Current status and challenges for cell-cultured milk technology: a systematic review

Cellular agriculture is an innovative technology for manufacturing sustainable agricultural products as an alternative to traditional agriculture.While most cellular agriculture is predominantly centered on the production of cultured meat,there is a growing demand for an understanding of the production techniques involved in dairy products within cellular agriculture.This review focuses on the current status of cellular agriculture in the dairy sector and technical challenges for cell-cultured milk production.Cellular agriculture technology in the dairy sector has been classified into fermentation-based and animal cell culture-based cellular agriculture.Currently,various companies synthesize milk components through precision fermentation technology.Nevertheless,several startup companies are pursuing animal cell-based technology,driven by public concerns regarding genetically modified organisms in precision fermentation technology.Hence,this review offers an up-to-date exploration of animal cell-based cellular agriculture to produce milk components,specifically emphasizing the structural,functional,and productive aspects of mammary epithelial cells,providing new information for industry and academia.

Effect of Butternut Squash (Cucurbita moschata) Seed Powder on the Chemical and Rheological Properties of Stirred Cultured Camel Milk and Yoghurt

Research shows that producing fermented camel milk is hard because of the milk’s inability to form a firm coagulum, attributed to low levels of κ-casein and ꞵ-lactoglobulin and the large casein micelle size, leading to a weak network of casein formation. In an effort to address this issue, researchers turned to corn starch as a thickening agent, discovering that a concentration of 2.0% effectively improved the viscosity and significantly reduced syneresis in stirred camel milk yoghurt and cultured camel milk. This study explores alternatives to corn starch, focusing on butternut squash seeds as a promising substitute due to their hydrocolloid composition. By incorporating butternut squash (Cucurbita moschata) seed powder (BSSP) as a thickening agent, this study aimed at enhancing the chemical and rheological properties of stirred camel milk yoghurt and cultured camel milk. Fermented camel milk was prepared using 4 litres of camel milk, 2% starter cultures (thermophilic culture for yoghurt and mesophilic aromatic culture for stirred cultured camel milk) and BSSP 0.0% (negative control), 0.4%, 0.8%, 1.2%, 1.6%, 2.0% mixed with 0.4% gelatin. 2.0% corn starch mixed with 0.4% gelatin was used as a standard for comparison. Results showed that increasing the BSSP level significantly (p < 0.05) decreased the moisture content while increasing the total solid content of stirred fermented camel milk products. There was an increase in ash content with an increase in BSSP levels. There was a significant (p < 0.05) reduction in the pH, with an increase in BSSP levels in stirred fermented camel milk samples. Increasing the concentration of BSSP from 0.4% to 2.0% resulted in a significant (p < 0.05) increase in viscosity and a reduction in syneresis of stirred camel milk yoghurt and stirred cultured camel milk samples. This study demonstrated that BSSP effectively enhances the viscosity, reduces syneresis and increases acidity in stirred fermented camel milk products during storage.

Investigation of the Acceptability of Cultured Meat in University Students

Background: Over the past 20 years, cultured meat has drawn a lot of public attention as a potential solution to issues with animal husbandry, including inadequate use of natural sources, improper animal welfare practices, and possible risks to public health and safety. The novel method of producing meat through culture reduces the need for animals to produce muscle fiber, thereby obviating the necessity for animal slaughter. Apart from its ethical advantages, cultured meat presents a possible way to fulfill the expanding need for food among growing populations. The purpose of this research was to find out whether Turkish students would be willing to pay for and accept cultured meat. Technique: Method: 371 university students who willingly consented to fill out a questionnaire and provide demographic data make up the research sample. Questions from previous studies on the acceptability of cultured meat were compiled to create the survey. The research’s data collection took place in March and April of 2022. The research was completed in June 2022 after the data had been processed and analyzed. Results: The results showed that the majority of participants were female and had omnivorous eating habits. Based on the results of the Bonferroni correction test, students with a higher intention to purchase and consume cultured meat were those who received economics and business education. Students with two years of university education had a higher overall survey score than those with four years of education (p < 0.05). Furthermore, it is discovered that there is a negative correlation between the participants’ ages and their Factor 2 (using cultured meat as an alternative to industrial meat) and Factor 3 (consuming and purchasing it) section points (r = -109, p = 0.036) (r = -0.121, p = 0.019). In conclusion, university students generally have a negative outlook on health-related issues, such as eating cultured meat as an alternative.

Sequential extraction of RNA,DNA and protein from cultured cells of the same group

BACKGROUND Efficient extraction of nucleic acids and proteins(ENAP)from cells is a prerequisite for precise annotation of gene function,and has become laboratory routine for revealing the mysteries of life.However,cell samples are often from different culture dishes,resulting in inevitable experimental errors and sometimes poor repeatability.AIM To explore a method to improve the efficiency of ENAP,minimizing errors in ENAP processes,enhancing the reliability and repeatability of subsequent experimental results.METHODS A protocol for the sequential isolation of RNA,DNA,and proteins from the same cultured HepG2 cells using RNAzol reagent is presented here.The first step involves culturing HepG2 cells to the exponential phase,followed by the sequential isolation of RNA,DNA,and proteins from the same cultured cells in the second step.The yield of nucleic acids and proteins is detected in the third step,and their purity and integrity are verified in the last step.RESULTS The procedure takes as few as 3-4 d from the start to quality verification and is highly efficient.In contrast to the existing kits and reagents,which are primarily based on independent isolation,this RNAzol reagent-based method is characterized by the sequential isolation of RNA,DNA,and proteins from the same cells,and therefore saves time,and has low cost and high efficiency.CONCLUSION The RNA,DNA,and proteins isolated using this method can be used for reverse transcription-polymerase chain reaction,polymerase chain reaction,and western blotting,respectively.

Analysis and Evaluation of Nutrient Composition in the Wild, Pond- and Lake-Cultured Topmouth Culter (Culter alburnus)

Research was conducted on topmouth culter (Culter alburnus) grown in ponds and lakes as well as wild types in order to determine their chemical composition and nutritional value. There are three types of fish that differ in their proximate composition, amino acids, fatty acids, and minerals. Wild fish had a significantly lower crude lipid contents than cultured fish (P P ∑PUFAs) showed an obviously opposite trend. As compared with cultured fish, wild fish had significantly higher levels of n-3 PUFAs, arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahxaenoic acid (DHA) (P P > 0.05), except for Na, Fe and Se. In conclusion, diet composition and external aqueous environment may determine the differences between wild and cultured topmouth culter.

Establishment and evaluation of the primary cultured tibial osteoblast model of broiler chicks

Osteoblasts are considered as a major factor contributing to bone development and mineralization,however,few studies have been done to establish and evaluate the primary cultured tibial osteoblast model of broiler chicks.Therefore,in the present study,two experiments were conducted to establish and evaluate the primary cultured tibial osteoblast model of broiler chicks.In experiment 1,osteoblasts were isolated from the tibia of one-day-old Arbor Acre male broiler chicks using the explant method and identified through the cell morphology,alkaline phosphatase(ALP)and alizarin red staining.Experiment 2 was carried out to evaluate the vitality and mineralization of primary cultured tibial osteoblasts of broilers on days 4,8,12,16,20,24,28 and 32 after incubation,respectively.The results from experiment 1 demonstrated that primary cultured tibial osteoblasts of broilers showed a spindle-shaped,triangular or polygonal morphology.More than 95%of the cells were stained blue-black after ALP staining,and mineralized nodules were formed after 4 days of continuous incubation.In experiment 2,lactate dehydrogenase(LDH)activity stayed at a relatively stabilized level although incubation time affected(P=0.0012)it during the whole culture period.Additionally,incubation time affected(P≤0.0001)the number and proportion of the area of mineralized nodules.They increased linearly and quadratically(P<0.04)with the increase of incubation time,and remained at a stabilized level from 24 to 32 days of incubation.The estimates of the optimal incubation time were 17 and 26 days based on the best fitted broken-line or quadratic models(P<0.0001)of the number and proportion of the area of mineralized nodules,respectively.These results indicate that the primary cultured tibial osteoblast model of broilers has been established successfully by the explant method,and it showed typical osteoblast morphology and characteristics of ALP activity and mineralization,and could maintain a relatively stabilized vitality from 4 to 32 days of incubation;and the optimal incubation time of primary tibial osteoblasts was 17 to 26 days.Therefore,it could be used to further study the underlying mechanisms of bone development and mineralization of broiler chicks.

Repetitive administration of cultured human CD34+cells improve adenine-induced kidney injury in mice

BACKGROUND There is no established treatment to impede the progression or restore kidney function in human chronic kidney disease(CKD).AIM To examine the efficacy of cultured human CD34+cells with enhanced proliferating potential in kidney injury in mice.METHODS Human umbilical cord blood(UCB)-derived CD34+cells were incubated for one week in vasculogenic conditioning medium.Vasculogenic culture significantly increased the number of CD34+cells and their ability to form endothelial progenitor cell colony-forming units.Adenineinduced tubulointerstitial injury of the kidney was induced in immunodeficient non-obese diabetic/severe combined immunodeficiency mice,and cultured human UCB-CD34+cells were administered at a dose of 1×106/mouse on days 7,14,and 21 after the start of adenine diet.RESULTS Repetitive administration of cultured UCB-CD34+cells significantly improved the time-course of kidney dysfunction in the cell therapy group compared with that in the control group.Both interstitial fibrosis and tubular damage were significantly reduced in the cell therapy group compared with those in the control group(P<0.01).Microvasculature integrity was significantly preserved(P<0.01)and macrophage infiltration into kidney tissue was dramatically decreased in the cell therapy group compared with those in the control group(P<0.001).CONCLUSION Early intervention using human cultured CD34+cells significantly improved the progression of tubulointerstitial kidney injury.Repetitive administration of cultured human UCB-CD34+cells significantly improved tubulointerstitial damage in adenine-induced kidney injury in mice via vasculoprotective and anti-inflammatory effects.

Propagation of Abies beshanzuensis by Water Cultured Medium

[Objective] The experiment aimed to explore the influences of phytohormones (ABT and IAA) and nutrient solution on rooting of Abies beshanzuensis M.H.Wu by water cultured medium. [Method] The Abies beshanzuensis M.H.Wu were treated by water (CK), 10 mg/L ABT+ water, 10 mg/L IAA+ water, 10 mg/L ABT+ hoagland solution, 10 mg/L IAA+ hoagland solution, then the rooting process was observed and the formation rate of callus, rooting rate, number of rooting, and root length were investigated and analyzed. [Result] ABT and IAA had obvious influences on callus induction, rooting rate and the number of root of Abies beshanzuensis M.H.Wu by water culture, so they were suitable to be used in water propagation of Abies beshanzuensis M.H.Wu. The treatments of phytohormones had no regular influences on the longest root length and average root length. The nutrient solutions would not generate obvious influence on propagation of Abies beshanzuensis M.H.Wu at firstly stage, but they generated influence on root growth after rooting. [Conclusion] The research provided new ideas for propagation of Abies beshanzuensis M.H.Wu, which could make it out of endangerment situation quickly.

Expression of Tissue Transglutaminase in Cultured Bovine Trabecluar Meshwork Cells

Summary: To study whether cultured bovine trabecluar meshwork cells (BTMC) are capable of expressing tTG in protein and at mRNA level, BTMC were cultured in vitro and passaged three times, then the cells were transferred onto or cultured on sterile cover or submitted to isolation of RNA with Trizol, and the expression of tTG was detected by immunohistochemical technique and reverse transcription polymerase chain reaction (RT-PCR) respectively. Our results showed that tTG immunostaining was positive in the cytoplasm and rarely in the nucleus of cultured BTMC. No immunostaining was seen in the negative control. Moreover, a single RT-PCR amplified product whose sequence and size were in accordance with our known parameters was obtained. The expression of tTG in cultured BTMC was confirmed in protein and at mRNA level. BMTC is available more readily for the investigation of the relationship between tTG and primary open-angle glaucoma.

Experimental study of bioartificial liver with cultured human liver cells

AIM To establish an extracorporeal bioartificial liver support system (EBLSS) using cultured human liver cells and to study its support effect for fulminant hepatic failure (FHF).METHODS The liver support experiment of EBLSS consisting of aggregates cultured human liver cells, hollow fiber bioreactor, and circulation unit was carried out in dizhepatic dogs.RESULTS The viability of isolated hepatocytes and nonparenchymal liver cells reached 96%. These cells were successfully cultured as multicellular spheroids with synthetic technique. The typical morphological appearance was retained up to the end of the artificial liver experiment. Compared with the control dogs treated with EBLSS without liver cells, the survival time of artificial liver support dogs was significantly prolonged. The changes of blood pressure, heart rate and ECG were slow. Both serum ammonia and lactate levels were significantly lowered at the 3rd h and 5th h. In addition, a good viability of human liver cells was noted after 5 h experiment.CONCLUSION EBLSS playing a metabolic role of cultured human hepatocytes, is capable of compensating the function of the liver, and could provide effective artificial liver support and therapy for patients with FHF.

Cultured meat from muscle stem cells: A review of challenges and prospects

Growing muscle tissue in culture from animal stem cells to produce meat theoretically eliminates the need to sacrifice animals. So-called ＂cultured＂ or ＂synthetic＂ or ＂in vitro＂ meat could in theory be constructed with different characteristics and be produced faster and more efficiently than traditional meat. The technique to generate cultured muscle tissues from stem cells was described long ago, but has not yet been developed for the commercial production of cultured meat products. The technology is at an early stage and prerequisites of implementation include a reasonably high level of consumer acceptance, and the development of commercially-viable means of large scale production. Recent advancements in tissue culture techniques suggest that production may be economically feasible, provided it has physical properties in terms of colour, flavour, aroma, texture and palatability that are comparable to conventional meat. Although considerable progress has been made during recent years, important issues remain to be resolved, including the characterization of social and ethical constraints, the fine-tuning of culture conditions, and the development of culture media that are cost-effective and free of animal products. Consumer acceptance and confidence in in vitro produced cultured meat might be a significant impediment that hinders the marketing process.

Challenges and prospects for consumer acceptance of cultured meat

Consumer acceptance of cultured meat is expected to depend on a wide diversity of determinants ranging from technologyrelated perceptions to product-specific expectations, and including wider contextual factors like media coverage, public involvement, and trust in science, policy and society. This paper discusses the case of cultured meat against this multitude of possible determinants shaping future consumer acceptance or rejection. The paper also presents insights from a primary exploratory study performed in April 2013 with consumers from Flanders（Belgium）（n=180）. The concept of cultured meat was only known（unaided） by 13% of the study participants. After receiving basic information about what cultured meat is, participants expressed favorable expectations about the concept. Only 9% rejected the idea of trying cultured meat, while two thirds hesitated and about quarter indicated to be willing to try it. The provision of additional information about the environmental benefits of cultured meat compared to traditional meat resulted in 43% of the participants indicating to be willing to try this novel food, while another 51% indicated to be ‘maybe＇ willing to do so. Price and sensory expectations emerged as major obstacles. Consumers eating mostly vegetarian meals were less convinced that cultured meat might be healthy, suggesting that vegetarians may not be the ideal primary target group for this novel meat substitute. Although exploratory rather than conclusive, the findings generally underscore doubts among consumers about trying this product when it would become available, and therefore also the challenge for cultured meat to mimic traditional meat in terms of sensory quality at an affordable price in order to become acceptable for future consumers.

Cloning of the non-structural gene 3 of hepatitis C virus and its inducible expression in cultured cells

AIM To study the inducible expression of hepatitis C virus ns3 gene (HCV ns3) in eukaryotic cells.METHODS The ns3 gene was obtained from plasmid pBns3 by polymerase chain reaction and inserted into the cloning vector pGEM-T. Then, the ns3 was subcloned into the vector pMSG to generate dexamethasone (DM)-inducible expression plasmid pMSG-ns3. CHO cells were transfected by pMSG-ns3 using calcium phosphate precipitation method and cultivated for 12 h-24 h. The transfected cells were induced with DM and the transient expression of NS3 protein was analyzed by ELISA and Western-blot methods.RESULTS After treated with 3×10-8mol/ L DM, the expression of NS3 was observed in the transfected CHO cells. A slightly higher level of NS3 was shown along with the time of DM treatment.CONCLUSION The inducible expressing vector pMSG-ns3 might be helpful for further studies of the characteristics of the ns3 gene in vivo.

Alternatives for large-scale production of cultured beef: A review

Cultured beef is a method where stem cells from skeletal muscle of cows are cultured in vitro to gain edible muscle tissue. For large-scale production of cultured beef, the culture technique needs to become more efficient than today＇s 2-dimensional（2D） standard technique that was used to make the first cultured hamburger. Options for efficient large-scale production of stem cells are to culture cells on microcarriers, either in suspension or in a packed bed bioreactor, or to culture aggregated cells in suspension. We discuss the pros and cons of these systems as well as the possibilities to use the systems for tissue culture. Either of the production systems needs to be optimized to achieve an efficient production of cultured beef. It is anticipated that the optimization of large-scale cell culture as performed for other stem cells can be translated into successful protocols for bovine satellite cells resulting in resource and cost efficient cultured beef.

Effect of endotoxin on fibronectin synthesis of rat primary cultured hepatocytes

AIM To investigate the effect of endotoxin on liver fibrosis and further define the role of hepatocytes in production of fibronectin in primary liver cell culture by endotoxin.METHODS After isolation and seeding of hepatocytes, the obtained cells were added to various doses (0, 5, 10, 15 and 20mg/L) of LPS treated culture media. The cells were collected and counted at various periods (0, 12, 24, 48, 72, 96, 120h). The concentrations of fibronectin were tested by electrophoresis.RESULTS The fibronectin levels tended to increase with prolongation of culture time. There was a sharp increase after 72h in 10 or 15 LPS treated group. The peak level of fibronectin was above 20mg/L. However, cell proliferation was inhibited during the course. Cell number of untreated control group (4.6±0.1×106) was about three-fold that of 20 LPS treated group (1.6±0.2×106) at 120h.CONCLUSION Hepatocytes have a potent ability to produce fibronectin stimulated by endotoxin, suggesting that hepatocytes might participate in the process of liver fibrosis.

Structural Analysis of Water-soluble Polysaccharide PIP_1 Extracted from the Cultured Mycelium of Phellinus igniarius

Water-soluble crude polyseccharide（PIP） was extracted from cultured mycelium of the fungus Phellinus igniarius. After ethanol precipitation and sepharose CL-6B gel filtration, the fraction of PIP1 was obtained, which was shown to be a homogeneous polysaccharide by means of high-performance liquid chromatography. The structure of PIPt was determined by using several methods. C.,C analysis indicates that PIP1 is composed of the monosaccharides of glucose, galactose, and mannose. Their malar ratio is 3. 70： 4. 06： 1.00. The molar weight was estimated to be 17 kd via HPLC. IR, GC, partial hydrolysis with acid, pefiedate oxidation, Smith degradation, methylation, and GC-MS analysis were used for the structural analyses of PIP1. The results show that PIP1 has a small quantity of branch structure, The main glycosidic linkage of PIP1 has a β-configurafion. The main chain is made up of a large mass of glucose （ 1→3 ） and few mannose （ 1→4 ） ; the side chain is composed of glucose （ 1 →3 ） and galactose （ 1→6 ） ; the nonreduced end is composed of galactose and glucose. The side chains are branched at 6-0 of glucose（ 1→3,6） and mannose（1→4,6）. On an average, there are three branches among 20 residues. It is presumable that the existence of 1,3-linked Glc in the main and side chains is the main reason for its higher antitumor activity.

Effects of Estradiol on Cashmere Goat Primary Hair Follicles Cultured in vitro

[Objective] The aim of this study was to preliminarily explore the effects of estradiol on morphology and growth of cashmere goat primary hair follicles. [Method] Cashmere goat primary hair follicles were cultured in serum-free Williams E media supplemented with different doses of 17 β-E2 (0, 0.1, 1.0, 10.0, 100.0 nmol/L), and their growth rates and morphological changes were observed. [Result] The growth rate of 0.1 nmol/L 17 β-E2 group was quite comparable with that of the control group(0 nmol/L), but the 17 β-E2 with concentrations of 1.0, 10.0 and 100.0 nmol/L displayed different degrees of inhibition on the growth of hair follicles. Different morphological changes of hair follicles could also be discovered in different concentration treatments. [Conclusion] The study laid a certain foundation for exploring the regulation mechanism of estrogen on growth of cashmere goat hair follicles.

Effects of aminoguanidine on nitric oxide production induced by inflammatory cytokines and endotoxin in cultured rat hepatocytes

AIM: To study the effects of aminoguanidine (AG) and two L-arginine analogues N(omega)-nitro-L-arginine methyl ester (L-NAME) and N(omega)-nitro-L-arginine (L-NNA) on nitric oxide (NO) production induced by cytokines (TNF-alpha, IL-1 beta, and IFN-gamma) and bacterial lipopolysaccharide (LPS) mixture (CM) in the cultured rat hepatocytes, and examine their mechanisms action. METHODS: Rat hepatocytes were incubated with AG, L-NAME, L-NNA, Actinomycin D (ActD) and dexamethasone in a medium containing CM (LPS plus TNF-alpha, IL-1 beta, and IFN-gamma) for 24h. NO production in the cultured supernatant was measured with the Griess reaction. Intracellular cGMP level was detected with radioimmunoassy. RESULTS: NO production was markedly blocked by AG and L-NAME in a dose-dependent manner under inflammatory stimuli condition triggered by CM in vitro. The rate of the maximum inhibitory effects of L-NAME (38.9%) was less potent than that obtained with AG(53.7%, P 【 0.05). There was no significant difference between the inhibitory effects of AG and two L-arginine analogues on intracellular cGMP accumulation in rat cultured hepatocytes. Non-specific NOS expression inhibitor dexamethasone (DEX)and iNOS mRNA transcriptional inhibitor ActD also significantly inhibited CM-induced NO production. AG(0.1 mmol x L(-1)) and ActD (0.2 ng x L(-1)) were equipotent in decreasing NO production induced by inflammatory stimuli in vitro, and both effects were more potent than that induced by non-selectivity NOS activity inhibitor L-NAME (0.1 mmol x L(-1)) under similar stimuli conditions (P【0.01). CONCLUSION: AG is a potent selective inhibitor of inducible isoform of NOS,and the mechanism of action may be not only competitive inhibition in the substrate level, but also the gene expression level in rat hepatocytes.

The environmental prospects of cultured meat in China

To deal with concerns in China about environmental degradation and a growth in population accompanied by increased consumption of livestock products, a meat alternative is required. This study compared the environmental impacts of producing different protein sources for nutrition, including crops, livestock products, and cultured meat. The results showed that cultured meat has the lowest land use per unit of protein and unit of human digestible energy. China＇s crops have the lowest energy use and greenhouse gas（GHG） emissions per unit of energy and protein. The energy use in cultured meat production is slightly higher than that of current pork production in China, whereas GHG emissions are lower. It is concluded that the overall impact of replacing livestock products with cultured meat would be beneficial for China＇s environment and would potentially improve food security because less land is needed to produce the same amount of protein and energy.

Oxidative damage of primary cultured hippocampal neurons Does androgen have an antagonistic effect?

BACKGROUND： Evidence illustrates that androgen has a neuroprotective role. However, whether androgen also has the protective effect on hippocampal neurons during free radical mediated injury remains unclear. OBJECTIVE： To investigate the neuroprotective effect of androgen on hippocampal neurons during free radical damage. DESIGN, TIME AND SETTING： A controlled in vitro experiment was performed at the Department of Human Anatomy, Cell Culture Lab, and Neuroendocrinology Lab, Basic Medical School, Hebei Medical University from February to June 2009. MATERIALS： Testosterone was provided by Tianjin Jinyao Amino Acid Company, China. METHODS： Primary cultured neurons from 24 Sprague Dawley rats were randomly assigned into four groups： control, H202, testosterone, and testosterone （pre-added） plus H2O2 groups. MAIN OUTCOME MEASURES： The positive cell ratio of microtubule associated protein-Ⅱ and neuron specific enolase was determined by immunocytochemistry. Neuronal morphology was observed by hematoxylin-eosin staining and Nissl staining. Cell vitality and viability were determined using an inverted phase contrast microscope. The content of nitric oxide synthase, malondialdehyde, and superoxide dismutase were measured with a spectrophotometer. RESULTS： As compared with the control group, cell vitality and viability, and superoxide dismutase level were significantly decreased in the H202 group （P 〈 0.05）, while nitric oxide synthase and malondialdehyde levels were significantly increased （P 〈 0.05）. Neuronal vitality and viability as well as superoxide dismutase level in the testosterone plus H2O2 group were significantly greater than in the H2O2 group （P 〈 0.05）, and nitric oxide synthase and malondialdehyde levels were significantly less than in the H2O2 group （P〈 0.05）. CONCLUSION： Androgen partially reversed H2O2-induced neuronal damage and protected neurons.