consumption of meat and Jainism institutes an outright ban on meat. Consequently, India is home to more vegetarians than any other country. About 30% of India's 1.2 billion population practices lacto vegetarianism.[8] While many ancient Indian religions with vegetarian tenets persist throughout India and the Indian diaspora today, Buddhism and Buddhist vegetarian practices are now more widespread throughout other parts of Asia and manifest on other continents as well. Jainism Main article: Jain vegetarianism The food choices of Jains are based on the value of ahimsa (non-violence), and this makes the Jains to prefer food that inflict the least amount of violence Vegetarianism in Jainism is based on the principle of nonviolence (ahimsa, literally "non-injuring"). Vegetarianism is considered mandatory for everyone. Jains are either lacto-vegetarians or vegans.[9] No use or consumption of products obtained from dead animals is allowed. Moreover, Jains try to avoid unnecessary injury to plants and suksma jiva (Sanskrit for 'subtle life forms'; minuscule organisms). The goal is to cause as little violence to living things as possible, hence they avoid eating roots, tubers such as potatoes, garlic and anything that involves uprooting (and thus eventually killing) a plant to obtain food. Every act by which a person directly or indirectly supports killing or injury is seen as violence (hinsa), which creates harmful karma. The aim of ahimsa is to prevent the accumulation of such karma.[10] Jains consider nonviolence to be the most essential religious duty for everyone (ahinsÄ paramo dharmaḥ, a statement often inscribed on Jain temples). Their scrupulous and thorough way of applying nonviolence to everyday activities, and especially to food, shapes their entire lives and is the most significant hallmark of Jain identity. A side effect of this strict discipline is the exercise of asceticism,[11] which is strongly encouraged in Jainism for lay people as well as for monks and nuns. Jains do not practice animal sacrifice as they consider all sentient beings to be equal. Hinduism Main article: Diet in Hinduism While vegetarianism is an integral part of Hinduism,[12] there are a wide variety of practices and beliefs that have changed over time.[13] Some sects of Hindus do not observe vegetarianism,[14] while an estimated 33% of all Hindus are vegetarians.[15][16] Nonviolence Indian vegetarian thali North Indian style vegetarian thali. South Indian style vegetarian thali. The principle of nonviolence (ahimsa) applied to animals is connected with the intention to avoid negative karmic influences which result from violence. The suffering of all beings is believed to arise from craving and desire, conditioned by the karmic effects of both animal and human action. The violence of slaughtering animals for food, and its source in craving, reveal flesh eating as one mode in which humans enslave themselves to suffering.[17] Hinduism holds that such influences affect the person who permits the slaughter of an animal, the person who kills it, the person who cuts it up, the person who buys or sells meat, the person who cooks it, the person who serves it up, and the person who eats it. They must all be considered the slayers of the animal.[17] The question of religious duties towards the animals and of negative karma incurred from violence (himsa) against them is discussed in detail in Hindu scriptures and religious law books. Hindu scriptures belong or refer to the Vedic period which lasted till about 500 BCE according to the chronological division by modern historians. In the historical Vedic religion, the predecessor of Hinduism, meat eating was not banned in principle, but was restricted by specific rules. Several highly authoritative scriptures bar violence against domestic animals except in the case of ritual sacrifice. This view is clearly expressed in the Mahabharata (3.199.11–12;[18] 13.115; 13.116.26; 13.148.17), the Bhagavata Purana (11.5.13–14), and the Chandogya Upanishad (8.15.1). For instance, many Hindus point to the Mahabharata's maxim that "Nonviolence is the highest duty and the highest teaching,"[19] as advocating a vegetarian diet. The Mahabharata also states that adharma (sin) was born when creatures started to devour one another from want of food and that adharma always destroys every creature "[20] It is also reflected in the Manu Smriti (5.27–44), a traditional Hindu law book (DharmaÅ›Ästra). These texts strongly condemn the slaughter of animals and meat eating. The Mahabharata (12.260;[21] 13.115–116; 14.28) and the Manu Smriti (5.27–55) contain lengthy discussions about the legitimacy of ritual slaughter and subsequent consumption of the meat. In the Mahabharata both meat eaters and vegetarians present various arguments to substantiate their viewpoints. Apart from the debates about domestic animals, there is also a long discourse by a hunter in defence of hunting and meat eating.[22] These texts show that both ritual slaughter and hunting were challenged by advocates of universal non-violence and their acceptability was doubtful and a matter of dispute.[23] Lingayats are strict vegetarians. Devout Lingayats do not consume beef, or meat of any kind including fish.[24] Modern day In modern India, the food habits of Hindus vary according to their community or caste and according to regional traditions. Hindu vegetarians usually eschew eggs but consume milk and dairy products, so they are lacto-vegetarians. According to a survey of 2006, vegetarianism is weak in coastal states and strong in landlocked northern and western states and among Brahmins in general, 85% of whom are lacto-vegetarians.[25] In 2018, a study from Economic and Political Weekly showed that as few as one third of upper-caste Indians could be vegetarian.[26] Many coastal inhabitants are fish eaters. In particular, Bengali Hindus have romanticized fishermen and the consumption of fish through poetry, literature, and music. Hindus who eat meat are encouraged to eat Jhatka meat.[27][28] Animal sacrifice in Hinduism Animal sacrifice in Hinduism[29] (sometimes known as Jhatka Bali) is the ritual killing of an animal in Hinduism. The ritual sacrifice normally forms part of a festival to honour a Hindu god. For example, in Nepal the Hindu goddess Gadhimai,[30] is honoured every five years with the slaughter of 250,000 animals. This practice was banned from 2015.[31] Bali sacrifice today is common at the Sakta shrines of the Goddess Kali. However, animal sacrifice is illegal in India.[32] Buddhism Main article: Buddhist vegetarianism Buddhist influenced Korean vegetarian side dishes. The First Precept prohibits Buddhists from killing people or animals.[33] The matter of whether this forbids Buddhists from eating meat has long been a matter of debate, however, as vegetarianism is not a given in all schools of Buddhism. The first Buddhist monks and nuns were forbidden from growing, storing, or cooking their own food. They relied entirely on the generosity of alms to feed themselves, and were not allowed to accept money to buy their own food.[34][35] They could not make special dietary requests, and had to accept whatever food alms givers had available, including meat.[34] Monks and nuns of the Theravada school of Buddhism, which predominates in Sri Lanka, Thailand, Cambodia, Burma, and Laos, still follow these strictures today. These strictures were relaxed in China, Korea, Japan, and other countries that follow Mahayana Buddhism, where monasteries were in remote mountain areas and the distance to the nearest towns made daily alms rounds impractical. There, Buddhist monks and nuns could cultivate their own crops, store their own harvests, cook their own meals, and accept money to buy foodstuffs in the market. According to the Vinaya Pitaka, when Devadatta urged the Buddha to make complete abstinence from meat compulsory, the Buddha refused, maintaining that "monks would have to accept whatever they found in their begging bowls, including meat, provided that they had not seen, had not heard, and had no reason to suspect that the animal had been killed so that the meat could be given to them".
aggregate above their transition temperature Endotoxins were removed from the ELR by way of additional NaCl and NaOH treatments Finally the polymer was dialyzed against ultrapure water type I sterilized by filtration μm filters Nalgene and freezedried prior to storage The molecular weight and purity of the recombinamers were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS PAGE and matrix assisted laser desorption ionization time of flight mass spectrometry MALDITOF MS respectively The amino acid composition was further verified by high performance liquid chromatography HPLC and NMR spectroscopy by the Instrumental Techniques Laboratory of the University of Valladolid Endotoxin levels were measured using the Endosafe PTSTM test Charles River Differential Scanning Calorimetry DSC DSC experiments were performed using a Mettler Toledo e with a liquidnitrogen cooler Both temperature and enthalpy were calibrated against an indium standard Solutions were prepared by dissolving the ELRs in PBS pH at mg mL A μL aliquot of each solution Biomacromolecules Article DOI: acs biomac b Biomacromolecules − and its corresponding PBS reference were subjected to an initial isothermal stage min at °C to stabilize the temperature and state of the samples followed by heating from to °C at °C min The enthalpy values for endothermic processes were taken as negative and exothermic values as positive Particle Size and ζ Potential The particle size and ζpotential of the polymers were determined by dynamic light scattering DLS using a Zetasizer Nano ZS Malvern Instruments Ltd U K at a temperature of °C Solutions of both ELRs were prepared by dissolving the ELRs in PBS pH or ultrapure water type I pH when indicated The solutions were stored at °C overnight to allow complete dissolution of the recombinamers and filtered using a μm poly vinylidene difluoride PVDF syringe filter The samples were then incubated for min at °C to allow supramolecular assembly to occur and then introduced into polystyrene cuvettes and stabilized for min at the desired temperature For bovine serum albumin BSA interaction experiments ELRs were incubated in % BSA PBS for or h at °C after overnight dissolution and then filtered introduced into polystyrene cuvettes and stabilized for min at the desired temperature Autocorrelation functions were used to obtain the size distribution and polydispersity index PDI Z average mean nm and ζ potential mV were used for data analysis Three different samples were analyzed Transmission Electron Microscopy TEM Solutions were prepared by dissolving the ELRs in ultrapure water type I and kept at °C overnight to allow complete dissolution of the polymers The sample was incubated for min at °C to allow supramolecular assembly to occur and stained with uranyl acetate solution wt % to enhance the contrast of the nanoparticles on a carbon coated copper grid followed by solvent evaporation Samples were observed using a JEM electron microscope operating at kV Surface Tension by the Pendant Drop Technique The critical micellar concentration CMC of the different ELR solutions in both PBS and ultrapure water type I was determined from surface tension measurements derived from a drop shape analysis using the pendant drop technique The changes in the shape of the resulting drop at the air water interface upon increasing the ELR concentration previously stabilized at °C for min from a blank solution to μm were monitored using SCA software of a Data Physics OCA instrument which scaled the profile of the drop hanging from a straight precision dosing needle The drops μL at μL s were infused using a μL Gastight Hamilton syringe Three drops were analyzed per condition and the CMC was determined from the point of slope change after plotting the change in surface tension values versus log concentration of the ELRs Cell Culture MCF and Caco cells were maintained in MEM supplemented with % FBS mM glutamine % NEAA U mL penicillin and mg mL streptomycin at % CO and °C MDA MB cells were cultured in L medium supplemented with % FBS U mL penicillin and mg mL streptomycin at % CO and °C hMSC and HFF cells were cultured in DMEM supplemented with U mL penicillin mg mL streptomycin and and % FBS at % CO and °C respectively HUVEC cells were grown in Medium supplemented with % gentamicin amphotericin and LSGS at % CO and °C When required cells were detached using a solution of % trypsin−EDTA Cells were seeded onto well plates at a quantity of × cells per cm for tumor cells and × cells per cm for primary cells to maintain the same levels of confluence for all the cell lines overnight prior to the treatment Confocal Microscopy MDA MB cells were treated with fluorescein conjugated ELRs at mg mL for min Confocal images were taken using a Leica TCS SP confocal microscope Cellular nuclei were stained with ′ diamidino phenylindole DAPI Flow Cytometry MDA MB cells × cells in well plates were incubated with complete medium containing fluoresceinlabeled nanoparticles at mg mL for min Cells were washed with PBS trypsinized and centrifuged for s at rpm The supernatant was discarded and the pellet was resuspended in PBS Flow cytometry analysis was performed to assess the fluorescein internalized in cells Gallios flow cytometer Beckman Coulter Cell Viability MCF MDA MB Caco hMSC HFF and HUVEC were treated with ELRs at three different concentrations and mg mL for different times and min Live and dead staining LIVE DEAD viability cytotoxicity assay kit Invitrogen was used according to the manufacturer’s instructions Briefly a stock solution of the LIVE DEAD reagents μm of calcein AM and μm of EthD in mL of DPBS was prepared samples μL were distributed in each well and incubated for min in the dark and then the fluorescence intensity emission was measured at and nm after excitation at and nm SpectraMax M e Molecular Devices microplate reader Additionally photographic images of cultures were taken using a Nikon eclipse Ti SR Japan fluorescence microscope Three independent experiments each in triplicate were performed Cell Proliferation HFF hMSC and HUVEC cells were seeded in well plates After h incubation cells were treated with ELRs at two different concentrations and mg mL for h Confluence percentages were determined each h by Cytosmart OMNI software Cyotsmart The Netherlands Two independent experiments each in quadruplicate were performed Western Blot Caco and MDA MB cells were incubated with complete medium containing nanoparticles at mg mL for h Cells were lysed and protein concentrations measured using Bradford’s reagent Thus μg of protein was separated using standard SDS PAGE and transferred to PVDF membranes Blocking was performed with % defatted dry milk in PBS pH for h at room
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