Fosfomycin has the potential to be re-purposed as part of a combination therapy to treat neonatal sepsis where resistance to current standard of care (SOC) is common. Limited data exist on neonatal fosfomycin pharmacokinetics and estimates of bioavailability and CSF/plasma ratio in this vulnerable population are lacking.To generate data informing the appropriate dosing of IV and oral fosfomycin in neonates using a population pharmacokinetic analysis of plasma and CSF data.The NeoFosfo study (NCT03453177) was a randomized trial that examined the safety and pharmacokinetics of fosfomycin comparing SOC versus SOC plus fosfomycin. Sixty-one neonates received fosfomycin (100 mg/kg IV q12h for 48 h) and then they converted to oral therapy at the same dose. Two plasma pharmacokinetic samples were taken following the first IV and oral doses, sample times were randomized to cover the whole pharmacokinetic profile and opportunistic CSF pharmacokinetic samples were collected. A population pharmacokinetic model was developed in NONMEM and simulations were performed.In total, 238 plasma and 15 CSF concentrations were collected. A two-compartment disposition model, with an additional CSF compartment and first-order absorption, best described the data. Bioavailability was estimated as 0.48 (95% CI = 0.347–0.775) and the CSF/plasma ratio as 0.32 (95% CI = 0.272–0.409). Allometric weight and postmenstrual age (PMA) scaling was applied; additional covariates included postnatal age (PNA) on clearance and CSF protein on CSF/plasma ratio.Through this analysis a population pharmacokinetic model has been developed that can be used alongside currently available pharmacodynamic targets to select a neonatal fosfomycin dose based on an infant’s PMA, PNA and weight. In 2013, infection accounted for one-quarter of all neonatal deaths globally;P < 0.0001) to be susceptible to fosfomycin than empirical ampicillin/gentamicin therapy.,It has recently been shown that community-acquired Gram-negative bacteraemia isolates are significantly more likely  ran between March 2018 and March 2019 at Kilifi County Hospital in Kenya. Neonates aged 0 to 28 days old, weighing >1500 g and born at >34 weeks of gestation  with at least one sign of clinical sepsis and eligible to receive IV antibiotics  were included. Neonates were randomized to standard of care (SOC) consisting of ampicillin and gentamicin or SOC plus 100 mg/kg fosfomycin twice daily (q12h). Patients in the fosfomycin arm received a minimum of four IV fosfomycin doses  over 48 h at 12 h intervals. Once oral fluids were tolerated, IV fosfomycin was changed to oral fosfomycin  therapy at 100 mg/kg q12h. The IV dose was given as a slow push, while oral fosfomycin was given via oral syringe, spoon or nasogastric tube. Once reconstituted, fosfomycin was stored below 25°C. The dose of 100 mg q12h was selected for use in the NeoFosfo study based on the current age and weight-based neonatal dosing recommendations in the Fomicyt summary of product characteristics (‘SPC’) and the findings of Traunmuller ,Simulation-based sample size calculations were performed.JAC Online.Plasma and CSF samples were centrifuged at 3000 rpm for 5 min then separated and frozen (at −80°C) within 30 min of collection. Frozen samples were shipped to Analytical Services International Ltd, St Georges University of London, UK. Analysis of fosfomycin concentration in plasma and CSF samples was assessed via LC-MS/MS assay. The lower limit of quantification for plasma was 5 mg/L and for CSF was 1 mg/L. The method was fully validated according to EMA guidlines.Model-based estimation of PK parameters was undertaken using the first-order conditional estimation method with interaction (‘FOCEI’) in NONMEM .2 distribution, a drop in the log likelihood ratio of >6.64 per degree of freedom was needed to be significant at a level of P < 0.01 and >3.84 at a level of P < 0.05.One- and two-compartment structural models were compared. Inter-individual variability (IIV) was assumed to follow a log-normal distribution for clearance, volume and absorption rate constants, and a logit distribution for bioavailability. Estimation of IIV was evaluated for all parameters. An additive, a proportional and a combined error model were tested. In line with the χ,Allometric (weight) scaling was included using a fixed exponent of 0.75 on clearance terms and linear scaling on volume terms. A standard weight of 70 kg was used to enable comparison of parameter estimates with other studies. A previously published neonatal renal maturation function,M; fraction of clearance on the first day of life, set to day = 0; and θN, postnatal maturation rate constant). While the Rhodin maturation functionet al.et al.The ability of serum creatinine concentration (SCR) to explain and reduce IIV on clearance was tested according to Equation 6, where the measured SCR was standardized using typical serum concentration (TSCR) for age calculated based on the function published by Ceriotti ,V4) and the CSF/plasma ratio (UPTK).Having defined the plasma population PK model including covariate effects, an additional peripheral compartment was added to model the available CSF data.JAC Online) and the Discussion section].The initial modelling strategy aimed to estimate Q2 and UPTK, while the volume of the CSF compartment was fixed to 0.15 L/70 kg with linear weight scaling.i is the individual’s measured CSF protein level and 0.94 the population’s median CSF protein level. CSF protein was tested as a covariate on UPTK according to Equations 7 to 9, where PRn = 1000 simulations. A non-parametric bootstrap (n = 1000) was performed on the final model to test parameter robustness and derive parameter uncertainty. Perl-speaks-NONMEM (PsN)Decisions during model development were made based on the likelihood ratio test, goodness of fit (GOF) plotsUsing the final model parameter estimates, the half-life for each phase of the PK profile was calculated using methods reported by Upton.A hypothetical neonatal population of 10 000 subjects was created using observed demographics from the present study combined with data from an international multicentre neonatal observational study . The hypothetical population along with the final model were used to simulate fosfomycin plasma concentrations at steady-state for different dosing regimens. The simulations were all executed in R using the linpk package.T>MIC.T>MIC were defined , but, for the AUC/MIC ratio, previously published in vitro target values for Escherichia coli were considered , as, within a given individual, clearance is predicted to increase by 36% over this time frame (based on just this increase in PNA). To illustrate further the implications of PNA on predicted TA, plots analogous to Figure et al.SCR was not found to be a significant covariate on fosfomycin clearance. As with previous neonatal antimicrobial PK reports,et al.In vitro intestinal permeability studies suggest fosfomycin is absorbed via both the paracellular and transcellular routes with uptake mediated in part by the Na+-dependent phosphate transport system.,,,et al.,Oral bioavailability was estimated to be 0.48  and, with limited first-pass extraction, this is likely indicative of the fraction absorbed. Fosfomycin is a low molecular weight (138 g/mol), highly polar (ACD/logP = −2.98) phosphonic acid derivative, thereby demonstrating pH-dependent solubility and ionization in the gastrointestinal tract. Fosfomycin absorption is likely to be permeability limited, which is consistent with the class III Biopharmaceutics Drug Disposition Classification assigned by Benet et al.n = 35 subjects) and 10 g (n = 5 subjects) IV fosfomycin. The mean age of the subjects in Kuhnen et al.JAC Online and the associated fixed effects and residual errors, along with GOF plots, are provided in Figure The CSF data available from this study were not sufficiently rich to support estimation of Q2. Instead, Q2 was fixed using adult prior information. Kuhnen et al.CSF/AUCS is 0.18 (0.09–0.27). Since progression from sepsis to meningitis in neonates can be rapid, and less overt than in older children, fosfomycin’s good CSF penetration in this population is supportive of its potential role in empirical regimens for neonatal sepsis.The final population estimate of CSF UPTK in the NeoFosfo trial population was 0.32 . Nau Whilst we use demographic data from the neonatal observational study to simulate pre-term neonates and those weighing <1500 g  that were not included in our model building population, the mechanistic covariates used with biological priors on allometric weight and PMA scaling give some confidence in this extrapolation. We are assuming, however, that our observed PNA maturation effect will follow a similar trajectory in smaller neonates and, whilst this assumption may be reasonable, it could only be fully confirmed by collecting further data in this population. Nevertheless, this analysis indicates that reducing the dose in the first week of life due to short-term PNA maturation is required regardless of GA and/or weight.A caveat specific to the oral TA predictions concerns the use of a constant Ka in all subjects. Following oral dosing the Ka in an individual will have a significant impact on the shape of the oral PK profile and therefore this does introduce an element of additional uncertainty compared with the IV TA predictions.in vitro work to better define the target AUC/MIC ratio required to achieve a 2 log kill against pathogens typically responsible for neonatal sepsis is needed, alongside work to define relevant MIC breakpoints for bacteraemia and meningitis. Confirmatory PK data in pre-term neonates would also be useful to make firmer conclusions on dosing in this population.Finally, further i and CSF uptake of fosfomycin. The planning of follow-up fosfomycin trials in neonates will benefit from our model and TA simulations, which can be used to inform selection of a neonatal IV dose based on an infant’s PMA, PNA and weight, and, where relevant, an oral step-down dose personalized using likely pathogen MIC at 48 h.To the best of our knowledge, this is the first study to report model-based oral bioavailability from cross-over data in a neonatal antibiotic study and the first report of neonatal fosfomycin CSF penetration. PNA in addition to PMA was needed to describe immediate postnatal changes in CL distinct from gestational effects. We also establish a positive relationship between PROur prediction of the PK in smaller pre-term neonates  is based on extrapolation of fixed covariate effects that are well established for renally cleared drugs;in vitro work to better define target AUC/MIC ratios to achieve a 2 fold log kill against pathogens typically responsible for neonatal sepsis, alongside work to define relevant MIC breakpoints for bacteraemia and meningitis, would be beneficial.Further dkab083_Supplementary_DataClick here for additional data file.


Consideration of animal welfare in food choices has become an influential contemporary theme. Traditional animal welfare views about food have been largely restricted to direct and intentional harms to livestock in intensive animal agriculture settings. However, many harms to animals arising from diverse food production practices in the world are exerted indirectly and unintentionally and often affect wildlife. Here we apply a qualitative analysis of food production by considering the breadth of harms caused by different food production systems to wild as well as domestic animals. Production systems are identified that produce relatively few and relatively many harms. The ethical implications of these findings are discussed for consumers concerned with the broad animal welfare impacts of their food choices.Ethical food choices have become an important societal theme in post-industrial countries. Many consumers are particularly interested in the animal welfare implications of the various foods they may choose to consume. However, concepts in animal welfare are rapidly evolving towards consideration of all animals (including wildlife) in contemporary approaches such as “One Welfare”. This approach requires recognition that negative impacts (harms) may be intentional and obvious  but also include the under-appreciated indirect or unintentional harms that often impact wildlife . This is especially true in the Anthropocene, where impacts on non-human life are almost ubiquitous across all human activities. We applied the “harms” model of animal welfare assessment to several common food production systems and provide a framework for assessing the breadth (not intensity) of harms imposed. We considered all harms caused to wild as well as domestic animals, both direct effects and indirect effects. We described 21 forms of harm and considered how they applied to 16 forms of food production. Our analysis suggests that all food production systems harm animals to some degree and that the majority of these harms affect wildlife, not livestock. We conclude that the food production systems likely to impose the greatest overall breadth of harms to animals are intensive animal agriculture industries  that rely on a secondary food production system , while harvesting of locally available wild plants, mushrooms or seaweed is likely to impose the least harms. We present this conceptual analysis as a resource for those who want to begin considering the complex animal welfare trade-offs involved in their food choices. Many groups in modern societies are interested in the animal welfare consequences of food production. These groups span food producers, processors, retailers and policy makers, as well as consumers . Modern There is also much more to food ethics than just animal welfare. Consumers are also interested in the environmental footprints of various foods , the relThis knowledge gap may be related to the history of animal welfare science as a discipline, which is focused on the way direct and intentional actions of moral agents (humans) impact individual animals, rather than the unintentional or indirect processes that determine consequences for the largest numbers of animals . This trWe recognize that consumers need more than just a list of animal impacts associated with industrial activities to make ethical choices. Enumerating impacts is one task; assessing their scope and intensity is another. Assessment of harm intensity is beyond the scope of this paper, and our intent is not to show that consumers ought to eat in one way or another, even if they are exclusively concerned with animal welfare. Rather, our intent is to provide a resource for those who want to begin considering the very complex animal welfare trade-offs involved in their food choices. This is because, depending on a consumer’s philosophical persuasions, they may weigh some welfare impacts much more heavily than others, and we cannot explore all this variation here. Some consumers, for instance, may distinguish between intended and merely foreseen impacts, which would lead them to be more concerned about harms to hunted animals  than harms to wild animals around agricultural lands . Consumers may also assign different weights to animals based on their species preferences; some may weigh impacts on wild animals more heavily, which may make them particularly concerned about the wildlife trade; others may think that animals’ cognitive complexity is especially important, elevating the moral significance of impacts on mammals over most invertebrates. These and other disagreements will generate very different practical recommendations.Much animal welfare focus has traditionally been on human (anthropogenic) activities that directly and intentionally harm animals , but there has been much less awareness of activities that indirectly and/or unintentionally cause undesirable animal welfare outcomes . These uPongo spp.) , , 122], aPelecanus conspicillatus) . . 139]. An Canada . While mn Canada . Transposnowmelt ) and forsnowmelt ), and thsnowmelt . Aside fsnowmelt .The broad term “pollution” relates to the introduction of harmful materials into the environment. Types of pollution relevant to food production include chemical pollution, noise, and light pollution. Modern anthropogenic climate change is also a product of special types of pollution, but we will consider this separately in There are several forms of chemical pollution, whereby a wide range of anthropogenic activities lead to the deposition of harmful chemicals into the environment. Eutrophication is a specific type of pollution that occurs when a body of water becomes overly enriched with minerals and nutrients from the discharge of soil nutrients from the environment . ExposurAgricultural insecticides also have profound impacts on wildlife, such as vultures. Bird mortalities may also occur due to feeding on seeds treated with insecticides or other pesticides. This phenomenon has been observed around the world , from thSecondary poisoning or “relay toxicity” is a type of chemical pollution specific to the indirect effects on non-target wildlife species that consume the body of another type of wild animal poisoned intentionally. Typical examples affect wild predators that consume smaller animals deliberately poisoned by humans. For example, cases of non-target anticoagulant poisoning are common in birds of prey from residues in rodents killed to protect stored grain .Gyps spp.) feeding on livestock carcasses with diclofenac; an anti-inflammatory drug that is used commonly to treat cattle on the Indian sub-continent [Veterinary pharmaceuticals  used to treat livestock, working animals or farmed fish may become available to wildlife and cause deleterious effects. These compounds are known to be widespread environmental contaminants but knowledge of exposure patterns and possible effects in wildlife remain poorly characterized . One proontinent .Noise pollution is a more subtle form of pollution that can nonetheless cause profound disturbance to wild and domestic animals . The magCalyptorhynchus baudinii) from orchards in the south-west of Australia [Odocoileus virginianus) have also been affected by intensive row-crop agriculture in the USA, but quieter times  had minimal effects [Noises generated in food production clearly fit into this category of harms and may cause intentional or unintentional harm to animals. Intentional noise is a Type 2 harm and includes sources such as gas guns used to deter birds from damaging orchards. For example, gas guns are commonly used to deter Baudin’s cockatoos  refers to nocturnal anthropogenic light sources which may have profound effects on animals that are highly sensitive to natural lunar light levels . Daily, Sarcophilus harrisii) [A miscellaneous type of pollution is commonly associated with shooting: exposure of scavenging animals to toxic lead (Pb) fragments from lead-based bullets. This process has been shown to cause toxic lead exposure for taxa such as birds of prey  and mammarrisii)  that frearrisii) .Loxodonta cyclotis) associated with a sharp decline in fruiting as a result of climate change [Rhinoceros unicornis) in the Terai region of India and Nepal are also adversely affected by intensified flooding and habitat loss associated with climate change [The ways in which climate change may impact animal welfare are numerous and profound but are not always intuitive . These ie change . Greatere change . It is ae change .2) is produced by a wide range of human activities associated with agriculture, ranging from electricity production to power equipment and refrigeration, to the burning of fossil fuels for transport and distribution of food. The carbon footprints of many food types have been studied extensively in the interest of sustainability [2, are released in huge quantities by food production. Nitrous oxide (N2O) is an especially potent greenhouse gas that is created concurrently with CO2 and methane (CH4) during fertilizer production [2O [4) is a ubiquitous, apparently unavoidable side effect of fermentative fiber digestion by symbiotic microbiota in mammalian herbivores. Relatively large quantities of methane are produced by all ruminants  through eructation [All food production systems produce greenhouse gas emissions (GHGEs) to some degree  and agrinability . Other goduction . Fertilition [2O . Productuctation  but not uctation . The prouctation .Cervus elaphus) represent almost 85% of GHGEs for this hunting method [Other sources of GHGEs from food production are less obvious. For example, agricultural crop residue burning, as commonly practiced in countries such as India , contribg method . Some ang method .It is also worth noting that energy-intensive food production systems will harm animals even if GHGEs are avoided through the use of renewable energy; wind farms cause wild bird and bat injuries and mortalities , as do sCapra hircus) and fallow deer (Dama dama) [Apis mellifera) are used for commercial honey production in many places including Australia, where they represent a source of competition for floral resources with native nectar and pollen feeding insects such as the native Australian bee Hylaeus alcyoneus [Anodorhynchus leari) from preferred nesting hollows in Brazil [Rhinella marina), imported to Australia to provide biological control of cane beetles  in sugar cane crops. Cane toads have since contributed to declines in a variety of native reptile species across large swaths of eastern and northern Australia [Some farmed animals have the potential to become invasive species that cause subsequent harm to native species. Common livestock examples include pigs b, goats ma dama) , and leslcyoneus . Invasivn Brazil . Historiustralia .Cenchrus ciliaris), a globally significant invader now widespread across central Australia that was originally introduced as forage for rangeland cattle. Buffel infestation has widespread impacts on native animals in Australia, including reductions in floral diversity and increases in the intensity and frequency of wildfires that negatively affect faunal diversity [Invasive plants introduced as horticultural or fodder species may also become invasive weeds and harm animals through modifications to environments. A prominent example is buffel grass (iversity . Transpoiversity .Canis lupus) attacking cattle in the USA [Panthera pardus) attacking goats in southern Africa [Uncia uncia) attacking livestock in mountainous areas of central Asia [Arctocephalus pusillus doriferus) feeding on farmed Atlantic salmon  in Tasmania, Australia [Related to the effects of wild invasive species is the special case of predation that occurs when introduced species are kept as livestock or used as working animals . In the  the USA , dingoes the USA , leopardn Africa , and snoral Asia . The samustralia . Manipulustralia .Sardinops sagax) observed in southern Australia caused by the introduction of a herpesvirus from frozen fish fed to sea-caged southern bluefin tuna (Thunnus thynnus) [Eudyptula minor), which experienced increased mortality rates and failed to breed due to food shortage [The animal welfare impacts of infectious diseases are not well documented  but can thynnus) . Mortalithynnus) . Consequshortage .Dryland salinity associated with clearing and cropping or grazing is noticeable in many parts of the world such as south-west Australia and is associated with severe impacts on biodiversity . Food prRecurvirostra americana) chicks exposed to saline conditions. Birds raised in highly saline conditions exhibited increased activity, decreased feeding and preening, weight loss and dehydration [Triodia spp.), causing the decline of some native mammals and favoring of others [The ways in which salinity and desertification harm animals are numerous and sometimes subtle. Short-term single-species impacts are especially evident for waterbirds breeding in inland wetlands affected by salinization. One example comes from studies of the health and behavior of American avocet  abundances in the Congo Basin rainforest declined and diets shifted towards smaller prey species in regions near human settlements due to competition with bushmeat hunters [Depletion of natural resources may occur through overharvesting of wild plants, seaweed, fungi or animals . The sca hunters . Over-ha hunters . Type 4 We described a number of examples of the four broad categories of animal harms that may result from food production systems. Our analysis described three forms of Type 1 harms, four forms of Type 2 harms, five forms of Type 3 harms and nine forms of Type 4 harms. It is natural that the number of ways that animals may be negatively affected increases as we move from intentional and direct towards unintentional and indirect. In total, we identified at least 21 ways in which food production systems may harm animals. This list is undoubtedly incomplete, but we present it as a conceptual guide. We now move to brief summaries of the harms imposed by several types of food production, listed in order of those imposing fewer harms to those imposing the most. A summary of animal harms for each food production system is provided in Vaccinium corymbosum) picking in North America [Harms for harvesting of wild mushrooms, terrestrial plants or seaweeds are almost identical. Wild plant harvesting is perhaps the oldest form of human food production and a well-known contemporary example is blueberry  [Ursus americanus) [No Type 1 harms are associated with honey production, at least on the assumption that insects are not sentient. Honey is rarely processed so Type 2 harms related to food additives and food safety testing are minimal. Land clearing is not required. However, Type 2 and Type 3 harms have been documented in some places such as the USA, where conflict with omnivorous wildlife results in Type 2 harms through trapping of skunks (racilis)  and Typericanus) . Other Tricanus) . Type 4 For succinctness, this section includes commercial harvesting and other forms of hunting of land-based wildlife. Type 1 harms are absent unless domestic animals  are used to aid hunting. Type 2 harms are obvious from shooting or trapping wild animals. Type 3 harms are few and land clearing is not required. Game meat is rarely processed so Type 3 harms related to food additives and food safety testing are minimal. Other Type 3 harms depend on the use of packaging (plastic waste). One Type 4 harm commonly associated with shooting is lead poisoning of wild scavengers through ingestion of fragments from lead-based bullets. Another Type 4 pollution harm is caused by noise disturbance from gunfire, which may be minimized by the use of suppressors . HuntingA quite different set of harms apply to the type of consumptive wildlife use typical of “wet markets”. In this context, Type 1 harms are considerable, applying to wild animals taken into captivity in often cramped and stressful conditions . Type 2 Lutra perspicillata) to herd wild fish into nets in Bangladesh [For succinctness, this section includes all commercial fishing, whaling and marine mammal harvesting. Small-scale subsistence harvesting of marine animals by Indigenous peoples is not focused upon here. Type 1 harms are absent except for a few specialized practices, e.g., the use of domesticated otters , and this has been an exceedingly common occurrence worldwide with commercial fishing . AnotherVulpes vulpes) in the United Kingdom [This category comprises the raising of domestic poultry under extensive outdoor conditions for the purpose of egg production on pasture, forage, or food waste products. Type 1 harms are associated with domestication, including effects on breeding, occasional husbandry procedures and infectious disease events. In most cases, Type 1 harms also include those associated with animal transport, usually by land or air. Type 2 harms include lethal control of bird predators , and eg Kingdom . Food waThis type of livestock production typically involves free-ranging herbivores foraging on native unimproved (uncleared) vegetation over large geographical areas with minimal human husbandry . As well2 emissions associated with long-distance transport  of live animals and meat, freezing or refrigeration of meat as well as methane emissions from the species raised, which are almost exclusively ruminants [Crops are not usually fed to raised animals, nor are they commonly treated with veterinary chemicals. Fencing is sometimes, but not always, used to keep livestock within designated management units. However, when fencing is used, it is typically barbed wire that can cause harm to wildlife attempting to cross under or over it . Irrigatuminants . Fertili2 emissions from the transport of grain and stubble burning. In addition, fertilizer use leads to considerable production of the potent greenhouse gas N2O [Dryland cropping refers to all production of plant crops on dry land . Dryland cropping is typically characterized by plant monocultures of grasses, such as wheat and corn, or legumes. The food products derived from dryland cropping are unusually diverse as processing is common , making gas N2O . Croppin2O emissions from the production and use of fertilizer. Horticultural species may also give rise to invasive plants (weeds) that exert Type 4 impacts [This general title is used here to describe all forms of vegetable and fruit production, including orchards and vineyards. Type 1 harms are not generally incurred unless domesticated herbivores are used to constrain grass growth in orchards  , domesti impacts  or may a impacts . Food waThere are some notable exceptions to the suite of harms described above with some specialized forms of horticulture. Systems such as tropical agro-forestry may alleviate many Type 3 and Type 4 harms by not requiring complete land clearing, but allowing persistence of selected native shade trees . 2O from the production and use of fertilizer, as well as CO2 emissions from the transport of grain. Fertilizer use in irrigated cropping also leads to considerable production of N2O [Irrigated cropping refers to all production of plant crops where water is diverted from natural waterbodies to the site of domesticated plants. Forms include flood, channel, terrace, and drip irrigation. Irrigated cropping is typically characterized by plant monocultures of water-intensive species such as rice, vegetables, or fruit, as well as nuts such as almonds. Type 1 harms are not incurred. Some Type 2 harms may be largely avoided in environments in which wildlife damage to crops is minimal. On the other hand, Type 2 harms can be considerable if local wildlife cause crop damage, e.g., culling of ducks or rats over rice fields . Other Tn of N2O . Dammingn of N2O  and foodInsects, like vertebrate livestock, can be raised for human consumption and are eaten regularly by at least 2 billion people worldwide, with predictions for this number to increase in the near future . Althoug2 emissions associated with an energy-intensive meat production process.Cellular agriculture is the most contemporary  form of food production examined here, so some additional background is provided. This form of food production involves growing animal-based foods  from cell cultures, as opposed to harvesting animal products from the animals themselves . As with2 emissions from motorized boats and refrigeration of produce. Fish farming has also made a notable contribution to the release of invasive species [Cyprinus carpio) in Australia [This category includes the raising of fish in captivity for meat production in aquaculture or mariculture. For conciseness, we also include all farming of crustaceans  here. Fa species , e.g., custralia . A partiustralia  and theiThis category includes the raising of domestic poultry under intensive indoor conditions for the purpose of egg production whereby animals are raised exclusively on crops rather than pasture or forage. As such, all harms associated with dryland cropping are also associated with this food system. Considerable Type 1 harms are imposed that are associated with domestication. Additional harms are associated with confinement to restrictive housing (“cage laying”) . In mostUnlike rangeland pastoralism, extensive livestock grazing involves land clearing to farm the animal more productively. Type 1 harms are imposed on livestock through domestication, transport, and related husbandry practices. The extent of Type 1 harms will be minimized in production systems that incorporate slaughter on the property of origin and will be greatest for those systems that require multiple forms of transport . Type 2This category includes the raising of domestic mammals or birds for the purpose of meat production in intensive indoor barns or feedlots where animals are raised exclusively on crops rather than pasture or forage. As such, all harms associated with dryland cropping are also associated with this food system. Considerable Type 1 harms are those associated with domestication, including effects of breeding, loss of natural behavior, confined conditions, regular husbandry procedures and frequent infectious disease events . These TThis category refers to the husbandry of domestic cattle, buffalo, goats, sheep, or camels  to extract milk. Some dairy systems rely exclusively on rain-fed pasture (grass), but most systems use grain to feed lactating animals to some extent. As such, all harms associated with dryland (and often irrigated) cropping are also associated. Extensive Type 1 harms are imposed. These include selective breeding, intensive husbandry, lameness, physiological stress associated with extended lactation, common surgical procedures, intensively managed reproduction, and calf removal. In most cases, this also includes the Type 1 harms associated with livestock transport, usually by land but sometimes by sea or air.Type 2 harms include the familiar processes of slaughtering dairy animals for meat production after their use for milk production ends, and culling of male calves . Here, w2 emissions from electric-powered milking systems, refrigeration and freezing, and motorized transport, N2O emissions from fertilizer and methane emissions from almost exclusively ruminant livestock. Dairy farming may have made occasional contributions to the release of invasive species , dairy From the preceding sections, it is apparent that some food production techniques avoid imposing one type of harm but subsequently incur a different type of harm. In many cases, particularly for non-animal-based food production systems, intentional harms are avoided by not producing animals, but harms are imposed on wildlife that are unintentionally affected by producing or harvesting plants, mushrooms and/or seaweed.One example of trading off different types of harm can be seen in a comparison of professional terrestrial wildlife harvesting and dryland cropping, both of which may be realistic food-producing land uses for many semi-arid parts of the world . NeitherVexing harm conflicts can occur even when considering the best way to consume one type of food. Consider the trade-off between plastic waste (Type 3) and food waste (Type 4) for fresh produce . Forgoing plastic packaging from produce may reduce entanglement risks for wildlife but comes with a reduction in shelf life and the resultant increase in food waste , with faSome harms were inflicted in the past  while others are ongoing . For example, food safety testing  generallSo far in our discussion, all foods are treated equally regarding their nutritive value and efficiency of production, when in reality, they are not equal. Some food types have a relatively high content of energy, protein, and important nutrients , while others do not . Minimizing harms while maintaining (or increasing) food production will be a key global challenge in the future . For thiEubalaena australis) may produce approximately 30 tons of food [Mus musculus) plagues in Australian crop lands estimate that >500 mice are intentionally harmed per ton of useable wheat [Another crucial consideration when attempting any sort of quantitative comparison of animal welfare impacts is the number of animals affected . When th of food  for one le wheat , or ~15,As we suggested earlier , there iAt some point along this hierarchy of animal sentience, it may be sufficiently unlikely that certain animals have any capacity for welfare that it is appropriate not to factor their (possible) welfare in moral deliberations. If so, then there is a point at which the numbers become moot. So, while it might be important to consider trade-offs between, say, whales and mice, it may not be important to consider trade-offs between whales and insects. Not incidentally, this view provides support for the use of insect meal as a source of protein despite the vast numbers of insects that need to be slaughtered to produce significant quantities of protein but can be tempered depending on the food source(s) provided to the insects.When Type 3 and 4 harms are considered, and attempts are made to quantify unintentional harms to wildlife, much hinges on the amount of land (or water) area occupied by different production systems. Land area used by different production systems vary widely, with some industries  occupying negligible land area, and others  occupying enormous areas. For example, in Australia, rangeland pastoralism accounts for 71% of all agricultural land use, or approximately 50% of Australia’s land mass . Type 1 Given that all food production systems cause harm to animals, the logical question for a concerned consumer is which harms should be considered more important than others. Are domestic animals more important than wild animals? Are mammals more important than birds? Are endangered species more important than common species? Should intentional harms be avoided more strongly than unintentional harms? Is it better to harm a lot of animals only a little, or to harm only a few animals a lot? Should we prioritize the few animals likely to experience the most severe harms or the greatest number of animals likely to experience less severe harms? There are no scientific approaches that can answer whether it is better to kill one whale for 30 tons of meat versus killing 15,000 mice for 30 tons of grain. Instead, we must look to ethics to un-pack the questions being asked here.Some consumers might think that, due to differences in cognitive sophistication, less intense harms to orangutans as a result of palm oil production are still more weighty, morally speaking, than more intense harms to rodents in crop production. Others, however, might object to privileging species based on the degree of cognitive sophistication, insisting that only the capacity for pleasure and pain should count. These examples show that the first step is simply to make the catalogue of harms available for discussion, as we did here. Depending on the particular dietary options under consideration, further research will be required to determine how those choices should be made.Assuming an egalitarian position, whereby all sentient animals assume equivalent moral status, intensive animal industries that require extensive modifications to land use  are associated with the broadest range of animal harms, whereas harvesting wild plants, mushrooms and seaweed are associated with the narrowest range of harms. Some of the outputs of our analysis are less intuitive. For instance, when choosing between wildlife that has been professionally harvested or eating plants from intensive crop production , our breGiven the above analysis, what should consumers do? As we suggested at the outset, the answer is not as simple as choosing the food type with the least breadth of harms. That is only part of the animal welfare picture and animal welfare is only one of many ethical concerns relevant to food choices . UltimatFor our purposes, a consumer ethic is a theory about what consumers ought to do, all things considered. It is not simply a theory about what consumers ought to do given one particular objective, such as minimizing animal harms or carbon footprints, or one of the many other morally significant aims . As a reTo provide that ranking, a consumer ethic depends on more general theorizing about the relative moral significance of different kinds of harms, the relative moral significance of human and nonhuman animals, the moral significance of the environment, and much else besides. For instance, we might be concerned about how harm is distributed — that is, the overall pattern of harm. Maybe there are special reasons to be worried, morally speaking, about highly concentrated harms in the way that focus might be applied to overlapping kinds of oppression, or harms to groups that have suffered various injustices in the past (in the way concerns are amplified for harms to Indigenous human populations). So, for instance, while it might be the case that mice suffer a great deal when poisoned, and more than orangutans do as a result of habitat loss from palm oil production, the fact that orangutans are critically endangered and mice are not may mean that harms to orangutans deserve special attention — not because species losses are bad in itself, but because harms are being concentrated on a species that has already lost so much.There are also epistemic hurdles that consumers face. Modern consumers are confronted with an enormous amount of information and conflicting opinions about what they ought to purchase and eat, and even regulated information is often difficult for consumers to interpret. Consider, for instance, the gap between regulatory standards and public perceptions when it comes to labels like “organic”, “free range”  or “eco-On top of this, there are many people who do not have the luxury of choosing how their food is produced. And for most of those who do have that luxury, it only comes in degrees. Consumers at different points of the income scale have more and fewer opportunities to adjust their purchasing in response to moral considerations, which means that they face different sets of options from which they must make their selections. Each set leaves consumers with a distinct set of trade-offs that need to be adjudicated which, given the epistemic challenges just mentioned, can be highly cognitively taxing.A usable consumer ethic needs to provide people with some sense of what they ought to do when the relevant moral and empirical facts are difficult to assess. In other words, a good consumer ethic will make it possible to generate “rules of thumb” that, while delivering imperfect advice about how to shop and eat, provide advice that is good enough for navigating a fraught food system. Again, we take no position on any of the substantive questions here and we are not advocating for any particular consumer ethic, general ethical theory, or other moral position. Instead, we simply want to stress the importance of moral theorizing for generating a consumer ethic while noting that all food production systems cause unavoidable harm to some animals. It is indeed important to catalogue the breadth of harms to nonhuman animals, but that is only the first step.We recognize that there is enormous diversity in food production practices and that the conclusions we draw will not be universal. For example, many of the harms we identified are only relevant to certain geographic locations. Increasing use of recently developed techniques such as zero-tillage cropping  or lead-We also acknowledge that consumer choices are only one part of a complex picture determining how food is produced, and what harms are imposed on animals. In addition to consumers, the decisions made by food producers, processors, retailers and policymakers have strong influences . We furtOur analysis further highlights the close connection between animal welfare and biodiversity concerns when animal welfare is considered broadly to include Type 4 harms and wild animals are given due consideration. A philosophical conclusion may be that we should replace our current approach to ethics, where animal ethics and environmental ethics are often discussed in isolation from each other, and develop a coherent, over-arching ethic for animals that includes the harms we cause to other species, whether intentionally or unintentionally, and whether directly or indirectly . These tWe show that all food production systems harm animals and that most harms imposed by food production have unintentional or indirect effects on wildlife. Some food production systems incur very few animal harms , while animal agriculture systems that require the feeding of crops could be argued to impose the greatest range of harms to the greatest number of animals. We encourage affluent consumers to carefully consider their own ethical stance toward different animals and use consideration of harms as a guide for selecting food rather than relying on activism, marketing, or advocacy. With world food production systems facing a challenge to increase production by approximately 70% during the next 30 years , much wi


The sex chromosomes play central roles in determining the sex of almost all of the multicellular organisms. It is well known that meiosis in mammalian spermatogenesis produces ~50% Y- and ~50% X-chromosome-bearing sperm, a 1:1 ratio. Here we first reveal that the X-chromosome-encoded miRNAs show lower expression levels in the left testis than in the right testis in healthy mice using bioinformatics modeling of miRNA-sequencing data, suggesting that the Y:X ratio could be unbalanced between the left testis and the right testis. We further reveal that the Y:X ratio is significantly elevated in the left testis but balanced in the right testis using flow cytometry. This study represents the first time the biased Y:X ratio in the left testis but not in the right testis is revealed. The sex chromosomes play central roles in determining the sex of almost all multicellular organisms . In mammMore recently, however, it was reported that the balance of the Y:X ratio was shifted under specific genetic and environmental conditions ,4. For eWe had previously identified a number of sex-biased genes and microRNAs (miRNAs) and revealed that they show significant bias in biological functions, disease risks, and therapeutics ,12,13. MWe first obtained the miRNA expression dataset of the left and the right testes from three healthy mice from our previous study, which were quantified based on the unique molecular identifier (UMI). We then located the miRNAs in the X-chromosome of mouse genome from miRBase Database (version 22.1)  and thenEight- to ten-week-old healthy male mice on C57BL/6J background (weight 22–25 g) were used in this study. All animals were bred and housed locally at 24 ± 2 °C and were acclimated to standard laboratory conditions (12L:12D cycle) with free access to rodent feed and water. All procedures involving experimental animals were approved by the Institutional Animal Care and Use Committee of Peking University Health Science Center.A percentage of 4–5% isoflurane was used to induce anesthesia, and 1%–3% isoflurane was applied to keep the mice anesthesia in the whole operated process. The left and right epididymis were removed respectively, and then the caudal epididymis was excised and transferred to a dish containing 2 mL 37 °C prewarmed Enriched Krebs-Ringer bicarbonate medium  ,18,19 wiTM S6 System  and FlowJo software  were used for data acquisition and analysis. The data are shown as the ratio of X- or Y-CBS from the left or right caudal epididymis.Many techniques have been used to separate mammalian X- and Y-bearing sperm through the past decades, mainly including the H-Y antigen separation method , densityt-tests. p-values of <0.05 are considered statistically significant.The results are presented as the mean ± SEM (N = 7). Experimental groups are compared using the two-tailed unpaired Student’s p-value = 0.003, p-value = 1.86 × 10−5). Statistically, most of the miRNAs (65 vs. 27) exhibited higher expression levels in the right testis than those in the left testis . However, the Y:X ratio is about 1:1 (Y:X ratio = 0.96:1) in the right testis . These findings support the predictive results as well. Meanwhile, we carried out the statistical comparison in a different way, comparing the ratio of left X-CBS to right X-CBS, and the ratio of left Y-CBS to right Y-CBS  and environmental  conditions, according to our knowledge, this is the first time the interesting phenomenon of unbalanced Y:X ratio has been observed, especially in one-side testis in healthy mice. It is important to validate whether this finding is consistent when using other mammals, for example rats, dogs, and especially humans. Moreover, it is also important to explore why and how biased Y:X ratio occurs only in the left testis. We also think there may be a causal link between the X-miRNA expression level and spermatogenesis, which is being further studied and analyzed currently. Apart from that, we also considered many other directions. For example, is there any miRNA specifically expressed in one side of the epididymis or testis? Do some miRNAs related to reproduction present an unbalanced expression level? Do X- and Y-chromosome-specific genes exist in interactions and cross talks that relate to the interesting phenomenon? Finally, we believe this difference has biological significance in physiology and pathophysiology, which remains to be explored in the future.


This approach revealed that CSF stimulated the production of SCFA  across donors while significantly lowering the production of branched SCFA (bSCFA). In terms of community composition, CSF stimulated a broad spectrum of health-related Bifidobacterium species, which are potent acetate and lactate producers. At the same time, it lowered the abundance of opportunistic pathogenic Escherichia coli. Later, we explore the effect of colonic fermentation of CSF on the gut barrier and intestinal immunity in the Caco-2/THP1-blue™ cell co-culture model. Based on the study using SHIME technology platform, CSF showed protective effects on inflammation-induced intestinal epithelial barrier disruption in all donors. Also, the treatment of CSF showed pronounced anti-inflammatory properties by strongly inducing anti-inflammatory cytokines IL-6 and IL-10 and reducing pro-inflammatory cytokine TNF-α. These findings demonstrate a significant modulatory effect of CSF on intestinal gut microbiota. CSF-microbial fermentation products improved the gut barrier and controlled intestinal inflammation.The traditional Chinese medicine (TCM)–Chaihu Shugan Formula (CSF), consisting of several Chinese botanical drugs like Bupleurum, is derived from the ancient Chinese pharmacopeia. It has been used for more than thousands of years in various suboptimal health statuses and diseases induced by chronic stress based on empirical therapy. Recent studies confirm the role of CSF in the development of many diseases, including depression, stress-induced hepatic injury and tumors. However, little has been known about the mechanisms behind the health effects of CSF. Here, we investigate the influence of CSF on the modulation of the simulated colonic microbiota of five healthy donors, gut barrier integrity, and intestinal immunity by combining the simulator of the human intestinal microbial ecosystem (SHIME Bupleurum chineseDC (Chinese name Chai-hu), Pericarpium citri reticulatae (Chinese name Chen-pi), Ligusticum chuanxiong Hort (Chinese name Chuan-qiong), Rhizoma cyperi (Chinese name Xiang-fu), Fructus aurantii (Chinese name Zhi-ke), Radix paeoniae alba (Chinese name Bai-shao) and Glycyrrhiza uralensis Fisch (Chinese name Gan-cao). CSF was firstly recorded in the Chinese pharmacopeia called “Jing Yue Quan Shu”, written by a famous physician named Jingyue Zhang in 1,624. CSF was created and extensively used to treat digestive diseases and depression for five hundred years. It has been confirmed that CSF exhibits positive effects on the treatment of depression, depression-related diseases and other diseases, including stress-induced hepatic injury, Alzheimer’s disease, and tumors , an empirical medicine that developed for thousands of years, has established its own medical system differing from modern medicine and has been widely used in many Asian countries, especially in China . Emergind tumors . Studiesd tumors . In the d tumors . AlthougThe micro-organisms in the gut represent a biologically active community that lies at the interface of the host with its nutritional environment . The intA wide range of microbial structural components and metabolites directly interact with host intestinal cells to influence nutrient uptake and epithelial health. Both microbial associated molecular patterns (MAMPs) and bacteria-derived metabolites ) activate various signaling pathways, which dictate the inflammatory tone, energy balance, gut motility and appetite regulation that physiologically connect the gut, liver, muscle, and brain . SCFA prThe gut barrier consists of immune cells, intestinal bacteria, and epithelial cells held together by intercellular tight junctions. The intact gut barrier acts as a physical and chemical barrier and protects the host from the invasion of toxins, antigens and pathogenic bacteria . Gut barvia activation of monocyte chemoattractant protein (MCP)-1, which promotes the clearance of neutrophils. IL-6 is also able to inhibit the production of pro-inflammatory cytokines such as IL-1. Moreover, IL-6 has a positive effect on the regeneration of the intestinal epithelium and wound healing , switching a physiological “tolerogenic” inflammation into a detrimental pathological inflammation . An infl healing . On the  tissues .®) is an in vitro model, which offers an excellent experimental setup to mechanistically study the processes that affect the human gut microbiome. In this study, a down-scaled version of it focusing on the proximal colon allowed to specifically evaluate the capacity of CSF to modulate the gut microbial composition and activity , Pericarpium Citri Reticulatae , Rhizoma Chuanxiong , Rhizoma Cyperi , Fructus Aurantii , Radix Paeoniae Alba  and Radix Glycyrrhizae .Chaihu-shugan san formula (CSF) was prepared based on the medical classic Jing-Yue Quan-Shu. It was composed of seven botanical drugs, Radix Bupleuri . To prepare this formula, these raw botanical drugs were immersed in 10-time volume of distilled water for 30min and decocted for 2 h, and the supernatant was collected, filtered with gauze. The concentration of Chaihu-shugan san decoction is 5 g/L.®  is a dynamic model of the complete human gastrointestinal tract in a controlled in vitro setting. The reactor setup consists of five pH-controlled consecutive reactors, which sequentially stimulate stomach, small intestine, ascending colon, transverse colon, and descending colon  and an informed consent was obtained from all subjects involved in the study. The SHIMEng colon . The curng colon , as descng colon , with orpH value and short-chain fatty acid (SCFA) production, including acetate, propionate, butyrate and branched SCFA , were measured at the start of the incubation as well as after 48 h incubation . LactateTen samples were collected at the start (control condition only) and 20 samples after 48 h incubation  for microbial community analysis. DNA was isolated from a total of 30 samples according to the method described by Catalayud et al. . 16S-tarFor flow cytometry analysis, 10-fold serial dilutions of luminal samples were prepared in anaerobic Dulbecco’s Phosphate-buffered Saline (DPBS)  and stained with 0.001 mM SYTO24  for 15′ at 37°C in the dark. Samples were analyzed on a BD Facsverse  using the high-flow-rate setting. Bacteria were separated from medium debris and signal noise by applying a threshold level of 200 on the SYTO channel.The number of total bacterial cells was used to convert the proportional values obtained with Illumina into absolute quantities by multiplying relative abundances of any population (at any phylogenetic level) in a sample with the total cell counts obtained with fold change of the given sample.2 incubator .The human epithelial Caco-2 cells and human monocytic leukemia THP1 cells were obtained from American Type Culture Collection  and InvivoGen , respectively. Caco-2 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), and 1% (v/v) penicillin-streptomycin (PS). THP1 cells were cultured in Roswell Park Memorial Institute (RPMI)-1,640 nutrient mixture with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin (PS). THP1-Blue™ were obtained from THP1 human monocytes, which were stably transfected with a reporter construct expressing a secreted alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factor nuclear factor kappa B (NF-κB). Cells were grown in a COThe co-culture model was established as previously described by Daguet et al., 2016 . BrieflyThe Caco-2-bearing inserts (pore size 0.4 µM) were placed on top of the PMA-differentiated THP1-Blue™ cells . The apical compartment (containing the Caco-2 cells) was filled with sterile-filtered (0.22 μm) colonic batch suspensions. Caco-2 cells were treated apically with 12 mM sodium butyrate (NaB)  as the positive control. The basolateral compartment (containing the THP1-Blue™ cells) was filled with Caco-2 complete medium. Caco-2 cells were also exposed to Caco-2 complete medium in both chambers as control. The duration for the above treatments was 24 h, after which the TEER was measured in all wells (= 24 h time point). After subtracting the TEER of the empty insert, all 24 h values were normalized to their own 0 h value  and are presented as a percentage of the initial value.Escherichia coli K12, InvivoGen, San Diego, CA, United States) in combination with 1 µM hydrocortisone  and medium without LPS (LPS-) as controls. After LPS stimulation, the basolateral supernatants were collected for cytokine measurement  and for NF-κB activity, according to the manufacturers’ instructions. All treatments were done in triplicate. Cells were incubated at 37°C in a humidified atmosphere of air/CO2 .The basolateral supernatant was then discarded, and cells were stimulated at the basolateral side with Caco-2 complete medium containing ultrapure LPS. Cells were also stimulated at the basolateral side with 500 ng/ml ultrapure LPS  in the cell assay.The experimental controls were presented first in separate plots; these relate to the complete medium control (CM or LPS−), the lipopolysaccharide (LPS+)-treated cells and the Sodium butyrate (NaB) and hydrocortisone (HC) control. Concerning the TEER, the conditions CM and NaB were compared and statistical significance was calculated by using unpaired, two-tailed Student’s t-test. For the immune markers (cytokines/chemokines and NF-κB activity), all conditions  were compared to LPS+. Statistical significance was calculated by using one-way ANOVA with Dunnett’s multiple comparisons test against LPS+. The results concerning the colonic batch suspensions presented the average of all donors. All colonic incubations were taken as biological replicates .To evaluate differences in different groups, the treatment of each donor was compared to their non-treated blank control using a two-way ANOVA with Sidak’s multiple comparisons test and are represented by (*). (*), (**), (***) and (****) represent +) and SCFAs profiles . Monitoring the pH during a colonic incubation provides a good indication of the production of SCFA, lactate and ammonium (NH4+). pH decrease was observed in both the negative control and CSF treatment groups upon 48 h incubation (Fermentation activity correlated with ammonium (NH4cubation . It showcubation .p < 0.001), explaining the difference of the pH decreases between the control and CSF treatment groups above. Further investigation showed that the increased SCFA concentrations resulted from stimulatory effects on the 2–3 times higher production of acetate, propionate and butyrate by CSF treatment compared to the negative control  was significantly and dramatically higher than the control (33.3 mM) for each donor , Bacteroidetes (37.94%) and Firmicutes (47.12%) (Proteobacteria (0.36%) and Verrucomicrobia (0.41%) were present in lower abundances  . Proteobundances . MicrobiActinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, followed by Desulfobacterota and Verrucomicrobiota  while decreasing the proposition of Proteobacteria (from 19.19 to 6.6%) compared to that of the control. CSF didn’t obviously cause a difference in the proposition of Bacteroidetes and Firmicutes phyla compared to control , B. longum (from 1.02 to 3.38%) and B. (pseudo)catenulatum/B. kashiwanohense (from 0.65 to 2.49%). Further, members of the Eggerthellaceae family were also significantly enriched (from 0.80 to 1.55%) by the CSF treatment. Within Bacteroidetes phylum, a slight but significant increase was induced by CSF in the relative abundance of Bacteroidaceae family due to increases of OTUs identified as Bacteroides uniformis (from 2.68 to 3.73%) and Bacteroides eggerthii (from 0.42 to 1.47%). Within Firmicutes phylum, CSF exposure significantly stimulated the Acidaminococcaceae, Butyricicoccaceae, Erysipelotrichaceae, Lachnospiraceae Peptostreptococcaceae and Streptococcaceae families, with the most important enrichments including OTUs identified as Mediterraneibacter faecis/Ruminococcus torques (from 1.42 to 2.27%), Blautia faecis (from 1.61 to 2.08%). Furthermore, within the Ruminococcaceae family, OTUs identified as Faecalibacterium prausnitzii (from 0.34 to 0.56%) and Gemmiger formicilis (from 1.06 to 1.49%) consistently increased. On the contrary, the CSF treatment significantly lowered the abundance of Enterobacteriaceae family by decreasing OTU identified as Escherichia coli/Shigella flexneri/E. fergusonii/S. sonnei/S. boydii/Pseudescherichia vulneris (from 22.81 to 6.88%), a known pathobiont of the human colon. Therefore, CSF increased the absolute and relative abundance of gut bacteria associated with the production of SCFA , whereas it led to a decrease in gut bacterial taxa linked to opportunistic pathogens.At the family level, CSF exposure consistently stimulated a variety of families and OTUs across donors . Within in vitro Caco-2/THP1 co-culture model by the measurement of transepithelial electrical resistance (TEER). Caco-2/THP1 co-cultures were exposed to colonic batch suspensions (with or w/o CSF treatment) for 24 h. The controls [complete Caco-2 medium (CM) and sodium butyrate ] behaved as expected  .The effects of colonic batch suspensions on NF-κB activity, cytokine and chemokine release in the Caco-2/THP1 co-culture system were reported in p-value < 0.0001). Next, the pro-inflammatory cytokine IL-1β and TNF-α were examined. Colonic batch supernatants with or without CSF all increased the LPS-induced secretion of the pro-inflammatory cytokine IL-1β, compared to the LPS + control . Concerning pro-inflammatory cytokine TNF-α, there’s a significant decrease in secretion upon treatment with CSF compared to that in control . To conclude, in all donors, colonic fermentation of CSF resulted in increased pro-inflammatory cytokine IL-1β levels, while in contrast, secretion of pro-inflammatory cytokine TNF-α was reduced.By exploring the inflammatory effects induced by CSF fermentation, the secretion of anti-inflammatory cytokines IL-6 and IL-10, as well as the pro-inflammatory cytokine IL-1β and TNF-α, were tested. With or without CSF added, colonic batch suspensions all increased the secretion of the anti-inflammatory cytokines IL-6 and IL-10, compared to the LPS + control . In addi control , Left. F3, a significant decrease in CXCL10 and MCP-1 levels was shown , compared to their respective controls. In contrast, a significant increase was shown in donor D5 . CSF treatment did not affect CXCL10 and MCP-1 levels in donors D1, D2 and D4 (increased or decreased <20%). When looking at the average of all donors, after CSF treatment, CXCL10 and MCP-1 levels were not significantly affected, compared to the control .The CSF effect on the recruitment of leukocytes was investigated through the examination of chemokines CXCL10, IL-8 and MCP-1. When looking at the LPS-induced secretion of chemokines CXCL10 and MCP-1, colonic supernatants increased CXCL10 secretion while decreasing MCP-1 secretion . Interes1 and D3 compared to its control . However, CSF treatment did not affect IL-8 levels in all other donors  while significantly lowering the production of bSCFA. In terms of community composition, CSF fermentation stimulated a broad spectrum of health-related species, such as in vitro models–a short-term simulation of the human proximal colon combined with Caco-2/THP1 cell co-culture model. We demonstrated that CSF exposure modulated the gut microbiota composition, which significantly increased the abundance of health-promoting bacteria and subsequently resulted in increased production of the beneficial SCFA metabolites. Also, CSF and its metabolites showed a protective effect on gut barrier integrity. Moreover, this CSF-microbiome-related interaction decreased the secretion of certain pro-inflammatory biomarkers while increasing the secretion of some anti-inflammatory biomarkers. To the best of our knowledge, this is the first study evaluating the beneficial colonic microenvironment created by CSF and its metabolites in healthy people using in vitro models.In this study, the effect of traditional Chinese medicine–Chaihu Shugan Formula (CSF), consisting of several Chinese botanical drugs like Bupleurum, on the gut microbiome, gut barrier and intestinal immunity was evaluated using 4+) during a colonic incubation. A pH decrease during colonic incubation could be due to various factors, but mainly caused by three factors, acting singly or in combination: 1) stimulatory effects on the production of acidic metabolites (SCFA and/or lactate) upon substrate fermentation, which is the most probable cause of pH decrease during colonic incubation. 2) a fall of ammonia  a dein vitro model by the increased production of acetate, propionate and butyrate. SCFA is essential for the maintenance of gut and metabolic health by various effects (CSF fermentation significantly increased short-chain fatty acids (SCFA) secretion and concentrations  effects . Acetate effects . TherefoBifidobacterium, Bacteroides and Lactobacillus spp.) and is a primary metabolite produced from the fermentation of prebiotic substrates. Propionate can also be produced by different gut microbes, with the most abundant propionate producers being Bacteroides spp. (phylum = Bacteroidetes), Akkermansia muciniphila (phylum = Verrucomicrobia) and Veillonellaceae (phylum = Firmicutes). Butyrate is produced by members of the Clostridium clusters IV and XIVa (phylum = Firmicutes). In a process called cross-feeding, these microbes convert acetate and/or lactate  to the health-related butyrate. In our study, the CSF and its metabolites stimulated the enrichment of Actinobacteria, Bacteroidetes and Firmicutes, while with a significant decrease in Proteobacteria, which is consistent with the alteration in the production of SCFA and bSCFA. It indicated that CSF fermentation thrived the growth of health-promoting gut microbes while inhibiting the harmful bacteria, which subsequently promoted the production of SCFA but reduced the secretion of bSCFA.Acetate can be produced by many different gut microbes . The effects observed on the basolateral chamber (where the THP1 cells reside) are then mediated indirectly by signals produced by the Caco- 2 cells and/or by the transport of micro- and macro-molecules. The unique aspect of this approach resides in the fact that it allows evaluating the effect induced by CSF and the fermentation-derived metabolites produced by the gut microbiota during the digestive steps but not only by the pure CSF product. The combined


The pathophysiology of isolated coronary artery ectasia (CAE) with the coronary slow flow (CSF) phenomenon is still unclear. The purpose of this study was to investigate the risk factors for isolated CAE complicated with CSF.A total of 126 patients with isolated CAE were selected retrospectively. The patients were grouped into the no CSF (NCSF) group (n = 55) and the CSF group (n = 71) according to the corrected thrombolysis in myocardial infarction (TIMI) frame count (CTFC). Data on demographics, laboratory measurements, left ventricular ejection fraction (LVEF), left ventricular end-diastolic diameter (LVEDd), CTFC and diameters of three coronary arteries were collected.p = 0.004) and patients with a smoking history  were higher in the CSF group than in the NCSF group. The neutrophil-to-lymphocyte ratio (NLR) (2.08(1.68–3.21) vs. 1.89 ± 0.58, p = 0.001), mean diameter of coronary arteries (mean D) , and uric acid (URIC) level  were significantly higher in the CSF group, while the lymphocyte-to-monocyte ratio (LMR)  and albumin (ALB) level  were lower. Multivariable logistic analysis showed that the LMR , mean D  and URIC level  were independent predictors of CSF in CAE.The proportions of males (84.5% vs. 61.8%, The LMR was a negative independent predictor of CSF in isolated CAE, while URIC level and mean D were positive independent predictors. Isolated coronary artery ectasia (CAE) refers to the local diameter of a coronary artery being dilated more than 1.5 times the adjacent normal vessels and having no obvious obstruction , with a There is no consensus on the physiological mechanism of the above two diseases. Although a change in the vascular diameter will affect the velocity of flow, not all patients with CAE have CSF. Since the morbidity of these two diseases is low, large sample studies are lacking. Some small sample studies have suggested that the lymphocyte-to-monocyte ratio (LMR), neutrophil-to-lymphocyte ratio (NLR), albumin (ALB) level, high-sensitivity C-reactive protein (hs-CRP) level and uric acid (URIC) level are risk factors for CSF or CAE –3,6,8–12From January 2010 to December 2019, 541 patients who underwent coronary angiography in our hospital and were diagnosed with isolated CAE were selected. The inclusion criteria were as follows: the diameter of the local coronary artery was more than 1.5 times larger than that of the adjacent normal segment with a degree of stenosis < 20%, and cardiac ultrasound showed that the heart structure and left ventricular ejection function (LVEF) were normal. The exclusion criteria were as follows: acute coronary syndrome, coronary spasm, coronary artery bypass graft, valve disease, congenital heart disease, Kawasaki disease, left ventricular or ventricular septal hypertrophy, immunological disease, malignant tumour, severe cerebrovascular disease, severe hepatic or renal insufficiency , haematological system disease or haemoglobin < 90 g/L, and received steroid hormone treatment or acute inflammatory disease within 1 month. A total of 126 patients with isolated CAE were included and grouped into the CSF group (n = 71) and the no CSF (NCSF) group (n = 55).The methods used to assess coronary flow included the thrombolysis in myocardial infarction (TIMI) grade and the corrected TIMI frame count (CTFC). The TIMI grade is divided into four grades.The CTFC method was first described by Gibson et al. . The fraAge, sex, body mass index (BMI), history of hypertension, type 2 diabetes mellitus, medication, smoking and drinking, white blood cell count, neutrophil count, lymphocyte count, monocyte count, red blood cell count, haemoglobin count, blood cell specific volume, red blood cell distribution width, mean platelet volume (MPV), mean red blood cell volume, C reactive protein (CRP), high-sensitivity C reactive protein (hs-CRP), ALB, URIC, creatinine, total bilirubin (TBIL), indirect bilirubin (IBIL), triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), glycated haemoglobin (HbA1C), NT-proBNP, LVEF, and left ventricular end-diastolic diameter (LVEDd) were noted. The CTFC and vascular diameter were also assessed by two interventional physicians.Written informed consent was obtained from each patient included in this study. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the institution's ethics committee on research on humans.All analyses were performed using the Statistical Package for the Social Sciences version 22.0  and MedCalc software . Continuous variables are presented as the means ± standard deviations  or as the medians . Categorical variables are presented as absolute values and percentages. Comparisons of categorical variables between the two groups were performed using the chi-squared (χ2) test. The Kolmogorov–Smirnov test was performed to assess whether the variables were normally distributed. Student's t-test or the Mann–Whitney U test was used to compare the continuous variables between the two groups according to whether they were normally distributed. Spearman's rho correlation analysis was performed to describe the degree of correlation between the parameters related to the mean TFC. To identify the independent predictors of CSF, univariate and multivariate logistic regression analyses (backward LR method) were performed. The variables with P < 0.1 in the univariate analysis were incorporated into the multivariate logistic regression analysis. Receiver operating characteristic (ROC) curve analysis was performed to determine additional assets of parameters found to be independent predictors of CSF. The optimal cut-off value was calculated from the point of maximum sensitivity and specificity (Youden's index). A P < 0.05 was considered as statistically significant.p = 0.004) and patients with a smoking history  were significantly higher in the CSF group than in the NCSF group.The number of CAE cases in the LAD and LCX arteries was significantly higher in the CSF group; however, there was no statistically significant difference in terms of the number of CAE cases in the RCA between groups. The number of CAE in single vessel was higher in the NCSF group. However, the number of CAE in multiple vessels was higher in CSF than NCSF group.A comparison of the demographic and clinical characteristics is shown in Table p = 0.002), LCX artery (p = 0.001) and mean D (p < 0.001) were significantly larger in the CSF group; however, the diameter of the RCA was not significantly different between the groups. The LAD-CTFC(p < 0.001), LCX-CTFC(p < 0.001), RCA-CTFC(p < 0.001), Mean CTFC (p < 0.001), NLR (p = 0.001) and URIC level (p = 0.019) were higher in the CSF group, while the LMR (p < 0.001) and ALB level (p = 0.036) were significantly lower in the CSF group.A comparison of the laboratory variables between the two groups is shown in Table r = -−0.21, p = 0.026), ALB level  and male sex  were negatively correlated with CSF, whereas the diameters of the LAD artery , LCX artery  and RCA , as well as the mean diameter of coronary arteries , NLR  and URIC level , were positively correlated with CSF. Smoking history  was also positively correlated with CSF.In the Spearman correlation analyses Table , the LMRp = 0.001), an increased mean D  and the URIC level  were found to be independent predictors of CSF development.To further explore the independent predictor(s) of CSF, univariable and multivariable logistic regression model analyses were performed based on the correlation analysis results Table . We perfp < 0.001). It was revealed that using a cut-off level of an LMR < 4.11 predicted CSF with a sensitivity of 90.9% and a specificity of 40.85% . The URIC level was found to have an AUC of 0.616 (p = 0.026) with an optimal URIC cut-off value of 314.39 μmol/L   the inflammatory indicators were higher in the CSF group than in the NCSF group; 2) The LMR was a negetive independent predictor of CSF in isolated CAE, while URIC level and mean D were positive independent predictors.At present, the exact pathogenesis of CAE and CSF is not clear. Hypotheses suggest that chronic inflammation, atherosclerosis, endothelial dysfunction and oxidative stress may participate in the pathological processes of the two diseases –12,15. MMonocytes and lymphocytes are vital immune cells. They also play an important role in the process of atherosclerosis ,10,16–21[[]] and CSF [Because the LMR combines two kinds of immune cells, it has long been used as an indicator of the condition and treatment of cancer patients ,23. In r and CSF , and it  and CSF ,21,24. Y and CSF . found t and CSF  investig and CSF . found tIn previous studies, elevated URIC levels were associated with an activated inflammatory response in patients with STEMI and associated with poor short-term and long-term outcomes –27. MandBoth the LMR and URIC level play an exclusive role in inflammation and the early formation of atherosclerotic plaques and growth of the lipid core, suggesting that early severe inflammation and rapid atherosclerotic development in CAE patients might be an important pathogenesis for the CSF phenomenon.2^v, where Q is traffic, r is the radius, and v is flow velocity. It is known that when the traffic is constant, flow velocity is inversely proportional to the square of the radius. This was consistent with our study, showing that the mean D in the CSF group was larger than that in the NCSF group.The deep impression of CAE in angiography is local or diffuse vascular expansion. It is viewed in the cross-section of a coronary artery as circular according to the flow equation Q = πrSomaschini  found thThere were some limitations to this study. The first limitation relates to its retrospective design. Some risk factors, such as the matrix metalloproteinase family, tissue inhibitors of metalloproteinases ,35 and aPatients with isolated CAE combined with CSF have a high URIC level and a large diameter of coronary arteries and a low LMR. This study revealed that severe inflammatory reactions and atherosclerosis development might be targets for future pathogenesis studies in patients with isolated CAE combined with CSF.


R2 ≥ 0.89. The optimum composition was 8% SL, 22% GMS, 28% water, and 42% PO (w/w) to produce a mean firmness of 1.91 N, spreadability of 15.28 N s−1, and oil binding capacity (OBC) of 83.83%. The OBC of optimized organogel was 10% higher than commercial spread product, and no significant difference was observed in the mechanical properties (p > 0.05). The microstructure, as well as the rheological and thermal properties of the optimized organogel were characterized. Fourier transform infrared analysis indicated that hydrogen bonding and van der Waals interactions were the key driving forces for organogelation. The mixture of SL and GMS favored the formation of β′ + β form crystals with a predominance of the β′ form. These results have important implications for the development of PO-based organogel as a potential fat replacer in the production of low-fat spread. The present work investigated the interaction between soy lecithin (SL), glyceryl monostearate (GMS), and water in structuring palm olein (PO) to create an organogel having similar mechanical properties to commercial spread. Extreme vertices mixture design was used to optimize the composition of PO-based organogel. The resulting model showed a good fit to the predicted data with  Organogelation is a new way to structure liquid oil, by dissolving organogelators that could impart solid-like functional characteristics in an oil phase . The forA number of studies have been conducted on organogels toward food, cosmetic, and pharmaceutical applications in recent years . The larThe influence of different organogelators on the physical properties of organogels has been addressed previously. However, most of those studies focused on the use of a single organogelator to structure the oil, while the use of organogelator mixtures could further improve the rheological and physical properties of food products. According to Lupi et al. , organogMechanical attributes such as firmness, spreadability, and oil separation are key physical properties determining the shelf life and consumer acceptance of food products . IdentifTo eliminate trans fatty acids from the food system, there have been proposals to use palm oils and palm oil fractions to replace partially hydrogenated fats. Palm olein (PO) is fractionated from palm oil as a mixture of 45.3% saturated and 52.7% unsaturated fatty acids. It also contains various bioactive components including tocotrienols, tocopherols, and β-carotene . Due to Specifically, in order to formulate a PO-based organogel with mechanical properties comparable to commercial spread, the present study investigates the influence of specific components  on selected response variables of the organogel: the firmness, spreadability, and oil binding capacity (OBC). Firstly, the possible interactions between components in the mixed organogel were determined, and the composition was optimized using extreme vertices mixture design. This was followed by evaluation of the characteristics of optimized organogel, namely the microstructure, polymorphism, and structural and thermal properties. The optimized composition provides useful information for developing PO-based organogel as a fat replacer for a trans-fat-free spread product.R2  and no significant difference (p > 0.05) in the lack-of-fit test [R2, adjusted R2, predicted R2, and lack-of-fit, as shown in Mixture design has been widely used in food production for composition optimization. This process is faster and more economical, and all the potential factors could be evaluated simultaneously and systematically ,19,20,21fit test . Table 1R2 values for firmness, spreadability, and OBC models were observed to be 0.9412 (94%), 0.9699 (97%), and 0.8970 (90%), respectively. This indicates excellent fit of the generated polynomials to the experimental data, in which a high variability in the responding variables was attributed to the independent variables. These models showed no significant lack of fit (p > 0.05), suggesting that they fit the experimental data well. Contrarily, the adjusted R2 provides an adjustment to R2 based on the independent variables that are significant in the model, whereas the predicted R2 is used to evaluate how accurate the model is in predicting the response for future observations. All adjusted and predicted R2 values were found to be close to the actual R2, again showing the excellent fit of the models to the responses  are shown in X1) and GMS (X2) had high positive linear coefficients in the suggested regression models of firmness and spreadability (X3) increased, the firmness and spreadability decreased linearly. Increasing the amount of water caused instability in the organogel and eventually led to phase separation. A similar observation was reported by Angelico et al. [Among the mixture components, both SL , GMS × water (X2X3), and water × PO (X3X4) did not show significant effects (p > 0.05) in all regression models. This indicates that changes in these three combinations would not strongly impact the mechanical properties of the organogels.On the other hand, all the organogels prepared showed OBC values ranging from 50.03 to 91.53%. The OBC value increased with higher proportion of GMS in the formulation c. In a hX1) and GMS (X2), along with a small amount of water (X3) and PO (X4) in the formulation , and maximal OBC. These ranges were obtained for commercial spreads from different manufacturers. Based on these values, optimization of the organogel composition was performed using the multi-response optimizer function in Minitab software. In order to obtain the best balance among the different responses, a composite desirability (D) with values in the range of 0  to 1  was used to optimize various responses simultaneously [t-test was conducted to compare the experimental and predicted values for each response , moderate spreadability . No significant difference was observed in the firmness and spreadability values (p > 0.05) between SL/GMS/PO and the commercial spread. Meanwhile, the OBC of the former was 10% higher, indicating a better ability to entrap the oil and reduce the occurrence of syneresis (separation of oil). According to Codex Alimentarius, the fat content of a spread product must be less than 80% (WHO/FAO 2007). SL/GMS/PO, which contains 42% oil, meets the requirement for a low-fat spread. Therefore, further characterization of SL/GMS/PO was conducted to determine its physical properties prior to food application.Comparison was also made between the optimized organogel (SL/GMS/PO) and a commercial spread  was also observed in SL/PO, GMS/PO, and SL/GMS/PO organogels.FTIR analysis was conducted at room temperature (25 °C) to determine the intermolecular interactions amongst individual components within the organogel sample. Based on 2 were shifted slightly from 2848 and 2914 cm−1 to 2850 and 2916 cm−1 for organogels. These small peak shifts between pure organogelators and organogels were reported to be due to the presence of van der Waals interactions, which decrease the fluidity of the alkyl chains [l chains ,33. AlthThe XRD patterns of the organogels at ambient temperature are shown in Rheological analysis was performed to determine the viscoelasticity of SL/PO, GMS/PO, and SL/GMS/PO. This was done by evaluating the changes in the storage modulus (G′) and loss modulus (G″). G′ reflects the solid-like component in the rheological behavior, whereas G″ represents the viscosity of the material . In FiguThe thermal behavior of all the samples was studied by cooling the samples from 80 to 10 °C . At tempAt temperatures from 45 to 55 °C, a sudden increase in the complex modulus curve was observed in GMS/PO and SL/GMS/PO organogels, indicating the beginning of organogel crystallization. During this cooling process, the gel network structure began to form due to the increased interaction between the organogelator molecules and the aggregation of the molecules over the entire sample. The temperature at which a crossover is observed between G′ and G″ is known as the gelation point (Tg), where the system begins to transform from a free-flowing liquid phase to a gelled phase . The meaNevertheless, the crystallization and melting peaks of SL are not notable in the SL/PO thermogram. A similar observation was reported by Brad and Chen  and Yusuw/w), which produced an organogel with high OBC, strong gel network, and mechanical properties comparable to the commercial spread. This organogel (SL/GMS/PO) showed structural and rheological properties intermediate between those of SL/PO and GMS/PO. However, its crystal polymorph and thermal properties seem to be closer to GMS/PO. The physical properties of SL/GMS/PO were strongly affected by the proportions of SL and GMS used. These results contribute to the development of PO-based organogels formed using mixed organogelators. Further study is being carried out to improve the storage stability of SL/GMS/PO.In this study, extreme vertices mixture design was successfully utilized to optimize the composition of PO-based organogels using mixed organogelators to achieve desired mechanical properties for spreads. The optimum proportions obtained was 8% SL, 22% GMS, 28% water, and 42% PO  = 56) was acquired from MOI Foods Malaysia Sdn Bhd . Soy lecithin (SL) was purchased from R&M Chemicals , and glyceryl monostearate (GMS) was acquired from a local food ingredient supplier . A commercial spread product of reduced fat  was purchased from Tesco supermarket . This commercial spread product contains palm oil with a total fat content of 55.5%.X1), GMS (X2), water (X3), and PO (X4) were determined and analyzed using extreme vertices mixture design performed with Minitab 17 . The lower and upper weight limits for the SL, GMS, water, and PO contents were 6–30%, 4–30%, 10–40%, and 30–80%, respectively, summing to 100% in each final formulation. All the experimental runs, factor levels, and responding variables are presented in Y is the predicted response , X is the factor considered . The analysis of variance (ANOVA) was used to determine the quality of the response models. The statistical coefficients used to interpret the models were the coefficient of determination (R2), the adjusted R2, and the predicted R2. Besides that, the lack-of-fit test was applied to indicate the probability of the model’s failure to predict the experimental data. The value obtained should not be significant, where p > 0.05.Mixture design is commonly used to study the relationship between mixture components to determine the optimum composition using desirability approach ,19,21. IA total of 29 organogel formulations were prepared according to the extreme vertices mixture design . Each foThe mechanical strength of organogels was evaluated in terms of firmness and spreadability. Both analyses were performed using the TA-XT plus texture analyzer  at room temperature (25 °C). Twenty grams of the organogel sample was first filled in a glass bottle (5.8 cm × 45 mm diameter). The firmness of the sample was then determined with a cylindrical probe (P/0.5R with 1/2 in diameter). The measurements were done in the compression mode, in which the probe penetrated 12 mm into the sample (2 mm/s), and returned to the initial position at the same speed.For the spreadability test, a male 90° cone probe and female perspex cone (sample holder) were used . Prior tThe ability of the organogel to entrap oil was determined following the method described by Martins et al. . Five gr% OBC = 100% of released oilt-test was further conducted to compare the experimentally determined values with those predicted by the multiresponse optimizer.Multiresponse optimization was used to determine the optimum formulation of organogel based on composite desirability . Model vThe molecular, rheological, and thermal properties of the organogels (both the optimized and those using a pure organogelator) were studied. The organogels prepared using pure SL, pure GMS and the optimized organogel are coded ‘SL/PO’, ‘GMS/PO’, and ‘SL/GMS/PO’ samples, respectively. These organogels are prepared via fluid-filled fiber mechanism with 28% of water in SL/PO and GMS/PO.Microstructure images of the organogels were obtained under a polarized light microscope  equipped with a digital camera . Initially, the organogels were melted at 65 °C. One drop of the melted organogel was then deposited on a glass slide and covered with a coverslip. Prior to microscope observation, the slides were stored overnight at 25 °C to ensure proper crystallization. The organogels were observed at 40× magnification at room temperature (25 °C), and the images were obtained using NIS-Element Imaging Software .−1 resolution from 650 to 4000 cm−1 using a Varian 640-IR FTIR spectrophotometer . Each spectrum was composed of 32 scans at room temperature (25 °C) [Organogel samples were placed directly on the attenuated total reflection (ATR) crystal. FTIR spectra of the organogels were measured at 4 cm (25 °C) .k = 1.5418 Å) at the voltage and current of 40 kV and 40 mA, respectively. The samples were analyzed at 2θ angles of 5° to 50° with a scanning rate of 2° 2θ/min. The X-ray peak and its width at half maximum (FWHM) were calculated using Diffrac. EVA (Bruker) software [The polymorphic forms of the fat crystals were determined at 25 °C with a D8 Discover X-ray diffractometer  fitted with Cu-Kα radiation k = 1.58 Å at thRheological properties of the organogels were determined using a rheometer  equipped with a temperature control unit. Using a parallel plate geometry (PP 25/s), the strain sweep tests were performed at a constant frequency of 1 Hz and at strain amplitude varying from 0.1 to 100 Pa to investigate the linear viscoelastic region (LVR) of organogels. Subsequently, frequency sweep test was performed at a fixed strain (10 Pa) with frequency 0.1–10 Hz. Both strain sweep and frequency sweep tests were carried out at room temperature (25 °C). The temperature ramp test was performed at 1 Hz frequency and the temperature range from 10 to 80 °C with cooling rate 1 °C/min, to study the thermal sensitivity of organogels. The G′ and G′′ values  for each sample were obtained.The crystallization and melting thermograms were obtained by using a differential scanning calorimeter  equipped with a refrigerated cooling unit. About 10–12 mg of sample was loaded in a sealed pan, and an empty aluminum pan was used as reference. Experiments were conducted using a nitrogen flow rate of 20 mL/min. The samples were first heated at 80 °C for 10 min to remove all the fat crystal memory, followed by cooling (5 °C/min) to −50 °C and re-heating to 80 °C at the same rate .p < 0.05 level were used.Each organogel sample was prepared in duplicate, and each sample analysis was repeated at least twice. The results were represented as mean ± standard deviation (SD). Data were analyzed statistically by one-way ANOVA, and Tukey’s test using the Minitab 17 software. The significant differences at a