We propose a new hypothesis: feelings of back stiffness are a protective perceptual construct, rather than reflecting biomechanical properties of the back. This has far-reaching implications for treatment of pain/stiffness but also for our understanding of bodily feelings. Over three experiments, we challenge the prevailing view by showing that feeling stiff does not relate to objective spinal measures of stiffness and objective back stiffness does not differ between those who report feeling stiff and those who do not. Rather, those who report feeling stiff exhibit self-protective responses: they significantly overestimate force applied to their spine, yet are better at detecting changes in this force than those who do not report feeling stiff. This perceptual error can be manipulated: providing auditory input in synchrony to forces applied to the spine modulates prediction accuracy in both groups, without altering actual stiffness, demonstrating that feeling stiff is a multisensory perceptual inference consistent with protection. Together, this presents a compelling argument against the prevailing view that feeling stiff is an isomorphic marker of the biomechanical characteristics of the back.Does  We assume that these bodily feelings reflect the biological state of our body tissues – a ‘read-out’, so to speak, of somatosensory and visceral input – particularly when the feeling is located somewhere in the body, as it is for pain or stiffness. There is growing evidence for pain however, that it is highly modulated by a wide range of cognitive and contextual variables5. For example, visually manipulating the perceived size of one’s hand alters the pain experienced in experimental contexts6 and during movement of a chronically painful limb7, and illuminating a blue or red light in synchrony with delivering a noxious cold stimulus can transform the feeling evoked from uncomfortably cold to painfully hot5.Bodily feelings constitute a fundamental aspect of self-awareness and provide critical homeostatic functions – e.g., feeling cold makes one seek warmth9. Indeed, we now know that the sense of bodily ownership, the felt location, and the anthropometric characteristics of our body parts, are tightly linked to their physiological regulation in a bi-directional manner10. Extensive human data from both healthy and diseased populations have led to the proposal of the cortical body matrix theory11, the predictions of which are yielding new developments in our understanding and treatment of some pathological pain conditions12.That somatic input triggered by physiological responses is important in cognitive processes – so-called ‘embodied cognition’ – has recently been countered with the idea that bodily awareness  might directly modulate physiological regulation of body tissue in an anatomically specific way14, but which remains relatively ignored, is feeling stiff. Feeling joint stiffness is unanimously attributed to actually having stiff joints. Take for example having a ‘stiff back’. We assume that this feeling reflects altered force-response profiles of the tissues in our back . But does it? A feeling of joint stiffness has been shown to remain despite loss of the limb15 suggesting that the bodily feeling of stiffness can be independent to the biological state of the tissues.One feeling that is almost universally experienced, which impacts on our daily lives16 and is a primary target of interventions for many musculoskeletal conditions including back pain, arthritis, and neck pain. Worldwide, the most burdensome of these conditions is low back pain (LBP). Affecting ~10% of the world’s population (~632 million people), chronic LBP is also the leading cause of disability17. Despite increasing healthcare expenditure, clinical outcomes remain sub-optimal18, spurring a call to re-examine basic mechanisms associated with LBP and its symptoms19, most obviously stiffness.Feeling stiff is a significant predictor of disabilityHere we describe a series of experiments, the results of which compel us to rethink what it means to feel stiff, what determines how stiff we feel, and what ecological value feeling stiff might have. First, we aimed to establish whether feeling back stiffness is related to (i) actual stiffness of the back as reflected in force-response profiles, and (ii) a manifestation of actual stiffness that would offer a protective behavioural advantage – the ability to judge the magnitude (and change) of applied force to the back. Specifically, Study 1a interrogated the prevailing view that feelings of stiffness reflect objective biomechanical back stiffness. We hypothesised that if this theory is correct, subjective and objective back stiffness measures would closely relate, and people who feel back stiffness would objectively have a stiffer spine than those who do not. Study 1b evaluated whether people with feelings of back stiffness show protective perceptual responses – do they differ from those without feelings of stiffness at estimating how much force was applied to the spine and at detecting change in this force? We hypothesised that they would overestimate force, but be more sensitive to changes in force, suggestive of a protective profile.Second, we hypothesised that feeling stiff should be modulated by sensory cues that are unrelated to actual back stiffness. To investigate this, we exploited the brain’s predilection for cross-modal integration by synchronising mechanical loading of the back with two different auditory signals – the sound of a ‘creaky door’ and a gentle ‘whooshing’ sound. Given the common pairing of sounds  with movement of a stiff back, we anticipated that pairing sound with force applied to the spine would provide ecologically valid information that may alter protective perceptual responses. Specifically, Study 2 evaluated the empirical support for our new theory that feeling stiffness in the back represents a perceptual construct, which could be modified by additional body-relevant sensory information, such as sound. We hypothesised that sound would modify back perception and that this modification would be enhanced in those that report feeling back stiffness.20. For example, people underestimate the distance from their body to a pain-relieving switch, but only when they are able to reach for it20. The following series of experiments comprehensively interrogate both the prevailing view that feeling back stiffness equates to having objective back stiffness (Study 1a) and our new hypothesis of stiffness as a protective perceptual inference (Study 1b and Study 2).Overall, we postulate that feeling back stiffness represents a protective, perceptual inference: a bodily feeling created to provide the critical homeostatic function of preventing movement and potential re-injury. We do so based on the bi-directional, intimate relationship between perception and movement: when we move, we constantly receive sensory feedback, as a result of the movement, that informs our perception of body position, but we also perceive things in line with how we will actfeelings of back stiffness and 15 age- and gender-matched healthy controls without LBP or feelings of back stiffness. Table We recruited 15 people with chronic LBP who also reported having chronic 21, that provides a standardised force to the spine, measuring displacement of the application probe . We established how stiff participants’ backs ‘felt’  and how stiff their backs objectively were (customised device).Study 1a used an established, customised device, validated in humansfeelings of stiffness correspond to how stiff the back actually is, we used Spearman’s rho. We found no significant relationships between perceived and actual stiffness at any applied force level  or any stiffness measure (AvgStiff or TermStiff) in people with LBP and stiffness . Univariate test results from this analysis (stiffness measure specific) are presented in Table 2,27 = 2.45, p = 0.11, partial η2 = 0.153) and at 70 N  suggesting that this lack of objective stiffness difference holds across the applied force spectrum . We considered objective stiffness measures at the start and end of testing to evaluate for any between-group differences in biomechanical spine properties secondary to repeated tissue loading (factor: ‘time’). There were no significant differences between groups in either of the objective spinal stiffness measures at any applied force level Fig. . Specifi22 and difference detection23 tasks. Participants (n = 30) were advised that they would receive indentation forces between 50 N and 70 N to their back; these forces were applied in pairs . Participants were asked to estimate, as accurately as possible, the magnitude of force delivered (see Methods for training procedure). Estimation errors were calculated for forces applied at 60 N . We evaluated force estimation errors at 60 N at both the start and the end of testing to determine any between-group differences in perception of force following repeated tissue loading (factor: ‘time’). Given that participants with LBP might experience increased back soreness following repeated loading, which could feasibly influence the accuracy of their force estimation, this was important to consider.Study 1b used established force magnitude estimation1,28 = 0.102, p = 0.75, partial η2 = 0.004), no interaction between time and group , but a significant main effect of group . This force overestimation was consistent at the individual level , than healthy controls did . This enhanced ability to detect force differences was not because of differences in biomechanical properties of the spine: the change in objective stiffness that occurred at the discrimination threshold was no different between groups for either objective stiffness measure .As mentioned above, the forces were applied in pairs (two indentations 30 sec apart). In addition to magnitude estimation, participants were asked to identify whether the second indentation was the same or different than the first. Based on the answer, the force difference between the two indentations was adjusted . Repeating this procedure allowed us to identify the smallest difference in force that was reliably detected – the ‘force discrimination threshold’. We found that the groups differed in their ability to detect changes in force Fig. : people 54. Thus, Study 2 used established principles of multisensory integration to evaluate whether adding temporally congruent auditory information to the application of pressure to the spine would alter back perception. Participants (n = 20) estimated the amount of force received (expected forces between 50–70 N), but in all conditions, forces were applied at 60 N . We used force estimation as a surrogate for perceived stiffness to allow between-group comparison ; this was validated as a surrogate prior to this experiment (see Methods).That feelings of back stiffness were unrelated to objective biomechanical measures of back stiffness, but did relate to overestimation of force applied to the spine, supports the idea that feeling stiff may represent a protective perceptual construct. As such, providing additional sensory input that enhances or reduces the need to protect the spine may have the ability to modulate perception at the back. Given clinical observations that sound is often paired with movement , auditory input could feasibly modulate the need to protect the spine. Further, established multisensory interactions between somatosensory (‘feeling’) and auditory inputs suggests that modulation of perception is possible1,18 = 6.3, p = 0.005, partial η2 = 0.26), no effect of Group , no effect of Indent number , but a Condition x Indent number interaction . No other interactions were significant (p = 0.20–0.59). Post hoc ANOVA tests revealed that the conditions differed in FEE for Indent 1 . For Indent 1, force estimations in the creaky sound condition (1.25 ± 5.5 N) were significantly higher than in the control sound condition , where participants underestimated the force applied (−3.25 ± 4.6 N), but were no different from the no-sound condition . The control sound condition significantly differed from the no-sound condition , where participants overestimated force applied (3.40 ± 4.3 N).First, we evaluated whether the effects on perception of the back (and force applied to it) were specific to the type of sound used – if so, this would provide compelling evidence that perceptual alterations were not merely due to the use of sound alone. We compared force estimation error (FEE) between groups during no-sound, creaky sound (“creaky door”), and control sound (“whoosh”) conditions , no effect of Condition , no effect of Indent number , no main effect of Group , and no other significant interactions (p = 0.12–0.48). Post-hoc paired comparisons showed that the conditions differed in FEE for the last indentation : participants over-estimated force in the creaky condition (3.05 ± 4.8 N) relative to the creaky decrease condition, where force was under-estimated (−0.15 ± 4.9 N). No differences were found for the other indentations .Second, we evaluated whether perception would be modified in both groups in line with change in the auditory input over time by comparing the creaky sound to a ‘creaky decrease’ condition (volume decreased over 3 indentations). We hypothesised that the creaky sound condition would increase FEE while the creaky decrease condition would decrease FEE with repeated indentations, and that this effect would be heightened in those with back pain and stiffness. Indeed, we observed opposite effects on force perception: whereas the constant creaky sound condition resulted in increasingly larger overestimation of applied force over each indent, the creaky decrease condition resulted in decreased force estimations over time Fig. . HoweverImportantly, there were no differences between Conditions in objective back stiffness (AvgStiff and TermStiff) as a function of Indent number or Group for either of the above comparisons 30. In chronic LBP, treatment altered objective spinal stiffness measures and this alteration related to self-reported measures of disability31 – that is, actual back stiffness is changeable and may be important to some body-relevant perceptions. However, none of the previous studies evaluated whether the participants specifically had feelings of back stiffness, whereas we specifically recruited these participants. This makes direct comparison to our work difficult.Our results are in contrast to previous studies that found differences in objective spinal stiffness measures between those with and without acute/subacute LBP32 reduced tactile acuity at the back;34 impaired motor imagery of the back;35 and impaired trunk voluntary motor control36. Our work suggests that this dysfunction extends to the perception of force applied to the back. Changes in the perception of touch have been linked to changes of receptive fields and response profiles of primary sensory cortex (S1) neurons37 – so-called cortical reorganisation38 – raising the possibility that alterations in force perception might also represent cortical changes related to the back. That people with back pain are less accurate than healthy controls on a task requiring intact cortical proprioceptive representation supports this possibility35.Our work also contributes to the knowledge of perceptual dysfunction in chronic conditions. There is consistent evidence of perceptual abnormalities in people with LBP: alterations in perceived shape of the back;32. Superior force acuity yet inaccurate estimation of force magnitude, while seemingly incongruent, are together consistent with high attentional demands of pain39. Expecting pain can impair attentional disengagement41 which may uniquely facilitate sustained attention to the back in the LBP group where indentations occurred over the painful area. Indeed, indentation evoked pain in those with LBP and stiffness but not in healthy controls. Expectations of pain and changes in attentional focus  are relevant because acuity in somatosensory  change detection has been shown to be improved by both43 as has enhanced perception of threat44, which in other non-nociceptive paradigms increases protective reflexes45 – a finding that is largely consistent with protective force over-estimation seen here. Although the specific role of attention and expectation cannot be elucidated in our paradigm, that enhanced change detection extends to force magnitude in people with back pain is sensible and consistent with the ecological value associated with bodily protection. Given that this increased acuity in detecting differences in force was not accompanied by group differences in objective spinal stiffness suggests that it is more likely to reflect a top-down mechanism , rather than a bottom-up one .It is intriguing that people with LBP and stiffness could detect a smaller difference in force than healthy controls. It seems counter to findings of poor tactile acuity in people with LBPWhile it is possible that features of the force discrimination task, such an indentation duration and response timing, played a role in the outcome seen, the likelihood of this is low. Regarding indentation duration, the indentor advanced at a constant velocity, meaning that differences in force targets (50 N vs 100 N) took slightly different time durations to reach. However, given our small range in force targets (50N-70N) this difference was less than a maximum of 1.5 seconds, and post-test questioning revealed that all participants based their force estimation judgements solely on the feel of the force on their back. None reported using time (indentation duration) to make these judgements. Second, regarding response timing, there was a 30 second interval between receiving the first and second indentation for the forced choice paradigm (response: same or different). This timing was essential given viscoelastic properties of the spine. Given that both groups received training to become comfortable with this task suggests that the impact of response timing is likely minimal.46 – pain and stiffness often go together; adaptation to pain model47 – pain-related muscle activation changes alter trunk stiffness49 to prevent ongoing irritation of sensitive tissues; and the more recent link between motor effort and proprioception50 – painful movements are predicted to be more effortful , which in turn might make them feel stiffer. Together, these suggest that pain and stiffness likely compel similar behavioural responses that limit movement and thus re-injury. Such issues could be disentangled with further studies based on the current experimental paradigm. Indeed, further research is warranted to determine if this protective perceptual inference, e.g., a feeling of stiffness, results in protective behaviour such as movement avoidance in people with back pain and stiffness. That there was no difference between groups in force perceptual error over time suggests that perceptual differences are unlikely to be due to differences between groups in cognitive function .Our findings of a heightened protective response in people that report back pain and stiffness raise the possibility that the mechanisms that subserve pain also contribute to the perception of stiffness. Such a possibility would be predicted by several relevant theoretical frameworks, for example: associative learning52. Further, substantial communication exists between the primary auditory cortex and the primary somatosensory cortex53, and auditory-somatosensory interactions exist in the early stages of cortical processing54. Last, integration of body-relevant information also occurs in the premotor55 and posterior parietal cortex57 – the latter being an area that provides cognitive representations of our body and the space surrounding it.This work provides crucial information to suggest how conscious perceptions are created, relevant to multisensory integration in chronic painful conditions. Our results suggest that auditory information is integrated with temporally congruent pressure information to create a perceptual experience and that this effect is not merely due to sound alone, but rather reflects the nature of this auditory information. Indeed, we found that the type and context of congruent sound had specific and varying effects on the modulation of perceptual error in force magnitude estimation. It is well-established that multisensory neurons in the superior colliculus receive information from auditory, visual and somatosensory inputs58. Our work extends this finding by showing that the ability to integrate multimodal information and alter perception can also occur in a chronic pain state. Previous work has shown that touch and visual information can still be integrated in chronic pain: when a rubber hand illusion is applied people experience disownership of their painful limb in a similar manner to that observed in healthy controls59; the visual illusion of touch (using mirrored reflection of the good leg) improves tactile perception in the leg with chronic tactile deficits60. Our results extend these findings by confirming that this integration extends to audition and touch/pressure.Our discoveries support previous work that has shown that congruent auditory and tactile information can induce changes in the perceived properties of the hand: the so-called Marble Hand illusion61. Given the positive relationship between force estimation error and feelings of stiffness (the more stiff you feel – the greater the error), this raises the possibility that we may directly target feelings of stiffness with such auditory cues. Indeed, that we saw perceptual effects of sound in people with LBP and stiffness suggests that processes of multisensory integration are likely intact and thus may be a new and relevant treatment target. Finally, our work suggests that subjective and objective measures of stiffness are not proxies for one another, but likely capture different aspects of a similar domain. This raises the possibility that either self-perception or the physiological, objective aspects of back stiffness can be normal or abnormal. It follows then that exploring how various combinations of these states play out in intervention may reveal new mechanisms underlying back pain and/or approaches for its effective treatment.Our findings may have relevant implications for treatment. Improved outcomes in people with tendon pain are seen when exercises are paired with auditory cuesHere we show that a conscious experience of feeling stiff does not reflect true biomechanical back stiffness, but may rather represent a protective perceptual inference that may serve to reduce movement and re-injury. By showing that feelings of stiffness do not relate to biomechanical stiffness measures and that biomechanical measures of stiffness do not differ between those with and without feelings of stiffness, this suggests that information other than actual joint stiffness is influencing this perceptual inference. Although preliminary, our assertion that this interference is protective is substantiated by the heightened response to applied force in people with back pain and stiffness – they overestimate applied force magnitude but are more sensitive to detecting change. Last, synchronising auditory information in a way that conveys meaning to pressure applied to the back, modulates these protective responses in a specific manner dependent on the nature of the auditory information, and crucially, does so in the absence of changes to biomechanical properties of the spine. Future work is warranted to determine if this protective perceptual inference relates to protective behavioural responses .Study 1a/1b recruited 30 participants (methods/results separated for clarity) providing 80% power to detect a moderate effect (f = 0.25) for force estimation and objective stiffness . Study 2 evaluated 20 participants from Study 1, providing 80% power to detect a small-to-moderate effect (f = 0.23) of auditory stimuli on force estimation . These studies received ethical approval from the University of Alberta Human Research Ethics Board. All methods were carried out in compliance with the Declaration of Helsinki and all participants gave written informed consent. Informed consent was attained for publication of photographic and video images Fig. –S3. All Fifteen people  with chronic (>3 months) non-specific LBP and self-reported stiffness, and 15 age- and gender-matched healthy volunteers without any current LBP/stiffness  were recruited. See Supplementary Table 2 head size) in a standardised manner while participants were prone  drives probe movement, a compression-tension load cell  measures applied force, and a rotary encoder  measures probe displacement. The probe advances at 2 mm/sec, pauses at a preload of 5 N (held 1 sec), then continues until the set target force is reached (held 1 sec), and then is withdrawn from the back.A custom-designed, mechanical indentor applied rate-controlled anterior-posterior force to participants’ backs using a circular probe . Three familiarisation indentations (target force = 30 N) accustomed participants to the procedure and to a standardised breathing pattern . Following familiarisation, formal testing occurred where participants received a maximum of 20 indentations, ranging from 50–70 N.Participants completed baseline questionnaires . Two measures of spinal stiffness (N/mm) were calculated: average stiffness  and terminal stiffness After each indentation, participants rated the pain experienced on a 0–100 numerical rating scale .The relationships between perceived back stiffness and objective spinal stiffness  in the LBP group were assessed using Spearman’s rho. For the healthy control group, perceived stiffness ratings were negligible.Objective measures of spinal stiffness (AvgStiff and TermStiff) were analysed using a repeated measures Multivariate Analysis of Variance (RM MANOVA), comparing stiffness between groups for the first and last applied force at 60 N . If participants did not receive two indentations at 60 N, either two indentations at 59 N or 61 N were used ; this was matched between groups. A MANOVA was used to compare objective spinal stiffness measures at 55 N and 70 N between groups.This is what 50 N feels like”). Participants were instructed to use these as reference points for future force estimations.Prior to formal testing , the same participants were trained in estimating force magnitude. Indentations forces at 50 N, 60 N, and 70 N were provided and participants were verbally advised of the magnitude of each impending force , the magnitude of force delivered (force magnitude estimation measure).Indentations were delivered to participants’ backs in sets of two (‘force pair’), force estimations were provided for each indentation within the pair (indentations separated by 30 secs and force pairs separated by 1 min). Participants were also asked to indicate whether indentation 2’s force was the ‘same’ or different’ in magnitude to that of indentation 1 (force discrimination measure). Initial training included accustoming participants to this 30 second time interval.23 was used to increase and decrease the force difference between indentations of a pair. We started with a large force difference between indentations of a pair ; this difference decreased over subsequent indentation pairs until participants reported that they felt the forces to be of the same magnitude. Then the force difference between indentations of a pair was increased until participants reported the forces to be of different magnitudes. This process was repeated at least 3 times (maximum of 20 indentations), alternating between increasing and decreasing the force difference . The primary outcome was the FEE at 60 N .This was defined as the smallest difference in force between indentations of a pair at which participants could detect a difference in force magnitude  but could not detect a difference in force between indentations of a pair when the force difference was 1 N smaller . In this example, the force discrimination threshold is 5 N.This was expressed as the absolute change in spinal stiffness at the force discrimination threshold as a proportion of that force difference: /(force indent 2 – force indent 1). This allowed us to determine whether between-group differences in force discrimination threshold may be due to differences in objective spinal stiffness changes.To determine if FEE differed between groups or as a function of time, a 2 × 2 RM ANOVA was used . In participants not receiving two indentations at 60 N, 59 N or 61 N was used; this was matched between groups.10 transformation. For between group differences in normalised objective stiffness change, Mann Whitney U tests were used.Independent t-tests investigated whether groups differed if force discrimination thresholds. Force discrimination threshold data required log10 healthy control participants  and 10 chronic LBP and stiffness participants  were included in this study.Identical methodology was used for force application to the spine and for force estimation training. Participants were fitted with earbuds  and protection ear muffs to minimise any errant noise. Participants completed four conditions, involving 3 indentations each, in a randomised order: (1) no sound; (2) creaky sound (‘creaky door noise’); (3) control sound (‘whoosh’); and (4) creaky decrease (volume reduced by 50% over each of the three indentations). All sounds were temporally paired to both the descent and withdrawal of the probe that applied force to the back. A customised Labview program delivered auditory stimuli, such that the timing of the sounds could be set to match the indentation (sound timing information covertly collected in Study 1). The investigator monitored indentation-sound timing in real-time (using earbuds) for all indentations.Participants were instructed that they would receive forces anywhere between 50 and 70 N and asked to estimate, as accurately as possible, how much force they thought that they received. In reality, all forces applied were at 60 N .Trunk muscle activity during indentation was measured using sEMG. Established skin preparation techniques were used before Ag/AgCl dipolar disposable electrodes  were applied bilaterally to the external obliquus, the internal obliquus, and the erector spinae muscles using standardised locations (grounding electrode over the acromioclavicular joint). In a prone position , participants simultaneously contracted the truck musculature maximally  three times.FEE was calculated for each indentation (estimated magnitude minus 60 N) and used as a surrogate for feelings of stiffness to allow comparisons between groups . Using data from Study 1, we established that feelings of stiffness at baseline correlated significantly with FEE at 60 N .Objective spinal stiffness was measured in an identical manner to Study 1. For muscle activation, the raw sEMG signals were A/D converted (16 bit), full wave rectified and low pass filtered with a second order single pass Butterworth filter, using a cut-off frequency of 2.5 Hz. Customised Labview software , was used to calculate the average EMG amplitude over the indentation (marked by trigger points on the EMG file). The processed EMG data were then normalised to MVC amplitudes.To compare if the type of sound matters to force perception (FEE), a 3  × 3  × 2 (group) RM ANOVA was performed. To determine if the context of sound matters, a 2 (condition: creaky sound vs creaky decrease) × 3  × 2 (group) RM ANOVA was performed. When relevant, post hoc analyses using one-way RM ANOVAs and paired t-tests were completed (Holm-Bonferroni correction). Identical analyses were completed for objective spinal stiffness and for sEMG measures of muscle activation.Supplementary informationVideo S1Video S2Video S3


The transition from residency to independent practice presents unique challenges for physicians. New attending physicians often have unmet learning needs in non-clinical domains. An attending physician is an independent medical practitioner, sometimes referred to as a staff physician or consultant. Peer mentorship has been explored as an alternative to traditional mentorship to meet the learning needs of new attendings. In this article, the authors describe how a journal club for general internal medicine fellowship graduates helped ease the transition by facilitating peer mentorship. Journal club members were asked to bring two things to each meeting: a practice-changing journal article, and a ‘transition to practice’ discussion topic such as a diagnostic dilemma, billing question, or a teaching challenge. Discussions fell into three broad categories that the authors have termed: trading war stories, measuring up, and navigating uncharted waters. It is likely that physicians have a strong need for peer mentorship in the first few years after the transition from residency, and a journal club or similar discussion group may be one way to fulfil this. The transition from trainee to practising physician is an exciting and daunting time. Whether entering academic orcommunity practice, the change can be abrupt and isolating. In a teaching hospital, attending physicians typically leada team of medical students and residents on a clinical teaching unit. An attending physician is an independent medicalpractitioner, sometimes referred to as a staff physician or consultant. At the completion of training, new attendingphysicians are more confident with their clinical knowledge than with non-clinical domains such as communication,leadership, and teamwork , 2. NewAccess to mentorship may ease the transition for new attendings . In a trGroup peer mentorship is well suited to improve the transition to attending physician. Peer mentorship groups can address the unique learning needs of new attendings such as research skills, conflict resolution, and teaching skills –12. PeerThere is a paucity of data on the implementation of peer mentorship during the transition to attending physician. Most of the studies to date have involved structured peer mentorship groups which rely on senior faculty facilitators , 13. OneWhen three of us  graduated and began practising as attendings, we discovered a number of common clinical and non-clinical learning needs. Acting on the advice of a senior faculty mentor (PC), we started a journal club for recent general internal medicine fellowship graduates to address some of these needs. The purpose of the journal club was twofold: first, to facilitate continuing medical education by reviewing the medical literature; second, to provide a forum for peer mentorship by creating a safe space to share fears, discuss challenges, and bond over the joys and frustrations of starting independent practice.To meet these objectives, we asked members to bring two things to each meeting: a practice-changing journal article, and a ‘transition to practice’ discussion topic such as a diagnostic dilemma, billing question, or a teaching challenge. The most successful venue was a private room at a local pub, where we sat around a long wooden dining table and enjoyed food and drink. Although we briefly considered inviting senior faculty for their advice and mentorship, we decided to limit membership to new attendings to facilitate frank and open discussion.Beginning in December 2013, we hosted 10 journal club sessions over 14 months. All of the 14 graduates from our fellowship programme in general internal medicine in 2013 were invited to participate. An invitation was subsequently extended to 10 new general internal medicine graduates in 2014 and 6 new attendings who joined our institution from elsewhere. The journal club’s composition varied over time, and members participated in as few as 1 session and as many as 10. Approximately 5–10 individuals attended each session.We intentionally began each session with unstructured time of 30–60 min for socializing and informal discussion. Each member then presented his or her ‘practice-changing’ article and ‘transition-to-practice’ issue. There was additional time provided at the end of the session for discussion. Our meetings stretched well beyond the allotted two hours and often late into the evening, which we suspect reflects the unmet need for peer mentorship in our group. While confidentiality was not a specific precondition of our sessions, group members indicated in advance if they were discussing a topic that they preferred remain confidential. Discussion of specific patient cases was anonymous with identifying information omitted.We quickly observed that ‘transition to practice’ issues dominated over journal articles as topics of discussion. This likely occurred because new attendings feel more confident in their clinical knowledge than in other areas such as negotiation, conflict resolution, research, and teaching, which were the focus of the ‘transition to practice’ discussions –4, 6.Conceptually, the discussions on transition to practice fit into three distinct categories that we have termed: trading war stories, measuring up, and navigating uncharted waters Table . A war sThrough sharing these war stories, we taught each other about the art as well as the science of medicine. Some stories warned of potential pitfalls, while others solicited feedback, support, or reassurance. A few revealed potentially traumatic experiences, such as adverse patient outcomes, complaints to regulatory agencies, or legal proceedings. Perhaps sharing these stories in a supportive environment had therapeutic benefits for the tellers. Discussions often continued after the journal clubs, either in person, by e‑mail or by text message, and frequently with follow-up discussions at subsequent meetings.‘Measuring up’ was the second transition to practice theme at the journal club. Early-career physicians need to closely monitor and measure their progress against others. We compared all aspects of our clinical work, practice structure, academic commitments, continuing medical education strategies, and mentorship relationships Table . DiscussThe third theme was ‘navigating uncharted waters.’ New situations, or ‘disruptive novel elements,’ often arise unexpectedly in the early stages of one’s medical career . ExampleThe information shared at our meetings revealed a ‘hidden curriculum’ of learning to become an effective independent practitioner. The information we exchanged could be of local nature, such as which consultants to seek out or avoid at a particular hospital, or it could be of broader applicability, such as the optimal communication strategy to employ when phoning a surgical colleague at 3 A.M. to discuss a contentious case.In recent months, attendance at the journal club has waned slightly. Competing demands from work and personal lives may be a factor, or it may be that the need for intensive moral support has eased with our growing clinical confidence and evolving career trajectories. With increasing distance from our medical training, we are beginning to recognize common medical knowledge gaps and are considering focusing our meetings more specifically on continuing medical education. Inviting experienced clinicians to future meetings may be one way to accomplish this. We also wonder whether our programme could be expanded to the next generation of new attendings at the University of Toronto who might find this sort of programme useful. Nevertheless, the collegial peer relationships that developed in the group remain robust, and are perhaps one of the most important revenues from our journal club.We have learned that physicians have a keen need for peer mentorship in the first few years after the transition from residency. An informal discussion group such as a journal club may be one way to fulfil this need. By providing an invaluable forum for ‘trading war stories’, ‘measuring up’, and helping each other ‘navigate uncharted waters,’ our journal club facilitated peer mentorship. We are hopeful that new graduates both at the University of Toronto and elsewhere might consider picking up the baton to keep such programmes fresh and innovative even as we move on to more senior roles.


Despite technological advances, the long-term outcomes of endovascular aortic aneurysm repair (EVAR) are still debatable. Although most endograft failures after EVAR can be corrected with endovascular techniques, open conversion may still be required. A 70-year-old male patient presented at the emergency unit with abdominal pain. Twice, in the third and fourth years after the first repair, a stent graft had been placed over a non-adhesive portion of the stent graft due to type Ia endoleaks. In the most recent admission, a CT scan showed type III endoleak and ruptured aneurysm sac. On this occasion the patient underwent late open conversion. The failure was repaired with total preservation of the main endovascular graft body and interposition of a bifurcated dacron graft. This case demonstrates that lifelong radiographic surveillance should be considered in this subset of patients. Late open conversion following EVAR of ruptured abdominal aortic aneurysms can be performed safely.  Improvements in endovascular stent technology and increasing experience and technical skills have resulted in endovascular aortic aneurysm repair (EVAR) becoming the first-choice treatment for patients with infrarenal abdominal aortic aneurysms.  Despite technological advances, only short-term outcomes have been shown to be better with EVAR than with open repair. Early advantages of EVAR are less pain, shorter hospitalization, rapid recovery, and significantly lower 30-day mortality and morbidity rates,  Indications for re-intervention include endoleaks, migration, graft disconnection, stent fractures, graft thrombosis, and infection.  In this report, we present a case of late open conversion with a partial stent-graft explantation technique for ruptured abdominal aortic aneurysm (rAAA) after a third failed EVAR.  A 70-year-old, hypertensive, male patient presented at the emergency unit with a complaint of continuous crescendo abdominal pain. The patient had been treated 5 years previously with endovascular repair of a symptomatic infrarenal, fusiform, 119 mm diameter abdominal aortic aneurysm (AAA), at another institution. An initial surveillance scan at the 6-month follow-up, prior to admission, using computed tomography (CT) angiograms and abdominal plain radiographs, had shown no evidence of endoleak or sac enlargement. The patient had no further follow up until the third year after the first repair, and then, in the third and fourth years after the first repair, had been treated twice for stent migration and type Ia endoleak with placement of a stent graft over the non-adhesive portion of the stent graft   .  In the current admission, the patient had severe abdominal pain and hemodynamic instability. Expansion of the aneurysm sac (to 133 mm), type III endoleak (disconnection of main body from the bifurcated aortoiliac endovascular graft), and ruptured aneurysm sac were now evident on CT scan   . Open s The patient underwent late open conversion through a midline transperitoneal approach. After dissecting the suprarenal neck and common iliac arteries, proximal aortic control was achieved by cross-clamping the suprarenal aorta and distal back-flow control was achieved by exposure and clamping of the iliac arteries below the stent graft. The aneurysm sac was opened by longitudinal aortotomy, a large quantity of mural thrombus was removed, and the disconnection of the main graft body from the bifurcated aortoiliac endovascular graft was observed   . The ma The patient was discharged uneventfully from the hospital on the 10th postoperative day. A control CT angiography performed six months after surgical treatment showed no new lesions.  The process of aneurysm exclusion with EVAR, instead of surgery, offers some advantages including less pain, shorter hospitalization, rapid recovery, and significantly lower 30-day mortality and morbidity rates, especially in the high-risk patient.  Despite technological advances, the risk of aneurysm sac enlargement and rupture after EVAR cannot be completely avoided and, as a result, it is associated with a higher re-intervention rate than open repair. Although most endograft failures after EVAR are corrected endovascularly, late conversion to open surgery has been reported widely.  In 2002, The EUROSTAR registry reported 4291 patients with EVAR between 1996 and 2002 and an annual rate for late conversion to open repair of 2.1% (elective and emergency) with a combined perioperative mortality rate of 43.6%.  Hölzenbein et al.  Late conversion to open surgery can be needed due to many reasons, including aneurysm enlargement, with or without endoleak, stent-graft migration or disconnection, thrombosis or infection of the stent graft, and aneurysm rupture with hemodynamic instability.  Surgical exposure of the aneurysm can be achieved either through a transperitoneal or retroperitoneal approach. Both approaches have similar effectiveness in cases of late conversion. Another important element is the site of aortic cross-clamping. Infrarenal, suprarenal, or locations further above the fixation site  may be preferred.  In the current case, graft disconnection and the ruptured aneurysm sac were shown by CT scan. We preferred open conversion to endovascular revision because of hemodynamic instability and the anatomical difficulty of the previous stent graft. Late open conversion was performed through a midline transperitoneal approach with total preservation of the main body of the endovascular stent-graft and interposition of a bifurcated aortic Dacron graft.  In conclusion, this case demonstrates that lifelong radiographic surveillance is crucial in this subset of patients. We advocate that following EVAR of ruptured abdominal aortic aneurysm, late open conversion with partial endograft explantation can be performed safely and successfully.


The following scientists assisted the journal by reviewing manuscripts during the period November 1, 2016 to October 31, 2017.We gratefully acknowledge their critical evaluation and assistance in selecting articles for publication.


Arabidopsis plants store part of the carbon fixed by photosynthesis as starch to sustain growth at night. Two competing hypotheses have been proposed to explain this diel starch turnover based on either the measurement of starch abundance with respect to circadian time, or the sensing of sugars to feedback to the circadian oscillator to dynamically adjust the timing of starch turnover. We report a phase oscillator model that permitted derivation of the ideal responses of the circadian regulation of starch breakdown to maintain sucrose homeostasis. Testing the model predictions using a sugar-unresponsive mutant of Arabidopsis demonstrated that the dynamics of starch turnover arise from the circadian clock measuring and responding to the rate of change of cellular sucrose. Our theory and experiments suggest that starch turnover is controlled by the circadian clock acting as a dynamic homeostat responding to sucrose signals to maintain carbon homeostasis. Most organisms on the Earth possess circadian clocks that coordinate metabolism and physiology with periodic environmental changes. Synchronizing circadian rhythms to environmental cues occurs through phase adjustments of the circadian clock, in the process of entrainment, which optimizes the circadian regulation of diverse biological processes, such as growth, stress responses, and reproduction6. Arabidopsis stores a portion of the carbon fixed through sugars as insoluble starch, which accumulates during the day. At night, starch is broken down to provide sugars to sustain growth under dark. This process has several remarkable properties that are not easily explained within the context of known cellular signalling pathways. Starch abundance increases and decreases in an almost linear manner despite the exponential dynamics of cellular processes. Starch is consumed during night and reaches a minimum almost precisely at dawn regardless of changing photoperiod, due to regulation from the circadian oscillator8. The system adjusts the rate of change of starch accumulation and loss in response to seasonal changes of photoperiod, such that Arabidopsis plants in shorter photoperiods accumulate starch faster during the day and lose starch more slowly at night than those grown in longer photoperiods11. Lastly, early, or late onset of night, causes an immediate change in the rate of loss of starch abundance8.In Arabidopsis, an important output of the circadian system is the timing of the diel turnover of starch13. In the first, it is assumed that the abundance of starch is measured15, whereas in the other, it is assumed that sucrose is measured18. Both hypotheses consider that the circadian oscillator is required for the correct regulation of diel starch dynamics but with major differences in the assumed role of the clock. In the starch sensing models, the circadian clock is a passive timer used to measure the time of day15. In the second, sucrose feedbacks to the circadian clock to dynamically regulate the phase of the circadian oscillator17. There is strong experimental evidence for the role of the circadian oscillator in the timing of turnover of starch, which is critical to both hypotheses19. In addition, inhibition of starch degradation rate in response to elevated sucrose levels, mediated by trehalose-6-phosphate (Tre6P) supports the hypothesis that sugar-sensing contributes to the regulation of starch turnover20. However, because of the interrelationships between starch, sugars and the clock it has been difficult using experimental data alone to differentiate between the profoundly different assumptions of starch or sucrose sensing and the role of dynamical feedback from sucrose to the circadian clock.The molecular mechanisms that coordinate the turnover of starch with the external photoperiod remain unknown. Mathematical modelling aimed at providing a theoretical framework to consider the processes that regulate the diel turnover of starch has generated two competing hypotheses21 motivated us to examine further the hypothesis that the diel turnover of starch arises from sucrose-sensing and feedback to the circadian oscillator17. We tested this hypothesis using both modelling and experimental approaches. Because previous models considering the feedback of sucrose status to the circadian oscillator17 did not explicitly formalize the phase-dependent response of the circadian clock to sucrose, we developed a new phase oscillator model, which describes the adjustment of the circadian oscillator as a function of the phase at which the sucrose signal is received. The phase shift of the circadian clock by metabolism has also been reported in feeding and fasting cycles in mammals22 and rhythmic feeding cycles in cyanobacteria23, suggesting the role of metabolism as an important synchronizer of the circadian clock in diverse organisms. Using our model, we demonstrated how the circadian oscillator in Arabidopsis shows a phase advance in the subjective morning and a phase delay at night in response to sugar signals21. We then predicted that the metabolic regulation of the circadian clock contributes to appropriate carbon use in changing photoperiods. Predictions arising from the phase oscillator model were tested in a mutant in which the circadian oscillator does not respond to sugars. In wild type plants, sugars advance the phase of the circadian oscillator in the morning, but this response is absent in plants in which the circadian oscillator gene PSEUDO RESPONSE REGULATOR 7 is not functional (prr7-11)21. Using prr7-11 we demonstrate that dynamic sensing of sucrose by the circadian oscillator is required for the correct regulation of the diel turnover of starch in response to altered photoperiod.Our recent experimental finding that sugars can adjust the phase and period of the circadian oscillator of Arabidopsis21, we combined a phase oscillator model of the dynamical adjustment of the circadian system with a description of carbon metabolism. We first explain the model of carbon metabolism, which is similar to that we have used previously16 and perform extended analysis to demonstrate that linear starch dynamics can arise from the homeostatic regulation of sucrose in the cell17. In the model, the following assumptions about the dynamics for sucrose (St) and starch (Ct) are made. During the light period photoassimilates are produced at a constant rate11a. A fraction, γ, of those photoassimilates is partitioned into starch for storage, and the remaining portion, 1 − γ, is transformed into sucrose for use in respiration or transportation at rate H  is likely to be regulated by the balance between photoassimilated carbon and the rate of sucrose synthesis, which is mediated by the exchange of triose phosphate generated in the chloroplast for inorganic phosphate generated in the cytosol26, but this regulation solely cannot explain the control of carbon availability over the diel cycle29. To produce sucrose, starch is degraded at a rate of βt per unit surface area of the starch granule. Here, we call βt the “starch degradation rate.” In an original model17, we considered that all the rates for carbon partitioning (γ), sucrose transportation (H), and starch degradation (βt) oscillate under the influence of the circadian clock. Here we simplified the model by considering the constant rates for γ and H because oscillations of γ and H were less important to explain observed starch dynamics17. We normalized the length of a day as 1, and the fractions of light and dark periods in a day are given as τL and τD, respectively (τL + τD = 1). Based on these assumptions, the dynamics of sucrose and starch are represented with the following equations:To theoretically assess the diel turnover of starch in the light of empirical data demonstrating a feedback from sugar sensing to the circadian systemCtκ in Eqs , and the degradation process only occurs at the surface of each granule. Thus, we assume that starch degradation occurs in proportion to the surface area of starch granule represented as κ in Eqs  and 2)..Ctκ inBased on Eq. , we can a and H on the right-hand side of Eq. . Thus the same logic for sucrose homeostasis can be approximately applied to the total pool of soluble carbon, including glucose, fructose and organic acids. The emergent nature of the starch profile in our model represents a major difference from models in which starch sensing is assumed and specific chemical kinetics are assumed to realize the linear starch profiles14. Our finding that linear starch kinetics can arise as an emergent property of homeostasis of sugars is consistent with the empirical data that demonstrate that rather stable sucrose levels are found through the day although starch levels change drastically through the diel cycle28. The idealized relationship between starch and sucrose presented here is realized even under fluctuating environments due to a feedback mechanism from sucrose to the circadian clock to minimize sucrose changes, as we explain later.These results show that when the level of sucrose is constant, the starch profile is always linear and βt) required to achieve sucrose homeostasis in a given photoperiod (Supplementary Methods)17, and showed that it has a peak at dawn regardless of the magnitude of κ in Eqs  of the circadian oscillator, which can be formalized by the following phase oscillator dynamics:ω is the angular frequency, and ZL(φt) and ZS(φt) are the phase-dependent sensitivity functions for light and sugar signals, respectively. βt in Eqs φt. The magnitude of phase shift to a sucrose signal at a certain circadian time was determined empirically by a sugar pulse experiment using plants in which the endogenous sugars are depleted but not sufficiently to cause growth cessation  until φt = φ*, zero at φt = φ* (the break point), and negative (phase delay) after φt = φ* 21. The prr7-11 mutant provides the critical test for the model because there is no effect on the circadian period of CCA1:LUC luminescence under the standard circadian testing conditions of constant light (80 μmol m−2 s−1) when compared with wild-type  and a mutant in which the circadian clock does not respond to sugar signals , and a mutant lacking this response to sucrose would accumulate more starch than the wild-type Fig. . As pred SD Fig. , and whel-0 Fig. . When trT16 Fig. , rather T16 Fig. . The difEven when the feedback to the oscillator from sugar in both the day and night was considered in the model, it successfully predicted the change in starch accumulation and degradation rates during changing photoperiods  and sown on half-strength Murashige and Skoog  0.8% w/v bactoagar (Becton–Dickinson) media adjusted to pH 5.7 with 0.1 M HCl (Fisher Scientific). Seeds were stratified for 72–96 h in the dark at 4 °C before being entrained in 12 L:12D, 16 L:8D, or 8 L:16D photoperiods at 19 °C in Sanyo growth cabinets supplied with 100–120 µmol m−2 s−1 of white fluorescent light.Col-0 and 51. Whole rosettes from three 21- to 28-day-old plants were used as a single sample. Samples were harvested at four hour intervals, the fresh weight measured, immediately frozen in liquid N2, ground to a fine powder and kept at −80 °C until analysis.Starch was determined in the insoluble material after ethanolic extraction of soluble sugars, followed by enzymatic digestion with α-amyloglucosidase and α-amylasein vivo Plant Imaging System, 10-day-old seedlings were imaged every hour for 800 sec following a 200 sec delay for chlorophyll fluorescence to decay. Photon counts per second were extracted using IndiGO software. Red and Blue LED light was provided at 80 µmol m−2 s−1. The change in photoperiod from LD to continuous light for 11-day-old seedlings was automated using IndiGO.Arabidopsis was grown on media as described above, except that the plants were sown as clusters of five seedlings. Nine-day-old seedlings were dosed with 40 µl of 2 mM D-luciferin (nanolight) 1 h before dawn. Using a Berthold NightSHADE LB 985 52 and dosed as described above for luciferase measurements. Plants were transferred to constant low light (10 µmol m−2 s−1) at ZT0, and assay boats were transferred to liquid 0.5 MS + 90 mM sucrose for three hours to deliver a sugar pulse. Pulses were applied at intervals of 1.5 h during the first cycle in constant light. Following the sugar pulse assay, boats were briefly washed in liquid 0.5 MS to remove sugars and then returned to the assay plate. Luminescence imaging was automated every hour for four days as described above. The manual phase calculations and FFT-NLLS period estimations were performed using BRASS (amillar.org). Manual phase calculations were normalized to a 24 h period to give true phase (x):Arabidopsis was grown on custom assay boatsprr7-11) Arabidopsis. A set of starch time-series data was separated into the starch accumulation and the starch loss sections by the peak timing of starch concentration, and these two sections were analysed independently. For each section and genotype, linear regression was performed to assess starch level at dawn (intercept) and starch accumulation or decrease rate (slope). If the best-fit model estimated a negative intercept, we repeated the analyses by assuming that intercept is zero. We used analysis of covariance to test the statistical significance of the slope difference between Col-0 and prr7-11, which was represented as the synergetic effect of time × genotype. Detailed results of this analysis are presented in Supplementary Tables Linear regression was applied in order to compare the rates of starch accumulation or loss between wild type (Col-0) and mutant . Differential equations were solved numerically with the classical fourth-order Runge–Kutta method. Parameter values used for the analyses are listed in Supplementary Table A numerical analysis of the mathematical models was performed with Supplementary InformationSupplementary Datasets 1–6


In July of 1955, a group of around 20 Brazilian geneticists got together in a meeting andtook the decision to launch the Brazilian Society of Genetics . This Society flourished rapidly, and today it has close to one thousandmembers. Since its foundation, this association of scientists has worked hard andcontinuously to advance the quality of research in Genetics to international levels. Oneamong the several decisions taken 39 years ago, was that SBG should have its own societyjournal, to give more visibility to Genetics research done in Brazil. The initial title ofthe journal was Revista Brasileira de Genética, which, in 1995, was changed to BrazilianJournal of Genetics. Shortly thereafter, in 1998, its name then became Genetics andMolecular Biology, and all articles published since were exclusively in English. Thedecision was taken in view of the ongoing process of internationalization in the scientificpublications field, and now offered a platform also for international researchers to submitand publish their results in this Open Access journal. Since its beginning, the journalsucceeded to publish four issues every year, and it has and is clearly achieving its goalto be an attractive platform for good quality scientific contributions from all over theworld in the broad field of Genetics, Molecular Biology and Evolution.th anniversary, the SBG'sBoard of Directors and the GMB Editors decided that it would be an interesting moment togather scientific contributions honoring the Society. A call was launched, inviting severalleading Geneticists from Brazil and abroad, and the response was very positive to thisinitiative. Their respect to the SBG and to this journal is now manifest by high qualityreviews and original articles, which will be published in a series of GMB issues, startingwith the current one.One year ago, as the Society was celebrating its 60th anniversary of SBG, this issue of GMB(39.3) brings six articles, including four reviews and two original researches. Dr. Salzanooffers us an in-depth review of many aspects of human evolution, including importantcontributions from Latin American studies. Dr. Vieira and her group review the relevance ofmicrosatellites in plant sciences studies, discussing what they are, how they can bedetected, and applied to the field. The group of Dr. Diaz provides an interesting reviewthat covers recent findings about plant senescence mediated by abiotic and biotic stresses.Dr. Zatz and her group review their most important scientific contributions in the lastthirty years on neuromuscular disorders, in terms of gene discovery, diagnosis, counselingand also therapy. The group of Dr. Pena reports on a genotype-phenotype study identifyingmutations that result in the haploinsufficiency of the NARS gene, whichmay be responsible for clinical features of the Noonan Syndrome. Finally, the group of Dr.Tanuri describes a rapid test to evaluate the sensitivity of hepatitis C virus frominfected patients to protease inhibitors, with potential application in clinicaldiagnosis.Thus, for the celebration of the 60th anniversary offer a good overview on the breadth of Genetics researchdone in Brazil and in collaboration with international group leaders, and, together withthe other published works in this and the upcoming issues, will stimulate readers andgeneticists to appreciate even more this scientific society and its journal. Enjoy!We believe that this first and also the upcoming series of scientific articles dedicated tothe 60 Guest Editors of theSBG 60 years Special Series of Articles Carlos F.M. Menck and KlausHartfelder Editors of Genetics and Molecular BiologyFabrício Rodrigues dos Santos and Francisco Mauro Salzano


This special BMC Evolutionary Biology BGRS\SB-2016 issue is an integral part of the collection of manuscripts presented at “Bioinformatics of Genome Regulation and Structure\Systems Biology” (BGRS\SB-2016) conference which took place at August 29 - September 2, 2016 in Novosibirsk, Russia. Other issues in this collection include BMC Genetics , BMC Biohttp://conf.bionet.nsc.ru/bgrssb2016/) is 10th installment of the biannual BGRS event which has a long history starting from 1998 in Novosibirsk. The conference organized by Institute of Cytology and Genetics of Siberian Branch of the Russian Academy of Sciences (ICG SB RAS) has focuses on evolutionary biology, systems biology and bioinformatics, gene network analysis, post-genomics and sequencing technologies. Since 1998, BGRS series has grown to large international multi-conference forum where medical doctors, mathematicians, biologists could share their recent theoretical and experimental insights.International Conference BGRS\SB-2016 (http://conf.bionet.nsc.ru/ishg2016/en/), Symposium “Cognitive Sciences, Genomics and Bioinformatics” (CSGB-2016) (http://physiol.ru/csgb2016/), and the Second International Conference on the Mathematical Modeling and High-Performance Computing in Bioinformatics, Biomedicine and Biotechnology (MM-HPC-BBB-2016) (http://conf.bionet.nsc.ru/mm-hpc-bbb-2016/en/).In 2016, the BGRS included several parallel events and symposia: the international Symposium “Systems Biology and Biomedicine” (SBioMed-2016) , BMC Genetics [http://www.biomedcentral.com/bmcevolbiol/supplements/15/S1), and BMC Systems Biology .Since 2014, The BGRS Program Committee collaborates with BioMed Central on full-text thematic issues of relevant BMC journals. In recent years, BioMed Central had published several special issues based on best materials presented at the conference, including ones in BMC Genomics (http://vavilov.elpub.ru/jour/) (in Russian). This year, a special issue in “Molecular Biology” (Springer) was added to the list .Additionally, special issues on bioinformatics were published at the “Journal of Bioinformatics and Computational Biology”  and “VavCurrent issue of BMC Genomics presents reports on genome-wide studies in computational biology discussed at the BGRS\SB-2016 conference.The paper by Oleg Balanovsky et al.  opens thThe work of Vladimir Babenko and colleagues  utilizes70 family of the factors by quantitative investigation of mix-and-matching in two canonical ECFfamily members (σE and σW) and proposed an evolutionary anchored simple model which relates the size of σfactor regulon with the extent of mix-and-matching.Jelena Guzina and Marko Djordjevic  exploredin silico evolution of Drosophila gap gene regulatory sequence to demonstrate that elevated mutational pressure leads to produce regulatory sequence organizations with fewer, albeit on average stronger, functional transcription factor binding sites that support preservation of adjacent regulatory sequences.Aleksandra Chertkova and colleagues  employedLasius niger in urbanized areas may result from the expansion of the CYP9 cytochrome family, the diversification of DNA repair systems, and reduced odorant communication.The paper by Evgenii Konorov et al.  presentsFinally, Ilyas Jetybaev et al.  studied The works presented above employ a variety of evolutionary analysis techniques, from karyotyping to genome sequencing and then to in silico evolution and to building kinetic models.http://conf.bionet.nsc.ru/sbb2016/en/). The CSGB-2016 Symposium on cognitive sciences has presented special issue at IEEE Xplore Digital Library (http://ieeexplore.ieee.org/xpl/mostRecentIssue.jsp?punumber=7587661). “Journal of Bioinformatics and Computational Biology” (http://www.worldscientific.com/worldscinet/jbcb) continues traditional special issues after BGRS\SB on mathematical problems of bioinformatics and biomedicine as well as “Journal of Integrative Bioinformatics” . Experimental research manuscripts fallen into the scope of BGRS\SB2016 have been collated in Special Issue of “Molecular Biology” (Springer)  that will be published in 2017. In addition, special issue on bioinformatics and evolutionary biology is published at the “Vavilov Journal of Selection and Breeding” (http://www.bionet.nsc.ru/vogis).Returning back to the tenth anniversary BGRS\SB event, note that 2016’ thematic collection of the manuscripts is augmented by Special Issues covering various Satellite events, including already traditional Young Scientists School “Systems Biology and Bioinformatics” (SBB-2016) (http://www.bionet.nsc.ru/files/2016/conference/BGRS2016.pdf.The BGRS\SB-2016 Proceedings including “Evolutionary bioinformatics” section are available at the multi-conference web-site: Next BGRS conference will take place in 2018. Welcome to Novosibirsk!


Acute antinociceptive and reward acquisition of PPL-101 and PPL-103 were assessed in mice using the tail flick assay and conditioned place preference (CPP) paradigm, respectively. The reinforcing effects of these compounds were assessed in rats using the self-administration paradigm. In mice, PPL-101 and PPL-103 produced antinociception reaching maximal effects that were equivalent to morphine at approximately 1/3 and 1/10 of morphine’s dose, respectively. PPL-101-induced antinociception was attenuated following pretreatment with the kappa antagonist JDTic, but not the mu opioid antagonist beta-FNA. In mice, PPL-101 and PPL-103 produced dose-dependent decreases in activity, similar to other kappa agonists; however, they did not produce conditioned place aversion, and in fact elicited a trend toward CPP. In rats, neither PPL-101 nor PPL-103 were self-administered when substituted for morphine and PPL-101 attenuated morphine self-administration, when administered systemically prior to the self-administration session. Collectively, these results indicate that mixed opioid receptor partial agonists can produce potent antinociceptive activity with a lack of aversion in mice and without being self-administered in rats. Compounds with this profile could be superior analgesics with greatly reduced addiction liability and fewer side-effects compared to traditional opiates.Opiates are still the most effective and widely used treatments for acute and chronic pain. However, the problems associated with morphine and other standard opioid analgesics severely limit their effectiveness in the clinic. PPL-101 and PPL-103 derived from morphine and morphinan ring systems contain a chiral N-substituent, which confers it with a unique combination of high-binding affinities and partial agonist activities at mu, delta, and kappa opioid receptors, leading to unique  It is well known that activation of the different opioid receptors induces different pharmacological actions. Mu receptor activation induces antinociception, along with a decrease in respiration and gut motility, and significant abuse liability , 2. KappN-methyl with N-allyl or N-cyclopropylmethyl increases affinity for kappa and delta receptors without decreasing affinity at mu receptors , have antagonist activity at mu receptors but agonist activity at kappa, resulting in an analgesic compound without reward, but with dysphoria  and PPL-103 (α-methyl-cyclopropylmethyl-morphinan) are opiate derivatives that bind with high affinity to mu and kappa receptors, with slightly lower affinity to delta receptors Table . StudiesFor assessment of antinociception and conditioned place preference (CPP), male ICR mice weighing 25–30 g at the start of the experiment were used. Mice were group-housed under standard laboratory conditions using nestlets as environmental enrichment in their cages and were kept on a 12:12-h day/night cycle (lights on at 7:00 a.m.). For experiments examining antinociception, animals were housed 10/cage and for the CPP experiments animals were housed 4/cage. Testing was conducted during the animals’ light cycle between 9 a.m. and 2 p.m. Animals were handled for 3–4 days before the experiments were conducted. On behavioral test days, animals were transported to the testing room and acclimated to the environment for 1 h.For self-administration experiments, male Sprague-Dawley rats (200–225 g) were obtained from Charles River . Rats were housed in a room with a reverse 12-h light/12-h dark cycle (lights off at 7:30 a.m.). All self-administration experiments were conducted during the dark phase of the cycle. Animals were acclimated for 7 days with water and chow  and handled for 3 days before the experiments were conducted. Throughout all operant procedures, rats were food restricted and received 16–20 g of chow daily, to maintain 80% of free-feeding weight. Water was freely accessible.PPL-101 (α-methyl-cyclopropylmethyl-morphine) and PPL-103 (α-methyl-cyclopropylmethyl-morphinan) Figure , 30. MorFor studies in mice, PPL-101 and PPL-103 were dissolved in 3% DMSO and 0.5% aqueous hydroxypropylcellulose, whereas morphine hydrochloride, beta-FNA, and JDTic were dissolved in distilled water. All drugs except beta-FNA were injected in a volume of 0.1 ml/30 g subcutaneous (s.c.) Beta-FNA was made up as a 20 mg/kg solution and injected in a volume of 0.2 ml/30 g, s.c. to get the desired dose of 40 mg/kg. Controls received the appropriate volume of vehicle . Doses of morphine, PPL-101, PPL-103, JDTic, and beta-FNA are reported as the salt.via the i.p. route. The kappa agonist U-69,593 was suspended in 10% DMSO, 10% TWEEN 80, and 80% distilled water and given s.c. 15 min prior to the plantar test.For studies in rats, intravenous (i.v.) solutions of morphine, PPL-101, and PPL-103 were produced by dissolving drugs in 0.9% saline and then adjusted to pH 7.0–7.4 with 3 M sodium hydroxide. Morphine, PPL-101, and PPL-103 self-administration doses are reported as free base concentrations. Systemic solutions of PPL-101 and naloxone were made by dissolving drugs in 0.9% saline and were administered in a volume of 1 ml/kg, intraperitoneal (i.p.) JDTic was dissolved in distilled water and administered All receptors were individually expressed in CHO cells stably transfected with human receptor cDNA, The cells were grown in Dulbecco’s modified Eagle medium with 10% fetal bovine serum, in the presence of 0.4 mg/ml G418 and 0.1% penicillin/streptomycin, in 100-mm polystyrene culture dishes. For binding assays, the cells were scraped off the plate at confluence. Receptor expression levels were 1.6, 1.8, and 3.7 pmol/mg protein for the mu, kappa, and delta opioid receptors respectively.g for 15 min. The final pellet was resuspended in Tris, and the suspension incubated with [3H]DAMGO , [3H]Cl-DPDPE , or [3H]U-69,593  for binding to mu, delta, and kappa receptors, respectively. Non-specific binding was determined with 1 µM of unlabeled DAMGO , DPDPE , and ethylketocyclazocine (EKC), respectively. Samples were incubated for 60 min at 25°C in a total volume of 1.0 ml, with 15 µg protein per well. The reaction was terminated by filtration using a Tomtec 96 harvester  through glass fiber filters and radioactivity was counted on a Pharmacia Biotech beta-plate liquid scintillation counter . IC50 values were calculated using Graphpad/Prism , and Ki values were determined by the method of Cheng and Prusoff  were incubated with [35S]GTPγS (50 pM), GDP (10 µM), and the appropriate compound, in a total volume of 1.0 ml for 60 min at 25°C. Samples were filtered over glass fiber filters and counted as described for the binding assays. Statistical analysis was conducted using the program Prism. schedule during daily 2-h sessions  conducteN = 24) were divided in four groups of N = 6, so that the average morphine self-administration rate based on their lever pressing performance on the last 3 days of morphine training was the same across all groups. To examine whether PPL-101 would substitute for morphine, two groups of rats were given access to PPL-101 , while the third group was given access to vehicle , and the fourth group continued morphine (100 µg/kg/infusion) self-administration. This self-administration period was conducted under the FR1TO20 task and lasted for an additional seven sessions (testing over 5 days/week). Results are reported as number of infusions earned in 120 min. After a week on this reinforcement schedule, the task was switched to progressive ratio (PR) schedule that better measures motivational properties rather than rate of drug intake ((0.20 × inj. number)] − 5” ] − 5” . The PR N = 8) rats using FR1TO20 schedule. Then, rats were given access to PPL-103  according to a Latin square within-subject design, in which each dose was tested 4 days/week under the FR1TO20 task and under PR on the fifth day. During the following weeks, rats had access to a different PPL-103 dose. Results of FR sessions are reported as number of infusions earned in 120 min. Total responses and break point were the dependent variable for PR.To examine whether PPL-103 would substitute for morphine, six 2-h sessions of morphine self-administration were initially conducted in a new cohort of (N = 8) were used to assess the effectiveness of systemically administered PPL-101 in altering morphine lever pressing. PPL-101  was tested using a Latin-square counterbalanced within-subjects design. Following the establishment of a stable morphine self-administration, for the test session, animals were treated with the desired dose of PPL-101 (i.p.) 15 min prior to the self-administration session. Following each test session day, animals were allowed 1 day off, and a new baseline of morphine self-administration was then established over the following 2 days, prior to the subsequent test session. Data are reported as number of infusions obtained in the 120-min test session. Responses on the inactive lever were also recorded and served as an index of unspecific motor behavior.After restoring a new morphine self-administration baseline of responding, animals (N = 5) or PPL-101  for 1 week under the FR1TO20 task. The kappa antagonist JDTic  was administered to both groups of rats 2 h following the last training session  according to the method used by Hargreaves et al. . BrieflyTo confirm that JDTic (10 mg/kg) was producing its effect on self-administration by blocking kappa receptors, we examined whether U-69,593-induced antinociception could be blocked using the rats that underwent self-administration testing. Paw withdrawal latency was measured in rats that were pretreated with JDTic or vehicle after completion of the self-administration experiments. One week after the JDTic administration, rats received an injection of U-69,593  or vehicle and were tested for paw withdrawal latency 15 min post-U-69,593 administration.post hoc tests where appropriate. Global activity was analyzed using repeated measures ANOVAs with drug treatment  as a between-group variable and injection day (first versus third) as a repeated measure. Significant interactions were further analyzed with one way ANOVAs and Bonferroni post hoc tests. To examine sensitization or tolerance to the development of global activity, following a significant overall ANOVA, t-tests were used to compare data following the third injection relative to the first. For the CPP test day data, a difference score was calculated as time spent in the drug-paired compartment minus time spent in the vehicle-paired compartment. Vehicle animals spent the same amount of time in both compartments such that the difference score is no different than 0 s. Difference scores were analyzed using ANOVAs, and significant effects were further analyzed with post hoc tests. CPP was evident if animals spent significantly more time in their drug-paired compartment, resulting in a positive difference score relative to control animals.Antinociception in mice [% maximum potential effect (% MPE)] was quantified by the following formula: % MPE = 100 × [(test latency − baseline latency)/(15 − baseline latency)]. If the animal did not respond prior to the 15-s cutoff, a score of 100% was assigned. In initial assessment of PPL-101 and PPL-103, post-tail flick latency was analyzed using an ANOVA with drug treatment  as a between-group variable and post-injection time as the dependent measure. PPL-101 time course data were analyzed using repeated measures ANOVAs with drug treatment  as a between-group variable and post PPL-101-injection time  as the repeated measure followed by Bonferroni Post hoc comparisons were conducted where appropriate.In rats, self-administration data  were analyzed using repeated measures ANOVA with reinforcer  as between-subject variables and “day” as a repeated measure. PR data, number of infusions, was analyzed using a one-way ANOVA with drug/dose  as the between-subject variable. Systemic effect of PPL-101 on morphine self-administration was analyzed by a one-way ANOVA that used treatment as a within-subject factor. Long-term effects of JDTic on morphine and PPL-101 self-administration were analyzed by comparing lever pressing before and after treatment using a one-way ANOVA. Paw withdrawal data were examined using a two-way ANOVA with drug pretreatment (vehicle or JDTic) and treatment  as between-subject factors. P < 0.05.For all experiments, the level of significance was set at in vitro functional activity were determined at mu, kappa, and delta receptors for PPL-101, PPL-103, and several selective and non-selective reference compounds. As seen in Table 3 counterparts, such as morphine, which have considerable selectivity for the mu receptors GTPγS binding, is also affected by the N-substitution, as the α-methyl-CPM moiety induces a change in efficacy, so that PPL-101 and PPL-103 have low efficacy at mu and delta receptors and higher partial agonist activity at kappa receptors  = 44.4, P < 0.05]. The positive control morphine produced the maximal effect in increase in tail flick relative to vehicle controls. The doses of 0.3–10 mg/kg PPL101 produced dose-dependent antinociception where there was a significant increase in %MPE relative to vehicle controls at all doses tested and maximal effects were observed with 3–10 mg/kg PPL-101.In initial assessments of thermal nociception, PPL-101 and PPL-103 exhibited significant antinociceptive activity measured by tail flick latency, as shown in Figure F = 31.1, P < 0.05]. Maximal effects were observed following administration of the 1.0 and 3.0 mg/kg doses, whereas the 0.3 mg/kg dose produced a significantly reduced level of antinociception relative to the higher two doses.PPL-103 also showed potent antinociception at all doses tested  = 10.18; P < 0.0001]. The positive control morphine produced the anticipated increase in %MPE at all the time points compared to vehicle controls. Across time, %MPE produced by morphine was highest at the 0.5–2 h time points; however, there was a significant decrease in %MPE at the 4-h time point compared to the first time-point . PPL-101, at 0.3–10 mg/kg, produced a dose-dependent increase in %MPE that was significantly different from vehicle controls up to 4-h post-injection , whereas at the 1- and 2-h test points 1–10 mg/kg produced a significant increase in %MPE (P < 0.001). However, by the 4-h time point, only the highest dose of PPL-101 tested produced antinociceptive effects (P < 0.0001). The highest dose of PPL-101 produced similar levels of antinociception as the maximally effective dose of morphine tested and %MPE produced by 10 mg/kg PPL-101 was not significantly different than that produced by morphine at all time-points tested.The time course for PPL-101 antinociception is shown in Figure F = 5.95; P < 0.0001]. As evident in the figure, JDTic (gray triangles) and beta-FNA (black square) when administered alone 24-h prior to a vehicle administration and testing have no effect on tail flick latency in mice. Morphine antinociception was completely inhibited by the mu opioid antagonist beta-FNA but was not altered by the kappa antagonist JDTic. On the other hand, PPL-101-induced antinociception was inhibited by JDTic and not beta-FNA. Although beta-FNA did not attenuate PPL-101-induced antinociception, it potentiated antinociception produced by 0.3 mg/kg PPL-101 at the 1- to 4-h post-PPL-101 injection, time-points during which the 0.3 mg/kg dose alone did not significantly produce antinociception (P < 0.05). Thus, it seems that in general, the kappa antagonist JDTic and not beta-FNA attenuated PPL-101-induced antinociception, whereas beta-FNA potentiated PPL-101 antinociception at the lowest dose.To determine the relative involvement of mu and kappa agonist activity in eliciting PPL-101-induced antinociception, animals were pre-treated with mu antagonist beta-FNA 40 mg/kg) or kappa antagonist JDTic  compared to vehicle controls. Although in Figures P = 0.08 and 0.12 for PPL-101 and PPL-103, respectively), the post hocs did not reveal any significant difference between the groups that received PPL-101 or PPL-103 when compared to vehicle controls.The effect of PPL-101 and PPL-103 on acquisition of CPP is shown in Figures F = 7.9; P < 0.05] and PPL-103 . As shown previously, morphine administration produced an increase in global activity after the first drug injection relative to vehicle controls (P < 0.05). Furthermore, sensitization of morphine-induced global activity was also evident as an increase in activity following the third drug injection relative to the first drug injection (P < 0.05). On the other hand, the 3.0 mg/kg dose of PPL-101 and PPL-103 produced decreases in global activity relative to vehicle controls following the first and third drug injections (P < 0.05). Additionally, 3 mg/kg PPL-101 produced a further decrease in global activity following the third drug injection relative to the first drug injection (P < 0.05).The effect of PPL-101 and PPL-103 on global activity during conditioning training, after the first and third drug injections, is shown in Figures F = 3.2, P < 0.05] that was not accompanied by a significant interaction “session × reinforcer” . Post hoc analysis was therefore carried out on the collapsed factor of “reinforcer” . This analysis indicated that both doses of PPL-101 were not self-administrated as compared to 100 µg/kg morphine .Rats were initially trained to self-administer food pellets and then morphine sulfate at 100 µg/kg/infusion. Average responses on the morphine-associated lever over the last 3 days prior to PPL-101 self-administration were 29.0 ± 5.8, 27.2 ± 6.1, and 32.2 ± 12.6 (mean ± SEM). When PPL-101 (30 and 100 µg/kg/infusion) was substituted for morphine, rats exhibited a decrease in responding when compared to their counterparts that continued to self-administer morphine  = 2.6, P < 0.05]}. Post hoc analysis showed that lever pressing for both doses of PPL-103 was markedly decreased as compared to responses for morphine across all test sessions  was substituted for morphine using a within-subject Latin square design, the operant response under the FR-1 schedule clearly varied {reinforce × session interaction . Post hoc comparisons indicated that the number of infusions for vehicle and PPL-101 (30–100 µg/kg/infusion) were significantly decreased compared to the morphine group (P < 0.05). As with PPL-101, when responding under the PR schedule  = 5.8, P < 0.05]. Post hoc comparisons indicated that the number of infusions for vehicle and PPL-103 (30–100 µg/kg/infusion) were significantly decreased compared to the morphine group (P < 0.05).When responding under the PR schedule Figure C, similae Figure D, PPL-10These data collectively suggest that at the doses tested, PPL-101 and PPL-103 are not self-administered by rats to the same extent as morphine and in fact were similar to the vehicle. This was further demonstrated by the evidence that all groups returned to high level of morphine-taking behavior when morphine replaced 0.9% saline, PPL-101, or PPL-103.F = 2.8; P = 0.06]. The same analysis conducted on the inactive lever led to an overall effect of treatment . Post hoc comparisons revealed a significant increase in lever pressing in rats treated with PPL-101 0.3 mg/kg (P < 0.05), suggesting altered non-specific motor behavior only at this dose of PPL-101, while inactive lever responding did not change at higher doses.Experiments were carried out to determine whether systemic pretreatment with PPL-101 would reduce morphine self-administration. PPL-101  = 1.5, n.s.]. Similarly, average lever pressing of JDTic-treated rats self-administering PPL-101 (100 µg/kg/infusion) across 7 days was greatly reduced compared to morphine self-administration and not different from baseline lever pressing of the same rats across the last 3 days of PPL-101 self-administration prior to JDTic treatment . When morphine was substituted for PPL-101 in these JDTic-treated rats, the number of morphine infusions increased and was significantly higher compared to the number of infusions for PPL-101 in the same animals , whereas the original single treatment with JDTic by itself did not . ANOVA revealed “pre-treatment × treatment” interaction . Post hoc pairwise comparisons, confirmed that the single dose of JDTic used (10 mg/kg) was able to block the antinociceptive effect U-69,593 (P < 0.01) and return paw withdrawal latencies to the level of controls  was functioning as a kappa antagonist during the self-administration experiments, we examined whether U-69,593-induced antinociception could be blocked using the rats that underwent self-administration testing. On plantar test, 0.3 mg/kg U-69,593 (i.p.) successfully induced antinociception  and were approved by the IACUC of SRI International  and the IACUC at the Torrey Pines Institute for Molecular Studies .Participated in research design: LT, TK, and AC. Conducted experiments: TK, AC, WP, and NT. Contributed New Reagents or analytic tools: WC and JL. Performed data analysis: TK and AC. Wrote or contributed to the writing of the manuscript: LT, TK, and AC.The authors declare that this study received partial funding from Phoenix PharmaLabs. Authors, LT, JL, and WC, are affiliated with Phoenix PharmaLabs. LT participated in research design and contributed to the writing of the manuscript. JL and WC synthesized compounds and made them available for the study. All other authors declare no conflict of interest.


Evidence-Based Complementary and Alternative Medicine would like to express our concern with the article titled “Modes of Antiviral Action of Chemical Portions and Constituents from Woad Root Extract against Influenza Virus A FM1” published in Evidence-Based Complementary and Alternative Medicine in January 2016 [ Journal of Traditional Chinese Medicine in June 2016 by different authors [ Journal of Traditional Chinese Medicine article was accepted in August 2015, before the Evidence-Based Complementary and Alternative Medicine article was submitted in November 2015. Evidence-Based Complementary and Alternative Medicine is the same as Figures 2(c)–2(f) in the Journal of Traditional Chinese Medicine; Figure 2(b) shows the same results, though presented differently, as Figures 2(a) and 2(b); Figure 3 is identical, except that panel (b) is rotated 180° and panel (f) is different; Table 1 is identical to Table 3; Table 2 is identical to Table 5; Figure 4 shows the same results; all but one of the references also appear in the Journal of Traditional Chinese Medicine reference list. Although one article uses the term “woad root” and the other “Banlangen (Radix Isatidis),” these are the same plant.Around half of the text is the same; Figure 1 is identical; Figure 2(a) in Evidence-Based Complementary and Alternative Medicine authors said that the work comes from Jian-Hang Su's M.S. thesis and is not the same as that published in the Journal of Traditional Chinese Medicine; their article focuses on the root extract, while the Journal of Traditional Chinese Medicine article focuses on the dose response and pure compounds. The Journal of Traditional Chinese Medicine authors said that the work was from Weiyang Ye's 2011 Ph.D. thesis. Both groups of authors presented evidence to support their positions.TheWe asked the institutions of both groups of authors to investigate. The University of California, San Diego, found an investigation was not within its remit and we did not receive a response from the other institutions, Yantai Traditional Chinese Medicine Hospital, Yantai Yuhuangding Hospital, and Nanjing University of Chinese Medicine. Because the outcome of our internal investigation and the institutional investigations has been inconclusive, the journal will not retract the article at this time. However, readers should be aware of the duplication of these results.