This study aimed to explore the experiences of adult clients with hearing loss and their audiologists in using an online tool, the Living Well Tool (LWT), during initial audiology appointments. The LWT is designed to help clients identify when and where it is most important for them to communicate effectively and live well with hearing loss. A total of 24 adult clients with hearing loss and two audiologists participated in this study. Clients were invited to complete the LWT prior to their next audiology appointment, however, most clients chose to use the LWT in-session with their audiologist. Following the appointment, clients and audiologists participated in individual qualitative semi-structured interviews to explore their experiences of using the LWT, and the extent to which the LWT facilitated person-centred care. Qualitative analysis five key themes which reflected participants’ experiences and perceptions of using the LWT: (1) the LWT enhances audiological care; (2) the LWT supports person-centred audiological care; (3) the use of the LWT should be individualised; (4) users value comprehensiveness; and (5) users value accessibility. This study demonstrated that the LWT supported the provision of person-centred audiological care, providing a flexible, comprehensive and accessible means for audiologists to gain an understanding of their clients’ needs and preferences. However, it was also noted that the use of a tool must be individualised and accessible for all. Hearing loss occurs frequently in older adults and has numerous negative consequences, including reduced access to everyday spoken communication, social isolation, depression, poor mental health, and reduced quality of life . Due to Despite widespread acknowledgement of the importance of person-centred care in audiological rehabilitation, research suggests that hearing healthcare continues to have a biomedical focus. In a video observation study, clients and their significant others often raised psychosocial concerns related to communication difficulties during appointments, however these concerns were rarely addressed, with audiologists having a tendency to focus on providing technical information and progressing a discussion about hearing aids . AlthougeHealth can be used as a way of enhancing person-centred care in healthcare . eHealthThe adoption of technology by older adults in managing their healthcare is heavily influenced by the platform’s perceived usefulness , accessihttps://apps.idainstitute.com/apps/lw_en) (accessed on 1 July 2021). The LWT is designed to promote communication and living well with hearing loss for clients, using an active problem-solving approach to focus on relevant and important communication situations and the biopsychosocial needs of the client, rather than focusing solely on hearing aids and the hearing loss. The online LWT has been designed to empower clients to prepare for the rehabilitation process. This is achieved by encouraging them to think about their communication needs prior to their audiology appointment, with a series of photographs and open-ended questions helping them to identify when and where it is most important for them to communicate effectively and live well with hearing loss. Armed with this information, the client and their audiologist can work together to map out strategies for managing the hearing loss and communication goals in a person-centred manner to ultimately improve communication outcomes  an improvement on the way it [case history] was done before” (Client 14), even providing additional case history questions: “Sometimes you [audiologists] don’t always remember to ask all the questions even if you have a sort of template that’s being followed, so it’s [LWT] probably good” (Client 16).The LWT complements traditional information-gathering practices. Within the context of typical audiology appointments, participants noted that the LWT was useful in setting the agenda and guiding the case history: “That list of [communication strategies in LWT] gives you an idea of what options you could have available” (Client 15) and “It took the focus of the hearing aid to … this is what you can do to make it better for you, so head against a wall in a noisy restaurant, turn the music down, you know you get TV headsets for the television if you’re having trouble with Scottish accents” (Clinician 1). Some client participants noted that the LWT prompted them to identify additional communication situations to discuss with the audiologist: “I hadn’t thought about the supermarket … so it does jog the memory when it needs to” (Client 8).Participants also described how the LWT was used to frame discussions around various communication situations, communication strategies, and technological solutions for improving communication in these situations. Clients and clinicians alike noted the usefulness of the LWT in helping clients to identify potential communication strategies: “positioning [myself] to hear and situations where I needed assistance” (Client 19), while another described how the audiologist used the LWT to discuss hearing aid solutions: “she was making the point how it [hearing aid] copes in this sort of situation [shown in LWT] and therefore that it [hearing aid] would be beneficial to be in terms of being able to hear better in that sort of scenario” (Client 16). Finally, participants recognised the benefit of the LWT in keeping records for both the client and audiologist: “It leaves a permanent record [of what was discussed with audiologist] ‘cause you know if you have a meeting with someone you can talk for 15 min about the problems but have no record” (Client 3).Another participant described how the audiologist used information from the LWT to discuss communication strategies which could be used in social situations: “I probably would’ve been more informed [if I had completed the LWT prior]” (Client 1). One participant reflected “What the program [LWT] said to me was, this is like pre-empting your visit. The technician’s [audiologist] got some information about you” (Client 4), while others said “I probably I came [to the appointment] prepared to be saying ‘This is the problem I’m having’” (Client 16). Others saw how the LWT could be used to help clients identify their own hearing difficulties, and even the potential use of the LWT as a communication screener: “You could screen people using the device [LWT] rather than taking history and everything when you actually saw them … and those that have a problem need to come and those who don’t have a problem may not need to come … or may not need to come so urgently” (Client 15). Interestingly, client participants also saw the benefits of the LWT in preparing audiologists for appointments, for example: “They [audiologist] might be able to pre-empt the problem … before they see the patient … and save them [audiologist] time from asking all the questions … which would make the appointment more efficient” (Client 15).The LWT could enhance preparedness for the client and clinician. One of the key benefits outlined by participants was the potential application of the LWT in preparing clients for appointments and even facilitating the help-seeking process. Despite only a small number of clients competing the LWT prior to their first appointment, many reflected that completing the LWT pre-appointment would have been valuable to the process: “it doesn’t [take more time], because you would be doing COSI [Client Oriented Scale of Improvement] goals anyway” (Clinician 2) and in fact, “it [LWT] kept me on track … rather than 20 min trying to get to the point [of the client’s visit]” (Clinician 1). Client’s also noted that “it was really just a few minutes at the start of the appointment” (Client 18) and “we [client and audiologist] didn’t have to go through a long, tedious interview” (Client 8). Clients noted that the LWT could also be a helpful tool in improving session efficiency for certain clients: “I think it [LWT] could save a lot more time with people who were maybe were a little bit reticent” (Client 11).The LWT improves appointment efficiency. Despite clinicians expressing concern that, prior to using the LWT, it would take too much time to complete, they reflected after the study that “forced the clinician to ask questions and listen to answers” (Client 7). One client noted how using the LWT “probably merged a bit into a conversation … rather than sort of being too locked into a series of questions … ensuring the content was covered naturally” (Client 17). The clinician participants agreed that the LWT prompted them to ask additional questions about the client’s use of communication strategies: “It [LWT] actually gave you more information, like what they’re actually doing [strategies] … that’s something that I probably got a lot more information than I usually would” (Clinician 2).The LWT aids communication processes. Client participants highlighted how the LWT Theme 2: The LWT Supports Person-Centred Audiological care.Analysis of the client and clinician interviews identified several ways in which the LWT supported person-centred audiological care, including facilitating the client-clinician relationship, making the discussion of sensitive matters easier, helping clients identify the biopsychosocial impacts of their hearing disability, making clients the priority, and normalising hearing experiences to help clients identify strategies.“I think it [LWT] actually engaged the clinician and the patient, or client more closely” (Client 7) and “I think it [LWT] was creating more of a rapport … and just took away from the ‘I’m the audiologist and you’re the client” (Clinician 1). One participant even reflected that the tool enabled the relationship to begin even before the first appointment: “I felt like somebody was talking to me already … the bond that existed before you even walked in here, before you even met” (Client 5).The LWT facilitates the client-clinician relationship. Both client and clinician participants agreed that the LWT played a role in building the client-clinician relationship: “It [LWT] obviates the necessity of other people pointing out to you that you’ve got hearing problems … a computer is nicely non-personal … at least when you do this sort of thing, you have to come to terms with it yourself” (Client 13). The LWT was also thought to be helpful in sensitively raising communication difficulties that clients might feel uncomfortable about: “I can understand how somebody who was a little bit hesitant might find it really really good” (Client 11), and even help facilitate more thoughtful discussions with family members about difficult communication situations: “It [LWT] helps them [family members] understand the person with the problem … they [family members] might try to assist in certain ways and not be so critical when people didn’t pick up what they were saying” (Client 15).The LWT makes discussing sensitive matters easier. In the context of audiological rehabilitation, the LWT was described by the participants as an effective means of sensitively communicating the diagnosis of a hearing loss: “they [clients] do tend to say ‘I don’t think I have any trouble’” (Clinician 1), with one client even noting that “It’s good to see that there’s different ways of looking at it [hearing assessment] now [other than solely instrumental assessment]” (Client 20). In particular, the pictures of various communication situations prompted clients to think: “Oh yeah, I have that problem” (Client 16) and were helpful in making “the client more aware of the different ranges of hearing situations and how they are currently effective or non-effective” (Client 7). Importantly, the LWT was also effective in helping clients identify their communication strengths: “There are many situations in which there are no problems … made me aware that I’m competent in a lot of ways” (Client 20). This focus on communication strengths was noted to be a particularly important given that clients may not always “think of that [positive] perspective” (Client 15).The LWT can help clients identify the biopsychosocial impacts of their hearing disability. Both clients and clinicians found the LWT particularly helpful in prompting clients to think about the broad ranging impacts of their hearing loss, and the everyday difficulties they encounter as “I think it [LWT] was creating more of a rapport so that they knew I was focusing on what their needs were … which in turn got me to focus on ‘Ok, this is what you want solved’, as opposed to me presuming what they were there for.” (Clinician 1). The LWT was also identified as a means of putting the client in the driver’s seat: “They [the client] felt more in control of giving me the information rather than often I’m like, ‘Oh, so how do you find the phone?’ So more control, they have more control I think” (Clinician 2).Clients are the priority. The LWT was identified by participants as a method of ensuring the clinician stayed focused on the needs of the client: “I think it’s [LWT] effective in that it does relate the conditions you’re having evaluated to your real life situations” (Client 21). Further, participants described how the photographs served to reassure clients that other people also experienced communication difficulties: “By looking at the pictures and realising that there’s a lot of other people in those kinds of situations … that you’re not alone kind of thing” (Client 10).Relatability of photographs can normalise hearing experiences and help clients identify strategies. Throughout the interviews, clients discussed the relatability of the photographs used in the LWT, and the effectiveness of the photographs in identifying the everyday communication difficulties resulting from hearing loss: “The only one that remotely identified with me was the crowd scene or the party scene” (Client 12). Further, some participants noted that the photographs portrayed people of a homogenous ethnicity and age: “I think they were all Caucasian people” (Client 8).Despite these positive reports about the relatability of the photographs used in the LWT, it is important to note that not all clients could relate to the communication situations depicted in the photos: Theme 3: The use of the LWT should be Individualised.Consistent with the principles of person-centred care, there was wide acknowledgement amongst participants that the use of the LWT should be individualised according to client needs because there was variability in how the LWT was used. The results also highlighted that audiologists provided valuable guidance when using the LWT and that some users value in-person services.“most of my clients didn’t do it [LWT] at home” (Clinician 1) and “I don’t think I had any [LWTs] completed in advance” (Clinician 2). One of the audiologists gave her clients the choice of how they would like to complete it, noting that “When I make the appointment I say to them ‘I can either send it to you and you can do it online or we can do it together when you come in’” (Clinician 1). The most common method of completion of the LWT, therefore, was within the audiology appointment time. Although both clinicians indicated a preference for using the LWT at the beginning of appointments “as part of the case history” (Clinician 2), clients reflected that “it doesn’t seem to matter which part of the appointment you have it in … it wouldn’t matter if it was at the start or the end really” (Client 9).The LWT is used in Varied Ways. Although the online LWT was initially designed to be used by the client at home prior to appointments, and then discussed within the appointment in the context of the client’s hearing test results, analysis indicated that the LWT was used by clients and audiologists in varied ways. Despite most clients being sent an electronic link to the online LWT, “I don’t actually recall us sort of referring back to that [LWT] per se”. Interestingly, a number of clients noted that they saw potential in using the LWT again at a future point in time: “I know how to access it so if there’s a need for me to progress if for whatever reason, if there’s any further change to my situation, well … there is somewhere I can go to, there is somewhere I can source this information” (Client 5) and “I guess you could compare those [LWT responses] overtime” (Client 17).In regard to ongoing use of the LWT, most clients reported that following completion of the LWT, the audiologists did not commonly refer back to the LWT: “I didn’t have too many where they actually came along, which is disappointing” (Clinician 2). When family members did attend appointments where the LWT was used, one family member reported “I kept as quiet as I could possibly be [during the LWT discussion] … I didn’t say very much at all” (Family Member 4), indicating that they were not actively involved in the process. One of the clinicians did note however that she thought the LWT was helpful in provoking discussions about third-party disability. Interestingly, one of the audiologists reflected that the LWT could be used to manage dominant family members: “It would be interesting to do more of the tool with the significant other to see if it stops them from being the dominant one [and bring the focus] back to the client” (Clinician 1).Family Member Involvement was Varied. According to participant reports, family members played a limited role in the use of the LWT, most often due to low attendance of family members at audiology appointments: She [audiologist] spoke to each question” (Client 2) and “asked how I coped in the various situations” (Client 12). The level of input provided by audiologists varied between clients, with audiologists acknowledging that “some people just needed more … some guidance” and “it seems to work whichever way you do it” (Clinician 2). As such, while some clients reported that the audiologist took the lead and completed the tool on behalf of the client by asking them questions: “I didn’t enter anything” (Client 8),“I just gave answers [to the audiologist’s questions]” (Client 13), others reported that “she [audiologist] turned the screen around and gave the mouse to me so I was actually writing it … I pretty much went through it [LWT] on my own … I think she was more there as a support if I needed it” (Client 20). When guidance was provided, this most often included an explanation of various communication strategies such as asking people for clarification, and at the end of appointments linking the results of the hearing test to the LWT responses: “She [audiologist] did make connections between the situations that were identified on the website and the results of the tests” (Client 21). Clients noted a preference for the audiologist to enter client responses into the LWT: “It was probably easier if she [audiologist] uses it. She could put the information in faster than someone who is at home” (Client 15), however a number of clients also noted that while they were completing the LWT with the audiologist, they could not see the computer screen (Client 13). Overall, however, the majority of clients saw value in going through the tool with the audiologist: “it would be a bit helpful if I wanted to talk through issues with someone … if people are using it by themselves for a diagnosis then you might be entering difficult territory” (Client 17).Audiologists Provide Valuable Guidance when Using the LWT. Clients reported that their audiologist provided valuable guidance when using the LWT, with audiologists talking through the instructions with clients, describing the pictures, defining the communication situations, typing in client responses, and discussing these responses throughout the process: ““I’m not one of those people who likes talking to screens” (Client 12), as well as preferring to discuss the results directly with their audiologist: “It’s probably more personalised to do it [LWT] actually with the audiologist rather than do it and give it to the receptionist and then never see it again, and don’t know whether anybody’s even looked at it” (Client 8).Some LWT Users Value In-person Services. Despite the acknowledged benefits of having access to an online LWT, some clients acknowledged that they preferred to use the tool with their audiologist in an in-person appointment. Client preferences for in-person services centred around their comfort with using technology for healthcare: Theme 4: Users value Comprehensiveness.Analysis of the interviews revealed a common finding that the users of the LWT valued comprehensiveness in a tool such as the LWT, and as such, there was potential for the LWT to be further developed to enhance its application in audiological rehabilitation.“plenty of options” (Client 11), along with “a list of different perspectives” (Client 15).The LWT is Comprehensive. Overall, participants agreed that the LWT was a comprehensive tool which offered “the audiologist a bit of a better indication of what your problems are” (Client 3), especially “if you had a problem and you felt you wanted to do a few different things” (Client 5). Participants also agreed that the LWT could be more comprehensive by asking clients to identify more potential communication strategies.There is Potential for the LWT to Ask More. Participants did acknowledge however that the LWT had the potential to ask more information and to give more information to clients with hearing loss. Participants suggested that the tool could ask for more specific information to give “examples of what you can hear and what you can’t hear” (Client 3), and providing more background information about the communication scenarios “with maybe even more of a breakdown of the different scenarios rather than just the pictures … more clarification of the pictures” (Client 10).There is Potential for the LWT to Give More. Participants also noted that there was potential for the LWT to include more communication scenarios, more specific communication strategies, giving more Theme 5: Users Value Accessibility of the LWT.Participants identified the importance of being able to readily access tools such using technology such as the LWT. Participants thus emphasised the importance of having a tool which is easy to use and is aesthetically pleasing, especially given the potential challenges of using technology.“was very straight forward” (Client 8) and “pretty easy to understand” (Client 9), especially given its logical step-by-step approach. One of the clinicians noted however that “it’s not so intuitive” (Clinician 2) and may be difficult for some clients to use. This was especially true for clients with other co-occurring conditions such as vision loss, which could affect their access to the LWT, and therefore has implications for the design of the tool, including font size: “You might make the text [of the LWT] bigger for people with poor vision” (Client 15).Usability of the LWT is Valued. It was apparent from the interviews that the usability of the LWT is an important consideration in order to maximise accessibility for users. The majority of participants noted that the LWT “When you’re faced with a website that you don’t have to think about … it means it’s [the LWT] got to be good” (Client 11). Participants also reported that the “pictures were effective in picking up [communication] situations” (Client 21). Some participants did note however that “the photos were a bit busy … and quite small” (Client 17).The Aesthetics of the LWT Enhances its Accessibility. Overwhelmingly, the participants agreed that the LWT was “well presented” (Client 17) with its design and aesthetics positively enhanced it’s accessibility: “older, much older people [who] may have a little bit of difficulty with it … if you don’t understand the computer” (Client 10). Some participants even expressed a preference for the LWT to be also provided in a “printable format” (Client 2) so they could “fill it out” (Client 15) in paper form, especially given some clients reported some technical difficulties with sending the electronic copy of the completed LWT to their clinician. Other participants with limited computer experience noted however that despite this lack of technological experience, the LWT was easy to use.Technology can be Challenging. Given the online format of the LWT, participants did make reference to the inherent challenges of an online platform, especially for The present study investigated the experiences of clients with hearing loss and their audiologists in using an online audiological rehabilitation tool, the LWT, and specifically, the extent to which the tool facilitated person-centred audiological care. We aimed to explore the experiences of clients and their audiologists in using the tool and determine the extent to which the LWT facilitates the provision of person-centred care. In doing so, we explored the effectiveness of Living Well in focusing audiologists’ talk on communication in everyday life rather than just hearing aids when working with adults with hearing loss and their families. Results of this study produced two overarching ideas. The first of these was that Living Well embodied person-centred care, as indicated by themes 1–3; the second was that users value the website’s functionality, shown in themes 4 and 5.The Living Well tool embodied person-centred care as indicated by the themes LWT supports person-centred audiological care, LWT enhances audiological care, and Use of LWT should be individualised. Participants in the present study showed the Living Well tool is adaptable to individuals’ heterogeneity by describing the variability in tool operator , how the tool is used  and how family members were or could be involved. Previous research has identified the importance of taking a person-centred approach to healthcare ; this waParticipants described how the LWT facilitated communication, facilitating the relationship between clients and clinicians, and easing the discussion of sensitive matters. This description is consistent with  characteIn the present study, clients and clinicians alike commented on the ways in which the LWT integrated with existing models and activities of care without diminishing traditional practice. Both client and clinician participants reported that the LWT improved preparedness for audiological rehabilitation, and therefore promoted greater session efficiency. Importantly, participants also reported that the LWT did not add any more time to appointments. Identifying these benefits is important given clinicians report time as a barrier to implementing person-centred care ,29,30. IIn addition to enhancing the information that is received from clients, the LWT also has capacity to help clients gain additional insight; the LWT helped clients identify the biopsychosocial impact of their hearing disability. Many clients underestimate the impact of their hearing loss until rehabilitation has commenced. The graduated nature of most cases of hearing loss means many clients do not realise what they are increasingly missing out on  and the In recognising client strengths and providing individualised care, information and its presentation can be tailored to suit the user , which cAccessibility of such programs has been commented on extensively in the literature, which has found that designing for older users requires careful consideration of layout, use of blank space and proximity and choice of colours ,39, and In terms of clinical implications, the Living Well tool provides a range of options for clinicians in future. As shown in the present research, it can be administered within the clinic with the help of the clinician, or can be used at home, either alone or with a family member. Indeed, input from family members is strongly encouraged; research has shown family-centred care improves communication within families and reduces third-party disability ,44. FamiThe present study contained some limitations. Firstly, we had limited control over which participants were invited. Clients of the participating clinics were invited directly by the audiologist; accordingly, we have no knowledge of which clients were not invited or for what reasons. Secondly, many of the interviews were conducted in the clinic of the treating audiologist, which may have influenced the interview data. Despite assurances of confidentiality, clients may have been concerned their responses could be overheard by or provided to clinicians. We conducted interviews in the clinic to maximise response rates; however, future research may be able to circumvent potential influence on interview responses by conducting all interviews in a separate setting. Finally, we aimed to gain the perspectives of family members, yet only two clients brought family members to their appointments. Although low levels of family attendance in audiology are common , increasDespite these limitations, the present study was the first of its kind in examining client and clinician experiences of using this LWT, and thus has provided important insights into how much an online tool can be used to enhance audiological care. The LWT tool provided participants with a platform to discuss their hearing and communication needs with their audiologist, in a way that has promoted person-centred care via individualised, comprehensive, accessible care that is suitable for an older audience. The use of the LWT can be expanded to provide family-centred care by including the needs and perspectives of family members.


The epithelial-to-mesenchymal transition (EMT) is a unique process resulting in enhanced cell motility, invasiveness, and metastasis in cancer. The EMT is regulated by several transcription factors, including Snail and Slug, which exert crucial roles during cancer progression. We have studied the effects of Docetaxel as the first-line chemotherapy agent for prostate cancer, and Telmisartan as an anti-hypertensive drug on the expression level of Snail and Slug. In addition, the effects of Docetaxel, Telmisartan and their combination on cancer cell proliferation were investigated. The PC3, DU145, MDA-MB468, and HEK cell lines were used for this study. Quantitative RT-PCR analysis and MTT assay were used to study the expression of Snail and Slug level and cell proliferative assay, respectively. We found that a combination of Docetaxel + Telmisartan effectively inhibits the cell proliferation in cancerous cells in comparison with each drug alone (P<0.05). Furthermore, in these cell lines, Docetaxel, Telmisartan and their combination significantly diminished the expression level of Snail and Slug genes compared to control cells (P<0.001), however, in the HEK cell line, this effect was seen only in the combination group. Our data imply that Telmisartan and its combination with Docetaxel exert strong inhibitory effects on the expression level of Snail and Slug genes. Also, these drugs and their combination could inhibit cancer cell proliferation. In conclusion, the combination of Telmisartan and Docetaxel has the potential to suppress the metastasis of prostate and breast cancer cells. Cancer is the second leading cause of death and remains a source of increasing concern globally. Prostate cancer is the most prevalent malignancy in men worldwide that could be treated by several approaches including, prostatectomy, radiotherapy, and chemotherapy at the early stage . Each ofThe metastasis process in cancer involves multiple steps. In the initial stage, the cancerous cells detach from the primary tumor site and migrate to nearby blood or lymph vessels. In later stages, these cells migrate to distant places, reside there and proliferate. A crucial point in the metastases process attributes to the epithelial-to–mesenchymal transition (EMT). EMT is a unique programmed process by which epithelial cells achieve a mesenchymal phenotype. During EMT, the cancer cells deplete their polarity, and adhesion capability and acquire the migration ability as well as the invasive potential to other tissues. It is well known that EMT has a vital role in cancer progression and metastases .More specifically, the EMT process demonstrated various hallmarks, such as E-cadherin down-regulation, which is associated with decreasing in cell-cell adhesion, up-regulation of matrix metalloproteinase (MMPs), and high expression of mesenchymal-related proteins including Vimentin and N-cadherin to increase cell motility. Furthermore, a series of EMT inducers have been described, such as β-catenin, and the Snail family members . The SnaDocetaxel (DTX) as a therapeutic agent is commonly used to treat a broad range of human malignancies, including lung cancer, stomach, head and neck, breast, and prostate cancer -9. DTX iToday, angiotensin II receptor blockers (ARBs) are widely administrated to treat hypertension, chronic kidney disease, and heart failure. Previous investigations indicated that angiotensin II is correlated with cancer progression, and ARBs could antagonize angiotensin receptors, and inhibit tumor growth. Among these ARBs, Telmisartan (Tel) could inhibit cell proliferation and leads to apoptosis in the different types of cancer cells, including gastric, colon , and uroReagents and materials: RPMI-1640, fetal bovine serum(FBS), penicillin–streptomycin, and Trypsin/EDTA were obtained from Invitrogen Life Technologies . DTX and Tel were purchased from MedChem Express . All drugs were dissolved in dimethyl sulfoxide  and were diluted with a culture medium before each experiment. All primers were obtained from Metabion International AG (Germany).Cell cultures: The PC3, DU145 (Prostate cancer cell lines), MDA-MB468 (Breast cancer cell line), and HEK (Human Embryonic Kidney cell line) were purchased from the National Cell Bank of Iran . The cell lines were cultured in RPMI-1640 supplemented with 10% FBS, 100 U/ml of penicillin, and 100 μg/ml of streptomycin, in an incubator with 5% CO2 atmosphere at 37˚C. Cell-proliferative assay: The cells were seeded onto 96 well plate  and incubated in a CO2 incubator at 37˚C. After 24h, the cells were treated with DTX and Tel dissolved in DMSO. The final concentration of DMSO was 0.1%. Cell viability was determined after 48h incubation using  3--2,5-diphenyltetrazo-lium bromide (MTT) assay and presented as the percentage of the control group which was treated by medium cell culture [ culture .Analysis of Interactions, Combination Index: For evaluation the in vitro pharmacological drug interaction for DTX and Tel, combination index (CI) is determined [termined . For thitermined . The conRNA isolation and Quantitative Real-Time PCR (qRT-PCR): Expression of Snail and Slug genes were quantitated by qRT-PCR and Glucuronidase Beta (GUSB) gene was used as internal control gene. Cells were cultured in 25 cm2 flask (7×105cells in each flask) and after 24h, the cells were treated with DTX (0.01 nM), Tel (0.1 µM), or their combinations for 48h. Total RNA was isolated from the cells by TriPure isolation reagent  as described by the manufacturer’s protocol. After determination of RNA concentration by a Nanodrop 1000 spectrophotometer , the integrity of RNA was evaluated using gel electrophoresis. To prepare cDNA, a reverse transcriptase kit  was used and the cDNA was made from 1 μg of total RNA. QRT-PCR was carried out triplicate using SYBR green PCR master mix, and Rotor Gene Q-series Real-Time PCR Systems . The qPCR process was carried out for 40 cycles using the following protocol: each cycle included 95˚C for 10 min, 95˚C for 15 secs, 58˚C  or 62.9 (for Snail) for 30 secs, 72˚C for 30 secs and 72˚C for 10 min. The sequences of primers for the determination of Snail, Slug and GUSB genes expression are shown in Statistical Analyses: Data in the present study were derived from at least three independent experiments. Statistical significance among all groups was evaluated using One-way Analysis of Variance (ANOVA) followed by the Tukey test. P-value<0.05 was considered statistically significant. The Real-time PCR graphs were done using GraphPad Prism (Version 6.0), and analysis of Combination Index (CI) and related graphs were done, using CompuSyn software.Due to the potential toxicity of DTX, we examined the effect of combination treatment involving DTX+ Tel to reduce the DTX dose that necessary to show its anti-cancer effects. The cells were treated with a combination of DTX +Tel at varying concentrations. Treatment of PC3 cell line with DTX 0.01 nM+Tel 0.01 µM inhibited the cell viability by 55%, whereas DTX and Tel alone by the same concentration inhibited the cell viability by 29% and 32%, respectively (P<0.001). In addition, treatment of these cells with DTX 0.005 nM+Tel 0.005 µM had more inhibitory effect on cell viability compared to DTX 0.01 nM (P<0.05). Treatment of DU145 cells with DTX 0.1 nM+ Tel 0.1 µM inhibited the cell viability by 61%, whereas DTX and Tel alone by the same concentration inhibited the cell viability by 35% and 7%, respectively (P< 0.001). In MDA-MB468 cell line DTX 0.5 nM+ Tel 100 µM and DTX 1 nM+ Tel 100 µM inhibited the cell viability by 55% and 57% respectively, whereas DTX 1 nM diminished the cell viability by 39% (P<0.05).Our results showed that the use of combination treatment in doses much lower than the drugs IC50, for normal cells (HEK) had significant effect in inhibiting cell growth. So that, treatment of these cells with DTX 0.0005 nM+ Tel 0.001 µM, DTX 0.001 nM+ Tel 0.0005 µM, and DTX 0.001 nM+ Tel 0.001 µM had the significant inhibitory effects on cell viability compared to the Tel 0.001 µM treatment . Also, DTX 0.001 nM+ Tel 0.0005 µM significantly inhibited cell viability compared to the DTX in both doses of 0.0005 nM and 0.001 nM (P<0.05).Over the past century, many attempts have been made by researchers to quantify the dose-effect relationships of each drug on its own or the combinations to answer the question of whether or not the combination of two drugs can have a synergistic effect. To determine the effects  of the combination treatment on cell lines, we mathematically evaluated the results using Chou and Talalay method . For thiIn this study the expression level of Snail mRNA was also performed using the quantitative reverse transcription PCR (RT-qPCR). As shown in As shown in Prostate and breast cancers are the two types of malignancy in humans with the poor prognosis of their patients and high rate of mortality. The conventional chemotherapeutic agents, including Taxanes are still the gold standard among prostate and breast cancer treatment during the early and late stages . HoweverIn the present work, we surveyed the effects of combination treatment of DTX+Tel on prostate, breast cancer, and the HEK cell proliferation. According to our knowledge, this study is the first study about the anti-cancer and anti-metastatic effects of the combination treatment of Tel and DTX on cancer cells.The anti-proliferative effects of DTX and Tel via a dose-dependent manner were seen in these cell lines. However, the Tel IC50 value in the HEK cell line was much higher than that of cancer cell lines, which shows the toxicity of Tel on the HEK cell line as a normal cell line is remarkably lower than that of cancer cells. An important finding of our study is the significant effect of Tel in reducing the dose of DTX for inducing its anti-cancer effects. Overall, our results showed that combination treatment ameliorated the cytotoxic profile of DTX in PC3, DU145, MDA-MB468, and HEK cell lines compared with the drugs treatment alone.The results showed that the combination treatment in cancer cell lines was more effective than the drugs alone treatment. In some treatments that used for all cell lines CI values were below 1.0, which indicate the synergistic effects between two drugs at lower doses . Howeveret al. reported that stearidonic acid (an omega-3 fatty acid) could synergistically enhance the cytotoxic effects of DTX in the PC3 and DU145 cells [ et al [et al. demonstrated that Tel, via inducing S-phase arrest and reduction in expression of cyclin A2 and cyclin-dependent kinase 2 suppressed the proliferation of Esophageal Squamous Cell Carcinoma cell lines. Additionally, they reported that Tel inhibited tumor growth in vivo in a xenograft mouse model [in vivo and in vitro by promotion cell cycle arrest at the G0/G1 phase and inhibiting the phosphorylation of the tumor suppressor retinoblastoma (pRb) protein [in vitro lung cancer cell viability and decreased stemness. Furthermore, intratumoral administration of Actinomycin D+ Tel synergistically reduced tumor burden in xenograft mouse models compared to either drug alone. Tel via increasing the drug permeability in tumors has anti-cancer stem cell activity [Many studies have shown that the combination of DTX with some agents and drugs has reduced the clinical dose of DTX. Mansour 45 cells . Treatme45 cells . In agre45 cells . It has 45 cells , 24. Uem [ et al . reportese model . Tel sup protein . Green aactivity . Tel sigactivity .et al. demonstrated Tel as an agonist for PPAR-γ, can alleviate the EMT induced by oxalate and calcium oxalate crystals via expending antioxidant effects through the PPAR-γ-AKT/STAT3/p38 MAPK-Snail signaling pathway. Snail as a regulator of E-cadherin could bind to E-box elements within E-cadherin promoter and suppress the transcription of E-cadherin that finally leads to the loss of cell-cell adhesion in epithelial cells and increases the EMT development [et al. showed the expression of Slug mRNA mainly was at a high level in the PC3 cell line and downregulation of Slug, using specific siRNA induced a significant increase in E-cadherin expression and decreasing in cell migration through a matrigel matrix. Also, the cell viability is significantly diminished after Slug suppression in the PC3 cell line. Overexpression of either Snail or Slug is sufficient for EMT induction and progression in cultured epithelial cells [The results of the present study also showed, treatment of cancer cell lines, PC3, DU145, and MDA-MB468, with DTX (0.01 nM), Tel (0.1 µM), and their combination caused a significant reduction in the expression level of Snail and Slug mRNA compared to control group. The different outcome was achieved when HEK cells were treated with DTX (0.01 nM), Tel (0.1 µM), and their combination. Snail and Slug gene expression were diminished substantially in the combination group; however, the expression of these genes in monotherapy groups did not make significant difference in comparison with the control group. This finding indicated that Tel and DTX did not have significant effects on normal cells. To the best of our knowledge, the effects of Tel and its combination with DTX on the expression level of Snail and Slug (two transcription factors involved in cancer metastasis) in prostate and breast cancer cells have not been investigated yet. Liu elopment , 31. In al cells . TherefoIn conclusion, the results of our study showed that combination treatment of prostate and breast cancer cells with DTX+Tel significantly decreased cell viability. Using combination therapy to treat various cancers may help to diminish the administrated dose of chemotherapeutic agents and subsequently reduce the side effects of these drugs. Furthermore, a subsequent decrease in Snail and Slug genes expression was noted in PC3, DU145, and MDA-MB468 cells. However, these results were slightly different for HEK cells as a normal cell line after treatment with Tel and DTX in terms of Snail and Slug gene expression. Since increased Snail and Slug genes expression, as master proteins for EMT regulation, have an essential role in tumor metastasis, finding drugs with the ability to suppress the expression of these genes with minimum side effects can be considered as an important strategy for decreasing the EMT process and cancer cell invasion and migration. in vivo studies and randomized clinical trials are required to clarify its mechanism to establish the strategy of combination therapy for prostate and breast cancers.However, future The authors report no conflict of interest.


Background/Aim: Cancer research has been conducted using cultured cells as part of drug discovery testing, but conventional two-dimensional culture methods are unable to reflect the complex tumor microenvironment. On the other hand, three-dimensional cultures have recently been attracting attention as in vitro models that more closely resemble the in vivo physiological environment. The purpose of this study was to establish a 3D culture method for oral cancer and to verify its practicality.Materials and Methods: Three-dimensional cultures were performed using several oral cancer cell lines. Western blotting was used for protein expression analysis of the collected cell masses (spheroids), and H-E staining was used for structural observation. The cultures were exposed to cisplatin and cetuximab and the morphological changes of spheroids over time and the expression changes of target proteins were compared.Results: Each cell line formed spheroidal cell aggregates and showed enhancement of cell adhesion molecules over time. H-E staining showed tumor tissue-like structures specific to each cell line. Cisplatin showed concentration-dependent antitumor effects due to loss of cell adhesion and spheroid disruption in each cell line, while cetuximab exhibited antitumor effects that correlated with EGFR expression in each cell line.Conclusion: Spheroids made from oral cancer cell lines appeared to have tumor-like characteristics that may reflect their clinical significance. In the future, it may become possible to produce tumor spheroids from tissue samples of oral cancer patients, and then apply them to drug screening and to develop individualized diagnostic and treatment methods. However, the cellular environment under 2D culture is very different and often a poor reflection of the complex tumor microenvironment (TME) in vivoA commonly used in vivoIn recent years, a spheroid model produced by three-dimensional 3D)-culture has attracted attention as a better experimental model that is closer to the physiological environment D-culturein vitro to clarify their antitumor effects.Cisplatin is the most widely used cell-killing anticancer drug for the treatment of oral cancer in vitro disease model. In addition, in order to verify the practicality of this model, we compared the utility of 2D- and 3D-culture models for analyzing the response of different oral cancer cell lines to cisplatin and cetuximab.In the present study, we aimed to establish a 3D-culture model using oral cancer cells, and attempted to create a more useful The human oral squamous cell carcinoma lines SAS, HSC-3, HSC-4, and OSC-19 were obtained from JCRB Cell Bank. All of these cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM)  supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin. They were cultured in an atmosphere of 5% CO2 at 37 °C.Antibodies against EGFR , p-EGFR , E-cadherin , cleaved caspase-3 , caspase-3 , GAPDH  and Ki-67  were purchased from Cell Signaling Technology . HRP-conjugated anti-β-actin antibody (ab49900) was purchased from Abcam .Cisplatin (Randa® Injection) was obtained from Nippon Kayaku (Tokyo) and Cetuximab (Erbitux® Injection) was from MerckSerono (Tokyo).5 cells were seeded in each well and cultured in the same environment as described above for the 2D culture. The same seeding methods were used for all four oral cancer cell lines. Medium was changed every three days and maintained at a volume of 100 µl/well. However, to avoid accidental aspiration of spheroids, only half the total volume of medium was exchanged on each occasion. Spheroid microplates were placed in a humidified incubator set at 37 °C and 5% CO2. Images of spheroids were captured using a bright-field microscope .3D spheroids were cultured in 96-well ultra-low attachment (ULA) plate . 1.0×10Cisplatin was added to the formed spheroids in a stepwise concentration  24 h after cell seeding to the ULA plate Fig. A. CetuxiFor 2D culture, cells were seeded in a 96-well 2D-culture plate in DMEM supplemented with 10% FBS for 24 h, and then the drugs were added and the cells were cultured for 48 h.Cisplatin was added to the oral cancer cells under 2D and 3D culture in a half maximal inhibitory concentration (IC50) 24 h after cell seeding. PBS was added to the control group as a substitute for cisplatin. Cells and spheroids were detached or disassembled using Trypsin/EDTA at 24 h after the addition of cisplatin or PBS, and the number of cells was counted using a Countess® Automated Cell Counter (ThermoFisher Scientific). Cell viability was determined by converting the ratio of the number of cells in the cisplatin- and PBS-added groups. An MTT assay was performed to determine the IC50 of cisplatin on each oral cancer cell line under 2D culture.The whole cell lysate (WCL) was prepared as described previously WB was performed as described previously Pipetting or aspiration was used to remove as much media as possible without disturbing the spheroids. Spheroids were washed with PBS and fixed in 4% PFA for 30 min. Spheroids were additionally washed with PBS three times for 5 min each time and embedded in paraffin. Spheroid sections (3 µm thickness) were deparaffinized and hydrated through xylenes and a graded alcohol series. Antigen retrieval was performed by dunking in proteinase K for 5 min. Sections were additionally washed with Wash Buffer  two times for 5 min each time, and then endogenous peroxidase activity was blocked by treatment with blocking solution (Dako) for 30 min at RT, and the sections were incubated with primary antibodies.We analyzed the expression of Ki-67 and cleaved caspase-3 in 3D spheroids by immunohistochemical analysis. The sections were incubated with anti-Ki-67 antibody (dilution 1:200) or anti-cleaved caspase-3 antibody (dilution 1:1000) overnight at 4 °C, followed by treatment with avidin-biotin complex  for 60 min, and visualized with the use of a DAB substrate-chromogen solution . The staining results were observed with an optical microscope BX53 (Olympus).For 2D IHC, cells were cultured on coverslips coated with collagen and fixed in 4% paraformaldehyde in PBS for 10 min at RT. Fixed cells were permeabilized for 10 min in PBS containing 0.1% Triton X-100. Endogenous peroxidases were blocked for 30 min in 1.5% H2O2 for 30 min. For blocking non-specific reactions of primary antibodies, cells were incubated for 30 min at RT in 1% bovine serum albumin (BSA) in PBS containing 0.1% Tween 20. The applied primary antibodies were follows: anti-E-cadherin antibody (dilution 1:100) and anti-EGFR antibody (dilution 1:100). The process from immunostaining to imaging is the same as above.Spheroids sections were deparaffinized in a series of xylene for 15 min, rehydrated in graded ethanol solutions, and washed well in distilled water. Then sections were incubated in Mayer's hematoxylin solution for 7 min and rinsed in tap water until the water was colorless. Finally, after sequential treatment with hydrogen chloride and 80% ethanol solution, sections were incubated in eosin for 7 min.Statistical significance was calculated using Microsoft Excel. Differences between two sets of data were examined with a paired Student's t-test; values of p<0.05 were considered to indicate statistical significance. Data were expressed as means ± S.D. unless otherwise specified.We used two oral cancer cell lines, SAS and HSC-3, that differed with respect to their cell morphologies, proliferation style and rate. The developmental dynamics of each cell line in monolayer culture were diverse, and the cell morphology varied from polygonal to paracircular to spindle-shaped Fig. A. In ordThese data showed that forcing oral cancer cell lines with different characteristics into a 3D-culture environment resulted in a successively enhanced intercellular adhesion in both cell lines, while tumor growth-related molecules such as EGFR showed cell-specific differences in their increased or decreased expression.To clarify the histology of the recovered spheroids, we embedded spheroids at each time, sectioned the paraffin-embedded blocks, stained the sections with H-E and DAB staining targeting specific proteins, and observed them using a microscope Fig. , S2C, S4These data suggested that the structure of spheroids made from oral cancer cells is likely to reflect the differentiation level and characteristics of the derived cancer cells.We then assessed the morphological and molecular changes in the spheroid upon exposure to anticancer drugs. First, we evaluated the effect of cisplatin, which is widely used in oral cancer Fig. A. CisplaThese data suggested that the cytotoxic, apoptosis-inducing effect of cisplatin was characterized by spheroid disintegration associated with activation of caspase-3 and attenuation of E-cadherin.Next, we evaluated the efficacy of cetuximab, the only molecularly targeted drug available for oral cancer Fig. A. CetuxiThese data suggested that the EGFR expression-dependent antitumor effects of cetuximab were more prominently reproduced in 3D culture and were characterized by spheroid disintegration in association with attenuation of E-cadherin.Finally, we examined the molecular changes resulting from the exposure of oral cancer spheroids to the anticancer drugs Fig. . SAS sphThese data suggested that the drug responsiveness of spheroids could be evaluated histologically, and demonstrated the utility of oral cancer spheroids as a drug screening model.in vitro. As a result, although many drugs have shown good results in vitro, most have failed to transfer to in vivo models, and only a few of the newly approved anticancer drugs have shown antitumor activity in clinical trials There is a need to develop more effective treatments for many types of cancer. However, it is a major challenge to recreate the complex TME  in vitro studies have been well reported over the past few years in vivo and may help bridge the gap between 2D cell culture in vitro and living tissue in vivo, such as by maintaining the native cell phenotype and function in vivo models, including mouse models for drug screening in vitro systems to model tumor pathogenesis in vivo, including tumor-associated signaling pathways and chemotherapy resistance In cancer research, the benefits of using 3D models forThe role of intercellular adhesion proteins in tumor progression, invasion and metastasis, and treatment resistance is one of the most closely studied issues in cancer biology. Intercellular adhesion proteins are considered to be participants in the regulatory signaling cascade associated with cell proliferation Schmidt et al. reported that spheroid morphology in head and neck cancer is associated with E-cadherin and Ki67 expression in vitro analysis of the drug efficacy of paclitaxel and cisplatin were feasible using organoid-derived spheroids in vivo, indicating that the HCC in vivo was well simulated by the 3D spheroid model in vitro spheroids, which have characteristics and histology more similar to those of real clinical tumor samples, has the potential to help overcome cancer.Cell lines exhibiting an epithelial phenotype have also been shown to form more regular and uniform cell aggregates One of the objectives of this study was to compare the two culture systems in terms of the survival of oral cancer cells after treatment with cisplatin and cetuximab Fig. -5. PreviIt has been reported that tumor spheroids that appear identical in shape may exhibit differential sensitivity to drugs that cannot be explained by molecular or metabolic variables In this study, we generated spheroids from oral cancer cell lines, and their structures appeared to have properties similar to the corresponding clinical tumor histology. Three-dimensional culture has been an important tool for developing assays to determine the drug chemosensitivity of cells as a platform for preclinical or clinical drug screening. In the future, spheroids/organoids could be generated from tissue samples obtained from patients with oral cancer and applied to drug screening and the development of individualized diagnosis and treatment while maintaining cancer-specific functionality Fig. .Supplementary figures.Click here for additional data file.


Cancer develops in a multi-step process where environmental carcinogenic exposure is a primary etiological component, and where cell–cell communication governs the biological activities of tissues. Identifying the molecular genes that regulate this process is essential to targeting metastatic breast cancer. Ionizing radiation can modify and damage DNA, RNA, and cell membrane components such as lipids and proteins by direct ionization. Comparing differential gene expression can help to determine the effect of radiation and estrogens on cell adhesion. An in vitro experimental breast cancer model was developed by exposure of the immortalized human breast epithelial cell line MCF-10F to low doses of high linear energy transfer α particle radiation and subsequent growth in the presence of 17β-estradiol. The MCF-10F cell line was analyzed in different stages of transformation that showed gradual phenotypic changes including altered morphology, increase in cell proliferation relative to the control, anchorage-independent growth, and invasive capability before becoming tumorigenic in nude mice. This model was used to determine genes associated with cell adhesion and communication such as E-cadherin, the desmocollin 3, the gap junction protein alpha 1, the Integrin alpha 6, the Integrin beta 6, the Keratin 14, Keratin 16, Keratin 17, Keratin 6B, and the laminin beta 3. Results indicated that most genes had greater expression in the tumorigenic cell line Tumor2 derived from the athymic animal than the Alpha3, a non-tumorigenic cell line exposed only to radiation, indicating that altered expression levels of adhesion molecules depended on estrogen. There is a significant need for experimental model systems that facilitate the study of cell plasticity to assess the importance of estrogens in modulating the biology of cancer cells. Cancer develops in a multi-step process where environmental carcinogen exposure is a primary etiological component, and where cell adhesion governs the biological activities of tissues . DespiteThe breast epithelium depends on interactions between epithelial cells and the extracellular matrix with the underlying stroma that works as a unit that continually communicates with each other for their structural integrity and specific function ,6.There are two forms of radiation such as non-ionizing and ionizing. Non-ionizing radiation can come from a variety of natural sources, such as the sun and lighting, as well as man-made sources, such as those utilized in industrial/medical applications and wireless communications . IonizinIonizing radiation has the unique property of randomly penetrating different tissues and cells, harming them according to the dose absorbed rather than just by the cells exposed . By direAnother factor associated with radiation is hypoxia, a hallmark of solid tumors; it means that is a major obstacle to the effectiveness of radiation therapy; thus, authors  have shoTranscriptional regulators have been identified to induce epithelial-to-mesenchymal transition (EMT) which plays an important role in development and malignancy . EMT-indCadherins are hormonally controlled and play a physiological role throughout mammary development; however, when these genes are altered, they might lead to pathological effects . Then, tDSC3 is a member of the cadherin superfamily of calcium-dependent cell adhesion molecules, an important component of desmosomes and its expression is down-regulated in breast cancer cell lines and primary breast tumors  and 10 μg/mL and 5% equine serum , 0.5 μg/mL hydrocortisone  and 0.02 μg/mL epidermal growth factor  were added [re added ,72,73,74Alpha-model: Exponentially growing MCF-10F cells were plated 3 days before irradiation at a density of 3 × 105 cells in 60 mm diameter stainless steel rings with a 6 μm mylar bottom. Cells were irradiated with graded doses of 150 KeV/μm 4He ions accelerated with the 4 Me V van de Graaff accelerator at the Columbia University Radiological Research Facilities, as described previously [4He ions were prepared by subculturing for 10–15 passages and 12–14 weeks between doses. Irradiated cultures were subcultured immediately to determine growth kinetics and expanded in culture to assay for transformed phenotypes and, at the same time, samples were frozen as future stock. The remaining cells were then sampled for various transformed phenotypes and further passaged for additional radiation treatment. Cells were subsequently cultured in the presence or absence of E. Irradiated cultures were assayed for cell growth kinetics, anchorage-independent growth, invasiveness, tumorigenicity, and determination of BRCA1, BRCA2, and RAD51 protein expression.−8; (c) a malignant and non-tumorigenic cell line (60/60 cGy) named Alpha3 (A3); (d) a tumorigenic cell line (60/60 cGy plus estrogen) named Alpha5 (A5); and (e) the Tumor2 cell line (T2) derived from a xenograft of the Alpha5 cell line injected into nude mice. These cell lines were cultured in the presence or absence of estrogen for periods of up to 10 months , the scAffymetrix HG-U133A Plus 2.0 GeneChip microarray gene expression analysis: The breast cancer model (Alpha-model) consists of: (i) MCF-10F, (ii) Estrogen, (iii) Alpha3, (iv) Alpha5, and (v) Tumor2 cell lines used to analyze gene expression by the Affymetrix U133A oligonucleotide microarray . Arrays were quantitatively analyzed for gene expression using the Affymetrix GeneChip Operating Software (GCOS) with a dual global scaling option in the Genes@Work software platform of the discovery algorithm SPLASH  with a false discovery rate of 0.05 [ of 0.05 . The AffGene expression analysis and statistical analysis. TIMER2.0, a web source of information, systematically evaluates the clinical impact of different immune cells in various cancer types through three components: Immune Association, Cancer Exploration, and Immune Estimation, each with several modules such as the Gene module in the Immune Association component that provided the statistical analysis carried out by Spearman’s p test. The Cancer Exploration component has the Gene_DE module that provided statistical analysis carried out by the Wilcoxon test; The Gene_Outcome module with the analysis carried out by the Z-Score test, and the Gene_Corr module provided the statistical analysis carried out by the Spearman’s test [n’s test . The clin’s test . This mop < 0.05 was considered significant.UCSC Xena, another web source of information, provided the statistical significance computed by the One-way Anova test. UCSC Xena web source allows users to explore functional genomic data sets for correlations between genomic and/or phenotypic variables and focuses on integrative visualization of multi-omics datasets across different genomic contexts, including genes, genomic elements, or any genomic region, for both coding and non-coding parts of the genome . A p < 0Overall, this study supports the idea that the expression of adhesion molecules could be used as the prognostic biomarker for the early detection of human breast cancer cases. In this study, molecular signatures that play key roles in cell adhesion were targeted. To determine the role of adhesion molecules as potential prognostic markers there is a significant need for experimental model systems. Such a model would facilitate the study of this plasticity and the importance of the adhesion state in modulating the biology of cancer cells. Such an experimental breast cancer model allowed us to analyze genes associated with cell adhesion that could help to improve the clinical outcome of chemotherapy. However, in vitro studies are not sufficient to explain in vivo situations. For that reason, in cancer, prognostic factors are important for early diagnosis as well as to get an efficient treatment to prevent the risk of overtreatment. Cell dependency must be considered in future treatment planning and the molecular and clinical features are important for radiotherapy. Thus, using gene technology and molecular information is possible to improve therapies and reduction of side effects. Therefore, these findings will provide new insight into breast cancer-related fields.


WT) and mutant-type  motor neuron-like amyotrophic lateral sclerosis (ALS) cell lines. Cytotoxicity induced by glutamate, lipopolysaccharides, and H2O2 reduced viability of cell; however, the addition of paeonol improved cell viability against neurotoxicity. The [3H]paeonol uptake was increased in the presence of H2O2 in both cell lines. Paeonol recovered ALS model cell lines by reducing mitochondrial oxidative stress induced by glutamate. The transport of paeonol was time-, concentration-, and pH-dependent in both NSC-34 cell lines. Kinetic parameters showed two transport sites with altered affinity and capacity in the MT cell line compared to the WT cell line. [3H]Paeonol uptake increased in the MT cell line transfected with organic anion transporter1 (Oat1)/Slc22a6 small interfering RNA compared to that in the control. Plasma membrane monoamine transporter (Pmat) was also involved in the uptake of paeonol by ALS model cell lines. Overall, paeonol exhibits neuroprotective activity via a carrier-mediated transport system and may be a beneficial therapy for preventing motor neuronal damage under ALS-like conditions.Paeonol is a naturally occurring phenolic agent that attenuates neurotoxicity in neurodegenerative diseases. We aimed to investigate the antioxidant and protective effects of paeonol and determine its transport mechanism in wild-type (WT; NSC-34/hSOD1 Other unlabeled reagent-grade chemicals, including paeonol, carnitine, cimetidine, 1-methyl-4-phenylpyridinium ion (MPP+), quinidine, verapamil, edaravone, clonidine, tramadol, acetyl-l-carnitine (ALC), tetraethyl ammonium (TEA), para-amminohippuric acid (PAH), and glutamic acid, were obtained from Sigma Aldrich . Lipopolysaccharide (LPS) was acquired from List Biological Laboratories .The radiolabeled labeling agent  assay was conducted. ALS model cell lines were treated with glutamate (2 mM) and LPS (20 ng/mL) and Hf 550 nM . For theMitoTracker  staining (CMS-ROS) was performed to measure the mitochondrial membrane potential in the motor neuron cell line of ALS . The celOn the other hand, active caspase-3 (Cas-3) immunoreactivity was determined in the motor neuron cell line of ALS. Fixed cells were incubated for 30 min in a blocking solution  and were further incubated with anti-cleaved caspase-3 rabbit antibody  at 4 °C for 24 h. After washing 3 times with PBS solution, the slides were incubated with goat anti-rebbit-488  at RT for 2 h. Photo-images were taken by using an Olympus epifluorescence microscopy . The semi-quantitative analysis of immunoreactivity was performed by the ImageJ program.3H]paeonol uptake study was performed using a previously reported method [3H]paeonol (347 nM) to each well with or without the addition of the inhibitor at 37 °C for 5 min [The [d method . Prior tor 5 min . Ice-chior 5 min . For pro3H]paeonol in NSC-34 cell lines, the Michaelis–Menten constant (Km) and the maximum uptake rate (Vmax) were estimated using Equation (1):3H]paeonol at 5 min and C is the concentration of unlabeled paeonol.For kinetic analysis of paeonol uptake in the MT cell lines.These results prompted us to further analyze the uptake of Oat1 substrate and transporter inhibitors, including PAH as an Oat1 substrate, and probenecid and furosemide, which are Oat1 inhibitors D. Paeono3H]paeonol uptake in disease model cell lines (50 value of edaravone at 0.05–10 mM and ibuprofen at 0.05–5 mM in the MT cell line. Edaravone, an antioxidant and neuroprotective agent, exhibited inhibition in a concentration-dependent manner with the value of IC50 3.77 mM, and ibuprofen, an anti-inflammatory drug, also showed concentration dependency inhibition with the value of IC50 1.15 mM (We examined the interactions of drugs owing to their potential inhibitory effects on the paeonol increased in ALS model cell lines compared to that in the untreated cells, and the addition of cold paeonol resulted in a further increase in the uptake of paeonol in both cell line. Furthermore, glutamate significantly increased the uptake of paeonol in the MT cell line. In the NSC-34 cell lines, the paeonol transport was time-dependent and increased linearly with time up to 5 min. The uptake rate of [3H]paeonol was lower in the MT cell line in comparison to that in the WT cell lines. Our results showed the pH-dependent uptake of [3H]paeonol in NSC-34 cell lines. The uptake of paeonol significantly increased in an alkaline pH 8.4 as compared to the control pH of 7.4 in both cell lines. The results obtained are similar to the previous study of paeonol in TR-iBRB cell lines paeonol uptake. OCT/OCTN transporter function modulators, including MPP+, verapamil, and quinidine [+ was mainly transported by dopamine transporters to dopaminergic neurons [+ antiporter in the blood–brain barrier [3H]paeonol uptake in both cell lines. Edaravone, a free radical scavenger, exerts neuroprotective effects by blocking lipid peroxidation paeonol. Alterations in Oat and OATP functions or levels have been reported in liver disease and cancer paeonol. Previous kinetic analysis suggested that Oat1 contributes to the uptake of PAH in rat renal slices [3H]paeonol uptake in the MT cell line. Moreover, furosemide, a diuretic drug, has demonstrated a modulating effect on the expression of the Oat1 transporter paeonol uptake in the disease model cell line model. Ibuprofen, an anti-inflammatory drug, has an IC50 value of 1 mM in glioma cells [To improve the clinical applicability of drugs in the disease state, we evaluated the inhibitory concentrations of the drugs. Ibuprofen and edaravone concentrations of 1.15 and 3.77 mM, respectively, were required to inhibit by half [ma cells , and edama cells .In the current study, we investigated the neuroprotective and antioxidant activities of paeonol against neurotoxicity induced by inflammatory cytokines in ALS cell lines. Paeonol pretreatment was found to reduce mitochondrial oxidative stress. The OCTN1, OAT1, and Pmat transporter showed altered behavior in the MT cell line. The transport characteristics of paeonol differed between the ALS cell lines. Our data indicate that Pmat may be responsible for the uptake of paeonol in the WT and MT ALS cell line. The findings imply that paeonol may serve as a potential neuroprotective and stress-reducing agent for the curative therapy of ALS.


N-hydroxysuccinimide (NHS). The effect of the nZVI ratio in the synthesized nanohybrids was studied at three different ratios of GO : nZVI, 1 : 1, 1 : 5 and 1 : 10. SEM/EDS, HRTEM, STEM/EDS, XRD, Raman, FTIR, and TGA analyses were conducted to provide physical and chemical properties of the adsorbents. The performance of nZVI and GO–nZVI nanohybrids as an adsorbent have been studied for methylene blue (MB) removal from an aqueous solution with an initial concentration of 12 mg L−1 at adsorbent dosages of 0.1, 0.3, 0.5, and 1 mg mL−1. Results indicated that GO–nZVI (1 : 5) provided the highest MB removal (99.1%) by using 10 mL of the 1 mg mL−1 adsorbent. After regeneration of the GO–nZVI (1 : 5) nanohybrids with ethanol, 84.3%, 67.2%, and 63.0% of MB removal were achieved in the first to third regeneration cycle. Results also showed that the GO–nZVI nanohybrids were not affected by aggregation compared to nZVI.GO and nZVI have been used for removing different contaminants from aqueous solution; however, difficulty in the separation of GO, and the aggregation propensity of nZVI particles prevent them from having efficient practical applications. In this study, a green synthesis method was performed to prepare nanohybrids of GO and nZVI to provide an adsorbent with high adsorption efficiency that can be removed from aqueous solution easily by magnetic separation. GO–nZVI nanohybrids were synthesized by using biocompatible cross linkers named 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and  A green synthesis method was used to prepare GO–nZVI nanohybrids to provide an adsorbent with high adsorption efficiency that can be removed from aqueous solutions easily by magnetic separation. Furthermore, nZVI's magnetic properties allow for its easy separation and recovery from the waste stream for regeneration and reuse. However, nZVI suffers significantly from aggregation due to its high van der Waals and magnetic attraction forces; this leads to a decrease in pollutant removal efficiency by the decline in reactive surface area.4Nano-scale zero valent iron , the nanohybrids exhibit superior performances in removing contaminants due to a combined enhancement of the adsorptive and reductive capacities.8 Such enhancement of contaminant removal ability of rGO–nZVI nanohybrids have accelerated the research on their various synthesis procedures.Aggregation of nZVI particles can be prevented either by modifying their surface using surfactant and polymers, or by loading these nanoparticles on a solid support.e.g., rGO–nZVI or GO–nZVI) has become attractive for exploiting the synergistic activities of the component materials to enhance the desired functionality or to achieve the multifunctional properties; unique property alterations and emergent new properties in the nanohybrids  can lead to uncertain fate and toxicity upon exposure to the environment.9 Thus, there is a need of employing safer-by-design approach for synthesizing multifunctional next generation of nanohybrids. Typical synthesis methods for graphene supported nZVI include chemical reduction, chemical vapor deposition (CVD), sonochemical treatment, hydrothermal or solvothermal processes, plasma-based technique, and spray pyrolysis – all of which essentially produce rGO–nZVI from GO and iron salt precursors.5 Many of these processes use various harmful reducing agents including sodium borohydride, hydrazine, and benzene. Most of these conventional synthesis methods co-reduce GO while depositing nZVI particles from iron salt precursor on rGO support. However, GO exhibits higher contaminant removal capacity than rGO for certain specific contaminants e.g., heavy metals,10 cationic dyes.11 Furthermore, while nZVI is deemed to be non-toxic due to their high aggregation propensity, it is unknown how their toxic behavior would change upon the formation of rGO–nZVI nanohybrids. This becomes particularly important since rGO has shown to be more toxic to aquatic organisms12,13 and mammalian cells14 when compared to GO. Our recent study has shown that rGO–nZVI, synthesized by the most commonly used chemical co-reduction technique, shows higher toxicity than nZVI (data not shown).15 Thus, safer strategies to conjugate nZVI onto graphene supports need to be pursued.While synthesis of multicomponent carbon–metal nanohybrids (16 Here we used well-known carbodiimide crosslinker chemistry using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) crosslinking agents. EDC-NHS coupling chemistry has been prevalent majorly in drug delivery and nanomedicine literature17–20 due to their excellent biocompatibility and also the ability for nanoparticle surface modification and conjugation. EDC can easily bond with the carboxylic acid groups of GO and then can lead to esterification upon addition of NHS. nZVI has a high affinity for amine functional groups21,22 and thus will probably attach to the NHS, providing a GO–nZVI hybrid linked via EDC-NHS chain. This well-established EDC-NHS coupling chemistry has been previously used in conjugating iron oxide nanoparticles with graphene for enabling contaminant removal; however, those involve surface modification of iron oxide nanoparticles using amine functional groups.23,24 Furthermore, no reports of using EDC-NHS crosslinkers have been reported for conjugating nZVI directly on the GO surfaces to synthesize GO–nZVI nanohybrids.This study presents a new method of conjugating pre-synthesized nZVI onto GO nanosheets using a biocompatible crosslinker – a technique that has the potential to create safer GO–nZVI nanohybrids. Crosslinkers can conjugate materials to each other based on the presence of particular moieties that are able to react with the specific kinds of functional groups.i.e., GO : nZVI ratio of 1 : 1, 1 : 5, and 1 : 10. Physical and chemical properties of the synthesized nanohybrids were characterized by high-resolution transmission electron microscopy (HRTEM), scanning electron microscope (SEM), energy dispersive X-ray spectroscopy (EDS), scanning transmission electron microscopy coupled with energy dispersive X-ray spectroscopy (STEM/EDS), X-ray diffraction (XRD), thermogravimetric analysis (TGA), and Raman and Fourier transform infrared (FTIR) spectroscopy. The ability of the prepared GO–nZVI nanohybrids to remove contaminants from aqueous media was evaluated by performing removal of methylene blue (MB) dye and was compared to the MB removal ability of nZVI only.In this study, GO and nZVI without any other surface modification, for the first time, were conjugated using biocompatible EDC-NHS crosslinkers to synthesize GO–nZVI nanohybrids. Three different sets of GO–nZVI nanohybrids were synthesized using different precursor ratios Single layer graphene oxide  and nZVI  were purchased from US Research Nanomaterials, Inc . First, 50 mg of GO was dispersed in 100 mL of deionized (DI) water using a microtip-based ultrasonic dismembrator  for 3 h on pulse-mode  at an amplitude of 50 and input power of 15–18 W. The sonication was performed under temperature control using ice bags (15 °C). Then, the suspension was mechanically stirred at 150 rpm using an open drum mixer (Dayton Inc.) while 25 mg of EDC (BioXtra) and 20 mg of NHS (98% purity), both purchased from Sigma Aldrich , were slowly added to the suspension. The obtained suspension was mixed for 30 min at ambient temperature. Then, 50, 250, or 500 mg of nZVI was added to the suspension for synthesizing GO–nZVI nanohybrids with different ratios GO : nZVI of 1 : 1, 1 : 5, and 1 : 10, respectively. The suspension was kept mixing for 1 h at 80 °C using an open drum mechanical mixer to avoid the possibility of magnetic attraction during the nanohybrid synthesis. Then, the suspension was filtered through a 0.22 μm polyvinylidene fluoride (PVDF) filter , and the solid residue was washed five times with ethanol and DI water. Finally, solid residue on the filter paper was dried at 65 °C in an oven before further use, and the solid final products were named GO–nZVI (1 : 1), GO–nZVI (1 : 5), and GO–nZVI (1 : 10) based on corresponding precursor ratios.High-resolution TEM (HRTEM) images of GO, nZVI, GO-NHS/EDC, and GO–nZVI nanohybrids were obtained using a JEOL JEM 2010 transmission electron microscope  operating at 200 kV to determine the physical morphology of the synthesized nanohybrids and the parent nanomaterials. In order to prepare the TEM samples, 1 mg of each sample was sonicated in 20 mL of ethanol (100%) for 15 min, then two drops of the suspensions were placed on carbon-coated copper grids , and left for 20 min to dry before placing on the TEM sample holder. Microscale morphologies and elemental composition of the samples were characterized using a Hitachi SU70 Field Emission Scanning Electron Microscope  at 20 kV coupled with an Oxford energy dispersive spectrometer . The nanomaterial samples for SEM/EDS characterization were placed on a stub covered with double-sided adhesive carbon tape. Then, a thin layer of gold (∼20 nm) was deposited on the samples using DV-502 thin film deposition system . More accurate quantification of the chemical composition along with nanoscale morphology characterization was performed using highly advanced Tecnai G2 30 TWIN scanning transmission electron microscope coupled with an energy dispersive X-ray spectroscope (STEM/EDS) operated at 300 kV. This STEM/EDS system was used to acquire the high angle angular dark field (HAADF) images and EDS maps. EDS maps were obtained for 20 minutes per frame using an EDAX TEAM silicon drift detector in the STEM. Brightness and contrast were adjusted with no major image alterations. The same TEM grids prepared for HRTEM imaging were used for STEM/EDS analyses.i.e., in aqueous media for 5 min using a bath sonicator  to mimic a condition similar to adsorbent usage for MB removal. A similar dispersion of nanohybrids was sonicated for 1 h  at high energy using a microtip based ultrasonicator  at an amplitude of 50. In both cases, after the sonication, the nanohybrids were separated using the magnet, and the iron content of the remaining aqueous media was measured using an Inductively Coupled Plasma Optical Emission Spectrometer .10 mg of the nanohybrids was dispersed in 10 mL of DI water The magnetic properties of the nZVI and GO–nZVI nanohybrids were measured at room temperature by a vibrating sample magnetometer .−1 within a range from 5° to 90°. Vibrational frequencies and structural changes of the samples were obtained using a Raman microscope  with an incident laser wavelength of 514 nm. Functional groups of the samples were characterized using a Fourier-transform infrared (FTIR) spectroscopy instrument  with 4 cm−1 resolution within the range of 400–3500 cm−1. The samples' thermal durability was studied by performing a thermogravimetric analysis  under nitrogen purging and with a heating rate of 10 °C min−1 from room temperature up to 900 °C.ULTIMA IV X-ray diffractometer  equipped with Cu laser source was used to characterize the crystal structures and phases of the samples. The powdered samples were placed on a glass slide and then put inside the XRD sample holder before scanning at a rate of 2° min−1 was prepared. Each 10 mL MB solution was poured in a 20 mL vial, where nano-adsorbents i.e., the three different sets of GO–nZVI nanohybrids or bare nZVI at different amounts  were added to the MB solution. Then, the mixture was bath sonicated  for 5 min and after that, the nano-adsorbents were separated using a simple ceramic block magnet with dimensions of 0.38 × 0.88 × 1.88 in3 . The concentration of MB solutions after each adsorption test was determined by measuring their light absorbance values at 665 nm wavelength using a Cary 60 UV-vis spectrophotometer , and the obtained results were compared with the absorbance of the prepared reference MB solutions of known concentrations. MB concentration in each case was computed based on a calibration curve prepared earlier following the Beer–Lambert law which states that the concentration of the solution and the light absorbance values are linearly proportional. Each of the adsorption experiments was performed in triplicates, and the average values of removal efficiency along with standard deviation were calculated.A stock solution of MB  in DI water with the initial concentration of 12 mg Li.e., the highest MB removal was selected for performing regeneration and reuse. After the MB adsorption experiment was performed and the adsorbents were magnetically separated, the remaining MB solution in the vial was decanted while keeping the adsorbent separate using the magnet. Then, 10 mL pure ethanol was added to the vial, magnet was taken away, and the mixture of adsorbent and ethanol was bath sonicated for 5 min. Then the adsorbents were separated using the magnet, and ethanol was decanted to retrieve the regenerated adsorbents for further reuse in MB removal. Three regeneration and reuse cycles were performed to determine the recyclability of the adsorbent. The regeneration steps are presented using a schematic briefly  of bare nZVI particles was found to be 38.6 ± 17.1 nm – a value consistent with previous studies.27,28i.e., when GO : nZVI is 1 : 1 or 1 : 5), the nZVI particle sizes are significantly smaller than bare nZVI particles and were less aggregated due to their attachment on dispersed sites on GO surface. However, with more nZVI content in case of GO–nZVI (1 : 10), the GO surface is completely covered and/or overwhelmed with the nZVI, which leads to high extent of nZVI aggregation and provides particle sizes close to those of bare nZVIs similar to the previous studies.29,30EDS analysis coupled with SEM imaging was first used to examine the gross elemental compositions of the samples at the microscale. EDS spectra showing the elemental distribution of the samples along with corresponding SEM images featuring their bulk morphology are provided in Fig. S3.28,31,32Moreover, the peak intensity for iron in the EDS spectra increased with increasing nZVI loading in the nanohybrids. To further confirm and quantify the elemental compositions of the samples at the nanoscale, STEM microscopy was performed to obtain high angle annular dark field (HAADF) images along with EDS mapping. HAADF images for GO, GO-NHS/EDC, nZVI, and GO–nZVI  are presented respectively in 33 Therefore, it can be concluded that the synthesized nanohybrids have a good stability, and may not cause concerns about environmental health and safety issues while being used as an adsorbent.The stability of the nanohybrids was assessed by measuring the released iron ion from the nanohybrids after 5 min of bath sonication (representing adsorbent usage condition) and 1 h of high-energy microtip sonication in aqueous media (representing an extreme condition). Results indicated that at adsorption representing condition of 5 min bath sonication, the concentration of iron released to the aqueous media reached 0.004 and 0.012 ppm for GO–nZVI (1 : 5) and GO–nZVI (1 : 10), respectively. At extreme condition of 1 h microtip sonication, the concentration of iron released to the aqueous media reached 0.151 and 0.233 ppm for GO–nZVI (1 : 5) and GO–nZVI (1 : 10), respectively. Based on these results, the amounts of the released iron from the nanohybrids are negligible at adsorption relevant conditions. Although after sonicating of the nanohybrids at extreme conditions during high energy microtip sonication, the released amounts of iron increased significantly compared to the bath sonication, the amounts are still lower than 0.3 ppm which is the maximum contaminant limit of iron in the drinking water.−1, respectively, which refers to high magnetic attraction for all of the samples. These values are in agreement with previous literature reports34–36 and guaranteed successful magnetic separation of the nanoparticles after adsorption. The magnetic property of the nanohybrids is lower than the pure nZVI because of the presence of GO. As expected, GO–nZVI (1 : 10) shows higher SM compared to GO–nZVI (1 : 5).Fig. S537 nZVI particles inhibit the formation of van der Waals and π–π stacking bond between the GO sheets causing the suppression of the graphitic peaks.38,39 The XRD pattern of GO-NHS/EDC shows three new peaks at 35.4°, 38.5° and 40.3° which were not observed for GO and can be closely attributed to the diffraction peaks of succinimide (JCPDS card no. 00-007-0745). The absence of diffraction peaks of NHS (JCPDS card no. 00-036-1688) suggests no residual NHS is present in GO-NHS/EDC. This indicates the successful formation of NHS active ester on GO surface confirming activation of GO surface by proper crosslinking of NHS and EDC.40 Also, the indicative peak of GO in GO/NHS-EDC is broader than in GO maybe due to some degree of aggregation of GO-NHS/EDC and NHS/EDC bonding on the GO surface.41 The samples of nZVI and GO–nZVI hybrids show strong characteristic peaks for nZVI at 44.75° and 82.55°.42,43 Moreover, the characteristic peaks of iron-oxide at 2θ = 30.11°, 35.43°, 43.22°, 53.62°, 57.14°, 62.78°, 65.03° and 74.24° for nZVI and GO–nZVI hybrids indicate the presence of Fe3O4i.e., the oxide-shell around nZVI particles observed in HRTEM and HAADF images.5,42,44 XRD patterns of GO-NHS/EDC and GO–nZVI (1 : 1) showed two peaks at 35.4° and 40.3° that can be attributed to the part of NHS/EDC crosslinking. However, these peaks cannot be seen in other hybrids as they are suppressed by high iron concentration.via TGA are presented in 45 The second step was from 100 to 300 °C where about 47.5 wt% of weight loss happened due to the decomposition of labile oxygen functional groups like carboxyl groups.45 The less steep slope of weight loss above 300 °C (15.2 wt%) can be attributed to the removal of more stable oxygen functional groups, and the release of CO and CO2 during char formation.45–48 Additional weight loss in the case of GO-NHS/EDC (at ∼400–600 °C) can be ascribed to the decomposition of amino silane coupling agent.49,50 The increasing residual ash content for GO–nZVI (1 : 1), GO–nZVI (1 : 5), and GO–nZVI (1 : 10) i.e., 65%, 87%, and 94% of their initial mass, respectively, suggest increasing nZVI particle loading for these nanohybrids.51Thermal properties and stabilities of GO, GO-NHS/EDC, and GO–nZVI nanohybrids observed 2 carbon atoms and the D band corresponding to defects and disorders caused by sp3 carbon atoms in graphitic sheets can be observed at ∼1607 cm−1 and ∼1368 cm−1, respectively in the samples containing GO.52 For nZVI particles, the Raman shifts are observed at 218, 283, 398, 487, 606, and 1315 cm−1 due to Fe–O stretching vibration.53 The intensity ratio of D to G (ID/IG) can be used to measure the defect and disorder degree in the graphitic matrix.54 The ID/IG values of GO and GO-NHS/EDC were 0.90 and 0.84, respectively, indicating lower defects in the graphitic matrix for cross-linked GO compared to GO. The presence of characteristic Raman peaks of iron in the nanohybrids samples further confirms the impregnation of iron nanoparticles onto the graphene sheets.30 In nanohybrids, the graphitic carbon peaks were flushed away due to high coverage of nZVI particles on the graphene sheets. As a result, ID/IG values of these nanohybrids were not quantifiable. The obtuse Raman peak at ∼1315 cm−1 observed in these nanohybrids, which is close to characteristic ID graphitic peak, was actually from the Raman signal of hybridized nZVI particles.55Raman spectra of GO, nZVI, GO-NHS/EDC, and GO–nZVI nanohybrids are presented in −1 in GO and GO-NHS/EDC spectra indicate stretching vibrations from C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O, skeletal vibrations from unoxidized graphitic domains (CC), and C–O stretching vibrations, respectively.56 Three new characteristic bands at 1585 cm−1 (–NH primary amide), 1257 cm−1 (C–N stretch of aromatic amines), and 1233 cm−1  appeared after crosslinking of GO with NHS/EDC and also in GO–nZVI nanohybrids.57 In the nZVI spectrum, the bands at 443, 547, and 686 cm−1 can be assigned to Fe–O bonds, and the band at 888 cm−1 can be assigned to the Fe–O–H bending vibration of α-FeOOH particle.58–60 These characteristic iron signals can also be seen in GO–nZVI nanohybrids indicating the presence of nZVI particles in the synthesized nanohybrids.59 The iron signals weakened in the case of nanohybrids compared to bare nZVI, which can be attributed to the presence of GO support in the nanohybrids. These results indicate the successful conjugation of GO and nZVI.21,22 amine groups are nucleophile meaning that they can donate a pair of electron to form new covalent bonds.61 Thus, GO-NHS/EDC, which contains amine groups  and GO–nZVI (1 : 10) showed effective magnetic separation capacity, with separation from the aqueous solution between 4–5 minutes. However, the magnetic property of GO–nZVI (1 : 1) was not strong enough for quick and effective magnetic separation of the adsorbents. The light absorption spectra of MB solution before and after adsorption by using different amount of these adsorbents are presented in Fig. S6.†Although GO can remove MB from the aqueous solution, its separation from the solution is difficult and requires high-speed centrifugation.−1 of GO–nZVI (1 : 5) as an adsorbent. Similarly, 58.2–92.6% MB removal was achieved by using 0.1–1 mg mL−1 GO–nZVI (1 : 10) as an adsorbent. However, the highest concentration of bare nZVI i.e., 1 mg mL−1 could only remove ∼62% MB from the solution. GO–nZVI (1 : 5) showed the highest MB removal percentage suggesting that not only did it have sufficient nZVI content for easy and quick magnetic separation, but also it had optimum nZVI particle content on the GO surface that resulted in reduced sizes of nZVI particles  which make it difficult to compare the results across the literature. Compared to the study results presented in Table S1,−1 of GO–nZVI (1 : 5) was able to remove 91.8% of MB from 12 ppm MB solution while 0.4 mg mL−1 of nZVI-bamboo64 was able to remove 92.3% MB from 10 ppm MB solution in 120 min. This provides the evidence for the rapid removal of contaminants using the synthesized GO–nZVI nanohybrids in this study.Removal percentage of MB from aqueous solution using nZVI, GO–nZVI (1 : 5), and GO–nZVI (1 : 10) are presented in s Fig. S2 resistin65 In previous studies, acetic acid,66 hydrochloric acid,67 methanol,68 and ethanol66 have been used for the regeneration of nZVI or magnetic GO. In this study, ethanol was selected for MB desorption from the nanohybrids as it is a non-toxic solvent.6970The ability to regenerate and reuse an adsorbent multiple times is directly related to its economic value.GO–nZVI nanohybrids with different GO : nZVI ratios have been synthesized using biocompatible crosslinker NHS/EDC to provide adsorbents with superior ability for contaminant adsorption and magnetic separation, compared to nZVI only. Among the different ratios, GO–nZVI (1 : 5) exhibited the highest MB removal (up to ∼99%) with fast magnetic separation. This can be attributed to the synergistic contribution by GO and nZVI due to the non-aggregated and smaller sized nZVI. Thus, this study indicates the significance of regulating the nZVI particle loading amount in optimizing the performance of the nanohybrids. Further studies are needed to experimentally determine the biocompatibility and ecotoxicity of these newly synthesized hybrids in order to confirm its applicability as a safer-by-design nanohybrid.The authors declare that they have no conflict of interests.RA-009-C8RA08386J-s001