The impact of the use of video resources in primarily paper-based problem-based learning (PBL) settings has been widely explored. Although it can provide many benefits, the use of video can also hamper the critical thinking of learners in contexts where learners are developing clinical reasoning. However, the use of video has not been explored in the context of interactive virtual patients for PBL.A pilot study was conducted to explore how undergraduate medical students interpreted and evaluated information from video- and text-based materials presented in the context of a branched interactive online virtual patient designed for PBL. The goal was to inform the development and use of virtual patients for PBL and to inform future research in this area.An existing virtual patient for PBL was adapted for use in video and provided as an intervention to students in the transition year of the undergraduate medicine course at St George’s, University of London. Survey instruments were used to capture student and PBL tutor experiences and perceptions of the intervention, and a formative review meeting was run with PBL tutors. Descriptive statistics were generated for the structured responses and a thematic analysis was used to identify emergent themes in the unstructured responses.Analysis of student responses (n=119) and tutor comments (n=18) yielded 8 distinct themes relating to the perceived educational efficacy of information presented in video and text formats in a PBL context. Although some students found some characteristics of the videos beneficial, when asked to express a preference for video or text the majority of those that responded to the question  expressed a preference for text. Student responses indicated that the use of video slowed the pace of PBL and impeded students’ ability to review and critically appraise the presented information.Our findings suggest that text was perceived to be a better source of information than video in virtual patients for PBL. More specifically, the use of video was perceived as beneficial for providing details, visual information, and context where text was unable to do so. However, learner acceptance of text was higher in the context of PBL, particularly when targeting clinical reasoning skills. This pilot study has provided the foundation for further research into the effectiveness of different virtual patient designs for PBL. Virtual patients are interactive online tools that present learners with simulated patient encounters . They arThe use of virtual patients has been linked to the development of learners’ clinical reasoning skills  by allowResearch into the effective use of simulation for learning has identified the benefits of feedback and repetitive practice -11. VirtSt George’s, University of London in the United Kingdom developed a range of virtual patients for use in problem-based learning (PBL) . This waIn 2010, St George’s, University of London transformed their PBL curriculum, replacing the paper cases with interactive online virtual patients and delivering this throughout the academic year ,18. ThisThe educational community is taking an increasingly holistic view of the role of technology in education, acknowledging that an effective learning exercise depends greatly on the way that is implemented and the context within which it is implemented ,20. EllaThe need to situate a virtual patient resource within the activities that make use of them is not unique to virtual patients; a similar approach is required to guide the use of any technical artifact used in a learning activity. The primarily Web-based nature of virtual patient resources allows additional forms of media, such as images and video, to be easily incorporated within the virtual patient and their effectiveness should also be considered in the context of the activities in which they are used.Although there have been several studies that have explored the impact of using video within traditional PBL curricula -27, therOur starting hypothesis was that virtual patients, and particularly branching virtual patients, are better suited to developing clinical reasoning skills than traditional PBL ,30,31 anWe created an educational intervention in the form of an adapted virtual patient case, in which the early stages of the case replaced text content with video. This virtual patient case was introduced into one week of the PBL curriculum for undergraduate medical students at St George’s, University of London.We used a mixed-methods approach ,33 to caThe primary participants for the study were undergraduate medical students enrolled in the transition year at St George’s, University of London. This was the second year of the program for graduate entry students and the third year for undergraduate entry students and was the point at which these 2 program streams joined.Students undertook a program of PBL tutorials during the transition year. Running twice a week, there were 18 themed tutorials, each of which took place over 3 separate timetabled 3-hour sessions. For practical reasons, a student cohort was divided into 2 streams, with the groups completing the PBL tutorials at different stages of the year. Students from one stream (stream A) received the intervention in October 2013. Because PBL sessions were a mandatory component of the course, all students in the selected stream participated in the learning activity. Participation in the feedback activity was encouraged, but was not mandatory. All survey responses were anonymous.Each PBL group consisted of 8 or 9 students, a mix of graduate entry and undergraduate entry learners. The PBL sessions were facilitated by tutors who also participated in the study. Their role was noninterventional; they did not teach but guided the session to ensure that the groups stay focused and covered the requisite learning objectives. Each PBL group was facilitated by a tutor who worked with the same learner groups throughout the year. The role of a tutor in PBL requires a specific approach and a particular set of guidelines must be followed; all PBL tutors received training in their roles and PBL techniques, but were not selected on the basis of any subject knowledge. The tutorial in which the intervention was introduced was conducted in the same manner as any other PBL session; experienced PBL tutors did not require or receive any specific training related to the intervention. However, they were informed before the date that the intervention would be introduced and given the opportunity to raise questions about the study with the research team.The study was reviewed and approved by the chair of the St George’s, University of London Ethics Review Board and approved by the undergraduate program course director. To provide assurance that students received no advantage through either participation or nonparticipation, all students were provided with access to both the video and text-based versions of the tutorial after the PBL session had taken place using the institutional learning management system. The video virtual patient was reviewed by the module leads before the intervention was delivered to confirm that the content was suitable for use, accurate, and that all the required information for the case was still delivered. The module leads agreed that the content was suitable and gave their consent to its use.One section of a preexisting virtual patient PBL case was adapted by replacing the textual information with video clips. We used the following predetermined criteria to select a suitable virtual patient case for adaptation from the existing St George’s, University of London PBL curriculum:The case had to have a narrative that could be staged using the equipment and performers available in the St George’s Advanced Patient Simulator simulation and skills training center. This eliminated cases that took place primarily in nonclinical environments.The part of the case to be filmed also had to require learners to interpret only information that could be effectively shown and visualized through video, meaning sections of the case which required analysis of detailed test results, scans, or other similar information were not considered suitable for video representation.The timing of the case in the curriculum was also a critical factor; it was necessary that it took place early in the year so that students were not so familiar with text-based cases that it would prejudice their perceptions against a change to video.Having reviewed all the available virtual patient cases against these criteria to determine their suitability for adaptation, we selected the second part of a 3-part virtual patient case regarding a patient suffering an abdominal aortic aneurysm. Considering the established typology for virtual patients , the selWe storyboarded the first 9 stages of this for filming, taking in the first 2 decision points that learners were required to negotiate during the PBL session. This was the first case that was scheduled to run during the academic year, helping to ensure that the learners were not already too familiar with the text-based tutorials used elsewhere in the curriculum in advance. Each clip was designed to provide both the scenario narrative and the relevant information that learners needed to make effective patient-management decisions.The video material was created in partnership with the St George’s Advanced Patient Simulator center because they had the facilities to create an approximation of the required settings. The videos were recorded in simulation rooms from 4 angles, with sound captured from room-mounted and individual wireless microphones worn by performers.The filming was completed over 2 half-day sessions using volunteer actors and 5 cameras. Four cameras were fixed-viewpoint cameras available in the simulation center, whereas an additional portable camera was used to capture close-up shots and other viewpoints. The simulation center was used to stage scenes representing a recovery room, an operating theater, and a ward environment. Approximately 4 hours of footage was captured from each camera and this was edited down into 9 video clips varying from between 45 seconds to 4 minutes in length. These video clips were then embedded into the virtual patient case, replacing the text in this part of the tutorial. Examples of the video clips are provided in Teaching sessions took place in dedicated small-group teaching rooms; students and tutors were arranged around a table with an Internet-connected computer workstation attached to an interactive SMART Board  and projWe conducted this mixed-methods study using structured and unstructured data gathered from 3 sources: a student survey, a survey of PBL tutors, and a discussion and review session with PBL tutors.We developed the survey instrument from an established instrument for exploring student experiences using virtual patients -37. QuesThe experiences and thoughts of the PBL tutors were captured through a review session and a distinct survey instrument tailored to the tutor experience. The survey instrument was based on those developed in a previous study relating to tutor perceptions of virtual patients in a virtual world  and adapWe analyzed the unstructured free-text data from the survey responses and reviewed session transcripts using a theoretical thematic analysis approach . The datU tests to identify if there were any statistically significant differences between the 2 groups.We analyzed the structured responses by converting Likert scale matrix values to ordinal form  and by generating descriptive statistics. We categorized data by course of study (undergraduate entry or graduate entry) to control for any bias caused by differing levels of experience among student participants and we used 2-tailed Mann-Whitney Out of 158 students registered to attend the PBL session, 119 responded to the student survey giving a response rate of 75.3%, although some students did not answer every question. We considered this response rate to be satisfactory and attributed noncompletion of the survey to a combination of absence from the session or students electing not to provide feedback. In addition, responses were not received from 2 of the 18 student groups, suggesting that the tutors responsible for collecting the student responses in those rooms had either not distributed or simply did not return the survey instruments. In total, students provided 274 open-text comments in their survey responses.The tutor survey received 8 responses, a response rate of 44% (8/18), with 21 open-text comments. In both datasets, responses that were null, “n/a,” or simply “no” when asked for further details were excluded. Due to the small sample size (n=8) for the PBL tutor survey response, we determined that generating descriptive statistics for this would be of little value and unreliable, although the unstructured tutor responses were included in the qualitative analysis. The low response rate for the PBL tutor survey was attributed to the timing of the survey data collection because it took place at the end of the session when tutors had a number of other tasks to complete, such as gathering materials and feedback from students, meaning that time for them to complete the survey was scarce. It was also noted that the tutor survey had been provided in the information pack provided to tutors, which also included the student survey forms. The 2 instruments were both printed on white paper and were, therefore, not immediately distinguishable. Although tutors had been previously briefed, a possible explanation for the low response rate was that they were unaware the tutor survey had been included.Of the 16 groups that returned responses, 7 reached a consensus on the number of times each video was watched, 6 stated that they had watched each video once on average and 1 group reported having watched each video twice. No consensus was reached for the other 9 groups, with responses ranging between 1 and 2 viewings.The descriptive statistics for the Likert scale items from the student participants are shown in U tests on the datasets to test whether the distributions for graduate entry and undergraduate entry students were significantly different at the 5% level (P<.05)  see . In eachOur thematic coding process drew on the unstructured responses of both tutors and students, and led to 67 distinct codes grounded in the data during the open-coding process. Five codes were automatically generated using ATLAS.ti  for the We found quite varied perspectives on the effectiveness and desirability of using video as part of the PBL process. For instance, 55 comments indicated that the video had had a positive impact, whereas 20 statements described a negative effect. Those students who described a positive engagement with video noted that it provided the scenario with more “immediacy” and “involvement.” Several students raised the countervailing idea that text “allows more room for imagination.”Z) because of the ordinal and dependent datasets to test whether the median values were different for video and text (P<.05), indicating that the use of video did not significantly impact on student responses to this statement when compared to their response for text.The structured data reflected a similar lack of consensus when considering the merits of using video to heighten engagement. We noted before the intervention that the role of the student was altered by the introduction of video; when using text the student was addressed as if they were the doctor, whereas in the video the student was at most observing the doctor. We anticipated that this may have had a negative impact on student engagement with the scenario, although there were other more positive factors, such as providing richer visual information and context. To explore this effect in more detail, individual students were asked whether they felt they had to make the same decisions as a doctor and whether they felt they were the doctor caring for the patient for both the video and text components of the virtual patient. We used sign tests  of the students felt that they on average watched each video twice or more than twice, which also had the effect of slowing the pace of the session. Comments also considered this in relation to the greater difficulty in reviewing video compared to text, stating that they had to “repeatedly watch” the video. It was suggested that providing a text transcript would alleviate this issue by eliminating the need for repeated viewings.Many students commented on ways in which they felt the use of video made the scenario more real. The comments reflected a broad range of reasons, but with a common thread that the use of video “brought it to life.” Some comments related to the students being able to identify themselves and their role in the scenario: “it made it more real as if I was present whilst the whole situation was happening.” Another felt that video “portrayed the urgency of the decision that needed to be made.”A particularly common observation among the students was that the video provided additional visual cues that were not present in the text. The nature of these visual cues varied greatly, some students identified the impact of seeing the social interactions taking place or of seeing reactions and facial expressions in the video. Others mentioned the importance of observing the environment in which the patient encounter occurred, saying that it “contextualizes the scenario” and provided “clues about what is in environment, IV drips, blood, etc.” Additionally, some students described the impact that these visual clues had on the decision-making process and stated that “visualizing blood loss on the video altered our initial opinion on what to do next.”A number of the participants (both tutor and student) suggested that video could provide particular advantages over text in representing clinical procedures. A significant component of the video we used showed a simulated surgical procedure to treat an abdominal aortic aneurysm. However, we should be clear that the clip was not intended to teach the procedure, but to further the narrative of the scenario. Nevertheless, its inclusion triggered responses indicating that video was perceived by students to be a superior way of learning about such procedures compared to text, comments including “some of the practical procedures (ie surgery) are better explained and understood if it was demonstrated ‘in action’ in the form of a video.” Other comments recognized the benefits of seeing video of procedures, but questioned the value of embedding them within patient scenarios, stating that it would be just as valuable to link to YouTube videos showing the same procedures.We asked student participants whether they felt that the video was effective and whether they preferred video or text. The responses to these questions showed similar patterns regardless of year of study, with a majority of students feeling that the use of video was effective . HoweverSeveral students suggested that an optimum arrangement would be to have a combination of video and text; one student suggested “a mix so memorable but also easy to understand.” Another agreed, commenting that a combined approach “would allow us to see the key case easily whilst seeing a more realistic scenario in the video.” Others asked for a text transcript of the content in the video. Other views indicated that the combination approach would best be achieved by providing the video first then the text equivalent.Many tutors and students commented on the quality of the video resource and the impact that it had on the session. A particular concern of many was the perceived poor sound quality, with participants stating that the sound was “hard to hear,” “muffled,” and “occasionally distorted.” Examination of the video clips revealed that there was audible distortion present in the original recording at 2 points in the videos. Inspection of the rooms in which the sessions took place also revealed that the character of the frequency response of the audio systems in those rooms had the effect of muffling the speech in the clips and accentuating the background noise making the speech less clear than had been apparent when preparing the clips using headphones.Additional comments were made about the quality of the video relating to the editing of the clips  and, in particular, the quality of the acting. The actors in the clips were volunteers from the project team and as a consequence were perceived to lack proficiency. It was felt by some that the acting quality distracted from the learning task and that “random clips of poor acting doesn’t translate information effectively.”Our study investigated the impact of video clips replacing related text content on a PBL session run using an interactive virtual patient. In particular, we considered the ability of students to process and evaluate the information provided in the video. What became clear on analysis of the results was, although a number of themes emerged, the findings showed a diversity of opinions among students. When asked the question explicitly, a majority of the student participants indicated that they preferred text-based virtual patients for PBL, while acknowledging the benefits of using video in certain circumstances. The contrast between the majority of students’ stated preference for text, yet their widespread identification of the benefits of video, may potentially be explained by understanding the motivation and challenges faced by today’s medical students, who are required to assimilate a significantly increased volume of knowledge and to continue to do so throughout their professional development without a comparable increase in time . Sobral Participants recognized the value that video provided in terms of engagement and the provision of visual information, commenting that it brought the scenario to life for them. However, the additional challenges that video brought were also demonstrated clearly in our results; the widespread belief among participants was that information was harder to identify in a video clip. Existing studies identify that the use of video makes the initial stages of critical thinking more challenging for undergraduate students ,26,29, pThe time taken during the learning activity was shown to be a priority issue among participants, with many responses noting that video slowed the pace of the PBL session. Groups were generally unable to agree on the extent of this effect, however, with only a minority of groups able to reach a consensus on how many viewings of each video were required. Moreover, video was considered harder to review multiple times to identify the information; repeat viewings had to always be watched by the whole group because an individual could not rewatch the video in isolation. Text, on the other hand, provided scrolling was not necessary, allowed all the information to be visible on screen at one time and could be reviewed independently by individuals without requiring the whole group’s attention. This loss of individual agency over the resource when wanting to review the information compounded the difficulty that learners faced in effectively evaluating the information provided and was a key factor in student perception of its effectiveness. This supports the conclusions reached in previous studies , which iStudent perceptions of the technical qualities of the video also impacted upon the perceived effectiveness of the resource. In videos where the students perceived the audio quality or the proficiency of the actors in the clip to be deficient, many commented that it had a negative effect on their learning. The key point from this is that video is not intrinsically educationally useful (or not) because production quality, editing, and a number of other contextual factors can separately impact its utility.Existing work has proposed specific design principles for virtual patients , which iHowever, the claim that video is more authentic is potentially a contentious one. Differing perspectives on what is represented by authenticity make it difficult to validate claims of video being more authentic; the concept of “thick” authenticity suggested by Shaffer and Resnick  identifiHowever, if effectively targeted to information where it is well-suited, our results indicate that the use of video can be an effective complement to text in PBL activities. It was widely commented in our study that video was well-suited to demonstrating procedures in a way that text cannot. A combined approach, favored by many of the participants, in which a combination of text and video is used, would provide a means to focus the use of video on areas of the virtual patient where it was beneficial to the intended learning activity, while using text to efficiently deliver learning in the areas where it is most effective. The provision of a text transcript that accompanies the video would run counter to the principles proposed by Mayer’s cognitive theory of multimedia learning , who poiThis study represents a first step toward investigating the effects of such interventions, piloting the use of video within an interactive virtual patient designed for PBL. However, there are a number of limitations to the study:The findings from this study are based on a single intervention and setting. Further research with a greater number of similar interventions will be required to validate the findings and to establish the broader generalizability.Although the student survey instrument was based on an existing validated instrument ,36, bothOur focus on experience and perception rather than quantifiable outcome measures  reflects the broad and relatively unstructured nature of PBL outcomes  and the Although the use of self-reported data would be a potential limitation for a more quantitative study , this was not a significant concern given the proximity of survey completion to the educational event, the use of multiple sources of data, and the pilot nature of the study.Although several students identified that videos of procedures would be beneficial and provide value, the piloted intervention did not include instructional material targeting knowledge of procedures, instead including a simulated procedure to serve the narrative of the scenario. Therefore, the suggestion that video may be well-suited to targeting this area has not been tested in this study and should be the focus of future inquiry.There was widespread dissatisfaction with the quality of the sound in the video clips that were developed for the intervention and this seemed to have had a substantial impact on learner perceptions of the utility of the resource. There was some suggestion that the sound on the clips was not clearly audible using the equipment in the PBL rooms and that this may have exacerbated some of the effects we observed. Learners played clips multiple times to fully understand the provided information may have slowed the PBL process more than it otherwise would have done. Any future studies should include procedures to test the media in the PBL rooms as well as on individual workstations to ensure that it plays with sufficient clarity in that environment.We felt that learner perceptions of the number of times they had played video clips was a relatively crude and unreliable measure and that in future work we would seek to use log data to track the number of times a clip had been played.Finally, this study examined the effect of the intervention at a specific institution, where all the learner participants in the study were previously familiar with the PBL process and had experience of text-based scenarios as a baseline experience. More research is required before the conclusions reached here can be safely generalized to other educational settings. We also urge caution in generalizing our findings to other types of learning activity in which learning may be self-directed or lecture-based. Indeed, our finding that the circumstances of the PBL activity reconfigured the utility of the video and text resources would suggest that the utility and efficacy of video material should be explicitly tested for different educational activities and settings.This pilot study introduced video clips into a virtual patient resource, replacing existing text content, and tested it with undergraduate learners as part of an interactive online PBL session. We explored the impact that this intervention had on participants’ ability to access and evaluate the information provided in the resource and their perceptions of how effective the approach was. The results identified both positive and negative effects from the introduction of the intervention. Students identified value in the video resource, but when asked to state a preference, the majority chose the text-based resource. Course of study (graduate entry or undergraduate entry), and accordingly varying levels of experience, did not impact learners’ stated preference.Our results lead us to conclude that a combination approach may be a superior one within the context of undergraduate PBL. Using video only for elements to which it is particularly suited  may reduce any negative impact on the pace of the learning activity and would reduce any extraneous cognitive load introduced by video that might reduce the efficacy of the learning resource. Further research is necessary, in particular larger scale studies using a greater number of virtual patient interventions and contexts. However, despite the provisional nature of our findings, we have illustrated the context dependency of the perceived value of different multimedia components in a small-group PBL setting and in doing so we have developed a richer understanding of the role of educational multimedia in health professional education.


Cancer immunotherapy has finally come of age, demonstrated by recent progress in strategies that engage the endogenous adaptive immune response in tumor killing. Occasionally, significant and durable tumor regression has been achieved. A giant leap forward was the demonstration that the pre-existing polyclonal T cell repertoire could be re-directed by use of cloned T cell receptors (TCRs), to obtain a defined tumor-specific pool of T cells. However, the procedure must be performed with caution to avoid deleterious cross-reactivity. Here, the use of engineered soluble TCRs may represent a safer, yet powerful, alternative. There is also a need for deeper understanding of the processes that underlie antigen presentation in disease and homeostasis, how tumor-specific peptides are generated, and how epitope spreading evolves during tumor development. Due to its plasticity, the pivotal interaction where a TCR engages a peptide/MHC (pMHC) also requires closer attention. For this purpose, phage display as a tool to evolve cloned TCRs represents an attractive avenue to generate suitable reagents allowing the study of defined pMHC presentation, TCR engagement, as well as for the discovery of novel therapeutic leads. Here, we highlight important aspects of the current status in this field. Most TCRs bind pMHC ligands in a semi-conserved diagonal orientation with the somatically derived CDR3 loops located centrally atop the bound peptide, and the germ-line encoded variable CDR1 and CDR2 loops positioned over the MHC α helices . Upon ac context . Such sc context –27. It c context –30. Clea context . However context . Also, t context . Here, e context , 35. Suc context , 37. In T cell receptors are membrane anchored proteins, and it is challenging to obtain sufficient quantities of recombinant soluble TCRs for molecular studies. A variety of approaches have therefore been adopted, including formation of single chain (sc) TCR, an analog to scFv antibody (Ab) fragments, and fusion of the extracellular TCR domains to other proteins; i.e., maltose binding protein, thioredoxin, human constant kappa domain, or leucine zippers –42. Howein vitro selection has been successfully applied to a wide range of protein classes .A final question is whether or not one actually needs to apply ACT to achieve optimal clinical benefit. ACT is demanding as it relies on massive nescence –83. Epitnescence , 84, 85.nescence , 86. Sucnescence . The useUndoubtedly, improved phage display technology will continue to be a driver in providing engineered TCRs, which will be powerful tools to monitor and elucidate specific pMHC complexes, as well as creating novel specificities suitable for safe use in the clinic.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.


Human eyes move continuously, even during visual fixation. These “fixational eye movements” (FEMs) include microsaccades, intersaccadic drift and oculomotor tremor. Research in human FEMs has grown considerably in the last decade, facilitated by the manufacture of noninvasive, high-resolution/speed video-oculography eye trackers. Due to the small magnitude of FEMs, obtaining reliable data can be challenging, however, and depends critically on the sensitivity and precision of the eye tracking system. Yet, no study has conducted an in-depth comparison of human FEM recordings obtained with the search coil (considered the gold standard for measuring microsaccades and drift) and with contemporary, state-of-the art video trackers. Here we measured human microsaccades and drift simultaneously with the search coil and a popular state-of-the-art video tracker. We found that 95% of microsaccades detected with the search coil were also detected with the video tracker, and 95% of microsaccades detected with video tracking were also detected with the search coil, indicating substantial agreement between the two systems. Peak/mean velocities and main sequence slopes of microsaccades detected with video tracking were significantly higher than those of the same microsaccades detected with the search coil, however. Ocular drift was significantly correlated between the two systems, but drift speeds were higher with video tracking than with the search coil. Overall, our combined results suggest that contemporary video tracking now approaches the search coil for measuring FEMs. Gaze position can indicate the locus of attention and provide information about visual processing strategies –5. MeasuResearchers have investigated FEMs for over 50 years ,20–24. HAs stated earlier, the reliable measurement of microscopic eye movements (i.e. microsaccades and ocular drift (hereafter drift)) is critical to FEM research, but no studies have conducted an in-depth comparison of human FEM recordings obtained with the search coil method (the gold standard for measuring FEMs) versus those obtained with contemporary, state-of-the art video trackers.Previous human studies compared search coil recordings with a variety of video tracking recordings, but they did not investigate FEMs –33,37,38It is also worth noting that human experimental setups typically stabilize the head with a head and/or chinrest, thereby allowing substantially more head motion than in headpost-implanted primates. In addition, whereas in non-human primates search coils are surgically implanted in the sclera of the eye, in human subjects they are embedded in a contact lens that is rested on top of the eye. The differential effects (or lack thereof) of both techniques on eye movement recordings have been debated ,33,34. THere we provide an in-depth study of human FEMs (microsaccades and drift) measured simultaneously with a search coil and a state-of-the-art video tracker . One important feature of the present study is that it was conducted in experimental conditions that are characteristic of most human research on FEMs  participated in the experiment. All of them had healthy eyes  and no visual complaints when they wore their refractive corrections. The subjects did not wear eye glasses or corrective lenses during the experiment, but had no difficulties seeing, or fixating their gaze on, the fixation target. No subjects experienced previous corneal surgery. These experiments were performed in approval of the Cleveland Veterans Affairs Medical Center Institutional Review Board and in conformity with the Declaration of Helsinki. All participants gave written informed consent.Before starting the experiment, a licensed physician applied a drop of topical anesthetic to the eye that was to have the contact lens-mounted search coil. The contact lens was then placed in the participant’s eye.Participants sat in a custom-designed chair with head support 1.2 m away from a tangent screen, and fixated their gaze on a small red laser spot presented at the center of their field of view. The experiment started with the five-point calibration procedure built into EyeLink 1000 , and consisted of 2 trials of one minute each.We used the EyeLink 1000 eye tracking system (SR Research) to obtain binocular eye position from participants 1, 3, 4, and 5 and monocular eye position (right eye) from participant 2 . We note that, even though binocular eye position was available from 4 subjects, we only compared eye movement data collected from the same eye in both recording systems (video tracking and search coil method). We did this to avoid potential confounding factors when interpreting differences between the two systems; for instance, the search coil may influence saccadic metrics  and oculWe recorded eye position monocularly for each subject . Horizontal and vertical eye movements were measured with 1.83 m field coils  that used a rotating magnetic field in the horizontal plane and an alternating magnetic field in the vertical plane. We put a head coil on to measure head rotations. Thus, the eye-in-head rotations are the difference between the eye (gaze) coil signal and the head coil signals. Search coils were calibrated before each experimental session using a protractor device .The raw analog output of EyeLink was sampled and recorded using the search coil system at 500 Hz. This allowed perfect synchronization of the search coil and video tracker data. The video tracker and search coil data received the same processing (post and online); thus differences between the data sets are not due to differences in processing methods.To avoid aliasing, the analog search coil signals  were passed through Krohn—Hite Butterworth filters (bandwidth 0–150 Hz) before digitization at 500 Hz with 16-bit resolution. The digitized search coil and EyeLink signals were then filtered with an 80-point software filter .The raw signals from both sets of recorded data were calibrated offline using the data from the five-point calibration procedure built into EyeLink 1000. To do this, we performed robust linear fits (using Matlab’s built in robustfit function) for each data set. Thus, each system was calibrated the same way and any differences between the data sets are not due to differences in calibration.A word of caution is necessary concerning analog recordings with EyeLink: analog data transfer may substantially degrade data quality compared to digital transfer via the link built into the EyeLink . To ensuSome saccades are followed by a fast small saccadic, oppositely directed, eye movement called overshoot, which is often more prominent for the eye that moves in the abducting direction . Unlike We displayed the horizontal and vertical eye positions . A cross hair, targeting the eye position, was displayed on each axis and could be moved with high precision control  with the mouse, allowing the experienced investigator to select and click (with the mouse) on the start sample and subsequently select and click on the stop sample. If a microsaccade was followed by an overshoot, the duration of the microsaccade was later refined to include the overshoot. Manual detection precluded the potential confound that any observed differences between data sets could be due to differences in the performance of an objective algorithm. For instance, the presence of many false alarms/negatives in one dataset ..Fig 3). We also detected saccades with a modified version of the algorithm developed by Engbert and Kliegl –52 with The most popular contemporary microsaccade-detecting algorithm used here, and developed by Engbert and Kliegl , relies We defined microsaccade magnitude as the magnitude of the 2-D vector resulting from the largest possible horizontal and vertical vectors made up from the samples comprising the microsaccade. Overshoot magnitude was defined similarly, as the magnitude of the 2-D vector resulting from the largest possible horizontal and vertical vectors made up from the samples comprising the overshoot. We defined microsaccade displacement as the magnitude of the vector from the beginning sample to the end sample of the microsaccade . We noteAll microsaccade and overshoot properties were calculated from the raw data using custom Matlab code. The microsaccade main sequence slopes were calculated by fitting a line to the peak-velocity/magnitude relationship by total least-squares (i.e. errors were assumed to be in the independent and dependent variables).We considered a video tracker microsaccade and a search coil microsaccade to be one and the same if they had any time overlap in both recording systems Figs –8. We coDrift periods were defined as the eye-position epochs between microsaccades, overshoots, and blinks. We removed 10 ms from the start and end of each drift period (because of imperfect detection of blinks and microsaccades), and filtered the remaining eye-position data with a low-pass Butterworth filter of order 13 and a cut-off frequency of 30 Hz . We remoρ) between all the eye position data samples  of eye position during ocular drift. To compare horizontal ocular drift between the two systems, we calculated Spearman's correlation coefficient . An experienced investigator (author M.B.M.) manually identified blinks, microsaccades, and overshoots from each recording system's data. Drift was defined as the eye movements occurring between microsaccades and blinks . Upon inspecting the magnitude distributions of common and mutually exclusive microsaccades, we found that the discrepancy was largely constrained to microsaccades smaller than 0.5 deg ; 89% of -1; video tracker: 102 ± 5 s-1; t(3) = 4.12, p = 0.025). These results indicate that video tracker and search coil measurements of microsaccade properties differ in some ways.To determine whether the properties of those microsaccades detected with both recording systems might differ, we measured each property using the same method for each dataset. We detected an average of 110 microsaccades per subject. We found that the peak/mean velocity of microsaccades detected with the video tracker was significantly larger than in their search coil counterparts . Microsaρ = 0.58 ± 0.09; average horizontal ρ = 0.51 ± .09) but highly significant . We also found that 2D video tracker drift speeds were significantly higher than those measured with the search coil  = 7.01, p = 0.02). Whereas the ocular drift speed was higher for the video tracker data than for the search coil data, neither the 2D displacement  = 1.74, p = 0.22) nor the 2D distance traversed by the eye during the drift  = 1.48, p = 0.27) differed significantly between the two systems.We compared the horizontal and vertical ocular drift  and a search coil system. Our results indicate substantial agreement between the microsaccades and drift measured with each recording system. On average, 95% of microsaccades detected with the search coil were also detected with the video tracker, and 95% of microsaccades detected with the video tracker were also detected with the search coil. Microsaccade and drift properties measured with each system differed in some ways, however. Video tracker microsaccade magnitudes, peak/mean velocities, overshoot sizes, and main sequence slopes were significantly higher than their search coil counterparts. Horizontal and vertical ocular drift were significantly correlated between the two systems, but drift speeds were significantly larger for video tracker than for search coil data. We discuss possible explanations for our combined findings next.The small disagreement in microsaccade detection across the two systems could be due to noise and/or classification errors by the experienced investigator tagging the microsaccades (author M.B.M.). The larger peak/mean velocities of microsaccades detected with the video tracker are consistent with previous studies showing larger peak/mean velocities of saccades  with video-oculography than with search coil methods ,31,32,34Some prior research has argued that video-oculography systems may produce less reliable measurements due to their low sampling rates . This exPrevious studies have also proposed that the contact lens used with the search coil technique applies an artificial load that slows down eye motion, or that contact lens slippage results in a low-pass filtered version of the true eye velocity . The resAn alternative explanation put forward by , and theOcular drift was moderately, but significantly, correlated between the two systems, but drift speeds were higher for video tracker than for search coil data. Increased drift speeds as measured with the video tracker are probably due to increased noise compared to the search coil method . Variations in pupil size may also affect drift speeds measured with a video tracker ,61, but A recent video tracking study found that drift speeds increased with time-on-task in human subjects . Drift sIt is worth noting as well that, whereas true drift speeds may be lower than those measured with the video tracker, it could also be that true drift speeds are higher than those measured with the search coil technique.Noise complicates microsaccade detection and lowers the accuracy of microsaccade and drift properties measurements. Here, recording conditions were not ideal for video tracking . Furthermore, the RMS was higher for the EyeLink data than the search coil data, suggesting higher levels of noise . Even so, there was substantial agreement between the two systems. Measurements in more ideal conditions might result in even closer agreement between EyeLink and search coil measurements. It must be noted that automated saccade detection algorithms may require different parameters for optimal saccadic detection under different levels of noise (i.e. as in ). Such oWe found substantial agreement between FEM measurements with EyeLink 1000 and the search coil technique. Whereas some of the measured microsaccade and drift properties did differ between the two systems, this should not matter for most studies using only one system, however .Microsaccade detecting ability is critical to FEM studies , and we show here that microsaccade detection is comparable for the EyeLink and the search coil system. Further, drift measurements with EyeLink were significantly correlated with those from the search coil. Thus, contemporary video tracking technology now approaches the search coil for measuring FEMs, while interfering less with natural vision and being less invasive.These results are consistent with recent research showing comparable detection of saccades and fixation disparity with EyeLink 1000 and a DPI tracker  . This cut-off value originated in studies finding that the distribution of saccadic sizes during fixation declined sharply around 12 arc min (, but see


Beauveria bassiana lipase with potential for biodiesel production. The His-tagged B. bassiana lipase A (BbLA) was produced in Komagataella pastoris in buffered methanol medium (BMM) induced with 1% methanol at 30°C. Purified BbLA was activated with 0.05% Triton X-100 and presented optimum activity at pH 6.0 and 50°C. N-glycosylation of the recombinant BbLA accounts for 31.5% of its molecular weight. Circular dichroism and molecular modeling confirmed a structure composed of α-helix and β-sheet, similar to α/β hydrolases. Immobilized BbLA was able to promote transesterification reactions in fish oil, demonstrating potential for biodiesel production. BbLA was successfully produced in K. pastoris and shows potential use for biodiesel production by the ethanolysis reaction.Lipases (EC 3.1.1.3) comprise a biotechnologically important group of enzymes because they are able to catalyze both hydrolysis and synthesis reactions, depending on the amount of water in the system. One of the most interesting applications of lipase is in the biofuel industry for biodiesel production by oil and ethanol (or methanol) transesterification. Entomopathogenic fungi, which are potential source of lipases, are still poorly explored in biotechnological processes. The present work reports the heterologous expression and biochemical characterization of a novel  Microbial lipases (EC 3.1.1.3) constitute a biotechnological important enzyme group because of their versatile properties and the relative ease of large-scale production compared to animal and vegetable homologues . LipasesResearch on lipase applications is currently focused on the area of biofuels, since production of biodiesel is a promising substitute for diesel oil. Biodiesel can be produced from renewable resources such as vegetable oils, animal oil, and residual oil from industrial and domestic kitchens. In order to this application, lipases in aqueous-restricted environments and the presence of ethanol or methanol results in a transesterification reaction called alcoholysis, giving rise to fatty acid esters .The use of lipases in biodiesel production is advantageous as compared to alkaline chemical catalysis, because the enzymatic route does not result in saponification reactions and the glycerin and biodiesel recovery is simpler with higher purity. In addition, the catalyst recovery is easy when the lipase is immobilized on inert supports, the energy cost is lower since lipases generally operate under mild temperature conditions, and as a consequence the environmental impact is significantly reduced . HoweverMetarhizium anisopliae  methodology. For this purpose, 1.0 mL of Trizol was used for each 50–100 mg of mycelium. Tubes were homogenized until mycelium was completely dissolved and then, incubated for 5 min at room temperature. Samples were quickly homogenized after the addition of 200 μL of chloroform per 1 mL of Trizol used. Subsequently, samples were incubated at room temperature for 10 min and centrifuged at 12,000 g, 4°C for 15 min. The aqueous phase was transferred to another clean tube. A volume of 500 μL of isopropanol was added to the samples. After 3 h at room temperature, samples were centrifuged again at 12,000 g, 15 min, 4°C and the pellet was washed with 75% ethanol. Samples were homogenized in vortex and centrifuged at 7,000 g, 5 min, 4°C. The supernatant was discarded. After drying for 30 min at room temperature, the pellets were resuspended in 100 mL DEPC-water and incubated for 10 min at 55°C. Total RNA was quantified in NanoDrop spectrophotometer (Thermo Scientific) and analyzed in 1.5% agarose gel, in 1x TAE buffer at 80 V for 2 h. After checking the quality of the samples in the gel, mRNA was used to synthesize the complementary DNA (cDNA) using OligodT20 and Invitrogen SuperScript® III Reverse Transcriptase Kit (Life TechnologiesTM) according to the manufacturer’s specifications.a medium  supplemeBeauveria bassiana cDNA was used as template for PCR amplification of the gene encoding B. bassiana Lipase A (BblA) using primers – BblAF, (5′-NNNNNNCTCGAGAAAAGAGAGGCTGAAGCTCTTTCTGCGGACATTGACGG-3) and BblAR, (5’-NNNNNNGAATTCAACATCCATACTCTCAGCAAGAAG CTT-3). The underlined sequences correspond to the XhoI and EcoRI sites, respectively. The full-length BblA coding sequence was PCR amplified by heating at 95°C for 10 min, followed by 35 cycles of 95°C for 15 s, 52°C for 40 s, 72°C for 3 min and final incubation at 72°C for 7 min. The PCR product was purified and digested with XhoI and EcoRI and cloned into the expression plasmid pPIC9K  to produce the recombinant protein fused to the α-factor secretion signal and to the 6His extension at the N- and C-terminus, respectively (BblA) was transformed into electrocompetent DH5α Escherichia coli, and isolated plasmid DNA was purified and the nucleotide sequence determined. The BbLA nucleotide sequence was analyzed using the available tools at http://www.expasy.org/.BblA  was linearized with MssI (PmeI) (Thermo Scientific) and used to transform competent cells of the strain K. pastoris GS115 by electroporation at 1720 V, 5.5 ms (Ʈ), using an electroporator Eporator . K. pastoris GS115/pPIC9CTBbla recombinants were grown on solid Minimal Dextrose (MD) plate medium for 3 days. Ninety-six colonies were transferred to Minimal Methanol Tributyrin plate medium  and incubated for approximately 3 days at 30°C until halos of tributyrin hydrolysis appeared.The pPIC9CT--5% biotin; 100 mM phosphate buffer, pH 6.0; 1% glycerol), and cultured in an orbital shaker (200 rpm) at 30°C. After 16 h, the cells were collected and resuspended in 100 mL buffered methanol-complex medium  in an Erlenmeyer flask of 1 L, with addition of 1% methanol every 24 h. Culture was incubated for up to 144 h at 30°C and 200 rpm, the culture medium was collected by centrifugation and the production of the recombinant protein was analyzed by SDS–PAGE  BbLA in 10 mM phosphate buffer was purified by hydrophobic chromatography on Octyl-sepharose resin. BbLA was eluted with increased Triton X-100 concentrations up to 1%. In both protocols, fractions containing enzyme were dialyzed in 10 mM phosphate buffer. Protein purity was checked using 12% SDS–PAGE, and protein concentration was assayed according to Culture supernatants of BbLA-G2 transformant was concentrated and buffer exchanged using Hollow Fiber Cartrige, 50000 MMWC, in QuixStand Benchtop  concentration system. Concentrated BbLA was purified by two protocols: (I) BbLA, added 500 mM NaCl, was purified by IMAC-CupNPB solution (2.0 mM p-nitrophenyl butyrate (pNPB) in 0.05% Triton X-100 and 25 mM sodium phosphate at pH 7.0) at 40°C. The increase in absorbance at 380 nm (ε = 4.430 M-1cm-1) resulting from pNP release was used to calculate enzymatic activity, given as μmol of pNPB hydrolyzed per minute per mL of enzyme (U/mL) under the described conditions. Kinetic parameters were determined for pNPB hydrolysis, in the best conditions for BbLA activity. Software GraphPad Prism 6.0 was used to calculate the Vmax and kM values.The catalytic activity assay, modified from 2 Rotational Central Composite Design. The pH (and encoded) values tested were 4.59 (-1.41), 5.0 (-1), 6.0 (0), 7.0 (+1) and 7.41 (+1.41), and temperature (and encoded) values were 35.9°C (-1.41), 40°C (-1), 50°C (0), 60°C (+1) and 64.1 °C (+1.41). Enzyme activity was evaluated discontinuously, where 50 μL of the reaction was added to 50 μL of supersaturated sodium tetraborate, after 1 and 2 min reaction. pNPB hydrolysis was measured at 410 nm (ε = 3.420 M-1cm-1) in a SpectraMax 96-well plate Spectrophotometer (Molecular Devices). Three central point repetitions were added to verify the analysis reproducibility. A confidence level of 95% was considered in the analysis of the variables effect. A polynomial equation was constructed for response surface analysis, and the Student’s t-test was used to verify the statistical significance of the regression coefficients. Statistical software Statistica v.12.0 was used to analyze the experimental data.The evaluation of the effect of surfactants on BbLA activity was made with 0.05% Triton X-100 was replaced by 0.05% Tween 20, Tween 80, Tergitol 40, CTAB or SDS. A control without surfactant was performed. Temperature and pH effects were evaluated by a 2f , with modifications to the manufacturer’s protocol. The deglycosylation reaction comprised 45 μL purified BbLA (80 ug protein) added to 5 μL 10X denaturation buffer, mixed and heated at 100°C for 10 min. Thereafter, 10 μL G5 reaction buffer, 0.5 μL of Endo Hf and 39.5 μL of MilliQ H20 were added. The reaction was incubated at 37°C for 24 h. Deglycosylation was evaluated using 12% SDS–PAGE.Deglycosylation of purified recombinant BbLA was performed by Endo HCircular dichroism spectra between 190 and 250 nm (far-UV CD) were measured using a JASCO 810 spectropolarimeter  at a protein concentration of 0.04 mg/mL in 0.1 cm path-length quartz cuvettes in 10 mM phosphate buffer pH 7.0. The mean of four accumulated spectra was measured, from which the spectrum of a buffer blank was subtracted.1. Homology modeling method (2. Based on the X-ray structure of EstA (PDB ID: 1UKC), which was selected as template because an X-ray structure of the more homologue lipase from B. bassiana was not available. The primary sequence of lipases (BbLA and EstA) were aligned with highest scoring lipase (B. bassiana ARSEF 2860) obtained by BLAST using MUSCLE 3.6  or Sepabeads-C18 (derivative named as BbLA-C18). The immobilization mixture was maintained at pH 7.0, overnight, at 25°C. The activity of the immobilized enzyme was measured by the Ethyl ester synthesis by BbLA was performed as described by 3COOH (80:20:0.1 v:v), pH 3. The UV detection was performed at 215 nm. Product yields were calculated from the pure peak areas of known quantities of EPA ethyl ester [retention time (RT) of 9 min] and docosahexaenoic acid (DHA) ethyl ester (RT of 12 min).Reactants and products were analyzed using a reverse-phase column  by RP-HPLC (Spectra Physic SP 100 coupled with a UV detector Spectra Physic SP 8450). Products were eluted at a flow rate of 1.5 mL/min with acetonitrile/water/CHAll data are the mean of at least three independent experiments.B. bassiana genome. The gene, named BblA (Beauveria bassiana lipase A) is 1,641 bp long and encodes a protein with 546 amino acid residues, including a 19-amino acid signal peptide. BbLA has a theoretical molecular weight and pI of 59.58 kDa and 5.22, respectively. The BblA cDNA without the signal peptide sequence was obtained by RT-PCR from B. bassiana cDNA with primers BblAF and BblAR. BblA was cloned into the expression vector pPIC9k_CT in-frame with the yeast α-factor secretion signal. The resulting plasmid was sequenced and named pPIC9CT-BblA. BblA is translated into a 554 amino acids protein (including a His tag at the C-terminus), which has the catalytic triad and the common consensus sequence (G-X-S-X-G) for lipases .A putative triacylglycerol lipase gene (GenBank access number EJP62207.1) was chosen from several genes described as lipase or lipase-like in the BblA was used to transform K. pastoris GS115. Ninety-six transformants were screened for the presence of hydrolysis halos in MM solid medium containing methanol and tributyrin . Three colonies that showed a higher D/d ratio (D/d) were selected. These clones were called BbLA F5, BbLA G2 and BbLA H1.Linearized pPIC9CT-Figure 3A). Transformant BbLA G2 was chosen for further studies because it showed the highest ratio D/d . BbLA showed better lipase expression in BMM medium after 120 h of methanol induction, demonstrating that buffering the culture medium and long induction times favor protein expression .After 120 h induction with 1% methanol in liquid medium, the culture supernatants from the three clones showed similar lipase activity . IMAC-Cu2+ showed a slightly higher purification factor, while the Octyl-sepharose chromatography showed higher recovery (Table 1). Therefore, the sample obtained from Octyl-sepharose purification was used for enzymatic characterization assays.Beauveria bassiana lipase A was activated by Triton X-100, Tween 20, Tween 80, Tergitol and CTAB, but was completely inactivated by SDS. The best condition for BbLA activation was with 0.05% Triton X-100 . A reduced second order model for BbLA activity is described by equation 1.A CCRD was used to analyze the optimal temperature and pH for BbLA reaction. For BbLA activity, the two variables showed a linear effect and temperature showed a quadratic effect . Therefore, this model was considered predictive and statistically significant at 95% confidence level. This result was analyzed by response surface methodology, and generated a 3D surface graph . These results led to the estimation of ideal temperature and pH reaction conditions for BbLA, which were 50°C and pH 6.0, corresponding to the central points assayed.M, Vmax, kcat and Kcat/KM of BbLA recombinant against pNPB, pH 6.0 at 50°C, were 0.5546 μM, 85.67 μmols.min-1.mg-1, 7.139 × 104.s-1, and 1.29 × 1011.M-1.s-1, respectively (Table 3). Hill coefficient was higher than 1.0, which suggested that BbLA can be an allosteric enzyme. Allosteric lipase modulation is not common, but it is described as possible and biologically relevant . Assuming that these forms represent the glycosylated and deglycosylated forms, respectively, then the saccharide content of the glycosylated BbLA is approximately 30%.Figure 7A). Nevertheless, the spectral feature generated is not characteristic of proteins exclusively formed by α-helices.The far-UV circular dichroism spectrum of purified BbLA at pH 7.0 presents a maximum at 194 nm and a minimum at 207 and 220 nm, which are spectral features typical of proteins rich in α-helix  , 153 in α-helices, 14 in 3-10 helix and 326 in other secondary structure motifs such as beta hairpins, beta bulges, beta turns, and gamma turns. The model of the enzyme is a monomer presenting a α/β fold comprised of four central stranded β sheet flanked by 22 α-helices. The superposition between BbLA and EstA structures  generates an RMSD score of 0.37. The catalytic triad is formed by Ser-Glu-His . Q6ED33) , shares Beauveria bassiana lipase A was immobilized on Duolite and C18-Sep by reversible anionic and hydrophobic interaction, respectively. Derivatives generated from this immobilization were named BbLA-Duolite and BbLA-C18, and had 8.65 and 12.35 U/g of support, respectively. Duolite and C18-Sep immobilized 83 and 98% of the BbLA present in the crude extract. BbLA-Duolite and BbLA-C18 were dried and added to a reaction medium free of water, in order to favor the transesterification reaction.Table 4).Eicosapentaenoic and DHA acids are abundant in sardine oil, and fatty acid ester formation after 24 h reaction revealed BbLA-C18 produced 7.35-fold more EPA-ester and 13.61-fold more DHA-ester than produced by BbLA-Duolite after 48 h reaction. However, BbLA-Duolite was more selective than BbLA-C18  for lipases. This sequence was the only consensus sequence identified in studies on non-redundant sequences of lipases deposited in the public data banks. The serine residue present in this pentapeptide is the nucleophilic amino acid present in the catalytic triad  pH 7.0, with 1.2% methanol added every 24 h, and 144 h induction  and hydrophobic interaction chromatography (Octyl-sepharose). The BbLA recovery after IMAC purification (39.13%) was superior to that observed for GCLI (13%) and GCLII (14%) for IMAC purification of Geotrichum candidum lipase expressed in Saccharomyces cerevisiae (Bacillus licheniformis lipase (64%) also fused to a His-tag and expressed in Escherichia coli  for CALB (3.0), TLL (29.0) and RML (13.0), immobilized on C18-Sep. Catalyst selectivity was influenced by the nature of the support used for immobilization, where TLL-C18 was more specific than TLL-Duolite and the selectivity were 29 and 12, respectively . It is nBiodiesel production by BbLA-C18 may be improved through the study of several factors, such as increasing the amount of enzyme on the support, changing the organic solvent used, altering the temperature and the efficiency of transesterification on other oils. The present results demonstrate that the search for new enzyme sources together with the modulation of the activity by enzyme immobilization are valuable strategies for the development of biotechnological routes for biofuels production.B. bassiana Lipase A was expressed as a heterologous protein in K. pastoris, and the biochemical characteristics of the BbLA show potential for biodiesel production. The differential immobilization, by anionic and hydrophobic interaction, can generate products with different proportions of EPA and DHA esters, at mild temperatures, reducing the energy costs for biodiesel production.A This work is part of the doctorate thesis of ACV and she is the main author. AC, RF-M, RW, and FT had collaboration in part of the experiments with molecular biology and fungal expression (enzyme assays optimum temperatures and heterologous protein expression). RW performed the circular dichroism. CC performed the molecular modeling BbLA and its analysis. BP, GF-L, FF and MGP had collaboration in the immobilization and application of enzyme. JJ designed some experiments; MLTMP contributed with the experimental design and the final manuscript.http://journal.frontiersin.org/article/10.3389/fmicb.2015.01083The Supplementary Material for this article can be found online at: Click here for additional data file.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.


Molecular dissection of hepatocellular adenomas has brought forward a diversity of well-defined entities. Their distinction is important for routine practice, since prognosis is tightly related to the individual subgroup. Very recent activity has generated new details on the molecular background of hepatocellular adenoma, which this article aims to integrate into the current concepts of taxonomy. Hepatocellular adenomas (HCA) are rare, benign liver tumors in young- and middle-aged people, frequently occurring in women with a long lasting history of oral contraceptives . ObesityClinically, HCAs may indolently present as a palpable tumor or they may ultimately lead to hemorrhage and shock . MalignaTherapeutic management of HCA is not yet standardized and stands outside the scope of this article. In a nutshell, however, first-line therapy of small lesions in females comprises withdrawal of medical products containing estrogen. For males, in general, and for females with tumors exceeding a cross-section diameter of 5 cm, surgical resection is recommended .During the past decade, Zucman-Rossi’s group has been achieving tremendous progress in characterizing HCA on a molecular basis. They defined four subcategories of HCA substituting three groups with genetically-defined tumorigenesis. Mutations inactivating hepatocytic nuclear factor 1 α (HNF1α) are responsible for 35% of HCA (H-HCA) ,11,12, mHNF1α gene ,6314,63]As a matter of importance, the gatekeeper TERT is sought-after as a therapeutic target. However, anti-TERT vaccines failed to induce a T-cell response in patients with advanced HCC . Future CTNNB1-mutations deserve special attention pointing to tumors with high-risk of malignant progression. In this connection, the role of TERT promoter mutations triggering malignancy in HCA is unique. Future research will reveal whether modern translational studies will have an impact on clinical care and the evolution of new pharmaceutics.Since the past decade, a great deal of molecular work has been shining a light on benign hepatocellular tumors and has let on about an unexpected genetic diversity. Thereby, a number of genetic events have been described , among w


Using tumor xenograft experiments, we also demonstrated that lincRNA-p21 inhibited HepG2 cell growth in vivo and lincRNA-p21 contributed to sorafenib-induced growth regression of HepG2 cell in vivo. Further mechanism analysis revealed that lincRNA-p21 promoted ER stress both in vitro and in vivo, which facilitated apoptosis of hepatocellular carcinoma cells. Finally, we demonstrated that ER stress accounted for lincRNA-p21 effects on apoptosis, proliferation and in vivo growth of hepatocellular carcinoma. These findings implicate that lincRNA-p21 is a potential prognostic factor and therapeutic target for human hepatocellular carcinoma.LincRNA-p21 is a downstream long non-coding RNA (lncRNA) transcript of p53. LincRNA-p21 serves as a repressor in p53-dependent transcriptional responses and participates in diverse biological processes, including apoptosis, cell cycle, metabolism and pluripotency. However, the role of lincRNA-p21 in human hepatocellular carcinoma remains to be defined. Here in this work, we demonstrated that lincRNA-p21 acted as a tumor suppressive lncRNA in human hepatocellular carcinoma. We firstly found the downregulation of lincRNA-p21 level in human hepatocellular carcinoma tissues, and showed that low expression of lincRNA-p21 was associated with high disease stage and predicted poor survival. Further we showed that lincRNA-p21 knockdown promoted proliferation and colony formation of HepG2, Huh7 and Bel-7042 cells  Hepatocellular carcinoma (HCC) causes more than 500,000 deaths each year worldwide . HoweverEndoplasmic reticulum (ER) stress has emerged as a major site of cellular homeostasis regulation, particularly in the unfolded protein response, which plays a major role in cancer and many other diseases . Severalvia several mechanisms ranging from repressing genes in the p53 transcriptional network to regulating mRNA translation and protein stability [et al. [et al. [Long non-coding RNAs (lncRNAs) have recently been found to be pervasively transcribed in the human genome. Aberrant expression of several lncRNAs was found to be involved in the carcinogenesis of human HCC , 14. Thetability . The phy [et al.  reported [et al.  showed t [et al. , 19. Herp = 0.0166) and stage (p = 0.0023), and vascular invasion , we firstly tested the expression profile of this lincRNA in normal liver tissues and tissues from HCC. The results showed that lincRNA-p21 level was significantly reduced in tissues from HCC than that from normal liver tissues Figure . We alsoin vitro. We knocked down lincRNA-p21 by lentivirus-mediated shRNAs in liver cancer cell lines  patients with full case history between May 2001 and October 2007 were enrolled in the present study. Fresh HCC or adjacent normal liver tissues were obtained and stored at −80°C before use. The patients were recruited at Eastern Hepatobiliary Surgery Hospital, Second Military Medical University . All HCC samples were histopathologically re-evaluated independently by two pathologists before further analysis. Healthy liver samples were obtained from donor livers used for transplantation that were pathologically evaluated before transplantation. Further patient information is included in Table Total RNA was extracted from cells or fresh tissues with TRIzol (Invitrogen # 10296010). cDNA was synthesized from two μg of total RNA with One-Step RT-PCR Kit (TaKaRa # RR086A). Then, q-PCR was performed with the SYBR Green (TaKaRa # DRR420A) on an ABI-7500 RT-PCR system (Applied Biosystems). The primers were listed in The liver cancer cell lines HepG2, Huh7, Hep3B, LM9, Bel-7402 and SMMC-7721 were cultured as described previously . Normal Sh-lincRNA-p21 and control shRNA (sh-Ctrl) lentivirus particles were purchased from GenePharma. The shRNA sequences targeting lincRNA-p21 is shown in Cell proliferation ability of the liver cancer cells was monitored by CCK-8 Cell Proliferation/Viability Assay Kit (Sigma # 96992) in according to the guidelines.For liver cancer cell colony formation assay, liver cancer cells were suspended in 1.5 ml complete medium supplemented with 0.45% low melting point agarose (Invitrogen # 16520-050). The cells were placed in 35 mm tissue culture plates containing 1.5 ml complete medium and agarose (0.75%) on the bottom layer. The cells were cultured for 14 days. Cell colonies were stained with 0.005% crystal violet and analyzed using a microscope. The colony number in each well was calculated.6) in 100 μl of a 1:1 mixture of culture medium and growth factor–reduced Matrigel were implanted subcutaneously into the forelegs of 4- to 5-week-old male BALB/c athymic nu/nu mice . When the tumors reached approximately 7–10 mm in diameter, they were prepared to form a brei and then injected subcutaneously into nude mice. Tumor growth was monitored by tumor weight at the end of study (Four weeks for in vivo tumor growth). The study was approved by the Animal Research Ethics Committee of Second Military Medical University. The methods were carried out in accordance with the approved guidelines.Equal numbers of HepG2 cells stably expressing either control or lincRNA-p21 knockdown, U6 or lincRNA-p21 overexpression vectors (5 × 10STOP (Roche #04906845001). 30 μg total proteins were subjected to 12% SDS–polyacrylamide gel. After electrophoresis, the proteins were transferred to PVDF membranes, which were then blocked with 5% milk for 2 hours. The membranes were then probed with primary antibody for IRE1 (Abcam # ab37073), CHOP , GAPDH (Santa Cruz #sc32233), GRP78 (Abcam # ab32618), p-eIF2α , eIF2α , DUOX1 (Abcam #ab78919), p-PERK , PERK  at 4°C overnight, and then the membranes were washed with TBST and incubated with HRP-conjugated secondary antibodies (Santa Cruz # sc-2030) for 1.5 hours and finally washed and visualized using Chemiluminescent ECL reagent (Beyotime # P0018).Cells or fresh tissues were lysed in RIPA lysis buffer with mixture of protease inhibitors (Beyotime #ST506) and PhosApoptosis of liver cancer cells was evaluated with fluorescence-activated cell sorting (FACS) assay. FACS analysis was conducted with an Annexin V-FITC Apoptosis Detection Kit (Abcam # ab14086) according to the manufacturer's protocol. A FACS Calibur flow cytometer was used for data analysis.Dihydroethidium  was used for detecting ROS generation in cardiomyocytes as per the manufacturer's protocol.t test. For more than two groups, one-way or two-way ANOVA analysis were applied. The correlation of lincRNA-p21 levels with patients' clinicopathological variables was analyzed by the χ2 test or Fisher's exact test. The Kaplan-Meier method was used to estimate overall and disease-free survival. Survival differences according to lincRNA-p21 expression were analyzed by the log-rank test. Linear regression analysis was performed to analyze the relation between lincRNA-p21 level and AFP serum level as well as ER stress markers in patients with HCC. P values of less than 0.05 were considered statistically significant.Values were expressed as Mean ± SEM. Statistical differences between two groups were determined using unpaired or paired Student's


To explore how the concept of lay epidemiology can enhance understandings of how drinkers make sense of current UK drinking guidelines.Qualitative study using 12 focus groups in four sites in northern England and four sites in central Scotland. Participants were 66 male and female drinkers, aged between 19 and 65 years, of different socio‐economic backgrounds. Data were analysed thematically using a conceptual framework of lay epidemiology.Current drinking guidelines were perceived as having little relevance to participants' drinking behaviours and were generally disregarded. Daily guidelines were seen as irrelevant by drinkers whose drinking patterns comprised heavy weekend drinking. The amounts given in the guidelines were seen as unrealistic for those motivated to drink for intoxication, and participants measured alcohol intake in numbers of drinks or containers rather than units. Participants reported moderating their drinking, but this was out of a desire to fulfil work and family responsibilities, rather than concerns for their own health. The current Australian and Canadian guidelines were preferred to UK guidelines, as they were seen to address many of the above problems.Drinking guidelines derived from, and framed within, solely epidemiological paradigms lack relevance for adult drinkers who monitor and moderate their alcohol intake according to their own knowledge and risk perceptions derived primarily from experience. Insights from lay epidemiology into how drinkers regulate and monitor their drinking should be used in the construction of drinking guidelines to enhance their credibility and efficacy. Low‐risk drinking guidelines are a widely used alcohol policy internationally and enjoy strong support from the alcohol industry and governments as a non‐regulatory intervention which does not directly limit individual freedoms. They are argued to have several functions, which include communicating evidence on risks of drinking to the public to inform consumption choices and providing an objective definition of risky drinking for use in medical practice Researchers have questioned the value and effectiveness of drinking guidelines One approach to addressing this deficit is for research and practice to give greater attention to lay epidemiology, which comprises knowledge and beliefs about health and causation of disease which are constructed primarily from subjective experience, observation of family and social networks and media sources Lay epidemiology fits within a social constructivist ontology which considers social phenomena—in this case illness—not as biological or natural ‘givens’ as in objectivist understandings of the social world, but as concepts that emerge from social interactions, which hold different meanings depending on the cultural and historical contexts in which they are produced and experienced While lay epidemiology has been used to understand failures to comply with public health guidance in contexts such as smoking n = 66) were conducted in England and Scotland (six in each) in February and March 2014. Independent market research consultants identified, recruited door‐to‐door and received informed consent from participants who were purposively sampled to be male and female drinkers  aged 19–65, of lower and higher socio‐economic backgrounds (measured using occupation of the household's highest earner). Each focus group comprised participants of the same gender and similar ages and socio‐economic backgrounds , which aims to assess the impact and effectiveness of alcohol control policies in each country. Twelve focus groups ranging in size from three to six participants  were also circulated to explore reactions to alternative guidelines. The drinking guidelines which were shown to the participants are displayed in Table The focus groups were audio‐recorded and transcribed verbatim. The transcripts were analysed inductively using a coding frame which combined the categories in the topic guide with those which emerged from initial reading of the transcripts. The conceptual tool of lay epidemiology also guided our analysis, in that particular attention was paid to how participants referred to their own rationales for moderating their alcohol consumption. Transcripts were coded to the emerging categories using NVivo version 10 software, and the coding framework was checked and revised following discussions between M.L. and D.E. in order to agree that the names given to codes accurately reflected the meaning in the transcripts. Once all transcripts had been coded, categories which contained similar meanings were then grouped into overarching themes In general, the drinking guidelines were disregarded by participants and there appear to be three primary interrelated factors which may help to explain this. These are: (1) a disconnect between the guidelines, which are focused on regular drinking, and the participants' tendency to drink irregularly, including occasional binge drinking; (2) a disconnect between the amounts given in the guidelines and participants' typical consumption levels; (3) difficulties measuring and monitoring units. While participants disregarded the guidelines, they referred to other rationales constructed from their own risk perceptions to monitor their drinking, demonstrating how lay epidemiology informed and addressed their perceived risks of alcohol.1Participant ID number corresponds to the focus group of which the participant was a member. See Table ). The guidelines were seen as more relevant for ‘people who drink every day probably [people agreeing] more than people who just drink at the weekend’ (E1). As most participants drank only at weekends, a weekly guideline was preferred, because: ‘then you can do it to suit yourself during the week I would say’ (S5). Some felt that as they did not drink during the week, they did not need to monitor their consumption at the weekend:I probably wouldn't consider it if I was drinking at the weekend and it was my only night of relaxing with a drink during the week, I probably wouldn't care if I was over the limit or not because it wouldn't be greatly and I'd just think that I don't do it often enough (S3).Participants interpreted the guidelines within the context of their own behaviour and justified disregarding them by referring to their own rationales for moderating their consumption. For instance, participants of all ages felt that having a guideline for daily use was unhelpful, as they did not drink on most days: ‘[b]ecause we don't drink every day you just don't take it in’ .The perception that the guidelines are targeted at frequent drinkers rather than occasional bingers may have informed the participants' preference for the Australian and Canadian guidelines, which include separate guidelines for regular drinking and for single occasion drinking. This ‘two‐guideline approach’ was seen as more flexible and of more relevance:Participants' preferences for guidance on heavy, single‐occasion drinking coheres with their positive responses to a more detailed, but less commonly displayed (e.g. not on product labels), version of the UK guidelines which advises people to leave 48 hours without drinking after a heavy drinking session. However, while participants commented that they did not drink following such occasions, this was due less to health concerns or guidance and more a reflection of practical issues, such as needing to go to work, childcare responsibilities or recovering from hangovers: ‘I wouldn't say you'd pay attention to it because they're saying it, you just pay attention because your body's telling you’ (S2).The daily UK guidelines were seen as broadly realistic for people who drank every day, but because the majority of participants did not drink regularly throughout the week but drank a great deal at weekends, a recommendation of not regularly drinking more than 2–3 units for a woman or 3–4 units for a man was seen as unrealistic: ‘[t]heir too much is not our too much. According to that, their too much is like ‘I've only just started’ to be honest’ (E4); ‘If I'm having a drink and I'm out, I don't want to stop at three drinks really if I'm having, if I'm at a night out, I'm not going out to get drunk but you know, three drinks is gone, especially three skinny glasses, well that's not even a bottle of wine is it when you're out?’ (S5).For many participants, one of the main motivations of drinking was to get drunk, and the guidelines were interpreted within that context. Consequently, they were disregarded as they were not seen to acknowledge this motivation. While the single‐occasion guidelines of Australia and Canada have higher ‘limits’, these were still regarded as unrealistically low: ‘like say five units, it don't seem like a lot, that just seems like a normal, like a normal drink, like I'd say five, I'd say binge drinking should be way more than five’ (E6); ‘[i]t's probably not really, like the average adult I wouldn't have thought five units would have made them particularly drunk’ (S3). While some participants acknowledged that exceeding the guidelines might lead to harm, rejection of the guidelines was rationalised by balancing risks against the perceived social benefits of drinking: ‘I think I would have to accept that if you drank more than the guidance that was given there then you could argue that there is a greater chance of harm as a consequence of drinking. But to, I think we'd have to weigh that up against the desire to be sociable and so to some there's a, you know, you're prepared to take that [risk] aren't you? (E2)’; ‘I'm aware that… binge drinking… has some detrimental potential effects, but I, if you like, waiver that in favour of going out and socializing and having a good time’ (E6).although I sort of appreciate the long term like risks of drinking a lot I'm not particularly bothered because I just think I'm 21, I'm at university so after, as soon as I finish university it will probably slow down a lot when I have a full‐time job so I don't really think about it in the long term either (E2).Other participants acknowledged the health risks while continuing to drink above the guidelines, as they felt that ‘it would never happen to them’. Rationales offered by participants to explain this view reflected lay interpretations of key epidemiological concepts such as relative and absolute risk. This included appraising their alcohol intake against others who drank more than them: ‘I'm probably a fool to myself; I never, I just palm it off as if it's never going to happen to me because I don't drink in excess. I always look further down… to people who are drinking every day kind of thing’ (E4). Attitudes towards drinking in relation to long‐term health conditions did not generally appear to be linked strongly to age or gender, although one participant from the 19–24 age group regarded himself as not at risk, as he anticipated drinking less as he got older and associated health harms with drinking heavily over a long period of time:And there's like a whole culture of buying everyone drinks so that the people who shove like one in my face and it's like yeah, yeah, yeah… no one's thinking ‘oh how many units have I had?’ (E2).[w]ell that's it as well, especially if you're at home you're not going to have a unit are you, not what a pub would serve you, so their units, [because] they do measure them, would be different to our units at home, so it would be quite different (E5).While awareness of the use of units in the drinking guidelines was high, participants reported that they thought about their own alcohol consumption in terms of bottles or drinks, rather than units: ‘you just count the drink, you don't count the units or anything’ (S3); ‘I think it would be better if it was like two to three cans or two to three whatever it was, or one bottle of whatever’ (E4). Many participants identified practical barriers to sticking to a limited number of units in both the on and off‐trade:Generally, participants regarded measuring alcohol in standard container sizes as more viable, and questioned the usefulness of units in guidelines: ‘I think when people say ‘units’ a lot of people are going to go, “oh I don't know about [those] units”, whereas if you went, “oh you can only have five cans a day” they'd be like, “oh okay, five cans”’ (E4).Despite generally rejecting units as a way of monitoring their drinking, participants gave clear examples of how they regulated their drinking in other ways, which were relevant to their motivations for drinking and broader social values. Strategies for self‐regulation included not drinking at all, drinking more slowly and limiting the number of drinks consumed. Other participants spoke of ‘knowing their own limits’ and bodies, and sometimes regulated themselves by switching to different types of drink according to how they believed they would feel the next day: ‘I could have vodkas all night and I'd wake up and I might feel a bit ropey but wine makes me feel like I'm like hit by a bus’ (S3); ‘I know when to stop drinking because I… know that if I have another one I'm going to be ill. So you know’ (E6).However, this regulation was informed more by their commitment to fulfilling valued responsibilities such as childcare and employment and was unrelated to the long‐term health risks which typically inform drinking guidelines: ‘I usually let the wife have a sleep in on a Saturday morning. So I get up early and do all the baby things so I don't want to be getting up feeling awful and with a thick head’ E6); ‘I'm a child minder so I'd be very conscious not to be really drunk obviously and yeah, if I'm going out and driving and you know, I'll just stick to one drink, if that, because I kind of think sometimes it's better to have nothing at all, don't risk it sort of thing’ (S3). Consequently ‘regulation’ did not always mean limiting the amount of alcohol consumed for long‐term health consequences, but limiting the immediate negative effects or consequences of drinking which would be felt the following day.In general, participants perceived that the guidelines lacked relevance to their drinking practices for three primary reasons. First, daily guidelines were seen as irrelevant by drinkers whose drinking patterns comprised heavy weekend drinking. Secondly, the amounts given in the guidelines were seen as unrealistic for those motivated to drink for intoxication. Thirdly, participants measured alcohol intake in numbers of drinks or containers rather than units. However, despite generally disregarding the guidelines, participants regulated their drinking in ways, and for reasons, which were meaningful to them.As a holistic approach to understanding health and illness, lay epidemiology assesses health risks in the context of a wide range of additional concerns, including the perceived benefits of risky activities, and individual and societal norms, attitudes and values. For example, participants explained their disregard for guidelines with reference to their motivations for and experiences of drinking, which often focused on pleasure and intoxication and the perceived failure of guidelines to account for this While the participants in this study did not generally adhere to the drinking guidelines, they were not impervious to the risks of alcohol consumption and monitored and moderated their drinking in ways that made sense to them. For instance, participants monitored their consumption in numbers of drinks rather than units, a finding which supports claims that units are a flawed metric for people who measure consumption in ways more meaningful to them, which include physiological and emotional experiences Viewed within a framework of lay epidemiology, which understands that people's health behaviour decisions are made within the context of their knowledge, experience and values, the participants acted rationally The disjuncture between the guidelines' concern with long‐term health issues and the participants' primary focus on social problems also reflects long‐standing debates over what sort of a problem alcohol is, whose responsibility it is to solve it, and how this should be done n = 66) from two parts of the United Kingdom, and given the similar approaches taken to drinking guidelines across high income countries To our knowledge, this is the first study in any country to examine qualitatively the interpretation and use of drinking guidelines by the general adult population. While the sample is not representative of the whole UK population, and caution should be exercised in generalizing from the findings, our research drew upon perspectives from a diverse sample of drinkers (A number of implications emerge from our findings. First, the disconnect between drinking practices and guidelines may be addressed partly by separate regular and single occasion guidelines as used in Canada and Australia but not currently in the United Kingdom Drinking guidelines derived from, and framed within, solely epidemiological paradigms lack relevance for adult drinkers who monitor and moderate their alcohol intake according to their own knowledge and risk perceptions derived primarily from experience. Insights from lay epidemiology into how drinkers regulate their drinking should be used in the construction of drinking guidelines in order for them to have enhanced credibility and efficacy.None.