Saccharomyces cerevisiae is a powerful system for the expression of genome-wide or combinatorial libraries for diverse types of screening. However, expressing large libraries in yeast requires high-efficiency transformation and controlled expression. Transformation of yeast using electroporation methods is more efficient than chemical methods; however, protocols described for electroporation require large amounts of linearized plasmid DNA and often yield approximately 106 cfu/µg of plasmid DNA. We optimized the electroporation of yeast cells for the expression of whole-genome libraries to yield up to 108 cfu/µg plasmid DNA. The protocol generates sufficient transformants for 10–100× coverage of diverse genome libraries with small amounts of genomic libraries (0.1 µg of DNA per reaction) and provides guidance on calculations to estimate library size coverage and transformation efficiency. It describes the preparation of electrocompetent yeast cells with lithium acetate and dithiothreitol conditioning step and the transformation of cells by electroporation with carrier DNA. We validated the protocol using three yeast surface display libraries and demonstrated using nanopore sequencing that libraries’ size and diversity are preserved. Moreover, expression analysis confirmed library functionality and the method’s efficacy. Hence, this protocol yields a sufficient representation of the genome of interest for downstream screening purposes while limiting the amount of the genomic library required.  Saccharomyces cerevisiae is a widely used method of genetic modification whereby exogenous DNA is inserted into a cell. Yeast cells can express proteins in their native folds and undergo post-translational modifications, offering further benefits over other bacterial recombinant methods [6 to 109 plasmids or DNA fragments [The transformation of  methods . Intact  methods . Despiteragments . Transforagments . A high-ragments . Althougragments .Trypanosoma brucei, Trypanosoma cruzi, and Giardia lamblia for yeast surface display [We aimed to generate a protocol for efficient transformation of assembled genome-wide or combinatorial libraries for expression in yeast. We used libraries to express genomic fragments of parasitic pathogens— display , but the display ,6,7. S. cerevisiae EBY100 strain using the Aga1p-Aga2p protein display system [8 cfu/µg DNA, yet only requires 0.1 µg of DNA per reaction, thus reducing the amount of DNA and transformation reactions required for whole genomic library representation. In our approach, libraries were cloned in pYD1 vector for galactose induction and surface expression in y system . The play system . We optiBefore beginning the experiment, plasmid DNA was obtained using Mini-Prep kits and further cleaned up using size-selection magnetic beads. The cleanup resulted in DNA with purity of approximately 1.8 260/280 and 2.0 260/230 (see note 1). We optimized this protocol by using 100 ng of DNA of libraries constructed in the pYD1 vector to minimize library usage. This system uses a galactose-inducible promoter (GAL1) to induce the expression of surface proteins. For the preparation of electrocompetent cells we used D-sorbitol, calcium chloride (CaCl), lithium acetate (LiAc), and dithiothreitol (DTT) because they were tested and shown to result in high transformation efficiencies . TransfoWe recommend electroporating yeast without plasmid and growing on YPD to ensure that yeast can survive treatment with buffers or electroporation. We also recommend growing yeast electroporated without plasmid on SD/-trp or respective selection marker to ensure that there is no contamination or unspecific growth associated with the yeast selection system. To evaluate transformation efficiency, yeast should be electroporated with a known plasmid (ex. pYD1) and grown on SD/-trp. If all controls, as seen in Nucleospin plasmid mini-prep kit ;Next Generation Sequencing (NGS) clean-up and size selection magnetic beads .Pre-sterile barrier tips, 1000 µL ;Saccharomyces cerevisiae Meyen ex E.C. Hansen, EBY100 strain ;Yeast extract ;Peptone ;Dextrose anhydrous ;D-Sorbitol ;Calcium chloride ;Lithium acetate ;Dithiothreitol .10.Pre-sterile barrier tips, 1000 µL ;11.Pre-sterile barrier tips, 200 µL ;12.Pre-sterile barrier tips, 10 µL ;13.pYD1 vector ;14.Agar A ;15.Cuvettes plus 2 mm gap ;16.Semi-micro cuvette, PS ;17.Falcon 50 mL Conical Centrifuge Tubes ;18.Tissue Culture-Treated Dishes, 100 mm × 20 mm .Glycerol ;Microcentrifuge tubes, 2 mL, with screw caps .Yeast nitrogen base w/o AAs, w/o ammonium sulphate ;DO supplement -trp ;Ammonium sulphate ;D-(+)-raffinose pentahydrate ;D-Galactose .Bunsen burner ;® basic pH meter ;AB15 Accumet® J-E BioSafe High Performance Centrifuge ;AvantiStackable incubator shaker ;Visible spectrophotometer V1-200 ;Gene Pulser Xcell Microbial System ;Vortex mixer ;Freezer −80 °C .DAY ONE(A)6 clones, then 107 (10-fold) to 108 (100-fold) yeast transformants are required. In our experience, aiming for 10-fold, or more, for the library size increases the library representation in yeast , to obtain 10 to 100-fold the library size. If the library size is 10Calculate the number of required library transfections:7–108 cfu/µg DNA using this protocol. (B)S. cerevisiease EBY100 strain culture.Prepare and sterilise all solutions. CRITICAL STEP The 0.1 M LiAc/10 mM DTT filter sterilized solution should be made fresh on the day of electroporation;Prepare at least 4 SD/-trp dex agar plates per library transfection.S. cerevisiease EBY100 strain from working stock plate (see notes 2 and 3) and inoculate into 5 mL of sterile YPD. Allow to grow for approximately 7 h in an incubator shaker at 30 °C whilst shaking at 225 rpm.Take 1 colony (~2 mm) of 600 of 0.02–0.07 and allow to grow overnight (16 h) at 30 °C whilst shaking at 225 rpm (see note 4).Add the 5 mL culture to 95 mL fresh YPD to reach an ODPrepare solutions  and 2.DAY TWO(A)600 of the overnight culture, the optimal OD600 range is between 1.6 and 1.8 after 16 h in a 30 °C incubator whilst shaking at 225 rpm. This will depend on the doubling time of your yeast (see note 5).Prepare 100 mL EBY100 culture in YPD per 4 electroporation reactions. Measure the ODg for 4 min, at 4 °C and remove the media.Collect EBY100 cells by centrifugation at 2000× 2O per 100 mL of culture. Centrifuge at 2000× g for 4 min at 4 °C. Repeat this step once.Ressuspend by vortexing the cell pellet in 50 mL sterile ice-cold ddHg for 4 min at 4 °C.Ressuspend by vortexing the cell pellet once in 50 mL ice-cold electroporation buffer per 100 mL of culture. Centrifuge at 2000× Resuspend by vortexing the cell pellet in 20 mL of fresh, 0.1 M LiAc/10 mM DTT (pre-warmed to 30 degrees) per 100 mL of culture. Transfer the cell suspension to a covered Erlenmeyer flask and condition the yeast by shaking at 225 rpm for 20 min at 30 °C (see note 6).g for 4 min at 4 °C.Collect cells by centrifugation at 2000× g for 4 min at 4 °C.Ressuspend by vortexing the cell pellet in 50 mL ice-cold electroporation buffer per 100 mL of culture. Centrifuge at 2000× 9 cells, which is sufficient for 4 electroporation reactions of 200 µL each (2.1 × 108 cells per reaction).Resuspend by vortexing the cell pellet in ice-cold electroporation buffer to achieve a final volume of 1.4 mL per 100 mL of culture. Keep the cells on ice until electroporation. There should be approximately 1.5 × 10Preparation of electrocompetent cells:(B)Prepare 5 mL of 1:1 Sorbitol:YPD per electroporation and store on ice.For each electroporation prepare 0.1 µg of genomic library DNA, 25 µg of Salmon Sperm DNA and 200 µL of electrocompetent cells. Mix gently by pipetting and transfer to a pre-chilled 2 mm cuvette. Prepare a separate negative transformation control using 0.1 µg of pYD1 plasmid per 200 µL electrocompetent cells (see note 7).Incubate the cells on ice for 5 min.Electroporate cells at 2.5 kV, 25 µF, 200 Ω with a Gene Pulser Xcell Microbial System. The time constant ranges should be between 3.1 and 4.2 ms.Immediately add 1 mL of pre-chilled Sorbitol:YPD to the cuvette. Transfer the cells to a 50 mL centrifuge tube containing 3 mL of Sorbitol:YPD. An additional flush of the cuvette with another 1 mL of sorbitol:YPD will help recover cells from the cuvette. Adjust the final volume to 5 mL Sorbitol:YPD solution. CRITICAL STEP Keep the cells on ice until all the electroporations are finished and cells are resuspended in Sorbitol:YPD. Allow transformed cells to recover for 1 h by placing the tubes in an incubator shaker at 30 °C whilst shaking at 225 rpm.g for 4 min, at 4 °C.Harvest cells by centrifugation at 2000× 2O to eliminate any trace of YPD. Centrifuge at 2000× g for 4 min at 4 °C. Repeat this step twice.Resuspend by vortexing the cell pellet in 10 mL ddHResuspend by vortexing cells in 4 mL SD/-trp dex.OPTIONAL STEP: Prepare serial dilutions of two samples of each library transfection in SD/-trp dex and plate 100 µL of 1:50 and 1:100 dilutions on SD/-trp dex agar plates for calculation of transformation efficiency. Invert plates and place them in 30 °C incubator for 3–5 days, until colonies are visible.600 approximately 0.1–0.2. A volume of roughly 150 mL is typically used as a 1:30 dilution that reaches the expected OD600.The cultures transformed with the same genomic library can be pooled together and resuspended in a 1:30 dilution of SD/-trp dex media to an ODAllow the culture to expand for 48 h by incubating on a platform shaker at 30 °C whilst shaking at 225 rpm.600 of the culture to ensure the culture has expanded (see note 8). The OD600 is expected to be approximately 1.5–1.8. At this point, transformed cells can be frozen or surface protein expression can be induced.Check the ODTransformation of electrocompetent cells with genomic library:(C)g for 4 min at 4 °C.Harvest cells by centrifugation at 2000× Resuspend by vortexing pellet in 1 mL of SD/-trp dex with 25% glycerol per 10 mL of culture. The glycerol causes the cells to freeze slowly reducing damage caused by the formation of ice crystals.Freeze cells in a −80 °C freezer.Freezing transformed yeast cells.3.DAY FIVE(A)Check the SD/-trp dex agar plates for visible colonies and count the total number of colonies on the plates. Count plates from dilutions resulting in 30 to 300 colonies.Calculate the transformation efficiency using the following formula :7 to 108.Typical transformation efficiencies range between 10Calculate the transformation efficiency and genome coverage of the YSD library:(B)1.Thaw each vial of transformed yeast in 37 °C water bath for 30 s.2.6 cells and recover in 5 mL YPD at 30 °C and 225 rpm for 2 h or until an OD600 of 1.Take 200 µL of cells, ~6 × 103.g for 4 min at 4 °C.Harvest cells by centrifugation at 2000× 4.2O and centrifuge at 2000× g for 4 min at 4 °C to remove any trace of YPD. Repeat this step twice.Ressuspend by vortexing cells in 10 mL ddH5.600 of 0.4 and allow the culture to grow for 4 h or until an OD600 of ~1 in an incubator shaker at 30 °C shaking at 225 rpm.Resuspend by vortexing cells in 20 mL selective media (SD/-trp dex) to an ODThawing transformed yeast cells:(C)1.g for 4 min at 4 °C.Harvest cells by centrifugation at 2000× 2.2O and centrifuge at 2000× g for 4 min at 4 °C. Repeat this step once.Ressuspend by vortexing cells in 10 mL ddH3.Resuspend by vortexing the cell pellet in 20 mL SD/-trp/raffinose (raf) media. Grow at 225 rpm, 30 °C for 2 h to adapt cells to raf.4.g for 4 min at 4 °C.Harvest cells by centrifugation at 2000× 5.Resuspend by vortexing pellet in 20 mL of SD/-trp/raf/3% galactose. Incubate for at least 16 h at 225 rpm 30 °C to induce the expression of the library (see note 9).6.OPTIONAL STEP To validate protein expression by Western blot, take an aliquot of induced cells, lyse the cells and extract proteins according to protocol . PerformInduction of YSD:The quality of the DNA used can greatly affect transformation efficiencies; thus, cleaning up the DNA with magnetic beads is highly recommended.All steps manipulating the yeast should be carried out in sterile conditions, such as in the vicinity of a flame.Always use an EBY100 colony from a fresh (<4 weeks old) working stock plate. Avoid selecting too small or too large colonies. If a fresh plate is unavailable, streak a new one from freezer stock EBY100 and not from a previous working stock plate.Yeast will grow faster with better aeration. It is recommended to shake them at 220–270 rpm whilst growing.We strongly recommend performing a growth curve of your EBY100 to establish the doubling time of EBY100 in the conditions specific to the user’s laboratory.We have found that conditioning the cells in LiAc/DTT for longer than 30 min significantly decreases transformation efficiency.Changing the amount of DNA in electroporation can affect transformation efficiency.At this transformation efficiency, approximately 99.5% of the cells die, which typically shows as clumps of black debris in the culture. It does not affect the procedure.Yeast galactose induction requires media with no glucose. In this example, the Aga1p-Aga2p display system using pYD1 and EBY100 yeast strain uses a GAL1-activated promoter, which is repressed by the presence of glucose and induced by galactose. A 2% raf is used as an alternative carbon source in the SD/-trp dex media to avoid affecting GAL1 induction.To avoid diluting the media by adding galactose prepared in water, consider preparing 30% galactose stock in SD/-trp dex media. D-galactose solubility in water is 32%.7 to 108 transformants/µg of library DNA with the average transformation efficiency being 4.6 × 107 transformants/µg of library as seen in 7 transformants/µg DNA, which can be seen in Trypanosoma cruzi, Trypanosoma brucei, and Giardia lamblia. The average transformation efficiency was 3.05 × 107, 2.72 × 107, and 4.62 × 107 transformants/µg DNA, respectively, as seen in E. coli (used to generate the library) and the genomic library extracted from S. cerevisiae (used to express the library) using Oxford Nanopore Sequencing to confirm the complete transformation of the library size. We found a Pearson correlation of 0.866, suggesting maintenance of library size as seen in Our transformation protocol gave a transformation efficiency of 10T. cruzi library (TcLib) was validated by Western blot. A smear is seen when yeast is grown in the presence of galactose inducing protein expression. This is not seen in the absence of galactose. In In We tested concentrations of DNA ranging from 0.1 to1.5 µg and found 0.1 µg to be ideal as it provides high transformation efficiency while using little DNA. We still recommend optimizing DNA concentrations as DNA preparations and differences in plasmids may affect efficiency. We also recommend purifying DNA with magnetic beads; see Note 1.a.2O. Dissolve 10 g yeast extract and 20 g peptone in 850 mL MilliQ Hb.Adjust pH to 6.5 with 1 M HCl or 1 M NaOH.c.2O to final volume of 900 mL.Add MillQ Hd.Autoclave for 20 min at 121 °C. e.Wait to cool, then add 100 mL of sterile 20% dextrose under sterile conditions  and store at 4 °C. YPDa.2O. Dissolve 1.7 g yeast nitrogen base, 5 g ammonium sulphate, 0.74 g -trp drop out supplement in 850 mL MillQ Hb.Adjust pH to 5.8. c.2O to final volume of 900 mL. Add MillQ Hd.Autoclave for 20 min at 121 °C. e.Wait to cool, then add 100 mL of sterile 20% dextrose under sterile conditions and store at 4 °C. SD/-trp dexa.2O. Dissolve 1.7 g yeast nitrogen base, 5 g ammonium sulphate, 0.74 g -trp drop out supplement in 850 mL MillQ Hb.Adjust pH to 5.8. c.Add 20 g agar. d.2O to final volume of 1 L. Add MillQ He.Autoclave for 20 min at 121 °C and store at room temperature.f.Wait to cool, then add 100 mL of sterile 20% dextrose under sterile conditions and store at 4 °C. SD/-trp dex agara.2O. Dissolve 1.7 g yeast nitrogen base, 5 g ammonium sulphate, 0.74 g -trp drop out supplement in 850 mL MillQ Hb.Adjust pH to 5.8. c.2O to final volume of 900 mL. Add MillQ Hd.Autoclave for 20 min at 121 °C. e.Wait to cool, then add 100 mL of sterile 20% raffinose under sterile conditions  and store at 4 °C. SD/-trp/rafa.2O. Add NaOH drop by drop if necessary to dissolve all the sugar. Dissolve 200 g dextrose in 900 mL MillQ Hb.Adjust to pH 6.5.c.2O to final volume of 1 L and filter sterilize. Store at 4 °C. Add MillQ H20% Dextrosea.2O. Add NaOH drop by drop if necessary to dissolve all the sugar. Dissolve 200 g galactose in 900 mL MillQ Hb.Adjust to pH 5.8.c.2O to final volume of 1 L and filter sterilize. Store at room temperature. Add MillQ H20% Galactose (see note 10)a.2O. Add NaOH drop by drop if necessary to dissolve all the sugar. Dissolve 200 g raffinose in 900 mL MillQ Hb.Adjust to pH 5.8c.2O to final volume of 1 L and filter sterilize. Store at room temperature. Add MillQ H20% Raffinosea.1 M sorbitolb.21 mM CaClc.Autoclave for 20 min at 121 °C or filter sterilize and store at 4 °C.Electroporation buffera.M Lithium Acetateb.10 mM DTTc.Make fresh on the morning of electroporation. 1 M stock solutions of each LiAc and DTT can be made in advance, filter sterilized, and used to make the conditioning buffer when necessary. Conditioning buffer


In mouse, it was discovered that resveratrol (Res) enhanced osteoporosis (OP) by boosting osteogenesis. Besides, Res can also have an impact on MC3T3-E1 cells, which are crucial for the control of osteogenesis and thus increase osteogenesis. Although some articles have discovered that Res enhanced autophagy to promote the value-added differentiation of MC3T3, it is unclear exactly how this affects the process of osteogenesis in mouse. Therefore, we will show that Res encourages MC3T3-E1 proliferation and differentiation in mouse pre-osteoblasts and further investigate the autophagy-related mechanism for this impact.(1) MC3T3-E1 cells were separated into blank control group and various concentrations  of group in order to determine the ideal Res concentration. In the Res group, Cell Counting Kit-8 (CCK-8) was used to measure the proliferation activity of pre-osteoblasts in mice in each group after resveratrol intervention. Alkaline Phosphatase (ALP) and alizarin red staining were used to gauge the degree of osteogenic differentiation, and RT-qPCR was used to measure the expression levels of Runx2 and OCN in the osteogenic differentiation ability of the cells. (2) In the experiment, four groups were set up: the control group, 3MA group, Res group, and Res + 3MA group. To examine cell mineralization, ALP and alizarin red staining were utilized. RT-qPCR and Western blot detection of cell autophagy activity levels and osteogenic differentiation capacity in each group following intervention.(1) Resveratrol might increase the number of mice pre-osteoblast, with the impact being most pronounced at 10µmol/L (P < 0.05). The nodules developed substantially more often than in the blank control group, and Runx2 and OCN expressions significantly increased (P < 0.05). (2) In contrast to the Res group, after 3MA purine blocked autophagy, the Res + 3MA group’s alkaline phosphatase staining and the development of mineralized nodules were reduced. Runx2, OCN, LC3II / LC3I expression decreased, p62 expression increased (P < 0.05).The present study partially or indirectly demonstrated that Res may, through increased autophagy, induce osteogenic differentiation of MC3T3-E1 cells.The online version contains supplementary material available at 10.1186/s12906-023-03943-8. Osteoporosis (OP) is a common metabolic bone disease in postmenopausal women and the elderly . OsteopeAnti-osteoporosis medications that inhibit bone resorption and encourage bone formation are becoming more common in the development of anti-osteoporosis medications . TraditiRecent studies have found that autophagy is an important physiological process to maintain the dynamic balance of cells by removing damaged organelles and proteins . FurtherMouse pre-osteoblast subclone 14 (MC3T3-E1 Subclone 14) was purchased from the Cell Bank of Chinese Academy of Sciences. Res was purchased from Aladdin Reagent Company, China, product number CAS No. 501-36-0. HPLC grade (≥ 94%), α-MEM medium was purchased from HyClone, USA. Fetal bovine serum (FBS) was purchased from Gibco, USA. Runx2 antibody (20700-1-AP), BMP-2 (66383-1-lg), Beclin1 antibody (11306-1-AP), LC3 antibody (14600-1-AP), p62 antibody (18420-1-AP) and β-Actin antibody (66009-1-lg) were purchased from Proteintech. Cell proliferation and toxicity test kit (CCK-8 kit) purchased from Beijing Solarbio Science & Technology Co., Ltd. Alkaline phosphatase (ALP) detection kit (P0321S) and BCIP / NBT alkaline phosphatase chromogenic kit (C3206) were purchased from Shanghai Beyotime Biotechnology Co., Ltd. 3-Methyladenine (3-MA) was purchased from Shanghai source leaf organisms.Arrangement 10 mg Res was broken down in 438 µl DMSO to plan 100 mmol/L Res stock arrangement, which was stuffed and put away at -20 °C fridge. The Res stock arrangement was weakened to the accompanying fixations: 0.01, 0.1, 1, 10, 100µmol/L, utilizing α-MEM medium .2 hatchery. At the point when the cells develop to logarithmic stage, the cells are processed and afterward passaged. The cell development state was firmly noticed. At the point when the cell development combination rate arrived at 80%, the cells were passaged once, and the third and fourth ages of cells were taken for tests.MC3T3-E1 cells were immunized in α-MEM medium  and refined at 37 °C, 5% COThe technique was utilized to distinguish the impact of medications on cell expansion function MC3T3-E1 cells developed to 80% conjunction, processed with a stomach related arrangement containing 0.25% trypsin, and made into cell suspension. The cells were cultivated in a 96-well plate with 3000 cells/well and refined in a 5% CO2 hatchery at 37 °C. Following 24 h of cell connection, 200µL of α-MEM culture medium containing various convergences of Res  was supplanted, and the cells were refined for 24 and 48 h. 10µL CCK-8 arrangement was added to each well and brooded in the hatchery for 2 h. The OD value at 450 nm was detected by microplate reader.4 cells / well evenly spread, divided into blank control group and 0.01, 0.1, 1, 10, 100µmol / L Res group. Cells in each group were treated with different doses of Res for 24 h, and then replaced with osteogenic induction solution containing 50 mg /L ascorbic acid and 10 mol/L β-glycerophosphoric acid for 7 days [MC3T3-E1 cells were blown and mixed evenly, with 12-well plates set to 5 × 10r 7 days , osteoge4/well in 12-well plates and separated into clear benchmark group and 0.01, 0.1, 1, 10, 100µmol/L Res gatherings. Cells in each group were treated with different doses of Res for 24 h, and then replaced with osteogenic induction solution containing 50 mg /L ascorbic acid and 10 mol/L β-glycerophosphoric acid for 21 days [The MC3T3-E1 cells were blown and blended well. The cells were set to 5 × 10 21 days . In the 5 cells/well. The cells were partitioned into clear benchmark group and 0.01, 0.1, 1, 10, 100µmol/L Res bunch. After 1 day of culture, cells were cultured in osteogenic induction medium. The MC3T3-E1 cells were washed two times with PBS, and RIPA lysis cushion was added to lyse the cells for protein extraction. The protein was isolated by SDS-PAGE, moved onto PVDF film and brooded with relating essential and auxiliary antibodies. The antibodies utilized incorporate p62, Beclin1, LC3 essential neutralizer and relating auxiliary immune response. The film was set in a chemiluminescence imaging framework, and the outcomes were examined by ImageJ programming to compute the dark worth of each band.The MC3T3-E1 cells were blown and blended, and the cells were equally spread on a 6-well plate with 1 × 105/well in a 6-well plate. After 24 h of hatching with normal α-MEM, the medium was supplanted by osteogenic enlistment medium . On days 7 and 14, quality investigation was completed on the cells. More or less, the cells were processed with trypsin, and the complete RNA was extricated utilizing Trizol re-specialist . Right away, cDNA was orchestrated from the extricated mRNA through turn around record response utilizing Prime Content RT reagent Pack . Then, Next, RT-PCR was con-ducted utilizing a SYBR Green RT-PCR unit  and ABI Stage One Or more Ongoing PCR Framework . The examples were rehashed multiple times, and every one of the above tests were per-framed under the maker’s directions. The preliminary groupings of every quality were displayed in Supplementary Table The outflow of osteogenesis qualities including Runx2, OCN were distinguished through Continuous quantitative PCR (RT-qPCR), and GAPDH was utilized as a source of perspective quality. MC3T3-E1 cells were refined on the frameworks with a thickness of 1 × 10The ideal mediation convergence of Res (10µmol/L) was chosen as the intercession grouping of ensuing tests, and 5 mmol/L 3-Methyladenine (3MA) was added to hinder autophagy. The examination was isolated into control bunch, 3MA bunch (5 mmol/L), Res bunch (10µmol/L), Res bunch (10µmol/L) + 3MA (5 mmol/L).4 cells/well in a 12-well plate, and 500µL of the comparing arrangement was added to each well as per the 2.4.(1) Basic phosphatase staining was utilized to notice the osteogenic capacity of cells: The cells were equitably plated at 5 × 104 cells/well in a 12-well plate, and 500µL of the relating arrangement was added to each well as per the 2.5.(2) Perception of mineralized knobs by alizarin red staining: The cells were uniformly plated at 5 × 105 cells/all things considered, and 100µL of the relating arrangement was added to each well as per the 2.6.(3) Identification of Beclin1, LC3 and p62 protein articulation in cells by Western smudge: The cells were equally plated in 6-well plates at 1 × 10Measurable investigation SPSS 20.0 programming was utilized for factual examination. The trial information were communicated as X ± S. All examinations were rehashed multiple times. The mean correlation between the two gatherings was performed by free examplet test. The mean examination between numerous gatherings was performed by one-way investigation of change. Further pairwise examination was performed by LSD test. P < 0.05 was viewed as measurably huge.Firstly, the effect of Res on the proliferation of mouse MC3T3-E1 was investigated. The MC3T3-E1 cells were treated with 0.01, 0.1, 1, 10, 100 µ / L Res for 24 and 48 h Respectively, as shown in Fig. Alkaline phosphatase (ALP) is an early marker of osteogenic differentiation. The higher the ALP activity, the more mature the differentiation . After 7Extracellular matrix mineralization is considered to be one of the important markers of late maturation of osteoblast differentiation . FollowiRunx2 also plays an important role in the early stage of osteogenic differentiation of cells. It is a downstream transcription factor of many osteogenic related pathways. Reduction will lead to a decrease in the level of osteogenic differentiation . OCN is As shown in the Fig. In order to further study whether Res enhances the osteogenesis of MC3T3-E1 cells through autophagy, the resulting tests were isolated into four gatherings: Control group, 3-methyladenine (3MA) group, Res group, Res + 3MA group. The above four groups were cultured in osteogenic differentiation medium at the same time. After 7 and 21 days, the results of ALP and alizarin red staining were as follows: compared with the control group, the degree of ALP and mineralization staining was significantly reduced after adding 3MA, indicating that 3MA inhibited the expression of ALP and the mineralization level of extracellular matrix, while the addition of Res could significantly promote the staining level of ALP and alizarin red, and the combined use of 3MA significantly inhibited the promoting effect of Res. The effect of Res on osteogenic differentiation of MC3T3-E1 cells was significantly decreased Fig. A-B. FiguDuring autophagy, the microtubule associated protein light LC3I is converted into LC3II to initiate autophagy body formation and autophagy, whereas the substrate for autophagic degradation of p62 binds to LC3 on the membrane of autophagosome during autophagy formation and is subsequently degraded by autophagosome , 30. TheOP is a metabolic bone disease in which osteoclasts are more active in bone resorption than osteoblasts in bone formation . ImbalanTo clarify how Res works to treat OP, we decided the impact of Res on the expansion and separation of pre-osteoblast MC3T3-E1 cells and the job of autophagy. Firstly, we demonstrated that Res could promote the proliferation of MC3T3-EI cells by CCK8 assay, this is consistent with the results of Liu Xu and others . SecondlAutophagy is eukaryotic cells through autophagy to intracellular abnormal proteins, damaged or aging organelles, pathogens and other degradation, its metabolites such as amino acids, nucleotides and re-used by cells, material synthesis, energy metabolism and other life activities . AutophaIn summary, we found that Res can promote osteoblast differentiation and promote bone formation through cell experiments. The potential mechanism is to enhance the differentiation of MC3T3-E1 cells into osteoblasts by promoting autophagy. Our work will add to the mechanistic study and clinical treatment of postmenopausal OP therapy. Of course, there are still some limitations in the experiment. The experiment only did cell verification, and lacked certain in vivo experimental verification. We will further study the relevant mechanisms later.The present study partially or indirectly demonstrated that Res may, through increased autophagy, induce osteogenic differentiation of MC3T3-E1 cells.Below is the link to the electronic supplementary material.Additional file 1: Supplementary Table 1. Primer sequences of osteogenesis-related genes.Additional file 2.


Limbal stem/progenitor cells (LSPCs) play a crucial role in maintaining corneal health by regulating epithelial homeostasis. Although PM2.5 is associated with the occurrence of several corneal diseases, its effects on LSPCs are not clearly understood.In this study, we explored the correlation between PM2.5 exposure and human limbal epithelial thickness measured by Fourier-domain Optical Coherence Tomography in the ophthalmologic clinic. Long- and short-term PM2.5 exposed-rat models were established to investigate the changes in LSPCs and the associated mechanisms.We found that people living in regions with higher PM2.5 concentrations had thinner limbal epithelium, indicating the loss of LSPCs. In rat models, long-term PM2.5 exposure impairs LSPCs renewal and differentiation, manifesting as corneal epithelial defects and thinner epithelium in the cornea and limbus. However, LSPCs were activated in short-term PM2.5-exposed rat models. RNA sequencing implied that the circadian rhythm in LSPCs was perturbed during PM2.5 exposure. The mRNA level of circadian genes including Per1, Per2, Per3, and Rev-erbα was upregulated in both short- and long-term models, suggesting circadian rhythm was involved in the activation and dysregulation of LSPCs at different stages. PM2.5 also disturbed the limbal microenvironment as evidenced by changes in corneal subbasal nerve fiber density, vascular density and permeability, and immune cell infiltration, which further resulted in the circadian mismatches and dysfunction of LSPCs.This study systematically demonstrates that PM2.5 impairs LSPCs and their microenvironment. Moreover, we show that circadian misalignment of LSPCs may be a new mechanism by which PM2.5 induces corneal diseases. Therapeutic options that target circadian rhythm may be viable options for improving LSPC functions and alleviating various PM2.5-associated corneal diseases.The online version contains supplementary material available at 10.1186/s12989-023-00540-y. Corneal epithelium on the surface of the cornea acts as a natural barrier and prevents the ocular surface from environmental stimuli . CornealLimbal stem/progenitor cells (LSPCs), located in the corneoscleral transition zone, are the primary source of corneal epithelial cells, essential for maintaining corneal epithelial homeostasis . LPSCs aPM2.5, one of the main hazardous components of air pollution, specifically referring to fine particulate matter with a diameter less than 2.5 microns, has been previously linked to ocular surface diseases such as dry eye, pterygium, and conjunctivitis –12. We pRecently, a few studies have shown that PM2.5 exposure disrupts circadian rhythm and causes functional or metabolic disorders in different organs , 16. CirIn this study, we analyzed the correlation between PM2.5 exposure and the limbal impairment in humans, explored changes in LSPCs and their microenvironment using PM2.5-exposure rat models, and investigated the role of circadian rhythm in LSPCs under PM2.5 exposure.As depicted in the meta-analysis Fig. , there i3 and ≥ 35 µg/m3. There was no significant difference in age and gender ratio between individuals in the two groups  in this group (regions with PM2.5 concentration above 35 µg/m3) as compared with that in the other group (regions with PM2.5 concentration below 35 µg/m3), while there was no significant change in minimum values . Further, the rate of thinning was − 0.7875 μm/(µg•m− 3), which indicated that for every 1 µg/m3 increase in PM2.5 concentration, the average thickness of corneal limbal epithelium decreased by 0.7875 μm , and the ocular surface damage worsened with increasing exposure time  in the PM2.5 group, which is generally expressed in epidermis-related tissues Fig. E, sugges+ cells   in rat tears throughout the PM2.5 exposure period. Interestingly, H2O2 in tears was elevated after 2 days of PM2.5 exposure but was not significantly different from control after 1 week and was even lower than control after 3 weeks , suggesting that oxidative phosphorylation may be involved in the activation of LSPCs, but it may not be the most critical factor in the dysfunction of LSPCs and disruption of corneal homeostasis.Besides, oxidative phosphorylation has also been suggested to be an important factor contributing to PM2.5-induced diseases –22. We eThe limbal microenvironment plays a critical role in providing a specialized stem cell niche for LSPCs and supporting their functions. However, the association between circadian disruption, LSPC dysfunction, and the impact on the limbal microenvironment following PM2.5 exposure remains unclear. Therefore, the present study also investigated the responses of the limbal microenvironment including the corneal neural network, limbal vasculature, and immune cell infiltration in short- or long-term exposure to PM2.5, respectively.The immunofluorescence staining of TUBB3 showed that long-term exposure to PM2.5 significantly decreased the density of subbasal nerve fibers Fig. A. LimbalIn addition, the EvansBlue leakage assay was performed to evaluate limbal vascular permeability. The vascular leakage increased in a dose-dependent manner after 2-day PM2.5 exposure Fig. C. Howeve+ cells that were located in the limbal stroma  according to the 5-year average annual PM2.5 concentration of their permanent residence regions between 2018 and 2022. Individuals with ocular surface diseases, surgeries or trauma history, and systemic diseases were excluded. Moreover, individuals with a long history of living away from home, smoking, and occupational dust exposure were also excluded. Differences in age composition ratio and male/female ratio between the groups were insignificant.We collected Fourier-domain Optical Coherence Tomography (FD-OCT)  corneal pattern images of patients in the outpatient clinic of Zhejiang University Eye Hospital from 2018 to 2022. According to the Chinese Ambient Air Quality Standards (GB3095-2012), the PM2.5 concentration limit for residential areas, mixed commercial and traffic residential areas, cultural areas, general industrial areas, as well as rural areas is 35 µg/mA previous study conducted by Feng et al.  showed tThe PM2.5 extraction process was as previously reported . BrieflyText. S1. For the short-term PM2.5 exposure group, both eyes of rats were administered with 5 µL eye drops of PM2.5 (1 mg/mL in PBS) for 2 days at the same frequency. Rats that were administered with eye drops of PBS for the same days were established as control groups. N = 35 in each group of long-term models. N = 20 in each group of short-term models. Male Sprague-Dawley rats  were obtained from SLAC Laboratory Animal Co., Ltd. , housed in a standard environment with a temperature of 20 to 24 °C, a humidity of 50 to 60% under a 12-hour light-dark cycle and provided with food and water without limitation. Rats were randomized into 4 groups. For the long-term PM2.5 exposure group, both eyes of rats were administered with 5 µL eye drops of PM2.5 (1 mg/mL in PBS) for 21 days at a frequency of 4 times per day. The actual daily dose was 20 µg. The rationale for the exposure treatment in rat models was described in  Rat corneal epithelium defects were explored after 0, 2, 7, 14, and 21 days of administration using the Fluorescein sodium strips (Meizilin). After anesthesia by intraperitoneal injection of 0.3% pentobarbital sodium (50 mg/kg), fluorescein sodium strips were moistened with PBS and placed in conjunctival sacs of the lower lids of rats. The eyes of the rats were gently closed several times and the staining of the ocular surface was examined with a slit lamp under cobalt blue light.To evaluate tear film stability and ocular surface damage, tear secretion was measured after 0, 2, 7, 14, and 21 days of administration using Schirmer’s test. After anesthesia, a graded sterile phenol red cotton thread for a tear test (Meizilin) was gently placed in the conjunctival sac of the lower lid. Rat eyes were closed for 60 s and the length of the wetting thread was measured in millimeters.2O2 in rat tears was measured after 0, 2, 7, 14, and 21 days of administration. After anesthesia, 10 µl of PBS was applied to the ipsilateral eye of all rats, and recovered after wetting the eye surface. The level of H2O2 was measured using H2O2 Detection Kit (mlbio#m1076343).HVessel leakage of limbal vessel after 0, 2, 7, 14, and 21 days of administration was measured using Evans blue. Evans blue  was injected via rat tail veins(n = 5), circulated for 2 h, and final blood samples for each rat were obtained. After anesthesia and opening the chest cavity, rats were perfused with 37 °C PBS via the left ventricle until the color of the fur lightened and the liquid flowing from the right atrium was clean. Both eyes were immediately enucleated and bisected at the equator, after which the corneas with limbus were dissected under a microscope. The corneas were dried at 4 °C overnight and cornea dry weights were measured. The cornea specimen was incubated in 0.2 ml formamide (Macklin# F809511) at 70 °C overnight to extract the Evans blue dye. The extract and blood samples were centrifuged at 17,000 g for 15 min and 60 µl of the supernatant was used to measure absorbance at 630 nm. A standard curve of Evans blue in formamide was established to calculate the concentrations of dyes. Vessel leakage levels were calculated using the following formula:For limbal epithelium RNA sequencing, rats were sacrificed after 2 days of administration. Eyeballs were collected(n = 3) and rinsed by PBS containing 50 µg/mL gentamicin and 1.25 µg/mL amphotericin B. Limbal segments were separated from eyeballs using ophthalmic scissors and the remaining conjunctiva, iris, and corneal endothelium was removed. The intact limbal epithelium sheet from the limbal segment was isolated by digestion at 37 °C for 20 min with 10 mg/ml Dispase II  in a modified embryonic stem cell medium made of 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium containing 5 ng/ml epidermal growth factor, 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml sodium selenite, 0.5 µg/ml hydrocortisone, 30 ng/ml cholera toxin B, 50 µg/ml gentamicin, 1.25 µg/ml amphotericin B and 10% fetal bovine serum.For H&E staining and immunofluorescence staining, rats were sacrificed after 2 or 21 days of administration. Eyeballs were obtained and directly embedded in optimum cutting temperature compound (OCT) in liquid nitrogen and sectioned to 8 μm using a Leica CM1950. Extra eyeballs were stored in liquid nitrogen for a long time.Sections were fixed, incubated with Hematoxylin, washed, stained with Eosin, dehydrated in 95% and 100% Ethanol, incubated with Xylene, and mounted.For the measurement of the cornea, we measured the average epithelial thickness within 400 microns of the central cornea using IPP6.0 software. For the measurement of the limbus, we examined the thickness of the limbal epithelium located at the transition zone of the corneal epithelium and conjunctival epithelium. The maximum values of the distance between the upper and lower margins of the limbal epithelium were calculated by IPP6.0 software.The rat cornea sections were soaked in PBS for 10 min to remove OCT and fixed with 4% paraformaldehyde for 15 min. After incubation with 10% goat serum and 0.2% TritonX-100 (Sigma Aldrich) for 1 h at room temperature, samples were incubated with primary antibodies at 4℃ overnight. The next day, they were incubated with secondary antibodies at room temperature for 1 h, labeled with 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (Sigma Aldrich), and covered with coverslips. Observation and imaging of samples were performed using the Leica TCS SP8 confocal microscope . The antibodies used in this study were: Ki67 , K12 , K10 , and CD45 .Whole-mount immunofluorescence examination was performed to assess vascular and nerve morphologies, and tight junction. Rat corneas containing limbus were dissected from the eyeballs’ posterior aspect of the corneoscleral rim using fine scissors. After removing the iris and retina, corneas were fixed in 4% paraformaldehyde on ice for 1 h, followed by permeabilization and blocking for 1 h. Cornea specimens were incubated with primary antibodies at 4℃ overnight, and then probed with secondary antibodies for 1 h at room temperature and nuclei staining the next day. Anti-rabbit CD31 antibody  was used for vascular staining, and anti-rabbit ZO-1 antibody  was used for tight junction staining. For cornea nerve staining, corneas were incubated with NorthernLights™ NL557-conjugated mouse monoclonal anti-TUBB3  at 4℃ overnight, and their nuclei were stained. The cornea was cut into 4 flaps, the endothelial side was turned upwards and covered with coverslips. The Leica TCS SP8 confocal microscope (Leica) was used to observe and image the corneal whole mount splices. The density of vascular and neural networks and the mean fluorescence intensity of tight junction were analyzed by Image J2 v2.3.0.The total RNA of limbal epithelium sheets was extracted by FlaPure Animal Tissue Total RNA Extraction Kit (Genesand# RE705). Nanodrop ND-1000 and Agilent 5400 were used to measure the concentration, integrity, and purity of RNA, and qualified samples were used for the subsequent RNA-seq. RNA-seq was performed by Novogene Co., LTD .p-value < 0.001 and | log2(fold change) | > 0.7. GO enrichment analysis and KEGG pathways were implemented by the clusterProfiler R package, and terms with corrected p-value < 0.05 were considered significantly enriched by the DEGs. PPI network among DEGs was analyzed on the website of STRING (https://cn.string-db.org/).The protocols used were adopted from our previous work . BrieflyAfter isolation of total RNA of limbal epithelium, reverse transcription was performed using PrimeScript™ RT Master Mix Kit , and Synthesized cDNA was amplified in duplicate on an ABI Prism 7500 Fast Sequence Detection System (Thermo Fisher Scientific) using TB Green® Premix Ex Taq™ II . Samples were denatured at 95 °C for 30s, followed by 40 cycles at 95 °C for 3s, 60 °C for 30s. The primers used were listed in Table t-test was used to compare the mean values between two groups, while the means for multiple groups were compared by one-way ANOVA. Spearman’s correlation analysis was used to assess the association between PM2.5 concentrations and limbal epithelial thickness. p < 0.05 was the threshold for statistical significance.Data are expressed as mean ± standard deviation and were analyzed using GraphPad Prism 9  and SPSS version 26 . The unpaired Below is the link to the electronic supplementary material.Additional file 1: Table S1. Mean/maximum/minimum value of limbal epithelium thickness in the two groups. *p < 0.05, **p < 0.01.Additional file 2: Table S2 Detail of Spearman analysis table. *p < 0.05.Additional file 3: Table S3 The primers used for qPCR.Additional file 4: Supplemental figures Fig. S1. Meta-analysis of fine particulate matter on corneal diseases. This meta-analysis was divided into the following two parts according to the data types in the study (binary variables or continuous variables). (A) 6 studies were plotted in a forest of binary variables and odds ratios (OR) were calculated . (B) 5 studies were plotted in a forest of continuous variables and regression coefficients (β) were calculated . The final merged results indicated that fine particulate matter exposure is positively associated with corneal disease or related symptoms. Fig. S2. Ocular surface fluorescein sodium staining in short-term PM2.5 exposure rat model by slit lamp examination (N = 6 per group). Fig. S3. Schirmer’s test revealed that tear secretion was not notably affected by PM2.5 exposure after 2 days (N = 6 per group). Fig. S4. Limbal vascular morphology of short-term PM2.5 exposure rat model  (N = 3 in each group). Fig. S5. Corneal innervation of short-term PM2.5 exposure rat model  (N = 3 in each group).Additional file 5: text. S1 The rationale for the exposure treatment rat models.


From pH-dependent experiments, the maximum adsorption efficiency was found to be at pH 8. Isotherm and kinetic studies were performed and found to follow Langmuir isotherm and pseudo-second order kinetics, respectively. The maximum adsorption capacity of CT@MNPs was calculated to be 45.5 mg of U(vi) per g of nanoparticles (NPs). Recyclability studies suggest that over 94% sorption was retained even after four consecutive cycles. The sorption mechanism was explained by the point of the zero-charge experiment and the XPS measurement. Additionally, calculations using density functional theory (DFT) were carried out to support the experimental findings.U( vi). Over 94% adsorption is still retained ever after 4th cycle. The experimental adsorption was also investigated and supported by DFT calculations.Here we report bio-functionalized magnetite nanoparticles (MNPs) for the adsorptive removal of U( To become a part of the industrial revolution, developing and developed countries are upgrading a variety of industries, such as textiles and pharmaceuticals, causing a rise in waste levels within the environment. The increase in waste increases water pollution since the trash from industry is directly discharged into water resources, such as rivers, ponds, and the sea. As a result of water contamination, aquatic life is in peril and at the same time, the quality of drinking water is deteriorating. To overcome issues regarding this, an immediate action is urgently needed. Aside from pollutants, wastewater can contain a variety of hazardous bacteria, such as 2 In drinking water, the permissible limit of uranium is 0.03 mg L−1 .3,4 Uranium is generally present in groundwater as a soluble uranyl (UO22+) ion. In addition to the uranyl ion, other forms of uranium, such as UF6, UO2F2, UO2(NO3)2, UO2Cl2, uranyl acetate, sulphate, and carbonates,2 can also exist in groundwater depending on the prevailing conditions. Uranium-contaminated water generally affects the kidney5 and therefore suitable techniques, which are low cost,6 environmentally friendly,7 and efficient,8 must be developed to remove U(vi).Uranium is a well-known radioactive element for nuclear energy as well as a highly chemo-toxic element.vi) removal.9–15 Nanomaterials are one of the greatest discoveries of all time, with applications in almost every field. Several NPs have been chosen earlier to remove U(vi) from water.16–19 Magnetite nanoparticles (MNPs) are unique among nanomaterials because they are superparamagnetic and hence easily separable using a simple static magnetic field. They are also less cytotoxic to both the aquatic and human environments.20 Earlier several magnetic composites have been used for uranium removal but only a few report natural products as surface modifiers.21,22 MNPs with proper coating can also act as an antifouling agent.23,24 It was reported that A. marina extract-coated iron NPs act as good antifouling agents.25Cinnamomum tamala (CT) leaf is the Indian spice, called “Indian bay leaf” or “tejpatta” and is a traditional medicine used in the treatment of scabies, anal diseases, rectum, heart troubles, bad taste, ozena, diarrhea, etc.26 Since CT leaf has antioxidant, as well as antibacterial properties,26,27 it might also be used as an antifouling agent for ships and boats in marine and freshwater system, in future.28,29 The CT leaf extract (aqueous) contains kaempferol and eugenol (as the main ingredient) and many flavonoids,30,31 which contain various coordinating groups, such as hydroxyl (–OH) and carbonyl  scale" fill="currentColor" stroke="none"><path d="M80 1160 l0 -40 40 0 40 0 0 -40 0 -40 40 0 40 0 0 -40 0 -40 40 0 40 0 0 -40 0 -40 40 0 40 0 0 -40 0 -40 40 0 40 0 0 -40 0 -40 40 0 40 0 0 -40 0 -40 40 0 40 0 0 80 0 80 -40 0 -40 0 0 40 0 40 -40 0 -40 0 0 40 0 40 -40 0 -40 0 0 40 0 40 -40 0 -40 0 0 40 0 40 -40 0 -40 0 0 40 0 40 -80 0 -80 0 0 -40z M560 520 l0 -40 -40 0 -40 0 0 -40 0 -40 -40 0 -40 0 0 -40 0 -40 -40 0 -40 0 0 -40 0 -40 -40 0 -40 0 0 -40 0 -40 -40 0 -40 0 0 -40 0 -40 -40 0 -40 0 0 -40 0 -40 80 0 80 0 0 40 0 40 40 0 40 0 0 40 0 40 40 0 40 0 0 40 0 40 40 0 40 0 0 40 0 40 40 0 40 0 0 40 0 40 40 0 40 0 0 80 0 80 -40 0 -40 0 0 -40z"/></g></svg>C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O), hence this extract was used as a surface modifier in nanoparticle synthesis.32 The CT extract was also employed for reducing metal ions to produce metal nanoparticles.33Various recently-developed materials exhibit high efficiency in U(vi) from contaminated water. To the best of our knowledge, this is the first time CT-coated MNPs have been explored for U(vi) removal. Because CT@MNPs are superparamagnetic and insoluble, they may be easily separated following wastewater treatment. Furthermore, they are nontoxic,29 making them preferable to other CT@NPs.30,32–35In this study, we have synthesized and characterized eco-friendly CT extract-coated MNPs (CT@MNPs) and used them for the removal of U(2.2.12(NO3)2·nH2O] in MilliQ water. CT leaves were collected from the local area of Cooch Behar Panchanan Barma University, Cooch Behar. MilliQ water was used to prepare all aqueous solutions. Various instruments were used for performing different experiments including an ultra-sonicator (Qsonica Sonicator); pH meter ; MilliQ plant from Labconco, Water Pro/Ro. The experimental data, viz. hydrodynamic size, and zeta potential analysis were obtained using a dynamic light scattering (DLS) instrument obtained from Anton Paar Litesizer 500; Fourier-transform infrared (FTIR) spectra were measured on a Thermo Scientific Nicolet IS50 instrument; powder X-ray diffractometry (pXRD) patterns were obtained on a Rigaku Ultima IV X-ray diffractometer; magnetization studies were performed on an alternating gradient field magnetometer (AGM), PMC MicroMag 2900 Series; high-resolution transmission electron microscope (TEM) images were obtained on a Tecnai G2 instrument, and thermogravimetric analysis (TGA) were performed on an SDT650 instrument.All chemicals were analytical grade and were purchased either from Sigma-Aldrich or a local manufacturer. The simulated uranium-contaminated wastewater was prepared by dissolving uranyl nitrate 2+, [(UO2)(OH)]+, [(UO2)(OH)2], and [(UO2)3(OH)5]+.46 When pH increases further from 8 to 10%, the removal was decreasing, probably due to the formation of uranium in its negatively charged forms: UO2(CO3)22− and UO2(CO3)34−.45,46 In all further experiments, we kept the solution pH at 8.The coprecipitation method was used to prepare CT@MNPs under aerobic conditions at room temperature and in aqueous solutions. Crude extract of CT leaf generally contains kaempferol and eugenolvi) after successful adsorption was found to be 0.010 mg L−1, which is less than the permissible value of U(vi) concentration according to WHO and BIS guidelines (0.030 mg L−1), indicating that the synthesized NPs is effective in removing uranium to a safer level. The results were fitted into various kinetics models and were found to be the best fit for pseudo-second-order suggesting that the sorption process is chemisorption.47 Other kinetic data are shown in Fig. S3 and Table S1.†Time-dependent sorption measurements were carried out to calculate the equilibration period and data are shown in vi) solutions (1–100 mg L−1). The results of the experiment are shown in vi) occurs for the CT@MNPs.48 The calculated maximal sorption capacity for CT@MNPs is 45.5 mg of U(vi) per g. Other isotherms are plotted in Fig. S4†In order to find the adsorption capacity of the material, studies were carried out with different initial concentrations of U(vi). The experimental findings are shown in 3 (sodium bicarbonate), Na2CO3 (sodium carbonate), and HNO3 (nitric acid) solutions were chosen for the desorption study. To, 1 mg of NPs containing 28 μg of sorbed uranium 10 mM; 1 mL solutions of each reagent were added. After the combination was sonicated for one hour, the NPs were removed from the solution, and the concentration of the uranium in the solution was determined. EDTA, NaHCO3, Na2CO3, and HNO3 were shown to desorb 89, 85, 86, and 83% of the uranium, respectively. This implies that CT@MNPs can be recycled after sorption and that uranium can be removed and used for a variety of purposes. The binding of uranium with the EDTA ligand is a chelating type and EDTA binds very effectively with uranium. CO32− is known as a strong binder of uranium. The addition of HNO3 changes the pH of the system and, therefore, uranium at low pH is extracted. Hence, respective reagents were chosen to find better regeneration and reusability results.We also checked the recyclability of the material and found that even after 10 cycles the material could sorb >70% of U sorption from waste environmental matrices.In order to use any material in an industry, it is important that the material fulfill many requirements. The foremost of these are non-toxic, environmentally friendly, low cost, and efficient. To calculate the cost, a complete cost analysis of the synthesized sorbent Table S3 was out,i.e., some type of strong interaction existed between U(vi) species and the nanoparticles. Hence, to establish the mechanism of binding of the U(vi) with the sorbent, XPS and XRD studies were carried out. As the surface of the CT@MNPs was surrounded by various coordinating sites such as –OH, after deprotonation, the surface was completely negatively charged , thus it can be concluded that the positively charged uranium ions were electrostatically adsorbed over the surface of CT@MNPs. XPS study (vi) ions were efficiently bound to the CT@MNPs surface by a strong force of attraction (electrostatic attraction). Similar results were reported by Xu et al.52 and Zhang et al.55 From i.e., on the investigation of high-resolution XPS spectra, two peaks were found: 392.01 eV (U 4f5/2), 381.03 eV (U 4f7/2), which were also reported by Ouyang et al.56 and Wang et al.57 A single contribution of the U(vi) state with binding energies of 392 eV (U 4f5/2) and 381 eV (U 4f7/2) could be used to match the XPS spectra (58 By comparing vi) was adsorbed on CT@MNPs. These two new peaks arise due to the U(vi) deposition over the nanoparticles.48,59From the kinetic study, it was found that the adsorption phenomena involved chemisorption, PS study  revealed spectra , showing4.60 utilizing the PW-SCF code to perform periodic density functional theory (DFT) calculations on the Fe3O4 (111) surface. In the present study, we have chosen the Fe3O4 (111) surface since previous studies showed that it has a predominantly naturally-cleaved surface with high thermodynamic stability at ambient conditions.61,62 Furthermore, Yu et al. within a DFT+U scheme showed that compared to the other naturally grown surfaces of Fe3O4, (111) surface has higher stability.63 In addition, the (111) surface is more active for reactions.64 As the CT leaf extract is rich in eugenol, we have analyzed its role in the attachment of uranyl to the surface of Fe3O4 (111) surface with eugenol. At higher pH, different oligomeric species such as (UO2)2(OH)22+, (UO2)3(OH)5+, (UO2)4(OH)7+, and (UO2)3(OH)7− were found in the solution but all these structures are complicated and quite large. From previous reports, it was found that [UO2(H2O)5]2+ is a potential candidate to study the adsorption of organic molecules.65 Hence to study the adsorption behavior, in the present work, we have chosen [UO2(H2O)5]2+. For optimization, the convergence threshold on the total energy of 10−6 (a.u.) and the force of 10−3 (a.u.) were set. Wavefunctions were given a kinetic energy cutoff of 64 Ry, while charge density was given a cutoff of 570 Ry. The simulation was conducted utilizing the approximate generalized gradient method (GGA) with Perdew–Burke–Ernzerh (PBE)66 and the Marzari–Vanderbilt smearing technique with a smearing threshold of 0.02 and a 1 × 1 × 1 k-point mesh.67 Additionally in this computation, we used the same lattice parameter as described previously.68 The experimental value of 8.394 Å for the standard cell (PBE+U) marginally overestimated the lattice parameter  for U = 3.5 eV, which is consistent with previously reported data.68To investigate the uranyl ion adsorption behavior on CT@MNPs, we have adopted the quantum Espresso's projector augmented wave (PAW) technique69 The lattice cell dimensions were a = 18.0121 and b = 15.599 Å, including a vacuum zone having a thickness larger than 30 Å in the c direction to ensure no contact between the slabs has been optimized and further used for uranyl adsorption calculations.To optimize the computational costs, the bottom two atomic layers were kept frozen while the remaining layers were completely relaxed during the calculations.oct terminated Fe3O4 (111) surface is illustrated in 3O4 (111) surface happed through the hydroxyl group of eugenol with an O–U bond. UO bonds for the attached uranyl experience a longer bond length of 1.87 Å than its normal value (1.796 Å).70 Previous studies showed that the outer-sphere association of foreign ions with the hexahydrated uranyl ions takes place with a consequent displacement of water molecules coordinated by the uranium until the limiting complex is formed.71 As the –O–H bond of the eugenol interacts with U(vi) ion and UO interacts with the iron atom of Fe3O4, thereby, UO bond is longer than its usual value.70 These facts essentially illustrate that uranyl adsorption is facilitated by eugenol-modified Fe3O4 (111) surface.The ground state energy-minimized geometry of uranyl adsorbed eugenol-modified Fe5.vi) from water. The synthesized NPs have a maximum adsorption capacity of 45.5 mg of U(vi) per g of the sorbent. Kinetic studies suggested that 99% of U(vi) was adsorbed within 8 h. The maximum sorption occurred at pH 8 and the results were explained by the interaction between the sorbent surface and sorbate. The binding of U(vi) over the surface of CT@MNPs was shown using XPS measurements. Furthermore, DFT calculation demonstrates the mode of binding of the uranyl ion and indicates that the adsorption is facilitated by the eugenol-modified Fe3O4 (111) surface. Both theoretical and experimental results suggest that the NPs are effective for uranium removal.In this study, we developed bio-functionalized MNPs to remove uranium(The data of this study are available upon request from the corresponding author(s).Chanchal Das: investigation, formal analysis, and writing—original draft; Narendra Nath Ghosh: theoretical investigation, analysis, and writing draft; Vandana Pulhani; investigation, formal analysis, and writing draft; Goutam Biswas and Pallavi Singhal: methodology, funding acquisition, supervision, and writing—review and editing.The author declares that there is no conflict of interest regarding this work.RA-013-D3RA00799E-s001


Calcium homeostasis modulator 1 (CALHM1) is a voltage-dependent channel involved in neuromodulation and gustatory signaling. Despite recent progress in the structural biology of CALHM1, insights into functional regulation, pore architecture, and channel blockade remain limited. Here we present the cryo-EM structure of human CALHM1, revealing an octameric assembly pattern similar to the non-mammalian CALHM1s and the lipid-binding pocket conserved across species. We demonstrate by MD simulations that this pocket preferentially binds a phospholipid over cholesterol to stabilize its structure and regulate the channel activities. Finally, we show that residues in the amino-terminal helix form the channel pore that ruthenium red binds and blocks. The voltage-dependent CALHM1 channel is critical in neuromodulation and gustatory signaling. The authors present cryo-EM structures of human CALHM1 in apo- and blocked-states. They explore effects of lipid binding on channel structure and activity. CALHM1 is the recently identified member of a large-pore channel that permeates ions, including Ca2+, Na+, K+, and Cl−, and small molecules, such as ATP, in a voltage-dependent manner2. CALHM1 activity has been demonstrated to modulate neuronal activity and the accumulation level of amyloid-beta4. Furthermore, the ATP efflux through the CALHM1 channel in type II gustatory cells and the subsequent purinergic signaling has been shown to facilitate the perception of sweet, bitter, and umami taste sensations5. Mutations in the calhm1 gene have been identified as a risk factor for the early onset of Alzheimer’s disease in some population groups3. A number of structures of CALHM1 and the other CALHMs  became available by single-particle cryo-EM recently9. In all cases, the CALHM proteins assemble as multi-oligomers, where CALHM1, 2, 4, 5, and 6 proteins assemble as an 8-mer, 11-mer, 10-11-mers, 11-13-mers, and 10-11-mers, respectively. These oligomeric states are controlled mainly by interactions between the C-terminal helices (CTHs) of neighboring protomers as well as between transmembrane domains (TMD) 2 and TMD41. Voltage-gated ion channel currents and ATP conductance have been definitively shown for CALHM1 and CALHM1/3 heteromeric channels10. CALHM2, CALHM4, and CALHM6 have been reported not to form functional channels by several groups10. One study reported a macroscopic current of CALHM2 in the presence of EGTA, which is attenuated by ruthenium red (RuR)6. Whether CALHM2 forms functional channels or binds RuR remains inconclusive. Thus far, there are three published cryo-EM structures of CALHM1 channels from non-mammalian species;13 however, a number of fundamental questions remain: (1) Do mammalian CALHM1s have similar protomer architectures and oligomeric assembly patterns? (2) What are critical structural motifs that regulate channel activity? and (3) Where is the channel pore and how does ruthenium red (RuR) block the CALHM1 bona fide voltage-dependent channel activity? Here, we address these shortfalls by obtaining the structures of human and chicken CALHM1 channels in the same condition for a direct comparison, and a human CALHM1 potentiating mutant in the presence and absence of RuR. We show the overall structural similarity between the human and chicken CALHM1 channels, a phospholipid-filled hydrophobic pocket critical for structural stability and functions, and the channel pore composed of the aminoterminal helices (NTHs), which RuR can physically block.Large-pore channels conduct ions, osmolytes, and metabolites in various tissues and organs to mediate cellular signaling and homeostasis11, expressed them in human embryonic kidney (HEK) 293 S GnTI- cells, purified and reconstituted into lipid nanodiscs. Our single-particle cryoEM resulted in the octameric chCALHM1Δct and hCALHM1Δct structures, resolved at the overall resolutions of 3.36 and 3.76 Å, respectively , despite low overall sequence identity among species, may facilitate an octameric assembly during biogenesis.To compare structures and assembly patterns of mammalian and non-mammalian CALHM1 channels, we determined the cryo-EM structure of human CALHM1, as well as that of chicken CALHM1 prepared using an identical protocol (see Methods). Here, we designed the equivalent C-terminal truncation constructs to the killifish CALHM1 (chCALHM1Δct and hCALHM1Δct), previously shown to boost the expression levelig. Fig. . As is tig. Fig. . In factig. Fig. . It is wig. Fig. . Such a PDB-6VAM; EMD-21143) protomers are structurally similar, indicating the truncation of residues after CTH does not alter protein folds  Fig. , illustrlds Fig. . The str11. In the current study, no lipid was supplemented during purification; thus, the phospholipid-like molecule is either carried from the expression host cell (HEK293S GnTI-) or the process of the lipid nanodisc reconstitution. The observations above led us to speculate that a phospholipid may be the physiological lipid that occupies and stabilizes the hydrophobic pocket.Inspection of the hCALHM1Δct and chCALHM1Δct structures reveals a hydrophobic pocket ~15 Å in length  and 10 Å wide, located between TMD3 and TMD4 of one subunit and TMD2 of the neighboring subunit Fig. . This poTo assess our structure-based hypothesis, we estimated the binding stability of a phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and cholesterol by coarse-grained molecular dynamics (CG-MD) simulations on hCALHM1Δct embedded into a membrane bilayer consisting of a 10:3 ratio of POPC to cholesterol Fig. . During To understand the functional role of the lipid-filled hydrophobic pocket, we generated tryptophan point mutations of the pocket forming residues and assessed their functional effects by measuring voltage-gated currents by whole-cell patch-clamp electrophysiology Fig. . Specifi16  by NHS-SS-biotin followed by the pull-down by Strepavidin-Sepharose, elution by DTT, and detection by western blot Fig. . The Ile16 Fig. . This as16 Fig. . We obse16 Fig. , consist16 Fig. .Overall, the mutations above alter both channel functions and cell surface expressions, indicating the hydrophobic pocket to be a critical motif for controlling the CALHM1 activities. The impact of mutations on cell surface expression and current density varies depending on their location. Specifically, Ala199Trp causes an increase in surface expression, while Leu67Trp, Val112Trp, Ala116Trp, and Thr196Trp result in decreased expression at different levels, and Val192Trp shows no detectable expression. Regarding current density, Ile109Trp leads to an increase, whereas Leu67Trp, Val112Trp, Ala116Trp, and Thr196Trp exhibit similar levels, and Val192Trp shows no current at all. Comparison of current density with cell surface expression suggests that the Ile109Trp and Ala199Trp mutations result in upregulation and downregulation of channel activities, respectively . In contrast, Val192Trp is structurally disruptive, as our cryo-EM structure suggests it could cause steric clashes with Leu120, Trp189, Leu193, and Val63 Fig. . FurtherI109W∆ct), we conducted single-particle cryo-EM on this mutant. We found that overexpression of the hCALHM1I109W∆ct in HEK293 cells resulted in low cell survival, likely due to toxicity caused by the upregulated channel activities. This issue was circumvented by adding the channel blocker, RuR, to the culture media, which allowed protein expression and purification. We obtained the structure of hCALHM1I109W∆ct in lipid nanodiscs and in the presence of RuR at 3.91 Å overall resolution with imposed C8 symmetry , supporting the cryo-EM observation that the Ile109Trp mutation stabilizes the POPC binding   Fig.  and withct) Fig.  the poreI109W∆ct structure provided a potential mechanism underlying the channel functional upregulation. The Ile109Trp is positioned to form stronger interactions with Asn21 and Ala28 on TMD1 than the wildtype, thereby strengthening the interaction between TMD3 and TMD1. The more ordered TMD1 facilitates the interaction between the lipid and Phe19 located in the linker between TMD1 and NTH to contribute to ordering and positioning the pore-forming NTH in the ‘upright’ manner, as observed in the cryoEM structure , indicating that TMD1 and NTH move relative to TMD2-4 and CTH to control ion channel functions.Further inspection of the hCALHM1ure Fig. . AlthougOur cryo-EM structures and functional analyses above provided insights into the pattern of lipid binding and its crucial role in channel functions. Indeed, our cryo-EM structures suggested that the binding strength of the integrated phospholipid could control the dynamics of the CALHM1∆ct protein. Therefore, to further investigate the contribution of lipid binding to overall protein stability, we conducted all-atom unbiased MD simulations with either POPC (8 × 100 ns), cholesterol (9 × 100 ns) or in an apo, non-lipid bound state (9 × 100 ns) in each of the eight hydrophobic pockets on the NTH containing hCALHM1∆ct homology model. The population density of Cα atom RMSDs Fig.  for the I109W∆ct in the presence of RuR showed oblong density consistent with the size of RuR in the central pore surrounded by the NTHs , indicating that the binding of RuR only affects the structure of the central pore comprised of TMD1 and NTH  permitted a range of motion. The Ile109Trp mutation and RuR together appear to stabilize the positioning of TMD1 and NTH, allowing us to resolve the RuR-blocked pore , ryanodine, Piezo, and two-pore domain potassium (K2P) channels, and the calcium mitochondrial uniporter16. Following bacmid generation, Sf9 cells were used to amplify the recombinant baculovirus. HEK293 GnTI- cells were cultured in FreeStyle 293 expression media supplemented with 2% FBS and at 37 °C. Cells were infected with baculovirus at a density of 4 × 106 cells/ml at a v:v ratio of 1:20. Following infection, the cultures were moved to 30 °C and supplemented with 5 mM sodium butyrate and 20 µM RuR (for the hCALHM1I109W∆ct and RuR-hCALHM1I109W∆ct samples), and harvested 48 h after infection. The cell pellets corresponding to expressed chicken CALHM1∆ct were resuspended in 20 mM Tris pH 8.0, 150 mM NaCl, 1 mM PMSF and then C12E8 (Anatrace) was added to a final concentration of 1%. Following 2 h of solubilization at 4 °C, the lysate was clarified by two rounds of centrifugation: the first one at 4,550 g for 20 min (4 °C), and the second at 186,000 g for 1 h at 4 °C. The clarified supernatant was loaded onto a Strep–Tactin Sepharose column (IBA) followed by 20 column volumes of washing with 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.01% C12E8 (wash buffer) and elution using the wash buffer supplemented with 3 mM desthiobiotin. The purified chicken CALHM1∆ct was concentrated to ~3.9 mg ml−1 at 4 °C using 100-kDa MWCO Amicon concentrators (Millipore) before reconstitution into nanodiscs. For reconstitution into nanodiscs, soybean polar extract, MSP2N2 and the purified chicken CALHM1∆ct protein, at final concentrations of 0.75, 0.3, and 0.4 mg/ml, respectively, were mixed for 1 h at 4 °C, followed by detergent removal by SM2 Bio-Beads (BioRad) overnight (~12 h). MSP2N2 was expressed and purified as previously described32. The nanodisc reconstitution reactions were pooled and concentrated and run over SEC using a Superose 6 10/300 (Cytiva) in 20 mM Tris pH 8.0, 150 mM NaCl. Peak fractions were pooled and concentrated to ~0.5–2.3 mg/ml, supplemented with 1 mM EDTA. To remove RuR from the hCALHM1I109W∆ct sample, the Strep-Tactin resin bound protein was washed first in buffer supplemented with 5 mM EGTA pH 7.0, then 20 mM CaCl2, and finally 40 mM EDTA pH 8.0, followed by extensive washing in 20 mM HEPES-NaOH pH 7.5, 200 mM NaCl. The RuR-hCALHM1I109W∆ct sample was supplemented with 100 μM RuR after loading on the Strep-Tactin column. The purified hCALHM1∆ct, RuR-free hCALHM1I109W∆ct, and RuR- hCALHM1I109W∆ct complex samples were concentrated to ~1.0-3.2 mg ml−1 at 4 °C using 100-kDa MWCO Amicon concentrators (Millipore) before reconstitution into nanodiscs. For reconstitution into nanodiscs, soybean polar extract, MSP2N2 and the purified human CALHM1 protein, at final concentrations of 0.75, 0.3 and 0.4 mg ml−1, respectively, were mixed for 1 h at 4 °C, followed by detergent removal by SM2 BioBeads (BioRad) overnight (~12 h). The beads were removed and the solution was further purified by size-exclusion chromatography using a Superose 6 10/300 column  in 20 mM HEPES-NaOH pH 7.5, 200 mM NaCl. The buffer for hCALHM1∆ct and hCALHM1I109W∆ct was supplemented with 1 mM EDTA and the RuR- hCALHM1I109W∆ct sample was supplemented with 50 μM RuR and 1 mM EGTA. Peak fractions were pooled and concentrated to ~0.5-1.3 mg/ml for cryo-EM grid preparation.Chicken CALHM1∆ct , human CALHM1∆ct , and human CALHM1∆ct-Ile109Trp , were cloned into the pFWgfpCMV, the baculovirus transfer vector harboring sequences for the CMV promoter, the WPRE 5’ UTR sequence, and EGFP, modified from pFp10I109W∆ct in nanodisc (4 μl) and RuR-hCALHM1I109W∆ct in nanodisc (4 μl) complexes were applied to glow-discharged gold grids (R1.2/1.3 UltrAuFoil 300 mesh). The grids used for the hCALHM1I109W∆ct in nanodisc samples had been treated with 0.01% β-OG immediately beforehand. The human CALHM1∆ct in nanodisc (3.5 μl) was applied to 1.2/1.3 400 mesh C-flat carbon-coated copper grids (EMS). Grids with human and chicken CALHM1∆ct were blotted for 4 s with blot force 7 at 85% humidity and 15 °C before plunge freezing into liquid ethane using a Vitrobot Mark IV (ThermoFisher). Grids with the RuR-free and RuR-hCALHM1I109W∆ct samples were blotted for 1.8 s at 85% humidity and 20 °C before plunge freezing into liquid ethane using an EM GP (Leica Microsystems). Datasets were collected using a Titan Krios operated at an acceleration voltage of 300 keV and the GATAN K3 direct electron detector coupled with the GIF quantum energy filter (Gatan) controlled by EPU v2.10.0.5 software (ThermoFisher). Movies were recorded with a pixel size of 0.856 Å, an exposure time of 1.8 s over 30 frames and a dose rate of 2 e − /Å2/frame. For the human and chicken CALHM1∆ct data sets, the program Warp (v1.0.9) was used to align movies, estimate the CTF and pick particles33. Two-dimensional classification was performed using cryoSPARC (v3) software and ab initio 3D map generation, 3D refinement, 3D classification were performed using Relion 3.1 software35. Per-particle CTF refinement and B-factor sharpening were then performed using cisTEM (v1.0.2) software36. For the RuR-free and RuR-hCALHM1I109W∆ct datasets, cryoSPARC was used to align movies, estimate the CTF, and pick particles as well as perform two-dimensional classification and ab initio 3D map generation, 3D refinement, and 3D classification34. Focused 3D classification was performed using Relion 3.1 software35, prior to nonuniform refinement using Cryosparc. Modeling was performed in the programs UCSF Chimera (v1.14) and Coot (v0.893), using the published structure of chicken CALHM1 as a starting model (PDB code: 6VAM)37. The final models were refined against the cryo-EM maps using PHENIX (v1.14) real-space refinement with secondary structure and Ramachandran restraints38. The FSCs were calculated by phenix.mtriage or within cryoSPARC38. Local resolutions were estimated using the program ResMap (v1.1.4)39. Superpositions of atomic models and subsequent RMSD calculations were performed in Pymol v2.5 using the align algorithm over Cαs unless otherwise specified40. Data collection and refinement statistics are summarized in Supplementary Table The chicken CALHM1∆ct in nanodisc (3.5 μl), RuR-free hCALHM1I109W∆ct, Cterminally fused to the 1D4-tag were subcloned into the pFWgfpCMV vector. Adherent HEK293 cells in DMEM + 10 % FBS were transfected  with the plasmids, and the cells were analyzed after ~36–48 h. Electrophysiological recordings were obtained from the transfected HEK293 cells using the whole-cell configuration of the patch-clamp technique41 at room temperature (22–24 °C). Patch pipettes were made from 1.50 OD/0.86 ID borosilicate glass capillaries  using a P-97 micropipette puller (Sutter Instruments) and polished with an MF-830 microforge (Narishige) to a final resistance of 2–6 MΩ when backfilled with the internal solution. The pipette solution for the Ala116Trp, Ala199Trp, Leu67Trp, Ile109Trp, Thr196Trp, Val112Trp, and Val192Trp hCALHM1∆ct was (in mM) 147 NaCl, 10 EGTA and 10 HEPES, pH 7.0 with NaOH, and for the Gln10Arg, Gln13Arg, and Gln16Arg CALHM1I109W∆ct mutants contained (in mM) 110 Cs-gluconate, 30 CsCl, 5 HEPES, 5 BAPTA, 4 NaCl, 2 MgCl2, 0.5 CaCl2, 2 ATP-Na, 0.3 GTP-Na; pH 7.35 with CsOH. Cells were held at −60 mV and superfused with a bath solution, containing (in mM): 150 NaCl, 3 KCl, 10 HEPES, 1 CaCl2, and 0.01 EDTA-Na, at pH 7.4 with NaOH  or 147 NaCl, 13 glucose, 2 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, pH 7.3 with NaOH . Data were acquired and lowpass-filtered with an AxoPatch 200B patch-clamp amplifier (Molecular Devices). Signals were fed to a Pentium-based PC through an Axon Digidata 1550B interface board (Molecular Devices). hCALHM1109W∆ct currents for all the constructs were elicited by application of 1 s steps from −100 mV to +100 mV in 20 mV increments, measured at the end of the step pulse, and normalized to cell capacitance. RuR  was applied using a fast perfusion system  until reaching the steady-state of the effect and hCALHM1109W∆ct amplitudes at the end of the +60 mV depolarizing step were measured before (control) and after drug application. The pClamp software  was used for stimulus generation, data display, acquisition, storage, and analysis. GraphPad Prism 9 software was used for visualization and statistical analysis. Paired or unpaired (with Welch’s correction) two-tailed ttests were used to assess the statistical significance of RuR (control vs. drug), RuR concentration-response, or comparison between WT and single-point mutants,  or a corresponding volume of buffer was added to half of each sample. The samples were nutated at 4 °C for 1 h. Tris-HCl pH 8.0 was added to a final concentration of 50 mM to quench the reaction. Following a wash step in PBS, the cells were lysed in 1% C12E8 (30 min nutation at 4 °C) and the lysate clarified by centrifugation . Samples of total cell input were analyzed by western blot. Streptavidin slurry (Sigma) was added and the lysate was incubated under rotation for 2 h at 4 °C. The resin was then washed and incubated in buffer supplemented with 100 mM DTT at room temperature for 2 h to elute bound protein. The elution was analyzed by FSEC and by western blot. For FSEC analysis, 50 µl of eluate was mixed with 1 µl of 0.06 mg/ml of anti-1D4FITC conjugated antibody prior to injection on a Superose 6 10/300 column equilibrated in 20 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.01% C12E8 running at 0.4 ml/min with excitation/emission wavelengths at 494/521 nm. Samples were subjected to western blot using monoclonal anti-1D4 (University of British Columbia) and anti-mouse horseradish peroxidase-conjugated antibody (Amersham) using dilutions of 1:5000 (of a 1 mg/ml anti-1D4 stock) and 1:20000, respectively in 0.5% nonfat dry milk powder, 20 mM Tris pH 8.0, 150 mM NaCl, 0.05% Tween. In addition, input samples of whole cell content were also probed using mouse monoclonal horseradish peroxidase-conjugated anti-β-actin antibody (Proteintech) at a dilution of 1:20000 in 5% nonfat dry milk powder, 20 mM Tris pH 8.0, 150 mM NaCl, 0.05% Tween. Protein bands were detected by enhanced chemiluminescence on X-ray film . Normalization using β-actin was used to compare protein expression levels between mutants. Two  or three  independent biological replicates were analyzed by western blot.42. Modeller v.10.1 was also used to generate the hCALHM1∆ct system that included the NTHs by reverting Trp109 from RuR-hCALHM1I109W∆ct to isoleucine. RuR was not included in the simulations. Gromacs 2021.2 pdb2gmx43 was used to protonate the structures at a pH of 7.4. The resulting atomistic models were converted into MARTINI coarse-grained models using the martinize v2.6 script44 and embedded into a membrane bilayer with a 10:3 ratio of POPC to cholesterol, solvated with water and a NaCl concentration of 0.15 M with additional net-charge neutralizing ions using the insane.py script45. The full coarse-grained system was subsequently energy minimized with the steepest descent algorithm. Tolerance was set to a value of 10 kJ/mol/nm, with an initial step size of 0.01 nm. Restraints were applied to all protein beads with a force constant of 1000 kj/mol/nm2. Following energy minimization, the system was equilibrated in the isothermal-isobaric ensemble for 2 ns with a timestep of 10 fs and harmonic restraints applied to all protein beads with a force constant of 1000 kj/mol/nm2. This was followed by a second round of equilibration for 2 ns where restraints were maintained on protein backbone beads only. The neighbor list was generated with a Verlet cut-off scheme and updated every 20 timesteps. Verlet-buffer tolerance was 0.005 Kj/mol/ps. Coulombic interactions were treated with reaction-field and a 1.1 nm cut-off. The relative dielectric constant was set to 15 and 0 for the reaction field. The Van der Waals cut-off was set to 1.1 nm with a potential-shift-verlet modifier. The system was coupled to a heat-bath by using the v-rescale temperature coupling algorithm. Temperature was set to 323 K. Pressure was maintained with the Berendsen barostat at 1 bar with semi-isotropic coupling and a 12 ps coupling constant. During production runs, restraints were applied to backbone protein beads with a force constant of 1000 kj/mol/nm2. Production runs were 4.2 μs, 5.5 μs and 4.3 μs respectively with a timestep of 0.02 ps for the hCALHM1∆ct system that included NTHs (built as a homology model based on the RuR-hCALHM1I109W∆ct structure). Three 6 μs simulations were performed for the NTH lacking hCALHM1∆ct system which were used to spawn initial umbrella sampling configurations. Temperature coupling was the same as specified for equilibration steps but with ParrinelloRahman pressure coupling. A pressure of 1 bar and coupling constant of 12 ps was maintained. All simulations performed herein were done using Gromacs version 2021.2.Missing heavy atoms and hydrogen atoms of the cryo-EM determined structure were added using modeller v.10.1x-axis with a force constant of 1000 kJ/mol/nm. Windows were initially spaced ~0.5 Å apart using a python script written by Dr. Owen Vickery (DOI: 10.5281/zenodo.3592318) with additional windows placed at undersampled regions. Each window was simulated for a total of 1 µs, with the first 200 ns discarded as equilibration. Each window was restrained with an umbrella potential of 1000 kJ/mol/nm apart from certain windows at saddle points on the free energy surface, where the force constant was increased to 2000 kJ/mol/nm. A temperature of 323 K was used for umbrella sampling windows. All other physical parameters were the same as for unbiased CG simulations. POPC PMFs were initiated from a representative frame of an unbiased CG simulation for the respective system wherein POPC had bound to the putative binding site. One POPC molecule was selected and steered in the x-dimension, laterally along the plane of the bilayer as depicted in Fig. Potential of mean force calculations for cholesterol were instigated from a coarse-grained conversion of the cholesterol bound atomistic model via martinize. Initial umbrella windows were generated by steering a single cholesterol molecule laterally across the bilayer along the 46. Eight cholesterol molecules were modeled into the EM density using Coot v 0.893 and Phenix v1.1438. For the POPC bound system, a single POPC molecule was docked into the proposed binding pocket using Vina and then duplicated for each of the eight potential binding sites using Phenix 1.247. The top scoring pose in which the phosphocholine moiety was positioned towards the pore was selected as this represented the main binding mode seen consistently during unbiased CG simulation  and semi-isotropic Berendsen pressure coupling (1 ps tau)54. Electrostatic interactions were treated with the Particle Mesh Ewald (PME) algorithm and a cubic spline interpolation of 4 with 0.1 grid spacing for the fast Fourier transform55. Isothermal compressibility was set to 4.5e−5 bar−1. Temperature was set to 298 K. Hydrogen atoms were constrained with LINCS holonomic constraints56. The time step used was 2 fs with heavy atom restrains of 1000 kJ/mol/nm2. Production runs of each equilibrated system was then initiated for 100 ns, with Cɑ atom restrains of 1000 kj/mol/nm2. Nosé-Hoover was used for temperature and pressure coupling with respective coupling constants of 0.5 and 1 ps at 298 K58. Analysis of RMSDs were done with in-house python scripts and used MDAnalysis v2.0.060. NTH containing hCALHM1∆ct with POPC, cholesterol and apo-state systems were simulated for 100 ns per repeat with 8, 9 and 9 independent repeats per system respectively.The fixed atomistic structure described earlier as the input for coarse grained simulations, was embedded in a pure POPC bilayer using the inflategro.pl scriptFurther information on research design is available in the Supplementary InformationPeer Review FileReporting Summary