Adolescents and young adults (AYAs) diagnosed with cancer undergo a range of cancer treatments with intension of being cured. It is well-documented that cancer treatment induces a risk of infertility due to ovarian damage. In recent years fertility preservation options for female AYAs with cancer have developed with opportunities for pregnancy after completion of cancer treatment. Despite international guidelines, a high level of evidence underpins the insufficiency of fertility counselling towards this patient group. The aim of this qualitative study was to examine the experience of fertility counselling from the AYAs perspective, and we strive to increase the attention towards the need of improvement and developing national guidelines at international level to ensure adequate and uniform fertility information.Background: Adolescents and young adults (AYAs) with cancer are at risk of therapy-induced infertility. The importance of initial and specialized fertility counselling to this patient group is undisputed. Despite international guidelines, oncofertility counselling is still inadequate. The purpose of this study was to examine how female AYA cancer patients and survivors experienced initial and specialized oncofertility counselling, and to present their specific suggestions on how to improve the oncofertility counselling. Methods: Twelve individual semi-structured interviews were performed with AYAs aged 20–35 with cancer or who were survivors. Participants were recruited via a youth support centre and social organization for AYAs with cancer. Data was analysed using thematic analysis. Results: Three main themes were found: Support is needed for navigating the fertility information jungle; The doctor’s approach determines the content of the patient consultation; Inadequate and worrying information causes mistrust and frustration. Conclusion: Results indicate a continuing problem regarding insufficient oncofertility counselling to AYAs with cancer. To ensure adequate and uniform information, especially in the initial oncofertility counselling, national guidelines for oncology specialists are suggested including multidisciplinary effort and collaboration between oncology and fertility specialists in mind. In addition, participants suggest focus on communication skills. Adolescents and young adults (AYAs) with cancer are considered to be a patient group with physical, emotional and social challenges specific to their age, throughout treatment and in survivorship . A canceIt is well known that one consequence of cancer treatment, especially alkylating chemotherapy and radiation therapy, is the risk of reduced fertility—for both genders ,6,7,8. HInternational guidelines recommend that initial, pre-treatment oncofertility counselling should be provided to young, fertile cancer patients as soon as possible upon diagnosis, and health professionals working in this field must be prepared for this conversation ,10,13,14AYAs with cancer and survivors are concerned about their fertility ,17,18, aDespite international guidelines and AYAs’ need for fertility counselling, initial oncofertility counselling is not always conducted systematically to patients treated in oncological and haematological settings, even though being offered specialized oncofertility counselling is associated with increased quality of life for AYA survivors . New eviIn Denmark, approximately 900 women a year aged 18–39 are diagnosed with cancer . At the The aim of this study was twofold: (1) to examine how female AYA cancer patients and survivors experienced initial and specialized oncofertility counselling; and (2) to present their specific suggestions on how to improve the oncofertility counselling.The study was carried out during September 2020 at ”Kræftværket”, the youth support centre and social organization for AYAs with cancer at the University Hospital of Copenhagen, Rigshospitalet.Inclusion criteria were female cancer patients and survivors aged 18–39. Recruitment was facilitated through Kræftværket, via a written invitation posted on a closed Facebook group managed by Kræftværket. The closed Facebook group has approximately 320 active members, all AYAs with cancer or survivors. At the time of recruitment, twelve participants who meet the inclusion criteria signed up to take part in the study. The participants were aged 20–35 (mean age: 28).The interview guide comprised questions to elicit thoughts about future family planning and pregnancies, expectations of oncofertility counselling, and experiences of the counselling during and after cancer treatment see .Twelve individual semi-structured interviews were performed and lasted, on average, 33 min (between 23 and 59 min). As data saturation  was achiThe analysis of this study was inspired by a thematic analysis approach rooted in Malterud’s systematic text condensation  and BrauThe project was approved 1 September 2020 by the local Data Protection Agency (P-2020-849). The participants were informed about the aim and design of the study, including information about anonymity and that they could withdraw their consent at any time if they wished without adverse consequences for their treatment. They were also informed that the interviews would be recorded electronically. Written informed consent was obtained from all participants.In this article, doctors at the respective oncology and haematology departments are defined as oncology specialists. Doctors at the fertility clinic are defined as fertility specialists.Oncofertility counselling is divided into initial counselling and specialized counselling. Initial counselling is primarily facilitated by oncology specialists and deals with the risk of decreased fertility due to cancer treatment and, if indicated, the patient is referred to a fertility unit at the same hospital. The specialized counselling, regarding fertility preservation options and subsequent follow-up after cancer treatment, is facilitated by fertility specialists.Characteristics of the study population are presented in Three themes were identified, as presented in Most of the participants in this study indicated that initial oncofertility counselling had not been offered by the oncology specialists at diagnosis or at subsequent consultations. The AYAs independently had to request specific information about the opportunity for fertility preservation. Likewise, some participants had to ask for referral to the fertility unit to receive this information. During the cancer treatment period, very sparse fertility information was offered by the oncology specialists:“you have learned from being through such a long process, that you have to knock on the door yourself, and phone and ask and follow up”Several participants felt somewhat responsible for their own treatment regarding fertility. This led to an overwhelming level of active decision-making. This often included dilemmas regarding choice of chemotherapy, administration of hormone substitution and finding an oocyte donor. There was no guidance from the oncology specialists. They also described having to be self-reliant and that they learned to take the initiative if they wanted oncofertility counselling:“…as a patient you are told ‘we are in control of your chemo and we are in control of your blood tests, we are in control of everything’, but the Goserelin , you just have to keep track of it yourself … it was almost as if my fertility was my own responsibility”Falling between two different departments and facilitating the communication on your own about fertility preservation issues were experiences shared among several participants. They felt that they were sent back and forth between different departments when, e.g., side effects of hormone replacement therapy arose. This led to the feeling of being in the middle of a negotiation about which department should be responsible for handling the given issue.“Where there has been, as I said, a mess-up, that’s when the gynaecologist took over. She should not have done that. I should probably have just been to the fertility doctor. So, I would say the fertility doctor could probably have done the same for me, given me hormones and whatever else was needed … you are tossed back and forth between gynaecologists and the fertility clinic”Most participants felt obliged to seek initial oncofertility counselling elsewhere—and felt that the search for information was like navigating a jungle because they did not know their opportunities. In particular, the Internet, including closed Facebook groups, was used to gain information about fertility preservation, along with options regarding adoption, oocyte-donation and surrogacy. Moreover, at Kræftværket, health professionals and other AYAs were informative when the participants sought information. The participants also received information at “theme meetings” about fertility that were arranged by Kræftværket. A senior doctor from the fertility unit took part and gave information in plenary and answered questions. Some participants sought information from their relatives, who themselves had experience of oncofertility counselling or among their private networks:“so I called an acquaintance of the family [ed. haematologist at another hospital] and said I simply need someone to tell me how to make head or tail of this, because I cannot really find out what’s going on and I am given different messages … so, yeah, I felt compelled to call another doctor because I did not trust what the oncology specialists said”During cancer treatment, most of the participants experienced that fertility was a topic the oncology specialists avoided. The participants were not asked about their thoughts or concerns about fertility, and this most obvious issue, from the participants’ perspective, was not addressed. At the first consultation with oncology specialists, some participants were given very brief information about the risks of infertility from cancer treatment, without further explanation or the chance to ask questions. Some even experienced that, when fertility was mentioned by the patient, the conversation was shut down, and there was no further dialogue on the topic:“the same day that I get the diagnosis, the doctor also says something in the direction of ‘well, and we cannot manage to take some eggs out and that’s how it is with leukaemia, and we just have to move on from here’. So, it was a bit of an inserted sentence now, it was only because I really felt it had been shut down from day one that I did not ask more about it”Many participants described that the oncology specialists’ approach or attitude when fertility information was brought up by the patient had a significant impact on the conversation, which often closed it down. The oncology specialist’s attitude when fertility was addressed by the patient was described by several participants as embarrassed, annoyed or judgmental.“The doctor became a little embarrassed, he did not quite know how to react”And “I feel, in a way, that my doctor gets sort of annoyed—’you cannot be a mother when you have a tumour like this in your head … it is not good for you to get pregnant because you will hurt yourself and the tumour can grow when you get pregnant’” .Some participants argued that the oncology specialist’s gender at some point influenced the approach to initial oncofertility counselling. Female doctors more often seemed to understand the importance of the issue, and initiated conversations about fertility—contrary to the male doctors, who were more likely to avoid the topic.“The doctor just said ‘my patients are usually over the age of 60 …’ you have to find someone else to ask,’ because he  did not know”And “Before I arrived, she  had actually tried to get hold of someone up at the fertility clinic so I could talk to someone even the same day, because they were probably well aware that, when you are 25, it is something that means a lot to you” .The participants did not question the sincere dedication and professional skills of the oncology specialists regarding the patient’s cancer treatment. However, the risk of infertility was for several participants the most overwhelming concern at diagnosis and during treatment. Therefore, the lack of sufficient initial oncofertility counselling had a huge impact on their experience of the medical consultation during and after cancer treatment. This was especially the case when the participants tried to initiate a conversation about fertility without being met with adequate answers or an accommodating approach. One of the participants stated that the oncology specialists were pessimists, and the fertility specialists were optimists: “Oncologists have a very pessimistic view of it and very much a sort of dead-set view of it. They do not have that down at the fertility clinic. I also think they know how happy a baby can make a woman”In contrast to this, the participants who were referred to the fertility specialists for specialized oncofertility counselling, expressed being met with understanding, taken seriously and were reassured that the fertility specialists would help them as much as possible with fertility preservation and subsequent fertility treatment, if necessary. Moreover, the fertility specialists were up-to-date regarding the plans for the participant’s cancer treatment and knew about the risks of infertility related to the various chemotherapy regimens. This led to a great sense of relief in relation to the participants’ concerns and allowed them to focus on the cancer treatment itself and convalescence. One participant argued that, even though she unfortunately did not have any fertility preservation options due to the location of the cancer and the need for acute chemotherapy, the fact that the fertility specialists had taken the time and presented her the information as to why it was impossible, led to her being able to accept the risk of infertility:“I felt like the opportunities I have at least heard of before, they went through them for me. It was then also explained why it was not an option, so regardless of whether you liked the answer you got or not, you were then at least clear that they had considered all the options that could have been possible”Finally, some participants mentioned that, even though they had a huge need for oncofertility counselling, it could be difficult to have an open conversation about this, especially if their relatives e.g., parents or a relatively new romantic partner participated—especially if they had never discussed this or told their parents about their family planning dreams. This was particularly difficult if the participant was unprepared for the topics of the consultation and if the risk of infertility suddenly was brought up. One participant, whose mother joined the consultations, argued that this led to a mother-daughter conflict, and the patient chose to exclude her mother from her further fertility treatment plans:“then you have to also hear her [her mother’s] views. So, it crosses a line to have to tell about my baby plans, I also think it’s a bit annoying actually … so therefore I have not told her that I am now going for fertility treatment”Most of the participants argued that they had received an inadequate level of initial information regarding fertility from the oncology specialists and it had not been explained why the cancer treatment increased the risk of infertility. Written information regarding cancer treatment and fertility preservation options either did not exist or was too generalized. Participants with germinal cell-type ovarian cancer experienced a lack of written fertility information and received written information regarding testicular cancer and fertility preservation options, because the oncology specialists did not have specific written information for AYAs with this type of ovarian cancer. The participants did not benefit from this material—and felt insignificant as a minority group, in contrast to the larger group of elderly menopausal women who are diagnosed with ovarian cancer:“I don’t think I’ve received much advice about it … I’ve received the same treatment as if I’d had testicular cancer, so they only had these information brochures and so on that they had for testicular cancer patients”If the participants received information, it was often too generalized and described all the options in detail. The participants argued that reading about options that were irrelevant for them, made them insecure about whether the right choice had been made for them regarding fertility preservation.“In the written information you get, all the possibilities are given ... but of course, you don’t have all the possibilities”Several participants with cryopreserved ovarian tissue were uncertain of the circumstances surrounding the cryopreserved tissue, e.g., the location of their stored tissue and the length of time the tissue would be preserved. This led to uncertainty about the time range for fertility treatment after cancer treatment, which they didn’t feel the doctors took into account during the specialized fertility counselling. Moreover, the participants were in doubt about whether they still belonged to the fertility unit or not, and where to obtain information, since most of them only had been in contact with the fertility unit once or twice.“The week after the operation it was like ’where is it really that my ovary is now?’ Is it frozen somewhere? It’s in a freezer ... I did not know where it was or who I should get it from if I wanted to use it”The majority of the participants had experienced receiving divergent information regarding fertility, especially from the oncology specialists. In this case, it was difficult for them to clarify which answer was the correct one. This led to a great deal of frustration and the feeling of being left alone without knowing where to turn. The divergent information concerned, e.g., how long the patient had to wait before trying to become pregnant after cancer treatment. The participants also mentioned conflicting information regarding whether the chemotherapy regime they had received would increase the risk of infertility. One participant had been through several regimes since childhood, and her mother had on numerous occasions asked the paediatric oncology specialists about the risk of infertility and been told that there was no risk. When, at age 20, the patient again had to undergo a new chemotherapy regime, she asked the oncology specialists herself about the risk of infertility. She was told that her ovaries had already been damaged due to previous chemotherapy. This shocked both the participant and her mother and led to a feeling of mistrust towards the doctors when given new information:“The oncologist said this summer that my ovaries were already affected because I had had such strong chemo as an 8-year-old. And my mother was in shock—then she said that it couldn’t be right, because then the doctor said it was not going to affect them at all”Several participants experienced that the information they received was not only incorrect but also frightening. One participant was briefly informed about cancer treatment and the risk of infertility at her first consultation with an oncology specialist, before the patient had been informed that she had cancer:“After surgery on the ovary, I had a conversation at hospital XX where they said it was just a normal cyst, but we’re just passing it on to Hospital YY for safety’s sake. And then I came to talk at Hospital YY, where I just thought it was like that … and then they talked to me like I knew I had cancer ... yes but it was actually cancer you had, you have to start chemo on Monday, it was sort of ‘wow’”Another participant, who had not received fertility preservation, was informed incorrectly, by an oncology specialist, that her oocytes had been genetically damaged from the chemotherapy and she would have to wait two years before trying to become pregnant. This information frightened her, because she had been reassured prior to cancer treatment that this was not the case, and now it was too late, if the new information was true. On asking other oncology specialists, no one could give her an accurate answer as to whether it was true or not.In the interviews, the participants were asked to give suggestions for possible improvements to the oncofertility counselling. The participants were free to come up with suggestions of any kind; thus, both suggestions for initial and specialized fertility counselling were proposed. The specific suggestions, along with essences from the themes regarding needs and wishes for fertility counselling, are presented in In this study, we found that most participants did not receive the fertility counselling, whether initial nor specialized, that they wished. Many of the participants felt as if they were falling between departments, not knowing which department was responsible for different aspects of the fertility counselling. This testifies to a major gap between oncology and fertility specialists and that bridging this gap is essential, in order to improve AYAs’ experiences of responsibility for addressing the initial and the specialized content of oncofertility counselling ,31. In lSeveral participants in our study experienced a condescending approach or response from the oncology specialists that negatively influences their relationship, which leads to dissatisfaction with the initial oncofertility counselling. Previous studies support our findings, arguing that oncology specialists’ personal assumptions can potentially have implications for the patient’s well-being and cause great concerns if the approach is considered to be patronizing ,31,32. MThe reason for avoidance or vague initial fertility information may be a lack of oncofertility knowledge ,31,34 onAs regards to the content of written and spoken information handed out on the wards, our study showed that the participants often experience a lack of sufficient written and verbal fertility information. Written information is either non-existent or too generalized. The importance of accurate and individualized written information is consistent with previous studies that argue that receiving timely, detailed, and accurate spoken and written information is essential ,45. MoreOn the positive side, in our study we found that those few participants who received specialized fertility counselling in addition to initial fertility information from the oncology specialists expressed greater satisfaction and comfort. When specialized fertility information was given, the participants were more likely to accept their current situation and felt more satisfied with the oncofertility counselling compared to those who received inadequate fertility information. Moreover, a focus on post-cancer treatment follow-up with fertility assessment and sexual and reproductive health counselling was more likely to be conducted when the AYAs were referred to fertility specialists. This had a positive impact on their experience of oncofertility counselling. The need for greater access to fertility clinics is described in the literature ,44,46,47The suggestions for fertility counselling proposed by the participants in the current study are consistent with recommendations and studies in the field, e.g., regarding doctors’ communication skills and psychosocial and family support ,40,44. AA strength in this study is the use of qualitative interviews, which allowed for an in-depth examination of the participants’ experiences of oncofertility counselling. Moreover, the study participants represented a wide variation in demographics, including a balanced age distribution and relationship status—including single AYAs and those in relationships. The population represented five of the most common cancers in AYAs in Denmark . Fifty pThe study was limited by being conducted at a single centre, limiting general statements on how widespread in Denmark is the lack of initial and specialized oncofertility counselling. However, international studies have also addressed this issue ,52.The study was undertaken at a regular hospital ward transferable to other national and international hospital settings with the same patient population, which strengthen the external validity of the study .With the international definition of AYAs being age 15–39  a possibResults in this single centre, qualitative study indicate a continuing problem regarding insufficient oncofertility counselling to AYAs with cancer. In our setting, this implies the need for further improvement to ensure uniform and adequate information, especially in the initial oncofertility counselling. National clinical guidelines for oncology specialists are suggested, including information about cancer treatment and fertility, fertility preservation options and characteristics of patients who should be referred to fertility specialists. We believe, it is necessary to be aware of the importance of the multidisciplinary effort and the collaboration between oncology and fertility specialists at time of diagnosis, during cancer treatment and post-treatment. In addition, participants in this study, suggest focus on verbal and written information along with communication upskilling to improve oncofertility counselling.


The selectivity in the simultaneous detection of ascorbic acid (AA), dopamine (DA), and uric acid (UA) has been an open problem in the biosensing field. Many surface modification methods were carried out for glassy carbon electrodes (GCE), including the use of graphene oxide and amino acids as a selective layer. In this work, molecular dynamics (MD) simulations were performed to investigate the role of serine oligomers on the selectivity of the AA, DA, and UA analytes. Our models consisted of a graphene oxide (GO) sheet under a solvent environment. Serine tetramers were added into the simulation box and were adsorbed on the GO surface. Then, the adsorption of each analyte on the mixed surface was monitored from MD trajectories. It was found that the adsorption of AA was preferred by serine oligomers due to the largest number of hydrogen-bond forming functional groups of AA, causing a 10-fold increase of hydrogen bonds by the tetraserine adsorption layer. UA was the least preferred due to its highest aromaticity. Finally, the role of hydrogen bonds on the electron transfer selectivity of biosensors was discussed with some previous studies. AA radicals received electrons from serine through hydrogen bonds that promoted oxidation reaction and caused the negative shifts and separation of the oxidation potential in experiments, as DA and UA were less affected by serine. Agreement of the in vitro and in silico results could lead to other in silico designs of selective layers to detect other types of analyte molecules. The discovery of graphene has opened the era of two-dimensional nanomaterials for different uses. Graphene is a two-dimensional sp2 hybridized carbon atom packed in a hexagonal lattice ,2,3. DueMany studies showed that using a cyclic voltammetry (CV) or differential pulse voltammetry (DPV) with glassy carbon electrodes (GCE) can detect AA, DA, and UA up to the micromolar limit of detection ,21,22. HIn this study, to gain more insight into the selective mechanisms of the polymerized L-serine and graphene oxide decorated on the GCE in a solvent environment, atomistic molecular dynamics (MD) simulations were performed for the model systems  consistiSix atomistic MD simulations were performed: (a) GO + AA, (b) GO + DA, (c) GO + UA, (d) GO + SE + AA, (e) GO + SE + DA, and (f) GO + SE + UA . The firFor systems decorated with serine tetramers, tetraserines lying across the GO formed a primary layer over the surface. The tetraserine layer selectively interacts with some analyte molecules and thus shields them away from the GO surface and facilitates the stacking between analyte molecules. n) as a function of distance from the surface were calculated between the analyte molecules and the GO surface for the systems of AA, DA, and UA adsorbed on GO without serine tetramers  was 8.63 for AA at the 0.48 nm distance, while the higher Cn of 9.93 was found for DA at the same distance. For UA, 9.73 molecules formed the first adsorption layer, and the remaining 2.36 UA molecules were adsorbed in the second layer. Binding constants were found at 0.62, 1.16, and 1.03 M−1 for AA, DA, and UA, respectively, suggesting that UA was the most energetically favorable analyte for the bare GO. The planar structure of UA could increase the surface area for binding and promote the van der Waals contacts. n at the first adsorption layer was decreased to 4.63, and the binding constant was substantially dropped to 0.15 M−1. Similar to AA, the decreased first RDF peak of DA on the GO surface when decorated by tetraserines was observed along with the higher probability distribution at a further distance. This also demonstrated the shielding effects of tetraserines, but the effects on DA were less significant than those on AA. The Cn at the first adsorption layer for DA on the GO surface was decreased to 7.80, and the binding constant was decreased to 0.45 M−1. For UA, however, only a slight decrease of the first RDF peak was observed, suggesting that the binding of UA on the GO surface was mostly unaffected by tetraserine. Only a small decrease of the Cn at the first adsorption layer and the binding constant were found at 9.69 and 1.01 M−1, respectively. The absorption of analytes on the GO surface was affected by the presence of tetraserine, as shown in n. Comparing the RDF profiles of AA, DA, and UA on the tetraserine surfaces in −1 for AA binding was significantly higher than the 0.18 M−1 for DA and 0.11 M−1 for UA binding. Coordination numbers and binding constants calculated from the first adsorption layers on GO were in concurrence with the analysis of minimum distances, in which AA molecules were the most affected and the UA molecules were the least affected by tetraserine decoration. UA/kAA = 1.66 M−1) were found for systems without tetraserines when considering the binding constants. On the other hand, clear differences (kUA/kAA = 6.73 M−1) in the binding constants were found for systems with tetraserines. These results suggested that tetraserine was unequally affected and was selective for the binding of AA, UA, and DA, which was due to the intrinsic properties of the analyte molecules. The high affinity of AA binding on the tetraserine surface shown in In the previous section, our MD simulations and RDF analysis illustrated the selective adsorption of AA, DA, and UA analytes on the GO surface decorated by tetraserines. The selectivity was contributed by the binding preference of AA on tetraserines and UA on the GO surface. Due to a large number of polar functional groups, a tetraserine in n and a carbonyl (C=O) oxygen atom of the neighboring amino acids  oxygens was found at 0.22 nm. Now, consider the distances between hydrogen bond donors in an AA molecule  oxygens of tetraserine as acceptors, the amine group at the tetraserine backbone and the hydroxyl group at the tetraserine sidechain acted as donor atoms for hydrogen bonding but were less dominant than the carboxyl (COO-) acceptors.Hydrogen bond analysis was then performed between the analytes and the tetraserine decorated substrates by counting the number of events that meet these criteria: (1) the distance between a donor atom and an acceptor atom was less than 0.35 nm, and (2) bending angle made by a group of donor-hydrogen-acceptor was less than 30°. It was found that the number of hydrogen bonds between the analytes and GO in 680 chlorophylls was facilitated by hydrogen bonding of the tyrosine with a deprotonated histidine residue. The phenol group of the tyrosine received electrons from the imidazole ring of the histidine and gave away the proton. After the first charge transfer, the phenol became a phenoxyl radical, for which the adiabatic ionization and the oxidation potential for the electron transfer to P680 became lower [In this work, atomistic molecular dynamics simulations were used to provide atomistic details on the selectivity of a GO electrode modified by serine oligomers for the simultaneous detections of the AA, DA, and UA molecules. Our model systems consisted of a small GO flake and several analyte molecules. Adsorption of analyte molecules of the GO flakes was monitored either in the presence or in the absence of tetraserines. It was found that the role of intrinsic molecular properties of the three analytes promoted the hydrogen bonding with the tetraserines, which became a selective layer. Molecular mechanisms of the enhanced selectivity by serine oligomers addressed by the MD simulations were in concurrence with our differential pulse voltammetry results of simultaneous detection of AA, DA, and UA. The largest negative shift found for the oxidation potential of AA corresponded to the highest number of hydrogen bonds with serine oligomers. Meanwhile, the smallest negative shift found for the oxidation potential of UA corresponded to the lowest number of hydrogen bonds with serine oligomers see . The higme lower . Analogome lower . TherefoAgreement of the in vitro and in silico results demonstrated the importance of the intrinsic properties, e.g., local polarity of functional groups, on the binding specificity and sensitivity of the electrode to the analytes. In addition, the simulation techniques had been validated for other designs of selective layers to detect other analytes, based-on the electro-polymerization of other biopolymers. However, not all molecular features of the real electrode system in vitro were depicted in the current in silico molecular model due to the limited simulation computing resource. Calibration of the model with the larger experimental dataset was also needed for more accurate prediction. 3 SPC [The 3D structure of a model graphene oxide nanosheet with dimensions of 20.94 nm × 22.56 nm in 3 SPC  solvated3 SPC , each sy3 SPC  at 300 K3 SPC . After all simulations were completed, water molecules were deleted from the trajectory files. Minimum distances were measured between the closest atom pairs from each analyte molecule and the GO surface to monitor the adsorption of each molecule on the bare GO and the tetraserine-decorated GO surface. Then, radial distribution functions (RDF) and coordination numbers (Cn) were calculated as a function of closest distance from the GO surface for the GO + AA, GO + DA, and GO + UA systems without serine tetramers. RDF and Cn of analyte molecules were calculated both from the GO surface and from serine tetramers for the GO + SE + AA, GO + SE + DA, and GO + SE + UA systems decorated by serine tetramers. To further quantify the adsorption of analyte molecules, the binding constant (k) was calculated from the cumulative average number of analytes at the first adsorption layer (N) by using Equation (1). Numbers of hydrogen bonds formed (1) between analytes and GO and (2) between tetraserine and GO were also calculated along all the trajectories. The cut-off distance between the donor atom and the acceptor atom of a hydrogen bond was set to 0.35 nm, and the bending angle cut-off was set to 30°. Finally, the RDF between hydrogen bond donor atoms of the analytes and all the oxygen atoms of the tetraserines was also calculated to display the contribution of each atom to selective binding of the analytes on the tetraserine surface.


Virus propagation is stimulated by (1) clonal expansion of infected cells and (2) de novo infection. Viral gene expression is induced by the transactivator protein Tax, which recruits host factors like positive transcription elongation factor b (P-TEFb) to the viral promoter. Since HTLV-1 gene expression is repressed in vivo by viral, cellular, and epigenetic mechanisms in late phases of infection, HTLV-1 avoids an efficient CD8+ cytotoxic T-cell (CTL) response directed against the immunodominant viral Tax antigen. Hence, therapeutic strategies using latency reversing agents (LRAs) sought to transiently activate viral gene expression and antigen presentation of Tax to enhance CTL responses towards HTLV-1, and thus, to expose the latent HTLV-1 reservoir to immune destruction. Here, we review strategies that aimed at enhancing Tax expression and Tax-specific CTL responses to interfere with HTLV-1 latency. Further, we provide an overview of LRAs including (1) histone deacetylase inhibitors (HDACi) and (2) activators of P-TEFb, that have mainly been studied in context of human immunodeficiency virus (HIV), but which may also be powerful in the context of HTLV-1.Human T-cell leukemia virus type 1 (HTLV-1), the cause of adult T-cell leukemia/lymphoma (ATLL), is a retrovirus, which integrates into the host genome and persistently infects CD4 Human T-cell leukemia virus type 1 (HTLV-1) is a highly oncogenic retrovirus causing adult T-cell leukemia/lymphoma (ATLL) or inflammatory diseases like HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in up to 10% of infected individuals ,3,4,5. WHTLV-1 poses as the etiological agent of malignant and inflammatory diseases, which develop after a clinical latency of years to decades. The aggressive T-cell malignancy ATLL and the inflammatory neurological conditions HTLV-1-associated HAM/TSP are of major concern . Moreove+ ATLL in Japan  is FDA approved and was initially used to treat alcoholism ,234. In tent HIV . MoreoveAnother potential compound for retroviral latency reversal is trichostatin a TSA; . LuciferThe last P-TEFb releasing agent discussed here is an analog of cytidine named 5-Azacytidine AzaC; . As a nuIn conclusion, besides the use of HDACi, P-TEFb releasing agents represent a potent alternative for reversing the latency of HTLV-1. These compounds offer the possibility to release P-TEFb from the HMW complex or the LMW complex. Since released P-TEFb could complex with Tax to promote viral transcription, it is reasonable to study the potential of P-TEFb-modulating agents either individually or in combination with other LRAs to reactivate HTLV-1 from latency.Thus far, HDACi and activators of P-TEFb have been outlined as LRAs. Beyond these agents, other stimuli can also activate HTLV-1 transcription. These include mitogens, sirtuin inhibitors, and extracellular factors. Moreover, also metabolic circumstances affect HTLV-1 latency. These regulators of HTLV-1 latency will be briefly reviewed in this section.T-cell activating mitogens are known for their potency to enhance Tax protein levels in HTLV-1 infected T-cell lines . Among tA commonly used phorbol ester to stimulate retroviral LTRs, among them the HTLV-1 LTR, is PMA. PMA acts as a diacylglycerol analog to activate protein kinase C (PKC) ,241. How2+ ionophore ionomycin. Ionomycin is a T-cell activator and stimulates latent viral transcription. Moreover, ionomycin can act synergistically with PMA to activate PKC [Apart from that, PKC triggers reactive oxygen species (ROS) production, thereby causing DNA damage and cell stress ,245. As vate PKC ,247. Sinvate PKC ,248.+-dependent class III of HDACs has been briefly introduced in The non-zinc-dependent but rather NADIt has been observed that SIRT 1 is overexpressed in ATL patients and acts as an antiapoptotic molecule in malignant cells. This is supported by the finding that the SIRT 1/2 inhibitor sirtinol caused growth and cell cycle arrest and apoptosis in PBMCs from ATL patients and HTLV-1 positive cell lines . Furthermore, sirtinol blocked NF-κB activity by inhibiting its translocation into the nucleus ,250,251.Tax mRNA [Interestingly, SIRT 1 can suppress Tax activation of the HTLV-1 LTR. However, SIRT 1 inhibitors, such as sirtinol and Ex527, can reverse this effect . AdminisTax mRNA . Novel sTax mRNA . Therefo+ reporter cells [Tax transcription [Once HTLV-1 infected PBMCs are introduced into culture, they display strong spontaneous transcriptional reactivation ,256. Of er cells  (Table 3er cells . These oer cells ,254,255.cription ,255. FurYet, it is unclear whether increased viral transcription also results under any conditions in enhanced viral protein expression and antigen presentation. For HIV, several studies have described post-transcriptional blocks, for HTLV-1, this is unknown ,132.The treatment of HTLV-1-infected patients with HDACi or related compounds does not specifically target virus-infected cells. Rather, other cells like CTLs themselves may also be affected by the treatment and may be functionally impaired. Thus, individual compounds have to be monitored more closely.Enhanced viral transcription may result in increased viral replication and worsening of the phenotype if the CTL response does not work properly. Since viral reactivation also poses the risk that a yet repressed but harmful HTLV-1-infected T-cell clone proliferates upon treatment, close monitoring of patients would be required.It is unclear whether the combination of antiretroviral therapy with different compounds used for the Kick and Kill strategy could improve clinical parameters comparable to the approaches used in HIV reactivation.The outcome of viral reactivation may differ depending on the viral integration sites in the human genome.Viral reservoirs are largely unknown for HTLV-1. Most studies performed thus far monitored the impact of LRAs on peripheral blood mononuclear cells. However, whether and how LRAs may impact viral gene expression in other cell types and tissues and how accessible these viral reservoirs are, is not understood.The impact of LRAs on HBZ expression and HBZ-specific CTLs remains to be elucidated in more detail.Despite promising results in cell culture experiments and in ex vivo analyses of fresh peripheral blood mononuclear cells from HTLV-1-infected patients, the first clinical trials using HDACi did not lead to a sustained reduction of the proviral load, nor could the patients be cured. However, until now, most trials in HTLV-1-infected patients have been focusing on valproate, which is known to be far less potent than the newer classes of HDACi that have been developed in the meantime . Thus, fSumming up, more work is needed to gain novel insights into host factors and chemical compounds required for manipulating HTLV-1 gene expression, and thus, the immunogenicity of HTLV-1. This holds important implications for the Kick and Kill strategy to relieve the disease burden of ATLL and HAM/TSP.


The relationship between hyperuricemia, gout, and renal disease has been investigated for several years. From the beginning, kidney disease has been considered a complication of gout; however, the viewpoints changed, claiming that hypertension and elevated uric acid (UA) levels are caused by decreased urate excretion in patients with renal impairment. To date, several examples of evidence support the role of hyperuricemia in cardiovascular or renal diseases. Several mechanisms have been identified that explain the relationship between hyperuricemia and chronic kidney disease, including the crystal effect, renin–angiotensin–aldosterone system activation, nitric oxide synthesis inhibition, and intracellular oxidative stress stimulation, and urate-lowering therapy (ULT) has been proven to reduce renal disease progression in the past few years. In this comprehensive review, the source and physiology of UA are introduced, and the mechanisms that explain the reciprocal relationship between hyperuricemia and kidney disease are reviewed. Lastly, current evidence supporting the use of ULT to postpone renal disease progression in patients with hyperuricemia and gout are summarized. Hyperuricemia is described as elevated levels of serum uric acid (UA), which is more commonly known as gout, an acute, symptomatic disease where urate crystals deposit in the joint and cause subsequent inflammatory arthritis. Gout, which is defined as “a progressive metabolic disease characterized by symptomatic hyperuricemia and the deposition of monosodium urate crystals in the joints and soft tissues due to an imbalance in UA uptake, synthesis, or excretion” , is the CKD is defined as abnormalities of the kidney structure or function presented for more than 3 months, according to the Kidney Disease Improving Global Outcome guidelines . Kidney Elevated serum UA levels are exacerbated by diets high in fat, protein, or nucleic acid components. Among these products, purine nucleic acid ingestion exhibited the greatest dietary influence on blood UA levels ,11. The UA is a product of the purine nucleic acid metabolic pathway. Apart from the enzymes involved in purine nucleotide degradation, xanthine oxidase (XO), also known as xanthine oxidoreductase, plays an important role in the biosynthesis of UA. XO is not only present in the plasma but is also widely distributed in various organs including the liver, gut, lung, kidney, heart, and brain, is normally present as an inactive nicotinamide adenine dinucleotide-dependent cytosolic dehydrogenase precursor, and is processed to its active form via oxidation or proteolytic modification . This enIn most mammalian species, UA is not the end product of the purine nucleic acid metabolic pathway. UA is further metabolized to the more soluble allantoin by uricase and readily excreted in the urine. However, humans lack the functional uricase enzymes, making UA the final degradation product of the metabolic pathway . In humaHyperuricemia should be considered as a combined result of UA overproduction and underexcretion by the kidneys; therefore, all of the genetic variations involved in UA metabolism and elimination could contribute to hyperuricemia.Several single-locus mutations are known to cause hyperuricemia via UA overproduction. Hypoxanthine-guanine phosphoribosyltransferase (HPRT) is an enzyme that recycles hypoxanthine and guanine. HPRT malfunction results in excessive UA production. Lesch–Nyhan syndrome, an X-linked disorder of HPRT deficiency, is characterized by UA overproduction resulting in precocious hyperuricemia, UA nephrolithiasis, spasticity, and mental retardation . PhosphoAs mentioned in the previous paragraph, urate was mainly excreted by the kidney and gastrointestinal tract in humans, so hyperuricemia can result from renal and gut underexcretions of UA . Thus, iIn addition to the proteins directly involved in UA metabolism and excretion, the mechanisms indirectly influencing urate excretion might also affect serum UA levels. Autosomal dominant tubulointerstitial kidney disease (ADTKD), also called medullary cystic kidney disease or familial juvenile hyperuricemic nephropathy, is a group of disorders characterized by precocious hyperuricemia, hyperuricosuria, and progressive CKD. Among these disorders, ADTKD caused by mutation in the uromodulin gene  is the most common form of ADTKD and is also known to be involved in its pathogenesis. UMOD is produced exclusively in the thick ascending limbs (TAL) of the loop of Henle, which is involved in intracellular trafficking of the furosemide-sensitive Na-K-2Cl cotransporter and the renal outer medullary potassium channel. Mutations in the UMOD gene cause decreased UMOD production and subsequently decrease the expression of the furosemide-sensitive Na-K-2Cl cotransporter in the apical membrane of the TAL. Decreasing furosemide-sensitive Na-K-2Cl cotransporter expression induces mild natriuresis and volume contraction. To compensate for these changes, the proximal tubule increases sodium reabsorption, which in turn increases proximal urate reabsorption following sodium absorption ,33,34.By identifying the genetic variants of hyperuricemia and gout and investigating its pathogenesis, the prediction of treatment responsiveness, for example, ULT, in hyperuricemia and gout is not impossible, making personalized and precision medication more feasible in the near future.According to cross-sectional case-control studies, hyperuricemia and gout have been reported to be comorbid with multiple diseases, including coronary artery disease, hypertension, type 2 DM, or CKD, and are independent risk factors for premature death ,36,37. GAs mentioned previously, the relationship between hyperuricemia and renal impairment was investigated as early as the 1960s via a postmortem analysis. At the time, the presence of kidney disease in gout patients was considered to be one of the complications of gout and was attributed to the presence of urate crystals in the kidney, leading to the extensive use of the term gouty nephropathy. However, in the 1980s, the viewpoint of the relationship between gout and renal impairment led us to propose that both of these diseases were the consequence of hypertension, causing an initial decline in the renal function and the subsequent elevation of serum UA levels due to decreased urate excretion via the kidneys. Based on this theory, the treatment of hyperuricemia in patients with kidney disease was only recommended to manage gout, renal stones, or, in rare cases, the tumor lysis syndrome. The treatment of asymptomatic hyperuricemia was not recommended at that time.However, the investigators again redefined the relationship between hyperuricemia and renal impairment during the millennium. After reviewing epidemiological studies, Johnson et al. suggested reconsidering the possibility that hyperuricemia plays a role in directly contributing to cardiovascular or renal diseases. These studies suggest that chronic hyperuricemia is strongly associated with chronic tubulointerstitial disease and a consequent decrease in renal function ,40,41. IAccording to experimental studies focused on the identification of mechanistic pathways, several mechanisms exist that might explain why serum urate or urinary UA might cause CKD. By understanding these important pathophysiological processes, investigators can design clinical trials more specifically and interpret the outcomes of clinical trials more accurately.The crystalline effect was discovered initially and is the most widely known negative impact of UA. Glomerular ultrafiltration can be enriched with minerals, proteins, or drug metabolites, which tend to supersaturate under volume depletion or specific urine pH levels. Tumor lysis syndrome, heat stress, and rhabdomyolysis can induce an abundant UA release from cells and transiently increase serum UA levels. The acute elevation of serum UA levels subsequently increases urinary urate levels and precipitates urate crystal formation in the renal tubular lumen, especially in acidic urine or dehydration status ,51. In aIn contrast to acute events, long-standing moderate supersaturation can promote crystal formation and tubule obstruction over extended periods of time and even lead to tissue remodeling ,53,54. SMazzali et al. revealed that rats with hyperuricemia developed hypertension after 3 weeks, which can be prevented or improved through the administration of allopurinol or benziodarone. According to the findings of this animal study, it was concluded that hyperuricemia itself can cause hypertension and renal injury via a crystal-independent mechanism, which can lead to hypertension and renal function impairment through the stimulation of the RAAS and inhibition of nitric oxide synthesis ,61,62,63In addition to the RAAS, UA activates other vasoconstrictors, such as endothelin and thromboxane, and suppresses vasodilatory pathways such as NO ,68. The Although most previous gout studies have mentioned the extracellular effects of UA, mainly the inflammatory response or toxic effects via crystalline deposition in joint spaces or renal tubules, more recent experimental studies have shown that the primary metabolic and renal effects of UA are mediated by intracellular urate ,73. IntrSeveral ULT drugs have been approved in the past few years, which can be classified into three main categories: drugs inhibiting UA synthesis (xanthine oxidase inhibitors (XOi), for example, allopurinol, febuxostat, and topiroxostat); drugs increasing UA excretion ; and drugs enabling systemic metabolic hydrolysis of UA . Considering that the main mechanism of uricosuric agents is to increase renal urate excretion, these agents might increase the risk of adverse renal events due to the elevated urinary UA levels by increasing the renal clearance of UA ,77. MoreAllopurinol, a structural isomer of hypoxanthine, was the first ULT introduced in 1966 and remains the standard treatment for patients with hyperuricemia and gout. Although allopurinol is the most frequently prescribed ULT, it is a relatively weak XOi that is rapidly metabolized to oxypurinol and renally cleared. No more than 50% of patients can achieve serum UA levels lower than 6 mg/dL under an allopurinol dose of 300 mg/day, according to previous reports ,79,80. I2, 95% confidence intervals [CI] −0.9 to 7.1 mL/min/1.73 m2), while it abrogated the increase in serum creatinine levels from the baseline in three trials   3.1 mL/min/1.73 m0 mg/dL) . Several1.73 m2) . In a su1.73 m2) .2 of the body surface area in the preceding year had been randomly assigned to allopurinol treatment or the placebo groups. This study revealed that ULT with allopurinol did not slow the decline in the eGFR compared with the placebo in CKD patients with a high risk of progression [2 of the body surface area randomly assigned to allopurinol treatment or the placebo groups. The authors concluded that no meaningful clinical benefits of serum urate reduction with allopurinol were observed on kidney outcomes among patients with type 1 DM with early to moderate diabetic kidney disease [However, two recent, large, placebo-controlled trials (CKD-FIX and PERL) revealed no benefit from ULT with allopurinol in slowing down CKD progression. In the CKD-FIX study, patients with CKD stage 3 or stage 4 with a urinary albumin to creatinine ratio >265 or an eGFR decrease of at least 3.0 mL/min/1.73 mgression . In the  disease . These t disease ,89,90,91 disease , and patFebuxostat is a selective, nonpurine-type XOi approved by the U.S. Food and Drug Administration in 2009. Febuxostat was reported to be significantly more potent than allopurinol and was more efficacious at doses of 80 to 120 mg to achieve serum urate levels <6 mg/dL compared to an allopurinol dose of 100–300 mg daily ,93. Unli2; 95% CI −1.62 to 6.33) among CKD stage 3 and stage 4 patients than those in the placebo group [p < 0.0001), and the subgroup analysis showed that the renal benefit of febuxostat was consistent across most patient subgroups and/or propensity score-matched cohorts. The adjusted hazard ratio was 0.65 and 0.66 for long-term dialysis and long-term dialysis or death with febuxostat use, respectively. This study revealed that in the CKD stage 5 predialysis patients, febuxostat users had a lower risk of progression to long-term dialysis or death compared to allopurinol users [In 2019, a meta-analysis enrolling 11 clinical trials with 1317 participants revealed that febuxostat not only showed a significant reduction in serum UA levels but was also associated with a higher eGFR (MD 2.36 mL/min/1.73 mbo group . In the bo group . Hsu et ol users . Chung eol users .Although the hepatic metabolism characteristic made febuxostat seem to be a better choice than allopurinol, especially in patients with renal function impairment, it is difficult for clinicians to not notice the results of the CARES study published in 2018. The CARES investigators reported a multicenter, double-blind, noninferiority trial involving patients with gout and cardiovascular disease who were randomly assigned to receive allopurinol or febuxostat. In 6190 patients, febuxostat was noninferior to allopurinol with respect to the rates of adverse cardiovascular events; however, the all-cause and cardiovascular mortalities were higher with febuxostat than that with allopurinol. Despite this finding, this study had a high discontinuation rate, with a trial regimen discontinuation rate of 56.6% and discontinued follow-up rate of 45.0% ,101,102.Besides the traditional therapeutic agent for hyperuricemia, the specific urate reabsorption inhibitor had been investigated as the new therapeutic option for gout and hyperuricemia patients. After that the URAT1 inhibitors had been proved to show no inferior efficacy in lowing serum urate level comparing to the existing ULT ,111, sevStack et al. had revealed that lowering the serum urate by combining febuxostat and verinurad can reduce the serum urate concentration and albuminuria in patients with type 2 DM, albuminuria, and hyperuricemia, and the effect was rapid and the improvement was sustained for 24 weeks . AlthougHyperuricemia can aggravate renal function impairment through several mechanisms, including the direct toxic effect and inflammasome-mediated inflammatory response induced by urate crystallization, activation of the RAAS, and increased intracellular oxidative stress by stimulating NADPH oxidase. ULT has shown its efficacy in slowing the GFR decline in patients with CKD based on the meta-analysis results. Considering the concerns of allopurinol use in patients with renal impairment and severe adverse events, hepatically metabolized febuxostat seems to be a better choice of ULT in patients with renal impairment with noninferior efficacy on lowering the serum urate level, slowing the eGFR decline, and cardiovascular risk. The strategy to combine xanthine oxidase inhibitor with a urate reabsorption inhibitor might also offer a new opportunity on declined CKD progression in patients with hyperuricemia. However, the definite treatment goal of lowering the serum urate level to slow down renal function impairment and prevent cardiovascular risk in patients with hyperuricemia is still ambiguous, and the data from large and long-term trials are expected to identify the goal of serum UA levels and appropriate doses required for ULT.


Adult mammalian hearts show limited capacity to proliferate after injury, while zebrafish are capable to completely regenerate injured hearts through the proliferation of spared cardiomyocytes. BMP and Notch signaling pathways have been implicated in cardiomyocyte proliferation during zebrafish heart regeneration. However, the molecular mechanism underneath this process as well as the interaction between these two pathways remains to be further explored. In this study we showed BMP signaling was activated after ventricle ablation and acted epistatic downstream of Notch signaling. Inhibition of both signaling pathways differentially influenced ventricle regeneration and cardiomyocyte proliferation, as revealed by time-lapse analysis using a cardiomyocyte-specific FUCCI (fluorescent ubiquitylation-based cell cycle indicator) system. Further experiments revealed that inhibition of BMP and Notch signaling led to cell-cycle arrest at different phases. Overall, our results shed light on the interaction between BMP and Notch signaling pathways and their functions in cardiomyocyte proliferation during cardiac regeneration. Myocardial infarction (MI) is a leading cause of death worldwide Zebrafish cardiac regeneration involves multiple signaling pathways Notch signaling pathway also plays important roles in cardiac regeneration Tg(vmhc:mCherry-NTR) with metronidazole (MTZ) at 3 days post fertilization (dpf) in situ hybridization (WISH) staining revealed that gene expressions of several BMP signaling components were dramatically increased in ablated hearts compared to that in control hearts at 5 dpf/ 2 days post MTZ-treatment (dpt)  Fig. A-D'. BMPTg(Bre:dGFP) which expressed destabilized GFP in BMP-activated cells Tg larvae displayed weak GFP signal in the ventricle at 4 dpf, besides other extracardiac signals. After ventricle injury, BMP signaling was activated in the ablated heart, mainly in the atrium, at 1 dpt. The signal intensity was gradually enhanced in the atrium and extended to the ventricle at 2 dpt  larvae. The results suggested that BMP signaling was strongly activated in myocardium during ventricle regeneration, while a weaker epicardial BMP signal could be detected occasionally  that inhibition of BMP signaling by DM treatment did not affect the activation of Notch signaling in the endocardium around atrioventricular canal  or early stage (0-2 dpt) of ventricle regeneration significantly reduced the heart recovery rates  with Tg(vmhc:mCherry-NTR) so that cardiomyocytes in S/G2/M phases would be labelled with green fluorescence  and recorded the change in zGem+ cardiomyocyte pattern by 12-hour-interval living imaging  fish. The overlapped signals of pH3 and EdU with zGem+ could help to eliminate the interference caused by proliferative cells in adjacent tissues, such as endocardium, epicardium and blood cells. Proliferating cardiomyocytes were in different cell-cycle stages , Tg(Bre:dGFP), Tg(tp1:dGFP), Tg(myl7:mAG-zGeminin).Zebrafish were maintained under standard conditions in situ hybridization was performed as previously described notch1b, nkx2.5, gata4, tbx20. The probes for bmp10, bmpr1aa, id2b and smad1 were amplified by PCR with the following primers: bmp10 F-GCAGCCAGCAAGTAAGAGGA and R-GGTAGAGCAGGGAGATGGGA, bmpr1aa F-TAGCCAACCCCAATGCTTAC and R-GCCCATTTGTCTCGCAGGTAT, id2b F-TCGTGCCGAGTTTACCG and R-GCAATACCATACAGCTCCAGAT, smad1 F-CACGCTTCCGTAACCCACTCC and R-GCTCAAACATTCGGCATACACCT.Whole-mount Tg(vmhc:mCherry-NTR) larvae were treated with 6 mM MTZ  in E3 water at 3 dpf for four hours as previously described The larvae were incubated with 500 μM EdU for 1 h in E3 water with 2% DMSO to facilitate EdU solubilization. After pulse labeling, larvae were rinsed with E3 water, anaesthetized with 0.2% tricaine and fixed in 4% PFA. The CLICK-IT reaction for EdU labeling was performed according to the manufacturer's instruction (Thermo Fisher Scientific).Immunofluorescence staining on whole-mount larvae was performed as previously described Live imaging and immunostaining images were obtained using a Zeiss LSM880 confocal microscope or an Olympus IX83 inverted microscope. The numbers of differentially marked cardiomyocytes were counted with ZEN software or cellSens software.P < 0.05 determined by Student's t-test between two groups, ANOVA analysis between more than two groups or Binomial test in quantification of the percentage of recovered hearts.Values were presented as mean ± SD. Statistical significance was defined as a threshold of


Human T-lymphotropic virus 1 (HTLV-1) infection may lead to the development of Adult T-cell leukemia/lymphoma (ATLL). To further elucidate the pathophysiology of this aggressive CD4+ T-cell malignancy, we have performed an integrated systems biology approach to analyze previous transcriptome datasets focusing on differentially expressed miRNAs (DEMs) in peripheral blood of ATLL patients.Datasets GSE28626, GSE31629, GSE11577 were used to identify ATLL-specific DEM signatures. The target genes of each identified miRNA were obtained to construct a protein-protein interactions network using STRING database. The target gene hubs were subjected to further analysis to demonstrate significantly enriched gene ontology terms and signaling pathways. Quantitative reverse transcription Polymerase Chain Reaction (RTqPCR) was performed on major genes in certain pathways identified by network analysis to highlight gene expression alterations.hsa-let-7a, hsa-let-7g, hsa-mir-181b, hsa-mir-26b, hsa-mir-30c, hsa-mir-186, hsa-mir-10a, hsa-mir-30b, and hsa-let-7f between ATLL patients and healthy donors. Further analysis revealed the first 5 of DEMs were directly associated with previously identified pathways in the pathogenesis of HTLV-1. Network analysis demonstrated the involvement of target gene hubs in several signaling cascades, mainly in the MAPK pathway. RT-qPCR on human ATLL samples showed significant upregulation of EVI1, MKP1, PTPRR, and JNK gene vs healthy donors in MAPK/JNK pathway.High-throughput in silico analysis revealed 9 DEMs The results highlighted the functional impact of a subset dysregulated microRNAs in ATLL on cellular gene expression and signal transduction pathways. Further studies are needed to identify novel biomarkers to obtain a comprehensive mapping of deregulated biological pathways in ATLL. Human T lymphotropic virus 1 (HTLV-1) is a single-stranded positive-strand RNA virus, which primarily infects CD4+ T-cells in humans . A subpoThe pX region of HTLV-1 genome encodes two important regulatory proteins HBZ and Tax . The subThe ATLL specific genomic signature is not only reflected by the pre-transcriptional and transcriptional modification of gene expression. Infection with HTLV-1 virus and development of ATLL has been associated with significant dysregulation of microRNA (miRNAs) transcriptome in host cells, despite HTLV-1 not having any genome-encoded miRNAs , 19–21. Here we have conducted an integrated approach to analyze gene expression profiling studies to elucidate the anomalies in miRNA gene regulation system in infected cells through high-throughput analysis of previous transcriptomic datasets in the literature. The enriched pathways and highlighted genes in this study revealed novel disruptions in cell signaling cascades which were then confirmed by real time-PCR.https://www.ncbi.nlm.nih.gov/geo) and Array Express (https://www.ebi.ac.uk/arrayexpress) by the end of 2018 to find datasets relevant to the expression levels of miRNA in ATLL patients and healthy donors. Fig. We searched the public domains, Gene Expression Omnibus (http://mirdb.org/miRDB/) [The selected datasets were pre-processed using GEOquerry package implemented in R programming language (Version 4.0.3). The datasets were normalized using log2 transformation in Affy package and were then integrated with MetaDE package in R programming language to identify DEMs. The proportion of DEMs with a p-value of less than 0.001 were considered for further analysis. Subsequently, the gene targets for selected DEMs were determined by miRDB online database (/miRDB/) . The assdegree and betweenness centrality measures were computed to determine hub genes. Lastly, the PPINs were reconstructed and visualized using Gephi (0.9.1) based on calculated gene hubs.The STRING database version 11.0 was employed to construct protein-protein interactions network (PPIN) for gene targets of each DEMs based on literature sources for protein-protein interactions. The interactions included for network construction in this study include physical and functional interactions, high-throughput experiments, co-expression, genomic context, databases, and text-mining. The primary PPINs analysis was conducted using NetworkAnalyzer in Cytoscape 3.5.1 . Genes wGene enrichment analysis was carried out to enrich hub genes in each DEMs in EnrichR web tool using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways , 30. A cThe blood samples were collected from 8 ATLL patients and 10 healthy subjects between 2019 and 2020 from Shariati Hospital, Tehran, Iran. All samples were collected after acquiring informed consent from the patients or their next of kin when appropriate. A standardized clinical checklist, comprising demographic information and the diagnosis of ATLL was evaluated by a trained hematologist. None of the included patients were receiving chemotherapy and/or anti-cancer drugs. All methods were carried out in accordance with the relevant guidelines and regulations. The enzyme-linked immunosorbent assay  was used to perform the serology test for HTLV-1. PCR was then employed to confirm the serology results . The incp-value of less than 0.05 was considered to be significant.Total RNA was extracted from fresh whole blood utilizing TriPure isolation reagent . cDNA was synthesized using RT-ROSET Kit  and SYBR Green-based  and subsequently, RT-qPCR was performed, according to the manufacturers’ instructions. The following primers were utilized to determine the expression levels of JNK, EVI1, MKP, and PTPRR and to confirm HTLV-1 infected cells in the samples: EVI1 (forward primer (FP): 5′-TCGTCGCCTCATTCTGAACTGGAA-3′, reverse primer (RP): 5′-ACTGCCATTCATTCTCTCCTCCACA-3′) MKP  PTPPR  JNK  HBZ  5’LTR . The relative two standard curves real-time PCR was performed on the cDNA samples using Q-6000 machine . The GAPDH gene was utilized to normalize the mRNA expression levels respectively, as well as to control the error between samples . The outAfter removal of redundancy, application of inclusion and exclusion criteria, and quality control using MetaQC package in R, 3 datasets namely, GSE28626 , GSE3162The primary analysis of microarray datasets identified hsa-let-7a, hsa-let-7g, hsa-mir-181b, hsa-mir-26b, hsa-mir-30c, hsa-mir-186, hsa-mir-10a, hsa-mir-30b, and hsa-let-7f as DEMs in ATLL patients compared to normal individuals. The target genes for each mentioned DEM were identified using miRDB. The analysis of networks using centrality parameters was utilized to select nodes with higher degree and betweenness as hub genes.The PPINs were constructed for each DEM separately using STRING to highlight the relationship between the target genes Fig. . The netThe Hub genes of all defined DEMs were enriched to reveal biological pathways associated with the PPINs. The analysis highlighted the following pathways for each DEM: hsa-let-7a: Endometrial cancer, Colorectal cancer, Renin secretion, Cocaine addiction, TNF signaling pathway, Human T-cell leukemia virus 1 infection, Thyroid hormone signaling pathway, Human cytomegalovirus infection, Renal cell carcinoma, Cortisol synthesis and secretion.hsa-let-7g: Endometrial cancer, Colorectal cancer, Human T-cell leukemia virus 1 infection, Circadian rhythm, Thyroid hormone signaling pathway, Toxoplasmosis, TNF signaling pathway, Hedgehog signaling pathway, Human cytomegalovirus infection, Chronic myeloid leukemia;hsa-mir-181b: Endocrine and other factor-regulated calcium reabsorption, Endometrial cancer, TNF signaling pathway, Renal cell carcinoma, Acute myeloid leukemia, Thyroid hormone signaling pathway, Human T-cell leukemia virus 1 infection, Toll-like receptor signaling pathway, Small cell lung cancer, Graft-versus-host disease.hsa-mir-26b: Endometrial cancer, Small cell lung cancer, Renal cell carcinoma, Melanoma, Amyotrophic lateral sclerosis (ALS), Chronic myeloid leukemia, Human T-cell leukemia virus 1 infection, Renin secretion, Cocaine addiction, Colorectal cancer.hsa-mir-30c: Renin secretion, Endocrine and other factor-regulated calcium reabsorption, TNF signaling pathway, Osteoclast differentiation, Endometrial cancer, Th17 cell differentiation, Human T-cell leukemia virus 1 infection, Cocaine addiction, Aldosterone-regulated sodium reabsorption, Renal cell carcinoma.hsa-mir-186: Endocrine and other factor-regulated calcium reabsorption, Endometrial cancer, Renal cell carcinoma, Acute myeloid leukemia, Thyroid hormone signaling pathway, Salmonellosis, Chronic myeloid leukemia, Pancreatic cancer, Colorectal cancer, Th17 cell differentiation.hsa-mir-10a: TNF signaling pathway, Cocaine addiction, Circadian rhythm, Small cell lung cancer, Endocrine and other factor-regulated calcium reabsorption, Endometrial cancer, Renal cell carcinoma, Chronic myeloid leukemia, Cortisol synthesis and secretion, Parathyroid hormone synthesis, secretion and action.hsa-mir-30b: Endocrine and other factor-regulated calcium reabsorption, Endometrial cancer, Cocaine addiction, Thyroid hormone signaling pathway, Chronic myeloid leukemia, Human cytomegalovirus infection, Long-term depression, Salivary secretion, Renin secretion, Salmonella infection.hsa-let-7f: Endometrial cancer, Renin secretion, Renal cell carcinoma, Colorectal cancer, Human cytomegalovirus infection, Thyroid hormone signaling pathway, Long-term depression, Cocaine addiction, Prostate cancer, Neurotrophin signaling pathway. The following five DEMs were directly associated with HTLV-1 related pathogenic pathways: hsa-let-7a, hsa-let-7g, hsa-mir-181b, hsa-mir-26b, hsa-mir-30c , MAP kinase  Phosphatidylinositol-3-kinase  pathways among others.EVI1 , MKP , PTPRR , and JNK  in ATLL patients compared to healthy controls  and the major regulators of MAPK/JNK in ATLL vs healthy controls. The results of this study provide further insight into the dysregulated biological processes in ATLL. Further studies are needed to identify novel and reliable biomarkers, and prognostic factors and to obtain a comprehensive mapping of deregulated biological pathways in ATLL.


Patients with acute optic neuritis typically present with acute loss of vision. We describe a case of a young lady of 25 years of age with blurring of vision in the upper visual field of the right eye with otherwise intact visual acuity as the only presenting symptom. Although altitudinal visual field defect is not unknown to be associated with acute optic neuritis, it is generally considered a relatively uncommon occurrence. Our case illustrates an unusually unique occurrence of upper altitudinal visual field defect in association with unaffected visual acuity as the sole presenting symptom of acute idiopathic unilateral optic neuritis. When an altitudinal visual field defect is a presenting feature, besides the usual vascular and compressive causes, optic neuritis should be remembered in the list of differential diagnoses. Inflammatory optic neuropathy, or optic neuritis (ON), is the most common cause of optic nerve injury in young adults. ON has multiple etiologies, including demyelinating, infectious, and autoimmune causes. It can also occur independently as idiopathic optic neuritis.A variety of visual field defects can be found in optic neuritis. The Optic Neuritis Treatment Trial (ONTT) identified different types of visual field defects at the baseline in the affected eyes. Diffuse visual field loss was present in 66% of the affected eyes and central field loss in 27%. Altitudinal visual field abnormality which is classically believed to be highly characteristic of the ischemic optic neuropathy occurred in 8% of the affected eyes at the baseline in ONTT . Acute onset altitudinal visual field defect, which involves loss of visual sensation in the horizontal half of the visual field, often vascular in origin, can rarely be caused by compressive neuropathy due to a tumor or aneurysm . Acute oPatients with optic neuritis typically present with acute unilateral loss of vision usually in association with impaired color vision. We describe a case of a young lady of 25 years of age who presented with acute onset of blurred vision in the upper visual field with no subjective changes either in visual acuity or color vision. Humphry’s visual field plot confirmed the presence of superior altitudinal scotoma in the affected eye. The local and systemic investigations suggested the idiopathic nature of acute optic neuritis. Our case therefore aims to illustrate an unusual scenario of subjective blurring in the superior visual field from altitudinal scotoma as a presenting feature of acute unilateral idiopathic optic neuritis.A 25-year-old Bahraini female presented to the eye clinic with blurring of vision in the upper field of her right eye of four days duration. There was no history of preceding febrile illness, immunization, trauma, or any past systemic or neurological disease or any medications. Her past ocular history included laser correction in both the eyes for a mild myopic refractive error four years ago. There was no significant family history. Her visual acuity on presentation was 6/6 in each eye. Ishihara test revealed a mildly impaired color vision in the right eye and fully normal color vision in the left eye. Ocular movements showed a full range but were associated with some pain on supraduction of the right eye.Anterior segment and adnexal examinations were unremarkable. A relative afferent pupillary defect grade two was noted in the right eye. Fundus examination revealed normal-looking optic disc, retinal and vasculature details. Her visual field plot, 24-2 on Humphrey’s field analyzer, revealed in the right eye a superior altitudinal visual field defect Figure . The felSystemic evaluation including full neurological evaluation and ENT status were within normal limits. Brain and orbit MRI scans were unremarkable. Blood investigations including full blood count, renal function tests, erythrocyte sedimentation rate, C-reactive protein, serum folate and B 12 levels were all also within normal. Serum autoimmune markers such as anti-cardiolipin antibodies, antinuclear antibody (ANA) screen and double-stranded DNA were also within normal range. Serological infective screening that included TP-antibody for syphilis, anti-hepatitis C virus (HCV), HIV AB/AG combo and hepatitis B surface antigen were all non-reactive. Serum anti-aquapore-4 antibody test also returned negative. Electrophysiological study of visual-evoked cortical potential (VEP) revealed delayed latencies suggestive of optic neuritis. Two days after the presentation, her visual acuity deteriorated to 6/15 at which point she was started on a five-day course of high dose intravenous methylprednisolone 1 gm daily under the care of a neurophysician. On her review four days after the completion of the treatment, that is on the 11th day from the initial presentation, she was noted to have total recovery of the visual acuity, full restoration of the color vision on Ishihara’s testing and complete disappearance of the altitudinal field defect on Humphrey’s perimetry Figure .Inflammatory optic neuropathy, or optic neuritis (ON), which can be defined as inflammation of the optic nerve due to various causes, is the most common optic neuropathy under 50 years among general ophthalmic practice . Optic nAltitudinal visual field defect in optic neuritis, though not completely unknown, is a relatively uncommon feature of optic neuritis. The patients included in the Optic Neuritis Treatment Trial (ONTT) presented with various types of visual field defects in the affected eye, with two-thirds showing diffuse defects and one-third with localized field defects. Keltner et al.  observedIt is suggested that either the inflammation of the optic nerve itself or the secondary perfusion defect caused by the inflammation of the optic nerve may lead to the altitudinal field defect in optic neuritis . Chin anA number of randomized, controlled, double-blind trials of corticosteroid treatment of optic neuritis were evaluated in a meta-analysis in 2012. One randomized and controlled trial, the Optic Neuritis Treatment Trial, had a major effect on the current standard of treatment. In this trial, oral prednisone treatment at a dose of 1 mg/kg body weight/day for 14 days was compared with intravenous methylprednisolone treatment at 1000 mg/day for three days followed by oral prednisolone (1 mg/kg BW) for 11 days, and with placebo treatment. Treatment with intravenous methylprednisolone, which was not blinded, led to a more rapid recovery of vision, but the final outcome with respect to visual acuity, fields, and perception of contrast and color was no better than with oral prednisone alone, or indeed with placebo. Similar results were found in earlier and later studies as well; thus, it was concluded in the meta-analysis of 2012 that faster recovery is the sole benefit of steroid treatment. Among the patients in the Optic Neuritis Treatment Trial who were treated only with low-dose oral prednisolone, early recurrences within six months were twice as common as in the placebo group. Since the publication of these findings, low-dose oral prednisolone alone has been considered to be contraindicated for patients with typical optic neuritis .The patients in the Optic Neuritis Treatment Trial who received intravenous methylprednisolone for three days all also received oral prednisolone over the ensuing 11 days. It is unclear whether this is necessary, and the guidelines leave the question open. Some authorities do not give oral prednisolone after intravenous methylprednisolone. Some current neurological and ophthalmological guidelines advocate treatment of optic neuritis with methylprednisolone at a dose of 500-1000 mg/day for three to five days .For patients with ON whose brain lesions on magnetic resonance imaging indicate a high risk of developing clinically definite multiple sclerosis, treatment with immunomodulators  may be considered. Intravenous immunoglobulin treatment of acute ON has been shown to have no beneficial effect .Recovery of visual function in acute optic neuritis is known to occur within the first month of the onset. In the ONTT, 79% of the participants had started to improve by three weeks and 93% by five weeks [In summary, we described a case of acute idiopathic unilateral optic neuritis with the subjective complaint of upper altitudinal visual field defect as a sole presenting feature in the presence of normal visual acuity. Optic neuritis is a multifaceted disease that may have atypical clinical presentations. The clinician should be familiar with atypical presentations which should prompt other differential diagnoses without ruling out the diagnosis of acute optic neuritis. If atypical features are present, urgent further investigations are indicated to exclude the differential diagnoses. Our case of acute idiopathic unilateral optic neuritis had an unusual presenting symptom of upper visual field blurring in the presence of unaffected visual acuity. When an altitudinal visual field defect is a presenting feature, besides the usual vascular and compressive causes, optic neuritis should be remembered in the list of differential diagnoses. Further imaging to exclude other intracranial or intraorbital pathologies is warranted. Brain MRI is important diagnostically and prognostically in monitoring the future development and progression of demyelinating disease. Last but not the least, in the case of young healthy female patients, diagnostic work up and co-management with a neuromedical team would ensure an optimal work up and the appropriate future follow up necessary for such patients.


The lateral ankle sprain (LAS) is one of the most common injuries in everyday and sports activities. Approximately 20–40 % of patients with LAS develop a chronic ankle instability (CAI). The underlying mechanisms for CAI have not yet been clearly clarified. An inadequate rehabilitation after LAS can be speculated, since the LAS is often handled as a minor injury demanding less treatment. Therefore, the aims of this retrospective study were to determine the CAI rate depending on age and sex and to identify possible determinants for developing CAI.Between 2015 and 2018 we applied the diagnostic code “sprain of ankle” (ICD S93.4) to identify relevant cases from the database of the BG Klinikum Duisburg, Germany. Patients received a questionnaire containing the Tegner-Score, the Cumberland Ankle Instability Tool (CAIT) and the Foot and Ankle Disability Index. Additionally, there were questions about the modality and beginning of therapy following LAS and the number of recurrent sprains. There was a total of 647 completed datasets. These were divided into a CAI and non-CAI group according to a CAIT cut-off-score with CAI ≤ 24 and non-CAI > 24 points, representing one out of three criteria for having CAI based on international consensus.p < .05). There was a significantly higher CAIT score in patients having no recurrent sprain compared to patients having 1–3 recurrent sprains or 4–5 recurrent sprains (p < .001).The overall CAI rate was 17.3 %. We identified a higher CAI rate in females and within the age segment of 41 to 55 years. A later start of therapy (> 4 weeks) after acute LAS significantly increases ankle instability in CAIT (Females over 41 years show a higher CAI rate which implies to perform specific prevention programs improving ankle function following acute LAS. A delayed start of therapy seems to be an important determinant associated with the development of CAI. Another contributing factor may be a frequent number of recurrent sprains that are also linked to greater levels of subjective ankle instability. Therefore, we would recommend an early start of functional therapy after acute LAS in the future to minimize the development of CAI. The lateral ankle sprain (LAS) is one of the most common musculoskeletal injuries in everyday and sports-related activities . In the The medical treatment, work loss as well as a loss of productivity lead to high socioeconomic costs, especially with recurrent sprains and long-term problems . EpidemiOne possible explanation for the high instability rate is an insufficient rehabilitation and/or a too early return to intense sports and workloads , 11. Up Therefore, the aims of this four-year retrospective study were firstly to determine the CAI rate depending on age and sex, and secondly to identify possible determinants or contributing factors  for developing CAI and negative long-term consequences. Both are relevant goals to evaluate and possibly optimize current treatment strategies after incurring acute LAS.A retrospective study design was used Fig. . BetweenAll patients of the identified cases received a postal questionnaire containing the Tegner-Score and patient-related outcome measures (PROMs) including the Cumberland Ankle Instability Tool (CAIT) and the Foot and Ankle Disability Index (FADI). Additionally, there were questions about the received modality of therapy , the beginning of therapy , as well as the number of recurrent sprains . The appropriate answers were marked with a cross by the patients.The Tegner-Score aims to determine the level of physical activity in patients using different grades on a numerical scale. Its values range from zero to ten, with zero representing being bedridden and ten representing doing competitive sport at a professional level . CAIT ann = 198) and additionally performed phone interviews (n = 449) led to a total of 647 completed datasets. The sample consisted of 381 male and 266 female participants. Participants were divided into three age groups . Furthermore, they were divided into a CAI and non-CAI group according to the predefined cut-off-score of CAIT ≤ 24, representing at least one out of three selection criteria for having CAI based on international consensus guidelines [The received questionnaires (idelines . Subjectp < .05), the Mann Whitney U test was performed to assess differences between the CAI and non-CAI groups in the FADI. Independent t-tests were used to analyze the effects of the reported modality of therapy (yes: received vs. no: not received) on the CAIT score. Relative differences in the beginning of therapy between the CAI and non-CAI group were performed using frequency analysis. A one-way ANOVA was used to determine the effects of the beginning of therapy on the CAIT score and to assess the effects of the number of recurrent sprains on the CAIT score. The Tukey post-hoc test was used for pairwise comparisons between the different starting times of therapy. Homogeneity of variance was verified with the Levene’s test (p > .05). In case homogeneity of variance was violated, a Welch ANOVA using Games-Howell post-hoc analysis was applied. Statistical analysis was done using SPSS Statistics . Data are presented as mean ± SD unless otherwise stated.Descriptive statistics using frequency analysis were performed to calculate the percentage CAI rate. Since the FADI was not normally distributed, according to the Kolmogorov-Smirnov-Test (p < .05).The Tegner score and the frequency of LAS were highest in the younger age group of 14–25 years (46 %) and gradually decreased with advancing age showing the lowest Tegner score and frequency in the older age group of 41–55 years (25 %) (Tables p < .001). Games-Howell post-hoc analysis revealed a significantly higher CAIT score in patients having no recurrent sprain compared to patients having 1–3 recurrent sprains (p < .001) or 4–5 recurrent sprains (p < .001)  = 81.379, p < .05) showing a higher CAIT score with an immediate start compared to > 4 weeks following acute LAS (p < .05)  we collected relevant epidemiological information on the distribution of the CAI rate. We identified a higher CAI rate in females and persons in the age group of 41 to 55 years. We could further show that a later beginning of therapy after acute LAS is associated with increased functional impairments of the ankle. Therefore, early treatment following acute LAS seems to be an effective means in preventing CAI.The overall CAI rate was about 17 % and thus slightly lower than the CAI rates reported in literature ranging between 20 and 40 % , 10, 19.According to our patients sex we found a higher CAI rate in females (21.2 %) than in males (10.3 %). The females also had higher CAI rates in each of the three age groups. Literature shows that there is in general a higher incidence of ankle sprains in females compared with males  . Yet theDoherty et al.  state thWith regard to the reported treatment modalities, there were no significant differences in the CAIT score between the received and non-received forms of therapy. Thus, based on the conditions of this study, the functional CAIT outcome does not seem to be generally determined by a specific modality of therapy. As this was a surprising result, we assumed that other factors might play a decisive role on subjective ankle joint function. In this respect, we found that the CAIT score significantly differed between patients receiving their therapy immediately and those who received therapy after more than four weeks. The latter had a worse outcome because of the delayed start of the therapy. This is supported by previous research showing superior treatment effects on subjective ankle function and prevention of CAI in patients with early functional bracing and exercise therapy compared to short-term immobilization , 19, 33.To increase the number of valid PROMs we performed an additional telephone interview. The individual communication might have influenced the perception and response behavior of patients which could have affected the outcome variables. As this was a retrospective study, there is generally a susceptibility to errors since the data might be biased due to the patient’s inaccurate recollection of events. That is why relevant information about the quality, intensity, duration and frequency of treatment after acute LAS is currently missing, since we decided to ask more simple questions to improve data quality. However, knowledge of the missing data could have affected the study results. The assignment into a CAI and a non-CAI group is based on only one selection criterion, the cut-off score of CAIT ≤ 24 for the definition of CAI. Future studies investigating CAI patients should therefore consider all three criteria recommended by the International Ankle Consortium . Given dAccording to this study, females and older age groups (41–55 years) have a higher risk for developing CAI which implies to focus on specific prevention or therapy programs improving ankle function. Moreover, patients reporting a later start of therapy after acute LAS (> 4 weeks) have a worse functional CAIT outcome, irrespective of the received treatment modality. Thus, a delayed beginning of therapy following acute LAS seems to be an important determinant associated with the development of CAI. A further contributing factor for CAI, suggested by literature, is the number of recurrent sprains which could also be supported by our data. Therefore, we highly recommend an early start of functional therapy after acute LAS in the future to minimize the LAS recurrence rate and the development of CAI. The therapy should be guided by four impairment domains, identified in patients with CAI . These d


Hepatocellular carcinoma (HCC), the most common type of primary liver cancer, is an aggressive tumor with a high mortality rate because of the limited systemic and locoregional treatment modalities. The development and progression of HCC depend on epigenetic changes that result in the activation or inhibition of some signaling pathways. The mTOR signaling pathway is essential for many pathophysiological processes and is considered a major regulator of cancer. Increasing evidence has shown that epigenetics plays a key role in HCC biology by regulating the mTOR signaling pathway. Therefore, epigenetic regulation through the mTOR signaling pathway to diagnose and treat HCC will become a very promising strategy. Hepatocellular carcinoma (HCC) is the fifth most common malignancy in men and the seventh most common malignancy in women worldwide and results in more than 700,000 deaths each year –3. FurthκB, RAF/MEK/ERK, JAK-STAT, Wnt, Notch, and MAPK as well as mTOR pathways [Epigenetics is a heritable phenomenon that affects gene expression without altering the DNA sequence . Epigenepathways –10. Morepathways . Among tmTOR is involved in multiple signaling pathways that regulate cell proliferation, autophagy, and apoptosis in vivo . There aIn a review, we systematically summarize information regarding the regulation of the mTOR signaling pathway by factors mutated in epigenetic inheritance for the diagnosis and treatment of HCC .Noncoding RNA (ncRNA) refers to RNA that does not encode proteins . AdvanceMiRNAs are a diverse family of highly conserved ncRNAs with sizes that range from approximately 18 to 25 nucleotides. MiRNAs pair by complete or incomplete base complementation with the 3′ untranslated region of mRNA. This pairing leads to the degradation or inhibited translation of the target mRNA, with subsequent effects on protein expression. MiRNAs participate in the processes of cell growth, differentiation, development, proliferation, apoptosis, and metabolism , 22. MiRHCC is the third leading cause of cancer-related death worldwide, and its incidence continues to rise. Although cirrhosis underlies most cases of HCC, many molecular pathways are closely associated with HCC carcinogenesis . For exaIn addition to miRNA-mediated regulation of the classical PI3K/AKT/mTOR signaling pathway, miRNAs can also affect HCC development through other mTOR signaling pathways.In HCC, upregulation of miR-18a and miR-25 is associated with poor patient survival and promotion of HCC cell line proliferation. Sanchez-Mejias et al.  analyzedZhou et al.  found thMeanwhile, the experimental results of Dong et al.  showed tThese studies indicate miRNAs can affect the progression of liver cancer by regulating the mTOR signaling pathway and provide a new approach for the diagnosis and treatment of liver cancer.Among the various types of ncRNAs, lncRNAs have received increasing attention. LncRNAs are defined as transcripts of more than 200 nucleotides that lack protein-encoding ability . LncRNAsWith the development of next-generation sequencing, more long noncoding RNAs (lncRNAs) were found. Initially, lncRNAs were considered as “noisy” transcripts or “dark matter” . An incrSeveral studies have shown that lncRNAs also regulate other mTOR signaling pathways in liver cancer. Ma and colleagues  found thWei and colleagues  found siLi et al.  found foα/mTOR signaling pathway. The authors showed that HIF1A-AS1 is involved in regulating the progression of HCC and provided a potential direction for future HCC treatment strategies.The lncRNA HIF1A-AS1 is overexpressed in HCC tissues and is associated with tumor size, TNM stage, and lymph node metastasis. Hong et al.  found thThese reports indicate that dysregulation of lncRNAs can activate the mTOR signaling pathway to regulate the development of HCC, taking the understanding and potential use of lncRNAs in the diagnosis and treatment of liver cancer patients to a new level.CircRNAs are a novel class of ncRNAs characterized by a covalent closed-loop structure without a 5′ cap structure or a 3′ polyA tail . Since tThe relationship between circRNAs and HCC has become a research hotspot in the past two years. CircRNAs were shown to have important regulatory roles in HCC development, and progression. In HCC, some circRNAs act as oncogenes and can promote the proliferation and migration of cancer, while other circRNAs act as tumor suppressors and can induce apoptosis of HCC cells. Although some studies have demonstrated that circRNAs can affect the progression of HCC, the roles of circRNAs on HCC remain largely unknown . TherefoCircRNA can affect the development and progression of HCC through the mTOR signaling pathway . For exaSun et al.  found thZheng et al.  found thAlthough few reports have shown that circRNAs play a role in HCC through the mTOR signaling pathway, future studies clarifying their role in HCC development may lead to new approaches for the diagnosis and treatment of HCC.DNA methylation is an important epigenetic modification that occurs mainly at the CpG islands of DNA and involves either hypermethylation or hypomethylation. Aberrant DNA methylation in cancer has been heralded as a promising target for the development of powerful diagnostic, prognostic, and predictive biomarkers . For exaHistone modifications, as a class of epigenetic regulatory mechanisms that regulate gene expression, have received increasing attention because their modification pattern changes are closely related to the development of a variety of malignancies . For exaThe SWI/SNF complex, originally discovered in yeast 20 years ago, is a family of multi-subunit complexes that use the energy of ATP hydrolysis to remodel nucleosomes. Chromatin remodeling processes mediated by the SWI/SNF complex are essential for the regulation of gene expression in a variety of cellular processes, including differentiation and proliferation . Many stMultiple studies have demonstrated that epigenetic changes play an important role in the development and progression of HCC. The mTOR pathway is involved in the growth and proliferation of HCC cells, and epigenetic regulation through mTOR signaling will affect HCC progression. As supported by increasing evidence, epigenetic regulators through the mTOR signaling pathway as ideal therapeutic targets for HCC are a potent future research direction. Although there have been major breakthrough in epigenetics in the treatment of HCC, some questions remain unanswered. For example, studies linking histone methylation and acetylation with the mTOR signaling pathway are scarce. Future studies should pursue this research direction to expand the exploration of strategies for HCC treatment targeting the mTOR pathway.