Premature ovarian insufficiency (POI) can result in lower fertility and shorten the female reproductive span. Bushen-Culuan Decoction (BCD) is a traditional Chinese medication utilized for treating POI for many years. We previously observed that BCD protects against further deterioration of the ovarian reserve of POI patients, however, the underlying mechanism has not been well studied. Our investigation seeks to evaluate the effect of BCD on POI induced by Tripterygium wilfordii polyglycosidium (TWP) and the likely mechanistic pathways, which we hypothesize may involve the Nrf2/ARE pathway. The body weights, estrous cycle, serum hormone levels, histological follicular analysis and quantification, levels of oxidative stress biomarkers in the ovarian tissue of POI mice models were evaluated. Western blotting and RT-PCR enabled quantification of the components of the Nrf2/ARE pathway. Our results showed that BCD restored hormonal profiles and estrous cycles of POI mice similar to those observed in healthy controls. BCD reduced the numbers of atretic follicles while increasing the number of primordial follicles. BCD facilitated lower 8-OHdG and MDA levels while increasing levels of key antioxidant enzymes including GSH-Px, CAT, and SOD. Furthermore, TWP increased Bach 1, Nrf2, and Keap 1 expressions at the translational level, while decreased that of HO-1. BCD treatment also promoted nuclear translocation rates of Bach 1 and Nrf2, suppressed Keap 1 protein expression, as well as raised HO-1 protein expression. Taken together, BCD likely augments ovarian reserve by activating the Nrf2/ARE signaling pathway, which stimulated higher levels of antioxidants and suppressed oxidative stress. BCD may be an important therapeutic compound in POI. As an increasing proportion of women in modern society postpone childbearing due to lifestyle, career and financial constraints, premature ovarian insufficiency (POI) has become an increasingly common clinical occurrence. POI mainly occurs in women younger than 40 years old who experience early depletion of ovarian follicular reserve . POI is Tripterygium wilfordii polyglycosidium (TWP) is well known for its role in treating inflammatory and autoimmune conditions. However, this component is notorious for inflicting severe ovarian toxicity, resulting in premature menopause and sterility . PreviouOxidative stress arises when reactive oxygen species (ROS) overwhelms the protective capacity of intrinsic antioxidant mechanisms, resulting in DNA, protein, and fat destruction of cell. Byproducts of oxidative stress damage include 8-hydroxy-deoxyguanosine (8-OHdG), which represents oxidized DNA, and malondialdehyde (MDA), a lipid peroxidative byproduct, all of which impairs cellular homeostasis . The nucIn physiological conditions, Nrf2 is primarily localized in the cytoplasm with a relatively low nuclear level. This is because Nrf2 is incorporated into the Keap1-Cul3 complex in the cytoplasm, resulting in increased proteasomal degradation and ubiquitination . ConversPOI therapy and prevention may be alleviated by traditional Chinese medicines (TCM). TCM has a long history of being used to treat POI in China. Based on TCM theory, POI is believed to originate in the Chong and Ren Meridians, with key pathogenic factors being Ben-Xu (kidney insufficiency) and Biao-Shi (blood stasis). Therefore, the principle of TCM treatment for POI is lies in activating blood circulation as well as tonifying the kidney. The Bushen-Culuan Decoction (BCD) is made from a combination of 12 herbs: Gou Qi Zi (Lycii fructus), Tu Si Zi (Cuscutae Semen), Sang Ji Sheng , Nv Zhen Zi (Ligustri Lucidi Fructus), Chuan Niu Xi (Cyathulae Radix), Dang Gui (Angelica sinensis), Xu Duan (Dipsaci Radix), Chi Shao (Paeoniae Radix Rubra), Ze Lan (Lycopi Herba), Pu Huang (Typhae Polien), Dan Shen , and Xiang Fu (Cyperi Rhizoma). Previous clinical trials have been showed that BCD can alleviate the clinical symptoms of POI, improve gonadal hormone profiles while elevating antral follicular counts in POI patients . Given tn = 100, weighing 18–20 g each) and 6-week-old CD-1 mice  were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Certificate number: SCXK (Beijing) 2017-0033). Animals were reared in specific-pathogen-free facilities under controlled light-dark exposure (12-h each of dark and light), temperatures (20–24°C), and humidity (45%–70%). Ad libitum food and water were provided. Animals were handled strictly adhering to the Guidelines for the Care and Use of Laboratory Animals. Ethical approval was obtained from the Ethics Committee of Xi Yuan Hospital of China Academy of Chinese Medical Sciences.Female Balb/c mice ]. BCD component herbs were all verified and conformed to the standard of quality in the 2015 Chinese Pharmacopoeia. Herbs were authenticated based on the following parameters: thin-layer chromatography, chemical, microscopic, and morphological identification, as well as detection of heavy metal and deleterious elements. A pharmaceutical company  was enlisted to produce BCD granules made out of a concentrated mixture of the components presented in BCD granule stability and quality were evaluated utilizing a high-performance liquid chromatography (HPLC) system  equipped with a Waters Xbridge-C18 analytical column . Mobile phase A indicated the acetonitrile organic phase, while mobile phase B indicated the ultrapure water (containing 0.1% phosphoric acid) aqueous phase. Mobile phases were altered based on the following: 0–5 min, 10% (phase A); 5–35 min, 10%–20% (phase A); 35–50 min, 20%–30% (phase A); 50–55 min, 30%–37% (phase A), 55–60 min, 37%–10% (phase A). The flow rate was 1.0 ml/min and the UV spectrum was calibrated to 230 nm. Specnuezhenide (CAS: 39011-92-2), cyasterone (CAS: 17086-76-9), ferulic acid (CAS: 537-98-4) and isorhamnetin-3-O-neohesperidoside (CAS: 55033-90-4) were procured from National Institutes for Food and Drug Control , and hyperoside (CAS: 482-36-0), asperosaponin VI (CAS: 39524-08-8), peoniflorin (CAS: 23180-57-6) and typhaneoside (CAS: 104472-68-6) were obtained from Shanghai Standard Technology Co., Ltd. . The detection wavelength of BCD was identified based on retention time compared with reference substance.The chemical constituents of TWP tablets  were normalized and evaluated as Qu L.’s research . TWP, BCMice were allowed 5 days of adaptive diets and has their estrous cycles continuously observed for 14 days. Eighty female mice noted to have relatively regular estrous cycles (5–6 days) were selected, with 20 mice each designated in the hormone replacement therapy (HRT), BCD, Model, and Blank groups. In accordance with previous literature and our initial modeling experiments, mice in the HRT, BCD, and Model groups were intragastrically fed 40 mg/kg/d TWP suspension every morning at 8 a.m. for 30 days to induce POI . The BlaAt the end of the experimental period, all mice models were starved overnight before being weighed and anesthetized the following morning with intraperitoneal 1% pentobarbital sodium (50 mg/kg) injections. Prior to euthanizing the animals, we extracted serum samples for further hormonal ELISA tests. The thymus, lung, cardiac, liver, spleen, kidney, uterus and ovary were then dissected to determine the organ index (organ index = organ weight/body weight × 100%). One ovary from each mouse was frozen in liquid nitrogen and stored at −80°C for further analysis while the other ovary was fixed with 4% paraformaldehyde (PFA) for histology and immunostaining.Ten mice chosen at random from every group had their estrous cycle checked daily and body weights monitored every 4 days during the experimental period. Estrous cycle staging was done by vaginal smear cytological examination every morning between 9 a.m. and 10 a.m. Smears were obtained by flushing 20 µl of sterile NS into the vaginal cavity, spreading the flushed fluid on a glass slide, and fixing the samples with 95% ethanol for 30 min. The Wright-Giemsa Stain solution  was used to stain the slides, which were then rinsed with running water, air-dried, and then observed using a light microscope at ×100 magnification.2, CUSABIO, CSB-E05109), luteinizing hormone , and follicle stimulating hormone  were evaluated using mouse-specific ELISA kits strictly following instructions provided by the manufacturer. The sensitivity of these kits was defined as the lowest mouse concentration that could be differentiated from zero and the CV% was less than 15%. Results were based on the 450 nm absorbance.Hormonal assays to determine the levels of serum anti-mullerian hormone , inhibin-B , estradiol  for further morphological observation and quantification of ovarian follicles. Two independent investigators quantified follicles by counting the number of follicles on whole ovarian three serial sections. Primordial follicles were visualized using ×400 magnification while all primary, secondary and antral follicles were observed at ×200 magnification.Ovarian tissue was homogenized in PBS to produce 10% ovarian homogenate, before being centrifuged for 10 min at 4°C and 3,000 rpm to collect the supernatant. 8-OHdG , MDA  and CAT  levels, and the activities of GSH-Px  and SOD  in the ovarian homogenate supernatant were performed in accordance with protocols provided by the manufacturer.−∆∆Ct method was utilized in calculating relative gene expression levels against endogenous control gene beta actin.Ovarian expression of the Bach 1, Nrf2, Keap 1, and HO-1 genes were evaluated using real-time qPCR. Fifty milligrams of ovarian tissues with TRIzol reagent (Invitrogen) on ice before RNA extraction that was carried out based on instructions provided by the manufacturer. Eight microliter RNA was used for 1% agarose gel electrophoresis. Twenty microliter of TIANScript reverse transcriptase (RT) mix, sequence-specific PCR primers, and RNA were combined for analysis using the Prism 7500 sequence detection system. Final results were evaluated using the ABI Prism 7500 SDS software  and a SYBR FAST qPCR Kit Master Mix (×2) Universal kit (Biosystems). The NCBI-Primer was used to design all primer sequences shown in . The 2−∆Proteins were extracted from the four different mice groups. The cytoplasm and nucleus proteins were isolated using a Nucleus-cytoplasmic protein preparation kit . The BCA Protein Assay Kit (CoWin Biosciences) was used to quantify ovarian lysate protein concentrations. Proteins were separated via electrophoresis and immunoblotted onto polyvinylidenefluoride (PVDF) membranes. A 5% bovine serum albumin solution (BSA) was added to the membranes for an hour before being incubated at 4°C overnight with the following primary antibodies: Bach 1, Nrf2 , Keap 1 , HO-1 , β-actin  and Histon H3 . The next morning, we added horseradish peroxides (HRP)-conjugated secondary antibody to incubate at room temperature for another 40 min. Signals were detected by enhanced chemiluminescence and imaged with a Gel Image system ver.4.0 . Protein levels were normalized first before measurement of the bands using the ImageJ 6.0 software program.n = 20 each) into BCD and control groups randomly after 3 days of adjustable feeding. The night before gavage, mice were fasted and only drank water. The BCD group mice were fed with the maximal concentration and maximal volume of BCD solution daily by intragastric administration, while control group mice underwent the same procedure but with NS instead. All mice were closely observed for 2 weeks for any potential adverse effects. Mice were weighed daily and their average daily feed intakes (ADFI) were recorded. After the 2-week observation period, mice were fasted overnight before they were anesthetized for further evaluation of their internal organs.Forty 6-week-old CD-1 mice  each weighing more than 18 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Certificate number: SCXK (Beijing) 2017-0033). All the mice were divided equally , ferulic acid , typhaneoside , hyperoside , isorhamnetin-3-O-neohesperidoside , specnuezhenide , cyasterone , and asperosaponin VI . The representative chromatography was depicted in p < 0.05). Conversely, the ovarian index was elevated in the BCD and HRT groups (p < 0.05). The ovarian indices of the BCD and HRT groups were not significantly different , however, these same mice possessed higher FSH level and ratio of FSH to LH in comparison to the Blank group . BCD intervention resulted in raised AMH, INHB, and E2 levels in comparison to the Model group . BCD intervention also resulted in lower FSH level and FSH to LH ratio . Conversely, mice in the HRT group displayed increasing levels of AMH and INHB, but decreased FSH level and FSH to LH ratio . The differences between INHB and E2 levels of the BCD and HRT groups achieved statistical significance (p < 0.01)  .p < 0.01), while HRT and BCD exposure restored them (p < 0.01) (p < 0.01). Mice exposed to BCD had lower atretic follicles (p < 0.01), with higher antral follicle and corpus luteum numbers in contrast to the Model group . We also observed variabilities in corpus luteum and atretic follicles between the HRT and BCD groups (p < 0.05) ( < 0.01) . Preantr < 0.01) . The Mod < 0.05) .Uterine sections from the Blank group also had normal histopathological structures, specifically the adventitia, tunica muscularis, and mucous layer which made up almost two-thirds of uterine wall. There were also large numbers of normal glands. However, after TWP treatment, uterine sections from the Model group showed endometrium obvious thinning with decreased numbers of glandular organs. In comparison with the Model group, the endometrium was significantly thickened whilst a large number of glands were visible in BCD group. The histopathological structure in HRT group was also improved compared to Model group .p < 0.05, p < 0.01), but lower levels of CAT, GSH-Px and SOD compared to the Blank group . BCD intervention resulted in decreased 8-OHdG and MDA levels, in addition to raised levels of CAT, GSH-Px, and SOD, in contrast to the Model group (p < 0.01). However, the level of MDA was decreased and SOD was increased in HRT group . MDA, CAT, and GSH-PX levels between BCD and HRT groups were statistically significant   .p < 0.01), although the differences between these three groups were not statistically significant (p < 0.05) and HO-1 mRNA expressions to be decreased in the Model group compared to the Blank group. Mice exposed to BCD caused lower Keap 1 mRNA expression (p < 0.05) but higher HO-1 mRNA expression in contrast to the Model group (p < 0.05). There were significant differences in terms of HO-1 mRNA expression between the HRT and BCD groups (p < 0.05)  .p < 0.05, p < 0.01) and increased the nuclear translocation rate of Bach 1 in the Model raised (p < 0.01). Compared to the Model group, Bach 1 expression in the nucleus and nuclear translocation rate were suppressed in the BCD and HRT groups (p < 0.01). Conversely, in the Model group, there was a rising trend of cytoplasmic Nrf2 expression but decreased nuclear Nrf2 expression (p < 0.05). There was also lower Nrf2 nuclear translocation rates (p < 0.01). After BCD and HRT treatment, Nrf2 expression in nucleus and nuclear translocation rate were raised  (p < 0.05) (p < 0.05)  . Further < 0.05) . HO-1 ex < 0.05) .A preliminary experiment was confirmed that the dose used in the following formal toxicity tests was nonlethal. Mice of BCD group was given 5.50 g/ml BCD solution 40 ml/kg orally three times a day with an interval of 4 h between each dose. The current human clinical dosage of BCD was 2.75 g/kg. Based on the acute toxicity test, we found that the maximum oral tolerance dosage in mice was 660.38 g/kg, which was equivalent to 240 times of the human clinical dosage. During the test, none of the mice died and none developed symptoms suggestive of acute toxicity. We also demonstrated no significant difference between the two groups in terms of body weight and ADFI . FurtherDespite the increasing incidence of POI, little is known regarding its exact pathogenesis. It is thought that several factors including endocrine dysfunction, immune dysregulation, and genetics may influence the occurrence of POI. Exposure to chemotherapy drugs and TWP are the two most common causes of POI in women of childbearing age. TWP is a Tripterygium wilfordii extract that exerts anti-tumour, anti-inflammatory, and immunosuppressive properties. This compound has been shown to be beneficial in autoimmune renal impairment, systemic lupus erythematosus, and rheumatoid arthritis. TWP is notorious for causing significant reproductive system toxicity, resulting in ovarian failure and dysgenesis. In addition, results from both clinical trials and animal studies indicate that TWP is an ideal drug for establishing a POI mouse model . The curPOI causes decline in ovarian function and is a state of hypergonadotropic hypogonadism. FSH, a gonadotropin, is produced in the pituitary gland and acts on FSH receptors on the granulosa cells of the ovary. FSH stimulates follicle recruitment, growth, and maturation, and is associated with E2 production. FSH modulates the female reproductive physiology and prepares the ovary for ovulation in response to the LH surge . ClinicaAMH is a growth factor that is specifically produced by the granulosa cells of early growing ovarian follicles. AMH can regulate or suppress the number of primordial follicles to be activated and is predictive of the quantity of remnant follicles in the ovaries . Since AThe normal estrous cycle comprises of four phases: proestrus, estrus, metestrus, and diestrus, all of which are governed by the controlled release of gonadal hormones such as FSH, LH, and E2 . The curIt has been reported that the antral follicle count (AFC), which is the amount of ovarian antral follicles, strongly correlates with the serum AMH levels and the number of primordial follicles. The AFC serves as an important marker of ovarian reserve . TherefoTWP-stimulated oxidative stress is essential in POI pathogenesis. There is a fine balance between antioxidant scavenger enzymes and reactive oxygen species (ROS) under normal circumstances. Any factor that causes oxidant accumulation or depletion of antioxidants will result in oxidative stress . The excThe Nrf2/Keap1 signaling pathway is a critical defence mechanism against oxidative stress . Nrf2 isWe demonstrated that TWP treatment remarkably increased mRNA expression of Bach 1 and Nrf2, findings that mirror those of previous studies that demonstrated Triptolide, a major active ingredient extracted from TWP, to be able to induce Nrf2 overexpression in protecting against kidney cell death . When tiTo conclude, the present study highlights the protective effects of BCD on TWP-triggered ovarian injury in mice. BCD may offer this protection by restoring the ovarian index, as well as AMH, INHB, and E2 levels, attenuating FSH level and FSH/LH ratio increases, improving the estrous cycle, and lastly by rescuing the primordial follicle pool through normalization of 8-OHdG, MDA, CAT, GSH-PX, and SOD levels. We hypothesize that BCD enhances oxidant defense systems and reduces oxidative damage by modifying the Nrf2/Keap 1 signaling pathway. BCD appears to be a useful therapeutic candidate in preventing and treating TWP-induced POI.


The facile preparation of the PVA bottlebrushes was performed via selective hydrolysis of the acetate of the poly(vinylacetate) (PVAc) side chains of the PVAc bottlebrush precursors. Ice-bindingpolymer side-chain density was shown to be crucial for nucleationactivity, with less dense brushes resulting in colder nucleation thandenser brushes. This bio-inspired approach provides a synthetic frameworkfor probing heterogeneous ice nucleation and a route toward definedsynthetic nucleators for biotechnological applications.Ice binding proteins (IBP) have evolved to limit thegrowth ofice but also to promote ice formation by ice-nucleating proteins (INPs).IBPs, which modulate these seemingly distinct processes, often havehigh sequence similarities, and molecular size/assembly is hypothesizedto be a crucial determinant. There are only a few synthetic materialsthat reproduce INP function, and rational design of ice nucleatorshas not been achieved due to outstanding questions about the mechanismsof ice binding. Poly (PVA) is a water-soluble syntheticpolymer well known to effectively block ice recrystallization, bybinding to ice. Here, we report the synthesis of a polymeric ice nucleator,which mimics the dense assembly of IBPs, using confined ice-bindingpolymers in a high-molar-mass molecular bottlebrush. Poly-basedmolecular bottlebrushes with different side-chain densities were synthesized  Davies and co-workers reported recombinantmutated INPs, but it was essential to study activity while attachedto the host organism (E.coli) to obtainice nucleation functional data.5Ice-binding proteins recognize and bindice faces, leading to controlover their formation  or growth . AFPs are well studied and have defined ice-bindingsites to engage ice either directly or 8 Synthetic polymers can be produced with excellentcontrol over architecture and composition, allowing molecular shapeand composition to be tuned to probe the material/ice interface. Themost active and widely studied ice-binding polymer is poly(PVA).10 PVA’s ice recrystallization inhibition(IRI) activity can be tuned by varying molecular weight,11 shape,13 and assembly into particles.14 PVA binds to ice via hydrogenbonding of the hydroxyl groups,16 but is unique in thatmost other hydroxylated polymers do not show this function.17 Antifreeze (glyco)proteins, in contrast, havehydrophobic segments that bind to ice (rather than the glycan hydroxyls),20 underpinning the concept that a molecular mechanisms can lead tosimilar measurable macroscopic effects.In addition to IBPs, it has emerged that synthetic materials canalso bind ice, reproducing the macroscopic properties of IBPs—presentingopportunities not only for biomedical applications but also to understandIBP function.22 Feldspar is a potent ice nucleator,23 and its functionappears to be caused by defects rather than the bulk structure.24 Graphene nanomaterials can also nucleate ice,25 and the size of the flakes proves crucial.26 The critical ice nucleus size is predicted tobe ∼20 nm for nucleation at −5 °C,27 supporting the need for aggregates or molecularly largespecies. Arrays of cholesterol crystals28 and fractions of lignin  have also beenreported to have some activity,29 alongsidemarine biogenic particles.30 Ice-nucleatingmacromolecules of protein and polysaccharide origin have also beenreported,31 but their chemical structures remain unknown.Considering the successof mimicking the macroscopic property ofice recrystallization inhibition, there remain remarkably few nonproteinice nucleators, none of which have been designed from the bottom-up.Silver iodide, which has a very similar structure to hexagonal ice,is probably the best known.27In general, effective ice nucleators are thought to have a goodlattice match to ice, meaning that water molecules bind to the nucleatorin spatial locations similar to those they would occupy in ice. Accordingto classical nucleation theory (CNT), the barrier to ice nucleationis due to the energetic cost of forming an interface between supercooledwater and ice. By templating ice, a nucleator allows the formationof a smaller ice critical cluster reducing the energy barrier to nucleationand causing ice to form at warmer temperatures. For ice nucleationto occur at warmer temperatures, more significant nucleation sitespossessing a lattice match to ice, or at least an ability to bindwater in an icelike structure, are required.32 and modeling evidence.3 Small IBPs (∼2 nm) nucleate ice at cold temperatures, nearto where homogeneous ice nucleation occurs32 but ice-nucleating proteins (INPs) maintain a coherent lattice matchto ice across nucleation sites spanning as many as 30 individual INPs,nucleating ice at −2 °C:3 Thiscorresponds to 50 nm x 50 nm nucleation sites. Ogawa et al. reportedthat linear poly, which is known to bind ice, is capableof nucleating ice at temperatures a few degrees above the homogeneousnucleation temperature but did not show a molecular-weight effectin the range tested.33 Taken together,the above shows an exciting possibility that a chemically definedsynthetic ice binder could be engineered to become an ice nucleatorif sufficient size, density, and placement of the ice binders canbe accessed. A synthetic ice nucleator would enable the first truestructure–property relationships to be determined, withoutusing heterogeneous mixtures.There are currently no design rules for ice nucleators, but somekey observations have been made. There is evidence that when the sizeof an AFP increases, it can become an INP supported by experimentalvia a combination of ring-opening metathesis polymerization (ROMP) andreversible addition–fragmentation chain-transfer (RAFT) polymerization,using “grafting-to” and “grafting-through”approaches. We demonstrate that densely grafted PVA bottlebrush polymersexhibit ice nucleation activity and are the first example of a syntheticpolymer ice nucleator. Lower-density brush copolymers had reducedactivity, demonstrating that confinement of the PVA is essential.These results show that synthetic ice nucleators can be accessed bymacromolecular engineering.Here, we introduce an ice nucleatorplatform based on well-definedpoly molecular brushes obtained 2IMes)(pyr)2(Cl)2Ru = CHPh), cis-5-norbornene-exo-2,3-dicarboxylic anhydride (95%), exo-5-norbornene carboxylic acid (97%), N-boc-ethylenediamine(≥98%), 1-hydroxypyrrolidine-2,5-dione (98%), ethyl vinyl ether(EVE) (≥99%), trifluoroacetic acid , mPEG10K-succinimidyl carboxymethyl ester , poly(ethylene glycol)methyl ether methacrylate , 4-cyano-4-(phenylcarbonothioylthio)pentanoicacid (97%), 4,4′-azobis ,poly , hydrazine hydrate ,lithium aluminum hydride solution ,1-hydroxybenzotriazole hydrate , triethylamine, PBS (tablets), and sucrose (≥99.5%) werepurchased from Sigma-Aldrich. The monomer vinyl acetate was also purchased from Sigma-Aldrich and passed through a columnof basic alumina to remove inhibitors prior to use. Potassium ethylxanthate (98%) was obtained from Alfa Aesar. N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride was purchased from Carbosynth. 2-Bromo-2-methyl-propionic acid (≥98%)and lanolin  was purchased from Acros Organics. Paraffinoil  was purchased from VWR International. Solventsof toluene , dichloromethane , N,N-dimethylformamide, and tetrahydrofuran  were also purchased from Sigma-Aldrich. Dialysis membranes(MWCO = 3.5/300 kDa) were purchased from Spectra/Por. Formvar-carbon-coated(300 mesh) copper grids were purchased from EM Resolutions. Photo-polymerizationreactions of vinyl acetate were conducted using an EvoluChem PhotoRedOxTemperature Controlled Box fitted with an EvoluChem LED spotlight(P201-18-2 450–455 nm) with total irradiance of 30 mW cm–2 and light beam angle of 25° operating at a wavelengthof λ = 450–455 nm. Ultrapure water used for buffers wasof Milli-Q grade (18.2 mΩ resistance).All chemicals were used as supplied unlessotherwise stated. ROMP catalyst G3 ((H2(g) for 30 min, and the sealedvial was incubated at 37 °C with magnetic stirring under 460nm light irradiation for 8 h. After that time, polymerization wasquenched by removing the sealing and exposing it to air. An aliquotof crude polymerization mixture was withdrawn for 1H NMRin CDCl3 for conversion and Mn,NMR analysis. The reaction was rapidly cooled inliquid nitrogen and precipitated into diethyl ether. The polymer wasreprecipitated into hexane from THF twice to yield a pale-yellow stickypolymer product that was further dried under vacuum. Mn,NMR was calculated by end-group analysisby comparing the integrations of the −(CH2)2 signals  of NHS group with those of the corresponding signals of the −CH signal  of the polymerbackbone. 1H NMR : δ (ppm)= 5.05–4.76 5.05–4.76 , 2.83 , 2.17–1.94 CH3 of polymer side chain), 1.94–1.58 ,1.34 . Conversion = 69%, Mn,NMR = 18400 g mol–1 . SEC (5 mM NH4BF4 in DMF) Mn,SEC RI= 22800 g mol–1, ĐM,SEC RI = 1.6.2-(Ethoxycarbonothioylthio)-2-methylpropanoicacid NHS-ester  and vinyl acetate (VAc) (8.46g (9.06 mL), 98.2 mmol, 300 eq) were dissolved in 2.1 mL of dioxanein a 20 mL vial (80% w/w solids content). The resulting solution wasdegassed by sparging with N50 homopolymer via solutionROMP is described. A stock solution of 100 mg/mL of G3 in CHCl3 and a solution of 173 mg of NB-NBoc  in3.3 mL of CHCl3 were first prepared. Then, 100 μLof G3 stock solution  was added to the vialof NB-NBoc monomer solution with rapid stirring, and polymerizationwas allowed to proceed at room temperature for 1 h . The polymerization was then quenchedby the addition of a few drops of ethyl vinyl ether, and P(NB-NBoc)50 homopolymer was precipitated from diethyl ether, isolatedby vacuum filtration, and dried under vacuum prior to 1H NMR and SEC analyses. The procedure was repeated for [NB-NBoc]/[G3]ratios of 100, 200, and 400. 1H NMR  Mn,theo. =15,300 g mol–1. SEC (DMF + 5 mM NH4BF4) Mn,SEC = 17,600g mol–1, ĐM,SEC = 1.10.A typical procedure for the synthesisof P(NB-NBoc)50 homopolymer  was dissolved in a 1:1 TFA/DCM solution (5 mL) and stirredat room temperature for 4 h. The polymer reaction mixture was concentratedto dryness under reduced pressure to afford a brown residue that wasprecipitated by the addition of diethyl ether. Removal of the solventresulted in the isolation of the amino-deprotected polymer productas a brown solid (P(NB-NH2)50). The resulting polymer wasthen dissolved in DI water and dialyzed against DI water  for approximately 14 h and lyophilized toafford the polymer products as brown solid. The procedure was repeatedfor P(NB-NBoc)100, 200, and 400 homopolymers. SEC characterizationof the resulting deprotected poly(amino exo-norbornene imide) polymerswas not possible due to high sample–column interactions. 1H NMR : δ(ppm) = 8.04–7.92 , 5.73–5.56, 5.56–5.33 , 3.74–3.48 , 3.48–3.24 , 3.24–2.83 CHCH C(O)of polymer backbone), 2.83–2.56, 2.13–1.80 , 1.62–1.32.Next, P(NB-NBoc)g-PVA210)-stat-P(NB-NH2))50 by “grafting-to”is as follows: NHS-PVAc210 ,P(NB-NH2)50 , andEDC.HCl  were dissolved in 6.5 mL of anhydrousDMF, followed by the addition of Et3N , and the reaction mixture was stirred at 70 °Cfor 16 h. An aliquot of the crude reaction mixture was then takenfor SEC analysis in DMF (+0.1% LiBr). The resulting polymer mixturewas precipitated twice in diethyl ether. The resulting P(NB-NH2)50-g-PVAc210 branchedcopolymer was then dissolved in methanol (2 mL) following by the additionof hydrazine hydrate solution  in astoppered vial. The reaction mixture was stirred at room temperatureovernight. The resulting graft copolymer was then dissolved in DIwater and dialyzed against DI water  for approximately 2 weeks to ensure full removal of unfunctionalizedPVA210 and lyophilized to afford the polymer product as spongy pale-yellowsolid. Procedure was repeated for the synthesis of P((P(NB-NH)-g-PVA210)-stat-P(NB-NH2))100, 200, 400 and control sample P((P(NB-NH)-g-PEG227)-stat-P(NB-NH2))200. Complete hydrolysis was confirmed by 1H NMRanalysis in DMSO-d6. 1H NMR: δ (ppm) = 4.74–4.63, 4.58–4.41 , 4.32–4.18 ,3.99–3.73 , 1.71–1.14 .A representativesynthesis of P((P(NB-NH)-2(g) for 30 min, and the sealed vialwas incubated at 37 °C with magnetic stirring under 460 nm lightirradiation for 8 h. After that time, polymerization was quenchedby removing the sealing and exposing it to air. An aliquot of crudepolymerization mixture was withdrawn for 1H NMR in CDCl3 for conversion and Mn,NMR analysis. The reaction was rapidly cooled in liquid nitrogenand precipitated into diethyl ether. The polymer was re-precipitatedinto hexane from THF twice to yield a pale-yellow sticky polymer productthat was further dried under vacuum. Mn,NMR was calculated by end-group analysis by comparingthe integrations of the −(CHCH) signals  of norbornene group with those of the correspondingsignals of the −CH signal  of the polymer backbone. 1H NMR : δ (ppm) = 6.28 , 5.05–4.76 , 2.83 , 2.17–1.94 CH3 of polymer side chain), 1.94–1.58 ,1.34 . Conversion = 67%, Mn,NMR = 18,400 g mol–1 . SEC (5 mM NH4BF4 in DMF) Mn,SEC RI = 17,900 g mol–1, ĐM,SEC RI = 1.40.2-(Ethoxycarbonothioylthio)-2-methylpropanoic acid exo-5-norbornene-2-methylamide and vinyl acetate (VAc) (8.24 g (8.82 mL),95.7 mmol, 300 eq) were dissolved in 2.1 mL of dioxane in a 20 mLvial (80% w/w solids content). The resulting solution was degassedby sparging with N20-g-PVAc208 bottlebrush polymer by ROMP“grafting-through” is as follows: NB-PVAc208  was dissolved in 1.9 mL of anhydrousTHF in a 3 mL vial equipped with a magnetic stirring bar. To the vialwas added 100 μL of a 2.0 mg mL–1 stock solutionof G3 in THF . The reaction mixturewas stirred at room temperature for 2 h. The polymerization was thenquenched via addition of a few drops of ethyl vinylether. An aliquot of the crude reaction mixture was then taken forSEC analysis in DMF (+0.1% LiBr). The resulting polymer, PNB20-g-PVAc208, was isolated by precipitationfrom diethyl ether and was dried under vacuum.A representative synthesis of a PNB20-g-PVAc210 (50 mg) bottlebrushpolymer was then dissolved in methanol (0.5 mL) followed by the additionof and hydrazine hydrate solution  ina stoppered vial. The reaction mixture was stirred at room temperatureovernight. The resulting polymer was then dissolved in DI water anddialyzed against DI water  for approximately1 week to ensure full removal of unfunctionalized PVA208 and lyophilized to afford the polymer products as a white solid.The procedure was repeated for the synthesis of PNBn-g-PVAc210 . Completehydrolysis was confirmed by 1H NMR analysis in DMSO-d6. 1H NMR : δ (ppm) = 4.74–4.63 ,4.58–4.41 , 4.32–4.18 , 3.99–3.73, 1.71–1.14 .The resultingPNB20 , ACVA , and 17 mL of DMF. Thevial was then sealed and deoxygenated using three successive cyclesof freeze–pump–thaw to remove O2(g). Thevial was placed into an aluminum heating block, which had been preheatedto 70 °C to initiate polymerization. After 24 h, the polymerizationwas quenched by exposing the vial to air and submerging it into liquidN2. An aliquot was withdrawn for the determination of monomerconversion by 1H NMR spectroscopy. The polymer was precipitatedinto diethyl ether from dioxane twice to yield a white polymer productthat was further dried under vacuum. 1H NMR : δ (ppm) = 3.88–3.51 , 2.48–1.58 CH2 of polymer backbone),1.41–0.76 CH2 of polymer backbone), Mn,theo = 200,000 g mol–1. SEC (5 mM NH4BF4 in DMF) Mn,SEC RI = 134,300 g mol–1, ĐM,SEC RI = 2.0.Avial was charged with 4-cyano-4-(phenylcarbonothioylthio)pentanoicacid , PEGMA35 Poly(norbornene)s bearing primaryamines were first synthesized via an N-Boc-protected intermediate monomer , initiated by Grubbs third-generation bispyridylcomplex (G3), [M]/[G3] of 50, 100, 200, 400. Full characterizationby 1H NMR and size exclusion chromatography (SEC) analysisis shown in Figures S17 and S18. Deprotectionof P(NB-NBoc)n with trifluoroacetic acid(TFA) gave the desired poly(amino norbornene imide) P(NB-NH2)n homopolymers .Our initial hypothesis was thatdensely packed PVA side chains,to mimic the regular arrays of IBPs which lead to nucleation, wereessential. A “grafting-to” approach was first employed,which results in low grafting densities due to steric repulsions betweenbulky side chains 1A.34,35 N-Hydroxysuccinimide-poly(vinylacetate)(NHS-PVAc) was then prepared by photo-RAFT/MADIX to maximize end-groupfidelity and was subsequently grafted to P(NB-NH2) homopolymers.See the Synthetic Procedures section in the Supporting Information  -g-PVAc210)-stat-P(NB-NH2))n  graft copolymerswere characterized by SEC analysis revealing bimodal peaks at high-molecular-weightdirection. 210 sidechain (dashed black curve) and the resultant graft copolymers, inwhich the peak positions of the graft copolymers completely shiftedto higher molecular weights. A significant amount of unfunctionalizedNHS-PVAc210 was also apparent for all graft copolymer formulations.Grafted side chains (%) of the resulting graft copolymers from SECanalysis were estimated to be ca. 20–40% (35 The acetate protecting groups were quantitively removed by an excessof hydrazine hydrate to yield P((P(NB-NH)-g-PVA210)-stat-P(NB-NH2))n graft copolymers that were purified by dialysis(MWCO = 300 kDa) to remove unfunctionalized PVA210 andcharacterized by 1H NMR and FTIR spectroscopic analysis.ω-. 20–40% 2, due to2)n (38 Dry-state TEM imagesof P((P(NB-NH)-g-PVA210)-stat-P(NB-NH2))n confirmed thepresence of small cylindrical nanostructures (S22). Importantly,no large aggregates were observed in the TEM images, indicating thatneither graft copolymers were spontaneously self-assembled into higher-orderstructures when dissolved in aqueous media at room temperature. AFManalysis also showed the formation of some spherical particles (S23). While the PVA graft copolymers appear tobe spherical in the AFM images, they likely exist as cylindrical nanostructuresin aqueous environments  due to theiranisotropic dimensions, as has been reported for brush polymers insolution measured using X-ray or neutron scattering analyses.40 Moreover, brush polymers have been shown to de-wet various substratesdue to unfavorable surface interactions, causing them to present asglobules in AFM images.41The solution behavior of the resulting PVA graft copolymerswasinvestigated by dynamic light scattering (DLS), transmission electronmicroscopy (TEM), and atomic force microscopy (AFM). DLS analysisshowed that graft copolymer samples exhibited an increase in lengthupon increasing the backbone of P(NB-NH2)n 1C. The fExo-5-norbornene-2-methylaminewas synthesized in this case , as it was demonstrated by Matsonand co-workers that the choice of anchor group dramatically affectsoverall ROMP propagation rates with norbornene anhydride-based macromonomersleading to poorer chain extensions and conversions when targetinghigh Mw bottlebrushes.43Next, a seriesof high-density PVA bottlebrush polymers were preparedusing ROMP “grafting-through” approach using a norbornene-endpoly(vinyl acetate) macromonomer (NB-PVAc), exo-5-norbornene-2-methylamide was synthesizedand used to prepare an NB-PVAc208 macromonomer via photo-initiated RAFT/MADIX polymerization . Bottlebrush polymers were synthesized through ROMP of thenorbornene amide end group on the PVAc macromonomer by varying themolar ratio of G3 catalyst ([NB-PVAc208]/[G3]) to achievePNBn-g-PVAc208 bottlebrush polymers .A norbornyl xanthate chain-transfer agent (NB-CTA)of 2-(ethoxycarbonothioylthio)-2-methylpropanoicacid 208 macromonomer. However,the long low-molecular-weight fraction could also be explained bythe premature termination of ROMP polymers. Large ĐM values can be primarily attributed to the employmentof a high Mw of PVAc macromonomer witha relatively large dispersity (nM) determined by SEC analysis were evidently lowerthan theoretically expected values determined from polymerizationconversions. This discrepancy in theoretical and observed nM likely originates from the branched natureof the bottlebrush polymers, leading to reduced hydrodynamic volumesrelative to linear polymers of the same molecular weight and thusincreased retention on the SEC columns. Acetate protecting groupswere removed by hydrazine hydrate, as confirmed by 1H NMRand FTIR analyses to yield PNBn-g-PVA208  bottlebrush polymersthat were dialyzed (MWCO = 300 kDa) to ensure removal of unreactedNB-PVA208 macromonomer.SEC analysis revealed low-molecular-weight shoulders, which maycorrespond to unreacted NB-PVAcspersity 3 as welln-g-PVA208  also confirmedthe presence of small cylindrical nanostructures (S26). AFM analysis again showed the formation of sphericalparticles, which is likely due to unfavorable surface interactionsof PVA side chains with silicon surface, causing them to present asglobules (S27). Since PVA’s side-chainlength is by far larger than bottlebrush backbones, it may appearspherical/ellipsoid, but further investigation is beyond the scopeof this study as the extended local bottlebrush structure is shown(below) to be sufficient for the intended application.DLS analysis also confirmed the formation of anisotropicparticlesand exhibited an increase in size upon increasing the backbone, asexpected 2C.Dry-sglobules 5 and S2720-g-PVA208 and 54 °C for PNB40-g-PVA208 (g-PVA210)-stat-P(NB-NH2))400 that exhibited a decrease intransmittance at 75 °C, confirming that the side chain densityis having a significant impact on the solution behavior of these polymers.DLS temperature ramp experiment for dense bottlebrush polymers showeda stepwise increase in the size of the dissolved nanostructures thatoccurred as the solution was heated above 40 °C  behavior was also observed for dense bottlebrush polymersprepared by ROMP “grafting-through”. This has not previouslybeen reported, due to the absence of high-density PVA copolymers,and was hence explored. It is, however, anticipated that the backbonehydrophobicity for bottlebrushes prepared by “grafting-through”will lead to some self-assembly which may contribute to this behavior.Variable-temperature ultraviolet–visible (UV–vis) spectroscopyshowed a cloud point of approximately 51 °C for PNBg-PVA208 3A. No apve 40 °C 3B. Linea46 Smaller MGS(mean grain size) means more ice recrystallization inhibition activity. –1. It has been previously reported that increasingthe molecular weight of PVA increases IRI activity11 explaining these observations, although architecture-relatedenhancements cannot be fully ruled out. Voets and co-workers havepreviously prepared an acrylate-based PVA bottlebrush polymer usingATRP, which also showed no significant change in IRI compared to linear.13 In this previous work, there was no investigationof ice nucleation activity (see below). Note it was not possible tocompare against linear PVA of the same MW, as linear PVA becomes increasinglyinsoluble in aqueous solutions at high MW, justifying our nonlinearsynthetic approach to access PVA, which could not be obtained by conventionalmethods. The ROMP-derived backbones P(NB-NH2)n  were testedas a control (2) was significantly lessthan this previous report but is not negligible.47With this panel of bottlebrush polymers prepared,and their solutionproperties evaluated, the ice-binding interactions were then investigated,by assessment of their IRI  activity4A. This N-hydroxysuccinimide-poly(ethyleneglycol) (NHS-PEG227) and grafted into the presynthesizedpoly(amino norbornene imide) P(NB-NH2)200 homopolymerto yield P((P(NB-NH)-g-PEG227)-stat-P(NB-NH2))200 . A PEG methacrylatemonomer (PEGMA20) was also employed to prepare P(PEGMA20)200 homopolymer . In both cases, nonlinear PEG controls were shownto have no appreciable IRI activity. Ice shaping (in sucrose solutions)showed that the PVA graft copolymers and bottlebrushes lead to morefaceting of ice crystals, compared to linear PVA homopolymer (S32–S35), as the large polymers can spanmultiple crystals as seen previously for PVA-grafted nanoparticles14 and AFP dendrimers.48 Single crystal assays (using a nanoliter osmometer) in contrastdid not show shaping, suggesting that steric confinement does impactthe ice/polymer interface .A PEG-based graft copolymer  wasalso synthesized using an 32 To achieve ice nucleation at −5 °C, a nucleator is predictedto support a cap-shaped cluster possessing a diameter of around 10nm.32 As shown in With the confirmation of ice binding(shown above), ice nucleationmeasurements could be undertaken. Pure liquid water can supercoolto temperatures as low as −40 °C. It is thought that heterogeneousice nucleators must template an ice critical cluster of a size predictedby classical nucleation theory (CNT).Figures S37 and S38, SupportingInformation) were frozen in a differential scanning calorimeter (DSC)by cooling at 1 °C/min, using a technique similar to that ofMarcolli et al.49Tf. Marcolli etal. found a Tf of −36.5 °Cfor droplets for emulsions containing pure water droplets of roughlythe same size, while Ogawa et al. found aTf of −37.5 °C for a pure water emulsion of smaller droplets.We determined Tf for pure Milli-Q waterto be −36.9 °C. This temperature is taken to representthe onset of homogeneous ice nucleation in our system. Similar emulsionscontaining 2 mg mL–1 linear and bottlebrush PVAsamples were prepared so that each emulsion droplet will contain morethan a million of even the largest bottlebrush. As shown in 4000 increased Tf by 3.5 °C comparedto pure water (i.e ΔTf = 3.5 °C).For linear PVA210, a ΔTf of 3.2 °C was determined. This is qualitatively consistentwith the findings of Ogawa et al., who found that the mass proportionof PVA present in an experiment determined the shift in nucleationtemperature, rather than molar concentration .33 The ΔTf observed for linear PVA is larger than thatmeasured by Ogawa et al. who found a maximum value of approximately2 °C for 2 mg mL–1 PVA solutions. The reasonfor this disparity is not clear. We observed that low-density bottlebrushesnucleated at lower temperatures than linear PVA, while high-molecular-weighthigh-density bottlebrushes nucleated at warmer temperatures, witha maximum ΔTf of 4.5 °C forPNB40-g-PVA208, equivalentto a nucleation onset of −32.4 °C. For both high- andlow-density bottlebrushes, a trend to higher nucleation temperatureswas observed as the total molecular weight of the polymers increased,noting the large error in SEC of bottlebrushes.Two ice nucleation measurementtechniques were employed here. Toinvestigate the ice nucleation ability at temperatures close to thehomogeneous freezing temperature, water-in-oil emulsion droplets ofroughly 2 pL  andP(PEGMA20)200 nucleated at temperatures slightlywarmer temperatures than the instrument background. As neither ofthese polymers can bind ice, they provide a negative control for thePVA bottlebrushes. In contrast, the various PVA bottlebrushes wereobserved to all nucleate ice at temperatures higher than the freezingbackground and higher than the linear PVAs, showing that this macromolecularengineering approach to high-density PVA does lead to polymeric icenucleating agents.Due to the variabilityof background measurements in this typeof experiment, great care needs to be applied in interpreting results.The two linear PVAs (PVAg-PVA210)-stat-P(NB-NH2))n  showedintermediate activity, while the densely grafted PNBn-g-PVA208  were more active, suggesting that dense presentationof PVA side chains is essential for nucleation activity—thesetrends are shown in Figure S39. With themuch smaller droplets frozen in the DSC measurements, where each dropletcontains molecules of the order of 106, the low-densitybottlebrushes nucleated ice less well than linear PVA, while the high-densitybottlebrushes nucleated ice better than linear PVA.The less densely grafted P((P(NB-NH)-40-g-PVA208 was observedto nucleate ice at average temperatures as warm as−19.2 °C, which is unprecedented for a fully syntheticsoluble material. We have previously reported that large (>200nm)polymer nanoparticles with a poly(vinyl pyrrolidone), PVP, coronaalso nucleated ice at similar temperatures.52 The large size and dense display in those particles may be related,but the particles did not have variable density coronas so the structure–propertyrelationship was not clear. These particles did show weak ice binding(and IRI) and hence agree with the results here. Soluble extractsfrom pollen, which are thought to be polysaccharides, have been reportedto nucleate ice, but they have not been characterized.31 It is worth noting that the largest bottlebrushesdid show some aggregation in solution, so the contribution of a smallnumber of larger aggregates is impossible to exclude, in this initialreport.High-densityPNB53 The mechanism(s) of heterogeneousice nucleation is not fully understood, even for the long-studiedice nucleation protein from Pseudomonas syringae. This is in part due to the challenge of studying membrane-anchoredproteins, and that isolation of these as pure protein domains doesnot recapitulate the 3D assembly. Protein expression techniques onlyallow for linear extension (in a single protein), whereas this polymericstrategy provides defined macromolecules with multiple ice bindingdomains in a constrained manner, therefore providing experimentalevidence that crowding and size are essential for nucleation, in agreementwith modeling of IBPs.3 CNT suggests thata nucleation site must have a diameter of between 2 and 5 nm, to nucleateice at −19 °C, with the size depending on the strengthof the interaction with the ice critical cluster. The bottlebrushesare each at least 100 nm across and therefore larger than the icecluster. It seems likely that the occasional patch of bottlebrushsurface assumes a conformation that is sufficiently icelike to actas a nucleation site. In our ice nucleation experiments, we kept themass of polymer per droplet the same while varying the size of thepolymers.There is evidence that ice nucleating protein activityis relatedto other ice-binding proteins based on their size and packing.54 We tentatively suggest that the lesser curvature of the larger bottlebrushesincreases the likelihood that they harbor an active site, and thatfor more rigid conformation, high-density bottlebrushes similarlyfavor the emergence of larger flat areas of icelike structure in thepolymer surfaces. While the magnitude of ice nucleation activity observedhere is much less than that for ice nucleating proteins, the abilityto sequentially modify a bottom-up synthesized chemical nucleatorwill enable new avenues of discovery in the study of ice nucleators,not possible with biological materials, or the poorly defined inorganicnucleator.This means that our experiments using larger bottlebrushes,whichinduce ice nucleation at higher temperatures, had less polymer surfacearea in contact with water. Simulations suggest that curved surfacesnucleate ice less well than flat surfaces.Here, we report a synthetic polymer capableof nucleating ice,inspired by how ice-binding proteins assemble into ice-nucleatingstructures. Our design was based on molecular bottlebrushes with (icebinding) PVA side chains, with the side-chain density tuned usingROMP to enable “grafting-through” (high density) or“grafting-to” (low density). The bottlebrush polymerswere fully characterized, confirming the confinement of the PVA chainsin the higher-density brushes, which also gave rise to LCST-type behavior,not seen for linear equivalents. The bottlebrush polymers were foundto bind ice and retain their potent ice-recrystallization inhibitionactivity, with a slight increase relative to linear PVA. However,due to the limited solubility of high-MW linear PVA, this effect couldbe due to the size increase, rather than a pure architectural effect.Using ice nucleation assays, we observed that high-density PVA bottlebrushessignificantly increased the ice nucleation temperature compared toother materials. Lower-density PVA brushes also favored nucleationin microliter droplet freezing assays, although they nucleated iceat colder temperatures than linear PVA in an emulsion freezing experimentusing picoliter scale droplets. We hypothesize that larger bottlebrushesare more likely to possess an ice nucleation site due to their lowdegree of curvature and that the conformational rigidity of high-densitybottlebrushes is again expected to favor the formation of flat, icelikewater–polymer interfaces that can act as ice nucleation sites.The role of aggregates of these brushes could not be excluded. Ourstrategy of controlling side-chain density would be challenging withprotein engineering tools alone. These materials may guide the developmentof new ice nucleators for cryobiology as synthetic probes to elucidatefundamental mechanisms of action and show that programmable ice nucleationfrom bottom-up synthesis may be achievable.


Occupational allergy has been described in employees working in contact with mealworms in pet stores, live fish bait or infested stored grains and recently, in mealworm farming for animal feed and human consumption. Mealworm allergens linked to occupational allergy are troponin C, cockroach-like allergen, tropomyosin, arginine kinase, early-staged encapsulation inducing- and larval cuticle proteins.We report a case of occupational mealworm allergy and studied the culprit component.Diagnosis was done by skin prick, specific IgE, basophil activation and lung function testing. Allergen purification was performed by anion-exchange chromatography and immunoblotting with patient IgE. Allergens were identified by in-gel trypsin digest and tandem mass spectrometry. Allergenicity and specificity further confirmed by IgE inhibition and passive basophil activation experiments.We describe a new case of occupational mealworm allergy in a laboratory worker, with sensitization to different developmental stages and derivates of the mealworm. In basophil activation tests, the majority of patient's basophils (69%–91%) degranulated upon stimulation with the lowest concentration of mealworm extracts (0.16 µg/ml). Despite strong sensitization to mites, the patient did not show cross-reactivity to other insects. We were able to identify alpha-amylase as the main allergen and through inhibition experiments, we demonstrated that low amounts (0.1 µg/ml) of this allergen could strongly inhibit mealworm specific IgE by 79.1%. Moreover, passive BAT experiments demonstrated the IgE-alpha-amylase interaction to be functional, inducing up to 25.5% degranulation in healthy donor basophils.Alpha-amylase can be identified as the responsible allergen in this specific case of occupational mealworm allergy. Tenebrio molitor is part of the Tenebrionidae or black beetle family. Mealworm farming has been of interest on a rather small scale in the context of fish bait and pet feed  at 15%–20% (w/v) depending on the structure of the material. Extracts were shaken for 1–2 h at room temperature and centrifuged at 700 g for 10 min at 4°C. After centrifugation, the supernatant was filtered through a 0.22 µM filter  and frozen at −20°C until further use. House dust mite extract was purchased from Greer laboratories , dissolved in PBS, filtered through a 0.22 µM filter and stored at −20°C. Protein quantification was achieved by a bicinchoninic acid assay (BCA) using the Pierce™ BCA Protein Assay Kit  according to the instructions of the manufacturer.via an ImmunoCAP fluorescence enzyme immunoassay on a Phadia 1,000 analyzer (Thermofisher). The following specific IgE tests were performed: mealworm (o211), birch pollen (t3), Bet v 1 (t215), Timothy grass (g6), Der p 1 (d202), Der p 2 (d203), Der p 10 (d205), Der p 23 (d209), Dermatophagoides farinae (d2), Euroglyphus maynei (d74), Acarus siro (d70), Tyrophagus putrescentiae (d72), cockroach (i6) and shrimp (f24). In inhibition experiments, the stimulus was added to serum 1:4, followed by overnight incubation at 4°C. As a baseline value, a condition where we added buffer (PBS or purification solvents), was included. This baseline level of specific IgE was used to calculate a percentage of inhibition.Specific IgE tests were performed 2, 3.5 mM MgCl2, 1 mg/ml human serum albumin (HSA), 0.5 mM glucose and 60 ng/ml interleukin (IL)-3  at pH 7.4. Stimulation of basophils was induced by adding 30 µl of stimulus in 150 µl whole blood followed by incubation for 25 min at 37°C. As IgE-mediated control, we used polyclonal goat anti-human IgE antibodies  and N-formyl-methionyl-leucyl-phenylalanine  as IgE- and non-IgE-mediated positive controls, respectively. Basophil stimulation buffer was used as a negative control. The reaction was stopped at 4°C for 5 min followed by incubation with fluorochrome-linked antibodies for 25 min at 4°C including anti-CD123-PE (111 ng/ml), anti-HLA-DR-AlexaFluor 647 (111 ng/ml) and anti-CD63-FITC (444 ng/ml) . Subsequently, red blood cells were lysed by adding 2 ml of lysis buffer (BD) for 10 min at room temperature in the dark. After washing with 2 ml of PBS, cells were fixed in 1% paraformaldehyde. Basophils were analyzed on an LSRFortessa flow cytometer equipped with FACSDiva software (BD). Basophils were identified as CD123+ and HLA-DR− cells and degranulation was defined by detection of CD63 expression on basophils.Whole blood samples were drawn using lithium/heparin tubes  and experiments were started within 1 h. Each stimulus was diluted in basophil stimulation buffer containing 20 mM HEPES, 133 mM NaCl, 5 mM KCl, 7.5 mM CaCl6 cells) of a healthy nonallergic donor were isolated through density gradient centrifugation over a ficoll gradient . PBMCs were then stripped of surface-bound IgE by adding acidic stripping buffer containing 0.9% NaCl, 50 mM KCl and 13.4 mM lactic acid. After incubation on ice for 5 min, neutralization buffer was added, containing 0.5% HSA, 12 mM Tris-HCl (pH 8) in Roswell Park Memorial Institute (RPMI) 1,640 Medium (Thermofisher). Immediately after neutralization, cells were centrifuged at 1,200 g for 5 min at 4°C and washed in 2 ml PBS. Cells were then incubated for 1 h at 37°C with serum of the patient, diluted 1:1 in either PBS or an inhibitory stimulus, pre-incubated overnight at 4°C. After incubation of cells with patient serum, approximately 2.5 × 105 to 5.105 sensitized donor cells were stimulated and stained per condition, identical to the standard basophil activation test but without the red blood cell lysing step.For passive BAT, peripheral blood mononuclear cells . After washing, SuperSignal™ West Femto substrate was used to develop western blots (Thermofisher). For silver stains, the same SDS-page protocol was followed. Silver staining was performed using SilverQuest™ Silver Staining Kit (Thermofisher) according to the instructions of the manufacturer.Extracts or purified allergens were diluted in sample buffer containing 0.125 mM Tris (Sigma-Aldrich), 4% sodium dodecyl sulfate , 20% glycerol  and 10% 2-mercaptoethanol . Diluted samples were loaded on 10% Tris-glycine gels  and ran in running buffer containing 192 mM glycine , 25 mM Tris and 0.1% SDS at pH 7.4. Proteins were transferred to polyvinylidene fluoride (PVDF) membranes using transfer buffer (Bio-Rad) and membranes were blocked for 1 h in 5% (w/v) bovine serum albumin  in wash buffer containing 20 mM Tris, 150 mM NaCl, 0.1% (w/v) Tween 20  at room temperature. After three wash steps of 5 min at room temperature, membranes were incubated overnight at 4°C in patient serum diluted 1:10 in blocking buffer. After three wash steps, membranes were incubated with mouse anti-human IgE antibody . Excess antibody was washed away (5 times) and membranes were finally incubated for 1 h at room temperature with goat anti-mouse IgE antibody coupled to horseradish peroxide  and an AKTA purifier . The loading buffer consisted of 20 mM Tris at pH 8 and elution buffer consisted of 20 mM Tris and 1M NaCl at pH 8. Elution fractions were screened by SDS-page  and stained with silver  or Coomassie blue . Potential candidates were further tested in immunoblot experiments using patient samples, as described before. Allergens were finally identified by in-gel trypsin digestion and nano scale liquid chromatography followed by tandem mass spectrometry by AlphaLyse .Collection of blood samples and clinical data was performed in accordance with a prospective study protocol approved by the Ethics Committee Research UZ/KU Leuven (study no° S60734 and S65293). The patient and healthy controls provided written informed consent prior to participation.Tenebrio molitor  and Hermetia illucens (black soldier fly). Initial symptoms included ocular pruritus and rhinorrhea upon close contact with insects, but developed into coughing and wheezing later on. Symptoms improved during weekends or holidays, suggesting an occupational factor. Moreover, the patient attempted consumption of mealworm once and experienced immediate tingling of the mouth, without swelling of lips or tongue.A 34-year-old female laboratory worker presented to the allergy department of the University Hospitals Leuven due to gradually worsening asthmatic symptoms, presumably upon occupational contact with two insect species: Schistocerca gregaria, Locusta migratoria, Blaptica dubia and Acheta domesticus) were all negative. SPT were positive for multiple aero-allergens including grass, rye, alder and birch pollen and both European and American house dust mite. Specific IgE  confirmed sensitization to mealworm, various pollen (birch and timothy grass) and mites . Component-resolved diagnostics demonstrated presence of specific IgE towards Der p 1, Der p 2, Der p 23 but not to the potentially cross-reactive allergen Der p 10 (tropomyosin). The house dust mite sensitization was already present two years before the onset of insect-related symptoms, but house dust mite-related symptoms were controlled by preventive dust measures. There were no signs of seasonal rhinoconjunctivitis, despite pollen sensitization.No commercial extracts were available for allergy diagnosis, leading to in-house generation of water soluble protein extracts of mealworm  and otheBaseline spirometry was normal, but reversible airway obstruction was observed, as the forced expiratory volume in 1 s (FEV1) improved significantly by 17.4% after administration of salbutamol. The fraction of exhaled nitric oxide was elevated to an intermediate level of 57.2 parts per billion. The lung function test results were considered compatible with asthma.Schistocerca gregaria, Locusta migratoria, Blaptica dubia and Acheta domesticus) were negative for our patient (Subsequent basophil activation tests (BAT), demonstrated reactivity to all derivatives of the mealworm in comparable extract concentrations (0.167 µg/ml to 167 µg/ml,  patient , corresp patient .Through immunoblotting, we attempted to pinpoint the causative allergen responsible for this occupational allergy. The patient showed the strongest IgE binding to a 55 kDa protein in in-house generated mealworm, beetle, larvae and dried mealworm extracts . Anion eAdditionally, we performed passive BATs in which basophils of a healthy non-allergic donor were stripped of their IgE and loaded with the patient's IgE . Up to 2We described a novel case of mealworm allergy in a laboratory employee, working in a facility involved in the optimization of mealworm farming, presenting with rhino-conjunctivitis and asthma upon mealworm exposure. The patient had detectable mealworm specific IgE in serum and positive SPT to mealworm, beetle, larvae, faeces, dried mealworm and the shedded skin of the mealworm. BAT experiments confirmed the broad sensitization to all developmental stages and derivatives of the insect. Moreover, despite presence of strong sensitization to mites, the patient did not demonstrate any IgE towards tropomyosin, often suggested as a cross-reactive allergen between insects and mites. The role of pan-allergens as culprits for the observed mealworm allergy was further excluded through SPT and BAT experiments which confirmed the absence of cross-sensitization to other insects.Through immunoblotting, ion-exchange chromatography and in-gel trypsin digest followed by MS/MS analyses, we identified alpha-amylase as the main allergen in this patient. Alpha-amylase is a digestive enzyme of the mealworm and is known to be an allergen in different insect species including mites  andFurther supporting the allergenicity of mealworm alpha-amylase, we showed that our patient's IgE, when loaded on healthy control basophils, induced degranulation when cross-linked by alpha-amylase. Moreover, alpha-amylase strongly inhibited mealworm specific IgE and reactivity towards mealworm in passive BAT. In summary, our results demonstrate for the first time that alpha-amylase is a potential culprit allergen in occupational mealworm allergy.


A link between hind hoof balance and pathologies in the hind limb of horses is emerging. However, the timeline of causation remains elusive, meaning a lack of treatment protocols within the veterinary and farriery industries. One area suggested as a link between the hind hoof balance and pathologies is hind limb posture. This study aimed to test the theory of the hoof being a neuro-sensory organ responsible for informing equine stance and the suggestion of a functional link between the hoof balance and hind limb posture. All horses presenting with negative plantar angles, a severe form of long toe, low heel conformation, also presented with a canted-in posture which was changed by farriery intervention. The implications for practice suggest that both the hind hoof balance and the pathologies in the hind limb and trunk could be resultant from the presenting posture, therefore showing the importance of a multi-profession approach to managing both hoof balance and higher pathologies, and the importance of correction in the treatment of postural dysfunction and pathology within the hind limb and trunk. Postural assessment should become part of farriery protocol and be incorporated into intervention decisions. Links between poor hind hoof balance, pathologies in the hind limb and associated altered posture have been suggested but not quantified. The hoof is proposed as a neuro-sensory organ responsible for informing equine stance with implications for musculoskeletal health in the hind limb and trunk of the horse. This study aims to quantify equine limb posture and its relationship with hoof balance. Twelve horses presenting with negative plantar angles were photographed and limb posture documented before and after the creation of positive plantar angles and improved three-dimensional proportions around the centre of rotation of the distal-interphalangeal joint, using farriery prosthetics. The results showed that horses presenting with negative plantar angles had canted-in postures and that farriery intervention had a significant effect on hind limb orientation in seven of these horses. There was a significant difference in metatarsal angle pre and post intervention with the mean for pre intervention being 81.3° ± 5.1 and post intervention being 88.0° ± 3.8 in the right hind and 74.4° ± 3.7 and 87.1° ± 2.9 in the left hind. The findings of this study support the hypothesis that the hoof balance informs equine stance and can play a role in affecting limb posture. The associations between hoof conformation, lameness, degenerative disease, and catastrophic injury have been widely documented ,6,7,8,9,Although these previous studies only mentioned posture as an observation, it has been suggested that posture is the important link and possible reason for referred pathology, with altered posture indicated as a possible cause of changes in hind hoof morphology . In suppWhilst conformation is the length and shape of bones, posture is how the horse orientates and supports those structures. Limb orientations, such as canted-in, cow hock and base narrow have been measured as conformation markers, with a scoring system . HoweverDistorted neural signalling from three main proprioceptive regions is associated with ACP : dental Recent studies indicating that change in posture is associated with poor hind hoof balance have focused on negative plantar angles ,5 and thNPLA is an extreme presentation of long toe/low heel hooves, recently documented as the most common hind hoof pathology . NegativThe progression from long toe/low heel to NPLA, due to the canted-in posture, has been suggested (5), however, other variables must be considered. Some studies ,25,26 suNPLA and the relationship between hoof balance/proportions and limb orientation are worth investigating for welfare and performance optimisation. The whole horse is connected from head to toe; therefore, changes in forces, load and strains on the hind hoof are not limited to the foot, but affect the entire leg, and presumably the rest of the body.The aim of this study was to establish a correlation between hind hoof imbalance in the dorso-plantar plane and canted-in hind limb position, using metatarsal angle as a quantitative indicator. Specifically, this study hypothesised that farriery intervention would create a positive plantar angle and external hoof-pastern axis alignment, therefore affecting metatarsal position. The proposal for the study received ethical review by a university institution prior to the farriery work being undertaken. The horses were referred for intervention by their veterinary surgeons. A convenience sample of twelve horses of different breeds, ages, height, and weights  , wedge pads (www.3dhoofcare.com)  or graduated duo ellipse (Hand made by YS) , hinds were used, with frog support padding, to provide elevation to establish an improved hoof pastern axis [As all horses in this study presented with a grade 2 or above NPLA, remedial heel support was necessary. The choice of prosthetic was determined for each horse depending on the cost for the owner. Formahoof  was taken from a line created from the bony ridge of the metatarsal, visible at the most proximal and distal ends of the metatarsal, hock angle was taken from the intersection of the dorsal surface of the metatarsal and the dorsal surface of the tarsus (HA), and coronet angle (CA) was measured taking a line through the most caudal and dorsal points of the hairline at the coronet band. The pictures were uploaded into Microsoft PowerPoint  and lines were put onto the images manually following parallel to the relevant anatomical points . A protractor was used to measure HA and P-DW. An angle measuring app able to measure angles with an accuracy of ±0.2° to ±0.3° was used to measure MA . This was repeated on both sides of the horse.Plantar angle (PA), proximal phalanx to middle phalanx angle difference (P1-P2), middle phalanx to distal phalanx angle difference (P2-P3), and P-DW were measured from lateral radiographs. The radiographs were uploaded into Microsoft PowerPoint, the centre of rotation (COR) of each joint was marked, the axis of each bone was drawn through the COR and the difference in the angle of the axis was collected  Figure . The P-Dp = 0.05.The numerical data obtained were subjected to the Anderson Darling normality test to determine if they were normal or non-parametric. Wilcoxon signed-rank tests were used to establish significant differences pre and post farriery intervention. A Pearson′s correlation test was used to measure the strength of the relationships between variables in the larger dataset. The alpha value was set at In the pre intervention group, radiographically, the hinds presented with a larger difference in p2-p3 angle difference than p1-p2. The mean pre intervention p1-p2 angle difference for the right was −10.5° ± 3.1, the left was −9.9° ± 2.7. The mean pre intervention angle for p2-p3 angle difference was −13.9° ± 4.5 for the right and −16.5° ± 5.6 for the left  p = 0.000) and left (p = 0.000). There was a significant difference in CA pre and post intervention for the right but not the left hind. The mean CA pre intervention was 29.7° ± 5.3 for the right and 31.3° ± 3.7 degrees for the left, post intervention averages were 22.7° ± 3.2 and 23.6° ± 3.2, respectively. There was a significant positive correlation between CA and P-DW on both the right (p = 0.001) and left (p = 0.004). There was a significant difference in MA pre and post intervention for both the right and left hind with the average for pre intervention being 81.3° ± 5.1 and post intervention being 88.0° ± 3.8 in the right hind and 74.4° ± 3.7 and 87.1° ± 2.9 in the left hind  and the left hinds . There was a significant negative correlation between p-DW and MA for both the right  and left . There was a negative correlation between PA and MA for the right  but a positive non-significant correlation for the left . There was a significant negative correlation between PA and P-DW on the right  but not on the left . There was a significant negative correlation between P-DW and POBPL on both the right  presenting with NPLA presented with ACP, which was altered after farriery intervention (n = 7)  . While previous studies discussed the biomechanical implications of NPLA possibly being responsible for the link to higher pathologies ,5, this Anecdotally, sighting CA to the fore limb has been used as a gauge for PA, this study showed there was no significant correlation between the PA and CA, although the photos were focused on the hind end to address parallax so testing this theory was not possible. However, there was a strong correlation between P-DW and CA, showing CA as a measure of digit alignment. Considering the changes in limb position with hind hoof balance, shown by this study, the accuracy of sighting CA to the forelimb would depend on limb position, therefore actual CA could be more accurate in suggesting PA. This study found a difference in average CA pre and post intervention of 31.3° ± 3.7 and 23.6° ± 3.2 in the left hind with similar findings in the right hind, although the changes were not statistically significant in the right hind. Recent unpublished research  found thThere was also a negative correlation between PA and MA for the right, but a positive non-significant correlation for the left, but there was a significant negative correlation between P-DW and MA, meaning that the data may suggest that P-DW is a more significant measurement then the absolute PA for postural changes. Considering that normal PA is variable, anywhere between 2° and 10°, it could be suggested that although a horse that has a normal PA of 2°, absent of the morphological changes associated with NPLA, may have a −2° PA, pre-intervention, with a difference of −4°, this would cause less of an increase in tendon strain and therefore have a reduced effect on posture than a horse who has a normal PA of 10°, with a 1° PA, pre-intervention, having a difference of −9° from its normal. This study suggests that the amount of angle change from the horse physiological norm has a more proprioceptive effect than whether the PA is negative. Recent studies ,5 have fIt was also noted that one of the horses still presented with a broken P-DW, while the other subjects all presented with 1 degree or less P-DW. This subject also still presented with a MA closer to the average of the pre-treatment group. This also supports the suggestion that a BBHPA should be added to further studies to see if the negative correlation between P-DW and MA continues with a positive PA but BBHPA. Farriery intervention to address hoof proportions and create a positive PA had a significant effect on P-DW and MA. Knowing that hoof proportions affect the distal interphalangeal joint over the proximal interphalangeal joint ,26, decrP-DW was negatively correlated with MA, this suggests that hoof balance directly affects digit alignment and subsequently limb orientation. However, the mechanism can only be assumed, as this does not prove primary causation, as the question remains whether NPLA is a result of excess heel load from ACP. Measuring alterations in regional hoof loading, due to ACP, this resulting in changes to hoof growth and appearance, was beyond the scope of this study. Further research to quantify changes in load on the hoof with changes in limb orientation is warranted to clarify the two-way relationship between hoof balance and limb orientation.The farriery intervention outlined in this study created an improved P-DW by providing elevation while maintaining frog support, and further longitudinal studies could research the efficacy of frog support as both a prevention and treatment method. Hoof morphology is a factor of changes in magnitude and direction of forces acting on the foot , furtherAll the horses in this study were shod. Previous studies have reported better hoof proportions in the barefoot population in their study  and gradIt could be suggested, in accordance with theoretical reviews ,12, thatAlthough there was a significant change in right HA, the changes were smaller than expected and there was no significant difference in left HA pre and post intervention, despite a significant negative correlation between P-DW and MA and significant changes in MA and POBPL. This also suggests, due to the reciprocal apparatus, that the stifle angle was not significantly changed. Although this study did not measure higher joint changes, this points toward the change in limb orientation coming from higher structures, likely the pelvis, sacro-illiac or lumbo-sacral joint . This could explain why, anecdotally, many horses with NPLA are suggested to have sacroiliac region pain. Considering preliminary findings which suggested a link between thoracolumbar extension and over-riding spinous processes , researcThis study suggests, in agreement with previous studies , that thAlthough this study begins to outline some relationships between hind hoof balance and hind limb posture, the small number of cases may have affected the significance of certain findings, and further research with larger numbers is required for clarification. As a pilot study undertaken in the field, there were variables outside of the researchers’ control. The radiographs were already collected so correct image acquisition could only be assumed. Some radiographs did not have the metatarsal visibly vertical to have as a reference for correct limb position and stance can influence radiographic measurements of hoof balance . Post inPrevious studies measuring joint angles placed the horses against a marked wall and used skin markers to accurately measure angles in the limb and ensure exact distance from the animal . The resThe results of this study suggest that hoof balance in the dorso-plantar plane and farriery intervention to change hind hoof proportions and digit alignment have a direct influence on hind limb posture. Further research into posture as a factor in the link between NPLA and higher pathologies is warranted, to outline the pathogenesis of ACP, as the key to elucidating pathogenesis. In this study, hoof balance was shown to play a role in limb orientation, and this could prove to be an important factor in both the genesis and amelioration of the relationship. Further studies quantifying the changes in the direction and magnitude of forces on both the hoof and musculoskeletal system from ACP are needed.


Additionally, omeprazole uncompetitively inhibits dexamethasone metabolism by the CYP3A2 enzyme activity with a Ki = 39.175 ± 0.230 µM and IC50 = 78.351 ± 0.460 µM. These results suggest that the tested inhibitors would not exert a significant effect on the CYP3A2 isoenzyme responsible for the co-administered dexamethasone drug’s metabolism in vivo.The role of individual cytochrome P450 (CYPs) responsible for the drug metabolism can be determined through their chemical inhibition. During the pandemic, dexamethasone and remdesivir with omeprazole were used for the treatment of COVID-19, while Ibuprofen was taken to treat the symptoms of fever and headache. This study aimed to examine the potency of ibuprofen remdesivir, and omeprazole as inhibitors of cytochrome P450s using rat liver microsomes in vitro. Dexamethasone a corticosteroid, sometimes used to reduce the body’s immune response in the treatment of COVID-19, was used as a probe substrate and the three inhibitors were added to the incubation system at different concentrations and analysed by a validated High Performance Liquid Chromatography (HPLC) method. The CYP3A2 isoenzyme is responsible for dexamethasone metabolism in vitro. The results showed that ibuprofen acts as a non-competitive inhibitor for CYP3A2 activity with K The drug-metabolizing enzyme system, also known as the CYP450s superfamily, is responsible for the biotransformation of a large number of endogenous  and exogenous compounds  . The useCYP3A4 is primarily accountable for 6β-hydroxylation of testosterone, cortisol, progesterone, and androstenedione . CYP3A2 COVID-19 is a mainly self-limited disease and up to 20% of the cases will develop severe symptoms such as hypercoagulation, acute respiratory distress syndrome, pneumonia, and multiorgan system dysfunction. COVID-19 infection has resulted in >1.2 million deaths among 47 million recorded cases . VariousThere are no data relating to the effects of ibuprofen, remdesivir, and omeprazole on dexamethasone metabolism (CYP3A2 activity). Our previous study showed that aspirin exhibits a weak competitive inhibition of CYP3A2 isoenzyme activity and has little potential to cause drug–drug toxicity .Thus, in this report, we aimed to study the potential inhibitory impacts of these three compounds on the activity of CYP3A2 isoenzymes in rat liver microsomes using dexamethasone as a probe substrate in the presence of different concentrations of inhibitor drugs. These findings could provide significant information for the safe co-administration of these drugs in clinical settings.The HPLC method was developed and validated according to the ICH guidelines in terms of specificity, linearity, accuracy, and precision. Separation of dexamethasone, 6β-hydroxydexamethasone, inhibitors , and internal standard were attained using conditions presented in 2) of 0.99. For dexamethasone, good intra-sample and inter-sample precision were achieved with %RSD (% relative standard deviation) < 5%.The obtained calibration curves for dexamethasone and 6β-hydroxydexamethasone  showed gGood intra–day and inter-day precision was also obtained with %RSD < 10% for 6β-hydroxydexamethasone. The standard deviation ranged between 0.01% and 3.13%. The percentage recovery values for dexamethasone were between 81.68% and 112.52% and for 6β-hydroxydexamethasone between 93.13% and 119.38% (ICH acceptance criteria: %Recovery: 80–120%), representing good accuracy.The developed method could be optimally performed with slight variations in peak areas, retention time, and peak heights. The calculated LOD and LOQ for dexamethasone were 5.60 µM and 16.98 µM, respectively. LOD and LOQ for 6β-hydroxydexamethasone were calculated to be 0.06 µM and 0.19 µM, respectively.The optimised incubation time was 40 min . For optm, Vmax, % inhibition, and Clint values (±SD).Rat liver microsomes (0.5 mg/mL) were incubated with various concentrations of dexamethasone (10–50 µM) and ibuprofen (0–100 µM) in the presence of a NADPH regenerating system for 40 min at 37 °C. The metabolite was extracted using ethyl acetate and diethyl ether. The rates of 6β-hydroxydexamethasone formation were determined using High-Performance Liquid Chromatography (HPLC) technique. i (inhibition constant) and IC50  values. m, Vmax, Clint, IC50, and Ki values (±SD).Incubation of probe substrate dexamethasone (10–50 µM) with multiple remdesivir concentrations (0–100 µM) in RLMs showed that remdesivir inhibits CYP3A2 activity as a mixed inhibitor . These pm, Vmax, Clint, IC50, and Ki values (±SD) are determined and summarised in An enzyme study was carried out to determine the CYP inhibition by omeprazole in RLMs with different concentrations of probe substrates (10–50 µM) in the absence and presence of omeprazole (inhibitor). The data were taken as a mean of triplicate. The Lineweaver–Burk plot and Michaelis–Menten plot  show theThe inhibition of CYP450 enzymes by a number of drugs causes potential clinical consequences . The useThis study provided the first complete in vitro data that enable us to see the interactions of drugs used in COVID-19 treatment and investigate the inhibitory effects of ibuprofen, remdesivir, and omeprazole on CYP3A2 activity, using a High-Performance Liquid Chromatography.max) decreased compared to the Vmax of uninhibited reaction (negative control assay), while Michaelis constant (Km) stayed the same, as the inhibitor is not competing with the substrate for the active site. The ibuprofen concentrations used (50–200 µM) are similar to the amounts found in plasma (49–242 µM), and cause 50% inhibition of CYP3A2 enzyme activity (IC50) , in which V is the observed velocity, V0 is uninhibited velocity, I is the concentration of inhibitor and S is the slope factor.The type of CYP3A2 inhibition for remdesivir inhibitor was believed to be a mixed type of inhibition based on the Lineweaver–Burk plots shape, AIC, SC, and standard error. The ICm, Vmax, and Clint. The following equation was used to calculate these parameters: V = Vmax × [S]/[S] + Km (1 + [I]/Ki), in which Vmax is the maximal rate of the reaction, V is the observed velocity, S is the slope factor, Ki is an inhibitory constant, Km is Michaelis–Menten constant, and I is the inhibitor concentration.For omeprazole, inhibition was considered as uncompetitive inhibition. The Lineweaver–Burk plot was used to assess the inhibition parameters such as KIn summary, an in vitro study using rat liver microsomes emphasizes the inhibition of CYP3A2 enzyme activity by ibuprofen, remdesivir, and omeprazole, as these drugs have been used in COVID-19 treatment. Our data demonstrated that ibuprofen, remdesivir, and omeprazole possibly inhibit CYP3A2 enzyme activity as a non-competitive, mixed and uncompetitive inhibition mode, respectively. The outcomes of this research guide the safe use of dexamethasone and other COVID-19 drugs  in healthcare screening. More in vivo trials should be performed to assess the safe administration of taking ibuprofen, remdesivir, and omeprazole drugs with dexamethasone for patient care.