Background: Immune-modulating therapies impart positive outcomes in a subpopulation of cancer patients. Improved delivery strategies and non-invasive monitoring of anti-tumor effects can help enhance those outcomes and understand the mechanisms associated with the generation of anti-tumor immune responses following immunotherapy.Methods: We report on the design of a microneedle (MN) platform capable of simultaneous delivery of immune activators and collection of interstitial skin fluid (ISF) to monitor therapeutic responses. While either approach has shown promise, the integration of the therapy and diagnostic arms into one MN platform has hardly been explored before. MNs were synthesized out of crosslinked hyaluronic acid (HA) and loaded with a model immunomodulatory nanoparticle-containing drug, CpG oligodinucleotides (TLR9 agonist), for cancer therapy in melanoma and colon cancer models. The therapeutic response was monitored by longitudinal analysis of entrapped immune cells in the MNs following patch retrieval and digestion.Results: Transdermal delivery of CpG-containing NPs with MNs induced anti-tumor immune responses in multiple syngeneic mouse cancer models. CpG-loaded MNs stimulated innate immune cells and reduced tumor growth. Intravital microscopy showed deposition and spatiotemporal co-localization of CpG-NPs within the tumor microenvironment when delivered with MNs. Analysis of MN-sampled ISF revealed similar immune signatures to those seen in the bulk tumor homogenate, such as increased populations of macrophages and effector T cells following treatment.Conclusions: Our hydrogel-based MNs enable effective transdermal drug delivery into immune cells in the tumor microenvironment, and upon retrieval, enable studying the immune response to the therapy over time. This platform has the theranostic potential to deliver a range of combination therapies while detecting biomarkers. Microneedles (MNs) have emerged as promising platforms in the drug delivery field, enabling self-administration, needle-free penetration, and minimally invasive access to a complex immune milieu unique to the skin Despite their promise for therapeutic and diagnostic purposes, MNs encompassing both abilities within the same platform —as a theranostic tool— have rarely been explored. Combining both compartments requires designs that allow for high drug loading and maximal sampling volume of biomarkers. MNs have been recently designed to detect soluble biomarkers such as nucleic acids in vivo delivery of CpG continues to be challenging due to high hydrophilicity and rapid degradation Unmethylated cytosine-phosphate-guanine (CpG) oligonucleotides (ODNs) are well-described innate immune effectors capable of instigating potent anti-tumor responses by selectively engaging toll-like receptors (TLRs) expressed by innate immune cells Figure ).In this study, we aimed to leverage the theragnostic potential of our Hyaluronic Acid-based MN platform for anti-cancer purposes by (1) delivering nano-encapsulated drugs in the vicinity of tumors to suppress tumor growth while simultaneously (2) sampling ISF from the TME to monitor the response to the therapy . To form the MNs, the modified HA polymer containing primary amines was crosslinked with 8-Arm-NHS-polyethylene glycol (PEG) containing a succinimidyl functional group, allowing for spontaneous hydrogel formation . Once the MN hydrogel backbone was formed, the nano-encapsulated drugs were incorporated by centrifugation, and the MN patch was finalized by adding a poly lactic-co-glycolic acid (PLGA) back layer. The resulting MNs could be digested on-demand in less than 5 min when incubated with a 10 mM TCEP solution  phosphine (TCEP)  as a model delivery vehicle to protect the ODNs and enhance their cellular internalization. To establish the C6-CR3-to-CpG ratio required for the complete complexation of the nucleic acids, we used a gel retardation assay . Here, the presence of cationic groups in the C6-CR3 polymer allowed electrostatic binding to the negatively charged phosphate groups of the CpG-ODNs. The gel retardation assay revealed that CpG-ODNs migration was impeded at weight ratios of 50:1 C6-CR3:CpG. Therefore, we used this formulation throughout the rest of the study. Analysis by dynamic light scattering (DLS) confirmed the encapsulation of the CpG into monodisperse particles (PDI = 0.12 ± 0.02) with a hydrodynamic diameter of 63 ± 9 nm.  that remained relatively unchanged for more than a week at room temperature . Analysis of the particles via transmission electron microscopy (TEM) confirmed their sphere-like morphology and size as per DLS measurements . CpG-NPs were positively charged  attributed to the high density of protonated groups in the polypeptides .TLR9 agonists, including CpG ODN 1826, were used as a model agent to prove the therapeutic merit of the MN platform as a delivery device and to monitor the TME response to the therapy. TLR9 agonists have been shown to inhibit tumor growth in pre-clinical studies with well-described mechanisms of action Figure ). Cells were incubated with varying concentrations of fluorescent CpG-NPs (from 0.0001 nM to 1000 nM) for 24 h in a complete growth medium, confirming over 90% of NP internalization at 10 nM and half-maximal effective concentration (EC50) of 0.78 nM. We also studied the ability of CpG-NPs to stimulate the NF-κB response, being the central signaling pathway for the induction of pro-inflammatory responses Figure ). The NF-κB response was explicitly triggered by the CpG-NPs, as negative control NPs  formulated with the same polymer —the tri-arginine-modified PBAE polymer (C6-CR3)— and with comparable features in terms of size and surface charge (Table S1), yielded negligible activation levels. In addition, we confirmed that the CpG-NPs remained biologically active once released from the MNs . Finally, cytotoxicity of CpG-NPs was studied by MTS cell proliferation assay using HEK293 TLR9 reporter cells. CpG-NP cytotoxicity  was only observed at ~20-fold higher concentration (>150nM) than that required to stimulate TLR9-dependent NF-kB transcriptional activity (less than 6 nM) .We next investigated the ability of CpG-NPs to be internalized by murine TLR9-reporter HEK 293 cells . Quantification of the mechanical strength of the MNs by compression testing did not reveal significant differences between empty MN patches and those loaded with NPs , supporting our previous findings that the inclusion of therapeutics such as chemokines or cytokines in the MN matrix did not have an impact on its physical properties ). Lastly, we confirmed the successful release of CpG-NPs from the MNs. We assessed the release kinetics of fluorescently labeled CpG-NPs when incubated under physiologically relevant conditions  in vitro . We confirmed that 70% of the CpG-NPs were released within the first 24 h of incubation and that a plateau was achieved by 72 h. Release studies in vivo showed that half of the NP cargo was delivered from the MNs within 24 h (Table S2). MNs retrieved from mice were digested, and the fluorescence associated with the remaining CpG-NPs inside the MN matrix was compared to the baseline levels recorded from MNs prior to implantation. In light of these findings, a total of 2 µg CpG per MN patch was loaded in subsequent in vivo studies to reach a therapeutic target of 1 µg CpG per mouse following 24 h-MN administration.We next examined the properties of the theranostic MN platform and its behavior when integrating the NPs into the hydrogel matrix, forming the MNs for delivery into the dermal milieu surrounding the tumors. Here, we proposed the use of MNs with a length of 600 µm and a base width of 300 µm which others have demonstrated to allow for transdermal drug delivery in superficial skin cancers + MHCII+ and BMDMs phenotype was confirmed as CD11b+ F4/80+ by flow cytometry. We first studied the ability of BMDMs and BMDCs to efficiently internalize CpG-NPs, and found EC50 values of 2.6 nM and 22.5 nM, respectively . To test the downstream cellular response to CpG-NPs, BMDCs were treated for 24 h with CpG-NPs or CpCCtrl-NPs, and the expression of cell activation markers was determined by flow cytometry. A 10% increase of cells expressing CD86+MHCIIhi  was observed in BMDCs treated with CpG-NPs compared to CpCCtrl-NPs. In addition, no significant CD86+MHCIIhi population increase was observed when comparing the levels of activation in CpCCtrl-NP-treated BMDCs and the untreated ones, corroborating that TLR9-specific activation only occurred in the presence of CpG . Similar results were observed in BMDMs since an increase in the ratio of pro-inflammatory to anti-inflammatory macrophages was detected when BMDMs were treated with 10 nM of CpG-NPs compared to untreated or CpCCtrl-NP controls (pro-inflammatory M1-like BMDMs were defined as CD86+ cells and anti-inflammatory M2-like as CD206+ cells) . These results confirmed that CpG-NPs stimulate the maturation and activation of BMDCs and BMDMs. Following TLR engagement, activated BMDMs are known to produce a wide range of cytokines and chemokines to promote anti-tumor immunity Figure ). The immunosuppressive IL-10 cytokine was also upregulated following TLR stimulation, in agreement with the literature We next studied the capability of CpG-NPs to activate murine bone marrow-derived dendritic cells (BMDCs) and bone marrow-derived macrophages (BMDMs). BMDCs and BMDMs were generated following published guidelines in vivo studies in mice to validate the theranostic ability of the MN platform. We first used an orthotopic murine model of melanoma (B16-F10) as a clinically relevant superficial tumor offering easy access via the transdermal route. We initially studied the therapeutic effect of the MN platform by examining the ability of the CpG-NPs to instigate antitumor immunity when delivered with the MNs . Five days-post tumor inoculation, B16-F10 tumor-bearing mice were administered with MNs loaded with CpG-NPs (CpG-NP MNs), MNs loaded with CpCCtrl-NPs (CpCCtrl-NP MNs) or empty MNs. The therapeutic regimen consisted of treatment every three days for five cycles (q3dx5). Treatment with CpG-NPs MNs resulted in a significant delay in tumor growth  and a corresponding increase in the survival time compared to the control groups (CpCCtrl-NP MNs and empty MNs) . The therapeutic benefit of CpG-NP MNs, when delivered transdermally, was evidenced by the 3-fold lower tumor volume at the end of the study [≈ 800 mm3 in the control groups  versus ≈ 200 mm3 in the treatment group ]. In addition, we observed that 1 μg of CpG-NPs per mouse was well tolerated as no body weight loss was observed during the treatment period . In agreement with previous reports Following the characterization of the MN platform, we pursued Figure ), which revealed a relatively homogeneous distribution of CpG-NPs from the patch into the neighboring tumor tissue and around the MN puncture sites . Single-cell analysis of CpG-NP uptake indicated accumulation in H2B-mApple+ tumor cells and non-malignant phagocytic host cells consistent with known morphology and localization of tumor-associated macrophages in this model Figure ).After confirming the potential of the MNs in a mouse model of melanoma, we studied the spatiotemporal distribution of CpG-NPs in the TME when delivered using MNs by intravital microscopy (IVM). To do so, we used the syngeneic MC38 cancer model with growth characteristics and fluorescent protein expression ideal for intravital microscopy. MC38 tumor cells expressing H2B-mApple nuclear-localized fluorescent protein were implanted into the dorsal skinfold window chambers (DSWC) in C57BL/6J mice Figure ) and extended overall humane survival . IVM also revealed a robust tumor response using the DSWC, where the loss of fluorescent signal reflected a loss of viable mApple+ tumor cells over time after a single CpG-NP MNs application . Finally, NP tumoral distribution and release kinetics from the patch was investigated using IVM, showing the release of NPs into the tumor tissue over the 24 h application duration and extended NP retention within the tumor tissue — as seen at 48 h, when MN patches were removed for analysis . In contrast, NPs injected intratumorally presented a more localized distribution , and by 24 h post-injection, 95% of the NPs had cleared from the tumor tissue .We next studied the therapeutic efficacy of transdermal delivery of CpG NPs using the subcutaneous MC38 colon cancer model. Tumors responded similarly to orthotopic B16-F10 tumors: treatment with CpG-NP MNs inhibited tumor growth . In addition, we characterized the composition and the phenotype of the immune cell populations in the TME and tumor-draining lymph nodes (tdLNs) in MC38 tumor-bearing mice two days after a single administration of CpG-NP MNs, with a particular focus on early immune responders. Expression of CD80 by DCs (CD11c+ MHCII+) in the TME and tdLNs was significantly increased in the animals treated with CpG-NP loaded MNs . A similar trend was observed for the macrophages (CD11b+ F4/80+), where an increase in CD86 expression was observed in treated mice compared to the control group receiving empty MNs . These results support that CpG TLR-agonists are potent stimulators of DC and macrophage maturation and drive the expression of co-stimulatory receptors, including CD80 or CD86 +NK+) cells were observed in tumors administered CpG-NP MNs, in agreement with prior literature showing that TLR-agonists such as CpG motifs can induce NK cell lytic activity to support anti-tumor immunity Figure ). We also observed higher frequencies of CD4+ T cell infiltrates in the treated group compared to the control group . Lastly, we examined how transdermal delivery of CpG-NPs affected downstream expression of proinflammatory cytokines and chemokines known to mediate innate and adaptive immune cell activation. Analysis of the tumor lysates confirmed that CpG-NP MNs increased the expression of interferon gamma (IFN-γ) in the TME compared to control mice receiving empty MNs. In addition, tumors treated with CpG-NP MNs showed increased expression of leukocyte chemokines, including the monocyte chemoattractant protein 1 (MCP-1) and the C-X-C motif chemokine ligand 1 (CXCL1), and pro-inflammatory cytokines such as interleukin 1 beta (IL-1β) responsible for immune cell recruitment Figure ). Overall, these results demonstrate the immunomodulatory potential of the MN platform, leading to a pro-inflammatory TME phenotype.To understand the mechanisms by which the transdermal delivery of CpG-NPs resulted in enhanced antitumor activity, we performed immunohistochemical analysis of MC38 tumors following therapy . In agreement with the macroscopic findings, immunohistochemical staining for H&E and Ki-67 showed less actively proliferating cells in tumors treated with CpG-NP MNs than in the empty MNs control group . The entire MN matrix was composed of highly swellable and digestible HA hydrogel that allowed us to capture and analyze roughly 1,000 CD45+ cells per patch in a theoretical volume of 3 mm3 . Next, we were able to further gate this subset cellular population by flow cytometry to examine the cellular frequencies of the main contributors to anti-tumor immunity, such as macrophages, and compare them with those found in tumor lysates . Flow cytometric quantification of the cellular suspensions recovered from MNs on day two post-treatment confirmed an increased percentage of macrophages (CD11b+ F4/80+ CD45+) in the MN-recovered ISF from treated mice compared to that of the control group . The same trend was observed in the corresponding tumors, which registered increased frequencies of macrophages in mice administered with CpG-NPs , supporting a correlation between cells extracted from MN patches and the bulk TME in response to the immunotherapy.We next investigated the diagnostic capacity of the MN platform and its ability to extract ISF for minimally-invasive monitoring of the TME. We aimed to utilize the MN platform to longitudinally monitor the immune changes in the TME profile in ISF, which we hypothesized would shift from immunosuppressive to pro-inflammatory following TLR engagement. Following CpG-NPs therapy administration, empty MNs were applied for 24 h and digested to analyze proximal responders of TLR stimulation . Mainly, we focused on the population of tumor-infiltrating lymphocytes, using the CD8+/CD4+ T cell ratio as a prognostic biomarker of responsiveness to immunotherapy + T cell population in treated mice compared to the control group, which was evident in all the samples analyzed over time . Also, an increase in the CD8+/CD4+ ratio following immunotherapy was observed, although not statistically significant, compared to untreated mice (pooled frequencies from day 11 and day 14 post-treatment initiation) . Here, the correlation between the T cell signature in the TME and the response to therapy could only be established qualitatively due to the low frequencies of effector T cells inherent to the MC38 model In a second study, we focused on monitoring the adaptive immune responses that may evolve more gradually, particularly examining effector and helper T cells. Here, CpG-NP MNs were serially administered following the same therapeutic regimen as for the efficacy studies (q3dx5). Cellular changes in the MN-sampled ISF were analyzed by flow cytometry , enabling their localization at the tumor site, as observed by IVM. Following MN administration, the tumor burden was reduced when treating an orthotopic melanoma cancer model and a subcutaneous colorectal cancer model. Mechanistic studies confirmed the activation of DCs and macrophages in response to TLR engagement and the remodeling of the TME towards an immune-stimulatory state, which was evidenced by the increased levels of pro-inflammatory cytokines and chemokines in tumor lysates. MNs facilitated longitudinal monitoring of the changes in the TME, focusing on the innate arm of the immune system at early time points followed by monitoring of the adaptive responses. We reported an increase in macrophages in MN-sampled ISF after treatment that correlated with the immune profile in tumors, as well as an average increase in the infiltrates of effector and helper T cells. We foresee that integrating therapeutic and diagnostic functionalities of hydrogel-based MNs in a single, theranostic patch will open up prospective clinical avenues for enhanced patient management, offering minimally invasive intervention and allowing for multiplex analysis of cellular biomarkers involved in anti-tumor immunity.All reagents and solvents were purchased from Sigma Aldrich unless otherwise stated. Sodium hyaluronate (60 kDa) was obtained from LifeCore Medical with a purity of at least 95%. NHS-terminated 8-arm PEG was purchased from Creative PEG Works. Microneedle PDMS custom-made molds  were obtained from Blueacre Technology. Arginine peptide (H-Cys-Arg-Arg-Arg-NH2) was obtained from CPC Scientific with a purity of at least 90%. ODN 1826 (CpG) and control ODN 1826 were purchased from InvivoGen.Polymers were synthesized in accordance with previous work To assess CpG complexation, different CpG to C6-CR3 ratios (w/w) (between 1:1 and 400:1) were studied. CpG-NP were freshly prepared and loaded in 4% E-Gel Precast Agarose Gels (Thermo Fisher), run following the manufacturer's instructions, and visualized in fluorescence mode.NPs were generated by mixing equal volumes of CpG-ODN 1826 at 0.4 mg/mL and C6-CR3 polymer at 20 mg/mL in sodium acetate buffer (AcONa) at 12.5 mM, followed by 5 min incubation at room temperature (RT). Next, this mixture was nano precipitated with 2.5 volumes of PBS and incubated for 20 min at RT to form NPs. The final CpG-NPs were concentrated by centrifugal filtration (10kDa MWCO) and sterilized by filtration (0.22 μm). Similarly, CpCCtrl-NPs were formulated following the same procedure but using a CpC-ODN 1826 control.Dynamic light scattering (DLS) was performed to determine the size and surface charge of the NPs. Briefly, 100 μL of CpG-NP or CpCCtrl-NPs were diluted with 900 μL of PBS and analyzed using a Zetasizer Nano ZS equipped with a He-Ne laser (λ = 633 nm) at a scattering angle of 137° . NP size was also analyzed by transmission electron microscopy (TEM) by the microscopy core at the Koch Institute for Integrative Cancer Research.2 polymer was synthesized following a previously described procedure 2 was purified by dialysis, freeze-dried, and stored at -20° C. HA-SS-NH2 molecular structure was characterized by 1H-NMR using D2O as a solvent (400 MHz Varian NMR spectrometer).The HA-SS-NHMNs were fabricated as described in our previous work Mus musculus skin melanoma (B16-F10 from ATCC) and colon carcinoma  FBS, 1% (v/v) P/S, 5% (v/v) GlutaMAX and recombinant murine macrophage-colony-stimulating factor . The flasks were supplemented with the additional medium on day three and day six. BMDMs phenotype was confirmed as CD11b+ F4/80+ by flow cytometry. To generate BMDCs, 2x106 bone marrow cells were added to non-tissue culture-treated Petri dishes and cultured in 10 mL of RPMI-1640 supplemented with 10% (v/v) FBS, 1% (v/v) P/S and recombinant murine granulocyte-macrophage colony-stimulating factor . Plates were replenished with fresh, supplemented media on day three, six and every other day after that until use. Cell harvesting was performed as follows: BMDMs were detached using AccumaxTM (Sigma), and BMDCs were loosely adherent and could be collected by simple washing. BMDCs phenotype was confirmed as CD11c+ MHCII+ by flow cytometry.The tibias and femurs of female C57BL/6 mice (aged 8-12 weeks) were isolated and flushed to harvest bone marrow and obtain a progenitor cell population. To generate BMDMs, 4-6×105 cells per well and incubated with the CpG-NP, CpCCtrl-NP, free CpG, or free CpCCtrl at concentrations ranging from 0 nM to 6 nM. At 24 h post-treatment, NF-κB activity was determined using the HEK-Blue DetectionTM reagent (InvivoGen) according to the manufacturer's instructions.HEK-Blue mTLR9 cells were seeded in 96-well plates at 2 × 104 cells per well. Cells were treated with different concentrations of CpG-NPs and CpCCtrl-NP for 24h, and cell viability was assessed using the MTS assay (Promega) as instructed by the manufacturer. Cells were incubated for up to 2 h, and absorbance was measured at 490 nm using a plate reader.HEK-Blue mTLR9 cells were seeded in 96-well plates at 2 × 105 cells per well and incubated with fluorescently labeled CpG-NP at CpG concentrations ranging from 0.0001 to 100 nM. After 4 h, CpG-NPs were removed, cells were washed, trypsinized, and fixed with 4% (v/v) paraformaldehyde for 10 min. CpG-NPs internalization was determined by flow cytometry.HEK-Blue mTLR9 cells, BMDCs, BMDMs were seeded in 24-well plates at 1 × 105 cells per well and incubated with CpG-NP or CpCCtrl-NP at 10nM. After 24 h, the supernatant was removed, and cells were then collected and analyzed by flow cytometry. The following antibodies were used: CD11c BV421 (clone N418), MHCII BV605 (clone M5/114.15.2), CD80 FITC (clone 16-10A1), CD86 PE (clone GL-1), F4/80 BUV395 (clone T45-2342), CD11b BV421 (clone M1/70), CD86 FITC(clone GL-1), CD80 APC (clone 16-10A1), CD206 PE (clone C068C2). Live cells were gated using LIVE/DEADTM (Thermo Fisher) near-IR (cat. no. L34976). In addition, CCL2 (MCP-1), CXCL1 (KC), CXCL10 (IP-10)), TNF-α and IL10 were analyzed from the BMDM supernatant using a custom LegendplexTM panel (BioLegend) as per the manufacturer's instructions.BMDCs or BMDMs were seeded in 24-well plates at 1 × 10in vitro by incubating the MNs in PBS for 24 h and recording the weights upon recovery. Swelling percentage was obtained using the following equation where: W(i.): MN weight following incubation and AW0: Average of MNs weights prior incubation.Mechanical strength of the MNs when empty or NP-loaded were measured using a micro-force test station with a mechanical sensor . MN patches were placed on the surface of the platform with the needle-like projections facing up. The displacement and force applied to the MNs were recorded from the moment the sensors touched the uppermost tip of the MNs until a maximum force of ≈100 N was reached. Force-travel curves of MNs arrays were obtained by correlating the compressive strain or displacement (%) with the compressive stress (kPa), and Young's modulus was determined from the slope (GPa). Swelling ability of the MNs was evaluated ex = 640 nm, λem = 680 nm).To assess the release of CpG-NPs from the HA MNs, CpG-NPs were conjugated with Alexa Fluor 647 before the microneedle fabrication. CpG-NP-loaded MNs were placed in Eppendorf tubes, immersed with PBS (1 mL), and incubated under rotation at 37 °C. 100 µL of releasing media was replaced at a predetermined time point, and fluorescence of the release NPs was measured by microplate reader [Infinite M Plex multimodal plate reader (TECAN)] (λin vitro studies or retrieved from mice were incubated with 10 mM Tris (2-carboxyethyl) phosphine (TCEP) solution in PBS at pH 7.4 with rotation at 37 °C for 5 min. The recovered cellular suspension was filtered with a 70 µm cell strainer (BD Biosciences) to remove any impurities and further processed by flow cytometry. Digestion of the MNs following incubation was confirmed by fluorescence microscopy using fluorescently-labeled MNs. To confirm on-demand digestibility, fluorescently-labeled MNs fabricated with AF647-conjugated HA were incubated with either the TCEP solution or PBS as control. Next, the supernatants were collected, and the levels of fluorescence were analyzed in a microplate reader .HA-based MNs from ex = 640 nm, λem = 680 nm). Finally, fluorescence measurements were compared to the theoretical ones and the percentage of total loading capacity was obtained.First, MNs were loaded with a solution of fluorescently-labeled NPs on top of the mold as described here. After drying and demolding, the resulting MNs were digested by incubating them in digestion buffer  and the fluorescence of the digested MNs was quantified in a microplate reader . Release profile was obtained by subtracting the fluorescence levels from those registered from control MNs that had not been applied.HA-MNs were loaded with CpG-NPs labeled with Alexa Fluor 647 (2 µg of CpG/MN patch) and subsequently applied on the back of healthy C57BL/6 mice (2 MN patches/mice) for increasing durations of time . Next, MNs were recovered and digested as described herein. Fluorescence of the resulting MN suspensions (associated with the remaining NPs still in the MN matrix) was analyzed in a microplate reader  were purchased from Charles River. Mouse procedures were conducted at the Koch Institute for Integrative Cancer Research at the Massachusetts Institute of Technology (MIT) under the protocol approved for this study by the Institutional Animal Care and Use Committee (IACUC). Intravital microscopy studies were performed at the Massachusetts General Hospital under a protocol approved for this study by the Institutional Animal Care and Use Committee (IACUC), using female C57BL/6J mice (8-12 weeks old) provided by Jackson Labs.6 cells in 100 μL of HBSS were injected subcutaneously into the right flank of the mice. For orthotopic B16-F10 cells, 5×105 cells in 50 μL of HBSS were injected intradermally into the right flank of the mice. Five days post-tumor induction, mice were administered empty MNs (negative control), CpCCtrl-NP-loaded MNs (negative control) or CpG-NP-loaded MNs (treatment group) 5 times, three days apart (n = 10 mice per group). MNs were administered on top of the tumor by thumb-pressing them against the skin or in the surrounding areas and secured with medical-grade tape . MNs were retrieved 24 h post-administration. Tumor size was measured every other day via caliper measurements (n = 6), and the tumor volume was calculated using the equation V = (Length × Width × Height)/π ÷ 6. Body weight was measured contemporaneously with tumor volume. Mice were euthanized when tumors reached a volume of 1000 mm3 or when poor body condition was observed.Two different tumor models were used. For mApple-expressing MC38 cells, 1×10Subcutaneous MC38 tumors were established in female C57BL/6 mice (6-8 weeks old) as previously described and two studies were conducted. Mechanistic analyses were performed after mice were administered CpG-NP loaded MNs (1 µg delivered per patch) or empty MNs either once or five times  depending on the cellular populations of interest. To test the diagnostic potential of the MNs, a single dose of CpG-NPs (1 µg in 25 µL of PBS) was administered intratumorally. On the day of mechanistic analysis, tumors were harvested, chopped into < 0.5 mm fragments, and digested in a solution of HBSS supplemented with collagenase I, II, and IV (100 ng/mL), and DNase I (100 μg/mL) for 2 h at 37 °C. tdLNs were harvested and mechanically dissociated. Single-cell suspensions of tumors and tdLNs were filtered through a 40 μm nylon cell strainer. Tumor cells were further treated with ACK Lysing Buffer (Gibco) for 1 min. Cells were washed, filtered through a 40 μm nylon cell strainer, and counted. MNs recovered from mice were digested as described before. The following anti-mouse antibodies were used for flow cytometry were purchased from BioLegend: CD45 APC-Cy7 (clone 30-F11), NK-1.1 BV711 (clone PK136), CD45 BV785 (clone 30-F11), CD11b BV421 (clone M1/70), CD86 BV510 (clone GL-1), CD80 BV711 (clone 16-10A1), CD206 PE (clone C068C2), MHCII BV605 (clone M5/114.15.2), CD11c APC (clone N418). The following anti-mouse antibodies were purchased from BD Biosciences: CD3 BB700 (clone 17A2), CD4 BUV395 (clone GK1.5), CD8a BUV737 (clone 53-6.7), F4/80 BUV395 (clone T45-2342), CD80 BUV737 (clone 16-10A1). Live cells were gated using LIVE/DEADTM (Thermo Fisher) aqua (cat. no. L34966), green (cat. no. L34970), or near-IR (cat. no. L34976). Stained cells were analyzed by flow cytometry using a BD LSRFortessaTM flow cytometer (BD Biosciences), and all data were analyzed using FlowJo software (Flowjo LLC). Samples yielding low cell counts due to experimental constraints were removed prior to the analysis.Half of the harvested tumors for immunophenotyping on day two were processed to analyze the cytokine/chemokines expression profile. Tissues were homogenized with T-PER™ Tissue Protein Extraction Reagent  containing 1% Halt protease and phosphatase inhibitors . Then, the samples were incubated at 4 °C for 30 min following centrifugation to remove the cell/tissue debris. The supernatant was collected for total protein quantification and cytokine analysis. Levels of IFNγ, CCL2 (MCP-1), CXCL1 (KC), and IL-1β were analyzed using a custom Legendplex™ panel (BioLegend) as per the manufacturer's instructions. Samples rendering bulk protein concentrations below the limit of detection by micro-BCA assay were not considered for analysis.3 tumor sections were harvested on the day of mechanistic analysis and were embedded in O.C.T. in plastic base molds for tissue embedding. Samples were flash-frozen and preserved at -80 °C until sectioning. Tumors were cryosectioned into 20 μm-wide tissue sections, and proliferation was assessed via H&E and Ki-67 staining. The microscopic images were processed using the Aperio ImageScope 12.3.3 software (Leica).Skin tissue sections were processed and imaged by the Hope Babette Tang Histology facility at the Koch Institute of Integrative Cancer Research at MIT . Briefly, 1 cmIn vivo microscopy was performed using an Olympus FV1000 multiphoton/confocal imaging system following procedures described previously 5 MC38-H2B-mApple cells in 50 μL PBS were injected under the fascia two days after DSWC implantation surgery and imaged ~7 days later upon tumor formation. MN patches were applied onto the tissue within the window chamber, and glass coverslips were replaced over the patch to protect the tissue. 24 h later, patches were removed, and tissue was imaged after adding saline and replacing the coverslip. An XLFluor X2 air objective  was used for low-magnification imaging of the entire tumor and MN delivery region. In contrast, higher-magnification imaging was conducted with an XLUMPLFLN ×20 water immersion objective . Sequentially scanned images were acquired with 559- and 633-nm diode lasers and a DM405/488/559/635-nm dichroic beam splitter in vitro experiments, a minimum of n = 3 biological replicates were used per condition in each experiment. The unpaired Student's t-test evaluated the statistical difference between the two measurements. A Post hoc test for one-way ANOVA was used to test the statistical difference between sets of measurements. For in vivo experiments, a minimum of n=4 biological replicates were used per condition in each experiment. Survival was estimated using Kaplan-Meier methodology and comparisons were made using the log-rank test. No specific pre-processing of data was performed before statistical analyses. Data were analyzed by Grubbs' test for statistical outliers, which were pre-defined using an alpha value of 0.01. Differences between groups were considered significant at p-values below 0.05 .Statistical analyses were performed using Graph-Pad Prism 8 (GraphPad Software). For


Bananas are among the world’s main economic crops and one of the world’s most-selling fresh fruits. However, a great deal of waste and by-products is produced during banana harvesting and consumption, including stems, leaves, inflorescences, and peels. Some of them have the potential to be used to develop new foods. Furthermore, studies have found that banana by-products contain many bioactive substances that have antibacterial, anti-inflammatory, and antioxidant properties and other functions. At present, research on banana by-products has mainly focused on various utilizations of banana stems and leaves, as well as the extraction of active ingredients from banana peels and inflorescences to develop high-value functional products. Based on the current research on the utilization of banana by-products, this paper summarized the composition information, functions, and comprehensive utilization of banana by-products. Moreover, the problems and future development in the utilization of by-products are reviewed. This review is of great value in expanding the potential applications of banana stems, leaves, inflorescences, and peels, which will not only help to reduce waste of agricultural by-product resources and ecological pollution but will also be useful for the development of essential products as alternative sources of healthy food in the future. Bananas are classified as part of ‘Magnoliophyta’, class ‘Liliopsida’, order ‘Zingiberales’, family ‘Musaceae’, and genus ‘Musa’  and are Recently, there has been a change in how banana by-products are managed, with a focus on transforming them into value-added goods. The diverse bioactive chemicals with potential health-promoting benefits have reignited interest in banana by-products. The use of banana by-products for value-added purposes to meet demand in areas such as food alternatives, feed, renewable energy, textiles, and fiber composites is a constant challenge .Banana stems and leaves can be used as resources for, e.g., fertilization, fodder, energy, and fiber, including food applications such as coloring, flavoring, livestock feed, and non-food applications such as wastewater treatment, biofertilizers, textiles, paper, and composites ,8. Banan3), bio-oil is thought to have financial advantages over other thermal conversion processes [Bio-oil is a high-density oxygenated liquid that can be used to replace liquid fossil fuels. Due to its comparatively high density 1200 kg/m00 kg/m3,Taib et al.  conducteBanana stems are rich in macronutrients such as potassium (K), calcium (Ca), phosphorus (P), magnesium (Mg), and sodium (Na): Potassium and sodium together play an important role in maintaining the acid–base balance in the body, calcium and phosphorus are the most important components of human bones and teeth, and magnesium has a role in the body in maintaining muscle contractility and nerve stress . Banana Bananas are rich in dietary fiber, and they have received attention in recent years due to their significant health-promoting effects . DietaryA definite drawback of the many techniques recognized for gingival depigmentation is gingival repigmentation. A method that can lengthen the time until pigmentation reappears is required to get around this. Numerous studies using antioxidant-rich plant extracts have demonstrated their antimelanogenic effects. Sowmya et al.  treated The potential value of banana by-products is driving the development of processing technologies to create products for the food and non-food industries . CompostIn the study by Ding et al. , straw oAccording to the study of Amarnath and Balakrishnan , banana Oleforuh-Okoleh et al.  fed broiClostridium thermophilus and Clostridium thermosaccharolyticus [The stems and leaves of bananas are also potential substrates for biofuel production by co-culture of cellulolytic olyticus . It is rolyticus . Taer etolyticus  successfThe chemical composition of banana fiber is mainly cellulose, hemicellulose and lignin; with its biodegradability and good chemical and mechanical properties, banana fiber has potential applications in the sanitary napkin, textile, pulp and paper industries , its cheFor decades, banana fibers obtained from banana pseudostems have been used as a raw material for the production of textiles for traditional handicrafts and clothing. Currently, the global export value of the textile and clothing industry is increasing year after year, which means that there is a huge demand for textile fiber materials. The enzymatic degumming of natural plant fibers using microbial strains is a promising method for processing textile fibers . AnotherThe ratio of essential amino acids to nonessential amino acids required by the human body in banana inflorescences is 0.54 , which iResearch on the carbohydrates in banana inflorescences is still in its infancy. However, a large amount of carbohydrates were detected . BhaskarAccording to Oliveira et al. , banana Although there were reports of different fatty acid levels in banana inflorescences, their primary fatty acids were somewhat similar. Sheng et al.  found thThe mineral makeup of banana inflorescences is balanced between macro and micro elements. Among them, potassium is the most abundant macronutrient, ranging between 33.52 and 55.2 mg/kg . The nexIt has been discovered that the banana inflorescence is abundant in polyphenols: phenolic acids such gallic acid, protocatechuic acid, p-hydroxylbenzoic acid, syringic acid, vanillic acid, gentisic acid, caffeic acid, vanillin, and ferulic acid were found ,43,44,45Banana inflorescences have been shown to exhibit a variety of biological functions with health-promoting potential. Particularly, the antioxidant properties of the inflorescence have drawn much interest. In addition, it has been proved that the inflorescence may possess antidiabetic, anticancer, cardioprotective, and other biologically active functions, which will be covered in the sections that follow. Many medical studies show that consuming antioxidants reduces the risk of diseases like diabetes, cancer, and cardiovascular disease . Banana v/v) as the solvent. The application of green technologies for extraction has been studied by some researchers. For instance, Madeline Correa et al. [Schmidt et al.  investiga et al.  optimizea et al. . These mIn drug discovery, the ability of extracts or compounds to block carbohydrate-hydrolyzing enzymes is typically seen as a sign of their potential antidiabetic effect. The isolated components of the banana inflorescence have been shown to have variable degrees of inhibitory action on α-amylase and α-glucosidase. For example, Ramu et al.  found thStudies have shown that banana inflorescence extracts have potential anticancer effects on a group of human cancer cells. Two human colorectal cancer cell lines, HT29 and HCT116, were especially sensitive to the ethanol extracts of the banana inflorescence when compared with other cancer cell ,56,61. THowever, the exact molecular mechanism of action of the cytotoxic constituents in the inflorescence is unknown. Preliminary findings suggested that cell death may be caused by the induction of apoptosis . In the 50 values of 169.52 and 217.45 μg/mL, respectively. The angiotensin I-converting enzyme (ACE) converts angiotensin I to angiotensin II, a strong vasoconstrictor that is important for blood pressure management. Angiotensin II has been linked to the advancement of vascular problems in diabetes as a key activator of insulin resistance. Therefore, ACE inhibition appears to be a new treatment option for diabetes management.Research showed that the expression of pro-inflammatory genes is increased by oxidized low density lipoprotein (LDL), which leads to monocyte recruitment into the vascular endothelial cells of a defective blood vessel wall that has been damaged by free radicals. Inhibiting LDL oxidation is critical in the treatment of cardiovascular disease and atherosclerosis. Arun et al.  found thOne study showed that the soluble dietary fiber from inflorescences had a higher cholesterol-absorbing capacity than the insoluble dietary fiber . The preBacillus cereus, Bacillus subtilis, and Staphylococcus aureus) and Gram-negative bacteria  and some pathogenic fungi  [w/v), and a period of 3 h offered the ideal conditions for extracting chemicals with anti-microbial activity from the banana inflorescence. Red blood cells were used for testing. Divya et al. [Apart from the biological activities already mentioned, the banana inflorescence has been proven to have other biofunction, such as the treatment of prostate enlargement and the improvement of urinary disorders . Banana lbicans) ,58,63. Ta et al.  discovera et al. . Divya ea et al.  also invAlthough banana inflorescence extracts have been shown to have many health-promoting biological activities; however, there is still a lack of information from clinical studies to support its alleged health benefits. Additional mechanistic research on some of the extracts, components, and metabolites existed in banana inflorescences could shed more light on their potential health-promoting qualities.Like other parts of the banana plant, the inflorescence has been used to cure a number of diseases. Banana inflorescence has been used for a variety of medical purposes around the globe, generally for the treatment of ailments like diabetes, bronchitis, ulcers, heavy menstruation, and menstrual cramps in the past. For instance, indigenous healers in India think that the juice from the banana inflorescence is helpful for reducing blood sugar levels, and banana inflorescence can be made into a preparation used to treat diabetes . IndigenBanana inflorescence extract can be employed as a nutritive supplement in food products. Most often, banana inflorescence or banana inflorescence extract used as a food additive. However, it has a significant impact on the physical, chemical, and sensory properties of food products, the appropriate amount of inflorescence or its extract to be added to food must be studied.It is interesting to note that in meat production, banana inflorescence has been combined with chicken flesh to create shredded banana inflorescence-chicken meat . The resThe chemical features of banana peel proteins have been uncovered, including amino acid profiles (UPLC) and secondary structural properties . Banana Essential fatty acids (EFAs) have been regarded as functional ingredients and nutraceuticals. EFAs are the basis for the building blocks of cells as well as the creation of a number of physiologically active substances. Numerous research studies have demonstrated the significance of EFAs in a variety of metabolic processes . EFAs haAccording to findings from recent literature, banana peels are a good source of polyunsaturated fatty acids including linolenic and linoleic acid, which make up more than 40% of the total fatty acids. A linoleic acid-rich diet showed reduced liver fat and a slightly improved metabolic status without any signs of inflammation ,72.Studies have shown that banana peels are rich in mineral elements such as calcium, phosphorus, and potassium, which have a variety of health benefits, such as supporting organ and body function regulation, acting as coenzymes in various enzyme systems, facilitating other biochemical and physiological processes, and preventing degenerative neurological diseases . OguntoyDietary fiber has been shown to prevent a range of diseases including cardiovascular disease, diverticulosis, constipation, colon cancer, obesity, and diabetes . The peeCompared with other fruits, banana peel contains an abundance of phenolics, essential secondary metabolites . PhenoliIn the future, there are two clear directions for research into the use of banana peels as a cheap and abundant source of phenolic compounds: (a) the development of affordable and effective technologies for the recovery of phenolic compounds; and (b) the use of phenolic compounds as functional ingredients in food and pharmaceutical products.When compared with other fruits, banana peels have higher concentrations of phenols, an essential secondary metabolite with antioxidant characteristics. In banana peel, a number of phenolic compounds are present, including gallic acid, catechin, epicatechin, gallocatechin, and anthocyanins . AdditioThe antioxidant capacity of banana peel extracts and components is evaluated using multi-mechanistic antioxidant tests. They are crucial for determining the capacity of the chemical constituents in banana peel extracts to reduce metal ion chelation, as determined by the FRAP assay, and scavenge free radicals like ABTS and DPPH. The phenolic chemicals present in banana peels include four subgroups, which are flavonols, flavan-3-ols, hydroxycinnamic acids, and catecholamines. In the study by Vu et al. , the antBacillus cereus, Staphylococcus aureus, Bacillus subtilis, and Escherichia coli [Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Bacillus species, and Klebsiella pneumoniae [Bacillus megaterium, Bacillus subtilis, and Staphylococcus aureus) and Gram-negative bacteria  [Banana peel extracts have also been reported to show varying degrees of antibacterial activity. Previous investigations found that the antibacterial properties of banana peels can be used to successfully inhibit the activity of hia coli . Accordihia coli , banana eumoniae . Banglada boydi) .Enterococcus faecalis biofilms that are cytotoxic to human lung cancer cells. The flavonoid content of banana peel is associated with its anti-cancer activity. Flavonoids inhibit the activity of ROS scavenging enzymes, induce apoptosis, and bring the cell cycle to a standstill, thereby inhibiting tumor production. The pharmacological functions and mechanisms of action in banana by-products are shown in The anti-disease benefits of banana peel actives are gaining traction. In the study by Dahham et al. , the extCurrently, banana peels are used to enhance the physicochemical and nutritional qualities of food products such as pastries, meat products, and jellies . The phyIn order to produce fish patties, Zaini et al.  substituWith the increasing human demand for food of animal origin, the demand for animal feed has risen. It is urgent to increase the productivity of feeds with higher nutritional value to overcome the problem of limited sources and high prices of raw materials for animal feed production. Banana peel is high in the main nutrients such as protein, fat, and carbohydrates, which account for 91.50% of its dry weight, and also contains a large amount of crude fiber . TherefoEssien et al.  reportedTraditionally, banana peels were simply broken down to replenish soil nutrients and used as fertilizer. Due to the higher requirements of modern agriculture for biological fertilizers and the advancement of agricultural production, many types of organic fertilizers have been produced from banana peels . Under bAspergillus niger to ferment banana peels for 7 days; it produced 1.60 OD of biomass and the ethanol output was about 7.45% v/v (ranged from 0.20 to 0.82 mg/cm3). Banana peels can also be used to create biogas. Pisutpaisal et al. [Banana peel is a good raw material for ethanol, biogas, hydrogen, and solid biochar production. Itelima et al.  used a cl et al.  analyzedl et al.  demonstrBanana waste can be transformed directly or through processing into materials suitable for the elimination of metal ions. Metals such as copper, lead, cadmium, zinc, and chromium have been shown to be adsorbed by banana peel cellulose and foam carbon produced from banana peels . Color rAn edible food wrapper produced from banana peels by Santhoskumar et al. , who shoIt has been discovered that banana peels contain bioactive compounds like flavonoids, phlobatannins, tannins, glycosides, alkaloids, and terpenoids that have a variety of biological and pharmacological effects, including antibacterial, antihypertensive, antidiabetic, and anti-inflammatory effects .The peel of the banana has been used as a herbal medicine for a number of conditions, including burns, ulcers, coughs, and diarrhea. For instance, banana peels are used to make wound ointment to reduce burn swelling and pain . StudiesThe comprehensive use of banana by-products can not only reduce waste and environmental pollution but also enhance the economic efficiency of the banana industry. However, there are still many problems in terms of utilization efficiency and technological means and solutions. A number of difficulties have been highlighted. In transforming banana by-products into a viable agricultural commodity, the focus should always be on converting banana by-products into high-value processed raw materials or products that match market demands and have significant economic benefits .Banana is a fruit that is closely related to people’s lives and is subject to a variety of pests and diseases during production, which can cause serious yield losses or even crop failure . TherefoBanana by-products have been proven to have great potential for use, but due to the large volume of banana stems and leaves and the water content of more than 90%, collection and transportation from the field would be very costly. It would also require a great deal of storage space and is not easy to transport. There are currently no effective techniques and equipment for collecting, transporting, and storing banana by-products, which has led to a large amount of residues being disposed of directly in the fields . The colIn order to make these unprocessed materials available for industrial-scale processing, standardized collection systems and transport systems must be established. This is particularly the case with regard to the establishment of appropriate by-product collection facilities for the preservation and sorting of these by-products according to their type and quality, as well as storage systems to prevent the degradation of valuable components. It is urgent to develop a set of technologies and equipment for the collection, transport, and storage of banana waste, for the post-processing utilization of the residue resources.The deep processing of banana by-products lacks processing technologies and equipment, the reliability and safety of the equipment is low, the energy consumption is extremely high, and many key technologies are yet to be broken through . The colThe properties of banana by-products themselves also limit the application of banana by-products, taking banana peel as an example. Banana peels are rich in active polyphenols, but polyphenols have poor stability and are easily oxidized. The soft texture of banana peel, rich in pectin, sugars, and tannins, not only makes it difficult to store but also makes it very susceptible to browning during processing. In addition, it may cause deterioration in nutrition and flavor, which affects the quality of the product. Therefore, the characteristics of bananas themselves make it difficult to maintain the original flavor and nutrition during processing, which affects the industrialization of the product . The bioAlthough recycling and using agricultural by-products to create commercially viable and revenue-generating products is not a new concept, maximizing the use of abundantly available resources to reduce solid waste emissions and create added value is still considered crucial. Banana by-products such as stems, leaves, inflorescences, and peels are good raw materials for the food and non-food industries, with a variety of potential applications . The utilization of banana by-products can have positive effects on protecting the environment and developing value-added foods for human being.However, banana by-products safety needs to be addressed further to meet market demand. There were various techniques such as transcriptomics, genomics, metabolomics, and proteomics can be used to learn more about the molecular mechanisms of banana by-products for health and to better understand how the phytochemicals in banana by-products function as functional components. Additionally, in order to make these raw materials accessible for processing on an industrial scale, standardized processes for the collection, transportation, storage, and processing of banana by-products must be established. Creating value from waste products like banana by-products is arguably one way to ensure that ecological agriculture and food systems are sustainable and beneficial to the environmental for future generations.


CaSR is also expressed in mammals including rodent taste cells and is involved in sensing kokumi, a rich, savory quality that enhances the intensities of salty, sweet, and umami tastes. In this study, we focused on chicken CaSR (cCaSR) since calcium is an essential nutrient that is necessary for making eggshell and for the extremely rapid initial growth of bones. First we confirmed that cCaSR is expressed in taste cells. Next we cloned the cCaSR gene from kidney and transiently transfected human embryonic kidney 293 T (HEK293T) cells with the recombinant cCaSR, or empty vector and looked for the agonists and allosteric modulators (including kokumi substances) of cCaSR by Ca2+ imaging. We found that cCaSR was activated by extracellular Ca2+ and Mg2+ in a dose dependent manner. Several L-amino acids and kokumi substances such as glutathione enhanced the response of cCaSR. In addition, NPS2143 as a negative allosteric modulator of human CaSR negatively modulated the response of cCaSR. These results suggest that cCaSR can sense extracellular Ca2+ and Mg2+ as well as positive and negative allosteric modulators. Taken together, the results imply that CaSR might be a multifunctional receptor for calcium, amino acids, and kokumi substances in chicken. The present finding that functional CaSR is expressed in the chicken oral tissues will allow us to further elucidate the physiological role of CaSR in the chickens' taste sense, and to create new feeds that will contribute to the poultry industry.In vertebrates, the extracellular calcium-sensing receptor (CaSR) plays a key role in calcium homeostasis by sensing slight changes in extracellular Ca It is known that chickens show a high affinity to calcium3. However, it is unknown whether chickens use taste to discriminate feed containing calcium.Calcium is a very important nutrient for chickens. Low levels of calcium in the diet induce decreases in egg production, eggshell strength, hatchability, and body weight gain4. Extracellular Ca2+ is the principal agonist of CaSR5, and other orthosteric agonists include various divalent and trivalent cations, including Mg2+, Al3+, Sr2+, Mn2+, Ni2+, Gd3+, and Ba2+, as well as polyamines , aminoglycoside antibiotics , and some peptides 7. Furthermore, functions of CaSR can be regulated by many allosteric modulators such as aromatic L-amino acids , calcimimetics , and calcilytics 7.The extracellular calcium sensing receptor (CaSR), a G-protein–coupled receptor belonging to Family C, is abundantly expressed in the parathyroid glands, kidney, gut, and bone and plays a key role in regulating the release of parathyroid hormone in response to circulating blood calcium concentrations in mammals8, and it was speculated that CaSR is also a calcium-magnesium taste receptor candidate gene9. Indeed, CaSR is expressed in taste cells in rodents11. Moreover, it has been reported that CaSR functions as the receptor of a specific flavor quality, kokumi12. Although kokumi substances have only a slight flavor in water, they were found to markedly increase thickness, continuity, and mouthfulness of taste when added to an umami solution or other food13. These kokumi substances, such as glutathione , are perceived through CaSR in mice and humans12.Tordoff et al. revealed that T1R3 is a calcium-magnesium taste receptor in mice23, it is unknown whether chicken oral tissues have sensors for calcium and kokumi flavors. Thus, the purpose of this study was to elucidate the sensing mechanisms of calcium taste and kokumi quality in chickens. We analyzed CaSR expression in chicken oral tissues and investigated whether cCaSR is activated by Ca2+, Mg2+, L-amino acids, and kokumi substances by using Ca2+ imaging assay by using cCaSR-expressing cells.Elucidation of the functions of chicken CaSR (cCaSR) in oral tissues may reveal fundamental knowledge with which to improve chicken feeds and enhance the efficiency of egg and meat production. Although it has been reported that chickens have umami, bitter, and fat taste receptors24. The present study showed that CaSR mRNA expression levels were significantly higher in the base of oral cavity than in the palate and the tongue tip (P < 0.01)  database , with a difference of only two bases  strain). The translated product of the cloned cCaSR gene matched that of the NCBI database (XP_416491.6).We cloned the cCaSR/pcDNA5/FRT were specifically labeled by the antiserum  cells transfected with c2, the ratio of the relative fluorescence units (RFU), reflecting the cytosolic Ca2+ concentration, was increased in cCaSR-expressing HEK293T cells in a dose dependent manner .Although RFU was increased slightly by only 2.5 mM CaClAla Fig. . Howeverkokumi substances such as GSH, γ-Glu-Cys, and γ-Glu-Val-Gly enhanced cCaSR activities with 2.5 mM CaCl2 .cCaSR was activated by 10 mM CaClrst Fig. A. That wred Fig. B. We ananly Fig. A and 10 143 Fig. B. There 143 Fig. C. These 2+ concentration, were increased in cCaSR-expressing HEK293T cells after stimulation with 2.5 mM CaCl2  compared with that of Gallus Gallus, the translated amino acids were not changed from those of Gallus Gallus. Because Gallus Gallus is an ancestor species of chickens, it is estimated that CaSR is an essential receptor for living, and this important molecule has been preserved even after a long period of selection pressure. In addition, when the amino acid sequence of CaSR in chicken was compared with those in other animal species, the homology percentages were about 85% with humans, rats, and mice (data not shown). These results also implied that CaSR is an important receptor from an evolutionary standpoint.We cloned the 2+ imaging, we confirmed the responses of cCaSR-expressing cells to CaCl2 and MgCl2 solutions in a dose dependent manner . Because extracellular Ca2+ activated cCaSR-expressing cells in lower concentrations than did extracellular Mg2+, Ca2+ is thought to be the main orthosteric agonist in cCaSR as it is in hCaSR. Diaz et al. reported that cCaSR is activated by Ca2+ and Mg2+30. The present study showed an identical result. The binding sites of extracellular Ca2+ in hCaSR are five extracellular domain regions31. In cCaSR, only one amino acid (Ser-388) in Site 4 was mutated to Thr among these important sites. Since other Ca2+-binding sites were not changed, it is reasonable that cCaSR can sense Ca2+ as observed in the present study.Through Caner Fig. . These r2 in cCaSR-expressing cells , a chicken umami taste receptor candidate, is activated by L-Ala and L-Ser but not by L-Pro in a heterologous expressing system32, chicken T1R1/T1R3 activities for other L-amino acids have not been reported. Furthermore, because there are few T1R1/T1R3 co-expressing cells in chicken taste cells28, it is possible that CaSR plays a certain role as an amino acid receptor in chickens. On the other hand, since the functions of chicken T1R1 and T1R3 homodimers have not been elucidated, further studies of chicken oral amino acid receptors are needed.In addition, L-Phe, L-Trp, and L-Ala enhanced the responses of CaCllls Fig. . Under tkokumi substances, GSH, γ-Glu-Cys, and γ-Glu-Val-Gly, on cCaSR by using Ca2+ imaging. These compounds were found to enhance the responses of cCaSR-expressing cells to CaCl2  conditions, these compounds did not show any response, suggesting that these compounds are positive allosteric modulators. Although the mutations of T145A/S170T in hCaSR reduced the responses to the potent γ-glutamyl peptide, S-methylglutathione, and L-Phe33, cCaSR does not have mutations in these portions. The present study revealed that cCaSR could receive kokumi substances as well as hCaSR.In the present study, we examined the effects of three representative known Cl2 Fig. . Under t2+. Especially, it has been reported that the mutations of Phe-668 (helix II), Arg-680 (helix III), Phe-684 (helix III), Glu-837 (helix VII), Phe-688 (helix III), and Ile-841 (helix VII) reduced the negative modulatory effects of NPS2143 to hCaSR35. These binding sites of NPS2143 are also the same in cCaSR, and there are only six amino acid residue differences in the seven-transmembrane regions between hCaSR and cCaSR. These six amino acid residues are not the known binding sites of NPS2143. Thus, it is reasonable that NPS2143 functions as a negative allosteric modulator of cCaSR.We also examined the effects of NPS2143 on cCaSR and found that NPS2143 functions as a negative allosteric modulator also in the case of cCaSR. NPS2143 is expected to serve as a negative allosteric modulator of cCaSR in various studies. It has been thought that NPS2143 binds to a seven-transmembrane region of hCaSR and negatively modulates the responses to extracellular Ca2. The results were identical to the previous study of hCaSR36. These results also suggested that the functions of cCaSR are similar to that of hCaSR and that CaSR is fundamental receptor from birds to humans.A representative calcimimetic, cinacalcet HCl, activated cCaSR as well as other cCaSR ligands found in the present study. Furthermore, the responses of cCaSR by cinacalcet HCl were enhanced by 1.5 mM CaCl37. However, the calcium taste receptor has not been fully elucidated. Tordoff et al. revealed that T1R3 is involved in the calcium and magnesium taste sense in mice8. Their study confirmed that T1R3 knockout mice prefer CaCl2 and MgCl2 solutions rather than water, whereas wild-type mice preferred water and were repelled by these solutions. Because T1R3 knockout mice lost their repellence to CaCl2 and MgCl2 solutions and showed a preference for them, it is supposed that there are other calcium and magnesium taste-related molecules besides T1R3. Tordoff et al. mentioned the hypothesis that CaSR may be involved in calcium taste by making a heterodimer with T1R39, but there is no clear evidence as to whether CaSR functions as a calcium taste receptor. On the other hand, although chickens show an affinity for calcium, the molecular mechanisms underlying this preference have been almost unknown. The present study implied one molecular mechanism by which CaSR may be involved in calcium taste sense in chickens.Calcium and magnesium are essential nutrients for animals and play various roles in their bodies. There is a lot of evidence that various species, not just chickens, have an appetite and a taste sense for calciumkokumi substances in chicken oral tissues. Further studies should be conducted to confirm whether the ligands of cCaSR are recognized as taste compounds in chickens by behavioral tests. It is hoped that the present results will propel further studies to fully elucidate calcium taste receptors, amino acid sensors, and kokumi flavor sensors in chickens. Furthermore, the fact that the ligands of cCaSR are almost the same as those of hCaSR implies that CaSR is an important and universal taste molecule among mammals and birds.In summary, it was estimated that CaSR might function as a multifunctional receptor for calcium taste, several amino acids, and 2·2H2O, MgCl2·6H2O, L-Phe, L-Trp, L-Ala, GSH, and NPS2143 hydrochloride were purchased from Sigma-Aldrich , and γ-Glu-Cys and γ-Glu-Val-Gly were purchased from Hokkaido System Science . For Ca2+ imaging by using Fluo 4-AM, these compounds except MgCl2·6H2O were dissolved in Ca2+-free and low-Mg2+ (0.5 mM) bath solution containing 140 mM NaCl, 5 mM KCl, 0.5 mM MgCl2, 10 mM HEPES, and 10 mM glucose at pH 7.4, adjusted with NaOH, to make stock solutions. MgCl2·6H2O was dissolved in a Mg2+-free and low-Ca2+ (0.5 mM) bath solution containing 140 mM NaCl, 5 mM KCl, 0.5 mM CaCl2, 10 mM HEPES, and 10 mM glucose at pH 7.4, adjusted with NaOH, to make stock solutions. These stock solutions were diluted with a low-Ca2+ and low-Mg2+ bath solution containing 140 mM NaCl, 5 mM KCl, 0.5 mM CaCl2, 0.5 mM MgCl2, 10 mM HEPES, and 10 mM glucose at pH 7.4, adjusted with NaOH just before each experiment. The low-Ca2+ and low-Mg2+ bath solution was used as a basic bath solution for Ca2+ imaging.CaCl2+ imaging by using Fura 2-AM, cinacalcet HCl was dissolved in dimethyl sulfoxide (DMSO) and stored at − 20 °C. The DMSO stock solution (20 mM) was diluted with a low-Ca2+ (0.5 mM) and low-Mg2+ (0.5 mM) bath solution containing 140 mM NaCl, 5 mM KCl, 0.5 mM CaCl2, 0.5 mM MgCl2, 10 mM HEPES, and 10 mM glucose at pH 7.4, adjusted with NaOH.Cinacalcet HCl was purchased from FUJIFILM Wako Pure Chemical Corporation . For CaqPCR analysis and immunohistochemistry (IHC). A 20-week-old chick was used for cCaSR cloning from kidney. This study was carried out in accordance with the Guide for Animal Experiments issued by Kyushu University, the Law Concerning the Human Care and Control of Animals , the Japanese Government Notification on the Feeding and Safekeeping of Animals , and the ARRIVE guidelines. It was approved by the committee for Laboratory Animal Care and Use at Kyushu University .Fertilized eggs of the RIR strain were obtained from the National Livestock Breeding Centerʼs Okazaki station , and the hatching chicks were used for the present study. One-week-old chicks were used for RT-qPCR, PowerUp SYBR Green Master Mix  was used according to the manufacture’s instructions. Primers were designed with the aid of the NCBI nucleotide database and are shown in Table qPCR reactions were done by StepOnePlus real time PCR system (Thermo Fisher Scientific). Each assay was performed in triplicate. To check the amplifications of unintended targets, we confirmed the single peaks in the melting curve analysis in all samples. After the RT-qPCR, to confirm whether the RT-qPCR products of the basal of oral cavity by using CaSR primer pair are identical with cCaSR sequence, these RT-qPCR products were electrophoresed by agarose gel, and the target single DNA bands around 120 bp were extracted from the agarose gel by using NucleoSpin Gel and PCR Clean-up (Takara Bio). The DNA sequences were confirmed by the Fasmac sequencing service .Total RNA was isolated from the palate, tongue tip, and the base of oral cavity with the use of FastGene RNA Premium Kit  according to the manufacture’s instructions, and genome DNA was removed with the use of DNase I (NIPPON Genetics). First-strand cDNA was synthesized using PrimeScript RT reagent Kit with gDNA Eraser  according to the manufacture’s instructions. For 21. The chicks were euthanized with an overdose of pentobarbital sodium solution, and the base of oral cavity and kidney were excised. For IHC analyses of the base of oral cavity, the tissues were embedded in Tissue-Tek O.C.T. Compound  and freshly frozen at − 80 °C. Frozen sections were cut at 10 µm thickness on a Leica CM1850 cryostat  and mounted on Matsunami adhesive silane (MAS)-coated glass slides .Tissues were collected and frozen sections prepared as described previously with some modification26. The secondary antibodies used were Alexa Fluor 488 goat anti-rabbit IgG (1:500), Alexa Fluor 488 donkey anti-rabbit IgG (1:500), Alexa Fluor 594 goat anti-rabbit IgG (1:500), and Alexa Fluor 594 goat anti-mouse IgG (1:500) (Thermo Fisher Scientific).The primary antibodies used were custom-made rabbit polyclonal anti-chicken CaSR antiserum   and mouse monoclonal anti-vimentin antibody  used in a previous study21. The sections were fixed with acetone for 10 min at 4 °C and dried for 60 min at RT. After rehydration with phosphate-buffered saline (PBS), nonspecific staining was blocked by 3% normal goat serum /PBS with 0.3% Triton X-100 (PBS-X) for 30 min at RT. The sections were then incubated with the primary antibodies in 1% NGS/PBS-X overnight at 4 °C. The sections were rinsed with PBS and incubated with the secondary antibodies in 1% NGS/PBS-X for 60 min at RT. Following rinses with PBS, the sections were mounted using VECTASHIELD with DAPI (Vector Laboratories). A confocal laser scanning microscope system  with NIS-Element AR3.2 was used for the observations.A double-fluorescence IHC examination of frozen sections of the base of oral cavity was performed as described in a previous studyFresh kidney was fixed in 4% paraformaldehyde in PBS for 50 min and dehydrated with sucrose solutions . Kidney frozen block embedded in FSC 22 Frozen Section Media  was sliced into 7 μm sections and placed onto silane-coated glass slides. After washing with PBS-X, then incubated in 1.5% normal donkey serum (NDS)/PBS-X for 1 h, then the sections were incubated with the primary antibody, anti-chicken CaSR antiserum (1:500), in 1.5% NDS/PBS-X overnight at 4 °C. The sections were rinsed with PBS-X and incubated with the secondary antibody, Alexa Fluor 488 donkey anti-rabbit IgG (1:500), in 1.5% NDS/PBS-X for 2 h at RT. Following rinses with PBS-X, the sections were mounted using VECTASHIELD with DAPI (Vector Laboratories). A confocal laser scanning microscope system  was used for the observations.CaSR plasmid, we isolated total RNA from kidney because CaSR expression in kidney is rich in other animals38, then synthesized first-strand cDNA by reverse transcription. The deduced ORF of cCaSR was amplified and sequenced. The PCR primers were designed based on the NCBI nucleotide databases of the predicted Gallus Gallus CaSR (XM_416491.7). The PCR product of the ORF was subcloned into the pcDNA5/FRT (Thermo Fisher Scientific) vector by using the In-Fusion HD Cloning Kit (Takara Bio). The entire sequence of cCaSR derived from RIR chicks was compared with the genomic NCBI database of the predicted Gallus Gallus CaSR.To construct the c2.HEK293T cells  were maintained in Dulbecco’s Eagle’s medium  containing 10% fetal bovine serum  and penicillin–streptomycin solution (× 100)  at 37 °C and 5% CO28. HEK293T cells were transfected with either empty vector pcDNA5/FRT or cCaSR/pcDNA5/FRT by using ScreenFect A  on coverslips coated with poly-d-lysine (0.1 mg/mL). After transfection, the cells were incubated for 48 h at 37 °C in 5% CO2. The HEK293T cells were then washed with PBS and fixed with − 20 °C methanol for 10 min. After another wash with PBS, nonspecific staining was blocked by 3% NGS/PBS for 30 min at RT. The cells were then incubated with the rabbit anti-chicken CaSR antiserum (1:1000) in 1% NGS/PBS overnight at 4 °C.Culture, transfection, and immunocytochemistry on HEK293T cells were performed as described previously with some modificationsThe cells were then washed with PBS, incubated with Alexa Fluor 594 goat anti-rabbit IgG (1:500) in 1% NGS/PBS for 90 min at RT, and mounted with VECTASHIELD with DAPI. A fluorescence microscope  was used for the observations.2+ imaging experiments by using Fluo 4, HEK293T cells were transfected with either empty vector pcDNA5/FRT for mock cells or cCaSR/pcDNA5/FRT by using ScreenFect A on coverslips coated with poly-d-lysine (0.1 mg/mL). After transfection, the cells were incubated for 48 h at 37 °C and 5% CO2. The cells were then loaded with 1.25 μM Fluo 4-AM solution for 30 min at 37 °C and 5% CO2 in the dark. Fluo 4-AM solution was prepared according to the manufacturer’s instructions . The coverslips were washed with the low-Ca2+ and low-Mg2+ bath solution, and Fluo 4 fluorescence was measured in that solution using a confocal laser-scanning microscope . The coverslips were mounted in a chamber connected to a gravity flow system to deliver various stimuli. Chemical stimulation was applied by running the bath solution containing various chemical reagents. The activity of the intracellular Ca2+ indicator (Fluo 4) was confirmed by responses to 0.1% Triton X-100.For the Ca2+ imaging experiments by using Fura 2, HEK293T cells were transfected with either empty vector pcDNA5/FRT for mock cells or cCaSR/pcDNA5/FRT by using ScreenFect A on 96-well clear bottom black plate (Thermo Fisher Scientific) coated with poly-d-lysine (0.1 mg/mL). After transfection, the cells were incubated for 48 h at 37 °C and 5% CO2. Then, we loaded Fura 2-AM solution per well in accordance with the manufacturer’s manual (Dojindo Laboratories). After incubation for 30 min at 37 °C and 5% CO2, calcium imaging was performed using a multimode microplate reader . The assay was carried out at about 37 °C, and stimulating solutions dissolved in the low-Ca2+ and low-Mg2+ bath solution were injected automatically. Cell activity was analyzed by the value of the ratio of fluorescence intensity excited at 340 nm and 380 nm before and after injection.For the Cat-test, or two-way repeated measures ANOVA followed by unpaired t-test using the IBM SPSS Statistics , and differences with P values < 0.05 were considered significant.The data are expressed as means ± SD or SE. Statistical analyses were performed using the one-way factorial ANOVA followed by post-hoc Tukey HSD test, unpaired


Although β-catenin signaling cascade is frequently altered in human cancers, targeting this pathway has not been approved for cancer treatment.High-throughput screening of an FDA-approved drug library was conducted to identify therapeutics that selectively inhibited the cells with activated β-catenin. Efficacy of iron chelator and mitochondrial inhibitor was evaluated for suppression of cell proliferation and tumorigenesis. Cellular chelatable iron levels were measured to gain insight into the potential vulnerability of β-catenin-activated cells to iron deprivation. Extracellular flux analysis of mitochondrial function was conducted to evaluate the downstream events of iron deprivation. Chromatin immunoprecipitation, real-time quantitative PCR and immunoblotting were performed to identify β-catenin targets. Depletion of iron-regulatory protein 2 (IRP2), a key regulator of cellular iron homeostasis, was carried out to elucidate its significance in β-catenin-activated cells. Online databases were analyzed for correlation between β-catenin activity and IRP2-TfR1 axis in human cancers.Iron chelators were identified as selective inhibitors against β-catenin-activated cells. Deferoxamine mesylate, an iron chelator, preferentially repressed β-catenin-activated cell proliferation and tumor formation in mice. Mechanically, β-catenin stimulated the transcription of IRP2 to increase labile iron level. Depletion of IRP2-sequered iron impaired β-catenin-invigorated mitochondrial function. Moreover, mitochondrial inhibitor S-Gboxin selectively reduced β-catenin-associated cell viability and tumor formation.β-catenin/IRP2/iron stimulation of mitochondrial energetics is targetable vulnerability of β-catenin-potentiated cancer.The online version contains supplementary material available at 10.1186/s12967-023-03914-0. CTNNB1), or loss-of-function mutations of its destruction complex, such as adenomatous polyposis coli (APC), AXIN1 and AXIN2, lead to β-catenin stabilization and eventually unrestrained transcriptional activity  2%Input Sample − C[T] IP Sample), C[T] = Threshold cycle of PCR reaction.Β-catenin ChIP-seq data analysis was conducted using our published datasets (GSE165853). For the bioinformatic analysis of ChIP-seq data, Bowtie2 was used for alignment, MACS2 with FDR < 0.01 was for enriched peaks identification, and ChIPseeker was for peaks annotation. Peaks enrichment distribution analysis was performed by Deeptools as previously described . ChIP asSite1-F: 5′-GTGAGCGCTGTGATGCAATA-3′.Site1-R: 5′-CCTTGGTGCTGTTGGTATCTG-3′.Site2-F: 5′-TAGCCAACGGTGTACAGGAG-3′.Site2-R: 5′-TGAGTCTGGATTTGCGATGC-3′.Site3-F: 5′-GCATCGCAAATCCAGACTCA-3′.Site3-R: 5′-TTGATCCACTGGCTTCCTGAC-3′.Site4-F: 5′-GATGCCTGTGGTAACAAGCG-3′.Site4-R: 5′-ATTGGCTCCAGCTATCCGTT-3′.Site5-F: 5′-GCAAGAGCTGGGGTATTAGCA-3′.Site5-R: 5′-GGAGTTGTCTGGTTTGCCCT-3′.6β-cateninΔ(ex3)/+ MEFs suspended in 100 μL PBS or 2 × 106 HuH7 in 100 μL PBS containing 50% Matrigel  were subcutaneously inoculated in the right dorsal flank of nude mice. When tumor volumes reached 100 mm3, mice were randomly assigned to different groups. The treatment group was intraperitoneally injected with 10 mg/kg S-Gboxin, or 400 mg/kg DFO every other day and control group was injected with equal volume of vehicle. Tumor volumes and body weights were measured every other day and tumor volumes were calculated by the formula: volume = length × width2/2.All animal studies were performed following the protocols approved by the Animal Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College . Female BALB/c nude mice (4–6 weeks) were purchased from HFK Bio-Technology . 1 × 10CTNNB1 and IRP2 or TFRC, cell line analysis was conducted with Dependency Map , and human sample analysis was performed using Gene Expression Profiling Interactive Analysis 2.0  [http://ualcan.path.uab.edu/analysis-prot.html) [IRP2 promoter (https://jaspar.genereg.net) [For gene expression correlation between ail.php) . UALCAN ot.html) . JASPAR reg.net) .P < 0.05 was considered as statistically significant.Data were expressed as mean ± SD. All data were independently repeated for at least 3 times. Statistical analysis was carried out using a two-tailed unpaired t-test with GraphPad Prism software. CTNNB1 gene, to establish constitutive β-catenin-activated MEFs (β-cateninΔ(ex3)/+) [β-cateninΔ(ex3)/+ cell viability /+ MEF viability /+) . We thenCTNNB1-mutated MHCC97H and SNU398 cells to DFO treatment /+ MEFs, manifesting as reduced tumor volumes and tumor weights, with negligible impact on body weights in nude mice /+ MEFs /+ MEFs were more resistant to erastin-induced ferroptosis /+ MEFs had higher LIP /+ MEFs /+ MEFs and HepG2 /+ MEFs /+ MEFs  to drive target gene expression . We thersted LIP . β-catenIrp2 in β-cateninΔ(ex3)/+ MEFs /+ MEFs. Similar to iron chelators, direct suppression of mitochondrial metabolism with oligomycin  or S-Gboxin (ATP synthetase inhibitor) preferentially hindered β-cateninΔ(ex3)/+ MEF proliferation /+ MEFs with minimal impact on mouse body weights  . NuclearIron plays various roles such as mitochondrial metabolism, DNA synthesis, cell cycle progression and redox homeostasis, deprivation of iron may cause various pathological changes . Iron chΒ-catenin-activated cells exhibit an augmented dependency on iron availability. Oncogenic β-catenin transactivates IRP2 to enhance mitochondrial function via raising intracellular LIP. Β-catenin/IRP2/iron-boosted mitochondrial energetics is essential for cell proliferation and tumor growth. Intervention of iron, IRP2 or mitochondrial function abolishes β-catenin-activated cancers. Since DFO is an old drug, its efficacy can be readily tested for the treatment of oncogenic β-catenin-related tumors.Additional file 1: Figure S1. Iron addition has no impact on cell proliferation of WT and β-cateninΔ(ex3)/+ MEFs. Figure S2. β-catenin-activated MEFs are resistant to ferroptosis induction. Figure S3. β-catenin is positively correlated with IRP2 and TfR1. Table S1. Mutations of cell lines.


Artificial intelligence (AI) represents a transformative advance in the practice of gastrointestinal endoscopy, with giant strides already made in a short space of time. In the last 5 years alone, the publication of a number of high‐profile randomised trials now give a glimpse of endoscopy's symbiotic future with AI. Computer‐aided detection (CADe) technology has been shown to increase adenoma detection rates,AI technologies which support a competent endoscopist to detect and diagnose polyps at the level of an expert are now already being installed in an increasing number of endoscopy units worldwide. However, despite the abundance of AI technologies in CADe and CADx, there are still opportunities to refine the capabilities of AI to provide further benefit to users.In this issue,While polyp detection has been the ‘tip of the spear’ for AI in gastroenterology, this paper gives a nice glimpse of some of the broader capabilities of AI which will impact our practice in the future. The concept of whether false positive alerts during AI polyp detection could cause distractions (or other unintended consequences) at certain moments is an important one that should be a topic for future study. Some would question whether the efforts taken by the authors to automate CADe deactivation is truly necessary, especially when AI can be manually toggled on/off with a simple button press during instrument passage for polypectomy. However, the concept of automatically disabling AI is appealing and forms the basis for future development. There is still a technical leap between ‘recognizing’ the instrument using computer vision, and actually integrating this into the user experience practically during colonoscopy with AI polyp detection. For instance, does the presence of an instrument on the screen toggle the AI off for 5 s, 10 s, or on a frame‐by‐frame basis, which would be annoying for the user, if the AI blinks on and off depending on how clearly the instrument is seen on the screen? These questions, which are centred on ‘user interface’ design, are critical to address for such technology to be practical.This study raises important questions and future research priorities. First, what is the best design for visual +/− auditory alarms to minimize alert fatigue (and distractions) while maximizing support for physicians? Second, as additional AI tools come along in future, how should complex AI‐generated data be presented without clogging up the endoscopy monitor and causing cognitive load? Thoughtful user interface design for AI systems in endoscopy will be critical so that physicians can integrate complex data during live endoscopy, without being distracted from the task at hand.An AI tool to recognize endoscopic instruments could have several applications beyond just auto‐disabling polyp detection. Auto‐population of endoscopy reports is perhaps the most exciting avenue, especially if specific instruments and interventions can be recognized and documented with high accuracy. Radio‐frequency identification embedded instruments combined with AI recognition of interventions might offer a particularly robust approach to procedure reporting along with device invoicing. This could help deliver an AI process stream, whereby lesions can be detected, characterised, followed by documentation of tools used  and their therapies, which could all be compiled within the endoscopy report. If accurately done, this could not only help reduce the overwhelming burden of electronic documentation for physicians, but can improve quality and consistency of procedure documentation, and performance metric reporting.The AI endoscopy interface of the next decade will represent the culmination of innumerable small but important steps. The effort by Brand et al. gives a glimpse of the incremental innovations which will be a critical part in our AI journey for gastrointestinal endoscopy.


SGO1, which encodes for Shugoshin1, have problems with normal pacing of the heart and gut.Cohesinopathies is a term that refers to/covers rare genetic diseases caused by mutations in the cohesin complex proteins. The cohesin complex is a multiprotein complex that facilitates different aspects of cell division, gene transcription, DNA damage repair, and chromosome architecture. Shugoshin proteins prevent the cohesin complex from premature dissociation from chromatids during cell division. Patients with a homozygous missense mutation in sgo1 gene. Homozygous sgo1 mutant embryos display various phenotypes related to different organs, including a reduced heart rate accompanied by reduced cardiac function. In addition, sgo1 mutants are vision‐impaired as a consequence of structurally defective and partially non‐functional photoreceptor cells. Furthermore, the sgo1 mutants display reduced food intake and early lethality.To study the role of shugoshin during embryo development, we mutated the zebrafish We have generated a zebrafish model of Sgo1 that showed its importance during organ development and function. Shugoshin 1 ((SGO1) is part of the cohesion complex and its gene is mutated in patients with CAID syndrome. Here we generated a zebrafish loss‐of‐function mutation for sgo1 and describe that these mutants have defects in cardiac function and eye development leading to early lethality. Patients are asymptomatic at birth, but they start to develop symptoms as early as 6 years of age.CdLS, the most common form of cohesinopathy, is a multisystem disease with a prevalence of 1–3 per 30 000 live births.Sgo1 loss‐of‐function mutation die in utero indicating that Sgo1 is required during early development. Haploinsufficiency for Sgo1 results in viable mice, but these display genome instability manifested as mis‐segregation of chromosomes, resulting in enhanced tumorgenesis.sgo1 loss‐of‐function mutant using CRISPR/Cas9 genome‐editing technology. While sgo1−/− larvae develop normally through the first 3 days post‐fertilization (3 dpf), they show a reduced embryonic heart rate, accompanied by reduced end‐diastolic volume, ejection fraction, stroke volume and cardiac output at 5 dpf. Furthermore, eye size of sgo1−/− larvae is smaller, retinal lamination is impaired and photoreceptor outer segment morphology is disturbed. The responsiveness to Light ON transitions is also impaired and the larvae have an overall reduced mobility. Due to these defects, early lethality of these mutants progresses within the first 2 weeks of life.Little is known about the role of SGO1 during development. Mice homozygous for a 22.1shugoshin‐like 1 gene annotated as sgo1 (ENSDART00000169729), which is expressed during embryo development . At 3 dpf sgo1−/− larvae have a normal size and develop without any visible phenotypes  were unable to ingest the tracer  were able to ingest the tracer within 24 hours of feeding, albeit at a significantly lower level as compared to their wild‐type siblings  are disrupted in sgo1−/− embryos . Furthermore, we observed that sgo1−/− larvae displayed an irregular morphology of photoreceptor outer segment , and occasionally regions within the retina with a complete absence of photoreceptor cells.To study photoreceptor function at the molecular level, we investigated the subcellular localization of the photopigment rhodopsin. Rhodopsin is present at the rod outer segments of wildtype larvae as expected ; however, rhodopsin is significantly mislocalized to the photoreceptor cell bodies of in situ hybridization to detect sgo1 mRNA expression in 2 dpf wild‐type embryos. We observed a weak but consistent expression of sgo1 in the ciliary marginal zone (CMZ)  Figure . The retE Figure , with tht Figure . These rsgo1−/− larvae show a significantly less pronounced light ON‐response compared to their wild‐type siblings as shown by a significant decrease in distance covered after a dark‐to‐light transition  by exposing larvae to alternating light–dark transitions. n Figure . Altogetsgo1 loss‐of‐function mutant and studied its role in zebrafish development. We found that sgo1−/− larvae at 5 dpf have a significant decrease in heart rate as well as functional cardiac defects resulting in reduced cardiac output. In addition, phenotypical changes in the eye were observed due to disruption in photoreceptor layer and reduced sensitivity to dark/light cycle. Furthermore, the sgo1−/− larvae show a reduced ability to ingest food, which may be related to either one of the other phenotypes observed such as malformed jaw, impaired vision, and a general asthenia. Together, this results in lethality of the sgo1−/− larvae by 2 weeks after fertilization. The cardiac arrhythmia phenotype reported here for sgo1−/− larvae resembles somewhat the arrhythmias seen in CAID patients with a homozygous p.(Lys23Glu) mutation in SGO1. On the contrary, the reduced vision of sgo1−/− fish larvae we report here has not been reported for CAID patients. This could indicate that the missense mutation in SGO1 in CAID patients acts as a hypomorphic mutation while the zebrafish sgo1 deletion reported here causes a much stronger or complete loss of Sgo1 activity. Indeed, the sgo1 heterozygous larvae display the cardiac arrhythmia phenotype  strain zebrafish using CRISPR/Cas9 technology. One‐cell‐stage zebrafish embryos were microinjected with an injection mixture containing of : 150 ng/μL nuclear Cas9 (nCas9) mRNA, 20–40 ng/μL sgRNA and 10% (v/v) Phenol Red. Each putative founder adult fish was crossed with a wild‐type adult fish (F1), F1 fish were out‐crossed to wild type at least two times prior to starting the experiments. Homozygous fish were generated by inbreeding heterozygous mutant carriers. The sequence of the sgRNA is GGGGAGCGCACAGCCTAAAG and genotyping primers are as follows: forward: TGTGTGAATGTGTCTTGACAGG and reverse: AAATCAAAGGTGTCGTTTCCTC. sgo1hu13334/+ will be referred to as sgo1+/− in the remainder of the study.The 3.3sgo1+/− fish incross were used for total RNA extraction using TRIZOL . Prior to RNA extraction, embryos were separated phenotypically as siblings or sgo1−/− (phenotype described in the main text). In vitro synthesis of cDNA was carried out with RT Superscript III (Life Technologies/Thermo Fisher Scientific). The obtained cDNA was used as template for qPCR using CFX Connect96 Real‐Time System  and Luminaris HiGreen qPCR Master Mix (ThermoFisher Scientific). Expression of sgo1 was assessed using primers 5′‐CAGAAGAAGAGCTTCCAGCAG‐3′ and 5′‐GGCCAGAGCTTTATTGTTGG‐3′, respectively, positioned on exon 1 and exon 2 of sgo1. Relative gene expression (2−∆∆Ct) was calculated after normalization of sgol1 expression to the average expression observed in sibling embryos using eef1g as a reference gene (primers used: 5′‐TCGTCTGAAGATTGCGAGTG‐3' and 5′‐ACCCTGGTAAGCTGGAACCT‐3′).Embryos at 5 dpf from an adult 3.42). Stroke volume (SV) by: EDV‐ESV. Ejection fraction (EF) by: SV/EDV. Cardiac output (CO) by: SV*Heart rate.Embryos were placed in 200 μM of 1‐phenyl‐2‐thiourea (PTU) 20–24 hours post fertilization (hpf) to prevent pigmentation. At 3 and 5 dpf, embryos were embedded in 0.3% agarose prepared in E3 medium containing 16 mg/mL MS‐222. Recordings were performed at 150 frames per seconds (fps) using a high‐speed inverted light microscope at 28°C. Whole larvae were then genotyped. Heart rate measurements and contractility parameters were analyzed using ImageJ . Hemodynamic parameters such as volumes and surface areas were analyzed using ImageJ by drawing an ellipse on top of the ventricle at end‐diastole and end‐systole. Per heart, six ellipses were analyzed: three at diastole and three at systole. Values were averaged. ImageJ provided the values for the minor and major axis of each ellipse. End diastolic and end systolic volume (EDV/ESV) were calculated by: (1/6)*(π)*(major axis)* according to the standard Hubrecht fish facility feeding protocol. The fluorescently labelled food was prepared by mixing 100 mg of powdered larvae feed  with 150 μL of fluorescently labelled beads  (Invitrogen) on a watch glass with 50 μL demi water, then spreading the paste on a watch glass in a thin layer to dry in the dark. The fluorescently labelled tracer was then scraped off the watch glass and crushed to a fine powder to store at 4°C in the dark. At day 7, larvae were administered the fluorescent tracer at a concentration of 2 mg per petri dish, in accordance with a published study.Larvae from a 3.6sgo1+/− incross were fixed overnight at 4°C in fixation and cartilage staining solution . Fixed larvae were dehydrated in 70%, 95%, and 100% EtOH (10 minutes each), then stored in 4°C until imaging. Before imaging, larvae were genotyped by tailfin clips to separate sgo1−/− from wild‐type siblings. Larvae were washed in 100% MeOH and placed in Murray's solution  for imaging. Brightfield imaging was performed on a Zeiss Axioplan microscope.Larvae at 5 dpf from a 3.7sgo1+/− incross were fixed at 5 dpf in 4% paraformaldehyde (dissolved in phosphate buffer containing 4% sucrose) overnight at 4°C. Genotyping was performed by tailfin clips to separate homozygous mutants from wild‐type larvae. Larvae were washed in PBS for 30 minutes at room temperature, then dehydrated in ethanol , washed in butanol at room temperature (3× 30 minutes), kept in paraffin 3× 30 minutes at 58°C before embedding in paraffin. Larvae were sectioned transversely at 6 μm thickness using a microtome (Leica RM2035). Hematoxylin and Eosin (H&E) staining was performed on sections according to standard laboratory protocol for paraffin sections. Images were acquired using a Leica DM4000 B LED upright automated microscope.Larvae from a 3.8In situ hybridization was performed on 2 dpf wild‐type embryos using sgo1 probe as previously described.sgo1+/− incross were genotyped at 3 dpf by tailfin clips to separate into homozygous mutants and wild types. All embryos were dehydrated in EtOH at 70%, 96%, and 100%, then washed in monomer technoit 8100 (technovit + hardnerI). Embryos in technovit 8100 were embedded on coverslip and left to polymerize overnight at 4°C. Sectioning was performed using Leica 2050 microtome using a triangle glass knife with a mounted waterbath. Sections of 7 μm in thickness were transferred on a noncoated slide and dried overnight at room temperature. Slides were stained for 5 minutes with 0.1% Neutral red , airdried and cover slipped with Pertex (histolab 00801‐EX). Imaging of stained sections was performed using Leica DM4000 B LED upright automated microscope.3.9sgo1−/− and wild‐type larvae cryosections of 7 μm thickness as previously described.The rhodopsin staining  was performed on pre‐genotyped (by tailfin clips) 5 dpf 3.10sgo1+/− incross were fixed at 3 dpf in 4% paraformaldehyde and genotyped by tailfin clips to separate homozygous mutant from wild‐type larvae. Three washes of 10 min were performed using 4% sucrose (in phosphate buffer) followed by an 30 minute incubation at room temperature in 30% sucrose (in phosphate buffer). Larvae were embedded in tissue‐freezing medium , frozen on dry ice, and kept at −80°C. Cryo‐sectioning using Cryostar NK70  was performed to obtain 7 μm thin sections. Staining of sgo1−/− and wild‐type larvae was performed. Using Roche In Situ Cell Detection Kit (TMR red# 12156792910). Sections were rinsed in PBT (PBS +0.1% Tween 20), incubated with collagenase Type II  for 30 minutes at room temperature, then blocked for 1 hour at RT with PBT supplied with DMSO and FBS, incubation with Anti‐phospho Histone H3 (Ser10) at 1:100  was performed overnight at 4°C. Slides were washed multiple times in PBT and incubated in labeling solution from the kit, supplied with the secondary antibody Alexa 555 Anti‐rabbit (1:500)  and DAPI (1:1000) for 1 hour at RT, washed multiple times in PBT and coverslip was added. Imaging was performed using Olympus Slideview VS200 digital slide scanner.Embryos from a 3.11sgo1+/− incross were genotyped by tailfin clips at 3 dpf to separate homozygous mutant from wild‐type larvae. At 5 dpf, larvae were placed separately in a 48 well plate  to record their motility and behavior to periods of 50 minutes dark and 10 minutes light using an automated video‐tracking system after 1 hour of acclimatizing in the dark. This method of imaging and video analysis was adjusted from the VMR, as previously described.Larvae from a 3.12t‐test, with the exception of the Kaplan‐Meier curve, where Log‐rank (Mantel‐Cox) test was used. All data are presented as mean ± SEM, and P < .05 was considered significant. *P ≤ .05, **P ≤ .01, ***P ≤ .001, ****P ≤ .0001, n.s. P > .05. n denotes the number of fish used per dataset.Statistical analysis and drawing of graphs and plots were carried out in GraphPad Prism . Differences between two groups were analyzed using the unpaired Student's Sarah Kamel: Conceptualization ; data curation ; formal analysis ; methodology ; visualization ; writing – original draft . Sanne Broekman: Data curation ; formal analysis ; visualization . Erwin van Wijk: Conceptualization ; funding acquisition ; supervision ; writing – review and editing . Federico Tessadori: Quantitative RT‐PCR experiment and interpretation of data. Jeroen Bakkers: Main funding, study design, data interpretation, supervision, manuscript reviewing and editing.Movie S1 High speed video played in slow motion of sgo1+/+ embryo at 5 dpfClick here for additional data file.Movie S2 High speed video played in slow motion of sgo1+/− embryo at 5 dpfClick here for additional data file.Movie S3 High speed video played in slow motion of sgo1−/− embryo at 5 dpfClick here for additional data file.