The role of Ribonucleotide reductase (RR) subunits in different cancers has been intensively studied in our laboratory. RRM2B was identified as a p53-inducible RR subunit that involves in various critical cellular mechanisms such as cell cycle regulation, DNA repair and replication, and mitochondrial homeostasis, etc. However, little is known about the p53-independent regulation of RRM2B in cancer pathology. In this study, we discovered tumor suppressor FOXO3 as the novel regulator of RRM2B. FOXO3 directly bound to and transcriptionally activated the promoter of RRM2B, and induced the expression of RRM2B at RNA and protein levels. Moreover, Overexpression of RRM2B and/or FOXO3 inhibited the proliferation of cancer cells. The cancer tissue microarray data also demonstrated a strong correlation between the co-expression of FOXO3 plus RRM2B and increased disease survival and reduced recurrence or metastasis in lung cancer patients. Our results suggest a novel regulatory control of RRM2B function, and imply the importance of FOXO signaling pathway in DNA replication modulation. This study provides the first time evidence that RRM2B is transcriptionally and functionally regulated independent of p53 pathway by FOXO3, and it establishes that FOXO3 and RRM2B could be used as predictive biomarkers for cancer progression. Human ribonucleotide reductase (RR) plays a vital role in DNA synthesis and repair by catalyzing the de novo conversion of ribonucleoside diphosphates to deoxyribonucleoside diphosphates , 2. In pThe p53 tumor-suppressor pathway is largely defective in numerous human tumors -27. RRM2Forkhead box O 3 (FOXO3) is one of the most comprehensively characterized members of the FOXO family of transcription factors -31. UndeWe have sought to identify p53-independent regulators of RRM2B, and we hypothesized that FOXO3 regulates RRM2B. To test this, we introduced a FOXO3-targeting siRNA into cancer cells and examined whether downregulating FOXO3 affects RRM2B levels. To verify the p53-independent effect of FOXO3, we used p53-deficient cancer cells: HeLa (p53-degraded), H1299 (p53-null), and MDA-231 (p53-mutated) cells. RRM2B protein was downregulated in all 3 cell lines when FOXO3 expression was knocked down Figure . Next, tTo understand how RRM2B is regulated under p53-deficient conditions further, we examined the promoter region of the RRM2B gene by using the programs TFSEARCH and PROMO. Several putative transcription factor-binding sites were identified within the 10-kb upstream region of the promoter. Among these sites, 3 putative FHREs (TTGTTTAC) were present upstream of the RRM2B ATG site; the presence of these sites, starting at −964, −2965, and −5583 bp, increased the possibility that the FOXO signaling pathway regulates RRM2B Figure .We hypothesized that FOXO3 regulates RRM2B activity through the promoter of RRM2B, and tested this by performing luciferase-reporter assays in p53-negative H1299 cells. A control experiment demonstrated that FOXO3(A)3 3)  activateTo confirm that the FHRE −964 site in the RRM2B promoter is specific for FOXO3-induced activation, we performed site-directed mutagenesis to mutate the reporter constructs; the mutated bases are underlined in Figure We also confirmed that FOXO3 was responsible for inducing the reporter activity. When the FOXO3 siRNA was applied in the reporter assays, the luciferase activity induced by (A)3 was substantially reduced compared to the control level, which verified that the RRM2B promoter activity was specifically activated by FOXO3 Figure . In addiCollectively, the results demonstrated that FOXO3 uses the FHRE in the RRM2B promoter and activates RRM2B transcription in a p53-independent manner.in vitro DNA pull-down assays. Briefly, 35S-FOXO3 protein was synthesized and incubated with biotinylated double-stranded FHREs asindicated; DNA oligos derived from the RRM2B −964 site in the promoter region (RRM2B FHRE WT); DNA oligos containing a mutated RRM2B −964 site (RRM2B FHRE Mut); or, as a positive control, a recognized FHRE derived from the promoter of manganese superoxide dismutase (MnSOD). DNA-protein complexes were detected when the RRM2B and MnSOD oligos containing the wild-type FHRE site were used  assays by using H1299 cells, as described previously . The resTo investigate the consequence of FOXO3-mediated RRM2B regulation, we established 3 pairs of H1299 stable cell lines that expressed a control vector or RRM2BshRNA, a GFP control or an RRM2B-expressing vector, and EGFP or EGFP-FOXO3. The expression levels of proteins in the cells were examined using Western blot analysis Figure . Next, cP < 0.0001), and high expression of nuclear FOXO3 coincided with RRM2B expression in cancer patients   was performed using a lung-tissue microarray (n = 63) and the expression levels of RRM2B and nuclear FOXO3 were analyzed. Figure s Figure . The Kap) Figure . Moreove) Figure .Finally, we examined the difference between the demographics (sex and age) of the FOXO3/RRM2B low/low and high/high groups, and the results indicated no statistically significant differences (data not shown). The detailed calculation methods used are listed in Materials and Methods section.Collectively, the results confirmed that FOXO3 is a novel regulator of RRM2B and revealed that FOXO3 can bind to the promoter region of the RRM2B gene and induce the transcription of RRM2B. Furthermore, FOXO3 and RRM2B inhibited the growth of cancer cells, and in accordance with these results, the IHC results obtained using tissues of human cancer patients indicated that coexpression of FOXO3 and RRM2B is correlated with increased survival and a reduced death rate and cancer recurrence or metastasis in lung-cancer patients.Since RRM2B was first identified as a p53 target, we have been considerably interested in determining whether the presence of wild-type p53 affects the FOXO3-dependent regulation of RRM2B. We examined the level of p53 expression in cancer patient samples used in Figure RRM2B was identified as a critical p53-inducible RR subunit that can be regulated by p53 and p73 . HoweverAlthough several FOXO regulatory mechanisms have been identified, the serine/threonine kinase AKT is still considered to be a key regulator of the FOXO pathway . RecentlFOXO3 and p53 share several common features. The subcellular localization and transcriptional activity of both proteins can be regulated by posttranslational modifications such as ubiquitination and phosphorylation , 40. TheAs a key RR subunit, RRM2B plays a vital role in DNA synthesis and repair ,38. It wCell lines (ATCC) were cultured in DMEM medium containing 10% fetal bovine serum (Omega Scientific Inc.) and penicillin and streptomycin. All cells were incubated at 37°Cwith 5% CO2. Virus generation was described . Stable pECE-FOXO3 (A)3 plasmid, EGFP, and EGFP-FOXO3 were kind gifts from Dr. Kato, Dr. Rama  and Dr. Cells were seeded onto the 96 well plates as 1500-2000 cells/well and then grow in the incubator for 72 hours before analysis. The number of the viable cells in proliferation was analyzed according to the manufacturer's instructions (Promega).n” represents an independent experiment done in duplicate or triplicate. Please find other statistical information in the “Statistical methods” section.Cells were seeded onto the 6 well plates as 100 cells/well, and then the cells were incubated for 10 days with medium changed every 3-4 days. When harvested, cells were rinsed with 1xPBS and then fixed and stained with 0.5% crystal violet in methanol: acetic acid (3:1) for 5 mins. Then, plates were rinsed and dried. Colonies were scored and analyzed for relative cell proliferation. In the bar charts, each “Lipofectamine 2000 or RNAiMAX (Invitrogen) were used for plasmid or siRNA transfection. Cells were harvested 48 hours after transfection of plasmids. siRNA treatment of cells was carried out using siRNA non-targeting control , or FOXO3 siRNA (Santa Cruz) for 72 hours at 10-30 nM. siRNA sequences are listed in n” represents an independent experiment done in duplicate or triplicate. Please find other statistical information in the “Statistical methods” section.Reporter assay was performed as described . Cells wEndogenous chromatin immunoprecipitation (ChIP) assay was performed with H1299 cells and anti-FOXO3 antibody (Santa Cruz) or IgG control antibody according to EZ-ChIP manufacturer's instructions (Millipore) . PCR wasCells were seeded on coverslips, fixed with 3.7% formaldehyde and permeabilized with 0.5% Tween20, stained with primary anti-FOXO3 (Santa Cruz), anti-RRM2B (Rockland), γ-H2AX (Active Motif) and secondary antibodies, washed, stained with DAPI as nuclear control, and then mounted onto the slides as described . The sli35S-MET.Details of the DNA pull-down assay was performed as described . SequencCell extracts were prepared with TNN buffer and analyzed as described . AntibodTotal RNAs were isolated by using Trizol following the manufacturer's protocol. Then, RNA was reverse transcribed (Quanta) to obtain cDNA for PCR. cDNA was subjected to real-time PCR using the SYBR Green PCR reagents kit (Stratagene). Experiments were performed in triplicate for each data point. GAPDH was used as a control for normalization. Primers are listed in the Student T-test was used for P value calculation. * stands for P < 0.05 if not specified. In all the bar charts, each “n” represents an independent experiment done in duplicate or triplicate for data with standard deviation, and the representative results are shown in this article. For the tissue microarray samples (n = 63), in order to assess if there is any difference between the demographics of the Low/Low group and the High/High group, Chi-Square test was conducted for the categorical variables while independent sample T-Test was utilized for the continuous variables. Calculation and comparison of survival functions was conducted using Kaplan-Meier method. The differences in the time to event distribution between the interested groups were examined using the log-rank test.


Graft failure is a major cause of morbidity in patients with burns, resulting in increased length of hospital stay and increased number of operations. At our regional burns unit we collated the data from anaesthetic charts of patients admitted to our burns ICU who required skin grafting. The aim was to analyse whether any anaesthetic variables contribute to graft failure.Thirty-five patients were included in the analysis with a total of 191 operations. These were a combination of debridement, split skin grafts (SSG) and change of dressings. All patients were admitted to our burns ICU between January 2009 and October 2013. Exclusion criteria were death prior to discharge and initial surgery at a different hospital. Sixteen patients had good graft viability (Group A) and 19 patients had poor graft viability (Group B). Logistical regression was performed using SPSS (Version 22.0). Hosmer and Lemeshow testing was used to confirm goodness of fit. Independent variables were age, sex, preoperative haemoglobin, intraoperative fluid resuscitation, blood products, inotropes, volatile agents and temperature. Poor graft viability was defined as requiring at least one additional skin graft. Analysis was performed on all operations and then by subtype of operation .P = 0.035, 95% CI). When analysis was performed on only SSG and debridement operations, colloids remained significant  and metarminol use was found to significantly contribute  to poor graft viability. Overall use of inotropes was not significant between the two groups. Other variables including minimum and maximum temperature, preoperative haemoglobin and blood transfusion were not found to be significant.There was no significant difference in age, %total burn surface area or Belgian Outcome Burns Injury score between the groups. For all operation data, use of colloids was found to significantly contribute towards poor graft viability (Our tesults suggest that the use of colloids is a contributor to poor graft viability in burns. This was found to be independent of temperature and overall inotrope use; however, the use of metarminol may be a contributing factor.


Robotic devices have been utilized in gait rehabilitation but have only produced moderate results when compared to conventional physiotherapy. Because bipedal walking requires neural coupling and dynamic interactions between the legs, a fundamental understanding of the sensorimotor mechanisms of inter-leg coordination during walking, which are not well understood but are systematically explored in this study, is needed to inform robotic interventions in gait therapy.In this study we investigate mechanisms of inter-leg coordination by utilizing novel sensory perturbations created by real-time control of floor stiffness on a split-belt treadmill. We systematically alter the unilateral magnitude of the walking surface stiffness and the timing of these perturbations within the stance phase of the gait cycle, along with the level of body-weight support, while recording the kinematic and muscular response of the uperturbed leg. This provides new insight into the role of walking surface stiffness in inter-leg coordination during human walking. Both paired and unpaired unadjusted t-tests at the 95 % confidence level are used in the approriate scernario to determine statistical significance of the results.We present results of increased hip, knee, and ankle flexion, as well as increased tibialis anterior and soleus activation, in the unperturbed leg of healthy subjects that is repeatable and scalable with walking surface stiffness. The observed response was not impacted by the level of body-weight support provided, which suggests that walking surface stiffness is a unique stimulus in gait. In addition, we show that the activation of the tibialis anterior and soleus muscles is altered by the timing of the perturbations within the gait cycle.This paper characterizes the contralateral leg’s response to ipsilateral manipulations of the walking surface and establishes the importance of walking surface stiffness in inter-leg coordination during human walking. Robot-assisted gait therapy has been explored as an alternative to conventional physiotherapy because robots can perform many repetitions with high accuracy . RepetitA limitation of the robotic devices is that they do not consider mechanisms of inter-leg coordination and how the sensory feedback from one leg affects the motion of the other leg. Instead, the state of the art devices impose motion on the impaired limb. Human walking, in addition to running and stair climbing, requires inter-limb coordination and neural coupling . A recenInvestigation of the role of afferent sensory feedback to gait control mechanisms of inter-leg coordination usually involve sensory perturbations and the analysis of their effects. Various platforms and protocols have been used to investigate bilateral reflex mechanisms during different phases of the gait cycle –21, withOne significant limitation of the previous studies is that the sensory perturbations presented in the previous experiments were almost exclusively caused by dropping the walking surface, which causes a disruption in both force and kinesthetic kinematic feedback. When the walking surface is dropped there is a change in leg kinematics, and the force feedback on the bottom of the foot is lost as the foot loses contact with the walking surface. These types of perturbations do not provide any separation of those two sources of sensory feedback, and do not allow further in-depth investigation of the role of force and kinesthetic feedback in gait. In order to answer important questions on inter-leg coordination and sensorimotor control, it is desirable, therefore, to differentiate force and kinesthetic feedback. Adjustment of the stiffness of the walking surface is a unique way to achieve this differentiation, since stepping on a low stiffness platform continues to provide force feedback but affects kinematics.A few studies have utilized compliant surfaces in researching sensorimotor mechanisms in human locomotion including while stepping on/off, hopping, or walking on a compliant surface –32. WhilMoreover, another limitation of all of the previously mentioned works, including those utilizing compliant surfaces, is that the previous studies have failed to separate the mechanisms of inter-leg coordination from that of balance support. As a result, mechanical perturbations and sudden load changes would have likely triggered reflex mechanisms and vestibular responses to maintan balance and stability. However, little is known whether the bilateral activations are exclusively caused by the mechanisms required for body stabilization and balance maintenance, or if it is also brought about from inter-limb coordination and mechanisms of gait. This lack of knowledge leaves a significant gap in our understanding of sensorimotor control of gait and the effect of surface stiffness on gait mechanisms.In this paper we utilize the unique capabilities of the Variable Stiffness Treadmill (VST) system , 34 to iIn order to understand the role of surface stiffness in inter-leg coordination during human walking, a variety of unilateral stiffness perturbations were induced using the Variable Stiffness Treadmill (VST) system , 34 showx from the pivot joint, is dependent on the coefficient of stiffness S of the linear spring and the moment arm through which it exerts a force  walked on the treadmill for at least 200 gait cycles while being supported with approximately 30 % BWS. A value of 30 % BWS was chosen because this level of support has been given in other studies , the stiffness was immediately dropped to 1 of 3 values: 10, 50 or 100 kN/m. The low stiffness perturbation began approx. 130 ms after heel-strike and lasted for the duration of the left leg stance phase (i.e. until toe-off) after which the stiffness was commanded back to 1000 kN/m for the next n number of steps. A graphical representation of the timing and magnitude of the stiffness perturbations is shown in Fig. For this experiment, five healthy subjects  walked on the treadmill for at least 100 gait cycles while being supported with the selected level of BWS. Similar to the previous experiment, the surface underneath the left leg was commanded to maintain a stiffness of 1000 kN/m and then, after a random number n of steps, where n∈, the stiffness was immediately dropped to 60 kN/m approx. 130 ms after heel-strike. The perturbation lasted for the duration of the left leg stance phase after which the stiffness was commanded back to 1000 kN/m for the next n number of steps. A walking surface stiffness of 60 kN/m was used for each level of BWS and was chosen because it is an intermediate value in the range of stiffness perturbations used in the first experiment. A graphical representation of the timing and magnitude of the stiffness perturbation for this experiment is shown in Fig. This experiment was broken up into four sub-experiments, where the only difference between each sub-experiment was the level of BWS  provided to the subject. Five healthy subjects  walked on the treadmill for at least 150 gait cycles while being supported with approximately 30 % BWS. A value of 30 % BWS was chosen to provide some balance support and to allow for comparison with the first experiment. The surface underneath the left leg was commanded to maintain a stiffness of 1000 kN/m for 30 gait cycles at the beginning of the experiment. Then, after a random number n of steps, where n∈, the stiffness immediately dropped to a level of 60 kN/m when the middle of the subject’s left foot reached a certain percentage of the left stance phase. A stiffness magnitude of 60 kN/m was chosen in order to be consistent with the second experiment. The perturbation began at one of four locations  that was randomly selected and lasted until the end of the left leg stance phase after which the stiffness was commanded back to 1000 kN/m for the next n number of steps. A graphical representation of the timing and magnitude of the stiffness perturbations for this experiment is shown in Fig. This experiment was similar to the first experiment, except that instead of changing the magnitude of the stiffness perturbation, the timing of a stiffness perturbation of constant magnitude (60 kN/m) was altered. In this experiment, five healthy subjects [age 25 ± 3.6 years, weight 756 ± 165 The data analysis of the kinematic and muscular response of the unperturbed leg was the same for each of the three experiments described above. In all of the experiments, kinematic data for both legs were obtained at 140 Hz using the previously mentioned infrared camera system that tracked 12 (6 on each leg) infrared LEDs placed as pairs on the thigh, shank, and foot. The muscle activity of the unperturbed leg was obtained using surface electromyography (EMG) via a wireless surface EMG system  and recorded at 2000 Hz. Electrodes were placed on the tibialis anterior (TA) and soleus (SOL) of the right leg. Raw EMG signals were processed by finding the moving root mean square envelope of each signal with a 250 ms window. After computing the EMG linear envelope, the data were normalized to the maximum value of that EMG signal. EMG electrodes were not placed on the left leg because the focus of this work is to understand inter-leg coordination in human walking by investigating the response of the unperturbed leg to unilateral stiffness perturbations. Therefore, even though the left leg was directly perturbed through the stiffness change of the left walking surface, the analyses for the rest of the paper will be focused on the effects of the perturbation on the response of the contralateral leg. Moreover, we have shown in previous studies that the ipsilateral leg kinematics are significantly affected by stiffness perturbations , 34. ThuThe kinematic and EMG data corresponding to the gait cycles of normal conditions and the cycles pertaining to the perturbations were found and normalized temporally to percent gait cycle in order to eliminate discrepancies due to natural variations in gait patterns . The data of each gait cycle was resampled at each 0.1 % of the gait cycle (approximately 1.5 ms). The first 30 gait cycles and the cycles in between perturbations during the normal conditions are included in the unperturbed data set. One or two cycles (depending on which of the three experiments) following a perturbation are not included in the unperturbed set in order to eliminate any residual effects from the perturbation. This results in normalized kinematic and EMG signals as a function of percent gait cycle, where 0 and 100 % correspond to the heel-strike of the left leg.unpaired t-test at each time instance. The unpaired t-test was selected in this case because it is a comparison of two independent distributions (i.e. gait cycles with and without perturbation) which have similar variances but different sample sizes. In order to evaluate the significance of perturbations across subjects, the paired t-test was used. Two values (the mean amplitude of cycles with and without perturbations) from each subject were used to test the significance of response to the perturbation at each time instance. Each test was performed at the 95 % confidence level and any potential Type I errors from tests being performed at each 0.1 % of the gait cycle were eliminated by only concluding significance if at least 40 tests (i.e. 4 % of the gait cycle) in a row indicated significance.Two different t-tests were utilized to establish statistical significance of the results. In order to evaluate the significance of recorded responses in both kinematics and EMG when compared to the normal condiction, statistical significance was determined using an unadjusted A latency of response in each experiment is calculated from the beginning of the perturbation until there is a statistically significant difference between the TA EMG magnitude recorded during the perturbation and normal conditions.As mentioned previously, the results for this experiment have been presented before , 37, butThe experimental data of the response of the unperturbed leg shows a systematic evoked response in both kinematics and muscular activity. The majority of these evoked changes begin near 22 % and then converge back to the normal walking pattern later in the gait cycle. The evoked response was statistically significant at the 95 % confidence level for a two sample unpaired t-test when comparing the data for each perturbation level to the rigid  condition. Colored bars indicating when significant changes are observed are included in Fig. Moreover, the response in the contralateral TA is scalable such that as the magnitude of the perturbation increases , there are increased changes in TA activation. The significant increase in TA activation with lower walking surface stiffness is shown by using a paired t-test to compare the mean TA values at two levels of stiffness for all subjects. For example, the evoked TA EMG during the lowest stiffness perturbations 10 kN/m) is significantly greater than the medium stiffness perturbations (50 kN/m) from 16.8 to 42.5 percent of the gait cycle. This is shown in the first row of Table 0 kN/m isWe first present the evoked muscle activity of the TA in the perturbed leg for all levels of BWS for comparison and validation with the first experiment. The normalized EMG amplitude for the TA (mean and standard deviation) for all perturbed and unperturbed gait cycles pertaining to each level of BWS for a representative subject is shown in Fig. Similar to experiment 1, there is a statistically significant increase in TA activity between approx. 20 to 40 % of the gait cycle, as well as occasionally at other times later in the gait cycle (such as shortly after heel-strike of the right leg). The significant evoked muscle activity during swing phase is observed for all levels of BWS and is consistent across subjects. The time (in percent gait cycle) of when significant TA EMG is first seen  for all subjects is shown in Table The kinematic and muscular response due to variation in the onset of the low stiffness perturbations for a representative subject is shown in Fig. The results presented in this paper indicate that walking surface stiffness is a significant stimulus in gait. Moreover, the timing of evoked muscle activity in the contralateral leg suggests that the feedback may be modulated by supra-spinal neural circuits. Discussion of the importance of walking surface stiffness in gait and the potential application in robot-assisted gait therapy will be presented below.As mentioned previously, stiffness control provides a unique way to perturb leg kinematics while allowing the foot to maintain contact with the walking surface. The repeatability (consistency across subjects and experiments) and scalability (significant increase in EMG activity with decreasing stiffness) suggest that the stiffness of the walking surface is an important stimulus in gait.Moreover, the consistent latency of evoked muscle activity after the perturbation in the results suggest that supra-spinal neural circuitry is stimulated through sudden low stiffness perturbations. The latency averaged across subjects and experiments resulted in a mean of 202 ± 60 ms. A delay of this duration corresponds to transcortical circuitry  suggestiIn addition, the timing of the evoked EMG in both the TA and SOL within the gait cycle suggests that supraspinal structures modify the amplitude of the neuromuscular response to sensory stimuli created by sudden changes in surface stiffness but does not initiate activation of the muscles in gait. This is consistent with the theory that supraspinal structures are not responsible for generating basic gait motor patterns through cyclical flexion and extension of the joints, but rather in modulating these basic gait patterns with descending inputs . As can In addition to all of the above, the experiment performed with different levels of BWS (experiment 2) suggests that stiffness is an important stimulus because the low stiffness perturbations evoke the same amplitude of contralateral TA activity despite changes in load force created by changes in BWS, as was shown in Figs. There is a need to develop and investigate therapeutic protocols that utilize inter-limb coordination , and thiMoreover, the evoked TA activity during swing phase that is presented in this study is an exciting result from a clinical perspective. A main deficiency in stroke survivors is insufficient TA activity (which is the primary muscle creating dorsiflexion) in the swing phase which results in decreased dorsiflexion (toe-up motion). Insufficient dorsiflexion during walking, referred to as drop-foot, is a problem that many impaired walkers suffer from, and is the leading cause of after-stroke falls . Since cms from the beginning of the perturbation to the evoked response that was observed during all experiments suggests the existence of supra-spinal mechanisms of inter-leg coordination. Moreover, the magnitude and latency of evoked muscle activity did not vary with different levels of body-weight support which suggests that walking surface stiffness is an important stimulus in gait independent of the level of body-weight support that is provided.This paper presents results of evoking kinematic and muscular changes in the contralateral leg of healthy subjects using unilateral low stiffness perturbations of the walking surface. By systematically altering the magnitude and timing of the stiffness perturbations, along with the level of body-weight support, we provide new insight into the role of surface stiffness in inter-leg coordination during human walking. The presented study demonstrates the importance of the stiffness of the walking surface as a stimulus in human gait. This importance is indicated by systematic changes in contralateral tibialis anterior activation, as well as lower limb joint angles, in response to unilateral stiffness perturbations. In addition, a latency greater than 200 The results presented in this paper provide novel insight into the role of sensory feedback, specifically surface stiffness, in inter-leg coordination during human gait. Moreover, the repeatability and scalability of the evoked muscle activity presented in this paper suggest the feasibility of using walking surface stiffness perturbations in gait rehabilitation. Future work will include testing of this hypothesis with hemi-plegic stroke patients and other impaired walkers.


Pongo pygmaeus) Facial Action Coding System (OrangFACS), we demonstrate that facial movements are more intense and more complex when recipient attention is directed towards the sender. Therefore, production of the playface is not an automated response to play (or simply a play behaviour itself) and is instead produced flexibly depending on the context. If sensitivity to attentional stance is a good indicator of intentionality, we must also conclude that the orangutan playface is intentionally produced. However, a number of alternative, lower level interpretations for flexible production of signals in response to the attention of another are discussed. As intentionality is a key feature of human language, claims of intentional communication in related primate species are powerful drivers in language evolution debates, and thus caution in identifying intentionality is important.Primate facial expressions are widely accepted as underpinned by reflexive emotional processes and not under voluntary control. In contrast, other modes of primate communication, especially gestures, are widely accepted as underpinned by intentional, goal-driven cognitive processes. One reason for this distinction is that production of primate gestures is often sensitive to the attentional state of the recipient, a phenomenon used as one of the key behavioural criteria for identifying intentionality in signal production. The reasoning is that modifying/producing a signal when a potential recipient is looking could demonstrate that the sender intends to communicate with them. Here, we show that the production of a primate facial expression can also be sensitive to the attention of the play partner. Using the orangutan ( Nonhuman primate facial expressions (as well as their human counterparts) have long been considered to be hard-wired, emotional displays e.g., . Facial not intentional, unless systematic evidence suggests otherwise.Efforts to understand the evolution of human language, the production of which is highly dependent on intentionality and related abilities such as theory of mind , often sintended or not, researchers have attempted to use various observable behaviours to distinguish intentional communication from unintentional, automated communication. Leavens and colleagues  claimed to demonstrate intentionality can be applied rigorously to the production of a prototypical orangutan facial expression. The relaxed open mouth display  modify their facial signals depending on the attentional stance of another individual (the recipient) during spontaneous play interactions, and thus meet one of the established criteria for intentionality. We also recorded additional variables to control for potential confounds of more intense play when face-to-face, and responses to the recipient’s facial expression. We recorded playfaces during social play, and examined the influence of (1) the recipients orientation towards the sender, (2) the recipient’s facial expression, and (3) intensity of play, on the complexity  and intensity (extent of mouth opening) of the playface.The goal of this study was to use OrangFACS  to deterThe spontaneous dyadic play of 20 orangutans was observed in total. Nineteen individuals  featured as focal individuals in the analysis . Twelve individuals were housed at the nursery of the Sepilok Orangutan Rehabilitation Centre (SORC), Malaysia. Inside enclosures consisted of cages where the orangutans stayed overnight either individually or in pairs, and in larger groups during the day. They were taken outdoors (outside their cages) for several hours in the morning and afternoon as part of their training programme where they were filmed during spontaneous play. The remaining eight individuals were semi-free ranging as they had been previously released by SORC into the Kabili Sepilok Forest Reserve. They lived in this forest area during day and night, and were filmed during spontaneous play. Feeding took place three times per day. The nursery-housed orangutans were fed inside their cages. The released orangutans obtained the food from feeding platforms in the forest (provided by SORC), but they were also showing natural foraging behaviours. There was no interaction between the individuals from SORC and the Forest Reserve during the data collection period.A total of 12 h of spontaneous play behavior was extracted from 39 h of ad libitum  social iThe video footage was then coded frame-by-frame (25 FPS) using Adobe Premiere Pro CS4 v.4 and Mangold Interact software. In each play dyad, one of the individuals was randomly chosen as the focal individual. All open mouth facial expressions (OMF) were identified using a broad, inclusive operational definition based on OrangFACS  or AU27 (mouth stretch) to avoid a priori assumptions about the form of play facial expressions). Any OMFs with poor visibility, where the onset was not visible, or where there was physical biting were discarded. OMFs were treated as separate events if the mouth was fully closed for at least 2 s between movements.The following binomial factors were coded for every OMF, and when any behaviour was not clearly discernable it was marked as unscorable:(1)Facial orientation: whether the individuals were facing each other and had an unobstructed view of each other’s face within an angle of 45 degrees of head rotation ((2)Recipient facial expression: whether the recipient individual displayed an OMF at any point during the duration of the focal OMF (OMFR or nOMFR).(3)Play intensity: the speed, strength and degree of physical contact of play behaviour between focal and recipient individuals during an OMF (low or high). Play bouts including resting, temporary breaks from play and slow grappling were classed as low intensity play. Play bouts containing chasing, gnawing, grappling, hitting and wrestling were classed as high intensity.FACS  are useful tools to quantify subtle changes in primate facial signals. The first FACS was developed as an anatomically based observational tool for the measurement of facial movement in humans , and hasK = 0.66, P < 0.0005) and non-focal OMF . Wexler’s index , and can be considered very good agreement.To test for data coding consistency, 30 clips of play were extracted at random from the footage and coded  at both the beginning and end of the project (one year apart). Intra-reliability was measured using Cohen’s Kappa . Good ag’s index  was usedp-values, which explains the impact of factors on the outcome variable as compared to each other. All the statistical analyses were computed in R 3.1.1  were used to analyse our nested data, with defined linear hierarchical groups. The GLMM analysis allowed us to include random factors to control for the fact that: (1) the data set contained missing values , (2) individuals appeared a different number of times as both focals and non-focals, (3) more than one OMF was collected from each play bout and/or from the same individual, and (4) not all individuals played together (since the play was spontaneous). We controlled for multiple observations of the same individuals from the same group by adding the identity of the individuals involved in the interaction nested within groups as a random factor and also added a third random factor to control for repeated dyad composition, thus avoiding pseudoreplication . The fun R 3.1.1 .T = − 2.20, N = 19, P < 0.05). Note that more OMFs were observed than coded . Therefore, as the start time of all AUs within each OMF is approximately the same time in both conditions , the length of the OMF cannot be a factor influencing the number of cumulated AUs, and AUs do not accumulate over time.As the goal was to compare features of the OMF produced during FTF and Not FTF play, we controlled for the fact that OMF durations differed between conditions (and so the number of AUs produced could differ as a function of time rather than condition if they accumulate over time). We compared the onset latencies of AUs in FTF and Not FTF play in a random subset sample using Wilcoxon matched-pairs signed-rank test. We found no significant difference between the mean latency of AUs onset in FTF (mean latency ± SD: 0.12 s ± 0.13) and Not FTF (mean latency ± SD: 0.14 s ± 0.21) conditions  varied depending on facial orientation, recipient facial expression and play intensity, we calculated GLMM with Poisson error distribution and log function. The total number of AUs in each OMF was used as a response factor and the identity of focal and recipient individuals as well as the play bout number were entered as random factors. Facial orientation (FTF vs Not FTF), recipient facial expression (OMFR vs nOMFR) and play intensity (low vs high) were entered as fixed factors. There was no overdispersion in the data set or collinearity in the factors.F3 = 417.26, P < 0.001. Removal of any of the three factors from the full model resulted in a significant change in the model’s AIC, since all the factors were strongly influencing the facial movement complexity of the focal individual during play behaviour : OMF produced when the recipient was facing the sender contained a greater number of AUs . Facial expression of the recipient was not significant (P = 0.06), but as the AIC was lowered significantly when this was taken out of the model, it had a weak positive effect on the model: OMF produced when the recipient also produced an OMF contained a greater number of AUs . Play intensity was also not significant as a fixed factor (P = 0.74). OMF produced when the play intensity was high or low contained a similar number of AUs , but did significantly improve the model fit . Each of the factors alone represented a significantly better fit when compared to the null model  and thus had some impact on complexity of OMFs.In the full model, facial orientation had a significant positive effect as a fixed factor  depending on facial orientation, recipient facial expression and play intensity, we calculated GLMM with binomial error distribution and logit link function. AU26 versus AU27 was imputed as the binary response factor. The identity of focal and recipient and the play bout number were added to the model as random factors. The model was slightly overdispersed , so we added an OMF-level random factor (1 | OMF), where OMF is a vector from 1 to the total number of observations (247) F1 = 17.279, P < 0.001; recipient facial expression vs null model ANOVA: F1 = 138.01, P < 0.001; play intensity vs null model ANOVA: F1 = 16.374, P < 0.001). When comparing the full best model to the null model, the result was also highly significant .The full model retaining all factors provided the best fit for the data see . The fulP < 0.05), being more associated with the stronger AU27 movement than AU26. However, when comparing models, recipient facial expression also had a strong (positive) significant influence , and play intensity had a weak negative influence  on the display of AU26 versus AU27. As low intensity was set as the baseline, a negative influence indicates that more intense play increased the likelihood of AU27 vs AU26. Therefore, playfaces were more likely to contain the more intense AU27 when the recipient was facing the sender (FTF), when the recipient produced an OMF, and when play was more intense  were more intense and contained more component movements. Although previous studies have interpreted similar findings as evidence of intentional communication, we have a more cautious interpretation. These findings may not demonstrate intentionality beyond doubt, but do show that production of the playface is not an automated response to play (or simply a play behaviour itself) and instead is highly flexible and dependent on subtle characteristics of the social context.could demonstrate that the sender is intending to communicate . If we accept this logic, and thus accept that this behavioural marker is a sufficient demonstration of intentional production, we must conclude that, like primate gestures, orangutan playfaces are produced intentionally.Modified and increased production of visual signals as a function of the recipient’s attentional stance has been used as sufficient criteria for intentionality in previous studies of primate gesture e.g., , and as It is possible, however, that sensitivity to attentional stance when producing signals demonstrates a degree of flexibility of production  without the need to attribute goal-directed, purposeful communication. The decision to produce a playface may be voluntary in the same sense that the decision to bite, eat or run may be voluntary in some way—the animal is capable of inhibiting or increasing the behaviour if conditions suggest such an approach might be sensible. Although part of the continuum of intentionality, this is not the same as goal-directed, purposeful intention to communicate to another.There are also other explanations that could be considered. First, face-to-face play could simply be more arousing and stimulate the production of more play (of which the playface is a component). Play intensity did have a weak positive impact on the intensity of the playface in our model, but also a negative impact on the complexity of the playface. Therefore, play intensity does seem to be one contributory factor (on intensity at least), but is not strong and does not influence the composition of the signal. Thus, increased playface production during face-to-face play is not simply a function of face-to-face play being more intense. Second, playfaces might be stronger during visual contact with the play partner due to reflexive mimicry of the partner’s facial expression  have long been seen as rather fixed, biologically based expressions of emotion, reflecting the internal state of the sender, a theoretical stance reinforced since 10.7717/peerj.827/supp-1Supplemental Information 1Click here for additional data file.


To protect from erosion of the tube in glaucoma drainage device (GDD), the tube is covered by a biologic tissue which is roofed by the conjunctiva. Sclera, pericardium, dura mater and cornea are available as a patch graft. Drawbacks of some of these materials may include high cost and poor appearance. The purpose of this study is to report the long-term outcomes of partial thickness corneal grafts to cover the tube and prevent its exposure, in GDD surgeries.This was a retrospective review of all patients who underwent Ahmed glaucoma valve implantation and had a minimum follow-up of 12 months. The tube was covered by a 300-micron partial thickness corneal graft taken either from a previous Descemet stripping endothelial keratoplasty procedure or cut from a whole corneal graft button unsuitable for keratoplasty.Forty-four patients  were enrolled. The partial thickness corneal grafts maintained clarity throughout follow-up with satisfactory cosmetic results. Mild conjunctival retraction occurred in 4 eyes (8.9 %) between 1 and 12 months after the surgery. Corneal graft melting occurred in 3 (6.7 %) eyes. Tube exposure and additional surgery to re-patch or suture the conjunctiva over the tube was needed in 1 (2.2 %) eye. None of the patients had graft infection or immunologic rejection.Partial thickness corneal grafts have favorable long-term outcome as a patch for GDD tubes with low rates of tube exposure and other complications. Glaucoma drainage device (GDD) surgeries are traditionally performed after failed trabeculectomy or in cases where trabeculectomy is at high risk for failure, such as in the management of complex glaucomas . The morDemographic and perioperative data were collected through a retrospective record review of all patients who underwent GDD surgery with partial thickness corneal graft covering between 2007 and 2011 at the Tel Aviv Sourasky Medical Center. Inclusion criteria were age above 18 years at the time of surgery and a minimum follow-up of 12 months. Patients were excluded if they had undergone a previous GDD surgery or if they had a past or current ocular surface disease that could affect the healing of the covering conjunctiva. Patients who had concomitant surgeries, such as pars plana vitrectomy or cataract surgery, were also excluded.Corneas were obtained from the Tel Aviv Sourasky Medical Center Lions Eye Bank after being approved for transplantation. Corneas were stored in Eusol-C Corneal Storage Media . Tissue evaluation included serology for hepatitis B and C, as well as for HIV. All surgeries were performed by one surgeon (RR) who was also responsible for the clinic visits that included intraocular pressure (IOP) measurements with Goldmann applanation tonometry  before and periodically after surgery. The study followed the tenets of the Declaration of Helsinki and was approved by the ethics committee of the Tel-Aviv Sourasky Medical Center.All GDDs were implanted through a fornix-based incision using the FP7 Ahmed glaucoma valve , with the plate secured 8–12 mm from the corneoscleral limbus. Thirty-three eyes (73.3 %) underwent superotemporal GDD implantation, 11 eyes (24.4 %) underwent inferotemporal implantation, and 1 eye (2.2 %) underwent inferonasal implantation.After priming, the tube was inserted through a scleral track posterior to the limbus by a 23-gauge needle, and introduced by an inserter into the anterior chamber. Viscoelastic was injected into the anterior chamber to maintain its proper depth. The partial thickness corneal graft, which was used to cover the tube, was sutured to the sclera with four 10-0 nylon sutures. The conjunctiva was closed meticulously with 8-0 vicryl and 10-0 nylon sutures, taking care to ensure that it fully covered the corneal graft.2 and expired tissue [7–60 days from harvesting]).A partial thickness corneal graft was taken either from a previous Descemet stripping endothelial keratoplasty (DSEK) procedure or cut from a whole corneal graft button. Corneal grafts prepared for a DSEK procedure were cut by an automated microkeratome system  with a 300 micron head passed to create a lamellar dissection. This formed an anterior cap mainly composed of stroma and a posterior endothelial lamellar donor graft. The posterior corneal lamella was used for the DSEK procedure, while the anterior corneal lamella was either discarded or used to cover the tube in the GDD surgery of a different patient. In other cases, a partial thickness corneal graft was prepared from a full-thickness corneal graft that was unsuitable for keratoplasty .All the data were recorded on Microsoft Excel™ spreadsheets. A paired-samples P < 0.001) than the preoperative IOP values (Table Forty-four patients (45 eyes), comprised of 25 males and 19 females with a mean postoperative follow-up of 27.6 ± 11.4 months (range 12–60 months) fulfilled the inclusion criteria and were included in the analysis. Their demographic characteristics and clinical data are summarized in Table Thirty-three (73.3 %) partial thickness corneal grafts were retrieved from previous DSEK procedures and 12 grafts (26.7 %) were prepared during the GDD surgery from full corneal grafts that were unsuitable to serve as an optical corneal graft. The mean corneal patch diameter was 8.3 ± 0.2 mm (range 8.0–.0 mm). The conjunctiva that covered the corneal patch graft had a button hole during surgery in 1 case (2.2 %). The conjunctival edges were approximated by 8-0 vicryl sutures. There were no other intra-operative complications.None of the patients had graft infection or immunologic rejection and the partial thickness corneal grafts maintained clarity throughout follow-up. There were no GDD-related complications, such as diplopia, inflammatory reaction in the conjunctiva or plate exposure. Four eyes (8.9 %) had conjunctival retraction between 1 and 12 months after the surgery. As the conjunctival retraction was minimal, Seidel test was negative, and the tube was well-covered by the patch graft, these patients were observed without treatment. Conjunctival erosion with tube exposure occurred in 1 case (2.2 %) with superotemporal GDDs. In this patient the corneal patch graft was also melting 1 month after surgery Table . The patPartial thickness corneal patch grafts covering GDD tubes have favorable long-term outcomes. According to our results, these grafts are associated with a 6.7 % rate of corneal graft melting and a 2.2 % rate of tube exposure. Only one case necessitated additional surgery to cover the exposed tube. A GDD transports aqueous humor from the eye through a tube to an episcleral plate located in one of the quadrants of the globe. The aqueous diffuses through the capsular wall and is absorbed by periocular lymphatics and capillaries , 9. PostConjunctival retraction leading to patch graft exposure is usually considered a minor complication, as long as the tube is well covered by the patch graft . This coDirect contact between the conjunctiva and the tube will cause the tube to erode the overlying conjunctiva over time and become exposed , puttingConsiderations in the choice of patch graft material include biocompatibility, availability, immunologic safety, ease of use, cost and cosmetic appearance . The useThe biologic material used as a patch graft in GDD surgeries has at least some role in preventing tube erosion , but disWhile a patch graft must be completely covered by the conjunctiva during the surgery, conjunctival laceration or button-hole formation may occur . Simple Limitations of this study include its retrospective nature, the relative small sample size and the lack of a control group with which to compare our results. Prospective randomized studies comparing partial thickness cornea to other patch graft materials are needed in order to determine which patch is less prone to conjunctival erosion and subsequent tube exposure. Cost considerations, the ideal thickness of the patch graft and its cosmetic appearance should also be taken into consideration. Since our analysis included only patients with follow-up of ≥12 months, complications occurring in patients with shorter follow-up were not reported, and this could have been a confounding factor. Although the mean postoperative follow-up time was more than 2 years, it was only 12 months in four patients. It is not clear whether a longer follow-up would reveal more cases of conjunctival dehiscence or corneal graft melting, but we consider it most probable that more of them would emerge over time. Some authors argued that these complications usually occur within the first 15-month postoperative period . Graft mA partial thickness corneal graft is a useful patch graft material for covering a GDD. It is associated with low rates of conjunctival retraction, graft melting, tube exposure and need for additional surgery.


There is considerable inter-individual variability in self-selected intensity or running speed. Metabolic cost per distance has been recognized as a determinant of this personal choice. As biomechanical parameters have been connected to metabolic cost, and as different running patterns exist, we can question their possible determinant roles in self-selected speed. We examined the self-selected speed of 15 terrestrial and 16 aerial runners, with comparable characteristics, on a 400 m track and assessed biomechanical parameters and ratings of pleasure/displeasure. The results revealed that aerial runners choose greater speeds associated with shorter contact time, longer flight time, and higher leg stiffness than terrestrial runners. Pleasure was negatively correlated with contact time and positively with leg stiffness in aerial runners and was negatively correlated with flight time in terrestrial runners. We propose the existence of an optimization system allowing the connection of running patterns at running speeds, and feelings of pleasure or displeasure. Summary: Aerial and terrestrial human running patterns self-select different pleasure running speeds. We suggest the existence of an optimization system allowing association of runners' stride with running speed. In the TER group, a large correlation between feeling score and tf  was observed. It is of note that there are only unclear relationships for tc  and kleg  in TER and for tf  in AER.AER runners chose a greater speed , and is fully described elsewhere  and TER (N=15) did not show significant differences  and the maximal leg length deformation, i.e. leg spring compression (ΔL) calculated with the centre of mass displacement (Δz), using the following equations:Twenty meters of an optical measurement system  sampling at 1000 Hz was used to record ve model , as the t-tests were used to compare participants' characteristics, self-selected speed, pleasure-displeasure, and biomechanical parameters  between AER and TER. Correlation coefficients were used to assess whether the feeling score of pleasure-displeasure correlated with the self-selected speed and biomechanical parameters for AER and TER separately. The following criteria were adopted to interpret the magnitude of the correlation between the different measures: ≤0.1, trivial; >0.1-0.3, small; >0.3-0.5, moderate; >0.5-0.7, large; >0.7-0.9, very large; and >0.9-1.0, almost perfect  and Hopkins spreadsheets (http://www.sportsci.org).Descriptive statistics are presented using mean±s.d. values and correlation statistics using mean±90% confidence limits (c.l.). Student  perfect . If the


In recent years, there have been numerous calls for global institutions to develop and enforce new international laws. International laws are, however, often blunt instruments with many uncertain benefits, costs, risks of harm, and trade-offs. Thus, they are probably not always appropriate solutions to global health challenges. Given these uncertainties and international law’s potential importance for improving global health, the paucity of synthesized evidence addressing whether international laws achieve their intended effects or whether they are superior in comparison to other approaches is problematic.Ten electronic bibliographic databases were searched using predefined search strategies, including MEDLINE, Global Health, CINAHL, Applied Social Sciences Index and Abstracts, Dissertations and Theses, International Bibliography of Social Sciences, International Political Science Abstracts, Social Sciences Abstracts, Social Sciences Citation Index, PAIS International, and Worldwide Political Science Abstracts. Two reviewers will independently screen titles and abstracts using predefined inclusion criteria. Pairs of reviewers will then independently screen the full-text of articles for inclusion using predefined inclusion criteria and then independently extract data and assess risk of bias for included studies. Where feasible, results will be pooled through subgroup analyses, meta-analyses, and meta-regression techniques.The findings of this review will contribute to a better understanding of the expected benefits and possible harms of using international law to address different kinds of problems, thereby providing important evidence-informed guidance on when and how it can be effectively introduced and implemented by countries and global institutions.CRD42015019830PROSPERO The online version of this article (doi:10.1186/s13643-016-0238-0) contains supplementary material, which is available to authorized users. Framework Convention on Tobacco Control (2003) and the International Health Regulations (2005) [In recent years, there have been numerous calls for global institutions to develop and enforce new international laws in the realm of health. These international laws, which regulate relations between and within countries, vary widely in their subject matter. Focus areas for these calls have included alcohol , antimic Control 03 and thAgreement on Trade-Related Aspects of Intellectual Property Rights (TRIPS). This law serves as the de facto global regulating policy for all forms of intellectual property, including copyrights, patents, and trademarks. The rules set out by this agreement have major implications for the medical product industry and thereby influence global access to medicines [Although few international laws have been adopted specifically to promote human health, many international laws have possible indirect effects on health, as they may impact the social determinants of health . Examples of social determinants of health include armed conflict, employment, empowerment, environment, finance, human rights, poverty, sanitation, social policies, trade, and water supply , 22. Oneedicines , 24.While many stakeholders have advocated for international laws that provide solutions to complex global health challenges –19, otheGiven these uncertainties associated with international law, and its ever-increasing popularity as a potential tool for global change, there is surprisingly little synthesized evidence on whether international laws achieve their intended results or whether they are superior to other available instruments for coordinating international actors. The current lack of synthesized evidence on the effectiveness of international law has posed a significant barrier to understanding the potential value, feasibility, and applicability of international law for global health and related challenges.A scoping review by Hoffman and Røttingen (2015) attempted to delineate the effects of international law by qualitatively summarizing 90 quantitative impact evaluations of different international laws . That reWhat are the effects of international law on health behaviors and outcomes?What are the effects of international law on the social determinants of health?Upon what factors are effects of international law conditional?This planned systematic review will build on the approach and findings of that recent scoping review, applying subgroup analysis, meta-analysis, and meta-regression techniques to synthesize all available quantitative research evidence on the effects of international law on health and its social determinants. Specifically, this review aims to answer the following three questions:Framework Convention on Tobacco Control (FCTC), which is a global health treaty providing an internationally coordinated response to combatting tobacco use [What are the effects of the FCTC on health behaviors and outcomes like tobacco consumption, lung cancer rates, and life expectancy?What are the effects of the FCTC on social determinants of health like tobacco advertising bans, tobacco taxes, and smoke-free workplace policies?Upon what factors are the effects of the FCTC conditional, such as a country’s development status, regulatory capacity, and government ownership of tobacco companies?For example, in the context of the acco use , the revThis systematic review will boost current understanding on whether and how countries and global institutions might employ international law as a population health intervention for addressing today’s pressing global health challenges.We will conduct this systematic review, meta-analysis, and meta-regression adhering to the following protocol and will report any changes to the protocol that arise as we proceed. The methods and design of this systematic review are based on recommendations from the Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA-P) statement . See AddAll quantitative impact evaluations that measure the effect of an international law on outcomes related to health or its social determinants will be included. These may include experiments , quasi-experiments , and observational designs .Given that international law only imposes obligations on states , “participants” will include all current and former countries, formally recognized as states by the international community, which is best indicated by membership in the United Nations or a similar multilateral organization. We will interpret this definition broadly to include jurisdictions that are often viewed as states by some entities .Any implementation of international law related to health or its social determinants between at least two states will be considered as an intervention. International law is commonly referred to as agreements, charters, conventions, declarations, exchange of notes, memorandums of understanding, modus vivendi, protocols, treaties, and at least 30 other names. It is defined as the “rules and principles of general application dealing with the conduct of states and of international organizations and with their relations inter se, as well as with some of their relations with persons” . Randomipeople , places , products , or policies . As this is an exploratory review, we have not specified exact outcomes a priori and have no reason to believe that one outcome will be more important than another. We will base the outcomes upon what data is available in the set of included studies and treat all outcomes as primary outcomes. However, we will preferentially extract and report outcomes that are found to have more data.To be included, a study has to measure the effect of international law on an objective and quantifiable outcome related to health or its social determinants, which, according to the World Health Organization’s Commission on the Social Determinants of Health (2008), include armed conflict, employment, empowerment, environment, finance, human rights, poverty, sanitation, social policies, trade and water supply, and among other factors . OutcomeThe search strategy for this review was developed in consultation with one health sciences librarian at McMaster University and one at the University of Toronto. The following ten electronic bibliographic databases were searched from inception to July 2014: MEDLINE, Global Health, Cumulative Index to Nursing and Allied Health Literature (CINAHL), Applied Social Sciences Index and Abstracts, International Bibliography of Social Sciences, International Political Science Abstracts, Social Sciences Abstracts, Social Sciences Citation Index, Public Affairs Information Service International , and Worldwide Political Science Abstracts. To find gray literature, we also conducted an additional search in ProQuest’s Dissertations and Theses electronic database. No language, geography, or date restrictions were applied to the searches.The exact search conducted in MEDLINE is presented in Table Is the study a quantitative impact evaluation?Are the participants current or former countries?Is there an international law being discussed?Are the effects of an international law being measured? This systematic review will include any published or unpublished study that aims to quantitatively measure the impact of an international law’s effect on any health-related behavior or outcome, including social determinants of health . StudiesTwo reviewers will independently evaluate titles and abstracts to determine whether or not each article might meet eligibility criteria. The initial title and abstract screening process will make use of the aforementioned four criteria to assess whether articles will be assessed for inclusion at the full-text screening stage. For all studies included after title and abstract screening, four pairs of reviewers will independently screen the full-text of articles for inclusion, applying the same four criteria. A codebook containing examples for all four inclusion criteria will be developed to help guide reviewers through the screening process. Prior to full-text screening, multiple rounds of calibration exercises will be conducted to ensure that there is near-perfect consistency among reviewers. Each round of calibration will be followed by a meeting with all reviewers to resolve any discrepancies. All disagreements that arise during full-text screening will be documented and resolved through discussion and consensus; if consensus cannot be reached, the principal investigator (SJH) will settle disagreements Fig. .Fig. 1PRAdditional records found in the recent scoping review  will go Search results from all databases will be aggregated in the reference management software EndNote, which will also be used to remove duplicate citations.A statistician has been contracted to aid in developing the data abstraction form so as to ensure comprehensive extraction methods that will allow for pooling and analysis of results. Additionally, calibration exercises will be conducted to ensure that there is consistency between reviewers. These exercises will serve to pretest the form before it is used to extract data from eligible studies. An online data abstraction program (Distiller SR) will be used to maximize efficiency—it will be customized for this task using the criteria of a standardized form. Two pairs of reviewers will independently extract data from each eligible study. Data extracted will include general study information, methodology, intervention details, and outcome data. Any disagreements will be documented and resolved through discussion and consensus; when consensus cannot be reached, the principal investigator will settle disagreements.Studies that are consistent with the inclusion criteria will be assessed for bias using Cochrane’s ROBINS-I tool for non-randomized studies of interventions . We do nIn presenting the evidence, the Grading of Recommendations Assessment, Development and Evaluation (GRADE) system of rating the quality of evidence will be used, as recommended by the Cochrane Collaboration .Three types of analyses are currently planned, resulting in at least three sets of results: (1) point estimates and confidence intervals for the impact of international laws in each field on any outcome; (2) point estimates and confidence intervals for the impact of international laws from any field on particular outcomes; and (3) a list of the conditions thus far found to potentially mediate the impact of international laws. Meta-analysis will facilitate the first two lines of inquiry, while meta-regression will help answer the third.In the earlier scoping review of 90 quantitative impact evaluations , most stIn principle, our aim is to be liberal in this judgment: that is, we are prepared to pool results from a relatively broad range of populations, interventions, and outcomes. Having done that, we can examine the variability in results to determine the extent to which the data support the assumption regarding similar effects across populations, interventions, and outcomes. We anticipate substantial variability and will address this through meta-regression analysis (see below).Since we know that the studies will measure outcomes of interest in different units, the effect size will be calculated using the difference in mean values divided by the pooled standard deviation of the comparators . This calculation will result in a measure of the effect known as the standardized mean difference (SMD) . For each study, the SMD is calculated as the difference in mean outcome between groups divided by the standard deviation of outcome among participants, as represented in the following equation:The resulting standard deviations are used to standardize the mean differences to a single scale, as well as in the computation of study weights using the inverse variance method. This method assumes that between-study variation in standard deviations reflects only differences in measurement scales and not differences in the reliability of outcome measures or variability among study populations .I2 measure.The SMD from each study is pooled to provide a summary measure in standard deviation units with an associated confidence interval. Random effects meta-analysis will be used, as this approach is conservative in that it considers both within- and between-study differences in calculating the error term. We will examine the extent of heterogeneity statistically using chi-square tests of heterogeneity and of inconsistency using the In interpreting the results, a rule of thumb suggests that 0.2 standard deviation units represents a small effect, 0.5 a moderate effect, and 0.8 a large effect. In addition to this rule of thumb, to facilitate our interpretation, we will convert the SMDs to measures of effect typically used for binary outcomes. In principle, this approach assumes that data are normally distributed, allowing calculation of the probability that results are greater than equal to a particular threshold. These probabilities allow calculation of odds ratios and risk differences. There are a number of methods available to conduct the conversion from continuous to binary outcomes. We will use the approach described by Furukawa .Meta-regression analysis will be used to systematically explore the reasons for different effect sizes and outcomes across studies. Meta-regressions are similar to standard regressions, in which an outcome variable is predicted according to the values of one or more explanatory variables. In meta-regression, the dependent variable is the effect estimate . The independent or explanatory variables are characteristics of studies that might influence the size of the intervention effect. In meta-regression, larger studies have more influence on the relationship than smaller studies, since studies are weighted by the precision of their respective effect estimate. We will use a random-effects meta-regression in which the residual heterogeneity among intervention effects not captured by the independent explanatory variables is incorporated in the same way as in a random effects meta-analysis.The regression coefficients obtained from a meta-regression analysis will describe how the outcome variable  changes with a unit increase in the explanatory variable . The statistical significance of the regression coefficient is a test of whether there is a linear relationship between intervention effect and the explanatory variable.The meta-regression will allow us to test existing theories in international law and international relations about when law matters and to provide global policymakers with evidence-informed guidance on when different types of international laws may be most helpful (and when they may even be harmful). Among international lawyers and international relations scholars, the effectiveness of international law stands as a subject of great debate. Elucidating the conditions upon which international law has effects, and what these effects may be, will allow for a strengthened understanding of the way in which international law works.Specifically, informed by a new analytic framework we have developed , 37, we In the absence of direct evidence, the aforementioned analytic framework outlines four criteria intended to assist decision-makers to ensure that new international laws are likely to yield positive effects. It takes into account the uncertain benefits, costs, risks of harm, and trade-offs involved with the implementation of international law. Specifically, the four criteria for assessing international laws are as follows: (1) there must be a significant transnational dimension to the addressed problem; (2) the coercive nature of treaties should be justified by the goals of the proposed solution; (3) there must be a reasonable likelihood that the treaty will achieve benefits; and (4) the treaty should represent the best commitment mechanism among competing alternatives . This syBy policy domain: Analysis across policy domain of international laws such as environment, finance, health, human rights, humanitarianism, and trade will allow for comparison of magnitude of effects of laws operating in different areas of intervention.By enforcement mechanism: Level or robustness of enforcement mechanisms embedded in treaties or used by states to encourage compliance such as legal penalties, monetary fines, and “naming and shaming” may impact the magnitude of its effects.By impact/indicator measure: Categorization of studies by impact measures such as economic growth, health status, and trade flows allows for comparison of the effects of international treaties on the same indicators.By scope of the intervention: This may provide insight into the relationship between the number of countries that are party to an international law and the impact measured on outcomes, such as between an international law binding a small group of countries versus a law adopted through the United Nations which may have almost 200 state parties.By study design: Robustness of analytical methods may impact the magnitude of effects determined in each study, and categorization by study design will be necessary when we attempt to pool results.By time period: This may give insight into whether there was enough time to measure an impact and whether international laws adopted at different time periods might have been more successful.By forum: This may provide information on whether international laws have differing effects depending on the process through which they are created and the organization that may have hosted its negotiation and enforcement, such as the United Nations General Assembly, World Health Organization, and World Trade Organization, or without a formal organization.In addition to the meta-regression, we will also do more traditional subgroup analyses. These analyses are currently planned for the following attributes to ascertain effects of different types of international laws under different circumstances:This review will use a rigorous systematic methodology and will represent a novel attempt at investigating an important question that has not yet benefitted from systematic synthesis techniques. While the review will build on a recent scoping review , it is uThis systematic review has several strengths. First, it addresses a particularly salient policy problem, as there exists a widespread and active discussion on the development of new global health laws , 39. TheAn important limitation of the study is its breadth of potential outcomes; the review is very broad, includes many studies, and considers a wide range of factors influenced by international laws. Our application of meta-analysis and meta-regression techniques to these diverse studies and outcomes may be affected by the significant heterogeneity among the pool of eligible studies. This may pose a challenge in comparing the success of different international laws across outcomes as there is much expected heterogeneity between the effects of the laws included. However, our calculation of effect size using SMD will allow us to pool results across different units. Our rigorous approach for measuring heterogeneity will ensure that we only pool results where feasible and appropriate; for example, heterogeneity will be measured prior to meta-analysis. Further, we will continue to have ongoing discussions with statisticians to plan for an efficient and optimal abstraction process in which we collect data that will demonstrate meaningful results. Another important limitation is that while our review includes theses and dissertations, we may have overlooked studies that were not indexed such as government documents or civil society reports.


The spider fang is a natural injection needle, hierarchically built from a complex composite material comprising multiscale architectural gradients. Considering its biomechanical function, the spider fang has to sustain significant mechanical loads. Here we apply experiment-based structural modelling of the fang, followed by analytical mechanical description and Finite-Element simulations, the results of which indicate that the naturally evolved fang architecture results in highly adapted effective structural stiffness and damage resilience. The analysis methods and physical insights of this work are potentially important for investigating and understanding the architecture and structural motifs of sharp-edge biological elements such as stingers, teeth, claws and more. Fangs are segments of the spider mouthparts, which are used to inject venom into prey and are required to sustain large mechanical loads. Here, the authors perform experiment-driven simulations, so to understand the correlation between the multiscale structural gradients and the biomechanical function of the fang. Natural structures adapt their mechanical properties to a large variety of needs, which may be competing, primarily by varying the spatial distributions of their components156781014159171819Spiders belong to the arachnid class of arthropods. They mostly feed on insects, another class of arthropods. Their cuticular fangs are part of the spider mouthparts called chelicerae and are connected to their basal segment by a joint and a pair of muscles. Among their several functions such as those in cleaning behaviour and the widening and opening of the egg sac to release the spiderlings, the mechanically most demanding function of the cheliceral fangs is to puncture the cuticle of prey insects in order to inject the venom212224Cupiennius salei)Here we investigate the structural mechanics of the fangs of a large wandering spider (Computer tomography (μCT) reconstruction  was used to analyse the macroscopic architecture of the spider fang. The fang has a curved shape similar to the cat’s claw and includes a venom canal that spans from its base up to close to the tip .R  and the fang’s local wall thickness w(θ) are then extracted geometrically from the μCT data. They show a monotonic linear increase from the fang’s tip to its base =w(θ)=aθ(θ/90)+a0 is considered, where a0 represents the geometry at the tip (θ=0) and aθ represents the steepness of the conical structure—that is, the structural gradient of the fang. Variations in the fang-like model architecture are addressed by variations in the structural gradient (aθ), with a0 typically being small (a0/R≪1).The circular shape of the spider fang indicates that when a moment is applied at the centre of the circle, the tip of the fang is displaced in the circumferential direction, and a penetration force (tangent to the centreline) is generated at the tip. However, the biting mechanism of the spider is more complex23K1, K2 and K3  analytical structural mechanics (beam theory)Abaqus 6.12). The analytical approach relies on the assumption that the structural deformations are dominated by the normal and torsional stresses, whereas the effect of shear stresses is assumed to be negligible. These assumptions are usually considered adequate for slender configurations with mild structural gradients. The FE approach, on the other hand, adequately solves the set of differential equations governing the mechanical problem without any simplifying assumptions. aθ) of the fang-like model on the orientation and magnitude of its stiffness. The case of aθ=0 represents a curved needle-like architecture with no structural gradient, and aθ=0.3 represents a steep conical architecture in which the venom canal and the fang wall are much thicker at the base than at the tip. K1 direction becomes closer to being tangent to the tip, and K1 is therefore identified as the ‘penetrating stiffness’ of the fang. In accordance, K2 and K3 are defined as ‘perpendicular stiffness’ parameters. K1>>K2≈K3) and that all stiffness parameters monotonically increase as the structural gradient increases. Interestingly, the needle-like fang architecture (aθ=0) exhibits the lowest penetration stiffness, oriented at ~\n30° to the tip direction. This ‘conventional’ engineering solution is therefore less efficient than are the conical configurations.Our stiffness analysis assumed the fang to be fixed at its base, a valid approximation because of the elongated shape of the fang see . Mechanid K3 see . K1 and –y plane  and are aθ<0.05) the analytical and FE results are compatible, indicating that bending and twisting moments dominate the deformations. In this case, significant stresses and strains are localized close to the base of the fang deforming the entire structure  the stresses and deformations are localized more towards the tip, and the rest of the structure is effectively non-stressed and non-deformed , aθ=0.14) and wall thickness (aθ=0.06), and an imperfect axis symmetric cross-sectional architecture  increases and that the maximal stress for the non-graded needle-like architecture (aθ=0) is within an order of magnitude higher than for the conical architectures. Moreover, the stress maximum is shifted from close to the base for the needle-like structure towards the tip for the conical structure.In addition to the stiffness variations, the structural gradients in the fang architecture affect the stress distribution and therefore strength and resilience. To evaluate these effects, von-Mises stress distribution maps  were calaθ≈0.05. Note, however, that the stress intensity in the natural configuration is higher than in the fang-like model, possibly because of the thinner walls and wider canal of the natural fang, which were not considered in the model.The stress distribution in the natural fang is shown in So far, we focused on the macroscopic structural gradients of the spider fang and considered the material to be homogeneous and isotropic. However, as the rest of the spider’s cuticular exoskeleton, the fang is composed of a composite material of stiff chitin nanofibrils and a more compliant proteinous matrix23Analytical mechanics of composite material methods and a bottom-up modelling approach30The chitin–protein architecture has a significant effect on the elastic properties of the layers composing the fang wall. This is demonstrated in β and ω, to describe the layered fang wall architecture  on the K1 and K3 stiffness parameters. The results for K2 are almost identical to those for K1 and therefore not shown. The penetration stiffness of the fang increases as the parallel-fibred portion in the fang wall increases , whereas the perpendicular stiffness increases as the plywood portion increases  provides the highest penetration stiffness but the lowest perpendicular stiffness. A fully plywood fang architecture (ω→1) exhibits the opposite features. ω→0). Note that at the biologically relevant configurations  considered, variations in the properties of the fang material such as the fibril-to-matrix stiffness ratio have only mild effects on the stiffness parameters of the fang structure =α at the tip and ω(θ=90)=1−α at the base of the fang (0≤α≤1). Whereas α=0 represents a fang with plywood architecture at the tip and a parallel-fibred architecture at the base, α=0.5 represents a non-graded configuration of equally thick plywood and parallel-fibred layers all along the fang (ω=0.5). The gradients in the fibril orientation only mildly affect the penetration stiffness of the fang but strongly affect its perpendicular stiffness (α→1)α=0.5).Experimental observations indicate that the elastic modulus and hardness of the fang material are significantly enhanced close to the fang tip, and that the degree of fibril alignment along the fang axis decreases from the tip to the base of the fangtiffness . The natA spider’s fang has to safely sustain high mechanical loads. Although most of its structural features are well documentedOn the macroscopic scale, the curved fang structure enables the spider to attack from different directions and to hold its prey like a claw3233233On the material level, the fang is a chitin–protein composite, combining both unidirectional  and quasi-isotropic (rotated-plywood) architectures. While non-curved biological structures typically are required for a high axial stiffness and usually include unidirectional fibre arrangements only31The multi-scale structural gradients discussed here are complementary regarding the fang’s mechanical functionality, which requires both high penetration stiffness and moderate perpendicular stiffness. The macroscopic structural gradients increase the fang’s specific stiffness in all directions; however, the perpendicular stiffness is much lower than the penetration stiffness. The base-to-tip gradient in the plywood-to-parallel composite architecture of the fang significantly enhances the perpendicular stiffness. This effect is further amplified by the thin plywood surface layer coating the fang material. Matrix hardening of the composite material near the tip3839In conclusion, it appears that the multiscale graded structural architecture of the spider fang is well adapted to support its biomechanical functionalities. The present analysis emphasizes the importance of considering different structural scales in an effort to understand the overall mechanical properties of biological structures. Revealing the mechanical significance of the spider fang architecture may contribute to the understanding of the selective pressures that have driven the evolution of a variety of sharp-edge biological structures, ranging from scorpion stinger to mammoth tusk. The present work may also promote innovative designs of bioinspired injection devices for engineering  and biomedical  applications.B.B. and Y.P. conceived the project. B.B.-O. designed the research, performed the analysis and wrote the manuscript. Y.P., F.G.B. and P.F. co-wrote the manuscript. All authors discussed the results and commented on the manuscript.How to cite this article: Bar-On, B. et al. Multiscale structural gradients enhance the biomechanical functionality of the spider fang. Nat. Commun. 5:3894 doi: 10.1038/ncomms4894 (2014).Supplementary Figures 1-4, Supplementary Notes 1-2 and Supplementary References


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