Stem cell technology and embryonic stem cell models are of great interest in biomedical research since they provide deeper insights into, e.g., neurogenesis and early mammalian brain development. Despite their great scientific potential, the reliable establishment of three-dimensional embryoid bodies (EBs) remains a major challenge, and the current lack of standardization and comparability is still limiting a broader application and translation of stem cell technology. Among others, a vital aspect for the reliable formation of EBs is optimizing differentiation protocols since organized differentiation is influenced by soluble inducers and EB size. A microfluidic biochip array was employed to automate cell loading and optimize directed neuronal and astrocytic differentiation protocols using murine P19 embryoid bodies to facilitate reliable embryonic stem cell differentiation. Our gravity-driven microfluidic size-controlled embryoid body-on-a-chip system allows (a) the robust operation and cultivation of up to 90 EBs in parallel and (b) the reproducible generation of five increasing sizes ranging from 300 µm to 1000 µm diameters. A comparative study adds two differentiation-inducers such as retinoic acid and EC23 to size-controlled embryoid bodies to identify the optimal differentiation protocol. Our study revealed a 1.4 to 1.9-fold higher neuron and astrocyte expression in larger embryoid bodies (above 750 µm) over smaller-sized EBs (below 450 µm), thus highlighting the importance of EB size in the establishment of robust neurodevelopmental in vitro models.  The global trend towards improved in vitro three-dimensional tissue models in biomedical research has led to an increased application of advanced stem cell technologies using iPSC- and embryonic stem cells. Due to their inherent pluripotency and self-renewal capacity, embryonic stem cells (ESCs)–the inner cell mass of a blastocyst–provide an indispensable and powerful tool in basic and applied research. By cultivating embryonic stem cells in three-dimensional 3D) cellular aggregates, early stages of embryonic development can be recapitulated in vitro in the form of so-called embryoid bodies (EBs). These in vitro derived structures give rise to all three primary germ layers, including the ectoderm, the mesoderm, and the endoderm ,2,3. TheD cellulaDespite this tremendous translational potential of embryonic stem cell technology, many technical challenges still persist, including the reproducible generation of identical embryoid bodies, a fact that still hampers their mainstream application and limits their therapeutic power. In other words, a significant challenge in achieving homogeneous lineage-specific differentiation from heterogeneous embryoid bodies to date is associated with the inability to properly control cellular microenvironments ,10,11. I(1)optimal cultivation conditions over several days,(2)precise control of nutrient and gas exchange,(3)user-friendly cell loading capacities,(4)parallel embryoid body production and harvesting option,(5)simple operation to ensure tissue maintenance.One cell culture technology capable of precisely regulating microenvironments of stem cells is called organ-on-a-chip technology, where 3D-cell assemblies, spheroids, and organoids can be cultured inside microfluidic devices under near-physiological conditions ,31,32,33In this study, we have employed a microfluidic embryoid body-on-a-chip array to study, in detail, the effects of embryoid body size on both neuronal and glial differentiation. Additionally, the cooperative impact of the two chemical inducers retinoic acid (RA) and its photostable synthetic analog EC23, are tested in a dose-dependent manner over a differentiation period of 14 days. Retinoic acid is chosen in this study because it is essential in the differentiation process of the adult hippocampus and has long been recognized as a robust neuronal differentiation-inducing molecule in vitro ,35,36,372 humidified atmosphere. The medium was changed every three days, and cells were passaged at 90% confluency. After a washing step with Dulbecco’s Phosphate-Saline Buffer , cells were enzymatically detached with 3 mL 0.25% Trypsin-EDTA  and subsequently pelleted at 140 × g for five minutes. The supernatant was aspirated, and the cell suspension was adjusted to required cell densities with a complete α-MEM medium. P19 cells were passaged twice or three times per week at a ratio of 1:4–1:6.The murine embryonal carcinoma cells line P19  was maintained in Minimum Essential Medium Alpha Modification  supplemented with 7.5% newborn calf serum , 2.5% fetal bovine serum , and 1% antibiotic/antimycotic solution  under standard cell culture conditions at 37 °C in a 5% COAs described previously, the master mold and microfluidic biochip array fabrication were conducted . In brie2 humidified atmosphere and placed on a rocker platform set to a flow rate of 4 μL/min at 1° tilting angle and 1 rpm with medium exchange every two days. Chips were initially coated with Lipidure® CM52006  using 100 µL ethanolic Lipidure® solution to induce EB formation and inhibit cell outgrowth. After evaporation at 80 °C, chips were filled with 70% ethanol and subsequently exchanged washed three times with PBS supplemented with 1% antibiotic/antimycotic solution  under sterile conditions. Finally, the channels were flushed twice with cell culture media and filled with P19 cell suspensions in a complete α-MEM medium. Chips were maintained and incubated at 37 °C in a 5% COA dye exclusion assay based on a solution containing 1.0 mg/mL Hoechst 33,342  and 2.0 mM ethidium bromide homodimer-1  in PBS was performed to determine cell viability. EBs were rinsed with 200 µL of the staining solution two times and incubated for 30 min at 37 °C. To avoid photobleaching, all steps were done in the dark. Fluorescence signals were analyzed by fluorescence imaging on an IX83 live-cell microscope setup  using DAPI  and TRITC filter sets . After three days post-seeding, EBs were treated with 0.5 µM, 1.0 µM, and 10.0 µM of RA  or EC23  for two or four days. After exposure, media was changed to neuronal differentiation medium containing 1× Neurobasal Medium , 2% B27 supplement , 0.5 mM L-Glutamine  and 1% antibiotic/antimycotic solution  and exchanged every second day. For static experiments, 200 µL of 5000 cells/mL were seeded in 96-ultralow attachment plates  and treated as described previously. After two- and four-day induction, the EBs were transferred to 0.1% gelatin-coated microscope slides  with 18 mm well sizes (0.5 mm chamber height). +; with MgCl2 and CaCl2, Sigma-Aldrich, Vienna, Austria) overnight at 4 °C. The next day, EBs were washed with DPBS+ and permeabilized with 0.2% Triton X-100  for 15 min at RT. After blocking for two hours with 1% bovine serum albumin , primary antibodies for neuronal microtubule-associated protein 2 (MAP2) and astrocytic glial fibrillary acidic protein (GFAP) were diluted in blocking solution 1:1000 and 1:250 and incubated at 4 °C overnight. Specimens were washed twice for 15 min with DPBS+ on a shaker platform at 600 rpm  and incubated with goat anti-rabbit 555  and goat anti-chicken 488 secondary antibody  solution for one hour at RT. Samples were washed twice for 15 min with DPBS+, and nuclei were counterstained for one hour with 2 mg/mL DAPI  diluted in DPBS+. After a final DPBS+ washing step, samples were transferred onto a microscope slide and mounted with a Vectashield® soft embedding mounting medium . For immunofluorescence staining, the EBs at day one, seven, and 14were fixed with 4% paraformaldehyde in Dulbecco’s phosphate-buffered saline  and a high-resolution ORCA-Flash 4.0 camera . For imaging the whole cultivation channel, MIA scans were conducted using 4× and 10× magnification. All images were processed by ImageJ  to quantify the spheroidal diameter of the embryoid bodies. To investigate spheroid size and morphology, bright-field images were taken by using an IX83 microscope  equipped with temperature, COFor viability determination of embryoid bodies, image backgrounds were subtracted , and mean fluorescence intensities of embryoid bodies were calculated as described in Equation (1):To evaluate the differentiation pattern, the fluorescence intensities of the specific antibodies were set in relation to DAPI. For that, the image channels of each picture were merged, and the signals were measured by using the built-in RGB measure plugin of ImageJ. For morphometric analysis of neuronal and astrocytic networks of EBs, AngioTool (Version 0.6a) was used with optimized analysis parameters .p values < 0.05were considered as statistically significant . The data are presented as the mean of experiments ± standard deviation (SD).Statistical significance was tested using Student’s t-test, ordinary one-way ANOVA, two-way ANOVA, or mixed-effect analysis performed by GraphPad Prism 8 . Although the Vitamin A derivatives RA and EC23 are commonly used for triggering stem cell differentiation to a neuronal phenotype , elevateFollowing the verification of suitable embryoid body viabilities, the ability of RA and EC23 to induce P19-stem cell differentiation into neurons and astrocytes was investigated in subsequent sets of experiments. Similar to RA, the photostable analog EC23 resulted in a time-resolved increase in MAP2 positive cells, independent of the induction strategy. However, strong concentration-dependent effects were observed in embryoid bodies treated with 10 µM EC23, revealing no significant differences between untreated and treated EBs at day 14 of differentiation for both induction scenarios, indicating a RA receptor saturation effect see A–C. Intep < 0.01) but not for RA, while astrocytes responded similarly for both inducers (p < 0.01). Additionally, neuronal branching was improved significantly in the presence of RA (p < 0.0021), and both treatment strategies improved astrocytic branching compared to the untreated control embryoid bodies (p < 0.01). Moreover, process length was significantly increased only for astrocytes and not neurons (p < 0.05). Furthermore, neural network length was significantly pronounced for neurons when embryoid bodies were treated with RA p < 0.001). A similar comparative analysis between neural morphology parameters of embryoid bodies is shown in Next, neural morphometric parameters including network area, degree of branching, process length, and overall network length were investigated for both inducers. 5, 2.5 × 105, and 5.0 × 105 cells/mL. However, increasing cell densities of 7.5 × 105 cells/mL, 1.0 × 106 cells/mL and 3.0 × 106 cells/mL produced excellent yields of 91 ± 9%, 98 ± 5% and 96 ± 6%, respectively. Interestingly, the overall lower average yield of ca. 90% using a seeding density of 7.5 × 105 cells/mL was caused by limited ability to generate embryoid bodies  inside the smallest microwells of 300 µm diameters, as shown in p > 0.01) using three seeding densities of 7.5 × 105 cells/mL, 1.0 × 106 cells/mL, and 3.0 × 106 cells/mL. While no discernable influence of seeding density on embryoid body growth was detected, cell viability was affected in the presence of high cell seeding densities over time. As an example, using the highest seeding density of 3.0 × 106 cells/mL yielded the lowest and gradually stronger declining viability values, particularly in smaller spheroid sizes and significant size-dependent differences (p < 0.05), while the lower 7.5 × 105 cells/mL did not show any toxicities (p = 0.49) independent of cultivation time and increasing embryoid body sizes. Notably, the lowest initial seeding density also showed better reproducibility and size control throughout the entire cultivation period. Consequently, the final set of experiments covering on-chip neuronal and astrocytic differentiation analysis using various embryoid body sizes was conducted using an initial cell seeding density of 7.5 × 105 cells/mL. An initial cell seeding optimization study was conducted to identify the ideal P19 cell density needed to reproducibly load each microwell with identical cell numbers inside the microfluidic biochip array. p < 0.001) and 1.6 ± 0.2 (p < 0.001) fold change for EC23 at day 14 for 500 µm wells. Similar concentration-independent effects were observed for embryoid bodies that have been generated in 900 µm sized microcavities (p < 0.05 for RA and EC23). It is important to highlight that embryoid bodies generated and differentiated within 900 µm microcavity displayed significantly higher MAP2 levels than the smaller 500 µm embryoid body size, thus confirming the direct influence of size on cellular differentiation status. In turn, untreated embryoid bodies cultivated in 500 µm microcavities showed no GFAP signal and completely lacked any astrocyte cell population. In contrast, RA and EC23-treated embryoid bodies showed a significant concentration-independent increase of astrocytes (p < 0.01), as shown in In a final set of experiments, embryoid body size-related effects on neural and astrocytic differentiation were monitored over a total cultivation time of 14 days post-induction with either RA or EC23 inducers. As shown in our differentiation optimization studies see , a four-Embryoid bodies serve as an advanced cell-based tool to treat congenital neural disorders and regenerate neural tissues lost as a result of neurodegenerative diseases and injuries . HoweverOne biophysical parameter that affects the differentiation efficiency and rate of embryonic stem cells is linked to the embryoid body size and its three-dimensional architecture , therebyIn conclusion, this platform demonstrates a facile approach to manipulate embryoid body size and their microenvironment enabling new opportunities to efficiently adapt stem cell culture conditions and direct cell fate for robust neurodevelopmental in vitro studies.


The global burden of colorectal cancer is high. Chemotherapy has been the backbone of colorectal cancer therapy for decades. Toxic side effects and frequently occurring drug resistances remain challenging problems. Therefore, exploring natural compounds with low or even no toxicity holds great potential. However, natural curcumin is poorly absorbed, limiting its clinical use. Therefore, our focus was to screen different molecular types of colorectal cancer to find the ones with the highest sensitivity to curcumin. We observed very individual responses to curcumin for various colorectal cancer cell lines. Most curcumin-sensitive cell lines were of the microsatellite-stable molecular type, and expressed high baseline levels of the IκBα protein. Contrarily, curcumin-resistant lines were mainly microsatellite instable, with low baseline IκBα levels. Considering all of the data obtained, we conclude that patients with microsatellite-stable tumors and high baseline IκBα protein expression would benefit from treatment with novel curcumin formulations and derivatives.Clinical utilization of curcumin in colorectal cancer (CRC) was revived as a result of the development of novel curcumin formulations with improved bioavailability. Additionally, identification of biomarkers for curcumin sensitivity would also promote successful clinical applications. Here, we wanted to identify such biomarkers in order to establish a predictive model for curcumin sensitivity. Thirty-two low-passage CRC cell lines with specified tumor characteristics were included. Curcumin suppressed cell proliferation, yet sensitivity levels were distinct. Most curcumin-sensitive CRC cell lines were microsatellite stable and expressed high levels of IκBα. The predictive capacity of this biomarker combination possessed a statistical significance of 72% probability to distinguish correctly between curcumin-sensitive and -resistant CRC cell lines. Detailed functional analyses were performed with three sensitive and three resistant CRC cell lines. As curcumin’s mode of action, inhibition of NF-κB p65 activation via IκBα was identified. In consequence, we hypothesize that novel curcumin formulations—either alone or, more likely, in combination with standard therapeutics—can be expected to prove clinically beneficial for CRC patients with high IκBα expression levels. In the database “Global Cancer Observatory”, the recent statistics for 2020 revealed that 1,931,590 individuals for both genders and all ages combined were diagnosed with colorectal cancer (CRC), thus accounting for approximately 10.0% of newly diagnosed cancer cases. More than 935,000 (9.4%) deaths of CRC patients per year make it the second most common cancer-related cause of death [However, chemotherapy is still the backbone of clinical CRC management. Toxic side effects and drug resistances remain very challenging problems, thus limiting the efficacy of therapy and largely affecting patients’ quality of life ,5,6; theCurcuma longa (turmeric), which belongs to the ginger family [Curcumin, a natural yellow coloring agent, is a phytochemical derived from r family ,8; it har family , and hasr family ,10.cyclin D1, Bcl-2, Bcl-xL, COX-2, and MMP-9.In fact, most of the anti-inflammatory and antitumor properties of curcumin are considered to be mediated via blocking of the NF-κB pathway’s activation. This pathway has been shown to be upregulated in CRC, and is a major cause of drug resistance ,12. ZhouHowever, Wong et al.  reported® and liposomal complexes, which are currently under investigation—promise the possibility of a “revival” of this very old natural compound for oncological purposes [However, novel drug formulations—such as Merivapurposes ,16,17. IAs various CRC cell lines differ in their response to treatments due to genomic and phenotypic alterations and molecular mechanisms ,20,21,22The goal of the present study was to (1) assess the sensitivity of CRC cell lines with defined molecular alterations to curcumin, (2) identify the mode of action by mechanistically analyzing sensitive CRC cell lines, (3) identify biomarkers associated with curcumin sensitivity, and (4) predict the sensitivity of additional CRC cell lines to curcumin by establishing a predictive model using the previously identified biomarkers.A total of 32 low-passage patient-derived cell lines, established from 30 colon cancer (CC) and 2 rectal cancer (RC) patients, were used in the present study . CRC pat50 of curcumin ranged from 6.69 μM to 18.49 μM. Values of 0.17 μM to 80.95 μM for 5-FU, 0.04 μM to 12.80 μM for irinotecan, and <0.01 μM to 8.72 μM for oxaliplatin were observed [50 values of irinotecan and oxaliplatin were significantly lower than that of curcumin in CRC cells, with all p-values < 0.001. Using Spearman’s correlation analysis, no significant correlation between curcumin and any of the standard drugs was identified; the correlation coefficients were −0.12 for 5-FU (p = 0.545) (p = 0.446) (p = 0.089)  A, −0.15 = 0.446) B, and −0= 0.089) C; the laAs shown in 50 concentrations. The correlation coefficient was −0.72 and p was < 0.001 , and even an obvious dose-dependent response to curcumin was observed (p < 0.05). Cell death increased to 80% dead cells in the HROC357 and HROC69 cell lines when treated with 20 μM curcumin (p < 0.05) , 10 μM, 15 μM, and 20 μM curcumin for 72 h. As expected, cell death in HROC24, HROC113, and HROC285 was not significantly affected, even at the highest curcumin concentration of 20 μM. Conversely, the cell death rate was significantly higher in all three curcumin-sensitive cell lines compared to the control group (observed A. In HRO < 0.05) A. p < 0.001 (Confluent curcumin-sensitive (HROC18 and HROC357) and -resistant (HROC24 and HROC87) cells were treated with 0 µM (control) and 10 μM curcumin overnight, scratched, and the wounds’ diameters were subsequently measured daily for up to 5 days. Migration was not affected by curcumin in HROC24 and HROC87; however, in HROC18 and HROC357 cells, the cells’ ability to migrate was consistently weaker after 10 μM curcumin treatment compared to controls,  < 0.001 B. After incubation with 25 μM curcumin for 24 h, the immunoblot showed that the expression of the NF-κB p65 target protein survivin significantly decreased in HROC18, HROC24, and HROC357 cells, but not in HROC285 cells. The expression of Bcl-xL—another NF-κB p65 target protein—was unchanged in all cell lines except in curcumin-sensitive HROC18 cells, where it showed a lower expression. In addition, we also observed that curcumin induced accumulation of the DNA damage marker ɣH2AX in all four CRC cell lines—especially in curcumin-sensitive HROC357 cells .p < 0.05, p < 0.05). The increase in IκBα expression after exposure to 20 μM curcumin was as high as 70% , 10 μM, 15 μM, and 20 μM curcumin treatment for 24 h. In general, IκBα levels increased in all six CRC cell lines with rising curcumin concentrations A. Treatmr = −0.943, p = 0.017). Similar inverse correlations were obtained for 15 μM (r = −0.986 (p < 0.001)) and 20 μM curcumin (r = −0.928 (p = 0.006)), confirming that the cell viability is negatively associated with the IκBα expression level in CRC cell lines after curcumin treatment.Next, correlations of cell viability and IκBα expression levels after curcumin treatment  were investigated. For curcumin treatment at 10 μM concentration, cell viability and IκBα levels in the CRC cell lines were strongly correlated B, and haWe further measured the expression of NF-κB p65, as well as cytokines and chemokines induced by NF-κB p65 . After iMoreover, no significant decrease in IL-6 secretion B was obsBased on the above results, we hypothesized that curcumin exhibits its anticancer effects via IκBα, which inhibits the NF-κB p65 nuclear translocation. In order to verify this, CRC cells were incubated with 50 µM caffeic acid phenethyl ester (CAPE)—an inhibitor of NF-κB—for 24 h. After that, an increase in IκBα levels was consistently seen in HROC18 and HROC24 cells A, and ceFurthermore, we evaluated the IL-8 and CXCL-1 levels in CRC cells after incubation with curcumin and CAPE for 24 h. A significant decrease in IL-8 and CXCL-1 levels was consistently found in HROC18 and HROC24 cells compared to controls C,D. In HAPC, K-Ras, B-Raf, PIK3CA, TP53, ATM, SMAD4, RAD50, POLE, and PTEN—were investigated as possible biomarkers associated with the IC50 values of curcumin. CRC cell lines with mutated SMAD4 were significantly more sensitive to curcumin . No further correlations were found  than in microsatellite-instable (MSI) CRC cell lines  (p = 0.008) (Stratified by microsatellite (MS) status, the IC= 0.011) A. This s= 0.008) B.p = 0.035), with a 72% probability to correctly distinguish curcumin-sensitive from curcumin-resistant CRC cell lines  curve presented a statistically significant predictive value (area under the ROC curve (AUC) = 0.72, 95%CI: 0.54–0.90, ll lines C. APC, K-Ras, B-Raf, PIK3CA, TP53, ATM, SMAD4, RAD50, POLE, and PTEN are usually considered to be relevant CRC biomarkers [SMAD4 to be associated with curcumin sensitivity. Agarwal et al. [SMAD4 and TP53; however, SMAD4 was not considered as a biomarker with the ability to predict the sensitivity of CRC cells to curcumin. Even though 30 patient-derived low-passaged CRC cell lines with known SMAD mutational status—consisting of 4 mutated and 26 wild-type cell lines—were analyzed in the present study, this point needs to be further clarified in future studies using an even larger number of suited CRC cell models.The present study is the first report on the identification of a potential biomarker for curcumin sensitivity in CRC cells. Mutations in omarkers ,30,31,32omarkers . Here, wl et al.  reportedMMR gene expression levels are regarded as important biomarkers in cancer progression, for prognosis of outcome, and even for assessment of the efficacy of adjuvant chemotherapy [More, the MS status and otherapy ,36,37. IShakibaei et al.  reportedMMR genes, have a high risk of cellular carcinogenesis caused by elevated oxidative stress [Lynch syndrome (LS) patients, who are characterized by germline mutations in four of the e stress ,44,45. He stress . Since de stress , it is rHowever, several studies have come to differing conclusions. Majumdar et al. reported a better effect of curcumin together with resveratrol on MSI-positive HCT116 cells, but not on HT29 cells, which are MSS . Wei et 50 values were found in MSI compared to MSS CRC cell lines. In contrast to the contradictory studies mentioned above, all cell lines used in the present study were below passage number 50, and can thus still be considered as closely mirroring the biology of the original patients’ tumors [In our analysis, curcumin could decrease cell viability and induce cell death in all 32 CRC cell lines tested. However, higher curcumin IC’ tumors .Moreover, the key inhibitory protein of NF-κB p65 in the NF-κB signaling pathway—IκBα—was for the first time identified as an important biomarker with the potential to precisely predict curcumin sensitivity. We found that curcumin-sensitive cell lines expressed significantly higher levels of IκBα even without treatment, while curcumin-resistant cell lines frequently expressed low baseline IκBα levels. Mechanistically, the NF-κB signaling pathway represents the classical pathway responsible for the anticancer action of curcumin. Resistance to radiation therapy in most patients has been linked to an activation of the NF-κB pathway, and Sandur et al. suggested that inhibiting this pathway could resensitize tumors to radiotherapy . Indeed,Deregulation of NF-κB and its downstream targets has been proposed as an important targetable mechanism in CRC therapy resistance . IκBα isDifferent gene expression patterns have been identified in oxaliplatin-resistant and -sensitive CRC cell lines after treatment with curcumin plus oxaliplatin. Compared to oxaliplatin-sensitive CRC cells, curcumin inhibited the NF-κB p65 level in oxaliplatin-resistant cells more effectively . SimilarTaken together, experimental evidence has proven that curcumin treatment is associated with inhibition of NF-κB p65, activation of IκBα, suppression of IκBα phosphorylation, and downregulation of NF-κB-p65-related antiapoptotic and cell-proliferation-activating proteins ,59. It i50 of curcumin and the baseline IκBα level in CRC cell lines was observed, along with a strong association of cell viability with the IκBα expression levels after curcumin treatment. Surprisingly, IκBα expression levels were higher in MSS compared to MSI CRC cell lines. When combining IκBα levels with MS status as a biomarker for predicting curcumin sensitivity, the combination could correctly distinguish the curcumin-sensitive CRC cell lines from curcumin-resistant CRC cell lines with an approximately 72% predictive power. The major finding of the present study—that MSS CRC cells have higher IκBα levels, and are more sensitive to curcumin treatment—is novel and surprising. Nevertheless, since these data were obtained with a relatively large number of low-passaged cell lines, even allowing for statistical comparison, they can be considered to be relevant. Moreover, a significant correlation between the ICMMR gene expression was negatively correlated with inflammation-associated transcription factor (including NF-κB) expression [Our findings clearly raise the question of whether there is a link between the NF-κB pathway and MSI status. MSI cancers are prototypically hypermutated, and are well known to possess a variety of immune escape mechanisms to evade high immune pressure . Among tpression . Anotherpression . More impression . In conclusion, data from different studies indicate that cancer responses to curcumin—especially CRC—are mediated by inhibition of NF-κB p65 via IκBα. This additionally seems to be linked to the MMR system. However, the exact mode of action still needs to be determined. n = 32) were established and characterized as previously described [2 and 37 °C). Cells below passage 50 were used in order to ensure comparability of molecular characteristics between parental tumor and tumor-derived cell lines. Directly patient-derived and patient-derived xenograft (PDX)-derived CRC cell lines , 1 nM–5000 µM 5-FU, 0.1 nM–1000 µM irinotecan, and 1.3 nM–20 µM oxaliplatin  [A total of 1.0–1.5 × 10Subsequently, cells were washed with PBS, then stained using 0.2% crystal violet solution . After that, cells were again washed twice and allowed to dry completely. Photos were taken, and stained biomaterial present in the wells was quantified by dissolving in 1% SDS (AppliChem) and measuring the absorption at 570 nm (reference: 620 nm) ,66. Cell50 values of standard drugs from our previous work. These values were obtained using the method described above. In detail, the cited IC50 values of 5-FU and irinotecan in HROC87 and HROC113 were from [50 of irinotecan in HROC18 was from [50 of oxaliplatin in HROC57 was from [It is worth mentioning that we cited the results of ICere from , in HROCere from , and in ere from ; the IC5was from  and in Hwas from ; while twas from , and in was from . 5 CRC cells per well were seeded in a 6-well plate and left for incubation overnight. Cultures were exposed to curcumin and CAPE  as single or combination treatments for 72 h. Cell death was evaluated by apoptosis assay using Annexin V-FITC  and propidium iodide  labeling and flow cytometric analysis on a FACSCalibur device  with CellQuest™ Pro software (Becton Dickinson), and 20,000 events were recorded for each measurement. The data were analyzed using the following formula: 100% − (Annexin V- PI-) % [A total of 2 × 10- PI-) % .5 CRC cells were fixed using 400 μL of 2% Formafix  followed by an incubation in 400 μL of 1 × buffer P  for 10 min. For staining, cells were incubated in 100 μL of 1 × buffer P with or without 5 μL of PE-anti- IκBα antibody , and protected from light for 30 min at 4 °C. IκBα expression was analyzed by flow cytometry as described above, and 10,000 events were recorded for each measurement.In order to evaluate the baseline IκBα level, 5 × 10To assess the IκBα levels in cells after treatment, cells were pretreated with 5% FCS medium containing curcumin and CAPE as single or combination treatments for 24 h, prior to PE-anti- IκBα staining.Fully confluent CRC cells were incubated in medium without FCS, with and without the addition of 10 μM curcumin, for 24 h. Afterwards, the monolayers were scratched using a 20 μL pipette tip to generate a wound in the cell layer. The wounds’ diameters were measured daily for up to 5 days using a light microscope . 5 CRC cells were allowed to adhere in a 6-well plate overnight. Medium containing 5% FCS with or without 25 μM curcumin was changed, and cells were incubated for 7 days. Supernatants were collected and measured by ELISA according to the manufacturer’s recommendations for IL-6 (cat: 31670069) and IL-8 (cat: 31670089) , as well as CXCL-1 . In order to test the effects of CAPE on IL-8 and CXCL-1 levels, cells were incubated with curcumin, CAPE, or a combination of curcumin and CAPE for 24 h prior to measurement.A total of 5 × 105 CRC cells per well were seeded in a 6-well plate. After incubation overnight, the medium was changed to 5% FCS medium plus/minus 25 μM curcumin. After 24 h, cells were pelleted and collected, and immunoblots were performed using anti-survivin , anti-ɣH2AX , anti-Bcl-xL , and anti-NF-κB p65 . A total of 5 × 10r). Student’s t-test and ANOVA were used to compare the mean differences between groups, and linear regression was used for comparison of trends by the slopes of regression lines. All results were taken from at least three independent measurements. The predictive ability of biomarkers for curcumin sensitivity was analyzed using the ROC, and is presented as the AUC and the 95% confidence interval. p-Values lower than 0.05 were considered significant. All statistical analyses were performed using GraphPad Prism 6.0 .Cell viability at gradient concentrations of curcumin was plotted by a heatmap visualization technique using the TBtools software . PearsonIn summary, this study demonstrated that curcumin could inhibit CRC cell viability, increase cell apoptosis, and suppress cell migration. The 32 CRC cell lines analyzed varied greatly in their response to curcumin. The primary focus was to identify the subset of CRC cell lines that are highly sensitive to curcumin. By unraveling common features of the responsive cell lines, possible biomarkers were identified; these were subsequently suggested as predictors of curcumin response. Most curcumin-sensitive CRC cell lines had the following characteristics: MSS and/or high baseline IκBα expression levels. Meanwhile, curcumin-resistant CRC cell lines were mostly MSI, and showed low baseline IκBα levels. The predictive ability of this biomarker combination was statistically significant, with a predictive power of around 72% in a classical ROC/AUC analysis.These data further fuel the ongoing efforts to improve the bioavailability of curcumin in order to pave the way for successful clinical application. Based on our large panel of patient-derived low-passaged CRC cell models with known sensitivity and biomarker expression, novel curcumin-derived candidate drugs could now easily be screened. Since the HROC model collection also includes a large number of matching in vivo PDX models, even the complete preclinical drug testing could be managed.Based on the data obtained so far, we postulate that MSS patients with high baseline IκBα expression levels can benefit from clinical treatment with novel curcumin formulations and derivatives. Further studies will also have to analyze the combination of curcumin with standard chemotherapeutics.


Background: During myocardial infarction (MI), billions of cardiomyocytes are lost. The optimal therapy should effectively replace damaged cardiomyocytes, possibly with stem cells able to engraft and differentiate into adult functional cardiomyocytes. As such, cardiac atrial appendage stem cells (CASCs) are suitable candidates. However, the presence of elevated levels of advanced glycation end products (AGEs) in cardiac regions where CASCs are transplanted may affect their regenerative potential. In this study, we examine whether and how AGEs alter CASCs properties in vitro. Methods and Results: CASCs in culture were exposed to ranging AGEs concentrations (50 µg/mL to 400 µg/mL). CASCs survival, proliferation, and migration capacity were significantly decreased after 72 h of AGEs exposure. Apoptosis significantly increased with rising AGEs concentration. The harmful effects of these AGEs were partially blunted by pre-incubation with a receptor for AGEs (RAGE) inhibitor (25 µM FPS-ZM1), indicating the involvement of RAGE in the observed negative effects. Conclusion: AGEs have a time- and concentration-dependent negative effect on CASCs survival, proliferation, migration, and apoptosis in vitro, partially mediated through RAGE activation. Whether anti-AGEs therapies are an effective treatment in the setting of stem cell therapy after MI warrants further examination. Coronary heart disease (CHD) remains the leading cause of mortality and morbidity worldwide, with myocardial infarction (MI) being the most common form of CHD . MI resuMuch research effort has been put into unravelling the therapeutic potential and mechanisms of cell therapy with bone marrow cells (BMCs). Bone marrow contains hematopoietic stem cells (HSCs), endothelial progenitor cells (EPCs), and mesenchymal stem cells (MSCs) . Clinica+ stem cell population in ischemic conditions may be, in this context, beneficial [In the last years, our research group discovered a new type of cardiac stem cells named ‘cardiac atrial stem cells’ (CASCs). In contrast to other stem cells, CASCs display extraordinary cardiomyogenic differentiation properties, making them a promising candidate for cardiac regeneration . Isolatineficial ,10. CASCneficial . This gineficial ,12.In MI patients, levels of advanced glycation end-products (AGEs) are increased . AGEs arad libitum. In total, 62 female Sprague-Dawley rats  were used.Animal studies were conducted in accordance with the EU Directive 2010/63/EU for animal experiments and approved by the Local Ethical Committee for Animal Experimentation . All animals were kept in a temperature-controlled environment  with a 12 h–12 h light-dark cycle. They were fed a standard pellet diet with water available 2, 1 mM CaCl2, 11.8 mM Na-HEPES, 10 mM glucose, 20 mM taurine, pH 7.4), and the right atrial appendages were collected. The extracted right atrial appendage tissue was minced in pieces of ~1 mm3, washed with phosphate buffered saline (PBS), and enzymatically dissociated for 30 min in Hank’s Balanced Salt Solution containing 0.6 WU/mL collagenase NB 4  and 20 mM CaCl2. ALDH+ cells were stained according to the Aldefluor kit . ALDH+ cells were defined as CASCs and were flow-sorted (BD FACS Aria) in X-VIVO 15 media  supplemented with 20% fetal calf serum (FCS) and 2% penicillin/streptomycin (P/S). Isolated CASCs were seeded in 6-well plates at a density of 60,000 cells per well and incubated at 37 °C in a humidified incubator with a 5% CO2 atmosphere. Medium was changed every 2 to 3 days. When CASCs reached 80% confluence, they were harvested using trypsin. For all experiments, passage 1 CASCs were used.CASCs were harvested from the right atrial appendages, as described before . BrieflyAGEs were prepared as previously described . BrieflyProliferation and survival assays were executed, with a propidium iodide (PI) assay as described before by Gervois et al. and Lo Monaco et al. ,23. BrieCASCs were seeded in a 12-well plate at a density of 50,000 cells per well in X-VIVO medium with 10% FCS and 2% P/S. Five conditions were added to the medium: 400 µg/mL BSA, 50 µg/mL, 100 µg/mL, 200 µg/mL, and 400 µg/mL AGEs. After an incubation period of 72 h, the conditioned CASCs were harvested using trypsin and used for a transwell migration assay. In the ThinCerts  with a porous membrane of 8 µm pore size, 100,000 cells per condition were seeded in X-VIVO medium with 0% FCS and 2% P/S. ThinCerts were placed onto 24-well plates containing X-VIVO medium with 2% FCS and 2% P/S. After 24 h of migration, the ThinCerts were fixated with 4% paraformaldehyde (PFA) for 15 min and incubated with 0.1% crystal violet for 30 min. Cells that did not migrate were removed at the top side of the ThinCerts, after which the amount of transmigrated CASCs was quantified with AxioVision 4.6 software . Data were normalized to data obtained with 400 µg/mL BSA.® Caspase-3/7 Green Apoptosis Assay Reagent . Five conditions were added to the medium: 400 µg/mL BSA, 50 µg/mL, 100 µg/mL, 200 µg/mL, and 400 µg/mL AGEs. CASCs cultured in X-VIVO medium without FCS and 2% P/S were used as a positive control. Experiments were performed in triplicate. Images were taken after 24, 48, and 72 h of incubation using the IncuCyte® S3 Live-Cell Analysis System . Analysis of the area occupied by apoptotic cells was performed using the IncuCyte® SX1 Live-Cell Analysis System . Data were normalized to the positive control .CASCs were seeded in a 96-well plate at a density of 10,000 cells per well in X-VIVO medium with 2% FCS and 2% P/S. To study apoptosis, a caspase assay was performed using the IncuCyte2 incubator with the RAGE antagonist FPS-ZM1 . After 2 h’ pre-incubation, 400 µg/mL AGEs were added. After 24, 48, and 72 h, proliferation and survival were evaluated as described above. After 72 h of incubation, pre-conditioned CASCs were harvested and used for a transwell migration assay as described above. RAGE was inhibited to assess the contribution of RAGE activation in proliferation, survival, and migration of CASCs. Briefly, CASCs were pre-incubated at 37 °C in a 5% COp < 0.05 was considered statistically significant.Statistical analyses were performed using GraphPad Prism 9.0.0 software. Normal distribution of data was assessed with the Shapiro–Wilk test. Normally distributed data were subjected to a one-way ANOVA test with repeated measurements, followed by the Holm–Sidak’s Multiple Comparison test. When data were not normally distributed, the non-parametrical Friedman test was used followed by the Dunn’s Multiple Comparison test. All data are expressed as mean ± standard error of the mean (SEM). A value of As shown in As shown in To elucidate the effect of different AGEs concentrations  on CASCs apoptosis, a caspase assay was performed. The percentage of cells expressing caspase 3/7 was measured at different time points: 24 A, 48 FiB, and 72CASCs migration capacity was evaluated with a transwell migration assay after 72 h of incubation with different concentrations of AGEs. In To evaluate the contribution of RAGE activation in the observed deleterious effects of AGEs, proliferation, survival, and migration of CASCs were assessed after the incubation with the RAGE antagonist FPS-ZM1. Before exposure to 400 µg/mL AGEs, CASCs were pre-incubated for 2 h with 10 or 25 µM FPS-ZM1. Application of FPS-ZM1 alone (10 and 25 µM) did not affect the proliferative capacity nor the survival of CASCs .p = 0.06).CASCs proliferation A–C was ep = 0.07). Survival A–C was ep = 0.07).Representative examples of CASCs migration after 72 h incubation with 400 µg/mL AGEs and FPS-ZM1 are presented in Our study is the first to show that AGEs affect CASCs properties, namely survival, proliferation, migration, and apoptosis in vitro. Our data demonstrate that these effects are partially mediated through RAGE activation in a dose-dependent manner. Circulatory AGEs levels are significantly elevated in patients with acute MI ,25. HoweIn our study, we tested a broad range of AGEs concentrations (50 to 400 µg/mL). The AGEs concentration used in other in vitro studies investigating the effect of AGEs on stem cells, ranges from 15 to 500 µg/mL . There iEven if CASCs transplantation shows promising potential for cardiac regeneration post-MI, survival and regenerative capacity of cells remains an issue. Ischemic areas are known to be a hostile environment with increased levels of oxidative stress, inflammation, and fibrosis combined with increased AGEs tissue levels. Whether AGEs would affect the regenerative capacity of CASCs was unknown but could be important knowledge in the context of cardiac regeneration and the promising regenerative capacities of CASCs . In our It has been shown that the underlying mechanisms in which AGEs execute their negative effects on organ function are dependent and/or independent of RAGE receptor activation . Studies®  as an anti-diabetic therapy, was stopped due to financial issues [In vivo confirmation on the use of anti-AGEs therapies and their potential added value to stem cell transplantation after MI need to be confirmed in an animal model before this can be translated to the clinic. It has been shown in multiple in vitro studies that the PPARγ inhibitor rosiglitazone ,42, MAPKl issues . No othel issues ,48. In al issues . Howeverl issues ,42,43,44l issues ,51, arthl issues , and Parl issues . sRAGE sWe found that AGEs had a time- and concentration-dependent, gradual effect on CASCs’ properties, by increasing apoptosis and by reducing survival, proliferation, and migration in vitro. The working mechanisms behind these effects remain to be further investigated, although we have shown that RAGE activation is an important contributor of these AGEs-related negative effects. Whether targeting AGEs in vivo could improve CASCs’ therapeutic capacity after MI, remains to be further investigated.


Even though exenatide is known to be chemically and physically unstable at pH 7.5, there lacks a systematic evaluation of the impact of pH and excipients on the peptide solution stability. In this study, we established analytical methods to measure the chemical and physical degradation of the peptide in solution. Exenatide remained relatively stable at pH 4.5 when incubated at 37 °C. At pH 5.5–6.5, degradation was driven by oxidation, while driven by deamidation at pH 7.5–8.5. Significant aggregation of exenatide at pH 7.5 and 8.5 was detected by size exclusion chromatography and dynamic light scattering. Each pH value greater than 4.5 exhibited unique profiles corresponding to a loss of α-helical content and an increase in unordered structures. The addition of sugars, including mannitol, sorbitol and sucrose, conferred small protective effects against peptide aggregation when incubating at pH 7.5 and 37 °C, as measured by size-exclusion chromatography and dynamic light scattering. The results of this study will be useful for investigators developing generic exenatide products, peptide analogs and novel exenatide drug delivery systems.Exenatide, a glucagon-like peptide-1 receptor agonist, is the active pharmaceutical ingredient in Byetta Teva Pharmaceuticals reached an agreement with AstraZeneca allowing them to manufacture and commercialize a generic version of Byetta® as of October 2017 but has yet to reach the market due to its pending ANDA FDA approval [® [Exenatide, also known as exendin-4, is a 39-amino acid glucagon-like peptide-1 (GLP-1) receptor agonist that acts as an incretin mimetic for the treatment of type II diabetes mellitus ,3,4,5. Enjection ,7. In 20approval ,11. Asidroval [® ,13,14. Sroval [® ,15,16,17® [A recently published FDA guidance on ANDA submissions for generic synthetic peptides highlights the importance of characterizing product-related impurities that may affect their safety, immunogenicity and effectiveness . Five pe® .Thus, understanding the chemical and physical stability of exenatide is critical to the development of potential generic exenatide drug products and novel extended-release formulations. Identifying the peptide’s potential mechanisms of degradation allows for the optimization of formulation and selection of appropriate manufacturing conditions to avoid the formation of product impurities ,21. At p® Pharmaceuticals . All other materials were purchased from commercial suppliers.Exenatide powder was generously provided by Amnealw/v mannitol, sorbitol or sucrose) were dissolved in 30 mM HEPES buffer (pH 7.5). These solutions were then used to reconstitute exenatide powder. Excipient samples were compared against exenatide reconstituted in 30 mM acetate buffer (pH 4.5) containing 4.3% (w/v) mannitol, the negative control. Samples were incubated at 37 °C for 30 min to induce conformational equilibrium. Reconstituted exenatide samples at various pH and excipient conditions were subjected to 4 weeks of incubation at 37 °C, removed from the incubator at 0, 1, 2 and 4 weeks and immediately analyzed. No significant changes in pH were observed over the incubation.Exenatide solutions were prepared at a concentration of 0.5 mg/mL in various 30 mM buffer solutions according to their respective buffer capacities, including sodium acetate (pH 4.5), sodium citrate (pH 5.5), sodium phosphate (pH 6.5), HEPES (pH 7.5) and HEPBS (pH 8.5). To investigate the impact of excipients, salt (154 mM NaCl) or sugars (4.3% 2O (0.1% TFA) at a flow rate of 1 mL/min, with a gradient ramping from 30 to 55% ACN (0.1% TFA) over the course of 25 min. The column temperature was held at 40 °C. Exenatide samples were detected from UV absorbance chromatograms that were extracted at 280 nm.Exenatide and its chemical degradation impurities were separated by hydrophobicity by reverse-phase liquid chromatography on a C4-Pack column  with a Waters 2595 Alliance System  on interfaced to a 2995 Photodiode Array Detector . Samples were filtered and injected at a concentration of 0.125 mg/mL at a volume of 50 µL and delivered using a mobile phase of acetonitrile (ACN) (0.1% TFA)/H2O (0.05% TFA) at a flow rate of 0.3 mL/min with a gradient ramping from 30 to 55% ACN (0.05% TFA) over the course of 25 min. The column temperature was also held at 40 °C. The MS parameters included a capillary voltage between 1.2 and 2.0 kV, a ToF-MS range of 300–3200 m/z, a drying-gas temperature of 325 °C, a drying-gas flow rate of 12 L/min, a nebulizer pressure of 45 psi, and a fragmentor voltage of 225 V. The total ion chromatogram (TIC) was detected at 280 nm, with impurity peak mass extractions generated by Qualitative Analysis Mass Hunter Software version b0705 .Exenatide chemical degradation impurities were separated by reverse-phase liquid chromatography on the same C4-Pack column and analyzed by a dual electrospray ionization equipped Agilent 6520 Accurate-Mass Q-ToF . Samples were filtered and injected at a concentration of 0.125 mg/mL at an injection volume of 20 µL and delivered using a mobile phase of ACN (0.05% TFA)/HRs) were correlated with their MWs, imported onto a semi-log plot with the generated equation, that were used to estimate the MW of exenatide monomer and aggregate peaks (not shown). Exenatide samples were detected from UV absorbance chromatograms that were extracted at 280 nm.Exenatide physical degradation impurities were separated by molecular weight using size exclusion chromatography on a Superdex Increase 75 10/300 GL column  with a Waters 2707 autosampler  connected to a 1525 binary HPLC pump , interfaced with a 2489 UV/Vis detector . Samples were filtered and injected at a concentration of 0.125 mg/mL at a volume of 50 µL and isocratically delivered with a pH 7.4 PBS mobile phase at a flow rate of 1 mL/min over the course of 25 min. Molecular weights of exenatide peaks were determined from a standard calibration, generated from the injection of Uracil (MW 114 Da), Aprotinin (MW 6.5 kD), Cytochrome C (MW 14.5 kD), Carbonic Anhydrase (MW 19.5 kD) and BSA (MW 65 kD). Maximum peak height retention times for each standard . A total of 100 µL aliquots of undiluted exenatide (0.5 mg/mL) were placed into low-volume TruView cuvettes  at an undiluted concentration of 0.5 mg/mL and analyzed. Attenuation and accumulation numbers were automatically optimized for each sample by the instrument prior to analysis. Particle size distributions were reported by volume and combined into 0.3–10, 10–100, 100–500, 500–1000 and 1000+ nm size ranges to match our instrument’s limit of detection specification ranges .Intrinsic fluorescence was used to determine the exenatide’s tertiary structure. Undiluted exenatide samples were placed into a QS 1.5 mm quartz cuvette  and analyzed at a concentration of 0.5 mg/mL on a SpectraMax M3  plate/cuvette reader. The fluorescence was measured using a wavelength emission range of 280–450 nm and an excitation wavelength of 270 nm while obtaining fluorescence at 6 flashes per reading.Circular dichroism was used to determine exenatide’s secondary structure. Exenatide samples were analyzed at a concentration of 0.125 mg/mL on a J-815 . Instrument sample temperature was held at 37 °C using a Peltier attachment. Circular dichroism spectra were averaged from 5 collected scans over a wavelength range of 245 to 195 nm, with an accumulation rate of 20 nm/min and a data integration time (DIT) of 4 s. Percent contributions from α-helix, β-sheet and unordered secondary structures were quantified using a third-party Spectra Manager Suite (JASCO) add-in, CDPro analysis. In this software, the CONTIN method was chosen, using soluble-membrane protein 56 (SMP 56) as the reference protein for secondary structural analysis.Liquid chromatography-mass spectrometry (QToF) was implemented to analyze the formation of exenatide chemical impurities. A short-term study was performed by incubating exenatide, reconstituted at pH 8.5, at an elevated temperature of 60 °C for 24 h. ® is commercially formulated in an acetate buffer solution at pH 4.5. On the other hand, rapid, pH-dependent degradation of exenatide was observed at pH 6.5, 7.5 and 8.5. We also observed a loss of total AUCs, indicating some precipitation occurring at pH 7.5 and 8.5 after 2 and 4 weeks, decreasing to 71.9 ± 10.1% and 70.9 ± 9.8% relative to day 0, respectively  were indicators of unfolding through increased solvent exposure of Trp A–C. ExenCircular dichroism is useful in combination with intrinsic fluorescence, providing further information on changes of underlying secondary structural content, including α-helical, β-sheet and unordered content. Exenatide samples were qualitatively measured by circular dichroism spectra D–F and t® formulation).Sugar excipients are generally known to offer protection against aggregation during freeze/thaw cycles and long-term storage, while other excipients, such as salt, may induce aggregation ,26. The Chemical degradation profiles, in terms of parent peak A and oxip < 0.05 for all solutions), indicating some minor protective abilities of these excipients against physical degradation. Physical degradation of exenatide at pH 7.5 in the presence of various excipients was mainly driven by aggregation.Physical degradation profiles, in terms of monomers D and aggFurther characterization of peptide aggregates was performed using dynamic light scattering. Our data showed that exenatide was stable for the negative control, with particles remaining in the 0.3–10 nm size range over the incubation. At pH 7.5, mannitol, sorbitol and sucrose exhibited stabilizing effects, where 100% of particles were retained in the 0.3–10 nm size range after 4 weeks of incubation . On the Subsequent structural analysis was performed by intrinsic fluorescence and circular dichroism. All pH 7.5 formulations experienced a large red shift of maximum emission wavelength and increase in maximum fluorescence intensity after 4 weeks, indicative of Trp-cage unfolding A,D. CD PThe goals of this study were to identify and characterize exenatide’s chemical and physical degradation using accelerated stability testing to elucidate exenatide’s potential underlying degradation mechanisms. To do so, we developed and implemented several orthogonal analytical techniques to investigate the impact of pH and the addition of excipients at elevated pH  over the course of long-term incubation at 37 °C. Chemical impurities were investigated using reverse-phase liquid chromatography and identified by liquid chromatography-mass spectrometry (QToF). Physical impurities were analyzed by size-exclusion chromatography, with a subsequent examination of particle size distributions by dynamic light scattering. Tertiary and secondary structures were analyzed by intrinsic fluorescence and circular dichroism. Overall, our study provides a valuable body of information regarding exenatide degradation that can be potentially useful for the approval of generic exenatide versions and the development of novel sustained-release formulations.Over the course of incubation, exenatide appeared to undergo a rapid, pH-dependent degradation at pH 6.5 and above while remaining relatively stable at lower pH. Analogously, endogenous GLP-1 has also shown degradation when pH was slightly increased, though pH was adjusted on a smaller scale . While tOur experiments investigating the impact of different excipients on exenatide’s stability at pH 7.5 revealed the limited ability of excipients to stabilize exenatide as well as their varied effects on exenatide’s degradation profiles. Of the excipients tested, only mannitol, sorbitol and sucrose had minor protective effects against physical degradation, where they slightly prevented monomer loss. Additionally, these excipients appeared to prevent particle size-shifting and even appeared to prevent oligomer formation relative to the formulation without excipient and with salt. Even so, these excipients were unable to prevent chemical degradation. The main impurities observed were aggregation and deamidation for all formulations after 4 weeks of incubation. While we determined that exenatide’s degradation kinetics were pH-dependent, we have not fully defined the mechanism of peptide aggregation. Currently, we propose that degradation occurs through a combination of chemical and physical degradation . At high


Trypanosoma brucei and is part of the dynein regulatory complex. TbTrypanin knockdown parasites showed motility defects in procyclic forms; however, silencing in bloodstream forms was lethal. Since TbTrypanin mutants show drastic phenotypic changes in mammalian stages, we decided to evaluate if the Trypanosoma cruzi ortholog plays a similar role by using the CRISPR-Cas9 system to generate null mutants. A ribonucleoprotein complex of SaCas9 and sgRNA plus donor oligonucleotide were used to edit both alleles of TcTrypanin without any selectable marker. TcTrypanin −/− epimastigotes showed a lower growth rate, partially detached flagella, normal numbers of nuclei and kinetoplasts, and motility defects such as reduced displacement and speed and increased tumbling propensity. The epimastigote mutant also showed decreased efficiency of in-vitro metacyclogenesis. Mutant parasites were able to complete the entire life cycle in vitro; however, they showed a reduction in their infection capacity compared with WT and addback cultures. Our data show that T. cruzi life cycle stages have differing sensitivities to TcTrypanin deletion. In conclusion, additional work is needed to dissect the motility components of T. cruzi and to identify essential molecules for mammalian stages.The flagellum of Trypanosomatids is an organelle that contributes to multiple functions, including motility, cell division, and host–pathogen interaction. Trypanin was first described in  Trypanosoma cruzi is a protozoan parasite of the Trypanosomatidae family, which bears a group of early diverging eukaryotes. This parasite is the etiological agent of Chagas disease, a potentially life-threatening illness that affects about 6–8 million people around the world (Trypanosoma cruzi epimastigotes (a replicative form) differentiate into metacyclic trypomastigotes (MTs), the infective and non-replicative forms in triatomine insects. MTs are released within insect feces and, once introduced into a host, can invade almost all nucleated cells. Once inside the host cells, they differentiate into amastigotes, a replicative form containing a short flagellum. After a period of multiplication intracellularly, the amastigote forms transform into bloodstream trypomastigotes; these cells egress the host cells, cross the extracellular matrix, and swim in a crowded and viscous environment (blood) to be able to reach different cell types of the mammalian organism . The PFR and axoneme are surrounded by the membrane flagellum and the PFR is linked to microtubules 4 to 7 of the axoneme similar to that in T. brucei  medium supplemented with heat-inactivated fetal bovine serum (10%), hemin, and penicillin/streptomycin  and incubated for 2 h at 28°C. After that, the culture was diluted 100-fold in TAU3AAG medium  and incubated for 72 h at 28°C. Then, we calculated the MT yield as previously reported . At least 100 random parasites for each cell line were analyzed using fluorescence microscopy.To do differential counting of nuclei and kinetoplast per cell, logarithmic phase parasites were DAPI stained as follows: 5 × 106 parasites/ml) were fixed with 70% methanol for 16 h, washed twice with PBS, resuspended in 100 µl of 2× PI solution , and added to 100 µl of PBS. These samples were analyzed by flow cytometry using the FACSCanto II equipment, and data were analyzed using the FlowJo 7.6 version software.Epimastigote forms at logarithmic phase  was performed using EuPaGDT (http://grna.ctegd.uga.edu/), selecting T. cruzi Dm28c (TriTryDB-28) as a reference genome and the Cas9 nuclease from Staphylococcus aureus (SaCas9)  as query, against all available genomes at TriTrypDB. The BlastN output was manually curated.Single guide RNA (sgRNA) design to target (SaCas9) . To ensuin-vitro transcription, we constructed a plasmid containing a T7 promoter, the SaCas9 sgRNA scaffold, and Hepatitis Delta Virus ribozyme (HDV), which we named pT7-SaCas9sgRNA-BsaI  and sgRNA-Trypa442-Minus (AAACAATCCGCATCTCATGACTCTC) and cloning in the BsaI sites of pT7-sgRNA scaffold-HDV, pT7-SaCas9-sgRNA-Trypa442 (in-vitro transcription using MEGAscript™ T7 Transcription Kit (Thermo Scientific), following the instructions of the manufacturer. sgRNA quantity and quality were confirmed by Nanodrop™ quantification and 2% agarose gel electrophoresis.For sgRNA TcTrypanin expression, the full-length coding sequence without the stop codon of TcTrypanin was PCR amplified with Trypa-For-XbaI (5′-AAATCTAGAATGCCACCAAAGGCGGTTCGTG-3′) and Trypa-Rev-BamHI (5′-AAAGGATCCCGACAATTCCCGCCGTCAGAAA-3′) oligonucleotides, using genomic DNA from the Dm28c clone as a template. The PCR product was digested with XbaI/BamHI and cloned at the same restriction sites in the plasmids pTREX-Amastin::GFP-Neo  donor. We electroporated epimastigote cultures twice with an interval of 7 days between each electroporation, following the electroporation conditions previously described  is 1,402 bp long and, after BamHI restriction, generates two bands (926 and 428 bp) detected on agarose gel. The resulting restriction digested fragments (restriction fragment length polymorphism—RFLP) was detected by ethidium bromide staining, and visualization was done with the UVP Bioimaging system.Wild-type epimastigotes of Dm28c were chosen to obtain mutant parasites with both alleles of escribed . After 4TcTrypanin null mutant clones (TcTrypanin −/−).After confirmation of gene editing, transfected parasites were cloned by limiting dilution in a 96-well ELISA plate to obtain clonal populations. To improve parasite growth during the cloning step, 90 μl of conditioned medium (media from the log-phase culture were recovered by centrifugation followed by supernatant filtering) was added to each well, and the plate was maintained in a wet chamber at 28°C and monitored each week for clone growth. One month later, we collected several clones and tested them with RFLP, as described above, to confirm the TcTrypanin −/−, TcTrypanin-addback (AB), and WT parasite were collected and counted in a Neubauer chamber. Ten microliters of cell culture at a density of 5 × 106–1 × 107 parasites/ml of the cell was placed on a microscope slide and filmed for at least 30 s at 5 frames per second using a Leica AF6000 Modular System microscope at ×20 magnification using darkfield illumination. Cell swimming was analyzed as previously described  BSA for 1 h at room temperature (RT). The parasites were incubated with a dilution 1:100 of the monoclonal antibody 2F6 (mAb 2F6), which recognizes a flagellar protein of ~70 kDa (TcTrypanin −/− (pTREX-TcTrypanin::GFP), WT (pTREX-TcTrypanin::GFP), and WT (pTREX-GFP) were fixed with paraformaldehyde 4%, coated on poly-lysine coverslips for 20 min, mounted with Fluoromount-G™ Mounting Medium with DAPI (Thermo Fisher), and visualized in a Nikon confocal microscope.Cell cultures of  ~70 kDa , for 16 6) of TcTrypanin −/− (pTREX-TcTrypanin::GFP), WT (pTREX-TcTrypanin::GFP), and WT (pTREX-GFP) were coated on poly-lysine coverslips and incubated for 20 min at RT. To preserve the parasite cytoskeleton, 40 μl of cold PEME  + Triton X-100 (1% v/v) was added and incubated for 10 s and washed twice with PBS Epimastigotes  buffer on ice for at least 5 min. The samples were centrifuged at 16,000×g for 20 min at 4°C. The pellet (cytoskeleton-enriched fraction) and supernatant fractions were collected in two microtubes. The pellet was washed twice with ice-cold PEME, resuspended in 50 μl of cold PEME, and stored at −20°C until the Western blot assay.To extract the epimastigote cytoskeleton in cell suspension, WT Dm28c expressing GFP or g. The cell pellet was washed with PBS and resuspended in SDS-PAGE loading buffer to have 1 × 107 parasites per loading in a polyacrylamide gel. After SDS-PAGE, the proteins were transferred to a PVDF membrane, which was blocked with 5% non-fat milk and incubated with polyclonal anti-GFP  at 4°C for 16 h. After the first antibody incubation, the membrane was washed three times with PBS + Tween 0.05% (v/v) and incubated with anti-rabbit conjugated with peroxidase  at 37°C for 1 h. Finally, the membrane was washed three times with PBS + Tween and antibody recognition was detected using the ECL Chemiluminescence Kit (Thermo Fisher) and X-ray film. Images were acquired exposing X-ray films on the UVP Bioimaging system.Total cell extracts were obtained by harvesting epimastigotes followed by centrifugation at 3,000×4) were placed in a 24-well plate containing coverslips and cultured in RPMI 1640 (Thermo Fisher) supplemented with 5% fetal bovine serum (FBS) in 5% CO2. WT, TcTrypanin −/−, and TcTrypanin −/− AB tissue-cultured derived trypomastigotes (TCTs) were harvested from supernatants from previously infected cultures and counted in a Neubauer chamber. Infection assays were performed using an infection ratio of 10 parasites per LLC-MK2 cell (MOI of 10:1) during 2 or 4 h. After the infection period, cells were washed with 1× PBS three times to remove extracellular forms, and fresh RPMI media supplemented with 5% of FBS was added. After 4 days of infection, the TCT release was analyzed by counting in a Neubauer chamber. TCT releasing was determined by harvesting TCTs 4, 5, 6, and 7 days after infection in the same conditions.Cultures of LLC-MK2 cells were treated with trypsin (0.05%) (Thermo Fisher) and washed twice with PBS. Cells  sequence and TcTrypanin (T. cruzi Dm28c clone—ID: C4B63_48g99). As expected, phylogenetic analysis shows that the Trypanosoma rangeli SC58 strain (ID: TRSC58_03641) is the closet related Trypanin among trypanosomatids followed by the T. brucei sequence (ID: Tb927.10.6350), sharing 76.32% identity (TcTrypanin presents higher divergence when compared with Leishmania species (55.41% to 57.62% identity). For additional evidence of its conservation, we searched for conserved Pfam domains using the SMART software (−60) starting at position 216 to 415, similar to its orthologs   reaching 1.2 × 107 parasites/ml, and stationary phase from day 7 to 10. TcTrypanin disruption is not essential for epimastigote survival, which allowed us to select fully edited clones. However, when we assessed the parasite epimastigote growth profile, TcTrypanin disruption interfered with parasite growth compared with WT cells. The reduction on TcTrypanin −/− growth is small, only becoming significant on the 7th day . The addback culture, named TcTrypanin-AB, was tested for TcTrypanin::HA expression. As shown in TcTrypanin::HA expression. The growth defect was reversed by TcTrypanin::HA overexpression in TcTrypanin −/− addback culture  compared with WT cells (34.96% ± 5.16%) and TcTrypanin −/− AB (35.5% ± 6.64%) (TcTrypanin −/− epimastigotes (6.5% ± 0.7%) compared with the WT cells (10% ± 1.4%), though it was not statistically significant  analysis. Similar to what was described for the nockdown , TcTrypaTcTrypanin disruption can interfere with parasite size (cell body area) and flagellum length, we performed the measurement of the flagellum from images obtained by immunofluorescence using a monoclonal antibody (clone 2F7) previously described as a flagellum marker , and the addback rescued the flagellum length (9.62 ± 3.29 µm) , and addback cells recovered their normal size (12.35 ± 3.15 µm2)  at the expected size. Furthermore, cell cytoskeleton preparation of parasite suspension followed by Western blot detection of TcTrypanin::GFP confirmed its presence in the cytoskeleton fraction , while the TcTrypanin∷HA addback culture (62.87 ± 43.79 µm) recovered the path length defect (TcTrypanin −/− mutant (2.867 ± 1.59 µm/s) was reduced compared with WT (5.793 ± 3.63 µm/s), with the addback partially recovering swimming speed (4.617 ± 2.68 µm/s) cultures . The addback of TcTrypanin::HA partially recovered the WT phenotype, by showing 14.28% ± 2.81% , while a non-significant difference was detected between WT and addback cultures   and cross obstacles during a natural infection  using conventional knockout strategy (unpublished data). However, to disrupt TcTrypanin in T. cruzi Dm28c, we performed genome editing with CRISPR/Cas9. For T. cruzi, successful editing can be achieved by different methods such as the stable expression of SpCas9 followed by the transient transfection with in-vitro-transcribed sgRNA (SpCas9 and sgRNA (SpCas9 followed by the transient transfection of a DNA template for the sgRNA expression directed by the T7 promoter (SaCas9 + sgRNA) protein SaCas9  mutants have detached flagella, and the epimastigote mutants are able to differentiate into infective metacyclic forms (reduced compared with WT). However, the infected cells do not release tissue culture-derived trypomastigotes, only amastigote-like forms with short flagella.arasites . Cytokin failure ; again, y defect . A previy defect . In thisT. brucei cell motility seems to shape SM (TcTrypanin −/− by CRISPR-Cas9 or differing morphogenesis in T. brucei. Flagellum attachment zone proteins (FAZ) form a complex that maintains flagellum attached to the cell body, but that also regulates the cell cycle and coordinate flagellum length, and FLA1BP silencing in T. brucei reduces flagellum length  , and T. shape SM . Sun et m length . HutchinTcTrypanin disruption has a higher impact on TCT infectivity depending on the time of infection (4 vs. 2 h) compared with wild-type parasites, suggesting that motility might be important for parasites to find host cells. Hence, mutant parasites need more time to infect a cell host and will likely be more exposed to the immune system during an in-vivo infection. For T. brucei, motility impairment leads to higher targeting by the higher clearance of antibodies . These data highlight the need for a large-scale functional genomics approach of flagellar components of T. cruzi.We described that The original contributions presented in the study are included in the article/JS-G, BB, NS-M, LVM, JSM, LAP-L, LCSM, and RW performed the experiments. JS-G drafted the manuscript. JS-G, NS-M, and WD critically revised the manuscript. All authors contributed to the article and approved the submitted version.The authors were supported by the Fundação Araucária  , National Counsel of Technological and Scientific Development (CNPq), and CAPES agency . LM has scholarship from CAPES, and JS-G and WD have scholarships from CNPq. This work was also supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), grants 2018/09948-0 and 2020/07870-4 to NS-M, and by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), grants 424729/2018-0 to NSM. We also thank the Wellcome Trust (211075/Z/18/Z) for funding RW research group.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


There is a growing recognition of the impact of gender and the social determinants of health on the clinical course of people living with HIV (PLHIV). However, the relative contribution of these factors to clinical outcomes of PLHIV is incompletely defined in many countries. This study was performed to gain a greater understanding of the non-clinical determinants of prognosis of PLHIV in Myanmar.Selected demographic, behavioural and socioeconomic characteristics of outpatients at two specialist HIV hospitals and one general hospital in Yangon, Myanmar were correlated with their subsequent clinical course; a poor outcome was defined as death, hospitalisation, loss to follow-up or a detectable viral load at 6 months of follow-up.3, 138 (62.4%) were male. Socioeconomic disadvantage was common: the median (interquartile range (IQR) monthly per-capita income in the cohort was US$48 (31–77); 153 (69.9%) had not completed high school. However, in a multivariate analysis that considered demographic, behavioural, clinical factors and social determinants of health, male gender was the only predictor of a poor outcome: odds ratio : 2.33 . All eight of the deaths and hospitalisations in the cohort occurred in males (p = 0.03).221 consecutive individuals with advanced HIV commencing anti-retroviral therapy (ART) were enrolled in the study; their median CD4 T-cell count was 92 (44–158) cells/mmMen starting ART in Myanmar have a poorer prognosis than women. Expanded implementation of gender-specific management strategies is likely to be necessary to improve outcomes.The online version contains supplementary material available at 10.1186/s12981-021-00364-w. This isGender also has a significant impact on the clinical outcomes of PLHIV . GloballThis pilot study was performed to determine the demographic and socioeconomic characteristics of PLHIV in Myanmar and the contribution of these factors to short-term morbidity and mortality. The study’s objective was to gain a greater understanding of the non-clinical determinants of prognosis to inform a more comprehensive approach to the local care of PLHIV.This study included participants of a randomized controlled trial (RCT) which was being performed at 3 ART clinics  in Yangon, Myanmar’s largest city.3 or WHO stage 3 or 4 disease), with no contraindication to IPT , peripheral neuropathy or allergy to isoniazid).The RCT examines immediate versus deferred isoniazid preventive therapy (IPT) in PLHIV with advanced disease. The RCT commenced in January 2018 and is continuing at the time of writing. Participants were eligible for the RCT if they were ART naïve adults (≥ 18 years) with advanced HIV (CD4 cell count < 200 cells/mmpro forma. Household income data were collected in Myanmar Kyat (MMK), but are expressed here in United States Dollars (USD) using an exchange rate of 1 USD to 1300MMK. The participants were then followed sequentially. The primary endpoint for the study was a poor clinical outcome, which was defined as death, hospitalisation, loss to follow-up or a detectable viral load (≥ 40 copies/mL) at 6 months.RCT participants were identified in the outpatient ART clinics of the three study sites and enrolled into the study after written, informed consent. Between January and October 2019, participants were also invited to participate in this study. After study doctors obtained written, informed consent for this substudy, demographic, socioeconomic, and selected clinical data were collected using a dedicated Data were de-identified, entered into an electronic database .There were 221 patients enrolled into this study, their median  age was 37 (32–46) and 138 (62.4%) were male. Their baseline characteristics and clinical course, stratified by gender, are presented in Table The demographic and socioeconomic characteristics of the men and women were similar. The median (IQR) age of the men was 37 (31–46) compared with 36 (33–44) among women (p = 0.95). A similar proportion of men and women were unmarried (66/138 (47.8%) versus 40/83 (48.2%), p = 0.95), a similar proportion had completed high school (43/136 (31.6%) versus 23/83 (27.7%), p = 0.54) and a similar proportion lived outside Yangon (38/138 (27.5%) versus 16/83 (19.3%), p = 0.17). The number of people living at home was comparable for men and women (median (IQR): 3 (2–4) versus 3 (2–5), p = 0.90) as was the monthly per-capita income (median (IQR): USD 46.15 (30.77–76.92) versus USD 51.28 (28.04–76.92), p = 0.90).However, men were more likely to be employed than women (119/138 (86.2%) versus 49/83 (59.0%), p < 0.0001). Men were also more likely to report hazardous alcohol use (47/137 (34.1%) versus 0/83, p < 0.0001), more likely to smoke tobacco (54/128 (42.2%) versus 2/83 (2.4%), p < 0.0001) and to chew betel (70/118 (59.3%) versus 7/82 (8.5%), p < 0.0001). The difference in intravenous drug use (3/138 (2.2%) versus 0/83) did not reach statistical significance (p = 0.29).2, p = 0.29) as was the mid-upper arm circumference (MUAC) (25 (22–27) versus 25 (22–27) cm, p = 0.99). The median CD4 cell count was comparable (92 (41–157) versus 92 (48–158) cells/mm3, p = 0.61), however men had less advanced clinical disease at enrolment (median (IQR) WHO stage 1 (1–3) versus 2 (1–3), p = 0.02) and a higher haemoglobin level (median (IQR) 12 (10–13.2) versus 10 (9–11) g/dL, p = 0.0001).The median (IQR) Body Mass Index (BMI) of men and women was similar (19.3 (17.2–22.2) versus 18.9 (16.1–22.3) kg/mOf the 221 patients enrolled in the study, there were 40  patients who were not able to have 6-month viral load testing; in 13 (32.5%) this was due to the evolving global COVID-19 pandemic which prevented clinic attendance for testing. Although none of these 40 patients died, were hospitalised, or were lost to follow-up, the absence of follow-up virological assessment precluded analysis of these patients using the pre-specified primary endpoint.Among the remaining 181 patients, 94 (52%) had a poor outcome  versus 0/83 women, p = 0.03), but there was no gender difference in loss to follow-up (10/138 (7.3%) versus 6/83 7.2%), p = 1.0). A detectable viral load was also more common in men than women (55/90 (53.5%) versus 20/61 (23.8%), p = 0.01). Gender was the only predictor of a poor outcome in univariate analysis  per-capita monthly income was only USD 48 (31–77)) in a country where the median per-capita monthly income is USD 51 [In contrast, the association between gender and outcomes seen in the cohort was striking. The association between male gender and poor outcome in PLHIV is consistent with previous work performed in Myanmar and other countries , 11–13. This is a pattern seen in numerous health conditions, not just HIV. Despite the fact that in many countries men generally have significantly greater privilege and opportunity than women, this advantage does not necessarily translate into better health outcomes . The mosIt is estimated that 87% (78–95) of women ≥ 15 are receiving ART in Myanmar compared with 70% (62–80) of men ≥ 15 years of age . In a coEven though the study only enrolled patients with advanced disease—who were generally profoundly socioeconomically disadvantaged—six months after starting ART, less than 3% had died and less than 7% lost to follow up. This suggests that once patients in Myanmar are able to access ART they usually do well. This may be explained by the fact that all HIV care in Myanmar is free of charge, that there is increasing access to contemporary ART (over 80% of this cohort received a dolutegravir-based regimen) and that medication adherence is generally excellent . The GloThis study has limitations. The lack of association between the social determinants of health and outcomes seen in this small cohort (which increases the likelihood of a type 2 error) does not suggest that these factors can be ignored. There is a significant body of work that demonstrates their importance and larger studies have shown their impact on HIV-related outcomes , 11, 24.Socioeconomic disadvantage was common in this cohort of PLHIV in urban Myanmar, however conventional SDH were not associated with important clinical outcomes during 6-months of follow-up. Gender had more prognostic utility and is likely linked to health-related behaviours that result from community level gender norms in Myanmar. Addressing and modifying these gender norms is challenging, however these data demonstrate that health care workers in Myanmar may need to provide HIV care that recognizes the contribution of gender to the clinical course if they are to deliver optimal outcomes.Additional file 1. Dataset.


The natural compound curcumin has been shown to have therapeutic potential against a wide range of diseases such as cancer. Curcumin reduces cell viability of renal cell carcinoma (RCC) cells when combined with TNF-related apoptosis-inducing ligand (TRAIL), a cytokine that specifically targets cancer cells, by helping overcome TRAIL resistance. However, the therapeutic effects of curcumin are limited by its low bioavailability. Similar compounds to curcumin with higher bioavailability, such as demethoxycurcumin (DMC) and 3,5-bis(2-fluorobenzylidene)-4-piperidone (EF24), can potentially have similar anticancer effects and show a similar synergy with TRAIL, thus reducing RCC viability. This study aims to show the effects of DMC and EF24 in combination with TRAIL at reducing ACHN cell viability and ACHN cell migration. It also shows the changes in death receptor 4 (DR4) expression after treatment with these compounds individually and in combination with TRAIL, which can play a role in their mechanism of action. Kidney cancer was responsible for 430,000 new cases and almost 180,000 deaths worldwide in 2020 ,6. Post-TRAIL is a cytokine from the tumor necrosis factor (TNF) superfamily that selectively induces apoptosis in cancer cells without displaying toxicity towards healthy cells ,11,12,13Curcuma longa (Turmeric), has demonstrated anti-inflammatory, antioxidant, immunomodulatory, anti-tumor and renoprotective activities [Curcumin -1,7-bis (4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), the primary bioactive extract from tivities , while btivities ,23. It ctivities . Recentltivities . Despitetivities .Another turmeric extract, DMC, displayed similar efficacy at inhibiting proliferation and progression in several tumor cell lines  and grea10 concentration , treatment with DMC decreases cell viability to approximately 76.69% compared to the vehicle control. A similar trend is observed following treatment with increasing concentrations of EF24 (3–48 μM), as ACHN cell viability decreases to approximately 18.69% after treatment with the IC10 concentration of EF24 (21.36 µM) (compared to vehicle control)  b.ACHN cells were pretreated using DMC or EF24 and a concentration of 50 ng/mL TRAIL was added after 4 h. Cell viability was analyzed after 72 h using an MTT assay. Cell viability of ACHN decreases after combination treatment with DMC and TRAIL in a dose-dependent manner. Cell viability decreases up to 32.73% using a 12 µM concentration of DMC in combination with TRAIL See a. After A scratch cell migration assay was performed using ACHN cells treated with either DMC or EF24. Images were taken at 0 and 72 h post-scratch and scratched, cell-free area was measured to see if treatment slowed scratch closure compared to control a. Scratc10 concentrations of DMC or EF24 individually and in combination with TRAIL showed an increase in DR4 protein expression after treatment with Curcumin, DMC, DMC + TRAIL, EF24 and EF24 + TRAIL , led to a reduction of ACHN cell viability to 76.69% compared to the vehicle control. After treatment with the synthetic curcuminoid EF24, ACHN cell viability decreased in a dose-dependent manner as well , PBS  and cell medium. For the experiments, 6-well plates were seeded at a density of 2 × 105 cells/well 24-well plates were seeded at a density of 4 × 104 cells/well.A human RCC cell line, ACHN  was used. ACHN cells were cultured in Minimum Essential Medium  with 1% Penicillin-streptomycin  and 10% fetal bovine serum . Cells were stored at 37 °C and 5% CO10 and IC50 concentrations of DMC (10.94 µM and 25.30 µM +/− 1.89 µM SEM) and EF24 (21.36 µM and 30.60 µM +/− 2.39 µM SEM), previously calculated using RPTEC/TERT1 (ATCC). In order to ensure that the treatments are not toxic to a high percentage of healthy kidney cells, the IC10 concentrations were chosen to proceed with. A treatment time of 72 h was chosen, as it was the time at which the maximum decrease in cell viability in RPTEC prior to cell doubling time was seen.For the experiments, cells were pre-treated with either DMC , and EF24  both in DMSO  and 50 ng/mL of TRAIL  (in PBS) was added to wells after 4 h. The IC2. MTT  solution was diluted using pre-warmed cell media to a working concentration of 0.5 mg/mL and 500 µL of working MTT solution was added to each well after old medium was removed. The plate was incubated in the dark for 2 h at 37 °C. The MTT solution was discarded and 500 µL of DMSO was added to each well and plate was incubated for 1h at room temperature. Absorbance was read at 570 nm using CLARIOstar plate reader .Cells were treated (see Methods above) in duplicate using increasing concentrations of DMC or EF24. Plate was incubated for 72 h at 37 °C, 5% COCells were cultured until a confluent monolayer was formed. Each well was scratched vertically down the center using a p200 pipette tip. Cells were then washed with PBS and treated (see Methods above). Images were taken using an Olympus E50 microscope and scratch width was recorded for time point 0 h measurements. Cells were incubated at 37 °C and images and measurements were recorded at 24, 48 and 72 h.Cells were cultured and treated in 6-well plates. After 72 h treatment, whole cell lysates were performed and frozen at −20 °C until used. Electrophoresis was performed using 12% gels at 80 V/110 V using the BioRad MiniProtean Electrophoresis system. Wet transfer was performed using a nitrocellulose membrane and the BioRad mini trans-blot cell system on ice at 100 V for 90 min. Membranes were blocked using a 5% Marvel in TBS-T solution and incubated at 4 °C overnight with DR4  primary antibody diluted in a 5% Marvel in TBS-T solution (1:2000). Membranes were then washed with TBS-T  and incubated with IRDye 800CW goat anti-rabbit IgG  secondary antibody  at room temperature, covered in foil for 1 h. Membranes were washed three times, 5 min each and detected using the Odyssey CLx system .p value of 0.05 or smaller was deemed statistically significant. Statistical significance was represented by * (p < 0.05); ** (p < 0.01); *** (p < 0.001); **** (p < 0.0001).Migration assay scratch area calculation and densitometry analysis were preformed using ImageJ Fiji . GraphPaCurcumin has been studied for its anti-cancer effects, but due to its low bioavailability, its therapeutic use is limited. This study shows that the curcumin analogues with better bioavailability, DMC and EF24, decrease in vitro cell viability and cell migration of RCC cells (ACHN). These effects are further enhanced when in combination with TRAIL, pointing to DMC and EF24 as potential compounds to overcome TRAIL resistance in kidney cancer cells. The mechanisms are yet to be elucidated; however, an increase in TRAIL receptor expression (DR4) may play a role in kidney cancer cell resensitization to TRAIL. Further analysis of protein expression of pro- and anti-apoptotic proteins, such as caspases and proteins from the Bcl-2 family, may potentially help understand the mechanism of action of these compounds in the ACHN cell line.