Colorectal cancer (CRC) has been the fourth leading cause of cancer-related mortality worldwide. Owing to clonal evolution and selection, CRC treatment needs multimodal therapeutic approaches and due monitoring of tumor progression and therapeutic efficacy. Liquid biopsy, involving the use of circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and exosomes, may offer a promising noninvasive alternative for diagnosis and for real-time monitoring of tumor evolution and therapeutic response compared to traditional tissue biopsy. Monitoring of the disease processes can enable clinicians to readily adopt a strategy based on optimal therapeutic decision-making. This article provides an overview of the significant advances and the current clinical and biological significance of CTCs, ctDNA, and exosomes in CRC, as well as a comparison of the main merits and demerits of these three components. The hurdles that need to be resolved and potential directions to be followed with respect to liquid biopsies for detection and therapy of CRC are also discussed. With a global incidence of about 1.4 million individuals and 693,900 deaths reported in 2012, colorectal cancer (CRC) is the third most common type of cancer and the fourth most common cause of cancer deaths worldwide , 6. WithTherefore, screening and early detection of CRC remain clinical dilemmas and are critically important for raising the chances of a more suitable treatment leading to better long-term survival. Several approaches involving colonoscopy, sigmoidoscopy, fecal occult blood test (FOBT) , 8, seruTherefore, it is necessary to identify ideal bio-markers that can be used for the early diagnosis, detection of recurrence, and monitoring of metastasis for CRC. Using body fluid samples, liquid biopsies may be the ideal approach for the detection of CTCs or for the products of primary or metastatic tumors. The advent of liquid biopsy as an alternate, easy, quick, convenient, and minimally invasive method has shown tremendous potential to help clinicians achieve early diagnosis of cancer and guide decision-making during the course of treatment. It is expected to be an informative or easily accessible tool to provide comprehensive information of tumors beyond the invasive tissue biopsies. The main approaches to liquid biopsies include the detection of CTCs , analysiThis review summarizes and discusses the current clinical and biological significance of CTCs, ctDNA, and exosomes in CRC. Additionally, the review covers significant advances and limitations of liquid biopsy in the clinical applications, discusses the hurdles that need to be resolved, and provides potential directions for the detection and therapy of CRC.Over the past few decades, many procedures have been developed for liquid biopsy ranging from the use of CTCs and ctDNA or RNA to that of exosomes and platelets , 17 detein situ, contributed to the metastasis through plakoglobin-dependent intercellular adhesion [CTCs are circulating tumor cells that are shed into the bloodstream from tumors and have the potential to cause metastatic lesions , 19. Theadhesion .Although the underlying advantages of CTCs make it a promising tool to help monitor the dynamic course of disease, there are some potential limitations in the existing detection techniques –29 and fCirculating free DNA (cfDNA) is a natural phenomenon and is thought to originate from the apoptosis and necrosis of normal and tumor cells, and secretion of tumor cells has also been suggested as a potential source , 35. TheSEPT9 DNA (mSEPT9) for detecting CRC in a screening population was conducted by Church et al. [Many studies have demonstrated that the level of cfDNA is substantially higher in patients with cancer than that in healthy individuals or patients with benign diseases, and it seems to increase with tumor stage , 42. ctDh et al. . Resultsh et al. . Although et al. . Therefoh et al. .Exosomes, which are well known for intercellular communication, are small membrane vesicles (30–100 nm) released from diverse cell types under both normal and pathological conditions, that can horizontally transfer functional biomolecules  to recipient cells –50. The 9 vesicles/mL in blood, the number of exosomes secreted by tumor cells correlates to their malignant behavior [BRAF (V600E) mutation and anti-epidermal growth factor receptor (EGFR) mutation, using allele-specific polymerase chain reaction (AS-PCR). The findings demonstrated that exoDNA could reflect the mutational status of the parental cell lines [Previous studies by Peinado and colleagues have demonstrated that exosomes played vital roles in vascular leakiness, inflammation, and bone-marrow progenitor cell recruitment during pre-metastatic niche formation and metastasis itself . Subsequbehavior , 53. Thell lines . HoweverHaving discussed the advantages and disadvantages of CTCs, ctDNA, and exosomes in different aspects, it is clear that these hold promise for researchers attempting to monitor tumor-specific changes during the course of cancer. This may also help clinicians to carry out noninvasive real-time assessment in various clinical settings, including diagnosis, therapy monitoring, and prognosis Table .A milestone in CTC research (CellSearch™) in patients with metastatic breast cancer (MBC) was reported in The New England Journal of Medicine in 2004 . The resDeneve et al. demonstrated that the CTC counts were significantly higher in mesenteric blood than in the peripheral blood, which meant that the liver acts as a filter for CTCs, and the follow-up analysis showed that localized colon-cancer patients with high CTC counts have an unfavorable outcome n =60) . By cont . By conTM CTC technique. Biophenotypic  CTCs as well as mesenchymal CTCs positively correlated with both clinical stage and lymph node metastasis and distant metastasis, suggesting that CTCs displaying a mesenchymal phenotype would denote a more aggressive and metastatic potential [Recently, a large cohort of 1203 patients was evaluated for the expression of EMT markers in CTCs by using CanPatrolotential . Their fotential .in vitro angiogenesis and tumors in immune-deficient mice [in vitro culture, and molecular characterization of CTCs should divulge the prognosis of mCRC as well as monitor the drug response, in addition to early detection of disease progression with new metastasis.Cancer stem cells are capable of seeding, recirculation, and evolution in metastatic clones, which causes clinical progression of the disease . The firent mice . By estaent mice . The cytent mice . TherefoRAS or BRAF mutation status in primary or metastatic tumors. The mutation status of KRAS, BRAF, and PI3KCA could affect the treatment response to EGFR monoclonal antibodies or small molecule inhibitors and have treatment-independent prognostic value [KRAS discordance between CTCs and primary tissue cancer after 100% pure cell recovery and sequencing [KRAS, BRAF, and PIK3CA hotspot mutations were analyzed by a label-free platform. Researchers found a concordance of 78.2% for KRAS, 73.9% for BRAF, and 91.3% for PIK3CA mutations [National Comprehensive Cancer Network (NCCN) highly recommends that patients with mCRC should be tested for the ic value , 64, 65.quencing . The unequencing . In a reutations . In geneThus, in primary or non-metastatic CRC, the existence of CTCs might indicate poor prognosis; while in advanced or metastatic CRC, there should be a positive correlation between the level of CTCs with the tumor progression and poor outcomes; CTCs can guide treatment decisions and assess treatment responses during the course of therapy; the molecular analysis of CTCs may predict drug resistance and the selection of anticancer drugs . GazzaniDiehl et al. applied BEAMing to quantify ctDNA in CRC patients undergoing multimodality therapy. Compared to CEA, the levels of ctDNA were more impressive in the prediction of recurrence (P=0.03), which certified that fluctuations of ctDNA could be used to monitor the course of therapy in patients with mCRC undergoing surgery or chemotherapy . In anotWIF1 (WNT inhibitory factor 1) and NPY (neuropeptide Y) was significantly higher in the tumor tissue compared to that in normal tissue and these methylated ctDNA (Met ctDNA) were detectable throughout the tumor progression, with their fraction being correlated to the tumor stage [WIF1 and NPY could instead be tumor-specific mutations. Recently, Bedin et al. found that the adenoma and methylated cfDNA in patients with CRC, showed a higher quantity of ALU83 and ALU244 [OSMR and SFRP1 methylation of cfDNA was also significantly higher in advanced CRC compared to that in the adenoma and control samples. This combination can improve the diagnostic efficiency and prognosis for CRC.Until date, several studies have focused on the detection of methylated DNA in patients with CRC. Garrigou and colleagues reported that hypermethylation of or stage . Therefod ALU244 . In thisKRAS mutations are frequent drivers of acquired resistance to cetuximab in CRC, and indicated that the emergence of KRAS mutant clones can be detected, non-invasively, months prior to radiographic progression [KRAS and BRAF in CRC tumor tissue, using routine gold-standard methods, and in ctDNA, using a quantitative PCR-based method. cfDNA analysis showed 100% specificity and sensitivity for the BRAF V600E mutation and 98% specificity and 92% sensitivity with a concordance value of 96% for the KRAS mutation, compared with a tumor-tissue analysis [Misale et al. demonstrated, for the first time, that gression . They algression . The samgression . They algression . The firanalysis . To concanalysis .EGFR antibodies, exhibit pulsatile levels of mutant KRAS. Results revealed that the CRC genome adapts dynamically intermittent drug schedules and provided a molecular explanation for the efficacy of rechallenge therapies based on EGFR blockade [KRAS gene, KRAS wild type status in the primary tumor is a prerequisite for treatment modality of patients with mCRC with targeted therapy apart from the best supportive care alone [KRAS or BRAF alleles in the plasma at baseline and before each cycle of the third-line treatment with cetuximab and irinotecan. cfDNA and KRAS levels was found to decrease from baseline to cycle 3 and increased in progress (P = 0.08), while the loss of mutations was associated to the benefit of treatment, whereas the appearance of mutations during therapy may be correlated to acquired resistance in primary wild-type disease [KRAS mutation in wild-type tumors indicated a shift for the poor progression, while some patients with CRC had tumors that contained mutation but wild-type KRAS in plasma proved to have responded better to the therapy [As a result of clonal evolution and selection , the ctDblockade . Becausere alone , 73. The disease . The app therapy .Most published studies of ctDNA have evaluated a single type of tumor mutation; however, numerous cancers have been reported with several undefined mutations. Further, the detection of a known anomalous gene could not completely reflect the heterogeneity of the tumor or replace the gold standard for tissue biopsy. A study explored the feasibility of Ion Torrent PGM (IonPGM) targeted NGS on cfDNA of mCRC patients undergoing colorectal liver metastasectomy . The panAlthough increasing the sequencing depth of NGS could make the results more accurate, such an approach will be more time-consuming and costly, which is an obstacle for practical clinical application. However, whole genome sequencing with an appropriate depth of cfDNA could be available for clinical purposes, which can remedy the restrictions of dPCR. Because of the 0.01% error rate of DNA polymerases in the process of amplification, PCR or NGS-based methods that depend on amplification are limited. The newer sequencing technologies such as nanopore sequencing do not require nucleotides, polymerases, or ligases and have the potential of generating very long read-lengths , which might be a more competitive and portable technology for clinical applications , 79.in vitro [Lately, exosomes have greatly attracted researchers’ attention as potential biomarkers of cancer. A study, performed at Kanazawa University on microarray-based profiling of exosomal microRNAs (miRNAs) in sera from patients with primary CRC, healthy controls and cancer cell lines, validated CRC-associated exosomal miRNAs in an independent sample set using quantitative real-time RT-PCR . They foin vitro . To summin vitro . The stuin vitro .TP53 changes the tumor microenvironment and promotes canceration via the endosomal sorting complex required for transport (ESCRT)-dependent exosome secretory machinery [Recently, Yanyu Chen et al. presented a quantitative proteomics analysis of purified exosomes from the serum of patients with CRC and normal volunteers, and identified 918 proteins with an overlap of 725 Gene IDs in the Exocarta proteins list . There wachinery . A comprachinery .KRAS CRC cells can be transferred to wild-type cells to induce cell growth and migration [KRAS regulates the composition of the secreted miRNAs, researchers compared small RNAs of cells and matched exosomes from isogenic CRC cell lines differing only in the KRAS status [KRAS status prominently affects the miRNA profile in cells and their corresponding exosomes, and cellular trafficking of miRNAs is sensitive to neutral sphingomyelinase (nSMase) inhibition in mutant KRAS cells as well as that the transfer of miRNAs between cells can functionally alter gene expression in recipient cells [KRAS mutations, which indicated a widespread effect of mutant KRAS on circRNA abundance [The delivery of exosomes to recipient cells could induce cell migration, inflammation, immune responses, angiogenesis, invasion, pre-metastatic niche formation, and metastasis , 84, 85.igration , 87. To bundance . CircRNAbundance . Exosomebundance .™ Lung (ALK) from Exosome Diagnostics Company has been approved by the FDA in the United States and has become the world's first noninvasive test using exosomal RNA-based liquid biopsy. As one of the promising diagnostic and therapeutic tools for CRC in future, research on the mechanism of exosomes will lay the foundation for a better clinical application.As miRNAs may find applications in molecular therapies for the treatment of CRC , exosomeTumorigenesis is the result of multiple factors implicated in the destruction of balance between oncogenes and anti-oncogenes. With approaches such as that of liquid biopsy, which are rapid, convenient, and minimally invasive, cancers can be detected from body fluid samples repeatedly, bypassing the need of tumor tissue biopsy while allowing clinicians to monitor the response to therapies and recurrence in real-time.Although the early detection strategies and the guidance for decision-making during therapies based on liquid biopsy are promising, there have several drawbacks that need to be overcome before they can be applied clinically: 1) The surge in various detection technologies to identify CTCs, ctDNA, or exosomes, urgently calls for the implementation of standard guidelines, internal quality control (IQC) and external quality assessment (EQA). 2) Researchers need to find universal signatures from CTCs that cover any stage or type of cancer, thereby improving the sensitivity and specificity of the detection methods. 3) Large prospective clinical trials involving multicenter studies are needed to validate the clinical significance for detection and prognosis. 4) Complementary assessment of both CTCs and ctDNA will be more superior for the noninvasive diagnosis and prognosis monitoring of CRC. 5) There is an imposing need to explain the inconsistent results between liquid biopsy results and imaging examinations or tissue biopsies. Thus, liquid biopsies could help clinicians make better treatment-related decisions, provided that the above mentioned hurdles are overcome.Fortunately, investigators have found that platelets might play a role in tumorigenesis and progression. Ratajczak and colleagues demonstrated that microparticles secreted by platelets induce angiogenesis and metastasis in both lung and breast cancer , 17. SomIn summary, owing to its significant benefits, liquid biopsy can be used as a feasible detection method for a variety of solid tumors and clinical diseases. Despite the existence of several challenges, we believe that the development of ideal detection methods is in progress. Our group has designed and conducted the first nationwide EQA of NGS-based targeted sequencing by laboratories in China in 2015 . In addi


Antimicrobial resistance is a public health challenge supplemented by inappropriate prescribing, especially for an upper respiratory tract infection in primary care. Patient/carer expectations have been identified as one of the main drivers for inappropriate antibiotics prescribing by primary care physicians. The aim of this study was to understand who is more likely to expect an antibiotic for an upper respiratory tract infection from their doctor and the reasons underlying it.n = 1509) and four focus groups. The outcome of interest was expectation and demand for an antibiotic from a doctor when presenting with a cold or flu.This study used a sequential mixed methods approach: a nationally representative cross sectional survey  contains supplementary material, which is available to authorized users. Antibiotic resistance is a serious and growing problem, caused in part through overuse and misuse of antibiotics . There iStudies suggest prescribers are influenced by a desire to maintain or establish positive relationships with patients, and prescribe according to their perceptions of patient expectations , 13. PatLow education levels, age, low socioeconomic status and ethnicity was found to be associated with variations in knowledge, attitudes and behaviours regarding antibiotic use , 13, 14.Several studies indicated that a high proportion of patients (ranging from 33.5 to 53.1 %) expected an antibiotic prescription for a URTI , 15. ExpIn Australia, NPS MedicineWise plays a role in developing health education and campaigns for health professionals and consumers. In 2012, NPS MedicineWise implemented a five-year program to encourage prudent use of antibiotics in the community. Continuous evaluation of this program shows that although there is a high overall knowledge of antibiotic resistance, inappropriate prescribing remains high. There is a need to understand the factors and circumstances that influence patient expectations and demands in the Australian context to develop future public health campaigns. The aim of this study was to understand who expects an antibiotic for an URTI from their doctor and the reasons underlying it.We used a sequential mixed methods approach to understand the characteristics of patients who expect antibiotics for an URTI and the underlying reasons for this expectation. The preliminary phase used a cross sectional survey to estimate the proportion and the characteristics of the population that expect an antibiotic from their doctor for an URTI. The second phase employed focus groups with a purposefully selected sample in an effort to further explore common themes related to expectations for an antibiotics and socio-cultural norms as well as possible communication messages. The use of a mixed methods approach strengthened the validation of the study.The NPS MedicineWise National Consumer Survey is an annual cross-sectional survey conducted with a random sample from the Australian population to assess general public knowledge and awareness of a range of quality use of medicines and medical tests issues. For this study, the results from the survey conducted between April and May 2014 were used. The survey was conducted using an online panel. Invitation emails were sent to a selection on the external panel with a link to the online survey. This process was repeated over five batches until sample size was reached. A random sample of 1,509 Australian consumers aged 16+ years were selected proportionate to geographic location. Quotas were used to ensure representative distribution across geographic locations, gender and age categories.The survey consisted of 22 questions about health and medicines. Respondents were asked questions about demographics and medicines they take, and then were asked several questions about their knowledge, beliefs and expectations for an antibiotic. The outcome of interest was self-reported expectation for an antibiotic from a doctor if presenting with a cold or flu. The survey questions are available in Additional file Four semi-structured focus group interviews were conducted in August 2014. Focus groups were conducted with groups that were more likely to ask for antibiotics in the quantitative survey results and those identified by general practitioners as more ‘demanding patients’ in a previous study. The discussion guide is available in Additional file The response rate for the survey was 22 %. Table n = 1057), agreed that antibiotics kill bacteria. However, 33 % (n = 497) also agreed that antibiotics kill viruses  correctly identified antibiotics as effective against bacteria and not viral infections.Most respondents, 70 % n = 057, agren = 1053) reported having heard of the term antibiotic resistance, whilst 10.2 % were unsure (n = 154). Age and educational level were associated with having heard of antibiotic resistance (p < 0.001). A higher proportion  correctly agreed or strongly agreed with the statement ‘Bacteria can become resistant to antibiotics’ .Overall 69.8 % of respondents n = 053 reporn = 1112) were aware that taking antibiotics when they don’t need them means that they are less likely to work in the future  agreed that they have to take a full course of antibiotics or they may not work in the future, and the proportion agreeing increased with age.Among respondents 73.7 % n = 112 were n = 284) of respondents reported that they expect their doctor to prescribe antibiotics if they have a cold or flu, with the most significant predictors including speaking a language other than English at home (p < 0.002) and younger age groups (p < 0.028). Of these patients, not all voiced their expectation for a prescription to their doctor. Overall only 16.9 % (n = 254) of respondents say they would directly ask their doctor for antibiotics when they have a flu or cold. However the proportion is higher among those that speak a language other than English at home (p < 0.01). The proportion of respondents who would directly ask their doctor for antibiotics is positively associated with educational level (p < 0.048) and age. In terms of knowledge, people who knew antibiotics didn’t kill viruses and agreed that taking an antibiotic when one is not needed means they won’t work in the future were also less likely to expect or demand antibiotics .Only 19.5 % The low socioeconomic group showed a low level of understanding of how antibiotics work for what conditions, and the difference between viral and bacterial induced illnesses. No one in the group knew what antibiotic resistance was but they all had heard of the term before the focus group. This group believed that antibiotics in general are more efficacious and cheaper compared to over the counter cold and flu medication and they did not understand why they would not ask for antibiotics if they were ill. Furthermore, they expressed that if the doctor withheld antibiotics, it was not because of medical reasons but rather that the doctors disliked antibiotics or wanted to save the government money.“Antibiotics, Love it. Shame, you have to go to a doctor to get prescribed them, you can’t just go to the pharmacist. Because it kills things very quickly. When you see them go green then you know they need antibiotics. Because you know they have an infection. I run to the doctor because I know they need antibiotics and that is what will treat them. In 2 days they will be fine.” “My mother, she’ll wake up, and if she feels sick she takes antibiotics. She is always at the pharmacy, and has a whole cupboard full. She will take it when there is nothing seriously wrong with her, as in no temperature. And she gives me a hard time when I don’t give my kids antibiotics.” The Arabic speaking group also showed a low understanding of antibiotics and thought antibiotics were for general illnesses. There was also a low level of understanding of antibiotic resistance in that the group, who also strongly believed that the body becomes resistant and there will always be other stronger antibiotics for them to switch to later if they developed resistance.“They (antibiotics) kill the germs and make you feel better. They give you your strength back a lot quicker. You’re not sick for too long. It cuts the length of the germ that’s in your body so you are re-gaining your health a lot quicker. You are feeling a lot better about yourself and you’re not taking too much time off work. If the kids aren’t sick they aren’t missing out on school and they aren’t having to catch up. There are a lot of benefits to it …” “If you take it too often then you become resistant to it and there’s always that thought in my mind that I might really need it one day. You kind of do need to power on and so that’s in the back of my mind.” All the participants in the mothers with young children group knew that antibiotics killed bacteria only and had some understanding of antibiotic resistance. However, they expected, and at times demanded, antibiotics for their children, especially if their child had a similar illness previously or the child was sick for longer than expected. There was also a common belief in this group that an antibiotic prescription would prevent the other children falling ill and will shorten the duration of the child’s illness.“The last time was a year ago, I had a really, really bad throat infection. I went to see a doctor to check it’s a throat infection and she said yes. This is a doctor that tends not to prescribe antibiotics. You talk about it all with your friends and the general belief is that antibiotics would kill the bad bacteria but it might also take away your good bacteria as well but at the same time you need to fix the problem. A colleague of mine would say while taking antibiotics it’s good to take other stuff like yoghurt and for her it works but for other people it may not work. So I had a very bad throat infection and I had a temperature and so I went to see the same doctor and she said my throat was bad. It could be bacterial but it could be viral, she said if it is causing you discomfort you may’s well take it because there is a 50 % chance that you could get fixed which she prescribed to me and I took it and it was only a weak one and it did work. It got rid of my throat infection and it did work and of course it could work on others but you eat nutritious food, pick up information about yoghurt and stuff like that. For me it did work, there are times when I’m sure I need antibiotics but I’d rather be cautious and go without it.” All the participants in the Chinese speaking group knew that antibiotics killed bacteria only and had a reasonable understanding of antibiotic resistance. They expected antibiotics if they had a serious illness or there was a chance of a bacterial infection. For this group, the belief that their illness is serious and they have a chance of developing an additional infection were their main reasons for expecting an antibiotic.“If you go to a doctor you want to walk away with something. It’s not just advice and to go home and rest. I don’t have to see the doctor to do that.” Some doctors push antibiotics all the time, some refuse to give it. I don’t really understand the reasons. They always say it’s viral. It frustrates me I have to tell my GP I’ve waited for an hour to see him and I don’t want to have to come back again in a few days and wait again! I really need a script for myself or my kids. They will give me a script anyway, and sometimes say don’t fill it unless you get worse. To be honest, I fill it straight away. Why take the risk of getting worse? I want myself or my kids to get well quickly!” “A script for antibiotics was considered a necessary tangible benefit to compensate for the time and effort invested to visit the doctor and waiting in the waiting area. This was one of the reasons patients demanded antibiotics as it meant the patient could avoid the inconvenience of visiting the doctor again if their condition deteriorated. This theme was common across the four groups.We found in all four groups communication gaps between GPs’ advice and participants’ understanding. This was evident mainly in two areas: the viral nature of their condition and delayed prescribing.“The GP doesn’t know what illness I have, I have to insist on antibiotics.” hen the GP doesn’t know what is wrong with me or my child they just say ‘it’s viral’.” ‘WConsumers were confused about what symptoms required antibiotics and what a viral condition was. Most participants reported that they couldn’t understand why their doctor wouldn’t prescribe them antibiotics and suggested that their condition was viral. There was also skepticism among the participants that because the doctor wasn’t able to diagnose them appropriately; hence the diagnosis with a viral condition.There was further confusion among the groups regarding delayed prescribing around the reason for the delay or waiting period. All reported that they filled the prescription straight away and couldn’t understand the logic behind the request to delay.“No I fill the script straight away because I have always had trouble with my throat.” “To be honest, I go straight to the chemist. I don’t listen to him because I want to get out of the terrible sickness. I get the antibiotics cause I know it helps me.” Examples of responses given when GPs tell them not to fill the prescription until they’re worse or in a few days:“I was thinking of the person over-using and not the community. It’s more like drugs. That person over used (it) and so he is going to get the problem (antibiotic resistance), it’s not everyone else is going to get the problem.” “In future, if (antibiotics) doesn’t help anymore. You need to take a stronger pill. But that’s in the future; you have to focus on this point in time.” “I know about it (antibiotic resistance) but antibiotics work for me. Is there an alternative for antibiotics? But as long as there is no other option and it works for me, I’m going to keep taking it.” “If it’s (antibiotic resistance) true I wonder if it’s worldwide or Australia wide. I think it would be worldwide. But in third world country they don’t have antibiotics so they would have basic bugs but they would be stronger too.” All the participants mentioned side effects as a consequence of taking antibiotics but this didn’t deter them. No one mentioned antibiotic resistance until prompted and didn’t realise that they personally could contribute or be affected by the issue. They perceived that personally they and their families had a low or negligible risk from antibiotic resistance. The issue of antibiotic resistance was an issue for future generations or an issue in developing countries and they themselves or their families would never be affected by its consequences.This sequential mixed methods approach combines survey and focus group data to understand the characteristics of patients who expect antibiotics for an URTI and the reasons underlying their expectation. Despite good levels of knowledge with regards to the effect of antibiotics and the risks of developing antibiotic resistance, we found 19.5 % of surveyed consumers reported that they would still expect the doctor to prescribe antibiotics for a cold or flu and 16.9 % would ask a doctor to prescribe antibiotics. The reason for this remains unclear and requires further study, but anecdotal evidence suggests that this may be associated with a long standing culture of not leaving the doctor empty handed.The survey results indicated that people under 65 years of age, never attended university/technical college and who speak a language other than English at home were more likely to expect or demand antibiotics for a cold; although this was lower than in other studies. This could be due to the significant underrepresentation of CALD groups in our survey sample and the slight over representation of respondents who had attended university or technical college. However, this would be an issue affecting other surveys as well.Three of the four groups had a clear expectation of receiving antibiotics for a cold or flu if presenting to a doctor. The main reasons given for this expectation is antibiotics help them feel better, prevention of potential deterioration of illness, previous successful experience and investment of time and money to consult a doctor. This is in keeping with other studies that found patients/carers lack an accurate understanding of the nature of antibiotics and the development of antibiotic resistance , 15–17. There was evidence of misconceptions among patients about what doctors mean. Most respondents felt that if the GP said that their illness was viral it indicated that the doctor didn’t know what was wrong with them and or didn’t want to give them a prescription. One study found that some expectations are shaped by their experience of widespread availability of antibiotics in their home country and that they hold substantial misconceptions about antibiotics use .Misconceptions concerning delayed prescribing and the need to wait before using the prescription were reported in this study and have been described elsewhere  AdvocateRespondents’ misconception that the human body can become resistant to antibiotics has been reported in other studies , 16. ThiThe study has several limitations. The consumer survey was based on an online panel and therefore is limited to participants on the panel population. However, sampling methods ensured the participants were representative of the general population. Another potential limitation of the study is that the number of respondents rating their experienced state of health as excellent is lower than the general population. This can indicate that the respondents are more frequently exposed to the health care system and therefore more frequently in need of antibiotics. The response rate was only 22 %, but this is considered typical for online population surveys in Australia . The selTo our knowledge, this is currently the largest Australian study exploring consumers’ knowledge and expectations for antibiotics when presenting with an URTI. This study has identified several key motivations for why patients expect antibiotics and their lack of understanding of personal consequences of antibiotic resistance that have not been addressed by recent public health campaigns. Several misconceptions amongst patients and their understanding of why GPs do or do not prescribe antibiotics indicate an urgent need to bridge the communication gap.Primary care physicians will need to be trained to develop better communication strategies for dealing with patients. There is an urgent need for the core message of future public health campaigns to be focused on the personal consequences of taking antibiotics inappropriately and the implications of antibiotic resistance for the general public. Key messages should focus on the immediate and dire repercussions of antibiotic resistance for individuals and their families in the short term.


In recent decades, the Kingdom of Saudi Arabia has seen an exponentially growing antibiotic resistance, which is exacerbated by the use of antibiotics without a prescription and other various factors. However, no published data are available on factors influencing non-prescription use of antibiotics among the general public in Saudi Arabia using an in-depth interview technique.Semi-structured interviews were carried out with 40 Saudi participants from the Eastern Province of Saudi Arabia, selected via snowball sampling technique. Participants were enrolled based on the following inclusion criteria: 18 years of age or older and had self-medicated themselves with antibiotics in the past two years. Data collection was continued until data saturation was attained. Interviews were audiotaped, transcribed verbatim and analysed using NVivo 10 software.Participants  had a mean (SD) age of 30 years (10.2). Self-medication with antibiotics was associated with various inappropriate antibiotic use behaviours and negative outcomes such as antibiotic resistance, treatment failures and adverse events. Interviews revealed that different reasons contribute to the rise of self-medication with antibiotics, ranging from difficulty accessing healthcare services, participant’s cultural beliefs and practices, lack of knowledge about antibiotics and antibiotic resistance, and weak regulatory enforcement.The findings of the present study will aid in generating data that may provide an insight when designing future interventions to promote public health awareness regarding safe and effective use of antibiotics.The online version of this article (10.1186/s12889-018-6088-z) contains supplementary material, which is available to authorized users. Although antibiotics represent one of the major improvements in public health, their misuse and overuse, as a result of self-medication and irrational prescribing and dispensing, may lead to an increased risk of antibiotic resistance –3. Self-Inappropriate self-medication practices with antibiotics may include insufficient dosage and/or frequency, the antibiotic spectrum being either too narrow or too broad, delayed antibiotic therapy in critical patients, unnecessary use of antibiotic for the wrong ailments, and receiving antibiotics from different sources such as patient’s stock, friends and family, without a medical consultation. Patients/consumers may mistakenly make self-diagnosis, with consequences such as failure to recognise symptoms that may be an indication for another disease, and failure to treat the disease effectively. In addition, when a bacterial infection is not presented, patients can expose themselves to the risks associated with antibiotics. Inappropriate utilisation of antibiotics can also lead to increased healthcare costs, poor health outcomes and increased risk of antibiotic resistance, adverse drug reactions and contraindications , 8.Although self-medication with antibiotics (SMA) is common in developing countries, with an overall prevalence of 38.8% (95% CI: 29.5–48.1)  and in two days you will be fine.” [P5].“Augmentin can be used for everything.” [P8].Another participant assumed that the power of antibiotics could deal with all her health problems:“I take Augmentin with me when I travel just in case me or my family get sick [laughs].” [P11].This belief led some participants to take antibiotics with them when travelling abroad “just in case” they or their family get sick:“It’s a 100% immunity booster and can provide quick relief.” [P25].For one participant, it was particularly interesting to find that he completely believed that antibiotics increase a person’s immunity and are effective in providing immediate relief:“When I travel, [in case] I get sick, especially in countries that do not allow people to buy antibiotics without a prescription, I bring along with me from home an antibiotic which has a short-course duration to help me recover quickly.” [P12].Another participant said that she always considered packing a short course of antibiotics in order to treat or prevent any possible infection that could ruin her holiday, especially when visiting countries that do not allow travellers to buy antibiotics without a prescription:“When I encounter an infection which requires an antibiotic during the holy month of Ramadan, I use Azithromycin [as] this will help me to recover quickly and continue my fasting.” [P12].Having a strong belief in the curative power of antibiotics had led one participant to use a short course of antibiotics during the holy month of Ramadan when she became ill, since this approach enabled her to recover quickly without the need to break her fasting:“I got flu while travelling and I bought some Augmentin. I used it for 10 – 14 days without benefit because it was manufactured in the United Arab Emirates and it was not original [it was generic]. When I came back to my country, I went to buy original Augmentin from the pharmacy.” [P1].An interesting cultural belief which emerged related to certain brands of antibiotics having a better quality and more curative power than others. For example, some participants believed that Western brands of antibiotics were somehow more effective at relieving symptoms than the local alternatives, which in the end led to inappropriate use of antibiotics:People wanting to store antibiotics at home for later use and sharing them with family members were common practices among study participants. Sharing of antibiotics allowed people to demonstrate care for one another and tackle the issue of sickness as a unit. It also allowed them to bypass accessing healthcare services in an effort to obtain treatment for common problems. Participants indicated that they shared antibiotics usually with close family members and less usually with neighbours, friends and work colleagues. Participants seemed to recognise that buying antibiotics without a medical consultation was against Saudi law, but, because the behaviour was consistent with Saudi culture, they did not seem troubled by this. Two participants made the following observations in relation to this:“I had flu and the doctor gave me an antibiotic and it worked for me. Then, I went back and recommended it to my family. It is not only me, even my mother tried an antibiotic two years ago and she found it useful, and then she advised the whole family to use it.” [P36].“No need to see a doctor over a minor thing that I experienced before. Well, I can’t remember exactly what the antibiotic is but my whole family use it and [it] is always available at home; its’ a big box with white and purple colours [Augmentin].” [P24].The third sub-theme to emerge was related to participants’ previous success with antibiotics. When they experienced symptoms that they had experienced before, they sought the treatment that had been successful previously. The participants reported that they had usually sought medical advice from a healthcare provider, often a doctor, for the first episode of a specific illness. If they had a subsequent episode with a similar symptom complex, they felt comfortable with a self-diagnosis. Therefore, because they are relying on previous experience with similar symptoms to identify the condition, diagnosis and treatment, self-medication seems appropriate, a behaviour which may have substantial public health implications. This is illustrated in the following scenarios:“I used to go to the doctor but later I treat myself by my own experience. Let’s say that I went to a doctor once and he gave me an antibiotic and I found it effective, I use it again if needed. No need to go to the doctor again.” [P3].“It is pink but I don’t remember the name. The doctor a long time ago gave it to me and I started to buy it from the pharmacy whenever I have a sore throat.” [P23].“I remember that I bought Augmentin over the counter and [it] caused an allergy for my son and I stopped it immediately.” [P5].“Once, I used an antibiotic and I had a fast heart rate after taking it. Well, I [was] supposed to take it for seven days but I used it only for five days.” [P2].Such practice has led to the development of intolerable side effects and allergies among some of the study participants, which they reacted to and dealt with in different ways – by early discontinuation of antibiotics before completing the course or by modifying the way they took their antibiotics. These reactions were always self-guided:“If I used the antibiotics without a prescription and it is written that you should take three tablets, I used to take two tablets to be on the safe side and avoid the side effects.” [P16].Fear of side effects had led some of the participants to deliberately take less of their antibiotics without consulting a doctor, in order to avoid these adverse effects. One participant declared:“Once I took a topical antibiotic for my skin infection; I used it all the time. I thought that because it is topical would have [fewer] side effects than oral. So, it would be OK if I used it as much as I wanted.” [P13].Another two participants reported that they tend to use topical antibiotics to avoid side effects because they believed that applying antibiotics to the skin does not cause as many side effects as swallowing antibiotics that circulate through the body. One participant stated:“Once I feel better, I stop the antibiotic although the person has to take the whole course.” [P8].Stopping antibiotics when feeling better or as soon as symptoms disappeared was also a common practice among some study participants in order to avoid side effects. One participant said:“Antibiotics are medicines that are used to treat different infections. Some are used to treat influenza and other are used to treat infections, etc.” [P21].Based on the knowledge part of the questionnaire (i.e. section number 2) that assesses knowledge about antibiotics and antibiotic resistance, 60% (24/40) of participants reported sub-optimal knowledge (i.e. where intermediate and poor knowledge were combined) of antibiotics. More than half of the participants  had misconceptions about antibiotics as they believed that they are effective in treating viral infections. One participant voiced:“I sometimes don’t take the full treatment course; for example, if the treatment is for five days, I take the antibiotic for three days and then I stop it once I feel better.” [P21].A majority of participants  held misconceptions that antibiotics can be purchased and taken without a doctor’s prescription. Thirty-eight percent (15/40) of participants incorrectly believed that it is safe to switch between antibiotics during the course of a single infectious disease. Fifty-five percent of participants (22/40) did not know that they should not stop taking a course of antibiotics as soon as they feel better. One participant declared:One-third (12/40) of participants thought that it is fine to take a missed antibiotic dose with the next dose. Others  incorrectly believed that taking many types of antibiotics at the same time during the course of a single illness will result in quick recovery. Forty percent of participants (16/40) held the misconception that antibiotics are the same as anti-inflammatories.“We should not repeat using the same antibiotic again if we experience another [case of] flu, because our body will get used to it, so we have to change it.” [P22].Many participants  were not very sure about what antibiotic resistance is, nor its causes or consequences, when they were asked what they know about antibiotic resistance. Some  thought that over-use of antibiotics might be the cause of antibiotic resistance and reduction of their future effectiveness. Others  believed that bacterial resistance would be increased when using antibiotics not according to the doctor’s advice. One participant mistakenly believed that using the same antibiotic again for flu symptoms would increase antibiotic resistance:“I know that, if you do not take [an] antibiotic as instructed, you might develop antibiotic resistance. So, what you have to do is either you change [the] antibiotic to another one or you reduce the strength of [the] antibiotic, say from 500 to 250 mg.” [P37].Another participant thought that changing from one antibiotic to another or reducing the strength of an antibiotic will help in reducing antibiotic resistance:Long waiting time for consultation and difficulty getting appointments were barriers weighted against the benefits of visiting a doctor for these seemingly uncomplicated and common infections. The participants reported that they would consult a physician in case of emergency situations or critical conditions. Some participants noted that they called family members who were healthcare professionals to provide an expert opinion when they had concerns about the accuracy of their self-diagnosis. This was still seen as a less-time consuming and easier approach than consulting a physician, as outlined in the following quotes:“When you go to the government hospitals, you need a long time to see a doctor, either waiting during or waiting for the appointment. Sometimes, [it takes] one or two months to see a doctor.” [P3].“My father is a pharmacist so, when I have an infection, I do consult my father. It is faster and less time consuming than seeing a doctor.” [P1].Some participants perceived their doctors to take full charge during the consultation process, making the ultimate decision about therapy without explaining the process or considering the views/preferences of the patient, which led them to self-medicate. This is illustrated in the following scenario:“The doctor doesn’t say much; he would only prescribe the medicine and that’s it! He does not look at my medical file. Even when I try to discuss something with him, he just doesn’t care or pay attention. So, my family and I decided to go directly to the pharmacy and get what we need from there.” [P13].Some women reported that they could not access healthcare services easily to consult a doctor due to transportation issues because, until recently, women in Saudi were not allowed to drive. One participant reported that she usually depends on her husband to bring a certain antibiotic for her:“I cannot remember the name of the antibiotic, but we always use it. My husband usually brings it for me since he has the experience and he has the car as well [laughs]. I just tell him to bring the antibiotic from the pharmacy and he brings it for me.” [P6].“Since I live in Qatif and the hospital is in Khobar and I don’t have transportation, I usually buy the antibiotic from the pharmacy that is beside my home.” [P9].Another participant said that she used to buy antibiotics from the pharmacy since the hospital is far away from her home and she might not always have proper transportation to go there:Some participants indicated that, since antibiotics are available and easily accessible in the pharmacies, people will buy them directly from there without consulting a doctor, because the enforcement of regulations is weak and antibiotics are often sold without a prescription:“Since it is readily available in pharmacies and once you request it, you receive it just like in a grocery store, I will buy it from the pharmacy or I will just send my driver to bring it.” [P19].“They are available everywhere and you can get them easily. The law is not strictly enforced to prevent [the] selling [of] antibiotics without prescriptions.” [P23].“I said, ‘If I’m feeling OK, why do I have to get an antibiotic from the pharmacy?’ He said, ‘Why not? You have insurance; you can keep the antibiotic with you just in case you need it for later use.’ I was really surprised when he said this.” [P28].For one participant, it was particularly interesting to find that a pharmacist encouraged her to get an antibiotic from his pharmacy without prescription, despite the fact that she did not need it, just because she has insurance – as illustrated in the following quote:Some participants declared that one of the reasons influencing people to self-medicate with antibiotics is low economic status and lack of health insurance, which might be contributing further to the difficulty in accessing the healthcare system. They explained that people lacking health insurance or those who have a low income tend to buy antibiotics directly from community pharmacies to avoid the trouble and costs of visiting a doctor, although this might be a burden on their budgets. They observed that the cost of a hospital visit is more expensive than that of an antibiotic, although the antibiotics are not cheap:“I’m not going to go to the physician, if I already know my symptoms and what medicine heals me. If I go, they will charge me just for the consultation, and then I have to buy the prescription and that’s another cost with more money. It is easier and cheaper to go and buy an antibiotic straightway, if I know what medication I’m going to take and will work for me.” [P12].“I think, nowadays, most people go to self-medication either because they don’t have health insurance, and they have to pay money sometimes that they cannot afford, and sometimes it is a burden on their budget and so they self-medicate.” [P3].Another reason which reflects a cultural norm and contributes to self-medication with antibiotics is the stigma of getting an infection upon going to hospital, making people less likely to access the healthcare system. This was mentioned by one of the participants who believed that a person might develop a more severe infection when going to a hospital, which enhances her tendency to self-medicate with antibiotics:“Once [upon] a time, there was a period when you got an infection such as corona etc. upon going to the hospital, which enhances my desire to avoid visiting a doctor and getting an avoidable infection.” [P7].This article presents the first study exploring a wide range of factors influencing self-medication practice, and providing a more inclusive picture of such practices and the circumstances in which they arise, providing novel evidence that SMA among this particular population is problematic in Saudi Arabia. Only four other studies have addressed the problem of SMA in Saudi Arabia –18; howeIn this study, the frequency of SMA among participants was high and it was associated with various inappropriate antibiotic use behaviours such as altering or stopping antibiotic-taking, either when feeling better (to relieve the body) or when feeling worse . This shows that customers are not passive recipients of care. Rather, they sometimes develop their own way of taking their antibiotics based on their circumstances or what makes sense to them. Thus, assessing participants’ beliefs, fears and expectations and exploring their practices regarding antibiotic use are necessary to prevent irrational use of antibiotics.An exploration of factors influencing self-medication behaviour among study participants revealed many similarities with evidence from the Middle Eastern literature , althougIn terms of reasons for SMA specific to this particular population, the current study identified some beliefs and practices about antibiotic use that have not been adequately described in the literature to date, such as beliefs participants held about Western brands of antibiotics and topical antibiotics, taking antibiotics while travelling abroad or being away from home for ‘just in case’ purposes, and using a short course of antibiotics during the holy month of Ramadan or while travelling abroad to enable quick recovery. In addition to these beliefs and practices, the stigma of getting an infection upon going to hospital to consult a doctor, and perceptions about healthcare providers and healthcare system, such as paternalism and ignoring the patient’s perspective during medical consultations, issues relating to the organisation of the healthcare system and the impact of this on access to healthcare services or antibiotic supply appeared to be findings specific to the Saudi population. If not addressed, these factors may lead to unanticipated adverse events, complications of incorrect use or misuse, delay in seeking professional help and antibiotic resistance. By uncovering factors influencing self-medication behaviour, the study can provide insight when designing future interventions to promote safe, rational and effective use of antibiotics.Strengthening the role of pharmacists from traditional drug dispenser to more effective healthcare provider is required to improve the rational use of antibiotics. Pharmacists are the key healthcare providers with the training, skills and knowledge associated with the profession required to minimise self-medication behaviours. Thus, it is vital to recognise and use their potential. Pharmacists in many European countries already have the ability to take on additional responsibilities and roles to promote the rational use of antibiotics. For example, many new forms of rapid diagnostics/point-of-care testing (PoCT) are being developed that will give simple-to-use and inexpensive approaches of deoxyribonucleic acid/ribonucleic acid (DNA/RNA)-based recognition of potentially multiple pathogens from a single sample . These nThis study provided evidence that 60% of participants had insufficient knowledge about antibiotics. There was also no or little recognition of the impact of SMA on antibiotic resistance. The insufficient knowledge about antibiotics could be translated into various inappropriate antibiotic use practices and negative outcomes such as antibiotic resistance, treatment failures and toxicity. Studies from the Middle Eastern literature have reported similar knowledge deficits among Middle Eastern populations . The insSince patients frequently visit pharmacies to obtain antibiotics for self-medication, pharmacists are well placed to improve rational use of antibiotics among pharmacy customers. The community pharmacists should take an active role in different public-health initiatives relating, for instance, to the restriction of irrational dispensing of antibiotics and enhancing public awareness of the importance of stopping self-medication without correct diagnosis and of the rising issue of antibiotic resistance infection in the society , 24. WitSince antibiotics in Middle Eastern countries can be obtained without prescription, research on effective strategies to limit unnecessary antibiotic use should target doctors’, pharmacists’ and patients’ knowledge and behaviours. Thus, addressing the knowledge gap and implementing multifaceted behaviour change interventions are likely to be effective. Malta, for example, has introduced a European Antibiotic Awareness Day  in an atThe key to successful strategies for managing antibiotic resistance is to promote behaviour modification besides providing relevant information on proper antibiotic use . BehavioPre-contemplation: where an individual is unaware that his/her current behaviour (e.g. SMA) constitutes a problem and thus has no intention of changing it.Contemplation: the individual is thinking about changing the risky behaviour but is not yet committed.Preparation: the individual has an intention to change the behaviour and is starting to make plans about how to change it.Action: the individual is actually attempting to change the behaviour.Maintenance: the individual is six months abstinent from the risky behaviour and is attempting to prevent relapse.A well-studied model that can be used to explain or predict behaviour change is the Transtheoretical Model (TTM) . A wide After classifying a patient into one of these stages, the pharmacist can develop appropriate interventions to assist the patient in moving towards the next stage. Individuals may cycle through the stages several times before achieving long-term behaviour change. Rewards, reminders and monitoring techniques may be needed for individuals in later stages of behaviour change, but, for patients in earlier stages, discussion of available OTC alternatives and consciousness raising that focuses on the disadvantages of SMA and advantages of stopping this ‘habit’ are needed. However, the application of TTM to influence self-medication practice among the general public is limited, and its efficacy within this context remains unclear. When applied correctly, TTM may provide the necessary approach to promote sustained behaviour change among targeted populations.This study also detected the important role of patients’ families in their infection management in general and in antibiotic-taking in particular. Families were frequently quoted as an important source of support, providing advice and supply of antibiotics to participants. In line with published studies in Kuwaiti culture , Saudi pLaw enforcement and stricter governmental policy regarding the sale of antibiotics without prescription in retail pharmacies and irrational antibiotic usage are also needed. In Chile, for example, after drawing up prescription-only regulations, consumption of oral antibiotics in the community pharmacies remarkably decreased . In SaudRobust antimicrobial use surveillance systems are essential to combat antibiotic resistance. In most European countries, there is a long tradition of monitoring the use of medicines but this should also be implemented in Middle Eastern countries. Antimicrobial stewardship courses in the pharmaceutical and medical postgraduate and undergraduate curricula are also required. The information provided in these courses should stress the importance of the roles of the pharmaceutical and medical professions in fostering the rational use of antibiotics.Strengths: (1) this article has presented the first study exploring a wide range of factors affecting self-medication practice, and providing a more comprehensive picture of such practices and the circumstance in which they arise. Limitations: (1) the sample of this research comprised Saudi participants living in the Eastern Province of Saudi Arabia; therefore, results may not be transferrable to the whole country, or to non-Saudis; (2) this study only involved the general public or community members and did not include other suppliers of antibiotics, such as pharmacists and doctors; (3) some participants were a little anxious about being recorded. This was apparent with a few participants who were anxious about the digital recorder for cultural reasons as they were not sure who would be listening to their voice. However, the researcher reassured them that she was the only one who would listen to the recording, and explained to them that data would be anonymous and confidential, and that it would be destroyed upon completion of the study. Once participants realised this, they were happy for the interview to be recorded. It is worth noting that, for future research purposes, careful attention must be paid to this issue as some patients may find it intimidating to be recorded and may not feel free to provide their honest views in such circumstances.Further studies are needed to design, implement and then evaluate culturally sensitive and effective interventions that are tailored to the target audience in whom behaviour change is required in order to decrease antibiotic self-medication practices. Currently, there are no published results of interventions to prevent this practice, which has important implications for public health and the development of antibiotic resistance . This caThe study indicates that SMA is common practice in Saudi Arabia. By uncovering factors influencing self-medication behaviour among the public, the study can provide insight when developing future interventions to improve prudent, safe and effective use of antibiotics.Additional file 1:Interview schedule or interview guide. (DOCX 63 kb)


Cre-mediated genetic lineage tracing in nr2f1a; nr2f2 double mutants reveals that tcf21+ progenitor cells, which can give rise to ventricular CMs and PM, more frequently become ventricular CMs potentially at the expense of posterior PMs in nr2f1a; nr2f2 mutants. Our studies reveal insights into the molecular etiology that may underlie developmental syndromes that share heart, neck and facial defects as well as the phenotypic variability of congenital heart defects associated with NR2F mutations in humans.Multiple syndromes share congenital heart and craniofacial muscle defects, indicating there is an intimate relationship between the adjacent cardiac and pharyngeal muscle (PM) progenitor fields. However, mechanisms that direct antagonistic lineage decisions of the cardiac and PM progenitors within the anterior mesoderm of vertebrates are not understood. Here, we identify that retinoic acid (RA) signaling directly promotes the expression of the transcription factor Nr2f1a within the anterior lateral plate mesoderm. Using zebrafish  NR2F2 are associated with variable types of congenital heart defects in humans. Our recent work demonstrates that zebrafish Nr2f1a is the functional equivalent to Nr2f2 in mammals and promotes atrial development. Here, we identify that zebrafish nr2f1a and nr2f2 have redundant requirements at earlier stages of development than nr2f1a alone to restrict the number of ventricular CMs in the heart and promote posterior pharyngeal muscle development. Therefore, we have identified an antagonistic mechanism that is necessary to generate the proper number of cardiac and pharyngeal muscle progenitors in vertebrates. These studies provide evidence to help explain the variability of congenital heart defects from NR2F2 mutations in humans and a novel molecular framework for understanding developmental syndromes with heart and craniofacial defects.Many developmental syndromes include both congenital heart and craniofacial defects, necessitating a better understanding of the mechanisms underlying the correlation of these defects. During early vertebrate development, cardiac and pharyngeal muscle cells originate from adjacent, partially overlapping progenitor fields within the anterior mesoderm. However, signals that allocate the cells from the adjacent cardiac and pharyngeal muscle progenitor fields are not understood. Mutations in the gene  In vectively , 7, whicectively , 10. Howectively . Thus, tTBX1 underlies DiGeorge Syndrome, which is characterized by congenital outflow tract and craniofacial defects [Tbx1, loss of Tcf21 in mice results in less severe outflow tract and PM defects [Musculin/MyoR [tbx1 mutants have outflow tract and craniofacial defects [tcf21+ progenitors contribute to both ventricular cardiomyocytes (CMs) and PMs [tcf21 in zebrafish is required for the development of almost all PMs [Given the proximity of the cardiac and PM progenitor fields within the anterior mesoderm of vertebrates, there is significant overlap in the expression of conserved regulators of these lineages. The transcription factors Tbx1 and Tcf21, in particular, share expression in cardiac and PM progenitors and are required to promote their development –14. In h defects . Further defects , 12. Witlin/MyoR . As in m defects , 18. Fur and PMs . However all PMs . Thus, aCiona has shed some light on transcriptional signals that drive cardiac and PM fate decisions within distinct precursors of the SHF [There is evidence that the origin of bi-potent SHF cardiac and PM progenitors is conserved in chordates . Work in the SHF . Despite the SHF –26. HoweNR2F1 and NR2F2, overlaps during early embryonic development as well as later in atrial CMs of the heart [Nr2f1 and Nr2f2 becoming predominantly expressed in neural and mesendodermal tissues, respectively [Nr2f2 knockout (KO) mice have morphologically smaller atria and sinus venosus [Nr2f2 KO mice studies using a Myh6:Cre suggest a later role for Nr2f2 in maintaining atrial CM identity [nr2f2 mutants are not early embryonic lethal and do not have overt cardiovascular defects through at least two weeks of development [nr2f1a mutants indicates that it is the functional homolog of Nr2f2 in mammals with respect to early heart development [nr2f1a mutants have smaller atria due to a requirement within atrial CMs to concomitantly promote atrial differentiation and limit the size of the atrioventricular canal (AVC) [NR2F1 and NR2F2 are redundantly required for atrial differentiation in human iPSC-derived atrial cells [NR2F2 have been associated with variable types of human congenital heart defects (CHDs), in particular atrial septal defects (ASDs) and atrioventricular septal defects (AVSDs), but surprisingly also left ventricular outflow tract obstruction (LVOTO) [Nr2f mutant models has provided insight into the molecular etiology of CHDs affecting the atria and AVC, the mechanisms underlying the observed phenotypic variability of CHDs, in particular the origins of ventricular malformations, in humans with NR2F2 mutations are not understood.NR2F proteins  are highly conserved orphan nuclear receptor transcription factors whose expression is RA-responsive in many tissues of all vertebrates –30. In mhe heart –32. Despectively , 29. Anaectively –36. With venosus . Conditielopment , 38, ourelopment . Specifial (AVC) . NR2F1 a (LVOTO) , 41. Thenr2f1a expression within the ALPM of zebrafish embryos and that retinoic acid receptors (RARs) can bind an absolutely conserved, yet unconventionally localized, response element. Using zebrafish mutants for both nr2f1a and nr2f2, we find redundant functions at earlier developmental stages in restricting ventricular CM and promoting PM specification, independent of the later requirement for nr2f1a in promoting atrial differentiation. Cre-mediated genetic lineage tracing shows that tcf21+ progenitors more frequently become ventricular CMs and less frequently contribute to skeletal muscle within the posterior PM in nr2f1a; nr2f2 mutant embryos. Our results support a novel antagonistic mechanism that controls allocation of ventricular CM and PM progenitors within the anterior mesoderm of vertebrates and may help explain the correlation of craniofacial and heart defects as well as the variability found in CHDs associated with NR2F2 mutations in humans.Here, we identify that RA signaling directly regulates nr2f1a as an RA-responsive gene within the ALPM of zebrafish embryos  treatment  of nr2f1a , was increased at the 20s stage  and PMs, since their progenitors intermingle with the cardiac progenitor population within the anterior mesoderm of zebrafish [nr2f1a; nr2f2 mutants at 48 hpf and 96 hpf, developmental time points when these cells have respectively differentiated [nr2f1a; nr2f2 mutant embryos carrying the kdrl:EGFP transgene  and hyoid (2nd) arch derived muscles were also often smaller and disorganized compared to WT and nr2f1a mutant siblings  to permanently label cells that have expressed tcf21+ (nr2f1a homozygous mutants (nr2f1amut) coupled with nr2f2 heterozygosity (nr2f2het) or nr2f2 mutant homozygosity (nr2f2mut) were analyzed together (referred to as nr2f1a-2mut), because our data suggest loss of a single WT nr2f2 allele in nr2f1a mutants produces similar ventricular CM and PM defects as loss of both WT alleles in nr2f1a mutants. Consistent with what has been reported [st and 2nd arches, we found a decrease in the frequency of contribution to the pp in the nr2f1a-2mut embryos  mutant embryos that are indistinguishable from nr2f1a mutant embryos with respect to the heart and blood pooling. We also have not found evidence for compensatory expression of any nr2f genes in the nr2f1a mutants , which has been proposed to be homologous to the ALPM-derived trapezius neck muscles in mammals [Recent clonal analysis in mice has suggested there are common cardio-pharyngeal progenitors that contribute progeny to the neck muscles, the arterial pole and atria that are distinct from other cardio-pharyngeal populations of the SHF . Our dat mammals –60. Ther mammals , these rCiona [tcf21+, as well as nkx2.5+ progenitors, can give rise to ventricular CMs and PMs [nr2f1a; nr2f2 mutant embryos are less dramatic than with loss of RA signaling, in neither case are the defects subtle enough that a very rare population of bi-potent progenitors is likely being affected. Instead, we favor a model where there is a larger population of progenitors within the ALPM that have the potential to become either ventricular CMs and PM, with signals such as Nr2f proteins functioning downstream of RA signaling to influence their allocation into one of these populations.Given the existence of bi-potent cardio-pharyngeal progenitors in mice and Ciona , 19, 20,Ciona –8. While and PMs , it is nOverall, our study provides valuable insight into the requirements of Nr2f genes in vertebrate cardiac and cranial muscle development. These studies may help us to further understand molecular and genetic etiology controlling phenotypic variability of CHDs as well as developmental syndromes that have congenital malformations concomitantly affecting the heart, head, and neck muscles in humans.All zebrafish husbandry and experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Cincinnati Children's Hospital Medical Center.Tg(kdrl:nlsEGFP)ubs1 [Tg(kdrl:EGFP)la116, TgBAC(−36nkx2.5:ZsYellow)fb7 [Tg(actc1b:GFP)zf13 [Tg(tcf21:nucEGFP)pd41 [Tg(tcf21:CreERT2)pd42 [Tg(ubi:LOXP-AmCyan-STOP-LOXP-ZsYellow)fb5 [Tg(myl7: LOXP-AmCyan-STOP-LOXP-ZsYellow)fb2 [Tg(myl7:h2afva-mCherry)sd12 [Tg(hsp70l:EGFP-VP16-RAR-)c1004 [nr2f1ael512 and nr2f2el506 [Adult zebrafish were raised and maintained under standard laboratory conditions. Transgenic lines used were: GFP)ubs1 , Tg(kdrlllow)fb7 , Tg(actcGFP)zf13 , Tg(tcf2GFP)pd41 , Tg(tcf2RT2)pd42 , Tg(ubi:llow)fb5 , Tg(myl7llow)fb2 , Tg(myl7rry)sd12  and Tg(hR-)c1004 . Mutant 2f2el506 , 39.zsyellow (ZDB-EFG-110824-1), egfp (ZDB-EFG-070117-1), nr2f1a (ZDB-GENE-980526-115), nr2f2 (ZDB-GENE-990415-252), vmhc (ZDB-GENE-991123-5), nkx2.5 (ZDB-GENE-980526-321), dlx2a (ZDB-GENE-980526-212), tbx1 (ZDB-GENE-030805-5), tcf21(ZDB-GENE-051113-88), and klf2a (ZDB-GENE-011109-1) were used. Area measurements were performed using ImageJ.Single and two-color whole mount ISH were performed using NBT/BCIP (Roche) and INT/BCIP (Roche), as previously reported . DigoxygNr2f genes plus a 10kb region 5’ and 3’ to the genes were taken from Ensembl (ensembl.org) and aligned using mVista (http://genome.lbl.gov/vista/mvista/submit.shtml). Locations, excluding exons, in which there was over 50% conservation between any of the sequences were analyzed for the presence of RARs. Conserved sequences were input into NHRscan (http://www.cisreg.ca/cgi-bin/NHR-scan/nhr_scan.cgi) to identify potential RAR binding sites.Sequences for zebrafish, mouse, and human β-actin expression and data were analyzed using the 2-ΔΔCT Livak Method. All experiments were performed in triplicate. Primer sequences for β-actin were reported previously [Total RNA isolation and RT-qPCR was performed using previously reported methods . Brieflyeviously , 75. Allnr2f1a and nr2f2 expression, embryos were treated with CHX , RA , and DEAB  at tailbud stage for 1 hour. For analysis of vmhc expression in nr2f1a; nr2f2 mutants, embryos were treated with 0.05 μM RA at the 3s stage until the 20s stage. Drugs were washed out 3X with embryo water then the embryos were fixed in 4% formaldehyde for analysis. Vmhc-stained embryos were genotyped following imaging. Tamoxifen  was administered in 30 mL of blue water with 0.003% PTU in petri dishes to embryos at 30%-50% epiboly until embryos were analyzed or through 2 days of development.All drug treatments were administered to embryos in 2 mL of blue water with drug at specified concentrations in a glass vial with 25–30 embryos/vial at 28.5°C. For analysis of Tg(hsp70l:VP16-RAR:EGFP)c1004 adults were crossed to WT adult zebrafish. The resulting embryos were collected at tailbud stage and heat-shocked at 37°C for 30 minutes. Transgenic embryos were sorted from their non-transgenic control siblings by the presence of GFP. Embryos (n = 100) were dechorionated and fixed in 1% formaldehyde 2 hours after heat-shock. Cells were lysed by gentle pipetting in cell lysis buffer. Nuclei were lysed and DNA was sheered by sonication with glass beads to 200-600bp fragments. Dynabeads (Invitrogen) were used to pull down GFP tagged proteins with ChIP-grade polyclonal anti-GFP antibody (Abcam ab290) per manufactures instructions. Samples were de-crosslinked and qPCR was used to quantify the fold difference in enrichment of the DR1 RARE in the nr2f1a promoter and the known DR5 RARE in the Cyp26a1 promoter as compared to a nr2f1a promoter region not containing a RARE. Expression levels were standardized to the no antibody control signal and data were analyzed using the 2-ΔΔCT Livak Method. Primer sequences for cyp26a1 ChIP-PCR were reported previously [nr2f1a DR1 ChIP-PCR and control are indicated in ChIP-qPCR was performed essentially as previously reported . Hemizygeviously . Primer nr2f1a DR1 site (GTGTCAAAGTTCA), the nr2f1a DR1 site with a targeted mutation in the second half site of the DR1 abolishing the direct repeat (GTGTCAAAGTCAT), and a previously reported Cyp26a1 DR5 site [myc-rarab was in the pCS2+MT. Zebrafish RXRba was cloned into pCS2p+. Proteins for EMSA were made using the TnT SP6 Quick Coupled Transcription/Translation System (Promega). Protein samples were gently mixed with LI-COR tagged probes and incubated at room temperature for 20 minutes. 4% polyacrylamide gels were run for 2 hours at 150 V. Gels were imaged using an Odyssey CLx LI-COR imager.EMSA was performed essentially as previously reported . OligonuDR5 site . A complEmbryos were fixed for 1 hour at room temperature in 1% formaldehyde in PBS in 3 ml glass vials. Embryos were washed 1X in PBS and then 2X in 0.2% saponin/1X PBS, followed by blocking in 0.2% saponin/0.5%sheep serum/1X PBS (Saponin blocking solution) for one hour. AMHC (S46) and MHC  primary antibodies  were incubated at 1:10 in Saponin blocking solution. Rabbit polyclonal DsRed antibody (Clontech), to detect mCherry, and Living colors anti-RCFP (Clontech), to detect ZsYellow, were used at a 1:1000 dilution. Rabbit anti-GFP (Abcam) was used at 1:500. Rabbit anti-Nkx2.5 (Gene Tex) was used at 1:250. Mouse anti-pHH3 (Abcam) was used at 1:1000. All secondary antibodies were used at dilutions of 1:100. Antibody information is also listed in DR1-ef1a construct contains 165 base pairs (bp) of the nr2f1a promoter and 5’UTR adjacent to 193 bp of a minimal elongation factor 1a (ef1a) promoter (green). The nr2f1a-DR1 construct contains 371 bp that include the promoter and 5’UTR containing the conserved DR1 site. The pGL3-12XRARE-tk vector and dual luciferase assays were reported and performed in HEK293 cells as described previously [The promoter fragments for both reporters used were cloned into the Kpn and HindIII sites of the pGL3 (Promega) multiple cloning site. The eviously .t-test or Mann-Whitney test. To compare 3 or more conditions are different, we performed ANOVA analysis. To determine if two proportions were statistically distinct we performed a Chi-squared test or Fisher’s exact test. Statistical analysis was performed using GraphPad Prism. A p value < 0.05 was considered statistically significant for all analysis.To compare two groups, we performed a Student’s S1 FigpGL3-DR1-ef1a construct has nucleotides -60 of the nr2f1a promoter (blue) through +105 of 5’UTR (gray), which includes the including the DR1 site (yellow box) cloned adjacent to a minimal elongation factor 1a (ef1a) promoter (green) (358 bp). The pGL3-nr2f1a-DR1 construct contains nucleotides -266 through +105 (371 bp) of the promoter and 5’UTR containing the conserved DR1 site. Blue indicates nr2f1a promoter sequences. Red boxes indicate predicted TATA boxes. (B) Luciferase assays testing RA responsiveness in HEK 293 cells. FF–firefly luciferase. RL—renilla luciferase. The previously reported pGL3-12XRARE-tk plasmid [(A) Schematic of the two constructs placed into pGL3. The  plasmid  was used(TIF)Click here for additional data file.S2 FigNr2f2 expression in the ALPM of control, DEAB-treated, and RA-treated embryos. View is dorsal with anterior left. Arrows indicated anterior and posterior limits of expression in control and RA-treated embryos.(A-C) (TIF)Click here for additional data file.S3 Figklf2a. Frontal views of hearts in nr2f1awt; nr2f2wt, nr2f1amut; nr2f2wt, nr2f1amut; nr2f2het, and nr2f1amut; nr2f2mut embryos. v–ventricle. a–atrium. Arrows indicate the length of klf2a expression within the hearts.(A-D) ISH for the endocardial atrioventricular canal marker (TIF)Click here for additional data file.S4 Fignkx2.5 in nr2f1awt; nr2f2wt, nr2f1amut; nr2f2wt, nr2f1amut; nr2f2het, and nr2f1amut; nr2f2mut embryos at the 16s stage. Dorsal view with anterior up. 160 embryos were examined with ≥9 embryos examined for each condition. Although we observed a trend in the expansion of nkx2.5 expression when assaying area of expression similar to vmhc, due to inherent variability in nkx2.5 expression and the low numbers of embryos, it was not statistically significant.  IHC for Nkx2.5 and pHH3 in nr2f1awt; nr2f2het and nr2f1amut; nr2f2mut embryos at the 16s stage. Confocal images of the ventro-lateral side of the embryo. Dorsal is right and anterior up. A single side of each embryo was used for analysis. (G) Number of Nkx2.5+ cells in control and nr2f1a; nr2f2 mutant embryos. (H) Percentage of pHH3+/Nkx2.5+ in control and nr2f1a; nr2f2 mutant embryos. For quantification of Nkx2.5+ and pHH3+/Nkx2.5+ cells, nr2f1a homozygous mutants (nr2f1amut) coupled with nr2f2 heterozygosity (nr2f2het) or nr2f2 mutant homozygosity (nr2f2mut) were analyzed together (referred to as nr2f1a-2mut), because our data suggest loss of a single WT nr2f2 allele in nr2f1a mutants produces a similar increase in ventricular CMs as double mutants. Nr2f1a-2ctrl includes any combination of nr2f1a and nr2f2 WT and heterozygous alleles. nr2f1a-2ctrl (n = 23) and nr2f1a-2mut (n = 9) for G and H.(A-D) ISH for the cardiac progenitor marker (TIF)Click here for additional data file.S5 Figvmhc in control (untreated), RA-treated nr2f1awt; nr2f2het, and RA-treated nr2f1amut; nr2f2mut embryos at the 20s stage. Control embryos were not genotyped. (D) Percentage of embryos with the genotypes found that lacked vmhc expression (n = 16) or had vmhc expression (n = 16). Although a RA-treated nr2f1awt; nr2f2het is shown in B, nr2f1a-2ctrl includes any combination of nr2f1a and nr2f2 WT and heterozygous alleles. Fisher’s exact test was used to compare the frequency of embryos with two nr2f1amut alleles found in each condition.(A-C) ISH for (TIF)Click here for additional data file.S6 Fignr2f1a-2ctrl and nr2f1a-2mut embryos. Numbers indicated arches. Anterior is to the right. PAAs in (TIF)Click here for additional data file.S7 Fignr2f1awt; nr2f2wt, nr2f1amut; nr2f2wt, nr2f1amut; nr2f2het, and nr2f1amut; nr2f2mut embryos at 75 hpf. Views are lateral with anterior to the left and dorsal up. (E) Percentage of nr2f1actrl; nr2f2ctrl (n = 7), nr2f1amut; nr2f2wt (n = 16), nr2f1amut; nr2f2het (n = 28), and nr2f1amut; nr2f2mut (n = 28) embryos with loss of posterior and malformed PMs at 75 hpf.(A-D) PMs in (TIF)Click here for additional data file.S8 Figtbx1 (red) and nr2f1a (blue) in the ALPM of an embryo at the 8s stage. Image is a dorsal view with anterior rightward of a flat-mounted embryo. (B-E) ISH for tcf21 in the ALPM of nr2f1awt; nr2f2wt, nr2f1amut; nr2f2wt, nr2f1amut; nr2f2het, and nr2f1amut; nr2f2mut embryos at the 18s stage. (F-I) ISH for the neural crest marker dlx2a in nr2f1awt; nr2f2wt, nr2f1amut; nr2f2wt, nr2f1amut; nr2f2het, and nr2f1amut; nr2f2mut embryos at the 18s stage. For B-I, views are dorsal with anterior up.(A) ISH for (TIF)Click here for additional data file.S9 Fignr2f1a-2ctrl and nr2f1a-2mut embryos.(A) Percentage of embryos with 1 and >1 ventricular CM. (B) Percentage of embryos with labeled CMs that had labeled atrial CMs. (C) Mean number of labeled atrial CMs in (TIF)Click here for additional data file.S10 Fignr2f1b, nr2f2, nr2f5, nr2f6a, and nr2f6b in nr2f1a mutants at 48 hpf does not show compensatory expression.RT-qPCR for (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


We apply in-plane and out-of-plane magnetic fields to proximity-coupled Bi2Se3 and find that the in-plane field creates a spatially oscillating superconducting order parameter in the junction as evidenced by the emergence of an anomalous Fraunhofer pattern. We describe how the anomalous Fraunhofer patterns evolve for different device parameters, and we use this to understand the microscopic origin of the oscillating order parameter. The agreement between the experimental data and simulations shows that the finite momentum pairing originates from the coexistence of the Zeeman effect and Aharonov–Bohm flux.Unconventional superconductivity arising from the interplay between strong spin–orbit coupling and magnetism is an intensive area of research. One form of unconventional superconductivity arises when Cooper pairs subjected to a magnetic exchange coupling acquire a finite momentum. Here, we report on a signature of finite momentum Cooper pairing in the three-dimensional topological insulator Bi 2Se3 and attribute it as a signature of finite momentum Cooper pairing.Unconventional superconductivity may emerge from the interplay between strong spin–orbit coupling and magnetism. Here, Chen et al. report an anomalous Fraunhofer pattern in three-dimensional topological insulator Bi However, introducing magnetism can change the stability of the BCS superconducting state, thereby destroying superconductivity or, as in unconventional superconductors, altering the pairing symmetry2. The potential for unconventional superconductivity at the confluence of magnetism and superconductivity has made it an area of great theoretical and experimental interest. One such example of an unconventional superconducting state is Fulde–Ferrell–Larkin–Ovchinnikov (FFLO) superconductivity, which was proposed as a way for maintaining superconductivity even beyond the critical Zeeman field4. Despite the intensive search for an FFLO superconductor in various types of materials such as heavy fermion compound CeCoIn56 and BEDTTTF-based organic superconductors9, FFLO superconductivity still remains a controversial subject12.In the conventional Bardeen–Cooper–Schrieffer (BCS) theory of superconductivity, Cooper pairs form an isotropic condensate with a zero center-of-mass momentum15. In particular, time-reversal invariant topological insulators (TIs), whose surface states are massless Dirac fermions, are proposed to be an attractive candidate for unconventional superconductivity that carries finite momentum pairing16. To the best of our knowledge, experimental signatures of finite momentum Cooper pairs in TIs have mainly been sought after in the electron-doped regime of the two-dimensional (2D) TI HgTe quantum wells17, but the surface states of three-dimensional (3D) TIs also provide unique advantages to engineering finite momentum pairing. The Dirac cones on the surfaces are non-degenerate and have spin-momentum locking. As a consequence, the Fermi surface of the Dirac cone shifts uni-directionally under the application of an in-plane magnetic field to the surface, which can lead to an FFLO state16. Even though transport measurements in normal 3D TIs are often complicated by the presence of bulk carriers, there is experimental consensus that the metallic surface state dominates transport in a proximity-coupled TI even when the bulk is not depleted22.To better hunt for unconventional superconductivity, there have been proposals for utilizing materials with strong spin–orbit interaction coupled to a conventional s-wave superconductor. This is predicted to stabilize an FFLO superconducting state: the spin–orbit coupling lifts the degeneracy in the Fermi surfaces of the material and introduces an anisotropy to the surfaces that makes it more amenable to a finite momentum phaseBz = 0 out to finite magnetic field values and (2) introducing asymmetries between positive and negative values of Bz in the Fraunhofer patterns. We show that the intensity transfer is suggestive of a spatially oscillating superconducting order parameter phase, which we call a finite momentum shift; we propose two potential origins for this finite momentum shift; and we demonstrate that asymmetries in the transport signal come from the sample geometry. Simulations show a close match between experimental data and finite momentum Cooper pair theory.To this end, we study the experimental signatures of Cooper pairs in a superconductor (S)-3D TI-S Josephson junction subjected to in-plane and out-of-plane magnetic fields. We probe the phase of Cooper pairs by generating Fraunhofer patterns with an out-of-plane field, and we find that adding an in-plane field distorts the Fraunhofer patterns by (1) transferring the intensity of superconductivity from the central Fraunhofer peak at 2Se3 flakes that are mechanically exfoliated from crystals with a bulk carrier density n ~ 5 × 1017 cm−3  as the superconducting material.Our devices consist of BiBz is applied to the superconducting junction to generate a Fraunhofer pattern. In the absence of any in-plane fields, the devices exhibit a central peak with maximum critical current Ic and decaying side peaks, as is expected for a conventional Fraunhofer pattern. The nodes of our Fraunhofer pattern are at d is the effective electrode spacing that takes into account flux focusing  and measure the differential resistance dV/dI as a function of Bz and By, where lower resistance corresponds to higher critical current, similar to in ref. 17. The evolution of the Fraunhofer pattern for device 1 is shown in Fig. By is applied, the Fraunhofer pattern is shifted along Bz. This is evident as an overall tilt of the 2D differential resistance map in Fig. By–Bz plane, this type of misalignment would cause a similar shift in the Fraunhofer pattern when a field is applied in any in-plane direction. Because we do not see a corresponding shift when an in-plane field perpendicular to current direction Bx is applied, we can exclude sample misalignment as a cause for the shift of the Fraunhofer pattern.When an in-plane field along the current direction By = 0, the intensity of superconductivity is maximum at the central lobe, but as By is increased, the intensity is transferred outwards to higher values of Bz. The emergence of this anomalous side branch becomes more evident if the tilt in the 2D differential resistance map is removed by rotating the graph until the lobe minima are vertical, as shown in Fig. Besides the shift to the Fraunhofer pattern, we also observe additional side branch features in the evolution of the Fraunhofer pattern: at Bz and By on the superconducting order parameter phase. For the following discussion we look primarily at the surface contribution because we find that the bulk order parameter decays much more rapidly than the surface contributions in the junction, which means the supercurrent is predominantly carried by the surface states .To determine the origin of the evolution of the Fraunhofer pattern, we begin by considering the mesoscopic effects of magnetic fields By results in a phase modulation given by an Aharonov–Bohm phase 26. For a bulk electron, the phase will be determined by its trajectory inside the flake, and we find that the bulk electrons will acquire a negligible net phase modulation  and ϕ2(x2) are the phases of the order parameters of superconductor 1 and 2 at the coordinates x1 and x2, respectively, along the width of the junction. Based on the phase difference between the two leads, we can model the total transport current along the 27. This analysis is equivalent to summing up all possible quasi-classical trajectories of electron transport, so the total transport current isW1(2) is the width of the superconducting lead 1(2), Δϕ is the overall phase difference between the superconductors, and d is the distance between the two superconductors. Using Eq.  in the differential resistance map with slope m. These side branches are visible in the simulation for a symmetric Josephson junction in Fig. By. This feature is known to be a key signature of finite momentum pairing and distinguishes the system from typical BCS superconductivity29.Figure sing Eq.  and illum of the side branches reflects the relative contributions of finite momentum pairing due to ZME and FME as a function of By. In the simulations, the slope is defined by a line between the origin and the nth side lobe as n becomes large  on thickness, where m is extracted from the data (black) and calculated using theoretical predictions for finite momentum pairing due to ZME alone (red), FME alone (blue), and ZME and FME together (purple).We examine the slope dependence on TI thickness across multiple samples, where devices 2–4 are shown in Fig. sing Eq. . Figure It is clear that the dominant contribution to the finite momentum shift comes from the FME. In the thickest sample, in particular, the FME closely predicts the slope in the experiment since more flux is enclosed in the flake and the orbital effect therefore has a more significant contribution. However, there are some deviations between the data and the FME curves. First, some of the normalized slopes do not follow the monotonically decreasing trend predicted by the FME theory. The deviations are caused by variations in the sample: in real samples, there are variations in chemical potentials, which would result in the We also find that the FME is not enough to predict the observed slope from the experiment on its own even when the error bars of slope calculation are taken into account. In fact, the ZME contribution needs to be considered for the theory to more closely match the data, which means that the finite momentum pairing due to the shifted Dirac cones is non-negligible. We calculate a curve that takes into account an additional ZME value (purple) that demonstrates that by introducing the ZME contribution to the theory, the theory curve is shifted closer to the experimental data. Therefore, our data are generally better explained by the coexistence of both FME and the unconventional ZME, which is more closely related to the FFLO state.17, it was found that a flux through the electrode seemed to contribute to the phase modulation in the system. However, the normalized slopes are not correlated with the thickness of the device leads in our experiment. This suggests that the relevant flux for the finite momentum pairing is the flux through the flake.We also looked at the potential phase contribution from inserting a flux through the superconducting leads. In the supplementary section of a previous workBy is applied to an ideal junction, we also consider the effect of asymmetries in the junction geometry and on the evolution of the Fraunhofer pattern. Due to the fact that typical sample fabrication can result in imperfect device configurations and flux focusing effects, it is important to understand what happens to the Fraunhofer pattern as the devices deviate from the ideal junction. For example, as reported in ref. 30, asymmetric features between positive and negative values of Bz often appear in Fraunhofer patterns and can be attributed to a combination of device-dependent factors such as disorder and the microscopic structure of the device. We consider some sources of asymmetries in the device configuration to model the effect of these asymmetries on the transport signal.In addition to simulating the Fraunhofer evolution as W1 and W2. To model this effect, we introduce the width asymmetry factor α, which is the ratio of the two superconducting lead widths and satisfies W1 = αW2 in Eq.  in Eq. :J0:After deriving the eigenstate and the energies and taking just the positive eigenstate , we can use these results to solve for the surface propagator, In the canonical quasi-classical approximation, we assume that axi term represents oscillations in the surface propagator due to the Zeeman effect.This is the final expression for the surface propagator, where the eβ = gμB. The bulk energy eigenvalue is given asWe now solve for the bulk propagator. To do so, we follow a similar procedure to the one used to solve for the surface propagator. Using Supplementary Eq.  a phase due to a trajectory with no reflections so that the quasiparticle is transferred directly from one superconducting contact into the other and (2) a phase due to a trajectory that reflects off of the bottom surface more than once before being transferred into the other contact. If there are no reflections, the bulk quasiparticle will follow the same trajectory as the surface quasiparticles. In this case, the bulk electron should gain the same phase as the surface electron:Unlike the surface electrons, the set of classical trajectories of the bulk electrons can be characterized by the number of reflections off the bottom surface (Supplementary Figure z = 0 to z = t and the bottom half will have the opposite modulation from the top half of the flake. In this case, the net phase cancels each other out:If the bulk quasiparticle is reflected from the bottom surface at least one time, it will follow a continuous path from As a result, bulk electrons with reflection-less trajectories gain the same FME effect as surface electrons while the bulk electrons with reflected trajectories do not experience the FME and only contribute to the conventional Fraunhofer pattern.The data that support the findings of this study are available from the corresponding author on reasonable request.Supplementary Information