Lactaseibacillus paracasei L-26, Lactobacillus casei 431, Lactobacillus acidophilus LA-5, Lactaseibacillus rhamnosus and Bifidobacterium animalis ssp. lactis BB-12) from sheep’s milk with inulin, apple fiber and inulin, or apple fiber and control samples were submitted to in vitro digestion in the mouth, stomach and small intestine. The bioavailability of calcium in the ice cream samples ranged from 40.63% to 54.40%, whereas that of magnesium was 55.64% to 44.42%. The highest bioavailability of calcium and magnesium was shown for the control samples. However, adding 4% inulin reduced the bioavailability of calcium by about 3–5% and magnesium only by about 5–6%. Adding 4% apple fiber reduced the bioavailability of calcium by as much as 6–12% and magnesium by 7–8%. The highest bioavailability of calcium was determined in ice cream with L. paracasei, and the highest bioavailability of magnesium was determined in ice cream with L. casei. The bioavailability of phosphorus in ice cream ranged from 47.82% to 50.94%. The highest bioavailability of phosphorus (>50%) was in sheep ice cream fermented by B. animalis. In the control ice cream, the bioavailability of potassium was about 60%. In ice cream with inulin, the bioavailability of potassium was lower by 3–4%, and in ice cream with apple fiber, the bioavailability of potassium was lower by up to 6–9%. The bioavailability of potassium was significantly influenced only by the addition of dietary fiber. The results of the study confirmed the beneficial effect of bacteria on the bioavailability of Ca, Mg and P.To determine the potential bioavailability of macroelements , probiotic ice cream samples ( Macronutrients occur in sheep’s milk in substantially higher concentrations than in cow’s or goat’s milk  .In milk, micelles are bound by calcium phosphate and small amounts of magnesium and citrate . MicelleThe mineral composition may differ from the content in dairy products. It was found that such differences may be the consequence of the processing factors applied, including heat treatment, homogenization or pasteurization, fermentation, type of starter culture and fermentation time, as well as storage conditions and time ,8,9. TheMinerals are mainly absorbed in the small intestine. The bioavailability of minerals strongly depends on a human’s age and health status, the type of product consumed, the form of the compound, the interaction between minerals contained in the product, technological processing and pH. The bioavailability of minerals is significantly influenced by probiotic bacteria, gastrointestinal microflora and prebiotics .Enzymes in the small intestine do not digest prebiotics such as inulin and apple fiber, and these prebiotics stimulate the growth and activity of probiotic bacteria by undergoing fermentation ,14, playProbiotic bacteria colonizing the intestines are believed to contribute to the bioavailability of calcium and magnesium ,20,21,22Lactaseibacillus paracasei L-26, Lactobacillus casei 431, Lactobacillus acidophilus LA-5, Lactaseibacillus rhamnosus and Bifidobacterium animalis ssp. lactis BB-12) from sheep milk with inulin, apple fiber and inulin, or apple fiber and control samples were subjected to in vitro enzymatic digestion.As a popular dessert consumed worldwide, dairy ice cream can be an excellent carrier of minerals. Adding probiotic bacteria, inulin and apple fiber could potentially help improve the bioavailability of macronutrients, a crucial issue. There is no information on the bioavailability of Ca, K, Mg and P from sheep’s milk ice cream manufactured from mixes fermented by probiotic strains. The differences in milk composition  of different species mean that further research is needed to determine the most appropriate types of milk and milk products to improve the bioavailability of micro- and macronutrients ,23,24. T−1), mucin from the porcine stomach (type II), pepsin from the porcine gastric mucosa (250 U mg−1 solid), porcine bile extract and pancreatin from the porcine pancreas (8 × USP specifications) were provided by Sigma-Aldrich .Digestive enzymes: heat-stable α-amylase , di-potassium hydrogen phosphate , sodium chloride pure p.a. ≥99.9% , hydrochloric acid (12 M HCl) and sodium hydroxide (1 M NaOH) were supplied by Chempur . All of the reagents used were of analytical reagent grade. Nitric acid (HNO3) was provided by Sigma Aldrich . EDTA disodium salt (EDTA Na2 0.01 M), sodium bicarbonate , sodium bicarbonate (NaHCO3 0.5 M) and sodium dodecyl sulfate (0.1%) were also purchased from Sigma-Aldrich . MRS agar and peptone media were purchased from Biocorp . The cellulose membrane for dialyzing  was purchased from Sigma-Aldrich .Reagents: di-sodium hydrogen phosphate anhydrous pure p.a. ≥99.0% , 100% micronized apple fiber  and white sugar  were used as additives.Raw sheep’s milk for the production of ice cream mixtures was obtained from a farm in Wyżne, Podkarpacie, Poland . Inulin . Freeze-dried commercial starter cultures used in the dairy industry are not genetically modified.Probiotic bacteria used for fermentation: w/w) and fermented for ten hours in an incubator , and then cooled to 5 °C and conditioned for 12 h. The ice cream mixtures were frozen in a DeLux 48,816 freezer  for 40–50 min at −22 °C [The process of producing ice cream from sheep’s milk according to the method described by Kowalczyk et al.  is inclut −22 °C . Next, tThe procedure of simulated digestion in the gastrointestinal tract was carried out according to the methods presented by Buniowska et al.  and Came2HPO4, 0.19 g K2HPO4, 8 g NaCl, 100 mg L−1 mucin and 150 mg L−1 α-amylase with enzyme activity of 200 U L−1, dissolved in 1 L distilled water) were transferred to a dark glass bottle. Using HCl (12 mol L−1) or NaOH (1 mol L−1) buffers, the mixture was adjusted to pH 6.75 ± 0.20 and then incubated in a shaker at 37 °C and 90 rpm for 10 min. In particular, 10 min is an extended time relative to the average time food is in the mouth during chewing, but this value ensured repeatability between samples. After the oral stage, the simulation of the gastric phase was initiated. In this stage, 13.08 mg of pepsin was added to the sample mixed with saliva, and the pH value was reduced to 2.0 ± 0.20 by adding HCl (12 mol L−1). The sample was placed in a shaker for 2 h at 37 °C and 90 RPM.To simulate the oral stage, 50 mL of sample and 5 mL of saliva enzyme solution (2.38 g Na−1) and bile salt (25 g L−1), changing the pH value to 7.00 ± 0.20 (12 M HCl M or 1 M NaOH). The previously prepared cellulose dialysis membranes (25 mm wide and 30 cm long) were conditioned  at a temperature of 100 °C for 10 min and rinsed five times with deionized water, filled with 25 mL of NaHCO3 (0.5 M) and placed in the sample until a pH value of 7.00 ± 0.20 was reached. During this process, the pH gradually adjusted, simulating intestinal conditions. The incubation was continued for another 2 h (37 °C/90 RPM). To complete the digestion process, the sample was placed in an ice bath for 10 min. Macroelements that diffuse into the semi-permeable dialysis membrane are potentially absorbed into the bloodstream, known as dialysate. Using a dialysis membrane eliminates the problems that occur during the dialysis of soluble and insoluble compounds [To induce simulated intestinal digestion, the contents from the oral and gastric stages were mixed with 5 mL of pancreatin (4 g Lompounds . The solBioavailability (%) refers to the percentage of the mineral compound tested that remains in the dialyzed intestinal fraction relative to the original undigested sample (before digestion), according to the following equation:After seven days, samples of the product and each stage and dialysate were frozen and stored at −45 °C until further analysis (about 12–14 days).Macroelements in ice cream samples were measured before digestion and after each stage of in vitro digestion using optical emission spectrometry with horizontal plasma and detection capabilities both along and across the plasma flame  (ICP-OES) using a Thermo iCAP Dual 6500 spectrometer  according to the method of Znamirowska et al. .−1 dry matter.The instrument was calibrated with certified standards  covering macronutrient concentrations  of 10,000 ppm. The method was validated using certified reference material (NIST-1515). Results were expressed in mg 100 g3 using a Milestone Ethos Ultra-wave-One mineralizer . The required amount of sample was placed in Teflon vessels, after which it was topped up with 8 mL of nitric acid and sealed tightly. The rotary mineralizer was topped up with a blank sample (8 mL of HNO3) during the mineralization process for all samples. The analyzed samples were mineralized for one hour using the temperature build-up algorithm, according to the procedure for this type of biological samples, not exceeding 200 °C. After mineralization, the samples were brought to room temperature, and the contents were poured into Falcon tubes (50 mL) topped up with demineralized water.Before the determination, the test samples were subjected to out-of-pressure mineralization in 65% HNO−1 (assuming detection of the measuring apparatus at 10 ppb).The detection level for each element was not less than 0.1 mg kg p ≤ 0.05).Statistical analysis was performed using Statistica v. 13.1 . The mean and standard deviation were statistically calculated from the obtained results. A one- and two-factor ANOVA analysis of variance was performed. The significance of differences between the means was determined by Tukey’s test . In comparison, the phosphorus content in these groups of ice cream ranged from 153.35 mg 100 g−1 to 157 mg 100 g−1. Adding inulin does not significantly increase the calcium and phosphorus content of ice cream. On the contrary, apple fiber contains 15.96 mg 100 g−1 of calcium and 51.80 mg 100 g−1 of phosphorus, resulting in a proportional increase in calcium and phosphorus in FLC (L. casei), FLA (L. acidophilus), FBB12 , FLP (L. paracasei) and FLR (L. rahamnosus) ice cream.In all control ice cream groups , the calcium content ranged from 199.47 mg 100 g2HPO4) in saliva. In humans, the amount of saliva secreted under resting conditions is 0.5 mL per minute, while after intense secretory stimulation with food, it can increase to 10 mL per minute [In the mouth, ice cream samples are mixed with saliva and thus diluted, which contributes to a non-significant decrease in calcium concentration in all samples . On the r minute .When the ice cream enters the stomach during fasting, it comes into contact with a small amount (~50 mL) of gastric juice, which for an adult will have a pH between 1 and 2 . However−1 to 151.21 mg 100 g−1 (p ≤ 0.05) calcium content was determined only in samples fermented by L. rhamnosus: the IFLR ice cream with inulin and fiber and FLR ice cream with fiber compared to control CLR ice cream and ILR ice cream with inulin. In other ice cream groups, added fiber had no effect on calcium content. In contrast, a significant effect of added apple fiber on the phosphorus content of the gastric phase was found in FLA (fermented by L. acidophilus) and FBB12 ice cream  compared to their control counterparts. Also notable was the tendency in the remaining ice cream with apple fiber to maintain higher phosphorus concentrations than those in samples without fiber.Therefore, when the pH value is lower than 4 in the stomach, it should be sufficient to dissolve all calcium and phosphate from casein micelles . Possibl 100 g−1 , represeThe rapidity of gastric emptying depends on many factors, including volume flow restrictions, caloric density, pH and rheological properties . PhosphoL. casei to 50.94% in the FBB12 group with fiber addition fermented by B. animalis. Adding apple fiber did not significantly reduce the bioavailability of phosphorus in ice cream fermented by L. casei, L. paracasei and L. rhamnosus, nor did it significantly increase bioavailability in ice cream with L. acidophilus (FLA) and B. animalis (FBB12), compared to control counterparts. The highest bioavailability of phosphorus (>50%) was found in sheep’s milk ice cream fermented by B. animalis. The remaining sheep’s milk ice creams had lower phosphorus bioavailability by 1–2.5%.The bioavailability of phosphorus in the ice cream ranged from 47.82% in FLC ice cream with fiber fermented by B. animalis) to 54.40% in control CLP samples fermented by L. paracasei  show that the bioavailability of calcium from sheep’s milk ice cream is significantly influenced by the two factors analyzed, which are the fiber (p = 0.0000) and the type of bacteria (p = 0.0001), as well as interactions between these factors (p = 0.0006). Also, a study by Bosscher et al. [The bioavailability of calcium in the tested ice cream ranged from 40.63% in FBB12  or bound to a soluble organic molecule. The auxiliary organs supporting intestinal digestion and absorption are the pancreas, liver and gallbladder [Most calcium (about 65%) is absorbed at pH 6.5–7.5. It should be noted that calcium is not absorbed from the stomach . In ordelbladder . Calciumlbladder . In the lbladder ,49. A stlbladder  on food Lactobacillus salivarius could increase Ca2+ uptake, although exposure of Caco-2 cells to probiotics has no effect on Ca2+ transport.Our study of the effect of applied probiotic strains on calcium bioavailability also supports the results of Sharifi-Rad et al. , where o−1 of magnesium, which occurs in the form of soluble compounds  and colloidal compounds . Only about 15% of magnesium is presented in an ionized form. [−1 in control ice cream with L. casei (CLC) to 17.52 mg 100 g−1 in control ice cream with L. paracasei (CLP). Adding apple fiber significantly increased the magnesium content of FLC (L. casei), FLA (L. acidophilus), FBB12 , FLP (L. paracasei) and FLR (L. rhamnosus) ice cream by about 0.5–1.4 mg 100 g−1 due to the presence of magnesium in apple fiber (Sheep milk provides 16–18 mg 100 gle fiber . In contL. casei (CLC) to 44.42% in the group with fiber fermented by L. acidophilus (FLA). The bioavailability of magnesium was highest in the control samples, where it always exceeded 50%. The highest bioavailability was determined in the control CLC ice cream fermented by L. casei. The addition of fiber had an essential role in modifying the bioavailability of magnesium. Hussain et al. [L. acidophilus), 7.48% in FLC (fermented by L. casei) and 7.25% in the FLR (fermented by L. rhamnosus) group. Ice cream with fiber addition, FBB12 fermented by B. animalis and FLP fermented by L. paracasei, showed 5–6% lower magnesium bioavailability than controls.The bioavailability of magnesium from sheep’s milk products is poorly studied. In our study, the bioavailability of magnesium in sheep’s milk ice cream ranged from 55.64% in the control ice cream fermented by n et al.  indicateLactobacillus spp. consumed with Dutch-type cheese increased Mg (~18%) and Ca (~2.5%) availability in vitro [Bielik et al.  considerin vitro . Similarin vitro .p = 0.0001), as confirmed by a two-factor analysis of variance (ANOVA). The calculations show that the bioavailability of magnesium from sheep’s milk ice cream is significantly influenced by both factors analyzed, including the fiber (p = 0.0011) and the interactions between these factors (p = 0.0005).This implies that the bacterial strain conducting the fermentations of the ice cream mixes also significantly affects magnesium bioavailability , while the type of bacteria (p = 0.6731) and interactions between these factors (p = 0.7456) were not significant.In our study, the bioavailability of potassium was higher in control ice cream than in ice cream with fiber . In the The bioavailability of potassium in whole fruits and vegetables can be as high as 50% to 60%, and there is a lack of evidence linking higher consumption of fruits and vegetables to higher serum potassium concentrations . The bioIn general, considering macronutrients in food matrices and their changes during digestion is crucial for allowing the optimal use of macroelements in the diet. The results of the study confirmed the beneficial effect of the bacterial strain on the bioavailability of Ca, Mg and P and, thus, their support for the human gut microbiome. Further research should address the action of specific bacterial species and their effect on improving or preventing micronutrient deficiencies. Similarly, a better understanding of the underlying mechanisms by which the gut microbiota influences host micronutrient uptake and absorption would enable a postbiotic approach to match micronutrient availability with host needs.


Salvia genus contain widely distributed species which have been used in folk medicine as well as pharmaceutical and food industries.The plants from Salvia species (14 plants) was identified using gas chromatography-mass spectrometry (GC–MS). Also, the inhibitory activity of all essential oils (EOs) was evaluated toward α-glucosidase and two types of cholinesterase (ChE) using spectrophotometric methods. The in vitro α-glucosidase inhibition assay was performed by the determination of p-nitrophenol (pNP) obtained from the enzymatic dissociation of p-nitrophenol-α-D-glucopyranoside (pNPG) as the substrate. In vitro ChE inhibitory assay was conducted based on the modified Ellman’s procedure using the measurement of 5-thio-2-nitrobenzoic acid produced from the hydrolysis of thiocholine derivatives as the substrate, in the presence of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE).The chemical composition of 12 native Iranian trans-β-caryophyllene were the most abundant compounds in all EOs. The yield of EOs extracted from the plants were also calculated in the range of 0.06 to 0.96% w/w. Herein, α-glucosidase inhibitory activity of 8 EOs was reported for the first time and among all, S. spinosa L. was found to be the most potent inhibitor (90.5 inhibition at 500 μg/mL). Also, the ChE inhibitory activity of 8 species was reported for the first time and our results showed that the BChE inhibitory effect of all EOs was more potent than that of AChE. The ChE inhibition assay indicated that S. mirzayanii Rech.f. & Esfand. collected from Shiraz was the most potent inhibitor .Totally, 139 compounds were detected and caryophyllene oxide and Salvia species of Iran could be considered in the development of anti-diabetic and anti-Alzheimer's disease supplements.It seems that native  Salvia species have attracted a lot of attention [Salvia officinalis L. (sage) is used for food preservation, particularly meat and cheese [Salvia sclarea L., Salvia hispanica L., and Salvia divinorum Epling & Játiva are cultivated in many parts of the world due to their commercial interests [Plants and their extracts, essential oils (EOs), and secondary metabolites have been widely used in the pharmaceutical, food, cosmetic, and perfume industries due to valuable medicinal, flavoring, and preserving properties. Among them, ttention ; for exad cheese . In thisnterests , 4.Salvia is one of the most important and largest genera of the Lamiaceae family containing approximately 900 distinct species worldwide  [Salvia have depicted a wide range of biological activities including antibacterial, carminative, diuretic, spasmolytic, anti-inflammatory, antioxidant, anti-cancer, anti-diabetic, anxiolytic, and sedative properties. Also, the genus has long been considered in folk medicine to deal with different ailments such as epilepsy, cancer, malaria, bronchitis, tuberculosis, hepatitis, anti-diabetic, dementia, and dysmenorrhea [Salvia plants have been extensively used for different therapeutic purposes in Persian medicine. For example, S. officinalis has been used as a diuretic, carminative, wound healing, and asthma-treating agent [regions)  and wideregions) . The extenorrhea -10. Furtng agent -13. Todang agent .S. officinalis EO exhibited anti-diabetic properties by reducing blood glucose up to 60% and elevating stored glycogen in the liver up to 43.7% [S. officinalis EO in the full flowering stage which inhibited the enzyme with an IC50 = 22.24 μg/mL, compared to acarbose as a standard (IC50 = 12.31 μg/mL) [Diabetes Mellitus (DM) is an endocrine disease which can impair carbohydrate metabolism due to insulin deficiency or insulin resistance . The gloto 43.7% . Assagga1 μg/mL) .Salvia species have also been linked to neuroprotective characteristics. Generally, the small size and lipophilicity of EOs constituents allow them to easily pass the blood–brain barrier and therefore could be suggested as a potential strategy for the treatment of neurodegenerative disease [Salvia lavandulifolia Vahl. and S. officinalis significantly improved memory performance and thereby, could be considered in aromatherapy [S. lavandulifolia EO was found to be a selective AChE inhibitor with an IC50 value of 3 µg/mL while inhibiting BChE by 22% at 0.5 mg/mL [S. lavandulifolia EO reduced the activity of AChE in the striatum of rats, however, not in their hippocampus or cortex [Salvia potentillifolia Boiss. & Heldr. ex Benth. EO indicated inhibitory activity against the BChE by 65.7% inhibition at the concentration of 200 µg/mL and the corresponding activity on AChE was obtained as 21.9% inhibition at the same concentration, compared with galantamine as a reference  [ disease . Nowaday disease . The cho disease . In thisatherapy . Further.5 mg/mL . Also, Sr cortex . Moreovectively) .Salvia genus EOs and α-pinene, β-pinene, germacrene D, spathulenol, bicyclogermacrene, 1,8-cineole, camphor, borneol, α-thujone and β-thujone, thymol, caryophyllene, and caryophyllene oxide have been commonly determined as the most prominent components [Unlike synthetic substances, which are often based on a single active component, EOs include a variety of compounds that interact synergistically or additively with each other to either reduce the risk of drug resistance or boost the effectiveness of the treatment . Severalmponents , 32.Salvia species were investigated, and they were evaluated for their α-glucosidase and ChE inhibitory activity to develop dual natural anti-diabetic and anti-AD agents as AD has been considered as type 3 diabetes and the role of insulin resistance in inducing impaired brain glucose metabolism, neurodegeneration, and cognitive impairment has been comprehensively discussed in the literature [The scientific research of numerous plant species to find new natural bioactive agents is a time-consuming and resource-intensive procedure. As a result, researchers are now receiving assistance in their quest to identify active pharmaceutical ingredients in medicinal plants, which were previously used to treat illnesses in a simple and cost-effective manner. In this study, constituents of EOs of 14 plants from 12 Iranian native terature .Saccharomyces cerevisiae; EC3.2.1.20, 20 U/mg), acetylcholinesterase , butyrylcholinesterase , p-nitrophenyl α-D-glucopyranoside (p-NPG), 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB), acetylthiocholine iodide (ATCI), and butyrylthiocholine iodide (BTCI) were provided from Sigma-Aldrich.α-Glucosidase  was hydro-distilled for 4 h using a Clevenger-type apparatus according to European Pharmacopoeia (2020). Anhydrous sodium sulfate was added to the isolated EO to remove the water. All EOs were stored in a dark sealed vial at 4 °C for further experiments. The yield of extraction was reported as w/w%.7–C21) were injected in the same manner to compare calculated retention indices with those in authentic references. For more accurate identification, the mass spectra of each compound were reconciled with the NIST database [GC–MS analysis was performed on a 7890B Agilent gas chromatograph including a DB-5 column  and a 5977A Agilent mass spectrometer. 1 μL of diluted samples (with ethyl acetate) was injected into the injection site. The temperature program was scheduled as follows: the initial temperature of the oven was 40˚C (held for 7 min) and programmed to reach 140˚C with a rate of 10 ˚C /min, eventually reached 250˚C with a rate of 3˚C /min and held for 7 min at this temperature. Helium with 99.99% purity was utilized as a carrier gas (flow rate: 1 mL/min). Also, the ionization voltage of the detector was set at 70 eV. To determine the components, normal alkanes (Cdatabase  and Adamdatabase .Saccharomyces cerevisiae).The α-glucosidase inhibitory activity of EOs was evaluated based on the previously described method  using α-Inhibitory activity against AChE  and BChE  was performed using modified Ellman's method .P values < 0.05 were considered statistically significant.The GraphPad Prism software was used to carry out statistical analysis. Data comparisons were performed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons as the post-hoc test. S. reuterana and S. mirzayanii (Jahrom region), respectively.The yield of isolation of EOs from each species was reported in Table Salvia spp. EOs. However, in the case of S. multicaulis, monoterpenes were the most abundant components.According to the GC–MS analysis, 139 components were identified in the isolated EOs from 14 plants as recorded in Table trans-β-caryophyllene (24.12%)  were the highest in value. It is worth mentioning that caryophyllene oxide was detected as the main compound in 7 Salvia spp. S. sharifii and S. sclarea EOs contained the corresponding compound as 21.11 and 22.26%, respectively, which were more significant than the others. Moreover, linalool, α-terpineol, trans-β-caryophyllene, spathulenol, and caryophyllene oxide were ubiquitous in all isolated EOs.The GC–MS analysis also revealed that among all EOs components Table , sclareoSalvia EOs were reported in Table The percentage inhibition values for α-glucosidase and ChE inhibitory activity of the S. spinosa EO exhibited the strongest activity (90.5% inhibition) and EOs of S. virgata and S. reuterana were also able to block the enzyme with high percentage inhibition (89.7% inhibition). Additionally, the EOs of S. mirzayanii collected from Darab and S. hypoleuca showed a weak α-glucosidase inhibitory activity with percentage inhibition values of 25.5%, and 22.7%, respectively, while S. mirzayanii from Jahrom displayed no activity.It was perceived that 8 EOs expressed a notable inhibitory effect toward α-glucosidase at 500 μg/mL, compared with acarbose. More specifically, In vitro ChE inhibitory assay of the 14 investigated EOs indicated moderate to remarkable activity toward both AChE and BChE at 500 μg/mL, however, they were less active than donepezil Table .S. mirzayanii collected from Shiraz revealed the highest inhibitory effect on AChE and BChE , and S. mirzayanii collected from Jahrom (30.81% and 64.76%) and Darab (23.86% and 63.02%), as well as S. syriaca (15.8% and 52.1%), showed good activity. S. verticillata and S. multicaulis had a negligible effect toward AChE (0.5% and 1.1% respectively) and S. palaestina and S. hypoleuca had no significant inhibitory effect on BChE . Furthermore, S. sharifii (42%), S. santolinifolia (44.1%), and S. multicaulis (44.1%) were found to be moderate inhibitors of BChE.S. multicaulis EO was the most selective BChE inhibitor (SI = 88.2). Meanwhile, the three populations of S. mirzayanii EOs exhibited no noticeable selectivity in the inhibition of ChEs.Finally, the selectivity index (SI) of the tested EOs in the inhibition of BChE over AChE showed that trans-γ-bisabolene, β-copaen-4-α-ol, and valeranone were detected only in the specific species. However, caryophyllene oxide, spathulenol, linalool, α-terpineol, and trans-β-caryophyllene were detected in all EOs. Additionally, caryophyllene oxide (0.38–22.26%), trans-β-caryophyllene (0.72–24.12%), spathulenol (0.94–17.62%), germacrene D (0–16.33%), and δ-cadinene (0–11.72%) were generally found to be the main components in this series of EOs. Comparing the major components of the studied Salvia spp. with those reported in the literature  was more potent than acarbose (IC50 = 9.6 mg/mL) [S. spinosa EO was evaluated, which demonstrated moderate inhibitory activity with an IC50 value of 43.79 µg/mL, compared with acarbose (IC50 = 17.1 µg/mL) [S. virgata nor S. sclarea EOs were active toward α-glucosidase [According to the literature review, α-glucosidase inhibitory activity of 6 mg/mL)  which is1 µg/mL) . On the p-cymene, borneol, γ-terpinene, and phytol have shown good inhibitory effect on α-glucosidase. For example, Carum carvi L. and Coriandrum sativum L. EOs containing high amounts of these compounds showed very potent α-glucosidase inhibitory activity, compared with acarbose [S. santolinifolia, S. verticillata and S. multicaulis EOs, respectively; they may be effective in inducing α-glucosidase inhibitory activity.EOs possessing high amounts of acarbose  which isAChE accounts for roughly 80% of cholinesterase activity in the normal brain, with BChE making up the remaining 20%. However, in severe AD, AChE activity may decline to 55–67% of baseline levels in particular brain regions, whereas BChE activity rises. Also, BChE may contribute to the accumulation of β-amyloid plaques that occur in the early phases of AD progression . TherefoS. sharifii, S. santolinifolia, S. reuterana, S. spinosa, S. palaestina, S. virgata, S. hypoleuca, and S. mirzayanii EOs are reported for the first time and those of S. sclarea, S. verticillata, S. multicaulis, and S. syriaca EOs were previously investigated [To the best of our knowledge, cholinesterase inhibitory activity of the stigated , 61-63.S. sclarea which were exposed to different fertilizers. The EOs of plants treated with organic and chemical fertilizers were able to inhibit the enzyme with the percentage inhibition of 76.0% and 45.1%, respectively at the concentration of 1 mg/mL. However, S. sclarea EO exhibited 32.8% inhibition toward BChE at the concentration of 500 μg/mL, in our study. On the other hand, only the plant EO treated with chemical fertilizers were able to inhibit the AChE , while in our study, S. sclarea EO inhibited this enzyme less than 10% at 500 μg/mL.Orhan et al.  studied S. verticillata and S. wiedemannii EOs. In agreement with our results, S. verticillata EO had a weak inhibitory effect on the ChEs , however, inconsistent with our results, the EO inhibited the AChE more strongly than the BChE [Kunduhoglu et al. reported the cholinesterase inhibitory activity of the BChE .S. syriaca EO demonstrated AChE and BChE inhibitory activity stronger than the reference  [S. syriaca EO showed significant inhibition on the ChEs, but they couldn't exceed donepezil as the standard.ntamine) , while cS. multicaulis EO was able to inhibit AChE and BChE by 25.6% and 71.2%, respectively, at the concentration of 100 μg/mL [As reported by Akdeniz et al., 00 μg/mL , but theS. leriifolia, α-pinene and 1,8-cineole showed BChE inhibitory activity with IC50 values of 0.87 and 0.93 mM, respectively [According to the study of Loizzo et al. on the EO of ectively . Also, gectively . Althoug50 values of 54.7 and 47.5 µM against AChE and BChE, compared with donepezil with IC50 values of 0.15 and 5.8 µM, respectively) [S. mirzayanii and S. syriaca EOs have α-terpinyl acetate in higher amounts than other EOs, which induced higher ChEs inhibitory properties particularly more potent BChE inhibitory activity.According to the Chowdhury and Kumar report, α-terpinyl acetate was introduced as a natural monoterpenoid with potent ChE inhibitory activity (ICctively) . These rOrhan et al. showed that geraniol inhibited the BChE by 55.4% over 15.3% for AChE . HoweverS. mirzayanii and S. syriaca EOs containing high levels of that compound, showed good activity against BChE. Also, the amount of mesitylene as one of the main components of S. syriaca EO (9.13%) was found to be important for higher BChE inhibitory activity. However, further study is needed to determine whether these compounds are responsible for BChE inhibitory property.It seems that the presence of δ-cadinene is also important for inducing BChE inhibitory activity as trans-β-caryophyllene, spathulenol, caryophyllene oxide, and linalool. Bonesi et al. studied the ChE inhibitory activity of Cordia gilletii De Wild. EO suggesting that trans-β-caryophyllene is playing an important role in BChE inhibitory activity [50 values of 51.6 and 66.7 µg/mL against AChE and BChE, respectively) [On the other hand, no correlation was detected between the ChE inhibitory activity of the EOs and limonene, activity . Moreovectively) . HoweverS. multicaulis EO possessing high amounts of trans-β-caryophyllene (19.02%), borneol (12.52%), α-pinene (9.43%), and carvacrol (5.66%).According to the literature, AChE and BChE have approximately 65% amino acid sequence identity . HoweverComparing wild plants with cultivated ones, demonstrated that cultivated counterparts offer numerous advantages to the pharmaceutical industry, including fewer chemical changes, a more manageable supply chain, minimized batch variations, and stable raw material prices, albeit still higher than those for wild plants . BecauseS. mirzayanii cultivated in Shiraz, showed the strongest activity toward ChEs while the S. spinosa EO was the most potent inhibitor of α-glucosidase. It seems that they can be a potential and guaranteed source for the industrial production of new medicinal agents to control T2DM and AD.In this work, the EO of Salvia species EOs by GC–MS led to the identification of 139 compounds. The S. spinosa EO showed high inhibitory activity toward α-glucosidase, while three S. mirzayanii EOs exhibited the strongest inhibitory effects on ChEs. Plants in the genus Salvia, especially S. mirzayanii and S. spinosa which were cultivated in Shiraz, can be considered as natural resources for industrial production of important supplements to be effective as a treatment for AD and T2DM. However, it should be mentioned that further studies are required to determine the responsible bioactive components for desired biological activities. Also, the toxicology and bioavailability profile of EOs are in high demand.This study aimed to find natural and safe resources for the treatment of two common diseases; T2DM and AD, as many people with Type 2 diabetes have shown a higher risk of developing AD and anti-diabetic agents have been recently found to be active in the treatment of AD. Evaluation of 14


Cystatin C-based estimates of GFR outperform creatinine-based estimates in predicting clinical outcomes and identifying patients at higher risk, particularly in elderly and non-white ethnic groups. Cystatin C offers advantages over creatinine as a marker of kidney function. It is not influenced by non-kidney factors and provides more accurate estimation of GFR, aiding in the early detection of kidney disease and predicting adverse outcomes. Incorporating cystatin C into routine kidney function assessment may improve patient risk stratification and guide clinical decision-making. However, widespread adoption of cystatin C testing requires increased availability and accessibility in clinical laboratories. Further research and implementation efforts are needed to fully realize the potential of cystatin C in kidney function assessment and improving patient outcomes.Kidney function assessment is crucial for diagnosing and managing kidney diseases. Glomerular filtration rate (GFR) is widely used as an indicator of kidney function, but its direct measurement is challenging. Serum creatinine, a commonly used marker for estimating GFR (eGFR), has limitations in accuracy and sensitivity. Cystatin C, a protein freely filtered by the glomerulus, has emerged as a promising alternative marker for kidney function. It is unaffected by muscle mass and shows stronger associations with cardiovascular disease and mortality than creatinine. Various equations have been developed to estimate GFR using creatinine or cystatin C alone or in combination. The CKD-EPI Kidney function can, especially amongst nephrologists, refer to a whole variety of biological functions of the kidney: excretion, filtration or clearance as well as metabolic or endocrine physiological roles. However, following the discovery that “pure” filtration occurs at the glomerulus of the nephron, the glomerular filtration rate (GFR) is often considered the best overall index of kidney function in health and disease. Unfortunately, true GFR cannot be measured directly and requires the assessment of the clearance of either exogenous or endogenous filtration markers. These markers should have particular chemical and physiological qualities to allow for an accurate and reliably measured GFR. That is, to be as close to the true GFR as possible. Whether they be exogenous or endogenous, all should be chemically inert in vivo, freely filtered by the glomerulus, neither secreted nor reabsorbed by the tubules and eliminated solely by the kidneys .Whilst there are many suggested markers for estimating the glomerular filtration rate (eGFR)—such as beta-trace protein, beta2-microglobulin and cystatin C, which has garnered lots of interest in recent years—the most common method for calculating eGFR worldwide is through creatinine measurement (eGFRcr), which has become more reliable with standardization to isotope dilution mass spectrometry (IDMS)-traceable techniques and the development of more robust eGFR equations. However, it is an imperfect tool for estimating GFR as it tends to overestimate GFR and underestimate the presence or severity of chronic kidney disease (CKD) in older individuals or those with lower-than-average muscle mass for their age. Moreover, it is insensitive in detecting early kidney disease. Serum creatinine measurement and the subsequent calculation of eGFR through derived equations have long been the mainstay of laboratory assessments of GFR; however, creatinine is not an ideal exogenous marker of GFR, being only freely filtered. As new creatinine-based equations have been developed and become more accurate, parallel efforts have been in play to identify, verify and validate new exogenous markers and the eGFR equations derived.creat 45–59 mL/min/1.73 m2 who do not have markers of kidney damage, measuring cystatin C is suggested to confirm the diagnosis of CKD. If the eGFRcys or combination eGFRcreat-cys (creatinine and cystatin) is <60 mL/min/1.73 m2, the diagnosis of CKD is confirmed, but if it is >60 mL/min/1.73 m2, the diagnosis of CKD is not confirmed. However, the National Institute for Health and Care Excellence (NICE) acknowledges the lack of high-quality evidence and currently does not make positive recommendations for the use of cystatin C equations to estimate GFR in routine clinical practice due to the discord between eGFRcreat and eGFRcys, particularly in older adults ..cys was In practice, serum creatinine is widely used in the majority of patients who have blood tests measured routinely, and therefore, many clinicians will likely feel comfortable interpreting the results. Concentrations are now routinely measured using enzymatic methods, although the older colorimetric Jaffe reaction is still in use. A reference material  is available for creatinine and most widespread methods are calibrated against the IDMS to reduce error and maximise comparability of measurements. Due to the more recent introduction of a reference material (ERM-DA471/IFCC—2011) for cystatin C, the measurements of all methods are now traceable; manufacturers have markedly improved accuracy and the between-method agreements of cystatin C measurements since 2014, thus allowing for greater confidence in estimating GFR, which relies on cystatin C .The mainstay of cystatin C measurement is through particle-enhanced immunoassays, via either a turbidometric immunoassay (PETIA) or nephelometric immunoassay (PENIA) . The adv2 [Studies have demonstrated that the cystatin-C-based estimates of GFR are more effective predictors of clinical outcomes than creatinine-based eGFR and identify more patients who are at increased risk in elderly ,41,42,432 ,47,48,492 ,47.The Health, Aging, and Body Composition cohort study, focusing on elderly patients aged 70–79 years, revealed a significant association between increasing levels of cystatin C and an elevated risk of mortality. This association remained consistent for both cardiovascular mortality and mortality from other causes, but not for cancer-related mortality. Conversely, after adjusting for a range of factors, creatinine levels were not found to be associated with mortality. Therefore, cystatin C emerged as a robust and independent risk factor for mortality in the elderly population .In another study called the Cardiovascular Health Study, which involved over 4000 patients who were elderly without chronic kidney disease (CKD), eGFR (estimated glomerular filtration rate) was measured using the MDRD equation and cystatin C. The study found strong associations between cystatin C concentrations and several adverse outcomes, including death (hazard ratio (HR 1.33), incident heart failure (HR 1.28), stroke (HR 1.22), and myocardial infarction (HR 1.20). On the other hand, serum creatinine concentrations exhibited weaker associations with each of these outcomes and only predicted cardiovascular death. Participants without CKD but with elevated cystatin C concentrations had a four-fold risk of progressing to CKD within a four-year follow-up period ,42,43.2 when compared with creatinine, and eGFRcys was on average lower than eGFRcreat by 13 mL/min 1.73 m2. This study identified a CKD population at elevated risk for mortality, incident heart failure, and incident atherosclerotic cardiovascular disease that was not detected by creatinine.As previously mentioned, unlike creatinine, cystatin C is unaffected by race or genetic ancestry. Supporting this, a large prospective study on individuals who are South Asian in the UK Biobank by Debbie Chen  found thMore research is needed to identify other high-risk ethnic groups where cystatin C measurements may help identify and stratify the risk of mortality and morbidity.cys and eGFRcys-creat below 85 mL/min/1.73 m2 were associated with increased risks of death from any cause and cardiovascular disease. Current guidelines and practice in CKD define a threshold of 60 mL/min/1.73 m2 with eGFRcreat, suggesting that cystatin C or a combination calculation eGFR can detect patients at risk of cardiovascular disease, particularly patients with deteriorating renal function much earlier, and well above what is currently considered normal renal function [Multiple studies have found that eGFRfunction .cys and eGFRcreat-cys to traditional risk factors for atherosclerotic diseases significantly enhanced the accuracy of predictions for all-cause mortality, as well as fatal and non-fatal cardiovascular disease [cys, whereas the inclusion of eGFR based on creatinine did not contribute to improved discrimination. In this particular cohort study, patients with an estimated glomerular filtration rate based on serum creatinine levels below 60 mL/min/1.73 m2 exhibited either low or high risks of future cardiovascular disease and mortality. Conversely, eGFR based on cystatin C effectively stratified the risk levels in these individuals. These findings indicate that, without cystatin C testing, eGFR alone inadequately distinguishes the broader risks associated with mild chronic kidney disease based solely on serum creatinine levels. Irrespective of the eGFR based on creatinine, the 10-year probabilities of cardiovascular disease and mortality were low when the eGFRcys was equal to or greater than 60 mL/min/1.73 m2. However, with eGFRcys below 60 mL/min/1.73 m2, the 10-year risks were nearly doubled in older adults and more than doubled in younger adults. The use of eGFRcys demonstrated better discriminatory ability for assessing the risk of cardiovascular disease and mortality compared to eGFR based on creatinine. It is important to note that these findings pertain specifically to individuals with a normal urine albumin concentration (<30 mg/mmol), as outcomes were not compared in participants with albuminuria exceeding 30 mg/mmol.In a comprehensive study conducted within the UK Biobank, which included over 400,000 participants, the addition of both eGFR disease ,46. NotaIn a separate analysis conducted on participants with CKD compared to those without CKD in the MESA (multi-ethnic study of atherosclerosis) and CHS , adjusted hazard ratios for mortality were assessed. In the MESA study, the hazard ratio for mortality was 0.80 for CKD identified by creatinine only, 3.23 for CKD identified by cystatin C only, and 1.93 for CKD identified by both markers. Similarly, in the CHS study, the adjusted hazard ratios were 1.09, 1.78, and 1.74, respectively. This pattern was consistent across various outcomes, including cardiovascular disease, heart failure, and kidney failure. It was observed that an unfavourable prognosis was specifically associated with the subset of individuals who were identified as having CKD according to the cystatin C-based equation .p < 0.001). Additionally, when evaluating the ROC-plot area-under-the-curve values, cystatin C demonstrated a closer resemblance to the reference test for GFR (0.926) compared to creatinine . Notably, immunonephelometric methods of the cystatin C assay exhibited significantly stronger correlations than other assay methods .Dharnidharka conducted a meta-analysis of 54 studies to assess the accuracy of cystatin C and creatinine in comparison to the reference standard of GFR . The resCystatin C demonstrated the ability to reclassify patients in CKD 3A categories to milder or non-CKD levels of eGFR, which can have several positive impacts. This reclassification may alleviate the burden on healthcare systems, reduce unnecessary referrals in certain patient groups and allow for healthcare efforts to be focused on individuals at the highest risk. Peralta, in a study conducted in 2011, proposed the development of a triple-marker panel utilizing cystatin C in combination with creatinine and albuminuria . In theicys but a decreased eGFRcreat, their risk level was similar to individuals with a normal eGFR. The author suggests that specifically targeting patients with CKD as defined by creatinine and assessing their cardiovascular risk would require only a small number of cystatin C tests. However, on the other hand, participants who were classified as having a decreased eGFRcys but not eGFRcreat were also found to be at a higher risk for adverse events. Detecting these individuals would necessitate screening a significantly larger number of people compared to confirming a decreased GFR alone.In cases where patients had a normal eGFRThere are three main obstacles to incorporating routine cystatin C testing: the cost, accessibility, and the level of clinical awareness and understanding of test results. Cystatin C testing is approximately ten times more costly than creatinine testing, with a price of GBP 2.50 (USD 3.00) per test compared to GBP 0.25 (USD 0.30) for creatinine .In a retrospective analysis of primary care laboratory requests in Oxfordshire, England, which represented a population of 600,000 individuals, 22,240 people with stable stage 3a CKD and no proteinuria were identified . Since tCystatin C has advantages over creatinine as a marker of kidney function. It is produced at a constant rate by all nucleated cells, and freely filtered by glomeruli, making it less susceptible to individual patient characteristics such as age, gender and muscle mass. Moreover, cystatin C is not affected by non-kidney factors such as diet, drugs and inflammation, which can affect creatinine levels. As a result, cystatin C is a more reliable and sensitive marker of renal function, especially in populations where creatinine measurements may be less accurate.A growing body of evidence supports the use of cystatin C in the diagnosis and management of chronic kidney disease. Studies have shown that cystatin C is more accurate than creatinine in detecting the initial stages of chronic kidney disease and in predicting the risk of adverse outcomes such as kidney failure and death. Furthermore, cystatin C has been shown to be a better predictor of cardiovascular disease and mortality than creatinine, suggesting that it may have broader clinical utility beyond renal function assessment.Given the advantages of cystatin C over creatinine, national efforts should be made to improve the availability and accessibility of routine cystatin C testing. Unpublished data  shows that external quality assessment samples are provided for creatinine and cystatin C to 605 and 17 laboratories, respectively. This is despite all major platform manufacturers providing access to their own cystatin C assay or allowing for third party assays to be run on their analysers.2 (G3a), who may be missed by creatinine-based estimates of GFR, and for patients who are at increased risk of kidney disease due to factors such as diabetes, hypertension, family history or deteriorating renal function. Additionally, patients of non-white or mixed ethnic backgrounds, as well as those with increasing age or frailty, may benefit from cystatin C testing, as these factors can affect creatinine levels and lead to inaccurate estimates of GFR. Finally, patients at weight or raw muscle mass extremes, including those with dystrophic diseases, may also benefit from cystatin C testing, as these conditions can affect creatinine production and clearance.This will be especially important for patients with mild kidney disease of eGFR 45–59 mL/min/1.73 m


The Arctic Ocean’s Beaufort Gyre (BG) is a wind-driven reservoir of relatively fresh seawater, situated beneath time-mean anticyclonic atmospheric circulation, and is covered by mobile pack ice for most of the year. Liquid freshwater accumulation in and expulsion from this gyre is of critical interest due to its potential to affect the Atlantic meridional overturning circulation and due to the importance of freshwater in modulating vertical fluxes of heat, nutrients and carbon in the ocean, and exchanges of heat and moisture with the atmosphere. Here, we investigate the hypothesis that wind-driven sea ice transport into/from the BG region influences the freshwater content of the gyre and its variability. To test this hypothesis, we use the results of a coordinated climate response function experiment with four ice-ocean models, in combination with targeted experiments using a regional setup of the MITgcm, in which we rotate the surface wind forcing vectors (thereby changing the ageostrophic component of these winds). Our results show that, via an effect on the net thermodynamic growth rate, anomalies in sea ice transport into the BG affect liquid freshwater adjustment. Specifically, increased ice import increases freshwater retention in the gyre, whereas ice export decreases freshwater in the gyre. Our results demonstrate that uncertainty in the ageostrophic component of surface winds, and in the dynamic sea ice response to these winds, has important implications for ice thermodynamics and freshwater. This sensitivity may explain some of the observed inter-model spread in simulations of Beaufort Gyre freshwater and its adjustment in response to wind forcing. The Beaufort Gyre is the largest reservoir of liquid freshwater content (FWC) in the Arctic Ocean  of sea ice into/out of the region. In particular, we hypothesise that net ice import into the gyre region should bolster Beaufort Gyre liquid FWC, while net ice export should act to drain liquid FWC. We investigate this hypothesis both under natural variability and under forced change .Sea ice growth rate is determined by a combination of thermodynamic and dynamic processes  pattern. In this scenario, whether ice converges into the regions or diverges, depends on the angle between ice flow and isobars. Note that there are other potential scenarios in which ice transport into/out from the region could occur.In the atmospheric and oceanic boundary layers, friction breaks the geostrophic balance, causing flow to deviate from isobars. The observed flow, mentclass2pt{minimG, Coriolis parameter, f, and the roughness length, G, Surface wind is rotated anticlockwise relative to geostrophic wind in the northern hemisphere, and clockwise in the southern hemisphere. Observed turning angles show a considerable range: values of 6ean Gray . There aean Gray , implyinair-ice angle) also varies substantially with these parameters  the former turns to the left, while the latter turns to the right, the cross-isobaric angle of ice flow tends to be relatively small, though it shows considerable spread in observations, as well as systematic spatial, seasonal, and long-term variability  is the time integral of the characteristic impulse response function. The latter can be convolved with a time history of the forcing to estimate a time history of the response, as per linear response theory  experiment . Two anomaly patterns were used: an anticyclonic pattern based on a strengthened Beaufort Sea High  total freshwater content, as Carmack ; 2 sea iBasic information about the participating models is shown in Table The models are all ice-ocean models, with prescribed atmospheric forcing, including near-surface winds, air temperatures, humidity, downward longwave and shortwave radiation, and precipitation , eddy-mediated isopycnal slumping, and effective ice-ocean governor strength. Here, however, we focus on the potential role of sea ice transport in explaining some of the observed differences Fig. c, d.Fig.Two models show approximately no response in ice volume import under BGP: the Alberta NEMO model and the MITgcm  climatology, and examine how an angular modification to the surface winds changes that climatology. Next, we investigate the system under forced change: we compare the results of the standard CRF experiments as described in Sect. In the MITgcm control run (CTRL), sea ice at the end of winter has a mean thickness of 2–3 m in the gyre region Fig. a, with iThe integrated FWC within the gyre region is shown in Fig. Comparing the MITgcm CTRL and CTRL10 runs highlights the impact of the 10The 1000 FWC Fig. a. As sucFWC Fig. .Spatially integrated changesWe now investigate the response of Beaufort Gyre FWC to wind forcing, and the hypothesised role of sea ice transport in influencing this response. Fig. entclass1pt{minimaIn the BGP CRF experiment, the imposed anticyclonic winds Fig. a induce ars Fig. ; note hoars Fig. , detectaars Fig. . There imentclass2pt{minimThe BGP10 CRF . These exceed the resulting anomalies in FWC. The lower magnitude of change in FWC is most likely due to the fact that the sink terms of FWC are FWC-dependent , and a sink due to eddy fluxes  changing the cross-isobaric angle of surface winds by 10ogy Fig. ; (b) depogy Fig. . We suggogy Fig. ; accuratogy Fig. . Our res


Nat. Mater.2020, 19, 663−66832015533) and Komatsu et al. . With our present calorimetric studyon the transition from pure cubic ice to hexagonal ice we are ableto clarify the value of the enthalpy change ΔHc→h to be −37.7 ± 2.3 J mol–1. The transition temperature is identified as 226 K, much higherthan in previous work on ice Isd. This is due to a catalyticeffect of hexagonal faults on the transition, but even more importantlydue to a relaxation exotherm that was not properly identified in thepast.The preparation ofpure cubic ice without hexagonal stackingfaultshas been realized only recently by del Rosso et al. ( While hexagonal ice shows an ABABstacking (where A is a mirror image of B), cubic ice consists of layersin an ABCA arrangement. However, all “cubic ices” studiedin the past actually contain significant numbers of hexagonal stackingfaults, depending on the mother phase they crystallized from.4 In retrospect, all these ices should have beentermed stacking-disordered ice (ice Isd), but not ice Ic.5 Studying the relative stabilitiesbetween the three different ice I polytypes has so far not been possiblein experiments due to the unavailability of pure ice Ic. Especially the enthalpy difference between ice Ic andice Ih ΔHc→h isnot known, but of fundamental interest. While it is settled that iceIh is the stable phase in the bulk at atmospheric conditions,simulations on mW water by Lupi et al. show that, for very small sizes, in fact ice Isd is more stablethan both ice Ic and ice Ih at 230 K.6 This is rationalized in terms of entropy, specificallyentropy gain through stacking-disorder, and supported by experimentalwork from Mayer & Hallbrucker,7 whofirst showed that ice Isd first nucleates from rapidlycooled liquid droplets at <200 K, before transforming to stableice Ih. While there is considerable computational workon thermodynamic stability of the ice I polytypes, utilizing differentwater potentials  as well as ab initio methods,17 experimental data on the Gibbs free energy, enthalpy, and entropyare notoriously difficult to access. Hondoh et al.19 approached this question applying X-ray topography to study formationand annihilation of stacking faults and reported a free energy costof 0.31 mJ m–2 (16.5 J mol–1 6) at 253 K, associated with the annihilation ofa hexagonal stacking fault in ice Ic.Cubic ice , this study isaimed at the open question about the enthalpy difference between iceIc and ice Ih, ΔHc→h.In terms ofenthalpy difference, numerous calorimetric studiesare available that have scrutinized the transition from differentstacking-disordered ices I70 K see 1, prior 32 or noctilucent/polar mesospheric clouds (PMCs) at even higher altitudesin Earth’s mesosphere (∼80 km).33 While there has been consensus that ice Isd exists inEarth’s atmosphere in addition to predominant ice Ih,38 likely based on preferential formation of small stacking-disorderedcrystallites via homogeneous nucleation,6 a recent study has shown that cubic ice crystallites preferentiallyform via heterogeneous nucleation on graphene or h-BN at ∼110K,39 a plausible mechanism at mesosphericconditions.In addition to therelevance for thermodynamics ofice I polytypes,this study has further implications for our understanding of fundamentalprocesses in Earth’s atmosphere, e.g., the formation of coldcirrus clouds in the upper troposphere2O cubic ice transformed from XVII, that is, void of hexagonalstacking faults.2We here present differential scanning calorimetry(DSC) measurementsof the cubic to hexagonal ice transition, starting from pure Hc between 160 and 250 K. c. For stacking-disordered hexagonalice the 111 peak is usually significantly broadened and shows a shoulderat ∼22.8° which originates from hexagonal stacks. In ourcase, there is a tiny peak at ∼23.00°, where the intensityis less than 3% of the 111 Bragg peak intensity. Other Bragg peaksof ice Ih are below the noise level of the measurement, which cp(T)in –1 clearly showsthat the Ic to Ih transition appears as a sharpexothermic feature centered around 231 K, with an onset temperature Tonset of 226 K .In addition, we performed DSC scans of ice Ic at heatingrates of 10 and 50 K min–1, successively  takesplace prior to the transition itself. As evident in c to Ih transition. Employing heating rates of 10–50 K min–1 allows for clear separation of the relaxation exothermfrom the Ic to Ih transition in the presentstudy. That is, the prepeak is related to the powdering of the sample,size of grains, etc., but unrelated to the inherent rearrangementof cubic to hexagonal stacking sequences.Furthermore, our heating experimentsof cubic ice exhibit a broadand weak exothermic feature prior to the polytypic transition, centeredaround ∼193 K 1a. It shHc→h(ice Isd) in these studies is less negative, closer tozero, than the apparent ΔHc→h(ice Isd) reported by the authors. We mark the studieswhich likely overestimated the enthalpy change by (*) in 28 performed heating scans of differentsamples of stacking-disordered ice Isd, crystallized fromice V and ice VI, respectively, at ∼0.17 K min–1 and observed only one exotherm between ∼175 and 220 K. Thiscan be rationalized by the low heating rate and a catalytic effectof the hexagonal stacking faults within the cubic ice sample, loweringthe onset temperature of the cubic to hexagonal transition. As a result,the two exothermic events ((i) relaxation and (ii) transformationof ice Ic to ice Ih) merge to one. Therefore,the value of ΔHc→h was overestimatedto be ca. −50 J mol–1,28 very close to the sum of the two heats reported here. Asimilar catalytic effect was also found in a Raman spectroscopy studyof the ice Ic-Ih transformation in the samepure cubic ice samples as studied here,45 where an even slower heating ramp  was applied, and the transformation was completed at temperaturesbelow 200 K. That is, even the tiny amount of hexagonal stackingfaults noted in 29 likely reported a too-large (negative) enthalpychange, even though their value (−37 ± 1 Jmol–1) coincides with the value presented here. However, in light of thepresent study, their integration limits were chosen too broadly. Consideringtheir Figure 12 of ref  and ice Ih (ph),a key question for supersaturation above ice clouds and the mechanismof cloud formation.46 Based on the Clausius–Clapeyronequation, the ratio of the vapor pressures is often approximated32 asWith the presentfinding of ΔHc→h into c is only 3% higher thanthe one of ice Ih at 170 K and only 2% higher at 230 K.In terms of absolute vapor pressure of cubic ice, we can then usethe vapor pressure reported for ice Ih by Marti & Mauersberger47 as well as Mauersberger & Krankowsky.48 Adding the 3% and 2%, the absolute vapor pressureabove ice Ic calculates as 7.19 × 10–4 Pa at 170 K and 10.2 × 10–4 Pa at 230 K.The vapor pressure of ice Isd particles in clouds willinevitably be between the two values, i.e., even closer to the oneof ice Ih. Any form of stacking-disordered ice containinga significant amount of hexagonal stacking faults will inevitablyshow a less negative ΔHc→h and accordingly an even smaller difference in vapor pressure comparedto pure hexagonal ice. That is, our result is inconsistent with measurementsreporting a difference in vapor pressure of ∼10% between iceIsd and ice Ih,49 with the needof clarification and more direct measurements of the vapor pressureof ices Ic and Isd in the future. Calorimetricstudies providing values more negative than −37.7 ± 2.3J mol–1 are affected by relaxation effects and peakmerging without proper separation from the exotherm related to thetransition itself  a transition temperature above240 K was found. It would be very interesting to study a sample preparedin that manner calorimetrically, but so far such samples have notbeen recovered for ex situ measurements. Knowledge of the phase behaviorof the different polytypes of ice I, e.g., their stability with respectto each other combined with remote sensing, will facilitate new insightsof the thermal history of celestial bodies.53Moreover, the measured onset temperatures of thecubic to hexagonalice transition of 220, 226, and 230 K at 10, 30, and 50 K minc was obtainedby heating a powder of iceXVII to 150 K. The precursor material ice XVII was previously obtainedby means of a suitable thermal treatment of hydrogen hydrate samplesin C0 structure.54 In totalwe recorded nine heating scans at 10, 30, and 50 K min–1 of samples of ∼10–20 mg of cubic ice in aluminumcrucibles using a DSC8000 by PerkinElmer. After thefirst heating scan, the sample (after transformation to ice Ih) was cooled down to ∼93 K and reheated in a secondheating scan. This second scan (of ice Ih) was used forbaseline correction . Powder


This study focuses on the conversion of solid CO2 into pellets using ram extrusion, a widely used technique. The length of the die land (DL) in this process plays a critical role in determining the maximum extrusion force and the density of dry ice pellets. However, the influence of DL length on the characteristics of dry ice snow, known as compressed carbon dioxide (CCD), remains understudied. To address this research gap, the authors conducted experimental trials using a customized ram extrusion setup, varying the DL length while keeping the other parameters constant. The results demonstrate a substantial correlation between DL length and both the maximum extrusion force and dry ice pellets density. Increasing the DL length leads to a decreased extrusion force and optimized pellet density. These findings provide valuable insights for optimizing the ram extrusion process of dry ice pellets and improving waste management, energy efficiency, and product quality in industries utilizing this technique.The management of waste materials, particularly non-biodegradable substances such as plastics and composites, is an increasingly pressing issue. Energy efficiency in industrial processes is crucial throughout their life cycle, including the handling of materials such as carbon dioxide (CO Management of waste materials is becoming a topical issue for researchers and a growing challenge for civilisation. This concerns in particular the materials that are not inert to the natural environment, such as plastics ,2 and coWaste materials that have a considerable effect on the natural environment include carbon dioxide . As can Carbon dioxide has many uses in its solid state. Its temperature-related physical characteristics  ,13 are dIn order to be used in these applications, carbon dioxide must be turned into pellets. Ram extruders are the machines used for this purpose. The ram extruder’s working system has been extensively described in the literature, for example in  where thFC) equals the resistance force (RF), depending on the geometry of the die that the material is pushed through [Z axis. The transversal shape of this portion of the die cavity approximates the shape of the pellets that leave the die. After the completion of the densification process, the compaction piston is withdrawn to its initial position.The process of extruding dry ice pellets is carried out in the sequence of stages presented in  through . While mσ0) may substantially exceed the material’s cohesion. Thus, we can adopt a non-zero absolute value of the deformation tensor (ε). With constant transverse dimensions over the whole DL length, in axes other than the Z axis, ε equals zero. If the transversal dimensions of the pressed material are constant, the phenomenon of stress relaxation comes into play. This phenomenon is described, for example, with the Maxwell model for linear viscoelastic response. It combines a purely elastic body with an ideally viscous body, represented by a spring and a damper  resulting from the material relaxation phenomenon is time-dependent. In this case, t depends on the DL length (DLl) and the speed of movement of the material   and the inS to outS ratio . Now Equation (2) may be rewritten as follows:According to Biszczanik et al. 2021), on the way through the convergent section, the density of pressed dry ice does not change considerably [21, on thDLt in Equation (1) yields:Substituting ε after leaving the die. Note that the value of ε has a direct bearing on the extrudate density (ρ), a parameter indicated as one of the dry ice pellets quality indicators [According to Biszczanik et al. 2021), dry ice pellets show elastic behaviour [21, dry iHowever, the Maxwell model does not take into account the material sublimation phenomenon. Note that the sublimation rate may vary considerably depending on the friction between the DL section’s sides and the pressed material.Reports from the investigation of the influence of the DL length and surface condition on the end product density and process forces can be found in the literature . However2 into a solid form. The material crystallises in this way. The density of the obtained loose dry ice is 550 kg/m3 [Dry ice powder is obtained by expanding the atmospheric pressure of liquid carbon dioxide stored in a tight container at −18 degrees Celsius and 20 bar pressure. This rapid expansion to the atmospheric pressure results in an adiabatic process and converts liquid CO50 kg/m3 .For the purposes of this experimental research, loose dry ice was stored in a 30 L insulated DRICY dry ice storage container  manufactIn the process comprising the compression and extrusion phases, loose dry ice was pushed by a ram through a single-cavity die. The experimental setup was built by the authors specifically for the verification of limit stress in the dry ice extrusion process. In this research, this test setup was mounted in the grips of the MTS Insight 50 kN test frame manufactured by MTS Systems Corporation . The test setup is described in detail in earlier reports .ZF) exerted on the 20 mm dia. ram. A 50 kN accuracy class 0.01 strain gauge sensor was used for this measurement, supplied as standard with the MTS Insight 50 kN test frame.The measured variable was the force l, which varied from 0 mm to 50 mm. The geometrical parameters of some of the tested dies are illustrated below  were used in the first stage. Next, measurements were carried out on dies with smaller DLl intervals falling in the range determined based on the previously obtained results. The details are given in For the purpose of the investigation subject, it was required to determine the The force measurement method used in this study was described by Biszczanik et al. (2023) . Dry iceThe specific test setup elements were cooled prior to measuring in order to reduce the effect of temperature by minimising the loss of the material mass through sublimation. The parts were cooled by submersion in dry ice to obtain a temperature approximating the temperature of the test material itself. The compaction chamber, along with the die and the ram, were cooled for 30 min before the tests. Next, during the tests, these parts were re-cooled for 10 min after every three consecutive material extrusion cycles (approx. every 6 min) in order to keep the temperature of the test setup close to the dry ice temperature.The MTS Insight 50 kN test frame equipped, with the TestWork4 software version 4.0, makes it possible to simultaneously record the extrusion force and the ram displacement. The first operation performed before the proper measurements were complete was to plug the end of the die placed in the chamber by pouring, compacting, and extruding the material several times (these results were ignored). As a result, the densified material remained in the die, which prevented the dry ice from getting through the die while filling the chamber with it. Only after cohesive pellets extruded through the die were obtained, could the proper testing start. In the course of the tests, the compaction chamber was filled with loose dry ice, the amount being 20 g pre-weighed on the Keller PCB10000-1 scale  with 0.1 g accuracy. In the next step, the program was initialized, moving the ram at a speed of 5 mm/s into the chamber and stopping 1 mm before the die edge (to prevent its possible damage), and simultaneously recording the force values and displacements. Ten tests were carried out for each die.CmaxF) are described in the groups assigned to the tested single-cavity dies.The recorded maximum forces (m0) is compared with m1 (the weight of the same sample), immersed in the test liquid of known density (ρTL). The test setup is shown in The hydrostatic method was chosen considering the sublimation of the tested material and the irregular shape of the produced pellets. In this method, the dry weight of the sample  and a Testo 440 multimeter  (8) to read the sensor output signal.Isopropyl alcohol was used to allow for the cooling of the liquid to a very low temperature. The value of ρTL and ρCCD, based on the measured values of m0, m1, and TTL. The equations used to calculate ρTL and ρCCD are given below:The equations derived in the previous studies  allowed CmaxF and ρCCD between the data groups. STATISTICA version 13.3, a program of TIBCO Software Inc. was used for the statistical analysis of the experimental data. In all, the comparison one-way test was applied with statistical significance, determined by the value of p below 0.05.One-way ANOVA, along with Tukey’s post hoc test, was used to assess the mean differences of The statistical significance of the differences between the data in the DL0, DL10, DL20, DL30, DL40 and DL50 populations was verified by means of the Tukey post hoc test. For this purpose, the null hypotheses of ANOVA were first verified.CmaxF, the value of p varied from 0.11 to 0.80. For ρCCD, the range was 0.16–0.62. The values obtained for each of the tested populations exceed 0.05, which confirms the normality of the data.The Shapiro–Wilk test was applied in the first step to verify the normality of the data in the populations. In the case of p was 0.54 for CmaxF and 0.84 for ρCCD, indicating the homogeneity of variance in both cases.Next, Levene’s test was used to verify the homogeneity of data distributions. The value of DLl equal to zero and the other dies under analysis. The general population data are given in The analysis of variance with Tukey’s post hoc test showed statistically significant differences between the results obtained for the die with an DLls of 5 mm and 2.5 mm. In this way, it was determined that the DLl variation range should be limited to the range of 0–2.5 mm.We decided to carry out additional tests for DLls in the range of 0–1.5 mm are different from other populations at a statistically significant level. The key statistical data obtained for these two tests are given in In the next step, the tests were carried out for dies with DLs equal to 0, 1, 1.5, 2 and 2.5 mm, respectively. The same, as in the previous assessment, Shapiro–Wilka and Levene’s tests were used to verify the ANOVA null hypotheses. This verification confirmed the normality of data in the respective populations and the homogeneity of variance. The statistical significance analysis showed that the data obtained for the dies with ρCCD, as a function of DLl, we see a decrease in the variation of ρCCD values for DLls greater than 1.5 mm. Therefore, we can assume that the stress resulting from the plastic deformation of the sample was reduced to a level at which its effect on the geometrical parameters of the pellets becomes ignorable.Analysing the change of DLl for the relaxation to take place in the compacted carbon dioxide should be taken at 1.5 mm. Substituting these parameters in Equation (3), it is now possible to calculate DLt:Based on this conclusion, the minimum value of σ) calculated with Equation (1), the value of stress inside the pellets is reduced to a level at which it no longer causes any significant deformation of the extrudate.Due to the exponential growth of stress  [ρ [3).In the literature, η and E) . Considend E) [ρ ,30, the This experimental research showed the need for a cylindrical section in dry ice compaction and extrusion dies. It was found to have a considerable bearing on the extrudate density and extrusion force required in the process. The indicated information has also been confirmed in the case of the densification process of other particulate materials  ).3 and a ram speed of 5 mm/s, the minimum relaxation time was 0.78 s. This allowed for a reduction of the stress induced inside the pellets to a level at which it no longer caused any significant deformation of the extrudate and, consequently, no density change.For the extrudate density of ca. 1540 kg/mThe outcome of this research could serve as the basis for further investigations of the dry ice relaxation phenomenon. The authors plan to use the Maxwell model described in this article and also the Kelvin–Voigt model to develop a material model for use in numerous studies ,32. SuppThe future objective is to advance the research and development efforts to improve the energy efficiency of industrial and geological processes used to increase the density of solid carbon dioxide ,34.


Background and Objectives: Fractures of the proximal humerus are common, particularly in elderly populations. Anatomical locking plates target stabilization with a multitude of screws spanning into the humeral head. Sound implant placement and screw length determination are key for a successful clinical outcome but are difficult to obtain from planar X-rays. A novel implant positioning technology for proximal humerus plating (Xin1) outputs screw lengths suggestions and plate position based on hole projections in conventional X-ray images. This study investigated the performance of a prototype Xin1 system in a postmortem (in vitro) experiment as well as in a clinical handling test. Materials and Methods: For in vitro testing, twelve shoulders from six anatomical specimens were randomized into two groups to compare the Xin1 technique to the conventional operation in terms of surgical precision, procedure time and X-ray exposure. For the clinical trial, 11 patients undergoing plating of the proximal humerus were included. The aim was to investigate clinical handling of the Xin1 marker clip and to retrospectively evaluate the system performance in a real-life fracture situation. Image pairs before and after insertion of the proximal screws were retrospectively processed to investigate the influence of potential bone fragment shifts on the system output. Results: In the postmortem experiment, the use of the system significantly improved the surgical precision (52% error reduction), procedure time (38% shorter) and radiation exposure (64% less X-rays). Clinical handling demonstrated seamless embedding of the marker clip into existing clinical workflows without adverse events reported. Retrospective X-ray analysis on six eligible patients revealed differences in the calculated screw lengths of ≤2 mm before and after screw insertion for five patients. In one patient, the screw lengths differed up to 8 mm, which might indicate displacement of the head fragment. Conclusions: Results suggest a strong potential of the Xin1 assistance technology to enhance the surgical procedure and patient outcomes in the rising incidence of osteoporotic humeral fractures. Robust performance in a real-life fracture situation was observed. In-depth validation of the system is, however, needed before placing it into clinical practice. Proximal humerus fractures are among the most common injuries ,2. PlateAdequate plate positioning  and deteWe recently proposed a simple and generic tracking technology (Xin1—Implant Positioning Assistance) utilizing conventional radiographic images to determine 3D positions and orientations of implants, instruments and anatomy . The sysThe purpose of this study was (1) to assess the performance of the Xin1 system for application at the humerus in comparison to the manual procedure in terms of accuracy, X-ray exposure and procedure time in a postmortem study; and (2) to investigate performance and handling of the system in a clinical fracture situation by retrospectivly comparing the system output before and after screw insertion as a measure for the influence of potential bone fragment shifts.The Xin1 technology is a generic, X-ray-based tracking and navigation concept, designed to assist implant positioning in various orthopedic applications. The underlying principle is based on feature extraction from projections of cylindrical holes in X-ray images for determining the spatial alignment of the implant and anatomy. It was first described by Windolf et al.  for the For proximal humeral plating, a stainless-steel marker comprising three cylindrical holes arranged in a defined pattern and two polymer clamps is clipped on a drill sleeve which is attached to a conventional anatomic locking plate  . The nosFor performance assessment, the Xin1 system was compared to the conventional free-hand method in 12 intact, non-fractured shoulders from 6 formalin-embalmed, full-body specimens , which were pre-used for medical student courses. The experiment was performed at the Department of Anatomy of the Medical University of Innsbruck, Austria. Left and right shoulders were randomized into Xin1 and conventional groups. All operations were performed by a single operator (DK), who was at the time the resident surgeon with moderate surgical experience. For both groups, a deltopectoral approach was created and a PHILOS plate was prepositioned and fixed with a cortex screw in the elongated combi-hole F-level, . A diffeFor the conventional procedure, the plate position and screw lengths were estimated via repeated fluoroscopy and by use of a length probe  with a target TJD of 5 mm. Eight screws  were placed using the screw insertion guide . Only screw hole 7 on the D-level was left empty. Plate and screw placement was verified via fluoroscopic imaging.For the Xin1 procedure, a marker clip was additionally attached to the drill sleeve . After pFor the TJD accuracy measurement, the humeral head was dislocated from the joint, and the screw tip-to-joint distances were measured including the cartilage layer for all screws by inserting a K-wire into the over-drilled hole until touching the screw tip. The insertion depth was then marked with forceps and measured with a caliper . CartilaNon-parametric independent sample tests (Mann–Whitney U) were performed to compare both techniques regarding accuracy (TJD and TJD error), procedure time, number of fluoroscopic images and radiation time. All analyses were performed using SPSS software version 27 . For all tests level of significance was set to α = 0.05.Eleven patients undergoing plate fixation osteosynthesis with PHILOS were enrolled in a focused registry at a single medical center . The clinical trial was approved by the local ethical committee and the Federal Agency for Medicines and Health Products Belgium  and was registered at clinicaltrials.gov (NCT03427112). Informed consent was obtained from all participants before surgery. The standard surgical protocol was maintained, except for attaching an Xin1 marker clip after plate prepositioning. The marker clip was manufactured from medical-grade stainless steel and PEEK (Polyetheretherketon) and was designed for reuse in a standard hospital surgical instruments workflow including autoclaving and cleaning. Intraoperative X-rays from two stages, before and after screw placement, were stored in a DICOM  format and were retrospectively evaluated with the Xin1 software. No system feedback was provided to the surgeon during operation. Placed screw lengths during surgery were noted for all operations.Image pairs from both stages were formed from the available X-rays and processed with the Xin1 software. An image pair was found valid if the view angle between the images exceeded 15°. So that several valid pairs could be formed, the pair with the largest view angle was selected. Screw lengths and plate positions were computed from the selected pairs for both stages. The plate position is calculated in relation to the anatomy, defined as the offset of the calculated center of the humeral head from the central screw trajectory  in cranial–caudal and dorsal–ventral directions . To investigate the effect of potential head displacement during screw insertion in a real fracture situation, the differences in screw length before and after fixing the plate with screws . The time reduction compared to the conventional technique was largest during a screw placement with 59% , compared to 21:41 ± 4:31 min for the Xin1 technique. This total time reduction of 38% when using Xin1 was statistically significant (with 59% .p = 0.002). The largest X-ray reduction was found during the screw placement phase with 84% (p = 0.002).The number of fluoroscopic images was on average 30.3 ± 5.7 (mean ± SD) with the conventional technique and 10.8 ± 2.5 images with Xin1. This reduction of 64% when using the Xin1 system was statistically significant (with 84% . Accordip = 0.053). However, the absolute error of the screw length deviating from the target TJD of 5 mm was on average 2.2 ± 1.6 mm for the conventional technique and 1.0 ± 0.7 mm with Xin1. The error, hence, diminished by 52% when using the Xin1 system, which was statistically significant (p < 0.001). In each study group 48 screws were placed. Two joint perforations occurred in the conventional groups, while in the Xin1 group no perforations were observed. The mean tip–joint distance (TJD) was 5.8 ± 2.6 mm (mean ± SD) with the conventional technique, and 5.1 ± 1.3 mm when using Xin1 . This diEleven proximal humerus fractures  from eight female and three male patients were operated by five different surgeons. The average age of patients at surgery was 67 years and ranged between 47 and 84 years. Fracture types (AO classification) were: 1× 11A2, 1× 11A3, 3× 11B1, 1× 11B2, 4× 11C1 and 1× 11C3. With one exception, where a Philips Zenition 70 system  was used, all intraoperative images were acquired with a Siemens Arcadis Varic  C-arm. Handling of the marker clip in the hospital environment and during surgery was feasible in all patients and did not interfere with hospital processes or surgical procedures. No adverse events concerning the use of the marker clip were recorded for the 11 performed operations. For the retrospective image analysis, five out of eleven patients needed to be excluded for various reasons: for example, one patient was excluded due to the intraoperative use of a wrong drill sleeve, leading to the mispositioning of the marker clip. For two patients, no images after screw insertion were stored to the C-arm. For another two patients, the image pair view angle was too narrow for calculation.Xray images of the remaining six patients underwent retrospective evaluation. Robust marker detection, humeral head segmentation and screw length determination were achieved in all image pairs. The view angle between the images of a pair was on average 27° (range 19°–34°). The calculated screw lengths before and after screw placement differed on average by 1.2 ± 1.7 mm. In five out of six patients, this difference was ≤2 mm for all screws. In one patient (#10), differences up to 8 mm were found. p < 0.001, A total of thirty-seven screws were placed in the six evaluated patients. Used screw lengths ranged from 32 to 50 mm. The Xin1 calculation before screw placement correlated significantly with the actual screw lengths implanted , the plate was displaced by 10 mm in the dorsal–ventral direction after screw placement according to the Xin1 measurement .The Xin1 implant positioning system, as introduced in , uses exAnatomical locking plates such as the PHILOS system stabilize proximal humerus fractures by spanning a multitude of screws in the often-osteoporotic head segment. It is aimed to anchor the screw tips in the subchondral bone region, close to the cartilage layer. The task is surgically demanding due to an eminent risk of screw penetration into the joint space while trying to judge an oblique screw path inside a spherical anatomical structure from planar X-ray projections. On the other hand, too-short screws miss anchorage . FailureIn the scope of this work, the prototype Xin1 system was evaluated in terms of performance in both postmortem and clinical settings. The system estimates the plate position and calculates all required screw lengths to reach into the subchondral bone area from a pair of conventional C-arm images. Xin1 outperformed the conventional freehand technique in terms of surgical precision (52% error reduction), procedure time (38% shorter) and radiation exposure of OR personnel and the patient (64% less X-rays). Particularly for medical professionals, the continuous exposure to ionizing radiation during surgery is a frequently addressed problem ,12,13. TThe postmortem experiment was performed on full-body specimens with compromised soft tissue mantle due to embalmment and previous use. However, we believe that the experiment carried high relevance for judging the technology before entering into clinical testing. One methodological issue, though, could not be neglected, which was the absence of bone fractures. In a second phase, we hence conducted a clinical registry on fracture patients to investigate the potential effect of bone fragment shifts on the system output, and at the same time to analyze the clinical handling of the Xin1 marker clip. Safe handling was shown in all 11 included patients by the absence of device-related adverse events. To minimize risks to the patient at the current state of system development by potentially falsified system outputs, X-ray image analysis was performed in a retrospective fashion without providing system information to the operator during surgery. The missing feedback in the operating theatre reduced risks but also led to a high number of patients, who needed to be excluded from the image-based evaluation (five out of eleven). These dropouts are deemed to be artifacts from the retrospective study design and may be avoided in the future when real-time feedback is provided to the operator. A stereotactic system always relies on an angular spread of at least two view ports to reconstruct a 3D situation . WithoutBesides these general observations, the clinical study confirmed the feasibility of the marker clip to be embedded into hospital and surgical workflows. The system software demonstrated robust performance in presence of real-life bone fractures. Certain head fragment shifts due to manipulations during the screw insertion were suggested by differences in the computed screw lengths and measured plate position before and after screw insertion. Shifts were generally small, but in one case they reached 10 mm in the dorsal–ventral direction. Clinical consequences such as screw perforation into the joint cannot be excluded in such cases. It is therefore proposed to perform a verification measurement with the Xin1 system after the head fragment has been fixed in place with the first screw. On a critical note, it cannot be ruled out that the measured differences are artifacts related to inaccuracies of the system rather than describing actual bone fragment shifts. In this regard, the segmentation procedure to determine the position of the humeral head, still relying on user input, might play an important role. Inaccurate determination of the humeral head boundaries will impact the accuracy of the system. Fully automated segmentation may be desired in the future. In a previous bench experiment , the angStudy limitations are summarized as the following: The postmortem experiment was performed on unfractured embalmed body donors with compromised soft tissues. The effect of intact soft tissues and fractured proximal humerus was hence difficult to estimate from this sub-study. Furthermore, the tip–joint distance evaluation assumed a constant cartilage layer thickness, which naturally varies between locations and individuals. Procedure time assessment did not take system setup and takedown durations into account, and thus represents only a net value. The clinical registry allows only for preliminary conclusions at this early development stage because of the missing system feedback in the operating room. This also led to a considerable number of dropouts lowering the sample size for image evaluation. As desired for a clinical handling investigation, several surgeons shared the surgeries. This, on the one hand, gave valuable insights about marker clip usage in different hands, but might have contributed to the large number of excluded datapoints for image analyses. However, several confounders regarding the Xin1 approach for proximal humerus plating could be identified from the studies, including: potential marker movements, risk for use of wrong implants and instruments, omitted use of a screw guide, bone fragment shifts due to surgical manipulation, a too-narrow X-ray view angle or the segmentation procedure of the humeral head requiring manual interaction. Furthermore, some technical limitations rendering validation and regulation of such assistance devices difficult are to be mentioned. Optical image distortion of older image intensifiers can drastically influence the system accuracy. Flat panel-based C-arms solve this issue and currently replace image intensifiers around the globe. Still, interfacing to the imaging modalities remains an issue. A variety of analog and digital output protocols make connecting external devices difficult. Important parameters such as flip or rotation flags are not transmitted. The ability to run third party image processing software directly on the C-arm would be an important prospect for future developments, dramatically increasing usability and effectiveness of assistance technologies such as Xin1.This study demonstrated the potential of a newly proposed assistance technology for implant positioning in proximal humerus plating using conventional X-ray projections. In an in vitro setting, the system significantly improved surgical precision, reduced X-ray exposure and shortened surgery time. In a clinical handling test, the device demonstrated seamless embedding into existing clinical workflows and robust performance in a real-life fracture situation. However, certain issues crystallized and need to be addressed before placing such system into clinical practice. A strong potential to improve patient outcomes in the rising incidence of osteoporotic humeral fractures is eminent.