This paper describes the protocols and results of the experiments for the estimation of the mechanical impedance of the humans’ lower leg in the External–Internal direction in the transverse plane under non-load bearing condition and with relaxed muscles. The objectives of the estimation of the lower leg’s mechanical impedance are to facilitate the design of passive and active prostheses with mechanical characteristics similar to the humans’ lower leg, and to define a reference that can be compared to the values from the patients suffering from spasticity. The experiments were performed with 10 unimpaired male subjects using a lower extremity rehabilitation robot  capable of applying torque perturbations to the foot. The subjects were in a seated position, and the Anklebot recorded the applied torques and the resulting angular movement of the lower leg. In this configuration, the recorded dynamics are due mainly to the rotations of the ankle’s talocrural and the subtalar joints, and any contribution of the tibiofibular joints and knee joint. The dynamic mechanical impedance of the lower leg was estimated in the frequency domain with an average coherence of 0.92 within the frequency range of 0–30 Hz, showing a linear correlation between the displacement and the torques within this frequency range under the conditions of the experiment. The mean magnitude of the stiffness of the lower leg (the impedance magnitude averaged in the range of 0–1 Hz) was determined as 4.9 ± 0.74 Nm/rad. The direct estimation of the quasi-static stiffness of the lower leg results in the mean value of 5.8 ± 0.81 Nm/rad. An analysis of variance shows that the estimated values for the stiffness from the two experiments are not statistically different. The ankle is the first major joint that transfers the ground reaction forces to the rest of the body during walking. Reaction forces allow for propulsion and the changing of direction of gait. Activities of daily leaving (ADLs) involve different tasks requiring the lower leg to function in all the anatomical planes. For example, during normal walking, walking on inclined planes, turning around corners, avoiding obstacles, and climbing/descending stairs, the lower leg dynamics change continuously to accommodate for these different maneuvers and conditions of agile gait. Studies of four representative daily activities show that, depending on the activity, turning steps may account for an average of 25% (range 8–50%) of all daily steps  in both external–internal (EI) and inversion–eversion (IE). It has been shown that during a sidestep cutting maneuver at normal walking speed, the ankle torque in the lateral direction at the push off phase increased more than six times compared to walking on a straight path  in the sagittal plane  ranging from 22.4 to 30.0). The subjects gave written consent to participate in the experiment, which was approved by the Michigan Tech Institutional Review Board.−6 m mounted on the traction drives. Torque is measured by current sensors (Burr-Brown 1NA117P), which provide a measure of motor torque with a nominal resolution of 2.59 × 10−6 Nm.A wearable lower extremity rehabilitation robot capable of applying controlled torque perturbations to the lower leg, Anklebot, was used to apply torque perturbations in EI direction. The Anklebot records the applied torques and the angular displacement of the lower leg as the result of the applied perturbations as described in detail by Roy et al. . The AnkThe Anklebot has been previously used for estimation of the ankle mechanical impedance in both DP and IE  and the actuation force of each actuator . To calculate the lower leg impedance and the quasi-static stiffness, both the angles and torques were required. Equations EI) and applied torques (τEI) in the EI direction based on the kinematics model of the experiment setup shown in Figure Figure During the experiments, the subjects were instructed to remain relaxed with no muscle contraction. The Anklebot was set to generate pseudo-random voltage inputs with a bandwidth of 100 Hz. The voltage inputs to the actuators were similar in magnitude but with opposite signs, generating lower leg rotations in EI. The ankle range of motion in EI during straight walking at normal speed is near 0.26 rad , is the transfer function:The Anklebot generated random torque inputs to the foot, which resulted in the rotation of the lower leg in the transverse plane. This system is properly defined as a mechanical admittance that admits torque inputs (τ) and generates motion output (θ). Linear dynamics were assumed based on small angular displacements of the applied perturbations (0.065 rad rms) with respect to the range of motion of the ankle in the EI (near 0.26 rad) during normal walking  is defined as the inverse of the admittance:The impedance In the experiments, the impedance function correlates the input angles to the output torques in EI:K = 10 Nm was added to the Anklebot controller to hold the foot near its central position and avoid its drift. This value was determined by trial and error to properly hold the foot in its central position for all the users during the experiments. Therefore, the total measured torque τ is the sum of the proportional controller torque and the human torque:o is the lower leg angle in the neutral position. In the beginning of each test, the foot is centered and the encoders are set to 0, resulting in the neutral position θo to be always 0. Combining and simplifying Eqs During the experiments, a proportional controller with gain Z(f) in Eq. f) and θEI(f) are the torque and angle measurements from the experiment. To calculate the impedance function, the Matlab’s® built in function tfestimate was used. The function tfestimate finds a transfer function based on the quotient of the cross power spectral density of the torques and angles and the auto power spectral density of the torques. The tfestimate was used with a Hamming window of 512 samples, 50% overlap, and evaluated with a fast Fourier transform length of 1024 samples, resulting in a spectral resolution of 0.19 Hz. The coherence between the input angle and output torque was calculated with the Matlab’s® function mscohere with the same parameters as the tfestimate function. The mscohere is a function of frequency with values between 0 and 1 that indicates how well the input correlates to the output at each frequency. The coherence is a function of the power spectral density of the angles, the power spectral density of the torques, and the cross power spectral density of the angles and torques. A final step was required to separate the Anklebot dynamics from the subject’s lower leg dynamics. The Anklebot, shoe, and lower leg share the same motion, while the torque measurement is the sum of the torques required to move all of them together. As a result, the estimated dynamic impedance in Eq. (ankle is the difference between the estimated impedance functions resulted from the two experiments with the human participants (Zlower leg+Anklebot+shoe) and without the human participants (ZAnklebot+shoe):Solving for e in Eq.  is the sThe experiments for the estimation of the quasi-static stiffness were performed with the same setup used for the estimation of the dynamic impedance of the lower leg. The quasi-static experiments were performed to validate the results obtained from the impedance estimation experiments. During the quasi-static experiments, the Anklebot was operated in a position control mode with a stiffness of 2177 Nm/rad and damping of 100 Nms/rad. These values were found experimentally to generate smooth operation of the Anklebot. Each experiment consisted of rotating the lower leg for 0.4 rad  in the internal direction followed by a 0.4 rad rotation in the external direction and back to the central position with a constant speed of 0.4 rad/s. This cycle was repeated 10 times without any pause. Below the break frequencies of the estimated dynamic impedance, the visco-elastic elements play the dominant role. The quasi-static experiment is focused on the dynamics of the lower leg in very low frequencies; thus, the speed of the quasi-static experiments should be low. However, very low speed would require long experimental time and caused non-linearities due to stiction in the Anklebot actuators. The loading speed of 0.4 rad/s was selected experimentally, as it is was found to minimize the effects of the Anklebot’s actuator stiction for a smooth operation. The loading speed of 0.4 rad/s is equivalent to 0.06 Hz, or about 1.74% of the break frequency .The recorded angles and torques were filtered with a low pass filter with a cutoff frequency of 1 Hz. For each subject, the lower leg crossed the reference angle (0 degrees) 10 times. To estimate the slopes of each segment (the quasi-static stiffness of the lower leg in Nm/rad), a second-order polynomial was fit to each segment in a least square sense. The 10 segments obtained from each test subject were averaged to obtain a representative quasi-static stiffness value for that subject.Figure ® function tfest for the impedance results of each subject. The tfest function estimates a transfer function from frequency domain data using the prediction error minimization approach to estimate the transfer function’s coefficients. From the second-order model, the inertia, damping, and stiffness of each subject’s lower leg were estimated as shown in Table 2. The average dynamic impedance transfer function was determined as Z(s) = 0.37/(s2 + 6.08s + 12.6).At very low frequencies 0–1 Hz), the magnitude plot of the dynamic impedance provides information about the quasi-static stiffness of the lower leg. The quasi-static stiffness magnitude for each test subject averaged over 0–1 Hz is shown in Table  Hz, the The quasi-static stiffness of the lower leg in EI was estimated directly for each subject and the results are shown in Table p-value was found to be 0.53 showing that the results obtained from the three methods are statistically similar.To compare the results of the quasi-static stiffness obtained from the two methods, only the data where the lower leg rotations were within ±0.065 rad was used. The angular motion in the lower leg within the range of ±0.065 rad is near linear when plotted against the estimated torque as shown in Figure Experimental and analytical methods were presented for the estimation of the mechanical impedance of the human lower leg in the transverse plane. The presented results are bound to the experiment conditions, including suspended lower leg under no load with no muscle contraction with a 90° bent knee. In this configuration, the rotations of the ankle’s talocrural and the subtalar joints are dominant in the recorded dynamics, and any contribution of the tibiofibular joints and knee joint are also measured. Similar to the mechanical impedance of the lower leg in sagittal and frontal planes, the lower leg impedance in the transverse plane showed a behavior close to a second degree function. A second-order mechanical impedance is a function of inertia, visco-elastic properties, and stiffness of the muscles and tendons of the lower leg that contribute to the rotation of the lower leg in the transverse plane. While in higher frequencies, the effects of the inertia are dominant; in the low frequencies, the visco-elastic properties of the lower leg due to the passive characteristics of the soft tissues are dominant. Visco-elastic elements have near constant stiffness below the break frequency, generating the observed near constant impedance magnitude plot at low frequencies. A break frequency would separate high- and low-frequency regions, and its average was determined to be 4.4 ± 0.22 Hz for the populations of this study. The slope of the average magnitude plot was 46.6 ± 1.5 dB/decade that is consistent with the results obtained in DP and IE  at all frequencies up to 30 Hz with a minimum of 0.85 at 0.89 Hz, indicating that lower leg impedance in EI is well characterized by linear models under the given experimental conditions. This coherence is similar to the reported coherence for the estimated ankle mechanical impedance function in DP and IE directions , and an average moment of inertia of 0.37 ± 0.03 kg.mp-value was found to be 0.53, indicating no statistically significant difference between the results.The quasi-static stiffness from direct estimation was 5.81 ± 0.81 Nm/rad compared to 4.9 ± 0.74 Nm/rad for the quasi-static stiffness averaged over the impedance magnitude in the range of 0–1 Hz, and 4.66 Nm/rad ± 0.70 from the estimated transfer function stiffness. The ANOVA was used to compare the results of the three methods. The The presented results can be used on the evaluation of patients suffering from spasticity due to stroke or patients with multiple sclerosis. It can be used to quantify the level of impairment and monitor the progress during the course of treatment. In addition, the results of the study provide a base for furthering our understanding of the lower leg musculoskeletal dynamics. During walking, the lower leg will be under varying load during the stance phase. Additionally, the lower leg’s muscles co-contractions would change the lower leg impedance during the stance phase. The presented experiment and analysis method will be extended to estimate the mechanical impedance of the lower leg with different muscle activation levels, and different knee and ankle angles to define a relationship between muscle activation levels and the mechanical impedance of the lower leg. This may provide design parameters for the development of ankle–foot prostheses with characteristics comparable to the human lower leg. This is particularly important since designing protocols for the estimation of the mechanical impedance of the lower leg or ankle in the transverse plane during walking is challenging. This is evident from the recent studies on estimation of the mechanical impedance of the ankle in the sagittal and frontal planes during stance phase of gait (Lee et al., This paper described the protocols and results for the estimation of the dynamic mechanical impedance and the quasi-static stiffness of the human lower leg in the EI rotation direction. Under the conditions of this experiment, the participants’ lower legs were suspended under no load, with knees bent 90°, and relaxed muscles. In the presented configuration, the recorded lower leg dynamics are due to the rotations of the ankle’s talocrural and the subtalar joints, and any contribution of the tibiofibular joints and knee joint. For dynamic mechanical impedance estimation, pseudo-random torque perturbations were applied to the foot, causing its movement in the transverse plane. For the quasi-static stiffness estimation, ramp perturbations with a constant velocity of 0.4 rad/s were applied to the foot, generating the quasi-static motion of the foot in the transverse plane. The dynamic mechanical impedance of the lower leg was estimated in frequency domain with an average coherence of 0.92 within the frequency range of 0–30 Hz, implying a linear correlation between the recorded angular displacement and the torques. The mean magnitude of the stiffness from the averaged impedance of the lower leg in the range of 0–1 Hz was 4.9 ± 0.74 Nm/rad. The results of the experiment for direct estimation of the quasi-static stiffness of the lower leg resulted in the means value of 5.8 ± 0.81 Nm/rad. An ANOVA determined that the difference in the estimated values for the stiffness from the two experiments were not statistically significant.EF contributed in developing the hardware, experiments, data analysis, and writing the material. GR contributed on the experiments and data analysis. MR contributed on the data analysis and writing the material.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Formica polyctena: nurses, foragers and reverted nurses. Sets of ten ants belonging to the same class were tested in “double nests” made of two interconnected test tubes, one kept in darkness and the other exposed to light. The number of ants present in the illuminated zone of each nest (ni) was recorded on 10 sample points at 30 min intervals. The values of ni were lower in nurses than in foragers and reverted nurses and decreased as a function of time in all three groups. Nurses differed from foragers with respect to the dynamics of dark–light choice behavior, but reverted nurses did not differ in that respect either from nurses, or from foragers. Reverted nurses and foragers did not differ significantly from each other with respect to the overall level of avoidance of illuminated zone, nor with respect to the dynamics of dark–light choice behavior. This implies that behavioral reversion is not accompanied by the return of illumination responses of workers of F. polyctena to the state characteristic for nurses.Social insect workers usually start adult life from intranidal tasks and then switch to extranidal activities, but this process may be reversed: foragers may switch again to intranidal brood care. The transition forager – reverted nurse is known as the behavioral reversion. Ant foragers are known to avoid illuminated zones less strongly than intranidal workers, but illumination responses of reverted nurses were so far never investigated. We compared dark–light choice behavior of three classes of workers of the red wood ant  Social insect workers as a rule start their adult life from intranidal tasks and then switch to extranidal activities Otto . ForagerApis mellifera L). Extensive research devoted to that phenomenon revealed that honeybee behavioral reversion is not limited to the induction of brood care behavior, but is also accompanied by other modifications of worker behavior . We choose that species, as phenotype modifications accompanying behavioral reversion were already investigated in F. polyctena . Nurses and foragers were collected separately on 15th April 2008 and 28th May 2008, respectively. In spring queens, nurses and brood of F. polyctena can be easily collected from the top parts of their mounds. A large colony fragment consisting of about 10–12 thousands of workers including nurses, abundant brood (eggs and larvae) and numerous (about 730) dealated queens was collected from the top of the nest mound together with the nest material. Foragers (about 7000) were collected from the trails at a distance of 3–4 m from the mound. The collected ants were transferred to laboratory and housed in artificial nests.Workers used in the experiment were collected from a colony of The ants collected from the mound were housed in a large nest composed of three large (38 cm × 30 cm × 15 cm) rectangular open Perspex containers connected by means of narrow tubes (Nest A). The walls of the nest were coated with Fluon® (PTFE), a substance providing silky smooth surface and, hence, commonly used in myrmecological research to prevent the ants from escaping from artificial nests. The floor of the nest was covered by a thin layer of fine sand. Two out of three interconnected boxes contained artificial brood chambers made of numerous large glass test tubes filled partly with water trapped in by means of a cotton plug to provide a humidity gradient allowing the ants to choose the preferred humidity conditions. The tubes were covered from above by a sheet of aluminum foil to assure darkness. The third box served as a foraging area. The ants had ad libitum access to water  and to carbohydrate and protein food .Foragers taken from the trails were housed in a similar nest (Nest B) made of two smaller (30 × 20 × 15 cm) interconnected containers. One of these boxes contained test tubes serving as artificial nest chambers, the other served as the foraging area. A week after their collection in the field foragers were divided into two groups, each counting approximately 3500 individuals. One of these groups was transferred to yet another nest (Nest C) in order to be subjected to the induction of behavioral reversion. The Nest C was composed of a single box (30 cm × 20 cm × 15 cm) containing several artificial nest chambers covered with aluminum foil to assure darkness. The ants were fed in the same way and kept in the same conditions as the ants from the nests A and B. The second group of foragers continued to be housed in the Nest B. All the nests were kept at a stable ambient temperature (24 °C) and relative humidity of the air (48–58 %) and exposed to a natural rhythm of daylight and darkness supplemented by artificial white light illumination delivered at 12:12 LD.The induction of behavioral reversion was carried out on 15–23 June 2008. On each day a relatively large amount (about 100 ml) of worker pupae taken from the nest A was gently placed on the floor of the nest C. After about 24 h, when all the pupae were already transported by the ants to test tubes acting as artificial nest chambers, the tubes were taken out and their contents (pupae and ant workers) were gently transferred to a cylindrical glass container  with the walls covered with Fluon®. The workers which moved away from the brood pile were put again in the nest C. All newly eclosed callows were also removed to avoid possible future difficulties in distinguishing them from the reverted nurses. The pupae and the workers engaged in brood care  were then placed in a new nest, also made of a single box (30 cm × 20 cm × 15 cm). This procedure was repeated on four successive days (16–19 June 2008). As a consequence, we created a series of nests (D1-D4), each of them containing the reverted nurses that switched to brood care on a particular day. Each nest contained 6 test tubes equipped with water reservoirs and covered by aluminum foil. Drinking water and food was provided in a similar way as in the case of the remaining nests. The state of the nests was monitored daily. We removed newly eclosed callows and, if necessary, added new worker pupae taken from the nest A to maintain an approximately constant volume of brood. The reverted nurses were tested after 7 days from the moment of the creation of the nest (D1-D4) to which they were transferred after having been found to engage in brood care in the nest C.We tested dark–light choice behavior of three groups of ants: nurses, foragers and reverted nurses. Nurses and reverted nurses were taken, respectively, from among workers present in the brood chambers of the nest A housing the main colony fragment collected from the mound, and the nests from the series D1-D4 housing groups of reverted nurses created a week earlier. In each case the tubes acting as brood chambers were taken out of the nest and their contents were gently transferred to a cylindrical container  with the walls covered with Fluon®. The ants that walked away from the brood pile were removed. The remaining ants were left to settle during 30 min. After that time only the ants that settled on the brood pile in direct contact with the pupae were selected for the dark–light choice tests.Foragers were selected from among workers present in the foraging area of the nest B. They were captured by means of a forceps and transferred directly to a cylindrical container similar to that used in the process of the selection of both types of nurses. They were then left to settle during 30 min and then started to be transferred to the nests used in the dark–light choice tests.F. polyctena was tested by means of a bioassay proposed by Korczyńska and Godzińska  was placed in the double nest (five workers in each arm) and then the number of workers present in the illuminated half of the nest was counted at 30 min intervals. Altogether, 10 sample points were taken for each nest. The first measurement was carried at 10.30 a.m., about 30 min after the moment when all the workers tested on this particular day were already placed in the double nests. All manipulations necessary to prepare the experimental situation were carried out in parallel by several persons to minimize the differences between the time spent by the ants in the double nests prior to the initial measurement. Ants from various experimental groups were placed in the double nests in parallel.The tests were carried out on four successive days (23–26 June 2008). On each day we tested an equal number of the double nests per group . Finally we analyzed the data obtained for 40 sets of 10 ants for each experimental group. Only the tests during which no worker died were taken into account in the analysis of the data. Worker mortality proved to be very low: only 3 foragers and 1 nurse were found dead after the end of the test.i) was the number of ants present in the illuminated zone of each nest on a particular sample point (1–10). The dependence of that variable on ant group  and on time from the start of the test (10 successive sample points) was analyzed by means of Repeated Measures ANOVA with group as a between-subject variable, time as a within-subject variable and the interaction between these two variables. The overall analysis of the data obtained for all three groups was followed by pairwise comparisons  also carried out by means of Repeated Measures ANOVA with probabilities adjusted by means of Bonferroni correction. All these analyses were carried out by means of the software SPSS.The main dependent variable analyzed in this study  and on time from the start of the test . The values of the variable ni denoting the number of ants staying in the illuminated zone of the dark–light choice nest were significantly higher in the case of nurses than in the case of both classes of ants that had already passed the transition from intranidal to extranidal tasks, and decreased as a function of time in all three groups , a highly significant effect of the within-subject effect „time” , and a significant interaction „group” x „time” . This last result implies that the dynamics of dark–light choice analyzed as a function of time from the start of the test differed between the compared groups.The behavior of workers of ups Fig. . The ovevs foragers) also carried out by means of Repeated Mesures ANOVA yielded more precise information about the differences between the tested ant groups.Subsequent pairwise comparisons  and „time”  and a significant interaction „group” x „time” . Such a constellation of significant effects implies that nurses and foragers differed not only with respect to the overall level of avoidance of illuminated zones, but also with respect to the dynamics of their dark–light choice behavior (changes of ni as a function of time from the start of the test). Nurses were withdrawing from illuminated zones more rapidly than foragers.The comparison nurses vs foragers discovered highly significant effects of „group”  and „time” , but interaction „group” x „time” was not significant . This implies that although the overall level of avoidance of illuminated zones differed between nurses and reverted nurses, the dynamics of dark–light choice behavior did not differ significantly between these two ant groups.The comparison of nurses with reverted nurses discovered highly significant effects of „group” , highly significant effect of „time” , and no significant interaction „group” x „time” . This implies that neither the overall level of avoidance of illuminated zones, nor the dynamics of dark–light choice differed between reverted nurses and foragers.Lastly, the comparison of reverted nurses with foragers discovered no significant effect of „group”  from the start of the reversion process, but, nevertheless, their illumination responses remained similar to those of foragers. However, our reverted nurses were not subjected to constant forced exposure to darkness, as in the experiment of Wehner et al. (F. polyctena (Menzel and Lange Our present results can also be compared with the data obtained by Wehner et al.  in his cr et al. , but to F. polyctena that did/did not pass the transition from intranidal to extranidal tasks are in concordance with our present knowledge concerning age-, task- and experience-related neuroanatomical changes taking place in the visual system of ant brains (Kühn-Bühlmann and Wehner F. polyctena remained largely similar to that of foragers, which implies that in that ant species modifications of illumination preferences accompanying the transition from intranidal to extranidal tasks are largely retained even if the individual returns back to dark intranidal environment and engages in intranidal brood care.Behavioral differences between workers of


Standardisation is required in research, so are approval procedures for advanced therapy medical products and other procedures for articular cartilage therapies. The process of creating samples needs to be reproducible.The aim of this study was to design, create and validate instruments (1) to create reproducible and accurate defects and (2) to isolate samples in the shape of osteochondral cylinders in a quick, reliable and sterile manner.Adjustable instruments were created: a crown mill with a resolution of 0.05 mm and a front mill to create defects in articular cartilage and subchondral bone. The instruments were tested on knee joints of pigs from the slaughterhouse; 48 defects were created and evaluated. A punching machine was designed to harvest osteochondral plugs. These were validated in an in vivo animal study.The instruments respect the desired depth of 0.5 and 1.5 mm when creating the defects, depending on whether the person using the instrument is highly experienced : 0.390 mm; 0.512 mm and 1.403 mm; CI: 1.305 mm; 1.502 mm) or less so . Eighty samples were taken from knee joints of Göttingen Minipigs with this punching technique. The time needed for the harvesting of the samples was 7.52 min (±2.18 min), the parallelism of the sides of the cylinders deviated by −0.63°  and the surface of the cartilage deviated from the perpendicularity by 4.86° . In all assessed cases, a sterile procedure was observed.Instruments and procedures for standardised creation and validation of defects in articular cartilage and subchondral bone were designed. Harvesting of samples in the shape of osteochondral cylinders can now be performed in a quick, reliable and sterile manner. The presented instruments and procedures can serve as helpful steps towards standardised operating procedures in the field of regenerative therapies of articular cartilage in research and for regulatory requirements. Guidelines for the determination of therapeutic outcomes require reproducible and validated protocols to be observed. Standards would also be helpful to satisfy regulatory requirements for regeneration therapy of articular cartilage tissue in preclinical tests in vivo and in vitro , 2.There are some specific challenges in preparing and isolating samples before and after the healing time regarding different types of analyses and the stated scientific hypotheses. Biomechanical tests in particular require specimens with a clear geometry. This is necessary in order to keep the specimen in place and allow free access for biomechanical testing devices such as indentation rods –6. The sFor the synthesis of regenerated articular cartilage tissue, a standardisation of the procedures is required. Most of the studies use defect models analysing regenerative concepts –16, but The regeneration of defects in the articular cartilage is described and investigated among others by different depths according to the hypothesis of the particular study design. One can roughly distinguish between partial and full thickness defects  which caHowever, the depth of the defects should be stable in dimension, homogenous in shape and conform to the same principle. The sides should be straight and the bottom of the defect should be perpendicular to the sides.Therefore, a cutting device is required which produces smooth surfaces on the cartilage and the bone. It must be capable of working in a quick, reproducible manner and irrespective of the user’s expertise in order to guarantee standardisation in an in vivo model to produce advanced therapy medicinal product (ATMP). It also needs to be suited for scientific assessment and has to meet regulatory guidelines.In the field of research of tissue engineering of articular cartilage, osteochondral blocks or plugs are often used with the subchondral bone as a fixation and the cartilage layer on top as protruding tissue to be examined. Thus, the isolation of samples can become a challenge when new tissue has to be isolated for testing. The time for preparation should be as short as possible to avoid changes arising after opening the joint.Samples for biomechanical and other tests have to be of a specific size, depending on the test procedure. Several biomechanical tests require samples of a size varying between 4 mm  and 6 mmThe aim of the study was to establish a reproducible design to create and isolate regenerative articular cartilage samples in large animals applicable in and ex vivo and in vitro.In particular, instruments had to be designed and constructed allowing (1) the creation of defects and (2) the isolation of the area of the regenerated tissue after healing. The operating sequence can also be applied to other species, either in vitro, in vivo and ex vivo.We constructed and built tools for the creation of reproducible chondral or osteochondral defects in vivo and in vitro in joints of animals.The instrument for creating the defects in articular cartilage consists of two different tools Fig. . The so-The outer sleeve of the front mill is placed in the groove and the inner section is turned clockwise, until the inbuilt stop respects the depth defined by the crown mill Fig. . ResiduaThe hypothesis for the validation of the newly created instrument was twofold:Firstly, we wanted to investigate if the tool could deliver the expected depth of a defect in articular cartilage or bone after the desired dimension was preset at the instrument. Secondly, differences between an experienced and a less experienced practised operator were to be addressed.n = 12) and 1.5 mm (n = 12) in depth, respectively, were placed at random in each trochlea.To validate the tool for creating defects with defined depths, we used trochleae of the knee joints of pigs (age approximately 5 months) from the local slaughterhouse. Defects with 0.5 mm ; the other one was a scientist (biologist GR) with an assumed lower skill level in using tools.The defects were cast with alginate . As the stability of alginate is limited, a second cast with dental cement  was performed at the top of the alginate body. Thus, the defects became solid enough to be able to perform a measuring procedure to determine their depths, thus avoiding measuring faults through contact with a digital gauge . The casts were mounted in a tap holder which was adjusted to the vertically positioned digital gauge for measuring Fig. .Fig. 4MeThe measurement algorithm was based on the idea that the first groove made by the crown mill represents the point of reference. Thus, the area in the middle of the defect that was created by the front mill and the untouched cartilage surface can be calculated as the difference in height according to the point of reference. The defects were measured in four positions  at the three levels  three times. Taking the means of the triplicates, the average depth of one defect was calculated with regard to the depth of the groove created by the crown mill and the area created by the front mill. The measurements of the twelve defects with the same defined depth provided the average depth and the corresponding statistical deviation.t test procedure in the SAS 9.3 program . The level of significance was α = 0.05.The depths were calculated by Excel 2010  in terms of differences, and the values of the results are shown graphically by box blots created by the Origin 8.6G software . Differences in the results of both operators were tested by the There are some issues to be considered and overcome when osteochondral samples have to be taken out of the joint, in particular from the bone after a longer healing period.The articular cartilage layer is supported by the subchondral bone lamella which can develop into a rigid structure –33 durinThe isolation of the samples has to be easy and reliable despite all critical situations described above.The presented construction for harvesting samples of articular cartilage after healing in vivo is based on a solid frame. A bench vise is mounted which is movable below and allows the adjustment of the fixed sample under the vertically mounted punch in a correct, perpendicular position in relation to the articular surface.In addition, the concentric position of the punch has to be adjusted with reference to the outer rim of the circular defect where the new tissue is expected, and only the new tissue has to be examined. In the presented equipment, the osteochondral plugs which have to be harvested need a diameter of 5 mm, whereas the defect is created with a diameter of 6 mm. The punch is driven into the tissue (cartilage and bone) by hand supported by a lever mechanism Fig. . We moisIn some cases, the osteochondral plug remains in the punch when retracting it. The punch Fig.  is equipWe applied the harvesting tools in a study using a large animal model with artificial articular defects in both facets of the trochlea at both sides according to Gotterbarm et al. . The stuWe determined (1) the time needed to create an osteochondral plug, (2) the parallelism of the sides of the plug, (3) the deviation of its surface from its symmetric axis that should be 0° regarding the addressed 90°, and (4) the sterility of the procedure if sterile samples are needed.We started timing when the adjustment of the defect to the punch above began after the separated trochlea was fixated in the bench clamp and stopped the time when the osteochondral plug was positioned under the camera taking pictures  for analysing the shape and geometry of the plug. The camera was mounted on a frame  parallel to the base where the plug was placed on its side. Thus, the optical-parallaxes were avoided, and a contactless measurement became possible.The geometry of the osteochondral plugs was analysed through the pictures taken after harvesting. For this purpose, pictures were taken from four different angles, and a particular view of the side of the plug that seemed the least straight was taken for evaluation. The geometry of the plug was analysed with the OpenOffice 3.4 Draw program .Both sides of the plug form a positive angle towards the cartilage at the top if the tangents at the sides converge, a negative angle vice versa.Eighty plugs, harvested from both trochleae of 10 Göttingen Minipigs, were analysed. Sterility was checked by swabs  at the surfaces of the remaining trochlea and analysed. The swabs were incubated according to the microbiological and infectiological quality standards ) of the German Society for Hygiene and Microbiology (Deutsche Gesellschaft für Hygiene und Mikrobiologie (DGHM)) between 2 and 10 days both in an anaerobic and aerobic environment . PositivThe time needed for harvesting the plugs was calculated in Excel for the mean and standard deviation. We calculated the means and 95 % confidence intervals (CI) by SAS 9.3 for the parallelism of the sides of the plug and the deviation of the edge of the surface of the plugs from their centreline.p = 0.018). The centres of the defects created by the front mill showed a low deviation from −0.013 mm (less) to 0.014 mm (deeper)  Fig. .Fig. 6AnThe shapes of the defects show a perpendicular junction of the plain at the bottom and the sides of the cylindrical groove Fig..Fig. 7HiThe plugs were isolated after 7.42 ± 2.28 min in the mean.The sides showed a mean deviation of −0.63°  in 80 cases with a value of 0° for parallelism; thus, the punch and the whole apparatus worked consistently for all examined specimens. A critical geometry is the relation of the slope of the articular surface to the axis of the cylindrically shaped plug, which should be 90° in the best case. Deviations were calculated with 4.86°  from the rectangularity Fig. .n = 80).Sterility was given in all cases  or traiThe presented instruments work independently of the force applied  as they However, we have not compared our results to the results achieved through conventional techniques, neither before nor after healing processes. But we assume that the new technique we developed is less prone to influences such as the expertise of the surgeon executing the operation. We also believe that the new technique is likely to be a lot faster. Another disadvantage of the new technique could be that only defects of one predetermined diameter can be created during any one operation. Changing the diameter is thus not possible while operating. As the instruments were designed for the use in preclinical studies, where the size of the defect is determined before the operation, this is not a disadvantage. As our previous tests have shown, so far the diameter cannot be smaller than 4 mm. However, it is possible to create a crown mill and corresponding front mill of a larger diameter than the 6 mm we used.Histopathological examination is indicated as the predominant outcome measurement tool in OA , 25, 16 The presented punching procedure allows the exact harvesting of regenerated tissue or tissue originating from other locations, as the used apparatus allows the individual adjustment of the punch as desired. In combination with a 6-mm defect in diameter, a 5-mm sample in diameter can be extracted exactly as shown (Fig. In conclusion, we can provide new instruments and procedures for standardisation of creating defects in vitro and in vivo and for exact harvesting of samples in a quick, secure and sterile manner.


Cosmopolites sordidus. In a “two patches” experiment, we used radiofrequency identification tags to study the C. sordidus movement response to patch conditions. We showed that local and neighboring densities of conspecifics affect the movement rates of individuals but that the density‐dependent effect can be either positive or negative depending on the relative densities of conspecifics in local and neighboring patches. We demonstrated that sex ratio also influences the movement of C. sordidus, that is, the weevil exhibits nonfixed sex‐biased movement strategies. Sex‐biased movement may be the consequence of intrasexual competition for resources  in females and for mates in males. We also detected a high individual variability in the propensity to move. Finally, we discuss the role of demographic stochasticity, sex‐biased movement, and individual heterogeneity in movement on the colonization process.Understanding the mechanisms underlying the movements and spread of a species over time and space is a major concern of ecology. Here, we assessed the effects of an individual's sex and the density and sex ratio of conspecifics in the local and neighboring environment on the movement probability of the banana weevil,  Movement, defined as a change in the spatial location of an individual .However, theoretical studies that focus on the evolution of sex‐biased movement have generally assumed that movement is a fixed trait in individuals, which is not always the case (Bowler and Benton C. sordidus (Germar). In an experiment that used populations of C. sordidus and focused on the departure stage of movement, we tested the theoretical prediction that sex‐biased movement strategies tend to balance intra‐ and intersexual competition. We studied the effects of individual phenotype (sex) and local and neighboring patch conditions (variations in conspecific densities and sex ratios) on the movement probability of C. sordidus in a “two patches” environment using RFID methods. This experiment determined (1) how the local and neighboring densities of conspecifics affect the movement rates of individuals; (2) how the local and neighboring sex ratios affect the movement rates of individuals; (3) whether banana weevils exhibit sex‐biased movement to balance intra‐ and intersexual competition; and (4) the individual variability in movement behavior.The current paper aims to understand the factors that influence the movement decisions and therefore the colonization potential of the banana weevil Cosmopolites sordidus is the most important insect pest of bananas and plantains. This weevil originated in the Indo‐Malaysian region  and the other end of the line was attached to the individual with cyanoacrylate glue ; a previous study showed that this method of tagging did not affect weevil mobility  was placed in the center of each patch and was changed after 10 days. Movements of individuals were recorded by antennas located on the corridor and 10 cm distant from each patch; the antennas were connected to RFID readers . Tag detection was restricted to a mean distance of 3.74 cm (±SE 0.86) from the antenna by using each antenna at 10% of its capacity. The experimental system was maintained in a climate‐controlled room at 25°C and with a relative humidity of 85% and a 12‐h/12‐h photoperiod .The experimental system consisted of two plastic patches (each patch was 50 cm in diameter) that were linked by a corridor (a transparent plastic tube) that was 1 m long, 7 cm wide, and 4 cm high. The patches were filled with washed sand, and the corridor was located to enable movement of weevils between the sand surface and the corridor. One plantain bulb  were created by varying the initial number of individuals from 4 to 50 in the patch of release. The initial sex ratio was 1:1 for each treatment . To minimize the effect of individual variability in behavior, we used each individual only one time, and we replicated each treatment 10 times  or four times . We alternated the patch where C. sordidus individuals were placed between replicates. Each replicate was started 5 min before the beginning of the artificial night by placing the desired number of individuals randomly around the plantain bulb in the release patch. The same individual could not be detected by the same antenna during the following 60 sec. The recorder registered the individual identification tag number and time of detection.We defined the local patch as the patch where an individual is present at time xi(t) is the number of individuals in patch i at time t. To determine xi(t), we performed an algorithm that divided the data according to the time of night. For each replicate, we retrieved the position of each marked weevil, and consequently we knew the exact local density xi(t) and the local sex ratio of patch i at any time t. From the population dynamics of the patches in the original data, we calculated the local density xi(t) of each patch as the mean of all the local densities observed in the time interval  in patch i. We applied the same procedure for calculation of the sex ratio. We assigned the value 1 when the weevil had moved between patches during Δt and the value 0 when the weevil had not moved between patches. We thus assumed that the decisions of individuals to move were related to xi(t). The algorithm was performed for Δt values of 5, 10, 15, 20, 25, 30, and 60 min to assess the robustness of the method and consistency between results.Local density p as a function of the linear combination of an intercept, fixed effects (xβ′), and random effects (zb′), which are assumed to follow a normal distribution centered at 0 and with variance σ2. A global GLMM took the general form of: We used generalized linear mixed models (GLMMs) to analyze the effects of patch conditions and sex on the probability of movement. Binomial GLMMs describe the logit of the response variable Probability of movement was analyzed using GLMMs with binomial error  between covariates using the variance inflation factor (VIF) method , local density , neighboring density , local sex ratio , and neighboring sex ratio . Quadratic terms of the hour of the night significantly affected the probability of movement . The probability of movement was also significantly affected by interactions between local and neighboring densities , between local density and local sex ratio , between neighboring density and neighboring sex ratio , and between neighboring density and local sex ratio . The sex of an individual did not significantly affect its probability of movement , but was retained in the final model because of the significant interaction between sex and local sex ratio . The age of bulbs did not significantly affect the probability of movement  and was removed from the final model. The probability of movement was not significantly affected by the interactions between local density and neighboring sex ratio  or between sex and neighboring sex ratio , and these terms were removed from the final model. Banana weevils responded linearly to the density of individuals both in the local and the neighboring patch, in that the probability of movement from the local patch decreased with local density and increased with neighboring density .According to GLMMs, the probability that an individual moved was significantly affected by the hour of the night  may explain the balanced sex ratio generally observed in large natural populations of banana weevils that reside in the same field , but because individuals were tested only once in the experiment, we cannot make inferences about the possible existence of disperser and nondisperser phenotypes in this species. Individual‐based modeling (IBM) is well suited to simulate the dynamics of the spatial spread of a species .Table S2. Likelihood ratio tests for the significance of adding “individual” and “replicate” random effects to the global model (in bold).Table S3. Model selection based on the deviance information criterion (DIC)  across the range of Δt values from 5 to 20 min.Table S5. Estimates of the best GLMMs (backward selection using likelihood ratio tests) across the range of Δt values from 25 to 60 min.Click here for additional data file.Figure S1. Experimental design for the study of the behavior of Cosmopolites sordidus, the banana weevil.Figure S2. Influence of the local densities and local sex ratio  on the probability of Cosmopolites sordidus movement.Figure S3. Influence of the neighboring densities and local sex ratio  on the probability of Cosmopolites sordidus movement.Figure S4. Influence of the neighboring densities and neighboring sex ratio  on the probability of Cosmopolites sordidus movement.Click here for additional data file.


Various clinical and radiological criteria have been suggested to choose one of the numerous techniques in surgical treatment of hallux valgus and rigidus. We hypothesized that the surgeons' professional background will influence that choice depending on specialization, age, type and institution of training as well as his orthopaedic cultural orientation. Since Switzerland is characterized by regional languages (the most important being German and French), we were interested to learn if the linguistic differences had an influence on the orientation of the surgeons towards e.g. Anglo-American or French surgical traditions and/or sources of literature on the subject.A survey was e-mailed to all members of the Swiss Orthopaedic Society (SGOT-SSOT). Questions were asked regarding respondents’ demographics as well as their preferred treatment for 3 separate cases of (1) moderate and (2) severe hallux valgus and (3) hallux rigidus. The responses were collected and statistically analyzed.Two hundred thirty of 322 respondents completed the survey(response rate 46 %). as they perform foot surgery on a regular base; 39 % were members of the Swiss Orthopaedic Foot and Ankle Society (SFAS). Selected surgical treatments differed as follows: in joint sparing procedures older and busier surgeons were more likely to use Chevron osteotomies, however more than 50 % preferred a Scarf-type of osteotomy. Along the so-called "Rösti-Graben" separating the French from the German speaking part of Switzerland no significant difference was found in the choice of operation technique.Nevertheless the fact being a member of SFAS showed significant differences in technical choice in case 2 and 3.There are significant associations between the surgeons’ age, expertise and training and their preferred operative intervention. Considerable differences in the surgical management were found in the practice of the general orthopaedic surgeons 72 and the foot and ankle specialists. The cultural background and training is not mirroring the classical Swiss east west discrepancy. Despite the large number of surgical options available for hallux valgus, only a small number were preferred by the majority of surgeons.The online version of this article (doi:10.1186/s12891-015-0751-7) contains supplementary material, which is available to authorized users. Hallux valgus and hallux rigidus are common conditions for which numerous operative interventions have been described in the literature –4. VarioSwitzerland is divided into several distinct cultural and linguistic regions that were formed through the variable influences of the surrounding empires  over time. An influence of the language difference on literature search, decision-making and practice due to membership of the surgeon in e.g. French or Anglo-American professional organization could not a priori be excluded.Thus we presented the questions of the Australian survey to Swiss orthopaedic surgeons aiming to identify factors that influenced their choice of treatment with special emphasis on that demographic peculiarity .A survey was electronically mailed out to members of the Swiss Society of Orthopaedic Surgery and Traumatology  including fellows and registrars in the orthopaedic training program. Participants completed the survey questionary online via a dedicated website which collected and collated the responses. Translations into the three main languages  were available.The first question of the survey was if the respondent performed foot and ankle surgery; a positive response allowed them to complete the remainder of the survey.Three separate cases were presented in the survey. Expecting a higher response rate the X-rays illustrating the cases were accompanied with only brief information on patient history in order to minimize the time needed for the completion of the survey. In Figs. Case 1 ; principal Swiss region of medical training ; type of institution of practice ; age and membership in the SFAS (Swiss Foot and Ankle Society).The responses were collated and then organised into an appropriate format for transfer to a statistical programme, SAS version 9.2 . Statistical analysis was performed to obtain percentages of all of the responses and chi squared tests were undertaken to investigate for significant statistical relationships between responses and demographic variables.There was no need for an ethical approval as the survey did not concern direct patient data according to our institutional review board  and the local ethical committee (EKNZ). The study was completed by medical professionals. See questionnaire here . Seventy-one percent (230) of these stated to perform surgery on the foot and ankle, while 29 % did not (74).So the response rate of the survey was actually 35 %. Demographic factors as well as participants case loads, type of training and practice are highlighted in Table p <0.001). See details in Table For case 1  distal Chevron was the most commonly chosen procedure (41 %). 78 % would perform a distal soft tissue (McBride) procedure in addition. Scarf osteotomy was the next most commonly chosen procedure (36 %). The correction was more likely to be accompanied by a McBride procedure 78 %) and was more likely to be preferred by members of SFAS  and by those who were less than 50 years old . 21 % preferred first metatarsophalangeal joint fusion with 80 % of those choosing a plate and screw construct for their fixation. See details in Table In case two  a Lapidus procedure was the most commonly preferred 31 %). It was more likely to be performed by members of the SFAS . See details in Table Case 3 (first MTPJ arthritis), first MTPJ fusion was the treatment of choice for the majority of respondents (78 %)  were performing foot and ankle surgery on a regular base.In a recent survey of academic American orthopaedic foot and ankle surgeons in mild bunion cases 87 % preferring a distal metatarsal osteotomy, followed by a more proximal osteotomy and in 10 % augmented by an additional Akin osteotomy .Compared to the in 2012 published survey of Australian orthopaedic surgeons we found less parallels in age and training as well as geographic/cultural differences as expected .It was interesting to note that the classical east–west differences in cultural and language in Switzerland is not correlated with the type of treatment chosen in forefoot surgery. Nevertheless being a member of the SFAS did appear to be a significant factor in the choice of the type of correction preferred in the more severe cases with a larger proportion of SFAS members choosing a Lapidus for case 2 or a MTP-I-joint fusion in the hallux rigidus over other type of operations .The scarf osteotomy has been described as a more involved and complex osteotomy than other types  and as such may be more commonly performed by the surgeon who more regularly performs, or has a more dedicated interest in foot and ankle surgery such as members of the SFAS. Many argue the merits of the scarf osteotomy are that it can provide significant degree of correction with less risk of metatarsal head avascular necrosis and better healing because of the biomechanical stability of the osteotomy .In case 1 it was noted that a proximal Chevron and a scarf osteotomy was associated with a higher rate of distal soft tissue procedure than the distal type of osteotomy. Fifty percent of the foot and ankle surgeons (F + A) performed a scarf while 52 % of the general surgeons did so.In case 2 with the severe bunion over 50 % of the F + A surgeons but only a third of the general orthopaedic surgeons would have chosen a scarf osteotomy in combination with distal soft tissue releases equal distribution of around 80 %. A Lapidus procedure was the preferred method to fix this condition in 32 % of all SGOT members while only 3 % of the F + A specialists did choose this here. The main difference was actually seen in the comparison of the french swiss to the German speeking swiss orthopaedic surgeons. It is clearly seen that the Lapidus procedure is more often used in the french part than in the German speeking.Another demographic variable which we found to be associated with differing choices of treatments was the age of the surgeon. We grouped people into either older or younger than 50 years of age. In case 1, a Mitchell osteotomy was more likely to be performed by those older than fifty. In case 2, a Keller’s excision arthroplasty was more likely to be performed by those surgeons older than fifty whilst in case 3, a Cheilectomy alone was more likely to be performed by those greater than 50 years of age. These three findings were interesting as they may represent a change not only in the teaching and training of orthopaedic surgeons in Switzerland over the past years but possibly also a change in the concepts of aetiology and patho-physiology of hallux valgus as a condition.Joint replacement arthroplasty was not a very common choice for the management of case 3  in 2 % of the F + A surgeons and 5 % of the general orthopaedic surgeons, with most respondents preferring to perform a first MTPJ fusion.With all of the available treatments described in the literature for hallux valgus it was interesting to note in our study that there were only a few procedures which were preferred by the majority of the swiss orthopaedic surgeons. Five procedures  accounted for the major percentage of preferred treatments for each of the three case examples , 18, 24.The other interesting finding was the significant differences in the practise of general orthopaedic surgeons and F + A specialists. The F + A specialists tend to perform more modern surgeries such as the scarf osteotomy with additional distal soft tissue releases and usually don’t perform arthroplasties in the highly loaded first MTP joint.There are significant associations between the surgeons’ age, expertise and training and their preferred operative intervention. Considerable differences were found in the practice of the general orthopaedic surgeons and the foot and ankle specialists. The cultural background and training is not demonstrating the expected classical Swiss east–west discrepancy. Despite the large number of surgical options available for hallux valgus, only a small number were preferred by the majority of surgeons. While we are all anecdotally aware that lesser deformity is treated with distal osteotomies and more severe deformity with a proximal osteotomy, we are aware of only limited data in the literature that verifies this. We could show differences in the swiss orthopaedic population in regard to the membership of the specialist’s society but the “Rösti-Graben” seems not to be as deep as mostly seen in the cultural and social correlation.


Iris normalization is an important stage in any iris biometric, as it has a propensity to trim down the consequences of iris distortion. To indemnify the variation in size of the iris owing to the action of stretching or enlarging the pupil in iris acquisition process and camera to eyeball distance, two normalization schemes has been proposed in this work. In the first method, the iris region of interest is normalized by converting the iris into the variable size rectangular model in order to avoid the under samples near the limbus border. In the second method, the iris region of interest is normalized by converting the iris region into a fixed size rectangular model in order to avoid the dimensional discrepancies between the eye images. The performance of the proposed normalization methods is evaluated with orthogonal polynomials based iris recognition in terms of FAR, FRR, GAR, CRR and EER. Owing to differences in the image capturing distance and in the illuminationed environment, the size of the subject’s pupil and the iris region of interest in the captured images are highly diverging. An accumulation of this, there is discrepancies of the same eye images owing to stretches of the iris. Additional criteria that cause dilation are eye rotary motion, revolving camera and head inclination. Such a deformation of the iris texture broadens intra-class dissimilarities and raises the FRR.A survey of the existing normalization techniques found in the literature is presented in the following section.From the perspective of iris texture feature extraction, the normalization techniques are classified into six categories such as Linear model Daugman , 2011, vr and normalized the angular resolution into  according to the step angle 0.8°. Joung et al.  axis, the extreme positions at lower end  and higher end  are extracted from the pupil border points. Similarly for y(major) axis, the extreme positions at lower end  and higher end  are extracted from pupil border points. The distance, x_dist, between the extreme positions in x axis are computed as followsFirst let us consider the pupil and limbus border points present in localized image of the iris image. The radius of irregular pupil border is estimated from the pupil border points with the following steps. Along the y_dist, between the extreme positions in y axis are computed as followsSimilarly, the distance, rp is determined asIn this work, the radius of the pupil border The center of the pupil border, rl). A circle is subsequently fitted throughout every point on the contour that are within a distance of (rl ± 10) pixels from the center of the pupil rl) of the limbus. When the limbus is detected in the corner of the eye, all six points preferred to calculate the rough limbus radius may not be positioned on the limbus border; some may be positioned on the eyelid. As the eyelid border is nearer to the center of the pupil, an approximate limbus radius is off-center and the same is demonstrated in Fig. rl) and it increases the accuracy even in the case of off-angle iris.After determining the coarse radius of pupil border, the radius of limbus border is calculated for iris normalization. If the obstruction owing to either the upper or lower eyelids is significant, in that case the circle that fits every point on the extorted contour will be positioned within the exact border of the limbus. For this reason, merely the points resting on the border of the limbus are modeled in this research work to calculate the radius and center of the limbus. To guarantee this, six points at the following angles  are chosen from the extorted contour as well as their mean distance from the center of the pupil is calculated and exploited as the coarse radius of the limbus (The maximum degree (360°) is divided into small units of angle increment factor that enables to reach each limbus border positions of the iris. The circumference The distance between the pupil border and limbus border is normalized to . The resolution angle of increment θ by rl, the variable-size rectangular resolution iris image for the plane  is obtained. Also the degree of rotation (360°) is calibrated in such a way that it reaches each position in the limbus border.By increasing the angle θ as 0, xi as yi as n from limbus border to pupil border with the length xs, ys) and  as start and end positions respectively of a line identity and traverse along the line and obtain each position co-ordinates in the iris Region of Interest (ROI) by decrementing n by 1. The starting point  of tracing process is computed as followsAfter determining the resolution angle of increment, the sampling points are extracted by tracing points from limbus border to pupil border. Let us initialize xe, ye) of tracing process is computed asThe end point (x(minor) axis width is computed asy(major) axis width is computed asThe R[n][θ]. Consider  and  as start and end positions respectively of a line identity and traverse along the line and obtain each position co-ordinates in the iris ROI by decrementing n by 1. If the absolute value of dx is larger than dy, the point R[n][θ] of the slope m and y_intercept b is computed using the Eqs.  is computed asxs as xs + dx, continue the following process step through xs + dx and n − 1 times and until xs becomes xe.Considering dx is not larger than dy and the value of dy is not equal to 0, the point R[n][θ] from the slope m is computed as follows using the Eqs.  is computed asys as ys + dx, continue the following process step through ys + dx and n − 1 times and until ys becomes ye.The b  is obtained. After fixing the resolution angle of increment, the sampling points are extracted by tracing points from pupil border to limbus border. Initialize θ as 0, n as 0, xi as yi as n from pupil border to limbus border with the length xs, ys) of tracing process is computed asxe, ye) of tracing process is computed asFirst let us consider the pupil and limbus border points present in iris localized image. The radius of the pupil and the limbus border are computed as described in section “θ becomes 360°.In this way, points from pupil border to limbus border for each position are traced and stored in angular resolution array step The algorithm of the proposed fixed-size normalization is given hereunder:With a view to extract iris texture feature, the normalized iris is further subjected to the orthogonal polynomials to extract the transformed coefficients  matching scheme  core (TM) i7 CPU 965@3.20 GHz system with 4.00 GB RAM. These outcomes are obtained and tabulated in Table For an unbiased comparison, the orthogonal polynomials based iris recognition has been experimenting with the proposed variable-size normalization model and proposed fixed-size normalization model using images in BITIRIS database. For recognition, a query image was matched against the entire database of stored iris representations. The results of the matching process yield the highest similarity across the registered images for each class were chosen as the matching iris. The performance of the proposed variable-size normalization model and fixed-size normalization models are evaluated with orthogonal polynomials based iris recognition system in terms of standard FAR, FRR and CRR. The empirical outcomes of the proposed variable size normalization for the identification with FAR, FRR and CRR are tabulated in Table It is clear from Table For verification, ROC curves are drawn by plotting the GAR as a function of the FAR in semi-logarithmetic scale. The ROC curves of proposed variable-size normalization model and proposed fixed-size normalization model are plotted for the BITIRIS database and these outcomes are presented in Fig. It is exemplified from the Fig. The EER of the proposed variable-size normalization model and proposed fixed-size normalization model is observed from the ROC curve and are tabulated in the Table It is evident from Table Similarly, the proposed iris normalization schemes are also used to authenticate the person in other database images viz CASIA V 1.0, CASIA V 3.0 Interval, BATH, MMU V 1.0 and MMU V 2.0. The performance of the proposed iris normalization schemes is evaluated with standard FAR, FRR and CRR for various database images and their outcomes are tabulated in Table In this paper, two different normalization methods are proposed that compensate the change in size of the iris due to the action of stretching or enlarging the pupil in iris acquisition process and camera to eyeball distance. In the first method, the variable dimension is used for irregular iris images to avoid under the samples near the limbus border. In the second method, the fixed dimension is used for irregular iris images with a rectangular model to circumvent the dimensional discrepancies among the iris images. The proposed normalization methods are compared along with the orthogonal polynomials based iris recognition and analyzed to enhance the normalization stage. The impacts of proposed variable-size normalization versus rectangular normalization on extracted features are presented. From the empirical outcomes, it is examined that the variable-size normalization scheme performs better than the fixed-size normalization approach in terms of matching. It is concluded that the proposed variable-size normalization makes orthogonal polynomials based iris recognition system more robust to the illumination variations than the proposed fixed-size normalization model.


Is to determine willingness to pay(WTP)/demand for the retreatment of Insecticide treated mosquito nets for malaria control in Ethiopia.A community based cross-sectional study was conducted in Gursum district in Eastern Ethiopia. A total of 335 households were surveyed using a pre-tested structured questionnaire. Multivariable regression analyses using the Tobit model were used to test the theoretical validity of elicited WTP.About 159(76.4 %) of them have received a treated insecticide when they obtained. One hundred twenty five (60.4 %) know that the net should be retreated. Around 110(50.7 %), 80(36.9 %) and 27(12.4 %) of the participants feel that the current price of ITNs as negotiable/ not as such expensive, expensive and cheap. About 306(96.5 %) of them reported that they support that ITNs be given freely and 257(82.9 %) were mentioned that the retreatment service should be provided without charge. The WTP amounts ranged from 0 to 10.4 USD. The mean with SD of the respondents from open ended elicitation method for willingness to pay was 1 USD and 1.53 USD. The reduced tobit regression models showed that average income more than 10.4 USD per month and those household who live within a distance in 30 min to the health facility were the determinant for willingness to pay.The mean with SD of the respondents for willingness to pay was 1 USD and 1.53 USD. Average monthly income and those household who live within a distance in 30 min to the health facility were determinant for willingness to pay. Government and other development partners should seek a mechanism to make a subsidy or free of charge for the retreatment services. Differential treatment from the public to address the poor and vulnerable households and those who are living far distance from the local health facility is warranted. According to WHO, there were an estimated 219 million cases of malaria (range 154–289 million) and 660 000 deaths (range 610 000–971 000) in 2010 .Access to bed nets or their delivery, acceptability and compliance with net use are the other critical issues in the success of any ITNs programme. Aside from access to mosquito nets and insecticides, one of the problems in the large-scale implementation of the ITNs programme is re-treatment of the net . ITN useIn Ethiopia, almost 68 % of the 73 million populations in the country live in malarious areas covering almost 75 % of the land. The strategy for selective vector control measure was ITNs distribution based on segmented market approach and through health facilities and campaigns with prioritizing high risk areas and population group. The target for ITNs is to achieve distribution of 2 ITNs per household (on average) in 90 % of the ITNs targeted areas by 2007 .p < 0.01) for willingness to pay  as revealed by people’s purchase decisions , or by determining their stated or hypothetical level of willingness to pay (WTP) for the nets through the contingent valuation method (CVM) . In thisIn another studies, it was documented that hat potential commercial ITN markets will be undermined if free nets are widely distributed, leaving communities with even poorer access once donor funds run out  and in AIn multivariate analysis, income level has a greater contribution with willingness to pay for retreatment of ITN. This is evidenced that in Tanzania high rates of net purchase in towns was observed but much lower rates in rural areas, especially among the people with lowest incomes whose children have the poorest health  and reacIn our study, distance to the health facility was associated with willingness to pay for the retreatment of ITN. Similar finding has been reported in other studies as well. Non-financial barriers, including distance from health services, and opportunity costs of lost time at work, may also be underlying factors for malaria infection in the poor . In one The study has some limitations. It is difficult to elucidate/disclose the exact income of the household or at individual level and cross-sectional nature of the study for making causality and inference. On top of that, the WTP approach creates a hypothetical market and tries to capture how much they would be willing to pay for the benefits the malaria retreatment provides. Hence, the WTP methods by itself might lead to undervaluation or inflation of the value of the service based on the approach employed and the measure, whether the community understands or imagines the problem, their experience. Though the approach inherently depend on the value that the community gives to the benefit from the service, the findings can be widely used to design policies, prioritize intervention actions, implement cost sharing arrangements etc. As to the knowledge of the authors, this is the first of its kind to determine the WTP for retreatment of ITN services in the country and gives a shade of light for the policy makers to take appropriate decision. There has been discussions on whether public health interventions like malaria net or retreatment can be seen as a purely public good, or marketable good. Given the public importance of these interventions and their abundance in the market at least in many countries, that might not be an easy task to get a best fit. Nonetheless, that doesn’t matter as such in a valuation experiment with a hypothetical setting. And most importantly, an aggregate value by the community for such a service is pertinent in designing policies and strategies in malaria control and treatment. As the study time has passed, we didn’t anticipate what happened recently.p < 0.01) for willingness to pay. So the government and other development partners should see the mechanism to make a subsidy or free of charge for the services hence the community can pay more value for the retreatment of ITN since free distribution of ITNs is not a solution unless retreatment services were available to the community to be implemented. Differential treatment from the public to address the poor and vulnerable households and those who are living far distance from the local health facility is warranted and applying other method of evaluation for estimation of willingness to pay may be warranted to probe and synthesize.In conclusion, the mean with SD of the respondents for willingness to pay was 1 USD and 1.53 USD. Average income more than 10.4 USD per month and those household who live within a distance in 30 min to the health facility were all statistically significant (


When biodegradation starts, the cube will shrink as a consequence of the erosion of the six surfaces. Therefore, the first phenomenon of biodegradation is surface erosion that can be measured as a decrease in thickness per unit of time per unit area (cm day−1 cm−2). In this simple model, suppose a constant erosion rate of 0.0095 cm day−1 cm−2. With this erosion rate it takes about 100 days to have a complete biodegradation of the plastic cube. After 16 days, each side of the cube will be reduced to 0.85 cm  and 0.39 g of plastic will have being assimilated .Most biodegradable plastics are water-insoluble solid materials under normal environmental conditions . Is a percentage of 39% a substantial biodegradation level? If referred to the Polyester X 39% sounds incomplete. But if we consider more carefully the matter, it is the cube that it is still to reach a complete biodegradation. Many polymeric chains of the Polyester X have been already fully biodegraded. Therefore, the biodegradation percentage of a solid polymer does not provide us with relevant information on the mass that has been depolymerised rather it is just a ratio between the mass that has been metabolized and the original mass. More accurately the biodegradation of the residual cube was 0%, while biodegradation of the missing part was 100%.Assume that the process is stopped at this point. The cube is still a plastic item, intact, apart from the surface, a thin, partly degraded layer where the degradation processes is “frozen” in an intermediate phase. A reduction in molecular weight and a loss of material properties has happened  but without weight loss  of 50,000. The moles of polymeric chains present in 0.39 g are: 0.39 g/ = 7.8 × 10−6 mol. This means that: 7.8 × 10−6 × 6.022 × 1023 = 4.70 × 1018 macromolecules have been biodegraded. Still, 7.35 × 1018 macromolecules are intact, in the cube. With an erosion rate of 0.0095 cm day−1 cm−2, a total of 1.14 × 1017 molecules are eroded and biodegraded from each cm2, every day.Assume the amount of polymer that is biodegraded. This is not just dictated by the biodegradation level, since this is a ratio. The number of macromolecules metabolized after 16 days, when the 1 g cube has reached a nominal biodegradation level of 39%, is 390 times higher than the number of macromolecules metabolized from a 1 mg totally biodegraded (100%) sample in 100 days. Therefore, it is clear that neither the test duration nor the biodegradation level are relevant, only the metabolized mass.We will now approach the core argument of this paper: ecotoxicity assessment of biodegradable plastics. The established approach is the following. A plastic sample is made to biodegrade in soil under controlled conditions up to a given extent. Then, a sample of soil is collected and tested with a proper array of ecotoxicity tests. In parallel, a sample of soil that has not been exposed to the plastic is tested with the same approach, as a control. The plastic is assumed to be safe for the environment if no difference between the soils are detected. The higher the sensitivity of the exotoxicity tests, the higher the concentration of the potential ecotoxic chemicals, the higher will be the robustness of this assumption. Therefore, the biodegradation test must be set up with the purpose to maximize the release of the potential toxic chemicals in the soil sample. The chance of detecting potential ecotoxic chemicals, depends on the The consequence of this is that the duration of the test procedure used to prepare the soil for subsequent ecotoxicity testing can be reduced  on the condition that the original mass of plastic and the biodegradation rate are both high. A high mass of plastic provides a high amount of macromolecules available for biodegradation . The biodegradation rate is controlled by the available surface which, in turn, is controlled by the granulometry of the plastics. Thus, plastics must be milled before testing. Best results are obtained by converting the plastics into very thin films, and then fragmenting the films via cryogenic milling.This approach is applicable when: the full biodegradation of the test material has been already proven; the material is isotropic. A material that is made in layers will not behave as the homogeneous “cube” shown in our example. All the layers must be subjected to biodegradation in this case. A material whose biodegradability is not known must be totally degraded before applying the ecotoxicity testing.The necessity of testing high concentrations of test material can in turn be problematic. The sudden addition of high amounts of biodegradable materials represents a disturbance to the soil environment. It is important to note that fresh organic matter  will cause transient “toxic” effects in soil if added in high amounts  the ecotoxicity testing should be carried out on samples of soil where the test plastic material has been added in high concentration ; (ii) the plastic material should be milled in order to get a high surface and a higher biodegradation rate (to reduce testing duration); (iii) a soil where a GRAS material  has been biodegraded should be used as a control, to take into account the effects due to soil disturbance, that could otherwise be mistaken as toxic effects caused by the plastic.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The data have been recorded in stroboscopic mode at controlled and varied phase between the acoustic frequency generator and the synchrotron bunch train. At each time delay (phase angle), the coherent far-field diffraction pattern in the small-angle regime is inverted by an iterative algorithm to yield the local instantaneous surface height profile along the optical axis. The results show that periodic nanoscale dynamics can be imaged at high temporal resolution in the range of 50 ps (pulse length). By integration of the intensity along i.e.Next, we apply further specifications and assumptions geared towards the present experiment. First we have to keep in mind that the illuminated area on the sample surface is highly anisotropic at glancing incidence angle i.e. the constraint that the measured data impose on the solution. This modulus constraint is thus described by an operator In the far-field limit we record the modulus squared of the Fourier transform, a priori knowledge about the SAW-induced height profile, in particular regarding the expected wavelength of the SAW , it is reasonable to assume a corresponding smoothness of The second constraint is formulated in real space. Since the SAW fills the entire illuminated field of view, the standard constraint of a compact support, applicable for the error reduction or hybrid input–output algorithms  Sampling: Given a discrete set of data points as measured by a pixel detector, equations (4)−1 , corresponding to a sampling interval of (b) Uniqueness: A one-dimensional iterative algorithm of GS type as given by equations (4)i.e. it is generally not solvable uniquely  shows the (unit-less) height profile c) shows the excellent agreement of the reconstructed diffraction pattern (in the last iteration before application of the modulus constraint) compared to the input data of the simulation.Fig. 2d). Thus we can conclude that the algorithm described above is in principle able to reconstruct data of the type collected in this experiment.Finally, two reconstructions from a noise-free and a noisy data set  scheme between the synchrotron radiation pulses and the SAW dynamics, and the coherent nanofocus setup. Each part will be discussed separately in the following subsections. All parameters relevant to this experiment are summarized in Table 13.1.et al., 1999i.e. surface waves with elliptical motion of lattice atoms in a plane given by the surface normal and the propagation direction. Standing SAWs of Rayleigh type are generated by two interdigital transducers (IDTs) placed at opposing ends of the acoustically active region (sound path) on the substrate, and matched in frequency and phase. The sound path in between the two IDTs is 8 mm long and 1 mm wide  (stroboscopic illumination). For the 40-bunch filling mode, a bunch repetition rate of et al., 2013Data acquisition is based on the pump–probe scheme. The periodic SAW excitation (pump) is synchronized to the PETRA III (probe) synchrotron radiation pulses. The coherent diffraction pattern is then accumulated by repeated probe pulses at constant time delay 3.3.et al., 2010et al., 2011i.e. 50 µm in the horizontal and 75 µm in the vertical direction. The sample was positioned into the focus of the KB beam and the zero angle was calibrated by recording the specularly reflected beam at various angles of incidence The experiment was carried out at P10 at the synchrotron facility PETRA III at DESY Photon Science , using the coherent nanofocus endstation GINIX (Göttingen Instrument for Nano-Imaging with X-rays)  for data collected with the Pilatus pixel detector. The spacing of the satellite peaks increases with decreasing angle, as expected from the conversion of (1) A rocking (angular) scan in the range b) shows examples of data at different SAW powers et al., 2000et al., 2004(2) An RF power scan in the range of −23 to −7 dBm for all angles b) shows that no satellite peaks are observed when the SAW is powered off, as expected. All measurements that do not involve a power scan have been performed at The lower inset in Fig. 4c) and 4d) show time- (phase-)dependent data for four selected values of ϕ recorded with the sCMOS and Pilatus detectors, respectively. In both cases, a strong dependence of the satellite intensity on the phase is observed, as expected. The minimum in intensity corresponds to the SAW at the instant of zero crossing, i.e. zero amplitude, whereas a maximum corresponds to the SAW at largest amplitude. To quantify this dependence, the intensities of the specular peak and of the (nonspecular) satellite peaks were integrated, as illustrated in Fig. 4d) (red and blue dashed lines).(3) A scan of the SAW phase ϕ in the range e) shows the resulting traces as a function of phase ϕ. The integrated intensity of the second diffraction order is 90° out of phase with respect to the specular reflectivity. The maximum of the SAW amplitude corresponds to a maximum in the nonspecular signal, while the specular signal is decreased by a Debye–Waller-like effect. At this point it is important to note that the satellite intensity is never zero for any given value of the phase ϕ, while it is at zero when the SAW device is powered off. This suggests that the SAW contains propagating modes in addition to the desired standing wave component, most likely due to a mismatch in signal amplitude of the two IDTs. Since the propagating modes are not matched to the phase of the SR pulse, this signal component is washed out over the data accumulation interval, resulting in an offset of the ϕ scan.Fig. 45.d) can be used to illustrate the reconstruction of a time-dependent height profile based on the coherent reflectivity data. In a preprocessing step, the data of each diffraction pattern were integrated over a ten-pixel-wide line along −1 corresponding to an incident angle So far, we have not exploited the fact that the data were recorded under coherent illumination. In this section, we show that the one-dimensional height profile of the SAW et al., 2013−1, 1− was used, similar to the reconstruction of simulated data. Comparing different implementations and parameters of the bandwidth algorithm, it was found that constraining the SAW bandwidth only in the central range of illuminated surface area, while leaving the phase unconstrained in the tails of the beam, stabilized the convergence.For the reconstruction, we constrained the illumination wavefield to that obtained in numerical simulations . The reconstructed and measured diffraction patterns in Fig. 5b) agree well in amplitude and position of the satellite peaks. Convergence is achieved after 50 iterations as monitored through the logarithmic error metric c).The result of the reconstruction for one exemplary phase is shown in Fig. 5Central to our investigation is the reconstruction of time-resolved data. Thus, we apply the reconstruction procedure to the entire set of diffraction profiles, and the unit-less height function 6.In this work we have addressed the reconstruction of time-resolved coherent diffraction data recorded in reflection geometry, as a somewhat technical step towards the goal of time-resolved coherent diffraction imaging. To this end, we have chosen surface acoustic waves (SAWs) excited on a piezoelectric substrate as a well characterized and controlled model system for aperiodic processes, which can be synchronized to the synchrotron pulses. The example shows that the height profile function of the SAW can be reconstructed with a temporal sampling of 178 ps and sub-ångström accuracy for the SAW amplitude. For the reconstruction, a dedicated algorithm was devised, based on a bandwidth constraint suitable for the current application of well controlled SAW frequency. Note that no support constraint was used. Oversampling was achieved by use of a KB focusing.a priori knowledge can be expected to be necessary in this case. Finally, the surface field of view in this demonstration was rather small compared to the SAW wavelength. More interesting configurations of SAW patterns in the presence of surface defects, SAW reflectors or splitters can be addressed in follow-up experiments. To this end, an adjustable beam size in combination with suitable detector pixel sizes is needed. A first step towards the illumination of larger fields of view by shifting the sample to a defocus position has already been made, but reconstruction of the data recorded in the corresponding holographic mode has to date not been achieved. This issue should also be addressed in following experiments.Notwithstanding the significant potential of the method for reconstruction of time-dependent interface profiles, there are several restrictions and points to note about the experimental parameters. First, However, the last argument can also be turned around. Focused coherent beams with cross sections in the range of a few hundred nanometres are ideally suited to probe functional devices with strong structural heterogenieties, for example SAW-based frequency filtering devices, by scanning the beam over the desired region of interest. Experiments in both transmission and reflection geometry are possible, but the strong phase shift induced by interface profiles leads to particularly high signal levels in reflection. Finally and more generally, we have shown that a combination of CXDI with a suitable timing scheme offers significant potential to study surfaces and interfaces a high spatial and temporal resolution.