Subsequently, the calcium ion (Ca2+) contained in the matrix layer penetrated the tip layer for cross-linking, leaving the drug in the cross-linked network. The patches have adequate mechanical strength to pierce the skin; then, the matrix layer is dissolved, leaving the tip layer to achieve sustained release. Additionally, the TS-MNs encapsulating EXT retained high activity during long-term storage at room temperature. The pharmacokinetic results indicated that the plasma concentrations of EXT were sustained for 48 h in the EXT MN group, which agreed with the in vitro release test. Furthermore, they had high relative bioavailability (83.04%). Moreover, the hypoglycemic effect was observed to last for approximately 24 h after a single administration and remained effective after multiple administrations without drug resistance. These results suggest that the TS-MNs are a promising depot for the sustained delivery of encapsulated EXT.Daily administration of multiple injections can cause inconvenience and reduce compliance in diabetic patients; thus, microneedle (MN) administration is favored due to its various advantages. Accordingly, the two-layer sustained-release MNs (TS-MNs) were fabricated by encapsulating exenatide (EXT) in calcium alginate (CA) gel in this work. The TS-MNs were composed of a sodium alginate (SA) tip and a water-soluble matrix-containing calcium chloride (CaCl Diabetes, a metabolic disease characterized by hyperglycemia, will affect 700 million people in 2045 . TypicalAs micron-sized structures, unlike other transdermal delivery methods, MNs can break through the skin barrier and only pierce the epidermis in a minimally invasive manner without damaging neurons in the dermis, thereby minimizing pain associated with transdermal delivery, showing improved skin permeability and enhancing transdermal delivery ,11,12. AAlginate is a naturally occurring high-molecular polysaccharide polymer that is ordinarily obtained from the cell walls of brown seaweed. The polymer has several unique advantages, including low toxicity, correspondingly low cost, biocompatibility, nonimmunogenicity, and biodegradation that have let it be extensively investigated and used for numerous biomedical and pharmaceutical applications ,19. Addi2 as the matrix material. Gelation of SA occurs rapidly in the presence of Ca2+; therefore, the in situ gel can hardly be poured into the mold to produce MNs due to the lack of fluidity [2+ was poured after removing excess solution. The Ca2+ contained in the matrix layer penetrated the tip layer for cross-linking, leaving the drug in the cross-linked network to achieve the sustained-release effect. After insertion, cross-linked MN tips were embedded in the skin after the matrix layer dissolved, followed by slow drug release [2 concentration on gel content and drug diffusion and SA concentration on tip height and implantation efficiency were studied. Moreover, the insertion capability, in vitro release ability, activity, and stability of TS-MNs were investigated, and the activity in vivo was also evaluated.In this study, the TS-MNs were first fabricated by using SA as the MN tip material and soluble PVA/PVP/CaClfluidity . Accordi release . Simulta2) were purchased from Yinghai Fine Chemical Factory . Fluorescein isothiocyanate (FITC) was bought from Beijing InnoChem Science & Technology Co., Ltd. . Acetonitrile (HPLC grade) was purchased from Thermo Fisher Scientific . STZ was acquired from Coolaber Technology Co., Ltd. . Exendin-4 enzyme immunoassay (ELISA) kits were obtained from Phoenix Pharmaceuticals, Inc. . Porcine cadaver skin was purchased from Kaikai Science and Technology Trading .EXT was obtained from Changchun Baiyi Pharmaceutical Co., Ltd. . Sodium alginate  was acquired from Huanghai Biopharmaceutical Co., Ltd. . Polyvinyl alcohol  was obtained from Jiangxi Alpha Hi-Tech Pharmaceutical Co., Ltd. . Polyvinylpyrrolidone  was acquired from Boai NKY Pharmaceuticals Co., Ltd. . Polydimethylsiloxane (PDMS) was obtained from Dow Corning . Rhodamine B and calcium chloride (CaClSprague Dawley (SD) male rats  were supplied by the SPF Biotechnology Co., Ltd. . All rats were housed under specific pathogen-free conditions with free access to food and water. The Institutional Animal Care and Utilisation Committee of the Technical Institute of Physics and Chemistry, CAS  approved the study protocol.2 matrix material. The tip material was obtained by dissolving 3% (wt) SA in an aqueous solution with an EXT content of 2% (wt). To attain the matrix material, the excipients consisting of 17.5% (wt) PVA, 17.5% (wt) PVP, and 4% (wt) CaCl2 were dissolved in deionized water. The MN fabrication process is depicted in The novel TS-MNs were composed of SA tip material encapsulating EXT and soluble PVA/PVP/CaCl2 powder was added to the above matrix solution. One milliliter of the acquired matrix solution (PVA/PVP/CaCl2) was put into EP tubes. The sodium alginate MNs (SA-MNs) were fabricated based on 0). In addition, SA-MNs were placed vertically in EP tubes containing 1 mL matrix solution, with the tip facing down (e). The gel content was achieved by using Equation (1) [A series of 25%, 30%, and 35% (wt) matrix solution was obtained by dissolving PVA and PVP (mass ratio 1:1) in a specific mass of deionized water. Subsequently, 4% (wt) CaCling down . Subseqution (1) .(1)Gel 2+, and the dye diffusion state was observed visually after 0 h and 10 h. To make the diffusion phenomenon more practical, the TS-MNs encapsulating rhodamine B were applied, which was attained based on the preparation method in To observe the diffusion intuitively of drugs in MNs in matrix solution with different concentrations, rhodamine B with a distinct color was selected as the model drug . The cas2 concentrations was obtained by adding 1%, 2%, 3%, and 4% (wt) CaCl2 powders to PVA/PVP matrix solution with a concentration of 35% (wt). The TS-MNs were acquired according to the preparation method described in e1). The gel content was similarly acquired from Equation (1).Matrix material containing different CaCln = 4). The total number of tips (Qt) was recorded and the TS-MNs were inserted into rat abdominal skin using a homemade applicator (20 N/cm2) for 30 s. The rat abdomen was wrapped with medical tape. After staying for 30 min, the TS-MNs were removed from the rat and observed under a microscope, and the number of tips (Qr) that were not implanted into the skin was recorded. The implantation efficiency was calculated based on Equation (2).The TS-MNs were prepared in the same way as described in 3-Na2CO3 solution was acquired by dissolving 0.80 g Na2CO3 in 5 mL water and 0.63 g NaHCO3 in 45 mL water, and they were mixed and adjusted to pH = 9.0; 10 mg/mL EXT solution was obtained by dissolving 0.03 g EXT in 3 mL (V0.15 M NaCl:V0.15 M NaHCO3-Na2CO3 = 9:1) solution. Then, 0.4 mg FITC was placed in a glass bottle containing 3 mL EXT solution and reacted with magnetic stirring at 4 °C for 12 h away from light. The reacted FITC-EXT solution was placed in a dialysis bag, and dialyzed at 4 °C away from light. Water was changed twice a day until no fluorescence was detected. The acquired FITC-EXT solution was filtered and entered the HPLC system for quantification. The experimental conditions for HPLC were similarly depicted in First, 0.15 M NaCl solution was obtained by dissolving 0.44 g NaCl in 50 mL water; 0.15 M NaHCO2) for 30 s. The skin after treatment of MNs was placed on an agarose hydrogel (2% wt) to keep it moist for 30 min; then, the residual MNs were removed from the porcine skin. The porcine skin surface and the residual MNs were photographed with a fluorescence microscope.The structural shape of TS-MNs was analyzed using a stereomicroscope  and a fluorescence microscope . To assess the skin insertion ability, TS-MNs loaded with FITC-EXT were inserted into porcine skin ex vivo (800 μm thick) using a homemade applicator . The procedures and conditions of the experiment were detailed in previous studies .To prepare histological sections, the porcine skin area treated with TS-MNs containing FITC-EXT was cut off, embedded with OCT complex at −80 °C for several minutes, and then placed in a cryostat mold for 20 min. The frozen OCT-skin was cut into 15 μm slices by a freezing microtome  and placed on a cover glass. The samples were viewed using a fluorescence microscope.2PO4 solution (pH = 4.5) and (B) acetonitrile at the following gradient elution: 1–12 min, B: 35–50%; 12–17 min, B: 50%; 17–18 min, B: 50–35%; and 18–25 min, B: 35%. The injection volume was 20 μL, the flow rate was 1 mL/min, and the wavelength was detected at 218 nm.The contents of EXT encapsulated in the TS-MNs were released by dispersing one patch in 1 mL PBS solution and vortex for 8 h. Then, the solution was filtered and entered the HPLC  system for analysis. A YMC Triart C18  column was applied to separate EXT at 40 °C. Chromatographic separation was adopted by the mobile phases (A) 0.05 M KHThe release of EXT encapsulated in MNs was carried out using the dialysis bag method. Specifically, the tips of TS-MNs were scraped off with a scalpel and placed in the dialysis bag; then, the dialysis bag was hung in a supported conical flask containing 2 mL PBS (pH = 7.4) receiving the solution during incubation at 37 °C with gentle magnetic stirring . The entt is the cumulative release rate of the drug within time t; k is the release rate constant; n is a constant; A is a simplified constant.To study the kinetics and mechanism of EXT release from the TS-MNs, the in vitro release data were fitted into three common mathematical models ,32. The The secondary structure of EXT was determined by a CD spectrophotometer . The method for extracting EXT from TS-MNs was similarly described in n = 6): (1) the subcutaneous (SC) injection group and (2) the EXT MN group. Each animal in the SC injection group was given 10.00 μg/0.5 mL EXT solutions subcutaneously using a 1 mL syringe. In the EXT MN group, two patches  were inserted into the skin of each rat using a homemade applicator (20 N/cm2) for 30 s. The MNs were removed from the rat after 30 min. Blood samples were collected from the caudal vein at time points . They were collected in a heparin sodium tube, centrifuged at 3000 rpm for 10 min, and then stored at −20 °C before analysis. The plasma EXT level was quantified using an ELISA kit according to the instructions. The relative bioavailability (RB) was calculated by Equation (6):The rats received adaptive feeding for one week after arrival, and they were anesthetized with ether. Their abdominal hair was removed and treated with depilatory cream. During the experiment, the animals were given optional food and water. The rats were randomly divided into two groups , and fasting blood glucose ≥ 16.7 mmol/L was considered indicative of diabetes .n = 6): the EXT MN group, the blank MN group, and SC injection group. After a detachment of the abdominal hair, the rats in the three groups were treated with EXT MNs , blank MNs (two patches for each), or SC injections (10.00 µg/0.5 mL EXT solution for each), respectively. The MNs were removed from the rat after 30 min. Blood was collected at 0, 0.5, 1, 2, 3, 4, 6, 8, 12, 20, 24, 30, and 36 h after administration, and the blood glucose level was detected by a glucose meter. A normal diet and water were given after blood glucose was measured at 12 h.To evaluate the efficacy of a single administration in diabetic rats, nonfasting diabetic rats were randomly divided into three groups (n = 4) with EXT MN, blank MN, and SC injection. All animals in the three groups received short-term treatment for 6 days and were administered the same dose as a single administration at 9:30 a.m. daily. During the period after daytime administration, the rats were fed a free diet. The blood glucose level was measured by a biochemical analyzer at 0, 4, and 12 h after administration, and overnight fasting for 12 h was continued after the last glucose measurement daily.Ultimately, the therapeutic efficacy of MNs for multiple administrations was assessed. The overnight fasting diabetic rats were randomly grouped  concentration on the gel content and the diffusion of rhodamine B were first investigated under the condition of constant CaCl calcium A, and th calcium B. The re2+, more than 90% CA gel content was formed in the 25%, 30%, and 35% matrix solution after 10 min. Before 60 min, the CA gel content increased with the extension of reaction time, although the increment was small, and there was no significant change in the subsequent trend. Besides, different matrix solution concentrations had no significant effect on CA gel content, probably due to the fast cross-linking process; thus, the matrix solution concentration did not affect the CA gel content. Consequently, the cross-linking rate was not affected by the concentration of the matrix solution, but drug diffusion was affected. Accordingly, PVA/PVP (mass ratio 1:1) matrix solution with a concentration of 35% was selected as the second layer to prepare TS-MNs.The influence of the matrix solution with different concentrations on CA gel content at different times is shown in 2 to the 35% mixed matrix solution. According to 2 concentration was 1%. When the CaCl2 concentration exceeded 2%, the content increased slightly, reaching above 96% in all instances. When the CaCl2 concentration increased to 4%, the content reached 98.44 ± 2.76%. Then, with increasing CaCl2 concentration, the CA gel content remained unchanged.The influence of calcium concentration in the upper matrix solution on CA gel content was further discussed when other conditions were consistent. The calcium concentration affected the CA gel content, and the content could be calculated by cross-linking with SA by adding different concentrations of CaCl1–D1)). As shown in 2+. To simplify the delivery method and allow rapid MN detachment, the time required for the patch to stick to the skin needs to be reduced [The effect of SA concentration on the relationship between tip height and implantation efficiency was analyzed. After the TS-MNs dissolved in water, the water-soluble matrix material was dissolved, and the cross-linked CA tip remained. Accordingly, the tip height of MNs could be measured directly by fluorescence microscopy A–D. Thir reduced . ConsequTo evaluate the morphology and skin-piercing capability of the TS-MNs, FITC, a fluorescent dye, was grafted onto EXT and then encapsulated into the TS-MNs. After grafting, the concentration of FITC-EXT solution was 3.64 mg/mL. The TS-MNs loaded with FITC-EXT are shown in To investigate the release behavior of encapsulated EXT from TS-MNs, the in vitro release of MNs with various SA: EXT ratio was carried out. The polymer-to-drug ratio may affect the behavior of drug release . As depiThe fitting results in Peptide drugs are unstable, especially in the fabrication process, and experience a variety of instability factors, resulting in peptide aggregation, degradation, and inactivation . To evalmax) in the EXT MN treatment group and SC injection group was 13.60 ± 2.95 ng/mL and 16.68 ± 4.87 ng/mL, respectively. The peak levels (Tmax) were reached in both groups 0.5 h after administration. Additionally, the plasma concentration of EXT after treatment with MNs could be detected for 48 h, which agreed with the in vitro release test. With SC injection as a reference, the RB of TS-MNs encapsulating EXT was 83.04% based on calculated AUC values and intake doses, indicating that EXT released by the MNs entered the systemic circulation well.The TS-MNs encapsulating EXT were embedded in normal mice to investigate EXT pharmacokinetics compared with SC injection. The pharmacokinetic curve and correlative pharmacokinetic parameters after administration are illustrated in The blood glucose levels versus time profiles after a single administration are displayed in To investigate the availability of EXT in regulating blood glucose in the short term, a daily administration was performed for six days. Blood glucose levels after multiple doses are shown in 2 concentration, and SA concentration. By analyzing the morphology and properties of the TS-MNs, EXT was clustered at the tips; then, the TS-MNs had sufficient mechanical strength to penetrate the skin so that the tips could be implanted into the skin for sustained release. Subsequently, in vitro release indicated that CA gel content affected drug diffusion. In addition, the release behavior of EXT from the TS-MNs conformed to the first-order kinetics. Additionally, the EXT activity was protected after MN preparation and remained above 85% after two months in the most severe 40 °C conditions. Finally, the activity of TS-MNs in vivo was evaluated. Pharmacokinetic experiments showed that the concentration of EXT could be detected within 48 h in the EXT MN group, indicating that part of the EXT was still released slowly from the gel. The hypoglycemic effect was observed to last for approximately 24 h after a single administration and remained effective after multiple administrations without drug resistance. Consequently, the TS-MNs provide tremendous convenience for patients to self-administer drugs.The TS-MNs encapsulating EXT were successfully prepared. In this work, the gel content, drug diffusion, tip height, and implantation efficiency were evaluated by optimizing the cross-linking time, matrix solution concentration, CaCl


APC-mutated colorectal cancer. nuPRISM expands our ability to captureaberrant nuclear morphological and functional traits associated withdistinctive signal transduction and subcellular localization-drivenfunctional processes with substantial resolution and high throughput.Genome-wide loss-of-functionscreens are critical toolsto identifynovel genetic regulators of intracellular proteins. However, studyingthe changes in the organelle-specific expression profile of intracellularproteins can be challenging due to protein localization differencesacross the whole cell, hindering context-dependent protein expressionand activity analyses. Here, we describe nuPRISM, a microfluidicschip specifically designed for large-scale isolated nuclei sorting.The new device enables rapid genome-wide loss-of-function phenotypicCRISPR-Cas9 screens directed at intranuclear targets. We deployedthis technology to identify novel genetic regulators of β-cateninnuclear accumulation, a phenotypic hallmark of  nuPRISM is a high-throughput nuclearscreening platformleveraging microfluidics and CRISPR genome-wide screens that identifiesnovel nuclear targets undetected by conventional platforms. Due to their broad applicability and ease of use in studyinginfection, cancer biology, and deciphering novel drug targets, genome-widephenotypic CRISPR screens are continuously subjected to a myriad ofmodifications to boost their potential.6Advances in cell sorting methodologieshave enabled multidimensionalanalysis of CRISPR-edited cell populations by integrating advancedsequencing, imaging, and cell sorting technologies through fluorescence-activatedcell sorting (FACS)-based screens, single-cell functional genomics(scFG) screens, and arrayed screens using microscopy as a read-out.9 However, studying the functionalimpact of a given protein of interest within a subcellular compartment,such as the nucleus or mitochondria, is often complicated by differencesin subcellular localization-dependent protein activity as well asthe background noise stemming from other biochemical processes theprotein is involved in.12 Multifunctional intracellularproteins with protein activities across the whole cell are especiallychallenging and necessitate the development of sophisticated cellsorting strategies to delineate the functional relevance of the dynamicprotein expression changes.Intracellular proteinexpression-based phenotypic screens are crucialin disease-biology research.14Conventional intracellular proteindetection techniques, such asthe immunoblotting and enzyme-linked immunosorbent assay (ELISA),have been developed to track intracellular proteins of therapeuticrelevance across different subcellular compartments. However, thesemethods are hindered by multiple limitations in the study of dynamicprotein translocations due to a complex workflow, the amount of samplerequired for robust signal detection, and the variability of experimentalprocedures across different settings that can affect biologicallyrelevant phenotypic read-outs.16 Moreover, the challengesrelated to the spectral imaging detection of specific proteins andspecificity to antibodies, especially those expressed over multiplesubcellular compartments, limit the use of flow cytometry or immunohistochemistryto precisely detect intracellular protein phenotypic changes.17High-throughputprotein immunoassays that can be used as screeningtools have addressed some of these limitations through robust quantificationsand sensitivity, but the costly instrumentation, complex workflowvariability, and applicability across a limited number of proteinshave constrained their use.adenomatous polyposiscoli (APC), which drive 80% of the sporadiccases of colorectal cancer. APC is part of the β-catenin destructioncomplex that targets β-catenin for proteasome-mediated degradation.In APC-mutated cells, β-catenin escapes degradationand accumulates in the nucleus, which leads to the activation of β-catenintarget genes in a ligand-independent fashion, increasing the proliferationand invasiveness of the tumors  to enable more accurate characterization,26 as well as allowing ultrasensitive detectionof surface proteins at single cell resolution within heterogeneouscell mixtures.27 Equally important, microscaletechnologies have enabled cell barcoding and sequencing providinga high-performance cellular characterization approach.28 Isotachophoresis is another capability thathas allowed the isolation and purification of cytoplasmic nucleicacids at a single cell level.29 Thus, microfluidicimmunomagnetic sorting is an attractive approach for further development.Advances in microfluidics have presented new opportunities to analyzebiological processes with high throughput.Here, we describe nuPRISM—a microfluidics-driven functionalgenomics platform—as the first high-throughput cellular nucleiscreening platform that enables the profiling of targeted proteinexpression with single nucleus resolution. As a proof-of-concept,we deployed the microfluidic nuPRISM chip to study levels of nuclearβ-catenin accumulation in SW480 colorectal cancer cells. Throughthe use of whole-genome phenotypic CRISPR screening to discover geneticregulators of nuclear β-catenin levels, we identified and validatedtwo novel positive regulators of β-catenin nuclear accumulation.The flexibility and high-throughput capacity of nuPRISM create a powerfultool to characterize nuclear phenotypes and track the subcellularlocalization differences of intranuclear markers at single-nucleusresolution.APC-mutated cells, the process of β-catenin degradationis hindered, leading to constitutive activation of the Wnt signalingpathway, targeting β-catenin to the nucleus and contributingto invasiveness and proliferation seen in colorectal cancer cells.Blocking β-catenin nuclear localization by targeting regulatorsof this process may represent a novel and personalized therapeuticstrategy for the treatment of colorectal cancers harboring APC mutation.In normal colorectal cells, the destructioncomplex  promotes β-catenin phosphorylation1A, thereAPC mutations lead to nuclear accumulation of β-catenin to enhancesignaling, but a substantial portion of the cytoplasmic β-cateninand the membrane-bound pools of β-catenin remain unchanged.As such, conventional cell sorting schemes would fail to detect phenotypicshifts in nuclear β-catenin levels.Conventional cell sorting-based CRISPRphenotypic screens haveshown great promise in discovering novel modulators and functionallyimportant intracellular protein targets. However, organelle-specificchanges in protein subcellular localization are often difficult totrack using conventional methods, creating a biological “blindspot” that can be overlooked. For example, –1 using thenuPRISM chips. The enrichment of sgRNAs in each replicate was determinedby NGS deep sequencing. In total, we processed ∼9 × 108 nuclei on parallelized nuPRISM chips powered by syringe pumps.This arrayed setup enabled efficient sorting, requiring a total sortingtime of around 9 h to process the entire whole-genome CRISPR screen.Our nuPRISM platformovercomes this by sorting the nuclei postsubcellular fractionation, enabling the detection of quantitativechanges in the nuclear pool of β-catenin 1B. To peThe nuPRISM chip sorts nuclei based on the loading of iron oxidebeads conjugated with a biotinylated antibody. The microfluidic chiparchitecture was optimized with reduced dimensions from our previousPRISM chip. This design allows us to obtain a high recovery rate ofnuclei. Unlike other magnetic sorting systems like MACS, the recoveryrate is increased given the high flow rate used that prevents nucleifrom clumping. Also, the nuPRISM chip allows the separation of low,medium, and high populations, which is beneficial if the medium populationis the target population or the application requires collection ofa certain specific population instead of the positives and negatives.For nuclear β-catenin expression-based sorting, colorectal cancerSW480 cell nuclei were first labeled with biotinylated anti-β-cateninantibody and then conjugated with antibiotin magnetic nanobeads 1C. The mTo accommodatethe application of cellular nuclei sorting, we configuredthe height of the flow channel in the nuPRISM chip to be 50 μm.Since a typical cell nucleus has a diameter of 6–10 μm,this optimized channel height prevents nuclei from clogging the flowchannel and allows for high sample throughput while avoiding structuraldamage that can cause nuclei leakage. The optimized setup also enhancesdevice sensitivity, enabling nuclei with low/no β-catenin expressionto be easily collected in the low/zero outlet.Supporting Information Figure 7).To enrich subpopulations of interest, we first optimized the flowrate to get the majority of the population expressing β-cateninto be collected in the medium outlet at ∼80% while retaining∼10% in the high and the low outlets . To evaluate the sensitivity of thenuPRISM method to detect population phenotypic drift, we transducedSW480 cells (exhibiting elevated levels of nuclear β-cateninaccumulation) using four different single-guide RNAs (sgRNAs) targetingCTNNB1 and processed the CRISPR-edited pools of isolated SW480 cellularnuclei using the nuPRISM platform. In parallel, we also performedFACS-based nuclei sorting for a direct comparison of the two methods.Detection of β cateninlow cells was comparable betweenboth methods, while the recovery using nuPRISM (∼85%) was considerablyhigher . The higher recovery rate of the nuPRISM platform compared to FACSis mainly due to the ability to collect nuclei in Lo-bind tubes inreal time rather than collecting them in FACS tubes then transferringagain to Lo-bind tubes for subsequent processing. This significantlydecreases recovery rates owing to the “sticky” natureof nuclei especially when collecting large populations as in CRISPRscreens. Also, the microfluidic chips’ high flow rate, narrowdimensions, and preconditioning by 1% Pluronic acid prior to sortingprevents nonspecific nuclei attachment.The immunomagneticcellular nuclei sortingis facilitated by magneticnanobeads conjugated to antibodies that bind to the intracellularprotein target based on protein expression levels as validated byTEM (microfluidic immunomagnetic cell sorting (MICS) platform,a high-throughput microfluidics platform that we previously used toperform a genome wide CRISPR screen in HAP1 cells to identify CD47regulators.4 We designed the nuPRISM asa new microfluidic chip with optimized architectural parameters. Theseadjustments allow for high-speed profiling of isolated cellular nucleibased on the preferred intracellular target protein expression. Weperformed a series of tests to optimize the sorting rates and demonstratethe sensitivity of the new device to nuclear β-catenin expression changes upon transduction of the SW480 cells with sgRNAstargeting CTNNB1 (β-catenin and CTNNB1-knockout nucleiat a high flow rate (2.5 mL h–1). However, we havenoticed the sensitivity of nuPRISM outperforms FACS at a higher CTNNB1KO ratio and becomes more comparable as we increase the positive CTNNB1population. This is mainly attributed to autofluorescence signalsemitted by SW480 cells and the fixation and permeabilization reagentsused prior to sorting to allow antibody intracellular staining. SincenuPRISM relies solely on magnetic beads and magnetic guide deflection,this issue is eliminated as it is unlikely that magnetic beads wouldbind to a nucleus with no protein expression as shown in TEM images. Moreover,we could recover the nuclei without structural damage to its membrane, whichcan hinder the downstream processing.Based on the cell profilingcapabilities of our existing g CTNNB1 2C. We acAPC-mutated colorectal cancer cells, we testeda panel of colorectalcancer cell lines to identify an ideal cell line for the whole-genomeCRISPR screen . As expected, most of the nuclei from cells expressing wildtype APC ended up in the low outlet due to the low accumulationof endogenous nuclear β-catenin. On the contrary, nuclei ofcells with APC mutation ended up in the medium and high outlets due to the higher level ofnuclear β-catenin accumulation .To identify β-catenin nuclear regulatorsin The SW480 cell line exhibited the optimumratios of the high, medium,and low nuclear β-catenin expressing cells under basal conditions,ideal for identification of negative and positive regulators of β-cateninnuclear retention. To further assess the performance of the nuPRISMdevice, we mixed β-catenin wildtype and CTNNB1-knockout cellsin predefined ratios to assess the profiling ability of the chip.Upon nuclei sorting, we observed accurate recovery that correspondedto the enriched population by flow cytometry and nuPRISM 2C.-catenin expressing subpopulation enrichments.Havingestablished nuPRISM as a reliable cellular nucleus sortingstrategy, we transduced the SW480 cells using the lentiviral TKOv3CRISPR library for a genome-scale loss-of-function phenotypic CRISPRscreen. The CRISPR-edited cell population was propagated for approximatelyseven doublings and was subsequently processed as three independentreplicates for microfluidic sorting in parallel with FACS sorting.The isolated nuclei were sorted for the highest 15% and lowest 15%βSupporting Information Figure 4B).30 The Gini index was below 0.2 in each, indicatingan even distribution of sgRNA and a high-quality screen.The enrichment of sgRNAs in each of the nuclei subpopulations ofinterest was determined by the MAGeCK algorithm post-NGS deep sequencing.We also assessed the evenness of sgRNA reads across the three replicatesin both high and low outlets of the chip using the Gini index (NCSTN (Nicastrin).We performed gene set enrichment analysis (GSEA) using hits with anFDR < 30% to determine significantly enriched cellular pathways.Toidentify regulators of β-catenin nuclear accumulation,we compared the sgRNA read counts between the enriched and unsortednuclei populations 3A,C. BotSupporting Information Figure 6). To assess theimpact on nuclear β-catenin accumulation, we designed a minipanel of sgRNAs targeting the candidate hits. We selected two sgRNAfor each of the top positive regulators from the nuPRISM and the FACSscreens as well as two sgRNAs targeting NCSTN asthe top negative regular of β-catenin accumulation.We confirmed reduced nuclear β-catenin levels after transductionof sgRNAs other than the ones used in the screen targeting ELOF1, CAB39, and OVOL2 as determined by intracellular flow cytometry and Western blotting. As expected, the lossof NCSTN expression increased levels of nuclear β-catenin .Loss ofELOF1 expression showed a similar profile as the CAB39effect on Wnt target genes, but minimal or no change was observedin the expression of AXIN2. Using intracellular immunofluorescence,we also assessed the effect of loss of CAB39 expression on β-cateninnuclear accumulation and found a significant reduction of the nuclearβ-catenin signal 4E,F. FinSupporting Information Figure 5). Consideringβ-catenin’s role in the EMT process, these results furthersupport the role of nuclear β-catenin accumulation in regulatingthis process.The invasive behavior of unmodified SW480 cells and CRISPR-editedSW480 cells with ELOF1 KO, CAB39 KO, OVOL2 KO, or NCSTN KO cells wastested using a Transwell cell invasion assay. A significantly highernumber of NCSTN knockout cells migrated from the top to the bottomof the membrane compared to the control group 4G. NotabSupporting Information Figure 1A).34 Taking the positive role of USP7 in Wnt−β-catenin signaling-mediatedgrowth of colorectal carcinoma cells into consideration, we hypothesizedthat this axis might play a role in ELOF1-mediated decrease in β-cateninnuclear accumulation.Recently, it was reported that ELOF1 plays a crucialrole in recruitingUV Stimulated Scaffold Protein A (UVSSA) to lesion-stalled Pol II.UVSSA recruits the deubiquitylating enzyme USP7, protecting CSB (Cockaynesyndrome group B (CSB) protein) from proteasomal degradation mediatedby the ubiquitin-selective segregase VCP (p97) . Moreover, treating cells with USP7 inhibitors (XL177Aand P5091) significantly affected cell growth and β-cateninnuclear accumulation . These results suggest that the ELOF1-UVSSA-USP7-nuclear β-cateninaxis was partly involved in the observed cytotoxic activity of XL177Aand P5091.Nevertheless, both XL177A and P5091 treatments(5 μM) decreasedthe accumulation of nuclear β-catenin as determined by the intracellularflow cytometry staining in SW480 cells 5B. Also,Genome-wide phenotypic CRISPR screens aretypically used to isolatewhole cell populations with either high or low expression levels ofa given target of interest, analyze sgRNA enrichments across subpopulationsof interest, and identify genetic modulators that regulate the expressionof the protein of interest. To date, no platform has been explicitlyadopted for cellular nuclei screening. Conventional phenotypic screenssort cells based on total protein expression across the entire cell,irrespective of the organelle-specific differences in protein expressionand/or localization. This significantly impacts the accuracy and thespecificity of the hits as the majority of multifunctional proteinsusually exhibit differential subcellular localization across differentorganelles that corresponds to specific signaling outcomes and/orprotein activity.APC-mutant colorectal cancercells. Nuclear β-catenin accumulation in APC-mutant colorectal cancers is therapeutically relevant given itscentral role in Wnt signaling. Normally, cytoplasmic β-cateninis actively degraded upon interaction with the destruction complexand maintained at a low level without a Wnt ligand. However, in APC-mutant cells, the aberrant signaling causes β-cateninto escape degradation routes and accumulate in the nucleus. This shuttlingactivates specific Wnt target genes in parallel with the T-cell factor/lymphoidenhancer factor (TCF/LEF) family of transcription factors implicatedin tumor relapse and resistance due to their functional role in colorectalcancer cancer-initiating cell survival.19 Moreover, this accumulation has been shown to cause radioresistancein colorectal cancer stem cells correlating with higher Wnt activation.35 These lines of evidence postulate the possibilityof using β-catenin nuclear accumulation as a prognostic markerfor CRC prognosis as it has a significant role in colorectal cancerprogression.Our nuPRISM platform enables the discoveryof targets regulatingprotein expression within the nucleus instead of the whole cell. High-throughputcellular nuclei sorting was applied to investigate the nuclear accumulationof β-catenin in APC gene mutation as the primary regulator of β-cateninnucleartranslocation, there is a lack of consensus that it is the sole justificationfor the heterogeneous nuclear accumulation and localization in theexamined samples of APC-mutant tumors. Some additionalproposed mechanisms involve the extracellular matrix through activationof integrin-associated kinases, which causes repression of E-cadherinand ultimately the nuclear localization of β-catenin that eventuallydictates the behavior of the colon cancer cell.38 Also, trefoilfactors have been implicated in the nuclear shuttling of β-cateninin colorectal cancer irrespective of APC-mutationstatus.39 Another study suggests the CRM1mediated pathway is an additional independent nuclear transport mechanismof β-catenin.40Despite the compelling evidence supporting APC-mutant colorectal cancer cells. Notably, loss of ELOF1 expressiondid not affect the levels of AXIN2.In the proof-of-conceptwhole-genome phenotypic CRISPR screen,we identified and validated two novel candidates (ELOF1 and CAB39)that showed a significant reduction of nuclear β-catenin accumulation.Interestingly, upon the knockout of these genes, we observed decreasedexpression of major Wnt signaling pathways related to genes such asCLDN1, CCND1, CDH2, CTNNB1, LEF1, MYC, and MMP7. All of these geneshave been previously implicated as targets of the Wnt signaling pathwayin colorectal cancer cells, and their reduced expression is usuallyassociated with reduced cancer cell growth rate and proliferation.Thus, their decreased expression strengthens the role of our candidatehits in suppressing the aberrant phenotype in We also observed a markedshift in the expression of EMT processgenes, consistent with the invasion and metastasis of adenocarcinomasthat frequently arise from the loss of epithelial characteristicsand gain of mesenchymal capacities of colorectal tumor cells. Accordingly,we saw a change in the number of migrating cells when we performedthe Matrigel assay.Desmoglein, alphaE-catenin,Collagen alpha-1(IV), and a decrease in mesenchymal markers such as Slug, Snail, Vimentin, Mucin 1, Zinc finger E-box-bindinghomeobox 1 (ZEB1), these results demonstrate a loss of epithelialand gain of mesenchymal characteristics which is a known feature ofthe APC-mutant tumor cells. ELOF1 function in transcription-couplednucleotide excision repair (TC-NER) is to promote the recruitmentof the TC-NER factors UVSSA and TFIIH to repair transcription-blockinglesions. UVSSA recruits the deubiquitylating enzyme USP7, protectingCSB from proteasomal degradation mediated by the ubiquitin-selectivesegregase VCP (p97).34The Matrigel invasion assay showed a significantincrease in SW480NCSTN KO cells with corresponding higher levels of nuclear β-catenin,while a lower number of migrating cells were observed for SW480 ELOF1KO,CAB39 KO, or OVOL2 KO cells, all of which exhibit lower expressionlevels of nuclear β-catenin. Combined with a significant increasein epithelial markers such as APC-mutated colorectal cancers by restoring β-cateninubiquitination, inhibiting tumor growth as evident in xenograft modelsgrowth suppression.37 We postulated that the ELOF1-UVSSA-USP7axis is involved in the reduced β-catenin nuclear accumulationwith ELOF1 being a regulator of the process. After we knocked outELOF1, we saw decreased expressions of UVSSA, USP7, and CTNNB1. Moreover,targeting USP7 with small molecule inhibitors (XL177A and P5091) selectivelyinhibited the growth of mutant APC-colorectal cancercells which are characterized by enhanced nuclear β-cateninaccumulation but not wildtype APC epithelial colorectalcells. These results are consistent with the previous reports thatsuggest the role of USP7 in colorectal cancer is APC-mutation-dependent.42Moreover,it has been shown that USP7 acts as a Wnt signaling inhibitorin APC-mutant colorectal cancer. Overall, this study demonstratesthe functionality and potential for nuPRISM to gain widespread implementationin the realm of drug discovery, especially for challenging intranuclearprotein targets. The ability to profile the nuclear-specific functionalphenotype of a protein at a single-nucleus resolution will open newavenues for targeted therapeutics development in colorectal cancerand other disease settings.Taken together, nuPRISMis a novel cellular nucleus sorting platformthat merges advances in microfluidics technologies and phenotypicCRISPR screening to discover regulators of nuclear accumulation ofproteins in a high-throughput manner. In our proof-of-concept nuclearβ-catenin screen, we executed a genome-wide CRISPR phenotypicscreen in under 9 h while maintaining the specificity of the nuclei’santibody binding and nuclei structural integrity. We identified ELOF1and CAB39 as novel nuclear β-catenin accumulation regulatorsand devised a novel sorting strategy that could be leveraged to target


However, the highest ΔmaxI equal to 384.20 ± 16.06 µA was obtained using GA-GOx-SAM/DGNs/GR electrode. ΔmaxI for biosensors based on the GA-GOx-SAM/DGNs/GR electrode was 1.41 times higher than for the GA-GOx/DGNs/GR, whereas the linear dynamic range from 0.1 to 10 mM was the same using all three GOx immobilization methods. The limit of detection using GA-GOx-SAM/DGNs/GR and GA-GOx/DGNs/GR electrodes was 0.019 and 0.022 mM, respectively. The ability to detect glucose in the serum by developed biosensors was evaluated.In recent years, many efforts have been made to develop rapid, sensitive and user-friendly glucose biosensors for monitoring blood glucose concentration in patients. In this study, the electrochemical glucose biosensors based on graphite rod (GR) electrode electrochemically modified with dendritic gold nanostructures (DGNs) and glucose oxidase (GOx) were developed. Phenazine methosulfate was used as a soluble redox mediator. Three GOx immobilization methods: adsorption on DGNs and cross-linking with glutaraldehyde (GA) vapour (GA-GOx/DGNs/GR), covalent immobilization on DGNs modified with 11-mercaptoundecanoic acid self-assembled monolayer (SAM) (GOx-SAM/DGNs/GR) and covalent immobilization on SAM with additional cross-linking with GA vapour (GA-GOx-SAM/DGNs/GR), were used. It was determined that GA significantly improved the stability of the enzyme layer. The difference of maximal current generated during the enzymatic reaction (Δ An important task of modern medicine is the comprehensive and accurate diagnosis of diseases and monitoring concentration of the biologically important substances in body fluids. Today, diabetes mellitus, a chronic metabolic disease commonly known as diabetes, has become one of the greatest health problems in the world . In a heVarious analytical methods, mainly optical and electrochemical, are used to determine the glucose content in biological fluids ,4. Among4 [Among different shaped GNs, the dendritic gold nanostructures (DGNs) have been greatly successful due to their superhydrophobicity , unique 4 .The selection of the best enzyme immobilization method on the electrode is of high importance in the development of well-functioning amperometric glucose biosensors. The most commonly used enzyme immobilization methods are adsorption or covalent attachment, whereas cross-linking of the enzyme on the electrode by specific reagents, covalent immobilization or entrapment in the conducting polymer layer, and encapsulation in nanoparticles or microcapsules are also applied ,25,26. AThe aim of this study was to compare different GOx immobilization methods on DGNs in order to develop a stable, selective, and sensitive amperometric glucose biosensor that functions well in real samples. The electrochemical deposition of DGNs on the electrode surface was optimized using the CPA method. Three GOx immobilization methods, namely, simple adsorption on DGNs and cross-linking with GA vapour (first method), covalent immobilization on DGNs modified with 11-mercaptoundecanoic acid SAM (second method), and covalent immobilization on SAM with additional cross-linking with GA vapour (third method) were used and the performance of the developed glucose biosensors was characterized.−1 protein) and ascorbic acid C6H8O6 were received from Sigma-Aldrich . Phenazine methosulfate (PMS), uric acid C5H4N4O3, and citric acid C6H8O6 were obtained from AppliChem GmbH . D-(+)-Glucose, D-(+)-galactose, D-(+)-xylose, D-(+)-mannose, and hydrogen tetrachloroaurate (III) trihydrate (HAuCl4·3 H2O) were purchased from Carl Roth GmbH&Co . Potassium nitrate (KNO3), potassium chloride (KCl), and N-hydroxysuccinimide (NHS) were acquired from MERCK KGaA . Sodium acetate trihydrate, 11-Mercaptoundecanoic acid (MUA), methanol, D-(+)-Saccharose and 1-ethyl-3-(3-dimethylaminopropyl)carbodimide hydrochloride (EDC) were obtained from Sigma-Aldrich Chemie GmbH . Sodium citrate was received from Penta  and 25% glutaraldehyde (GA) from Fluka Chemie GmbH . Graphite rods (GR)  were purchased from Sigma-Aldrich  and alpha alumina powder  from Electron Microscopy Sciences . The sodium acetate (SA) buffered solution (0.05 M CH3COONa) with 0.1 M KCl was prepared by mixing sodium acetate trihydrate and potassium chloride. A GOx solution of 40 mg mL−1 concentration was prepared in SA (pH 6.5). Before investigations, the glucose solution was allowed to stay overnight for the formation of α-β optical isomers at equilibrium. All other chemicals used in the present study were either analytically pure or of higher quality. All solutions were prepared using deionized water purified with the water purification system Millipore S.A. .Glucose oxidase  (FE-SEM). Energy-dispersive X-ray spectroscopy (EDS) using scanning electron microscope Hitachi TM3000  at 15 kV accelerating voltage was applied for determination of the chemical compositions of prepared electrodes.The electrochemical deposition of DGNs and electrochemical measurements were performed using computerized potentiostat/galvanostat Autolab PGSTAT30  driven by NOVA1.9 software. The electrochemical cell was composed of a GR electrode (unmodified and premodified) as a working electrode, Ag/AgCl/KCl2. The GR electrode was immersed into an electrochemical cell filled with an aqueous solution consisting of 10 mM HAuCl4 and 0.1 M KNO3, and an electrochemical deposition of DGNs was performed at a constant −0.2 V potential vs. Ag/AgCl/KCl3M for 200 or 400 s. After DGNs synthesis, the electrode was rinsed with deionized water and dried at room temperature. The surface of GR electrodes modified by electrochemically deposited DGNs (DGNs/GR) was characterized by a FE-SEM.The graphite rods were broken and mechanically polished with fine (P120), very fine (P320), and finally, with ultra-fine grit (P2000) sandpaper followed by GR electrodes washing with ethanol and UHQ water and drying at room temperature. Then, the side surface of GR electrodes was isolated with a silicone tube in order to avoid its contact with solution in the electrochemical cell. The working surface area of GR electrodes is 0.071 cm2SO4 electrolyte solution. Cyclic voltammogram was recorded in the potential range from 0 to +1.4 V vs. Ag/AgCl/KCl3M using the scan rate of 0.1 V/s. The integrated peak of gold oxide reduction and the conversion factor of 386 μC cm−2 [To determine the electroactive surface area of electrodeposited DGNs, cyclic voltammogram was performed in 0.5 M H μC cm−2  were use μC cm−2 .−1 GOx solution for 30 min and dried at room temperature (+20 ± 2 °C). Then, the working electrode was kept for 15 min in a closed vessel over a 25% solution of GA at room temperature. The selection optimal time for the cross-linking of GOx with the GA vapour is presented in −1 GOx solution for 30 min to covalently immobilize GOx. In the third method, GOx was immobilized the same way as it was done in second method, but additional cross-linking with the GA vapour was applied. Thus, the GOx-SAM/DGNs/GR electrode was kept for 15 min in a closed vessel over a 25% solution of GA at room temperature. Before all electrochemical measurements, GOx-modified electrodes were washed with distilled water to remove non cross-linked enzymes. All working electrodes were stored in a closed vessel over SA buffer at +4 °C before use. Prior to electrochemical measurements, the electrodes were washed with UHQ water.Three different GOx immobilization methods on the GR electrode premodified with electrochemically deposited DGNs were used and compared in this study. Namely, GOx was immobilized by simple adsorption on DGNs and cross-linked with the GA vapour (GA-GOx-DGNs/GR) (first method), covalently on DGNs modified with 11-MUA SAM (GOx-SAM/DGNs/GR) (second method), and covalently on 11-MUA SAM with additional cross-linking with the GA vapour (GA-GOx-SAM/DGNs/GR (third method). In case of the first method, the DGNs/GR electrode was immersed in a tube filled with 40 mg mL2 (the reduced form of PMS) is reoxidized on the surface of the working electrode (2 are transferred to the positively charged electrode in two ways: directly to the GR electrode and through GNs [3M were recorded at glucose concentrations in the range from 0.1 to 100 mM. The current response under certain concentrations of glucose (ΔI) was evaluated as the difference of registered steady anodic currents after the addition of glucose and baseline. All investigations were carried out at +20 ± 2 °C temperature in a 5 mL volume electrochemical cell stirring solution with a magnetic stirrer (120 rpm). The results of all electrochemical measurements are reported as the mean values of three independent experiments.The amperometric signals of biosensors using GR electrodes premodified with DGNs and GOx by three immobilization methods were evaluated in 0.05 M SA buffer with 0.1 M KCl (pH 6.0) and PMS. Firstly, a steady baseline current was reached, and then different concentrations of glucose were added. During the enzymatic oxidation of β-D-glucose, two molecules of flavin adenine dinucleotide (FAD) tightly bound to each GOx subunit are reduced. The artificial redox mediator PMS present in the electrochemical cell is involved in the reoxidation of the GOx redox center, and the resulting PMSHlectrode . Electroough GNs . CurrentImax) and the apparent Michaelis constant (KM(app.)) were, correspondingly, the parameters a and b of the hyperbolic function y = ax/(b+x), which was used for the approximation of the results. Parameters of the enzyme-catalyzed reaction were calculated using Sigma-Plot software (version 11.00). The calibration curve parameters, such as correlation coefficient (R) and determination coefficient (R2), were evaluated. The LOD was calculated as the lowest concentration of the analyte, which gives an analytical signal greater than the background value plus 3σ.Amperometric signals showed hyperbolic dependence on glucose concentrations and it was in good agreement with the Michaelis–Menten kinetics. The difference of maximal currents generated during the enzymatic reaction  was selected for this study. It is obviously seen that amperometric signals obtained at glucose concentrations of 5, 25 and 50 mM were similar for both DGNs electrodeposition times . However2SO4 was performed for the calculation of the electroactive surface area of electrodeposited DGNs. It is obvious that electrochemical gold oxidation and further reduction take place. The gold electroactive surface area was calculated to be equal to 1.03 ± 0.15 cm2.Additionally, cyclic voltammetry measurement  in 0.5 M3M. The amperograms for all three types of electrodes are presented in maxI equal to 19.21 ± 0.75 µA (R = 0.9864) was obtained using the second GOx immobilization method, in which GOx was covalently immobilized on SAM/DGNs/GR without additional crosslinking (GOx-SAM/DGNs/GR). The 14.2 times higher ΔmaxI equal to 272.06 ± 8.69 µA (R = 0.9938) was obtained using the first GOx immobilization method when GOx was adsorbed on DGNs and cross-linked with GA vapour (GA-GOx/DGNs/GR). The KM(app.) was equal to 26.56 ± 2.42 mM, and 20 times higher ΔmaxI equal to 384.20 ± 16.06 µA (R = 0.9965) was obtained using the third method, in which covalent GOx immobilization and a further 15 min of lasting cross-linking with 25% vapour of GA were used. In this case, the KM(app.) was a little bit higher and equal to 35.23 ± 3.76 mM. The highest current response and ΔmaxI were obtained after GOx covalent immobilization on the 11-MUA SAM with additional crosslinking by the GA vapour (GA-GOx-SAM/DGNs/GR). ΔmaxI for the glucose biosensor based on the GA-GOx-SAM/DGNs/GR electrode was 1.41 times higher than for the GA-GOx/DGNs/GR electrode, whereas the linear dynamic range (LDR) from 0.1 to 10 mM was the same using all three GOx immobilization methods  in comparison to smaller enzymes, such as horseradish peroxidase, and random distribution of amino groups on the surface of enzyme [Summarizing the results obtained, we can state that the performance of glucose biosensor is better using GR electrode premodified with DGNs and GOx by the third method. The requirement of additional cross-linking of GOx covalently immobilized on the SAM was also observed by other authors. The 0.05% f enzyme . The comRepeatability is one of the important characteristics of biosensors showing the ability to generate similar results during multiple measurements of the same quantity of analyte. Despite the fact that the highest current response was registered by the GA-GOx-SAM/DGNs/GR electrode, the GA-GOx/DGNs/GR electrode was also evaluated. In the repeatability study, the current response was recorded by detecting 25 mM glucose 10 times by adding glucose to the electrochemical cell. As shown in Serum glucose levels for people with diabetes is much wider than in healthy people—from 2 to 30 mM. Thus, the suitability of GA-GOx-SAM/DGNs/GR and GA-GOx/DGNs/GR electrodes was tested for glucose determination in diluted human serum. Before the use, serum was taken from the freezer, thawed, and filtered. Then, it was diluted five times with 0.05 M SA buffer (pH 6.0), containing 0.1 M KCl.maxI was equal to 312.58 ± 18.34 µA (R = 0.9985), whereas when using the GA-GOx/DGNs/GR electrode, it was 244.12 ± 8.79 µA (R = 0.9960). Consequently, the KM(app.) were 40.42 ± 5.72 mM and 25.22 ± 2.64 mM, respectively. Despite the difference in the value M(app.)of K, the LDRs (The hyperbolic relationships between registered currents in the diluted serum and added glucose concentrations in the range from 0.1 to 100 mM were obtained for the two types of electrodes A. It is the LDRs B were thThe selectivity of the developed biosensors was tested after addition of 25.0 mM of galactose, fructose, xylose, and mannose. These substrates had no effect on the registered current responses. Only the addition of 25.0 mM concentration of glucose gives a measurable, current response. Furthermore, the influence of ascorbic and uric acids as possible interfering substances on the determination of glucose was determined . The preA little impact of tested electrochemically active compounds on the registered current response using electrodes modified with GOx according different protocols was observed; however, in most cases the signal difference was less than 10% except in the highest concentrations of ascorbic acid. Additionally, less impact of interfering species was registered by the GA-GOx-SAM/DGNs/GR electrode. Thus, the developed biosensors are suitable for the glucose detection in the serum, and the higher signal to glucose is registered when GOx is covalently immobilized on the 11-MUA SAM and additionally cross-linked with GA. Furthermore, the impact of interfering species on the analytical signals can be reduced by use of electrically conducting polymers formed over the tested electrodes ,37.I% calculated for both types of biosensors at 50 mM glucose gradually decreased over time. A more rapid decrease in the current response was observed up to 5 days using the GA-GOx-SAM/DGNs/GR electrode, whereas for GA-GOx/DGNs/GR, it takes longer, up to 12 days. After 12 days 73.25% of the initial current response was retained for the GA-GOx-SAM/DGNs/GR electrode and 66.20% for the GA-GOx/DGNa/GR electrode. The GA-GOx-SAM/DGNs/GR electrode was found to have better stability compared to the GA-GOx/DGNs/GR electrode. It might be due to more tightly bound enzyme with proper spatial orientation on more biocompatible surface, so GOx molecules longer are active and stable during the electrochemical measurements and washing setups.The stability of glucose biosensors based on GA-GOx-SAM/DGNs/GR and GA-GOx/DGNs/GR electrodes was tested during the 32-day period . The ΔI%In this work, the electrochemical deposition of DGNs on a GR electrode at a constant potential in time was optimized. Additionally, it was shown that the selection of GOx immobilization on DGNs method has a great importance on the performance of the developed glucose biosensors in the buffer solution and in the serum. The application of GA to cross-link GOx after covalent immobilization on the SAM significantly improved the sensitivity and stability of the biosensing layer, and repeatability of the current response after multiple detection of glucose with the developed biosensor. Such an immobilization method minimizes the loss of enzyme during continuous amperometric measurements in solution with mixing; however, DGNs are quite fragile and can be damaged or removed from the surface together with enzymes under inappropriate experimental conditions. Furthermore, multilayers of GOx covalently immobilized on gold nanostructures are a very promising direction for improving the analytical parameters of biosensors. In order to simplify the operation of such biosensors, a reagent-less system must be constructed, where the redox mediator will be immobilized on the surface of DGNs/GR electrodes.


The distribution and mobility of proteins inside the living cell can be used to differentiate cancer from normal cells. This review highlights differential protein distribution of two exemplary proteins, beta-catenin and CapG, and their role in gynecologic cancers. Recognizing differential protein distribution in cancer cells may have diagnostic and therapeutic implications.sequence and expression of specific proteins. There are only a few examples, however, showing that cancer and normal cells can be differentiated based on the altered distribution of proteins within intracellular compartments. Here, we review available data on shifts in the intracellular distribution of two proteins, the membrane associated beta-catenin and the actin-binding protein CapG. Both proteins show altered distributions in cancer cells compared to normal cells. These changes are noted (i) in steady state and thus can be visualized by immunohistochemistry—beta-catenin shifts from the plasma membrane to the cell nucleus in cancer cells; and (ii) in the dynamic distribution that can only be revealed using the tools of quantitative live cell microscopy—CapG shuttles faster into the cell nucleus of cancer cells. Both proteins may play a role as prognosticators in gynecologic malignancies: beta-catenin in endometrial cancer and CapG in breast and ovarian cancer. Thus, both proteins may serve as examples of altered intracellular protein distribution in cancer and normal cells.It is well-established that cancer and normal cells can be differentiated based on the altered  Cells are individual compartments that differ in composition from the exterior. These compartments are generally maintained by lipid membrane systems that prevent homogenous mixing of interior and exterior. Furthermore, various intracellular compartments exist that may or may not be enclosed by lipid membranes. These compartments limit the exchange of molecules and permit the buildup of concentration gradients, but also employ mechanisms that allow transport across membranes and thus the exchange between compartments. Thereby, certain molecules distribute differentially between cellular compartments as well as exterior and cell interior. Examples of differential molecular distribution are symmetry-breaking and polarization events during embryonic development. Similarly, shifts in compartmentalization occur during carcinogenesis. Although less studied, these shifts in compartmentalization could be used to differentiate normal and cancer cells. steady-state distribution, the other one differences in the dynamic distribution. The former can be visualized using immunohistochemistry, while the latter can only be made visible using the tools of quantitative fluorescence live-cell microscopy. Both protein re-distribution scenarios discussed here have been described in gynecologic malignancies and may play a role as prognosticators in these cancers; beta-catenin in endometrial cancer and CapG in breast and ovarian cancer. Cancer has been recognized as a disease of the genome. Experimental emphasis has been placed on analyzing its genome, differences in genomic sequences and protein expression in normal and cancer. Rapid developments in sequencing capabilities facilitated these studies including the Human Genome Project  and The 2-dependent cell adhesion. Hence, the name catenin (catena—Latin chain) linking E-cadherin to cytoskeletal structures [Beta-catenin is a dual function, or so-called moonlighting protein. It has two major roles in the cell: It is part of (i) the cell adhesion complex E cadherin and (ii) the canonical Wnt/beta-catenin signaling pathway. In the late 1980s, beta-catenin was discovered as a structural protein of the E-cadherin complex, a key molecule of Caructures . Drosophila and was known as Wingless (Wg). Wingless is the main component of the Wnt signaling pathway. The name Wnt is a portmanteau from Wingless and Int-1. It was further recognized that the armadillo protein, a main component of the Wnt signaling pathway in Drosophila, is structurally and functionally homologous to the mammalian beta-catenin. In the early 1980s, Nusse and Varmus discovered a new mouse proto-oncogene, named Int1 (integration 1) . Later iJun, c-Myc, and CyclinD-1. If Wnt does not bind to the Frizzled receptors, the beta-catenin destruction complex is not inactivated, but instead phosphorylates beta-catenin which results in its ubiquitylation and ultimately the proteasomal degradation of beta-catenin.In the canonical Wnt/beta catenin pathway, Wnt glycoproteins bind to Frizzled (Fzd) receptors and Lrp co-receptors on the plasma membrane which results in the phosphorylation and thereby inactivation of the beta-catenin destruction complex. The multi-protein beta-catenin destruction complex consists of APC (adenomatous polyposis coli), axin, CK1α , and GSK3β (glycogen synthase kinase 3 beta). Once the destruction complex has been deactivated, beta-catenin accumulates in the cytoplasm and then shuttles to the cell nucleus where it binds to the transcription factor complex Tcf/Lef (T-cell factor/lymphoid enhancement factor). Multiple nucleocytoplasmic import and export mechanisms have been described. Target genes include Alterations on any level of the Wnt/beta-catenin signaling pathway can promote cancer development: overexpression of the Wnt ligands, alterations in the Fzd receptor or Lrp co-receptor, mutations in the destruction complex, and mutations in beta-catenin itself. A prime example for alterations in the destructions complex are APC mutations resulting in decreased beta-catenin phosphorylation. Mutations in APC can cause familial adenomatous polyposis (FAP), the main hereditary factor in colorectal cancer. It was noted that the retention and not the complete loss of the first 20 amino acid repeat of APC that can bind beta-catenin and regulate its activity to some extent is favoring colon cancer formation. The unrestricted activity of beta-catenin by the complete loss of inactivating APC, however, results in further increased signaling and risk of cell death. This was postulated as the ‘just-right’ signaling model . In a ‘tAside from its role in colon cancer, beta catenin was described to play a role in many cancers including breast cancer and the breast cancer microenvironment ,8,9,10, CTNNB1 mutations in the high frequency of 52%. Most mutations were found in exon 3 of the beta-catenin gene, which is thus a mutational hotspot. A mutation in this gene location results in a missense of the GSK3β consensus site, thus prevents the destruction complex from binding and stabilizes beta-catenin. This stabilization results in the re-distribution of beta-catenin from the plasma membrane to the cell nucleus. Wright et al. found this mutation in 16% of all endometrioid endometrial adenocarcinomas [p < 0.0001) of exon 3 mutations identified by sequencing with the beta-catenin redistribution into the cell nucleus detected by immunohistochemistry in endometrial cancer. Nuclear beta-catenin immunohistochemistry stain showed a 91% sensitivity and an 89% specificity. The positive and negative predictive value in this study were 86% and 93%, respectively [In 2013, The Cancer Genome Atlas (TCGA) suggested a subclassification of endometrial cancers into four groups based on their molecular profiles [rcinomas . The redectively . Thus, IBased on clinical data, the prognostic relevance of a beta catenin mutation and redistribution in endometrial cancer is still discussed. However, limited data suggest that a beta-catenin mutation in endometrial cancer increases the risk of recurrence in low-risk and low-stage endometrial cancers including the risk of distant recurrences ,16. To e2-dependent the rapidly growing plus ends of actin filaments, by placing a ‘cap’ on actin filaments (capping like Gelsolin). CapG does not sever [CapG is a relatively small 39 kDa actin-binding protein of the Gelsolin family ,18. In cot sever  or nucleot sever . Cytoplaot sever ,21. The ot sever ,23. The In comparison with beta-catenin, few is known about the role of CapG in cancer. CapG was described within the German Human Genome project to be overexpressed in breast and ovarian cancer ,25. It mAs in other cells, CapG distributes diffusively in both the cell nucleus and cytoplasm of the highly invasive breast cancer cell line MDA-MB-231 and the nearly normal breast epithelial cell line MCF-12A. Transfected with CapG-GFP, there is no apparent difference in fluorescence intensity in cytoplasm and nucleus in both cell lines. However, the nucleocytoplasmic shuttling in these two cell lines was found to be different . CapG-GFed below .F(t)=RoiThe experimentally determined fluorescence intensity over time F(t) B can be a is the fraction of fluorescence intensity initially bleached, b the fraction that recovers over time, and In a single-exponential function, olecules C. The reIn fact, ubgroups . The proBeta-catenin in endometrial cancer and CapG in breast cancer are examples of differential protein distributions in normal and cancer cells. Beta-catenin shows a difference in its steady-state distribution and can be analyzed using immunohistochemistry. CapG shows a difference in its dynamic distribution between the cytoplasm and the cell nucleus that can only be revealed by quantitative fluorescence live-cell microscopy . To our Preventing shifts in compartmentalization could be used therapeutically. Currently, there are ongoing clinical trials exploring the interference with intracellular shuttling and in particular nucleocytoplasmic shuttling. Selinexor, a synthetic small molecule, blocks the nuclear export protein exportin 1 (XPO1) or also so-called chromosomal maintenance region (CRM1) by interfering with the Ran GTP-GDP cycle. It is not fully understood how far this interference results in anti-cancer effects. It has been hypothesized that XPO1 exports particularly tumor suppressor proteins from the cell nucleus so that they cannot exert their tumor suppressive effects. It is unclear if there are other consequences of the disruption of this highly conserved export mechanism on the cellular level. In initial clinical trials Selinexor was used for various cancers including multiple myeloma and lymphoma. A phase II trial studied Selinexor in heavily pretreated gynecological cancers . The SIEThe biophysical techniques of quantitative fluorescence microscopy may help identify and analyze differences in molecular distributions. These techniques are especially well suited for the analysis of dynamic molecular distributions that are not accessible to classic immunohistochemistry. In this respect, quantitative fluorescence microscopy may contribute to personalized medicine and its current analyses of genome sequence and protein expression levels.Beta-catenin shifts its steady-state distribution in endometrial cancer cells from the plasma membrane to the cell nucleus which can be shown using immunohistochemistry. CapG exhibits faster nucleocytoplasmic shuttling in breast cancer cells than in normal breast epithelial cells as shown in a proof-of-principal study using quantitative live-cell microscopy. Intracellular protein compartmentalization and mobility can be used to differentiate normal from cancer cells and may have diagnostic, prognostic, and therapeutic implications.


P525L MNs and a preferential binding of the mutant protein to introns flanking downregulated circRNAs and containing inverted Alu repeats. For a subset of circRNAs, FUSP525L also impacts their nuclear/cytoplasmic partitioning, confirming its involvement in different processes of RNA metabolism. Finally, we assess the potential of cytoplasmic circRNAs to act as miRNA sponges, with possible implications in ALS pathogenesis.Deregulation of RNA metabolism has emerged as one of the key events leading to the degeneration of motor neurons (MNs) in Amyotrophic Lateral Sclerosis (ALS) disease. Indeed, mutations on RNA-binding proteins (RBPs) or on proteins involved in aspects of RNA metabolism account for the majority of familiar forms of ALS. In particular, the impact of the ALS-linked mutations of the RBP FUS on many aspects of RNA-related processes has been vastly investigated. FUS plays a pivotal role in splicing regulation and its mutations severely alter the exon composition of transcripts coding for proteins involved in neurogenesis, axon guidance, and synaptic activity. In this study, by using in vitro-derived human MNs, we investigate the effect of the P525L FUS mutation on non-canonical splicing events that leads to the formation of circular RNAs (circRNAs). We observed altered levels of circRNAs in FUS Fused in sarcoma (FUS) is a DNA/RNA-binding protein playing crucial roles in RNA metabolism and in numerous cellular processes, especially in nerve cells ,2,3. MutCircular RNAs (circRNAs) belong to the long non-coding RNA family, and they hold high stability because of their unique closed structure. They are evolutionarily conserved RNAs and are broadly expressed across the animal kingdom ,25. EvenIn this study, we explored the transcriptome of progenitor and mature MNs derived from induced pluripotent stem cells (iPSCs) to assess the effect of the P525L FUS mutation on circRNA biogenesis. This mutation is associated with a strong delocalization of the protein in the cytoplasm and is linked to one of the most severe forms of familial ALS ,43. We oTo identify circRNAs expressed in human motor neurons (MNs) and determine how their levels are modulated in an ALS background, we reanalyzed Total RNA-seq data (GEO accession number GSE94888) previously published  by usingWT and FUSP525L)  ≥ 1 in at least one condition) ; notablycircRNAs . When weWT or FUSP525L pure MNs and carried out a differential expression analysis to detect circRNAs deregulated in the cells carrying the mutant protein. We found 121 deregulated circRNAs  and, among them, the majority (86) were downregulated in the mutant condition  (The plot in  to 0.1) . This fiThe Gene Ontology (GO) term enrichment analysis performed using the DAVID tool  was condWT and FUSP525L MNs [P525L MNs  and circDLC1 , since they hold interesting features. CircSLC8A1 was already described as being deregulated in another neurodegenerative disorder  possibl disease  and it iificance b; howeveificance c. Moreovificance .In order to exclude that circRNA downregulation was due to differences in MNs maturation or FUS (WT and P525L) expression between the two conditions, we also measured the levels of CHAT, ISLET1, and FUS mRNAs in WT and mutant MNs. Similar levels of these mRNAs were observed in both conditions, thus confirming that circRNA deregulation is a real consequence of the presence of ALS-linked FUS mutation .When assessed for circularity, all the putative circRNAs showed resistance to the RNase R treatment, albeit to different extent, while the linear counterparts were almost completely degraded a. circSLH517Q (76 downregulated and 226 upregulated circRNAs) and ALS samples (363 downregulated and 271 upregulated circRNAs); however, a very poor overlap was observed between the circRNAs deregulated in these datasets and in the FUSP525L MNs , suggesting a major contribution of transcriptional regulation in these latter conditions. Notably, transcriptional defects have been already observed in MNs carrying the H517Q FUS mutation [We next investigated whether the variation of the levels of the studied circRNAs was specifically caused by the P525L mutation or if it was a more general feature linked to the ALS condition. To this aim, we analyzed two additional RNA sequencing datasets (NCBI GEO: GSE203173 and dbGaP: phs000747) obtained from in vitro derived MNs carrying the H517Q FUS mutation and from sporadic ALS post-mortem tissues , respectively ,63. As s525L MNs . Among tmutation .These results lead to conclude that the P525L mutation causes a specific deregulation of a subset of circRNAs not observed in other ALS conditions and strengthen the indication that the P525L-linked deregulation mainly occurs at the post-transcriptional level.p-value < 0.05, two-tailed Student’s t-test), except for circSMARCA5 and circZNF124, which resulted in being located predominantly in the nucleus  and/or CircDlc1 also binds to miR-217 which, among the others, targets HNRNPA3. Notably, reduced levels of HNRNPA3 protein in C9orf72 ALS cases have been associated with enhanced repeat-dependent toxicity . MoreoveAnother intriguing aspect of circRNA sponge activity is that, upon circularization, new binding sites for miRNAs might be generated at the back-splicing junction. Interestingly, we found that this specific region of circCARHSP1 is targeted by miR-24-3p and miR-345-5p. Notably, miR-24-3p regulates neuronal differentiation and promotes neurite growth ,76, whilFinally, we decided to validate the predicted circRNA sponge activity by downregulating circDLC1 in MNs through siRNA treatment and by using quantitative Real-Time PCR to evaluate the expression of its putative targets as preliminary screening. Among the predicted targets listed in CircRNAs are rediscovered molecules that are widely expressed and conserved in the animal kingdom ,25. TheyBy looking at circRNAs deregulated in the mutant MN samples, we have enlarged the repertoire of FUS targets in human neuronal cells, possibly contributing to the unraveling of molecular mechanisms pertaining ALS that are still not completely understood. In particular, we discovered that the FUS mutation leads to the deregulation of 121 circRNAs in MN cells. Notably, in most cases their linear counterparts do not show the same trend, suggesting that mutant FUS is acting at the post-transcriptional level.Since circRNAs may control the expression of their host genes, GO term enrichment analysis was carried out on genes producing deregulated circRNAs. We found interesting functional categories such as transcription, apoptosis, cell adhesion, and chemical synaptic transmission, which are all described as being altered in ALS ,46,51,82WT and FUSP525L MNs [P525L MNs. Significant preferential binding of mutant FUS was observed for introns of downregulated circRNAs, usually coinciding with the presence of inverted Alu repeats. The A-to-I editing enzyme ADAR1 has been described to preferentially act on such repeats [Considering the described ability of FUS to affect circularization by binding intronic sequences , we anal525L MNs  to exami repeats ,57,58,59 repeats . Indeed, repeats . The absNevertheless, among the circRNAs affected by FUS mutation, only the downregulated ones showed high Alu editing levels in their flanking intronic regions compared to unaffected circRNAs. Further analyses are indeed required to eventually clarify the molecular mechanisms behind the concomitant presence of the P525L FUS protein and edited Alu repeats in the introns of downregulated circRNAs. At this stage, we might just speculate that the presence of mutant FUS on introns enriched for edited Alu repeats allows the recruitment of additional factors that eventually exacerbate the inhibitory effect of editing on circularization.The study of the subcellular localization of a subset of downregulated circRNAs showed that only two of them have a clear nuclear localization, while all the others are preferentially located in the cytoplasm. P525L mutation causes a profound cytoplasmic delocalization of FUS with consequences on the expression of many genes through a gain of function mechanism ,84,85,86H517Q in vitro derived MNs and ALS post-mortem tissues) allows to conclude that the biogenesis of the circRNAs characterized in this study is specifically affected by P525L FUS mutation, since they do not vary or do not show the same trend of variation in the other two conditions. Moreover, differently from what is observed in FUSP525L MNs, a higher percentage of deregulation likely occurs at the transcriptional levels both in FUSH517Q MNs and ALS tissues.The analysis of two additional RNA sequencing datasets obtained from different types of ALS samples  that may serve as circular-specific binding sites for miRNAs. Such mode of interaction was indeed detected for circCARHSP; miRNAs bound to its BSJ, miR-345-5p and miR-24-3p, regulate the expression of many genes involved in the endo-lysosomal pathway and autophagy . ALS is a complex and multifactorial disease involving different pathological processes; nowadays, we are well aware that the comprehension of the molecular mechanisms which lead to the alteration of such processes may represent an actual opportunity to foresee clinical treatments. We believe that the present study, even if preliminary, contributes to add insights to this complex scenario by including other players in the regulatory networks involved in the disease and by assessing how the coordinate action of different ncRNAs (circRNAs and miRNAs) may affect the molecular pathogenesis of ALS.RNA-Seq reads were initially trimmed using the Trimmomatic software  to removThe Venn diagrams showing the localization of circRNAs with respect to gene features were generated using the jvenn tool . The comcpm function from the edgeR R package [To quantify the expression of circRNA species and linear counterparts, we generated a matrix containing the read counts of both the back-splicing events and their cognate linear splicing events; the read counts of cognate linear splicing events were calculated summing all the reads mapping linearly on both the splice junctions involved in back-splicing. Events not having two or more reads in at least two samples were filtered out. The  package  was empl package  was useddesign <- model.matrix (~ experiment + condition); model fitting and testing was performed using glmFIT and glmLRT functions. Given the low number of reads used for testing, we decided to use the p-value instead of false discovery rate to select for differentially expressed events, setting the significance threshold value to 0.05 for back-splicing events and to 0.1 for linear splicing events; we chose to use a more relaxed cutoff for linear splicing events to obtain a set of circRNAs whose deregulation was less likely to be due to the concordant change in expression of the host gene.To identify those circRNAs that were differentially expressed between two conditions motor neurons, circRNAs and linear splicing events were initially filtered by removing those not supported by at least 2 reads in a number of samples equal to the minimum number of replicates. edgeR R package was employed to perform differential expression analysis on the resulting count matrix; an additive model was fitted to adjust for baseline differences between the three independent differentiation experiments: p-value < 0.05 were considered as significantly enriched.The DAVID web server  was usedWT and FUSP525L PAR-CLIP data produced from in vitro-derived human MNs [To gain insight into the intronic determinants regulating the biogenesis of the circRNAs whose expression is altered upon the FUS mutation, we focused on the 1 kb-long regions upstream and downstream the circRNAs tested for differential expression; among the deregulated circRNAs, we analyzed only those whose linear counterpart does not vary significantly in the same direction. To assess FUS binding in the intronic regions, we used the publicly available FUSuman MNs . Flankinuman MNs . To evalTo evaluate the presence of inverted Alu repeats in the circRNA intronic regions, we first extracted the coordinates of Alu elements from the UCSC genome browser  hg38 RepWT and FUSP525L samples [--outSAMtype BAM SortedByCoordinate --outFilterType BySJout --outFilterMultimapNmax 1 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --outFilterMismatchNmax 999 --outFilterMismatchNoverLmax 0.04; finally, the RNAEditingIndexer tool [t-tests were conducted to evaluate the Alu editing index difference between deregulated and non-deregulated circRNAs.To calculated the Alu editing index, preprocessed FASTQ files produced from Total RNA-Seq analysis of MN FUS samples  were giv samples  in order samples  with parxer tool  was emplxer tool  also use–very-sensitive option. For each circular RNA, we selected those read pairs that were aligned to the same extended subsequence, with at least one of them mapping across the original 3′ end. The final circular RNA sequence was then assigned by choosing the transcript subsequence with the highest number of mapping read pairs, retaining only the first 25 nucleotides from the extension. Such extended circular RNA sequences were used for miRNA-binding site prediction, performed using the miRanda tool [p-value cutoff equal to 0.01. The results of all these analyses were integrated to construct a circRNA–miRNA–mRNA network, in which miRNAs are connected to putative circRNA sponges, to known/predicted downregulated targets and to the enriched categories to which at least one of their targets belongs. Cytoscape version 3.9.1 [In order to infer the full sequences of circular RNAs for miRNA-binding prediction, we first intersected the coordinates of their back-splicing junctions with those of the exons belonging to transcripts expressed in MN samples  using BEnda tool  with defnda tool . circRNAnda tool  web resonda tool  and intenda tool  and miRDnda tool , which anda tool , an onlinda tool . miRNA-3nda tool  and/or anda tool  level. Tnda tool , providion 3.9.1  was usediPSCs, stably transfected with epB-NIL, an inducible expression vector containing the Ngn2, Isl1, and Lhx3 transgenes , were ma2. After 2 days of 1 μg/mL doxycycline (Thermo Fisher Scientific) induction in DMEM/F12 medium , supplemented with 1× Glutamax (Thermo Fisher Scientific), 1× NEAA (Thermo Fisher Scientific), and 0.5× Penicillin/Streptomycin (Sigma-Aldrich), the doxycycline induction is maintained for additional 3 days in Neurobasal/B27 medium , containing 5 μM DAPT and 4 μM SU5402 . At day 5, cells were dissociated with Accutase and plated on Matrigel-coated dishes at a density of 100,000 cells/cm2. Ten micrometers of rock inhibitor were added for the first 24 h after dissociation. Neuronal cultures were maintained in neuronal medium  for 7 days.To obtain spinal MNs, iPSCs were differentiated as previously described . BrieflyDownregulation of circDLC1 was achieved by siRNA transfection at day 5 of differentiation and cells were collected after 3 days. A mix of two siRNAs targeting the back splicing junction of circDLC1 and the control siRNA scramble see  were useNucleus/cytoplasm fractionation was performed on an enriched MN population (day 5 of the iPSCs-NIL differentiation). After treating the cells with accutase for 10 min at 37 °C, the reaction was stopped using DMEM-F12 and the cells were centrifuged at 1200 rpm for 5 min. The pellet was washed twice with DPBS, and the solution was centrifuged for 5 min at 1200 rpm. The supernatant was completely removed, and the cellular pellet was treated with the PARIS kit , according to the manufacturer’s instructions.−ΔΔCt, or as 2−ΔCt. DNA amplification was monitored with an ABI 7500 Fast qPCR instrument. Data analysis was performed using the SDS Applied Biosystem 7500 Fast Real-Time PCR system software. Oligonucleotides are listed in Total RNA was extracted using the Direct-zol Miniprep RNA Purification Kit  and reverse-transcribed using the SuperScript™ VILO™ cDNA Synthesis Kit , all according to the manufacturer’s instructions. RNaseR treatment was performed on 1 μg of total RNA extracted from MNs at day 12 of differentiation; 2U of RNaseR  was used and the reaction was carried out for 15 min at 37 °C; the RNA was then extracted and reverse-transcribed as described above. One picogram of a DNA spike-in molecule was added to each reaction for quantitative real-time PCR (qPCR) normalization. DNA spike-in was produced from the multiple cloning site in pcDNA3.1(-) . qPCR was performed by using PowerUp SYBR Green Master Mix (Thermo Fisher Scientific) following the manufacturer’s suggested protocol. Target gene expression was obtained by normalizing target quantity per housekeeping gene ATP5O and GAPDH quantity used as a control gene and the Relative RNA quantity was calculated as the fold change, 2


Bacteroides fragilis (ETBF) has emerged as a gut microbiome pathogen that can promote colitis associated cancer in humans. ETBF secretes the metalloprotease, B. fragilis toxin (BFT), which can induce ectodomain cleavage of E-cadherin and IL-8 secretion through the β-catenin, NF-κB, and MAPK pathways in intestinal epithelial cells. However, it is still unclear whether E-cadherin cleavage is required for BFT induced IL-8 secretion and the relative contribution of these signaling pathways to IL-8 secretion. Using siRNA knockdown and CRISPR knockout studies, we found that E-cadherin cleavage is required for BFT mediated IL-8 secretion. In addition, genetic ablation of β-catenin indicates that β-catenin is required for the BFT induced increase in transcriptional activity of NF-κB, p65 nuclear localization and early IL-8 secretion. These results suggest that BFT induced β-catenin signaling is upstream of NF-κB activation. However, despite β-catenin gene disruption, BFT still activated the MAPK pathway, suggesting that the BFT induced activation of the MAPK signaling pathway is independent from the E-cadherin/β-catenin/NF-κB pathway. These findings show that E-cadherin and β-catenin play a critical role in acute inflammation following ETBF infection through the inflammatory response to BFT in intestinal epithelial cells.Enterotoxigenic  Bacteroides fragilis (ETBF) is an obligate anaerobic bacterium that causes diarrheal disease in livestock and humans  . CTNNB1 gRNA-lentiCRISPR v2 vector was co-transfected with pMD2.G  and psPAX2  into HEK293T cells to produce lentiviruses containing CTNNB1 gRNA-lentiCRISPR v2. After transfection, virus containing media was harvested at 48 and 72 h after transfection and concentrated using Lenti-X™ concentrator  in accordance with the manufacturer’s instructions. Concentrated viruses were used to infect with cells (HT29/C1) in cultured media for 24 h, and then the virus containing media was removed. Infected cells were then selected with puromycin . All the experiments were conducted under the approval of the Institutional Biosafety Committee of Yonsei University MIRAE Campus (IBC No. 201908-P-004-01). For analyzing genomic DNA of β-catenin, CTNNB1-gRNA lentiCRISPR v2 containing HT29/C1 cells were harvested, and genomic DNA was isolated using phenol:chloroform:isoamyl alcohol (25:24:1). To confirm CTNNB1 from genomic DNA, we proceeded with nested PCR. The broad primer sequences were forward 5′–GTA AGA GTA TTA TTT TTC ATT GCC–3′ and reverse 5′–CTG TAA TAC AGG AAT TCA GAA AC–3′, and the nested primer sequences were forward 5′–GTT GTG GTG AAG AAA AGA GAG–3′ and reverse 5′–GGG TAG ACA TTC TGA AAC TAC TCC–3′. After CTNNB1 amplification was confirmed, genomic DNA sequences were determined by Cosmogentech .Three plasmids  were purchased from Addgene  and individually transformed into Cells were grown on sterilized glass coverslips and fixed with 4% paraformaldehyde solution  at 4 °C for 10 min, washed with PBS, and permeabilized with 0.2% Triton X-100 at RT for 20 min. Regarding immunostaining, cells were blocked with 1% bovine serum albumin in PBS for 30 min and stained with a 1 μg of primary antibody in 1% BSA-PBS solution overnight at 4 °C. Then, the cells were labeled with 1:1000 dilution of the appropriate fluorochrome labeled secondary antibodies ) in PBS at RT for 2 h. Finally, cells were washed three times with PBS and mounted with mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) . Images were captured with Carl Zeiss LSM710 confocal microscope .® 2000 . After 48 h of transfection, the cells were incubated with BFT supernatant, TNF-α , recombinant IL-1β  or lipopolysaccharide  for 24 h. Then, the reporter assay was conducted using the Dual-Luciferase® reporter assay system  and the luciferase activity was determined using a GloMax® 20/20 luminometer . Each luciferase activity was normalized with Renilla luciferase activity.Cells were seeded in 24-well plates and incubated for 24 h before transfection. Reporter constructs (NF-κB luciferase and Renilla luciferase plasmid)  were transfected using LipofectamineCell supernatants were collected and stored at −80 °C until analysis. Cytokine levels of IL-8 were determined using a commercialized ELISA kit  following the manufacturer’s instructions.t-test and p < 0.05 was considered to be statistically significant .Data in the bar graphs are presented as mean ± SEM. All statistical analyses were performed using GraphPad Prism 7.0 software . All data were analyzed using an unpaired p < 0.005)  C. The la< 0.005) C. These CTNNB1) in HT29/C1 cells using the CRISPR-Cas9 gene-editing system. Two different guide RNAs for CTNNB1 knockout were designed and cloned into a lentiviral vector. These CTNNB1 knockout lentiviral vectors were packaged and named as lenti-CTNNB1 KO#1 (KO#1) and lenti-CTNNB1 KO#2 (KO#2). To confirm that the CTNNB1 was disrupted, CTNNB1 was examined by nested PCR and sequencing analysis in two gene edited independent cell clones to confirm that the CTNNB1 gene was disrupted. In KO#1 and KO#2, 13 and 4 base pairs were deleted, respectively (CTNNB1 affected cellular proliferation. Cellular proliferation of β-catenin knockout HT29/C1 (KO#1 and KO#2) cells was lower than that of wild type HT29/C1 cells   with rET and rNT for 6 h, and IL-8 secretion was analyzed by ELISA. In contrast to WT HT29/C1 cells, IL-8 secretion was ablated in β-catenin knockout HT29/C1 cells treated with rET ( 0.0001) A. In a p 0.0001) B. Taken p < 0.05)  A. This r < 0.05) B. To con < 0.05) C. These p < 0.0001) and in β-catenin knockout cells (p < 0.0001)  C, althou 0.0001) C. Taken Colitis associated cancer is a type of colorectal cancer that is preceded by inflammation and can be observed in inflammatory bowel disease (IBD) patients . In coloB. fragilis (NTBF) of normal flora and acts as a tumor initiator by inducing colitis and DNA damage [ETBF has emerged as a human enteric pathogen prominently contributing to IBD and colon tumorigenesis . Recent A damage ,38,39. BA damage ,41. TherA damage . In addiA damage . Since BFour key observations were made in the current study. First, BFT induced IL-8 secretion was dependent on the presence of E-cadherin. Second, BFT induced IL-8 secretion was dependent on the presence of β-catenin. Third, activation of the NF-κB pathway is dependent on and occurs downstream of β-catenin. Fourth, MAPK activation is partially dependent on the presence of β-catenin. It has been previously reported that the β-catenin/NF-κB/MAPK signaling pathways are activated in response to BFT treatment . It was E-cadherin plays an important role in physiologic cellular processes and cancer progression. Loss of E-cadherin is a well known feature of developmental epithelial–mesenchymal transition (EMT), and its loss in cancer plays a crucial role in the transition to malignant cancer . In addiTo elucidate the associations within BFT induced cellular signaling, the CRISPR-Cas9 gene editing system has the advantage of generating permanent, inherited knockout cell lines . In thisActivation of the NF-κB pathway was ablated in β-catenin knockout cells. However, activation of MAPK signaling was unaffected. Regarding IL-8 secretion, early IL-8 secretion (6 h) was diminished to control levels in β-catenin deficient cells. However, late IL-8 secretion (24 h) was greatly decreased but detectable in β-catenin deficient cells. Considering that the MAPK signaling pathway was activated in β-catenin deficient cells, it is possible that early IL-8 secretion is due to β-catenin activation, whereas late IL-8 secretion can be attributed to MAPK activation. Regardless, it is clear that the β-catenin pathway contributes overwhelmingly to BFT induced IL-8 secretion. Our data suggest that that two independent signaling pathways, namely, the β-catenin/NF-κB and MAPK pathways, are independently activated by BFT.Our current study does have limitations. First, we examined the BFT mediated E-cadherin/β-catenin/NF-κB/IL-8 secretion signal cascade in a single colonic epithelial cell line, HT29/C1. Second, we did not elucidate the mechanisms of MAPK signaling induced by BFT. The independent activation of the β-catenin/NF-κB pathway and the MAPK pathway may be due to the presence of two different cognate receptors for BFT. Sears and colleagues proposed that the putative candidate receptor of BFT is the G protein coupled receptor 35 (GPR35). GPR35 is highly expressed in BFT responsive cells and the knockdown of GPR35 in HT29/C1 resulted in β-arrestin dependent loss of BFT induced effects . ConsideIn general, MAPK activation is associated with IL-8 secretion through the transcription factor AP-1 . ConsistIn the current study, we did not examine whether BFT induces late IL-8 secretion via AP-1 activation in β-catenin knockout cells. In addition, lithium chloride (LiCl) treatment would be helpful to understand the exact molecular pathways involved in BFT induced inflammatory signals, such as β-catenin, NF-κB and MAPK signaling. Similar to BFT, LiCl is known to activate the β-catenin, NF-κB, and MAPK pathways, resulting in IL-8 secretion ,60, so dIn summary, the results from this study demonstrate that β-catenin is required for BFT induced early IL-8 secretion and the subsequent nuclear translocation of NF-κB p65 in HT29/C1 cells. However, MAPK activation is independent of BFT induced β-catenin signaling. The results from this study outline several associations between BFT induced cellular mechanisms and help uncover the effects of ETBF on colon epithelial cells and colorectal cancer.