Objective: Perceptual alternations evoked by binocular rivalry (BR) reflect cortical dynamics strongly dependent on the excitatory–inhibitory balance, suggesting potential utility as a biomarker for epileptogenesis. Therefore, we investigated the characteristics of BR in patients with idiopathic generalized epilepsy (IGE) and potential associations with clinical variables.Methods: Sixty-two healthy controls (HCs) and 94 IGE patients completed BR task. Perceptual alternation rates were compared between HC and IGE groups as well as among the HC group and IGE patients stratified according to the presence or absence of interictal activity on the ambulatory electroencephalogram (EEG), termed the abnormal ambulatory EEG group  and normal ambulatory EEG group , respectively.Results: The IGE patients demonstrated a slower rate of BR perceptual alternation than HC subjects . The alternation rate also differed among the HC, AB-AEEG, and N-AEEG groups , and post hoc comparisons indicated a significantly slower alternation rate in the AB-AEEG group compared with the N-AEEG and HC groups . Stepwise linear regression revealed positive correlations between the BR alternation rate and both the ambulatory EEG status  and Montreal Cognitive Assessment score . Receiver operating characteristic curve analysis of the BR alternation rate distinguished AB-AEEG from N-AEEG subjects with 90.00% sensitivity and 76.90% specificity . Alternatively, Montreal Cognitive Assessment score did not accurately distinguish AB-AEEG from N-AEEG subjects and the area under the receiver operating characteristic curve combining the BR alternation rate and Montreal Cognitive Assessment score was not markedly larger than that of the BR alternation rate alone . K-fold cross-validation was used to evaluate the predictive performance of BR alternation rate, MoCA score, and the combination of both, which yielded average AUC values of 0.870, 0.584 and 0.847, average sensitivity values of 89.36, 92.73, and 91.28%, and average specificity values of 62.25, 13.42, and 61.78%, respectively. The number of interictal epileptiform discharges was significantly correlated with the alternation rate in IGE patients . A forward stepwise linear regression model identified the number of interictal epileptiform discharges  as an independent factor associated with BR alternation rate in these patients.Conclusion: These results suggest that interictal epileptiform discharges are associated with disruptions in perceptual awareness, and that the BR may be a useful auxiliary behavioral task to diagnosis and dynamically monitor IGE patients with interictal discharge. When incongruent images are presented to the two eyes, perceptual awareness spontaneously alternates every few seconds between one image and the other rather than forming a stable composite –3. This Binocular rivalry is a specific example of a more general phenomenon termed bistable perception. Since the stimulus remains constant, the spontaneous perceptual alternation characteristic of bistable perception reflects inherent dynamic operations in the brain and thus may offer a tool to distinguish normal from abnormal neural dynamics. Functional imaging techniques and electrophysiology have revealed that as the perceptual awareness of one image (precept) is suppressed, there is a concomitant reduction in response amplitude within brain regions processing that image, and that this reduction is reversed as the percept regains dominance , 5. BraiThe major underlying pathological mechanism in epilepsy is an imbalance between glutamatergic excitation and γ-aminobutyric acid (GABA)-ergic inhibition –24, whicEpileptiform discharges can be viewed as an eruption of excess excitatory drive  that is As the balance between γ-GABAergic inhibitory mechanisms and glutamatergic excitatory mechanisms underlies both IGE and BR, BR may be altered in IGE and thus serve as a useful diagnostic or prognostic marker. It is therefore of interest to explore the features of BR in IGE patients and the associations between BR changes and relevant clinical factors.Ninety-four patients with IGE  were enrolled from the Department of Neurology, First Affiliated Hospital of Anhui Medical University, Hefei, Anhui Province, China. Sixty-two healthy control (HC) subjects  were also enrolled, all students or social personnel. IGE and HC subjects were matched for age, gender, and years of education. All patients were clinically diagnosed with IGE based on seizure history, ambulatory EEG, and neuroimaging as defined by the Commission on Classification and Terminology of the International League Against Epilepsy . IGE is Exclusion criteria were (1) history of cerebral infarction, space-occupying intracranial lesions, infection, or brain trauma; (2) intracranial disease as evidenced by computed tomography or magnetic resonance imaging; (3) long-term use of medications other than antiseizure medicines (ASMs); (4) history of epilepsy correction surgery; (5) current psychiatric conditions such as moderately severe anxiety, depression, and history of drug abuse; or (6) a history of severe chronic physical illness.During the experiment, all IGE patients were in the interictal state, and all participants were naive to the purpose of the study. Finally, 94 patients with IGE were enrolled, 64 with abnormal ambulatory EEG (AB-AEEG group) as defined by the presence of interictal discharges and 30 with normal ambulatory EEG (N-AEEG group). The 64 patients with abnormal AEEG included 23 with no ASM history and 41 who had received ASMs. Twenty-three patients were new-onset cases with no ASM history, while all others had demonstrated good ASM adherence with no seizures for at least 3 months. Detailed information about ASMs is presented in . Some IGAll participants completed the Montreal Cognitive Assessment (MoCA) to confirm normal cognition as well as the Beck Depression Inventory (BDI) and Hamilton Anxiety Scale (HAMA) to assess current depression and anxiety symptoms. The BR test was performed within 2 h before 24 h ambulatory EEG monitoring. All assessments were conducted by the same trained assessor.2, sine wave 0.9, background luminance 30 cd/m2, and black frame was 1.6° × 1.6°.The BR task was conducted in a dimly lit closed room. Lenovo laptop  was used to display the visual stimuli. Stimulus presentation and trial timing were controlled by Psychtoolbox running in MATLAB . SubjectTo ensure participants understood the BR task, they received instructions and a practice session before starting the actual BR trials. Subjects were instructed to focus on the center of the screen and press a right response button when the green radial raster image was perceptually predominant and a left response button when the red concentric circular raster image was predominant. Following the practice session, three BR trials were conducted, each lasting 120 s, with an inter-trial interval of 3 min.In each test session, subjects also completed three pseudo-randomly programmed catch trials to ensure accurate performance of the binocular rivalry task. In the catch trials, two monocular stimuli were alternately presented separately, with each stimulus between 0.5 and 5 s, to roughly simulate the transformation in perception experienced during rivalry. The response was considered correct only when the subject pressed the correct button in response to the stimulus within the time window of 800 ms. To verify that the subjects understood and accurately completed the task, the average correct response rate of the catch trials was recorded.Perceptual rivalry rate was counted as the number of rivalry conversions (right to left button transitions and vice versa) divided by the total viewing time (in seconds). Participants were instructed to view all stimuli passively, without attempting to control their perceptions. The behavioral indicator of binocular rivalry was the alternation rate (in Hz).th-75th quartile], and nominal variables as numbers and percentages. Group means were compared by independent samples t-tests or one-way ANOVA in the case of two or more than two groups, respectively. The chi-square test was used to compare ratios among groups. The non-parametric Mann-Whitney U test was used to compare the BDI score and HAMA score between two groups, and non-parametric test  was used to compare the BDI score and HAMA score among the three groups, expressed as M . Pearson and Spearman correlation analyses were used to examine within-subject reliability for the perceptual alternation rate and the associations between alternation rate and various demographic and clinical characteristics. In the correlation analysis, Pearson or Spearman correlation analysis was selected according to whether the two related variables followed a normal distribution. Multivariable linear regression analysis was used to explore the effects of demographic and clinical characteristics  on the alternation rate. The ambulatory EEG-related variables were the number of IEDs, total duration of IEDs, and presence or absence of IEDs. The medication-related variable was the ASM used .Numeric variables are presented as mean ± standard deviation or median  is an independent factor influencing BR alternation rate in patients with IEDs . These results indicate that BR alternation rate can be used to distinguish IGE cases complicated with IEDs. Similarly, Pitts et al. , which confirmed that IEDs on the ambulatory EEG is an independent predictor of slower BR alternation rate. ROC analysis showed that a slow BR alternation rate predicted the presence of an abnormal ambulatory EEG with an AUC value of 0.881 and 90.00% sensitivity and 76.90% specificity. We also evaluated the diagnostic efficiency of the MoCA score using ROC, which yielded an AUC value of 0.566, and s et al.  demonstrTo examine the relationships between BR alternation rate and both the number and total duration of IEDs, we conducted Pearson correlation analyses and found that the number of IEDs was significantly associated with alternation rate in IGE patients, while there was no correlation between alternation rate and total duration of IEDs. Moreover, a forward stepwise linear regression model adjusting for potential confounding factors  confirmed that the number of IEDs was an independent factor associated with the BR rate in patients with IEDs.There are several possible mechanisms for the slower rate of binocular rivalry in IGE patients with higher IED frequency. First, interictal discharges may transiently disrupt perceptual dynamics, thus slowing the rate of BR. Tong et al.  proposedSecond, IEDs may cause an acute decline in cognitive function –53, whicThird, the ASMs taken by these patients may affect BR rate indirectly by disrupting cognitive function. ASMs are known to slow cognitive function, including valproic acid , benzodiFourth, IEDs may disrupt excitatory circuits, inhibitory circuits, or both required for perceptual switching. Epileptic seizures arise from an imbalance between glutamatergic excitation and GABAergic inhibition –24. BinoAnxiety and depression are common psychiatric comorbidities in epilepsy, with incidence rates far exceeding those of the general population –62. AccoPrevious studies have suggested that BR alternation rate may be influenced by participant mood. For instance, Bajwa et al.  reportedWhile this study clearly demonstrates that IEDs interfere with BR alternation and that the BR alternation rate can predict the presence of IEDs, there are several limitations. First, the IED frequency detected by a single 24-h AEEG trial may not be truly representative as IED rates can fluctuate substantially over weeks or months . HoweverWe demonstrate that an abnormally slow BR alternation rate is strongly associated with the presence of interictal discharges among IGE patients. The number of IEDs is an independent factor influencing the BR rate. These results suggest that interictal activity disrupts perceptual awareness, and that the BR may be a valuable auxiliary behavioral task to diagnose and dynamically monitor IGE patients with IEDs.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Anhui Medical University Ethics Committee. Written informed consent to participate in this study was provided by the participant's legal guardian/next of kin.JW, YJ, XY, and KW: conceived and designed the experiments. JW, WD, QW, and XY: performed the experiments. JW, WD, and YJ: analyzed the date and wrote the initial manuscript. JW and WD: finished the final manuscript with guidance from YJ and KW. All authors contributed to the article and approved the submitted version.This study was supported by the National Natural Science Foundation of China (81601187) and the Natural Science Foundation of Anhui Province, China (KJ2019A0289).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Lactobacillus and Lacticaseibacillus strains and the chemical, physical and organoleptic properties of dairy ice cream during storage. The obtained results contribute to a more practical application of different probiotic strains for the fermentation of ice cream mixes and the possibility of using apple fiber in ice cream production. Moreover, the study’s findings showed that symbiotic ice cream with acceptable physicochemical and organoleptic characteristics might be produced from sheep milk of the Olkuska breed.Ice cream may be used as a carrier to deliver probiotics and prebiotics. In this study, we decided to investigate the possibility of using sheep milk from the Olkuska breed for ice cream manufacture and evaluate the viability of −1 than in their equivalents with inulin only. These differences persisted during the storage of the ice cream. After fermentation of the ice mixes, the bacterial cell count ranged from 10.62 log cfu g−1 to 12.25 log cfu g−1. The freezing process reduced the population of probiotic bacteria cells in ice cream with inulin from 0.8 log cfu g−1 in ice cream with Lactobacillus acidophilus, 1.0 log cfu g−1 in ice cream with Lacticaseibacillus paracasei and 1.1 log cfu g−1 in ice cream with Lacticaseibacilluscasei. Freezing the varieties with apple fiber also resulted in a reduction of viable bacterial cells from 0.8 log cfu g−1 in ice cream with L. paracasei and Lb. acidophilus to 1 log cfu g−1 in ice cream with L. casei, compared to the results after fermentation. The highest percentage overrun was determined in ice cream with L. paracasei and Lb. acidophilus. Ice cream with L. casei was characterized by significantly lower overrun on the 7th and 21st days of storage. Although L. paracasei ice cream had the highest overrun, it did not cause a significant reduction in the probiotic population during storage. After seven days of storage, the first drop differed significantly depending on the type of bacteria used for fermentation of the mixture and the addition of apple fiber. L. casei ice cream had a longer first drop time than L. paracasei and Lb. acidophilus ice cream. Partial replacement of inulin with apple fiber resulted in a significant darkening of the color of ice cream mixes. Depending on the type of bacteria used for fermentation, the addition of apple fiber decreased the value of the L* parameter. Ice cream mixes and ice cream with inulin and apple fiber were characterized by a high proportion of yellow. Partial replacement of inulin with apple fiber reduced the hardness of ice cream compared to inulin-only ice cream. Moreover, the panelists found that ice cream with inulin was characterized by a sweeter taste than ice cream with apple fiber. Moreover, the addition of apple fiber favorably increased the flavor and aroma perception of the mango-passion fruit. Therefore, the milk of Olkuska sheep could be successfully used for the production of symbiotic dairy ice cream.The aim of this study was to determine the possibility of using Olkuska sheep milk for the production of ice cream with probiotics and prebiotics. The study examined the effect of the storage and type of bacteria used for the fermentation of ice cream mixes and partial replacement of inulin with apple fiber on the physicochemical properties, viability of probiotic cultures and organoleptic properties of sheep’s milk ice cream stored at −22 °C for 21 days. The addition of apple fiber reduced the pH value of ice cream mixes before fermentation. In ice cream mixes and ice cream with apple fiber, the lactic acid content was higher by 0.1–0.2 g L The parameters of sheep milk are influenced by various factors including genetic, physiological and environmental. Polish sheep breeds, such as Olkuska, are well adapted to local environmental conditions. This breed is characterized by resistance to diseases and demanding environmental conditions, moreover good milk yield .Sheep milk is characterized by a higher content of total solids compared to cow and goat milk. Moreover, sheep milk is characterized by a high content of micro- and macroelements, vitamins, protein and fat .1, αS2, β and κ-casein represent 6.7%, 22.8%, 61.6% and 8.9%, respectively. The remaining 15% of milk proteins includes major whey proteins β-lactoglobulin and α-lactalbumin as well as other protein constituents [The protein content of raw milk differs among the species and the sheep raw milk has the higher protein content (5.5%). According to Balthazar et al.  the totatituents .Milk proteins have a wide range of functional properties such as emulsifying, thickening, gelling and foaming. Milk proteins promote the formation and stabilization of oil droplets in emulsions or air bubbles in foams in food formulations . MoreoveRaw milk from different species remains an element of human nutrition. Therefore, the free and total amino acid profile of milk from different species plays a crucial role for both milk producers and processors as well as consumers to achieve innovative new product design, complexity, flavor and functionality . The chaSheep milk could be considered a substitute for cow’s milk for allergy sufferers. Specific antibodies in milk-allergic individuals (IgE) poorly recognize the αS1-casein, αS2-casein and β-casein protein fractions from goat and sheep milk, which is not observed with cow milk ,7. NeverSheep’s milk ice cream can be an excellent carrier of bioactive compounds, including probiotic cultures and prebiotic ingredients. The high protein level and fat in sheep’s milk allows for the production of ice cream with a higher density, which translates into better protection of probiotic cells against oxygen access during ice cream storage and during passage through the gastrointestinal tract .6–109 cfu g−1 is required for a therapeutic effect [Lactobacillus and Bifidobacterium are the most commonly used probiotic strains in food production [Probiotics added to food benefits by restoring the intestinal microflora balance ,11,12 wic effect ,14,15. Pc effect ,17,18. Loduction ,20. Few oduction ,22. Inuloduction  due to toduction ,25 in adoduction .Recent studies indicate that prebiotic compounds may also increase the bioavailability of milk protein . SimultaSheep milk ice cream with the addition of prebiotics such as inulin and apple fiber, apart from the health benefits and properties stimulating the growth of probiotic bacteria, is an innovative product with functional properties. There are few publications on the production of ice cream from sheep’s milk with probiotic strains and prebiotics in the scientific literature. Therefore, the study aimed to determine the possibility of using Olkuska sheep milk for the production of ice cream with probiotics and prebiotics.Raw morning and cooled (4 °C) sheep’s milk for the production of ice cream was purchased from the farm “Owcza Zagroda”  in June 2021. A flock of 35 Olkuska breed sheep were kept on the farm. Green fodder and hay with the addition of cereals  from the breeder’s farm were used for feeding animals. Milking was conducted by hand. Before ice cream manufacture, milk was filtrated to remove dirt and foreign particles.The chemical composition of milk was determined in the chemical composition analyzer of milk and dairy products Bentley B-150 .Lacticaseibacillus paracassei L-26, Lacticaseibacillus casei 431 and Lactobacillus acidophilus LA-5.The following materials were used to prepare the ice cream mixture: inulin ; apple fiber  composed of 100% micronized apple fiber; white sugar ; mango-passion fruit flavor essence  with the composition: natural and identical to raw mango and passion fruit flavors, citric acid E330 and mango juice. Probiotic bacteria  were used to produce the inoculum: Sheep’s milk (84.9%), sugar (11%) and flavor essence (0.1%) were mixed and then divided into two parts. Inulin (4%) was added to the first part of the blend and divided into three groups: CP, CC and CA. Inulin (2.5%) and apple fiber (1.5%) were added to the second part and analogously divided into three groups: CPF, CCF and CAF. The milk with additives was mixed and homogenized with a homogenizer  at 60 °C with a pressure of 20 MPa, and then pasteurized at 85 °C, 1 min. After heat treatment, it was cooled to 37 °C.Lacticaseibacillus paracasei L-26, the CC and CCF groups were inoculated with Lacticaseibacillus casei 431. The CA and CAF groups were inoculated with Lactobacillus acidophilus LA-5. Each starter probiotic culture was previously inoculated in sheep’s milk at 40 °C for five hours. After five hours, 5% (w/w) of inoculum containing 9 log CFU of g−1 bacteria was added to the ice mixes. The fermentation process of the ice cream mixes was carried out in an incubator  at 37 °C for 10 h. After cooling to 5 °C, it was conditioned at the specified temperature for 12 h in an incubator and then froze in a DeLux 48816 freezer  for 40–50 min with a freezing temperature down to −22 °C. The prepared ice cream was packaged in 100 mL plastic cups and stored at −22 °C for twenty-one days.The CP and CPF groups were inoculated with a monoculture The experiment was repeated on three occasions.®Solids Pro-ISM  with an integrated temperature sensor. The content of lactic acid was determined (g of lactic acid L−1) by titration of samples of ice cream mixes and dissolved ice cream with 0.1 N NaOH  according to the method of Jemaa et al. [The chemical composition of the ice cream mixes was determined using a Bentley B-150 Milk and Milk Product Analyzer . The determination of the pH value was performed with a FiveEasy digital pH meter  with an electrode InLaba et al. . The mela et al. . Ice crea et al. . Five saLacticaseibacillus paracasei L-26, Lacticaseibacillus casei 431, Lactobacillus acidophilus LA-5) was determined in the ice mix, then immediately after freezing and on the 7th and 21st days of storage at −22 °C. 10 g of each sample was diluted in 90 mL of sterile peptone water solution (0.1%) . Serial dilutions from 1 log CFU g−1 to 8 log CFU g−1 were made. The inoculation was performed by the plate-deep method using MRS agar  and incubated anaerobically in a vacuum desiccator at 37 °C for 72 h using the GENbox anaer . The cultured probiotic colonies were counted with a colony counter . The result was expressed as log CFU g−1. Five samples were tested for each ice cream variant, and it was repeated was repeated in triplicate.The number of probiotic strains  using the CIElab system. The following parameters were measured: L*—brightness of ice cream , a*  and b* , C—color saturation and purity and h° as a hue of color. Before analysis, the device was calibrated on a white and black reference standard ,35. FiveOrganoleptic assessment for six groups of ice cream was carried out by a trained team of 15. The samples of ice cream encoded (with a random three-digit code) were analyzed on a nine-point linear scale with marginal, structured markings. The left end of the scale indicated the following features: not very characteristic (appearance); soft (hardness); sandy (smooth); immediate (spreadability); dark (color); hardly perceptible  . The rigp < 0.05).The mean and standard deviation were calculated using Statistica v. 13.1 . One, two and three-way ANOVA was performed. The significance of differences between the mean values was verified with the Turkey test  and carbohydrates (18.39%) in the control milk ice cream was demonstrated by Ismail et al. . Moreove% and carLactobacillus and Lacticaseibacillus growth is 5.5–6.0 [ 5.5–6.0 . The add 5.5–6.0 ,44.L. paracasei. Most likely, it results from the specificity of L. paracasei because this strain, unlike L. casei, ferments lactose to produce L (+) lactic acid, and about 50% of cells also can ferment inulin [As in our study, in the study by Favaro-Trindade et al. , the addt inulin .L. casei and CA from Lb. acidophilus  and interactions between bacterial type and storage time. However, the combined effect of these three factors on the lactic acid content turned out to be insignificant.The highest amount of lactic acid was found in the mixture of CPF with apple fiber fermented by Similar results were obtained by Ismail et al.  in ice cLb. acidophilus and Bifidobacterium lactis), and their results suggest that the addition of inulin stimulates growth Lb. acidophilus and Bifidobacterium lactis, which improved their survival. Therefore, partial replacement of inulin with apple fiber in our research might also contribute to the differentiation of growth and survival of probiotic bacteria cells.The pH value and acidity of probiotic products can significantly affect the survival of probiotic bacteria cells in fermented ice cream. The addition of growth promoters and prebiotics such as inulin has been shown in many studies to significantly improve the viability of probiotic organisms . Akin etThe number of bacterial cells in the mixes and ice cream depending on the type of bacteria, storage time and the addition of apple fiber is presented in −1 to 12.25 log cfu g−1. The addition of apple fiber did not significantly affect the growth of L. paracasei in the CPF ice cream mix compared to CP. In turn, this addition contributed to an increase in the number of Lb. acidophilus cells by approximately one log cfu g−1 in CAF mixtures with apple fiber compared to the CAF mixture with inulin only. It should be mentioned that Lb. acidophilus, especially, has a high cytoplasmic buffering capacity (pH 3.72–7.74), which allows it to resist cytoplasmic pH changes and obtain stability under acidic conditions [After fermentation of the ice cream mixes, bacteria cells ranged from 10.62 log cfu gnditions ,54. Acconditions  and Haynnditions , and Taknditions , this stL. casei with inulin. However, the addition of apple fiber resulted in a reduction in the number of cells by about 1 log CFU g−1 in the CCF mixture. These studies indicate that a vital problem is selecting probiotic bacteria for the fermentation of sheep’s milk mixtures because partial replacement of inulin with apple fiber changed the fermentation conditions  and differentiated the number of probiotic cells after fermentation.In this study, the highest number of bacterial cells was determined in the CC-fermented mixture with The resistance of probiotics to freezing damage varies among probiotic strains. Microorganisms that show a better ability to survive under freezing conditions can dehydrate without rupturing the cytoplasmic membranes. Such cells can reduce the number and growth of intracellular ice crystals and thus reduce the heat transfer of their cells; both factors minimize microbial cell damage .−1 in CA ice cream, 1.0 log cfu g−1 in CP ice cream and 1.1 log cfu g-1 in CC ice cream with inulin. This means that the survival rate of bacteria when freezing sheep’s milk ice cream mixes depends on the probiotic strain. Lb. acidophilus showed the best survival and the lowest reduction of the population after freezing. However, it should be added that this strain multiplied the least when fermenting the CA mix, which resulted in the lowest number of Lb. acidophilus cells in CA after fermentation. This is essential information for ice cream producers because fermentation should be extended in industrial production or the starter dose of bacteria increased to obtain a more significant number of Lb. acidophilus cells. These treatments are needed to obtain a comparable number of bacterial cells to those obtained in Lb mixtures: L. paracasei and L. casei.In our research, the freezing process reduced the population of probiotic bacteria cells in all ice cream groups. After freezing, significant cell count reductions were observed by 0.8 log cfu g−1 in CPF and CAF to 1 log cfu g−1 in CCF, compared to the results after fermentation. This means that the reduction in the probiotic population in apple fiber ice cream due to freezing is similar to their inulin counterparts in CCF and CAF ice cream and less only in CPF ice cream. Additionally to the damage caused by probiotic cell freezing, the inclusion of oxygen in the mixture (aeration process) could result in an additional reduction in the number of viable probiotic bacteria cells since the oxygen content, and the redox potential  is essential. Most of the probiotic Lactobacillus and Lacticaseibacillus strains are organisms derived from the intestines with microaerophilic or anaerobic metabolism. Therefore, molecular oxygen and high redox potential values are critical factors for these bacteria [Freezing mixtures with the addition of apple fiber also resulted in a reduction of viable bacterial cells from 0.8 log CFU gbacteria ,59,60.−1 compared to the number of bacterial cells after freezing. The most intense reduction in the log cycle (1.2 log cfu g−1) was found in CC and CCF ice cream with L. casei compared to the number of cells determined after freezing. The highest survival after seven days of ice cream storage was characterized by L. paracasei, where the reduction in the number of bacterial cells was only 0.8 log cfu g−1 in CP ice cream and 0.9 log cfu g−1 in CPF ice cream with apple fiber, compared to the number of cells determined after freezing.After seven days of freezing storage at −22 °C, the viability of the probiotics in all ice cream groups decreased, and the population was reduced by another 0.8–1.2 log cfu g−1 compared to the 7th day of storage. In contrast, probiotic ice cream stored at −20 °C for 90 days, tested by Turgut and Cakmakci [Lb. acidophilus with 0.38 log CFU g−1 at this time. Furthermore, in the study by Salem et al. [−1, respectively, for Lb. acidophilus, B. bifidum, Lb. reuteri, Lb. gasseri and Lb. rhamnosus within 12 weeks of frozen storage (−26 °C).In our study, extending the storage time of ice cream from 7 to 21 days did not significantly affect the number of bacterial cells in all ice cream groups. After seven days and 21 days of ice cream storage, the lowest number of bacterial cells was determined in CA and CAF ice cream. After 21 days, the CAF ice cream showed a population reduction of only 0.1 log CFU gCakmakci , showed m et al. , the numL. paracasei was characterized by the best survival rate, which predisposes it to be used to produce probiotic ice cream from sheep’s milk. The conducted 3-factor ANOVA confirms that the survival and the number of probiotic bacteria cells are significantly influenced by all tested factors  and their interactions.The highest number of bacterial cells after 21 days of ice cream storage was determined in CP and CPF ice cream . L. para−1, which means that the ice cream can be classified as a probiotic food [−1, the daily therapeutic dose of probiotics is about 8–9 log cfu g−1 to compensate for losses during the digestive process. In our research, all ice cream groups meet these recommendations for 21 days of storage.After 21 days of storing ice cream, the number of bacterial cells in each group of ice cream exceeded 8 log cfu gtic food ,64. Accotic food , and at L. paracasei and Lb. acidophilus. L. casei ice cream was characterized by significantly lower overrun on the 7th and 21st days of storage. Although the L. paracasei ice cream had the greatest overrun, it did not cause a significant reduction in the probiotic population during storage.Detailed analysis of the influence of the bacteria used for fermentation on the aeration of ice cream  shows thThe literature reports that grape, peach and apple pomace is valuable source of pectin, especially pectin with many methoxylated groups, which have favorable gelling properties . This tyw/w) was 46.5% and 42.5%, respectively [According to Feizi et al. , aeratioectively .According to Soukoulis et al. , prebiotL. casei had a longer first drop time than that of L. paracasei and Lb. acidophilus. Extending the storage time from 7 to 21 days significantly reduced the first drop time in all ice cream groups by 57 s in CP, 93 s in CPF, 131 s in CC, 28 s in CCF and 68 s in CA 70 s in CAF. Furthermore, in a study by Bahram-Parvar et al. [It should be added that the time of the first drop after seven days of storage in the freezer differed significantly depending on the type of bacteria used to ferment the mixture and the addition of apple fiber. The ice cream fermented with r et al. , the melr et al.  found thThe conducted research shows that the addition of apple fiber significantly shortens the total melting time. It is most likely related to the properties of apple fiber resulting from a lower water retention capacity than inulin. Inulin is a hygroscopic substance; it reacts with water molecules limiting their free movement and stabilizing the ice mixture, reducing meltability . AccordiSeveral researchers have reported conflicting results about the effects of inulin on the melting properties of ice cream. Akbari et al.  and AkalHowever, the presence of pectin in apple fiber may also contribute to the cryopreservation of ice cream by controlling the mobility of water molecules in the non-frozen aqueous phase due to its thermodynamic incompatibility with the current separation phase proteins . Goh et L. casei ice cream, where the melting time was 1206 s shorter in the CCF ice cream with apple fiber compared to the CCF ice cream with inulin. On the other hand, CP and CPF ice cream fermented with L. paracasei had a significantly lower total melting time. In these ice cream, the addition of apple fiber reduced the total melting time by 488 s compared to CP ice cream with inulin only. Extending the shelf life from 7 to 21 days significantly reduced the total melting time only in L. casei-fermented ice cream (CC and CCF) and Lb. acidophilus (CA and CAF). However, in ice cream with L. paracasei, the storage time did not significantly affect the total melting time.In our study, the difference in total melting time was most significant in the The results describing the color parameters of ice cream and ice cream mixes are presented in L. paracasei, while the slightly darker CC mixes fermented with L. casei and CA with Lb. acidophilus. Partial replacement of inulin with 1.5% of apple fiber resulted in a significant darkening of the color of ice cream mixes. Depending on the type of bacterial mixtures used for fermentation, the addition of apple fiber increased the value of the L* parameter from 13.2 to 16.6.The brightest was the CP mixes fermented with After seven days of freezing storage in CPF and CAF ice cream, the L* brightness of the ice cream increased compared to the blends. The remaining ice cream  also tended to increase the brightness of the ice cream, but the differences were not significant. This color change in ice cream samples was due to ice crystals and air bubbles inside the ice cream formed during whipping during freezing .According to Nozière et al. , inulin The a*  parameter in CP, CC and CA ice cream mixes and ice cream with inulin only took negative values, while CPF, CCF and CAF mixes and ice cream with apple fiber-positive values. This means that in ice cream mixes and ice cream with inulin only , a greater proportion of green color was observed, while in mixes and ice cream with apple fiber , the red color was dominant. After the 7th day of storage, the proportion of green color in ice cream with inulin  increased compared to the a* parameter determined in ice cream mixes . Moreover, in the ice cream with apple fiber, an intensification of the red color was observed compared to the a* value determined in the mixes .Lb. acidophilus. On the other hand, the most yellow CP mixes and ice cream fermented by L. paracasei. This effect is confirmed by the studies of Acevedo-Martinez et al. [Extending the storage time of ice cream with apple fiber from 7 to 21 days increased the proportion of red color. The analyzed ice cream mixes and ice cream with inulin and apple fiber were characterized by a high ratio of yellow (+ b*). The minor balance of yellow was found in the CA ice cream mix fermented by z et al. , which iA 1.5% addition of apple fiber resulted in the intensification of the yellow color in CPF, CCF and CAF mixtures. Moreover, after 7 and 21 days of freezing storage, the yellow color was found. These results are confirmed by the analysis of variance, which shows that the type of bacteria, storage time and addition of apple fiber and the interactions of these factors significantly influenced the intensity of yellow color. Furthermore, when analyzing the results of the C color saturation and the h° hue of ice cream mixes and ice cream, a significant effect of apple fiber was found.Apple fiber is characterized by a beige color, which is formed during the drying of apple pomace. During drying, the enzyme polyphenol oxidase causes oxidation of polyphenols resulting in browning of the powder. The same effect is obtained from the non-enzymatic browning reaction, i.e., the reaction of sugars with amino acids, occurring at high speed at an elevated temperature and with a water content of about 30% ,85. The The results of the organoleptic evaluation of ice cream on days 7 and 21 of freezer storage are presented in The most characteristic appearance of milk ice cream was that of ice cream with CA and CP inulin, both after 7 and 21 days of freezing storage. This is probably because the use of apple fiber caused a significant reduction in L* brightness, and the fiber ice cream was darker and redder than inulin only ice cream. The ANOVA, also, confirms that the addition of fiber had a significant effect on the appearance of sheep’s milk ice cream.In the Akalin et al.  study, iIce cream hardness depends on the aeration and size of the ice crystals. The hardness is the greater the larger the ice crystals, while with the increase in overrun, the hardness decreases ,88. MoreAccording to Franck , the addThe addition of apple fiber in our study reduced the hardness of ice cream compared to ice cream with inulin only, but these differences were not significant. According to Bahram-Parvar et al. , hardnesLb. acidophilus on both dates. This is most likely due to the low lactic acid content of 0.50–0.51 g L−1 in CA ice cream compared to other ice cream groups. Moreover, the panelists, analyzing the taste of sheep’s milk ice cream (According to Abdullah et al.  and Soukce cream , found tL. paracasei, L. casei, Lb. acidophilus, the addition of inulin and apple fiber did not introduce any foreign taste and smell to the ice cream. Furthermore, according to Salem et al. [As in the studies by Turgut and Cakmaci  also in m et al. , no foreL. paracasei and Lb. acidophilus. The best survival rate was found for L. paracasei. Results demonstrated the effects of using apple fiber and various types of bacteria in milk ice cream, mainly probiotics that have not yet been studied and used in sheep’s milk ice cream. Partial replacement of inulin with apple fiber is, also, a way to reduce production costs.Symbiotic ice cream with acceptable physicochemical and organoleptic characteristics may be produced from sheep milk of the Olkuska breed. Moreover, partial replacement of inulin with apple fiber contributed to increased overrun in the ice cream and to a shorter run-off time of the first drop and the total melting time. The addition of apple fiber stimulated the growth of


Conventional evaluation of the stability of bone screws focuses on pullout strength, while neglecting lateral migration resistance. We measured pullout strength and lateral migration resistance of bone screws and determined how these characteristics relate to screw stability of locking plate (LP) and dynamic compression plate (DCP) fixation. Pullout strength and lateral migration resistance of individual bone screws with buttress, square, and triangular thread designs were evaluated in polyurethane foam blocks. The screw types with superior performance in each of these characteristics were selected. LP and DCP fixations were constructed using the selected screws and tested under cyclic craniocaudal and torsional loadings. Subsequently, the association between individual screws’ biomechanical characteristics and fixation stability when applied to plates was established. Screws with triangular threads had superior pullout strength, while screws with square threads demonstrated the highest lateral migration resistance; they were selected for LP and DCP fixations. LPs with square-threaded screws required a larger force and more cycles to trigger the same amount of displacement under both craniocaudal and torsional loadings. Screws with triangular and square threads showed no difference in DCP fixation stability under craniocaudal loading. However, under torsional loading, DCP fixation with triangular-threaded screws demonstrated superior fixation stability. Lateral migration resistance is the primary contributor to locking screw fixation stability when applied to an LP in resisting both craniocaudal and torsional loading. For compression screws applied to a DCP, lateral migration resistance and pullout strength work together to resist craniocaudal loading, while pullout strength is the primary contributor to the ability to resist torsional loading. Plates and screws can be secured to fractured long bones of arms and legs, thus providing a stable environment for bone healing. Locking and compression screws are the two most commonly used types of screws in the treatment of long bone fractures.The use of bone screws to stabilize orthopedic plates is a widely applied technique in fracture treatment5. Hip fracture screw failure rates vary between 3 and 5%3, while those for proximal humeral fractures vary between 15 and 40%5. It has been suggested that at least one million pedicle screw failures occur annually worldwide due to loosening and/or migration; and while some failures do not have significant clinical consequences, some may need immediate and costly revision surgery6.Despite favorable clinical results, the number of clinically reported screw failures is high9; however, previous studies have only evaluated holding power in pure axial pullout experiments15, that represents the exclusive standardized method to test the anchorage strength of medical bone screws16. In research of the pedicle screw, pullout strength has been found to be an unsuitable predictor of screw loosening, because it does not simulate physiological loading conditions18. Cyclic fatigue loading tests, also referred to as toggle tests, are considered more suitable for pedicle screw testing20. The loading of the human arms and/or legs during daily activity is highly complex, and plate/screw constructs experience both vertical  and horizontal (screw pullout) forces. Therefore, using a pullout test alone to evaluate bone screw stability cannot realistically simulate the in vivo loading conditions of plate/screw constructs to predict its real fixation strength. Our previous research on screw loosening mechanisms revealed that high radial stress around the screw is the primary reason for screw loosening21. Lateral migration resistance, the ability to resist lateral movements of the screw during physiological loading of the bone-implant construct, has great potential to become an important performance metric when evaluating the fixation stability of a screw design. Therefore, lateral migration resistance, another important characteristic of bone screws, should also be considered when evaluating their fixation stability for long bone fracture plate fixation.To overcome some of these difficulties, cement augmentation and improved screw design have been used to increase the holding power of bone screws22. Which characteristic of bone screws  primarily determines the fixation stability of each screw type remains unclear, and this knowledge would be useful in guiding us to apply appropriate tests to the development of new screw designs and the validation of existing screws. Therefore, the aim of this study was to clarify the association between the characteristics of individual bone screws and the fixation stability of plate/screw constructs. We hypothesized that lateral migration resistance of bone screws has a higher association with the fixation stability of plate/screw constructs than pullout strength.Locking screws and compression screws are under different loading patterns during physiological loading because of different plate/screw interfaces (locking and non-locking)3,  adhering to ASTM F-1839–08, were used23. This density was chosen because it has similar biomechanical strength to normal cancellous bone, which has been validated by screw pullout testing in previous studies26.Locking screws and compression screws with three different kinds of thread designs  were manufactured from 316 LVM stainless steel. All screws were self-tapping screws with a thread pitch of 2.0 mm, major and minor diameters of 4.5 mm and 3.2 mm, respectively, a screw shaft length of 38 mm, and had the same cutting flute design. The 4.5 mm screws were used to be compatible with the standard instrumentation for the 4.5 mm AO cortical screw by relying on identical 3.2 mm diameter drill bits. Locking plates (LP) and dynamic compression plates (DCP) were specially designed for the above locking and compression screws and were manufactured from 316 LVM stainless steel . In the DCP fixation model, all the compression screws were tightened with a maximum torque of 1 Nm (50% of the stripping torque)30 by the same surgeon manually with a calibrated torque wrench .For the screw stability test when applied to a plate, polyurethane foam blocks were cut into rectangular blocks measuring 30 × 30 × H60 mm to represent the proximal fragment of a long bone fracture. All specimens were pre-drilled in two planned locations with a 3.2 mm drill bit by a drill press. One end of the plate was secured to the rectangular foam block using two of the same type of screws to build the fracture plate fixation construction, and the other end of the plate that did not attach to the bone was fixed vertically on the load cell (0–1000 N) . Since different thread types alter the contact surface area with the bone, and thus the pullout strength and lateral migration resistance of the screw34, this study used identical screws that only differed in thread type . The result of pullout test in this study was consistent with the finding of a biomechanical pullout test performed by Kim that triangle shape thread showed higher pullout strength than buttress thread and square thread pedicle screws in synthetic cancellous bone34.Evaluation of the fixation stability of bone screws often focuses on simple axial pullout, which rarely simulates physiological loading conditions, while lateral migration resistance, another important biomechanical characteristic for bone screws, has been neglected20; however, they did not consider lateral migration resistance as an influencing factor of screw fixation stability as we did in our study.The experimental design in this study enabled us to clarify the association between a screw’s biomechanical characteristics and its stability during plate fixation to determine which screw characteristic is the primary contributor to fixation stability when applied to a plate. In the LP/locking screw constructs, screws with superior lateral migration resistance had better fixation stability under both cyclic craniocaudal loading and torsional loading; however, screws with superior axial pullout strength did not have better fixation stability under either type of loading. This suggests that fixation stability of the locking screw is directly related to its lateral migration resistance, while axial pullout strength is not involved in resisting cyclic craniocaudal or torsional loading. This is consistent with the finding of Kueny et al. in a pedicle screw biomechanical study, in which the difference in axial pullout strength among different pedicle screws was not reflected in the more physiologically relevant fatigue testingIn the DCP/compression screw constructs, screws with superior lateral migration resistance but inferior axial pullout strength had similar fixation stability under cyclic craniocaudal loading to screws with superior axial pullout strength but inferior lateral migration resistance. This indicates that, in compression screws, both lateral migration resistance and axial pullout strength are working together to resist cyclic craniocaudal loading. Because compression screws are not locked with the screw hole of the plates, when craniocaudal loading is applied to the bone, the plates oppositely push the screws’ head upward so that the whole screw has a trend of unilateral migration and axial pullout. Screws with superior axial pullout strength, but not those with superior lateral migration resistance, had better fixation stability under torsional loading. This suggests that axial pullout strength is the primary contributor to fixation stability of the compression screw, while lateral migration resistance is not involved in resisting torsional loading.Axial pullout tests have been commonly used as the standard test to predict the fixation strength of medical bone screws. According to the results of this study, screws with good axial pullout resistance do not necessarily have better fixation stability. Axial pullout tests can only be used to predict the axial pullout strength of a screw, not its fixation stability, which suggests that an axial pullout test alone is insufficient to evaluate the fixation stability of a screw. The lateral migration test described in this study is simple and repeatable and should be an important factor in bone screw evaluation. This study underscores the importance of using different tests to evaluate different screw types under different loading conditions. When evaluating the craniocaudal and torsional loading stability of a locking screw, a lateral migration test should be used. When evaluating the craniocaudal loading of a compression screw, both a lateral migration and axial pullout test should be used. When evaluating the torsional loading stability of a compression screw, an axial pullout test should be used.28. For cyclic craniocaudal loading, we started with 100–200 N and increased the load by 50 N every 100 cycles to simulate the weight bearing of walking activity experienced by the lower extremities. A similar loading pattern was used in a study of pedicle screw fixation stability35. For torsional loading, we started with ± 1 Nm and increased by 0.5 Nm every 100 cycles to simulate the torque loading of a long bone during daily activity. The 1 Nm starting value was chosen because it is 10% of the maximum torque that the 30 × 30 × H60 mm polyurethane foam blocks could withstand, as tested in a pilot study. Clinically, screw loosening is defined as a radiolucency more than 1 mm surrounding the screw36. However, most clinical cases show no significant clinical consequences to a 1 mm radiolucency6. In this study, 5 mm and 10 degree endpoints were selected to evaluate the fixation stability of the screws at significant failure. The load and number of cycles required to achieve 1, 2, 3, 4, and 5 mm displacement and the torque and number of cycles required to achieve 2, 4, 6, 8 and 10 degree torsion were recorded to quantify the fixation stability of the screws.This study attempted to elucidate the fixation stability of different screw types under two specific loads on long bones. Cyclic craniocaudal loading and torsional loading were used to simulate the normal physiological loads of humans during daily activity26. The polyurethane foam is microstructurally similar to human bone and exhibits similar mechanical properties while avoiding inconsistencies due to interindividual variations in human bones. Second, this study used cyclic craniocaudal loading and torsional loading to simulate physiological loading, which cannot fully replicate the highly complex loading conditions of human arms and legs during daily activity. However, the loadings used in this study are already able to simulate most daily activity loading. Third, simulated bone with normal density was tested in this biomechanical study as a representative. Bone densities representing osteopenia and osteoporosis will be tested in a further study to strengthen the conclusion of this study. Fourth, the use of two-screws-per-segment fixation instead of using three screws and the use of polyurethane specimens without a cortical shell were unable to represent the real clinical environment. However, the choice of such experimental model allowed us to recreate under in vitro conditions the worst possible clinical situation, in which the bone could not hold the screws firmly, to better evaluate the fixation stability of different screw designs. Finally, because of the unique design of this study, the accompanying underpowered nature of the study should be considered. The conclusions of this study are drawn considering these limitations.There are limitations to this study. First, biomechanical studies with polyurethane foam blocks cannot fully replicate in vivo conditions and therefore, such limitations must be considered when attempting to draw conclusions. While synthetic bone models are a consistent medium for mechanical testing, they do not perfectly simulate normal human bone. What is more, the polyurethane foam models used in this study have previously been accepted as a substitute for cadaveric bone in biomechanical testingThis biomechanical study demonstrates that lateral migration resistance of a locking screw is the primary contributor to fixation stability in resisting both craniocaudal and torsional loading. For compression screws, lateral migration resistance and axial pullout strength work together to resist cyclic craniocaudal loading, while the ability to resist torsional loading is mainly provided by axial pullout strength.


Compound 20p dose-dependently induced small heterodimer partner (SHP) mRNA and decreased cytochrome P450 7A1 (CYP7A1) in the liver at 10 and 30 mg/kg, respectively, which were used as FXR agonist markers. Compound 20p significantly increased the plasma levels of GLP-1 as a TGR5 agonist, and a high concentration of GLP-1 lowered blood glucose levels. We confirmed that compound 20p was a non-bile acid FXR/TGR5 dual agonist.Although several potent bile acid Farnesoid X receptor (FXR) and Takeda G-protein-coupled receptor 5  dual agonists such as INT-767 have been reported, no non-bile acid FXR/TGR5 dual agonist has been investigated to date. Therefore, we attempted to discover potent non-bile acid FXR/TGR5 dual agonists and identified some non-bile acid FXR/TGR5 dual agonists, such as isonicotinamide derivatives in vitro assay. Compound  Metabolic syndromes, including insulin resistance, hypertension, dyslipidemia, and obesity, are closely associated with the pathogenesis of NAFLD2. Approximately 20–30% NAFLD patients develop nonalcoholic steatohepatitis (NASH)3, which can lead to cirrhosis and hepatocellular carcinoma5.Type 2 diabetes and nonalcoholic fatty liver disease (NAFLD) are serious hepatic disease events7. The expression levels of FXR are high in the liver, intestine, kidney, and adrenal glands10. The bile acid (BA), chenodeoxycholic acid (CDCA), is an endogenous ligand against FXR. BAs play key roles in regulating cholesterol and bile acid homeostasis11. Activation of FXR in the liver induces a downregulation of CYP7A1 gene transcription. CYP7A1 regulates the conversion of free cholesterol to BAs, and the downregulation of CYP7A1 upregulates a small heterodimer partner (SHP)12.The farnesoid X receptor , a member of the nuclear receptor (NR) superfamily, is a ligand-induced transcription factor2) is the first steroidal FXR agonist. In the combination with ursodeoxycholic acid, it reduces alkaline phosphatase levels in patients with primary biliary cirrhosis13. A proof-of-concept study reported that OCA significantly improves insulin sensitivity and reduces liver inflammation and fibrosis markers, such as alanine aminotransferase (ALT) and enhanced liver fibrosis score, in patients with type 2 diabetes and NAFLD14. OCA can also be used for the treatment of patients with NASH. It has been reported that 25 mg/day of the FXR ligand, OCA, improves NAFLD activity score (NAS) and fibrosis status in the livers of noncirrhotic, nonalcoholic steatohepatitis patients. However, OCA causes pruritus as a clinical adverse event15.Obeticholic acid , have been reported. GW4064 is a selective nonsteroidal FXR agonist19 that significantly reduces free fatty acid and triglyceride levels in db/db mice20.Several potent and selective FXR agonists21. Activation of TGR5 by BA stimulates glucagon-like peptide-1 (GLP-1) secretion from intestinal enteroendocrine L cells by increasing intracellular cAMP concentration24. A TGR5 agonist reduces lipopolysaccharide (LPS)-induced release of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin (IL)-1, IL-6, and IL-8. Thus, TGR5 agonists may have broad therapeutic applications, from the treatment of metabolic disorders to liver inflammatory diseases25.Takeda G-protein-coupled receptor 5  is a class A G-protein-coupled receptor (GPCR) that is expressed in the liver, skeletal muscle, intestine, and brown adipose tissues3), a steroidal FXR/TGR5 dual agonist, decreases serum cholesterol and triglyceride levels in diabetic db/db mice and streptozotocin-induced type 1 diabetes model mouse26. Moreover, INT-767 reduces the production of inflammatory markers, such as IL-1beta, cytokine, and TNF-α, in diabetic db/db mice and apolipoprotein E-deficient mice28. Treatment with INT-767 for 16 weeks significantly improves histological parameters as compared to OCA treatment. Furthermore, INT-767 leads to a significant reduction in NAS and fibrosis in ob/ob NASH mice model as compared to OCA29.INT-767 . The carboxylic acid of GW4064 forms an electrostatic interaction with Arg331 in helix 5. A cocrystal structure of compound 7 with the active site of FXR has been reported (PDB code 1OSH). However, the insertion regions of 15 residues between helices 1 and 3 are completely disordered31. The interactions between FXR LBD and compound 7 can be divided into two sets. The first set of interactions stabilizes the position of hexyl ring, which contacts Ile339 and Leu344. The second set comprises a biaryl ring and dimethylamine group. The dimethylamine group interacts with Trp454. The side chain of Trp454 is shifted by the dimethylamine group as compared to PDB code 3DCT. The biaryl group stabilizes His447 and Trp469.Crystallographic studies have enabled a clear understanding of the interaction between FXR agonists and the ligand binding domain (LBD), making FXR an attractive target. A cocrystal structure of GW4064 with the active site of FXR protein has been reported (PDB code 4DCT)32. An isonicotinamide compound has been identified as a non-bile acid TGR5 agonist  was reported as a specific and potent partial agonist against FXR with no significant agonist activity against TGR531. The indole derivative 8 is a TGR5 agonist , in which cells were transfected with an FXR luciferase reporter system. Cells were incubated with the test compounds for 22 h, and potency was assessed using a multi-mode microplate reader. OCA 3 was set as 100% TGR5 activation37.TGR5 agonist activity was evaluated by transfecting CHO cells with TGR5 using the Discover X expression kit . Cells were incubated with test compounds for 0.5 h, and intracellular cAMP levels were measured using the multi-mode microplate reader. The response to 10 µM INT-767 7 was divided into four fragments, dimethylaminophenyl group (A), center phenyl linker (B), cyclohexyl group (C), and aniline group (D), to explore the structure activity relationship (SAR). A chimera strategy between compound 7 and isonicotinamide compound 8 enabled the modulation of cyclohexyl (C) and aniline (D) groups to rapidly assess the C/D region of molecule. To investigate the activities of some chimera compounds between compound 7 and isonicotinamide compound 8 C/D phenyl ring, we synthesized compounds 13a-13j, as shown in Fig. 7 was synthesized and optimized to incorporate a 3-methyl acrylate moiety for potent FXR agonist activity38. The 2-methyl isonicotinamide group is important for TGR5 agonist activity34, and ortho-substituted anilides, such as 2-methyl-4-fluoroanilide, in the center aniline group exhibit sub-nanomolar TGR5 agonist activities in the cAMP assay33.Compound 13a showed a strong FXR agonist activity with full efficacy while compounds 13b and 13c significantly decreased the FXR activity. The methyl group at 2-position and the position of nitrogen in C region are important for FXR agonist activity. Unfortunately, although a methyl acrylate moiety at the meta position in D region showed a potent FXR agonist activity, compounds 13a and 13b were inactive against TGR5. Compound 13e, which is a similar type of compound 8, showed a strong TGR5 agonist activity. Compounds 13f and 13g, however, exhibited decreased TGR5 activities. These results mimic the following SARs of Novartis compounds34: (1) the position of methyl group is important and (2) a pyridine isomer leads to a complete activity loss in the C region. To explore the disubstituted analogs in D region, we investigated 2, 3-, 2, 5-, and 3, 5-disubstituted analogs. Compound 13h decreased TGR5 potency; however, 13i showed a weak FXR/TGR5 dual agonist activity. The methyl acrylate group plays a critical role in FXR agonist efficacy, and the 2-methyl group in D region exhibited an agonist activity against TGR5. A significant breakthrough was achieved with the 3, 5-disubstituted aniline analog. Compound 13j exhibited a potent FXR/TGR5 dual agonist activity.Compound 13j and 13k showed dual potencies against both FXR and TGR5. However, 13m, 13n, and 13o exhibited remarkably reduced activities against TGR5. The 2, 5-di- or 2, 3, 5-trisubstituted compounds 13p, 13q, and 13r exhibited less potency in FXR agonist assay whereas they demonstrated good potency against TGR5. The 2-methyl-3-substituted compound 13s showed a moderate FXR/TGR5 agonist activity. The 2-fluoro-3-substituted compounds 13t and 13u only showed strong FXR agonist activities, with a similar potency as OCA. These results suggested that the 3, 5-halogenated substitution pattern was essential for optimal FXR/TGR5 dual agonist potency.Next, we investigated the effect of substitution pattern on aniline moiety (part D), as shown in Supplementary Fig. 13v and 13w significantly decreased FXR potencies with an equipotent TGR5 agonist potency. In contrast, the 3-halogenated compounds, 13x and 13y, retained FXR potencies and reduced TGR5 potencies. The trifluoromethylpyridinyl group 13z decreased the potencies against both FXR and TGR5. The 3-methoxy group of 13aa showed a weak dual agonist activity. The pyridazine compound 13ab did not have any activity against either FXR or TGR5. A lack of nitrogen in C ring of compounds 13ac, 13ad, 13ae, 13af, and 13ag showed strong FXR activities. Compound 13ag exhibited an FXR agonist activity of 136.5%, but it demonstrated a significantly low potency against TGR5. The results of Supplementary Figs. We next sought to improve the dual potency against FXR/TGR5 by altering the substitution patterns on C ring . Compound 20a exhibited a significantly increased FXR agonist activity but decreased TGR5 agonist activity. Similar trends were observed in 20d, 20e, and 20f, which were more potent partial FXR agonists and weak TGR5 agonist. The bicyclo[2.2.2]octyl group fills the hydrophobic pocket in FXR LBD. We assumed that the bicyclic ring system would fill the central portion of binding pocket, and the bulkiness and tight conformation would cause a conformational resistance of Trp454. Merk et al. recently reported that a partial agonist DM175 (9) causes an outward movement of Trp45448. Compound 7 also moved Trp454 outward. LY2562175 (6) is also a partial agonist49.Some FXR agonist compounds with cyclohexyl or bicyclo groups in the B ring have been reportedity Fig. . AlthougFinally, we targeted the substitution of part A because the substituent would interact with Trp454. Additional SAR investigations extensively explored the substitutions of aryl rings  in 20p and 20q provided a further improvement in FXR/TGR5 dual agonist potency. These compounds exhibited strong dual agonist potencies. Compound 20r showed the most potent TGR5 activity, although its activity against FXR was less potent. Compound 20s exhibited a high potency against FXR. Introduction of a nitrogen atom in A ring (20u) decreased the dual agonist potency. Replacement of the nitrile group with chloro group in compound 20v decreased the agonist activity. Compounds 20p and 20q were promising candidates for FXR/TGR5 dual agonist.Compound 7 is a potent FXR partial agonist in cell-based assays31. Compound 7 has been reported to possess a crystal structure of human FXR LBD31. The complex has a disordered 15-residue insertion region between helices 1 and 3. Compound 7 and its derivatives are partial agonists of FXR due to their decreased abilities to recruit coactivator SRC-1 as compared to that of the typical full agonist GW4064. The most notable impact of a partial agonist is the outward movement of Trp454 (Trp458 in PDB 1OSH) in compound 7 and the FXR-LBD complex31. The cocrystal structure indicated that this is the mechanistic difference between an agonist and partial agonist. A partial agonist causes an outward movement of Trp454 and destabilizes a loop connecting helix 12. In the agonistic conformation, the ligand interacts with Trp454 and His447, and His447 makes an edge-to-face stacking interaction with Trp469 in helix 12 (AF2), thus, stabilizing it.Compound 48. Binding of DM175 causes an outward movement of Trp454, which is driven by site occupation by the tert-butyl moiety. The binding mode of DM175 is obviously different from the structure of the complex containing full agonists such as OCA, CDCA, and GW4064.Merk et al. recently reported the partial agonist DM175 and its cocrystal structure with FXR LBD20p into the X-ray structure of FXR LBD , which stabilizes Trp458 and recruits the co-activator. Compound 7 causes an outward movement of Trp458, thus, inducing a partial agonist activity against FXR. Ivermectin, a widely used antiparasitic drug with a distinct chemical structure from GW4064 and compound 7, promoted coactivator recruitment by FXR. Ivermectin enhanced the interaction of FXR with various coactivator LXXLL motifs from the family of steroid receptor coactivators such as SRC1, SRC2, and SRC3. Since its interaction is less than that of GW4064, ivermectin is a partial agonist50. However, ivermectin also induces the recruitment of corepressor NCoR-2 by FXR, which is not exhibited by GW4064. The results indicate a partial agonist activity of ivermectin with unique properties that modulate coregulator recruitment. The cocrystal structure of ivermectin with a corepressor instead of a coactivator showed a disordered helix 12 and loop between helices 11 and 12. Additionally, helix 12 is shortened such that the structure contains no activator but a corepressor. Therefore, the cocrystal structure between ivermectin and FXR-LBD is the antagonist form.The agonist mechanism of action for FXR LBD is through interaction with Trp469 and His447. Moreover, the interaction with Trp454 plays an important role between FXR LBD and agonist ligands. We examined the computational molecular docking of 20p and 20q were used to measure the EC50 value. The potencies of 20p  and 20q  were stronger than those of OCA and INT-767  consisting 60 kcal% fat and 0.1% methionine by weight for 1 week52. This model was reported an elevated ALT level, which was a parameter to detect liver injury. Histopathological results indicated hepatocellular steatosis after 1 week. CDAHFD was fed for 6 weeks, and fibrosis developed in C57BL/6J mice.Pharmacokinetic profiles of the bicyclo compounds 20p and 20q . Compound 7 is an FXR agonist, and it increases SHP mRNA in the liver of mice53. Both compounds, 20p and 20q, dose-dependently induced SHP mRNA in the liver at 100, 30, and 10 mg/kg . 20p and 20q significantly increased the plasma levels of GLP-1  and cellular and NR functional assays for GPR40, GPR120, GPR119, LXR alpha, LXR beta, CAR, PPAR alpha, PPAR delta, PPAR gamma, and PXR (EC50 > 30 µM) are included in additional data of S4 Supplementary information.Compound 20p  as a non-bile acid FXR/TGR5 dual agonist. The potency of 20p was stronger than those of OCA and INT-767 in vitro assay. Compound 20p was evaluated in C57BL/6 J mice, that were administered CDAHFD consisting of 60 kcal% fat and 0.1% methionine by weight for one week. Elevated ALT levels were observed in the model after one week of initiation of feeding, and histopathological results indicated hepatocellular steatosis after 1 week52. Moreover, after feeding CDAHFD for 6 weeks, fibrosis was developed in C57BL/6J mice52. Compound 20p dose-dependently induced SHP mRNA and decreased CYP7A1 expression in the liver. The compound 20p significantly increased the GLP-1 level in C57BL/6J mice. The in vivo glucose-lowing study indicated that a single oral dose of 20p significantly reduced blood glucose levels. These results indicate that 20p is an FXR/TGR5 dual agonist.We identified 50 values of agonist activities against FXR and TGR5 for some isonicotinamide derivatives were below 1 µM. An in vivo glucose-lowering study reported that a single oral dose of 20p significantly reduces blood glucose levels. Compound 20p significantly increased the GLP-1 level. Intestine-restricted FXR agonist compound 7 is known to stimulate serum GLP-1 secretion in wild-type C57BL/6J mice for 7 days but not in TGR5-/- or FXR-/- mice54. In our model, a single dose of compound 7 did not increase serum GLP-1 in the wild-type mice. It has been reported that compound 7 modulates gut microbiomes and turns taurochenodeoxycholic acid into lithocholic acid (LCA) by the gut microbiomes, and the LCA stimulates TGR5 expression and TGR5-mediated cAMP signaling and increases GLP-1 secretion54. The GLP-1 secretion of compounds 20p and 20q would be a direct action of TGR5 agonist activities via TGR5.Although several potent steroidal FXR/TGR5 dual agonists have been reported, no non-steroidal FXR/TGR5 dual agonist has been reported to date. A variety of isonicotinamide derivatives were designed, synthesized, and evaluated as a series of highly potent non-bile acid FXR/TGR5 dual agonists. The EC36.To directly evaluate the in vitro agonist activity towards FXR, test compounds were evaluated in CHO cells transfected with FXR responsive luciferase reporter . Cells were incubated with the test compounds for 22 h, and potency was assessed using Multi-Mode Microplate Reader . Efficacies are reported relative to OCA, which was set as 100% FXR activation at a concentration of 1 µM. Each compound was tested in duplicate, and the average value was reported2 for 22 h. To prepare luciferase detection reagent (LDR), the detection substrate was gently mixed with detection buffer, media contents were removed from each well, and 100 µL LDR was added to each well of the assay plate. The assay plate was allowed to rest at room temperature for 5 min, and luminescence was quantified using the Multi-Mode Microplate Reader 36.The cell recovery medium  and compound screening medium  were removed from the freezer and thawed in a water bath at 37 °C. Test compounds were dissolved in dimethyl sulfoxide , and the treatment medium was prepared and diluted with CSM to achieve a final concentration of 0.2% total DMSO. The reporter cells were thawed by transferring 3.3 mL CRM at 37 °C into tubes of frozen cells. The tube containing reporter cells was recapped, and it was immediately place in a 37 °C water bath for 5 min. The reporter cells were gently inverted several times, and 100 µL cell suspension was dispensed into each well. One hundred microliters of treatment media was added into wells. Subsequently, the assay plate was kept at 37 °C, and it was incubated with 5% CO3 was set as 100% TGR5 activation37.TGR5 agonist activity was evaluated by transfecting CHO cells with TGR5 using the Discover X expression kit . Cells were incubated with the test compounds for 0.5 h, and intracellular cAMP levels were measured using Multi-Mode Microplate Reader . The response to 10 µM INT-767 50 value of compounds 20p and 20q were determined by using a 4 parameter logistic equation, Y = min + (max − min)/(1 + 10^(n * (log10(EC50) − log10(conc)))), in nonlinear least squares fitting using the Microsoft Excel Solver55.Each compound was tested at concentrations of 10, 1, or 0.1 µM in duplicate (n = 2), with the average value shown. EC52.Test compounds were orally administered to 7-week old male C57BL/6J mice , that were administered CDAHFD  consisting 60 kcal% fat and 0.1% methionine by weight for 7 days. After four hours of administration, the liver was removed, and total RNA was extracted using a Pure Link RNA Mini Kit . The concentration of total RNA was measured using Micro Sample Spectrophotometer , and cDNA was synthesized using ReverTra Ace qPCR RT Master Mix . Real-time PCR was performed using a CFX96 Touch Real-Time PCR Analysis System , and the mRNA expression levels of SHP and CYP7A1, the target genes of FXR, were measured using the intercalator method20p and 20q were administered orally to overnight-fasted C57 mice . One hour later, all mice were challenged with 2 g/kg glucose, and blood samples were collected after 15 min of glucose challenge test. Plasma GLP-1 levels were measured using the GLP-1 ELISA Kit Wako, High Sensitive 56.Measurement of GLP-1 secretion: To measure plasma GLP-1 levels, 100 mg/kg of compounds 20p and 20q or vehicle were administered orally to overnight-fasted C57 mice . One hour later, all mice were challenged with 2 g/kg glucose, and blood samples were collected after 15 min of glucose loading. The blood glucose levels were measured using LabAssay Glucose 56.To measure blood glucose levels, 100 mg/kg of test compounds Study protocols were designed and refined by taking reduction of animal use into consideration. The study protocol was approved by the Animal Care and Utilization Committee of Fuji Yakuhin Research Laboratories, and all methods complied. We carried out in the compliance with ARRIVE guidelines. A statement to confirm that all methods were carried out in accordance with relevant guidelines and regulations.General synthesis procedures were conducted as described in Supplementary S5.13 was conducted as described in Supplementary Fig. The general synthesis of isonicotinamide derivative 40.Substituted-phenyl-cycloalkyl compounds were synthesized as described in Supplementary Fig. S443. Some substituted-phenyl-bicyclooctane compounds were synthesized47.Substituted-phenyl-bicyclooctane analogs were synthesized as described in Supplementary Fig. 61. Using an iron-based organometallic catalyst, such as trisiron (III) (Fe(acac)3), enables the formation of the C–C bond between tertiary alkyl carboxylic acids and aryl magnesium coupling61. The iron-catalyzed cross-coupling of RAE was a good opportunity for us to optimize the A ring region with bicyclooctane derivatives. We synthesized C–C coupling of the N-hydroxyphthalimide-based ester compound 35 derived from 4-(methoxycarbonyl) bicyclooctane-1-carboxylic acid 31 with Fe(acac)3 and aryl magnesium bromide 36 in the presence of 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone, as shown in Supplementary Fig. The direct cross-coupling of substituted aromatic ring (A region) and bicyclooctane (B region) is effective for the synthesis and evaluation of bicyclooctane compounds. Baran et al. recently reported a cross-coupling method of Redox-Active Ester (RAE) with organometallic species such as a borane, nickel, and iron-catalystSupplementary Information.