The intercalated IL-anions promoted LDH swelling in monomers and LDH delamination during the course of in-situ polycondensation, which led to the production of PBAT/LDH nanocomposites with intercalated and exfoliated morphology containing well-dispersed LDH nanoplatelets. The prepared nanocomposite films showed improved water vapor permeability and mechanical properties and slightly increased crystallization degree and therefore can be considered excellent candidates for food packaging applications.Currently, highly demanded biodegradable or bio-sourced plastics exhibit inherent drawbacks due to their limited processability and end-use properties . To overcome all of these shortcomings, the incorporation of lamellar inorganic particles, such as layered double hydroxides (LDH) seems to be appropriate. However, LDH delamination and homogenous dispersion in a polymer matrix without use of harmful solvents, remains a challenging issue, which explains why LDH-based polymer nanocomposites have not been scaled-up yet. In this work, LDH with intercalated ionic liquid (IL) anions were synthesized by a direct co-precipitation method in the presence of phosphonium IL and subsequently used as functional nanofillers for in-situ preparation of poly (butylene adipate- Currently, engineering plastics are designed as sustainable materials with an adjustable lifetime in order to minimize their impact on the environment. The challenging issue is thus to prepare advanced plastics either with (i) increased durability or (ii) easy and fast degradability by means of abiotic factors  and/or biological  attack. The first are hi-performance materials with a long service life, while the latter are often designed as biodegradable polymers for packaging with a typical life-time shorter than one year.co-terephthalate) (PBAT) is a typical representative of aliphatic-aromatic copolyesters, which provide balance between biodegradability of aliphatic segments and thermo-mechanical properties of aromatic units. PBAT is a flexible material with high elongation at break and thus suitable for coatings and film applications x+(An−)x/n·mH2O, where M2+ and M3+ are divalent  and trivalent  layer cations, respectively, and An− is the exchangeable inorganic anion  in the interlayer space with x being the relative substitution rate generally ranging between 0.20 < x < 0.33 resulting in M2+/M3+ ratios of 2/1–4/1 . Th. Th−1 , (006), and (009) planes, respectively. The (003) reflection of pristine LDH corresponded to the basal spacing value of 0.77 nm, which is typical for unmodified Mg-Al LDH [The XRD measurements enabled us to indicate whether the IL-anions were intercalated into the basal spacing of LDH. The XRD pattern of pristine LDH a showed g-Al LDH ,35,36.θ range—3.39°, 3.05°, and 2.19° for LDH-phosphate, LDH-decanoate, and LDH-phosphinate, respectively  reflection in the lower 2ectively , denoteser anion . Assuminer anion , then th authors  or by th32− decomposition, and dehydroxylation of the metal hydroxides proceed simultaneously.Thermogravimetric (TG) and derivative weight TG curves of all LDH samples display an initial weight loss (8–13 wt %) between 50 °C and 250 °C  due to t32−-intercalated into Mg-Al LDH was always present as a contaminant. The PBAT nanocomposites containing LDH-phosphate  revealing that the layered particles were neither exfoliated nor intercalated during the in-situ polycondensation. On the contrary, a complete disappearance of the reflection peak at a low angle range (2θ < 4°) was observed for all PBAT nanocomposites with the modified LDH . In such conditions, the permeation, diffusion, and solubility mechanisms in the system resembled gas transport in liquids. Crystallinity of the neat PBAT film (8%) was slightly enhanced by the presence of LDH fillers (<13%—see the DSC result below). However, such low overall crystallinity in the PBAT/LDH nanocomposites has a negligible effect on the evolution of gas transport properties [The permeability coefficients of all samples increase in the order of O2 << H2O . The peroperties .Since the permeability of gas and water vapor molecules was directly proportional to diffusion and solubility coefficients, the more dominating process can be determined based on the diffusion and solubility selectivities. In the cases of neat PBAT and PBAT/LDH nanocomposites, the gas and water vapor permeabilities were preferably driven by solubility in PBAT rather than by diffusion  . General2 and H2O [2 and H2O are much higher than those of O2, which further results in significantly faster permeation of these polar molecules through PBAT materials as compared to O2. Neutral oxygen interacted weakly with PBAT and therefore relatively high selectivities for CO2/O2 gases were obtained.It seems that the relatively high polarity of the PBAT backbone caused by the presence of ester groups promotes strong interactions between PBAT and polar molecules such as CO2 and O2 permeations by acting as a physical barrier, decreasing diffusion but only negligibly affecting solubility, which is in agreement with the theory of transport properties of polymeric materials filled with impermeable particles [The LDH fillers in PBAT nanocomposites were shown to slightly lower COarticles ,45. Gas articles .2 and O2 transport properties, the neat PBAT exhibited high water vapor permeabilities , where Ps and Pp are WVP of PBAT/filler (in our case PBAT/LDH) nanocomposite and neat PBAT, respectively, are depicted in Contrary to COies WVP, , which lies WVP, a. In conies WVP,  was seleOur results are promising compared to the results so far published in the literature. They show the most significant improvement in the water vapor barrier properties of PBAT/LDH-phosphate nanocomposites, especially considering the relatively low nanofiller content (1.5 wt %). The materials prepared in our last study displayed similar WVP results only when higher amounts (2 wt %) of IL-modified LDH was incorporated into the PBAT matrix . In thatOther lamellar fillers  hagT) values increased with the increasing content of the modified LDH containing IL-anions, which indicates homogenous nanofiller dispersion in the PBAT matrix and LDH delamination into individual LDH layers thus reducing mobility of PBAT chains. In contrast, the incorporation of pristine LDH showed no influence on gT due to poor nanofiller dispersion in the PBAT matrix. The increase in crystallinity (cX) and the decrease in melting temperature (mT) of PBAT with increasing content of modified LDH indicates that the particles act as heterogeneous nucleating agents promoting the PBAT crystallite growth [e growth ,9,15.dT5%) of the PBAT matrix  was used for synthesis of IL-anion intercalated LDH. Using this technique, bis (2-ethylhexyl) phosphate (IL-phosphate), decanoate (IL-decanoate), and bis  phosphinate (IL-phosphinate) anions were successfully introduced into the interlayer space of LDH compounds. The produced LDH show the increased interlayer spacing and high content of IL-anions. The results surprisingly show that LDH-IL prepared by this technique practically does not contain surface-bonded IL (in contrast to anion exchange technique used in our last study ). The wa2 and O2 permeability reduction while the water vapor permeation was significantly decreased for all PBAT/IL-modified LDH nanocomposites. From this point of view, the produced PBAT/LDH nanocomposites are considered excellent candidates for food packaging applications.In the next step, LDH with intercalated IL-anions were shown to delaminate readily in a mixture of monomers during in-situ polycondensation. The produced PBAT/LDH nanocomposites exhibited exfoliated morphology either with homogenously dispersed LDH nanoplatelets in the PBAT matrix  or formation of LDH-rich domains in the PBAT matrix . Moreover, the IL-phosphinate modifier was shown to ensure the strongest LDH-PBAT affinity resulting in optimized mechanical performances. The presence of IL-anion intercalated LDH exhibited a relatively low effect on CO


The literature investigating female and male medical students’ differing career intentions is extensive. However, medical school experiences and their implications for professional identity formation and specialty choice have attracted less attention. In this study we explore the impact of medical school experiences on students’ specialty preferences, investigate gender similarities and differences, and discuss how both might be related to gender segregation in specialty preference.interested and uninterested in a specialty. Utilizing a sequential mixed methods design, their responses were analyzed qualitatively to create categories that were compared quantitatively.In a questionnaire, 250 Swedish final-year medical students described experiences that made them Similar proportions of women and men became interested in a specialty based on its knowledge area, patient characteristics, and potential for work-life balance. These aspects, however, often became secondary to whether they felt included or excluded in clinical settings. More women than men had been deterred by specialties with excluding, hostile, or sexist workplace climates . In contrast, more men had been discouraged by specialties’ knowledge areas .Male and female undergraduates have similar incentives and concerns regarding their career. However, the prevalence of hostility and sexism in the learning environment discourages especially women from some specialties. To reduce gender segregation in specialty choice, energy should be directed towards counteracting hostile workplace climates that explain apparent stereotypical assumptions about career preferences of men and women. Despite a preponderance of women in medical schools there remains an unequal distribution of men and women over specialties and in medical leadership , 2. A stIn parallel, there is research showing how experiences during clinical training, such as role modeling or exposure to harassment, affect students’ sense of belonging and (dis)identification on different wards, thus driving them to avoid particular specialties –16. MostInclusion is also fundamental to professional identity formation . In ordeAttitudes towards gender in the educational environment have important implications for medical students’ clinical experiences. Female students are more likely to encounter sexism such as gender related prejudice, gender discrimination, and sexual harassment, than are their male peers , 26, 27.Researchers have suggested that the cumulative effect of overt and covert forms of sexism in the learning environment create an adverse climate for female students that dampens their confidence, participation, and aspirations , 28, 29.In a questionnaire to Swedish final year medical students we included open-ended questions about concrete experiences that made them interested versus uninterested in a specialty. Our aim was to explore the impact of medical school experiences on students’ specialty preferences, to investigate similarities and differences between men and women regarding the character and consequences of experiences described, and to discuss how this might be related to gender segregation in specialty preference.This study is part of the project ‘Gender Challenges in Medical Education’, aimed at investigating medical students’ attitudes and thoughts about gender-related questions . A studyThe study was conducted at the medical school at Umeå University in Northern Sweden. In Sweden, paid parental leave and subsidized childcare encourages women and men to share paid and unpaid work . At presUndergraduate medical education in Sweden comprises 5.5 years, including 3 years of clinical training. The undergraduate period is followed by an 18–24 month internship that is required before applying for a residency position.During the clinical phase of their program, medical students in Umeå rotate between different wards at the university hospital, local hospitals, and health care centers in the region. The proportion of female medical students in Umeå has been 50–60% in the past 10 years. According to questionnaires administered to all students in 2006–2009 a large majority came from a middle class background and less than 10% had parents who were born outside of Scandinavia .: “Can you describe an event that made you interested in working in a certain specialty?” and: “Can you describe an event that made you uninterested in working in a certain specialty?”. Subsequent questions and scales measuring respondents’ attitudes to gender issues in medicine are not included in this analysis.Between 2011 and 2013, final-year medical students at Umeå University were invited to respond to the anonymous questionnaire “Gender in medical education”. After demographic queries, the survey continued with two open-ended questionsStudents were informed about the study when attending compulsory lectures, unrelated to the study, and participation was voluntary. No attempt was made to reach students who did not attend the lecture where the questionnaire was administered. The lecture, however, was mandatory and we have no reason to believe that the absent students differed in any specific way from those present.Out of 404 students  registered during the study period, 344 students were present at the lectures and therefore invited, and 305 of them stayed on to fill out the questionnaire. After exclusion of 55 students who did not answer either of the two open questions 250 remained , 104 men), giving a response rate of 73%.The Regional Ethics Committee in Umeå approved the study: (dnr 2011–262-31 M.). The return of students’ completed questionnaires was considered as consent for participation.p-value < 0.05 was considered significant.Pearson’s chi-square test was used for comparing socio-demographics for responders and non-responders. A We adopted a simple sequential mixed-method design to explore students’ experiences and observe similarities and differences between men and women . It was Prior to the analysis, students’ responses were ‘blinded’ from socio-demographic information, e.g. gender. However, a few participants referred to themselves as either a man or a woman in a way that was not possible to blind. The last author (JA) read one third of the answers to get an overview of the content, conducted an open coding and created a preliminary coding schedule. Answers from 100 students were then read, coded and categorized independently by the four Swedish-speaking authors . In joint sessions, the coding and labeling of categories was then compared, discussed, and reformulated. Based on this, answers from another 50 students were read, categorized, and discussed. This procedure was repeated one more time, at which point the elaborated categories seemed consistent and varied enough to capture the variety of answers. In this way a final coding schedule of eight categories was established. Thereafter, the first author (EK) reread and encoded all answers, i.e., conducted the main coding. To facilitate understanding and presentation, related categories were grouped into three themes.To check the reliability of the main coding, two of the authors  individually encoded 160 randomly chosen answers (80 respectively about experiences inducing interest and disinterest). Each answer was encoded according to the categories in the final coding schedule and the codings were then compared to the first authors coding. A total of 68 discrepancies were identified and these were spread among authors as well as answers about interest and disinterest. With three authors , 160 answers, and eight categories the percentage of discrepancies was less that 2% (68/(160 × 8 × 3) = 0,018). Thus, the inter-rater reliability test showed high consensus, suggesting that the categories were clear and consistent.p-value < 0.01 was considered significant. The number of words in the answers was also counted and compared between men and women (range and mean).When the main coding was completed and all answers sorted into categories, we created a chart in SPSS (version 24.0 for Mac OSX). If a category was coded in an answer it was marked with ‘1’, otherwise with ‘0’. The proportions of answers coded with ‘1’ within each category were compared between inspiring and deterring experiences, and between men and women, and tested using the Pearson chi-square test. A Below we present students’ socio-demographics, followed by the themes and categories developed in the qualitative analysis of free text answers. Finally, we present the statistical analysis of gender patterns in the categories.Men and women had similar socio-demographics, as shown in Table The answers varied in level of detail and depth. Most responses coded for more than one category. The results comprised three themes; ‘The character of work suits me’, ‘Inspiring and inclusive workplace’, and ‘Matches my work-life priorities’. Each theme consists of two to three categories. The content and examples of all categories are outlined in Table In our presentation of themes, more space is given to the second theme ‘Inspiring and inclusive workplace’. The reason is that this theme in particular conveyed examples of students’ educational experiences and the relational settings in which professional identity formation is situated. The answers within the other two themes were often shorter, included less variation, and conveyed simple arguments and views rather than accounts of concrete experiences. Still all three themes are important and were often interconnected.In the excerpts, details about wards and staff have been modified to ensure confidentiality. For example, quotes pertaining to specific surgical specialties e.g.: ‘general surgery’, ‘orthopedics’, etc., have been changed to ‘surgery’ or ‘surgical specialty’.‘Knowledge area and practice’: Participants recounted specific work tasks that intrigued them or matched their perceived talent, like ‘the discussions’ and the ‘detective work’ of internal medicine, or the surgical ‘craftsmanship’. Interest in specific work tasks, however, was often initiated, or maintained by invitations to hands-on participation:I was interested in surgery before I started medical school, but what kept me interested was that I got the opportunity to try a lot on my own. (woman)Correspondingly, being a passive bystander rendered the same knowledge area or work assignments being labeled as ‘boring’, ‘monotonous’, and ‘deterring’.‘Patient characteristics and patient contact’: Rewarding patients were portrayed as grateful, or ‘giving a lot back’ – spanning from particular age groups, like children and elders to people with concrete problems that could be cured or ‘fixed’. Meeting patients with severe or incurable diseases, or trauma victims, could also make a lasting impression.When I met a patient with chronic pain, who had very little drive and who was totally uninterested in working to reach forward. For some reason, this provoked me. (man)‘Values and care ideology.’ Some participants considered whether they shared values and care ideology (e.g. patient-centered care) with physicians in a particular specialty. Others wanted to make a difference by working in medically underserved areas of the world. Workplaces where values were at odds with those of the student were described as dissuading:I lose interest when all that matters is how fast you finish off the operation list - not how well you take care of the patients. (w)Conversely, patients suffering from ‘multiple morbidities’, diffuse symptoms, or those who were ‘unmotivated’ or ‘ungrateful’ were discouraging for some students:‘Workplace climate’: Often participants explained that even though patients and work tasks were important, the workplace climate was paramount when opting for a specialty:The clinic and my co-workers will be of greater importance than the work itself. (w)Students were attracted by nonhierarchical wards, well functioning cooperation, and staff who seemed to enjoy their job and supported each other – factors that often were interconnected. However, workplace climates, characterized as ‘hierarchical’ and ‘tough’, and with senior physicians who treated colleagues of lower rank poorly were described as reasons for not choosing a certain specialty. Most examples concerned male-dominated and surgical wards where students also noted prevalence of ‘abuse of power’, ‘sexist jokes’, ‘macho jargon’, and ‘macho attitudes’; behaviors with which they disagreed:Chauvinistic and macho atmosphere reoccurred on morning rounds. Sexist jokes were told and I lost interest completely for that clinic. I just couldn’t stand it there. (w)There were also negative accounts of competitive rather than supportive attitudes, as well as accusations and conflict rather than solidarity where ‘not showing weakness’ and ‘not asking for help’ were encouraged. Likewise, settings where residents were expected to be wholly devoted to work and have no other interests were deterring.Since I have seen what the jargon is like at several surgical clinics and how unpleasant many surgeons are, I am no longer interested in working there although I still think it is an exciting subject. (m)Overall, accounts of workplace climate did not explicitly concern gender, although ‘macho jargon’ and ‘sexist jokes’ was mentioned. Still, some women overtly discussed gender related issues. Seeing specific challenges facing women in male-dominated clinics, for example difficulty gaining respect, worried them:The only female physician expressed her disappointment over that the other physicians did not prioritize the ward. The atmosphere became very tense, and she got very little support. I do not want to work as the sole female physician in my team. (w)‘Supervision and participation’: Participants’ interest was spurred by experiences of being seen, included, and taken seriously by supervisors:I had good contact with a supervisor who gave me the “right” space to work independently, combined with good support. As a result I had fun during my placement and thus aroused interest in the specialty. (w)Positive attention, like constructive feedback and praise, or being offered a job, made long lasting impressions. Active participation, and getting to do practical tasks were also important. This student described his overwhelming first experience in the operating room:I went from being totally uninterested to being quite interested in surgery the first day of the clinical placement. I got to assist on a surgery and meanwhile I got fantastic tutoring from the surgeon. (m)Lack of supervision or participation on the other hand made a negative impression, as did being ‘used as free labor’, or feeling ‘excluded’ or ‘ignored’:I lost interest already at the first morning meeting when all the surgeons treated us students like we were invisible, and also ignored pretty much everything the interns and everyone else said. (m)Some respondents disclosed how they had been subjected to domination techniques, or felt humiliated by supervisors. Such treatment had the power to literally ‘destroy’ clinical placements. Experiences of neglect were also detrimental and some students, particularly women, described such incidents as gender related:I lose interest when I feel spoken to as if I'm not a potential future colleague at work - usually from a male physician. (w)‘Role models’: Role models depicted as caring and empathic towards patients and students, who seemed enthusiastic and dedicated even after many years within the profession, were highlighted as important for specialty preferences:When I meet an experienced physician that is still excited about patients and being a supervisor. (m)Negative role models in contrast were those who demonstrated cynicism and detachment towards their work. They belittled students and staff, and tried to escape their responsibilities as physicians and supervisors. Other accounts pertained to physicians who treated patients insensitively and harshly:When a surgeon during the morning rounds in an abstruse and cold-hearted way notified a patient that he had cancer, a patient who had lost his wife in cancer a couple of years earlier. (w)Some students disclosed that experiencing a bad environment compelled them to revise previous career plans:‘Workload’: Working office hours and being in control of one’s work schedule, often related to the potential of combining paid work with family and leisure time, was generally considered preferable. Poor organization, heavy workloads, and seeing residents and consultants staying on past regular hours, and working nightshifts scared the students:It doesn’t matter how interesting a specialty is, if I have to be awake at night I will not be able to appreciate it. I don’t want to have to ‘live through’ a shift. I want each day to be enjoyable and rewarding. (w)‘Development possibilities’: Some accounts pertained to the potential for professional development in a specialty, for example, ‘platforms for improving communication skills’, ‘promising technical innovations’, and research possibilities. Some also gave negative reports of ‘scarce development possibilities’ or ‘expectations to work without pay’.Most participants  responded to both of the open-ended questions, resulting in 220 answers regarding experiences inducing interest and 224 answers about experiences inducing non-interest Figures  and non-p = 0.013). Workplace climate was the third most common incentive for women. Among men, the third most prevalent incentives were ‘Supervision and participation’ and ‘Role models’ (both M = 19%). Compared with men, women gave longer answers including more categories. As a result, even though ‘Supervision and participation’ and ‘Role models’ did not rank among the top three incentives among women, they still occurred just as frequently as for men (at 18 and 19% respectively).Men and women showed a similar pattern with no significant differences found when comparing the relative proportions of men and women describing experiences that fostered interest in a specialty Fig . Examplep < 0.001). There was also a significant gender difference in the category ‘Knowledge area and practice’ which was the most common deterrent among men but the third most common deterrent for women . The category ‘Supervision and participation’ ranked second (at 28%) among women’s deterring experiences, followed by ‘Workload’ (W = 23%). Among men, the second and third most common deterrents were ‘Workload’ (M = 20%), closely followed by ‘Supervision and participation’ (M = 19%).Gender patterns were more disparate for deterrent experiences Fig . The larWe explored how medical students’ specialty preferences are shaped by educational experiences, and investigated similarities and differences between men and women regarding the character and consequences of experiences described. Male and female participants shared common inspiring experiences pertaining to knowledge area, their own talent, and patient groups. However, participants’ impressions of workplace climate, supervision, and participation were often described to be of greater importance in their specialty considerations. These experiences rendered feelings of inclusion or exclusion and sometimes outweighed other experiences during clinical practice. Among women, the most common reason for avoiding a specialty was related to experiences of hierarchical, hostile, and sexist workplace climates. In contrast, perceived lack of interest in the knowledge area of a given medical field was the most common reason for men to avoid a specialty.Earlier studies have showed that it is mainly women who consider patient orientation, while men focus on technical challenges –8. By exEven if specialty preference at first seemed to be about students selecting it was just as much a matter of feeling selected and welcomed at a workplace. Knowledge area was the most common factor that made students, women and men alike, interested in a specialty. However, the fact that more women than men described experiences of a hostile and unwelcoming workplace climate made it more of a male privilege to choose a specialty according to interest in its knowledge base. In keeping with previous research, there were more examples of unwelcoming workplace climates from male-dominated and surgical specialties , 27. ThuA sexist jargon and difficulties for women gaining respect described by our participants, convey the message that women are less worthy and less capable than men , 29. It Publicly espoused professional values in Swedish health care include respect, empathy, and gender equality . Our resLave and Wenger  propose Another example of resignation concerned work-life priorities. Our students were dissuaded from specialties where they saw residents struggling with high workloads and expectations to prioritize work above all else. Work-life balance is often suggested to be an issue for women , 5, 8, ball students with good opportunities to pursue their career goals, and improve their professional development.Even if students can discard specialties where supervisors display unprofessional behaviors and sexism they still cannot avoid interaction with these physicians during compulsory clinical training. Although it was primarily women who raised concerns about an adverse workplace climate, quite a few men had similar complaints and changes in the climate would probably benefit the professional development of all students, even those who are not apparently troubled by the problem. By creating welcoming workplace climates, medical schools and other stakeholders in the future medical workforce can therefore provide However, this might constitute a challenge, as research shows that elusive gendered inequities are often communicated by well-intended people who are unaware of their harmful conduct , 29. TheWe used free-text answers from a questionnaire, analyzed by means of mixed methods, to explore and compare how male and female medical students experiences from clinical practice might affect their specialty preferences. The use of qualitatively evolved categories grounded in the data when performing statistical analyses helped us highlight what the students themselves chose to focus on instead of using pre-defined categories.The questionnaire enabled us to represent a large number of students and their multiple realities and to search for gender patterns. The response rate was high and it was similar for women and men, reflecting the distribution of women and men at the Umeå medical school. Although in-depth answers from students were lacking, the answers were given spontaneously by all without having to probe. However, a limitation inherent in using a questionnaire is that it precludes researchers from asking respondents to clarify comments.In research focusing on differences between women and men there is always a risk of exaggerating gender differences. Thus, the open-ended questions were placed at the beginning of the questionnaire, in order to lower the risk of biasing responses by subsequent questions concerning gender. Furthermore, we did not search for gendered patterns until after categories were outlined, a procedure we believe limited the risk of exaggerating gender differences caused by researchers’ expectations. Finally, since women’s answers were, on average, longer and included more categories than did men’s we considered which categories were the most common in women and men’s answers respectively, rather than focusing only on the gender differences in each category separately.Our findings are confined to the perceptions of students from one Swedish medical school. Even though Sweden ranks high on international measures of gender equality , partici(Un)professional beliefs, values, norms, and behaviors among supervisors and staff shape the clinical experiences of medical students, affecting their professional identity formation and how they imagine their future careers. Although male and female undergraduates have similar incentives and concerns regarding career, they enter clinical environments that tend to be more hostile for women, resulting in them feeling less welcomed or even excluded. Irrespective of students’ interests and aptitudes, feelings of inclusion or exclusion in specific workplaces, likely affect subsequent specialty choices. Thus, the understanding of gender as merely a socio-demographic factor, which primes women for ‘family friendly’ specialties and part-time work, and men for technical specialties and prestigious careers, needs to be revised. Future research and debates about gender segregated specialties should be directed towards countering problematic workplace climates that still make it more of a male privilege to choose specialties according to interests.


Activation of the T cell receptor (TCR) on the T cell through ligation with antigen-MHC complex of an antigen-presenting cell (APC) is an essential process in the activation of T cells and induction of the subsequent adaptive immune response. Upon activation, the TCR, together with its associated co-receptor CD3 complex, assembles in signaling microclusters that are transported to the center of the organizational structure at the T cell-APC interface termed the immunological synapse (IS). During IS formation, local cell surface receptors and associated intracellular molecules are reorganized, ultimately creating the typical bull's eye-shaped pattern of the IS. CD6 is a surface glycoprotein receptor, which has been previously shown to associate with CD3 and co-localize to the center of the IS in static conditions or stable T cell-APC contacts. In this study, we report the use of different experimental set-ups analyzed with microscopy techniques to study the dynamics and stability of CD6-TCR/CD3 interaction dynamics and stability during IS formation in more detail. We exploited antibody spots, created with microcontact printing, and antibody-coated beads, and could demonstrate that CD6 and the TCR/CD3 complex co-localize and are recruited into a stimulatory cluster on the cell surface of T cells. Furthermore, we demonstrate, for the first time, that CD6 forms microclusters co-localizing with TCR/CD3 microclusters during IS formation on supported lipid bilayers. These co-localizing CD6 and TCR/CD3 microclusters are both radially transported toward the center of the IS formed in T cells, in an actin polymerization-dependent manner. Overall, our findings further substantiate the role of CD6 during IS formation and provide novel insight into the dynamic properties of this CD6-TCR/CD3 complex interplay. From a methodological point of view, the biophysical approaches used to characterize these receptors are complementary and amenable for investigation of the dynamic interactions of other membrane receptors. T cells play an important role in the execution of the adaptive immune response by regulating the activity of innate and other adaptive immune cells or directly executing effector functions, such as killing by cytotoxic T cells. In general, for T cells to execute their function, they need to become activated by antigens through interaction with an antigen-presenting cell (APC). Crucial to this activation is the interaction between the T cell receptor (TCR) on the T cell surface and the peptide-Major Histocompatibility Complex (pMHC) on the APC surface. Immediately after recognition of the pMHC, the TCR, associated with the CD3 receptor complex, combines with co-stimulatory receptors CD4/CD8 and CD28 on the T cell membrane in small so-called TCR microclusters where signaling is initiated , 2. DuriCD6 is one of the cell surface co-receptors on the T cell membrane involved in T cell activation. CD6 is a transmembrane glycoprotein, part of the scavenger receptor cysteine-rich superfamily (SRCR-SF), that is expressed on thymocytes, mature T cells, a subset of B cells and NK cells, and brain parenchymal cells –18. On tAlready early on, it was clear that CD6 was involved in T cell activation in mature T cells, since monoclonal antibodies targeting CD6 were able to induce T cell activation, subsequent T cell proliferation and IL-2 receptor expression , 24. SinMultiple data hint at an interaction, either direct or indirect, between CD6 and the TCR. Co-precipitation studies have indicated that rat CD6 associates with protein kinases Lck, Fyn, ZAP-70, and Itk: protein kinases that also interact with and co-precipitate with the TCR or are part of the LAT signalosome , 32. ThiCD6 gene, together with the gene for its ligand ALCAM, was identified as a susceptibility locus and a potential target for treatment of multiple sclerosis  Microscopy, combined with biochemical or immunological assays, such as supported lipid bilayers , have be*105 Jurkat cells were transfected at 1325 Volt  with 3 μg of DNA in 100 μl Resuspension buffer. After transfection cells were seeded in 2 ml of 1640 RPMI with 10% FCS and 1% U-glut. Antibiotics were added after 3 h. Stable cell lines were sorted on RFP or GFP expression on a FACSAria cell sorter (BD Biosciences), and cells were maintained in complete RPMI medium as described above, additionally supplemented with 500 ng/ml geneticin .Jurkat E6.1 lymphoma T cells were maintained in 1640 RPMI (PAA) supplemented with 10% Fetal Calf Serum (Greiner Bio-one), 1 mM Ultra-glutamine  and antibiotics . Jurkat cell lines stably expressing CD6-RFP, CD6-GFP, or LifeAct-GFP were obtained by electroporation using the Neon Transfection System for Electroporation (Invitrogen) according to the manufacturer's guidelines. Shortly, 5The following primary antibodies were used: Mouse IgG2A-anti-human CD3 antibodies clone T3B and clone OKT-3 (both referred to in the text as αCD3), and Mouse IgG1 anti-human LFA-1 antibody TS2/4 were obtained from in-house hybridoma production. Mouse IgG1 anti-human phospho-tyrosine (P-Tyr-100), both unconjugated and conjugated to Alexa488, was obtained from Cell Signaling Technology; Mouse IgG1 anti-human CD6  was obtained from BD Biosciences. The following secondary antibodies were used: Goat anti-Rabbit-IgG(H+L)-Alexa647 and Goat-anti-Mouse-IgG1-Alexa488 (both from Invitrogen). Neutravidin-TexasRed was obtained from Thermo Fisher Scientific. For use in immunofluorescence staining, anti-CD3 antibody clone OKT-3 was biotinylated  at RT for 1.5 h, with a molecular ratio of IgG:Biotin at 1:15. Following the same procedure, for use in supported lipid bilayers, anti-human CD3 antibody OKT-3 was simultaneously biotinylated and conjugated to ATTO647 Carboxylic Acid, Succinimidyl ester (ATTO-TEC) at a molecular ratio of IgG:Biotin:dye at 1:15:15. In both cases, purification was performed with Zeba Desalting columns (Thermo Fisher Scientific). For preparation of supported lipid bilayers, the lipids POPC  and Biotin Capped PE , both from Avanti Polar Lipids Inc, were used, together with the fluorescent lipophilic tracer DiI . For inhibition of actin polymerization Cytochalasin D (CytoD) was used . The CD6-GFP plasmid was generated by cloning CD6 from the CD6-RFP construct into peGFP-N1 (Clontech) . LifeAct2O and dried under a N2 stream. A glass microscope slide was cleaned by rinsing consecutively with distilled H2O, 70% ethanol and 100% acetone, and was dried under a N2 stream. The stamp was then manually pushed on the cleaned glass microscope slide for 20 s and removed, after which the stamped area was back-filled with 20 μg/ml fibronectin  in PBS for 1 h at RT. The microscope slide was washed in PBS and incubated with 1% BSA for 30 min to block all uncoated glass surface. The slide was subsequently washed with PBS and dried under a N2 stream before cell seeding.PDMS stamps containing a regular pattern of 5 μm circular spots were prepared as described earlier . PDMS st2 stream. SLBs were prepared by spin coating  solution and sonified for 15 min at RT after which they were rinsed with ultra clean water and ethanol and dried under a N coating . To formImmunofluorescent staining of CD3 was performed on wildtype Jurkat T cells on microprinted antibody spots. Immunofluorescent staining of phospho-tyrosine was performed on wildtype Jurkat T cells on microprinted antibody spots and on wildtype Jurkat T cells on SLBs. LFA-1 staining was performed on wildtype Jurkat T cells on SLBs. Cells were seeded on spots or SLBs for 15–30 min at 37°C. Samples were washed with PBS and subsequently fixed with 4% PFA in PBS for 15 min at RT. After fixation, samples were blocked for 1 h with blocking solution (PBS/3% BSA/10 mM glycine/1% human serum) at RT. For CD3 and phospho-tyrosine staining on antibody spots and for phospho-tyrosine staining on SLBs, permeabilization was performed simultaneously with blocking by adding 0.1% saponin to the blocking solution. 0.1% saponin was added to all subsequent antibody staining solutions. For LFA-1 staining, after blocking, cells on SLBs were incubated with primary Mouse IgG1 anti-human LFA-1 antibody TS2/4 and subsequently Goat-anti-Mouse-IgG1-Alexa488. For phospho-tyrosine staining, after blocking/permeabilization, cells on antibody spots were incubated with Mouse IgG1 p-Tyr-100-Alexa488; cells on SLBs were incubated with primary Mouse IgG1 p-Tyr-100 and subsequently Goat-anti-Mouse-IgG1-Alexa488. For CD3 staining, after blocking/permeabilization, cells on antibody spots were incubated with OKT3-biotin and subsequently NeutrAvidin-Texas-Red. After immunofluorescence staining, samples on microprinted antibody spots were washed with phosphate buffer and embedded in Mowiol (Sigma-Aldrich). Immunofluorescence samples on SLBs were not embedded but imaged in PBS directly after preparation. To study the effect of inhibition of actin polymerization on IS formation, CD6-GFP Jurkat cells were taken from culture and incubated in HBSS with or without 0.5 μM Cytochalasin D for 15 min at 37°C at a concentration of 800,000 cells per ml. Next, cell suspensions were added onto αCD3-containing SLBs, reaching a final cell concentration of 400,000 cells per ml. Samples were incubated for 30 min at 37°C. After incubation, samples were fixed with 4% PFA in PBS for 15 min at RT. Finally samples were washed once and imaged in PBS directly after preparation. CD6-GFP, αCD3-ATTO647, DiI, and brightfield signals were acquired. Samples of cells seeded on microprinted antibody spots were imaged on an Olympus FV1000 confocal laser scanning microscope with a 60 × 1.35 NA oil immersion objective. Samples of cytochalasin D treated cells on SLBs were imaged using a Leica DMI6000 widefield microscope equipped with a HC PL APO 63 × 1.40 NA oil immersion objective. Samples of LFA-1 and phospho-tyrosine staining in cells on SLBs were imaged with TIRF microscopy at an Olympus IX-71 wide field fluorescence microscope equipped with a 3-line TIRF system and a Hamamatsu ImagEM EM-CCD camera equipped with a PL APO 60 × /1.4 NA oil immersion TIRF objective.Live cell imaging in cells on SLBs was performed at 37°C with TIRF microscopy at the Olympus TIRF microscope setup described above. Prior to live cell imaging, Jurkat cells (LifeAct-GFP or CD6-GFP) were washed with PBS and resuspended in HBSS. Cells were added to the SLBs at the microscope, in a final concentration of 400,000 cells per ml HBSS. Directly after adding the cells, both cells and αCD3-ATTO647coupled to the SLBs were imaged. Images were acquired at a frame rate of 300 ms/frame or 1 s/frame with an exposure time of 10–100 ms.Dynal CD3 beads coated with mouse monoclonal anti-CD3 antibody (Invitrogen) or fibronectin-coated beads, all with a diameter of 4.5 μm, were used for bead experiments. Jurkat CD6-GFP cells were seeded on fibronectin-coated coverslips in imaging medium . Subsequently, beads were added to the cells in a concentration of 5 μM. Imaging of the CD6-GFP signal and the brightfield channel of cells with beads was performed on a Zeiss LSM510 meta confocal laser scanning microscope equipped with a PL APO 63 × /1.4 NA oil immersion objective. Cells were imaged at RT to slow down internalization of the beads.0 using a method that is known as double normalization . Photobleaching was performed at 100% laser power by scanning the bleached ROI for two iterations, yielding a total bleach time of 0.10 s and an average fluorescence loss of ~50%. Recoveries were collected with time intervals of 200 ms using 488 nm excitation. Fluorescence intensity data for the bleached ROI and a control ROI were calculated using LSM software (Zeiss). After background correction and normalization to tlization , the sinI(t) is the intensity in the bleached ROI at time t, A is the mobile fraction, and τ is the characteristic recovery time. The halftime recovery t0.5 was calculated with:where For FRAP measurements on cells in contact with beads, Jurkat CD6-GFP cells were resuspended in phenol red-free medium, incubated with αCD3-coated beads and seeded on fibronectin-coated surfaces. FRAP was performed using a 2 × 1 μm rectangular ROI. Photobleaching was performed at 100% laser power by scanning the bleached ROI for 20 iterations, yielding a total bleach time of 1 s and average fluorescence loss of ~50%. Recoveries were collected with time intervals of 100 ms using 488 nm excitation. After background correction and single normalization, FRAP curves were fitted using the Ellenberg fitting  with theI(t) is the fluorescence intensity as a function of time, Ifinal the final intensity reached after complete recovery, w the width of the rectangular ROI, and D is the one-dimensional diffusion constant. Recovery halftime t0.5 was calculated using the formula . The fraction of cells forming an immunological synapse on SLBs upon Cytochalasin D treatment was determined by manual counting. Cells having formed an immunological synapse were defined as CD6-GFP positive cells, also visible in brightfield, on top of SLB (DiI-positive area), overlaying an αCD3 positive cluster. In bead experiments, CD6 enrichment was determined as the ratio between CD6-GFP fluorescence intensity of the membrane area of cell that was in contact with the bead and the fluorescence intensity in an equal sized ROI in the membrane of the cell at the opposite side of bead contact.Image analysis was performed using Fiji Image J . To quan Image J . A ROI it-test was applied. To compare three or more groups, one-way ANOVA with post-hoc Tukey's Multiple Comparison test or Kruskal–Wallis test with post-hoc Dunn's Multiple Comparison test was applied. Differences were considered statistically significant at p < 0.05.Statistical analysis was carried out with GraphPad Prism version 5.03. Data are presented as mean ± standard deviation for bar plots and median ± interquartile range for box plots. To compare two groups, a paired/unpaired Although CD6 has been recognized as a TCR co-receptor, the nature of the interaction with the TCR/CD3 complex has not been resolved. To provide a biophysical characterization of the interplay of these receptors during IS formation, we first studied CD6 and TCR/CD3 (co-)localization using antibody spots created with microcontact printing Figure  52). Wi. Wi52). To investigate the recruitment of CD6 upon cross-linking of the TCR/CD3 complex, we created Jurkat T cells stably expressing CD6-RFP or CD6-GFP. Total cell surface expression of CD6 as well as GFP and RFP expression in CD6-GFP cells, CD6-RFP cells and wildtype cells was determined with flow cytometry  Figure . To studWhen a T cells engages contact with a stimulating antigen-presenting cell, the TCR/CD3 complex is transported to the center of the immunological synapse (IS) formed at the interface between these cells. CD6 has been previously shown to co-localize with the TCR/CD3 complex in the central supramolecular activation cluster (cSMAC) of this IS , 35. HowTo assess whether Jurkat T cells formed an IS on these αCD3-containing SLBs, wildtype cells were allowed to interact with and spread on the SLBs. After fixation, the αCD3 antibody in the lipid bilayer was visualized to localize TCR/CD3 complexes. Representative brightfield images overlaying αCD3 signal are shown in Supplementary Figure Next, we investigated the dynamics of CD6-TCR/CD3 interplay during synapse formation. To this end, Jurkat cells expressing CD6-GFP were imaged during spreading on and engagement of contact with SLBs using live cell TIRF microscopy Figure . Within The actin cytoskeleton provides a dynamic mechanical framework to spatially organize the IS, and the radial transport of TCR/CD3 microclusters is dependent on retrograde actin flow , 11, 53.To better understand CD6 mobility in a cell-cell contact model, magnetic beads coated with αCD3 or with FN were added to CD6-GFP Jurkat T cells seeded on a FN-coated surface and CD6 enrichment at the cell-bead interface was determined. Brightest point reconstructions of confocal image stacks of CD6-GFP show that CD6 was a threefold more enriched to αCD3-coated beads than to fibronectin-coated beads Figures . Next, CIn this study, we applied different experimental techniques to characterize the interplay between CD6 and the TCR/CD3 complex. We show that CD6 and the TCR/CD3 complex are co-recruited to stable stimulatory clusters, both in Jurkat T cells seeded on antibody spots and in Jurkat T cells in contact with αCD3-coated beads. This association to TCR/CD3 applies to only a fraction of the CD6 population, as FRAP measurements on CD6-GFP (both in cells on αCD3 antibody spots or in cells in contact with αCD3-coated beads) indicate that more than half of the CD6-GFP population was still mobile. If the interaction was transient, a reduction in recovery time but no change in immobile fraction would have been expected. This partial association of CD6 with TCR/CD3 confirms previous reports by Gimferrer and colleagues which showed a partial association using co-precipitation . AlthougNext to recruitment to static ligands, we exploited SLBs where αCD3 could freely diffuse in the lateral plane. This setup allowed us to visualize CD6 dynamics during IS formation. We found that CD6 co-localizes with TCR microclusters on the Jurkat T cell membrane during IS formation. These CD6-TCR/CD3 microclusters were transported toward the cSMAC of the IS, which finally resulted in CD6-TCR/CD3 co-localization in the mature IS, as reported previously by us and others , 35. SinFurthermore, in all set-ups we have used antibodies directed against CD3 to induce TCR/CD3 clustering and triggering. Although this is an artificial way of inducing T cell activation, it has been shown that stimulating CD3, without presence of an MHC-antigen complex, can sufficiently induce IS formation in Jurkat T cells . FurtherThe actin cytoskeleton provides a dynamic mechanical framework to spatially organize the IS, and the radial transport of TCR microclusters depends on retrograde actin flow , 11, 53.Next to SLP-76, CD6 also interacts with the actin-binding adaptor protein syntenin-1 . SynteniDetailed investigation of the organization of CD6, TCR/CD3, SLP-76 during IS formation using super-resolution imaging, such as Sherman and colleagues showed for the TCR, LAT, ZAP-70, and SLP-76 , could pAlthough the data presented here further substantiate the interplay between CD6 and TCR/CD3 and indicate that this co-recruitment already occurs in TCR microclusters prior to stable IS formation, it still remains a subject of debate whether CD6 signaling plays a stimulatory or inhibitory role in T cell activation. On the one hand, many studies employing monoclonal antibodies or soluble CD6 to target CD6 or its interaction with ALCAM have underlined the stimulatory role of CD6 in T cell activation and proliferation –29. On tThe raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.MM, AC, and CF designed the study. MM performed microcontact printing experiments. BJ, JW, and RB assisted with microcontact printing. SM performed lipid bilayer experiments. BJ assisted in cell culture, lipid bilayer experiments and image analysis. FC and JK performed magnetic tweezer and beads experiments. MM, SM, and FC analyzed data with support of JtR. SM, MM, AC, and CF wrote manuscript, with input from all authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


At present, the state-of-the-art approaches of Visual Question Answering (VQA) mainly use the co-attention model to relate each visual object with text objects, which can achieve the coarse interactions between multimodalities. However, they ignore the dense self-attention within question modality. In order to solve this problem and improve the accuracy of VQA tasks, in the present paper, an effective Dense Co-Attention Networks (DCAN) is proposed. First, to better capture the relationship between words that are relatively far apart and make the extracted semantics more robust, the Bidirectional Long Short-Term Memory (Bi-LSTM) neural network is introduced to encode questions and answers; second, to realize the fine-grained interactions between the question words and image regions, a dense multimodal co-attention model is proposed. The model’s basic components include the self-attention unit and the guided-attention unit, which are cascaded in depth to form a hierarchical structure. The experimental results on the VQA-v2 dataset show that DCAN has obvious performance advantages, which makes VQA applicable to a wider range of AI scenarios. Visual Question Answering (VQA) is a multimodal research task that aims to answer questions related to the given image. Compared with other multimodal learning tasks , the core of which is the Dense Co-Attention (DCA) layers stacked in depth. Each DCA layer consists of two parallel question self-attention units, an image self-attention unit, and a guided-attention unit. The self-attention unit aims to carry out intra-modal interactions, while the guided-attention unit is used to realize the inter-modal interactions between the image regions and question words. Compared with the single-layer self-attention unit in MCAN, two parallel question self-attention units can extract more fine-grained question features. When the question features are used to guide the image, more accurate image features can be obtained. Experimental results on the benchmark VQA-v2 dataset  demonstrAn improved multimodal co-attention model is proposed by stacking the self-attention unit and the guided-attention unit. It can not only describe the interactions between multimodalities in a more effective way but also take account of the dense self-attention in each modality. Compared with the existing scheme MCAN, DCAN achieves higher precision.Ablation studies on VQA-v2 are conducted to explain the effectiveness of DCAN. The qualitative evaluation results demonstrate how it generates reasonable attention to questions and images.In summary, the main contributions of this paper are as follows:The rest of this paper is organized as follows—the related work is introduced in When looking at an image, the focus is necessarily on a certain part of the image. In other words, when shifting eyes to another place, attention is also shifting along with the movement of the eyes. In this sense, when people notice a target or scene, the distribution of attention within the target or at each spatial location in the scene is different. With reference to the way the human brain processes information, the attention mechanism is introduced in deep learning, which can quickly select useful information from large amounts of data. A series of methods based on the attention mechanism came into being, but these methods are not the same.Reference  had achiMultimodal feature fusion ,34 referAt present, the fusion methods include the method based on linear fusion and the method based on bilinear pooling. The former includes feature connection and element multiplication and other linear operations. The latter is expressed as the outer product of two vectors. However, the dimension of the feature obtained by the ordinary exterior product is the square of the original feature’s size, making the subsequent classification model large. Therefore, the academic community has made various improvements to the bilinear pooling method, which significantly reduces the dimension of features. Kim et al.  put forwV, K, and Q, respectively. The attention function on all queries is performed simultaneously. The attended feature F is given by:The input of the scaled dot-product attention  includesh paralleled heads, and each head corresponds to a scaled dot-product attention function. On each projection of the values, keys, and queries, the attention function is executed in parallel, resulting in output values of dimension h = 8 parallel heads are applied to reduce each head’s dimensionality, and the total calculation consumption is the same as that of full-dimensional single-head attention. Additionally, To further enhance the representation capacity of the attended features, multi-head attention is presented in Reference . Multi-hThe pointwise feed-forward layer is a forward neural network, which uses several weight coefficients E and P represent question features and image features respectively. The input feature i-th head concerning image features. The feed-forward layer transforms the attended image features further. The final feature is obtained as follows:Both Self-Attention (SAtt) unit  and GuidGuided-attention unit takes the question features and image features as input, which represents question-guided attention or image-guided attention. Correspondingly, the output feature represents the image features guided by the question or the question features guided by the image. As shown on the right side in This section demonstrates DCAN in detail, the main structure of which is shown in The questions and answers are encoded by Bi-LSTM. In It is assumed that M, it can be encoded as When encoding the answers, a similar method as the question encoding method is adopted. Supposing that an answer has words with the number of Inspired by bottom-up attention , Faster d represents the dimensionality of each feature in each image. Considering better performance, lower cost and computational efficiency, The output feature is As can be seen in Firstly, taking the original question features L is set to 6. SGAtt means the image self-attention is carried out firstly, then the question-guided attention is performed.Secondly, the original image features are input to a layer of the self-attention unit to model self-attention of the image. Then the obtained image features are fed to the guided-attention unit together with the question features in the above step. For each SGAtt unit, the output feature of each layer is defined as Equation (15):P is taken as an example, the final attended feature L is the number of layers stacked by DCA layers, namely After co-attention learning, the question features and image features contain abundant information about the attention weights of words and regions. Therefore, a two-layer multi-layer perceptron (MLP) is designed as an attention reduction model, which can obtain the attended features of both the question and the image. If the image feature C is the joint representation of question and image. In this paper, C is input into a non-linear layer, and the score of each candidate answer is predicted by linear mapping.s is the score of the candidate answer, After calculating the final image features The binary cross-entropy (BCE) is employed as the loss function to train the classifier of N answers.In this section, DCAN is evaluated on the VQA-v2 dataset. Firstly, the dataset is introduced, and then experimental demonstrations and results are highlighted. Finally, the qualitative analysis is presented.The VQA-v2 dataset is based on MSCOCO , which cN answers as N classes and set the number of answers to 3000. The dropout ratio in each fully connected layer is set to 0.1 to prevent overfitting. Due to GPU memory limitation, the batch size of the model is set to 64, and 13 epochs of training are performed. Finally, the best epoch is chosen in the validation set.The question feature In this section, some ablation experiments are conducted on the VQA-v2 dataset to verify the effectiveness of DCAN. For a fair comparison, all models use bottom-up object features, which are extracted from Faster R-CNN. The ablation studies are trained on the train set to save the training time, and the results are evaluated on the validation set.As shown in h is 16, accuracy is no longer improved. Considering the training time, we set h = 8 in our bes model.To explore the effect of the number of heads in multi-head attention on the accuracy, we set the number of heads As shown in To explore the effect of the depth of DCA on the accuracy, we set the number of DCA layer In this section, DCAN is compared with state-of-the-art methods under the same experimental settings. We use the train set, vg set, and validation set to train all models, where vg represents the augmented training samples from Visual Genome. First of all, the first part of It can be seen from Secondly, to further verify the effectiveness of DCAN, the second part of In this section, some results of the DCAN are visualized in This paper focuses on fine-grained interactions between multimodalities in VQA tasks. An effective Dense Co-attention Networks (DCAN) for the VQA task is developed, the core of which is a dense co-attention model. It consists of six layers of self-attention units and guided-attention units, namely, six layers of SAtt (E)-SGAtt , which achieves the fine-grained and simultaneous understanding of both images and questions. Moreover, to better capture the relationship between words that are relatively far apart and make the extracted semantics more robust, Bi-LSTM is adopted in the question encoding phase to encode the bidirectional semantic features of the question. Compared with the existing method MCAN, DCAN can make use of the complex correlation between multimodal features in a more effective way and extract more discriminative features for images and questions. This exploration of modeling dense intra- and inter-modality interactions has been applied to intelligent transportation , intelli


P = 0.007). A significant difference was found between different degrees of lens subluxation and the length of surgical time and complications. At follow-up, only two eyes (1.98%) were identified to have developed retinal detachments. In conclusion, better visual outcomes can be achieved when patients received an early operation with MCTR implantation.Marfan syndrome (MFS) is a hereditary disease with an incidence of 0.3% in the general population. Approximately 60% of MFS patients with FBN1 gene mutation will suffer ectopia lentis (EL) from the age of 3. With the development of EL, severe loss of vision will accrue because of lens tilt and glaucoma. Cionni modified capsular tension rings (MCTR) has been applied in the surgery for EL in MFS patients. To evaluate visual acuity and safety of using MCTR during lens subluxation surgery in MFS patients, 66 MFS patients (110 eyes) were included in our study, with the mean duration of follow-up of 4.7 months (SD 1.76 months). The capsular bags were preserved in 101 eyes (91.81%) with MCTR implantation. There was an overall significant improvement in BCVA at 1-month follow-up which was maintained at 3 months. Multivariable linear regression revealed that older age at first visit was associated with greater postoperative BCVA at the 1-month follow-up ( The mutation of the fibrillin-1 (FBN-1) gene located on chromosome 15, which is the main component of elastic matrix microfibrils is acknowledged as the main cause of MFS with a detection rate of 97% among MFS patients4.Marfan syndrome (MFS) is an autosomal dominant disorder affecting several systemic systems, such as the cardiovascular and ophthalmologic system. The prevalence has been estimated to be 1 in 10,000 to 20,000 individuals without geographic, ethnic, or gender predilection6. Maumenee et al. 7 reported that EL was stably accrued in 12.5% of MFS children before the age of 3 and 45% of those at the age of 4 to 5, which could be the initial presenting sign of MFS. Other well-recognized ocular manifestations are myopia, early cataract, glaucoma, and retinal detachment9.Affected individuals present with typical manifestations of MFS during all phases of life, including descending aortic root aneurysm and ectopia lentis (EL). EL occurs in approximately 60% of MFS patients10. The lack of FBN-1 in the capsular bags and zonules as well as in the iris and sclera of MFS patients, which are the two ideal positions for IOL fixation, results in difficulty in the fixation of Scleral- and iris-fixated intraocular lens (IOL)12. As a consequence, intra-operative and postoperative complications have been reported13, including iris capture and lens decentration due to deficient FBN-1 in the iris and sclera. These pathological changes make EL in the setting of MFS to be one of the most challenging anterior segment surgeries.Without adequate zonular support for the lens, EL may cause a large refractive error, a partially phakic visual axis, and an anisometropia. Furthermore, the risk of developing glaucomatous damage, secondary microspherophakia, as well as endothelial compromise, can also be highly ascended by severe EL11. Moreover, intra-operatively during this procedure, the needle needs to be passed through the vascular uveal tissue that may result in bleeding and great trauma14. From a survey participated by 185 MFS patients with EL and RD, 21% with RD had prior lens surgery11. Furthermore, at 7–10 years after transscleral suture-fixated IOL implantation, IOL dislocation has been observed in 24% of patients due to breakage in the polypropylene suture14.The transscleral suture-fixated IOL implantation has previously been accepted as an ideal operative method for the surgical correction of EL. However, it is associated with a high risk of serious complications such as retinal detachment (RD) or glaucoma15. MCTR can provide adequate support and correct the decentration of capsular bags in the presence of progressive zonular degradation. As a simplified operation, MCTR can be implanted into capsule bags with greater ease and minimal trauma. It can also be easily fixated to the sclera with 1 or 2 sutures without compromising the integrity of the capsular bag. Other advantages of MCTR implantation, such as minimizing vitreous loss and allowing placement of posterior chamber IOL (PC-IOL) in capsular bags have also been reported16. Furthermore, the long-term risk of postoperative complications including retinal detachment, glaucoma, and IOL dislocation can also be reduced by implanting MCTR in eyes with subluxation of the lens secondary to MFS17. However, the evidence is still lacking in the practice of MCTR implantation for the subluxation of the lens with its visual outcomes in the MFS cohort.When performing anterior segment procedures, preservation of capsular bags by implanting the Cionni modified capsular tension rings (MCTR) has become a preferred practice instead of transscleral fixated IOL implantation for MFS patientsIn this study, we aimed to evaluate the visual outcomes of MFS patients following MCTR implantation in eyes with a subluxated lens. Length of surgical time, complications of the surgery, and predictive factors for postoperative best-corrected visual acuity (BCVA) were also investigated.From June 2018 to June 2020, a total of 252 MFS patients with congenital EL at the Eye and ENT Hospital of Fudan University were retrospectively reviewed and screened for suitability for this study. All of these patients had MFS confirmed by Ghent-2 criteria. Patients with microspherophakia, keratoconus, previous history of ocular surgery, uveitis, corneal disease, glaucoma, retinal detachment, or use of contact lenses in the 2 weeks prior to the examinations were excluded from this study. Finally, 66 (110 eyes) MFS patients with EL who were suitable for surgical intervention were included in this study. The family and medical histories of all patients were carefully recorded. A flow chart summarizing the selection of the study cohort was illustrated in Fig. This study was approved by the Human Research Ethics Committee of the Eye and ENT Hospital of Fudan University that adhered to the tenets of the Declaration of Helsinki. All the participants have given their written informed consent and informed consent for the publication of the images and data, of which the participants under the age of 18 years were provided through their legal guardians. This study was an extension of our randomized controlled trial (ChiCTR2000039132).In accordance with previously published reports, data on patient demographics, preoperative ocular parameters measured by Pentacam AXL system, best-corrected visual acuity (BCVA) during the first visit and 1-, 3-, and 6-month postoperative follow-ups, degree of lens subluxation, and surgery time were collected. Intra-operative and postoperative complications were recorded, including iris dysgenesis, vitreous prolapse, a peripheral extension of the tear, retinal detachment, posterior capsular opacification (PCO), and anterior capsule opacification (ACO). The logarithm of the minimum angle of resolution (logMAR) units was used to describe BCVA.Fig. For analyses, eyes were divided into 3 groups based on the BCVA: Group 1: logMAR < 0.3 ; group 2: 0.3 < logMAR < 1 ; group 3: logMAR > 1 . The degrees of lens subluxation were stratified into 3 broad groups  with a rotating Scheimpflug camera was used to measure the biological characters of the eyes in MFS patients.All patients were examined by experienced ophthalmologists with data recorded as the means of three repeated measurements.All surgeries of scleral-fixated MCTR implantation with intraocular lenses (IOL) were performed by one surgeon (Dr. YX Jiang). A 2.6 mm clear corneal incision was made after general anesthesia. A continuous curvilinear capsulorrhexis was created. To stabilize the capsular bag, 2–4 capsular hooks  were applied. Soft nucleus and cortical aspiration were performed using an irrigation/aspiration handpiece (Alcon Laboratories Inc.) under a low flow rate with an infusion bottle height of 65 cm. After the capsular bag was refilled with DisCoVisc , MCTR was positioned in the area of maximum zonular weakness with the fixation eyelet through the main incision into the expanded capsular bag. The MCTR was sutured with 9–0 polypropylene to the sclera, 1.5 mm posterior to the corneal limbus. The suture needle was passed around the interlamellar sclera four times with a modified knotless Z-suture technique. The suture was tightened and cut. The end of the suture was spontaneously retracted to the interlamellar sclera. Then, an acrylic 1-piece foldable IOL  selected by the surgeon was implanted into the capsular bag. After aspirating residual OVD by I/A handpiece, the main corneal wound was sutured with 10–0 nylon suture while the overlying conjunctiva was closed with 8–0 vicryl suture.Postoperatively, Cravit Eye Drops  and Pred Forte Eye Drops  were applied three times daily for 1 month. Also, 0.1% pranoprofen  was applied three times daily for 1 month, followed by weekly tapering.t-test, one-way ANOVA, and Mann–Whitney U-test were used for analyzing the data between the modes of different groups.The average standard deviation (SD) was used to describe continuous variables. Categorical variables were described by number and proportion as appropriate. To confirm the normal distribution of the variables, the Kolmogorov–Smirnov test was used. The Chi-square test, Student’s Univariable and multivariable line regression analyses were performed to identify the predictors of BCVA at 1-month, 3-month, and 6-month postoperatively.The cohort for this study consisted of 66 110 eyes) MFS patients with EL. However, transscleral suture-fixated IOL was performed in 9 eyes (8.18%) instead of the originally-planned MCTR implantation because of intra-operative complications. The baseline preoperative ocular characteristics of the study participants (101 eyes) who underwent MCTR implantation were shown in Table 10 eyes MAfter reviewing the surgery video of 103 EL eyes in this cohort (video data of 6 eyes with MCTR implantation was not available), a peripheral extension of the tear occurred in 7 eyes (6.8%), of which 2 developed further to a posterior extension. As a result of the peripheral extension of the tear, MCTR could not be implanted or had to be removed in 6 patients. In preoperative EL, the vitreous loss occurred in 7 eyes (6.8%) with severe zonular weakness, the breakup of the anterior vitreous membrane occurred in 5 eyes, and vitreous prolapse occurred locally in intra-operative traction of capsular bags in 2 eyes. After vitrectomy of 5 eyes, MCTR could still be maintained and IOLs were implanted into capsular bags. Transscleral suture-fixated IOL was performed on 2 eyes with vitreous loss. Iris dysgenesis appeared in 5 eyes (4.85%). Surgery time and intra-operative complications were analyzed according to the different degrees of lens subluxation, as illustrated in Table Retinal detachment (RD) occurred in 2 MFS patients (1.94%) postoperatively, first in a 10-year-old girl at 1 month after MCTR implantation, and another occurred in the left eye of a 24-year-old man on the second day after surgery. After detachment surgery and posterior capsulotomy by YAG laser, his BCVA was 20/40 at the 3-month follow-up. PCO was found in 22 eyes (21.36%) and ACO was found in 11 eyes (10.68%) at 6-month follow-ups. Dislocation of IOL-capsular bag complex was observed in 6 eyes (5.94%) with no obvious influence of visual outcome in 4 eyes while anterior capsulotomy by YAG laser was performed in 2 eyes compared with ACO. There were no cases of postoperative endophthalmitis or vitreous in the anterior chamber.P = 0.028, paired t-tests.). In the 28 eyes at 6-month follow-ups, there was no significant difference in the BCVA between postoperative 3-month and 6-month . MFS patients were divided into two groups by age: the “children” group with 71 eyes (age < 15 years) and the “adults” group with 30 eyes (age ≥ 15 years). There was no significant difference in the BCVA during the first visit and 1-, 3-, and 6-month follow-ups between the “children” group and “adults” group or “mild”, “moderate” and “severe” group 22. Besides, the capsular tension ring (CTR) has been commonly recognized as a rewarding and effective procedure in localizing zonular weakness. However, it does not provide accurate decentration of the capsular bag in the presence of progressive zonular dialysis in MFS patients, whereby further decentration or even dislocation of the IOL-CTR-Bag Complex Subluxation may be encountered23.Patients with EL may present primarily with fluctuating vision, blurred vision, or monocular diplopiaMCTR has been recognized as an excellent device in providing good stabilization of capsular bags with zonular dialysis. As MFS represents a progressive disease with advancing age, MCTR implantation has been chosen for MFS patients in this study. Our study has shown that MCTR preserved the capsular bags safely in MFS patients and consistent surgical outcomes were observed.In our study group, over 98% of eyes showed an improvement in BCVA at 1-month while the increase of BCVA slowed down after 3-month follow-ups, and tended to be stable after 3 months as no significant difference was observed between the 3- and 6-month follow-ups. The treatment of PCO and amblyopia are essential to achieve good visual acuity. In our cohort, the treatment of amblyopia that gradually improved vision acuity was recommended one month after surgery. Conversely, PCO that developed after surgery might have negated the improved visual acuity brought by amblyopia therapy and finally contributed to the stable BCVA at 3- to 6-month follow-ups.Our analysis has found a negative correlation between age and BCVA (logMAR units) at 1-month follow-up. Univariable and multivariable line regression analyses revealed that with the additional age, BCVA (logMAR units) at 1-month follow-up would decrease, which suggested that older patients achieved a better visual outcome. Severe amblyopia is more common in MFS children with EL than the individuals who developed EL in adulthood, which may result in poorer visual prognosis in MFS children of whom with possible inherent severe phenotypes than the adults. Meanwhile, a significant difference was observed in BCVA between the first visit and 1-, 3-, and 6-month follow-ups, which indicated that better preoperative BCVA was correlated with better visual outcomes after implantation of MCTR. Although there was no significant correlation between degrees of lens subluxation and BCVA after surgery, a significant correlation was demonstrated in our analysis between degrees of lens subluxation and surgery time. These correlation results suggest that implantation of MCTR should be considered early especially when an EL has occurred.In our study, the majority  had MCTR implantation with IOL while transscleral suture-fixated IOL was performed in 9 eyes (8.18%) as a result of intra-operative complications. The most common intra-operative complications that occurred were a peripheral extension of the tear and prolapse of vitreous, which were the main reasons for not proceeding with MCTR implantation but a transscleral suture-fixated IOL was performed instead. Significant differences were observed between the varying degrees of lens subluxation and risks of prolapse of vitreous and peripheral extension of the tear, which suggested that patients with severe lens subluxation are at high risk of surgical failure. Moreover, the severity progression of EL was associated with increased surgical time, indicating the increased operative difficulty that further enhancing the risk of intraoperative complications such as a peripheral extension of the tear. Although ideal BCVA was achieved in the 9 eyes operated with transscleral suture-fixated IOL, the risk of postoperative complications also increased. Therefore, these results have further emphasized that in the EL with rapidly deteriorating BCVA, the corrective operation should be expedited to reduce the risk of potential intra-operative and postoperative complications.25 have reported a high risk of PCO of 60%–84% from a few months to a few years following CTR implantation in children with EL. In our study, posterior capsular opacification was observed in 22 eyes, and ACO was found in 11 eyes (10.68%) at 6-month follow-up, and 9 (93.9%) of these needed an anterior capsulotomy by YAG laser as a precautionary measure in our practice. Comparatively, the lower rates of PCO in our study might be due to the shorter length of follow-up. Retinal detachment is another potential vision-threatening complication of MFS. Without appropriate intervention, the risks of 5–11% in retinal detachments have been reported in MFS patients, with particularly higher risk (8–38%) among those with prior intraocular surgery17. In this context, MCTR minimizes pulling forces to the vitreous base especially crucial in the setting of MFS. In our study, RD was only observed in 2 MFS patients (1.94%) postoperatively, which is lower than reported in the literature.Some postoperative complications have been observed during the follow-ups in our cohort, with PCO being the most common complication observed in 21.36% of eyes. Previous studiesThere were some limitations to this study. Firstly, it was limited by a fixed, relatively small sample size and rather short and variable follow-ups due to the COVID-19 pandemic in 2020. Secondly, the retrospective methodology and the lack of a control group for comparison might have resulted in biases in our findings. Despite these, our finding of a significant association between the varying degrees of lens subluxation and intro-operative complications lays the foundation for our future study, in addition to a longitudinal study for long-term postoperative complications.In conclusion, MCTR implantation contributes to an obvious improvement in BCVA. The preoperative BCVA and age are predictors of BCVA outcome postoperatively. The severity of EL determines the length of surgical time and risks of intraoperative complications, with ACO and PCO being the main postoperative complications in the short-term follow-up. Therefore, early operation with MCTR is safe and necessary for MFS patients with EL.