr. Needham and Ho 1970: 319)

The emperor appreciated this reasoning but soon afterwards fell ill and died from poisoning. Palace eunuchs supposedly used poisonous elixirs to assassinate Muzong's young successor Emperor Jingzong (r. 824827) (Needham et al. 1976: 151, 182).

The next Tang emperor to die from elixir poisoning was Wuzong (r. 840846). According to the Old Book of Tang, "The emperor [Wuzong] favoured alchemists, took some of their elixirs, cultivated the arts of longevity and personally accepted (Taoist) talismans. The medicines made him very irritable, losing all normal self-control in joy or anger; finally when his illness took a turn for the worse he could not speak for ten days at a time." Chancellor Li Deyu and others requested audiences with the emperor, but he refused and subsequently died in 846 (Needham and Ho 1970: 319).

Wuzong's successor Emperor Xunzong (r. 846849) astonishingly also died of elixir poisoning. Xunzong made himself the patron of some Daoists who concocted immortality elixirs of vegetable origin, possibly because his father Wuzong had died from metallic and mineral elixir poisoning (Needham et al. 1976: 146). The New Book of Tang records that the emperor received a wine tincture of ivy (, Hedera helix) that the Daoist adept Jiang Lu claimed would turn white hair black and provide longevity. However, when the emperor heard that many people died a violent death after drinking ivy tincture, he stopped taking it. Jiang was publicly shamed and the emperor granted his request to search in the mountains for the right plant, but he never appeared again (Needham et al. 1976: 147). According to the 890 Dongguan zuoji (Record of Memorials from the Eastern Library), "A medical official, Li [Xuanbo], presented to the emperor [Xuanzong] cinnabar which had been heated and subdued by fire, in order to gain favour from him. Thus the ulcerous disease of the emperor was all attributable to his crime." (tr. Needham and Ho 1970: 319).

Besides emperors, many Tang literati were interested in alchemy. Both Li Bai (Waley 1950: 5556) and Bai Juyi (Ho, Goh, and Parker 1974) wrote poems about the Cantong qi and alchemical elixirs. Other poets, including Meng Haoran, Liu Yuxi, and Liu Zongyuan also referred to elixir compounding in their works (Pregadio 2000: 171).

The influential Tang physician and alchemist Sun Simiao's c. 640 alchemical Taiqing zhenren dadan (Great Purity Essentials of Elixir Manuals for Oral Transmission) recommends 14 elixir formulas he found successful, most of which seem poisonous, containing mercury and lead, if not arsenic, as ingredients (Needham et al. 1976: 133). Sun's medical c. 659 Qianjin yifang (Supplement to the Thousand Golden Remedies) categorically states that mercury, realgar, orpiment, sulphur, gold, silver, and vitriol are poisonous, but prescribed them in much larger amounts for elixirs than for medicines. In contrast to drinking soluble arsenic (as in groundwater), when powdered arsenic is eaten "astonishing degrees of tolerance can be achieved", and Sun Simiao might have thought that when human beings reached to a level "approaching that of the immortals their bodies would no longer be susceptible to poison" (Needham et al. 1976: 135).

Tang alchemists were well aware of elixir poisoning. The c. 8th9th century Zhenyuan miaodao yaole (Synopsis of the Essentials of the Mysterious Dao of the True Origin) lists 35 common mistakes in elixir preparation: cases where people died from eating elixirs made from cinnabar, mercury, lead, and silver; cases where people suffered from boils on the head and sores on the back by ingesting cinnabar prepared by roasting together mercury and sulphur, and cases where people became seriously ill through drinking melted "liquid lead" (Needham and Ho 1970: 330). The c. 850 Xuanjie lu (Record of Mysterious Antidotes)which is notable as the world's oldest printed book on a scientific subjectrecommends a potent herbal composition that serves both as an elixir and as an antidote for common elixir poisoning (Needham and Ho 1970: 335). The procedure to make Shouxian wuzi wan (Five-herbs Immortality-safeguarding Pills) is to take 5 ounces each of Indian gooseberry, wild raspberry, dodder, five-flavor berry, and broadleaf plantain and pound them into flour. Mix it with boxthorn juice and false daisy juice and dry. Heat almonds and good wine in a silver vessel, and add foxglove, tofu, and "deer glue". Combine this with the five herbs, and dry into small pills. The usual dosage is 30 pills a day taken with wine, but one should avoid eating pork, garlic, mustard, and turnips when taking the medicine (Needham and Ho 1970: 335).

During the Tang period, Chinese alchemists divided into two schools of thought about elixir poisoning. The first altogether ignored the poison danger and considered the unpleasant symptoms after taking an elixir as signs of its efficacy. The c. 6th century Taiqing shibi ji (Records of the Rock Chamber) described away the side effects and recommended methods of bringing relief.

After taking an elixir, if your face and body itch as though insects were crawling over them, if your hands and feet swell dropsically, if you cannot stand the smell of food and bring it up after you have eaten it, if you feel as though you were going to be sick most of the time, if you experience weakness in the four limbs, if you have to go often to the latrine, or if your head or stomach violently achedo not be alarmed or disturbed. All these effects are merely proofs that the elixir you are taking is successfully dispelling your latent disorders. (tr. Needham and Lu 1974: 283)

Many of these symptoms are characteristic of metallic poisoning: formication, edema, and weakness of the extremities, later leading to infected boils and ulcers, nausea, vomiting, gastric and abdominal pain, diarrhea, and headaches (Needham and Lu 1974: 283). For relieving the side-effects when the elixir takes effect, the Taiqing shibi ji recommends that one should take hot and cold baths, and drink a mixture of scallion, soy sauce, and wine. If that does not bring relief, then one should combine and boil a hornets' nest, spurge, Solomon's seal, and ephedra into a medicine and take one dose (Needham and Ho 1970: 331).

The second school of alchemists, admitted that some metal and mineral elixir constituents were poisonous and tried either to neutralize them or to replace them with less dangerous herbal substances (Needham et al. 1976: 182). For instance, the 8th-century Zhang zhenren jinshi lingsha lun (The Adept Zhang's Discourse on Metals, Minerals, and Cinnabar) emphasized the poisonous nature of gold, silver, lead, mercury, and arsenic, and described witnessing many cases of premature death brought about by consuming cinnabar. Zhang believed however that the poisons could be rendered harmless by properly choosing and combining adjuvant and complimentary ingredients; for example gold should always be used together with mercury, while silver can only be used when combined with gold, copper carbonate, and realgar for the preparation of the jindan Golden Elixir (tr. Needham and Ho 1970: 331). Many Tang alchemical writers returned to the fashion of using obscure synonyms for ingredients, perhaps because of the alarming number of elixir poisonings, and the desire to dissuade amateur alchemists from experimenting on themselves (Needham et al. 1976: 138). By the end of the Yuan dynasty (12711368), the more cautious alchemists had generally changed the elixirs ingredients from minerals and metals to plants and animals (Ho and Lisowski 1997: 39).

The late Tang or early Song Huangdi jiuding shendan jingjue (Explanation of the Yellow Emperor's Manual of the Nine-Vessel Magical Elixir) says, "The ancient masters (lit. sages) all attained longevity and preserved their lives (lit. bones) by consuming elixirs. But later disciples (lit. scholars) have suffered loss of life and decay of their bones as the result of taking them." The treatise explains the secret ancient methods for rendering elixir ingredients harmless by treating them with wine made from chastetree leaves and roots, or with saltpeter and vinegar. Another method of supposedly removing the poison from mercury was to put it in three-year-old wine, add sal ammoniac and boil it for 100 days (Needham and Ho 1970: 3323).

Two rulers died from elixir poisoning during the Five Dynasties period (907979) of political turmoil after the overthrow of the Tang dynasty. Zhu Wen or Emperor Taizu (r. 907912), the founder of the Later Liang dynasty, became seriously incapacitated as a result of elixir poisoning, and fell victim to an assassination plot. Li Bian or Emperor Liezu (r. 937943), the founder of the Southern Tang kingdom, took immortality elixirs that made him irritable and deathly ill (Needham et al. 1976: 180).

The Daoist adept Chen Tuan (d. 989) advised two emperors that they should not worry about elixirs but direct their minds to improving the state administration, Chai Rong or Emperor Shizong of Later Zhou in 956, and then Emperor Taizu of Song in 976 (Needham et al. 1976: 194).

After its heyday in the Tang dynasty Daoist alchemy continued to flourish during the Song dynasty (9601279) period. However, since six Tang emperors and many court officials died from elixir poisoning, Song alchemists exercised more caution, not only in the composition of the elixirs themselves, but also in attempts to find pharmaceutical methods of counteracting the toxic effects. The number of ingredients used in elixir formulas was reduced and there was a tendency to return to the ancient and difficult terminology of the Cantongqi, perhaps to conceal the processes from rash and ignorant operators. Psycho-physiological neidan alchemy became steadily more popular than laboratory waidan alchemy (Needham et al. 1976: 208).

During the Song dynasty, the practice of consuming metallic elixirs was not confined to the imperial court and expanded to anyone wealthy enough to pay. The author and official Ye Mengde (10771148) described how two of his friends had died from elixirs of immortality in one decade. First, Lin Yanzhen, who boasted about his health and muscular strength, took an elixir for three years, "Whereupon ulcers developed in his chest, first near the hairs as large as rice-grains, then after a couple of days his neck swelled up so that chin and chest seemed continuous." Lin died after ten months of suffering, and his doctors discovered cinnabar powder had accumulated in his pus and blood. Second, whenever Xie Renbo "heard of anyone who had some cinnabar subdued by fire he went after it, caring nothing about the distance, and his only fear was that he would not have enough." He also developed ulcers on the chest. Although his friends noticed changes in his appearance and behavior, Xie did not recognize that he had been poisoned, "till suddenly it came upon him like a storm of wind and rain, and he died in a single night." (tr. Needham and Ho 1970: 320)

The scientist and statesman Shen Kuo's 1088 Dream Pool Essays suggested that mercury compounds might be medicinally valuable and needed further studyforeshadowing the use of metallic compounds in modern medicine, such as mercury in salvarsan for syphilis or antimony for visceral leishmaniasis. Shen says his cousin once transformed cinnabar into an elixir, but one of his students mistakenly ate a leftover piece, became delirious, and died the next day.

Now cinnabar is an extremely good drug and can be taken even by a newborn baby, but once it has been changed by heat it can kill an (adult) person. If we consider the change and transformation of opposites into one another, since (cinnabar) can be changed into a deadly poison why should it not also be changed into something of extreme benefit? Since it can change into something which kills, there is good reason to believe that it may have the pattern-principle [li] of saving life; it is simply that we have not yet found out the art (of doing this). Thus we cannot deny the possibility of the existence of methods for transforming people into feathered immortals, but we have to be very careful about what we do. (tr. Needham and Ho 1970: 327).

Su Shi (10371101), the Song dynasty scholar and pharmacologist, was familiar with the life-prolonging claims of alchemists, but wrote in a letter that, "I have recently received some cinnabar (elixir) which shows a most remarkable colour, but I cannot summon up enough courage to try it." (tr. Needham and Ho 1970: 320).

The forensic medical expert Song Ci was familiar with the effects of metal poisoning, and his c. 1235 Collected Cases of Injustice Rectified handbook for coroners gives a test for mercury poisoning: plunge a piece of gold into the intestine or tissues and see if a superficial amalgam forms. He also describes the colic, cramps, and discharge of blood from arsenic poisoning, and gives several antidotes including emetics.

Ming dynasty (13681644) authorities strongly disapproved of immortality elixirs, but the Jiajing Emperor (r. 15211567) supposedly died from consuming them. The emperor was interested in the art of immortality and put great confidence in Daoist physicians, magicians, and alchemists. One named Wang Jin , who was appointed a Physician-in-Attendance in the Imperial Academy of Medicine, convinced the emperor that eating and drinking from vessels made of alchemical gold and silver would bring about immortality, but it only resulted in his death. Wang fled but was caught and exiled to the frontiers in 1570 (Needham et al. 1976: 212).

Li Shizhen's classic 1578 Compendium of Materia Medica discusses the historical tradition of producing gold and cinnabar elixirs, and concludes, "(the alchemists) will never realise that the human body, which thrives on water and the cereals, is unable to sustain such heavy substances as gold and other minerals within the stomach and intestines for any length of time. How blind it is, in the pursuit of longevity, to lose one's life instead!" (tr. Needham and Ho 1970: 325326). In another section, Li criticizes alchemists and pharmacologists for perpetuating the belief in mercury elixirs.

I am not able to tell the number of people who since the Six Dynasties period (3rd to 6th centuries) so coveted life that they took (mercury), but all that happened was that they impaired their health permanently or lost their lives. I need not bother to mention the alchemists, but I cannot bear to see these false statements made in pharmacopoeias. However, while mercury is not to be taken orally, its use as a medicine must not be ignored. (tr. Needham and Ho 1970: 325326)

The Qing dynasty Yongzheng Emperor (r. 17221735) was the last Chinese ruler known to die from elixir poisoning. He was a superstitious man, affected by portents and omens, and a firm believer in Daoist longevity techniques. Taking immortality elixirs is thought to have caused his sudden death in 1735 (Zelin 2002: 229).

The Chinese tradition of using toxic heavy metals in elixirs of immortality has historical parallels in Ayurvedic medicine. Rasa shastra is the practice of adding metals and minerals to herbal medicines, rasayana is an alchemical tradition that used mercury and cinnabar for lengthening lifespan, rasevara is a tradition that advocated the use of mercury to make the body immortal, and samskara is a process said to detoxify heavy metals and toxic herbs.

The historians of Chinese science Joseph Needham and Ho Peng-Yoke wrote a seminal article about poisonous alchemical elixirs (1959, 1970). Based upon early Chinese descriptions of elixir poisoning, they decisively demonstrated a close correspondence with the known medical symptoms of mercury poisoning, lead poisoning, and arsenic poisoning. Compare the historical descriptions of Jin Emperor Ai (d. 365) who "no longer knew what was going on around him" and Tang Emperor Wuzong (d. 846) who was "very irritable, losing all normal self-control in joy or anger ... he could not speak for ten days at a time" with the distinctive psychological effects of mercury poisoning: progressing from "abnormal irritability to idiotic, melancholic, or manic conditions" (1970: 327). Needham and his collaborators further discussed elixir poisoning in the Science and Civilisation in China series, particularly Needham and Lu Gwei-djen (1974), and Needham, Ho, and Lu (1976).

Although Chinese elixir poisoning may lead some to dismiss Chinese alchemy as another example of human follies in history, Ho Peng-Yoke and F. Peter Lisowski note its positive aspect upon Chinese medicine. The caution given to elixir poisoning later led Chinese alchemy to "shade imperceptibly" into iatrochemistry, the preparation of medicine by chemical methods, "in other words chemotherapy" (1997: 39).

A recent study found that Chinese emperors lived comparatively short lives, with a mean age at death of emperors at 41.3, which was significantly lower than that of Buddhist monks at 66.9 and traditional Chinese doctors at 75.1. Causes of imperial death were natural disease (66.4%), homicide (28.2%), drug toxicity (3.3%), and suicide (2.1%). Homicide resulted in a significantly lower age of death (mean age 31.1) than disease (45.6), suicide (38.8), or drug toxicity (43.1, mentioning Qin Shi Huang taking mercury pills of immortality). Lifestyles seem to have been a determining factor, and 93.2% of the emperors studied were overindulgent in drinking alcohol, sexual activity, or both (Zhao et al. 2006: 1295). The study does not refer to the Chinese belief that the arsenic sulphides realgar and orpiment, frequently used in immortality elixirs, had aphrodisiac properties (Needham and Lu 1974: 285).

A significant question remains unanswered. If the insidious dangers of alchemical elixir poisoning were common knowledge, why did people continue to consume them for centuries? Joseph Needham and his collaborators suggested three hypothetical explanations, and Michel Strickmann proposed another.

Needham and Lu's first explanation is that many alchemical mineral preparations were capable of giving an "initial exhilaration" or transient sense of well-being, usually involving weight loss and increased libido. These preliminary tonic effects could have acted as a kind of "bait" inveigling an elixir-taker deeper into substance intoxication, even to the point of death (1974: 282). Chinese medical texts recorded that realgar (arsenic disulphide) and orpiment (arsenic trisulphide) were aphrodisiacs and stimulated fertility, while cinnabar and sulphur elixirs increased longevity, averted hunger, and "lightened the body" (namely, qngshn , which is a common description of elixir effects) (1974: 285).

Wine, as mentioned above, was both prescribed to be drunk when taking elixir pills and to relieve the unpleasant side-effects of elixir poisoning. Needham and Lu further suggest the possibility that elixir alchemy included hallucinogenic drugs, tentatively identifying the busi zhi yao "drug of deathlessness" as fly-agaric and busi zhi shu "tree of deathlessness" as birch (1974: 117). The elixir that Tao Hongjing's disciple Zhou Ziliang took to commit suicide "probably had hallucinogenic and toxic mushrooms" (1974: 296). In the present day, realgar wine is traditionally consumed as part of the Dragon Boat Festival.

The apparent incorruptibility of an elixir-taker's corpse is Needham and Lu's second explanation for the persistent belief in immortality elixirs. They suggest that in some cases a body did not decompose because the deceased had died from mercury or arsenic poisoning, which is forensically known to often preserve a corpse from decay. For a believer in Daoist immortality drugs, even when an elixir-taker had unmistakably died, if the corpse was comparatively undecomposed, that could be interpreted as proof that the adept had become a xian immortal, as well as evidence for the alchemical elixir's efficacy. (1974: 298).

Terminal incorruptibility was an ancient Chinese belief associated with jade, gold, and cinnabar. The Baopuzi says, "When gold and jade are inserted into the nine orifices, corpses do not decay. When salt and brine are absorbed into flesh and marrow, dried meats do not spoil. So when men ingest substances which are able to benefit their bodies and lengthen their days, why should it be strange that (some of these) should confer life perpetual?" The abolition of decay was believed to demonstrate the power of elixirs, "the corruptible had put on incorruptibility" (Needham and Lu 1974: 284). Chinese jade burial suits are a better known example of using a mineral to preserve corpses.

There is a possibility that Sun Simiao (above) died from taking mercury elixirs (Needham and Ho 1970: 330). According to Sun's hagiography in the 10th-century Xuxian zhuan (Further Biographies of the Immortals), after his death in 682 there was no visible sign of putrefaction, "After more than a month had passed there was no change in his appearance, and when the corpse was raised to be placed in the coffin it was as light as (a bundle of) empty clothes." (tr. Needham and Lu 1974:298).

The incorruptibility stories about elixir users were not all myth, and recent archeological evidence showed that the ancient Chinese knew how "to achieve an almost perpetual conservation". The 1972 excavation of a tomb at Mawangdui discovered the extremely well-preserved body of Xin Zhui or Lady Dai, which resembled that of "a person who had died only a week or two before" (Needham and Lu 1974: 303304). A subsequent autopsy on her corpse found "abnormally high levels" of mercury and lead in her internal organs (Brown 2002: 213).

Needham and Lu's third justification for taking poisonous elixirs is a drug-induced "temporary death", possibly a trance or coma. In the classic legend (above) about Wei Boyang drinking an elixir of immortality, he appears to die, subsequently revives, and takes more elixir to achieve immortality.

The Baopuzi describes a Five Mineral-based multicolored Ninefold Radiance Elixir that can bring a corpse back to life: "If you wish to raise a body that has not been dead for fully three days, bathe the corpse with a solution of one spatula of the blue elixir, open its mouth, and insert another spatula full; it will revive immediately." (tr. Ware 1966:82).

A Tang Daoist text prescribes taking an elixir in doses half the size of a millet grain, but adds, "If one is sincerely determined, and dares to take a whole spatula-full all at once, one will temporarily die [zns ] for half a day or so, and then be restored to life like someone waking from sleep. This however is perilous in the extreme." (tr. Needham and Lu 1974: 295).

Michel Strickmann, a scholar of Daoist and Buddhist studies, analyzed the well-documented Shangqing School's alchemy in the Maoshan revelations and in the life of Tao Hongjing, and concluded that scholars need to reexamine the Western stereotype of "accidental elixir poisoning" that supposedly applied to "misguided alchemists and their unwitting imperial patrons". Since Six Dynasties and Tang period Daoist literature thoroughly, "even rapturously", described the deadly toxic qualities of many elixirs, and Strickmann proposed that some of the recorded alchemical deaths were intentional ritual suicide (1979: 191). Two reviewers disagreed about Strickmann's conclusions. The first questions why he defends the logic of alchemical suicide rather than simply accepting the idea of accidental elixir poisoning, and says Tao Hongjing never experimented with alchemy seriously enough to achieve suicide himselfbut fails to mention Strickmann's prime example: Tao's disciple Zhou Ziliang whom Shangqing deities reportedly instructed to prepare a poisonous elixir and commit suicide in order to achieve immortality (Chen 1981: 547). The second describes Strickmann's chapter as "one of the most thorough and useful" in the volume, and says he proves that it is "almost ludicrous to assume that a Taoist (commoner or emperor) could have died from accidental elixir poisoning" (Cass 1982: 9293).

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Chinese alchemical elixir poisoning - Wikipedia

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Synthesis of proteins by automated flow chemistry – Science Magazine

Fully synthetic whole proteins in reach

Solid-phase peptide synthesis of homogeneous peptides longer than about 50 amino acids has been a long-standing challenge because of inefficient coupling and side reactions. Hartrampf et al. used an automated chemistry platform to optimize fast-flow peptide synthesis and were able to produce fully synthetic single-domain proteins (see the Perspective by Proulx). The targets included proinsulin and enzymes such as barnase and a version of HIV-1 protease containing multiple noncanonical amino acids. Refolded peptides were nearly indistinguishable from recombinant proteins, and the synthesized enzymes had activity close to that of their ribosomally synthesized counterparts. This method will enable fast, on-demand synthesis of small proteins with a vastly expanded pool of precursor amino acids.

Science, this issue p. 980; see also p. 941

Ribosomes can produce proteins in minutes and are largely constrained to proteinogenic amino acids. Here, we report highly efficient chemistry matched with an automated fast-flow instrument for the direct manufacturing of peptide chains up to 164 amino acids long over 327 consecutive reactions. The machine is rapid: Peptide chain elongation is complete in hours. We demonstrate the utility of this approach by the chemical synthesis of nine different protein chains that represent enzymes, structural units, and regulatory factors. After purification and folding, the synthetic materials display biophysical and enzymatic properties comparable to the biologically expressed proteins. High-fidelity automated flow chemistry is an alternative for producing single-domain proteins without the ribosome.

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Synthesis of proteins by automated flow chemistry - Science Magazine

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Oncoprotein SND1 hijacks nascent MHC-I heavy chain to ER-associated degradation, leading to impaired CD8+ T cell response in tumor – Science Advances

INTRODUCTION

Exploring the strategies for tumor immunotherapy is highly dependent on the discovery of molecular mechanisms of tumor immune escape. Tumor cells can escape immune response through loss of antigenicity and/or immunogenicity or by coordinating a suppressive immune microenvironment. Therefore, distinct therapeutic strategies may be required, depending on the mechanisms. Tumor immunotherapy strategies mediated by T cells rely on the functional competence of multiple immunological elements. For example, therapeutic monoclonal antibodies designed to disrupt inhibitory signals received by T cells through the Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and Programmed cell death protein 1 (PD-1) have been demonstrating long-term survival benefits for some patients with metastatic melanoma (1, 2). However, not all tumors appear to respond effectively. The heterogeneity of cancer suggests the necessity to explore the additional immunoregulatory mechanisms. Defect in the surface expression of major histocompatibility complex class I (MHC-I) molecules is one of the most important reasons for tumor immune escape due to decreased recognition by CD8+ T cells, which has been found in approximately 20 to 60% of common solid cancers, including melanoma and lung, breast, renal, prostate, and bladder cancers, (3, 4). The molecular mechanisms underlying these changes vary according to the tumor type. These alterations can be genetic or regulatory at the transcriptional or posttranscriptional level (511).

Staphylococcal nuclease and tudor domain containing 1 (SND1) is a newly identified oncoprotein that is highly expressed in almost all the detected different tumor cells (1214). SND1 was first identified as a transcriptional coactivator for Epstein-Barr virus nuclear antigen 2. It is a ubiquitously expressed and highly conserved protein in mammals and plays important physiological roles in a variety of cellular processes (15). It comprises a tandem repeat of four staphylococcal nuclease (SN)like domains (referred to as SN domains) at the N terminus and a fusion of a Tudor domain with a partial SN domain at the C terminus (referred to as TSN domain) (16). Our current studies and others studies have demonstrated that SND1 regulates the differentiation and migration of different tumor cells via variant signal pathways at the cellular level (1719). For example, SND1 expression is up-regulated in breast cancer tissues, and it associates with transforming growth factor signaling pathway to promote epithelial-mesenchymal transition in breast cancer (20). SND1 regulates the cadherin switch for epithelial-mesenchymal transition in ovary SKOV3 cells (17). However, the fundamental impact of SND1 on the tumorigenesis in vivo is largely unknown. In the present study, we demonstrate that in tumor cells, SND1 is a novel endoplasmic reticulum (ER)associated protein hijacking the nascent heavy chain (HC) of MHC-I to ER-associated degradation (ERAD) process. With reduced expression of MHC-I on tumor cell membrane, it would be easy for tumor cells to orchestrate a cancer-favored immune microenvironment and escape immune response.

To investigate the fundamental role of oncoprotein SND1 in tumor proliferation, we performed affinity purification and mass spectrometry to identify SND1-associated proteins from cellular extracts of HeLa cells with stable expression of SND1-FLAG. As shown in Fig. 1A, a group of ER-related proteins were coprecipitated with SND1, including human leukocyte antigenA (HLA-A; the HC of human MHC-I), Valosin-containing protein (VCP), SEC61 translocon subunit alpha (SEC61A), and ribosomal protein L7a (RPL7A). As MHC-I molecule plays essential roles in antigen presentation, we therefore focused on investigating the relationship between SND1 and HLA-A. Coimmunoprecipitation (Co-IP) assay was performed to verify the association of SND1 and HLA-A in HeLa cells. As shown in Fig. 1B, the endogenous HLA-A was efficiently associated with ectopically overexpressed SND1-FLAG. In addition, anti-HC10 antibody that could specifically recognize immature (unfolded/partially folded) conformation of HLA-A, HLA-B, and HLA-C was used to detect the in vivo physical association of endogenous SND1 and HLA-A. The endogenous SND1 was efficiently coimmunoprecipitated with anti-HC10 antibody (Fig. 1C) and vice versa. HLA-A or HLA-B was coimmunoprecipitated with anti-SND1 antibody (Fig. 1D). Consistently, the Duolink assay [Fig. 1E, red dots indicate the proximity ligation assay (PLA) probe signal] and immunofluorescence assay (Fig. 1F) further confirmed the cellular colocalization of SND1 and HLA-A. We then mapped the interaction domain between SND1 and HLA-A by glutathione S-transferase (GST) pull-down assay. The bacterially produced GST-fusion protein containing full-length SND1 (GST-SND1), SN domain (GST-SN), TSN domain (GST-TSN) (as indicated in Fig. 1G), or GST alone was purified using glutathione agarose beads and used to incubate with HLA-A in vitrotranslated from rabbit reticulocytes. As shown in Fig. 1G, the full length of SND1 or SN domain, but not TSN domain, efficiently associated with HLA-AFLAG. Likewise, the GST-fusion proteins containing full-length HLA-A (GSTHLA-A), A1 domain (GSTHLA-AA1), A2 domain (GSTHLA-AA2), A3 domain (GSTHLA-AA3), C domain (GSTHLA-AC) (as indicated in Fig. 1H), or GST alone were used to incubate with recombinant histidine-tagged SND1 (His-SND1) purified from Escherichia coli. As shown in Fig. 1H, the full length of HLA-A, domain A1, domain A3, or domain C, but not domain A2, efficiently associated with His-SND1. To further consolidate the molecular interface required for the interaction between SND1 and HLA-A, FLAG-tagged different domain deletion mutants of SND1 were generated. Immunoprecipitation analysis in HeLa SND1-KO (knockout) cells demonstrated that the SN3 region of SND1 was required for the interaction of HLA-A (Fig. 1I). These data prompted us to interrogate the three-dimensional conformation for the complex of SND1 and HLA-A. Because the complex structure was not determined experimentally, we performed docking and molecular dynamics simulation to predict their associated conformation (2123). The resulting structure (Fig. 1J) showed that the interacting interface was located between the SN3 region of SND1 and domains A1 and A3 of HLA-A. The electrostatic interaction between the basic and acidic amino acids on SN3 region and A3 domain might play an important role in the association process. The key residues on the interface included K484, K496, K490, K401, K450, and R384 in SN3 domain and E232, E229, and D227 in A3 domain. The interaction between K401-E232, K496-E229, and K496-D227 were identified to be vital for the association of SND1 with HLA-A. These results indicated that SND1 could physically interact with the immature form of HC in partially folded/unfolded conformation, which raises the question about the functional association of SND1 with nascent HC of MHC-I.

(A) Immunopurification and mass spectrometry of SND1-containing protein complexes. Cellular extracts from HeLa cells stably expressing SND1-FLAG were immunopurified with anti-FLAG affinity beads and eluted with FLAG peptide. The elutes were resolved on SDS-PAGE and silver-stained. The protein bands on the gel were recovered by trypsinization and analyzed by mass spectrometry. (B) Co-IP analysis of the association between SND1 and HLA-A. Whole-cell extracts from HeLa cells with SND1-FLAG expression were immunoprecipitated with anti-FLAG beads, followed by Western blot with antibodies against the HLA-A. (C) Co-IP analysis of the association between SND1 and HC10. Whole-cell extracts from HeLa cells were immunoprecipitated with anti-HC10, followed by immunoblot (IB) with antibodies against the SND1. (D) Cellular extracts from HeLa cells were immunoprecipitated with anti-SND1 antibody, followed by Western blot with antibodies against the indicated proteins. (E) Duolink in situ PLA was adopted for detecting the association between SND1 and HLA-A. Two PLA probes were designed to respectively recognize either mouse or rabbit antibody against SND1 or HLA-A. Immunoglobulin G (IgG) was used as staining control. Scale bar, 20 m. (F) Immunostaining and confocal microscopic analysis of subcellular colocalization of SND1 and HLA-AFLAG (C terminus) in HeLa cells. HeLa cells were fixed and immunostained with antibodies against the indicated proteins. Scale bar, 10 m. (G) GST pull-down analysis of the bacterially produced GST-fusion protein containing full-length SND1 (GST-SND1), SN domain (GST-SN), and TSN domain (GST-TSN) involved in the interaction with in vitrotranslated HLA-A from rabbit reticulocytes. Coomassie blue staining for GST-fusion proteins refers to fig. S1A. aa, amino acid. (H) GST pull-down analysis of the different domains of HLA-A involved in the interaction with SND1. The His-SND1 and sample of GST-tagged different domains of HLA-A were purified from E. coli bacteria cells. Coomassie blue staining for GST-fusion proteins refers to fig. S1B. (I) Immunoprecipitation analysis of the domains involved in the interaction between SND1 and HLA-A with FLAG-tagged deletion mutants of SND1 purified from HeLa SND1-KO cells. The immunoprecipitation of FLAG refers to fig. S1C. (J) The spatial conformation of SND1-HLA-A complex predicted by the database of ZDOCK (http://zdock.umassmed.edu/) was further analyzed using the Gromacs package. The structural stability and binding energy refer to fig. S1 (E and F).

Since the nascent HC is synthesized on the ER membrane and matured in the ER lumen (9), it raises the question of where the interaction of SND1 and HC occurs. By analyzing and comparing our previous mass spectrometry data from Jurkat cells with the present data from HeLa cells, we found 221 proteins (fig. S2A) in the overlapped set of SND1-associated proteins, including ribosomal (RPLs or RPSs) or ER-associated proteins, such as HLA-A, SEC61A, ribosome binding protein 1 (RRBP1), VCP, signal recognition particle 72 (SRP72), etc. We then performed immunofluorescence to investigate the cellular colocalization of SND1 and ER-associated proteins. As shown in Fig. 2A, SND1 colocalized with RRBP1 (a ribosome receptor on ER; top) and SEC61A (a core component of ER translocation channel; middle). The colocalization of HLA-A and RRBP1 (bottom) was used as positive control. It indicated that SND1 was a potential ER-associated protein.

(A) Immunostaining for cellular colocalizations, followed by confocal microscopic analysis by using antibody against SND1, RRBP1, SEC61A, and HLA-AFLAG. Scale bar, 10 m. (B) HeLa cells were transfected with the ER reporter plasmids, GFG, HLA-SP-GFG, UGGT1-SP-GFG, GAPDH-NP-GFG, and SND1-NP-GFG, respectively. Western blot for molecular weight of these GFG-tagged fusion proteins expressed in HeLa cells. (C) Colocalizations of these GFG-tagged fusion proteins with SEC61A were detected by confocal microscopy. UGGT1 was used as a positive control for ER-associating protein, while GAPDH was used as a negative control. Scale bar, 20 m. Fluorescence intensity profiles of regions indicated by short lines are shown in the bottom. (D) Co-IP by antibody against SEC61A for interaction with SND1-GFP or SND1-NP/-GFP in HeLa cells transfected with either SND1-GFP (lane 3) or SND1-NP/-GFP vector (lane 4). (E) Co-IP by antibody against FLAG for interaction with SND1-NP in HeLa cells transfected with either GFG (lane 3) or SND1-NP-GFG vector (lane 4). (F) Ectopically increased expression of either SND1-GFP or SND1-NP/-GFP in SND1-KO HeLa cells followed by Western blot for SND1 and HLA-A expression. WT, wild type.

ER lumen or secretory proteins containing an N-terminal signaling peptide (SP) composed of hydrophobic amino, which is recognized by SRP and subsequently cleaved by signal peptidase in the ER lumen (24), such as HLA-A and UGGT1 (a glucosyltransferase in ER lumen) (as illustrated in fig. S2B). We noticed a hydrophobic amino acid sequence at N terminus (NP) of SND1 protein. To investigate whether it is an SP, we developed an ER reporter assay by constructing a pair of two ER luminal reporter vectors. One is named GFG containing green fluorescent protein (GFP)FLAGGFP sequences after the multiple cloning sites that could insert the sequence of designated peptide. The other one is GFG-KDEL with an additional sequence of peptide KDEL following GFP-FLAG-GFP sequences. KDEL is a specific peptide sequence at the C terminus of ER lumen proteins that keeps the protein in the ER lumen (25). Using these vectors, we constructed a series of plasmids containing the SP of HLA-A (HLA-ASPGFG and HLA-ASPGFG-KDEL) or UGGT1 (UGGT1SPGFG and UGGT1SPGFG-KDEL), respectively, as positive control of ER-associated proteins. The N-terminal sequence of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (GAPDH-NP-GFG and GAPDHNPGFG-KDEL) was used as negative control of ER-associated proteins. The N-terminal sequence of SND1 was inserted to construct SND1-NP-GFG and SND1NPGFG-KDEL (fig. S2B). The plasmids were transfected into HeLa cells, respectively, and Western blot was performed to observe whether the S(N)P could be cleaved from the S(N)P-GFG fusion protein by signal peptidase (Fig. 2B). The molecular mass of SND1-NP-GFG(-KDEL) (lanes 7 and 8) was heavier than that of the GFG(-KDEL) control (lanes 1 and 2) (molecular mass is about 55 kDa), HLA-ASPGFG(-KDEL) (lanes 3 and 4), or UGGT1-SP-GFG(-KDEL) (lanes 5 and 6) but at the same level as negative control GAPDH-NP-GFG(-KDEL) (lanes 9 and10). It demonstrated that the SP of HLA-ASPGFG(-KDEL) and UGGT1-SP-GFG(-KDEL) was successfully cleaved from the fusion protein by signal peptidase, but not the NP of SND1-NP-GFG(-KDEL) or GAPDH-NP-GFG(-KDEL). All these data indicate that SND1 is an ER-associated protein but not an ER lumen protein since the NP of SND1 is not an SP. Therefore, it is likely that SND1 associates with HLA-A on ER membrane.

To further clarify the localization of SND1, the above X-S(N)P-GFG plasmids were transfected into HeLa cells, respectively; then, the colocalization of the X-S(N)P-GFG with endogenous SEC61A and RRBP1 was detected by immunofluorescence assay. Fluorescence intensity profiles were used to quantify the degree of localization. As shown in Fig. 2C and fig. S2C, the GFG control protein did not colocalize with SEC61A (Fig. 2C, a) or RRBP1 (fig. S2C, a). HLA-ASPGFG or UGGT1-SP-GFG was well colocalized with SEC61A (Fig. 2C, b and c) and RRBP1 (fig. S2C, b and c). There was no obvious colocalization of GAPDH-NP-GFG with SEC61A (Fig. 2C, d) or RRBP1 (fig. S2C, d). SND1-NP-GFG was colocalized with SEC61A (Fig. 2C, e) or RRBP1 (fig. S2C, e) respectively, which is in accordance with the results of S(N)PGFG-KDEL (fig. S2D). It is understandable that the colocalization of HLA-ASPGFG or UGGT1-SP-GFG with SEC61A and RRBP1 is due to the distribution of the GFG in the ER lumen cleaved by SP. How to explain the colocalization of SND1-NP-GFG with SEC61A and RRBP1?

In eukaryotes, ER membranelocated SEC61 translocation complex is the core component of the translocon that transports proteins to the ER (26). We thus performed Co-IP assay to explore the potential interaction of SND1 with SEC61A. HeLa cells were transfected with the expression plasmid containing full-length SND1 tagged with GFP (SND1-GFP) or N-terminal peptidedeficient SND1 tagged with GFP (SND1-NP/-GFP), respectively. As shown in Fig. 2D, SEC61A coprecipitated with ectopically overexpressed SND1-GFP (lane 3) but not SND1-NP/-GFP (lane 4). In addition, the endogenous SEC61A was coprecipitated with anti-FLAG for SND1-NP-GFG (Fig. 2E, lane 4) but not the GFG alone (lane 3). All these data suggest that the NP of SND1 is required for the efficient interaction of SND1 and SEC61A. Consistently, fig. S2E showed that the full-length SND1-GFP, but not SND1-NP/-GFP, was well colocalized with endogenous SEC61A. In HeLa cells with deletion of endogenous SND1, the protein level of HLA-A was reduced with ectopically overexpressed SND1-GFP in a dose-dependent manner (Fig. 2F, lanes 3 and 4), while there was no obvious alteration with overexpressed SND1-NP/-GFP (lanes 5 and 6). Together, it is likely that SND1 is an ER-associated protein anchored on ER membrane by binding SEC61A upon where nascent MHC-I HC is caught.

To determine the relevance between SND1/HC association and the presentation of MHC-I molecules, we carried out flow cytometry to detect the protein level of HLA-A/B/C on the surface of tumor cells with deletion of SND1 (SND1-KO) by CRISPR-Cas9 system. As shown in Fig. 3A, the protein level of HLA-A/B/C molecules was increased in two different SND1-KO HeLa cell clones. Likewise, the similar changes were observed in two SKOV3 ovarian cancer cell clones with deletion of SND1 (shSND1#1 and shSND1#2) (fig. S3B). The Western blot further confirmed that the protein level of HLA-A was enhanced in the cells with deletion of SND1 (Fig. 3B, left), while it is reduced in the cells with ectopic overexpression of SND1 (Fig. 3B, right). However, there was no obvious alteration at the mRNA level (fig. S3A). The same results were observed in SKOV3 cells (fig. S3, B to F). We then detected the half-life of endogenous HLA-A in HeLa cells with treatment of cycloheximide (CHX). As shown in Fig. 3C, HLA-A was gradually degraded in parental HeLa cells [wild type (WT)]; however, the degradation of HLA-A was noticeably retarded in SND1-KO cells. In addition, the protein level of HLA-A was significantly increased in the cells pretreated with proteasome inhibitor MG132 (Fig. 3D, lane 2) but not in the cells pretreated with lysosomal inhibitor chloroquine (Fig. 3D, lane 3). Therefore, we examined the ubiquitylation of HLA-A to determine whether SND1-promoted HLA-A destabilization is via ubiquitin mediatedproteasome pathway. As shown in Fig. 3E, compared with the control cells (lanes 1 and 3), the ubiquitylation of ectopically overexpressed HLA-A was obviously decreased in SND1-KO cells (lane 4) but relatively increased in the cells with overexpression of SND1 (SND1-HA) (lane 2). On the basis of the aforementioned data, it is likely that SND1 leads to HLA-A degradation through the ubiquitin-proteasome pathway.

(A) Two clones of HeLa cells with stable depletion of SND1 by CRISPR-Cas9 system were analyzed by flow cytometry for human MHC-I using antibody simultaneously against HLA-A/B/C. (B) Two clones of HeLa cells with stable depletion of SND1 by CRISPR-Cas9 system and HeLa cells stably expressing SND1-FLAG were collected, followed by Western blot using antibodies against HLA-A. (C) The effect of KO SND1 on the half-life of HLA-A was evaluated in HeLa cells treated with CHX (50 g/ml) and harvested at the indicated time point, followed by Western blot. The protein half-life curves were obtained by quantifying relative intensities. (D) HeLa cells with ectopic HLA-A expression were pretreated with proteasome inhibitor MG132 (10 mM) or lysosomal inhibitor chloroquine (100 mM) for 8 hours and subjected to Western blot with antiHLA-A antibody. DMSO, dimethyl sulfoxide. (E) WT and SND1-KO HeLa cells were transfected with HLA-AFLAG and treated with MG132 (10 mM) for 8 hours. Cellular extracts were immunoprecipitated with anti-FLAG, followed by Western blot with anti-ubiquitin (Ub) antibody. (F) HeLa cells were cotransfected with control vector or SND1-HA and HLA-AFLAG or with control small interfering RNA (siRNA) or SND1 siRNA and HLA-AFLAG, and whole-cell lysates were collected and immunoprecipitated with anti-FLAG, followed by Western blot with indicating antibodies. (G) The Duolink in situ PLA was adopted for detecting the direct association between HLA-A and calnexin or 2-microglobulin (2m) in the presence of SND1-HA or in the absence of SND1. Scale bar, 20 m. The signal dots were calculated and plotted. *P < 0.05 and ****P < 0.0001, by unpaired t test.

In ER lumen, the nascent unfolded HLA-A can be initially retained by a key chaperone, calnexin, to ensure proper folding and quality control before ultimate assembly with 2-microglobulin (2m) to form mature MHC-I (27). The catching of nascent HLA-A by SND1 may have an impact on the association of calnexin and HLA-A and lead to a misfolding process of HLA-A. Immunoprecipitation assay revealed that the binding efficiency of HLA-A to calnexin, and HLA-A to 2m, was remarkably reduced in the presence of ectopically overexpressed SND1 (Fig. 3F, left) but was obviously increased in the absence of SND1 (right). According to the red dots of PLA probe signal from confocal images, Duolink assay (Fig. 3G) further validated that the binding efficiency of calnexin and 2m to HLA-A was decreased by overexpression of SND1 (SND1-HA), while it was significantly increased by deletion of SND1 (si-SND1). Moreover, glycosylation sitemutated HLA-A (N110Q) is not able to associate with both calnexin and 2m but sufficiently interacts with SND1 (fig. S3H). These observations suggest that SND1 hindering the normal assembly process of MHC-I in the ER lumen, consequently guiding the nascent HLA-A for degradation.

Misfolded or nascent HC, which fails to achieve the native conformation in complex with 2m, is dislocated from ER to cytosol and ubiquitinated for ERAD process (28). To investigate the underlying mechanisms of SND1-mediated HC degradation, we used affinity purification and mass spectrometry to identify HLA-Aassociated proteins in HeLa cells with stable expression of HLA-AFLAG. There were 278 affinity-purified proteins (fig. S4A) overlapping in both HLA-Aassociated and SND1-associated proteins. These overlapped proteins were further filtered by Kyoto Encyclopedia of Genes and Genomes analysis (fig. S4B). The top-ranking proteins include SND1, HLA-A, VCP, calnexin (Fig. 4A), SEC61A, etc. (fig. S4C).

(A) Immunopurification and mass spectrometry of HLA-Acontaining protein complexes. Cellular extracts from HeLa cells stably expressing HLA-AFLAG were immunopurified with anti-FLAG affinity beads and eluted with FLAG peptide. The elutes were resolved on SDSpolyacrylamide gel electrophoresis (SDS-PAGE) and silver-stained. The protein bands on the gel were recovered by trypsinization and analyzed by mass spectrometry. HLA-Ainteracted proteins were highlighted. (B) HeLa cells were coimmunoprecipitated by HLA-A antibody and subjected to Western blot by antibody against VCP. (C) HeLa cells were coimmunoprecipitated by SND1 antibody and subjected to Western blot by antibody against VCP. (D) Duolink assay followed by confocal microscopic analysis for direct molecular interactions among SND1, VCP, and HLA-A. IgG was used as a negative control. Scale bar, 20 m. (E) HeLa cells were cotransfected with control vector or SND1-HA and HLA-AFLAG or cotransfected with control siRNA or SND1 siRNA and HLA-AFLAG, and whole-cell lysates were collected and immunoprecipitated with anti-FLAG, followed by Western blot with anti-SND1, anti-VCP, anti-VIMP, and anti-HRD1 antibodies. Results of input were shown in fig. S4D. (F) HeLa cells were cotransfected with vector or HRD1-HA and HLA-AFLAG with the treatment of MG132. Cellular extracts were immunoprecipitated with anti-FLAG, followed by Western blot with anti-ubiquitin antibody.

As VCP plays essential roles in ERAD, we then validated the association of VCP, SND1, and HLA-A. Co-IP experiments revealed that VCP was able to physically interact with both HLA-A (Fig. 4B) and SND1 (Fig. 4C) in vivo. Moreover, Duolink assay (Fig. 4D) further demonstrated the association of these three proteins. Comparatively, the binding ability of SND1 and HLA-A (a) or HLA-A and VCP (b) was stronger than SND1 and VCP (c). It was reported that VIMP (the cofactor of VCP) and HRD1 (the E3 ligase) are the key participants in the degradation of HLA-A (29); meanwhile, the interaction of HRD1 with SND1 was found in our present study by Co-IP (fig. S4E). We therefore investigated the correlation of the SND1 expression and the association of VCP/VIMP/HRD1 with HLA-A. As shown in Fig. 4E, the interaction of VCP, VIMP, and HRD1 to HLA-A was remarkably increased in the presence of ectopically overexpressed SND1 (left) but was largely decreased in the absence of SND1 (right). Furthermore, compared with the control cells, the ubiquitylation of ectopically overexpressed HLA-A was obviously increased in the cells with overexpression of HRD1 (HRD1-HA) (Fig. 4F). These observations suggest that SND1 sequestrates the nascent HC of MHC-I and redirects it to the ERAD pathway for proteasomal degradation.

To explore the consequence of SND1-mediated HC degradation in vivo, we used murine syngeneic tumor models on the C57BL/6 background by using two murine cancer cell lines, B16F10 melanoma cells and MC38 colon adenocarcinoma cells. B16F10SND1-KO and MC38SND1-KO cell clones with deletion of SND1 were obtained by using CRISPR-Cas9 system. Consistent with previous results, both Western blot detection (Fig. 5A) and flow cytometry analysis (Fig. 5B) demonstrated that the protein level of H2Kb (HC of mouse MHC-I) was increased in different B16F10SND1-KO cells. The same results were observed in MC38SND1-KO cells (fig. S5, A and B). We then subcutaneously inoculated 5 105 parental or B16F10SND1-KO cells into the flank of C57BL/6 mice, and the tumor growth was monitored in the following days. Compared with the parental B16F10 tumor, the growth of B16F10SND1-KO cells with SND1 deficiency was markedly slow in terms of the developmental kinetics (Fig. 5C and fig. S5C). Consistently, the tumor size (Fig. 5D for B16F10 and fig. S5D for MC38) and weight (Fig. 5E for B16F10 and fig. S5E for MC38) were also smaller in SND1-KO cells with SND1 deletion than those in the control parental cells. As MHC-I molecules play essential roles in tumor antigen presentation for CD8+ T cellmediated immune response, we thus investigated the infiltration of CD8+ T cells in tumor tissues by immunofluorescence and flow cytometry. Compared with the parental tumor tissue, there were more CD8+ T cells (red) infiltrated in B16F10SND1-KO (Fig. 5F) and MC38SND1-KO tumor tissues (fig. S5F). The flow cytometry analysis further revealed that the infiltration of CD45.2+ leukocytes (Fig. 5, G and H) and CD8+ T cells (Fig. 5, G and I) was significantly increased in the B16F10SND1-KO tumor tissue and the MC38SND1-KO tumor tissue (fig. S5, G to I) compared with the parental tumor tissue. Specifically, the proportion of CD8+ T cells among CD45.2+ leukocytes was significantly increased in the B16F10SND1-KO tumors (Fig. 5J) and MC38SND1-KO tumors (fig. S5J). Besides, we also detected the potential exhaustion of CD8+ T cells in tumor tissues. Compared with the control parental, there was no apparent discrepancy in the percentage of PD-1+ CD8+ T cells with deletion of SND1 (Fig. 5K). Furthermore, we clarified that SND1 deficiency in mice melanoma (fig. S6, A to D) and colon carcinoma (fig. S6, E to H) resulted in no significant changes in regulating cell proliferation, apoptosis, or cell cycle in vitro. We also performed the experiments by using RAG-1 (recombination activating gene 1) KO mice (Rag-1/ mice) that lack mature T and B cells to investigate whether the absence of SND1 would affect tumor growth in vivo. We initially inoculated 5 105 WT or SND1-KO B16F10 cells subcutaneously into the flank of C57BL/6 WT mice and Rag-1/ mice. By comparison between WT cells and SND1-KO B16F10 cells inoculated in either C57BL/6 WT mice or Rag-1/ mice (fig. S6, I to K), we found that the size and weight of SND1-KO tumors were significantly decreased in the group of C57BL/6 WT mice, whereas in the group of Rag-1/ mice, the tumors were comparable between WT and SND1-KO B16F10 cells in terms of the tumor size and weight. Together, these observations imply that deletion of SND1 in tumor cells is likely to promote CD8+ T cellmediated cellular immune responses in the tumor microenvironment.

(A) Three clones of B16F10 cells with stable depletion of SND1 by CRISPR-Cas9 system were collected, followed by IB for murine MHC-I (H2Kb). (B) Two clones of B16F10 cells with stable depletion of SND1 by CRISPR-Cas9 system were analyzed by flow cytometry for murine MHC-I (H2Kb/H2Db) using antibody simultaneously against H2Kb/H2Db. (C to E) 5 105 of either WT or SND1-KO B16F10 cells were subcutaneously transplanted into C57BL/6 mice. The tumor growth was monitored at the indicated times. C57BL/6 mice were sacrificed at day 11. Tumors were removed and photographed. The tumor tissues were weighed and plotted. Data are presented as means SD; n = 5 tumors for each group. *P < 0.05, two-tailed t test. (F) Immunofluorescence images of CD4+ T and CD8+ T cells in B16F10 tumor sections (scale bar, 20 m). (G) C57BL/6 mice injected with equal numbers of WT or SND1-KO B16F10 cells were sacrificed at day 11. The digested tumor suspensions stained with antibodies against CD8 and CD45.2 (pan-leukocyte marker) were subjected to flow cytometry. (H to J) Percentages of infiltrating CD45.2+ cells and CD8+ T cells among total tumor tissuederived cells and the percentage of infiltrating CD8+ T cells among total CD45+ leucocytes. n = 5 tumors for each group. *P < 0.05 and **P < 0.01, by unpaired t test. The experiments were performed and repeated at least three times, independently. (K) The percentage of infiltrating PD-1+ CD8+ T cells among total CD8+ T cells. n = 5 tumors for each group. n.s., not significant.

Moreover, in light of our observation that SN domain of SND1 is responsible for the association of SND1 with MHC-I HC, it is tempting to speculate the crucial function of SN domain in vivo. Our supplementary data support that the rescue of SN domain of SND1 significantly increased the tumor growth through mobilizing less CD8+ T cells infiltrating in tumors [fig. S7, A to H (B16F10) and I to P (MC38)].

To further clarify the influence of high expression of SND1 on CD8+ T cellmediated cellular immune responses in tumor, we used transgenic OT-I mice. OT-I mice are ovalbumin (OVA)specific T cell receptor transgenic (OT-I) mice whose CD8+ T cells could recognize the specific peptides (257 to 264 SIINFEKL) of chicken OVA, a surrogate tumor antigen that can be conveniently used to investigate CD8+ T cellmediated immune responses directed against the OVA antigen (30). Meanwhile, B16F10-OVA cells and MC38-OVA cells were constructed by stably expressing the OVA antigen, which is able to be presented by MHC-I complex and specifically recognized by CD8+ T cells derived from OT-I mice. The identical expression of OVA was observed in both B16F10-OVA WT cells and B16F10-OVA SND1-KO cells with SND1 deletion (Fig. 6A), as well as in MC38-OVA WT and MC38-OVA SND1-KO cells (fig. S8A). The flow cytometry analysis demonstrated that higher level of MHC-I was detected in B16F10-OVA SND1-KO (Fig. 6B) or MC38-OVA SND1-KO cells (fig. S8B), which suggested that more OVA peptides might be presented in tumor cells in the absence of SND1. To interrogate the in vivo effect of antigen presentation, we subcutaneously inoculated equal numbers of B16F10-OVA WT or B16F10-OVA SND1-KO cells into OT-I mice. The tumor volume was monitored accordingly. The growth curve illustrated that SND1 deficiency in B16F10-OVA cells markedly inhibited the tumor growth in vivo (Fig. 6C). At day 19, the tumors were resected and the size and weight were measured. With deletion of SND1 (B16F10-OVA SND1-KO), the tumor size (Fig. 6D) and weight (Fig. 6E) were remarkably smaller than those of the control tumor (B16F10-OVA). The flow cytometry assay revealed that more CD45.2+ cells (Fig. 6F and G) and CD8+ T cells (Fig. 6F and H) were infiltrated in the tumor tissue of B16F10-OVA with SND1 deficiency (B16F10-OVA SND1-KO), and the ratio of CD8+ T cells to CD45.2+ leukocytes was significantly increased (Fig. 6I). These results further verified the impact of SND1 on MHC-I/antigenic peptide presenting in tumor cells and consequently affecting the infiltration of cytotoxic CD8+ T cells in tumor tissue.

(A) B16F10 cells with stable depletion of SND1 by CRISPR-Cas9 system were stably transfected with OVA vector, followed by IB. (B) B16F10-OVA with SND1 deficiency was analyzed by flow cytometry for murine MHC-I (H2Kb/H2Db). (C to E) OT-I mice were injected with equal numbers of WT or SND1-KO B16F10-OVA cells, and tumor growth was observed over time. Then tumors were removed, photographed, and weighted. *P < 0.05 and **P < 0.01. (F) Flow cytometry was used for the analysis of CD45.2+ leucocyte and CD8+ T cell infiltration in tumor tissues. (G to I) Percentages of infiltrating CD45.2+ leucocytes and CD8+ T cells among total tumor tissuederived cells and the percentage of infiltrating CD8+ T cells among total CD45.2+ leucocytes. n = 5 tumors for each group. **P < 0.01 and ***P < 0.001, by unpaired t test. (J) CD8+ T cells were purified from spleens of tumor-bearing OT-I mice and stimulated with 257 to 264 (SIINFEKL) peptide of OVA for 24 hours. Percentages of IFN+CD8+ T cells among total CD8+ T cells in the culture system were measured by flow cytometry. (n = 5, **P < 0.01). The experiments were repeated two times independently. (K) CD8+ T cells recognizing specific peptide of OVA (SIINFEKL) were purified from spleens of OT-I and then cocultured with WT or SND1-KO B16F10 cells stably expressing OVA (CD8+ T:B16F10-OVA, 10:1). Representative images were taken under a bright field at different time points. Scale bar, 20 m. (L) In vitro comparison of cytolysis rates against CD8+ T cells purified from spleens of OT-I mice between WT and SND1-KO B16F10-OVA cells at different cell rates of CD8+ T (effector cells) to B16F10 (target cells) with/without antiMHC class I antibodies (Ab) (E:T, 5:1, 10:1, 15:1, or 20:1). A lactate dehydrogenasereleasing cytotoxicity assay was performed to measure the cytolysis efficiency of CD8+ T cells on tumor cells. Each bar represents mean SD for biological triplicate experiments. ****P < 0.0001, two-way analysis of variance (ANOVA).

To further examine the contribution of SND1 to cytotoxic CD8+ T cellmediated immune response in tumor, we isolated CD8+ T cells from spleen of OT-I mice bearing B16F10-OVA-WT or B16F10OVASND1-KO tumor, respectively, and tested the cytotoxic CD8+ T cell population. As shown in Fig. 6J, the amount of interferon- (IFN)producing CD8+ splenic T cells was significantly increased in SND1-KO group compared to WT control, suggesting that SND1 deficiency in tumor cells promoted and activated the OVA-specific CD8+ T cell response in the peripheral immune organ. To examine whether the enhanced antigen presentation in the SND1-KO cells results in robust recruitment and activation of CD8+ T cellmediated cytotoxicity in the tumor environment, CD8+ T cells were isolated from OT-I mice and cocultured with tumor cells expressing OVA in the presence or absence of SND1. Representative time-lapse images showed that B16F10-OVA cells with SND1 deficiency were recognized and killed by more cytotoxic CD8+ T cells after 12 hours, while the parental B16F10-OVA cells survived after 12 hours with less aggregated CD8+ T cells (Fig. 6K). Furthermore, the amount of lactate dehydrogenase released from lysed target cells was used as indicator for cytolysis. Accordingly, the cytolysis value was higher in SND1-KO group in an E/T cell ratiodependent manner and significantly abolished with the treatment of MHC-blocking antibodies (AF6-88.5) (Fig. 6L, red), which indicated an efficient cytotoxic effect mediated by CD8+ T cells. Consistently, the same results were also observed in MC38-OVA cells (fig. S8, C and D). Collectively, these findings illustrated that SND1 impaired tumor antigen presentation to cytotoxic CD8+ T cells and sabotaged the CD8+ T cellmediated cellular immune response, supporting our previous data that deletion of SND1 in tumor cells promoted CD8+ T cellmediated cellular immune response, and, as a result, inhibited the tumor growth in vivo.

To illustrate the significant relevance of high SND1 expression with tumor immune response in human, we screened the Tumor Immune Estimation Resource (TIMER) database at http://cistrome.org/TIMER/, a comprehensive resource for systematic analysis of immune infiltrates across diverse cancer types from The Cancer Genome Atlas (31). We analyzed the correlation of SND1 expression and the infiltration of CD8+ T cells in melanoma and colon adenocarcinoma (fig. S9, A and C). The results showed that SND1 expression was moderately negatively correlated with infiltration level of CD8+ T cells in melanoma (r = 0.247, P = 1.68 107) and colon adenocarcinoma (r = 0.393, P = 2.10 1016).

In addition, we analyzed the correlation of SND1 with cancer patients prognosis by using PrognoScan database at http://dna00.bio.kyutech.ac.jp/PrognoScan/index.html (32). Notably, SND1 expression significantly affects the prognosis in melanoma. The cohort of melanoma (GSE19234) included 38 samples at different stages of melanoma and showed that high SND1 expression was significantly associated with poorer prognosis [Overall survival (OS) hazard ratio (HR) = 7.39, 95% confidence interval (CI) = 1.51 to 36.30, Cox P = 0.000912] (fig. S9B). Consistently, similar trend between SND1 expression with prognosis was observed in colorectal cancer (GSE17536; OS HR = 1.49, 95% CI = 0.69 to 3.22, Cox P = 0.314186) (fig. S9D). Since SND1 was negatively associated with the infiltration of CD8+ T cells and the survival of patients, we speculated that targeting SND1 might be a potentially therapeutic approach to enhance immune response and suppress tumor growth.

In conclusion, as shown in the working model (fig. S9E), SND1, localized on the membrane of ER in tumor cells, is able to hijack MHC-I HC from normally assembling and physically associating with its chaperone calnexin in the ER lumen at an early stage of ER processing, thereby leading MHC-I HC to the proteasomal pathway of ERAD promoted by VCP, cofactor VIMP, and E3 ligase HRD1 and sensitizing tumor to the diminished immune surveillance with decreased cytotoxic CD8+ T cells. Thus, SND1 profoundly facilitates immune evasion from tumor immune microenvironment through inhibition of antigen presentation and that this effect is mediated by down-regulation of MHC-I HC molecule (left). On the other hand, the absence of SND1 in either melanoma or colon carcinoma from subcutaneously tumor-bearing WT mouse results in growth inhibition and promotes tumor inflammation with more cytotoxic CD8+ T cells (right). Genetic ablation of SND1 in OVA-expressed tumors on OT-I mice induces sufficient antigen presentation to cytotoxic CD8+ T cells and enhances antitumor immunity. Therefore, SND1 determines the fate of MHC-I HC maturation and orchestrates a cancer-favored immune microenvironment. This model proposes the blockade of SND1MHC-I HC axis in tumors as a viable option for immune system against cancer.

In the present study, we demonstrate that SND1 promotes immune escape of tumor cells through inhibition of MHC-I antigen presentation pathway, leading to impaired antitumor CD8+ T cell response in tumor microenvironment. Physiologically, the nascent unfolded HC of MHC-I would be stabilized by the chaperone calnexin before association with the 2m in ER. Here, we revealed that SND1 physically interacted with the nascent HC of MHC-I molecule in tumor cells. Instead of promoting the assembly of MHC-I molecule, SND1 recruits the nascent HC to VIMP/VCP complex for ERAD pathway. As a result, the MHC-I expression on tumor cell membrane is reduced, leading to impaired CD8+ T cell activation in tumor microenvironment.

SND1 is highly expressed in various cancers and is newly identified as a novel oncoprotein. We have previously reported that SND1 plays important physiological roles in a variety of cellular processes (1719, 33). By using various methodologies, we identified cytoplasmic SND1 as an ER-associated protein and physically interacting with the nascent HC on the ER membrane in tumor cells. The biogenesis of transmembrane proteins requires the activity of the SEC61 complex, in which the subunit SEC61A has been proposed to act as a gate for the membrane insertion of nascent polypeptides (34). The MHC-I HC is synthesized on membrane-associated ribosomes and inserted cotranslationally into the ER through the translocon composed of SEC61 complex. In ER lumen, the nascent HC associates with its chaperones and glycosylation-related enzymes to generate a properly folded glycoprotein, and the formation of HC-2m dimers indicates the maturity of MHC-I molecules (35). We have previously reported that SND1 protein is composed of SN and TSN domain (35). Here, we illustrated that SND1 protein is an ER-associated protein containing a functional N-terminal sequence (NP) that could associate with SEC61A on ER membrane. The predicted spatial conformation three-dimensional model of SND1-HLA-A complex is consistent with the mapping data, corroborating that SN domain of SND1 could physically interact with the A1 and A3 domain of HC. It indicates a fundamental and preceding role for SND1 at the early phase of HC assembly in the ER machinery. We further reveal that SND1 efficiently facilitates HC disassociation with calnexin and 2m, which interrupt the regular maturation and assembly of MHC-I molecule.

Previous studies have demonstrated that unfolded or misfolded HC that fails to form a proper structure can be recognized, dislocated, and degraded by the ERAD machinery (28). Accordingly, we, by far, are able to investigate how SND1 manages to hijack the nascent HLA-A for further degradation process on ER membrane. Although MHC-I HC is highly polymorphic and its potential ubiquitylation sites are variable (3), it is worthwhile to study the molecular mechanisms of how SND1 determines the fate of MHC-I HC precisely via the ubiquitin proteasome system. As a central element of the ubiquitin-proteasome system, VCP plays a key role in ERAD (36). During dislocation from the ER, misfolded or misassembled MHC-I HC as an ERAD substrate is ubiquitylated on the cytosolic side of the ER membrane and is degraded by the cytosolic proteasome (36). In the present study, we identified VCP, cofactor VIMP, and E3 ligase HRD1 of ERAD components as potential interactors of SND1, as revealed by mass spectrometric and coimmunoprecipitated analysis. Moreover, the results revealed that in the presence of SND1, more MHC-I HC was directed to the ERAD pathway for degradation.

We used mice model bearing tumors, especially transgenic OT-I mice, to illustrate the in vivo consequences of the tumor growth and immune response. With the absence of SND1 in either melanoma or colon carcinoma, the inoculated subcutaneous tumor growth was markedly inhibited but the amount of infiltrated CD8+ T cells in the tumor tissue was greatly increased. In accordance with the evidence from human database, we deduce that the highly expressed SND1 sabotages tumor antigen presentation to cytotoxic CD8+ T cell, thereby creating an immune niche with impaired surveillance that favors tumor growth.

Underlying the physiological relevance of our findings, the protein level of SND1 and MHC-I HC was negatively correlated with each other in human cervical and ovarian cancer cells, as well as in murine melanoma and colon cancerous cells. It is known that SND1 was robustly overexpressed in a variety of tumorigenic tissues and relatively highly expressed in normal tissues, and it was suggested that SND1 was an attractive target for anticancer therapy and a potent tumor marker (16). For more than a decade, it has been recognized that intact antigen presentation machinery, including MHC expression, in malignant cells is critical for T celldependent antitumor immunity because HLA-I antigen expression in tumors directly correlates with the degree of tumor T cell infiltration inside the tumor nests (3, 37). More recently, this knowledge has been underscored by findings showing that MHC class I molecule can be used as an independent prognostic factor for colorectal cancer and for predicting the efficacy of immunotherapy in bladder cancer and chemotherapy in ovarian cancer (1, 5, 38, 39). However, there is a long way to go before the different molecular mechanisms responsible for MHC-I alterations are precisely defined in different tumor types. For example, the mechanisms responsible for total MHC-I loss in about 60% of patients with breast cancer, in 50% of patients with prostate cancer, in 15% of patients with laryngeal cancer, or in 40% of patients with gastric cancer are yet to be identified (2, 6, 39, 40). However, it was proposed that during tumor development, tumors are heterogeneous with both HLA-positive and HLA-negative cells at early stages and are infiltrated by lymphocytes and M1 macrophages as a part of an active antitumor T helper 1 response (40). Thus, it is necessary to analyze tumor HLA expression and monitor HLA changes taking place during immunotherapy to understand how, when, and why the MHC/HLA alterations occur. In a way, our current study provides direct evidence for the idea of how the expression of MHC-I HC was regulated by an endogenously expressed protein SND1, the mechanism of which could be extendedly applied in the nonmalignant cells that may explain when and how MHC-I HC was altered during tumor initiation. Thus, SND1 could be a potential therapeutic target, at least for the treatment of malignancies with MHC-I defects in which the MHC-I is not genetically compromised.

To sum up, SND1 profoundly facilitates immune evasion from tumor immune microenvironment, and this effect is mediated by reducing the expression of MHC-I HC molecule. As a newly identified ER-associated protein, SND1 is able to hijack nascent MHC-I HC that is guided to ERAD-related proteasomal pathway, thereby impairing the proper assembling of HC with 2m in the ER lumen and sensitizing tumor cells to a diminished immune surveillance with abolished antigen presentation to cytotoxic CD8+ T cells. This novel finding may shed light on orchestrating the cancer-favored immune microenvironment via blockade of SND1MHC-I HC axis in tumors as a viable option for immune system against cancer.

HeLa, B16F10, and MC38 cells were obtained from the American Type Culture Collection (ATCC) and cultured using the standard conditions according to the ATCC instructions. B16F10 and HeLa cells were cultured in Dulbeccos modified Eagles medium [Biological Industries (BI)] supplemented with 10% fetal bovine serum (FBS; BI), and MC38 cells were cultured with RPMI 1640 (BI) supplemented with 10% FBS. The human SKOV3 cell line was purchased from China Infrastructure of Cell Line Resources (Beijing, China), and SKOV3 cells were cultured with McCoys 5A Medium (Sigma-Aldrich) supplemented with 10% FBS. All cell lines were cultured under an atmosphere of 5% CO2 at 37C. All of the cells were authenticated by examination of morphology and were confirmed to be mycoplasma-free.

Cells were transiently transfected with a Cas9 and single-guide RNA expression plasmid encoding puromycin resistance. The CRISPR-transfected cells will thus acquire transient resistance to puromycin, and the guide sequences were described as using the optimized CRISPR design at http://crispr.mit.edu. We confirmed that SND1-KO cells were not sensitive to puromycin after expansion, indicating a transient expression of CRISPR-Cas9 system in those cells.

Six- to 8-week-old male C57BL/6 mice were originally purchased from the Academy of Military Medical Sciences. OT-I transgenic mice, whose T cell receptor was designed to recognize OVA residues 257 to 264, were provided by Z. Dong from Tsinghua University. RAG-1 KO mice (Rag-1/ mice) that lack mature T and B cells were purchased from Nanjing Biomedical Research Institute of Nanjing University. The gene phenotype was routinely confirmed by polymerase chain reaction (PCR) using specific primers. All animal procedures were approved by the Committee on the Use and Care of Animals of Tianjin Medical University.

For xenograft experiments, B16F10 cells or MC38 cells (5 105) (WT and SND1-KO) were subcutaneously transplanted into the flank of C57BL/6 mice or Rag-1/ mice. Tumor height and width were measured with a caliper every 2 to 3 days to calculate tumor volume (= width2 height 0.523). Mice were sacrificed when tumors reached maximum allowed size (15 mm in diameter) or when signs of ulceration were evident. Likewise, 5 105 of B16-OVA or MC38-OVA cells were subcutaneously transplanted into the flank of OT-I mice. In all experiments, the initial implantation was conducted to animals at the age of 6 to 8 weeks.

Several plasmids were obtained from corporations including pCIpA102-G-HLA-A2-GFP (Addgene plasmid, no. 85162) and pcDNA3.1+ (Invitrogen, V79020), and several plasmids were gifts, including the following: pLV-IRES-Puro, pET-28a-c (+) vector from L. Shi (Tianjin Medical University), and CRISPR-Cas9 constructs px462 from X. Wu (Tianjin Medical University). Lentivirus plasmids expressing SND1 short hairpin RNA (shRNA) and the vector plasmid pLKO were purchased from MilliporeSigma (SHCLNG-NM_014390). The target sequences of shSND1 are shown as previously described (17). The FLAG-tagged or HA-tagged SND1 carried by pLV-IRES-Puro vector or pcDNA3.1+ were amplified from cDNA of HeLa cells or B16F10 cells with specific PCR primers. For exogenous HLA-A expression, the full length of HLA-A cloned from pCIpA102-G-HLA-A2-GFP with a FLAG tag at the C terminus was inserted into the lentiviral vector pLV-IRES-Puro. The HLA-A(N110Q)-FLAG mutant plasmid carried by pLV-IRES-Puro vector was constructed by the GENEWIZ Inc. The SN domain (SN-HA, 1 to 660 amino acids) or TSN domain (TSN-HA, 661 to 910 amino acids) of SND1 (mouse) protein with an HA tag at the C terminus was inserted into the lentiviral vector pLV-IRES-Puro. The FLAG-tagged full-length and truncation mutants of SND1 (human) were carried by pCMV-Blank vector for transient transfection. The pGEXT-4T-1 plasmids containing full length of SND1, SN domain, or TSN domain were generated as previously described (35). The pGEXT-4T-1 plasmids were inserted with full length of HLA-A(GST-HLA-A, 1 to 365 amino acids) and fragments (GSTHLA-AA1, 25 to 114 amino acids; GSTHLA-AA2, 115 to 206 amino acids; GSTHLA-AA3, 207 to 298 amino acids; and GSTHLA-AC, 299 to 365 amino acids) for GST pull-down assay.

An ER reporter plasmid was constructed with fusion protein (GFP-FLAG-GFP) and Eco RI/Xho I sites at the N terminus, which could be inserted with different signal peptides, such as HLA-A, UGGT, GAPDH, and SND1. KDEL, a target peptide sequence located on the C terminus, which prevents protein from secreting from the ER, was added at the N terminus of the fusion protein. Full length and deletion of N-terminal peptides (1 to 35 amino acids) of SND1 were inserted into the vector pcDNA3.1+ with a GFP tag at the C terminus.

Tumors were removed from sacrificed mice and digested by collagenase I (1.5 mg/ml) and deoxyribonuclease I (100 g/ml; Solarbio) in RPMI 1640 for 1 hour at 37C. The cell suspensions were passed through 70-m filters (Falcon) to remove undigested tumor tissues, and then, the erythrocytes were removed by ACK lysis buffer. Cell suspensions were incubated in mouse Fc block (anti-CD16/CD32, BioLegend) before staining. Fluorochrome-conjugated anti-mouse CD45.2 (clone 104, eBioscience), CD8a (clone 53-6.7, BD Biosciences), IFN (clone XMG1.2, BioLegend), and PD-1 (clone RMP1-30, BioLegend) antibodies were used following the manufacturers protocol. Flow cytometry results were analyzed using FlowJo software.

Cells were collected and lysed with radioimmunoprecipitation assay buffer on ice. Protein concentration was determined by the bicinchoninic acid assay. Equal amounts of protein from each cell lysate were subjected to 10% SDSpolyacrylamide gel electrophoresis (SDS-PAGE). The resolved proteins were transferred to polyvinylidene difluoride membranes and blotted with indicated antibodies. -Actin or GAPDH was used as an internal control.

Lysates from HeLa cells with or without SND1-FLAG expression were immunoprecipitated using Anti-FLAG M2 affinity beads (Sigma-Aldrich, A2220) for 12 hours at 4C with constant rotation. After extensive washing with phosphate-buffered saline (PBS) plus 0.1% Tween, the bound proteins were eluted with excess FLAG peptides for 12 hours at 4C with constant rotation, concentrated with Amicons (Ultra-0.5, 3 kDa, MilliporeSigma), and then visualized by silver staining on 8% SDS-PAGE. The distinct protein bands on the gel were recovered by trypsinization and analyzed by mass spectrometry.

Cellular lysates were prepared by incubating the cells in cell lysis buffer [50 mM tris-HCl (pH 8.0), 150 mM NaCl, 0.2% Nonidet P-40, and 2 mM EDTA] in the presence of protease inhibitor cocktail for 20 min at 4C with vortex every 5 min, followed by centrifugation at 13,000 rpm for 10 min at 4C. For immunoprecipitation, about 1 mg of protein was incubated with control [immunoglobulin G (IgG)] or specific antibodies (1 to 2 g) for 12 hours at 4C with constant rotation. A total of 50 l of 50% protein A/G agarose beads was then added, and the incubation was continued for an additional 6 hours. Beads were then washed five times using the PBS with 0.1% Tween. Between washes, the beads were collected by centrifugation at 2000 rpm for 5 min at 4C. The precipitated proteins were eluted from the beads by resuspending the beads in 2.5 SDS-PAGE loading buffer and boiling for 10 min. The boiled immune complexes were subjected to SDS-PAGE, followed by immunoblotting with appropriate antibodies.

Cells were fixed for 10 min at room temperature with 4% paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature. Samples were then blocked in 3% bovine serum albumin and incubated consecutively with primary antibodies to SND1/calnexin (ProteinTech, 60265-1-Ig/10427-2-AP) and the appropriate secondary antibodies coupled to Alexa Fluor 488 or 594 (Invitrogen). Cells were covered with a drop of 4,6-diamidino-2-phenylindole (DAPI) for 5 min. After washing with PBS, slides were mounted for observation. Confocal images were captured on Leica DMi8 using a 63 oil objective. The fluorescence intensity profiles of the targeted regions were obtained with a Leica DMi8 microscope and LAS X version 3.5.5.

Duolink assay was performed using the Duolink In Situ Red Starter Kit Mouse/Rabbit following the manufacturers instructions (Sigma-Aldrich, DUO92004), and its basic protocols of fixation, retrieval, and permeabilization are the same as immunofluorescence. Samples were incubated with blocking solution for 1 hour at 37C in a humidity chamber and then overnight at 4C with anti-SND1 mouse antibody and anti-calnexin rabbit antibody. Slides were then incubated for 1 hour at 37C with a mix of the MINUS (anti-mouse) and PLUS (anti-rabbit) PLA probes. Hybridized probes were ligated using the ligation-ligase solution for 30 min at 37C and then amplified using the amplification-polymerase solution for 100 min at 37C. Slides were covered with a drop of DAPI (Invitrogen) for 5 min and mounted for observation with a Leica DMi8 confocal microscope.

GST-fusion proteins that contained full length or truncations of SND1 and HLA-A were produced in BL21 E. coli and purified using glutathione agarose beads. Recombinant proteins (HLA-A) were expressed with rabbit reticulocyte lysate (TNT systems, Promega) according to the manufacturers recommendation. His-SND1 was purified in BL21(DE3) E. coli and purified using HIS-Select Nickel Affinity Gel (Sigma-Aldrich) according to the standard procedures.

In GST pull-down assays, the bead-bound GST-fusion proteins were incubated with in vitro transcribed/translated products at 4C for 10 hours. The beads were then washed five times with binding buffer containing 75 mM NaCl and detected by Western blotting.

OT-I mice were injected with WT or SND1-KO B16F10-OVA cells, and CD8+ T cells were collected by microbeads (no. 130-116-478, Miltenyi Biotec), restimulated ex vivo with SIINFEKL peptide (OVA-derived peptide being presented through the MHC class I molecules; no. S7951, Sigma-Aldrich) for 20 hours, and lastly cultured with brefeldin A (no. 420601, BioLegend) for 4 hours. Then, the cells were subjected to flow cytometric analysis, and the percentages of IFN+CD8+ T cells among total CD8+ T cells was measured by flow cytometry.

A cytotoxicity detection kit (R&D Systems) was used to measure the cytolysis rate elicited by CD8+ T cells against different tumor cells. B16F10-OVA or MC38-OVA (WT or SND1-KO) cells (2 104) were cocultured with CD8+ T cells in an E/T cell ratiodependent manner (1 105/2 105/3 105/4 105) isolated from OT-I mice with or without the treatment of MHC-blocking antibodies (AF6-88.5; 0.25 g for 1 105 CD8+ T cells) in sterile 96-well tissue culture plates for 12 hours. The microplate was centrifuged at 250g for 10 min, and supernatant was removed to incubate with reaction mixture. The absorbance of the samples was measured at 490 or 492 nm with an enzyme-linked immunosorbent assay reader.

The amount of lactate dehydrogenase released from lysed target cells was used as an indicator for cytolysis. Cytolysis rate (percentage) was calculated on the basis of the following equation: cytotoxicity (%) = (effector/target cell mix effector cell control low control)/(high control low control) 100. Under the same conditions, B16F10-OVA or MC38-OVA (WT or SND1-KO) cells (1 105) were cocultured with CD8+ T cells (1 106) in sterile 24-well culture plates, and images were taken under a bright field using a Leica microscope at different time points.

The three-dimensional structure of SND1 and HLA-A were generated using I-TASSER. Then, the ZDOCK website was used to predict the possible binding conformation between SND1 and HLA-A. The structure with the lowest binding free energy was chosen as the initial binding conformation, which was further optimized using molecular dynamics (MD) simulation. MD simulation was carried out with Gromacs package (version 5.0.1) and GROMOS96 54A7 force field. The binding complex of SND1 and HLA-A was first placed in a cube box with a minimum distance of 1.0 nm from the edge of box. Then, the complex was solvated with TIP3P water molecules and neutralized with the addition of Na+ and Cl ions. A total of 1000 steps steepest descent energy minimization was conducted to remove local contacts, and the equilibration was performed including 1-ns NVT (Constant volume) and 5-ns NPT (Constant pressure) relaxations. Last, 50-ns MD simulations were performed at 300 K and 1.0-atm pressure under periodic boundary conditions. In all simulation steps, the SHAKE algorithm was applied to constrain all bonds involving hydrogen atoms, Particle mesh Ewald method was used to treat long-range electrostatics, and a cutoff distance of 1.0 nm was used for short-range electrostatic and van der Waals. On the basis of the resulting MD trajectory, we can analyze the structural stability of the complex, calculate the binding free energy using Molecular Mechanics Poisson Boltzmann Surface Area (MM-PBSA) method and identify key residues to the association process.

The correlation between SND1 and cancer immune infiltrates was investigated via TIMER (https://cistrome.shinyapps.io/timer/). The strength of correlations was evaluated using the Spearman correlation test; the Spearman coefficient was considered to indicate poor correlation if <0.2, moderate if <0.4, relatively strong if <0.6, strong if <0.8, and very strong if >0.8. P values <0.05 were considered statistically significant. The correlation between SND1 expression and survival in different cancers was analyzed by the PrognoScan database (http://dna00.bio.kyutech.ac.jp/PrognoScan/index.html). The threshold was adjusted to a Cox P < 0.05.

Data from biological triplicate experiments are presented with error bars as means SD unless otherwise noted. Two-tailed unpaired t test was used to compare two groups of data, and analysis of variance (ANOVA) with Bonferronis correction was used to compare multiple groups of data used for statistical analysis. All of the statistical testing results were determined using GraphPad Prism 7.0 software.

Acknowledgments: We thank Z. Dong from Tsinghua University for sharing the OT-I mouse model. Funding: This work was supported by grant 31125012 from the National Science Foundation for Distinguished Young Scholars of China (to J.Y.); grant IRT13085 from the Innovation Team Development Plan of the Ministry of Education (to J.Y.); grant 31300709 (to X.W.), grants 31870747 and 31370749 (to J.Y.), and grant 81572882 (to Z.Y.) from the National Natural Science Foundation of China; and the High-Level Innovation and Entrepreneurship Team of Tianjin Talent Development Special Support Plan (to J.Y.). This work also received support from grant YJSCX201814 (to Y.W.) from the Postgraduate Innovation Fund of 13th Five-Year Comprehensive Investment, Tianjin Medical University. Author contributions: Y.W., X.W., and J.Y. generated the initial idea and conducted key experiments. Y.W., X.C., H.L., C.H., and L.X. performed the research. Y.W., X.W., X.C., and J.Y. analyzed the data and wrote the manuscript. Y.Z., T.Z., and K.Z. helped with the bioinformatics analysis. J.Y., X.Y., and Z.Y. critically revised the manuscript for important intellectual content. L.G., X.L., J.H., Y.R., W.Z., and X.S. provided administrative, technical, or material support. J.Y. supervised the study. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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Oncoprotein SND1 hijacks nascent MHC-I heavy chain to ER-associated degradation, leading to impaired CD8+ T cell response in tumor - Science Advances

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How to do PewDiePies workout for abs and biceps: Cheap equipment and other YouTubers to follow! – HITC – Football, Gaming, Movies, TV, Music

Although PewDiePie deleted his Twitter account awhile back, he was recently all over the social media space a couple of days ago thanks to a shredded picture posted to Instagram by his wife Marzia. This shredded image resulted in fans wanting to know his biceps and abs workout routine so they can do it as well, and thankfully the Swedish YouTube star has shared his routine and methods. Here youll discover how to do the workout as well what cheap equipment you can buy and other YouTubers you can follow to expand upon Pewds advice.

PewDiePie has been a controversial figure on YouTube over the years for some and he has explained some of his heated acts as him being pretty irresponsible in the past. However, he has lately been one of the more honest and straightforward celebrities on the mega platform and this has helped continue his popularity despite him no longer being the horror video game squealer he was before.

His transformation is shown by him acting a lot wiser and more mature, but his transformation is also now embodied by his ripped body. And here youll discover how to do his workout routine for abs and biceps with even cheap equipment.

PewDiePie has shared his five day dumbbell workout routine for abs, biceps, and more.

According to PewDiePie, his five day dumbbell workout routine for abs, biceps, and more begins on Monday with him focusing heavily on his chest and finishing on his shoulders.

Tuesday is a leg day where he does squats, dead-lifts, and lunges, meanwhile Wednesday is reserved for pull exercises.

Thursday is another leg day whereas Friday is a mix of both push and pull exercises.

Although the YouTuber didnt show himself performing any of the exercises, he did share a diagram of the moves he consistently performs.

Of course, anyone will tell you that to build muscle and to burn fat you need to do a lot more than just lift weights.

PewDiePie himself admitted this by stating that he is now eating a greater amount of protein. Not only that, but he has also largely quit alcohol with the exception of social gatherings.

You can buy workout equipment used by PewDiePie to follow his five day dumbbell workout routine.

His DTX Fitness Folding Weight Bench is currently unavailable on Amazon, but you can find other just as good benches for as cheap as 109.99.

PewDiePie says he uses a PowerBlock Sports Series Interchangeable Dumbbell that goes up to 90 pounds, and you can buy one of these from the Powerblock website.

If you really want to hone in on your abs, a machine you could buy is a wonder core for just 89.99. This is a great piece of equipment which allows you to do multiple ab exercises as well as even arms.

You could also instead buy an adjustable Power Tower for the same price. This is an extremely effective tool as it allows you to do ab exercises as well pull ones.

As for weights, you can do PewDiePies pull and lift five day dumbbell workout routine with dumbbells or his Power Blocks, but you may wish to invest in a barbell with a set of weight plates.

This is because it helps you become stronger and lift heavier thanks to both your arms sharing and lifting the load.

For squats and other leg exercises you may want to buy some resistance bands for extra tension.

Lastly, PewDiePie also states that he uses Wrist Wraps to help prevent injury when lifting and these can be bought for as cheap as 9.

If youre interested in changing your figure like PewDiePie there are other YouTubers you can watch for workout routines.

Athlean X is particularly good as he shares routines that can be done at home as well as in the gym, with expensive equipment or with just DIY resources such as a towel.

WWE wrestler Sheamus is also good as he showcases a wide variety of different workout routines from heavy lifting to crossfit. And yes, a lot of his can be performed at home too.

If you want to burn body fat, then youll also be interested in performing HIIT exercises as these burn more calories than lifting weights.

YouTubers/figures who are helpful in this area include Joe Wicks as well as crossfits Lauren Fisher who has her own virtual fitness classes.

In other news, TikTok: What is the Pause Challenge? And how can I do it?

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How to do PewDiePies workout for abs and biceps: Cheap equipment and other YouTubers to follow! - HITC - Football, Gaming, Movies, TV, Music

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Nanomedicine Market: Industry Analysis and forecast 2026: By Modality, Diseases, Application and… – Azizsalon News

Nanomedicine Market was valued US$ XX Bn in 2018 and is expected to reach US$ XX Bn by 2026, at CAGR of XX% during forecast period of 2019 to 2026.

Nanomedicine Market Drivers and Restrains:Nanomedicine is an application of nanotechnology, which are used in diagnosis, treatment, monitoring, and control of biological systems. Nanomedicine usages nanoscale manipulation of materials to improve medicine delivery. Therefore, nanomedicine has facilitated the treatment against various diseases. The nanomedicine market includes products that are nanoformulations of the existing drugs and new drugs or are nanobiomaterials. The research and development of new devices as well as the diagnostics will become, more effective, enabling faster response and the ability to treat new diseases are likely to boost the market growth.

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The nanomedicine markets are driven by factors such as developing new technologies for drug delivery, increase acceptance of nanomedicine across varied applications, rise in government support and funding, the growing need for therapies that have fewer side effects and cost-effective. However, long approval process and risks associated with nanomedicine (environmental impacts) are hampering the market growth at the global level. An increase in the out-licensing of nanodrugs and growth of healthcare facilities in emerging economies are likely to create lucrative opportunities in the nanomedicine market.

The report study has analyzed revenue impact of covid-19 pandemic on the sales revenue of market leaders, market followers and disrupters in the report and same is reflected in our analysis.

Nanomedicine Market Segmentation Analysis:Based on the application, the nanomedicine market has been segmented into cardiovascular, neurology, anti-infective, anti-inflammatory, and oncology. The oncology segment held the dominant market share in 2018 and is projected to maintain its leading position throughout the forecast period owing to the rising availability of patient information and technological advancements. However, the cardiovascular and neurology segment is projected to grow at the highest CAGR of XX% during the forecast period due to presence of opportunities such as demand for specific therapeutic nanovectors, nanostructured stents, and implants for tissue regeneration.

Nanomedicine Market Regional Analysis:Geographically, the Nanomedicine market has been segmented into North America, the Europe, Asia Pacific, Latin America, and Middle East & Africa. North America held the largest share of the Nanomedicine market in 2018 due to the rising presence of patented nanomedicine products, the availability of advanced healthcare infrastructure and the rapid acceptance of nanomedicine. The market in Asia Pacific is expected to expand at a high CAGR of XX% during the forecast period thanks to rise in number of research grants and increase in demand for prophylaxis of life-threatening diseases. Moreover, the rising investments in research and development activities for the introduction of advanced therapies and drugs are predicted to accelerate the growth of this region in the near future.

Nanomedicine Market Competitive landscapeMajor Key players operating in this market are Abbott Laboratories, CombiMatrix Corporation, General Electric Company, Sigma-Tau Pharmaceuticals, Inc, and Johnson & Johnson. Manufacturers in the nanomedicine are focusing on competitive pricing as the strategy to capture significant market share. Moreover, strategic mergers and acquisitions and technological innovations are also the key focus areas of the manufacturers.

The objective of the report is to present a comprehensive analysis of Nanomedicine Market including all the stakeholders of the industry. The past and current status of the industry with forecasted market size and trends are presented in the report with the analysis of complicated data in simple language. The report covers all aspects of the industry with a dedicated study of key players that includes market leaders, followers and new entrants by region. PORTER, SVOR, PESTEL analysis with the potential impact of micro-economic factors by region on the market are presented in the report. External as well as internal factors that are supposed to affect the business positively or negatively have been analyzed, which will give a clear futuristic view of the industry to the decision-makers. The report also helps in understanding Nanomedicine Market dynamics, structure by analyzing the market segments and project the Nanomedicine Market size. Clear representation of competitive analysis of key players By Type, Price, Financial position, Product portfolio, Growth strategies, and regional presence in the Nanomedicine Market make the report investors guide.

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Scope of the Nanomedicine Market:

Nanomedicine Market by Modality:

Diagnostics TreatmentsNanomedicine Market by Diseases:

Oncological Diseases Infectious Diseases Cardiovascular Diseases Orthopedic Disorders Neurological Diseases Urological Diseases Ophthalmological Diseases Immunological DiseasesNanomedicine Market by Application:

Neurology Cardiovascular Anti-Inflammatory Anti-Infectives OncologyNanomedicine Market by Region:

Asia Pacific North America Europe Latin America Middle East AfricaNanomedicine Market Major Players:

Abbott Laboratories CombiMatrix Corporation General Electric Company Sigma-Tau Pharmaceuticals, Inc Johnson & Johnson Mallinckrodt plc. Merck & Company, Inc. Nanosphere, Inc. Pfizer, Inc. Teva Pharmaceutical Industries Ltd. Celgene Corporation UCB (Union Chimique Belge) S.A. AMAG Pharmaceuticals Nanospectra Biosciences, Inc. Arrowhead Pharmaceuticals, Inc. Leadiant Biosciences, Inc. Epeius Biotechnologies Corporation Cytimmune Sciences, Inc.

MAJOR TOC OF THE REPORT

Chapter One: Nanomedicine Market Overview

Chapter Two: Manufacturers Profiles

Chapter Three: Global Nanomedicine Market Competition, by Players

Chapter Four: Global Nanomedicine Market Size by Regions

Chapter Five: North America Nanomedicine Revenue by Countries

Chapter Six: Europe Nanomedicine Revenue by Countries

Chapter Seven: Asia-Pacific Nanomedicine Revenue by Countries

Chapter Eight: South America Nanomedicine Revenue by Countries

Chapter Nine: Middle East and Africa Revenue Nanomedicine by Countries

Chapter Ten: Global Nanomedicine Market Segment by Type

Chapter Eleven: Global Nanomedicine Market Segment by Application

Chapter Twelve: Global Nanomedicine Market Size Forecast (2019-2026)

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Amid the COVID-19 crisis and the looming economic recession, the Nanotechnology in Drug Delivery market worldwide will grow by a projected US$124.7…

driven by a revised compounded annual growth rate (CAGR) of 22. 6%. Nanocrystals, one of the segments analyzed and sized in this study, is forecast to grow at over 20. 2% and reach a market size of US$83.

New York, May 27, 2020 (GLOBE NEWSWIRE) -- Reportlinker.com announces the release of the report "Global Nanotechnology in Drug Delivery Industry" - https://www.reportlinker.com/p05621749/?utm_source=GNW 1 Billion by the end of the analysis period. An unusual period in history, the coronavirus pandemic has unleashed a series of unprecedented events affecting every industry. The Nanocrystals market will be reset to a new normal which going forwards in a post COVID-19 era will be continuously redefined and redesigned. Staying on top of trends and accurate analysis is paramount now more than ever to manage uncertainty, change and continuously adapt to new and evolving market conditions.

As part of the new emerging geographic scenario, the United States is forecast to readjust to a 17% CAGR. Within Europe, the region worst hit by the pandemic, Germany will add over US$4.1 Billion to the regions size over the next 7 to 8 years. In addition, over US$3.7 Billion worth of projected demand in the region will come from Rest of European markets. In Japan, the Nanocrystals segment will reach a market size of US$3 Billion by the close of the analysis period. Blamed for the pandemic, significant political and economic challenges confront China. Amid the growing push for decoupling and economic distancing, the changing relationship between China and the rest of the world will influence competition and opportunities in the Nanotechnology in Drug Delivery market. Against this backdrop and the changing geopolitical, business and consumer sentiments, the worlds second largest economy will grow at 25.2% over the next couple of years and add approximately US$20.2 Billion in terms of addressable market opportunity. Continuous monitoring for emerging signs of a possible new world order post-COVID-19 crisis is a must for aspiring businesses and their astute leaders seeking to find success in the now changing Nanotechnology in Drug Delivery market landscape. All research viewpoints presented are based on validated engagements from influencers in the market, whose opinions supersede all other research methodologies.

Competitors identified in this market include, among others, AbbVie Inc.; Aquanova AG; BlueWillow Biologics; Camurus AB; Celgene, Inc.; Ceramisphere Health Pty Limited; Cristal Therapeutics; CYTIMMUNE SCIENCES, Inc.; EnColl Corporation; EyePoint Pharmaceuticals; Lena Nanoceutics Ltd.; Nanobiotix; NanoCarrier Co., Ltd.; NanOlogy LLC; Nanospectra Biosciences, Inc.; Parvus Therapeutics Inc.; Selecta Biosciences; Starpharma Holdings Limited; Taiwan Liposome Co., ; Tarveda Therapeutics

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NANOTECHNOLOGY IN DRUG DELIVERY MCP-7MARKET ANALYSIS, TRENDS, AND FORECASTS, JUNE 2CONTENTS

I. INTRODUCTION, METHODOLOGY & REPORT SCOPE

II. EXECUTIVE SUMMARY

1. MARKET OVERVIEW Nanotechnology, A Critical Part of Healthcare Reform Recent Market Activity Nanomedicine, Revolutionizing the Basics of Medicine A Peek Into the Evolving Role of Nanoparticles in Nanomedicine Nanocrystals Continue to Gain Momentum as Alternatives to Traditional Nanocarriers Nanocarriers Enable Targeted Drug Delivery Systems to Improve Therapeutic Outcomes Advanced Liposomes Enable Low Soluble Drugs to Achieve Targeted Delivery Multifunctional Dendrimers Present an Ideal Structure for Targeted Drug Delivery High Drug Loading Capacity of PAMAM Dendrimers Bodes Well for Targeted Drug Delivery Systems Nanotechnology-Based Strategies for siRNA Grows in Popularity NEM Devices as Drug Delivery Vehicles Nanotechnology - In Pursuit of Co-Delivery of Drugs Nanoemulsions Begin to Make a Mark Nanotechnology Finds Increasing Role in Fighting Infectious Diseases List of Select Nanotechnology-based Antimicrobial Drugs in Clinical Use Nanotechnology Opening New Avenues in Antiretroviral Therapy Demonstrated Activity of Select Nanotechnology- Delivered Antiretroviral Therapies Nanotechnology in Delivery of CNS Therapeutics Market Outlook Global Competitor Market Shares Nanotechnology in Drug Delivery Competitor Market Share Scenario Worldwide (in %): 2020 & 2029 Impact of Covid-19 and a Looming Global Recession 2. FOCUS ON SELECT PLAYERS AbbVie Inc. (USA) Aquanova AG (Germany) BlueWillow Biologics (USA) Camurus AB (Sweden) Celgene, Inc. (Canada) Ceramisphere Health Pty Limited (Australia) Cristal Therapeutics (The Netherlands) CYTIMMUNE SCIENCES, INC. (Italy) EnColl Corporation (USA) EyePoint Pharmaceuticals (USA) Lena Nanoceutics Ltd. (UK) NanOlogy LLC (USA) NanoCarrier Co., Ltd. (Japan) Nanobiotix (France) Nanospectra Biosciences, Inc (USA) Parvus Therapeutics Inc. (USA) Selecta Biosciences (USA) Starpharma Holdings Limited (Australia) Taiwan Liposome Co. (Taiwan) Tarveda Therapeutics (USA) 3. MARKET TRENDS & DRIVERS Growing Need for Alternative Approaches to Conventional Chemotherapy Creates Opportunities for Nanoparticles Stimuli Responsive Polymeric Micelles Promise Enhanced Therapeutic Effect for Hydrophobic Anticancer Drugs Inorganic Nanocarriers Facilitate High Payload Capacity and Co -Delivery Platforms for MDR Cancer Therapy Solid Lipid Nanoparticles Provide Increased Physical Stability in Targeted Drug Delivery Pulmonary Delivery of Nanoparticle-Based Drugs Receives Increased Interest Inhalable Liposome Formulations Attract Research Interest in Pulmonary Delivery SLNs in Pulmonary Delivery of Drugs Market Challenges Increasing Environmental and Health Concerns Limited Success and Scaling Up Issues Pose a Major Hurdle to Further Advancement Higher Concentration of Research in Academia limits Commercialization Collaborations Assume Importance Nanomedicine Regulation 4. GLOBAL MARKET PERSPECTIVE Table 1: Nanotechnology in Drug Delivery Global Market Estimates and Forecasts in US$ Million by Region/Country: 2020-2027 Table 2: Nanotechnology in Drug Delivery Market Share Shift across Key Geographies Worldwide: 2020 VS 2027 Table 3: Nanocrystals (Technology) World Market by Region/Country in US$ Million: 2020 to 2027 Table 4: Nanocrystals (Technology) Market Share Breakdown of Worldwide Sales by Region/Country: 2020 VS 2027 Table 5: Nanocarriers (Technology) Potential Growth Markets Worldwide in US$ Million: 2020 to 2027 Table 6: Nanocarriers (Technology) Market Sales Breakdown by Region/Country in Percentage: 2020 VS 2027 III. MARKET ANALYSIS GEOGRAPHIC MARKET ANALYSIS UNITED STATES Market Facts & Figures US Nanotechnology in Drug Delivery Market Share (in %) by Company: 2020 & 2025 Market Analytics Table 7: Nanotechnology in Drug Delivery Market in US$ Million in the United States by Technology: 2020-2027 Table 8: United States Nanotechnology in Drug Delivery Market Share Breakdown by Technology: 2020 VS 2027 CANADA Table 9: Nanotechnology in Drug Delivery Market Analysis in Canada in US$ Million by Technology: 2020-2027 Table 10: Canadian Nanotechnology in Drug Delivery Market Share Breakdown by Technology: 2020 VS 2027 JAPAN Table 11: Japanese Medium & Long-Term Outlook for Nanotechnology in Drug Delivery Market in US$ Million by Technology: 2020-2027 Table 12: Japanese Nanotechnology in Drug Delivery Market Percentage Share Distribution by Technology: 2020 VS 2027 CHINA Table 13: Nanotechnology in Drug Delivery Market Estimates and Forecasts in China in US$ Million by Technology: 2020-2027 Table 14: Nanotechnology in Drug Delivery Market in China: Percentage Share Analysis by Technology for 2020 and 2027 EUROPE Market Facts & Figures European Nanotechnology in Drug Delivery Market: Competitor Market Share Scenario (in %) for 2020 & 2025 Market Analytics Table 15: European Nanotechnology in Drug Delivery Market Demand Scenario in US$ Million by Region/Country: 2018-2025 Table 16: European Nanotechnology in Drug Delivery Market Share Shift by Region/Country: 2020 VS 2027 Table 17: European Nanotechnology in Drug Delivery Market Assessment in US$ Million by Technology: 2020-2027 Table 18: Nanotechnology in Drug Delivery Market in Europe: Percentage Breakdown of Sales by Technology for 2020 and 2027 FRANCE Table 19: French Nanotechnology in Drug Delivery Market Estimates and Projections in US$ Million by Technology: 2020-2027 Table 20: French Nanotechnology in Drug Delivery Market Share Breakdown by Technology: 2020 VS 2027 GERMANY Table 21: German Nanotechnology in Drug Delivery Latent Demand Forecasts in US$ Million by Technology: 2020-2027 Table 22: German Nanotechnology in Drug Delivery Market Share Breakdown by Technology: 2020 VS 2027 ITALY Table 23: Nanotechnology in Drug Delivery Market Estimates and Forecasts in Italy in US$ Million by Technology: 2020-2027 Table 24: Nanotechnology in Drug Delivery Market in Italy: Percentage Share Analysis by Technology for 2020 and 2027 UNITED KINGDOM Table 25: United Kingdom Medium & Long-Term Outlook for Nanotechnology in Drug Delivery Market in US$ Million by Technology: 2020-2027 Table 26: United Kingdom Nanotechnology in Drug Delivery Market Percentage Share Distribution by Technology: 2020 VS 2027 SPAIN Table 27: Nanotechnology in Drug Delivery Market Analysis in Spain in US$ Million by Technology: 2020-2027 Table 28: Spanish Nanotechnology in Drug Delivery Market Share Breakdown by Technology: 2020 VS 2027 RUSSIA Table 29: Nanotechnology in Drug Delivery Market in US$ Million in Russia by Technology: 2020-2027 Table 30: Russian Nanotechnology in Drug Delivery Market Share Breakdown by Technology: 2020 VS 2027 REST OF EUROPE Table 31: Rest of Europe Nanotechnology in Drug Delivery Market Assessment in US$ Million by Technology: 2020-2027 Table 32: Nanotechnology in Drug Delivery Market in Rest of Europe: Percentage Breakdown of Sales by Technology for 2and 2027 ASIA-PACIFIC Table 33: Asia-Pacific Nanotechnology in Drug Delivery Market Estimates and Forecasts in US$ Million by Region/Country: 2020-2027 Table 34: Asia-Pacific Nanotechnology in Drug Delivery Market Share Analysis by Region/Country: 2020 VS 2027 Table 35: Asia-Pacific Nanotechnology in Drug Delivery Market Estimates and Projections in US$ Million by Technology: 2020-2027 Table 36: Asia-Pacific Nanotechnology in Drug Delivery Historic Market Analysis in US$ Million by Technology: 2020 VS 2027 AUSTRALIA Table 37: Australian Nanotechnology in Drug Delivery Latent Demand Forecasts in US$ Million by Technology: 2020-2027 Table 38: Australian Nanotechnology in Drug Delivery Market Share Breakdown by Technology: 2020 VS 2027 INDIA Table 39: Nanotechnology in Drug Delivery Market Analysis in India in US$ Million by Technology: 2020-2027 Table 40: Indian Nanotechnology in Drug Delivery Market Share Breakdown by Technology: 2020 VS 2027 SOUTH KOREA Table 41: Nanotechnology in Drug Delivery Market in South Korea: Recent Past, Current and Future Analysis in US$ Million by Technology for the Period 2020-2027 Table 42: Nanotechnology in Drug Delivery Market Share Distribution in South Korea by Technology: 2020 VS 2027 REST OF ASIA-PACIFIC Table 43: Rest of Asia-Pacific Medium & Long-Term Outlook for Nanotechnology in Drug Delivery Market in US$ Million by Technology: 2020-2027 Table 44: Rest of Asia-Pacific Nanotechnology in Drug Delivery Market Percentage Share Distribution by Technology: 2020 VS 2027 LATIN AMERICA Table 45: Latin American Nanotechnology in Drug Delivery Market Trends by Region/Country in US$ Million: 2020-2027 Table 46: Latin American Nanotechnology in Drug Delivery Market Percentage Breakdown of Sales by Region/Country: 2020 and 2027 Table 47: Nanotechnology in Drug Delivery Market Estimates and Forecasts in Latin America in US$ Million by Technology: 2020-2027 Table 48: Nanotechnology in Drug Delivery Market in Latin America : Percentage Analysis by Technology for 2020 and 2027 ARGENTINA Table 49: Argentinean Nanotechnology in Drug Delivery Market Assessment in US$ Million by Technology: 2020-2027 Table 50: Nanotechnology in Drug Delivery Market in Argentina: Percentage Breakdown of Sales by Technology for 2020 and 2027 BRAZIL Table 51: Brazilian Nanotechnology in Drug Delivery Market Estimates and Projections in US$ Million by Technology: 2020-2027 Table 52: Brazilian Nanotechnology in Drug Delivery Market Share Breakdown by Technology: 2020 VS 2027 MEXICO Table 53: Mexican Nanotechnology in Drug Delivery Latent Demand Forecasts in US$ Million by Technology: 2020-2027 Table 54: Mexican Nanotechnology in Drug Delivery Market Share Breakdown by Technology: 2020 VS 2027 REST OF LATIN AMERICA Table 55: Nanotechnology in Drug Delivery Market in US$ Million in Rest of Latin America by Technology: 2020-2027 Table 56: Rest of Latin America Nanotechnology in Drug Delivery Market Share Breakdown by Technology: 2020 VS 2027 MIDDLE EAST Table 57: The Middle East Nanotechnology in Drug Delivery Market Estimates and Forecasts in US$ Million by Region/Country: 2018-2025 Table 58: The Middle East Nanotechnology in Drug Delivery Market Share Breakdown by Region/Country: 2020 and 2027 Table 59: The Middle East Nanotechnology in Drug Delivery Market Analysis in US$ Million by Technology: 2020-2027 Table 60: The Middle East Nanotechnology in Drug Delivery Market Share Breakdown by Technology: 2020 VS 2027 IRAN Table 61: Iranian Medium & Long-Term Outlook for Nanotechnology in Drug Delivery Market in US$ Million by Technology: 2020-2027 Table 62: Iranian Nanotechnology in Drug Delivery Market Percentage Share Distribution by Technology: 2020 VS 2027 ISRAEL Table 63: Israeli Nanotechnology in Drug Delivery Market Assessment in US$ Million by Technology: 2020-2027 Table 64: Nanotechnology in Drug Delivery Market in Israel: Percentage Breakdown of Sales by Technology for 2020 and 2027 SAUDI ARABIA Table 65: Nanotechnology in Drug Delivery Market Estimates and Forecasts in Saudi Arabia in US$ Million by Technology: 2020-2027 Table 66: Nanotechnology in Drug Delivery Market in Saudi Arabia: Percentage Share Analysis by Technology for 2020 and 2027 UNITED ARAB EMIRATES Table 67: Nanotechnology in Drug Delivery Market in the United Arab Emirates: Recent Past, Current and Future Analysis in US$ Million by Technology for the Period 2020-2027 Table 68: Nanotechnology in Drug Delivery Market Share Distribution in United Arab Emirates by Technology: 2020 VS 2027 REST OF MIDDLE EAST Table 69: Rest of Middle East Nanotechnology in Drug Delivery Latent Demand Forecasts in US$ Million by Technology: 2020-2027 Table 70: Rest of Middle East Nanotechnology in Drug Delivery Market Share Breakdown by Technology: 2020 VS 2027 AFRICA Table 71: Nanotechnology in Drug Delivery Market in US$ Million in Africa by Technology: 2020-2027 Table 72: African Nanotechnology in Drug Delivery Market Share Breakdown by Technology: 2020 VS 2027 IV. COMPETITION

Total Companies Profiled: 43 Read the full report: https://www.reportlinker.com/p05621749/?utm_source=GNW

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Amid the COVID-19 crisis and the looming economic recession, the Nanotechnology in Drug Delivery market worldwide will grow by a projected US$124.7...

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13 Habits Linked to a Long Life (Backed by Science)

Many people think that life expectancy is largely determined by genetics.

However, genes play a much smaller role than originally believed. It turns out that environmental factors like diet and lifestyle are key.

Here are 13 habits linked to a long life.

The link between calorie intake and longevity currently generates a lot of interest.

Animal studies suggest that a 1050% reduction in normal calorie intake may increase maximum lifespan (1).

Studies of human populations renowned for longevity also observe links between low calorie intake, an extended lifespan, and a lower likelihood of disease (2, 3, 4).

What's more, calorie restriction may help reduce excess body weight and belly fat, both of which are associated with shorter lifespans (5, 6, 7).

That said, long-term calorie restriction is often unsustainable and can include negative side effects, such as increased hunger, low body temperature, and a diminished sex drive (3).

Whether calorie restriction slows aging or extends your lifespan is not yet fully understood.

Nuts are nutritional powerhouses.

They're rich in protein, fiber, antioxidants, and beneficial plant compounds. Whats more, theyre a great source of several vitamins and minerals, such as copper, magnesium, potassium, folate, niacin, and vitamins B6 and E (8).

Several studies show that nuts have beneficial effects on heart disease, high blood pressure, inflammation, diabetes, metabolic syndrome, belly fat levels, and even some forms of cancer (9, 10, 11, 12).

One study found that people who consumed at least 3 servings of nuts per week had a 39% lower risk of premature death (13).

Similarly, two recent reviews including over 350,000 people noted that those who ate nuts had a 427% lower risk of dying during the study period with the greatest reductions seen in those who ate 1 serving of nuts per day (14, 15).

When it comes to anti-aging strategies, turmeric is a great option. Thats because this spice contains a potent bioactive compound called curcumin.

Due to its antioxidant and anti-inflammatory properties, curcumin is thought to help maintain brain, heart, and lung function, as well as protect against cancers and age-related diseases (16, 17, 18, 19, 20, 21, 22).

Curcumin is linked to an increased lifespan in both insects and mice (23, 24, 25).

However, these findings have not always been replicated, and no human studies are currently available (26, 27).

Nevertheless, turmeric has been consumed for thousands of years in India and is generally considered safe.

Consuming a wide variety of plant foods, such as fruits, vegetables, nuts, seeds, whole grains, and beans, may decrease disease risk and promote longevity.

For example, many studies link a plant-rich diet to a lower risk of premature death, as well as a reduced risk of cancer, metabolic syndrome, heart disease, depression, and brain deterioration (28, 29, 30, 31).

These effects are attributed to plant foods nutrients and antioxidants, which include polyphenols, carotenoids, folate, and vitamin C (32).

Accordingly, several studies link vegetarian and vegan diets, which are naturally higher in plant foods, to a 1215% lower risk of premature death (33, 34).

The same studies also report a 2952% lower risk of dying from cancer or heart, kidney, or hormone-related diseases (33, 34).

Whats more, some research suggests that the risk of premature death and certain diseases increases with greater meat consumption (35, 36, 37).

However, other studies report either nonexistent or much weaker links with the negative effects seeming specifically linked to processed meat (38, 39).

Vegetarians and vegans also generally tend to be more health-conscious than meat eaters, which could at least partly explain these findings.

Overall, eating plenty of plant foods is likely to benefit health and longevity.

It should come as no surprise that staying physically active can keep you healthy and add years to your life (40).

As few as 15 minutes of exercise per day may help you achieve benefits, which could include an additional 3 years of life (41).

Furthermore, your risk of premature death may decrease by 4% for each additional 15 minutes of daily physical activity (41).

A recent review observed a 22% lower risk of early death in individuals who exercised even though they worked out less than the recommended 150 minutes per week (42).

People who hit the 150-minute recommendation were 28% less likely to die early. What's more, that number was 35% for those who exercised beyond this guidance (42).

Finally, some research links vigorous activity to a 5% greater reduction in risk compared to low- or moderate-intensity activities (43).

Smoking is strongly linked to disease and early death (44).

Overall, people who smoke may lose up to 10 years of life and be 3 times more likely to die prematurely than those who never pick up a cigarette (45).

Keep in mind that it's never too late to quit.

One study reports that individuals who quit smoking by age 35 may prolong their lives by up to 8.5 years (46).

Furthermore, quitting smoking in your 60s may add up to 3.7 years to your life. In fact, quitting in your 80s may still provide benefits (44, 46).

Heavy alcohol consumption is linked to liver, heart, and pancreatic disease, as well as an overall increased risk of early death (47).

However, moderate consumption is associated with a reduced likelihood of several diseases, as well as a 1718% decrease in your risk of premature death (47, 48).

Wine is considered particularly beneficial due to its high content of polyphenol antioxidants.

Results from a 29-year study showed that men who preferred wine were 34% less likely to die early than those who preferred beer or spirits (49).

In addition, one review observed wine to be especially protective against heart disease, diabetes, neurological disorders, and metabolic syndrome (50).

To keep consumption moderate, it is recommended that women aim for 12 units or less per day and a maximum of 7 per week. Men should keep their daily intake to less than 3 units, with a maximum of 14 per week (51).

It's important to note that no strong research indicates that the benefits of moderate drinking are greater than those of abstaining from alcohol.

In other words, there is no need to start drinking if you don't usually consume alcohol.

Feeling happy can significantly increase your longevity (52).

In fact, happier individuals had a 3.7% reduction in early death over a 5-year study period (53).

A study of 180 Catholic nuns analyzed their self-reported levels of happiness when they first entered the monastery and later compared these levels to their longevity.

Those who felt happiest at 22 years of age were 2.5 times more likely to still be alive six decades later (54).

Finally, a review of 35 studies showed that happy people may live up to 18% longer than their less happy counterparts (55).

Anxiety and stress may significantly decrease your lifespan.

For instance, women suffering from stress or anxiety are reportedly up to two times more likely to die from heart disease, stroke, or lung cancer (56, 57, 58).

Similarly, the risk of premature death is up to three times higher for anxious or stressed men compared to their more relaxed counterparts (59, 60, 61).

If you're feeling stressed, laughter and optimism could be two key components of the solution.

Studies show that pessimistic individuals have a 42% higher risk of early death than more optimistic people. However, both laughter and a positive outlook on life can reduce stress, potentially prolonging your life (62, 63, 64, 65).

Researchers report that maintaining healthy social networks can help you live up to 50% longer (66).

In fact, having just 3 social ties may decrease your risk of early death by more than 200% (67).

Studies also link healthy social networks to positive changes in heart, brain, hormonal, and immune function, which may decrease your risk of chronic diseases (68, 69, 70, 71, 72).

A strong social circle might also help you react less negatively to stress, perhaps further explaining the positive effect on lifespan (73, 74).

Finally, one study reports that providing support to others may be more beneficial than receiving it. In addition to accepting care from your friends and family, make sure to return the favor (75).

Conscientiousness refers to a person's ability to be self-disciplined, organized, efficient, and goal-oriented.

Based on data from a study that followed 1,500 boys and girls into old age, kids who were considered persistent, organized, and disciplined lived 11% longer than their less conscientious counterparts (76, 77).

Conscientious people may also have lower blood pressure and fewer psychiatric conditions, as well as a lower risk of diabetes and heart or joint problems (78).

This might be partly because conscientious individuals are less likely to take dangerous risks or react negatively to stress and more likely to lead successful professional lives or be responsible about their health (79, 80, 81).

Conscientiousness can be developed at any stage in life through steps as small as tidying up a desk, sticking to a work plan, or being on time.

Both coffee and tea are linked to a decreased risk of chronic disease.

For instance, the polyphenols and catechins found in green tea may decrease your risk of cancer, diabetes, and heart disease (82, 83, 84, 85, 86).

Similarly, coffee is linked to a lower risk of type 2 diabetes, heart disease, and certain cancers and brain ailments, such as Alzheimer's and Parkinson's (87, 88, 89, 90, 91, 92).

Additionally, both coffee and tea drinkers benefit from a 2030% lower risk of early death compared to non-drinkers (93, 94, 95, 96).

Just remember that too much caffeine can also lead to anxiety and insomnia, so you may want to curb your intake to the recommended limit of 400 mg per day around 4 cups of coffee (97, 98).

It's also worth noting that it generally takes six hours for caffeine's effects to subside. Therefore, if you have trouble getting enough high-quality sleep, you may want to shift your intake to earlier in the day.

Sleep is crucial for regulating cell function and helping your body heal.

A recent study reports that longevity is likely linked to regular sleeping patterns, such as going to bed and waking up around the same time each day (99).

Sleep duration also seems to be a factor, with both too little and too much being harmful.

For instance, sleeping less than 57 hours per night is linked to a 12% greater risk of early death, while sleeping more than 89 hours per night could also decrease your lifespan by up to 38% (100, 101).

Too little sleep may also promote inflammation and increase your risk of diabetes, heart disease, and obesity. These are all linked to a shortened lifespan (102, 103, 104, 105).

On the other hand, excessive sleep could be linked to depression, low physical activity, and undiagnosed health conditions, all of which may negatively affect your lifespan (106).

Longevity may seem beyond your control, but many healthy habits may lead you to a ripe, old age.

These include drinking coffee or tea, exercising, getting enough sleep, and limiting your alcohol intake.

Taken together, these habits can boost your health and put you on the path to a long life.

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How to live to 100 – Business Times

FROM 1960 till 2020, there has been a 28-fold increase in the number of centenarians. The path to longevity is strewn with false promises of expensive elixirs, exotic supplements, and stem cell rejuvenation. Human longevity is a complex interplay between the genes, the environment and lifestyle.

Genes and longevity

The study of human longevity genes is a developing science. Scientists estimate that between 15 and 30 per cent of the variation in human life span is determined by genes, but it is not clearly understood which genes are relevant, and how they contribute to longevity. In 2015, Ancestry, a genealogy and genetics company, partnered Calico, a Google spinoff, to study data from more than 54 million families and their family trees representing six billion ancestors, and were able to tease out a set of pedigrees that included over 400 million people. These individuals were connected to one another by either a parent-child or a spouse-spouse relationship.

In 2018, they published their results in Genetics, a journal of the Genetics Society of America. The study found that the lifespan of spouses were more similar and better correlated than in siblings of opposite gender. The study concluded that life span heritability is likely 7 per cent or less, and hence the contribution of genes to longevity is even lower.

Although genes seem to have only a small influence on lifespan, they appear to play a larger role in centenarians. Hence, there are a few genetic factors that do give you a headstart in the journey to longevity.

Being a first-degree relative of a centenarian makes it more likely for you to remain healthy longer and to live to an older age than your peers. First-degree relatives are less likely at age 70 years to have the age-related diseases that are common among older adults.

Women generally live longer than men , and the number of female centenarians is more than fourfold higher than that of male centenarians. It is thought that this is due to a combination of social and biological factors. Studies on mammals and Korean eunuchs has shown that the removal of testosterone at a young age was correlated with an increase in lifespan.

Genetic studies show that centenarians have a lower genetic risk of having heart disease, stroke , high blood pressure, high cholesterol, Alzheimer's disease and decreased bone mineral density. A study on Chinese centenarians published in 2013 showed that 55 per cent have normal systolic blood pressure, 82 per cent had normal diastolic blood pressure and less than 20 per cent were on long term medication. Hence, centenarians appear to have genes that reduce that risk of age-related chronic illnesses.

Biological clock

Epigenetics is the study of changes in organisms caused by modification of gene expression rather than alteration of the genetic code itself. One of the major mechanisms in which epigenetics manifest itself is by the process of DNA methylation, which involves the chemical modification of the DNA, thereby modifying the gene function and expression.

Through this process, certain genes can be silenced or activated and potentially impact age-related diseases such as cancer, osteoarthritis, and neurodegeneration. The biological or epigenetic clock in centenarians show a decrease in DNA methylation age, indicating that they are biologically younger than their chronological age. There is also data to suggest that although circadian rhythms deteriorate during ageing, they seem to be well preserved in centenarians, including preserved sleep quality.

Environment and longevity

Environmental factors have a large impact on longevity. Better living environment, clean food, clean water, good sanitation, reduction of infectious diseases, and access to better healthcare have resulted in significant improvement in human longevity.

Using Italy as an example of the impact of a better living environment, the average life expectancy went up from 29 years in 1861 to 84 years in 2020. The number of centenarians in Italy increased from 165 in 1951 to more than 15,000 in 2011, and the incidence of deaths occurring in those less than 60 years of age, decreased from 74 per cent in 1872 to less than 10 per cent in 2011 .

The continuous increase in lifespan in recent decades is mainly due to the advances in medical science. It is estimated that medical advances have allowed an increase in lifespan of five years in the last two decades and additional two years in the last decade.

When comparing two countries at different stages of development in 1950, the average life expectancy increase of 11 years from 68 years in 1950 to 79 years in 2020 in the USA, which was more developed in 1950, was much less remarkable than the increase of 3114 years in average life expectancy from 43 years in 1950 to 77 years in 2020 in China, which was less developed in 1950. The significant improvement in the living environment in China has contributed to the narrowing in the average life expectancy between those living in the US and China.

Lifestyle and longevity

In addition to environmental factors, lifestyle factors have an important impact on longevity. A study of more than 300,000 individuals over 7.5 years showed that individuals with social relationships have more than 50 per cent greater probability of survival compared with those with few and poor social interactions.

A study on centenarians in Utah in the US between 2008 and 2015 suggested that sleep, life satisfaction and social attachment were significant predictors of days lived. There is an extricable linkage between lifestyle and socioeconomic status. The term socioeconomic status as used in longevity studies encompass all the factors that can impact longevity including wealth, geography, education, occupation, ethnicity, cultural environment, neighbourhood environment, quality of healthcare and quality of diet. It is well established that the socioeconomic status of an individual will have a major impact on health and longevity.

A study on more than 120, 000 individuals by researchers from Harvard, published in the Circulation journal in April 2018, identified five low-risk lifestyle factors for increased life expectancy. They were: no smoking, non obese ( body mass index of 18.5 to 24.9 kg/m2), exercise (at least 30 minutes per day of moderate to vigorous physical activity, including brisk walking), low-risk alcohol consumption (5 to 15 gm/day for women and 5 to 30 gm/day for men), and a high score for healthy diet.

In this study, the projected life expectancy at age 50 years was on average 14.0 years longer among female Americans with five low-risk factors compared with those with zero low-risk factors; for men, the difference was 12.2 years.

These findings are consistent with a study on Chinese centenarians in which less than 20 per cent were smokers and less than 40 per cent drank alcohol. Hence, in general, most centenarians do not smoke, do not drink alcohol or are low-risk alcohol drinkers, are sociable, friendly, cope well with stress, are satisfied with life, have healthy diets and sleep well.

In summary, the main drivers of longevity in the first eight decades of life are the socioeconomic environment and lifestyle choices. Beyond the eighties, the inheritance of genes that defer age-related chronic diseases and a younger biological clock will help to propel these individuals beyond a hundred years.

This series is produced on alternate Saturdays in collaboration with Singapore Medical Specialists Centre

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